*Article* **Isolation and Structure Determination of Echinochrome A Oxidative Degradation Products**

**Natalia P. Mishchenko 1,\*, Elena A. Vasileva 1, Andrey V. Gerasimenko 2, Valeriya P. Grigorchuk 3, Pavel S. Dmitrenok <sup>1</sup> and Sergey A. Fedoreyev <sup>1</sup>**


Academic Editor: Marina DellaGreca

Received: 18 September 2020; Accepted: 16 October 2020; Published: 18 October 2020

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