A New α-Cyclopiazonic Acid Alkaloid Identified from the Weizhou Island Coral-Derived Fungus Aspergillus flavus GXIMD 02503

WANG Jiamin, LI Zhichao, ZHANG Yanting, CHEN Chunmei, CHEN Weihao,GAO Chenghai, LIU Yonghong, ,TAN Yanhui, and LUO Xiaowei, *

A New-Cyclopiazonic Acid Alkaloid Identified from the Weizhou Island Coral-Derived FungusGXIMD 02503

WANG Jiamin1), #, LI Zhichao3), #, ZHANG Yanting2), CHEN Chunmei1), CHEN Weihao1),GAO Chenghai2), LIU Yonghong1), 2),TAN Yanhui3), and LUO Xiaowei2), *

1),,,510301,2),,530200,3),,,541004,

A newoxygenated tricyclic cyclopiazonic acid (CPA) alkaloid, asperorydine Q (1), along with seven known compounds, namely, asperorydines O (2) and J (3), speradine H (4), cyclopiamides A (5) and H (6), saadamysin (7), and pyrazinemethanol (8), were isolated from the coral-associatedGXIMD 02503. The structures were elucidated by physicochemical pro- perties and comprehensive spectroscopic data analysis. Compounds 1?5 and 7?8 exhibited potent inhibition of lipopolysaccharide (LPS)-induced nuclear factor-B (NF-B) with the IC50values ranging from 6.5 to 21.8μmolL?1. In addition, the most potent one, pyrazinemethanol (8), dose-dependently suppressed receptor activator of NF-B ligand (RANKL)-induced osteoclast differentiation without obvious cytotoxicity in bone marrow macrophages cells (BMMCs), suggesting it is a promising lead compound for the treat- ment of osteolytic diseases.

coral-derived fungi;; cyclopiazonic acid; NF-B; osteoclastogenesis

1 Introduction

The coral-derived microorganisms have proven their va- lue as a source of bioactive compounds (Sang., 2019).The funguswith ubiquitous marine, ter- restrial, or symbiotic sources,known as mutagenic myco- toxins production (Yang., 2019),is a rich origin of diversified secondary metabolites with significant bioac- tivities (Wu., 2018; Liu., 2019b), including the common-cyclopiazonic acid (CPA) alkaloids (Vaidet., 2017). Since the first CPA was reported as a mycotoxinfrom the fungusin 1968 (Chen.,2021), approximately 50 CPA-type alkaloids have been hi- therto found in the predominant fungal genera ofand(Ostry., 2018).CPA alkaloids are characterized with three main structural units: an in- dole, a dimethylallyl, and two acetic acids, and have been reported with attractive biological activities, including cy- totoxicity (Hymery., 2014), insecticidal activity (Ma., 2015), neurotrophic activity (Liu., 2018), and Ca2＋-ATPase inhibitory activity (Walsh., 2013), which have attracted great attention among synthetic and biosyn-thetic chemists (Liu., 2019a; Zhurakovskyi., 2019; Ahmad., 2020).

In our ongoing search for novel bioactive compounds from marine-derived fungi (Luo., 2019, 2020, 2021; Tan., 2020), the fungusGXIMD 02503 with interesting HPLC-DAD profiles of its EtOAc extract was isolated from a coral, which was collected from the Weizhou Island coral reef in Guangxi Zhuang Au- tonomous Region, China. Further chemical investigation of its extracts led to the isolation and identification of a new oxygenated tricyclic CPA alkaloid, asperorydine Q (1), along with seven known compounds (2?8). Herein, their isola- tion, structure elucidation, and bioactivity are described in detail.

2 Materials and Methods

2.1 General Experimental Procedure

Optical rotations were acquired by an Anton Paar MPC 500 polarimeter (Anton Paar, Graz, Austria). ECD spectra were measured on a Chirascan Circular Dichroism spec- trometer (Applied Photophysics Ltd., Leatherhead, UK). UV and IR spectra were recorded on a Shimadzu UV-2600 PC spectrometer and an IR Affinity-1 spectrometer(Shimadzu Corporation, Nakagyo-ku, Kyoto, Japan, re- spectively). The NMR spectra were measured on a Bruker Avance spectrometer (Bruker BioSpin, F?llanden, Switzer-land) operating at 700MHz for1H NMR, 175MHz for13C NMR, using TMS as an internal standard. HR-ESI-MSspectra were acquired by a Bruker miXis TOF-QII massspectrometer (Bruker BioSpin, F?allanden, Switzerland). Semi-preparative HPLC was performed on a Hitachi Pri- maide (Hitachi, Tokyo, Japan) using an ODS column (YMC- pack ODS-A, YMC Co. Ltd., Japan, 10mm×250mm, 5μm, 12nm). Thin layer chromatography (TLC) and column chromatography (CC) were performed on plates precoat- ed with silica gel GF254(10?40μm) and over silica gel(200?300 mesh) (Qingdao Marine Chemical Factory, Chi- na), respectively. All solvents employed were of analyti- cal grade (Tianjin Damao Chemical and Industry Factory, China). The artificial sea salt was a commercial product(Guangzhou Haili Aquarium Technology Company, China).

2.2 Fungal Collection and Fermentation

The strain GXIMD 02503 was isolated from a coralcollected from the Weizhou Island coral reef in Guangxi Zhuang Autonomous Region, China, in March 2019. It was identified asby referring to sequence analysis of the internal spacer (ITS) regions ofthe rDNA (GenBank accession MT510157 and MT510158), and a voucher specimen has been deposited in our labora- tory. The strainGXIMD 02503 was cultured on MB-agar plates (malt extract 15g, artificial sea salt 15g, and agar 20g in 1.0L tap distilled H2O) at 25℃ for 7 days. Massive fermentation ofGXIMD 02503 was car- ried out in the liquid medium (mannitol 2.0%, maltose 2.0%, glucose 1.0%, corn steep liquor 0.1%, MSG 1.0%, KH2PO40.05%, MgSO4·7H2O 0.03%, yeast extract 0.3% and sea salt 1.5%, pH 7.4) employing with 300mL×100 Erlenme- yer flasks (1L) at room temperature for 40 days. Then all the cultures were overlaid and extracted with EtOAc to yield a brown extract (50g).

2.3 Extraction and Isolation

The crude extract was separated into ten fractions (Frs. 1–10) by silica gel vacuum liquid chromatography (VLC) using a gradient solvent system with CH2Cl2/MeOH (, 1:0–1:1) based on TLC (GF254) analysis. Fr. 4 was further divided into nine subfractions (Frs. 4-1–4-9) by ODS silica gel chromatography eluting with MeOH/H2O (10–100%). After repeating the above operation, Fr. 4-4 was further divided into four subfractions (Frs. 4-4-1–4-4-4) by ODS silica gel chromatography eluting with MeOH/H2O (10%–100%). Fr. 4-4-1 was then purified by semiprepa- rative HPLC (60% MeOH/H2O, 2mLmin?1, 230nm) toafford 1 (2.4mg,R18min), 2 (2.8mg,R22min), 3 (2.8mg,R29min). Fr. 4-6 was purified by semipreparative HPLC (60% MeOH/H2O, 2mLmin?1, 230nm) to afford 4 (2.7mg,R15min). Additionally, eight subfractions (Frs. 3-1–3-8) were obtained from Fr. 3ODS silica gel chroma- tography eluting with MeOH/H2O (10%–100%). Fr. 3-1 was further divided into five subfractions (Frs. 3-1-1–3-1-5)ODS silica gel chromatography eluting with MeOH/H2O (10%–100%). Fr. 3-1-2 was then purified by semipreparative HPLC (50% MeOH/H2O, 2mLmin?1, 230nm) to afford 8 (16.5mg,R23min). Fr. 5 was further di- vided into twelve subfractions (Frs. 5-1–5-12) by ODS silica gel chromatography eluting with MeOH/H2O (10%–100%). Compound 7 (11.6mg,R11min) was isolated from Fr. 5-3 by semipreparative HPLC (60% MeOH/H2O, 2mLmin?1, 230nm). Fr. 6 was further divided into thir- teen subfractions (Frs. 6-1–6-13) by ODS silica gel chro- matography eluting with MeOH/H2O (10%–100%). Fr. 6-6 was further divided into six subfractions (Frs. 6-6-1–6-6-6) by ODS silica gel chromatography eluting with MeOH/ H2O (10%–100%). And Fr. 6-6-4 was subsequently sepa- rated by semipreparative HPLC (60% MeOH/H2O, 2mLmin?1, 230nm) to afford 5 (7.7mg,R15min) and 6 (1.0mg,R12min).

2.4 ECD Calculation

The theoretical ECD curves of 2 were calculated by the Gaussian 16 software. Conformational searches were car- ried out by means of the Spartan’14 software using a Mole- cular Merck force field (MMFF). Low-energy conformers with a Boltzmann distribution over 1% were chosen for ECD calculations by TD-DFT method at the B3LYP/6- 311+G (d, p)//B3LYP/6-31+G (d) level in MeOHby adopt- ing 30 excited states. The ECD spectra were generated by the SpecDis 1.71 under a half band width of 0.3eV to fa- cilitate comparison to the experimental data.

2.5 Spectral Data

Asperorydine O (2): Yellow powder; []25 D?20 (0.10, MeOH); UV (MeOH) λmax(log) 218 (3.52), 278 (3.02), 412 (2.93)nm; ECD (5.6í10?4molL?1, MeOH) λmax(Δ) 215 (?5.16), 246 (+5.84), 282 (+2.78), 335 (+0.94), 412 (?2.43)nm; IR (film)max3318, 2922, 1701, 1612, 1321, 1204cm?1; HR-ESI-MS/[M+H]+peak at 359.1606 (calculated for C19H23N2O5, 359.1607).1H NMR (700MHz, CD3OD):H7.37 (1H, dd,=8.6, 7.3Hz, H-13), 6.62 (1H, d,=8.6Hz, H-14), 6.49 (1H, d,=8.0Hz, H-12), 3.88 (3H, overlapped, H3-16), 3.33 (1H, m, H-10b), 2.96 (1H, dd,=17.9, 3.5Hz, H-10a), 2.92 (3H, s, N-CH3), 2.56 (1H, dd,=6.7, 3.5Hz, H-9), 2.25 (3H, s, H3-18), 1.68 (3H,s, H3-19), 1.21 (3H, s, H3-20);13C NMR (175MHz, CD3OD)C204.2 (C-17), 193.5 (C-4), 174.9 (C-6), 169.6 (C-15), 154.8 (C-2), 145.0 (C-11), 138.0 (C-13), 115.2 (C-12), 113.9 (C-3), 110.2 (C-14), 77.9 (C-5), 64.4 (C-8), 54.3 (C-16), 49.8 (C-9), 30.1 (C-18), 29.4 (1N-CH3), 25.7 (C-10), 28.3 (C-19), 22.3 (C-20).

2.6 Bioassay

The isolated compounds (1?8) were evaluated for their inhibitory activities of lipopolysaccharide (LPS)-induced NF-B activation in RAW264.7 cells by luciferase repor- ter gene assay as described previously (Tan., 2020). In brief, the RAW264.7 cells stably transfected with a lu- ciferase reporter gene were plated in 96-well plates, and then pretreated with these compounds (10μmolL?1) and BAY11-7082 (NF-B inhibitor as positive control, 5μmolL?1, Sigma-Aldrich) for 30min, followed by 5μgmL?1LPSstimulation for 8h. Cells were harvested, and luciferase ac- tivities of the triplicate tests were measured by the luci-ferase assay system (Promega, Madison, WI, USA). The dose-dependent effects of compounds (50, 20, 10, 5, and 1μmolL?1) on LPS induced NF-B luciferase activity were also detected by the same assay.

In order to study the effect of the potent NF-B inhibi- tor (8) on osteoclastogenesis, pyrazinemethanol (8) (1–4 μmolL?1) was added in bone marrow macrophages cells(BMMCs, extracted from the femurs of C57BL/6 mice) with macrophage-stimulating factor (M-CSF) (50ngmL?1) and RANKL (100ngmL?1) stimulation for 3 days. Then the cells were fixed and stained to detect tartrate-resistant aci- dic phosphatase activity (TRAP) and the images were pho-tographed by using an inverted microscope (Nikon, Japan). CCK-8 kit was used to evaluate the cytotoxic effects of 8on BMMCs as described previously(Tan., 2020). BMMCs (1×105cellsmL?1) with M-CSF (50ngmL?1) were seeded with or without 8 (0.1, 1, 5, 10μmolL?1) for 72h. Cell viability was carried out as a percentage of the con- trol. Data were expressed as the mean±SD and analyzedusing GraphPad Prism 7.0 software (San Diego, CA, USA). Statistical differences among groups were performed using one-way analysis of variance (ANOVA) with Bonferronitest. A-value of <0.05 was considered statisti- cally significant.

3 Results and Discussion

3.1 Structural Determination

The fermentation broth ofGXIMD 02503 was extracted with EtOAc for three times. The whole extract was then partitioned and purified by repeated column chro- matography. And the HPLC-DAD-guided purification fi- nally led to the discovery of eight compounds (Fig.1), in- cluding a new CPA alkaloid, asperorydine Q (1), along with seven known compounds (2?8), which were identi- fied as asperorydines O (2) (Xiang., 2021) and J (3) (Liu., 2018), speradine H (4) (Hu., 2014), cy- clopiamides A (5) (Holzapfel., 1990) and H (6) (Xu., 2015), saadamysin (7) (Lin., 2008), and pyra- zinemethanol (8) (Ran., 2020)by comparison with literature data.

Fig.1 Structures of compounds 1–8.

Asperorydine Q (1)was isolated as yellow powder with the molecular formula of C19H22N2O6as determined by HRESIMS peak at/375.1553 [M+H]+. The IR spec- trum indicated the presence of hydroxy (3385cm?1) and carbonyl (1684cm?1) groups. The1H NMR data (Table 1) aided with HSQC spectrum of 1 revealed CPA character- istics with three aromatic protons, assigned to H-12 (H6.50, d,=8.0Hz), H-13 (H7.38, m), H-14 (H6.62, d,=8.6Hz); one methine, H-9 (H2.59, dd,=6.7, 3.5Hz); two methylenes, H2-10 (H2.97, dd,=17.9, 3.5Hz; 3.33, d,=6.8Hz) and H2-16 (H3.90, overlapped); and four singlet methyls, attributed to 1N-CH3(H2.92), H3-19 (H1.68), H3-20 (H1.21); and one methoxy group, H3-18 (H3.69). Analysis of13C NMR data of 1 classified the nine- teen carbons into four methyls (C52.7, 29.4, 28.2, 22.1), two methylenes (C45.4, 25.7), four methines (C138.1, 115.6, 110.3, 49.8), as well as nine nonprotonated carbons (C193.4, 175.0, 169.5, 168.5, 154.8, 145.0, 114.0, 77.8, 64.4). The above NMR data highly resembled those of the co-isolated asperorydine J (3).The only obvious difference was the occurrence of a hydroxyl group located at C-5 (C77.8) in 1 instead of a hydrogen in 3 (C54.0). This deduc- tion was verified by the HMBC correlations (Fig.2a) from H-9 and H2-10 to C-5, and the strongly deshielded chemi- cal shift of C-5. The NOESY correlation (Fig.2a) of H-9and H3-19 allowed the same orientation of H-9 and Me-19.

The absolute configuration of 1 was determined by com- parison between the calculated and experimental ECD cur- ves, in combination with biosynthetic considerations. Com- pound 1 shared the nearly identical experimental ECD cur- ve (positive Cotton effects at 245 and 280nm, and nega- tive ones at 215 and 410nm) with that of the very recent- ly reported co-isolated asperorydine O (2) (Xiang., 2021), revealing the (5, 9)-configuration of 1. This de- duction was also further confirmed by the well-matchedexperimental and/or calculated ECD curves of 1 and 2 (Fig.2b).

Table 1 1H (700MHz) and 13C (175 MHz) NMR data of 1 (CD3OD)

Fig.2 Structural assignments of asperorydine Q (1).Key HMBC, 1H-1H COSY, and NOESY correlations of 1 (a). Experimental and/or calculated ECD spectra of 1–2 (b).

3.2 Effects on Osteoclast Differentiation of the Compounds

Osteolytic disease is a pathologic condition character- ized by the imbalance of two coordinating and opposite as- pects, bone formation by osteoblastogenesis and bone re- sorption by osteoclastogenesis (Zhou., 2020). Target- ing osteoclast differentiation is a therapeutic strategy for osteolytic diseases (Tan., 2020). The differentiation and formation of osteoclasts are regulated by several sig- naling pathways, while the critical pathway is induced by RANKL secreted mainly by osteocytes (Hong., 2020). During our course of screening osteoclast differentiation inhibitors from marine natural products, all the isolated com-pounds (1?8) were primarily evaluated for their inhibitory activities of lipopolysaccharide (LPS)-induced NF-B acti-vation in RAW264.7 cells (Fig.3a). Interestingly,compounds 3 and 8 significantly and dose-dependently suppressed NF-B activation with the IC50values of (8.6±1.3) and (6.5±1.4)μmolL?1(Fig.3b), respectively. In addition, compounds1, 2, 4, 5, and 7 exhibited moderate inhibitory activities of NF-B activation, with IC50values of (14.1±1.5), (21.8±1.9), (17.4±1.7), (11.3±2.0), and (10.7±1.3)μmolL?1, re-spectively. Meanwhile, the positive control, BAY11-7082, showed inhibitory activities of NF-B activation with an IC50value of (1.5±1.4)μmolL?1.However, compound 6 was found to be inactive (IC50>50μmolL?1).

Since NF-B plays an important role in RANKL-in- duced osteoclast differentiation (Hong., 2020; Tan., 2020; Zhou., 2020), the most potent compound, pyrazinemethanol (8), was further evaluated for its effects on RANKL-induced osteoclastogenesis, and the results showed that 8 suppressed RANKL induced osteoclast dif- ferentiation in BMMCs in a dose-dependent manner with- out an obvious cytotoxicity (Figs.3c?e). Taken together, compound 8 can be a promising osteoclast differentiation inhibitor for the treatment of osteoclast-related diseases.

Fig.3 The inhibitory effects of compounds 1–8 on LPS-induced NF-κB activation in RAW264.7 cells and pyrazineme- thanol (8) suppresses RANKL-induced osteoclast differentiation.NF-κB inhibitions of 1–8 at 10μmolL?1 (a) and gradient concentrations (b). C, concentration of compounds (μmolL?1). Representative images of osteoclasts from BMMCs treated with 8 (1–4μmolL?1) for 3 days, TRAP-positive multinucleated cells were regarded as osteoclasts (c) and quantified (d). Cell viability of 8 at different concentrations on BMMCs for 72h were measured by cell counting kit 8 assay (e). All ex- periments were performed at least three times. The data are presented as the mean±SD of representative experiments. ### P<0.001 vs. control group; * P<0.05, ** P<0.01, *** P<0.001 vs. LPS or RANKL group. Scale bars, 500μm.

4 Conclusions

Chemical investigation of the cultures of the Weizhou Island coral-derived fungusGXIMD 02503 led to the identification of a new oxygenated tricyclic CPA al- kaloid, asperorydine Q (1), along with seven known com- pounds (2?8). Compounds 1?5 and 7?8 exhibited potent inhibition of LPS-induced NF-B activation in RAW264.7 cells with the IC50values ranging from 6.5 to 21.8μmolL?1. Notably, the most potent one, pyrazinemethanol (8), dose- dependently suppressed RANKL-induced osteoclast diffe- rentiation without obvious cytotoxicity in BMMCs, sug- gesting a promising lead compound for the treatment ofosteolytic diseases. Our findings would enrich chemical context of the fungusand expand the structuraland biological diversity of CPA alkaloids and/or pyrazinone derivatives.

Acknowledgements

This work was supported by the Natural Science Foun- dation of Guangxi (No. 2020GXNSFGA297002), the Spe- cific Research Project of Guangxi for Research Bases and Talents (No. AD20297003), the Special Fund for Bagui Scholars of Guangxi (Y. Liu), the National Natural Sci- ence Foundation of China (Nos. U20A20101, 22007019), the Key State Laboratory Talent Project of Guangxi Nor- mal University (No. CMEMR 2019-A05), and the Open Project of CAS Key Laboratory of Tropical Marine Bio- resources and Ecology (No. LMB20211005).

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February 20, 2021;

April 6, 2021;

September 21, 2021

? Ocean University of China, Science Press and Springer-Verlag GmbH Germany 2022

#The two authors contributed equally to this work.

. E-mail: luoxiaowei1991@126.com

(Edited by Qiu Yantao)