Isolation and Structure Determination of Echinochrome A Oxidative Degradation Products

Natalia P Mishchenko; Elena A Vasileva; Andrey V Gerasimenko; Valeriya P Grigorchuk; Pavel S Dmitrenok; Sergey A Fedoreyev

doi:10.3390/molecules25204778

Isolation and Structure Determination of Echinochrome A Oxidative Degradation Products

Molecules. 2020 Oct 18;25(20):4778. doi: 10.3390/molecules25204778.

Authors

Natalia P Mishchenko¹, Elena A Vasileva¹, Andrey V Gerasimenko², Valeriya P Grigorchuk³, Pavel S Dmitrenok¹, Sergey A Fedoreyev¹

Affiliations

¹ G.B. Elyakov Pacific Institute of Bioorganic Chemistry, Far Eastern Branch of the Russian Academy of Sciences, Vladivostok 690022, Russia.
² Institute of Chemistry, Far Eastern Branch of the Russian Academy of Sciences, Vladivostok 690022, Russia.
³ Federal Scientific Center of the East Asia Terrestrial Biodiversity, Far-Eastern Branch of Russian Academy of Sciences, Vladivostok 690022, Russia.

Abstract

Echinochrome A (Ech A, 1) is one of the main pigments of several sea urchin species and is registered in the Russian pharmacopeia as an active drug substance (Histochrome^®), used in the fields of cardiology and ophthalmology. In this study, Ech A degradation products formed during oxidation by O₂ in air-equilibrated aqueous solutions were identified, isolated, and structurally characterized. An HPLC method coupled with diode-array detection (DAD) and mass spectrometry (MS) was developed and validated to monitor the Ech A degradation process and identify the appearing compounds. Five primary oxidation products were detected and their structures were proposed on the basis of high-resolution electrospray ionization mass spectrometry (HR-ESI-MS) as 7-ethyl-2,2,3,3,5,7,8-heptahydroxy-2,3-dihydro-1,4-naphthoquinone (2), 6-ethyl-5,7,8-trihydroxy-1,2,3,4-tetrahydronaphthalene-1,2,3,4-tetraone (3), 2,3-epoxy-7-ethyl-2,3-dihydro-2,3,5,6,8-pentahydroxy-1,4-naphthoquinone (4), 2,3,4,5,7-pentahydroxy-6-ethylinden-1-one (5), and 2,2,4,5,7-pentahydroxy-6-ethylindane-1,3-dione (6). Three novel oxidation products were isolated, and NMR and HR-ESI-MS methods were used to establish their structures as 4-ethyl-3,5,6-trihydroxy-2-oxalobenzoic acid (7), 4-ethyl-2-formyl-3,5,6-trihydroxybenzoic acid (8), and 4-ethyl-2,3,5-trihydroxybenzoic acid (9). The known compound 3-ethyl-2,5-dihydroxy-1,4-benzoquinone (10) was isolated along with products 7-9. Compound 7 turned out to be unstable; its anhydro derivative 11 was obtained in two crystal forms, the structure of which was elucidated using X-ray crystallography as 7-ethyl-5,6-dihydroxy-2,3-dioxo-2,3-dihydrobenzofuran-4-carboxylic acid and named echinolactone. The chemical mechanism of Ech A oxidative degradation is proposed. The in silico toxicity of Ech A and its degradation products 2 and 7-10 were predicted using the ProTox-II webserver. The predicted median lethal dose (LD₅₀) value for product 2 was 221 mg/kg, and, for products 7-10, it appeared to be much lower (≥2000 mg/kg). For Ech A, the predicted toxicity and mutagenicity differed from our experimental data.

Keywords: HPLC–DAD–MS; NMR; echinochrome A; histochrome; oxidative degradation.

MeSH terms

Animals
Chromatography, High Pressure Liquid
Humans
Magnetic Resonance Spectroscopy
Naphthoquinones / chemistry*
Naphthoquinones / isolation & purification
Naphthoquinones / metabolism
Naphthoquinones / pharmacology
Oxidation-Reduction / drug effects
Oxidative Stress / drug effects*
Sea Urchins / chemistry*
Spectrometry, Mass, Electrospray Ionization

Substances

Naphthoquinones
histochrome
echinochrome A