CS255180B1 - Method of preparative isolation of (6-3H) -thymine - Google Patents

Abstract

(6- H) -thymine is used as a tracer in biochemical, biological and medical research. Furthermore, it serves as a starting material for the synthesis of more complex biologically active substances, i.e. (6-3H) -thymidine, respectively. 5'-phosphates (6H) -thymidine. The aim of the solution was to provide yields of (6-3H) -thymine, shorten the separation cycle and improve the quality of the final product. Higher performance is due to the use of high performance liquid chromatography, which makes it possible to obtain pure (6-3H) -thymine with a higher yield in a shorter production cycle. This yielded 85-90% (6-3H) -thymine based on the content of the reaction mixture. The radiochemical purity and chemical purity is 97-98%.

Description

The invention relates to a process for the preparative isolation of (6- H) -thymine from the reaction mixture after its preparation by chemical synthesis (cf. AO 192 238).

The usual methods of isolating the compound from the reaction mixtures after its synthesis are methods of preparative paper chromatography. Especially in the preparation of (6H) -thymine of high molar activity, this isolation method has a number of drawbacks. In particular, insulation losses (30%) occur and the isolated compound is not chemically pure. In today's requirements for high molar activity of radionuclide-labeled compounds, the insulation losses in the application of standard procedures are no longer negligible economically. The low degree of chemical purity of preparations isolated by preparative paper chromatography is mainly due to the presence of extracts from chromatographic paper or impurity from solvents. The application of (6- H) -thymine thus prepared in the enzymatic syntheses of (6- H) -thymidine, (6- H) -thymidine-5'-monoO phosphate and (6- H) -thymidine-5'-triphosphate cannot be excluded total loss of catalytic activity of enzyme preparations.

Another shortcoming of paper chromatography falls into the field of safety of work with radioactive substances. Handling radioactive compounds when applied to chromatographic papers, self-chromatography, autoradioohromatography and elution essentially increases the possibility of contamination of the environment and people. Moreover, preparative paper chromatography is a time-consuming method.

The hitherto used technological process for the isolation of (6- H) -thymine from the reaction mixture after its chemical synthesis is carried out by the method of paper chromatography on Whatmann-3 chromatography paper in the system butanol-propanol-ammonia-water (7: 5: 7: 2). _f , the thymine in this system is 0.45. Identification of radioactive compounds in ohromatograms is performed by comparing them with inactive standards. The band corresponding to radioactive thymine is excised and eluted with redistilled water. Since the desired radiochemical purity is not achieved, the paper chromatography should be repeated in another developing system, further increasing the losses of the isolated preparation.

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The principle of the preparative isolation of (6- H) -thymine (having a molar activity of more than 0.8 GBq / / mmol by elution high performance chromatography according to the present invention consists in the 3 mixture of compounds formed in the preparation of radioactive (6- H) -thymine The silica gel surface (tetrapotassium silicic acid H 2 SiO 4) is covered with octadecyl groups and the residual silanols are blocked with methyl groups. C-18.

After dosimetry and radiochemical purity determination, the eluted radioactive (6- ³ H) -thymine is diluted to expedition concentration and shipped directly, or used for other chemical syntheses.

The advantages of the novel process are that the yield of the radioactive product is 20% higher in the long-term average than when it is isolated by the above-mentioned preparative paper chromatography method.

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A major advantage of the isolation of (6- H) -thymine according to the present invention is the possibility of obtaining a product of high radiochemical purity (min. 97%) and in the absence of chemical impurities. This makes it possible to apply the thus isolated (6H) -thymine as the starting radioactive substrate for the (6HH) -thymidine and (6H) -5-thymidine phosphates by appropriate enzymatic techniques without inhibiting the catalysis enzymes. Another advantage is to reduce the production cycle time of the separation.

Separation by liquid chromatography takes place in a liquid medium in a closed semi-automated system. There is no need for autoradiography and drying of chromatographic papers, which can easily be contaminated with radioactivity. This significantly increases the safety of work during the preparation of (6- ² H) -thymine.

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For clarification, an example of a procedure for isolating radioactive (6 H) -thymine from the reaction mixture after hydrogenation of inactive 6-bromothymine (30 mg) in 1M KOH (0.4 mL) with tritium gas (185 GBq) is given below. 4 mg of 10% Pd on activated carbon is used as catalyst. After chemical synthesis, the labile bound tritlm is removed from the reaction mixture by lyophilization. The catalyst is removed by centrifugation. 1M HCl was added to the reaction mixture to give a final pH of 6-7. The total radioactivity of the sample prepared in this way would be 65,285 MBq, of which 91.2% (6- ³ H) -thymine. The chemical composition of this mixture was: 38.4% thymine, 56.3% 6-bromothymine, 5.3% unidentifiable substance. Due to the capacity of the separation column, the reaction mixture was divided into 3 parts and each part was separated separately. The column was packed with domestic SEPARON SIX-C-18 sorbent and was washed with a 0.6% aqueous solution of C 1 H 4 OH at a flow rate of 100 ml / h for 120 minutes before loading the sample. After loading the sample, the column is washed with the same solution at the same flow rate. At this flow rate, radioactive material of unknown composition began to elute after 36 minutes and absorbed in the UV region of 254 nm. After 50 minutes, a second radioactive substance of unknown composition began to elute, which also absorbs in the UV region of 254 nm. After 87 minutes, (6H) -thymine began to elute, which eluted for 23 minutes. Radioactive (6- H) -thymine was eluted in a volume of 40 ml. After 3 (6H) -thymine elution was completed, the column was washed with 96% C2H5OH. This eluent displaced unreacted 6-bromothyrate 187 minutes after the beginning of the wash. The average radiochemical purity of 3 3 (6H) -thymine was 97.3%. 52,052 MBq of (6H) -thymine was obtained, which represents a yield of 87.4%.

Claims (1)

SUBJECT OF THE INVENTION A process for the preparative isolation of (6- H) -thymine from a reaction mixture by elution high performance liquid chromatography, characterized in that the mixture of compounds resulting from the preparation of (6- ³ H) -thymine is separated on a column of porous silica-based ion exchanger whose surface is covered octadecyl groups using a 0.6% aqueous solution of pure ethanol as eluent.