HU Yiwei, ZHAO Kai,ZHU Junheng,QI Xin,CHEN Weihao, SONG Yingying,PANG Xiaoyan, 2), LIU Yonghong, 2), 3), and WANG Junfeng, 2), 3), *

New Neuraminidase Inhibitory Alkaloids from the Mangrove Soil-Derived Fungussp. SCSIO 41305

HU Yiwei1), 3), ZHAO Kai1),ZHU Junheng1),QI Xin1),CHEN Weihao1), 3), SONG Yingying1), 3),PANG Xiaoyan1), 2), LIU Yonghong1), 2), 3), and WANG Junfeng1), 2), 3), *

1) CAS Key Laboratory of Tropical Marine Bio-Resources and Ecology, Guangdong Key Laboratory of Marine Materia Medica, Innovation Academy of South China Sea Ecology and Environmental Engineering, South China Sea Institute of Oceanology, Chinese Academy of Sciences, Guangzhou 510301, China 2) Southern Marine Science and Engineering Guangdong Laboratory (Guangzhou), Guangzhou 511458, China 3) University of Chinese Academy of Sciences, Beijing 100049, China

New alkaloid, ()-2-(hydroxyimino)-4-methylpentanamide (1) and a new cyclopentano[] pyridine, 4-hydroxy-7-meth- yl-6,7-dihydro-5-cyclopenta[c]pyridin-5-one (2), together with ten known compounds (3 – 12) were isolated from the mangrove soil-derived fungussp. SCSIO 41305. Extensive spectroscopic analysis and X-Ray crystallographic analysis were used to elucidate the structure of ()-2-(hydroxyimino)-4-methylpentanamide (1), including its absolute configuration. All the isolated compounds (1–12) were evaluated for their antimicrobial and enzyme inhibitory activities against acetylcholinesterase (AChE), neuraminidase (NAs), and phosphatidylinositol 3-kinase (PI3K). Among them, compounds 1 and 3 exhibited strong neuraminidase inhibitory activity with IC50values of 12.04, 1.92 μmol L?1(IC5020 μmol L?1for oseltamivir acid), while compounds 5, 6, 8, and 10 showed moderate neuraminidase inhibitory activity, and compounds 6 – 10 displayed weak enzyme inhibitory activities against PI3K.

; alkaloid; pyridine; neuraminidase; acetylcholinesterase

1 Introduction

Mangroves are woody plant communities that grow in tropical and subtropical coastal and estuarine intertidal zones, a land-to-sea transitional ecosystem in an intertidal environment of high salinity, frequent tides, strong winds, high temperatures, strong UV radiation and anoxic sludge (Xu., 2015). Mangrove fungi, due to their unique growth environment, have relatively special biosynthetic pathways and enzymatic reaction systems for their secondary metabolites, which often result in drug lead compounds with novel chemical structures and significant and diverse biological activities. Mangrove fungi are an important source of active functional molecules with good bioactivity and unique chemical structures, and the current secondary metabolites of mangrove fungal origin are diverse and have good anti-tumor (Li., 2019), anti- bacterial (Cai., 2019) and enzyme inhibitory activities (Meng., 2016).

In our continuing research for novel natural products from mangrove soil-derivedfungi, (Wang., 2014, 2015a, 2015b) a fungussp. SCSIO 41305, showed plentiful secondary metabolites. A new alkaloid (1), and a new cyclopentano[] pyridine (2), together with ten known compounds (3-12), which were identified as bacillibactin (3) (Li., 2017), altechromone A (4) (Lee., 2001), anomalin B (5) (Abdel., 2003), 1,3,5,6-tetrahydroxy-8-methyl-xanthone (6) (Belofsky., 1998), anomalin A (7) (Abdel., 2003), 1,3,6-trihydroxy-8-methylxanthone (8) (Mutanyatta., 2003), 3,4,8-trihydroxy-6-meth-oxy-1-me- thylxanthone (9) (Wang., 2014), caloxanthone E (10) (Iinuma., 1995), aloesone (11) (Speranza., 1985), and alternariol (12) (Tan., 2008) were isolated from the rice solid culture (Fig.1). Herein, we reported the isolation, structural elucidation, and activities of these structures.

2 Materials and Methods

2.1 General Experimental Procedure

Optical rotations were measured on a MCP-500 polarimeter (Perkin-El-mer, Inc., Waltham, MA). X-ray diffraction intensity data were collected on Agilent Xcalibur Nova single-crystal diffractometer using Cu Kα radiation. UV spectra were recorded on a Shimadzu UV-2401PC spectrometer.1H,13C NMR, DEPT, and 2D-NMR spectra were recorded on the Avance-700 and Avance-500 spectrometers (Bruker BioSpin, Fallanden, Switzerland) using TMS as internal standard and chemical shifts were recorded as δ-values. HRESIMS spectra were recorded on a Bruker maXis Q-TOF mass spectrometer in positive/ne- gative ion mode. Thin-layer chromatography (TLC) and column chromatography (CC) were performed on plates precoated with silica gel GF254 (10 – 40 μm) and over silica gel (200 – 300 mesh) (Qingdao Marine Chemical Factory, Qingdao, China), and Sephadex LH-20 (Amersham Biosciences, Uppsala, Sweden), respectively. All solvents were used of analytical grade (Tianjin Fuyu Chemical and Industry Factory, Tianjin, China). Semipreparative High-performance liquid chromatography (HPLC) was performed using an ODS column (YMC- pack ODS-A, 10 × 250 mm, 5 μm, 2 mL min?1). The artificial sea salt was a commercial product (Guangzhou Haili Aquarium Technology Company, China).

Fig.1 Chemical structures of compounds 1 – 12.

2.2 Fungal Material and Fermentation

The fungal strainsp. SCSIO 41305 was isolated from the mangrove soil, which was collected from the Fuli Mangrove Bay Wetland Park, Haikou, Hainan Province of China. It was identified according to its morphological characteristics and internal spacer (ITS) gene sequences (GenBank accession no. ON359828). A voucher specimen has been deposited in at our laboratory at ? 80℃. The producing strain was stored on MB agar (malt extract 15 g, sea salt 10 g, agar 16 g, H2O 1 L, pH 7.4 – 7.8) slants at 28℃ for 7 d. Massive fermentation ofsp. SCSIO 41305 was carried out in the liquid medium (mannitol 2.0%, MSG 1.0%, maltose 2.0%, yeast extract 0.3%, glucose 1.0%, corn steep liquor 0.1%, MgSO4·7H2O 0.03%, KH2PO40.05%, and sea salt 3.5%, pH 7.5) employing with 300 mL × 100 Erlenmeyer flasks (1 L) by shaker fermentation at 28℃ for 9 d. Then all the cultures were overlaid and extracted with EtOAc to yield a brown extract (23 g).

2.3 Extraction and Isolation

The crude extract was separated into seven fractions (Frs. 1–7) by combiflash nextgen 300+ using a gradient solvent system with MeOH/H2O (5% – 100%) based on own system analysis. Fr. 2 was subjected to vacuum liquid chromatography on an ODS silica gel column using step-gradient elution with MeOH/H2O (5% – 100%) to se- parate into seven fractions (Frs. 2-1 – 2-7) based on TLC properties. Fr. 2-2 was applied to a Sephadex LH-20 column eluted with MeOH, and then was divided into six parts (Frs. 2-2-1 – 2-2-6). Fr. 2-2-2 was further purified with semi-preparative HPLC (20% CH3OH/H2O, 3 mL min?1) to yield 2 (1.78 mg,R= 28.4 min). Fr-2-2-3 was further separated with semi-preparative HPLC (45% CH3OH/H2O), 2.5 mL min?1) to gain 1 (15.71 mg,R= 14.8 min). Fr. 2-3 was subjected to a Sephadex LH-20 column eluted with MeOH and reversed-phase C-18 MPLC with MeOH/H2O (10% – 100%) to afford six sub- fractions (Fr. 2-3-1 – Fr. 2-3-6). Fr. 2-3-3 was further purified with semi-preparative HPLC (45% CH3OH/H2O with 0.4‰ trifluoroacetic acid, 2.5 mL min?1) to gain 11 (3.19 mg,R= 34.0 min). Fr. 2-3-6 was further purified with semi-preparative HPLC (50.5% CH3OH/H2O, 3 mL min?1) to obtain 10 (31.67 mg,R= 7.8 min). Fr. 2-5 was subjected to a Sephadex LH-20 column eluted with MeOH, and reversed-phase C-18 MPLC with MeOH/H2O (10%– 100%) to get seven sub-fractions (Fr. 2-5-1 – Fr. 2-5-7). Fr. 2-5-6 was subjected by semi-preparative HPLC (28.5% CH3CN/ H2O, 3 mL min?1) to afford 3 (2.79 mg,R= 27.3 min), 4 (1.61 mg,R= 16.8 min), 5 (9.53 mg,R= 20.8 min), 6 (9.35 mg,R= 32.0 min) and 7 (2.23 mg,R= 52.5 min). Fr. 2-5-7 was further purified with semi-preparative HPLC in 3 mL min?1with different mobile phase to obtain 8 (9.84 mg,R= 28.2 min, 35% CH3CN/H2O), 9 (3.25 mg,R= 29.1 min, 45% CH3CN/H2O) and 12 (2.90 mg,R= 31.8 min, 55% CH3OH/H2O), respectively.

2.4 Spectral Data

()-2-(hydroxyimino)-4-methylpentanamide (1): White crystal; UV (MeOH) λmax(log ε) 206.4 (4.11); IR (film) νmax3327, 2957, 2833, 1674, 1456, 1418, 1022, 826, 762, 648, 600 cm?1;1H NMR (DMSO-6, 700 MHz) and13C NMR (DMSO-6, 175 MHz) data, see Table 1; HR-ESI- MS/[M+H]+peak at 145.0967 (calcd for C6H13N2O2, 145.0972).

4-hydroxy-7-methyl-6,7-dihydro-5-cyclopenta[c]pyridin-5-one (2): Yellow solid; UV (MeOH) λmax(log ε) 205.6 (4.45), 247.6 (3.71), 314.2 (3.27); IR (film) νmax3341, 2916, 2849, 1653, 1541, 1456, 1018, 667, 600 cm?1;1H NMR (DMSO-6, 700 MHz) and13C NMR (DMSO-6, 175 MHz) data, see Table 1; HR-ESI-MS/[M–H]-peak at 162.0555 (calcd for C9H8NO2, 162.0561).

2.5 X-Ray Crystallographic Analysis

Crystallographic Data (CDCC: 2169023) for ()-2-(hy- droxyimino)-4-methylpentanamide (1): Moiety formula: C6H12N2O2(M = 144.18 g mol?1), triclinic, space group P-1 (no. 2),= 5.0511(4) ?,= 5.6913(4) ?,= 13.7542(9) ?,= 98.423(5)?,= 93.358(6)?,= 105.221(6) ?,= 375.46(5) ?3,= 2,= 100.00(10) K, μ(Cu Kα)= 0.800 mm?1,= 1.275 g cm?3, 2745 reflections measured (13.082? ≤ 2Θ ≤ 147.75?), 1437 unique (int= 0.0269,sigma= 0.0373) which were used in all calculations. The final1was 0.0459 (I > 2σ(I)) and2was 0.1255.

2.6 Antimicrobial Activity Assay

Compounds 1-12 were tested for antibacterial activities against five pathogenic bacteria of(ATCC 29213), methicillin-resistant(MRSA),(ATCC 29212),(ATCC 25922) as well as(ATCC 13883) and antifungal activities against five plant pathogen fungi (,,,, and) using the agar filter-paper diffusion method.

2.7 Enzyme Inhibitory Assays

All compounds (1–12) were tested for neuraminidase inhibitory activity by using Neuraminidase Inhibitors Screen Kit (Beyotime Biotechnology, China). Briefly, 10 μL of purified N1-typed neuraminidase was added to 70 μL of detection buffer, followed 115 by adding 10 μL of a test compound and 10 μL of neuraminidase substrate sequentially. After incubation at 37℃ for 30 min, the fluorescence intensity was measured at an excitation wavelength of 340 nm and an emission wavelength of 535 nm using a microplate reader.

The AChE inhibitory activity was determined by a classical spectrophotometric method (Yang., 2019). Briefly, the enzymatic reaction was conducted in a 200 μL volume reaction system consisting of 0.1 U mL?1AChE (Sigma-Aldrich, CAS No.: 9000-81-1), 6.25 mmol L?15,5’-dithiobis (2-nitro-benzoic acid) (DTNB, Sigma- Aldrich, CAS No.: 69-78-3), 6.25 mmol L?1acetylthiocholine iodide (Sigma-Aldrich, CAS No.: 1866-15-5) and PBS (pH 8.0) in 96-well microplates. DMSO solutions of the tested compounds were added to the assay solution and incubated for 20 min, followed by the measurement of absorbance at 405 nm with a microplate reader. All test and control assays were corrected by blanks for nonenzymic hydrolysis. Tacrine was used as the positive control and all assays were performed in three replicates.

PI3K proteins were expressed and purified as previously reported (Gong., 2017). All compounds were carried out in black 384-well microtiter plates (PerkinElmer, Boston, MA, USA) using the 3-step PI3K homogenous time-resolved fluorescence (HTRF) bioassay (Millipore, Burlington, MA, USA) following the manufacturer’s instructions. Briefly, 0.5 μL of compounds were preincubated with 14.5 μL of enzyme and PIP2substrate for 10 min, before addition of 5 μL of ATP to achieve a final ATP concentration of 10 μmol L?1. The total reaction volume was 20 μL, and the reaction was allowed to proceed for 45 min at room temperature before the addition of stop solution and detection mixture provided in the kit (Millipore). The plates were then incubated for 3 h in the dark and read using an EnVision Multilabel Plate Reader (PerkinElmer; 320/620/665). All assays were performed in duplicate to confirm reproducibility.

3 Results and Discussion

3.1 Structural Detemination

Compound 1 was afforded as white crystal. It had the molecular formula of C6H12N2O2as inferred from HRESIMS at/145.0967 [M+H]+(calcd. for 145.0972) and NMR data (Table 1). The1H NMR data (Table 1) atH2.35 (2H, d,= 7.3 Hz, H2-3), 1.91 (1H, m, H-4), 0.84 (6H, d,= 6.8 Hz, H3-5, 6) and13C NMR data (Table 1) atC31.6 (C-3), 25.9 (C-4), 22.6 (C-5, 6) and HMBC (Fig.2a) correlations between H-4 and C-3/C-5/C-6 as well as COSY correlations of H2-3/H-4/H3-5/H3-6 suggested that 1 contained an isobutyl group. In the HMBC spectrum (Fig.2a), correlations from H2-3 to C-1, C-2 and C-4, and from 7-OH and 1-NH2to C-2, suggested the existence of 2-(hydroxyimino) butanamide unit. Fortunately, single crystals were obtained for compound 1 in MeOH. And then single-crystal X-ray diffraction experiment of 1 not only allowed the above deductions but also determined theconfiguration of oxime moiety (Fig.2b). Therefore, 1 was determined as ()-2-(hydroxyimino)-4- methylpentanamide.

Fig.2 (a) COSY and key HMBC correlations of compound 1. (b) ORTEP drawings of compound 1.

Table 1 1H and 13C NMR Data for 1 and 2 (700, 175 MHz, DMSO-d6, δ ppm)

Compound 2 was obtained as yellow solid, and it possessed the elemental composition of C9H9NO2, (6 degrees of unsaturation) as established by a protonated molecule at162.0555 in the HRESIMS spectrum and its13C NMR data (Table 1). Its1H NMR data showed two olefinic protons atH7.53 (brd,= 1.8 Hz, H-3), and 6.24 (brs, H-1), one methine proton atH2.98 (m, H-7), one methylene atH2.58 (dd,= 17.9, 7.5 Hz, H-6a), and 1.92 (dd,= 17.9, 3.3 Hz, H-6b), and one methyl atH1.19 (d,= 7.1 Hz, H3-8). The13C NMR spectra (Table 1) exhibited 9 carbon resonances, including four quaternary carbons (including one ketone, one oxygenated and two olefinic), three methines (including two olefinic), one methylenes, and one methyls. The two methines atC146.9 (C-3) and 115.0 (C-1) and three quaternary carbons atC171.1 (C-4), 159.2 (C-7a) and 133.8 (C-4a) suggested a 1,4a,7a-trisubstituted pyridine nucleus that was further supported by HMBC correlations from H-1 to C-3 and C-4a, and from H-3 to C-4 and C-4a.1H-1H COSY from H3-8 to H2-6 through H-7 and HMBC correlations from H2-6 to C-5 indicated a CH3-CH-CH2-C=O moiety in the molecule. These data indicated a cyclopentano[] pyridine skeleton that was further supported by the key HMBC correlations of H3-8 to C-7a, of H-7 to C-7a, of H2-6 to C-7a, and of H-1 to C-4a and C-7 (Fig.3). Compared with 3-hydroxy-5-methyl-5,6-dihydro-7-cyclo- penta[]pyridin-7-one (Dai., 2015), the C-5 (C198. 1) and C-7a (C133.8) of 2 were shifted downfield resulting from the conjugate effects and the electron-with- drawing effects of 4-OH. Notably, the specific optical rotation value of 2 was almost zero, suggesting a racemic mixture. Subsequent chiral HPLC analysis of 2 showed that compound 2 was a pair of enantiomers with about a 1:1.5 ratio. However, it was difficult for the enantiomers to be baseline separated under the chromatographic conditions. Thus, the structure of 2 was elucidated as 4-hy- droxy-7- methyl-6,7-dihydro-5-cyclopenta[c]pyri-din-5-one (2).

Fig.3 COSY and key HMBC correlations of compound 2.

All the isolated compounds were evaluated for their antibacterial, antifungal and enzyme inhibitory activities against acetylcholinesterase (AChE), neuraminidase (NAs), and phosphatidylinositol 3-kinase (PI3K). In the test of enzyme inhibitory activity against neuraminidase, oseltamivir acid was selected as a positive control with IC50value of 20 μmol L?1. Compounds displaying inhibitory effects against-glycosidase with values > 50% at 100 μg mL?1were further evaluated their IC50values. Compounds 1 and 3 showed more potent inhibitory activity than oseltamivir acid with IC50values of 12.04, 1.92 μmol L?1and compounds 5, 6, 8, and 10 exhibited moderate inhibitory activity of 83.30%, 91.46%, 75.72% and 77.46% at 100 μg mL?1, respectively. Tacrine as the positive control in the test of enzyme inhibitory activity against AChE showed inhibition ratio of 96% at 0.33 μmol L?1. Only compound 6 showed weak enzyme inhibitory activities against AChE of 52.14% at 50 μg mL?1. ZSTK474 was selected as a positive control with IC50value of 45.4 nmol L?1in the test of enzyme inhibitory activity against PI3K. Compounds 6-10 showed weak enzyme inhibitory activities against PI3K with IC50values of 1.07, 4.41, 1.93, 2.90, and 3.32 μmol L?1, respectively. None of the isolated compounds displayed inhibitory activity against the selected five plant pathogenic fungi and five pathogenic bacteria at concentration of 50 μg mL?1.

To investigate the inside interactions between compound 3 and NAs, a preliminary molecular docking study was conducted in AutoDock Vina. The highest scoring conformation was obtained as shown in Fig.4a. Compound 3 was fully embedded in the protein. The analysis of the predicted result revealed that compound 3 could tightly bind inside the active pocket of NAs with the calculated binding energy of ?10.4 kcal mol?1. The trithreo- nine lactones with three benzene rings groups of 3 stre- tched into the catalytic pocket (Fig.4b) which consisted of eleven residues. This catalytic pocket was the same as that oseltamivir and zanamivir acid would bind to when exerting its inhibitory effect against NAs (Russell., 2006). In the active site, ten hydrogen bonds between 3 and ten residues Arg-118, Ser-246, Asn-247, Asn-294, Tyr-347, Asn-369, Arg-371, Arg-371, Ile-427 and Lys- 432 with the lengths of 3.62, 2.80, 3.07, 3.83, 4.02, 2.75, 3.20, 3.87, 3.76 and 2.28 ?, respectively, were observed in 3D molecular binding mode (Fig.4b). In addition, the benzene ring with two hydroxyls of 3 formed a π-π T-shaped interaction with Tyr-347 as well as the trithreonine lactones of 3 formed two salt bridges interaction with Arg-118 and Arg-371. All above interactions contributed to the strong anchoring of 3 to the binding site of NAs, and provided an inside perspective of the action of 3, and a better understanding of the enzyme inhibitory activity.

Fig.4 (a) 3D molecular surface of NAs docked with compound 3. (b) Detailed binding mode of compound 3 with the residues in catalytic pocket of NAs.

4 Conclusions

In this study, a new mycotoxin, ()-2-(hydroxyimino)- 4-methylpentanamide (1) and a new cyclopentano[] pyridine, 4-hydroxy-7-methyl-6,7-dihydro-5-cyclopea- nta[c]pyridin-5-one (2), together with ten known com- pounds (3 – 12), were isolated from the marine-derived fungussp. SCSIO 41305. Extensive NMR spectroscopic analysis and X-Ray crystallographic analysis were used to elucidate the structure of ()-2-(hydro- xyimino)-4-methylpentanamide (1), including its absolute configuration. All the isolated compounds (1 – 12) were evaluated for their antimicrobial activities and enzyme inhibitory activities against AChE and NAs. Among them, only compound 6 showed weak inhibitory activity against AChE. Compounds 1, 3 exhibited strong neuraminidase inhibitory activity with IC50values of 12.04, 1.92 μmol L?1(IC5020 μmol L?1for oseltamivir acid), while compounds 5, 6, 8, and 10 showed moderate neuraminidase inhibitory activity. Furthermore, an in silico molecular docking study was also performed between 3 and NAs.

Acknowledgements

This work was financially supported by the Guangdong Basic and Applied Basic Research Foundation (Nos. 2021 A1515011523, 2021B1515120046), the Guangdong MEPP Funds (No. GDNRC[2021]48), the Finance Science and Technology Project of Hainan Province (No. ZDKJ2020 18), National Natural Science Foundation of China (No. 41776169). We are grateful to Drs. Xiao, Z. H., Sun, A. J., Zheng, X. H., Zhang, Y., and Ma, X., in the analytical facility at SCSIO for recording spectroscopic data.

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(May 19, 2022;

January 2, 2023;

February 20, 2023)

? Ocean University of China, Science Press and Springer-Verlag GmbH Germany 2023

. E-mail: wangjunfeng@scsio.ac.cn

(Edited by Ji Dechun)