Diterpenoids and Triterpenoids from the Resin of Bursera microphylla

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Diterpenoids and Triterpenoids from the Resin of Bursera microphylla and Their Cytotoxic Activity Federica Messina,† Massimo Curini,† Chiara Di Sano,‡ Claudia Zadra,§ Giulia Gigliarelli,† Luisa Alondra Rascón-Valenzuela,⊥ Ramón Enrique Robles Zepeda,*,⊥ and Maria Carla Marcotullio*,† †

Department of Pharmaceutical Sciences, University of Perugia, Via del Liceo, 1-06123 Perugia, Italy Department of Oncohematology, University of Perugia, Santa Maria Hospital, Via Tristano di Joanuccio, 1-05100 Terni, Italy § Department of Pharmaceutical Sciences, University of Perugia, Borgo XX Giugno, 74-06121 Perugia, Italy ⊥ Department of Chemical and Biological Science, University of Sonora, Hermosillo, Sonora, Mexico ‡

S Supporting Information *

ABSTRACT: A chemical study of the nonpolar fraction of a methanol-soluble extract of Bursera microphylla resin yielded a variety of di- and triterpenoids. In total, 15 compounds were isolated, of which three are new, namely, malabaricatrienone (1), malabaricatrienol (2), and microphyllanin (3). The antiproliferative activity of the major compounds was evaluated in different murine cancer cell lines (M12.C3.F6 and RAW264.7) and human cancer cells (A549, HeLa, and PC-3). The new compounds (1− 3) did not show significant antiproliferative activity. The known compounds ariensin (4), burseran (5), and dihydroclusin diacetate (6) were effective against the RAW264.7 cell line, with IC50 values in the micromolar range.

T

The aim of the present study was to determine the terpenoid composition of B. microphylla resin methanol extract and to study the antiproliferative activity of the isolated compounds. The new terpenoids malabaricatrienone (1), malabaricatrienol (2), and microphyllanin (3) were isolated along with the known substances β-caryophyllene, caryophyllene oxide, verticillene, 5-epi-ent-verticillol, mansumbinone, betulonic acid, oleanonic acid, 16,20-dihydroxydammarenone, and the lignans ariensin (4), burseranin, burseran (5), and dihydroclusin diacetate (6). The crude methanolic extract of B. microphylla resin was partitioned with n-hexane (see the Experimental Section) to obtain a nonpolar extract. Fractionation of this n-hexane extract afforded three new compounds, namely, two triterpenoids (1 and 2) and a diterpenoid (3). Compound 1 was obtained as a white solid with [α]22D +21.1. Its molecular formula, C30H48O, was deduced by elemental analysis indicating seven degrees of unsaturation. The IR spectrum showed absorption bands for a carbonyl group (1702 cm−1). 1H NMR spectroscopic analysis of 1 in CDCl3 (Table 1) revealed the presence of eight distinct methyl signals (δ 0.97, 0.99, 1.04, 1.06, 1.59, 1.61, 1.63, 1.68) and three olefinic protons (δ 4.97, 5.09, 5.12), each appearing as a triplet (J = 7.5, 7.9, and 6.9 Hz, respectively). In the 1H NMR spectrum, a methylene group at δ 2.72 (t, J = 6.8 Hz) and a

he plant genus Bursera is distributed in the southwestern USA, most of Mexico, and Central American tropical forests toward northwestern South America.1 The genus is endemic to the tropical dry forest of Mexico, where 70 species are present,2 and these are characterized by their exudates that arise from a system of resin canals.3 Chemical investigation of the members of this genus has resulted in the isolation of different lignans with cytotoxic and antiproliferative activity4 and terpenoids with antiproliferative5 and antimicrobial activity.6,7 The essential oils and exudates of Bursera spp. have shown anti-inflammatory activity.8−10 Bursera microphylla A. Gray (Burseraceae), commonly known as “elephant tree” or “torote blanco”, is largely distributed in the Sonoran Desert in Mexico. In folk medicine, B. microphylla is steeped in alcoholic beverages to make a tincture for gum sores, cold sores, and abscessed teeth. The dried stems and leaves are used in a tea to relieve painful urination and as a stimulating expectorant for bronchitis, while the exudate has been used to treat venereal diseases.11 The Seri ethnic group in the Sonoran Desert uses different parts of the plant (leaves, fruits, bark, and exudate) for treating several illnesses, such as sore throats, for headache, and for wound healing.12 Studies on B. microphylla leaf and twig essential oils showed them to be constituted mostly (89%) by terpenoids,13 with α- and β-phellandrene being the most abundant compounds. The essential oil of the oleo-gum-resin showed β-caryophyllene as the major component.14 From the ethyl acetate resin extract, burseran and deoxypodophyllotoxin have been isolated.15,16 © XXXX American Chemical Society and American Society of Pharmacognosy

Received: February 3, 2015

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DOI: 10.1021/acs.jnatprod.5b00112 J. Nat. Prod. XXXX, XXX, XXX−XXX

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IR spectrum showed absorption bands for a hydroxy group (2926 cm−1). A careful examination of NMR spectra (Table 1) showed that compound 2 is closely related to 1. The 13C NMR and JMODXH spectra showed the presence of an oxygenated carbon (δ 79.2) and the absence of a carbonyl group. The location of the hydroxy group was confirmed by HMBC correlations between δH 3.19 (H-3) and δC 38.7 (C-4), 28.1 (C-28), and 15.6 (C-29). The relative configuration at C-3 was deduced by interpretation of the coupling constants and the NOESY correlations between H-3 and δH 0.64 (H-5) and 0.95 (H-28). Particularly diagnostic to establish the β disposition of H-13 were the couplings between CH3-19 (β-oriented) and CH3-30 and H-13. According to these data, compound 2 (malabaricatrienol) was identified as the previously unreported (13βH)-malabarica-14E,17E,24-trien-3β-ol. Microphyllanin (3) was isolated as a colorless oil with [α]26D −27.8. Its molecular formula, C22H36O3, was deduced by HRTOFMS, indicating five degrees of unsaturation, of which two were associated with two trisubstituted double bonds. The IR spectrum established the presence of an ester (1729 cm−1). The remaining oxygen was confirmed to be involved in an oxirane ring moiety, as characterized by the signals at δH 2.80 (t, J = 4.5 Hz) and δC 62.6 (C-7) and 59.9 (C-8). Analysis of the 1H and 13C NMR spectra (Table 2) confirmed the presence of an acetoxy group [δH 2.00 (3H, s), δC 22.7, 170.5]. In the 1H NMR spectrum (in CDCl3), microphyllanin (3) showed the presence of two olefinic protons at δH 5.29 (dd, J = 3.1, 9.0 Hz) and 5.08 (t, J = 7.9 Hz), two vinylic methyls (δH 1.67 and 1.58, each as a singlet), two methyls appearing as doublets centered at δH 0.94 and 0.95 (J = 6.8 Hz), and another methyl at δH 1.26 (s). Between 2.80 and 2.40 ppm there were several multiplets that were difficult to define. However, in C6D6, the presence of a broad doublet occurred at δH 3.06 (J = 16.5 Hz), along with a doublet of doublet at δH 2.61 (J = 9.3, 16.5 Hz) and a septet at δH 2.72 (J = 6.8 Hz). These signals, together with the presence of two methyl doublets, clearly showed the occurrence of an isopropyl group. These findings supported for compound 3 a 14-membered cembrane skeleton. COSY correlations (Figure S23, Supporting Information) allowed the establishment of spin-system sequences from H-2 to H-3, from H-5 to H-6, and from H-9 to H-11, H-13, and H-14 and confirmed the presence of the isopropyl group. HMBC correlations (Figure S26, Supporting Information) were shown for H-2 with C-1, C-3, and C-4, H-3 with C-5, H-7 with C-6, H-9 with C-6, H-10 with C-11, and H-16 with C-13. NOESY correlations (Figure 2) enabled the relative configuration of compound 3 to be determined. When H-7 was arbitrarily assigned as α-oriented, NOE enhancements were observed between H-7 and H-3, H9b and H-11, H-19 and H-5a, and CH3-18 and both H-2 protons. From these observations and by examining a molecular model, compound 3 was established as rel(1S,7S,8S)-1-acetoxy-7,8-epoxy-cembra-3E,11E-diene and was named microphyllanin. The antiproliferative activity of the new compounds (1−3) and some of the known compounds was evaluated against three human cancer cell lines, namely, A549 (lung cancer), HeLa (cervix cancer), and PC-3 (prostate cancer), and against the murine cell lines M12.C3.F6 (B cell lymphoma) and RAW264.7 (macrophages transformed by virus Abelson leukemia). None of the new compounds (1−3) showed antiproliferative effects against any of the cells used. Among the known compounds, only dihydroclusin diacetate (6) was shown to be active against murine cell line M12.C3.F3 (IC50

second one characterized by signals centered at δ 2.55 (ddd, J = 16.0, 10.7, and 7.5 Hz) and 2.38 (ddd, J = 10.6, 7.1, and 3.1 Hz) could be recognized. The 13C NMR spectrum of 1 (Table 1), supported by a JMODXH (J-modulated spin echo) experiment, indicated the presence of a carbonyl carbon (δ 217.7), three nonprotonated sp2 carbons (δ 131.4, 135.1, 138.6), three sp2 methines (δ 123.3, 123.9, 124.4), three sp3 quaternary carbons (δ 36.3, 45.0, 47.4), three sp3 methines (δ 55.2, 55.5, 58.9), nine sp3 methylenes (δ 20.4, 20.9, 26.4, 26.8, 27.1, 34.2, 35.9, 39.2, 39.7), and eight methyls (δ 15.1, 16.2, 17.7, 19.7, 21.1, 24.3, 25.7, 26.6). All these features suggested a triterpenoidal tricyclic skeleton. Analysis of the 1D and 2D NMR (HMQC, H−H COSY, HMBC) data allowed the structure of compound 1 to be determined (Table 1). COSY data were used to establish the spin system sequence from H-1 to H-2, from H-5 to H-6 and H-7, and from H-9 to H-11, H-12, and H-13 and showed a clear coupling among the olefinic protons H-15 and H-17 with the methylene at C-16.17 Furthermore, coupling was evident between the olefinic proton in C-24 and H-23 and between the latter and H-22. Particularly diagnostic for the structure of the C-13 side chain were the couplings between H15 and methyl-18, between H-17 and CH3-21, and between H24 and Hs-26 and Hs-27 (Figure S5, Supporting Information). These last features are typical for a residual isoprenoid side chain. Further support for these assignments was provided by HMBC correlations observed between H-29 and C-4 and C-5, H-30 and C-7, C-9, and C-13, H-19 and C-10, H-13 and C-18, H-18 and C-14, and H-21 and C-20 and C-22 (Figure S8, Supporting Information). All these data were indicative for the tricyclic triterpenoid malabaricane structure of compound 1.18 The relative configuration of compound 1 was established through analysis of its NOESY spectrum. NOESY interactions were observed between CH3-30 and the 1,3-diaxially juxtaposed CH3-19 and CH3-29 (Figure 1). This evidence confirmed the relative stereochemistry trans−anti−trans for the ring system and excluded the trans−syn−trans isomalabaricane skeleton.19 Furthermore, NOESY correlations between H-13 and CH3-30 were used to establish compound 1 as (13βH)-malabarica14E,17E,24-trien-3-one, and it was named malabaricatrienone. Compound 2 was obtained as a white solid with [α]24D +13.2. Its molecular formula, C30H50O, was deduced by elemental analysis, indicating six degrees of unsaturation. The B

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Table 1. NMR Data for Compounds 1 and 2a malabaricatrienone (1) carbon

δC, type

1a 1b 2a 2b 3 4 5 6a 6b 7a 7b 8 9

39.2, CH2 34.2, CH2 217.7, 45.0, 55.2, 20.4,

C C CH CH2

35.9, CH2

malabaricatrienol (2)

δH (J in Hz) 1.53, 1.78, 2.38, 2.55,

δC, type

m ddd (10.8,7.1, 3.3) ddd (10.6, 7.1, 3.1) ddd (16.0, 10.7, 7.5)

38.8, CH2 27.3, CH2 79.2, CH 38.7, C 55.8b, CH 19.0, CH2

1.22, dd (11.9, 2.9) 1.38−1.53, m 1.53−1.60, m 1.00−1.05, m 1.53−1.60, m

36.6, CH2

47.4, C 55.5, CH

1.33, dd (12.0, 7.8)

45.2, C 55.9,b CH

10 11

36.3, C 20.9, CH2

1.53, m

36.5, C 20.8, CH2

12a 12b 13 14 15 16 17 18 19 20 21 22 23 24 25 27 26 28 29 30

26.4, CH2 58.9, 138.6, 123.9, 27.1, 123.3, 19.7, 15.1, 135.1, 16.2, 39.7, 26.8, 124.4, 131.4, 17.7, 25.7, 26.6, 21.1, 24.3,

CH C CH CH2 CH CH3 CH3 C CH3 CH2 CH2 CH C CH3 CH3 CH3 CH3 CH3

1.53−1.60, m 1.90−2.02, m 2.14, d (9.2) 4.97, 2.72, 5.12, 1.61, 0.97,

26.5, CH2 58.9, 138.8, 123.7, 27.1, 123.4, 19.7, 15.3, 135.0, 16.2, 39.8, 26.8, 124.3, 131.3, 17.7, 25.7, 28.1, 15.6, 24.7,

t (7.5) t (6.8) t (6.9) s s

1.63, s 1.90−2.02, m 2.02−2.11, m 5.09, t (7.9) 1.59, 1.68, 1.06, 1.04, 0.99,

s s s s s

CH C CH CH2 CH CH3 CH3 C CH3 CH2 CH2 CH C CH3 CH3 CH3 CH3 CH3

δH (J in Hz) 1.51, 1.05, 1.60, 1.63, 3.19,

m m m m dd (9.9, 6.1)

0.64, 1.42, 1.48, 1.42, 1.52,

bd (10.7) m m m m

1.25, m 1.28, m 1.27, m 1.46, m 1.50−1.85, m 2.02, m 4.97, 2.72, 5.15, 1.60, 0.78,

t (6.9) m t (7.3) s s

1.63, s 1.86−2.05, m 1.45−1.54, m 5.10, t (6.9) 1.60, 1.68, 0.95, 0.84, 0.95,

s s s s s

Chemical shifts: δ values are given in ppm with reference to the signal of CDCl3 (δ 7.26 ppm) for 1H and to the center peak of the signal of CDCl3 (δ 77.1 ppm) for 13C. bInterchangeable values. a

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EXPERIMENTAL SECTION

General Experimental Procedures. Optical rotations ([α]D) were measured on a JASCO DIP-1000 digital polarimeter. ATR-FTIR spectra (5000−600 cm−1) were recorded using a Bruker Tensor 27 spectrometer equipped with an ATR accessory ZnSe crystal cell and taken with 2 cm−1 resolution and a sampling time of 50 scans. The FTIR experiments were carried out with a Tensor 27 FTIR spectrometer, equipped with an HTS-XT accessory for rapid automation of the analysis (Bruker Optics GmbH, Ettlingen, Germany). NMR spectra were recorded using a Bruker Avance DPX-400 spectrometer operating at frequencies of 400 MHz (1H) and 100 MHz (13C). The spectra were measured in CDCl3 and C6D6. The 1 H and 13C NMR chemical shifts (δ) were expressed in ppm with reference to the solvent signals [CDCl3: δH 7.26 and δC 77.1, C6D6: δH 7.16 and δC 128.0]. Coupling constants are given in Hz. The LCQTOF system was an Agilent 6540 UHD accurate-mass quadrupole time-of-flight (Agilent Technologies, Santa Clara, CA, USA) with a dual jet stream electrospray ionization source and an Agilent 1290 Infinity LC system. The LC consisted of a binary pump with integrated vacuum degasser, high-performance well-plate autosampler,

Figure 1. Key NOESY correlations for malabaricatrienone (1) and malabaricatrienol (2).

2.5 μM), while ariensin (4), burseran (5), and dihydroclusin diacetate (6) were active against the RAW246.7 murine cell line (IC50 9.8, 0.4, and 0.2 μM, respectively). C

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min and 25 psi, respectively. The fragmentor voltage was set to 150 V and the nozzle voltage to 800 V. The sheath gas flow was set at 9 mL/ min, and the sheath gas temperature at 300 °C. GC analyses were performed on a Hewlett-Packard HP 6890 gas chromatograph equipped with a Hewlett-Packard MS 5973 mass selective detector and a fused silica capillary column (HP-5MS; 30 m × 0.25 mm i.d., 0.25 μm film thickness). Column chromatography was performed using Merck silica gel 60 (70−230 mesh ASTM). Fractions were monitored by TLC (silica gel 60 F254, Merck), and spots on TLC were visualized under UV light and after staining with p-anisaldehyde− H2SO4−EtOH (1:1:98) followed by heating at 110 °C. Combustion analyses were carried out on a Fison EA1108 elemental analyzer. Plant Material. The collection of botanically certified resin samples was performed by Ing. José Jesús Sánchez Escalante, head of the Herbarium of Universidad de Sonora, Hermosillo, México, in Bahiá de Kino in January 2013. Resin was obtained of natural exudates present in the bark of the plant. It was stocked in 1.5 mL polyethylene tubes that were sterile and free of contaminants. Five different trees of Bursera microphylla were sampled. A voucher specimen of the species was deposited at Herbarium of Universidad de Sonora (No. 22039), and a voucher specimen of resin (No. BM-1) was deposited at the Department of Pharmaceutical Sciences, University of Perugia. Extraction and Isolation. The ground resin (4.0 g) was extracted by maceration in MeOH (3 × 100 mL) at room temperature for 24 h. After filtration, the organic solutions were concentrated at 40 °C to give a crude MeOH extract, which was diluted with H2O (50 mL) and sequentially partitioned with n-hexane (3 × 50 mL) and CH2Cl2 (3 × 50 mL). The hexane fraction was evaporated under vacuum, obtaining 0.90 g of extract. An aliquot of this extract (0.65 g) was purified over a SiO2 gel chromatography column using a gradient of n-hexane−Et2O (0−7%), followed by n-hexane−Et2O−CH2Cl2 from 18:1:1 to 2:2:1, collecting 7 mL fractions that were evaluated by TLC and combined as a result of their similar appearance, yielding 18 pooled fractions (Fr1− Fr18). Fr2 (30 mg) was analyzed by GC-MS and showed the presence of 3% β-caryophyllene, 22% α-humulene, and 60% verticillene. Fr15 (70 mg) was fractionated by SiO2 gel column chromatography using CH2Cl2−Et2O (9:1), leading to ariensin (4) (40 mg), betulonic acid (10 mg), and a mixture (15 mg) of unidentified compounds. Fr16 after purification on SiO2 gel chromatography and elution with CH2Cl2− Et2O (9:1) gave 2 mg of burseranin and 2 mg of oleanonic acid. Finally, column chromatography purification of Fr18 and elution with CH2Cl2−Et2O (8:2) gave 10 mg of dihydroclusin diacetate (6) and 3 mg of 16,20-dihydroxydammarenone. Malabaricatrienone (1): white solid; [α]22D +21.1 (c 0.35, CHCl3); IR νmax (ATR) 2963, 2933, 1702, 1452, 1381 cm−1; 1H and 13C NMR, see Table 1; anal. C 84.84%, H 11.39%, calcd for C30H48O, C 84.62%, H 11.50%. Malabaricatrienol (2): white solid; [α]24D +13.2 (c 0.63, CHCl3); IR νmax (ATR) 2962, 2926, 1449, 1380 cm−1; 1H and 13C NMR, see Table 1; anal. C 84.44%, H 11.81%, calcd for C30H50O, C 84.28%, H 11.93%. Microphyllanin (3): colorless oil; [α]24D −27.8 (c 0.35, CHCl3); IR νmax (ATR) 2960, 2929, 1729, 1240 cm−1; 1H and 13C NMR, see Table 1; EIMS m/z 288 [M − AcOH]+ (62), 273 (18), 245 (36), 121 (100), 93 (87); HRTOFMS m/z 348.2680 [M+] (calcd for C20H36O3, 348.2664). Cell Lines. The A549 (human alveolar adenocarcinoma), PC-3 (human prostatic adenocarcinoma), and HeLa (human cervix cancer) cell lines were purchased from the American Type Culture Collection (ATCC, Rockville, MD, USA). The M12.C3.F6 (murine B cell lymphoma) and RAW 264.7 (murine macrophages transformed by virus Abelson leukemia) cell lines were provided by Dr. Emil A. Unanue (Department of Pathology and Immunology, Washington University, St. Louis, MO, USA). Cells were cultured in DMEM supplemented with 5% FBS (Sigma, St. Louis, MO, USA). Experiments were conducted when the cell cultures were approximately 70% confluent. Cell Viability Assays. Cell viability was evaluated by the MTT [3(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium] reduction assay, with some modifications.20 Briefly, cells (1 × 104 per well, 50 μL)

Table 2. NMR Data for Compound 3 carbon

δC, typea

1 2a 2b 3 4 5a 5b 6a 6b 7 8 9a 9b 10a 10b 11 12 13a 13b 14a 14b 15 16 17 18 19 20 CO CH3

90.9, C 32.8, CH2 120.1, CH 135.0, C 36.8, CH2 26.0, CH2 62.6, CH 59.9, C 39.7, CH2 23.8, CH2 123.4, CH 136.6, C 34.5, CH2 33.5, CH2 34.3, 16.1, 17.8, 16.5, 16.8, 15.7, 170.5, 22.7,

CH CH3 CH3 CH3 CH3 CH3 C CH3

δH (J in Hz)b

δH (J in Hz)c

2.45−2.60, m 2.76, m 5.29, dd (3.1, 9.0)

2.61, dd (9.3, 16.5) 3.06, bd (16.5) 5.54, dd (4.5, 8.2)

2.19−2.25, m 2.27−2.31, m 1.64, m 1.80, m 2.80, t (4.5)

1.99−2.15, m 2.24−2.31, m 1.73−1.85, m

1.04, 2.08, 1.93, 2.17, 5.08,

1.21, 2.11, 1.95, 2.20, 5.21,

m m m m t (7.9)

2.95, t (6.9) m m td (5.6, 13.9) m t (6.9)

1.95, m 1.79, m 1.72, m 2.09, m 2.45−2.60, m 0.94, d (6.8) 0.95, d (6.8) 1.67, s 1.26, s 1.58, s

1.96, m 2.06, m 2.11−2.18, m 2.22−2.33, m 2.72, sept (6.8) 1.04, d (6.8) 1.05, d (6.8) 1.61, s 1.25, s 1.57, s

2.00, s

1.85, s

Chemical shifts: δ values are given in ppm with reference to the signal of CDCl3 (δ 77.1 ppm). bChemical shifts: δ values are given in ppm with reference to the signal of CDCl3 (δ 7.26 ppm). cChemical shifts: δ values are given in ppm with reference to the signal of C6D6 (δ 7.16 ppm). a

Figure 2. Key NOESY correlations for microphyllanin (3). and thermostated column compartment modules. The column was a 100 × 2.1 mm Peptide Aeris C18, 1.7 μm (Phenomenex, Bologna, Italy), with the column temperature set at 40 °C and the injection volume 8 μL. Two mobile phases (A: water containing 10 mL of ammonium acetate; B: acetonitrile) were utilized with a gradient from 0% B to 100% B within 5 min. The flow rate was set to 300 μL/min, and the eluate was introduced into the mass spectrometer by means of an Agilent Dual Jet Stream electrospray ionization (ESI) unit in the positive and negative mode. Source parameters were the following: the capillary voltage was set to 4000 V, the ion source temperature was 300 °C, and nitrogen was used as nebulizing and collision gas at 10 L/ D

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were placed in each well of a 96-well plate. After 24 h incubation at 37 °C in an atmosphere of 5% CO2 to allow cell attachment, aliquots (50 μL) of medium containing a range of concentrations of 200 to 0.05 μM of the test substances were added, and the cell cultures were incubated for 48 h. Preliminary experiments established that use of DMSO concentrations ranging from 0.06% to 2.0% in the cell cultures caused no cell damage. Previously, substances were dissolved in DMSO and subsequently diluted in culture medium. In the last 4 h of incubation time, the cells were washed with PBS. Fresh culture medium and 10 μL of a MTT solution (5 mg/mL) were added to each well. The cell viability was assessed by the ability of metabolically active cells to reduce tetrazolium salt to the colored formazan. The formazan crystals formed were dissolved with acidic isopropyl alcohol. The absorbance of the samples was measured with an ELISA plate reader (iMark microplate absorbance reader, Bio-Rad, Laboratories, D.F., Mexico), using a test wavelength of 570 nm and reference wavelength of 650 nm. Proliferative cells were expressed in terms of percentages, where the optical density measured from vehicle-treated cells was considered 100% of proliferation. Antiproliferative activity of substances was determined as IC50 values (IC50 was defined as the concentration of substance required to inhibit cell proliferation by 50%) using GraphPad Prism 5 (GraphPad Software, Inc., San Diego, CA, USA). Values of proliferation of at least three experiments, with six doses, in triplicate, were log transformed and normalized, and nonlinear regression analysis was used to generate a dose−response curve to calculate IC50 values. The differences in means were analyzed using one-way analysis of variance (one-way ANOVA) followed by Tukey’s test (Sigma Stat 3; Systat Software Inc., CA, USA). Doxorubicin was included as a positive control and for comparison purposes. IC50 values of 0.3 ± 0.01, 1.3 ± 0.2, 0.63 ± 0.08, 1.78 ± 0.12, and 3.18 ± 0.31 were calculated for M12.C3.F6, HeLa, RAW 264.7, A549, and PC-3 cells, respectively.

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ASSOCIATED CONTENT

S Supporting Information *

Purification details, NMR spectra of the new compounds (1− 3). The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jnatprod.5b00112.

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AUTHOR INFORMATION

Corresponding Authors

*Tel: +52-662-2592162. Fax: +52-662-2592159. E-mail: [email protected] (R. E. Robles Zepeda). *Tel: +39-075-585-5100. Fax: +39-075-5855116. E-mail: [email protected] (M. C. Marcotullio). Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The authors acknowledge financial support from Fondazione Cassa di Risparmio di Terni e Narni [Project “Resine dal Messico-Resins from Mexico” (2014)] and from CONACYT, Grant 83462. We thank Prof. P. Sassi and Dr. R. M. Pellegrino (University of Perugia) for the use of ATR−FTIR and LCQTOF facilities.

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REFERENCES

(1) Espinosa, D.; Llorente, J.; Morrone, J. J. J. Biogeogr. 2006, 33, 1945−1958. (2) Becerra, J. X. Proc. Natl. Acad. Sci. U.S.A. 2005, 102, 10919− 10923. (3) Becerra, J. X.; Venable, D. L.; Evans, P. H.; Bowers, W. S. Am. Zool. 2001, 41, 865−876. E

DOI: 10.1021/acs.jnatprod.5b00112 J. Nat. Prod. XXXX, XXX, XXX−XXX