Introduction

1. A New Antituberculosis Dihydroagarofuranoid Sesquiterpene from the Stems of Microtropis fokienensis Tsung-Hsien Choua, Ih-Sheng Chen a, Chien-Fang Peng b, Lin-Yang Cheng c, Ya-Chih Chang c, Jih-Jung Chen c,* a Graduate Institute of Pharmaceutical Sciences, Kaohsiung Medical University, Kaohsiung, 807, Taiwan b Faculty of Biomedical Laboratory Sciences, College of Health Science, Kaohsiung Medical University, Kaohsiung 807, Taiwan c Graduate Institute of Pharmaceutical Technology & Department of Pharmacy, Tajen University, Pingtung 907, Taiwan Introduction Microtropis fokienensis Dunn1)(Celastraceae) is a small shrub that grows in high altitude forests throughout southern China and Taiwan. Various dihydroagarofuranoid sesquiterpenes, sesquiterpene alkaloids, and triterpenes are widely distributed in plants of the family Celastraceae. Many of these compounds exhibit insecticidal, anti-inflammatory, anti-AIDS, and antitumor activities. In our continuing studies on the antitubercular constituents of Formosan plants, over 400 species have been screened for in vitro antituberculosis activity to date, and Microtropis fokienensis has been found to be one of the active species. In our previous studies2), we have reported four new dihydroagarofuranoid sesquiterpenes, fokienagarofuran A (10), fokienagarofuran B (11), fokienagarofuran C (12), and fokienagarofuran D (13) from the stem of this plant. Continuing investigation of the EtOAc-soluble fraction of the stem of M. fokienensis led to the isolation of a new b-dihydroagarofuranoid sesquiterpene, 8-acetoxymutangin (1), along with eight known compounds, mutangin (2), vanillin (3), p-hydroxy-benzaldehyde (4), syringic acid (5), 2,6-dimethoxy-p-benzoquinone (6), b-sitosterol (7), and a mixture of 1-tetracosanol (8) and 1-hexacosanol (9). The structures of this new compound was determined through spectral analyses. The structural elucidation of this new compound and antitubercular activities of all isolates will be discussed in this symposium. 8-acetoxymutangin (1)* R = OAc mutangin (2) R = H 1H-NMR date (CDCl3,400MHz) of 1 Significant NOESY correlations of 1 8-Acetoxymutangin (1) Amorphous powder HR-ESI-MS: C35H40O11, found: 659.2701 [M+H]+, calcd: 659.2703 ESI-MS m/z (%): 659 ([M+H]+, 100). IRνmax(KBr) cm-1: 1748 (C=O), 1723 (C=O). UV lmax(MeOH) nm (log ε): 230 (4.14), 274 (3.15), 281 (3.08). CD (MeOH): 236.7 (－13.9), 220.8 (＋5.33). [a] D25+ 85.4° (c 0.12, CHCl3). 13C-NMR date (CDCl3,100MHz) of 1 Significant HMBC correlations of 1 10* R1 = OBz, R2 = OAc, R3 = OBz 11* R1 = H, R2 = OAc, R3 = OBz 12* R1 = OBz, R2 = OAC, R3 = isobutanoyloxy 13* R1 = R2 = R3 = OBz *New compound ★New compound reported before Key words Microtropis fokienensis, Celastraceae, stem, dihydroagarofuranoid sesqui-terpenes, 8-acetoxymutangin, antitubercular activity. Fokienagarofuran A (10) Amorphous powder HR-FAB-MS: C40H43O11, found: 699.2802 [M+H]+, calcd: 699.2806 FAB-MS m/z (%): 699 ([M+H]+, 28). IRνmax(KBr) cm-1: 1746 (C=O), 1716 (C=O). UV lmax(MeOH) nm (log ε): 231 (4.12), 274 (3.13), 281 (3.06). [a] D25+ 32.4° (c 0.15, CHCl3). Results and Discussion The antituberculosis effects of the isolated compounds from the stems of M. fokienensis were tested in vitro against Mycobacterium tuberculosis 90-221387. The antituberculosis activity data are shown in Table. The clinically used antituberculosis agent, ethambutol, was used as the positive control. From the results of our antituberculosis tests, the following conclusions can be drawn regarding these isolated compounds: (a) Among the b-dihydroagarofuranoid sesquiterpene analogues (1 and 2), compound 1 with an 8-acetoxy moiety exhibited stronger antituberculosis activity than 2 against M. tuberculosis 90-221387. (b) 8-Acetoxymutangin (1) is the most effective among the isolated compounds, with an MIC of 10.0 mg/ml against M. tuberculosis 90-221387. (c) The compounds 3–5, 7, 8, and 9 showed no antituberculosis activities. (d) Agents acting specifically against M. tuberculosis have been less well characterized than other antimicrobial drugs. However, it is thought that several of them owe their activity to selective effects on the unique structure of the mycobacterial envelope. The positive control, ethambutol, inhibits arabinosyl transferases. These enzymes bring about the polymerization of arabinose to form arabinan, a polysaccharide component of the core polymers of the mycobacterial cell wall. The structures of 1 and 2 with b-dihydroagarofuranoid sesquiter-pene skeleton are quite different from those of known antituberculosis agents, e.g. ethambutol, isoniazid, and artemisinin. Thus, the detail action mechanisms of 1 and 2 need further experiments to elucilate. 1H-NMR date (CDCl3,400MHz) of 10 13C-NMR date (CDCl3,100MHz) of 10 Fokienagarofuran B (11) Amorphous powder HR-ESI-MS: C40H43O11, found: 601.2417 [M+Na]+, calcd: 601.2413 ESI-MS m/z (%): 601 ([M+Na]+, 100). IRνmax(KBr) cm-1: 1725 (C=O). UV lmax(MeOH) nm (log ε): 231 (4.20), 274 (3.14), 281 (3.07). [a] D25 + 46.8° (c 0.12, CHCl3). Fokienagarofuran C (12) Amorphous powder HR-FAB-MS: C38H47O11, found: 679.3122 [M+H ]+ calcd: 679.3119 FAB-MS m/z (%): 679 ([M+H]+, 100). IRνmax(KBr) cm-1: 1720 (C=O). UV lmax(MeOH) nm (log ε): 231 (4.25), 274 (3.15), 281 (3.07). [a] D25+ 34.5° (c 0.18, CHCl3). 1H-NMR date (CDCl3,500MHz) of 11 13C-NMR date (CDCl3,125MHz) of 11 Fokienagarofuran D (13) Amorphous powder HR-ESI-MS: C45H44O11, found: 783.2784 [M+Na]+, calcd: 783.2781 ESI-MS m/z (%): 783 ([M+Na]+, 100). IRνmax(KBr) cm-1: 1749 (C=O), 1721 (C=O). UV lmax(MeOH) nm (log ε): 231 (4.11), 273 (3.12), 280 (3.05). [a] D25 + 35.4° (c 0.13, CHCl3). Antitubercular effects of compounds isolated from the stems of Microtropis fokienensis against Mycobacterium tuberculosis 90-221387 1H-NMR date (CDCl3,400MHz) of 12 13C-NMR date (CDCl3,100MHz) of 12 3 R1 = H, R2 = H, R3 = OCH3 4 R1 = R2 = R3 = H 5 R1 = OH, R2 = R3 = OCH3 6 a Ethambutol was used as a positive control. Reference 1.Lu SY, Yang YP. Celastaceae in Flora of Taiwan; 2en ed.; Editorial commi-ttee of the Flora of Taiwan, Taipei, Taiwan, 1993; Vol. 3, 640-660. 2.Chen JJ, Chou TH, Duh CY, Chen IS. Cytotoxic Dihydroagarofuranoid Sesquiterpenes from the Stem of Microtropis fokienensis. J. Nat. Prod. 2006, 69, 685-688. 13C-NMR date (CDCl3,125MHz) of 13 1H-NMR date (CDCl3,500MHz) of 13 Acknowledgement This work was supported by a grant from the National Science Council of the Republic of China (NSC 93-2320-B-127-003). CH3(CH2)nCH2OH 1-Teteracosanol (8) n = 22 1-Hexacosanol (9) n = 24 7