2.2.1. 6-Hydroxy-*N*-acetyl-β-oxotryptamine (**1**)

6-Hydroxy-*N*-acetyl-β-oxotryptamine (**1**) was not cytotoxic against neuroblastoma Neuro2a cell up to 100 μM. This compound scavenged DPPH radicals by 48% at 100 μM (Table 2).


**Table 2.** Radical scavenging and cytotoxicity activities of compounds **1**–**6**.

Melatonin-like compound **1** showed a statistically significant reduction of reactive oxygen species (ROS) level on 18% in the neuronal 6-OHDA-treated cells in the in vitro experiment (Figure 2). Melatonin (**1a**), a well-known antioxidant and neuroprotective compound, was used to compare with **1**. It decreased ROS formation in the 6-OHDA-treated neuronal cell stronger in comparison with **1**.

**Figure 2.** Influence of compounds **1**–**6** on reactive oxygen species (ROS) formation in Neuro2a cells treated with 6-hydroxydopamine (6-OHDA) for 30 min. **\*** Difference between data for compounds and for 6-OHDA was statistically significant with *p* ≤ 0.05.

The neuroprotective effect of **1** was shown both in the Neuro2a cells treated 1 h before, as well as 1 h after, adding of 6-OHDA by 23% and 28%, respectively, at a concentration of only 10 μM. Melatonin (**1a**) did not increase the viability of cells treated with neurotoxin in this experiment (Figure 3a). Our experiments showed that 6-hydroxy-*N*-acetyl-β-oxotryptamine (**1**) more effectively protected Neuro2a cells against 6-OHDA-induced neuronal death, in comparison with melatonin (**1a**).

**Figure 3.** Neuroprotective effects of compounds **1**–**6** on Neuro2a cells treated with 6-OHDA (50 μM). All compounds were added to the cell suspension 1 h before treatment with 6-OHDA or 1 h after treatment with 6-OHDA. (**a**) Viability of the 6-OHDA-treated cells incubated with compounds **1** and **1a** at 10 μM; (**b**) Viability of 6-OHDA-treated cells incubated with compounds **2** and **3** at 10 μM; (**c**) Viability of 6-OHDA-treated cells incubated with compounds **4** and **5** at 10 μM; (**d**) Viability of the 6-OHDA-treated cells incubated with compound **6** (1 and 10 μM) \* Difference between data for compounds and for 6-OHDA was statistically significant with *p* ≤ 0.05.

In the PQ-induced PD model, compounds **1** and **1a** (at concentration of 10 μM) were more effective in comparison with their influence in the 6-OHDA-induced model, and decreased ROS formation in the PQ-treated cells by 35% and 22%, respectively (Figure 4). As a result, increase of the PQ-treated cell viability, by 40% and 24%, was observed (Figure 5).

**Figure 4.** Effects of compounds **1**–**6** on ROS formation in Neuro2a cells treated with paraquat (PQ) (500 μM) for 1 h. All compounds were added to the cell suspension 1 h before treatment with PQ. **\*** Difference between data for compounds and for PQ was statistically significant with *p* ≤ 0.05.

**Figure 5.** Neuroprotective effects of compounds **1**–**6** on Neuro2a cells treated with PQ (500 μM). All compounds were added to the cell suspension 1 h before treatment with PQ. **\*** Difference between data for compounds and for PQ was statistically significant with *p* ≤ 0.05.

2.2.2. 3-*O*-Methylorsellinic acid (**2**) and 8-methoxy-3,5-dimethylisochroman-6-ol (**3**)

3-*O*-Methylorsellinic acid (**2**) and 8-methoxy-3,5-dimethylisochroman-6-ol (**3**) were not cytotoxic against neuroblastoma Neuro2a cells up to 100 μM.

3-*O*-Methylorsellinic acid (**2**) at 100 μM scavenged 10% DPPH radicals in our experiments (Table 2). For orsellinic acid and its derivatives, 2,20-azinobis(3-ethylbenzothiozoline-6-sulfonate cation (ABTS·+) scavenger activities were recently reported [32]. 8-Methoxy-3,5-dimethylisochroman-6-ol (**3**) in DPPH assay was not very effective; also, at 100 μM concentration, it reduced the free radical value by 35%, as we reported earlier [33]. Nevertheless, in the 6-OHDA-treated Neuro2a cells, compounds **2** and **3** at a concentration of 10 μM significantly decreased ROS formation by 30% and 45% respectively (Figure 2).

3-*O*-Methylorsellinic acid (**2**) statistically significantly increased 6-OHDA-treated cell viability by 26%, when the compound was added to cells 1 h before adding 6-OHDA. When compound **2** was added to cells 1 h after adding 6-OHDA, it had no neuroprotective effect.

8-Methoxy-3,5-dimethylisochroman-6-ol (**3**) statistically significantly increased the neuroblastoma cell viability in the 6-OHDA-induced PD model by about 55%, regardless of when it was added to cells (Figure 3b). Compounds **2** and **3** were effective at a concentration of only 10 μM.

On the other hand, compounds **2** and **3** did not exhibit any protective effect on based on the viability of cells treated with PQ, despite the fact that they reduced ROS formation in these cells (Figures 4 and 5).
