Setosphaerine A: A New Chlorinated Polyketide Isolated From the Entomogenous Fungus Setosphaeria rostrata

Abstract

A new chlorinated polyketide setosphaerine A (1), together with 3 known ones penicipyran D (2), 7-O-demethymonocerin (3), and monocerin (4), was isolated from the entomogenous fungus Setosphaeria rostrata. The structure of compound 1 was established on the basis of 1D/2D Nuclear Magnetic Resonance (NMR) spectroscopic and High Resolution Electro Spray Ionization Mass Spectra (HR-ESI-MS) data. Compounds 1 to 4 showed moderate cytotoxicity against 3 human tumor cell lines MCF-7, MGC-803, and Hela with IC₅₀ values ranging from 33 to 243 nM.

Insect entomogenous fungi have proved to be a rich and important source of secondary metabolites with a variety of chemical structures and biological activities. ^{1 -4} They are an ecologically specialized group of micro-organisms, ^5,6 which, during the long-time co-evolution with their hosts, have had more chances to acquire unique traits in their physiology and metabolite pathways. ^7,8 In our continuing search for more natural-product-derived bioactive metabolites, we have investigated a cytotoxic MeOH extract of a rice culture of Setosphaeria rostrata, an entomogenous fungus isolated from the tissue of Harmonia axyridis. Fractionation of this extract led to the isolation of a new chlorinated polyketide setosphaerine A (1) and 3 known ones (2-4).

The molecular formula of setosphaerine A (1) was determined as C₁₆H₁₉O₆Cl by the negative High Resolution Electro Spray Ionization Mass Spectra (HR-ESI-MS) ion at m/z 341.0794 [M−H]⁻ (calcd for C₁₆H₁₈O₆Cl, 341.0797), requiring 7 degrees of unsaturation. The Infrared Spectroscopy (IR) spectrum exhibited absorption bands for hydroxyl (3423 cm⁻¹) and carbonyl (1735 cm⁻¹) groups. The ¹H Nuclear Magnetic Resonance (NMR) spectrum (Table 1) suggested the presence of 1 methyl group [δ_H 0.94 (3H, t, J = 7.0 Hz)], 4 methylene groups [δ_H 2.72 (1H, dd, J = 14.4, 4.8 Hz), 2.64 (1H, dd, J = 14.4, 8.4 Hz), δ_H 1.59 (2H, m), and δ_H 1.30-1.53 (4H, m)], 2 methine groups [δ_H 4.26 (1H, m) and δ_H 3.85 (1H, m)], and 2 olefinic protons [δ_H 6.73 (1H, s) and δ_H 6.44 (1H, s)]. The ¹³C NMR spectrum of 1 (Table 1) showed 16 carbon resonances, including 1 methyl group (δ_C 14.4), 4 methylene groups (δ_C 45.4, 43.4, 41.4, and 19.9), 2 oxygenated methine groups (δ_C 68.8 and 67.2), 2 olefinic carbons (δ_C 103.5 and 103.0), and 7 quaternary carbons (δ_C 167.3, 163.1, 162.7, 157.7, 137.7, 107.2, and 100.4). The ¹H and ¹³C NMR data of 1 showed close similarity to those of exserolide F, ⁹ which was isolated from Exserohilum sp., except for an extra chlorine atom in 1. The isocoumarin skeleton with 2 hydroxyl groups at C-6 and C-8 was established by Heteronuclear Multiple Bond Correlation (HMBC) correlations of H-4/C-3, C-5, C-6, and C-8a (Figure 1), and further supported by the typical Polyketide Synthase biosynthetic pathway of 1. ¹⁰ A rare chlorine atom was assigned at C-5, supported by the HMBCs of H-4/C-5 and H-7/C-5. The location of the side chain was confirmed at C-3 based on the HMBC correlations of H-9/C-3 and C-4 and H-10/C-3. Therefore, the structure of setosphaerine A was determined as 1 (Figure 2).

Table 1

¹³C NMR (150 MHz) and ¹H NMR (600 MHz) Data of Compound 1.

Position	1 (in CD3OD)
	δC	δH (mult., J in Hz)
1	167.3, C
3	157.7, C
4	103.5, CH	6.73, s
4a	137.7, C
5	107.2, C
6	163.1 a,b , C
7	103.0, CH	6.44, s
8	162.7a,b, C
8a	100.4, C
9	43.4, CH2	2.72, dd, (14.4, 4.8), 2.64, dd, (14.4, 8.4)
10	67.2, CH	4.26, m
11	45.4, CH2	1.59, m
12	68.8, CH	3.85, m
13	41.4, CH2	1.30-1.53 c , m
14	19.9, CH2	1.30-1.53 c , m
15	14.4, CH3	0.94, t, (7.0)

^a,b May be interchanged.

^c The signals were overlapped.

Figure 1

Key Heteronuclear Multiple Bond Correlation (HMBC) (arrows) and COSY (bold) correlations for compound 1.

Figure 2

Chemical structures of compounds 1 to 4.

Compounds 2 to 4 were identified as penicipyrans D, ^2,11 7-O-demethymonocerin, ^3,12 and monocerin, ^4,13 by comparison of their NMR data with those reported in the literature.

All isolated compounds were evaluated for their cytotoxic activities against 3 human tumor cell lines MCF-7, MGC-803, and Hela using the MTT Assay method; paclitaxel was selected as the positive control. The results (Table 2) revealed that compounds 1 to 4 showed moderate cytotoxicity against 3 human tumor cell lines MCF-7, MGC-803, and Hela with IC₅₀ values ranging from 33 to 243 nM.

Table 2

Cytotoxic Activities of Compounds 1 to 4.

Compound	IC50 (nM)
	MCF-7	MGC-803	Hela
1	243 ± 4.25	80 ± 3.4713	42 ± 1.90
2	158 ± 3.58	103 ± 5.80	43 ± 0.35
3	52 ± 1.14	85 ± 5.75	33 ± 1.59
4	182 ± 2.14	138 ± 8.45	55 ± 2.02
Paclitaxel	11 ± 0.22	18.5 ± 1.55	9 ± 1.74

Experimental

General

Optical rotations, Perkin-Elmer 341 spectropolarimeter; IR, Perkin-Elmer 577 instrument; UV, UV-210 spectrometer; NMR, Bruker AM-600 spectrometer; HR-MS, Bruker apex-ultra 7.0T spectrometer; column chromatography (CC), silica gel (SiO₂; 200-300 mesh; Sephadex LH-20 gel (25-100 μm); and Thin Layer Chromatography, silica gel GF₂₅₄ plates.

Fungal Material and Fermentation

The strain LGWB-10 was isolated from H. axyridis collected in Baoding, Hebei Province of China. The voucher specimen of the fungus is deposited at Key Laboratory of Medicinal Chemistry and Molecular Diagnosis of the Ministry of Education, College of Life Science of Hebei University. Setosphaeria rostrata was cultured on a culture dish of potato dextrose agar (PDA; 20.0 g glucose, 18.0 g agar, 200.0 g potato in 1 L of distilled H₂O) at 28°C for 6 days, and then inoculated into a 500 mL Erlenmeyer flask containing 200 mL of Potato D-glucose medium (20.0 g glucose, 200.0 g potato in 1 L of distilled H₂O). Flask cultures were incubated at 25°C on a rotary shaker at 120 rpm/min for 4 days. Fermentation was carried out in 100 Erlenmeyer flasks (500 mL), each containing 80 g of rice (grains)/100 mL of distilled H₂O; 5 mL of culture liquid was transferred as seed into each flask and incubated at 25°C for 40 days.

Extraction and Isolation

The fermented material was extracted 3 times with methanol (20 L for each time), and the methanol extract was concentrated in vacuo to yield a yellow oily residue (125.0 g). This was subjected to silica gel CC and eluted with a gradient of PE/EtOAc (100:0, 90:10, 80:20, 60:40, 50:50, 40:60, 20:80, 10:90, and 0:100 (v/v)) to obtain nine fractions Frs. 1-9. Fr. 6 (1.6 g) was repeatedly purified by CC and Sephadex LH-20 (MeOH) to afford compounds 1 (6.6 mg), 2 (5.3 mg), 3 (9.2 mg), and 4 (5.8 mg).

Compound 1

White powder

UV (MeOH) λ _max (log ε): 245 (4.90).

IR (KBr) v _max: 3423, 2954, 2915, 2848, 1735, 1465, 1032, 1018, 853 cm⁻¹.

¹H- and ¹³C- NMR: Table 1.

HR-ESI-MS: m/z 341.0794 [M+H]⁺ (calcd for C₁₆H₁₈O₆Cl, 341.0797).

Cytotoxicity Assay

The cells were cultured in Roswell Park Memorial Institute (RPMI1640, Hyclone, Logan, UT, United States, MCF-7) and Dulbecco’s modified Eagle’s medium (DMEM, Hyclone, Logan, UT, United States, MGC-803, Hela) supplemented with 10% fetal bovine serum (FBS, Hyclone, Logan, UT, United States) at 37°C under an atmosphere of 5% CO₂, and were seeded on each well of 96-well plates containing 100 µL of tumor cells suspension (5 × 10⁴ cells/mL). After 48 hours, the test compounds from Dimethyl Sulfoxide (DMSO) (Sigma, St. Louis, MO, United States) dissolved stock solution (2 µL [2 mg/mL]) was added to each well with final concentrations of 40, 20, 10, 5, and 2.5 µg/mL , and incubated for another 24 hours. Twenty microliters of MTT solution (1 mg/mL, Beijing Cellchip Biotechnology Co., Ltd) was added to each well, and the plate was incubated for 4 hours under the same conditions. Then, the plate was centrifuged, and the supernatants were removed and cells dissolved in 100 µL of DMSO to determine the IC₅₀ values. The absorbance in control and drug-treated wells was measured in a Microplate Reader (Thermo Scientific, United States) at 570 nm (emission) wavelength. All experiments were carried out in triplicate and repeated twice. The cytotoxicity was expressed as IC₅₀ values (50% inhibitory concentration).

Supplemental Material

Supporting information - Supplemental material for Setosphaerine A: A New Chlorinated Polyketide Isolated From the Entomogenous Fungus Setosphaeria rostrata

Supplemental material, Supporting information, for Setosphaerine A: A New Chlorinated Polyketide Isolated From the Entomogenous Fungus Setosphaeria rostrata by Wen-Bin Gao, Sha-Sha Liu, Ying-Chao Tian, Qian Dong, Jun Zhang, Jian-Chun Qin, and Du-Qiang Luo in Natural Product Communications