The endophyte P. chrysogenum QEN-24S displayed obvious activity against the pathogen fungus A. brassicae in our initial dual culture test (Figure 1). This endophyte was therefore submitted to a large scale fermentation for bioactive compounds isolation. The EtOAc extract derived from the rice culture of the fungal strain was suspended in MeOH-H₂O (9:1, v/v) and was extracted with n-hexane to remove the non-polar fraction. The MeOH-soluble fraction displayed moderated inhibitory activity against the pathogenic fungus A. brassicae. This fraction was then subjected to various separation procedures by column chromatography on silica gel, Lobar LiChroprep RP-18, and Sephadex LH-20, to afford four new (1–3, and 5) and one known secondary metabolites (4), as shown in Figure 2. By detailed analysis of the spectroscopic data, the structure for the known compound 4 was determined as 1-(2,4-dihydroxy-6-methylbenzoyl)-glycerol (4) [17].

Compounds 1 and 2 were obtained as colorless oils. The IR spectrum of 1 showed absorption bands for OH (3405 cm⁻¹) and C=O (1716 cm⁻¹) functionalities in the molecules. The low-resolution ESI-MS displayed ion peaks at m/z 337 [M + Na]⁺ and 651 [2M + Na]⁺. Its molecular formula was determined as C₁₈H₃₄O₄ on the basis of positive HR-ESI-MS, indicating two degrees of unsaturation. The ¹³C-NMR (Table 1) along with the DEPT experiments revealed the presence of 18 carbon atoms including one carbonyl carbon, five sp³ methines, nine sp³ methylenes, and three methyl groups. The remaining one unsaturation demonstrated that 1 is a monocyclic compound. The ¹H-¹H COSY correlations revealed the presence of a contiguous sequence of the proton signals comprising from H-2 to H-16, as shown in Figure 3. Further ¹H-¹H COSY correlations from H₃-17 to H-12 and from H₃-18 to H-10 unambiguously demonstrated two methyl groups H₃-17 and H₃-18 attached to C-12 and C-10, respectively. The HMBC correlations from H-2 and H-3 to C-1 established the linkage of C-1 to C-2 and C-3. Finally, the chemical shifts of C-5 (δ_C 75.68, d) and C-1 (δ_C 170.33, s) established the connection of C-1 and C-5 via an ester linkage to form a tetrahydropyran-2-one moiety as shown in Figure 3.

The relative configuration of the two chiral centers C-3 and C-5 in the tetrahydropyran ring was proposed by comparison of its ¹³C-NMR data with those published for its analogues [18] sharing common partial structures from C-1 to C-8 (Figure 4). The relative configurations of C-3 and C-5 were deduced as 3β and 5α, respectively, as shown in Figure 2, according to the corresponding chemical shifts as shown in Figure 4. This was supported by the fact that no NOE correlations could be observed between H-3 and H-5 in the NOESY experiment. However, the relative configuration for C-10, C-12, and C-15 in the side chain remains unknown. Based on the above evidence, the structure for compound 1 was established as 4β-hydroxy-6α-(10-hydroxy-5,7-dimethylundecyl)tetrahydropyran-2-one, which was named as penicitide A, as shown in Figure 2.

The absolute configuration at C-15 of penicitide A (1) was determined to be R by application of modified Mosher’s method, as shown in Figure 5. However, due to the dehydration between H-2 and OH-3 during the acylation, attempts to determine the absolute configurations for C-3 and C-5 failed.

Compound 2 was assigned the molecular formula C₁₈H₃₄O₅, having one oxygen unit more than 1, on the basis of positive HR-ESI-MS experiments. Detailed comparison of the ¹H- and ¹³C-NMR data (Table 1) revealed that the structure of 2 was very similar to 1. However, the methine carbon signal at δ_C 30.11 (C-12) in the ¹³C-NMR spectrum of 1 was replaced by an oxygenated quaternary carbon at δ_C 72.37 in 2. Accordingly, the methine proton at δ_H 1.45 in 1 disappeared in the ¹H-NMR spectrum of 2. Moreover, the doublet signal at δ_H 0.85 (J = 6.4 Hz) for H₃-17 in 1 was replaced by a downfield singlet at δ_H 1.15 in the ¹H-NMR spectrum of 2. The above evidences indicated that an OH substituent at C-12 in 2. The HMBC correlations from H-11 and H₃-17 to C-12 supported this conclusion. The configuration for the chiral centers at C-3 and C-5 was determined to be the same as that of 1 by NOESY experiment, as well as by detailed comparison of the NMR data with that of 1. The structure for compound 2 was therefore established as 4β-hydroxy-6α-(7,10-dihydroxy-5,7- dimethylundecyl)-tetrahydropyran-2-one as shown in Figure 2, which was named penicitide B.

Penicitides A and B (1 and 2) bear a unique 10-hydroxy- or 7,10-dihydroxy-5,7-dimethylundecyl moiety substituting at C-5 of the α-tetrahydropyrone ring. This structure feature is not reported previously among natural products. These compounds appear to be α-tetrahydropyrone polyketides that are derived from a mixed-precursor biosynthesis including acetate and propionate building blocks [19]. Propionate polyketides are well known components from marine invertebrates and actinomycetes [20,21], but they have been rarely isolated from fungi as far as we know.

Compounds 3 and 4 were obtained as a colorless oily mixture. Attempts to separate the two compounds by different CC steps as well as by semi-preparative HPLC with different solvent systems failed. Fortunately, compound 3 could be distinguished from 4, aided by 2D NMR experiments including ¹H-¹H COSY, HSQC, and HMBC, and by their different ratio (1:3) as indicated by the ¹H- and ¹³C-NMR spectra. Most NMR signals are well-resolved. The structure of compound 4 was readily identified as 1-O-(2,4-dihydroxy-6-methylbenzoyl)-glycerol by detailed NMR spectral data analysis (Table 2) and comparison with our recent report [17]. Low-resolution ESI-MS displayed pseudo-molecular ion peaks at m/z 265 [M + Na]⁺ and 507 [2M + Na]⁺ for both compounds. The molecular formula was determined as C₁₁H₁₄O₆ on the basis of positive HR-ESI-MS, suggesting five degrees of unsaturation. The structure of 3 was independently assigned by analysis of the ¹H- and ¹³C-NMR data (Table 2) as well as by HSQC and HMBC correlations. The only difference between the two isomers was the connectivity of the glycerol moiety with the 2,4-dihydroxy-6-methylbenzoyl unit. The symmetric nature of the NMR data for the glycerol moiety (H-1/H-3 and C-1/C-3) in 3 as well as the observed ³J correlation from H-2 to C-4 in the HMBC spectrum (Figure 3) established the linkage of 2,4-dihydroxy-6-methylbenzoyl group to C-2 of the glycerol unit. Thus, the structure of compound 3 was assigned as 2-(2,4-dihydroxy-6-methylbenzoyl)-glycerol, as shown in Figure 2.

Compound 5 was obtained as colorless oil. Its IR spectrum showed absorption bands for OH (3178 cm⁻¹) and C=O (1727 cm⁻¹) functionalities in the molecules. The low-resolution ESI-MS displayed ion peaks at m/z 245 [M + H]⁺, 267 [M + Na]⁺, and 511 [2M + Na]⁺. Its molecular formula was determined as C₁₂H₂₀O₅ on the basis of positive HR-ESI-MS. The planar structure was determined mainly by comparison with the literature reports of the known compounds (E)-3,8-dihydroxy-3,7- dimethyl-6-octenoic acid (6) [22] and (S,E)-3-hydroxy-3,7-dimethyl-6-octenoic acid (7) [23] as well as by the NOESY spectral data. Detailed comparison of the ¹H-NMR data of 5 (Table 3) with that of 6 [22] revealed that these two structures were very similar, except an extra acetyl methyl signal at δ_H 2.07 was observed in the ¹H-NMR spectrum of 5. Further analysis of the ¹³C-NMR data of 5 indicated that an extra acetyl unit was attached to the monoterpene skeleton of 6. The location of the acetyl unit was clearly determined at C-8 by the long range correlations from the acetoxyl methyl proton to C-8 in the HMBC spectrum. The observed NOE correlation from H-6 to H-8 in the NOESY spectrum revealed that the double bond was in trans-geometry. Finally, the absolute stereochemistry at C-3 was assigned to be S by comparison of the optical rotation of 5 ([α]_D²⁰ = +1.4, MeOH) with that of 7 ([α]_D²⁵ = +1.8, MeOH) [23].

Compound 5, which was named penicimonoterpene, pertains to a class of naturally occurring monoterpene derivatives named citronellic acid formed by the oxidation of citronellal with a carboxyl group at C-1 and an olefinic double bond at C-6 as shown in Figure 2, which has been isolated from ascomycete [23] and fungus [24].

The biological activity of the isolated compounds 1–5 was examined in antifungal and cytotoxicity bioassays. In the initial antifungal screening, penicimonoterpene (5) displayed potent activity against pathogen A. brassicae with an inhibition zone of 17 mm in diameter at the concentration of 20 μg/disk, while penicitide A (1) displayed moderate activity with an inhibition zone of 6 mm in diameter at the concentration of 20 μg/disk (Table 4). The cytotoxicity evaluation was also performed and the results indicated that penicitide A (1) possessed activity against HepG2 cell line with the IC₅₀ of 32 μg/mL. The other compounds displayed no appreciable activity.

Column chromatography was performed on commercial silica gel (Qingdao Haiyang Chemical Group Co., China; 200–300 mesh), Lobar LiChroprep RP-18 (40–63 μm; Merck), and Sephadex LH-20 (Pharmacia). Optical rotations were measured on an Optical Activity AA-55 polarimeter. UV Spectroscopic data were obtained on a Lengguang Gold Spectrumlab 54. IR spectra were performed on a JASCO FT/IR-4100 Fourier Transform infrared spectrometer. NMR spectra were acquired on a Bruker Avance-500 spectrometer at 500 MHz for ¹H and 125 MHz for ¹³C. MS data were recorded on a VG Autospec-3000 mass spectrometer.

The endophytic fungus Penicillium chrysogenum QEN-24S was isolated, by use of a standard procedure as in our previous report [9], from the inner tissue of an unidentified marine red algal species of the genus Laurencia collected from the Weizhou Island of southern China sea. Fungal identification was carried out by use of a molecular biological protocol by DNA amplification and sequencing of the ITS region as in our previous report [9]. The sequence data derived from the fungal strain has been submitted to and deposited at GenBank with accession number GU985086. A BLAST search result showed that the sequence was similar (99%) to the sequence of P. chrysogenum (compared to AY373903.1 GI: 34809383). The strain is preserved at the Key Laboratory of Experimental Marine Biology, Institute of Oceanology of the Chinese Academy of Sciences, with accession number QEN-24S.

Mass growth of the fungus for the isolation and identification of new metabolites was carried out in Erlenmeyer flasks (1 L each). The fungus was grown on rice solid medium (to 100 g commercially available rice, 0.6 g of peptone and 100 mL of distilled water was added, then kept overnight prior to autoclaving) at room temperature under static conditions for 30 days.

The rice culture (10 flasks) of the fungal strain P. chrysogenum QEN-24S was extracted with EtOAc. The crude extract obtained was dried and partitioned between n-hexane and 90% MeOH. The 90% MeOH-soluble material (2 g) was subjected to column chromatography (CC) over silica gel, eluting with different solvents of increasing polarity from petroleum ether (PE) to MeOH to yield 10 fractions (Frs. 1–10) on the basis of TLC analysis. Fr. 5 (0.3 g) was further purified by CC on silica gel eluting with a CHCl₃-MeOH gradient (from 80:1 to 1:1), Sephadex LH-20 (MeOH), and Lobar LiChroprep RP-18 with a H₂O-MeOH gradient (from 1:1 to 0:1) to afford 1 (10.5 mg) and a CC-unseparable mixture of 3 and 4 (11.1 mg). Fr. 8 (0.4 g) was further purified by CC on silica gel by eluting with a CHCl₃-MeOH gradient (from 80:1 to 5:1), Sephadex LH-20 (MeOH), and preparative TLC (plate: 20 × 20 cm, developing solvents: CHCl₃-MeOH, 10:1) to afford 2 (5.4 mg) and 5 (12.4 mg).

Penicitide A (1). Colorless oil; [α]_D²⁰: +42.9 (c 0.14, MeOH); IR (KBr) ν_max 3405, 2927, 2865, 1716, 1457, 1376, 1253, 1064, 937, 852, 725, 586 cm^{−1; 1}H- and ¹³C-NMR data, see Table 1; ESI-MS m/z 337 [M + Na]⁺, 651 [2M + Na]⁺; HR-ESI-MS: m/z 337.2345 [M + Na]⁺ (calcd for C₁₈H₃₄O₄Na⁺, 337.2354).

Penicitide B (2). Colorless oil; [α]_D²⁰: +100.0 (c 0.08, MeOH); IR (KBr) ν_max 3394, 3297, 2931, 2861, 1724, 1635, 1458, 1377, 1057, 802, 489 cm^{−1; 1}H- and ¹³C-NMR data, see Table 1; ESI-MS m/z 353 [M + Na]⁺, 683 [2M + Na]⁺; HR-ESI-MS: m/z 353.2297 [M + Na]⁺ (calcd for C₁₈H₃₄O₅Na⁺, 353.2303).

2-(2,4-Dihydroxy-6-methylbenzoyl)-glycerol (3). Colorless oil; UV λ_max (MeOH) nm (log ɛ): 214 (4.79), 264 (4.50); ¹H- and ¹³C-NMR data, see Table 4; ESI-MS m/z 265 [M + Na]⁺, 507 [2M + Na]⁺; HR-ESI-MS: m/z 265.0689 [M + Na]⁺ (calcd for C₁₁H₁₄O₆Na⁺, 265.0688).

Penicimonoterpene (5). Colorless oil; [α]_D²⁰: +1.4 (c 0.83, MeOH); IR (KBr) ν_max 3178, 2969, 2938, 1727, 1442, 1381, 1238, 1025, 949, 852 cm^{−1; 1}H- and ¹³C-NMR data, see Table 5; ESI-MS m/z 245 [M + H]⁺, 267 [M + Na]⁺, 511 [2M + Na]⁺; HR-ESI-MS: m/z 267.1203 [M + Na]⁺ (calcd for C₁₂H₂₀O₅Na⁺, 267.1208).

(S)-(+)-α-Methoxy-α-(trifluoromethyl)phenylacetyl chloride (10 μL) and 4-(dimethylamino)-pyridine (2 mg) were added to penicitide A (1, 1.1 mg) which was dissolved in dry pyridine (400 μL). The mixture was kept at room temperature for 12 h. The acylation product was purified by preparative TLC on silica gel [eluent: petroleum ether/EtOAC (4:1, v/v)] to yield corresponding (R)-Mosher ester 1r. Treatment of 1 (1.2 mg) with (R)-MTPA-Cl (10 μL) as described above yielded the corresponding (S)-Mosher ester 1s.

Dual culture test of P. chrysogenum QEN-24S against A. niger and A. brassicae was carried out as literature report [26]. Pathogens and endophyte P. chrysogenum QEN-24S were inoculated at the periphery of the PDA-medium plate (200 g potato, 20 g dextrose, 20 g agar, and 1 L sea water), and then cultured at 27 °C for 5 days.

Antifungal assay against A. niger and A. brassicae was carried out using the well diffusion method [27]. Amphotericin B (AMPB) was used as positive control.

The cytotoxic activities against NCI-H460 (human non-small cell lung cancer), SMMC-7721 (human hepatoma), SW1990 (human pancreatic cancer), DU145 (human prostate carcinoma), HepG2 (human hepatocellular liver carcinoma), Hela (human epithelial carcinoma), and MCF-7 (human breast adenocarcinoma) cell lines were determined according to previously reported methods [28].