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Penicilindoles A−C, Cytotoxic Indole Diterpenes from the MangroveDerived Fungus Eupenicillium sp. HJ002 Cai-Juan Zheng,†,# Meng Bai,†,# Xue-Ming Zhou,† Guo-Lei Huang,† Tai-Ming Shao,‡ You-Ping Luo,† Zhi-Gang Niu,† Yan-Yan Niu,† Guang-Ying Chen,*,† and Chang-Ri Han*,‡ †
Key Laboratory of Tropical Medicinal Plant Chemistry of Ministry of Education, College of Chemistry and Chemical Engineering, Hainan Normal University, Haikou, Hainan 571158, People’s Republic of China ‡ Hainan Institute of Science and Technology, Haikou 571126, People’s Republic of China S Supporting Information *
ABSTRACT: Three new indole diterpenes, penicilindoles A− C (1−3), were isolated from the mangrove-derived fungus Eupenicillium sp. HJ002. Their planar structures and absolute configurations were determined by interpretation of NMR spectroscopic data, HR-ESIMS, and X-ray diffraction analysis using Cu Kα radiation. The cytotoxic and antibacterial activities were evaluated in vitro; penicilindole A (1) showed cytotoxic activity against human A549 and HepG2 cell lines with IC50 values of 5.5 and 1.5 μM, respectively.
I
7.2 Hz, H-8), 7.07 (dd, J = 8.0, 7.2 Hz, H-7), 6.99 (dd, J = 8.0, 7.2 Hz, H-6), and 6.99 (s, H-2). One olefinic proton at δH 5.21 (m, H-23), one oxygenated proton at δH 4.21 (m, H-19), and five methyls at δH 1.74 (s, 26-Me), 1.71 (s, 25-Me), 1.16 (s, 27Me), 1.00 (s, 29-Me), and 0.89 (d, J = 6.4 Hz, 28-Me) were also observed. The 13C and DEPT NMR spectra displayed 28 signals, including eight aromatic carbons, two olefinic carbons, seven methylenes carbons, three methines, five methyls, and two quaternary carbons. These 1H and 13C NMR spectroscopic data indicated that 1 possessed an indole diterpenoid skeleton and was very similar to the indole diterpenoid nominine isolated from the sclerotia of the fungus Aspergillus nomius.2 The obvious differences were the appearance of one methyl signal at δH 1.16 (s) for 27-Me and the disappearance of one terminal double-bond signal at δH 4.92 (br s) and 4.81 (br s) for H-12 and H-27 in the 1H NMR spectrum.2 Furthermore, in the 13C NMR data signals from one methyl carbon at δC 32.6 (CH3) for 27-Me and one oxygenated carbon at δC 74.1 (C) for C-12 were observed, instead of the two olefinic carbons at δC 107.7 (CH2) for C-12 and 148.9 (C) for C-27 found in nominine,2 indicating the terminal double bond of the latter was replaced by a methyl and a hydroxy group in 1. These results were confirmed by the 1H−1H COSY, HMQC, and HMBC data (Figure 1). The relative configuration of 1 was determined by interpretation of the NOESY data (Figure 2). The correlations of H-11 to H-16, H-16 to H-19, and 27-Me to H-11 suggested that H-11, H-16, H-19, and 27-Me were on the same face. The correlation between 28-Me and 29-Me indicated that the two methyls were on the opposite face to H-11, H-16, H-19, and 27-Me. To support the above deduction and determine the absolute configuration of 1, an X-ray crystal
ndole diterpenes are a class of natural products possessing an indole nucleus connected to a cyclized diterpene unit, produced by a variety of fungi including Aspergillus,1,2 Dichotomomyces,3 Emericella,4 and Penicillium.5 Some indole diterpenes are bioactive, showing insecticidal,2,6 antibactierial,1 and pollen tube growth inhibition activities.7 Flavinins and nominine analogues of these compounds are relatively rare, being derived biogenetically from a common digeranyindole precursor.2,5,8,9 Marine-derived fungi have become important sources of novel structural and bioactive metabolites,10 and many bioactive compounds have been found in our own research into marine-derived fungi metabolites.11−15 Eupenicillium sp. HJ002 isolated from the medicinal mangrove Xylocarpus granatum Koenig, showed significant cytotoxic activity against the human A549 cell line. A chemical investigation of the EtOAc extract of the fermentation broth led to the identification of three new nominine analogues, penicilindoles A−C (1−3). Herein, we described the isolation, structure elucidation, and bioactivities of these compounds.
Compound 1 was isolated as colorless crystals. Its molecular formula of C28H41NO2 (nine degrees of unsaturation) was determined by HR-ESIMS. The 1H NMR spectrum displayed the typical pattern of a 3-substituted indole moiety with five aromatic protons at δH 7.56 (d, J = 8.0 Hz, H-5), 7.30 (d, J = © XXXX American Chemical Society and American Society of Pharmacognosy
Received: August 4, 2017
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DOI: 10.1021/acs.jnatprod.7b00673 J. Nat. Prod. XXXX, XXX, XXX−XXX
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that H-11, H-16, H-19, H-22, and 27-Me were on the same side of the structure, and 28-Me and 29-Me were on the opposite side. The relative configuration of C-20 and the absolute configuration of 3 can be tentatively determined based on biosynthetic correlation with 1, while it cannot be confirmed according to the NOESY and other NMR data. Consequently, the absolute configuration of 3 was assumed as 11S, 12R, 15S, 16R, 19S, 20S, and 22S. The structure of 3 was proposed as penicilindole C. Penicilindoles A−C (1−3) are new indole diterpenes, and there appears to be a parallel biogenetic relationship between nominine and 1−3. Compounds 1−3 are proposed to be derived from the same intermediate as paxilline by a different cyclization process.2,5,8,9,16 Compounds 1 and 2 were cytotoxic against human A549, HeLa, and HepG2 cell lines, with IC50 values ranging from 1.5 to 47.2 μM (Table 2). Their antibacterial activity against four terrestrial pathogenic bacteria and two marine pathogenic bacteria was also tested; no activity was observed at the concentration of 20 μM.
Figure 1. Key 1H−1H-COSY (bold blue) and HMBC correlations (H → C) of 1−3.
structure with a Flack parameter of 0.0(2) was obtained (Figure 3). The absolute configuration of 1 was confirmed as 11S, 12R, 15S, 16R, 19S, and 20S, and 1 was named penicilindole A. Compound 2 was isolated as colorless crystals. Its molecular formula of C28H39NO2 (10 degrees of unsaturation) was determined by HR-ESIMS. Comparison of the 1D NMR data (Table 1) between 2 and 1 indicated that they shared the same basic skeleton. The 1H NMR spectroscopic data for 2 were almost identical to those of 1 (Table 1), with the exception of the absence of an oxygenated methine signal. Moreover, the 13 C NMR resonance of C-19 was shifted from δC 70.3 (CH) in 1 to δC 219.3 (C) in 2, suggesting the oxygenated methine group in 1 was replaced by a carbonyl group in 2. The 2D NMR data allowed the complete assignment for 2 (Figure 1). The relative configuration of 2 was based on the NOESY correlations as indicated in Figure 2. The NOESY correlations of H-11 to H-16 and 27-Me, 28-Me to 29-Me indicated that 28Me and 29-Me were placed on the opposite side to H-11, H-16, and 27-Me. In order to determine the relative configuration of C-20 and the absolute configuration of 2, X-ray analysis was performed showing the absolute configration of 2 to be 11S, 12R, 15S, 16R, and 20S (Figure 3), and 2 was named penicilindole B. Compound 3 was isolated as a white powder, with the molecular formula of C28H39NO2 (10 degrees of unsaturation) determined by HR-ESIMS. The 1D NMR spectroscopic data for 3 closely resembled those of 1. The most obvious difference was the oxygenated methine at δH 4.54 (m) and δC 77.1 (CH) for C-22 in 3 replacing the methylene at δH [2.21 (m) and 2.42 (m)] and δC 26.8 (CH2) in 1. These, combined with the HRESIMS data, implied that the methylene at C-22 of 1 was replaced by an oxygenated methine to form a five-membered ring (C-19 to C-22) in 3. This conclusion was confirmed by the 2D NMR and HR-ESIMS data. The relative configuration of 3 was identified by the NOESY correlations of H-22 to H-19, H19 to H-11 and H-22, H-11 to 27-Me and H-16, H-16 to 27-Me and H-19, and 29-Me to 28-Me (Figure 2), which indicated
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EXPERIMENTAL SECTION
General Experimental Procedures. Melting points were determined on an X-6 micromelting point apparatus and were uncorrected. Optical rotations were measured on a JASCO P-1020 digital polarimeter. IR spectra were recorded on a Thermo Nicolet 6700 (using KBr disks) spectrophotometer (Thermo Scientific, Madison, WI, USA). 1D and 2D NMR spectra were measured on a Bruker AV-400 (Bruker Corporation, Switzerland) instrument with tetramethylsilane as the internal standard. HR-ESIMS spectra were obtained on a Bruker Daltonics Apex-Ultra 7.0 T (Bruker Corporation, Billerica, MA, USA) and a Q-TOF Ultima Global GAA076 LC mass spectrometer. Single-crystal data were measured by an Agilent Gemini Ultra X-ray single crystal diffractometer (Cu Kα radiation). Preparative HPLC were used for an Agilent 1260 prep-HPLC system with a Waters C18 analytical HPLC column (4.6 × 250 mm, 5 μm) and a semipreparative column (9.4 × 250 mm, 7 μm). Sephadex LH-20 (Pharmacia Co. Ltd., Sandwich, UK) and silica gel (200−300 and 300−400 mesh, Qingdao Marine Chemical Factory, Qingdao, China) were used for column chromatography (CC). All solvents were purchased from Xilong Chemical Reagent Factory (Guangzhou, China). Mycology. The fungal strain Eupenicillium sp. HJ002. was isolated from the mangrove Xylocarpus granatum Koenig collected in the South China Sea in August 2015. It was deposited in China General Microbiological Culture Collection Center, CGMCC, Beijing, China, with the CGMCC code 13373. The fungus was identified according to its morphological traits and a molecular protocol by amplification and sequencing of the DNA of the ITS region of the rRNA gene as described previously.12 It was identified as Eupenicillium sp. according to morphologic traits and molecular identification. Its 557 base pair ITS sequence had 99% sequence identity to that of Eupenicillium sp.
Figure 2. Key NOESY correlations [(solid ↔) β-orientations and (dashed ↔) α-orientations] of 1−3. B
DOI: 10.1021/acs.jnatprod.7b00673 J. Nat. Prod. XXXX, XXX, XXX−XXX
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Figure 3. ORTEP view of the X-ray structures of 1 and 2.
Table 1. 1H (400 MHz) and 13C (100 MHz) NMR Data of 1−3 (δ in ppm, J in Hz) in CD3OD 1
2
3
position
δC
δH (J in Hz)
δC
δH (J in Hz)
δC
δH (J in Hz)
1 2 3 4 5 6 7 8 9 10
122.3 119.6 128.9 119.2 119.3 122.1 112.0 137.9 23.5
6.99, s
122.5 117.9 128.9 119.4 119.3 122.4 112.0 137.9 24.2
7.06, s
122.4 118.5 128.4 119.0 119.4 122.5 112.1 138.1 23.9
7.06, s
11 12 13
47.7 74.1 37.0
14
29.7
15 16 17
41.1 32.0 26.6
18
31.4
19 20 21
70.3 48.0 31.5
22
26.8
23 24 25 26 27 28 29
128.5 130.8 26.0 18.1 32.6 16.9 19.2
7.56, d (8.0) 6.99, dd (8.0, 7.2) 7.07, dd (8.0, 7.2) 7.30, d (7.2) 3.10, br d (16.8) 3.00, dd (16.8, 6.4) 2.50, m 1.45, 1.76, 1.25, 1.89,
m m m m
2.26, 1.25, 1.67, 1.35, 1.90, 4.21,
m m m m m m
1.94, m 2.21, m 2.42, m 5.21, m 1.71, s 1.74, s 1.16, s 0.89, d (6.4) 1.00, s
7.48, br d (8.0) 6.99, ddd (8.0, 8.0, 0.8) 7.07, ddd (8.0, 8.0, 0.8) 7.30, br d (8.0) 2.54, br d (17.2) 3.14, dd (17.2, 6.8) 3.06, dd (6.8, 1.2)
47.9 72.6 37.0 29.1 46.2 31.3 32.8 40.3
1.57, 1.92, 1.42, 1.92,
m m m m
2.78, 1.54, 2.00, 1.88, 2.72,
m m m m m
46.6 73.2 35.9 29.6
2.81, dd (17.2, 3.6) 3.15, dd (17.2, 3.2) 2.34, m 1.45, 1.69, 1.02, 1.33,
m m m m
39.0 32.1 27.1
2.13, m 1.65, m
26.3
1.62, m 3.70, m
219.3 62.3 32.5
1.79, m
82.2 53.2 38.4
28.2
2.38, m
77.1
128.3 131.0 25.9 18.1 32.1 16.0 18.1
5.21, m
126.2 136.8 25.9 18.4 30.8 16.4 20.8
1.71, s 1.73, s 1.22, s 0.89, d (6.4) 0.68, s
7.59, d (7.6) 7.01, dd (8.0, 7.6) 7.09, dd (8.0, 8.0) 7.31, d (8.0)
2.50, m 1.81, m 4.54, m 5.44, brd (8.0) 1.76, s 1.74, s 1.21, s 0.86, d (6.4) 0.94, s
medium in seawater, using 1 L Erlenmeyer flasks each containing 300 mL of medium, and incubated at 26 °C without shaking for 4 weeks. The cultures were filtered through cheesecloth, and the filtrate was extracted with EtOAc (3 × 15 L, 24 h each). The organic extracts were
ph511. The sequence data have been submitted to GenBank with the accession number JQ828843. Fermentation, Extraction, and Isolation. The fungal strain Eupenicillium sp. HJ002 was cultivated in 15 L of potato glucose liquid C
DOI: 10.1021/acs.jnatprod.7b00673 J. Nat. Prod. XXXX, XXX, XXX−XXX
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BIOLOGICAL ASSAYS Cytotoxic activities of all compounds against human A549, HeLa, and HepG2 cell lines were evaluated by the MTT method.17 5-Fluorouracil and adriamycin were used as positive controls. Antibacterial activities were determined against four terrestrial pathogenic bacteria, Escherichia coli (ATCC 25922), Staphylococcus aureus (ATCC 25923), methicillin-resistant Staphylococcus aureus MRSA (ATCC 33591), and Bacillus cereus (ATCC 11778), and two marine pathogenic bacteria, Vibrio parahemolyticus (ATCC17802) and V. alginolyticus (ATCC17749), by the microplate assay method.18 Ciprofloxacin was used as the positive control.
Table 2. Cytotoxic Activity for Compounds 1 and 2 IC50 (μM) compound
A549
HeLa
HepG2
1 2 5-Fua adriamycinb
5.5 18.6 36.8 0.002
23.3 20.0 28.0 0.1
1.5 47.2 76.9 0.1
Note
a 5-Fluoracil used as positive control. bAdriamycin used as positive control.
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concentrated in vacuo to yield an oily residue (6.2 g), which was subjected to silica gel CC (petroleum ether, EtOAc v/v, gradient 100:0−0:100) to generate six fractions (Fr. 1−Fr. 6). Fr. 3 was isolated by CC on silica gel eluted with petroleum ether−EtOAc, then subjected to Sephadex LH-20 CC eluting with CHCl3−MeOH (v/v, 1:1), and further purified by using HPLC on an ODS semipreparative column (Waters C18, 9.4 × 250 mm, 7 μm, 2 mL/min) eluted with 80% MeOH−H2O to obtain 3 (1.5 mg). Fr. 4 was subjected to silica gel CC eluted with petroleum ether−EtOAc and Sephadex LH-20 CC eluting with mixtures of CHCl3−MeOH (v/v, 1:1) and then further purified by using HPLC on an ODS semipreparative column (Waters C18, 9.4 × 250 mm, 7 μm, 2 mL/min) eluted with 75% MeOH−H2O to obtain 1 (6.4 mg). Fr. 5 was purified by repeated Sephadex LH-20 CC (MeOH) and HPLC on an ODS semipreparative column (Waters C18, 9.4 × 250 mm, 7 μm, 2 mL/min) eluted with 72% MeOH−H2O to obtain 2 (7.2 mg). Penicilindole A (1): colorless crystals; [α]25D +29.6 (c 0.35, MeOH); mp 92.8−93.4 °C; UV (MeOH) λmax (log ε) 221 (3.28), 282 (2.59), 290 (2.43); IR (KBr) νmax 3427, 2949, 1636, 1455, 1377, 1234, 1121, 737 cm−1; 1H and 13C NMR see Table 1; HR-ESIMS m/z 424.3204 [M + H]+ (calcd for C28H42NO2, 424.3210). Penicilindole B (2): colorless crystals; [α]25D +20.5 (c 0.30, MeOH); mp 98.6−99.2 °C; UV (MeOH) λmax (log ε) 220 (3.00), 246 (2.51), 312 (2.32) nm; IR (KBr) νmax 3543, 1737, 1652, 1278, 1103, 816 cm−1; 1H and 13C NMR see Table 1; HR-ESIMS m/z 422.3048 [M + H]+ (calcd for C28H40NO2, 422.3054). Penicilindole C (3): white powder; [α]25D +34.6 (c 0.42, MeOH); UV (MeOH) λmax (log ε) 224 (4.02), 246 (3.60), 312 (2.82) nm; IR (KBr) νmax 3518, 1712, 1602, 1250, 1129, 1086, 802 cm−1; 1H and 13C NMR see Table 1; HR-ESIMS m/z 422.3050 [M + H]+ (calcd for C28H40NO2, 422.3054). X-ray Crystallographic Analysis of 1 and 2. Colorless crystals of 1 and 2 were obtained from MeOH. Single-crystal X-ray diffraction data were collected on a Xcalibur, Atlas, Gemini ultra diffractometer with Cu Kα radiation (λ = 1.541 80 Å) at 120.01(10) K, respectively. The structure was solved by direct methods (ShelXS) and refined with the ShelXL refinement package using least squares minimization. All nonhydrogen atoms were refined anisotropically, and all hydrogen atoms were placed in idealized positions and refined relatively isotropically with a riding model. Crystallographic data of 1 and 2 have been deposited in the Cambridge Crystallographic Data Centre with the deposition numbers CCDC 1561610 and 1561611, respectively. Copies of the data can be obtained, free of charge, on application to the Director, CCDC, 12 Union Road, Cambridge CB21EZ, UK [fax: + 44-(0)1223-336033, or e-mail:
[email protected]]. Crystal data for 1: C28H41NO2, Mr = 423.62, orthorhombic, a = 12.119 07(2) Å, b = 13.202 9(3) Å, c = 15.399 4(3) Å, α = 90°, β = 90°, γ = 90°, V = 2624.00(8) Å3, space group P212121, Z = 4, Dx = 1.142 mg/mm3, μ(Cu Kα) = 0.540 mm−1, and F(000) = 928. Independent reflections: 4336 (Rint = 0.0616). The final R1 values were 0.05046, wR2 = 0.1067 (I > 2σ(I)). Flack parameter = 0.0(2). Crystal data for 2: C28H39NO2, Mr = 421.60, orthorhombic, a = 12.111 05(14) Å, b = 13.124 45(18) Å, c = 15.256 55 (18) Å, α = 90°, β = 90°, γ = 90°, V = 2625.04(5) Å3, space group P212121, Z = 4, Dx = 1.155 mg/mm3, μ(Cu Kα) = 0.548 mm−1, and F(000) = 920. Independent reflections: 4344 (Rint = 0.0279). The final R1 values were 0.0323, wR2 = 0.0746 (I > 2σ(I)). Flack parameter = −0.01(17).
ASSOCIATED CONTENT
S Supporting Information *
The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jnatprod.7b00673. X-ray crystallographic data of compounds 1 and 2 (ZIP) 1 H NMR, 13C NMR, DEPT, HMQC, COSY, HMBC, NOESY, and HR-ESIMS spectra of compounds 1−3 (PDF)
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AUTHOR INFORMATION
Corresponding Authors
*Tel: +86-898-65889422. Fax: +86-898-65889422. E-mail:
[email protected] (G.-Y. Chen). *Tel: +86-898-65889422. Fax: +86-898-65889422. E-mail:
[email protected] (C.-R. Han). ORCID
Cai-Juan Zheng: 0000-0003-1779-8736 Author Contributions #
C.-J. Zheng and M. Bai contributed equally to this study.
Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS This work was supported by the National Natural Science Foundation of China (31760093, 21462015, and 21662012), Marine Science and Technology Program of Hainan Province (2015XH06), Program for Innovative Research Team in University (IRT-16R19), and Hainan Province Natural Science Foundation of Innovatie Research Team Project (No. 2016CXTD007).
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REFERENCES
(1) Qiao, M. F.; Ji, N. Y.; Liu, X. H.; Li, K.; Zhu, Q. M.; Xue, Q. Z. Bioorg. Med. Chem. Lett. 2010, 20, 5677−5680. (2) Gloer, J. B.; Rinderknecht, B. L.; Wicklow, D. T.; Dowd, P. F. J. Org. Chem. 1989, 54, 2530−2532. (3) Liu, T.; Meyer, S. L. F.; Chitwood, D. J.; Chauhan, K. R.; Dong, D.; Zhang, T. T.; Li, J.; Liu, W. C. J. Agric. Food Chem. 2017, 65, 3127−3132. (4) Kawai, K.; Nozawa, K.; Nakajima, S. J. Chem. Soc., Perkin Trans. 1 1994, 13, 1673−1674. (5) Li, C.; Gloer, J. B.; Wicklow, D. T. J. Nat. Prod. 2003, 66, 1232− 1235. (6) TePaske, M. R.; Gloer, J. B.; Wicklow, D. T.; Dowd, P. F. J. Nat. Prod. 1992, 55, 1080−1086. (7) Kimura, Y.; Nishibe, M.; Nakajima, H.; Hamasaki, T.; Shigemitsu, N.; Sugawara, F.; Stout, T. J.; Cladry, J. Tetrahedron Lett. 1992, 33, 6987−6890.
D
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(8) TePaske, M. R.; Gloer, J. B.; Wicklow, D. T.; Dowd, P. F. Tetrahedron 1989, 45, 4961−4968. (9) Gloer, J. B.; Tepaske, M. R.; Sima, J. S.; Wicklow, D. T.; Dowd, P. F. J. Org. Chem. 1988, 53, 5457−5460. (10) Blunt, J. W.; Copp, B. R.; Keyzers, R. A.; Munro, M. H. G.; Prinsep, M. R. Nat. Prod. Rep. 2017, 34, 235−294. (11) Zheng, C. J.; Shao, C. L.; Guo, Z. Y.; Chen, J. F.; Deng, D. S.; Yang, K. L.; Chen, Y. Y.; Fu, X. M.; She, Z. G.; Lin, Y. C.; Wang, C. Y. J. Nat. Prod. 2012, 75, 189−197. (12) Zheng, C. J.; Shao, C. L.; Wu, L. Y.; Chen, M.; Wang, K. L.; Zhao, D. L.; Sun, X. P.; Chen, G. Y.; Wang, C. Y. Mar. Drugs 2013, 11, 2054−2068. (13) Zhou, X. M.; Zheng, C. J.; Chen, G. Y.; Song, X. P.; Han, C. R.; Li, G. N.; Fu, Y. H.; Chen, W. H.; Niu, Z. G. J. Nat. Prod. 2014, 77, 2021−2028. (14) Huang, G. L.; Zhou, X. M.; Bai, M.; Liu, Y. X.; Zhao, Y. L.; Luo, Y. P.; Niu, Y. Y.; Zheng, C. J.; Chen, G. Y. Mar. Drugs 2016, 14, 177. (15) He, K. Y.; Zhang, C.; Duan, Y. R.; Huang, G. L.; Yang, C. Y.; Lu, X. R.; Zheng, C. J.; Chen, G. Y. J. Antibiot. 2017, 70, 823−827. (16) Tang, M. C.; Lin, H. C.; Li, D. H.; Zou, Y.; Li, J.; Xu, W.; Cacho, R. A.; Hillenmeyer, M. E.; Garg, N. K.; Tang, Y. J. Am. Chem. Soc. 2015, 137, 13724−13727. (17) Scudiero, D. A.; Shoemaker, R. H.; Paull, K. D.; Monks, A.; Tierney, S.; Nofziger, T. H.; Currens, M. J.; Seniff, D.; Boyd, M. R. Cancer Res. 1988, 48, 4827−4833. (18) Pierce, C. G.; Uppuluri, P.; Teistan, A. R.; Wormley, F. L. J.; Mowat, E.; Ramage, G.; Lopez-ribot, J. L. Nat. Protoc. 2008, 3, 1494− 1500.
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