- Email: [email protected]
Ingol diterpenoids as P-glycoprotein-dependent multidrug resistance (MDR) reversal agents from Euphorbia marginata
Journal Pre-proofs Ingol diterpenoids as P-glycoprotein-dependent multidrug resistance (MDR) reversal agents from Euphorbia marginata Yao Zhang, Run-Zhu Fan, Jun Sang, Yi-Jing Tian, Jia-Qi Chen, Gui-Hua Tang, Sheng Yin PII: DOI: Reference:
S0045-2068(19)31697-9 https://doi.org/10.1016/j.bioorg.2019.103546 YBIOO 103546
To appear in:
Bioorganic Chemistry
Received Date: Revised Date: Accepted Date:
10 October 2019 19 December 2019 21 December 2019
Please cite this article as: Y. Zhang, R-Z. Fan, J. Sang, Y-J. Tian, J-Q. Chen, G-H. Tang, S. Yin, Ingol diterpenoids as P-glycoprotein-dependent multidrug resistance (MDR) reversal agents from Euphorbia marginata, Bioorganic Chemistry (2019), doi: https://doi.org/10.1016/j.bioorg.2019.103546
This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
© 2019 Published by Elsevier Inc.
Ingol diterpenoids as P-glycoprotein-dependent multidrug resistance (MDR) reversal agents from Euphorbia marginata Yao Zhang,† Run-Zhu Fan,† Jun Sang, Yi-Jing Tian, Jia-Qi Chen, Gui-Hua Tang, and Sheng Yin*
School of Pharmaceutical Sciences, Sun Yat-sen University, Guangzhou, 510006, China *Corresponding author. E-mail: [email protected] Author Contributions †These
authors contributed equally to this work.
1
Abstract Twenty new ingol diterpenoids, euphornans AT (120), representing a rare class of C-19-oxidated and H-2, H-3 -oriented ingols, were isolated from the seeds of Euphorbia marginata. Their structures including absolute configurations were elucidated by extensive spectroscopic analysis, ECD analysis, and single crystal X-ray diffraction. Compounds 120 were screened for the multidrug resistance (MDR) reversal activity on P-glycoprotein (Pgp)-dependent MDR cancer cell line HepG2/ADR, and 11, 14, and 18 were identified as potent MDR modulators that could enhance the efficacy of anticancer drug adriamycin to ca. 20 folds at 5 M. The Pgp inhibition mechanism and brief structureactivity relationships (SARs) of these compounds were also discussed.
Keywords: Euphorbia marginata; Ingol diterpenoids; Multidrug resistance (MDR); MDR modulators
2
1. Introduction Ingols are a subgroup of lathyrane diterpenoids featuring a 5/11/3 carbon ring system with a 4,15-epoxy ring. Until now, more than 100 ingols have been exclusively isolated from the Euphorbia plants (Euphorbiaceae), and most of them shared the basic structural features of cis-fused 5/11/3 ring system, 4,15-epoxy, -oriented H-2 and H3, C-14 ketocarbonyl, and 5, with the structural diversity mainly arising from the various ester substituents on the 11-membered macro ring [1,2]. In recent years, their fascinating structures and intriguing biological activities have attracted considerable interest from the scientists in this research field [3-5]. Multidrug resistance (MDR) designates a phenomenon where resistance to one drug is accompanied by resistance to drugs that are structurally and functionally unrelated. The development of MDR is one of the leading causes of treatment failure in the chemotherapy of malignant tumors. A primary mechanism of MDR is the overproduction of P-glycoprotein (Pgp) in the plasma membranes of resistant cells, where the Pgp acts as an energy-dependent efflux pump, reducing the intracellular accumulation of anticancer drugs [6]. Thus, compounds with Pgp inhibitory activities are considered as promising MDR reversal agents when coadministered with an anticancer drug in cancer therapy. Euphorbia marginata Pursh. (Euphorbiaceae) is an ornamental plant native to south America, but now is naturalized throughout most parts of China [7]. To date, there is only one publication related to the chemistry of this plant, reporting a hydroxyingol derivative from its seeds [8]. As part of our continuing efforts to discover MDR reversal 3
agents from the Euphorbiaceae plants [9,10], we undertook a detailed chemical investigation of E. marginata, which led to the isolation of twenty new ingol diterpenoids. All these compounds were screened for the reversal activity on Pgpdependent MDR cancer cell line HepG2/ADR, and three compounds were identified as potent MDR modulators. Herein, we describe the isolation, structure elucidation, the MDR reversal activities, and the structureactivity relationships of these compounds. 2. Materials and methods 2.1. General experimental procedures Melting points were measured using an X-4 melting instrument and uncorrected. Optical rotations were determined on a Rudolph Autopol I automatic polarimeter. ECD spectra were obtained on an Applied Photophysics Chirascan spectrometer and UV spectra on a Shimadzu UV-2450 spectrophotometer. IR spectra were determined in KBr disks on a Bruker Tensor 37 infrared spectrophotometer. NMR spectra were measured with a Bruker AM-400 spectrometer at 25 C. ESIMS and HRESIMS were carried out on a Waters Micromass Q-TOF spectrometer. X-ray data were obtained on an Agilent Xcalibur Nova X-ray diffractometer. Semipreparative HPLC was performed with a Shimadzu LC-20 AT equipped with an SPD-M20A PDA detector. A YMC-pack ODSA column (250 10 mm, S-5 m, 12 nm) was used for HPLC purification. Silica gel (100−200 and 300−400 mesh, Qingdao Haiyang Chemical Co., Ltd.), MCI gel (CHP20P, 75−150 m, Mitsubishi Chemical Industries Ltd.), reversed-phase C18 (RpC18) silica gel (12 nm, S-50 m, YMC Co., Ltd.), D101 macroporous adsorptive resins (Sinopharm Chemical Reagent Co., Ltd) and Sephadex LH-20 gel (Amersham 4
Biosciences) were used for column chromatography (CC). All solvents were of analytical grade (Guangzhou Chemical Reagents Company, Ltd.). 2.2. Plant material The seeds of Euphorbia marginata were collected in Suqian, Jiangsu Province, P. R. China, in June 2018, and were authenticated by one of the author (G. H. Tang). A voucher specimen (accession number: YBC201806) has been deposited at the School of Pharmaceutical Sciences, Sun Yat-sen University. 2.3. Extraction and isolation The powder of dried seeds E. marginata (7.5 kg) was extracted with 95 EtOH (3 20 L) at room temperature to give 860 g of crude extract. The extract was suspended in H 2 O (3 L) and partitioned with petroleum ether (PE, 3 3 L). The PE extract (470 g) was separated on a D101 macroporous adsorptive resins eluted with a MeOHH 2 O gradient (3:7 10:0) to afford five fractions (Fr. V). Fr. III (4.5 g) was applied to silica gel CC (PEEtOAc, 30:1 0:1) to give four fractions (Fr. IIIaIIId). Separation of Fr. IIIc (2.8 g) by Rp-C 18 silica gel CC eluted with MeOH/H 2 O (4:6 10:0) yielded four fractions (Fr. IIIc1IIIc4). Fr. IIIc1 (184 mg) was chromatographed over silica gel column (PEEtOAc, 20:1 1:1) to give 1 (100 mg) and 19 (27 mg). Fr. IIIc2 (150 mg) was purified on semipreparative HPLC (CH 3 CN/H 2 O, 70:30, 3 mL/min) to give 8 (50 mg, t R 17 min). Fr. IIIc3 (1.6 g) was loaded onto a Sephadex LH-20 column using MeOH as eluent to give 2 (1.4 g). Fr. IIIc4 (280 mg) was subjected to silica gel CC (PEEtOAc, 30:1 0:1), followed by HPLC (MeCN/H 2 O, 70:30, 3 mLmin) afforded 4 (50 5
mg, t R 12.5 min) and 5 (35 mg, t R 15 min). Separation of Fr. IIId (900 mg) by silica gel CC (PEEtOAc, 30:10:1) afforded four fractions (Fr. IIId1IIId4). Fr. IIId3 (50 mg) was purified using HPLC (MeCN/H 2 O, 50:50, 3 mL/min) to give 6 (6 mg, t R 17 min) and 14 (4 mg, t R 19 min). Fr. IIId4 (150 mg) was loaded onto a Sephadex LH-20 column eluted with MeOH, and further purified by HPLC (MeCN/H 2 O, 55:45, 3 mLmin) as mobile phase to afford 7 (3.1 mg, t R 10 min) and 3 (18 mg, t R 12 min). Fractionation of Fr. IV (57g) by silica gel CC (PEEtOAc, 1:0 0:1) led to five fractions (Fr. IVaIVe). Fr. IVc (4.6 g) was subjected to MCI gel CC eluted with a MeOHH O gradient (6:4 10:0) to yield six fractions (Fr. IVc1IVc6). Fr. IVc1 (200 mg) was chromatographed successively over a Sephadex LH-20 eluted with MeOH, and silica gel column (PECH2Cl2, 5:1 0:1) to afford 17 (100 mg) and 18 (15 mg). Fr. IVc2 was loaded onto a Sephadex LH-20 column eluted with MeOH, and further purified by HPLC (MeCN/H 2 O, 65:35, 3 mLmin) to give 9 (18 mg, t R 12 min) and 20 (4 mg, t R 14 min). Fr. IVc3 (112 mg) was applied to silica gel CC (PEEtOAc, 30:1 1:1), followed by Sephadex LH-20 CC using MeOH to yield 15 (18 mg) and 16 (55 mg). Fr. IVc4 (200 mg) was subjected to silica gel CC (PEEtOAc, 25:1 1:1), and further purified by HPLC (MeCN/H 2 O, 60:40, 3 mLmin), to obtain 12 (9.2 mg, t R 15 min), 11 (5.3 mg, t R 16 min), and 13 (5.7 mg, t R 19 min ). Fr. IVc5 (300 mg) was separated by Sephadex LH-20 CC using MeOH to give 10 (50 mg). 2.4. Spectroscopic data
6
Euphornan A (1): colorless crystals (petroleum ether/EtOAc: 5:1), mp 230232 C; []20D 11.3 (c 0.3, MeCN); UV (MeCN) max (log ) 193 (4.59), 262 (3.52) nm; IR (KBr) max 2938, 1735, 1591, 1371, 1228, 1021 cm1; 1H and 13C NMR data for the diterpene moiety see Tables 1 and 3; 1H NMR data for the acyloxy groups: 3-OAc [H 2.08 (3H, s)], 7-OAc [H 2.04 (3H, s)], 8-OAc [H 1.97 (3H, s)], 12-OAc [H 1.74 (3H, s)], 19-ONic [H 9.10 (1H, s), 8.71 (1H, br s), 8.21 (1H, d, J 7.9 Hz), and 7.35 (1H, dd, J 7.8, 4.9 Hz)]; 13C data for the acyloxy groups: 3-OAc (C 169.4, 20.8), 7-OAc (C 168.9, 20.5), 8-OAc (C 170.0, 20.8), 12-OAc (C 170.1, 20.6), 19-ONic (C 164.7, 153.3, 150.4, 136.9, 126.1, and 123.2); HRESIMS m/z 656.2704 [M H] (calcd for C34H42NO12, 656.2702). Euphornan B (2): colorless oil; []20D 36.7 (c 0.3, MeCN); UV (MeCN) max (log
) 195 (4.71), 223 (4.28), 263 (3.45) nm; IR (KBr) max 2935, 1733, 1591, 1370, 1229, 1023 cm1; 1H and 13C NMR data for the diterpene moiety see Tables 1 and 3; 1H NMR data for the acyloxy groups: 3-OAc [H 2.12 (3H, s)], 7-OAc [H 2.10 (3H, s)], 8-OBz [H 7.90 (2H, d, J 6.9 Hz), 7.42 (1H, t, J 7.4 Hz), and 7.29 (2H, t, J 7.7 Hz)], 12OAc [H 1.86 (3H, s)], 19-ONic [H 8.95 (1H, s), 8.65 (1H, br s), 7.99 (1H, d, J 9.0 Hz), and 7.22 (1H, dd, J 7.8, 4.9 Hz)]; 13C NMR data for the acyloxy groups: 3-OAc (C 169.4, 20.7), 7-OAc (C 168.7, 20.5), 8-OBz (C 165.4, 133.2, 129.5 2, 129.1, and 128.3 2), 12-OAc (C 170.1, 20.7), 19-ONic (C 164.5, 152.9, 150.3, 136.7, 126.0, and 123.0); HRESIMS m/z 718.2846 [M H] (calcd for C39H44NO12, 718.2858). Euphornan C (3): white amorphous powder; []20D 14.3 (c 0.3, CH2Cl2); UV (MeCN) max (log ) 194 (4.79), 219 (4.48), 263 (3.86) nm; IR (KBr) max 2922, 1735, 7
1591, 1373, 1277, 1230, 1021 cm1; 1H and 13C NMR data for the diterpene moiety see Tables 1 and 3; 1H NMR data for the acyloxy groups: 3-OAc [H 1.72 (3H, s)], 7-ONic [H 9.23 (1H, s), 8.83 (1H, br s), 8.32 (1H, d, J 8.0 Hz), and 7.46 (1H, dd, J 7.8, 4.9 Hz)], 8-OAc [H 2.04 (3H, s)], 12-OAc [H 1.81 (3H, s)], 19-ONic [H 9.16 (1H, s), 8.77 (1H, br s), 8.26 (1H, d, J 7.9 Hz), and 7.39 (1H, dd, J 7.8, 4.9 Hz)]; 13C NMR data for the acyloxy groups: 3-OAc (C 169.4, 20.0), 7-ONic (C 163.4, 153.9, 150.4, 137.3, 125.8, and 123.6), 8-OAc (C 170.2, 21.0), 12-OAc (C 170.2, 20.7), 19-ONic (C 164.8, 153.4, 150.6, 137.0, 126.2, and 123.3); HRESIMS m/z 719.2795 [M H] (calcd for C38H43N2O12, 719.2811). Euphornan D (4): colorless oil; []20D 13.0 (c 0.3, MeCN); UV (MeCN) max (log
) 193 (4.29), 257 (3.50) nm; IR (KBr) max 2937, 1734, 1591, 1370, 1230, 1023 cm1; 1H
and 13C NMR data for the diterpene moiety see Tables 1 and 3; 1H NMR data for
the acyloxy groups: 3-OAc [H 2.10 (3H, s)], 7-OAc [H 2.08 (3H, s)], 8-OiBu [H 2.48 (1H, m), 1.16 (3H, d, J 4.0), and 1.14 (3H, d, J 4.0)], 12-OAc [H 1.73 (3H, s)], 19ONic [H 9.12 (1H, s), 8.74 (1H, br s), 8.22 (1H, d, J 7.9 Hz), and 7.37 (1H, dd, J 7.8, 4.9 Hz)]; 13C NMR data for the acyloxy groups: 3-OAc (C 169.5, 20.8), 7-OAc (C 168.8, 20.5), 8-OiBu (C 176.2, 33.9, 18.8, and 18.6), 12-OAc (C 170.2, 20.7), 19ONic (C 164.8, 153.3, 150.4, 136.9, 126.2, and 123.2); HRESIMS m/z 684.3009 [M H] (calcd for C36H46NO12, 684.3015). Euphornan E (5): clorless oil; []20D 10.7 (c 0.3, MeCN); UV (MeCN) max (log ) 192 (4.55), 258 (3.49) nm; IR (KBr) max 2937, 1734, 1592, 1370, 1230, 1023 cm1; 1H and 13C NMR data for the diterpene moiety see Tables 1 and 3; 1H NMR data for the 8
acyloxy groups: 3-OAc [H 2.11 (3H, s)], 7-OAc [H 2.08 (3H, s)], 8-OMeBu [H 2.33 (1H, dd, J 13.8, 6.9), 1.67 (1H, m), 1.45 (1H, m), 1.16 (3H, d, J 6.9), and 0.89 (3H, t, J 7.5)], 12-OAc [H 1.74 (3H, s)], 19-ONic [H 9.14 (1H, s), 8.76 (1H, br s), 8.24 (1H, d, J 7.9 Hz), and 7.38 (1H, dd, J 7.8, 4.9 Hz)]; 13C NMR data for the acyloxy groups: 3-OAc (C 169.5, 20.7), 7-OAc (C 168.7, 20.6), 8-OMeBu (C 176.1, 40.9, 26.7, 16.1, and 11.5), 12-OAc (C 170.2, 20.7), 19-ONic (C 164.9, 153.3, 150.5, 137.0, 126.3, and 123.3); HRESIMS m/z 698.3174 [M H] (calcd for C37H48NO12, 698.3171). Euphornan F (6): colorless oil; []20D 29.4 (c 0.3, MeCN); UV (MeCN) max (log
) 195 (4.71), 223 (4.28), 262 (3.45) nm; IR (KBr) max 3387, 2929, 1730, 1594, 1371, 1276, 1232, 1023 cm1; 1H and 13C NMR data for the diterpene moiety see Tables 1 and 3; 1H NMR data for the acyloxy groups: 3-OAc [H 2.16 (3H, s)], 8-OBz [H 8.00 (2H, d, J 8.3 Hz), 7.46 (1H, t, J 7.4 Hz), and 7.34 (2H, t, J 7.4 Hz)], 12-OAc [H 1.82 (3H, s)], 19-ONic [H 8.98 (1H, s), 8.67 (1H, br s), 8.02 (1H, d, J 9.9 Hz), and 7.24 (1H, overlap)]; 13C NMR data for the acyloxy groups: 3-OAc (C 170.3, 20.8), 8OBz (C 165.8, 133.4, 129.6 2, 129.3, and 128.5 2), 12-OAc (C 170.3, 20.8), 19ONic (C 164.6, 152.9, 150.4, 136.8, 126.1, and 123.2); HRESIMS m/z 676.2749 [M H] (calcd for C37H42NO11, 676.2752). Euphornan G (7): colorless oil; []20D 40.7 (c 0.3, MeCN); UV (MeCN) max (log
) 194 (4.64), 223 (4.20), 263 (3.38) nm; IR (KBr) max 3445, 2930, 1733, 1593, 1280, 1233, cm1; 1H and 13C NMR data for the diterpene moiety see Tables 1 and 3; 1H NMR data for the acyloxy groups: 7-OAc [H 2.14 (3H, s)], 8-OBz [H 7.93 (2H, d, J 7.7 Hz), 7.44 (1H, t, J 7.4 Hz), and 7.31 (2H, t, J 7.6 Hz)], 12-OAc [H 1.84 (3H, s)], 9
19-ONic [H 8.96 (1H, s), 8.65 (1H, br s), 8.00 (1H, d, J 7.9 Hz), and 7.22 (1H, dd, J 7.8, 5.0 Hz)]; 13C NMR data for the acyloxy groups: 7-OAc (C 169.6, 21.1), 8-OBz (C 165.5, 133.3, 129.6 2, 129.3, and 128.4 2), 12-OAc (C 170.2, 20.8), 19-ONic (C 164.6, 152.9, 150.4, 136.8, 126.1, and 123.1); HRESIMS m/z 676.2751 [M H] (calcd for C37H42NO11, 676.2752). Euphornan H (8): colorless oil; []20D 30.7 (c 0.3, MeCN); UV (MeCN) max (log
) 195 (4.77), 223 (4.32), 257 (3.56) nm; IR (KBr) max 3451, 2932, 1729, 1593, 1371, 1235, 1023 cm1; 1H and 13C NMR data for the diterpene moiety see Tables 1 and 3; 1H
NMR data for the acyloxy groups: 7-OBz [H 8.00 (2H, d, J 7.7 Hz), 7.55 (1H, t,
J 7.2 Hz), and 7.42 (2H, t, J 7.6 Hz)], 8-OAc [H 1.99 (3H, s)], 12-OAc [H 1.75 (3H, s)], 19-ONic [H 9.07 (1H, s), 8.64 (1H, br s), 8.20 (1H, d, J 7.8 Hz), and 7.31 (1H, dd, J 7.5, 5.0 Hz)];
13C
NMR data for the acyloxy groups: 7-OBz (C 165.1,
133.3, 129.6, 129.5 2, and 128.5 2), 8-OAc (C 170.2, 20.9), 12-OAc (C 170.2, 20.7), 19-ONic (C 164.6, 153.1, 150.3, 137.0, 126.2, and 123.3); HRESIMS m/z 676.2756 [M H] (calcd for C37H42NO11, 676.2752). Euphornan I (9): colorless oil; []20D 19.0 (c 0.3, MeCN); UV (MeCN) max (log ) 196 (4.79), 228 (4.31), 273 (3.09) nm; IR (KBr) max 2937, 1737, 1603, 1370, 1227, 1020 cm1; 1H and 13 C NMR data for the diterpene moiety see Tables 1 and 3; 1H NMR data for the acyloxy groups: 3-OAc [H 2.12 (3H, s)], 7-OAc [H 2.10 (3H, s)], 8-OAc [H 2.00 (3H, s)], 12-OAc [H 1.76 (3H, s)], 19-OBz [H 7.97 (2H, d, J 7.1 Hz), 7.53 (1H, t, J 7.3 Hz), and 7.42 (2H, t, J 7.6 Hz)]; 13C NMR data for the acyloxy groups: 3-OAc (C 169.5, 20.9), 7-OAc (C 168.9, 20.6), 8-OAc (C 170.1, 20.9), 12-OAc (C 10
170.2, 20.7), 19-OBz (C 166.2, 132.8, 130.3, 129.4 2, and 128.3 2); HRESIMS m/z 677.2557 [M Na] (calcd for C35H42O12Na, 677.2568). Euphornan J (10): colorless oil; []20D 48.7 (c 0.3, MeCN); UV (MeCN) max (log
) 196 (4.85), 229 (4.46), 273 (3.30) nm; IR (KBr) max 2933, 1739, 1719, 1602, 1370, 1271, 1228 cm1; 1H and 13 C NMR data for the diterpene moiety see Tables 1 and 3; 1H
NMR data for the acyloxy groups: 3-OAc [H 2.12 (3H, s)], 7-OAc [H 2.09 (3H,
s)], 8-OBz [H 7.93 (2H, d, J 7.2 Hz), 7.46 (1H, t, J 7.4 Hz), and 7.33 (2H, t, J 7.7 Hz)], 12-OAc [H 1.77 (3H, s)], 19-OBz [H 7.78 (2H, d, J 7.2 Hz), 7.44 (1H, t, J 7.6 Hz), and 7.28 (2H, t, J 7.2 Hz)]; 13C NMR data for the acyloxy groups: 3-OAc (C 169.5, 20.5), 7-OAc (C 168.7, 20.6), 8-OBz (C 165.5, 133.2, 129.5 2, 129.3, and 128.4 2), 12-OAc (C 170.1, 20.7), 19-OBz (C 165.9, 132.5, 130.1, 129.3 2, and 128.0 2); HRESIMS m/z 739.2729 [M Na] (calcd for C40H44O12Na, 739.2725). Euphornan K (11): colorless crystals (EtOH), mp 156158 C; []20D 21.0 (c 0.3, MeCN); UV (MeCN) max (log ) 195 (4.86), 229 (4.35), 273 (3.24) nm; IR (KBr) max 2933, 1736, 1372, 1270, 1238 cm1; 1H and 13 C NMR data for the diterpene moiety see Tables 2 and 3; 1H NMR data for the acyloxy groups: 3-OAc [H 1.58 (3H, s)], 7-OBz [H 8.05 (2H, d, J 7.8 Hz), 7.61 (1H, t, J 7.4 Hz), and 7.49 (2H, t, J 7.7 Hz)], 8OAc [H 2.02 (3H, s)], 12-OAc [H 1.79 (3H, s)], 19-OBz [H 7.99 (2H, d, J 7.2 Hz), 7.54 (1H, t, J 7.4 Hz), and 7.42 (2H, t, J 7.6 Hz)]; 13C NMR data for the acyloxy groups: 3-OAc (C 169.3, 19.8), 7-OBz (C 164.6, 133.5, 129.9, 129.5 2, and 128.6 2), 8-OAc (C 170.3, 21.0), 12-OAc (C 170.2, 20.7), 19-OBz (C 166.2, 132.9, 130.3, 129.5 2, and 128.3 2); HRESIMS m/z 739.2704 [M Na] (calcd for C40H44O12Na, 11
739.2725). Euphornan L (12): colorless oil; []20D 16.9 (c 0.3, MeCN); UV (MeCN) max (log
) 196 (4.72), 228 (4.17), 273 (2.96) nm; IR (KBr) max 2933, 1740, 1370, 1272, 1232, 1024 cm1; 1H and 13C NMR data for the diterpene moiety see Tables 2 and 3; 1H NMR data for the acyloxy groups: 3-OAc [H 2.13 (3H, s)], 7-OAc [H 2.10 (3H, s)], 8-OiBu [H 2.51 (1H, m), 1.18 (3H, d, J 5.8), and 1.17 (3H, d, J 5.8)], 12-OAc [H 1.72 (3H, s)], 19-OBz [H 7.97 (2H, d, J 7.7 Hz), 7.53 (1H, t, J 7.4 Hz), and 7.41 (2H, t, J 7.7 Hz)]; 13C NMR data for the acyloxy groups: 3-OAc (C 169.5, 20.6), 7-OAc (C 168.9, 20.8), 8-OiBu (C 176.3, 34.0, 18.9, and 18.6), 12-OAc (C 170.2, 20.7), 19-OBz (C 166.3, 132.8, 130.3, 129.4 2, and 128.2 2); HRESIMS m/z 705.2875 [M Na] (calcd for C37H46O12Na, 705.2881). Euphornan M (13): colorless oil; []20D 15.0 (c 0.3, MeCN); UV (MeCN) max (log
) 195 (4.75), 228 (4.20), 272 (3.52) nm; IR (KBr) max 2935, 1738, 1454, 1370, 1231 cm1; 1H and 13C NMR data for the diterpene moiety see Tables 2 and 3; 1H NMR data for the acyloxy groups: 3-OAc [H 2.12 (3H, s)], 7-OAc [H 2.09 (3H, s)], 8-OMeBu [H 2.34 (1H, dd, J 13.8, 6.9), 1.67 (1H, m), 1.46 (1H, m), 1.16 (3H, d, J 6.9), and 0.89 (3H, t, J 7.4)], 12-OAc [H 1.71 (3H, s)], 19-OBz [H 7.97 (2H, d, J 8.1 Hz), 7.53 (1H, t, J 7.4 Hz), and 7.41 (2H, t, J 7.6 Hz)]; 13C NMR data for the acyloxy groups: 3-OAc (C 169.5, 20.6), 7-OAc (C 168.8, 20.8), 8-OMeBu (C 176.2, 40.9, 26.7, 16.2, and 11.5), 12-OAc (C 170.3, 20.7), 19-OBz (C 166.2, 132.8, 130.4, 129.4 2, and 128.2 2); HRESIMS m/z 719.3042 [M Na] (calcd for C38H48O12Na, 719.3038). 12
Euphornan N (14): colorless oil; []20D 30.0 (c 0.3, MeCN); UV (MeCN) max (log
) 197 (4.83), 229 (4.42), 273 (3.21) nm; IR (KBr) max 3363, 2928, 1713, 1643, 1272, 1023 cm1; 1H and 13 C NMR data for the diterpene moiety see Tables 2 and 3; 1H NMR data for the acyloxy groups: 3-OAc [H 2.16 (3H, s)], 8-OBz [H 8.03 (2H, d, J 7.8 Hz), 7.51 (1H, t, J 7.4 Hz), and 7.38 (2H, t, J 7.7 Hz)], 12-OAc [H 1.74 (3H, s)], 19-OBz [H 7.82 (2H, d, J 7.8 Hz), 7.47 (1H, t, J 7.5 Hz), and 7.31 (2H, t, J 7.7 Hz)]; 13C NMR data for the acyloxy groups: 3-OAc (C 170.3, 20.8), 8-OBz (C 165.9, 133.4, 129.7 2, 129.4, and 128.5 2), 12-OAc (C 170.3, 20.7), 19-OBz (C 166.1, 132.6, 130.3, 129.3 2, and 128.1 2); HRESIMS m/z 697.2639 [M Na] (calcd for C38H42O11Na, 697.2619). Euphornan O (15): colorless oil; []20D 57.0 (c 0.3, MeCN); UV (MeCN) max (log
) 197 (4.86), 229 (4.51), 273 (3.33) nm; IR (KBr) max 3461, 2932, 1719, 1452, 1370, 1272, 1230, 1025 cm1; 1H and 13C NMR data for the diterpene moiety see Tables 2 and 3; 1H NMR data for the acyloxy groups: 7-OAc [H 2.13 (3H, s)], 8-OBz [H 7.96 (2H, d, J 7.2 Hz), 7.49 (1H, t, J 6.7 Hz), and 7.35 (2H, t, J 7.8 Hz)], 12-OAc [H 1.76 (3H, s)], 19-OBz [H 7.80 (2H, d, J 7.1 Hz), 7.45 (1H, t, J 7.4 Hz), and 7.30 (2H, t, J 7.8 Hz)]; 13C NMR data for the acyloxy groups: 7-OAc (C 169.6, 21.1), 8OBz (C 165.6, 133.3, 129.6 2, 129.3, and 128.4 2), 12-OAc (C 170.3, 20.7), 19OBz (C 166.0, 132.6, 130.2, 129.3 2, and 128.1 2); HRESIMS m/z 709.2411 [M Cl] (calcd for C38H42O11Cl, 709.2421). Euphornan P (16): amorphous solid; []20D 29.0 (c 0.3, CH2Cl2); UV (MeCN) max (log ) 196 (4.96), 229 (4.49), 273 (3.32) nm; IR (KBr) max 3452, 2925, 1727, 1372, 13
1265, 1021 cm1; 1H and 13 C NMR data for the diterpene moiety see Tables 2 and 3; 1H
NMR data for the acyloxy groups: 7-OBz [H 8.04 (2H, d, J 7.5 Hz), 7.58 (1H, t,
J 7.4 Hz), and 7.46 (2H, t, J 7.6 Hz)], 8-OAc [H 1.99 (3H, s)], 12-OAc [H 1.74 (3H, s)], 19-OBz [H 7.96 (2H, d, J 7.4 Hz), 7.52 (1H, t, J 7.4 Hz), and 7.40 (2H, t, J 7.6 Hz)]; 13C NMR data for the acyloxy groups: 7-OBz (C 165.2, 133.4, 129.7, 129.6 2, and 128.6 2), 8-OAc (C 170.3, 20.9), 12-OAc (C 170.3, 20.6), 19-OBz (C 166.2, 132.8, 130.2, 129.4 2, and 128.2 2); HRESIMS m/z 697.2615 [M Na] (calcd for C38H42O11Na, 697.2619). Euphornan Q (17): colorless oil; []20D 49.3 (c 0.3, MeCN); UV (MeCN) max (log
) 196 (4.70), 230 (4.25), 273 (3.08) nm; IR (KBr) max 2937, 1739, 1369, 1228, 1025 cm1; 1H and 13 C NMR data for the diterpene moiety see Tables 2 and 3; 1H NMR data for the acyloxy groups: 3-OAc [H 2.09 (3H, s)], 7-OAc [H 2.06 (3H, s)], 8-OBz [H 7.96 (2H, d, J 7.2 Hz), 7.53 (1H, t, J 7.3 Hz), and 7.40 (2H, t, J 7.8 Hz)], 12-OAc [H 2.08 (3H, s)], 19-OAc [H 1.65 (3H, s)]; 13C NMR data for the acyloxy groups: 3OAc (C 169.4, 20.7), 7-OAc (C 168.7, 20.5), 8-OBz (C 165.3, 133.2, 129.6 2, 129.5, and 128.4 2), 12-OAc (C 170.1, 21.0), 19-OAc (C 170.3, 20.2); HRESIMS m/z 677.2553 [M Na] (calcd for C35H42O12Na, 677.2568). Euphornan R (18): colorless oil; []20D 16.8 (c 0.3, MeCN); UV (MeCN) max (log
) 196 (4.71), 230 (4.16), 273 (2.88) nm; IR (KBr) max 2930, 1739, 1370, 1245, 1026 cm1; 1H and 13 C NMR data for the diterpene moiety see Tables 2 and 3; 1H NMR data for the acyloxy groups: 3-OAc [H 1.56 (3H, s)], 7-OBz [H 8.03 (2H, d, J 7.7 Hz), 7.61 (1H, t, J 7.2 Hz), and 7.48 (2H, t, J 7.4 Hz)], 8-OAc [H 2.05 (3H, s)], 12-OAc 14
[H 2.14 (3H, s)], 19-OAc [H 2.00 (3H, s)]; 13C NMR data for the acyloxy groups: 3OAc (C 169.3, 19.7), 7-OBz (C 164.5, 133.5, 129.8, 129.5 2, and 128.6 2), 8-OAc (C 170.3, 21.0), 12-OAc (C 170.3, 21.1), 19-OAc (C 170.6, 20.7); HRESIMS m/z 677.2559 [M Na] (calcd for C35H42O12Na, 677.2568). Euphornan S (19): colorless oil; []20D 24.7 (c 0.3, MeCN); UV (MeCN) max (log
) 193 (4.52), 218 (4.11), 258 (3.44) nm; IR (KBr) max 2934, 1736, 1370, 1230, 1024 cm1; 1H and 13C NMR data for the diterpene moiety see Tables 2 and 3; 1H NMR data for the acyloxy groups: 3-OAc [H 1.68 (3H, s)], 7-ONic [H 9.20 (1H, s), 8.81 (1H, br s), 8.30 (1H, d, J 7.7 Hz), and 7.45 (1H, dd, J 7.8, 4.9 Hz)], 8-OAc [H 2.04 (3H, s)], 12-OAc [H 2.12 (3H, s)], 19-OAc [H 1.98 (3H, s)]; 13C NMR data for the acyloxy groups: 3-OAc (C 169.4, 20.0), 7-ONic (C 163.4, 153.8, 150.3, 137.3, 125.8, and 123.6), 8-OAc (C 170.2, 20.9), 12-OAc (C 170.3, 21.0), 19-OAc (C 170.5, 20.6); HRESIMS m/z 656.2701 [M H] (calcd for C34H42NO12, 656.2702). Euphornan T (20): colorless oil; []20D 6.0 (c 0.3, MeCN); UV (MeCN) max (log
) 197 (4.35) nm; IR (KBr) max 2937, 1736, 1368, 1227, 1024 cm1; 1H and 13C NMR data for the diterpene moiety see Tables 2 and 3; 1H NMR data for the acyloxy groups: 3-OAc [H 2.11 (3H, s)], 7-OAc [H 2.08 (3H, s)], 8-OMeBu [H 2.33 (1H, dd, J 13.8, 6.9), 1.67 (1H, m), 1.46 (1H, m), 1.15 (3H, d, J 6.9), and 0.88 (3H, t, J 7.4)], 12OAc [H 2.12 (3H, s)], 19-OAc [H 1.98 (3H, s)]; 13C NMR data for the acyloxy groups: 3-OAc (C 169.5, 20.7), 7-OAc (C 168.8, 20.6), 8-OMeBu (C 176.1, 40.9, 26.7, 16.1, and 11.5), 12-OAc (C 170.3, 21.1), 19-OAc (C 170.7, 20.6); HRESIMS m/z 657.2871 [M Na] (calcd for C33H46O12Na, 657.2881). 15
2.5. X-ray crystal structure analysis Crystallographic data for euphornan A (1). C34H41NO12; M 655.68;orthorhombic, space group P212121 (no. 19), a 10.33870 (10) Å, b 17.30960 (10) Å, c 18.9050 (2) Å, V 3383.22 (5) Å3, Z 4, T 100 K, (Cu Kα) 0.816 mm1, Dcalc 1.287 g/cm3, 33801 reflections measured (6.924° 2Θ 144.238°), 6678 unique (Rint 0.0313, Rsigma 0.0206) which were used in all calculations. The final R1 was 0.0266 (I 2 (I)) and wR2 was 0.0680 (all data). Flack parameter 0.03 (3). Crystallographic data for the structure of 1 have been deposited in the Cambridge Crystallographic Data Centre (deposition number: CCDC 1905570). Crystallographic data for euphornan K (11). C40H44O12; M 716.75; orthorhombic, space group P212121 (no. 19), a 14.88910 (10) Å, b 16.00270 (10) Å, c 17.77030 (10) Å, V 4234.05 (5) Å3, Z 4, T 100 K, (Cu Kα) 0.687 mm1, Dcalc 1.124 g/cm3, 43500 reflections measured (7.434° 2Θ 154.784°), 8828 unique (Rint 0.0297, Rsigma 0.0212) which were used in all calculations. The final R1 was 0.0342 (I 2 (I)) and wR2 was 0.0909 (all data). Flack parameter 0.02 (3). Crystallographic data for the structure of 11 have been deposited in the Cambridge Crystallographic Data Centre (deposition number: CCDC 1913396). 2.6. Cell culture HepG2/ADR (doxorubicin-resistant human liver hepatocellular carcinoma cell line) cells were cultured in RPMI1640 complete growth mediums (Gibco BRL, USA), supplemented with 10 fetal bovine serum (FBS) (Gibco BRL, USA), 100 IU/mL penicillin, and 100 g/mL streptomycin (HyClone, USA). The cells were incubated at 16
37 C in a humidified atmosphere with 5 CO2. 2.7. Cytotoxicity analysis Cytotoxicity was determined using a MTT colorimetric assay as previously described [9]. 2.8. MDR reversal activity HepG2/ADR cells were seeded into 96-well plates at a density of 5 103/well and incubated with premeditated concentration of reversal agents (0.5 M, 1 M, or 5 M) at a carbon dioxide incubator for 1 h, then the cells were incubated with ADR (6 concentration gradients) together for an additional 48 h. The cytotoxicities of ADR with or without reversal agents were analyzed using the MTT assay. Verapamil and Tariquidar were used as positive controls, and DMSO was used as a negative control. The reversal fold is calculated as a ratio of IC50
(ADR)
to IC50
(Pgp inhibitor ADR).
All
experiments were performed at least three times. 2.9. Intracellular accumulation of Rho123 HepG2/ADR cells were seeded into 96-well plates at a density of 5 103well and were incubated with given concentration of compounds and 10 M Rho123 in the dark at 37 C for 90 min. The cells were washed twice with ice-cold PBS after centrifugation, resuspended in PBS buffer, and analyzed by flow cytometry. Tariquidar was evaluated at 5 M and parallel time as a positive control. The fluorescence intensity of intracellular Rho123 was measured with a flow cytometer in FL1 channel (Em 525 nm). Fold of control (The fluorescence of compound treated cell Background)(The fluorescence of Rho123-only cell Background). 17
−
3. Results and discussion The air-dried powder of the seeds of E. marginata was extracted with 95 EtOH at room temperature to give a crude extract, which was suspended in H2O and partitioned with PE. Various column chromatographic separations of the PE extract afforded compounds 120 (Fig. 1). Compound 1 was obtained as colorless crystals. Its molecular formula C34H41NO12 was determined by the HRESIMS ion at m/z 656.2704 [M H] (calcd for 656.2702), corresponding to 15 indices of hydrogen deficiency (IHDs). The 1H NMR spectrum revealed the presence of a nicotinoyl group [H 9.10 (1H, s), 8.71 (1H, br s), 8.21 (1H, d, J 7.9 Hz), and 7.35 (1H, dd, J 7.8, 4.9 Hz)], eight methyl groups [H 2.08 (s), 2.06 (s), 2.04 (s), 1.97 (s), 1.74 (s), 1.28 (s), 0.98 (d, J 7.2 Hz), and 0.88 (d, J 6.8 Hz)], two oxymethylene protons [H 4.28 (1H, d, J 12.0 Hz) and 3.91 (1H, d, J 12.0 Hz)], four oxymethine protons [H 5.25 (1H, d, J 5.5 Hz), 5.23 (1H, s), 4.94 (1H, dd, J 10.2, 3.6 Hz), and 4.57 (1H, d, J 8.7 Hz)], an olefinic proton [H 5.61 (1H, s)], and a series of aliphatic multiplets. The 13C NMR spectrum, in combination with DEPT experiments, resolved 34 carbon resonances attributable to a ketocarbonyl (C 206.8), five ester carbonyls (C 170.1, 170.0, 169.4, 168.9, and 164.7), a pyridine ring [C 153.3, 150.4, 136.9, 126.1, and 123.2], a trisubstituted double bond (C 138.7 and 115.3), eight methyls, two sp3 methylenes (one oxygenated), eight sp3 methines (four oxygenated), two oxygenated sp3 tertiary carbons, and a quaternary carbon. As 11 of the 15 IHDs were accounted for by a ketocarbonyl, five ester carbonyls, a pyridine ring, and a double bond, the remaining four IHDs required that 1 was tetracyclic. The aforementioned 18
information was characteristic of ingol-type diterpenoids [11,12]. The 2D structure of 1 was established by detailed analysis of its 2D NMR data. Two spin systems, H2-1/H2/H-3 (H3-16) and H-7/H-8/H-9/H-11/H-12/H-13/H3-20, were firstly established by the 1H1H
COSY correlations (Fig. 2a). The connectivities of these two fragments, the
hydrogen-free carbons, and the substituents were achieved by analysis of the key HMBC correlations (Fig. 2a). Briefly, the HMBC correlations from H2-1 and H-3 to C4 (C 70.6) and C-15 (C 73.0) generated a five-membered carbon ring with a characteristic 4,15-expoxy ring [13,14]. The cyclopropane ring was located at C-9 and C-11 by HMBC correlations from H3-18 and H2-19 to C-9, C-10, and C-11. The C-4 was linked to C-7 via 5 by HMBC correlations of H-5/C-4 and H3-17/C-6 and C-7. The ketocarbonyl (C-14) was linked to C-13 by HMBC correlations from H-13 and H320 to C-14. Although no direct HMBC correlations were available to connect C-14 to C-15, the still unassigned carbons of C-15 and C-14 as well as the remaining one IHD required the connection between C-14 and C-15 to generate the 11-membered macro ring. Four acetoxyl groups were attached to C-3, C-7, C-8, and C-12, respectively, by HMBC correlations from the oxymethines to the corresponding ester carbonyls. The nicotinoyloxy group was attached to C-19 by HMBC correlation from H2-19 (H 4.28 and 3.91) to the carbonyl of nicotinoyl group. Thus, the gross structure of 1 was established. The relative configuration of 1 was established by analysis of the NOESY data (Fig. 2b) and 1H−1H coupling constants. The chemical shifts of H2-1 (H 2.21 and 1.97) and the coupling constant of H-2H-3 (J 5.5 Hz) indicated that H-2 and H-3 were 19
oriented [15]. In addition, the NOE correlations of H-8/H-7, H-13, and H-19b, and H12/H-19b revealed that H-7, H-8, H-12, and H-13 were cofacial and arbitrarily designated as -oriented. Thus, the NOE correlations of Me-18H-9 and H-11 assigned H-9, H-11, and Me-18 as -oriented. The geometry of the 5(6) double bond was assigned as E by NOE correlation of H-5/7-acetoxyl. Finally, the structure of 1 including absolute configuration (2R,3R,4S,7R,8S,9S,10R,11R,12R,13R,15R) was confirmed by a single crystal X-ray crystallographic analysis using anomalous scattering of Cu K radiation [Flack parameter 0.03 (3)] (Fig. 3). Compound 1 was given the trivial name euphornan A. Compound 2 possessed a molecular formula of C39H43NO12, as determined by the HRESIMS ion at m/z 718.2846 [M H] (calcd for 718.2858). The 1D NMR spectra of 2 were very similar to those of 1, with the only difference being the replacement of an acetyl group in 1 by a benzoyl group in 2. The location of the benzoyl group was assigned at OH-8 by the HMBC correlation from H-8 (H 4.95) to the benzoyl carbonyl (C 165.4). Thus, 2 was assigned as depicted and was given the trivial name euphornan B. Compound 3, a white amorphous powder, possessed a molecular formula of C38H42N2O12 as determined by the HRESIMS ion at m/z 719.2795 [M H] (calcd for 719.2811). The 1D NMR data of 3 were very similar to those of 1 except for the presence of an additional nicotinoyl group in 3 instead of an acetyl group in 1. The additional nicotinoyl group was located at OH-7 by the HMBC correlation from H-7 (H 5.55) to the nicotinoyl carbonyl (C 163.4). Therefore, the structure of 3 was 20
determined as depicted and was named euphornan C. Compound 4 had the molecular formula C36H45NO12, as determined by HRESIMS ion at m/z 684.3009 [M H] (calcd for 684.3015). The NMR data of 4 showed high similarity to those of 1, with the only difference being the presence of an isobutyryl group in 4 instead of the acetyl group in 1. The HMBC correlation from H-8 (H 4.59) to the isobutyryl carbonyl (C 176.2) assigned the isobutyryl group at OH-8. Thus, the structure of 4 was identified as shown and was given the trivial name euphornan D. Compound 5 exhibited a molecular formula of C37H47NO12 as determined by the HRESIMS ion at m/z 698.3174 [M H] (calcd for 698.3171). The 1H and 13C NMR spectra of 5 were very similar to those of 4, with the only difference being the replacement of the isobutyryl group in 4 by a 2-methylbutanoyl group in 5. The location of the 2-methylbutanoyl group was assigned at OH-8 by the HMBC correlation from H-8 (H 4.61) to the 2-methylbutanoyl carbonyl (C 176.1). Compound 5 was thus assigned as depicted and was named euphornan E. The molecular formula of 6 was deduced as C37H41NO11 from its HRESIMS data (m/z 676.2749 [M H], calcd for 676.2752). The NMR data of 6 showed high similarity to those of 2 excepted for the absence of an acetyl group and the upfieldshifted H-7 signal (H 4.40 in 6; H 5.35 in 2), indicating that 6 was a 7-O-deacetylated derivative of 2. The above described deduction was further secured by detailed analyses of its 2D NMR data. Therefore, the structure of 6 was determined as depicted and was given the trivial name euphornan F. Compound 7 displayed a molecular ion at m/z 676.2751 [M H] (calcd for 21
676.2752), consistent with a molecular formula of C37H41NO11. The 1H and 13C NMR data of 7 closely resembled those of 2 except for the absence of an acetyl group and the severely upfield-shifted H-3 signal (H 4.06 in 7; H 5.31 in 2), indicating that 7 was a 3-O-deacetylated derivative of 2. This was further confirmed by detailed 2D NMR analysis. Compound 7 was thus assigned as depicted and was named euphornan G. Compound 8 had the same molecular formula as that of 7. Its 1D NMR data were very similar to those of 7 except for the slight differences arising from H-7 (H 5.33 in 7; H 5.55 in 8) and H-8 (H 4.97 in 7; H 4.72 in 8), suggesting that the locations of the acetyl group at OH-7 and the benzoyl group at OH-8 in 7 were switched in 8. This was further confirmed by the HMBC correlations from H-7 to the benzoyl carbonyl (C 165.1) and from H-8 to the acetyl carbonyl (C 170.2). Hence, the structure of 8 was determined as shown and was given the trivial name euphornan H. Compound 9 exhibited a molecular formula of C35H42O12, as determined by HRESIMS ion at m/z 677.2557 [M Na] (calcd for 677.2568). The 1D NMR data of 9 showed high similarity to those of 1, with the only difference being the occurrence of a benzoyl group in 9 instead of a nicotinoyl group in 1. The benzoyl group was located at OH-19 by the HMBC correlation from H2-19 (H 4.30 and 3.91) to the benzoyl carbonyl (C 166.2). Therefore, compound 9 was determined as depicted and named euphornan I. Compounds 10 and 11 shared the same molecular formula C40H44O12, as established by HRESIMS. The NMR spectra of them were very similar to those of 9, with the main difference being the presence of an additional benzoyl group in 10 and 11 instead of 22
the acetyl group in 9. The positions of benzoyl group were assigned at OH-8 and OH7 in 10 and 11, respectively, by the HMBC correlations from the oxymethines to corresponding ester carbonyls. The structure of 11 including the absolute configuration (2R,3R,4S,7R,8S,9S,10R,11R,12R,13R,15R) was further confirmed by a single crystal X-ray crystallographic analysis using anomalous scattering of Cu K radiation [Flack parameter 0.02 (3)]. Hence, the structures of 10 and 11 were established as depicted and were given the trivial names euphornan J and euphornan K, respectively. Compound 12 had a molecular formula of C37H46O12, as determined by the HERSIMS ion at m/z 705.2875 [M Na] (calcd for 705.2881). The 1H and 13C NMR data of 12 were very similar to those of 9 except for the appearance of an isobutyryl group in 12 instead of the acetyl group in 9. The isobutyryl group was located at OH-8 by the HMBC correlation from H-8 (H 4.61) to the isobutyryl carbonyl (C 176.3). Thus, the structure of 12 was defined as shown and was named euphornan L. Compound 13 gave a [M Na] ion at m/z 719.3042 (calcd for 719.3038) in the HRESIMS, corresponding to the molecular formula C38H48O12. The 1D NMR data of 13 bore a resemblance to those of 9 except for the presence of a 2-methylbutanoyl group in 13 instead of the acetyl group in 9. The 2-methylbutanoyl group was located at OH8 by the HMBC correlation from H-8 (H 4.61) to the 2-methylbutanoyl carbonyl (C 176.2). Thus, 13 was assigned as depicted and was given the trivial name euphornan M. Compounds 1416 had the same molecular formula of C38H42O11, as determined by the HERSIMS data. The 1D NMR spectra of these isomers bore a high resemblance, 23
with the differences being due to the different locations of two benzoyl groups and two acetyl groups. For 14, the HMBC correlations from H-3 (H 5.36) and H-12 (H 5.05) to acetyl carbonyls at C 170.3 and 170.3, respectively, located the acetyl groups at OH3 and OH-12, while HMBC correlations from H-8 (H 4.82) and H2-19 (H 4.28 and 4.03) to the benzoyl carbonyls at C 165.9 and 166.1, respectively, positioned the benzoyl groups at OH-8 and OH-19. Similarly, in 15, two acetyl groups were located at OH-7 and OH-12, and the two benzoyl groups were attached at OH-8 and OH-19. For 16, two acetyl groups were located at OH-8 and OH-12, and two benzoyl groups were located at OH-7 and OH-19. Thus, compounds 1416 were deduced as depicted, and were given the trivial names euphornan N, euphornan O, and euphornan P, respectively. Compounds 17 and 18 possessed the same molecular formula C35H42O12 as that of 9. Analysis of their 1D NMR data revealed that they are structurally related isomers differentiating in acyl locations. In comparison with 9, the 8-OAc and 19-OBz in 9 were switched in 17, as indicated by the HMBC correlations from H-8 (H 4.88) to the benzoyl carbonyl (C 165.3) and from H2-19 (H 4.05 and 3.72) to the acetyl carbonyl (C 170.3). The 7-OAc and 19-OBz in 9 were switched in 18, as indicated by the HMBC correlations from H-7 (H 5.45) to the benzoyl carbonyl (C 164.5) and from H2-19 (H 4.25 and 3.61) to the acetyl carbonyl (C 170.6). Therefore, 17 and 18 were determined as depicted, and were named euphornan Q and euphornan R, respectively. Compound 19 had the molecular formula C34H41NO12, as determined by HRESIMS ion at m/z 656.2701 [M H] (calcd for 656.2702). The NMR spectra of 19 were very 24
similar to those of 18 except for the replacement of the benzoyl group in 18 by a nicotinyl group in 19. A HMBC correlation from H-7 (H 5.49) to the nicotinyl carbonyl (C 163.4) positioned the nicotinyl group at OH-7. The structure of compound 19 was thus determined as shown and was named euphornan S. Compound 20 exhibited a molecular formula of C33H46O12, as established by HRESIMS ion at m/z 657.2871 [M Na] (calcd for 657.2881). The 1H and 13C NMR spectra of 20 were very similar to those of 17, with main difference being the appearance of a 2-methylbutanoyl group instead of a benzoyl group in 17. The location of the 2-methylbutanoyl group was assigned at OH-8 by the HMBC correlation from H-8 (H 4.57) to the 2-methylbutanoyl carbonyl (C 176.1). Thus, the structure of 20 was determined as shown and was given a trivial name euphornan T. Compounds 120 shared the same relative configuration as indicated by their similar 1D NMR and NOESY data. Furthermore, the 5(6) double bond in 220 was assigned to have the same geometry by the NOE correlation between H-5 and 7-acyloxy and/or by comparing their chemical shifts of C-6 and C-7 with those of 1. Their absolute configurations were further established by CD analysis [16,17] (Supporting Information, Fig. S1). In the 190400 nm region, the ECD curves for all compounds showed a similar tendency, with Cotton effects being around 307 nm (), 260 nm (), 226 nm (), and 197 nm (). With the absolute configurations of 1 and 11 in hand, the absolute configurations of 120 were assigned as 2R, 3R, 4S, 7R, 8S, 9S, 10R, 11R, 12R, 13R, 15R, which were consistent with the biogenetic origin of the ingol diterpenoids [5,14]. 25
As lathyrane diterpenoids were widely reported as P-glycoprotein (Pgp) inhibitors in multidrug resistance (MDR) cancer therapy [18,19], compounds 120 were screened for their reversal ability on Pgp-dependent MDR cancer cell line HepG2/ADR. Firstly, the intrinsic cytotoxicities of 120 were determined using the MTT method, and all compounds showed no obvious cytotoxicity (IC50 50 M) in HepG2/ADR cell line. Then, the MDR reversal assay was performed by combination of 5 M of tested compounds with various concentrations of anticancer drug adriamycin (ADR). Verapamil (Vrp) and tariquidar (Tar), the first- and third-generation Pgp inhibitors, respectively, were used as positive controls. As shown in Fig. 4A, the co-administration of most of the tested compounds significantly enhanced the cytotoxicity of ADR, and 14 compounds (2, 46, 915, 17, 18, and 20) exhibited greater reversal activity than verapamil (detailed data see Supporting Information, Table S1). Among them, 11, 14, and 18 with comparable activity to tariquidar were further chosen for a doseeffect dependence study at the concentrations of 0.5, 1, and 5 M. As shown in Fig. 4B, all the three compounds exhibited a good dose-dependent manner, and reversed the sensitivity of anticancer drug adriamycin to ca. 20 folds at 5 M. To further confirm the MDR reversal ability of these compounds were related to Pgp modulation mechanism, 14 was selected for the Rho123 efflux assay. As shown in Fig. 4C, 14 could dose-dependently increase the intracellular accumulation of Rho-123, a substrate of Pgp efflux pump, suggesting that compounds reversed the sensitivity of MDR cancer cell line via the inhibition of Pgp. The different substitution patterns of these ingol diterpenoids made them a good set 26
of homologues to evaluate structureactivity relationships (Fig. 5). In general, the acylation of OH-3 was beneficial to the activity, as shown by 2 vs 7, 10 vs 15, and 11 vs 16. On the contrary, the acylation of OH-7 led to a decrease in activity (6 vs 2 and 14 vs 10), and the benzoyl at OH-7 showed greater activity than acetyl at OH-7 (11 vs 9). The acylation patterns of OH-8 showed little influence on the activity, as shown by 13 vs 12 and 10. Comparison of 14 vs 6, 13 vs 5, and 15 vs 7, it can be inferred that the replacement of nicotinoyl by benzoyl at OH-19 remarkably improved the activity. 4. Conclusions In summary, twenty new ingol diterpenoids (120) were isolated from the seeds of Euphorbia marginata, which is a chemically rarely investigated Euphorbiaceae plant. Although more than 100 ingols have been reported from Euphorbia species so far, most of them possess a -oriented H-2 and H-3 on the five-membered carbon ring and bear the geminal methyls (Me-18 and Me-19) at C-10 of the cyclopropane ring. 120 stand for a rare C-19-oxidated and H-2, H-3 -oriented subgroup in this diterpenoid class. It is possible that their peculiar structures render them the potent MDR reversal activity. The current study not only enriched the structural categories of ingol class, but also provided a potential structural motif in future MDR drug development. Declaration of competing interest The authors declare no competing financial interest. Acknowledgments The authors thank the Natural Science Foundation of China (81722042, 81573302, and 81973195) for providing financial support for this work. Appendix A. Supporting Information MS, IR, 1D and 2D NMR spectra for compounds 120. Supplementary data 27
associated with this article can be found online at
28
References [1] Q.W. Shi, X.H. Su, H. Kiyota, Chemical and pharmacological research of the plants in genus Euphorbia, Chem. Rev. 108 (2008) 42954327. [2] A. Vasas, J. Hohmann, Euphorbia diterpenes: isolation, structure, biological activity, and synthesis (20082012), Chem. Rev. 114 (2014) 85798612. [3] V. Ravikanth, V.L.N. Reddy, T.P. Rao, P.V. Diwan, S. Ramakrishna, Y. Venkateswarlu, Macrocyclic diterpenes from Euphorbia nivulia, Phytochemistry 59 (2002) 331335. [4] M.O. Fatope, L. Zeng, J.E. Ohayagha, J.L. McLaughlin, New 19-acetoxyingol diterpenes from the latex of Euphorbia poisonii (Euphorbiaceae), Bioorg. Med. Chem. Lett. 4 (1996) 16791683. [5] N.D. Zhao, X. Ding, Y. Song, D.Q. Yang, H.L. Yu, T.A. Adelakun, W.D. Qian, Y. Zhang, Y.T. Di, F. Gao, X.J. Hao, S.L. Li, Identification of ingol and rhamnofolane diterpenoids from Euphorbia resinifera and their abilities to induce lysosomal biosynthesis, J. Nat. Prod. 81 (2018) 12091218. [6] B. Sarkadi, L. Homolya, G. Szakács, A. Váradi, Human multidrug resistance ABCB and ABCG transporters: participation in a chemoimmunity defense system, Physiol. Rev. 86 (2006) 1179−1236. [7] Flora of China Editorial Committee, Flora of China. Science Press: Beijing, 1997, Vol. 44, pp 55. [8] K.B. Sarah, G.R. Michael, Concerted use of homo- and heteronuclear 2D NMR in the elucidation of the structure of a novel pentaester of 19-hydroxyingol from 29
Euphorbia marginata seeds, Magn. Reson. Chem. 30 (1992) 632636. [9] J.Y. Zhu, R.M. Wang, L.L. Lou, W. Li, G.H. Tang, X.Z. Bu, S. Yin, Jatrophane diterpenoids as modulators of P-glycoprotein-dependent multidrug resistance (MDR): advances of structureactivity relationships and discovery of promising MDR reversal agents, J. Med. Chem. 59 (2016) 63536369. [10] W. Li, Y.Q. Tang, J. Sang, G.H. Tang, S. Yin, Jatrofolianes A and B, two highly modified lathyrane diterpenoids from Jatropha gossypiifolia, Org. Lett. DOI: 10.1021/acs.orglett.9b04029. [11] H.J. Opferkuch, E. Hecker, Ingol-a new macrocyclic diterpene alcohol from Euphorbia ingens, Tetrahedron Lett. 37 (1973) 36113614. [12] L.J. Lin, A.D. Kinghorn, 8-methoxyingol esters from the latex of Euphorbia hermentiana, Phytochemistry 22 (1983) 27952799. [13] W.Y. Qi, W.Y. Zhang, Y. Shen, Y. Leng, K. Gao, J.M. Yue, Ingol-type diterpenes from Euphorbia antiquorum with mouse 11-hydroxysteroid dehydrogenase type 1 inhibition activity, J. Nat. Prod. 77 (2014) 14521458. [14] X.L. Li, Y. Li, S.F. Wang, Y.L. Zhao, K.C. Liu, X.M. Wang, Y.P. Yang, Ingol and ingenol diterpenes from the aerial parts of Euphorbia royleana and their antiangiogenic activities, J. Nat. Prod. 72 (2009) 10011005. [15] J.A. Marco, J.F. Sanz-Cervera, A. Yuste, Ingenane and lathyrane diterpenes from the latex of Euphorbia canariensis, Phytochemistry 45 (1997) 563570. [16] S. Y. Wang, G.Y. Li, K. Zhang, H.Y. Wang, H.G. Liang, C. Huang, J. Huang, J.H. Wang, B.F. Yang, New ingol-type diterpenes from the latex of Euphorbia 30
resinifera, J. Asian. Nat. Prod. Res. 21 (2019) 10751082. [17] Z.Y. Yin, Y. Dai, P. Hua, Z.J. Sun, Y.F. Cheng, S.H. Yuan, Z.Y. Chen, Q. Gu, Discovery of diverse diterpenoid scaffolds from Euphorbia antiquorum and their activity against RANKL-induced osteoclastogenesis, Bioorg. Chem. 92 (2019) 103292. [18] M. Reis, R.J. Ferreira, M.M.M. Santos, D.J.V.A. dos Santos, J. Molnar, M.J.U. Ferreira, Enhancing macrocyclic diterpenes as multidrug-resistance reversers: structure−activity studies on Jolkinol D derivatives, J. Med. Chem. 56 (2013) 748760. [19] W. Jiao, Z.M. Wan, S. Chen, R.H. Lu, X.Z. Chen, D.M. Fang, J.F. Wang, S.C. Pu, X. Huang, H.X. Gao, H.W. Shao, Lathyrol diterpenes as modulators of Pglycoprotein dependent multidrug resistance: structure-activity relationship studies on Euphorbia factor L3 derivatives, J. Med. Chem. 58 (2015) 37203738.
31
20
O 2 15
16
3
O
19
11
7
6
OAc H
O
OBz
OAc H
O
OAc
18
H
8
5
ONic
10
9
17
4
R 1O
1 2 3 4 5 6 7 8
13 12
14
1
OAc H
H
O
OR3
R 2O R1
R2
R3
Ac Ac Ac Ac Ac Ac H H
Ac Ac Nic Ac Ac H Ac Bz
Ac Bz Ac iBu MeBu Bz Bz Ac
OR3
R 1O
9 10 11 12 13 14 15 16
R2
Ac Ac Ac Ac Ac Ac H H
Ac Ac Bz Ac Ac H Ac Bz
OR3
R 1O
R 2O
R1
H
O
R3
R1 Ac Ac Ac Ac
17 18 19 20
Ac Bz Ac iBu MeBu Bz Bz Ac
R 2O R2 Ac Bz Nic Ac
O
O
O
O
R3 Bz Ac Ac MeBu
N iBu
MeBu
Bz
Nic
Fig. 1. Structures of compounds 120.
N O
O 14
1 16
13
2
O 3
O
12
4
O 18
H
9
17
16
8 6
5
19
10
11
15
O
20
O
O
20
7
O
O
O
1
14 15
2 3
H
4
OAc
O
AcO H
13
5
12
H
11
17
O
H
6
7
H
H H
H 8
OAc
9
19
10
ONic H
18
H OAc
O a
Fig. 2. Key 1H1H COSY (
b
) and HMBC (
correlations of 1 (b).
32
) correlations of 1 (a); Key NOE (
)
C20 C20
C12 C12
C1
C15
C1
C13 C19
C10
C3
C3
C4 C5
C6
C7
C17
C4 C5
C9 C6 C7
C8
1
C8
11
Fig. 3. ORTEP diagram of 1 and 11.
33
C19
C10 C18
C18 C9
C17
C11
C16
C2 C16
C14
C13
C2
C11
C14
C15
Fig. 4. (A) Co-administration of compounds (at 5 M) enhanced the cytotoxicity of anticancer drug adriamycin (ADR) on HepG2/ADR. (B) The reversal effects of 11, 14, and 18 on the sensitivity of HepG2/ADR at different concentrations (0.5, 1, and 5 M). Reversal fold is calculated as a ratio of IC50 (ADR) to IC50 (ADR Cmpd). *P 0.05, **P 0.01, and ***P 0.001 (C) Inhibitory effects of 14 on the accumulation of rhodamin123 (Rho-123) in HepG2/ADR.
34
Fig. 5. SARs of ingol diterpenoids on Pgp inhibition
35
Table 1. 1H NMR data for the diterpene moiety of compounds 110 in CDCl3 ( in ppm, J in Hz) No.
1a
2a
3a
4a
5a
6b
7b
8a
9a
10a
1
2.21, dd (13.4,
2.27, dd (13.5,
2.26, dd (13.5,
2.24, dd (13.4,
2.25, dd (13.5,
2.29, dd (13.3,
2.26, dd (16.3,
2.24, dd (16.0,
2.25, dd (13.6,
2.27, dd (13.5,
10.4)
10.4)
10.4)
10.6)
10.6)
10.7)
13.2)
13.4)
10.4)
10.4)
1.97, m
2.02, m
2.00, m
1.99, dd (13.6,
2.00, dd (13.5,
2.02, dd (13.2,
1.97, m
1.93, m
2.01, m
2.02, dd (13.6,
6.9)
7.0)
7.1)
1
6.9)
2
2.08, m
2.14, m
2.11, m
2.11, m
2.12, m
2.14, m
1.98, m
1.93, m
2.13, m
2.14, m
3
5.25, d (5.5)
5.31, d (5.5)
5.25, d (5.5)
5.28, d (5.1)
5.28, d (5.3)
5.36, d (5.3)
4.06, d (3.5)
3.99, d (3.9)
5.29, d (5.6)
5.30, d (5.5)
5
5.61, s
5.70, s
5.77, s
5.64, s
5.63, s
5.93, s
6.07, s
6.20, s
5.65, s
5.70, s
7
5.23, s
5.35, s
5.55, s
5.27, s
5.26, s
4.40, s
5.33, s
5.55, s
5.28, s
5.36, s
8
4.57, d (8.7)
4.95, d (10.7)
4.74, d (8.9)
4.59, d (9.1)
4.61, d (8.3)
4.84, d (10.7)
4.97, d (10.6)
4.72, d (10.5)
4.62, d (10.0)
4.94, d (10.7)
9
1.46, d (9.8)
1.67, m
1.61, m
1.50, m
1.52, m
1.86, t (10.0)
1.73, m
1.66, m
1.49, m
1.64, m
11
1.46, d (9.8)
1.54, m
1.59, m
1.51, m
1.52, m
1.52, t (10.1)
1.53, m
1.54, m
1.48, m
1.53, m
12
4.94, dd (10.2,
5.06, dd (10.7,
5.03, dd (10.3,
4.97, dd (10.3,
4.98, dd (10.7,
5.04, dd (10.9,
5.06, dd (10.9,
4.99, dd (10.9,
5.00, m
5.07, dd (10.7,
3.6)
3.8)
3.7)
3.5)
3.3)
3.0)
3.6)
3.3)
13
2.86, m
3.02, m
2.96, m
2.89, m
2.91, m
2.98, m
3.03, m
2.98, m
2.93, m
3.01, m
16
0.88, d (6.8)
0.94, d (6.8)
0.89, d (6.8)
0.91, d (6.8)
0.92, d (6.8)
0.95, d (6.6)
1.04, d (6.5)
0.98, d (6.2)
0.92, d (6.9)
0.93, d (6.9)
17
2.04, s
2.16, s
2.18, s
2.08, s
2.10, s
2.10, s
2.17, s
2.16, s
2.09, s
2.16, s
18
1.28, s
1.37, s
1.36, s
1.31, s
1.34, s
1.35, s
1.36, s
1.30, s
1.32, s
1.37, s
19a
4.28, d (12.0)
4.26, d (12.3)
4.36, d (12.0)
4.39, d (12.0)
4.39, d (12.0)
4.29, d (12.1)
4.28, d (12.1)
4.34, d (11.9)
4.30, d (12.0)
4.24, d (12.1)
19b
3.91, d (12.0)
4.07, d (12.1)
3.99, d (12.0)
3.87, d (12.0)
3.89, d (12.0)
4.07, d (12.0)
4.05, d (12.2)
3.95, d (12.0)
3.91, d (12.0)
4.03, d (12.1)
20
0.98, d (7.2)
1.07, d (7.2)
1.06, d (7.2)
1.01, d (7.2)
1.02, d (7.2)
1.07, d (7.0)
1.06, d (7.2)
1.01, d (7.1)
1.03, d (7.3)
1.06, d (7.2)
aat
400 MHz; bat 500 MHz.
36
3.8)
Table 2. 1H NMR data for the diterpene moiety of compounds 1120 in CDCl3 ( in ppm, J in Hz) No.
11a
12a
13b
14a
15a
16a
17a
18b
19a
20b
1
2.24, dd (13.4,
2.26, dd (13.2,
2.26, dd (13.6,
2.29, dd (13.3,
2.26, dd (16.4,
2.24, dd (16.1,
2.24, dd (13.5,
2.23, m
2.24, dd (13.5,
2.26, dd (13.7,
10.3)
10.4)
10.7)
10.5)
13.1)
13.2)
10.4)
10.3)
10.7)
2.01, m
2.01, dd (13.6,
2.01, dd (13.8,
2.02, m
1.96, m
1.94, m
2.01, dd (13.5,
2.00, m
2.01, m
2.02, m
7.0)
7.0)
1
6.9)
2
2.12, m
2.14, m
2.13, m
2.15, m
1.98, m
1.94, m
2.12, m
2.11, m
2.10, m
2.12, m
3
5.25, d (5.6)
5.30, d (5.1)
5.29, d (5.4)
5.36, d (5.4)
4.04, d (4.8)
3.99, d (4.6)
5.28, d (5.7)
5.24, d (5.3)
5.23, d (5.3)
5.29, d (5.5)
5
5.77, s
5.66, s
5.64, s
5.94, s
6.06, s
6.18, s
5.66, s
5.74, s
5.73, s
5.62, s
7
5.49, s
5.30, s
5.27, s
4.42, s
5.35, s
5.55, s
5.31, s
5.45, s
5.49, s
5.23, s
8
4.75, d (10.6)
4.61, d (9.6)
4.61, d (9.5)
4.82, d (10.7)
4.96, d (10.8)
4.74, d (10.6)
4.88, d (10.8)
4.69, d (10.7)
4.68, d (9.4)
4.56, d (9.6)
9
1.67, m
1.50, m
1.52, m
1.84, m
1.71, m
1.66, m
1.56, m
1.59, m
1.50, m
1.44, m
11
1.58, m
1.51, m
1.50, m
1.51, m
1.52, m
1.52, m
1.44, m
1.50, m
1.49, m
1.44, m
12
5.05, dd (10.8,
5.00, dd (9.7,
4.99, dd (9.5,
5.05, dd (11.0,
5.07, dd (11.0,
5.02, dd (11.0,
4.99, dd (10.8,
4.98, d (11.0)
4.96, dd (10.5,
4.93, dd (9.7,
3.9)
4.2)
3.9)
3.6)
3.8)
3.7)
3.9)
3.8)
3.9)
13
3.00, m
2.93, m
2.92, m
2.97, m
3.02, m
3.00, m
2.97, m
2.98, m
2.94, m
2.90, m
16
0.87, d (6.9)
0.93, d (6.8)
0.93, d (6.9)
0.94, d (6.8)
1.03, d (6.6)
0.98, d (6.3)
0.91, d (6.9)
0.87, d (6.7)
0.87, d (6.6)
0.93, d (6.9)
17
2.18, s
2.11, s
2.11, s
2.10, s
2.18, s
2.17, s
2.13, s
2.16, s
2.15, s
2.10, s
18
1.37, s
1.33, s
1.34, s
1.35, s
1.36, s
1.32, s
1.18, s
1.20, s
1.17, s
1.17, s
19a
4.34, d (12.0)
4.38, d (12.0)
4.37, d (12.0)
4.28, d (12.1)
4.27, d (12.1)
4.32, d (12.0)
4.05, d (12.2)
4.25, d (12.0)
4.21, d (12.0)
4.31, d (12.1)
19b
3.97, d (12.0)
3.83, d (12.0)
3.84, d (12.0)
4.03, d (12.0)
4.01, d (12.1)
3.92, d (12.0)
3.72, d (12.2)
3.61, d (12.1)
3.59, d (12.0)
3.46, d (12.1)
20
1.06, d (7.2)
1.03, d (7.2)
1.03, d (7.2)
1.06, d (7.2)
1.05, d (7.3)
1.02, d (7.2)
1.06, d (7.3)
1.09, d (7.1)
1.07, d (7.1)
1.06, d (7.2)
aat
400 MHz; bat 500 MHz.
37
Table 3. 13C NMR data for the diterpene moiety of compounds 120 in CDCl3 ( in ppm) No.
1a
2a
3a
4a
5a
6b
7b
8a
9a
10a
11a
12a
13b
14a
15a
16a
17a
18b
19a
20b
1
32.7
32.8
32.8
32.7
32.8
32.9
32.2
32.2
32.8
32.9
32.9
32.8
32.7
32.9
32.2
32.2
32.8
32.8
32.7
32.7
2
32.5
32.6
32.6
32.5
32.6
32.6
33.0
32.9
32.6
32.6
32.7
32.6
32.6
32.6
33.0
32.9
32.5
32.6
32.5
32.6
3
76.9
77.0
76.8
77.0
77.1
77.1
76.6
76.1
77.1
77.1
76.8
77.1
77.1
77.1
76.6
76.3
77.0
76.7
76.8
77.1
4
70.6
70.6
70.6
70.7
70.7
71.2
72.5
72.7
70.7
70.7
70.8
70.7
70.8
71.2
72.5
72.6
70.6
70.8
70.6
70.7
5
115.3
115.3
115.8
115.3
115.2
115.0
116.8
117.4
115.4
115.3
115.2
115.3
115.1
115.0
116.7
117.1
115.3
115.1
115.7
115.1
6
138.7
139.1
138.5
138.9
139.2
141.2
137.9
137.3
139.0
139.2
139.1
139.0
139.2
141.1
137.9
137.5
139.0
138.9
138.5
139.1
7
75.8
76.4
76.9
75.7
76.1
76.0
76.9
76.5
76.0
76.4
76.7
75.8
76.1
75.9
76.9
76.6
76.4
76.7
77.0
76.1
8
70.6
70.8
70.8
70.7
70.6
73.7
71.0
71.1
70.7
70.9
70.8
70.7
70.6
74.0
71.1
71.0
70.8
70.7
70.7
70.5
9
24.7
25.4
25.2
24.7
24.8
24.1
25.5
25.2
24.9
25.3
25.3
24.7
24.8
24.0
25.3
25.2
25.2
25.1
25.0
24.7
10
22.0
22.2
22.2
22.1
22.2
22.1
22.3
22.0
22.3
22.4
22.3
22.3
22.3
22.3
22.5
22.2
22.1
22.1
22.1
22.0
11
30.8
31.0
31.0
31.0
31.0
31.2
31.1
31.0
30.8
31.0
31.0
31.0
30.9
31.2
31.0
30.9
31.0
31.0
31.0
31.0
12
68.8
69.0
68.8
68.9
68.9
69.2
69.2
69.1
68.8
68.9
68.8
68.8
68.8
69.1
69.1
69.0
69.0
69.0
68.9
69.1
13
43.6
43.9
43.7
43.7
43.7
43.9
43.7
43.4
43.8
43.9
43.8
43.7
43.7
43.9
43.7
43.4
43.7
43.6
43.5
43.6
14
206.8
206.9
206.8
206.9
207.0
207.1
207.3
207.2
207.1
207.0
207.2
207.2
207.2
207.2
207.5
207.4
207.0
207.2
206.9
207.2
15
73.0
73.1
73.2
73.1
73.1
73.2
73.3
73.3
73.2
73.2
73.3
73.2
73.1
73.2
73.2
73.2
73.1
73.2
73.1
73.1
16
12.2
12.3
12.2
12.3
12.3
12.3
12.0
12.0
12.3
12.3
12.3
12.3
12.3
12.3
12.0
12.0
12.3
12.3
12.2
12.3
17
17.5
17.5
17.6
17.5
17.5
17.5
17.6
17.6
17.6
17.6
17.5
17.6
17.6
17.6
17.6
17.6
17.5
17.5
17.5
17.5
18
24.4
24.6
24.6
24.5
24.6
24.5
24.6
24.5
24.6
24.7
24.8
24.7
24.7
24.6
24.7
24.6
24.4
24.5
24.4
24.4
19
65.5
65.5
65.5
65.8
65.9
66.2
65.7
65.7
65.1
64.9
65.1
65.3
65.4
65.4
65.1
65.1
64.2
64.4
64.3
64.8
20
12.9
12.8
13.1
13.0
13.1
13.0
13.1
13.1
13.0
12.9
13.0
13.1
13.1
13.1
13.2
13.1
13.0
13.2
13.2
13.2
aat
C
100 MHz; bat 125 MHz
38
Graphical abstract
39
Highlights
Twenty new ingol diterpenoids were isolated from Euphorbia marginata.
11, 14, and 18 were identified as potent multidrug resistance (MDR) modulators.
The mechanism of these MDR modulators was related to Pgp inhibition.
The brief structure-activity relationships of these ingols were discussed.
40
S0045-2068(19)31697-9 https://doi.org/10.1016/j.bioorg.2019.103546 YBIOO 103546
To appear in:
Bioorganic Chemistry
Received Date: Revised Date: Accepted Date:
10 October 2019 19 December 2019 21 December 2019
Please cite this article as: Y. Zhang, R-Z. Fan, J. Sang, Y-J. Tian, J-Q. Chen, G-H. Tang, S. Yin, Ingol diterpenoids as P-glycoprotein-dependent multidrug resistance (MDR) reversal agents from Euphorbia marginata, Bioorganic Chemistry (2019), doi: https://doi.org/10.1016/j.bioorg.2019.103546
This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
© 2019 Published by Elsevier Inc.
Ingol diterpenoids as P-glycoprotein-dependent multidrug resistance (MDR) reversal agents from Euphorbia marginata Yao Zhang,† Run-Zhu Fan,† Jun Sang, Yi-Jing Tian, Jia-Qi Chen, Gui-Hua Tang, and Sheng Yin*
School of Pharmaceutical Sciences, Sun Yat-sen University, Guangzhou, 510006, China *Corresponding author. E-mail: [email protected] Author Contributions †These
authors contributed equally to this work.
1
Abstract Twenty new ingol diterpenoids, euphornans AT (120), representing a rare class of C-19-oxidated and H-2, H-3 -oriented ingols, were isolated from the seeds of Euphorbia marginata. Their structures including absolute configurations were elucidated by extensive spectroscopic analysis, ECD analysis, and single crystal X-ray diffraction. Compounds 120 were screened for the multidrug resistance (MDR) reversal activity on P-glycoprotein (Pgp)-dependent MDR cancer cell line HepG2/ADR, and 11, 14, and 18 were identified as potent MDR modulators that could enhance the efficacy of anticancer drug adriamycin to ca. 20 folds at 5 M. The Pgp inhibition mechanism and brief structureactivity relationships (SARs) of these compounds were also discussed.
Keywords: Euphorbia marginata; Ingol diterpenoids; Multidrug resistance (MDR); MDR modulators
2
1. Introduction Ingols are a subgroup of lathyrane diterpenoids featuring a 5/11/3 carbon ring system with a 4,15-epoxy ring. Until now, more than 100 ingols have been exclusively isolated from the Euphorbia plants (Euphorbiaceae), and most of them shared the basic structural features of cis-fused 5/11/3 ring system, 4,15-epoxy, -oriented H-2 and H3, C-14 ketocarbonyl, and 5, with the structural diversity mainly arising from the various ester substituents on the 11-membered macro ring [1,2]. In recent years, their fascinating structures and intriguing biological activities have attracted considerable interest from the scientists in this research field [3-5]. Multidrug resistance (MDR) designates a phenomenon where resistance to one drug is accompanied by resistance to drugs that are structurally and functionally unrelated. The development of MDR is one of the leading causes of treatment failure in the chemotherapy of malignant tumors. A primary mechanism of MDR is the overproduction of P-glycoprotein (Pgp) in the plasma membranes of resistant cells, where the Pgp acts as an energy-dependent efflux pump, reducing the intracellular accumulation of anticancer drugs [6]. Thus, compounds with Pgp inhibitory activities are considered as promising MDR reversal agents when coadministered with an anticancer drug in cancer therapy. Euphorbia marginata Pursh. (Euphorbiaceae) is an ornamental plant native to south America, but now is naturalized throughout most parts of China [7]. To date, there is only one publication related to the chemistry of this plant, reporting a hydroxyingol derivative from its seeds [8]. As part of our continuing efforts to discover MDR reversal 3
agents from the Euphorbiaceae plants [9,10], we undertook a detailed chemical investigation of E. marginata, which led to the isolation of twenty new ingol diterpenoids. All these compounds were screened for the reversal activity on Pgpdependent MDR cancer cell line HepG2/ADR, and three compounds were identified as potent MDR modulators. Herein, we describe the isolation, structure elucidation, the MDR reversal activities, and the structureactivity relationships of these compounds. 2. Materials and methods 2.1. General experimental procedures Melting points were measured using an X-4 melting instrument and uncorrected. Optical rotations were determined on a Rudolph Autopol I automatic polarimeter. ECD spectra were obtained on an Applied Photophysics Chirascan spectrometer and UV spectra on a Shimadzu UV-2450 spectrophotometer. IR spectra were determined in KBr disks on a Bruker Tensor 37 infrared spectrophotometer. NMR spectra were measured with a Bruker AM-400 spectrometer at 25 C. ESIMS and HRESIMS were carried out on a Waters Micromass Q-TOF spectrometer. X-ray data were obtained on an Agilent Xcalibur Nova X-ray diffractometer. Semipreparative HPLC was performed with a Shimadzu LC-20 AT equipped with an SPD-M20A PDA detector. A YMC-pack ODSA column (250 10 mm, S-5 m, 12 nm) was used for HPLC purification. Silica gel (100−200 and 300−400 mesh, Qingdao Haiyang Chemical Co., Ltd.), MCI gel (CHP20P, 75−150 m, Mitsubishi Chemical Industries Ltd.), reversed-phase C18 (RpC18) silica gel (12 nm, S-50 m, YMC Co., Ltd.), D101 macroporous adsorptive resins (Sinopharm Chemical Reagent Co., Ltd) and Sephadex LH-20 gel (Amersham 4
Biosciences) were used for column chromatography (CC). All solvents were of analytical grade (Guangzhou Chemical Reagents Company, Ltd.). 2.2. Plant material The seeds of Euphorbia marginata were collected in Suqian, Jiangsu Province, P. R. China, in June 2018, and were authenticated by one of the author (G. H. Tang). A voucher specimen (accession number: YBC201806) has been deposited at the School of Pharmaceutical Sciences, Sun Yat-sen University. 2.3. Extraction and isolation The powder of dried seeds E. marginata (7.5 kg) was extracted with 95 EtOH (3 20 L) at room temperature to give 860 g of crude extract. The extract was suspended in H 2 O (3 L) and partitioned with petroleum ether (PE, 3 3 L). The PE extract (470 g) was separated on a D101 macroporous adsorptive resins eluted with a MeOHH 2 O gradient (3:7 10:0) to afford five fractions (Fr. V). Fr. III (4.5 g) was applied to silica gel CC (PEEtOAc, 30:1 0:1) to give four fractions (Fr. IIIaIIId). Separation of Fr. IIIc (2.8 g) by Rp-C 18 silica gel CC eluted with MeOH/H 2 O (4:6 10:0) yielded four fractions (Fr. IIIc1IIIc4). Fr. IIIc1 (184 mg) was chromatographed over silica gel column (PEEtOAc, 20:1 1:1) to give 1 (100 mg) and 19 (27 mg). Fr. IIIc2 (150 mg) was purified on semipreparative HPLC (CH 3 CN/H 2 O, 70:30, 3 mL/min) to give 8 (50 mg, t R 17 min). Fr. IIIc3 (1.6 g) was loaded onto a Sephadex LH-20 column using MeOH as eluent to give 2 (1.4 g). Fr. IIIc4 (280 mg) was subjected to silica gel CC (PEEtOAc, 30:1 0:1), followed by HPLC (MeCN/H 2 O, 70:30, 3 mLmin) afforded 4 (50 5
mg, t R 12.5 min) and 5 (35 mg, t R 15 min). Separation of Fr. IIId (900 mg) by silica gel CC (PEEtOAc, 30:10:1) afforded four fractions (Fr. IIId1IIId4). Fr. IIId3 (50 mg) was purified using HPLC (MeCN/H 2 O, 50:50, 3 mL/min) to give 6 (6 mg, t R 17 min) and 14 (4 mg, t R 19 min). Fr. IIId4 (150 mg) was loaded onto a Sephadex LH-20 column eluted with MeOH, and further purified by HPLC (MeCN/H 2 O, 55:45, 3 mLmin) as mobile phase to afford 7 (3.1 mg, t R 10 min) and 3 (18 mg, t R 12 min). Fractionation of Fr. IV (57g) by silica gel CC (PEEtOAc, 1:0 0:1) led to five fractions (Fr. IVaIVe). Fr. IVc (4.6 g) was subjected to MCI gel CC eluted with a MeOHH O gradient (6:4 10:0) to yield six fractions (Fr. IVc1IVc6). Fr. IVc1 (200 mg) was chromatographed successively over a Sephadex LH-20 eluted with MeOH, and silica gel column (PECH2Cl2, 5:1 0:1) to afford 17 (100 mg) and 18 (15 mg). Fr. IVc2 was loaded onto a Sephadex LH-20 column eluted with MeOH, and further purified by HPLC (MeCN/H 2 O, 65:35, 3 mLmin) to give 9 (18 mg, t R 12 min) and 20 (4 mg, t R 14 min). Fr. IVc3 (112 mg) was applied to silica gel CC (PEEtOAc, 30:1 1:1), followed by Sephadex LH-20 CC using MeOH to yield 15 (18 mg) and 16 (55 mg). Fr. IVc4 (200 mg) was subjected to silica gel CC (PEEtOAc, 25:1 1:1), and further purified by HPLC (MeCN/H 2 O, 60:40, 3 mLmin), to obtain 12 (9.2 mg, t R 15 min), 11 (5.3 mg, t R 16 min), and 13 (5.7 mg, t R 19 min ). Fr. IVc5 (300 mg) was separated by Sephadex LH-20 CC using MeOH to give 10 (50 mg). 2.4. Spectroscopic data
6
Euphornan A (1): colorless crystals (petroleum ether/EtOAc: 5:1), mp 230232 C; []20D 11.3 (c 0.3, MeCN); UV (MeCN) max (log ) 193 (4.59), 262 (3.52) nm; IR (KBr) max 2938, 1735, 1591, 1371, 1228, 1021 cm1; 1H and 13C NMR data for the diterpene moiety see Tables 1 and 3; 1H NMR data for the acyloxy groups: 3-OAc [H 2.08 (3H, s)], 7-OAc [H 2.04 (3H, s)], 8-OAc [H 1.97 (3H, s)], 12-OAc [H 1.74 (3H, s)], 19-ONic [H 9.10 (1H, s), 8.71 (1H, br s), 8.21 (1H, d, J 7.9 Hz), and 7.35 (1H, dd, J 7.8, 4.9 Hz)]; 13C data for the acyloxy groups: 3-OAc (C 169.4, 20.8), 7-OAc (C 168.9, 20.5), 8-OAc (C 170.0, 20.8), 12-OAc (C 170.1, 20.6), 19-ONic (C 164.7, 153.3, 150.4, 136.9, 126.1, and 123.2); HRESIMS m/z 656.2704 [M H] (calcd for C34H42NO12, 656.2702). Euphornan B (2): colorless oil; []20D 36.7 (c 0.3, MeCN); UV (MeCN) max (log
) 195 (4.71), 223 (4.28), 263 (3.45) nm; IR (KBr) max 2935, 1733, 1591, 1370, 1229, 1023 cm1; 1H and 13C NMR data for the diterpene moiety see Tables 1 and 3; 1H NMR data for the acyloxy groups: 3-OAc [H 2.12 (3H, s)], 7-OAc [H 2.10 (3H, s)], 8-OBz [H 7.90 (2H, d, J 6.9 Hz), 7.42 (1H, t, J 7.4 Hz), and 7.29 (2H, t, J 7.7 Hz)], 12OAc [H 1.86 (3H, s)], 19-ONic [H 8.95 (1H, s), 8.65 (1H, br s), 7.99 (1H, d, J 9.0 Hz), and 7.22 (1H, dd, J 7.8, 4.9 Hz)]; 13C NMR data for the acyloxy groups: 3-OAc (C 169.4, 20.7), 7-OAc (C 168.7, 20.5), 8-OBz (C 165.4, 133.2, 129.5 2, 129.1, and 128.3 2), 12-OAc (C 170.1, 20.7), 19-ONic (C 164.5, 152.9, 150.3, 136.7, 126.0, and 123.0); HRESIMS m/z 718.2846 [M H] (calcd for C39H44NO12, 718.2858). Euphornan C (3): white amorphous powder; []20D 14.3 (c 0.3, CH2Cl2); UV (MeCN) max (log ) 194 (4.79), 219 (4.48), 263 (3.86) nm; IR (KBr) max 2922, 1735, 7
1591, 1373, 1277, 1230, 1021 cm1; 1H and 13C NMR data for the diterpene moiety see Tables 1 and 3; 1H NMR data for the acyloxy groups: 3-OAc [H 1.72 (3H, s)], 7-ONic [H 9.23 (1H, s), 8.83 (1H, br s), 8.32 (1H, d, J 8.0 Hz), and 7.46 (1H, dd, J 7.8, 4.9 Hz)], 8-OAc [H 2.04 (3H, s)], 12-OAc [H 1.81 (3H, s)], 19-ONic [H 9.16 (1H, s), 8.77 (1H, br s), 8.26 (1H, d, J 7.9 Hz), and 7.39 (1H, dd, J 7.8, 4.9 Hz)]; 13C NMR data for the acyloxy groups: 3-OAc (C 169.4, 20.0), 7-ONic (C 163.4, 153.9, 150.4, 137.3, 125.8, and 123.6), 8-OAc (C 170.2, 21.0), 12-OAc (C 170.2, 20.7), 19-ONic (C 164.8, 153.4, 150.6, 137.0, 126.2, and 123.3); HRESIMS m/z 719.2795 [M H] (calcd for C38H43N2O12, 719.2811). Euphornan D (4): colorless oil; []20D 13.0 (c 0.3, MeCN); UV (MeCN) max (log
) 193 (4.29), 257 (3.50) nm; IR (KBr) max 2937, 1734, 1591, 1370, 1230, 1023 cm1; 1H
and 13C NMR data for the diterpene moiety see Tables 1 and 3; 1H NMR data for
the acyloxy groups: 3-OAc [H 2.10 (3H, s)], 7-OAc [H 2.08 (3H, s)], 8-OiBu [H 2.48 (1H, m), 1.16 (3H, d, J 4.0), and 1.14 (3H, d, J 4.0)], 12-OAc [H 1.73 (3H, s)], 19ONic [H 9.12 (1H, s), 8.74 (1H, br s), 8.22 (1H, d, J 7.9 Hz), and 7.37 (1H, dd, J 7.8, 4.9 Hz)]; 13C NMR data for the acyloxy groups: 3-OAc (C 169.5, 20.8), 7-OAc (C 168.8, 20.5), 8-OiBu (C 176.2, 33.9, 18.8, and 18.6), 12-OAc (C 170.2, 20.7), 19ONic (C 164.8, 153.3, 150.4, 136.9, 126.2, and 123.2); HRESIMS m/z 684.3009 [M H] (calcd for C36H46NO12, 684.3015). Euphornan E (5): clorless oil; []20D 10.7 (c 0.3, MeCN); UV (MeCN) max (log ) 192 (4.55), 258 (3.49) nm; IR (KBr) max 2937, 1734, 1592, 1370, 1230, 1023 cm1; 1H and 13C NMR data for the diterpene moiety see Tables 1 and 3; 1H NMR data for the 8
acyloxy groups: 3-OAc [H 2.11 (3H, s)], 7-OAc [H 2.08 (3H, s)], 8-OMeBu [H 2.33 (1H, dd, J 13.8, 6.9), 1.67 (1H, m), 1.45 (1H, m), 1.16 (3H, d, J 6.9), and 0.89 (3H, t, J 7.5)], 12-OAc [H 1.74 (3H, s)], 19-ONic [H 9.14 (1H, s), 8.76 (1H, br s), 8.24 (1H, d, J 7.9 Hz), and 7.38 (1H, dd, J 7.8, 4.9 Hz)]; 13C NMR data for the acyloxy groups: 3-OAc (C 169.5, 20.7), 7-OAc (C 168.7, 20.6), 8-OMeBu (C 176.1, 40.9, 26.7, 16.1, and 11.5), 12-OAc (C 170.2, 20.7), 19-ONic (C 164.9, 153.3, 150.5, 137.0, 126.3, and 123.3); HRESIMS m/z 698.3174 [M H] (calcd for C37H48NO12, 698.3171). Euphornan F (6): colorless oil; []20D 29.4 (c 0.3, MeCN); UV (MeCN) max (log
) 195 (4.71), 223 (4.28), 262 (3.45) nm; IR (KBr) max 3387, 2929, 1730, 1594, 1371, 1276, 1232, 1023 cm1; 1H and 13C NMR data for the diterpene moiety see Tables 1 and 3; 1H NMR data for the acyloxy groups: 3-OAc [H 2.16 (3H, s)], 8-OBz [H 8.00 (2H, d, J 8.3 Hz), 7.46 (1H, t, J 7.4 Hz), and 7.34 (2H, t, J 7.4 Hz)], 12-OAc [H 1.82 (3H, s)], 19-ONic [H 8.98 (1H, s), 8.67 (1H, br s), 8.02 (1H, d, J 9.9 Hz), and 7.24 (1H, overlap)]; 13C NMR data for the acyloxy groups: 3-OAc (C 170.3, 20.8), 8OBz (C 165.8, 133.4, 129.6 2, 129.3, and 128.5 2), 12-OAc (C 170.3, 20.8), 19ONic (C 164.6, 152.9, 150.4, 136.8, 126.1, and 123.2); HRESIMS m/z 676.2749 [M H] (calcd for C37H42NO11, 676.2752). Euphornan G (7): colorless oil; []20D 40.7 (c 0.3, MeCN); UV (MeCN) max (log
) 194 (4.64), 223 (4.20), 263 (3.38) nm; IR (KBr) max 3445, 2930, 1733, 1593, 1280, 1233, cm1; 1H and 13C NMR data for the diterpene moiety see Tables 1 and 3; 1H NMR data for the acyloxy groups: 7-OAc [H 2.14 (3H, s)], 8-OBz [H 7.93 (2H, d, J 7.7 Hz), 7.44 (1H, t, J 7.4 Hz), and 7.31 (2H, t, J 7.6 Hz)], 12-OAc [H 1.84 (3H, s)], 9
19-ONic [H 8.96 (1H, s), 8.65 (1H, br s), 8.00 (1H, d, J 7.9 Hz), and 7.22 (1H, dd, J 7.8, 5.0 Hz)]; 13C NMR data for the acyloxy groups: 7-OAc (C 169.6, 21.1), 8-OBz (C 165.5, 133.3, 129.6 2, 129.3, and 128.4 2), 12-OAc (C 170.2, 20.8), 19-ONic (C 164.6, 152.9, 150.4, 136.8, 126.1, and 123.1); HRESIMS m/z 676.2751 [M H] (calcd for C37H42NO11, 676.2752). Euphornan H (8): colorless oil; []20D 30.7 (c 0.3, MeCN); UV (MeCN) max (log
) 195 (4.77), 223 (4.32), 257 (3.56) nm; IR (KBr) max 3451, 2932, 1729, 1593, 1371, 1235, 1023 cm1; 1H and 13C NMR data for the diterpene moiety see Tables 1 and 3; 1H
NMR data for the acyloxy groups: 7-OBz [H 8.00 (2H, d, J 7.7 Hz), 7.55 (1H, t,
J 7.2 Hz), and 7.42 (2H, t, J 7.6 Hz)], 8-OAc [H 1.99 (3H, s)], 12-OAc [H 1.75 (3H, s)], 19-ONic [H 9.07 (1H, s), 8.64 (1H, br s), 8.20 (1H, d, J 7.8 Hz), and 7.31 (1H, dd, J 7.5, 5.0 Hz)];
13C
NMR data for the acyloxy groups: 7-OBz (C 165.1,
133.3, 129.6, 129.5 2, and 128.5 2), 8-OAc (C 170.2, 20.9), 12-OAc (C 170.2, 20.7), 19-ONic (C 164.6, 153.1, 150.3, 137.0, 126.2, and 123.3); HRESIMS m/z 676.2756 [M H] (calcd for C37H42NO11, 676.2752). Euphornan I (9): colorless oil; []20D 19.0 (c 0.3, MeCN); UV (MeCN) max (log ) 196 (4.79), 228 (4.31), 273 (3.09) nm; IR (KBr) max 2937, 1737, 1603, 1370, 1227, 1020 cm1; 1H and 13 C NMR data for the diterpene moiety see Tables 1 and 3; 1H NMR data for the acyloxy groups: 3-OAc [H 2.12 (3H, s)], 7-OAc [H 2.10 (3H, s)], 8-OAc [H 2.00 (3H, s)], 12-OAc [H 1.76 (3H, s)], 19-OBz [H 7.97 (2H, d, J 7.1 Hz), 7.53 (1H, t, J 7.3 Hz), and 7.42 (2H, t, J 7.6 Hz)]; 13C NMR data for the acyloxy groups: 3-OAc (C 169.5, 20.9), 7-OAc (C 168.9, 20.6), 8-OAc (C 170.1, 20.9), 12-OAc (C 10
170.2, 20.7), 19-OBz (C 166.2, 132.8, 130.3, 129.4 2, and 128.3 2); HRESIMS m/z 677.2557 [M Na] (calcd for C35H42O12Na, 677.2568). Euphornan J (10): colorless oil; []20D 48.7 (c 0.3, MeCN); UV (MeCN) max (log
) 196 (4.85), 229 (4.46), 273 (3.30) nm; IR (KBr) max 2933, 1739, 1719, 1602, 1370, 1271, 1228 cm1; 1H and 13 C NMR data for the diterpene moiety see Tables 1 and 3; 1H
NMR data for the acyloxy groups: 3-OAc [H 2.12 (3H, s)], 7-OAc [H 2.09 (3H,
s)], 8-OBz [H 7.93 (2H, d, J 7.2 Hz), 7.46 (1H, t, J 7.4 Hz), and 7.33 (2H, t, J 7.7 Hz)], 12-OAc [H 1.77 (3H, s)], 19-OBz [H 7.78 (2H, d, J 7.2 Hz), 7.44 (1H, t, J 7.6 Hz), and 7.28 (2H, t, J 7.2 Hz)]; 13C NMR data for the acyloxy groups: 3-OAc (C 169.5, 20.5), 7-OAc (C 168.7, 20.6), 8-OBz (C 165.5, 133.2, 129.5 2, 129.3, and 128.4 2), 12-OAc (C 170.1, 20.7), 19-OBz (C 165.9, 132.5, 130.1, 129.3 2, and 128.0 2); HRESIMS m/z 739.2729 [M Na] (calcd for C40H44O12Na, 739.2725). Euphornan K (11): colorless crystals (EtOH), mp 156158 C; []20D 21.0 (c 0.3, MeCN); UV (MeCN) max (log ) 195 (4.86), 229 (4.35), 273 (3.24) nm; IR (KBr) max 2933, 1736, 1372, 1270, 1238 cm1; 1H and 13 C NMR data for the diterpene moiety see Tables 2 and 3; 1H NMR data for the acyloxy groups: 3-OAc [H 1.58 (3H, s)], 7-OBz [H 8.05 (2H, d, J 7.8 Hz), 7.61 (1H, t, J 7.4 Hz), and 7.49 (2H, t, J 7.7 Hz)], 8OAc [H 2.02 (3H, s)], 12-OAc [H 1.79 (3H, s)], 19-OBz [H 7.99 (2H, d, J 7.2 Hz), 7.54 (1H, t, J 7.4 Hz), and 7.42 (2H, t, J 7.6 Hz)]; 13C NMR data for the acyloxy groups: 3-OAc (C 169.3, 19.8), 7-OBz (C 164.6, 133.5, 129.9, 129.5 2, and 128.6 2), 8-OAc (C 170.3, 21.0), 12-OAc (C 170.2, 20.7), 19-OBz (C 166.2, 132.9, 130.3, 129.5 2, and 128.3 2); HRESIMS m/z 739.2704 [M Na] (calcd for C40H44O12Na, 11
739.2725). Euphornan L (12): colorless oil; []20D 16.9 (c 0.3, MeCN); UV (MeCN) max (log
) 196 (4.72), 228 (4.17), 273 (2.96) nm; IR (KBr) max 2933, 1740, 1370, 1272, 1232, 1024 cm1; 1H and 13C NMR data for the diterpene moiety see Tables 2 and 3; 1H NMR data for the acyloxy groups: 3-OAc [H 2.13 (3H, s)], 7-OAc [H 2.10 (3H, s)], 8-OiBu [H 2.51 (1H, m), 1.18 (3H, d, J 5.8), and 1.17 (3H, d, J 5.8)], 12-OAc [H 1.72 (3H, s)], 19-OBz [H 7.97 (2H, d, J 7.7 Hz), 7.53 (1H, t, J 7.4 Hz), and 7.41 (2H, t, J 7.7 Hz)]; 13C NMR data for the acyloxy groups: 3-OAc (C 169.5, 20.6), 7-OAc (C 168.9, 20.8), 8-OiBu (C 176.3, 34.0, 18.9, and 18.6), 12-OAc (C 170.2, 20.7), 19-OBz (C 166.3, 132.8, 130.3, 129.4 2, and 128.2 2); HRESIMS m/z 705.2875 [M Na] (calcd for C37H46O12Na, 705.2881). Euphornan M (13): colorless oil; []20D 15.0 (c 0.3, MeCN); UV (MeCN) max (log
) 195 (4.75), 228 (4.20), 272 (3.52) nm; IR (KBr) max 2935, 1738, 1454, 1370, 1231 cm1; 1H and 13C NMR data for the diterpene moiety see Tables 2 and 3; 1H NMR data for the acyloxy groups: 3-OAc [H 2.12 (3H, s)], 7-OAc [H 2.09 (3H, s)], 8-OMeBu [H 2.34 (1H, dd, J 13.8, 6.9), 1.67 (1H, m), 1.46 (1H, m), 1.16 (3H, d, J 6.9), and 0.89 (3H, t, J 7.4)], 12-OAc [H 1.71 (3H, s)], 19-OBz [H 7.97 (2H, d, J 8.1 Hz), 7.53 (1H, t, J 7.4 Hz), and 7.41 (2H, t, J 7.6 Hz)]; 13C NMR data for the acyloxy groups: 3-OAc (C 169.5, 20.6), 7-OAc (C 168.8, 20.8), 8-OMeBu (C 176.2, 40.9, 26.7, 16.2, and 11.5), 12-OAc (C 170.3, 20.7), 19-OBz (C 166.2, 132.8, 130.4, 129.4 2, and 128.2 2); HRESIMS m/z 719.3042 [M Na] (calcd for C38H48O12Na, 719.3038). 12
Euphornan N (14): colorless oil; []20D 30.0 (c 0.3, MeCN); UV (MeCN) max (log
) 197 (4.83), 229 (4.42), 273 (3.21) nm; IR (KBr) max 3363, 2928, 1713, 1643, 1272, 1023 cm1; 1H and 13 C NMR data for the diterpene moiety see Tables 2 and 3; 1H NMR data for the acyloxy groups: 3-OAc [H 2.16 (3H, s)], 8-OBz [H 8.03 (2H, d, J 7.8 Hz), 7.51 (1H, t, J 7.4 Hz), and 7.38 (2H, t, J 7.7 Hz)], 12-OAc [H 1.74 (3H, s)], 19-OBz [H 7.82 (2H, d, J 7.8 Hz), 7.47 (1H, t, J 7.5 Hz), and 7.31 (2H, t, J 7.7 Hz)]; 13C NMR data for the acyloxy groups: 3-OAc (C 170.3, 20.8), 8-OBz (C 165.9, 133.4, 129.7 2, 129.4, and 128.5 2), 12-OAc (C 170.3, 20.7), 19-OBz (C 166.1, 132.6, 130.3, 129.3 2, and 128.1 2); HRESIMS m/z 697.2639 [M Na] (calcd for C38H42O11Na, 697.2619). Euphornan O (15): colorless oil; []20D 57.0 (c 0.3, MeCN); UV (MeCN) max (log
) 197 (4.86), 229 (4.51), 273 (3.33) nm; IR (KBr) max 3461, 2932, 1719, 1452, 1370, 1272, 1230, 1025 cm1; 1H and 13C NMR data for the diterpene moiety see Tables 2 and 3; 1H NMR data for the acyloxy groups: 7-OAc [H 2.13 (3H, s)], 8-OBz [H 7.96 (2H, d, J 7.2 Hz), 7.49 (1H, t, J 6.7 Hz), and 7.35 (2H, t, J 7.8 Hz)], 12-OAc [H 1.76 (3H, s)], 19-OBz [H 7.80 (2H, d, J 7.1 Hz), 7.45 (1H, t, J 7.4 Hz), and 7.30 (2H, t, J 7.8 Hz)]; 13C NMR data for the acyloxy groups: 7-OAc (C 169.6, 21.1), 8OBz (C 165.6, 133.3, 129.6 2, 129.3, and 128.4 2), 12-OAc (C 170.3, 20.7), 19OBz (C 166.0, 132.6, 130.2, 129.3 2, and 128.1 2); HRESIMS m/z 709.2411 [M Cl] (calcd for C38H42O11Cl, 709.2421). Euphornan P (16): amorphous solid; []20D 29.0 (c 0.3, CH2Cl2); UV (MeCN) max (log ) 196 (4.96), 229 (4.49), 273 (3.32) nm; IR (KBr) max 3452, 2925, 1727, 1372, 13
1265, 1021 cm1; 1H and 13 C NMR data for the diterpene moiety see Tables 2 and 3; 1H
NMR data for the acyloxy groups: 7-OBz [H 8.04 (2H, d, J 7.5 Hz), 7.58 (1H, t,
J 7.4 Hz), and 7.46 (2H, t, J 7.6 Hz)], 8-OAc [H 1.99 (3H, s)], 12-OAc [H 1.74 (3H, s)], 19-OBz [H 7.96 (2H, d, J 7.4 Hz), 7.52 (1H, t, J 7.4 Hz), and 7.40 (2H, t, J 7.6 Hz)]; 13C NMR data for the acyloxy groups: 7-OBz (C 165.2, 133.4, 129.7, 129.6 2, and 128.6 2), 8-OAc (C 170.3, 20.9), 12-OAc (C 170.3, 20.6), 19-OBz (C 166.2, 132.8, 130.2, 129.4 2, and 128.2 2); HRESIMS m/z 697.2615 [M Na] (calcd for C38H42O11Na, 697.2619). Euphornan Q (17): colorless oil; []20D 49.3 (c 0.3, MeCN); UV (MeCN) max (log
) 196 (4.70), 230 (4.25), 273 (3.08) nm; IR (KBr) max 2937, 1739, 1369, 1228, 1025 cm1; 1H and 13 C NMR data for the diterpene moiety see Tables 2 and 3; 1H NMR data for the acyloxy groups: 3-OAc [H 2.09 (3H, s)], 7-OAc [H 2.06 (3H, s)], 8-OBz [H 7.96 (2H, d, J 7.2 Hz), 7.53 (1H, t, J 7.3 Hz), and 7.40 (2H, t, J 7.8 Hz)], 12-OAc [H 2.08 (3H, s)], 19-OAc [H 1.65 (3H, s)]; 13C NMR data for the acyloxy groups: 3OAc (C 169.4, 20.7), 7-OAc (C 168.7, 20.5), 8-OBz (C 165.3, 133.2, 129.6 2, 129.5, and 128.4 2), 12-OAc (C 170.1, 21.0), 19-OAc (C 170.3, 20.2); HRESIMS m/z 677.2553 [M Na] (calcd for C35H42O12Na, 677.2568). Euphornan R (18): colorless oil; []20D 16.8 (c 0.3, MeCN); UV (MeCN) max (log
) 196 (4.71), 230 (4.16), 273 (2.88) nm; IR (KBr) max 2930, 1739, 1370, 1245, 1026 cm1; 1H and 13 C NMR data for the diterpene moiety see Tables 2 and 3; 1H NMR data for the acyloxy groups: 3-OAc [H 1.56 (3H, s)], 7-OBz [H 8.03 (2H, d, J 7.7 Hz), 7.61 (1H, t, J 7.2 Hz), and 7.48 (2H, t, J 7.4 Hz)], 8-OAc [H 2.05 (3H, s)], 12-OAc 14
[H 2.14 (3H, s)], 19-OAc [H 2.00 (3H, s)]; 13C NMR data for the acyloxy groups: 3OAc (C 169.3, 19.7), 7-OBz (C 164.5, 133.5, 129.8, 129.5 2, and 128.6 2), 8-OAc (C 170.3, 21.0), 12-OAc (C 170.3, 21.1), 19-OAc (C 170.6, 20.7); HRESIMS m/z 677.2559 [M Na] (calcd for C35H42O12Na, 677.2568). Euphornan S (19): colorless oil; []20D 24.7 (c 0.3, MeCN); UV (MeCN) max (log
) 193 (4.52), 218 (4.11), 258 (3.44) nm; IR (KBr) max 2934, 1736, 1370, 1230, 1024 cm1; 1H and 13C NMR data for the diterpene moiety see Tables 2 and 3; 1H NMR data for the acyloxy groups: 3-OAc [H 1.68 (3H, s)], 7-ONic [H 9.20 (1H, s), 8.81 (1H, br s), 8.30 (1H, d, J 7.7 Hz), and 7.45 (1H, dd, J 7.8, 4.9 Hz)], 8-OAc [H 2.04 (3H, s)], 12-OAc [H 2.12 (3H, s)], 19-OAc [H 1.98 (3H, s)]; 13C NMR data for the acyloxy groups: 3-OAc (C 169.4, 20.0), 7-ONic (C 163.4, 153.8, 150.3, 137.3, 125.8, and 123.6), 8-OAc (C 170.2, 20.9), 12-OAc (C 170.3, 21.0), 19-OAc (C 170.5, 20.6); HRESIMS m/z 656.2701 [M H] (calcd for C34H42NO12, 656.2702). Euphornan T (20): colorless oil; []20D 6.0 (c 0.3, MeCN); UV (MeCN) max (log
) 197 (4.35) nm; IR (KBr) max 2937, 1736, 1368, 1227, 1024 cm1; 1H and 13C NMR data for the diterpene moiety see Tables 2 and 3; 1H NMR data for the acyloxy groups: 3-OAc [H 2.11 (3H, s)], 7-OAc [H 2.08 (3H, s)], 8-OMeBu [H 2.33 (1H, dd, J 13.8, 6.9), 1.67 (1H, m), 1.46 (1H, m), 1.15 (3H, d, J 6.9), and 0.88 (3H, t, J 7.4)], 12OAc [H 2.12 (3H, s)], 19-OAc [H 1.98 (3H, s)]; 13C NMR data for the acyloxy groups: 3-OAc (C 169.5, 20.7), 7-OAc (C 168.8, 20.6), 8-OMeBu (C 176.1, 40.9, 26.7, 16.1, and 11.5), 12-OAc (C 170.3, 21.1), 19-OAc (C 170.7, 20.6); HRESIMS m/z 657.2871 [M Na] (calcd for C33H46O12Na, 657.2881). 15
2.5. X-ray crystal structure analysis Crystallographic data for euphornan A (1). C34H41NO12; M 655.68;orthorhombic, space group P212121 (no. 19), a 10.33870 (10) Å, b 17.30960 (10) Å, c 18.9050 (2) Å, V 3383.22 (5) Å3, Z 4, T 100 K, (Cu Kα) 0.816 mm1, Dcalc 1.287 g/cm3, 33801 reflections measured (6.924° 2Θ 144.238°), 6678 unique (Rint 0.0313, Rsigma 0.0206) which were used in all calculations. The final R1 was 0.0266 (I 2 (I)) and wR2 was 0.0680 (all data). Flack parameter 0.03 (3). Crystallographic data for the structure of 1 have been deposited in the Cambridge Crystallographic Data Centre (deposition number: CCDC 1905570). Crystallographic data for euphornan K (11). C40H44O12; M 716.75; orthorhombic, space group P212121 (no. 19), a 14.88910 (10) Å, b 16.00270 (10) Å, c 17.77030 (10) Å, V 4234.05 (5) Å3, Z 4, T 100 K, (Cu Kα) 0.687 mm1, Dcalc 1.124 g/cm3, 43500 reflections measured (7.434° 2Θ 154.784°), 8828 unique (Rint 0.0297, Rsigma 0.0212) which were used in all calculations. The final R1 was 0.0342 (I 2 (I)) and wR2 was 0.0909 (all data). Flack parameter 0.02 (3). Crystallographic data for the structure of 11 have been deposited in the Cambridge Crystallographic Data Centre (deposition number: CCDC 1913396). 2.6. Cell culture HepG2/ADR (doxorubicin-resistant human liver hepatocellular carcinoma cell line) cells were cultured in RPMI1640 complete growth mediums (Gibco BRL, USA), supplemented with 10 fetal bovine serum (FBS) (Gibco BRL, USA), 100 IU/mL penicillin, and 100 g/mL streptomycin (HyClone, USA). The cells were incubated at 16
37 C in a humidified atmosphere with 5 CO2. 2.7. Cytotoxicity analysis Cytotoxicity was determined using a MTT colorimetric assay as previously described [9]. 2.8. MDR reversal activity HepG2/ADR cells were seeded into 96-well plates at a density of 5 103/well and incubated with premeditated concentration of reversal agents (0.5 M, 1 M, or 5 M) at a carbon dioxide incubator for 1 h, then the cells were incubated with ADR (6 concentration gradients) together for an additional 48 h. The cytotoxicities of ADR with or without reversal agents were analyzed using the MTT assay. Verapamil and Tariquidar were used as positive controls, and DMSO was used as a negative control. The reversal fold is calculated as a ratio of IC50
(ADR)
to IC50
(Pgp inhibitor ADR).
All
experiments were performed at least three times. 2.9. Intracellular accumulation of Rho123 HepG2/ADR cells were seeded into 96-well plates at a density of 5 103well and were incubated with given concentration of compounds and 10 M Rho123 in the dark at 37 C for 90 min. The cells were washed twice with ice-cold PBS after centrifugation, resuspended in PBS buffer, and analyzed by flow cytometry. Tariquidar was evaluated at 5 M and parallel time as a positive control. The fluorescence intensity of intracellular Rho123 was measured with a flow cytometer in FL1 channel (Em 525 nm). Fold of control (The fluorescence of compound treated cell Background)(The fluorescence of Rho123-only cell Background). 17
−
3. Results and discussion The air-dried powder of the seeds of E. marginata was extracted with 95 EtOH at room temperature to give a crude extract, which was suspended in H2O and partitioned with PE. Various column chromatographic separations of the PE extract afforded compounds 120 (Fig. 1). Compound 1 was obtained as colorless crystals. Its molecular formula C34H41NO12 was determined by the HRESIMS ion at m/z 656.2704 [M H] (calcd for 656.2702), corresponding to 15 indices of hydrogen deficiency (IHDs). The 1H NMR spectrum revealed the presence of a nicotinoyl group [H 9.10 (1H, s), 8.71 (1H, br s), 8.21 (1H, d, J 7.9 Hz), and 7.35 (1H, dd, J 7.8, 4.9 Hz)], eight methyl groups [H 2.08 (s), 2.06 (s), 2.04 (s), 1.97 (s), 1.74 (s), 1.28 (s), 0.98 (d, J 7.2 Hz), and 0.88 (d, J 6.8 Hz)], two oxymethylene protons [H 4.28 (1H, d, J 12.0 Hz) and 3.91 (1H, d, J 12.0 Hz)], four oxymethine protons [H 5.25 (1H, d, J 5.5 Hz), 5.23 (1H, s), 4.94 (1H, dd, J 10.2, 3.6 Hz), and 4.57 (1H, d, J 8.7 Hz)], an olefinic proton [H 5.61 (1H, s)], and a series of aliphatic multiplets. The 13C NMR spectrum, in combination with DEPT experiments, resolved 34 carbon resonances attributable to a ketocarbonyl (C 206.8), five ester carbonyls (C 170.1, 170.0, 169.4, 168.9, and 164.7), a pyridine ring [C 153.3, 150.4, 136.9, 126.1, and 123.2], a trisubstituted double bond (C 138.7 and 115.3), eight methyls, two sp3 methylenes (one oxygenated), eight sp3 methines (four oxygenated), two oxygenated sp3 tertiary carbons, and a quaternary carbon. As 11 of the 15 IHDs were accounted for by a ketocarbonyl, five ester carbonyls, a pyridine ring, and a double bond, the remaining four IHDs required that 1 was tetracyclic. The aforementioned 18
information was characteristic of ingol-type diterpenoids [11,12]. The 2D structure of 1 was established by detailed analysis of its 2D NMR data. Two spin systems, H2-1/H2/H-3 (H3-16) and H-7/H-8/H-9/H-11/H-12/H-13/H3-20, were firstly established by the 1H1H
COSY correlations (Fig. 2a). The connectivities of these two fragments, the
hydrogen-free carbons, and the substituents were achieved by analysis of the key HMBC correlations (Fig. 2a). Briefly, the HMBC correlations from H2-1 and H-3 to C4 (C 70.6) and C-15 (C 73.0) generated a five-membered carbon ring with a characteristic 4,15-expoxy ring [13,14]. The cyclopropane ring was located at C-9 and C-11 by HMBC correlations from H3-18 and H2-19 to C-9, C-10, and C-11. The C-4 was linked to C-7 via 5 by HMBC correlations of H-5/C-4 and H3-17/C-6 and C-7. The ketocarbonyl (C-14) was linked to C-13 by HMBC correlations from H-13 and H320 to C-14. Although no direct HMBC correlations were available to connect C-14 to C-15, the still unassigned carbons of C-15 and C-14 as well as the remaining one IHD required the connection between C-14 and C-15 to generate the 11-membered macro ring. Four acetoxyl groups were attached to C-3, C-7, C-8, and C-12, respectively, by HMBC correlations from the oxymethines to the corresponding ester carbonyls. The nicotinoyloxy group was attached to C-19 by HMBC correlation from H2-19 (H 4.28 and 3.91) to the carbonyl of nicotinoyl group. Thus, the gross structure of 1 was established. The relative configuration of 1 was established by analysis of the NOESY data (Fig. 2b) and 1H−1H coupling constants. The chemical shifts of H2-1 (H 2.21 and 1.97) and the coupling constant of H-2H-3 (J 5.5 Hz) indicated that H-2 and H-3 were 19
oriented [15]. In addition, the NOE correlations of H-8/H-7, H-13, and H-19b, and H12/H-19b revealed that H-7, H-8, H-12, and H-13 were cofacial and arbitrarily designated as -oriented. Thus, the NOE correlations of Me-18H-9 and H-11 assigned H-9, H-11, and Me-18 as -oriented. The geometry of the 5(6) double bond was assigned as E by NOE correlation of H-5/7-acetoxyl. Finally, the structure of 1 including absolute configuration (2R,3R,4S,7R,8S,9S,10R,11R,12R,13R,15R) was confirmed by a single crystal X-ray crystallographic analysis using anomalous scattering of Cu K radiation [Flack parameter 0.03 (3)] (Fig. 3). Compound 1 was given the trivial name euphornan A. Compound 2 possessed a molecular formula of C39H43NO12, as determined by the HRESIMS ion at m/z 718.2846 [M H] (calcd for 718.2858). The 1D NMR spectra of 2 were very similar to those of 1, with the only difference being the replacement of an acetyl group in 1 by a benzoyl group in 2. The location of the benzoyl group was assigned at OH-8 by the HMBC correlation from H-8 (H 4.95) to the benzoyl carbonyl (C 165.4). Thus, 2 was assigned as depicted and was given the trivial name euphornan B. Compound 3, a white amorphous powder, possessed a molecular formula of C38H42N2O12 as determined by the HRESIMS ion at m/z 719.2795 [M H] (calcd for 719.2811). The 1D NMR data of 3 were very similar to those of 1 except for the presence of an additional nicotinoyl group in 3 instead of an acetyl group in 1. The additional nicotinoyl group was located at OH-7 by the HMBC correlation from H-7 (H 5.55) to the nicotinoyl carbonyl (C 163.4). Therefore, the structure of 3 was 20
determined as depicted and was named euphornan C. Compound 4 had the molecular formula C36H45NO12, as determined by HRESIMS ion at m/z 684.3009 [M H] (calcd for 684.3015). The NMR data of 4 showed high similarity to those of 1, with the only difference being the presence of an isobutyryl group in 4 instead of the acetyl group in 1. The HMBC correlation from H-8 (H 4.59) to the isobutyryl carbonyl (C 176.2) assigned the isobutyryl group at OH-8. Thus, the structure of 4 was identified as shown and was given the trivial name euphornan D. Compound 5 exhibited a molecular formula of C37H47NO12 as determined by the HRESIMS ion at m/z 698.3174 [M H] (calcd for 698.3171). The 1H and 13C NMR spectra of 5 were very similar to those of 4, with the only difference being the replacement of the isobutyryl group in 4 by a 2-methylbutanoyl group in 5. The location of the 2-methylbutanoyl group was assigned at OH-8 by the HMBC correlation from H-8 (H 4.61) to the 2-methylbutanoyl carbonyl (C 176.1). Compound 5 was thus assigned as depicted and was named euphornan E. The molecular formula of 6 was deduced as C37H41NO11 from its HRESIMS data (m/z 676.2749 [M H], calcd for 676.2752). The NMR data of 6 showed high similarity to those of 2 excepted for the absence of an acetyl group and the upfieldshifted H-7 signal (H 4.40 in 6; H 5.35 in 2), indicating that 6 was a 7-O-deacetylated derivative of 2. The above described deduction was further secured by detailed analyses of its 2D NMR data. Therefore, the structure of 6 was determined as depicted and was given the trivial name euphornan F. Compound 7 displayed a molecular ion at m/z 676.2751 [M H] (calcd for 21
676.2752), consistent with a molecular formula of C37H41NO11. The 1H and 13C NMR data of 7 closely resembled those of 2 except for the absence of an acetyl group and the severely upfield-shifted H-3 signal (H 4.06 in 7; H 5.31 in 2), indicating that 7 was a 3-O-deacetylated derivative of 2. This was further confirmed by detailed 2D NMR analysis. Compound 7 was thus assigned as depicted and was named euphornan G. Compound 8 had the same molecular formula as that of 7. Its 1D NMR data were very similar to those of 7 except for the slight differences arising from H-7 (H 5.33 in 7; H 5.55 in 8) and H-8 (H 4.97 in 7; H 4.72 in 8), suggesting that the locations of the acetyl group at OH-7 and the benzoyl group at OH-8 in 7 were switched in 8. This was further confirmed by the HMBC correlations from H-7 to the benzoyl carbonyl (C 165.1) and from H-8 to the acetyl carbonyl (C 170.2). Hence, the structure of 8 was determined as shown and was given the trivial name euphornan H. Compound 9 exhibited a molecular formula of C35H42O12, as determined by HRESIMS ion at m/z 677.2557 [M Na] (calcd for 677.2568). The 1D NMR data of 9 showed high similarity to those of 1, with the only difference being the occurrence of a benzoyl group in 9 instead of a nicotinoyl group in 1. The benzoyl group was located at OH-19 by the HMBC correlation from H2-19 (H 4.30 and 3.91) to the benzoyl carbonyl (C 166.2). Therefore, compound 9 was determined as depicted and named euphornan I. Compounds 10 and 11 shared the same molecular formula C40H44O12, as established by HRESIMS. The NMR spectra of them were very similar to those of 9, with the main difference being the presence of an additional benzoyl group in 10 and 11 instead of 22
the acetyl group in 9. The positions of benzoyl group were assigned at OH-8 and OH7 in 10 and 11, respectively, by the HMBC correlations from the oxymethines to corresponding ester carbonyls. The structure of 11 including the absolute configuration (2R,3R,4S,7R,8S,9S,10R,11R,12R,13R,15R) was further confirmed by a single crystal X-ray crystallographic analysis using anomalous scattering of Cu K radiation [Flack parameter 0.02 (3)]. Hence, the structures of 10 and 11 were established as depicted and were given the trivial names euphornan J and euphornan K, respectively. Compound 12 had a molecular formula of C37H46O12, as determined by the HERSIMS ion at m/z 705.2875 [M Na] (calcd for 705.2881). The 1H and 13C NMR data of 12 were very similar to those of 9 except for the appearance of an isobutyryl group in 12 instead of the acetyl group in 9. The isobutyryl group was located at OH-8 by the HMBC correlation from H-8 (H 4.61) to the isobutyryl carbonyl (C 176.3). Thus, the structure of 12 was defined as shown and was named euphornan L. Compound 13 gave a [M Na] ion at m/z 719.3042 (calcd for 719.3038) in the HRESIMS, corresponding to the molecular formula C38H48O12. The 1D NMR data of 13 bore a resemblance to those of 9 except for the presence of a 2-methylbutanoyl group in 13 instead of the acetyl group in 9. The 2-methylbutanoyl group was located at OH8 by the HMBC correlation from H-8 (H 4.61) to the 2-methylbutanoyl carbonyl (C 176.2). Thus, 13 was assigned as depicted and was given the trivial name euphornan M. Compounds 1416 had the same molecular formula of C38H42O11, as determined by the HERSIMS data. The 1D NMR spectra of these isomers bore a high resemblance, 23
with the differences being due to the different locations of two benzoyl groups and two acetyl groups. For 14, the HMBC correlations from H-3 (H 5.36) and H-12 (H 5.05) to acetyl carbonyls at C 170.3 and 170.3, respectively, located the acetyl groups at OH3 and OH-12, while HMBC correlations from H-8 (H 4.82) and H2-19 (H 4.28 and 4.03) to the benzoyl carbonyls at C 165.9 and 166.1, respectively, positioned the benzoyl groups at OH-8 and OH-19. Similarly, in 15, two acetyl groups were located at OH-7 and OH-12, and the two benzoyl groups were attached at OH-8 and OH-19. For 16, two acetyl groups were located at OH-8 and OH-12, and two benzoyl groups were located at OH-7 and OH-19. Thus, compounds 1416 were deduced as depicted, and were given the trivial names euphornan N, euphornan O, and euphornan P, respectively. Compounds 17 and 18 possessed the same molecular formula C35H42O12 as that of 9. Analysis of their 1D NMR data revealed that they are structurally related isomers differentiating in acyl locations. In comparison with 9, the 8-OAc and 19-OBz in 9 were switched in 17, as indicated by the HMBC correlations from H-8 (H 4.88) to the benzoyl carbonyl (C 165.3) and from H2-19 (H 4.05 and 3.72) to the acetyl carbonyl (C 170.3). The 7-OAc and 19-OBz in 9 were switched in 18, as indicated by the HMBC correlations from H-7 (H 5.45) to the benzoyl carbonyl (C 164.5) and from H2-19 (H 4.25 and 3.61) to the acetyl carbonyl (C 170.6). Therefore, 17 and 18 were determined as depicted, and were named euphornan Q and euphornan R, respectively. Compound 19 had the molecular formula C34H41NO12, as determined by HRESIMS ion at m/z 656.2701 [M H] (calcd for 656.2702). The NMR spectra of 19 were very 24
similar to those of 18 except for the replacement of the benzoyl group in 18 by a nicotinyl group in 19. A HMBC correlation from H-7 (H 5.49) to the nicotinyl carbonyl (C 163.4) positioned the nicotinyl group at OH-7. The structure of compound 19 was thus determined as shown and was named euphornan S. Compound 20 exhibited a molecular formula of C33H46O12, as established by HRESIMS ion at m/z 657.2871 [M Na] (calcd for 657.2881). The 1H and 13C NMR spectra of 20 were very similar to those of 17, with main difference being the appearance of a 2-methylbutanoyl group instead of a benzoyl group in 17. The location of the 2-methylbutanoyl group was assigned at OH-8 by the HMBC correlation from H-8 (H 4.57) to the 2-methylbutanoyl carbonyl (C 176.1). Thus, the structure of 20 was determined as shown and was given a trivial name euphornan T. Compounds 120 shared the same relative configuration as indicated by their similar 1D NMR and NOESY data. Furthermore, the 5(6) double bond in 220 was assigned to have the same geometry by the NOE correlation between H-5 and 7-acyloxy and/or by comparing their chemical shifts of C-6 and C-7 with those of 1. Their absolute configurations were further established by CD analysis [16,17] (Supporting Information, Fig. S1). In the 190400 nm region, the ECD curves for all compounds showed a similar tendency, with Cotton effects being around 307 nm (), 260 nm (), 226 nm (), and 197 nm (). With the absolute configurations of 1 and 11 in hand, the absolute configurations of 120 were assigned as 2R, 3R, 4S, 7R, 8S, 9S, 10R, 11R, 12R, 13R, 15R, which were consistent with the biogenetic origin of the ingol diterpenoids [5,14]. 25
As lathyrane diterpenoids were widely reported as P-glycoprotein (Pgp) inhibitors in multidrug resistance (MDR) cancer therapy [18,19], compounds 120 were screened for their reversal ability on Pgp-dependent MDR cancer cell line HepG2/ADR. Firstly, the intrinsic cytotoxicities of 120 were determined using the MTT method, and all compounds showed no obvious cytotoxicity (IC50 50 M) in HepG2/ADR cell line. Then, the MDR reversal assay was performed by combination of 5 M of tested compounds with various concentrations of anticancer drug adriamycin (ADR). Verapamil (Vrp) and tariquidar (Tar), the first- and third-generation Pgp inhibitors, respectively, were used as positive controls. As shown in Fig. 4A, the co-administration of most of the tested compounds significantly enhanced the cytotoxicity of ADR, and 14 compounds (2, 46, 915, 17, 18, and 20) exhibited greater reversal activity than verapamil (detailed data see Supporting Information, Table S1). Among them, 11, 14, and 18 with comparable activity to tariquidar were further chosen for a doseeffect dependence study at the concentrations of 0.5, 1, and 5 M. As shown in Fig. 4B, all the three compounds exhibited a good dose-dependent manner, and reversed the sensitivity of anticancer drug adriamycin to ca. 20 folds at 5 M. To further confirm the MDR reversal ability of these compounds were related to Pgp modulation mechanism, 14 was selected for the Rho123 efflux assay. As shown in Fig. 4C, 14 could dose-dependently increase the intracellular accumulation of Rho-123, a substrate of Pgp efflux pump, suggesting that compounds reversed the sensitivity of MDR cancer cell line via the inhibition of Pgp. The different substitution patterns of these ingol diterpenoids made them a good set 26
of homologues to evaluate structureactivity relationships (Fig. 5). In general, the acylation of OH-3 was beneficial to the activity, as shown by 2 vs 7, 10 vs 15, and 11 vs 16. On the contrary, the acylation of OH-7 led to a decrease in activity (6 vs 2 and 14 vs 10), and the benzoyl at OH-7 showed greater activity than acetyl at OH-7 (11 vs 9). The acylation patterns of OH-8 showed little influence on the activity, as shown by 13 vs 12 and 10. Comparison of 14 vs 6, 13 vs 5, and 15 vs 7, it can be inferred that the replacement of nicotinoyl by benzoyl at OH-19 remarkably improved the activity. 4. Conclusions In summary, twenty new ingol diterpenoids (120) were isolated from the seeds of Euphorbia marginata, which is a chemically rarely investigated Euphorbiaceae plant. Although more than 100 ingols have been reported from Euphorbia species so far, most of them possess a -oriented H-2 and H-3 on the five-membered carbon ring and bear the geminal methyls (Me-18 and Me-19) at C-10 of the cyclopropane ring. 120 stand for a rare C-19-oxidated and H-2, H-3 -oriented subgroup in this diterpenoid class. It is possible that their peculiar structures render them the potent MDR reversal activity. The current study not only enriched the structural categories of ingol class, but also provided a potential structural motif in future MDR drug development. Declaration of competing interest The authors declare no competing financial interest. Acknowledgments The authors thank the Natural Science Foundation of China (81722042, 81573302, and 81973195) for providing financial support for this work. Appendix A. Supporting Information MS, IR, 1D and 2D NMR spectra for compounds 120. Supplementary data 27
associated with this article can be found online at
28
References [1] Q.W. Shi, X.H. Su, H. Kiyota, Chemical and pharmacological research of the plants in genus Euphorbia, Chem. Rev. 108 (2008) 42954327. [2] A. Vasas, J. Hohmann, Euphorbia diterpenes: isolation, structure, biological activity, and synthesis (20082012), Chem. Rev. 114 (2014) 85798612. [3] V. Ravikanth, V.L.N. Reddy, T.P. Rao, P.V. Diwan, S. Ramakrishna, Y. Venkateswarlu, Macrocyclic diterpenes from Euphorbia nivulia, Phytochemistry 59 (2002) 331335. [4] M.O. Fatope, L. Zeng, J.E. Ohayagha, J.L. McLaughlin, New 19-acetoxyingol diterpenes from the latex of Euphorbia poisonii (Euphorbiaceae), Bioorg. Med. Chem. Lett. 4 (1996) 16791683. [5] N.D. Zhao, X. Ding, Y. Song, D.Q. Yang, H.L. Yu, T.A. Adelakun, W.D. Qian, Y. Zhang, Y.T. Di, F. Gao, X.J. Hao, S.L. Li, Identification of ingol and rhamnofolane diterpenoids from Euphorbia resinifera and their abilities to induce lysosomal biosynthesis, J. Nat. Prod. 81 (2018) 12091218. [6] B. Sarkadi, L. Homolya, G. Szakács, A. Váradi, Human multidrug resistance ABCB and ABCG transporters: participation in a chemoimmunity defense system, Physiol. Rev. 86 (2006) 1179−1236. [7] Flora of China Editorial Committee, Flora of China. Science Press: Beijing, 1997, Vol. 44, pp 55. [8] K.B. Sarah, G.R. Michael, Concerted use of homo- and heteronuclear 2D NMR in the elucidation of the structure of a novel pentaester of 19-hydroxyingol from 29
Euphorbia marginata seeds, Magn. Reson. Chem. 30 (1992) 632636. [9] J.Y. Zhu, R.M. Wang, L.L. Lou, W. Li, G.H. Tang, X.Z. Bu, S. Yin, Jatrophane diterpenoids as modulators of P-glycoprotein-dependent multidrug resistance (MDR): advances of structureactivity relationships and discovery of promising MDR reversal agents, J. Med. Chem. 59 (2016) 63536369. [10] W. Li, Y.Q. Tang, J. Sang, G.H. Tang, S. Yin, Jatrofolianes A and B, two highly modified lathyrane diterpenoids from Jatropha gossypiifolia, Org. Lett. DOI: 10.1021/acs.orglett.9b04029. [11] H.J. Opferkuch, E. Hecker, Ingol-a new macrocyclic diterpene alcohol from Euphorbia ingens, Tetrahedron Lett. 37 (1973) 36113614. [12] L.J. Lin, A.D. Kinghorn, 8-methoxyingol esters from the latex of Euphorbia hermentiana, Phytochemistry 22 (1983) 27952799. [13] W.Y. Qi, W.Y. Zhang, Y. Shen, Y. Leng, K. Gao, J.M. Yue, Ingol-type diterpenes from Euphorbia antiquorum with mouse 11-hydroxysteroid dehydrogenase type 1 inhibition activity, J. Nat. Prod. 77 (2014) 14521458. [14] X.L. Li, Y. Li, S.F. Wang, Y.L. Zhao, K.C. Liu, X.M. Wang, Y.P. Yang, Ingol and ingenol diterpenes from the aerial parts of Euphorbia royleana and their antiangiogenic activities, J. Nat. Prod. 72 (2009) 10011005. [15] J.A. Marco, J.F. Sanz-Cervera, A. Yuste, Ingenane and lathyrane diterpenes from the latex of Euphorbia canariensis, Phytochemistry 45 (1997) 563570. [16] S. Y. Wang, G.Y. Li, K. Zhang, H.Y. Wang, H.G. Liang, C. Huang, J. Huang, J.H. Wang, B.F. Yang, New ingol-type diterpenes from the latex of Euphorbia 30
resinifera, J. Asian. Nat. Prod. Res. 21 (2019) 10751082. [17] Z.Y. Yin, Y. Dai, P. Hua, Z.J. Sun, Y.F. Cheng, S.H. Yuan, Z.Y. Chen, Q. Gu, Discovery of diverse diterpenoid scaffolds from Euphorbia antiquorum and their activity against RANKL-induced osteoclastogenesis, Bioorg. Chem. 92 (2019) 103292. [18] M. Reis, R.J. Ferreira, M.M.M. Santos, D.J.V.A. dos Santos, J. Molnar, M.J.U. Ferreira, Enhancing macrocyclic diterpenes as multidrug-resistance reversers: structure−activity studies on Jolkinol D derivatives, J. Med. Chem. 56 (2013) 748760. [19] W. Jiao, Z.M. Wan, S. Chen, R.H. Lu, X.Z. Chen, D.M. Fang, J.F. Wang, S.C. Pu, X. Huang, H.X. Gao, H.W. Shao, Lathyrol diterpenes as modulators of Pglycoprotein dependent multidrug resistance: structure-activity relationship studies on Euphorbia factor L3 derivatives, J. Med. Chem. 58 (2015) 37203738.
31
20
O 2 15
16
3
O
19
11
7
6
OAc H
O
OBz
OAc H
O
OAc
18
H
8
5
ONic
10
9
17
4
R 1O
1 2 3 4 5 6 7 8
13 12
14
1
OAc H
H
O
OR3
R 2O R1
R2
R3
Ac Ac Ac Ac Ac Ac H H
Ac Ac Nic Ac Ac H Ac Bz
Ac Bz Ac iBu MeBu Bz Bz Ac
OR3
R 1O
9 10 11 12 13 14 15 16
R2
Ac Ac Ac Ac Ac Ac H H
Ac Ac Bz Ac Ac H Ac Bz
OR3
R 1O
R 2O
R1
H
O
R3
R1 Ac Ac Ac Ac
17 18 19 20
Ac Bz Ac iBu MeBu Bz Bz Ac
R 2O R2 Ac Bz Nic Ac
O
O
O
O
R3 Bz Ac Ac MeBu
N iBu
MeBu
Bz
Nic
Fig. 1. Structures of compounds 120.
N O
O 14
1 16
13
2
O 3
O
12
4
O 18
H
9
17
16
8 6
5
19
10
11
15
O
20
O
O
20
7
O
O
O
1
14 15
2 3
H
4
OAc
O
AcO H
13
5
12
H
11
17
O
H
6
7
H
H H
H 8
OAc
9
19
10
ONic H
18
H OAc
O a
Fig. 2. Key 1H1H COSY (
b
) and HMBC (
correlations of 1 (b).
32
) correlations of 1 (a); Key NOE (
)
C20 C20
C12 C12
C1
C15
C1
C13 C19
C10
C3
C3
C4 C5
C6
C7
C17
C4 C5
C9 C6 C7
C8
1
C8
11
Fig. 3. ORTEP diagram of 1 and 11.
33
C19
C10 C18
C18 C9
C17
C11
C16
C2 C16
C14
C13
C2
C11
C14
C15
Fig. 4. (A) Co-administration of compounds (at 5 M) enhanced the cytotoxicity of anticancer drug adriamycin (ADR) on HepG2/ADR. (B) The reversal effects of 11, 14, and 18 on the sensitivity of HepG2/ADR at different concentrations (0.5, 1, and 5 M). Reversal fold is calculated as a ratio of IC50 (ADR) to IC50 (ADR Cmpd). *P 0.05, **P 0.01, and ***P 0.001 (C) Inhibitory effects of 14 on the accumulation of rhodamin123 (Rho-123) in HepG2/ADR.
34
Fig. 5. SARs of ingol diterpenoids on Pgp inhibition
35
Table 1. 1H NMR data for the diterpene moiety of compounds 110 in CDCl3 ( in ppm, J in Hz) No.
1a
2a
3a
4a
5a
6b
7b
8a
9a
10a
1
2.21, dd (13.4,
2.27, dd (13.5,
2.26, dd (13.5,
2.24, dd (13.4,
2.25, dd (13.5,
2.29, dd (13.3,
2.26, dd (16.3,
2.24, dd (16.0,
2.25, dd (13.6,
2.27, dd (13.5,
10.4)
10.4)
10.4)
10.6)
10.6)
10.7)
13.2)
13.4)
10.4)
10.4)
1.97, m
2.02, m
2.00, m
1.99, dd (13.6,
2.00, dd (13.5,
2.02, dd (13.2,
1.97, m
1.93, m
2.01, m
2.02, dd (13.6,
6.9)
7.0)
7.1)
1
6.9)
2
2.08, m
2.14, m
2.11, m
2.11, m
2.12, m
2.14, m
1.98, m
1.93, m
2.13, m
2.14, m
3
5.25, d (5.5)
5.31, d (5.5)
5.25, d (5.5)
5.28, d (5.1)
5.28, d (5.3)
5.36, d (5.3)
4.06, d (3.5)
3.99, d (3.9)
5.29, d (5.6)
5.30, d (5.5)
5
5.61, s
5.70, s
5.77, s
5.64, s
5.63, s
5.93, s
6.07, s
6.20, s
5.65, s
5.70, s
7
5.23, s
5.35, s
5.55, s
5.27, s
5.26, s
4.40, s
5.33, s
5.55, s
5.28, s
5.36, s
8
4.57, d (8.7)
4.95, d (10.7)
4.74, d (8.9)
4.59, d (9.1)
4.61, d (8.3)
4.84, d (10.7)
4.97, d (10.6)
4.72, d (10.5)
4.62, d (10.0)
4.94, d (10.7)
9
1.46, d (9.8)
1.67, m
1.61, m
1.50, m
1.52, m
1.86, t (10.0)
1.73, m
1.66, m
1.49, m
1.64, m
11
1.46, d (9.8)
1.54, m
1.59, m
1.51, m
1.52, m
1.52, t (10.1)
1.53, m
1.54, m
1.48, m
1.53, m
12
4.94, dd (10.2,
5.06, dd (10.7,
5.03, dd (10.3,
4.97, dd (10.3,
4.98, dd (10.7,
5.04, dd (10.9,
5.06, dd (10.9,
4.99, dd (10.9,
5.00, m
5.07, dd (10.7,
3.6)
3.8)
3.7)
3.5)
3.3)
3.0)
3.6)
3.3)
13
2.86, m
3.02, m
2.96, m
2.89, m
2.91, m
2.98, m
3.03, m
2.98, m
2.93, m
3.01, m
16
0.88, d (6.8)
0.94, d (6.8)
0.89, d (6.8)
0.91, d (6.8)
0.92, d (6.8)
0.95, d (6.6)
1.04, d (6.5)
0.98, d (6.2)
0.92, d (6.9)
0.93, d (6.9)
17
2.04, s
2.16, s
2.18, s
2.08, s
2.10, s
2.10, s
2.17, s
2.16, s
2.09, s
2.16, s
18
1.28, s
1.37, s
1.36, s
1.31, s
1.34, s
1.35, s
1.36, s
1.30, s
1.32, s
1.37, s
19a
4.28, d (12.0)
4.26, d (12.3)
4.36, d (12.0)
4.39, d (12.0)
4.39, d (12.0)
4.29, d (12.1)
4.28, d (12.1)
4.34, d (11.9)
4.30, d (12.0)
4.24, d (12.1)
19b
3.91, d (12.0)
4.07, d (12.1)
3.99, d (12.0)
3.87, d (12.0)
3.89, d (12.0)
4.07, d (12.0)
4.05, d (12.2)
3.95, d (12.0)
3.91, d (12.0)
4.03, d (12.1)
20
0.98, d (7.2)
1.07, d (7.2)
1.06, d (7.2)
1.01, d (7.2)
1.02, d (7.2)
1.07, d (7.0)
1.06, d (7.2)
1.01, d (7.1)
1.03, d (7.3)
1.06, d (7.2)
aat
400 MHz; bat 500 MHz.
36
3.8)
Table 2. 1H NMR data for the diterpene moiety of compounds 1120 in CDCl3 ( in ppm, J in Hz) No.
11a
12a
13b
14a
15a
16a
17a
18b
19a
20b
1
2.24, dd (13.4,
2.26, dd (13.2,
2.26, dd (13.6,
2.29, dd (13.3,
2.26, dd (16.4,
2.24, dd (16.1,
2.24, dd (13.5,
2.23, m
2.24, dd (13.5,
2.26, dd (13.7,
10.3)
10.4)
10.7)
10.5)
13.1)
13.2)
10.4)
10.3)
10.7)
2.01, m
2.01, dd (13.6,
2.01, dd (13.8,
2.02, m
1.96, m
1.94, m
2.01, dd (13.5,
2.00, m
2.01, m
2.02, m
7.0)
7.0)
1
6.9)
2
2.12, m
2.14, m
2.13, m
2.15, m
1.98, m
1.94, m
2.12, m
2.11, m
2.10, m
2.12, m
3
5.25, d (5.6)
5.30, d (5.1)
5.29, d (5.4)
5.36, d (5.4)
4.04, d (4.8)
3.99, d (4.6)
5.28, d (5.7)
5.24, d (5.3)
5.23, d (5.3)
5.29, d (5.5)
5
5.77, s
5.66, s
5.64, s
5.94, s
6.06, s
6.18, s
5.66, s
5.74, s
5.73, s
5.62, s
7
5.49, s
5.30, s
5.27, s
4.42, s
5.35, s
5.55, s
5.31, s
5.45, s
5.49, s
5.23, s
8
4.75, d (10.6)
4.61, d (9.6)
4.61, d (9.5)
4.82, d (10.7)
4.96, d (10.8)
4.74, d (10.6)
4.88, d (10.8)
4.69, d (10.7)
4.68, d (9.4)
4.56, d (9.6)
9
1.67, m
1.50, m
1.52, m
1.84, m
1.71, m
1.66, m
1.56, m
1.59, m
1.50, m
1.44, m
11
1.58, m
1.51, m
1.50, m
1.51, m
1.52, m
1.52, m
1.44, m
1.50, m
1.49, m
1.44, m
12
5.05, dd (10.8,
5.00, dd (9.7,
4.99, dd (9.5,
5.05, dd (11.0,
5.07, dd (11.0,
5.02, dd (11.0,
4.99, dd (10.8,
4.98, d (11.0)
4.96, dd (10.5,
4.93, dd (9.7,
3.9)
4.2)
3.9)
3.6)
3.8)
3.7)
3.9)
3.8)
3.9)
13
3.00, m
2.93, m
2.92, m
2.97, m
3.02, m
3.00, m
2.97, m
2.98, m
2.94, m
2.90, m
16
0.87, d (6.9)
0.93, d (6.8)
0.93, d (6.9)
0.94, d (6.8)
1.03, d (6.6)
0.98, d (6.3)
0.91, d (6.9)
0.87, d (6.7)
0.87, d (6.6)
0.93, d (6.9)
17
2.18, s
2.11, s
2.11, s
2.10, s
2.18, s
2.17, s
2.13, s
2.16, s
2.15, s
2.10, s
18
1.37, s
1.33, s
1.34, s
1.35, s
1.36, s
1.32, s
1.18, s
1.20, s
1.17, s
1.17, s
19a
4.34, d (12.0)
4.38, d (12.0)
4.37, d (12.0)
4.28, d (12.1)
4.27, d (12.1)
4.32, d (12.0)
4.05, d (12.2)
4.25, d (12.0)
4.21, d (12.0)
4.31, d (12.1)
19b
3.97, d (12.0)
3.83, d (12.0)
3.84, d (12.0)
4.03, d (12.0)
4.01, d (12.1)
3.92, d (12.0)
3.72, d (12.2)
3.61, d (12.1)
3.59, d (12.0)
3.46, d (12.1)
20
1.06, d (7.2)
1.03, d (7.2)
1.03, d (7.2)
1.06, d (7.2)
1.05, d (7.3)
1.02, d (7.2)
1.06, d (7.3)
1.09, d (7.1)
1.07, d (7.1)
1.06, d (7.2)
aat
400 MHz; bat 500 MHz.
37
Table 3. 13C NMR data for the diterpene moiety of compounds 120 in CDCl3 ( in ppm) No.
1a
2a
3a
4a
5a
6b
7b
8a
9a
10a
11a
12a
13b
14a
15a
16a
17a
18b
19a
20b
1
32.7
32.8
32.8
32.7
32.8
32.9
32.2
32.2
32.8
32.9
32.9
32.8
32.7
32.9
32.2
32.2
32.8
32.8
32.7
32.7
2
32.5
32.6
32.6
32.5
32.6
32.6
33.0
32.9
32.6
32.6
32.7
32.6
32.6
32.6
33.0
32.9
32.5
32.6
32.5
32.6
3
76.9
77.0
76.8
77.0
77.1
77.1
76.6
76.1
77.1
77.1
76.8
77.1
77.1
77.1
76.6
76.3
77.0
76.7
76.8
77.1
4
70.6
70.6
70.6
70.7
70.7
71.2
72.5
72.7
70.7
70.7
70.8
70.7
70.8
71.2
72.5
72.6
70.6
70.8
70.6
70.7
5
115.3
115.3
115.8
115.3
115.2
115.0
116.8
117.4
115.4
115.3
115.2
115.3
115.1
115.0
116.7
117.1
115.3
115.1
115.7
115.1
6
138.7
139.1
138.5
138.9
139.2
141.2
137.9
137.3
139.0
139.2
139.1
139.0
139.2
141.1
137.9
137.5
139.0
138.9
138.5
139.1
7
75.8
76.4
76.9
75.7
76.1
76.0
76.9
76.5
76.0
76.4
76.7
75.8
76.1
75.9
76.9
76.6
76.4
76.7
77.0
76.1
8
70.6
70.8
70.8
70.7
70.6
73.7
71.0
71.1
70.7
70.9
70.8
70.7
70.6
74.0
71.1
71.0
70.8
70.7
70.7
70.5
9
24.7
25.4
25.2
24.7
24.8
24.1
25.5
25.2
24.9
25.3
25.3
24.7
24.8
24.0
25.3
25.2
25.2
25.1
25.0
24.7
10
22.0
22.2
22.2
22.1
22.2
22.1
22.3
22.0
22.3
22.4
22.3
22.3
22.3
22.3
22.5
22.2
22.1
22.1
22.1
22.0
11
30.8
31.0
31.0
31.0
31.0
31.2
31.1
31.0
30.8
31.0
31.0
31.0
30.9
31.2
31.0
30.9
31.0
31.0
31.0
31.0
12
68.8
69.0
68.8
68.9
68.9
69.2
69.2
69.1
68.8
68.9
68.8
68.8
68.8
69.1
69.1
69.0
69.0
69.0
68.9
69.1
13
43.6
43.9
43.7
43.7
43.7
43.9
43.7
43.4
43.8
43.9
43.8
43.7
43.7
43.9
43.7
43.4
43.7
43.6
43.5
43.6
14
206.8
206.9
206.8
206.9
207.0
207.1
207.3
207.2
207.1
207.0
207.2
207.2
207.2
207.2
207.5
207.4
207.0
207.2
206.9
207.2
15
73.0
73.1
73.2
73.1
73.1
73.2
73.3
73.3
73.2
73.2
73.3
73.2
73.1
73.2
73.2
73.2
73.1
73.2
73.1
73.1
16
12.2
12.3
12.2
12.3
12.3
12.3
12.0
12.0
12.3
12.3
12.3
12.3
12.3
12.3
12.0
12.0
12.3
12.3
12.2
12.3
17
17.5
17.5
17.6
17.5
17.5
17.5
17.6
17.6
17.6
17.6
17.5
17.6
17.6
17.6
17.6
17.6
17.5
17.5
17.5
17.5
18
24.4
24.6
24.6
24.5
24.6
24.5
24.6
24.5
24.6
24.7
24.8
24.7
24.7
24.6
24.7
24.6
24.4
24.5
24.4
24.4
19
65.5
65.5
65.5
65.8
65.9
66.2
65.7
65.7
65.1
64.9
65.1
65.3
65.4
65.4
65.1
65.1
64.2
64.4
64.3
64.8
20
12.9
12.8
13.1
13.0
13.1
13.0
13.1
13.1
13.0
12.9
13.0
13.1
13.1
13.1
13.2
13.1
13.0
13.2
13.2
13.2
aat
C
100 MHz; bat 125 MHz
38
Graphical abstract
39
Highlights
Twenty new ingol diterpenoids were isolated from Euphorbia marginata.
11, 14, and 18 were identified as potent multidrug resistance (MDR) modulators.
The mechanism of these MDR modulators was related to Pgp inhibition.
The brief structure-activity relationships of these ingols were discussed.
40