Mycobacterium tuberculosis Growth Inhibition by Constituents of Sapium haematospermum Girma M. Woldemichael, †,⊥ Maria-Teresa Gutierrez-Lugo, † Scott G. Franzblau, ‡ Yuehong Wang, ‡ Enrique Suarez, § and Barbara N. Timmermann* ,† Department of Pharmacology and Toxicology, Division of Medicinal and Natural Products Chemistry, College of Pharmacy, University of Arizona, 1703 E. Mabel Street, Tucson, Arizona 85721-0207, Institute for Tuberculosis Research (M/C 964), College of Pharmacy, University of Illinois at Chicago, 833 S. Wood Street, Chicago, Illinois 60612-7231, and INTA, Instituto Nacional de Recursos Biolo ´ gicos, Las Caban ˜ as y Los Reseros s/n, 1712 Villa Udaondo, Castellar Buenos Aires, Argentina Received July 31, 2003 Four novel compounds consisting of two new pimaranes, lecheronol A (1) and lecheronol B (2), an acylated cycloartane, 3-O--lauroyl-cycloart-(23E)-en-25-ol (10), and a highly oxygenated novel chalconoid, R,,3,4,5,2′,4′,6′-octahydroxydihydrochalcone (12), were isolated along with seven known triterpene derivatives and three flavonol glucosides from Mycobacterium tuberculosis growth-inhibiting fractions of the CH 2 Cl 2 /MeOH (1:1) extract of the aerial parts of Sapium haematospermum. Compounds 1, 3 (3R- hydroxyolean-12-ene), 8 [3R-hydroxylup-20(29)-en], and 9 (cycloartanol) were found most active, with MIC values of 4, 12.2, 13.4, and 8 µg/mL, respectively. Cytotoxicity tests in Vero cells for compounds 1, 3, 8, and 9 gave IC 50 values of 104.8, 127.2, 127.2, and 102.4 µg/mL, respectively. Sapium haematospermum Mu ¨ ll (Euphorbiaceae) is a plant native to drier regions of South America usually found growing as a small tree up to 5 m tall. In the locality where the plant was collected in northern Argentina, it is commonly known as “lecheryn” and is traditionally used in the treatment of teeth ailments. Some species of the genus Sapium, especially Chinese tallow (Sapium seb- iferum), have been the focus of investigations owing to the potential of sapium fat as a substitute for cocoa butter. 1 Phytochemical investigations on other species in the genus have so far led to the identification of an anti-inflammatory alkaloid, 2 antihypertensive phenolic compounds, 3 toxic phorbol esters, 4 toxic cucurbitacins, 5 and hydrolyzable tannins and flavonoids. 6 The CH 2 Cl 2 /MeOH (1:1) extract of the aerial parts of S. haematospermum was included in an initial screening carried out as part of an effort at discovery of bioactive agents from dryland biodiversity of Latin America under the International Cooperative Biodiversity Group (ICBG) program. In the present study, this extract was found active in inhibiting the growth of Mycobacterium tubercu- losis and, thus, merited further chemical investigation. Below we describe compounds obtained from bioactive fractions of the extract and evaluation of their inhibitory effect on the growth of M. tuberculosis. Results and Discussion On discovery that the CH 2 Cl 2 /MeOH (1:1) extract of S. haematospermum inhibited growth of M. tuberculosis with a MIC value of 25 µg/mL, bioassay-directed fractionation of the extract was undertaken as described in the Experi- mental Section. This resulted in the identification of 3R- hydroxyolean-12-ene (3), 7a 3-hydroxyolean-12-ene (4), 7b 3R-hydroxyurs-12-ene (5), 7c 2R,3,23-trihydroxyolean-12- en-28-oic acid (6), 7d 3-hydroxylup-20(29)-ene (7), 7e 3R- hydroxylup-20(29)-ene (8), 7f cycloartanol (9), 8 stigmast-5- en-3-O--D-glucopyranoside (11), 9 kaempferol-3-O--D- glucopyranoside (13), 10 and quercetin-3-O--D-glucopyran- oside (14) 10 as known compounds from bioactive fractions. Compounds 1, 2, 10, and 12 were identified as novel on the basis of analysis of spectroscopic data.Compound 1 was isolated as a white powder from one of the bioactive frac- tions after extensive chromatographic purification. Its molecular composition, C 20 H 30 O 3 , was then inferred from HRFABMS. The IR spectrum for this compound indicated the presence of a ketone and hydroxyl groups through characteristic absorptions. The combined analysis of its 13 C and DEPT NMR spectra then revealed the presence of 20 carbons assigned to four methyls, four methylenes, two methines, two quaternary carbons, two tertiary carbinols, one carbonyl, and four olefinic carbons. These, together with the molecular composition, suggested that 1 possessed 6 degrees of unsaturation. The presence of two π-bonds and a ketone function further indicated that the compound was a tricyclic diterpene. Olefinic methine and quaternary carbon signals observed in the 13 C NMR spectrum at δ 128.6 (C-14) and 137.6 (C-8), respectively, confirmed the presence of a trisubstituted double bond, while those at δ 144.5 (C-15) and 115.5 (C-16) in the 13 C NMR and at δ 5.94 (H-15), 4.90 (H-16a), and 5.03 (H-16b) in the 1 H NMR spectra made the presence of an exocyclic vinyl group readily apparent. All these indicated that compound 1 possessed a pimara-8(14),15-diene skeleton. Also identifi- able in the 1 H and 13 C NMR spectra were, however, two signals at δ 3.99 (br s, H-1) and 3.29 (dd, J ) 12.2 and 2.2 Hz, H-12), compatible with two hydroxy-bearing methines, and a signal ascribable to a ketone group at δ 211.1 (C-3). On the basis of extensive selective 1D-TOCSY experiments, the spin systems H 1 -H 2 ,H 5 -H 6 -H 7 ,H 9 -H 11 -H 12 , and H 14 -H 15 -H 16 were identified. Connections between the identified spin systems were then established through analysis of the HMBC spectrum as shown in Table 1. In addition, 3 J C,H correlations between one of the methyls in the gem-dimethyl group (C 18 -C 4 -C 19 , also identified using the HMBC spectrum) and both C 3 and C 5 , between H 16 and C 13 , between H 15 and C 17 , and between H 14 and both C 17 and C 7 enabled the specific location of groups that were * To whom correspondence should be addressed. Tel: (520) 626-2481. Fax: (520) 626-2515. E-mail: btimmer@pharmacy.arizona.edu. † University of Arizona. ‡ University of Illinois at Chicago. § Instituto Nacional de Tecnologia Agropecuaria. ⊥ Present address: MTDP, National Cancer Institute (Frederick), Bldg. 562, Rm. 201/301, Frederick, MD 21701. 598 J. Nat. Prod. 2004, 67, 598-603 10.1021/np0303411 CCC: $27.50 © 2004 American Chemical Society and American Society of Pharmacognosy Published on Web 03/20/2004