**2. Results and Discussion**

The ethanol extract of a sponge *Inflatella* sp., collected in the Sea of Okhotsk, was concentrated and partitioned between distilled water and CHCl3. The obtained organic layer was further separated using a combination of column chromatography and normal- or reverse-phase HPLC to yield compounds **1**–**14** (Figure 1). To identify the structures of isolated compounds, along with the absolute stereochemistry, their NMR and HRESI MS characteristics were analyzed and compared with previously published data [13–18].

**Figure 1.** Structures of compounds **1**–**14**.

Compound **1** was isolated as white amorphous powder with molecular formula C28H46O2, established from (+) HRESI MS (*m/z* 437.3393 [M + Na]+) and 13C NMR spectroscopic data (Figures S2

and S4; Table 1). The 1H and 13C NMR spectra (Tables 1 and 2; Figures S3 and S4) brought out signals characteristics of steroidal derivatives. Namely, the 1H NMR (Table 2) and HSQC (Figure S7) spectra allowed to refer signals of five methyl groups as belonging to Me-18 (δ<sup>H</sup> 0.71, s; δ<sup>C</sup> 12.4), Me-19 (δ<sup>H</sup> 0.92, s; δ<sup>C</sup> 14.9), Me-21 (δ<sup>H</sup> 0.96, d, *J* = 6.5 Hz; δ<sup>C</sup> 18.9), Me-26 (δ<sup>H</sup> 1.03, d, *J* = 6.8 Hz; δ<sup>C</sup> 22.1), and Me-27 (δ<sup>H</sup> 1.02, d, *J* = 6.8 Hz; δ<sup>C</sup> 22.2). The chemical shifts at δ<sup>H</sup> 4.12, m; δ<sup>C</sup> 67.4, (HC-3), at δ<sup>H</sup> 5.57, dd, *J* = 9.8, 2.7 Hz; δ<sup>C</sup> 133.5, (HC-6) and at δ<sup>H</sup> 5.63, dd, *J* = 9.8, 1.6 Hz; δ<sup>C</sup> 133.4 (HC-7) along with oxygenated non-protonated carbon signal δ<sup>C</sup> 74.2 (C-5), suggested the 3β,5α-6-ene steroidal core [13,14]. It was supported by close similarity of the corresponding 13C NMR signals of **1** and known cholest-6-ene-3β,5α-diol while the epimoric compound with 3β,5β-6-ene core was quite different [13]. As the signals at δ<sup>H</sup> 4.66 (s) and 4.72 (s) showed an exo-methylene double bond (δ<sup>C</sup> 106.3 and 157.1), the 24(28)-ergostene side-chain was deduced for compound **1**. Therefore, the structure of new compound **1** was established as 24-methylcholesta-6,24(28)-diene-3β,5α-diol.


**Table 1.** 13C NMR data of Compounds **1–4** (δ in ppm, CDCl3).

<sup>a</sup> Spectra recorded at 125.76 MHz; <sup>b</sup> spectra recorded at 176.04 MHz.

Compound **2** has the molecular formula C28H46O2 that was determined by (+) HRESI MS *m/z* 437.3393 [M + Na]+ and 13C NMR (Figures S2 and S9; Table 1). The NMR data of **2** (Tables 1 and 2; Figures S8–S11) also confirmed its steroidal framework. At first, they showed the same type of side-chain in the structure **2** as in **1** by the presence of very close chemical shifts in the spectra of both compounds. The remaining signals were attributed to Me-18 (δ<sup>H</sup> 0.69, s; δ<sup>C</sup> 11.9), Me-19 (δ<sup>H</sup> 1.03, s; δ<sup>C</sup> 20.2), 4α-OH (δ<sup>H</sup> 4.06, m; δ<sup>C</sup> 75.2), and trisubstituted 5(6)-double bond (δ<sup>H</sup> 5.74, dt, *J* = 5.5, 2.2 Hz; δ<sup>C</sup> 117.8, HC-6 and δ<sup>C</sup> 142.2, C-5) of the previously described 4α-hydroxylated tetracyclic system. Really, the both 4α-hydroxy- and 4β-hydroxycholesterols were obtained and well characterized during the investigation of cholesterol autoxidation [15]. As a result of the thorough comparison of 1D and 2D

NMR experimental data of **2** with published values, the structure of this new oxysterol was deduced as 24-methylcholesta-5,24(28)-diene-3β,4α-diol.


**Table 2.** 1H NMR data of Compounds **1–4** (δ in ppm, *J* in Hz, CDCl3).

<sup>a</sup> Assignments were made with the aid of the 1H-1H COSY and HSQC spectra; <sup>b</sup> spectra recorded at 500.13 MHz; <sup>c</sup> spectra recorded at 700.13 MHz.

The structure of the compound **3** corresponds to the molecular formula C26H42O2 (HRESI MS *m/z* 409.3081 [M + Na]+) (Figures S2 and S14; Table 1). It has a common Δ5-3β,7α-diol steroidal core that has been confirmed by the signals of Me-18 (δ<sup>H</sup> 0.70, s; δ<sup>C</sup> 11.9), Me-19 (δ<sup>H</sup> 1.00, s; δ<sup>C</sup> 18.2), two methines, connected with hydroxy groups (δ<sup>H</sup> 3.59, m; δ<sup>C</sup> 71.4, HC-3 and δ<sup>H</sup> 3.85, br s; δ<sup>C</sup> 65.3, HC-7), and the 5(6)-double bond (δ<sup>H</sup> 5.60, dd, *J* = 5.3, 1.6 Hz; δ<sup>C</sup> 123.9, HC-6 and δ<sup>C</sup> 146.2, C-5) [16]. However, the molecular formula and characteristic signals of the 22*E*-double bond (δ<sup>H</sup> 5.18, dd, *J* = 15.3, 6.6 Hz; δ<sup>C</sup> 133.6, HC-22 and δ<sup>H</sup> 5.27, dd, *J* = 15.3, 6.6 Hz; δ<sup>C</sup> 134.9, HC-23), as well as Me-26,27 signals (δ<sup>H</sup> 0.94, d, *J* = 6.7 Hz; 6H; δ<sup>C</sup> 22.7) showed that this new steroid was characterized by the presence of the 22*E*-unsaturated 24-*nor*-side chain [17] (Tables 1 and 2; Figures S13–S18). Finally, the structure of new oxysterol **3** was determined as (22*E*)-24-*nor*-cholesta-5,22-diene-3β,7α-diol (**3**) that was also in good agreement with ROESY and HMBC data (Figures S16 and S18).

The C26H42O2 molecular formula ((+) HRESI MS *m/z* 409.3081 [M + Na]+) of compound **4** together with similar NMR spectral characteristics (Tables 1 and 2; Figures S2, S19–S24) suggested the presence of a side-chain the same as in compound **3**. The core parts of these compounds also demonstrated a set of close 1H NMR signals except for the chemical shift values, constants, and multiplicity of H-6 (δ<sup>H</sup> 5.29, t, *J* = 2.2 Hz) and H-7 (δ<sup>H</sup> 3.85, dt, *J* = 8.3, 2.2 Hz). Both mentioned signals were similar to those described previously [16,18] and suggested the 7β-OH stereochemistry in **4**. All these data along with confirmation by ROESY and HMBC spectra (Figures S22 and S24) allowed to establish the (22*E*)-24-*nor*-cholesta-5,22-diene-3β,7β-diol (**4**) structure of this new oxysterol.

Additionally, the remaining ten isolated compounds **5**–**14** were identified as known oxygenated steroidal derivatives by spectroscopic methods and comparison with reported data including NMR spectra [16,18–20]. Most of them were described as naturally occurring metabolites possessing diverse biological activities. Thus, (22*E*)-cholesta-5,22-diene-3β,7α-diol (**5**) was originally isolated from the marine sponge *Cliona copiosa* [16], and demonstrated anti-inflammatory, analgesic and gastroprotective activities as a component of ethanolic fraction of gorgonian *Eunicella singularis* [21]. The (22*E*)-cholesta-5,22-diene-3β,7β-diol (**6**) [16] deterred starfish predators [22]. Selective activity towards DNA repair-deficient yeast mutants and cytotoxicity towards wild-type P-388 murine leukemia cells [23] were showed for 24-methylene-5-cholestene-3β,7α-diol (**7**) [18]. Moreover, the compound **7** was noted as a potential drug development candidate for Alzheimer's disease due to inhibitory potential against butyrylcholinesterase (BuChE) with IC50 9.5 μM [24]. The 24-methylene-5-cholestene-3β,7β-diol (**8**) [18] was completely inactive in the screening for DNA-damaging agents in the RAD 52 yeast assay (while its epimer **7** was active with IC50 7 μg/mL), and showed moderate cytotoxic activity (IC50 31 μM) in the Vero cell assay, indicating that 7β-OH compound acted by a different mechanism in comparison with its α-counterpart [25]. Both compounds **7** and **8** were also noted as constituents of the royal jelly of honeybees [26].

To the best of our knowledge there are not any reported bioassay results in relation to (22*E*,24*S*)-24-methylcholesta-5,22-diene-3β,7α-diol (**9**) [16], (22*E*,24*S*)-24-methylcholesta-5,22-diene-3β,7β-diol (**10**) [16,18], and (22*E*,24*R*)-24-methylcholesta-5,22-diene-3β,7α-diol (**11**) [16,19]. 7β-Hydroxycholesterol (**12**) isolated from the Red Sea grass *Thalassodendron ciliatum* displayed an inhibitory activity against breast carcinoma cell line MCF-7 (IC50 18.6 ± 0.72 μM) and liver carcinoma cell line Hep G2 (IC50 25.4 ± 0.38 μM). However, it did not show the anti-inflammatory action on carrageenan-induced rat hind paw edema model [27]. When human THP-1 macrophages were exposed with an atheroma-relevant mixture of 7β-hydroxycholesterol (**12**) and 7-ketocholesterol followed by proteome analysis, the alterations in macrophage proteome were indicated with a significant differential expression of 19 proteins [28].

3β-Hydroxy-24-methylene-5-cholesten-7-one (**13**) [18] exhibited the potent inhibitory activity on the interleukin-6 production, with 54.0% inhibition at 10 μM and IC50 9.4 ± 1.2 μM [29]. 24-Methylenecholest-4-ene-3β,6β-diol (**14**) [20] demonstrated the cytotoxic activity against the leukemia P-388 cell line with an IC50 1 μg/mL [30].

Encouraged with the short literature review presented above, that shows different attractive bioactivities of oxysterols, we have made an attempt to evaluate the action of the isolated compounds **1**–**14** on viability of Neuro2a cells and reactive oxygen species (ROS) formation in these cells. In fact, neuroblastoma cells treated by 6-hydroxydopamine (6-OHDA) are used as a cell model of PD [31].

Compounds **4**, **5**, **9** and **12** did not show any notable effects on Neuro2a cell viability. Compounds **1**, **2**, **7**, **8**, **13** and **14** demonstrated slight cytotoxic activity at concentration 100 μM and decreased viability of Neuro2a cells on 25%, 17%, 44%, 27%, 38% and 33%, respectively. It is of interest that all of them have the same structural peculiarity, being the 24 (28)-unsaturated derivatives of ergostane series. No compounds decreasing cell viability more than 50% were found. At concentration of 10 μM, the oxysteroids **1**–**14** were non-toxic against these neuronal cells, and were used in next experiments at the non-toxic concentrations (Figure S25). Moreover, compounds **3**, **6**, **10** and **11** increased the viability of Neuro 2a cells in comparison with non-treated cells, when MTT cell viability test was used (Figure 2a and Figure S25).

**Figure 2.** Compounds **3**, **6**, **10** and **11**: (**a**) Caused a statistically significant overestimation of MTT reduction in MTT cell viability assay; (**b**) did not statistically significant affect activity of nonspecific esterase in fluorescein diacetate cell viability test. \* Statistically significant differences (*p* ≤ 0.05) between results for control cells and cells incubated with these compounds.

Applied MTT assay is one of the most widely exploited approaches in research for measuring cell proliferation, viability and drug cytotoxicity. In living cells, the water-soluble yellow dye MTT is reduced to a dark purple (blue-magenta) colored formazan precipitate, which can be analyzed colorimetrically after dissolving in an organic solvent. It was shown, that the MTT reduction site is not only mitochondria. Non-mitochondrial, cytosolic and microsomal MTT reduction makes the major contribution to an overall reduction. Changes in the activity of dozens of the mitochondrial and non-mitochondrial oxidoreductases, cellular metabolic and energy perturbations, and oxidative stress may significantly impact the MTT assay read out [32].

To study action of the tested compounds on cells in details, we additionally used fluorescein diacetate (FDA) assay based on nonspecific esterase activity measuring and thus examined the influence of compounds **3**, **6**, **10** and **11** on proliferation or/and viability of Neuro 2a cells [33]. Compounds **3**, **6**, **10** and **11** did not increase the fluorescence intensity in FDA assay in comparison with control and therefore did not influence significantly on nonspecific esterase activity in Neuro2a cells (Figure 2b). Hence, we could conclude that the observed increasing of MTT reduction was not caused by the influence of tested compounds on cell proliferation.

In fact, the overestimation in MTT assay of the compounds **3**, **6**, **10** and **11** could be caused by alternative metabolic processes. For example, the overestimation reported for rottlerin was explained by dissipation of the inner mitochondrial membrane potential, acceleration of electron transfer and increasing of dehydrogenases activity, oxygen consumption and NADH oxidation [33]. On the other hand the polyphenolic antioxidant resveratrol exhibited increasing of MTT-reducing activity without a corresponding increasing of living cells number [34]. The ability of resveratrol to down-regulate NADPH-oxidase leading to decreased ROS production and thereby provide a protective effect in cardiovascular and neurodegenerative diseases is also well known [35].

As the above reviewed reports described the influence of compounds on intracellular ROS formation, we investigated the effect of compounds **1**–**14** on ROS formation in Neuro 2a cells by short-time 2 ,7 -dichlorodihydrofluorescein diacetate (H2DCF-DA) test. Oxysterols **1**–**4**, **8**, **10** and **13** at concentrations of 1 or/and 10 μM slightly decreased the ROS level in Neuro2a cells by 12–16%, while the increasing in ROS formation was not detected for all the studied compounds (Figure S26).

PD is the one of the most common age-related motoric neurodegenerative disease, regardless of countries and regions [36]. Pathogenesis of PD includes neuronal death as a result of oxidative stress involved intracellular level of ROS increasing. In this reason, compounds exhibited ROS-scavenger activities could be interesting as neuroprotective agents. All isolated compounds **1**–**14** were studied in 6-OHDA-induced Neuro2a cell model of PD (Figure S26). Only compounds **3**, **4** and **11** affected on viability of 6-OHDA-treated cells (Figure 3a) and ROS formation in these cells (Figure 3b).

**Figure 3.** Influence of compounds **3**, **4** and **11** in 6-OHDA-treated Neuro2a cells: (**a**) on cell viability; (**b**) on ROS formation. **\*** Statistically significant differences (*p* ≤ 0.05) between results for 6-OHDA-treated cells and cells incubated with compounds.

As a result, compound **3** increased the viability of 6-OHDA-treated cells by 18% (at the dose of 10 μM) and 22% (1 μM), while compounds **4** and **11** increased cell viability by 28% (10 μM) and 18% (10 μM), correspondingly. All these compounds decreased ROS formation in 6-OHDA-treated cells to normal value in similar manner. Thus, compound **4** exhibits the essential neuroprotective activity in 6-OHDA-induced model of Parkinson's disease, probably due to ROS scavenging effect. Oxysterols **3**, **6**, **10** and **11** may positively influence on metabolic processes in the Neuro2a cells because they show the overestimation of survival in MTT assay.
