*2.1. Isolation and Structure Elucidation of Echinochrome A Oxidative Degradation Products*

According to our observations, in dry form and in the absence of oxygen in solution, the pharmaceutical substance Ech A remains stable for several years. 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. In aqueous solutions, the Ech A sodium derivative (Histochrome) readily hydrolyzes and oxidizes (Figure S2, Supplementary Materials). Therefore, 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. In this work, we studied the oxidation of Ech A sodium derivative in air-equilibrated aqueous solutions.

An HPLC method coupled with diode-array detection (DAD) and mass spectrometry (MS) was developed and validated to monitor the degradation of Ech A and to support the peak identification procedure. The eluent system consisting of H2O (A)/MeCN (B) with the addition of 0.2% AcOH in a gradient mode provided acceptable separation of Ech A and its oxidation products. The developed LC method demonstrated good linearity, and the correlation coefficient for Ech A was found to be 0.9987. The limit of detection (LOD) and limit of quantification (LOQ) of Ech A were found to be 22 and 72 ng/mL, respectively. The analytical area of this method was established by the range of experimental data satisfying the linear model. For Ech A (**1**), the corresponding range was determined to be 72–600 ng/mL. The accuracy and reproducibility of the quantification procedure was evaluated according to the results obtained for Ech A, shown in Table S1 (Supplementary Materials). The detection wavelength 254 nm was chosen on the basis of the fact that all target compounds display intense absorption in the region of 230–270 nm.

A solution of Ech A sodium derivative from an ampoule was diluted approximately 50-fold with distilled water saturated with atmospheric oxygen, pH 7.2. In this solution, the molar ratio of Ech A to dissolved O2 was 3:1. HPLC–DAD–MS analysis showed that, after 1 h in the air-equilibrated aqueous solution, the first oxidation product of Ech A (**1**) was compound **2,** with a retention time of 7.79 min (Figure 2). The high-resolution electrospray ionization mass spectrum (HR-ESI-MS) of compound **2** presented a peak at *m*/*z* [M − H]<sup>−</sup> 299.0399 (calculated for [C12H11O9] − 299.0409). The increase in molecular weight of 34 Da compared to Ech A indicates that compound **2** contained two additional hydroxyl groups in the molecule. In its absorption spectrum, there was no absorption band at 468 nm that is characteristic of Ech A, and absorption bands at 256, 321, and 391 nm were present, indicating a decrease in the length of the conjugation chain in the molecule and, therefore, that oxidative changes affected the quinonoid ring of Ech A. According to NMR data, the structure of compound **2** was previously established by us as 7-ethyl-2,2,3,3,5,6,8-heptahydroxy-2,3-dihydro-1,4-naphthoquinone (Figures S12–S17, Supplementary Materials) [23].

**Figure 2.** HPLC profiles of Histochrome (Ech A) oxidation products. Unmarked peaks are natural impurities of the Ech A substance [24].

The presence of four aliphatic hydroxyl groups was confirmed by the preparation of tetramethyl ether of compound **2** (*m*/*z* [M − H]<sup>−</sup> 355.1035, calculated for C16H19O9 − 355.1029) by methylation with methyl iodide according to the procedure in [25] (Figure S3, Table S2, Supplementary Materials), which also confirmed the structure of bis-*gem*-diol for compound **2**.

It is interesting to note that, in the mass spectrum of the chromatographic peak with a retention time of 7.79 min, along with the main peak at *m*/*z* [M − H]<sup>−</sup> 299, there were low-intensity peaks with mass values *m*/*z* [M − H]<sup>−</sup> 263, 281, 237, and 253. These peaks were observed in all cases when the ESI mass spectrum of compound **2** was obtained. On the basis of HR-ESI-MS (Table 1), structures of compounds **3**–**6** were predicted (Figure 3).


**4** 6 C12H10O8 281.0294 281.0303 **5** 2 C11H10O6 237.0397 237.0405 **6** 3 C11H10O7 253.0356 253.0356

**Table 1.** High-resolution electrospray ionization mass spectrometry (HR-ESI-MS) characteristics of the chromatographic peaks of Ech A oxidation products at retention time 7.79 min.

**Figure 3.** Primary oxidation products of Ech A (**1**); structures **3**–**6** were proposed on the basis of HR-ESI-MS data.

Prior to our studies, according to published data, the first oxidation product of Ech A was attributed to structures such as the dihydrate of 5,6,8-trihydroxy-7-ethyl-1,2,3,4-tetrahydronaphthalene-1,2,3,4 -tetraone (**3**) [26] or monohydrate of 2,3-dihydro-2,3,5,6,8-pentahydroxy-2,3-epoxy-7-ethyl-1,4-naph thoquinone (**4**) [27]. These compounds were present in combination with compound **2**. A compound with the brutto-formula C12H10O8 can exist both in the form of structure **4** and in the form of keto-*gem*-diols **4a** and **4b** (Figure 4). The presence of those compounds in the mixture of oxidation products was previously recorded by us using 1H- and 13C-NMR spectroscopy [23].

**Figure 4.** Structures of Ech A oxidation products with brutto-formula C12H10O8.

For compounds **5** and **6**, structures 2,3,4,5,7-pentahydroxy-6-ethylinden-1-one and 2,2,4,5,7 pentahydroxy-6-ethylindane-1,3-dione were predicted, respectively. It is known that di- and polycarbonyl vicinal compounds are prone to hydration; therefore, they are often isolated in the form of *gem*-diols, and, in our case, compound **2** was predominant.

Additionally, structures of compounds **3**–**6** were confirmed by obtaining their methyl derivatives by methylation with both methyl iodide and diazomethane, the MS data for which are provided in Tables S2 and S3 (Supplementary Materials).

Compounds **3**–**6** were extremely reactive, as their formation was accompanied by the formation of an intermediate ene-diol radical, superoxide anion radical, hydroxyl radical, and hydrogen peroxide. It was not possible to isolate them from the mixture of Ech A oxidation products, and they continued the chain reaction of oxidation of bis-*gem*-diol **2**, even if O2 was removed from the reaction medium. In the process of developing technology for the preparation of a Histochrome for injections (0.2 mg/mL Ech A), we observed that even a small amount of O2 entering the drug solution in sealed ampoules led to the appearance of product **2**. Even the subsequent use of an inert medium (argon) did not stop the process of Ech A oxidation, which led to the formation of products **2**–**10** and continued until the complete discoloration of the red-brown Histochrome solution.

Twenty hours after the start of the reaction, the opaque dark red solution became transparent yellow red. HPLC–MS analysis showed that approximately 50% of Ech A was consumed during this time (Figure 2 and Figure S2, Supplementary Materials). Unreacted Ech A was removed from the aqueous solution by extraction with chloroform, and the oxidation products were extracted with ethyl acetate. Low-pressure reversed-phase chromatography on Toyopearl HW-40 gel of ethyl acetate extraction revealed five oxidation products of Ech A with retention times of 7.79 (**2**), 5.32 (**7**), 5.67 (**8**), 6.89 (**9**), and 8.56 (**10**) min (Table 2). The absorption spectra of compounds **7**–**9** contained absorption bands due to π → π\* transitions in the benzenoid core in the region of 310–370 nm, but there were no absorption bands associated with π → π\* transitions in the quinonoid core in the region of 460–540 nm, which indicated a cleavage of the quinonoid ring (Table 2, Figure 5).

**Table 2.** HPLC coupled with diode array detection (DAD) and MS parameters of Ech A (**1**) and its oxidation products **2** and **7**–**10**.


**Figure 5.** Absorption spectra of Ech A (**1**) and its oxidation products **2** and **9**.

It turned out that compound **7** was quite unstable in an acidic environment, and, after vacuum evaporation in fractions with compound **7**, product **11** was formed. According to the ESI-MS spectrum, product **11** had an *m*/*z* 251 [M − H]−, which was 18 Da less than mass of compound **7**. The absorption band at 385 nm in the absorption spectrum of compound **11** indicated a longer π → π\* transition chain in its molecule compared to compound **7**. In the 1H-NMR spectrum of compound **11**, we observed a triplet (δ<sup>H</sup> 1.24) and a quartet (δ<sup>H</sup> 2.78) of an ethyl substituent, a broadened singlet of two hydroxyl groups (δ<sup>H</sup> 5.33), and a singlet of the hydroxyl group bound to carbonyl (δ<sup>H</sup> 12.84) (Figure S18, Supplementary Materials). The 13C-NMR spectrum of compound **11** contained 11 carbon signals: two signals for the ethyl group (δ<sup>C</sup> 12.8 and 17.2), three quaternary carbons (δ<sup>C</sup> 106.3, 108.2, and 121.4), and seven quaternary carbons bound to oxygen (δ<sup>C</sup> 151.2, 158.0, 159.9, 161.6, 171.0, and 177.8) (Figure S19, Supplementary Materials). In the HMBC spectrum of compound **11**, protons of the ethyl group (δ<sup>H</sup> 2.78) showed correlations with δ<sup>C</sup> 121.4, 159.0, and 161.6 (Figure S20, Supplementary Materials). These spectral data were insufficient to establish the structure of **11**; however, it turned out that compound **11** easily formed crystals and, as such, X-ray analysis was used to establish its structure. Compound **11** showed polymorphism, with two types of crystals obtained from the same system of solvents (EtOH–CHCl3 = 1:5 *v*/*v*). Recrystallization from these solvents simultaneously provided crystals as dark-red plates (α-form) and as orange prisms (β-form) (Figure 6). Red crystals were predominant (about 90%). Both crystal forms were a crystalline hydrate of **11** (Tables S4–S6, Supplementary Materials).

**Figure 6.** Molecular structure of echinolactone (**11**) in different crystal types (hydrogen bonds are shown as dotted lines).

The α-form of **11** crystallized as monoclinic system with the space group *P*21/*c* and cell parameters *a* = 4.7823(6) Å, *b* = 7.9520(9) Å, *c* = 14.4705(17) Å, and *Z* = 4, and the final *R*-value was found to be 0.0512 (Table S4, Supplementary Materials). The β-form of **11** crystallized as a triclinic system with the space group *P*¯ı and cell parameters *a* = 4.7823(6) Å, *b* = 7.9520(9) Å, *c* = 14.4705(17) Å, and *Z* = 2, and the final *R*-value was found to be 0.0407 (Table S4, Supplementary Materials).

In the α- and β-forms of molecule **11**, all atoms with the exception of the CH3 carbon atom of the ethyl group C12 were located in the same plane (Figure 6). The deviation from the plane did not exceed 0.138(2) Å. The main difference between the two forms was the dimensional orientation of the hydrogen atom H7 of the carboxyl group, which allowed us to consider the molecules of α- and β-forms as stereoisomers (Figure 6); in the α-form, this atom participates in the formation of an intramolecular hydrogen bond, while, in the β-form, it participates in an intermolecular hydrogen bond. In both crystalline forms of **11**, all the corresponding C–C and C–O bonds had close values (Table S5, Supplementary Materials). The torsion angles of C6–C7–C11–C12 in the α- and β-forms were 96.3(3)◦ and 83.4(2)◦, respectively. The intermolecular hydrogen bonds of molecule **11** with H2O molecules played a decisive role in the formation of crystalline structures. In the α-form, the H2O molecules were linked to each other by O*w*–H···O*w* hydrogen bonds in an infinite chain along the [0 0 1] direction, and they combined isolated **11** molecules into a three-dimensional framework (Figure S21, Supplementary Materials). The coordination number of O atoms of H2O molecules in α-C11H8O7·H2O was 4. In the β-form, molecules of **11** were joined by O7–H7···O3 bonds in pairs into a centrosymmetric bimolecular associate, and H2O molecules distributed their hydrogen bonds only between molecules of **11**, combining pairs into flat ribbons that were infinite along the [1 –1 0] direction (Figure S22, Supplementary Materials). Ribbons were packed in corrugated layers parallel to the plane [0 0 1] (Figure S23, Supplementary Materials). The coordination number of O atoms of H2O molecules in <sup>β</sup>-C11H8O7·H2O was 3. The triclinic <sup>β</sup>-form of C11H8O7·H2O had a slightly higher density (1.664 g/cm3) at a temperature of T = 173(2) K than the monoclinic α-form (1.642 g/cm3) and could formally be considered as more stable.

On the basis of X-ray data, **11** was assigned the structure of 7-ethyl-5,6-dihydroxy-2,3 dioxo-2,3-dihydrobenzofuran-4-carboxylic acid, and this compound was named echinolactone.

Compounds **7** and **8** turned out to be unstable under conditions of repeated chromatographic separation; therefore, to establish their structures, their stable methyl derivatives with *m*/*z* [M − H]<sup>−</sup> 297 and 239, respectively, were obtained by methylation with diazomethane.

On the basis of the 1H- and 13C-NMR spectra of the dimethyl ether of compound **7**, methyl ether of compound **8,** and of compound **9** (Table 3, Figure 7), it could be concluded that these compounds retained the same substituents as the Ech A benzenoid ring: an ethyl substituent, a free hydroxyl group, and hydroxyl groups, the protons of which were bound by an intramolecular hydrogen bond with the corresponding carbonyl groups, but the carboxyl group appeared. The differences in the NMR spectra of the Ech A oxidation products consisted of the chemical shifts of the substituents next to the carboxyl group (Table 3); therefore, to establish the structure of compounds **7**–**9**, it was necessary to establish the nature of these substituents.

**Figure 7.** Structures of dimethyl ether of compound **7**, methyl ether of compound **8**, and of compound **9**.


**Table 3.** NMR data of dimethyl ether of compound **7** (500 MHz for 1H and 126 MHz for 13C, δ, ppm, *J*/Hz), methyl ether of compound **8**, and of compound **9** (700 MHz for 1H and 176 MHz for 13C, δ, ppm, *J*/Hz).

In the dimethyl ether of compound **7**, the proton of the hydroxyl group at C-6 (δ<sup>H</sup> 10.49) was hydrogen-bonded to the carbonyl of the ester group at C-9 (δ<sup>H</sup> 169.1), and the proton of the hydroxyl group at C-3 (δ<sup>H</sup> 11.30) was hydrogen-bonded to the carbonyl of the methylcarboxy group at C-10 (δ<sup>C</sup> 186.8) (Figures S24–S26, Supplementary Materials). The signal at δ<sup>C</sup> 162.9 ppm in the 13C-NMR spectrum corresponded to the carbonyl of the ester group of the methylcarboxy fragment. Two singlets with an integrated intensity of 3H each at δ<sup>H</sup> 3.82 and 3.88 in the 1H-NMR spectrum corresponded to the protons of methoxy groups at C-9 and C-11. In the 13C-NMR spectrum of compound **7** dimethyl ether, there were two corresponding signals at δ<sup>C</sup> 52.2 and 53.0 ppm. According to an analysis of the NMR spectra of the dimethyl derivative, the structure of compound **7** was established as 4-ethyl-3,5,6-trihydroxy-2-oxalobenzoic acid.

The 13C-NMR spectrum of methyl ether of compound **8** contained a signal in a low field at δ<sup>C</sup> 195.2, the chemical shift of which was characteristic for the aldehyde carbon atom (Table 3, Figure S28, Supplementary Materials). The singlet at δ<sup>H</sup> 10.43 in the 1H-NMR spectrum of this compound corresponded to the proton of the aldehyde group. According to NMR data of its methyl derivative (Figures S27–S34, Supplementary Materials), the structure of compound **8** was established as 4-ethyl-2-formyl-3,5,6-trihydroxybenzoic acid.

The 1H-NMR spectrum of compound **9** contained a singlet signal of the aromatic proton at C-6 (δ<sup>H</sup> 6.88), which corresponded to a signal at δ<sup>C</sup> 104.8 ppm in the 13C-NMR spectrum (Table 3, Figures S35 and S36, Supplementary Materials). The chemical shift of the proton of the hydroxyl group at C-5 (δ<sup>H</sup> 7.54) indicated that it was not bound by an intramolecular hydrogen bond as in compound **8**. Thus, the structure of compound **9** was established as 4-ethyl-2,3,5-trihydroxybenzoic acid (Figures S35–S39, Supplementary Materials).

On the basis of the NMR data of compound **10** and its methyl derivative (Figures S40–S48, Supplementary Materials), the structure of **10** was established as 3-ethyl-2,5-dihydroxy-1,4 benzoquinone. This compound was previously described by Moore et al. as a natural pigment of sea urchins of the genus *Echinothrix* [28]. However, it is likely that compound **10** was one of the most stable oxidation products of Ech A obtained by the authors during the storage and repeated chromatographic separation of sea urchin extracts. According to the conditions of our experiment, the oxidation process was stopped when half of the Ech A was oxidized; thus, a very small amount of **10** was isolated.
