CH643858A5 - 9-Hydroxyethoxymethylguanine derivatives and processes for their preparation - Google Patents

Description

The invention relates to 9- (2-hydroxyethoxymethyl) guanine monophosphate and pharmaceutically acceptable salts of this compound. The invention further relates to 25 processes for the preparation of this compound and its pharmaceutically acceptable salts as well as pharmaceutical preparations which contain this compound.

It is known that 9- (2-hydroxyethoxymethyl) derivatives of purines are effective against viruses, in particular different types of DNA-RNA viruses, as could be shown in both in vitro and in vivo experiments Swiss Patent No. 626,363. These compounds are particularly effective against cowpox and herpes viruses, including herpes simplex, herpes zoster and 35 chickenpox viruses in mammals, which cause diseases such as keratitis herpetica in rabbits and encephalitis herpetica in mice.

It has now been found that the monophosphate ester of 9- (2-hydroxyethoxymethyl) guanine is not only as active as the nonphosphorylated compound, but moreover, as a particular advantage, it has a much greater solubility, at least in the pH range from 1 to 7.5 , in comparison with the corresponding unphosphorylated compound.

45 The invention therefore relates to a 9-hydroxyethoxy-methylguanine derivative of the formula I.

(I),

nium and hydrogen are preferred, those compounds of the formula I in which Z is sodium or ammonium and W are hydrogen being particularly preferred.

In the definition of W and Z above, the polyvalent cations are referred to as calcium / 2, magnesium / 2 and aluminum / 3, which means that each

\\

n '

0

CH2.O.CH2.CH2.O-p-0H

Oh

0

o.ch .ch2.0-p-0z

5

643 858

one equivalent of the cation is to be considered, i.e. that the cation is divided by its valence, e.g. Ca ++ / 2, Mg ++ / 2 and Al +++ / 3. This is to indicate that calcium or magnesium cations are ionically linked to two phosphate oxygen atoms, while aluminum is linked to three phosphate oxygen atoms.

The invention further provides processes for the preparation of a compound of the formula I, which are characterized in that a) a compound of the formula II

OH

(II)

ch ^ .0.ck .ch .oh ^ 2 2

with a phosphorylating agent to give a compound of the formula I in which W and Z are both hydrogen, or b) a compound of the formula III "

(ml

0

ch2.0.ch2.ch2-0-

p-oh oh wherein either M is a 6-hydroxy group; and

G is a group which can be converted into an amino group or can be replaced by an ammonium group or a corresponding atom by selective ammonolysis; or G is a 2-amino group; and

M denotes a group which can be converted into a hydroxy group or can be replaced by a hydroxy group or a corresponding atom by selective hydrolysis, is converted into a compound of the formula I by selective ammonolysis or by selective hydrolysis and optionally the compound of the formula I obtained in which W and Z both are hydrogen , with a base containing the desired cation or a corresponding salt to give a compound of the formula I in which W and / or Z are a pharmaceutically acceptable cation.

The compounds of the formula III "comprise the compounds of the formulas III and III 'in claims 11 and 15.

When carrying out process variant a), derivatives of phosphoric acid in which 1 to 3 hydroxyl groups by halogen atoms, e.g. Chlorine, are preferred as the phosphorylating agent, with particular mention being made of phosphorus oxychloride. Also suitable as phosphorylating agents are derivatives of phosphoric acid in which up to two hydroxyl groups are substituted by alkoxy groups, which in turn can be substituted, e.g. Benzyloxy groups, replaced s, are suitable. Such phosphorus halogen derivatives or phosphates are generally used under the usual neutral or alkaline conditions. When using the latter compounds, activation in general, for example by carbodiimides, such as iodicyclohexylcarbodiimide, is recommended unless the compounds are used in the form of the anhydride.

If phosphoric acid derivatives in which at least two of the hydroxyl groups are replaced by halogen are used as the phosphorylating agent, it is necessary, after the reaction with the compound of the formula II, for the unbound halogen atoms to be dissolved by mild aqueous hydrolysis, for example using a molar equivalent of water in one to remove water-miscible solvents such as alcohol. If alkoxy groups substituted or unsubstituted with a phosphate are introduced in the reaction, these can be split off in a suitable medium in the presence of bases in a subsequent process step. Aromatically substituted alkoxy groups, such as, for example, benzyloxy, 25 can also be removed by hydrogenolysis, preferably in the presence of a catalyst, by the methods customary for reductive cleavage.

A preferred method for the phosphorylation of compounds of the formula II is to react this compound with phosphorus oxychloride in the presence of a trialkyl phosphate and preferably at a temperature of 0 ° C. or below.

Further advantageous methods for producing the monophosphate include the reaction of a compound of the formula II with phosphorus oxychloride in dry pyridine.

The compound of the formula II can be regarded as a precursor in the synthesis of compounds of the formula I and can be prepared according to the method described in Swiss patent no.

626 363 described processes are produced. The conversion of compounds of formula III "according to process variant b) can be achieved in various ways. In formula III" G means, for example, a halogen atom or a mercapto or alkylthio group, e.g. a methylthio group, this can be converted into an amino group by ammonolysis. This method, like other methods, belongs to the prior art and is described in "Heterocyclic Compounds-Fused Pyrimidines Part II Purines, edited by D. J. Brown, published by Wiley-Interscience (1971)".

If M represents a halogen atom or a mercapto or alkylthio group, these groups can be converted 55 into a hydroxyl group with the aid of hydrolytic methods as described in the previously mentioned book.

The compounds of the formula III "can be regarded as precursors in the synthesis of compounds of the formula I and can be prepared in an analogous manner to process variant 60 a). The starting compounds for process variant a) can in turn be analogous to those described in Swiss Patent No. 626 363 described methods can be obtained.

Pharmaceutically acceptable salts of 9- (2-hydroxy-65-ethoxymethyl) guanine monophosphate can be obtained by neutralizing the monophosphate in its acidic form with an equivalent amount of a base, e.g. a hydroxide, bicarbonate or carbonate which contains the desired cation,

643 858

6

e.g. Contains sodium, potassium, ammonium, lithium, calcium, magnesium or aluminum. Alternatively, these salts can also be prepared by exchange reactions by treating a salt of the monophosphate with a solution, preferably an aqueous solution, of a salt which contains the desired cation. For example, the weakly soluble barium salt of 9- (2-hydroxyethoxymethyl) guanine monophosphate in aqueous suspension can be treated with sodium sulfate and the barium can be removed as barium sulfate, which is very difficult to dissolve, sodium 9- (2-hydroxyethoxy methyl) guanine monophosphate remains in solution.

The invention further relates to pharmaceutical preparations which contain a compound of the formula I together with a pharmaceutically acceptable carrier.

Solid, liquid or gaseous materials which are inert, medically acceptable and compatible with the active component are suitable as pharmaceutically acceptable carriers for the production of pharmaceutical preparations. The pharmaceutical preparations so produced can e.g. orally, parenterally, in the form of suppositories or pessaries, or locally, e.g. in the form of ointments, creams, aerosols or powders and in the form of eye or nose drops, the type of formulation depending on whether the preparations are to be used for the treatment of internal or external virus infections.

For the treatment of internal infections, the dosage of the compounds of formula I is generally 0.1 to 250, preferably 1.0 to 500 mg / kg body weight of the mammal, calculated as free phosphate. If the compounds of the formula I are used in human medicine, their use in the form of single doses is recommended. These are administered, for example, several times a day, one or more individual doses being used in each case. The amount of active ingredient per single dose is generally 1 to 800, preferably 1 to 250, and in particular 10 to 200 mg, per single dose.

For parenteral use or for topical use in the form of drops, e.g. for the treatment of eye infections, the compounds of formula I in the form of aqueous solutions, generally at a concentration of 0.1 to 10, preferably 0.1 to 7, and in particular 0.2 to 5, w / v%. Tobe offered.

Another way of treating infections of the eye or other external tissues, e.g. of the mouth or skin, consists in the use of solutions, ointments or creams for topical application. In this case, the active substance concentration can generally be about 0.1 to 10%, preferably 0.3 to 6%, and in particular 3%.

Viral infections in mammals can be treated by administering to the subject to be treated an antivirally effective non-toxic amount of a compound of formula I. The term “antivirally effective non-toxic amount” is to be understood as a predetermined amount which is sufficient to effectively fight the virus in vivo.

The following examples serve to explain the invention.

example 1

9- (2-Hydroxyethoxymethyl) guanine monophosphate

To a suspension of 20 mg of 2-chloro-9- (2-hydroxy-ethoxymethyl) hypoxanthine in 0.3 ml of triethyl phosphate, 0.03 ml of phosphorus oxychloride are added all at once with stirring at -8 ° C. Then you let the temperature

rend to 0 ° C for 30 minutes and then stir the reaction mixture for 40 minutes at 0 ° C and then for 50 minutes at + 5 ° C. Then the reaction mixture is poured onto ice and the pH is adjusted to pH 7 with 2N s potassium hydroxide. The resulting solution is extracted twice with 2 ml of chloroform. The pH of the aqueous phase is adjusted to pH 8 to 8.5 with 2N potassium hydroxide, after which 105 mg of barium acetate are added. The resulting barium phosphate precipitate is removed by filtration. The supernatant solution is treated with a large excess of ethanol, as a result of which crude barium-2-chloro-9- (2-hydroxyethoxymethyl) hypoxanthine monophosphate is precipitated. The solid is collected by filtration and suspended in ethanol. The ethanolic suspension is then heated on a steam bath for a few minutes, cooled and filtered. The collected precipitate is washed with anhydrous ether and dried and yields 26 mg barium-2-2o chloro-9- (2-hydroxyethoxymethyl) hypoxanthine monophosphate.

3.96 mg of ammonium sulfate are added with stirring to a suspension of 7 mg of barium-2-chloro-9- (2-hydrooxyethoxymethyl) hypoxanthine monophosphate in 0.5 ml of water. The mixture is stirred at room temperature for 15 minutes and then cooled in an ice bath. The precipitated barium sulfate is removed by filtration and washed out with 1 ml of water and 10 ml of ethanol. The combined filtrates and washings are evaporated under reduced pressure, after which the resulting residue is dissolved in 3 ml of methanol. The methanolic solution is transferred to a “Teflon” lined stainless steel bomb tube, after which 8 ml of methanol, saturated with gaseous ammonia at an ice bath temperature, are also introduced into the bomb tube. The bomb tube is sealed, placed in an oven and held there for 4 hours at a temperature of 122 ° C .; then the bomb tube is quickly cooled and opened. The solvent is then concentrated to a minimum volume. The remaining reaction mixture is applied in spots to «Eastman Chromatogram» cellulose thin-layer plates, which are then developed in an n-propanol: water mixture (70:30 v / v). The bands at Rf 0.16 and 0.34 are cut out, suspended in 0.6 ml Tris buffer at pH 8 and then the cellulose is removed by filtration.

It was shown by enzymatic dephosphorylation with alkaline phosphatase that these bands 9- (2-hydroxy-ethoxymethyl) guanine monophosphate and 2-chloro-9- (2-50 hydroxyethoxymethyl) hypoxanthine monophosphate, which lead to 9- (2- Hydroxyethoxymethyl) guanine or 2-chloro-9- (2-hydroxyethoxymethyl) hypoxanthine are dephosphorylated. To the filtrate were added 2 µl of E. coli alkaline phosphates, after which the mixture was heated at 32 ° C for 2 hours. It was then examined by thin layer chromatography on “Eastman Chromatogram” cellulose plates in the three solvent systems listed below:

(a) n-propanol: water (70: 30v / v)

(b) water

(c) n-Propanol concentrated ammonium hydroxide: water (60:30:10 v / v)

65

There were two spots in each system corresponding to 9- (2-hydroxyethoxymethyl) guanine (A) and 2-chloro-9- (2-hydroxyethyloxymethyl) hypoxanthine (B).

7

(»43 858

Solvent system

Rf (A)

Rf (B)

Rf of the reaction product

(a)

0.51

0.64

0.51 and 0.65

(b)

0.68

0.97

0.67 and 0.97

(c)

0.51

0.71

0.51 and 0.71

Example 2

9- (2-HydroxyethoxymethyI) guanine monophosphate

0.76 ml of phosphorus oxychloride are added with stirring to a mixture of 0.225 g of 9- (2-hydroxy-ethoxymethyl) guanine and 5 ml of triethyl phosphate which has cooled to -10 ° C. The temperature of the reaction mixture is then allowed to rise to 0 ° C. over 30 minutes and this temperature is maintained for 2 hours. The reaction mixture is then poured onto a mixture of ice and water and the pH is adjusted to pH 7 with 2N potassium hydroxide. The resulting solution is extracted twice with chloroform and once with ether. The pH of the remaining aqueous solution is adjusted to 7.1 with 2N potassium hydroxide, after which the solution is lyophilized. The resulting white solid is dissolved in 7 ml of water, after which 7 ml of methanol are added in order to precipitate the inorganic salts, which are subsequently removed by filtration. 70 ml of acetone are added to the filtrate, a white resin being precipitated. The resin is dissolved in 7 ml of water, mixed with 7 ml of ethanol and the resulting mixture is then filtered. Then a large excess of acetone (70 ml) is added to precipitate the resin again. The resin is dissolved in about 20 ml of ethanol and the solvent is removed by a flash evaporator. 2.6 g of a white powder are obtained, which consists of a mixture of inorganic salts and the desired phosphate. The solid is dissolved in 10 ml of water, placed on a "Bio-Gel" P-2 column (particle size 0.038 to 0.074 mm, 2.7 x 90 cm) and eluted with water. The greater part of the monophosphate was eluted in a volume of 50 ml after 166 ml of the eluate had been collected. This could be demonstrated by thin layer chromatography on “Eastman Chromatogram” cellulose in n-propanol: water (70:30 v / v). The following values were obtained: Rf = 0.26 for 9- (2-hydroxyethoxymethyl) guanine phosphate and Rf = 0.11 for potassium 9- (2-hydroxyäthoxymethyl) guanine phosphate. The eluate was lyophilized to give 0.28 g of a solid containing 0.2 g of the monophosphate as confirmed by ultraviolet spectroscopy.

Example 3

Ammonium 9- (2-hydroxyethoxymethyl) guanine monophosphate

0.28 g of 9- (2-hydroxyethoxymethyl) guanine phosphate are dissolved in 30 ml of water and the pH of the solution is adjusted to 6 using 6N hydrochloric acid. The product is adsorbed on 14 ml of packed charcoal (Fischer 5-690B, grain size 0.074 to 0.305 mm, washed with acid and deactivated with toluene). The activated carbon is washed thoroughly with water and eluted with 70 ml of 50% aqueous ethanol, which contains 2% concentrated ammonium hydroxide. The solvent is evaporated under reduced pressure to give 0.048 g of ammonium 9- (2-hydroxyethoxymethyl) guanine monophosphate, Rf = 0.30 on Eastman cellulose in n-propanol: water (70:30 v / v) .

Example 4

tablet

Sodium 9- (2-hydroxyethoxymethyl) -

■ guanine phosphate

100 mg

Lactose

200 mg

Strength

50 mg

Polyvinyl pyrrolidone

5 mg

Magnesium stearate

4 mg

total weight

359 mg

Example 5

Solution for ophthalmic use N atrium-9- (2-hydroxyethoxymethyl) guanine phosphate 1.0 g

Sodium chloride, analytical grade 0.9 g

Thimerosal (o- (ethyl mercurithio) benzoic acid) 0.001 g of purified water to 100 ml pH, adjusted to 5.5 to 7.5

Example 6

injectable solution sodium 9- (2-hydroxyethoxymethyl) guanine phosphate

0.775 g sterile, pyrogen-free phosphate buffer (pH 7) ad 25 ml Example 7

Disodium 9- (2-hydroxyethoxymethyl) guanine phosphate

54 ml of phosphorus oxychloride are added with stirring to a mixture of 25 g of 9- (2-hydroxyethoxymethyl) guanine and 250 ml of triethyl phosphate, cooled to -30 to -20 ° C., for 3 hours. The temperature of the reaction mixture is allowed to rise to 0 ° C. over 45 minutes and the reaction mixture is kept at this temperature for a further 45 minutes. Then the reaction mixture is poured into a mixture of ice and water, whereupon the pH is adjusted to about 1 with 2N sodium hydroxide. The resulting solution is extracted once with chloroform and once with ether. The pH of the remaining aqueous solution is adjusted to 6.8 with 2N sodium hydroxide and then to 7.3 with 10N sodium hydroxide, a final volume of 2.5 liters being obtained.

The neutralized solution was applied to a column containing 2000 g of Dowex 1 x 8 which had been compared with 50 mmol KHCOî. The mixture was then eluted with 30 liters of a 50 to 500 m-molar KHCOa solution with a linear gradient, after which it was washed with 30 liters of a 500 m-molar KHC03 solution. The fractions containing the product were pooled and most of the KHCO3 was separated by adding Dowex 50-H + and removing the CO2 under vacuum. The volume was reduced to 2 liters in vacuo, after which the product was precipitated at 4 ° C. by adding 10 liters of acetone. 55 g of the dried precipitate was applied to a 10: 110 cm "Bio-Gel" P-2 column and eluted with water; 22 g of solid were obtained. The material was crystallized at 4 ° C from an aqueous solution at pH 3 as the hydrogen salt. Further material was obtained from the mother liquor by crystallization from 20% ethanol at pH 3. The hydrogen salt was dissolved in the smallest possible volume of water, whereupon the solution was brought to pH 8.5 with NaOH. This was followed by precipitation with 2 volumes of ethanol at 4 ° C. The precipitate thus produced was dissolved in 100 ml of water and reprecipitated with 9 volumes of ethanol at 4 ° C. 15.1 g of the disodium salt of 9- (2-hydroxyethoxymethyl) guanine monophosphate were obtained in the form of the dihydrate.

The purity was confirmed by elemental analysis, high pressure liquid chromatography and UV spectroscopy.

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643 858

8th

Empirical formula: CsHi2N506P2Na2H20

Calc .: C 24.94%; H 3.66%; N 18.19%; P 8.04% Found: C 25.20%; H 3.63%; N 18.10%; P 7.89%.

UV spectra

Solvent Imax.

0.1 M HCl 254

pH 7 249

0.1M NaOH 255-264

E À min. shoulder

12 970 225 272 14 060 219 266 11 760 228

Purity determined by high pressure liquid chromatography: 99%

Ratio: base / phosphate = 1.00 / 1.01

B

Claims (20)

643 858 2nd PATENT CLAIMS 1. 9-Hydroxyethoxymethylguanine derivative of the formula I 0 ch2-o.ch2-ch2-o-p-oz ÒW (I), where W and Z, which are the same or different, are each a 2. Process for the preparation of a 9-hydroxyethoxy or a pharmaceutically acceptable cation 20 methylguanine derivative of the formula I. mean. Oh H2r • n 0 -N ch2. 0. ch2. ch2. o-p-oz L (I) in which W and Z, which are the same or different, each have a meaning, characterized in that a compound atom or a pharmaceutically acceptable cation of the formula II oh n h2N n ' _n ■ (ii) : H2-O.CH2.ch2.OH with a phosphorylating agent to give a compound of formula I and optionally the resulting compound of formula I, in which W and Z are both hydrogen, with a base containing the desired cation or a corresponding salt to a compound of formula I, in which W and / or Z is a pharmaceutically acceptable cation. 3rd 643 858 a hydroxide, bicarbonate or carbonate containing the desired cation is used as the base. 3. The method according to claim 2, characterized in that a phosphoric acid derivative is used as the phosphorylating agent. 4th wherein W and Z, which are the same or different, each represent a hydrogen atom or a pharmaceutically acceptable cation M wherein G is a 2-amino group; and M denotes a group which can be converted into a hydroxyl group or can be replaced by a group or a corresponding atom by selective hydrolysis, is converted into a compound of the formula I by selective hydrolysis and, if appropriate, the compound of the formula I obtained in which W and Z are both hydrogen, with a the desired cation-containing base or a corresponding salt to give a compound of the formula I in which W and / or Z are a pharmaceutically acceptable cation. 4. The method according to claim 3, characterized in that one uses a phosphoric acid derivative in which 1 to 3 hydroxyl groups are replaced by halogen atoms. 5. The method according to claim 4, characterized in that phosphorus-xychloride is used as the phosphoric acid derivative. 6. The method according to claim 3, characterized in that a phosphoric acid derivative in which up to two hydroxyl groups by alkoxy groups, which- 55 on the one hand can carry substituents, are replaced, used. 7. The method according to claim 5, characterized in that reacting the compound of formula II with phosphorus oxychloride in the presence of a trialkyl phosphate at a temperature of 0 ° C or below. 60 8. The method according to claim 5, characterized in that the compound of formula II is reacted with phosphorus oxychloride in dry pyridine. 9. The method according to any one of claims 2 to 8, characterized in that one for transferring a 65 Compound of the formula I, in which W and Z both represent hydrogen, into the corresponding compound, in which W and / or Z represent a pharmaceutically acceptable cation, 10. The method according to claim 9, characterized in that the desired cation is a sodium, potassium, Is ammonium, lithium, calcium, magnesium or aluminum cation. 11. A process for the preparation of a 9-hydroxyethoxy-methylguanine derivative of the formula I. 0 0.ch "-. Ch" .0- • oz ÒW (I), wherein W and Z, which are the same or different, each represent a hydrogen atom or a pharmaceutically acceptable cation, characterized in that a compound of formula III 0 ch2-0.ch .ch .o-p-oh Oh (iii), where 40 M is a 6-hydroxy group; and G is a group which can be converted into an amino group or can be replaced by an ammonium group or a corresponding atom by selective ammonolysis, is converted into a compound 45 of the formula I by selective ammonolysis and, if appropriate, the compound of the formula I obtained in which W and Z are both hydrogen, with a the desired cation-containing base or a corresponding salt is converted into a compound of the formula I in which W and / or Z are a pharmaceutically acceptable cation. 12. The method according to claim 11, characterized in that one of a compound of formula III, where M is a 6-hydroxy group and G is a halogen atom or a mercapto or alkylthio group, and these are subjected to ammonolysis. 13. The method according to claim 11 or 12, characterized in that for converting a compound of formula I, wherein W and Z are both hydrogen, in the corresponding combination, in which W and / or Z are a pharmaceutically acceptable cation, as a base the desired cation containing hydroxide, bicarbonate or carbonate used. 14. The method according to claim 13, characterized in that the desired cation is a sodium, potassium, ammonium, lithium, calcium, magnesium or aluminum cation. 15. A process for the preparation of a 9-hydroxyethoxy-methylguanine derivative of the formula I. 643 858 16. The method according to claim 15, characterized in that one starts from a compound of formula III ', in which M is a halogen atom or a mercapto or alkylthio group and G is a 2-amino group, and this is subjected to hydrolysis. 17. The method according to claim 15 or 16, characterized in that for converting a compound of formula I, wherein W and Z are both hydrogen, into the corresponding compound, wherein W and / or Z are a pharmaceutically acceptable cation, as a base the desired cation containing hydroxy, bicarbonate or carbonate used. 18. The method according to claim 17, characterized in that the desired cation is a sodium, potassium, ammonium, lithium, calcium, magnesium or aluminum cation. 19. A pharmaceutical preparation containing a verbinoh wherein W and Z, which are the same or different, each represent a hydrogen atom or a pharmaceutically acceptable cation. Examples of pharmaceutically acceptable cations are sodium, potassium, lithium, calcium / 2, magnesium / 2, aluminum / 3 or ammonium. Compounds of the formula I in which Z is sodium, potassium or ammonium mean, characterized in that a compound of formula III ' (iii 1), of formula I together with a pharmaceutically acceptable carrier. 20th