Pyran Rings Containing Polyketides from Penicillium raistrickii

Abstract

Five new pyran rings containing polyketides, penicipyrans A–E (1–5), together with the known pestapyrone A (6), were isolated from the saline soil-derived Penicillium raistrickii. Their structures were determined by interpretation of NMR and HRESIMS data. The absolute configurations of compounds 4 and 5 were established by the modified Mosher’s method and single-crystal X-ray diffraction analysis, respectively. These compounds possessed high structural diversity including two α-pyrones (1, 2), three isocoumarins (3, 4, 6), and one dihydropyran derivative (5). Among them, Compound 5 exhibited cytotoxicity against HL-60 and K562 cell lines with IC₅₀ values of 4.4 and 8.5 μM, respectively.

1. Introduction

Natural products are intriguing and a promising source of anticancer drugs [1]. Those produced by organisms living in special environments, such as high pH, high/low temperature, high salty, etc., represent a group of compounds possessing unique chemical scaffolds that are important for finding new drug leads [2,3,4,5]. The saline soil broadly distributes near the seaside, which features a complex ecosystem with high salt and pH, and low nutrients, due to the interaction of land and sea [6]. To adapt to special environments, fungal strains inhabiting saline niches represent an excellent biosynthesizer with great potential to produce structurally diverse and biologically important secondary metabolites.

In the course of our ongoing search for bioactive natural products from saline soil-derived fungi [7,8], a P. raistrickii strain isolated from saline soil collected from the coast of Bohai Bay in Zhanhua, China, was selected for further study because of the potent anti-proliferative activity of its EtOAc extract. In the previous study, a series of new spiroketals with cytotoxcity were obtained from the same strain [9,10]. Encouraged by the findings, we carried out a study that lead to the isolation of five new metabolites with pyran moiety, including two α-pyrones (1, 2), two isocoumarins (3, 4), and one dihydropyran derivative (5), called penicipyran A–E (1–5) (Figure 1) [11]. Herein, we report the isolation, structure elucidation, and cytotoxicity of these compounds.

2. Results

Penicipyran A (1) was obtained as a colorless needle. HRESIMS produced an ion peak at m/z 231.0659 [M − H]⁻, indicating a molecular formula of C₁₃H₁₂O₄. The ¹H NMR data (

Table 1. ¹H and ¹³CNMR data for 1 (DMSO-d₆), 2 (acetone-d₆) and 3 (DMSO-d₆).

Position	1 a		2 b		3 a
	δC	δH (J in Hz)	δC	δH (J in Hz)	δC	δH (J in Hz)
1	165.3, C		166.2, C		166.3, C
2	97.5, C		98.3, C
3	164.5, C		164.7, C		149.2, C
4	103.6, CH	6.17, s	104.1, CH	6.28, s	108.4, CH	7.21, s
5	155.7, C		156.6, C		103.4, CH	6.69, s
6	120.2, C		112.5, C		163.8, C
7	155.4, C		156.7, C		111.0, C
8	113.3, CH	6.76, d (8.2)	100.4, CH	6.34, s	159.9, C
9	130.5, CH	7.16, dd (8.2, 7.5)	159.3, C		98.7, C
10	120.6, CH	6.73, d (7.5)	109.0, CH	6.30, s	135.7, C
11	137.8, C		139.6, C		8.1, CH3	2.04, s
12	19.4, CH3	2.15, s	19.4, CH3	2.17, s	162.1, C
13	8.5, CH3	1.83, s	7.8, CH3	1.92, s
OH-3		11.21, s
OH-7		9.73, s
OH-8						11.39, br s

^a The ¹H (400 M Hz) and ¹³C (100 M Hz); ^b The ¹H (500 M Hz) and ¹³C (125 M Hz).

) showed the presence of two methyl groups (δ_H 2.15, 1.83, 3H, each), three vicinal aromatic protons of an ABC spin system at δ_H 7.16 (1H, dd, J = 8.2, 7.5 Hz), 6.76 (1H, d, J = 8.2 Hz), and 6.73 (1H, d, J = 7.5 Hz), an isolated olefinic proton at δ_H 6.17, and two phenolic hydroxyl protons at δ_H 11.21 and 9.73. The ¹³C NMR spectrum displayed 13 carbon signals. Eleven of them were aromatic or olefinic carbons, including a carbonyl and three oxygenated ones, and two of them were methyl carbons. An α-pyrone moiety was deduced from the HMBC correlations from H-13 to C-1, C-2, and C-3, and from H-4 to C-2, C-3, and C-5, as well as the chemical shifts of C-1–C-5. The IR absorptions at 1652 and 1562 cm⁻¹ and UV maximum absorption at 291 nm also supported the presence of the α-pyrone moiety [12]. The HMBC correlation (Figure 2) from H-4 to C-6 suggested a linkage between the 1,2,3-trisubstituted phenyl ring and the α-pyrone moiety from C-5 to C-6. To satisfy the molecular formula, two hydroxyl groups were assigned to C-3 and C-7, respectively, which was confirmed by their chemical shifts (Table 1), as well as the HMBC correlations (Figure 2). Thus, the structure of 1 was established.

Penicipyran B (2) was isolated as colorless needles. Its molecular formula was assigned to be C₁₃H₁₂O₅ on the basis of negative HRESIMS ion at m/z 247.0604 [M − H]⁻, which was one more oxygen atom than that of 1. The IR and UV spectra of Compounds 1 and 2 were almost identical, indicating they were structural analogues. The ¹³C NMR spectrum of 2 also showed 13 carbon signals (Table 1), which was highly similar to those of 1, except for the presence of an oxygenated aromatic quaternary carbon instead of an aromatic methine in 1. Those findings suggested 2 was a hydroxylated derivative of 1. The additional hydroxyl in 2 was assigned at C-9, supported by the observation of two isolated aromatic hydrogen signals at δ_H 6.34 and 6.30, and the chemical shift of C-9. The structure was further confirmed by the HMBC spectrum (Figure 2).

Penicipyran C (3) was acquired as a pale yellowish amorphous powder. Its molecular formula C₁₁H₈O₆ was assigned based upon the HRESIMS ion at m/z 235.0246 [M − H]⁻. The UV spectrum (absorption peaks at 340, 330, 304, and 254 nm) was similar to that of 6,8-dihydroxyisocoumarin-3-carboxylic acid [13]. In the IR spectrum, the broad absorption peak at 3600–2500 cm⁻¹ indicated the presence of a carboxyl group. The ¹H NMR spectrum displayed two isolated aromatic hydrogen signals at δ_H 7.21 (1H, s) and 6.69 (1H, s), and one aromatic methyl at δ_H 2.04 (3H, s). The ¹³C NMR spectrum showed ten sp² carbon signals and one aliphatic carbon signal (Table 1). Carefully comparing the data of 3 with those of 6,8-dihydroxyisocoumarin-3-carboxylic acid and pestapyrone A (6) [14], likewise isolated in this study, revealed that the presence of a carboxylic acid group in 3 in place of a hydroxymethyl group in 6. Finally, the HSQC and HMBC spectra were used to confirm the structure of 3.

Penicipyran D (4), a colorless needle, was assigned the molecular formula C₁₅H₁₈O₆ based on the HRESIMS ion at m/z 293.1015 [M − H]⁻. The UV (absorption peaks at 333, 280, 247, and 240 nm) and IR (absorptions at 1672, 1621, 1582 and 1510 cm⁻¹) spectra of 4 were almost the same as those of 6 and peneciraistin D [10], which suggested they possessed the same isocoumarin moiety. The NMR data (

Table 2. ¹H and ¹³C NMR data for 4 (DMSO-d₆) and 5 (acetone-d₆).

Position	4 a			5 b
	δC	δH (J in Hz)	δC	δH (J in Hz)
1	166.3, C		152.5, C
2			124.8, CH	6.24, dd (9.7, 2.5)
3	154.6, C		127.9, CH	6.09, ddd (9.7, 6.5, 2.6)
4	105.3, CH	6.47, s	31.4, CH2	2.34, ddd (18.0, 6.1, 3.5); 2.16, ddt (18.0, 10.3, 2.5)
5	101.5, CH	6.44, s	71.6, CH	4.11, m
6	163.5, C		20.3, CH3	1.34, d (6.2)
7	109.6, C		100.6, CH	5.86, s
8	159.9, C		139.3, C
9	97.8, C		111.1, C
10	136.4, C		163.5, C
11	7.9, CH3	2.01, s	108.8, C
12	41.7, CH2	2.47, m	162.4, C
13	64.8, CH	4.02, m	108.6, CH	7.08, s
14	46.4, CH2	1.40, m	193.8, CH	10.08, s
15	62.7, CH	3.83, m	6.5, CH3	2.04, s
16	24.5, CH3	0.90, d (6.1)
OH-6/10		10.84, s		12.87, s
OH-8/12		11.33, s		9.32, s
OH-13		4.72, d (5.6)
OH-15		4.38, d (4.9)

^a The ¹H (400 M Hz) and ¹³C (100 M Hz); ^b The ¹H (500 M Hz) and ¹³C (125 M Hz).

) also strongly supported that Compound 4 contained the same isocoumarin moiety as 6, except additional signals for two hydroxyls at δ_H 4.72 (1H, d, J = 5.6 Hz) and 4.38 (1H, d, J = 4.9 Hz), two oxygenated methines, two methylenes, and a methyl group were observed in the spectra. The ¹H-¹H COSY data (Figure 2) established the presence of a 2,4-dihydroxyl pentyl moiety. Finally, the HMBC correlations from H-12 to C-3 and C-4 indicated the connection between pentyl moiety and isocoumarin moiety, which established the planar structure of 4.

The coupling constants of H_a-14 [δ_H 1.61 (1H, ddd, J = 14.2, 8.8, 3.3 Hz)] and H_b-14 [δ_H 1.56 (1H, ddd, J = 14.2, 9.0, 3.4 Hz)] in the ¹H NMR spectrum (MeOH-d₆, 500 MHz) (Figure S28) revealed two hydroxyls were on the opposite sides [15]. In order to determine its absolute configuration, 4 was treated with (R)- and (S)-MTPA chlorides to produce its (S)- and (R)-MTPA esters (4a and 4b), respectively. According to the distributions of the Δδ_S-R values (Figure 3) for 1,3-anti-diol model as reported [16,17,18], the positive Δδ_S-R values of C-13 and C-15 suggested their absolute configurations to be 13R and 15S.

Penicipyran E (5) was obtained as yellow needles. Its molecular formula C₁₅H₁₆O₄ was established by HRESIMS at m/z 259.0965 [M − H]⁻, which was in agreement with the ¹H and ¹³C NMR data. An aldehyde, two phenolic hydroxyls, an aromatic methyl and an isolated aromatic hydrogen proton were observed in the ¹H and ¹³C NMR spectra (Table 2). The 1D NMR data were similar to those of 4, except two major differences were observed: (1) the observation of an aldehyde group (δ_C 193.8, δ_H 10.08) in 5 instead of an ester carbonyl in 4; (2) a -CH₂-CHOH- moiety in 4 was replaced by a double bond (δ_C 124.8, δ_H 6.24; δ_C 127.9, δ_H 6.09) in 5. These findings indicated that there was a different polyketide cyclization pattern for Compound 5, forming a hydro-pyran ring at the terminus instead of the benzoenol lactone in 4. The structure of 5 was further conformed by comprehensive 2D NMR analyses (Figure 2) and single crystal X-ray diffraction analyses (Figure 4), which finally established the absolute configuration of 5 as 1Z, 6S.

Compounds 1–6 were tested for their cytotoxicity against a panel of cancer cell lines (A549, HL-60, and K562) using previously described methods [8,10], with doxorubicin as a positive control (IC₅₀s: 0.42, 0.15, and 0.33 μM, respectively). Compounds 5 showed cytotoxicity against HL-60 and K562 cell lines with IC₅₀ values of 4.4 and 8.5 μM, respectively, while the other compounds were inactive (IC₅₀ > 20 μM).

3. Materials and Methods

3.1. General Experimental Procedures

Melting point (mp) data were obtained with an XRC-1 micro-melting point apparatus (Sichuan University Scientific Instrument Factory, Chengdu, China). Optical rotations were determined on an Autopol V Plus digital polarimeter (Rudolph Research Analytical, Hackettstown, NJ, USA). UV spectra were recorded on a TU-1091 spectrophotometer (Beijing Purkinje General Instrument Co., Ltd., Beijing, China). CD spectra were acquired with a Chirascan spectropolarimeter (Applied Photophysics Ltd., Leatherhead, UK). IR spectra were collected on a Nicolet 6700 spectrophotometer (Thermo Scientific, Waltham, MA, USA) using an attenuated total reflectance (ATR) method. NMR spectra were recorded on a Bruker Avance 400 (Bruker Biospin Group, Karlsruhe, Germany) or Bruker AVIII 500 spectrometer (Bruker Biospin Group, Karlsruhe, Germany) with TMS as the internal standard. Crystal structure determination was carried out on a Bruker Smart 1000 CCD X-ray diffractometer (Bruker Biospin Group, Karlstuhe, Germany). HRESIMS was measured on a 1200RRLC-6520 Accurate-Mass Q-TOF LC/MS mass spectrometer (Agilent Technologies, Ltd., Palo Alto, CA, USA). Semipreparative HPLC was performed on a SHIMADZU LC-6AD Liquid Chromatography (Shimadzu Corporation, Kyoto, Japan) with an SPD-20A Detector using an ODS column (HyperClone 5 μ ODS (C₁₈) 120 Å, 250 mm × 10 mm, Phenomenex, 4 mL/min). Sephadex LH-20 (Ge Healthcare Bio-Sciences AB, Uppsala, Sweden).

3.2. Fungal Material

The working strain, P. raistrickii (Genbank accession No. HQ717799), was isolated from the saline soil collected from the coast of Bohai Bay in Zhanhua, Shandong Province of China, in August 2008. The fungus was identified on the basis of sequence analysis of the ITS region of the rDNA. It was deposited at Department of Chemistry, Binzhou Medical College, Yantai. The fermentation and extraction of P. raistrickii were described in a previous article [10].

3.3. Extraction and Isolation

The extract (42 g) of P. raistrickii was subjected to a silica gel (200–300 mesh) column eluted with a stepwise gradient of petroleum ether/chloroform (2:1, 1:1, 1:2, and 0:1, v/v), and chloroform/methanol mixtures (100:1, 50:1, 20:1, 10:1, and 0:1, v/v) to yield eight fractions (Fractions 1–8). Fraction 6 (3.8 g) was passed through an ODS column (25–40 μm, Merck, Darmstadt, Germany) eluting with a H₂O/CH₃OH gradient to yield eighteen subfractions (Subfractions 6-1–6-18). Subfraction 6-5 (0.2 g) was purified by semipreparative HPLC on an ODS column using MeOH/H₂O (4:6, v/v; 1 L of water was added to 2 mL of trifluoroacetic acid; 4 mL/min) as the eluting solvent to yield 1 (13 mg, t_R 22.1 min) and 6 (26 mg, t_R 25.8 min). Subfraction 6-2 (0.1 g) was subjected to semipreparative HPLC (MeOH/H₂O, 3:7, v/v; 1 L of water contained 2 mL of trifluoroacetic acid; 4 mL/min) to obtain 2 (21 mg, t_R 24.6 min). Compounds 3 (7 mg) and 4 (22 mg) were obtained from Fraction 4 (1.1 g) through Sephadex LH-20 eluting with methanol. Fraction 2 (6.3 g) was fractionated using an ODS column eluting with a H₂O/CH₃OH gradient to yield fourteen subfractions (Subfractions 2-1–2-14). Subfraction 2-8 (0.3 g) was purified on Sephadex LH-20 using methanol as eluant to yield Compound 5 (16 mg).

Penicipyran A (1): colorless needles; mp 204–206 °C (MeOH); UV (MeOH) λ_max (log ε) 291 (3.90), 214 (4.37) nm; IR (ATR) ν_max 2935, 2619, 1652, 1612, 1562, 1464, 1402, 1375, 1222, 1134, 1063, 960, 910, 878, 828, 779, 752, 738, 701 cm⁻¹; ¹H and ¹³C NMR data, see Table 1; HRESIMS m/z: 231.0659 [M − H]⁻ (calcd. for C₁₃H₁₁O₄, 231.0652).

Penicipyran B (2): colorless needles; mp 245–248 °C (MeOH); UV (MeOH) λ_max (log ε) 299 (3.67), 219 (4.04); IR (ATR) ν_max 3335, 2931, 2598, 1699, 1646, 1606, 1549, 1471, 1404, 1374, 1340, 1274, 1250, 1223, 1120, 1055, 990, 949, 904, 881, 830, 753, 733, 706 cm⁻¹; ¹H and ¹³C NMR data, see Table 1; HRESIMS m/z 247.0604 [M − H]⁻ (calcd. for C₁₃H₁₁O₅, 247.0601).

Penicipyran C (3): pale yellowish amorphous power (MeOH); UV (MeOH) λ_max (log ε) 340 (3.80), 330 (3.77), 304 (3.79), 254 (4.50), 205 (4.04) nm; IR (ATR) ν_max 3600–2500 (broad), 1729, 1662, 1614, 1515, 1435, 1392, 1347, 1247, 1198, 1149, 1123, 1087, 977, 889, 827, 788, 760 cm⁻¹; ¹H and ¹³C NMR data, see Table 1; HRESIMS m/z 235.0246 [M − H]⁻ (calcd. for C₁₁H₇O₆, 235.0248).

Penicipyran D (4): colorless needles; mp 222–224 °C (MeOH); ${\left[\mathsf{\alpha }\right]}_{\mathrm{D}}^{20}$ −21.9 (c, 0.05, CH₃OH). UV (MeOH) λ_max (log ε) 333 (3.39), 280 (3.46), 247 (4.28), 240 (4.23), 201 (3.98) nm; CD (c, 0.5 mg/mL, MeOH) λ_max (Δε) 336 (−3.93), 289 (−7.65) nm; IR (ATR) ν_max 3126, 2966, 1672, 1621, 1585, 1510, 1462, 1439, 1348, 1259, 1206, 1176, 1152, 1104, 1058, 986, 834, 790, 715, 693 cm⁻¹; ¹H NMR (MeOH-d₄, 500 MHz) δ_H 6.35 (2H, s, H-4 and 5), 4.23 (1H, m, H-13), 4.03 (1H, m, H-15), 2.66 (1H, dd, J = 14.5, 5.0 Hz, H_a-12), 2.59 (1H, dd, J = 14.5, 8.1Hz, H_b-12), 2.09 (3H, s, H-11), 1.61 (1H, ddd, J = 14.2, 8.8, 3.3 Hz, H_a-14), 1.56 (1H, ddd, J = 14.2, 9.0, 3.4 Hz, H_a-14), 1.21 (3H, d, J = 6.3 Hz, H-16); ¹H (400 MHz) and ¹³C NMR (100 MHz) data, see Table 2; HRESIMS m/z 293.1015 [M − H]⁻ (calcd. for C₁₅H₁₇O₆, 293.1020).

Penicipyran E (5): yellow needles; mp 168–170 °C (MeOH); ${\left[\mathsf{\alpha }\right]}_{\mathrm{D}}^{20}$ +94.3 (c, 0.14, CH₃OH). UV (MeOH) λ_max (log ε) 371 (3.88), 322 (4.14), 272 (4.45), 215 (4.05); CD (c, 0.5 mg/mL, MeOH) λ_max (Δε): 330 (+4.78), 289 (+1.76), 270 (−12.78), 212 (+15.75) nm; IR (ATR) ν_max 3374, 2917, 2849, 1630, 1599, 1464, 1429, 1397, 1335, 1246, 1122, 1111, 1075, 1053, 962, 851, 801, 768, 750, 727, 674 cm⁻¹; ¹H and ¹³C NMR data, see Table 2; HRESIMS m/z 259.0965 [M − H]⁻(calcd. for C₁₅H₁₅O₄, 259.0965).

3.4. X-ray Crystal Data for Penicipyran E (5)

Penicipyran E was obtained as a yellow needle from MeOH: molecular formula C₁₅H₁₆O₄; M_r = 260.28, triclinic, space group P1, a = 8.5057(5) Å, b = 8.5593(5) Å, c = 9.4977(5) Å, α = 102.001(3)°, β = 101.812(3)°, γ = 97.7165(17)°, V = 650.64(6) ų, Z = 2, T = 293 (2) K, μ (Cu Kα) = 0.792 mm⁻¹, D_calc = 1.329 g/cm³, F (000) = 276, 4030 reflections measured (9.80 ≤ 2Θ ≤ 132.00), 2600 unique (R_int = 0.0129) which were used in all calculations. The final R₁ was 0.0427 [I ≥ 2σ (I)] and wR₂ was 0.1318 (all data). The crystal structure of Compound 5 was solved by direct method SHELXS-97 and expanded using difference Fourier techniques, refined by the program SHLXL-97 and full-matrix least-squares calculations. Crystallographic data for the structure of penicipyran E have been deposited with the Cambridge Crystallographic Data Centre as supplementary publication CCDC 1512473. These data can be obtained free of charge from the Cambridge Crystallographic Data Centre via www.ccdc.cam.ac.uk/data_request/cif.

3.5. Preparation of (R)- and (S)-MTPA Esters of Penicipyran D (4)

As our previously reported method [10], (R)-MTPA chloride (20 μL) and DMAP (50 μg) were added to a solution of 4 (2.0 mg) in pyridine (0.5 mL). The mixture was stirred at 28 °C for 12 h. After the addition of water (1 mL) and extraction with EtOAc, the extract was evaporated and the residue was subjected to HPLC on an ODS column (MeOH/H₂O, 90:10, 4 mL/min) to obtain the (S)-MTPA ester (4a). The same procedure was used to prepare the (R)-MTPA ester (4b) with (S)-MTPA chloride. ¹H NMR (CDCl₃, 400 MHz) data of pentyl chain in 4a: δ_H 5.33 (1H, m, H-13), 5.10 (1H, m, H-15), 2.71 (2H, m, H-12), 1.98 (2H, m, H-14), 1.28 (3H, J = 5.2 Hz, H-16); ¹H NMR (CDCl₃, 400 MHz) data of pentyl chain in 4b: δ_H 5.14 (1H, m, H-13), 5.06 (1H, m, H-15), 2.72 (2H, m, H-12), 1.93 (2H, m, H-14), 1.32 (3H, d, J = 5.2 Hz, H-16).

4. Conclusions

Penicipyrans (1–6) are members of a large family of pyran-containing natural products, including flavones, coumarins, isocoumarins, pyrones, xanthones, spiroketals, and others. Those compounds have a wide range of biological activities, including antitumor [19,20,21], antimicrobial [22], anti-inflammatory [23], and protective activities of the central nervous system (CNS) [24,25], which provide attractive chemicals for drug discovery [26]. In this research, five new pyran-bearing compounds, penicipyran A–E, were isolated from the extract of P. raistrichii. Penicipyran E (5) showed cytotoxicity against HL-60 and K562 cell lines. This finding provides a series of pyran-bearing chemicals that may be applied for the analyses of structure activity relationships of analogues in the future.