New alkaloids and isocoumarins from the marine gorgonian-derived fungus Aspergillus sp. SCSIO 41501

Abstract Two new β-carboline alkaloids, aspergillspins A-B (1–2), three new quinolone alkaloids, aspergillspins C-E (3–5), and two new isocoumarins, aspergillspins F-G (6–7), together with four known alkaloids were isolated from the marine gorgonian-derived fungus Aspergillus sp. SCSIO 41501. Their structures were identified by spectroscopic analysis, and the absolute configurations of several chiral carbons in 2 and 3 were further established by quantum chemical calculations of the electronic circular dichroism (ECD) spectra. Their cytotoxic and antibacterial activities were also evaluated. Graphical Abstract


Results and discussion
Aspergillspin A (1) was obtained as deep yellow oil. Its molecular formula was determined to be C 16 H 10 N 2 O 3 on the basis of its HRESIMS signal at m/z 279.0771 [M þ H] þ . The 1 H NMR spectrum showed eight aromatic protons (d H 7.35, 7.56, 7.60, 7.66, 7.80, 8.34, 8.36, 8.50) and one exchangeable proton (d H 11.62). The 13 C NMR spectrum showed 16 low-field carbon signals, including seven methines, eight quaternary carbons and one carbonyl group. These date indicated that 1 had a b-carboline alkaloid skeleton (Wang et al. 2012;Xie et al. 2013;Rao et al. 2006), which was supported by the 1 H-1 H COSY and HMBC spectral data ( Figure S55). In addition, the HMBC spectrum showed correlations of H-4 with C-2/C-3/C-5/C-6, and H-2 with C-1/C-3/C-4, which indicted a six-membered lactone attached on C-6 of the b-carboline skeleton. So, the structure of 1 was determined as shown.
Aspergillspin B (2) was isolated as yellow oil. Its molecular formula was determined as C 16 H 18 N 2 O 4 by HRESIMS (m/z 303.1348 [M þ H] þ ). The 1 H and 13 C NMR spectral data of 2 were similar to that of 1, and the obvious difference between them was the substituent at C-6 of the b-carboline skeleton. The COSY spectrum ( Figure S55) showing as equential correlation of H-1/H-2/H-3/H-4/H-5, and the HMBC spectrum ( Figure  S55) exhibiting correlations of H-4 and H-5 with C-6, which indicated a 1,2,3,5-tetrahydroxypentyl group attached on C-6 as shown. The chemical shifts for the geminal protons of H 2 -4 were differentiated at d H 2.24 and 2.50, which indicated a syn 1,3-diol unit between C-3 and C-5 in 2 (Wu et al. 2014;Kikuzaki et al. 1992;Huang et al. 2018). Usually, the chemical shifts for the geminal protons of anti1,3-diol unit overlap (Wu et al. 2014;Kikuzaki et al. 1992;Huang et al. 2018). The coupling constant between H-2 and H-3 (J H-2/H-3 ¼ 5.5 Hz) suggested an anti relative configuration at C-2/C-3 (Chlipala et al. 2010;Hwang et al. 2014). The syn/anti relative configuration for C-3/C-5 and C-2/C-3 was also supported by the 13 C chemical shift of C-3 (d C 68.9) that was consistent to the reported universal NMR databases (Kobayashi et al. 2001;Benowitz et al. 2001;Kobayashi et al. 2000).The planar structure of 2 was the same as that of tangutorids E-F (Zhao et al. 2017) and 1-(1,3,4,5-tetrahydroxypent-1-yl)-b-carbolines (Diem and Herderich 2001), however, the relative structures of the three known compounds were not identified, and according to the coupling constants of the hydrogens of their side chains, we inferred that 2 was a diastereomer of the three known compounds.
The absolute configuration of C-5 was determined by ECD calculation. In order to save computation time, we simplified the structure as model in Figure S55. Three conformers were obtained from Spartan software with low-energy conformers within a 10 kcal/mol window, which were further geometry-optimized at the B3LYP/6-31G level single point energies were calculated at M062X/dex2TZVP using the Gaussian 16 program. The ECD spectra of three conformers were calculated using the TD-DFT method at the B3LYP/6-311G level. The experimental ECD spectra of 2 did not match well with the calculated spectrum ( Figure S57). However, the final Boltzmann factor-weighted theoretical ECD spectrum in the range of 240-300 nm showed acceptable similarity to the experimental ECD spectrum ( Figure S57). Thus, the absolute configuration of C-5 was tentatively established as R. Correspondingly, the absolute configurations of C-3 and C-2 were tentatively assigned as S and R, respectively. Aspergillspin C (3) was obtained as a light yellow solid, and its molecular formula C 16 H 16 N 2 O 5 was determined by HRESIMS (m/z 339.0949 [M þ Na] þ ). The 1 H and 13 C NMR spectral data showed the presence of one oxygenated methyl, three methylenes, one oxygenated methine, one 1,2-di-substituented benzene ring, two carbonly groups, and one a, b-unsaturated ketone group. These data suggested that 3 had a 4-quinolone skeleton (Takayama et al. 1994;Jadulco et al. 2014). The suggestion was supported by the COSY spectrum ( Figure S56) showing correlations of H-11 with H-10/H-12, and H-10 with H-9/H-11, and the HMBC spectrum ( Figure S56) showing correlations of NH with C-7/C-9a, H-9 with C-9a/C-12a/C-13. In addition, the 1 H-1 H COSY spectrum showing a sequential correlation of H-2/H-3/H-4/H-5, and HMBC spectrum showing correlations of H-3 with C-1/C-2/C-4/C-5, H-4 with C-3/C-5, H-5 to C-1/C-3/C-4/C-7, indicted a six-membered ring lactam attached to C-7. Thus, the planar structure of 3 was determined as shown.
The absolute configuration of C-2 in 3 was determined by ECD calculations. Nine conformers were obtained from the Spartan software, and among then, four conformers with low energy were further calculated by using DFT at the B3LYP/6-31G(d) level. The final Boltzmann factor-weighted theoretical ECD spectrum was reasonably similar to the experimental ECD spectrum ( Figure S57). Thus, the absolute configuration of C-2 in 3 was established as R.
Aspergillspin D (4) was isolated as a yellow solid and had the molecular formula C 18 H 22 N 2 O 3 as determined by HRESIMS. The 1 H and 13 C NMR spectral data of 4 were very similar to those of quinolactacin C1 (Clark et al. 2006), and the only obvious difference between them was one additional oxygenated ethyl group instead of one hydroxy group attached to C-3 in 4, which was supported by the HMBC spectrum ( Figure S56) showing a correlation of H-1'' (d H 3.12, 3.49) with C-3 and the 1 H-1 H COSY spectrum ( Figure S56) showing correlation of H-1'' with H-2''. The trans configuration between-OCH 2 CH 3 and CH 3 -1' was determined by the high field-shifted chemical shift of CH 3 -1' (d H 0.55) that was induced by the anisotropic effect of the pyrrolo-quinolone ring (Kim et al. 2001).
Aspergillspin E (5) had the same molecular formula C 18 H 22 N 2 O 3 as 4. The 1 H and 13 C NMR spectral data of 5 were greatly similar to those of 4, and the only obvious difference between them was the chemical shifts of CH 3 -1', H-2', and H-3'. The 1D and 2D NMR spectral data revealed that 5 had the same planar structure as 4. The ECD spectra of 4 and 5 are almost mirror images ( Figure S58), which indicated that the configuration of C-3 in 4 and 5 was opposite. The cis configuration between-OCH 2 CH 3 and CH 3 -1' was determined by the relatively low-field chemical shift of CH 3 -1' (d H 1.12) that wasn't induced by anisotropic effect of the pyrrolo-quinolone ring (Kim et al. 2001). These above data indicated that 4 and 5 were epimers at C-3.
Aspergillspin F (6) was obtained as a brown solid and had the molecular formula C 11 H 10 O 6 as established by HRESIMS. The 1 H and 13 C NMR spectral data showed the presence of two acyl groups, one oxygenated methine, one methylene, one methyl, and one penta-substituted benzene ring. These data were very similar to those of 7hydroxymellein (Pontius et al. 2008). Further analysis of its 2D NMR data revealed the planar structure of 6 as shown, and displayed that the only obvious difference between 6 and 7-hydroxymellein was the presence of an additional carboxyl group at C-6 in 6. The absolute configuration of C-3 in 6 was proposed to be R by comparing the optical rotations of 6 (  (Devys et al. 1980), and (-)-(3R)-5-hydroxymellein (Devys et al. 1994).
Compounds 1-3 and 6-7 were also evaluated for their cytotoxicity against human carcinoma HL60, HepG2, and MCF-7 cell lines by MTT methods and for their antibacterial activity against Bacillus subtilis and Escherichia coli by standard disc diffusion assay. Unfortunately, none of them showed activity in these assays.

General experimental procedures
The procedures were the same as previous reported. 4

Fermentation and extraction
The procedures were the same as previous reported (Ma et al. 2017).