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Mar. Drugs 2017, 15(2), 46; https://doi.org/10.3390/md15020046

Article
Zosteropenillines: Polyketides from the Marine-Derived Fungus Penicillium thomii
1
Elyakov Pacific Institute of Bioorganic Chemistry, Far Eastern Branch of Russian Academy of Sciences, 159 Prospect 100‐letiya Vladivostoka, Vladivostok 690022, Russia
2
School of Natural Science, Far Eastern Federal University, 8 Sukhanova St., Vladivostok 690090, Russia
3
Institute of Chemistry, Far Eastern Branch of Russian Academy of Sciences, 159 Prospect 100‐letiya Vladivostoka, Vladivostok 690022, Russia
4
Laboratory of Experimental Oncology, Department of Oncology, Hematology and Bone Marrow Transplantation with Section Pneumology, Hubertus Wald‐Tumorzentrum, University Medical Center, 20246 Hamburg, Germany
*
Authors to whom correspondence should be addressed.
Academic Editor: Russell Kerr
Received: 28 December 2016 / Accepted: 13 February 2017 / Published: 17 February 2017

Abstract

:
Twelve new polyketides, zosteropenillines A–L (112), together with known polyketide pallidopenilline A (13), were isolated from the ethylacetate extract of the fungus Penicillium thomii associated with the seagrass Zostera marina. Their structures were established based on spectroscopic methods. The absolute configuration of zosteropenilline A (1) as 4R, 5S, 8S, 9R, 10R, and 13S was determined by a combination of the modified Mosher’s method, X-ray analysis, and NOESY data. Absolute configurations of zosteropenillines B–D (24) were determined by time-dependent density functional theory (TD-DFT) calculations of ECD spectra. The effect of compounds 13, 7, 8, 10, and 11 on the viability of human drug-resistant prostate cancer cells PC3 as well as on autophagy in these cancer cells and inhibitory effects of compounds 1, 2, and 810 on NO production in LPS-induced RAW 264.7 murine macrophages were examined.
Keywords:
marine fungi; Penicillium thomii; polyketide decalin derivative; X-ray; ECD spectra

1. Introduction

Marine-derived fungi are a prolific source of new secondary metabolites, many of which are biologically active [1,2,3]. In previously published works, we described the isolation and identification of 10 new meroterpenoids–austalides and seven new 6,6-spiroketals–sargassopenillines A–G from fungi Penicillium thomii and P. lividum associated with the brown alga Sargassum miyabei [4,5] and four new eudesmane-type sesquiterpenes–thomimarines A–D from P. thomii KMM 4667 associated with the sea grass Zostera marina [6]. Recently, we have isolated 11 new polyketides, pallidopenillines that have a polyketidic decalin scaffold from P. thomii associated with the brown alga Sargassum pallidum [7]. During our ongoing search for new natural compounds from marine-derived fungi, we investigated the strain P. thomii KMM 4674 isolated from the seagrass Z. marina. Herein, we report the isolation, structure elucidation and biological assay results of 12 new polyketides containing “decalin” motif produced by this fungus (Figure 1).

2. Results and Discussion

2.1. Structure Elucidation

The molecular formula of 1 was established to be C15H22O3 from a HRESIMS peak at m/z 273.1461 [M + Na]+ and by 13C NMR analyses. A close inspection of 1H NMR, 13C NMR, DEPT, and HSQC data of 1 (Table 1 and Table 2) revealed the presence of two methyl (δH 1.26, δC 23.2 and δH 1.03, δC 18.4) groups, four methylenes (δC 39.9, 32.7, 28.7), including one oxygen-bearing (δH 4.11, 3.89, δC 60.9), and seven methines (δH 1.89, δC 37.5, δH 1.41, δC 40.2, δH 1.65, δC 48.6, δH 2.09, δC 62.8, δH 6.24, δC 130.1, δH 5.69, δC 131.6), together with one oxygen-bearing (δH 2.92, δC 77.9), one carbonyl carbon (δC 210.2), and one oxygenated quaternary carbon (δC 74.7). These data and five unsaturated degrees from the molecular formula suggested that compound 1 possessed three rings. COSY-45 data and HMBC correlations (Figure 2) from H-4 (δH 2.09) to C-5 (δC 37.5), C-10 (δC 48.6), C-14 (δC 23.2), from H-5 (δH 1.89) to C-4 (δC 62.8), C-6 (δC 28.7), C-10, C-13 (δC 74.7), from H2-6 (δH 1.28, 1.14) to C-5, C-7 (δC 32.7), C-8 (δC 40.2) and C-10, from H-7a (δH 1.78) to C-5, C-6, C-8 and C-15 (δC 18.4), from H-7b (δH 1.08) to C-9 (δC 77.9), from H-8 (δH 1.41) to C-7, C-9 and C-10, from H3-15 (δH 1.03) to C-7, C-8 and C-9, from H-9 (δH 2.92) to C-10, C-11 (δC 130.1), C-15, from H-10 (δH 1.65) to C-11, C-12 (δC 131.6), from H-11 (δH 6.24) to C-13, from H-12 (δH 5.69) to C-4, C-13, and C-14 revealed the presence of a decalin moiety and established a Δ11 double bond and the location of methyl groups at C-8 and C-13 in 1.
HMBC correlations from H-1a (δH 4.11) to C-2 (δC 39.9), C-3 (δC 210.2) and C-13, from H-2a (δH 2.64) to C-1 (δC 60.9), C-3, from H-2b (δH 2.21) to C-3, C-4 established a saturated γ-pyrone derivative moiety in 1. NOESY cross-peaks H3-14 (δH 1.26)/H-4 (δH 2.09), H-1β (δH 3.89); H3-15 (δH 1.03)/H-9 (δH 2.92); H-5/H-2β (δH 2.64), H-6β (δH 1.28), H-7β (δH 1.08), H-9; H-10 (δH 1.65)/H-6α (δH 1.14), H-8 (δH 1.41), H-4; H-9/H3-15, H-5 and H-4/H-6α, H-10, H3-14 indicated a trans-ring fusion of the A and B rings, cis-ring fusion of the B and C rings, the α-orientation of H3-14 and 9-OH, and the β-orientation of H3-15. The planar structure and relative configuration of 1 were unequivocally confirmed by X-ray analysis, which was carried out for a single crystal obtained by recrystallization from acetonitrile–water (Figure 2 and Figure S9).
Esterification of 1 with (R)- and (S)-MTPA chloride occurred at the C-9 hydroxy group to give the (S)- and (R)-MTPA esters 1a and 1b, respectively. The observed chemical shift differences Δδ (δS − δR) (Figure 3) indicated the 9R configuration. These data and observed NOESY correlations determined the absolute stereostructure of 1 as 4R, 5S, 8S, 9R, 10R, and 13S. Compound 1 was named zosteropenilline A.
The molecular formula of 2 was established to be C15H22O3 from a HRESIMS peak at m/z 273.1474 [M + Na]+ and by 13C NMR analyses. The general features of the 13C NMR of 2 resembled those of 1 with the exception of the C-9–C-11 and C-15 carbon signals. COSY and HSQC spectra of 2 revealed the partially connectivity sequence of the protons in the A ring as CH2(15)-CH(8)-CH2(9)-CH(10). These data and HMBC correlations from H-9b (δH 0.93) to C-7 (δC 29.1), C-8 (δC 40.5), C-10 (δC 41.4), C-11 (δC 134.4), and C-15 (δC 68.1); and from H2-15 (δH 3.50, 3.47) to C-7, C-8 and C-9, established the A ring structure, lacking the hydroxy group at C-9, and indicated the presence of a hydroxymethyl group at C-8 in 2. The NOESY cross-peaks H-4 (δH 2.07)/H-10 (δH 1.74), H3-14 (δH 1.26); H-8 (δH 1.63)/H-9α (δH 1.91), H-10; H-5 (δH 1.78)/H-9βH 0.93); H-9β/H2-15 (δH 3.50, 3.47) indicated the trans-ring fusion of the A and B ring, cis-ring fusion of the B and C ring, and the β-orientation of hydroxymethyl group at C-8. Compound 2 was named zosteropenilline B.
The molecular formula of 3 was established to be C15H22O3 from a HRESIMS peak at m/z 273.1463 [M + Na]+ and by 13C NMR analyses. The 1H and 13C NMR spectra (Table 1 and Table 2) for this compound were very similar to those obtained for zosteropenilline B (2) with the exception of the C-6–C-10 and C-15 carbon and proton signals. The HMBC correlations H-9a (δH 1.75)/C-8 (δC 69.7), C-10 (δC 36.9); H3-15 (δH 1.25)/C-7 (δC 24.9), C-8, C-9 (δC 44.5) established the structure of 3 including the hydroxy and methyl groups at the C-8 position. The relative configuration of 3 was defined based on the observed NOESY correlations (Figure S27). Compound 3 was named zosteropenilline C.
The molecular formula of 4 was established to be C15H22O2 from a HRESIMS peak at m/z 257.1510 [M + Na]+ and by 13C NMR analyses. The 1H and 13C NMR data (Table 1 and Table 2) observed for 4 closely resembled those obtained for zosteropenilline B (2) with the exception of the C-7–C-9 and C-15 carbon and proton signals. The mutual correlations from H3-15 to C-7 and C-9 established the structure of the A ring and location methyl group at C-8. Compound 4 was named zosteropenilline D.
The absolute configurations of zosteropenillines B–D (24) were determined on the basis of the ECD spectroscopy data. The geometry of the stable conformations of compounds 14 were optimized using general procedure, described in the experimental section of the paper. The internal rotations of the hydroxyl groups as well as the inversions of the six-membered rings A and C were accounted for during fulfilled conformational analysis. We found that for all these compounds ring C can exist in two stable conformations: “chair” and “boat”. The “chair” conformation is more stable for about ΔE# (chair → boat) ≈ 2 kcal·mol−1 and the barrier for interconversion between conformations is ΔE# (chair → boat) ≈ 6 kcal·mol−1. Analogously, ring A can also exist in “chair” and “boat” conformations. The “chair” conformation is more stable for about ΔE# (chair → boat) ≈ 10 kcal·mol−1 and the barrier for interconversion between conformations is ΔE# (chair → boat) ≈ 14 kcal·mol−1 (Figure 4 and Figure 5).
To obtain theoretical ECD spectra the excitation energies and the rotatory strengths for 14 were calculated using time-dependent density functional theory (TDDFT) with PBE1PBE exchange-correlation functional and cc-pvTz basis set [8]. A comparison of statistically averaged ECD spectra for 14 with corresponding experimental spectra is presented in Figure 6. The comparison of all theoretical spectra with the experimental ones showed that all spectra are qualitatively similar in the region λ ≥ 200 nm, where the pronounced Cotton effects occur, thus proving 4R, 5S, 8S, 9R, 10R, 13S absolute configuration for 1; 4R, 5S, 8S, 10R, 13S for 2; 4R, 5S, 8R, 10R, 13S for 3; 4R, 5S, 8S, 10R, 13S for 4.
The molecular formula of 5 was established to be C15H24O3 from a HRESIMS peak at m/z 283.1538 [M − H] and by 13C NMR analyses. The COSY-45 data and HMBC correlations from H-4 (δH 2.11) to C-5 (δC 38.5), C-6 (δC 29.0), C-10 (δC 53.5), from H-5 (δH 1.95) to C-6, C-10, from H-6a (δH 1.33) to C-4 (δC 63.7), C-7 (δC 31.8) and C-10, from H-7a (δH 1.73) to C-5, C-9 (δC 77.2), from H3-15 (δH 1.06) to C-7, C-8 (δC 39.2), C-9, from H-9 (δH 3.29) to C-5, C-11 (δC 67.3), from H-10 (δH 1.61) to C-5, C-9, C-11, from H-11 (δH 4.38) to C-12 (δC 72.7), and from H3-14 (δH 1.36) to C-4, C-12, C-13 (δC 77.5) revealed the presence of a decalin moiety in 1 and established the location of methyl groups at C-8 and C-13 and hydroxy groups at C-9, C-11 and C-12. HMBC correlations from H-1a (δH 4.22) to C-2 (δC 37.9), C-3 (δC 206.0) and C-13, from H-2a (δH 2.67) to C-1 (δC 60.9), C-3, from H-4 (δH 2.11) to C-2 and C-3 established a saturated γ-pyrone derivative in 5 (Figure S40). The relative configuration of 5 was assigned based on NOESY cross-peaks (Figure 7) and 1H-1H coupling constants (Table 2). Observed NOESY correlations and magnitudes of the vicinal coupling constants between H-4 and H-5; H-5 and H-10; H-9 and H-10; H-10 and H-11; H-11 and H-12 indicated a trans-ring fusion of the A and B rings, cis-ring fusion of the B and C rings, the α-orientation of H3-14, 9-OH, 11-OH groups and the β-orientation of H3-15 and 12-OH groups. Compound 5 was named zosteropenilline E.
The molecular formula of 6 was established to be C15H24O4 from a HRESIMS peak at m/z 291.1579 [M + Na]+ and by 13C NMR analyses. The 13C NMR data of 6 matched those for 1 with the exception of the C-1–C-3 and C-13–C-14 carbon signals. The structure of the decalin moiety and location of the methyl groups at C-8 and C-13 and oxygen functions at C-9 and C-13 were established as for zosteropenilline A (1) based on COSY and HMBC correlations. The COSY-45 data and HMBC correlations from H2-1 (δH 3.91) to C-2 (δC 48.6) and C-3 (δC 217.6), from H2-2 (δH 2.86, 2.76) to C-1 (δC 57.6) and C-3, and from H-4 (δH 2.59) to C-3 indicated the presence of a 3-hydroxy-1-oxopropyl residue (C-1–C-3 residue numbering) at C-4. The relative configuration of 6 was assigned on the basis of a NOESY experiment and 1H-1H coupling constants (Table 2). Compound 6 was named zosteropenilline F. Zosteropenilline F is an epimer of the pallidopenilline A (13) which was earlier isolated from the P. thomii associated with the brown alga Sargassum pallidum [7]. The 1H and 13C NMR data observed for 6 closely resembled those obtained for pallidopenilline A (measured in CDCl3) (Figures S58–S68) with the exception of the C-4, C-13 and C-14 carbon and proton signals The molecular formula of 7 was established to be C15H24O3 from a HRESIMS peak at m/z 275.1621 [M + Na]+ and by 13C NMR analyses. The 13C NMR data (Table 3) observed for 7 closely resembled those obtained for zosteropenilline D (4) with the exception of the C-1–C-3 and C-13–C-14 carbon signals. The structure of the decalin moiety, location of the methyl groups at C-8 and C-13, oxygen functions at C-13 and 3-hydroxy-1-oxopropyl residue at C-4 were established by HMBC and COSY correlations (Figures S74 and S75). The relative configuration of 7 was assigned based on NOESY correlations H-4 (δH 2.89)/H-10 (δH 1.84); H-10/H-8 (δH 1.46) and H-5 (δH 1.48)/H3-14 (δH 1.20) and H3-15 (δH 0.89) recorded in CDCl3 and 13-OH (δH 4.95)/H-4 correlation in spectrum of 7 recorded in DMSO-d6 (Figure S77). Compound 7 was named zosteropenilline G.
The molecular formula of 8 was established to be C15H24O4 from a HRESIMS peak at m/z 291.1571 [M + Na]+ and by 13C NMR analyses. The 1H NMR, 13C NMR, DEPT and HSQC spectra of 8 (Table 3 and Table 4) displayed obvious signals for two methyl (δH 1.60, δC 20.9 and δH 1.02, δC 17.8) groups, four methylenes (δC 43.6, 30.5, 31.9) including one oxygen-bearing (δH 3.85, 2H, δC 57.9), seven methines (δH 1.69, δC 37.9, δH 1.47, δC 39.2, δH 1.28, δC 49.6, δH 2.92, δC 61.7, δH 3.25, δC 82.3, δH 4.32, δC 73.2, δH 5.53, δC 129.0,) including two oxygen-bearing, one carbonyl carbon (δC 214.0) and one sp3 quaternary carbon (δC 131.5). The structure of a 1,2,3,4-tetrasubstituted cyclohexane ring and side chain in 8 were found by extensive NMR spectroscopy (1H and 13C NMR, COSY, HSQC and HMBC) as for zosteropenillines E (5) and F (6). HMBC correlations from H-4 (δH 2.92) to C-5 (δC 37.9), C-12 (δC 129.0), C-13 (δC 131.5), from H-10 (δH 1.28) to C-5, C-11 (δC 73.2), C-12, from H-11 (δH 4.32) to C-12, C-13, and from H3-14 (δH 1.60) to C-4 (δC 61.7), C-12, C-13 established a B ring structure, Δ12 double bond and the location of the hydroxy group at C-11. The relative configuration of 8 was assigned based on NOESY cross-peaks H-4/H-10; H-5 (δH 1.69)/H-9 (δH 3.25), H-11, H3-15 (δH 1.02); H-8 (δH 1.47)/H-10; H-9/H-11 and 1H-1H coupling constants (Table 4). Compound 8 was named zosteropenilline H.
The molecular formula of 9 was established to be C15H22O3 from a HRESIMS peak at m/z 273.1462 [M + Na]+ and by 13C NMR analyses. Structures of an A ring and side chain in 9 were established as for zosteropenillines E (5) and F (6). Observed long-range COSY correlations H-4 (δH 3.15)/H2-14 (δH 4.99, 4.59), H-12 (δH 6.20)/H-14b. and HMBC correlations from H-4 (δH 3.15) to C-13 (δC 140.8), C-14 (δC 112.8), from H-9 (δH 2.89) and H-10 (δH 1.85) to C-11 (δC 130.6) and from H2-14 (δH 4.99, 4.59) to C-4 (δC 59.6), C-12 (δC 129.3), C-13 indicated the presence of a diene system at the C-11 and C-13(14) in 9. The relative configuration of 9 was assigned based on the NOESY correlations H-4/H-10; H-8 (δH 1.43)/H-10 and H-5 (δH 1.68)/H-9, H3-15 (δH 1.05). Compound 9 was named zosteropenilline I.
The molecular formula of 10 was established to be C15H24O4 from a HRESIMS peak at m/z 291.1573 [M + Na]+ and by 13C NMR analyses. The 13C NMR data (Table 3) observed for 10 matched the data reported for zosteropenilline G (7) with the exception of the C-7–C-9 and C-15 carbon signals. These data, together with the molecular mass difference of 16 mass units between 7 and 10, indicated the presence of the hydroxymethyl group at C-8 in 10 instead of a methyl group. NOESY cross-peaks H-4 (δH 2.92)/H-6b (δH 0.95), H-10 (δH 1.87); H-5 (δH 1.52)/H-7b (δH 1.02), H-9b (δH 0.82), H3-14 (δH 1.20); H-7b/H-9b, H2-15 (δH 3.47, 3.45); H-8 (δH 1.61)/H-6b, H-10 indicated the trans-ring fusion of the A and B rings, a β-orientation of the side chain, H3-14 and hydroxymethyl group at C-8. Compound 10 was named zosteropenilline J.
The molecular formula of 11 was established to be C15H22O5 from a HRESIMS peak at m/z 305.1370 [M + Na]+ and by 13C NMR analyses. The UV spectrum exhibits a λmax at 242 nm (log ε 3.46), consistent with the enone system in 11. The COSY and HSQC spectra of 11 revealed the partial connectivity sequences of the protons in the A ring as CH(5)-CH2(6)-CH2(7)-CH(8) and CH2(15)-CH(8)-CH2(9). These data and HMBC correlations from H-9a (δH 2.60) to C-7 (δC 27.9), C-8 (δC 41.1) and C-15 (δC 67.4), from H-9b (δH 2.01) to C-10 (δC 166.3), from H2-15 (δH 3.58, 3.54) to C-7, C-8 and C-9 (δC 38.3) established the structure of A ring. Long range correlations from H-4 (δH 3.01) to C-5 (δC 39.4), C-10, C-12 (δC 200.3) and C-13 (δC 74.2), from H-11 (δH 5.90) to C-5 and C-13 and from H3-14 (δH 1.19) to C-4 (δC 61.6), C-12, C-13 determined the structure of B ring and indicated the 10-en-12-one position for the enone chromophore in 11. The structure of side chain in 11 was found by NMR spectroscopy as for zosteropenilline F (6). Observed NOESY correlations H-5 (δH 2.89)/H-9b (δH 2.01), H3-14; H2-15/H-9b indicated these protons to be on the same side of the molecule. These data and the magnitude of the coupling constant of the H-4 signal (δH 3.01, J = 9.8 Hz) indicated H-4 and 13-OH to be on the other side of molecule. Compound 11 was named zosteropenilline K.
The molecular formula of 12 was established to be C15H24O5 from a HRESIMS peak at m/z 307.1501 [M + Na]+ and by 13C NMR analyses. The structure of the A ring in 12 was established based on COSY and HMBC correlations as for zosteropenilline K (11). HMBC correlations from H-11 (δH 5.60) to C-5 (δC 40.3), C-9 (δC 37.2), C-12 (δC 68.0), C-13 (δC 72.4), from H-12 (δH 4.36) to C-4 (δC 57.7), C-10 (δC 144.5), C-11 (δC 118.8), C-13, and from H3-14 (δH 1.20) to C-4, C-12, C-13 established the structure of B ring, location oxygenated functions at C-12, C-13 and methyl group at C-13 in 12. The relative configuration of 12 was defined based on NOESY correlations H-5/H3-14; H3-14/H-12; H-4/H-6b; H-6b/H-8 and the magnitude of the coupling constant of the H-4 signal (δH 3.09, J = 10.0 Hz). Compound 12 was named zosteropenilline L. Besides the new zosteropenillines (112), a known pallidopenilline A (13) was also isolated from this fungus. For the first time, pallidopenilline A was found in fungus P. thomii KMM 4675 [7].

2.2. Bioassay Results

Compounds 2, 8, and 10 induced a moderate downregulation of NO production in LPS-stimulated macrophages at non-cytotoxic concentration of 10.0 µM. Respectively, NO levels in these cells were decreased by 27.7 ± 1.8, 20.6 ± 1.2, and 22.3 ± 3.8 percent, in comparison with LPS-pretreated control cells. The most effective substance was compound 2, which exhibited the maximal pronounced inhibition of NO formation in lipopolysaccharide-stimulated RAW 264.7 cells (Figure 8).
We have investigated the effect of the isolated zosteropenillines 13, 7, 8, 10, and 11 on the viability of human drug-resistant prostate cancer cells PC3 as well as on autophagy in these cancer cells. Induction of autophagy is a well-characterized phenomenon in prostate cancer, frequently associated with cancer cell survival and resistance to therapy [9,10]. Therefore, novel compounds that effectively inhibit prosurvival autophagy are of high interest [10,11]. p62 (or SQSTM) is a cargo protein that targets and binds other proteins to deliver them to autophagosomes for selective autophagy. During autophagy autophagosomes fuse with lysosomes and intra-autophagosomal components are degraded by lysosomal hydrolases. Therefore, an increased level of p62 has previously been associated with inhibition of autophagy flux. In our experiments MTT analysis revealed all compounds to be non-cytotoxic up to 100 µM (data not shown). Consequently, we investigated the effect of the zosteropenillines on the expression of autophagy-related cargo protein p62. Western blotting analysis revealed a slight increase in p62 levels in PC3 cells treated with all the investigated substances (Figure 9), suggesting the drug-induced inhibition of autophagy. The effect on p62 expression was similar to the effect of Bafilomycin A1 (Baf)—a well-established autophagy inhibitor. These results suggest that the investigated compounds are able to inhibit autophagy at non-cytotoxic concentrations and may sensitize human cancer cells to cytotoxic anticancer drugs.

3. Materials and Methods

3.1. General Experimental Procedures

Optical rotations were measured on a Perkin-Elmer 343 polarimeter (Perkin Elmer, Waltham, MA, USA) in MeOH. UV spectra were recorded on a Specord UV VIS spectrometer (Carl Zeiss, Jena, Germany) in MeOH and in n-hexane. IR spectra were determined on a Specord M 82 (Carl Zeiss) in CHCl3. ECD spectra were measured with a Chirascan-Plus CD Spectrometer (Leatherhead, UK) in MeOH in n-hexane. X-ray crystallographic analysis was carried out on a Bruker Kappa APEX2 diffractometer with graphic-monochromated Mo Kα radiation. 1H and 13C NMR spectra were recorded in CDCl3 and in DMSO-d6 on a Bruker Avance-500 (Bruker BioSpin GmbH, Rheinstetten, Germany) and Avance III-700 (Bruker BioSpin GmbH, Rheinstetten, Germany) spectrometers operating at 500.13 MHz and 125.77 MHz and 700.13 and 176.04 MHz, respectively, using TMS as an internal standard. HRESIMS spectra were obtained on an Agilent 6510 Q-TOF LC mass spectrometer (Agilent Technologies, Santa Clara, CA, USA). Low-pressure liquid column chromatography was performed using Si gel L (50/100 μm, Sorbpolimer, Imid, Krasnodar, Russia). Plates (4.5 × 6.0 cm) pre-coated with Si gel (5–17 μm, Sorbfil, Imid, Krasnodar, Russia) were used for thin layer chromatography. Preparative HPLC was carried out on a Shimadzu LC-20 (Shimadzu, USA Manufacturing, Canby, OR, USA) and Agilent 1100 (Agilent Technologies, Ratingen, Germany) chromatographs using an YMC ODS-AM (YMC Co, Ishikawa, Japan) (5 μm, 10 × 250 mm), YMC Si (YMC Co, Ishikawa, Japan) (5 μm, 10 × 250 mm), Supelco Discovery C-18 (Supelco Analytical, Bellefonte, PA, USA) (5 μm, 4.6 × 250 mm), Ultrasphere Si (Beckman Coulter Inc., Brea CA, USA) (5 μm, 4.6 × 250 mm) and Kromasil 3-CelluCoat (Kromasil, Bohus, Sweden) (4.6 × 150 mm) columns and Shimadzu RID-20A (Shimadzu Corporation, Kyoto, Japan) and Agilent 1100 (Agilent Technologies, Ratingen, Germany) refractometers.

3.2. Fungal Strain

The strain of the fungus Penicillium thomii KMM 4674 was isolated from superficial mycobiota of the rhizome sea-grass Zostera marina (Sea of Japan) and was identified on the basis of morphological evaluation.

3.3. Cultivation of P. thomii

The fungus was grown stationary at 22 °C for 21 days in 20 Erlenmeyer flasks (500 mL), each containing 20 g of rice, 20 mg of yeast extract, 10 mg of KH2PO4, and 40 mL of natural sea water (Marine Experimental Station of G.B. Elyakov Pacific Institute of Bioorganic Chemistry, Troitsa (Trinity) Bay, Sea of Japan) by Dr. Natalya Kirichuk.

3.4. Extraction and Isolation

At the end of the incubation period, the mycelia and medium were homogenized and extracted with EtOAc (1 L). The obtained extract was concentrated to dryness. The residue (2.46 g) was dissolved in H2O–EtOH (4:1) (100 mL) and was extracted with n-hexane (0.2 L × 3) and EtOAc (0.2 L × 3). After evaporation of the EtOAc layer, the residual material (2.26 g) was passed over a silica column (3 × 14 cm), which was eluted first with n-hexane (200 mL) followed by a step gradient from 5% to 50% EtOAc in n-hexane (total volume: 5 L). Fractions of 250 mL were collected and combined on the basis of TLC (Si gel, toluene–isopropanol 6:1 and 3:1, v/v).
The n-hexane–EtOAc (75:25) eluate (146 mg) was purified on an YMC ODS-AM column eluting with MeOH–H2O (80:20) to yield fractions A (15.4 mg), B (10 mg), C (2.3 mg), D (3.3 mg), and individual compound 7 (8.4 mg). Fraction A was purified on a Supelco C-18 eluting with CH3CN–H2O (25:75) to yield 3 (2.3 mg). Fraction B was purified on a Kromasil 3-CelluCoat column eluting with CH3CN–H2O (25:75) to yield 4 (3.0 mg). Fraction C was purified on an Ultrasphere Si column eluting with EtOAc–n-hexane–EtOH (50:50:5) to yield 9 (1.8 mg). Fraction D was purified on an Ultrasphere Si eluting with EtOAc–n-hexane–EtOH (100:100:5) to yield 5 (0.5 mg). The n-hexane–EtOAc (65:35) eluate (283 mg) was purified on a YMC ODS-AM column eluting with MeOH–H2O (65:35) to yield 1 (11 mg), 6 (3.0 mg), 8 (6.1 mg), 10 (34.6 mg), 11 (4.6 mg) and 13 (100 mg). The n-hexane–EtOAc (60:40) eluate (200 mg) was purified on a YMC ODS-AM column eluting with MeOH–H2O (65:35) to yield 2 (83 mg) and on a Supelco C-18 eluting with CH3CN–H2O (80:20) to yield 12 (1.6 mg).

3.5. Spectral Data

Zosteropenilline A (1): colorless crystals; [ α ] D 20 −125.0 (c 0.1, MeOH); UV (n-hexane) low intensity band; CD (c 0.05, n-hexane), λmax (∆ε) 202 (−6.31), 300 (−0.77), 310 (−0.73), 321 (−0.40) nm; IR (CHCl3) νmax 3462, 2958, 2928, 1703, 1648, 1601, 1444, 1397, 1120, 1034 cm−1; 1H and 13C NMR data, see Table 1 and Table 2, Supplementary Figures S1–S8; HRESIMS [M + Na]+ m/z 273.1461 (calcd. for C15H22O3Na 273.1461), [M + H]+ m/z 251.1636 (calcd. for C15H23O3 251.1642).
Zosteropenilline B (2): colorless crystals; [ α ] D 20 −161.6 (c 0.12, MeOH); UV (MeOH low intensity band; CD (c 0.07, MeOH), λmax (∆ε) 201 (−1.55), 291 (−0.23), 332 (+0.03), 365 (−0.03) nm; IR (CHCl3) νmax 3460, 2957, 2919, 1701, 1602, 1455, 1385, 1108, 1055 cm−1; 1H and 13C NMR data, see Table 1 and Table 2, Supplementary Figures S11–S19; HRESIMS [M + Na]+ m/z 273.1474 (calcd. for C15H22O3Na 273.1461), [M − H] m/z 249.1496 (calcd. for C15H21O3 249.1496).
Zosteropenilline C (3): white solid; [ α ] D 20 −170.0 (c 0.1, MeOH); UV (MeOH) low intensity band; CD (c 0.07, MeOH), λmax (∆ε) 204 (−9.86), 297 (−1.63) nm; IR (CHCl3) νmax 3604, 2973, 2927, 1705, 1602, 1453, 1375, 1140, 1044 cm−1; 1H and 13C NMR data, see Table 1 and Table 2, Supplementary Figures S20–S27; HRESIMS [M + Na]+ m/z 273.1463 (calcd. for C15H22O3Na 273.1461), [M − H] m/z 249.1499 (calcd. for C15H21O3 249.1496).
Zosteropenilline D (4): amorphous; [ α ] D 20 −56.0 (c 0.06, MeOH); UV (MeOH) low intensity band; CD (c 0.06, MeOH), λmax (∆ε) 204 (−4.01), 296 (−0.96) nm; IR (CHCl3) νmax 2956, 2929, 1706, 1602, 1456, 1376, 1121, 1069 cm−1; 1H and 13C NMR data, see Table 1 and Table 2, Supplementary Figures S28–S35; HRESIMS [M + Na]+ m/z 257.1510 (calcd. for C15H22O2Na 257.1512).
Zosteropenilline E (5): amorphous; [ α ] D 20 −16.0 (c 0.05, MeOH); UV (MeOH) low intensity band; CD (c 0.05, MeOH), λmax (∆ε) 206 (−0.27), 293 (−0.21), 299 (−0.22), 305 (−0.19), 339 (−0.03); 1H and 13C NMR data, see Table 1 and Table 2, Supplementary Figures S36–S42; HRESIMS [M − H] m/z 283.1538 (calcd. for C15H23O5 283.1551).
Zosteropenilline F (6): white solid; [ α ] D 20 +9.1 (c 0.11, MeOH); UV (MeOH) low intensity band; CD (c 0.07, MeOH), λmax (∆ε) 194 (+0.70), 204 (−1.14), 230 (−0.12), 293 (+0.08), 319 (−0.02) nm; IR (CHCl3) νmax 3621, 2976, 2928, 1702, 1602, 1450, 1390, 1046 cm−1; 1H and 13C NMR data, see Table 1 and Table 2, Supplementary Figures S43–S50; HRESIMS [M + Na]+ m/z 291.1579 (calcd. for C15H24O4Na 291.1567), [2M + Na]+ m/z 559.3242 (calcd. for 2C15H24O4Na 559.3241).
Zosteropenilline G (7): white solid; [ α ] D 20 −15.3 (c 0.034, MeOH); UV (MeOH) λmax (log ε) low intensity band; CD (c 0.05, MeOH), λmax (∆ε) 199 (−1.85), 218 (−1.07), 244 (+0.11), 287 (+0.57) nm; IR (CHCl3) νmax 3623, 3459, 2884, 1704, 1670, 1602, 1455, 1391, 1056 cm−1; 1H and 13C NMR data, see Table 3 and Table 4, Supplementary Figures S69–S77; HRESIMS [M + Na]+ m/z 275.1621 (calcd. for C15H24O3Na 275.1618), [M − H] m/z 251.1646 (calcd. for C15H23O3 251.1653).
Zosteropenilline H (8): amorphous; [ α ] D 20 +83.4 (c 0.11, MeOH); UV (MeOH) low intensity band; CD (c 0.06, MeOH), λmax (∆ε) 209 (+3.62), 295 (+1.98) nm; IR (CHCl3) νmax 3617, 3489, 2959, 2929, 1703, 1602, 1456, 1383, 1055 cm−1; 1H and 13C NMR data, see Table 3 and Table 4, Supplementary Figures S78–S85; HRESIMS [M + Na]+ m/z 291.1571 (calcd. for C15H24O4Na 291.1567), [2M + Na]+ m/z 559.3236 (calcd. for 2C15H24O4Na 559.3241).
Zosteropenilline I (9): white solid; [ α ] D 20 +80.0 (c 0.07, MeOH); UV (MeOH) low intensity band; CD (c 0.04, MeOH), λmax (∆ε) 204 (−6.61), 247 (−1.13) nm; 1H and 13C NMR data, see Table 3 and Table 4, Supplementary Figures S86–S93; HRESIMS [M + Na]+ m/z 273.1462 (calcd. for C15H22O3Na 273.1461), [2M + Na]+ m/z 523.3029 (calcd. for 2C15H22O3Na 523.3030).
Zosteropenilline J (10): colorless crystals; [ α ] D 20 −52.9 (c 0.71, MeOH); UV (MeOH) low intensity band; CD (c 0.06, MeOH), λmax (∆ε) 196 (−3.81), 213 (+0.35), 238 (+0.05), 295 (−2.15) nm; IR (CHCl3) νmax 3605, 3497, 2959, 2929, 1702, 1602, 1456, 1383, 1061 cm−1; 1H and 13C NMR data, see Table 3 and Table 4, Supplementary Figures S94–S101; HRESIMS [M + Na]+ m/z 291.1573 (calcd. for C15H24O4Na 291.1567), [2M + Na]+ m/z 559.3236 (calcd. for 2C15H24O4Na 559.3241).
Zosteropenilline K (11): amorphous; [ α ] D 20 +11.1 (c 0.09, MeOH); UV (MeOH) λmax (log ε) 242 (3.46) nm; CD (c 0.06, MeOH), λmax (∆ε) 193 (0.75), 219 (−4.62), 244 (+0.65), 258 (−0.13), 279 (0.46), 294 (0.41), 318 (0.35) nm; IR (CHCl3) νmax 3617, 3464, 2958, 2927, 1702, 1671, 1653, 1604, 1456, 1390, 1059 cm−1; 1H and 13C NMR data, see Table 3 and Table 4, Supplementary Figures S102–S109; HRESIMS [M + Na]+ m/z 305.1370 (calcd. for C15H22O5Na 305.1359).
Zosteropenilline L (12): amorphous; [ α ] D 20 −54 (c 0.05, MeOH); UV (MeOH) low intensity band; CD (c 0.05, MeOH), λmax (∆ε) 196 (−1.29), 210 (+0.14), 298 (−1.12), 349 (+0.04) nm; 1H and 13C NMR data, see Table 3 and Table 4, Supplementary Figures S110–S117; HRESIMS [M + Na]+ m/z 307.1501 (calcd. for C15H24O5Na 307.1516).
Pallidopenilline A (13): colorless crystals; [ α ] D 20 −21. (c 0.1, MeOH); UV (MeOH) λmax (log ε) low intensity band; CD (c 0.1, MeOH), λmax (∆ε) 196 (−9.89), 213 (+1.14), 294 (−1.78) nm; 1H and 13C NMR data, Supplementary Figures S51–S65; HRESIMS [M + Na]+ m/z 291.1575 (calcd. for C15H24O5Na 291.1567).

3.6. Preparation of (S)-MTPA and (R)-MTPA Esters of 1

To a solution of 1 (1.75 mg) in pyridine were added 4-dimethylaminopyridine (a few crystals) and (R)-MTPA-Cl (1.2 μL) at room temperature, and the mixture was stirred for 1.5 h. After evaporation of the solvent, the residue was purified by HPLC on an YMC Si (5 μm, 10 × 250 mm) column eluted with n-hexane–acetone (85:15) to afford the (S)-MTPA ester of 1 (1a). The (R)-MTPA ester of 1 (1b) was prepared similarly manner using (S)-MTPA-Cl. NMR data of (R, S)-MTPA esters of 1 (Figure S10). HRESIMS (1a) m/z 489.1864 [M + Na]+ (calcd. for C25H29F3O5Na, 489.1859), m/z 955.3830 [2M + Na]+ (calcd for C50H58F6O10Na, 955.3826); HRESIMS (1b) m/z 489.1858 [M + Na]+ (calcd. for C25H29F3O5Na, 489.1859), m/z 955.3815 [2M + Na]+ (calcd for C50H58F6O10Na, 955.3826).

3.7. X-ray Crystal Data for 1

Experimental intensity data for C15H22O3 were collected at T = 296(2) K (α-C15H22O3) on a BRUKER Kappa APEX2 diffractometer with graphite monochromated Mo Kα radiation (λ = 0.71073 Å). Intensity data were corrected for absorption using the multi-scan method. The structures were solved using direct methods and refined by least-squares calculation in anisotropic approximation for non-hydrogen atoms. Hydrogen atoms were placed at idealized positions and refined using a riding model. Data collection, reduction, and refinement of the lattice parameters were performed using the Apex II software package [12]. All calculations were performed with SHELXL/PC program [13]. Selected bond lengths and hydrogen bonds are listed in the Supplementary Materials, Figure S8. Supplementary crystallographic data (accession numbers CCDC 1522593) can be obtained free of charge from the Cambridge Crystallographic Data Center via http://www.ccdc.cam.ac. uk/data_request/cif (or from the Cambridge Crystallographic Data Centre, 12 Union Road, Cambridge, UK; fax: + 44 1223 336 033 or email: [email protected]).

3.8. Quantum Chemical Modeling

The conformational analyses of 1 in a hexane solvent and for 24 in a CH3OH solvent were done using density functional theory (DFT) with the exchange-correlation functional PBE1PBE [14], the polarization continuum model (PCM) [15] and 6-311+G(d) split-valence basis set, implemented in the Gaussian 03 package of programs [16]. The molecular cavity was modeled according to unified force field (radii = UFF). The statistical weights (gim) of different conformations of 14 were obtained according to Equation (1):
g i m = e Δ G i m / R T i e Δ G i m / R T
where the summation was done over all possible conformations of 14 and ∆Gim = GiGm; G = Eel + Gtr,T + Grot,T + Gvib,T is a sum of electronic, translational, rotational, and vibrational contributions to the Gibbs free energy, calculated at temperature T = 298.15 K; the subscript “m” denotes conformation, for which G is minimal. The excitation energies and the rotatory strengths were calculated using time-dependent density functional theory (TDDFT). Theoretical ECD spectra (∆εcalc(λ)) were simulated as a superposition of Gauss-type functions, individual for each transition from electronic ground state to the i-th calculated excited electronic state (1 ≤ i ≤ 50). The same value ξ = 0.20 eV for the bandwidths at 1/e peak heights was used. The total theoretical ECD spectra were obtained after statistical averaging:
ε c a l c ( λ ) = i g i · Δ ε i , c a l c ( λ )
The scaled theoretical and experimental ECD spectra were obtained according to Equation (3):
ε s c a l e d ( λ ) = Δ ε ( λ ) | Δ ε ( λ p e a k ) |
where the denominator |∆ε(λpeak)| is a modulo of the peak value for the chosen characteristic band in the corresponding ECD spectrum.

3.9. Measurement of Nitric Oxide Content

RAW 264.7 murine macrophages were plated into 96-well microplates and incubated at 37 °C with 5% CO2 for 2 h. After adhesion, cells were incubated with tested compounds (10.0 µM) for 24 h. For endogenously generated NO detection studies, the cells were coincubated with 10 µM of FA–OMe fluorescent probe for 8 h [17]. Prior to fluorescence registration, the cells were washed three times with PBS and then bathed in 200 µL/well of PBS. Green fluorescence of cells was registered at λex = 460 nm and λem = 524 nm. In each experiment LPS from E. coli serotype 055:B5 (1.0 µg/mL, Sigma-Aldrich, St. Louis, MO, USA) were used as a positive control. Fluorescent intensity was measured using plate reader PHERAstar FS (BMG Labtech GmbH, Ortenberg, Germany).

3.10. Statistical Analysis

All assays were performed at least in triplicate. The results are expressed as the mean ± standard error (SD). A Student’s t-test was used to evaluate the data with the significance level of p < 0.05. The means and standard errors for each treatment were calculated and plotted using SigmaPlot 3.02 software (Jandel Scientific, San Rafael, CA, USA).

3.11. Cell Lines and Culture Conditions

The human prostate cancer cells line PC-3 was purchased from ATCC. Cells were cultured according to the manufacturer’s instructions in 10% FBS/RPMI media (Invitrogen, Paisley, UK) containing penicillin/streptomycin (Invitrogen). Cells were continuously kept in culture for a maximum of three months, and were routinely inspected microscopically for stable phenotype and regularly checked for contamination with mycoplasma. Cell line authentication was recently performed by DSMZ (Braunschweig, Germany) using highly polymorphic short tandem repeat loci [18].

3.12. In Vitro MTT-Based Drug Sensitivity Assay

The in vitro cytotoxicity of individual substances was evaluated using the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay, which was performed as previously described [19].

3.13. Western Blotting

Western blotting was performed as described before [19]. For detection of the proteins following primary and secondary antibody were used: anti-SQSTM/p62 (#5114, 1:1000, Cell Signaling Technology, Danvers, MA, USA), anti-α-tubulin (T5168, 1:10,000, Sigma-Aldrich, St. Louis, MO, USA), anti-mouse IgG-HRP (NXA931, 1:10,000, GE Healthcare, Little Chalfont, UK), anti-rabbit IgG-HRP (Cell Signaling Technology, Danvers, MA, USA, #7074, 1:5000). Signals were detected using the ECL chemiluminescence system (Thermo Scientific, Rockford, IL, USA) according to the manufacturer’s protocol. Relative optical density of the signal intensity of the bands was quantified with Quantity One 4.6 software (Bio-Rad, Hercules, CA, USA) and normalized first, against corresponding signal of α-tubulin (loading control), and second, against the signal of control sample. Mean values of two independent experiments ± SEM are presented. SEM values were calculated using GraphPad Prism software v. 5.01 (GraphPad Prism software Inc., La Jolla, CA, USA).

4. Conclusions

Zosteropenillines A–L (112) belong to the series of lovastatin-related polyketides that are formed by an enzymatic intramolecular Diels–Alder cycloaddition of a linear hexaketide precursor [20,21,22]. Polyketides containing “decalin” moiety exhibit diverse biological properties such as anticancer, antimicrobial, neurotrophic, and growth-stimulating activity [23,24,25,26,27]. Twelve new polyketides, named zosteropenillines A–L (112), have been isolated from the extract of the marine-derived fungus Penicillium thomii. The absolute configuration zosteropenilline A (1) was determined by a combination of the modified Mosher’s method, X-ray analysis, and NOESY data. Absolute configurations of zosteropenillines B–D (24) were determined by time-dependent density functional theory (TD-DFT) calculations of ECD spectra. Zosteropenillines B, G, and I at a non-toxic concentration of 10.0 µM induced a moderate downregulation of NO production in macrophages stimulated with LPS.

Supplementary Materials

1H, 13C, DEPT, COSY-45, HSQC, HMBC and NOESY spectra of all compounds are available online at www.mdpi.com/1660-3397/15/2/46/s1.

Acknowledgments

The study was supported by grant No. 14-14-00030 from the Russian Science Foundation.

Author Contributions

Shamil Sh. Afiyatullov supervised research and wrote the manuscript; Elena V. Leshchenko designed the project, isolated compounds and elucidated their structure, discussed results, and wrote the manuscript; Alexandr S. Antonov and Maria P. Sobolevskaya isolated and recrystallized compounds; Anatoly A. Udovenko performed X-ray experiments; Mikhail V. Pivkin cultivated the fungus; Roman S. Popov performed MS experiments; Vladimir A. Denisenko performed NMR experiments; Dmitrii V. Berdyshev calculated ECD spectra, discussed the results, and helped to write the manuscript; Evgeny A. Pislyagin evaluated inhibitory effects on NO production; Gunhild von Amsberg designed bioassays, raised financial support for biological experiments with human cells, and discussed the results; Sergey A. Dyshlovoy performed and designed bioassays with human cells, discussed the results, and helped to write the manuscript. The authors are grateful to the Far Eastern Center of Structural Research for performing the X-ray investigation.

Conflicts of Interest

The authors declare no conflict of interest.

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Figure 1. Structures of zosteropenillines A–L (112).
Figure 1. Structures of zosteropenillines A–L (112).
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Figure 2. (A) Key HMBC and COSY correlations of 1; (B) Crystal structure of 1.
Figure 2. (A) Key HMBC and COSY correlations of 1; (B) Crystal structure of 1.
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Figure 3. Δδ (δS − δR) values (in ppm) for the (S)- and (R)-MPTA esters of 1.
Figure 3. Δδ (δS − δR) values (in ppm) for the (S)- and (R)-MPTA esters of 1.
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Figure 4. The inversion of A-ring 4R, 5S, 8S, 9R, 10R, 13S of 1.
Figure 4. The inversion of A-ring 4R, 5S, 8S, 9R, 10R, 13S of 1.
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Figure 5. The inversion of C-ring for 4R, 5S, 8S, 9R, 10R, 13S of 1.
Figure 5. The inversion of C-ring for 4R, 5S, 8S, 9R, 10R, 13S of 1.
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Figure 6. The normalized experimental and statistically averaged ECD spectra. (A) 4R, 5S, 8S, 9R, 10R, 13S-1; (B) 4R, 5S, 8S, 10R, 13S-2; (C) 4R, 5S, 8R, 10R, 13S-3; (D) 4R, 5S, 8S, 10R, 13S-4. The half-width bands ΔEexcitation = 0.20 eV were used for the simulations of individual bands in theoretical spectra for each conformer.
Figure 6. The normalized experimental and statistically averaged ECD spectra. (A) 4R, 5S, 8S, 9R, 10R, 13S-1; (B) 4R, 5S, 8S, 10R, 13S-2; (C) 4R, 5S, 8R, 10R, 13S-3; (D) 4R, 5S, 8S, 10R, 13S-4. The half-width bands ΔEexcitation = 0.20 eV were used for the simulations of individual bands in theoretical spectra for each conformer.
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Figure 7. Energy-minimized 3D model of 5 with key NOESY correlations.
Figure 7. Energy-minimized 3D model of 5 with key NOESY correlations.
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Figure 8. Effect of compounds 1, 2, and 810 on NO level in RAW 264.7 murine macrophages co-incubated with LPS from E. coli. The compounds were tested at a concentration of 10 µM. Time of cell incubation with compounds is 24 h at 37 °C. * p < 0.01.
Figure 8. Effect of compounds 1, 2, and 810 on NO level in RAW 264.7 murine macrophages co-incubated with LPS from E. coli. The compounds were tested at a concentration of 10 µM. Time of cell incubation with compounds is 24 h at 37 °C. * p < 0.01.
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Figure 9. The effect on p62 expression. The expression of p62 in PC3 cells treated with the compounds 13 (A) and 7, 8, 10, 11 (B). Cells were treated for 48 h, then proteins were extracted and both p62 and α-tubulin levels were detected by Western blotting. The signals’ intensity was quantified with Quantity One. Cells treated with the autophagy inhibitors bafilomycin A1 (Baf, 100 nM) were used as a positive control. The values are presented as mean ± SEM.
Figure 9. The effect on p62 expression. The expression of p62 in PC3 cells treated with the compounds 13 (A) and 7, 8, 10, 11 (B). Cells were treated for 48 h, then proteins were extracted and both p62 and α-tubulin levels were detected by Western blotting. The signals’ intensity was quantified with Quantity One. Cells treated with the autophagy inhibitors bafilomycin A1 (Baf, 100 nM) were used as a positive control. The values are presented as mean ± SEM.
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Table 1. 13C NMR spectroscopic data (δ in ppm) for zosteropenillines A–F (16).
Table 1. 13C NMR spectroscopic data (δ in ppm) for zosteropenillines A–F (16).
Position1 *2 *3 *4 **5 **6 *
160.9, CH260.9, CH260.9, CH260.9, CH260.9, CH257.6, CH2
239.9, CH239.9, CH239.9, CH239.9, CH237.9, CH248.6, CH2
3210.2, C210.4, C210.3, C210.9, C206.0, C217.6, C
462.8, CH62.9, CH62.7, CH63.0, CH63.7, CH61.8, CH
537.5, CH39.5, CH38.9, CH39.3. CH38.5, CH37.4, CH
628.7, CH228.8, CH238.7, CH229.3, CH229.0, CH229.4, CH2
732.7, CH229.1, CH224.9, CH234.9, CH231.8, CH232.9, CH2
840.2, CH40.5, CH69.7, C32.8, CH39.2, CH40.6, CH
977.9, CH35.2, CH244.5, CH240.9, CH279.2, CH77.8, CH
1048.6, CH41.4, CH36.9, CH41.9, CH53.5, CH49.1, CH
11130.1, CH134.4, CH134.4, CH130.5, CH67.3, CH128.6, CH
12131.6, CH130.9, CH131.1, CH134.8, CH72.7, CH133.0, CH
1374.7, C75.0, C75.0, C75.2, C77.5, C69.1, C
1423.2, CH323.3, CH323.3, CH323.3, CH322.0, CH328.8, CH3
1518.4, CH368.1, CH231.5, CH322.3, CH319.1, CH318.4, CH3
* Chemical shifts were measured at 125.77 MHz in CDCl3; ** Chemical shifts were measured at 176.04 MHz in CDCl3.
Table 2. 1H NMR spectroscopic data (δ, J in Hz) for zosteropenillines A–F (16).
Table 2. 1H NMR spectroscopic data (δ, J in Hz) for zosteropenillines A–F (16).
Position1 **2 *3 *4 **5 **6 *
1a: 4.11, ddd (12.0, 8.0, 1.2)
b: 3.89, td (12.0, 3.0)
a: 4.11, ddd (11.8, 8.0, 1.0)
b: 3.89 td (12.0, 3.0)
a: 4.11, ddd (11.8, 8.0, 1.0)
b: 3.89, td (12.0, 2.9)
a: 4.11, ddd (11.7, 8.0, 1.4)
b: 3.89, td (12.0, 3.0)
a: 4.22, dd (12.0, 9.0)
b: 3.91, td (12.0, 4.0)
a: 3.91, ddd (11.7, 6.2, 4.2)
b: 3.87, ddd (11.7, 6.2, 4.2)
2a: 2.64, ddd (14.0, 12.0, 8.0)
b: 2.21, ddt (14.0, 3.0, 1.2)
a: 2.66, ddd (14.1, 12.1, 8.0)
b: 2.21, ddt (14.1, 3.0, 1.3)
a: 2.66, ddd (14.0, 12.1, 8.0)
b: 2.21, ddt (13.9, 2.9, 1.3)
a: 2.68, ddd (14.0, 12.0, 3.0)
b: 2.21, ddt (14.0, 3.0, 1.5)
a: 2.67, ddd (15.0, 12.0, 9.0)
b: 2.21, dd (15.0, 4.0)
a: 2.86, ddd (18.4, 6.2, 4.2)
b: 2.76, ddd (18.4, 6.2, 4.4)
42.09, dd (11.8, 1.2)2.07, dd (11.3, 1.2)2.10, dd (11.3, 1.2)2.04, dd (11.5, 1.6)2.11, d (11.7)2.59, d (11.4)
51.89, tdd (11.8, 10.6, 3.2)1.78, m1.75, m1.75, m1.95, m1.93, tdd (11.4, 10.4, 3.0)
6a: 1.28, m
b: 1.14, qd (13.0, 3.3)
a: 1.41, dq (13.2, 3.2)
b: 1.14, qd (13.2, 3.5)
a: 1.69, m
b: 1.41, m
a: 1.34, dq (13.3, 3.6)
b: 1.11, qd (13.0, 3.8)
a: 1.33, m
b: 1.11, m
a: 1.50, m
b: 1.15, qd (12.7, 3.2)
7a: 1.78, dq (12.7, 3.3)
b: 1.08, qd (12.7, 3.6)
a: 1.85, dq (12.9, 3.4)
b: 1.01, qd (12.9, 3.7)
a: 1.45, m
b: 1.19, m
a: 1.75, m
b: 0.96, m
a: 1.73, m
b: 1.03, m
a: 1.77, m
b: 1.12, qd (13.0, 3.3)
81.41, m1.63, m 1.48, m1.48, m1.42, m
92.92, td (9.9, 5.8)a: 1.91, dq (12.6, 3.5)
b: 0.93, q (12.3)
a: 1.75, m
b: 1.28, m
a: 1.77, m
b: 0.87, q (12.2)
3.29, t (9.0)2.92, t (9.9)
101.65, tt (10.4, 2.3)1.74, m2.15, ddd (13.3, 10.2, 3.5)1.71, m1.61, td (10.8, 9.0)1.73, tt (10.3, 2.3)
116.24, dd (10.0, 2.4)5.67, dd (9.8, 1.5)5.63, brs5.59, dd (9.9, 2.5)4.38, t (10.5)6.11, dd (10.0, 2.0)
125.69, dd (10.0, 2.6)5.61, dd (9.8, 2.5)5.63, brs5.65, dd (9.8, 1.0)3.78, d (10.5)5.64, dd (10.0, 2.6)
141.26, s1.26, s1.26, s1.25, s1.36, s1.30, s
151.03, d (6.5)a: 3.50, dd (10.3, 6.3)
b: 3.47, dd (10.3, 6.3)
1.25, s0.91, d (6.6)1.06, d (6.5)1.03, d (6.5)
9-OH1.55, d (6.3)
* Chemical shifts were measured at 500.13 MHz in CDCl3; ** Chemical shifts were measured at 700.13 MHz in CDCl3.
Table 3. 13C NMR spectroscopic data (δ in ppm) for zosteropenillines G–L (712).
Table 3. 13C NMR spectroscopic data (δ in ppm) for zosteropenillines G–L (712).
Position7 *8 **9 *10 **11 *12 **
158.1, CH257.9, CH258.0, CH258.0, CH257.9, CH258.0, CH2
249.3, CH243.6, CH242.1, CH249.2, CH248.9, CH249.5, CH2
3214.3, C214.0, C213.5, C214.1, C211.2, C213.2, C
463.5, CH61.7, CH59.6, CH63.4, CH61.6, CH57.7, CH
541.3, CH37.9, CH40.3, CH41.5, CH39.4, CH40.3, CH
629.4, CH230.5, CH229.9, CH228.7, CH232.4, CH232.1, CH2
734.8, CH231.9, CH232.6, CH228.9, CH227.9, CH228.1, CH2
832.9, CH39.2, CH40.4, CH40.6, CH41.1, CH41.1, CH
940.9, CH282.3, CH78.3, CH35.2, CH238.3, CH237.2, CH2
1042.1, CH49.6, CH47.2, CH41.5, CH166.3, C144.5, C
11131.9, CH73.2, CH130.6, CH131.5, CH120.0, CH118.8, CH
12133.7, CH129.0, CH129.3, CH134.0, CH200.3, C68.0, CH
1373.0, C131.5, C140.8, C72.9, C74.2, C72.4, C
1425.9, CH320.9, CH3112.8, CH225.9, CH321.8, CH320.5, CH3
1522.3, CH317.8, CH318.4, CH368.1, CH267.4, CH267.8, CH2
* Chemical shifts were measured at 125.77 MHz in CDCl3; ** Chemical shifts were measured at 176.04 MHz in CDCl3.
Table 4. 1H NMR spectroscopic data (δ, J in Hz) for zosteropenillines G–L (712).
Table 4. 1H NMR spectroscopic data (δ, J in Hz) for zosteropenillines G–L (712).
Position7 *8 **9 *10 **11 *12 ***
1a: 3.90, ddd (11.7, 7.1, 3.7)
b: 3.86, ddd (11.7, 6.6, 3.7)
3.85, m (2H)a: 3.91, ddd (11.5, 7.1, 3.9)
b: 3.85, ddd (11.5, 6.8, 4.2)
a: 3.91, ddd (11.2, 6.9, 3.6)
b: 3.87, ddd (11.2, 6.7, 3.7)
a: 3.96, ddd (11.5, 7.7, 3.7)
b: 3.82, ddd (11.5, 6.7, 3.9)
a: 3.95, ddd (11.3, 7.5, 3.5)
b: 3.82, ddd (11.3, 6.4, 3.6)
2a: 3.04, ddd (18.0, 6.8, 3.8)
b: 2.60, ddd (18.0, 7.1, 3.8)
a: 2.69, ddd (18.2, 6.6, 4.3)
b: 2.64, ddd (18.2, 6.3, 4.3)
a: 2.83, ddd (18.3, 6.8, 3.8)
b: 2.65, ddd (18.3, 6.8, 4.0)
a: 3.06, ddd (18.0, 7.1, 3.7)
b: 2.61, ddd (18.0, 7.0, 3.7)
a: 3.14, ddd (18.0, 6.5, 3.6)
b: 2.66, ddd (18.0, 7.6, 3.9)
a: 3.05, ddd (18.1, 6.5, 3.6)
b: 2.69, ddd (18.0, 7.6, 3.7)
42.89, d (11.5)2.92, m3.15, dtd (12.1, 2.6, 0.9)2.92, d (11.6)3.01, d (9.8)3.09, d (10.0)
51.48, m1.69, m1.68, m1.52, dq (11.7, 2.7)2.89, m2.55, m
6a: 1.70, m
b: 0.93, m
a: 1.57, dq (13.2, 3.5)
b: 1.17, qd (13.1, 3.5)
a: 1.53, m
b: 1.14, m
a: 1.79, m
b: 0.95, qd (12.3, 3.6)
a: 1.91, dq (12.9, 3.0)
b: 1.15, qd (13.1, 3.3)
a: 1.74, m
b: 1.12, m
7a: 1.71, m
b: 0.96, m
a: 1.70, m
b: 1.03, qd (13.2, 3.5)
a: 1.77, m
b: 1.10, m
a: 1.79, m
b: 1.02, qd (12.4, 3.6)
a: 1.84, dq (13.1, 3.2)
b: 1.29, m
a: 1.79, m
b: 1.12, m
81.46, m1.47, m1.43, m1.61, m1.74, m1.64, m
9a: 1.73, m
b: 0.77, q (12.4)
3.25, t (9.3)2.89, t (9.9)a: 1.87, m
b: 0.82, q (13.0)
a: 2.60, dq (14.0, 2.5)
b: 2.01, m
a: 2.39, dq (13.5, 2.5)
b: 1.77, m
101.84, m1.28, dt (11.9, 9.3)1.85, t (10.0)1.87, m
115.42, brs4.32, dq (8.9, 2.1)6.20, brs5.44, brs5.90, brs5.60, dt (5.9, 2.1)
125.42, brs5.53, q (1.8)6.20, brs5.44, brs 4.36, dd (5.9, 1.7)
141.20, s1.60, brsa: 4.99, dd (2.8, 1.3)
b: 4.59, dt (2.3, 0.9)
1.20, s1.19, s1.20, s
150.89, d (6.5)1.02, d (6.5)1.05, d (6.5)a: 3.47, dd (10.6, 6.6)
b: 3.45, dd (10.6, 6.6)
a: 3.58, dd (10.5, 5.8)
b: 3.54, dd (10.5, 5.8)
a: 3.50, dd (10.5, 5.9)
b: 3.48, dd (10.4, 6.2)
* Chemical shifts were measured at 500.13 MHz in CDCl3; ** Chemical shifts were measured at 700.13 MHz in CDCl3.
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