Method of 9-(s) or 9-(rs)-(3-hydroxy-2-phosponylmethoxypropyl)adenine production

The present invention provides a process for the preparation of 9- (S1- or 9- (RS) - (3-hydroxy-2-phosphonylmethoxypropyl) adenine.

- (S) - (3-Hydroxy-2-phosphorylmethoxypropyl-adenine (HPMPA) is a nucleotide analogue that has a strong antiviral effect, specifically directed against DNA viruses, characterized by its action on herpesviruses, including those strains and their mutants, which resist previously known virostatics, such as acyclovir (cf. No. 263 952). The racemate of the compound has a similar effect, while its (R) -enantemometer as well as the (R) -, (S) - and (RS) form of the isomeric 9- (2-hydroxy-3-phosphonylmethoxypropyl Jadenine) are almost ineffective. the problem to be solved is therefore the preparation of the isomerically pure 2-isomer of HPMPA [(Sj-form or racemate), or a mixture of both isomers with the highest content of active form.

HPMPA has so far been prepared, for example, by reaction of N⁸,O³-disubstituted 9- (2,3-dihydroxypropyl) adenines with p-toluenesulfonyloxymethanephosphonic acid diester, followed by cleavage of the protecting and ester groups (cf. No. 263 951) or by reaction of N⁸-substituted 9- (2,3-dihydroxypropyljaidenine with chloromethylphosphonyldichloride and subsequent alkaline hydrolysis (cf. No. 233 665).

Although the first of these methods provides pure 2'-phosphonylmethoxy derivative HPMPA, it requires the prior introduction of protecting groups into both the heterocyclic ring and the 3 'position, as well as their subsequent removal.

The second method is simpler, but always provides a mixture of both isomers, of which the desired HPMPA is present in the mixture at most 30-40%. Both isomers need to be separated and the by-product is no longer usable. Therefore, neither of the two prior art processes is a technically advantageous way of preparing the active ingredient.

These disadvantages are overcome by the present invention for a process for the preparation of 9- (S) - and 9- (RS) - (3-hydroxy-2-phosphonylmethoxypropyl) adenine of the formula I

<img file="CS263953B1_D0001.tif" />

CH-CH-CH-OH, och_ofp (o) (oh)_of (I) and its alkali metal and ammonia salts, characterized in that a mixture of isomeric chloromethylphosphonyl esters of 9- (Sj- and 9- (RSj- (2,3-dihydroxypropyl) adenine of formula II)

<img file="CS263953B1_D0002.tif" />

(II) where

R¹ is hydrogen and

R² is -P (O) (OH) CH 2 Cl or R 5²is hydrogen and R is¹ is -P (O) (OHJCH 2 Cl group), dissolves to a final concentration of 25 to 150 mmol.^_1%, based on the compound of formula II, in a mineral or organic acid having a concentration of 0.5 to 10 moles. 1^_1, preferably in hydrochloric acid, the mixture is maintained at a temperature of 40 to 80 ^TOC for 8 to 24 h, after removal of excess acid, the compound of formula II (R)¹ R 2 is hydrogen, R 3² is -P (Oj (OHJCH 2 Cl group) isolated by ion-exchange chromatography or hydrophobized slag, heated in aqueous solutions to a final concentration of 1 mol.^-1 up to 6 moles. l · '¹ alkali hydroxides at 40 to 100 ° C and the compound of formula I is obtained by neutralization and desalting.

The invention is based on the fundamental improvement and streamlining of the known process for the preparation of the compound of formula I and its 3'-isomer, i.e. 9- (2-hydroxy-3-phosphonylmethoxypropyl kernin) cyclic esters of formula II in an alkaline environment according to US Patent No. 233,665 .

Preparation of compounds of formula I by reaction of 9- (2,3-dihydroxypropyl adenine) with chloromethanesulfonyldichloride in pyridine (cf. author's certificate no. 233 665) or its hydrolysis product with an equimolar amount of water in pyridine [I. Rosenberg, A. Holý, Collect. Chem. Commun., 50, 1, 507 (1985)] provides the 3'-isomer of Formula II (R¹ R 2 is hydrogen, R 3² is —P (Oj (OHJCH2Cl group) (hereinafter referred to as the 3'-IIj) required for the preparation of the compound of formula I only in minor amounts (30 to 40%) relative to the unused 2'-isomer [II, R]² R 2 is hydrogen, R 3¹ is a —P (O) (OH) CH 2 Cl group] (hereinafter 2'-11).

The present invention provides the isomerization of the latter 2'-II isomer to the 3'-isomer of formula 3'-II, which takes place in aqueous solutions of organic, but preferably mineral acids (hydrochloric acid, sulfuric acid, etc.) at slightly elevated temperatures. This process results in the equilibrium of a high mixture (75-80 percent) of a 3'-isomer of formula 3'-II equal to 263953. The composition of this mixture is dependent on the acid concentration and reaction time. These data are documented in Table 1. The resulting mixture does not change when the mixture is treated in an acidic or slightly alkaline medium.

The mixture of compounds of formula II thus enriched in the 3'-isomer of formula 3'-II can be effectively separated into the individual isomers after removal of the excess acid by various means, most preferably ion exchange chromatography on strongly acidic cation exchange or hydrophobized silica gel, or can be used directly as an enriched mixture for cyclization . Separation at the level of compounds of formula II is much more efficient than chromatographic purification of isomers of compound of formula I.

A further advantage of the present invention is that the 2'-II isomer can be re-converted by isomerization to the 3'-II compound in an acidic environment, thereby increasing the degree of utilization of the starting material and other reagents.

The isomerization of the mixture of compounds of formula II proceeds equally well regardless of the method for its preparation. The isomerically pure 3'-O-chloromethanesulfonyl ester of formula 3'-II is subjected to an alkaline treatment according to the conditions of the original procedure (cf. No. 233 665) to give an isomerically pure product of formula I (HPMPA); 2'-II similarly gives the pure 3'-isomer of the compound of formula I [9- (2-hydroxy-3-phosphonylmethoxypropyl) adenine. Further purification of the compound of formula (I) is no longer necessary since isomerization does not occur upon reaction with the hydroxide. The insulation is therefore limited to removing excess hydroxide and alkaline chloride by desalting, for example on cation exchangers.

There is no racemization or configuration change at the 2 'carbon atom during the process. The process of the present invention does not require special equipment or chemicals and is a significant improvement in the state of the art for the preparation of Formula I. High purity of the product, high yield and increased process efficiency are particularly important. The separation of the isomeric compounds of the formula II can be easily achieved even on a large scale by preparative liquid chromatography with the appropriate volume of the stationary phase and with suitably selected column parameters.

In the following, the process according to the invention is exemplified without limiting it in any way.

Example 1

A mixture of 522 mg (2.5 mmol) of 9- (RS) - (2,3-dihydroxypropyl) adenine, 10 ml of dimethylformamide and 3 ml of dimethylformamide dimethyl acetal is stirred in a sealed flask for 16 h at room temperature and evaporated at 40 ° C / 13 Pa. Dissolve in 20 ml 50% aqueous pyridine and add about 1 g solid carbon dioxide, evaporate after 1 h and co-distill 3 times with 20 ml pyridine at 40 ° C / 13 Pa. To a solution of 1.87 g (10 mmol) of chloromethanephosphonyl chloride in 2.0 ml of pyridine is added at 0 ° C 180 µl of water, after 30 min. in an ice bath, the mixture is filtered under exclusion of moisture to the above residue and the mixture is allowed to stand at room temperature for 2 hours. 50 ml of 1 mol. AND¹ triethylammonium hydrogen carbonate, pH 7.5, evaporated at 40 ° C / 2 kPa and the residue was evaporated three times with 50 ml ethanol under the same conditions.

The residue is left to stand at room temperature for 16 hours in 100 ml of 5% aqueous ammonia and evaporated to dryness at 40 ° C / 2 kPa. According to liquid chromatography analysis:

column 4 X 200 mm Separon CGX C18 ACS (10 µm), mobile phase 5% methanol in 0.05 mol.

. AND^-1 triethylammonium hydrogen carbonate,

PH 7.5 (v / v, elution rate 0.7 mL min^-1detection at 254 nm contained a mixture of 95.5% of a mixture of compounds II containing 33% of 3'-H (t = 18.8 min) and 67% of 2'-II (t = 14.7 min). This residue is introduced in a 20 ml water column onto a 100 ml ion exchange column (e.g. Dowex 50 X8) in H.⁺and the column was eluted with water (2-3 ml. min.)^-1(300 mL) and 2.5^!% aqueous ammonia (1 liter). The ammonia eluate is evaporated at 40 ° C / 2 kPa and the residue in 40 ml of 1 mol. AND"¹hydrochloric acid was heated to 40 degrees Celsius for 18 h. The mixture is applied to 100 ml cation exchange resin (e.g. Dowex 50 X8) in H⁺-cycle and the column eluted with water (3 ml / min)¹) when eluate was detected at 254 nm.

The main UV-absorbing fraction was evaporated at 40 ° C / 2 kPa (76% 3'-II content, 24% 2'-II content) and the residue was heated to 80 degrees Celsius in 40 ml of 2 moles for 6 h. AND"¹ sodium hydroxide. After cooling, the mixture is neutralized by addition of an ion exchanger (Dowex 50 X8) in an H + -cycle to pH 7.0, filtered, washed with 100 ml of water and the filtrate is evaporated at 40 ° C / 2 kPa. precipitation from^:5 ml of methanol by adding 100 ml of ether. After suctioning and drying under vacuum, 700 mg of the disodium salt of the racemic compound of the formula I (81%) is obtained, containing 76% of the substance I and 24% of its 3'-isomer. Example 2

627 mg (3 mmol) of 9- (S) - (2,3-dihydroxypropyl) adenine was converted according to Example 1 into a mixture of compounds of formula II which was isomerized by heating with 40 ml of 10 moles.¹ hydrochloric acid at 60 ° C for 8 hours. The mixture is directly loaded onto a 200 ml cation exchange column (e.g., Dowex 50 X8) in H⁺and the column was eluted with water (3 mL / min). A mixture of the compounds of formula II containing 81% 3'-II is obtained from the main UV-absorbing strip. This residue was dissolved in 10 ml of water by addition of trlethylamine and the solution was concentrated in vacuo at 40 ° C / 2 kPa to a volume of 1.5 ml. The 3 µL portions of this solution are applied to a column 8 of XX 500 mm octadecyl-silica gel (eg Separon SIX C18, 7 µm) equilibrated with 0.05 mol. 1^_1 triethylammonium hydrogen carbonate, pH 7.5.

The column is eluted first with the same buffer to remove salts and impurities and then with a 10% methanol solution in the same buffer (elution rate 2 ml / min.) The eluates of the two isomers II are collected separately. Evaporation at 40 ° C / 2 kPa The purity determined by liquid chromatography (see Example 1) is greater than 99% in both cases The evaporations of the two isomers are separately treated with 10 ml of 2 mol each.¹ sodium hydroxide according to Example 1. 500 mg (56% based on the mixture of compounds II) of HPMPA of formula I are obtained as the disodium salt with a content of more than 99%.

SUBJECT

Process for the preparation of 9- (S) - and 9- (RS) - (3-hydroxy-2-phosphonylmethoxypropyl adenine of the formula I)

<img file="CS263953B1_D0003.tif" />

CHoCH-CH-OH

I Z.

α H, P (δ) (OH).

(I) and their alkali metal and ammonia salts, characterized in that a mixture of the isomeric chloromethylphosphonyl esters of 9- (Sj- and 9- (RS) - (2,3-dihydroxypropyl adenine) of formula II nh₂

Table 1

Effect of hydrochloric acid concentration on isomerization of a mixture of compounds of formula II (25 mmol¹) after 24 h at 37 ° C

Concentration%

HCl 2'-II 3'-II (mol^_1)

<td> 0</td><td> 84,6</td><td> 15,4</td>

<td> 0,1</td><td> 79,0</td><td> 21,0</td>

<td> 0,2</td><td> .74,0</td><td> 26,0</td>

<td> 0,5</td><td> 57,7</td><td> 42,3</td>

<td> 1,0</td><td> 27,0</td><td> 73,0</td>

<td> 2,0</td><td> 21,0</td><td> 79,0</td>

<td> 5,0</td><td> 15,0</td><td> 85,0</td>

<td> 10,0</td><td> 13,0</td><td> 87,0</td>

<td colspan="3">Note:</td>

<td>When performing</td><td>isomerization</td><td>at 2.5 mole. 1¹</td>

<td colspan="3">sulfuric acid under otherwise identical conditions, the 3'-II content after 24 h is 81%.</td>