RU2617851C1 - Tritiated on the acetyl and holinum fragments acetylcholin - Google Patents

Abstract

FIELD: chemistry.

SUBSTANCE: invention relates to novel labeled tritium on acetyl and choline fragments acetylcholine of the formula CH₃COOCH₂CH₂N⁺(CH₃)₃Br^-, Which can be used in the study of physiologically active compounds.

EFFECT: improved properties.

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Description

The invention relates to the field of organic chemistry and may find application in analytical chemistry and biological research.

When studying physiologically active compounds, their labeled analogues are necessary.

It is known that the replacement of atoms of compounds with their analogues labeled with hydrogen isotopes does not lead to a change in any properties of the initial compound (Evans EA - Tritium and its compounds London Butterworths, 1974, p. 48) [1].

In addition, it was shown that the reactions of deuterium and tritium with substances deposited on the catalyst surface occur with similar rates and the same selectivity, and the ratio of the rates of these reactions approaches unity (Zolotarev Yu.A., Dadayan A.K., Kost N. V., Voevodina M.E., Sokolov O.Yu., Kozik BC, Scar S.I., Azev V.N., Bocharov E.V., Bogachuk A.P., Lipkin V.M., Myasoedov N .F. Quantitative analysis of the peptide HLDF-6-amide and its metabolites in the tissues of laboratory animals using their labeled with tritium and deuterium derivatives // Bioorganic chemistry I, 2015, Volume 41, No. 6, pp. 644-656 and Zolotarev Yu. A., Dadayan A.K., Borisov Yu. A., Kozik VS, Nazimov IV, Ziganshin RH, Bocharov EV, Chizhov AO, Myasoedov NF New Development in the Solid-State Isotope Exchange with Spillover Hydrogen in Organic Compounds // J. Phys. Chem. C. 2013. V. 117. P. 16878-16884) [2, 3].

It was also shown in [1] that in biological studies, the distribution of tritium in the molecules of labeled preparations plays a crucial role. The work emphasizes that in the study of drug metabolism, the stability of the position of tritium is one of the most important conditions of a biological experiment. For each specific study, a certain distribution of tritium in the molecules of the labeled preparations is required. A chemically stable bound tritium atom can become mobile upon the interaction of a tritium-containing compound with an enzyme. As a result, the tritium atom can be lost, despite the complete safety of the carbon skeleton of the molecule. If tritium is in another fragment of the molecule of the substance or there are several of them in the molecules of the substance, then the probability of obtaining more reliable data is significantly increased. It is important not only the distribution of tritium, but even its stereochemical conformation. For example, it is shown that the conversion of cholesterol-4- ^{14 C-3} H 7a- cholic acid in the body in vivo rat leads to a loss of at least 93% of the tritium position 7. However, when using cholesterol-4- ¹⁴ C-7β- ³ N all tritium remained in cholic acid.

Known acetylcholine of the formula I:

CH ₃ COOCH ₂ CH ₂ N ⁺ (CH ₃ ) ₃ Br ^-

Acetylcholine is a chemical transmitter of nerve excitation in the ends of nerve fibers, for which it serves as a mediator. Acetylcholine does not have a strict selective effect on a variety of cholinergic receptors. The peripheral nicotine-like action of acetylcholine is associated with its participation in the transmission of nerve impulses from preganglionic fibers to postganglionic fibers in the autonomic nodes, as well as from motor nerves to the striated muscle. In small doses, it is a physiological transmitter of nervous excitation, in large doses it can cause persistent depolarization in the synapse region and block transmission of excitation (Rockland, KS Brain. In Α.Ε. Kazdin (Ed.), Encyclopedia of psychology Vol. 8, pp. 447-455. 2000. Washington, DC: American Psychological Association [4])

However, its tritium-labeled analogue, simultaneously containing a label for acetylcholine and choline fragments of acetylcholine, is not described.

The technical result achieved by the present invention is to expand the range of labeled analogues of physiologically active compounds.

This technical result is achieved by obtaining tritium-labeled acetylcholine of formula I:

CH ₃ COOCH ₂ CH ₂ N ⁺ (CH ₃ ) ₃ Br ^-

The following is an example implementation of the invention.

Example I

A solution of 3 mg of CH ₂ ClCOOCH ₂ CH ₂ N ⁺ (CH ₃ ) ₃ Br ^- in 0.1 ml of acetonitrile was added to 120 mg of Lindlar (Fluka) catalyst, acetonitrile was removed by evaporation on a rotary evaporator. The residue was crushed and lyophilized. An aliquot of the dry mixture (82 mg) was transferred to the reaction vial. Then, the ampoule was evacuated to a pressure of 0.1 Pa, filled with gaseous tritium to a pressure of 400 hPa and kept at a temperature of 190 ° С for 7 min. Excess tritium gas was removed by vacuum. The substance from the catalyst was separated by filtration, extraction was carried out with methanol (5 × 2 ml). Labile tritium was removed several times, dissolving the substance in methanol (5 × 2 ml) and evaporating the latter. The radioactivity of the isolated product is about 150 mCi.

Preparative purification was carried out by HPLC on a Reprosil pur DIOL column, 10 × 150 mm, 5 μm, system: acetonitrile-methanol-25 mm ammonium acetate (pH 5.6) - acetic acid (60: 20: 40: 0.05), flow rate - 2 ml / min, retention time - 8.14 min, yield - 70%, molar radioactivity - 20 Ci / mmol.

The analysis of the labeled preparation was performed on a Reprosil Pur Diol column, 4 × 150 mm, 3 μm, system: acetonitrile-methanol-25 mm ammonium acetate (pH 5.6) - acetic acid (60: 20: 40: 0.05), flow rate - 1 ml / min, retention time - 2.43 min, radiochemical purity - 99%.

Example II

A solution of 3 mg of Br CH ₂ COOCH ₂ CH ₂ N ⁺ (CH _{3) 3} Br ^- in 0.1 mL of acetonitrile was added to 120 mg of Lindlar catalyst (Fluka), the acetonitrile was removed by rotary evaporation. The residue was crushed and lyophilized. The subsequent procedures were similar to those described in example I. The yield of acetylcholine was 40-45%, the molar radioactivity was 25-30 Ci / mmol.

Example III

A solution of 3 mg CCl ₃ COOCH ₂ CH ₂ N ⁺ (CH ₃ ) ₃ Br ^- in 0.1 ml of acetonitrile was added to 120 mg of Lindlar (Fluka) catalyst, acetonitrile was removed by evaporation on a rotary evaporator. The residue was crushed and lyophilized. The subsequent procedures were similar to those described in example I. The yield of acetylcholine was 2–4%, and the molar radioactivity was 35–40 Ci / mmol.

It was found that 84% of tritium is included in acetyl and 16% in choline.

Thus, a new labeled compound was obtained in which both the acetyl and choline fragments of acetylcholine contained tritium.

Claims (3)

Labeled with tritium on acetyl and choline fragments acetylcholine of the formula: CH ₃ COOCH ₂ CH ₂ N ⁺ (CH ₃ ) ₃ Br ^-