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Journal of Chemistry, Vol. 43 (3), P. 375 - 378, 2005 A Furanolabdane Diterpene Alcohol from Alpinia tonkinensis Gagnep. Received 24th-June-2004 Phan Minh Giang1, Hideaki Otsuka2, Phan Tong Son1 1 Faculty of Chemistry, College of Natural Science, Vietnam National University 2 Graduate School of Biomedical Sciences, Hiroshima University, Japan summary A furanolabdane diterpene alcohol was isolated from the rhizomes of the endemic Vietnamese medicinal plant Alpinia tonkinensis Gagnep. (Zingiberaceae). Its absolute stereostructure was established to be (12S)-15,16-epoxy-8(17),13(16),14-labdatrien-12-ol from spectroscopic data and by applying the modified Mosher’s method. It is first report on the natural occurrence and absolute stereochemistry of the diterpene which was previously known as a synthetic intermediate. Keywords: Alpinia tonkinensis; Zingiberaceae; furanolabdane diterpenoid; absolute stereostructure; modified Mosher’s method. Alpinia tonkinensis Gagnep. (Zingiberaceae) is an endemic medicinal plant to Vietnam and has been only found in some provinces (Nam Ha, Ninh Binh, Vinh Phuc) in North Vietnam [1, 2] The plant was also introduced to Guangxi and Hainan of China [3]. The rhizomes of the plant were known to contain a large amount of essential oil [4]. Therefore, the plant possesses antifungal and antibacterial properties and is used as a natural antibiotic and a condiment in Vietnam. Previously, we investigated the chemical constituents of the hydrodistilled essential oil from the rhizomes of A. tonkinensis; myrcene, 1,8-cineol and camphor were found as the major components [4]. This paper describes the isolation and structural elucidation of the absolute configuration of a furanolabdane diterpene alcohol 1 (Fig. 1) which has not previously reported as natural products. Compound 1, obtained as a white amorphous powder with a positive optical O 15 16 14 13 12 OH 11 20 17 1 9 10 2 3 5 4 19 8 H 7 6 18 Fig. 1: Chemical Structure of Compound 1 rotation [[ ]23D +15.5o (c 1.3, in CHCl3)], gave the molecular formula C20H30O2 (negative-ion high-resolution (HR)FAB-MS): see Experimental). The 13C-NMR of 1 (table 1) displayed six sp2 carbons which were attributed to a monosubstituted furane ring and an exocyclic double bond. The assignments were confirmed by the proton signals of the 1H-NMR spectrum of 1 (table 1) [ 4.72 (brs), 4.88 (d, 375 1.4) of the exocyclic double bond, 6.42 (brd, 1.2), 7.41 (t, 1.2) and7.34 (brs) of the furane ring]. A secondary alcohol group was found to be in the structure of 1 on the basis of the signal at H 4.71, C 66.3 (based on the correlation in the HMQC spectrum of 1). Furthermore, three singlet signals of tertiary methyl groups ( 0.70, 0.78, and 0.83) were seen in the 1H-NMR spectrum of 1. Taken all together, 1 was suggested as a labdane-type diterpenoid with an exocyclic double bond at the 8-position. Comparison of the 13C-NMR spectroscopic data of 1 with the known labd-8(17)-enes [5, 6] disclosed the location of the hydroxyl group in the alicyclic side chain (C-11 C-16). The coupling constant of the secondary carbinol proton (dd, J = 9.6, 4.8) clearly indicated the location of the group at C-12 since the furane ring was attached to C-13. The HMBC spectrum of 1, as shown in Fig. 2, was in full agreement with the suggested structure. Generally, the labdane-type diterpenes are characteristic of the genus Alpinia [7]. The diterpene 1, therefore, was also assigned to the normal labdane-type diterpenoids and determined to be 15,16-epoxybut the 8(17),13(16),14-labdatrien-12 -ol, absolute stereochemistry at the 12-position remained to be elucidated. Literature search showed the presence of two diastereoisomeric hydroxides, prepared semisynthetically [8, 9], owing to the asymmetric center at the 12-position, but their absolute stereochemistry remained in doubt. Compound 1 showed the NMR data (table 1) superimposable with those of one of the diastereomers, and thus is the first reported natural product possessing 15,16-epoxy8(17),13(16),14-labdatrien-12-ol structure. Table 1: 1H-NMR and 13C-NMR Spectroscopic data of compounds 1 (CDCl3, in ppm, J in parentheses in Hz)a) C/H 1 2 3 4 5 6 7 O OH Fig. 2: HMBC Correlations (H C) of Compound 1 376 1 13 C 38.9 19.4 42.1 33.6 55.4 24.4 38.3 8 9 10 11 149.1 53.1 39.6 12 13 14 15 16 17A B 18 19 20 66.3 128.9 108.3 143.5 139.7 32.0 106.7 33.5 21.7 14.6 1 H 0.80 m 1.70 brd (13.1) 1.44 m 1.55 qt (13.8, 3.2) 1.10 ddd (13.8, 13.3, 4.2) 1.35 brd (13.3) 0.98 dd (12.6, 2.8) 1.69 m 1.31 qd (12.6, 4.2) 1.90 m 2.37 ddd (12.6, 4.2, 2.3) 1.42 brd (11.2) 1.93 m 1.90 m 4.71 dd (9.6, 4.8) 6.42 brd (1.2) 7.41 t (1.2) 7.34 brs 4.72 brs 4.88 d (1.4) 0.83 s 0.78 s 0.70 s a) Assignments were made on the basis of the HMQC and HMBC experiments The secondary function of the alcohol group at C-12 allowed us to determine the absolute stereochemistry at C-12 applying the modified Mosher’s method [10]. Treatment of 1 with (R)and (S)- -methoxy- -trifluoromethylphenylacetic acid (MTPA) in the presence of 1-ethyl3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) and 4-dimethylaminopyridine (DMAP) afforded the 12-(R)- and 12-(S)-MTPA esters (1a and 1b, respectively) [11] The chemical shifts for the proton signals of 1a and 1b were assigned by comparison with the spectroscopic data of 1 and 1H-1H COSY experiments. The differences in the 1H chemical shifts between the diastereomers 1b and 1a ( = S R) indicated an S-configuration for C-12 and therefore the absolute stereostructure of 1 was determined to be (12S)-15,16-epoxy8(17),13(16),14-labdatrien-12-ol. In the NOESY spectrum of 1 (Fig. 3), no NOEs were observed between H-12 and H-9, and between H-12 and H3-20. The NOE interaction between H-12 and H-17A ( 4.72; H-17B appeared at 4.88 showing a NOE interaction with H-7 at 2.37) (dotted line) was possible but uncertain to be confirmed in CDCl3 since their overlapping signals ( 4.71 and 4.72, respectively). HO H H Optical rotation was measured on a Union Giken PM-101 digital polarimeter. 1H-NMR (500 MHz) and 13C-NMR (100 MHz) spectra were obtained on JEOL JNM-ECP 500 and -400 NMR spectrometers, JEOL JNM respectively, with tetramethylsilane as an internal standard. Negative-ion mode spectrum of 1 was recorded on a JEOL JMS SX-102. Preparative HPLC was carried out with JASCO PU-1580 pump and UV-2075 Plus detector (set at 210 nm) on an YMC ODS column (150×20 mm i.d.) at the flow rate of 5 ml/min. Silica gel 60 (0.063 - 0.200 mm, Merck) was used for open column chromatography. TLC was carried out on pre-coated TLC plates (Kieselgel 60 F254, MERCK Art. 5747), and detected by spraying with 10% H2SO4 in 50% EtOH followed by heating on a hot plate at 200oC. (R)- and (S)MTPAs were purchased from Nacalai Tesque Co., Ltd. Plant material The fresh rhizomes of A. tonkinensis Gagnep. (4 kg) were collected in Vinh Phuc Province, Vietnam, in August 2003. The plant was identified by Mr Nguyen Quoc Binh, a botanist of the Institute of Ecology and Biological Resources, Vietnam National Center for Natural Science and Technology, Hanoi, Vietnam. A voucher specimen (No. VN1295) was deposited in the Herbarium of the same Institute. Extraction and Isolation H CH3 H CH3 HA H HB H H H H H H3C H H H H H Fig. 3: NOESY Correlations of Compound 1 Experimental General Procedure The powdered dried rhizomes of A. tonkinensis were extracted three times with MeOH by percolation at room temperature. Removal of the solvent from the combined MeOH extracts gave a black sirup (22.3 g). This sirup was suspended in water and partitioned with solvents of increasing polarity (n-hexane, EtOAc, and 1-BuOH). The n-hexane-soluble fraction (5.5 g) was subjected to a silica gel column chromatography eluted with n-hexaneEtOAc solvent systems (9 : 1, 8 : 1, 4 : 1 and EtOAc), six fractions were collected on the basis of TLC spots. Fraction 4 (160 mg) was chomatographed on silica gel using n-hexaneEtOAc (9 : 1) as solvent system, the main 377 subfraction was purified by HPLC on an ODS column using MeOH H2O (4 : 1) as solvent system to afford compound 1 (12 mg). (12S)-15,16-epoxy-8(17),13(16),14labdatrien-12-ol (1): Amorphous powder. [ ]23D +15.5o (c 1.3, CHCl3). 1H-NMR (CDCl3) and 13 C-NMR (CDCl3): Table 1. HRFAB-MS (negative-ion mode) m/z : 301.2189 (Calcd for C20H29O2: 301.2168). Preperation of 12-(R)- and 12-(S)-MTPA esters of 15,16-epoxy-8(17),13(16),14labdatrien-12-ol (1a and 1b, respectively): see reference 11. Acknowledgements: This research was supported by the International Foundation for Science, Stockholm, Sweden, through a Grant to Dr. Phan Minh Giang, and the Basic Research Program in Natural Science of Vietnam. References 1. Pham H. H. An Illustrated Flora of Vietnam, Published by the Author, Montreal, Tome III, Fascicle 1, P. 547 (1993). 378 2. Nguyen Q. B. Vietnam Journal of Biology, 16(4), P. 143 - 145 (1994). 3. Flora Online: http://flora.huh.harvard.edu. 4. Phan M. G., Phan T. S. and Koenig W. A. Flavour and Fragrance Journal, submitted paper. 5. H. Itokawa, S. Yoshimoto and H. Morita. Phytochemistry, 27, 435 (1988). 6. H. Itokawa, H. Morita, I. Katou, K. Takeya, A. J. Cavalheiro, R. C. B. De Olivera, H. Ishige and M. Motidome. Planta Medica, 54, 311 (1988). 7. S. Tesaki, H. Kikuzaki, S. Yonemori and N. Nakatani. J. Nat. Prod., 64, P. 515 - 517 (2001). 8. M. Jung, I. Ko and S. Lee. J. Nat. Prod., 61, 1394 - 1396 (1998). 9. J. Willamizar, J. Fuentes, F. Salazar, E. 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