Crotonkinins A and B and Related Diterpenoids from Croton tonkinensis as Anti-inflammatory and Antitumor Agents Ping-Chung Kuo, † Yuh-Chiang Shen, ‡ Mei-Lin Yang, # Su-Hui Wang, † Tran Dinh Thang, ¶ Nguyen Xuan Dung, ⊥ Po-Cheng Chiang, § Kuo-Hsiung Lee, § E-Jian Lee, 4 and Tian-Shung Wu* ,#,| Department of Biotechnology, National Formosa UniVersity, Yunlin 632, Taiwan, Republic of China, National Research Institute of Chinese Medicine, Taipei 112, Taiwan, Republic of China, Department of Chemistry, National Cheng Kung UniVersity, Tainan 701, Taiwan, Republic of China, Department of Chemistry, Vinh UniVersity, Vinh City, Vietnam, Department of Chemistry, College of Natural Sciences, Hanoi National UniVersity, Hanoi 10000, Vietnam, Natural Products Research Laboratories, School of Pharmacy, UniVersity of North Carolina, Chapel Hill, North Carolina 27599, Neurophysiology Laboratory, Neurosurgical SerVice, Departments of Surgery and Anesthesiology, and Institute of Biomedical Engineering, National Cheng Kung UniVersity, Medical Center and Medical School, Tainan, Taiwan, Republic of China, and Department of Applied Chemistry, ProVidence UniVersity, Taichung 433, Taiwan, Republic of China ReceiVed July 31, 2007 Cytotoxicity-guided phytochemical investigation of a methanolic extract of Croton tonkinensis afforded two new kaurane diterpenoids (1, 2) and 10 known ent-kaurane-type diterpenoids (3-12). The structures of 1 and 2 were based on analysis of spectroscopic and mass spectral data. Compounds 3-12 were identified by comparison of their spectroscopic and physical data with those reported in the literature. Selected compounds from this plant were examined for cytotoxic and anti-inflammatory activities. Compounds 4 and 9 showed the highest cytotoxic activity against the tested tumor cell lines. Compounds 3, 4, 6, 8, 9, and 11 had IC 50 values less than 5 µM and were more potent than the nonspecific NOS inhibitor L-NAME in inhibiting LPS-induced NO production. The genus Croton (Euphorbiaceae) includes about 300 species that are widely distributed throughout tropical regions. 1 C. tonki- nensis Gagnep. is a tropical shrub native to Northern Vietnam, where it has been commonly used to treat stomachache, abscesses, impetigo, gastric and duodenal ulcers, malaria, urticaria, leprosy, psoriasis, and genital organ prolapse. 2–4 Prior phytochemical investigations showed that this species contains steroids and ent- kaurane diterpenoids. 5–10 The crude extract of C. tonkinensis was also found to show significant cytotoxicity against MCF-7, NCI- H460, and SF-268 tumor cell lines. On the basis of the above studies, we selected C. tonkinensis as a target of our integrated program aimed at new drug discovery. Cytotoxicity-monitored fractionation of CH 2 Cl 2 -solubles of a MeOH extract from the whole plant of C. tonkinensis led to the isolation of two new kaurane diterpenoids, crotonkinins A (1) and B (2), together with 10 known diterpenoids. Herein, we report the structural determination of the new diterpenoids, evaluation of cytotoxic and anti-inflammatory bioactivities, and structure-cytotoxicity relationships of the isolated compounds. Results and Discussion Air-dried and powdered whole plants of C. tonkinensis were extracted with hot MeOH and concentrated. The MeOH extract was suspended in MeOH-H 2 O (90:10) and partitioned with n-hexane. The MeOH-H 2 O fraction was further fractionated between CH 2 Cl 2 and H 2 O to afford CH 2 Cl 2 - and H 2 O-solubles, respectively. Cytotoxicity-guided purification of the CH 2 Cl 2 fraction yielded two new compounds (1 and 2) and 10 known diterpenoids (3–12). Crotonkinin A (1) was isolated as optically active colorless needles (mp 175–178 °C, [R] 25 D +90.5), and its molecular formula was determined as C 20 H 30 O 2 , with six degrees of unsaturation as established by HRFABMS (m/z 303.2322 [M + H] + ). IR absorption bands at 3363 and 1689 cm -1 were assignable to hydroxy and carbonyl functionalities, respectively. Analysis of 13 C NMR (Table 1), DEPT-135, and HMQC spectroscopic data revealed that 1 contains one carbonyl group, four quaternary carbons, including one olefinic carbon, four methines, including one oxygenated carbon, eight methylenes, including one olefinic carbon, and three methyl groups. The 1 H NMR spectrum of 1 displayed characteristic signals for three methyl groups, two terminal olefinic protons, an oxygenated methine proton, two methylene groups, and a methine. The 1 H- 1 H COSY spectrum revealed connectivities for H-1 to H-3, H-5 to H-6, and H-9 to H-14. In the HMBC spectrum, long-range proton-carbon correlations from CH 3 -18 to C-3, -4, and -5; CH 3 - 20 to C-1, -5, and -9; CH 2 -6 to C-5 and -7; and H-9 to C-8, -10, -11, and -14 established the ABC rings of the kaurane diterpenoid skeleton. These data also indicated the presence of a unique 14- hydroxy-7-one unit in the molecule. Moreover, HMBC 2 J- and 3 J- correlations of CH 2 -17 to C-13, -15, and -16; CH 2 -15 to C-7, -8, * To whom correspondence should be addressed. Tel: 886-6-2757575, ext. 65333. Fax: 886-6-2740552. E-mail: tswu@mail.ncku.edu.tw. † National Formosa University. ‡ National Research Institute of Chinese Medicine. # National Cheng Kung University. ¶ Vinh University. ⊥ Hanoi National University. § University of North Carolina. 4 National Cheng Kung University Medical Center and Medical School. | Providence University. J. Nat. Prod. 2007, 70, 1906–1909 1906 10.1021/np070383f CCC: $37.00  2007 American Chemical Society and American Society of Pharmacognosy Published on Web 12/28/2007