Penicisulfuranols A–F, Alkaloids from the Mangrove Endophytic

Article pubs.acs.org/jnp

Penicisulfuranols A−F, Alkaloids from the Mangrove Endophytic Fungus Penicillium janthinellum HDN13-309 Meilin Zhu,† Xiaomin Zhang,† Huimin Feng,† Jiajia Dai,† Jing Li,† Qian Che,† Qianqun Gu,† Tianjiao Zhu,*,† and Dehai Li*,†,‡ †

Key Laboratory of Marine Drugs, Chinese Ministry of Education, School of Medicine and Pharmacy, Ocean University of China, Qingdao 266003, People’s Republic of China ‡ Laboratory for Marine Drugs and Bioproducts of Qingdao, National Laboratory for Marine Science and Technology, Qingdao 266237, People’s Republic of China S Supporting Information *

ABSTRACT: Six new epipolythiodioxopiperazine (ETP) alkaloids, penicisulfuranols A−F (1−6), were isolated from the mangrove endophytic fungus Penicillium janthinellum HDN13-309. All structures including absolute configurations were elucidated on the basis of comprehensive spectroscopic data and ECD calculations. They belong to the unusual family of ETPs containing sulfur atoms on both α- and β-positions of amino acid residues and a rare 1,2-oxazadecaline core moiety. In addition, compounds 1−6 also possess a rare spiro-furan ring and 1−3 showed cytotoxicity with IC50 values ranging from 0.1 to 3.9 μM. IC50 values ranging from 0.1 to 3.9 μM. Herein, the isolation, structure elucidation, and biological activities are described.

E

pipolythiodiketopiperazines (ETPs) are a broad class of fungal toxins (gliotoxin, chaetocin, chetomin, etc.) characterized by a disulfide (or other polysulfide) moiety connected to the diketopiperazine ring.1 Generally, the polysulfide bridge in ETPs locates on α-carbons of the two constitutive amino acid residues.1 Relatively, the subgroup possessing sulfur atoms on both α- and β-carbons is reported rarely, and they are often embedded in a 1,2-oxazadecaline moiety. Since the initial discovery of gliovirin from Gliocladium virens in 1983,2 about 15 cases in this subgroup have been reported, including FA-2097,3,4 DC1149B, DC1149R,5 iododithiobrevamide,5 pretrichodermamide A,6 adametizines A and B7 (also reported by Orfali et al. at almost the same time and named N-methylpretrichodermamide B and pretrichodermamide C8), outovirins A−C (outovirin B has the same structure as adametizine B),9 and peniciadametizines A−C10 from various fungi such as Trichoderma sp., Aspergillus sp., Eupenicillium sp., and Penicillium sp. (Figure S1 in the Supporting Information, SI). They show extensive bioactivities including antibacterial (pretrichodermide A), antifungal (outovirin C), and anti-inflammatory (gliovirin) effects and cytotoxicity (N-methylpretrichodermamide B). During our search for novel bioactive secondary metabolites from mangrove endophytic fungi,11,12 the Penicillium janthinellum strain HDN13-309, isolated from the root of Sonneratia caseolaris, was selected for its cytotoxic activity against the P388 cell line (with an inhibitory rate of 81% for the EtOAc extract at a concentration of 100 μg/mL). Chemical investigations of the fermentation led to the isolation of six new alkaloids, named penicisulfuranols A−F (1−6),13 which belong to the subtype of ETPs discussed above. In addition, compounds 1−6 possess an additional rare spiro-furan ring, with only one case (peniciadametizine A) reported.10 Compounds 1−3 were cytotoxic, with © 2016 American Chemical Society and American Society of Pharmacognosy

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RESULTS AND DISCUSSION The fungus P. janthinellum HDN13-309 was cultured (20 L) under static conditions at 28 °C. The EtOAc extract (8 g) was fractionated by column chromatography including silica gel, Sephadex LH20, C18, and semipreparative HPLC ODS columns, which led to the isolation of compounds 1−6. Penisulfuranol A (1) was obtained as a pale yellow solid. The molecular formula was determined as C21H21ClN2O8S2 on the basis of HRESIMS analysis, indicating 12 degrees of unsaturation. The 1D NMR data indicated three methyls (with two of Received: May 26, 2016 Published: December 19, 2016 71

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Table 1. 1H (500 MHz) and13C (125 MHz) NMR Data of 1−3 in DMSO-d6 1 no.

δC, type

1 2 3

165.2, C 67.8, C 32.4, CH2

4 5 6 7 8 9 1′ 2′ 3′ 4′ 5′ 6′ 7′ 8′ 9′ 10′ 11′ 12′ 4-OH 5-OH 8-OH

70.4, C 68.9, CH 131.8, CH 126.6, CH 64.5, CH 86.5, CH 161.9, C 99.7, C 51.1, CH 115.9, C 119.3, CH 107.2, CH 154.6, C 133.5, C 150.2, C 28.8, CH3 56.7, CH3 60.7, CH3

2 δH (J in Hz)

a 2.14, d (16.0) b 2.19, d (16.0) 4.88, s 5.62, d (10.3) 5.55, d (10.3) 4.44 d (7.4) 4.09, d (7.4)

5.23, s 6.86, d (8.3) 6.67, d (8.3)

2.98, s 3.78, s 3.82, s 5.64, s

δC, type 165.7, C 69.4, C 31.2, CH2 71.2, C 75.0, CH 130.0, CH 129.3, CH 65.0, CH 86.6, CH 162.2, C 99.8, C 51.2, CH 116.1, C 119.4, CH 107.3, CH 154.7, C 133.5, C 150.4, C 28.9, CH3 56.8, CH3 60.8, CH3

5.46, d (6.9)

3 δH (J in Hz)

a 2.02, d (16.3) b 2.12, d (16.3) 4.17, d (2.4) 5.41, d (10.3) 5.44, d (10.3) 4.29, br s 3.96, d (8.7)

5.20, s 6.85, d (8.4) 6.66, d (8.4)

2.97, s 3.78, s 3.81, s 5.05, s 5.16, d (5.0) 5.27, d (6.9)

δC, type

δH (J in Hz)

163.6, C 76.9, C 36.7, CH2

a 2.06, d (15.1) b 2.50, d (15.1)

72.1, C 73.8, CH 129.9, CH 129.0, CH 65.2, CH 87.4, CH 161.4, C 101.3, C 65.1, CH 118.8, C 118.3, CH 106.4, CH 154.3, C 133.3, C 150.6, C 28.7, CH3 56.6, CH3 60.9, CH3

4.19, d (2.1) 5.41, d (10.4) 5.46, d (10.4) 4.28, br s 4.02, d (7.1)

5.89, s 6.73, d (8.2) 6.60, d (8.2)

3.05, s 3.76, s 3.72, s 5.74, s 5.35, d (5.0) 5.26, d (6.6)

them oxygenated), one methylene, eight methines (with four aromatic/olefinic carbons), and nine nonprotonated carbons (with three sp3 hybrid carbons and six sp2 carbons including two carbonyls) (Table 1). The 1D NMR spectral features were similar to those of adametizine A (also named N-methylpretrichodermamide B),7,8 which possessed an α, β-disulfide bridge, indicating that they share a similar skeleton, which was further supported by the COSY and HMBC correlations (Figure 1 and Table S1 in the SI). In comparison to adametizine A, an additional furan ring was suggested to be formed by connecting C-2′

and C-9′ via an oxygen atom, similar to peniciadametizines A,10 based on the chemical shifts of C-2′ and C-9′ (δC 99.7 and 150.2 in 1 vs δC 100.8 and 149.7 in peniciadametizines A) as well as the molecular formula. The relative configuration of 1 was deduced by the data from NOESY experiment (Figure 2). The NOESY correlations of H-3a/H-8/H-10′/H-3′ in 1 indicated the relative configurations of C-2, C-8, and C-3′. The NOESY correlations between H-5 and H-9 and between 4-OH and H-9 suggested a cis relationship. In addition, the large coupling constant of H-8 and H-9 (7.4 Hz)

Figure 1. Key COSY and HMBC correlations of 1, 3, and 5.

Figure 2. Key NOESY correlations of 1, 3, and 5. 72

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further confirmed the trans orientation of H-8 and H-9. However, the relative configuration of the spiro carbon (C-2′) was unsolved due to a lack of diagnostic correlation. Considering the rigidity of the spiro-fused benzofuran−piperazinedione ring system, the relative configuration of 1 was assigned as 2R*, 4R*, 5R*, 8R*, 9S*, 2′R*, 3′R*. To determine the absolute configuration of 1, density functional theory (DFT) calculations performed at the B3LYP/6-31+G(d) level were used to generate electronic circular dichroism (ECD) spectra for a set of the lowestenergy conformers of each isomer (Figure S4 and Table S2 in the SI). According to the good agreement of the Boltzmann-weighted ECD curve of (2R,4R,5R,8R,9S,2′R,3′R)-1 with the experimental one (Figure 3), the absolute configuration of 1 was determined as 2R, 4R, 5R, 8R, 9S, 2′R, 3′R.

Figure 4. ECD spectrum of compounds 1−3.

Penicisulfuranol F (6) was isolated as a pale yellow powder. The molecular formula was deduced as C22H26N2O8S2 according to HRESIMS analysis. The 1D NMR data showed high similarity to those of 5. The only difference between them was the disappearance of signals for the methoxy group of C-7 in 6, which was further confirmed by the exhibition of signals of H-7 (δH 6.96 and δC 113.1) (Table 2). To check if compounds 4−6 were artificial products, the fresh EtOAc extract was analyzed by HPLC. All of compounds 4−6 could be detected (Figure S5 in the SI). The cytotoxicity of compounds 1−6 was evaluated against HeLa and HL-60 cell lines (Table 3). Compounds 1−3 were active against those cells with IC50 values ranging from 0.1 to 3.9 μM, while 4−6 were inactive (IC50 > 30 μM), indicating that the disulfide bridge plays an essential role in the activity of these compounds.

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Figure 3. B3LYP/6-31+G(d)-calculated ECD spectra of 1 and its enantiomer (red and blue) and the experimental ECD spectrum of 1 (black) (σ = 0.26 eV).

EXPERIMENTAL SECTION

General Experimental Procedures. Optical rotations were measured with a JASCO P-1020 digital polarimeter. UV spectra were recorded on a Beckman DU640 spectrophotometer. IR spectra were obtained with a Nicolet NEXUS 470 spectrophotometer in KBr discs. CD spectra were measured on a JASCO J-715 or Chirascan CD spectropolarimeter. NMR spectra were recorded on JEOL JNMECP 600 and Bruker-400 spectrometers using tetramethylsilane as an internal standard, with chemical shifts recorded as δ values. ESIMS was measured on a Micromass Q-TOF Ultima Global GAA076 LC mass spectrometer. HRESIMS was obtained with a Micromass EI-4000 (Autospec-Ultima-TOF). Isolation and Identification of Fungal Material. The mangrove plant Sonneratia caseolaris was collected from Hainan Province in 2012. After surface sterilization with 70% EtOH for 15 s, the root of S. caseolaris was rinsed in sterile water. Then the root was aseptically cut into small pieces and pressed onto potato dextrose agar plates. By classical microscopic analysis, the fungus strain HDN13-309 was identified as Penicillium janthinellum (GenBank accession number KM659023) by ITS sequence. A voucher specimen is deposited in our laboratory at −20 °C. The working strain was prepared on potato dextrose agar slants and stored at 4 °C. Fermentation and Extraction. The fungus P. janthinellum HDN13-309 was cultured under static conditions at 28 °C in 1000 mL Erlenmeyer flasks containing 300 mL of liquid medium containing maltose (2%), mannitol (2%), glucose (1%), sodium glutamate (1%), yeast extract (0.3%), corn syrup (0.1%), KH2PO4 (0.05%), and MgSO4· 7H2O (0.03%) dissolved in naturally collected seawater (Huiquan Bay, Yellow Sea, Qiangdao, China). After 4 weeks of cultivation, the whole broth (20 L) was filtered through cheesecloth to separate supernatant and mycelia. The former was extracted three times with EtOAc, while the latter was extracted three times with acetone and concentrated under reduced pressure to afford an aqueous solution, which was then extracted three times with EtOAc. All EtOAc solutions were combined and concentrated under reduced pressure to obtain the whole crude extract (8 g).

Penicisulfuranols B (2) and C (3) were obtained as pale yellow powders with molecular formulas C21H22N2O9S2 and C21H22N2O9S3 on the basis of HRESIMS analysis. The 1D NMR data were similar to those of 1, indicating that they shared the same skeleton. The major differences between them were the appearance of a hydroxy group (δH 5.16/5.35 in 2/3) located at C-5, confirmed by the HMBC correlations from 5-OH to C-5 and C-6 (Figure 1), indicating the Cl atom in 1 was replaced by a hydroxy group. Considering the MS data and the chemical shift differences of C-2 and C-3′ between 2 and 3 (δC 69.4 and 51.2 in 2 vs 76.9 and 65.1 in 3), it is suggested that compound 3 was the trisulfide analogue of 2 (Table 1). The NOESY correlations, coupling constants, and similar chemical shifts indicated that they shared the same relative configurations as 1 (Figure 2 and Figure S3 in the SI). The absolute configurations of compounds 2 and 3 were also determined to be the same as 1 based on similar ECD spectra (Figure 4). Penicisulfuranols D (4) and E (5) were obtained as pale yellow powders and were assigned the molecular formulas C23H27ClN2O8S2 and C23H28N2O9S2 on the basis of HRESIMS analysis. Detailed analysis of 1D NMR data along with the molecular formula indicated that compounds 4 and 5 showed a striking resemblance to compounds 1 and 2, respectively, except for the presence of two S-methyls (δH 1.96 and 2.26 in 4; δH 2.00 and 2.26 in 5). The S-methyl groups were located on C-2 and C-3′, respectively, according to the HMBC correlations from H3-10 to C-2 and from H3-13′ to C-3′ (Table 2, Figure 1, and Figure S2 in the SI). The absolute configurations of 4 and 5 were deduced to be the same as 1 and 2 on the basis of NOESY correlations (Figure 2 and Figure S3 in the SI), coupling constants (Table 2), and biogenetic consideration. 73

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Table 2. 1H (500 MHz) and 13C (125 MHz) NMR Data of 4−6 in DMSO-d6 4 δC, type

no. 1 2 3

162.9, C 66.5, C 33.1, CH2

4 5 6 7 8 9 10 1′ 2′ 3′ 4′ 5′ 6′ 7′ 8′ 9′ 10′ 11′ 12′ 13′ 4-OH 5-OH 8-OH

71.6, C 67.4, CH 126.7, CH 130.9, CH 64.5, CH 88.8, CH 15.2, CH3 157.9, C 100.6, C 55.1, CH 119.9, C 119.4, CH 107.0, CH 153.5, C 132.6, C 150.4, C 28.5, CH3 60.7, CH3 56.7, CH3 15.2, CH3

5 δH (J in Hz)

δC, type 163.2, C 66.8, C 32.3, CH2

1.95, d 2.47, d

72.6, C 64.9, CH 128.3, CH 130.0, CH 74.4, CH 89.2, CH 15.0, CH3 158.0, C 100.7, C 55.0, CH 119.4, C 120.1, CH 107.0, CH 153.5, C 132.7, C 150.3, C 28.6, CH3 56.7, CH3 60.7, CH3 15.4, CH3

4.88, d 5.55, d (10.3) 5.61, d (10.3) 4.35, br s 4.09, d (7.4) 2.26, s

4.89, s 6.91, d (8.3) 6.69, d (8.3)

2.79, s 3.77, s 3.71, s 1.96, s 5.57, s 4.98, d (5.7)

IC50 (μM) no.

HeLa

HL-60

0.5 3.9 0.3 >30.0 >30.0 >30.0 0.5

0.1 1.6 1.2 >30.0 >30.0 >30.0 0.2

a 1.77, d (15.6) b 2.43, d (15.6) 4.19, s 5.49, d (10.4) 5.42, d (10.4) 4.19, br s 3.96, d (6.7) 2.26, s

4.91, s 6.92, d (8.2) 6.70, d (8.2)

2.98, s 3.78, s 3.71, s 2.00, s 5.10, br s 5.31, br s 4.85, br s

δC, type 163.2, C 66.8, C 32.4, CH2 72.6, C 74.4, CH 130.0, CH 128.4, CH 64.8, CH 89.1, CH 15.1, CH3 158.0, C 99.8, C 55.5, CH 127.4, C 116.9, CH 123.2, CH 113.1, CH 143.8, C 146.1, C 28.6, CH3 56.1, CH3 15.6, CH3

δH (J in Hz)

a 1.78, d (15.5) b 2.43, d (15.5) 4.20, m 5.43, d (10.3) 5.50, d (10.3) 4.19, m 3.98, d (6.9) 2.29, s

4.98, s 6.88, d (7.9) 7.01, t (7.9) 6.96, d (7.9)

2.77, s 3.80, s 2.03, s 5.11, s 5.30, d (5.0) 4.90, d (5.4)

3510, 1660, 1625, 1606, 1445, 1384, 1098, 1030, 709, 670, 652 cm−1; 1H and 13C NMR data, see Table 1; HRESIMS m/z 551.0335 [M + Na]+ (calcd for C21H21ClN2O8S2Na, 551.0320). Penicisulfuranol B (2): [α]21D −34.3 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 200 (3.42) nm; ECD (0.94 × 10−3 M, MeOH) λmax (Δε) 304 (+0.38), 261 (−4.06), 244 (−1.73), 217 (−17.38) nm; IR (KBr) νmax 3511, 1661, 1625, 1606, 1446, 1383, 1098, 1030, 709, 670, 652 cm−1; 1H and 13C NMR data, see Table 1; HRESIMS m/z 511.0842 [M + H]+ (calcd for C21H23N2O9S2, 511.0839). Penicisulfuranol C (3): [α]21D −42.8 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 202 (3.31) nm; ECD (0.74 × 10−3 M, MeOH) λmax (Δε) 263 (−2.90), 243 (6.03), 218 (−13.67), 204 (−5.62) nm; IR (KBr) νmax 3515, 1663, 1623, 1610, 1424, 1384, 1093, 1010, 709, 670, 652 cm−1; 1H and 13C NMR data, see Table 1; HRESIMS m/z 565.0388 [M + Na]+ (calcd for C21H22N2O9S3Na, 565.0380). Penicisulfuranol D (4): [α]21D +0.5 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 202 (3.31) nm; IR (KBr) νmax 3515, 1663, 1623, 1610, 1424, 1384, 1093, 1010, 709, 670, 652 cm−1; 1H and 13C NMR data, see Table 2; HRESIMS m/z 576.1246 [M + NH3]+ (calcd for C23H31ClN3O8S2, 576.1236). Penicisulfuranol E (5): [α]21D +0.8 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 200 (3.05) nm; IR (KBr) νmax 3510, 1660, 1625, 1606, 1445, 1384, 1098, 1030, 709, 670, 652 cm−1; 1H and 13C NMR data, see Table 2; HRESIMS m/z 563.1138 [M + Na]+ (calcd for C23H28N2O9S2Na, 563.1128). Penicisulfuranol F (6): [α]21D +1.2 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 210 (3.20) nm; IR (KBr) νmax 3503, 1658, 1623, 1601, 1438, 1384, 1093, 1035, 709, 671, 652 cm−1; 1H and 13C NMR data, see Table 2; HRESIMS m/z 533.1024 [M + Na]+ (calcd for C22H26N2O8S2Na, 533.1023). Computation Section. Conformational searches were performed employing the systematic procedure implemented in Spartan’14,14 using the MMFF (Merck molecular force field). All MMFF minima were reoptimized with DFT calculations at the B3LYP/6-31+G(d) level using

Table 3. Cytotoxicity of Compounds 1−6

1 2 3 4 5 6 adriamycin

6 δH (J in Hz)

Purification. The crude extract was applied to a silica gel (300− 400 mesh) column and was separated into 10 fractions (fraction 1 to fraction 10) with a step gradient elution of MeOH−CH2Cl2. Among them, fractions 4−10 showed cytotoxic activity on the P388 cell line by microscopic examination. Then fraction 4 was purified by Sephadex LH20, giving five subfractions (fraction 4-1 to fraction 4-5). Fraction 4-5 was cytotoxic through microscopic examination and was separated into 10 parts (fraction 4-5-1 to fraction 4-5-10) by chromatography on a C18 column using stepwise gradient elution with 40−90% MeOH−H2O. Fraction 4-5-8 was applied on semipreparative HPLC (45:55 MeOH− H2O, 4 mL/min) to afford compound 1 (20 mg). Fraction 5 was further purified by semipreparative HPLC (50:50 MeOH−H2O, 4 mL/min) to yield compound 4 (8 mg). Then fraction 6 was separated and purified by semipreparative HPLC (50:50 MeOH−H2O) to obtain compounds 3 (5 mg) and 5 (20 mg). Compounds 2 (8 mg) and 6 (7 mg) were obtained from fractions 7 and 10 separately by Sephadex LH20 (CH3OH) and semipreparative HPLC (50:50 MeOH−H2O and 30:70 CH3CN−H2O). Penicisulfuranol A (1): [α]21D −32.0 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 200 (3.05) nm; ECD (0.95 × 10−3 M, MeOH) λmax (Δε) 291 (+1.67), 259 (−2.31), 216 (−7.69), 209 (−6.88) nm; IR (KBr) νmax 74

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the Gaussian09 program.15 The geometry was optimized starting from various initial conformations, with vibrational frequency calculations confirming the presence of minima. Time-dependent DFT calculations were performed on lowest-energy conformations (>5% population) for each configuration using 20 excited states and using a polarizable continuum model for MeOH. ECD spectra were generated using the program SpecDis16 by applying a Gaussian band shape with a 0.26 eV width and a blue or red shift to facilitate comparison to the experimental data. Biological Assay. Cytotoxic activities of 1−6 were evaluated against HeLa (human cervical carcinoma cell line) (using the SRB method) and HL-60 (human promyelocytic leukemia cells) (using the MTT method). Adriamycin was used as the positive control, and the IC50 values are shown in Table 3. The detailed methodologies for biological testing have been described in a previous report.17,18

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(10) Liu, Y.; Mándi, A.; Li, X. M.; Meng, L. H.; Kurtán, T.; Wang, B. G. Mar. Drugs 2015, 13, 3640−3652. (11) Zhang, G. J.; Sun, S. W.; Zhu, T. J.; Lin, Z. J.; Gu, J. Y.; Li, D. H.; Gu, Q. Q. Phytochemistry 2011, 72, 1436−1442. (12) Guo, W. Q.; Li, D.; Peng, J. X.; Zhu, T. J.; Gu, Q. Q.; Li, D. H. J. Nat. Prod. 2015, 78, 306−310. (13) Li, D. H.; Zhu, M. L.; Gu, Q. Q.; Zhu, T. J.; Li, J.; Che, Q. (Faming Zhuanli Shenqing) CN105037397A, 20151111, 2015. (14) Spartan’14; Wavefunction Inc.: Irvine, CA, 2013. (15) Frisch, M. J.; Trucks, G. W.; Schlegel, H. B.; Scuseria, G. E.; Robb, M. A.; Cheeseman, J. R.; Scalmani, G.; Barone, V.; Mennucci, B.; Petersson, G. A.; Nakatsuji, H.; Caricato, M.; Li, X.; Hratchian, H. P.; Izmaylov, A. F.; Bloino, J.; Zheng, G.; Sonnenberg, J. L.; Hada, M.; Ehara, M.; Toyota, K.; Fukuda, R.; Hasegawa, J.; Ishida, M.; Nakajima, T.; Honda, Y.; Kitao, O.; Nakai, H.; Vreven, T.; Montgomery, J. A., Jr.; Peralta, J. E.; Ogliaro, F.; Bearpark, M.; Heyd, J. J.; Brothers, E.; Kudin, K. N.; Staroverov, V. N.; Kobayashi, R.; Normand, J.; Raghavachari, K.; Rendell, A.; Burant, J. C.; Iyengar, S. S.; Tomasi, J.; Cossi, M.; Rega, N.; Millam, J. M.; Klene, M.; Knox, J. E.; Cross, J. B.; Bakken, V.; Adamo, C.; Jaramillo, J.; Gomperts, R.; Stratmann, R. E.; Yazyev, O.; Austin, A. J.; Cammi, R.; Pomelli, C.; Ochterski, J. W.; Martin, R. L.; Morokuma, K.; Zakrzewski, V. G.; Voth, G. A.; Salvador, P.; Dannenberg, J. J.; Dapprich, S.; Daniels, A. D.; Farkas, Ö .; Foresman, J. B.; Ortiz, J. V.; Cioslowski, J.; Fox, D. J. Gaussian 09, Revision A.1; Gaussian, Inc.: Wallingford, CT, 2009. (16) Bruhn, T.; Hemberger, Y.; Schaumlöffel, A.; Bringmann, G. SpecDis, Version 1.53; University of Wuerzburg: Germany, 2011. (17) Du, L.; Feng, T.; Zhao, B. Y.; Li, D. H.; Cai, S. X.; Zhu, T. J.; Wang, F. P.; Xiao, X.; Gu, Q. Q. J. Antibiot. 2010, 63, 165−170. (18) Mosmann, T. J. Immunol. Methods 1983, 65, 55−63.

ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jnatprod.6b00483. Structures of known gliovirin-like compounds, key 2D correlations of 1−6, the computational calculation details of 1, and 1D and 2D NMR spectra of 1−6 (PDF)

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AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected] (T. Zhu). *Tel: 0086-532-82031619. Fax: 0086-532-82033054. E-mail: [email protected] (D. Li). Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was financially supported by the National Natural Science Foundation of China (21372208, 81673450, and 41676127), the Shandong Provincial Natural Science Fund for Distinguished Young Scholars (JQ201422), AoShan Talents Program Supported by Qingdao National Laboratory for Marine Science and Technology (2015ASTP-ES09), NSFC-Shandong Joint Fund for Marine Science Research Centers (U1406402), and Fundamental Research Funds for the Central Universities (201564026). We also thank Dr. Wei Xu (University of California, Los Angeles) for help with the manuscript preparation.

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DOI: 10.1021/acs.jnatprod.6b00483 J. Nat. Prod. 2017, 80, 71−75