Indole alkaloids from marine-derived fungus Aspergillus sydowii SCSIO 00305

Marine microorganisms have proved to be an important source of pharmacologically active metabolites, and a growing number of marine-derived fungi have been reported to produce metabolites with unique structures and interesting biological activities.^{1, 2} The genus Aspergillus (Moniliaceae), with over 180 species, has attracted considerable attention as a rich source of alkaloids, terpenoids, xanthones, polyketides and etc, some of which showed antifungal, antibacterial, anti-HIV and cytotoxic activities.^{3, 4, 5} In order to obtain new bioactive metabolites from marine fungi, we investigated on the marine fungal strain Aspergillus sydowii SCSIO 00305 isolated from a healthy tissue of Verrucella umbraculum. Bioassay-guided fractionation led to the isolation of a new indole diketopiperazine alkaloid, cyclotryprostatin E (1), together with nine known ones, [4-(2-methoxyphenyl)-1-piperazinyl][(1-methyl-1H-indol-3-yl)]-methanone (2), cyclotryprostatin B (3),⁶ fumiquinazoline D (4),⁷ fumitremorgin B (5),⁸ fumiquinazoline C (6),⁷ fumiquinazoline B (7),⁷ fumiquinazoline A (8),⁷ fumiquinazoline F (9),⁷ fumiquinazoline G (10)⁷ from a culture broth of the strain. The structures of compounds (1) and (2) were characterized by spectroscopic data interpretation. Compound (2) was a synthetical compound, however, no reference for it. The NMR data and biology source of (2) were reported for the first time. We present herein the fermentation, isolation, structure elucidation and cytotoxicity of compounds (1) and (2).

Compound (1) was obtained as pale yellow powder with the molecular formula C₂₃H₂₉N₃O₆ deduced from NMR spectra and positive HRESIMS (found 466.1953 [M+Na]⁺, calculated 466.1954). The UV bands (221, 293 nm) and IR absorptions at 3400, 3312, 1664 and 1653 cm⁻¹ indicated the presence of conjugated system, hydroxyl and carbonyl groups. The ¹H NMR spectrum of (1) showed two methyl groups (δ_H 1.34 and 1.47), two methoxyl groups (δ_H 3.41 and 3.83), and three aromatic protons [δ_H 7.46 (d, J=9.0 Hz, H-16), 6.75 (dd, J=2.5, 9.0 Hz, H-17), 6.95 (d, J=2.5 Hz, H-19)]. The ¹³C and DEPT NMR spectra showed signals for 23 carbons, including two methyls (δ_C 29.1, 31.5), four methylene groups (δ_C 22.7, 30.9, 46.4 and 50.8), one oxygenated methine carbon (δ_C 77.6), two oxygenated quaternary carbons (δ_C 71.2 and 87.3), eight olefinic carbons, and two amide carbonyl groups (δ_C 167.8 and 169.0). These NMR data of (1) showed similarity to those of (3),³ which suggested that (1) was an indole diketopiperazine alkaloid. A spin coupling system of H₂-7/H₂-8/H₂-9 in the ¹H-¹H COSY spectrum (Figure 1), combined with HMBC correlations from H-16 to C-14/C-18/C-20, from H-19 to C-15/C-17/C-18/C-20, from H-3/H₂-7 to C-5, and from H-6 to C-11 (Figure 1), further confirmed the suggestion. Comparison of ¹H- and ¹³C NMR data of (1) with those of cyclotryprostatin B (3) revealed that a tri-substituted double bond (δ_C 123.5 and 137.9) in (3) was replaced by an oxygenated quaternary carbon (δ_C 71.2) and a methylene group (δ_C 50.8) in (1). In the HMBC spectrum (Figure 1), correlations from H₃-23/H₃-24/H-3 to C-21/C-22 suggested the assignment of C-21 (δ_C 50.8) and C-22 (δ_C 71.2). The relative stereochemistry of (1) was determined by the NOESY spectrum, the magnitude of ¹H-¹H COSY coupling constants, and comparison of the ¹³C NMR data of (1) with those of (3). The observed NOE correlations between H-3 and H₃-24/H₂₋21, between H₃-24 and H₂-21, and between H-6 and H-7α/H-8α, together with nearly identical carbon chemical shift of C-12 (δ_C 87.3 in (1) and 84.7 in (3)) and C-13 (δ_C 77.6 in (1) and 76.8 in (3)), indicated that (1) had the same relative configuration as 3.

Figure 1

Key correlations of COSY (bold line) and HMBC (arrow) of 1.

Compound (2) was isolated as pale yellow crystals with the molecular formula C₂₁H₂₃N₃O₂ deduced from HRESIMS (m/z at 350.1865 [M+H]⁺, calc. 350.1869). The ¹H-, ¹³C-, and DEPT NMR spectra displayed 18 carbon signals, including one conjugated carbonyl group (δ_C 168.9), nine methines, one methoxyl (δ_H 3.84, δ_C 56.0), one methyl (δ_H 3.84, δ_C 33.3), one methylene (δ_C 52.4), and five quaternary carbons. Three spin coupling systems (H-5/H-6/H-7/H-8, H-20/H-21/H-22/H-23, and H-13(17)/H-14(18)) deduced from ¹H-¹H COSY spectrum, together with HMBC correlations from H-5 to C-3/C-7/C-9, from H-2 to C-3/C-4/C-9, and from H-8 to C-4/C-6 suggested the existence of one indolyl group, one piperazinyl group, and one 1,2-disubstituted phenyl group. One methyl group and one methoxyl group were deduced to be located at N-1 and C-19, respectively, from the observation of HMBC correlations from δ_H 3.84 (6H, s) to C-2/C-9/C-19. This information coupled with the key HMBC correlations from H-2, H-13 and H-17 to C-11 and from H-14 and H-16 to C-18 enabled us to establish the structure of (2) as shown in Figure 2. We proposal that chorismate acid may be a biogenetic precursor for (2).

Figure 2

Structures of 1–3.

Compounds (1) and (2) were screened for their cytotoxicity against A549 (lung cancer cell line), A375 (human melanoma cell line) and Hela (Human cervical carcinoma cell) cell lines, using the MTT method with cis-platin as positive control. Compound (2) showed significant cytotoxicity against A375 cell lines with IC₅₀ (half maximal inhibitory concentration) value of 5.7 μM. Compound (1) had no obvious cytotoxicity towards the above mentioned three cell lines.

Experimental procedure

Taxonomy

The fungus A. sydowii SCSIO 00305 was isolated from a healthy tissue of V. umbraculum collected from Sanya, Hainan Province, China, and was identified by Dr Xiaoyong Zhang, and a voucher specimen (A. sydowii SCSIO 00305) has been deposited in the RNAM Center for Marine Microbiology, South China Sea Institute of Oceanology, Chinese Academy of Sciences.

Fermentation, isolation and identification of compounds

The fungus strain A. sydowii SCSIO 00305 was cultivated in 500 ml Erlenmeyer flasks containing 100 ml of the production medium composed of glucose 1%, maltose 2%, mannitol 2%, yeast extract 0.3%, monosodium glutamate 1%, MgSO₄·7H₂O 0.03%, KH₂PO₄ 0.05% and sea water 5 l (pH 7.2 before sterilization), and cultured without shaking at 28 °C for 20 days. The EtOAc extract of mycelia (800 mg) was chromatographed on RP-C 18 column using gradient elution from 5% MeOH/H₂O to100% MeOH, to give four fractions. Fraction A (MeOH/H₂O, 35% v/v elution, 100 mg) was further purified by semi-preparative reversed-phase HPLC (MeOH/H₂O 45 % v/v, 3 ml/min, detector 230 nm) to yield (5) (t_R=23.1 min), (6) (t_R=28.0 min), (8) (t_R=32.5 min). Fraction B (MeOH/H₂O, 55% v/v elution, 120 mg) was further purified by semi-preparative reversed-phase HPLC (MeOH/H₂O 50% v/v, 3 ml/min, detector 230 nm) to yield (1) (t_R=16.6 min), (4) (t_R=18.9 min), (9) (t_R=22.1 min) and (10) (t_R=23.8 min). Fraction C (MeOH/H₂O, 75% v/v elution, 250 mg) was further purified by reversed-phase HPLC (60%CH₃CN/H₂O, 3 ml/min, detector 254 nm) to yield (2) (t_R=12.8 min), (3) (t_R=15.2 min) and (7) (t_R=20.0 min).

Cyclotryprostatin E (1): pale yellow powder; [α]_D²⁵ +28.35 (c 0.23, CH₃OH); UV (CH₃OH) λ_max (log ɛ) 209 (2.91), 251 (2.03); ¹H and ¹³C NMR data see Table 1; IR (KBr) ν_max 3298, 2980, 2914, 1665, 1450, 1418, 1249, 1158, 1110, 1028 cm⁻¹; ESIMS m/z 466 [M+Na]⁺; HRESIMS m/z 466.1953 [M+Na]⁺, calculated for C₂₃H₂₉N₃O₆Na m/z 466.1954).

Table 1 ¹H- and ¹³C-NMR Data of 1 and 2^a

[4-(2-methoxyphenyl)-1-piperazinyl](1-methyl-1H-indol-3-yl)-methanone (2): pale yellow powder; [α]_D²⁵ +33.33 (c 0.06, CH₃OH); UV (CH₃OH) λ_max (log ɛ) 286 (3.84); ¹H and ¹³C NMR data see Table 1; IR (KBr) ν_max 3428, 2932, 2831, 1612, 1534, 1500, 1471, 1434, 1238, 1154, 1139, 747 cm⁻¹; ESIMS m/z 466 [M+Na]⁺; HRESIMS m/z 350.1871 [M+H]⁺, calculated for C₂₁H₂₃N₃O₂ m/z 350.1868).