Chloropupukeananin, the First Chlorinated Pupukeanane

Transcription

1 Chloropupukeananin, the First Chlorinated Pupukeanane Derivative and Its Precursors from Pestalotiopsis fici Ling Liu, Renrong Tian, Shuchun Liu, Xulin Chen, *, Liangdong Guo, and Yongsheng Che *, Key Laboratory of Systematic Mycology & Lichenology, Institute of Microbiology, Chinese Academy of Sciences, Beijing, , People s Republic of China, and State Key Laboratory of Virology, Wuhan Institute of Virology, Chinese Academy of Sciences, Wuhan, , People's Republic of China Contents Page 1) Experimental Section 2-6 2) Figure S1. 3) Figure S2. 4) Figure S3. 5) Figure S4. 1 H NMR spectrum of chloropupukeananin (1; 400 MHz, acetone-d 6 ) 7 13 C NMR spectrum of chloropupukeananin (1; 100 MHz, acetone-d 6 ) 8 1 H NMR spectrum of iso-a82775c (3; 400 MHz, acetone-d 6 ) 9 13 C NMR spectrum of iso-a82775c (3; 100 MHz, acetone-d 6 ) 10 * To whom correspondence should be addressed. Tel: Fax: cheys@im.ac.cn. Institute of Microbiology. Wuhan Institute of Virology. 1

2 Experimental Section General Procedures. Optical rotations were measured on a Perkin-Elmer 241 polarimeter, and UV data were recorded on a Hitachi U-2800 spectrophotometer. IR data were recorded using a Bruker Vertex 70 spectrophotometer. 1 H and 13 C NMR data were acquired with a Bruker Avance-400 spectrometer using solvent signals (acetone-d 6 ; H 2.05/ C 29.8, 206.1) as references. The HMQC and HMBC experiments were optimized for and 8.0 Hz, respectively. ESIMS data were recorded on a Bruker Esquire 3000 plus spectrometer, and HRESIMS data were obtained using a Bruker APEX III 7.0 T spectrometer. Fungal Material. The culture of P. fici was isolated by one of authors (L.G.) from the branches of an unidentified tree in suburb of Hangzhou, in April, The isolate was identified (by L.G.) and assigned the accession number W106-1 in the culture collection at the Institute of Microbiology, Chinese Academy of Sciences, Beijing. The fungal strain was cultured on slants of potato dextrose agar (PDA) at 25 o C for 10 days. Fermentation was carried out in four 500 ml Fernbach flasks each containing 100 g of rice. Spore inoculum was prepared by suspension in sterile, distilled H 2 O to give a final spore/cell suspension of /ml. Distilled H 2 O (100 ml) was added to each flask, and the contents were soaked overnight before autoclaving at 15 lb/in. 2 for 30 min. 1 After cooling to room temperature, each flask was inoculated with 5.0 ml of the spore inoculum and incubated at 25 o C for 40 days. Extraction and Isolation. The fermented rice substrate was extracted repeatedly with MEK (3 300 ml), and the organic solvent was evaporated to dryness under vacuum to afford 2.8 g 2

3 of crude extract. The extract was fractionated by silica gel VLC using petroleum ether-etoac gradient elution. The fraction (116 mg) that was eluted with 30 % EtOAc was subsequently fractionated by Sephadex LH-20 column chromatography using CHCl 3 -CH 3 OH (1:1) as eluents. The active fraction (33 mg) was further separated by semipreparative reversed-phase HPLC (Kramosil C 18 ; 10 µm; mm; 2 ml/min) to afford chloropupukeananin (1; 10.0 mg; t R 17.0 min; 70% MeOH in H 2 O over 2 min, 70 85% over 20 min). The fractions that were eluted with 15% (140 mg), 20% (120 mg) EtOAc were fractionated again by Sephadex LH-20 column chromatography using CHCl 3 -CH 3 OH (1:1) as eluents. Purification of these fractions using different gradients afforded iso-a82775c (2; 15.0 mg; t R 15 min; 50 to 70% CH 3 OH in water over 20 min) and the known compound pestheic acid (3; 16.0 mg; t R 20 min; 20 to 100% CH 3 OH in water over 25 min). Chloropupukeananin (1): colorless powder; [] D +32 (c 0.1, CH 3 OH); UV (CH 3 OH) max 246 ( 15900) nm; IR (neat) max 3443, 2960, 2929, 1746, 1730, 1662, 1643, 1571, 1449, 1368, 1262, 1190, 1092 cm -1 ; 1 H, 13 C NMR, and HMBC data, see Table 1; HRESIMS obsd m/z [M + Na] +, calcd for C 33 H 35 ClO 11 Na, X-ray Crystallographic Analysis of 1. 2 Upon crystallization from acetone-h 2 O (99:1) using the vapor diffusion method, colorless crystals were obtained for 1, and a crystal of 1 ( mm) was separated from the sample and mounted on a glass fiber, and data were collected using a Rigaku Saturn CCD area detector with graphite monochromator and Mo-Ka radiation, = Å at 113(2) K. Crystal data: C 36 H 43 ClO 13, M = , space group monoclinic, P ; unit cell dimensions a = (2) Å, b = (14) Å, c = (3) Å, V = (5) Å 3, Z = 2, D calcd =

4 mg/m 3, µ = mm -1, F(000) = 760. The structure was solved by direct methods using SHELXL-97 3 and refined using full-matrix least-squares difference Fourier techniques. All non-hydrogen atoms were refined with anisotropic displacement parameters, and all hydrogen atoms were placed in idealized positions and refined as riding atoms with the relative isotropic parameters. Absorption corrections were applied with the Crystalclear. 4 The measurements yielded 8242 independent reflections after equivalent data were averaged, and Lorentz and polarization correction were applied. The final refinement gave R 1 = and wr 2 = [I > 2 (I)]. Iso-A82775C (2): oil; [] D +224 (c 0.1, CH 3 OH); 1 H NMR (acetone-d 6, 400 MHz) 5.96 (1H, d, 3.2, H-12), 5.14 (1H, t, 7.5, H-4), 4.93 (1H, s, H14-b), 4.84 (1H, s, H-14a), 4.26 (1H, d, 8.6, H-16), 4.12 (1H, d, 6.3, OH-8), 4.00 (1H, ddd, 9.7, 6.3, 5.5, H-8), 3.88 (1H, d, 8.6, OH-16), 3.25 (1H, s, H-7), 2.80 (1H, dd, 15, 7.5, H-5b), 2.32 (1H, ddd, 16, 9.7, 3.2, H-9b), 2.15 (1H, dd, 16, 5.5, H-9a), 2.05 (1H, dd, 15, 7.5, H-5a), 1.70 (3H, s, H-1), 1.70 (3H, s, H-15), 1.64 (3H, s, H-2); 13 C NMR data (acetone-d 6, 100 MHz) 18.0 (C2), 19.7 (C15), 25.9 (C1), 31.1 (C9), 33.6 (C5), 63.3 (C7), 65.9 (C6), 68.6 (C8), 68.6 (C16), 98.1 (C12), (C10), (C14), (C4), (C3), (C13), (C11); ESIMS obsd m/z [M+ Na] +. Pestheic acid (4): The 1 H, 13 C NMR, and ESIMS data were fully consistent with literature values. 5 Antimicrobial Bioassay. Antimicrobial bioassay was conducted in triplicate by following National Center for Clinical Laboratory Standards (NCCLS) recommendations. 6 The bacterial strain was obtained from China General Microbial Culture Collection (CGMCC) and grown on Mueller-Hinton agar (MHA). The inocula 4

5 were prepared from broth culture that was incubated at 37 o C for 24 h, and the final spore suspension was 10 6 cells/ml. Test samples (10 mg/ml as stock solution in DMSO and serial dilutions) were transferred to 96-well clear plate in triplicate, and the suspension of test organism was added to each well achieving a final volume of 200 µl (antimicrobial peptide AMP was used as positive control). After incubation at 37 o C for 24 h, the absorbance at 595 nm was measured with a microplate reader (TECAN), and the inhibition rate was calculated and plotted versus test concentrations to afford the IC 50. The MIC was defined as the lowest test concentration that completely inhibited the growth of test organism. 7,8 HIV-1 Replication Inhibition Assay. The HIV-1 replication inhibition assay was determined by p24 antigen capture ELISA. 9 C8166 cells were exposed to HIV-1 (MOI = 0.058) at 37 o C for 1.5 h, washed with PBS to remove free viruses, and then seeded into a 96-well microtitre plate at cells per well in the absence or presence of test compound (indinavir sulfate was used as positive control). After 4 days, the supernatant was collected and inactivated by 0.5% Triton X-100. The supernatant was diluted three times, added to the plate coating with anti-p24 McAb (provided by Dr. Bin Yan, Wuhan Institute of Virology, China), and incubated at 37 o C for 1 h. After washing 5 times with PBST, the HRP labeled anti-p24 antibody (provided by Dr. Bin Yan, Wuhan Institute of Virology, China) was added and incubated at 37 o C for 1 h. The plate was washed 5 times with PBST, followed by adding OPD reaction mixture. The assay plate was read at 490 nm using a microplate reader within 30 min, and the inhibition rate and the IC 50 based on p24 antigen expression level were calculated. 5

6 Reference and Notes 1. Che, Y.; Gloer, J. B.; Wicklow, D. T. J. Nat. Prod. 2002, 65, Crystallographic data for compound 1 have been deposited with the Cambridge Crystallographic Data Centre (deposition number CCDC ). Copies of the data can be obtained, free of charge, on application to the director, CCDC 12 Union Road, Cambridge CB2 1EZ, UK (fax: or deposit@ccdc.cam.ac.uk). 3. Sheldrick, G. M. SHELXL-97, Program for X-ray Crystal Structure Solution and Refinement; University of Göttingen: Göttingen: Germany, Rigaku Corporation. Crystal Clear Software User's Guide, Molecular Structure Corporation Shimada, A.; Takahashi, I.; Kawano, T.; Kimura, Y. Z. Naturforsch. 2001, 56B, NCCLS 2002, NCCLS document M27-A2; NCCLS: Wayne, PA. 7. Khera, S.; Woldemichael, G. M.; Singh, M. P.; Suarez, E.; Timmermann, B. N. J. Nat. Prod. 2003, 66, Yamaguchi, H.; Uchida, K.; Nagino, K.; Matsunaga, T. J. Infect. Chemother. 2002, 8, Zhang, G. H.; Wang Q.; Chen J. J.; Zhang X. M; Tam S. C.; Zheng Y. T. Biochem. Biophys. Res. Commun. 2005, 334,

7 Figure S1. 1 H NMR Spectrum of Chloropupukeananin (1; 400MHz, Acetone-d 6 ) 7

8 Figure S2. 13 C NMR Spectrum of Chloropupukeananin (1; 100 MHz, Acetone-d 6 ) 8

9 Figure S3. 1 H NMR Spectrum of Iso-A82775C (3; 400 MHz, Acetone-d 6 ) 9

10 Figure S4. 13 C NMR Spectrum of Iso-A82775C (3; 100 MHz, Acetone-d 6 ) 10