2.4.2. Antimicrobial Activity

All synthesized tetracyclic conjugates **17a**–**b**–**20a**–**b** and 5-hydroxy-1,4-naphthoquinone (juglone) were screened by the agar diffusion method for antibacterial activity against two strains of Gram-positive bacteria (*S. aureus* ATCC 21027; *B. cereus* ATCC 10702), two strains of Gram-negative bacteria (*P. aeruginosa* ATCC 27853; *E. coli* K-12), and antifungal activity against fungus *C. albicans* KMM 453 from the Collection of Marine Microorganisms (KMM) of the G.B. Elyakov Pacific Institute of Bioorganic Chemistry. Commercial antibacterial drugs (vancomicin and gentamicin) and antifungal (clotrimazol) drugs were used as positive controls. Compound concentrations and diameter of inhibition zone are presented in Table 3.

**Table 3.** Antimicrobial activity (zone inhibition) of oxathiine fused 1,4-naphthoquinonethioglucoside tetracycles.


Zones within 20–40 mm mean strong antibiotic activity, zones up to 20 mm—moderate, ≤10 mm—weak, +—insignificant (− means that the study was not conducted).

Conjugates **17a**–**c** with a 7,10-dimethoxynaphthoquinone core were inactive to Gram-positive and Gram-negative bacteria in concentrations of 100 μg/well. Only one of these conjugates—**17d**, based on l-arabinose, revealed weak activity against *C. albicans*. Conjugates **18c**,**d** with two chlorine atoms in their naphthoquinone skeleton showed moderate activity against Gram-positive strains *S. aureus* and *B. cereus* at a concentration of 100 μg/well, but also were not active to Gram-negative bacteria and fungus *C. albicans*. As evidenced from Table 3, tetracycles **19b**–**d**, bearing a hydroxyl group at C-10 atom of naphthoquinone core, showed various antimicrobial activity levels to Gram-positive bacteria from weak activity for tetracycle **19b**, to strong activity for compound **19d**.

Sugar tetracylic conjugates **20a**–**d**, with a hydroxyl group at the C-7 atom of their naphthoquinone core, constituted the most effective set of antimicrobials among tested compounds **17a**–**d**–**20a**–**d**. All these compounds, **20a**–**d**, have an inhibition zone diameter of 22–40 mm at concentration 100 μg/well and kept the value of inhibition zone in the range of 10 to 25 mm for the concentration 10 μg/well. At a concentration of 10 μg/well, the most effective conjugates with d-xylose **20c** and l-arabinose **20d** showed antimicrobial activity comparable with antibiotics vancomicin and gentamicin, and these ones retained residual effect upon dilution to 1 μg/well for Gram-positive strains. Surprisingly, 5-hydroxy-1,4-naphthoquinone (juglone) showed moderate activity against only *P. aeruginosa*. Thus, heterocyclization with 1-thiosugars leads to an increase of antibiotic activity, especially against the *S. aureus* strain.

The antimicrobial activity for the most active compounds, **19d** and **20a**–**d**, was also determined against *S. aureus* by the minimum inhibitory concentration (MIC) using the broth microdilution method. As it follows from Table 4, juglone derivatives **19d** and **20d** with d-arabinose moiety showed highest antibacterial activity with a MIC of 6.25 μM. The worse activity for **19d** in the agar medium test is probably associated with interactions with agar molecules in the medium. In comparison to non-tumor mouse epithelial Jb6 Cl 41-5a cells, no selective inhibitory activity on bacterial cells was observed, as SI < 1.


**Table 4.** Minimum inhibitory concentration (MIC, μM) and selectivity index (SI) of testing compounds against *S. aureus*.
