Method of 9-(s)-(3-hydroxy-2-phosphonylmethoxypropyl)adenine production

The present invention provides a process for the preparation of 9- (S) - (3-hydroxy-2-phosphorylmethoxypropyl) adenine.

9- (S) - (3-Hydroxy-2-phosphonylmethoxypropyl) adenine (HPMPA) is a novel antiviral preparation with specific activity against DNA viruses, independent of the action of the virus-specific enzyme thymidine kinase (US-A-No. 233 665). The preparation of this compound to date consists in separating mixtures of HPMPA with its 3'-isomer, prepared by the reaction of 9- (S) - (2,3-dihydroxypropyl Jadenine) with chloromethanephosphonyl dichloride and subsequent alkaline hydrolysis (cf. . 233 665) or by the separation of isomeric chloromethanephosphonyl derivatives of DHPA and alkaline hydrolysis of the pure 3-isomer (CSN 263 953, No. 263 954) or finally by condensation of 3'-O-protected DHPA derivatives with p-toluenesulfonyloxymethanephosphonic acid diester, followed by reaction of the intermediate and trimethyl halogeinisilane and cleavage of protecting groups (MS Auto-certificate No. 263 951). The first alternative is subject to efficient separation techniques, thereby limiting the amount of HPMPA available in a single production cycle, while the second method requires the preparation of protected DHPA derivatives that are not readily available, as well as the preparation of an organophosphorus derivative (cf. use of expensive and sensitive trimethylbalogensilane. In doing so, the reaction must be such that racemization does not occur because only the (S) -enantiomer of HPMPA is the biologically active form of this derivative.

The disadvantages of these methods are eliminated and their particular advantages are utilized by the present invention for a process for the preparation of 9- (S) - (3-hydroxy-2-phosphonylmethoxypropyl) adenine of the formula (I).

AND

CH₂CH-OCH₂P (0)₂

CH_OFOH (1) comprising 9- (S) - (2,3-dihydroxypropyl) -N⁴-benzoyladenine of formula II

NlK0C₆H₅

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Ol ·!

(U) reacts with di- (p-methoxyphenyl) phenylmethyl chloride of formula III

C6H5 (p-CH3OC6H4) 2C \

Cl (III) in pyridine at 5 ° C to 30 ° C, crystalline intermediate of formula IV obtained by extraction of the hydrolyzed mixture, preferably with chloroform

<img file="CS263956B1_D0002.tif" />

(IV) is reacted with 1.05 to 1.2 molar equivalents of benzoyl cyanide and trimethylamine in acetonitrile as solvent at 15 ° C to 30 ° C, treated with an aqueous solution of a volatile organic acid, preferably trifluoroacetic acid, and neutralization and removal of the volatiles in vacuo was partitioned between chloroform and water. Crystallization from the chloroform fraction obtained by N⁶,O²Dibenzoyl-9- (S - (2,3-dihydroxypropyl) adenine V)

<img file="CS263956B1_D0003.tif" />

OCOC_&amp;H_£ (V) is reacted with 2 molar equivalents, based on the compound of formula V, of chloromethanephosphonyl dichloride of formula VI

C1CH2P (O) C12 (VI) in triethylphosphate as solvent and at 0 ° C to 30 ° C, precipitated with ether, heated to 60 ° C to 80 ° C with 2 moles. 1^_1 sodium hydroxide, deionized on a strongly acidic cation exchanger and the compound of formula I is purified by chromatography, preferably on a medium basic anion exchange resin.

The process according to the invention is based on the specific preparation of the new N⁶,O²The process is based on (S) -DHPA, available, e.g., according to the literature (A. Holy, Collect. Czech. Chem. Commun. 40, 187 (1975)) in large quantities; and is based on the introduction of an acid-labile dimethoxytrityl group at the O position³side chain. To this end, the exocyclic amino group of adenine must be protected beforehand by benzoylation, either directly by reaction (S-DHPA in pyridine sequentially with trimethylchlorosilane and benzoyl chloride according to the general method (see GS Ti, BL Gaffney, RA Jones: J. Amer. Chem. Soc. 104, 1316 (1982)), or by benzoylation of the 2 ', 3'-O-isopropylidene derivative of (S) -DHPA (intermediate of preparation (S) -DHPA), followed by controlled ammonolysis of dibenzoate and finally acid hydrolysis protecting the dioxolane groups. Originated N⁶-benzoyl- (S-DHPA of formula II provides easily and in quantitative yield a 3'-O-dimethoxytrityl derivative of formula IV by reaction with dimethoxytrityl chloride in pyridine. Dimethoxytrityl chloride of formula III is available by Grignard reaction from p-bromoanisole, e.g. in Nucleic Acid Chemistry (WW Zorbach, RS Tipson, eds.), Vol 1, p 5.25, Interscience, New York (1968). The intermediate of formula IV, which contains a single unsubstituted hydroxyl group, is readily isolated from the reaction mixture by crystallization from the chloroform extract of the hydrolyzed reaction mixture, so that no additional purification is required. The remaining hydroxyl group is benzoylated in the next reaction step, e.g. benzoyl chloride in pyridine, but preferably for this purpose a reaction with a small excess of benzoyl cyanide and triethylamine in acetonitrile is used (A. Holy, M. Soucek; Tetrahedron Letters 1971, 185). The reaction mixture is then subjected to acid hydrolysis catalysed by any volatile acid without isolation of the product; preferably trifluoroacetic acid is recommended for this purpose. The progress of cleavage of the dimethoxytrityl group is readily controlled, for example, by thin layer chromatography on silica gel. After completion of this reaction, the mixture was neutralized and evaporated in vacuo. The key reaction product of formula V is obtained from the reaction mixture by extraction with chloroform and crystallization. Thus, the compound of formula V is obtained in a pure state without racemization and in a final yield of more than 50-60% based on the starting material (Sj-DHPA).

This specifically protected compound contains a single hydroxyl group at the 3 'side chain position that is available for the formation of the chloromethanephosphoinyl ester (HPMPA synthesis principle according to US-A-233 233). This reaction is preferably carried out according to the method of Art. aut. osv. No. 6,263,935, by reacting a compound of Formula V with commercially available chloromethanephosphonyldichloride of Formula VI in triethylphosphate at room temperature. The mixture is freed from triethyl phosphate and excess reagent by ether precipitation and the resulting volatile crystalline ester chloride is directly reacted with an alkali hydroxide solution (e.g. sodium hydroxide). This involves the cleavage of the benzoyl groups and the itramolecular reaction of the chloromethanephosphonate at the 3 'position with the released hydroxyl group at the 2' position (cf. auto. Number 233 665). Since migration of ester-linked chloromethanephosphonic acid is excluded under reaction conditions, the only reaction product of HPMPA of formula I is accompanied by excess hydroxide, alkaline chloride, benzoic acid and possibly small amounts (S-DHPA) in the reaction mixture. of formula I and (S) -DHPA) is most readily separated from the mixture on a strongly acidic cation exchange column (e.g. Dowex 50), the benzoic acid is eluted from the column with aqueous methanol and the adenine derivatives are subsequently released with dilute aqueous ammonia. Finally, purification of the compound of Formula I is carried out on a column of a medium basic anion exchange resin (e.g. Sephadex A-25 in volatile buffer or Dowex 1 in acetic acid).

For storage, the pure HPMPA of Formula I obtained is preferably converted to an alkaline, e.g. sodium, salt by exchange on a cation exchange resin in an appropriate cycle. The isomeric purity of the compound of formula I thus prepared exceeds 99%.

The procedure is equally applicable to the preparation of 2'-O-phosphonylmethyl derivatives derived from (R) -DHPA, (RS J-DHPA and other 2,3-dihydroxypropylderivatives of the heterocyclic bases). high yields in individual steps and only requires double isolation of intermediates. Although using costly dimethoxytrityl chloride (which can be recovered), it is feasible in hundreds of grams and provides HPMPA without the need for a high-efficiency HPMPA isomer separation device.

¹ In the following, the invention is illustrated by way of example without limiting the synthesis of HPMPA.

Example 1

9- (S) - (2,3-dihydroxyprspyl) adenine (16.5 g of monohydrate, i.e. 72.7 mmol) is dried by repeated distillation with pyridine (3 X 100 ml) at 2 kPa, suspended (20 min in ultrasonic mode). bath) in 400 ml of pyridine and with stirring 50.6 ml of trimethylchlorosilane are added. The mixture was stirred for 1 h, benzyl chloride (45.8 ml) was added, stirred at room temperature for 2 h and cooled in an ice bath. 79 ml of ice-water are added dropwise and after stirring for 5 minutes, 176 ml of 26% aqueous ammonia are added rapidly so that the temperature inside the mixture does not exceed 25 ° C. After 30 min. stirring without cooling, the solution is evaporated at 2 kPa, the residue is extracted with 100 ml of ethyl acetate in 250 ml of water and the aqueous residue is left to crystallize at 0 ° C for 48 h. The crystalline product is filtered off with suction, washed with 3 X 20 ml of ice water. and 250 ml of ether are added with stirring. (After 30 min. the stirring is filtered off with suction, washed with ether and dried at 13 Pa. 18.5 g [81% percent] N are obtained⁶-benzoyl-9- (S) - (2,3-dihydroxypropyl) adenine of formula II, mp 198-199 ° C. For C15H15N5O3 (313.3) calculated:

% C, 57.50;% H, 4.82;% N, 22.36; found:

% C, 57.43;% H, 4.83;

To a solution of 17.1 g (55 mmol) of this material in 600 mL of pyridine was added 21.4 g (61.6 mmol) of dimethoxytrityl chloride and the mixture was stirred at room temperature for 18 h. 5 ml of methanol are added and after stirring for 3 hours, 20 g of sodium hydrogen carbonate are added dropwise and 500 ml of water are added dropwise. The resulting solution was extracted with chloroform (4 X 500 mL), the extract was washed with water (2 X 500 mL), and dried over sodium sulfate. The solution is evaporated at 2 kPa, the residue codistilled with toluene (5 X 300 ml), dissolved in 800 ml of toluene and evaporated at 2 kPa to start crystallization. The product precipitated by standing overnight at 4 ° C after the addition of 1500 ml of petroleum ether is suction filtered, washed with a mixture of benzene-petroleum ether (70 ml, 1: 9, v / v), petroleum ether (100 ml) and dried at 13 Pa. Yield 31 g (78.7%) of III.

This product (43.3 mmol) is dissolved in 400 ml of acetonitrile, 7 ml of triethylamine and 6.6 g (50 mmol) of benzoyl cyanide in 100 ml of acetonitrile are added, the mixture is left to stand at room temperature overnight. 4 ml of methanol and after 20 min. the mixture was cooled with ice. While stirring, 3.85 ml of trifluoroacetic acid is added dropwise, followed by a mixture of 10.6 ml of trifluoroacetic acid and 100 ml of water. The solution is allowed to stand for 4 hours without cooling, recooled with ice, neutralized with 21 ml of triethylamine and evaporated at 2 kPa. The residue was codistilled with dioxane-toluene (1: 1, v / v) (3 X 200 mL) and dissolved in 550 mL of boiling chloroform. 1 liter of benzene was added to the solution and allowed to crystallize at 4 ° C overnight. The precipitated product is filtered off with suction, washed with benzene (2 X 50 ml), petroleum ether and dried at 13 Pa. 16.1 g (89%) of (IV) are obtained.

This product (38.5 mmol) was suspended (20 min in an ultrasonic bath) in 200 ml of triethyl phosphate, cooled with ice and added with stirring - 6 ml (60 mmol) of chloromethanephosphoinyldichloride. The resulting solution was allowed to stand overnight at room temperature, with vigorous stirring, 1200 ml of ether and suspension were added for · 10 min. Stir suction and wash with ether (3 X 200 mL). The product is dried for 5 hours at 20 mbar over potassium hydroxide, dissolved in 400 ml of 2 moles. 1^_1 sodium hydroxide solution and the solution was heated to 75 ° C for 6 h. Cool, neutralize by adding Dowex 50X8 (H⁺) and the suspension is poured onto a column of 400 ml of the same cation exchanger. The column was eluted with 5 L of methanol-water (1: 1, v / v) followed by 3 L of 5% (w / v) aqueous ammonia.

The ammoniacal eluate is evaporated at 2 kPa and the residue is chromium, atographed on a 500 ml column of Sephadex A-25 in an HCO3-cycle eluting with a gradient (0 to 0.3 mol).^-1A total of 4 1) triethylammonium bicarbonate pH 7.5. The fractions of production were detected by UV-absorbing eluate, pooled, evaporated at 20 mbar and freed from the buffer by codistillation with methanol (5 X 100 ml). The product was converted to the sodium salt by filtration through a 200 ml column of Dowex 50X8 (Na⁺), eluting with water. The eluate is evaporated at 2 kPa, dried by codistillation with ethanol (3 X 50 ml) and suction filtered from ethainol-ether (1: 9, 200 ml) and dried at 13 Pa. 10.0 g of the disodium salt of the compound of formula I are obtained, purity (HPLC) higher than 99.5%.

Example 2

To a solution of 10.0 g (40 mmol) of 2 ', 3^!-O-Isopropyllden-9- (S) - (2,3-dihydroxypropyl) adefin in 150 ml of pyridine was added 0.5 g of 4-dimethylaminopyridine, cooled with ice and 9.3 ml (80 mmol) of benzoyl chloride was added in one portion. The mixture was stirred at 0 ° C for 2 h and allowed to stand at 0 ° C for 2 days. Methanol (10 ml) was added, evaporated at 50 psi (1/2 kPa) for 1 h, chloroform (500 ml) was added, washed with water (3 X 200 ml), the chloroform extract was dried over sodium sulfate, evaporated and the residue crystallized from ethanol. 12.9 g (70%) of N are obtained⁶dibenzoyl derivative, mp 210-211 ^QC.

This product was dissolved in 240 mL of dioixane, 40 mL of conc. aqueous ammonia, after 30 min. It is evaporated at 2 kPa, codistilled with dioxane (2 × 50 ml) and crystallized from ethyl acetate (petroleum ether to turbidity). 10 g of the N &amp; apos; -benzoyl derivative are obtained, mp 146-147³C. This product (9.0 g) was allowed to stand in a mixture of 80 ml dioxane and 80 ml 0.25 mol. AND¹ of sulfuric acid at room temperature for 24 h, neutralized with triethylamine, evaporated at 20 mbar, the residue in 200 ml of chloroform is filtered off with suction, washed with chloroform and the precipitate is air-dried. The chloroform filtrate was washed with water (2.times.50 ml), dried over sodium sulfate and evaporated at 20 mbar. The residue was combined with the first crystalline fraction and crystallized from ethanol. 4.9 g (61 percent) of N are obtained^(B)-benzoyl-9- (S) - (2,3-dihydroxypropyl) adenine, mp 198-199 ° C. A further reaction of this material was carried out as in Example 1. The (S) -HPMPA sodium salt was obtained in a yield of 45% of the latter N-benzoyl derivative.

Subject

A process for the preparation of 9- (S) - (3-hydroxy-2-phosphonylmethoxypropyl) adenine of the formula I