Three Pairs of New Spirocyclic Alkaloid Enantiomers From the Marine-Derived Fungus Eurotium sp. SCSIO F452

Three pairs of new spirocyclic alkaloid enantiomers eurotinoids A–C (1–3), as well as a known biogenetically related racemate dihydrocryptoechinulin D (4) were isolated from a marine-derived fungus Eurotium sp. SCSIO F452. Their structures were determined by spectroscopic analyses and electronic circular dichroism (ECD) calculations. Compounds 1 and 2 represent the first two “meta” products from a non-stereoselective [4 + 2] Diels-Alder cycloaddition presumably between an enone group of a diketopiperazine alkaloid and a diene group of a benzaldehyde derivative via a new head-to-tail coupling mode biosynthetically, while 3 and 4 were “ortho” products. Their enantiomers exhibited different antioxidative and cytotoxic activities. The modes of action were investigated by a preliminary molecular docking study.


INTRODUCTION
Marine fungi are widely known as a rich source producing structurally diverse and biologically active natural products, which attract people's great interests in pharmaceutical, agrochemical, and food industries (Richards et al., 2012;Martins et al., 2014;Blunt et al., 2018). The fungal genus Eurotium, known as the teleomorph of Aspergillus, has been found to produce diketopiperazine (DKP) alkaloids, prenyl benzaldehyde derivatives, anthraquinones, and so on, which display a wide range of biological activities such as antibacterial, antioxidative, insecticidal, and binding with opioid and cannabinoid receptors activities (Gao et al., 2011;Du et al., 2012Du et al., , 2017Gomes et al., 2012;Meng et al., 2015). Motivated by the fascinating secondary metabolites produced by the fungi Eurotium spp., we have launched a chemical investigation on a marine-derived fungus isolated from a South China Sea sediment sample, Eurotium sp. SCSIO F452, and found a series of prenylated DKPs, prenyl benzaldehyde derivatives, especially three pairs of spirocyclic diketopiperazine-anthraquinone enantiomers variecolortins A-C (Wang et al., 2013;Zhong W.-M. et al., 2018). In order to enrich and improve the chemical diversity of this fungus, we carried out a further chemical study on the Eurotium sp. SCSIO F452, and disclosed three pairs of new spirocyclic alkaloid enantiomers eurotinoids A-C (1-3), as well as a known biogenetically related racemate dihydrocryptoechinulin D (4) (Gao et al., 2012). Structurally, compounds 1-4 feature a cyclohexene core presumably deriving from a key non-stereoselective [4 + 2] Diels-Alder cycloaddition between an enone group of a diketopiperazine alkaloid and a diene group of a benzaldehyde derivative. Interestingly, compounds 1 and 2, 3 and 4 occurred as two pairs of diastereomers. The molecular structures of all the compounds including absolute configurations were unambiguously assigned by comprehensive spectroscopic data and quantum chemical electronic circular dichroism (ECD) calculations. Herein we report the isolation, structure elucidation, plausible biosynthetic pathway, antioxidative and cytotoxic bioactivities, and molecular dockings of 1-4.

General Experimental Procedures
Optical rotations were measured with an MCP 500 automatic polarimeter (Anton Paar) with MeCN as solvent. UV spectra were recorded on a UV-2600 spectrometer (Shimadzu). IR spectra were measured on an IR Affinity-1 spectrometer (Shimadzu). 1 H, 13 C NMR, DEPT, and 2D NMR spectra were recorded on the AVANCE III HD 700 (Bruker). Circular dichroism spectra were measured with a Chirascan circular dichroism spectrometer (Applied Photophysics). HRESIMS spectra data were recorded on a MaXis quadrupole-time-offlight mass spectrometer. Thin layer chromatography (TLC) was performed on plates precoated with silica gel GF 254 (10-40 µm). Column chromatography (CC) was performed over silica gel (200-300 mesh and 300-400 mesh) (Qingdao Marine Chemical Factory) and octadecylsilyl silica gel (ODS) (50 µm, YMC). High performance liquid chromatography was performed on an Agilent 1260 HPLC equipped with a DAD detector, using an ODS column (YMC-pack ODS-A, 10 × 250 mm, 5 µm), two chiral-phase columns (Daicel chiralpak IA and IC, 4.6 × 250 mm, 5 µm). All solvents used in CC and HPLC were of analytical grade (Tianjin Damao Chemical Plant, Tianjin, China) and chromatographic grade (Oceanpak, Sweden), respectively. Fractions were monitored by TLC and spots were visualized by heating silica gel plates sprayed with 10% H 2 SO 4 in EtOH.

Fungal Material
The fungal strain used in this investigation was isolated from a South China Sea sediment sample (17 • 29.804'N, 110 • 0.292'E) at a depth of 158 m in May, 2010. It was identified as Eurotium sp. SCSIO F452 according to a molecular biological protocol by DNA amplification and sequencing of the ITS region (deposited in GenBank, accession no. JX481973). The working strain was prepared on potato dextrose agar slants modified with seawater instead of distilled water and stored at 4 • C. A reference culture was maintained at −80 • C in RNAM Centre for Marine Microbiology, South China Sea Institute of Oceanology, Chinese Academy of Sciences.

Fermentation and Extraction
The fungus Eurotium sp. SCSIO F452 was cultured and extracted as previously described (Zhong W.-M. et al., 2018).

Computational Details
Molecular Merck force field (MMFF) calculations were done using Spartan'14 program (Wavefunction Inc., Irvine, CA, USA). Density functional theory (DFT) and time-dependent density functional theory (TDDFT) calculations were performed with Gaussian09 program package (Frisch et al., 2010). In order to reduce the computational cost, the truncated structures ( Figure S5) were used in ECD calculations of compounds (12S,28R,31R)-1 ′ , (12S,28S,31S)-2 ′ , and (12R,28R,31R)-3 ′ , corresponding to innate compounds (12S,28R,31R)-1, (12S,28S,31S)-2, and (12R,28R,31R)-3. For conformational analysis, the conformers generated by a MMFF conformational search in an energy window of 10 kcal/mol were subjected to geometry optimization using the DFT method at the B3LYP/def2-SVP level (Lee et al., 1988;Becke, 1993;Weigend and Ahlrichs, 2005). Frequency calculations were run at the same level to estimate their relative thermal ( E) and free energies ( G) at 298.15 K. Energies of the low-energy conformers in MeCN were re-calculated at the M06-2X/def2-TZVP level. Solvent (MeCN) effects were taken into account by using polarizable continuum model (IEFPCM). The TDDFT calculations were performed using the hybrid PBE1PBE (Perdew et al., 1996;Adamo and Barone, 1999) and M06-2X (Zhao and Truhlar, 2008) functionals, and the Ahlrichs' basis sets TZVP (Schäfer et al., 1994) and/or def2-SVP. The number of excited states is 36 for each of the compound. The ECD spectra were generated by the program SpecDis (Bruhn et al., 2013) using a Gaussian band shape from dipole-length dipolar and rotational strengths. The equilibrium population of each conformer at 298.15 K was calculated from its G using Boltzmann statistics. The calculated spectra of compounds were generated from the low-energy conformers according to the Boltzmann weighting of each conformer in MeCN solution.

Biological Assays
The details of the experimental procedures for antioxidative and cytotoxic bioassays were similar to those presented in our former paper (Zhong W.-M. et al., 2018).

Molecular Docking Study
The detail procedures of molecular docking study were presented in Supporting Information File.
The Diels-Alder (DA) reaction is classified as a [4 + 2] cycloaddition in the pericyclic reaction involving a 1,3-diene and a dienophile to afford a six-membered ring with four contiguous stereocenters (Minami and Oikawa, 2016). Hundreds of natural products (NP) containing carbocycles or heterocycles are potentially biosynthesized by DA reaction. With the advancements in whole-genome sequencing and searching tools of biosynthetic gene clusters of secondary metabolites, some Diels-Alderases, such as SpnF, PyrE3, Pyrl4, AbyU, and PvhB, have been identified and substantiated to be of great importance in the biosynthesis of NPs (Takao et al., 2005;Kim et al., 2011;Fage et al., 2015;Tian et al., 2015;Byrne et al., 2016;Tan et al., 2019). Besides, some NPs like spirotriscoumarins A and B (Tang et al., 2016), artemisians A-D (Xue et al., 2017), and alpininoids A-E (Liu et al., 2018) are proposed to be biosynthesized by spontaneous DA cycloaddition. A plausible biogenetic route of 1-4 is proposed in Scheme 1. The enone group of neoechinulin B, an indole DKP alkaloid, was supposed to be the dienophile to couple with the diene group of a benzaldehyde derivative, isodihydroauroglaucin, forming the four pairs of enantiomers 1-4 through a key non-stereoselective [4 + 2] Diels-Alder cycloaddition. To be specific, the dienophile undergone a DA reaction with the diene by a head-to-tail approach to produce 1 and 2, and by a head-to-head mode to yield 3 and 4, respectively. Notably, neoechinulin B and isodihydroauroglaucin both have been isolated from this fungus in previous chemical study (Wang et al., 2013;Zhong W.-M. et al., 2018), which supported the biosynthetic hypothesis. To the best of our knowledge, only four such kind of spirocyclic alkaloids have been reported, namely, cryptoechinuline B (Gatti, 1976), cryptoechinuline D (Gatti, 1976), dihydrocryptoechinulin D (Gao et al., 2012), and effusin A (Gao et al., 2012), all isolated from fungi. Structurally, the former three reported compounds, together with compound 3 were all tend to be "ortho" products, while 1 and 2 were represented as the first two "meta" products belonging to this kind of spirocyclic alkaloids.

CONCLUSIONS
In conclusion, three pairs of new spirocyclic alkaloid enantiomers eurotinoids A-C (1-3), as well as a known biogenetically related racemate dihydrocryptoechinulin D (4), were isolated from a marine-derived fungus Eurotium sp. SCSIO F452. Their structures including absolute configurations were determined by extensive spectroscopic data and ECD calculations. Compounds 1 and 2 were represented as the first two "meta" products biosynthetically from a non-stereoselective [4 + 2] Diels-Alder cycloaddition presumably between an enone group of a diketopiperazine alkaloid and a diene group of a benzaldehyde derivative via a new head-to-tail coupling mode biosynthetically. All the enantiomers were evaluated antioxidative and cytotoxic activities. A preliminary molecular docking study provided an inside perspective of the action of their different biological activities.

AUTHOR CONTRIBUTIONS
WZho performed the isolation, purification, characterization and evaluation the antioxidative activities of all the compounds, and prepared the manuscript. JW contributed to the structure elucidation and revised the manuscript. XW performed the ECD calculations. TF and XH performed the molecular docking experiments. YC and WZha contributed to the determination of cytotoxic activities. QZ and ZH contributed to the isolation of the compounds and determination of antioxidative activities. SZ, LL, and FW designed and supervised the research and revised the manuscript.