CS248907B1 - 9-(aminoalkyl)-8-hydroxyadenines and method of their preparation - Google Patents

Abstract

The present invention provides 9- (aminoalkyl) - -8- / hydroxyadenines of formula I NH2 Si -OH / 1 / CH-IcHfi NH, lnCH3R3t wherein R 3 and R 2 is hydrogen, alkyl or hydroxyalkyl, wherein alkyl has a number of carbons one to four, R1 is hydrogen or hydroxy and n is 0 or 1. Further, the present invention provides a method for their preparation the preparation consisting of the reaction of 9- / hydroxyalkyl / adenines with an aqueous solution of bromine a following heating of the resulting 9- / hydroxyalkyl / -8-bromoadenines with aqueous solutions ammonia to 80-150 ° C. Substances in question they serve as ligands for the preparation of modified ones polymer dextran gels to affinity chromatography of S-adenosyl-L-homocysteine hydrolase.

Description

The invention relates to 9- (aminoalkyl) -8-hydroxyadenines and to a process for their preparation.

For efficient purification and isolation of enzymes, so-called affinity chromatography is successfully used, which consists in the formation of a strong, specific and reversible binding of the enzyme to polymeric carriers with bound low molecular ligands having the character of modified substrates, products or inhibitors.

To isolate the important enzyme S-adenosyl-L-homocysteine hydrolase by affinity chromatography, 8- (3-aminopropylamino-adenosine) E has been used as ligands to date. O. Kajander, A.M. Raina: Biochem. J. 193, 503 (1981), but not sufficiently effective, or 9- (RS) - (3/2) - (3-aminopropylamino) -2 (3) -hydroxypropyl) -3-hydroxyadenines. AO Nos. 231238 and 231239), which are sufficiently effective, but their preparation requires the use of expensive and high-boiling 1,3-diaminopropane.

The above-mentioned disadvantages are overcome by the alkyl / -8-hydroxyadenines of the general object of the invention, which are based on the novel 9- / amino

NH ₂ y

/Ir

R ¹ - CH / CHR ² / nch / R ³ / NH 2

Wherein R and R are hydrogen, alkyl or hydroxyalkyl, R is hydrogen or hydroxy and n is 0 or 1.

The present invention further provides a process for the preparation of a compound of formula (I) comprising: 9- (hydroxyalkyl) adenine of formula (II) nh ₂

/ 11 /,

R ¹ - CH - / CHR ² / _n CH / R ³ / OH wherein R ¹ to R ¹ and _n have the same meaning as in formula I reacted with bromine in an aqueous solution or suspension, at a temperature of 0 ° C to 30 ° C and the resulting 9- (hydroxyalkyl) -8-bromoadenine of formula III

(111), wherein R ¹ -R ¹ and n are the same as in Formula I, are heated with an aqueous ammonia solution containing 20 to 30 wt. in mass ratios 1:10 to 1:40 to 80 to 150 ° C.

An advantage of the process for the preparation of the compounds of the formula I according to the invention is that the substitution of the 8-position substituent of the compounds of the formula III and the exchange of the hydroxyl group of their side chain with an amino group take place in a single reaction step. This reaction is conditional upon the possibility of the formation of cyclic intermediates / A. Bare, Collect. Czeohoslov. Chem. Communs. 48, 1910 (1983)] and is at the same time limited to the preparation of compounds of formula I wherein the amino group of the side chain is in the alpha or beta position relative to the carbon atom that binds the adenine nucleus.

The starting compounds of formula (II) are generally readily available, for example by alkylation of adenine:. Bare, Collect. Czechoslov. Chem. Communs. 43, 3103 (1978); 43, 3444 (1978);

43, 2054 (1978); 44, 593 (1979); 47, 173 (1982).

The reaction of these compounds with bromine readily takes place in an aqueous medium, both homogeneous and heterogeneous, with a small excess (20-50%) of bromine, and provides the hydrobromides of compounds of formula III as the only reaction products.

These compounds can be used directly for the reaction with ammonia, but the more active compounds of the formula III can be isolated, thereby eliminating the risk of formation of hardly removable color impurities in the compounds of the formula I.

This isolation is carried out after evaporation of the water from the reaction mixture under vacuum (e.g. 2 to 2.5 kPa at temperatures of 30 to 50 ° C (by careful and accurate neutralization of the reaction mixture with concentrated (0.5 to 4.0 moles ⁴ ) aqueous solutions of alkali hydroxides (preferably lithium hydroxide), which in most cases eliminates little soluble product of formula III.

In those cases where the compounds of formula III are more or more soluble in water, the dried residue of the neutralized reaction mixture can be easily extracted with chloroform or mixtures thereof with methanol or ethanol, or they can advantageously be obtained from the neutralized reaction mixture by deionization on a cation exchange resin. which, after being washed with water, is slightly alkalinized by the addition of a volatile amine (preferably ammonia) and the compound of formula III is obtained by evaporation of this eluate.

The reaction of the compounds of formula III with ammonia is preferably carried out by heating with concentrated aqueous ammonia, in suspension or solution, without external stirring. Since the pressure in the closed reaction vessel does not reach high values at the temperatures used, simple, conventional low pressure reaction vessels resistant to aqueous ammonia or glass thick-walled reactors can be used for this purpose.

The progress of the reaction and the purity of the reaction products are preferably checked by paper chromatography or thin layer chromatography on silica gel, in both cases in a system of 80% aqueous 2-propanol-concentrated aqueous ammonia (9: 1); detection is performed in ultraviolet light and the compounds of formula I are detected by spraying a solution of ninhydrin (purple spots).

The reaction affords all of the compounds of formula I as the only reaction products. Evaporation of the reaction mixture yields a mixture of I or its hydrobromide with ammonium bromide. This product can be used directly for binding to polymeric carriers / according to U.S. Pat. AO No. 248908) or the compound of formula I can be obtained in pure form by chromatography on, for example, silica gel or cellulose in a mixture of 80% aqueous 2-propanol-conc. aqueous ammonia (9: 1) or octadecylsilica gel in water.

In the following, the invention is illustrated by means of exemplary embodiments for the general procedure, ie, without limitation to all types of compounds of formula I.

Example 1

To a solution of 1 ml of bromine in 150 ml of water is added 10 nmol of the compound of formula II and the mixture is stirred in a sealed vessel at 18-24 ° C for 16-24 hours. The suspension is then evaporated to 40 ° C / kPa to dryness. Dissolve in 100 ml of water. The solution was neutralized by stirring at pH-meter with 4 mol / l ⁴ of sodium or lithium hydroxide solution to pH 7.0 (0.95 to 7.05) and the mixture was cooled at 0 ° C for 1-2 h.

The precipitated product of formula III is filtered off with suction, washed with water (200 ml), acetone (100 ml) and ether (100 ml) and dried at 10 to 15 Pa over phosphorus pentoxide.

A suspension of 5 mmol of the compound of formula III in 50 ml of concentrated aqueous ammonia (25 to 29% NH 4) is heated in a steel cylinder at 100 to 110 ° C for 6-8 h. After cooling, the clear pink solution is evaporated at 40 ° C. The residue is dissolved in 20 ml of a mixture of 80% aqueous 2-propanol-conc. aqueous ammonia (9: 1) and loaded onto a column (80 x 4 cm) of crystalline cellulose in the same system.

The column was eluted (20 ml / h with the same system) and fractions (20 ml) were analyzed by paper chromatography in the same system. The product fractions were combined, evaporated at 40 ° C / 20 mbar, evaporated to ethanol (2 x 20 mL) under the same conditions, and the residue was crystallized from methanol, adding ether to turbidity.

The compound of formula I obtained is suction filtered, washed with ether and dried at 10 to 15 Pa over sodium hydroxide or soda lime. The mother liquors contain an additional portion of the pure compound of formula I as the hydrogen carbonate, which can be re-subjected to the same chromatography or precipitated with ether and isolated as the hydrogen carbonate. The yield and properties of the compounds of formula I thus obtained are shown in Table 2.

Example 2

The compound of formula (II) (10 mol) is reacted with bromine and treated as in Example 1. If the compound of formula (III) is water-soluble and does not precipitate out of water, proceed as follows: neutral aqueous solution of the residue of the reaction mixture is applied to a column (200 to 250 ml) of cation exchange resin (preferably Dowex 50) in acid form and the column is washed with 1 l of water.

The ion exchanger is then suspended in 400 ml of water and dilute (1: 2, v / v) aqueous ammonia is added with stirring so that the pH does not exceed 8 until the value has changed for 30 min.

The suspension was filtered off with suction and washed with boiling water (0.5 to 1.0 L). The filtrate is evaporated at 40 ° C / 2 kPa and the residue is crystallized from ethanol or 80% aqueous ethanol. The product is filtered off with suction and dried at 10-15 Pa over phosphorus pentoxide. The properties and yield of the compounds of formula III thus obtained are shown in Table 1. The procedure is as in Example 1.

Tabulkal

Preparation and Properties of 9- (hydroxyalkyl) -8-bromadenines of Formula III

Starting material II (mmol) R ¹ R ² R ³ n Yield % Method ^ / Melting point ° C SI S2 30.0 H H 0 58 AND 238 to 239 0.76 0.72 10.0 H H H 1 62 (B) 205 to 207 0, 59 0.63 40.0 H OH H 1 92 AND 260 0, 63 0.50 6.0 H OH CH 1 75 (B) 224 0, 72 0.68 2.5 H OH CH 2 OH ^J 1 95 (B) 260 0.60 0.48 15.0 H - 0 70 (B) 191 to 192 0, 58 0.63 4.0 ch ₂ oh - H ³ 0 50 AND 235 to 236 0, 68 0.57 Comment: a / 'Ultraviolet spectrum substances III / pH 2 and 7 /: 'X' max 267 nm / f 'max 18,000 /;

According to Example 1 (A), according to Example 2 (B);

^{C //} 80% aqueous 2-propanol-conc. Aqueous ammonia / 9: 1 // S1 // Paper chromatography; chloroform-methanol (4: 1 // S2 // silica gel thin layer).

Table 2

Preparation and Properties of 9-Aminoalkyl / -8-Hydroxyadenines of Formula I

Starting material III / mmol / R ¹ R ² R ³ n Yield % Melting point Mass ^{Cy /} Spectrum / M ⁺ / 10.0 H - H 0 57 251 to 252 0.38 194 5.0 H H H 1 48 217 to 218 0.38 208 15.0 H OH H 1 63 174 0.30 224 2.5 H OH ^CH 3 1 54 168 to 170 0.46 238 2.5 H OH CH ₂ OH 1 42 149 0.35 254 7.5 H - ^CH 3 0 64 175 to 176 0.53 208 2.5 CH ₂ OH - H 0 56 145 0.40 224

Notes: Ultraviolet spectra of compounds of formula I: pH 2, λ 270 nm (£ 11,000), max max λ max ²⁸² ™ / £ max ¹⁰ ° ⁰⁰ /? ^{H 7} '* max ²⁷⁰ ™> £ max ¹³ ° ⁰⁰ /> P ^{h 12} ' max ^28Onm / £ max ¹⁴ ° ⁰⁰ / '

80% aqueous 2-propanol-conc. aqueous ammonia (9: 1) paper chromatography (tc) molecular peak.

Claims (2)

1-9- (Aminoalkyl ⁾ -8-hydroxyadenines of the general formula I- ^1- ch-CHR ² -nCH (R ³ -n 2 where R ¹ and R ³ are up to hydrogen, alkyl or hydroxyalkyl, wherein the alkyl has a carbon number of one 2 four, R is hydrogen or hydroxy and n is 0 or 1. 2. A process for the preparation of the compounds of the formula I according to claim 1, characterized in that 9- (hydroxyalkyl) adenine of the formula II is prepared. R @ ¹ --CH-- / CHR @ ² -CH _n / n ³ / OH Wherein R to R and n have the same meaning as in Formula I, react with bromine in an aqueous solution or suspension, at a temperature of 0 ° C to 30 ° C to form the 9- (hydroxyalkyl) -8-bromoadenine of formula III ¹ -CH- (CHR 2) _n -CH (R 3) -OH Wherein R to R and n have the same meaning as in formula I, are heated with an aqueous ammonia solution containing 20 to 30 wt. in mass ratios 1:10 to 1:40 to 80 to 150 ° C.