This invention relates to a new process for the preparation of certain optically active purine substituted cyclopentene derivatives and novel intermediates used in this process. In particular, the invention describes the synthesis of the IR-cis isomer of carbovir, [l'R^'Sl-Σ-amino^-CA-^ydroxymethylJ-Σ-cyclopenten-l-ylj-l^ -dihydro -6H-purin-6-one, an antiviral agent.

GB-A-2217320 discloses a group of antiviral purine substituted cyclopentene derivatives including the IR-cis isomer of carbovir, [l'R, ^I S]-2-amino-9-[4-(hydroxymethyl)-2-cyclopenten-l-yl]-l, 9- dihydro-6H-purin-6-one, a compound of the formula (I)

0

together with processes for their preparation.

The compound of formula (I) (also referred to hereinafter as (-)-carbovir) has been found to have potent activity against human immunodeficiency virus (HIV) associated with acquired immune deficiency syndrome (AIDS) [see Vince, R., et al. , Biochem. Biophys.

Res. Commun., 156(2), 1046 (1988)]. There is however a need for improved synthetic routes to (-)-carbovir from relatively inexpensive starting materials.

We have now found that (-)carbovir may conveniently be prepared from an optically pure 6-oxabicyclo[3.1.0]hex-3-ene derivative of formula (II)

(wherein R ^** represents a hydroxyl protecting group).

The present invention thus provides in one aspect a process for the preparation of a compound of formula (I) and physiologically acceptble salts thereof which comprises the steps of (a) reacting a compound of formula (II) with 2,6-diaminopurine to produce a 1-OH 0 compound of formula (III) below (b) reacting the said compound of formula (III) to replace the 1-OH group by hydrogen to form a compound of formula (V) below and (c) reacting said compound of formula (V) to convert the 2,6-diamino purine base therein to the desired guanine base with removal of the R protecting group after step (b) or at a suitable stage in step (c) to produce (-)carbovir, with salt formation *-_5 as an optional subsequent step.

In a particular embodiment of this invention, step (a) above may conveniently be effected by reacting a compound of formula (II) with 2,6-diaminopurine in the presence of a suitable base to give a compound of formula (III)

_Q (wherein R ¹ is as defined above). Bases which may be used include alkali metal hydrides, such as sodium hydride, and the reaction may be effected in the presence of a suitable solvent, conveniently dimethylformamide when sodium hydride is the base. It may be

35

desirable to carry the reaction out at an elevated temperature (e.g. 50 ^ϋ -120°C).

In another particular embodiment of this invention, step (b) above may conveniently be effected by (i) reacting the compound of formula (III) to convert the 1-OH group to a leaving group removable by reduction (e.g. by homolytic reduction) and (ii) reducing said compound to replace the leaving group by a hydrogen atom.

A particularly convenient means of effecting step (b) may be to react the compound of formula (III) with a compound of formula HalC(=S)0R (where Hal is a halogen atom, e.g. chlorine, and R ² is C _jL _ _b alkyl, aryl such as phenyl, heteroaryl such as imidazole or C _i _ ₆ alkylaryl such as p-tolyl) to provide a compound of formula (IV)

(wherein 0R represents a group OC^SjOR and R ¹ and R are as defined previously), and thereafter reducing the compound of formula (IV) using, for example, an alkyltin hydride (e.g. tri-n-butyltin hydride) in the presence of a radical initiator such as a peroxide, azobisisobutyronitrile or light to provide a compound of formula (V)

(wherein R ^l is as defined above).

The reaction of a compound (III) to"introduce the R ^J group in the compound (IV) may be effected in the presence of a suitable base such as an amine (e.g. pyridine or 4-dimethylaminopyridine) and in a solvent such as a halogenated hydrocarbon (e.g. dichloromethane), conveniently at a reduced temperature (e.g. about ~30 to -10 ^U C).

The reduction reaction to provide a compound of formula (V) may conveniently be carried out in a suitable solvent, such as pyridine when an alkyltin hydride is the reducing agent.

In another particular embodiment of this invention, step (c) above may conveniently be effected by a series of reactions (l)-(6) as illustrated in the following Scheme.

(VI )

(4 )

(VII) (VIII )

[wherein R ¹ * represents an acyl protecting group such as acetyl] Reaction (1) may be effected, for example, by treating the compound of formula (V) with a suitable acylating agent such as acetic anhydride, under conditions whereby the R ¹ protecting group is removed, for example in the presence of a Lewis acid catalyst such as boron trifluoride etherate.

Reaction (2) may be effected under conditions suitable for the removal of the protecting group R ¹ without affecting the rest of the

molecule. The group R ¹ and conditions for the removal of R ¹ are defined below.

Reaction (3) may be effected, for example, by treating (15,4R)- 4-[2,6-diamino-9H-purin-9-yl]-2-cyclopentenemethanol with a suitable acylating agent such as acetic anhydride.

Reaction (4) may be effected, for example, by treating the compound of formula (VI) with methanolic ammonia, conveniently at ambient temperature.

Reaction (5) may be effected, for example, by hydrolysis, and conveniently by treating the compound of formula (VII) with a suitable acid, for example nitrous acid (conveniently prepared in situ from acetic acid and sodium nitrite).

Reaction (6) may be effected, for example, by treating the compound of formula (VIII) with methanolic ammonia, conveniently at ambient temperature. It is to be understood that R ^L may represent any suitable hydroxyl protecting group which can be removed utilising conventional reagents and without adversely affecting the rest of the molecule. Suitable readily removable hydroxyl protecting groups will be familiar to those skilled in the art. Such groups are disclosed in, for example, 'Protective Groups in Organic Chemistry' , Ed. 3. F. W. McOmie (Plenum Press, 1973) and 'Protective Groups in Organic Synthesis' by Theodora W. Greene (3ohn Wiley and Sons, 1981). Examples of suitable hydroxyl protecting groups include aralkyl groups such as p-methoxybenzyl or benzyl which may conveniently be removed under mild acid conditions, for example using a Lewis acid such as boron tribromide at low temperature (e.g. ~78 ^ϋ C).

It is to be further understood that each of the individual steps in the multistep process herein for preparing (-)carbovir from a compound of formula (II) and sequential combinations of such steps represent particular aspects of the present invention.

(-)Carbovir may be prepared according to the present multistep process as an optically and chemically pure product. The present process provides a particularly convenient method for preparing (-)carbovir for use on a laboratory or an industrial scale.

Compounds of formula (II ) may be prepared by reacting a compound of formula (IX)

(wherein L is a suitable leaving group and R ¹ is as defined above) to eliminate HL, followed, where necessary, by separating the desired product from any by-products formed.

Examples of suitable leaving groups will be within the knowledge of persons skilled in the art. Particular examples of the atom or group L included 0H,0S02R ^S (where R ⁵ represents alkyl, for example C _j ,_ ₆ alkyl such as methyl, aryl, for example phenyl or tolyl, or trifluoromethyl) or halogen (e.g. bromine or iodine).

Suitable conditions for the conversion of compounds of formula (IX) to compounds of formula (II) will of course depend upon the nature of the leaving group L. Thus, for example, when L represents OH the elimination reaction may be effected by methods such as those referred to in "Compendium of Organic Synthetic Methods", Eds. I. T. Harrison and S. Harrison, Wiley-Interscience, 1971, pp. 484-488. When L represents a halogen atom the elimination reaction may be effected by methods such as those referred to in "Compendium of Organic Synthetic Methods, Eds. I. T. Harrison and S. Harrison, Wiley-Interscience, 1971, pp. 507-510.

We have found that compounds of formula (IX) in which L represents 0S0 R ⁵ (where R ⁵ is as defined previously) are particularly convenient precusors to the compounds of formula (II). Such compounds may be converted to compounds of formula (II) by treating the appropriate compound of formula (IX) with a base, for example an alkali metal alkoxide (e.g. sodium methoxide) in an alcoholic solvent such as methanol or a quaternary ammonium salt in a solvent such as an ether (e.g. tetrahydrofuran) . When a quaternary ammonium salt is used this is conveniently a tetraalkylammonium halide, for example a tetrabutylammoniu halide such as tetrabutylammonium fluoride. The

elimination reaction may be carried out at any suitable temperature and conveniently at ambient temperature.

It will be appreciated that some of the aforementioned elimination reactions may yield a mixture of the desired compound of formula (II) and the corresponding 6-oxabicyclo[3.1.θ]hex-2-ene compound. Where a mixture of products results the desired compound of formula (II) may be isolated by conventional separation techniques, e.g. by column chromatography.

The compounds of formula (IX) in which L represents OH are either known compounds described by S.M.Roberts et al. in 3. Chem. Soc,

Perkin I, 1988, p 549 or may be prepared by methods analogous to the methods described therein for the preparation of the known compounds of formula (IX) in which L represents OH. Compounds of formula (IX) in which L represents a different leaving group may be prepared from compounds of formula (IX) in which L is OH by conventional means. Thus, for example, compounds of formula (IX) in which L represents a group OSO^ ^* ' (where R ^{5^} is as defined previously) may be prepared from compounds of formula (IX) wherein L is OH by reaction with a sulphonyl halide of the formula R S0 ₂ Hal (where R ^? and Hal are defined previously), to give compounds of formula (IX) in which L represents 0S0 ₂ R • Such reactions may be effected in a solvent such as a halogenated hydrocarbon (e.g. dichloromethane) conveniently at a temperature in the range ~10 ^ϋ to +50 ^U C (e.g. about 0°C). The reaction preferably takes place in the presence of a base such as an amine (e.g. triethylamine or 4-dimethylaminopyridine) or a mixture of amine bases.

It will be appreciated that compounds of formulae (II) to (IX) will have the stereochemistry assigned for the specific compounds relating thereto described in the Examples section hereinafter.

Intermediates of formulae (III) and (IV) are novel compounds and form a further aspect of the present invention.

Salts (e.g. physiologically acceptable salts) of the compound of formula (I) may be prepared from the corresponding free base according to the methods described in GB-A-2217320.

The following examples illustrate the present invention but should not be construed as a limitation of the invention. All temperatures are in °C.

Intermediate 1

[35-(lα,2β,3α,5α)]-2-((Phenylmethoxy)methyl)-6-oxabic yclo[3.1.0]-hexan -3-ol, methanesulphonyl ester

To a stirred, ice-chilled solution of [3S-(lα,2β,3α,5α)]-2- ((phenylmethoxy)methyl)-6-oxabicyclo[3.l.Oj-hexan-S-ol ¹ (3.53g) in dichloromethane (70ml) under nitrogen, was added 4-dimethylamino- pyridine (53g) followed by triethylamine (2.7ml). Methanesulphonyl chloride (1.37ml) was then added dropwise, and the solution was stirred for 45mins at 0 ^υ . The reaction was quenched by addition of water and the layers were separated. The organic layer was further washed with water, saturated aqueous sodium bicarbonate solution, and brine. The organic phase was dried over anhydrous sodium sulphate, filtered and evaporated, giving a residue which crystallised. The crystals were triturated with ether, affording the title compound (3.87g), m.p. 81-83 ^u ; [α] ³ + 51.89 ^u (c = 1.55, CHCl _d ); ^l NMR (CDCl _d ) δ 7.2-7.4, 5H, m; 5.10, 1H, t; 4.4-4.6, 2H, m; 3.4-3.7, 4H, m; 3.0, 3H, s; 2.75, 1H, t; 2.30, 2H, d.

1. S.M.Roberts et al., 3. Che . Soc. , Perkin I, 1988,549.

Intermediate 2 [15-(lα,2β,5α)]-2-((Phenylmethoxy)methyl)-6-oxabicyclo[3. 1.03-hex-3- ene

To a stirred solution of Intermediate 1 (2.61g) in tetrahydrofuran (30ml), under nitrogen, was added tetrabutylammonium fluoride solution (52.5ml of a 1M solution in tetrahydrofuran) and the solution was allowed to stand at ambient temperature for 2 days. After this time, a further 35ml of the tetrabutylammonium fluoride solution was added, and the solution was again allowed to stand at ambient temperature for a further one day. The solution was evaporated to about one third of its volume, and the residue was taken

up in chloroform, washed with water and brine, and then dried over anhydrous magnesium sulphate. After filtration and evaporation, the residue was purified by column chromatography (Merck 7734 silica gel, eluted with petrol-diethyl ether 1:1) to give the title compound as a light brown oil (1.58g). ^** H NMR(CDC1 ₃ ) δ 7.3-7.4, 5H, m; 6.23, 1H, m; 5.95, 1H, m; 4.5-4.6, 2H, m; 3.90, 1H, m; 3.80, 1H, m; 3.55, 1H, dd; 3.30, 1H, t; 3.08, 1H, m; 1R (CHC1 ₃ ) 1485, 1450, 1358cm ^{" *•} ; MS (+ve CI, CH^) m/e 203 (MH+), 185, 91(B+)-

Intermediate 3

(15,2S,55)-2-[2,6-Diamino-9H-purin-9-yl]-5-(phenylmethoxy )-methyl-3- cyclopentenol

To a magnetically stirred suspension of sodium hydride (864mg) in di ethylformamide (190ml) under nitrogen was added 2,6-diaminopurine (8.20g). The mixture was heated at 85° for 20min and allowed to cool to ambient temperature. 15-Crown-5 (1ml) was added, followed by a solution of Intermediate 2 (7.45g) in dimethylformamide (40ml). The mixture was heated at 100 ^u for 3h and allowed to cool to ambient temperature overnight. The solvent was removed, the residue dissolved in ethyl acetate and the solution washed with water. The combined aqueous phase was extracted with ethyl acetate, the extract combined with the organic phase, the total organic solution washed with brine, dried over anhydrous magnesium sulphate and evaporated. The residue was purified by column chromatography (Merck 7734 silica gel, eluant chloroform-methanol 9:1) to afford the title compound

(6.49g); ^X H NMR (CDC1 ₃ ) δ 7.39,lH,s; 7.18-7.40, 5H, m; 6.18, 2H, bs; 6.03, 1H, m; 5.80, 1H, m 5.38, 2H, bs; 5.25, 1H, m; 4.50, 2H, s; 4.31, 1H, t; 3.50-3.70, 2H, m; 3.03, 1H, m; MS (+ve CI, CH ₄ ) m/e 353 (MH÷), 151,91 (B+).

Intermediate 4

(IS ,2S ,55 )-2-[2 , 6-Diamino-9H-purin-9-yl]-5- (phenylmethoxy )-methyl-3- cyclopentenol-phenoxythiocarboxylate

To a magnetically stirred solution of Intermediate 3 (6.46g) in d i c h l o r o m e t h a n e ( 1 90 m l ) u n d e r n i t r o g e n wa s a d d e d 4-dimethylaminopyridine (6.61g) . The solution was cooled to -25°,

phenyl chlorothionofor ate (6.29g) added dropwise over lOmin and the mixture stirred at -20°C for lh, then at -20° to 15° for 0.75h. The reaction was quenched by addition of water. The phases were separated and the organic layer washed with saturated aqueous sodium bicarbonate solution. The organic solution was dried over anhydrous magnesium sulphate, filtered and the solvent removed. The residue was purified by column chromatography (Merck 7734 silica gel, eluant chloroform-methanol 19:1) to give the title compound (8.28g); ^l H NMR (CDC1 ) δ 7.68, IH, s; 7.20-7.45, 8H, m; 7.08, 2H, d; 6.11, IH, m; 5.92, IH, t; 5.86, IH, m; 5.73, IH, m; 5.39, 2H, bs, 4.78, 2H, bs; 4.55, 2H, s; 3.70-3.85, 2H, m; 3.26, IH, m.

Intermediate 5

(1'R,4'5)-9-[4-(Phenylmethoxy)methyl-2-cyclopenten-l-yl]- 9H-2,6- purinediamine

To a magnetically stirred solution of Intermediate 4 (8.17g) in pyridine (150ml) under nitrogen was added azobisisobutyronitrile (250mg) and tributyltin hydride (9.72g). The mixture was heated at 95° for 3h and evaporated to dryness. The residue was purified by column chromatography (Merck 7734 silica gel, eluant chloroform-methanol 19:1) to give the title compound (2.17g). Crystallisation from dichloromethane afforded pure title compound m.pt. 166-7°; L°-J _D -85.7° (c = 0.98, CHCl ; ^X H NMR (CDCl _d ) δ 7.60, IH, s; 7.20-7.40, 5H, m; 6.15, IH, m; 5.84, IH, ; 5.58, 2H, bs; 5.56, IH, ; 4.80, 2H, bs; 4.52, 2H, s; 3.49, 2H, m; 3.09, IH, m; 2.80, IH, dt; 1.68, IH, dt.

Intermediate 6

(15,4R )-4-[2,6-Diamino-9H-purin-9-yl3-2-cyclopentenemethanol To a magnetically stirred suspension of Intermediate 5 (199mg) in dichloromethane (10ml) under nitrogen at -78° was added a solution of boron tribromide (2.36ml, 1M) in dichloromethane, dropwise. The mixture was stirred at -78° for 1.25h and then allowed to warm to ambient temperature. The reaction was quenched by addition of aqueous ammonia (10ml, 2M). The phases were separated and the aqueous layer

extracted with dichloromethane. The combined organic solution was washed with brine, dried over anhydrous magnesium sulphate, filtered and the solvent removed.- The residue was purified by column chromatography (Merck 7734 silica gel, eluant chloroform-methanol 4:1) and the crude product crystallised from ethyl acetate to give the title compound (25mg) as a white powder; ^X H NMR (d ⁶ DMSO) δ 7.61, IH, s; 6.70, 2H, bs; 6.12, IH, m; 5.88, IH, m; 5.80, 2H, bs; 5.38, IH, ; 4.79, IH, t; 3.45, 2H, m; 2.87, IH, m; 2.60, IH, dt; 1.60, IH, dt; IR (Nujol) 3332, 2924, 1640cm ^* - ¹ .

Intermediate 7

(IS,4R)-4-[2,6-Diamino-9H-purin-9-yl3-2-cyclopentenemetha nol

To a magnetically stirred solution of the product of Example 2(b) below (54mg) in dioxan (3.4ml) was added aqueous sodium hydroxide (6.75ml, 2N). The mixture was stirred at ambient temperature for 5h. The pH was adjusted to pH8 by addition of hydrochloric acid (2N) and the mixture evaporated. The residue was triturated with ethanol and the salt that remained removed by filtration. The filtrate was evaporated and the residue purified by column chromatography (Merck 7734 silica gel, eluant chloroform-methanol 9:1) to afford the crude product as a colourless gum. This was triturated with diethyl ether and then recrystallised from ethyl acetate to give the title compound (36mg) as a white solid; 'H NMR (d ⁶ DMSO) δ 7.61, IH, s; 6.70, 2H,bs; 6.12, IH, m; 5.88, IH, m; 5.80, 2H, DS; 5.38, IH, m; 4.79, IH, t; 3.45, 2H, m; 2.87, IH, m; 2.60, IH, dt; 1.60, IH, dt; IR (Nujol) 3324, 2924, 1646cm- ^l .

Example 1

[1'R,4'S]-2-Amino-9-(4-(hydroxymethyl)-2-cyclopenten-l-yl )-l,9- dihydro-6H-purin-6-one [i.e. (-)carbovir]

(a) (l"R,4"S)-N-[6-Amino-9-(4-(acetoxy)methyl-2-cyclopenten-l-yl )-9H- purin-2-yl]-acetamide

(i) To a magnetically stirred solution of Intermediate 6 (50mg) in acetic anhydride (2ml) under nitrogen at 0° was added boron trifluoride etherate (0.1ml), dropwise. The mixture was stirred at 0 ^U for lOmin and then at ambient temperature for 3h. The reaction was

quenched by addition to saturated aqueous sodium bicarbonate solution and the product extracted into ethyl acetate. The combined organic extracts were washed with brine, dried over anhydrous magnesium sulphate, filtered and evaporated. The residue was triturated with diethyl ether and dried in vacuo to give the title compound (63mg) as a white solid; ¹ H NMR (CDC1 ₃ ) δ 10.08, IH, bs; 7.75, IH, s; 6.80, 2H, bs; 6.17, IH, m; 5.98, IH, m; 5.61, IH, m; 4.15, 2H, m; 3.20, IH, ; 2.87, IH, dt; 2.64, 3H, s; 2.07, 3H, s; 1.71, IH, dt. 0 (ii) To a magnetically stirred solution of Intermediate 5

(1.3g) in acetic anhydride (40ml) under nitrogen at 0° was added boron trifluoride etherate (1.89ml), dropwise. The mixture was stirred at 0° for lOmin and then at ambient temperature for 2h. The reaction was quenched by addition to saturated aqueous sodium bicarbonate solution 5 and the product extracted into ethyl acetate. The combined organic solution was washed with brine, dried over anhydrous magnesium sulphate, filtered and evaporated. The residue was azeotroped with toluene to remove acetic anhydride and then purified by column chromatography (Merck 7734 silica gel, eluant chloroform-methanol o 19:1) to give the title compound (699mg) as yellow solid; 'H NMR

(CDCl _j ) δ 10.08, IH, bs; 7.75, IH, s; 6.17, IH, m, 5.98, IH, m; 5.61, IH, m; 4.15, 2H, m; 3.20, IH, m; 2.87, IH, dt; 2.64, 3H, s; 2.07, 3H, s; 1.71, IH, dt; MS (+ve CI, NH ₃ ) m/e 331 (MH+, B+), 289.

5 (b) (l"R,4"5)-N-[6-Amino-9-(4-(hydroxymethyl)-2-cyclopenten-l-yl )-9H- purin -2-yl]-acetamide

To a magnetically stirred suspension of the product of part (a) above (480mg) in methanol (3ml) was added a saturated solution of ammonia in methanol (15ml). The mixture was stirred at ambient 0 temperature overnight. A further aliquot of methanolic ammonia (30ml) was added and stirring continued overnight. The solution was evaporated to dryness and the residue purified by column chromatography (Merck 7734 silica gel, eluant chloroform-methanol 92.5:7.5) to afford a crude product which was triturated with diethyl 5 ether and dried in vacuo to give the title compound (252mg) as a white amorphous solid; Η NMR (d ⁶ DMSO) δ 9.75, IH, bs; 7.94, IH, s; 7.18,

2H, bs; 6.13, IH, m; 5.90, IH , m; 5.48, IH, m; 4.72, IH , t; 3.45, 2H, m; 2.89, IH, m; 2.63, IH , dt; 2.22; 3H, s; 1.62, IH , dt.

(c) (1"R,4"5)-N-[l,9-Dihydro-9-(4-(hydroxymethyl)-2-cyclopenten- l- yl)-6-oxo-6H-purin-2-yl]-acetamide

To a magnetically stirred solution of the product of part (b) above (151mg) in glacial acetic acid (5ml) was added water (6ml) and sodium nitrite (290mg). The mixture was stirred at ambient temperature for 30min, a further aliquot of sodium nitrite (290mg) was added and stirring continued overnight. Excess nitrous acid was destroyed by addition of sulphamic acid, and the mixture neutralised by addition of aqueous sodium hydroxide solution (2N). The solvent was removed and the residue purified by column chromatography (Merck 7734 silica gel, eluant chloroform-methanol 9:1) to give the title compound (147mg) as a white solid; Η NMR (d ⁶ DMSO) δ 7.90, IH, s; 6.17, IH, m; 5.92, IH, m; 5.40, IH, m; 3.47, 2H, d; 2.90, IH, ; 2.62, IH, dt; 2.20, 3H, s; 1.62, IH, dt.

(d) [l'R,4'S]-2-Amino-9-(4-(hydroxymethyl)-2-cyclopenten-l-yl)- 1,9-dihydro-6H-purin-6-one

To a solution of the product of part (c) above (lOOmg) in o methanol (10ml) was added a saturated solution of ammonia in methanol (30ml) at 0°. The solution was stirred at ambient temperature overnight. The solution was evaporated and the residue purified by column chromatography (Merck 7734 silica gel, eluant chloroform-methanol 4:1) to afford the crude product. This was 5 crystallised from water to give the title compound (49mg) as a white solid. M.p. 248-251° (deco p); L ^α J _D -67.5° (c = 0.40, MeOH); ^l H NMR (d ⁶ DMSO) δ 10.58, IH, bs; 7.59, IH, s; 6.45, 2H, bs; 6.61, IH, m; 5.85, IH, m; 5.33, IH, m; 4.74, IH, t; 3.44, 2H, t; 2.87, IH, m; 2.58, IH , dt; 1.57, IH, dt. 0

5