Sclerotioloids A–C: Three New Alkaloids from the Marine-Derived Fungus Aspergillus sclerotiorum ST0501

Alkaloids, as one of the largest classes of natural products with diverse structures, are an important source of innovative medicines. Filamentous fungi, especially those derived from the marine environment, are one of the major producers of alkaloids. In this study, three new alkaloids, sclerotioloids A–C (1–3), along with six known analogs (4–9), were obtained under the guidance of the MS/MS-based molecular networking from the marine-derived fungus, Aspergillus sclerotiorum ST0501, collected from the South China Sea. Their chemical structures were elucidated by comprehensive analysis of the spectroscopic data, including 1D and 2D NMR and HRESIMS. Additionally, the configuration of compound 2 was unambiguously determined by X-ray single crystal diffraction, and that of compound 3 was determined by the TDDFT-ECD approach. Sclerotioloid A (1) represents the first example of 2,5-diketopiperazine alkaloid with a rare terminal alkyne. Sclerotioloid B (2) showed the inhibition of NO production induced by lipopolysaccharide (LPS), with an inhibition rate of 28.92% higher than that of dexamethasone (25.87%). These results expanded the library of fungal-derived alkaloids and further prove the potential of marine fungi in the generation of alkaloids with new scaffolds.


Introduction
In the past few years, natural products isolated from marine-derived fungi have aroused great interest due to their unique structures, interesting the pharmacological and biological properties [1]. Among the marine-derived fungi, Aspergillus is the largest source of fungal natural products. The growth of marine-derived fungi research rose from 2015 to 2019, with natural products from marine-derived fungi sources already accounting for nearly half (47%) of the total number of new marine natural products reported by 2019 [2]. Additionally, a large part of the compounds isolated from Aspergillus show anti-bacterial, anti-cancer, anti-inflammatory, anti-tuberculosis, and cytotoxic activities [3]. Metabolites of marine-derived fungi, such as polyketides, alkaloids, terpenes, lactones and peptides, are rich sources of biologically active natural products [4]. It was reported that alkaloids represent about 18% of all the marine natural products in 2019 [2]. Recent studies of marine fungal metabolites looking for bioactive compounds suggest that they have the potential to become a source of new drugs [5,6]. For example, plinabulin is a 2,5-diketopiperazine alkaloid isolated from the marine-derived fungus Aspergillus sp., which was first used in clinical trials for the treatment of non-small-cell lung cancer (NSCLC) [7]. Ecteinascidin-743 (ET743) is a novel anti-tumor drug on the market [8]. Aspergillus sclerotiorum have potential to biosynthesize alkaloids, though alkaloids are the most bioactive metabolites that possess marked antimicrobial and cytotoxic activities [9]. Sclerotiamides C and F, which are isolated from the A. sclerotiorum LZDX-33-4, a fungus derived from a gorgonian coral (LZDX-33), showed a significant inhibitory effect against a panel of tumor cell lines with IC 50 values ranging from 1.6 to 7.9 µM [10]. A new cytotoxic indole-3-ethenamide isolated from A. sclerotium PT06-1 showed moderate (3.0 mM) and weak (27 mM) cytotoxicity toward A-549 and HL-60 cells, respectively [11].
During our ongoing research for new bioactive secondary metabolites from marinederived fungi in the South China Sea [12][13][14][15], plenty of marine-derived alkaloids have been isolated. For example, emestrins L and M were obtained from the marine-derived fungus A. terreus RA2905. Emestrin M displayed antibacterial activity against Pseudomonas aeruginosa ATCC 27,853, with a minimum inhibitory concentration (MIC) value of 64 µg/mL [16]. Three new indole-diketopiperazine alkaloids, spirotryprostatin G and cyclotryprostatins F and G, were obtained from fungal strain HBU-136, and among them, spirotryprostatin G exhibited cytotoxicity against the HL-60 cell line, with an IC 50 value of 6.0 µM, while cyclotryprostatins F and G exhibited cytotoxicity against the MCF-7 cell line, with IC 50 values of 7.6 and 10.8 µM, respectively [17].
The fungal strain A. sclerotiorum ST0501 was isolated from the inner part of an unidentified sponge, GDST-2013-05, collected from the South China Sea. In the previous work, three new 2,5-diketopiperazine alkaloids, speramide C, 3,21-epi-taichunamide F and 2-epi-amoenamide C, were obtained from the same fungus strain. Speramide C represents the first prenylated indole alkaloid with an ethylene oxide ring on the isopentenyl side chain [18]. However, in this paper, we discovered three new alkaloids (1-3) and six known analogs (4-9) from ethyl acetate extract of A. sclerotiorum ST0501 based on the MS/MS molecular network. Herein, we report the isolation, structure elucidation and biological activities of these isolated compounds.

Elucidation of Chemical Structures
In order to fully explore the alkaloids of ST0501, molecular networking analysis was performed on the crude ethyl acetate extract (EtOAc) of solid rice and wheat cultures of A. sclerotiorum ST0501. The results show that there are signals associated with alkaloids. The relationship between compound 2 and compound 6 is close. Based on the indication of molecular networking (Figure 1), three unreported (1-3) and six previously described alkaloids (4-9) (Figure 2) were isolated by using a combination of column chromatography including silica gel, octadecylsilyl, Sephadex LH-20 columns and semi-preparative HPLC. The structures of gartryprostatin C (4) [19], stephacidin A (5) [20], sclerotiamide (6) [21], notoamide B (7) [22], speramide C (8) [18] and stephacidin B (9) [23] were elucidated by comparison of their spectroscopic data with those previously reported in the literature. Hz, H-6) and one acetylenic proton (δ H 3.10, t, J = 2.4 Hz, H-9). The 13 C NMR spectrum, in combination with HSQC spectra, revealed the presence of fourteen carbons, including two amide carbonyl (δ C 165.7, C-5 and δ C 163.4, C-2), six aromatic carbons (δ C 133.4, C-11; δ C 129.4, C-13 and C-15; δ C 128.8, C-14; δ C 128.6, C-12 and C-16), two olefinic carbons (δ C 129.9, C-3 and δ C 120.8, C-10), two alkynyl carbons (δ C 78.2, C-8 and δ C 74.5, C-9) and two methylene sp 3 (δ C 44.5, C-6 and δ C 32.9, C-7). The correlations between H-1 and H-6 in the 1 H-1 H COSY spectrum and the HMBC correlations between NH-1 and C-3 and between H-6 and C-2/C-5 revealed the existence of a 2,5-diketopiperazine ring. Furthermore, the HMBC correlations between H-10 and C-2/C-3/C-16 indicated that the benzene ring was connected to C-3 of the 2,5-diketopiperazine ring through C-10. Based on the HMBC correlations between H-7 and C-3/C-5/C-9, the alkynyl group was connected to N-4 of the 2,5-diketopiperazine ring ( Figure 3). According to the (Z)-vinyl proton chemical shifts of H-7 (δ H 7.03) in nocazine C [24] and H-7 (δ H 6.54) in (3S,6E)-3benzyl-6-benzylidenepiperazine-2,5-dione [25], the ∆ 3,10 double bond in 1 was deduced to have a Z configuration due to the relative downfield shift of H-10 (δ H 7.10), where the (Z)-vinyl proton had a larger downfield shift than that of the (E)-vinyl proton because of the deshielding effect of the carbonyl in 2,5-diketopiperazine [24][25][26][27]. Therefore, the structure of 1 was elucidated.     Sclerotioloid A (1) was isolated as a yellow powder. Its molecular formula C14H12N2O2 was deduced from its HRESIMS m/z 263.0794 [M+Na] + (calcd for C14H12N2O2Na, 263.0797) and 1D NMR data analysis, suggesting ten degrees of unsaturation. The IR spectrum of 1 featured typical absorption bands for alkyne (2361 cm −1 ) and  164.5, C C-12, C- 13 12 52.5, OCH3 3.81 (s) C- 11 13 139.5 CH 7.84 (s) C-11, C-14 C- 15 14 131.5, C C- 13 15 130.    Sclerotioloid B (2) was also isolated as a yellow powder. The HRESIMS spectrum of 2 indicates a molecular formula of C 17 H 18 N 2 O 4 based on the prominent peak [M+Na] + at m/z 337.1160 (calcd for C 17 H 18 N 2 O 4 Na, 337.1164), as well as 1D NMR data analysis, requiring ten degrees of unsaturation. The IR spectrum of 2 featured typical absorption bands for alkyne (2361 cm −1 ) and conjugated ketone (1557 cm −1 ). The 1 H NMR and 13 C NMR spectroscopic data of 2 (Table 2) showed high similarity with 1, except for the appearance of one methoxyl protons resonating at δ H 3.81 (H-12) and one methyl protons resonating at δ H 1.77 (H-1), as well as the additional carbonyl carbon resonating at δ C 169.0 (C-2). The existence of an acetamide group was suggested by the HMBC correlations between NH-3 and C-2, between H-1 and C-2 and between H-4 and C-5, as well as the COSY correlation between NH-3 and H-4. The additional HMBC correlations between H-12 and C-11 and between H-13 and C-11/C-15 proved that the benzene ring is connected to the methyl acrylate portion at C-13 ( Figure 4). Finally, a single crystal of 2 was obtained after one week of slow crystallization in 95% MeOH (H 2 O) at 4 • C by optimizing the conditions. Therefore, the structure of 2 was confirmed by X-ray crystal diffraction analysis, and this further proved the Z configuration of the ∆ 10,13 double bond.      13 C NMR and HSQC data revealed the presence of seventeen carbons, including two amide carbonyl (δ C 167.6, C-2 and δ C 160.7, C-5), ten sp 2 resonated between δ C 158.2 and δ C 114.3, one methylene sp 3 (δ C 64.3, C-17), one methine sp 3 (δ C 50.3, C-6) and three methyl (δ C 25.4, C-19; δ C 19.2, C-7; δ C 18.0, C-18) carbons. The COSY correlations between NH-1 and H-6 and between H-6 and H-7 and the HMBC correlations between NH-4 and C-2/C-5/C-6, between H-6 and C-2/C-5/C-7 and between H-7 and C-2/C-6 revealed the existence of the 2,5-diketopiperazine ring. The COSY correlations between H-6 and H-7 and the HMBC correlations between H-7 and C-6 revealed that the methyl group is connected to C-6 of the 2,5-diketopiperazine ring. The COSY correlations between H-13 and H-14 and the HMBC correlations between H-12 and C-13/C-14 and between H-14 and C-10 revealed the existence of 1,4-disubstituted benzene ring. The HMBC correlations between H-8 and C-2/C-10 and between H-10 and C-3 indicate that the 2,5-diketopiperazine ring is connected to C-9 of the benzene ring through C-8. The COSY correlations between H-16 and H-17 and between H-and to H-18 and the HMBC correlations between H-17 and C-12/C-16, between H-18 and C-15/C-16 and between H-19 and C-15/C-16, indicate that the cis-2-pentene is connected to C-12 of the benzene ring ( Figure 5). Therefore, the planar structure of 3 was elucidated as a new diketopiperazine derivative. The configuration of the double bond in 3 was established by the analysis of its NOESY spectrum ( Figure 5). The NOESY spectrum did not show any correlation between H-16 and H-19, confirming the E configuration of the double bond between C-15 and C-16. In addition, there was no NOESY correlation between H-8 and 4-NH, suggesting Z configuration of the double bond between C-3 and C-8. The absolute configuration of C-6 in 3 was determined by the TDDFT-ECD approach. The experimental ECD spectrum of 3 matched well with the calculated ECD spectrum of S-3 ( Figure 6) and further supported its absolute configuration.

Cytotoxicity Assay
Compound 3 was screened for inhibitory activity on 20 human tumor cell lines at a concentration of 20 μM. However, it showed no observable activities against the abovementioned tumor cell lines (Table S1).

Cytotoxicity Assay
Compound 3 was screened for inhibitory activity on 20 human tumor cell lines at a concentration of 20 µM. However, it showed no observable activities against the abovementioned tumor cell lines (Table S1).

Anti-Inflammatory Activity Assays
Compounds 1-3 were screened for NO production inhibitory activity. Compound 2 showed inhibition of NO production induced by lipopolysaccharide (LPS), with an inhibition rate of 28.92 ± 3.49% (Table S2). Compound 2 has a higher inhibition rate than dexamethasone does, which makes it promising as a good anti-inflammatory drug.
Compared with other alkaloid compounds, the activity of compounds 1-3 is not good, and other activity models need to be further screened.

Fungal Material
The fungal strain, A. sclerotiorum ST0501, was isolated from the inner part of an unidentified sponge, GDST-2013-05, collected from the South China Sea (Guangdong, China) in May 2013. The fungal identification was performed by analysis of its morphological characteristics and ITS region of the rDNA [19]. The sequence data were submitted to Genbank with accession number MT534582. The strain was deposited in a −80 • C refrigerator in the laboratory.

LC-MS/MS and Molecular Networking Analysis
LC-MS/MS was performed using a Waters series 2695 HPLC instrument coupled with an amaZon SL ion trap Mass spectrometer (Bruker, Karlsruhe, Germany), with a Xchange C18 column (Acchrom Co., CO, USA) 250 mm × 4.6 mm, 5 µm, 0.5 mL/min). The organic portion was dissolved in MeOH at 10 mg/mL, filtered through a Gracepure C18 SPE cartridge and analyzed by LC-MS/MS. Ten µL aliquot of each sample was injected and eluted with a gradient program of MeOH-H 2 O (0.1% formic acid) (0-20 min 10-100%, 21-25 min 100%; 1.0 mL/min). Mass spectra were obtained in positive ESI mode and with an automated fully dependent MS/MS scan from 100 to 1000 Da. MS/MS data were converted digitally to mzXML files using Filezilla software. The molecular networking was performed using the GNPS data analysis workflow using the spectral clustering algorithm [28]. The spectral networks were imported into Cytoscape 3.9.1 and visualized using the force-directed layout.  Table 2.

Spectroscopic and Spectrometric Data
X-ray Crystallographic Analysis of Compounds 2. Colorless crystals of 2 suitable for X-ray diffraction were obtained from 95% MeOH (H 2 O) by slow evaporation. The crystal data were collected at 293 K using an Agilent Gemini Ultra (Agilent, PA, USA) diffractometer with Cu Kα radiation (λ = 1.54184 Å). The structure was solved using CrysAlis Pro Table 3.

Biological Assay
Cytotoxic activity against human cancer cell lines was evaluated following the CCK-8 assay [29]. Twenty cell lines were used, including human lung cancer cells ( Antibacterial activity was evaluated following the standards recommended by Pierce [30], with ampicillin sodium as a positive control. The antifungal bioassay was conducted following the standards recommended by the Clinical and Laboratory Standards Institute [30], with amphotericin B used as a positive control. Compounds The DPPH scavenging assay was performed using the method described by Aquino et al. [31]. The reaction mixture consisted of freshly prepared DPPH dissolved in ethanol (100 µmol/L) mixed with different concentrations of the tested compound. The reaction mixture was incubated for 20 min at room temperature in the dark, and the optical density was recorded at 517 nm.
The bioassay for NO production inhibitory activity was conducted as described by Xia et al. [32]. The mouse macrophages were seeded in 96-well plates. In each well, LPS (1 µg/mL) was added after treating with or without the tested compound for 24 h. The NO production in the supernatant was detected by the Griess reaction. The absorbance at 540 nm was measured with a microplate reader. The NO concentration and the inhibitory rate were calculated as a calibration curve. Dexamethasone was used as the positive control. Experiments were performed in triplicate, and the data are described as mean ± SD of three independent experiments.

Conclusions
In conclusion, nine alkaloids, including three new ones, were obtained by using MS/MSbased molecular networking for continuous investigation of the marine-derived fungus, A. sclerotiorum ST0501. Sclerotioloid A (1) represents the first example of 2,5-diketopiperazine alkaloid with a rare terminal alkyne. Additionally, the absolute configuration of compound 2 was unambiguously determined by single crystal X-ray analysis. Compound 2 showed the inhibition of NO production induced by lipopolysaccharide (LPS). Compound 3 displayed weak proliferation inhibitory activity against human chronic myeloid leukemia cells K-562 and human renal clear cell adenocarcinoma cell 786-O. Therefore, the potential of the marinederived fungus, A. sclerotiorum ST0501, to produce novel bioactive secondary metabolites is worthy of further exploration.