Alismanin A, a Triterpenoid with a C 34 Skeleton from Alisma orientale as a Natural Agonist of Human Pregnane X Receptor

Transcription

1 Supporting information Alismanin A, a Triterpenoid with a C 34 Skeleton from Alisma orientale as a Natural Agonist of Human Pregnane X Receptor Chao Wang,,+ Xiao-Kui Huo,,+ Zhi-Lin Luan,,+ Fei Cao, Xiang-Ge Tian, Xin-Yu Zhao, Cheng-Peng Sun,*, Lei Feng, Jing Ning, Bao-Jing Zhang, Xiao-Chi Ma*,, College of Pharmacy, College (Institute) of Integrative Medicine, Dalian Medical University, Dalian , China Key Laboratory of Pharmaceutical Quality Control of Hebei Province, College of Pharmaceutical Sciences, Hebei University, Baoding , China Basic Medical College, Dalian Medical University, Dalian , China + These authors contributed equally to this work. 1

2 Content Experimental details... 3 General Experimental Procedures Plant Material Extraction and Isolation X-ray Crystal Structure Determination of ECD and 13 C NMR calculation PXR Activation Bioassay Docking References... 7 Table S1. Calculated 13 C NMR chemical shifts of Table S2. 1 H (600 MHz, MeOH-d 4 ) and 13 C NMR (150 MHz, MeOH-d 4 ) data of 2 and Figure S1. Binding model of compounds 1 (A) and 2 (B) in the PXR ligand binding pocket Figure S2. 1 H NMR spectrum of 1 (600 MHz, MeOH-d 4 ) Figure S3. 13 C NMR spectrum of 1 (150 MHz, MeOH-d 4 ) Figure S4. HSQC spectrum of 1(600 MHz, MeOH-d 4 ) Figure S5. HMBC spectrum of 1 (600 MHz, MeOH-d 4 ) Figure S6. COSY spectrum of 1 (600 MHz, MeOH-d 4 ) Figure S7. NOESY spectrum of 1 (600 MHz, MeOH-d 4 ) Figure S8. HRESIMS spectrum of Figure S9. ECD spectrum of Figure S10. 1 H NMR spectrum of 2 (600 MHz, MeOH-d 4 ) Figure S C NMR spectrum of 2 (150 MHz, MeOH-d 4 ) Figure S12. HSQC spectrum of 2(600 MHz, MeOH-d 4 ) Figure S13. HMBC spectrum of 2 (600 MHz, MeOH-d 4 ) Figure S14. COSY spectrum of 2 (600 MHz, MeOH-d 4 ) Figure S15. NOESY spectrum of 2 (600 MHz, MeOH-d 4 ) Figure S16. HRESIMS spectrum of Figure S17. 1 H NMR spectrum of 3 (600 MHz, MeOH-d 4 ) Figure S C NMR spectrum of 3 (150 MHz, MeOH-d 4 ) Figure S19. HSQC spectrum of 3(600 MHz, MeOH-d 4 ) Figure S20. HMBC spectrum of 3 (600 MHz, MeOH-d 4 ) Figure S21. NOESY spectrum of 3 (600 MHz, MeOH-d 4 ) Figure S22. HRESIMS spectrum of Figure S23. ECD spectrum of Figure S24. Lowest energy conformers for (5R,8S,9S,10S,11S,14R,20R) Figure S25. Lowest energy conformers for (5R,8S,9S,10S,14R,17S,20R,23S,24S)

3 Experimental details General Experimental Procedures. Optical rotations were measured on a Perkin-Elmer 241 polarimeter. UV spectra were recorded on a JASCO V-650 spectrophotometer. The NMR spectra were recorded on a Bruker-600 spectrometer. Chemical shifts are in δ (ppm), and coupling constants (J) in Hz. HRESIMS spectra were measured on an Agilent 1100 series LC/MSD ion trap mass spectrometer. High-performance liquid chromatography (HPLC) analyses were performed on a UItimate 3000 HPLC system equipped with a photodiode array detector and a quaternary pump system and a column compartment. Preparative HPLC was performed on an Elite P2300 instrument with an Elite UV2300 detector and a YMC C 18 column (250 mm 10 mm, 5 μm). All solvents were obtained from Tianjin Kemiou Chemical Reagent Company (Tianjing, China), MeOH for HPLC analysis were chromatographic grade (Merck, Darmstadt, Germany). Silica gel ( mesh) for column chromatography (CC) were purchased from Qingdao Marine Chemical Factory (Qingdao, People s Republic of China). Plant Material. Dried rhizomes of A. orientale were purchased in January 2013 from Beijing Tongrentang Co., Ltd., China, and identified by Prof. Jing-Ming Jia, Shenyang Pharmaceutical University. A voucher specimen ( P) has been deposited in the herbarium of the Department of Medicinal Chemistry, Dalian Medical University. Extraction and Isolation. The dried rhizomes of A. orientale (4.0 kg) were extracted with 80% EtOH (3 2 h 10 L) at 95 to afford a residue after solvent removal in vacuo. The residue was 3

4 suspended in H 2 O (5 L), and extracted with petroleum ether (3 5 L), CHCl 3 (3 5 L), EtOAc (3 5 L), and n-buoh (3 5 L), successively. The CHCl 3 extract (240 g) was separated by a silica gel column, eluted with CHCl 3 MeOH (100:1 4:1), to afford fractions Fr.3 (3.7 g) was subjected to silica gel column chromatography eluted with petroleum ether-etoac (from 100:1 to 1:1) to obtain five subfractions Fr.3A-Fr.3E. Fr.3A (500 mg) was purified through preparative HPLC (80% 90% MeOH-H 2 O), yielding compound 3 (2.0 mg). Fr.4 (5.1 g) was separated by silica gel column chromatography eluted with petroleum ether-etoac (from 100:1 to 1:1) to obtain three subfractions Fr.4A-Fr.4C. The purification of Fr.4B (320 mg) has led to the isolation of compounds 1 (3.0 mg) and 2 (8.1 mg). Aslimanin A (1): amorphous powder; [α] 25 D 18.3 (c 0.06, CH 2 Cl 2 ); UV (CH 2 Cl 2 ) λ max (log ε) 236 (3.8), 263 (3.3) nm; ECD (c 0.6, MeOH) nm (Δε) 207 ( 1.62), 222 (+1.47), 290 (+1.45); 1 H (600 MHz, MeOH-d 4 ) and 13 C NMR (150 MHz, MeOH-d 4 ) data, see Table 1; HRESIMS m/z [M + NH 4 ] + + (calcd for C 34 H 50 NO ). Aslimanin B (2): amorphous powder; [α] 25 D (c 0.05, CH 2 Cl 2 ); UV (CH 2 Cl 2 ) λ max (log ε) 207 (3.9) nm; 1 H (600 MHz, MeOH-d 4 ) and 13 C NMR (150 MHz, MeOH-d 4 ) data, see Table 2; HRESIMS m/z [M + Na] + (calcd for C 29 H 48 NaO ). Aslimanin C (3): amorphous powder; [α] 25 D 98.3 (c 0.06, CH 2 Cl 2 ); UV (CH 2 Cl 2 ) λ max (log ε) 236 (3.9) nm; ECD (c 0.6, MeOH) nm (Δε) 209 (+2.79), 237 ( 12.79), 289 (+3.14), 338 ( 1.03); 1 H (600 MHz, MeOH-d 4 ) and 13 C NMR (150 MHz, MeOH-d 4 ) data, see Table 2; HRESIMS m/z [M + H] + + (calcd for C 32 H 49 O ). 4

5 X-ray Crystal Structure Determination of 2 The data were collected on an SuperNova, Dual, AtlasS2 diffractometer using monochromatized Cu Kα radiation. The structure was solved by direct methods using SHELXL2016. All H atoms were refined using the riding model. All non-h atoms were refined anisotropically. Crystallographic data have been deposited in the Cambridge Crystallographic Data Centre (CCDC number ). Copies of the data can be obtained, free of charge, from the CCDC website ( Crystal Data: C 29 H 48 O 3, M = orthorhombic (MeOH), size mm 3, a = (2) Å, b = (3) Å, c = (3) Å, α = 90, β = 90, γ = 90, V = (15) Å 3, T = (10) K, space group P , Z = 8, μ (Cu Kα) = mm -1, F(000) = , 2θ range for data collection from to , reflections measured, unique (Rint = , Rsigma = ) which were used in all calculations. The final R 1 was (I > 2σ(I)) and wr 2 was The Flack parameter was 0.06(6). The largest difference peak and hole were and e Å 3. ECD and 13 C NMR calculation. Conformational searches were performed using MMFF94S force field for 1 and 3. All geometries (50 lowest energy conformers) with relative energy from 0-10 kcal/mol used in optimizations at the B3LYP/6-31G(d) level using Gaussian09 package. The B3LYP/6-31G(d)-optimized conformers with relative energy from 0 to 4.6 kcal/mol were then re-optimized at the B3LYP/6-311+G(d) level. Based on the optimized geometries, the NMR and ECD calculations were performed at the 5

6 B3LYP/6-311G+(2d,p) level of theory. 1-5 PXR Activation Bioassay. The expression vector of hpxr and the hpxr XREM-driven luciferase reporter plasmid (CYP3A4XREM-luciferase) has been described previously. 6 Using the lipofectamine 3000 (Invitrogen, U.S.A.), the human hepatoma HepG2 cell line was transfected with expression and reporter plasmids, together with pgl4.74 (Promega, China) as an internal standard. The cells were treated with individual compounds at the final concentration of 10 nm after an overnight incubation. Rifampicin, the commonly used hpxr agonist, was applied as positive control at the final concentration of 10μM. The luciferase activities were measured by using the Dual-luciferase Reporter Assay System (Promega, China). The co-transfected plasmid results were normalized by dividing the Firefly luciferase signal by the Renilla luciferase signal. Docking. The crystal structure of PXR was taken from Protein Data Bank (PDB ID: 4X1F). The missing residues from 177 to 194 was added by homology modelling using modeler (version 9.17). The modelled PXR conformation was relaxed by performing 5000 step energy minimization and equilibrated by performing additional 1000 ps molecular dynamics (MD) simulations in aqueous solution at 310 K and 1atm. MD simulations were carried out using NAMD2 software, with CHARMM 22 force field with CMAP correction and TIP3P water model. It was found that these residues (177 to 194) formed a loop. The 3D structures of the compounds were prepared and Gasteiger-Hückel charges were added with Sybyl software. Each ligand was subjected to energy minimization with Tripos force filed parameters. Blind docking was carried out using AutoDock 4.2 program. The 3D docking grid was sufficiently large to cover the protein. The non-protein molecules in the original PDB file was deleted prior to 6

7 docking. A total 100 conformations of each ligand were searched using Lamarckian generic algorithm, and the final docking conformations were clustered into different number of clusters in terms of the root-mean-square deviations of the ligand within the binding pocket of the receptor. References 1. A. G. Kutateladze, O. A. Mukhina, J. Org. Chem. 2015, 80, A. G. Kutateladze, O. A. Mukhina, J. Org. Chem. 2015, 80, A. G. Kutateladze, O. A. Mukhina, J. Org. Chem. 2014, 79, H. Yu, W. X. Li, J. C. Wang, Q. Yang, H. J. Wang, C. C. Zhang, S. S. Ding, Y. Li, H. J. Zhu, Tetrahedron 2015, 71, P. He, X. F. Wang, X. J. Guo, C. Q. Zhou, S. G. Shen, D. B. Hu, X. L. Yang, D. Q. Luo, R. Dukor, H. J. Zhu, Tetrahedron Lett. 2014, 55, C. Zhou, E. J. Poulton, F. Grun, T. K. Bammler, B. Blumberg, K. E. Thummel, D. L. Eaton, Mol. Pharmacol. 2007, 71,

8 Table S1. Calculated 13 C NMR chemical shifts of 1 position δ C (Exp.) δ C (Calcd.) Δδ (0.7) (0.7) (3.0) (0.2) (0.4) (1.5) (1.9) (2.8) (3.1) (4.7) (0.2) (3.9) (2.3) (2.5) (3.0) (0.7) (0.4)

9 Table S2. 1 H (600 MHz, MeOH-d 4 ) and 13 C NMR (150 MHz, MeOH-d 4 ) data of 2 and No. δ C δ H (J in Hz) δ C δ H (J in Hz) td (12.2, 2.5) m 2.10 m 1.77 m ddd (15.5, 12.2, 6.5) ddd (15.4, 12.1, 6.7) 2.24 ddd (15.5, 9.6, 2.5) 2.24 dd (15.4, 9.4) m br d (12.7) m m 1.30 m 1.46 dd (12.8, 5.6) m m 1.30 m 1.41 m d (10.6) br s ddd (16.6, 10.6, 5.8) br d (10.4) dd (13.6, 5.8) br d (10.4) 2.04 m ddd (15.0, 9.4, 5.8) m 1.34 m 1.35 m m m 2.01 m 1.65 m s s s s m m d (7.0) d (7.0) m m 1.27 m 1.69 m m m m d (3.5) d (6.8) s d (6.8) s s s s s s s 23-OAc

10 s Figure S1. Binding model of compounds 1 (A) and 2 (B) in the PXR ligand binding pocket. 10

11 Figure S2. 1 H NMR spectrum of 1 (600 MHz, MeOH-d 4 ) Figure S3. 13 C NMR spectrum of 1 (150 MHz, MeOH-d 4 ) 11

12 Figure S4. HSQC spectrum of 1(600 MHz, MeOH-d 4 ) 12

13 Figure S5. HMBC spectrum of 1 (600 MHz, MeOH-d 4 ) Figure S6. COSY spectrum of 1 (600 MHz, MeOH-d 4 ) 13

14 Figure S7. NOESY spectrum of 1 (600 MHz, MeOH-d 4 ) Figure S8. HRESIMS spectrum of 1 14

15 Figure S9. ECD spectrum of 1 15

16 Figure S10. 1 H NMR spectrum of 2 (600 MHz, MeOH-d 4 ) 16

17 Figure S C NMR spectrum of 2 (150 MHz, MeOH-d 4 ) Figure S12. HSQC spectrum of 2(600 MHz, MeOH-d 4 ) 17

18 Figure S13. HMBC spectrum of 2 (600 MHz, MeOH-d 4 ) 18

19 Figure S14. COSY spectrum of 2 (600 MHz, MeOH-d 4 ) 19

20 Figure S15. NOESY spectrum of 2 (600 MHz, MeOH-d 4 ) Figure S16. HRESIMS spectrum of 2 20

21 Figure S17. 1 H NMR spectrum of 3 (600 MHz, MeOH-d 4 ) Figure S C NMR spectrum of 3 (150 MHz, MeOH-d 4 ) 21

22 Figure S19. HSQC spectrum of 3(600 MHz, MeOH-d 4 ) Figure S20. HMBC spectrum of 3 (600 MHz, MeOH-d 4 ) 22

23 Figure S21. NOESY spectrum of 3 (600 MHz, MeOH-d 4 ) 23

24 Figure S22. HRESIMS spectrum of 3 Figure S23. ECD spectrum of 3 24

25 25

26 26

27 27

28 Figure S24. Lowest energy conformers for (5R,8S,9S,10S,11S,14R,20R)-1 28

29 Figure S25. Lowest energy conformers for (5R,8S,9S,10S,14R,17S,20R,23S,24S)-3 29