CS248003B2 - Production method of 9-hydroxyethoxymethylguanine derivatives - Google Patents

Abstract

The object of the present invention is a method production of 9-hydroxyethoxymethylguanine derivatives of formula I, wherein W and Z are meanings of the same or different and mean hydrogen atom or pharmaceutically acceptable a cation characterized in that it is a compound of formula II is reacted with phosphorus chloride at a temperature of from -30 ° C to + 25 ° C, after which to form a halophosphoric compound hydrolyses to form a compound of formula I wherein both W and Z are atoms hydrogen and optionally further converting the compound of formula (I) wherein W and Z are in which case hydrogen, another compound, wherein one of W and Z, or both the symbols mean pharmaceutically acceptable cation by reaction with a base, or with another salt containing the corresponding cation. Compounds produced by the process of the invention have antiviral activity and can be used as drugs.

Description

The present invention relates to a process for the preparation of 9-hydroxyethoxymethylguanine derivatives as an antiviral agent, in particular 9- (2-hydroxyethoxymethyl) guanine monophosphoric ester, and in particular to the corresponding pharmaceutically acceptable salts.

It is known that 9- (2-hydroxyethoxymethyl) derivatives of purine compounds have antiviral activity against both deoxyribonucleic acid and ribonucleic acid viruses, both in vitro and in vivo, see British Patent No. 1,523,865. have particular efficacy as antiviral agents against vaccines and herpes viruses, including herpes simplex, zoster and varicella in mammals, which cause diseases such as herpes keratitis in rabbits and herpes encephalitis in mice.

It has now been found that the monophosphoric ester of 9- (2-hydroxyethoxymethyl jguanine) is not only as potent as a non-phosphorylated compound, but also has a selective advantage in that it is much more soluble at least at a pH range of 1 to 7.5 when compared to a non-phosphorylated compound.

The present invention provides a process for the preparation of the 9-hydroxyethoxymethylguanine derivatives of formula (I)

Oil

I ¹¹

CH-O-Cl-1-CH-O-P-OZ.

2 '2'

O W (1) wherein W and Z have the same or different meanings and are a hydrogen atom or a pharmaceutically acceptable cation, characterized in that the compound of formula II

CH ₂ -CH .O.CH _{of a} »OH (II) is reacted with phosphorus oxychloride at a temperature from -30 ° C to +25 ° C, after which the resulting compound halogenfosforečná hydrolyzed to give a compound of formula I wherein both W Z is hydrogen, and optionally further converts a compound of formula I wherein W and Z are both hydrogen to a compound wherein one of W and Z or both is a pharmaceutically acceptable cation by reaction with a base or another salt containing the corresponding cation.

The pharmaceutically acceptable cations are selected from the group consisting of sodium, potassium, lithium, calcium, magnesium or aluminum equivalent, or an ammonium cation.

Compounds of formula I wherein Z is sodium, potassium or ammonium, and W is hydrogen are preferred, and particularly preferred are compounds of formula I wherein Z is sodium or ammonium, and W is hydrogen.

Polyvalent cations within the meaning of W and Z are also referred to as Ga / 2, Mg / 2, and Al / 3, and this is to indicate that it is a valence cation, ie Ca ²⁺ / 2, Mg ²⁺ / 2 and Al ³⁺ / 3, and this corresponds to the fact that calcium or magnesium cations are ionically bound to 2 phosphate oxygen, and aluminum to 3.

A preferred process for phosphorylating intermediates of the invention is reacting a compound of formula II with phosphoryl chloride in the presence of a phosphoric acid trialkyl ester, and preferably at a temperature of about 0 ° C or less.

Another suitable process for preparing the monophosphoric ester is by reacting the compound of formula II with phosphoryl chloride in anhydrous pyridine.

The compound of formula (II) may be prepared by one of the methods described in British Patent No. 1,523,865.

Pharmaceutically acceptable salts of 9- (2-hydroxyethoxymethyl) guanine monophosphoric ester can be prepared by neutralizing the acidic monophosphoric ester form with an equivalent amount of a base such as hydroxide, bicarbonate, carbonate, etc. containing the desired cation such as sodium, potassium, ammonium, Otherwise, such salts may also be prepared using exchange reactions where the monophosphoric ester salt is treated with a solution, preferably an aqueous salt solution containing a suitable other cation. 9- (2-hydroxyethoxymethyl) guanine with an aqueous sodium sulfate suspension, the barium is thus removed as insoluble barium sulfate, leaving the sodium salt of the 9- (2-hydroxyethoxymethyl) guanine monophosphoric ester remaining in solution.

Also described are pharmaceutical compositions comprising a compound of Formula I together with a pharmaceutically acceptable carrier.

Pharmaceutically acceptable carriers are materials useful in formulating the active ingredient and can be solid, liquid or even gaseous, if otherwise inert, therapeutically acceptable and compatible with the active ingredient. The pharmaceutical preparations may be administered orally, parenterally or in the form of suppositories, or they may be administered topically in the form of ointments, creams, aerosols or powders, or they may be administered as eye or nose drops, depending on whether the product is used for treatment. internal or external viral disease.

For internal infections, the compounds of formula I are administered in doses of from 0.1 to 250 mg per kg, calculated as free monophosphoric ester, and are preferably administered in the range of 1.0 to 50 mg per kg of mammalian body weight, and humans may be used in unit dosage form, administered in single or multiple doses, for example, several times a day in an amount of 1-800 mg per dose, preferably 1-250 mg per dose, most preferably 10-200 mg per dose.

For parenteral administration or topical administration in the form of drops, for example in ocular infections, the compound of formula I may be present in an aqueous solution at a concentration of from about 0.1 to 10% (w / v), preferably 0.1 to 7% most preferably 0.2 to 5%.

Otherwise, solutions, ointments or creams may be used as topical formulations in infections of the eye or other external organs, e.g. The concentration ranges from 0.1 to 10%, preferably 0.3 to 6%, most preferably 3%.

The compounds of this invention are used to treat mammalian viral diseases by administering an effective and nontoxic antiviral amount of a compound of Formula I. The term "effective and nontoxic antiviral amount" refers to a predetermined amount of anti-viral agent active in vivo.

The activity of the compounds of the invention was tested by inhibition assay on a plate. The inhibition assay was performed on VERRO cells, in which a confluent monolayer of cells was infected with sufficient virus to form a confluent coating on the plate. After the addition of a solid coating layer, a filter paper disc impregnated with a test compound solution (normally 50 µg / disc) is placed in the center of the coating layer. The cultures are incubated at 37 ° C for 96 to 120 hours in an atmosphere of 5% carbon dioxide. After this time, the cells are fixed with 10% formalin and then stained with 0.05% methyl violet.

The results of the inhibition test are evaluated according to the following scale:

Inhibition plate zones

- zone greater than 25 mm in diameter

-j- zone 5–25 mm in diameter ± zone 0–5 mm in diameter - negative.

In this test, the compounds of the invention were found to exhibit activity evaluated as -) - | -

When testing the compounds by plate reduction assay, the confluent monolayer of cells is infected with the virus as in the inhibition assay, and the test compound is incorporated into the solid coating in two-fold dilutions. Test results expressed as ED 50, that is, doses effective at 50% plates of the appropriate dilution, showed that no virus was present if the compound was 0.2 µΐοsolar.

The present invention is described in more detail in the following examples.

Example 1

9- (2-Hydroxyethoxymethyl) guanine monophosphate ester

To a stirred mixture of 0.225 g of 9- (2-hydroxyethoxymethyl) guanine and 5 ml of triethyl phosphate, 0.76 ml of phosphoryl chloride was added while cooling to -10 ° C, then the reaction mixture was allowed to rise to 0 ° C over 30 minutes. The reaction mixture is kept at this temperature for two hours, poured onto ice-water, adjusted to pH 7 by addition of 2N potassium hydroxide solution, extracted twice with chloroform and once with ether. The solid is dissolved in 7 ml of water and the inorganic salts precipitated by the addition of 7 ml of methanol, which is filtered off, 70 ml of acetone are added to the filtrate, precipitated. This gives a white gum which is dissolved in 7 ml of water. After addition of 7 ml of ethyl alcohol, the reaction mixture is filtered and a large excess of acetone is added. The gum was precipitated again (70 ml) to the filtrate, which was dissolved in about 20 ml of ethyl alcohol, the solvent was distilled off vigorously, and 2.6 g of white powdery solid, a mixture of inorganic salts and the desired phosphoric acid ester, was obtained. . This solid is dissolved in 10 ml of water and applied to a gel column (2.7 by 90 cm, particle size 200-400 mesh); eluting with water elutes the largest portion of the monophosphoric ester in a volume of about 50 ml after collecting 166 ml of eluate, as can be seen from the thin-layer chromatography of the cellulose in the n-propyl alcohol-water system (70:30, v / v);

0.26 for 9- (2-hydroxyethoxymethyl) guanine monophosphoric ester, a

0.11 for the corresponding potassium salt.

By lyophilization of the eluate, 0.28 g of solid is isolated which, according to ultraviolet spectra, contains 0.2 g of monophosphoric ester product.

Example 2

9- (2-hydroxyethoxymethyl) guanine monophosphoric ester ammonium salt

Dissolve 0.28 g of 9- (2-hydroxyethoxymethyl) guanine monophosphoric ester in 30 ml of water and adjust the pH to 6 by adding 6 N hydrochloric acid solution. The product is adsorbed onto 14 ml of activated carbon in a column (particle size 50 to 200). The charcoal is washed well with water and the column is eluted using 70 ml of 50% aqueous ethanol containing 2% concentrated aqueous ammonia solution, after which the solvent is distilled off under reduced pressure to give 0.048 g of ammonium monophosphoric salt. 9- (2-hydroxyethoxymethyl) guanine ester having an Rf value of 0.30 on cellulose in n-propyl alcohol-water (70:30, v / v).

Example 3

Tablet composition sodium monophosphate ester

9- (2-hydroxyethoxymethyl) guanine 100 mg lactose 200 mg starch 50 mg polyvinylpyrrolidone 5 mg magnesium stearate 4 mg total weight 359 mg

Example 1 4

Ophthalmic solution 9- (2-hydroxyethoxymethyl) guanine monophosphoric ester sodium 1.0 g sodium chloride, p. A. 0.9 g thimerosal 0.001 g purified water, make up to 100 ml pH adjust to 5.5 to 7.5

Example 5

Solution for injection 9- (2-hydroxyoxyethoxymethyl) guanine monophosphate ester 0.775 g sterile, pyrogen-free phosphate buffer, pH 7, make up to 25 ml

Example 6

9- (2-Hydroxyethoxymethyl) guanine monophosphate ester disodium salt

54 ml of phosphoryl chloride are introduced over 3 hours into a mixture of 25 g of 9- (2-hydroxyethoxymethyl) guanine and 250 ml of phosphoric acid triethyl ester, cooled to -30 to -20 [deg.] C., with stirring. The reaction mixture is kept at this temperature for a further 45 minutes, poured into ice-water, and the pH is adjusted to about 1 by the addition of 2 N sodium hydroxide solution. The mixture was adjusted to 6.8 with 2 N sodium hydroxide solution and then 7.3 with 10 N sodium hydroxide solution to a 2.5 liter cooking volume.

The neutralized solution was applied to a column containing 2000 g of ion exchange resin equilibrated using a 50 mM potassium bicarbonate solution. Elution is performed with a 30 liter linear gradient of 50-500 mM potassium bicarbonate solution followed by washing using 30 liter 500 mM potassium bicarbonate. The fractions containing the product were combined, and the largest portion of potassium bicarbonate was removed by addition of an ion exchange resin and evacuation of the resulting carbon dioxide. The volume is then reduced to 2 liters by concentration in vacuo, and under cooling to 4 ^and C the product is precipitated by the addition of 10 liters of acetone. 55 g of dried precipitate is applied to a 10 cm 110 cm gel-containing column and 22 g of solid are isolated by elution with water. This is recrystallized at 4 ^and C as the hydrogen salt from an aqueous solution at pH 3, further material fractions are obtained from the mother liquors by crystallization from 20% ethanol at pH 3. The hydrogen salt is dissolved in a minimum amount of water by adding sodium hydroxide solution to pH raised to 8.5, and 2 volumes ethainetlu precipitates under cooling at 4 ^C C a solid which was dissolved in 100 ml of water, precipitating with 9 volumes of ethanol at 4 ° C gave 15.1 g of the dihydrated disodium salts of 9- (2-hydroxyethoxymethyl) guanine monophosphoric ester.

The purity of the substance was confirmed by elemental analysis, high pressure liquid chromatography with ultraviolet spectra.

For C 6 H 12 N 5 O 69 2N and 2H 2 O calculated:

% C, 24.94;% H, 3.66;% N, 18.19;% P found *

% C, 25.20;% H, 3.63;% N, 18.10

Ultraviolet spectra:

Solvent A _max 6 Amin inflex

0.1 M HCl 254 12 970 255 272 pH 7 209 14 060 219 266

0.1 M NaOH 255-264 11760 228

Purity according to HPLC analysis was 99%. Base to phosphate ratio - 1.00 / 1.01.

Claims (6)

A process for the preparation of a 9-hydroxyethoxyinethylguanine derivative of the general formula I CH.-O-CH-CH-O-P-OZ OW (/) wherein W and Z have the same or different meanings and are a hydrogen atom or a pharmaceutically acceptable cation, characterized in that the compound of formula II CH 2 O.CH 2 CH 2 OH (II) is reacted with phosphoryl chloride at a temperature of from -30 ° C to + 25 ° C, whereupon The resulting halogenophosphorous compound hydrolyzes to form a compound of formula I wherein both W and Z are hydrogen, and optionally further converting a compound of formula I wherein W and Z are both hydrogen to a compound wherein one of W's and Z or both represent a pharmaceutically acceptable cation by reaction with a base or other salt containing the corresponding cation. 2. A process according to claim 1, wherein the compound of formula II is treated with phosphoryl chloride in the presence of a phosphoric acid trialkyl ester at a temperature of 0 ° C to -30 ° C. 3. The process of claim 1, wherein the compound of formula II is treated with phosphoryl chloride in anhydrous pyridine. 4. A process as claimed in any one of claims 1 to 3, wherein the base to be treated with a compound of formula I wherein both W and Z are hydrogen is a hydroxide, bicarbonate or carbonate containing the desired pharmaceutically acceptable cation. 5. A process according to claim 4, wherein sodium, potassium, ammonium, calcium, magnesium or aluminum are used as a suitable cation. 6. The process of claim 4 wherein lithium is used as a suitable cation.