Gelsekoumidines A and B: Two Pairs of Atropisomeric Bisindole Alkaloids from the Roots of Gelsemium elegans Wei Zhang a,b, Wei Xu a, Gui-Yang Wang a, Xue-Ying Gong a, Ni-Ping Li a, Lei Wang a,b,*, Wen-Cai Ye a,b, a Institute of Traditional Chinese Medicine & Natural Products, and JNU-HKUST Joint Laboratory for Neuroscience & Innovative Drug Research, College of Pharmacy, Jinan University, Guangzhou 510632, People s Republic of China b Guangdong Province Key Laboratory of Pharmacodynamic Constituents of TCM and New Drugs Research, Jinan University, Guangzhou 510632, People s Republic of China 1
Contents 1. General experimental procedures... 3 2. Plant material... 3 3. Extraction and isolation of 1-2... 3 4. Physico-chemical constants of 1-2... 4 5. X-Ray crystallographic analysis of 1a... 6 6. Dynamic HPLC analyses of 1a and 1b... 7 7. Quantum chemical ECD calculation method... 8 8. Bioassay methods... 19 Figure S5. The UV of compound 1 in MeOH... 21 Figure S6. The IR (KBr disc) of compound 1... 21 Figure S7. The HR-ESI-MS of compound 1... 21 Figure S8. The 1 H NMR spectrum for compound 1 in CD 3 OD... 22 Figure S9. The 13 C NMR spectrum of compound 1 in CD 3 OD... 22 Figure S10. The DEPT-135 spectrum of compound 1 in CD 3 OD... 22 Figure S11. The 1 H- 1 H COSY spectrum of compound 1 in CD 3 OD... 23 Figure S12. The HSQC NMR spectrum of compound 1 in CD 3 OD... 23 Figure S13. The HMBC spectrum of compound 1 in CD 3 OD... 24 Figure S14. The NOESY spectrum of compound 1 in CD 3 OD... 24 Figure S15. 1 H NMR spectra for compound 1 in CDCl 3, DMSO-d 6, CDOD 3, and pyridine-d 5... 25 Figure S16. The UV of compound 2 in MeOH... 26 Figure S17. The IR (KBr disc) of compound 2... 26 Figure S18. The HR-ESI-MS of compound 2... 26 Figure S19. The 1 H NMR spectrum for compound 2 in CD 3 OD... 27 Figure S20. The 13 C NMR spectrum of compound 2 in CD 3 OD... 27 Figure S21. The DEPT-135 spectrum of compound 2 in CD 3 OD... 27 Figure S22. The 1 H- 1 H COSY spectrum of compound 2 in CD 3 OD... 28 Figure S23. The HSQC spectrum of compound 2 in CD 3 OD... 28 Figure S24. The HMBC spectrum of compound 2 in CD 3 OD... 29 Figure S25. The NOESY spectrum of compound 2 in CD 3 OD... 29 2
1. General experimental procedure Melting points were obtained on an X-5 micro melting point apparatus without correction (Fukai Instrument, Beijing, China). Optical rotations were determined using a JASCO P-1020 digital polarimeter (Jasco, Tokyo, Japan) at 25 C. UV spectra were recorded on a Jasco V-550 UV/VIS spectrophotometer (Jasco, Tokyo, Japan). IR spectra were determined on a Jasco FT/IR-480 plus Fourier transform infrared spectrometer (Jasco, Tokyo, Japan) using KBr pellets. ECD spectra were obtained on a Jasco J-810 spectropolarimeter (Jasco, Tokyo, Japan) at room temperature. HR-ESI- MS was carried out on Agilent 6210 LC/MSD TOF mass spectrometer (Agilent Technologies, CA, USA). NMR spectra were measured on Bruker AV-400 and AV- 500 spectrometers (Bruker, Switzerland) with TMS as an internal standard. Singlecrystal data were performed using Oxford-Diffraction SuperNova diffractometer and Cu K radiation. Column chromatography (CC) were performed on Sephadex LH-20 (Pharmacia Biotech AB, Uppsala, Sweden), silica gel (200-300 mesh; Qingdao Marine Chemical Inc., Qingdao, P. R. China), and ODS (YMC, Kyoto, Japan). Preparative HPLC was performed on an Agilent 1260 Chromatograph equipped with a G1311C pump and a G1315D photodiode array detector (Agilent Technologies, CA, USA) with a Waters Xbridge C 18 OBD reversed-phase column (19 250 mm, 5 mm, USA). All solvents used in HPLC were of chromatographic grade (Fisher Scientific, New Jersey, USA). 2. Plant material The roots of Gelsemium elegans were collected from Conghua city, Guangdong province of P. R. China, in October of 2014. A voucher specimen (No. 2014102001) identified by Prof. Guang-Xiong Zhou (Jinan University) was deposited in the Institute of Traditional Chinese Medicine & Natural Products, Jinan University, Guangzhou, P. R. China. 3. Extraction and isolation of 1-2 The air-dried G. elegans (18 kg) were pulverized and extracted with 95% EtOH. The extract (1.7 kg) was suspended in H 2 O and acidified with 5% HCl to ph 3. The acidic suspension was partitioned with CHCl 3 to remove the neutral components. 3
Then the aqueous layer was basified with 10% aqueous ammonia to ph 9 and extracted with CHCl 3 to obtain a total alkaloid fraction. The alkaloid fraction (220 g) was subjected to silica gel column chromatography using CHCl 3 -MeOH (100:0 0:100, v/v) as gradient eluent to afford eleven fractions (Frs. A-K). Fr. D (16.5 g) was separated by ODS CC with MeOH-H 2 O (1:9 to 10:0, v/v) to yield six fractions (Frs. D1-D6). Fr. D2 (3.2 g) was chromatographed on Sephadex LH-20 (CHCl 3 -MeOH, 1:1, v/v) and then was purified by preparative HPLC (ACN-H 2 O, 40:60, v/v) to afford compounds 1 (36.6 mg) and 2 (15.4 mg). 4. Physico-chemical constants of 1-2 Compound 1: light-yellow crystals (MeOH-H 2 O); mp 198-199 ; [ ] 25 D = - 105 (c = 0.50, CHCl 3 ); 1 H and 13 C NMR data, see Table 1; UV (MeOH) λ max (log ε): 208 (3.15), 272 (2.97), 302 (2.27) nm; IR (KBr) ν max : 3285, 2925, 1719, 1643, 1588, 1466, 1322, 1109, 866, 754, 682 cm -1 ; HR-ESI-MS: m/z 661.3022 [M+H] + (calcd for C 39 H 41 N 4 O 6, 661.3021); ECD (MeCN) λ max (Δε) 236 (+ 8.3), 268 (+ 23.0), 217 (- 12.0), 299 (- 40.6). Compound 2: amorphous powder; [ ] 25 D = - 92.8 (c = 0.50, CHCl 3 ); 1 H and 13 C NMR data, see Table S1; UV (MeOH) λ max (log ε): 208 (3.28), 266 (2.28), 306 (2.95) nm;ir (KBr) ν max : 3434, 2935, 1725, 1618, 1470, 1326, 1207, 1118, 1062, 946, 757, 624 cm -1 ; HR-ESI-MS: m/z 645.3078 [M+H] + (calcd for C 39 H 41 N 4 O 5, 645.3072); ECD (MeCN) λ max (Δε) 228 (+ 17.5), 267 (+ 15.5), 299 (- 46.3). Table S1. 1 H (500 MHz) and 13 C NMR (125 MHz) Data of 2 in CD 3 OD ( in ppm, J in Hz) No. 2b 2a a) H C a) H C 2-186.6-186.9 3 5.09 br s 72.5 5.09 br s 72.5 5 4.05 d (8.4) 60.5 4.64 d (8.4) 54.3 6 2.66 d (8.4) 39.3 2.69 d (8.4) 41.3 2.57 2.56 7-64.0-64.3 8-141.7-141.9 9 7.56 125.9 7.56 125.9 10 7.51 128.0 7.51 127.9 4
11 7.50 130.0 7.50 ddd 130.1 12 7.65 br d (7.6) 122.5 7.65 br d (7.6) 122.5 13-154.5-154.4 14 3.16 33.7 3.17 33.7 1.47 1.48 15 3.99 29.6 3.99 29.6 16 2.95 d (4.7) 40.5 2.95 d (4.7) 40.5 17 3.83 65.9 3.83 66.3 4.47 4.46 18 6.95 d (11.5) 130.1 6.96 d (11.5) 130.0 19 6.35 d (11.5) 119.2 6.37 d (11.5) 119.0 20-147.5-148.7 21 8.24 s 164.3 8.01 s 165.7 22 2.92 s 29.9 3.06 s 29.9 2' - 173.0-173.0 3' 3.68 76.4 3.68 76.3 5' 4.49 73.9 3.37 d (8.6) 74.0 6' 2.55 38.4 2.59 38.6 2.15 2.15 7' - 57.6-57.5 8' - 133.4-133.4 9' 7.56 125.9 7.59 125.8 10' 7.11 ddd (7.7, 7.7, 0.9) 124.4 7.12 ddd (7.7, 7.7, 0.9) 124.6 11' 7.30 ddd (7.7, 7.7, 0.9) 129.4 7.32 ddd (7.7, 7.7, 0.9) 129.4 12' 6.95 br d (7.7) 107.8 6.96 br d (7.7) 107.8 13' - 139.3-139.1 14' 2.34 29.5 2.34 29.4 2.33 2.33 15' 3.57 40.6 3.61 40.5 16' 2.61 41.0 2.61 41.0 17' 4.35 62.6 4.35 62.6 4.32 4.32 18' 1.92 s 13.6 1.95 s 13.6 19' - 135.2-135.4 20' - 182.9-182.8 21' 3.93 s 64.1 3.91 s 64.0 a) Overlapped signals were reported without designating multiplicity. 5
5. X-Ray Crystallographic analysis of 1a Crytallographic data for 1a have been deposited with the Cambridge Crystallographic Data Centre as supplementary publication no. CCDC 1561616. Copies of the data can be obtained, free of charge, on application to the Director, CCDC, 12 Union Road, Cambridge CB2 IEZ, UK (fax: +44-(0)1223-336033 or email: deposit@ccdc.cam.ac.uk). Table S2. Crystal data and structure refinement for 1a Empirical formula C 39 H 40 N 4 O 6 Formula weight 660.75 Temperature Wavelength Crystal system 100.00(10) K 1.54184 Å Monoclinic Space group P 1 21 1 Unit cell dimensions a = 12.6396(3) Å = 90 b = 20.1301(4) Å = 91.5813(17) c = 14.1840(3) Å = 90 Volume 3607.54(12) Å 3 Z 4 Density (calculated) 1.334 Mg/m 3 Absorption coefficient 0.805 mm -1 F(000) 1528 6
Crystal size 0.2 0.16 0.1 mm 3 Theta range for data collection 3.813 to 74.020 Index ranges -15<=h<=14, -25<=k<=24, -17<=l<=17 Reflections collected 26285 Independent reflections 13180 [R(int) = 0.0498] Completeness to theta = 67.684 99.30 % Absorption correction Semi-empirical from equivalents Max. and min. transmission 1.00000 and 0.34621 Refinement method Full-matrix least-squares on F 2 Data / restraints / parameters 13180/1/963 Goodness-of-fit on F 2 1.022 Final R indices [I>2sigma(I)] R 1 = 0.0759, wr 2 = 0.2056 R indices (all data) R 1 = 0.0821, wr 2 = 0.2176 Absolute structure parameter 0.07 (15) Extinction coefficient n/a Largest diff. peak and hole 0.804 and -0.590 e.å -3 6. Dynamic HPLC analyses of 1a and 1b The isomers 1a and 1b isolated by HPLC were respectively analyzed at different time (0 h, 0.5 h, 1 h, and 24 h; room temperature). The HPLC/UV spectra were obtained by an Agilent 1260 instrument equipped with a DAD detector (Agilent 1260, DADVL, USA) and a Waters Xbridge C 18 OBD (4.6 250 mm, 5 μm). The column temperature was controlled at 298 K by an Agilent 1260 TCCVL (USA).The mobile phase was ACN-H 2 O (40:60, v/v) (λ = 210 nm; flow rate: 1 ml/min). 7
Figure S1. Dynamic HPLC spectra of 1a and 1b 7. Quantum chemical ECD calculation method Computational data of 1 Figure S2. Calculated and experimental CD spectra of 1 8
The systematic random conformational analysis of the enantiomers of compound 1 was performed in the SYBYL 8.1 program by using MMFF94s molecular force field, which afforded 32 conformers of 1, with an energy cutoff of 10 kcal mol -1 to the global minima. All the obtained conformers were further optimized using DFT at the B3LYP/6-31+G(d) level in gas phase by using Gaussian09 software, [1] and 30 conformers of 1 were selected. All of the optimized stable conformers were used for TDDFT computation of the excited stats at the same levels, with the consideration of the first 30 excitations. The overall ECD curves of 1 were weighted by Boltzmann distribution of each conformer (with a half-bandwidth of 0.3 ev), with a UV correction of 10 nm. The calculated ECD spectra of 1 were subsequently compared with the experimental one. The ECD spectra were produced by SpecDis 1.6 software. [2] Figure S3. Key molecular orbitals involved in important transitions regarding the ECD spectra of conformer 25 in the gas phase at the B3LYP/6-31+G(d) level. 9
Table S3. Key transitions and their related rotatory and oscillator strengths of conformer 25 of 1 at the B3LYP/6-31+G(d) level in the gas phase. HOMO is 175 No. Energy (cm -1 ) Wavelength (nm) R (length) Osc. Strength Major contribs 1 29407.1776 340.0530352-155.6875 0.4123 HOMO->LUMO (79%), HOMO- >L+1 (18%) 2 32306.7608 309.5327341 22.8301 0.0163 H-1->LUMO (88%) 3 32775.37216 305.1071381-156.5385 0.29 H-2->LUMO (22%), HOMO- >LUMO (11%), HOMO->L+1 (58%) 4 33719.04736 296.5682836 46.8551 0.04 H-4->LUMO (37%), H-3->LUMO (13%), H-2->LUMO (31%) 5 34002.14992 294.0990503 253.0407 0.1474 H-4->LUMO (24%), H-2->LUMO (45%), HOMO->L+1 (11%) 6 35329.74768 283.0475918-10.8034 0.0072 HOMO->L+2 (95%) 7 36275.84256 275.6655475 28.8883 0.0274 H-6->LUMO (11%), H-4->LUMO (10%), H-3->LUMO (53%) 8 36933.18896 270.7591811 0.5784 0.0149 H-1->L+2 (80%) 9 37066.27136 269.7870499 1.4678 0.0018 H-5->LUMO (14%), HOMO->L+4 (65%) 10 37551.82048 266.298674-17.8351 0.0049 H-6->LUMO (18%), H-5->LUMO (29%), H-4->LUMO (12%), H-3- >LUMO (19%) 11 37594.56816 265.9958736 12.7203 0.0057 H-6->LUMO (21%), H-5->LUMO (24%), HOMO->L+4 (31%) 12 37800.24096 264.5485782-0.1788 0.0005 H-1->L+1 (89%) 13 38103.50752 262.4430309-17.5976 0.012 HOMO->L+3 (88%) 14 38645.51584 258.7622337-7.5748 0.109 H-2->L+1 (90%) 15 39328.67216 254.2674199-49.098 0.0497 H-9->LUMO (28%), H-7->LUMO (23%), H-1->L+3 (21%) 16 39639.19776 252.2755395-20.6143 0.0573 H-9->LUMO (15%), H-1->L+3 (45%) 17 39793.25072 251.2988966 8.8163 0.0036 H-5->LUMO (20%), H-5->L+1 (28%), H-3->L+1 (23%), H-2->L+4 (18%) 10
18 39919.88064 250.501751 4.3056 0.0122 HOMO->L+5 (88%) 19 40335.25904 247.9220473-14.1756 0.0027 H-9->LUMO (21%), H-8->LUMO (42%), H-6->LUMO (13%) 20 40448.17744 247.2299281 3.9827 0.005 H-9->LUMO (25%), H-8->LUMO (13%), H-7->LUMO (39%) 21 40565.12864 246.5171524-7.4574 0.0018 H-12->LUMO (13%), H-8->LUMO (40%), H-6->L+1 (11%) 22 40828.0672 244.9295469 0.5171 0.0003 HOMO->L+6 (92%) 23 41125.68784 243.1570273 67.3354 0.0247 H-9->L+2 (56%), H-9->L+3 (10%) 24 41322.48848 241.9989785 7.5998 0.0204 H-12->LUMO (12%), H-4->L+1 (10%), H-3->L+1 (36%) 25 41985.4808 238.1775749-8.4738 0.0042 HOMO->L+7 (79%) 26 42346.81968 236.1452424 13.3297 0.0029 H-10->LUMO (22%), H-6->L+1 (24%), H-5->L+1 (10%), H-4->L+1 (24%) 27 42413.76416 235.7725186 1.7757 0.004 H-4->L+1 (49%), H-3->L+1 (20%) 28 42459.73808 235.5172324 0.1984 0.0001 H-2->L+2 (98%) 29 42624.27632 234.6080887-2.5795 0.0074 H-1->L+5 (54%) 30 42742.84064 233.9573096 4.0289 0.0032 H-12->LUMO (23%), H-10->LUMO (40%) Table S4. Z-matrices and Cartesian coordinates of conformer 25 of 1 Row Tag Symbol Bond Angle Dihedral X Y Z 1 1 C 7.951627-1.783353-0.392879 2 2 C 1.3978039 7.545637-2.724785-1.343002 3 3 C 1.4021144 120.99492 6.20824-2.802425-1.756845 4 4 C 1.3886487 117.04397 0.098375 5.31676-1.905381-1.183329 5 5 C 1.4011241 123.19481 0.2331412 5.696601-0.947184-0.234262 6 6 C 1.3900217 118.97787-0.498494 7.025714-0.888167 0.168399 7 7 N 1.4033087 128.63301-179.3684 3.937357-1.776956-1.407002 8 8 C 1.3855634 113.22501-173.3564 3.381031-0.677732-0.772972 9 9 C 1.5311686 109.8496 179.80688 4.47814-0.121075 0.186896 10 10 C 1.5662023 109.82463-121.9708 4.74009 1.396144-0.10019 11 11 O 1.4352657 110.86-69.68691 5.735969 1.920329 0.790564 12 12 C 1.5457537 119.68967 162.90217 3.551724 2.383771-0.141829 13 13 C 1.4435691 116.78186-78.48896 5.306346 2.239225 2.131318 14 14 C 1.5249198 114.43184-3.114803 3.815808 2.036052 2.381113 15 15 C 1.540975 114.42107-44.31781 2.903257 2.571782 1.260913 11
16 16 C 1.5669574 108.43892 112.61171 4.153918-0.444626 1.685412 17 17 C 1.5412208 118.82956 158.97121 3.32244 0.565032 2.50064 18 18 N 1.4658354 112.96337 71.831181 1.911401 0.595608 2.104777 19 19 C 1.2885334 109.37123-106.4907 1.676479 1.67598 1.443017 20 20 C 1.479764 123.47111 178.86129 0.353129 2.012155 0.87255 21 21 H 1.0943138 106.79365 42.552499 5.217183 1.45413-1.083319 22 22 O 1.2178038 124.439 170.73297 2.244636-0.27967-0.955178 23 23 H 1.0982322 105.54053-170.3514 3.376016 0.218082 3.54125 24 24 H 1.0932195 113.07977-85.4587 2.669603 3.634174 1.369807 25 25 H 1.0977942 107.24347-165.4979 3.570531 2.548154 3.320657 26 26 O 1.4318994 107.46116-169.7101 4.043681 3.626523-0.655523 27 27 C 1.511394 116.97619 164.65315 0.135847 3.444644 0.442346 28 28 C 5.611449 108.65394-176.884-5.158517 4.002535-1.331584 29 29 C 1.4006272 113.60593-93.60655-5.307429 3.834727-2.714126 30 30 C 1.4005658 120.83067 67.505453-5.09628 2.58615-3.312504 31 31 C 1.3924221 117.92234-0.366076-4.727361 1.523699-2.491568 32 32 C 1.4069887 121.6728-0.385247-4.560039 1.685745-1.103994 33 33 C 1.3903268 120.14786 1.2899066-2.923114-0.51284 4.789088 34 34 N 1.4199453 126.28814 179.22238-4.485101 0.187029-2.90493 35 35 C 1.2882285 106.8375-178.1376-4.209379-0.487844-1.84283 36 36 C 1.5134897 107.38399 178.53415-4.198315 0.332402-0.531046 37 37 C 1.5033111 123.88573-173.4301-3.750761-1.91944-1.854941 38 38 C 1.5371008 107.31039 119.61041 - -1.959607-1.202211 2.359715 39 39 C 1.5525006 107.66452 56.724086-2.487948-1.346194 0.21819 40 40 C 1.5216663 107.20643-67.76319-0.134622 0.043289 2.791365 41 41 C 1.5699906 111.70251-118.6596-5.265988-0.162201 0.508335 42 42 C 1.579733 109.28219 54.136836-3.62681-2.100747 1.011411 43 43 O 1.4478365 109.37369-119.4196 - -2.734042-1.125805 4.699979 44 44 C 1.5373834 111.91955-151.0534-4.646222-1.078839 1.575674 45 45 N 1.4793437 112.13-171.02-5.67394-1.716404 2.427592 46 46 C 1.4606404 122.24596 26.515524-7.035-1.959892 1.956742 47 47 C 1.4299325 115.25988-107.6559 - -3.201903 0.148432 4.250387 48 48 C 1.3539599 125.69022-105.6373-1.918831 1.14936 0.248712 12
49 49 C 1.3588739 118.26299-16.02083-1.020785 0.75902 0.569278 50 50 C 1.3650014 117.74263-150.0859 - -2.016037 3.715476 5.335113 51 51 O 1.2255782 125.31811-1.759012-4.234437-1.827926 4.220618 52 52 H 1.1060481 112.88411 178.40579-6.169854-2.463287 4.286893 53 53 O 1.3821948 120.91777-14.99411 3.336025-2.375769-2.498004 54 54 C 1.4408021 111.03653 117.28738 2.368441-3.35836-2.080623 55 55 H 1.0864464 119.82406-179.6669 8.993924-1.736308-0.089944 56 56 H 1.0868329 119.78306 179.65201 8.274437-3.407062-1.7726 57 57 H 1.0849999 121.82207 179.29121 5.880568-3.520241-2.501557 58 58 H 1.0854967 120.43814 178.94095 7.34936-0.144063 0.889416 59 59 H 1.0904029 109.95247-55.31627 2.809413 2.042501-0.86397 60 60 H 1.0980715 106.56235-124.9372 5.576877 3.289131 2.305305 61 61 H 1.0990763 107.64442 120.32844 5.902375 1.630565 2.825761 62 62 H 1.0962115 108.94322-75.88555 3.667839-1.425629 1.740548 63 63 H 1.0957856 106.71114 38.195819 5.115516-0.553896 2.199353 64 64 H 0.9731245 107.7891-40.91711 4.916755 3.788082-0.257262 65 65 H 1.0988951 111.28557-60.19545 0.252157 4.137051 1.287695 66 66 H 1.0974728 111.48857 58.794116 0.864997 3.750889-0.318575 67 67 H 1.0914777 112.34681 179.16738-3.605044 0.020407 0.857915 68 68 H 1.0869787 96.269181 140.12148-5.330607 4.979088-0.886329 69 69 H 1.0870171 119.54755-112.8256-5.595726 4.683393-3.329152 70 70 H 1.0862068 121.62668 179.30119-5.219018 2.438859-4.381656 71 71 H 1.0881369 121.32468 178.8202-4.683901 3.065277 0.56083 72 72 H 1.0941351 109.3092-2.487082-3.737601-2.286422-2.885612 73 73 H 1.0952012 109.62751-63.29297-1.653636-1.37847-1.80487 74 74 H 1.0962264 110.47771 178.71233 - -2.988676-1.16326 1.983933 75 75 H 1.0911123 109.06704 173.25944-1.542129-1.471944 0.747472 76 76 H 1.0955859 108.42284-31.47877-5.69325 0.713541 1.009166 77 77 H 1.0925717 108.33391 84.709307 - -0.652429-0.037479 6.075603 78 78 H 1.0957034 108.46994 117.36587-3.177078-2.591141 1.88194 79 79 H 1.0949313 107.5389 74.229577-4.068553-0.446922 2.258209 80 80 H 1.0946129 111.57411 55.033461 - -2.541208 1.029273 7.042095 81 81 H 1.0936466 108.58133 174.12413-7.571094-2.530441 2.720378 13
82 82 H 1.096336 111.86434-66.62922-7.580622-1.024824 1.783834 83 83 H 1.0999935 110.09861-75.44363-3.532681-4.025095 0.017136 84 84 H 1.0932777 105.47572 169.91126-5.133257-3.631012 0.629745 85 85 H 1.085844 116.77506 174.94224-2.263076 2.148536-0.000687 86 86 H 1.0855424 115.35221-1.348502-0.239047 0.039166 1.084249 87 87 H 1.093472 110.31128 63.566574 1.555345-2.879548-1.528088 88 88 H 1.0922428 104.49695-177.9755 1.991742-3.784439-3.013119 89 89 H 1.0958511 110.56585-59.36332 2.846439-4.135905-1.474132 Computational data of 2 The systematic random conformational analysis of the enantiomers of compound 1 was performed in the SYBYL 8.1 program by using MMFF94s molecular force field, which afforded 28 conformers of 1, with an energy cutoff of 10 kcal mol -1 to the global minima. All the obtained conformers were further optimized using DFT at the B3LYP/6-31+G(d) level in gas phase by using Gaussian09 software, [1] and 21 conformers of 1 were selected. All of the optimized stable conformers were used for TDDFT computation of the excited stats at the same levels, with the consideration of the first 30 excitations. The overall ECD curves of 1 were weighted by Boltzmann distribution of each conformer (with a half-bandwidth of 0.3 ev), with a UV correction of 10 nm. The calculated ECD spectra of 1 were subsequently compared with the experimental one. The ECD spectra were produced by SpecDis 1.6 software. [2] 14
Figure S4. Key molecular orbitals involved in important transitions regarding the ECD spectra of conformer 18 in the gas phase at the B3LYP/6-31+G(d) level. Table S5. Key transitions and their related rotatory and oscillator strengths of conformer 18 of 1 at the B3LYP/6-31+G(d) level in the gas phase. HOMO is 171 No. Energy (cm -1 ) Wavelength (nm) R (length) Osc. Strength Major contribs 1 29441.85968 339.6524577-33.0486 0.2618 HOMO->LUMO (78%), HOMO- >L+1 (18%) 2 32136.57664 311.1719121 1.8252 0.0006 H-1->LUMO (95%) 3 32605.188 306.6996577-51.4037 0.1173 H-2->LUMO (44%), HOMO->L+1 (46%) 4 33687.59152 296.8452047 179.6158 0.3047 H-4->LUMO (20%), H-2->LUMO (32%), HOMO->L+1 (23%) 5 34595.77808 289.0526115-13.3871 0.1504 H-4->LUMO (31%), H-3->LUMO (26%), H-2->LUMO (19%) 6 36081.4616 277.1506352-29.9282 0.0237 H-5->LUMO (10%), H-4->LUMO 15
(17%), H-3->LUMO (42%) 7 36334.72144 275.2188431-68.3826 0.0456 HOMO->L+2 (77%) 8 36788.81472 271.8217501-3.2273 0.0071 H-1->L+1 (26%), H-1->L+2 (49%) 9 36883.9888 271.1203513-4.2133 0.0115 H-7->LUMO (22%), H-5->LUMO (28%), HOMO->L+4 (17%) 10 37212.25872 268.7286487 12.0951 0.0039 H-1->L+1 (67%), H-1->L+2 (22%) 11 37341.30832 267.7999366 4.2899 0.0026 H-5->LUMO (19%), HOMO->L+3 (10%), HOMO->L+4 (56%) 12 38086.56976 262.5597438-1.2578 0.0887 H-2->L+1 (73%) 13 38301.92128 261.0835088 43.7432 0.0357 H-7->LUMO (25%), H-5->LUMO (22%), H-2->L+1 (17%) 14 38841.50992 257.4565206-41.8424 0.0363 H-8->LUMO (13%), H-6->LUMO (15%), H-1->L+3 (12%), HOMO- >L+3 (29%) 15 39118.16 255.6357456 1.0297 0.0002 H-6->LUMO (12%), H-3->L+1 (24%), H-2->L+4 (10%), HOMO- >L+3 (22%) 16 39147.19616 255.4461361-2.8943 0.0046 H-3->L+1 (16%), H-2->L+4 (10%), HOMO->L+3 (22%) 17 39661.78144 252.1318921-30.8072 0.063 H-8->LUMO (10%), H-1->L+3 (30%), HOMO->L+5 (31%) 18 39797.28352 251.2734316 3.7609 0.0073 H-1->L+3 (17%), HOMO->L+5 (51%) 19 40294.12448 248.1751404-6.3296 0.0031 H-10->LUMO (10%), H-8->LUMO (27%), H-6->LUMO (35%) 20 40696.59792 245.7207853 7.3968 0.0018 H-11->LUMO (18%), H-11->L+1 (10%), H-7->LUMO (23%), H-7- >L+1 (17%), H-5->L+1 (10%) 21 40883.71984 244.5961385 4.9316 0.0057 HOMO->L+6 (81%) 22 41102.2976 243.2954016 11.9036 0.007 H-10->LUMO (42%), H-9->LUMO (20%) 23 41294.25888 242.1644139 0.6919 0.001 H-9->LUMO (72%) 24 41380.5608 241.6593639 47.0012 0.0169 H-8->LUMO (11%), H-8->L+2 (37%) 25 41604.78448 240.3569716 0.1824 0.0093 H-4->L+1 (58%) 26 41766.09648 239.4286477 8.5801 0.0036 H-11->LUMO (12%), H-10->LUMO 16
(16%), H-5->L+1 (13%), H-4->L+1 (10%), H-3->L+1 (22%) 27 42075.00896 237.6707753-16.2515 0.0147 HOMO->L+7 (70%) 28 42170.9896 237.1298396 5.9013 0.0014 H-1->L+3 (12%), H-1->L+4 (44%), H-1->L+5 (23%) 29 42479.90208 235.4054391-1.4149 0.002 H-1->L+4 (39%), H-1->L+5 (37%) 30 42505.712 235.2624984-4.5892 0.0019 H-2->L+2 (86%) Table S6. Z-matrices and Cartesian coordinates of conformer 18 of 2 Row Tag Symbol Bond Angle Dihedral X Y Z 1 1 C -8.097954 0.83797 1.379146 2 2 C 1.3980772-7.897228 2.217145 1.489624 3 3 C 1.4022981 121.00503-6.683954 2.80385 1.102089 4 4 C 1.3888858 117.04454 0.1327175-5.702594 1.955759 0.605422 5 5 C 1.4010138 123.14305 0.1689919-5.879018 0.571738 0.478142 6 6 C 1.3900338 119.06352-0.525935-7.085246 0.00556 0.873933 7 7 N 1.4046322 128.6466-179.4485-4.409054 2.27603 0.161381 8 8 C 1.3879832 112.71634-171.8285-3.766114 1.201032-0.436536 9 9 C 1.5299774 109.89511-179.7673-4.610172-0.064538-0.092834 10 10 C 1.5698957 110.8685-125.3643-4.935841-0.905641-1.377769 11 11 O 1.4326349 108.54976-72.59316-5.617932-2.100829-0.979372 12 12 C 1.5397731 118.13181 160.12823-3.782669-1.228599-2.34565 13 13 C 1.4370396 116.34034-90.00506-4.791564-3.24052-0.690749 14 14 C 1.5247652 114.39674 13.969156-3.310002-2.919109-0.527722 15 15 C 1.544359 113.57547-54.4244-2.706176-2.162034-1.73075 16 16 C 1.5678333 107.67452 111.40735-3.962677-0.934193 1.039664 17 17 C 1.5409141 118.74591 163.1899-2.909206-1.990003 0.65252 18 18 N 1.4666508 111.48667 71.263673-1.649041-1.364621 0.237856 19 19 C 1.2872515 109.21151-105.5482-1.534539-1.450209-1.041433 20 20 C 1.4815279 122.2158-178.3875-0.346556-0.938884-1.764045 21 21 H 1.0950766 106.74654 39.231282-5.686783-0.342354-1.941672 22 22 O 1.2184703 123.94648 167.01257-2.743104 1.298501-1.091227 23 23 H 1.0984301 105.81371-171.1969-2.72128-2.57356 1.563944 24 24 H 1.0964307 111.9751-87.7099-2.341782-2.846914-2.505551 17
25 25 H 1.0982013 107.33182-175.3571-2.780972-3.872441-0.396073 26 26 C 1.5094844 117.26952-152.6718-0.542258-0.504484-3.196365 27 27 C 4.1302232 110.55778 110.51832 1.30559-2.059721 3.03888 28 28 C 1.4005017 124.87576 73.877406 1.956077-3.28527 3.229409 29 29 C 1.3995218 120.64002 89.773613 3.122187-3.591869 2.518884 30 30 C 1.3929459 117.89992-0.626318 3.60089-2.647875 1.613336 31 31 C 1.4066567 121.95854-1.231012 2.934826-1.43004 1.385477 32 32 C 1.3911136 119.69853 3.0506761 1.797595-1.117893 2.123357 33 33 N 1.4181541 125.87023 176.14507 4.801376-2.748277 0.865062 34 34 C 1.2873278 106.96485-175.5802 4.935797-1.64213 0.220405 35 35 C 1.5208064 107.3818 176.62557 3.780176-0.625369 0.410418 36 36 C 1.5030497 123.30207-176.867 6.057058-1.381141-0.745937 37 37 C 1.5336504 107.71982 123.36954 5.43239-1.012965-2.097351 38 38 C 1.549376 107.3034 55.959096 4.490145 0.1931-1.856213 39 39 C 1.5301868 108.05256-68.12126 3.307567-0.290045-1.013864 40 40 C 1.5756532 109.41709-117.4924 4.259951 0.704218 1.10662 41 41 C 1.5775875 109.2211 55.126391 5.302142 1.356872-1.166954 42 42 O 1.4485475 108.89564-115.6411 6.878574-0.304627-0.231623 43 43 C 1.5368308 112.76028-154.2859 4.551335 1.829869 0.101705 44 44 N 1.4792019 112.15004-171.129 5.196949 2.996288 0.742554 45 45 C 1.4607223 122.30757 26.465655 6.002628 2.874331 1.954873 46 46 C 1.4298666 115.2024-107.7838 6.756317 0.939523-0.925642 47 47 C 1.3561981 118.65679-108.4239 2.091868-0.403477-1.604188 48 48 C 1.3605463 117.56025 27.203698 0.832136-0.895121-1.085918 49 49 C 1.3646607 117.67826-150.5018 4.966906 4.226449 0.198419 50 50 H 1.1060027 112.89131 178.59925 5.466644 5.042479 0.753035 51 51 O 1.2259262 125.33265-1.545024 4.28772 4.444971-0.798501 52 52 O 1.384553 120.30211-17.69573-4.085142 3.582624-0.162471 53 53 C 1.4411585 110.95026 122.38604-2.971718 4.047921 0.625389 54 54 H 1.0865756 119.80396-179.6451-9.047951 0.40468 1.679832 55 55 H 1.0869116 119.77305 179.71111-8.691921 2.848815 1.877996 56 56 H 1.0850045 121.84103 179.2299-6.517968 3.8739 1.170473 57 57 H 1.0856432 120.19729 179.10572-7.247124-1.062897 0.769932 58 58 H 1.0977194 105.44616-175.4266-4.24994-1.741301-3.196404 59 59 H 1.090575 109.78811-60.18443-3.348526-0.302746-2.724688 18
60 60 H 1.1004058 108.13957-108.2813-4.920927-3.971912-1.502675 61 61 H 1.0980969 106.53141 136.67989-5.194124-3.694615 0.224434 62 62 H 1.0965324 108.87694-72.16842-3.5249-0.267686 1.792328 63 63 H 1.0954832 106.38257 42.187546-4.786291-1.461256 1.533575 64 64 H 1.0920965 112.68351-168.4775 0.402175-0.332993-3.717241 65 65 H 1.0969541 111.41579-47.94606-1.097357-1.256379-3.770671 66 66 H 1.0976766 110.35599 70.895614-1.12285 0.426183-3.23718 67 67 H 1.0865928 73.778677-41.49851 0.402641-1.834266 3.599716 68 68 H 1.0871506 119.64173-90.93899 1.557248-4.000178 3.944768 69 69 H 1.086244 121.75226 178.25749 3.656691-4.524901 2.672776 70 70 H 1.0871564 121.56163 178.7269 1.271131-0.176601 1.986558 71 71 H 1.0939905 109.15098 0.7904755 6.694153-2.268544-0.804598 72 72 H 1.0956785 109.47442-63.89501 4.867183-1.868414-2.483692 73 73 H 1.0960967 110.72904 178.08593 6.210146-0.778611-2.833287 74 74 H 1.094009 109.18289 174.31351 4.105251 0.54638-2.817413 75 75 H 1.0951327 108.09187-34.70828 3.464036 1.042691 1.778387 76 76 H 1.0925484 107.9675 81.093882 5.128511 0.466293 1.725213 77 77 H 1.0957477 108.57105 116.36741 5.323909 2.216772-1.845748 78 78 H 1.0948013 107.4814 74.161088 3.588565 2.205702-0.259423 79 79 H 1.0945954 111.58364 55.107481 6.809304 2.144975 1.830564 80 80 H 1.0936643 108.57758 174.19027 6.45343 3.846616 2.172912 81 81 H 1.096177 111.82119-66.57755 5.396554 2.57775 2.81877 82 82 H 1.0933637 105.49589 169.63052 7.282305 1.671888-0.307239 83 83 H 1.0999874 110.04726-75.72125 7.295227 0.891588-1.883374 84 84 H 1.0891285 113.99467-4.263682 2.067186-0.058887-2.637072 85 85 H 1.0813374 115.91016 1.426559 0.806765-1.2646-0.069979 86 86 H 1.0922182 104.47638-177.8419-2.83386 5.086483 0.316642 87 87 H 1.0958919 110.47155-59.25182-3.21061 3.997713 1.693747 88 88 H 1.0932873 110.39004 63.720149-2.074973 3.464857 0.399199 8. Bioassay methods MTT assay method The cell viability of Macrophage RAW 264.7 cells was performed by MTT assay. Cells were cultured in DMEM containing 10% FBS (V/V) with 19
penicillin (100 U/mL) and streptomycin (100 U/mL) at 37 C with 5% CO 2 (V/V). Cells at the logarithmic phase were seeded in 96-well plates at 4 10 4 cells per well. After preincubation overnight, cells were treated with various concentrations of compounds and cultured for 24 h. Then, 40 μl of 5 mg/ml MTT solu-tions was added to each well and incubated for 4 h. After complete removal of the medium, 100 μl of DMSO was added to each well to dissolve the formazan crystals. And the optical density (OD) was rec-orded at 570 nm. Viability rate = the average OD value of the treatment group/the average OD value of the control group. Determination of LPS-Induced NO Production in Macrophage RAW 264.7 Cells. The macrophage RAW 264.7 cells were cultivated in DMEM containing 10% FBS (V/V) with penicillin (100 U/mL) and streptomycin (100 U/mL) at 37 C in a humidified atmosphere with 5% CO 2 (V/V). The cells were allowed to grow in 96-well plates with 1 10 4 cells to treat test compounds. Cells were pre-incubated for 1 h in the absence or presence of compounds before the addition of LPS for 24 h. Then, the culture supernatant (100 μl) was incubated with a Griess reagent (100 μl, Sigma) at room temperature for 10 min. The absorbance was measured at 550 nm against a calibration curve with sodium nitrite standards. All the experiments were performed in three independent replicates. 20
Figure S5. The UV of compound 1 in MeOH Figure S6. The IR (KBr disc) of compound 1 Figure S7. The HR-ESI-MS of compound 1 21
Figure S8. 1 H NMR spectrum for compound 1 in CD 3 OD [1a (blue) and 1b (red)] Figure S9. The 13 C NMR spectrum of compound 1 in CD 3 OD Figure S10. The DEPT-135 spectrum of compound 1 in CD 3 OD 22
Figure S11. The 1 H- 1 H COSY spectrum of compound 1 in CD 3 OD Figure S12. The HSQC spectrum of compound 1 in CD 3 OD 23
Figure S13. The HMBC spectrum of compound 1 in CD 3 OD Figure S14. The NOESY spectrum of compound 1 in CD 3 OD 24
a b c d Figure. S15. 1 H NMR spectra for compound 1 in CDCl 3 (a), DMSO-d 6 (b), CDOD 3 (c), and pyridine-d 5 (d). 25
Figure S16. The UV of compound 2 in MeOH Figure S17. The IR (KBr disc) of compound 2 Figure S18. The HR-ESI-MS of compound 2 26
Figure S19. 1 H NMR spectrum for compound 2 in CD 3 OD [2a (blue) and 2b (red)] Figure S20 The 13 C NMR spectrum of compound 2 in CD 3 OD Figure S21. The DEPT-135 spectrum of compound 2 in CD 3 OD 27
Figure S22. The 1 H- 1 H COSY spectrum of compound 2 in CD 3 OD Figure S23. The HSQC spectrum of compound 2 in CD 3 OD 28
Figure S24. The HMBC spectrum of compound 2 in CD 3 OD Figure S25. The NOESY spectrum of compound 2 in CD 3 OD 29
Reference [1] Gaussian 09, Revision A.02, 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, 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, 2009. [2] T. Bruhn, A. Schaumlöffel, Y. Hemberger, G. Bringmann, SpecDis version 1.60, University of Wuerzburg, Germany, 2012. 30