HU194565B - Process for production of salts of 9-/1,3-dihydroxi-2-propoximethil/-guanin-chained-phosphor esther and antivirus compounds containing such compounds - Google Patents

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Description

FIELD OF THE INVENTION The present invention relates to novel 9- (1,3-dihydroxy-2-propoxymethyl) -glycol ring-linked los (transfer salts and antiviral compositions containing such compounds).

Viral infections are widespread and have many symptoms. Some viral infections are easily overcome by the body's defense mechanisms, while other, more serious infections can lead to permanent damage, such as blindness or even death. One group of viruses that cause serious infections are herpes viruses.

The drugs currently used to treat viral infections are often ineffective or, if they are effective, require high doses and / or continuous use, leading to serious side effects. Therefore, there is a need for an effective antiviral agent that is effective at lower doses than currently available drugs and thus reduces potential side effects.

No. 4,199,574. Patent U.S. discloses the compounds of formula (A) wherein X is sulfur or oxygen, ^R1 is hydrogen, halogen, hydroxy, alkoxy radical, azido, thio, alkylthio, amino, alkyl-amino group or a dialkylamino R ² is hydrogen, halogen, alkylthio, acylamino, amino or azido, R ³ is hydrogen, straight-chain or cyclic alkyl, hydroxyalkyl, benzyloxyalkyl or phenyl, R ⁴ is hydrogen, hydrogen, hydroxy or alkyl, RS ^is hydroxy, amino, alkyl, hydroxy-alkyl, benzyloxy, benzoyloxy, benzoyloxymethyl group, szulfamoiloxi group, a phosphate group, carboxy-propionyloxy group, a C1-8, a linear or cyclic acyloxy group such as an acetoxy group or an -NHCO group - a substituted carbamoyl group of formula Z wherein Z is alkyl, aryl or aralkyl optionally substituted with one or more sulfonyl, amino or carbamoyl groups or halogen, R ⁶ is hydrogen or alkyl, provided that when X is oxygen and R ² , R ³ , R ⁴ and R ⁶ are hydrogen, R ⁵ is hydrogen or hydroxy, then R ¹ cannot be amino or methylamino.

The known compounds of formula (A) and their pharmaceutically acceptable acid addition salts have antiviral activity. See also Tetrahedron Letters, 21, 327-330 (1980); No. 4,323,573 U.S. Patent No. 4,972; European Patent Application.]

It has been found that the cyclic phosphate esters of 9- (1,3-dihydroxy-2-propoxymethyl) -guanine, and especially the salts thereof, have particularly potent antiviral activity. According to the present invention, compounds of formula I, in which B represents a pharmaceutically acceptable salt-forming ion, preferably an alkali metal or ammonium ion, are prepared by:

a) reacting a compound of formula Xb wherein Y represents a lipophilic amino group, with a condensing agent and subjecting the resulting product to chromatography in the presence of a buffer containing ion B, preferably an alkali metal or aminomonium ion; obsession

b) reacting the 9- (1,3-dihydroxy-2-propoxymethyl) -guanine of formula (X) with a phosphorylating agent and hydrolyzing the resulting product with a strong base containing an ion B, preferably an alkali metal or anonium ion.

Examples of pharmaceutically acceptable salt-forming ions include ammonium, sodium, potassium, lithium, calcium, magnesium, and the like. It can be mentioned. Particularly preferred are potassium, sodium and ammonium.

The compounds of formula (I) have an intrinsic activity. in warm-blooded and cold-blooded animals, particularly mammals, birds and fish, but primarily humans. For example, the compounds of formula I have outstanding activity against the following viruses: herpes simplex virus I and II, and similar viruses such as cytomegalovirus, Epstein-Barr virus and chicken pox Zoster virus, and hepatitis B viruses such as hepatitis B.

Veterinary and human pharmaceutical preparations containing the compounds of formula (I) and one or more excipients customary in the pharmaceutical art are prepared by methods known per se. These methods and standard carriers are described in E. W. Martin, Reinington's Pharmaceutical Sciences, Mark Publ. Vo., 15th Edition, 1975. Liposomes may also be used for the pharmaceutical preparation of compounds of formula I in a manner known per se (e.g., F. Koka et al., Ann. Port. Biophys, Bioeng., 9, 467-508 (1980), S. E. Schullery et al., Biochemistry, 19, 3919-3923 (1980) and G. Gregoriadis et al., Liposomes in Biological Systems, John Wiley and Sons, 1980].

The compounds of formula (I) may be administered parenterally (e.g., by intravenous, subcutaneous, intreperitoneal or intramuscular injection), orally, intranasally or rectally, or may be administered topically.

For oral or parenteral administration of the pharmaceutical compositions, the dosage of the compound of formula (I) is 0.1 to 300 mg / kg, preferably 1.0 to 30 mg / kg. In the treatment of humans, a dosage unit dosage form is used, wherein the dosage unit is 10 to 500 mg of the compound of formula I, which is administered 1 to 5 times daily.

For oral administration, fine powders or granules may also contain diluents, dispersants and / or surfactants. Administration can be in water or syrup; when dry, in capsules or non-aqueous solution or suspension, which may also contain suspending agents; in the form of tablets, which may also contain binders and lubricants; or in a suspension in water or a sugar solution. If desired, the pharmaceutical composition may also contain flavoring, preservative, suspending, thickening or emulsifying agents.

Preferred are tablets and granules, which may also be coated.

The compound of formula (I) is present in the pharmaceutical composition in an amount of 0.1 to 99% by weight. The remaining 1-99.9% by weight of the vehicle is present. The pharmaceutical composition Gyo-2194,565 preferably contains 10-95% by weight of the compound of formula (I).

For parenteral administration or, for example, for the treatment of ocular infection, an aqueous solution of the compounds of the formula I in a concentration of 0.1 to 10%, preferably 0.1 to Ί%, may be used. The solution may also contain antioxidants, buffers and other suitable additives.

For the treatment of infections of the eye or other external tissues, such as the mouth and skin, it is also preferable to treat the infected body part of the patient locally. Vcgyüicic of formula (1)! containing ointments, cream, aerosol or powder. A suitable pharmaceutical preparation for this purpose is an ointment and a cream.

For example, a water-soluble ointment base may be used as the carrier for the ointment, while an oil / water cream base may be used as the cream. The concentration of the compounds of the formula I in the above pharmaceutical compositions is generally 0.01 to 10%, preferably 0.1 to 7%, particularly preferably 4.0%.

For the treatment of viral infections, the compounds of formula (I) may also be used in sustained release formulations such as those disclosed in U.S. Pat. No. 4,217,898. U.S. Pat.

For use in aerosol form, the active ingredient is preferably provided in a finely divided form, together with a surfactant and a propellant. The concentration of the active ingredient in the aerosol is generally 0.01 to 20% by weight, preferably 0.04 to 1.0% by weight.

Surfactants should be non-toxic and preferably soluble in the vehicle. For example, the following surfactants may be used: C 6 -C 22 fatty acids such as caproic acid, octanoic acid, lauric acid, palmitic acid, stearic acid, linoleic acid, linoleic acid, oleostearic acid or oleic acid, with aliphatic polyhydric alcohol or glyceryl, esters or partial esters of sorbitol or sorbitol-derived hexitan anhydrides (known as the "Span" trademark of sorbitan esters) and polyoxyethylene and polyoxypropylene derivatives of these esters. Mixed esters such as mixed or natural glycerides may also be used.

Preferred surfactants are sorbitan oleate such as Arlacel C (sorbitan sesquioleate), Span 80 (sorbitan monooleate) and Span 85 (sorbitan trioleate).

The surfactant comprises 0.1 to 20%, preferably 0.25 to 5%, by weight of the pharmaceutical composition. The remainder of the formulation is the propellant.

At room temperature, liquefied powertrainers are generally gases that liquefy under pressure. Suitable liquefied propellants include alkanes having up to 5 carbon atoms, such as butane and propane; and fluorinated or fluorinated and chlorinated alkanes available under the trademark "Freon". Mixtures of the above materials may also be used.

In preparing the aerosol, the container with a suitable valve is filled with a propellant containing a finely divided active ingredient and a surfactant. The components are maintained under increased pressure until the aerosol is dispensed by depressing the valve.

The compounds of formula (I) and pharmaceutical compositions containing them may also be used for the treatment of non-human mammals, poultry such as hens and turkeys, and cold-blooded animals such as fish. For example, the compounds of formula (I) and pharmaceutical compositions containing them are effective against the following non-human viruses:

Cancer Herpes Virus 1

Guinea pig herpes virus 1

Lagomorph herpesvirus 1

Eacan hcrpcs virus 1 Pheasant hcrpcs virus 2 (Marck's disease)

Turkey Herpes Virus 1 Duck Herpes Virus 1

Diphtheria herpesvirus 1

Equine herpes virus 3

Bovine herpes virus 1

Bovine herpes virus 2

Bovine herpes virus 3

Bovine hcrpc virus 4

Cell herpes virus 1

Seitic herpes virus 2

Rat herpes virus 1

Monkey herpes virus 1

Monkey Herpes Virus 2

Mole herpes virus 1

Canine herpes virus 1

Cat herpes virus 1

Equine herpes virus 1

Horse Herpes Virus 2

Viral diseases of poultry, such as Marek's disease, are inhibited and / or cured by methods known in the veterinary art using the compounds of formula (I). For example, the animals are injected with a pharmaceutical composition containing a compound of formula (I) or added to the feed or drinking water.

Fish in a limited area such as a pool, aquarium, or tank can also be treated for viral infections such as those caused by herpes-like viruses such as channel dwarf cat virus (CCV), herpes virus salomons, Nerka virus, and the like. During the treatment, the compound is either added directly to the water of the pool, aquarium or tank or mixed into the diet.

The course of treatment and the dosage employed will, of course, depend on the condition of the individual or animal being treated and the judgment of the physician.

Compounds of formula (I) may be prepared via compounds of formula (X), which may be synthesized according to Scheme B. The compound of formula (V), which is used as an intermediate in Scheme B, may be prepared as described in Scheme A.

As shown in Scheme A, the compound of formula (III) is prepared by dropwise addition of epichlorohydrin to a solution of an optionally substituted benzyl alcohol alkali metal salt, preferably its sodium salt, at a temperature of 0-100 ° C, preferably 1540 ° C. Suitable solvents are, for example, dimethylformamide, dimethylacetamide, hexamethylphosphoric acid amide, dimethylsulfoxide, sulfulane, tetrahydrofuran or dioxane. The reaction mixture is stirred for 10-24 hours, preferably 12-18 hours, at a temperature of 0-100 ° C, preferably 20-50 ° C.

The compound 3A (H1) is chloromethylated by bubbling anhydrous hydrogen chloride gas into a solution of the chemical and paraformaldehyde in a halogenated hydrocarbon such as dichloroethane, chloroform, dichloromethane or 1,1,2-trichloroethane at a temperature between 0 ° C and 25 ° C, preferably At 0 ° C. The hydrogen chloride gas is introduced for 0.5 to 3 hours, preferably for 1 to 2 hours, until the paraformaldehyde is dissolved. The solution is kept at 0-10 ° C for 12-48 hours, preferably at 0-5 ° C for 16-24 hours. In this way, the compound of formula IV is obtained.

The compound of formula (V) is prepared by reacting an alkali metal acetate, such as sodium acetate, with a solution of the compound of formula (IV). Suitable solvents are, for example, dimethylformainide, tetrahydrofuran, dimethylacetamide, hexamethylphosphoric acid amide, dinethylsulfoxide, sulfolane or dioxane, at a temperature between 0 ° C and 45 ° C, preferably 0-25 ° C. The solution is then stirred for 5-24 hours, preferably 10-18 hours, at 10-30 ° C, preferably 15-25 ° C.

In Scheme B, compound (VII) is prepared by boiling guanine (VI) with acetic anhydride in the absence of solvent for 1024 hours, preferably for 12-18 hours.

The N, ² , 9-diacetylguanine of formula (VII) is reacted with the compound of formula (V) in the absence of a solvent or in a solvent such as dioxane or sulfolane with a catalytic amount of acid such as bis (p-nitrophenyl) phosphate, , methylphosphonic acid or dichloroacetic acid, preferably in the presence of bis (p-nitrophenyl) phosphate, at 75 to 200 ° C, preferably at 110 to 180 ° C. Generally, 0.8 to 1.2 moles of compound (V) are used for each mole of compound (VU).

The benzyl protecting groups of the resulting compound (VIII) are removed by catalytic hydrogenation. As a catalyst, for example, a suspension of palladium on carbon is added to a solution of compound (VIII). The solvent is, for example, aqueous methanol. The hydrogen gas is contacted with the solution at a pressure of 15200 psi, preferably 30-80 psi. In this way, the compound of formula IX is obtained.

Compound (X) is prepared by deacetylating Compound (IX) with a base dissolved in an alcohol such as methanol. The solution of the compound of formula (IX) and the base is stirred for 5 to 36 hours, preferably 10 to 24 hours, at 10 to 30 ° C, preferably 15 to 25 ° C.

From the compound of formula (X), the compound of formula (Xb), which is the starting compound of process (a) of the present invention, wherein Y is a lipophilic amino port, can be synthesized by the synthesis of 9- (1,3-dihydroxy -2-propoxymethyl) -guanine can be prepared via f-nionorophate-ester in the following manner:

In Scheme C, where R is benzoyl or triphenylmethyl, the compound of formula (X) is reacted with a silyl protecting group such as tert-butyldimethylsilyl chloride in a solvent such as dimethylformamide, N-methyl. pyrrolidone, pyridine, etc. The reaction mixture is stirred at -5 ° C to room temperature, preferably 0 ° C, for 0.5 to 4 hours, preferably 2 hours. The resulting syrupy residue is added to water to give compound (XI).

Compound (XI) containing lricidylanone in a solvent such as pyridine, lutidyluben or dichloromethane is reacted with benzoyl chloride at a temperature of -5 to 10 ° C, preferably 0 ° C.

Allow the reaction mixture to warm to room temperature. After 18-36 hours, preferably 24 hours, the solvent is evaporated off and the crude products of formula XII are partially purified by methods known per se, for example by chromatography.

θ The compound of formula (XIII) is prepared by treating the compound of formula (ΧΠ) with an acid such as 80% acetic acid at 50-100 ° C, preferably at 80 ° C for 1-6 hours, preferably 3-4 hours. This treatment removes the silyl protecting group.

g Λ Compound of Formula (XIV): wherein Wa is - l '(OII) ₂ Lysate - Phosphorylating Agent

is prepared from the compound of formula (XIII) by treatment with, for example, pyrophosphoryl chloride. Compound (XIII) in a solvent such as ethyl acetate, acetonitrile, cresol, etc., preferably ethyl acetate, is cooled to a temperature between 5 ° C and 10 ° C, preferably 0 ° C, and a phosphorylating agent such as pyrophosphoryl chloride is added. . After 1-4 hours, preferably 1.5-3 hours, the reaction is quenched with ice. The reaction mixture is neutralized by the addition of a base such as sodium hydrogencarbonate. The compound of formula (XIV) is recovered and treated with a base such as ammonium hydroxide, sodium hydroxide and the like. and hydrolyzing the resulting compound of formula (,), wherein Wa is -P (OH) ₂ , to isolate it in the alkali salt thereof,

II

Ü known in the art, preferably by chroniography. For example, compound (Γ) isolated as a disodium salt is converted to the free acid by passing the solution containing the salt through an ion exchange resin. Such an ion exchange resin is Dow Chemical Co. Dowex

50-X8 product can be used. The free acid is converted to a non-polar salt with a lipophilic amine, such as 4-inorpholino-N, N'-dicyclohexylcarboxamidine [described by J.Am. Chem. Soc., 83, 649 (1961)] by treatment in a solvent mixture, for example, a mixture of pyridine and water.

The compound of formula Xb obtained above and a solvent such as pyridine, lutidine and the like. a mixture of a condensing agent such as dicyclohexylcarbodiimide (which, inter alia,

Available from Chemical Co.) with a solvent such as pyridine, lutidine and the like. After completion of the addition, the reaction mixture is refluxed for 1.5 to 4 hours, preferably 2 to 3 hours. The resulting cyclic phosphate ester (I) is isolated in the form of a salt such as potassium or trialkylammonium salt after preparative high pressure liquid chromatography and elution with potassium phosphate buffer or a trialkylammonium carbonate solution. The compounds of formula (I) according to process (b) of the invention may also be prepared directly from compounds of formula (X) by reacting a compound of formula (X) with a phosphorylating agent. To a suspension of the compound of formula (X) in a solvent such as anhydrous acetonitrile is added a solution of a phosphorylating agent such as pyrophosphoryl chloride in a solvent such as anhydrous acetonitrile at room temperature for 30-90 minutes, preferably 60 minutes. After the addition is complete, the reaction mixture is stirred further, followed by the addition of ice and further stirring at room temperature for 1060 minutes, preferably 20-45 minutes. A concentrated base such as ammonium hydroxide is added and the solution is concentrated in vacuo. The residue was dissolved in water (about 50 mL), passed through a pad of activated carbon, washed with water and eluted with water / ethanol containing ammonium hydroxide. The eluate was evaporated to dryness and the residue was recrystallized from aqueous ethanol. In this way, the cyclic phosphate ester of 9- (1,3-dihydroxy-2-propoxymethyl) -guanine (I) is obtained in the form of the monoammonium salt. The compound melts at 230-233 ° C with decomposition.

The invention is further illustrated by the following examples, without limiting the scope thereof. The preparation of starting compounds is described in I-Vili. preparation.

Preparation I

Preparation of 1,3-Di-O-beazylglycerol

Sodium hydride (50 g dispersion in mineral oil) (100 g, 2.08 mol) was washed twice with 1 L of hexane and dried under nitrogen. Anhydrous dimethylformamide (1.5 L) was added followed by benzyl alcohol (400 mL) at a rate such that the temperature remained below 50 ° C. Addition of benzyl alcohol lasts for 2 hours. Then, 92.5 g (1 mol) of epichlorohydrin was added dropwise over 0.5 hour while maintaining the reaction temperature below 40 ° C with ice-cooling. The solution was stirred for 16 hours at 21 ° C and then for 2.5 hours at 50 ° C. The dimethylformamide was evaporated under reduced pressure and the oily residue was dissolved in 8.5 liters of diethyl ether. The organic occluder was washed with 2 L of water, 2 L of 2% hydrochloric acid, 2 L of 1% sodium bicarbonate solution and 1 L of aqueous sodium chloride solution, dried over anhydrous sodium sulfate and evaporated. Fractionation of the residual brown oil afforded 147.8 g of 1,3-di-O-benzylglycerol, b.p. 170-180 ° C / 133 Pa.

II. pREPARATION

Preparation of 1,3-Di-O-benzyl-2-O-chloromethylglycerol g (55 mmol) of 1,3-di-O-benzylglycerol (

Prepared according to Preparation I) and anhydrous hydrogen chloride gas was bubbled into a solution of paraformaldehyde (3.3 g, 110 mmol) in 175 ml of 1,2-dichloroethane at 0 ° C.

1.5 hours. The solution is stored in a stoppered flask for 21 hours at 4 ° C. The solution was then dried over anhydrous magnesium sulfate while heating to 21 ° C, filtered and evaporated. In this way, 17.5 g

1,3-di-O-benzyl-2-O-chloromethyl-glycerol is obtained.

III. pREPARATION

Preparation of 2-O-Acetoxymethyl-1,3-di-O-benzylglycerol

To a solution of 17.5 g (55 mmol) of 1,3-di-O-benzyl-2-octylmethylglycerol (prepared according to Preparation II) in 400 ml of dimethylformamide was added 6 g under a drying tube at 0 ° C. of sodium acetate. The solution was heated to 21 ° C and stirred for 15 hours using a magnetic stirrer. The solvent was evaporated under reduced pressure and the oily residue was dissolved in 455 g of diethyl ether. The ethereal solution was washed once with 750 mL of water, twice with 250 mL of water and once with 250 mL of brine, dried over anhydrous sodium sulfate and evaporated. In this way, 19 g of 2-O-acetoxymethyl- 3-di-O-benzylglycerol is obtained in the form of an oil.

ARC. pREPARATION

Preparation of N, ² , 9-Diacetylguanine 300 g of acetic anhydride were added to g (0.132 mol) of guanine and the reaction mixture was refluxed for 16 hours. The mixture was cooled and the excess acetic anhydride was evaporated under reduced pressure. The residue was recrystallized from dimethyl sulfoxide to give 25.6 g of N, ² , 9-diacetylguanine.

Preparation V.

A. Preparation of TV ^2- Acetyl-9- (1,3-dibenzyloxy-2-propoxymethyl) -guanine

15.61 g (66 mmol) of ^N2, 9-diacetylguanine (which was prepared according to IV. Preparation), 19 g (55 mol) of 2-O-acetoxymethyl-l, 3-di-O-benzyl-gliccriii ( (prepared according to Preparation 111), a mixture of 0.5 g of bis (p-nitrophenyl) phosphate and 150 ml of diethyl ether. The solvent was evaporated and the residue was held in an oil bath at 175 ° C for 1.5 hours under a stream of nitrogen. Column chromatography (1: 9 methanol: dichloromethane as eluant) followed by recrystallization of the product from ethyl acetate. 4.76 g of N ² -acetyl-9- (1,3-dibenzyloxy-2-propoxymethyl) -guanine are obtained, m.p. 145-146 ° C.

Β. Preparation of N ² -A cetyl-9- (1,3-dihydroxy-2-propoxymethyl) -guanine

To a solution of 4.62 g (9.67 mmol) of N ² -acetyl-9- (1,3-dibenzyloxy-2-propoxymethyl) -guanine in 150 ml of methanol and 40 ml of water was added 20% palladium hydroxide / carbon catalyst suspended in 10 ml of water. . The mixture was hydrogenated in a Parr hydrogenator at 413 kPa for 38 hours, then filtered through celite and the filtrate was evaporated. The resulting white solid was recrystallized from methanol / ethyl acetate to give 1.4 g of N ² -acetyl-9- (1,3-dihydroxy-2-propoxymethyl) guanine, m.p. 205-208 ° C.

The mother liquor was further reduced by adding 1 g of 10% palladium on carbon in a mixture of 150 ml of methanol and 50 ml of water at 413 kPa for 47 hours. A total of 2.11 g of N ² -acetyl-9- (1,3-dihydroxy-2-propoxymethyl) -guanine is obtained.

C. Preparation of 9- (1,3-Dihydroxy-2-propoxymethyl) -gitamn

721.9 mg (2.4 mmol) of N ² -cetyl-9- (1,3-dihydroxy-2-propoxymethyl) -guanine was stirred for 17 hours at 21 ° C.

194,565 ml of methanolic ammonia solution. The solution was concentrated and the residual white solid was recrystallized from methanol. 582.3 mg of 9- (1,3-dihydroxy-2-propoxymethyl) -guanine were obtained, m.p. 250 ° C (dec.).

VI. pREPARATION

A. A mixture of 3.0 g of 9- (1,3-dihydroxy-2-propoxymethyl) -guanine, 2.0 g of imidazole, 2.13 g of tert-butyldinylethylsilyl chloride and 40 ml of anhydrous dimethylformamide was added for 2 hours. Stir at 0 ° C and evaporate in vacuo. The syrup-like residue is taken up in water to give a crude product. The solid was filtered, washed with water, then toluene and dried in vacuo.

B. To a solution of the dried crude mono-tert-butyldimethylsilyl derivative (prepared as in A above) in pyridine (100 mL) at 0 ° C is added 5 mL of benzoyl chloride. The reaction mixture was allowed to warm to room temperature (21 ° C), allowed to stand for 24 hours and then concentrated in vacuo. The residue was chromatographed on a silica gel column (300 g), eluting with a gradient of 2 liters of dichloromethane and 2 liters of 5% methanol in dichloromethane. 3.5 g of a crude yellow-brown foam consisting mainly of 9- [1-benzoyloxy-3- (tert-butyldimethylsilyloxy) -2-propoxymethyl] -N ² -benzoyl-guanine are obtained.

C. The crude product obtained in (B) above is directly treated with 80 ml of 80% acetic acid at 80 ° C.

3.5 hours. After evaporation of the solvent, the residue was chromatographed on a column of silica gel (500 g) using dichloromethane (8% methanol) as the solvent. Concentration of the appropriate fractions afforded 611 mg of pure 9- (1-benzoyloxy-3-hydroxy-2-propoxymethyl) -N ² -benzoylguanine as a foam.

VII. pREPARATION

8.18 g of 9- (1,3-dihydroxy-2-propoxymethyl) -guanine, 21.7 g of monomethoxytrityl chloride, 13.3 ml of triethylamine, 0.08 g of 4-dimethylaminopyridine and 100 µl of ! The dimethylformamide mixture was stirred under anhydrous conditions at 40 ° C for 2 hours, then quenched with methanol and concentrated in vacuo. The crude product was dissolved in ethyl acetate (500 mL), washed with aqueous sodium bicarbonate (500 mL, 300 mL), water (300 mL), dried over anhydrous magnesium sulfate and concentrated in vacuo. The crude product was heated in 800 mL of ethanol, cooled and filtered. The filtrate was concentrated in vacuo and the residue was chromatographed on a silica gel column (500 g) eluting with methanol / dichloromethane (1: 4). The product is crystallized from ethanol. 11.2 g of N ² -monomethoxytrityl-9- (1-inonoinetoxytrityl-3-hydroxy-2-propoxymethyl) -guanine are obtained, m.p. 159-160 ° C.

VIII. pREPARATION

To a suspension of 463 mg of 9- (1-benzoyloxy-3-bidroxy-2-propoxyethyl) -N ² -benzoylguanine (prepared according to Preparation VI) in 10 ml of ethyl acetate cooled to 0 ° C was added 0 75 ml of pyrophosphoryl chloride. After 2 hours, quench the reaction with ice and adjust the pH to 5 with solid sodium bicarbonate. This causes the phosphorylated product to precipitate out of solution as an oily substance. The supernatant liquid was decanted and the residue was dissolved in water and extracted with 10% (v / v) butanol in dichloromethane. The aqueous phase is evaporated in vacuo to leave an almost pure, fully protected phosphorylated product. This product was treated with 10 mL of concentrated ammonium hydroxide for 16 hours at 40 ° C. The solution was concentrated in vacuo and the remaining wet solid was dissolved in water and extracted with dichloromethane (10% v / v). The aqueous phase is then applied to a 2.54 cm diameter and 76 cm high column packed with Sephadex G-10 resin and the column is eluted with water. The fractions containing pure product were combined, evaporated and lyophilized. There was thus obtained 276 mg of the sodium ammonium salt of 9- (1,3-dihydroxy-2-propoxymethyl) -guanine-1- (monophosphate) ester as an amorphous substance, m.p. 215-220 ° C.

Example 1

180 mg of 9- (1,3-dihydroxy-2-propoxyl-glycyl) -guanine-1- (monophosphate) ester disodium salt were converted to the free acid by passing through a 10 mL bovine column packed with Dowex 50-X8 cation exchange resin as eluent. After evaporation of the water, 170 mg of free acid remain as a colorless glass. This residue was dissolved in a mixture of 10 ml of pyridine and 5 ml of water containing 138 mg of 4-morpholino-N, N''-dicyclohexylcarboxamidine and the solution was concentrated in vacuo. After the addition of 10 ml of pyridine, the residue is again evaporated and the pyridine is added and evaporated twice. A solution of the foam formed in 60 ml of pyridine was added to a solution of 194 mg of dicyclohexylcarbodiimide in 50 ml of pyridine under reflux for 45 minutes. After the addition was complete, the reaction mixture was refluxed for an additional 2.75 hours and then concentrated in vacuo. The residual white solid was dissolved in water (30 mL), extracted three times with an equal volume of ether and concentrated in vacuo to about 1 mL. The concentrated solution was purified by preparative high performance liquid chromatography using multiple injections. The column, 1 cm in diameter and 25 cm in height, contains Partisii SAX-10 resin. The column was eluted for 20 minutes with potassium dihydrogen phosphate buffer (pH 6.8), the concentration of which was changed from 0.02 M to 0.08 M. Fractions containing pure cyclic phosphate were combined and evaporated. The solid (535 mg) was desalted by passing through a column 5 cm in diameter and 60 cm in height filled with Sephadex G-10 resin using water as eluent. The pure desalted product is lyophilized from water. 82 mg

9- (1,3-Dihydroxy-2-propoxymethyl) -guanine cyclic phosphate ester is obtained in the form of the monopotassium salt, m.p. 250 ° C (dec.).

Example 2

Λ. 1.70 g of N ² -mono- (4-ineloxy) -1-trityl-9- [1-mono- (4-methoxy) -trityloxy-3-hydroxy-2-propoxymethyl] -guanil, 2.68 g of dicyclohexylcarbodiimide, A mixture of 0.13 M cyanoethyl phosphate and 50 ml pyridine was stirred at 21 ° C for 2.5 days,

The reaction was quenched with water (194,565) followed by 10 ml of water. The solution was concentrated in vacuo and the residue treated with concentrated ammonium hydroxide (80 mL) for 2 hours at 60 ° C and then concentrated again under vacuum. The residue was dissolved in 80% by volume acetic acid and the solution was heated at 80 ° C for 2 hours, then allowed to stand at 21 ° C for 16 hours and concentrated in vacuo. The residue was taken up in 80 ml of water and the solution was extracted with dichloromethane (3 x 50 ml). The aqueous phase was filtered, evaporated to a volume of about 10 ml, basified with concentrated ammonium hydroxide and applied to a column of 5.5 cm diameter and 40 cm height filled with Sephadex G-10 gel. The column was eluted with water and the fractions containing pure product were evaporated. Amorphous 9 (1-monophosphate-3-hydroxy-2-propoxymethyl) -guanine (520 mg) was obtained as its diammonium salt.

B. 520 mg of 9- (1-monophosphate-3-hydroxy-2-propoxymethyl) -guanine diammonium salt is converted to the free acid by passing through a 45 mL column packed with Dowex 50-X8 cation exchange resin. One liter of water is used as the extender. The aqueous solution was evaporated and the residue was dissolved in a mixture of 25 ml of pyridine, 10 ml of water and 350 mg of 4-morpholino-N, N'-dicyclohexylcarboxamidine and the solution was concentrated in vacuo. To the residue was added pyridine (10 ml), evaporated and the latter two operations repeated two more times. The resulting foam was dissolved in pyridine (150 mL) and added dropwise over 1 hour to a refluxing solution of dicyclohexylcarbodiimide (500 mg) in pyridine (120 mL). After the addition was complete, the reaction mixture was refluxed for a further 2.5 hours and then concentrated in vacuo. The residual white solid was dissolved in water (100 mL), extracted with dichloromethane (3 x 30 mL) containing 10% (v / v) butanol and extracted with diethyl ether (30 mL). The aqueous solution was concentrated in vacuo to a volume of 5 ml and the concentrated solution was applied to a 2.5 cm diameter and 40 cm high column packed with DEAE Sephadex A-25 carbonate gel. The column was eluted with a linear concentration gradient using 1 L water and 1 L 0.6 M triethylammonium bicarbonate (pH 1.6). The eluate containing the pure product was lyophilized to give 282 mg of an amorphous solid.

The resulting solid was dissolved in 2 mL of water and applied to a 5 mL column containing Dowex 5O-8X cation exchange resin. The column is then washed with water until the liquid flowing from the column has no ultraviolet absorbance. This requires about 200 ml of water. The washings were made basic with concentrated ammonium hydroxide and then concentrated in vacuo. The residue was crystallized from aqueous ethanol. In this way, the 1,3-cyclic phosphate ester of 9- (1,3-dihydroxy-2-propoxymethyl) -guanine is obtained in the form of the monoammonium salt in an amount of 203 mg; mp: 230-233 ° C.

Example 3

The following describes the preparation of typical pharmaceutical compositions. The pharmaceutical compositions contain a biologically active compound of formula (I).

4. Preparation for topical application

Active ingredient 0.2-2 g

Span 60 2 g

Tween 60 2 g

Mineral oil 5 g

Petrolatum 10 g

Methyl paraben 0.15 g

Propoyl paraben 0.05 g

BHA (butylated hydroxyanisole) 0.01 g

Water up to 100 ml

The components listed, except water, are mixed and heated to 60 ° C with stirring. Water at 60 ° C was then added with vigorous stirring to give 100 g of cream, which was cooled to room temperature.

The following formulation is suitable for intraperitoneal and intranasal injection purposes.

B. IP and IM injectable preparation

Active ingredient 0.5 g

Propylene glycol 20 g

Polisthenyl glycol 20 g

Tween 80 1 g

0.9% sodium chloride solution up to 100 ml

The active ingredient is derived from a mixture of propylene glycol, polytylene glycol and Tween 80. While stirring, 0.9% aqueous sodium chloride solution was added to give a volume of 100 ml, which was filtered through a 0.2 micron membrane filter and filled into ampoules under sterile conditions.

The following pharmaceutical preparation is suitable for intravenous injection.

C. IV. a pharmaceutical composition for administration

Active ingredient 0.5 g

0.9% sodium chloride solution 100 g

The active ingredient is dissolved in 100 ml of 0.9% aqueous sodium chloride solution with stirring. The resulting solution was filtered through a 0.2 micron pore size membrane filter and filled into ampoules under sterile conditions.

D. Tablet

Active ingredient 200 parts by weight

Magnesium, iumstearate 3 parts by weight

Starch 30 parts by weight

Equine weight of lactose

PVP (polyvinylpyrrolidone) 3 parts by weight

The listed components are mixed and granulated using methanol as solvent. The granulate is dried and converted into tablets using a suitable tabletting machine. (Each tablet contains 200 mg of active ingredient.)

194,565

Example 4

The exceptional antiviral activity of the compounds of formula I is demonstrated by the assay described below.

In HEp-2 cell cultures, the herpes simplex 2 virus G strain for infection is prepared. The virus was adsorbed for 1 hour, then fresh media was added to the cells and incubated at 35C until all cells were infected. The cell suspension was frozen at -70 ° C, allowed to thaw, and centrifuged to remove cell debris. The supernatant liquid was aliquoted and stored frozen at -70 ° C until use.

A ^6.7 -fold dilution of supernatant fluid infects 50% of the cell culture in HEp-2 cells (CCID10). Dilution of this liquid at ^3.7 times 10% kills 50% of the mice (LCS0).

Groups of 20 to 20 Swiss Webster female mice weighing 15 to 17 g were infected by intraperitoneal administration of 0.2 ml EMEM. This volume of fluid contains enough virus to give each mouse 10 times the LC ₅₀ value. _50, the LD 10 sz.ercsénél 10 ^°, more or less infected ⁵ -dei virus egeTáblázat

W, Wa meaning Dose, Number of Surviving Mice Mean Survival) total mg / kg total number of mice fall in formula day

control 0/28 9.1 ± 1.7 W = Η O 20 *. 13/20 11.4 ± 2.3 Wa = (HO) P- 10 8/20 U 6 ± 2.7 OH 5 5/20 11.5 ± 2.8 W = Wa = * 20 6/20 14,0i5.1 SHE II (HO) P 10 4/20 12.8 ± 4.8 OH 5 8/20 14.2 ± 5.8 W + Wa = * 20 10/20 16.3 ± 5, O SHE II 10 '3/20 12.2 ± 4.7 <HO) P = cyclic 5 8/20 14.7 ± 5.5

* LD _S0 greater than 20 mg / kg.

Records are used to verify virulence to make sure that the model used is appropriate.

6 hours after infection, treatment with the test compounds is started. Each mouse group is dosed orally with 5 mg / kg, 10 mg / kg and 20 mg / kg, respectively. The control group, also consisting of 20 mice, was given saline orally. The treatment was repeated 24 hours after infection.

In the assay described above, the compounds of formula I exhibit antiviral activity. The data obtained is shown in the table below.

Claims (4)

A process for the preparation of the 9- (1,3-dihydroxy-2-propoxymethyl) guanine cyclic phosphate ester salts of the formula (I), wherein B represents an ion forming a pharmaceutically acceptable salt, preferably an alkali metal or ² θ ammonium ion. characterized by a) a (Xb) a compound of formula wherein Y represents an amino group lipolil - is reacted with a condensing agent, and the buffer containing the product from the B ion, ²⁵ preferably alkali or ammonium ion in the presence subjected to chromatographic purification; obsession b) reacting 9- (1,3-dihydroxy-2-propoxyethyl) -guanine of formula (X) with a phosphorylating agent and then hydrolyzing the resulting product with a strong base containing ion B, preferably an alkali metal or ammonium ion. Method 2 (a) according to claim 1, characterized in that dicyclohexylcarbodiimide is used as the tip condensing agent. 35 3. Process b) according to claim 1, characterized in that pyrophosphoryl chloride is used as the phosphorylating agent. A process for the preparation of pharmaceutical compositions having particular antiviral activity, comprising combining a compound of formula (I) according to claim J, wherein B is as defined in claim 1, with a pharmaceutically acceptable carrier and / or other pharmaceutical excipient. into a composition.