Phenylspirodrimane Derivatives From Cultures of the Fungus Stachybotrys chartarum YIM DT 10079

Abstract

A new phenylspirodrimane derivative, stachartin F (1), and 2 known secondary metabolites stachybonoid E (2) and stachybonoid F (3) were isolated from cultures of the tin mine tailings-associated fungus Stachybotrys chartarum YIM DT 10079. Their structures were determined with the help of extensive spectroscopic analyses and absolute configuration of compound 1 was rationalized by quantum chemical calculations of the electronic circular dichroism spectra.

The phenylspirodrimanes, structurally characterized by the fusion of a drimane-based sesquiterpene with a phenyl moiety, are known as characteristic secondary metabolites of the fungus Stachybotrys chartarum. These compounds have exhibited diverse pharmacological activities, such as antihyperlipidemic activities, ¹ anti-HIV activity, ² osteoclast differentiation inhibitor, ³ and antimalarial activity. ⁴ As part of our ongoing search for novel molecules from extremophiles, ^5,6 S. chartarum YIM DT 10079 was isolated from a soil sample collected from the Datun tin mine tailings area, Yunnan, China. An EtOAc extract of cultures of S. chartarum YIM DT 10079 was subjected to investigation, which resulted in the isolation of a new phenylspirodrimane derivative, stachartin F (1), and the known stachybonoid E (2) and stachybonoid F (3) ⁷ (Figure 1). Herein, we report the isolation and structure elucidation of these compounds, and stachartin F (1) was tested for its cytotoxicity against 5 human cancer cell lines.

Figure 1

Chemical structures of 1 to 3 from Stachybotrys chartarum YIM DT 10079.

Compound 1 was isolated as a white powder. Its molecular formula was established as C₂₈H₃₉NO₆ by high-resolution electrospray ionization mass spectrometry (HR-ESI-MS) at m/z 508.2671 [M+Na]⁺ (calcd 508.2670), indicating 10 degrees of unsaturation. Infrared Spectroscopy (IR) absorption bands at 3429, 1725, and 1658 cm⁻¹ implied the presence of hydroxy and carbonyl functionalities. Comparison of the ¹H and ¹³C Nuclear magnetic resonance spectroscopy (NMR) data (Table 1) with those of chartarlactam H¹ showed the presence of the same phenylspirodrimane skeleton, except the side chain moiety on the nitrogen was replaced by a methyl butyrate group in 1, which was further confirmed by the Heteronuclear Multiple Bond Correlation (HMBC) correlations (Figure 2) from H₃-5″ (δ _H 3.54) and H₂-2″ (δ _H 1.79) to C-4″ (δ _C 173.1) and from H₂-1″ (δ _H 3.35) to C-7′ (δ _C 49.3) and C-8′ (δ _C 166.1), and ¹H-¹H correlation spectroscopy (COSY) correlations of H-2″ with H-1″ and H-3″ (δ _H 2.31). Its relative configuration was determined by the Rotating frame overhauser enhancement spectroscopy (ROESY) correlations of H-3/H₃-14, H₃-14/H₃-15, H₃-15/H-8, and H₃-13/H-5, H-5/Há-7, Há-7/H₃-12 determined that H-3, H₃-14, and H₃-15 as being in β-orientations, while H₃-13, H-5, and H₃-12 as being in á-orientations (Figure 2). Consequently, compound 1 was established as stachartin F. The absolute configuration of 1 was determined by means of electronic circular dichroism (ECD). As shown in Figure 3, the calculated ECD spectrum of 1 matched well with the experimental one, suggesting the absolute configuration of 1 to be 3R, 5S, 8R, 9R, 10S.

Table 1

¹H and ¹³C NMR Data for Compound 1.

No.	δ H	δ C	No.	δ H	δ C
1a 1b	1.69 (brt) 0.89 (brd)	23.9 t	1′		112.5 s
2a2b	1.79 (m)1.36 (m)	25.1 t	2′		156.5 s
3	3.15 (m)	73.4 d	3′	6.31 (s)	101.4 d
4		37 .4 s	4′		144.2 s
5	2.05 (dd, 12.2, 1.5)	39.3 d	5′		105.9 s
6a6b	1.45 (m)1.38 (m)	20.6 d	6′		158.2 s
7a	1.47 (m)	30.5 t	7′	4.22	49.3 t
8	1.75 (m)	36.7 d	8′		166.1 s
9		98.3 s	1″	3.35 (m)	40.8 t
10		41.8 s	2″	1.79 (m)	23.3 t
11a11b	3.00 (d,16.1)2.63 (d,16.1)	30.9 t	3″	2.31 (m)	30.9 t
12	0.59 (d, 6.6)	15.5 q	4″		173.1 s
13	0.87(s)	28.7 q	5″	3.54 (s)	51.4 q
14	0.78 (s)	22.6 q	3-OH	4.09 (s)
15	0.92 (s)	16.0 q	3′-OH	9.94 (s)

Figure 2

Selected 2D NMR correlations of 1.

Figure 3

Experimental and calculated electronic circular dichroism for compound 1.

Compound 1 was evaluated for its cytotoxicity against 5 human cancer cell lines using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) method as reported previously. ⁸ Cisplation (Sigma, United States) was used as the positive control. The compound was inactive (IC₅₀ values >40 µM).

Experimental

General

Optical rotations, a Horiba SEPA-300 polarimeter; NMR, Avance III 600, Bruker DRX-500, and Bruker AM-400 spectrometers; HR-ESI-MS, an API-Qstar-Pulsar-1 spectrometer.

Fungal Material and Cultivation Conditions

Stachybotrys chartarum was isolated from a soil sample collected from the Datun tin mine tailings area, Yunnan, P.R. China. A voucher specimen (No. YIM DT 10079) was deposited at Yunnan Institute of Microbiology, Yunnan University. The culture medium consisted of glucose (1.0%), peptone from porcine meat (0.5%), yeast powder (0.5%), KH₂PO₄ (0.1%), and MgSO₄·7H₂O (0.02%). Fermentation was carried out on a shaker at 200 rpm for 15 days.

Extraction and Isolation

The culture broth (100 L) of S. chartarum YIM DT 10079 was filtered, and the filtrate was extracted 3 times with EtOAc, while the mycelium was extracted 3 times with CHCl₃/MeOH (1:1). The EtOAc layer together with the mycelium extraction was concentrated under reduced pressure to give a crude extract. The extract was subjected to column chromatography over silica gel (200-300 mesh) eluted with a gradient of CHCl₃/MeOH (1:0→0:1) to obtain 6 fractions (1-6). Fraction 3 eluted with CHCl₃/MeOH (80:1) was separated repeatedly by Sephadex LH-20 (CHCl₃/MeOH 1:1) to afford 1 (16.0 mg), 2 (25.0 mg), and 3 (200.0 mg).

Cytotoxicity Assay

Five human cancer cell lines, breast cancer SK-BR-3, hepatocellular carcinoma SMMC-7721, human myeloid leukemia HL-60, pancreatic cancer PANC-1, and lung cancer A-549 cells, were used in the cytotoxic assay. Cells were cultured in RPMI-1640 or in DMEM medium (Hyclone, United States), supplemented with 10% fetal bovine serum (Hyclone, United States) in 5% CO₂ at 37°C. The cytotoxicity assay was performed according to the MTT method in 96-well microplates. ⁸ Briefly, 100 µL of adherent cells were seeded into each well of 96-well cell culture plates and allowed to adhere for 12 hours before addition of test compounds, while suspended cells were seeded just before drug addition with initial density of 1 × 10⁵ cells/mL. Each tumor cell line was exposed to the test compound at concentrations of 0.0625, 0.32, 1.6, and 8 ìM in triplicates for 48 hours, with cisplatin (Sigma, United States) as positive control. After compound treatment, cell viability was detected and a cell growth curve was graphed. IC₅₀ values were calculated by Reed and Muench’s method. ⁹

Computational Methods

All Discrete fourier transformation (DFT) and Time dependent density functional theory (TD-DFT) calculations were carried out at 298 K in the gas phase with Gaussian 09. Conformational searches were carried out at the molecular mechanics level of theory employing MMFF force fields. ^10,11 The conformers with relative energy within 10 kcal/mol of the lowest-energy conformer were selected and further geometry optimized at the B3LYP/6-311++G(2d,p) level. All the lowest-energy conformers, which correspond to 99% of the total Boltzmann distribution, were selected for ECD spectra calculation. The Boltzmann factor for each conformer was calculated based on Gibbs free energy. Vibrational analysis at the B3LYP/6-311++G(2d,p) level of theory resulted in no imaginary frequencies, confirming the considered conformers as real minima. TD-DFT was employed to calculate excitation energy (in nm) and rotatory strength R in dipole velocity form, at the B3LYP/6-311++G(2d,p) level.

Stachartin F (1)

White powder.

$[\alpha {]}_{\mathrm{D}}^{26}$ : −6.0 (c 0.35, MeOH).

¹H and ¹³C NMR: Table 1.

HR-ESI-MS: m/z 508.2671 [M+Na]⁺ (calcd for C₂₈H₃₉NNaO₆, 508.2670)

Supplemental Material

Supplementary material - Supplemental material for Phenylspirodrimane Derivatives From Cultures of the Fungus Stachybotrys chartarum YIM DT 10079

Supplemental material, Supplementary material, for Phenylspirodrimane Derivatives From Cultures of the Fungus Stachybotrys chartarum YIM DT 10079 by Zhang-Gui Ding, Jian-Hai Ding, Jiang-Yuan Zhao, Ming-Gang Li, Dong-Bao Hu, Xian-Jun Jiang, Shao-Jie Gu, Fei Wang and Meng-Liang Wen in Natural Product Communications