WO1990010012A1

WO1990010012A1 - Nucleoside analogues

Info

Publication number: WO1990010012A1
Application number: PCT/GB1990/000277
Authority: WO
Inventors: Christopher Mcguigan
Original assignee: University College London
Priority date: 1989-02-23
Filing date: 1990-02-22
Publication date: 1990-09-07
Also published as: JPH04505005A; EP0460045A1; AU5165790A

Abstract

A nucleoside analogue, as defined by formula (I), for use as a pharmaceutical, where B is an organic base, R1O is a leaving group, R2O is a leaving group, or an alkyloxy, aryloxy, substituted alkyloxy or substituted aryloxy group, and R3 is selected from hydrogen; halide; nitro; azide; substituted or unsubstituted amine, almine, alkoxy, aryloxy, acyloxy, sulphide, sulphone, sulphoxide, sulphonate, alkyl, alkenyl, alkynyl or heterocyclic group.

Description

NUCLEOSIDE ANALOGUES

This invention relates to nucleoside analogues and, in particular, to nucleoside phosphate triesters and processes for their preparation. Such compounds are useful in the treatment of viral infection.

Nucleoside analogues of general formula (I) are currently of considerable interest for use as therapeutic agents in the treatment of viral infection and, in particular, in the treatment of infection by human immunodeficiency virus (HIV) , the symptoms of which are known collectively as acquired immunodeficiency syndrome (AIDS) .

adenine, thymine, guanine, or cytosine,

(I)

Particular examples include 2* ,3'-dideoxycytidine (ddC) (B = cytosine, A = H) , 2' ,3'-dideoxyadenosine (ddA) (B = adenine, A = H) , and 3'-azidothymidine (AZT) (B = thymine, A = N₃).

AZT (Mitsuya et al. , 1985) has found widespread clinical use as an inhibitor of human immunodeficiency virus (HIV) in the treatment of AIDS.

Other nucleoside analogues have found widespread use in the treatment of a number of viral infections; for example, 9- ø-D-arabinofuranosyladenine (araA) in the treatment of herpes simplex encephalitis and disseminated herpes zoster

(North et al.I, 1979) . AIDS was first recognised as a distinct clinical entity in 1981 (Gottlieb et al., 1981). The main target in anti-AIDS treatment has been the causative agent itself, the HIV virion. In particular, being a retrovirus, HIV depends on a unique viral enzyme, reverse transcriptase (RT) , to proliferate. This enzyme has long been considered an attractive target for an attack on retroviruses (Smith et al., 1974; Chandra et al., 1977).

The mode of action of AZT (Scheme I below) as an inhibitor of HIV in lymphocytes has been studied in detail (Furman et al., 1986). In common with other nucleoside analogues, AZT requires conversion to its 5 ' -triphosphate ( arqar et al., 1984; Cooney et al., 1986). Thus, following transport of the nucleoside across the cell membrane, the nucleoside is monophosphorylated by a nucleoside kinase enzyme present in the cell. Further kinase enzymes convert the monophosphate to the corresponding triphosphate product, which is the bioactive form. The bioactive form efficiently and selectively inhibits the HIV reverse transcriptase and its incorporation into DNA results in termination of DNA synthesis.

BIOACTIVE FORM

I INHIBITION OF HIV RT

REACTION SCHEME I

However, nucleoside analogues suffer from a number of problems in relation to their anti-viral activity. First, the compounds are rapidly deactivated. For example, deactivation of nucleosides may occur by cleavage of the glycosidic bond by phosphorylase enzymes. Phosphorylases are known to cleave the glycosidic bond in natural nucleosides (Stryer, 1981). Furthermore, phosphorylases have been specifically implicated in the degradation of nucleoside analogues with therapeutic applications (Birnie et al., 1963; Saffhill et al., 1986).

In addition, where the base portion (B) of the nucleoside is adenine, guanine or cytosine, the nucleosides may be deactivated by deaminase enzymes. Deaminases cause the loss of the amine group from the base portion (B) of the nucleoside. For example, adenosine deaminase mediates in the deactivation of araA by converting it to arahypoxanthine (Bryson et al., 1976 and Haskell, 1977). In an effort to overcome this major problem, potent inhibitors of deaminase enzymes have been sought (Cha, 1976; Schaeffer et al., 1974) . However, whilst the therapeutic effect of the nucleoside compounds may be improved in the presence of deaminase inhibitors (Agarwal et al., 1978; Sloan et al., 1977) , the inhibitors themselves may have undesirable toxic side effects (North et al.II, 1979).

In an alternative approach to overcoming the problem of deactivation by deaminase enzymes, deamination resistant compounds have been sought. For example, a major substrate requirement of adenosine deaminase is a free 5'- hydroxyl group (Bloch et al., 1967). Many 5'- modified adenosine nucleosides have been prepared and are indeed resistant to adenosine deaminase (Declercq et al., 1977).

A second problem leading to poor clinical response to the nucleosides results from dependence on nucleoside kinase to effect monophosphorylation of the nucleoside. Poor intracellular phosphorylation may result in a poor clinical response to the nucleoside. In some cases a dependence on the virally-coded kinases is advantageous since it leads to enhanced antiviral selectivity (Fur an et al., 1979). However, in most cases it is deleterious. There are now many reports of the absence, low activity or deletion of the kinase leading to a poor clinical response to the nucleoside analogue (Reichard P. et al., 1962; Morse P.A. et al., 1965; and Bapat A.R. et al; 1983) . Indeed, the development of clinical resistance to AZT has recently been noted (Larder et al. 1989) .

A further problem relating to the clinical use of nucleosides is their poor physical properties, in particular their low solubility in water and poor membrane penetrability.

The problems associated with nucleosides mentioned above have prompted investigation of bio-active phosphorylated nucleosides as chemotherapeutic agents in their own right. However, little, if any clinical benefit arises from the use of pre-formed nucleoside monophosphate in comparison to the corresponding nucleosides (Heidelberger C. et al., i960). This is commonly attributed to poor membrane penetration of the charged monophosphate and the rapid extracellular cleavage to the corresponding nucleoside (Posternak, 1974; Lichtenstein. et al., 1960; Liebman et al., 1955).

More recently, the use of uncharged phosphate triester nucleoside derivatives (II) as more lipophilic and therefore more membrane soluble pro-drugs of the nucleotides have been reported (Farquhar D. et al., 1983 and 1985; Hunston R.N. et al., 1984 and Chawla R.R. et al., 1984; Declercq et al., 1988) . The therapeutic utility of such compounds is, however, disappointing.

The triester compounds (II) show increased stability to deactivation by enzymes such as deaminases and may be expected to possess the desired lipophilicity to facilitate crossing of the cell membrane. However, once inside the cell, in order to function as an HIV inhibitor according to Reaction Scheme I, the compounds require hydrolytic cleavage of the two "R0-"groups.

There remains, therefore, a need for chemical compounds which fulfil the desired criteria of improved resistance to enzymatic deactivation, reduced kinase dependence and improved physical characteristics.

It is now postulated that the disappointing bio-activity of triester compounds (II) is a consequence of the cell's inability to effect hydrolytic cleavage. This is probably a consequence of the general lack of triesterase activity in cells.

Derivatives of nucleoside phosphate triesters are known as chemical intermediates in the selective phosphorylation, at the 5' position of natural nucleosides, for example, using the bulky phosphorylating agent bis(2,2,2-trichloroethyl) phosphorochloridate (Franke, A. et al; Broom et al 1983 and 1984) . Free nucleotides can be produced from the resulting phosphate triester product by treatment with zinc dust. In addition a compound of formula (II) , where R¹ and R² are Cl₃CCH₂-, A is -OAc and B is thymine has been reported (Broom et al . 1984).

In a first aspect the present invention provides a nucleoside analogue, as defined by the formula below, for use as a pharmaceutical:

where B is an organic base ^X0 is a leaving group

R²0 is a leaving group or alkyloxy, aryloxy, substituted alkyloxy or substituted aryloxy group, and

R³ is selected from hydrogen; halide; nitro; azide; substituted or unsubstituted amine, alkoxy, aryloxy, acyloxy, sulphide, sulphone, sulphoxide, sulphonate, alkyl, alkenyl, alkynyl or heterocyclic group.

As used herein, the term "leaving group" connotes a group wherein the substituents of the group facilitate hydrolytic cleavage of a bond adjacent to the group.

The nature of the leaving group may be varied widely. The leaving group may thus include oxygen-containing groups (ketone, aldehyde, ester, acid, substituted amide, ether or alcohol) , nitrogen-containing groups (for example amine or nitro groups) , aryl groups (for example aryl groups substituted with unsaturated groups, such as nitro, for example paranitrophenyl) , alkenes/alkyne-containing groups.

Preferably, the leaving group is a substituted lower alkyloxy group.

As used herein, the term "lower alkyl group" connotes a C_± to C₁₀ branched or unbranched alkyl group, more preferably C. to C₃, most preferably C₂. In nucleoside analogues of the invention, the base portion (B) may be any organic base; for example, purine or pyrimidine bases. Preferably however, the base is adenine, thymine, guanine or cytosine. Most preferably the base is thymine.

When RιO is not the same as R2O it will be appreciated that the phosphate group is an asymmetric chiral centre. Consequently, the compound may be a single diastereomer or a mixture of diastereomers with respect to the phosphate chiral centre. The biological activity of the individual or mixed diastereomers may be different. Preferably, the compounds of the present invention are single diastereomers. More preferably, the compounds of the present invention are the most biologically active diastereomers.

R¹ and R² may together form a cyclic group provided that at least one linkage to the phosphorus atom is susceptible to hydrolytic cleavage. Preferably however R¹ and R² do not together form a covalently bonded cyclic group.

Ε^O or R²0 must be a leaving group, preferably a substituted lower alkyloxy group. Preferably both R O and R²0 are leaving groups in which case R¹ and R² may be the same or different.

Optionally, R² may be an alkyl, aryl, substituted alkyl or substituted aryl group chosen to optimise the properties of the compound. R may be a straight or branched chain substituted or unsubstituted lower alkyl group. In this way, one group only (R 0) may be used to confer lability and the second (R²0) may be modified at will to alter the physical properties such as lipophilicity and thus, for example, the membrane transport characteristics of the compound. R¹ and/or R² may be a group having the formula:

R⁴

wherein R⁴ R⁵ and R⁶ are the same or different and are selected from electron withdrawing groups or -H, provided that at least one electron withdrawing group is present and n is an integer from 0 to 5.

The electron-withdrawing group may be any group capable of rendering R²0 or R²0 a leaving group. For example the electron withdrawing group may be a halogen, an oxygen- containing group (such as a ketone, an aldehyde, an acid, and ester, an amide or an alcohol) , a nitrogen-containing group (such as an amine or a nitro group) , or an alkene or alkyne-containing group.

R⁴, R⁵ and/or R⁶ may be halogen such as -F, -Cl, -Br or -I.

Preferably R⁴, R⁵ and R⁶ are the same and are halogen, most preferably -Cl or -F.

n is preferably 1.

In particularly useful nucleoside analogues of the invention, R¹ and R² are the same and are CC1₃CH₂- (a bis(trichloroethyl) phosphate ester of a nucleoside analogue) or CF₃CH- - (a bis(trifluoroethyl) phosphate ester of a nucleoside analogue) .

Preferably, either or both R¹ and R² may contain an ester group. Preferably, R¹ and/or R² are a group of the formula 0

7 " 8

R O-C-CHR - wherein

R⁷ is a substituted or unsubstituted lower alkyl group and R⁸ is a substituted or unsubstituted lower alkyl group or

-H.

More preferably, R¹ and/or R² is selected from ethyl glycolyl (R⁷ is ethyl and R⁸ is -H) or methyl lactyl (R⁷ and R⁸ are methyl) .

It is a surprising result and a particular feature of the present i •nventi•on that R3 may be selected from a wide variety of groups whilst the nucleoside analogue retains potent antiviral activity, even when the parent nucleoside containing R³ is inactive.

Preferably, R may be hydrogen, halide, amino, azide, alkoxy, sulphonate or acyloxy. More preferably, R³ may be hydrogen, fluoride, amino, or azide. More preferably, R³ is azide.

It will be appreciated that varying the individual substituents R 1O, R O, R3 and B enables the nucleoside analogue's properties to be tuned to the optimum combination for biological activity. For example, modification of the structure may enhance the selectivity of hydrolysis in the infected cell; the substituents may also be _. chosen to enhance the physical characteristics of the nucleoside analogue, for example to increase the lipophilicity and thereby enhance its transport across the cell membrane or to increase the solubility of the nucleoside analogue.

The nucleoside analogues of the present invention may be particularly useful in the treatment of AIDS. The nucleoside analogues of the present invention have been shown in in vitro assays to be excellent inhibitors of HIV proliferation. Thus, an assay in which the nucleoside analogues of the present invention, suitable host cells, and HIV are incubated together, indicates that the IC₅₀ of the compounds (i.e. concentration of the compound required to produce a 50% reduction in the formation of HIV antigen) is less than 40μM and may be much less than lμM. Enhanced inhibition may be observed in an assay in which the compounds and host cells are preincubated prior to addition of HIV.

In particular, it has been noted that while the nucleoside analogues of the present invention are excellent in vitro inhibitors of HIV proliferation they present low toxicity towards uninfected cells.

It is believed that the compounds of the present invention overcome the above-mentioned problems associated with the bioactivity of nucleoside analogues in a number of ways.

Firstly, the compounds possess enhanced stability towards deactivation; secondly, the phosphorylated structure of the compounds leads to a reduced dependence on kinase enzymes to phosphorylate the nucleoside; and thirdly, the uncharged nature of the compounds enables them to cross the lipophilic cell membranes.

In particular, it is postulated that once the uncharged compounds have been transported across the cell membranes they are readily hydrolysed, possibly by enzymic means. The resulting phosphate diester may then be further hydrolysed by, for example, phosphodiesterase enzymes or chemical means, to yield the corresponding monophosphate. The monophosphate is then a substrate for transformation by kinase enzymes to the corresponding triphosphate, as shown in Reaction Scheme I. Thus, the bioactive form of the nucleoside is produced. It is worthy of note that the nucleotide may be released, rather than the nucleoside. This may be the origin of the finding herein reported, that phosphate derivatives of inactive nucleosides may be active as anti-viral agents. This may correspond to an inability of host nucleoside kinase enzymes to phosphorylate highly modified nucleosides to the corresponding δ'-monophosphates. If, however, the monophosphate of such a nucleoside is released intracellularly, by way of the phosphate triesters herein disclosed, this might then be a good substrate for further phosphorylation to the bio-active triphosphate.

It is not intended to limit this disclosure to these postulates explaining the surprisingly efficacious nature of the compounds of the present invention.

A second aspect of the present. invention provides a nucleoside analogue per se as defined in the first aspect of the present invention with the proviso that when R¹ and R² are C1-CCH-- then R³ is not OAc.

A third aspect of the present invention provides a process for the preparation of a nucleoside analogue as defined in the second aspect of the present invention.

The nucleoside analogue as defined in the second aspect of the present invention may be prepared according to the scheme outlined in Reaction Scheme II, where R⁷ is a chemical precursor to R³.

Reaction Scheme II

A fourth aspect of the present invention provides a pharmaceutical composition comprising a nucleoside analogue as defined in the first aspect of the present invention in association with a pharmaceutically acceptable excipient.

A fifth aspect of the present invention provides a nucleoside analogue as defined in the first aspect of the present invention in a form suitable for parenteral or oral administration.

A sixth aspect of the present invention provides a process for the preparation of a pharmaceutical composition comprising bringing a nucleoside analogue as defined in the first aspect of the present invention into association with a pharmaceutically acceptable excipient.

A seventh aspect of the present invention provides a method of treatment comprising the administration, to a human or animal in need of such treatment, of an effective amount of a nucleoside analogue as defined in the first aspect of the present invention.

Preferably, the seventh aspect of the present invention provides a method of treatment of a viral infection. More preferably, the viral infection is human immunodeficiency virus.

An eighth aspect of the present invention provides use of a nucleoside analogue as defined in the first aspect of the present invention for the manufacture of a medicament for the treatment of a viral infection.

Preferably the viral infection is human immunodeficiency virus.

A ninth aspect of the present invention provides a pharmaceutically acceptable salt or addition compound of a nucleoside analogue as defined in the first aspect of the present invention.

The present invention will now be described by reference to specific embodiments.

Example 1

Preparation of 3 ' -Az idothymid ine-5 ' -bis ( 2 . 2 . 2 - trichloroethyl) phosphate , UCL30

3 • -azidothymidine-5 ' -bis (2 , 2 , 2-trichloroethyl) phosphate was prepared using essentially the method of Franke et al .

3 ' -azidothy idine (0.2g, 0.75mmol) was dissolved in anhydrous pyridine (30 mL) , and bis(2,2,2-trichloroethyl) phosphorochloridate (0.57g, 1.49mmol, 2 equivs.) added with vigorous stirring, at ambient temperature. After stirring for 5 hours, the reaction was quenched with water (27_/.L, l.49mmol), and the solvent then removed under reduced pressure. The crude product was purified by column chromatography on silica, using chloroform as the eluant. Pooling and evaporation of appropriate fractions gave the product, (0.27g, 59%).

³¹P nmr 5(CDC1₃) -3.97,

¹H nmr ό^"(CDCl-) 9.67(1H, bs, NH) , 7.28(1H, s, H6) , 6.15(1H, t, HI', J=6.7HZ), 4.67(4H, d, CH-OP, J=7.2Hz), 4.45(2H, m, H5'), 4.38(1H, m, H3') , 4.04(1H, m, H ') , 2.49(2H, m, H2') , 1.91(3H, s, CH₃) .

¹³C nmr <S(CDC1₃) 163.81(C2), 150.81(C4), 135.48(C6),

111.70(C5), 94.38(CC1₃, d, J=10.4Hz), 85.50(C1'), 81.77(04', d, J=8.5HZ), 67.76(C5', d, J=5.1HZ), 60.05(C3'), 37.20(C2'),

12.57(CH₃), (CH-OP peak at Ca. 77ppm, obscured by solvent).

¹³C nmr £(CH₃0D) 165.19(C2), 151.11(C4), 137.04(C6), 111.14(C5), 95.00(CC1₃, d, J=10.9Hz), 85.83(C1'), 82.27(C4', d, J=8.4HZ), 77.52(CH₂OP, d, J=4.1Hz), 68.56(C5', d, J=5.4HZ), 60.59(C3;), 36.58(C2'), 11.83 (CH₃) .

Found C 27.28%, H 2.61%, P 4.95%; C₁₄H-₆C1₆N₅0₇P requires C 27.57%, H 2.64%, P 5.07%.

HPLC: Waters system, using a 25cm x 4.6mm Partisil 5 silica column, and a mobile phase of 98% ethyl acetate/2% petroleum spirit, with a flow rate of 2.0 cm / in. Detection was by UV, at 254nm; no AZT observed. Example 2

Preparation of 3 ' -Azidothymidine-5 '-bis(2 , 2.2- trifluoroethyl) phosphate. UCL42.

3'-Azidothymidine (0.2g, 0.75mmol) was dissolved in anhydrous pyridine (30mL) , and bis(2,2,2- trifluoroethyl)phosphorochloridate (0.42g, 1.49mmol, 2 equivs.) added with vigorous stirring, at ambient temperature. After stirring for 5 hours, the reaction was quenched with water (27μL, 1.49mmol), and the solvent then removed under reduced pressure. The crude product was purified by column chromatography on silica, using chloroform as the eluant. Pooling and evaporation of appropriate fractions gave the product, (0.25g, 65%).

³¹P nmr ό^"(CDCl₃) -2.52

¹H nmr <S(CDC1-) 9.90(1H, bs, NH) , 7.22(1H, s, H6) , 6.12(1H, t, HI', J=6.3HZ), 4.41(4H, quintet, CH₂OP, J=7.8Hz), 4.36(3H, m, H5' , H3') , 4.04(1H, m, H4 ' ) , 2.41(2H, m, H2•) , 1.82(3H, s, CH₃) .

¹³C nmr <5(CDC1-) 163.99(C2), 150.37(C4), 135.53(C6), 121.10(CF₃, qd, 3=211 . 6 , 8.9Hz), 111.61(C5), 85.60(C1'), 81.70(C4', d, J=7.9Hz), 67.54(C5',d, J=5.8Hz), 64.14(CH₂OP, qd, J=38.2, 4.1Hz), 60.86(C3'), 37.03(C2'), 12.16(CH₃). Found C 32.33%, H 3.07%, N 13.18%, P 6.22%; ^C ₁₄ ^H ₁₆ ^F ₆ ^N ₅°₇ ^{P re<}3^{uires c} 32.21%, H 3.32%, N 13.41%, P 5.93%.

HPLC: Waters system, using a 25cm x 4.6mm Partisil 5 silica column, and a mobile phase of 98% ethyl acetate/2% petroleum spirit, with a flow rate of 2.0cm³/min. Detection was by UV, at 254nm; no AZT observed. EXAMPLE 3

3 '-Azidothvmidine 5 '-(ethyl qlvcolyl propyl) phosphate,, UCL52.

Ethyl glycolyl propyl phosphorochloridate (1.10g, 4mmol) was added to 3 '-azidothymidine (0.20g, 0.75mmol) in anhydrous pyridine (lO L) with stirring at room temperature. After 14d, water (ca 2mL) was added and the solvent removed under reduced pressure, the last traces being co-evaporated with toluene (3 x 20mL) . The crude product was purified by chromatography on silica eluted with 3% methanol in chloroform, (0.13g, 37%) . δ_p (CDC1₃) 0.776, 0.362 (1:1); ό"_H (CDC1₃) 9.73(1H, s, NH) , 7.39/7.38(1H, d, H6) , 6.23(1H, t, HI'), 4.58 (2H, m, glycolyl CH-) , 4.39(lH, m, H3 ' ) , 4.34 (2H, m, H5'), 4.18(2H, m, CH₃CH-CH-) 4.04(2H, q, CH-CH-O) , 3.98(1H, m, H4¹), 2.43(2H, m, H2 ' ) , 1.90(3H, d, 5-Me) , 1.65(2H, m, CH₃CH₂CH₂) , 1.23 (3H, t, CH₃CH₂0) , 0.90(3H, t, CH-CH-CH-) ; 5_C (CDC1₃) 167.79/167.72 (d, glycolyl C=0, J=4.4/4.8Hz) , 163.91(C2), 150.39(C4), 135.19/135.07 (C6) , 111.40/111.35(C5) , 84.52/84.47 (Cl^« ) , 82.14(d, C4 ' , J=8.3Hz), 70.13/70.06(d, CH-_jCH-CH-, J=6.6/6.5Hz) , 66.57/66.44 (d, C5' , J=5.8/5.3HZ) , 63.72/63.64(d, glycolyl CH-, J=4.3/5.1Hz) , 61.68(CH₃CH₂0) , 60.11/60.05(C3 ^• ) , 37.40/37.33 (C2 ' ) , 23.42(d, CH₃CH-CH₂, J=7.0HZ), 13.97 (CH₃CH-0) , 12.29/12.26(5-Me) , 9.77(CH₃CH₂CH₂) ; EIMS m/e 476(MH⁺), 373 (MH⁺ - C₄H_?0₃) , 350(MH⁺ -thymine), 329 (M⁺-EtGlycO -PrO) , 307 (MH⁺ - thymine - N₃H) , 126(thymineH⁺) , 81(C₅H₅0, base peak); HPLC 50+250 x 4.6mm Spherisorb column. Eluant of water (A), 5% water in acetonitrile (B) ; 5% B (t=0-10min) then a linear gradient to 90% B (t=30min) . Retention time= 20.02min.

EXAMPLE 4

3 '-Azidothvmidine 5'-(S-methyl lactyl propyl) phosphate.

UCL66.

S-Methyl lactyl propyl phosphorochloridate (0.69g, 2.81mmol) was added to 3 '-azidothymidine (0.25g, 0.94mmol) and N- methylimidazole (0.61g, 7.5mmol) in dry tetrahydrofuran

(5mL) with stirring at room temperature. After 48h the solvent was removed under reduced pressure, the residue dissolved in chloroform (30mL) and extracted with saturated sodium bicarbonate solution (15mL) and water (lO L). The combined aqueous layers were back extracted with chloroform (15mL) , and the combined organic phases dried over magnesium sulphate and concentrated to a small volume under reduced pressure. Addition to petroleum ether (bp 30-40 °C; 500mL) and storage at -20 °C for 17h gave the crude product as a gum. The ether was decanted and the gum purified by chromatography on silica (60g) eluted with 0.5% methanol in chloroform. Further purification was achieved using a second chromatographic column, eluted with neat chloroform. Pooling and evaporation of appropriate fractions, followed by extraction into isopropanol, gave the product (0.20g, 45%) . δ (CDC1₃) -2.168, -2.311 (4:5) ; $_H (CDC1-) 10.08/10.03(1H, S, NH) , 7.43/7.39(1H, d, H6) , 6.25(1H, m, Hl^»), 4.94(1H, m, lactyl CH) , 4.36(1H, m, H3 ' ) , 4.32(2H, m, H5') , 4.10(1H, m, H4'), 4.02(2H, m, ^'CH-OP) , 3.74(3H, s, OMe) , 2.40(1H, m, H2 ' ) , 2.30(1H, m, H2 • ) , 1.90/1.89(3H, d, 5-Me) , 1.69(2H, m, CH.-CH-) , 1.54(3H. t, lactyl Me) , 0.94(3H, m, CH.-CH-) ; 5_ (CDCl₃) 170.87/170.51 (d, lactyl C=0, J=3.7/4.6HZ) , 163.95/163.91(C2) , 150.42/150.38 (C4) , 135.03/135.00(C6) , 111.46/111.33 (C5) , 84.44(01'), 82.18/81.97(d, C4 ' , J=8.2/8.0Hz) , 72.21/72.05 (d, lactyl CH, J=5.2Hz), 70.16/69.66(d, CH-OP, J=6.1/5.2Hz) , 66.57/66.19(d, C5^«, J=6.1/5.6HZ) , 60.16/59.91(C3^») , 52.51/52.44 (OMe) , 37.36/37.24(C2') , 23.36(m, CH₃CH₂) , 19.97/18.96(d, lactyl Me, J=5.4/5.6HZ) , 12.26(5-Me), 9.77/9.73 (CH-CH₂) ; EIMS m/e 476(MH⁺), 416(M⁺-C0₂Me) , 373 (MH⁺-MeLacO) ^' , 350(M⁺-thymine) , 329(M⁺-MeLac0 -PrO) , 307 (MH⁺-MeLac0 -PrO -N₃H) , 250(AZT⁺- OH) , 185(C₄H_0₃ phosphate^"1"), 126(thymineH⁺) , 81(C-H.O, base peak); Found C 42.99%, H 5.71%, N 14.41%, P 6.47%, C₁₇H₂₆N₅0₉P requires C 42.95%, H 5.51%, N 14.73%, P 6.52%; HPLC 50+250 x 4.6mm Spherisorb column. Eluant of water (A), 5% water in acetonitrile (B) ; 5% B (t=0-10min) then a linear gradient to 90% B (t=30min) . Retention time= 22.33min. EXAMPLE 5

3 '-Azidothvmidine 5'-(S-methyl lactyl 2,2.2-trichloroethyl) phosphate. UCL73.

S-Methyl lactyl 2,2,2-trichloroethyl phosphorochloridate (1.16g, 3.49mmol) was added to 3 '-azidothymidine (0.25g, 0.94mmol) and N-methylimidazole (0.61g, 7.5mmol) in dry tetrahydrofuran (5mL) with stirring at room temperature. After 48h the solvent was removed under reduced pressure, the residue dissolved in chloroform (30mL) and extracted with saturated sodium bicarbonate solution (15mL) and water (lOmL) . The combined aqueous layers were back extracted with chloroform (15mL) , and the combined organic phases dried over magnesium sulphate and concentrated to a small volume under reduced pressure. Addition to petroleum ether (bp 30-40°C; 500ml) and storage at -20^CC for 17 h gave the crude product as a gum. ^' The ether was decanted and the gum purified by chromatography on silica (60g) eluted with 0.5% methanol in chloroform. Further purification was achieved by extraction into isopropanόl; to yield the product (0.38g, 79%). 5_H (CDC1₃) 10.3/10.2(1H, s, NH) , 7.31/7.30(1H, d, H6) , 6.17 (H, m, HI'), 4.90(1H, m, lactyl CH) , 4.65/4.64(2H, d, CC1₃CH₂, J=1.5/1.7Hz) , 4.40(1H, , H3') , 4.30(2H, m, H5'), 3.85-4.00(lH, m, H4') , 3.71(3H, s, OMe), 2.38(1H, m, H2'), 2.36(1H, m, H2'), 1.84(3H, m, 5-Me) , 1.53/1.51(3H, d, lactyl Me, J=7.1/7.0Hz) ; HPLC 50+250 x 4.6mm Spherisorb column. Eluant of water (A), 5% water in acetonitrile (B) ; 5% B (t=0-10min) then a linear gradient to 90% B (t=30min) . Retention time = 23.57min.

EXAMPLE 6

3'-Azidothvmidine 5'-di(S-methyl lactyl) phosphate. UCL74.

Di-(S-methyl lactyl) phosphorochloridate (0.647g, 2.25mmol) was added to 3'-azidothymidine (O.lg, 0.37mmol) and N- methylimidazole (0.25g, 3mmol) in dry tetrahydrofuran (3mL) with stirring at room temperature. After 48h the solvent was removed under reduced pressure, the residue dissolved in chloroform (30mL) and extracted with saturated sodium bicarbonate solution (15mL) and water (lOmL) . The combined aqueous layers were back extracted with chloroform (15mL) , and the combined organic phases dried over magnesium sulphate and concentrated to a small volume under reduced pressure. Addition to petroleum ether (bp 30-40°C; 500mL) and storage at -20°C for 17h gave the crude product as a gum. The ether was decanted and the gum purified by chromatography on silica (60g) eluted with 0.5% methanol in chloroform. Pooling and evaporation of appropriate fractions gave the product (0.17g, 89%). δ (CDC1₃) -2.709; <S_H (CDC1₃) 9.45(1H, s, NH) , 7.48(1H, S, H6) , 6.31(1H, t, HI'), 5.08(1H, m, lactyl CH) , 4.93(1H, m, lactyl CH) , 4.85(1H, m, H3') , 4.45(2H, m, H5' ) , 4.08(1H, m, H4') , 3.79(3H, s, OMe), 3.77(3H, s, OMe), 2.44(1H, m, H2•) , 2.37(1H, in, H2'), 1.94(3H, d, 5-Me, J=1.2Hz), 1.60(3H, dd, lactyl Me), 1.58(3H, dd, lactyl Me); δ_Q (CDC1-) 171.09(d, lactyl C=0, J=3.3Hz), 170.68 (d, lactyl C=0, J=3.9 Hz), 163.91(C2), 150.46(C4), 135.23(C6), 111.59(C5), 84.58(C1'), 82.27(d, C4', J=8.4HZ), 72.73(d, lactyl CH, J=5.2Hz), 72.53 (d, lactyl CH, J=5.1Hz), 66.96(d, C5• , J=5.9Hz), 60.28(C3^«), 52.69(OMe), 37.51(C2'), 19.07(d, lactyl Me, J=6.9Hz), 18.87(d, lactyl Me, J=7.1Hz), 12.43(5-Me); EIMS m/e 519(M⁺), 518(M⁺-H), 460(M⁺-C0₂Me) , 416(M⁺-methyl lactylO) , 394(M⁺-thymine) , 373(M⁺-methyl lactylO -N₃H) , 351(M⁺-thymine -N₃H) , 313(M⁺-2 x methyl lactylO), 250(AZT⁺- OH) , 185(methyl lactyl phosphate), 81(C₅H₅0, base peak); Found C 42.12%, H 5.10%, N 13.14%, P6.00%, -_gH-.N₅O.-P requires C 41.62%, H 5.05%, N 13.48%, P 5.96%; HPLC 50+250 x 4.6mm Nucleosil 100 silica column. Eluant of dichloromethane (A) , methanol (B) ; 1% B (t=0min) then a linear gradient to 10% B (t=20min) . Retention time = 11.209 min. EXAMPLE 7

3'-Azidothvmidine 5'-(ethyl αlvcolyl S-methyl lacty_l) phosphate. UCL75.

Ethyl glycolyl S-methyl lactyl phosphorochloridate (1.62g, 5.62mmol) was added to 3'-azidothymidine (0.25g, 0.94mmol) and N-methylimidazole (0.61g, 7.5mmol) in dry tetrahydrofuran (5mL) with stirring at room temperature. After 48h the solvent removed under reduced pressure, the residue dissolved in chloroform (30mL) and extracted with saturated sodium bicarbonate solution (15mL) and water (lO L) . The combined aqueous layers were back extracte_d with chloroform (15mL) , and the combined organic phase dried over magnesium sulphate and concentrated to a small volume under reduced pressure. Addition to petroleum ether (bp 30- 40°C; 500mL) and storage at -20°C for I7h gave the crude product as a gum. The ether was decanted and the gum purified by chromatography on silica (60g) eluted with 0.5% methanol in chloroform. Pooling and evaporation of appropriate fractions gave the product (0.42g, 86%). δ (CDC1₃) -1.605, -2.369 (3:1); δ^ (CDCl..) 8.83/8.81(₁H, s, NH) , 7.49/7.46(lH, S, H6) , 6.30(1H, m, HI'), 5.04(1H, m, lactyl CH) , 4.70(1H, , H3') , 4.50(4H, m, H5• , glycolyl CH₂), 4.25(2H, q, CH₃CH₂) , 4.07(1H, , H4') , 3.87/3.79₍3H, S, OMe), 2.40(2H, m, H2'), 1.94/1.93(3H, d, 5-Me) , 1.598/1.595(3H, m, lactyl Me), 1.30(3H, t, CH₃CH-) , 5_C (CDC1₃) 167.90(lactyl C=0) , 167.89(glycolyl C=0) , 163.61(C2) , 150.25(C4) , 135.49/ 133.21 (C6) , 111.62/111.47(C5), 84.59(C1^«), 82.32/82.20(d, _C4• , J=8.4/8.3HZ) , 72.75/72.70(d, lactyl CH, J=5.4/5.5Hz₎ , 67.02/66.90(d, C5' , J=5.8Hz), 64.07/63.81(d, glycolyl CH-., J=5.1/5.0HZ), 61.91/61.83(CH₃CH₂), 60.24/60.15(C3'₎ , 52.73(OMe), 37.53/37.49(C2'), 19.07/18.96(d, lactyl Me, J=6.6/6.9Hz), 14.12(CH₃CH₂) , 12.41/12.37(5-Me) ; EIMS m/e 519(M⁺), 416(M⁺-C₄H₇0₃), 393(M⁺-thymineH) , 386 (M⁺-ethyl glycolyl -OMe), 373(M⁺-C₄H₇0--N-H) , 351(M⁺-thymine-N₃H) , 313(M⁺- 2 X C₄H₇0₃) , 250(AZT⁺-0H) , 185(C₄H₇0₃phosphate⁺) , 126(thymineH⁺) , 81(C₅H.O, base peak); Found C 41.45%, H 5.03%, N 12.86%, P 6.25%, -_gH-.N_gO-^P requires C 41.62%, H 5.05%, N 13.48%, P 5.96%; C₁₈H-₆N₅0--P. [H-0]^- requires C 40.91%, H 5.15%, N 13.25%, P 5.86%; HPLC 50+250 X 4.6mm Spherisorb column. Eluant of water (A) , 5% water in acetonitrile (B) ; 5% B (t=0-10min) then a linear gradient to 90% B (t=30min) . Retention time=20.928min.

Example 8

Preparation of 3 '-acetylthvmidine-5 ' -bis (2.2.2- trichloroethyl) phosphate. UCL58

This was prepared using essentially the method of Franke et al .

Bis(2,2,2-trichloroethyl) phosphorochloridate (0.53g, 1.40mmol) was added to a solution of 3'-acetylthymidine (0.20g, 0.70mmol) in anhydrous pyridine (20mL) , with stirring at ambient temperature. After 3 hours, water (25μL) was added, and the solvent removed under reduced pressure. The residue was purified by chromatography on silica, with elution by chloroform. Pooling and evaporation of appropriate fractions gave the product as a white solid (0.32g, 72%), (m.pt. 63-5°C).

³¹P nmr δ(CDCl₃) -4.64.

¹H nmr ό^"(CDCl-) 8.99(1H, bs, NH) , 7.40(1H, d, H6, J=1.0Hz), 6.34(1H, dd, HI', J=8.4, 5.5Hz), 5.36(1H, m, H3^») , 4.64(4H, d, CH-OP, J=7.2HZ), 4.51(2H, m, H5') , 4.18(1H, m, H4') , 2.40(1H, m, H2¹), 2.18(1H, m, H2') , 2.08(3H, s, OAc) , 1.90(3H, d, 5-CH₃, J=1.0Hz) .

¹³C nmr δ(CDCl₃) 170.57(CH₃C0) , 163.64(C2), 150.64(C4) ,134.86(C6) , 111.94(C5), 94.38(CCl₃, d, J=10.0Hz), 84.64(C1'), 82.56(C4', d, J=7.2Hz), Ca. 77(CH₂OP, obscured by solvent) , 74.10(C3^«) , 68.48(C5' , d, J=5.9HZ) , 37.06(C2^») , 20.96(CH-CO) , 12.60(5-CH₃) .

Found C 30.20%, H 2.98%, N 3.85%, P 4.76%;

C-_gH.-Cl_gN-O_gP.H-O requires C 29.79%, H 3.28%, N 4.34, P 4.80%

HPLC: Waters system, using a 50+250cm x 4.6mm Spherisorb OD52 5μM silica column, and a gradient controlled mobile phase of A=99.5% water/0.5% acetonitrile, B=95% acetonitrile/5% water, with a flow rate of 1.0 cm³/min.

The mobile phase was 20% B at 0-30 min. then 80% B.

Detection was by UV, at 254nm; retention time=30.907 min.

Example 9

Preparation of

3-methanesulphonylthymidine-5'-bis(2.2.2-trichloroethyl) phosphate. UCL60

This was prepared essentially by the method of Crossland and Servis.

A solution of methanesulphonyl chloride (0.048g, 0.42mmol) in anhydrous (5mL) was added to a solution of thymidine 5'- bis(2,2,2-trichloroethyl) phosphate (0.19g, 0.32mmol) and triethylamine (0.045g, 0.45mmol) in anhydrous dichloromethane (20mL) with stirring at -20°C. After stirring for 30 minutes at ambient temperature the reaction mixture was washed with saturated sodium bicarbonate solution (4 x 50mL) , and saturated brine (2 x 50mL) . The organic phase was dried (MgS0₄) , and the solvent removed under reduced pressure, to yield the product as a white solid (O.llg, 52%), (m.pt. 72-4°C).

³¹P nmr δ(CDCl-) -4.80. ¹H nmr 5(CDC1₃) 8.92(1H, bs, NH) , 7.30(1H, d, H6) , 6.29(1H, dd, HI', J=7.2, 5.6Hz), 5.36(1H, m, H3 ') , 4.65(4H, d, CH₂OP, J=6.4HZ), 4.42(3H, m, H4 ' , H5^• ) , 3.10(3H, S, OMs) , 2.56(1H, m, H2'), 2.36(1H, m, H2' ) , 1.91(3H, d, 5-CH₃) ,

¹³C nmr 5(CH₃OD) 165.30(02), 151.18(04), 137.18(06),

111.26(05), 95.05(CC1₃, d, J=11.2Hz), 86.27(01'), 82.73(C4, d, J=7.6HZ), 77.65(CH₂OP, d, J=4.3Hz), 68.16(C5', d, J=6.0Hz), 59.98(03'), 37.49(OMs), 37.10(02'), 11.77(5-CH₃) .

Found C 27.11%, H 2.92%, N 3.79%, P 4.29%;

C₁₅H₁₉C1₆N₂0-₀PS requires C 27.17%, H 2.96%, N 4.22, P 4.67%.

HPLC: Water systems, using a 50+250cm x 4.6mm Spherisorb OD52 5μM silica column, and a gradient controlled mobile phase of A=99.5% water/0.5% acetonitrile, B=95% acetonitrile/5% water, with a flow rate of 1.0cm³/min. The mobile phase was 20% B at 0-30 min. then 80% B. Detection was by UV, at 254nm; retention time=30.545 min.

Example 10

Preparation of 3 ' -O-Methy lthymidine-5 ' bis (2 . 2 .2- trichloroethyl ) phosphate . UCL78

This was prepared essentially by the method of Franke et al.

Bis(2,2,2-trichloroethyl)phosphorochloridate (0.35g, 0.93mmol) was added to a solution of 3'-O-methylthymidine (0.15g, 0.62mmol) in anhydrous pyridine (8mL) with stirring at ambient temperature. After 17 hours the solvent was removed under reduced pressure, and the residue was purified by chromatography on silica, with elution by chloroform. Pooling and evaporation of appropriate fractions gave the product as a white solid (0.32g, 87%).

³¹P nmr ό"(CDCl-) -5.46. ^XH nmr 5(CDC1₃) 7.31(1H, s, H6) , 6.22(1H, m, HI'), 4.65(4H, , CH₂OP) , 4.40(2H, m, H5' ) , 4.11(2H, m, H3 ' , H4 ' ) , 3.33(3H, s, OCH₃) , 2.40(1H, m, H2'), 2.10 (lH,m,H2'), 1.91(3H, S, CH₃) .

¹³C nmr 5(CDCl₃) 163.41(02), 150.04(04), 135.01(C6), 111.23(05), 94.15(CC1₃, d, J=10.4Hz), 85.02(01'), 81.92(04', d, j=7.8Hz), 80.01(03'), Ca.77(CH-OP, m, obscured by solvent), 68.31(C5', d, J=6.2Hz), 58.69(0CH₃), 36.32(C2'), 12.27 (CH₃) .

Found C 30.66%. H 3.10%, N 4.47%, Cl 35.60%; C.-H-^Cl_gN-O_gP requires C 30.08%, H 3.20%. N 4.68%. Cl 35.51%.

HPLC: Water systems, using a 50+250cm x 4.6mm Spherisorb 0D52 5μM silica column, and a gradient controlled mobile phase of A=99.5% water/0.5% acetonitrile, B=95% acetonitrile/5% water, with a flow rate of 1.0 cm³/min. The mobile phase was 20% B at 0-30 min. then 80% B. Detection was by UV, at 254nm; retention time=30.192 min.

Comparative Example A

A range of phosphate triester compounds were synthesised and their HIV inhibition activity was determined in the following _Ln vitro assays.

Primary Testing

1. 10 TCD50 HTLV III (RF) is added to the total number of cells required (10⁷ - 10⁸) and absorbed to the cells for 90 min. at 37°C.

2. Cells are washed three times in PBSA to remove unabsorbed virus and resuspended in the required volume of growth medium.

3. The cells (2xl0⁵/l.5ml) are then cultured in 6 ml tu_bes with drugs at two concentrations (100 and 1 μM) for 72h.

4. 200 μl of tissue culture supernatant from each sample is assayed for HIV antigen using a commercial ELIS_A.

5. Controls: (I) untreated infected cells;

(II) infected cells treated with AZT/ddC etc.

Secondary Evaluation (Titration)

1 and 2. (absorption and washing).

3. cells (2xl0⁵/1.5ml) are then cultured in 6 ml tubes with drugs at half log dilutions (10 - 0.001 μM) for 72h.

4. Assayed for HIV by ELISA. Toxicitv Assay

This procedure is carried out simultaneously with the secondary evaluation of active compounds.

1. Cells (2xl0⁵/1.5ml) are cultured in 6 ml tubes with drugs only at half log dilutions (100 - 0.01 μM) for 72h.

2. Cells are washed with PBSA and resuspended with ¹⁴C- protein hydrolysate in 100 μl and incubated overnight.

3. The cells are harvested, washed and ¹⁴C incorporation measured.

The compounds synthesised conformed to the following general formula:

where B is adenine, thymine

X and Y are unsubstituted alkyl-O- Z is -OH, -OMS, -OAC, -OEt, -N, -H

The assay results are summarised in Table I in which IC₅₀ (μM) for each compound is the micro olar concentration of that compound required to inhibit HIV antigen formation by 50%.

Compounds UCL2 and UCL3 are thymidines, UCL4 to 6 are acetyl thymidines, UCL7 to 9 are mesyl thymidines, UCL31 and 32 are ethyl thymidines, UCL29 and 33 are dideoxy adenosines and UCLl, 25 and 26 are azido thymidines with simple alkoxy X and Y groups.

TABLE : I

UCL No. X Y Z B ^IC5*n0

UCL2 PrO PrO OH T >100

UCL3 BuO BuO OH T >100

UCL4 EtO EtO OAc T >100

UCL5 PrO EtO OAc T >100

UCL6 BuO BuO OAc T >100

UCL7 EtO EtO OMs T >100

UCL8 PrO PrO OMs T >100

UCL9 BuO BUO OMs T >100

UCL31 PrO PrO EtO T >100

UCL32 BuO BuO EtO T >100

UCLl PrO PrO ^N3 T >100

UCL25 EtO EtO ^N3 T >100

UCL26 BuO BuO ^N3 T >100

UCL29 BuO BuO H A >100

UCL33 PrO Pro H A >100

These results show in every case an IC₅₀ of greater than 100 μM, indicating generally poor anti-HIV activity. Indeed no anti-HIV activity was noted at the highest concentration studied.

Examples 11 to 20

The compounds prepared as described in Examples 1 to 10 respectively, were tested in the in vitro assay described in Comparative Example A above. The results are given in Table II below. TABLE II

UCL No. R^X0 R²0 R³ B IC 5-_f1

UCL30 TCEO TCEO ^N3 T 0.6

UCL42 TFEO TFEO ^N3 T 0.2

UCL52 PrO EG ^N3 T 0.5

UCL66 PrO ML ^N3 T 7

UCL73 TCEO ML ^N3 T 0.4

UCL74 ML ML ^N3 T 0.09

UCL75 EG ML ^N3 T 0.3

UCL58 TCEO TCEO OAc T 15

UCL60 TCEO TCEO OMs T 5

UCL78 TCEO TCEO OMe T 35

where

EG = ethyl glycolyl

ML = methyl lactyl

These results show that compounds in which at least one of R^xO and R²0 is a leaving group are extremely effective as anti-HIV compounds.

The invention is described by way of example only and modifications of detail may be made within the scope of the invention.

REFERENCES

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Claims

1. A nucleoside analogue, as defined by the formula below, for use as a pharmaceutical:

where B is an organic base,

R²0 is a leaving group, or an alkyloxy, aryloxy, substituted alkyloxy or substituted aryloxy group, and R³ is selected from hydrogen; halide; nitro; azide; substituted or unsubstituted amine, alkoxy, aryloxy, acyloxy, sulphide, sulphone, sulphoxide, sulphonate, alkyl, alkenyl, alkynyl or heterocyclic group.

2. A nucleoside analogue for use as a pharmaceutical according to claim 1, wherein the leaving group is a substituted lower alkyloxy group.

3. A nucleoside analogue for use as a pharmaceutical according to claim 1 or 2, wherein R¹ or R is a group of the formula:

R⁴

wherein

R⁴, R⁵ and R⁶ are the same or different and are selected from electron withdrawing groups or -H, provided that at least one electron withdrawing group is present, and

n is an integer from 0 to 5.

4. A nucleoside analogue for use as a pharmaceutical according to claim 3 wherein R⁴, R⁵, and/or R⁶ are halogen atoms.

5. A nucleoside analogue for use as a pharmaceutical according to claim 3 or 4 wherein n=l.

6. A nucleoside analogue for use as a pharmaceutical according to any one of claims 3 to 5 wherein R⁴, R⁵ and R⁶ are -Cl or -F.

7. A nucleoside analogue for use as a pharmaceutical according to any one of the preceding claims wherein R¹ and/or R² contains an ester group.

8. A nucleoside analogue for use as a pharmaceutical according to claim 7 wherein R¹ and/or R² is a group of the formula

9. A nucleoside analogue for use as a pharmaceutical according to claim 8 wherein R¹ and/or R² is selected from ethyl glycolyl and methyl lactyl.

10. A nucleoside analogue for use as a pharmaceutical according to any one of the preceding claims wherein R²0 and

R²0 are both leaving groups.

11. A nucleoside analogue per se as defined in any one of claims 1 to 10 with the proviso that when R¹ and R² are

C1₃CCH₂- then R³ is not OAc. Cl₃CCH₂- then R³ is not OAc.

12. A pharmaceutical composition comprising a nucleoside analogue as defined in any one of claims 1 to 10 in association with a pharmaceutically acceptable excipient.

13. A pharmaceutical composition according to claim 12 in a form suitable for parenteral and oral administration.

14. A process for the preparation of a pharmaceutical composition comprising bringing a nucleoside analogue as defined in any one of claims 1 to 10 in association with a pharmaceutically acceptable excipient.

15. A method of treatment comprising the administration, to a human or animal in need of such treatment, of an effective amount of a nucleoside analogue as defined in any one of claims 1 to 10.

16. Use of a nucleoside analogue as defined in any one of claims 1 to 10 for the manufacture of a medicament for the treatment of a viral infection.

17. A pharmaceutically acceptable salt or addition compound of a nucleoside analogue as defined in any one of claims 1 to 10.