Penichrypyrone A: A New γ-Pyrone Derivative from the Sponge-Derived Fungus Penicillium chrysogenum LS18

γ-Pyrones (also 3-pyrones), structurally belonging to the class of the 6-membered cyclic unsaturated compounds containing the vinylog of a lactone, ¹ representing a large number of natural products widespread with pharmacological effects including anticancer, ² cytotoxicity, ^3,4 antifungal activity, ^5,6 as well as inducing adiponectin production. ⁷ In the course of our ongoing investigation to find novel fungal bioactive compounds, a new γ-pyrone, penichrypyrone A (1), together with 4 known compounds, conichaetone E (2), janthinone (3), mrlactonel (4), and aspergillusene A (5) (Figure 1), were isolated from a sponge-derived fungus Penicillium chrysogenum LS18 obtained from the sponge Haliclona sp. collected from the Linshui, Hainan Province, China. In this paper, we described the isolation, structure elucidation, and biological activities of these isolates.

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Figure 1

Structures of compounds 1-5.

Penichrypyrone A (1) was obtained as a colorless syrup. HRESIMS data of the minor metabolite 1 gave an [M+H]⁺ peak at m/z 223.1686 suggesting a molecular formula of C₁₄H₂₂O₂ and a requirement of 4 degrees of unsaturation, which were entirely consistent with the spectroscopic data. The IR spectrum indicated the presence of methyl (2962 and 1380 cm⁻¹) and unsaturated carbonyl (1661 cm⁻¹) groups (see Supplemental Material). In combination with NMR spectra of 1, it can be deduced that 1 contained γ-pyrone moiety. ^8,9 The ¹H NMR spectrum of 1 indicated the presence of 1 aromatic proton at δ _H 6.06 (s, H-3), as well as a 1,3-dimethylpentyl moiety at δ _H 3.08 (m, H-7), 1.77, 1.22 (2H, m, H-8), 1.14 (m, H-9), 1.25, 1.15 (2H, m, H-10), 0.86 (3H, d, J = 6.1 Hz, H-12), and 0.84 (3H, t, J = 7.2 Hz, H-11) (Table 1). Analysis of ¹³C NMR spectrum indicated 14 carbons including signals for 5 sp² carbons δ _C 180.4 (C-4), 167.6 (C-6), 164.5 (C-2), 119.7 (C-5), and 112.4 (C-3) assignable to a 4H-pyran-4-one moiety; 5 methyl groups δ _C 19.7 (C-15), 19.7 (C-12), 19.2 (C-13), 11.4 (C-11), and 9.3 (C-14); 2 methylenes δ _C 41.3 (C-8) and 29.9 (C-10); and 2 methines δ _C 33.0 (C-7) and 32.5 (C-9). The HMBC correlations from H₃-14 (δ _H 1.96, s) to C-4, C-5, and C-6, from H-15 (δ _H 2.22, s) to C-2 and C-3, and from H-3 to C-5 established a 2,5-dimethyl-γ-pyrone fragment (Figure 2). The HMBC correlations from H₂-8 to C-9 and C-10, from H-11 to C-9, as well as the ¹H-¹H COSY correlations of H₃-11/H₂-10, H-9/H₃-12, H₂-8/H-7, and H-7/H₃-13, established a 1,3-dimethylpentyl fragment. Additionally, the H₂-8 (δ _H 1.77, m, 1.22, m) and C-6 have a strong HMBC correlation, indicating that the 1,3-dimethylpentyl unit was connected to the C-6 position of the 2,5-dimethyl-γ-pyrone moiety (Figure 2). The absolute stereochemistry of 1 was determined by comparing the optical rotation value (+19) with that of aglajne-2 acetate (+46), ¹⁰ which had had the similar side chain with 1. Thus, the structure of 1 was tentatively assigned as (7S,9S)-3,6-dimethyl-2-(4-methylhexan-2-yl)-4H-pyran-4-one, named penichrypyrone A. Although a patent was issued by our group in 2018, compound 1 had not formally been reported with its detail physical and spectroscopic properties.

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Table 1

¹H (600 MHz, CDCl₃) and ¹³C (150 MHz, CDCl₃) NMR Data, ¹H-¹H COSY and HMBC Correlations for 1.

C/H	δ H [mult. (J in Hz)]	δ C	HMBC	COSY
2		164.5 C
3	6.06 s	112.4 CH	2, 5, 15
4		180.4 C
5		119.7 C
6		167.6 C	1, 3, 4, 6	6
7	3.08 m	33.0 CH	8, 13	8, 13
8	1.77 m, 1.22, m	41.3 CH2	6, 7, 9, 10, 12, 13	7
9	1.14 m	32.5 CH	11, 12	12
10	1.25 m, 1.15 m	29.9 CH2	8, 9, 11, 12	11
11	0.84 t (7.2)	11.4 CH3	9, 10	10
12	0.86 d (6.1)	19.7 CH3	8, 9, 10	9
13	1.19 d (6.9)	19.2 CH3	6, 7, 8	7
14	1.96 s	9.3 CH3	4, 5, 6
15	2.22 s	19.7 CH3	2, 3

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Figure 2

Key COSY and HMBC correlations of compound 1.

The structures of 4 known compounds, conichaetone E (2), ¹¹ janthinone (3), ^12,13 mrlactonel (4), ¹⁴ and aspergillusene A (5), ¹⁵ were identified by comparison of spectroscopic data with those reported. The isolated compounds were evaluated for their antioxidant activity. And all the compounds were found to be inactive (IC₅₀ > 128 µg/mL).

Experimental

General Experimental Procedures

NMR spectra were recorded on a DirectDrive2 600 MHz NMR spectrometer (Agilent, Santa Clara, CA, United States) at 25°C and Auto Triple Resonance (Agilent). HRESIMS data was measured using a Waters Q-TOF Premier LC/MS spectrometer (Waters Co., Milford, MA, United States). The UV spectrum was recorded on a Thermo Evolution 201 spectrophotometer (Thermo Scientific, MA, United States). IR spectrum was recorded on a Nicolet iS5 FT-IR spectrophotometer (Thermo Scientific). Column chromatography was conducted with silica gel (200-300 mesh, Qingdao Marine Chemical Inc., Qingdao, China). Medium-pressure liquid chromatography (MPLC) was performed on a FLEXA Purification System (Agela Technologies, China) using a ODS column. The HPLC equipment (Alliance 2695, Milford, MA, United States) was equipped with a Waters 2996 detector and Empower System.

Fungal Materials

The fungus LS18 was isolated from the sponge-derived fungus, obtained from the sponge Haliclona sp. collected from Linshui, Hainan Province, China. On the basis of ITS rRNA gene sequence similarity, the strain was identified as P. chrysogenum. The strain specimen in PDA medium was deposited at the College of Food and Pharmaceutical Sciences, Ningbo University, China.

Culture, Extraction, and Isolation

The strain was directly cultivated in a seawater-based PDB medium (potato 200 g/L, glucose 20 g/L, sea salt 37 g/L, and H₂O 1 L). After 2 days of incubation at 25°C with 180 rpm agitation, the large-scale fermentation was conducted in 50 × 1 L Erlenmeyer flasks containing 400 mL PDB medium, each of which was followed by inoculation with 40 mL of the seed culture. After 14 days of fermentation, 20 L of culture broth was obtained. The whole broth was extracted with 20 L of EtOAc for 3 times. After removal of the solvents by rotary evaporator, 12.6 g of crude extract was obtained.

Then, the crude extract was chromatographed over silica gel and eluted with petroleum ether-EtOAc (20:1, 10:1, 10:3, 2:1, 1:1, 1:2, and 0:1) stepwise gradient elution to generate 6 fractions (Fr.1-Fr.6). Fr. 2 (2.22 g) was applied to MPLC using a gradient elution of MeOH/H₂O (20%-60%, 20 mL/min, 100 minutes, UV detection at 220 nm) to give 4 subfractions Fr.2A-Fr.2D. Subfraction Fr.2B (256 mg) was further separated by semipreparative HPLC (33% ACN/H₂O) to yield 1 (1.6 mg). Subfraction Fr.2.C (409.2 mg) was further purified by semipreparative HPLC (37% ACN/H₂O) to obtain 2 (7.8 mg). Fr.2.D (189.4 mg) was further purified by semipreparative HPLC (36% ACN/H₂O) to afford 3 (4.6 mg), 4 (5.6 mg), and 5 (4.8 mg).

Penichrypyrone A (1)

Colorless syrup.

[α]:_D ²⁵ +19 (c 0.03, MeOH).

IR (KBr): ν_max 3344, 2962, 2928, 2887, 2857, 1733, 1661, 1607, 1457, 1411, 1380, 1250, 1202, 1180, 1135, 1084, 1039, 856, 802 cm⁻¹.

UV λ _max (MeOH) nm (log ε): 204.5 (4.35), 254 (4.31).

¹H NMR (600 MHz, CDCl₃) δ _H: 6.06 (1H, m, H-3), 3.08 (1H, m, H-7), 2.22 (3H, s, H-15), 1.96 (3H, s, H-14), 1.77, 1.22 (2H, m, H-8), 1.25, 1.15 (2H, m, H-10), 1.19 (3H, d, J = 6.9 Hz, H-13), 1.14 (1H, m, H-9), 0.86 (3H, d, J = 6.1 Hz, H-12), 0.84 (3H, t, J = 7.21 Hz, H-11).

¹³C NMR (150 MHz, CDCl₃) δ _C: 180.4 (C-4, C), 167.6 (C-6, C), 164.5 (C-2, C), 119.7 (C-5, C), 112.4 (C-3, CH), 41.3 (C-8, CH₂), 33.0 (C-7, CH), 32.5 (C-9, CH), 29.9 (C-10, CH₂), 19.7 (C-15, CH₃), 19.7 (C-12, CH₃), 19.2 (C-13, CH₃), 11.4 (C-11, CH₃), 9.3 (C-14, CH₃).

HRESIMS: m/z 223.1686 [M+H]⁺ (calcd. for C₁₄H₂₂O₂; 223.1693).

Antioxidant Assays

The antioxidant property of 5 compounds was determined by DPPH free radical scavenging assay. All the compounds were diluted to various concentrations and added to the methanolic solution of DPPH (0.2 mM). The mixture (200 µL/well) was added to 96-well plates, shaken, and kept for 20 minutes at room temperature. The absorbance was measured at 517 nm by a microplate reader. Vitamin C was used as the positive control. And the DPPH free radical scavenging activity was calculated by using the following equation: DPPH free radical scavenging activity (%) = [(absorbance of the control-absorbance of the sample)/absorbance of the control] ×100.

Supplemental Material

Supporting information - Supplemental material for Penichrypyrone A: A New γ-Pyrone Derivative from the Sponge-Derived Fungus Penicillium chrysogenum LS18

Supplemental material, Supporting information, for Penichrypyrone A: A New γ-Pyrone Derivative from the Sponge-Derived Fungus Penicillium chrysogenum LS18 by Xiaoping He, Lu Ren, Lijian Ding, Jianzhou Xu, Bin Zhang, Weiyan Zhang, and Shan He in Natural Product Communications