*2.3. Predicted Toxicity of Echinochrome A and Its Oxidation Products*

Obtaining information on the toxicity of compounds and their impurities is an important part of the drug design development process. However, for impurities in particular, this information cannot be obtained experimentally. In this case, in silico studies assist in evaluating the results.

The potential toxicity of Ech A and its oxidative degradation products was assessed with the webserver ProTox-II. This virtual lab predicts the toxicity of small molecules on the basis of a total of 33 models for the prediction of various toxicity endpoints such as acute toxicity, hepatotoxicity, cytotoxicity, carcinogenicity, mutagenicity, immunotoxicity, adverse outcome (Tox21) pathways, and toxicity targets [36].

The results of the predicted toxicity of the original Ech A molecule and its oxidative degradation products are shown in Table 4.


**Table 4.** Oral toxicity prediction results for Ech A and its degradation products. LD50, median lethal dose.

The predicted LD50 value for the first major oxidation product **2** was 221 mg/kg (toxicity class III), and the predicted acute toxicity for other oxidation products **7**–**10** was much lower (≥2000 mg/kg, toxicity class IV–V), suggesting they cannot lead to serious toxic effects.

As shown above, structures **2** and **7**–**10** were unambiguously defined as nontoxic and there was no doubt about the predicted toxicity results for these compounds. However, for derivatives of the naphthazarin (5,8-dihydroxy-1,4-naphthoquinone) structure, to which Ech A belongs, it is not so simple. Under various conditions, Ech A (**1**) can exist as a mixture of four tautomeric forms; however, only the 1,4-naphthoquinonoid forms Ech-B (ethyl in the benzene ring) and Ech-Q (ethyl in the quinonoid ring) are energetically favorable (Figure 9) [23,37,38].

**Figure 9.** Tautomeric forms of Ech A: Ech-B (ethyl in the benzene ring) and Ech-Q (ethyl in the quinonoid ring).

As seen in Table 4, the Ech-B and Ech-Q formulas loaded in ProTox-II showed dramatically different results for acute toxicity. For the Ech-B form, a toxic LD50 of 16 mg/kg and toxicity class II were predicted; for Ech-Q, the LD50 was 487 mg/kg and the toxicity class IV was predicted. According to our experimental data for the determination of intraperitoneal acute toxicity of the Ech A substance in outbred mice, the LD50 was found to be 153.7 mg/kg (Table S7, Supplementary Materials) and toxicity class III was determined, which was somewhere between the predicted values for Ech-B and Ech-Q, confirming the benzenoid-quinonoid equilibrium of Ech A. However, the structure of bis-*gem*-diol **2** and of other products **3**–**6** indicated that only one of the possible tautomeric forms (Ech-B) of Ech A was involved in the oxidation process.

We established the experimental cytotoxicity for two compounds, Ech A (**1**) and bis-*gem*-diol **2**. The cytotoxicity of **1** and **2** was estimated by methylthiazolyltetrazolium bromide (MTT) assay using pig embryo kidney (PK) cells and African green monkey kidney (Vero) cells. For Ech A (**1**), 50% inhibition of cell viability was observed at 54.4 mkg/mL and 60.5 mkg/mL in PK and Vero cell lines, respectively [39]. For compound **2**, this value was found to be 140 mkg/mL in Vero cells; thus, it had a weaker toxic effect on normal cells.

With confidence scores of 0.77 and 0.82 for Ech-B and Ech-Q, respectively, it was predicted that these compounds had mutagenic activity (Table 4). However, as shown in a comprehensive study of the mutagenic properties of the Histochrome drug carried out in accordance with the requirements of the Pharmacological Committee of the Russian Ministry of Health, Histochrome in the range of 1.0–10 mg/kg does not have the ability to induce chromosomal damage in the bone marrow cells of C57BL/6 mice, nor does it lead to an increase in the level of spontaneous gene mutations in *Drosophila* or induce gene mutations in *Salmonella typhimurium* (Tables S8–S10, Supplementary Materials). These results allowed us to conclude that Ech A does not exhibit mutagenic activity, at least in the range of therapeutic doses. However, there is published evidence of mutagenic activity of Ech A [33]. In this publication, the source of the drug was not clearly indicated, and neither were its preparation method or purity. This once again confirms that the standardization of drug substances is very important. For the implementation of its medicinal properties, not only the structure of the active substance is important, but also the properties of the drug, determined by the technological process used for its production.
