Diterpenoids from Azorella madreporica and Their Antibacterial Activity Carlos Areche 1 , Inmaculada Vaca 1 , Luis A. Loyola 2 , Jorge Borquez 2 , Juana Rovirosa 1 , Aurelio San-Martín 1 1 Department of Chemistry, Faculty of Sciences, University of Chile, Santiago, Chile 2 Department of Chemistry, Faculty of Basic Sciences, University of Antofagasta, Antofagasta, Chile Abstract ! Two new diterpenoids, mulin-12-en-16-al-20-oic acid and 13-α- hydroxy-mulin-11-en-14-one-20-oic acid, were isolated from Azorella madreporica. Their structures were identified on the ba- sis of one-dimensional and two-dimensional NMR experiments. Their antibacterial activity was also tested. Key words Azorella madreporica · Apiaceae · diterpenoids · mulinane · antibacterial activity Supporting information available online at http://www.thieme-connect.de/ejournals/toc/plantamedica In previous studies, some diterpenoids with four skeletal classes, such as azorellane [1], mulinane [2], yaretane [2], and madrepor- ane [3] have been isolated from Azorella madreporica Clos. (Apia- ceae). In the course of our investigation of medicinal plants from Chile, we report the isolation and identification of two new mu- linane diterpenoid acids, 1 and 2, from Azorella madreporica and their antibacterial activity. HREIMS at m/z 318.2193 and 13 C NMR of 1 showed the molecular formula C 20 H 30 O 3 . The presence of a conjugated aldehyde was shown by 1 H and 13 C NMR spectrum (δ H 9.35 s and δ C 193.4 d). IR and 13 C NMR (3400–2500 cm -1 and δ C 178.2 s) indicated the presence of one carboxyl group. The comparison with the NMR spectra of mulinolic acid [4] and mulin-11,13-dien-20-oic acid [6] suggested that 1 is a mulinane-type diterpenoid. In the HMBC spectrum, the proton signal at δ 9.35 (H-16) had cross-peaks with δ 153.8 (C-12), δ 147.9 (C-13), and δ 18.3 (C-14); the proton signal at δ 6.70 (H-12) showed long-range correlations with the carbon signals at δ 193.4 (C-16), δ 42.6 (C-9), and δ 18.3 (C-14), and the methyl group at δ 1.08 (H 3 -17) showed correlations with δ 42.6 (C-9), δ 41.8 (C-7), and δ 30.6 (C-15), indicating that the double bond and the aldehyde group were located on C-12 and C-16. This analysis established the planar structure of 1. The relative config- uration of 1 was assigned by NOESY experiments. A NOE correla- tion observed between H-9 and H 3 -17 indicated that the rings B/ C must be cis-fused, which is in agreement with the stereochem- istry of mulinane-type diterpenoids [4, 6–8]. Based on these find- ings, we assigned the structure of 1 as mulin-12-en-16-al-20-oic acid. The molecular formula of compound 2 was proved to be C 20 H 30 O 4 (m/z 334.2145) based on HREIMS and 13 C NMR data. The IR and 13 C NMR data showed the presence of a hydroxyl group (3380 cm -1 and δ C 77.9 s), a ketone group (1710 cm -1 and δ C 212.0 s), and a carboxyl group (1690 cm -1 and δ C 179.8 s). Two deshielded carbons at δ C 132.9 d and 131.8 d were assigned to a disubstituted olefinic double bond. The comparison of the 13 C NMR spectrum of 2 with the one of mulinolic acid [4] permitted us to identify its mulinane skeleton. In the HMBC spectrum, the proton signal at δ 5.83 (H-11) showed long-range correlations with the carbon signals at δ 77.9 (C-13), δ 51.7 (C-10), and δ 38.6 (C-8); the proton signal at δ 5.62 (H-12) had cross-peaks with δ 212.0 (C-14), δ 47.6 (C-9), and δ 27.5 (C-16), and the methyl group at δ 1.44 (H 3 -16) showed correlations with δ 212.0 (C-14), δ 131.8 (C-12), and δ 77.9 (C-13), indicating that the hydroxyl group and the ketone group were at C-13 and C-14. In addition, the proton signal at δ 2.23 (H-9) correlated with δ 131.8 (C-12) and δ 47.9 (C-15) confirming the olefinic double bond. On the basis of these findings, the planar structure of 2 was determined (l " Fig. 1). The main correlations in the NOESY spectrum were observed be- tween H-9 and H 3 -17 as well as between H 3 -16 and H 3 -17, which indicated their relative configuration. Based on the spectral data described above, the structure of 2 was established as 13α-hy- droxy-mulin-11-ene-14-one-20-oic acid. In an agar diffusion test, paper disks containing 50, 100, and 200 μg of compounds 1 or 2 did not produce significant zones of inhibition against five phytopathogenic bacterial strains. These results are in agreement with those described by Wächter et al. [9], which suggest that the keto function in position C-14 is not essential for the antibacterial activity of mulinane diterpenoids. In addition, our results also suggest that the keto function in po- sition C-16 and the hydroxyl group in position C-13 are not im- portant for the antibacterial activity. Material and Methods ! Azorella madreporica Clos. (Apiaceae) was collected in March 2007 in the III region of Chile and identified by Clodomiro Marti- corena, Biology Department, Universidad de Concepción. A voucher (N° 3545) was deposited at the herbarium of the Univer- sidad de Concepción, Concepción, Chile. Azorella madreporica Clos. (5.0 kg) was extracted with MeOH at room temperature (3 × 8 L, 72 h each) to give a gum (295 g). This Fig. 1 The structures of compounds 1–5. 1749 Areche C et al. Diterpenoids from Azorella madreporica … Planta Med 2010; 76: 1749–1751 Letters