Absolute Configuration of Dihydro-β-agarofuran Sesquiterpenes from

Aug 19, 2016 - Chemoprevention of human cancer appears to be a feasible strategy for cancer control, especially when chemopreventive intervention is ...
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Absolute Configuration of Dihydro-β-agarofuran Sesquiterpenes from Maytenus jelskii and Their Potential Antitumor-Promoting Effects Nayra R. Perestelo,† Ignacio A. Jiménez,† Harukuni Tokuda,‡ Jesús T. Vázquez,† Eiichiro Ichiishi,§ and Isabel L. Bazzocchi*,† †

Instituto Universitario de Bio-Orgánica Antonio González, Departamento de Química Orgánica, and Instituto Canario de Investigación del Cáncer, Universidad de La Laguna, Avenida Astrofísico Francisco Sánchez 2, 38206 La Laguna, Tenerife, Spain ‡ Organic Chemistry in Life Science, Division of Food Science and Biotechnology, Graduate School of Agriculture, Kyoto University, Kyoto 606-8502, Japan § Department of Internal Medicine, International University of Health and Welfare Hospital, Nasushiobara, Tochigi 329-2763, Japan S Supporting Information *

ABSTRACT: Chemoprevention of human cancer appears to be a feasible strategy for cancer control, especially when chemopreventive intervention is involved during early stages of the carcinogenesis process. As a part of our ongoing research program into new chemopreventive agents, herein are reported the isolation, structural elucidation, and biological evaluation of 10 new (1−10) and three known (11−13) sesquiterpenes with a dihydro-β-agarofuran skeleton from the leaves of Maytenus jelskii Zahlbr. Their stereostructures have been elucidated by means of spectroscopic analysis, including 1D and 2D NMR techniques, ECD studies, and biogenetic considerations. The isolated metabolites and eight previously reported sesquiterpenes (14−21) were screened for their antitumor-promoting activity using a short-term in vitro assay for Epstein−Barr virus early antigen (EBV-EA) activation induced by 12-O-tetradecanoylphorbol-13-acetate (TPA). Six compounds from this series (4, 5, 11, and 13−15) were found to exhibit higher efficacies than β-carotene, used as reference inhibitor for EBV-EA activation. In particular, promising antitumor activity was observed for compound 5, exhibiting inhibition even at the lowest concentration assayed (10 mol ratio/TPA). Preliminary structure−activity relationship analysis revealed that the acetate, benzoate, and hydroxy groups are the most desirable substituents on the sesquiterpene scaffold for activity in the EBV-EA activation assay.

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prevention, defined as slowing the process of carcinogenesis, appears to be a feasible alternative for cancer control,21 and the Epstein−Barr virus early antigen (EBV-EA) activation assay efficiently evaluates chemopreventive activity in vitro.22,23 Several classes of natural products have been used to demonstrate that chemoprevention is an effective strategy in the search for novel therapeutic agents for cancer.24 Among these, sesquiterpenoids with a dihydro-β-agarofuran skeleton have been reported as promising cancer chemopreventive agents.25 A previous report on Maytenus jelskii Zahlbr. (Celastraceae), a shrub that grows in the forests of Bolivia, Peru, and Ecuador, identified a sesquiterpene as having promising antitumor-promoting activity in vitro and also cancer chemopreventive effects in an in vivo two-stage carcinogenesis model.26 These findings warranted a more in-depth study of this species as a source of potential cancer chemopreventive agents.

esquiterpenoids with a dihydro-β-agarofuran (5,11-epoxy5β,10α-eudesm-4(14)-ene) skeleton are a family of naturally occurring metabolites isolated mainly from Celastraceae species,1 although, recently, they have also been reported from Santalaceae2 and Pinaceae3 species. They are considered as privileged structures4 due to their structural diversity and wide range of biological activities. Sesquiterpenes have shown insecticidal/antifeedant,5 photosynthetic inhibition,6 antifungal,7 antiplasmodial,8 anti-Mycobacterium,9 antiHIV,10 neuroprotective,11 cytotoxic,12 reversal of multidrug resistance,13 and cancer chemopreventive14 properties. These structural and biological features have made them attractive synthetic targets15,16 and have generated medicinal and pharmacological interest.17 The Epstein−Barr virus (EBV) is a cancer-causing virus and is involved in several lymphoproliferative disorders18 and cancers,19 including Hodgkin’s disease, Burkitt’s lymphoma, nasopharingeal carcinoma, gastric carcinoma, and HIV-related central nervous system lymphomas. EBV infection is also linked to autoimmune diseases such as multiple sclerosis.20 Chemo© XXXX American Chemical Society and American Society of Pharmacognosy

Received: May 23, 2016

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DOI: 10.1021/acs.jnatprod.6b00469 J. Nat. Prod. XXXX, XXX, XXX−XXX

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The present work deals with the isolation and structural elucidation of 10 new (1−10) and three known (11−13) dihydro-β-agarofuran sesquiterpenes. Their sterostructures were determined by means of spectroscopic and spectrometric techniques. Compound 10 shows a polyhydroxy sesquiterpene core with a hydroxy group in an unusual position in its basic skeleton. The isolated compounds, except for 9 due to the small quantity isolated, and some previously reported sesquiterpenes (14−21),27 were evaluated for their inhibitory effects on EBV-EA induced by the tumor promoter 12-Otetradecanoylphorbol-13-acetate (TPA), a test to identify potential cancer chemopreventive agents.

Article

RESULTS AND DISCUSSION

Compound Structure Elucidation. Thirteen dihydro-βagarofuran sesquiterpenes (1−13) were isolated from the EtOH extract of the leaves of M. jelskii. The structure and absolute configuration of the new compounds (1−10) were elucidated by spectroscopic and spectrometric methods. Compound 1 was obtained as a colorless, amorphous solid and gave a molecular formula of C36H39O13 from its HREIMS. The IR spectrum showed characteristic absorption bands for hydroxy (3526 cm−1), ester (1737, 1638 cm−1), and aromatic (758, 712 cm−1) groups. The EIMS exhibited peaks attributable to the presence of methyl (M+ − 15, m/z 679), alcohol (M+ − 15 − 18, m/z 661), acetate (M+ − 15 − 60, m/z 619, CH 3 COOH), benzoate (M+ − 15 − 122, m/z 557, C6H5COOH, 105 m/z, C7H5O+), and cinnamate (M+ − 15 − 148, m/z 531, C 8 H 7 COOH, 131 m/z, C 8 H 9 CO + ) functionalities. This was confirmed from the 1H and 13C NMR spectra (Tables 1 and 2), which included signals for 12 protons in the aromatic region (δH 6.44−7.75) and 12 aromatic carbons (δC 128.1−134.2), two olefinic carbons (δC 117.5 and 145.9), and two carboxyl groups at δC 164.7 and 167.9, assigned to benzoyl and cinnamoyl moieties. In addition, signals corresponding to three acetyl groups [δH 1.77 (s), δC 20.3 (q), 170.3 (s); δH 2.15 (s), δC 21.2 (q), 169.9 (s); and δH 2.30 (s), δC 20.8 (q), 170.1 (s)] were observed. Its 1H NMR spectrum (Table 1) also showed an ABM system with signals at δH 5.16 (d, JAB = 3.0 Hz, H-3), 5.29 (dd, JBA = 3.0 Hz, JBM = 11.3 Hz, H-2), and 6.25 (d, JMB = 11.3 Hz, H-1), an AMX system with signals at δH 5.09 (d, JAM = 6.0 Hz, H-9), 4.55 (dd, JMX = 2.8 Hz, JMA = 6.0 Hz, H-8), and 2.43 (d, JXM = 2.8 Hz, H7), and a singlet for an oxymethine proton at δH 5.40 (H-6), whose multiplicity is due to the dihedral angle between H-6 and H-7 being close to 90°. Signals corresponding to a methyl group at δH 1.49 (Me-14) attached to a carbon at δC 69.8 (C-4) bearing a hydroxy group and those for three methyl groups at δH 1.54 (Me-12), 1.58 (Me-15), and 1.68 (Me-13) were confirmed from the 13C NMR spectrum (Table 2). These data

Table 1. 1H NMR Spectroscopic Data for Compounds 1−10a H

1

2

3

4

5

6

1

6.26, d (11.2) 5.34, dd (3.2, 11.2) 5.23, d (3.2)

6.28, d (11.2)

3

6.25, d (11.3) 5.29, dd (3.0, 11.3) 5.16, d (3.0)

6.39, d (11.1) 5.41, dd (2.3, 11.1) 4.03, d (2.3)

6.35, d (11.0) 5.39, dd (2.6, 11.0) 4.00, br s

6.27, d (11.1) 5.32, dd (2.7, 11.1) 3.99, br s

5.35, dd (2.8, 11.1) 4.02, br s

4 6

3.55, s OH 5.40, s

4.00, s OH 4.42, s

2.73, q (7.5) 5.49, s

2.73, q (7.6) 4.56, s

2.67, q (7.6) 5.45, s

2.68, q (7.6) 5.40, s

7 8

2.43, d (2.8) 4.55, dd (2.8, 6.0)

2.51, d (2.5) 4.39, dd (2.5, 6.2)

3.36, s OH 1.85 α, d (12.8) 2.51 β, dd (5.0, 12.8) 2.27, m 4.45, m

2.51, d (2.7) 5.60, dd (2.7, 6.2)

2.38, d (2.7) 5.40, dd (2.7, 6.3)

2.49, d (2.6) 5.58, dd (2.6, 6.2)

9 12 13 14 15

5.09, 1.54, 1.68, 1.49, 1.58,

5.07, 1.60, 1.72, 1.80, 1.57,

5.12, 1.35, 1.66, 1.47, 1.51,

5.36, d (6.2) 1.53, s 1.65b 1.21, d (7.5) 1.65b

5.26, d (6.3) 1.60b 1.60b 1.36, d (7.6) 1.59, s

5.33, 1.51, 1.62, 1.19, 1.57,

2.22b 2.37 α, ddd (2.9, 6.8, 16.0) 2.22b β 4.77, d (6.8) 1.46, s 1.45, s 1.19, d (7.6) 1.56, s

2

a

d (6.0) s s s s

d (6.2) s s s s

5.31, dd (3.1, 11.2) 5.27 d (3.1)

d (5.8) s s s s

d (6.2) s s d (7.6) s

7 6.36, d (11.1)

8 6.36, d (11.2) 5.38, dd (3.0, 11.2) 3.99, dd (3.0, 9.8) 4.62, d (9.8) OH 2.70, q (7.7) 4.49, d (3.5)

2.08, m 2.19, m

4.70, 1.50, 1.44, 1.37, 1.58,

d (5.9) s s d (7.7) s

9

10

6.26, d (11.0)

6.26, d (11.1)

5.29, dd (2.7, 11.0) 3.99, br s

5.31, dd (3.0, 11.1) 5.27, d (3.0)

2.68, q (7.6) 5.42, s

2.28b 2.39 α, ddd (3.0, 6.5, 16.0) 2.28b β 5.00, d (6.5) 1.48, s 1.46, s 1.17, d (7.6) 1.51, s

1.89 α, br d (11.4) 2.58 β, br d (2.2, 11.4) 2.27, m

4.98, 1.37, 1.35, 1.46, 1.48,

d (6.3) s s s s

Spectra recorded in CDCl3 at 400 MHz. bSignals without multiplicity assignments were overlapping resonances deduced by HSQC experiments. B

DOI: 10.1021/acs.jnatprod.6b00469 J. Nat. Prod. XXXX, XXX, XXX−XXX

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Table 2. 13C NMR Spectroscopic Data for Compounds 1−10a C 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 a

1 67.5, 67.9, 75.9, 69.8, 90.5, 77.9, 53.9, 70.2, 74.2, 49.4, 85.2, 30.4, 26.0, 23.6, 20.5,

CH CH CH C C CH CH CH CH C C CH3 CH3 CH3 CH3

2 67.6, 67.9, 74.8, 71.2, 90.6, 78.5, 55.0, 70.7, 74.4, 48.4, 85.2, 30.9, 26.7, 23.9, 20.7,

CH CH CH C C CH CH CH CH C C CH3 CH3 CH3 CH3

3 67.7, 68.4, 75.1, 69.5, 88.9, 32.5, 47.9, 71.2, 74.8, 48.2, 84.0, 31.0, 24.7, 23.9, 20.0,

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

4 68.0, 70.7, 73.0, 40.9, 90.7, 76.7, 51.1, 68.3, 71.3, 52.3, 85.6, 30.9, 26.5, 15.7, 19.6,

CH CH CH CH C CH CH CH CH C C CH3 CH3 CH3 CH3

5 68.0, 70.8, 73.2, 40.6, 92.1, 76.1, 54.1, 68.7, 72.0, 50.9, 85.5, 30.9, 26.6, 16.3, 19.8,

6

CH CH CH CH C CH CH CH CH C C CH3 CH3 CH3 CH3

67.7, 70.6, 72.9, 40.8, 90.7, 77.0, 52.3, 68.3, 71.7, 51.0, 85.5, 30.8, 26.4, 15.6, 19.6,

CH CH CH CH C CH CH CH CH C C CH3 CH3 CH3 CH3

7 68.5, CH 71.8, CH 73.0, CH 41.0, CH 90.8, C 79.3, CH 47.4, CH 31.5, CH2 72.5, CH 52.6 C 85.0, C 30.2, CH3 25.7, CH3 15.8, CH3 19.6, CH3

8 68.8, 71.3, 73.1, 40.5, 90.3, 77.7, 48.5, 31.8, 72.8, 52.4, 85.0, 30.4, 26.0, 16.3, 19.5,

9

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

68.1, 70.8, 73.1, 41.0, 90.8, 79.3, 47.5, 31.6, 72.7, 52.6, 85.2, 30.2, 25.9, 15.8, 19.7,

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

10 67.5, 68.4, 75.1, 69.9, 88.1, 37.6, 77.6, 38.1, 73.7, 47.1, 82.5, 21.6, 26.4, 24.0, 19.5,

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

Spectra recorded in CDCl3 at 100 MHz. Data based on DEPT, HSQC, and HMBC experiments. bOverlapping signals.

indicated that 1 is a polyester sesquiterpene with a 1,2,3,4,6,8,9heptasubstituted dihydro-β-agarofuran skeleton. The regiosubstitution of 1 was determined by an HMBC experiment, showing three-bond correlations between the carboxyl signals of the acetate groups at δC 170.3, 170.1, and 169.9 and the resonances at δH 5.29 (H-2), 5.16 (H-3), and 5.40 (H-6), respectively. The carboxyl signal of the cinnamate group at δC 167.9 was correlated with the signal at δH 5.09 (H9), whereas the carboxyl signal of the benzoate group at δC 164.7 was linked to the resonance at δH 6.25 (H-1). The relative configuration of 1 was established on the basis of the coupling constants and confirmed by a ROESY experiment (Figure 1). Thus, J1,2 (11.3 Hz), J2,3 (3.0 Hz), and J8,9 (6.0 Hz)

Table 3. Circular Dichroism Data of Compounds 1−4, 7, and 10 (MeCN) compound 1 2 3 4 7 10

λext, nm (Δε) 270 269 270 235 270 269

(+12.6) (+14.5) (+15.1) (+21.8) (+17.6) (+14.5)

λext, nm (Δε = 0) 241 241 239 225 242 242

λext, nm (Δε) 227 226 224 220 228 227

(−11.9) (−13.6) (−12.1) (−5.7) (−16.1) (−14.4)

Figure 1 was calculated as +78° by molecular mechanic calculations (PC Model).29 Therefore, the structure and a b s o l u t e c o nfi guration of 1 was es t ablished as (1R,2S,3S,4S,5S,6R,7R,8S,9R,10R)-2,3,6-triacetoxy-1-benzoyloxy-9-trans-cinnamoyloxy-4,8-dihydroxydihydro-β-agarofuran. Compounds 2 and 3 gave molecular formulas of C35H40O12 and C35H40O11, respectively, from the HREIMS and 13C NMR data. Their 1H and 13C NMR data (Tables 1 and 2) revealed similar structural features to those of compound 1, with the main differences being the replacement of the acetate group at C-6 in 1 by a hydroxy group (δH 4.42, s; δC 78.5, CH) in compound 2 and the occurrence of an aliphatic methylene group (δH 2.51, dd, J = 5.0, 12.8 Hz, H-6β; δH 1.85, d, J = 12.8, H-6α) in compound 3. 2D NMR experiments (COSY, ROESY, HSQC, and HMBC) and CD studies (Table 3) allowed the complete and unambiguous assignments of the chemical shifts and the regiosubstitution. The structure and absolute configuration of compounds 2 and 3 were proposed as (1R,2S,3S,4S,5S,6R,7R,8S,9R,10R)-2,3-diacetoxy-1-benzoyloxy9-trans-cinnamoyloxy-4,6,8-trihydroxydihydro-β-agarofuran and (1R,2S,3S,4S,5R,7S,8S,9R,10R)-2,3-diacetoxy-1-benzoyloxy-9trans-cinnamoyloxy-4,8-dihydroxydihydro-β-agarofuran, respectively. Compound 4 was obtained as a colorless, amorphous solid. The 1 H and 13C NMR spectra combined with mass spectrometric data were used to establish its structure as that of a 1,2,3,6,8,9-hexasubstituted dihydro-β-agarofuran sesquiterpene polyester, containing three acetate and two benzoate groups. The NMR spectra showed that the characteristic resonances of the tertiary hydroxy group at C-4 (around δH 3.55, δC 69.8) in the above compounds (1−3) were replaced by those of an aliphatic methine group (δH 2.73, δC 40.9). The

Figure 1. Selected NOE effects (solid line) and CD exciton coupling (dashed line) for compound 1.

values indicated a trans relationship between H-1/H-2 and a cis relationship between H-2/H-3 and H-8/H9, and NOE correlations were observed from Me-15 to H-2/H-6/H-8/H-9 as well as between Me-14 and H-2/H-3/H-6. The geometry of the disubstituted double bond of the cinnamate moiety at C-9 was determined as trans by the large vicinal coupling constant value (J = 15.9 Hz) of the olefinic proton signals at δH 6.44 and 7.44. The absolute configuration of 1 was deduced by the circular dichroism exciton chirality method.28 The ECD spectrum showed a Davidoff-type split curve with a first positive Cotton effect at 270 nm (Δε +12.6) and a second negative effect at 227 nm (Δε −11.9) (Table 3), due to the coupling of the transition moments of the benzoate and cinnamate chromophores at C-1α and C-9β. The dihedral angle between these chromophores for the configuration shown in C

DOI: 10.1021/acs.jnatprod.6b00469 J. Nat. Prod. XXXX, XXX, XXX−XXX

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Figure 2. Relative ratio of Epstein−Barr virus early antigen activation induced by 12-O-tetradecanoylphorbol-13-acetate (TPA, 20 ng/mL, 32 pmol/ mL) in the presence of compounds 1−8 and 10−21 at the selected concentration of 100 mol ratio/TPA. Percentages relative to the positive control value (100%) (n = 3): (black dots) β-carotene (C) used as reference inhibitor; (gray dots) compounds more active than β-carotene; (striped dots) compounds less active than β-carotene.

same polyhydroxy core as 4, for which the absolute configuration was established by an ECD study. The spectroscopic data showed 7, 8, and 9 to be closely structurally related to the known isolated compound 11.30 Consequently, the stereostructure elucidations of these molecules were aided greatly by comparison of their values. A set of 2D NMR spectra was acquired for each metabolite in order to gain the complete and unambiguous assignment of the 1 H and 13C NMR chemical shifts as listed in Tables 1 and 2. The same stereochemistry previously assigned to 11 was also confirmed for 7, 8, and 9 by ROESY experiments. The structure and absolute configuration of compound 7 were established as (1R,2R,3R,4R,5S,6R,7R,9S,10R)-2,6-diacetoxy-1benzoyloxy-9-trans-cinnamoyloxy-3-hydroxydihydro-β-agarofuran by ECD (Table 3), whereas those for compounds 8 and 9 were assumed based on these substances having the same polyhydroxy sesquiterpene core as 7. Compound 10 was obtained as a colorless lacquer. Its molecular formula was established as C35H40O11 from its HREIMS, and the IR spectrum indicated the presence of ester and hydroxy functionalities. The 1H and 13C NMR spectra (Tables 1 and 2) combined with mass spectrometric data were used to established its structure as that of a dihydro-βagarofuran sesquiterpene polyester containing two acetates, a benzoate, a cinnamate, and two hydroxy groups. Its NMR spectra indicated, as the most significant difference from those of the above-described compounds, the replacement of the resonances assigned to the methine group at C-7 (around δH 2.50, δC 55.0) by the signal of a quaternary carbon at δC 77.6, suggesting the presence of a tertiary hydroxy group. The position of this hydroxy group was confirmed by a HMBC experiment, showing three-bond correlations from the proton resonances at δH 1.37 (Me-12), 1.35 (Me-13), and 4.98 (H-9) to the carbon resonance at δC 77.6. The ROESY experiment showed NOE correlations from Me-15 (δH 1.48) to H-2 (δH 5.31), H-3 (δH 5.27), H-9 (δH 4.98), and H-6α (δH 1.89), and the configuration of the tertiary alcohol was assumed as α due

complete assignments of the chemical shifts of 4 (Tables 1 and 2) were achieved by 2D NMR experiments. Thus, the regiosubstitution was determined by the long-range 2,3JC,H correlations observed from H-2 (δH 5.41), H-6 (δH 5.49), and H-8 (δH 5.60) to the carboxyl signals of the acetate groups at δC 170.6, 169.2, and 168.8, respectively, whereas the carboxyl signals of the benzoate groups at δC 163.9 and 165.3 were linked to the proton resonances at δH 6.39 (H-1) and 5.36 (H9), respectively. The J1,2 (11.1 Hz), J2,3 (2.3 Hz), and J8,9 (6.2) values indicated a trans relationship between H-1/H-2 and a cis relationship between H-2/H-3 and H/8-H-9. NOE correlations from H-2 to Me-14/Me-15 and correlations between H-3 and Me-14 as well as from H-8 to H-9/Me-15 were observed in a ROESY experiment, confirming the relative configuration of 4. The structure and absolute configuration were established for 4 as (1R,2R,3R,4R,5S,6R,7R,8S,9R,10R)-2,6,8-triacetoxy-1,9-dibenzoyloxy-3-hydroxydihydro-β-agarofuran by the electronic circular dichrosim (ECD), showing a Davidoff-type split curve with a first positive Cotton effect at 235 nm (Δε +21.8) and a second negative effect at 220 nm (Δε −5.7) (Table 3). The HREIMS of compounds 5 and 6 gave molecular formulas of C33H38O11 and C32H35O13, respectively, and their 1D NMR spectra (Tables 1 and 2) resembled those of compound 4. The main differences were that the resonances assigned to the acetate group at C-6 in compound 4 were replaced by those for a hydroxy group in 5, along with the shift of the signal corresponding to H-6 from δH 5.49 to 4.56. A comparison of the NMR data of compound 6 with those of 4 showed the replacement of the resonances assigned to the benzoate group at C-1 by those corresponding to a furoate group (δH 6.38, 7.29, 7.70; δC 109.2, 118.3, 143.3, 147.3, 160.4). The regiosubstitution pattern of compounds 5 and 6 was established by the long-range 2,3JC,H HMBC couplings, and the relative configuration previously assigned to compound 4 was also confirmed for 5 and 6 by ROESY experiments. Their absolute configurations were assumed based on their having the D

DOI: 10.1021/acs.jnatprod.6b00469 J. Nat. Prod. XXXX, XXX, XXX−XXX

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to the β orientation of the agarofuran ring. This is the first report of a polyhydroxy dihydro-β-agarofuran sesquiterpene core oxidized at the C-7 position. The structure and absolute configuration of 10 were established as (1R,2S,3S,4S,5R,7R,9S,10R)-2,3-diacetoxy-1-benzoyloxy-9-trans-cinnamoyloxy4,7-dihydroxydihydro-β-agarofuran by analysis of its ECD spectrum (Table 3). Inhibition of Epstein−Barr Virus Early Antigen Activation. The isolated sesquiterpenes (1−8, 10−13) and the previously reported compounds 14−2127 were examined for their antitumor-promoting effects toward activation of the EBV-EA induced by the tumor promoter TPA in Raji cells. Compound 9 could not be assessed due to the insufficient sample isolated. The inhibitory effects on the activation of the virus at concentrations of 10, 100, 500, and 1000 mol ratio/ TPA of the test compound and the viability of the Raji cells were evaluated (Table S20, Supporting Information). The wellknown cancer chemopreventive agent β-carotene31 was used as a positive control. All the compounds exhibited dose-dependent inhibitory activities, and the viability percentage of Raji cells treated with the tested compounds was more than 60% at the highest concentration of 1000 mol ratio/TPA (Table S20, Supporting Information), indicating their cytotoxicity to be moderately potent against the in vitro cell line. Six compounds from this series (4, 5, 11, and 13−15) exhibited relative ratio of EBV-EA activation with respect to TPA (100%) values of 79.5%, 76.3%, 81.0%, 79.3%, 81.9%, and 82.2%, respectively, and were more effective than β-carotene (82.7% mol ratio/ TPA, means 17.3% inhibition) at a concentration of 100 mol ratio/TPA (Figure 2). In particular, compound 5 was the most promising compound from this series (100.0, 69.9, 23.7, and 2.2% inhibition at 1000, 500, 100, and 10 mol ratio/TPA, respectively, Table S20, Supporting Information), since it gave an inhibitory effect even at the lowest concentration of 10 mol ratio/TPA, at which the β-carotene did not show any activity. In general, this series of compounds showed slightly greater activity than the previously reported sesquiterpenes from M. jelskii.26 The influence of the substitution pattern of the dihydro-βagarofuran sesquiterpene skeleton on the potential antitumorpromoting activity was analyzed, based on the results of the in vitro assays at 100 mol ratio/TPA concentration (Figure 2). This structure−activity relationship study revealed the following trends: (a) the replacement of an acetate group by a hydroxy group at C-6 increased the activity (88.6% in 1 vs 85.0% in 2; 79.5% in 4 vs 76.3% in 5); (b) the replacement of a cinnamate group by a benzoate group at C-9 also increased the activity (86.2% in 7 vs 81.0% in 11), and (c) the substitution at C-1 of a benzoate group by a furoate group decreased the activity (79.5% in 4 vs 87.0% in 6). These findings are in agreement with previous data26 that indicated an inverse correlation between the observed activity and the presence of cinnamate, furoate, and nicotinate ester types and suggested that acetate, benzoate, and hydroxy functional groups are the most desirable substituents on the dihydro-β-agarofuran sesquiterpene core for their potential use as chemopreventive agents.

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were obtained in CH3CN on a JASCO V-560 spectrophotometer, and CD spectra were recorded in absolute EtOH on a JASCO J-600 spectropolarimeter. IR (film) spectra were measured on a Bruker IFS 55 spectrophotometer. 1H (400 MHz) and 13C NMR (100 MHz) spectra were recorded at 300 K on a Bruker Avance 400 spectrometer; the chemical shifts are given in δ (ppm) with residual CDCl3 (δH 7.26, δC 77.0) as internal reference and coupling constants in Hz; DEPT, COSY, ROESY (spin lock field 2500 Hz), HSQC, and HMBC (optimized for J = 7.7 Hz) experiments were carried out with the pulse sequences given by Bruker. EIMS and HREIMS were obtained on a Micromass Autospec spectrometer. Silica gel 60 (particle size 15−40 and 63−200 μm, Macherey-Nagel) and Sephadex LH-20 (Pharmacia Biotech) were used for column chromatography, while silica gel 60 F254 (Macherey-Nagel) was used for analytical or preparative TLC, and nanosilica gel 60 F254 (Macherey-Nagel) for high-performance TLC (HPTLC). Compounds used for CD were purified by highperformance TLC and eluted with a mixture of n-hexane−EtOAc (6:4). Plant Material. Maytenus jelskii was collected in December 2006 in Urubamba Province, Cuzco, Perú. The plant was identified by Professor Alfredo Tupayachi Herrera, and a voucher specimen (CUZ 29845) is deposited at the Herbarium of Missouri Botanical Garden, St. Louis, MO, USA. Extraction and Isolation. The dried leaves (790 g) of M. jelskii were sliced into chips and extracted with EtOH in a Soxhlet apparatus for 48 h. Evaporation of the solvent under reduced pressure provided 171.1 g of crude extract, which was partitioned into a CH2Cl2−H2O (1:1, v/v) solution. The CH2Cl2 (61.2 g) fraction was submitted to vacuum-liquid chromatography on a silica gel column, using mixtures of n-hexane−EtOAc (10:0 to 0:10) of increasing polarity as eluant to afford 36 fractions, which were combined into nine fractions (A−I) on the basis of their TLC profiles. Preliminary NMR studies revealed that two fractions, F and G, were found to be rich in sesquiterpenes, and these were investigated further. Fraction F was chromatographed on Sephadex LH-20, using as eluent mixtures of n-hexane−CHCl3− MeOH (2:1:1) to provide 24 fractions, which were combined into six fractions (F1−F6). Fractions F3 and F4 were chromatographed by silica gel flash column chromatography eluted in a step gradient manner with CH2Cl2−Me2CO (from 10:1 to 7:3) and further purified by preparative TLC with n-hexane−1,4-dioxane (6:4), n-hexane−Et2O (2:8), and CH2Cl2−Me2CO (8:2) to give compounds 1 (6.5 mg), 2 (5.0 mg) 4 (6.1 mg), 5 (5.3 mg), 10 (5.7 mg), 1232 (6.7 mg), and 1333 (13.5 mg). Fraction G was chromatographed on Sephadex LH-20 (nhexane−CHCl3−MeOH, 2:1:1) to provide 20 fractions, which were combined into five fractions (G1−G5). Fraction G4 was purified by silica gel flash column chromatography (CH2Cl2−Me2CO, from 10:1 to 8:2) and further purified by preparative TLC with n-hexane−1,4dioxane (6:4), heptane−Et2O (1:9), and CH2Cl2−Et2O (8:2) to yield compounds 3 (4.5 mg), 6 (7.1 mg), 7 (3.4 mg), 8 (2.0 mg), 9 (1.0 mg), and 1130 (40.0 mg). (1R,2S,3S,4S,5S,6R,7R,8S,9R,10R)-2,3,6-Triacetoxy-1-benzoyloxy9-trans-cinnamoyloxy-4,8-dihydroxydihydro-β-agarofuran (1): colorless, amorphous solid; [α]20D +132.8 (c 0.6, CHCl3); UV (EtOH) λmax (log ε) 274 (4.2), 216 (3.9) nm; CD (CH3CN) λext (Δε), see Table 3; IR νmax (film) 3526, 2960, 2926, 1737, 1638, 1163, 1110, 1028, 758, 712 cm−1; 1H NMR (CDCl3, 400 MHz) δ OAc-2 (1.77; 3H, s), OAc-6 (2.15; 3H, s), OAc-3 (2.30; 3H, s), OBz-1 [7.31 (2H, t, J = 7.6 Hz, H-3, H-5), 7.48 (1H, t, J = 7.4 Hz, H-4), 7.75 (2H, d, J = 7.7 Hz, H-2, H-6)], OCinn-9 [6.44 (1H, d, J = 15.9 Hz, H-α), 7.42 (3H, m, H-3, H-4, H-5), 7.44 (1H, d, J = 15.9 Hz, H-β), 7.55 (2H, m, H-2, H-6)], for other signals, see Table 1; 13C NMR (CDCl3, 100 MHz) δ OAc-2 (20.3 CH3, 170.3 C), OAc-3 (20.8 CH3, 170.1 C), OAc-6 (21.2 CH3, 169.9 C), OBz-1 [128.1 (2 x CH, C-3, C-5), 129.0 (2 × CH, C-2, C-6), 129.4 (C, C-1), 132.9 (CH, C-4), 164.7 (C, COO−)], OCinn-9 [117.5 (CH, C-α), 128.1 (2 × CH, C-2, C-6), 128.6 (2 × CH, C-3, C-5), 130.2 (CH, C-4), 134.2 (C, C-1), 145.9 (CH, C-β), 167.9 (C, COO−)], for other signals, see Table 2; EIMS m/z 679 [M+ − CH3] (8), 661 (7), 634 (2), 619 (1), 557 (1), 531 (14), 504 (2), 429 (1), 321 (2), 253 (2), 165 (3), 131 (100), 105

EXPERIMENTAL SECTION

General Experimental Procedures. Optical rotations were measured on a PerkinElmer 241 automatic polarimeter in CHCl3 at 20 °C, and the [α]D values are given in 10−1 deg cm2/g. UV spectra E

DOI: 10.1021/acs.jnatprod.6b00469 J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products

Article

CH, C-2, C-6), 129.2 (C, C-1), 132.5 (CH, C-4), 164.3 (C, COO−)], OBz-9 [127.9 (2 × CH, C-3, C-5), 129.6 (C, C-1), 130.2 (2 × CH, C2, C-6), 132.8 (CH, C-4), 165.6 (C, COO−)], for other signals, see Table 2; EIMS m/z 610 [M+] (1), 595 (7), 578 (2), 535 (2), 473 (2), 428 (2), 413 (2), 291 (1), 247 (2), 105 (100); HREIMS m/z 610.2422 [M+] (calcd for C33H38O11, 610.2414). (1R,2R,3R,4R,5S,6R,7R,8S,9R,10R)-2,6,8-Triacetoxy-9-benzoyloxy1-(3-furoyloxy)-3-hydroxydihydro-β-agarofuran (6): colorless, amorphous solid; [α]20D +13.1 (c 5.2, CHCl3); UV (EtOH) λmax (log ε) 274 (3.5), 202 (4.3) nm; IR λmax (film) 3481, 2930, 2857, 1749, 1718, 1277, 1180, 1098, 1045, 759, 714 cm−1; 1H NMR (CDCl3, 400 MHz) δ OAc-8 (1.82; 3H, s), OAc-2 (1.90; 3H, s), OAc-6 (2.16; 3H, s), OFu-1 [6.38 (1H, s, H-4), 7.29 (1H, s, H-5), 7.70 (1H, s, H-2)], OBz9 [7.46 (2H, m, H-3, H-5), 7.59 (1H, m, H-4), 8.02 (2H, d, J = 7.6 Hz, H-2, H-6)], for other signals, see Table 1; 13C NMR (CDCl3, 100 MHz) δ OAc-8 (20.3 CH3, 168.8 C), OAc-2 (20.6 CH3, 170.6 C), OAc-6 (20.9 CH3, 169.2 C), OFu-1 [109.2 (CH, C-4), 118.3 (C, C-3), 143.3 (CH, C-5), 147.3 (CH, C-2), 160.4 (C, COO−)], OBz-9 [127.9 (2 × CH, C-3, C-5), 129.0 (C, C-1), 130.1 (2 × CH, C-2, C-6), 132.9 (CH, C-4), 165.4 (C, COO−)], for other signals, see Table 2; EIMS m/z 627 [M+ − CH3] (4), 515 (15), 231 (3), 189 (7), 105 (100), 95 (11); HREIMS m/z 627.2089 [M+ − CH3] (calcd for C32H35O13, 627.2078). (1R,2R,3R,4R,5S,6R,7R,9S,10R)-2,6-Diacetoxy-1-benzoyloxy-9trans-cinnamoyloxy-3-hydroxydihydro-β-agarofuran (7): colorless, amorphous solid; [α]20D +62.6 (c 0.3, CHCl3); UV (EtOH) λmax (log ε) 277 (4.2), 223 (4.3) nm; CD (CH3CN) λext (Δε), see Table 3; IR λmax (film) 3480, 2928, 1741, 1716, 1662, 1273, 1246, 1167, 1140, 759, 715 cm−1; 1H NMR (CDCl3, 400 MHz) δ OAc-2 (1.90; 3H, s), OAc-6 (2.15; 3H, s), OBz-1 [7.29 (2H, t, J = 7.8 Hz, H-3, H-5), 7.47 (1H, t, J = 7.4 Hz, H-4), 7.76 (2H, d, J = 7.5 Hz, H-2, H-6)], OCinn-9 [6,47 (1H, d, J = 16,0 Hz, H-α), 7.42 (3H, m, H-3, H-4, H-5), 7.45 (1H, d, J = 16.0, Hz, H-β), 7.59 (2H, m, H-2, H-6)], for other signals, see Table 1; 13C NMR (CDCl3, 100 MHz) δ OAc-2 (20.7 CH3, 170.6 C), OAc6 (21.1 CH3, 169.7 C), OBz-1 [128.1 (2 × CH, C-3, C-5), 129.1 (2 × CH, C-2, C-6), 129.7 (C, C-1), 132.6 (CH, C-4), 164.6 (C, COO−)], OCinn-9 [118.2 (CH, C-α), 128.0 (2 × CH, C-2, C-6), 128.6 (2 × CH, C-3, C-5), 130.0 (CH, C-4), 134.4 (C, C-1), 148.8 (CH, C-β), 165.7 (C, COO−)], for other signals, see Table 2; EIMS m/z 620 [M+] (1), 605 (15), 578 (43), 560 (1), 545 (5), 483 (1), 458 (1), 430 (7), 231 (3), 220 (5), 131 (100), 105 (91); HREIMS m/z 620.2604 [M+] (calcd for C35H40O10, 620.2621). (1R,2R,3R,4R,5S,6R,7R,9S,10R)-2-Acetoxy-1-benzoyloxy-9-transcinnamoyloxy-3,6-dihydroxydihydro-β-agarofuran (8): colorless, amorphous solid; [α]20D +115 (c 0.2, CHCl3); UV (EtOH) λmax (log ε) 279 (3.9), 223 (4.2) nm; IR λmax (film) 3436, 2963, 2927, 1732, 1637, 1265, 1109, 801, 759, 711 cm−1; 1H NMR (CDCl3, 400 MHz) δ OAc-2 (1.91; 3H, s,), OBz-1 [7.30 (2H, t, J = 7.8 Hz, H-3, H5), 7.46 (1H, m, H-4), 7.76 (2H, d, J = 7.1 Hz, H-2, H-6)], OCinn-9 [6,47 (1H, d, J = 16,0 Hz, H-α), 7.43 (3H, m, H-3, H-4, H-5), 7.46 (1H, m, H-β), 7.56 (2H, m, H-2, H-6)], for other signals, see Table 1; 13 C NMR (CDCl3, 100 MHz) δ OAc-2 (20.7 CH3, 170.8 C), OBz-1 [128.5 (2 × CH, C-3, C-5), 129.8 (2 × CH, C-2, C-6), 129.9 (C, C-1), 132.4 (CH, C-4), 164.7 (C, COO−)], OCinn-9 [118.2 (CH, C-α), 128.1 (2 × CH, C-2, C-6), 129.1 (2 × CH, C-3, C-5), 129.9 (CH, C4), 134.4 (C, C-1), 147.7 (CH, C-β), 165.9 (C, COO−)], for other signals, see Table 2; EIMS m/z 578 [M+] (12), 563 (2), 545 (1), 430 (1), 396 (1), 248 (2), 131 (100), 105 (97); HREIMS m/z 578.2109 [M+] (calcd for C33H38O9, 578.2123). (1R,2R,3R,4R,5S,6R,7R,9S,10R)-2,6-Diacetoxy-9-benzoyloxy-1-(3furoyloxy)-3-hydroxydihydro-β-agarofuran (9): colorless, amorphous solid; [α]20D +36.3 (c 11.0, CHCl3); UV (EtOH) λmax (log ε) 274 (3.6), 202 (4.3) nm; IR λmax (film) 3480, 2928, 2856, 1743, 1718, 1245, 1234, 1099, 759, 715 cm−1; 1H NMR (CDCl3, 400 MHz) δ OAc-2 (1.91; 3H, s), OAc-6 (2.15; 3H, s), OFu-1 [6.33 (1H, s, H-4), 7.26 (1H, s, H-5), 7.65 (1H, s, H-2)], OBz-9 [7.43 (2H, t, J = 7.4 Hz, H-3, H-5), 7.54 (1H, t, J = 8.5 Hz, H-4), 8.00 (2H, d, J = 7.8 Hz, H-2, H-6)], for other signals, see Table 1; 13C NMR (CDCl3, 100 MHz) δ OAc-2 (20.7 CH3, 170.7 C), OAc-6 (21.1 CH3, 169.7 C), OFu-1 [109.0 (CH, C-4), 118.5 (C, C-3), 143.2 (CH, C-5), 147.1 (CH, C-2),

(68); HREIMS m/z 679.2381 [M+ − CH3] (calcd for C36H39O13, 679.2391). (1R,2S,3S,4S,5S,6R,7R,8S,9R,10R)-2,3-Diacetoxy-1-benzoyloxy-9trans-cinnamoyloxy-4,6,8-trihydroxydihydro-β-agarofuran (2): colorless, amorphous solid; [α]20D +63.5 (c 0.4, CHCl3); UV (EtOH) λmax (log ε) 274 (4.2), 216 (4.5) nm; CD (CH3CN) λext (Δε), see Table 3; IR λmax (film) 3470, 2957, 2925, 1736, 1276, 1241, 1163, 1111, 1026, 758, 712 cm−1; 1H NMR (CDCl3, 400 MHz) δ OAc-2 (1.79; 3H, s), OAc-3 (2.33; 3H, s), OBz-1 [7.32 (2H, t, J = 7.3 Hz, H3, H-5), 7.50 (1H, t, J = 7.5 Hz, H-4), 7.75 (2H, d, J = 7.7 Hz, H-2, H6)], OCinn-9 [6.47 (1H, d, J = 15.9 Hz, H-α), 7.44 (3H, m, H-3, H-4, H-5), 7.46 (1H, d, J = 15.9 Hz, H-β), 7.56 (2H, m, H-2, H-6), for other signals, see Table 1; 13C NMR (CDCl3, 100 MHz) δ OAc-2 (20.3 CH3, 170.3 C), OAc-3 (20.9 CH3, 170.1 C), OBz-1 [128.1 (2 × CH, C-3, C-5), 129.1 (2 × C, C-2, C-6), 129.3 (C, C-1), 132.9 (CH, C-4), 164.7 (C, COO−)], OCinn-9 [117.4 (CH, C-α), 128.1 (2 × CH, C-2, C-6), 128.6 (2 × CH, C-3, C-5), 130.3 (CH, C-4), 134.1 (C, C1), 145.9 (CH, C-β), 168.0 (C, COO−)], for other signals, see Table 2; EIMS m/z 637 [M+ − CH3] (10), 619 (28), 611 (3), 577 (2), 489 (6), 321 (1), 279 (1), 131 (100), 105 (68); HREIMS m/z 637.2281 [M+ − CH3] (calcd for C34H37O12, 637.2285). (1R,2S,3S,4S,5R,7S,8S,9R,10R)-2,3-Diacetoxy-1-benzoyloxy-9trans-cinnamoyloxy-4,8-dihydroxydihydro-β-agarofuran (3): colorless, amorphous solid; [α]20D +116.1 (c 0.4, CHCl3); UV (EtOH) λmax (log ε) 274 (4.4), 223 (4.2) nm; CD (CH3CN) λext (Δε), see Table 3; IR λmax (film) 3521, 2961, 2928, 1737, 1278, 1244, 1026, 757, 711 cm−1; 1H NMR (CDCl3, 400 MHz) δ OAc-2 (1.79; 3H, s), OAc-3 (2.30; 3H, s), OBz-1 [7.33 (2H, t, J = 7.7 Hz, H-3, H-5), 7.49 (1H, m, H-4), 7.78 (2H, d, J = 7.2 Hz, H-2, H-6)], OCinn-9 [6,49 (1H, d, J = 16,0 Hz, H-α), 7.43 (3H, m, H-3, H-4, H-5), 7.54 (1H, d, J = 16.0, Hz, H-β), 7.58 (2H, m, H-2, H-6)], for other signals, see Table 1; 13C NMR (CDCl3, 100 MHz) δ OAc-2 (20.3 CH3, 170.1 C), OAc-3 (20.9 CH3, 170.3 C), OBz-1 [128.0 (2 × CH, C-3, C-5), 129.1 (2 × CH, C2, C-6), 129.2 (C, C-1), 133.0 (CH, C-4), 164.8 (C, COO−)], OCinn9 [117.7 (CH, C-α), 128.0 (2 × CH, C-2, C-6), 128.6 (2 × CH, C-3, C-5), 130.1 (CH, C-4), 134.3 (C, C-1), 145.7 (CH, C-β), 168.3 (C, COO−)], for other signals, see Table 2; EIMS m/z 621 [M+ − CH3] (1), (14), 595 (13), 577 (2), 473 (17), 413 (1), 249 (2), 195 (12), 131 (91), 105 (100); HREIMS m/z 621.2307 [M+ − CH3] (calcd for C34H37O11, 621.2336). (1R,2R,3R,4R,5S,6R,7R,8S,9R,10R)-2,6,8-Triacetoxy-1,9-dibenzoyloxy-3-hydroxydihydro-β-agarofuran (4): colorless, amorphous solid; [α]20D +31.8 (c 1.0, CHCl3); UV (EtOH) λmax (log ε) 230 (4.4) nm; CD (CH3CN) λext (Δε), see Table 3; IR λmax (film) 3477, 3016, 2928, 1738, 1369, 1278, 1235, 1106, 1026, 756, 711 cm−1; 1H NMR (CDCl3, 400 MHz) δ OAc-8 (1.86; 3H, s), OAc-2 (1.88; 3H, s), OAc-6 (2.19; 3H, s), OBz-1 [7.28 (2H, t, J = 7.6 Hz, H-2, H-6), 7.45 (1H, t, J = 7.6 Hz, H-4), 7.60 (2H, d, J = 7.3 Hz, H-2, H-6)], OBz-9 [7.45 (2H, t, J = 7.7 Hz, H-3, H-5), 7.61 (1H, t, J = 7.2 Hz, H-4), 7.60 (2H, d, J = 7.3 Hz), 8.03 (2H, d, J = 7.8), H-2, H-6], for other signals, see Table 1; 13 C NMR (CDCl3, 100 MHz) δ OAc-8 (20.3 CH3, 168.8 C), OAc-2 (20.6 CH3, 170.6 C), OAc-6 (20.9 CH3, 169.2 C), OBz-1 [127.8 (2 × CH, C-3, C-5), 129.0 (C, C-1), 129.1 (2 × CH, C-2, C-6), 132.6 (CH, C-4), 163.9 (C, COO−)], OBz-9 [127.9 (2 × CH, C-3, C-5), 129.4 (C, C-1), 130.2 (2 × CH, C-2, C-6), 132.9 (CH, C-4), 165.3 (C, COO−)], for other signals, see Table 2; EIMS m/z 637 [M+ − CH3] (4), 577 (2), 515 (2), 473 (2), 428 (1), 351 (1), 289 (1), 247 (1), 195 (3), 153 (5), 105 (100); HREIMS m/z 637.2267 [M+ − CH3] (calcd for C34H37O12, 637.2285). (1R,2R,3R,4R,5S,6R,7S,8S,9R,10R)-2,8-Diacetoxy-1,9-dibenzoyloxy-3,6-dihydroxydihydro-β-agarofuran (5): colorless, amorphous solid; [α]20D +48.9 (c 0.6, CHCl3); UV (EtOH) λmax (log ε) 230 (4.5) nm; IR λmax (film) 3463, 2957, 2928, 1734, 1640, 1369, 1280, 1242, 1112, 757, 711 cm−1; 1H NMR (CDCl3, 400 MHz) δ OAc-8 (1.83; 3H, s), OAc-2 (1.85; 3H, s), OBz-1 [ [7.30 (2H, t, J = 7.8 Hz, H-2, H6), 7.50 (1H, t, J = 7.6 Hz, H-4), 7.57 (2H, d, J = 8.1 Hz, H-2, H-6)], OBz-9 [7.47 (2H, t, J = 7.9 Hz, H-3, H-5), 7.61 (1H, t, J = 7.7 Hz, H4), 8.02 (2H, d, J = 7.9 Hz, H-2, H-6)], for other signals, see Table 1; 13 C NMR (CDCl3, 100 MHz) δ OAc-8 (20.7 CH3, 169.2 C), OAc-2 (21.0 CH3, 170.6 C), OBz-1 [127.8 (2 × CH, C-3, C-5), 129.0 (2 × F

DOI: 10.1021/acs.jnatprod.6b00469 J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products

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160.4 (C, COO−)],OBz-9 [127.8 (2 × CH, C-3, C-5), 128.6 (C, C-1), 130.1 (2 × CH, C-2, C-6), 132.9 (CH, C-4), 160.4 (C, COO−)], for other signals, see Table 2; EIMS m/z 569 [M+ − CH3] (1), 542 (17), 515 (2), 457 (1), 406 (2), 248 (4), 105 (100), 95 (33); HREIMS m/z 569.2009 [M+ − CH3] (calcd for C30H33O11, 569.2023). (1R,2S,3S,4S,5R,7R,9S,10R)-2,3-Diacetoxy-1-benzoyloxy-9-transcinnamoyloxy-4,7-dihydroxydihydro-β-agarofuran (10): colorless, amorphous solid; [α]20D +91.4 (c 0.4, CHCl3); UV (EtOH) λmax (log ε) 274 (4.3), 223 (4.2) nm; CD (CH3CN) λext (Δε), see Table 3; IR λmax (film) 3502, 2924, 2854, 1735, 1276, 1243, 1164, 1096, 758, 713 cm−1; 1H NMR (CDCl3, 400 MHz) δ OAc-2 (1.80; 3H, s), OAc-3 (2.30; 3H, s), OBz-1 [7.33 (2H, t, J = 7.8 Hz, H-3, H-5), 7.48 (1H, m, H-4), 7.85 (2H, d, J = 7.7 Hz, H-2, H-6)], OCinn-9 [6.41 (1H, d, J = 16.0 Hz, H-α), 7.42 (3H, m, H-3, H-4, H-5), 7.49 (1H, d, J = 16.0 Hz, H-β), 7.56 (2H, m, H-2, H-6)], for other signals, see Table 1; 13C NMR (CDCl3, 100 MHz) δ OAc-2 (20.3 CH3, 170.1 C), OAc-3 (20.9 CH3, 170.3 C), OBz-1 [128.1 (2 × CH, C-3, C-5), 129.1 (2 × CH, C2, C-6), 129.6 (C, C-1), 132.8 (CH, C-4), 164.8 (C, COO−)], OCinn9 [117.9 (CH, C-α), 128.0 (2 × CH, C-2, C-6), 128.6 (2 × CH, C-3, C-5), 130.0 (CH, C-4), 134.3 (C, C-1), 145.1 (CH, C-β), 165.8 (C, COO−)], for other signals, see Table 2; EIMS m/z 636 [M+] (1), 621 (14), 594 (1), 576 (1), 474 (1), 473 (8), 412 (2), 369 (2), 310 (4), 248 (2), 230 (8), 206 (12), 131 (82), 105 (100); HREIMS m/z 636.2585 [M+] (calcd for C35H40O11, 636.2571). In Vitro Epstein−Barr Virus Early Antigen Induction Assay. The inhibition of EBV-EA activation was assayed using Raji cells (virus nonproducer type), as described previously.14,22 Briefly, EBV genomecarrying lymphoblastoid cells (Raji cells, derived from Burkitt’s lymphoma) were cultivated in 10% fetal bovine serum in RPMI1640 medium (Nissui). Spontaneous activation of EBV-EA in the subline Raji cells was less than 0.1%. The cells (1 × 106/mol) were incubated for 48 h at 37 °C in 1 mL of a medium containing n-butyric acid (4 mmol), TPA (32 pmol) in dimethyl sulfoxide (DMSO) as inducer, and various amounts of test compounds in 5 μL of DMSO. Smears were made from the cell suspensions, and the activated cells that were stained by EBV-EA-positive serum from nasopharyngeal carcinoma patients were detected by an indirect immunofluorescence technique. In each assay, at least 500 cells were counted, and the number of stained cells (positive cells) present was recorded. Triplicate assays were performed for each compound. The average EBV-EA induction of the test compounds were expressed as a relative ratio to the control experiment (100%), which was carried out with only n-butyric acid (4 mmol) plus TPA (32 pmol). EBV-EA induction was around 35%. The viability of treated Raji cells was assayed by the Trypan Blue staining method.

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CO (Spain and FEDER funds from the EU) and by a Grant-inAid for Scientific Research (B-24300253) from the Ministry of Education, Culture, Sports, Science and Technology, Japan. Thanks are also due to Professor A. Tupayachi Herrera, University Nacional San Antonio Abad del Cuzco, Cuzco (Perú), for collecting and identifying the plant material.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jnatprod.6b00469. NMR spectra of compounds 1−10, ECD curves of compounds 1−4, 7, and 10, and relative ratio of Epstein−Barr virus early antigen in the presence of compounds 1−8 and 10−21 (PDF)

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REFERENCES

AUTHOR INFORMATION

Corresponding Author

*Tel (I. L. Bazzocchi): +34-922-318594. Fax: +34-922-318571. E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This study was supported by the projects SALUCAN03 Fundación CajaCanarias and SAF2015-65113-C2-1-R MINEG

DOI: 10.1021/acs.jnatprod.6b00469 J. Nat. Prod. XXXX, XXX, XXX−XXX

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DOI: 10.1021/acs.jnatprod.6b00469 J. Nat. Prod. XXXX, XXX, XXX−XXX