Pyran Rings Containing Polyketides from Penicillium raistrickii

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.


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.

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 1 H NMR data (Table 1) showed the presence of two methyl groups (δH 2.15, 1.83, 3H, each), three vicinal aromatic protons of an ABC spin system at δH 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 δH 6.17, and two phenolic hydroxyl protons at δH 11.21 and 9.73. The 13 C 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 13 C 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 δH 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

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 C 13 H 12 O 4 . The 1 H NMR data (Table 1) 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 13 C 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.

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 1 H NMR data (Table 1) showed the presence of two methyl groups (δH 2.15, 1.83, 3H, each), three vicinal aromatic protons of an ABC spin system at δH 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 δH 6.17, and two phenolic hydroxyl protons at δH 11.21 and 9.73. The 13 C 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 13 C 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 δH 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 Penicipyran B (2) was isolated as colorless needles. Its molecular formula was assigned to be C 13 H 12 O 5 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 13 C 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 C 11 H 8 O 6 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 1 H 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 13 C NMR spectrum showed ten sp 2 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 C 15 H 18 O 6 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) 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 1 H-1 H 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  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 ∆δ S-R values ( Figure 3) for 1,3-anti-diol model as reported [16][17][18], the positive ∆δ S-R values of C-13 and C-15 suggested their absolute configurations to be 13R and 15S.  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 ΔδS-R values ( Figure 3) for 1,3-anti-diol model as reported [16][17][18], the positive ΔδS-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 1 H and 13 C NMR data. An aldehyde, two phenolic hydroxyls, an aromatic methyl and an isolated aromatic hydrogen proton were observed in the 1 H and 13 C 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 (δC  An aldehyde, two phenolic hydroxyls, an aromatic methyl and an isolated aromatic hydrogen proton were observed in the 1 H and 13 C 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 -CH 2 -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.  Figure S28) revealed two hydroxyls were on the opposite sides [15]. In order to determine its absolute configuration,  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 1 H and 13 C NMR data. An aldehyde, two phenolic hydroxyls, an aromatic methyl and an isolated aromatic hydrogen proton were observed in the 1 H and 13 C 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 (δC 193.8, δH 10.08) in 5 instead of an ester carbonyl in 4; (2) a -CH2-CHOH-moiety in 4 was replaced by a double bond (δC 124.8, δH 6.24; δC 127.9, δH 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 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 (IC 50 s: 0.42, 0.15, and 0.33 µM, respectively). Compounds 5 showed cytotoxicity against HL-60 and K562 cell lines with IC 50 values of 4.4 and 8.5 µM, respectively, while the other compounds were inactive (IC 50 > 20 µM).

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].

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.