*3.4. HPLC Method Validation*

Linearity of the method was established by using methanolic Ech A solutions containing 50–1500 ng/mL. Each linearity sample was injected in triplicate. The calibration curve was constructed as linear regression analysis of the peak area versus concentration. The limits of detection (LOD) and quantification (LOQ) of Ech A were calculated as concentrations at which the signal-to-noise ratio was below 3 and 10, respectively. The accuracy of the method was established by recovery studies of Ech A samples (100, 250, 500 ng/mL); data are provided in the Supplementary Materials (Table S1). Accuracy was expressed as relative standard deviations (RSDs) and recoveries (%). Selectivity was confirmed through peak purity studies using a DAD detector.

#### *3.5. Oxidation Products Preparation and Isolation*

Histochrome from an ampoule (10 mg/mL Ech A, 5 mL) was diluted with distilled water (250 mL), balanced with atmospheric oxygen, to obtain a solution with an Ech A concentration of 0.2 mg/mL, pH 7.2. The oxidation was carried out in the light without stirring at ambient temperature. The process was monitored by HPLC–DAD–MS. When approximately 50% of the Ech A was consumed (after 20 h), the reaction was stopped by adding hydrochloric acid to pH 2. Unreacted Ech A was removed from solution by extraction with chloroform. The oxidation products were extracted with ethyl acetate, the solvent was removed under reduced pressure, and the residue was chromatographed on a Toyopearl HW-40 TSKgel column in a gradient of 10–50% ethanol containing 0.5% formic acid. From fractions eluted with 10–30% ethanol compounds were extracted with ethyl acetate and the solvent was evaporated. As a result, compounds **2**, **9**, and **10** were isolated. Fractions eluted with 30–50% ethanol were concentrated in vacuo at 50 ◦C; as a result, echinolactone **11** was obtained from the fraction of compound 7.

A small portion of oxidation products (10 mg) was dissolved in cold MeOH and treated with diazomethane in diethyl ether on ice as described [44]. The reaction process was controlled by HPLC–DAD–MS. After 1 h, the solvent was removed in vacuo, and the residue was subjected to column chromatography on Sephadex LH-20 eluting with CHCl3/EtOH 8:1. As a result, dimethyl ether of **7** and methyl ether of **8** were obtained.

*7-ethyl-2,2,3,3,5,6,8-heptahydroxy-2,3-dihydro-1,4-naphthoquinone* (**2**): C12H12O9; UV (ethanol) λmax 256, 320, 391 nm; ESI-MS *m*/*z*: 299 [M − H]−; HR-ESI-MS *m*/*z*: 299.0399 [M − H]<sup>−</sup> (calculated for [C12H11O9] − 299.0409).

*4-ethyl-3,5,6-trihydroxy-2-oxalobenzoic acid* (**7**); C11H10O8; UV (CH3CN–H2O) λmax 219, 271, 320 nm; ESI-MS *m*/*z*: 269 [M − H]−; HR-ESI-MS *m*/*z*: 269.0304 [M − H]<sup>−</sup> (calculated for [C11H9O8] − 269.0297).

*Dimethyl ether of compound* **7**: light-yellow powder; C13H14O8; UV (CH3CN–H2O) λmax 224, 250, 324, 384 nm; IR (CDCl3) νmax 3520, 3156, 2975, 2956, 2937, 2878, 2855, 1734, 1683, 1632, 1595 cm−1; ESI-MS *<sup>m</sup>*/*z*: 297 [M <sup>−</sup> H]−; 1H- and 13C-NMR (see Table 3).

*4-ethyl-2-formyl-3,5,6-trihydroxybenzoic acid* (**8**); C10H10O6; light-yellow powder; UV (CH3CN–H2O) λmax 270, 320 nm. ESI-MS *m*/*z*: 225 [M − H]−; HR-ESI-MS *m*/*z*: 225.0405 [M − H]<sup>−</sup> (calculated for [C10H9O6] − 225.0399).

*Methyl ether of compound* **8**: C11H12O6; light-yellow powder; UV (CH3CN–H2O) λmax 238, 303, 369 nm; IR νmax (CDCl3) 3011, 2959, 2935, 2878, 2865, 1668, 1634, 1611 cm−1. ESI-MS *m*/*z*: 239 [M − H]−; HR-ESI-MS *m*/*z*: 239.0570 [M − H]<sup>−</sup> (calculated for [C11H11O6] <sup>−</sup> 239.0555); 1H- and 13C-NMR (see Table 3).

*4-ethyl-2,3,5-trihydroxybenzoic acid* (**9**); C9H10O5; light-yellow powder; UV (CH3CN–H2O) λmax 228, 251, 333 nm; IR <sup>ν</sup>max (CCl4) 3352, 2960, 2928, 2875, 2855, 1642 cm−1. ESI-MS *m*/*z*: 197 [M <sup>−</sup> H]−; HR-ESI-MS *m*/*z*: 197.0452 [M − H]<sup>−</sup> (calculated for [C9H9O5] <sup>−</sup> 197.0450); 1H- and 13C-NMR (see Table 3).

*3-ethyl-2,5-dihydroxy-1,4-benzoquinone* (**10**); C8H8O4; light-yellow powder; UV (CH3CN–H2O) λmax 228 nm, literature 228 nm [28]; IR νmax (CDCl3) 3366, 2976, 2935, 2878, 1731, 1641 cm<sup>−</sup>1. ESI-MS *m*/*z*: 167 [M − H]−; HR-ESI-MS *m*/*z*: 167.0343 [M − H]<sup>−</sup> (calculated for [C8H7O4] <sup>−</sup> 167.0344). 1H-NMR (700 MHz, CDCl3) δ, ppm (*J*, Hz): 1.21 (3H, t, *J* = 7.5, CH3), 2.49 (2H, q, *J* = 7.5, CH2), 6.01 (1H, s, H), 7.54 (2H, s, OH); 1H- and 13C-NMR data of dimethyl ether of **10** (see Figures S43–S48, Supplementary Materials).

*Echinolactone* (7-ethyl-5,6-dihydroxy-2,3-dioxo-2,3-dihydrobenzofuran-4-carboxylic acid, **11**): C11H8O7; UV (ethanol) λmax (logε ): 215 (3.4), 331 (2.8), 385 (2.8) nm; IR νmax (CDCl3) 3483, 1838, 1698, 1581 cm<sup>−</sup>1; ESI-MS *<sup>m</sup>*/*z*: 251 [M <sup>−</sup> H]−; EI-MS *<sup>m</sup>*/*z*: 252 [M]+. 1H-NMR (300 MHz, CDCl3), <sup>δ</sup>, ppm: 1.24 (3H, t, *J* = 7.5, CH3), 2.78 (2H, dd, *J* = 7.5, CH2), 5.24, (1H, s, OH), 12.88 (1H, s, OH). 13C-NMR (75 MHz, CD3CN), δ, ppm: 13.3, 17.8, 106.9, 108.8, 121.9, 151.7, 158.6, 160.5, 162.2, 171.5, 178.4.

The main crystal data, data collection, and refinement parameters are presented in Table S4 (Supplementary Materials). The selected geometric parameters of **11** crystal forms are given in Table S5 (Supplementary Materials). The hydrogen-bond geometry parameters are listed in Table S6 (Supplementary Materials). Crystallographic data for the structures in this study were deposited to the Cambridge Crystallographic Data Center as supplementary publication No. CCDC 1,822,816 and 1822821; copies of the data can be obtained, free of charge, via an application to CCDC, 12 Union Road, Cambridge CB2 1EZ, UK, (fax: +44 1223 336,033 or e-mail: deposit@ccdc.cam.ac.uk).
