Cytotoxic and anti-HIV-1 constituents from leaves and twigs of Gardenia tubifera Vichai Reutrakul, a, * Chongkon Krachangchaeng, a Patoomratana Tuchinda, a, * Manat Pohmakotr, a Thaworn Jaipetch, b Chalobon Yoosook, c Jittra Kasisit, c Samaisukh Sophasan, d Kulawee Sujarit d and Thawatchai Santisuk e a Department of Chemistry, Faculty of Science, Mahidol University, Rama 6 Road, Bangkok 10400, Thailand b Department of Radiology, Faculty of Medicine, Siriraj Hospital, Mahidol University, Bangkok 10700, Thailand c Department of Microbiology, Faculty of Science, Mahidol University, Rama 6 Road, Bangkok 10400, Thailand d Department of Physiology, Faculty of Science, Mahidol University, Rama 6 Road, Bangkok 10400, Thailand e The Forest Herbarium, Department of Royal Forest, Phaholyothin Road, Bangkok 10900, Thailand Received 10 October 2003; revised 11 November 2003; accepted 4 December 2003 Abstract—Two new natural cycloartanes, tubiferolide methyl ester (1) and tubiferaoctanolide (2), together with the known coronalolide (3) and coronalolide methyl ester (4) have been isolated from leaves and twigs of Gardenia tubifera. In addition, a new flavone 5,3 0 ,5 0 -tri- hydroxy-7,4 0 -dimethoxyflavone (5), five known flavones 6 – 10 and hexacosyl 4 0 -hydroxy-trans-cinnamate (11) were also obtained from the same source. The structures were assigned on the basis of spectroscopic methods. Compounds 3, 7, 9, and 10 showed significant cytotoxic activities only in P-388 cell line. Compound 1 was cytotoxic against P-388, KB, Col-2 and Lu-1, while 4 was active in P-388 and BCA-1. Compounds 3 and 4 displayed significant anti-HIV activities in the HIV-1RT assay; compound 7 showed moderate activity in this assay. Compounds 5 – 10 were also found to be active in the DTat/Rev MC 99 syncytium assay. q 2003 Elsevier Ltd. All rights reserved. 1. Introduction Several species of plants in Gardenia genus (Rubiaceae) have been used ethnomedically in various countries, primarily for abortifacient 1 and contraceptive 1–3 purposes. Some species are used as a febrifuge 4 and a larvicide, 5 as well as for the treatment of headaches, 6 asthma 7 and malaria. 8 Extracts of various species exhibiting anti- implantation and abortifacient effects, 9 anti-ulcer, 10 anti- bacterial, 11 diuretic, 12 analgesic, 12 hypertensive, 12 and larvicidal activity 13 have been previously reported. Our investigations of G. coronaria and G. sootepensis 14 led to the isolation of four ring-A seco-cycloartanes, and the work on G. obtusifolia 15 yielded one ring-A seco-cycloartane, two cycloartanes and five flavones. Herein we describe the investigation of hexane and chloroform extracts of G. tubifera which led to the isolation of tubiferolide methyl ester (1), tubiferaoctanolide (2), coronalolide (3), coronalo- lide methyl ester (4), 5,3 0 ,5 0 -trihydroxy-7,4 0 -dimethoxy- flavone (5), 5,3 0 ,5 0 -trihydroxy-3,6,7,4 0 -tetramethoxyflavone (6), 5,7,4 0 -trihydroxy-6-methoxyflavone (7), 5,7,3 0 -trihy- droxy-6,4 0 ,5 0 -trimethoxyflavone (8), 5,3 0 -dihydroxy-7,4 0 ,5 0 - trimethoxyflavone (9), 5,3 0 -dihydroxy-6,7,4 0 ,5 0 -tetrameth- oxyflavone (10), and hexacosyl 4 0 -hydroxy-trans-cinnamate (11) (see Fig. 1). Compounds 1, 2 and 5 have not been isolated previously. The structures of all isolated com- pounds were elucidated on the basis of spectroscopic methods. The results from cytotoxic and anti-HIV-1 activity evaluations are also included. 2. Results and discussion Tubiferolide methyl ester (1) was shown to possess a molecular formula C 31 H 46 O 4 by HR-FABMS ([MþH] þ ¼ 483.3458). Its IR (CHCl 3 ) spectrum displayed two carbonyl absorptions at 1755 and 1734 cm 21 , corresponding to the CvO stretching of a,b-unsaturated lactone and CvO stretching of ester, respectively. The 1 H NMR spectral data of 1 (Table 1) were similar to those of coronalolide methyl ester (4) obtained from the same species and previously isolated from G. coronaria and G. sootepensis. 14 Compound 1 exhibited a characteristic pair of doublets at d 0.17 and 0.43 (J¼5.3 Hz each), assignable to C-19 methylene protons of the cyclopropane ring of a cycloartane triterpene. 16 – 23 Two doublets at d 6.34 (J¼2.5 Hz) and 5.74 (J¼2.1 Hz) were ascribed to H-28a and H-28b in exocyclic methylene g-lactone ring, while the signals of the b and g-methine protons of the lactone ring appeared at d 3.24 (br d, 0040–4020/$ - see front matter q 2003 Elsevier Ltd. All rights reserved. doi:10.1016/j.tet.2003.12.010 Tetrahedron 60 (2004) 1517–1523 * Corresponding authors. Tel.: þ66-02-2015152; fax: þ66-02-6445126; e-mail addresses: scvrt@mahidol.ac.th; scptc@mahidol.ac.th Keywords: Ring-A seco-cycloartane; Octanolide; Flavones; Cytotoxic activity; Anti-HIV activity.