Fitoterapia 103 (2015) 106–112
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Fitoterapia journal homepage: www.elsevier.com/locate/fitote
Four new tetramic acid and one new furanone derivatives from the plant endophytic fungus Neopestalotiopsis sp. Shasha Zhao a,d, Shenxi Chen a,d, Bo Wang b, Shubin Niu a,d, Wenping Wu c, Liangdong Guo a, Yongsheng Che b,⁎ a
State Key Laboratory of Mycology, Institute of Microbiology, Chinese Academy of Sciences, Beijing 100190, PR China State Key Laboratory of Toxicology & Medical Countermeasures, Beijing Institute of Pharmacology & Toxicology, AMMS, Beijing 100850, PR China c Novozymes (China) Investment Co. Ltd China Headquarters, 14 Xinli Road, Shangdi Zone, Haidian District, Beijing 100085, PR China d University of Chinese Academy of Sciences, Beijing 100049, PR China b
a r t i c l e
i n f o
Article history: Received 13 February 2015 Accepted in revised form 20 March 2015 Accepted 22 March 2015 Available online 27 March 2015 Chemical compound: Equisetin (PubChem CID: 54684703) Keywords: Neopestalotiopsis sp. Structure elucidation Tetramic acid Furanone Antibacterial activity
a b s t r a c t Four new tetramic acid analogues neopestalotins A–D (1–4), one new furanone derivative neopestalotin E (6), and the known compound hymenosetin have been isolated from the solid cultures of the plant endophytic fungus Neopestalotiopsis sp. The structures of the new compounds were determined mainly by nuclear magnetic resonance (NMR) experiments. The absolute configurations of 1 and 2 were assigned by circular dichroism (CD) data, whereas those of 3 and 4 were deduced by a combination of CD and heteronuclear long range coupling (HETLOC) data. Compound 2 showed modest antimicrobial activity against the Gram-positive bacteria, Bacillus subtilis, Staphylococcus aureus col, and Streptococcus pneumoniae. © 2015 Elsevier B.V. All rights reserved.
1. Introduction The plant endophytic fungi are microorganisms that live in normal tissues and organs of plants without causing apparent symptoms [1], which have been considered as an outstanding source of new bioactive secondary metabolites [2–4]. As a genus of the most widely distributed endophytic fungi, Pestalotiopsis spp. have been demonstrated to produce a variety of bioactive natural products [5–9]. The genus of Neopestalotiospsis was originally grouped to Pestalotiopsis, and was re-classified as a separate genus based on morphological and DNA data in 2014 [10]. Tetramic acids are a group of structurally unique natural products that have attracted much attention of natural products and organic chemists [11–16]. In the course of our continuing ⁎ Corresponding author. Tel.: +86 10 66932679; fax: +86 10 66931600. E-mail address: [email protected] (Y. Che).
http://dx.doi.org/10.1016/j.fitote.2015.03.023 0367-326X/© 2015 Elsevier B.V. All rights reserved.
efforts to find new bioactive secondary metabolites, a strain of Neopestalotiopsis sp. was subjected to chemical investigation. Bioassay-directed fractionation of the EtOAc extract prepared from the solid-substrate fermentation products resulted in the isolation of four new dacalin-tetramic acid (1–4) and one new furanone derivatives (6), which we named neopestalotins A–E (1–4 and 6), along with the known compound hymenosetin (5). Details of the isolation, structure elucidation, and antimicrobial activity of the new metabolites are reported herein. 2. Experimental 2.1. General Optical rotations were measured on a Rodolph Research Analytical Automatic Polarimeter, and UV data were obtained on a Shimadzu Biospec-1601 spectrophotometer. CD spectra
S. Zhao et al. / Fitoterapia 103 (2015) 106–112
were recorded on a JASCO J-815 spectropolarimeter using MeOH as solvent. IR data were recorded using a Nicolet MagnaIR 750 spectrophotometer. 1H and 13C NMR data were acquired with Bruker-500 spectrometer using solvent signals (CDCl3: δH 7.26/δC 77.2 ppm) as references. The HMQC and HMBC experiments were optimized for 145.0 and 8.0 Hz, respectively. ESIMS and 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 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 a rate of 1.03 spectra/s. HPLC separations were performed on an Agilent 1260 instrument (Agilent, USA) equipped with a variable-wavelength UV detector. 2.2. Fungal material The fungus was identified as Neopestalotiopsis sp. by one of the authors (L.G.) based on morphology and sequence (GenBank Accession No. KP745600) 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 cm × 0.5 cm) under aseptic conditions; 15 pieces were used to inoculate three Erlenmeyer flasks (250 mL), each containing 50 mL of medium (0.4% glucose, 1% malt extract, and 0.4% yeast extract; the final pH of the medium was adjusted to 6.5 and the mixture sterilized with an autoclave). Three flasks of the inoculated medium were incubated at 25 °C on a rotary shaker at 170 rpm for 5 days to prepare the seed culture. Fermentation was conducted 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 being autoclaved 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.
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silica gel and Sephadex LH-20 CC eluting with MeOH, and the resulting subfractions were further purified by semipreparative RPHPLC (73% MeOH in H2O with 0.1% formic acid for 30 min; 2 mL/min) to afford 3 (7.8 mg; tR 27.6 min). The fraction (250 mg) eluted with 70% EtOAc was separated by Sephadex LH-20 CC eluting with MeOH, and subsequent purification of this fraction by the same HPLC column (50% MeOH in H2O with 0.1% formic acid for 2 min, followed by 50–70% MeOH over 20 min; 2 mL/min) to afford 6 (0.8 mg; tR 15.2 min). Compound 6 was resolved by HPLC using a chiral column (CHIRALPAK AD-H column; 4.6 × 250 mm; 10% isopropanol in n-hexanes for 12 min; 1 mL/min) to afford two enantiomers, 6a (0.4 mg; tR 6.9 min) and 6b (0.4 mg; tR 7.4 min).
2.3.1. Neopestalotin A (1) pale yellow powder; [α]25D −304 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 274 (2.68) nm; CD (c 2.6 × 10−3 M, MeOH) λmax (Δε) 214 (−20.38), 250 (−2.13), 273 (−4.09) nm; IR (neat): νmax 3363 (br), 2964, 2932, 2917, 1689, 1602, 1565, 1451, 1376 cm−1; 1H, 13C NMR, and HMBC data see Table 1; ROESY correlations (CDCl3, 500 MHz) H-6 ↔ H3-12 and H3-17; H-3 ↔ H3-12 and H-14; H-13 ↔ H-11 and H3-15; NH ↔ H3-7′; HRESIMS m/z 386.2328 [M + H]+ (calcd. for C23H32NO4, 386.2326).
2.3. Extraction and isolation
2.3.2. Neopestalotin B (2) pale yellow powder; [α]25D −260 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 274 (2.62) nm; CD (c 2.3 × 10−3 M, MeOH) λmax (Δε) 209 (−2.32), 250(−0.18), 272 (−0.36) nm; IR (neat): νmax 3193 (br), 2964, 2919, 2853, 1741, 1689, 1605, 1561, 1451, 1245 cm−1; 1H and 13C NMR data see Table 1; HMBC correlations (CDCl3, 500 MHz) H-3 → C-2, 4, 5, 11, 12, 13, 14, 16; H-5 → C-3, 6, 7, 11, 16; H-6 → C-8, 11; H2-7 → C-5, 6, 8, 9, 17; H-8 → C-7, 9, 17; H2-9 → C-8, 10, 11, 17; H2-10 → C-8, 9, 11; H11 → C-2, 6, 7, 10, 12; H3-12 → C-1, 2, 3, 11; H-13 → C- 15; H-14 → C-3, 15; H3-15 → C-13, 14; H3-16 → C-3, 4, 5; H2-17 → C-7, 8, 9, 18; H3-19 → C-18; H-6′ → C-4′, 5′, 7′; H37′ → C-4′, 5′, 6′; NH → C-3′, 4′, 5′; ROESY correlations (CDCl3, 500 MHz) H-6 ↔ H3-12 and H2-17; H-3 ↔ H3-12 and H-14; H-13 ↔ H-11, H3-15; H-11 ↔ H-8; NH ↔ H3-7′; HRESIMS m/z 428.2433 [M + H]+ (calcd. for C25H34NO5, 428.2431).
The fermented material was extracted with EtOAc (4 × 1.0 L), and the organic solvent was evaporated to dryness under vacuum to afford a crude extract (15 g), which was fractionated by silica gel VLC using petroleum ether-EtOAc gradient elution. The fraction (400 mg) eluted with 35% EtOAc was separated by Sephadex LH-20 column chromatography (CC) eluting with MeOH. The resulting subfractions were purified using semipreparative RPHPLC (Agilent Zorbax SB-C18 column; 5 μm; 9.4 × 250 mm; 80% MeOH in H2O with 0.1% formic acid for 25 min; followed by 90% MeOH over 40 min; 2 mL/min) to afford 1 (13.0 mg, tR 21.7 min), 2 (9.1 mg; tR 35.2 min) and 5 (2.5 mg, tR 39.8 min). The fraction (570 mg) eluted with 50% EtOAc was separated by Sephadex LH-20 CC eluting with MeOH, and the resulting subfractions were further purified by the same HPLC column (90% MeOH in H2O with 0.1% formic acid for 30 min; 2 mL/min) to afford 4 (9.1 mg, tR 13.1 min). The fraction (1.0 g) eluted with 90% EtOAc was separated by
2.3.3. Neopestalotin C (3) pale yellow oil; [α]25D −238 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 249 (2.36), 287 (2.50) nm; CD (c 2.5 × 10−3 M, MeOH) λmax (Δε) 206 (−23.95), 273 (−5.57) nm; IR (neat): νmax 3364 (br), 2967, 2933, 2917, 1657, 1568, 1452, 1376 cm−1; 1H and 13C NMR data see Table 2; HMBC correlations (CDCl3, 500 MHz) H-3 → C-4, 5, 11, 12, 13, 14, 16; H-5 → C-3, 6, 7, 11, 16; H-6 → C-4, 5, 7, 11; H2-7 → C-6, 8, 9, 11; H2-9 → C-11; H2-10 → C-9, 11; H-11 → C-2, 12; H3-12 → C1, 2, 3, 11; H-13 → C-15; H-14 → C-3, 15; H3-15 → C-13, 14; H3-16 → C-3, 4, 5; H2-17 → C-7, 8, 9; H-5′ → C-2′, 4′, 6′, 7′; H-6′ → C-4′, 7′; H3-7′ → C-5′, 6′; NH → C-3′, 4′, 5′; ROESY correlations (CDCl3, 500 MHz) H-6 ↔ H3-12 and H2-17; H-3 ↔ H3-12 and H-14; H-13 ↔ H-11, H3-15; NH ↔ H3-7′; HRESIMS m/z 404.2434 [M + H]+ (calcd. for C23H34NO5, 404.2431).
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S. Zhao et al. / Fitoterapia 103 (2015) 106–112
Table 1 NMR data for 1–2. 1
2 a
Pos
δC , mult.
1 2 3 4 5 6 7
206.3, qC 51.4, qC 49.1, CH 132.8, qC 125.1, CH 34.2, CH 46.4, CH2
8 9 10
70.3, qC 39.4, CH2 23.5, CH2
11 12 13 14 15 16 17 18 19 2′ 3′ 4′ 5′ 6′ 7′ NH
40.0, CH 14.2, CH3 130.9, CH 127.9, CH 18.0, CH3 22.4, CH3 31.7, CH3
a b
167.4, qC 105.1, qC 180.6, qC 132.4, qC 110.0, CH 12.8, CH3
δHb
(J in Hz)
HMBC
a
δCa, mult.
3.48, d (9.5)
2, 4, 5, 11, 12, 13, 14, 16
5.12, s 2.22, m 1.77, m 1.29, m
3, 6, 7, 11, 16 4, 5, 7, 11 6, 8, 9, 11 5, 6, 9, 11
1.71, m 1.85, m 1.31, m 1.65, m 1.47, s 5.10, m 5.30, m 1.53, d (6.0) 1.59, s 1.25, s
8, 10, 11 8, 9, 11 6, 11 2, 6, 12 1, 2, 3, 11 15 3, 15 13, 14 3, 4, 5 7, 8, 9
5.86, m 1.94, d (7.5) 8.53, br
4′, 5′, 7′ 5′, 6′ 3′, 4′, 5′
206.2, qC 51.4, qC 49.1, CH 132.7, qC 125.2, CH 38.6, CH 36.9, CH2 37.8, CH 30.1, CH2 27.2, CH2 40.0, CH 14.2, CH3 130.9, CH 128.0, CH 18.0, CH3 22.4, CH3 69.6, CH2 171.5, qC 21.1, CH3 167.5, qC 105.1, qC 180.6, qC 132.5, qC 110.2, CH 12.8, CH3
δHb (J in Hz)
3.47, d (9.5) 5.18, s 1.81, m 1.89, m 0.95, m 1.79, m 1.85, m; 1.23,m 2.08, m 1.00, m 1.68, m 1.44, s 5.10, m 5.30, m 1.53, d (6.0) 1.58, s 3.93, d (6.0) 2.06, s
5.86, m 1.94, d (7.5) 9.13, br
Recorded at 125 MHz in CDCl3. Recorded at 500 MHz in CDCl3.
2.3.4. Neopestalotin D (4) pale yellow oil; [α]25D −224 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 249 (2.34), 287 (2.48) nm; CD (c 2.2 × 10−3 M, MeOH) λmax (Δε) 217 (−4.52), 267 (−2.68) nm; IR (neat): νmax 3296 (br), 2969, 2920, 2854, 1740, 1716, 1656, 1566, 1451, 1375, 1246 cm−1; 1H and 13C NMR data see Table 2; HMBC correlations (CDCl3, 500 MHz) H-3 → C-2, 4, 5, 11, 12, 13, 14, 16; H-5 → C-3, 6, 7, 11, 16; H-6 → C-4, 5, 9, 11; H2-7 → C-6, 8, 9, 11, 17; H-8 → C-7, 17; H2-9 → C-7, 10, 11, 17; H2-10 → C-6, 8, 9, 11; H-11 → C-2, 6, 10, 12; H3-12 → C-1, 2, 3, 11; H-13 → C-15; H-14 → C-3, 15; H3-15 → C-13, 14; H3-16 → C-3, 4, 5; H2-17 → C-7, 8, 9, 18; H3-19 → C-18; H-5′ → C-2′, 4′, 7′; H-6′ → C-4′, 7′; H3-7′ → C-5′, 6′; NH → C-3′, 4′, 5′; ROESY correlations (CDCl3, 500 MHz) H-6 ↔ H3-12 and H2-17; H-3 ↔ H3-12 and H-14; H13 ↔ H-11, H3-15; H-11 ↔ H-8; NH ↔ H3-7′; HRESIMS m/z 446.2533 [M + H]+ (calcd. for C25H36NO6, 446.2537). 2.3.5. Neopestalotin E (6) (E)-3-Methyl-5-oxo-5-((2,4,5-trimethyl-3-oxo-2,3dihydrofuran-2-yl)amino) pent-3-en-1-yl acetate (6): colorless oil; [α]25D −6.0 (c 0.1, MeOH) (6a [α]25D −30.0 (c 0.04, MeOH), 6b [α]25D +37.5 (c 0.04, MeOH)); UV (MeOH) λmax (log ε) 271 (2.43) nm; IR (neat): νmax 3310 (br), 3405, 2964, 2931, 1739, 1631, 1268, 1240 cm−1; 1H and 13C NMR data see Table 2; HMBC correlations (CDCl3, 500 MHz) H-6 → C-5, 8, 12; H2-8 → C-6, 7, 9, 12; H2-9 → C-7, 8, 10; H3-11 → C-10; H3-12 → C-6, 7, 8; H3-13 → C-1, 2, 3; H3-14 → C-2, 3; H3-15 → C-1, 4, 5; NH → C-5; ROESY correlations (CDCl3,
500 MHz) H-6 ↔ H-8, H2-9 ↔ H3-12; HRESIMS m/z 296.1495 [M + H]+ (calcd. for C15H21NO5, 296.1492). 2.4. Antibacterial assays Antibacterial bioassays were conducted in triplicate by following the National Center for Clinical Laboratory Standards (NCCLS) recommendations [17]. The bacterial strains Bacillus subtilis (ATCC 6633), Staphylococcus aureus col (CGMCC 1.2465), Streptococus pneumoniae (CGMCC 1.1692), and Escherichia coli (CGMCC 1.2340) were grown on Mueller–Hinton agar. Targeted microbes (3–4 colonies) were prepared from broth culture (37 °C for 24 h), and the final spore suspensions of bacteria in MHB medium, were 106 cells/mL. Test samples (10 mg/mL as stock solution in DMSO and serial dilutions) were transferred to a 96-well clear plate in triplicate, and the suspension of the test organisms was added to each well, achieving a final volume of 200 μL (ampicillin) was used as the positive control for the Gram-positive bacteria, and gentamycin for the Gramnegative bacteria. After incubation, the absorbance at 595 nm was measured with a microplate reader (TECAN). The MIC was defined as the lowest test concentration that completely inhibited the growth of the test organisms. 3. Results and discussion The molecular formula of neopestalotin A (1) was established as C23H31NO4 (nine degrees of unsaturation) on the basis of high resolution electrospray ionization mass
S. Zhao et al. / Fitoterapia 103 (2015) 106–112
109
Table 2 NMR data for 3, 4, and 6. Pos
3
4 a
δC , mult. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 2′ 3′ 4′ 5′ 6′ 7′ NH a b
200.9, qC 49.7, qC 49.5, CH 132.6, qC 125.1, CH 34.1, CH 46.4, CH2 70.4, qC 39.8, CH2 24.0, CH2 39.9, CH 13.8, CH3 130.5, CH 127.8, CH 17.9, CH3 22.3, CH3 31.7, CH3
179.0, qC 100.1, qC 191.8, qC 64.8, CH 68.5, CH 18.2, CH3
δHb
(J in Hz)
3.10, d (9.5) 5.12, s 2.23, m 1.76, m 1.30, m 1.70, m 1.82, m 1.38, m 1.64, m 1.45, s 5.09, m 5.23, m 1.54, d (6.0) 1.60, s 1.25, s
3.78, d (5.5) 4.05, m 1.19, d (6.0) 6.32, br
6 a
δC , mult. 200.8, qC 49.6, qC 49.5, CH 132.5, qC 125.1, CH 38.5, CH 36.8, CH2 37.8, CH 30.1, CH2 27.7, CH2 39.9, CH 13.6, CH3 130.4, CH 127.8, CH 17.8, CH3 22.2, CH3 69.5, CH2 171.5, qC 21.1, CH3 179.4, qC 100.0, qC 191.8, qC 64.8, CH 68.4, CH 18.2, CH3
δHb
(J in Hz)
3.07, d (9.5) 5.16, s 1.82, m 1.89, m 0.95, m 1.79, m 1.85, m; 1.24, m 2.03, m 1.07, m 1.70, m 1.42, s 5.12, m 5.22, m 1.52, d (6.0) 1.59, s 3.91, d (6.0)
δCa, mult. 201.3, qC 109.0, qC 181.4, qC 88.9, qC 164.9, qC 118.1, CH 153.9, qC 39.6, CH2 61.8, CH2 171.1, qC 21.1, CH3 18.7, CH3 6.1, CH3 14.9, CH3 23.0, CH3
δHb (J in Hz)
5.58, s 2.40, t (6.5) 4.18, t (6.5) 2.04, s 2.12, s 1.75, s 2.16, s 1.51, s
2.06, s
3.78, d (5.0) 4.04, m 1.18, d (6.0) 6.34, br
6.00, br
Recorded at 125 MHz in CDCl3. Recorded at 500 MHz in CDCl3.
spectrometry (HRESIMS) (m/z 386.2328 [M + H]+; Δ −0.2 mmu). Analysis of its 1H and 13C NMR nuclear magnetic resonance (NMR) data (Table 1) revealed one amide proton (δH 8.53), five methyl groups, three methylenes, three methines, two sp3 quaternary carbons with one oxygenated, eight olefinic carbons (four of which were protonated), and two carbonyls (δC 167.4 and 180.6, respectively). These data accounted for all the NMR resonances of 1 except two exchangeable protons and suggested that it was a tricyclic compound. Interpretation of the 1 H–1H COSY NMR data of 1 revealed the presence of three isolated spin systems of C-6′–C-7′, C-3–C-13–C-15, and C-5–C-7, with the C-11–C-9 fragment attached to C-6 (Fig. 2). In the
HMBC spectrum of 1, correlations from H3-16 to C-3, C-4, and C-5 indicated that C-4 is connected to both C-3 and C-5. HMBC correlations from H3-17 to C-7, C-8, and C-9 led to the connections of C-7 and C-9 to C-8. In turn, correlations from H3-12 to C-2, C-3, C-11, and C-1 indicated that C-1, C-3, C-11, and C-12 are all connected to C-2, completing the decalin moiety. Further HMBC correlations from H-7′ to C-5′ and from H-6′ to C-4′ and C-5′ connected both C-4′ and C-6′ to C-5′. HMBC cross peaks from the amide proton to C-3′, C-4′, and C-5′ established a pyrrolidine-2,4-dione unit. Considering the chemical shift values for C-1 (δC 206.3) and C-3′ (δC 105.1), as well as the unsaturation requirement of 1, the two olefinic carbons should be directed
Fig. 1. Structures of compounds 1–6.
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S. Zhao et al. / Fitoterapia 103 (2015) 106–112
Fig. 2. Key 1H–1H COSY (
), HMBC (
connected to each other. Therefore, the planner structure of 1 was established as shown in Fig. 1. The relative configuration of compound 1 was proposed by analysis of its ROESY data. ROESY correlation of amide proton with H3-7′ established a Z configuration of the C-5′/C-6′ double band, whereas those of H-3 with H-14 and of H-13 with H3-15 revealed an E configuration for the C-13/C-14 double band on the propenyl side chain. The E configuration of the C-1/C-3′ olefin was assigned based on the chemical shift values for C-2′ (δC 167.4) and C-3′ (δC 105.1) [11]. Further ROESY correlations of H-6 with H3-12 and H3-17, and of H3-12 with H-3 placed these protons on the same face of the decalin ring. Finally, a ROESY correlation of H-11 with H-13 placed these two protons on the opposite face of the ring system. On the basis of these data, the relative configuration of neopestalotin A (1) was established. The absolute configuration of neopestalotin A (1) was deduced by comparison of its CD spectrum with a model compound equisetin [18]. The CD spectrum of 1 showed three negative Cotton effects at 214, 250, and 273 nm, respectively, similar to those observed for equisetin. Combining the relative configuration established above, the 2S, 3R, 6S, 8R, 11R absolute configuration was deduced for 1. Neopestalotin B (2) was given the molecular formula C25H33NO5 (10 degrees of unsaturation) on the basis of its HRESIMS data (m/z 428.2433 [M + H]+; Δ −0.2 mmu), which is 42 mass units more than 1. The 1H and 13C NMR spectra of 2 revealed the presence of the same pyrrolidine-2,4-dione and decalin moieties as found in 1, except that the C-8 methyl group (δH/δC 1.25/31.7) was replaced by the acetylated hydroxymethyl unit (δH/δC 3.93/69.6; 2.06/21.1) and the hydroxyl group on C-8 was absent, which was confirmed by the chemical shift of C-8 (δC 37.8) and HMBC correlations from H2-17 to C-7, C-8, C-9, and C-18, and from H3-19 to C-18, suggesting that 2 is an analogue of 1. The relative configuration of 2 was deduced on the basis of ROESY data. ROESY correlations of H-6 with H3-12 and H2-17, and of H3-12 with H-3 placed these protons on the same face of the two six-membered rings, whereas that of ROESY correlations H-8 with H-11 placed these protons on the other face of the ring system, thereby allowing deduction of the relative configuration for 2. The absolute configuration of 2 was also determined by comparison the CD data with those of 1 and the known compound equisetin. Since the CD spectrum of 2 was
), and ROESY (
) correlations of 1 and 4.
nearly identical to that of 1, the 2S, 3R, 6S, 8S, 11R absolute configuration was proposed for neopestalotin B (2). Neopestalotin C (3) gave a pseudomolecular ion [M + H]+ peak at m/z 404.2434 (Δ − 0.3 mmu) by HRESIMS, consistent with the molecular formula C23H33NO5 (eight degrees of unsaturation). The 1H and 13C NMR data (Table 2) of neopestalotin C (3) are similar with those of neopestalotin A (1), except that the C-5′–C-6′ olefin was replaced by two methine units including one O-methine in 3, which was verified by relevant 1H–1H COSY and HMBC correlations and the high-field chemical shifts of C-5′ (δH/δC 3.78/64.8) and C-6′ (δH/δC 4.05/68.5). The relative configuration for the decalin in 3 was proposed to be the same as in 1 based on ROESY data. However, the Z configuration of the C-1/C-3′ olefin was established based on the chemical shifts of C-2′ (δC 179.4) and C-3′ (δC 100.1) [11], which were different from those in 1 and 2. ROESY correlation of the amide proton with H3-7′ revealed their proximity in space. The small coupling constant of 5.5 Hz observed for H-5′/H-6′ suggested a gauche-like configuration for H-5′ and H-6′. In addition, J-based configuration analysis is a proven strategy to determine the relative configuration for adjacent asymmetric centers, such as the C-5′–C-7′ side chain in 3–5 [19]. Therefore, the relative configurations of C-5′ and C-6′ can be represented by six staggered rotamers [19,20]. The small coupling constants of 2.64 and 0.84 Hz observed for 2 J (H-5′, C-6′) and 2 J (H-6′, C-5′), respectively, suggested the anti-like configurations between H-5′/OH-6′ and H-6′/NH. Collectively, the relative configurations of C-5′ and C-6′ were determined. However, effort to assign the absolute configuration of C-6′ in 3 and 4 using the modified Mosher's method was unsuccessful. Eventually, the CD spectrum of 3 (Fig. S13) showed negative Cotton effects at 230 and 280 nm, correlating with the 2S, 5′S absolute configuration [19]. Therefore, the 2S, 3R, 6S, 8R, 11R, 5′S, 6′R absolute configuration was proposed for 3. The elemental composition of neopestalotin D (4) was determined to be C25H35NO6 (nine degrees of unsaturation) by HRESIMS (m/z 446.2533 [M + H]+; Δ +0.4 mmu). The 1H and 13 C NMR data (Table 2) of 4 closely resembled to those of neopestalosetin C (3), except that the C-8 methyl group (δH/δC 1.25/31.7) was replaced by the acetylated hydroxymethyl unit (δH/δC 3.91/69.5; 2.06/21.1) and the OH-8 was absent. The high-field chemical shift of C-8 (δC 37.8) in the 13C NMR spectrum together with HMBC correlations from H2-17 to C-7,
S. Zhao et al. / Fitoterapia 103 (2015) 106–112
C-8, C-9, and C-18, and from H3-19 to C-18 supported the planar structure of 4. The relative configurations of the decalin and tetramic acid moieties in 4 were deduced to be the same as those in 2 and 3 respectively by analysis of the ROESY data and coupling constants. In addition, a strong ROESY correlation of the amide proton with H3-7′ and the small coupling constants of 5.0, 0.36, and 1.14 Hz observed for 3 J (H-5′, H-6′), 2 J (H-5′, C-6′), and 2 J (H-6′, C-5′), respectively, were similar to those found in 3, indicating that C-5′ and C-6′ has the same relative configuration in both compounds. Since the CD spectrum of 4 (Fig. S14) showed negative Cotton effects at 230 and 280 nm, the 2S, 3R, 6S, 8S, 11R, 5′S, 6′R absolute configuration was deduced for 4. Compound 5 was identified as hymenosetin, a second metabolite isolated from the ash dieback pathogen Hymenoscyphus pseudoalbidus, by comparison of its NMR and MS data with literature values [19]. Compound 6 was assigned a molecular formula of C15H21NO4 (six degrees of unsaturation) on the basis of the HRESIMS (m/z 296.1495 [M + H]+; Δ −0.3 mmu). Interpretation of the 1H and 13C NMR data (Table 2) revealed one amide proton (δH 6.00), five methyl groups, two methylenes (one of which is oxygenated), four olefinic carbons with one protonated, one sp3 quaternary carbons and three carbonyls. In addition, NMR resonances corresponding to an acetyl group (δH/δC 2.04/ 21.1, 171.1) was observed, which was supported by HMBC correlations from H2-9 (δH/δC 4.18/ 61.8) to the carboxylic carbon at 171.1 ppm. The HMBC correlations from H3-12 to C-6, C-7, and C-8, indicated that C-6, C-8, and C-12 were all attached to C-7. Correlations from H2-9 and H2-8 to C-7 led to the connection of C-8 and C-9. Further HMBC correlations from H-6 and amide proton to C-5 indicated that C-6 and NH are both connected to C-5, completing the same 5-amino-3methyl-5-oxopent-3-enyl acetate moiety as pestalotiopamide A [21]. Additional HMBC correlations from H3-13 to C-1, C-2, and C-3, and from H3-14 to C-2, and C-3 located both methyl groups (δH/δC 1.75/6.1; 2.16/14.9) at C-2 and C-3, respectively. HMBC cross peaks from H3-15 to C-1, C-4, and C-5 connected C-15 and NH to C-4. Considering the chemical shift of C-3 (δC 181.4) and C-4 (δC 88.9), and the unsaturation of 6, C-3 and C-4 should be attached to the remaining oxygen atom to form the 2,4,5-trimethylfuran-3-one moiety. On the basis of these data, the planar structure of 6 was established. The ROESY correlations of H-6 with H2-8, and of H3-12 with H2-9 established the E configuration of the double bond. However, the small measured specific rotation value of 6 ([α]25D −6.0, c 0.1, MeOH) imply that 6 may be a racemate. Subsequently, 6 was resolved by HPLC using a chiral column to afford two enantiomers, 6a and 6b (Fig. S15), and the absolute configurations of 6a (4S) and 6b (4R) were assigned by comparison of their specific rotation values ([α]25D −30.0 and + 37.5, respectively, c 0.04, MeOH) to that ([α]20D −13.6, c 0.18, CHCl3) of the model compound, (S)-5-Hexyl-5-methyl2,4(3H,5H)-furandione [22]. Compounds 1–4 were evaluated for antibacterial activity against B. subtilis (ATCC 6633), S. aureus col (CGMCC 1.2465), S. pneumoniae (CGMCC 1.1692), and E. coli (CGMCC 1.2340). Compound 2 showed inhibitory effect against B. subtilis, S. aureus col, and S. pneumoniae, with MIC values of 10, 20, and 20 μg/mL, respectively (the positive control ampicillin showed MIC values of 1.25, 0.16, and 10 μg/mL, respectively).
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Neopestalotins A–D (1–4) are new members of the 3decalinoyltetramic acid family of the natural products, which have been reported with various biological activities [23]. To our knowledge, compound 1 is the first dacalin-type tetramic acid derivative with a C-5′/C-6′ olefin unit. In addition, the presence of C-8 hydroxyl group is also rare in this class of metabolites, with pallidorosetin A as the only precedent [24]. Compound 2 possesses the same core structure as 1, but the substituents at C-8 were replaced by an acetylated hydroxymethyl unit. Compounds 3 and 4 differed from the known compound hymenosetin [19] by having different substituents at C-8. Compound 6 is a derivative of the known compound pestalotiopamide A [20], but differs in having a 2,4,5-trimethylfuran-3-one connected at the amide proton. The discovery of these compounds further demonstrated that the plant endophytic fungi are a valuable source for new bioactive natural products. Conflict of interest The authors declare no conflict interest. Acknowledgments We gratefully acknowledge financial support from the National Program of Drug Research and Development (2012ZX09301-003). Appendix A. Supplementary data Supplementary data to this article can be found online at http://dx.doi.org/10.1016/j.fitote.2015.03.023. Reference [1] [2] [3] [4] [5] [6]
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Contents lists available at ScienceDirect
Fitoterapia journal homepage: www.elsevier.com/locate/fitote
Four new tetramic acid and one new furanone derivatives from the plant endophytic fungus Neopestalotiopsis sp. Shasha Zhao a,d, Shenxi Chen a,d, Bo Wang b, Shubin Niu a,d, Wenping Wu c, Liangdong Guo a, Yongsheng Che b,⁎ a
State Key Laboratory of Mycology, Institute of Microbiology, Chinese Academy of Sciences, Beijing 100190, PR China State Key Laboratory of Toxicology & Medical Countermeasures, Beijing Institute of Pharmacology & Toxicology, AMMS, Beijing 100850, PR China c Novozymes (China) Investment Co. Ltd China Headquarters, 14 Xinli Road, Shangdi Zone, Haidian District, Beijing 100085, PR China d University of Chinese Academy of Sciences, Beijing 100049, PR China b
a r t i c l e
i n f o
Article history: Received 13 February 2015 Accepted in revised form 20 March 2015 Accepted 22 March 2015 Available online 27 March 2015 Chemical compound: Equisetin (PubChem CID: 54684703) Keywords: Neopestalotiopsis sp. Structure elucidation Tetramic acid Furanone Antibacterial activity
a b s t r a c t Four new tetramic acid analogues neopestalotins A–D (1–4), one new furanone derivative neopestalotin E (6), and the known compound hymenosetin have been isolated from the solid cultures of the plant endophytic fungus Neopestalotiopsis sp. The structures of the new compounds were determined mainly by nuclear magnetic resonance (NMR) experiments. The absolute configurations of 1 and 2 were assigned by circular dichroism (CD) data, whereas those of 3 and 4 were deduced by a combination of CD and heteronuclear long range coupling (HETLOC) data. Compound 2 showed modest antimicrobial activity against the Gram-positive bacteria, Bacillus subtilis, Staphylococcus aureus col, and Streptococcus pneumoniae. © 2015 Elsevier B.V. All rights reserved.
1. Introduction The plant endophytic fungi are microorganisms that live in normal tissues and organs of plants without causing apparent symptoms [1], which have been considered as an outstanding source of new bioactive secondary metabolites [2–4]. As a genus of the most widely distributed endophytic fungi, Pestalotiopsis spp. have been demonstrated to produce a variety of bioactive natural products [5–9]. The genus of Neopestalotiospsis was originally grouped to Pestalotiopsis, and was re-classified as a separate genus based on morphological and DNA data in 2014 [10]. Tetramic acids are a group of structurally unique natural products that have attracted much attention of natural products and organic chemists [11–16]. In the course of our continuing ⁎ Corresponding author. Tel.: +86 10 66932679; fax: +86 10 66931600. E-mail address: [email protected] (Y. Che).
http://dx.doi.org/10.1016/j.fitote.2015.03.023 0367-326X/© 2015 Elsevier B.V. All rights reserved.
efforts to find new bioactive secondary metabolites, a strain of Neopestalotiopsis sp. was subjected to chemical investigation. Bioassay-directed fractionation of the EtOAc extract prepared from the solid-substrate fermentation products resulted in the isolation of four new dacalin-tetramic acid (1–4) and one new furanone derivatives (6), which we named neopestalotins A–E (1–4 and 6), along with the known compound hymenosetin (5). Details of the isolation, structure elucidation, and antimicrobial activity of the new metabolites are reported herein. 2. Experimental 2.1. General Optical rotations were measured on a Rodolph Research Analytical Automatic Polarimeter, and UV data were obtained on a Shimadzu Biospec-1601 spectrophotometer. CD spectra
S. Zhao et al. / Fitoterapia 103 (2015) 106–112
were recorded on a JASCO J-815 spectropolarimeter using MeOH as solvent. IR data were recorded using a Nicolet MagnaIR 750 spectrophotometer. 1H and 13C NMR data were acquired with Bruker-500 spectrometer using solvent signals (CDCl3: δH 7.26/δC 77.2 ppm) as references. The HMQC and HMBC experiments were optimized for 145.0 and 8.0 Hz, respectively. ESIMS and 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 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 a rate of 1.03 spectra/s. HPLC separations were performed on an Agilent 1260 instrument (Agilent, USA) equipped with a variable-wavelength UV detector. 2.2. Fungal material The fungus was identified as Neopestalotiopsis sp. by one of the authors (L.G.) based on morphology and sequence (GenBank Accession No. KP745600) 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 cm × 0.5 cm) under aseptic conditions; 15 pieces were used to inoculate three Erlenmeyer flasks (250 mL), each containing 50 mL of medium (0.4% glucose, 1% malt extract, and 0.4% yeast extract; the final pH of the medium was adjusted to 6.5 and the mixture sterilized with an autoclave). Three flasks of the inoculated medium were incubated at 25 °C on a rotary shaker at 170 rpm for 5 days to prepare the seed culture. Fermentation was conducted 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 being autoclaved 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.
107
silica gel and Sephadex LH-20 CC eluting with MeOH, and the resulting subfractions were further purified by semipreparative RPHPLC (73% MeOH in H2O with 0.1% formic acid for 30 min; 2 mL/min) to afford 3 (7.8 mg; tR 27.6 min). The fraction (250 mg) eluted with 70% EtOAc was separated by Sephadex LH-20 CC eluting with MeOH, and subsequent purification of this fraction by the same HPLC column (50% MeOH in H2O with 0.1% formic acid for 2 min, followed by 50–70% MeOH over 20 min; 2 mL/min) to afford 6 (0.8 mg; tR 15.2 min). Compound 6 was resolved by HPLC using a chiral column (CHIRALPAK AD-H column; 4.6 × 250 mm; 10% isopropanol in n-hexanes for 12 min; 1 mL/min) to afford two enantiomers, 6a (0.4 mg; tR 6.9 min) and 6b (0.4 mg; tR 7.4 min).
2.3.1. Neopestalotin A (1) pale yellow powder; [α]25D −304 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 274 (2.68) nm; CD (c 2.6 × 10−3 M, MeOH) λmax (Δε) 214 (−20.38), 250 (−2.13), 273 (−4.09) nm; IR (neat): νmax 3363 (br), 2964, 2932, 2917, 1689, 1602, 1565, 1451, 1376 cm−1; 1H, 13C NMR, and HMBC data see Table 1; ROESY correlations (CDCl3, 500 MHz) H-6 ↔ H3-12 and H3-17; H-3 ↔ H3-12 and H-14; H-13 ↔ H-11 and H3-15; NH ↔ H3-7′; HRESIMS m/z 386.2328 [M + H]+ (calcd. for C23H32NO4, 386.2326).
2.3. Extraction and isolation
2.3.2. Neopestalotin B (2) pale yellow powder; [α]25D −260 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 274 (2.62) nm; CD (c 2.3 × 10−3 M, MeOH) λmax (Δε) 209 (−2.32), 250(−0.18), 272 (−0.36) nm; IR (neat): νmax 3193 (br), 2964, 2919, 2853, 1741, 1689, 1605, 1561, 1451, 1245 cm−1; 1H and 13C NMR data see Table 1; HMBC correlations (CDCl3, 500 MHz) H-3 → C-2, 4, 5, 11, 12, 13, 14, 16; H-5 → C-3, 6, 7, 11, 16; H-6 → C-8, 11; H2-7 → C-5, 6, 8, 9, 17; H-8 → C-7, 9, 17; H2-9 → C-8, 10, 11, 17; H2-10 → C-8, 9, 11; H11 → C-2, 6, 7, 10, 12; H3-12 → C-1, 2, 3, 11; H-13 → C- 15; H-14 → C-3, 15; H3-15 → C-13, 14; H3-16 → C-3, 4, 5; H2-17 → C-7, 8, 9, 18; H3-19 → C-18; H-6′ → C-4′, 5′, 7′; H37′ → C-4′, 5′, 6′; NH → C-3′, 4′, 5′; ROESY correlations (CDCl3, 500 MHz) H-6 ↔ H3-12 and H2-17; H-3 ↔ H3-12 and H-14; H-13 ↔ H-11, H3-15; H-11 ↔ H-8; NH ↔ H3-7′; HRESIMS m/z 428.2433 [M + H]+ (calcd. for C25H34NO5, 428.2431).
The fermented material was extracted with EtOAc (4 × 1.0 L), and the organic solvent was evaporated to dryness under vacuum to afford a crude extract (15 g), which was fractionated by silica gel VLC using petroleum ether-EtOAc gradient elution. The fraction (400 mg) eluted with 35% EtOAc was separated by Sephadex LH-20 column chromatography (CC) eluting with MeOH. The resulting subfractions were purified using semipreparative RPHPLC (Agilent Zorbax SB-C18 column; 5 μm; 9.4 × 250 mm; 80% MeOH in H2O with 0.1% formic acid for 25 min; followed by 90% MeOH over 40 min; 2 mL/min) to afford 1 (13.0 mg, tR 21.7 min), 2 (9.1 mg; tR 35.2 min) and 5 (2.5 mg, tR 39.8 min). The fraction (570 mg) eluted with 50% EtOAc was separated by Sephadex LH-20 CC eluting with MeOH, and the resulting subfractions were further purified by the same HPLC column (90% MeOH in H2O with 0.1% formic acid for 30 min; 2 mL/min) to afford 4 (9.1 mg, tR 13.1 min). The fraction (1.0 g) eluted with 90% EtOAc was separated by
2.3.3. Neopestalotin C (3) pale yellow oil; [α]25D −238 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 249 (2.36), 287 (2.50) nm; CD (c 2.5 × 10−3 M, MeOH) λmax (Δε) 206 (−23.95), 273 (−5.57) nm; IR (neat): νmax 3364 (br), 2967, 2933, 2917, 1657, 1568, 1452, 1376 cm−1; 1H and 13C NMR data see Table 2; HMBC correlations (CDCl3, 500 MHz) H-3 → C-4, 5, 11, 12, 13, 14, 16; H-5 → C-3, 6, 7, 11, 16; H-6 → C-4, 5, 7, 11; H2-7 → C-6, 8, 9, 11; H2-9 → C-11; H2-10 → C-9, 11; H-11 → C-2, 12; H3-12 → C1, 2, 3, 11; H-13 → C-15; H-14 → C-3, 15; H3-15 → C-13, 14; H3-16 → C-3, 4, 5; H2-17 → C-7, 8, 9; H-5′ → C-2′, 4′, 6′, 7′; H-6′ → C-4′, 7′; H3-7′ → C-5′, 6′; NH → C-3′, 4′, 5′; ROESY correlations (CDCl3, 500 MHz) H-6 ↔ H3-12 and H2-17; H-3 ↔ H3-12 and H-14; H-13 ↔ H-11, H3-15; NH ↔ H3-7′; HRESIMS m/z 404.2434 [M + H]+ (calcd. for C23H34NO5, 404.2431).
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Table 1 NMR data for 1–2. 1
2 a
Pos
δC , mult.
1 2 3 4 5 6 7
206.3, qC 51.4, qC 49.1, CH 132.8, qC 125.1, CH 34.2, CH 46.4, CH2
8 9 10
70.3, qC 39.4, CH2 23.5, CH2
11 12 13 14 15 16 17 18 19 2′ 3′ 4′ 5′ 6′ 7′ NH
40.0, CH 14.2, CH3 130.9, CH 127.9, CH 18.0, CH3 22.4, CH3 31.7, CH3
a b
167.4, qC 105.1, qC 180.6, qC 132.4, qC 110.0, CH 12.8, CH3
δHb
(J in Hz)
HMBC
a
δCa, mult.
3.48, d (9.5)
2, 4, 5, 11, 12, 13, 14, 16
5.12, s 2.22, m 1.77, m 1.29, m
3, 6, 7, 11, 16 4, 5, 7, 11 6, 8, 9, 11 5, 6, 9, 11
1.71, m 1.85, m 1.31, m 1.65, m 1.47, s 5.10, m 5.30, m 1.53, d (6.0) 1.59, s 1.25, s
8, 10, 11 8, 9, 11 6, 11 2, 6, 12 1, 2, 3, 11 15 3, 15 13, 14 3, 4, 5 7, 8, 9
5.86, m 1.94, d (7.5) 8.53, br
4′, 5′, 7′ 5′, 6′ 3′, 4′, 5′
206.2, qC 51.4, qC 49.1, CH 132.7, qC 125.2, CH 38.6, CH 36.9, CH2 37.8, CH 30.1, CH2 27.2, CH2 40.0, CH 14.2, CH3 130.9, CH 128.0, CH 18.0, CH3 22.4, CH3 69.6, CH2 171.5, qC 21.1, CH3 167.5, qC 105.1, qC 180.6, qC 132.5, qC 110.2, CH 12.8, CH3
δHb (J in Hz)
3.47, d (9.5) 5.18, s 1.81, m 1.89, m 0.95, m 1.79, m 1.85, m; 1.23,m 2.08, m 1.00, m 1.68, m 1.44, s 5.10, m 5.30, m 1.53, d (6.0) 1.58, s 3.93, d (6.0) 2.06, s
5.86, m 1.94, d (7.5) 9.13, br
Recorded at 125 MHz in CDCl3. Recorded at 500 MHz in CDCl3.
2.3.4. Neopestalotin D (4) pale yellow oil; [α]25D −224 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 249 (2.34), 287 (2.48) nm; CD (c 2.2 × 10−3 M, MeOH) λmax (Δε) 217 (−4.52), 267 (−2.68) nm; IR (neat): νmax 3296 (br), 2969, 2920, 2854, 1740, 1716, 1656, 1566, 1451, 1375, 1246 cm−1; 1H and 13C NMR data see Table 2; HMBC correlations (CDCl3, 500 MHz) H-3 → C-2, 4, 5, 11, 12, 13, 14, 16; H-5 → C-3, 6, 7, 11, 16; H-6 → C-4, 5, 9, 11; H2-7 → C-6, 8, 9, 11, 17; H-8 → C-7, 17; H2-9 → C-7, 10, 11, 17; H2-10 → C-6, 8, 9, 11; H-11 → C-2, 6, 10, 12; H3-12 → C-1, 2, 3, 11; H-13 → C-15; H-14 → C-3, 15; H3-15 → C-13, 14; H3-16 → C-3, 4, 5; H2-17 → C-7, 8, 9, 18; H3-19 → C-18; H-5′ → C-2′, 4′, 7′; H-6′ → C-4′, 7′; H3-7′ → C-5′, 6′; NH → C-3′, 4′, 5′; ROESY correlations (CDCl3, 500 MHz) H-6 ↔ H3-12 and H2-17; H-3 ↔ H3-12 and H-14; H13 ↔ H-11, H3-15; H-11 ↔ H-8; NH ↔ H3-7′; HRESIMS m/z 446.2533 [M + H]+ (calcd. for C25H36NO6, 446.2537). 2.3.5. Neopestalotin E (6) (E)-3-Methyl-5-oxo-5-((2,4,5-trimethyl-3-oxo-2,3dihydrofuran-2-yl)amino) pent-3-en-1-yl acetate (6): colorless oil; [α]25D −6.0 (c 0.1, MeOH) (6a [α]25D −30.0 (c 0.04, MeOH), 6b [α]25D +37.5 (c 0.04, MeOH)); UV (MeOH) λmax (log ε) 271 (2.43) nm; IR (neat): νmax 3310 (br), 3405, 2964, 2931, 1739, 1631, 1268, 1240 cm−1; 1H and 13C NMR data see Table 2; HMBC correlations (CDCl3, 500 MHz) H-6 → C-5, 8, 12; H2-8 → C-6, 7, 9, 12; H2-9 → C-7, 8, 10; H3-11 → C-10; H3-12 → C-6, 7, 8; H3-13 → C-1, 2, 3; H3-14 → C-2, 3; H3-15 → C-1, 4, 5; NH → C-5; ROESY correlations (CDCl3,
500 MHz) H-6 ↔ H-8, H2-9 ↔ H3-12; HRESIMS m/z 296.1495 [M + H]+ (calcd. for C15H21NO5, 296.1492). 2.4. Antibacterial assays Antibacterial bioassays were conducted in triplicate by following the National Center for Clinical Laboratory Standards (NCCLS) recommendations [17]. The bacterial strains Bacillus subtilis (ATCC 6633), Staphylococcus aureus col (CGMCC 1.2465), Streptococus pneumoniae (CGMCC 1.1692), and Escherichia coli (CGMCC 1.2340) were grown on Mueller–Hinton agar. Targeted microbes (3–4 colonies) were prepared from broth culture (37 °C for 24 h), and the final spore suspensions of bacteria in MHB medium, were 106 cells/mL. Test samples (10 mg/mL as stock solution in DMSO and serial dilutions) were transferred to a 96-well clear plate in triplicate, and the suspension of the test organisms was added to each well, achieving a final volume of 200 μL (ampicillin) was used as the positive control for the Gram-positive bacteria, and gentamycin for the Gramnegative bacteria. After incubation, the absorbance at 595 nm was measured with a microplate reader (TECAN). The MIC was defined as the lowest test concentration that completely inhibited the growth of the test organisms. 3. Results and discussion The molecular formula of neopestalotin A (1) was established as C23H31NO4 (nine degrees of unsaturation) on the basis of high resolution electrospray ionization mass
S. Zhao et al. / Fitoterapia 103 (2015) 106–112
109
Table 2 NMR data for 3, 4, and 6. Pos
3
4 a
δC , mult. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 2′ 3′ 4′ 5′ 6′ 7′ NH a b
200.9, qC 49.7, qC 49.5, CH 132.6, qC 125.1, CH 34.1, CH 46.4, CH2 70.4, qC 39.8, CH2 24.0, CH2 39.9, CH 13.8, CH3 130.5, CH 127.8, CH 17.9, CH3 22.3, CH3 31.7, CH3
179.0, qC 100.1, qC 191.8, qC 64.8, CH 68.5, CH 18.2, CH3
δHb
(J in Hz)
3.10, d (9.5) 5.12, s 2.23, m 1.76, m 1.30, m 1.70, m 1.82, m 1.38, m 1.64, m 1.45, s 5.09, m 5.23, m 1.54, d (6.0) 1.60, s 1.25, s
3.78, d (5.5) 4.05, m 1.19, d (6.0) 6.32, br
6 a
δC , mult. 200.8, qC 49.6, qC 49.5, CH 132.5, qC 125.1, CH 38.5, CH 36.8, CH2 37.8, CH 30.1, CH2 27.7, CH2 39.9, CH 13.6, CH3 130.4, CH 127.8, CH 17.8, CH3 22.2, CH3 69.5, CH2 171.5, qC 21.1, CH3 179.4, qC 100.0, qC 191.8, qC 64.8, CH 68.4, CH 18.2, CH3
δHb
(J in Hz)
3.07, d (9.5) 5.16, s 1.82, m 1.89, m 0.95, m 1.79, m 1.85, m; 1.24, m 2.03, m 1.07, m 1.70, m 1.42, s 5.12, m 5.22, m 1.52, d (6.0) 1.59, s 3.91, d (6.0)
δCa, mult. 201.3, qC 109.0, qC 181.4, qC 88.9, qC 164.9, qC 118.1, CH 153.9, qC 39.6, CH2 61.8, CH2 171.1, qC 21.1, CH3 18.7, CH3 6.1, CH3 14.9, CH3 23.0, CH3
δHb (J in Hz)
5.58, s 2.40, t (6.5) 4.18, t (6.5) 2.04, s 2.12, s 1.75, s 2.16, s 1.51, s
2.06, s
3.78, d (5.0) 4.04, m 1.18, d (6.0) 6.34, br
6.00, br
Recorded at 125 MHz in CDCl3. Recorded at 500 MHz in CDCl3.
spectrometry (HRESIMS) (m/z 386.2328 [M + H]+; Δ −0.2 mmu). Analysis of its 1H and 13C NMR nuclear magnetic resonance (NMR) data (Table 1) revealed one amide proton (δH 8.53), five methyl groups, three methylenes, three methines, two sp3 quaternary carbons with one oxygenated, eight olefinic carbons (four of which were protonated), and two carbonyls (δC 167.4 and 180.6, respectively). These data accounted for all the NMR resonances of 1 except two exchangeable protons and suggested that it was a tricyclic compound. Interpretation of the 1 H–1H COSY NMR data of 1 revealed the presence of three isolated spin systems of C-6′–C-7′, C-3–C-13–C-15, and C-5–C-7, with the C-11–C-9 fragment attached to C-6 (Fig. 2). In the
HMBC spectrum of 1, correlations from H3-16 to C-3, C-4, and C-5 indicated that C-4 is connected to both C-3 and C-5. HMBC correlations from H3-17 to C-7, C-8, and C-9 led to the connections of C-7 and C-9 to C-8. In turn, correlations from H3-12 to C-2, C-3, C-11, and C-1 indicated that C-1, C-3, C-11, and C-12 are all connected to C-2, completing the decalin moiety. Further HMBC correlations from H-7′ to C-5′ and from H-6′ to C-4′ and C-5′ connected both C-4′ and C-6′ to C-5′. HMBC cross peaks from the amide proton to C-3′, C-4′, and C-5′ established a pyrrolidine-2,4-dione unit. Considering the chemical shift values for C-1 (δC 206.3) and C-3′ (δC 105.1), as well as the unsaturation requirement of 1, the two olefinic carbons should be directed
Fig. 1. Structures of compounds 1–6.
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S. Zhao et al. / Fitoterapia 103 (2015) 106–112
Fig. 2. Key 1H–1H COSY (
), HMBC (
connected to each other. Therefore, the planner structure of 1 was established as shown in Fig. 1. The relative configuration of compound 1 was proposed by analysis of its ROESY data. ROESY correlation of amide proton with H3-7′ established a Z configuration of the C-5′/C-6′ double band, whereas those of H-3 with H-14 and of H-13 with H3-15 revealed an E configuration for the C-13/C-14 double band on the propenyl side chain. The E configuration of the C-1/C-3′ olefin was assigned based on the chemical shift values for C-2′ (δC 167.4) and C-3′ (δC 105.1) [11]. Further ROESY correlations of H-6 with H3-12 and H3-17, and of H3-12 with H-3 placed these protons on the same face of the decalin ring. Finally, a ROESY correlation of H-11 with H-13 placed these two protons on the opposite face of the ring system. On the basis of these data, the relative configuration of neopestalotin A (1) was established. The absolute configuration of neopestalotin A (1) was deduced by comparison of its CD spectrum with a model compound equisetin [18]. The CD spectrum of 1 showed three negative Cotton effects at 214, 250, and 273 nm, respectively, similar to those observed for equisetin. Combining the relative configuration established above, the 2S, 3R, 6S, 8R, 11R absolute configuration was deduced for 1. Neopestalotin B (2) was given the molecular formula C25H33NO5 (10 degrees of unsaturation) on the basis of its HRESIMS data (m/z 428.2433 [M + H]+; Δ −0.2 mmu), which is 42 mass units more than 1. The 1H and 13C NMR spectra of 2 revealed the presence of the same pyrrolidine-2,4-dione and decalin moieties as found in 1, except that the C-8 methyl group (δH/δC 1.25/31.7) was replaced by the acetylated hydroxymethyl unit (δH/δC 3.93/69.6; 2.06/21.1) and the hydroxyl group on C-8 was absent, which was confirmed by the chemical shift of C-8 (δC 37.8) and HMBC correlations from H2-17 to C-7, C-8, C-9, and C-18, and from H3-19 to C-18, suggesting that 2 is an analogue of 1. The relative configuration of 2 was deduced on the basis of ROESY data. ROESY correlations of H-6 with H3-12 and H2-17, and of H3-12 with H-3 placed these protons on the same face of the two six-membered rings, whereas that of ROESY correlations H-8 with H-11 placed these protons on the other face of the ring system, thereby allowing deduction of the relative configuration for 2. The absolute configuration of 2 was also determined by comparison the CD data with those of 1 and the known compound equisetin. Since the CD spectrum of 2 was
), and ROESY (
) correlations of 1 and 4.
nearly identical to that of 1, the 2S, 3R, 6S, 8S, 11R absolute configuration was proposed for neopestalotin B (2). Neopestalotin C (3) gave a pseudomolecular ion [M + H]+ peak at m/z 404.2434 (Δ − 0.3 mmu) by HRESIMS, consistent with the molecular formula C23H33NO5 (eight degrees of unsaturation). The 1H and 13C NMR data (Table 2) of neopestalotin C (3) are similar with those of neopestalotin A (1), except that the C-5′–C-6′ olefin was replaced by two methine units including one O-methine in 3, which was verified by relevant 1H–1H COSY and HMBC correlations and the high-field chemical shifts of C-5′ (δH/δC 3.78/64.8) and C-6′ (δH/δC 4.05/68.5). The relative configuration for the decalin in 3 was proposed to be the same as in 1 based on ROESY data. However, the Z configuration of the C-1/C-3′ olefin was established based on the chemical shifts of C-2′ (δC 179.4) and C-3′ (δC 100.1) [11], which were different from those in 1 and 2. ROESY correlation of the amide proton with H3-7′ revealed their proximity in space. The small coupling constant of 5.5 Hz observed for H-5′/H-6′ suggested a gauche-like configuration for H-5′ and H-6′. In addition, J-based configuration analysis is a proven strategy to determine the relative configuration for adjacent asymmetric centers, such as the C-5′–C-7′ side chain in 3–5 [19]. Therefore, the relative configurations of C-5′ and C-6′ can be represented by six staggered rotamers [19,20]. The small coupling constants of 2.64 and 0.84 Hz observed for 2 J (H-5′, C-6′) and 2 J (H-6′, C-5′), respectively, suggested the anti-like configurations between H-5′/OH-6′ and H-6′/NH. Collectively, the relative configurations of C-5′ and C-6′ were determined. However, effort to assign the absolute configuration of C-6′ in 3 and 4 using the modified Mosher's method was unsuccessful. Eventually, the CD spectrum of 3 (Fig. S13) showed negative Cotton effects at 230 and 280 nm, correlating with the 2S, 5′S absolute configuration [19]. Therefore, the 2S, 3R, 6S, 8R, 11R, 5′S, 6′R absolute configuration was proposed for 3. The elemental composition of neopestalotin D (4) was determined to be C25H35NO6 (nine degrees of unsaturation) by HRESIMS (m/z 446.2533 [M + H]+; Δ +0.4 mmu). The 1H and 13 C NMR data (Table 2) of 4 closely resembled to those of neopestalosetin C (3), except that the C-8 methyl group (δH/δC 1.25/31.7) was replaced by the acetylated hydroxymethyl unit (δH/δC 3.91/69.5; 2.06/21.1) and the OH-8 was absent. The high-field chemical shift of C-8 (δC 37.8) in the 13C NMR spectrum together with HMBC correlations from H2-17 to C-7,
S. Zhao et al. / Fitoterapia 103 (2015) 106–112
C-8, C-9, and C-18, and from H3-19 to C-18 supported the planar structure of 4. The relative configurations of the decalin and tetramic acid moieties in 4 were deduced to be the same as those in 2 and 3 respectively by analysis of the ROESY data and coupling constants. In addition, a strong ROESY correlation of the amide proton with H3-7′ and the small coupling constants of 5.0, 0.36, and 1.14 Hz observed for 3 J (H-5′, H-6′), 2 J (H-5′, C-6′), and 2 J (H-6′, C-5′), respectively, were similar to those found in 3, indicating that C-5′ and C-6′ has the same relative configuration in both compounds. Since the CD spectrum of 4 (Fig. S14) showed negative Cotton effects at 230 and 280 nm, the 2S, 3R, 6S, 8S, 11R, 5′S, 6′R absolute configuration was deduced for 4. Compound 5 was identified as hymenosetin, a second metabolite isolated from the ash dieback pathogen Hymenoscyphus pseudoalbidus, by comparison of its NMR and MS data with literature values [19]. Compound 6 was assigned a molecular formula of C15H21NO4 (six degrees of unsaturation) on the basis of the HRESIMS (m/z 296.1495 [M + H]+; Δ −0.3 mmu). Interpretation of the 1H and 13C NMR data (Table 2) revealed one amide proton (δH 6.00), five methyl groups, two methylenes (one of which is oxygenated), four olefinic carbons with one protonated, one sp3 quaternary carbons and three carbonyls. In addition, NMR resonances corresponding to an acetyl group (δH/δC 2.04/ 21.1, 171.1) was observed, which was supported by HMBC correlations from H2-9 (δH/δC 4.18/ 61.8) to the carboxylic carbon at 171.1 ppm. The HMBC correlations from H3-12 to C-6, C-7, and C-8, indicated that C-6, C-8, and C-12 were all attached to C-7. Correlations from H2-9 and H2-8 to C-7 led to the connection of C-8 and C-9. Further HMBC correlations from H-6 and amide proton to C-5 indicated that C-6 and NH are both connected to C-5, completing the same 5-amino-3methyl-5-oxopent-3-enyl acetate moiety as pestalotiopamide A [21]. Additional HMBC correlations from H3-13 to C-1, C-2, and C-3, and from H3-14 to C-2, and C-3 located both methyl groups (δH/δC 1.75/6.1; 2.16/14.9) at C-2 and C-3, respectively. HMBC cross peaks from H3-15 to C-1, C-4, and C-5 connected C-15 and NH to C-4. Considering the chemical shift of C-3 (δC 181.4) and C-4 (δC 88.9), and the unsaturation of 6, C-3 and C-4 should be attached to the remaining oxygen atom to form the 2,4,5-trimethylfuran-3-one moiety. On the basis of these data, the planar structure of 6 was established. The ROESY correlations of H-6 with H2-8, and of H3-12 with H2-9 established the E configuration of the double bond. However, the small measured specific rotation value of 6 ([α]25D −6.0, c 0.1, MeOH) imply that 6 may be a racemate. Subsequently, 6 was resolved by HPLC using a chiral column to afford two enantiomers, 6a and 6b (Fig. S15), and the absolute configurations of 6a (4S) and 6b (4R) were assigned by comparison of their specific rotation values ([α]25D −30.0 and + 37.5, respectively, c 0.04, MeOH) to that ([α]20D −13.6, c 0.18, CHCl3) of the model compound, (S)-5-Hexyl-5-methyl2,4(3H,5H)-furandione [22]. Compounds 1–4 were evaluated for antibacterial activity against B. subtilis (ATCC 6633), S. aureus col (CGMCC 1.2465), S. pneumoniae (CGMCC 1.1692), and E. coli (CGMCC 1.2340). Compound 2 showed inhibitory effect against B. subtilis, S. aureus col, and S. pneumoniae, with MIC values of 10, 20, and 20 μg/mL, respectively (the positive control ampicillin showed MIC values of 1.25, 0.16, and 10 μg/mL, respectively).
111
Neopestalotins A–D (1–4) are new members of the 3decalinoyltetramic acid family of the natural products, which have been reported with various biological activities [23]. To our knowledge, compound 1 is the first dacalin-type tetramic acid derivative with a C-5′/C-6′ olefin unit. In addition, the presence of C-8 hydroxyl group is also rare in this class of metabolites, with pallidorosetin A as the only precedent [24]. Compound 2 possesses the same core structure as 1, but the substituents at C-8 were replaced by an acetylated hydroxymethyl unit. Compounds 3 and 4 differed from the known compound hymenosetin [19] by having different substituents at C-8. Compound 6 is a derivative of the known compound pestalotiopamide A [20], but differs in having a 2,4,5-trimethylfuran-3-one connected at the amide proton. The discovery of these compounds further demonstrated that the plant endophytic fungi are a valuable source for new bioactive natural products. Conflict of interest The authors declare no conflict interest. Acknowledgments We gratefully acknowledge financial support from the National Program of Drug Research and Development (2012ZX09301-003). Appendix A. Supplementary data Supplementary data to this article can be found online at http://dx.doi.org/10.1016/j.fitote.2015.03.023. Reference [1] [2] [3] [4] [5] [6]
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