Commiphoratones A and B, Two Sesquiterpene Dimers from Resina Commiphora

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1 Commiphoratones A and B, Two Sesquiterpene Dimers from Resina Commiphora Jia-Wang Liu,,,,# Ying Liu,,# Yong-Ming Yan, Jing Yang, Xi-Feng Lu,*, Yong- Xian Cheng*,, Guangdong Key Laboratory for Genome Stability & Disease Prevention, School of Pharmaceutical Sciences, Shenzhen University Health Science Center, Shenzhen, , PR China State Key Laboratory of Phytochemistry and Plant Resources in West China, Kunming Institute of Botany, Chinese Academy of Sciences, Kunming , PR China Henan University of Chinese Medicine, Zhengzhou , PR China University of Chinese Academy of Sciences, Beijing , PR China 1

2 Contents of Supporting Information No. Page 1 Table S1. The 1 H and 13 C NMR data of 1 and Figure S1. 1 H NMR spectrum of 1 in CDCl Figure S2. 13 C NMR and DEPT spectra of 1 in CDCl Figure S3. HSQC spectrum of 1 in CDCl Figure S4. HMBC spectrum of 1 in CDCl Figure S5. 1 H- 1 H COSY spectrum of 1 in CDCl Figure S6. ROESY spectrum of 1 in CDCl Figure S7. HRESIMS of Figure S8. 1 H NMR spectrum of 2 in CDCl Figure S9. 13 C NMR and DEPT spectra of 2 in CDCl Figure S10. HSQC spectrum of 2 in CDCl Figure S11. HMBC spectrum of 2 in CDCl Figure S12. 1 H- 1 H COSY spectrum of 2 in CDCl Figure S13. ROESY spectrum of 2 in CDCl Figure S14. HRESIMS of Detailed isolation procedures X-ray crystallographic data of ECD calculations of Bioassay methods 19 2

3 Table S1. The 1 H and 13 C NMR data of 1 and 2 (δ in ppm) 1 H (800 MHz) and 13 C NMR (200 MHz) Data of 1 and 2 in CDCl 3 (δ in ppm, J in Hz) 1 2 position δ H δ C δ H δ C d (9.7) brs Ha:2.72 brd(11.9) 48.3 Ha:2.03 dd (13.4, 6.1) 40.2 Hb: 2.22 overlap Hb:1.24 dd (13.4, 6.1) m m Ha:2.98 brd(16.0) overlap 59.0 Hb:1.98 overlap s d (11.4) s s s s s s d(6.12) d (6.4) m m Ha:2.40 dd(16.3, 2.9) brs Hb:1.96 overlap m Ha:2.04 overlap overlap 54.0 Hb:1.11 overlap m m Ha: 1.48 m 22.1 Hb: 1.09 m brs 88.7 Ha: 1.75 m 41.9 Hb: 1.42 m m Ha: 2.30 d(13.4) 39.6 Hb: 1.93 d(13.4) m s d (6.9) d (7.5) d (6.6) d (6.5) s OCH s

4 Figure S1. 1 H NMR spectrum of 1 in CDCl 3. Figure S2. 13 C NMR and DEPT spectra of 1 in CDCl 3. 4

5 Figure S3. HSQC spectrum of 1 in CDCl 3. Figure S4. HMBC spectrum of 1 in CDCl 3. 5

6 Figure S5. 1 H- 1 H COSY spectrum of 1 in CDCl 3. Figure S6. ROESY spectrum of 1 in CDCl 3. 6

7 Figure S7. HRESIMS of 1. 7

8 Figure S8. 1 H NMR spectrum of 2 in CDCl 3. Figure S9. 13 C NMR and DEPT spectra of 2 in CDCl 3. 8

9 Figure S10. HSQC spectrum of 2 in CDCl 3. Figure S11. HMBC spectrum of 2 in CDCl 3. 9

10 Figure S12. 1 H- 1 H COSY spectrum of 2 in CDCl 3. Figure S13. ROESY spectrum of 2 in CDCl 3. 10

11 Figure S14. HRESIMS of 2. 11

12 Detailed isolation procedures 1. General experimental procedures Optical rotations were recorded on a Jasco P-1020 polarimeter. UV spectra were obtained on a Shimadzu UV-2401PC spectrometer. CD spectra were determined on a Chirascan instrument. NMR spectra were measured on a Bruker AV-600 or a AV-800 spectrometer, with TMS as an internal standard. ESIMS and HRESIMS were measured on an API QSTAR Pulsar 1 spectrometer. Silica gel GF254 (Qingdao Marine Chemical Inc., PR China) was used for preparative TLC. Silica gel ( mesh; Qingdao Marine Chemical Inc., PR China), C-18 silica gel (40 60 µm; Daiso Co., Japan), MCI gel CHP 20P ( µm, Mitsubishi Chemical Industries, Tokyo, Japan) and Sephadex LH-20 (Amersham Pharmacia, Sweden) were used for column chromatography. Semi-preparative HPLC was carried out using an Agilent 1200 liquid chromatograph equipped with an Agilent Zorbax SB-C 18 column (250 mm 9.4 mm, i.d., 5 µm). 2. Plant resins The medicinal materials of Resina Commiphora (myrrha) were obtained from Juhuacun Market of Material Medica, Kunming, Yunnan Province, PR China, in July The material was identified by Mr. Bin Qiu at Yunnan Institute of Materia Medica, and a voucher specimen (CHYX ) was deposited at the State Key Laboratory of Photochemistry and Plant Resources in West China, Kunming Institute of Botany, Chinese Academy of Sciences, PR China. 3. Extraction and isolation The dried resins (50 kg) were ground and soaked with 95% EtOH (180 L 48 h 3) to give a crude extract, which was suspended in warm water and extracted with EtOAc to afford an EtOAc soluble extract (8 kg). Six parts (Fr.A Fr.F) were obtained by using a silica gel column chromatography eluted with petroleum ether acetone (100:0, 100:1, 60:1, 40:1, 20:1, 5:1, 3:1, 1:1, 0:100) to process this EtOAc soluble extract. Then, Fr.B (2.4 kg) was passed through a silica gel column washed with petroleum ether EtOAc (100:0, 100:1, 60:1, 40:1, 20:1, 5:1, 3:1, 1:1) and petroleum ether acetone (5:1, 3:1, 1:1) to provide six portions (Fr.B.1 Fr.B.6). 12

13 Among them, Fr.B.5 (186.6 g) was further separated via MCI gel CHP 20P eluted with aqueous MeOH (55% 100%) to provide nine portions (Fr.B.5.1 Fr.B.5.9). Fr.B.5.5 (16.0 g) was submitted to a RP-18 column eluted with aqueous MeOH (50% 100%) to yield nine fractions (Fr.B Fr.B.5.5.9). Fr.B (0.49 g) was subjected to Sephadex LH-20 (MeOH) to yield two fractions of Fr.B (0.28 g). and Fr.B (0.10 g). Further purification of Fr.B (100.0 mg) by semipreparative HPLC eluted with aqueous MeCN (70%) afforded compound 1 (1.8 mg, t R = 12.6 min). Likewise, Fr.B.5.6 (21.2 g) underwent a RP-18 column eluted with aqueous MeOH (50% 100%) to yield six fractions (Fr.B Fr.B.5.6.6). Fr.B (5.71 g) was applied to a MCI gel CHP 20P column washed with aqueous MeOH (70% 100%) to provide three portions (Fr.B Fr.B ). Fr.B (0.76 g) was divided into five fractions (Fr.B Fr.B ) via Sephadex LH-20 (MeOH). Finally, Compound 2 (2.4 mg, t R = 19.5 min) was obtained from Fr.B by HPLC purification (aqueous MeCN, 85%). X-ray crystallographic data Crystal data for 1: C 30 H 32 O 6, M = , a = (3) Å, b = (5) Å, c = (7) Å, α = 90, β = (2), γ = 90, V = (15) Å 3, T = 100(2) K, space group P21, Z = 4, µ(cukα) = mm -1, reflections measured, 8092 independent reflections (R int = ). The final R 1 values were (I > 2σ(I)). The final wr(f 2 ) values were (I > 2σ(I)). The final R 1 values were (all data). The final wr(f 2 ) values were (all data). The goodness of fit on F 2 was Flack parameter = 0.15(10). The deposition number CCDC can be obtained free of charge from The Cambridge Crystallographic Data Centre via www. ccdc.cam.ac.uk/data request/cif. ECD calculations of 2 The theoretical calculations of compound 2 were performed using Gaussian 09 1 and figured using GaussView Conformation search using molecular mechanics calculations was performed in Discovery Studio 3.5 Client with MMFF force field with 20 kcal mol -1 upper energy limit. 3 The optimized conformation geometries and thermodynamic parameters of all selected conformations were provided. The predominant conformers were optimized 13

14 at B3LYP/6-31G(d,p) level. The theoretical calculation of ECD was performed using time dependent Density Functional Theory (TDDFT) at B3LYP/6-31G(d,p) level in MeOH with PCM model. 4 The ECD spectra of compound 2 were obtained by weighing the Boltzmann distribution rate of each geometric conformation. 5 The ECD spectra were simulated by overlapping Gaussian functions for each transition according to: The σ represented the width of the band at 1/e height, and E i and R i were the excitation energies and rotational strengths for transition i, respectively. R vel had been used in this work. (1) Gaussian 09, Revision C.01,M. J. Frisch, G. W. Trucks, H. B. Schlegel, G. E. Scuseria, M. A. Robb, J. R. Cheeseman, G. Scalmani, V. Barone, B. Mennucci, G. A. Petersson, H. Nakatsuji, M. Caricato, X. Li, H. P. Hratchian, A. F. Izmaylov, J. Bloino, G. Zheng, J. L. Sonnenberg, M. Hada, M. Ehara, K. Toyota, R. Fukuda, J. Hasegawa, M. Ishida, T. Nakajima, Y. Honda, O. Kitao, H. Nakai, T. Vreven, J. A. Montgomery, Jr., J. E. Peralta, F. Ogliaro, M. Bearpark, J. J. Heyd, E. Brothers, K. N. Kudin, V. N. Staroverov, T. Keith, R. Kobayashi, J. Normand, K. Raghavachari, A. Rendell, J. C. Burant, S. S. Iyengar, J. Tomasi, M. Cossi, N. Rega, J. M. Millam, M. Klene, J. E. Knox, J. B. Cross, V. Bakken, C. Adamo, J. Jaramillo, R. Gomperts, R. E. Stratmann, O. Yazyev, A. J. Austin, R. Cammi, C. Pomelli, J. W. Ochterski, R. L. Martin, K. Morokuma, V. G. Zakrzewski, G. A. Voth, P. Salvador, J. J. Dannenberg, S. Dapprich, A. D. Daniels, O. Farkas, J. B. Foresman, J. V. Ortiz, J. Cioslowski, and D. J. Fox, Gaussian, Inc., Wallingford CT, (2) GaussView, Version 5, Dennington, R.; Keith, T.; Millam, J. Semichem Inc., Shawnee Mission, KS, (3) Smith, S. G.; Goodman, J. M. J. Am. Chem. Soc. 2010, 132, (4) (a) Miertus, S.; Scrocc, E.; Tomasi, J. J. Chem. Phys. 1981, 55, 117. (b) Miertus, S.; Tomasi, J. J. Chem. Phys. 1982, 65, 239. (c) Cossi, M.; Barone, V.; Cammi, R.; Tomasi, J. Chem. Phys. Lett. 1996, 255, 327. (5) Tähtinen, P.; Bagno, A.; Klika, K. D.; Pihlaja, K. J. Am. Chem. Soc. 2003, 125,

15 16 15 O 2 14 H (S) 11' 4 4' 1 2' (S) (R) (R) 10 5' (R) 1' H (S) H (R) 6 H (R) 8 10' O H (S) 13' (S) 7' (R) H O 11 12' (Z) H 8' ' 2 OH 15' a b c Figure S15. Optimized geometries of predominant conformers for compound 2 at the B3LYP/6-31G(d,p) level in the gas phase. Table S2. Important thermodynamic parameters (a.u.) and Boltzmann distributions of the optimized compound 2 at B3LYP/6-31G(d,p) level in the gas phase Conformations E+ZPE G 2-a b c E+ZPE, G: total energy with zero point energy (ZPE) and Gibbs free energy in the gas phase at B3LYP/6-31G(d,p) level., %: Boltzmann distributions, using the relative Gibbs free energies as weighting factors % Table S3. Optimized Z-matrixes of compound 2 in the gas phase (Å) at B3LYP/6-31G(d,p) level 2-a 2-b C C C C C C C C C C

16 C C C C C C C C C C C C C C C C C C C C O O C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C O O H H O O O O C C H H H H H H H H H H H H H H H H H H H H H H H H H H H H H H H H H H H H H H H H H H H H H H H H

17 H H H H H H H H H H H H H H H H H H H H H H H H H H H H H H H H H H H H H H c C C C C C C C C C C C C C C C O C C C C C C C C C C C C C C C

18 O H O O C H H H H H H H H H H H H H H H H H H H H H H H H H H H H H H H H H H H H H H H H H H H

19 Bioassay methods Cell viability assay: Cell viability was determined by MTT assay. HepG2 cells were plated in 96-well plates at a density of cells/well and were cultured with medium for 24 h. After incubation for 48 h in the absence or presence of 5 µm, 10 µm, and 20 µm 1 or 2, a total of 10 µl of 0.5 mg/ml MTT solution was added into each well and the cultures were further incubated for 4 h under the same condition. After removing the medium, DMSO (100 µl) was added to dissolve the formazan crystals. The samples were detected at 490 nm by an enzyme-labeled microplate reader. RNA isolation and Q-PCR: Total RNA was extracted from HepG2 cells using Direct-zolTM RNA MiniPrep kit (ZYMO RESEARCH) according to the manufacturer s instructions. cdna was synthesized and amplified from 1 µg of total RNA using the PrimeScriptTM RT Master Mix (TaKaRa) as described in the manufacturer s protocol. Q-PCR analyses were performed with SYBR Premix Ex Taq II (TaKaRa) under the following conditions: 30 s at 95 C, 40 cycles at 95 C for 5 s and 60 C for 35 s. Gene expression was determined and normalized to the expression level of 36B4. The sequences of the primer for human 36B4 were 5 - TCTACAACCCTGAAGTGCTTGAT-3 and 5 -CAATCTGCAGACAGACACTGG; for human HMGCR were 5 -GAATGGCCCTAGAACTGTGC-3 and 5 - CAAAGAGCCCTGTGTGAATG-3 ;for human SQLE were 5 - CCTGAATCAGAAAATAAGGAGCA-3 and 5 - GCTTGTTTCTGAAATATTGGTTCC-3 ; for human LDLR were 5 - GCTTGTCTGTCACCTGCAAAT-3 and 5 -AACTGCCGAGAGATGCACTT-3. Western blot: After 2-day culturing with compounds, cells were lysed with RIPA lysis buffer (Beyotime) and centrifuged at 12,000 g for 5 min at 4 C. Lysates were subjected to SDS-PAGE; proteins were transferred onto a PVDF membrane and were probed with the indicated antibodies (Proteintech). Immunoreactive proteins were then visualized using ECL Western blotting detection reagent (Bio-rad). Signals on membranes were detected and quantified using ImageQuant LAS 4000 mini (GE Healthcare). 19