NO153260B - ANALOGY PROCEDURE FOR THE PREPARATION OF ANTIVIRAL EFFECTIVE 9-HYDROXYETOXYGUANINE DERIVATIVES - Google Patents

Description

The present invention relates to the production of a 9-hydroxyethoxymethylguanine derivative with the formula:

wherein W and Z are the same or different and each is a hydrogen atom or a pharmaceutically acceptable cation.

9-(2-Hydroxyethoxymethyl) derivatives of purines are known to have antiviral activity against various classes of DNA and RNA viruses both in vitro and in vivo experiments, see Norwegian patent no. 144,216. These compounds are particularly active as anti-viral agents against cowpox and herpes viruses, including simplex, zoster and varicella in mammals, which viruses cause diseases such 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 active as the non-phosphorylated compound, but also has the selective advantage of much greater solubility at at least a pH value of from 1 to 7, 5 compared to the corresponding non-phosphorylated compound.

The pharmaceutically acceptable cation may be selected

from a group comprising sodium, potassium, lithium, calcium/2, magnesium/2, aluminum/3 or ammonium. Compounds of formula I wherein Z is sodium, potassium or ammonium, and where

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

In the above definition of W and Z, the multivalent cations are expressed as calcium/2, magnesium/2 and aluminum/3, which should mean the cation divided by its valence, i.e. Ca<++>/2, Mg<+ +>/2 and Al<+++>/3. This should mean that calcium or magnesium cations are in ionic connection with two phosphate oxygen atoms and aluminum with three.

According to the present invention, the preparation of a compound of formula I comprises that:

(a) a compound of the formula:

is reacted with a phosphorylating agent to form a compound of formula I wherein both W and Z are hydrogen atoms; or

subjected to ammonolysis to a compound of formula I and, if desired, conversion of a compound of formula I wherein W and Z are both hydrogen to a compound wherein one or both of W and Z is a pharmaceutically acceptable cation, by reaction with a base or a salt containing the desired cation.

In method (a) are derivatives of phosphoric acid, which have one more

three hydroxy groups replaced by halogen atoms, e.g. chlorine,

such as phosphorus oxychloride, preferred for phosphorylation. Up to two of the hydroxy groups can also be substituted to form alkoxy groups, optionally containing further substitutions to form e.g. benzyloxy groups. Such phospho-halide derivatives or phosphates are used under the usual neutral or alkaline conditions, the latter preferably necessitating activation, e.g. with a carbodiimide, e.g. dicyclohexylcarbodiimide except when present in the form of the anhydride.

When at least two of the hydroxy groups in the phosphoric acid derivative

is replaced by halogen, after the reaction with the compound of formula II, it will be necessary to remove the free halogens by means of mild aqueous hydrolysis, e.g. using a molar equivalent of water in a water-miscible solvent, such as alcohol.

Substituted or unsubstituted alkoxy groups in a phosphate derivative can be hydrolyzed 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 conventional reductive cleavage techniques.

A preferred method for phosphorylating the intermediates involves reacting 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 0°C or lower.

Other useful methods for preparing the monophosphate are the reaction of a compound of formula II with phosphorus oxychloride in dry pyridine.

The compound with formula II can be considered an intermediate product in the synthesis of compounds with formula I and can be prepared according to the methods described in Norwegian patent no. 144,216.

Ammonolysis of a compound of formula III by

using method b) is described in "Heterocyclic compounds - Fused Pyrimidines Part II Purines", by D. J. Brown (1971) published by Wiley - Interscience.

Compounds of formula III can be regarded as intermediates in the synthesis of compounds of formula I and can be prepared analogously according to method (a), which compounds can in turn be prepared analogously according to the methods described in the above-mentioned British patent.

Pharmaceutically acceptable salts of 9-(2-hydroxyethoxymethyl)guanine monophosphate can be prepared by neutralizing the monophosphate in its acid form with an equivalent amount (ie, equinormal) of a base such as a hydroxide, bicarbonate, carbonate containing the desired cation, ie. sodium, potassium, ammonium, calcium, lithium, magnesium or aluminium. Alternatively, they can be produced by exchange reactions whereby a salt of the monophosphate is treated with a solution, preferably aqueous, of a salt containing the desired cation. The slightly soluble barium salt of 9-(2-hydroxyethoxymethyl)guanine monophosphate is treated e.g. in aqueous suspension with sodium sulfate to remove the barium in the form of the very insoluble barium sulfate, leaving sodium 9-(2-hydroxyethoxymethyl)-guanine monophosphate in solution.

A pharmaceutical preparation comprises a compound of formula I as defined above, together with a pharmaceutically acceptable carrier.

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 preparations can 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 for the treatment of internal or external viral infections.

For internal infections, the compounds of formula I are administered at dose levels of 0.1-250 mg per kg, calculated as the free phosphate, preferably in an amount of 1.0-50 mg per kg body weight, and is used in humans in a unit dosage form, administered e.g. sometimes daily in the form of one or more unit doses, in an amount of 1-800 mg per unit dose, preferably 1-250 mg per unit dose, and preferably in an amount of 10-200 mg per unit dose.

For parenteral administration or topical administration, as drops, e.g. for eye infections, the compounds of formula I may be present in aqueous solutions at a concentration of 0.1-10% w/v, preferably 0.1-7%, and preferably 0.2-5% w/v.

For infections in the eye or other external tissue, e.g. mouth and skin, alternatively topical solution, ointment or cream preparations are preferred. Concentrations of 0.1-10%

preferably 0.3-6%, and preferably 3%, can be used.

In the treatment of viral infections in mammals, an effective, non-toxic antiviral amount of a compound of formula I is administered. The term "effective, non-toxic antiviral amount" means a specific antiviral amount that is sufficient to be effective against the virus in vivo.

The following examples illustrate the invention.

Example 1

9-(2-Hydroxyethoxymethyl)guanine monophosphate

Phosphorus oxychloride (0.03 mL) was added in one more portion

a stirred suspension of 2-chloro-9-(2-hydroxyethoxymethyl)hypoxanthine (20 mg) in triethyl phosphate (0.3 mL) at -8°C. The temperature was allowed to rise to 0°C within 30 minutes. The reaction mixture was then stirred at 0°C for 40 minutes and at +5°C 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 (2x2 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, which resulted in the precipitation of impure 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 to give barium 2-chloro-9-(2-hydroxyethoxymethyl)hypoxanthine monophosphate (26 mg).

Ammonium sulfate (3.96 mg) was added to a stirred suspension of barium 2-chloro-9-(2-hydroxyethoxymethyl)hypoxanthine monophosphate (7 mg) in water (0.5 mL). The mixture was stirred at ambient temperature for 15 minutes and then cooled in an ice bath.

in

The precipitated barium sulfate was removed by filtration and washed

/\

\ with water (1 ml) and ethanol (10 ml). The combined filtrate and

the wax solutions were evaporated under reduced pressure and the resulting residue dissolved in methanol (3 mL). The methanolic solution was transferred to a "Teflon" lined, stainless steel container, and methanol (8 mL) saturated with gaseous ammonia at ice bath temperature was also added to the container. The sealed container was placed in an oven at 122°C for 4 hours, cooled and opened. Solvent was evaporated to a minimal volume. The remaining reaction mixture was spotted onto "Eastman i Chromatogram" cellulose thin layer chromatography sheets which were then developed in n-propanol:water (70:30 vol/vol). The bands at Rf 0.16 and 0.34 were removed, suspended in Tris buffer (0.6 ml) at pH 8

and the cellulose became filtration.

These bands were found to contain 9-(2-hydroxyethoxymethyl)guanine monophosphate and 2-chloro-9-(2-hydroxyethoxymethyl)hypoxanthine monophosphate by enzymatic dephosphorylation with alkaline phosphatase to 9-(2-hydroxyethoxymethyl)guanine and 2-chloro- 9-(2-hydroxyethoxymethyl) hypoxanthine, respectively. Alkaline phosphatase (2 yl) from

\ E. coli, was added to the filtrate and the mixture was heated at 32°C for 2 hours. It was then examined by thin-layer chromatography on "Eastman Chromatogram" cellulose sheets in three solvent cisterns:

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

(b) water

(c) n-propanol conc. ammonium hydroxide:water (60:30:10 vol/vol) with two spots found in each system, corresponding to 9-(2-hydroxyethoxymethyl)guanine (A) and 2-chloro-9-(2-hydroxyethoxymethyl)hypoxanthine (B ).

Example 2

9-(2-Hydroxyethoxymethyl)guanine monophosphate

Phosphorus oxychloride (0.76 mL) was added to a stirred, cooled (-10°C) 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 0°C within 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 then lyophilized. The resulting white solid was dissolved in water (7 mL) and methanol (7 mL) was added to precipitate the inorganic salts which were then removed by filtration. Acetone (70 mL) was added to the filtrate, resulting in the precipitation of a white gummy substance. This substance was dissolved in water (7 ml), ethanol (7 ml) was added and the mixture filtered. A large excess of acetone (70 ml) was added and this also precipitated a gummy substance. This material was dissolved in ethanol (ca. 20 ml) and the solvent was removed by flash evaporation to give a white powder (2.6 g) which was a mixture of inorganic salts and the desired phosphate. The solid was dissolved in water (10 mL), applied to a "Bio-Gel P-2" column (200-400 mesh, 2.7x90 cm) and eluted with water. Most of the monophosphate was eluted in a 50 ml volume after 166 ml of the eluate was collected, as shown by thin layer chromatography on "Eastman Chromatogram" cellulose in n-propanol:water (70:30 vol/vol); Rf = 0.26 for 9-(2-hydroxyethoxymethyl)guanine phosphate and Rf = 0.11

for potassium 9-(2-hydroxyethoxymethyl)guanine phosphate. The eluate was lyophilized and this gave 0.28 g of a solid which, by ultraviolet spectroscopy, was shown to contain 0.2 g of the monophosphate product.

Example 3

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 ("Fischer 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 monophosphate (0.048 g); Rf = 0.30 on "Eastman" cellulose paper in n-propanol: water (70:30 vol/vol).

Example 4

Disodium 9-(2-hydroxyethoxymethyl)guanine phosphate

Phosphorus oxychloride (54 mL) was added over 3 hours to a stirred, cooled (-30 to -20°C) mixture of 9-(2-hydroxyethoxymethyl)guanine (25 g) and triethyl phosphate (250 mL). The temperature of the reaction mixture was allowed to rise to 0°C within 45 minutes and was held at this temperature for a further 45 minutes. It was then poured into a mixture of ice and water and the pH was adjusted to approx. 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 sodium hydroxide and then to 7.3 with 10N sodium hydroxide, giving a final volume of 2.5 liters.

The neutralized solution was applied to a column containing 2000 g of "Dowex 1 x 8" equilibrated with 50 mM KHCO 2 . Elution was by 30 l linear gradient of 50-500 mM

KHCO 3 followed by a 30 in wash of 500 mM KHCO 3 . The fractions containing the product were combined and most of the KHCO was removed by addition of "Dowex 50-H" and removal of CO 2 under vacuum. The volume was reduced to 2 L in vacuo and the product was precipitated at 4°C by the addition of 10 L of acetone. The dried precipitate, 55 g, was applied to a 10 x 110 cm "Bio-Gel P-2" column and eluted with H 2 O. 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 crystallization from 20% ethanol at pH 3. The hydrogen salt was dissolved in a minimal volume of H20, brought to pH 8.5 with NaOH and precipitated with 2 volumes of ethanol at 4°C. This precipitate was dissolved in 100 ml. H 2 O and precipitated with 9 volumes of ethanol at 4°C, yielding 15.1 g of 9-(2-hydroxyethoxymethyl)guanine monophosphate disodium salt dihydrate.

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

Empirical formula: CgH^NgOgP 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%.

Purity by high-pressure liquid chromatography = 99% Base/phosphate ratio = 1.00/1.01.

Claims (1)

Analogous method for the preparation of a new, antivirally active 9-hydroxyethoxymethylguanine derivative with the formula: wherein W and Z are the same or different, and each is a hydrogen atom or a pharmaceutically acceptable cation, characterized in that (a) a compound of formula: is reacted with a phosphorylating agent to form a compound of formula I where both W and Z are hydrogen atoms, or (b) a compound of the formula: subjected to ammonolysis to a compound of formula I, and, if desired, converting a compound of formula I wherein W and Z are both hydrogen to a compound wherein one or both of W and Z is a pharmaceutically acceptable cation, by reaction with a base or a salt containing the desired cation.