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We investigated the composition of extracts derived from Guaiacum spp. (Zygophyllaceae), a group of neotropical tree species with varied uses in Central-and South American traditional medicine. Activity-guided fractionation of Guaiacum heartwood extracts led to the identification of four new spirocyclic lignans, named ramonanins A-D (1-4). The ramonanins exhibit cytotoxic activity against human breast cancer cell lines with an IC50 value of 18 μM and induce cell death via apoptotic mechanisms. The ramonanins are derived from four units of coniferyl alcohol and feature an unusual spirocyclic ring system. Several plant-derived cancer drugs were discovered by examining the use of plants in traditional medicine,1 , 2 for example etoposide and teniposide, which are derived from the lignan podophyllotoxin, isolated from Podophyllum spp. (Berberidaceae). Podophyllum was used by several American and Asian cultures for the treatment of skin cancers and warts.3 , 4 Here we report results from our chemical analysis of two neotropical tree species of the genus Guaiacum, which have a long history of use by indigenous societies of Central and South America to treat inflammatory diseases and cancer.5 , 6 In addition, Guaiacum extracts and "gum" have been employed extensively as antioxidant food additives.7 Correspondingly, the chemistry of Guaiacum species has been subject to numerous studies. 8 , 9 Results and DiscussionWe tested methanol and chloroform extracts from G. officinale and G. sanctum heartwood for cytotoxicity in a series of human cancer cell lines including lung, colon, cervical, and breast cancer cell lines. Alamar blue assays indicated that G. sanctum chloroform extracts affected cell viability of breast cell cancer lines at concentrations as low as 16 μg/mL, whereas lung, colon, and cervical cell lines were affected only at much higher concentrations of 125-250 μg/mL. Methanol extracts were generally less active. Cytotoxicity of the G. sanctum chloroform extracts was further characterized using sulforhodamine B * To whom correspondence should be addressed. Figure S1).We further investigated whether the ramonanins affect cell cycle progression by using fractional DNA content analysis. MD-MBA-231 cells were treated with ramonanin A or camptothecin, and cell cycle distribution was monitored via flow cytometry using fluorescence-activated cell sorting. We found that ramonanin A caused concentrationdependent decreases in S-phase events from 13% for untreated control cells to less than 4% for cells treated with 30 μg/mL ramonanin A or B (Figure 1). Furthermore, the decrease of S-phase populations was accompanied by an increase in the percentage of cells in sub-G0 phase. Therefore, it appears that the ramonanins strongly disrupt cell cycle progression at the G1/S phase transition. Furthermore, the results indicate that the ramonanins activate apoptotic cell death at a rate similar to that induced by camptothecin.For structure elucidation, samples of ramonanin A (1) and B (2) were analyzed using NMR spectrosc...