HETEROCYCLES

Short Paper | Regular issue | Vol. 89, No. 7, 2014, pp. 1662-1669
Received, 9th April, 2014, Accepted, 23rd May, 2014, Published online, 28th May, 2014.
DOI: 10.3987/COM-14-13004

■ Speradines B-E, Four Novel Tetracyclic Oxindole Alkaloids from the Marine-Derived Fungus Aspergillus oryzae

Xiao Hu, Qi-Wen Xia, Yang-Yang Zhao, Qiu-Hong Zheng, Qin-Ying Liu, Li Chen,* and Qi-Qing Zhang*

College of Chemistry and Chemical Engineering, Institute of Biomedical and Pharmaceutical Technology , Fuzhou University, No. 523, Gongye Road, Fuzhou City, 350002, China

Abstract

Four novel tetracyclic oxindole alkaloids, namely, speradines B (1), C (2), D (3), and E (4) were isolated from the marine-derived fungus Aspergillus oryzae. The structures of these compounds were elucidated through 1D and 2D nuclear magnetic resonance and high-resolution mass spectrometric analyses. Among these compounds, 1 and 4 showed weak cytotoxic effects on the hela cell line, with IC50 values of 57.2 and 73.4 μg/mL, respectively.

Indole-terpenes are fungal and bacterial secondary metabolites with unique biological activities such as inhibitory activity of calcium-activated potassium channels,1 antitumor activity,2 tremorgenic activity,3 potent and selective progesterone receptor agonistic activity,4 and anti-MRSA activity.5 Cyclopiazonic acid (CPA)-type alkaloids are one important group of indole-terpenes, and they usually contain three structural units: an indole, a dimethylallyl (DMA), and two acetic acids. Only six analogous natural products have been reported to date, including α-cyclopiazonic acid,6 iso-α-cyclopiazonic acid,7 β-cyclopiazonic acid,6 α-acetyl-γ-(β-indolyl)methyl-tetramic acid,8 speradine A,9 and 3-hydroxyl speradine A.10 In our search for novel anticancer compounds,11 a strain of Aspergillus oryzae showed significant cytotoxic activity. Further chemical study led to isolation and structure elucidation of four new CPA-type alkaloids (1–4), namely speradines B (1), C (2), D (3), and E (4). The isolation, structure elucidation, and bioactivity of compounds 1–4 are presented in the current study.

Compound 1, which was obtained as yellow oil, was analyzed to have the molecular formula C16H18N2O3 through positive high-resolution electrospray ionization mass spectroscopy (HRESIMS) (m/z: 287.1396 [M + H]+, Calcd. for C16H19N2O3, 287.1390). This compound was subjected to 1H and 13C NMR (Tables 1 and 2), DEPT and HMQC spectral analyses. Results showed that 1 had sixteen carbon signals, including three methyls, one methylene, five methines, and seven quaternary carbons. The plane structure of 1 was determined using COSY and HMBC spectrum analyses (Figure 2). The COSY correlations of H-8 with H-4 and H-9, and H-11 with H-10 and H-12 demonstrated the connections from H-4 to H-9 via H-8 and from H-10 to H-12 via H-11, respectively. The HMBC correlations of H-4 with C-3 and C-3a; H-8 with C-9a; H-9 with C-3a and C-9a; H-10 with C-9 and C-3a; H-11 with C-12a; and H-12 with C-3a connected rings A and B. The HMBC correlations of H-4 with C-5; H-6 with C-4, C-7, and C-8; H-8 with C-14; and H-14 with C-7 and C-15 linked rings C and B. Since the unsaturation degree of 1 was nine, an additional ring was necessary. After carefully comparing the chemical shifts of C-2, C-3, C-3a, C-12a, and Me-13 with those of speradine A,9 and considering the HMBC correlations from Me-13 to C-2 and C-12a, the last ring D was established and C-3 was oxidated. The NOESY correlations between H-4 and H-8 observed in 1 indicated that H-4 and H-8 were on the same side of the ring. The structure of 1 is shown in Figure 1.
Compound 2, which was obtained as yellow oil, was analyzed to have the molecular formula C20H22N2O5 through positive HRESIMS (m/z: 371.1606 [M + H]+, Calcd. for C20H23N2O5, 371.1601). The 1H and 13C NMR (Tables 1 and 2), DEPT, and HMQC spectral analyses revealed that 2 had twenty carbon signals, including four methyls, two methylenes, five methines, and nine quaternary carbons. The 1D-NMR data of 2 indicated a structure immensely similar to 1, except for the presence of an additional acylamino carbonyl (δC 168.0), a methylene (δC 53.6 and δH 3.34, 3.63), a ketone carbonyl (δC 200.5), and a methyl (δC 29.7 and δH 1.89). The HMBC correlations of H-17 with C-16 and C-18, and H-19 with C-17 demonstrated the connection of a four-carbon chain from C-16 to C-19. The HMBC correlations from H-15 to C-16 linked C-16 to N-6, which was consistent to the molecular formula of 2. The similar NOESY correlations suggested that the configuration of 2 was similar to that of 1. The structure of 2 is shown in Figure 1.

Compound 3, which was obtained as yellow oil, was analyzed to have the molecular formula C20H22N2O6 through negative HRESIMS (m/z: 385.1399 [M – H]–, Calcd. for C20H21N2O6, 385.1400). The 1H and 13C NMR (Tables 1 and 2), DEPT, and HMQC spectral analyses revealed that 3 had twenty carbon signals, including four methyls, two methylenes, five methines, and nine quaternary carbons. The 1D-NMR data of 3 indicated that 3 was nearly similar to 2, except for the appearance of a methoxy group (δH 3.51 and δC 52.1) and a carbonyl (δC 167.0), and for the disappearance of an acetyl group (δH 1.89 and δC 29.7, 200.5). The HMBC correlations from H-17 and H-19 to C-18 confirmed the substitution of the acetyl group by an ester group. The NOESY correlations between H-4 and H-8 suggested that the configuration of 3 was similar to those of 1 and 2.

Compound 4, which was obtained as orange powder, was analyzed to have the molecular formula C20H18N2O5 through positive HRESIMS (m/z: 389.1108 [M + Na]+, Calcd. for C20H18N2NaO5, 389.1113). The 1H and 13C NMR (Tables 1 and 2), DEPT, and HMQC spectral analyses revealed that 4 had twenty carbon signals, including four methyls, one methylene, four methines, and eleven quaternary carbons. The 1H and 13C NMR data of 4 and 3 were compared. The results showed four obviously downfield shifts, including C-3 (from δC 73.0 s to δC 125.8 s), C-4 (from δC 50.4 d to δC 125.5 s), C-8 (from δC 42.0 d to δC 152.8 s), and C-9 (from δC 25.1 t to δC 123.3 d). Since the degree of unsaturation of 4 was thirteen, which was two more than that of 3, two additional double bonds were necessary. Furthermore, taking into account one oxygen atom and four hydrogen atoms less than molecular formula of 3 and similar 2D NMR, compound 4 was deduced to be the 3,4-dehydrate and 8,9-dehydro derivative of 3.

To explain the biogenetic origin of speradines B, C, D, and E (1–4), a plausible biosynthetic pathway is proposed in Figure 3, which is very similar to the reported biosynthesis of CPA.12
The compounds 1–4 were tested for their cytotoxic effects on the Hela and MGC803 cell lines using the Sulforhodamine B (SRB) method.13 Compounds 1 and 4 demonstrated weak cytotoxicity against the HeLa cell line, with IC50 values of 0.20 and 0.20 mM, respectively.

EXPERIMENTAL
General Experimental Procedures. Optical rotations were obtained from an Anton Paar MCP-200 digital polarimeter. IR spectra were recorded on a Nicolet Avatar 670 spectrophotometer. 1H-NMR, 13C-NMR, DEPT spectra and 2D-NMR were recorded on a BRUKER BIOSPIN AVANCE III spectrometer using TMS as the internal standard. HRESIMS were obtained by an Agilent Q-TOF 6520 LC mass spectrometer. Semipreparative HPLC was performed using an ODS column (ODS-A, 10×250 mm, 5 µm) at 5 mL/min.
Fungal Material. The fungus A. oryzae was isolated from marine sediments collected from Langqi Island, Fujian, China. It was identified according to its morphological characteristics and ITS by Beijing Sunbiotech Co. Ltd, and preserved in our laboratory at −80 ˚C. The producing strain was prepared on Martin medium and stored at 4 ˚C.
Fermentation and Extraction. The fungus A. oryzae was cultured under static conditions at 28 ˚C for 30 days in 1000-mL conical flasks containing the liquid medium (400 mL/flask), composed of glucose (10 g/L), maltose (20 g/L), mannitol (20 g/L), monosodium glutamate (10 g/L), KH2PO4 (0.5 g/L), MgSO4·7H2O (0.3 g/L), yeast extract (3 g/L), and seawater. The fermented whole broth (40 L) was filtered through cheese cloth to separate supernatant from mycelia. The former was extracted two times with EtOAc to give an EtOAc solution, while the latter was extracted three times with acetone. The acetone solution was concentrated under reduced pressure to afford an aqueous solution. The aqueous solution was extracted two times with EtOAc to give another EtOAc solution. Both EtOAc solutions were combined and concentrated under reduced pressure to give a crude extract (42.3 g).
Purification. The crude extract of the fungus A. oryzae was separated into five fractions on a silica gel column using a step gradient elution of petroleum ether, CH2Cl2, and MeOH. Fraction A (6.7 g) was purified on a Sephadex LH-20 (CHCl3:MeOH, 1:2) to derive three subfractions. Subfraction A-2 (2.8 g) was further purified on a silica gel column through a step gradient elution of CH2Cl2 and MeOH to afford four subfractions. Subfraction A-2-1 (160 mg) was purified by a semipreparative HPLC (60% MeOH) to yield compound 4 (3.2 mg). Fraction C (5.4 g) was further purified on a Sephadex LH-20 (CHCl3:MeOH, 1:2) to afford two subfractions. Subfraction C-1 (3.1 g) was further purified on a silica gel column using a step gradient elution of CH2Cl2 and MeOH to afford four subfractions. Subfraction C-1-1 (130 mg) was purified by a semipreparative HPLC (45% MeOH) to yield compounds 1 (2.9 mg), 2 (5.7 mg), and 3 (3.2 mg).
Speradine B (1): yellow oil (CHCl3); [α]20D –31.1 (c 0.17, CHCl3); IR (KBr) νmax 3428, 2925, 1708, 1614, 1475, and 1368 cm–1; 1H and 13C NMR data (see Tables 1 and 2); HRESIMS (m/z: 287.1396 [M + H]+, calcd for C16H19N2O3, 287.1390).
Speradine C (2): yellow oil (CHCl3); [α]20D –272.9 (c 0.13, CHCl3); IR (KBr) νmax 3407, 2929, 1728, 1610, 1475, and 1307 cm–1; 1H and 13C NMR data (see Tables 1 and 2); HRESIMS (m/z: 371.1606 [M + H]+, calcd for C20H23N2O5, 371.1601).
Speradine D (3): yellow oil (CHCl3); [α]20D –40.6 (c 0.13, CHCl3); IR (KBr) νmax 3432, 2933, 1736, 1704, 1618, 1475, and 1340 cm–1; 1H and 13C NMR data (see Tables 1 and 2); HRESIMS (m/z: 385.1399 [M – H]–, calcd for C20H21N2O6, 385.1400.
Speradine E (4): orange powder (CHCl3); IR (KBr) νmax 3444, 2921, 1732, 1699, 1634, 1458, and 1328 cm–1; 1H and 13C NMR data (see Tables 1 and 2); HRESIMS (m/z: 389.1108 [M + Na]+, calcd for C20H18N2NaO5, 389.1113).
Biological Assays. The cytotoxic activity for the HeLa and MGC803 cell lines were evaluated by the SRB method. Doxorubicin was used as the reference drug.

ACKNOWLEDGEMENTS
This research was supported by the Chinese National Natural Science Fund (21102015 and 31201034), the Joint Project of Ministry of Sanitation and Ministry of Education in Fujian Province (WKJ-FJ-14), Natural Science Foundation of Fujian Province (2012J05138) and Key Project of Department of Science and Technology in Fujian Province (2012Y0017).

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