CS263956B1 - Method of 9-(s)-(3-hydroxy-2-phosphonylmethoxypropyl)adenine production - Google Patents

Abstract

The solution is in the field of new virostaitics and relates to a more preferred method of production anti-vigorously active 9- (S) - (3-hydroxy-2-phosphonylmethoxypropyl) adenine. The essence is reaction of non-benzoyl-9- (S) - (2,3-dihydroxypropyl) adenine with dimethoxytrityl chloride; following benzoylation with benzoyl cyanide and triethylamine and acidic cleavage with trifluoroacetic acid. N6, O2-dibenzoyl-9- (S1- (2,3-dihydroxypropyl) adenine) formed reacts with chloromethanephosphonyldichloride in triethylphosphate to protected ester chloride which is sodium hydroxide isomerically pure product, isolated as sodium salt.

Description

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

9- (S) - (3-Hydroxy-2-phosphonylmethoxypropyl iadenine) (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 the alkaline hydrolysis of the pure 3-isomer (CSN 263 263, No. 263 954) or finally by condensation of 3'-O-protected DHPA derivatives with p-toluenesulfonyloxymethanephosphonic acid diester, as follows reaction of the intermediate e with trimethyl halogeinisilane and cleavage of protecting groups (MS Auto-certificate No. 263 951) .The first alternative is conditioned by an efficient separation technique, which limits the amount of HPMPA available in a single production cycle, while the second method of preparation requires the preparation of protected DHPA derivatives that are not readily available, as well as the preparation of an organophosphoric derivative (Cs. aut. osv. No. 220,713) and the 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 exploited by the present invention by a process for the preparation of 9- (S) - (3-hydroxy-2-phosphonylmethoxypropyl) adenine of the formula I and A '

AND

CH ₂ CH-OCH ₂ P (O) (OH) ₂

CH ₂ OH (1), characterized in that 9- (S) - (2,3-dihydroxypropyl) -N ⁴ -benzoyladenine of the formula II

NlK0C ₆ H ₅

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

(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. Crystallisation from chloroform fractions obtained N ^6, O ² '-dibe.nzoyl-9- (S j - (2,3-dlhydroxypropyljadenin V

OCOC _{& £} H (V) is reacted with 2 molar equivalents relative to compound of Formula V, Formula VI chlormethanfosfonyldichloridu

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 soda deionisuje on strong acid cation exchanger and the substance of formula I is purified by chromatography, preferably on a medium-basic anion exchanger.

The process of the invention is based on the specific preparation of the new N ⁶ , O ² '-dibenzoyl derivative of (S) -DHPA of formula V. The process is based on (S) -DHPA, available, for example, from literature (A. Holy, Collect. Czechoslov. Chem. Commun., 40, 187 (1975)) in large amounts and is based on the introduction of an acid-labile dimethoxytrityl group at the O ³ 'side chain position. 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 1 316/1982 /) or benzoylation of 2 ', 3'-0-isopropylidene compound (S) -DHPA (an intermediate for preparing (S) -DHPA), following controlled amonolysou dibenzoate and finally the acid hydrolysis of the dioxolane protecting group. The N ⁶ -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, Ed.), Vol 1, page 5,25, Interscience, New York (1968). An intermediate of formula IV which contains a single unsubstituted hydroxyl group, it 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 the cleavage of the dimethoxytrityl group is readily controlled by, for example, 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 poloze side chain position that is available for the formation of chloromethanephosphoinylester (HPMPA synthesis principle according to US-A-A-233 665). 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 in the 3 poloze position with the hydroxyl group released in the 2 poloze position (MS No. 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. Also on a column of medium basic anion exchange resin (eg 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). Although it uses costly dimethoxytrityl chloride (which can be recovered), it is feasible on hundreds of grams and provides HPMPA without the need for a high-efficiency HPMPA isomer separation device.

¹ The invention is further illustrated in the example embodiment of the synthesis of HPMPA, without thereby in any way limiting.

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 bath). 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 stirring for 30 min., Suction filtered, washed with ether and dried at 13 Pa. 18.5 g [81%] of N ⁶ -benzoyl-9- (S) - (2,3-dihydroxypropyl) adenine of formula II, m.p. 199 ° C. For C 15 H 15 N 5 O 3 (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 hydroxide solution and heated for 6 h at 75 ° C. It is cooled, neutralized by the addition of 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 was evaporated at 20 mbar and the residue was chromatographed on a 500 ml column of Sephadex A-25 in an HCO 3 cycle using a gradient of 0 to 0.3 moles. ^-1 , total 4 L of 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 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, after standing for 1 h at 2 kPa to half volume, 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. Igramu to give 12.9 (70%) of N ⁶ -dibenzoylderivátu, bt '210-211 ^Q C.

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 N '-benzoylderivátu, mp 146-147 DEG C. This product ³ (9.0 g) is allowed to' stand in 80 ml of dioxane and 80 ml of 0.25 mol. I ¹ of sulfuric acid for 24 h at room temperature, neutralized with triethylamine and evaporated at 2 kPa, the residue in 200 ml of chloroform was filtered off with suction, the precipitate washed with chloroform and 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) ^B N -benzoyl-9- (S) - (2,3-dihydroxypropyl) adenine, mp 198-199 DEG. 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

Claims (1)

NH, NHCOC./4 AND CH ₂ CH-OCH ₂ P (O) (OH) ₂ Gjfc i ch-ch-ch.Jjc (p-ch, oc,.;; ..) OH ch ₂ oh. fO characterized in that 9- (S) - (2,3-dihydroxypropyl) -N ⁴ -benzoyladenine of formula II (II) 'is reacted with di- (p-methoxyphenyl) phenylmethyl chloride of formula III The CeHs are reacted with 1.05 to 1.2 molar equivalents of beuzoylcyanide 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 after neutralization and removal of the volatiles in vacuo was partitioned between chloroform and water. Crystallization from the chloroform fraction obtained by N ^H , O ^2' -dibenzoyl-9- (S) - (2,3-dihydroxypropyl adenine of formula V) QCOC.H (1) is reacted with 2 molar equivalents, based on the compound of formula (V), chloromethanephosphonyldl chloride of formula (VI) (p-CH3OC6H4) 2C C1CH2P (O) C12 (VI) Cl (III) w pyridine at 5 ° C to 30 ° C, extraction of the hydrolyzed mixture, preferably with chloroform, the crystalline intermediate of formula IV obtained in triethyl phosphate as solvent and at 0 ^° C to 30 ° C, precipitated with ether, the precipitated intermediate is heated to 60 ° C to 80 ° C with 2 moles. 1 ^_1 soda deionisuje on strong acid cation exchanger and the substance of formula I is purified by chromatography, preferably on a medium-basic anion exchanger.