Fitoterapia 92 (2014) 79–84
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Mono- and bis-furanone derivatives from the endolichenic fungus Peziza sp. Kun Zhang a,b,1, Jinwei Ren a,1, Mei Ge b, Li Li c, Liangdong Guo a, Daijie Chen b,⁎, Yongsheng Che d,⁎ a b c d
State Key Laboratory of Mycology, Institute of Microbiology, Chinese Academy of Sciences, Beijing 100101, PR China Shanghai Normal University, Shanghai 200234, PR China Institute of Materia Medica, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing 100050, PR China Beijing Institute of Pharmacology & Toxicology, Beijing 100850, PR China
a r t i c l e
i n f o
Article history: Received 17 August 2013 Accepted in revised form 24 October 2013 Accepted 25 October 2013 Available online 1 November 2013 Keywords: Endolichenic fungus Peziza sp. Furanones Electronic circular dichroism
a b s t r a c t Seven new mono- and bis-furanones, pezizolides A–G (1–7), have been isolated from the crude extract of the endolichenic fungus Peziza sp. inhabiting the lichen Xanthoparmelia sp. The structures of the new compounds were elucidated mainly by NMR and MS methods. The absolute configuration of 1–4 was assigned by the application of CD exciton chirality method, and 1 was further supported by electronic circular dichroism (ECD) calculations, whereas that of 5 was deduced by Snatzke's method following the relative configuration analysis of its acetonide. The cytotoxity and antimicrobial activity of compounds 1–7 were tested. © 2013 Elsevier B.V. All rights reserved.
1. Introduction Lichens are symbiotic combinations of fungi (mycobiont) and cyanobacteria (algae) [1]. Except for fungal mycobiont and some non-obligate microfungi, endolichenic fungi are also living in the thalli of lichens [2–6]. Endolichenic fungi inhabiting in the lichen thalli were similar to endophytes living in the intercellular spaces of healthy plant tissues, but the chemical diversity of this class of fungi remained largely unexploited [7–10]. In our present work, the fungus Peziza sp. was initially found in the surface of Xanthoparmelia which was collected from the Zixi Mountain of Yunnan Province, People's Republic of China. Peziza species are macrofungi commonly called cup fungi. To date, only a few secondary metabolites isolated and identified from this genus [11,12]. Fractionation of an EtOAc extract prepared from a solid-substrate fermentation culture afforded seven new mono- (5–7) and bis-furanone (1–4) derivatives. Details of the isolation, structure ⁎ Corresponding authors. Tel.: +86 10 66932679. E-mail addresses: [email protected] (D. Chen), [email protected] (Y. Che). 1 Authors contributed equally to this work. 0367-326X/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.fitote.2013.10.011
elucidation, and biological activity of these compounds are reported herein. 2. Experimental method 2.1. General Optical rotations were measured on a Perkin-Elmer 241 polarimeter, and UV data were obtained on a Shimadzu Biospec-1601 spectrophotometer. CD spectra were recorded on a JASCO J-815 spectropolarimeter. IR data were recorded using a Nicolet Magna-IR 750 spectrophotometer. 1H and 13C NMR data were acquired with AVANCE III-500 and NMR system-600 spectrometers using solvent signals (acetone-d6: δH 2.05/δC 29.8, 206.1; CDCl3: δH 7.26/δC 77.2) as references. The HMQC and HMBC experiments were optimized for 145.0 and 8.0 Hz, respectively. HRESIMS data were obtained using an Agilent Accurate-Mass-Q-TOF LC/MS 6520 instrument equipped with an electrospray ionization (ESI) source. The fragmentor and capillary voltages were kept at 125 and 3500 V, respectively. Nitrogen was supplied as the nebulizing and drying gas. The temperature of the drying gas was set at 300 °C. The flow rate of
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chromatography (CC) using 1:1 CH2Cl2–MeOH as eluents, and the resulting subfractions were further purified by semipreparative RP HPLC (Agilent Zorbax SB-C18 column; 5 μm; 9.4 × 250 mm; 28% CH3CN in H2O over 40 min; 2 mL/min) to afford 1 (67.9 mg, tR 31.3 min), 2 (12.8 mg, tR 29.6 min), 3 (11.0 mg, tR 27.7 min) and 4 (1.7 mg, tR 21.3 min). The fractions (340 mg) eluted with 3:2 PE–EtOAc were separated by Sephadex LH-20 CC eluting with MeOH. The resulting subfractions were purified by RP HPLC (Agilent Zorbax SB-C18 column; 5 μm; 9.4 × 250 mm) to afford 5 (4.5 mg, tR 26.0 min; 55%–65% MeOH in H2O for 30 min), 6 and 7 (2.1 mg and 9.0 mg, tR 10.3 min and 10.9 min, respectively; 65% MeOH in H2O for 30 min). 5 (1.5 mg) was derived and purified by RP preparative HPLC to yield the acetonide 5a (1.0 mg, tR 23.6 min; 20%–90% CH3CN in H2O for 30 min).
the drying gas and the pressure of the nebulizer were 10 L/min and 10 psi, respectively. All MS experiments were performed in positive ion mode. Full-scan spectra were acquired over a scan range of m/z 100–1000 at 1.03 spectra/s. 2.2. Fungal material The culture of Pezizales sp. was isolated from the Xanthoparmelia sp. collected from the ZiXi Mountain of Yunnan Province, People's Republic of China, in October, 2010. The isolation was identified by Dr. Wenchao Li on the basis of morphology and sequence (GenBank accession No. KC493353) analysis of the ITS region of the rDNA. The fungal strain was cultured on slants of potato dextrose agar (PDA) at 25 °C for 10 days. Agar plugs were cut into small pieces (about 0.5 × 0.5 × 0.5 cm3) under aseptic conditions; 15 pieces were used to inoculate three Erlenmeyer flasks (250 mL), each containing 50 mL of media (0.4% glucose, 1% malt extract, and 0.4% yeast extract; the final pH of the media was adjusted to 6.5 and sterilized by autoclave). Three flasks of the inoculated media were incubated at 25 °C on a rotary shaker at 170 rpm for 5 days to prepare the seed culture. Fermentation was carried out in 12 Fernbach flasks (500 mL), each containing 80 g of rice. Distilled H2O (120 mL) was added to each flask, and the contents were soaked overnight before autoclaving at 15 psi for 30 min. After cooling to room temperature, each flask was inoculated with 5.0 mL of the spore inoculum and incubated at 25 °C for 40 days.
2.3.1. Compound 1 Light yellow oil, [α]25 D + 16.0 (c 0.1, MeOH), UV (MeOH) λmax (log ε) 216 (0.55), 251 (0.55) nm; CD (c 6.2 × 10–4 M, MeOH) λmax (Δε) 220 (–6.35); IR (neat) νmax 3432 (br), 2980, 2938, 2876, 1756, 1668, 1618, 1450, 1386, 1347, 1224, 1112, 1085, and 1038 cm–1; 1H, 13C NMR, and HMBC data (see Table 1); HRESIMS m/z 267.1227 [M + H]+ (calculated for C14H19O5, 267.1227). 2.3.2. Compound 2 Light yellow oil, [α]25 D + 5.3 (c 0.3, MeOH); UV (MeOH) λmax nm (log ε): 217 (0.36), 245 (0.21) nm; CD (c 6.2 × 10–4 M, MeOH) λmax (Δε) 225 (–3.37); IR (neat) νmax 3412 (br), 2976, 2938, 2876, 1755, 1666, 1618, 1542, 1488, 1448, 1387, 1303, 1210, 1148, 1116, 1079, and 1043 cm–1; 1H and 13C NMR data (see Table 1); HRESIMS m/z 267.1228 [M + H]+ (calculated for C14H19O5, 267.1227).
2.3. Extraction and isolation The fermented material was extracted repeatedly with EtOAc (4 × 250 mL) for each flask, and the organic solvent was evaporated to dryness under vacuum to afford the crude extract (30.0 g), which was fractionated by silica gel VLC using petroleum ether (PE)–EtOAc–MeOH gradient elution. The fractions (1.06 g) eluted with 1:1 and 1:4 PE–EtOAc were combined and separated again by Sephadex LH-20 column
2.3.3. Compound 3 Colorless oil, [α]25 D + 10.3 (c 0.3, MeOH), UV (MeOH) λmax nm (log ε): 217 (0.55) nm; CD (c 6.0 × 10–4 M, MeOH)
Table 1 NMR data for 1–4. 1 Pos.
δCa
2 3 4 5 6 7 8 9 10 11 12a 12b 13 14 15 OH-13
173.3 122.8 159.8 71.1 119.9 132.0 19.2 22.2 38.7 83.4 42.7
a b c
69.0 176.6 26.8
2 b
δH (J in Hz)
4.79 6.24 6.82 1.82 2.70 1.97
(s) (dd, 15.7, 1.5) (m) (d, 6.7) (overlap) (overlap)
HMBC
2, 2, 3, 6, 3, 4,
3, 3, 8 7 4, 9,
c
4, 9 4, 7, 8
5, 10, 11 11, 12, 15
2.64 (overlap) 2.01 (overlap) 4.62 (t, 8.5)
11, 12, 14
1.51 (s) 5.01 (d, 3.9)
10, 11, 12 12, 13
Recorded at 125 MHz in acetone-d6. Recorded at 500 MHz in acetone-d6. Recorded at 125 MHz in acetone-d6.
10, 11, 13, 14, 15
δCa 173.3 122.9 159.8 71.2 119.9 132.1 19.3 21.9 39.9 82.8 42.2 68.7 176.6 25.6
3 δHb
(J in Hz)
4.81 6.25 6.84 1.83 2.69 1.99
(s) (dd, 15.7, 1.5) (m) (d, 6.7) (m) (t, 8.6)
2.52 (dd, 12.8, 8.6) 2.08 (dd, 12.7, 9.8) 4.68 (t, 9.1) 1.45 (s) 4.98 (d, 5.3)
δCa 174.6 128.2 158.3 70.9 25.7 21.4 14.0 21.8 39.1 83.7 42.0 68.6 176.2 26.63
4 δHb
(J in Hz)
4.64 2.23 1.53 0.92 2.51 1.79
(s) (t, 7.4) (m) (t, 7.3) (overlap) (t, 8.5)
2.53 (overlap) 2.12 (dd,13.3, 8.4) 4.58 (t, 8.5) 1.56 (s) 2.79 (s)
δCa 172.9 125.3 159.7 71.2 117.5 133.8 15.8 22.8 38.1 83.6 41.9 68.6 176.1 26.63
δHb (J in Hz)
4.74 5.87 5.98 1.67 2.51 1.82
(s) (t, 11.2) (m) (d, 6.8) (overlap) (dd, 10.2, 6.5)
2.10 (dd,13.3, 8.4) 2.50 (overlap) 4.56 (t, 8.6) 1.53 (s) 2.69 (s)
K. Zhang et al. / Fitoterapia 92 (2014) 79–84
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λmax (Δε) 222 (–4.75); IR (neat) νmax 3435 (br), 2962, 2936, 2873, 1748, 1669, 1455, 1386, 1348, 1294, 1262, 1221, 1177, 1137, 1113, 1079, and 1033 cm–1; 1H and 13C NMR data (see Table 1); HRESIMS m/z 269.1381 [M + H]+ (calculated for C14H21O5, 269.1384).
2.3.7. Compound 7 Colorless oil, UV (MeOH) λmax (log ε) 199 (1.53), 257 (0.33) nm, IR (neat) νmax 3436 (br), 2974, 2935, 2876, 1755, 1678, 1447, 1376, 1287, 1122, 1074, 1038, and 973 cm–1; 1H, 13 C NMR and HMBC data (see Table 2); HRESIMS m/z 179.1072 [M + H]+ (calculated for C11H15O2, 179.1067) (Fig. 1).
2.3.4. Compound 4 Colorless oil, [α]25 D + 9.2 (c 0.2, MeOH), UV (MeOH) λmax nm (log ε): 206 (0.58) nm; CD (c 6.2 × 10–4 M, MeOH) λmax (Δε) 222 (–5.68); IR (neat) νmax 3430 (br), 2979, 2938, 2873, 1755, 1667, 1638, 1619, 1450, 1413, 1386, 1353, 1293, 1262, 1226, 1131, 1094, and 1037 cm–1; 1H, 13C NMR data (see Table 1); HRESIMS m/z 267.1228 [M + H]+ (calculated for C14H19O5, 267.1227).
2.4. MTS assay [13] The assay was run in triplicate. In a 96-well plate, each well was plated with (2–5) × 103 cells (depending on the cell multiplication rate). After cell attachment overnight, the medium was removed, and each well was treated with 100 μL medium containing 0.1% DMSO, or appropriate concentrations of the test compounds and the positive control cisplatin (100 mM as stock solution of a compound in DMSO and serial dilutions; the test compounds showed good solubility in DMSO and did not precipitate when added to the cells). The plate was incubated for 48 h at 37 °C in a humidified, 5% CO2 atmosphere. Proliferation assessed by adding 20 μL of MTS (Promega) to each well in the dark, followed by a 90 min incubation at 37 °C. The assay plate was read at 490 nm using a microplate reader. The inhibition rate was calculated and plotted versus test concentrations to afford the IC50.
2.3.5. Compound 5 Light yellow oil, [α]25 D + 23.1 (c 0.2, MeOH), UV (MeOH) λmax nm (log ε): 211 (0.93), 253 (0.94) nm; CD spectrum of Mo2(OAc)4–complex subtracted (c 5.8 × 10–4 M, DMSO) λmax (Δε) 302 (+ 0.41); IR (neat) νmax 3419 (br), 2968, 2937, 2879, 1743, 1666, 1615, 1447, 1379, 1344, 1290, 1245, 1193, 1170, 1114, 1067, and 1044 cm–1; 1H, 13C NMR and HMBC data (see Table 2); HRESIMS m/z 235.0937 [M + Na]+ (calculated for C11H16O4Na, 235.0941). 5a 1H NMR (in CDCl3): 4.88 (H-5a, d, J = 15.2 Hz), 4.73 (H-5b, d, J = 15.2 Hz), 6.08 (H-6, m), 7.09 (H-7, m), 1.87 (H3-8, d, J = 5.6 Hz), 4.75 (H-9, d, J = 7.2 Hz), 3.75 (H-10, m), 1.66 (H2-11, m), 1.03 (H3-12, t, J = 6.2 Hz), 1.46 (H3-14, s), and 1.43 (H3-15, s). 1D NOE correlation (in CDCl3): H-9 → H3-15, H-10 → H3-14.
2.5. Antifungal assays Antifungal assays were conducted in triplicate following the National Center for Clinical Laboratory Standards (NCCLS) recommendations [14]. Bacillus subtilis (ATCC 6633), Staphylococcus aureus (CGMCC 1.2465) and Candida albicans (CGMCC 2.2086), were obtained from the China General Microbial Culture Collection (CGMCC) and were grown on PDA. Targeted fungi (3 or 4 colonies) were prepared from broth cultures incubated at 28 °C for 48 h, and the final suspensions contained 104 hyphae/mL (in PDB medium). Test samples (10 mg/mL as stock solution in DMSO and serial dilutions) were transferred to 96-well clear plates in triplicate, and the suspensions of the test organisms were added to each well to
2.3.6. Compound 6 Light yellow oil, UV (MeOH) λmax (log ε) 213 (1.84), 260 (2.02) nm; IR (neat) νmax 3435 (br), 2972, 2936, 2877, 1756, 1680, 1448, 1374, 1285, 1124, 1072, 1035, and 976 cm–1; 1H, 13 C NMR and HMBC data (see Table 2); HRESIMS m/z 179.1070 [M + H]+ (calculated for C11H15O2, 179.1067).
Table 2 NMR data for 5–7. 5 Pos.
δCa
2 3 4 5 6 7 8 9 10 11
173.2 122.7 161.0 70.4 120.3 132.5 19.3 71.0 76.1 26.9
12
10.8
a b c d e f
6 δHb
(J in Hz)
4.86 (s) 6.29 (d, 16.3) 6.87 (m) 1.82 (d, 6.6) 4.77 (d, 4.3) 3.58 (m) 1.59 (m) 1.50 (m) 0.97 (t, 7.4)
Recorded at 125 MHz in acetone-d6. Recorded at 500 MHz in acetone-d6. Recorded at 125 MHz in acetone-d6. Recorded at 125 MHz in CDCl3. Recorded at 500 MHz in CDCl3. Recorded at 125 MHz in CDCl3.
HMBC
2, 2, 3, 6, 3, 4, 9,
c
3, 4, 9 3, 7, 8 8 7 4, 5, 10, 11 11, 12 10, 12
10, 11
7
δC
d
173.4 120.7 150.7 68.6 118.6 133.3, 19.5 119.5 140.9 26.7 13.0
δHe
(J in Hz)
4.84 6.22 6.91 1.88 6.54 6.06 2.27
(s) (dd, 15.7, 1.6) (m) (d, 6.8) (d, 16.0) (m) (m)
1.09 (t, 7.5)
HMBC
2, 2, 3, 3, 3, 4, 9,
f
3, 4 4, 7, 8 8 6, 7 4, 5, 10, 11 11, 12 10, 12
10, 11
δCd
δHe (J in Hz)
HMBCf
173.3 122.8 156.8 70.7 118.4 132.8 19.3 30.2 124.6 129.4
4.63 6.08 6.83 1.85 3.15 5.38 5.57
2, 2, 3, 3, 3, 9, 9,
17.9
(s) (d, 14.6) (m) (d, 6.7) (d, 6.6) (m) (m)
1.68 (t, 6.4)
3, 4, 9 3, 8 8 6, 7 4, 5, 10, 11 11, 12 10, 12
10, 11
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7
13
11
O 14
15
3
4
9
O 2
12
10
O
OH
OH
OH 6
O
O
O
O
O
O O
O
5
O
1
3
1. 11S 2. 11R
4
8 6
7 3
OH 4
O 2
O
5
10
11
9
12
O
O OH
1
5
O
O
7
6 Fig. 1. Structures of isolated compounds 1–7.
achieve a final volume of 200 μL with Alamar blue (10 μL of 10% solution) added to each well as indicator. After incubation at 28 °C for 48 h, the fluorescence intensity was measured at Ex/Em = 544/590 nm using a microtiter plate reader. The inhibition rates were calculated and plotted versus the test concentrations to afford the MICs, which were defined as the lowest concentration that completely inhibited the growth of the test organisms. 3. Results and discussion Pezizolide A (1) was assigned the molecular formula C14H18O5 (six degrees of unsaturation) on the basis of HRESIMS, which was further supported by 1H and 13C NMR data (Table 1). Analysis of its 1H and 13C data revealed the presence of one exchangeable proton (δH 5.01), two methyl groups, four methylenes (one of which was oxygenated), one O-methine, one oxygenated sp3 quaternary carbon, four olefinic/aromatic carbons (two of which were protonated), and two carboxylic carbons (δC 173.3 and 176.6, respectively).
The 1H-1H COSY NMR data of 1 displayed three isolated spin-systems of C-6–C-8, C-9–C-10 and C-12–C-13–OH-13. HMBC correlations from H-7 to C-3 and from H-6 to C-2, C-3 and C-4 indicated C-3 attached to C-2, C-4 and C-6. Meanwhile, HMBC crossing peaking from H-5 to C-2, C-3, C-4, C-6 and C-9, together with the chemical shift values C-2 (δC 173.3) and C-5 (δC 71.1) revealed the existence of an unsaturation γ-lactone ring. HMBC correlations from H-9 to C-3, C-4, C-5, C-10 and C-11, from H-10 to C-4, C-9, C-11, C-12 and C-15, and from H-15 to C-10, C-11 and C-12 suggested that C-10, C-12 and C-15 were all bonded to C-11. Coupling from H-12 to C-10, C-11, C-14 and C-15, meanwhile correlations from H-13 to C-11, C-12 and C-14 completed the substructure of C-10–C-14. Considering the chemical shift of C-11 (δC 83.4) and C-14 (δC 176.6), C-11 attached to C-14 through an oxygen atom to form another γ-lactone ring to satisfy the unsaturation requirement of 1. Collectively, these data permitted assignment of the planar structure of 1. The relative configuration of 1 was deduced by J-based configurational analysis and 1D NOE experiment. The coupling
a) (38.04%)
b) (33.93%)
c) (11.68%)
d) (7.05%)
e) (5.66%)
f) (3.64%)
Fig. 2. The optimized conformers for enantiomers 8a and 8b.
K. Zhang et al. / Fitoterapia 92 (2014) 79–84
Fig. 3. CD spectrum of 2–4 in MeOH.
constant of 15.7 Hz observed between H-6 and H-7 revealed their trans relationship. The NOESY correlation between H-10 and H-13 placed these protons on the same face of γ-lactone ring. The absolute configuration of C-13 was assigned by application of the CD exciton chirality method. The CD spectrum of 1 showed negative Cotton effects at 220 (Δε 6.35), suggesting the 11R absolute configuration for 1 [15]. Comparison of the experimental and simulated electronic circular dichroism (ECD) spectra generated by time-dependent density functional theory (TDDFT) [16] supported the above assignment. Since the unsaturated γ-lactone ring (C-2–C-10) had insignificant effect on the CD spectrum of 1, a simplified 8 was used for ECD calculations (Fig. 2). A systematic conformational analysis was performed for 1 by the Molecular Operating Environment (MOE) software package using the MMFF94 molecular mechanics force field calculation. The MMFF94 conformational search followed by reoptimization using TDDFT at the B3LYP/ 6-31G(d) basis set level afforded two lowest-energy conformers for 1 (Fig. 2). The overall calculated ECD spectra of 1 were then generated by Boltzmann weighing of the two conformers with 38.04% and 33.93% populations, by their relative free energies. The absolute configuration of 1 was then extrapolated by comparison of the experimental and calculated ECD spectra of 8a and 8b (Fig. 2). The experimental
83
CD spectrum of 1 was nearly identical to the calculated ECD spectrum of (3R, 5R)-8b, both showing negative Cotton effects (CEs) in the regions of ~220 nm. Therefore, the absolute configuration of 1 was deduced to be 11R, 13R. The molecular formula of 2 was suggested to be C14H18O5 (six degrees of unsaturation) by analysis of HRESIMS and NMR data (Table 1). Analysis of the 1H and 13C NMR data of 2 revealed the similar structural features found in 1; detailed comparison with 1 disclosed that the different chemical shift values of H-12 (δH 2.52), C-10 (δC 39.9), and C-15 (δC 25.6). Analysis of 1H-1H COSY and HMBC NMR data revealed that the planar structure of 2 was the same as compound 1. The NOESY correlation between H-13 and H-15 placed these protons on the same face of γ-lactone ring. The absolute configuration of 2 was deduced to be 11S, 13R via the rule relating the Cotton effect (Fig. 3) for the γ-lactone moiety. The elemental composition of 3 was determined to be C14H20O5 (five degrees of unsaturation) on the basis of HRESIMS and NMR data (Table 2), two more protons than 1. The complete proton and carbon resonance assignment was achieved by analysis of 1D NMR and HMQC data. By comparison of its NMR data with those of 1, we can easily figure out the double bond between H-6 and H-7 in compound 1 was hydrogenized. 1D NOE correlation between H-13 and H-10 indicated their same orientation. The absolute configuration of 3 was also deduced to be the same as that of 1 based on the Cotton effect observed at ~220 nm in their CD spectra (Fig. 3). Pezizolide D (4) was assigned the same molecular formula as 1 by HRESIMS analysis. 1D NMR and 2D NMR spectra data (Table 1) analyzing indicated the same structural characteristic as 1. The coupling constant between H-6 and H-7 was 11.3 Hz which revealed their cis relationship, comparing the 15.7 Hz in 1 and 2. The structure of 4 was proposed to be 11R, 13R, based on the 1D NOE correlation between H-13 and H-10 and negative Cotton effect at ~220 nm (Fig. 3). Compound 5 was assigned the molecular formula of C11H16O4 (four degrees of unsaturation) on the basis of HRESIMS. Interpretation of its NMR data (Table 2) showed the presence of an unsaturation γ-lactone ring structural features as 1. 1H-1H COSY correlation displayed two independent
Fig. 4. CD spectrum of 5a in DMSO.
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spin-systems C-6–C-8 and C-9–C-12. HMBC correlations from H-9 to C-3, C-4, C-5, C-10 and C-11 defined C-9 as attached to C-4. On the basis of these data, the planar structure of 5 was established. The relative configuration of 5 was assigned by the derivatization of acetone fork/cross. To determine the relative configuration of the 9,10-diol in 5, compound 5 (1.5 mg) was treated with 2,2-dimethyoxypropane (1 mL) and pyridinium p-toluene sulfonate (1 mg), then stirred at 30 °C for about 6 h under N2 circumstance. The reaction solution was evaporated in vacuo and purified by reversed-phase preparative HPLC to yield the acetonide 5a (1.0 mg) [17]. In the 1D NOE experiment of 5a, irradiation of H-9 and H-10, NOE effect was observed unambiguous for the acetonide methyl signal (δH 1.43) and (δH 1.46) respectively, which indicated a threo relationship for H-9 and H-10 in 5. To determine the absolute configuration, a transition metal chelate reagent dimolybdenum tetraacetate [Mo2(OAc)4] was employed, which induced the CD (ICD) spectra and showed a positive Cotton effect at 300 nm, suggesting the 9R and 10S absolute configuration [18,19] (Fig. 4). The molecular formula of 6 was determined by HRESIMS to be C11H14O2 (four degrees of unsaturation), 34 mass units less than 5. 1H and 13C NMR data of 6 have the same characteristics as 5, except two more protonated olefinic carbons appeared; meanwhile the signals of methine connected to an oxygen atom were absent. A coupling constant of 16.0 Hz between H-9 and H-10 revealed their trans relationship. On the basis of these data, the structure of 6 was proposed. Compound 7 was assigned the same molecular formula as 6 by HRESIMS analysis. Comparison of their 1H and 13C NMR spectra data suggested the similar structural features as 6. 1 H-1H COSY and HMBC correlation revealed that the double bond between C-9 and C-10 moved to C-10 and C-11. Compound 1, 2 and 4 possessed the same plain structure; they were stereoisomers; 3 had the same carbon skeleton with 1, but the double bond between C6 and C7 was hydrogenated. Although mono-furanone derivatives were common in natural products [20,21], bis-furanone derivatives were rarely separated in fungus fermentation. Compounds 1–7 were tested for cytotoxicity against the following five human tumor cell lines, HeLa (cervical epithelial cells), A549 (lung carcinoma epithelial cells), MCF-7 (breast cancer cells), HCT116 (colon cancer cells), and T24 (bladder cancer cells), and antimicrobial activity against
B. subtilis (ATCC 6633), S. aureus (CGMCC 1.2465) and C. albicans (CGMCC 2.2086). On the other hand, compounds 1–7 did not show detectable cytotoxicity and antimicrobial activity at 20 μg/mL. Acknowledgements This work was financially supported by grants from the National Natural Science Foundation of China (81001380) and the Ministry of Science and Technology of China (2012ZX9301002-003) and (2012ZX09301-003). References [1] Paranagama PA, Wijeratne EMK, Burns AM, Marron MT, Gunatilaka MK, Arnold AE, et al. J Nat Prod 2007;70:1700–5. [2] Arnold AE. Fungal Biol Rev 2007;21:51–66. [3] Wang Y, Niu S, Liu S, Guo L, Che Y. Org Lett 2010;12:5081–3. [4] Paranagama PA, Wijeratne EMK, Burns AM, Marron MT, Gunatilaka MK, Arnold AE, et al. J Nat Prod 2007;70:1700–5. [5] Li G, Wang H, Zhu R, Sun L, Wang L, Li M, et al. J Nat Prod 2012;75: 142–7. [6] Wu W, Dai H, Bao L, Ren B, Lu J, Luo Y, et al. J Nat Prod 2011;74:1303–8. [7] Deng L, Niu S, Liu X, Che Y, Li E. Fitoterapia 2013;89:8–14. [8] Wang Q, Bao L, Yang X, Guo H, Ren B, Guo L, et al. Fitoterapia 2013;85: 8–13. [9] He J, Chen G, Gao H, Yang F, Li X, Peng T, et al. Fitoterapia 2012;83: 1087–91. [10] Wang Q, Bao L, Yang X, Guo H, Ren B, Guo L, et al. Fitoterapia 2012;83: 209–14. [11] Deffieux G, Baute R, Baute MA, Atfani M, Carpy A. Phytochemistry 1987;26:1391–3. [12] Umino K, Takeda N, Ito Y, Okuda T. Chem Pharm Bull 1974;22:1233–8. [13] Zhang N, Chen Y, Jiang R, Li E, Chen X, Xi Z, et al. Jiang, X. Autophagy 2011;7:598–612. [14] NCCLS. NCCLS document M38-A. Wayne PA: NCCLS; 2002. [15] Beecham AF, Melbourne. Tetrahedron Lett 1968;32:3591–4. [16] (a) Diedrich C, Grimme S. J Phys Chem A 2003;107:2524–39. (b) Crawford TD, Tam MC, Abrams ML. J Phys Chem A 2003;111: 12057–68. (c) Stephens PJ, Devlin FJ, Gasparrini F, Ciogli A, Spinelli D, Cosimelli B. J Org Chem 2003;72:4707–15. (d) Ding Y, Li X, Ferreira D. J Org Chem 2003;72:9010–7. (e) Berova N, Bari LD, Pescitelli G. Chem Soc Rev 2003;36:904–31. (f) Bringmann G, Bruhn T, Maksimenka K, Hemberger Y. Eur J Org Chem 2003;17:2717–27. [17] McCulloch MWB, Bugni TS, Concepcion GP, Coombs GS, Harper MK, Kaur S, et al. J Nat Prod 2009;72:1651–6. [18] Bari LD, Pescitelli G, Pratelli C, Pini D, Salvadori P. J Org Chem 2001;66: 4819–25. [19] Li J, Wu X, Ding G, Feng Y, Jiang X, Guo L, et al. Eur J Org Chem 2012;12: 2445–552. [20] Hein SM, Gloer JB, Koster B, Malloch D. J Nat Prod 2001;64:809–12. [21] Eklund AM, Wahlberg I. Acta Chem Scand 1994;48:850–6.
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Mono- and bis-furanone derivatives from the endolichenic fungus Peziza sp. Kun Zhang a,b,1, Jinwei Ren a,1, Mei Ge b, Li Li c, Liangdong Guo a, Daijie Chen b,⁎, Yongsheng Che d,⁎ a b c d
State Key Laboratory of Mycology, Institute of Microbiology, Chinese Academy of Sciences, Beijing 100101, PR China Shanghai Normal University, Shanghai 200234, PR China Institute of Materia Medica, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing 100050, PR China Beijing Institute of Pharmacology & Toxicology, Beijing 100850, PR China
a r t i c l e
i n f o
Article history: Received 17 August 2013 Accepted in revised form 24 October 2013 Accepted 25 October 2013 Available online 1 November 2013 Keywords: Endolichenic fungus Peziza sp. Furanones Electronic circular dichroism
a b s t r a c t Seven new mono- and bis-furanones, pezizolides A–G (1–7), have been isolated from the crude extract of the endolichenic fungus Peziza sp. inhabiting the lichen Xanthoparmelia sp. The structures of the new compounds were elucidated mainly by NMR and MS methods. The absolute configuration of 1–4 was assigned by the application of CD exciton chirality method, and 1 was further supported by electronic circular dichroism (ECD) calculations, whereas that of 5 was deduced by Snatzke's method following the relative configuration analysis of its acetonide. The cytotoxity and antimicrobial activity of compounds 1–7 were tested. © 2013 Elsevier B.V. All rights reserved.
1. Introduction Lichens are symbiotic combinations of fungi (mycobiont) and cyanobacteria (algae) [1]. Except for fungal mycobiont and some non-obligate microfungi, endolichenic fungi are also living in the thalli of lichens [2–6]. Endolichenic fungi inhabiting in the lichen thalli were similar to endophytes living in the intercellular spaces of healthy plant tissues, but the chemical diversity of this class of fungi remained largely unexploited [7–10]. In our present work, the fungus Peziza sp. was initially found in the surface of Xanthoparmelia which was collected from the Zixi Mountain of Yunnan Province, People's Republic of China. Peziza species are macrofungi commonly called cup fungi. To date, only a few secondary metabolites isolated and identified from this genus [11,12]. Fractionation of an EtOAc extract prepared from a solid-substrate fermentation culture afforded seven new mono- (5–7) and bis-furanone (1–4) derivatives. Details of the isolation, structure ⁎ Corresponding authors. Tel.: +86 10 66932679. E-mail addresses: [email protected] (D. Chen), [email protected] (Y. Che). 1 Authors contributed equally to this work. 0367-326X/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.fitote.2013.10.011
elucidation, and biological activity of these compounds are reported herein. 2. Experimental method 2.1. General Optical rotations were measured on a Perkin-Elmer 241 polarimeter, and UV data were obtained on a Shimadzu Biospec-1601 spectrophotometer. CD spectra were recorded on a JASCO J-815 spectropolarimeter. IR data were recorded using a Nicolet Magna-IR 750 spectrophotometer. 1H and 13C NMR data were acquired with AVANCE III-500 and NMR system-600 spectrometers using solvent signals (acetone-d6: δH 2.05/δC 29.8, 206.1; CDCl3: δH 7.26/δC 77.2) as references. The HMQC and HMBC experiments were optimized for 145.0 and 8.0 Hz, respectively. HRESIMS data were obtained using an Agilent Accurate-Mass-Q-TOF LC/MS 6520 instrument equipped with an electrospray ionization (ESI) source. The fragmentor and capillary voltages were kept at 125 and 3500 V, respectively. Nitrogen was supplied as the nebulizing and drying gas. The temperature of the drying gas was set at 300 °C. The flow rate of
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chromatography (CC) using 1:1 CH2Cl2–MeOH as eluents, and the resulting subfractions were further purified by semipreparative RP HPLC (Agilent Zorbax SB-C18 column; 5 μm; 9.4 × 250 mm; 28% CH3CN in H2O over 40 min; 2 mL/min) to afford 1 (67.9 mg, tR 31.3 min), 2 (12.8 mg, tR 29.6 min), 3 (11.0 mg, tR 27.7 min) and 4 (1.7 mg, tR 21.3 min). The fractions (340 mg) eluted with 3:2 PE–EtOAc were separated by Sephadex LH-20 CC eluting with MeOH. The resulting subfractions were purified by RP HPLC (Agilent Zorbax SB-C18 column; 5 μm; 9.4 × 250 mm) to afford 5 (4.5 mg, tR 26.0 min; 55%–65% MeOH in H2O for 30 min), 6 and 7 (2.1 mg and 9.0 mg, tR 10.3 min and 10.9 min, respectively; 65% MeOH in H2O for 30 min). 5 (1.5 mg) was derived and purified by RP preparative HPLC to yield the acetonide 5a (1.0 mg, tR 23.6 min; 20%–90% CH3CN in H2O for 30 min).
the drying gas and the pressure of the nebulizer were 10 L/min and 10 psi, respectively. All MS experiments were performed in positive ion mode. Full-scan spectra were acquired over a scan range of m/z 100–1000 at 1.03 spectra/s. 2.2. Fungal material The culture of Pezizales sp. was isolated from the Xanthoparmelia sp. collected from the ZiXi Mountain of Yunnan Province, People's Republic of China, in October, 2010. The isolation was identified by Dr. Wenchao Li on the basis of morphology and sequence (GenBank accession No. KC493353) analysis of the ITS region of the rDNA. The fungal strain was cultured on slants of potato dextrose agar (PDA) at 25 °C for 10 days. Agar plugs were cut into small pieces (about 0.5 × 0.5 × 0.5 cm3) under aseptic conditions; 15 pieces were used to inoculate three Erlenmeyer flasks (250 mL), each containing 50 mL of media (0.4% glucose, 1% malt extract, and 0.4% yeast extract; the final pH of the media was adjusted to 6.5 and sterilized by autoclave). Three flasks of the inoculated media were incubated at 25 °C on a rotary shaker at 170 rpm for 5 days to prepare the seed culture. Fermentation was carried out in 12 Fernbach flasks (500 mL), each containing 80 g of rice. Distilled H2O (120 mL) was added to each flask, and the contents were soaked overnight before autoclaving at 15 psi for 30 min. After cooling to room temperature, each flask was inoculated with 5.0 mL of the spore inoculum and incubated at 25 °C for 40 days.
2.3.1. Compound 1 Light yellow oil, [α]25 D + 16.0 (c 0.1, MeOH), UV (MeOH) λmax (log ε) 216 (0.55), 251 (0.55) nm; CD (c 6.2 × 10–4 M, MeOH) λmax (Δε) 220 (–6.35); IR (neat) νmax 3432 (br), 2980, 2938, 2876, 1756, 1668, 1618, 1450, 1386, 1347, 1224, 1112, 1085, and 1038 cm–1; 1H, 13C NMR, and HMBC data (see Table 1); HRESIMS m/z 267.1227 [M + H]+ (calculated for C14H19O5, 267.1227). 2.3.2. Compound 2 Light yellow oil, [α]25 D + 5.3 (c 0.3, MeOH); UV (MeOH) λmax nm (log ε): 217 (0.36), 245 (0.21) nm; CD (c 6.2 × 10–4 M, MeOH) λmax (Δε) 225 (–3.37); IR (neat) νmax 3412 (br), 2976, 2938, 2876, 1755, 1666, 1618, 1542, 1488, 1448, 1387, 1303, 1210, 1148, 1116, 1079, and 1043 cm–1; 1H and 13C NMR data (see Table 1); HRESIMS m/z 267.1228 [M + H]+ (calculated for C14H19O5, 267.1227).
2.3. Extraction and isolation The fermented material was extracted repeatedly with EtOAc (4 × 250 mL) for each flask, and the organic solvent was evaporated to dryness under vacuum to afford the crude extract (30.0 g), which was fractionated by silica gel VLC using petroleum ether (PE)–EtOAc–MeOH gradient elution. The fractions (1.06 g) eluted with 1:1 and 1:4 PE–EtOAc were combined and separated again by Sephadex LH-20 column
2.3.3. Compound 3 Colorless oil, [α]25 D + 10.3 (c 0.3, MeOH), UV (MeOH) λmax nm (log ε): 217 (0.55) nm; CD (c 6.0 × 10–4 M, MeOH)
Table 1 NMR data for 1–4. 1 Pos.
δCa
2 3 4 5 6 7 8 9 10 11 12a 12b 13 14 15 OH-13
173.3 122.8 159.8 71.1 119.9 132.0 19.2 22.2 38.7 83.4 42.7
a b c
69.0 176.6 26.8
2 b
δH (J in Hz)
4.79 6.24 6.82 1.82 2.70 1.97
(s) (dd, 15.7, 1.5) (m) (d, 6.7) (overlap) (overlap)
HMBC
2, 2, 3, 6, 3, 4,
3, 3, 8 7 4, 9,
c
4, 9 4, 7, 8
5, 10, 11 11, 12, 15
2.64 (overlap) 2.01 (overlap) 4.62 (t, 8.5)
11, 12, 14
1.51 (s) 5.01 (d, 3.9)
10, 11, 12 12, 13
Recorded at 125 MHz in acetone-d6. Recorded at 500 MHz in acetone-d6. Recorded at 125 MHz in acetone-d6.
10, 11, 13, 14, 15
δCa 173.3 122.9 159.8 71.2 119.9 132.1 19.3 21.9 39.9 82.8 42.2 68.7 176.6 25.6
3 δHb
(J in Hz)
4.81 6.25 6.84 1.83 2.69 1.99
(s) (dd, 15.7, 1.5) (m) (d, 6.7) (m) (t, 8.6)
2.52 (dd, 12.8, 8.6) 2.08 (dd, 12.7, 9.8) 4.68 (t, 9.1) 1.45 (s) 4.98 (d, 5.3)
δCa 174.6 128.2 158.3 70.9 25.7 21.4 14.0 21.8 39.1 83.7 42.0 68.6 176.2 26.63
4 δHb
(J in Hz)
4.64 2.23 1.53 0.92 2.51 1.79
(s) (t, 7.4) (m) (t, 7.3) (overlap) (t, 8.5)
2.53 (overlap) 2.12 (dd,13.3, 8.4) 4.58 (t, 8.5) 1.56 (s) 2.79 (s)
δCa 172.9 125.3 159.7 71.2 117.5 133.8 15.8 22.8 38.1 83.6 41.9 68.6 176.1 26.63
δHb (J in Hz)
4.74 5.87 5.98 1.67 2.51 1.82
(s) (t, 11.2) (m) (d, 6.8) (overlap) (dd, 10.2, 6.5)
2.10 (dd,13.3, 8.4) 2.50 (overlap) 4.56 (t, 8.6) 1.53 (s) 2.69 (s)
K. Zhang et al. / Fitoterapia 92 (2014) 79–84
81
λmax (Δε) 222 (–4.75); IR (neat) νmax 3435 (br), 2962, 2936, 2873, 1748, 1669, 1455, 1386, 1348, 1294, 1262, 1221, 1177, 1137, 1113, 1079, and 1033 cm–1; 1H and 13C NMR data (see Table 1); HRESIMS m/z 269.1381 [M + H]+ (calculated for C14H21O5, 269.1384).
2.3.7. Compound 7 Colorless oil, UV (MeOH) λmax (log ε) 199 (1.53), 257 (0.33) nm, IR (neat) νmax 3436 (br), 2974, 2935, 2876, 1755, 1678, 1447, 1376, 1287, 1122, 1074, 1038, and 973 cm–1; 1H, 13 C NMR and HMBC data (see Table 2); HRESIMS m/z 179.1072 [M + H]+ (calculated for C11H15O2, 179.1067) (Fig. 1).
2.3.4. Compound 4 Colorless oil, [α]25 D + 9.2 (c 0.2, MeOH), UV (MeOH) λmax nm (log ε): 206 (0.58) nm; CD (c 6.2 × 10–4 M, MeOH) λmax (Δε) 222 (–5.68); IR (neat) νmax 3430 (br), 2979, 2938, 2873, 1755, 1667, 1638, 1619, 1450, 1413, 1386, 1353, 1293, 1262, 1226, 1131, 1094, and 1037 cm–1; 1H, 13C NMR data (see Table 1); HRESIMS m/z 267.1228 [M + H]+ (calculated for C14H19O5, 267.1227).
2.4. MTS assay [13] The assay was run in triplicate. In a 96-well plate, each well was plated with (2–5) × 103 cells (depending on the cell multiplication rate). After cell attachment overnight, the medium was removed, and each well was treated with 100 μL medium containing 0.1% DMSO, or appropriate concentrations of the test compounds and the positive control cisplatin (100 mM as stock solution of a compound in DMSO and serial dilutions; the test compounds showed good solubility in DMSO and did not precipitate when added to the cells). The plate was incubated for 48 h at 37 °C in a humidified, 5% CO2 atmosphere. Proliferation assessed by adding 20 μL of MTS (Promega) to each well in the dark, followed by a 90 min incubation at 37 °C. The assay plate was read at 490 nm using a microplate reader. The inhibition rate was calculated and plotted versus test concentrations to afford the IC50.
2.3.5. Compound 5 Light yellow oil, [α]25 D + 23.1 (c 0.2, MeOH), UV (MeOH) λmax nm (log ε): 211 (0.93), 253 (0.94) nm; CD spectrum of Mo2(OAc)4–complex subtracted (c 5.8 × 10–4 M, DMSO) λmax (Δε) 302 (+ 0.41); IR (neat) νmax 3419 (br), 2968, 2937, 2879, 1743, 1666, 1615, 1447, 1379, 1344, 1290, 1245, 1193, 1170, 1114, 1067, and 1044 cm–1; 1H, 13C NMR and HMBC data (see Table 2); HRESIMS m/z 235.0937 [M + Na]+ (calculated for C11H16O4Na, 235.0941). 5a 1H NMR (in CDCl3): 4.88 (H-5a, d, J = 15.2 Hz), 4.73 (H-5b, d, J = 15.2 Hz), 6.08 (H-6, m), 7.09 (H-7, m), 1.87 (H3-8, d, J = 5.6 Hz), 4.75 (H-9, d, J = 7.2 Hz), 3.75 (H-10, m), 1.66 (H2-11, m), 1.03 (H3-12, t, J = 6.2 Hz), 1.46 (H3-14, s), and 1.43 (H3-15, s). 1D NOE correlation (in CDCl3): H-9 → H3-15, H-10 → H3-14.
2.5. Antifungal assays Antifungal assays were conducted in triplicate following the National Center for Clinical Laboratory Standards (NCCLS) recommendations [14]. Bacillus subtilis (ATCC 6633), Staphylococcus aureus (CGMCC 1.2465) and Candida albicans (CGMCC 2.2086), were obtained from the China General Microbial Culture Collection (CGMCC) and were grown on PDA. Targeted fungi (3 or 4 colonies) were prepared from broth cultures incubated at 28 °C for 48 h, and the final suspensions contained 104 hyphae/mL (in PDB medium). Test samples (10 mg/mL as stock solution in DMSO and serial dilutions) were transferred to 96-well clear plates in triplicate, and the suspensions of the test organisms were added to each well to
2.3.6. Compound 6 Light yellow oil, UV (MeOH) λmax (log ε) 213 (1.84), 260 (2.02) nm; IR (neat) νmax 3435 (br), 2972, 2936, 2877, 1756, 1680, 1448, 1374, 1285, 1124, 1072, 1035, and 976 cm–1; 1H, 13 C NMR and HMBC data (see Table 2); HRESIMS m/z 179.1070 [M + H]+ (calculated for C11H15O2, 179.1067).
Table 2 NMR data for 5–7. 5 Pos.
δCa
2 3 4 5 6 7 8 9 10 11
173.2 122.7 161.0 70.4 120.3 132.5 19.3 71.0 76.1 26.9
12
10.8
a b c d e f
6 δHb
(J in Hz)
4.86 (s) 6.29 (d, 16.3) 6.87 (m) 1.82 (d, 6.6) 4.77 (d, 4.3) 3.58 (m) 1.59 (m) 1.50 (m) 0.97 (t, 7.4)
Recorded at 125 MHz in acetone-d6. Recorded at 500 MHz in acetone-d6. Recorded at 125 MHz in acetone-d6. Recorded at 125 MHz in CDCl3. Recorded at 500 MHz in CDCl3. Recorded at 125 MHz in CDCl3.
HMBC
2, 2, 3, 6, 3, 4, 9,
c
3, 4, 9 3, 7, 8 8 7 4, 5, 10, 11 11, 12 10, 12
10, 11
7
δC
d
173.4 120.7 150.7 68.6 118.6 133.3, 19.5 119.5 140.9 26.7 13.0
δHe
(J in Hz)
4.84 6.22 6.91 1.88 6.54 6.06 2.27
(s) (dd, 15.7, 1.6) (m) (d, 6.8) (d, 16.0) (m) (m)
1.09 (t, 7.5)
HMBC
2, 2, 3, 3, 3, 4, 9,
f
3, 4 4, 7, 8 8 6, 7 4, 5, 10, 11 11, 12 10, 12
10, 11
δCd
δHe (J in Hz)
HMBCf
173.3 122.8 156.8 70.7 118.4 132.8 19.3 30.2 124.6 129.4
4.63 6.08 6.83 1.85 3.15 5.38 5.57
2, 2, 3, 3, 3, 9, 9,
17.9
(s) (d, 14.6) (m) (d, 6.7) (d, 6.6) (m) (m)
1.68 (t, 6.4)
3, 4, 9 3, 8 8 6, 7 4, 5, 10, 11 11, 12 10, 12
10, 11
82
K. Zhang et al. / Fitoterapia 92 (2014) 79–84 8
7
13
11
O 14
15
3
4
9
O 2
12
10
O
OH
OH
OH 6
O
O
O
O
O
O O
O
5
O
1
3
1. 11S 2. 11R
4
8 6
7 3
OH 4
O 2
O
5
10
11
9
12
O
O OH
1
5
O
O
7
6 Fig. 1. Structures of isolated compounds 1–7.
achieve a final volume of 200 μL with Alamar blue (10 μL of 10% solution) added to each well as indicator. After incubation at 28 °C for 48 h, the fluorescence intensity was measured at Ex/Em = 544/590 nm using a microtiter plate reader. The inhibition rates were calculated and plotted versus the test concentrations to afford the MICs, which were defined as the lowest concentration that completely inhibited the growth of the test organisms. 3. Results and discussion Pezizolide A (1) was assigned the molecular formula C14H18O5 (six degrees of unsaturation) on the basis of HRESIMS, which was further supported by 1H and 13C NMR data (Table 1). Analysis of its 1H and 13C data revealed the presence of one exchangeable proton (δH 5.01), two methyl groups, four methylenes (one of which was oxygenated), one O-methine, one oxygenated sp3 quaternary carbon, four olefinic/aromatic carbons (two of which were protonated), and two carboxylic carbons (δC 173.3 and 176.6, respectively).
The 1H-1H COSY NMR data of 1 displayed three isolated spin-systems of C-6–C-8, C-9–C-10 and C-12–C-13–OH-13. HMBC correlations from H-7 to C-3 and from H-6 to C-2, C-3 and C-4 indicated C-3 attached to C-2, C-4 and C-6. Meanwhile, HMBC crossing peaking from H-5 to C-2, C-3, C-4, C-6 and C-9, together with the chemical shift values C-2 (δC 173.3) and C-5 (δC 71.1) revealed the existence of an unsaturation γ-lactone ring. HMBC correlations from H-9 to C-3, C-4, C-5, C-10 and C-11, from H-10 to C-4, C-9, C-11, C-12 and C-15, and from H-15 to C-10, C-11 and C-12 suggested that C-10, C-12 and C-15 were all bonded to C-11. Coupling from H-12 to C-10, C-11, C-14 and C-15, meanwhile correlations from H-13 to C-11, C-12 and C-14 completed the substructure of C-10–C-14. Considering the chemical shift of C-11 (δC 83.4) and C-14 (δC 176.6), C-11 attached to C-14 through an oxygen atom to form another γ-lactone ring to satisfy the unsaturation requirement of 1. Collectively, these data permitted assignment of the planar structure of 1. The relative configuration of 1 was deduced by J-based configurational analysis and 1D NOE experiment. The coupling
a) (38.04%)
b) (33.93%)
c) (11.68%)
d) (7.05%)
e) (5.66%)
f) (3.64%)
Fig. 2. The optimized conformers for enantiomers 8a and 8b.
K. Zhang et al. / Fitoterapia 92 (2014) 79–84
Fig. 3. CD spectrum of 2–4 in MeOH.
constant of 15.7 Hz observed between H-6 and H-7 revealed their trans relationship. The NOESY correlation between H-10 and H-13 placed these protons on the same face of γ-lactone ring. The absolute configuration of C-13 was assigned by application of the CD exciton chirality method. The CD spectrum of 1 showed negative Cotton effects at 220 (Δε 6.35), suggesting the 11R absolute configuration for 1 [15]. Comparison of the experimental and simulated electronic circular dichroism (ECD) spectra generated by time-dependent density functional theory (TDDFT) [16] supported the above assignment. Since the unsaturated γ-lactone ring (C-2–C-10) had insignificant effect on the CD spectrum of 1, a simplified 8 was used for ECD calculations (Fig. 2). A systematic conformational analysis was performed for 1 by the Molecular Operating Environment (MOE) software package using the MMFF94 molecular mechanics force field calculation. The MMFF94 conformational search followed by reoptimization using TDDFT at the B3LYP/ 6-31G(d) basis set level afforded two lowest-energy conformers for 1 (Fig. 2). The overall calculated ECD spectra of 1 were then generated by Boltzmann weighing of the two conformers with 38.04% and 33.93% populations, by their relative free energies. The absolute configuration of 1 was then extrapolated by comparison of the experimental and calculated ECD spectra of 8a and 8b (Fig. 2). The experimental
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CD spectrum of 1 was nearly identical to the calculated ECD spectrum of (3R, 5R)-8b, both showing negative Cotton effects (CEs) in the regions of ~220 nm. Therefore, the absolute configuration of 1 was deduced to be 11R, 13R. The molecular formula of 2 was suggested to be C14H18O5 (six degrees of unsaturation) by analysis of HRESIMS and NMR data (Table 1). Analysis of the 1H and 13C NMR data of 2 revealed the similar structural features found in 1; detailed comparison with 1 disclosed that the different chemical shift values of H-12 (δH 2.52), C-10 (δC 39.9), and C-15 (δC 25.6). Analysis of 1H-1H COSY and HMBC NMR data revealed that the planar structure of 2 was the same as compound 1. The NOESY correlation between H-13 and H-15 placed these protons on the same face of γ-lactone ring. The absolute configuration of 2 was deduced to be 11S, 13R via the rule relating the Cotton effect (Fig. 3) for the γ-lactone moiety. The elemental composition of 3 was determined to be C14H20O5 (five degrees of unsaturation) on the basis of HRESIMS and NMR data (Table 2), two more protons than 1. The complete proton and carbon resonance assignment was achieved by analysis of 1D NMR and HMQC data. By comparison of its NMR data with those of 1, we can easily figure out the double bond between H-6 and H-7 in compound 1 was hydrogenized. 1D NOE correlation between H-13 and H-10 indicated their same orientation. The absolute configuration of 3 was also deduced to be the same as that of 1 based on the Cotton effect observed at ~220 nm in their CD spectra (Fig. 3). Pezizolide D (4) was assigned the same molecular formula as 1 by HRESIMS analysis. 1D NMR and 2D NMR spectra data (Table 1) analyzing indicated the same structural characteristic as 1. The coupling constant between H-6 and H-7 was 11.3 Hz which revealed their cis relationship, comparing the 15.7 Hz in 1 and 2. The structure of 4 was proposed to be 11R, 13R, based on the 1D NOE correlation between H-13 and H-10 and negative Cotton effect at ~220 nm (Fig. 3). Compound 5 was assigned the molecular formula of C11H16O4 (four degrees of unsaturation) on the basis of HRESIMS. Interpretation of its NMR data (Table 2) showed the presence of an unsaturation γ-lactone ring structural features as 1. 1H-1H COSY correlation displayed two independent
Fig. 4. CD spectrum of 5a in DMSO.
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spin-systems C-6–C-8 and C-9–C-12. HMBC correlations from H-9 to C-3, C-4, C-5, C-10 and C-11 defined C-9 as attached to C-4. On the basis of these data, the planar structure of 5 was established. The relative configuration of 5 was assigned by the derivatization of acetone fork/cross. To determine the relative configuration of the 9,10-diol in 5, compound 5 (1.5 mg) was treated with 2,2-dimethyoxypropane (1 mL) and pyridinium p-toluene sulfonate (1 mg), then stirred at 30 °C for about 6 h under N2 circumstance. The reaction solution was evaporated in vacuo and purified by reversed-phase preparative HPLC to yield the acetonide 5a (1.0 mg) [17]. In the 1D NOE experiment of 5a, irradiation of H-9 and H-10, NOE effect was observed unambiguous for the acetonide methyl signal (δH 1.43) and (δH 1.46) respectively, which indicated a threo relationship for H-9 and H-10 in 5. To determine the absolute configuration, a transition metal chelate reagent dimolybdenum tetraacetate [Mo2(OAc)4] was employed, which induced the CD (ICD) spectra and showed a positive Cotton effect at 300 nm, suggesting the 9R and 10S absolute configuration [18,19] (Fig. 4). The molecular formula of 6 was determined by HRESIMS to be C11H14O2 (four degrees of unsaturation), 34 mass units less than 5. 1H and 13C NMR data of 6 have the same characteristics as 5, except two more protonated olefinic carbons appeared; meanwhile the signals of methine connected to an oxygen atom were absent. A coupling constant of 16.0 Hz between H-9 and H-10 revealed their trans relationship. On the basis of these data, the structure of 6 was proposed. Compound 7 was assigned the same molecular formula as 6 by HRESIMS analysis. Comparison of their 1H and 13C NMR spectra data suggested the similar structural features as 6. 1 H-1H COSY and HMBC correlation revealed that the double bond between C-9 and C-10 moved to C-10 and C-11. Compound 1, 2 and 4 possessed the same plain structure; they were stereoisomers; 3 had the same carbon skeleton with 1, but the double bond between C6 and C7 was hydrogenated. Although mono-furanone derivatives were common in natural products [20,21], bis-furanone derivatives were rarely separated in fungus fermentation. Compounds 1–7 were tested for cytotoxicity against the following five human tumor cell lines, HeLa (cervical epithelial cells), A549 (lung carcinoma epithelial cells), MCF-7 (breast cancer cells), HCT116 (colon cancer cells), and T24 (bladder cancer cells), and antimicrobial activity against
B. subtilis (ATCC 6633), S. aureus (CGMCC 1.2465) and C. albicans (CGMCC 2.2086). On the other hand, compounds 1–7 did not show detectable cytotoxicity and antimicrobial activity at 20 μg/mL. Acknowledgements This work was financially supported by grants from the National Natural Science Foundation of China (81001380) and the Ministry of Science and Technology of China (2012ZX9301002-003) and (2012ZX09301-003). References [1] Paranagama PA, Wijeratne EMK, Burns AM, Marron MT, Gunatilaka MK, Arnold AE, et al. J Nat Prod 2007;70:1700–5. [2] Arnold AE. Fungal Biol Rev 2007;21:51–66. [3] Wang Y, Niu S, Liu S, Guo L, Che Y. Org Lett 2010;12:5081–3. [4] Paranagama PA, Wijeratne EMK, Burns AM, Marron MT, Gunatilaka MK, Arnold AE, et al. J Nat Prod 2007;70:1700–5. [5] Li G, Wang H, Zhu R, Sun L, Wang L, Li M, et al. J Nat Prod 2012;75: 142–7. [6] Wu W, Dai H, Bao L, Ren B, Lu J, Luo Y, et al. J Nat Prod 2011;74:1303–8. [7] Deng L, Niu S, Liu X, Che Y, Li E. Fitoterapia 2013;89:8–14. [8] Wang Q, Bao L, Yang X, Guo H, Ren B, Guo L, et al. Fitoterapia 2013;85: 8–13. [9] He J, Chen G, Gao H, Yang F, Li X, Peng T, et al. Fitoterapia 2012;83: 1087–91. [10] Wang Q, Bao L, Yang X, Guo H, Ren B, Guo L, et al. Fitoterapia 2012;83: 209–14. [11] Deffieux G, Baute R, Baute MA, Atfani M, Carpy A. Phytochemistry 1987;26:1391–3. [12] Umino K, Takeda N, Ito Y, Okuda T. Chem Pharm Bull 1974;22:1233–8. [13] Zhang N, Chen Y, Jiang R, Li E, Chen X, Xi Z, et al. Jiang, X. Autophagy 2011;7:598–612. [14] NCCLS. NCCLS document M38-A. Wayne PA: NCCLS; 2002. [15] Beecham AF, Melbourne. Tetrahedron Lett 1968;32:3591–4. [16] (a) Diedrich C, Grimme S. J Phys Chem A 2003;107:2524–39. (b) Crawford TD, Tam MC, Abrams ML. J Phys Chem A 2003;111: 12057–68. (c) Stephens PJ, Devlin FJ, Gasparrini F, Ciogli A, Spinelli D, Cosimelli B. J Org Chem 2003;72:4707–15. (d) Ding Y, Li X, Ferreira D. J Org Chem 2003;72:9010–7. (e) Berova N, Bari LD, Pescitelli G. Chem Soc Rev 2003;36:904–31. (f) Bringmann G, Bruhn T, Maksimenka K, Hemberger Y. Eur J Org Chem 2003;17:2717–27. [17] McCulloch MWB, Bugni TS, Concepcion GP, Coombs GS, Harper MK, Kaur S, et al. J Nat Prod 2009;72:1651–6. [18] Bari LD, Pescitelli G, Pratelli C, Pini D, Salvadori P. J Org Chem 2001;66: 4819–25. [19] Li J, Wu X, Ding G, Feng Y, Jiang X, Guo L, et al. Eur J Org Chem 2012;12: 2445–552. [20] Hein SM, Gloer JB, Koster B, Malloch D. J Nat Prod 2001;64:809–12. [21] Eklund AM, Wahlberg I. Acta Chem Scand 1994;48:850–6.
