2-Aminopurine derivatives and process for their preparation.

The present invention relates to compounds having antiviral activity, processes for their preparation and pharmaceutical compositions containing them.

The compound 9- (4-hydroxy-3-hydroxymethylbut-l -yl ) guanine of formula (A)

HO — CH ₂ — CH— CH — OH

is disclosed in Synthetic Communications, 2(6), 345-351 (1972) but no pharmaceutical activity has been indicated for the compound in this document. We have subsequently shown that the compound of formula (A) does have pharmaceutical activity, and this is disclosed in our Published European Pat. Appn. 0141 927.

We have now prepared a series of analogues of the compound of formula (A) which has useful oral absorption properties and is converted in vivo to the compound of formula (A) which has anti-viral activity.

According to the present invention there is provided a compound of formula (I)

or a salt thereof, wherein R^ and R2 are each independently hydrogen, acyl or phosphate, provided that when one of R^ or R2 is phosphate, the other is hydrogen; or Ri and R2 are joined together to form a cyclic acetal group, a cyclic carbonate group or a cyclic phosphate group.

Examples of acyl groups for Ri and R2 are those where the group R^O- or R2O- is a pharmaceutically acceptable ester group, such as a carboxylic ester group.

II

Suitable acyl groups for Ri and R2 are R3C- where R3 is Cι_5 al yl, C^_g alkoxy, or optionally substituted aryl.

As used herein the term 'aryl' includes phenyl which may be optionally substituted with one or two groups selected from Ci-g alkyl, Ci-e alkoxy or halo such as fluoro or chlorb.

Preferably R3 is methyl, ethyl, propyl, methoxy, or phenyl.

they alkyl)2

A suitable example of a compound of formula (I) is the compound where one of Ri or R2 is (H0)2-P- and

the other is hydrogen.

In the case of compounds of formula (I) wherein one of Rl or R2 is an acyl or phosphate group, the compound exists in two enantiomeric forms. The invention includes both enantiomers in isolated form and mixtures thereof.

The compounds of the invention may be in crystalline form or as hydrates and it is intended that both forms are encompassed by the expression 'compound of formula (I) ' used herein.

Salts of the compound of formula (I) are preferably pharmaceutically acceptable, but non-pharmaceutically acceptable salts are also within the scope of the present invention, since these are useful as intermediates in the preparation of pharmaceutically acceptable compounds.

Examples of pharmaceutically acceptable salts of the compound of formula (I) are acid addition salts formed with a pharmaceutically acceptable acid such as hydrochloric acid,orthophosphoric acid and sulphuric acid.

When the compound of formula (I) contains a phosphate group suitable salts include metal salts, such as aluminium, alkali metal salts such as sodium or potassium, alkaline earth metal salts such as calcium or magnesium and ammonium or substituted ammonium salts, for example those with lower alkylamines such as triethylamine, hydroxy-lower alkylamines such as 2-hydroxyethylamine, bis-(2-hydroxyethyl)-amine or tri-(2-hydroxyethyl)- amine.

Suitable compounds of formula (I) include?

2-amino-9-(4-hydroxy-3-hydroxymethylbut-l-yl)purine;

2-amino-9-(4-acetoxy-3-acetoxymethylbut-l-yl)purine;

2-amino-9-(4-acetoxy-3-hydroxymethylbut-l-yl)purine;

2-amino-9-(3-hydroxymethyl-4-methoxycarbonyloxybut

-l-yl)purine;

2-amino-9-[2-(2,2-dimethyl-l,3-dioxan-5-yl)ethyl] purine;

2-amino-9-(4-propionyloxy-3-propionyloxymethylbut-l-yl) purine;

2-amino-9-(4-butyryloxy-3-hydroxymethylbut-l-yl)purine;

2-amino-9-(4-benzoyloxy-3-hydroxymethylbut-l-yl)purine;

2-amino-9-(4-hydroxy-3-hydroxymethylbut-l-yl)purine 4'-phosphate;

2-amino-9-(4-hydroxy-3-hydroxymethylbut-l-yl)purine 4':

4' 'phosphate; and pharmaceutically aceptable salts thereof.

The compounds of the present invention are potentially useful in the treatment of infections caused by herpes viruses, such as herpes simplex type 1, herpes simplex type 2 and varicella zoster viruses.

Accordingly, the present invention also provides a compound of formula (I) or a pharmaceutically acceptable salt thereof, for use as an active therapeutic substance and in particular for use in the treatment of viral infections.

The compound of formula (I) wherein Ri and R2 are both hydrogen or a salt thereof may be prepared by hydrolysing the 1,3-dioxane ring of a compound of formula (II)

and subsequently, if necessary, converting the compound of formula (I) thus formed to the free base or to a different salt thereof.

Preferably the hydrolysis of the compound of formula (II) is carried out in acid medium, conveniently aqueous hydrochloric acid.

The compound of formula (II) is itself an example of a compound of formula (I) and may be prepared by reducing a compound of formula (III)

,

ct mixture.

T _h e interme _d iate compound of formula ⁽ III ⁾ may ^b e prepare _{d b} y treating a compound of formu ^l a ⁽ I ^V)

wit _h a compound of formula ^(V)

H

The reaction may be carried out in an inert organic solvent, preferably dimethylformamide, in the presence of an inorganic base, preferably potassium carbonate.

The compound of formula (IV) may itself be prepared by brominating a compound of formula (VI)

The reaction is preferably carried out by treating the compound of formula (VI) with carbon tetrabromide and triphenylphosphine in an organic, aprotic solvent such as dimethylformamide.

The compound of formula (VI) may itself be prepared by treating a compound of formula (VII)

HO-CH2 CH-(CH ₂ )2-OH (VII)

HO-CH2

with 2, 2-dimethoxypropane and p-toluenesulphonic acid in the presence of acetone or tetrahydrofuran.

Compounds of formula (I) wherein R^ and R2 are acyl groups or are joined together to form a cyclic carbonate group can be prepared by reduction of a compound of formula (VIII)

Cl

(CH ₂ ) ₂

R ₄ OCH ₂ - CH - CH ₂ OR ₅

(VIII )

wherein R and R5 are the same or different acyl groups, or R4 and R5 are joined together to form a cyclic carbonate group.

Suitable acyl groups for R ₄ and R ₅ include groups O

II

R3~C-as hereinbefore defined.

The reduction is suitably carried out under conditions described above for the reduction of a compound of formula (III).

Compounds of formula (I) wherein Ri and R2 are acyl groups can be converted to a compound of formula (I) wherein R]_ and or R2 are hydrogen by conventional deacylation or partial deacylation processes. For example, reaction with methanolic ammonia can be used to effect complete deacylation to yield compound of formula (I) wherein both Ri and R2 are hydrogen. Reaction with a mild base such as potassium carbonate can result in partial deacylation to produce a compound of formula (I) wherein one of R^ or R2 is hydrogen and the other is an acyl group.

Compounds of formula (VIII), may be prepared by treating the compound of formula (V) as hereinbefore defined, with a compound of formula (X)

in which R4 and R5 are as defined in formula (VIII) and Z is a leaving group such as Cl, Br, or I, preferably Br=

The compound of formula (V) is a known compound.

Compounds of formula (X) in which Z is bromine may be prepared by brominating a compound of formula (XI).

preferably by treatment with carbon tetrabromide and triphenylphosphine in an organic, aprotic solvent, such as dimethylformamide.

Compounds of formula (X) in which Z is Cl or I may be prepared in an analogous manner.

Compounds of formula (XI) in which R4 and R5 are the same and are acyl groups may be prepared according to the following schematic process:

⁽

(XI)

wherein R^ is a removable protecting group.

Suitably Rg is a group removable by hydrolysis or hydrogenolysis.

Preferably Rg is a group removable by hydrogenolysis such as benzyl. This group can be removed by conventional methods for example by using hydrogen in the presence of a palladium/carbon catalyst.

Compounds of formula (XI) wherein R4 and R5 are joined together to form a cyclic carbonate group may be prepared by reaction of a compound formula (XII)

(HOCH ₂ )2 CH (CH ₂ ⁾ 2 OR (XII)

wherein Rg is a hereinbefore defined with phosgene or 1,1' - carbonyldiimidazole, and thereafter if desired removing the protecting group Rg. The reaction is

suitably carried out in a dry organic solvent such as pyridine at a temperature of from 0° - 50°c, conveniently at ambient temperature.

The above described processes for preparing the compound of formula (III) and compounds of formula (VIII) are also disclosed in Published European European Patent Application No. 0141 927.

Compounds of formula (I) wherein Ri and/or R2 is acyl may be prepared by esterifying a compound of formula (I) wherein R^ and R2 is hydrogen by conventional methods. If necessary during the esterification process the -NH2 group and optionally also one of the -OR^.or -OR2 groups may be protected by a suitable protecting group such as trityl or monomethoxytrityl. The product is subsequently deprotected for example by treatment with acid such as acetic acid. For example, compounds of formula (I) wherein R^O- and/or R2O- is a carboxylic ester group may be prepared by reaction of a compound of formula (I) which has been optionally protected as described above with (a), an appropriate carboxylic acid chloride or (b) an appropriate carboxylic acid anhydride or (c) an appropriate carboxylic acid in the presence of a dehydrating agent •such as dicyclohexylcafbodiimide (DCCI).

Compounds of formula (I) wherein Ri and R2 form a cyclic carbonate group can be prepared by reaction of a compound of formula (I) wherein R^ and R2 are hydrogen and the H2 group is optionally protected; with phosgene or 1,1-carbonyldiimidazole, and thereafter if necessary deprotecting the product. Suitable protecting groups for the NH2 group include trityl and monomethoxytrityl as described above. The reaction is

suitably carried out in a dry organic solvent such as pyridine at a temperature of from 0°-50°C, conveniently at ambient temperature.

Compounds of formula (I) wherein one of R]_ or R2 is phosphate or Ri and R2 together form a cyclic phosphate can be prepared by treating a compound formula (XIII)

RgOCH _* CH- CH ₂ OR ₈

wherein R7 is a protecting group and Rg and Rg are hydrogen or a protecting group provided that one of Rg or Rg is hydrogen; with a phosphorylating agent and thereafter if desired deprotecting resultant product. When R3 and Rg are both hydrogen, a cyclic phosphate compound is produced. Suitable protecting groups for R7 and Rg or Rg are trityl or monomethoxytrityl. Deprotection of the resultant product can then be effected by treatment with acid such as acetic acid.

A suitable phosphorylating agent is phosphorus oxychloride, optionally in the presence of a base such as pyridine.

In addition, when one of Rg or Rg is a protecting group cyanoethyl phosphoric acid can be employed as a phosphorylating agent in order to produce a compound of formula (I) wherein one of Ri or R2 is phosphate.

The reaction product after treatment with cyanoethyl phosphoric acid is treated with aqueous ammonia, which yields the ammonium salt of the phosphate ester as the final product.

Compounds of formula (XIII) can be prepared by protection of a compound of formula (I) wherein R^ and R2 is hydrogen, for example by reaction with a trityl or monomethoxytrityl halide such as monomethoxytrityl chloride.

Alternatively compounds of formula (I) wherein Ri and R2 are joined together to form a cyclic phosphate can be prepared from a compound of formula (I) wherein one of Ri or R2 is phosphate and the other is hydrogen by cyclisation of the monophosphate for example using dicyclohexylcarbodiimide.

Compounds of formula (I) wherein one of R^ or R2 is acyl and the other is hydrogen or Ri and R2 together form a cyclic acetal can be prepared by reacting a compound of formula (I) wherein Ri and R2 are hydrogen with a compound of formula (XIV)

wherein R^Q i ^c ι_g alkyl, Rχi is Cχ_g alkyl, m is 0,1 or 2, and n is an integer of 2, 3 or 4 provided that m + n is equal to 4,

and thereafter, if n is 3 or 4, hydrolysing the product.

VThen a compound of formula (I) in which Ri and R2 is a cyclic acetal is required, a compound of formula (XIV) wherein m is 2 and n is 2 is employed. For example, when m is 2, n is 2 and RJ _^ is methyl, the product is the compound of formula (II) as hereinbefore defined. The reaction is suitably carried out in an inert organic solvent such as tetrahydrofuran or N,N-dimethylformamide, in the presence of an acid such as p-toluene sulphonic acid.

Where necessary the subsequent hydrolysis step is an aqueous hydrolysis preferably carried out in the presence of an acid such as p-toluene sulphonic acid.

Compounds of formula (XIV) are known compounds or can be prepared from known compounds by known methods.

Compounds of formula (I) or pharmaceutically acceptable salts thereof may be formulated for use in a pharmaceutical composition. Accordingly, in a further aspect of the invention, there is provided a pharmaceutical composition which comprises a compound of formula (I) or pharmaceutically acceptable salt thereof together with a pharmaceutically acceptable carrier or excipient.

A composition which may be administered by the oral route to humans "may be compounded in the form of a syrup, tablet or capsule. When the composition is in the form of a tablet, any pharmaceutical carrier suitable for formulating such solid compositions may be used, for example magnesium stearate, starch, lactose, glucose, rice, flour and chalk. The composition may

also be in the form of an ingestible capsule, for example of gelatin, to contain the compound, or in the form of a syrup, a solution or a suspension. Suitable liquid pharmaceutical carriers include ethyl alcohol, glycerine, saline and water to which flavouring or colouring agents may be added to form syrups. The compounds may also be presented with a sterile liquid carrier for injection.

The composition may also be formulated for topical application to the skin or eyes.

For topical application to the skin, the composition may be in the form of a cream, lotion or ointment. These formulations may be conventional formulations well known in the art, for example, as described in standard books of pharmaceutics and cosmetics, such as Harry's Cosmeticology published by Leonard Hill Books and the British Pharmacopaeia.

The composition for application to the eyes may be a conventional eye-drop composition well known in the art, or an ointment composition.

Preferably, the composition of this invention is in unit dosage form or in some other form that the patient may administer to himself a single dose. A suitable dosage unit might contain from 50 mg to 1 g of active ingredient, for example 100 to 500 mg.

Such doses may be administered 1 to 4 times a day or more usually 2 or 3 times a day. The effective dose of compound will in general be in the range of from 1.0 to 20 g/kg of body weight per day or more usually 2.0 to 10 mg/kg per day.

No toxicological effects are indicated at the above described dosage levels.

In a further aspect of the invention there is provided a method of treating viral infections in a human or non-human animal, which comprises administering to the animal an effective, non-toxic amount of a compound of formula (I) or a pharmaceutically acceptable salt thereof.

The following examples illustrate the invention.

F>: . _: u _* j] e — 1

2 -Ami no- 9- ( 4 -hy oxy-3-hy roxy i'u;lhylbut- 1 -yl ) pur i ne

Method A

To a solution of 2-amino-6-chloro-9-I2-(2,2-dimethyl-1 , 3-dioxan-5-yl)ethyl]purine (0.54g, 1.75mmol) in ethanol (10ml) and cyclohexene (20ml) was added 10% palladium-on- charcoal (400mg) and the solution was refluxed for 7 hours. A further quantity of catalyst (200mg) was added and the solution was refluxed overnight. The solution was filtered and washed through with methanol. To the filtrate was added hydrochloric acid (5M, 0.3ml) and water (0.7ml) and the solution was stirred for 30 minutes at room temperature. The solution was neutralised by addition of aqueous sodium bicarbonate and the solvent was removed. The residue was purified by column chromatography on silica gel eluting with chloroform-methanol (5:1, 4:1) to afford 2-amino-9-(4-hydroxy-3-hydroxymethylbut- 1-yl)purine as a crystalline solid (150mg, 36%), .p. 156- 158°C; λ ax (H ₂ 0) 242 and 303 n ; v ax (KBr) 3320, 3210, 1640, 1610, 1580, and 1430 cm ^"1 ; δ _R [ (CD ₃ ) ₂ SO] 1.47 (1H, m, 3'-H), 1.78 (2H, q, J 7.2Hz, 2'-H), 3.3-3.5 (4H, m, 2 x 4'-H), 4.12 (2H, t, J 7.4Hz, T-H), 4.42 (2H, t, J 5.2Hz, D ₂ 0 exchange¬ able, 2 x OH), 6.45 (2H, s, D ₂ 0 exchangeable, 2-NH ₂ ) , 8.06 (1H, s, 8-H) , and 8.56 (1H, s, 6-H) ; (Found: C, 50.61; H, 6.45; N, 29.62 %. ^c _1C) H _l5 N ₅ 0 ₂ requires: C, 50.62; H, 6.37; N, 29.52 %) .

Method B (alternative reduction reaction)

To a solution of ammonium formate in methanol (400mM, 3ml) were added 2-amino-6-chloro-9-t2-(2,2-dimethyl-1 ,3- dioxan-5-yl)ethyl]purine (90mg, 0.3mmol) and 10% palladium- on-charcoal (28mg) and the mixture was heated under reflux. After 1.5 hours reduction to 2-amino-9-[2-(2,2-dimethyl-1 ,3- dioxan-5-yl)ethyl]purine was complete.

- 1 3 -

Exaπple 2

9-(4-Acetoxy-3-acetoxymethylbut-1-yl)-2-aminopurine

A suspension of 9-(4-acetoxy-3-acetcκymethylbut-1-yl)-2-amino-6-chloropurin e (0.36g, I.QΠΠTDI) and 10% palladium-σn-charcoal (30πg) in methanol containing aππDnium formate ( 00πM, 10ml) was heated under reflux for 30 minutes. The mixture was allowed to cool, filtered and the solvent removed. The residue was taken up in water and the solution extracted twice with chloroform. The organic layers were combined, dried (magnesium sulphate) and the solvent removed to afford 9-(4-acetoxy-3-acetoxyπet_hylbut-1-yl)-2-am_i_nopuri_ne (0.29g, 90%) . Recrystallisation from ethyl acetate-hexane gave white shiny plates (0.25g, 78%) .p. 102-104°C; λ ax (MeOH) 222 (27,500), 244 (4,890), and 309 (7,160)nm; vmax (KBr) 3340, 3170, 1745, 1730, 1660, 1615 and 1580cm ^"1 ; δ _R (CDC1 ₃ ) 1.90-2.05 (3H, , 2'-H and 3'-H) , 2.07 (6H, s, 2 x CH ₃ ) , 4.15 (4H, d, J 5.2 Hz, 2x4'-H), 4.21 (2H, t, J 7.2Hz, 1 '-H) , 5.16 (2H, br s, 2-NH ₂ ) , 7.79 (1H, s, 8-H) , and 8.70 (1H, s, 6-H) ; (Found: C, 52.10; H, 6.00; N, 21.49%. C _l4 H ₁₉ N ₅ 0 ₄ requires C, 52.33; H, 5.96; N, 21.79%).

- ]9 -

rnpJ_e 3

2-Anύno-9-(4-hydroxy-3-hydroxymethylbut-1-yl)purine

To a suspension of 9-(4-ecetoxy-3-acetcκyπet_hylbut-1-yl)-2-amino-6- chloropurine (4.86g, 13.7ππol) in methanol (140ml) containing armonium formate (400ιιM) was added 10% palladium-on-charcoal (0.4g) and the mixture was heated under reflux for 40 minutes. After cooling the solution was filtered and the solvent removed. The residue was taken up in water and extracted with chloroform (100ml and 50ml) . The organic layers were combined, dried (magnesium sulphate) and the solvent removed. The residue, was dissolved in methanol saturated with ammonia at 0 C (150ml) and the solution was stirred for 20 hours. The solvent was removed and the residue suspended in chloroform (20ml) and filtered. The solid was recrystallised from isopropanol-water and a second recrystallisation was carried out from the mother liquors from ethanol (total 2.71g, 83%).

Ξxaπple 4

9-(4-Acetoxy-3-hydroxymethylfaut-1-yl)-2-aminopurine

To a solution of 9-(4-acetoxy-3-acetoxyπethylbut-1-yl)-2-aminopurine

(0.48g, 1.5TOTD1) in methanol (9ml) was added anhydrous potassium carbonate (14ιtg, O.limol) and the solution was stirred for 20 minutes.

Two drops of glacial acetic acid were added, the solution was filtered and the solvent was removed. The residue was purified by column chromatography on silica σel eluting with chlorofor -methanol (15:1,

10:1) to afford 9-(4-acetoxy-3-hydroxymethylbut-1-yl)-2-aminopurine as a white crystalline solid (124mg, 30%), m.p. 166-168°; v (KBr) 3440,

_. max

3220, 1720, 1650, 1615, and 1580cm ; δ _H [(CD^SO] 1.68 (1H, m, 3'-H) , 1.82 (2H, m, 2'-H) , 1.98 (3H, s, CH ₃ ) , 3.41 (2H, t, J4.8Hz, D ₂ 0 exchange gives d, CH ₂ 0H) , 3.9 - 4.05 (2H, AB part of ABX, J 10.9Hz and ^J AX ^{= J} BX ⁵ * ^8Hz ' CH ₂ OCO) , 4.12 (2H, t, J 7.2Hz, 1 '-H) , 4.62 (1H, t, J 5.0Hz, D D exchangeable, OH), 6.44 (2H, s, D ₂ 0 exchangeable, 2-NH ₂ ) , 8.07 (1H, s, 8-H), and 8.56 (1H, s, 6-H) ; (Observed M ⁺ , 279.1326. C ₁₂ H ₁₇ N ₅ 0 ₃ requires 279.1331).

Example 5

2-Amino-9-(3-hydroxymethy1-4-methoxycarbonyloxybut-1-yl)p irine

To a suspension of 2-a ino-9-(4-hydroxy-3-hydroxymethylbut-1-yl)Furine (237mσ, LOimol) in dry tetrahydrofuran (3ml) were added p_-toluene- sulphonic acid onohydrate (0.21g, I.IΠΠDI) and tetramethyl ortho- carbonate (0.53ml, 4.0mmol) and the mixture was stirred for 100 minutes. Water (0.8ml) was added and after a further 15 minutes the solution was neutralised by addition of aqueous sodium bicarbonate. The solvent was removed and the residue was extracted with chloroform-nethanol (3:1) . The solvent was removed and the residue was purified by column chromato- graphy on silica gel eluting with chlorofonrt-nethanol (10:1) to afford 2-amino-9-(3-hydroxymethyl-4-methoxycarbonyloxybut-1-yl)puri ne which was obtained as a white crystalline solid after trituration with ethyl acetate (65mg, 22%), m.p. 129 - 132°; v _v (KBr) 3440, 3220, 1745, 1650, 1615, and 1580cm ; 6 _R [(CD^SO] 1.73 (1H, m, 3'-H) , 1.81 (2H, , 2'-H) , 3.41 (2H, t, J 5.1Hz, D ₂ 0 exchange σives d, C ₂ OH) 3.68 (3H, s, CH ₃ ) , 4.0 - 4.2 (4H, m, CH ₂ OCO and 1 '-H) , 4.65 (1H, t, J 5.2Hz, D ₂ 0 exchangeable, OH), 6.44 (2H, s, D ₂ 0 exchangeable, 2-NH ₂ ) , 8.06 (1H, s, 8-H) , and 8.55 (1H, s, 6-H) ; (Observed M ⁺ , 295.1286, C^H^N^ requires 295.1280).

2-Amino-9-[2-(2,2 3imethyl-1 ,3-dioxan-5-yl)ethyl3purine

To a suspension of 2-amino-9-(4-hydroxy-3-hydroxymethylbut-1-yl)pαrine

(240mg, LOπtπol) in N,N-diιtethylforma ide (3ml) were added jo-toluenesulphonic acid onohydrate {21Qmg,1.1πτrol) and 2,2-dimethσxy- propane (0.62ml, S.Ommol) and the solution was stirred for 30 minutes.

Potassium carbonate (110mg, O.δmmol) was added and the solution was stirred for a further 30 minutes. Water (10ml) was added and the solution was extracted with chloroform (3 x 8ml) . Ihe organic layers were combined, dried (magnesium sulphate) and the solvent removed.

Trituration with toluene-ether afforded 2-amino-9-[2-(2,2-dimethyl-1 ,3- dioxan-5-yl)ethyl]purine as a white crystalline solid (262mg, 94%) which was recrystallised from ethyl acetate-hexane (216πg, 78%), m.p. 118 - 120 λ _av (MeOH) 221 (27,200), 244 (4,920), and 308 (7,130)nm; v _v (KBr)

3450, 3140, 1635, 1615, 1580, and 1435cm ; δ _H [ (CD ₃ ) ₂ SO] 1.26 (3H, s,

CH ₃ ) , 1.33 (3H, s, CH ₃ ) , 1.58 HH, m, 3'-H) , 1.74 (2H, q, J 7.1Hz, 2'-H) ,

3.54 (2H, dd, J 11.8Hz and 8.5Hz, 2 x H ) , 3.78 (2H, dd, J 11.8Hz and

4.4Hz, 2 x H ) , 4.07 (2H, t, J 7.2Hz, 1 '-H) , 6.46 (2H, s, D-0 exchangeabl eq

2-NH ₂ ), 8.09 (1H, s, 8-H) , and 8.56 (1H, s, 6-H) ; (Found: C, 56.09? H, 6.91; N, 24.88%. C -H .N^ requires C, 56.30; H, 6.91; N, 25.25%).

Exa ple 7

2-Amino-9-(4-propionyloxy-3-propionyloxymethylbut-1-yl)pu rine

A solution of 2-amino-9-(4-hydroxy-3-hydroxymethylbut-1-yl)purine

(0.21g, 0.9ιmol) , 4-di_methylami_nopyridine (10mg) and propionic anhydride

(0.64ml, 5.0mmol) in N,N-dimethylformamide (5ml) was stirred for 16 hours.

The solvent was removed and the residue was partitioned between aqueous sodium bicarbonate and chloroform. The organic layer was dried (magnesium sulphate) and the solvent was removed. The residue was purified by column chromatography on silica gel eluting with chloroform-methanol

(20:1) to give 2-amino-9-(4-propionyloxy-3-propionyloxymethylbut-1-yl)- purine (160m-τ, 51%) which was recrystallised from ethyl εcetate-hexane

(115πrg, 37%), m.p. 77.5 - 9°; j^ (EtOH) 222 (27,300), 244 (5,020), and 309 (7,110)nm; v (KBr) 3390, 3210, 1 ^" ?35, 1650, 1605, 1580, 1525, - max

1475, and 1425cm ; δ _p (CDC1 ₃ ) 1.14 (6H, t, J 7.6Hz, 2 x CH ₃ ) , 1.96 (3H, m, 2'-H and 3'-H), 2.34 (4H, q, J 7.6Hz, 2 x CH_ ₂ CH ₃ ) , 4.15 (4H, d,

J 5.5Hz, 2 x CH ₂ 00C) , 4.21 (2H, t, J 7.0Hz, 1 '-H) , 5.05 (2H, s, D ₂ 0 exchangeable, 2-NH ₂ ) , 7.77 (1H, s, 8-H) , and 8.69 MH, s, 6-H) ; (Observed M ⁺ 349.1752. C ₁₆ H _{23 5} 0 ₄ requires 349.1751).

9-(3-Hydroxymethyl-4-monome_thoxytrityloxybut-1-yl)-2-mon cmethoxy- tritylaminopurine (Example 8) and

9-(4-Hydroxy-3-hydroxymethylbut-1-yl)-2-monomethoxytrityl am_i_no- purine (Example 9)

To a suspension of 2-amino-9-(4-hydroxy-3-hydroxymethylbut-1-yl)purine (2.37g, 10ππol) in N,N-dimethyIformamide (40ml) containinq 4- imethyl- aminopyridine (30ιrg) and triethylamine (4.2ml) was added a solution of moncπethoxytrityl chloride (6.8g, 22ΠΠD1) in N,N-dimethylformamide (60ml) over a period of 40 minutes. The solution was stirred for a further 40 minutes, methanol (1ml) was added and the solvent was removed. The residue was taken up in chloroform and washed with water and dilute aqueous sodium bicarbonate. The organic layer was dried (magnesium sulphate) and the solvent was removed. The residue was purified by column chrαmatography on silica gel eluting with chloroform-πethanol mixtures (40:1 to 6:1).

The first product to elute was 9-(3-hydroxyπethyl-4-πmσmethoxytrityloxybut- 1-yl)-2-πon tethoxytritylaminopurine which was further purified by a second silica gel column eluting with chloroform-itethanol (40:1) and obtained as a colourless foam (3.34g, 43%); λ ^ (EtOH) 227 (47,400) and 312 (6,450)nm; (KBr) 3430, 1615, 1580, 1510, 1490, and 1415cm ^"1 ;

IllαX δ _H l(CD ₃ ) ₂ SO] 1.37 (2H, m, 2'-H), 1.49 (1H, m, 3'-H) , 2.8 - 2.9 (2H, m, CH ₂ 0C), 3.2 - 3.4 (2H, m, CH ₂ 0H) , 3.64 (5H, m, 1 '-H and OCH- _j ) , 3.73 (3H, s, 0CH ₃ ) , 4.40 (1H, t, J 5.0Hz, unexchangeable, OH), 6.7-7.4 (28H, m, Ar-H) , 7.46 (1H, s, D ₂ <-> exchangeable, ^' 2-NH) , 7.88 (1H, s, 8-H) , and 8.53 (1H, s, 6-H (Found: C, 77.28; H, 6.27; N, 8.94%. C^H^^requires C, 76.80; H, 6.06; N, 8. _'

The second product to elute was 9-(4-hydroxy-3-hydroxyιrεthylbut-1-yl)- 2-monoπe1±oxytritylaminopurine which was obtained as a white crystalline solid after trituratiσn and filtration from ether (2.07g, 41%) , m.p. 181 - 183°; λ _jnaχ (EtOH) 227 (36,000) and 312 (6,780)ran; v^ (KBr) 3390, 1615, 1580, 1525, 1510, 1490, and 1420cm ^"1 ; δ _R [(CD^SO] 1.30 (1H, m, 3'-H), 1.39 (2H, q, J 6.8Hz, 2'-H) , 3.15 - 3.35 (4H, m, 2 x 4'-H), 3.70 (3H, s, 0CH ₃ ), 3.76 (2H, t, J 7.2Hz, 1 '-H) , 4.33 (2H, t, J 5.1Hz, D ₂ 0 exchangeable, 2 x OH) , 6.8 - 7.4 (14H, m, Ar-H) , 7.52 (1H, s, D ₂ 0 exchang¬ eable, 2-NH), 7.97 (1H, s, 8-H) , and 8.52 (1H, s, 6-H) ; (Found: C, 70.49; H, 6.24; N, 13.41%. ^H^N^ requires C, 70.71; H, 6.13; N, 13.74%).

Example 10

2-Amino-9-(4-butyryloxy-3-hydroxymethylbut-1-yl) urine

To a solution of 9-(3-hvdroxyπethyl-4-παιαre^

2-monαnet_hoxvtritylamincρurine (0.70g, 0.9πιtol) and 4-d___methylamino- pyridine (lOmg) in N,N-dimethylformamide (5ml) was added butyric anhydride

(0.29ml, 1.8ιπτol) and the solution was stirred for 15 minutes. Methanol

(1ml) was added and the solvent was removed. The residue was taken up in 80% acetic acid (9ml) and the solution was stirred at 70° for

30 minutes. Water (2ml) was added and the solution was extracted with hexane (2 x 10ml) . The aqueous layer was retained and the solvent was removed. The residue was partitioned between saturated aqueous sodium bicarbonate and chloroform and the organic layer was dried (magnesium sulphate) and the solvent removed. The residue was purified by column chromatography on silica gel eluting with chloroform-methanol (16:1) to afford 2-amino-9-(4-butyryloxy-3-hydroxymethylbut-1-yl) urine which was obtained as a white crystalline solid after trituration with methanol

(188mg, 68%), m.p. 125 - 127°; λ „ (MeOH) 222 (27,600), 243 (4,830), and 308 (6,950)nm; v _w (KBr) 3190, 1730, 1640, 1620, and 1580cm ; δ„ max n

[(CD ₃ ) ₂ SO] 0.85 (3H, t, J 7.4Hz, CH ₃ ) , 1.50 (2H, sextet, J 7.3Hz, CH CΗ ₂ CH ₃ ) , 1.68 (1H, m, 3'-H) , 1.82 (2H, m, 2'-H), 2.23 (2H, t, J 7.4Hz, CH ₂ CH ₂ CH ₃ ) , 3.42 (2H, t, J 5.2Hz, D ₂ 0 exchange gives d, CH_ ₂ OH) , 3.95 - 4.1 (2H, ABX, J^g 11.0Hz, J^ = J _βχ 5.8Hz, CH ₂ OOC) , 4.12 (2H, t, J 7.3Hz, T-H), 4.62 (1H, t, J 4.9Hz, D D exchangeable, CH) , 6.44 (2H, s, D ₂ 0 exchangeable, 2-NH ₂ ) , 8.06 (1H, s, 8-H) , and 8.56 (1H, s, 6-H) ; (Found: C, 54.41; H, 6.91; N, 22.70%. ₁₄ H ₂₁ N ₅ 0 ₃ requires C, 54.71; H, 6.89; N, 22.79%).

Example 11

2-Amino-9-(4-benzoyloxy-3-hydroxymethylbut-1-yl)purine

To a solution of 9-(3-hydroxymethyl-4-mon nethoxytrityloxybut-1-yl)-2- monomethoxytritylaminopurine (0.70g, 0.9πrnol) and 4-di_methylaminopyridine (10mg) in ,N-di_methylformamide (5ml) was added benzoic anhydride (0.61g, 2.7mmol) and the solution was stirred for 1 hour. Methanol (1ml) was added and the solvent was removed. The residue was taken up in 80% acetic acid (9ml) and the solution was stirred at 80° for 20 minutes. Water (3ml) was added and the solution was extracted with hexane (2 x 10ml) . The aqueous layer was retained and the solvent was removed. The residue was partitioned between saturated aqueous sodium bicarbonate and chloro¬ form and the organic layer was dried (magnesium sulphate) and the solvent removed. The residue was purified by column chromatography on silica gel eluting with chloroform-methanol (14:1) to afford 2-amino-9-(4-benzoyloxy- 3-hydroxymethylbut-1-yl)purine which was obtained as a white crystalline solid after trituration with methanol (235ιrg, 76%), m.p. 116-118°; λ „ (MeOH) 223 (36,700) and 309 (6,680)nm; υ _v (KBr) 3320, 1710, 1610, and

1580cm ; δ„ I (CD.) -SO] 1.83 (1H, m, 3'-H) , 1.93 (2H, q, J 7.1Hz, 2'-H) , ri j __.

3.52 (2H, t, J 5.3Hz, D ₂ 0 exchange gives d, CH_ ₂ OH) , 4.19 (2H, t, J 7.0Hz, T-H), 4.2 - 4.3 (2H, ABX, J^ 11.0Hz, J = J _βχ 5.6Hz, CH ₂ OOC) , 4.69 (1H, t, J 5.2Hz, D ₂ 0 exchangeable, OH), 6.43 (2H, s, D ₂ 0 exchangeable, 2-NH ₂ ) , 7.5 - 7.9 (5H, m, C _g H ₅ ) , 8.10 (1H, s, 8-H) , and 8.55 (1H, s, 6-H) ; (Found: C, 59.20; H, 5.63; N, 20.82%. C ₁₇ H _ιg N ₅ 0 ₃ requires C, 59.81; H, 5.61; N, 20.52%) .

Exa ple 12

2-Amino-9-(4-hydroxy-3-hydroxymethylbut-1-yl)purine 4'-phosphate

To an ice-cooled solution of phosphorus oxychloride (0.10ml, 1.1ιmol) in pyridine (2ml) was added dropwise over 15 minutes a solution of 9-(3-hydroxymethy1-4-monσmethoxytrityloxybut-1-y1)-2-monome thoxy- tritylaminopurine (0.78g, I.Onmol) in pyridine (2ml). The solution was stirred for a further 5 minutes at 0 and then for 30 minutes at room temperature. The solution was added dropwise to a solution of sodium bicarbonate (0.5g, 6.0nmol) in water (7ml) . The solvent was removed and the residue was taken up in 80% acetic acid (10ml) and the solution was stirred at 70 for 25 minutes. The solvent was removed and the residue was taken up in water and brought to pH 6 by addition of ammonia. The solution was extracted twice with chloro¬ form and the solvent was removed. The residue was purified by preparative high pressure liquid chromatography on a C. _g reverse-phase μ-Bondapack column eluting with 3% methanol in anmonium acetate buffer (pH 4.5, 50nM) to afford 2-amino-9-(4-hydroxy-3-hydroxymethylbut-1-yl)- purine 4'-phosphate as a hygroscopic white powder (85ιrg, 25%) ; λ _„ rriix

(H,0) 220, 241, and 303nm; -υ (KBr) 3410, 1660, 1620, and 1580cm ^"1 ; max _H [(CD ₃ ) ₂ SO] 1.57 (1H, m, 3'-H) , 1.77 (2H, m, 2'-H) , 3.37 (2H, d, J 4.4Hz, CH ₂ OH) , 3.77 (2H, t, J 5.6Hz, CH ₂ OP) , 4.12 (2H, t, J 7.4Hz, T-H), 6.48 (2H, s, D ₂ 0 exchangeable, 2-NH ₂ ) , 8.08 (1H, s, 8-H) , and 8.54 (1H, s, 6-H) ; (Found: C, 35.53; H, 5.93; N, 22.24%. ^C 10 ^H 16 ^N 5°5 ^P * ^0,5NH 3 * H ₂ 0 requires C, 34.94; H, 5.72; , 22.41%).

Exaπple 13

2-Amino-9-(4-hydroxy-3-hydroxymethylbut-1-yl)purine 4':4"-phosphate

To an ice-cooled solution of phosphorus oxychloride (93μl, I.Oπtnol) in pyridine (2ml) was added dropwise over 45 minutes a solution of 9-(4-hydroxy-3-hydroxymethylbut-1-yl)-2-monomethoxytritylani nopurine (0.46g, 0.9mτol) in pyridine (4ml) . The solution was stirred for a further 20 minutes at room temperature and was then added dropwise to a solution of sodium bicarbonate (0.34g, 4.0ιtιrDl) in water (6ml). The solvent was removed and the residue was taken up in 80% acetic acid (9ml) and the solution was stirred at 70 for 25 minutes. The solvent was removed and the residue was taken up in water and brought to pH 6 by addition of aitmonia. The solution was extracted twice with chloro¬ form and the solvent was removed. The residue was purified by preparative high pressure liquid chromatography on a C.- reverse-phase y-Bondapack column eluting with 4% methanol in ai onium acetate buffer (pH 4.5, 50πM) to afford 2-amLno-9-(4-hydroxy-3-hydroxymethylbut-1-yl) purine 4 ^, :4" -phosphate as a white powder (225 cg, 75%); λ (H,0) 220, 242, and 303nm; v (KBr) 2900 - 3200 (br) , 1705, 1615, and 1580cm ;

Ht3X _H [(CD ₃ ) ₂ SO] 1.63 (1H, m, 3'-H) , 1.74 (2H, q, J 7.0Hz, 2'-H) , 3.80 (2H, q, J 9.2Hz, 2 x H I, 3.98 (2H, ddd, J 14.3, 10.9, and 3.5Hz, 2 x H ) , ax eq

4.08 (2H, t, J 7.1Hz, T-H), 6.51 (2H, s, D ₂ 0 exchangeable, 2- H ₂ ) , 8.10 (1H, s, 8-H) , and 8.56 (1H, s, 6-H) ; (Found: C, 36.41; H, 5.18; N, 22.38%. C ₁₀ H N ₅ O ₄ P . 0.3 NH... 1.5H ₂ C requires C, 36.25; H, 5.45; N, 22.40%).

BIO OG1CA DATA

Xanthine Oxidase Catalysed Oxidation of 2-Amino-9-(4-hydroxy- 3-hydroxymethylbut-1-yl)purine

To an aqueous solution of 2-amino-9-(4-hydroxy-3-hydroxy- methylbut-1-yl)purine (0.5mM, 0.7ml; pH7) was added bovine milk xanthine oxidase (20yl, 0.4 unit). Dissolved atmosphere oxygen was allowed to act as electron acceptor and changes in the UV spectrum were measured. After 4 minutes 25% conversion had occurred and after 2.5 hours conversion was essentially complete. The oxidation product was identified as 9-(4-hydroxy-3-hydroxymethylbut-1-yl)guanine by its UV spectrum and HPLC retention time.

(Incubation of 9-(4-hydroxy-3-hydroxymethylbut-1-yl) - guanine with xanthine oxidase under identical conditions resulted in no change over a 2 hour period.)

Oral Absorption of 2-Aminc^9-(4-hvdr >xy-3-hydroxyethylbut-l-yl)p\irine and 2-Aπd_no-9-(4-acetoxy-3-acetoxymethylbut-l-yl) purine and their

Conversion to 9- (4-hydroxy-3-hydroxymethylbut-1-yl) guanine in Mice

Procedure

2-Ami_no-9-(4-hydroxy-3-hydroxymethylbut-1-yl)pu ine, 2-amino~9- (4-acetoxy-3-acetoxymethylbut-1-yl)purine and 9-(4-hydroxy-3-hydroxy- methylbut-1-yl)guanine were administered fcy oral gavage (0.2πιτoles/kg in 0.1ml of 1% carboxymethyl cellulose) to 20g female Balb/C mice which had been starved for 18 hours. Fifteen, 60 and 180 minutes later, blood was collected from three mice per time point by cardiac puncture using heparinised syringes. Equal aliquots at each tijne were pooled and an equal voluπe of 16% trichloroacetic acid added. Following centrifugation (8,500g) to remove precipitated proteins, 0.5ml of supernatant was immediately added to 0.1ml of saturated sodium bicarbonate solution and the resulting mixture analysed by high performance liquid chromatography or stored at -20 C prior to analysis.

Results

Administered Compound Concentration of 9-(4-hydroxy-3-hydroxy- methylbut-1-yl)guan_a_ne (μq/ml) in blood at stated times after administration

15 min 1 hr 3 hr

9-(4-Hydroxy-3-hydroxy- _, 0.7 0.4 0.1 methylbut-1-yl)guanine

Expt. 1

[ 2-Amino-9-(4-hydroxy- 3.0 2.2 0.3 | 3-hydroxymethylbut-1- yl)purine

9-(4-Hydroxy-3-hydroxy- 1.3 1.0 0.4 methylbut-1-yl)guanine

2-Amino-9-(4-hydroxy- 4.6 2.8 0.6

Expt. 2 3-hydroxymethylbut-1- yl)purine

2-Amino-9-(4-acetoxy- 18.7 4.3 0.3 3-acetoxymethylbut-1- yl) urine

Administered Compound Concentration of 9-(4-hydroxy-3-hydroxy- methylbut-1-yl)guanine (μg/ml) in blood at stated times after administration

15 min 1 hr 3 hr

9-(4-Hydroxy-3-hydroxy- 1.4 1.1 0.5 methy but-1-yl)guanine

2-Amino-9-(4-hydroxy-3- 4.8 4.6 1.2 hydroxy ethylbut-1-yl) purine

9-(4-acetoxy-3- 12.9 5.1 0.3 ^r xpt. 3< hydroxymethyltut-1-yl- - 2-aminopurine

2-Amino-9-(3-hydroxy- 13.7 5.6 0.7 methyl-4- ethαxycar onyloxybut- τ-y ^] )purine

2-Amino-9-[2-(2,2- 8.4 2.8 0.8 dimethyl-l ,3-dioxan-5- yl)ethyl]purine

9-(4-Hydroxy-3-hydroxy- 1.1 0.9 0.4 methylbut-1-y1) uanine

2-Amino-9-(4-hydroxy-3- 3.5 4.0 0.8 hydroxymethylbut-1-yl) purine

2-Amino-9-(4-propionyloxy- 20.0 6.6 0.5 Expt. 3-propionyloxymethylbut-1- yl)purine

2-Amino-9-(4-butyryloxy-3- 16.2 7.1 0.5 hydroxymethylbut-1-yl) purine

2-Amino-9-(4-benzoyloxy-3- 16.0 6.6 0.3 hydroxymethylbut-1-yl) purine

Concentration of 9-(4-hydrαxy-

Administered Compound 3-hydroxymethylbut-1-yl) guanine(μg/ml) in blood at stated times after administration

15 min 1 hr 3 hr