3.4.5. Reactive Oxygen Species (ROS) Level Analysis

Cell suspensions (1 × <sup>10</sup><sup>3</sup> cells/well) were incubated during 1 h with 1 and 10 <sup>μ</sup>M solutions of the tested compounds. Non-treated and treated with 6-OHDA at concentration of 50 μM (Sigma-Aldrich, USA) cells were used as negative and positive controls, respectively. The portion (20 μL) of 2 ,7 -dichlorodihydrofluorescein diacetate (H2DCF-DA) stock solution (Molecular Probes, Eugene, OR, USA) with concentration of 100 mM was added in each well and the microplate was incubated for an additional 10 min at 37 ◦C. The intensity of dichlorofluorescin fluorescence was measured at λex = 485 nm, and λem = 518 nm with plate reader PHERAstar FS (BMG Labtech, Ortenberg, Germany). The data were processed by MARS Data Analysis v. 3.01R2 (BMG Labtech, Ortenberg, Germany). In other experiments, cells were incubated with compounds during 1 h. Then, 6-OHDA (50 μM) was added in each well for 30 min and ROS levels were measured. All obtained results were presented as percent of negative control data.

#### **4. Conclusions**

A sponge *Inflatella* sp. contains a variety of oxysterols differing from each other in positions of additional hydroxy or oxo groups of their tetracyclic core and in structures of side chains. Fourteen oxidized sterols, including four previously unknown compounds, were isolated. Structures of new oxysterols have been established. Previously known compounds were structurally identified by comparison of their NMR and MS spectra with those reported in literature. All of the obtained

compounds were studied in the 6-hydroxydopamine-induced cell model of Parkinson's disease. At least, one new oxysterol, (22*E*)-24-*nor*-cholesta-5,22-diene-3β,7β-diol (**4**), showed a substantial activity in this test and might be used for the further studies as a drug candidate.

**Supplementary Materials:** 1H and 13C spectra of new compounds **1**–**4** are available online at http://www.mdpi. com/1660-3397/16/11/458/s1, Figure S1: Experimental Section, Figure S2: HRESI MS Spectra (Positive Ion Mode) of compounds **1**–**4** in CDCl3, Figure S3: 1H NMR (500.13 MHz) spectrum of **1** in CDCl3, Figure S4: 13C NMR (125.76 MHz) spectrum of **1** in CDCl3, Figure S5: COSY NMR (700.13 MHz) spectrum of **1** in CDCl3, Figure S6: ROESY NMR (500.13 MHz) spectrum of **1** in CDCl3, Figure S7: HSQC NMR (700.13 MHz) spectrum of **1** in CDCl3, Figure S8: 1H NMR (700.13 MHz) spectrum of **2** in CDCl3, Figure S9: 13C NMR (125.76 MHz) spectrum of **2** in CDCl3, Figure S10: COSY NMR (700.13 MHz) spectrum of **2** in CDCl3, Figure S11: ROESY NMR (700.13 MHz) spectrum of **2** in CDCl3, Figure S12: HSQC NMR (700.13 MHz) spectrum of **2** in CDCl3, Figure S13: 1H NMR (700.13 MHz) spectrum of **3** in CDCl3, Figure S14: 13C NMR (176.04 MHz) spectrum of **3** in CDCl3, Figure S15: COSY NMR (700.13 MHz) spectrum of **3** in CDCl3, Figure S16: ROESY NMR (700.13 MHz) spectrum of **3** in CDCl3, Figure S17: HSQC NMR (700.13 MHz) spectrum of **3** in CDCl3, Figure S18: HMBC NMR (700.13 MHz) spectrum of **3** in CDCl3, Figure S19: 1H NMR (500.13 MHz) spectrum of **4** in CDCl3, Figure S20: 13C NMR (125.76 MHz) spectrum of **4** in CDCl3, Figure S21: COSY NMR (500.13 MHz) spectrum of **4** in CDCl3, Figure S22: ROESY NMR (500.13 MHz) spectrum of **4** in CDCl3, Figure S23: HSQC NMR (500.13 MHz) spectrum of **4** in CDCl3, S24: HSQC NMR (500.13 MHz) spectrum of **4** in CDCl3, Figure S25: Viability of Neuro2a cells, Figure S26 ROS formation in Neuro2a cells, Figure S27: Viability of Neuro2a cells treated with 6-OHDA.

**Author Contributions:** Conceptualization, S.A.K., E.G.L.; methodology, S.A.K., E.G.L.; formal analysis, S.A.K., E.G.L.; investigation, S.A.K., E.G.L. and E.A.Y.; data curation S.A.K., E.G.L.; writing—original draft preparation, S.A.K., E.G.L.; writing—review and editing, S.A.K., E.G.L. and V.A.S; visualization, S.A.K., E.G.L.; bioassay, E.A.Y; NMR data providing, A.I.K.; HRESI MS and EIMS spectra providing, R.S.P.; supervision, V.A.S.; project administration, V.A.S.; funding acquisition, V.A.S.

**Funding:** This work was supported by the Grant No. 17-14-01065 from the RSF (Russian Science Foundation).

**Acknowledgments:** The study was carried out on the equipment of the Collective Facilities Center, The Far Eastern Center for Structural Molecular Research (NMR/MS) of PIBOC FEB RAS.

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