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.
Highlights
Histochrome® is a solution of the sodium salt of naturally occurring quinone echinochromeA (7-ethyl-2,3,5,6,8-pentahydroxy-1,4-naphthoquinone) for intravenous injections and infusions, manufactured in ampoules (Supplementary Materials, Figure S1)
This was confirmed by us after a 3 year stability study of the Ech A substance and Histochrome preparation in ampoules closed under inert conditions
To provide the opportunity to establish primary oxidation products, we did not use the onerous conditions recommended in the ICH guidelines for degradation product studies
Summary
Histochrome® is a solution of the sodium salt of naturally occurring quinone echinochromeA (7-ethyl-2,3,5,6,8-pentahydroxy-1,4-naphthoquinone) for intravenous injections and infusions, manufactured in ampoules (Supplementary Materials, Figure S1). It is is used used in in cardiology cardiology for for the the treatment treatment of of coronary coronary heart heart disease disease and and Russia as as an drug It for restriction of the necrosis zone in myocardial infarction N002363/01), for restriction of the necrosis zone in myocardial infarction (state registration number P N002363/01), and for the and in in ophthalmology ophthalmology for the treatment treatment of of dystrophic dystrophic diseases diseases of of the the retina retina and and cornea, cornea, macular macular degeneration, primary open-angle glaucoma, diabetic retinopathy, hemorrhages to vitreous humor, humor, degeneration, primary open-angle glaucoma, diabetic retinopathy, hemorrhages to vitreous retina, and anterior chamber, and discirculatory disorders in the central artery and retinal vein retina, and anterior chamber, and discirculatory disorders in the central artery and retinal vein (state P N002363/02).
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