Six New Polyketide Decalin Compounds from Mangrove Endophytic Fungus Penicillium aurantiogriseum 328#

Six new compounds with polyketide decalin ring, peaurantiogriseols A–F (1–6), along with two known compounds, aspermytin A (7), 1-propanone,3-hydroxy-1-(1,2,4a,5,6,7,8,8a-octahydro-2,5-dihydroxy-1,2,6-trimethyl-1-naphthalenyl) (8), were isolated from the fermentation products of mangrove endophytic fungus Penicillium aurantiogriseum 328#. Their structures were elucidated based on their structure analysis. The absolute configurations of compounds 1 and 2 were determined by 1H NMR analysis of their Mosher esters; the absolute configurations of 3–6 were determined by using theoretical calculations of electronic circular dichroism (ECD). Compounds 1–8 showed low inhibitory activity against human aldose reductase, no activity of inducing neurite outgrowth, nor antimicrobial activity.


Results and Discussion
Peaurantiogriseol A (1, Figure 1) was obtained as a colorless solid and had a molecular formula of C16H26O3 as determined by HREIMS data (observed m/z 266.1878 M + , calculated 266.1876), requiring 4° of unsaturation. The 13 C-NMR and DEPT spectra (Table 1) indicated the presence of a carbonyl group (δ 215.4), two olefinic carbons, four sp 3 CH2 groups, five sp 3 CH groups, one sp 3 quaternary carbon atom, and three methyl groups. The 1 H-NMR and 1 H-1 H COSY spectra (Table 1 and Figure 2) showed the signals of a 3-oxopropanol system (δH 3.82/2.64), and a cis double bond signals (δH 5.91 d J = 10.6 Hz; 5.58 ddd J =10. 6,4.8,2.4 Hz). The remaining 2° of unsaturation supported a decalin segment in 1.
In the HMBC spectrum ( Figure 2), rich correlation data allowed us to unambiguously establish the locations of substituents on the decalin ring. A methyl singlet at δH 1.19 correlated with C-3 and C-5 respectively, which revealed that the methyl group, with the 3-oxopropanol side chain, was located at C-4 position. A methyl doublet signals at δH 0.75 (J = 8.4 Hz) correlated with C-13 and C-12, and another methyl doublet signals at δH 1.00 (J = 9.6 Hz) correlated with C-9 and C-7, revealing that the two methyl groups were located at C-8 and C-13 positions, respectively. Based on the HMBC correlations of H-11/C-9 and H-12/C-14, the cis double bond was easily assigned as C-11 and C-12. One hydroxyl group was identified at C-9 position based on the chemical shift of CH-9 (δ 2.89/79.3) and HMBC correlations.  Table S2). OH-1 of compound 1 was mainly esterified by S/R-MTPA-Cl based on the larger chemical shift of H-1. There was an esterified C-1 hydroxyl group in Mosher esters of 1, which were confirmed by their 19 F NMR spectra that showed 2 CF3 signals (Supplementary Materials Figure S8). The preferred conformations of Mosher esters of 1 dominating 1 H NMR spectroscopic features were that the 3-oxopropanol side chains with MTPA moiety were in equatorial position, and bend to C-13 position; CH2-1-O-C=O-CF3 substructure of Mosher esters of 1 were coplanar ( Figure 4). The shielding or deshielding effects of the phenyl rings of MTPA moiety on H-13 or H-14 were larger in its R-Mosher ester than that of S-Mosher ester. The absolute configuration of C-13 in 1 was deduced as S-configuration based on the positive chemical shift differences (Δδ SR ) of H-13 and negative chemical shift differences (Δδ SR ) of H-14 from corresponding Mosher esters [6,7]. Finally, the absolute configuration of 1 was confirmed as (4S,5S,8S,9R,10R,13S)-configuration (Figure 1) based. The absolute configuration of 1 was validated by the result that the experimental data and calculated ECD spectrum for (4S,5S,8S,9R,10R,13S)-configuration of 1 matched exactly ( Figure 5).    Peaurantiogriseol B (2, Figure 1) was obtained as a white solid and had a molecular formula of C16H26O3 based on HREIMS data (observed m/z 266.1875 M + , calculated 266.1876), same as compound 1. The 1 H-and 13 C-NMR spectra of 2 were very similar to those of 1 (Table 1), except for the absence of one oxygenated CH-9 group signal, and the change of a doublet signal at δ 1.00/18.7 to hydroxymethyl signals at δ 3.44/3.41/68.3. These results suggested the presence on compound 2 of a hydroxymethyl group at C-16 position. The 1 H-1 H COSY and HMBC correlations of 2 were also similar to those of 1 (Figure 2), which confirmed that an OH group was located at C-16.
The relative stereochemistry of 2 was established by its NOESY spectrum ( Figure 3). Its NOE data were very similar to those of 1.  Table S2), which were confirmed based on its 19 F NMR spectra (Supplementary Materials, Figure 13S); there were 3 CF3 signals in 19 F NMR spectra of Mosher esters of 2. The 1 H NMR spectroscopic features and preferred conformations of Mosher esters of 2 were the same as that of 1. Therefore, the absolute configuration of 2 was confirmed as (4S,5S,8S,10R,13S)-configuration ( Figure 1). The absolute configuration of 2 was validated by the result that the experimental data and calculated ECD spectrum for (4S, 5S, 8S, 10R, 13S)-configuration of 2 matched exactly ( Figure 5).
Peaurantiogriseol C (3, Figure 1) was obtained as a white solid and had a molecular formula of C16H26O3 based on HREIMS data (observed m/z 248.1770 [M − H2O] + , calculated for C16H24O2, 248.1771). The 1 H-and 13 C-NMR spectra of 3 were very similar to those of compound 2 (Table 1), except for the changes of hydroxymethyl signals at δ 3.41/68.3 to a methyl singlet at δ 1.22/31.0 in 3, and CH group signals at δ 1.58/41.0 to quaternary carbon atom signal at δ 70.2 in 3. These results suggested that compound 3 possesses a hydroxyl group at C-8 position, which were confirmed by the HMBC correlations from H-16 to C-7, and H-16 to C-9 ( Figure 2). The relative stereochemistry of 3 was the same as compound 2 based of its NOESY spectrum (Figure 3). The absolute configuration of compound 3 was determined by the result that the experimental data, showing a negative Cotton effect at 291 nm, and calculated ECD spectrum for (4S,5S,8S,10R,13S)-configuration of 3 matched exactly ( Figure 5).
Peaurantiogriseol D (4, Figure 1) had a molecular formula of C16H26O4 based on HREIMS data (observed m/z 282.1824 M + , calculated 282.1826), with one more oxygen atom than compound 3. The 1 H-NMR, 13 C-NMR, 1 H-1 H COSY, and HMBC correlations of 4 were very similar to those of 3 (Table 1, Figure 2), except for the change of a methyl doublet signal at δ 0.75 (d, J = 7.2 Hz) to singlet signal at δ 1.13 in 4, which suggested that compound 4 had an added OH group. The additional OH group was located at C-13 based on the chemical shift of CH-13 (δc 74.0) and HMBC correlations. The relative stereochemistry of 4 was established based on the result that the interatomic non-bonded distance of key atoms and NOESY correlation signals matched exactly in its 3D model ( Table 2, Me-eea-trans conformer). The relative stereochemistry of 4 was assigned as that methyl-14 and methyl-15 were equatorial; methyl-16 was axial; and the decalin ring was trans based on the interatomic non-bonded distance of key atoms less than 4 Å. The trans-fused decalin ring of 4 was supported by the coupling constant of CH-10 signal (tt, J = 11.8, 2.8 Hz). The absolute configuration of 4 was confirmed as (4R,5S,8S,10R,13R)-conformer by the result that the experimental data, showing a negative Cotton effect at 298 nm, and calculated ECD spectrum for (4R,5S,8S,10R,13R)-conformer of 4 matched exactly ( Figure 5).

Table 2.
The key NOE correlations of compound 4 and interatomic non-bonded distance of the key atoms in its main 3D conformers. Peaurantiogriseol E (5, Figure 1) had a molecular formula of C16H24O3 based on HREIMS data (observed m/z 264.1721 M + , calculated 264.1720), which was two mass units less than that of compound 2, requiring 5 degrees of unsaturation. The 1 H-NMR and 13 C-NMR data for 5 were similar to those of compound 2 ( Table 1). The most obvious difference between 5 and 2 was that the absence of one CH group signal at δ 2.06/39.9, and the change of a doublet signal at δ 0.72 to singlet at δ 1.18/20.5 in 5. These results suggested that a pyrone moiety was formed in 5 by an O-C bond at C-13 position, which was supported by the HMBC correlations from H-1 to C-13 (Figure 2), and, from H-2 to C-4.

Main 3D Conformers Me-aaa-Cis Me-eee-Cis Me-eea-Trans Distance (Å) Distance(Å) Distance(Å)
The relative stereochemistry of 5 was established by its NOESY spectrum (Figure 3). A cis-fused decalin ring in 5 was confirmed based on the NOE correlations of H-  were oriented on the same side by the NOE correlations between H-16 and H-10. The absolute configuration of 5 was determined based on the result that the experimental ECD spectrum, showing a negative Cotton effect at 298 nm, and calculated ECD spectrum for (4R,5R,8R,10R,13S)-configuration of compound 5 matched exactly ( Figure 5).
Peaurantiogriseol F (6, Figure 1) Table S1) [8]. The Compound 7 was identified as aspermytin A by comparison of its spectral data with [9]; both compound 7 and aspermytin A had the same NMR, MS and specific rotation data.
Optical rotation measurements were carried out using a Bellingham-Stanley 37-440 polarimeter (Bellingham Stanley Ltd., Kent, UK). UV spectra were determined using a UV-240 spectrophotometer (Shimadzu, Tokyo, Japan). ECD spectra were measured using a Chirascan Circular Dichroism Spectrometer (Applied PhotoPhysics, Surrey, UK). IR spectra were measured on a TENSOR37 spectrometer (Bruker Optics, Ettlingen, Germany). The 1 H-NMR and 13 C-NMR data were acquired using a Bruker Avance 400 spectrometer at 400 MHz for 1 H nuclei and 100 MHz for 13 C nuclei, a Bruker Avance III 500 MHz NMR spectrometer at 470 MHz for 19 F nuclei, and a Bruker Avance III 600 MHz NMR spectrometer at 600 MHz for 1 H nuclei and 150 MHz for 13 C nuclei (Bruker Biospin,Rheinstetten,German). TMS was used as an internal standard, and the chemical shifts (δ) were expressed in ppm or Hz. The EI mass spectra and high-resolution mass spectra were obtained using MAT95XP (ThermoFinnigan, Bremen, Germany) high resolution mass spectrometer and a LTQ-Orbitrap LC-MS (Thermo Fisher, Frankfurt, German). HPLC was performed using a 515 pump with a UV 2487 detector (Waters, Milford, MA, USA) and an Ultimate XB-C-18 column (250 mm × 10 mm, 5 μm; Welch, MD, USA). Normal pressure preparative column chromatography was carried out on RP-18 gel (25-40 μm, Daiso Inc., Osaka, Japan), silica gel (200-400 mesh, Qingdao Marine Chemical Inc., Qingdao, China), or Sephadex-LH-20 (GE Healthcare, Stockholm, Sweden) for reverse and direct phase elution modes, respectively. TLC was performed over F254 glass plates (Qingdao Marine Chemical Inc., Qingdao, China) and analyzed under UV light (254 and 366 nm).

Fungal Material
Endophytic fungus Penicillium aurantiogriseum 328# was isolated with PDA medium from the bark of Hibiscus tiliaceus collected in the Qi'ao Mangrove Nature Reserve of Guangdong Province, China and identified according to its morphological characteristics and the ITS region [11]. A voucher specimen is deposited in our laboratory at −20 °C.

Fermentation, Extraction and Isolation
Small agar slices bearing mycelia were placed in 1000 mL Erlenmeyer flasks containing rice medium (composed of 60 g rice, 80 mL distilled water, and 0.24 g sea salt); and incubated for 30 days at 28 °C. In total, 120 flasks of culture were obtained. Cultures were extracted with EtOAc. In total, 200 g crude extract was obtained by evaporation of EtOAc. The crude extract was suspended in H2O (2 L) and optimization, frequency analysis, and ECD spectrum prediction, were carried out with the density functional theory (DFT) and time-dependent density functional theory (TDDFT) methods in the Gaussian 09 software package [14]. The geometry optimizations were performed at the B3LYP/6-31+G (d) level in the gas phase. Based on the final optimized structure, the ECD spectra were calculated at the PBE1PBE-SCRF/6-311++g (d, p) level using the PCM solvent continuum models with methanol as a solvent. The theoretical predicted ECD spectra were fitted in the SpecDis software package.

Inhibition of Aldose Reductase
The method to examine the inhibition of aldose reductase was similar to the method used by Michael C. Van Zandt et al. [15]. Enzyme activity was measured by monitoring the rate of disappearance of NADPH at 340 nm. The reaction contents in a final volume of 300 μL were 6.6% w/v (NH4)2SO4, 33 mM NaH2PO4 (pH 6.6), 0.11 mM NADPH, 4.7 mM DL-glyceraldehyde, 0.59 μg of enzyme, 1% DMSO, and compound. Each assay was done in triplicate. Percent inhibition was calculated on the basis of enzyme activity in the presence or absence of compound.