New Meroterpenoids and α-Pyrone Derivatives Isolated from the Mangrove Endophytic Fungal Strain Aspergillus sp. GXNU-Y85

Two new meroterpenoids, aspergienynes O and P (1 and 2), one new natural compound, aspergienyne Q (3), and a new α-pyrone derivative named 3-(4-methoxy-2-oxo-2H-pyran-6-yl)butanoic acid (4) were isolated from the mangrove endophytic fungal strain Aspergillus sp. GXNU-Y85, along with five known compounds (5–9). The absolute configurations of those new isolates were confirmed through extensive analysis using spectroscopic data (HRESIMS, NMR, and ECD). The pharmacological study of the anti-proliferation activity indicated that isolates 5 and 9 displayed moderate inhibitory effects against HeLa and A549 cells, with the IC50 values ranging from 16.6 to 45.4 μM.


Introduction
Endophytic fungi, as a type of fungi, persist for an extended period in the healthy tissues and organs of plants, which can be regarded as a novel resource of microorganisms with high exploitation value and an important source of natural active substances [1,2].Therefore, the development and utilization of endophytic fungi can alleviate the shortage of resources and the destruction of ecological balance caused by extracting and separating a large number of beneficial bioactive products from plants, and also help protect rare and endangered plant resources [3].Some secondary metabolites isolated from endophytic fungi have also drawn attention owing to their superior biological activities.Consequently, these secondary metabolites might be used as promising lead compounds for drug discovery [4].
In the NOESY spectrum of 1 (Figure 3), there is a diagnostic association between the olefinic proton [δH 6.72 (m, H-13)] and the terminal methyl proton [δH 1.87 (s, H3-15)], and thus the geometry of the Δ 13,14 double bond was defined as Z.Furthermore, the NOESY signals of H-1/H-12β suggested that H-1 and H-12β were β-oriented.Meanwhile, the small value of JH-3/H-4 (2.5 Hz) demonstrated a cis-orientation of these protons.The final configuration of 1 was elucidated as (1S,2S,3R,4R)-1 by means of comparing its experimental ECD spectrum to those calculated (Figure 4).

Results of Antiproliferative Activity
The MTT method was used to evaluate the anti-proliferative activity of all isolates on five human cancer cell lines.Among them, 9 displayed moderate anti-proliferation activity on HeLa cancer cells (IC 50 =16.6µM); moreover, 5 displayed anti-proliferation activity on the HeLa and A549 cancer cell lines (IC 50 = 29.3µM and IC 50 = 45.4 µM, respectively).The other isolates displayed no notable anti-proliferation activity on five cancer cell lines (IC 50 > 50 µM).The positive control was etoposide (IC 50 : 15.7 µM for HeLa cells, 8.2 µM for A549 cells).

General Experimental Procedures
The NMR data were measured by Bruker 400 MHz and 600 MHz instruments (Bruker, Bremen, Germany).The HR-ESI-MS reports and the Optical rotations were collected using an LC-MS spectrometer (Agilent 6545 Q-TOF) and a JASCO P-2000 polarimeter (Jasco, Tokyo, Japan), respectively.The other instruments and materials employed in the experiments were the same as those in our previous reports [15].

Fungal Material
According to the sequence and morphology of the internal transcriptional spacer (ITS) of the strain, the fungal strain collected from fresh fruits of the mangrove plant Kandelia candel in the Beihai was identified and designated as GXNU-Y85.Subsequently, we obtained its registration number OR999402, as we submitted the ITSrDNA of GXNU-Y85 to GenBank.

ECD and NMR Calculations
The absolute stereochemistry of the new isolates was defined through the ECD and NMR calculations described in previous reports [16,17].The Supporting Information provides a detailed description of the process.

Conclusions
In the present study, three new compounds (1, 2, and 4), one new natural compound (3), and five previously reported isolates (5)(6)(7)(8)(9) were acquired from the fungal strain Aspergillus sp.GXNU-Y85 by various chromatographic techniques.Extensive spectroscopic data (HRESIMS, NMR, and ECD) and quantum chemical calculations were used to determine the structures of these new compounds.Biological activity studies revealed that compounds 5 and 9 showed moderate anti-proliferative activity against HeLa and A549 cells (IC 50 = 16.6-45.4µM).

Figure 5 .
Figure 5. Conformations of low-energy conformers of structures 2a and 2b in MeOH.

Figure 6 .
Figure 6.Regression analyses of experimental versus calculated 13 C NMR chemical shifts of model compounds 2a and 2b.

Figure 5 .
Figure 5. Conformations of low-energy conformers of structures 2a and 2b in MeOH.

Figure 5 .
Figure 5. Conformations of low-energy conformers of structures 2a and 2b in MeOH.

Figure 6 .
Figure 6.Regression analyses of experimental versus calculated 13 C NMR chemical shifts of model compounds 2a and 2b.

Table 1 .
1H and 13 C NMR Data for 1 and 2 in CD 3 OD.