Phytochemistry 71 (2010) 1395–1399 Contents lists available at ScienceDirect Phytochemistry journal homepage: www.elsevier.com/locate/phytochem Cytotoxic terpenoids from Nardophyllum bryoides Marianela Sánchez a, Marcia Mazzuca b, María José Veloso c, Lucía R. Fernández a, Gastón Siless a, Lydia Puricelli c, Jorge A. Palermo a,* a UMYMFOR, Departamento de Química Orgánica, Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires, Ciudad Universitaria, Pabellón 2 - (1428), Buenos Aires, Argentina b Dpto. Química, Facultad de Ciencias Naturales, Universidad Nacional de la Patagonia San Juan Bosco, Km 4 (9000) Comodoro Rivadavia, Chubut, Argentina c Research Area, ‘‘Angel H. Roffo” Institute of Oncology, University of Buenos Aires, Av. San Martín 5481 (C1417DTB), Buenos Aires, Argentina a r t i c l e i n f o Article history: Received 13 November 2009 Received in revised form 23 February 2010 Available online 20 May 2010 Keywords: Nardophyllum bryoides Astereae Terpenoid Seco-chiliolidic acid Seco-ent-halimane a b s t r a c t The investigation of the ethanol extract of fresh aerial parts of the Patagonian shrub Nardophyllum bryoides collected in the province of Chubut, Argentina, yielded eleven terpenoids. These include: three secoent-halimane diterpenoids (1–3), two ent-halimanes (4–5) and six pentacyclic oleanane and ursane triterpenoids (6–11). Four of these compounds (2, 6, 8 and 11) are hitherto unknown, while two others (1 and 4) have been previously reported but only as synthetic products. Several of these compounds showed moderate cytotoxicity against a human pancreatic adenocarcinoma cell line while compounds 4 and 5 were active at micromolar concentrations. The main component, seco-chiliolidic acid (1), could be isolated from this extract in large amounts, turning N. bryoides into a sustainable source of this bioactive compound. Ó 2010 Elsevier Ltd. All rights reserved. 1. Introduction Plant natural products from abundant species represent an attractive and sustainable source of starting materials for the preparation of new bioactive substances. In this context, we began to investigate the native flora of Argentina in search of readily accessible and abundant natural products which could serve as scaffolds for the preparation of novel structures. Among other local species, we examined a sample of Nardophyllum bryoides (Lam.) Cabrera collected in the province of Chubut. A preliminary chromatographic inspection of this extract indicated the presence of a major compound which could fit the abovementioned criteria. After identification of this compound, the rare seco-chiliolidic acid (1), and taking into account the lack of chemical information on the genus Nardophyllum, we undertook a complete study of the minor terpenoid components of N. bryoides. The genus Nardophyllum is native to South America and is widely distributed in the Argentinean and Chilean Patagonia and the Andes (Jakupovic et al., 1986; Bonifacino, 2005). This is a small genus that comprises only six species and is placed in the Chiliotrichum group, tribe Astereae (Bonifacino, 2005). From this genus, only Nardophyllum lanatum had been previously investigated (Jakupovic et al., 1986; Zdero et al., 1990). * Corresponding author. Tel./fax: +54 11 4576 3385. E-mail address: palermo@qo.fcen.uba.ar (J.A. Palermo). 0031-9422/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.phytochem.2010.04.019 Herein, we report the isolation and structural elucidation of three seco-ent-halimane diterpenoids (1–3), two ent-halimanes (4–5) and six pentacyclic oleanane and ursane triterpenoids (6–11) from an ethanolic extract of the fresh aerial parts of N. bryoides. Four of these compounds (2, 6, 8 and 11) are new, while two others (1 and 4) have been previously reported but only as semi-synthetic products (Jakupovic et al., 1986; Harde and Bohlmann, 1988). Two of these compounds showed cytotoxicity against a human pancreatic adenocarcinoma cell line at micromolar concentrations. To the best of our knowledge, this is the first chemical report for this species, and the first report on the biological activity of seco-chiliolidic acid and derivatives. Furthermore, secochiliolidic acid could be isolated from this extract in large amounts, turning N. bryoides into a sustainable source of this bioactive compound which will be used as starting material for synthetic transformations. 2. Results and discussion Fractionation of the ethanolic extract of fresh aerial parts of N. bryoides (see Section 4) led to the isolation of five diterpenoids of the ent-halimane and seco-ent-halimane families, and six pentacyclic triterpenoids belonging to the a and b-amyrin series (Fig. 1). The 1H NMR spectrum of compound 1, the major secondary metabolite in the extract, displayed the typical signals of a monosubstituted furan ring at 6.41, 7.42 and 7.47 ppm (Table 1). The 13C NMR spectroscopic data indicated the presence of a lactone 1396 M. Sánchez et al. / Phytochemistry 71 (2010) 1395–1399 Fig. 1. Structure of compounds 1–11. Table 1 1 H and 13C NMR spectroscopic data for compounds 1–3 in CDCl3, d in ppm. 1 2 1 13 1 1 26.0 27.2 2 32.7 2.12 m 2.04 m 2.22 m 3 4 5 6 179.6 128.1 128.3 21.6 7 29.6 8 9 10 11 36.4 52.1 41.6 45.2 12 13 14 15 16 17 18 19 20 OMe 70.7 124.4 108.6 144.0 140.2 16.0 20.9 20.6 177.4 – 1.82 1.74 1.50 1.38 3.61 – – 2.44 2.17 1.69 1.51 1.74 – 2.88 2.50 2.01 5.34 – 6.40 7.42 7.47 1.34 1.73 1.74 – – C a,b 3 13 H – – – 2.43 2.17 1.72 1.51 1.72 – 2.92 2.46 2.02 5.37 – 6.41 7.42 7.47 1.33 1.71 1.73 – – C br t (5.6) m m m s dd (11.6, 3.1) dd (13.0, 6.0) m dd (10.5, 6.0) dd (1.7, 0.6) t (1.7) br t (0.6) d (6.7) d (1.8) d (1.0) 32.0 63.1 129.1 126.7 21.2 29.8 36.3 52.2 42.2 45.1 70.5 124.3 108.6 143.6 139.8 15.9 20.8 20.6 177.5 – H 13 1 26.1 2.12 2.02 2.23 2.16 – – – 2.44 2.17 1.70 1.51 1.71 – 2.92 2.45 2.00 5.37 – 6.41 7.42 7.47 1.34 1.69 1.73 – 3.65 C m m m m m 32.6 173.8 128.6a 128.0a 21.4 m m m m s dd dd dd dd 29.6 (11.0, (12.9, (12.9, (10.3, 3.4) 6.0) 11.0) 6.0) dd (1.8, 0.9) t (1.8) br s d (6.8) d (2.0) d (1.0) 36.3 52.1 41.5 45.1 70.4 124.2 108.4 143.4 139.6 15.8 20.6b 20.5b 177.3 51.6 H m m m m m m m m s dd (11.9, 3.0) m m dd (10.6, 5.9) dd (1.8, 0.9) t (1.7) m d (6.7) d (2.0) d (1.2) s Assignments may be interchanged. carbonyl and two further oxygen bearing carbons. Additionally, the resonances of three methyl groups could be observed, one as a doublet and the other two corresponding to olefinic methyl groups at 1.71 and 1.73 ppm. A molecular formula C20H26NaO5 obtained by HRMS (m/z: 369.1690 [M + Na]+), indicated the presence of three rings in the structure, while a complete set of 2D NMR spectra established that 1 was a member of the 3,4-seco-ent-halimane family. Compound 1 was finally identified as seco-chiliolidic acid, previously reported by Jakupovic et al. (1986) from Chiliotrichium rosmarinifolium and Nardophyllum lanatum, but with incomplete NMR spectroscopic data. Full 1H and 13C NMR data obtained by detailed 2D experiments are listed in Table 1. The NMR spectra of 1–3 were quite similar. The difference between compounds 1 and 2 was the presence of a 2H-multiplet at 3.61 ppm in the 1H NMR spectrum of 2, suggesting the presence of a primary hydroxyl group instead of the carboxylic acid. This assumption was confirmed by the presence of an oxidized methylene carbon at d 63.1 in the 13C NMR spectrum of 2. The molecular formula deduced from the NMR data and MS was in agreement with the proposed structure. Thus, compound 2 was identified as the previously unreported seco-chiliolide alcohol. On the other hand, compound 3 was readily identified by NMR and MS data as the methyl ester of secochiliolidic acid, which was previously obtained as a semi-synthetic derivative of 1 (Jakupovic et al., 1986), and is reported herein for 1397 M. Sánchez et al. / Phytochemistry 71 (2010) 1395–1399 the first time as a natural product. A complete set of 2D NMR analyses allowed a detailed and complete assignment of all signals for this group of compounds. Compounds 4 and 5 belonged to the ent-halimane class, and were identified as 3a-hydroxy-5,10-didehydrochiliolide and 3ahydroxy-5,6-didehydrochiliolide, respectively. Compound 5 had been previously isolated from Chiliotrichium rosmarinifolium and Nardophyllum lanatum; however, compound 4 was obtained as an intermediate in the synthesis of 3a-hydroxy-5b,10b-epoxychiliolide and is reported here for the first time as a natural product (Jakupovic et al., 1986; Harde and Bohlmann, 1988). Based on their NMR spectra, compounds 6–11 were clearly characterized as pentacyclic triterpenoids. On the basis of its HRMS compound 6 had the molecular formula C30H50O3 (m/z: 481.3671 [M + Na]+) which indicated six degrees of unsaturation. The 1H NMR spectrum showed eight methyl signals, six as singlets at d 0.79, 0.96, 1.00, 1.03, 1.06 and 1.14 and two as doublets at d 0.76 (J = 6.3 Hz) and 1.00 (J = 6.3 Hz) (see Table 2). The presence of an olefinic proton was evident from the triplet at d 5.20 (J = 3.6 Hz), while a double doublet at 3.22 (J = 11.2, 5.1 Hz) indicated the presence of a C-3 hydroxyl group. These facts strongly suggested the structure of a pentacyclic triterpene with an ursane skeleton. The 13 C NMR spectroscopic data showed typical signals for the A, B and C rings of an a-amyrin derivative, and the presence of two additional oxidized carbons at 68.5 and 81.8 ppm. Both of them showed HMBC correlations with a methyl singlet at d 1.06 (assigned to C-28) indicating that the additional hydroxyls were probably located in the C and D rings (C-16 and C-22). The resonance at dC 68.5, dH: 4.55 (dd J = 11.2, 5.1 Hz) was assigned to C-16 due to COSY and HMBC correlations with the double doublet at 1.30 ppm (J = 13.0, 5.1 Hz, H-15). The assignment of H-15 in turn was confirmed by the HMBC correlations with the C-27 methyl; C-16 also displayed an HMBC correlation with a multiplet at d 1.45 (H-18). The remaining signal at dC 81.8, dH: 3.52 (dd J = 12.2, 4.2 Hz) was attributed to C-22 due to the COSY and HMBC correlations with the protons at d 1.65 and d 1.77 (H-21), and the HMBC correlation with the signal at d 1.45 (H-18). The relative stereochemistry at C-16 and C-22 was determined by the correlations observed in a phase-sensitive NOESY experiment. NOE correlations between H-16, H-27 and H-19, suggested a configurations for these protons, while correlations between H-22, H-18 and H-28, confirmed b configurations for these signals. Thus, compound 6 was identified as urs-12-ene-3b,16b,22a-triol, which, to the best of our knowledge, is a new compound. The spectroscopic data for compound 7 were in agreement with those reported for the oleanane longispinogenin (Tori et al., 1976). Compounds 8 and 6 had the same molecular formula (C30H50O2, m/ z: 481.3671 [M + Na]+). Although 8 had also an ursane skeleton, only one methyl doublet (Me-29) could be observed in the 1H NMR spectrum, since the Me-30 doublet multiplicity was masked due to strong coupling to H-20 which had exactly the same chemical shift, a fact that was evident in the DEPT–HSQC spectrum. As Table 2 1H and 13C NMR spectral data of compounds 6, 8 and 11 in CDCl3 in ppm. 6 8 13 1 1 39.0 2 3 4 5 6 27.5 79.3 39.0 55.4 18.5 7 33.0 8 9 10 11 40.3 47.1 37.0 23.7 12 13 14 15 126.1 137.3 43.1 36.3 16 68.5 1.67 1.02 1.62 3.22 – 0.72 1.57 1.42 1.57 1.39 – 1.49 – 1.95 1.93 5.20 – – 1.75 1.30 4.55 17 18 19 20 42.1 60.4 39.4 38.1 21 39.8 22 C H 11 13 1 38.7 1.65 1.01 1.62 3.23 – 0.74 1.57 1.43 1.58 1.41 1.49 1.91 C m m m dd (11.2, 5.1) m m m m m m m m t (3.6) m dd (13.0, 5.2) dd (11.2, 5.1) 27.3 79.0 38.8 55.2 18.2 32.7 46.5 46.9 36.8 23.3 125.4 136.8 43.6 36.4 13 H m m m dd (11.3, 5.0) m m m m m m m 39.2 27.5 79.2 38.9 55.5 18.6 32.9 40.5 47.7 37.1 23.6 68.7 5.13 – – 1.94 1.50 4.33 41.3 56.2 39.6 39.5 – 1.36 m 1.31 m 0.96 bs 39.3 58.8 39.4 37.5 29.8 1.55 1.26 2.57 1.13 1.01 0.79 0.95 1.05 1.15 3.11 4.15 0.78 0.96 39.3 81.8 – 1.45 m 1.38 m 1.1 m 0.99 m 1.77 t (3.8) 1.65 m 3.52 dd (12.2, 4.2) 30.4 23 24 25 26 27 28 28.3 15.8 15.9 17.0 25.0 19.0 1.00 0.79 0.96 1.03 1.14 1.06 s s s s s s 28.0 15.5 15.6 16.6 24.4 71.4 29 30 17.9 21.2 0.76 d (6.3) 1.00 d (6.3) 17.4 21.2 t (3.5) C m m dd (11.8, 4.3) m m m m s s s s s dd (11.0, 0.7) d (11.0) d (6.2) bs 125.4 138.9 42.7 26.3 21.0 1 H 1.65 1.01 1.62 3.23 – 0.74 1.56 1.41 1.56 1.36 – 1.53 – 1.93 1.53 5.14 – – 1.75 1.04 1.72 1.34 – 1.33 1.37 1.04 m m m dd (11.3, 4.8) dd (11.9, 1.4) m m m m m m m t (3.6) m m m m m m m 79.3 1.64 m 1.38 m 3.35 dd (11.3, 4.3) 28.5 15.9 16.0 17.1 23.6 24.8 1.00 0.80 0.96 1.02 1.09 0.97 17.7 21.3 0.79 d (6.2) 0.97 d (6.2) s s s s s s 1398 M. Sánchez et al. / Phytochemistry 71 (2010) 1395–1399 Table 3 ID50 values (lM) for the in vitro screening against human solid tumor cells. Cell line 1 2 3 4 5 6 11 Compound PANC1 LM3 20 28 25.1 20.0 31.6 14.1 2.5 3 0.3 40 63.1 25.1 44.7 0.8 for compound 6, a signal at dH: 4.33 (dd J = 11.8, 4.3 Hz) clearly established the presence of a C-16 hydroxyl group, while a pair of resonances at d 3.11 (dd J = 11.0, 0.7 Hz) and 4.15 (d J = 11.0 Hz) corresponding to a methylene, indicated the presence of an oxidized methyl carbon (dC 71.4). This was attributed to C28 due to the HMBC correlations with carbons at 30.4 (C-22), 41.3 (C-17) and 68.7 (C-16). A NOESY correlation between H-16 and Me-27 confirmed a b stereochemistry for the C-16 hydroxyl. In this way, compound 8 was identified as the previously unreported urs-12-ene-3b,16b,28-triol. The spectroscopic data for compounds 9 and 10 were in agreement with reported chemical shifts (Quijano et al., 1998; Siddiqui et al., 1986) for the oleanane maniladiol, and the ursane uvaol, respectively. Compound 11 had also an ursane skeleton and a molecular formula C30H50O2 (m/z: 465.3699 [M + Na]+). The 13C NMR spectrum showed two signals of oxidized carbons at d 79.2 and 79.3 which were assigned to C-3 and C-22. In particular, H-22 (d 3.35 dd (11.3, 4.3)) showed COSY correlations to both C-21 protons, as well as C-22 showed HMBC correlations with the same signals. On the other hand, a NOESY correlation between H-22 and Me-28 indicated that the hydroxyl group at C-22 was a. Thus, compound 11 was identified as urs-12-ene-3b,22a-diol, which is reported here for the first time. 2.1. Biological activity The biological activity of seco-chiliolidic acid and chiliolide derivatives had not been previously investigated. The effect of compounds 1–6 and 11 on cell growth was assayed on log phase unsynchronized monolayers of two different cell lines: LM3 (murine lung adenocarcinoma cells) and PANC1 (human ductal pancreatic carcinoma). The observed ID50 values (lM, PANC1, LM3) are depicted in Table 3. 3. Conclusions This work describes the terpenoid profile of N. bryoides, with a total of eleven isolated and completely characterized compounds. As a result of this work, four new compounds (a 3,4-seco-ent-halimane and three ursane triterpenoids) were identified and described. Two additional compounds were isolated for the first time as natural products, and the complete NMR spectroscopic data of seco-chiliolidic acid (1) were reported for the first time. It is interesting to note that 3,4-seco-ent-halimane compounds may be chemotaxonomic markers for the Chiliotrichum group, based on these and previous findings. However, a more thorough investigation on the chemistry of related species will be necessary to confirm this proposal. Interestingly, in a previous paper, Bohlmann proposed that compounds 1, 4 and 5 may arise from a common precursor, 3a,5a-dihydroxychiliolide, which was isolated from Chiliotrichium rosmarinifolium and Nardophyllum lanatum (Jakupovic et al., 1986). However, this compound could not be detected among the minor components of this extract. The preliminary cytotoxicity results indicated that compounds 4 and 5 were highly active against PANC 1 (pancreatic carcinoma) cell line. On the other hand, ursane tritrpenoid 11 was highly and selectively active towards the murine LM3 cell line. These promising results will have to be completed by a more extensive screening of other solid tumor cell lines as well as mechanistic studies. In the case of compound 1, although the activity can only be considered as moderate, the results are still important, since 1 is an abundant component of this extract (80 mg of HPLC purified substance from 100 g of fresh plant material) which can be transformed into more bioactive derivatives. These results, together with the fact that N. bryoides is a sustainable biological resource, suggests that 1 can be used as a suitable scaffold for structural modifications which will further lead to a structure–activity study, and eventually to large-scale preparation of derivatives with increased bioactivity. 4. Experimental 4.1. General Optical rotations were measured on a Perkin–Elmer 343 polarimeter, whereas 1H and 13C NMR spectra were acquired using a Bruker Avance-2 (500 MHz) and AC-200 (200 MHz) spectrometers, with CDCl3 as solvent. Proton chemical shifts were referenced to the residual signal of CHCl3 at d 7.26 ppm, with 13C NMR spectra referenced to the central peak of CDCl3 at 77.0 ppm. Homonuclear 1 H connectivities were determined by COSY experiments. The edited reverse-detected single quantum heteronuclear correlation (DEPT–HSQC) experiment allowed determination of carbon multiplicities, as well as one-bond proton-carbon connectivities, and the heteronuclear multiple bond correlation (HMBC) experiment allowed the determination of long-range proton-carbon connectivities. The relative stereochemistry was determined by gradient-enhanced NOESY experiments. All 2D NMR experiments were performed using standard pulse sequences. HRESI mass spectra were recorded using a MicrOTOF QII Bruker mass spectrometer. Reversed-phase vacuum flash chromatography was carried out on octadecyl functionalized silica gel (Aldrich Chemical Co.). HPLC separations were performed using HPLC-grade solvents, a Thermo Separations Spectra Series P100 pump, a Thermo Separations Refractomonitor IV RI detector and a Thermo Separations SpectraSeries UV 100 UV detector, HPLC-grade solvents and an YMC RP-18 (5 lm, 20 mm  250 mm) column. UV detection was performed at 220 nm. Sephadex LH-20 was obtained from Pharmacia Inc., TLC was carried out on Merck Sílicagel 60 F254 plates. TLC plates were sprayed with 2% vanillin in concentrated H2SO4. All other solvents were distilled prior to use. 4.2. Plant material Specimens of N. bryoides were collected at Escalante Department, Province of Chubut, Argentina in February 2008 (summer). A voucher specimen (HRP6865) was identified by María Elena Arce (Universidad Nacional de la Patagonia San Juan Bosco, Argentina) and was stored at the Herbario Regional Patagónico, Universidad Nacional de la Patagonia San Juan Bosco. 4.3. Extraction and isolation Ground aerial parts of fresh plant material (100 g) were extracted with EtOH (3  1l, 24 h each) at room temperature and evaporated at reduced pressure to yield a syrupy residue 7 g. The latter was partitioned between MeOH: H2O (9:1) and cyclohexane, with the polar phase (5.8 g) subjected to vacuum flash chromatography on reversed phase-silica gel column (H2O/MeOH gradient). The fraction eluted with MeOH: H2O 7:3 (NOT-3, 2.1 g) was then permeated through a Sephadex LH-20 column (2  50 cm), using M. Sánchez et al. / Phytochemistry 71 (2010) 1395–1399 MeOH as eluant. After TLC comparison, the fractions showing similar TLC patterns were pooled into eight groups (S1–S8). Fraction S5 (1.16 g) was purified by dry column flash chromatography on silica gel using a CH2Cl2/EtOAc gradient. The fraction eluted with CH2Cl2 (E1, 26 mg) was purified by preparative TLC with CH2Cl2: MeOH 95:5 as eluant to yield 3 (10 mg). Separation of E4 (120 mg, eluted with CH2Cl2: EtOAc, 6:4) by preparative HPLC with MeOH: H2O (7:3) as eluant afforded compounds 1 (80 mg), 2 (1.6 mg), 4 (1.1 mg) and 5 (8.0 mg). Separation of NOT-5 (eluted with MeOH: H2O 9:1, 226 mg) by preparative HPLC with MeOH: H2O (95:5) as eluant afforded 6 (2.7 mg) and two impure fractions: NAR-2 (15 mg) and NAR-4 (80 mg). NAR-2 was purified by preparative HPLC using CH3CN as eluant to yield 7 (1 mg) and 8 (1.2 mg). The same conditions were applied to the separation of NAR-4, which afforded compounds 9 (17 mg), 10 (4 mg) and 11 (1 mg). 4.3.1. Seco-chiliolide alcohol 2 Oil, [a] 71.7 (c 0.06, CHCl3)25D; for 1H and 13C NMR spectroscopic data, see Table 1; ESI–MS m/z [M + Na]+ 355.1868 (calc. for C20H28NaO4, 355.1879). 1399 10 nM, 100 nM, 1 lM, 10 lM, 100 lM and 1 mM of compound or vehicle (0.1% DMSO) for 3 days. Cell viability was assessed by reduction of the tetrazolium salt MTS [3-(4,5-dimethylthiazol2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt] to the formazan product in viable cells (Cell Titer 96 TM, Promega Corp.) as calculated by the 492/620 nm absorbance ratio. The ID50 (Inhibitory Dosis) was calculated from the log-phase of each growth curve. Acknowledgements This research was supported by grants from CONICET (PEI 6478), UBA (X 260 Programación 2004–2007), UNPSJB (PI 734) and ANPCyT (PICT (2003) 14321). We thank Mr. José Gallardo and Lic. Gernot Eskuche (UMYMFOR–CONICET) for recording some of the NMR spectra. M. Sánchez and G. Siless thank CONICET for postdoctoral and doctoral fellowships respectively. M. Mazzuca thanks Bioq. M. Barría for helping in the collection and preparation of plant material. Appendix A. Supplementary material 4.3.2. Urs-12-ene-3b,16b,22a-triol 6 White amorphous powder, [a]+11.8 (c 0.135, CHCl3)25D; for 1H and 13C NMR spectroscopic data, see Table 2; ESI–MS m/z [M + Na]+ 481.3671 (calc. for C30H50NaO3, 481.3652). Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.phytochem.2010.04.019. 4.3.3. Urs-12-ene-3b,16b,28-triol 8 White amorphous powder; for 1H and 13C NMR spectroscopic data, see Table 2; ESI–MS m/z [M + Na]+ 481.3671 (calc. for C30H50NaO3, 481.3652). Bonifacino, J., 2005. Nardophyllum cabrerae (Asteraceae: Astereae), a new species from Argentina and new taxonomic changes involving Nardophyllum Hook. Et. Arn. Taxon. 54, 688–692. Harde, C., Bohlmann, F., 1988. Synthesis of 3a-hydroxy-5b, 10b-epoxychiliolide, an isolabdane derivative from Chiliotrichium rosmarinifolium. Tetrahedron 44, 81– 90. Jakupovic, J., Banerjee, S., Bohlmann, F., King, R.M., Robinson, H.E., 1986. New diterpenes from Chiliotrichium rosmarinifolium and Nardophyllum lanatum. Tetrahedron 42, 1305–1313. Quijano, L., Rios, T., Fronczek, F.R., Fischer, N.H., 1998. The molecular structure of maniladiol from Baccharis salicina. Phytochemistry 49, 2065–2068. Siddiqui, S., Hafeez, F., Begum, S., Siddiqui, B.S., 1986. Kaneric acid, a new triterpene from the leaves of Nerium oleander. J. Nat. Prod. 49, 1086–1090. Tori, K., Yoshimura, Y., Seo, S., Sakurawi, K., Tomita, Y., lshii, H., 1976. Carbon-13 NMR spectra of saikogenins. Stereochemical dependence in hydroxylation effects upon Carbon-13 chemical shifts of oleanene-type triterpenoids. Tetrahedron Lett. 46, 4163–4166. Urtreger, A.J., Ladeda, V.E., Puricelli, L.I., Rivelli, A., Vidal, M.C., Lustig, E.S., Bal de Kier Joffé, E., 1997. Modulation of fibronectin expression and proteolytic activity associated with the invasive and metastatic phenotype in two murine mammary tumor cell lines. Int. J. Oncol. 11, 489–496. Zdero, C., Bohlmann, F., Niemeyer, H.M., 1990. Diterpenes from Nardophyllum lanatum. Phytochemistry 29, 1227–1230. 4.3.4. Urs-12-ene-3b,22a-diol 11 White amorphous powder; for 1H and 13C NMR spectroscopic data, see Table 2; ESI–MS m/z [M + Na]+ 465.3699 (calc. for C30H50NaO2, 465.3703). 4.4. Biological activity The effect of the different compounds on cell growth was assayed on log phase unsynchronized monolayers of two different cell lines: LM3 (murine lung adenocarcinoma cells) (Urtreger et al., 1997) and PANC1 (human ductal pancreatic carcinoma). Briefly, 2  103 cells/well from each cell line in complete medium were seeded in 96 multiwell plates. After 24 h, cells received References