Agar Diffusion Method

Test substances were dissolved in DMSO at concentrations of 1.0, 0.1, and 0.01 mg/mL. All strains were grown on trypticase-soy agar TSA (BBL) at 37 ◦C. After overnight incubation of each test organism in Petri dishes with the same medium, wells with a diameter of 7 mm were cut and 100 μL of the test substance solution was added. After incubation of the dishes at 37 ◦C for 24 h (for bacteria) and 48 h (for the fungus) at 28 ◦C, the diameters of the growth inhibition zones of pathogenic microorganisms (mm) were measured, including the diameter of the wells. Commercial antibacterial drugs (vancomicin and gentamicin) and antifungal (clotrimazol) drugs were used as positive controls.

## Determination of Minimum Inhibitory Concentration (MIC)

Stocks of substances were prepared in DMSO at a concentration of 10 mM. All studied compounds were tested in concentrations from 1.5 μM to 100 μM using twofold dilution. All tested compounds were add to the wells of the plates in a volume of 20 μL diluted in PBS (DMSO concentration < 1%). The bacterial culture of *S. aureus* ATCC 21027 (Collection of Marine Microorganisms PIBOC FEB RAS) was cultured in a Petri dish at 37 ◦C for 24 h on solid medium with the following composition; pepton—5.0 g/L, K2HPO4—0.2 g/L, glucose—1.0 g/L, MgSO4—0.05 g/L, yeast extract—1 g/L, agar—16.0 g/L, and distilled water—1.0 L. The pH of the medium was adjusted to 7.2–7.4 with NaOH solution.

The antimicrobial activity of the compounds was determined by the minimum inhibitory concentration (MIC) according to the method adopted by the Clinical and Laboratory Standards Institute (CLSI). Methods for dilution antimicrobial susceptibility tests for bacteria that grow aerobically followed the approved standard—tenth edition. CLSI document M07-A10, PA: Clinical and Laboratory Standards Institute; 2015, with a slight modification of the medium. The assays were performed in 96-well microplates in appropriate broth (pepton—5.0 g/L, K2HPO4—0.2 g/L, glucose—1.0 g/L, MgSO4—0.05 g/L, yeast extract—1 g/L, casein hydrolyzate—2.5 g/L, distilled water—1L). The *S. aureus* bacterial suspension had a concentration of 10 <sup>×</sup> 10<sup>9</sup> CFU/mL in the medium, 180 <sup>μ</sup>L was then added to each well of the microplates, followed by incubation at 37 ◦C for 24 h. The MIC value is defined as the lowest concentration of compounds resulting in the complete inhibition of bacterial growth by measuring the absorbance at 600 nm with a microplate reader (BioTek, Winooski, Vt, USA). All experiments were performed twice in triplicate. Gentamicin was used as a positive control in concentration 1 mg/mL; 1% DMSO solution in PBS as a negative. The selectivity index for S. *aureus* has been defined as the ratio of EC50 for normal cells and the MIC value for the bacterial culture (SI = EC50/MIC).

#### **4. Conclusions**

In summary, a series of new tetracyclic oxathiine-fused quinone-thioglycoside conjugates based on biologically active 1,4-naphthoquinones (chloro-, hydroxy-, and methoxysubstituted) have been synthesized, characterized, and evaluated for their cytotoxic and antimicrobial activities. It was shown that the most active compounds are tetracyclic conjugates of juglone (5-hydroxy-1,4-naphthoquinone), which showed high cytotoxic activity with EC50 in the range of 0.3 to 0.9 μM for all cancer and noncancer cell lines. Furthermore, for the first time the antimicrobial activity for this type of compounds was evaluated by the agar diffusion method. Among the tested conjugates, the activity of juglone derivatives with a d-xylose or l-arabinose moiety and hydroxyl group at C-7 position of naphthoquinone core was comparable with antibiotics vancomicin and gentamicin against Gram-positive bacteria *S. aureus* and *B. cereus*. In liquid media, the juglone-arabinosidic tetracycles showed highest activity with a MIC of 6.25 μM against *S. aureus* strain. Thus, the positive effect of heterocyclization with mercaptosugars on cytotoxic and antimicrobial activity for a group of 1,4-naphthoquinones was shown. The effect of chloro-, hydroxy-, and metoxysubstituents on teracycles activity was also studied and a significant effect of the hydroxy group on activity was shown. It can be assumed that further modification of such tetracycles may lead to new compounds with selectivity for cancer cells and great antimicrobial activity. **Supplementary Materials:** 1H and 13C-NMR spectra for synthesized compounds are available online.

**Author Contributions:** Y.E.S. and S.G.P. performed the chemical experiments; S.G.P. and Y.E.S. conceived and designed the chemical experiments; V.A.D. carried out the NMR analyses and analyzed the data, R.S.P. performed MS measurements; E.S.M., L.S.S., E.A.C. and A.R.C. performed bioactivity testing supervised by D.L.A. and V.V.M.; Y.E.S.—original draft preparation, S.G.P.—review and editing, D.L.A. and V.V.M.—supervision. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was funded by the Russian Foundation for Basic Research (Grant № 18-33-00492\_mol\_a) and particularly by the Russian Science Foundation (Grant № 19-14-00047).

**Acknowledgments:** We thank V. P. Glazunov for recording IR spectra.

**Conflicts of Interest:** The authors declare no conflict of interest.
