Supporting Information

Transcription

1 Supporting Information Leonuketal, a Spiroketal Diterpenoid from Leonurus japonicus Liang Xiong,, Qin-Mei Zhou,, Yike Zou, Ming-ua Chen, Li Guo,, Guan-Ying u,, Zhao-ua Liu, and Cheng Peng *,, School of Pharmacy and State Key Laboratory Breeding Base of Systematic Research, Development and Utilization of Chinese Medicine Resources, Chengdu University of Traditional Chinese Medicine, Chengdu , China Department of Chemistry, University of Pennsylvania, 231 S. 34 Street, Philadelphia, PA , United States Institute of Medicinal Biotechnology, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing , China Chengdu No.1 Pharmaceutical Co. Ltd., Chengdu , China

2 The List of Contents no. content page 1 Experimental Section S1 2 General Experimental Procedures S1 3 Plant Material S1 4 Extraction and Isolation S1 5 Synthesis of (R)- and (S)-MPA Esters of 1 (1a and 1b) S2 6 Figure S1. δ values (δ R - δ S, in ppm) for 1a and 1b S2 7 Table S1. 1 NMR Data (600Mz) for 1a and 1b in Me 2 C-d 6 S3 8 ECD Calculation of 1A and 1B S3 9 Figure S2. Proposed Absolute Configurations (1A and 1B) of 1 S4 10 Table S2. Energy Analysis of Conformers of Compound 1A S4 11 Table S3. Energy Analysis of Conformers of Compound 1B S5 12 Figure S3. B3LYP/6-31+G(d, p) ptimized 3 Lowest Energy Conformers of 1A and Their Relative Gibbs Free Energies (ΔG < 2 Kcal/mol) 13 Figure S4. B3LYP/6-31+G(d, p) ptimized 3 Lowest Energy Conformers of 1B and the Corresponding Relative Gibbs Free Energies (ΔG < 2 Kcal/mol) Figure S5. The Experimental ECD Spectra of 1 (Black) and the Calculated ECD Spectra of 1A (Red) and 1B (Blue) 15 Vasorelaxant Activity Assay S6 16 References S7 Figure S6. The 1 NMR Spectrum of Compound 1 in Me 2 C d 6 (600 Mz) S8 Figure S7. The 13 C NMR Spectrum of Compound 1 in Me 2 C d 6 (150 Mz) S9 19 Figure S8. The DEPT Spectrum of Compound 1 in Me 2 C d 6 S10 20 Figure S9. The SQC Spectrum of Compound 1 in Me 2 C d 6 S11 21 Figure S10. The 1-1 gcsy Spectrum of Compound 1 in Me 2 C d 6 S12 Figure S11. The MBC Spectrum of Compound 1 in Me 2 C d 6 S13 23 Figure S12. The NESY Spectrum of Compound 1 in Me 2 C d 6 S 24 Figure S13. The IR Spectrum of Compound 1 S15 25 Figure S. The UV Spectrum of Compound 1 S16 26 Figure S15. The (+)-RESIMS Spectroscopic Data of Compound 1 S 27 Figure S16. The CD Spectrum of Compound 1 in MeCN S 28 Figure S. The 1 NMR Spectrum of Compound 1a in Me 2 C d 6 (600 Mz) S19 29 Figure S. The 1-1 gcsy Spectrum of Compound 1a in Me 2 C d 6 S20 30 Figure S19. The 1 NMR Spectrum of Compound 1b in Me 2 C d 6 (600 Mz) S21 31 Figure S20. The 1-1 gcsy Spectrum of Compound 1b in Me 2 C d 6 S S5 S6 S6

3 Experimental Section General Experimental Procedures. ptical rotations were measured on a Rudolph Research Autopol III automatic polarimeter. IR spectra were recorded using a Nicolet 5700 FT-IR microscope instrument. CD spectra were recorded on a JASC J-815 CD spectrometer. NMR spectra were obtained using a Bruker-AVIIID-600 spectrometer with the solvent peaks used as the references. RESIMS spectra were measured using a Waters Synapt G2 DMS. Column chromatography (CC) was performed using silica gel ( mesh, Yantai Institute of Chemical Technology, Yantai, China), MCI gel CP 20P ( μm, Mitsubishi Chemical, Co., Japan), and Sephadex L-20 (Amersham Pharmacia Biotech AB, Uppsala, Sweden). PLC separation was performed using an instrument equipped with a Cometro 6000LDS pump, a Cometro 6000PVW UV/VIS detector, and an Ultimate ( mm 2 ) preparative column packed with C (5 μm). TLC was performed using glass precoated silica gel GF254 plates (Qingdao Marine Chemical Inc., Qingdao, China). Plant Material. The aerial parts of L. japonicus were collected in May of 2012 from a field in Wenjiang District, Chengdu City, Sichuan Province, China. The herb was identified by Prof. Min Li (Chengdu University of TCM, Sichuan, China). A voucher specimen (SYMC-05) was deposited at the School of Pharmacy, Chengdu University of TCM, China. Extraction and Isolation. The air-dried herb of L. japonicus (20 kg) was extracted three times with 95% Et (3 160 L) at room temperature for 72 h. The ethanolic extract was evaporated under reduced pressure to yield a dark brown residue (1.2 kg). The residue was suspended in 2 and then successively partitioned into EtAc (400 g) and n-bu (160 g) fractions. The EtAc extract was subjected to silica gel column using a gradient elution of petroleum ether acetone (100:1 0:1) to afford 19 fractions (F 1 F 19 ). Eluting with a step gradient of 50% 100% Me in 2, F 7 was separated by flash chromatography over MCI gel to give ten subfractions (F 7-1 F 7-10 ). F 7-3 was fractioned via silica gel column over petroleum ether acetone (20:1 1:1) to afford F F F S1

4 was further purified by preparative TLC (petroleum ether acetone 5:1) followed by RP semi-preparative PLC (85% Me in 2 ) to afford 1 (6.5 mg). Leonuketal (1): colorless gum; [α] 20 D 98.4 (c 0.05, Me); UV (Me) λ max (log ε) 260 (1.38) nm; CD (MeCN) 202 (Δε +2.13), 209 (Δε +0.40), 6.5 (Δε +1.51), (Δε 4.69) nm; IR (KBr) ν max 3362, 39, 2920, 2850, 87, 26, 1646, 68, 1287, 1119, 1027, 972, 919 cm -1 ; 1 NMR (Me 2 C-d 6, 600 Mz) data and 13 C NMR (Me 2 C-d 6, 150 Mz) data, see Table 1. (+)-R-ESIMS m/z [M+] + (calcd for C 35 7, ), [M+Na] + (calcd for C 34 7 Na, ), [M+K] + (calcd for C 34 7 K, ), and [2M+Na] + (calcd for C Na, ). Synthesis of (R)- and (S)-MPA Esters of 1 (1a and 1b). (R)- or (S)-MPA(10 mg) was added to a solution of compound 1 (1.2 mg), EDCI (10 mg), and DMAP (8.0 mg) in freshly distilled methylene chloride (3 ml) and stirred at room temperature overnight. The reaction mixture was separated by RP semipreparative PLC (90% Me in 2 ) to afford compounds 1a (1-R-MPA) or 1b (1-S-MPA). Then the 1 NMR and 1-1 gcsy spectra were measured. The chemical shifts differences between 1a and 1b were determined and showed in Figure S1. Applying the MPA determination rule based on the Δδ values, 1 the absolute configuration of C-15 was assigned as R. 1 NMR (Me 2 C-d 6, 600 Mz) data of 1a and 1b see Table S1. Figure S1. δ values (δ R - δ S, in ppm) for 1a and 1b. S2

5 Table S1. 1 NMR Data (600Mz) for 1a and 1b in Me 2 C-d 6. a no. 1a 1b dd (3.0, 2.4) 3.44 dd (3.0, 2.4) d (1.8) 2.85 d (2.4) 5a 2.26 ddd (13.2, 10.8, 8.4) 2.32 ddd (13.2, 10.8, 8.4) 5b 1.49 dddd (13.2, 10.8, 1.8, 1.8) 1.53 dddd (13.2, 10.2, 2.4, 6a 1.83 m 1.86 m 6b 1.78 m 1.82 m 8a 1.64 ddd (13.2, 4.2, 3.0) 1.76 ddd (13.2, 4.2, 3.0) 8b 1.25 ddd (13.2, 13.2, 4.8) 1.32 ddd (13.2, 13.2, 4.8) 9a 2. m 2. m 9b 1.98 m 1.99 m m 2.78 m d (4.2) 4.47 (4.2) s 5.30 s dd (9.0, 3.0) 5.03 dd (9.6, 3.6) 16a 2.08 m 2.02 m 16b 1.68 m 1.58 m 0.93 t (7.2) 0.73 t (7.2) 0.80 s 0.89 s s 1.25 s s 0.73 s 21a 3.80 dq (9.6, 7.2) 3.81 dq (9.6, 7.2) 21b 3.68 dq (9.6, 7.2) 3.69 dq (9.6, 7.2) 1.19 t (7.2) 1.20 t (7.2) Data for (R)-α-methoxyphenylacetyl unit in 1a: δ 7.51 (-2', 6'), 7.39 (-3', 5'), 7.36 (-4'), 4.97 (-7'), 3.48 (Me) ppm. Data for (S)-α-methoxyphenylacetyl unit in 1b: δ7.49 (-2', 6'), 7.39 (-3', 5'), 7.35 (-4'), 4.99 (-7'), 3.43 (Me) ppm. ECD Calculation of 1A and 1B. All quantum computations were performed by using Gaussian09 program package on an IBM cluster machine located at the igh Performance Computing Center of Peking Union Medical College. First, conformational analyses of 1A and 1B (Figure S2) were carried out by using Spartan 10 software with MMFF94 force field. 2 As a result, 6 and 15 low energy conformers were generated for 1A and 1B, respectively (Tables S2 and S3). The conformers with relative energies less than 2 kcal/mol were then submitted for the DFT optimizations and frequency S3

6 calculations (to confirm the energy minimal as well as generating set of thermodynamic data) at B3LYP/6-31+G (d,p) level by using the Gaussian 09 program. 3 The optimized structures were then used for the TDDFT calculations at B3LYP/ G (2d, 2p) level in gas phase to generate the excitation energies and the corresponding rotational strengths for the first 9 excited states. The three lowest energy conformers (Gibbs free energies ( G) less than 2 kcal/mol) of 1A (Figure S3) and 1B (Figure S4) were then submitted for ECD spectra simulations by using the Gausssum 2.25 program 4 (σ= 0.3 ev). The averaged spectra of 1A and 1B (Figure S5) were generated by using Boltzmann distribution according to the corresponding Gibbs free energies ( G). Figure S2. Proposed Absolute Configurations (1A and 1B) of 1. Table S2. Energy Analysis for the Conformers of Compound 1A. Conf. 1A-C1 1A-C2 1A-C3 1A-C4 1A-C5 1A-C6 MMFF energy B3LYP/6-31+G(d, p) Gibbs free energy ( K) ΔE (Kcal/mol) Boltzmann G Boltzmann ΔG (Kcal/mol) Distribution (artree) Distribution Table S3. Energy Analysis of Conformers of Compound 1B. Conf. MMFF energy B3LYP/6-31+G(d, p) Gibbs free energy ( K) ΔE (Kcal/mol) Boltzmann G ΔG (Kcal/mol) Boltzmann S4

7 Distribution (artree) Distribution 1B-C1 1B-C2 1B-C3 1B-C4 1B-C5 1B-C6 1B-C7 1B-C8 1B-C9 1B-C10 1B-C11 1B-C12 1B-C13 1B-C 1B-C A-C1 1A-C2 1A-C5 Figure S3. B3LYP/6-31+G(d, p) ptimized 3 Lowest Energy Conformers of 1A and Their Relative Gibbs Free Energies (ΔG < 2 Kcal/mol). 1B-C2 1B-C3 1B-C13 S5

8 Figure S4. B3LYP/6-31+G(d, p) ptimized 3 Lowest Energy Conformers of 1B and the Corresponding Relative Gibbs Free Energies (ΔG < 2 Kcal/mol). Figure S5. The Experimental ECD Spectrum of 1 (Black) and the Calculated ECD Spectra of 1A (Red) and 1B (Blue). Vasorelaxant Activity Assay. Contraction of isolated rat aorta was measured as described. 5 Briefly, aortic segments from SD rats were suspended in organ baths filled with a physiological solution [composition(mm): NaCl, 120; KCl, 4.6; K 2 P 4, 1.2; MgS 4, 1.2; NaC 3, 25; glucose, 10; CaCl 2, 2.5)] bubbled with a gas mixture of 95% 2 and 5% C 2, and maintained at 37 C. Contraction was evoked by a depolarizing 60 mm KCl solution. The amplitude of the contraction evoked in the presence of the tested compound was compared to the response measured in its absence. Methoxyverapamil was used as a positive drug. The EC 50 values of the test compound and methoxyverapamil were calculated from cumulative concentration tension curves by linear regression. Data were expressed as the mean ± SEM. Statistical significance was tested using Student s t-test with a P value of less than 0.05, indicating a significant difference between data sets. S6

9 References (1) Seco, J. M.; Quiñoá, E.; Riguera, R. Chem. Rev. 2004, 104, 1. (2) Spartan 10, Wavefunction, Inc.: Irvine, CA. (3) Frisch, M. J.; Trucks, G. W.; Schlegel,. B.; Scuseria, G. E.; Robb, M. A.; Cheeseman, J. R.; Scalmani, G.; Barone, V.; Mennucci, B.; Petersson, G. A.; Nakatsuji,.; Caricato, M.; Li, X.; ratchian,. P.; Izmaylov, A. F.; Bloino, J.; Zheng, G.; Sonnenberg, J. L.; ada, M.; Ehara, M.; Toyota, K.; Fukuda, R.; asegawa, J.; Ishida, M.; Nakajima, T.; onda, Y.; Kitao,.; Nakai,.; Vreven, T.; Montgomery, Jr., J. A.; Peralta, J. E.; gliaro, F.; Bearpark, M.; eyd, J. J.; Brothers, E.; Kudin, K. N.; Staroverov, V. N.; Kobayashi, R.; Normand, J.; Raghavachari, K.; Rendell, A.; Burant, J. C.; Iyengar, S. S.; Tomasi, J.; Cossi, M.; Rega, N.; Millam, J. M.; Klene, M.; Knox, J. E.; Cross, J. B.; Bakken, V.; Adamo, C.; Jaramillo, J.; Gomperts, R.; Stratmann, R. E.; Yazyev,.; Austin, A. J.; Cammi, R.; Pomelli, C.; chterski, J. W.; Martin, R. L.; Morokuma, K.; Zakrzewski, V. G.; Voth, G. A.; Salvador, P.; Dannenberg, J. J.; Dapprich, S.; Daniels, A. D.; Farkas,.; Foresman, J. B.; rtiz, J. V.; Cioslowski, J.; Fox, D. J. Gaussian 09, Revision C.01; Gaussian, Inc., Wallingford CT, (4) Gausssum 2.25: Boyle, N. M.; Tenderholt, A. L.; Langner, K. M. J. Comput. Chem. 2008, 29, (5) Kang, J.; Li, L.; Wang, D.; Wang,.; Liu, C.; Li, B.; Yan, Y.; Fang, L.; Du, G.; Chen, R. Phytochemistry 2015, 116, S7

10 Figure S6. The 1 NMR Spectrum of Compound 1 in Me 2 C d 6 S8

11 Figure S7. The 13 C NMR Spectrum of Compound 1 in Me 2 C d 6 S9

12 Figure S8. The DEPT Spectrum of Compound 1 in Me 2 C d 6 S10

13 Figure S9. The SQC Spectrum of Compound 1 in Me 2 C d 6 S11

14 Figure S10. The gcsy Spectrum of Compound 1 in Me 2 C d 6 S12

15 Figure S11. The MBC Spectrum of Compound 1 in Me 2 C d 6 S13

16 Figure S12. The NESY Spectrum of Compound 1 in Me 2 C d 6 S

17 Figure S13. The IR Spectrum of Compound 1 S

18 Figure S. The UV Spectrum of Compound 1 S16

19 Figure S15. The (+)-RESIMS Spectroscopic Data of Compound 1 S

20 Figure S16. The CD Spectrum of Compound 1 in MeCN S

21 R a R = (R)-MPA Figure S. The 1 NMR Spectrum of Compound 1a in Me 2 C d 6 S19

22 R a R = (R)-MPA Figure S. The 1-1 CSY Spectrum of Compound 1a in Me 2 C d 6 S20

23 R b R = (S)-MPA Figure S19. The 1 NMR Spectrum of Compound 1b in Me 2 C d 6 S21

24 R b R = (S)-MPA Figure S20. The 1-1 CSY Spectrum of Compound 1b in Me 2 C d 6 S