Mishchenko NP et al. (2020), Isolation and Structure Determination of Echino...

ECB-ART-49541

Molecules 2020 Oct 18;2520:. doi: 10.3390/molecules25204778.

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Isolation and Structure Determination of Echinochrome A Oxidative Degradation Products.

Mishchenko NP , Vasileva EA , Gerasimenko AV , Grigorchuk VP , Dmitrenok PS , Fedoreyev SA .

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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.

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References [+] :

Banerjee, ProTox-II: a webserver for the prediction of toxicity of chemicals. 2018, Pubmed

	Figure 1. Chemical structure of echinochrome A (7-ethyl-2,3,5,6,8-pentahydroxy-1,4-naphthoquinone, Ech A).
	Figure 2. HPLC profiles of Histochrome (Ech A) oxidation products. Unmarked peaks are natural impurities of the Ech A substance [24].
	Figure 3. Primary oxidation products of Ech A (1); structures 3–6 were proposed on the basis of HR-ESI-MS data.
	Figure 4. Structures of Ech A oxidation products with brutto-formula C12H10O8.
	Figure 5. Absorption spectra of Ech A (1) and its oxidation products 2 and 9.
	Figure 6. Molecular structure of echinolactone (11) in different crystal types (hydrogen bonds are shown as dotted lines).
	Figure 7. Structures of dimethyl ether of compound 7, methyl ether of compound 8, and of compound 9.
	Figure 8. Proposed scheme of the Ech A oxidative degradation process.
	Figure 9. Tautomeric forms of Ech A: Ech-B (ethyl in the benzene ring) and Ech-Q (ethyl in the quinonoid ring).