CA1278517C - Antiviral composition - Google Patents

Abstract

Abstract of the Disclosure A compound of the formula:

Description

;~78~ 7 - , This application is closely related to Canadian Application Serial No.~3i~y~ dated ~rch 5/ /~
(Agent's Docket No. 24205-715) filed on the same date.
This invention provides nucleoside analogs havinc cyclopentane rins which can be used as substitutes fo-purine nucleosides in the fields of biology, medicine or gene manipulation ana as an antiviral agent.
Derivatives of the compounds having the formula _~' Y
3~ 2 wherein Y is guanin-9-yl or adenin-9-yl are the e~amples of dideoxy analogs of purine nucleosides usec in determination of base sequence in DNA
[Proc.Nat.Acad.Sci.USA,74,4563(1977)~. However, 2',3'-dideoxy analogs of purine nucleosides are so susceptible to acids that cleavage occurs easily at the glycosyl linkage, which is a great difficult~ in their synthesis.
Recently it has been reported tllat 2',3'-dideoxy analogs of purine nucleosides can act as inhihitors o reverse transcriptase of virus origin, and hence these analogs have attracted attention as therapeutics in diseases due to RNA virus [Chemical and Engineering News, January 27, number 28(1986~].
Although, as mentioned above, dideoxy nucleosides and their carbocyclic analogs have been studied to some extent, there are still many aspects to be clarified. Therefore it is important to synthesize and evaluate various analogs. This invention intends to provide novel 2',3'-dideoxycarbocyclic nucleosides , ' ;,s-, ., ' ?., . .
. " :;.,~" .. . .. ..

78~i~L7 which can be used as antiviral agents or for other purposes.
The inventors completed this invention as the result of their researches under the circumstances described above to obtain novel and useful purine nucleoside analogs. That is, this invention relates to (lJ A compound of the formula (I~

~ 1' (I) 3 2' wherein R is a hydroxyl group which may be protected and Y is a purine base which may be protected, and the salts thereof, (2) a method of production of the compounds having the formula (I) and the salts thereof, which comprises subjecting a compound of the formula (II) 6~ 5~ Y
R ~ 1' (II) ~ ~ 2 3 l R2 R, wherein R is a hydroxyl group which may be protected, either R1 or R2 is a hydroxyl group and the other is a hydrogen, and Y is a purine base which may be protected, to reduction reaction of 2' or 3'-hydroxyl group, and (3) an antiviral agent containing a compound of the formula (I).
The protective groups of the hydroxyl group in the compounds of the formula (I) or (II) are not specifically limited as far as they are those used as protective groups in nucleoside chemistry. In this ~78~

invention the protective groups which are relatively stable under alkaline conditions are favorably used, such as alkylsilyl groups having 3-10 carbon atoms (e.g. t-butyldimethylsilyl), alkyl or alkoxy cyclic ethers having 4-10 carbon atoms (e.g. tetrahydrofuranyl and its derivatives having 4-7 carbon atoms, tetra-hydropyranyl and its derivatives having 5-8 carbon atoms such as methoxytetrahyropyranyl), alkoxyalkyl groups having 3-10 carbon atoms (e.g. ethoxyethyl, methoxyethyl), and trityl and alkoxy substituted trityl (e.g.monomethoxytrityi, dimethoxytrityl). When the protective group is an acyl group, the hydroxyl group can be protected in the form of aliphatic carboxylic acid ester (e.g. straight chain or branched with 1-lO
carbon atoms), or arylcarboxylic acid ester (e.g.
with 5-30 carbon atoms).
Purine bases represented by Y include various bases having purine ring skeleton which are used usua-lly in the field of nucleic acid chemistry. Such bases are exemplified by adenine, hypoxanthine, guanine, isoguanine, xanthine, 3 deazaadenine, 7-deazaadenine, 8~azaadenine, and 2,6-diaminopurine, and bound via the nitrogen atom at 9 position in the purine ring of the compound having the formula (I) or (II).
~he protective group of purine base in a compouncl of the formula (I) or (II), i.e. the amino protecti~Je group at 2 or 6 position is any of those which can be used usually in the field of nucleoside chemistry.
~or example an arylcarboxylic acid residue (with 5-30 carbon atoms) such as benzoyl for a protective group of adenine and an aliphatic carboxylic acid residue (straight chain or branched, with 2-lO
carbons) for a protective group of guanine are favora-bly used.
~or production of a compound of the formula (I) from a compound of the formula(II),preferably the hydroxyl ., ~ii .

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1~7~35~7 group at 2' or 3' position in a compound of formula (II) is thiocarbonylated at 0~80C or preferably at room temperature, followed by reduction with a tri(lower alkyl) tin hydride, such as tributyl tin hydride in the presence of an equivalent amount or an excess of an azo compound radical initiator, such as a,' azobisisobutylonitrile at 0-100C for 30 minutes to 2 hours, to give a 2',3'-dideoxy derivative having the formula (I). The thiocarbonylation can be favourably conducted by using thiocarbonyl diimidazole for thiocarbonylation, phenyl chlorothiocarbonate for phenoxythiocarbonylation. Further, S-methyldithiocarbonylation may be carried also by using the mixture of carbon bisulfide and methyl iodide. After this reduction, the 6'-hydroxyl protective group, for example, the 4,4'-dimethoxytrityl group is easily removed under acidic condition (e.g. treatment with acetic acid or IN hydrochloric acid at room temperature), and moreover the protective group of purine base can be removed under alkaline condition (e.g. concentrated ammoniac water, IN-sodium hydroxide, IM-sodium ethylate).
The compounds of formula (II) can be produced by, for example/ the following procedure: the compounds of formula (II) of which Y is an adenin-9-yl which may be protected can be produced also by the method described in Japanese Patent Application Laid-Open No. 62992/1975, Chemical & Pharmaceutical Bulletin 24,2624(1976) or Nucleic Acids Symposium Series, No. 16,141(1985). For example by the method described in Japanese Patent Application Laid-Open No.62992/1975 or Chemical & Pharmeceutical Bulletin 24,2624(1976), a compound in which in ~Z178~ L7 - 4a -which in the formula (II) Y is adenin-9-yl, either Rl or R2 is a hydroxyl group and the other is a hydrogen, and R is a hydroxyl group is obtained by using aristeromycin as starting compound;
and a compound in which Y in the formula (II) is N6-benzoyl-adenin-9-yl, R is a hydroxyl group protected with 4,4'-dimethoxy-trityl, and Rl is a hydrogen and R2 is a ~785~
_ 5 _ 24205-716 hydro.~yl group is obtained by the method described in Nucleic Acids Symposium Series described above. A
compound in which Y in the formula (II) is guanin-9-yl or hypoxanthin- 9-yl which may be protected, R is a hydro~yl group which may be protected, with a hydrogen at 2' position and a hydroxyl group at 3' position is obtained by the method described in Canadian Patent Application Ser. No. 520,946 (see the Reference Examples 1~8).
On the Gther hand, the compound of formula ~II) in which Y is 2,6-diaminopurine-9-yl, Rl is hydrogen, R2 is hydroxylgroup can be synthesized as follows:
Hydroxyl group of the compound in which Y is adenin-9-yl is protected, followed by Nl-oxidation with hydrogen peroxide or metachloro perbenzoic acid, and then the amino group at 6-pOsitioll is deaminated by nitrous acid, and is heated with phosphorus oxychloride (Japanese Patent Publication 4347/1967) to give the corresponding 2,6-dichloropurine-9-yl derivative. The chlorine at 6-position is substitutecl by amino group, and deamination is con-ducted by using sodium nitrite in aqueous acetic acid to give the product of 2-chloro-6-hydroxyl-9-yl. Thus obtained compound is subjected to amidation at 2-position and after chlorination at 6-position, the chlorine at this position is substituted by amino group to give the desired compound.
The salts of the compounds having the formula (I) in this invention include those formed with the amino group in the purine base and a mineral acid (e.g.hydrochloric acid, sulfuric acid, nitric acid), anorganic carbo~:ylic acid (e.g. acetic acid, lactic acid, tartaric acid, maleic acid, succinic acid) or an organicsulfonic acid (e.g. methanesulfonic acid, ethansulfonic acid, benzenesulfonic acid).

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The campounds of th~ formula (I) in ~hls lnven-tion provlde useful tools ~n gene clonin~. ~hat is, the analogs derlved ~rom the compounds of this inven-tion having cyclopentane ~ing are the ca~ocycllc analogs of purine-2',3'-dldeoxynucleotid~, and are ea~ ly aynthasized be~ause o~ ~b3Pnce of a glycosyl lin~cage; and the tripho~phate ~erivatives thereo m~y be used as age~a to 8 Op the DNA chs~n elongatlon in determlnation o~ DNA ~e~u~nc~.
On ~he othex hand, the compounds of the ~ormulA
~I) have antiviral ~ctiv~ty againa~ DNA viruse~ or RNA
viruses. ~here may be mentioned as DNA viruse4 herpe~virus group (9.g. herpes simplex virus ~ype I or II, cytomagalovlrus, Epstsln-9arr virus), adenovlru~
e (e.g. typ~ ), He~titis B vlrus or poxvirus7 ~s ~NA
viru3es hum~n immunodeficiency vlrus (HIV), which is a pathoger~
o~ ac~ired ~odeficiency syr~ome (A::DS), vesicular ~'camati-tls vlrus, feline leukemia viru~, equine ln~ectious anemic vlrus. Especially, the compound5 of the present 20 invention have patant antiviral activi~y aga~t RN~ viruses, in part~-cular H~rLv-III(~u;nan T-cell Lyny?hotro~ic vin~ tYPe ~ V~TLV.IU)~ which lr~ onc o~ H~l, as po33ible iniLibitor~ of reverse trælscriptase.
Accord.in~ly, .the c~mpound3 o~ the present lnven-tion may be u3ed for txeatment agalnst var~ou~ vixal ln~ectlons. A~ such viral in~ections, there may be mentioned acquired immunode~iciency ~yndrome7 herpes gimp:Lex ~type I or II), varicella, zostex, ker~titis, con~unctivitis or acute hepatitis varlous opportunistic in~ection~, mallgnant tumor or cen~ral nervous ~ymptoms which are brought out by vlral in~ectlon and immuno~e~iciency.
The compound~ of the formula (I) and the ~h~eutlcally acceptable ~alt~ thereof can be also u~0d as antiviral agent~ for treatment o~ viru~-induced diseaq~ ln animals, par-ticularly in mamalian animalY (laboratory an~als such a3 rabbit, rat ~nd mouse~ pet animals such a8 aOg and cat; human beingi livestock such as cattle, horse, sheep and pig).
In general for the above pourpose, a suitable effective dose of the compounds of the present invention is in the range of 30-500 mg per kg body weight per day, preferably in the range of 100-300 mg per kg body weight per day. The desired dose is generally presented as two, three or four more sub-doses administered at appropriate intervals throughout the day. Administration may be any suitable route including oral, rectal, nasal, topical (e.g.
buccal, sublingual), vaginal and parenteral (e.g.
subcutaneous, intramuscular, intravenous, intradermal).
The preferred route may vary with, for example, the condition and age of the recipient. While the present compounds can be administered alone, it is preferable to present them as part of pharmaceutical formulation.
The formulations of the present invention comprise at least one of the compounds of the formula (I), together with one or more acceptable carrier thereof and optionally other therapeutic ingredients.
The foxmulation may conveniently be presented in unit dosage form. Formulations containing the com-pound of the present invention for oral administration include discrete units such as capsules or tablets;
powder or granules; solution or suspension; or an oil-in-water liquid emulsion or a water-in-oil liquid emulsion etc.
A tablet may be made by compression or molding, 30 optionally with one or more accessory ingredients.
Compressed tablets may be prepared by compressing in a suitable machine the compound of the present invention in a form of a powder or granules, optionally mixed with a binder (e.g.hydroxypropyl-cellulose), lubricant (e.g. magne-3r sium stearate), inert diluent (e.g. starch), pre-servative, surface-active or dispersing agent.

~Z~ 7 Formulations for parenteral administration include aqueous and non-a~ueous sterile injection solutions which may contain anti-oxidants, buffers or, bacteriostats; and aqueous and non-aqueous sterile suspensions which may include suspending agents and thickening agents. The formulations may be presented in unit-dose or multi-dose containers, for example, sealed ampules and vials, and may be stored in a freeze-dried condition requiring only the addition of the sterile liquid carrier, for example water for injections, immediately prior to use. Extemporaneous injection solutions and sus-pensions may be prepared from sterile powders, gra-nules and tablets.
Formulations for topical administration include lozenges comprising the present compounds in a fla-voured basis, usually sucrose and acacia or traga-canth; pastilles comprising the present compound in an inert basis such as gelatin and glycerin, or sucrose and acacia; and monthwashes comprising the ingredient to be administered in a suitable liquid carrier.
Formulations for rectal administration may be presented as a suppository with a suitable base such as cacao butter.
Pormulations for vaginal administration may be presented as pessaries, tampons, creams, gels, pastes, foams or spray formulations containing in addition to the present compound such carriers as are known in the art to be appropriate.
Among the compounds of the formula (I), particularly 2',3'-dideoxyaristeromycin (the compound of Example 3) and 9-~(lS,4R)~4-hydroxymethyl-cyclopentan-l-yl]guanine (the compound of Example 4) inhibit potently growth of AIDS virus, and they are more useful compounds accordingly.

8~L7 In the following, Rference Examples, Examples and Test Example are described by which the invention is explained in the concrete.
Reference Example 1 ~ ynthesis of 9-[(lR,2S,3R,4R)-4-methyl-2-hydroxyl-3,6-(tetraisopropyldisiloxanyl)dioxycyclopentan-1-yl]-hypoxanthine The C-analog to inosine (10 g, 37.5 mmol) was dissolved in 200 ml of anhydrous DMF, 1,3-dichloro-1,1,3,3-tetraisopropyldisiloxane t13 ml, 41 mmol) and imidazole (11.3 g, 165 mmol) were 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 quickly with diethyl ether and dried to give the title compound as a white powder (17.2 g). A portion of the product was recrystallized from dichloromethane to give crystals, m.p. 135-138C.
Note: The C-analog to inosine is known by Chemical & Pharmaceutical Bulletin 24, 2624(1976).

1~7~

Reference Example 2 Synthesis of 9-[(lP~,2S,3~,4R)-4-methyl-2-phenoxy-thiocarbonyloxy-3,6-(tetraisopropyldisiloxanyl)-dioxycyclopentan-1-yl]hypoxantine S The compound obtained in Reference E~le 1 (11.2 g, 22.3 mmol1 was dissolved in 300 ml of anhydrous acetonitrile, dimethylaminopyridine (15.8 g, 53.5 mmol) and phenoxy-thiocarbcnyl 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 give a yellow syrup. This was purified by silica gel chromatography (90 g; solvent:
CHC13 and CHCl3/MeOH = 60/1) to give the title compound as a light-yellow grass-like substance (13.0 g) .
NMR(60MHz,CDCl3)~ ppm: 1.0-1.23 (28H, m), 2.13-2.43 (3H, m, H4', H5'1, 3.93-4.10 (2H, m, H6'), 4.80-5~20 (2H, m, Hl', H3'), 6.00-6.20 (H, m, H2'), 7.03-7.50 (5H, m), 7.87 (lH, s), 8.13 (lH, s) s;singlet, m;multiplet Reference Example 3 Synthesis of 9-[(lR,3S,4R)-4-methyl-3,6-(tetraisopropyldisiloxanyl)dioxycyclopentan-1-yl]-hypoxanthine Anhydrous toluene ~30 ml) was added to the compound obtained in Reference Example 2 (13.0 g, 20 mmDl), 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 80C and crystal-line ~ azobisisohutyronitrile ~AIBN) (820 mg) wasadced in 4 portlons at 15~minute intervals. A~ter 3 hours of heating with stirring, the solvent was removed under reduced pressure. The oil obtained was purified by silica gel chromatography (80 g; solvent: CHC13 and CHC13/MeOH = 60/1 to 30/1) to give the title compound as a colorless glass-like substance (10.4 g).
Recrystallization of a portion o~ the product from ethanol gave colorless needles, m.p. 200-202C.
NMR(60MHz,CDC13)~ ppm: 0.93-1.20 (28H, s), 1.97-2.5~ (5H, m, H2', H4', H5'), 3-80-4-07 (2H, m, H6'), 4.4~-5.27 (2H, m, Hl', H3'), 7.87 (lH, s), 8.20 (lH, s) Reference Example 4 Synthesis of 9-[(lR,3S,4R)-4-(monomethoxytrityloxy)-methyl-3-hydroxylcyclopentan-1-yl]-(1-methoxymethyl-hypoxantine) The compound obtained in Reference E~mple 3 (9.8 g, 19.8 mmol) was dissolved in 240 ml of anhydrous dioxane, sodium hydride (880 mg, 21.8 mmol) was quickly added to the solution with ice cooling and stirring and, then, the mixture was stirred at room temperature for 1.5 hours. Thereafter, methoxymethyl chloride (2 ml, 21.8 mmol) was quickly added to the mixture with ice cooling.
The whole 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 chloro~orm.
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 sul~ate) and concentrated under reduced pressure to give a syrup. Purification of the syrup by C18 silica gel chromatography (~ 5.3 x 7.0 cm; solvent: acetone water, 55%-80%) gave a colorless glass-like compound (8.5 g).

1~78~ 7 This compound (8.0 g) was dissolved in 32 ml of tetrahydrofuran (THF), tetrabutylammonium fluoride trihydrate (TBAF~3H2O) (10 g) was added, and the mixture was stirred at room temperature for 0.5 hour.
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 was deprived of the tetrabutylammonium salt by treatment on Dowex-50*resin (pyridine form 60 ml). The erfluent and water washings (240 ml) were combined and concen-trated and the concentrate was dehydrated azeotropically with three portions of pyridine. The residue was dlssolved in 100 ml of pyridine, monomethoxytrityl chloride (MMTrCl~ (5.4 g) was added and the mixture was stirred at 37C for 4 hours. The solvent was removed under reduced pressure and the oily residue was distributed between 0.1 M TEAB buffer (50 ml) and CHC13 (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 give a colorless syrup. 0.1 M-TEAB
buffer fraction and water washings were combined and concentrated, whereby the unmonomethoxytritylated compound was recovered. This compound was purified on HP-20 resin (190 ml; solvent: water and 30~ ethanol-water) and, after concentration and azeotropic distillation with pyridine, monomethoxytritylated in the same manner as mentioned above. Both the thus-obtained crops of the 0 title compound were combined and purified by silica gel chromatography (80 g; solvent: CHC13/MeOH = 100/1, 60/1, 50/1) to give a colorless glass-like product ~6.1 g). A solution of a portion of this product in di~
chloromethane, when added dropwise to n-hexane, gave a white powder-N~R(60MHz,CDC13)~ ppm: 1.87~2.70 (5H, m, H2', *Trade Mark .~
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H4', H5'), 3.20-3.40 (2H, m, H6'), 3.4~ (3H, s, CH30CH2), 3.80 (3H, s), 4.30-4.57 (lH, m, H3'), 4.87-5.10 (lH, m, H1'), 5.47 (2H, s, CH30CH2-N), 6.73-6.97 (2H, m), 7.17-7.53 (12H, m3, 7.73 (lH, s), 7.98 (lH, s) Reference Example 5 Synthesis of 1-L(lR,3S,4R)-4-(monomethoxytritylox~;)-methyl-3-hydro~ycyclopentan-1-ylJ-(4-carbamoyl-5-amino-imidazole) The compound obtained in Reference E~ple 4 (6.1 g, 10.7mmol) was dissolved in 490 ml of ethanol and, with heating under reflux, a warmed 5 M aqueous sodium hydroxide solution (130 ml) was added quickly.
Refluxing was continued for additional 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-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 give a syrup. Purification of this syrup by silica gel chromatography (90 g; solvent: CHC13/MeOH - 100/1 to 20/1) gave Zl colorless glass-like product (3.2 g).
solution of a portion of this product in chloroform, when added dropwise to n-pentane with stirring, gave a white powder.
Elemental analysis (%) for C30H32N404-0.5 H20, molecular weight 5~1.616 Calcula~ed : C, 69.08; H, 6.38; N, 10.74 Found : C, 69.14; H, 6.09; N, 10.54 NMR(lOOMHz,CDC131~ ppm: 1.36-2.52 (5H, m), 3.00-3.40 (3H, m, H6', OH), 3.77 (3H, ~), 4.12-4.60 (2H, m, H1', H3'), 4.80-5.28 (2H, bs, NH2), 5.64-6.44 (2H, bs, NH2), 6.76-6.94 (3T-T, m), 7.14-7.48 (12H, m) bs;broad singlet 1278~7 A _ t -Synthesis cf 1-[(lR,3S,4R)-~(monomethoxytritylox-)-methyl-3-hydroxycyclopentan-1-yl]-[4-carbamoyl-5-(N-benzoyl-S-methylisothiocarbamoyl)aminoimidazole]
The compound obtained in Reference E~mple 5 (0.88 g, 1.-mmol) was dissolved in 25 ml of anhydrous acetone and, with heating under reflux, a solution of benzovl isothiocyanate (260~1, 1.9 mmol) in acetone (8 ml) was added dropwise over 10 minutes, followed by refluxing for 50 minutes. The solvent was removed under reduced pressure and the light-yellow glass-like substance obtained was purified by silica gel chromatography (15 g; solvent: CHC13/~eOH = 50/1 to 30/1) to give 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 resultant syrup was converted to a homogeneous solution by addition of 12.5 ml of 0.2 N
NaOH and sonication. Dimethyl sulfate (130 ~1, 1.4 mmol) was added with stirring and, then, vigorous stirring was continued at room temperature for 1 hour.
The reaction mixture was mixed with CHCl~ (15 ml x 2) for partition, 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 chromatograp:ly (15 g; solvent CHC13/MeOH = 100/1 to 60/lj. A small amount of dichloromethane was added to the glass-like substance obtained, the mixture was added dropwise to hexane and the resultant precipitate was collected by centrifugation and dried to give the title compound as a powder (400 mg).

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lemental analysis (%) for C39H39N505S, molecular weight 689.835 Calcula~ed : C, 67.90; H, 5.70; N, 10.15;
Found : C, 67.45; H, 5.45; N, 9.89;
NMR(lOOMHz,CDC13)~ ppm: 1.34-2.60 (5H, m), 2.52 t3H, s, SCH3), 3.04-3.44 (2H, m, H6'), 3.79 (3H, s, OCH3), 4.08-4.44 (lH, m, H3'), 4.60-5.00 (lH, m, Hl'), 5.64 (lH, bs, NH2), 6.72-6.94 (3H, m), 7.12-7.52 (15H, m), 7.BO-7.96 (2H, m), 11.35 (lH, bs, NH) Reference Example 7 Synthesis of 9-[(lR,3S,4R)-4-monomethoxytrityloxy-methyl-3-hydroxycyclopentan-1-yl~guanine The compound obtained in Reference Example 6 (360 mg, 0.53 mmol) was added to a warmed 6 N sodium hyd~oxide (18 ml) and the mixture was heated under reflux for 1 hour. The product was extracted from the reaction mixture with CHC13, 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 (8 g; solvent: CHC13/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 resultant precipitate was collected by cen-trifugation and dried to give the title compound as a powder (210 mg).
Elemental analysis (%) for C31H31N504-1.0H20, molecular weight 555.633 Calculated : C, 67.01; H, 5.99; N, 12.60 Found : C, 6/.01; H, 5.69; N, 12.42 NMR(lOOMHz,DMSO-d6)~ ppm: 1.50-2.60 (5H, m~, 3.01 ~2H, bs), 3.98-4.20 (lH, m), 4.70-4.96 (2H, m), 6.37 (2H, bs, NH~), 6.82-7.46 (14H~ m), 7.68 (lH, s, H8), 10.60 (lH, bs, NH) 1~785~7 Reference Example 8 . _ Synthesis of 9-I(lR,3S,4R)-4-hydroxymethyl-3 hydroxy-cyclopentan-l-ylJguanine The compound obtained in Reference Example 7 (180 mg, 0.33 mmol) was dissolved in 10 ml of 80~ acetic acid and the solution was stirred at 40C for 4.5 hours. The solvent was removed under reduced pressure and, further, azeotropic distillation was conducted 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 was obtained the title compound as colorless crystals (41 mg), m.p. ~46-248C.
[~]25=+/.7~ (C=0.5, DMF) ~max (nm): (H~O); 255, 278 (sh) (H ) ; 257, 282 (OH ); 256 (sh), 273 Elemental analysis (~) for C11H15N5O3-0.5H2O-0.1C2H5OH, molecular weight 278.886 - Calculated : C, 48.24; H, 6.00; N, 25.11 Found : C, 48.61; H, 6.41; N, 25.40 ~IL2~78~

Example 1 N6-benzoyl-6'-O-(4,4'-dimethoxytrityl)-3'-O-[(imidazol-1-yl)-thiocarbonyl]-2'-deoxyaristeromycin N6-benzoyl-6'-O-(4,4'-dimethoxytrityl)-2'-deoxyaristeromycin (2.5 g) was dissolved in 10 ml ofdry dichloromethane, to which thiocarbonyl diimidazole (8.0 g) was added and stirred at room temperature for 20 hr. The reaction mixture was concentrated to dryness, and subjected to purification with silica gel chronatography (Kieselgel*60, Merck Co., 50 g, solvent:ethyl acetate), to give a light yellow glassy substance (yield 2~2 g).
NMR(90 MHz CDC13) ~ ppm: 3.80 (6H,S,2 CH30-), 8.35(1H,S,H8), 8.76(1H,S,H2).
Example 2 N6-benzoyl-6'-0-(4,4'-dimethoxytrityl)-2',3'-dideoxyaristeromycin The 3'-thiocarbonyl derivative obtained in Example 1 (2.0 g) was dissolved in 20 ml of dry dioxane, to which a solution of tributyl tin hydride (4.5 g) in dry dioxane (10 ml) was added dropwise with refluxing by heating. Meanwhile crystals of ~,~'-azobisisobutylonitorile (500 mg) were added little by little. The dropwise addition was completed in 20 minutes and refluxing was continued for further 2 hr. The solvent was evaporated off under reduced pressure, and the oily residue was purified with silica gel chromatography (40 g, solvent:CHC13), to give a colorless powder (1.1 g).
NMR(9OMHz, CDC13) ~ ppm: 3.80 (6H,S,2 CH30-), 4.80-5.20(1H,m,H1,), 3.15(2H,d,2H6,), 8.76(1H,S,H2), 9.10 (lH,S,-NH-C-).

Example 3 2',3'-dideoxyaristeromycin The compound obtained in Example 2 (1~0 *Trade Mark ;~ Y~.,.:.
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.....

~i~785~7 g) was dissolved in a small amount of pyridine, to which 50 ml of concentrated ammoniac water was added and heated in a pressure-proof tube at 60C for 5 hr.
The reaction mixture was concentrated to dryness, to which 80~ acetic acid (lO0 ml) was added and heated at 60C for 2 hr followed by concentration to dryness un~er reduced pressure. The residue was dissolved in water (100 ml) and washed twice with water. The aqueous layer was concentrated to dryness, and the residue was powderized in ether to give 2',3'-dideoxyaristeromycin (0.23 g).
UVlmHa2O (n~);260 Elemental analysis (~) for CllH15N5O~H2O
with molecular weight 251.29 Calc.: C;52.57, H;6.82, Nj27.87 Found: C;52.83, H;6.95, N;27.54 The thus obtained 2',3'-dideoxyaristeromycin was dissolved in the equivalent weight of lN-HCl. After concentration, ethanol was added to the solution and concentrated to dryness. This procedure was repeated several times. The residue was recrystralized from hot ethanol to give the crystals of the hydrochloric acid salt. m.p. 173 - 175C.
Elemental analysis (%) for CllH15N5O~HCl~l/2H2O
with molecular weight 278.73 Calc.: C;47.40, H;6.15, N;25.12, Cl;12.72 Found: C;47.98, H;6.06, N;24.87, Cl;12.71 [a]D5=-6.79(C=0.61, H2O) Example 4 The compound obtained in Reference Example 8 (2.5 g) was treated in a similar way to that in Examples 1, 2, and 3, to give a crystalline powder of 9-[(lS,4R)-4-hydroxymethylcyclopentan-1-yl]guanine (0.3 g). m.p. 269~C.
UV~max(nm):255,280(shoulder); UV~ma2x(nm):253,270 (shoulder); UVmax (nm):258 (shoulder), 270 Elemental analysis (~) for C11H152N5 with molecular weight 249.27 Calc.: C;53.00, H;6.07, N;28.10 Found: C;52.81, H;5. 86, N; 27. 83 [~]D =-4.74(C=0.57, DMF) 9-[(lS,4R)-4-hydroxymethylcyclopentan-1-yl]-hypoxanthine is obtained when a hypoxanthine derivative, instead of N6-benzoyl-6'~O-(4,4'-dimethoxytrityl)-3'-O-[(imidazol-l-yl)-thiocarbonyl]-2'-deoxyaristeromycin in Example 1 is treated in a similar way to that in Examples 1-3.
Elementary analysis (%) for C11H14N4O2 with molecular weight 234.25 Calc.: C;56.40, H;6.02, N;23.92 Found: C;56.81, H;6.33, N;24.25 Æxam~_e 5 9-[lS,4R)-4-hydroxymethylcyclopentan-1-yl]guanine (1) Synthesis of 9-[llR,3S,4R)-4-hydroxymethyl-3-hydroxylcyclopentan-1-yl]hypoxanthine The compound (12.4g, 20 m mol) obtained in Reference Example 3 was dissolved in 200 ml of toluene and to the mixture was added tetrabutylammonium fluoride (10.46 g, 40 m mol), followed by heating at 75C for 2 hr. The reaction solution was concentrated to dryness and dissolved in water. The solution was subjected to desaltation with 30 g of activated charcoal, the crude product was recrystallized from a mixture of methanol and ethylether to give colorless crystals (4.6 g). m.p. 170C.
30 Elemental analysis (%) for C11H14N4O3-H2O
with molecular weight 268.27 Calc.: C;49.25, H;6.01, N;20.88 Found: C;49.08, H;5.86, N;20.81 (2) Synthesis of 9-[(lR,3S,4R)-4-monomethoxytrityl-35 oxymethyl-3-hydroxylcyclopentan-1-yl]hypoxanthine 78~7 The crystaline compound (2.3 g, 9.2 m mol) obtained by the method (1) described above was dissolved in 100 ml of pyridine, followed by addition of monomethoxytritylchloride (3.1 g, 10 m mol) and then was stirred at room temperature for 5 hr. The reaction solution was purified with silica gel chromatography (80 g, solvent: CHC13/MeOH = 40/1 -6/1) to give powdery desired product (4.3 g). A part of the product was recrystalized from a mixture of chloroform and ether. m.p. 244 - 246C.
Elementary analysis (%) for C31H30N4O4~H2O
with molecular weight 531.60 Calc.: C;70.04, H;5.~8, N;10.54 Found: C;70.39, H;5.77, N;10.38 (3) Synthesis of 9-[(lS,4R)-4-monomethoxytrityloxy-methyl-cyclopentan-l-yl]hypoxanthine The compound (4.32 g, 8.27 m mol) obtained by the method (2) described above was dissolved in 70 ml of toluene, followed by addition of thiocarbonyl-diimidazol (2.2 g, 12.4 m mol) and stirred at room temperature for 5 hr. The reaction solution was concentrated to dryness, and the residue was purified with silica gel chromatography (80 g, solvent:
CHC13/MeO~ = 100~1 - 60/1) to give pale yellow powder (5 2 g). The thus obtained product was dis-solved in 90 ml of toluene and reacted with tributyl tin hydride (3.4 ml, 12.4 m mol) and~
azobisisobutylonitrile ~270 mg, 1.6 m mol) by the similar method as described in Reference Example 3, followed by purification with silica gel chlomatography (100 g, solvent: ethylacetate/MeOH =
9/1) to give the desired product (1.63 g). A part of the product was recrystalized from a mixture solution of methanol and ethyl ether. m.p. 175 - 177C.
Elementary analysis (~) for C31H30~4O3-1/2H2O
with molecular weight 515.60 ~78~

Calc.: C;72.21, H;6.06, N;10.87 Found: C;72.69, H;5.88, N;10.92 (4) Synthesis of 9-[(lS,4R)-4-hydroxymethyl-cyclopentan-1-yl]guanine The desired compound can be produced by similar methods as described in Reference Examples 4 to 8 using the compound obtained in the step (3) described above.
Exam~le 7 Tablets for oral administration of the antiviral composition according to the present invention are prepared as exemplified below:
Two hundred (200) mg of 2',3'-dideoxyaristromycin, 300 mg of lactose, 50 mg of starch and 2 mg of magnesium stearate are mixed in methanol and after removal of methanol with heating the mixture is molded into tablets.
ExamDle 8 Injection for antiviral composition is prepared as exemplified below:
~ive hundred (500) mg of 2',3'-dideoxyaristero-mycin is dissolved in 10 ml of sterilized water and adjusted to pH 6.0 with aqueous sodium hydroxide. The solution is filtered with sterilized fi].ter and sealed up in vial.
Text Examp~e 1 MATERIALS AND METHODS*
* Antimicrob. Agents Chemother,30,No.6,Dec.1986,933-937 Cells. An HTLV type I-carrying cell line, MT-4,and an HIVHTLV III-producing cell line, Molt 4/HI~HTL~ III' were used in this study. The cells were maintained in RPMI 1640 medium supplemented with 10% fetal calf serum, 100 IU of penicillin per ml, and 100 ~g of streptomycin per ml at 37C in a CO2 incubator.
Virus and virus infection. HIVHTL~

,:
.-r.4, . .

from culture supernatants of Molt-4/HIVHTLv III as previously described [Virolog~ 146, 272(1985)~. The titer of this virus preparation was 6 x 10 PFU/ml.
Infection of MT-4 cells with HIvHTLv~IIIwas made at multiplicity of infection of 0.002. Briefly, the cells were mixed with virus solution and incubated for 1 h at 37C. After adsorption, infected cells were washed and resuspended in fresh medium to a concentra-tion of 3 x 10 cells per ml. This concentration was cultured in both the presence and absence of various concentrations of carbocyclic 2',3'-dideoxynucleosides in a CO2 incubator at 37C for 6 days.
VHTLv_III~indUCed cytopathic effect HIVHTLV III-induced cytopathic effect was analvzed by mesuring the decrease in the number of viable cells.
The viable cells were counted by the trypan blue exclusion staining method.
Y HTLV-III antigen expression.
HTLV-III-infected MT-4 cells with virus-specific antigens were counted by an indirect [immuno-fluorescence (IF)] method. Briefly, methanol-fixed cells were incubated with diluted anti-HIVHTLv III
positive human serum for 30 min at 37~C. The pre-parations were then washed for 15 min withphosphate-~buffered saline. The cells were then incubated with fluorescein isothiocyanate-conjugated rabbit anti-human immunoglobulin G (Dakoppatts A/S, Copenhagen, Denmark) for 30 min at 37C and washed again with phosphate-buffered saline. More than 500 cells were counted under a fluorescence microscope, and the percentage of IF-positive cells was cal-culated.
From the assay mentioned above, it was confirmed that the compounds of the present invention have obvious anti-HIVHTLV-III aCtivity- In the case of ,~;~
, . ~ ..... .
s~:
. ;.....
.... .... .

~ ~78~

2',3'-dideoxyaristeromycin, the effective con-centration was 50-100 ~M and the cytotoxity was observed at 5Q0-1,000 ~Mr respectively.

Claims (5)

1. An antiviral composition containins an effective amount of compounc having the formula:

(wherein R is 2 hydroxyl group which may be protected and Y is a purine base which may be prctected), or a salt thereof and pharmacologically accept2Dle carrier.

2. The compcsition accordlns to claim 1, wherein the compound is 2',3'-dideoxycristeromycin.

3. The composition according to claim 1, wherein the compound is 9-[(1S,4R) -4-hydroxymethyl-cyclopentan-1-yl]guanine.

4. The composition according to claim 1, wherein the compound is 9-[(1S,4R)-4-hydroxymethyl-cvclopentan-1-yl]hypoxanthine.

5. The composition according to claim 1, which is for inhibiting the growth of AIDS virus.