HK1090060A - Nucleotide lipid ester derivatives - Google Patents

Description

Nucleotide lipid ester derivatives

The subject of the invention is specific nucleotide lipid esters of the general formula I below, their tautomers and their physiologically acceptable inorganic and organic acids and bases, processes for their preparation and medicaments containing these compounds as active ingredient,

wherein

R¹Is a linear or branched, saturated or unsaturated alkyl residue having 1 to 20 carbon atoms, which is optionally mono-or polysubstituted with the following substituents: halogen, C₁-C₆Alkoxy radical, C₁-C₆Alkylthio radical, C₁-C₆Alkoxycarbonyl group, C₁-C₆Alkylsulfinyl or C₁-C₆An alkyl sulfonyl group, a carboxyl group,

R²is hydrogen, a straight or branched, saturated or unsaturated alkyl chain having from 1 to 20 carbon atoms, which is optionally mono-or polysubstituted with the following substituents: halogen, C₁-C₆Alkoxy radical, C₁-C₆Alkylthio radical, C₁-C₆Alkoxycarbonyl or C₁-C₆An alkyl sulfonyl group, a carboxyl group,

R³is amino OR OR⁴Wherein R is⁴Is C₁-C₈An alkyl group, a carboxyl group,

x represents thio, sulphinyl or sulphonyl, and

y is an oxygen atom.

The amino group of an adenine residue in formula I may also be protected by well known amino protecting groups.

Since the compounds of the formula I contain asymmetric carbon atoms, all optically active forms and racemic mixtures of these compounds are also subject matter of the present invention.

Methods for the preparation of liponucleotides (liponucleotides) and their use as antiviral agents are described in J.biol.chem.265, 6112(1990) and EP-A-0,350,287. However, only the dimyristoyl and dipalmitoyl phosphatidyl residues, including their fatty acid ester structures, to which well-known nucleosides (e.g., AZT and DDC) are coupled are disclosed therein.

Nucleoside conjugates of thioether lipids with cytidine diphosphate, having antitumor activity and potentially useful in oncology, are described in j.med.chem.33, 1380, (1990).

Chem. pharm. Bull.36, 209(1988) describes 5' - (3-sn-phosphatidyl) nucleosides having antileukemic activity, and their enzymatic synthesis from the corresponding nucleosides and phosphorylcholine in the presence of phospholipase D having transferase activity.

In patent application WO 92/03462 thioether lipid conjugates are described which have antiviral activity, in particular for use in the treatment of HIV infections.

Methods for the synthesis of 2-chloro-9- (2 '-deoxy-2' -fluoro-. beta. -D-arabinofuranosyl) adenine are described in J.org.chem.34, 2632-2636(1969), patent application WO 01/60383 and U.S. Pat. No. 6,680,382.

The pharmacological activity of 2-chloro-9- (2 '-deoxy-2' -fluoro- β -D-arabinofuranosyl) -adenine as an inhibitor of DNA replication has also been described in Hematology 463(1999) and compared to other nucleosides.

Other halogenated vidarabines with anticancer activity are mentioned in patent applications US 5,384,310 and WO 92/20347.

The antiviral activity of such purine derivatives is shown in EP 0314011.

It is well known that 2-chloro-9- (2 '-deoxy-2' -fluoro- β -D-arabinofuranosyl) adenine (Clofarabine) is a product developed in clinical trials.

The compounds of formula I of the present invention incorporating the chemical structure of 2-chloro-9- (2 '-deoxy-2' -fluoro-. beta. -D-arabinofuranosyl) adenine have biological activities different from that of the parent nucleoside. In particular, the compounds of the present invention exhibit antitumor activity and can be used in pharmacologically relevant doses against tumors, may reduce one or more toxic side effects of the parent compound, and/or the covalently bound lipid moiety improves the bioavailability of the conjugated drug and thus appears to enhance the selectivity and efficacy of the compound.

The compounds of the invention have valuable pharmacological properties. In particular, they are useful in the treatment and prevention of malignancies, including cancer, sarcoma or leukemia.

The compounds of the present invention have enhanced efficacy/efficacy or lower toxicity for specific indications compared to unconjugated nucleoside derivatives currently used for the treatment of malignancies, and thus have a broader therapeutic window. In some embodiments of the invention, pharmaceutical compositions comprising these compounds may be administered continuously over an extended period of time. The compounds of the invention reduce the incidence of drug withdrawal or intermittent dosing due to the often adverse side effects of chemotherapeutic agents, as compared to their parent compounds. In addition, higher doses may be used because of the increased selectivity for tumor cytotoxicity and reduced toxic side effects.

The lipid ester compounds of the present invention are also useful in the treatment of autoimmune diseases, including multiple sclerosis, rheumatoid arthritis, lupus, systemic vasculitis, inflammatory bowel disease, scleroderma, and Sjorgen's syndrome.

The lecithin-like structure of the lipid fraction is required for the claimed improvement of the compounds of general formula I. It contributes to transmembrane permeability and resorption barrier, and the formula I conjugates show a deposition effect in different tissues.

The formation of lipid conjugates also helps to cross the blood brain barrier due to their better diffusion or active transport processes.

Likewise, the compounds of the present invention and their pharmaceutical preparations can be used for the treatment and prevention of the above-mentioned diseases alone or in combination with other drugs.

Examples of such additional drugs include the following: mitotic inhibitors such as colchicine, vinblastine, alkylating cytostatics such as cyclophosphamide, melphalan, malignane or cisplatin, antimetabolites such as folate antagonists (methotrexate) and purine and pyrimidine base antagonists (mercaptopurine, 5-fluorouridine, cytarabine), cytostatic-active antibiotics such as anthracyclines (e.g., doxorubicin, daunorubicin), hormonal agents such as fosfestrol, tamoxifen, taxanes such as paclitaxel, and other cytostatic/cytotoxic-active chemotherapeutic and biologic agents.

Embodiments of the present invention also include salts of the compounds of formula I, including alkali metal, alkaline earth metal, and ammonium salts of the phosphate group thereof. Examples of the alkali metal salt include lithium salt, sodium salt and potassium salt. Alkaline earth metal salts include magnesium and calcium salts, ammonium salts being salts containing ammonium ions which may be substituted up to four times by alkyl and/or aryl residues having 1 to 4 carbon atoms, for example benzyl residues. In this case, the substituents may be the same or different.

The compounds of formula I may contain basic groups, in particular amino groups, which may be converted into acid addition salts by suitable inorganic or organic acids. Possible acids for this purpose are, in particular, hydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric acid, fumaric acid, succinic acid, tartaric acid, citric acid, lactic acid, maleic acid or methanesulfonic acid.

In the general formula I, R¹Preferably represents may be C₁-C₆Alkoxy or C₁-C₆Straight chain C further substituted by alkylthio₈-C₁₆An alkyl residue. More specifically, R¹Representing a nonyl, decyl, undecyl, dodecyl, tridecyl, tetradecyl or pentadecyl residue. Preferably methoxy, ethoxy, butoxy and hexyloxy are R¹Possible substituents for the residue. At R¹Quilt C₁-C₆In the case of substitution of the alkylthio residue, it is understood that the group is specifically a methylthio, ethylthio, propylthio, butylthio and hexylthio residue.

Preferably R²Represents may be C₁-C₆Alkoxy or C₁-C₆Straight chain C further substituted by alkylthio₈-C₁₅An alkyl group. More specifically, R²Represents octyl, nonyl, decyl, undecyl, dodecyl, tridecyl or tetradecyl. Preferably methoxy, ethoxy, propoxy, butoxy and hexyloxy are R²Preferred of (1) C₁-C₆An alkoxy substituent. At R²Quilt C₁-C₆In the case of substitution of the alkylthio residue, it is understood that the group is specifically a methylthio, ethylthio, propylthio, butylthio, pentylthio and hexylthio residue.

Examples of preferred lipid moieties are the following groups:

wherein

R¹Is C₁₂H₂₅，

R²Is C₁₀H₂₁，

X is S, SO or SO₂And are and

y is O.

The most preferred compounds are [ 2-chloro-9- (2 ' -deoxy-2 ' -fluoro-. beta. -D-arabinofuranosyl) adenine ] -5 ' -phosphate- (3-dodecylthio-2-decyloxy) propyl, [ 2-chloro-9- (2 ' -deoxy-2 ' -fluoro-. beta. -D-arabinofuranosyl) adenine ] -5 ' -phosphate- (3-dodecylsulfinyl-2-decyloxy) propyl, [ 2-chloro-9- (2 ' -deoxy-2 ' -fluoro-. beta. -D-arabinofuranosyl) adenine ] -5 ' -phosphate- (3-dodecylsulfonyl-2-decyloxy) propyl and [2- Chloro-9- (2 '-fluoro- β -D-arabinofuranosyl) -6-methoxy-9H-purine ] -5' -phosphoric acid- (3-dodecylthio-2-decyloxy) propyl ester, [ 2-chloro-9- (2 '-fluoro- β -D-arabinofuranosyl) -6-methoxy-9H-purine ] -5' -phosphoric acid- (3-dodecylsulfinyl-2-decyloxy) propyl ester and [ 2-chloro-9- (2 '-fluoro- β -D-arabinofuranosyl) -6-methoxy-9H-purine ] -5' -phosphoric acid- (3-dodecylsulfonyl-2-decyloxy) ) Propyl ester.

The compounds of formula I can be prepared as follows:

1. reacting a compound of the following general formula II or a salt form thereof with a compound of the following general formula III in the presence of a suitable acid chloride (e.g. 2, 4, 6-triisopropylbenzenesulfonyl chloride) and a tertiary nitrogen base (e.g. pyridine or lutidine) in an inert solvent such as toluene or directly in anhydrous pyridine:

wherein R is¹、R²X and Y have the meanings indicated,

wherein R is³Is amino OR OR⁴Wherein R is⁴Is C₁-C₈The alkyl, 3' -hydroxy group may optionally be protected by an oxygen protecting group familiar to the skilled person, and the compound of formula II may be activated, optionally followed by subsequent conventional methods according to nucleoside chemistryBy removing the oxygen protecting group by hydrolysis when R is³In the case of amino in the compounds of the formula I, the OR in the 6-position of the purine is optionally substituted⁴The group is converted into an amino group,

or

Reacting an aliphatic alcohol (corresponding to formula II) with nucleoside-5' -monophosphate (corresponding to formula III) in the same manner as above, or

2. Reacting a compound of formula IV below with a compound of formula III in the presence of phospholipase D from streptomyces in an inert solvent such as chloroform in the presence of a suitable buffer:

wherein R is¹、R²X and Y have the abovementioned meanings,

wherein R is³Is amino OR OR⁴Wherein R is⁴Is C₁-C₈The alkyl, 3' -hydroxy group may optionally be protected by an oxygen protecting group familiar to the skilled person, optionally followed by reaction to remove the oxygen protecting group according to conventional methods of nucleoside chemistry, when R is³In the case of amino in the compounds of the formula I, the OR in the 6-position of the purine is optionally substituted⁴The group is converted to an amino group.

The compounds of formula II and formula IV are prepared in analogy to the methods described in Lipids 22, 947(1987) and J.Med.chem.34, 1377 (1991). The compounds of the formula III are prepared analogously to the methods described in J.org.chem.34, 2632-2636(1969), J.Med.chem.35, 397-401(1992) or WO 01/60383, with the proviso that R is prepared in two steps³Is amino or R³＝OR⁴. The first step involves the preparation of 2, 6-dichloro-9- (3 ', 5' -O-dibenzoyl-2 '-deoxy-2' -fluoro-. beta. -D-arabinofuranosyl) -9H-purine by reacting 2, 6-dichloropurine with a protected 2, 6-dichloropurine in the presence of a hindered potassium base, preferably potassium tert-butoxide or potassium tert-valerate, in a suitable solvent-deoxy-2-fluoro-alpha-D-arabinofuranosyl halide. Suitable protecting groups include benzoyl and acetyl. Suitable halides include bromine and chlorine. Suitable inert solvents include, but are not limited to, t-butanol, acetonitrile, dichloromethane, dichloroethane, t-amyl alcohol, tetrahydrofuran, or mixtures thereof. Preferred solvents include a mixture of acetonitrile, t-butanol and 1, 2-dichloroethane. Calcium hydride may optionally be added to the reaction mixture. The second step involves subjecting the 2, 6-dichloropurine nucleoside derivative to deprotection and aromatic nucleophilic substitution conditions, such as sodium hydroxide and C₁-C₈Alcohol or C₁-C₈Sodium alcoholate in corresponding C₁-C₈Alcohols (e.g., methanol with sodium methoxide, ethanol with sodium ethoxide, etc.) or other suitable non-alcoholic solvents to obtain the desired C of formula III₁-C₈6-alkoxy purine nucleoside compounds.

Compounds of formula I wherein X ═ sulfinyl or sulfonyl can be prepared as follows: reacting the corresponding compounds of the formula I in which X is sulfur with, for example, H₂O₂Acetic acid, or using a suitable compound of formula II or IV as starting material.

Other compounds of the invention are diphosphates of formula V, wherein n ═ 2, R¹、R²、R³X and Y have the same meanings as in formula I.

They can be prepared by reacting lipid phosphates (corresponding to formula II) with nucleoside-5' -monophosphates (prepared from nucleosides corresponding to formula III).

The lipid phosphate may be activated beforehand according to methods familiar to the skilled person.

Salts of the compounds of formula I are prepared by reacting the free acid with hydroxides, alkoxides or acetates of alkali or alkaline earth metals.

Can pass through non-pairsIsolation of the enantiomeric salts, or by optically active C starting from formula II₃Enantioselective synthesis of the lipid residue of the precursor, preparing the "enantiomer" of the lipid moiety of the compound of formula I.

Medicaments containing compounds of formula I for the treatment of cancer may be administered orally or parenterally in liquid or solid form. The usual administration forms can be, for example, tablets, capsules, coated tablets, syrups, solutions or suspensions.

Water is preferably used as an injection medium containing additives such as stabilizers, solubilizers and buffers, which are generally contained in injection solutions. Such additives are e.g. tartaric and citric acid buffers, ethanol, complexing agents such as ethylenediaminetetraacetic acid and its non-toxic salts, high molecular polymers such as liquid polyethylene oxide for viscosity control. Liquid vehicles for injection must be sterile and are preferably packaged in ampoules.

Solid carriers are, for example, starch, lactose, mannitol, methylcellulose, talc, highly disperse silicic acid, high molecular fatty acids, such as stearic acid, gelatin, agar-agar, calcium phosphate, magnesium stearate, animal and vegetable fats, solid high molecular polymers, such as polyethylene glycol, and the like. Formulations suitable for oral administration may include flavoring or sweetening agents, as necessary. The dosage depends on various factors such as the administration mode, species, age or individual condition.

The compounds of the invention may be adapted for oral or intravenous (i.v.) administration in an amount in the range of 0.1-100mg/kg body weight/day, preferably 0.2-80mg/kg body weight/day. In certain dosage regimens, the daily amount is divided into 2-5 administrations, each administration having an active ingredient content in the tablet ranging from 0.5-500 mg.

Also, the tablets may be sustained release to reduce the number of administrations, e.g. to 1-3 administrations per day. The active ingredient content of the sustained-release tablet can be in the range of 2-1000 mg. The active ingredient may also be administered by i.v. bolus injection or continuous infusion, in an amount generally in the range of 5-1000mg per day being sufficient.

In addition to the compounds mentioned in the examples, the following compounds of the formula I and their pharmacologically acceptable salts are further examples of compounds of the invention:

[ 2-chloro-9- (2 ' -deoxy-2 ' -fluoro- β -D-arabinofuranosyl) adenine ] -5 ' -phosphoric acid- (3-dodecylthio-2-decyloxy) propyl ester

[ 2-chloro-9- (2 ' -deoxy-2 ' -fluoro- β -D-arabinofuranosyl) adenine ] -5 ' -phosphoric acid- (3-dodecylsulfinyl-2-decyloxy) propyl ester

3- [ 2-chloro-9- (2 ' -deoxy-2 ' -fluoro- β -D-arabinofuranosyl) adenine ] -5 ' -phosphoric acid- (3-dodecylsulfonyl-2-decyloxy) propyl ester

[ 2-chloro-9- (2 ' -deoxy-2 ' -fluoro- β -D-arabinofuranosyl) adenine ] -5 ' -phosphoric acid- (3-undecylthio-2-decyloxy) propyl ester

[ 2-chloro-9- (2 ' -deoxy-2 ' -fluoro- β -D-arabinofuranosyl) adenine ] -5 ' -phosphoric acid- (3-undecylthio-2-undecyloxy) propyl ester

[ 2-chloro-9- (2 ' -deoxy-2 ' -fluoro-. beta. -D-arabinofuranosyl) adenine ] -5 ' -phosphate- (3-decylthio-2-dodecyloxy) propyl ester

[ 2-chloro-9- (2 ' -deoxy-2 ' -fluoro- β -D-arabinofuranosyl) adenine ] -5 ' -phosphoric acid- (3-dodecylthio-2-dodecyloxy) propyl ester

[ 2-chloro-9- (2 ' -deoxy-2 ' -fluoro- β -D-arabinofuranosyl) adenine ] -5 ' -phosphate- (3-decylthio-2-decyloxy) propyl ester

[ 2-chloro-9- (2 ' -deoxy-2 ' -fluoro- β -D-arabinofuranosyl) adenine ] -5 ' -phosphoric acid- (3-undecylsulfinyl-2-decyloxy) propyl ester

[ 2-chloro-9- (2 ' -deoxy-2 ' -fluoro- β -D-arabinofuranosyl) adenine ] -5 ' -phosphoric acid- (3-undecylsulfonyl-2-decyloxy) propyl ester

[ 2-chloro-9- (2 ' -deoxy-2 ' -fluoro-. beta. -D-arabinofuranosyl) adenine ] -5 ' -phosphoric acid- (3-undecylsulfinyl-2-undecyloxy) propyl ester

[ 2-chloro-9- (2 ' -deoxy-2 ' -fluoro- β -D-arabinofuranosyl) adenine ] -5 ' -phosphoric acid- (3-undecylsulfonyl-2-undecyloxy) propyl ester

[ 2-chloro-9- (2 ' -deoxy-2 ' -fluoro- β -D-arabinofuranosyl) adenine ] -5 ' -phosphoric acid- (3-tridecylthio-2-undecyloxy) propyl ester

[ 2-chloro-9- (2 ' -deoxy-2 ' -fluoro- β -D-arabinofuranosyl) adenine ] -5 ' -phosphoric acid- (3-tridecylthio-2-decyloxy) propyl ester

[ 2-chloro-9- (2 ' -deoxy-2 ' -fluoro-. beta. -D-arabinofuranosyl) adenine ] -5 ' -phosphoric acid- (3-tridecylsulfinyl-2-decyloxy) propyl ester.

Furthermore, the present invention includes analogs of the above exemplified compounds wherein the substituent at the 6-position of the purine is C₁-C₈Alkoxy, preferably methoxy. The compounds also have good pharmaceutical properties and are furthermore useful as intermediates for the preparation of the above exemplified compounds.

Example 1

Preparation of 2-chloro-6-methoxy-9- (2 '-deoxy-2' -fluoro-beta-D-arabinofuranosyl) -9H-purine

The first step is the preparation of 2, 6-dichloro-9- α -D- (3 ', 5' -O-dibenzoyl-2 '-deoxy-2' -fluoro- β -D-arabinofuranosyl) -9H-purine according to the following scheme:

initially, a 1000ml flask was charged with 2, 6-dichloropurine (12.65g, 66.9mmol), calcium hydride (2.43g, 57.7mmol) and acetonitrile (150ml) and stirred. In 5 pointsPotassium tert-butoxide solution (60.6ml, 60.6mmol, 1.0M in tert-butanol) was added over a period of time to give a viscous but still stirrable suspension. A solution of 3, 5-O-dibenzoyl-2-deoxy-2-fluoro-. alpha. -D-arabinofuranosyl bromide (26.88g, 63.5mmol) in 1, 2-dichloroethane (200ml) was added at ambient temperature over 45 minutes. After the addition was complete, the resulting mixture was stirred at ambient temperature for 16 hours. The mixture was filtered through celite, the flask and the solid were washed with acetonitrile (100 ml). Rotary evaporation to remove volatiles gave a yellow gum (38.1 g). Ethyl acetate (100ml) was added to check the pH at 8. Acetic acid (0.5ml) was added and the pH was checked again for 4. The resulting cloudy solution was filtered through Whatman541 filter paper. The flask and filter were washed with ethyl acetate (100 ml). No clarification of the solution was observed. The organic layer was washed with water (100ml) and then brine (100 ml). The organic layer was dried (MgSO₄) And concentrated by rotary evaporation and then by high vacuum pump to give a white foam (34.0 g). The resulting crude material was purified by column chromatography (silica gel 60, 230-400 mesh, diameter 14cm, height 14.5cm, 2232 ml). Fractions containing the purest product were concentrated by rotary evaporation, homogenized twice in methanol, filtered and washed with methanol to give a white solid (13.4g, 92.6% AUC) using a hexane/ethyl acetate gradient. Fractions of lower purity were combined, concentrated by rotary evaporation and re-purified by column chromatography to give a white solid (3.85g, 94.7% AUC). The overall recovery was 17.3g (56%). A portion of this was re-homogenized in methanol for characterization (97.9% AUC).

mp＝157-159℃.¹H NMR(DMSO-d₆)δ8.84(d，1H，J＝2.82Hz，H₈)，8.14-8.00(m，4H，Bz)，7.76-7.50(m，6H，Bz)，6.81(dd，1H，J＝18.2，3.9Hz，H_1’)，5.95(m，2H，H_3’)，5.91(dm，1H，J＝75.4Hz，H_2’)，4.84-4.79(m，3H，H_4’and H_5’).¹³C NMR(DMSO-d₆)165.4，164.8，152.7，151.6，150.3，146.7(d，J_(CF)＝4Hz)，133.9，133.4，130.3，129.6，129.2，128.7，128.6，128.5，92.9(d，J_(CF)＝192Hz)，82.8(d，J_(CF)＝16Hz)，78.9，76.2(d，J_(CF)＝28Hz)，63.7ppm.¹⁹F NMR(DMSO-d₆)-197.6(dt，J＝50，19Hz)ppm.IR(KBr)3431，3139，3063，2966，1726，1596，1272，1091，714cm^-1.UV(H₂O/MeCN)λmax₁ 214nm(0.94AU)，λmax₂ 231nm(0.90AU)，λmax₃ 273nm(0.37AU).

Mass spectrum (electrospray, positive) M/e [ M + H ]]⁺＝531。C₂₄H₁₇Cl₂FN₄O₅Calculated elemental analysis of (a): c, 54.25; h, 3.22; cl, 13.35; f, 3.58; n, 10.54. Measured value: c, 54.19; h, 3.11; cl, 13.20; f, 3.49; n, 10.52.

The second step was the preparation of 2-chloro-6-methoxy-9- (2 '-deoxy-2' -fluoro- β -D-arabinofuranosyl) -9H-purine according to the following scheme:

a500 ml flask was charged with protected 2, 6-dichloro-9-. alpha. -D- (3 ', 5' -O-dibenzoyl-2 '-deoxy-2' -fluoro-. beta. -D-arabinofuranosyl) -9H-purine (13.33g, 25.1mmol) and methanol (300 ml). The pH was adjusted to 9.5 with NaOH solution (2ml, 1.0N in water). The resulting suspension was stirred at ambient temperature for 16.5 hours. The pH was checked to be 5.5. More NaOH solution (11.3ml) was added (pH 11) and the resulting mixture was stirred at ambient temperature for 1.5 h. The pH was checked to be 6. TLC (10% EtOH/90% CH)₂Cl₂，UV₂₅₄) Shows 3 points, R_f0.28, 0.72 and 0.88, respectively. More NaOH solution (13.3ml) was added (pH 11). After stirring at ambient temperature for 5 minutes, the reaction mixture became a colorless clear solution which was judged to be complete by TLC after stirring for an additional 2.5 hours. Acetic acid (0.8ml) was added and the base (pH. apprxeq.5) was neutralized. Rotary evaporation gave a biphasic residue. Isopropanol (100ml) was added to give a white suspension. Removal by azeotropic rotary evaporationAnd (3) water. This step was repeated twice with more isopropanol (100 ml). When about 50ml remained in the flask, the rotary evaporation was stopped, the resulting suspension was filtered, and the flask and filter cake were washed with the filtrate, then with isopropanol (10 ml). The resulting solid was dried (50 ℃, 27torr, 16.5 hours). The weight of the solid was 5.58g (92.4% AUC). The filtrate was concentrated by rotary evaporation and then concentrated by a high vacuum pump. The residue weight was 6.79g (70.9% AUC). The resulting solid and residue were purified separately by column chromatography (silica gel 60, 230-400 mesh, 10% ethanol, 90% dichloromethane). The weight of the purified product from the crude solid was 4.62g (95.5% AUC). The weight of the purified product from the residue was 1.69g (98.1% AUC). The overall recovery was 6.31g (79%).

mp＝197-201℃.¹H NMR(DMSO-d₆)δ8.59(d，1H，J＝1.9Hz，H₈)，6.47(dd，1H，J＝12.8，4.9Hz，H_1’)，6.02(d，1H，J＝5.4Hz，3’-OH)，5.31(dt，1H，J＝52.5，4.5Hz，H_2’)，5.15(t，1H，J＝5.7Hz，5’-OH)，4.47，ddd，1H，J＝19.1，9.9，5.3Hz，H_3’)，4.13(s，1H，MeO)，3.90(dd，1H，J＝9.7，4.7Hz，H_4’)，3.75-3.64(m，2H，H_5’).¹³C NMR(DMSO-d₆)160.9，152.6，151.8，143.0，119.7，95.3(d，J_(CF)＝193Hz)，83.6(d，J_(CF)＝7Hz)，81.8(d，J_(CF)＝17Hz)，72.3(d，J_(CF)＝23Hz)，60.2，55.1ppm.¹⁹FNMR(DMSO-d₆)-199.1(ddd，J＝53，19，13Hz)ppm.IR(KBr)3438，3235，3113，2916，1599，1471，1389，1320，1238，1045，925，691cm^-1.UV(H₂O/MeCN)λmax₁ 210nm(1.00AU)，λmax₂ 257nm(0.67AU).

Mass spectrum (electrospray, positive) M/e [ M + H ]]⁺＝319。C₁₁H₁₂ClFN₄O₄Analytical calculation of (a): c, 41.46; h, 3.80; cl, 11.12; f, 5.96; n, 17.58. Measured value: c, performing a chemical reaction on the mixture to obtain a reaction product,41.70；H，3.36；Cl，11.12；F，5.75；N，17.54。

example 2

Preparation of [ 2-chloro-9- (2 ' -deoxy-2 ' -fluoro-. beta. -D-arabinofuranosyl) adenine ] -5 ' -phosphoric acid- (3-dodecylthio-2-decyloxy) propyl ester

The first step is the preparation of [ 2-chloro-9- (2 '-fluoro- β -D-arabinofuranosyl) -6-methoxy-9H-purine ] -5' -phosphoric acid- (3-dodecylthio-2-decyloxy) propyl ester according to the following scheme:

3.12g of 3- (3-dodecylthio-2-decyloxy) propyl phosphate are treated twice with 60ml of anhydrous pyridine and then concentrated by evaporation. The residue obtained is dissolved in 60ml of anhydrous pyridine at room temperature, treated with 3,80g of 2, 4, 6-triisopropylbenzenesulfonyl chloride (trisyl chloride) under nitrogen and stirred for 2 hours at 20 ℃. 2.00g of 2-chloro-9- (2 '-deoxy-2' -fluoro-. beta. -D-arabinofuranosyl) -6-methoxy-9H-purine were then immediately added and the resulting mass was stirred under nitrogen for 16 hours. The hydrolysis was carried out by adding 10ml of water, the resulting mixture was stirred at room temperature for a further 0.5 h, the solvent was removed in vacuo and then extracted twice with 20ml of toluene. The residue was stirred in tert-butyl methyl ether (160ml) at 40 ℃ for 0.5 h. After cooling to room temperature, the resulting pyridinium sulfonate salt was filtered off. The filtrate was washed twice with 40ml of 2N hydrochloric acid and then evaporated to dryness. The remaining 7.38g of slurry was used in the next step without further purification.

Samples of the above starting material were purified by column chromatography on Lichrospher 60 RPSelect B using methanol/40 mM aqueous sodium acetate (90: 10) as eluent. Fractions containing the product were evaporated and the resulting residue partitioned between 50ml of tert-butyl methyl ether and 10ml of 2N hydrochloric acid. The organic layer was evaporated and the residue was dissolved in a mixture of 5ml toluene and 5ml methanol. By addingSodium methoxide was added and the pH was adjusted to 7. The solvent was removed and the residue was dried in vacuo. To obtain [ 2-chloro-9- (2' -fluoro-beta-D-arabinofuranosyl) -6-methoxy-9H-purine]-5' -phosphoric acid- (3-dodecylthio-2-decyloxy) propyl ester sodium salt, as an amorphous solid, which is specific optically active [ alpha ] when melted at 65-75 ℃]₂₀ ^Hg436+31.9(c ═ 1.0, methanol solution).

¹H NMR(300MHz，DMSO-d₆)：8.5(s，1H，H₈)，6.6，(s(br)，1H，3’-OH)，6.4(dd，1H，H_1’)，5.3(dt，1H，H_2’)，4.4，(dt，1H，H_3’)，4.1(s，3H，OCH₃)，3.9-4.0，(m，3H，H_4’，POCH₂)，3.6，(m，1H，H_5’a)，3.6(m，1H，H_5’b)，3.3-3.4(m，3H，＞CHOCH₂-)，2.5-2.6(m，4H，CH₂SCH₂)，1.1-1.5(m，36H，-(CH₂)₉-，-(CH₂)₇-)，0.8(m，6H，CH₂-CH₃)；³J_{1’- H，2’-H}≈³J_{2’-H，3’-H}≈³J_{3’-H，4’-H}≈4.7Hz，³J_1’-H，F＝12.1Hz，²J_2’-H，F＝52.8Hz，³J_3’-H，F＝19.0Hz.

¹³C-NMR(75,0MHz，DMSO-d₆)：160.8，152.6，151.7(C-2，C-4，C-6)，142.9，(C-8)，119.6，(C-5)，94.9，(C-2’)，82.2，(C-4’)，81.6，(C-1’)，78.7，(O-CH＜)，73.7，(C-3’)，69.1，(CH₂-CH₂O-CH＜)，64.8，(C-5’)，63.4，(5’-O-P(O)₂OCH₂)，55.0，(6-CH₃)，32.1，32.3，(-CH₂SCH₂-)，20.0-31.2(-(CH₂)₉-，-(CH₂)₇-)，13.9，(2xCH₃)

³¹P NMR(121,5MHz，DMSO-d₆)：-0.46ppm

19F NMR(282MHz，DMSO-d₆)：-198.7ppm.

UV (methanol) lambda_max1 205.3nm，λ_max2 255.9nm，

Na⁺]，

Mass Spectrometry (FAB)^-)：m/z＝795[M-Na⁺]。

The second step is ammonolysis of crude [ 2-chloro-9- (2 ' -fluoro- β -D-arabinofuranosyl) -6-methoxy-9H-purine ] -5 ' -phosphate- (3-dodecylthio-2-decyloxy) propyl ester to give [ 2-chloro-9- (2 ' -deoxy-2 ' -fluoro- β -D-arabinofuranosyl) adenine ] -5 ' -phosphate- (3-dodecylthio-2-decyloxy) propyl ester:

this ammonolysis step was carried out in a stainless steel reaction kettle at 80 ℃.

The crude material (7.38g) was dissolved in 30ml of 7M NH₃In an ethanol solution (saturated at-5 ℃). After heating for 20 hours, [ 2-chloro-9- (2' -fluoro-. beta. -D-arabinofuranosyl) -6-methoxy-9H-purine was not detected]-5' -phosphoric acid- (3-dodecylthio-2-decyloxy) propyl ester methoxy derivative reactant. The resulting product was purified by column chromatography on Lichrospher 60 RPSelect B using methanol/40 mM aqueous sodium acetate (85: 15) as eluent. Fractions containing product were evaporated. The residue obtained is partitioned between 100ml of tert-butyl methyl ether and 50ml of 2N hydrochloric acid. The organic layer was evaporated, the residue was dissolved in 30ml of methanol mixture and the pH was adjusted to pH7 by addition of sodium methoxide (30% methanol solution). The solvent was removed and the residual sodium salt was dried in vacuo. The product (2.90g) was obtained in a total yield of 57% based on the conversion of 2-chloro-9- (2 '-deoxy-2' -fluoro-. beta. -D-arabinofuranosyl) -6-methoxy-9H-purine. The purity was determined by HPLC to be 93.6% (area). Melting point: 130 ℃ and 131 ℃.

MS(FAB^-)：m/z＝780

[ M-Na + ], UV (. lamda.max) 263.4nm.

¹H NMR(300MHz，DMSO-d₆)：8.2(s，1H，H₈)，7.7，(s(br)，1H，NH₂)，6,5，(s(br)，1H，3’-OH)，6.2(dd，1H，H_1’)，5.2(dt，1H，H_2’)，4.4，(dt，1H，Hz，H_3’)，3.8-4.0，(m，3H，H_4’，POCH₂)，3.6，(m，1H，-H_5a’)，3.6(m，1H，H_5’b)，3.3-3.5(m，3H，＞CHOCH₂-)，2.5-2.7(m，4H，CH₂SCH₂)，1.1-1.4(m，36H，-(CH₂)₉-，-(CH₂)₇-)，0.8(m，6H，CH₂-CH₃)；³J_{1’-H，2’-H}≈³J_{2’-H，3’-H}≈³J_{3’-H，4’-H}≈4.2Hz，³J_1’-H，F＝14.1Hz，²J_2’-H，F＝54Hz，³J_{3’- H，F}＝19.0Hz.

¹³C NMR(75,0MHz，DMSO-d₆)：156.8，153.3，150.1(C-2，C-4，C-6)，139.8，(C-8)，117.3，(C-5)，95.0，(C-2’)，81.8，(C-4’)，81.2，(C-1’)，78.8，(O-CH＜)，72.9，(C-3’)，69.1，(CH₂-CH₂O-CH＜)，64.8，(C-5’)，64.4，(5’-O-P(O)₂OCH₂)，32.1，31.3，(-CH₂SCH₂-)，22.1-29.7(-(CH₂)₉-，-(CH₂)₇-)，13.9，(2xCH₃)

³¹P NMR(121,5MHz，DMSO-d₆)：-0.48ppm

¹⁹F NMR(282MHz，，DMSO-d₆)：-198.7ppm.

Example 3

Method for preparing [ 2-chloro-9- (2 ' -deoxy-2 ' -fluoro-beta-D-arabinofuranosyl) adenine ] -5 ' -phosphoric acid- (3-dodecylthio-2-decyloxy) propyl ester from 2-chloro-9- (2 ' -deoxy-2 ' -fluoro-arabinofuranosyl) adenine

0.91g of 3-dodecylthio-2-decyloxy propyl phosphate are treated twice with 20ml of anhydrous pyridine and concentrated by evaporation. The residue obtained is dissolved in 20ml of anhydrous pyridine at room temperature, treated under nitrogen with 1.07g of 2, 4, 6-triisopropylbenzenesulfonyl chloride and stirred at 25 ℃ for 0.5 h. Then 0.5g of 2-chloro-9- (2 '-deoxy-2' -fluoro-arabinofuranosyl) adenine was immediately added and the resulting mass was allowed to stand under nitrogen for 20 hours. 5ml of water were added for hydrolysis, the mixture was stirred at room temperature for a further 0.3 h, the solvent was removed in vacuo and then extracted twice with 50ml of toluene. The residue was purified by column chromatography on Lichrospher 60 RPSelect B using methanol/40 mM aqueous sodium acetate (88: 12) as eluent. Fractions containing product were evaporated. The residue obtained is partitioned between 50ml of tert-butyl methyl ether and 10ml of 2N hydrochloric acid. The organic layer was evaporated and the residue was dissolved in a mixture of 5ml toluene and 5ml methanol. The pH was adjusted to pH7 by the addition of sodium methoxide. The solvent was removed and the residue was dried in vacuo.

The yield was 0.82g (62%) of a white powder.

The preparation of 3-dodecylthio-2-decyloxy propyl phosphate was carried out according to the method described in WO 92/03462.

Example 4

Preparation of [ 2-chloro-9- (2 ' -deoxy-2 ' -fluoro-. beta. -D-arabinofuranosyl) adenine ] -5 ' -diphosphate, (3-dodecylthio-2-dodecyloxy) propyl ester

The first step is the preparation of 2-chloro-6-methoxy-9- (2 ' -deoxy-2 ' -fluoro-5 ' -O-phosphate- β -D-arabinofuranosyl) -9H-purine from 2-chloro-6-methoxy-9- (2 ' -deoxy-2 ' -fluoro- β -D-arabinofuranosyl) -9H-purine:

the flask was charged with 2-chloro-6-methoxy-9- (2 '-deoxy-2' -fluoro- β -D-arabinofuranosyl) purine and triethyl phosphate (e.g., 2.3 ml/mmol nucleoside) under nitrogen. The resulting mixture is cooled (e.g., -25 deg.C) and POCl is added₃(e.g., 3 equivalents). Immediately after warming to ambient temperature, the mixture is stirred (e.g., for 3 hours). Ice (e.g., 1.4 g/mmol nucleoside) and water (e.g., 8.7 ml/mol nucleoside) are then added with stirring, and the resulting mixture is transferred to a separatory funnel. MTBE (e.g., 4.4 ml/mole nucleoside) was added, and the phases were separated after stirring. The organic phase is washed twice with water (e.g., 8.7 ml/mmol of nucleoside). The combined aqueous extracts are acidified with NaOH (e.g., 50% aqueous solution) to about pH2 and then stirred with activated charcoal (e.g., 5.7 g/mmol nucleoside) for a suitable period of time (e.g., 2 hours). The resulting mixture was filtered and the filtrate was discarded. Activated carbon is stirred with a mixture of MeOH (e.g., 4.4 ml/mmol nucleoside), ammonium hydroxide (concentrated) (e.g., 44 ml/mmol nucleoside), and water (e.g., 3.9 ml/mmol nucleoside) for a suitable period of time (e.g., 30 minutes), then filtered. This step is repeated (e.g. 5 times) and the filtrates obtained are combined. The combined filtrates were evaporated to give crude 2-chloro-6-methoxy-9- (2 ' -deoxy-2 ' -fluoro-5 ' -O-phosphate- β -D-arabinofuranosyl) -9H-purine. It is dissolved in water (e.g., 8.7 ml/mmol nucleoside) and treated with Dowex50WX8-100 (e.g., 4 g/mmol nucleoside) cationic resin under agitation for a suitable period of time (e.g., 30 minutes). The mixture is filtered, the resin is stirred with water (e.g. 9 ml/mmol nucleoside) and filtered. The resin is extracted with water (e.g. 4 times) and the combined aqueous filtrates are evaporated to give 2-chloro-6-methoxy-9- (2 '-deoxy-2' -fluoro-. beta. -D-arabinofuranosyl) -9H-purine (e.g. in 30-100% yield).

The second step is the ammonolysis of 2-chloro-6-methoxy-9- (2 ' -deoxy-2 ' -fluoro- β -D-arabinofuranosyl) -9H-purine to give 2-chloro-9- (2 ' -deoxy-2 ' -fluoro-5 ' -O-phosphate- β -D-arabinofuranosyl) adenine:

in a pressure vessel, 2-chloro-6-methoxy-9- (2 '-deoxy-2' -fluoro-. beta. -D-arabinofuranosyl) purine is dissolved in anhydrous ethanol under nitrogen cooling (e.g., -5 ℃). Ammonia gas was bubbled through the solution until a saturated solution was obtained. The system is then heated (e.g., to 80 ℃) for a suitable period of time (e.g., > 20 hours). The progress of the reaction was monitored by sampling and HPLC analysis. Upon completion of the reaction, the solvent was evaporated immediately to give crude 2-chloro-9- (2 ' -deoxy-2 ' -fluoro-5 ' - -O-phosphate- β -D-arabinofuranosyl) adenine.

The third step is to produce (2-chloro-9- (2 ' -deoxy-2 ' -fluoro-. beta. -D-arabinofuranosyl) adenine-5 ' -diphosphate, (3-dodecylthio-2-dodecyloxy) propyl ester, namely, by reacting morpholin-4-ylphosphonic acid mono- (3-dodecylthio-2-decyloxy) -1-propyl ester (morpholino of phosphoric mono- (3-didecyllmercapto-2-decyloxy) -1-propyl ester) with 2-chloro-9- (2 ' -deoxy-2 ' -fluoro-5 ' -O-phosphate- β -D-arabinofuranosyl) adenine:

mono- (3-dodecylthio-2-decyloxy) -1-propyl morpholin-4-ylphosphonate was prepared analogously to Bioorg.Med.chem., 7, 1195-propanole 1200, (1999), with mono- (3-dodecylthio-2-decyloxy) -1-propyl phosphate and morphiline dissolved in a mixture of water and tert-butanol (e.g., in a volume ratio of 1: 1). To this solution was added a solution of Dicyclohexylcarbodiimide (DCC) in t-butanol (e.g., approximately 4 molar excess of DCC relative to mono (3-dodecylthio-2-decyloxy) -1-propyl phosphate) and the reaction was refluxed (e.g., for 3.5 hours). The volume was reduced by evaporation and the mixture was then cooled to precipitate the morpholin-4-ylphosphonate.

The morpholin-4-ylphosphonate (e.g. 1.13mol/mol adenosine derivative) was prepared under nitrogen by adding anhydrous pyridine (e.g. 23 ml/mmol adenosine derivative) and 2-chloro-9- (2 ' -deoxy-2 ' -fluoro-5 ' -O-phosphate- β -D-arabinofuranosyl) adenine with stirring. The resulting mixture is stirred (e.g. at 40 ℃ for at least 16 hours) and then water (e.g. 4.5 ml/mmol adenosine derivative) is added and stirring is continued (e.g. for 1 hour). The solvent is evaporated and the product is purified by chromatography (e.g. on silica gel, using CHCl)₃MeOH and NH₄Elution of the OH (aqueous) mixture) to give (2-chloro-9- (2 ' -deoxy-2 ' -fluoro-. beta. -D-arabinofuranosyl) adenine-5 ' -diphosphate, [ 3-dodecylthio-2-dodecyloxy ] adenine]Propyl ester as a white solid (e.g., in 20-100% yield).

Example 5

Tablet formulation

1.50kg of the sodium salt of [ 2-chloro-9- (2 ' -deoxy-2 ' -fluoro-. beta. -D-arabinofuranosyl) adenine ] -5 ' -phosphoric acid- (3-dodecylthio-2-decyloxy) propyl ester,

1.42kg of microcrystalline cellulose was added,

1.84kg of lactose was added to the milk,

0.04kg of polyvinylpyrrolidone and

0.20kg magnesium stearate

Mixed in dry form, moistened with water and granulated. After drying, the mass is compressed into tablets weighing 500 mg.

Example 6

Injectable preparation

10.0kg of the sodium salt of [ 2-chloro-9- (2 ' -deoxy-2 ' -fluoro-. beta. -D-arabinofuranosyl) adenine ] -5 ' -phosphoric acid- (3-dodecylthio-2-decyloxy) propyl ester was dissolved in 500ml of sodium chloride physiological saline, and was dispensed in 5ml ampoules and sterilized. The solution can be used for intravenous injection.

Example 7

In vivo antitumor Activity of 2-chloro-9- (2 ' -deoxy-2 ' -fluoro- β -D-arabinofuranosyl) adenine ] -5 ' -phospho- (3-dodecylthio-2-decyloxy) propyl ("nucleotidic conjugates") and 2-chloro-9- (2 ' -deoxy-2 ' -fluoro- β -D-arabinofuranosyl) adenine ("nucleosides") in the human Colon cancer xenograft model (HCT-15)

The antitumor activity of the nucleotide conjugates and their corresponding nucleosides was compared in a human colon cancer xenograft HCT-15 model in nude mice.

Tumor-bearing mice were randomly assigned to each treatment group of 9 mice per group on day 7 after inoculation with HCT-15 tumor cells. Treatment was started on day 8. The nucleotide conjugates or nucleosides are administered intraperitoneally (ip) to the animals once a day for 5 consecutive days. The doses included 50% and 25% of the Maximum Tolerated Dose (MTD). Control animals were injected with the corresponding solvent (vehicle 1 or 2). On day 28, primary tumors were removed and tumor weights were determined. Median tumor weights are shown in table 1.

TABLE 1

Compound (I)	MTD	Dosage (mg/kg/injection)	Tumor weight (mg)	Tumor inhibition (%)
					Control I (untreated)	-	-	592
Control II (vehicle 1)	-	0	572
					Nucleosides*	25％	20	431	25％
Nucleosides*	50％	40	329	42％
					Control III (vehicle 2)	-	0	669
Nucleotide conjugates**	25％	63	257	62％
					Nucleotide conjugates**	50％	125	16	98％

^*2-chloro-9- (2 '-deoxy-2' -fluoro-beta-D-arabinofuranosyl) adenine

^**[ 2-chloro-9- (2 '-deoxy-2' -fluoro- β -D-arabinofuranosyl) adenine]-5' -phosphoric acid- (3-dodecylthio-2-decyloxy) propyl ester-example 2 or 3

At both doses, the antitumor efficacy of the nucleotide conjugates was significantly higher than that of the corresponding nucleosides (p < 0.01).

Claims (19)

1. A nucleotide derivative of the formula I, its tautomers, its optically active forms and racemic mixtures and physiologically acceptable salts of inorganic and organic acids or bases,

wherein

R¹Selected from the group consisting of straight or branched, saturated or unsaturated alkyl chains having from 1 to 20 carbon atoms, said alkyl chains being unsubstituted or substituted byAt least one substitution: halogen, C₁-C₆Alkoxy radical, C₁-C₆Alkylthio radical, C₁-C₆Alkoxycarbonyl group, C₁-C₆Alkylsulfinyl or C₁-C₆An alkylsulfonyl group;

R²selected from hydrogen, straight or branched, saturated or unsaturated alkyl chains having from 1 to 20 carbon atoms, said alkyl chains being unsubstituted or substituted at least once by: halogen, C₁-C₆Alkoxy radical, C₁-C₆Alkylthio radical, C₁-C₆Alkoxycarbonyl or C₁-C₆An alkylsulfonyl group;

R³is amino OR OR⁴Wherein R is⁴Is C₁-C₈An alkyl group;

x is selected from the group consisting of a sulfur atom, a sulfinyl group and a sulfonyl group;

y is oxygen;

wherein when R is³In the case of amino, the amino group may be unsubstituted or substituted with a known amino protecting group.

2. The nucleotide derivative of claim 1, wherein R¹Is straight chain C₈-C₁₅Alkyl, said alkyl being unsubstituted or substituted by C₁-C₆Alkoxy or C₁-C₆Alkylthio groups.

3. The nucleotide derivative of claim 1, wherein R²Represents a straight chain C₈-C₁₅Alkyl, said alkyl being unsubstituted or substituted by C₁-C₆Alkoxy or C₁-C₆Alkylthio groups.

4. The nucleotide derivative of any one of claims 1-3, wherein R³Is OCH₃。

5. The nucleotide derivative of any one of claims 1-4, wherein the compound is:

wherein X is thio, sulphinyl or sulphonyl.

6. The nucleotide derivative of any one of claims 1-3, wherein R³Is NH₂。

7. The nucleotide derivative of any one of claims 1-3 or 6, wherein said compound is:

wherein X is thio, sulphinyl or sulphonyl.

8. A pharmaceutical composition comprising at least one compound according to any one of claims 1 to 7 and a pharmaceutically acceptable adjuvant or vehicle.

9. A method of treating a malignant tumor comprising administering to a patient in need of such treatment an amount of a compound of any one of claims 1-7 effective to treat the tumor.

10. The method of claim 9, wherein the tumor is selected from the group consisting of a carcinoma, a sarcoma, or a leukemia.

11. A method of treating a malignant tumor comprising administering to a patient in need of such treatment an amount of the composition of claim 8 effective to treat the tumor, by itself or in combination with other anti-cancer agents.

12. A process for synthesizing a compound of formula Ia:

wherein R is¹Is a linear or branched, saturated or unsaturated alkyl residue having 1 to 20 carbon atoms, said alkyl residue being optionally mono-or polysubstituted with the following substituents: halogen, C₁-C₆Alkoxy radical, C₁-C₆Alkylthio radical, C₁-C₆Alkoxycarbonyl group, C₁-C₆Alkylsulfinyl or C₁-C₆An alkylsulfonyl group;

R²is hydrogen, a linear or branched, saturated or unsaturated alkyl chain having from 1 to 20 carbon atoms, said alkyl chain being optionally mono-or polysubstituted with the following substituents: halogen, C₁-C₆Alkoxy radical, C₁-C₆Alkylthio radical, C₁-C₆Alkoxycarbonyl or C₁-C₆An alkylsulfonyl group;

x is selected from the group consisting of a sulfur atom, a sulfinyl group and a sulfonyl group;

y is oxygen;

the method comprises the following steps:

(a) reacting 2, 6-dichloroadenine with an arabinofuranosyl derivative of the formula:

wherein R is⁵Is bromine or chlorine, R⁶And R⁷As a protecting group, to yield a dichloropurine nucleoside derivative of the formula:

(b) subjecting the dichloropurine nucleoside derivative to deprotection and aromatic nucleophilic substitution conditions to obtain a 6-alkoxy-2-chloropurine nucleoside derivative of general formula IIIb:

wherein R is⁴Is C₁-C₈An alkyl group;

(c) reacting said 6-alkoxy-2-chloropurine nucleoside derivative with an active form of a compound of the formula:

to obtain bound 6-alkoxy-2-chloropurine nucleotide derivatives of the general formula Ib:

(d) subjecting the bound 6-alkoxy-2-chloropurine nucleotide derivative to aminolysis conditions, thereby producing the following bound 2-chloroadenine derivative:

13. the method of claim 12, wherein the hindered potash base is potassium tert-butoxide or potassium tert-valerate.

14. The process of claim 12 wherein said solvent for the reaction of said 2, 6-dichloroadenine and said arabinofuranosyl derivative is a mixture of acetonitrile, t-butanol and 1, 2-dichloroethane.

15. The method of claim 12, wherein R⁴Is methyl.

16. The method of claim 12, wherein R⁵Is bromine.

17. The method of claim 12, wherein R⁶And R⁷Independently acetyl or benzoyl.

18. The method of claim 12, wherein R¹And R²Each being unsubstituted or substituted by C₁-C₆Alkoxy or C₁-C₆Alkylthio substituted straight chain C₈-C₁₅An alkyl group.

19. The method of claim 12, wherein R¹Is C₁₂H₂₅，R²Is C₁₀H₂₁。