KR20010023890A - Purine acyclonucleosides as antiviral agents - Google Patents

Abstract

The present invention relates to a method of using the compound of formula (1) or a salt thereof or a pharmaceutically acceptable derivative thereof for the treatment and / or prevention of hepatitis B virus infection.

Formula 1

In the above formula,

R ¹ is hydrogen, halogen, hydroxy, azido, alkoxy, aryloxy, thio, alkylthio, amino, alkylamino, hydrazino, hydroxylamino, benzyloxy, NRR 'or NRCOR',

R ² is hydrogen, halogen, hydroxy, azido, alkoxy, aryloxy, thio, alkylthio, amino, alkylamino, hydrazino, hydroxylamino, benzyloxy, NRR 'or NRCOR',

R and R 'are independently selected from hydrogen, alkyl and aryl.

Description

Purine acyclonucleosides as an antiviral agent {PURINE ACYCLONUCLEOSIDES AS ANTIVIRAL AGENTS}

Infection by human hepatitis B virus is a major public health problem due to its ability to cause acute and chronic infections. Chronic hepatitis B virus infection (hereinafter referred to as HBV) causes serious liver disease in the human body, often resulting in cirrhosis and hepatocellular carcinoma. At present, there are no effective therapies that successfully treat chronic HBV infection. More than 250 million carriers of HBV worldwide are not effective in the current vaccines.

Currently available recipes for HBV provide insufficient levels of efficacy or have adverse side effects. Thus, there has been a need for effective treatment of HBV.

Recently, it has been found that the compound of formula 1 is active against hepatitis B virus.

The present invention relates to a method of using purine acyclonucleosides as a medicament for the treatment and / or prophylaxis of hepatitis B, and to pharmaceutical compositions and novel purine acyclonucleosides for such uses.

Accordingly, the present invention provides a method of using the compound of formula 1 and salts or pharmaceutically acceptable derivatives thereof in the treatment and / or prophylaxis of hepatitis B virus infection.

In the above formula,

R ¹ is hydrogen, halogen, hydroxy, azido, alkoxy, aryloxy, thio, alkylthio, amino, alkylamino, hydrazino, hydroxylamino, benzyloxy, NRR 'or NRCOR',

R ² is hydrogen, halogen, hydroxy, azido, alkoxy, aryloxy, thio, alkylthio, amino, alkylamino, hydrazino, hydroxylamino, benzyloxy, NRR 'or NRCOR',

R and R 'are independently selected from hydrogen, alkyl and aryl.

These compounds have been found to exhibit surprisingly good activity in anti-hepatitis B assays.

R ¹ is preferably hydroxy or a group that can be converted to hydroxy in vivo.

R ² is preferably a group that can be converted to amino or amino in vivo.

The present invention also provides a method for treating or preventing hepatitis B virus infection comprising administering to a patient in need thereof a compound of formula 1, a salt thereof and a pharmaceutically acceptable derivative thereof. to provide.

The present invention also provides a compound of formula (1), salts thereof and pharmaceutically acceptable derivatives thereof useful for the treatment or prevention of hepatitis B virus infection.

In addition, the compounds of the present invention can be used in the manufacture of a medicament for the treatment or prevention of HBV. Accordingly, the present invention provides a pharmaceutical composition for the treatment or prophylaxis of HBV comprising a compound of formula 1, a salt thereof, and a pharmaceutically acceptable derivative and a pharmaceutically acceptable carrier or diluent.

The present invention also provides a method of using the compound of formula 1, a salt thereof, or a pharmaceutically acceptable derivative thereof in the manufacture of a medicament for the treatment or prophylaxis of HBV.

Salts of formula (I) are preferably pharmaceutically acceptable, but non-pharmaceutically acceptable salts are also within the scope of the present invention. This is also because it is usefully used as an intermediate in the preparation of pharmaceutically acceptable salts. Pharmaceutically acceptable salts may include conventional non-toxic salts or quaternary ammonium salts of the compounds, for example they may be formed from organic or inorganic acids or bases. Examples of such acid addition salts include, but are not limited to, acetic acid, propionic acid, citric acid, lactic acid, methanesulfonic acid, toluenesulfonic acid, benzenesulfonic acid, salicylic acid, ascorbic acid, hydrochloric acid, orthophosphoric acid, sulfuric acid, and hydrobromic acid. no. Base salts include, but are not limited to, those formed with pharmaceutically acceptable cations such as sodium, potassium, lithium, calcium, magnesium, ammonium and alkylammonium. These can be formed by treating the compound of formula 1 with a suitable metal hydroxide. In addition, basic nitrogen-containing groups include lower alkyl halides (eg, methyl, ethyl, propyl and butyl chlorides, bromide and iodide); It may be quaternized with reagents such as dialkyl sulfates (eg dimethyl and diethyl sulfate) and the like.

The compounds of the present invention may exist as crystalline forms or solvates (eg hydrates), both of which are within the scope of the present invention. Solvation methods are generally known in the art.

A pharmaceutically acceptable derivative is any pharmaceutically acceptable salt, hydrate that, when administered to a subject, can provide (directly or indirectly) a compound of Formula 1 or an antiviral active metabolite or residue thereof. , Prodrugs or any other compound. For example, compounds in which the semicyclic side-chain hydroxy group is replaced by phosphate esters fall into the category of pharmaceutically acceptable derivatives.

The term "prodrug" is used in the broadest sense and encompasses derivatives which are converted into the compounds of the invention in vivo. These derivatives are readily recognizable to those skilled in the art, such as compounds in which R ¹ is a group which can be converted to hydroxy in vivo or a group in which R ² can be converted to amino in vivo; Or compounds in which the semi-cyclic side chain free hydroxy group is converted to a specific group such as an ester, carbonate or carbamate and can be converted back to a hydroxy group in vivo. Prodrugs may include modifications of one or more of the functional groups of the compounds of the invention.

Throughout this specification, the phrase “groups that can be converted in vivo” as used in connection with other functional groups includes all functional groups or derivatives of those groups that can be converted to designated functional groups when administered to a mammal. Those skilled in the art will readily be able to determine which groups can be converted in vivo to designated functional groups through routine enzymatic or animal studies.

It is contemplated that some derivatives of compounds of Formula 1 may have asymmetric centers and therefore exist in one or more stereoisomeric forms. The invention can be applied to each of these individual forms and mixtures thereof. Alternatively, individual isomers may be prepared by asymmetric synthesis using chiral intermediates or enzymes.

R ¹ is preferably hydroxy or a group that can be converted to hydroxy in vivo.

R ² is preferably a group that can be converted to amino or amino in vivo.

Some of the compounds of formula (1) are novel and therefore the present invention provides compounds of formula (1a) and salts and pharmaceutically acceptable derivatives thereof:

In the above formula,

R ¹ is hydrogen, halogen, hydroxy, azido, alkoxy, aryloxy, thio, alkylthio, amino, alkylamino, hydrazino, hydroxylamino, benzyloxy, NRR 'or NRCOR',

R ² is hydrogen, halogen, hydroxy, azido, alkoxy, aryloxy, thio, alkylthio, amino, alkylamino, hydrazino, hydroxylamino, benzyloxy, NRR 'or NRCOR',

R and R 'are independently selected from hydrogen, alkyl and aryl,

Provided that 9- [3-hydroxy-2-hydroxymethylprop-1-yl] -guanine,

9- [3-hydroxy-2-hydroxymethylprop-1-yl] -adenine,

9-[(2-isopropyl-1,3-dioxan-5-yl) methyl] -guanine, and

9-[(2-isopropyl-1,3-dioxan-5-yl) methyl] -adenine is excluded from the compound of Formula 1a.

Compounds in which R ¹ is OH and R ² is NH ₂ in Formula 1 have been reported by Martin (1986) and the like to be inactive against herpes simplex virus type 1 and to be a poor substrate for the thymidine kinase of the virus.

The term alkyl, as used throughout this specification, is used alone or in the compound name (eg haloalkyl or alkyl acid), unless stated otherwise straight chain C _1-30 alkyl or branched C _3-30 alkyl, and branched chain Or both unbranched C _3-30 cycloalkyl. Unless otherwise defined, the groups may be saturated or unsaturated groups.

The term "aryl" refers to any compound comprising at least one aromatic ring or consisting of at least one aromatic ring. The aromatic ring may be carbocyclic, heterocyclic or pseudoaromatic, may be monocyclic or polycyclic and preferably has 2 to 20 carbon atoms. The aromatic ring may also contain one or more heteroatoms selected from N, S, O and P. Examples of suitable rings include benzene, biphenyl, terphenyl, quarterphenyl, naphthalene, tetrahydronaphthalene, 1-benzylnaphthalene, anthracene, dihydroanthracene, benzanthracene, dibenzanthracene, phenanthracene, perylene, pyridine, 4 -Phenylpyridine, 3-phenylpyridine, thiophene, benzothiophene, naphthothiophene, thianthrene, furan, pyrene, isobenzofuran, chromate, xanthene, phenoxatine, pyrrole, imidazole, pyrazole, pyrazine pyridine Midine, pyridazine, indole, indoliazine, isoindole, purine, quinoline, isoquinoline, phthalazine, quinoxaline, quinazoline, pteridine, carbazole, carboline, phenanthridine, acridine, phenanthrol Lean, phenazine, isothiazole, isoxazole, phenoxazine and the like, but are not limited to these, each of which may be optionally substituted. The term "quasiaromatic" refers to a ring system that is not strictly aromatic but stabilizes by electron localization and behaves like an aromatic ring. Examples of pseudoaromatic rings include, but are not limited to, furan, thiophene, pyrrole and the like.

The term alkoxy, as used throughout this specification, is used alone or in the compound name (e.g. haloalkoxy), unless stated otherwise straight chain C _1-30 alkoxy or branched C _3-30 alkoxy, and branched or unbranched Chain C _3-30 cycloalkoxy. Unless otherwise defined, the groups may be saturated or unsaturated groups.

Although not specifically mentioned, the term "ester" means a compound or derivative corresponding to an ester formed by the reaction of an alcohol with an organic acid, preferably a carboxylic acid. Particular preference is given to carboxylic acids which can form esters with amino acids and alkyl acids. Preferred amino acids are aliphatic amino acids such as valine and isoleucine, preferably L-form. Preferred alkyl acids include C ₂ -C ₄ alkyl acids, and fatty acids obtained from C ₁₁ -C ₂₂ , such as lauryl acid, myristoyl acid, palmitoyl acid, stearoyl acid, ecasanoylic acid, behenolic acid, myristol Acids, myristic acid, palmitoyl acid, palmitlideic acid, n6-octadecenoic acid, oleic acid, ellideic acid, erucic acid or brasidic acid.

Preferred groups of the compounds of Formula 1 and Formula 1a and pharmaceutically acceptable derivatives of the compounds of Formula 1 and Formula 1a are the compounds of Formula 4 and salts thereof:

In the above formula,

X is hydrogen or hydroxy,

R ⁵ and R ⁶ are the same or different and together with the oxygen atom to which they are attached form a hydroxy group, ester, carbonate, carbamate or thiocarbonate, preferably hydroxy or ester.

Examples of some compounds of Formula 4 are listed in Table 1 below.

X R ⁵ R ⁶ H H H OH H H H Acetyl Acetyl OH Acetyl Acetyl H Acetyl H OH Acetyl H H Ballyl Ballyl OH Ballyl Ballyl H Ballyl H OH Ballyl H H Ballyl Acetyl OH Ballyl Acetyl H Isoleucine Isoleucine OH Isoleucine Isoleucine H Isoleucine Acetyl OH Isoleucine Acetyl H Ballyl Stearyl OH Ballyl Stearyl H Ballyl Octadecenyl OH Ballyl Octadecenyl H Isoleucine Stearyl OH Isoleucine Stearyl H Isoleucine Octadecenyl OH Isoleucine Octadecenyl H Ballyl Palmityl OH Ballyl Palmityl H Ballyl Elydil OH Ballyl Elydil

X R ⁵ R ⁶ H Isoleucine Palmityl OH Isoleucine Palmityl H Isoleucine Elydil OH Isoleucine Elydil H H Stearyl OH H Stearyl H H Octadecenyl OH H Octadecenyl H H Palmityl OH H Palmityl H H Elydil OH H Elydil H H Collyl OH H Collyl H Ballyl Collyl OH Ballyl Collyl H Propionyl Propionyl OH Propionyl Propionyl H Isopropionyl Isopropionyl OH Isopropionyl Isopropionyl H H Butyryl OH H Butyryl H Butyryl Butyryl OH Butyryl Butyryl

In another aspect of the present invention there is provided a process for the preparation of a compound of formula 1, salts thereof and pharmaceutically acceptable derivatives. The compound may be prepared by reacting a purine derivative of Formula 2 with a compound of Formula 3 below.

The group R ¹ of the purine derivative of formula 2 may be any group listed as R ¹ for the compound of formula 1 or any group convertible to the group according to methods known in the art, such as chlorine, bromine or iodine , R ² may be any group listed as R ² for the compound of Formula 1 or any group convertible to the group according to methods known in the art. In formula (3), Z can be any group suitable as leaving group (eg methane sulfonate or bromine), and R ³ and R ⁴ include hydroxy or groups which can be converted to hydroxy such as ethers and esters do. Such conversion reactions are known to those skilled in the art. R ³ and R ⁴ may be joined together to form an optionally substituted 5- or 6-membered ring system.

Compounds of formula (2) can be purchased commercially or prepared using the methods described in the literature. Compounds of formula (3) can be prepared according to the processes described in the literature or the procedures described in steps A to C of Example 1 and steps A to E of the alternative route of Example 1 (see overview 1 and 2 respectively). Those skilled in the art will be able to make various modifications to this practically illustrated method.

Methods of preparing compounds of formula (3) outlined in outline 1 of Example 1 are particularly useful and may be applied more broadly to the synthesis of semicyclic nucleoside analogs than those outlined in certain examples. The general procedure of step B constitutes another aspect of the present invention. Thus, trialkylorthoesters, preferably triethylorthoacetate, are treated with about 1 equivalent under suitable conditions, followed by treatment with about 1 equivalent of water under appropriate conditions, followed by treatment with water under suitable conditions, thereby providing a symmetric triol of formula (5) Preferably, a symmetric triol in which n is 1 or 2 can be converted to the diester of the formula (6). The diester can then be separated by conventional methods, which is convenient to separate by carefully neutralizing the mixture with a mild base (eg sodium bicarbonate) and then extracting into an organic solvent. Those skilled in the art will be able to readily determine which conditions are suitable given the particular triol and trialkylorthoesters selected.

-OR = ester

Residual hydroxyl groups on the diester of Formula 6 may be converted to a leaving group Z to produce a diester compound of Formula 1 wherein n is 1.

Diester compounds including compounds of formula 6 (eg 2-hydroxymethyl-1,3-propanediol diacetate) and compounds of formula 3 (eg 3-acetoxy-2-acetoxymethylprop-1- One methanesulfonate) is novel and constitutes an additional aspect of the present invention. It is preferred that n is 1 in the compounds of formulas (5) and (6).

According to the conventional methods known in the art, the semicyclic hydroxyl groups of the compound of formula 1 can be easily converted into ester, ether or phosphate groups or mixtures of these groups on the semicyclic chain. In some instances, it may be useful to use protected intermediates of the compound of formula 1 to prepare the desired final derivative. Such intermediates can be prepared according to standard methods and can be removed according to standard methods (eg, the method described by Greene) when protecting groups are no longer needed. Examples of suitable protecting groups are trimethylsilyl and monomethoxytrityl groups.

Acylation and alkylation can be accomplished by conventional methods, such as those known in the art, or as described or cited in the Advanced Organic Chemistry 3rd Edition of March, published by Wiley-Interscience. Can be performed. Examples of suitable acylating agents for acylating the compound of formula 1 include carboxylic acid, acid halide and acid anhydride. The reaction is carried out in a conventional manner, for example in the presence of a coupling agent (e.g., N, N'-dicyclohexylcarbodiimide) and optionally a catalyst base (e.g. 4-dimethylaminopyridine) in a solvent such as pyridine, dimethylformamide and the like. It can be performed under. The reaction product can be separated by conventional methods. Examples of alkylating agents suitable for alkylating compounds of Formula 1 include alkyl halides (eg methyl, ethyl, propyl and benzyl chlorides, bromide and iodide) and dialkyl sulfates (eg dimethyl and diethyl sulfate).

In the case of amino acids or their functional equivalents (eg acid halides), it may be advantageous to use amino protected derivatives of amino acids or amino acid equivalents, such as benzyloxycarbonyl derivatives, to avoid side reactions. Such derivatives can be purchased commercially. Protecting groups can be removed using standard methods.

The acylation or alkylation reaction may produce a single derivative of compound (1) incorporating one or more acyl or alkyl groups or may produce a mixture of compounds incorporating one or more acyl or alkyl groups. The amount produced depends on many factors, such as the relative amounts and chemical nature of the reactants, the physical conditions of the reaction and the solvent system. Any mixture produced in this manner can be separated using standard techniques, such as chromatography.

It is contemplated that one of ordinary skill in the art would be able to produce derivatives of the compounds of formula 1 that may contain mixtures of different acyl and / or alkyl groups. Such derivatives fall within the scope of the present invention.

In addition, protected intermediates of the compounds of formula (1) may be used to prepare derivatives of formula (1) incorporating phosphate esters.

In addition, the compounds of the present invention can be usefully used in combination with known antiviral or antiretroviral agents or other agents used in the treatment of viral infections. Representative examples of these added agents include immunomodulators, immunostimulants and antibiotics. Examples of antiviral agents include AZT, 3TC, acyclovir, famcyclovir, ddI, ddC, gancyclovir, saquanivir, lobbyide, other nonnucleotide reverse transcriptase (RT) inhibitors and protease inhibitors. Examples of immunomodulators and immunostimulants include a variety of interleukins, cytokines, antibody preparations, hemotransmitters, and cytotransmitters. Examples of antibiotics include antifungal agents, antibacterial agents and antipneumocytis carini reagents.

When formulated at a fixed dosage, the combination product uses the compounds of the invention in the dosage ranges described below and other pharmaceutical actives within the validated dosage ranges. If a combination combination is not suitable, the compounds of the invention may be used sequentially with known antiviral agents, antiretroviral agents or pharmaceutical reagents.

An effective amount refers to an amount of active compound that is effective when one or several doses are administered to a patient to control concentrations of active compounds known to inhibit viral infections (eg, HBV) or to inhibit the virus. As such, this is defined by assays known to predict the clinical antiviral activity of chemical compounds, such as those described by Korba and Gerin.

As used herein, the term "virus infectious disease control" refers to slowing, disrupting, arresting or stopping the growth or replication of viruses, and does not necessarily remove all viruses.

Controlling viral infections is useful for the treatment and / or prevention of viral infections described above.

In the case of administering a compound of the present invention to a human, the daily dosage is usually determined by a physician, and the dosage is generally determined by the age, weight and response of each patient as well as the severity of the patient's condition. In general, suitable daily dosages of the compounds of the invention range from 0.1 to 50 mg, preferably from 0.5 to 10 mg, per kg of body weight of the patient. It is preferred that the predetermined dose be divided into two, three, four, five, six or more sub doses at appropriate intervals throughout the day. These sub doses may be administered in unit dosage form, eg, in a form containing 1 to 1000 mg, preferably 10 to 500 mg of the active ingredient per unit dosage form.

In addition, the compounds of the present invention as active ingredients include oral, rectal, intranasal, topical (eg buccal and sublingual), vaginal and parenteral (eg subcutaneous, intramuscular, intravenous, intradermal) It may be administered for treatment via any suitable route of administration. While oral administration is preferred, it will be appreciated that the preferred route of administration may vary depending upon the condition and age of the patient, the nature of the present invention and the active ingredient selected. For oral administration, prodrugs of the active compounds that are absorbed more effectively than the unmodified compound are generally preferred.

The composition of the present invention comprises a compound of formula (1), optionally a salt or other pharmaceutically acceptable derivative thereof, one or more pharmaceutically acceptable carriers, diluents or excipients thereof, and any other therapeutic agent. Carriers, diluents or excipients should each be pharmaceutically acceptable, meaning they are compatible with the other ingredients of the composition and do not harm the patient. The compositions include those suitable for oral, rectal, intranasal, topical (eg buccal and sublingual), vaginal and parenteral (eg subcutaneous, intramuscular, intravenous, intradermal) administration. The compositions may conveniently be presented in unit dosage form and may be prepared by methods known in the art of pharmacy. Such methods include the step of mixing the active ingredient with a monomer, diluent or excipient comprising one or more accessory ingredients. Generally, the composition may be prepared by uniformly and thoroughly mixing the active ingredient with a liquid carrier or finely divided solid carrier or both, and then molding the product into a product as necessary.

Compositions of the invention suitable for oral administration may be presented in discrete units, such as capsules, sachets or tablets, each containing the desired amount of active ingredient; In powder or granule form; In the form of a solution or suspension in an aqueous or non-aqueous liquid; Or in an oil-in-water liquid emulsion or a water-in-oil liquid emulsion. In addition, the active ingredient may be formulated as a pill, paste or paste.

Tablets may be made by compression or molding, optionally with one or more accessory ingredients. Compressed tablets may contain an active ingredient optionally mixed with a binder (e.g., an inert diluent, preservative, a disintegrant (e.g. sodium starch glycolate, crosslinked povidone, crosslinked sodium carboxymethylcellulose), a surface active agent or a dispersant). It can be produced by pressing in a suitable machine in the same free flow form. Molded tablets may be made by molding in a suitable machine a mixture of powdered compounds moistened with an inert liquid diluent. Tablets may be optionally coated or scored and formulated, such as using hydroxypropylmethyl cellulose, to release the active ingredient therein in various fractions slowly or at a controlled rate to exhibit the desired release behavior. Tablets may optionally be provided with enteric skin to release the active ingredient in other intestinal parts other than the stomach.

Compositions suitable for topical administration in the mouth include lozenges comprising the active ingredient in a flavored base (usually sucrose and acacia or tragacanth gum); Pastilles comprising the active ingredient in an inert base (eg, gelatin and glycerin) or sucrose and acacia gum; And mouthwashes comprising the active ingredient in a suitable liquid carrier.

Compositions suitable for rectal administration are provided in the form of suppositories containing suitable bases, including, for example, cocoa butter.

Compositions suitable for vaginal administration may be presented in the form of pessaries, tampons, creams, gels, pastes, foams or spray formulations which contain, in addition to the active ingredient, carriers known in the art as suitable.

Compositions suitable for parenteral administration include aqueous and non-aqueous isotonic sterile injectable solutions that may contain antioxidants, buffers, bacteriostatics, and solutes that make the composition into the recipient's blood and isotonic solution; And aqueous and non-aqueous sterile suspensions which may include suspending agents and thickening agents. The composition may be provided in a single dose enclosed container or in a multiple dose enclosed container such as ampoules and vials, and stored in a lyophilized state just by the addition of a sterile liquid carrier such as water for injection, immediately before use. Can be. Extemporaneous injection solutions and suspensions can be prepared from sterile powders, granules and tablets of the kind described above.

Preferred unit dosage compositions are those which contain the active ingredient in a daily dosage or daily dosage unit, a daily subdosage as described above or a suitable fraction thereof.

The compounds of the invention may also be provided in the form of veterinary compositions, which may be prepared, for example, by conventional methods in the art. Examples of such veterinary compositions include

(a) veterinary compositions for oral administration, for external application, such as drunks (eg, aqueous or non-aqueous solutions or suspensions); Tablets or ingots; Powders, granules or pellets for blending with feed; Paste for applying to the tongue,

(b) veterinary compositions for parenteral administration, such as by subcutaneous, intramuscular or intravenous injection, such as in the form of sterile solutions or suspensions,

(c) a veterinary composition for topical application, such as a cream, ointment or spray applied to the skin, or

(d) Veterinary compositions for intravaginal administration, such as pesalis, creams or foams.

In particular, in addition to the components mentioned above, the compositions of the present invention include other reagents customary in the art with respect to the type of composition in question (eg, sweeteners, thickeners and flavoring agents suitable for oral administration). can do.

The examples are provided to help further understand the present invention. The specific materials and conditions used are intended to illustrate the invention and are not intended to limit the reasonable scope of the invention.

2-amino-6-chloropurine in 98% purity was purchased from Chugai Boyeki Co., Ltd. 2-amino-6-chloropurine was converted to 2-amino-6-iodopurine according to the method of Bisacchi et al. Unless otherwise noted, reagents not cited were purchased and used as such.

All temperatures are given in degrees Celsius (° C.).

Example 1

Overview 1

Reaction procedure described in example 1

Step A

2-hydroxymethyl-1,3-propanediol

A modified Hardenden et al. Method was used. Triethylmethane tricarboxylate (9.68 g, 0.0417 mol) was added dropwise to a solution of borane methylsulfide complex (10 M) (14 ml, 0.14 mol) dissolved in toluene (65 ml) at low reflux under a nitrogen atmosphere. It was. The reaction mixture was refluxed for 7.5 hours under distillation of dimethyl sulfide. The reaction mixture was cooled to room temperature and methanol (40 ml) was added dropwise. The reaction mixture was stirred overnight at room temperature, then the solvent was removed and the residue was repeatedly evaporated with methanol. The residue was treated by chromatography on silica with 25% methanol in dichloromethane to give 2-hydroxymethyl-1,3-propanediol (3.18 g, 72%) as a light lemon oil. ¹ H nmr (DMSOd ₆ ) 1.60, quartet, J = 5.5 Hz, 1H; 3.40, t, J = 5.5 Hz, 2H; 4.31, t, J = 5.5 Hz, 3H.

Step B

2-hydroxymethyl-1,3-propanediol diacetate

2-hydroxymethyl-1,3-propanediol (3.18 g, 0.03 mol), trifluoroacetic acid (1.54 ml, 0.02 mol) dissolved in N, N-dimethylformamide (35 ml) under a nitrogen atmosphere Triethylorthoacetate (6.09 ml, 0.033 mol) was added to the solution. The reaction mixture was stirred for 1.5 hours and then water (0.63 ml, 0.035 mol) was added. The reaction mixture was further stirred for 1.5 h, then triethylorthoacetate (6.26 ml, 0.034 mol) was added. The reaction mixture was further stirred for 1.5 h, then water (1.74 ml, 0.097 mol) was added. After 1 hour, sodium bicarbonate was carefully added until the reaction mixture became neutral. After addition of water, the solution was extracted with dichloromethane (3x). The combined extracts were washed with water, dried over magnesium sulfate and concentrated to give 2-hydroxymethyl-1,3-propanediol diacetate (4.50 g, 79%) as a colorless oil. ¹ H nmr (CDCl ₃ ) 2.06, s, 6H; 2.18, quintet, J = 6Hz, 1H, 2.54, br s, 1H, 3.62, d, J = 6Hz, 2H; 4.15, d, J = 6 Hz, 4H.

Step C

3-acetoxy-2-acetoxymethylprop-1-yl methanesulfonate

Under a nitrogen atmosphere, a 2-hydroxymethyl-1,3-propanediol diacetate (4.49 g, 0.0236 mol) solution dissolved in dichloromethane (40 ml) was cooled to -5 ° C. Triethylamine (4.93 ml, 0.0354 mol) was added, followed by dropwise addition of a solution of methanesulfonyl chloride (2.19 ml, 0.0283 mol) dissolved in dichloromethane (20 ml). The reaction was stirred for 2 more hours at 03 and then warmed to room temperature. The reaction mixture was washed with 1.5 M hydrochloric acid (3x30 ml), washed with sodium bicarbonate solution (30 ml) and brine (30 ml), dried over magnesium sulfate and concentrated to 3-acetoxy in light brown oil. 2-acetoxymethylprop-1-yl methanesulfonate (5.02 g, 79%) was obtained. ¹ H nmr (CDCl ₃ ) 2.0, s, 6H; 2.48, quintet, J = 6Hz, 1H; 3.03, s, 3 H; 4.07-4.24, m, 4H; 4.29, d, J = 6 Hz, 2H.

Step D

9- [3-acetoxy-2-acetoxymethylprop-1-yl] -2-amino-6-iodopurine

3-acetoxy-2-acetoxymethylprop-1-yl methanesulfonate (5.02 g, 0.0187 mol), 2-amino-6-io, in N, N-dimethylformamide (250 ml) under nitrogen atmosphere A mixture of doprine (4.66 g, 0.0178 mol) and potassium carbonate (7.38 g, 0.0534 mol) was stirred at 60 ° C. for 2 days. N, N-dimethylformamide was removed under vacuum and the residue was extracted with ethyl acetate (3 x 100 ml) by addition of water. The extract was washed with water and brine, dried over magnesium sulfate and filtered through a silica plug while being thoroughly washed with ethyl acetate. The filtrate was concentrated to give crude product (5.96 g) as a yellow solid. This was recrystallized from methanol and diethyl ether to give light lemon colored 9- [3-acetoxy-2-acetoxymethylprop-1-yl] -2-amino-6-iodopurine (4.43 g, 55 %) Was obtained. ¹ H nmr (DMSOd ₆ ) 1.95, s, 6H; 2.56-2.78, m, 1 H; 3.90-4.10, m, 4H; 4.12, d, J = 7 Hz, 2H; 6.83, s, 2 H; 8.09, s, 1 H.

Step E

9- [3-hydroxy-2-hydroxymethylprop-1-yl] -guanine

9- [3-acetoxy-2-acetoxymethylprop-1-yl] -2-amino-6-iodolyurine (2.5 g, 0.00576 mol) in 1.5 M hydrochloric acid was heated under reflux for 3 hours. The reaction mixture was cooled to room temperature and the pH was adjusted to 14 with sodium hydroxide. The reaction mixture was further stirred for 1.5 hours and then neutralized with concentrated hydrochloric acid. The obtained precipitate was filtered off and then recrystallized from water to give 9- [3-hydroxy-2-hydroxymethylprop-1-yl] -guanine (1.267 g, 92%) as colorless fuzzy crystals. Mp 294-296 ° C. ¹ H nmr (DMSOd ₆ ) 1.9-2.05, m, 1H; 3.3, t, J = 5.5 Hz, 4H; 3.95, d, J = 7.0 Hz, 2H; 4.6, t, J = 5.5 Hz, 2H; 6.5, s, 2 H; 7.6, s, 1 H; 10.1, s, 1 H.

Example 1 (Alternative Path)

Outline 2

Schematic of an alternative reaction procedure

Step A

5,5-dicarboxyethyl-2-isopropyl-1,3-dioxane

5,5-dicarboxyethyl-2-isopropyl-1,3-dioxane was prepared using a modified Eliel et al. Method. Diethyl bis (hydroxymethyl) malonate (25.0 g, 0.113 mol), isobutylaldehyde (0.226 mol, 20.6 ml) and p-toluene sulfonic acid (0.300 g) in petroleum spirit (50 ml) were heated under reflux. The water was collected in a Dean-Stark apparatus. Petroleum spirit was removed and the product was distilled to give 5.5-dicarboxyethyl-2-isopropyl-1,3-dioxane (22.5 g, 73%) as a colorless oil. Bp 103 / 0.15 mmHg. ¹ H nmr (CDCl ₃ ) 0.9, d, J = 6.9 Hz, 6H; 1.2, t, J = 6.9 Hz, 3H; 1.3, t, J = 6.9 Hz, 3H; 1.7-1.85, m, 1 H; 3.9, d, JAB = 11.5 Hz, 2H; 4.15, q, J = 6.9 Hz, 2H; 4.22, d, J = 4.8 Hz, ¹ H; 4.3, q, J = 6.9 Hz, 2H; 4.7, d, JAB = 11.5 Hz, 2H.

Step B

5,5-dicarboxylic-2-isopropyl-1,3-dioxane

5,5-dicarboxy-2-isopropyl-1, from 5,5-dicarboxyethyl-2-isopropyl-1,3-dioxane (20.0 g, 72.9 mmol) according to the method of Eliel et al. 3-dioxane was synthesized. The crude product was recrystallized from ethyl acetate and petroleum spirit to give 5,5-dicarboxylic-2-isopropyl-1,3-dioxane (14.1 g, 80%) as a colorless solid. Mp 143.5-145 ° C. ¹ H nmr (DMSOd ₆ ) 0.8, d, J = 6.9 Hz, 6H; 1.55-1.75, m, 1 H; 3.8, d, JAB = 11.3 Hz, 2H; 4.3, d, J = 4.8 Hz, 1H; 4.5, d, JAB = 11.3 Hz, 2H; 12.8, br s, 2H.

Step C

5-carboxy-2-isopropyl-1,3-dioxane

5-carboxy-2-isopropyl-1,3-dioxane from 5,5-dicarboxylic-2-isopropyl-1,3-dioxane (15.7 g, 64.8 mmol) according to the method of Eliel et al. Was synthesized. The crude product was recrystallized from ethyl acetate and petroleum spirit to give 5-carboxy-2-isopropyl-1,3-dioxane (9.75 g, 76%) as a colorless solid. Mp 131-134 ° C. ¹ H nmr (DMSOd ₆ ) 0.8, d, J = 6.9 Hz, 6H; 1.6-1.8, m, 1 H; 2.7-2.85, m, 1 H; 3.65, t, JAB = 11.5 Hz, 2H; 4.15, t, JAB = 10.1 Hz, 2H; 4.2. t, J = 4.8 Hz, 1H.

Step D

5-hydroxymethyl-2-isopropyl-1,3-dioxane

Borane methylsulfide complex (9.0 ml) in a 5-carboxy-2-isopropyl-1,3-dioxane (8.75 g, 44.1 mmol) solution dissolved in anhydrous diethyl diethyl ether (65 ml) under nitrogen atmosphere. , 10 M, 90 mmol) was added. The reaction mixture was heated to reflux for 1 hour, then cooled to room temperature and quenched with water (20 ml) and methanol (30 ml). Methanol was removed and the aqueous phase was extracted several times with diethyl ether. The combined diethyl ether extracts were washed with water and brine, dried over magnesium sulfate, and the solvent was removed to give 5-hydroxymethyl-2-isopropyl-1,3-dioxane (6.50 g, 80) as colorless oil. %) Was obtained. ¹ H nmr (DMSOd ₆ ) 0.85, d, J = 7 Hz, 6H; 1.6-1.8, m, 1 H; 1.85-2.05, m, 1 H; 3.2, t, JAB = 11.3 Hz, 2H; 3.3-3.45, m, 4H; 4.15, d, J = 4.8 Hz, 1 H.

Step E

2-isopropyl-5- (methanesulfoxy) methyl-1,3-dioxane

2- from 5-carboxy-2-isopropyl-1,3-dioxane (7.30 g, 39.6 mmol) according to the method used for 3-acetoxy-2-acetoxymethylprop-1-yl methanesulfonate Isopropyl-5- (methanesulfoxy) methyl-1,3-dioxane was synthesized to give the product as a colorless oil (10.1 1 g, 97%). ¹ H nmr (DMSOd ₆ ) 0.85, d, J = 6.9 Hz, 6H; 1.6-1.8, m, 1 H; 2.15-2.35, m, 1 H; 3.2, s, 3 H; 3.5, t, JAB = 11.3 Hz, 2H; 4.0-4.15, m, 4H; 4.2, d, J = 4.8 Hz, 1 H.

Step F

2-amino-6-chloro-9-[(2-isopropyl-1,3-dioxan-5-yl) methyl] purine

2-isopropyl-5- (methanesulfoxy) methyl according to the method used for 9- [3-acetoxy-2-acetoxymethylprop-1-yl] -2-amino-6-iodopurine Colorless to synthesize 2-amino-6-chloro-9-[(2-isopropyl-1,3-dioxan-5-yl) methyl] purine from -1,3-dioxane (10.1 g, 38.5 mmol) A solid product (4.80 g, 40%) was obtained. Mp 204-205 캜. ¹ H nmr (DMSOd ₆ ) 0.85, d, J = 7 Hz, 6H; 1.6-1.75, m, 1 H; 2.4-2.55, m, 1 H; 3.45, t. J = 11.3 Hz, 2H; 3.85-4.0, m, 4 H; 4.2, d, J = 5 Hz, 1 H; 6.9, s, 2 H; 8.1, s, 1 H. ¹³ C nmr (DMSO-d ₆ ) 16.8, 31.9, 34.3, 41.4, 68.6, 104.5, 123.3, 143.1, 149.4, 154.2, 159.7

Mass spectrum: m / z 312 ((M + 1) +, 100%), 340 ((M + 29) +, 15), 314 ((M + 3) +, 30), 313 ((M + 2) +, 18), 276 (20). Exact Mass: Found 312.1209 (M + 1) < + >, C ₁₃ H ₁₉ N ₅ O ₂ Cl, calcd 312. 1227.

Step G

9- [3-hydroxy-2-hydroxymethylprop-1-yl] -guanine

2-amino-6-chloro-9-[(2-isopropyl-, according to the method used for 9- [3-hydroxy-2-hydroxymethylprop-1] -guanine (step E described above) 9- [1-hydroxy-2-hydroxymethylpropyl] guanine was synthesized from 1,3-dioxan-5-yl) methyl] purine (1.28 g, 4.11 mmol) to give a colorless solid product (0.60 g, 61%). Mp 285 ° C. Decomposition. ¹ H nmr (DMSOd ₆ ) 1.9-2.05, m, 1H; 3.3, t, J = 5.5 Hz, 4H; 3.95, d, J J = 7.0 Hz, 2H; 4.6, t, J = 5.5 Hz, 2H; 6.5, s, 2 H; 7.6, s, 1 H; 10.1, s, 1 H. ¹³ C nmr (DMSO-d ₅ ) 41.5, 43.6, 58.9, 116.3, 138.1, 151.3, 153.4, 156.8. Mass spectrum m / z 340 ((M + l) < + >, 100%), 368 ((M + 29) < + >, 20), 341 ((M + 2) < + >, 12).

Example 2

9- [3-hydroxy-2-hydroxymethylprop-1-yl] -2-amino-6-methoxypurine

9- [3-acetoxy-2-acetoxymethylprop-1] -2-amino-6-iodopurine (307 mg, 0.708 mmol), sodium hydroxide (7.75 g, 194 mmol), methanol (30 mL) ) And water (3 mL) were stirred at rt for 18 h. The reaction mixture was neutralized with aqueous HCl and the solvent removed by rotary evaporation. To this residue hot methanol was added and the solution was decanted and filtered through a short silica film. The filtrate was concentrated and recrystallized from water to give 9- [3-hydroxy-2-hydroxymethylprop-1-yl] -2-amino-6-methoxypurine (179 mg, quantitative) as colorless crystals. Got it. ¹ H nmr (DMSOd ₆ ) 1.93-2.16, m, 1H; 3.22-3.40, m, 4H; 3.95, s, 3 H; 4.19, d, J = 7 Hz, 2H; 4,69, t, J = 5 Hz, 2H; 6.45, s, 2 H; 7.79, s, 1 H.

Example 3

9- [3-acetoxy-2-acetoxymethylprop-1-yl] -2-amino-6-hydrazino-purine

9- [3-acetoxy-2-acetoxymethylprop-1-yl] -2-amino-6-iodopurine (300 mg, 0.69 mmol) under a nitrogen atmosphere, hydrazine hydrate (85%, 210 μl, 6.68 mmol) and ethanol (35 mL) were stirred under reflux for 3 hours and then at room temperature for 16 hours. The resulting colorless solid was separated by filtration, washed well with ethanol and dried in vacuo to give 9- [3-acetoxy-2-acetoxymethylprop-1-yl] -2-hydrazino-purine (196 mg, 84%) Got. ¹ H nmr (DMSOd ₆ ) 1.98, s, 6H; 2.54-2.77, m, 1 H; 3.87-4.08, m, 4 H; 4.05, d, J = 7 Hz, 2H; 4.40, br s, 2H; 5.89, br s, 2 H; 7.67, s, 1 H; 8.40, s, 1 H.

Example 4

2-amino-6-hydrazino-9- [3-hydroxy-2-hydroxymethylprop-1-yl] purine

9- [3-acetoxy-2-acetoxymethylprop-1-yl] -2-amino-6-iodopurine (300 mg, 0.69 mmol) under a nitrogen atmosphere, hydrazine hydrate (85%, 700 μl, 22.27 mmol) and ethanol (35 mL) were stirred at reflux for 6 hours and then at room temperature for 16 hours. The reaction mixture was concentrated to dryness and the residue was recrystallized from water to give 2-amino-6-hydrazino-9- [3-hydroxy-2-hydroxymethylprop-1-yl] purine (86 mg) in creamy crystalline form. , 49%). ¹ H nmr (DMSOd ₆ ) 1.90-2.12, m, 1H; 3.20-3.40, m, 4H; 3.98, d, J = 7 Hz, 2H; 4.42, br s, 2 H; 4.74, t, J = 5 Hz, 2H; 5.98, s, 2 H; 7.62, s, 1 H; 8.45, s, 1 H.

Example 5

2-amino-9- [3-hydroxy-2-hydroxymethylprop-1-yl] -6-iodopurine

Stopped mixture of 9- [3-acetoxy-2-acetoxymethylprop-1-yl] -2-amino-6-iodopurine (304 mg, 0.70 mmol) and methanolic ammonia (10 mL) Stir for 2 hours in the flask. The stopper was opened and the reaction mixture was left for 16 hours. The reaction mixture was filtered to give 2-amino-9- [3-hydroxy-2-hydroxymethylprop-1-yl] -6-iodopurine (201 mg, 82%) in the form of a colorless needle. ¹ H nmr (DMSOd ₆ ) 1.96-2.19, m, 1 H; 3.25-3.43, m, 4H; 4.01, d, J = 7 Hz, 2H; 4.61, t, J = 5 Hz, 2H; 6.84, s, 2 H; 8.02, s, 1 H.

Example 6

2,6-diamino-9- [3-hydroxy-2-hydroxymethylprop-1-yl] purine

Of 9- [3-acetoxy-2-acetoxymethylprop-1] -2-amino-6-iodopurine (299 mg, 0.69 mmol) and methanolic ammonia (10 mL) at 100 ° C. in a bomb The mixture was stirred for 18 hours. The reaction mixture was cooled to room temperature and a precipitate formed after 1 hour. This was separated by filtration and dried in vacuo to give 2,6-diamino-9- [3-hydroxy-2-hydroxymethylprop-1-yl] purine (128 mg, 78%) as colorless crystals. ¹ H nmr (DMSOd ₆ ) 1.90-2.10, m, 1 H; 3.20-3.40, m, 4H; 3.96, d, J = 6.5 Hz, 4.75, t, J = 5 Hz, 2H; 5.84, s, 2 H; 6.70, s, 2 H; 7.78, s, 1 H.

Example 7

2- (dimethylaminomethylene) amino-9- [3-hydroxy-2-hydroxymethylprop-1-yl] guanine

9- [3-hydroxy-2-hydroxymethylprop-1] -guanine (200 mg, 0.84 mmol), N, N-dimethylformamide dimethyl acetal (1.5 mL, 11.3 mmol) and N in a nitrogen atmosphere; The mixture of N-dimethylformamide (20 mL) was stirred for 2 days at room temperature. The solvent was removed in vacuo at 60 ° C. and the residue was recrystallized from ethanol to give 2- (dimethylaminomethylene) amino-9- [3-hydroxy-2-hydroxymethylprop-1-yl] guanine as colorless crystals. (180 mg, 73%) was obtained. ¹ H nmr (DMSOd ₆ ) 1.95-2.17, m, 1 H; 3.03, s, 3 H; 3.15, s, 3 H; 3.21-3.47, m, 4H; 4.03, d, J = 7 Hz, 2H; 4.62, t, J = 5 Hz, 2H; 7.74, s, 1 H; 8.53, s, 1 H; 11.26 br s, 1 H.

Example 8

2-amino-9-[(2-isopropyl-1,3-dioxan-5-yl) methyl] purine

2-amino-6-chloro-9-[(2-isopropyl-1,3-dioxan-5-yl) methyl] purine (350 mg, 1.12 mmol), triethylamine (172 μl, 1.23 under hydrogen atmosphere mmol), a mixture of 10% palladium on carbon (35 mg) and ethanol (5 mL) was stirred for 3 days. The reaction mixture was then filtered through GFA paper with repeated washing with dichloromethane. The filtrate was concentrated to dryness, dissolved in dichloromethane, washed with water (2 ×) and brine, dried over sodium sulfate and then concentrated to give 2-amino-9-[(2-isopropyl-) as a colorless solid. 1,3-dioxan-5-yl) methyl] purine (260 mg, 84%) was obtained. ¹ H nmr (DMSOd ₆ ) 0.83, d, J = 7 Hz, 6H; 2.35-2.63, m, 1 H; 3.45, t, J = 11 Hz, 2H; 3.87, d, J = 7 Hz, 2H; 3.82-3.95, m, 2 H; 4.17, d, J = 5 Hz, 1 H; 6.53, s, 2 H; 8.02, s, 1 H; 8.57, s, 1 H.

Example 9

2-amino-9-[(3-hydroxy-2-hydroxymethylprop-1-yl] purine

2-amino-9-[(2-isopropyl-1,3-dioxan-5-yl) methyl] purine (160 mg, 0.58 mmol) was stirred in trifluoroacetic acid (5 mL) for 2 hours. Thereafter, a few drops of water were added and the reaction mixture was further stirred for 3 hours. The solvent was removed in vacuo and the residue was neutralized with saturated sodium bicarbonate. The solution was concentrated to dryness and hot methanol was added. The solution was decanted and passed through a short silica column. The crude material was then treated by hplc chromatography using 2% acetonitrile aqueous solution as elution solvent. Fractions containing the desired product were combined and lyophilized to afford 2-amino-9- [3-hydroxy-2-hydroxymethylprop-1-yl] purine (70 mg, 55%) as a colorless fuzzy solid state. Got it. ¹ H nmr (DMSOd ₆ ) 2.00-2.20, m, 1H, 3.20-3.40, m, 4H; 4.06, d, J = 7 Hz, 2H; 4.68, br s, 2H; 6.53, s, 2 H; 8.00, s, 1 H; 8.57, s, 1 H.

Example 10

9- [3-hydroxy-2-hydroxymethylprop-1-]-guanine sodium salt

9- [3-hydroxy-2-hydroxymethylprop-1-yl] -guanine (147.5 mg, 0.616 mmol) was dissolved in aqueous sodium hydroxide (1.0 M, 616 μl, 0.616 mmol). The solution was filtered, washed with a little water and lyophilized to give 9- [3-hydroxy-2-hydroxymethylprop-1-yl] -guanine sodium salt (160.7 mg, quantitative) as a colorless fuzzy appearance. Obtained in a solid state. ¹ H nmr (DMSOd ₆ ) 1.78-2.02, m, 1H; 3.06-3.29, m, 4H; 3.91, d, J = 6 Hz, 2H; 5.16, br s, 2H; 5.42, br s, 2 H; 7.32, s, 1 H.

Example 11

9- [3-acetoxy-2-hydroxymethylprop-1-yl] -guanine

Trifluoroacetic acid in a solution of 9- [3-hydroxy-2-hydroxymethylprop-1-yl] -guanine (1.0 g, 4.18 mmol) in N, N-dimethylformamide (5 mL) under nitrogen atmosphere. (510 μl, 6.63 mmol) and triethylorthoacetate (790 μl, 4.31 mmol) were added to form a turbid mixture. The reaction was monitored by hplc method (5% acetonitrile aqueous solution) and more portions of triethylorthoacetate (720 μl, 3.92 mmol) were added dropwise until the reaction was complete. Water (243 μl, 13.5 mmol) was added and the reaction stirred for 1.5 hours more. The reaction mixture was neutralized with sodium bicarbonate and the solvent was removed at room temperature. The crude product was recrystallized from water to give a colorless product (901 mg). This was presorbed and treated by flash chromatography on silica gel eluting with 10%, 15% and 17% methanol in dichloromethane. Fractions containing the purified product were combined and concentrated to give 9- [3-acetoxy-2-hydroxymethylprop-1-yl] -guanine (520 mg, 44%) as a colorless solid. . ¹ H nmr (DMSOd ₆ ) 1.94, s, 3H; 2.20-2.40, m, 1 H; 3.26-3.43, m, 2 H; 3.84-4.03, m, 4 H; 4.82, t, J = 5 Hz, 1 H; 6.46, s, 2 H; 7.64, s, 1 H; 10.58, s, 1 H.

Example 12

9- [3-hydroxy-2-hydroxymethylprop-1-yl] -guanine triphosphate tetraammonium salt

Ludwig et al. Were used. A solution of 2-chloro-4H-1,3,2-benzodioxaphosphorin-4-one (150 mg, 0.74 mmol) in dry dioxane (2 mL) was dried with N, N-dimethylformamide (10 mL). And stirred 9- [3-acetoxy-2-hydroxymethylprop-1-yl] -guanine (187.8 mg, 0.668 mmol, in high temperature at 85 ° C. for about 7-8 hours in dry pyridine (2 mL). Dried) to the solution for about 5 minutes. Stirring was continued for 0.75 hours. Then, this stirred solution was dissolved in anhydrous N, N-dimethylformamide (2.5 mL) containing dry n-Bu ₃ N (0.75 mL), and bis [(tri-n-butyl) ammonium] pyrophosphate hemi DMF (590 mg, 1.0 mmol) was added dropwise.

After stirring for about 2.5 hours at room temperature, the yellow reaction solution was treated by dropwise addition of an iodine solution (9.5 mL) prepared by dissolving iodine (3.56 g) in pyridine (200 mL) and water (5 mL). Slight excess of iodine added degraded as soon as a few drops of 5% NaHSO ₃ solution was added.

After stirring at room temperature for about 1.5 hours, the solvent was removed at a temperature below 30 ° C. to give an orange-yellow oil. It was treated with 25 mL of water with vigorous stirring for 1 hour at room temperature. Concentrated NH ₄ OH (50 mL) was added and the reaction mixture was stirred at room temperature for 3 hours, then the NH ₄ OH solution was removed at 25 ° C. The semisolid product was treated twice with acetone to give a pale yellow solid (0.56 g).

The product (0.5 g) was dissolved in a small amount of water and centrifuged to a volume of 5 ml, which was then treated by HPLC chromatography with 0.5 ml of water as eluent (flow rate 12 ml / min). It was. Fractions between 13-14 minutes and 18-19 minutes were collected and lyophilized to give a pale yellow solid (320 mg).

The material was dissolved in water (4 mL) and it was chromatographed again in a 0.5 mL lot. For each fraction, head and tail sections of the peak containing triphosphate were taken. This was repeated four more times. Finally the product was dialyzed (100 MWCO tube, 6.5 h, H ₂ O) and then purified again by HPLC. By this treatment, a small amount of phosphorus impurities (small P nmr peak at δ + 0.93) due to some inorganic phosphates were predicted.

¹ H nmr (D ₂ O); δ 2.41, s, 1H; 3.60, d, J = 5.38 Hz, 2H; Overlapping doublet 4.00, d, J = 4.98 and 4.17 Hz, d, J = 5.76 Hz, 4H; 7.87, s, 0.8 H.

³¹ P nmr (D ₂ O): At nmr of crude triphosphate, phosphate peaks were best identified; That is, P _β (t, δ-21.16, J _PP = 19.66 Hz); P _α (double line of triplets at β-9.87 (J _PP 19.40 Hz, J _PH 5.39 Hz); Pα (d, δ-5.96; J _PP 20.01 Hz). ³¹ P nmr peaks widened, shifted by HPLC purification The purified sample has two broad ³¹ P peaks when δ-9.96 (Pα + Pα) and δ-22.07 (Pβ) are at an intensity ratio of 2: 1.

Example 13

2-amino-6-cyclopropylamino-9- [3-hydroxy-2-hydroxymethylprop-1-yl] purine

9- [3-acetyloxy-2-acetyloxymethylprop-1] -2-amino-6-iodopurine (557 mg, 1.24 mmol) and cyclopropylamine (1.0 mL, 14.4 in 80 ° C., Bomb) mmol) was stirred for 18 h. The reaction mixture was cooled to room temperature and concentrated to give crude orange oil (989 mg). Thereafter, part of this was treated by hplc chromatography using 3% acetonitrile aqueous solution as elution solvent. 2-amino-6-cyclopropylamino-9- [3-hydroxy-2-hydroxymethylprop-1-yl] in the form of a yellow oil which solidifies upon standing by combining the fractions containing the desired product and freeze drying. Obtained Purine. ¹ H nmr (DMSOd ₆ ) 0.55-0.76, m, 4H; 1.91-2.12, m, 1 H; 2.95-3.13, m, 1 H; 3.21-3.45, m, 4H; 3.97, d, J = 7 Hz, 2H; 4.75, t, J = 5 Hz, 2H; 5.91, br s, 2 H; 7.09, d, J = 5 Hz, 1 H; 7.62, s, 1 H.

Example 14

9- [3-acetyloxy-2-acetyloxymethylprop-1-yl] -2-aminopurine

9- [3-acetyloxy-2-acetyloxymethylprop-1-yl] -2-amino-6-iodopurine (5.0 g, 11.5 mmol) in ethanol (200 mL) according to the method of example 8 , 9- [3-acetyloxy-2-acetyloxymethylprop-1-yl] -2-aminopurine was obtained from triethylamine (1.76 mL, 12.65 mmol) and 10% palladium on carbon (500 mg). Yield: 2.33 g (66%). ¹ H nmr (DMSOd ₆ ) 1.93, s, 6H; 2.59-2.80, m, 1 H; 3.96, dd, J = 5.6 & 11.4 Hz, 2H; 4.03, dd, J = 5.6 & 11.4 Hz, 2H; 4.15, d, J = 7 Hz, 2H; 6.49; br s, 2 H; 8.04, s, 1 H; 8.57, s, 1 H.

Example 15

9- [3-acetyloxy-2-acetyloxymethylprop-1-yl] -guanine

A mixture of 9- [3-hydroxy-2-hydroxymethylprop-1-yl] -guanine (560 mg, 2.34 mmol) and 4-dimethylaminopyridine (50 mg) in acetic anhydride (15 mL) was stirred at room temperature. Stirred for 16 h. The reaction mixture was concentrated to dryness and the residue partitioned between water and chloroform. The obtained solid was separated by filtration and recrystallized from methanol to obtain 9- [3-acetyloxy-2-acetyloxymethylprop-1-yl] -guanine (593 mg, 78%) in a colorless crystal state. ¹ H nmr (DMSOd ₆ ) 1.97, s, 6H; 2.53-2.70, m, 1 H; 3.87-4.09, m, 6 H; 6.42, br s, 2 H; 7.67, s, 1 H; 10.56, br s, 1 H.

Examples 16 and 17

9- [2-L-valyloxymethyl-3-L-valyloxyprop-1-yl] -guanine bishydrochloride and 9- [3-hydroxy-2-L-valyloxyprop-1-yl] -Guanine hydrochloride

9- [3-hydroxy-2-hydroxymethylprop-1-yl] -guanine (3.02 g, 12.6 mmol) in N, N-dimethylformamide (75 mL) under N atmosphere, N-benzyloxycarr Bonyl-L-valyl-N-carboxy anhydride (ZL-valyl-NCA) (purchased from Isochem or SNPE North American Inc.) (3.69 g, 13.32 mmol) And a mixture of 4-dimethylaminopyridine (75 mg) at room temperature for 16 hours. HPLC analysis found the reaction to be incomplete. An additional fraction of ZL-valyl-NCA (1.84 g, 6.67 mmol) was added and the reaction mixture was stirred for a further 24 hours. It was concentrated to dryness and ethyl acetate was added to precipitate unreacted 9- [3-hydroxy-2-hydroxymethylprop-1-yl] -guanine. The filtrate was concentrated and preadsorbed on silica and then treated by flash chromatography on silica using 5%, 7%, 10% and 15% methanol in dichloromethane as the elution solvent. Fractions containing a single point on thin layer chromatography (tlc) were combined to give 9- [2- (N-benzyloxycarbonyl-L-valyloxymethyl) -3- (N-benzyloxycarbonyl-L in the colorless solid state. -Valyloxyprop-1-yl] guanine (2.54 g) ¹ H nmr (DMSO-d ₆ ) 0.86, d, 12H; 1.85-2.15, m, 2H; 2.51-2.70, m, 1H; 3.80 -4.18, m, 8H; 5.04, s, 4H; 6.41, br s, 2H; 7.31, br s, 10H; 7.59, s, 1H; 7.75, d, J = 8 Hz, 2H; 10.62, s, 1H.

The remaining fractions were combined and then treated again by chromatography using 5%, 7%, 10% and 15% methanol in dichloromethane as the elution solvent to give additional divalyl compound (229 mg) and a colorless solid (880 mg). Got. This was dissolved in dichloromethane, washed with sodium bicarbonate (2 ×) and brine, dried over magnesium sulfate, and dried to a colorless form of 9- [2- (N-benzyloxycarbonyl-L-valyloxymethyl) as a colorless foam. 3-hydroxyprop-1-yl] guanine (266 mg) was obtained. ¹ H nmr (DMSOd ₆ ) 0.86, d, J = 6.8 Hz, 6H; 1.88-2.11, m, 1 H; 2.20-2.39, m, 1 H; 3.21-3.43, m, 2 H; 3.78-4.10, m, 5H; 4.82, t, J = 5 Hz, 1 H; 5.04, s, 2 H; 6.46, br s, 2H; 7.34, br s, 5 H; 7.61, d, J = 3 Hz, 1 H; 7.65-7.75, m, 1 H; 10.59, br s, 1 H.

9- [2-L-valyloxymethyl-3-L-valyloxyprop-1-yl] guanine bishydrochloride

9- [2- (N-benzyloxycarbonyl-L-valyloxymethyl) -3- (N-benzyloxycarbonyl-L-valyloxyprop-1-yl] guanine (858 in ethanol (50 mL) Mg, 1.22 mmol), a mixture of 1 M aqueous hydrochloric acid (2.44 mL, 2.43 mmol) and 10% palladium on carbon (215 mg) was stirred under hydrogen atmosphere for 3 hours The reaction mixture was washed repeatedly with ethanol to remove GFA paper. The filtrate was concentrated to dryness at 40 ° C. The residue was filtered through a short column dissolved in water and packed with GFA paper, Celite 577 and more GFA paper The filtrate was lyophilized to cream Obtained 9- [2-L-valyloxymethyl-3-L-valyloxyprop-1-yl] guanine bis hydrochloride (517 mg, 83%) in the solid state ¹ H nmr (DMSO-d ₆ ) 0.87 -1.02, m, 12H; 2.05-2.30, m, 2H; 2.60-2.80, m, 1H; 3.85, dd, J = 4.6 & 11.1 Hz, 2H; 4.00-4.32, m, 6H; 6.59, s, 2H; 7.78, s, 1 H; 8.63, br s, 6 H; 10.78, br s, 1 H.

9- [3-hydroxy-2-L-valyloxymethyl-3-L-valyloxyprop-1-yl] guanine hydrochloride

9- [2- (N-benzyloxycarbonyl-L-valyloxymethyl) -3-hydroxyprop-1-yl] -guanine (212 mg, 0.45 mmol) in ethanol (15 mL), 1 M aqueous 9- [3-hydroxy-2-L-valyloxymethylprop-1-yl] guanine hydrochloride according to the method of Example 16 from hydrochloric acid (0.45 mL, 0.45 mmol) and 10% palladium on carbon (56 mg) Was obtained as a colorless fuzzy solid state product (162 mg, 96%). ¹ H nmr (DMSOd ₆ ) 0.96, dd, J = 7.2 & 11.9 Hz, 6H; 2.00-2.25, m, 1 H; 2.25-2.47, m, 1 H; 3.31-3.51, m, 2H; 3.82, dd, J = 4.6 & 13.5 Hz, 1H; 3.96-4.14, m, 4H; 4.91, br s, 1 H; 6.59, br s, 2 H; 7.69, s, 1 H; 8.52, br s, 3 H; 10.72, br s, 1 H.

Example 18

9- [3-acetoxy-2-L-valyloxymethyl) prop-1-yl] -guanine hydrochloride

9- [3-acetyloxy-2-hydroxymethylprop-1-yl] guanine (330 mg, 1.17 mmol) in N, N-dimethylformamide (20 mL) under nitrogen atmosphere, ZL-valyl-NCA ( 358 mg, 1.29 mmol) and 4-dimethylaminopyridine (30 mg) were stirred at room temperature for 2 days. The reaction mixture was concentrated to dryness, and the residue was preadsorbed on silica and then treated by flash chromatography using 10% methanol in dichloromethane. Fractions containing the desired product were combined and then dried to give 9- [3-acetoxy-2- (N-benzyloxycarbonyl-L-valyloxy methyl) prop-1-yl] guanine (in a colorless solid). 313 mg, 52%). ¹ H nmr (DMSOd ₆ ) 0.87, d, J = 7 Hz, 6H; 1.97, s, 3 H; 1.88-2.13, m, 1 H; 2.53-2.71, m, 1 H; 3.82-4.15, m, 7H; 5.04, s, 2 H; 6.43, br s, 2 H; 7.35, br s, 5H, 7.63, d, J = 3.6 Hz, 1H; 7.75, d, J = 8.1 Hz, 1 H.

9- [3-acetoxy-2- (N-benzyloxycarbonyl-L-valyloxymethyl) prop-1-yl] -guanine (201 mg, 0.39 mmol) in ethanol (10 mL), 1 M aqueous 9- [3-acetoxy-2-L-valyloxymethyl) prop-1-yl]-according to the method of Example 16 from hydrochloric acid (0.39 mL, 0.39 mmol) and 10% palladium on carbon (62 mg)- Guanine hydrochloride was obtained as a colorless fuzzy solid state product (162 mg, quantitative). ¹ H nmr (DMSOd ₆ ) 0.95, dd, J = 3.8 & 6.8 Hz, 6H; 1.99, s, 3 H; 2.03-2.25, m, 1 H; 2.57-2.78, m, 1 H; 3.84, dd, J = 4.7 & 10.2 Hz, 1H; 3.93-4. 23, m, 6H; 6.54, br s, 2 H; 7.71, s, 1 H; 8.33, br s, 3 H; 10.71, br s, 1 H.

Example 19

9- [3-hydroxy-2-palmityloxymethylprop-1-yl] guanine

Palmitoyl chloride (2.07 g, 7.53 mmol) was dissolved in anhydrous dichloromethane and made up to 10 ml in volume to serve as mother liquor.

Of 9- [3-hydroxy-2-hydroxymethylprop-1-yl] guanine (1.0 g, 4.18 mmol) in pyridine (20 mL) and N, N-dimethylformamide (10 mL) under nitrogen atmosphere. To the suspension was added a mother liquor of palmitoyl chloride (3.5 mL). The reaction mixture was stirred at rt for 16 h and an additional fraction of the mother liquor (3.5 mL) was added. The reaction mixture was stirred for 24 hours and the last fraction of mother liquor was added. The reaction mixture was stirred for 2 more days and the solvent was removed at 60 ° C. under vacuum. The crude solid (3.7 g) was presorbed and treated by flash chromatography on silica eluting with 5% to 23% methanol in dichloromethane. Fractions containing the purified product were combined to give 9- [3-hydroxy-2-palmityloxymethylprop-1-yl] guanine (599 mg) as a colorless solid. ¹ H nmr (DMSOd ₆ ) 0.85, t, J = 6.8 Hz, 3H; 1.12-1.38, m, 24 H; 1.33-1.60, m, 2H; 2.19, q, J = 7.2 Hz, 2H; 3.32-3.40, m, 2H; 3.95, t, J = 6 Hz, 2H; 4.07-4.20, m, 2 H; 4.87, br s, 1 H; 6.65, br s, 2H; 7.63, s, 1 H; 10.76, br s, 1 H.

Example 20

9- [3-palmityloxy-2-L-valyloxymethylprop-1-yl] guanine hydrochloride

9- [3-hydroxy-2-palmityloxymethylprop-1-yl] guanine (534 mg, 1.12 mmol), ZL-valyl-NCA in N, N-dimethylformamide (25 mL) under nitrogen atmosphere. A mixture of (620 mg, 2.24 mmol) and 4-dimethylaminopyridine (25 mg) was stirred at rt for 16 h. The solvent was removed in vacuo at 60 ° C. and the residue was partitioned between dichloromethane and water. The layers were separated and the aqueous layer was extracted twice more with dichloromethane. The combined organic layers were washed with saturated sodium bicarbonate solution (2 ×) and brine, dried over magnesium sulfate and concentrated to give crude product (509 mg) as a colorless oil. This was treated by flash chromatography on silica eluting with 5% methanol in dichloromethane. Fractions containing the purified product were combined to give 9- [3-palmityloxy-2- (N-benzyloxycarbonyl-L-valyloxymethyl) prop-1-yl] guanine (290 mg) as a colorless foamy solid. ) ¹ H nmr (DMSOd ₆ ) 0.75-0.95, m, 9H, 1.22, br s, 24H; 1.33-1.60, m, 2H; 1.90-2.15, m, 1 H; 2.23, t, J = 7.2 Hz, 2H; 2.50-2.75, m, 1 H; 3.85-4.14, m, 7H; 5.04, s, 2 H; 6.45, br s, 5H; 7.62, d, J = 3.8 Hz, 1 H; 7.74, d, J = 8.2 Hz, 1H; 10.66, br s, 1 H.

9- [3-palmityloxy-2- (N-benzyloxycarbonyl-L-valyloxymethyl) prop-1-yl] guanine (174 mg, 0.24 mmol) in ethanol (10 mL), 1 M aqueous 9- [3-palmityloxy-2-L-valyloxymethylprop-1-yl] guanine according to the method of Example 16 from hydrochloric acid (0.24 mL, 0.24 mmol) and 10% palladium on carbon (50 mg) Hydrochloride was obtained as a colorless fuzzy solid state product (124 mg, 84%). ¹ H nmr (DMSOd ₆ ) 0.78-0.98, m, 9H; 1.22, br s, 24 H; 1.37-1.58, m, 2H; 1.99-2.22, m, 1 H; 2.25, t, J = 7.2 Hz, 2H; 2.55-2.76, m, 1 H; 3.74, dd, J = 4.8 & 10.0 Hz, 1H; 3.90-4.20, m, 6H; 6.51, br s, 2H; 7.30-8.10, br s, 3 H; 7.69, s, 1 H; 10.88, br s, 1 H.

Example 21

9- [3-hydroxy-2-cholyloxymethylprop-1-yl] -guanine

Ethylchloroformate (400) in a solution of choline acid (1.71 g, 4.18 mmol) and diisopropylethylamine (567 mg, 4.39 mmol) in N, N-dimethylformamide (25 mL) at 10 ° C. under a nitrogen atmosphere. Μl, 4.18 mmol) was added. After stirring for 30 minutes, a mixture of 9- [3-hydroxy-2-hydroxymethylprop-1-yl] guanine (1.0 g, 4.18 mmol) in N, N-dimethylformamide (100 mg) was removed. Was added and the reaction mixture was stirred for 2 more days. HPLC analysis (70% aqueous methanol solution) found that about 20-30% of the product was formed. The reaction mixture was concentrated to dryness and methanol was added to precipitate unreacted 9- [3-hydroxy-2-hydroxymethylprop-1-yl] guanine. The filtrate was concentrated to dryness to give a yellow oil (2.0 g). This was purified by semi-preparative HPLC eluting with 70% aqueous methanol solution to obtain 9- [3-hydroxy-2-cholyloxymethylprop-1-yl] guanine (124 mg) as a colorless glassy solid. ¹ H nmr (DMSOd ₆ ) 0.57, s, 3H; 0.80, s, 3 H; 0.70-2.40, m, 27 H; 3.08-3.28, m, 2 H; 3.37, d, J = 5.2 Hz, 2H; 3.61, s, 1 H; 3.78, s, 1 H; 3.87-4.05, m, 5H; 4.12, d, J = 3.4 Hz, 1 H; 4.33, d, J = 4.3 Hz, 1 H; 4.82, t, J = 4.5 Hz, 1 H; 6.52, s, 2 H; 7.63, s, 1 H; 10.74, br s, 1 H.

Example 22

9- [3-Colyloxy-2-L-valyloxy-methylprop-1-yl] guanine hydrochloride

Crude 9- [3-hydroxy-2-cholyloxymethylprop-1-yl] -guanine (1.21 g, 1.92 mmol) in N, N-dimethylformamide (10 mL), ZL-valyl-NCA ( 0.99 g, 3.57 mmol) and 4-dimethylaminopyridine (20 mg) according to the method of Example 20 9- [2- (N-benzyloxycarbonyl-L-valyloxy-3-colyloxymethylprop- 1-yl] -guanine was prepared The crude material was treated by flash chromatography on silica eluting with 10% methanol in dichloromethane The fractions containing the purified product were combined to give 9- [in a colorless solid form. 2- (N-benzyloxycarbonyl-L-valyloxy-3-colylmethylprop-1-yl] guanine (258 mg) was obtained: ¹ H nmr (DMSO-d ₆ ) 0.57, s, 3H; 0.80 , s, 3H; 0.74-2.40, m, 34H; 2.50-2.78, m, 1H; 3.05-3.30, m, 2H; 3.60, s, 1H; 3.77, s, 1H; 3.83-4.20, m, 8H; 4.33 , d, J = 4.3 Hz, 1H; 5.04, s, 2H; 6.44, br s, 2H; 7.34, br s, 5H; 7.62, d, J = 3.7 Hz, 1H; 7.74, d, J = 8.2 Hz, 1H; 10.61, br s, 1H.

9- [2- (N-benzyloxycarbonyl-L-valyloxy-3-cholyloxymethylprop-1-yl] guanine (248 mg, 0.29 mmol) in ethanol (10 mL), 1 M aqueous hydrochloric acid ( 0.29 mL, 0.29 mmol) and 10% palladium on carbon (30 mg) were prepared according to the method of Example 16, according to the method of Example 16, to obtain 9- [3-collyloxy-2-L-valyloxy-methylprop-1-yl] guanine hydrochloride. Obtained as a cream solid product (185 mg, 84%).

¹ H nmr (DMSOd ₆ ) 0.57, s, 3H; 0.80, s, 3 H; 0.74-2.40, m, 34H; 2.50-2.78, m, 1 H; 3.05-3.30, m, 2H; 3.60, s, 1 H; 3.77, s, 1 H; 3.85, dd, J = 4.6 & 10.5 Hz, 1H; 3.93-4.26, m, 7H; 4.34, d, J = 4.0 Hz, 1 H; 6.56, br s, 2 H; 7.71, s, 1 H; 8.25-8.80, br s, 3H; 10.72, br s, 1 H.

Example 23

9- [2-Elyridyloxy-3-hydroxy-methylprop-1-yl] guanine

Elidoyl chloride (3.1 g, 9.70 mmol) and 9- [3-hydroxy-2- dissolved in anhydrous dichloromethane (7 mL), pyridine (25 mL) and N, N-dimethylformamide (12 mL) 9- [2-Elyridyloxy-3-hydroxy-methylprop-1-yl] guanine was prepared from hydroxymethylprop-1-yl] guanine (1.3 g, 5.43 mmol) according to the method of Example 19. Prepared. After operation, some of the elide acid was distilled off to give a crude yellow solid (2.14 g). This was treated by flash chromatography on silica eluting with 7.5% methanol in dichloromethane. Fractions containing the purified product were combined to give 9- [2-erylidyloxy-3-hydroxy-methylprop-1-yl] guanine (640 mg). ¹ H nmr (DMSOd ₆ ) 0.84, t, J = 6.7 Hz, 3H; 1.23, br s, 20 H; 1.30-1.59, m, 2H; 1.85-2.00, m, 4H; 2.20, t, J = 7.3 Hz, 2H; 2.22-2.41, m, 1 H; 3.36, d, J = 5.4 Hz, 2H; 3.85-4.07, m, 4H; 4.82, t, J = 5 Hz, 1 H; 5.23-5.48, m, 2H; 6.44, br s, 2 H; 7.63, s 1 H; 10.58, br s, 1 H.

Example 24

9- [2-stearoyloxy-3-valyloxymethyl) prop-1-yl] guanine hydrochloride

9- [2-erylidyloxy-3-hydroxymethylprop-1-yl] guanine (200 mg, 0.40 mmol), ZL-valyl-NCA (121 mg, 0.44 mmol) and 4-dimethylamino under nitrogen atmosphere The mixture of pyridine (5 mg) was stirred for 3 days at room temperature. An additional amount of ZL-valyl-NCA (40 mg, 0.14 mmol) was added and the reaction stirred at 40 ° C. for 6 hours and then at room temperature for 16 hours. The solvent was removed in vacuo at 60 ° C., the residue was pre-adsorbed and treated by flash chromatography on silica eluting with 6% methanol in dichloromethane to give 9- [2- (N-benzyl) as a colorless solid. Oxycarbonyl-L-valyloxy-3-erylidyloxy-methyl) prop-1-yl] guanine (174 mg, 60%) was obtained. ¹ H nmr (DMSOd ₆ ) 0.78-0.93, m, 9H; 1.22, br s, 20 H; 1.30-1.59, m, 2H; 1.89-2.13, m, 5H; 2.23, t, J = 7.2 Hz, 2H; 2.50-2.72, m, 1 H; 3.85-4.14, m, 6H; 5.04, s, 2 H; 5.24-5.45, m, 2H; 6.43, s, 2 H; 7.34, br s, 5 H; 7.61, d, J = 3.9 Hz, 1 H; 7.74, d, J = 8.1 Hz, 1H; 10.64, br s, 1 H.

9- [2- (N-benzyloxycarbonyl-L-valyloxy-3-erylidyloxy-methyl) prop-1-yl] guanine (162 mg, 0.21 mmol), 1 M in ethanol (10 mL) 9- [2-stearoyloxy-3-valyloxymethyl) prop-1-yl] from aqueous hydrochloric acid (0.22 mL, 0.22 mmol) and 10% palladium on carbon (60 mg) according to the method of Example 16 Guanine hydrochloride was obtained as a colorless fuzzy solid product (108 mg, 77%). ¹ H nmr (DMSOd ₆ ) 0.84, t, J = 6.3 Hz, 3H; 0.93, dd, J = 3.2 & 6.8 Hz, 6H; 1.22, br s, 28 H; 1.36-1.57, m, 2H; 1.98-2.23, m, 1 H; 2.25, t, J = 7.3 Hz, 2H; 2.55-2.75, m, 1 H; 3.77, dd, J = 4.6 & 10.0 Hz, 1H; 3.90-4.22, m, 6 H; 6.51, br s, 2H; 7.69, s, 1 H; 7.65-8.15, br s, 3 H; 10.48-10.82, br s, 1 H.

Example 25

2-amino-9- [2-L-valyloxy-3-L-valyloxymethylprop-1-yl] purine bis hydrochloride

2-amino-9- [3-hydroxy-2-hydroxymethylprop-1-yl] purine (9) (500 mg, 2.24 mmol), ZL-valyl-NCA (1.30 mg, 4.70 mmol) under nitrogen atmosphere ) And 4-dimethylaminopyridine (5 mg) were stirred at room temperature for 18 hours. The reaction mixture was concentrated to dryness and the residue was treated by chromatography on silica with ethyl acetate as the eluting solvent to give 2-amino-9- [2- (N-benzyloxycarbonyl-L-valyl) as a honey colored glassy solid. Oxymethyl) -3- (N-benzyloxycarbonyl-L-valyloxyprop-1-yl] purine (1.12 g, 73%) was obtained ¹ H nmr (DMSO-d ₆ ) 0.86, d, J = 6.7 Hz, 12H; 1.85-2.15, m, 2H; 2.57-2.78, m, 1H; 3.83-4.19, m, 8H; 5.04, s, 4H; 6.49, s, 2H; 7.34, br s, 10H; 7.76 , d, J = 8.2 Hz, 2H; 7.96, s, 1H; 8.59, s, 1H.

2-amino-9- [2- (N-benzyloxycarbonyl-L-valyloxymethyl) -3- (N-benzyloxycarbonyl-L-valyloxyprop-1-yl in ethanol (25 mL) ] Purine (500 mg, 0.73 mmol), 1 M aqueous hydrochloric acid (1.45 mL, 1.45 mmol) and 10% palladium on carbon (90 mg) according to the method of Example 16 according to the method of Example 16 2-amino-9- [2-L- Valyloxy-3-L-valyloxymethylprop-1-yl] purine bishydrochloride was obtained as a colorless fuzzy solid product (319 mg, 89%), ¹ H nmr (DMSO-d ₆ ) 0.87-1.03 , m, 12H; 2.03-2.27, m, 2H; 2.70-2.80, m, 1H; 3.83, dd, J = 4.8 & 11.2 Hz, 2H; 4.07-4.42, m, 6H; 6.50, s, 2H; 8.15- 8.88, br s, 3 H; 8.20, s, 1 H; 8.60, s, 1 H.

Example 26

9- [3-acetoxy-2-acetoxymethylprop-1-yl] -6-thioguanine

9- [3-acetoxy-2-acetoxymethylprop-1-yl] -2-amino-6-iodopurine (576 mg, 1.33 mmol) and thiourea in ethanol (7 mL) under nitrogen atmosphere. 100 mg, 1.33 mmol) was heated to reflux for 1 hour. The reaction mixture was cooled while washing with ethanol and the product was filtered off. The light lemon solid was dried in vacuo at 80 ° C. to obtain 9- [3-acetoxy-2-acetoxymethylprop-1-yl] -6-thioguanine (288 mg, 64%). ¹ H nmr (DMSOd ₆ ) 1.98, s, 6H; 2.52-2.74, m, 1 H; 3.87-4.13, m, 6 H; 6.77, s, 2 H; 7.88, s, 1 H; 11.89, s, 1 H.

Example 27

Antiviral activity

Antiviral activity in human cells infected with hepatitis B virus was tested according to the method of Korba and Gerin. Effective concentrations that inhibited viral replication by 50% and 90% were determined via the dose response curve. The results for some compounds of the invention are listed in Table 2 below.

Test compound EC ₅₀ μM EC ₉₀ μM Example 1 0.16 1.9 Example 13 4.4 13

Example 28

Bioavailability Test

Oral bioavailability of the various compounds of the invention was compared in the rats. Briefly, the compounds were administered by oral gavage at 0.2 mmol / kg body weight. The compounds were suspended in 1 ml of an aqueous excipient containing 1% carboxymethylcellulose and 0.05% Tween 80. Plasma was sampled for 8 hours to determine the concentration of the parent compound (in this case, the compound of Example 1) by the hplc method.

Plasma fractions of rats (150 μl) were acidified with 10% trichloroacetic acid (37.5 μl) and centrifuged at 3000 rpm for 10 minutes. The supernatant was filtered through a 0.22 μm cellulose acetate centrifugal filter. Samples (100 μl) were then injected into C18 Waters Symmetry HPLC columns (3.9 and 150 mm, 5 μm) equilibrated at 40 ° C. Two-component mobile phase (A-0.05% trifluoroacetic acid and 20 mM heptane sulfonic acid in distilled, deionized water; B-0.05% trifluoroacetic acid, 20 mM heptane sulfonic acid in distilled, deionized water and 70% acetonitrile) was pumped at a rate of 0.5 ml / min. The% of mobile phase component B increased linearly from 0% to 25% for 25 minutes. Analysis was performed by ultraviolet detection at 254 nm. The parent compound eluted at 17 minutes.

The area under the curve was measured by plotting drug concentration versus time pattern. This was compared to the area under the curve obtained by intravenous administration of the sodium salt of the parent compound to provide a measure of total bioavailability in%. The results are listed in Table 3 below.

Test compound Bioavailability% Example 1 2.8 Example 9 53.7 Example 14 66.2 Example 16 16.3 Example 17 33.1 Example 18 6.5 Example 20 21

Those skilled in the art may make various changes and / or modifications to the present invention without departing from the spirit of the invention. Therefore, the present embodiments and the detailed description are to be considered in all respects as illustrative of the present invention and are not intended to limit the present invention.

The steps, features, compositions and compounds are disclosed, referred to or indicated in the description and / or claims of the invention individually, in combination and in any two or more of the above steps or features.

Throughout this specification and the appended claims, unless otherwise indicated, the expression "comprise" or a variation thereof, "comprising" means that a component or step disclosed in the specification or components or steps thereof is disclosed. It is to be understood as referring to a group, but does not exclude other elements or steps or groups of elements or steps.

references

Antimicrob of Cullen et al. Agents Chemother., 41 (10), 2076-82 (1997)

Martin et al. J. Med. Chem. 29, 1384-1389 (1986)

Green T. Double U., Protective Groups in Organic Synthesis, John Wiley and Sons, Inc. (1991)

Bissach et al. J. Org. Chem. 60, 2902-2905 (1995).

Harden et al. J. Med. Chem. 33, 187-196 (1990)

Antiviral Research, 19, 55-70 (1992) by Corva and Gerin

Eliel et al. J. Am. Chem. Soc. 94 (1), 171-176 (1972)

Dun et al. J. Org. Chem. 55, 6368-6373 (1990)

Ludwig et al. J. Org. Chem., 54, 631-635 (1989)

Claims (14)

A method of using the compound of formula 1 or a salt thereof and a pharmaceutically acceptable derivative thereof for the treatment and / or prevention of hepatitis B virus infection: Formula 1 In the above formula, R ¹ is hydrogen, halogen, hydroxy, azido, alkoxy, aryloxy, thio, alkylthio, amino, alkylamino, hydrazino, hydroxylamino, benzyloxy, NRR 'or NRCOR', R ² is hydrogen, halogen, hydroxy, azido, alkoxy, aryloxy, thio, alkylthio, amino, alkylamino, hydrazino, hydroxylamino, benzyloxy, NRR 'or NRCOR', R and R 'are independently selected from hydrogen, alkyl and aryl. The method of claim 1, R ¹ is a hydroxy or a group which can be converted to hydroxy in vivo. The method according to claim 1 or 2, R ² is characterized in that it is amino or a group which can be converted to amino in vivo. The method according to any one of claims 1 to 3, Wherein the compound of Formula 1 or a salt thereof or a pharmaceutically acceptable derivative thereof is a compound of Formula 4 or a salt thereof: Formula 4 In the above formula, X is hydrogen or hydroxy, R ⁵ and R ⁶ are the same or different and together with the oxygen atom to which they are attached form a hydroxy group, ester, carbonate, carbamate or thiocarbonate. The method of claim 4, wherein R ⁵ and R ⁶ are the same or different and are characterized in that together with the oxygen atom to which they are attached form a hydroxy group or an ester. The method of claim 1, R ¹ is hydroxy and R ² is amino. A method of treating or preventing hepatitis B virus infection characterized by administering to a patient with hepatitis B virus infection an effective amount of a compound of formula (1) or a salt thereof or a pharmaceutically acceptable derivative thereof: Formula 1 In the above formula, R ¹ is hydrogen, halogen, hydroxy, azido, alkoxy, aryloxy, thio, alkylthio, amino, alkylamino, hydrazino, hydroxylamino, benzyloxy, NRR 'or NRCOR', R ² is hydrogen, halogen, hydroxy, azido, alkoxy, aryloxy, thio, alkylthio, amino, alkylamino, hydrazino, hydroxylamino, benzyloxy, NRR 'or NRCOR', R and R 'are independently selected from hydrogen, alkyl and aryl. A method of using a compound of formula 1 or a salt thereof or a pharmaceutically acceptable derivative thereof for the preparation of a medicament for the treatment or prevention of hepatitis B virus infection: Formula 1 In the above formula, R ¹ is hydrogen, halogen, hydroxy, azido, alkoxy, aryloxy, thio, alkylthio, amino, alkylamino, hydrazino, hydroxylamino, benzyloxy, NRR 'or NRCOR', R ² is hydrogen, halogen, hydroxy, azido, alkoxy, aryloxy, thio, alkylthio, amino, alkylamino, hydrazino, hydroxylamino, benzyloxy, NRR 'or NRCOR', R and R 'are independently selected from hydrogen, alkyl and aryl. A pharmaceutical composition for the treatment or prophylaxis of hepatitis B virus infection comprising a compound of formula 1 or a salt or a pharmaceutically acceptable derivative thereof and a pharmaceutically acceptable carrier or diluent: Formula 1 In the above formula, R ¹ is hydrogen, halogen, hydroxy, azido, alkoxy, aryloxy, thio, alkylthio, amino, alkylamino, hydrazino, hydroxylamino, benzyloxy, NRR 'or NRCOR', R ² is hydrogen, halogen, hydroxy, azido, alkoxy, aryloxy, thio, alkylthio, amino, alkylamino, hydrazino, hydroxylamino, benzyloxy, NRR 'or NRCOR', R and R 'are independently selected from hydrogen, alkyl and aryl. A compound of Formula 1a or a salt or pharmaceutically acceptable derivative thereof: Formula 1a In the above formula, R ¹ is hydrogen, halogen, hydroxy, azido, alkoxy, aryloxy, thio, alkylthio, amino, alkylamino, hydrazino, hydroxylamino, benzyloxy, NRR 'or NRCOR', R ² is hydrogen, halogen, hydroxy, azido, alkoxy, aryloxy, thio, alkylthio, amino, alkylamino, hydrazino, hydroxylamino, benzyloxy, NRR 'or NRCOR', R and R 'are independently selected from hydrogen, alkyl and aryl, However, among the compounds of the formula 9- [3-hydroxy-2-hydroxymethylprop-1-yl] -guanine, 9- [3-hydroxy-2-hydroxymethylprop-1-yl] -adenine, 9-[(2-isopropyl-1,3-dioxan-5-yl) methyl] -guanine, and 9-[(2-isopropyl-1,3-dioxan-5-yl) methyl] -adenine is excluded. The method of claim 10, 2-amino-9- [3-hydroxy-2-hydroxymethylprop-1-yl] purine, 9- [3-acetyloxy-2-acetoxymethylprop-1-yl] -2-aminopurine, or A compound characterized by being a salt or a pharmaceutically acceptable derivative thereof. A pharmaceutical composition comprising a compound of formula 1a according to claim 10 and a pharmaceutically acceptable carrier or diluent. The method of claim 7, wherein A method of administering a compound of Formula 1 or a salt or derivative thereof, together with another agent used for the treatment of viral infection. The method of claim 7, wherein Oral administration.