CS264223B1 - Method of n-(2-phospohonylmethoxyethyl) derivatives of heterocyclic bases production - Google Patents

Abstract

The solution is in the field of new analogues nucleotides and provides a more convenient method of production virostatik. The essence of the solution is a new way production of N9- (2-phosphonylmethoxyethyl) purines and N '- (2-phosphonylmethoxyethyl) pyrimidine, based for the reaction of the sodium salt of the corresponding free or N-protected heterocyclic base with diethyl ester 2-bromoethoxymethanephosphonic acid followed by cleavage of the resulting intermediate trimethylhalogensilane.

Description

The present invention provides a process for the preparation of N- (2-phosphonylmethoxyethyl) heterocyclic base derivatives 9- (2-Phosphonylmethoxyethyl) adenine (PMEA) is a novel virostatic with a broad spectrum of activity against DNA viruses (herpesviruses, poxviruses) and retroviruses (AO 263 952). In the group of herpesviruses, which are the cause of numerous diseases with dangerous complications, PMEA works similarly to 9; (S) - (3-hydroxy-2-phosphonylmethoxypropyl) adenine (HPMPA) even in cases where other virostatics, including acydovirus, often fail. Although the efficacy of ⁵ MEA is reduced over HPMPA, the first may offer some advantages because its preparation is not subject to enantiomeric homogeneity or isomeric uniformity of the preparation.

The prior art process for the preparation of PMEA consists in condensing N-protected 9- (2-hydroxyethyl) adenine with p-toluenesulfonyloxymethanephosphonic acid diesters, followed by deprotection and cleavage of ester-linked alkyl groups by treatment with trialkyl halogens (AO 263 951). This procedure requires prior preparation of 9- (2-hydroxyethyl) adenine (e.g., according to A. Holy, J. Kohoutkova, A. Merta, I. Votruba: Collect. Czechoslov. Chem. Commun. 51, 459 (1986)). N- (2-hydroxyethyl) derivatives of this type would be needed as starting materials for carrying out a similar reaction to prepare N- (phosphonylmethoxyethyl) derivatives derived from other heterocyclic bases. Further, said process requires the introduction of a protecting group at all positions of the heterocyclic base capable of reacting with sodium hydride.

This disadvantage is overcome by the general process for the preparation of N- (2-phosphonylmethoxyethyl) heterocyclic base derivatives of the general formula I in a solvent, preferably acetonitrile, the mixture is further decomposed in neutral or alkaline medium and the compound of the formula I is obtained from the mixture by desalting and ion exchange chromatography. Alternatively, the process of the invention may be modified by treating the mixture with an aqueous compound of 1-2 moles after reaction with the compound of Formula II. Γ ¹ sodium hydroxide solution at 60-100 ° C, and after neutralization and deionization it is further treated with trimethyl halogensilane.

A key intermediate of this process is a novel reactive substituted phosphonic acid derivative of formula (II), which is obtained by a process starting from readily available 1,3-dioxolane: by treatment with acetyl chloride or acetyl bromide (preferably in the presence of a catalyst such as zinc chloride). -dioxolane prepared in high yield 2-acetoxyethoxymethyl halide (see BA Arbuzov, Ε N. Ukhvatova: Zh. obshch. Khim. 29, 503 (19591), which is converted to the corresponding substituted phosphonic ester at elevated temperature by Arbuzov reaction at the elevated temperature. This reaction works better with triethylphosphite so that the reaction product obtained, isolated by fractional distillation of the reaction mixture, is the diethyl ester of formula IV

CH ₃ COOCH ₂ CH ₂ OCH ₂ P (OXOC ₂ H ₅ ) ₂ (IV)

For example, heterogeneous reaction of a compound of formula IV with ethanol in the presence of a strongly acidic cation exchanger in an acidic form (e.g., Dowex 50) can be advantageously used to cleave the acetyl group. The reaction proceeds at the boiling point of the mixture and can be equally well carried out in a homogeneous mixture if another suitable, e.g. mineral acid, is used. This reaction yields diethyl acid ester

2-hydroxyethoxymethanephosphonic formula V,

B - CH ₂ CH ₂ OCH ₂ P (O) (OH) ₂ (0

HOCH ₂ CH ₂ OCH ₂ P (O) (OC ₂ H ₃ ) ₂ (V) wherein B is a purin-9-yl or pyrimidin-1-yl residue and their alkali metal salts, characterized in that the sodium heterocyclic salt Purine or pyrimide base, N-acetyl, N-benzoyl-, N-dimethylaminomethylene- or O-methyl derivative reacted with 1 to 1.5 molar equivalents, calculated on a heterocyclic basis, of 2-bromoethoxymethanephosphonic acid diethyl ester of formula II

BrCH ₂ CH ₂ OCH ₂ P (O) (OC ₂ H ₅ ) ₂ (II) in dimethylformamide as a solvent at 80 ° C to 120 ° C, the resulting intermediate of formula III

B'-CH ₂ CH ₂ OCH ₂ P (O) (OC ₂ H ₅ ) ₂ (III) wherein B ^z has the same meaning as B in formula I or is a residue of N-acetyl-, Ν-benzoyl-, N-dimethylaminomethylene - or the O-methyl derivative of the base B in formula I is isolated by chromatography, preferably on silica gel, then reacted with 3 to 5 molar equivalents of trimethyl halogensilane relative to the compound of formula III, preferably trimethyl bromosilane, internally and possibly from the mixture high vacuum distillation; however, its isolation is not absolutely necessary. Said sequence yields the compound of formula (V) from 1,3-dioxolane in a sufficiently high yield, without special equipment and chemical requirements. The preparation can also be carried out on a larger scale without difficulty.

The compound of formula V can be converted to a reactive 2-halogeno derivative, e.g., a compound of formula II, with a variety of agents that do not attack the diester function of the molecule, e.g., by tetramethyl-α-haloenamines (A. Devos, J. Renion,

AM by Frisque-Hesbain, A. Colens. L. Ghozes, J. Chem. Soc. Chem. Comm. 1979, 1180), preferably by treatment with an agent formed from triphenylphosphine and carbon bromide or bromine (JPH Verheyden, JG Moffatt: J. Org. Chem. 37, 2 289 (1972)). The reaction is carried out in dimethylformamide or dioxane as solvent. A small excess of both components is used relative to the hydroxyethyl derivative of formula V, which must be distilled or de-ethanoled by other means (e.g. codestilation with toluene) prior to the reaction. The course of the reaction can be controlled, for example, by thin layer chromatography on silica gel. The reaction product of Formula II may be a mixture of 'O ¹ · A

Bo

It can be effectively isolated by chromatography, for example on silica gel, or it can be partially purified by precipitation of the reaction mixture with an ether which dissolves the compound of formula II. By repeating this procedure (re-extracting the residue of the ether solution with a mixture of petroleum ether and ether), the compound of formula II is sufficiently pure for further use. Purification of the compound of Formula II by high vacuum distillation is not desirable.

The heterocyclic base required for the reaction with the compound of formula II can be used after prior drying (e.g., by distillation with dimethylformamide in vacuum) directly. For substances containing basic amino groups (cytosine, guanine derivatives), and especially where the starting base is poorly soluble in dimethylformamide, it is advisable to protect the amino group in advance to avoid possible side reactions. N-acyl is suitable for this purpose. (N-acetyl, N-benzoyl) derivatives or N-dimethylaminomethylenderivatives obtained by reaction of the base with acetals of dimethylformamide. In those cases where substitution of the sodium salt of the heterocyclic base may lead to a mixture of isomers (eg, uracil, thymine derivatives), it is possible to ensure uniform formation of the N-isomer by, e.g.

O-alkyl derivative of a heterockylic base.

The heterocyclic base or derivative thereof (see above) is preferably converted to the sodium salt directly in the reaction medium - dimethylformamide - by reaction with an equimolar amount of sodium hydride. For N-protected bases, this reaction is better performed under ice-cooling. A solution of the compound of formula II in dimethylformamide is gradually added to the formed sodium salt at a slightly elevated temperature, and the reaction is carried out usually at 80-100 ° C with vigorous stirring. During the preparation of the sodium salt and during the actual reaction it is necessary to observe anhydrous conditions and to prevent the access of air humidity. The progress of the reaction can be easily controlled, for example, by thin layer chromatography on silica gel in chloroform / methanol mixtures. The composition of the reaction mixture usually does not change after 8 hours of reaction time.

After evaporation of the solvent in vacuo, the crude product is preferably obtained by extraction with chloroform from the residue of the reaction mixture. It is preferred at this stage to purify the intermediate of formula III, for example by chromatography on silica gel, from the unreacted base or by-products, but this is not a necessary condition for the further procedure. The isolated (mostly crystalline) diester of formula III is reacted with trimethylbromosilane or trimethyl iodosilane; A stable and readily available trimethylbromosilane has proven to be the most suitable for this purpose. The reaction is preferably carried out in acetonitrile at room temperature for 18-24 h. After removal of the solvent in vacuo, the reaction mixture is quenched in aqueous medium, e.g. with a solution of triethylamine in 50% aqueous acetonitrile or a volatile buffer, e.g. triethylammonium hydrogen carbonate. After re-evaporation, the mixture is subjected to the conditions necessary for cleavage of the protecting group, if present, the mixture is deionized and the product of formula I is finally purified by ion exchange chromatography. by anion exchange matography (eg Dowex 1 in acetic acid or Sephadex A-25 in triethylamoCS 264 223 B1 4 sodium hydrogen carbonate).

However, the crude intermediate of formula III, obtained by extracting the residue of the reaction mixture after alkylation of the sodium base salt, can be processed directly without purification: in which case one of the two ester functions of the compound of formula III is first hydrolysed under alkaline conditions. The product obtained, the monoester of formula VI

B-CH ₂ CH ₂ OCH ₂ P (OXOHXOC ₂ H ₅ ) (VI) is recommended to be isolated by ion exchange chromatography. Reaction with trimethyibromosilane in acetonitrile or dimethylformamide and further treatment, which is the same as in the first variant, also yields the pure product of the general formula:

and.

Said reaction can be used for the "natural" purine and pyrimidine bases, as well as for their various analogues, derived by substitution or substitution of typical functions (amino, oxo), system substitution, replacement of -CH = heterocycle groups by -N = or vice versa. The advantage is that there is no need to prepare and re-specifically protect N- (2-hydroxyethyl) derivatives of the respective heterocyclic bases; the intermediates of formula V can be prepared in large quantities and stored without special precautions. Also, the reactive bromo derivative of formula II is sufficiently stable at 0 ° C to -10 ° C for at least several weeks.

The resulting compounds of formula I can be stored as free acids; however, they may be sparingly soluble in water. Advantageously, alkali (sodium, lithium) salts obtained by neutralization or ion exchange are therefore recommended for storage.

The process according to the invention is further exemplified without limiting the preparation of the compounds of the formula (I).

Example 1.

To a solution of 3 g (10 mmol) of 2-hydroxyethoxymethanephosphonic acid diethyl ester in 30 ml of ice-cooled dimethylformamide was gradually added, with stirring, 2.63 g (10 mmol) of triphenylphosphine and 3.3 g of carbon tetrabromide. The mixture is allowed to stand for 15 h at room temperature, evaporated at 13 Pa and the residue re-evaporated under the same conditions with 50 ml of dimethylformamide. Separately, a solution of N ⁴ -acetylcytosine sodium salt is prepared by adding 330 mg of a 60% dispersion of sodium hydride in paraffin oil to a suspension of 1.25 g.

8.2 mmol) of N ⁴ -acetylcytosine in 50 ml of dimethylormamide, which was stirred at 100 ° C for 15 min. To this solution was added a solution of the previous reagent in 50 mL of dimethylformamide, the mixture was heated at 100 ° C for 40 min and then stirred at room temperature for 24 h. It is evaporated at 13 Pa, the residue is heated at 80 ° C in 100 ml of 1 mol for 8 h. 1% sodium hydroxide and deionized on a column of 200 ml of strongly acidic cation exchange resin (Dowex 50) in H ⁺ form. After washing the column with water, the cytosine derivative is eluted with 2.5% (o./o.) Aqueous ammonia, the eluate is evaporated and the residue is dried at 13 Pa. To this residue were added 30 ml of acetonitrile and 3.25 ml of methyl bromosilane triCS 264 223 B1, the solution was allowed to stand overnight at room temperature, evaporated at 20 mbar and the residue dissolved in 10% triethylamine solution (o./o.). in 50% aqueous acetonitrile (50 mL). After three hours, the mixture is re-evaporated at 2 kPa, deionized on a 50 ml cation exchange column as above and the ammoniacal eluate residue purified by medium-basic anion exchange chromatography (e.g. Sephadex A-25) eluting with a gradient of triethylammonium hydrogen carbonate, pH

7.5 (0-0.15 mol. I ^-1 ), the product is converted to the salt by passing the residue over a column of 50 ml cation exchange resin in Na ⁺ form, evaporated at 2 kPa and the residue is filtered off with a mixture of ethanol (5 ml). ) and ether (50 ml), washed with ether and dried at 13 Pa. Yield: 35% of 1- (2-phosphonylmethoxyethyl) cytosine sodium, homogeneous by liquid and paper chromatography in 2-propanol-conc. aqueous ammonia-water (7: 1: 2, o./o.).

Example 2

The reaction is carried out as in Example 1 using the sodium salt of 4-methoxy-5-methyl-2-pyrimidinone prepared by the reaction

1.54 g (10 mmol) of this base with 400 mg of a 60% dispersion of sodium hydride in 50 ml of dimethylformamide. After working up the reaction mixture 1 mol. Γ ¹ with sodium hydroxide according to Example 1, the alkaline mixture is neutralized by addition of a cation exchanger in H ⁺ form, the suspension is basified with triethylamine, filtered and the filtrate is evaporated at 2 kPa. The residue was dissolved in 10 ml of water and the solution was applied to a column of 200 ml octadecyl-silica gel (20 µm / equilibrated with 0.05 mol / ^l triethylammonium hydrogen carbonate, pH 7.5). The column is eluted with a linear gradient (total 21) of 0-30% (o./o.) Methanol in the same buffer, the product fraction (according to UV-absorption of the eluate) is evaporated, dried by forged distillation with ethanol at 2 kPa Pa over phosphorus pentoxide. The residue of this residue with trimethylbromosilane and further work-up is carried out according to Example 1 except for the cation exchange deionization. Chromatography on Sephadex A-25 and conversion to the sodium salt according to the procedure of Example 1 affords 1- (2-phosphonylmethoxyethyl) thymine sodium in a yield of 42%. The product is homogeneous according to the criteria of Example 1.

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Example 3

The compound of formula II is prepared by reacting 2-hydroxyethoxymethylphosphonic acid diethyl ester in 200 ml dioxane with 21 g (0.1 mol) with 26.3 g (0.1 mol) triphenylphosphine and 33 g of carbon tetrabromide under stirring for 24 h at 20 ° C. . The mixture is evaporated at 20 mbar, 250 ml of ether are added, the mixture is stirred for 20 min, decanted and the residue is again treated with a further 250 ml of ether. The combined ethereal solutions are evaporated at 2 kPa and the residue is extracted with ether-petroleum ether (1: 1, o./o., 300 ml), re-evaporated at 2 kPa and chromatographed on a column of 300 ml of silica gel in benzene. The combined product fractions were evaporated and dried at 13 Pa. Yield of 60-65% of the compound of formula II as a colorless oil.

A mixture of 1.35 g (10 mmol) of adenine and 0.24 g of sodium hydride in 80 ml of dimethylformamide is stirred for 1 hour at 80 DEG C. and a solution of 2.75 g (10 mmol) is added dropwise over 3 hours at 80 DEG C. of the compound of formula II in 10 ml of dimethylformamide, the mixture is heated at 80 DEG C. for a further 3 hours and evaporated at 13 Pa. The residue is extracted with boiling chloroform (400 ml total), the extract is evaporated at 2 kPa and purified by column chromatography on 200 ml of silica gel, eluting successively with chloroform and ethanol-chloroform (5: 95, o./o.). The compound of formula III (B '= adenin-9-yl) (methanol-chloroform liquid chromatography on silica gel, 1: 4, o./o. R _F = 0.57) was obtained from the combined fractions by crystallization from ethyl acetate - petroleum ether. 1.4 g (42.5%) of the compound of formula III (B ' -adenin-4-yl), m.p.

This product is dissolved in a mixture of 36 ml of acetonitrile and 2.4 ml of trimethylbromsilane, left to stand at room temperature for 16 h and evaporated at 20 mbar. The residue was codistilled with acetonitrile (3 x 20 mL) under the same conditions and dissolved in a mixture of acetonitrile-water (1: 1, 30 mL) and triethylamine (2 mL). After 30 min, it is evaporated at 2 kPa and chromatographed on a 120 ml column of Sephadex A-25 according to Example 1. Upon further conversion to the sodium salt (see Example 1), 9- (2-phosphonylmethoxyethyl) adenine sodium (PMEA) sodium salt is obtained in yield 85-90%, based on the compound of formula III, electrophoretic mobility at pH 7.0 = 0.75 (based on nauridine-3'-phosphate), UV spectrum. (pH 2): λ _max 261 nm, λ _max 15,900. R _f = 0.20 (see Example 1).

Example 4

The sodium salt of N ^with -dimethylaminomethylenadenine (10 mmol) is reacted with the compound of formula II according to Example 1. The sodium salt of 9- (2-phosphonylmethoxyethyl) adenine (PMEA) is obtained in 42% yield, formed on the starting heterocyclic base. The product (PMEA) is homogeneous according to the criteria of Example 3.

Example 5

The N ⁴ -benzoylcytosine sodium salt (10 mmol) is reacted with the compound of formula II under the conditions of Example 3. The compound of formula III (B '= N ⁴ -benzoylcytosin-1-yl) is isolated on a silica gel column in chloroform according to Example _(Rf = 0.68 in chloroform-ethanol, 1, o./o.) as an amorphous foam, in 67% yield. Further work-up was carried out according to Example 3. 89% (based on the compound of formula III) of 1- (2-phosphonylmethoxyethyl) cytosine (sodium salt) identical to the preparation obtained according to Example 1 were obtained.

Claims (2)

A process for the preparation of N- (2-phosphonylmethoxyethyl) heterocyclic base derivatives of the general formula I B-CH ₂ CH ₂ OCH ₂ P (OXOH) ₂ (I) wherein B is a purin-9-yl or pyrimidin-1-yl residue and their alkali metal salts, characterized in that the sodium salt of a heterocyclic purine or pyrimidine base , its Ν-acetyl-, N-benzoyl-, N-dimethylaminomethylene- or O-methyl derivative reacted with 1 to 1.5% molar equivalents, calculated on a heterocyclic basis, of 2-bromoethoxymethanephosphonic acid diethyl ester of formula II BrCH ₂ CH ₂ OCH ₂ P (OXOC ₂ H ₅ ) ₂ (II) in dimethylformamide as a solvent at 80 ° C to 120 ° C, the resulting intermediate of formula III B'-CH ₂ CH ₂ OCH ₂ P (OXOC ₂ H ₅ ) ₂ (III) wherein B 'has the same meaning as B in formula I or is a residue of N-acetyl-, Ν-benzoyl-, N-dimethylaminomethylene- or O The methyl base B derivative of formula I is isolated by chromatography, preferably on silica gel, then reacted with 3 to 5 molar equivalents of trimethyl halogensilane relative to the compound of formula III, preferably trimethyl bromosilane, in an inert aprotic solvent, preferably acetonitrile, a mixture is further decomposed in neutral or alkaline medium and the compound of formula I is obtained from the mixture by desalting and ion exchange chromatography. 2. Processes for the preparation of the compounds of the formula I according to claim 1, characterized in that after the reaction with the compound of the formula II, the mixture is treated with an aqueous 1-2 mol. Γ ¹ sodium hydroxide solution at 60-100 ° C, and after neutralization and deionization, further processed with trimethyl halogensilane according to point 1.