New Naphthalene Derivatives from the Mangrove Endophytic Fungus Daldinia eschscholzii MCZ-18

Five new naphthalene derivatives dalesconosides A–D, F (1–4, 6), a known synthetic analogue named dalesconoside E (5), and eighteen known compounds (7–24) were isolated from Daldinia eschscholzii MCZ-18, which is an endophytic fungus obtained from the Chinese mangrove plant Ceriops tagal. Differing from previously reported naphthalenes, compounds 1 and 2 were bearing a rare ribofuranoside substituted at C-1 and the 5-methyltetrahydrofuran-2,3-diol moiety, respectively. Their structures were determined by detailed nuclear magnetic resonance (NMR) and mass spectroscopic (MS) analyses, while the absolute configurations were established by theoretical electronic circular dichroism (ECD) calculation. Compounds 1, 3, 13–17 and 19 showed broad ranges of antimicrobial spectrum against five indicator test microorganisms (Enterococcus faecalis, Methicillin-resistant Staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa and Candida albicans); especially, 1, 16 and 17 were most potent. The variations in structure and attendant biological activities provided fresh insights concerning structure−activity relationships for the naphthalene derivatives.

synthetic analogue named dalesconoside E (5) (this is the first isolation from a natural source), as well as 18 known compounds (7-24) (Figure 1) were isolated and identified from the mangrove endophytic fungus Daldinia eschscholzii MCZ-18.These compounds were examined for antimicrobial and cytotoxic activities.Details of the isolation, structure elucidation, and antimicrobial and cytotoxic activities of these compounds are reported herein.
Dalesconoside E (5) was obtained as a red amorphous powder.Its molecular formula C 23 H 18 O 6 (i.e., differing from that of 4 by an additional OCH 3 group) was established from HRESIMS at m/z 391.1177 [M+H] + .The 1 H and 13 C NMR data (Tables 1 and 2) of 5 were similar to those of 4 except for the significant downfield shift of C-1(δ C 147.9) and the presence of a methoxy signal (δ H 3.96, s, δ C 56.0, q, 1-OCH 3 ) indicative of 5 was a methoxylated analogue of 4. The HMBC correlations (Figure 2) of 1-OCH 3 with C-1 revealed that the extra methoxy group is bound to C-1.Hence, 5 was 1-methoxydalesconoside D (Figures S33-S40).Compound 5 is here reported for the first time as a natural product.It was previously prepared via a two-step sequence involving the oxidative dimerization of hydroquinone monomethyl ethers [21].

Biological Activity
Compounds 1-24 were tested for antimicrobial activity against Pseudomonas aeruginosa, Enterococcus faecalis, methicillin-resistant Staphylococcus aureus and Escherichia coli as well as antifungal activity against Candida albicans.As shown in Table 3, all metabolites showed antimicrobial activity against at least one of the five tested indicator pathogens at the selected concentration of 50 µg/mL, but all of the antimicrobial activities were weaker than that of the positive control.Compounds 1, 3, 13-17 and 19 exhibited a wide range of antimicrobial activities; especially, 1 (against P. aeruginosa and E. coli), 16 (against E. coli) and 17 (against MRSA) were most potent, and their resistant strain with MIC values reached 6.25 µg/mL.The comparison between compounds 1 and 7 approved that the substitution of additional furanose was the critical structural component for its antimicrobial activity.The antibacterial results of the comparison of 2-16 suggested that the dihydronaphthalen-1-one nucleus may not be sensitive to the tested microbes in some cases, but the extra auxochromes, e.g., -OCH 3 , -OH or CH 3 enhanced the antibacterial effect of the compound against the tested bacteria and fungi.Meanwhile, when the hydroxyl group is oxidized to a carbonyl group to a certain extent, the antibacterial activity of the metabolite against some indicator bacteria is weakened.Comparing the antimicrobial activities of compounds 17-22 indicated that the O-containing heterocyclic substructure may increase the compound's effect on P. aeruginosa, E. faecalis, MRSA and E. coli.When the -OH and -OCH 3 groups are differently substituted on the benzene ring or the lactone/quionone of metabolites' isocoumarin backbone, different indicator bacteria have different promotion and inhibition.In general, the broad antimicrobial activities of these naphthalene derivatives support their continued investigation so as to develop a deeper understanding of structure−activity relationships and mechanism of antimicrobial action.

General Experimental Procedure
Optical rotation was recorded at 20 • C using a WYA-2S digital Abbe polarimeter (Shanghai Physico-optical Instrument Factory, Shanghai, China).UV spectral data were obtained from online UV spectra acquired on a Shimadzu UV-2401 PC spectrophotometer.NMR spectra were recorded on a Bruker AV-400 and AV-500 (Bruker Corporation, Fällanden, Switzerland) instrument with TMS as an internal standard.ESI-MS spectra were recorded on a VG Auto Spec-3000 mass spectrometer (VG, Manchester, UK).Highresolution ESI-MS were recorded on an Agilent 6210 mass spectrometer (Agilent Technologies, Waldbronn, Germany) employing peak matching.The ECD spectra were measured on a JASCO J-715 spectra polarimeter (Japan Spectroscopic, Tokyo, Japan).SephadexLH-20 (Beijing Biotopped Science & Technology Co., Ltd., Beijing, China), SiliaSphere C18 (Sili-Cycle, Quebec, Canada) and Silica gel (200-300 mesh, Qingdao Marine Chemical Factory, Qingdao, China) were used for column chromatography (CC).Thin-layer chromatography (TLC) was performed on precoated silica gel GF254 plates (Qingdao Marine Chemical Factory, Qingdao, China).

Fungal Material
The strain MCZ-18 was isolated from a fresh, healthy branch of Ceriops tagal collected from the Dong Zhai Gang-Mangrove Garden on Hainan Island, China.The fungus was isolated under sterile conditions from the inner tissue of the flower following an isolation protocol described previously [37] and identified as Daldinia eschscholzii (GenBank accession no.MH712260) by morphologic traits and molecular identification.A voucher strain was deposited in the School of Chemical Engineering and Technology, Hainan University, Haikou, China.

Fermentation, Extraction and Isolation
The Daldinia eschscholzii MCZ-18 strain was cultivated on an autoclaved rice solidsubstrate medium, consisting of 80 1 L Erlenmeyer flasks.Each flask included 80 g of rice, 0.24 g of sea salt and 80 mL of water.The culture was maintained at a temperature of 25 • C. Following a fermentation period of 29 days, the mycelia and rice were subjected to three extractions using EtOAc.The extracts underwent filtration and evaporation under decreased pressure to yield a crude extract of 75 g.The crude extracts were then submitted to vacuum liquid chromatography (VLC) on silica gel employing a step gradient of petroleum ether/ethyl acetate (1:0-0:1, v/v) to obtain five fractions (Fr.1-Fr.5).Fr.1 was eluted equivalently with petroleum ether to give 8 (200 mg).Fr.2 was eluted equivalently with petroleum ether / ethyl acetate (50:1, v/v) to obtain 7 (150 mg).Fr.3 was eluted with a step gradient of petroleum ether/ethyl acetate (20:1, 10:1, 5:1, 3:1, 1:1, v/v) to give 3 fractions (Fr.

ECD Calculations
The Monte Carlo conformational searches were carried out by means of the Spartan's 14 software (v1.1.4)using a Merck Molecular Force Field (MMFF).The conformers of 1-3 and 6 with a Boltzmann population of over 5% were chosen for ECD calculations, and then the conformers were initially optimized at B3LYP/6-31g (d, p) in gas.The theoretical calculation of ECD was conducted in MeOH using time-dependent density functional theory (TD-DFT) at the B3LYP/6-31+g (d, p) level for all conformers of compounds.Rotatory strengths for a total of 30 excited states were calculated.ECD spectra were generated using the program SpecDis 1.6 (University of Würzburg, Würzburg, Germany) and Graph-Pad Prism 5 (University of California San Diego, CA, USA) from dipole-length rotational strengths by applying Gaussian band shapes with sigma = 0.3 eV.

Antimicrobial Activity Assay
The microplate assay method was used to assess the antimicrobial activities of compounds 1-24 against four terrestrial pathogenic bacteria (Pseudomonas aeruginosa, Methicillin-resistant Staphylococcus aureus, Bacillus subtilis and Escherichia coli) and one pathogenic fungus (Candida albicans) (Guangdong Microbial Culture Collection Center,

Table 3 .
Antimicrobial activities of isolated compounds 1