CA1094062A

CA1094062A - 9-(2-hydroxyethoxymethyl) guanine monophosphate derivatives

Info

Publication number: CA1094062A
Application number: CA297,833A
Authority: CA
Inventors: Howard J. Schaeffer
Original assignee: Wellcome Foundation Ltd
Current assignee: Wellcome Foundation Ltd
Priority date: 1977-02-24
Filing date: 1978-02-24
Publication date: 1981-01-20
Also published as: IT7848192A0; FI68402C; AU3356078A; ATA134678A; CH643858A5; IL54130A0; IN149483B; IE780397L; FI68402B; SE7802140L; AR218718A1; MC1182A1; NO153260B; DD134098A5; PL114474B1; PT67706B; PH13996A; IE46210B1; ES467300A1; AR219735A1

Abstract

ABSTRACT

This invention relates to a 9-hydroxyethoxymethyl guanine derivative of formula (I)

Description

O~;Z

This invention relates to 9-(2-hydroxyethoxy-methyl)guanine monophosphate, in particular to pharmaceutically acceptable salts thereof. The invention also relates to methods of preparing this compound and its salts, and also to pharmaceutical compositions containing them.
9-(2-Hydroxyethoxymethyl) derivatives of purines are known to have antiviral activity against various classes of D~A and RNA viruses both in in vitro and in vivo experiments, see U.K. Patent No. 1,523,865. In parti-cular these compounds are active as antiviral agents against Vaccinia, and herpes viruses, including simplex, zoster and varicella in mammals, which viruses cause such diseases as herpetic keratitis in rabbits and herpetic encephalitis in mice.
It has now been found that the monophosphate ester of 9-(2-hydroxyethoxymethyl)guanine is not only as active as the unphosphorylated compound but also has the selective advantage of much greater solubility at least at a pH of from 1 to 7.5 compared with the correspond-ing unphosphorylated compound.
According to the present invention there is provided an 9-hydroxyethoxymethyl guanine derivative of formula (I~:

OH

Hz ~

CH2 . O ~ CH2 C~2 ~ I

- 2 -10~ 40fi Z YC/77 wherein W and Z are the same or different and each represents an hydrogen atom or pharmaceutically acceptable cation.
The pharmaceutically acceptable cation may be selected from a group comprising sodium, potassium, lithium, calcium/2, magnesium~2, aluminium/3 or ammonium.
Compounds of formula (I) wherein Z is sodium, potassium or ammonium and W is hydrogen are preferred, and compounds of formula (I) wherein Z is sodium or ammonium and W i5 hydrogen are particularly preferred.
In the above definition of W and Z, the polyvalent cations are expressed as calcium/2, magnesium/2 and aluminium/3, which is intended to mean the cation divided . ~ ~/2 and Al~*/3. This is meant by its valence le. Ca /2, Mg to indicate that calcium or magnesium cations are in ionic association with two phosphate oxygens, arld aluminium with three.
In a second aspect of the present invention there is provided a method of preparing a compound of formula (I), as de~ined above characterised in that:-(a) a compound of formula (II) OH

H2N ~ ~

~2-0-CH2-cH2.OH
is reacted ~ h a phosphorylating agent to produce a compound of formula (I) wherein l~oth ~ and Z are hydrogen lO9~0fiZ
atoms, (b) a compound of formula (III), M

N ~ ~ (III) G N N

CH20.CH2.CH2.O-P-OH
OH
wherein either M is a 6-hydroxy group and G is an atom or group that can be replaced or converted to an amino group by selective ammonolysis, or G is a 2-amino group and M is an atom or group that can be replaced or converted to an hydroxy group by selective hydrolysis, is converted to a compound of formula (I) and optionally converting a compound of formula (I) wherein W and Z are both hydrogen into a compound wherein either or both of W and Z is a pharmaceuti-cally acceptable cation, by reaction with a base or a salt containing the desired cation.
In method (a) derivativés of phosphoric acid having one to three hydroxy groups replaced by halogen atoms, e.g. chlorine, such as phosphorus oxychloride, are preferred for phosphorylation. Up to two of the hydroxy groups can also be substituted to form alkoxy groups optionally carrying further substitutions to form for instance benzyloxy groups. SUch phosphohalic derivatives or phosphates are applied under the usual neutral or alka-line conditions, the latter preferably requiring activation for instance by carbodiimide, e.g. dicyclohexylcarbodiimide except when it is presented in ~ Z yC/77 the form of the anhydride.
Where at least two of the hydroxy groups in the phosphoric acid derivative are replaced by halogen, then after reaction with the compound of formula (II) it is necessary to remove the free halogens by fairly mild aqueous hydrolysis, using for example a molar equivalent of water in a water miscible solvent such as alcohol.
Substituted or unsubstituted alkoxy groups introduced with a phosphate may be hydrolysed in a suitable aqueous medium in the presence of bases in a subsequent step. Aromatically substituted alkoxy groups such as benzyloxy can also be subjected to hydrogenolysis, preferably in the presence of a catalyst, according to the usual techniques of reductive cleaving.
A preferred method of phosphorylating the intermediate compounds of this invention involves reaction of a compound of formula ~II), as defined above, with phosphorus oxychloride in the presence of a trialkyl phosphate and preferably at a temperature of about 0C
or less.
Other useful methods for preparing the mono-phosphate include reaction of a compound of formula (II) with phosphorus oxychloride in dry pyridine.
Compound of formula (II) can be considered as intermediate in the synthesis of compounds of formula ~I) ~and can be prepared according to the methods described in ;Z

U.K. Patent No. 1,523,86S.
Conversion of a compound of formula (III), by method (b) can be achieved in several ways, for example, when G is a halogen atom, mercapto or an alkylthio group, such as methylthio, it can be converted to an amine group by ammonolysis. This method, together with other processes well known in the art can be found in "Heterocyclic compounds - Fused Pyrimidines Part II Purines ed. by D. J.
Brown (1971) published by Wiley - Interscience".
M can represent a halogen atom, mercapto or alkyl-thio group, which may be converted to an hydroxy group by hydrolytic methods described in the aforementioned book.
Compounds of formula (III) can be considered as intermediates in the synthesis of compounds of formula (I) and can be analogously prepared according to method (a), which compounds can in turn be analogously prepared accord-ing to the methods described in U.K. Patent No. 1,523,865.
Pharmaceutically acceptable salts of 9-(2-hydroxy-ethoxymethyl)guanine monophosphate may be prepared by neutralizing the monophosphate in its acidic form with an equivalent (i.e. equinormal) amount of a base such as an hydroxide, bicarbonate, carbonate which contains the desired cation, that is sodium, potassium, ammonium, calcium, lithium, magnesium or aluminium. Alternatively they may be prepared by exchange reactions whereby one salt of the monophosphate is treat~d with a solution, preferably aqueous, of a salt containing the desired cation. For example the slightly , ~ . . , ,, - . .

10~40fi2 soluble barium salt of 9-(2-hydroxyethoxymethyl)guanine monophosphate is treated in aqueou~ suspension with sodium sulphate to remove the barium as the very insoluble barium sulphate, leaving sodium 9-(2-hydroxyethoxymethyl)-guanine monophosphate in solution.
In another aspect of the invention there is provided a pharmaceutical composition comprising a compound of formula (I) as hereinbefore defined together with a pharmaceutically acceptable carrier therefor.
Pharmaceutically acceptable carriers are materials useful for the purpose of administering the composition, and may be solid, liquid or gaseous materials, which are otherwise inert and medically acceptable and are compatible with the active ingredient. These pharmaceutical composi-tions may be administered orally, parenterally, used as a suppository or pessary, applied topically as an ointment, cream, aerosol, or powder, or given as eye or nose drops, depending on whetHer the preparation is used to treat internal or external viral infections.
For internal infections the compound of formula (I) are administered at dose levels of 0.1 to 250 mg per kg, calculated as the free phosphate form, preferably 1.0 to 50 mg per kg of mammal body weight, and are used in man in a unit dosage form, administered for example a few times daily, as one or more unit doses in an amount of 1 to 800 mg per unit dose, preferably 1 to 250 mg per unit dose, most preferably 10 to 200 mg per unit dose.

lO ~ ~CH~2 yC/77 Por p~renteral administration, or administration topically as drops, e.g. for eye infections, compounds of formula (I) may be presented in aqueous solutions at a concentration of from about 0.1 to 10~ w/v, preferably 0.1 to 7%, most preferably 0.2 to 5% w/v.
Alternatively, for infections of the eye or other external tissue, e.g. mouth and skin, solution, ointment or cream topical formulations are preferred. Concentrations of from about 0.1 to 10~, preferably 0.3 to 6~, most preferably 3~ may be used.
In yet a further aspect of the invention there is provided a method of treating viral infections in mammals which comprises the administration of an effective non-toxic antiviral amount of a compound of formula (I) as hereinbefore defined. As used herein the term "effective non~toxic antiviral amount" is denoted to mean a predeter-mined antiviral amount sufficient to be effective ag&inst the virus in vivo.
The invention will now be illustrated with reference to the following Examples.

10~ ~~Z YC/77 9-(2-l~ydroxyethox~ethyl)guanine monophosphate Phosphorus oxychloride (0.03 ml) was added in one portion to a stirred suspension of 2-chloro-9-(2-hydroxy-ethoxymethyl)hypoxanthi~e (20 mg) in triethyl phosphâte (0.3 ml) at -8C. The temperature was allowed to rise to 0C over 30 minutes. The reaction mixture was then stirTed at 0C for 40 minutes and at ~5C for 50 minutes. It was then poured onto ice, and the pH was adjusted to 7 with 2N
potassium hydroxide. The resulting solution was extracted twice with chloroform (2 x 2 ml). The aqueous phase was adjusted to pH 8-8.5 with 2N potassium hydroxide, and barium acetate (105 mg) was added. The resulting barium phosphate precipitate was removed by filtration. The supernatant was treated with a large excess of ethanol, precipi~ating crude barium 2-chloro-9-(2-hydroxyethoxymethyl) hypoxanthine monophosphate. The solid was collected by filtration and suspended in ethanol. The ethanolic suspension was then heated on a steam bath for several minutes, cooled and filtered. The collected precipitate was washed with anhydrous ether and dried, giving barium 2-chloro-9-(2-hydroxyethoxymethyl)hypoxallthine monophosphate (26 mg).
Ammonium sulfate (3.96 mg) was added to a stirred suspension of barium 2-chloro-~-(2-hydroxyethoxymethyl) hypoxanthine monophosphate ~7 mg) in water (0.5 ml). The mixture was stirred at ambient temperature for 15 m;nutes and then cooled in an ice bath. The precipitated barium sulfate was remo~ed by filtration and wasTIed Wit}l water g 109 40fi 2 YC/77 (1 ml) and ethanol (10 ml). The combined filtrate and washings were evaporated under reduced pressure, and the resulting residue dissolved in methanol (3 ml).
The methanolic solution was transferred to a Teflo ~
lined stainless steel bomb~ and methanol ~ ml) saturated with gaseous ammonia at ice bath temperature was also added to the bomb. The sealed bomb was placed in a 122C
oven for 4 hours, chilled and opened. Solvent was evaporated to minimal volume. The residual reaction mixture was spotted on Eastman Chromatogram~ cellulose TLC
sheets which were then developed in n-propanol:water ~70:30 v/v). The bands at Rf 0.16 and 0.34 were excised, suspended in Tris buffer (0.6 ml) at pH 8, and the cellulose was removed by filtration.
These bands were shown to contain 9-(2-hydroxy-ethoxymethyl)guanine monophosphate and 2-chloro-9-(2-hydroxy-ethoxymethyl)hypoxanthine monophosphate by enzymatic dephosphorylation with alkaline phosphatase to 9-(2-hydroxy-ethoxymethyl)guanine and 2-chloro-9-(2-hydroxyethoxymethyl) hypoxanthine, respectively. Alkaline phosphatase (2 ~1) from E. coli was added to the filtrate and the mixture was heated at 32C for 2 hours. It was then examined by thin layer chromatography on Eastman Chromatogram~ cellulose sheets in three solvent systems:-(a) _-propanol:water (70:30 v/v) (b) water (c) n-propanol conc. ammonium hydroxide:water (60:30:10 v/v) yC/77 ` ` 10940fi2 two spots were present in each system, corresponding to ~-(2-hydroxyethoxymethyl)guanine (A) and 2-chloro-9-(2-hydroxyethoxymethyl)hypoxanthine ~B).

Solvent System Rf (A) Rf (B) Rf of 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 9-(2-Hydroxyethoxymethyl)guanine monophosphate Phosphorus oxychloride (0.76 ml) was added to a stirred, cooled (-10C) mixture of 9-(2-hydroxyethoxymethyl) guanine (0.225 g) and triethyl phosphate (5 ml). The temperature of the reaction mixture was allowed to rise to 0C over 30 minutes and was held at this temperature for 2 hours. It was then poured onto a mixture of ice and water, - and the pH was adjusted to 7 with 2N potassium hydroxide.
The resulting solution was extracted twice with chloroform and once with ether. The pH of the remaining aqueous solution was adjusted to 7.1 with 2N potassium hydroxide and was then lyophylized. The resulting white solid was dissolved in water (7 ml), and methanol (7 ml) was added to precipitate the inorganic sal~s which were then removed by filtration.
Acetone ~70 ml) was added to the filtrate, precipitating a white gum. The gum was dissolved in water (7 ml), ethanol (7 ml) added and the mixture filtered. A large excess of acetone (70 ml) was added, again precipitating the gum. The `` ` lO~Ofi2 gum was dissolved in ethanol (ca 20 ml) and the solvent was removed by flash evaporation, giving a white powder (2.6 g) which was a mixture of inorganic salts and the desired phosphate. The solid was dissolved in water B ~lo ml), applied to a Bio-Gel P-2 column ~200-400 mesh, 2.7 x 90 cm) and eluted with water. The majority of the monophosphate was eluted in a 50 ml volume after 166 ml of eluate had been collected, as shown by thin layer chromatography on Eastman Chromagram~ cellulose in _-propanol:water ~70:30 v/v); Rf = 0.26 for 9-~2-hydroxy-ethoxymethyl)guanine phosphate and Rf = 0.11 for potassium 9~2-hydroxyethoxymethyl)guanine phosphate. The eluate was lyophilized to give 0.28 g of a solid which was shown by ultraviolet spectroscopy to contain 0.2 g of mono-phosphate product.

Ammonium 9-~2-hydroxyethoxymethyl)guanine monophosphate 9-~2-Hydroxyethoxymethyl)guanine phosphate ~0.28 g) was dissolved in water ~30 ml) and the pH of the solution was adjusted to 6 with 6N hydrochloric acid. The product was adsorbed onto 14 ml of packed charcoal (Pischer 5-690B, 50-200 mesh, acid washed and deactivated with toluene). The charcoal was washed well with water and eluted with 70 ml of 50~ aqueous ethanol containing 2~ concentrated ammonium hydroxide. The solvent was evaporated under reduced pressure to give ammonium 9-~2-hydroxyethoxymethyl)guanine mono-phosphatc ~0.048 g); Rf = 0.30 on Eastman cellulose in ~ ~aJ~ n~

10~0~

n-propanol:water (70:~0 v/v).

EXAMPLE 4 - Tablet Sodium 9-(2-hydroxyethoxymethyl)guanine phosphate 100 mg Lactose 200 mg Starch . 50 mg Polyvinylpyrrolidone 5 mg Magnesium stearate 4 mg Total weight 359 mg EXAMPLE 5 - Ophthalmic Solution 10Sodium 9-(2-hydroxyethoxymethyl)guanine phosphate 1.0 g Sodium chloride, analytical reagent 0.9 g Thimerosal 0.001 g Purified water, ~.s. to lOO. ml pH adjusted to 5.5-7.5 EXAMPLE 6 - Injectable Solution Sodium 9-(2-hydroxyethoxymethyl)guanine phosphate 0.775 g Sterile, pyrogen-free, pH7 phosphate buffer ~s to 25 ml EXA~IPLE 7 Disodium 9~2-hydroxyethox~ymethyl)guanine phosphate 20Phosphorus oxychloride ~54 ml) was addcd over a 3 hour period to a stirred~ cooled (-30 to -20C) mixture of 9-(2-hydroxyethox~neth}rl)guanine (25 g) and triethyl 10~ 4~fi 2 YC/77 phosphate (250 ml). The temperature of the reaction mixture was allowed to rise to 0C over 45 minut~s and was held at that temperature for an additional 45 minutes.
It was then poured into a mixture of ice and water, and the pH was adjusted to about 1 with 2N sodium hydroxide.
The resulting solution was extracted once with chloroform and once with ether. The pH of the remaining aqueous solution was adjusted to 6.8 with 2N sodiuln hydroxide and subsequently to 7.3 with lON sodium hydroxide, giving a final volume of 2.5 liters.
The neutralized solution was applied to a column B containing 2,000g of Dowex 1 x 8 which had been equilibrated with 50 mM KHC03. Elution was by a 30 Q linear gradient of 50-500 mM KHC03 followed by a 30 ~ wash of 500 mM KH~03.
The fractions containing product were combined and most of the KHC03 was removed by adding ~owex~ 50-H and removing C02 under vacuum. The volume was reduced to 2 Q in vacuo and the product was precipitated at 4C by the addition of 10 of acetone. The dr ed precipitate 55 g was applied to a 10 x 110 cm Bio-Gel P-2 column and eluted with E~20. 22 g of solid was obtained. The material was recrystallized at 4~C
as the hydrogen salt from a pH 3 solution of water and more material was obtained from the mother liquor by crystalli~ing from 20~ ethanol at pH 3. The hydrogen salt was dissolved in a minimal ~ol~ne of H20 brought to pH 8.5 with NaOE~ and precipitated with 2 volume of ethanol at 4C. This preci-pitate was dissolved in 100 ml H20 and precipitated ~ith 9 volumec of ethanol at 4C to give 15.1 g of 9-~2-hy~roxy-~ rr~d~

10~?40fiZ
ethoxymethyl)guanine monophosphate disodium salt dihydrate.
Purity was confirmed by elemental analysis, high pressure liquid chromatography and W spectra.
Empirical formula : C8H12N5O6P 2Na 2H2O
Theory: 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%
W Spectra: Solvent ~ max ~ ~ min sh O.LM HCl 254 12970 225 272 ph 7 299 14060 219 266 0.lm NaOH 255-264 11760 228 High pressure liquid chromatography purity = 99%
Base/phosphate ratio = 1.00/1.01

Claims

The embodiments of the invention in which an exclusive property or privilege is claimed are defined as follows:

1. A method of preparing a 9-hydroxyethoxymethyl guanine derivative of formula (I) (I) wherein W and Z are the same of different and each represents a hydrogen atom or pharmaceutically acceptable cation, characterized in that:-(a) a compound of formula (II) (II) is reacted with a phosphorylating agent to produce a compound of formula (I) wherein both W and Z are hydrogen atoms;
(b) a compound of formula (III) (III) wherein either M is a 6-hydroxy group and G is an atom or group that can be replaced or converted to an amino group by selective ammonolysis, or G is a 2-amino group and M is an atom or group that can be replaced or converted to an hydroxy group by selective hydrolysis, is converted to a compound of formula (I); and optionally converting a compound of formula (I) wherein W and Z are both hydrogen into a compound wherein either or both of W and Z is a pharmaceutically acceptable cation, by reaction with a base or a salt containing the desired cation.

2. A method as claimed in claim 1(a) wherein the phosphorylating agent is a derivative of phosphoric acid.

3. A method as claimed in claim 2 wherein the derivative of phosphoric acid has one to three hydroxy groups replaced by halogen atoms.

4. A method as claimed in claim 3 wherein the phosphoric acid derivative is phosphorus oxychloride.

5. A method as claimed in claim 2 wherein up to two hydroxy groups in the phosphoric acid are substituted to form alkoxy groups, optionally carrying further substitutions.

6. A method as claimed in claim 4, wherein the com-pound of formula (II) is reacted with phosphorus oxy-chloride in the presence of a trialkylphosphate at a temperature of 0°C or less.

7. A method as claimed in claim 4, wherein the com-pound of formula (II) is reacted with phosphorus oxy-chloride in dry pyridine.

8. A method as claimed in claim 1(b), in which a com-pound of formula (III), wherein M is a 6-hydroxy group and G is a halogen atom, mercapto or an alkylthio group, is converted to a compound of formula (I) by ammonolysis.

9. A method as claimed in claim 1(b), in which a compound of formula (III) wherein M is a halogen atom, mercapto or alkylthio group and G is a 2-amino group, is converted to a compound of formula (I) by hydrolysis.

10. A method as claimed in claim 1, including a step of reacting a compound of formula (I) in which W
and Z are both hydrogen with a base selected from the group consisting of hydroxides, bicarbonates and carbonates having a pharmaceutically acceptable cation.

11. A method as claimed in claim 10, wherein the cation is selected from the group consisting of sodium, potassium, ammonium, calcium, lithium, magnesium and aluminium.

12. A method according to claim 1, wherein W and Z
are both hydrogen.

13. A method according to claim 1, wherein Z is sodium, potassium or ammonium and W is hydrogen.

14. A method according to claim 1, wherein Z is sodium or ammonium and W is hydrogen.

15. A method according to claim 1(b), of preparing 9-(2-hydroxyethoxymethyl)guanine monophosphate comprising ammonolysing 2-chloro-9-(2-hydroxyethoxymethyl)hypoxanthine monophosphate.

16. A method according to claim 1(a), of preparing 9-(2-hydroxyethoxymethyl)guanine mophosphate comprising phosphorylating 9-(2-hydroxyethoxymethyl)guanine.

17. A method according to claim 16, wherein said phosphorylating is carried out with phosphorus oxychloride.

18. A method according to claim 10, of preparing ammonium 9-(2-hydroxyethoxymethyl)guanine monophosphate comprising reacting 9-(2-hydroxyethoxymethyl)guanine monophosphate with ammonium hydroxide.

19. A method according to claim 10, of preparing disodium 9-(2-hydroxyethoxymethyl)guanine monophosphate comprising reacting 9-(2-hydroxyethoxymethyl)guanine monophosphate with sodium hydroxide.

20. A 9-hydroxyethoxymethyl guanine derivative of formula (I), as defined in claim 1, whenever prepared by the method of claim 1, 2 or 4, or by an obvious chemical equivalent.

21. A 9-hydroxyethoxymethyl guanine derivative of formula (I), as defined in claim 1, in which W and Z are both hydrogen, whenever prepared by the method of claim 12, or by an obvious chemical equivalent.

22. A 9-hydroxyethoxymethyl guanine derivative of formula (I), as defined in claim 1, wherein at least one of W and Z is selected from the group consisting of sodium, potassium, ammonium, calcium, lithium, magnesium and aluminium, whenever prepared by the method of claim 11, or by an obvious chemical equivalent.

23. A 9-hydroxyethoxymethyl guanine derivative of formula (I), as defined in claim 1, wherein Z is sodium, potassium or ammonium and W is hydrogen, whenever prepared by the method of claim 13, or by an obvious chemical equivalent.

24. A 9-hydroxyethoxymethyl guanine derivative of formula (I), as defined in claim 1, wherein Z is sodium or ammonium and W is hydrogen, whenever prepared by the method of claim 14, or by an obvious chemical equivalent.

25. 9-(2-Hydroxyethoxymethyl)guanine monophosphate, whenever prepared by the method of claim 15, 16 or 17, or by an obvious chemical equivalent.

26. Ammonium 9-(2-hydroxyethoxymethyl)guanine mono-phosphate, whenever prepared by the method of claim 18, or by an obvious chemical equivalent.

27. Disodium 9-(2-hydroxyethoxymethyl)guanine mono-phosphate, whenever prepared by the method of claim 19, or by an obvious chemical equivalent.