WO1995022330A1 - Antiviral agents - Google Patents

Abstract

The invention relates to a method for the treatment and/or prophylaxis of a Hepadnaviridae associated infection which comprises administration of an effective amount of a compound of Formula (1), wherein R1 is hydrogen, halogen, hydroxy, azide, optionally substituted alkyl, optionally substituted alkoxy, optionally substituted aryloxy, mercapto, optionally substituted alkylthio, optionally substituted amino, optionally substituted hydrazino or optionally substituted hydroxylamino; R2 is hydrogen, halogen, hydroxy, azide, optionally substituted alkoxy, optionally substituted aryloxy, mercapto, optionally substituted alkylthio or optionally substituted amino; R3 and R3' are the same or different and selected from hydrogen, optionally substituted alkyl, halogen, hydroxy, azide, optionally substituted alkoxy, optionally substituted aryloxy, mercapto, optionally substituted thio and optionally substituted amino; or R3 and R3' together form =O, =S, =NOH or =NOR, wherein R is optionally substituted alkyl; and R4, R5 and R6 are the same or different and selected from optionally substituted alkyl, optionally substituted aralkyl, halogen, hydroxy, azide, optionally substituted alkoxy, optionally substituted aryloxy, mercapto, optionally substituted alkylthio, optionally substituted amino, optionally substituted acyl, optionally substituted ester, cyano, carboxy and mono-, di- or tri- phosphate; two of R4, R5 and R6 are joined together to form a cyclic group; or R4, R5 and R6 are joined together to form a cyclic ortho ester group, salts thereof, pharmaceutically acceptable derivatives thereof, pro-drugs thereof, tautomers thereof and/or isomers thereof to a subject requiring said treatment and/or prophylaxis. The invention also relates to novel compounds of Formula (1), processes for their preparation and pharmaceutical or veterinary compositions containing them.

Description

ANTIVIRAL AGENTS

The present invention relates to purine acyclonucleosides, processes for f their preparation and their use as agents in the treatment or prophylaxis of

Hepadnaviridae associated infections, in particular hepatitis B.

Infection with human hepatitis B virus (hereinafter referred to as "HB V") is a major public health problem because of the ability of the virus to cause acute and chronic infections. Chronic hepatitis B virus infection causes serious liver disease in humans and frequently results in cirrhosis and hepatocellular carcinoma. Currently there is no effective therapy for the successful management of chronic HBV infections. The >250 million chronic HBV carriers throughout the world are unable to benefit from the commercial vaccine now available.

HBV is a member of the Hepadnaviridae family, of which several animal viruses have been identified. These include woodchuck hepatitis virus, ground squirrel, and duck hepatitis virus (hereinafter referred to as "DHBV") (W.S. Mason, G. Seal, and J Summers J Virology 36, 829-836(1980). These viruses share common biological features with HBV including the virion ultrastructure, genomic structure, and a unique mechanism of replication (I.D. Gust, C. J. Burrell, A. G. Coulepsis, W. S. Robinson and A. Zuckerman, Intervirology 25, 14-29 (1986)) and consequently may be used for assaying in vitro the activity of potential anti-HBV agents.

Hannah and Tolman in EP 0 381 531 disclose purine and pyrimidine acyclonucleosides and nucleotides stated to be particularly effective against the herpes group of viruses with special utility against cytomegalovirus(CMV) and varicella-zoster virus (VZV) and against retroviruses such as HTLV HI. EP 0 381 531 provides no evidence for the antiviral activity of the disclosed compounds even against the above viruses. However, Hannah et al in J Heterocyclic Chem., 26, 1261 (1989) give the activity of N-7 and N-9 substituted guanines against the Herpes simplex viruses HSV-1 and HSV-2. These results show that the N-7 substituted guanines disclosed are not active against these viruses. While the N-9 substituted guanines disclosed are mostly weakly active, _m the 3' methoxybutyl N-9 substituted guanine is not active. Bailey and Harnden in J. Chem. Soc. Perkin Trans. I (1988) 2767 independently reported the synthesis and activity of the N-9 substituted guanine compounds confirming that the 3' fluoro compound had good activity against HSV 1 and HSV 2 and that 3' methyl-, hydroxy-, and hydroxymethyl- substituted compounds had slight activity against HSV 1 and HSV 2. They reported that these compounds were not active against Influenza A virus, or para-influenza type 1 (Sendai ) virus.

This reported antiviral activity data show that the activity of a chemical compound does not translate from one virus to another even in a related virus group.

It has now been found that compounds of Formula (1) below are inhibitors of replication of duck and human hepatitis B virus and may be useful control agents for hepatitis B infections when used alone or with another antiviral agent.

According to one aspect of the present invention there is provided a method for the treatment and/or prophylaxis of a Hepadnaviridae associated infection which comprises administration of an effective amount of a compound of Formula (1):

(1) wherein R_j is hydrogen, halogen, hydroxy, azide, optionally substituted alkyl, optionally substituted alkoxy, optionally substituted aryloxy, mercapto, optionally substituted alkylthio, optionally substituted amino, optionally substituted hydrazino or optionally substituted hydroxylamino; R2 is hydrogen, halogen, hydroxy, azide, optionally substituted alkoxy, optionally substituted aryloxy, mercapto, optionally substituted alkylthio or optionally substituted amino;

R3 and R3' are the same or different and selected from hydrogen, optionally substituted alkyl, halogen, hydroxy, azide, optionally substituted alkoxy, optionally substituted aryloxy, mercapto, optionally substituted thio and optionally substituted amino; or R3 and R3' together form =0, =S, =NOH or =NOR, wherein R is optionally substituted alkyl; and

R4, R5 and R are the same or different and selected from optionally substituted alkyl, optionally substituted aralkyl, halogen, hydroxy, azide, optionally substituted alkoxy, optionally substituted aryloxy, mercapto, optionally substituted alkylthio, optionally substituted amino, optionally substituted acyl, optionally substituted ester, cyano, carboxy and mono-, di- or tri- phosphate; two of R4, R5 and R are joined together to form a cyclic group; or R4, R5 and Rg are joined together to form a cyclic ortho ester group,

salts thereof, pharmaceutically acceptable derivatives thereof, pro-drugs thereof, tautomers thereof and/or isomers thereof to a subject requiring said treatment and/or prophylaxis

Throughout this specification unless the context requires otherwise, the word "comprise", or variations such as "comprises" or "comprising", will be understood to imply the inclusion of a stated integer or group of integers but not the exclusion of any other integer or group of integers.

The term "Hepadnaviridae associated infection" is used herein in its broadest sense. A particular example of such an infection is human hepatitis B. The subject may be a human or an animal such as a domestic or wild animal, particularly an animal of economic importance.

An "effective amount" of the compound of Formula (1) is an amount sufficient to inhibit or reduce viral replication, generally by greater than 50% (as measured by viral DNA levels or reverse transcriptase activity).

The salts of the compound of Formula (1) are preferably pharmaceutically acceptable, but it will be appreciated that non-pharmaceutically acceptable salts also fall within the scope of the present invention, since these are useful as intermediates in the preparation of pharmaceutically acceptable salts. Examples of pharmaceutically acceptable salts include salts of pharmaceutically acceptable cations such as sodium, potassium, lithium, calcium, magnesium, ammonium and alkylammonium; acid addition salts of pharmaceutically acceptable inorganic acids such as hydrochloric, orthophosphoric, sulphuric, phosphoric, nitric, carbonic, boric, sulfamic and hydrobromic acids; or salts of pharmaceutically acceptable organic acids such as acetic, propionic, butyric, tartaric, maleic, hydroxymaleic, fumaric, citric, lactic, mucic, gluconic, benzoic, succinic, oxalic, phenylacetic, methanesulphonic, toluenesulphonic, benzenesulphonic, salicyclic, sulphanilic, aspartic, glutamic, edetic, stearic, palmitic, oleic, lauric, pantothenic, tannic, ascorbic and valeric acids.

By "pharmaceutically acceptable derivative" is meant any pharmaceutically acceptable salt, hydrate or any other compound which, upon administration to the subject, is capable of providing (directly or indirectly) a compound of Formula (I) or an antivirally active metabolite or residue thereof.

The term "pro-drug" is used herein in its broadest sense to include those compounds which are converted in vivo to compounds of Formula (1). For example, a compound of Formula (1) wherein R. and/or R~ are acyloxy substituted alkyl groups may be converted in vivo to a compound of Formula (1) wherein R. and/or R₅ as appropriate is a hydroxy substituted alkyl group.

The term "tautomer" is used herein in its broadest sense to include compounds of Formula (1) which are capable of existing in a state of equilibrium between two isomeric forms. Such compounds may differ in the bond connecting two atoms or groups and the position of these atoms or groups in the compound, for example, -OH and -SH substituents on the compound of Formula (1) may be replaced by =0 and =S substituents, respectively.

The term "isomer" is used herein in its broadest sense and includes structural, geometric and stereo isomers. As the compound of Formula (1) has a chiral centre, it is capable of existing in enantiomeric forms. The present invention includes enantiomers in isolated form and mixtures thereof. When R^, R5 and R6 are different then the present invention includes all enantiomers and diasteriomers of the compound of Formula (1) that may result from this situation in isolated form and mixtures thereof.

The term "halogen" denotes fluorine, chlorine, bromine or iodine, preferably chlorine or fluorine.

The term "alkyl" used either alone or in compound words such as "optionally substituted alkyl", "optionally substituted alkylthio" and "aralkyl" denotes straight chain, branched or cyclic alkyl, preferably C1.30 alkyl or cycloalkyl. Examples of straight chain and branched alkyl include methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl, tert-butyl, amyl, isoamyl, sec-amyl, 1,2-dimethylpropyl, 1,1- dimethylpropyl, hexyl, 4-methylpentyl, 1-methylpentyl, 2-methylpentyl, 3- methylpentyl, 1,1-dimethylbutyl, 2,2-dimethylbutyl, 3,3-dimethylbutyl, 1,2- dimethylbutyl, 1,3-dimethylbutyl, 1,2,2,-trimethylpropyl, 1,1,2-trimethylpropyl, heptyl, 5-methylhexyl, 1-methylhexyl, 2,2-dimethylpentyl, 3,3-dimethylpentyl, 4,4- dimethylpentyl, 1,2-dimethylpentyl, 1,3-dimethylpentyl, 1,4-dimethylpentyl, 1,2,3,- trimethylbutyl, 1,1,2-trimethylbutyl, 1,1,3-trimethylbutyl, octyl, 6-methylheptyl, 1- methylheptyl, 1,1,3,3-tetramethylbutyl, nonyl, 1-, 2-, 3-, 4-, 5-, 6- or 7-methyloctyl, 1-, 2-, 3-, 4- or 5-ethylheptyl, 1-, 2- or 3-propylhexyl, decyl, 1-, 2-, 3-, 4-, 5-, 6-, 7- and 8-methylnonyl, 1-, 2-, 3-, 4-, 5- or 6-ethyloctyl, 1-, 2-, 3- or 4-propylheptyl, undecyl, 1-, 2-, 3-, 4-, 5-, 6-, 7-, 8- or 9-methyldecyl, 1-, 2-, 3-, 4-, 5-, 6- or 7- ethylnonyl, 1-, 2-, 3-, 4- or 5-propyloctyl, 1-, 2- or 3-butylheptyl, 1-pentylhexyl, dodecyl, 1-, 2-, 3-, 4-, 5-, 6-, 7-, 8-, 9- or 10-methylundecyl, 1-, 2-, 3-, 4-, 5-, 6-, 7- or

8-ethyldecyl, 1-, 2-, 3-, 4-, 5- or 6-propylnonyl, 1-, 2-, 3- or 4-butyloctyl, 1-2- pentylheptyl and the like. Examples of cyclic alkyl include mono- or polycyclic alkyl groups such as cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, cyclononyl, cyclodecyl and d e like.

The term "alkoxy" used either alone or in compound words such as "optionally substituted alkoxy" denotes straight chain or branched alkoxy, preferably C_j_3o alkoxy. Examples of alkoxy include methoxy, ethoxy, n-propyloxy, isopropyloxy and the different butoxy isomers.

The term "acyl" used either alone or in compound words such as "optionally substituted acyl" denotes carbamoyl, aliphatic acyl group and acyl group containing an aromatic ring, which is referred to as aromatic acyl or a heterocyclic ring which is referred to as heterocyclic acyl, preferably

acyl. Examples of acyl include carbamoyl; straight chain or branched alkanoyl such as formyl, acetyl, propanoyl, butanoyl, 2-methylpropanoyl, pentanoyl, 2,2-dimethylpropanoyl, hexanoyl, heptanoyl, octanoyl, nonanoyl, decanoyl, undecanoyl, dodecanoyl, tridecanoyl, tetradecanoyl, pentadecanoyl, hexadecanoyl, heptadecanoyl, octadecanoyl, nonadecanoyl and icosanoyl; alkoxycarbonyl such as methoxycarbonyl, ethoxycarbonyl, t-butoxycarbonyl, t-pentyloxycarbonyl and heptyloxycarbonyl; cycloalkylcarbonyl such as cyclopropylcarbonyl cyclobutylcarbonyl, cyclopentylcarbonyl and cyclohexylcarbonyl; alkylsulfonyl such as methylsulfonyl and ethylsulfonyl; alkoxysulfonyl such as methoxysulfonyl and ethoxysulfonyl; aroyl such as benzoyl, toluoyl and naphthoyl; aralkanoyl such as phenylalkanoyl (e.g. phenylacetyl, phenylpropanoyl, phenylbutanoyl, phenylisobutyl, phenylpentanoyl and phenylhexanoyl) and naphthylalkanoyl (e.g. naphthylacetyl, naphthylpropanoyl and naphthylbutanoyl); aralkenoyl such as phenylalkenoyl (e.g. phenylpropenoyl, phenylbutenoyl, phenylmethacrylyl, phenylpentenoyl and phenylhexenoyl and naphthylalkenoyl (e.g. naphthylpropenoyl, naphthylbutenoyl and naphthylpentenoyl); aralkoxycarbonyl such as phenylalkoxycarbonyl (e.g. benzyloxycarbonyl); aryloxycarbonyl such as phenoxycarbonyl and naphthyloxycarbonyl; aryloxyalkanoyl such as phenoxyacetyl and phenoxypropionyl; arylcarbamoyl such as phenylcarbamoyl; arylthiocarbamoyl such as phenylthiocarbamoyl; arylglyoxyloyl such as phenylglyoxyloyl and naphthylglyoxyloyl; arylsulfonyl such as phenylsulfonyl and naphthylsulfonyl; heterocycUccarbonyl; heterocyclicalkanoyl such as thienylacetyl, thienylpropanoyl, thienylbutanoyl, thienylpentanoyl, thienylhexanoyl, thiazolylacetyl, thiadiazolylacetyl and tetrazolylacetyl; heterocyclicalkenoyl such as heterocyclicpropenoyl, heterocyclicbutenoyl, heterocyclicpentenoyl and heterocycUchexenoyl; and heterocyclicglyoxyloyl such as thiazolylglyoxyloyl and thienylglyoxyloyl.

The term "aryl" used either alone or in compound words such as "optionally substituted aryloxy" denotes single, polynuclear, conjugated and fused residues of aromatic hydrocarbons or aromatic heterocyclic ring systems. Examples of aryl include phenyl, biphenyl, terphenyl, quaterphenyl, phenoxyphenyl, naphthyl, tetrahydronaphthyl, anthracenyl, dihydroanthracenyl, benzanthracenyl, dibenzanthracenyl, phenanthrenyl, fluorenyl, pyrenyl, indenyl, azulenyl, chrysenyl, pyridyl, 4-phenylpyridyl, 3-phenylpyridyl, thienyl, furyl, pyrryl, pyrrolyl, furanyl, imadazolyl, pyrrolydinyl, pyridinyl, piperidinyl, indolyl, pyridazinyl, pyrazolyl, pyrazinyl, thiazolyl, pyrimidinyl, quinolinyl, isoquinolinyl, benzofuranyl, benzothienyl, purinyl, quinazolinyl, phenazinyl, acridinyl, benzoxazolyl, benzothiazolyl and the like. Preferably, the aromatic heterocylic ring system contains 1 to 4 heteratoms independently selected from N, O and S and containing up to 9 carbon atoms in the ring.

The term "ester" used either alone or in compound words such as "optionally substituted ester" denotes acyloxy, acylalkoxy or acyloxyaryl groups wherein the terms "acyl", "alkoxy" and "aryl" are as defined above.

The term "aralkyl" used either alone or in compound words such as "optionally substituted aralkyl" denotes arylalkyl groups wherein the terms "aryl" and "alkyl" are as defined above, such as, for example, benzyl.

The term "cyclic group" denotes the aryl groups defined above and non- aromatic ring systems which may also contain the additional heteroatom P. Examples of suitable cyclic groups include optionally substituted cyclic acetals, carbonates, alkyls, phosphates, or orthoesters.

In this specification "optionally substituted" means that a group may or may not be further substituted with one or more groups, preferably 1 to 5 groups selected from alkyl, alkenyl, alkynyl, aryl, halo, haloalkyl, haloalkenyl, haloalkynyl, haloaryl, hydroxy, alkoxy, alkenyloxy, aryloxy, carboxy, benzyloxy, haloalkoxy, haloalkenyloxy, haloaryloxy, nitro, nitroalkyl, nitroalkenyl, nitroalkynyl, nitroaryl, nitroheterocyclyl, azido, amino, alkylamino, dialkylamino, alkenylamino, alkynylamino, arylamino, diarylamino, benzylamino, dibenzylamino, acyl, alkenylacyl, alkynylacyl, arylacyl, acylamino, diacylamino, acyloxy, aldehydo, alkylsulphonyloxy, arylsulphonyloxy, heterocyclyl, heterocycloxy, heterocyclamino, haloheterocyclyl, alkylsulphenyl, arylsulphenyl, carboalkoxy, carboaryloxy, mercapto, alkylthio, arylthio, acylthio and phosphorus-containing groups. Preferably R., R- and/or R_fi in the compound of Formula (1) are optionally substituted alkyl, for example, alkyl substituted with hydroxy or optionally substituted alkoxy.

A preferred sub-class of compounds of Formula (1) have the Formula (la):

(la) wherein R_j, R2 and Rg are as defined above; and

R4'and R5' are the same or different and selected from hydrogen, optionally substituted alkyl, optionally substituted aralkyl, optionally substituted aminoacyl, optionally substituted acyl, optionally substituted ester and mono-, di- or tri- phosphate, provided that when one of R4', R_ζ' or R5 is or contains mono-, di- or tri- phosphate then the remaining groups are other than phosphate; two of R4', R5' and

R are joined together to form a cyclic acetal group, a cyclic carbonate group or a cyclic phosphate group; or R4', R5' and Rg are joined together to form a cyclic ortho ester group.

Thus, in an alternative aspect, the present invention provides a method for the treatment and/or prophylaxis of a Hepadnaviridae associated infection which comprises adminstration of an effective amount of a compound of Formula (la) defined above. The present invention also provides use of a compound of Formula (1) or

(la) as defined above in the manufacture of a medicament for the treatment and/or prophylaxis of Hepadnaviridae associated infection.

The present invention further provides a compound of Formula (1) or (la) as defined above for use in the treatment and/or prophylaxis of a Hepadnaviridae associated infection.

Most of the compounds of Formula (1) are novel per se. Thus in another aspect of the present invention there is provided a compound of the Formula (1) as defined above with the provisos that:

(a) when

R_j is benzyloxy;

R2 is amino; and

R4 and R5 together form 2,2-dimethyl-l,3-dioxane, then _β is other than fluoro, methoxy or hydroxy;

(b) when

R_j is hydroxy;

R2 is amino; and

R4 and R5 are hydroxymethyl, then R-g is other than fluoro, hydroxy, methoxy, methyl or hydroxymethyl;

(c) when

R_j is hydroxy;

R2 is amino; and

R4 and R5 together form

then Rg is other than fluoro, hydroxy or methoxy; or

(d) when

R_j is chloro;

R2 is amino; and

R4 and R5 together form 2,2-dimethyl-l,3-dioxane, then Rg is other than methyl or hydroxymethyl.

The present invention also provides a compound of Formula (la) as defined above with the provisos that: (a) when

Rj is benzyloxy;

R2 is amino; and

R4' and R5' together form

:CR₁₄ R₁₅

wherein R14 and R_j5 are methyl, then Rg is other than fluoro, methoxy or hydroxy;

(b) when

R_j is hydroxy; R2 is amino; and

R4' and R5' are hydrogen, then Rg is other than fluoro, hydroxy, methoxy, methyl or hydroxymethyl;

(c) when

R_j is hydroxy;

R2 is amino; and

R4' and R5' together form

wherein R is as defined above, then Rg is other than fluoro, hydroxy or methoxy; or

(d) when

Rj is chloro;

R2 is amino; and

R4' and R5' together form

CR₁ R₁₅

wherein R_j4 and R15 are methyl,

then Rg is other than methyl or hydroxymethyl.

Preferred Regroups are hydrogen, halogen, hydroxy, optionally substituted

alkoxy, optionally substituted hydroxylamino or optionally substituted hydrazino. Preferred R₂ groups are -NR Rg, wherein R is hydrogen, optionally

substituted alkyl or optionally substituted acyl and Rg is hydrogen or alkyl.

Preferred R^ groups are -alkylORg', fluorine, -ORg' or methyl, wherein Rg'

is hydrogen, optionally substituted alkyl, optionally substituted aminoacyl, optionally substituted acyl or mono-, di- or tri-phosphate.

Preferred R4', R5', Rg' and/or R7 groups are those selected from hydrogen, aminoacyl, R9- C (O)- and R9-C (S)- ,wherein Rg is hydrogen, optionally substituted alkyl, optionally substituted alkoxy, optionally substituted aryl, optionally substituted aralkyl, optionally substituted heterocyclyl or -NRJ_QR, . ,

wherein RJQ and R_{j j} are the same or different and are selected from hydrogen and alkyl or together with the nitrogen to which they are attached form a 3-7 membered heterocyclic ring.

RΛ R-\ Rg' and/or R7 may also represent other suitable acyl groups such

as those derived from amino acids. These amino acyl derivatives may be derived for example from naturally occuring amino acids, preferably neutral amino acids, that is, amino acids with one amino group and one carboxyl group. Examples of suitable amino acids include phenylalanine and aliphatic acids containing up to 6 carbon atoms such as glycine, alanine, valine and isoleucine. The amino acids may be D-, L-and DL amino acids, with the natural L-amino acids being most preferred. Suitable examples of phosphate groups for R, or RΛ or R_<- or R_<-' and/or

R₆ or Rg' form the group:

R_{1 2} O

R13 O P o

o

wherein Rj2 and R13 are the same or different and are selected from hydrogen,

alkyl, optionally substituted aryl, optionally substituted aralkyl and pharmaceutically acceptable cations, preferably hydrogen and pharmaceutically acceptable cations.

Suitable examples of a cyclic acetal group, a cyclic carbonate group or a

cyclic phosphate group formed when two of RΛ R^' and R^ or Rg' together form the groups:

wherein R, « s as defined above; R₁₄ and R_j5 are the same or different and are selected from hydrogen or C. , alkyl.

A suitable example of cyclic ortho ester groups formed when R. or RΛ or

R or R_<-' and/or R_fi or Rg' together form the group:

^ CR₁₄

wherein R, . is as defined above. A preferred group of compounds of Formula (la) are those wherein

R*. is hydrogen, halogen, hydroxy, alkoxy, aryloxy, mercapto, alkylthio, amino,

benzyloxy, hydroxy lamino or hydrazino, preferably R, is hydrogen, hydroxy or

C_j g alkoxy;

R2 is amino or acylamino;

Rg is CH₂-OR₆' or C₂H₄-OR₆';

RΛ R_<-' and/or R are the same or different and selected from hydrogen, halogen,

optionally substituted alkyl, optionally substituted aralkyl, optionally substituted ester, optionally substituted aminoacyl and optionally substituted acyl; or two of

R Λ R_<-' and/or R,' are joined together to form a cyclic acetal group or a cyclic

carbonate group.

Many of these preferred compounds act as prodrugs of the compound of Formula (2). That is they are converted in vivo to the compound of Formula (2):

Accordingly in a preferred aspect of the present invention the compound of Formula (1) is a compound of Formula (2) or pharmaceutically acceptable salts thereof, esters, ethers, acyl or aminoacyl derivatives thereof and/or pro-drugs thereof.

Some of the compounds of Formulae (1) and (la) may be prepared by known methods such as those disclosed by Hannah and Tolman in EP 0381531 and Heterocyclic Chem., 26, 1261 (1989) or by Bailey and Harnden in J Chem. Soc. Perkin Trans. I, (1988) 2767.

In a further aspect of the present invention, there is provided a process for the preparation of the novel compounds of Formula (la) which comprises the steps of: (a) reacting a compound of Formula (3):

(3) wherein

R_j and R2 are as defined above with a compound of Formula (4):

(4)

wherein R_a and R_j, may be the same or different and are selected from R4' and R5'as defined above, hydrogen and benzoyl;

R_c is the same as Rg as defined above, hydroxy, hydroxyalkyl or protected derivatives thereof; and

Z is a leaving group, to form a compound of Formula (6):

(6) wherein

R_j , R2, R_a, Rb and R_c are as defined above; and

(b) if necessary, converting the compound of Formula (6) to a compound of Formula (la).

It will be appreciated that the process of the invention may also be used to prepare the known compounds of Formulae (1) and (la).

In the compound of Formula (3), R, is preferably chlorine or benzyloxy, more preferably chlorine and R₂ is preferably amino or aminoacyl, more preferably amino. In the compound of Formula (4), protected hydroxy may be acyloxy or alkoxy; protected hydroxyalkyl may be acyloxyalkyl or alkoxyalkyl; and protected amino may be acylamino.

Suitable examples of the leaving group Z are methane sulfonate or bromine.

The compound of Formula (4) may be prepared from a compound of Formula (5):

(5) wherein R , R, and R are as defined above, either by activation of the hydroxyl

group to form the leaving group Z or by replacement of the hydroxyl group to give a leaving group Z. An example of the former process is treatment of the compound of Formula (5) with methane sulfonyl chloride under conventional conditions to form the compound of Formula (4) wherein Z is a methane sulfonyl group. An example of the latter process is the treatment of the compound of Formula (5) with carbon tetrabromide and triphenylphosphine in an organic, aprotic solvent, such as, dimethylformamide to prepare the compound of Formula (4) wherein Z is bromine.

Compounds of Formula (5) may be prepared by the methods disclosed by Hannah and Tolman in EP 0381531 and J Heterocyclic Chem., 26, 1261 (1989) and by Bailey and Harnden in J Chem. Soc. Perkin Trans. I, (1988) 2767 or modifications of these methods. The product of the reaction between the compound of Formula (3) and the compound of Formula (4) is a compound of Formula (6):

(6)

wherein R , R, and R are as defined above. The compound of Formula (6) may

also, depending on the nature of the groups R , R, and R , be a compound of

Formula (la).

Compounds of Formula (6) can be readily converted into compounds of Formula (1) or (la) using methods known in the art. Compounds of Formula (1) or (la) can be converted into other compounds of Formula (1) or (la) using similar known methods. Such known methods may include the removal of protecting groups, hydrogenation, aminolysis, hydrolysis, alkylation, acylation and phosphorylation.

In accordance with conventional processes known in the art, compounds of Formula (1) or (la) that have acyclic hydroxyl groups may be readily converted into the compounds of Formula (1) or (la) with either acyl or phosphate groups or a mixture of these groups on the acyclic chain. In some instances it may be useful to utilise protected intermediates of the compounds of Formula (1) or (la) in order to prepare the desired final product of Formula (la). Such intermediates may be prepared in accordance with known methods and when no longer required the protecting groups removed using known methods. Examples of suitable protecting groups are trimethylsilyl and monomethoxytrityl groups.

The acylation reaction of compounds of Formula (1) or (la) with acyclic hydroxyl groups may be carried out using an acylating agent containing a group

aminoacyl or R9- C (0)-, wherein R_g is as defined above.

Examples of other acylating agents suitable for use in the process are carboxylic acids, acid halides and acid anhydrides. The reaction may be carried out in a conventional manner, for example in a solvent such as pyridine or dimethylformamide, optionally in the presence of a coupling agent such as N,N'- dicyclohexylcarbodiimide and optionally in the presence of a catalytic base such as 4-dimethylaminopyridine. The product of the reaction may be isolated in a conventional manner.

In the case of amino acids or their functional equivalents, for example acid halides, it may be advantageous, in order to avoid side reactions, to use amino protected derivatives of the amino acid or amino acid equivalent, for example benzyloxycarbonyl derivatives. Such derivatives are commercially available. The protecting groups may be removed utilising standard procedures.

The acylation reactions may produce a single compound of Formula (1) or (la) incorporating one or more acyl groups or may produce a mixture of compounds of Formula (1) or (la) incorporating acyl groups. The outcome depends on a number of factors, such as the relative amounts and chemical nature of the reactants, the physical conditions of the reaction, and the solvent system. Any mixture produced in this way may be separated using standard techniques, preferably chromatography.

It will be appreciated that it is possible to produce compounds of Formula (1) or (la) which may have a mixture of different acyl and/or alkyl groups.

Protected intermediates of the compounds of Formula (1) or (la) may also be used to prepare compounds of Formula (1) or (la) incorporating phosphate . esters.

Compounds of Formula (1) incorporating cyclic monophosphates may be produced according to the known methods discussed above.

The present invention also extends to a pharmaceutical or veterinary composition for the treatment and/or prophylaxis of a Hepadnaviridae associated infection which comprises a compound of Formula (1) or (la) as defined above in association with a pharmaceutically or veterinarily acceptable carrier, diluent, adjuvant and/or excipient.

The compounds of the invention may be advantageously used in combination therapy with other antiviral agents such as Ganciclovir, Famcyclovir, Pencyclovir, Lamivudine and interferon. Hence a preferred method in accordance with the present invention utilises the compound of Formula (1) or (la), analogue or derivative in conjunction with another antiviral agent.

The compound of Formula (1) or (la), also referred to herein as the "active ingredient", may be administered for therapy by any suitable route, including oral, rectal, nasal, topical (including buccal and sublingual), vaginal and parenteral (including subcutaneous, intramuscular, intravenous and intradermal). Preferably, administration will be by the oral route, however it will be appreciated that the preferred route will vary with the condition and age of the subject and the chosen active ingredient.

The compositions of the present invention comprise at least one compound of Formula (1) or (la), together with one or more pharmaceutically acceptable carriers, diluents, adjuvants and/or excipients and optionally other antiviral or therapeutic agents. Each carrier, diluent, adjuvant and/or excipient must be pharmaceutically "acceptable" in the sense of being compatible with the other ingredients of the composition and not injurious to the subject. Compositions include those suitable for oral, rectal, nasal, topical (including buccal and sublingual), vaginal or parenteral (including subcutaneous, intramuscular, intravenous and intradermal) administration. The compositions may conveniently be presented in unit dosage form and may be prepared by any methods well known in the art of pharmacy. Such methods include the step of bringing into association the active ingredient with the carrier diluent, adjuvant and/or excipient which constitutes one or more accessory ingredients. In general, the compositions are prepared by uniformly and intimately bringing into association the active ingredient with liquid carriers, diluents, adjuvants and/or excipients or finely divided solid carriers or both, and then if necessary shaping the product.

Compositions of the present invention suitable for oral administration may be presented as discrete units such as capsules, sachets 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 or non-aqueous liquid; or as an oil-in-water liquid emulsion or a water-in-oil liquid emulsion. The active ingredient may also be presented as a bolus, electuary or paste.

A tablet may be made by compression or moulding, optionally with one or more accessory ingredients. Compressed tablets may be prepared by compressing in a suitable machine the active ingredient in a free-flowing form such as a powder or granules, optionally mixed with a binder (e.g inert diluent, preservative disintegrant (e.g. sodium starch glycolate, cross-linked polyvinylpyrrolidone, cross-linked sodium carboxymethyl cellulose) surface-active or dispersing agent. Moulded tablets may be made by moulding in a suitable machine a mixture of the powdered compound moistened with an inert liquid diluent. The tablets may optionally be coated or scored and may be formulated so as to provide slow or controlled release of the active ingredient therein using, for example, hydroxypropylmethyl cellulose in varying proportions to provide the desired release profile. Tablets may optionally be provided with an enteric coating, to provide release in parts of the gut other than the stomach.

Compositions suitable for topical administration in the mouth include lozenges comprising the active ingredient in a flavoured basis, usually sucrose and acacia or tragacanth gum; pastilles comprising the active ingredient in an inert basis such as gelatin and glycerin, or sucrose and acacia gum; and mouthwashes comprising the active ingredient in a suitable liquid carrier.

Compositions for rectal administration may be presented as a suppository with a suitable base comprising, for example, cocoa butter or a salicylate.

Compositions suitable for vaginal administration may be presented as pessaries, tampons, creams, gels, pastes, foams or spray formulations containing in addition to the active ingredient such carriers as are known in the art to be appropriate.

Compositions suitable for parenteral administration include aqueous and non-aqueous isotonic sterile injection solutions which may contain anti-oxidants, buffers, bacteriostats and solutes which render the composition isotonic with the blood of the intended recipient; and aqueous and non-aqueous sterile suspensions which may include suspending agents and thickening agents. The compositions may be presented in unit-dose or multi-dose sealed containers, for example, ampoules and vials, and may be stored in a freeze-dried (lyophilized) condition requiring only the addition of the sterile liquid carrier, for example water for injections, immediately prior to use. Extemporaneous injection solutions and suspensions may be prepared from sterile powders, granules and tablets of the kind previously described.

Preferred unit dosage compositions are those containing a daily dose or unit, daily sub-dose such as one or more unit dosage forms per day or an appropriate fraction thereof, of an active ingredient.

The compound of Formula (1) or (la) may also be presented for use in the form of veterinary compositions, which may be prepared, for example, by methods that are conventional in the art. Examples of such veterinary compositions include those adapted for:

(a) oral administration, external application, for example drenches (e.g. aqueous or non-aqueous solutions or suspensions); tablets or boluses; powders, granules or pellets for admixture with feed stuffs; pastes for application to the tongue;

(b) parenteral administration for example by subcutaneous, intramuscular or intravenous injection, e.g. as a sterile solution or suspension; or (when appropriate) by intramammary injection where a suspension or solution is introduced into the udder via the teat;

(c) topical application, e.g. as a cream, ointment or spray applied to the skin; or

(d) intravaginally, e.g. as a pessary, cream or foam. It should be understood that in addition to the active ingredients particularly mentioned above, the compositions of this invention may include other agents conventional in the art having regard to the type of composition in question, for example, those suitable for oral administration may include such further agents as binders, sweeteners, thickeners, flavouring agents disintegrating agents, coating agents, preservatives, lubricants and/or time delay agents.

Suitable sweeteners include sucrose, lactose, glucose, aspartame or saccharin. Suitable disintegrating agents include corn starch, methylcellulose, polyvinylpyrrolidone, xanthan gum, bentonite, alginic acid or agar. Suitable flavouring agents include peppermint oil, oil of wintergreen, cherry, orange or raspberry flavouring. Suitable coating agents include polymers or copolymers of acrylic acid and/or methacrylic acid and/or their esters, waxes, fatty alcohols, zein, shellac or gluten. Suitable preservatives include sodium benzoate, vitamin E, alpha-tocopherol, ascorbic acid, methyl paraben, propyl paraben or sodium bisulphite. Suitable lubricants include magnesium stearate, steric acid, sodium oleate, sodium chloride or talc. Suitable time delay agents include glyceryl monostearate or glyceryl distearate.

The invention will now be described with reference to the following non- limiting Examples. These Examples are not intended to limit the scope of the invention in any way. The term "active ingredient" as used in Examples 111 to 113 refers to the compound of Formula (1) or (la) or a pharmaceutically acceptable derivative thereof. All temperatures are in degrees Celsius (°C) unless otherwise stated. GENERAL

All melting points were determined on a Mettler FP51, an Electrothermal 9300 or a Reichert apparatus, and are uncorrected. Microanalyses were performed by the National Analytical Laboratories Pty. Ltd.. H n.m.r. specta were recorded on a Varian Gemini (200 MHz, Fourier mode), a Bniker (200 MHz, Fourier mode) or a Bruker (250 MHz, Fourier mode) n.m.r. spectrometer and 13c n.m.r. spectra were performed on the same instruments at 50 MHz. or 62.5 MHz. All chemical shifts are expressed in parts per million (ppm) downfϊeld from TMS (δ scale). The multiplicity patterns are designated as s (singlet), d (doublet), t (triplet), q (quartet), m (multiplet) and br (broad). High resolution mass measurements were recorded on a JEOL DX303 mass spectrometer. Merck silica gel (230-400 mesh ASTM) was employed in column chromatography. Unless otherwise stated, all organic extracts were dried over magnesium sulfate, filtered and solvents removed on a rotary evaporator. Unless otherwise specified, unreferenced reagents were obtained commercially and used as supplied.

EXAMPLE 1

9-[4-Acetoxy-3,3-bis(acetoxymethyl)-but-l-yl]-2-amino-6-chloropurine

Step A

2-Benzyloxyethanol

To potassium hydroxide (1900 g, 35 mol) was added 1,2-ethanediol (4800 ml, 87 mol). The mixture was heated to 90°. Benzyl chloride (4000 ml, 35 mol) was added dropwise over 3 hrs, maintaining the temperature at 90°. The mixture was then heated to 130° and held for 2 hrs. The mixture was then allowed to cool to room temperature. The product was separated into two batches and each half extracted with ethyl acetate (6 x 1000 ml) from water (8000 ml). The combined organic extract was backwashed with water, dried and the solvent removed. The crude product was distilled to give an oil. Yield 3800 g (72%). b.p. 123-127 8 mm Hg (Lit. 265 760 mm Hg). IH n.m.r. (CDCI3) δ 2.25, q; 3.60, m; 3.75, m;

4.56, s; 7.35, s.

Step B

2-Benzyloxyethyl methanesulfonate

To 2-benzyloxyethanol (30 g, 20 ml, 0.2 mol), triethylamine (1.4 eq., 39 ml) and dichloromethane (375 ml), immersed in an ice/water bath under an atmosphere of argon, was added dropwise a solution of methane sulfonyl chloride (1.1 eq., 17.1 ml, 0.22 mol) in dichloromethane (114 ml) over 100 min. The reaction mixture was allowed to stir at approximately 4° for an additional 1 hr. The mixture was then transfered to a separating funnel and washed successively with 400 ml portions of 2M HC1 (x 2), sat. aqueous sodium hydrogen carbonate, and 5% aqueous sodium chloride. The organic phase was dried and the solvent removed to give an oil. Crude yield 46.3 g (quant.). H n.m.r. (CDCI3) δ 3.05, s; 3.75, m;

4.41, m; 4.60, s; 7.35, s.

Step C

Diethyl (2-benzyloxyethyl)malonate

To a stirred suspension of NaH (83% in oil, 3.66 g, 0.13 mol) in THF (32 ml) immersed in an ice/water bath was added dropwise, over 90 min, diethyl malonate (33.76 g, 32.15 ml, 0.211 mol). To the resulting enolate solution was added dropwise 2-benzyloxyethyl methanesulfonate (25 g, 0.109 mol) and the mixture stirred at reflux for 11 hrs. The reaction was allowed to cool to room temperature, then transfered to a conical flask containing 2M HC1 (200 ml) and diethyl ether (300 ml) and stirred for 30 min. The phases were separated and the aqueous phase further extracted with ether (2 x 100 ml). The combined organic extracts were dried and the solvent removed to give an oil. The crude product was distilled.

Yield 25.83 g (81%). b.p. 150-15570.4 mm Hg. IH n.m.r. (CDCI3) δ 1.25, t; 2.23, q; 3.55, t; 4.60, t; 4.15, q; 4.50, s; 7.30, s. Step D

Diethyl hydroxymethyl(2-benzyloxyethyl)malonate

A mixture of diethyl (2-benzyloxyethyl)malonate (2 eq., 20.0 g, 67.9 mmol), formalin (37% w/v, 2.6 ml, 34.7 mmol), ethanol (4.0 ml) and potassium hydrogen carbonate (7.5 g, 74.9 mmol) in a 100 ml flask, under an atmosphere of argon, was stirred at room temperature for 3 days. The mixture was transfered to a separating funnel and 5% aqueous sodium chloride (130 ml) added, the product was extracted with diethyl ether (3 x 100 ml). The combined organic extracts were dried and the solvent removed. The crude product was preadsorbed onto silica and flash chromatographed, eluting with 1:9 ethanol:petroleum spirits (60-80°). Fractions containing the second peak were combined and the solvent removed to give an oil.

Yield 8.78 g (79.7%). IH n.m.r. (CDCI3) δ 1.24, t; 1.60, s; 2.33, t; 3.05, t; 3.61, t;

4.02, d; 4.18, q; 4.48, s; 7.70, s.

Step E

2-(2-Benzyloxyethyl)-2-hydroxymethylpropane-l,3-diol

To solid sodium borohydride (95%, 2.45 g, 61.5 mmol) in a 100 ml flask, under an atmosphere of argon, immersed in an ice/water bath with a reflux condenser, was added dropwise over 20 min a solution of diethyl hydroxymethyl(2- benzyloxyethyl)malonate (1.0 g, 3.08 mmol) in methanol (15 ml). The mixture was then gently heated to reflux and stirred overnight. The methanol was found to have completely evaporated, thus additional methanol was added and the mixture allowed to reflux for a further 17 hrs. The mixture was then cooled to room temperature and water (12 ml) was carefully added to the mixture and stirred for 30 min. The methanol was removed. The residue was then placed in a separating funnel and washed with chloroform (4 x 20 ml). The combined organic extracts were dried and the solvent removed, to give the diol, yield 0.09 g (14%). The aqueous phase was freeze dried and the residue refluxed in ethanol (100 ml) for 2 hrs. The hot ethanolic solution was then placed on a short silica column, conditioned with ethanol, and eluted with additional ethanol. The solvent was removed to give an oil. Crude yield 0.34 g (42%). IH n.m.r. (CDCI3) δ 1.68, t;

2.92, m; 3.63, s; 4.53, s; 7.33, s. Step F

l-Acetoxy-2,2-bis(acetoxymethyl)-4-benzyloxybutane

2-(2-Benzyloxyethyl)-2-hydroxymethylpropane-l,3-diol (25.2 g, 0.1 mol), acetic anhydride (100 ml) and 4-dimethylaminopyridine (200 mg), with a drying tube attached, were stirred at room temperature overnight. The acetic anhydride was removed under reduced pressure. The residue was added to water and repeatedly extracted with dichloromethane. The combined organic extracts were dried and the solvent removed to give an oil. Yield 36.3 g (94%). ^JH n.m.r. (CDCI3) δ 1.75, t;

2.05, s; 3.57, t; 4.05, s; 4.48, s; 7.35, s.

Step G

l-Acetoxy-2,2-bis(acetoxymethyl)-4-hydroxybutane

A mixture of l-acetoxy-2,2-bis(acetoxymethyl)-4-benzyloxybutane (3.86 g, 11.5 mmol), 10% palladium on carbon (0.1 g), ethanol (35 ml) and glacial acetic acid (1 ml), in a 100 ml flask under an atmosphere of hydrogen, was stirred at room temperature for 24 hrs. The mixture was filtered through a bed of celite®, washed with ethanol, the solvent removed, and the residue extracted from water (30 ml) with dichloromethane (30 ml). The organic phase was dried and the solvent removed to give an oil. Yield 3.42 g (quant.). IH n.m.r. (CDCI3) δ 1.73, t; 2.07, s;

3.77, t; 4.09, s.

Step H

l-Acetoxy-2,2-bis(acetoxymethyl)-4-methanesulfoxybutane

To l-acetoxy-2,2-bis(acetoxymethyl)-4-hydroxybutane (3.4 g, 12.3 mmol), triethylamine (1.1 eq., 2.4 ml) and dichloromethane (30 ml), in an ice/water bath, under an argon atmosphere, was added dropwise over 1 hr, a solution of methane sulfonyl chloride (1.2 eq., 1.15 ml) in dichloromethane (15 ml). The reaction mixture was then stirred at approximately 4° for 110 min. The reaction mixture was allowed to warm to room temperature. The mixture was placed in a separating funnel and washed successively with 50 ml portions of 2M HCl (x 2), sat. aqueous sodium hydrogen carbonate and 5% aqueous sodium chloride. The organic phase was dried and the solvent removed to give an oil. Crude yield 4.29 g (98%). H n.m.r. (CDCI3) δ 1.95, t; 2.08, s; 3.02, s; 4.07, s; 4.36, t.

Step I

9-[4-Acetoxy-3,3-bis(acetoxymethyl)but-l-yl]-2-amino-6-chloropurine

A mixture of 2-amino-6-chloropurine (97%, 1.1 eq., 2.25 g, 12.9 mmol), l-acetoxy-2,2-bis(acetoxymethyl)-4-methanesulfoxybutane (4.29 g, 12.1 mmol), potassium carbonate (4.1 g, 29.7 mmol) and dry dimethylformamide (30 ml) under an argon atmosphere was stirred at 35° for 22 hrs. The dimethylformamide was removed under reduced pressure. To the residue was added water (75 ml) and dichloromethane (75 ml), transfered to a separating funnel and the phases separated. The aqueous phase was further extracted with dichloromethane (2 x 75 ml). The combined organic extracts were dried and the solvent removed. The crude product was preadsorbed onto silica and flash chromatographed eluting with 1:99 methanol:ethyl acetate. Some fractions contained product that crystallized overnight and were filtered and kept separate, yield 2.01 g (39%). Additional fractions containing product and the mother liquor from the crystallized fractions were combined, the solvent removed and crystallized from ethyl acetate/methanol. Yield 0.54 g (49% total), m.p. 172°. IH n.m.r. (DMSO-dg) δ 1.98, t; 2.02, s; 4.04, s; 4.17, t; 6.88, s, br; 8.19, s. ¹ C n.m.r. (CDCI3) δ 22.8, 33.4, 41.0, 42.4, 65.8,

143.8, 153.5, 155.7, 161.0, 172.6.

Alternative synthesis of 2-(2-Benzyloxyethyl)-2-hydroxymethylpropane-l,3-diol

Step J

4-(Benzyloxy)butan-l-ol

Butanediol (901.5 g, 10 mol) and potassium hydroxide (85%, 264 g, 4 mol) were stirred, at room temperature, with a mechanical stirrer overnight. The mixture was then heated to 130° and vacuum applied to remove water. 49 g of water was condensed. The mixture was then cooled to 90° and benzyl chloride (506.4 g, 465 ml, 4 mol) added dropwise over 2 hrs. The mixture was stirred at 90° for 1 hr then heated to 130° and stirred for 1 hr. The mixture was allowed to cool to room temperature overnight. The mixture was dissolved in water (2000 ml) and dichloromethane (2000 ml), separated and the aqueous phase further extracted with dichloromethane (2 x 500 ml). The combined organic extract was backwashed with water, dried and the solvent removed. The crude product was distilled to give an oil. Yield 515.3 g (71.5%). b.p. 105-10670.2-0.3 mm Hg. 1H n.m.r. (CDCI3) δ 1.69, tt; 2.39, s; 3.52, t; 3.63, t; 4.52, s; 7.33, s. Step K

4-(Benzyloxy)butanal

To an open vessel, immersed in an ice/water bath, containing 4-(benzyloxy)butan- l-ol (100 g, 0.55 mol), 2,2,6,6-tetramethyl-l-ρiρeridiinyloxy, free radical (0.75 g, 4.8 mmol) dissolved in dichloromethane (500 ml), potassium bromide (5.0 g, 42 mmol) dissolved in sat. aqueous sodium hydrogen carbonate (250 ml) and tetrabutylammonium chloride (5.0 g, 18.0 mmol) was added dropwise, over 45 min, a solution of sodium hydrogen carbonate (46.5 g, 0.55 mol) and sodium chloride (17.5 g, 0.3 mol) in sodium hypochlorite (0.5M, 1120 ml, 0.56 mol). The mixture was stirred, at approximately 4°, for a further 45 min. The two phases were separated and the aqueous phase extracted with dichloromethane (x 3). The combined organic extracts were washed with sat. aqueous sodium hydrogen carbonate and brine, dried and the solvent removed. The crude product was purified by distillation to give an oil. b.p. Yield 89.0 g (90%). b.p. 57-7070.01 mm

Hg. !H n.m.r. (CDCI3) δ 1.95, tt; 2.55, t; 3.53, t; 4.50, s; 7.32, s; 9.80, s.

Step L

2-(2-Benzyloxyethyl)-2-hydroxymethylpropane-l,3-diol

To a mixture of paraformaldehyde (5.0 eq., 4.19 g, 0.14 mol) and tetrahydrofuran (115 ml), immersed in an ice/water bath, was added a cold (<10°) aqueous solution of sodium hydroxide (2 eq., 1M, 56.2 ml, 0.56 mol). The temperature was allowed to stabilise at approximately 4°. To the mixture was then added dropwise, over 15 min, a solution of 4-(benzyloxy)butanal (5.0 g, 0.028 mol) in tetrahydrofuran (115 ml). The mixture was stirred at 4° for a further 30 min and then at room temperature for 2 hrs. The tetrahydrofuran was removed by rotary evaporation. Water was added to the residue and the product extracted repeatedly with dichloromethane. The organic extract was washed with brine, dried and the solvent removed to give an oil. Yield 4.5 g. The aqueous phase was adjusted to pH 8 and extracted with dichloromethane (x 4) to give 0.5 g product. The aqueous phase was then adjusted to pH 0 and extracted with dichloromethane (x 4) to give 0.5 g of product. Total yield 5.5 g (82%). IH n.m.r. (CDCI3) δ 1.57, t; 3.33, s; 3.50, s;

4.43, s; 7.26, s.

EXAMPLE 2

9-[4-Hydroxy-3,3-bis(hydroxymethyl)but-l-yl]guanine

9-[4-Acetoxy-3,3-bis(acetoxymethyl)but-l-yl]-2-amino-6-chloropurine (2.4 g, 5.6 mmol), and 2M HCl (50 ml) were stirred at reflux for 2 hr. The mixture was cooled in an ice/water bath and 5% NaOH added dropwise until a precipitate formed. The solid was filtered and crystallized from water as the monohydrate. Yield 0.98 g. A second crop gave an additional 0.29 g (75% total), m.p. 294-297°. (@47min dec.) (Lit. 295-299° dec). [Found: 284.1357 Calc. for Ci 1H17N5O4 requires 284.1359]. *H n.m.r. (DMSO-dg ) δ 1.69, m; 4.04, m; 4.41, t; 6.42, s;

7.66, s; 10.52, s. ¹³C n.m.r. (D2O) δ 33.5, 45.7, 47.7, 66.3, 111.8, 141.5, 154.1, 159.6.

EXAMPLE 3

9-[4-Hydroxy-3,3-bis(hydroxymethyl)but-l-yl]guanine sodium salt

To a stirring suspension of 9-[4-hydroxy-3,3-bis(hydroxymethyl)-but-l-yl]guanine (0.5 g, 1.8 mmol) in water immersed in an ice/water bath was added slowly 0.1N NaOH (17.6 ml, 1.8 mmol). The mixture was allowed to warm to room temperature, filtered, washed with cold water and freeze dried overnight. Yield 0.54 g (99.3%). m.p. 257° dec. (Found: C, 38.45; H, 5.68; N, 19.03. Calc. for ^cll^H16^N5°4Na requires C, 38.43; H, 5.57; N, 19.05). IH n.m.r. (DMSO-dg) ) δ

1.64, m; 3.61, s, br; 3.96, m; 4.82, s, br; 7.36, s. ¹³C n.m.r. (D2O) ) δ 33.9, 43.1, 47.8, 66.4, 121.9, 142.5, 155.5, 165.3, 172.5.

EXAMPLE 4

9-(4-Acetoxy-3-acetoxymethyl-3-hydroxybut-l-yl)-2-amino-6-chloropurine

Step A

2-(2-Benzyloxyethyl)-2-propen-l-ol

A solution of diethyl 2-(benzyloxyethyl)malonate (10.0 g, 34 mmol), sodium hydride (80% in oil, 1.25 g, 41.7 mmol) and 1,2-dimethoxyethane (80 ml), in a 1000 ml flask with a condenser and thermometer, under an atmosphere of argon, was refluxed for 2 hrs. The mixture was cooled to room temperature and immersed in an ice/water bath. Lithium aluminium hydride (powder, 4.0 g, 105.4 mmol) was added portion-wise over 10 min. The mixture was heated to 55° and stirred for 2.5 hrs. The mixture was allowed to cool to room temperature and a solution of ethyl formate (25 ml, 0.31 mol) in diethyl ether (375 ml) was added and stirred for 15 min. A solution of aqueous sodium hydroxide (2M, 15 ml) was added and the mixture allowed to stir overnight at room temperature. The mixture was filtered and the product extracted with 1,2-dichloroethane. The organic extract was dried and the solvent removed. The crude product was distilled to give an oil. Yield 2.1 g (32%). b.p. 130-15071 mm Hg. *H n.m.r. (CDCI3) δ 2.43, t; 3.62, t; 4.53, s;

4.93, s; 5.07, s; 7.34, s.

Step B

4-Benzyloxy-2-hydroxymethylbutane-l,2-diol

A mixture of AD-mix (36.4 g), t-butyl alcohol (130 ml) and water (130 ml), in a 1000 ml flask, was stirred, at room temperature, until two clear phases were observed. The mixture was then immersed in an ice/water bath and 2-(2- benzyloxyethyl)-2-propen-l-ol (5.0 g, 26 mmol) was added. The mixture was stirred, at approximately 4°, for 3 hrs. Sodium sulfite (39 g, 0.31 mol) was added and the mixture allowed to warm to room temperature. Dichloromethane (300 ml) was added and the mixture stirred for 30 min. The phases were separated and the aqueous phase extracted with dichloromethane (2 x 300 ml). The combined organic extracts were dried and the solvent removed to give an oil. Yield 6.0 g

(quant.). n.m.r. (CDCI3) δ 1.83, m; 2.68, s, br; 3.36, s; 3.54, s; 3.70, m; 4.53, s;

7.33, s. Step C

l-Acetoxy-2-acetoxoxymethyl-4-benzyloxybutan-2-ol

A mixture of 4-benzyloxy-2-hydroxymethylbutane-l,2-diol (5.9 g, 26 mmol), acetic anhydride (50 ml) and dimethylaminopyridine (0.5 g, 4.1 mmol), in a 150 ml flask, were stirred, at room temperature, overmght. The acetic anhydride was removed under reduced pressure. To the residue was added water (100 ml) and diethyl ether (100 ml). The phases were separated and the aqueous phase further extracted with diethyl ether (2 x 100 ml). The combined organic extracts were dried and the solvent removed. The crude product was flash chromatographed eluting with ethyl acetate. Product containing fractions were combined and the solvent removed to give an oil. Yield 7.4 g (91%). !H n.m.r. (CDCI3) δ 1.90, t;

2.80, s; 3.74, t; 4.08, s; 4.10, s; 4.52, s; 7.33, s.

Step D

4-Acetoxy-3-acetoxoxymethyl-butane-l,3-diol

l-Acetoxy-2-acetoxoxymethyl-4-benzyloxybutan-2-ol (5.0 g, 16.1 mmol), 10% palladium on carbon (0.2 g), ethanol (50 ml) and glacial acetic acid (1 drop), in a 100 ml flask, under an atmosphere of hydrogen,, was stirred, at room temperature, for 3 days. The mixture was filtered through a bed of celite® then filter paper and the solvent removed to give an oil. Yield 3.26 g (92%). *H n.m.r. (CDCI3) δ 1.84, t; 2.09, s; 3.92, t; 4.12, s.

Step E

l-Acetoxy-2-acetoxoxymethyl-3-methanesulfoxybutan-2-oI

To 4-acetoxy-3-acetoxoxymethyl-butane-l,3-diol (3.20 g, 14.5 mmol), triethylamine (1.1 eq., 2.84 ml, 20.3 mmol) and dichloromethane (20 ml), immersed in an ice/water bath, under an atmosphere of nitrogen, was added dropwise, over 1 hr, a solution of methane sulfonyl chloride (1.1 eq., 1.35 ml, 16 mmol) in dichloromethane (15 ml). The mixture was stirred, at approximately 4°, for 90 min. The mixture was allowed to warm to room temperature, transfered to a separating funnel and washed successively with 50 ml portions of 2M HCl ( x 2), sat. aqueous sodium hydrogen carbonate, and 5% sodium chloride. The organic extract was dried and the solvent removed to give an oil. Yield 3.28 (75%). H n.m.r. (CDCI3) δ 2.41, t; 2.22, s; 3.20, s; 4.10, d; 4.45, t.

Step F

9-(4-Acetoxy-3-acetoxymethyl-3-hydroxybut-l-yl)-2-amino-6-chloropurine

A mixture of 2-amino-6-chloropurine (99%, 1.1 eq., 0.66 g, 3.8 mmol), 1-acetoxy- 2-acetoxoxymethyl-3-methanesulfoxybutan-2-ol (1.19 g, 3.5 mmol), potassium carbonate (1.2 g, 8.7 mmol) and dry dimethylformamide (15 ml), with a drying tube attached, was stirred, at room temperature, for 60 hrs. The dimethylformamide was removed under reduced pressure. To the residue was added water (25 ml), transfered to a separating funnel and extracted with dichloromethane (4 x 25 ml). The combined organic extracts were dried and the solvent removed. The crude product was preadsorbed onto silica and flash chromatographed eluting with 1:9 methanokdichloromethane. The major fraction was collected and the solvent removed (5072 x 10^~2 mm Hg). The product was crystallized from methanol/dichloromethane as fine white crystals, yield 72 mg. A second crop gave a further 107 mg (12.5% total). !H n.m.r. (DMSO-dg) δ 2.00, s;

3.94, m; 4.18, t; 5.32, s; 6.90, s, br; 8.14, s.

EXAMPLE 5

2-Amino-9-[3,3-bis(acetoxymethyl)but-l-yl]-6-chloropurine

Step A

Diethylmethyl(2-benzyloxyethyl)malonate

To sodium hydride (dry, 1.80 g, 75 mmol), under an atmosphere of nitrogen, was added a solution of diethyl (2-benzyloxyethyl)malonate (20.0 g, 68 mmol) in dioxane (400 ml). The mixture was heated at reflux for 3 hrs. The mixture was allowed to cool and methyl iodide (1.5 eq., 1.90 ml, 30.7 mmol) added and allowed to stir at room temperature for 1 hr and then gently heated and refluxed overnight. The solvent was removed. 0.4M HCl (186 ml) was added to the residue and the product extracted with diethyl ether (4 x 150 ml). The combined organic extracts were dried and the solvent removed. The crude product was distilled to give an oil. Yield 18.6 g (89%). b.p. 12571.5 x 10"² mm Hg. iH n.m.r. (CDCI3) δ

1.15, t; 1.38, s; 2.18, t; 3.48, t; 4.05, q; 4.39, s; 7.25, s.

Step B

2-Methyl-2-(2-benzyloxyethyl)propane-l,3-diol

A solution of diethyl methyl(2-benzyloxyethyl)malonate (18.0 g, 58.4 mmol) in methanol (270 ml) was added dropwise over 1.5 hrs to solid sodium borohydride (45.0 g, 1.19 mol), in a flask with a reflux condenser, mechanical stirrer, immersed in an ice/water bath and under an argon atmosphere. After addition was complete additional methanol (270 ml) was added and the reaction mixture refluxed for 20 hrs. The mixture was allowed to cool to room temperature and water (225 ml) carefully added and stirred for 1 hr. The methanol was removed and the water residue extracted with chloroform (3 x 220 ml). The combined organic extracts were backwashed with water (2 x 270 ml), dried and the solvent removed. The crude product was preadsorbed onto silica and flash chromatographed eluting with 6:4 ethyl acetate:petroleum spirits. The fractions containing the product were combined and evaporated to give an oil. Yield 7.35 g (56%). ^XH n.m.r. (CDCI3) δ

0.79, s; 1.76, t; 3.50, s; 3.63, t; 4.53, s; 7.34, s.

Step C

l-Acetoxy-2-acetoxyoxymethyl-4-benzyloxy-2-methylbutane

2-Methyl-2-(2-benzyloxyethyl)propane-l,3-diol (7.35 g, 32.8 mmol), acetic anhydride (76 nil) and 4-dimethylaminopyridine (152 mg, 18.6 mmol), in a stoppered flask, were stirred together at room temperature over the weekend. The excess acetic anhydride was removed, water (80 ml) and diethyl ether (50 ml) were added to the residue and stirred for 30 min. The phases were separated and the aqueous phase further extracted with diethyl ether (3 x 70 ml). The combined organic extracts were dried and the solvent removed. The crude product was distilled to give an oil. Yield 9.03 g (89%). b.p. 15071.5 x 10"² mm Hg. ^AH n.m.r. (CDCI3) δ 0.98, s; 1.71, t; 2.04, s; 3.55, t; 3.95, s; 4.49, s; 7.32, s. Step D

4-Acetoxy-3-acetoxymethyl-3-methyIbutan-l-ol

l-Acetoxy-2-acetoxymethyl-4-benzyloxy-2-methylbutane (9.0 g, 29.2 mmol), 10% palladium on carbon (1.13 g), ethanol (95 ml) and glacial acetic acid (1 drop), under an atmosphere of hydrogen, were stirred vigorously at room temperature for 17 hrs. The mixture was filtered through a bed of celite®, washed with ethanol and the solvent removed to give an oil. Yield 6.29 g (quant.). *H n.m.r. (CDCI3) δ 0.98, s; 1.61, t; 2.05, s; 3.71, t; 3.94, s.

Step E

l-Acetoxy-2-acetoxymethyl-4-methanesulfoxy-2-methylbutane

To 4-acetoxy-3-acetoxymethyl-3-methylbutan-l-ol (6.29 g, 28.8 mmol), triethylamine (1.5 eq., 6.12 ml, 43.9 mmol) and dichloromethane (85 ml), immersed in an ice/water bath and under an atmosphere of nitrogen, was added dropwise, over 30 min, a solution of methane sulfonyl chloride (1.2 eq., 2.72 ml,

35.1 mmol) in dichloromethane (30 ml). The mixture was stirred for a further 2 hrs, transfered to a separating funnel and washed successively with 60 ml portions of 2M HCl (x 3), sat. aqueous sodium hydrogen carbonate and 5% aqueous sodium chloride. The organic extract was dried and the solvent removed. The crude product was placed under vacuum (90° at 3.7 x 10^_5 mm Hg) to remove low boiling impurities to give an oil. Yield 7.4 g (87%). *H n.m.r. (CDCI3) δ 1.02, s; 1.85, t; 2.07, s; 3.02, s; 3.95, s; 4.32, t.

Step F

2-Amino-9-[3,3-bis(acetoxymethyl)but-l-yl]-6-chloropurine

A mixture of 2-amino-6-chloropurine (1.01 eq., 4.25 g, 25.1 mmol), l-acetoxy-2- acetoxymethyl-4-methanesulfoxy-2-methylbutane (7.4 g, 25.0 mmol), potassium carbonate (7.8 g, 56.4 mmol) and dry dimethylformamide (105 ml) under an argon atmosphere was stirred at room temperature for 40 hrs. The temperature was increased to 35° and stirred for a further 7 hrs. Additional 2-amino-6-chloropurine (0.5 g) was added to the mixture and stirred at 35° over the weekend. The dimethylformamide was removed under reduced pressure. Water (200 ml) was added to the residue and the product extracted with dichloromethane (5 x 100 ml). The combined organic extracts were washed with water (200 ml), dried and the solvent removed by rotary evaporation. The crude product was preadsorbed onto silica and flash chromatographed eluting with ethyl acetate. Fractions containing product were combined and the solvent removed to give a solid. Yield 6.4 g

(69%). m.p. 86°. iH n.m.r. (CDC1₃) δ 1.00, s; 1.82, m; 2.02, s; 3.95, s; 4.09, m;

5.06, s, br; 7.71, s.

EXAMPLE 6

9-[3,3-Bis(hydroxy--nethyl)but-l-yI]guanine

2-Amino-9-[3,3-bis(acetoxymethyl)but-l-yl]-6-chloropurine (6.40 g, 17.2 mmol) in 2M HCl (145 ml) was refluxed for 3 hrs. The mixture was allowed to cool to room temperature and was then immersed in an ice/water bath. Aqueous sodium hydroxide (19.2 g in 40 ml) was added and the mixture stirred, at room temperature, for 2 hrs. HCl was added until a precipitate formed (pH 5). The solid was filtered, washed with water and recrystallized from water. Yield 4.18 g (90%). m.p. 278° (Lit. 284-287°) ^JH n.m.r. (DMSO-dg) δ 0.82, s; 1.67, m; 3.25, d; 3.98, m; 4.50, t; 6.43, s, br; 7.70, s; 10.53, s, br. EXAMPLE 7

9-(4-Acetoxy-3-acetoxymethyl-3-fluorobut-l-yl)-2-amino-6-chloropurine

Step A

Diethyl (2-Benzyloxyethyl)fluoromalonate

To a stirred suspension of sodium hydride (95%, 9.4 g, 0.37 mol) in tetra¬ hydrofuran (dry, 1000 ml), immersed in an ice/ethanol bath, under an atmosphere of argon, was added dropwise, over 2.5 hr, a solution of diethyl (2- benzyloxyethyl)malonate (100 g, 0.34 mol) in tetrahydrofuran (400 ml). The solution was allowed at warm to room temperature and stirred overnight. The mixture was immersed in an ice/ethanol bath and dimethylformamide (70 ml) added then l-chloromethyl-4-fluoro-l,4-diazobicyclo[2.2.2]octane bis tetrafluoroborate (120.3 g, 0.34 mol) added in portions over 30 min and stirred at room temperature for 2 hrs. The mixture was added to diethyl ether (2000 ml) and washed successively with 1000 ml portions of 2M H2SO4 , water and sat. aqueous sodium hydrogen carbonate (500 ml). The organic extract was dried, filtered and the solvent removed to give an oil. Yield 102.4 g (96%). Rp 0.56 (1:9

Ethanol:Petroleum spirits (60-80°)). *H n.m.r. (CDCI3) δ 1.25, t; 2.52, dt; 3.66, t; 4.20, q; 4.45, s; 7.31, s. 13c n.m.r. (CDCI3) δ 14.1, 34.4, 34.8, 62.7, 64.1, 90.9, 94.8, 127.9, 128.0, 128.5, 138.1, 166.2, 166.7.

Step B

2-(2-Benzyloxyethyl)-2-fluoropropane-l,3-diol

To sodium borohydride (95%, 33.75 g, 847 mmol) in a 1000 ml flask, fitted with a thermometer, condenser, dropping funnel, mechanical stirrer and under an atmosphere of argon, was added dropwise, over 2.75 hrs, a solution of diethyl (2- benzyloxyethyl)fluoromalonate (13.5 g, 43.2 mmol) in methanol (200 ml). The mixture was then gently heated to reflux and stirred for 2.5 hrs. Heating was removed and the mixture stirred at room temperature overnight. Water (150 ml) was added carefully and stirred at room temperature until all excess sodium boro¬ hydride was destroyed and all salts dissolved. The methanol was removed and the product was extracted with chloroform (4 x 100 ml). The combined organic extracts were backwashed with water (70 ml), dried and the solvent removed to give an oil. Yield 7.04 g (71%). Rp 0.33 (4:6 Ethyl acetate:Hexane). H n.m.r.

(CDCI3) δ 2.07, dt; 2.64, t; 3.66, t; 3.71, dd; 3.75, dd; 4.54, s; 7.35, s.

Step C

l-Acetoxy-2-acetoxymethyl-4-benzyloxy-2-fluorobutane

2-(2-Benzyloxyethyl)-2-fluoropropane-l,3-diol (11.96 g, 52.4 mmol), acetic anhydride (150 ml) and 4-dimethylaminopyridine (250 mg, 2.0 mmol), in a 1000 ml flask with a drying tube, were stirred at 40° for 2 hrs. The acetic anhydride was removed. Water (100 ml) and dichloromethane (150 ml) were added to the residue and stirred virorously for 5 min. The phases were separated and the aqueous phase further extracted with dichloromethane (2 x 100 ml). The combined organic extracts were dried and the solvent removed to give an oil. Yield 18.4 g (quant.) approximately 14% w/w acetic anhydride impurity. ^XH n.m.r. (CDCI3) δ 2.08, dt;

2.08, s; 2.09, s; 3.62, t; 4.27, dd; 4.29, dd; 4.50, s; 7.33, s. Step D

4-Acetoxy-3-acetoxymethyl-3-fluorobutan-l-ol

A mixture of l-acetoxy-2-acetoxymethyl-4-benzyloxy-2-fluorobutane (86%, 18.4 g, 52.4 mmol), 10% palladium on carbon (700 mg), ethanol (120 ml) and glacial acetic acid (1 ml), in a 250 ml flask under an atmosphere of hydrogen, was stirred at room temperature for 3.5 hrs. The mixture was filtered through filter paper, then through a bed of celite® and the solvent and acetic acid removed overnight to give an oil. Yield 10.4 g (89%). *H n.m.r. (CDCI3) δ 2.03, dt; 2.10, s; 2.11, s; 3.85, t; 4.28, dd; 4.30, dd.

Step E

l-Acetoxy-2-acetoxymethyl-2-fluoro-4-methanesulfoxybutane

To a stirring mixture 4-acetoxy-3-acetoxymethyl-3-fluorobutan-l-ol (10.4 g, 46.8 mmol), triethylamine (1.4 eq., 9.13 ml, 65.5 mmol) and dichloromethane (100 ml) in a 500 ml flask, immersed in an ice/water bath under an atmosphere of argon, was added dropwise, over 2 hrs, a solution of methane sulfonyl chloride (1.2 eq., 4.35 ml, 56.2 mmol) in dichloromethane (50 ml). The mixture was stirred, at approximately 4°, for a further 100 min. The mixture was then transfered to a separating funnel and washed successively with 150 ml portions of 2M HCl (x 2), sat. aqueous sodium hydrogen carbonate and 5% sodium chloride. The organic extract was dried, filtered and the solvent removed to give an oil. Yield 13.85 g (98.6%). *H n.m.r. (CDCI3) δ 2.12, s; 2.24, dt; 3.04, s; 4.26, dd; 4.42, t.

Step F

9-(4-Acetoxy-3-acetoxymethyl-3---luorobut-l-yl)-2-amino-6-chloropurine

A mixture of 2-amino-6-chloropurine (1.1 eq., 4.11 g, 24.2 mmol), l-acetoxy-2- acetoxymethyl-2-fluoro-4-methanesulfoxybutane (6.62 g, 22.0 mmol), potassium carbonate (7.4 g, 53.5 mmol) and dimethylformamide (dry, 50 ml), in a flask with a drying tube attached, was stirred, at room temperature, for 18 hrs. The dimethylformamide was removed under reduced pressure. The residue was dissolved in water (150 ml) and dichloromethane added (100 ml). The phases were separated and the aqueous phase further extracted with dichloromethane (2 x 100 ml). The combined organic extract was dried and the solvent removed. The crude product was preadsorbed onto silica and flash chromatographed eluting with 1:99 methanokethyl acetate. Fractions containing pure product were combined and evaporated to give a solid. Yield 2.54 g. Additional less pure fractions were combined, evaporated and crystallized from methanol/ethyl acetate. Total yield

3.94 g (48%). m.p. 128-129°. [Found: 374.1030 Calc. for Ci4HιgClFN5C>4 requires 374.1031]. !H n.m.r. (CDCI3) δ 2.01, s; 2.30, dt; 4.28, d; 5.15, s, br; 7.78, s. 13c n.m.r. (CDCI3) δ 22.7, 34.3, 34.7, 40.3, 65.6, 66.1, 144.0, 151.3, 155.5, 160.9, 172.2.

EXAMPLE 8

9-(4-Acetoxy-3-acetoxymethyl-3-methoxycarbonylbut-l-yl)-2-amino-6- chloropurine

Step A

Diethyl tetrahydropyranyloxymethyl(2-benzyloxyethyl)malonate

To a solution of diethyl hydroxymethyl(2-benzyloxyethyl)malonate (56.17 g, 173 mmol) and dihydropyran (23.8 ml, 260 mmol), under an atmosphere of argon, was added a solution of pyridiniump-toluenesulfonate (4.36 g, 17 mmol) in dichloromethane (600 ml). The mixture was stirred at room temperature for 4 hrs. Diethyl ether (400 ml) was added and the mixture tranfered to a separating funnel and washed with brine (700 ml). The organic extract was dried and the solvent removed to give an oil. Yield 66.5 g (94%). iH n.m.r. (CDCI3) δ 1.19, t; 1.53, m, br; 2.39, t; 3.43-3.90, m; 3.58, t; 4.06-4.20, m; 4.43, s; 4.79-4.96, m; 7.30, s.

Step B

2-Tetrahydropyranyloxymethyl-2-(2-benzyloxyethyl)propane-l,3-diol

To lithium aluminium hydride (30.73 g, 0.81 mol) was added diethyl ether (dry, 600 ml). The mixture was stirred, at room temperature, for 10 min. Diethyl tetrahydropyranyloxymethyl(2-benzyloxyethyl)malonate (66.5 g, 0.162 mol) in diethyl ether (dry, 200 ml) was added dropwise, over 2 hrs. The mixture was gently heated and refluxed for 2 hrs. Sat. aqueous sodium sulfate (90 ml) was added dropwise to die reaction mixture. The mixture was filtered and the phases separated. The organic phase was dried and the solvent removed to give an oil.

Yield 53.6 g (quant.). !H n.m.r. (CDCI3) δ 1.52, br; 1.70, t; 2.75, s, br; 3.36-3.72, m; 3.56, s; 4.51, s; 4.68-4.95, m; 7.32, s. Step C l-Acetoxy-2-acetoxymethyI-4-benzyloxy-2-tetrahydropyranyloxymethyl butane

A mixture of 2-tetrahydropyranyloxymethyl-2-(2-benzyloxyethyl)propane-l,3-diol (17.3 g, 53.3 mmol), acetic anhydride (35 ml) and dimethylaminopyridine (100 mg, 0.8 mmol) was stirred, at room temperature, for 66 hrs. The acetic anhydride was removed by rotary evaporation. To the residue was added water and dichloromethane. The phases were separated and the aqueous phase further extracted with dichloromethane. The combined organic extract was dried and the solvent removed to give an oil. Yield 21.4 g (98%). !H n.m.r. (CDCI3) δ 1.58, br;

1.82, t; 2.04, s; 3.22-3.76, m; 3.57, t; 4.08, s; 4.48, s; 4.81-4.97, m; 7.32, s.

Step D

2,2-Bis(acetoxymethyl)-4-benzyloxybutan-l-ol

A solution of l-acetoxy-2-acetoxymethyl-4-benzyloxy-2- tetrahydropyranyloxymethylbutane (21.0 g, 51.4 mmol), pyridinium - toluenesulfonate (0.1 eq., 1.3 g, 5.14 mmol) in ethanol (410 ml) was stirred at 55° for 3 hrs. The solvent was removed by rotary evaporation. The crude product was preadsorbed onto silica and flash chromatographed eluting initially with 1:4 ethyl acetate:petroleum spirits, changing to 2:3 ethyl acetate:petroleum spirits when the impurities had eluted. Fractions containing the product were combined and die solvent removed to give an oil. Yield 9.05 g (54%). Rp 0.31 (1:4 etiiyl acetate:petroleum spirits) H n.m.r. (CDCI3) δ 1.77, t; 2.05, s; 3.05, s; 3.61, t; 4.04, s; 4.53, s; 7.33, s.

Step E

2,2-Bis(acetoxymethyl)-4-benzyloxybutanoic acid

A mixture of 2,2-bis(acetoxymethyl)-4-benzyloxybutan-l-ol (4.0 g, 12.3 mmol), pyridinium dichromate (3.5 eq., 16.0 g, 56.8 mmol) and dry dimethylformamide (64 ml), in a 250 ml flask, under an atmosphere of argon, was stirred, at room temperature, for 43.5 hrs. The mixture was added to water (100 ml) and the product extracted with dietiiyl ether (4 x 100 ml). The combined organic extract was backwashed with 5% aqueous sodium chloride, dried and the solvent removed to give an oil. Yield 3.47 g (83%). *H n.m.r. (CDCI3) δ 2.04, s; 3.58, t; 4.33, s;

4.46, s; 7.29, s; 8.63, s.

Step F

Methyl 2,2-Bis(acetoxymethyl)-4-benzyloxybutanoate

To 2,2-bis(acetoxymethyl)-4-benzyloxybutanoic acid (3.47 g, 10.3 mmol) in methanol (22 ml) and benzene (69 ml) in a 250 ml flask, at room temperature, was added (trimethylsilyl)diazomethane (2M in hexane, 1.3 eq., 6.7 ml, 13.4 mmol). Rapid evolution of nitrogen gas was observed. The mixture was stirred, at room temperature, for 1 hr. The solvent and by-products were removed. The crude product was distilled to give an oil. Yield 2.43 g (67%). b.p. 170-19071.5 x 10"⁴ mm Hg *H n.m.r. (CDCI3) δ 1.96, t; 2.03, s; 3.51, t; 3.59, s; 4.30, s; 4.43, s; 7.31, s.

Step G

Methyl 2,2-bis(acetoxymethyl)-4-hydroxybutanoate

A mixture of methyl 2,2-bis(acetoxymethyl)-4-benzyloxybutanoate (2.4 g, 6.8 mmol), 10% palladium on carbon (100 mg), ethanol (20 ml) and glacial acetic acid (2 drops), under an atmosphere of hydrogen, was stirred, at room temperature, for 19 hrs. The mixture was filtered through a bed of celite®, washed with hot ethanol, and the solvent removed. Yield 1.75 g (98%). !H n.m.r. (CDCI3) δ 1.91, t; 2.05, s; 2.35, t; 3.72, t; 3.73, s; 4.31, s.

Step H

Methyl 2,2-bis(acetoxymethyl)-4-methanesulfoxybutanoate

To methyl 2,2-bis(acetoxymethyl)-4-hydroxybutanoate (1.75 g, 6.7 mmol), triethylamine (1.1 eq., 1.3 ml, 7.4 mmol) and dichloromethane (18 ml), in an ice/water bath, under an argon atmosphere, was added dropwise over 1 hr, a solution of methane sulfonyl chloride (1.2 eq., 0.62 ml, 8.0 mmol) in dichloromethane (9.5 ml). The reaction mixture was then stirred, at approximately 4°, for 2 hrs. The reaction mixture was allowed to warm to room temperature, transfered to a separating funnel and washed successively with 30 ml portions of 2M HCl (x 2), sat. aqueous sodium hydrogen carbonate and 5% aqueous sodium chloride. The organic phase was dried and the solvent removed to give an oil.

Yield 2.09 g (92%). iH n.m.r. (CDCI3) δ 2.07, s; 3.00, s; 3.76, s; 4.30, s. Step I

9-(4-Acetoxy-3-acetoxymethyl-3-methoxycarbonylbut-l-yl)-2-amino-6- chloropurine

A mixture of 2-amino-6-chloropurine (97%, 1.1 eq., 1.14 g, 6.7 mmol) methyl 2,2- bis(acetoxymethyl)-4-methanesulfoxybutanoate (2.08 g, 6.1 mmol), potassium carbonate (2.7 g, 19.5 mmol) and dry dimethylformamide (17 ml) under an argon atmosphere was stirred at room temperature for 137 hrs. The dimetitiy lformamide was removed under reduced pressure. To die residue was added water (75 ml), transfered to a separating funnel and extracted with dichloromethane (2 x 75 ml). The combined organic extracts were dried and die solvent removed. The crude product was preadsorbed onto silica and flash chromatographed eluting with 1:99 methanol:ethyl acetate. Fractions containing product were combined and die solvent removed to give a solid. Yield 1.41 g (56%). Rp 0.64 (1:99

MethanokEtiiyl acetate) ^AH n.m.r. (DMSO-dg) δ 2.02, s; 2.22, t; 3.54, s; 4.15, t; 4.23, s; 6.93, s, br; 8.15, s. EXAMPLE 9

9-[4-Acetoxy-3,3-bis(isopropoxymethyl)but-l-yl]-2-amino-6-chloropurine

Step A

4-Benzyloxy-2,2-bis(isopropoxymethyl)butan-l-ol

A mixture of 2-(2-benzyloxyethyl)-2-hydroxymethylproρane-l,3-diol (10.0 g, 42 mmol), potassium hydroxide (dry powder, 11.6 g, 208 mmol), 2-bromopropane (4 eq., 20.4 g, 15.6 ml, 166 mmol) and tetrabutylammonium bromide (0.1 eq., 1.35 g, 4.2 mmol), in a 150 ml oven dried flask with a mechanical stirrer and a drying tube, was stirred at 35° for 6 hrs. Additional sodium hydroxide (1 eq., 1.66 g, 42 mmol) and 2-bromopropane (10.2 g, 7.8 ml, 83 mmol) were added. The mixture was stirred for 18 hrs, still at 35°, whereupon additional sodium hydroxide (1 eq., 1.66 g, 42 mmol) and 2-bromopropane (10.2 g, 7.8 ml, 83 mmol) were added and stirred for 26 hrs. The mixture was allowed to cool to room temperature. Water (200 ml) and dichlorometiiane (200 ml) were added and d e solution transfered to 2000 ml beaker. The mixture was neutralised by the dropwise addition of 2M HCl.

The phases were separated and die aqueous phase further extracted with dichloromemane (2 x 100 ml). The combined organic extracts were dried and the solvent removed. The crude product was chromatographed on silica eluting with

1:4 diediyl etheπhexane. Fractions containing the product were combined and die solvent removed to give an oil. Yield 2.02 g (15%). *H n.m-r. (CDCI3) δ 1.11, d;

1.67, t; 3.40, s; 3.42-3.63, m; 3.60, d; 4.50, s; 7.33, s.

Step B

l-Acetoxy-4-benzyloxy-2,2-bis(isopropoxymethyl)butane

4-Benzyloxy-2,2-bis(isopropoxymethyl)butan-l-ol (1.87 g, 6.6 mmol), acetic anhydride (35 ml) and 4-dimethylaminopyridine (100 mg, 0.8 mmol), with a drying tube attached, were stirred at room temperature for 27 hrs. The acetic anhydride was removed by rotary evaporation. The residue was added to water and extracted with dichloromethane (x 3). The combined organic extracts were dried and die solvent removed to give an oil. Yield 2.30 g (95%). H n.m.r. (CDCI3) δ 1.08, d; 1.75, t; 2.02, s; 3.29, s; 3.44, sept; 3.59, t; 4.05, s; 4.49, s; 7.33, s.

Step C

4-Acetoxy-3,3-bis(isopropoxymethyl)butan-l-ol

A mixture of l-acetoxy-4-benzyloxy-2,2-bis(isopropoxymethyl)butane (2.31 g, 6.3 mmol), palladium on carbon (10%, 0.1 g), etiianol (25 ml) and glacial acetic acid (2 drops), under an atmosphere of hydrogen, was stirred at room temperature for 5 hrs. The mixture was filtered through a bed of celite® and die solvent removed to give an oil. Yield 1.72 g (99%). *H n.m.r. (CDCI3) δ 1.14, d; 1.68, t; 2.05, s; 3.35, s; 3.53, sept; 3.66, t; 4.05, s.

Step D

l-Acetoxy-2 -bis(isopropoxymethyl)-4-methanesulfoxybutane

To 4-acetoxy-3,3-bis(isopropoxymethyl)butan-l-ol (1.70 g, 6.2 mmol), triethylamine (1.4 eq., 1.2 ml, 8.6 mmol) and dichloromethane (15 ml), in an ice/water bath, under a nitrogen atmosphere, was added dropwise over 45 min, a solution of methane sulfonyl chloride (1.2 eq., 0.57 ml, 0.85 g, 7.4 mmol) in dichloromethane (8 ml). The reaction mixture was then stirred, at approximately 4°, for 2 hrs. The reaction mixture was allowed to warm to room temperature. The mixture was placed in a separating funnel and washed successively witii 25 ml portions of 2M HCl (x 2), sat. aqueous sodium hydrogen carbonate and 5% aqueous sodium chloride. The organic phase was dried and die solvent removed to give an oil. Yield 2.14 g (98%). *H n.m.r. (CDCI3) δ 1.02, d; 1.88, t; 2.07, s; 3.00, s; 3.31, s; 3.48, sept; 4.04, s; 4.41, t.

Step E

9-[4-Acetoxy-3,3-bis(isopropoxymethyl)but-l-yl]-2-amino-6-chloropurine

A mixture of 2-amino-6-chloropurine (97%, 1.1 eq., 1.13 g, 6.64 mmol), l-acetoxy-2,2-bis(isopropoxymethyl)-4-methanesulfoxybutane (2.14 g, 6.04 mmol), potassium carbonate (2.7 g, 19.5 mmol) and dry dimethylformamide (17 ml), with a drying tube attached, was stirred, at room temperature, for 1 ldays. The dimethylformamide was removed under reduced pressure. To die residue was added water and extracted with dichloromethane (x 3). The combined organic extracts were dried and die solvent removed. The crude product was preadsorbed onto silica and flash chromatographed eluting with dietiiyl ether. Fractions containing product were combined and the solvent removed to give a sohd. Yield 1.11 g (43%). m.p. 116°. Rp 0.83 (Diethyl ether). [Found: 428.2093 Calc. for

C19H30CIN5O4 requires 428.2064]. iH n.m.r. (DMSO-dg) δ 1.42, d; 1.87, t;

2.01, s; 3.30, s; 3.46, sept; 3.95, s; 4.16, t; 6.86, s, br; 8.15, s. ¹³C n.m.r. (CDCI3) δ 22.9, 23.0, 34.1, 41.8, 43.7, 67.4, 70.8, 127.2, 144.4, 153.0, 155.7, 160.9, 172.9. EXAMPLE 10

9-[4-Hydroxy-3,3-bis(isopropoxymethyl)but-l-yl]guanine

9-[4-Acetoxy-3,3-bis(isopropoxymethyl)but-l-yl]-2-amino-6-chloropurine (170 mg, 5.6 mmol), and 2M HCl (5 ml) were stirred at reflux for 1 hr. The mixture was cooled in an ice/water bath and 5% NaOH added dropwise until precipitate formed. The solid was filtered, washed witii ice cold water an dried under reduced pressure at 60° for 2 days. Yield 90 mg (62%). [Found: 368.2317 Calc. for

C17H29N5O4 requires 368.229]. n.m.r. (DMSO-dg) δ 1.73, d; 2.02, m; 3.25, s; 3.35, s; 3.45, sept; 4.03, m; 4.48, t; 6.37, s, br; 7.63, s; 10.52, s. 13c n.m.r. (D₂0) δ 25.3, 34.0, 45.7, 46.8, 66.9, 72.9, 77.9, 111.7, 141.4, 154.1, 159.2, 159.5.

EXAMPLE 11

9-(4-Acetoxy-3-acetoxymethyl-3-isopropoxymethylbut-l-yl)-2-amino-6- chloropurine

Step A

2-(2-Benzyloxyethyl)-2-isopropoxymethylpropane-l,3-diol

A second product from Step A in Example 9 was eluted and d e solvent removed to give the diol as an oil, 1.87 g (16%). *H n.m.r. (CDCI3) δ 1.13, d; 1.74, t; 3.41, s; 3.55, s; 3.61, t; 4.54, s; 7.33, s.

Step B

l-Acetoxy-2-acetoxymethyl-4-benzyloxy-2-isopropoxymethylbutane

Prepared from 2-(2-benzyloxyethyl)-2-isopropoxymethylpropane-l,3-diol using the method of Step B, Example 9. Yield 2.30 g (95%). ^XH n.m.r. (CDCI3) δ 1.07, d; 1.78, t; 2.03, s; 3.31, s; 3.43, sept; 3.56, t; 4.06, s; 4.48, s; 7.33, s.

Step C

4-Acetoxy-3-acetoxymethyl-3-isopropoxymethylbutan-l-ol

Prepared from l-acetoxy-2-acetoxymethyl-4-benzyloxy-2- isopropoxymethylbutane using the method of Step C, Example 9. Yield 1.61 g (93%). iH n.m.r. (CDCI3) δ 1.16, d; 1.69, t; 2.05, s; 3.41, s; 3.57, m; 3.70, 14.04, s.

Step D

l-Acetoxy-2-acetoxymethyl-2-isopropoxymethyl-4-methanesulfoxybutane

Prepared from 4-acetoxy-3-acetoxymemyl-3-isopropoxymethylbutan-l-ol using die method of Step D, Example 9. Yield 2.15 g (quant.). ^JH n.m.r. (CDCI3) δ 1.12, d; 1.92, t; 2.07, s; 3.01, s; 3.34, s; 3.49, m; 4.05, s; 4.38, s.

Step E

9-(4-Acetoxy-3-acetoxymethyl-3-isopropoxymethylbut-l-yl)-2-amino-6- chloropurine

Prepared from l-acetoxy-2-acetoxymethyl-2-isopropoxymethyl-4- methanesulfoxybutane and 2-amino-6-chloropurine using the method of Step E,

Example 9. Yield 1.31 g (53%). m.p. 100°. *H n.m.r. (DMSO-dg) δ 1.05, d; 1.92, t; 2.01, s; 3.33, s; 3.46, m; 3.99, s; 4.16, t; 6.86, s, br; 8.16, s. ¹ C n.m.r. (CDCI3) δ

22.9, 23.9, 33.8, 41.4, 43.0, 66.6, 70.3, 74.4, 127.2, 144.1, 153.2, 155.6, 160.9, 172.7.

EXAMPLE 12

9-(4-Hydroxy-3-hydroxymethyl-3-isopropoxymethylbut-l-yl)guanine

Prepared from 9-(4-acetoxy-3-acetoxymethyl-3-isopropoxymethylbut- 1 -yl)-2- amino-6-chloropurine using the method of Example 10. Yield 259 mg (76%). m.p.

261°. iH n.m.r. (DMSO-dg) δ 1.07, d; 1.70, m; 3.26, s; 3.46, m; 4.03, m; 4.43, t;

6.40, s, br; 7.63, s; 10.56, s, br. 13c n.m.r. (D2O) δ 25.3, 33.7, 45.7, 47.2, 669.6, 72.9, 77.9, 111.8, 141.4, 154.1, 159.3, 159.5. EXAMPLE 13

9-[4-Acetoxy-3,3-bis(acetoxymethyl)but-l-yl]-2-a--mnopurine

A mixture of 9-[4-acetoxy-3,3-bis(acetoxymethyl)but-l-yl]-2-amino-6- chloropurine (5.98 g, 26.2 mmol), 10% palladium on carbon (0.5 g), triethylamine (1.1 eq., 2.35 ml, 27.5 mmol) and etiianol (120 ml), all in a 250 ml flask, under an atmosphere of hydrogen, was stirred vigorously at room temperature for 18 hours. The hydrogen was removed and the mixture filtered through a bed of celite® and die solvent removed by rotary evaporation. The crude product was dissolved in dichloromemane, washed twice with water, dried over magnesium sulfate, filtered and the solvent removed by rotary evaporation. The product was crystallized from methanol as a white crystalline solid. Yield 4.32 g (78.6%). m.p. 152-153°. (@l°C/min). (Found: C, 51.83; H, 5.92; N, 18.09;. Calc. for C₁₇H23N5θ requires C, 51.90; H, 5.89; N, 17.80;). [Found: 255.3654 Calc. for CχoHgN3 requires 253.2854]. 'H n.m.r. (DMSO-dg) δ 2.05, s; 4.05, s; 4.17, m; 6.45, s, br;

8.12, s; 8.58, s. 13c n.m.r. (CDCI3) δ 20.8, 31.3, 38.3, 40.3, 63.8, 128.2, 142.0, 149.6, 153.0, 159.7, 170.5. EXAMPLE 14

2-Amino-9-[4-hydroxy-3,3-bis(hydroxymethyl)but-l-yl]-6-methoxypurine

A mixture of 9-[4-acetoxy-3,3-bis(acetoxymethyl)but-l-yl]-2-amino-6- chloropurine (200 mg, 0.47 mmol), sodium hydroxide (4.26 g, 0.11 mol), methanol (15 ml) and water (1.6 ml) was stirred, at room temperature, for 30 min. The mixture was diluted witii water (8 ml), transfered to a beaker and neutralised by die addition of aqueous HCl. The solvents were removed by rotary evaporation and the residue refluxed in ethanol for 20 min. The ethanolic solution was applied to a short silica column and eluted witii hot etiianol. The eluent was collected and d e solvent removed by rotary evaporation. The crude product was crystallized from water. Yield 66 mg (48%). *H n.m.r. (DMSO-dg) δ 1.72, m; 4.11, m; 4.43, t;

6.39, s, br; 7.84, s.

EXAMPLE 15

2,6-Diamino-9-(4-hydroxy-3,3-bis(hydroxymethyl)but-l-yl)-purine

A mixture 9-[4-acetoxy-3,3-bis(acetoxymethyl)but-l-yl]-2-amino-6-chloropurine (100 mg, 0.2 mmol) and ammonia saturated methanol (20 ml), in a glass-lined stainless steel bomb, was stirred, at 100°, for 21 hrs. The mixture was allowed to cool to room temperature and die solvent removed by rotary evaporation. The solid residue was suspended in metiianol and filtered. The methanol was removed by rotary evaporation and die product crystalUzed from water. Yield 13 mg (20%). H n.m.r. (DMSO-dg) δ 1.71, m; 4.06, m; 4.45, t; 5.75, s, br; 6.64, s, br; 7.69, s.

EXAMPLE 16

2-Amino-6-hydroxyamino-9-[4-hydroxy-3,3-bis(hydroxyπιethyl)but-l- yl]purine

A mixture of 9-[4-acetoxy-3,3-bis(acetoxymethyl)but-l-yl]-2-amino-6- chloropurine (100 mg, 0.2 mmol), monohydroxylamine hydrochloride (0.49 g, 7.1 mmol), potassium hydroxide (dry powder, 0.46 g, 8.2 mmol) and etiianol (stored over 4A sieves, 8.2 ml), in oven dried glassware, was refluxed for 18 hrs. The mixture was allowed to cool to room temperature. The residue was dissolved in methanol/water and dietiiyl ether added until a precipitate formed. The solid was filtered and dried under reduced pressure at 60° for 1 hr. Yield 33 mg (47%).

[Found: 299.1465 Calc. for C11H19O4N4: 299.1468]. ^JH n.m.r. (DMSO-dg) δ

1.71, m; 4.07, m; 4.44, s, br; 6.83, s, br; 7.84, s.

EXAMPLE 17

2-An-dno-6-hydrazino-9-[4-hydroxy-3,3-bis(hydroxymethyl)but-l-yl]purine

A mixture of 9-[4-acetoxy-3,3-bis(acetoxymethyl)but-l-yl]-2-amino-6- chloropurine(1.0 g, 2.3 mmol), hydrazine hydrate (55%, 2.65 ml, 1.5 g, 46.7 mmol) and etiianol (100 ml) was stirred, at reflux, for 19 hrs. The mixture was allowed to cool to room temperature then allowed to stand at 4° for 1 hr. The solid precipitate was filtered and die product crystallized from water. Yield 290 mg

(43%). ^AH n.m.r. (DMSO-dg) δ 1.71, m; 4.06, m; 4.42, s, br; 4.44, t; 5.89, s, br;

7.69, s; 8.39, s, br.

EXAMPLE 18

9-[4-Acetoxy-3,3-bis(acetoxymethyl)but-l-yl]guanine

9-[4-Hydroxy-3,3-bis(hydroxymethyl)but-l-yl]guanine (1.0 g, 3.53 mmol), 4- dimemylaminopyridine (100 mg) and acetic anhydride (30 ml) were stirred at room temperature for 65 hrs. The acetic anhydride was removed in vacuo at 60°C. The crude residue was preadsorbed onto silica and flash chromatographed eluting witii 1:9 methanokdichloromethane. Fractions containing the pure compound were combined arid evaporated to give a sohd. Yield 0.29 g. Additional less pure fractions were combined evaporated and recrystallized from dichloromethane/metiianol. Total yield 0.99 g (69%). m.p. 242-245°. *H n.m.r. (DMSO-dg) δ 1.84-1.97, m; 2.02, s; 3.90-4.14, m; 4.02, s; 6.38, s; 7.71, s; 10.58, s, br.

EXAMPLE 19

9-[4-Hydroxy-3,3-bis(acetoxymethyl)but-l-yl]guanine

9-[4-Hydroxy-3,3-bis(hydroxymethyl)butyl-l-yl]guanine (5.0 g, 17.7 mmol), 4- dimemylaminopyridine (250 mg), acetic anhydride (2.34 ml, 24.8 mmol) in dimethylformamide (300 ml) were stirred at room temperature for 20 hrs. The dimethylformamide was removed in vacuo at 60°C. The crude residue was preadsorbed onto silica and flash chromatographed eluting with 1:9 methanol:di^'chloromethane. Fractions containing the pure 9-[4-hydroxy-3,3- bis(acetoxymethyl)but-l-yl]guanine were combined and evaporated to give a solid.

Yield 1.25 g (19%). m.p. 229.5-232.5°C. !H n.m.r. (DMSO-dg) δ 1.75-1.88, m;

2.01, s; 3.34, s; 3.39, d, 75 Hz; 3.88-4.10, m; 3.96, s; 4.89, t; 6.38, s; 7.69, s; 10.53, s.

EXAMPLE 20

9-[4-Acetoxy-3,3-bis(hydroxymethyl)but-l-yl]guanine

A second product from Example 19 was eluted and die solvent removed to give a solid. Yield 0.96 g (17%) m.p. 224-225°. !H n.m.r. (DMSO-dg) δ 1.66-1.80, m; 2.00, s; 3.35, s; 3.91, s; 3.95-4.09, m; 4.65, br s; 6.46, s; 7.66, s; 10.70, s.

EXAMPLE 21

A mixture of 1,1-carbonyldiimidazole (1.55 eq., 0.89 g, 5.5 mmol), 3- phenoxybenzoic acid (1.5 eq., 1.13 g, 5.3 mmol) and dry dimethylformamide (50 ml) was stirred at room temperature, under an atmosphere of nitrogen, for 75 minutes. 9-[4-Hydroxy-3,3-bis(hydroxymemyl)but-l-yl]guanine (1.0 g, 3.5 mmol) was added in one portion to die mixture and stirred for a further 24 hours. The DMF was removed under reduced pressure. The crude product was suspended in 1:2 methano edianol at 50°. The suspension was filtered and die filtrate preadsorbed onto silica and chromatographed, eluting witii 5:95 methanokethyl acetate. The product was eluted and the solvent removed to give a white solid.

Crude yield 1.21 g (39%). n.m.r. (DMSO-dg) δ 2.18, m, 2H; 4.16, m, 2H; 4.46, s, 6H; 6.28, s, 2H, br; 7.02-7.71, complex, 27H; 7.47, s, IH; 10.50, s, IH.

EXAMPLE 22

A second product from Example 21 was eluted and die solvent removed to give a solid. Yield 0.39 g (16%). m.p. 187-188°. (Found: C, 65.25; H, 4.56; N, 10.09.

Calc. for C37H33N5O5 requires C, 65.77; H, 4.92; N, 10.36) iH n.m.r. (DMSO- dg) δ 2.02, m, 2H; 4.13, m, 2H; 4.31, s, 4H; 6.35, s, 2H, br; 7.04-7.73, complex, 18H; 7.67, s, IH; 10.55, s, IH.

EXAMPLE 23

A third product from Example 21 was eluted and the solvent removed to give a solid. Yield 0.17 g (10%). !H n.m.r. (DMSO-dg) δ 1.84, m, 2H; 3.44, d, 75 Hz

4H; 4.07, m, 2H; 4.19, s, 2H; 4.74, t, 75 Hz, 2H; 6.42, s, 2H, br; 7.08-7.76, complex, 9H; 7.67, s, IH; 10.57, s, IH.

EXAMPLE 24

2-Amino-9-[3,3-bis(acetoxymethyl)but-l-yl]-6-chloropurine (0.14 g, 0.42 mmol) and a solution of metiianol saturated with ammonia at 4° (5.0 ml) were stirred at room temperature for 1 hr.The solvent was removed by rotary evaporation. HPLC analysis indicated that the reaction had not gone to completion. The crude product was purified by preparative reverse-phase HPLC, eluting witii a non-linear gradient of 25/75 CH3CN/H2O to 45/55 CH3CN/H2O. The product was eluted and die solvent removed to give a white solid. Yield 0.05 g (44%). m.p. 154-156°. iH n.m.r. (DMSO-dg) δ 0.83, s, 3H; 1.73, m, 2H; 3.27, d, 75.5 Hz, 4H; 4.09, m, 2H; 4.53, t, 75.5 Hz, 2H; 6.49, s, 2H, br; 8.08, s, IH; 8.56, s, IH.

EXAMPLE 25

A second product from Example 24 was eluted and the solvent removed to give a white solid. Yield 0.03 g (26%). m.p. 125-129°. ^AH n.m.r. (DMSO-dg) δ 0.90, s,

3H; 1.78, m, 2H; 2.01, s, 3H; 3.28, s, 2H; 3.86, s, 2H; 4.10, m, 2H; 4.78, s, IH, br; 6.48, s, 2H, br; 8.09, s, IH; 8.56, s, IH.

EXAMPLE 26

Prepared from 9-[4-Acetoxy-3,3-bis(acetoxymethyl)but-l-yl]-2-amino-6- chloropurine using the method of Example 24. H n.m.r. (DMSO-dg) δ 1.89, m,

2H; 2.02, s, 6H; 3.42, s, 2H; 3.98, s, 4H; 4.18, m, 2H; 4.93, s, IH; 6.89, s, 2H , br; 8.17, s, IH.

EXAMPLE 27

A second product from Example 26 was eluted and the solvent removed to give a solid that was crystalUsed from methanol. Yield 0.71 g (18%). m.p. 159-160°. *H n.m.r. (DMSO-dg) δ 1.81, m, 2H; 2.01, s, 3H; 3.94, s, 2H; 4.27, m, 2H; 4.67, t, 2H; 6.91, s, 2H, br; 8.15, s, IH. EXAMPLE 28

A third product from Example 26 was eluted and die solvent removed to give a white soUd that was precipitated from methanol. Yield 0.19 g. Second crop. Yield

0.47 g (19%). m.p. 207-214°. iH n.m.r. (DMSO-dg) δ 1.75, m, 2H; 3.36, d, 75

Hz, 6H; 4.16, m, 2H; 4.43, t, 75 Hz, 3H; 6.90, s, 2H, br; 8.13, s, IH.

EXAMPLE 29

Prepared from 2-amino-9- [4-hydroxy-3 ,3-bis(hydroxymethyl)but- 1 -yl]purine using the method of Example 21. n.m.r. (DMSO-dg) δ 1.93, m; 3.50, s; 4.20, m; 4.77, s; 6.43, s, br; 7.05-8.01, complex; 8.08, s; 8.54, s.

EXAMPLE 30

Prepared from 9-(4-acetoxy-3-acetoxymeti yl-3-isopropoxymethylbut- 1 -yl)-2- amino-6-chloropurine using the method of Example 13. m.p. 166-167°. ^XH n.m.r. (DMSO-dg) δ 1.07, d; 1.93, m; 2.03, s; 3.48, m; 4.01, s; 4.17, m; 6.45, s, br; 8.10, s; 8.58, s.

EXAMPLE 31

Prepared from 9-[4-hydroxy-3,3-bis(isopropoxymetiιyl)but-l-yl]guanine using the method of Example 18. m.p. 245-248°. ^AH n.m.r. (DMSO-dg) δ 1.08, d, 76 Hz, 12H; 1.81, m, 2H; 2.02, s, 3H; 3.29, s, 4H; 3.47, dt, 76 Hz, 2H; 3.95, s, 2H; 4.04, m, 2H; 6.36, s, 2H, br; 7.67, s, IH; 10.56, s, IH. 13c n.m.r. (DMSO-dg) δ 22.4, 23.7, 33.3, 43.1, 66.3, 69.8, 73.2, 118.4, 138.9, 152.8, 155.1, 158.6, 172.1.

EXAMPLE 32

9-[4-L-Valyloxy-3,3-bis(L-valyloxymethyI)but-l-yl]guanine

Step A

9-[4-(iV-Benzyloxycarbonyl-L-valyloxy)-3,3-bis(iV-benzyloxycarbonyl-L- valyloxymethyl)but-l-yl]guanine

A suspension of 9-(4-hydroxy-3,3-bis(hydroxymethyl)but-l-yl)guanine (2.00 g, 7.06 mmol) in dry dimemylformamide (60 ml) was warmed to 60° and stirred until the solid dissolved. N-Benzyloxycarbonyl-L-vaUne (7.20 g, 28.7 mmol), 4- dimethylaminopyridine (0.36 g, 2.9 mmol) and dicyclohexylcarbodiimide (7.14 g, 34.6 mmol) were then added and die resulting solution stirred at 22° for 3 days tiien the white soUd filtered off and washed with dimethylformamide. The filtrate was concentrated under reduced pressure, and die residual oil subjected to flash chromatography (Merck Kieselgel 60). Elution with a gradient from 5 to 15% methanol in dichloromediane gave 3 bands A, B and C, witii RF values of 0.75, 0.52 and 0.20 respectively.

Concentration of the eluate containing band A gave 9-(4-N-benzyloxycarbonyl-L- valyloxy-3,3-bis(N-benzyloxycarbonyl-L-valyloxymethyl)but-l-yl)guanine as a near colourless foam. Yield 0.82 g (12%). !H n.m.r. (DMSO-dg) δ 0.87, d, 77 Hz,

18H; 1.82-2.19, m, 5H; 3.98, t, 76 Hz, 2H; 4.15, br s, 2H; 4.93, d, 7 12 Hz, 3H; 5.05, d, 7 12 Hz, 3H; 6.35, br s, 2H; 7.14, s, 15H; 7.67, s, IH; 7.79, d, 77 Hz, 3H.

Step B

9-[4-L-Valyloxy-3,3-bis(L-valyloxymethyl)but-l-yl]guanine

A suspension of 9-[4-N-(benzyloxycarbonyl-L-valyloxy)-3,3-bis(N- benzyloxycarbonyl-L-valyloxymethyl)but-l-yl]guanine (0.50 g, 0.51 mmol), ethanol (20 ml), 10% palladium on carbon (0.40 g) and glacial acetic acid (2 drops) was stirred vigorously at room temperature under latin of hydrogen overnight then filtered d rough a pad of activated charcoal on celite. The pad was washed with ethanol, and d e combined filtrates diluted with toluene and concentrated under reduced pressure at 22° to give 9-(4'-valyloxy-3',3'- bis(valyloxymethyl)but-l-yl)guanine as a thermally unstable brown film. Yield

0.22 g (74%). iH n.m.r. (DMSO-dg) δ 0.80, d, 76.8 Hz, 9H; 0.85, d, 76.8 Hz, 9H; 1.64-2.00, m, 5H; 3.15, d, 75.3 Hz, 3H; 3.91-4.16, m, 8H; 6.59, br s, 2H; 7.67, s,

IH.

EXAMPLE 33

9-[4-Hydroxy-3,3-bis(L-valyloxymethyl)but-l-yl]guanine

Step A

9-[4-Hydroxy-3,3-bis(iV-benzyloxycarbonyl-L-valyloxymethyl)but-l- yl]guanine

Concentration of die eluate containing band B from Step A, Example 32 gave 9- [4- hydroxy-3,3-bis(N-benzyloxycarbonyl-L-valyloxymethyl)but-l-yl]guanine (1.78 g, 34%) as a near colourless foam. Yield 1.78 g (34%). ^AH n.m.r. (DMSO-d6) δ 0.87, br d, 76.6 Hz, 12H; 1.70-1.92, m, 2H; 1.92-2.14, m, 2H; 3.40, s, 2H (after D2θ exchange); 3.91-4.11, m, 8H; 4.88-5.10, m, 5H; 6.38, br s, 2H; 7.33, s, 10H; 7.66, s, IH; 7.73, d, 78.1 Hz, 2H; 10.54, s, IH.

Step B

9-[4-Hydroxy-3,3-bis(L-valyloxymethyl)but-l-yl]guanine

Prepared from 9-[4-hydroxy-3,3-bis(N-benzyloxycarbonyl-L-valyloxymethyl)but- l-yl]guanine using the method of Step B, Example 32. Yield

0.22 g, (68%). iH n.m.r. (DMSO-d6) δ 0.83, d, 77 Hz, 6H; 0.87, d, 77 Hz, 6H; 1.68-1.95, m, 4H; 3.12, d, J 8 Hz, 2H; 3.93-4.14, m, 6H; 6.55, br s, IH; 6.57, br s, IH; 7.67, 7.69, 2 peaks, IH. EXAMPLE 34

9-[4-Hydroxy-3-(hydroxymethyl)-3-(L-valyloxymethyl)but-l-yl]guanine

Step A

9-[4-Hydroxy-3-(hydroxymethyl)-3-(N-benzyIoxycarbonyl-L- valyloxymethyl)but-l-yl]guanine

Concentration of the eluate containing band C from Step A, Example 32 gave 9-[4- hydroxy-3-(hydroxymethyl)-3-(N-benzyloxycarbonyl-L-valyloxymethyl)but-l- yl)guanine as a colourless soUd. m.p 166-170°. Yieldl.15 g (32%). *H n.m.r. (DMSO-d6) δ 0.88, d, 77 Hz, 3H; 0.89 d, 77 Hz, 3H; 1.73-1.91, m, 2H; 2.00-2.25, m, IH; 3.36, s, 4H (after D2O exchange); 3.94-4.17, m, 5H; 4.65, br t, 75 Hz, 2H; 4.97, d, J 12 Hz, IH; 5.05, d, J 12 Hz, IH; 6.40, br s, 2H; 7.34, s, 5H; 7.66, s, IH; 7.70, d, J 8 Hz, IH; 10.53, s, IH.

Step B

9-[4-Hydroxy-3-(hydroxymethyl)-3-(L-valyloxymethyl)but-l-yl]guanine

Prepared from 9-[4-hydroxy-3-(hydroxymethyl)-3-(N-benzyloxycarbonyl-L- valyloxymethyl)but-l-yl]guanine using the method of Step B, Example 32. Yield

0.09 g (58%). iH n.m.r. (DMSO-d6) δ 0.82, d, 76.8 Hz; 0.87, d, 76.8 Hz; 1.61- 1.98, m, 3H; 3.16, d, 74.2 Hz; 3.38, s, 4H (after D2θ exchange); 3.97, s, 2H; 3.89- 4.11, m, 2H; 6.59, br s, 7.66, s, IH; 10.74, br s, IH.

EXAMPLE 35

9-[4-Acetoxy-3,3-bis(L-valyloxymethyl)but-l-yl]guanine

9-[4-Acetoxy-3,3-bis(iV-benzyloxycarbonyl-L-valyloxymethyl)but-l-yl]guanine

Prepared from 9-[4-hydroxy-3,3-bis(N-benzyloxycarbonyl-L-valyloxymethyl)but- l-yl]guanine using the method of Step F, Example 1. Yield 0.40 g (62%). *H n.m.r. (DMSO-d6) δ 0.91, d, 77 Hz, 12H; 0.83-2.15, m, 7H; 3.89-4.22, m, 8H; 4.97, d, 7 12 Hz, 2H; 5.07, d, 712 Hz, 2H; 6.38, br s, 2H; 7.37, s, 10H; 7.70, s, IH; 7.80, d, 78 Hz, 2H. 9-[4-Acetoxy-3,3-bis(L-valyloxymethyl)but-l-yl]guanine

Prepared from 9-[4-acetoxy-3,3-bis(N-benzyloxycarbonyl-L-valyloxymethyl)but- l-yl]guanine in methanol using the method of Step B, Example 32. Yield 0.14 g,

(74%). !H n.m.r. (DMSO-d6) δ 0.75-0.98, m, 12H; 1.73-2.05, m, 4H; 2.02, s, 3H; 3.05-3.24, m, 2H; 3.90-4.18, m, 8H; 6.49-6.65, m, 2H; 7.68, 7.71, 2 peaks, IH.

EXAMPLE 36

9-[4-Acetoxy-3-acetoxymethyl-3-L-valyloxymethylbutyl]guanine

OAc Step A

9-[4-Acetoxy-3-(acetoxymethyl)-3-(iV-benzyloxycarbonyl-L" valyloxymethyl)but-l-yϊ]guanine

Prepared from 9-[4-hydroxy-3-(hydroxymethyl)-3-(N-benzyloxycarbonyl-L- valyloxymethyl)but-lyl]guanine using the method of Step F, Example 1. Yield 0.26 g, (59%). !H n.m.r. (DMSO-dg) δ 0.87, d, 76.7 Hz; 1.81-2.22, m, 3H; 2.02, s, 6H; 3.88-4.18, m, 9H; 4.96, d, 7 12.5 Hz, IH; 5.04, d, 7 12.5 Hz, IH; 6.36, br s, 2H; 7.34, s, 5H; 7.70, s, IH; 7.77, d, 78 Hz, IH; 10.55, s, IH.

Step B

9-[4-Acetoxy-3-(acetoxymethyl)-3-(L-valyloxymethyl)but-l-yl]guanine

OAc

Prepared from 9-[4-acetoxy-3-(acetoxymethyl)-3-(N-benzyloxycarbonyl-L- valyloxymethyl)but-l-yl]guanine in methanol using the procedure of Step B, Example 32. m.p. 172-176° dec. Yield 0.16 g, (95%). iH n.m.r. (DMSO-d6) δ 0.82, d, 76.8 Hz, 3H; 0.87, d, 76.8 Hz, 3H; 1.77-2.05, m, 3H; 2.01, s, 6H; 3.16, d, J 5.4 Hz, IH; 3.95-4.17, m, 8H; 6.53, br s, 2H; 7.72, s, IH.

EXAMPLE 37

9-[4-Hydroxy-3,3-bis(D,L-2-a--ninobutyroxymethyl)but-l-yl]guanine

Step A

9-[4-Hydroxy-3,3-bis( V-benzyloxycarbonyl-D,L-2-aminobutyroxymethyl)but- l-yl]guanine

A suspension of 9-[4-hydroxy-3,3-bis(hydroxymethyl)but-l-yl]guanine (2.11 g, 7.45 mmol) in dry dimemylformamide (150 ml) was heated to 60° with stirring. D, L-N-benzyloxycarbonyl-2-aminobutyric acid (3.36 g, 0.015 mmol), 4- dimemylaminopyridine (0.185 g, 1.51 mmol) and dicyclohexylcarbodiimide (3.77 g, 0.018 mmol) were then added to the suspension while still at 60°. The resulting mixture was allowed to cool to room temperature and stirred for 6 days. Additional D, L-N-benzyloxycarbonyl-2-aminobutyric acid (1.66 g, 7.45 mmol) and dicyclohexylcarbodiimide (1.54 g, 7.45 mmol) were, added to the reaction mixture and stirring continued for 3 days at room temperature. Additional D, L-N- benzyloxycarbonyl-2-aminobutyric acid (1.66 g, 7.45 mmol) and dicyclohexylcarbodiimide (1.54 g, 7.45 mmol) were added to the reaction mixture and stirring continued for a further 11 days at room temperature. The white solid which formed was filtered off, washed witii dimemylformamide and the filtrate concentrated under reduced pressure. The filtrate was preadsorbed onto siUca and flash chromatographed eluting with 10:90 methanol-ethyl acetate to give two products. Yield 0.932 g (17%). m.p.97-99°. iH n.m.r., (DMSO-d6) δ 0.93, t, J 8.3 Hz, 3H; 1.73, m, 2H; 3.43, m; 4.07, m, 4H; 5.03, m, 3H; 6.5, s, br, 2H; 7.37, s, 5H; 7.67, s, IH; 7.77, J 8.3 Hz, d, IH; 10.6, s, br, IH. 13c n.m.r., (DMSO-dg ) 10.4, 24.0, 31.0, 41.5, 61.0, 55.4, 64.5, 65.5, 117.8, 127.8, 128.3, 136.7, 136.8, 150.1, 151.7, 153.4, 156.2, 156.7, 172.0

Step B

9-[4-Hydroxy-3,3-bis(D,L-2aminobutyroxymethyl)but-l-yl]guanine

Prepared from 9-[4-hydroxy-3,3-bis(N-benzyloxycarbonyl-D,L-2- aminobutyroxymethyl)but-l-yl]guanine by the memod of Step B, Example 32. ^AH n.m.r., (DMSO-dg) δ 0.87, t, 6H, 5.5 Hz; 1.50, m, 4H; 1.73, m, 2H; 3.17-3.50, complex, 2H; 3.40, s, IH; 3.80-4.17, m, 6H; 6.50, s, br, 2H; 7.67, 7.70, 2 peaks, IH.

EXAMPLE 38

9-[4-Hydroxy-3-hydroxymethyI-3-(Λ^-benzyloxycarbonyl-2-amino- butyroxymethylbut-l-yI]guanine

The second product from Step A, Example 37 was eluted and the solvent removed to give a white soUd. Yield 0.043 g (1.1%). mp 183.0-184.0°. ^lU-n.m.r., (DMSO- d6) δ 0.90, t, J 8.3 Hz, 3H; 1.73, m, 4H; 4.03, m, 5H; 4.67, t, 2H; 5.00, d, 2H; 6.43, s, br, 2H; 7.33, s, 5H; 7.67, s, IH; 7.73, d, J 8.3 Hz, IH; 10.53, s, br, IH. ^C n.m.r, (DMSO-d6 ) 12.3, 25.9, 33.0, 44.4, 57.3, 63.3, 67.4, 129.6, 130.14, 138.6, 138.8, 152.2, 155.2, 158.1, 158.6, 174.1.

EXAMPLE 39

9-[4-Hydroxy-3-(hydroxymethyl)-3-(2-amino-2,2-dimethylacetoxymethyl)but- l-yl]guanine

Step A

9-[4-Hydroxy-3-(hydroxymethyl)-3-( -benzyloxycarbonyl-2-amino-2,2- dimethylacetoxymethyl)but-l-yl]guanine

Prepared from 9-[4-hydroxy-3,3-bis(hydroxymethyl)but-l-yl]guanine and N- benzyloxycarbonyl-2-amino-2,2-dimedιylacetic acid using the method of Step A, Example 32. *H n.m.r. (DMSO-d6) δ 1.37, s, 6H; 1.67-1.84, m, 2H; 3.91-4.10, m,

4H; 4.61, br t, J Hz, 2H; 4.91, s, 2H; 6.41, br s, 2H; 7.25-7.44, m, 5H; 7.66, s, IH; 7.80, br s, IH.

Step B

9-[4-Hydroxy-3-(hydroxymethyl)-3-(iV-benzyloxycarbonyl-2-amino-2,2- dimethylacetoxymethyl)but-l-yl]guanine

Prepared from 9-[4-hydroxy-3-(hydroxymethyl)-3-(N-benzyloxycarbonyl-2- amino-2,2-dimed ylacetoxymethyl)but-l-yl]guanine using the method of Step B, Example 32. 1H n.m.r. (DMSO-d6) δ 1.20, s, 3H; 1.22, s, 3H; 1.59-1.90, m, 2H; 3.37, br s, 4H(after D2θ exchange); 3.94, s, 2H; 3.89-4.12, m, 2H, 6.54, br s, 2H; 7.65, 7.66, 2 peaks, IH; 10.16, br s, IH.

EXAMPLE 40

9-(4-Hydroxy-3,3-bis(iV-benzyIoxycarbonyl-L-alanyloxyπιethyl)but-l- yl)guanine

Prepared from 9-[4-hydroxy-3,3-bis(hydroxymethyl)but-l-yl]guanine and N- benzyloxycarbonyl-L-alanine using the method of Step A, Example 37. ^XH n.m.r., (DMSO-dg) δ 1.30, d, J 5.6 Hz, 6H; 1.80, m, 2H; 3.90-4.30, m, 8H; 4.87-5.13, m, 4H; 6.43, s, br, 2H; 7.33, s, 10H; 7.67, s, IH; 7.83 d, 2H, J 5.6 Hz; 10.57, s, br, IH.

EXAMPLE 41

9-[4-Hydroxy-3-(hydroxymethyl)-3-(L-alanyloxymethyl)but-l-yl)guanine, acetate salt

Step A

9-[4-Hydroxy-3-(hydroxymethyl)-3-(N-benzyloxycarbonyl-L- alanyloxymethyl)but-l-yl)guanine

Prepared from 9-[4-hydroxy-3,3-bis(hydroxymethyl)but-l-yl]guanine and N- benzyloxycarbonyl-L-alanine using the method of Step A, Example 37. ^H n.m.r., (DMSO-d6) δ 1.33, d, J 5.6 Hz, 2H; 1.77, m, 2H; 4.10, m, 5H; 4.70, t, 2H; 5.00, d, 2H; 6.43, s, br, 2H; 7.37, s, 5H; 7.67, s, IH; 7.77, J 5.6 Hz, d, IH; 10.57, s, br, IH. !3c n.m.r., (DMSO-dg ) 16.9, 31.1, 42.6, 49.4, 61.5, 65.0, 65.5, 116.5, 127.8, 128.3, 136.8, 137.0, 150.9, 153.3, 155.8, 156.8, 172.7

Step B

9-[4-Hydroxy-3-(hydroxymethyl)-3-(L-alanyloxymethyl)but-l-yl)guanine, acetate salt

Prepared from 9-[4-hydroxy-3-(hydroxymethyl)-3-(N-benzyloxycarbonyl-L- alanyloxymethyl)but-l-yl)guanine in methanol with one equivalent of acetic acid using the metiiod of Step B, Example 32. ^AH n.m.r., (DMSO-d6) δ 1.10, m, 3H; 1.70, m, 2H; 2.90, s, 3H; 3.33, complex, 5H; 4.03, m, 4H; 6.47, s, br, 2H; 7.67, s, IH.

EXAMPLE 42

Prepared using the method of Example 32, Step A, with N-benzyloxy carbonyl L- leucine. Yield 600 mg (16%). !H n.m.r. (DMSO-d6) δ 0.88, t, 6H; 1.41-1.79, m,

4H; 3.38, s, 4H; 4.05, m, 4H; 4.39 and 4.68(ratio 1:4), 2t, 2H; 5.01, s, 2H; 6.41, s, 2H; 7.34, s, 5H; 7.68, s, IH; 7.75, d, IH; 10.55, s, IH.

EXAMPLE 43

A second product from Example 42 was eluted, and the solvent removed to give a solid. Yield 1.13 g (21%). ^lU n.m-r. (DMSO-d6) δ 0.85, t, 12H; 1.41- 1.89, m, 8H; 3.19, d, IH; 3.41, s, br, 2H; 4.18, s, br, 8H; 5.01, m, 4H; 6.42, s, 2H; 7.31, s, 10H; 7.69, s, IH; 7.80, d, 2H; 10.61, s, IH.

EXAMPLE 44

9-(4-Hydroxy-3,3-bis(Λ^-benzyloxycarbonyl-L-prolyloxymethyI)but-l- yl)guanine

Prepared from 9-(4-hydroxy-3,3-bis(hydroxymethyl)but-l-yl)guanine and N- benzyloxycarbonyl-L proUne using the method of Step A, Example 32. ^AH n.m.r. (DMSO-dg) δ 1.63-2.06, m, 8H; 2.08-2.40, m, 2H; 3.25-3.52, m, 2H (after D2O exchange); 3.85-4.16, m, 9H; 4.24-4.44, m, 3H; 4.90-5.15, m, 5H; 6.38, br s, 2H; 7.16-7.42, m, 10H; 7.58, 7.63, 7.64, 7.68, 4 peaks, IH. EXAMPLE 45

9-[4-Hydroxy-3-(hydroxymethyl)-3-(iV-benzyloxycarbonyl-L- prolyloxymethyl)but-l-yI]guanine

Prepared from 9-(4-hydroxy-3,3-bis(hydroxymetiιyl)but-l-yl)guanine and N- benzyloxycarbonyl-L-proUne using the method of Step A, Example 32. ^AH n.m.r. (DMSO-d6) δ 1.65-2.07, m, 5H; 2.10-2.38, m, IH; 3.31-3.46, m, 5H (after D2O exchange); 3.90-4.15, m, 5H; 4.27-4.45, m, IH; 4.69, t, 76 Hz, 2H; 4.93-5.15, m, 2H; 6.42, br s, 2H; 7.22-7.44, m, 5H; 7.64, 7.68, 2 peaks, IH; 10.54, br s, IH.

EXAMPLE 46

9-[4-Hydroxy-3-(hydroxymethyl)-3-(L-phenylalanyloxymethyl)but-l- yl]guanine, acetate salt

Step A

9-[4-Hydroxy-3-(hydroxymethyl)-3-(iV-benzyloxycarbonyl-L- phenylalanyloxymethyl)but-l-yl]guanine

Prepared from 9-[4-hydroxy-3,3-bis(hydroxymethyl)but-l-yl]guanine and L- phenylalanine using the method of Step A, Example 37. m.p. 103-105°. !H n.m.r., (DMSO-d6) δ 1.73, m, 2H; 3.00, m, 2H; 4.07, m, 4H; 4.37, m, IH; 4.70, t, 2H; 4.97, s, 2H; 6.50, s, br, 2H; 7.27, m, 5H; 7.70, s, IH; 7.87, d, IH; 10.63, s, br, IH.

Step B

9-[4-Hydroxy-3-(hydroxymethyl)-3-(L-phenylalanyloxymethyl)but-l- yl]guanine, acetate salt

Prepared from 9-[4-hydroxy-3-(hydroxymethyl)-3-(N-benzyloxycarbonyl-L- phenylalanyloxymethyl)but-l-yl]guanine in methanol with one equivalent of acetic acid using the method of Step B, Example 32. ^AH n.m.r., (DMSO-d6) δ 1.67, m, 2H; 1.83, s, 3H; 2.80, m, 2H; 3.20-3.80, complex, 5H; 3.90, m, 4H; 6.50, s, br, 2H; 7.20, m, 5H; 7.67, s, IH.

EXAMPLE 47

Prepared from the crude product from step A of Example 46 using the method of Step F, Example 1. m.p. 147-148°. ^AH n.m.r., (DMSO-d6) δ 1.87, m, 2H; 2.03, s, 6H; 3.00, m, 2H; 3.90-4.20, complex, 8H; 4.33, m, IH; 4.97, s, 2H; 6.43, s, br, 2H; 7.30, m, 10H; 7.73, s, IH; 7.93,75.5 Hz, d, IH.

EXAMPLE 48

9-(5-Acetoxy-3,3-bis(acetoxymethyl)pentyl)-2-amino-6-chloropurine

Step A

l,5-Bis(benzyloxy)-3,3-bis(hydroxymethyl)pentane

Anhydrous etiier (100 ml) was carefully added to stirred litiiium aluminium hydride (3.54 g, 93 mmol) maintained under nitrogen. A solution of 1,5- bis(benzyloxy)-3,3-bis(ethoxycarbonyl)pentane (10.0 g, 23.3 mmol) in anhydrous ether (100 ml) was then added portionwise at such a rate that the mixture gently refluxed. The resulting suspension was heated to reflux for 5.5 hr then cooled and stirred at room temperature overnight. Ethyl acetate was then carefully added to destroy die remaining hydride, followed by ethanol then water until the soUd turned from grey to white. The soUd was tiien filtered off and washed witii hot THF (ca 200 ml). The combined filtrates were partially concentrated under reduced pressure, diluted with water and extracted with ether (3 x 200 ml). The combined organic phases were dried, filtered and concentrated under reduced pressure to give l,5-bis(benzyloxy)-3,3-bis(hydroxymethyl)pentane as an oil.

Yield 8.0 g (96% pure, 96%). iH n.m.r. (CDCI3): d 1.57, t, 76 Hz, 4H; 3.31, br s, 4H; 3.48, t, 76 Hz, 4H; 3.65, br s, 2H; 4.39, s, 4H; 7.05-7.31, m, 10H.

Step B

3,3-Bis(acetoxymethyl)-l,5-bis(benzyloxy)pentane

Prepared from l,5-bis(benzyloxy)-3,3-bis(hydroxymethyl)pentane using the method of Step F, Example 1. Yield 8.51 g (89%). b.p. 200-220° (oven temρerature)/cα 2-4 x 10"⁴ mm. *H n.m.r. (CDCI3) δ 1.68, t, 76 Hz, 4H; 1.95, s, 6H; 3.48, t, 76 Hz, 4H; 3.93, s, 4H; 4.39, s, 4H; 7.12-7.33, m, 10H.

Step C

5-Benzyloxy-3,3-bis(acetoxymethyl)-l-hydroxypentane

A suspension of 3,3-bis(acetoxymethyl)-l,5-bis(benzyloxy)pentane (5.00 g, 11.7 mmol), 10% paUadium on activated carbon (0.30 g) and ethanol (45 ml) was stirred vigorously under latin of hydrogen until 270 ml of hydrogen had been absorbed. It was then filtered tiiough ceUte, which was washed with additional ethanol. The combined filtrates were concentrated under reduced pressure to a pale yellow oil (3.95 g). Radial chromatography (4 mm plate, Merck Kieselgel 60PF254) of the crude oil (dichloromethane elution followed by ether elution) gave two chromophoric bands A and B with RF values of 0.63 and 0.33 (dichloromethane) respectively. Concentration of the eluate corresponding to band A gave 3,3-bis(acetoxymethyl)-l,5-bis(benzyloxy)pentane (0.55 g, 11% recovery) as a colourless oil with properties in accord with those described above. Concentration of the eluate corresponding to band B gave 5-benzyloxy-3,3- bis(acetoxymethyl)-l-hydroxypentane as a colourless oil. Yield 2.94 g (74%). ^AH n.m.r. (CDCI3) δ 1.67, t, 76.7 Hz, 2H; 1.78, t, 76.4 Hz, 2H; 2.04, s, 6H; 3.56, t, 7 6.4 Hz, 2H; 3.73, t, 76.7 Hz, 2H; 4.00, s, 4H, 4.49, s, 2H; 7.27-7.38, m, 5H.

Step D

l-Acetoxy-5-benzyloxy-3,3-bis(acetoxymethyl)pentane

Prepared from 5-benzyloxy-3,3-bis(acetoxymethyl)-l-hydroxypentane using the method of Step F, Example 1. Yield 3.35 g (99%). !H n.m.r. (CDCI3) δ 1.64-1.84, m, 4H; 2.03, s, 3H; 2.05, s, 6H; 3.55, t, 77 Hz, 2H; 4.00, s, 4H; 4.17, t, 77 Hz, 2H; 4.47, s, 2H; 7.19-7.41, m, 5H. Step E

l-Hydroxy-5-acetoxy-3,3-bis(acetoxymethyl)pentane

Prepared from l-acetoxy-5-benzyloxy-3,3-bis(acetoxymethyl)pentane using the method of Step G, Example 1. Yield 2.5 g (quant.). H n.m.r. (CDCI3) δ 1.65, dt, 7 3.3, 7.1 Hz, 2H; 1.73 dt, 71.7, 6.9 Hz, 2H; 1.99, s, 3H; 2.02, s, 6H; 3.70, t, 76.9 Hz, 2H; 3.96, s, 4H; 4.04-4.18, m, 2H.

Step F

5-Acetoxy-3,3-bis(acetoxymethyl)-l-methanesulphonyloxypentane

Prepared from l-hydroxy-5-acetoxy-3,3-bis(acetoxymethyl)pentane using the method of Step H, Example 1. Yield 3.2 g (98%). *H n.m.r. (CDCI3) δ 1.60-1.79, m, 2H; 1.85, t, 77.1 Hz, 2H; 1.98, s, 3H; 2.01, s, 6H; 2.98, s, 3H; 3.96, s, 4H; 4.02- 4.18, m, 2H; 4.30, t, 77.1 Hz, 2H. Step G

9-(5-Acetoxy-3,3-bis(acetoxymethyl)pent-l-yl)-2-amino-6-chIoropurine

Prepared from 5-acetoxy-3 ,3-bis(acetoxymethyl)- 1 -methanesulphonyloxypentane using the method of Step I, Example 1. Yield 1.58 g (40%). m.p 154-155°.^ n.m.r. ( DMSO-d6) δ 1.81, t, 77 Hz, 2H; 1.99, br t, 2H; 2.04, s, 3H; 2.06, s, 6H;

4.02, s, 4H; 4.10-4.29, m, 4H; 6.88, br s, 2H; 8.24, s, ¹ C n.m.r. (CDCI3) δ 22.8, 23.0, 31.7, 33.6, 40.8, 41.1, 62.3, 67.7, 127.0, 143.9, 153.2, 155.6, 161.3, 172.6, 173.2.

EXAMPLE 49

9-{2-[(3-hydroxymethyl)oxolan-3-yl]eth-l-yl}guanine

Prepared from 9-(5'-acetoxy-3^,,3'-bis(acetoxymethyl)pentyl)-2-amino-6- chloropurine using the method of Example 2. Yield 0.47 g (93%). m.p. 266-271°. ^AH n.m.r. (DMSO-d6) δ 1.49-1.75, m, 2H; 1.79-1.96, m, 2H; 3.29, d, 78 Hz, IH (after D2O exchange); 3.33, s, 2H; 3.56, d, 78 Hz, IH (after D2O exchange); 3.69, t, 77 Hz, 2H; 3.99, br t, 78 Hz, 2H; 4.89, br t, 75 Hz, IH; 6.42, br s, 2H; 7.72, s, IH; 10.53, br s, IH.

EXAMPLE 50

9-(5-Acetoxy-3,3-bis(acetoxymethyl)pent-l-yl)-2-aminopurine

Prepared from 9-(5'-acetoxy-3',3'-bis(acetoxymethyl)pentyl)-2-amino-6- chloropurine using the method of Example 13. Yield (quant.), m.p. 137-138°. ^AH n.m.r. (DMSO-d6) δ 1.78, t, 76.8 Hz, 2H; 1.83-2.04, m, 2H; 1.99, s, 3H; 2.01, s, 6H; 3.97, s, 4H; 4.05-4.24, m, 4H; 6.44, br s, 2H; 8.11, s, IH; 8.57, s, IH. EXAMPLE 51

9-(5-Hydroxy-3,3-bis(hydroxymethyl)pent-l-yl)-2-aminopurine

Prepared from 9-(5'-acetoxy-3',3'-bis(acetoxymethyl)pentyl)-2-aminopurine using the method of Example 24. Yield (quant.). !H n.m.r. (DMSO-d6) δ 1.49, t, 76.9 Hz, 2H; 1.66-1.76, m, 2H; 3.17, d, 75.1 Hz, 2H; 3.30, d, 75 Hz, 2H; 3.55, q, 74.8 Hz, 2H; 4.01-4.16, m, 2H; 4.49-4.62, m, 2H; 6.47, br s, 2H; 8.05, s, IH; 8.55, s, IH.

EXAMPLE 52

9-(5-Hydroxy-3,3-bis(hydroxymethyl)pent-l-yl)guanine

A mixture of 9-(5'-acetoxy-3',3'-bis(acetoxymethyl)pentyl)-2-amino-6- chloropurine (0.19 g, 0.43 mmol) and 50% aqueous formic acid (3 ml) was stirred and heated at 100° for 100 min then concentrated under reduced pressure. A chilled (0-5°) saturated solution of ammonia in methanol (20 ml) was then added to the residual oil and the mixture stirred at room temperature overnight. After concentration under reduced pressure, a further aUquot of ammonia saturated methanol (20 ml) was added and die solution stirred overnight then concentrated under reduced pressure. Recrystallisation (water) o the crude solid gave 9-(5'- hydroxy-3',3'-bis(hydroxymethyl)pentyl)guanine as cream needles. Yield 114 mg

(89%). m.p. 232-234° dec. tø n.m.r. ( DMSO-d6) δ 1.46, t, 76.9 Hz, 2H; 1.59- 1.73, m, 2H; 3.28, s, 4H; 3.49, t, 77.0 Hz, 2H; 3.89-4.04, m, 2H; 4.56, br s, 3H; 6.49, br s, 2H; 7.64, s, IH; 10.21-11.15, br s, IH.

EXAMPLE 53

A slurry of 9-[4-hydroxy-3,3-bis(hydroxymethyl)-but-l-yl]guanine (1 g) in 10 ml DMF, protected from moisture with Ar, was treated with 1 ml methyl orthoacetate followed by 0.35 ml trifluoroacetic acid. A clear solution was obtained which, after several minutes, deposited a white powder. The mixture was stirred at room temp, for 1 hr and then 3 ml triethylamine were added and the soUd collected and washed witii dietiiyl ether. It was then warmed to 100 ° in 10 ml DMF, cooled to ambient temperature, collected and washed with dichloromethane before drying in vacuo at 70 ° for several hr. Yield 0.77 g. !H n.m.r. (DMSO-dg) δ 1.28, s, 3H;

1.68, t, 2H; 6.44, s, 2H; 3.89, t, overlapping with 3.85, s, 8H; 7.71, s, IH; 10.56, s, IH.

EXAMPLE 54

To a suspension of 9-[4-hydroxy-3,3-bis(hydroxymethyl)-but-l-yl]guanine (1 g) and 0.1 g LiBr in 40 ml CH2CI2 was added 0.45 ml CF3CO2H followed by 2.5 ml 2,2-dimethoxypropane. After stirring for 1.5-2 days at room temp., the mixture was extracted with 100-150 ml 10% NaHCθ3 solution and die soUd collected and washed well with water. After drying at 80 ° under vacuum, the product (0.61 g) was obtained as a sUghtly greyish to white powder. ^XH n.m.r. (DMSO-dg) δ 1.29, d, 6H; 1.71, t, 2H; 3.46, s, 2H; 3.56, s, 4H; 3.99, t, 2H; 4.76 s, IH,; 6.42, s, 2H; 7.70, s, IH; 10.54, s, IH.

EXAMPLE 55

A solution of 9-[4-hydroxy-3,3-bis(hydroxymethyl)-but-l-yl]guanine (1 g) in 40 ml DMF at 145 °, protected from moisture with Ar, was treated dropwise with a solution of 0.58 g 1,1-carbonyldiimidazole in 25 ml DMF over 15-20 mins. After 2 hr the reaction (clear, pale yellow solution) was removed from the oil bath and the solvent stripped off to near dryness at 75 °. The product was stirred with several aliquots (50-75 ml) of CH2CI2, collected on a frit and washed several more times before air drying. Buff-yellow colour, yield 1.29 g. The washings contained ca. 0.25 g imidazole. iH n.m.r. (DMSO-dg) δ 1.74, m, 2H; 3.33, s, 4H; 4.00, m, 4H;

4.35 to 5.1(ca. 2H, three br. peaks, H/D exchange, OH); 6.43, s, 2H; 5.76, s, IH, imidazole; 7.01, s, IH, imidazole; 7.71, 7.68, 7.64, 2H, imidazole; 10.54, s, IH.

EXAMPLE 56

9-[4-Acetoxy-3,4-bis(acetoxymethyI)but-l-yl]-2-amino-6-methylaminopurine

9-[4-Acetoxy-3,4-bis(acetoxymethyl)but-l-yl]-2-amino-6-chloropurine (2.0 g, 4.7 mmol) and 33% methylamine in ethanol (560 ml, 4.7 mmol) in dichloromethane

(13 ml) and ethanol (7 ml) was stirred in a stoppered flask for 18 hrs. A further portion of 33% methylamine (1.12 ml, 9.4 mmol) was added and the reaction allowed to stir for a further 5 hrs. The solvent was removed in vacuo and the crude product preadsorbed onto siUca and flash chromatographed eluting with 1:19 methanokdichloromethane. Fractions 7-14 were combined and recrystalUzed from dichloromethane and methanol to give white crystals. Yield 0.70 g (36%). m.p.

154-156°. H n.m.r. (DMSO-d6) δ 1.85-2.00, m, 2H; 2.02, s, 9H; 2.80-2.95, br s,

3H; 3.95-4.13, m, 2H; 4.03, s, 6H; 5.76, s, 2H; 7.13, br s, IH; 7.71, s, IH.

EXAMPLE 57

9-[4-(12-Methoxydodecanoxy)-3,4-bis(hydroxymethyl)but-l-yl]-2- aminopurine

12-Methoxydodecanoic acid (1.72 g, 7.5 mmol) and l, -carbonyldiimidazole (1.34 g, 8.3 mmol) were stirred together in dry dimethylformamide (50 ml) for 1.5 hrs under a nitrogen atmosphere. To this solution was added 9-[4-hydroxy-3,4- bis(hydroxymethyl)but-l-yl]-2-aminopurine (2.0 g, 7.5 mmol) δissolved in dimethylformamide (80 ml) and 4-dimethylaminopyridine (0.22 g). The reaction mixture was stirred for 16 hrs, when hplc analysis indicated 50% completion. The solvent was removed in vacuo and the crude material was preadsorbed onto siUca and flash chromatographed eluting with 3:17 methanol:dichloromethane. Fraction 5 (1.18 g)was found to contain the desired product. This was further purified by hplc eluting with 2:3 acetonitrile:water to give the product as white crystals, m.p.

91-92°.lH n.m.r. (DMSO-d6)) δ 1.20,br s, 14H; 1.37-1.57, m, 4H; 1.73-1.86, m, 2H; 2.27, t, 77.2 Hz, 2H; 3.21, s, 3H; 3.28, t, 76.4 Hz, 2H; 3.36, s (after D2O exchange), 4H; 3.94, s, 2H; 4.08-4.22, m, 2H; 4.63-4.73, m, 2H; 6.46, s, 2H; 8.05, s, IH; 8.55, s, IH.

EXAMPLE 58

9-[4-Hydroxy-3,4-bis(12-methoxydodecanoxymethyl)but-l-yl]guanine

12-Methoxydodecanoic acid (1.63 g, 7.1 mmol) and l,r-carbonyldiimidazole (1.27 g, 7.8 mmol) were stirred together in dry dimethylformamide (70 ml) for 1.5 hrs under a nitrogen atmosphere. To this solution was added 9-[4-hydroxy-3,4- bis(hydroxymethyl)but-l-yl]guanine (1.0 g, 3.53 mmol) and 4- dimethylaminopyridine (0.20 g). The reaction mixture was stirred for 2 days. The solvent was removed in vacuo and the crude material was preadsorbed onto siUca and flash chromatographed eluting with 1:19 methanol:dichloromethane, 1:9 methanokdichloromethane and 3:17 methanol:dichloromethane. Fractions 17-21 were combined to give the product as white crystals, m.p. 174-176°. H n.m.r. (DMSO-d6) δ 1.21, s, 28H; 1.13-1.58, m, 8H; 1.74-1.89, m, 2H; 2.27, t, 77.2 Hz, 4H; 3.20, s, 6H; 3.27, t, 76.4 Hz, 4H; 3.36-3.44, m, 2H; 3.83-4.17, m, 2H; 3.95, s, 4H; 4.80-4.95, m, IH; 6.39, s, 2H; 7.67, s, IH; 10.55, br s, IH.

EXAMPLE 59

Prepared from 9-[4-hydroxy-3,4-bis(hydroxymethyl)but- 1 -yl]-2-aminopurine using the method of Example 57. !H n.m.r. (DMSO-d6)) δ 1.67-1.84, m, 2H; 3.33, s (after D2O exchange), 4H; 3.69, s, 2H; 3.98, s, 2H; 4.04-4.18, m, 2H; 4.61-4.75, m, 2H; 6.48, s, 2H; 7.01-7.06, m, IH; 7.29-7.34, m, IH; 7.41-7.47, m, IH; 8.00, s, IH; 8.56, s, IH.

EXAMPLE 60

9-[4-Hydroxy-3,4-bis(hydroxymethyl)butyl]guanine (1.0 g, 3.53 mmol) and 1,1'- carbonyldiimidazole (0.69 g, 4.24 mmol) were stirred together in dry dimethylformamide (60 ml) for 1.0 hr under a nitrogen atmosphere. To this solution was added methylamine (8.03M in ethanol, 0.53 ml, 4.24 mmol) and the reaction was stirred for 4 days. The solvent was removed in vacuo and the crude material was purified by hplc eluting with 1:19 acetonitrile: water to give the product as white crystals, m.p. 213-215°. *H n.m.r. (DMSO-d6) δ 1.65-1.82, m, 2H; 2.56, d, 74.6 Hz, 3H; 3.33, s (after D2O exchange), 4H; 3.90, s, 2H; 3.95- 4.17, m, 2H; 6.47, s, 2H; 6.90-7.06, m, IH; 7.64, s, IH.

EXAMPLE 61

H₂N

Prepared from 9-(4-acetoxy-3-acetoxymethyl-3-fluorobut- l-yl)-2-amino-6- chloropurine using the method of Example 2. [Found: 272.1162 Calc. for

C10H15FN5O3 requires 272.1159]. !H n.m.r. (DMSO-dg) δ 2.09, dt, 76, 18 Hz, 2H; 3.51, dd, 75, 18 Hz, 4H; 4.08, t, 76 Hz, 2H; 4.98, t, 75 Hz, 2H; 6.46, s, IH, br; 7.69, s, IH; 10.55, s, IH, br.

EXAMPLE 62

Prepared from 9-(4-acetoxy-3-acetoxymethyl-3-fluorobut- 1 -yl)-2-amino-6- chloropurine using the method of Example 17. m.p. 200-206°. ^AH n.m.r. (DMSO- dg) δ 2.09, dt, 76, 18 Hz, 2H; 3.51, dd, 75, 18 Hz, 4H; 4.12, t, 76 Hz, 2H; 4.41, s,

2H, br; 5.01, t, 76 Hz, 2H; 5.93, s, 2H, br; 7.72, s, IH; 8.38, s, IH.

EXAMPLE 63

Prepared from 9-(4-acetoxy-3-acetoxymethyl-3-fluorobut- 1 -yl)-2-aminopurine using the method of Example 24. iH n.m.r. (DMSO-dg) δ 2.16, dt, 7, 18 Hz, 2H; 2.51, s; 3.53, dd, 6, 18 Hz, 4H; 4.21, t, 7 Hz, 2H; 5.00, t, 6 Hz, 2H; 6.51, s, 2H, br; 8.08, s, IH; 8.56, s, IH. 13c n.m.r. (D2O) δ 34.2, 34.7, 43.7, 100.2, 103.6, 127.5, 142.1, 154.8, 158.2, 160.8.

EXAMPLE 64

Prepared from 9-(4-acetoxy-3-acetoxymethyl-3-fluorobut- 1 -yl)-2-amino-6- chloropurine using the method of Example 13. ^XH n.m.r. (DMSO-dg) δ 2.05, s, 6H; 2.34, m, 2H; 4.52, d, 720 Hz; 6.53, s, 2H, br; 8.13, s, IH; 8.58, s, IH.

EXAMPLE 65

Prepared from 9-(4-acetoxy-3-acetoxymethyl-3-fluorobut- l-yl)-2-amino-6- chloropurine using the method of Example 14. ^AH n.m.r. (DMSO-dg) δ 2.14, s, 6H; 2.34, m, 2H; 4.52, d, 720 Hz; 6.53, s, 2H, br; 8.13, s, IH; 8.58, s, IH.

EXAMPLE 66

Prepared from 9-(4-acetoxy-3-acetoxymethyl-3-fluorobut- 1 -yl)-2-amino-6- chloropurine using the method of Example 15. ^AH n.m.r. (DMSO-dg) δ 2.10, m,

2H; 4.10, m, 2H; 4.15, s, 4H; 5.15, m, 2H; 5.78, s, 2H, br; 6.64, s, 2H, br; 7.79, s, IH. EXAMPLE 67

Prepared from 9-(4-acetoxy-3-acetoxymethyl-3-fluorobut- 1 -yl)-2-amino-6- chloropurine using the method of Example 16. ^H n.m.r. (DMSO-dg) δ 2.17, dt, 8,

18 Hz, 2H; 3.53, dd, 6, 18 Hz, 4H; 4.22, t, 8 Hz, 2H; 5.02, t, 6 Hz, 2H; 6.94, s, 2H, br; 8.17, s, IH.

EXAMPLE 68

Prepared from 9-(4-acetoxy-3-acetoxymethyl-3-fluorobut- 1 -yl)-2-aminopurine using the method of Example 24. *H n.m.r. (DMSO-dg) δ 2.00, s 3H; 2.20, m; 3.61, dd, 6, 15 Hz, 2H, 4.16, d, 15 Hz, 2H; 4.22, m, 2H; 5.61, s, 2H, br; 7.78, s, IH; 8.51, s, IH. 13c n.m.r. (DMSO-d6) δ 20.2, 31.5, d, 21 Hz, 37.1, 61.8, d, 28 Hz, 64.0, d, 25 Hz, 95.0, d, 176 Hz, 127.2, 141.8, 148.6, 152.5, 159.6, 169.7.

EXAMPLE 69

Prepared from 2-amino-9-[3,3-bis(acetoxymethyl)but-l-yl]-6-chloropurine using the method of Example 14. m.p. 212° dec. iH n.m.r. (DMSO-dg) δ 0.80, s, 3H; 1.67, m, 2H; 1.70, s,

EXAMPLE 70

Prepared from 9-(4-acetoxy-3-acetoxymethyl-3-hydroxybut- 1 -yl)-2-amino-6- chloropurine using the method of Example 14. m.p. 188° dec. iH n.m.r. (DMSO- dg) δ 1.85, m, 2H; 3.51, s, 4H; 3.97, s, 3H; 4.13, m, 2H; 6.41, s, 2H, br; 7.84, s, IH.

EXAMPLE 71

Prepared from 9-(4-acetoxy-3-acetoxymethyl-3-methoxycarbonylbut- 1 -yl)-2- amino-6-chloropurine using the method of Example 13. m.p. 132-133°. iH n.m.r. (DMSO-dg) δ 2.02, s, 6H; 2.21, m, 2H; 3.53, s, 3H; 4.16, m, 2H; 4.24, s, 4H; 6.51, s, 2H, br; 8.07,- s, IH; 8.58, s, IH. 13c n.m.r. (D2O) δ 24.6, 33.3, 44.6, 56.2, 55.7, 66.0, 127.4, 142.1, 154.8, 158.1, 160.9, 179.8, 180.8.

EXAMPLE 72

Prepared from 9-(4-acetoxy-3-acetoxymethyl-3-fluorobut- 1 -yl)-2-aminopurine using the method of Example 24. iH n.m.r. (DMSO-dg) δ 1.81, m, 2H; 2.01, s, 3H; 3.95, s 2H; 4.16, m, 2H; 4.69, m, 2H; 6.48, s, 2H, br; 8.07, s, IH; 8.57, s, IH.

EXAMPLE 73

A second product from Example 72 was eluted and d e solvent removed to give a solid. m.p. 132-134°. iH n.m.r. (DMSO-dg) δ 1.89, m, 2H; 2.02, s, 6H; 3.99, s, 4H; 4.17, m, 2H; 4.95, s, IH, br; 6.47, s, 2H, br; 8.10, s, IH; 8.57, s, IH.

EXAMPLE 74

HoN

Prepared from 9-[4-acetoxy-3,3-bis(acetoxymethyl)but-l-yl]-2-aminopurine using the method of Example 24. m.p. 190-191°. (Found: C, 48.94; H, 6.59; N, 25.82;. Calc. for Cl 1H17N5O3 requires C, 49.43; H, 6.41; N, 26.20;). iH n.m.r. (DMSO- dg) δ 1.76, m, 2H; 3.38, d, 75 Hz, 6H; 4.18, m, 2H; 4.44, t, 75 Hz, 3H; 6.50, s, 2H, br; 8.06, s, IH; 8.57, s, IH. 13c n.m.r. (DMSO-d6) δ 32.6, 45.4, 63.9,

128.7, 144.2, 150.6, 154.7, 162.2.

EXAMPLE 75

9-[4-Hydroxy-3,3-bis(hydroxymethyl)but-l-yl]guanine (1.07 g, 3.8 mmol), 4-dimemylaminopyridine (100 mg, 0.8 mmol) and dimethylformamide (60 ml), were stirred at 0°. Di-t-butyl dicarbonate (3.74 g, 17.1 mmol) was added portionwise to the mixture and stirred at room temperature overnight. The dimethylformamide was removed under reduced pressure. HPLC analysis indicated a mixture of products. A portion of the crude product was purified by reverse phase HPLC, eluting with a non-Unear gradient of 10/90 CH3CN/H2O to 30/70 CH3CN/H2O. The first band was collected and concentrated under reduced pressure to give the product. iH n.m.r. (DMSO-d6) δ 1.42, s, 9H; 1.83, m, 2H; 3.96, s, 2H; 4.04, m, 2H; 5.0, m, 2H; 6.4, br s, 2H; 7.69, s, IH; 10.5 br s, IH. EXAMPLE 76

The second band collected from Example 75 was concentrated under reduced pressure to give the product. iH n.m.r. (DMSO-d6) δ 1.42, s, 18H; 1.83, m, 2H; 3.96, s, 4H; 4.04, m, 2H; 5.0, m, IH; 6.4, br s, 2H; 7.69, s, IH; 10.5 br s, IH.

EXAMPLE 77

The third band collected from Example 75 was concentrated under reduced pressure to give the product. ^JH n.m.r. (DMSO-d6) δ 1.42, s, 27H; 1.83, m, 2H; 4.02, s, 6H; 4.04, m, 2H; 6.4, br s, 2H; 7.69, s, IH; 10.5 br s, IH. EXAMPLE 78

9-[4-Hydroxy-3,3-bis(hydroxymethyl)but-l-yl]guanine (1.00 g, 3.5 mmol), 4-dimethylaminopyridine (100 mg, 0.8 mmol) and dimethylformamide (60 ml), were stirred at 0°. Dimethyl pyrocarbonate (2.27 ml, 21.2 mmol) was added dropwise to the mixture and stirred at room temperature overnight. The dimethylformamide was removed under reduced pressure. The crude product was dissolved in acetic anhydride (20 ml) and stirred at room temperature overnight. The acetic anhydride was removed under reduced pressure. HPLC analysis indicated a mixture of products. A portion of the crude product was purified by reverse phase HPLC, eluting with 20/80 CH3CN/H2O. The first band was collected and concentrated under reduced pressure to give die product. iH n.m.r. (DMSO-dg) δ 1.92, m, 2H; 2.03, s, 6H; 3.72, s, 3H; 4.04, br, 6H; 4.14, s, 2H; 6.4, br s, 2H; 7.72, s, IH; 10.6, br s, IH.

EXAMPLE 79

The second band collected from Example 78 was concentrated under reduced pressure to give the product. iH n.m.r. (DMSO-d6) δ 1.92, m, 2H; 2.03, s, 3H; 3.72, s, 6H; 4.04, br, 4H; 4.14, s, 4H; 6.4, br s, 2H; 7.72, s, IH; 10.6, br s, IH.

EXAMPLE 80

The third band collected from Example 75 was concentrated under reduced pressure to give the product. H n.m.r. (DMSO-d6) δ 1.92, m, 2H; 3.72, s, 9H; 4.04, m, 2H; 4.14, s, 6H; 6.4, br s, 2H; 7.72, s, IH; 10.6, br s, IH. EXAMPLE 81

To a stirred solution of methyl chloroformate (2.2 ml, 28.5 mmol) in pyridine (10 ml) at 0° was added dropwise 2-(2-benzyloxyethyl)-2-hydroxymethylpropane- 1,3-diol (1.05 g, 4.4 mmol) dissolved in pyridine (2 ml). The mixture was stirred under nitrogen overnight. The mixture was poured onto ice water (100 ml) and extracted with chloroform (3x100 ml). The organics were separated and concentrated under reduced pressure to give a yellow oil. The oil was dissolved in acetic anhydride (10 ml) with 4-dimethylaminopyridine (50 mg, 0.4 mmol) and stirred overnight. The mixture was poured onto water (100 ml) and extracted witii chloroform (3 x 100 ml). The organics were separated and concentrated under reduced pressure to give a yellow oil. Distillation (200°C, lxl0"5 bar) gave a mixture of 2,2-bis(methoxycarbonyloxymethyl)- 4-benzy loxybutan- 1 -ol and 2,2-bis(methoxy carbony loxymethy l)-4-benzyloxybut- 1-yl methoxy methanoate. The distilate was treated as per Example 1 steps G, H and I to give crude product. A portion of the crude product was purified by reverse phase HPLC, eluting with a non-Unear gradient of 10/90 CH3CN/H2O to 30/70 CH3CN/H2O. The first band was coUected and concentrated under reduced pressure to give the product. iH n.m.r. (DMSO-dg) δ 1.98, m, 2H; 2.02, s, 3H; 3.72, s, 6H; 4.04, s, 2H; 4.14, s, 4H; 4.16, m, 2H; 6.89, br s, 2H; 8.17, s, IH.

EXAMPLE 82

The second band collected from Example 81 was concentrated under reduced pressure to give the product. iH n.m.r. (DMSO-d6) δ 1.98, m, 2H; 3.72, s, 9H; 4.14, s, 6H; 4.16, m, 2H; 6.89, br s, 2H; 8.17, s, IH.

EXAMPLE 83

The crude product from Example 81 was treated as per Example 13 to give crude product. A portion of the crude product was purified by reverse phase HPLC, eluting with a non-linear gradient of 10/90 CH3CN/H2O to 30/70 CH3CN/H2O. The first band was collected and concentrated under reduced pressure to give the product. iH n.m.r. (DMSO-dg) δ 1.98, m, 2H; 2.02, s, 3H; 3.72, s, 6H; 4.04, s, 2H; 4.14, s, 4H; 4.16, m, 2H; 6.46, br s, 2H; 8.09, s, IH; 8.57, s, IH.

EXAMPLE 84

The second band collected from Example 83 was concentrated under reduced pressure to give the product. iH n.m.r. (DMSO-dg) δ 1.98, m, 2H; 3.72, s, 9H; 4.14, s, 6H; 4.16, m, 2H; 6.46, br s, 2H; 8.09, s, IH, 8.57, s, IH.

EXAMPLE 85

Prepared from 9-(4-acetoxy-3-acetoxymethyl-3-hydroxybut- 1 -yl)-2-amino-6- chloropurine using the method of Example 13. iH n.m.r. (DMSO-dg) δ 2.03, s, 6H and NCH2CH22H overlapping; 3.97, dd, 11Hz, 4H; 4.21, m, 2H; 5.34, s, IH; 6.50, s, 2H, br; 8.09, s, IH; 8.57, s, IH.

EXAMPLE 86

Isolated as a bi-product from Reaction 1 step I. CrystalUsed from methanol as a white crystalUne sold. ^JH n.m.r. (DMSO-dg) δ 1.92, m, 2H; 2.03, s, 6H; 2.07, s, 3H, 3.62, s, 2H; 4.00, s, 4H; 4.16, m, 2H; 5.22, s, 2H; 6.89, s, br, 2H; 8.17, s, IH. EXAMPLE 87

Prepared from Example 86 as per method described for Example 13. The product was crystalUsed from methanol as a white crystalUne soUd. iH n.m.r. (DMSO-dg) δ 1.93, m, 2H; 2.03, s, 6H; 2.07, s, 3H; 3.63, s, 2H; 4.01, s, 4H; 4.16, m, 2H; 5.23, s, 2H; 6.47, s, br, 2H; 8.09, s, IH; 8.58, s, IH.

EXAMPLE 88

Prepared from 9-(4-acetoxy-3-acetoxymethyl-3-methoxycarbonylbut- 1 -yl)-2- aminopurine using the method of Example 24. m.p. 121-123°. iH n.m.r. (DMSO- dg) δ 2.02, t, 77 Hz, 2H; 3.40, s, 2H; 3.61, t, 5 Hz, 4H; 4.11, t, 77 Hz, 2H; 4.85, t,

5 Hz, 2H; 6.49, s, 2H, br; 8.01, s, IH; 8.56, s, IH.

EXAMPLE 89

Prepared from 2-amino-9-[3,3-bis(acetoxymethyl)but-l-yl]-6-chloropurine using the method of Example 13. m.p. 125-9° dec. ^JH n.m.r. (DMSO-dg) δ 1.00, s, 3H;

1.85, m, 2H; 2.01, s, 6H; 3.90, s, 4H; 4.11, m, 2H; 6.46, s, 2H, br; 8.09, s, IH; 8.55, s, IH.

EXAMPLE 90

Prepared using the method of Example 14, with ethanol. Yield 400 mg ( 55%). iH n.m.r. ( DMSO-d6) δ 1.35, t, 3H; 1.72, m, 2H; 3.35, s, 6H; 4.12, m, 2H; 4.45, s, 3H; 4.47, q, 2H; 6.38, s, 2H; 7.85, s, IH.

EXAMPLE 91

Prepared using die metiiod of Example 14, with benzyl alcohol. Yield 640 mg (73%). iH n.m.r. (DMSO-dg) δ 1.72, m, 2H; 3.38, s, 6H; 4.12, m, 2H; 4.45, s, 3H; 5.50, s, 2H; 6.45, s, 2H; 7.30-7.55, m, 5H; 7.88, s, IH.

EXAMPLE 92

Prepared using the method of Example 32, Step A, with cyclohexane carboxylic acid. Yield 150 mg ( 11%). iH n.m.r. (DMSO-dg) δ 1.15-1.88, m, 12H; 2.20 to 2.35, m, IH; 3.35, s, 4H; 3.91, s, 2H; 4.05, m, 2H; 4.63, s, 2H; 6,40, s, 2H; 7.67, s, IH.

EXAMPLE 93

A second product from Example 92 was eluted, and the solvent removed to give a solid. iH n.m.r. (DMSO-d6) δ 1.12-1.90, m, 24H; 2.30, m, 2H; 3.41, d, 2H; 3.95, s, 4H; 4.05, m, 2H; 4.90, t, IH; 6.40, s, 2H; 7.70, s, IH; 10.58, s, IH.

EXAMPLE 94

Prepared using the method of Example 32, Step A, with heptanoic acid. iH n.m.r. (DMSO-dg) δ 0.87, t, 3H; 1.22, m, 6H; 1.51, m, 2H; 1.77, m, 2H; 2.30, t, 2H; 3.38, d, 4H; 3.95, s, 2H; 4.05, m, 2H; 4.41 and 4.65 (ratio 1:4), 2t, 2H; 6.43, s, 2H; 7.68, s, IH; 10.57, s, IH.

EXAMPLE 95

H₂N

A second product from Example 94 was eluted, and the solvent removed to give a soUd. iH n.m.r. (DMSO-d6) δ 0.85, t, 6H; 1.22, s, 12H; 1.50, m, 4H; 1.82, m, 2H; 2.29, t, 4H; 3.40, d, 2H; 3.97, s, 4H; 4.05, m, 2H; 4.93, t, IH; 6.47, s, 2H; 7.69, s, IH; 10.61, s, IH.

EXAMPLE 96

A third product from Example 94 was eluted, and the solvent removed to give a soUd. iH n.m.r. (DMSO-d6) δ 0.87, t, 9H; 1.22, s, 18H; 1.51, m, 6H; 1.92, m, 2H; 2.31, t, 6H; 4.02, s, 6H; 4.05, m, 2H; 6.41, s, 2H; 7.70, s, IH; 10.59, s, IH.

EXAMPLE 97

Prepared using the method of Example 19, with isobutyric anhydride. Yield 780 mg (31%). iH n.m-r. (DMSO-d6) δ 1.10, d, 6H; 1.77, m, 2H; 2.52, m, IH; 3.40, d, 4H; 3.92, s, 2H; 4.07, m, 2H; 4.42 and 4.67(ratio 1:3), 2t, 2H; 6.41, s, 2H; 7.69, s, IH; 10.58, s, IH.

EXAMPLE 98

A second product from Example 97 was eluted and die solvent removed to give a soUd. Yield 710 mg (24%). iH n.m.r. (DMSO-d6) δ 1.11, d, 12H; 1.87, m, 2H; 2.55, m, 2H; 3.42, s, 2H; 3.95, s, 4H; 4.05, m, 2H; 4.92, s, br, IH; 6.40, s, 2H; 7.71, s, IH; 10.61, s, IH.

EXAMPLE 99

Prepared using the method of Example 32, Step A, with isovaleric acid. Yield 805 mg (31%). iH n.m.r. (DMSO-d6) δ 0.91, d, 6H; 1.78, m, 2H; 2.00, m, IH; 2.20, d,

2H; 3.39, d, 4H; 3.95, s, 2H; 4.05, m, 2H; 4.67, t, 2H; 6.42, s, 2H; 7.69, s, IH; 10.57, s, IH.

EXAMPLE 100

A second product from Example 99 was eluted, and the solvent removed to give a soUd. Yield 513 mg (16%). iH n.m.r. (DMSO-dg) δ 0.91, d, 12H; 1.87, m, 2H;

1.97, m, 2H; 2.20, d, 4H; 3.42, s, 2H; 3.98, s, 4H; 4.06, m, 2H; 4.91, s, br, IH;

6.45, s, 2H; 7.70, s, IH; 10.63, s, IH.

EXAMPLE 101

A third product from Example 99 was eluted, and the solvent removed to give a solid. Yield 427 mg (11%). iH n.m.r. (DMSO-dg) δ 0.91, d, 18H; 1.71, m, 2H;

1.96, m, 3H; 2.21, d, 6H; 4.02, s, 6H; 4.05, m, 2H; 6.39, s, 2H; 7.71, s, IH; 10.59, s, IH.

EXAMPLE 102

Prepared using the method of Example 18. Yield 360 mg (73%). iH n.m-r. (DMSO-dg) δ 0.89, d, 6H; 1.91, m, 3H; 2.03, s, 6H; 2.20, d, 2H; 4.01, s, 6H; 4.05, m, 2H; 6.41, s, 2H; 7.72, s, IH; 10.61, s, IH.

EXAMPLE 103

9-[4-Acetoxy-3,3-bis(acetoxymethyl)but-l-yl]-2-amino-6-chloropurine (2.02 g, 4.72 mmol) was dissolved in a 7:3 solution of ethanokdichloromethane (60 ml). 40% Ethoxylamine in water (2.85 mis, 4 eq. 18.8 mmol) was added and the reaction mixture was heated to 45° for four days in a glass pressure sealed reaction vessel. Solvent was removed by rotary evaporation. The crude product was preadsorbed onto silica and flash chromatographed, eluting with 10% metiianol in dichloromethane. Fractions containing product were combined and the solvent removed by rotary evaporation. Yield 600 mg (28%). H n.m.r. (DMSO-d6) δ 1.25, t, 3H; 1.88, m, 2H; 2.03, s, 9H; 3.99, m, 4H; 4.02, s, 6H; 5.97 and 6.49(ratio 1:7), 2s, 2H; 7.51 and 7.80(ratio 1:7), 2s, IH; 9.64 and 10.22(ratio 1 :7), 2s, IH.

EXAMPLE 104

9-[4-Hydroxy-3,3-bis(hydroxymethyl)but-l-yl]guanine (1.0 g, 3.53 mmol), 4- dimethylaminopyridine (0.1 g) and acetic anhydride (60 ml) were stirred at 45° for 23 hours.The acetic anhydride was removed in vacuo at 60°.The crude residue was preadsorbed onto siUca and flash chromatographed eluting with methanol. Fractions containing the pure compound were combined and evaporated to give a soUd. The soUd was recrystalUzed from methanol.Yield 0.475 g (30%). m.p. 202-

205°. iH n.m.r. (DMSO-d6) δ 1.90-2.0, m, 2H; 2.05, s, 9H; 2.2, s, 3H; 4.05, s, 6H; 4.25-4.10, m, 2H; 8.05, s, IH; 11.7, s, lH,br; 12.0, s, IH, br. EXAMPLE 105

A mixture of l,l-carbonyldimidazole(3.3 eq, 1.89 g, 11.6 mmol), 2-furoic acid(3.0 eq, 1.19 g, 10.6 mmol) and dry dimemylformamide (60 ml) were stirred at room temperature, under an atmosphere of nitrogen for 90 minutes. 9-[4-Hydroxy-3,3- bis(hydroxymethyl)but-l-yl]guanine (1.0 g, 3.53 mmol), was added in one portion to die mixture and stirred for a further 24 hours. The solvent was removed under reduced pressure. The crude product was preadsorbed onto siUca and chromatographed, eluting with 10:90:0.2 methanol:dichloro-methane:acetic acid. The product was eluted and die solvent removed to give a white solid. The soUd was recrystalUzed from methanol /acetone.Yield 0.543 g (27%).m.p. 259-261°. iH n.m.r. (DMSO-d6) δ 2.2, m, 2H; 4.2, m, 2H; 4.4, s, 6H; 6.3, s, 2H; 6.65, s, 3H; 7.35, s, 3H; 7.7, s, IH; 7.95, s, 3H; 10.55, s, IH.

EXAMPLE 106

9-[4-Acetoxy-3,3-bis(acetoxymethyl)but-l-yl]guanine(0.5 g, 1.22 mmol),4- dimethylaminopyridine(0.5 g), n-caprylic anhydride(3 ml, 9.2 mmol) and dry dimethylformamide(l ml) were combined under nitrogen and stirred overnight at 70°C. The solvents were removed under reduced pressure at 95°C. The crude residue was preabsorbed onto silica and flash chromatographed eluting with 2:98:0.2 methanol:chloroform:acetic acid. Fractions containing the pure compound were combined, evaporated and recrystalUzed from ethyl acetate/petroleum spirit to give a white soUd. Yield 0.470 g (73%).m.p.l02-105°. iH n.m.r. (DMSO-dg) δ 0.8-0.9, m, 3H; 1.25, s, 10H, br; 1.45-1.65, m, 2H; 2.0, s, 11H; 3.35, s, IH; 4.0, s, 6H; 4.15, m, 2H; 8.05, s, IH.

EXAMPLE 107

Isolated from Example 1 Step I as a minor by-product. iH n.m.r. (DMSO-d6) δ 1.85-2.0, m, 2H; 2.05, s, 6H; 3.25, s, 3H; 3.35, s, 2H; 4.05, s, 4H; 4.1-4.25, m, 2H; 6.9, s, 2H; 8.2, s, IH.

EXAMPLE 108

A mixture of 9-[4-acetoxy-3-acetoxymethyl-3-methoxymethylbut-l-yl)-2-amino- 6-chloropurine (0.280 g, 0.62 mmol), 10% palladium on carbon (0.1 g), triethylamine (l.leq, 0.078 g, 0.6 ml) and ethanol (20 ml) were placed in a 100 ml flask,under an atmosphere of hydrogen. The mixture was stirred vigorously at room temperature for 2.5 hours. The mixture was filtered through glass fibre paper. The solution was evaporated and recrystalUzed from methanol to produce a white solid. Yield 0.08 g ( 31.6%). m.p. 136-138°.lH n.m.r. (DMSO-dg) δ 2.0- 1.85, m, 2H; 2.05, s, 6H; 3.25, s, 3H; 3.35, s, 2H; 4.0, s, 4H; 4.05-4.25, m, 2H; 6.5, s, 2H; 8.1, s, IH; 8.6, s, IH.

EXAMPLE 109

Prepared from 9-[4-hydroxy-3,3-bis(hydroxymethyl)but-l-yl]guanine using the method of Step A, Example 37. H n.m.r., (DMSO-d6) δ 1.73, m, 2H; 1.83, s, 3H; 2.93, m, 2H; 3.87-4.17, complex, 4H; 4.57 m, IH; 4.87, s, br, 2H; 6.50 s, br, 2H; 6.87, s, br, IH; 7.53, s, IH; 7.67, s, IH; 8.20, d, IH, J 5.3 Hz; 10.60, s, br, IH.

EXAMPLE 110 ANTI-HEPATITIS TESTING

1. Duck

(a) Experimental Animals, Virus and Cell Culture

One day old Pekin-Aylesbury cross-bred ducks congenitally infected witii an AustraUan strain of DHB V (CJ.Freiman and Y.E. Cossart, AustraUan Journal of Experimental Biology and Medical Science 64477-484 (1986)) were obtained from a commercial supplier. Ten to fourteen day old ducks were used to obtain primary duck hepatocyte(PDH) cultures. Sera from these animals were tested for DHBV DNA by the dot blot hybridization method (W.S. Mason, M.S. Halpern, J.M. England, G. Seal, J. Egan, L. Coates, C. Aldrich, and J. Summers, Virology 131 373-384 (1983)) and duckUngs with an intermediate virus litre [5-10xl0⁸ viral genome equivalents per ml] were selected for the preparation of congenitally infected primary duck hepatocyte (PDH) cultures. The genome of this strain of DHBV has been molecularly cloned. Hepatocytes were obtained by a modification of the method described by J.S. Tuttleman, J.C. Pugh, and J.Summers Journal of Virology 5B17-25 (1986). Ducks were anaesthetized witii ketamine (Parke-Davis, USA) at 30 mg per kg, the liver was surgically removed and perfused with 200 ml of pre-warmed (37°C) Hanks balanced salt solution (calcium and magnesium free) containing 0.5 mM EGTA followed by 200 ml of pre- warmed serum-free Eagle's Essential Medium (MEM) supplemented with 100 mg collagenase type 1 (Boehringer Mannheim, West Germany) and 2.5 mM CzCl. (Ajax Chemicals, AustraUa). A single cell suspension was prepared in MEM by gently extruding the perfused liver through a fine wire-gauze mesh. Hepatocytes were purified from the cell mass using Percoll density gradients (Pharmacia, Sweden) following a modification of the manufacturer's specifications. The gradient medium stock solution (SIP; stock isotonic Percoll) consisted of nine parts Percoll mixed witii one part 1.5 M NaCl solution. Percoll of the required density of 1.05 g/ml was then generated by diluting six parts SIP with four parts MEM at a final pH of 7.4. Five ml of hepatocyte ceU suspension was layered onto 30 ml of this solution and centrifuged at 20,000 rpm for 20 min at 20°C in a JA-20 fixed angle rotor (Beckman, USA). The bands of cells corresponding to die density of hepatocytes (1.07 - 1.09 g/cm³) were collected and washed in L 15 medium (CSL, AustraUa) supplemented with 5% fetal bovine serum (FBS) and counted in a haemocytometer. Cell viability was established using trypan blue dye exclusion.

Hepatocytes were diluted and subsequently seeded with L 15 complete (L 15) which consisted of L 15 media supplemented with 15 mM Tris, insuUn, glucose, hydrocortisone hemisuccinate, penicillin and streptomycin according to Tuttleman et al, supra and 5% FBS was also included. Hepatocytes were seeded at approximately 2.0 x 10⁶ cells per well into 6 well multiplates (Greiner, West Germany) or at approximately 0.5 x 10⁶ cells per well into 24 well plates (Costar, Cambridge Mass.). Hepatocytes were allowed to attach overnight before the first medium change (on day 1 post plating) and were maintained with L 15 complete media, at 37°C in a 5% C0₂ humidified incubator, for a total of 10 days The culture medium in both control and treated cultures (see below) was changed every second day. (b) Preparation of DHBV from Cell Cultures

Total intracellular viral DNA was extracted from cell lysates by a modification of the method of Tuttleman et al, supra. Cells were lysed in a solution containing 0.5% sodium dodecyl sulphate (SDS), 20 mM Tris-HCl (pH 7.4), 10 mM EDTA, 5 mM EGTA, and 150 mM NaCl. DNA was extracted from all samples by digestion with 200 ug per ml of proteinase K (International Biosciences Incorporated, USA) at 37°C for 1 hour, and deproteinised by extraction with an equal volume of phenoUchloroform (1:1), followed by chloroform. The aqueous phase was collected and adjusted to 0.2 M NaCl and the nucleic acids precipitated with 2 volumes of absolute ethanol at -20°C overnight. The pellets were washed in 70% ethanol and then air dried and finally redispersed in TE buffer (10 mM Tris-HCl (pH 8), 1 mM EDTA). (c) Preparation of radiolabelled DHBV DNA probes

A full length clone of the Australian strain of DHBV was propagated in E. coli and the plasmid extracted using standard techniques (J. Sambrook, E. F. Fritsch and T. Maniatis "Molecular Cloning: A Laboratory Manual" Second Edition Cold Spring Harbour Laboratory Press 1989). The cloned DHBV DNA sequences were excised from the plasmid by EcoRI digestion and were separated by preparative gel electrophoresis using a Prep-A-Gene DNA purification kit (Bio- Rad, Hercules Calif.) according to the manufacturer's recommendations. A 648 bp DNA fragment was also prepared by further digesting the EcoRI DHBV and purifying the smaller fragment as described above. Excised, purified DHBV DNA was radiolabelled with [α-³²P] dCTP using a NEN Random Primer Plus Extension kit (NEN Research Products, DuPont, Wilmington, USA) to a specific activity of 0.5-1.0 x l0⁹ cρm/mg.

(d) Analysis of DHBV DNA from Hepatocyte Cultures

DHBV DNA in cell culture was detected by slot-blot hybridization. Extracted DNA dissolved in TE buffer was diluted in 6x saUne sodium citrate (SSC) lxSSC is 0.15M NaCl + 0.15M sodium citrate, pH7.0), denatured by rapid boiUng and quenching then serially diluted in 6xSSC. A commercial slot-blot apparatus was used to apply the DNA samples to nitrocellulose hybridisation membranes (Hybond C extra, Amersham International, England). DNA was baked onto membranes at 80°C for 2 hours before pre-hybridisation in a buffer consisting of 50% deionised formamide, 6xSSC, 5mM NaH₂P0₄ (pH 6.5), 2 x Denhardt solution and 100 mg/ml of herring sperm DNA (Boeringer Manheim, Germany). After pre-hybridisation at 42°C for at least 3 hours in a hybridization oven (Hybaid, England) heat-denatured radio-labelled DHBV DNA probe was added to a concentration of at least 2xl0⁶cpm and hybridisation allowed to proceed overnight at 42°C. After hybridisation, membranes were washed twice in 2 x SSC-0.1% SDS at 24°C and twice in 0.1 x SSC/0.1% SDS for 30 min at 50°C to remove unbound probe. Radiolabelled DHBV probe bound to die air-dried filters was detected with the aid of intensifying screens by autoradiography at -70°C onto

X-OMAT RP film, (Eastman Kodak Co., USA).

(e) Preparation of test compounds

Where possible, stock solutions of test compounds were prepared in sterile deionised distilled water. For compounds witii poor aqueous solubility stock solutions were prepared in ceU culture grade dimethylsulphoxide (DMSO). Immediately before each test, dilutions of test compound stock solutions were prepared in deionised distilled water or DMSO at lOOx the final test concentration. These dilutions were then added to complete cell culture medium at the rate of 10ml per ml (a dilution of 1 in 100), so that the final concentration of distilled water or DMSO added in every case was constant at 1%, a concentration at which neither DMSO nor distiUed water had any effect on virus replication. For tests of antiviral activity, media containing a range of dilutions of test compound were tested against appropriate controls. Negative controls were drug-free media containing only 1% distilled water or DMSO ; positive controls were compounds previously tested and found to have reproducible anti-DHBV activity in this test system.

(f) Assay of viral repUcation

Following incubation total cellular DNA was extracted as described above (b), and the amount of DHBV DNA detected by slot-blot hybridization and autoradiography as in (d) above.

Areas of hybridization membranes corresponding to each sample were located by aUgnment with the autoradiographs, cut out and counted for ³²P in a gamma radiation counter.

DHBV DNA standards were used to estabUsh both the detection Umit, and prove that the relationship between the ³²P count and die amount of bound DHBV was linear over the range of interest. The extent of viral repUcation (measured as cpm bound ³²P bound DHBV probe detected) in the presence of test compounds is expressed as a percentage of viral repUcation in the control cultures. The effective concentration for 50% inhibition of repUcation is shown in Table 1.

TABLE 1 : DUCK HEPATITIS B ACTIVITY

Compoundof EC50 (μM) ExampleNo.

1 70

2 2

3 9

5 700

6 10

7 5

12 5

13 5

14 30

15 100

16 6

17 90

18 10

24 5

27 <5

28 6

32 <50

33 <100

50 3

51 25

53 <50

54 <50

61 7

63 200

64 8

65 10

66 20

67 70

68 10

69 20 70 600

72 3

73 10

74 3

Human

Tests of antiviral activity in human cells infected witii Hepatitis B were performed according to the method of Korba and Gerin reported in Antiviral Research, 19, 55-70 (1992) and the results are shown in Table 2. .

TABLE 2 : HUMAN HEPATITIS B ACTIVITY

Compound of EC50 (μM) EC90 (μM) Example No

2 1.0 5.8

6 1.5 11

62 0.6 4.7

74 0.16 1.1

EXAMPLE : 111 Tablet Formulations

The following formulation A may be prepared by wet granulation of the ingredients with a solution of povidone, followed by addition of magnesium stearate and compression.

Formulation A mg/tablet

(a) Active ingredient 250 250

(b) Lactose B.P. 210 26

(c) Povidone B.P. 15 9

(d) Sodium starch glycollate 20 12

(e) Magnesium stearate _5 _3

500 300

The following formulation B, may be prepared by direct compression of the admixed ingredients.

Formulation B mg/capsule or tablet Active ingredient 250 Pregelatinised starch NF15 150

400 Formulation C (Controlled release formulation)

This formulation may be prepared by wet granulation of the ingredients (below) with a solution of povidone followed by the addition of magnesium stearate and compression.

mg/tablet

(a) Active ingredient 500

(b) Hydroxypropylmethylcellulose 112

(methocel K4M Premium)

(c) Lactose B.P. ^•53

(d) Povidone B.P.C. 28

(e) Magnesium stearate _7

700

EXAMPLE : 112 Capsule Formulations

Formulation A

A capsule formulation may be prepared by admixing the ingredients of Formulation B in Example 4 above and filUng into a two-part hard gelatin capsule. Formulation B (infra) may be prepared in a similar manner.

Formulation B mg/capsule

(a) Active ingredient 250

(b) Lactose B.P. 143

(c) Sodium starch glycoUate 25

(d) Magnesium stearate _2 420

Formulation C (Controlled release capsule)

The following controlled release capsule formulation may be prepared by extruding ingredients a, b and c using an extruder, followed by spheronisation of the extrudate and drying. The dried pellets may then be coated witii release-controlUng membrane (d) and filled into a two-piece, hard gelatin capsule.

mg/capsule

(a) Active ingredient 250

(b) MicrocrystalUne cellulose 125

(c) Lactose B.P. 125

(d) Ethyl cellulose 13

513

EXAMPLE : 113 Injectable Formulation

Formulation:

Active ingredient 0.200 g

Hydrochloric acid solution, 0.1M qs to pH 5.0-7.0

Sodium hydroxide solution, 0.1M qs to pH 5.0-7.0

Sterile water qs to 10 ml

The active ingredient may be dissolved in most of the water at 35-40C. and the pH adjusted to between 5.0 and 7.0 with the hydrochloric acid or the sodium hydroxide as appropriate. The batch may then be made up to volume with the water and filtered through a sterile micropore filter into a sterile 10 ml amber glass vial (type 1) and sealed with sterile closures and overseals.

EXAMPLE 114

The tendency for many of the compounds of Formula (1) or (la) to act as pro-drugs of other compounds of Formula (1) or (la) is demonstrated in the

following example where various compounds of Formula (la) where R_fi is the

group -CH_^OR are orally absorbed and converted to the compound of Formula

(2), 9-[4-hydroxy-3,3-bis(hydroxymethyl)but-l-yl]guanine, in rats. This example is merely demonstrative of the ability of some compounds of Formula (la) to act as pro-drugs and is not intended in any way to limit the scope of the invention or

the scope of the nature of the groups R, , R₂, Rg, R4' or R₅' that may be present in

pro-drugs. Those skilled in the art will appreciate that other compounds of

Formula (la) where one or more of R« , R₂, Rg, R/ or R ' are different from the

exact examples shown may also act as pro-drugs of other compounds of Formula (la).

PROCEDURE

Compounds of Formula (la) were administered (50 mg/kg) by oral gavage (50 mg/ml of water) to groups of three Sprague-Dawley rats. The rats were housed separately in metabolic cages and urine was collected from each animal at 6 and 24 hours after treatment. The volume of urine was measured and recorded and aliquots taken. The aliquots were clarified by centrifugation and die supernatant transferred to a fresh tube and stored at -20°C until analysed.

Samples for analysis were allowed to warm to ~4°C, 100 μl was withdrawn and diluted to 10 ml with aqueous 40 mM ammonium acetate solution, pH 6.5. The diluted samples were filtered tiirough 0.2 μm membrane filters and analysed by high performance liquid chromatography.

In Table 3 below, the total number of moles of 9-[4-hydroxy-3,3- bis(hydroxymethyl)but-l-yl] guanine recovered in the urine is expressed as a percentage of the number of moles of compound administered. TABLE 3 : RAT METABOLIC STUDIES

Total proportion (%) of 9-[4-hydroxy-3,3- bis(hydroxymethyl)but- 1 -yl]guanine

Example No recovered at stated times after administration

(averaged results )

6 hours 24 hours

2 1.2 1.2

12 1.8 9.4

13 9.6 9.6

18 2.9 12.5

29 7.2 8.9

32 4.6 18.4

35 4.3 10.4

45 0.0 3.4

46 2.1 8.0

55 2.0 5.2

92 0.7 5.0

94 2.0 -

98 0.9 5.9

104 0.9 -

Claims

CLAIMS:

1. A method for the treatment and/or prophylaxis of a Hepadnaviridae associated infection which comprises administration of an effective amount of a compound of Formula (1):

(1) wherein R_j is hydrogen, halogen, hydroxy, azide, optionaUy substituted alkyl, optionally substituted alkoxy, optionaUy substimted aryloxy, mercapto, optionally substituted alkylthio, optionaUy substimted amino, optionaUy substituted hydrazino or optionally substituted hydroxylamino; R2 is hydrogen, halogen, hydroxy, azide, optionaUy substimted alkoxy, optionally substimted aryloxy, mercapto, optionaUy substituted alkylthio or optionally substituted amino;

R3 and R3' are me same or different and selected from hydrogen, optionally substituted alkyl, halogen, hydroxy, azide, optionally substituted alkoxy, optionaUy substimted aryloxy, mercapto, optionally substituted thio and optionaUy substituted amino; or R3 and R3' together form =0, =S, =NOH or =NOR, wherein R is optionaUy substituted alkyl; and

R4, R5 and Rg are the same or different and selected from optionally substituted alkyl, optionaUy substimted aralkyl, halogen, hydroxy, azide, optionally substituted alkoxy, optionaUy substituted aryloxy, mercapto, optionally substimted alkylthio, optionaUy substituted amino, optionally substituted acyl, optionally substituted ester, cyano, carboxy and mono-, di- or tri- phosphate; two of R4, R5 and Rg are joined together to form a cycUc group; or R4, R5 and Rg are joined togetiier to form a cycUc ortho ester group, salts thereof, pharmaceuticaUy acceptable derivatives thereof, pro-drugs thereof, tautomers thereof and/or isomers thereof to a subject requiring said treatment and/or prophylaxis.

2. A method according to Claim 1, wherein R4, R5 and/or Rg are optionally substituted alkyl or optionally substituted alkoxy.

3. A method according to Claim 1 or Claim 2, wherein R4, R5 and/or Rg are alkyl substituted with hydroxy.

4. A method according to any one of the preceding claims wherein the compound of Formula (1) has the Formula (la):

(la) wherein R_j, R2 and Rg are as defined in Claim 1; and R4'and R5'are d e same or different and selected from hydrogen, optionally substimted alkyl, optionally substimted aralkyl, optionally substimted aminoacyl, optionally substituted acyl, optionaUy substituted ester and mono-, di- or tri- phosphate, provided tiiat when one of R4', R5' or Rg is or contains mono-, di- or tri- phosphate then the remaining groups are other than phosphate; two of R4', R5' and

Rg are joined togetiier to form a cycUc acetal group, a cycUc carbonate group or a cycUc phosphate group; or R4', R5' and Rg are joined togetiier to form a cycUc ortho ester group.

5. A method according to any one of the preceding claims, wherein R_j is hydrogen, halogen, hydroxy, optionally substimted alkoxy, optionally substimted hydroxylamino or optionally substimted hydrazino.

6. A method according to any one of the preceding claims, wherein R2 is -NR Rg, wherein R7 is hydrogen, optionally substimted alkyl, optionaUy substituted aminoacyl or optionaUy substituted acyl and Rg is hydrogen or alkyl.

7. A method according to any one of the preceding claims, wherein Rg is -alkylORg', fluorine, -ORg' or methyl and Rg' is hydrogen, optionally substimted alkyl, optionally substituted acyl or mono-, di- or tri-phosphate.

8. A method according to Claim 6 or Claim 7 wherein R4', R5', Rg'

and/or R7 groups are those selected from hydrogen, aminoacyl, R9-C (O)- and R9- C (S)-, wherein R_g is hydrogen, optionally substituted alkyl, optionally

substituted alkoxy, optionaUy substituted aryl, optionaUy substituted aralkyl,

optionally substituted heterocyclyl or -NR, _QR. , , wherein Rχo and R_{j j} are the same

or different and are selected from hydrogen and alkyl or together with the nitrogen to which they are attached form a 3-7 membered heterocycUc ring.

9. A method according to any one of the preceding claims, wherein one of R4 or R4', R5 or R5' and/or Rgor Rg' form

R_{1 2} O

R_{1 3} O p o

wherein R 2 and Rχ3 are the same or different and selected from hydrogen, alkyl, aryl, aralkyl and a pharmaceutically acceptable cation.

10. A method according to any one of die preceding claims wherein two of R4 or R4', R5 or R5' and/or Rg or Rg' are joined together to form

CRuR -s • :C=0 or ^P(0)OR ₁₂

wherein Rχ2 is as defined above in Claim 9 and Rχ and R 5 are the same or different and are selected from hydrogen and Cχ.g alkyl.

11. A method according to any one of Claims 4 to 8, wherein R4', R5' and Rg or Rg' are joined together to form

wherein

Rχ4 is as defined in Claim 10.

12. A method according to Claim 4, wherein R_j is hydrogen, halogen, hydroxy, alkoxy, aryloxy, mercapto, alkylthio, amino, benzyloxy, hydroxylamino or

hydrazino; R2 is amino or acylamino; Rg is CH2-ORg'or G2H4OR ; R4', R5' and

Rg' are the same or different and are selected from hydrogen, optionally substimted alkyl, optionally substimted aralkyl, optionally substimted ester, optionaUy substimted aminoacyl and optionaUy substimted acyl; or two of R4', R5' and Rg' are joined together to form a cycUc acetal group or a cycUc carbonate group.

13. A method according to Claim 12 wherein R_j is hydrogen, hydroxy or alkoxy.

14. A method according to any one of Claims 1 to 8, 12 or 13 wherein the compound of Formula (1) or (la) has the Formula (2):

(2) or pharmaceuticaUy acceptable salts thereof, esters, ethers, acyl or aminoacyl derivatives thereof and or pro-drugs thereof.

15. A method according to any one of Claims 1 to 13 wherein d e compound of Formula (1) or (la) is:

9-(4-hydroxy-3-hydroxymemyl-3-isopropoxymethylbut- 1 - yl)guanine;

9-[4-acetoxy-3,3-bis(acetoxymethyl)but-l-yl]-2-aminopurine;

9-[4-acetoxy-3,3-bis(acetoxymethyl)but-l-yl]guanine;

2-amino-9-[4-(4' -phenoxy)phenylcarbonyl-3,3-bis(hydroxymethyl)but- 1 - yl]purine;

9-[4-L-valyloxy-3,3-bis(L-valyloxymethyl)but-l-yl]guanine;

9-[4-acetoxy-3,3-bis(L-valyloxymethyl)but-l-yl]guanine;

9-[4-hydroxy-3-(hydroxymethyl)-3-(N-benzyloxycarbonyl-L- proly loxy methy l)but- 1 -y 1] guanine;

9-[4-hydroxy-3-(hydroxymethyl)-3-(L-phenylalanyloxymethyl)but- 1 - yl]guanine, acetate salt;

9-[4-(iιnidazol-l-yl)-3,3-bis(hydroxymethyl)but-l-yl]guanine;

9-[4-cyclohexylcarbonyloxy-3,3-bis(hydroxymethyl)but-l-yl]guanine;

9-[4-hexylcarbonyloxy-3,3-bis(hydroxymethyl)but-l-yl]guanine; 9-[3,3-bis(isopropylcarbonyloxy)-4-hydroxybut-l-yl]guanine; or

9-[4-acetoxy-3,3-bis(acetoxymethyl)but-l-yl]-2-acetylamino-6- hydroxypurine.

16. A method according to any one of die preceding claims, wherein the Hepadnaviridae associated infection is Hepatitis B.

17. A method according to any one of die preceding claims, wherein the compound of Formula (1), (la) or (2) is administered in the form of two or more sub-doses per day.

18. A method according to Claim 15, wherein die sub-doses are administered in unit dosage forms.

19. Use of a compound of Formula (1), (la) or (2) as defined in any one of Claims 1 to 15 in the manufacture of a medicament for the treatment and/or prophylaxis of a Hepadnaviridae associated infection.

20. A compound of Formula (1), (la) or (2) as defined in any one of Claims 1 to 15 for use in the treatment and/or prophylaxis of a Hepadnaviridae associated infection.

21. A pharmaceutical or veterinary composition for the treatment and/or prophylaxis of a Hepadnaviridae associated infection which comprises a compound of Formula (1), (la) or (2) as defined in any one of Claims 1 to 15 in association witii a pharmaceuticaUy or veterinarily acceptable carrier, diluent, adjuvant and/or excipient.

22. A compound of Formula (1) as defined in any one of Claims 1 to 4, with the provisos that:

(a) when

R_j is benzyloxy;

R2 is amino; and and R4 and R5 together form 2,2-dimethyl-l,3-dioxane, then Rg is other than fluoro, methoxy or hydroxy;

(b) when

R_j is hydroxy;

R2 is amino; and

R4 and R5 are hydroxymethyl, then Rg is otiier than fluoro, hydroxy, methoxy, methyl or hydroxymethyl;

(c) when

R_j is hydroxy;

R2 is amino; and

R4 and R5 together form

then Rg is other than fluoro, hydroxy or methoxy; or

(d) when

Rj is chloro;

R2 is amino; and

R4 and R5 togetiier form 2,2-dimethyl-l,3-dioxane, tiien Rg is otiier than methyl or hydroxymethyl.

23. A compound of Formula (la) as defined in any one of Claims 4 to 13 with the provisos that: (a) when

R_j is benzyloxy;

R2 is amino; and

R4' and R5' together form

•CRi R15

wherein Rχ2 and Rχ3 are methyl, then Rg is otiier tiian fluoro, methoxy or hydroxy;

(b) when

R_j is hydroxy;

R2 is amino; and

R4' andR5' are hydrogen, then Rg is other than fluoro, hydroxy, methoxy, methyl or hydroxymethyl;

(c) when

R_j is hydroxy;

R2 is amino; and

R4' and R5' together form

then Rg is other than fluoro, hydroxy or methoxy; or (d) when

R_j is chloro;

R2 is amino; and

R4' and R5' togetiier form

R-i 4 R₁5

wherein R 2 and Rχ3 are methyl, then Rg is other than methyl or hydroxymethyl.

24. A compound of Formula (2) as defined in Claim 14 with the provisos as defined in Claim 23.

25. A compound as defined in Claim 15.

26. A process for the preparation of the compound of Formula (la) as defined in Claim 23 or Formula (2) as defined in Claim 24 which comprises the steps of:

(a) reacting a compound of Formula (3):

(3) wherein Rj and R2 are as defined in Claim 23 with a compound of Formula (4):

(4) wherein R_a and R^ may be the same or different and are selected from R4' and R_ζ'as defined in Claim 23, hydrogen and benzoyl;

R_c is the same as Rg as defined in Claim 23, hydroxy, hydroxyaUcyl or protected derivatives thereof; and

Z is a leaving group, to form a compound of Formula (6):

wherein

Rj, R2, R_a, R , and R are as defined above; and

(b) if necessary, converting the compound of Formula (6) to a compound of Formula (la) or (2).

27. A method for the treatment and/or prophylaxis of a Hepadnaviridae associated infection which comprises administration of an effective amount of a compound as defined in any one of Claims 22 to 25 to a subject requiring said treatment and/or prophylaxis.

28. A method according to Claim 27, wherein the Hepadnaviridae associated infection is Hepatitis B.

29. A method according to Claim 27 or Claim 28 wherein the compound is administered in the form of two or more sub-doses per day.

30. A method according to Claim 29, wherein the sub-doses are administered in unit dosage forms.

31. Use of a compound as defined in any one of Claims 22 to 25 in the manufacture of a medicament for the treatment and/or prophylaxis of a Hepadnaviridae associated infection.

32. A compound as defined in any one of Claims 22 to 25 for use in the treatment and/or prophylaxis of a Hepadnaviridae associated infection.

33. A pharmaceutical or veterinary composition for the treatment and/or prophylaxis of a Hepadnaviridae associated infection which comprises a compound as defined in any one of Claims 22 to 25 in association with a pharmaceutically or veterinarily acceptable carrier, diluent, adjuvant and/or excipient.