Oxygenated Pimarane Diterpenes from Kaempferia marginata Sanit Thongnest, † Chulabhorn Mahidol, †,‡ Somyote Sutthivaiyakit,* ,⊥ and Somsak Ruchirawat* ,†,‡ Chulabhorn Research Institute, Vipavadee Rangsit Highway, Bangkok 10210, Thailand, Chulabhorn Research Center, Institute of Science and Technology for Research and Development, Mahidol University, Salaya 73170, Thailand, and Department of Chemistry, Faculty of Science, Ramkhamhaeng University, Bangkok 10240, Thailand Received May 28, 2005 Six new diterpenes, (1R,2S,5S,7S,9R,10S,13R)-1,2,7-trihydroxypimara-8(14),15-diene (1), (1R,2S,5S,9S,- 10S,11R,13R)-1,2,11-trihydroxypimara-8(14),15-diene (2), (1S,5S,7R,9R,10S,11R,13R)-1,7,11-trihydrox- ypimara-8(14),15-diene (3), (1S,5S,9S,10S,11R,13R)-1,11-dihydroxypimara-8(14),15-diene (4), (5S,6R,9S,- 10S,13R)-6-hydroxypimara-8(14),15-diene-1-one (5), and (1R,2S,5S,7S,9R,10S,13R)-1,2-dihydroxypimara- 8(14),15-diene-7-one (6), along with four known diterpenes, have been isolated from the dichloromethane extract of whole plants of Kaempferia marginata. The structures were assigned by spectroscopic methods. The absolute configuration of 1 was established by the Mosher ester method. Substances obtained were evaluated against a panel of bioassays including antimalarial, antituberculous, and antifungal activity. Kaempferia marginata Carey (Zingerberaceae, local name: tup mup) is a Thai medicinal plant, and its roots have been used in the treatment of allergy, fever, and swollen leg. 1 Chemical constiuents that have been reported for the genus Kaempferia include cyclohexane oxide deriva- tives, 2 chalcone derivatives, 3 cinnamates, 4 diterpenes, 5,6 monoterpenes, 7 and flavonoids. 8 A preliminary biological assay on K. marginata indicated that the dichloromethane extract of whole plants exhibited activity against the malarial parasite Plasmodium falciparum with an IC 50 value of 26.4 µg/mL. In the present study on this plant we have isolated six new pimarane-type diterpenes, namely, (1R,2S,5S,7S,9R,10S,13R)-1,2,7-trihydroxypimara-8(14),- 15-diene (1), (1R,2S,5S,9S,10S,11R,13R)-1,2,11-trihydroxy- pimara-8(14),15-diene (2), (1S,5S,7R,9R,10S,11R,13R)-1,7,- 11-trihydroxypimara-8(14),15-diene (3), (1S,5S,9S,10S,11R,- 13R)-1,11-dihydroxypimara-8(14),15-diene (4), (5S,6R,9S,- 10S,13R)-6-hydroxypimara-8(14),15-diene-1-one (5), and (1R,2S,5S,7S,9R,10S,13R)-1,2-dihydroxypimara-8(14),15- diene-7-one (6), along with four known compounds, san- daracopimaradiene, 9 sandaracopimaradien-1R-ol, 10 2R- acetoxysandaracopimaradien-1R-ol, 5 and sandaracopimara- dien-1R,2R-diol. 5 We herein report the isolation, structure elucidation, and biological activity of these compounds. Results and Discussion Compound 1 was isolated as a white solid, mp 131-133 °C. A molecular formula of C 20 H 32 O 3 was determined from HRFABMS. The 13 C NMR spectrum indicated 20 carbon signals, including four methyls, five methylenes, seven methines, and four quaternary carbons. The 1 H NMR spectrum displayed signals characteristic for vinylic pro- tons at δ H 5.82 (1H, dd, J ) 17.4, 11.2 Hz, H-15), 4.95 (1H, dd, J ) 17.5, 1.3 Hz, H-16a), and 4.93 (1H, dd, J ) 11.2, 1.3 Hz, H-16b), as well as an olefinic proton at δ H 5.67. Three oxygenated methine groups were observed at δ H 4.00 (δ C 72.1, d), 3.91 (δ C 66.8, d), and 3.71 (δ C 74.8, d) in addition to four tertiary methyl group singlet signals at δ H 1.09, 0.99, 0.93, and 0.83. On the basis of the molecular formula and the presence of two unsaturated double bonds inferred from the 1 H and 13 C NMR data, it was apparent that three rings were present in the molecule. The long- range 1 H- 13 C NMR correlations between a methyl proton signal at δ H 1.09 and the 13 C signal at δ C 148.1(d) as well as 3 J correlations between a vinylic proton at δ H 4.95 and 4.93 and a quaternary 13 C signal at δ C 37.0 indicated a pimarane diterpene skeleton with a vinyl group attached to C-13. 11 The 1 H- 13 C HMBC NMR correlations between H-1/C-2, C-3, C-5, C-10, and C-20; H-3/C-2, C-18, and C-19; H-6/C-5, C-7, and C-10; and H-7/C-6, C-8, and C-14 placed the three hydroxyl groups at C-1, C-2, and C-7, respectively. The 3 J correlations between H-14/C-7, C-9, C-12, C-13, C-15, and C-17 indicated the double bond at C-8(14). The NOESY spectrum of 1 showed correlations between H-1, H-2, H 3 -18, and H 3 -20, indicating that both OH-1 and OH-2 adopt an R-orientation. NOE correlations between H-7/H-5 and H-9 as well as the large vicinal coupling constant (J ) 11.4 Hz) between H-6a and H-7R detected from the H-7 signal at δ H 4.00 (ddd, J ) 11.4, 5.5, and 1.0 Hz) further indicated the -oriented hydroxyl group at C-7. The NOE correlation between H 3 -17 and H-11 could also be ob- served. The absolute stereochemistry of 1 was determined using the Mosher ester method. The presence of vicinal hydroxyl groups on C-1 and C-2 was not favorable for the application of this method. The 1,2-diol was converted to the 1,2- acetonide prior to the esterification step, which was further treated with (S)-(+)- and (R)-(-)-R-methoxy-R-(trifluoro- methyl)phenylacetyl chloride (Experimental Section) at room temperature. MTPA (methoxytrifluoromethyl phen- ylacetic acid) esters of the 1,2-acetonide of 1, which showed the observed chemical shift differences (∆δ S-R ) indicated in Figure 1, unambiguously determined the absolute con- * To whom correspondence should be addressed. Tel: (662) 574-0622, ext. 1505. Fax: (662) 574-2027. E-mail: somsak@tubtim.cri.or.th. or somyote_s@yahoo.com. † Chulabhorn Research Institute. ‡ Chulabhorn Research Center, Mahidol University. ⊥ Ramkhamhaeng University. 1632 J. Nat. Prod. 2005, 68, 1632-1636 10.1021/np050186l CCC: $30.25 © 2005 American Chemical Society and American Society of Pharmacognosy Published on Web 11/02/2005