CS216563B1 - Process for preparing D-eritadenine specifically and non-specifically labeled with 14C radionuclide in the adenine ring - Google Patents

Abstract

The invention relates to a method for the preparation of D-eritadenine specifically and non-specifically labeled with the radionuclide 14C in the adenine ring. The substance is prepared by the reaction of adenine labeled with the radionuclide 14C with 2,3-O'-Cyclohexyl-D-erythronolactone in the presence of sodium or potassium carbonate and in dimethyl sulfoxide as a solvent and by acid hydrolysis. The product is purified by paper chromatography and electrophoresis. The substance is of importance in the biochemical study of D-eritadenine, which has a hypolipidemic and enzyme-inhibiting effect.

Description

SUMMARY OF THE INVENTION The present invention provides a process for the preparation of D-eritadenine of formula I (I), specifically and nonspecifically labeled with radionuclide (II) in the adenine ring.

D-Eritadenine (I) ((2R, 3R) - (4-adenin-9-yl) -2,3-dihydroxybutyric acid) was isolated from the edible wood decaying fungus Lentinus edodes shiitake (Chibata I., Okumura'K. Takeyama) S., Kotera

K. Experientia 25, 1237 (1969). This substance strongly lowers plasma cholesterol (Japanese Patent 7247398, 7221435, 5013799, 7208548, 7202259, 7204078), and is an effective inhibitor of S-adenosyl-L-homocysteine hydrolase, an enzyme involved in the regulation of methylation processes in living matter (Votruba I., Bare Ai Collection Czech Republic Chem. Commun., 45, 3039 (1980).

For the study of the mechanical effect of this substance and its pharmacokinetic and metabolic studies it is necessary to use D-eritadenine appropriately labeled with radionuclides. Previous syntheses of D-eritadenine (I) consist in the oxidation of 5- (adenin-9-yl) -5-deoxy-D-ribose or D-arabinoay by elemental oxygen in an alkaline medium (see Japanese patents and Kawazu U., Kanno T.). , Yamamure

S., Mizoguchi T., Saito S., J. Org. Chem. 38, 2687 (1973) or in oxideal 2,2-dimethyl-5i (adenin-9-yl) -1,3-dioxolane-4-methanol (Certificate No. 216562). These methods are not suitable for the preparation of a label because they require multi-step reaction procedures with isolecem1 intermediates. The only synthesis of radionuclide-labeled D-eritadenine so far described is the reaction of 4- (4,5-diaminopyrimidin-6-yl) amino-2,3-dihydroxy butyric acid with ¹⁴ C-formic acid (Kamiya T., Saito Y., Hashimoto 11 , Seki H., Tetrahedron Letters 1969, 4729). However, this method can only be used to produce 6 -? CD eritadenine with low specific activity.

D-eritadenine (1) was also prepared by condensing 2,3-O * -cyclohexylidene-D-erythronolactone of formula II

(II) with adenine sodium in dimethylformamide (Okumura K. et al., J. Org. Chem. 36, 1573 (1971)).

Even this variant is not advantageous for efficient preparation of labeled D-eritadenine, since adhering to the reaction conditions (adenime and sodium ion equilibrium, high concentration of reaction components) is difficult to work at microscale and the overall yield relative to adenine is not too high.

These drawbacks are overcome by the present invention, which comprises reacting radionuclide-labeled adenine with 10 to 20 equivalents of 2,3-O-cyclohexylidene-D-erythronolactone of formula II

0.

(II)

and powdered sodium or potassium carbonate in an amount of 25 to 50 wt. calculated on the compound of formula II in dimethylsulfoxide as solvent at temperatures of 60-140 ° C, after which the mixture is electrophoresed in an alkaline medium, preferably in triethylemin bicarbonate, and the obtained intermediate is reacted with an inorganic or organic acid and then purified successively by paper chromatography -propanol-conententated aqueous ammonia-water in the volume ratio 7: 1: 2, by paper electrophoresis in acetic or formic acid and then by paper chromatography in the system 1-butanol-ootonic acid-water in the volume ratio 10: 1: 3 ·

The reaction is simple and does not require special equipment. From a handling point of view and with respect to environmental contamination, an important feature of the process of the present invention is that the reaction is carried out in a single vessel and the entire mixture is subjected to cleaning operations directly and without loss. The use of sodium or potassium carbonate in place of the edenine sodium salt in heterogeneous reaction with the compound of formula II makes it possible to increase the excess of this agent without the risk of its decomposition and thereby substantially increase the overall conversion of adenine. An essential feature is also the use of dimethylsulfoxide as a solvent, which allows working in a low absolute concentration of reactants and the use of a higher temperature without the risk of leakage of radioactive substances by evaporation. In the reaction, the 3-isomer of the compound of formula I is formed as a by-product; its relative proportion is lower when working in dimethylsulfoxide.

An important feature of the invention is also the process for purifying the compound of formula (I): the entire reaction mixture is first freed from unreacted labeled adenine by alkaline electrophoresis (preferably in volatile triethylamine carbonate), which can be regenerated and acidic substances are isolated. By treatment with an acid (preferably volatile formic acid), the cyclohexylidene protecting group is cleaved off and the almost pure D-eritadenine contaminated with the 3-isomer is isolated by alkaline paper chromatography. Further purification of the 3-isomer of the compound of formula (I) is carried out by acid electrophoresis and acid-phase paper chromatography. For biological purposes, it is advisable to include, as a last step, ammoniacal chromatography, which deprives the acetic acid product and its esters. This procedure gives D-eritadenine of formula 1 of high (> 99%) radiochemical purity with a final yield of 55 to 70% (based on the C-adenine used).

The method according to the invention can be used with the same result for adenine labeled specifically on individual carbon atoms or for non-specific labeled adenine. This suggests the possibility of preparing C-D-eritadenine and a high specific activity sufficient for biological use both in vivo and in vitro. The process can also be used to prepare enantiomeric L-eritadenine by replacing the D-erythronolactone derivative of Formula II with a hantiomeric compound derived from L-erythronolactone. It is also possible to use the above process for the preparation

D-eritadenine labeled with nuclide C.

The starting compound of formula (IIe) is preferably used because it is relatively easy to prepare

I and get in a crystalline state. However, it can also be substituted for other substituted derivatives of D-erythronolakion at the 2,3-position protected by other cyclic acetal or ketal groups (ie, propylidene, benzylidene, ethoxymethylene, etc.). It is also possible to prepare totally labeled or labeled side-chain D-eritadenine using a radionuclide-labeled compound of formula II.

In the following, the invention is illustrated by way of examples, without being limited thereto.

EXAMPLE 1 ([beta] -c-Adenine) -D-eritadenine

A mixture of 10 MBq of β-adenine (814 MBq / mmol, 12.25 µmol), 20 mg of 2,3-O'-cyclohexylidene-D-erythronolactone, 5 mg of potassium carbonate and 250 µl of dimethylsulfoxide was heated at room temperature for 7 hours. 140 ° C in thick-walled, ground-glass tube. Place the mixture on a 16 cm strip of chromatography paper (Whatman No 30) and perform a paper electrophoresis at 20 V / cm in OC. For triethylammonium carbonate, pH 7.5 for 70 min. The mobile UV-absorbing strip of the product (78% ns adenine) was eluted with water (3-5 mL), evaporated to dryness in vacuo and the residue dissolved in 2 mL formic acid and heated at 37 ° C for 48 h. The mixture was evaporated in vacuo, the residue repeatedly evaporated with 3x5 ml of water in vacuo and the residue in 0.5 ml of 10% ammonia was incubated for 60 min at room temperature. The mixture was applied to a 2 cm strip on chromatography paper (Whatman No 3 10t) and chromatographed for 16 h in 2-propanol-concentrated ammonia-water (7: 1: 2). The product (Rj, 0.30-0.35) was eluted with water (2 to 3 mL), evaporated and vacuum evaporated and purified by paper electrophoresis (15V / cm, 60 min) on a 20x36 cm strip of chromatography paper (Whatman No 3 UM) in The product was eluted with water (2-3 mL), concentrated in vacuo and chromatographed on a 2 cm strip of paper (Whatman No 3 UM) in 1-butenol-acetic acid-water (10: 1). The spot of D-eritadenine (I) (78%) (Rp = 0.50) and its 3-isomer (Rp 0 0.40, 22%) was eluted with water (2 ml) and lyophilized. After re-chromatography in 2-propanol-concentrated aqueous ammonia-water (7: 1: 2), the product band was eluted with water and lyophilized to give 56-65% of the product β by radiochemical purity greater than 99%.

Example 2 ¹⁴ 0 £ β- -D-J Eritadenin

A mixture of 2.3 MBq of 8- ¹⁴ C-adenine (specific activity 174 MBq / molar, 12.25 µmol), 5 mg of sodium carbonate, 20 mg of 2,3-O-xyclohexylidene-D-erythronolactone and 250 µl. of dimethylsulfoxide was heated at 120 ° for 16 h and the mixture was treated as in Example 1. 70-72% of the product was obtained with a radiochemical purity greater than 99% of the chromatographic and electrophoretic properties identical to the non-radioactive material.

Claims (1)

Process for preparing D-eritadenine of formula I OF THE INVENTION HIM Hi (I) specifically and nonspecifically labeled by radionuclide removal in an adenine ring, characterized in that ae radionuclide-labeled adenine is reacted and 10 to 20 equivalents of 2,3-O-cyclohexylidene-D-erythronolactone of formula II (II) and scabrous % sodium or potassium carbonate in an amount of 25 to 50 wt. calculated on the compound of formula II in dimethylsulfoxide as a solvent at temperatures of 80 to 140 ° C, after which the mixture is subjected to electrophoresis in an alkaline medium, preferably in triethylamine bicarbonate, and the resulting intermediate is reacted with an inorganic or organic acid and then purified successively by paper chromatography 2-propanol-conc. Aqueous ammonia-water 7: 1: 2 by volume, by paper electrophoresis in acetic or formic acid, and then by paper chromatography in a 1: butanol-cyeeline-acetic-water system by volume 10: 1: 3 ·