*3.3. Cellular Toxicity*

Human lung adenocarcinoma epithelial cell line A549 cells were cultured at 37 ◦C in growth medium (DMEM with 10% fetal bovine serum) in 5% CO2, and then seeded into 96-well plates at a density of 1 <sup>×</sup> 105 cells/mL. Once the cells reached 80%–90% confluence, they were treated with or without 10 μM of various compounds at 37 ◦C for 12 h. Next, 10 μL Cell Counting Kit-8 (Dojindo Molecular Technologies, Kumamoto, Japan) solution was added to each well, and the plate was incubated for 2 h at 37 ◦C. Cell viability was determined by measuring the absorbance at 450 nm using a fluorimeter (Varioscan, Thermo, USA).

### **4. Conclusions**

We constructed a chemical library of the side-chain derivatives of eurotiumide A, which is a dihydroisocoumarin-type marine natural product. The antimicrobial evaluation of these compounds was conducted against MSSA, MRSA, and *P. gingivalis*. We discovered several compounds to be effective against these strains; among them, the isopentyl derivative **6** is especially more active against all three strains than **1**. Continuous research to clarify the modes of action of these derivatives is under way in our laboratory.

**Supplementary Materials:** The following are available online at http://www.mdpi.com/1660-3397/18/2/92/s1, 1Hand 13C-NMR charts of all new compounds.

**Author Contributions:** A.N. conceived and designed this research and analyzed the experimental data; H.S., T.N., M.H., S.N., and S.K. (Shuhei Kameyama) prepared compounds and collected their spectral data; S.K. (Sangita Karanjit) checked the experimental data; Y.F., N.H., G.K. and M.O. evaluated the antimicrobial activity; A.N., M.O. and K.N. wrote the paper; all of the authors reviewed and approved the manuscript. All authors have read and agreed to the published version of the manuscript.

**Funding:** This work was supported by JSPS KAKENHI Grant Nos. 17K08365 (A.N.), 18H02657 (M.O.), JP19H02851 (K.N.), and JP16H01156 (K.N.), as well as the Kurita Water and Environment Foundation. We also acknowledge Tokushima University for their financial support of the Research Clusters program of Tokushima University (No. 1802001).

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