Meroterpenoids and Isocoumarinoids from a Myrothecium Fungus Associated with Apocynum venetum

Four new meroterpenoids 1–4 and four new isocoumarinoids 5–8, along with five known isocoumarinoids (9–13), were isolated from the fungus Myrothecium sp. OUCMDZ-2784 associated with the salt-resistant medicinal plant, Apocynum venetum (Apocynaceae). Their structures were elucidated by means of spectroscopic analysis, X-ray crystallography, ECD spectra and quantum chemical calculations. Compounds 1–5, 7, 9 and 10 showed weak α-glucosidase inhibition with the IC50 values of 0.50, 0.66, 0.058, 0.20, 0.32, 0.036, 0.026 and 0.37 mM, respectively.


Introduction
Since the discovery of penicillin, fungi have been an important source of lead compounds for drug development, which have provided a lot of attractive natural products (NPs) with different biological activities [1][2][3]. With the increase of study on the terrestrial fungal NPs, more and more known compounds were isolated repeatedly. Therefore, many researchers turned their attention to the fungi isolated from specific habitats, such as the marine-derived fungi [4][5][6][7] and the fungi associated with the plants or animals [8][9][10][11].
The molecular formula of 4 was assigned as C 23 H 32 O 5 by the HRESIMS peak at m/z 411.2139 [M + Na] + ( Figure S58D), which was C 2 H 2 O less than that of 3. The similarity of the UV and NMR data between 3 and 4 (Table 1) suggested that 4 possesses the same skeleton as 3. Careful comparison of their 1 H and 13 C NMR spectra ( Figures S26-S31) showed that the acetyloxy group (δ C 21.0/δ H 1.98 and δ C 169.8) in 3 was replaced by a hydroxy group (δ H 4.13) in 4 ( Table 1). The NOESY data ( Figure 3 and Figure S32) suggested that 4 has the same relative configuration as 3. The ECD Cotton effects of 4 were nearly identical to those of 3 ( Figure 5), indicating the same absolute configurations of the corresponding stereogenic carbons. Thus, 4 was named myrothecisin D.
Mar. Drugs 2018, 16, x 4 of 12 Compound 3 was also obtained as a pale-yellow oil. Its molecular formula was determined as C25H34O6 according to the HRESIMS peak at m/z 453.2238 [M + Na] + ( Figure S58C). The 13 Table 1). Analysis of its 1D and 2D NMR (Figures S19-S24) data revealed the presence of a substituted benzene ring and a sesquiterpene unit, indicating 3 was an analogue of 1 and 2. Comparison of the 1 H and 13 C NMR spectra with those of 1 and 2 suggested a same pentasubstituted benzene ring. The structure of the sesquiterpene unit was slightly modified and was determined by the COSY (Figure S23 Figure S2). Therefore 3 was named myrothecisin C.
Compound 6 was obtained as a white powder. Its molecular formula was determined as C 12 H 10 O 7 based on the HRESIMS peak at m/z 265.0355 [M − H] − ( Figure S58F). The UV and 13 C NMR data of 6 ( Table 2) were similar to those of 5, indicating that they have the same isocoumarin scaffold. Comparison of their 1 H and 13 C data ( Figures S39-S43) indicated that the hydroxymethyl group (δ C/H 64.8/3.62&3.51, δ H 4.81) in 5 was replaced by the carboxyl group (δ C 173.4). This change was verified by the key HMBC ( Figure S44) correlations from H-11 to C-3/C-10/C-12. The absolute configuration of C-11 of 6 was determined as 11R-by comparison of the calculated and experimental ECD spectra ( Figure 4 and Figure S4). Therefore, 6 was name myrothelactone B.             Compound 7 was obtained as a white powder. Its molecular formula was determined as C 12 H 10 O 6 according to its HRESIMS peak at m/z 249.0408 [M -H] − (Figure S58G), which was only two hydrogen atoms less than that of 5. The difference observed in the NMR spectra of 7 and 5 was that the signals for hydroxymethine (δ C/H 68.8/4.66) in 5 were replaced by the signal of a carbonyl group (δ C-11 198.3) in 7 ( Table 2, Figures S45-S49). The HMBC ( Figure S50) correlations from H-3 (δ H 8.47) and H-12 (δ H 4.57) to C-11 further confirmed the structure of 7 which was name myrothelactone C ( Figure 2).
The α-glucosidase inhibitory activity of 1-13 was preliminarily investigated. Compounds 1-5, 7, 9 and 10 exhibited inhibitory activity against the human-sourced α-glucosidase recombinant expressed in Saccharomyces cerevisiae [31][32][33] with IC 50 values of 0.50, 0.66, 0.058, 0.20, 0.32, 0.036, 0.026 and 0.37 mM, while the IC 50 value of positive control acarbose was 0.47 mM. Due to the low activity, the deeper investigation of the mechanism and type of enzymatic inhibition as well as the binding mode were not done.

Collection and Phylogenetic Analysis
The fungus OUCMDZ-2784 was isolated from Apocynum venetum (Apocynaceae) collected from the estuary of Yellow River, Dongying, China. The leaves of the plant were washed with tap water and sterile distilled water in sequence. Then, it was cut into small pieces, which were then put into a centrifuge tubes filled with different concentrations of sucrose solution. These tubes were centrifuged at 1200 rpm for 20 min. Four zones were separated by improved discontinuous sucrose gradient centrifugation. The interface between the third and the fourth bands was deposited on a PDA (200 g potato, 20 g glucose, 20 g agar per liter of sea water) plate containing chloramphenicol (100 µg/mL) as a bacterial inhibitor and was then cultured at 28 • C for 3 days. A single colony was transferred to PDA agar media and was identified as Myrothecium sp. according to its morphological characteristics and 18S rRNA gene sequences (GenBank accession No. KF977010).

Cultivation and Extraction
Fungus OUCMDZ-2784 was prepared on PDA agar medium. Spores were incubated at 28 • C for 48 h on a rotary shaker with shaking at 120 rpm in a 500 mL cylindrical flask containing 150 mL liquid medium (20 g maltose, 20 g mannitol, 10 g glucose, 3 g yeast extract, 10 g monosodium glutamate per liter of sea water). The cultures were transferred to 350 × 1000 mL Erlenmeyer flasks and each containing 300 mL liquid fermentation media (1 g peptone, 10g soluble starch per liter of sea water, pH 7.0). The flasks were incubated at room temperature under static conditions for 30 days. The cultures were extracted three times by EtOAc and the combined EtOAc extracts were dried in vacuo to yield 20.1 g of extract.

Purification
The extract (20.1 g) was fractionated by VLC, eluting with a step gradient of CH 2 Cl 2 -petroleum ether (50-100%) and MeOH-CH 2 Cl 2 (0-50%) and five fractions (Fr.    were collected on a CCD area detector diffractometer with graphite monochromated Cu Kα radiation (λ = 1.54178 Å). The structure was solved by direct methods (SHELXS-97) and expanded using Fourier techniques (SHELXL-97). The final cycle of full-matrix least squares refinement was based on 1478 unique reflections (2θ < 50 • ) and 165 variable parameters and converged with unweighted and weighted agreement factors of R 1 = 0.0326, wR 2 = 0.0885 and R = 0.0880 for I > 2sigma(I) data. Absolute structure parameter: −0.2 (2). The deposited number of compound 5 in the Cambridge Crystallographic Data Centre is 980155.

α-Glucosidase Inhibitory Assays
The human-sourced α-glucosidase was recombinant expressed in the yeast Saccharomyces cerevisiae and the inhibitory effects of compounds 1-13 were tested using p-nitrophenyl-α-D-glucopyranoside (pNPG) as substrate [31][32][33]. The sample was dissolved in sodium phosphate buffer (PBS, pH 6.8) at three concentrations. 10 µL of the sample solution, 20 µL of 2.5 mM pNPG solution (in phosphate buffer) and 20 µL of PBS were mixed in a 96-well microplate and incubated at 37 • C for 5 min. A volume of 10 µL of α-glucosidase diluted to 0.2 U/mL by 0.01 M PBS was then added to each well. After incubating at 37 • C for 15 min, the absorbance at 405 nm was recorded by a Spectra max 190 micro plate reader (Molecular Devices Inc., San Jose, CA, USA). The blank was prepared by adding phosphate buffer instead of the α-glucosidase and the positive control was acarbose. Blank readings (no enzyme) were subtracted from each well and results were compared to the control. The inhibition (%) was calculated as [1 − (OD drug /OD blank )] × 100%. The IC 50 value was calculated as the compound concentration that is required for 50% inhibition and the IC 50 value of acarbose was 0.47 mM.