Speradines F–H, Three New Oxindole Alkaloids from the Marine-Derived Fungus Aspergillus oryzae

Abstract

A rare hexacyclic oxindole alkaloid, speradine F (1), together with two novel tetracyclic oxindole alkaloids, speradines G (2) and H (3), were isolated from the marine-derived fungus Aspergillus oryzae. Their structures were determined by spectroscopic methods and X-ray diffraction analysis. This study is the first report on cyclopiazonic acid (CPA)-type alkaloids with a hexacyclic skeleton.

It was not until Alexander Fleming discovered penicillin G from Penicillium notatum in 1928 that fungal microorganisms suddenly became a hunting ground for novel drug leads.^1,2) Hence, many pharmaceutical companies and research groups were motivated to start sampling and screening large collections of fungal strains for antibiotic,^2,3) antimycotic,^4–7) antiviral,⁸⁾ antitubercular,⁹⁾ anticancerous,^10,11) and other pharmacologically active agents.¹²⁾ The genus Aspergillus, which contains approximately 180 recognized species, has been proven a rich source of novel bioactive metabolites.^13,14) In our ongoing search for bioactive novel compounds from marine-derived fungi, a strain identified as Aspergillus oryzae was isolated from sediment samples that were collected from the estuary of Min River in China. The chemical study led to the isolation of a novel hexacyclic oxindole alkaloid, speradine F (1), and two novel tetracyclic oxindole alkaloids speradines G (2) and H (3) (Fig. 1), which own structures similar to speradines A–E.^15,16) In this paper, the isolation, structural elucidation and bioactivities of compounds 1–3 are reported. To the best of our knowledge, this study is the first report on cyclopiazonic acid (CPA)-type alkaloids with a hexacyclic skeleton.

Fig. 1. Structures of Compounds 1–3

Results and Discussion

Compound 1, which is trivially named as speradine F, was obtained as yellow crystal and was analyzed to have the molecular formula C₂₁H₂₂N₂O₇ through positive high-resolution electrospray ionization mass spectroscopy (HR-ESI-MS) (m/z: 415.1506 [M+H]⁺, Calcd for C₂₁H₂₃N₂O₇: 415.1500). Its NMR data (Tables 1, 2), combined with distortionless enhancement by polarization transfer (DEPT) and heteronuclear multiple quantum coherence (HMQC) spectrum analyses, revealed twenty-one carbon signals, including four methyls, one methylene, five methines, and eleven quaternary carbons. The planer structure of 1 was revealed through correlation spectroscopy (COSY) and heteronuclear multiple bond connectivity (HMBC) spectrum analyses (Fig. 2). The COSY correlations of H-8 with H-4 and H-9 as well as 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. The HMBC correlations of H-4 with C-3, H-9 with C-3a and C-9a, H-10 with C-3a and C-9, H-11 with C-9a, and H-12 with C-12a connected rings A and B together. The HMBC correlations of H-4 with C-5 and C-7, H-8 with C-14, and H-14 with C-7 and C-15 linked rings C to B. The HMBC correlations from H-13 to C-2 and C-12a, and from H-4 to C-2 linked ring D to rings A and B. The HMBC correlations from H-4 to C-3, C-5, and C-20 linked ring E to rings B and C. The HMBC correlations of H-15 with C-16 (4-bond correlation, weak) and 17-OH with C-16, C-17, and C-20 linked ring F to rings C and E. The HMBC correlations of H-19 with C-17 and C-18 linked the last acetyl group to ring F. Furthermore, nuclear Overhauser effect spectroscopy (NOESY) data and X-ray diffraction results help in determine the relative configuration of 1, as shown in Figs. 1, 3, and 5.

Table 1. ¹H-NMR Data (500 MHz, J in Hz and δ in ppm) of Compounds 1–3 in CDCl₃

Position	1	2	3
4	3.15 (1H, d, 8.3)	3.19 (1H, d, 9.4)
8	2.40 (1H, m)	2.27 (1H, ddd, 12.8, 9.4, 5.0)
9-1	2.68 (1H, dd, 13.6, 5.3)	2.98 (1H, dd, 13.3, 12.8)	8.03 (1H, s)
9-2	2.49 (1H, dd, 13.6, 12.6)	2.66 (1H, dd, 13.3, 5.0)
10	6.89 (1H, d, 7.7)	6.88 (1H, d, 7.7)	7.57 (1H, d, 8.0)
11	7.30 (1H, dd, 7.8, 7.7)	7.26 (1H, dd, 7.8, 7.7)	7.56 (1H, dd, 8.0, 5.8)
12	6.69 (1H, d, 7.8)	6.67 (1H, d, 7.8)	6.89 (1H, d, 5.8)
13	3.17 (3H, s)	3.17 (3H, s)	3.46 (3H, s)
14	1.75 (3H, s)	1.40 (3H, s)	1.92 (3H, s)
15	1.56 (3H, s)	1.32 (3H, s)	1.92 (3H, s)
17			4.25 (2H, s)
19	2.41 (3H, s)		2.37 (3H, s)
17-OH	6.48 (1H, br s)
3-OH or 6-NH		7.48 (1H, br s)
3-OH or 6-NH		5.38 (1H, br s)
5-OH or 20-OH	4.68 (1H, br s)

Table 2. ¹³C-NMR Data (125 MHz and δ in ppm) of Compounds 1–3 in CDCl₃

Position	1	2	3
2	177.3 s	177.3 s	164.9 s
3	83.0 s	70.5 s	125.7 s
3a	121.5 s	126.5 s	126.5 s
4	53.6 d	46.4 d	125.7 s
5	103.3 s	174.1 s	165.1 s
7	69.1 s	57.0 s	66.2 s
8	54.7 d	49.8 d	152.8 s
9	27.7 t	27.3 t	123.3 d
9a	137.7 s	138.1 s	132.4 s
10	122.0 d	121.0 d	120.4 d
11	132.0 d	130.7 d	131.6 d
12	106.9 d	106.5 d	105.1 d
12a	142.7 s	143.1 s	141.7 s
13	26.6 q	26.4 q	26.6 q
14	21.1 q	24.6 q	27.0 q
15	30.1 q	31.0 q	27.0 q
16	169.8 s		167.9 s
17	88.0 s		54.2 t
18	210.4 s		201.9 s
19	27.5 q		30.5 q
20	107.0 s

Fig. 2. Key COSY and HMBC Correlations of Compounds 1–3

Fig. 3. NOESY Correlations of Compounds 1 and 2

Compound 2, which is trivially named as speradine G, was obtained as orange oil and was analyzed to have the molecular formula C₁₆H₁₈N₂O₃ through positive HR-ESI-MS (m/z: 287.1388 [M+H]⁺, Calcd for C₁₆H₁₉N₂O₃: 287.1390). Its NMR data (Tables 1, 2), combined with DEPT and HMQC spectrum analyses, revealed sixteen carbon signals, including three methyls, one methylene, five methines, and seven quaternary carbons. The one dimensional (1D)-NMR data of 2 indicated that its structure is similar to that of 1, except for the disappearance of five carbons from C-16 to C-20, two obviously upfield shifts of C-3 (from δ_C 83.0 s to δ_C 70.5 s) and C-7 (from δ_C 69.1 s to δ_C 57.0 s), and an obviously downfield shift of C-5 (from δ_C 103.3 s to δ_C 174.1 s). The similar COSY, HMBC and NOESY correlations of 2 and 1 from rings A to D, and two left hydroxy or amidogen protons of 2 (δ_H 7.48 br s and 5.38 br s) suggested 2 was 3,6-hydrolytic and 5-oxidative degradation derivative of 1 (Fig. 1).

Compound 3, which is trivially named as speradine H, was obtained as orange powder and was analyzed to have the molecular formula C₂₀H₁₈N₂O₄ through positive HR-ESI-MS (m/z: 373.1162 [M+Na]⁺, Calcd for C₂₀H₁₈N₂NaO₄: 373.1159). Its NMR data (Tables 1, 2), combined with DEPT and HMQC spectrum analyses, revealed twenty carbon signals, including four methyls, one methylene, four methines, and eleven quaternary carbons. The 1D-NMR data of 3 indicated that its structure is similar to that of 1, except for the disappearance of C-20 (δ_C 107.0 s), an obviously upfield shift of C-17 (from δ_C 88.0 s to δ_C 54.2 t), and five obviously downfield shifts of C-3 (from δ_C 83.0 s to δ_C 125.7 s), C-4 (from δ_C 53.6 d to δ_C 125.7 s), C-5 (from δ_C 103.3 s to δ_C 165.1 s), C-8 (from δ_C 54.7 d to δ_C 152.8 s), and C-9 (from δ_C 27.7 t to δ_C 123.3 d). The similar COSY and HMBC correlations of 3 and 1 suggested rings A, D and the four-carbon chain from C-16 to C-19 were reserved. In view of the five obviously downfield shifts in rings B and C, the HMBC correlations of H-9 with C-3a, C-4, and C-5 (4-bond correlation, weak), H-12 with C-3 (4-bond correlation, weak), as well as H-14 with C-7 and C-8 suggested ring B was dehydrated and dehydrogenized, and C-5 of ring C was oxidated, which were consistent to its molecular formula and degree of unsaturation. So the structure of 3 was deduced as shown in Fig. 1.

To explain the biogenetic origin of speradines F–H (1–3), a plausible biosynthetic pathway is proposed in Fig. 4, which is very similar to the reported biosynthesis of CPA.^17,18)

Fig. 4. Plausible Biosynthetic Pathway of Compounds 1–3

Fig. 5. Molecular Structure of Speradine F (1) Obtained by X-Ray Analysis (ORTEP Drawing; Ellipsoids Are Drawn at 30% Probability Level)

The compounds 1–3 were tested for their cytotoxic effects on the Hela cell line using the Sulforhodamine B (SRB) method and on the HL-60 and K562 cell lines using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) method.¹⁹⁾ Unfortunately, the results showed that all of their IC₅₀ values were larger than 30 µg/mL.

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 tetramethylsilane (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 34 d 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 Si 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 afford three subfractions. Subfraction A-2 (2.8 g) was further purified on a Si gel column using a step gradient elution of CH₂Cl₂ and MeOH to afford four subfractions. Subfraction A-2-1 (160 mg) was purified by semipreparative HPLC (60% MeOH) to yield compounds 1 (4.2 mg) and 3 (3.3 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 Si gel column using a step gradient elution of CH₂Cl₂ and MeOH to afford four subfractions. Subfraction C-1-1 (130 mg) was purified by semipreparative HPLC (45% MeOH) to yield compound 2 (4.1 mg).

X-Ray Crystallography of 1

C₂₁H₂₂N₂O₇, molecular weight (MW)=414.41, space group P4₃2₁2, a=14.8878 (4) Å, b=14.8878 (4) Å, c=17.9410 (8) Å, V=3976.6 (2) ų, Z=8. The X-ray diffraction intensity data was collected on a Rigaku Saturn 724 CCD diffractometer with graphite-monochromater MoKα radiation (λ=0.71073 Å) by the ω scan technique. A total of 27183 reflections were collected, of which 3703 were independent (R_int=0.0428). The structure was solved by the direct method and refined by the full-matrix least-squares technique on F² to give R₁=0.0421, wR₂=0.1115, and S=1.051 for 3683 observed reflections (I>2σ(I)). H atoms bonded to C and O atoms were refined in idealized positions using the riding-model. All of the calculations were performed with the Siemens SHELXTL™ version 5 package of crystallographic software.

Supporting Information Available

The X-ray crystallographic data for the structure of 1 has been deposited at the Cambridge Crystallographic Data Centre (CCDC 996022). Copy of the data can be obtained free of charge by applying to CCDC, 12 Union Road, Cambridge CB2 1EZ, U.K. (fax: [phone]; e-mail: [e-mail]).

Speradine F (1): Yellow crystal (CHCl₃). [α]_D²⁰ −25.6 (c=0.13, CHCl₃). IR (KBr) cm⁻¹: 3428, 2925, 1699, 1614, 1475, 1373, 1299. ¹H- and ¹³C-NMR data (see Tables 1, 2). HR-ESI-MS m/z: 415.1506 [M+H]⁺ (Calcd for C₂₁H₂₃N₂O₇: 415.1500).

Speradine G (2): Orange oil (CHCl₃). [α]_D²⁰ −58.1 (c=0.17, CHCl₃). IR (KBr) cm⁻¹: 3273, 2929, 1724, 1679, 1614, 1475, 1368. ¹H- and ¹³C-NMR data (see Tables 1, 2). HR-ESI-MS m/z: 287.1388 [M+H] ⁺ (Calcd for C₁₆H₁₉N₂O₃: 287.1390).

Speradine H (3): Orange powder (CHCl₃). IR (KBr) cm⁻¹: 3436, 2921, 1736, 1704, 1634, 1458, 1319, 1287. ¹H- and ¹³C-NMR data (see Tables 1, 2). HR-ESI-MS m/z: 373.1162 [M+Na]⁺ (Calcd for C₂₀H₁₈N₂NaO₄: 373.1159).

Biological Assays

The cytotoxic activity for the HL-60 and K562 cancer cell lines was evaluated by the MTT method and the Hela cancer cell line was evaluated by the SRB method. Doxorubicin was used as the reference drug.

Acknowledgment

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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