**1. Introduction**

Currently, Parkinson's disease is one of the most common age-related motoric neurodegenerative diseases, regardless of countries and regions. This disease is characterized clinically by resting tremor, bradykinesia, rigidity, and postural instability, that significantly worsen the life quality of patients [1]. Pathogenesis of PD includes neuronal death as a result of oxidative stress involving the increase in the intracellular level of reactive oxygen species (ROS) and reactive nitrogen species. The hyperproduction of ROS may cause several forms of cell damage, such as increasing DNA damage, lipid, and protein peroxidation which can promote mitochondrial injury. In addition, some of the

studies show that ROS can cause mitochondrial dysfunction and activation of apoptosis-related death signaling, which lead to neuronal cell death. These findings show the requirement of using antioxidants as a therapeutic intervention in PD in addition to other protective agents [2]. To estimate the antioxidative properties of neuroprotective agents, both cell-free and cell assays may be used. One of the most popular cell-free tests in natural product antioxidant studies is the 2,2-diphenyl-1-picrylhydrazyl (DPPH) free radical scavenging assay [3]. A widely used cell test is ROS level determination using 2 ,7 -dichlorofluorescin diacetate, that is deesterified intracellularly, and turns into the highly fluorescent 2 ,7 -dichlorofluorescein upon oxidation [4].

PD etiology may be linked to several factors, including genetic susceptibility and environmental elements. Regarding environmental factors, several dopaminergic neurotoxins, including 6-hydroxydopamine (6-OHDA) and 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), have been identified. Moreover, some pesticides/herbicides, such as rotenone, paraquat (PQ), maneb (MB), and mancozeb (MZ), cause neurotoxicity, and induce a PD-like pathology. As a result, 6-OHDA and MPTP are common models used in PD research, and pesticide-based approaches have become secondary models of study [5,6].

It is known that marine fungi produce polyketides with antioxidant and neuroprotective properties [7–9]. For example, gentisyl derivatives aspergentisyls A and B, and auroglaucin-related compounds from the marine-derived *Aspergillus glaucus* exhibited a strong radical-scavenging activity in DPPH test, with an IC50 of 7.6–24.2 μM [7]. Terrestrol G, a dimeric derivative of gentisyl alcohol from the marine sediment-derived fungus *Penicillium terrestre*, exhibited DPPH-scavenging properties with an IC50 of 4.1 μM [10].

Diketopiperazines are widespread marine fungal products with different biological activities, including free radical scavenging and neuroprotection [11,12]. Gliotoxin from *Pseudallescheria* sp., neoechinulin E, and cryptoechinulin D, exhibited DPPH scavenger activity with IC50 values of 5.2, 46.0, and 23.6 μM, respectively [13,14]. Neoechinulin A from *Eurotium rubrum* protected PC12 cells from the cytotoxic influence of MPTP neurotoxin [15].

Some other metabolites of marine fungi also show a neuroprotective effect in the in vitro and in vivo models of Parkinson's disease [16]. Xyloketal B, from *Xylaria* sp., scavenged free radicals in DPPH assay, and protected PC12 cells against ischemia-induced cell injury and MPTP-induced neurotoxicity [17]. About 40 synthetic derivatives of xyloketal B were investigated in various in vivo Parkinson's models, and some of them showed significant activities [18]. Secalonic acid A from *Aspergillus ochraceus* and *Paecilomyces* sp. protected against MPTP-induced dopaminergic neuronal cell death in mouse PD model, in nigral neurons, and SH-SY5Y cells [19].

In this paper, we described the isolation and identification of new alkaloid and known polyketides from the marine fungus *Penicillium* sp. KMM 4672, known diketopiperazine alkaloid from *Aspergillus flocculosus*, polyketides reported earlier from *Aspergillus* sp. KMM 4676, as well as free radical scavenging and neuroprotective activities of these compounds in two cell models of Parkinson's disease induced by 6-hydroxydopamine and paraquat.

#### **2. Results and Discussion**

#### *2.1. Isolation and Identification of Compounds*

Recently, we have found new natural compounds whose structures have not been established, possibly due to their insufficient content, along with several new and known compounds from a marine algicolous fungus *Penicillium* sp. KMM 4672 [20,21]. A repeated cultivation of this fungus, in the same conditions, was carried out to obtain a sufficient amount of unidentified compounds. As a result, these compounds were identified as new melatonin derivative (**1**), known *o*-orsellinic acid (**2**) and isochromene (**3**) derivatives (Figure 1).

**Figure 1.** The structures of investigated compounds.

6-Hydroxy-*N*-acetyl-β-oxotryptamine (**1**) was isolated as a white solid. An (–)HRESIMS spectrum (Figure S8) of compound **<sup>1</sup>** contains a [M−H]– pseudomolecular peak at *<sup>m</sup>*/*<sup>z</sup>* 231.0772, which indicated a molecular formula of C12H12N2O3 (calcd for C12H11N2O3,231.0775), which corresponded to six double-bond equivalents. A careful inspection of NMR data of **1** (Table 1 and Figures S1–S7) revealed the presence of one acetyl methyl, one methylene, four sp2-methines, four quaternary sp2-carbons, one keto-group, and one amide carbonyl. In addition, the 1H NMR spectrum contains the signal of three heteroatom protons. The coupling constant values of NH-1 (*δ*<sup>H</sup> 11.55, d, *J* = 2.9 Hz), H-2 (*δ*<sup>H</sup> 8.17, d, *J* = 2.9 Hz), H-4 (*δ*<sup>H</sup> 7.89, d, *J* = 8.6 Hz), H-5 (*δ*<sup>H</sup> 6.68, dd, *J* = 8.6, 1.7 Hz), and H-7 (*δ*<sup>H</sup> 6.80, d, *J* = 1.7 Hz), together with the HMBC correlations from NH-1 to C-3a (*δ*<sup>C</sup> 118.3), from H-2 to C-3a and C-7a (*δ*<sup>C</sup> 137.6), from H-4 to C-3 (*δ*<sup>C</sup> 114.1), C-6 (*δ*<sup>C</sup> 154.0) and C-7a, from H-5 to C-3a and C-7 (*δ*<sup>C</sup> 97.1), from H-7 to C-3a and C-5, and from 6-OH (*δ*<sup>H</sup> 9.14) to C-5, C-6, and C-7, established the indole moiety with OH group at C-6. This suggestion was additionally proved by ROESY correlation H-1/H-7. The HMBCs from methylene H-2 (*δ*<sup>H</sup> 4.38) to C-1 (*δ*<sup>C</sup> 189.9) and C-4 (*δ*<sup>C</sup> 169.3), from H-3 (*δ*<sup>H</sup> 8.06) to C-4 , and from H-5 (*δ*<sup>H</sup> 1.90) to C-4 showed the side chain structure. The location of the side chain at C-3 was revealed by the ROESYs of H-2 with H-2 and H-4. Thus, the structure of **1** was elucidated to be very close to that of known *N*-acetyl-β-oxotryptamine [22]. Similar melatonin-like compounds are usually isolated from several bacterial species [23,24]. Recently, *N*-acetyl-β-oxotryptamine was reported from the medicinal basidiomycete *Inonotus vaninii* [25], and from ascomycete *Scopulariopsis* sp. [26]. To our knowledge, this study is the second case of isolation of related compounds from microfilamentous fungi.


**Table 1.** 1H and13C NMR data (700/176 MHz, *δ* in ppm, DMSO-*d*6) for 6-hydroxy-*N*-acetylβ-oxotryptamine (**1**).

Together with melatonin-related **1**, the well-known fungal polyketides 3-methylorsellinic acid (**2**) and 8-methoxy-3,5-dimethylisochroman-6-ol(**3**) were isolated from this fungus. Their structures were identified by comparing the NMR and MS data (Figures S9–S13) with previously reported data [27,28].

The chemical composition of extract of an ascidian-derived fungus *Aspergillus* sp. KMM 4676 was reported earlier [29,30]. *p*-Terphenyl polyketides, candidusin A (**4**) and 4"-dehydroxycandidusin A (**5**), were major metabolites of this fungus.

The 2,5-diketopiperazine alkaloid mactanamide (**6**) was isolated from the Vietnamese sediment-derived fungus *Aspergillus flocculosus*. The NMR data (Figures S14 and S15) for this compound were identical with earlier published data [31]. It is only the third case of isolation this compound.
