NO870541L - CARBOCYCLIC PURIN NUCLEOSIDES AND PROCEDURES FOR THEIR PREPARATION. - Google Patents

Abstract

The carbocyclic analogs of purine nucleosides represented by the formula: wherein R 1 and R 2 are each a hydroxyl group, which may be optionally protected, X is a hydrogen atom or a hydroxyl group, which may be optionally protected, and Y is guanin-9-yl, isoguanin-9-yl, or hypoxanthyl, are useful for the treatment of viral infections and as replacements for purine nucleosides in the field of genetic engineering, biochemistry, or the like. Methods for preparing these compounds are also described.

Description

The present invention provides carbocyclic analogues of purine nucleosides which are useful as substitutes for purine nucleosides in the field of genetic engineering, biochemistry and the like and as an antiviral agent.

As examples of carbocyclic analogues of purine nucleosides, known are aristeromycin with the formula:

where Y' is adenin-9-yl [Chemical Comunicatlons, 852 (1967)] and a compound of the above formula where Y' is hypoxanthine-9-yl [Chemical & Pharmaceutical Bulletin, 24, 2624

(1976)]. The compounds listed above are carbocyclic analogues (hereinafter sometimes abbreviated to "C analogues") of adenosine and inosine, respectively.

It has already been established that purine nucleoside analogs of the cyclopentanediol type, typically aristeromycin, can effectively serve as substrates in various enzyme systems involving common purine nucleosides [Journal of Biochemistry, 73, "945 (1973)].

Furthermore, C-analogues of purine nucleosides have recently attracted attention as antiviral agents [Science, 227, 1296

(1985)]. On the other hand, DNAs containing C-analogues of 2'-deoxypurine nucleotides have certain properties that can be advantageously exploited in gene manipulation compared to DNAs containing deoxyadenosine [Japanese Patent Applications 73822/1984 and 258789/1984].

However, C-analogues of deoxyguanosine and C-analogues of guanosine remain unexplored in many areas. Synthesizing and evaluating various additional C analogs is an important task.

Under these conditions, research has been carried out in connection with the present invention to obtain new and useful C-analogues of purine nucleosides and has arrived at the present invention.

According to the invention, thus provided:

(1) A compound with the formula:

where R^ and Rg are each independently a hydroxyl group, which may optionally be protected, X is a hydrogen atom or a hydroxyl group, which may optionally be protected, and Y is guanin-9-yl, isoguanin-9-yl or -hypoxanthine-9 -yl, provided that when X is a hydroxyl group, which may optionally be protected, Y is different from hypoxanthine-9-yl. (2) A process for the preparation of compounds of formula (I) which includes that a compound with which Ri and R2 are independently a hydroxyl group, which may optionally be protected, and X is a hydrogen atom or a hydroxyl group, which may optionally be protected, is subjected purine ring formation reaction. (3) An antiviral preparation containing a compound of the formula:

where X-L is a hydrogen atom or a hydroxyl group, and Y is guanin-9-yl, isoguanin-9-yl, or hypoxanthine-9-yl, provided that when X^ is a hydroxyl group, then Y is different from hypoxanthine-9-yl . (4) The preparation according to point (3) above, where X is a hydrogen atom and Y is guanin-9-yl.

Referring to the compounds of formula (I) or (II), when R 1 , R 2 and/or X is a protected hydroxyl group, the protecting group should preferably be stable under alkaline conditions. Thus, e.g. mention are made of alkylsilyl groups with 3-10 carbon atoms (e.g. dimethyl-t-butylsilyl), alkyl- or alkoxy-cyclic ethers with 4-10 carbon atoms (e.g. tetrahydrofuranyl and its alkyl derivatives with 4-7 carbon atoms, or tetrahydropyranyl and its alkyl derivatives with 5-8 carbon atoms such as methoxytetrahydropyranyl), alkoxyalkyl groups with 3-10 carbon atoms (e.g. ethoxymethyl, methoxyethyl), trityl and alkoxy-substituted trityl groups (e.g. monomethoxytrityl, dimethoxytrityl).

The compounds of formula (I) are obtained by subjecting the compounds of formula (II) to purine cyclization reaction. Purine formation can be effected using the reaction methods known for that purpose in heterocyclic chemistry. The compounds with the general formula (I) where Y is hypoxanthine-9-yl can e.g. is produced in the manner indicated below. The compounds of general formula (II) are thus reacted with a number of different esters (e.g. esters of straight or branched aliphatic carboxylic acids with 2-20 carbon atoms or aromatic carboxylic acids with 7-30 carbon atoms and alcohols with 1-10 carbon atoms) under heating in the presence of a sodium alcoholate (eg ethylate, methylate, tert-butylate) to obtain 2-alkyl- or 2-aryl-substituted C-analogues of inosine or 2'-deoxyinosine. On heating with formamide or formic acid, they give C-analogues of inosine or 2'-deoxyinosine, respectively. It is also possible to first prepare C-analogues of 2-mercaptoinosine or 2'-deoxy-2-mercaptoinosine by heating the compounds (II) with phenyl isothiocyanate or potassium xanthate, respectively, in pyridine and then derive from there 2-alkoxy (e.g. . methoxy, ethoxy, butoxy) or 2-substituted-amino (e.g. methylamino, dimethylamino, ethylamino, diethylamino, cyclohexylamino, morpholino) derivatives via the corresponding 2-halogen (e.g. chlorine, bromine) intermediates* ; For those compounds of formula (I) where Y is guanin-9-yl, the process comprising the above C-analogues of 2-mercaptoinosine or 2'-deoxy-2-mercaptoinosine as intermediates [Chem. Pharm. Bull., r9, 576 (1971 )] is used. Furthermore, according to the description in Nucleic Acids Res., 3, 251 (1976), one can also use the process which includes e.g. reaction of a compound of formula (II) with benzoyl isothiocyanate in boiling acetone, methylation of the resulting benzoylthiourea derivative with methyl iodide in an acetone-potassium carbonate mixture or with dimethyl sulfate in an aqueous sodium hydroxide solution and further heating of the methylation product for approx. 2-6 N sodium hydroxide, thereby effecting ring closure.

On the other hand, the benzoyl-methylisothiourea intermediate obtained in the above process, when heated for ring closure in an alkali solution of relatively low concentration, such as ca. 0.1-1 N sodium hydroxide, a compound of formula (I) where Y is isoguanin-9-yl.

Trityl groups and other ether-forming hydroxyl protecting groups in the compounds of formula (I) can be easily eliminated by a conventional method using various organic acids (e.g. acetic acid, trichloroacetic acid, dichloroacetic acid, benzenesulfonic acid, toluenesulfonic acid) or various inorganic acids [e.g. hydrochloric acid, sulfuric acid, various Lewis acids (eg zinc bromide, aluminum chloride, etc.)]. The hydroxyl-protecting silyl groups can be eliminated with acids as mentioned above, but can be removed under milder conditions using tetrabutylammonium fluoride.

The compounds of formula (II) to be used in the present method can be obtained by treating a compound of the formula:

where Ri, Rg and X have the meanings given above, and R3 is an alkoxymethyl group, with an alkali.

The alkoxymethyl group represented by R3 usually contains 2-7 carbon atoms and includes, among other things, methoxymethyl, ethoxymethyl, propoxymethyl and phenoxymethyl. The methoxymethyl group is particularly useful.

For the above-mentioned alkali treatment, an alkali hydroxide can preferably be used. Thus, e.g. the treatment is carried out by heating at a temperature in the range of approx. 60-200°C In approx. 1-5 M aqueous sodium hydroxide or potassium hydroxide solution or in a mixture of such an alkali solution and a water-soluble neutral organic solvent (e.g. alcohol, dioxane). In this case, the reaction time is usually from approx. 10 minutes to approx. 2 hours.

The compounds of formula (III) for use in the above-mentioned reaction are prepared by converting the adenine ring in aristeromycin to the hypoxanthine ring [Chem. Pharm. Bull., 24, 2624 (1976)], then introduction of a hydroxyl protecting group or groups represented by R-^, R 2 or/and X as required and further introduction of an alkoxymethyl group represented by R 3 .

The introduction of the above-mentioned hydroxyl protecting group can be carried out by means of a method known per se [e.g. Can. J. Chem., 60, 111 (1982)]. In the case that the compounds (III) have such a protective group or groups, the reaction products after ring cleavage of the hypoxanthine ring by the above-mentioned alkali treatment can be suitably purified. This introduction of a protective group is carried out e.g. by reaction with the -alkylsilyl chlorides, alkoxyalkyl chlorides or the trityl- and alkoxy-substituted trityl chlorides, or alkyl- or alkoxy-cyclic ethers in an organic solvent such as pyridine, dimethylformamide, dichloromethane, chloroform, dioxane, ethyl ether, acetonitrile, dimethyl sulfoxide or nitromethane .

The compounds of formula (III) where X is hydrogen can be obtained by protecting the hydroxyl group R-^ and/or R2 in advance, converting the hydroxyl group at position X into the thiocarbonylated forms (e.g. phenylthioformate ester form) and then converting these to the 2'-deoxy form by means of a radical reaction using an organotin hydride. For this purpose, the method described in J. Org. Chem., 47, 485 (1982) is used.

The introduction of an alkoxymethyl group represented by R3 can e.g. is performed using the method described in J. Am. Chem. Soc., 24, 2624 (1976). This introduction of the alkoxymethyl group advantageously enables the above-mentioned hypoxanthine ring cleavage to be carried out under relatively mild conditions and further enables easy elimination of said group in a later treatment step compared to the case where R3 is a simple alkyl group. In the above-mentioned manner, the compounds of formula (III) can be obtained.

The compounds of formula (I) produced according to the invention can be used in the field of genetic engineering or in biochemistry as equivalents to nucleosides containing guanine, isoguanine or hypoxanthine as a base. Thus, e.g. the phosphoric acid derivatives of the compounds of formula (I) in which Y is guanin-9-yl are used instead of dGTP which serves as a substrate for DNA polymerase. Thus obtained DNAs and containing such carbocyclic analogues of guanosine are useful in various fields of recombinant DNA technology.

On the other hand, the compounds of formula (I') according to the invention have strong antiviral activity against DNA viruses. "As such a virus can be mentioned the herpes virus group (e.g. herpes simplex virus type I or II, varicella-zoster virus), adenovirus (e.g. type 3) or vaccinia virus. The compounds show particularly strong antiviral activity against Adenovirus The compounds can be used as antiviral agents for the treatment of virus-induced disease in animals, especially in mammals (laboratory animals such as rabbits, rats and mice; pets such as dogs and cats; and humans).

For use in the above treatment, the compounds of the present invention can be administered orally or otherwise, either alone or in combination with a suitable pharmacologically acceptable carrier, excipient or diluent, in such dosage forms as powders, granules, tablets, capsules, solutions, ointments and injections . As carriers, mention may be made of lactose, starch, mineral oil, petroleum jelly, polyethylene glycol, propylene glycol, saline solution for injection and the like.

The dosage depends on the type of virus, symptoms, administration hosts and routes or the like. For the treatment of herpes virus-induced disease in e.g. an adult human, the compound is preferably administered into a vein at a daily dose level of approx. 1-10 mg, in a single dose or up to 3 divided portions. For the oral route, the compound is preferably administered at a level of about 10-100 mg per a dose in a single portion or up to three divided portions.

The following reference examples, working examples and test examples further illustrate the invention.

Example 1

Synthesis of 9-[(1R,2S,3R,4R)-4-methyl-2-hydroxyl3,6-(tetra-isopropyldisiloxanyl)dioxycyclopentan-1-yl]-hypoxanthine. The C analogue of inosine (10 g, 37.5 mmol) was dissolved in 200 mL of anhydrous DMF, 1,3-dichloro-1,1,3,3-tetraisopropyldisiloxane (13 mL, 41 mmol) and imidazole (11.3 g, 165 mmol) was added and the mixture was stirred at room temperature for 2.5 hours. The reaction mixture was added dropwise to 2 liters of water, and the precipitate was collected by filtration, washed with water, further washed rapidly with diethyl ether and dried to obtain the title compound as a white powder (17.2 g). A portion of the product was recrystallized from dichloromethane to obtain crystals, m.p. 135-138°C.

Example 2

Synthesis of 9-[(IR,2S,3R,4R)-4-methyl-2-phenoxy-thiocarbonyl-oxy-3,6-(tetraisopropyldisiloxanyl)-dioxycyclopentan-1-yl]hypoxanthine.

The compound obtained in Example 1 (11.2 g, 22.3 mmol) was dissolved in 300 mL of anhydrous acetonitrile, dimethylaminopyridine (15.8 g, 53.5 mmol) and phenoxythiocarbonyl chloride (5 g, 29 mmol) were added and the mixture was stirred at room temperature for 7 hours. The solvent was removed under reduced pressure and the residue was dissolved in 250 ml of chloroform. The solution was washed with 0.5 M potassium dihydrogen phosphate solution (250 ml x 2) and then with water (200 ml), dried (anhydrous sodium sulfate) and concentrated under reduced pressure to obtain a yellow syrup. This was purified by silica gel chromatography (90 g; solvent: CHCl 3 and CHCl 3 /MeOH = 60/1) to obtain the title compound as

a pale yellow glass-like substance (13.0 g).

NMR (60 MHz, CDCl3) S ppm: 1.0-1.23 (28H, m), 2.13-2.43 (3H, m, H4', H5'), 3.93, 4.10 ( 2H, m, H6'), 4.80-5.20 (2H,

m, E1'), 6.00-6.20 (H, m, H2 * ), 7.03-7.50 (5H, m), 7.87 (1H, s), 8.13 (1H, s) s; singlet, m; multiplet.

Example 3

Synthesis of 9-[(IR,3S,4R)-4-methyl-3,6-(tetraisopropyldi-siloxanyl)-dioxycyclopentan-1-yl]hypoxanthine.

Anhydrous toluene (30 mL) was added to the compound obtained in Example 2 (13.0 g, 20 mmol), followed by concentration under reduced pressure. The residue was dissolved in 300 ml of anhydrous toluene and nitrogen gas was bubbled into the solution for 20 minutes. After addition of tributyltin hydride (11 mL, 40 mmol), the solution was heated at 80°C and crystalline α,α'-azobisisobutyronitrile (AIBN) (820 mg) was added in 4 portions at 15 minute intervals. After 3 hours of heating with stirring, the solvent was removed under reduced pressure. The obtained oil was purified by silica gel chromatography (80 g; solvent: CHCl 3 and CHCl 3 /MeOH = 60/1 to 30/1) to obtain the title compound as a colorless glassy substance (10.4 g).

Recrystallization of part of the product from ethanol gave colorless needles, m.p. 200-202°C.

NMR (60 MHz, CDCl3) S ppm: 0.93-1.20 (28H, s), 1.97-2.53 (5H, m, H2', H4', H5'), 3.80-4 .07 (2H, m, H6'), 4.43-5.27 (2H, m, H1', H3'), 7.87 (1H, s), 8.20 (1H, s).

Example 4

Synthesis of 9-[ (IR , 3R,4R)-4-(monomethoxytrityloxy)-methyl-3-hydroxycyclopentan-1-yl]-(1-methoxymethylhypoxanthine)

The compound obtained in Example 3 (9.8 g, 19.8 mmol) was dissolved in 240 mL of anhydrous dioxane, sodium hydride (880 mg, 21.8 mmol) was rapidly added to the solution with ice-cooling and stirring and then the mixture was stirred at room temperature for 1.5 hours. Then, methoxymethyl chloride (2 mL, 21.8 mmol) was rapidly added to the mixture with ice-cooling. The entire mixture was stirred at room temperature for 3 hours.

The solvent was removed under reduced pressure and the oily residue was dissolved in 200 ml of chloroform. The solution was washed with 0.1 M triethylammonium bicarbonate (TEAB) buffer (pH 7.5, 100 ml x 2) and further with water (200 ml), dried (anhydrous sodium sulfate) and concentrated under reduced pressure to obtain a syrup. Purification of the syrup by C₁g silica gel chromatography (0 5.3 x 7.0 cm; solvent: acetone-water, 55°-80°) gave a colorless glassy compound (8.5 g).

This compound (8.0 g) was dissolved in 32 mL of tetrahydrofuran (THF), tetrabutylammonium fluoride trihydrate (TBAF-3H 2 O)

(10 g) was added and the mixture was stirred at room temperature for 0.5 h. The solvent was removed under reduced

pressure and the remaining oil was dissolved in 100 ml of water. The solution was washed with diethyl ether (100 mL x 2) and the tetrabutylammonium salt was removed by treatment on Dowex-50 resin (pyridine form 60 mL). The effluent and water washings (240 mL) were combined and concentrated and the concentrate was dehydrated azeotropically with three portions of pyridine. The residue was dissolved in 100 ml of pyridine, monomethoxytrityl chloride (MMTrCl)

(5.4 g) was added and the mixture was stirred at 37° C. for 4 hours. The solvent was removed under reduced pressure and the oily residue was partitioned between 0.1 M TEAB buffer (50 mL) and CHCl 3 (100 mL), the organic layer was washed with water (100 mL), dried (anhydrous sodium sulfate) and concentrated under reduced pressure and the concentrate was subjected to dehydration by azeotropic distillation with toluene to obtain a colorless syrup. 0.1 M-TEAB buffer fraction and water washings were combined and concentrated, removing the non-monomethoxy tritylated compound. This compound was purified on HP-20 resin (190 ml; solvent: water and 30% ethanol-water) and, after concentration and azeotropic distillation with pyridine, monomethoxy tritylated in the same manner as mentioned above. Both yields of the title compound thus obtained were combined and purified by si 1 isium dioxide gel chromatography (80 g; solvent: CHCl 3 /MeOH = 100/1, 60/1, 50/1) to obtain a colorless glass-like product (6.1 g) . A solution of a portion of this product in dichloromethane gave, on addition dropwise to n-hexane, a white powder.

NMR (60MHz, CDCl3) S ppm: 1.87-2.70 (5H, m, H2' , H4', H5' ), 3.20-3.40 (2H, m, H6' ), 3.43 (3H, s, CH30CH2), 3.80 (3H, s), 4.30-4.57 (1H, m, H3'), 4.87-5.10 (1H, m, B1'), 5 .47 (2H, s, CH30CH2-N), 6.73-6.97 (2H, m), 7.17-7.53 (12H, m), 7.73 (1H, s), 7.98 (1H, p).

Example 5

Synthesis of 1-[ (IR , 3S , 4R )-4-(monomethoxytrityloxy )-methyl-3-hydroxycyclopentan-1-yl]-(4-carbamoyl-5-aminoimidazole)

The compound obtained in Example 4 (6.1 g, 10.7 mmol) was dissolved in 490 mL of ethanol and, while heating under reflux, a heated 5 M aqueous sodium hydroxide solution (130 mL) was rapidly added. Reflux was continued for another 40 minutes. The solvent was then removed under reduced pressure. The oily residue was dissolved in 200 ml of chloroform and washed with water (100 ml x 2), then with 0.1 M M-TEAB buffer (100 ml x 2) and further with saturated aqueous sodium chloride solution (100 ml), dried ( anhydrous sodium sulfate ) and concentrated under reduced pressure to obtain a syrup. Purification of this syrup by silica gel chromatography (90 g; solvent: CHCl 3 /MeOH = 100/1 to 20/1) gave a colorless glassy product (3.2 g) .A solution of a portion of this product in chloroform gave, on addition dropwise to n-pentane with stirring, a

white powder.

NMR (100 MHz, CDCl 3 ) S ppm: 1.36-2.52 (5H, m), 3.00-3.40 (3H, m, H6', OH), 3.77 (3H, s), 4.12-4.60 (2H, m, B. 1', H3'), 4.80-5.28 (2H, bs, NH2), 5.64-6.44 (2H, bs, NH2) , 6.76-6.94 (3H, m), 7.14-7.48 (12H, m) bs; wide singlet

Example 6

Synthesis of l-[(IR , 3S , 4R )-4-hydroxymethyl-3-hydroxy-cyclo-pentan-1-yl]-(4carbamoyl-5-aminoimidazole)

The compound obtained in Example 5 (2.3 g, 4.4 mmol) was dissolved in 50 ml of 80% acetic acid and the solution was stirred at 40° C. for 7 hours. After concentration under reduced pressure, azeotropic distillation was carried out with toluene and then with ethanol. The residue was dissolved in 12 ml of ethanol. 130 ml of n-hexane ether (1:1, v/v) was added dropwise to the solution with stirring. The syrup thus obtained was purified by C₁g silica gel chromatography (10 g; solvent: water and 50% acetone-water). The residue obtained after removal of the solvent under reduced pressure was recrystallized from ethanol to obtain the title compound (0.93 g), m.p. 162-163"C. NMR (60 MHz,~<*>DMS0-d6+ D2O) S ppm: 1.67-2.67 (5H, m), 3.43-3.60 (2H, m, H6' ), 3.90-5.00 (2H, m), 7.23 (1H, s, H2); (DMS0-d6) 5.77 (2H, bs, NH2), 6.63 (2H, bs, NH2)

Example 7

Synthesis of l-[ (IR , 3S ,4R)-4-(monomethoxytrityloxy )-methyl-3-hydroxy cyclopent an -1 - y 1 ] - [ 4 - carbamoyl-5 - (N-benzoyl -S- methylisothiocarbamoyl)aminoimidazole]

The compound obtained in Example 5 (0.88 g, 1.7 mmol) was dissolved in 25 ml of anhydrous acetone and, while heating at reflux, a solution of benzoyl isothiocyanate (260 µl, 1.9 mmol) in acetone (8 ml) was added dropwise during 10 minutes, followed by reflux for 50 minutes. The solvent was removed under reduced pressure and the obtained light yellow glass-like substance was purified by silica gel chromatography (15 g; solvent: CHCl 3 /MeOH = 50/1 to 30/1) to obtain a light yellow glass-like compound (0.87 g) . A small amount of acetone was added to this compound (0.84 g, 1.2 mmol) and the resulting syrup was converted to a homogeneous solution by addition of 12.5 mL of 0.2N NaOH and sonication. Dimethyl sulfate (130 µl, 1.4 mmol) was added with stirring and then vigorous stirring was carried out at room temperature for 1 hour. The reaction mixture was mixed with CHCl 3 (15 mL x 2) for separation, the organic layer was washed with 0.1 M TEAB buffer (15 mL x 3) and then with saturated aqueous sodium chloride solution (20 mL), dried ( anhydrous sodium sulfate) and concentrated under reduced pressure, and the residue was purified by silica gel chromatography (15 g; solvent:

CHCl3/MeOH = 100/1 to 60/1). A small amount of dichloromethane was added to the obtained glassy substance, the mixture was added dropwise to hexane and the resulting precipitate was collected by centrifugation and dried to obtain the title compound as a powder (400 mg).

NMR (100 MHz, CDCl 3 ) S ppm: 1.34-2.60 (5H, m), 2.52 (3H, s, SCH3), 3.04-3.44 (2H, m, H6'), 3.79 (3H, s, 0CH3), 4.08-4.44 (1H, m, H3'), 4.60-5.00 (1H, m, R±' ), 5.64 (1H, bs, NH2), 6.72-6.94 (3H, m), 7.12-7.52 (15H, m), 7.80-7.96 (2H, m), 11.35 (1H, bs, NH).

Example 8 Synthesis of 1-[(1R,3S,4R)-4-hydroxymethyl-3-hydroxycyclopentan-1-y1]-[4-carbamoyl-5-(N-benzoyl-S-methylisothiocarbamoyl) aminoimidazole]

The compound obtained in Example 6 (815 mg, 3.4 mmol) was reacted with 1.1 equivalents of benzoyl isothiocyanate in acetone. The solvent was then removed under reduced pressure, 15 ml of acetone-CHCl 3 (2:1, v/v) was added and ether was further added. The resulting precipitate was collected by filtration and dried. The thus obtained powder (1.4 g) was dissolved in 35 ml of 0.2N sodium hydroxide, dimethyl sulfate (340 µl) was added and the mixture was stirred at room temperature for 1 hour. Acetic acid was added to the reaction mixture with ice-cooling (to pH 4-5), the product was extracted from the white, cloudy reaction mixture with n-butanol (20 mL x 3), the extract was washed with water (10 mL x 2), the solvent was removed under reduced pressure and the residue was purified by silica gel chromatography (10 g; solvent: water and 30% acetone-water) to obtain a pale yellow glassy substance (920 mg). Recrystallization from water gave the title compound as colorless crystals (570 mg);

m.p. 119-120T.

The following was confirmed: NMR (100 MHz, DMS0-d6) § ppm: 2.52 (3H, s, CH3), 7.34-7.94 (6H, m), 11.85 (1H, bs. NH) .

Example 9

Synthesis of 9-[(IR , 3S , 4R )-4-monomethoxytriethyloxy-methyl-3-hydroxycyclopentan-l-yl]guanine

The compound obtained in Example 7 (360 mg, 0.53 mmol) was added to heated 6N sodium hydroxide (18 mL) and the mixture was heated under reflux for 1 hour. The product was extracted from the reaction mixture with CHCl 3 and the extract was washed with 0.1 M TEAB buffer (30 mL) and then with saturated aqueous sodium chloride solution (30 mL), dried (anhydrous sodium sulfate) and subjected to silica gel chromatography in (8 g; solvent: CHCl3/MeOH = 40/1 to 6/1). To the thus obtained glass-like substance was added a small amount of acetone, the mixture was added dropwise to benzene and the resulting precipitate was collected by centrifugation and dried to obtain the title compound as a powder (210 mg).

NMR (100 MHz, DMS0-d6) S ppm: 1.50-2.60 (5H, m), 3.01 (2H, bs), 3.98-4.20 (1H, m), 4.70 -4.96 (2H, m), 6.37 (2H, bs, NH2), 6.82-7.46 (14H, m), 7.68 (1H, s, H8), 10.60 (1H , bs,

NH).

Example 10

Synthesis of 9-[(IR,3S,4R)-4-hydroxymethyl-3-hydroxycyclo-pentan-1-yl]guanine

The compound obtained in Example 9 (180 mg, 0.33 mmol) was dissolved in 10 ml of 80% acetic acid and the solution was heated at 40°C for 4.5 hours. The solvent was removed under reduced pressure and further azeotropic distillation was carried out twice with water. Water (10 mL) was added, the mixture was washed with ether (10 mL x 2) and the water was removed under reduced pressure. Thus, the title compound was obtained as colorless crystals (41 mg), m.p. 246-248°C.

Example 11

Synthesis of 9-[(IR,3S,4R)-4-monomethoxytrityloxy-methyl-3-hydroxyl-cyclopentan-1-yl]isoguanine

The compound obtained in Example 7 (585 mg, 0.85 mmol) was dissolved in 10 mL of ethanol, followed by the addition of 6 mL of 1N NaOH and 44 mL of water. The mixture was heated under reflux for 2 hours. After additional addition of 60 mL of 0.1N NaOH to the mixture, heating was continued under reflux for an additional 2 hours. The reaction mixture was then neutralized with IN HCl, the product was extracted with CHCl 3 , the extract was washed with saturated aqueous sodium hydroxide solution and then with water, the chloroform was removed under reduced pressure, and the syrup thus obtained was placed overnight in a refrigerator to obtain of the title compound as white crystals (200 mg; mp 245-247°C). Furthermore, the filtrate was purified by C₂₂-si₁₂₂ gel chromatography (10 g; solvent: 50-90% acetone-water).

NMR (100 MHz, DMS0-d6) § ppm: 1.46-2.46 (5H, m, 2H2', 2H5', H4'), 3.00-3.38 (4H, m), 3.75 (3H, s, CH3), 3.96-4.16 (1H, m, H3'), 4.68-4.94 (2H, m), 6.38 (1H, bs, NH), 6, 80-7.50 (14H, m), 7.77 (1H, s, H8)

Example 12

Synthesis of 9-[(IR , 3S , 4R )-4-hydroxymethyl-3-hydroxyl-cyclo-pentan-1-yl]isoguanine

The compound obtained in Example 11 (148 mg, 0.27 mmol) was dissolved in 11 ml of 80% acetic acid and the mixture was stirred at 45°C for 6 hours. Then the solvent was removed under reduced pressure, followed by azeotropic distillation with water. The reaction mixture in the form of an aqueous solution was washed with ether (10 ml x 2). The water was distilled off under reduced pressure and the resulting glassy substance was suspended in ethanol to obtain the title compound [69 mg, m.p. 162-165°C (decomposition)].

NMR (100 MHz, DMS0-D6+ D2O) S ppm: 1.10-2.60 (5H, m, 2H2', 2H5', H4 ' ), 3.40-3.60 ( 2H, m, 2H6 ' ) , 4.00-4.20 (1H, m, H3'), 4.60-5.10 (1H, m, Hi'), 7.90 (1H, s, Hg)

Example 13

Synthesis of the compound of Example 4 (alternative method)

The compound of Example 8 (420 mg) was dissolved in 10 ml of 6N sodium hydroxide and the solution was rapidly heated and refluxed for one hour. After cooling to room temperature, the reaction mixture was neutralized with 1N hydrochloric acid, purified by HP-20 chromatography (190 ml; solvent: water and 10% ethanol-water) and concentrated to obtain colorless crystals (174 mg), m.p. 246-248°C.

Reference example 1

Synthesis of 9-[ (IR , 2S , 3R , 4R )-4-monomethoxytrityloxy-methyl-2,3-(dimethylmethylene)dioxycyclopentan-1-yl]-hypoxanthine

The C-analog of inosine (10 g, 37.6 mmol) was suspended in 380 mL of acetone. To the suspension was added 2,2-dimethoxypropane (23 mL) and p-tosylic acid (9.3 g), and the mixture was stirred at 37°C for 3 hours. After addition of 40 ml of concentrated aqueous ammonia with ice-cooling and subsequent concentration under reduced pressure, the obtained insoluble material was collected by filtration, purified by Cg-silica gel chromatography (0 5.0 x 6.5 cm; solvent: 5% acetone-water) and recrystallized from ethanol to obtain 8.4 g of a crystalline compound. The filtrate from recrystallization 1 in the serification step was purified by silica gel chromatography (60 g; solvent: CHCl 3 /MeOH = 25/1 to 5/1) to obtain a certain amount of the compound as a s in rup.

The above-mentioned crystals (8.4 g, 27 mmol) were suspended in a small amount of pyridine. After azeotropic dehydration under reduced pressure, 150 ml of anhydrous pyridine was added to dissolve the residue. To the solution was added monomethoxytrityl chloride (9 g) and the mixture was left at room temperature for 13 hours. After addition of 20 ml of methanol, all the solvent was removed under reduced pressure and the residue was dissolved in 150 ml of CHCl 3 . The solution was washed with 0.1 M TEAB buffer (100 mL x 2) and then with water (100 mL), dried (anhydrous sodium sulfate) and concentrated by removing CHCl 3 under reduced pressure to precipitate the title compound as crystals (9 .4 g), m.p. 194-195<0>C. Likewise, the above-mentioned syrup was monomethoxy tritylated and the product was combined with the previously recrystallized mother liquor. The combined mixture was dissolved in 70 mL of CHCl 3 , the solution was added dropwise to 1 liter of n-hexane with stirring, and the resulting precipitate was collected by filtration and dried to obtain an additional 10.3 g of the title compound as a powder.

Reference example 2

Synthesis of 9-[ (IR , 2S , 3R , 4R )-4-monomethoxytrityloxy-methyl-2,3-(dimethylmethylene)dioxycyclopentan-1-yl]-(1-methoxymethyl-hypoxanthine)

The compound obtained in Reference Example 1 (19.1 g, 33 mmol) was dissolved in 360 mL of dioxane-dimethylformamide (DMF) (3:1, v/v), followed by the addition of 1.47 g of sodium hydride (60% in oil ) under ice-cooling. The mixture was immediately returned to room temperature and stirred for one hour. Then 3.2 ml of methoxymethyl chloride was added under ice-cooling and the mixture was stirred for 4 hours under ice-cooling. The solvent was removed under reduced pressure and the residue was dissolved in 200 ml CHCl3. The solution was washed with 0.1 M TEAB buffer (200 ml x 2) and then with water (200 ml), and dried (anhydrous sodium sulfate), and the solvent was removed under reduced pressure. The glassy substance thus obtained was crudely purified by silica gel chromatography (100 g; solvent: CHCl3/MeOH = 100/1 to 10/1 and further purified by Cg silica gel chromatography (0 5.3 x 7.0 cm; solvent: 55-90% acetone-water) to obtain 16.8 g of a glass-like substance, mp 97-102° C.

NMR (90 MHz, CDCl3) S ppm: 1.28 (3H, s, CH3), 1.54 (3H, s, CH3), 2.33-2.60 (3H, m, H4', H5') , 3.10-3.40 (2H, m, H6*),

3.43 (3H, s, CH20CH3), 3.79 (3H, S,

4.47-5.07 (3H, m E1', H2', H3'), 5.46 (2H, s, N-CH2-0), 6.79 (1H, s), 6.88 (1H , s), 7.13-7.57 (12H, m), 7.82 (1H, s), 8.04 (1H, s)

Reference example 3

Synthesis of 1-[(IR,2S,3R,4R)-4-monomethoxytrityloxy-2,3-(dimethylmethylene)dioxycyclopentan-1-yl]-(4-carbamoyl-5-aminoimidazole)

The compound obtained in Reference Example 2 (16.8 g, 26.5 mmol) was dissolved in ethanol (685 ml) and, under reflux, 137 ml of 5M sodium hydroxide, heated to 80°C, was rapidly added. Refluxing was continued for 20 minutes and then the ethanol was removed under reduced pressure. The product was extracted with 300 mL CHCl 3 and the extract was washed with water (300 mL x 2), 0.1 M TEAB buffer (300 mL) and saturated aqueous sodium chloride solution (300 mL) in that order, and dried (anhydrous sodium sulfate). Coarse purification by silica gel chromatography (100 g; solvent: CHCl 3 /MeOH = 100/1 to 50/1) followed by four repetitions of such silica gel chromatography gave 7.9 g of a colorless glassy substance, m.p. 107-112°C.

NMR (90 MHz, CDCl 3 ) S ppm: 1.27 (3H, s), 1.58 (3H, s), 1.90-2.67 (3H, m), 3.23-3.40 (2H , m), 3.80 (3H, s), 4.07-4.50 (3H, m), 5.54 (2H, bs, NH2), 6.80 (1H, s), 6.21 ( 2H, s), 7.20-7.57 (13H, m)

Reference example 4

synthesis of 1-[(1R,2S,3R,4R)-4-hydroxymethyl-2,3-(dimethyl-methylene)dloxycyclopentan-1-yl]-(4-carbamoyl-5-aminoimidazole)

(compound in reference example 4-1) and synthesis of 1-[ (IR , 2S , 3R , 4R )-4-hydroxymethyl-2 , 3-dihydroxycyclopentan-1-yl]-(4-carbamoyl-5-aminoimidazole) (compound In reference example 4-2)

The compound obtained in Reference Example 3 (7.9 g, 13.9 mmol) was dissolved in 120 ml of 80% acetic acid and the solution was stirred at 40°C for 7 hours. The reaction mixture was concentrated under reduced pressure and subjected to several repetitions of azeotropic distillation with water added, the residue was partitioned between water and ether, the aqueous layer was washed with ether and then concentrated under reduced pressure. The concentrate was purified by C 8 silicon dioxide gel chromatography. Fractions eluted with water and 5% acetone-water were collected, the solvent was removed under reduced pressure and azeotropic distillation with ethanol was repeated twice to obtain the compound of Reference Example 4-2 as a crystalline precipitate (0.34 g, mp 212 -214°C).

Separately, fractions eluted with 15% acetone-water were collected, the solvent was removed under reduced pressure and azeotropic distillation with ethanol was carried out to obtain the above compound of Reference Example 4-1 as crystals weighing 1.85 g, m.p. 169-171°C (decomposition).

Characteristic data for the compound in Reference Example 4-1:

NMR (100 MHz, DMS0-d6) S ppm: 1.26 (3H, s), 1.50 (3H, s), 1.70-2.40 (3H, m), 3.40-3.60 (2H, m), 4.16-4.82 (4H, m, R1' t H2', H3\ OH), 5.72 (2H, bs, NH2), 6.65 (2H, bs, NH2) , 7.26 (1H, s, H2).

Characteristic data for the compound in Reference Example 4-2:

NMR (100 MHz, DMS0-d6) S ppm: 1.00-2.40 (3H, m, H2', H5'), 3.38-3.54 (2H, m, H6'), 3.68 -4.34 (3H, m, E1', H3'), 4.56-5.00 (3H, m, OH), 5.69 (2H, bs, NH2), 6.63 (2H, bs, NH2), 7.22 (1H, s, H2)

Example 14

Synthesis of 1-[(IR,2S,3R,4R)-4-hydroxymethyl-2,3-(dimethylmethylene)dioxycyclopentan-1-yl]-5 (N-benzoyl-S-methyl slotio-carbamoyl) aminoimidazole]

The compound of Reference Example 4-1 (300 mg, 1 mmol) was suspended in 18 ml of acetone and, under reflux, an acetone solution (5 ml) containing 150 µl of benzoyl thiocyanate was added dropwise over 10 minutes. Refluxing was continued for a further hour, the solvent was then removed under reduced pressure and the residue was dissolved in 5 ml of CHCl 3 -acetone (1:1, v/v). Dropwise addition of the solution to 100 ml of n-hexane with stirring gave a precipitate which was collected by centrifugation and dried to form a pale yellow powder. This powder was dissolved in 10 ml of 0.2N sodium hydroxide and after adding 110 µl of dimethyl sulfate, the mixture was stirred for 1.5 hours. Several drops of acetic acid were added with ice-cooling (pH 6-7) and the resulting precipitate was extracted with CHCl 3 (10 mL x 2). The extract was washed with water (20 ml x 3), dried (anhydrous sodium sulfate) and purified by silica gel chromatography (11 g; solvent: CHCl 3 /MeOH =_100/1 to 40/1) to obtain a colorless glass-like substance. This was dissolved in 6 ml of dichloromethane and the solution was added dropwise to 100 ml of n-hexane with stirring. The resulting precipitate was collected by centrifugation and dried. Thus, the title compound was obtained as a white powder (320 mg).

NMR (100 MHz, CDCl3) S ppm: 1.30 (3H, s, CH3), 1.54 (3H, s, CH3), 2.0-2.52 (3H, m, H4 ' , H5 ' ) , 2.58 (3H, s, S-CH3), 3.75 (2H, d, H6'), 4.48-496 (3H, m, % ' , H2 ' , H3' ) , 5.85 ( 1H, bs, NH), 7.58 (1H, s, NH), 7.26-7.54 (4H, m), 7.80-7.94 (2H, m)

Example 15

Synthesis of 9-[( IR , 3S , 4R )-4-hydroxymethyl 1-2 , 3-(dimethyl-methylene)dioxycyclopentan-l-yl]guanine

The compound obtained in Example 14 (820 mg, 1.75 mmol) was added to 14 mL of 6N sodium hydroxide and the mixture was heated rapidly and stirred to dissolve it. After one hour of continued heating under reflux, the reaction mixture was neutralized with 1N hydrochloric acid with ice cooling. Coarse purification by C 16 -silica gel chromatography (10 g; solvent: water and 20% acetone-water), washing the aqueous eluate with CHCl 3 , concentration under reduced pressure and crystallization from water gave 410 mg of crystals, m.p. 287-288°C.

NMR (100 MHz, DMS0-d6+ D2O) S ppm: 1.26 (3H, s, CH3), 1.51 (3H, s, CH3), 1.84-2.40 (3H, m, H4* , H5*), 3.53 (2H, d, H6'), 4.46-5.04 (3H, m, H21', H2', H3'), 7^86 (1H, s, H8), ( 100 MHz, DMS0-d6) S ppm: 3.31 (1H, bs, OH), 6.63 (2H, s, NH2), 10.85 (1H, bs, NH)

Example 16

Synthesis of 9- [ (IR , 2S , 3R, 4R )-4-hydroxymethyl-2,3-dihydroxy-cyclopentan-l-yl]guanine

The compound obtained in Example 15 (150 mg, 0.46 mmol) was dissolved in 20 ml of 0.05N HCl and the solution was heated at 70°C for 20 minutes.

The reaction mixture was neutralized with 1N NaOH with ice-cooling to afford the title compound as crystals (91 mg), m.p. 268-270"C (decomposition).

Example 17

Synthesis of 9-[(IR , 3S , 4R )-4-hydroxymethyl-3-hydroxyl-cyclo-pentan-1-yl]hypoxanthine

In 200 ml of toluene, 12.37 g of 9-[(IR, 3S, 4R)-4-methyl-3, 6 - (tetraisopropyldisiloxanyl)dioxycyclopentan-l-yl]hypoxanthine obtained by the method in example 3, 10 g of tetrabutylammonium fluoride were dissolved was added to the solution and then the mixture was stirred at 75°C for 2 hours. The reaction mixture was concentrated to dryness, the residue was dissolved in water and absorbed on a column (30 g) of activated carbon. The material eluted from the column with 1 L of 50% ethanol-water was recrystallized from ether-methanol to give the title compound (2.6 g), m.p. 170°C.

Example 18

Tablets for oral administration of the antiviral preparation obtained by the invention are prepared as exemplified below: 10 mg 9-[(IR,3S,4R)-4-hydroxymethyl-3-hydroxyl-cyclopentan-1-yl]guanine (the compound in example 10) , 250 mg of lactose, 50 mg of starch and 2 mg of magnesium stearate are mixed in methanol and, after removal of the methanol, molding is carried out into tablets.

Example 19

Ointment for the antiviral preparation is prepared as exemplified below: 0.1 g of 9-[(IR,3S,4R)-4-hydroxymethyl-3-hydroxyl-cyclopentan-1-yl]guanine (the compound in Example 10), 40.0 g vaseline album, 18.0 g ethanol, 5.0 g sorbitan sesquioleate, 0.5 g polyoxyethylene lauryl alcohol ether, 0.1 g methyl p-hydroxybenzoate and 36.3 g purified water are mixed to form 0 .1% ointments.

Test example 1

Inhibitory effect of antiviral compounds against Herpes Simplex virus type 1.HF strain

Vero cells were infected with 100 TCID50 (50% tissue culture infected doses) of virus and cultured with various concentrations of the compounds. Antiviral activity (ID50; 50% inhibitory dose) was observed three days after infection, when the control culture without any drug showed 100% cytopathogenic effects. Cytotoxicity was observed with the uninfected control culture at the same time point. a) The concentration needed to reduce the viral cytopathogenic effect by 50%. b) Minimum concentration of compounds required to cause a visible disruption of normal cell morphology.

Test example ~* 2

Tests of antiviral drugs for antiadenovirus activity

FL cells were infected with 100 TCID50 (50% tissue culture infected doses) of virus and cultured with various concentrations of the compounds. Antiviral activity (ID5Q; 50% inhibitory dose) was observed eight days after infection, when the control culture without any drug showed 100% cytopathogenic effects. Cytotoxicity was observed with the uninfected control culture at the same time point.

Claims (11)

1. Compound, characterized by the formula: where R 1 and R 2 are each a hydroxyl group, which may optionally be protected, X is a hydrogen atom or a hydroxyl group, which may optionally be protected, and Y is guanin-9-yl, isoguanin-9-yl or hypoxanthine-9- yl, provided that when X is a hydroxyl group, which may optionally be protected, then Y is different from hypoxanthine-9-yl. 2. Compound according to claim 1, characterized in that R 1 and R 2 are each a hydroxyl group, X is a hydrogen atom and Y is guanin-9-yl. 3. Compound according to claim 1, characterized in that R 1 and R 2 are each a hydroxyl group, X is a hydrogen atom and Y is isoguanin-9-yl. 4. Compound according to claim 1, characterized in that R 1 and R 2 are each a hydroxyl group, X is a hydrogen atom and Y is hypoxanthine-9-yl. 5. Compound according to claim 1, characterized in that R]; , R 2 and X are each a hydroxyl group and Y is guanin-9-yl. 6. Procedure for the preparation of compounds of the formula: where Ri and Rg are each a hydroxyl group, which may optionally be protected, X is a hydrogen atom or a hydroxyl group, which may optionally be protected, and Y is guanin-9-yl, isoguanih-9-yl or hypoxanthine-9-yl, provided that when X is a hydroxyl group, which may optionally be protected, then Y is different from hypoxanthine-9-yl, characterized by subjecting a compound of the formula: where R 1 , R 2 and X have the meanings given above, for purine ring formation reaction. 7. Compound, characterized by the formula: where and R2 is a hydroxyl group, which may optionally be protected. 8. Antiviral preparation, characterized in that it contains a compound with the formula wherein Xi is a hydrogen atom or a hydroxyl group, and Y is guanin-9-yl, isoguanin-9-yl or hypoxanthine-9-yl, provided that when is a hydroxyl group, then Y is different from hypoxanthine-9-yl. 9. Preparation according to claim 8, characterized in that X is a hydrogen atom and Y is guanin-9-yl. 10. Procedure for the preparation of a compound of the formula: where Ri and Rg are each a hydroxyl group, which may optionally be protected, X is a hydrogen atom or a hydroxyl group, which may optionally be protected, and Y is guanin-9-yl, isoguanin-9-yl or hypoxanthine-9-yl, provided that when X is a hydroxyl group, which may optionally be protected, then Y is different from hypoxanthine-9-yl, characterized in that (A) reacts with a compound of the formula: where the symbols have the meanings given above, with an ester of a C-L-CiQ alcohol and a straight or branched C2 -C2o~ aliphatic carboxylic acid or a Cy-Cø g-aromatic carboxylic acid, by heating in the presence of an alkali metal alcoholate, for thereby forming a 2-alkyl- or 2-aryl-substituted C-analog of inosine or .2'-deoxyinosine of the formula: where R 3 is C 1 -C 19 -alkyl or C 1 -C 2 -aryl, and the other symbols have the meanings given above, (B) reacts the compound (II) with formamide or formic acid, thereby forming a C-analogue of inosine or 2'-deoxyinosine of the formula: where the symbols have the above meanings, (C) reacts compound (II) with alkyl or aryl isothiocyanate or alkali metal xanthate in tertiary amine solvent to form a C analogue of 2-mercaptoinosine or 2'-deoxy-2-mercaptoinosine, converts the C analog of 2-mercaptoinosine or 2'-deoxy-2-mercaptoinosine to its 2-chloro or 2-bromo intermediate, and converts the 2-chloro- or 2-bromo intermediate into the 2-alkoxy- or 2-substituted amino derivative with the formula: where R 4 is alkoxy or substituted amino, and the other symbols have the meanings given above, (D) reacts compound (II) with alkyl or aryl isothiocyanate or alkali metal xanthate in tertiary amine to form a C analog of 2-mercaptoinosine or 2'-deoxy-2-mercaptoinosine, converts the C analog of 2-mercaptoinosine or 2'-deoxy-2-mercaptoinosine to its 2-chloro or 2-bromo intermediate, and converts the 2-chloro or 2-bromo intermediate into a compound (I) where Y is guanin-9-yl, (E) reacts the compound (II) with benzoyl isothiocyanate in boiling acetone, methylates the resulting benzothiourea derivative with methyl iodide or dimethyl sulfate, and further heats the methylation product for approx. 2-6N sodium hydroxide solution, thereby forming a compound (I) where Y is guanine-9-yi. (F) reacts the compound (II) with benzoyl isothiocyanate in boiling acetone, methylates the resulting benzothiourea derivative with methyl iodide or dimethyl sulfate, and further heats the methylation product for approx. 0.1-1 N sodium hydroxide solution, thereby forming a compound (I) where Y is isoguanin-9-yl, or (G) reacts with a compound of the formula: where R-1 and R2 have the meanings given above, and Y' is hypoxanthine-9-yl, with phenoxythiocarbonyl chloride or ... -bromide to form a corresponding 2-phenoxythio-carbonyloxycyclopentane derivative, and treating the 2-phenoxycarbonyloxycyclopentane derivative with a trialkyltin hydride, for the formation of a compound (II) where X is hydrogen and Y is hypoxanthine-9-yl, and (H) if necessary, deprotecting any protected hydroxyl group as R₁ or R₂ or both in a product of any of the above methods (A)-(G). 11. Procedure for preparing a compound of the formula: where Ri and Rg are each a hydroxyl group, which may optionally be protected, characterized by treating a compound with the formula: where Ri and Rg have the meanings given above, and R3 is alkoxymethyl, with alkali.