Jatrophane Diterpenes from Euphorbia mellifera and Their Activity as

Article pubs.acs.org/jnp

Jatrophane Diterpenes from Euphorbia mellifera and Their Activity as P‑Glycoprotein Modulators on Multidrug-Resistant Mouse Lymphoma and Human Colon Adenocarcinoma Cells Inês Valente,† Mariana Reis,† Noélia Duarte,† Julianna Serly,‡ Joséph Molnár,‡ and Maria-José U. Ferreira*,† †

Research Institute for Medicines and Pharmaceutical Sciences (iMed.UL), Faculty of Pharmacy, University of Lisbon, Avenida Prof. Gama Pinto 1649-003, Lisbon, Portugal ‡ Department of Medical Microbiology and Immunobiology, Faculty of Medicine, University of Szeged, Dóm tér 10, H-6720 Szeged, Hungary S Supporting Information *

ABSTRACT: Three new macrocyclic jatrophane diterpenes, named euphomelliferine (1) and euphomelliferenes A (2) and B (3), and one new tetracyclic triterpene, 19(10→9)-abeo8α,9β,10α-tirucalla-5,25-diene-3β,24-diol (6, C-24 epimers), were isolated from the methanolic extract of Euphorbia mellifera. A known ingenane (7) and two jatrophane diterpenes (4 and 5) were also isolated. Their structures were elucidated by extensive spectroscopic methods, including 1D and 2D homo- and heteronuclear NMR experiments. Jatrophane diterpenes 1−3 and 5 were evaluated for their effects on the reversion of multidrug resistance (MDR) mediated by P-glycoprotein, by using the rhodamine-123 exclusion test, on human MDR1 gene-transfected mouse lymphoma cells (L5178Y MDR) and on human colon adenocarcinoma cells (COLO 320). The apoptosis-inducing activity of these compounds was also tested on COLO 320 cells, using the annexinV/propidium iodide assay. Diterpenes 1 and 2 displayed significant MDR reversing activity, in a dose-dependent manner, on both cancer cell models. The tested compounds did not induce apoptosis in the COLO 320 cells.

M

has been slow and challenging, and currently no reversal drugs are clinically available.3,4 Therefore, it is important to continue research in this area in order to discover new promising leads to improve chemotherapy of multidrug-resistant cancers. Euphorbia species have long been explored for their unique biologically active compounds, including macrocyclic diterpene polyesters and their polycyclic rearranged derivatives.5,6 The discovery of macrocyclic jatrophane and lathyrane diterpenes as a new class of potent modulators of P-gp has promoted increased interest in research of these compounds in this genus.7−13 Euphorbia mellifera Ait. (Euphorbiaceae) is an evergreen shrub endemic to Macaronesia and is particularly abundant in the Madeira archipelago. Apart from some previous studies on acetone extracts of this species, its chemical constituents have not been completely investigated.14,15 Continuing our search for novel MDR modulators from natural sources we herein report the isolation and structure elucidation of three new macrocyclic jatrophane-type diterpenes (1−3) and one new tetracyclic triterpene (6) from a methanolic extract of E.

ultidrug resistance (MDR) is one of the main clinical challenges for effective cancer chemotherapy. There are several mechanisms by which tumor cells develop resistance to cytotoxic agents. One of them is caused by the overexpression of ATP-binding cassette (ABC) proteins, such as Pglycoprotein (P-gp), multidrug-resistant protein 1 (MRP1), or breast cancer resistance protein (BCRP).1 These membraneembedded transport proteins employ the energy derived from ATP hydrolysis to efflux many chemically diverse compounds across the plasma membrane, including the anticancer agents, therefore decreasing their intracellular concentration.2 The best studied ABC transporter is P-glycoprotein, which is overexpressed in most cancer cells that present a resistant phenotype. One promising approach to overcome drug resistance is to inhibit the efflux activity of the protein with nontoxic compounds, usually named MDR modulators, inhibitors, or chemosensitizers. When used in combination with anticancer drugs, these compounds should be able to restore the effective intracellular concentration of the latter and improve the treatment outcome. Many MDR modulators, from natural or synthetic origins, have been reported, and some of them have reached the stage of clinical trials. However, progress in finding a potent and selective agent able to modulate ABC transporters © 2012 American Chemical Society and American Society of Pharmacognosy

Received: June 8, 2012 Published: October 25, 2012 1915

dx.doi.org/10.1021/np3004003 | J. Nat. Prod. 2012, 75, 1915−1921

Journal of Natural Products

Article

mellifera. Nine known compounds (4, 5, and 7−13) were also isolated. Jatrophane diterpenes 1−3 and 5 were investigated for their P-gp modulating effects on human MDR1-gene transfected mouse lymphoma cells (L5178Y MDR) and on human colon adenocarcinoma cells (COLO320). These compounds were also assayed for their activity as apoptosis inducers on COLO320 cells.

an olefinic carbon at 136.4, and an oxygenated carbon at 92.3. On the basis of the 12 degrees of unsaturation deduced from the molecular formula C32H42O10, a bicyclic diterpene skeleton was proposed for 1. These structural features were corroborated by two-dimensional NMR experiments (COSY, HMQC, and HMBC), which allowed unambiguous assignment of all the proton and carbon resonances. The HMQC and 1H−1H COSY spectra provided evidence for two proton spin-systems (denoted by bold lines in Figure S1, Supporting Information). Heteronuclear connectivities displayed in the HMBC spectrum of compound 1 between the quaternary carbons and the protons of these two spin systems allowed the linkage of the referenced fragments (Figure S1, Supporting Information). The 3 JC−H HMBC correlations also led to the location of the ester functions. Because all carbonyl groups could be related with oxymethine protons, the OH group was assigned to C-15. The relative configuration of 1 was deduced from analysis of the NOESY spectrum and the coupling constant pattern. Assuming an α-orientation for H-4 and a trans-linked cyclopentane ring, characteristic of all macrocyclic diterpenes isolated to date, the strong NOE correlations of H-4 with H-3 and H-1α provided evidence for α-orientation of these protons (Figure S2, Supporting Information). The β-orientation of H-2 was established by strong NOE cross-peaks between H-1β/H-2 and H-1α/CH3-16. The stereochemistry at C-2 and C-3 was also corroborated by J2,3 (2.0 Hz) and J3,4 (6.4 Hz) values, which were similar to those of related diterpenes (CH3-16α, H3α, J2,3 ≈ 3.5, J3,4 ≈ 7.0 Hz)16,17 and markedly different from those found in common jatrophane esters (CH3-16β, H-3α, J2,3 ≈ J3,4 ≈ 3.5 Hz).18,19 Further NOE cross-peaks detected between H-1β/CH3-20, CH3-20/H-12, H-12/CH3-18, and CH3-18/H-9 dictated their β orientation, while an α orientation of H-13, H-19, and H-8 was established by the NOE correlations of these protons with H-11. Moreover, the E geometry of the C-5/C-6 double bond was suggested by the absence of an NOE effect between H-5 and CH3-17. This was corroborated by the existence of NOESY cross-peaks between H-5/3-OBz, H-5/H-9, CH3-17/H-4, and CH3-17/H-8. Finally, the α orientation of H-7 was supported by NOE interactions between H-5/7-OAc and H-7/CH3-17. The remarkable diamagnetic shift observed for the resonance of the 7-OAc methyl group (δ 1.43), due to its location in the shielding cone of the aromatic ring, corroborated the configuration at this carbon. All the above data are in agreement with structure 1, corresponding to a new highly acylated jatrophane diterpene, which was named euphomelliferine. Compound 2 was isolated as a white, amorphous solid, [α]20 D −7.0. Its molecular formula was determined as C37H48O12 from the HRLSIMS, which showed a molecular ion at m/z 707.3021 [M + Na]+, indicative of 10 degrees of unsaturation. Characteristic IR absorption bands for ester carbonyl groups and an aromatic ring were observed. The 1H and 13C NMR data of 2 resembled those of 1, suggesting a similar jatrophane skeleton (Table 1). The spectroscopic data indicated the presence of two double bonds (one trans-disubstituted and one trisubstituted), five methyl groups, and one diastereotopic methylene group. The most remarkable differences were the replacement of the C-14 ketone signal at δC 213.6 by an oxymethine signal (δ 4.98, d, J = 2.8 Hz; δC 79.4) and the presence of two additional acetyl groups (at C-14 and C-15), along with changes in chemical shifts of the corresponding neighboring carbons.

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RESULTS AND DISCUSSION Air-dried and powdered E. mellifera plant material was exhaustively extracted with MeOH. The crude methanolic extract was suspended in ethanol, treated with Pb(OAc)2, and subjected to a liquid fractionation with EtOAc. Repeated column chromatography and HPLC of the EtOAc solubles yielded three new jatrophane diterpenes (1−3), a new tetracyclic triterpene (6), and nine known compounds. Compound 1 was obtained as a white, amorphous powder, and HRLSIMS showed a pseudomolecular ion at m/z 621.2663 [M + Na]+. The IR spectrum displayed absorption bands for OH (3532 cm−1) and ester carbonyl (1741 cm−1) groups, as well as characteristic aromatic ring absorptions (1450 and 716 cm−1). These structural features were also supported by the 1H NMR spectrum of 1, which showed the signals typical for one benzoyl (δ 7.50−8.13, 5 H) and three acetyl groups (δ 1.43, 2.07, and 2.12, Table 1). The spectrum also displayed signals for five methyl groups (two secondary at δ 1.26 and 1.37, two tertiary at δ 0.87 and 1.02, and one vinylic at δ 1.75) and four protons geminal to ester functions, three of them displayed as singlets (δ 4.91, 4.88, and 5.30) and one as a double doublet at δ 4.98 (J = 6.4 and 2.0 Hz). The spectrum also indicated the presence of a trans-disubstituted double bond (δ 5.37, d, J = 16 Hz and δ 6.00, dd, J = 16 and 8.4 Hz) and a trisubstituted one (δ 5.75, d, J = 10.4 Hz). A broad singlet at δ 2.82, without correlation in the HMQC spectrum, confirmed the existence of a free OH group in the molecule. In addition to signals assigned to ester groups, the 13C NMR spectrum displayed 20 carbon resonances discriminated by a DEPT experiment as five methyl groups, one methylene, 10 methines (four oxygenated at δC 69.2, 73.0, 78.4, and 84.3 and three olefinic at δC 117.7, 131.5, and 138.6), and four quaternary carbons, including a carbonyl group at δC 213.6, 1916



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Table 1. NMR Data for Compounds 1−3a euphomelliferine (1) position

δC

1α 1β 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 3-OBz 1′ 2′, 6′ 3′, 5′ 4′ 7-OAc

49.2, CH2

8-OAc 9-OAc

40.7, 84.3, 49.2, 117.7, 136.4, 78.4, 69.2, 73.0, 39.4, 138.6, 131.5, 53.0, 213.6, 92.3, 19.4, 16.1, 23.9, 21.5, 16.3, 165.6, 129.9, 129.8, 128.7, 133.2, 169.2, 20.1, 170.5, 21.2, 169.8, 21.2,

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

δH 1.68, 2.26, 2.54, 4.98, 3.63, 5.75,

dd (13.4, 8.8) dd (13.4, 8.0) m dd (6.4, 2.0) dd (10.4, 6.4) d (10.4)

4.91, s 4.88, s 5.30 s 5.37, d (16.0) 6.00, dd (16.0, 8.4) 3.18, m

1.26, 1.75, 1.02, 0.87, 1.37,

d (7.2) s s s d (7.2)

8.13, dd (8.0, 1.6) 7.50, br t (8.0) 7.55, br t (8.0) 1.43, s 2.07, s 2.12, s

14-OAc 15-OAc 15-OH a

euphomelliferene A (2) δC 43.5, CH2 37.2, 79.5, 48.9, 121.1, 131.4, 77.4, 68.2, 72.2, 39.4, 136.0, 130.8, 38.4, 79.4, 91.1, 13.3, 15.9, 22.4, 20.4, 20.1, 165.0, 130.0, 129.5, 128.5, 132.9, 168.8, 19.4, 170.2, 21.0, 169.2, 20.9, 171.4, 20.8, 169.4, 22.5,

euphomelliferene B (3)

δH

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

2.50, 2.50, 2.12, 5.41, 3.00, 5.68,

δC

m m m t (3.6) dd (9.6, 4.4) d (8.8)

48.5, CH2

4.92, br s 5.00, br s 5.11, br s 5.09, 5.65, 2.58, 4.98,

d (15.6) dd (16.0, 8.4) m d (2.8)

0.94, 1.89, 0.98, 0.87, 0.94,

d (6.4) s s s d (6.4)

7.95, d (7.2) 7.42, t (7.2) 7.52, t (7.2) 1.14, s 2.08, s 2.06, s 2.15, s

37.8, 82.7, 48.4, 124.1, 131.4, 79.2, 69.6, 73.6, 40.4, 135.5, 134.2, 39.9, 82.2, 84.1, 14.0, 16.1, 23.1, 20.9, 20.1, 167.2, 130.9, 129.6, 129.6, 134.2, 170.9, 20.9, 171.8, 20.9, 171.1, 19.9, 173.2, 20.9,

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

δH 2.10, 1.71, 2.19, 5.38, 3.08, 5.79,

dd (12.8, 7.6) t (12.8) br s br s dd (9.6, 4.0) d (9.6)

4.90, s 5.01, s 5.15, s 5.03, 5.92, 2.64, 4.93,

d (14.8) dd (15.6, 8.4) br s s

0.93, 1.83, 0.96, 0.83, 0.93,

m s s s m

8.08, d (7.2) 7.42, t (7.2) 7.59, t (7.2) 1.10, s 2.02, s 2.06, s 2.17, s

2.33, s

2.82, s

Spectra were measured in CDCl3 for compounds 1 and 2 and MeOD for compound 3 (1H 400 MHz, 13C 100.61 MHz; δ in ppm, J in Hz).

Compound 3, named euphomelliferene B, was obtained as a white, amorphous solid and had the molecular formula C35H46O11 as established by HRLSIMS. The IR spectrum had characteristic ester carbonyl and aromatic ring absorptions and an OH absorption band at 3373 cm−1. According to the NMR data, and taking into account the molecular formula, it was evident that compound 3, in comparison with 2, had an OH at C-15 instead of an acetyl group (Table 1). The structure of 3 was confirmed by extensive 2D NMR experiments, which allowed unambiguous assignments of all 1H and 13C signals. The stereochemistry of all tetrahedral centers was found to be identical to those of 2 (Figures S1 and S2, Supporting Information). Compound 6 was obtained as a white, amorphous powder, [α]20 D +2.0. Its molecular formula was determined as C30H50O2 by HRLSIMS, indicating six degrees of unsaturation. The 1H NMR spectrum of 6 displayed signals due to seven methyl groups (five tertiary at δ 0.80, 0.83, 0.86, 1.05, and 1.14, one secondary at δ 0.91, and one vinylic at δ 1.72). The spectrum also indicated the presence of two oxymethine protons at δ 4.01

These structural features were confirmed by the analysis of the 1H−1H-COSY, HMQC, and HMBC spectra, which allowed the unambiguous assignment of all proton and carbon resonances (Figure S1, Supporting Information). The relative configuration of compound 2 was deduced considering the NOESY spectrum and the coupling constant values, as well as by comparison of the NMR data with those of closely related compounds.20 Using the α orientation of H-4 as reference, the strong NOE interactions of this proton with H-2, H-3, H-13, and H-14 were consistent with the presence of β-oriented methyl groups at C-2 and C-13 and β-oriented acyl groups at C-3 and C-14. The stereochemistry of the remaining tetrahedral centers was found to be identical to those of 1 (Figure S2, Supporting Information). Compound 2 exhibited an acylation pattern similar to that of a jatrophane diterpene isolated from E. platyphyllos,20 and it was named euphomelliferene A. However, according to the differences in NMR data, particularly those concerning C-2, C-3, and C-16, it was concluded that these two compounds were epimers at C-2. 1917



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(d, J = 6.0 Hz) and 3.48 (br s) and the vinylic signals of a trisubstituted double bond at δ 5.63 (d, J = 5.6 Hz) and of an exocyclic double bond at δ 4.84 (br s) and 4.92 (br s). The 13C and DEPT NMR spectra showed signals for seven methyl groups, 10 methylenes, seven methines (two oxygenated at δC 76.8 and 76.4 and one sp2 at δC 121.8), and six quaternary carbons (two olefinic at δC 142.0 and 147.5). These data closely resemble those of euferol, a tetracyclic triterpene previously isolated from this genus.14 The major difference was found in the side chain signals due to the presence of an OH group at C24 and a double bond at C-25 in compound 6. The 13C NMR spectrum of 6 showed doubling of certain resonances, consistent with the presence of a mixture of C-24 epimers, although the physical separation of the two diastereomers could not be carried out. The relative configuration of 6 was determined by comparison of the NMR data with those reported in the literature for euferol14 and corroborated through a NOESY experiment. Accordingly to the spectroscopic data, the structure of 6 was undoubtedly assigned as 19(10→9)-abeo-8α,9β,10α-tirucalla-5,25-diene-3β,24-diol. Nine known compounds were also isolated and identified by comparison of their physical and spectroscopic data with those reported in the literature: serrulatin B (4),10 7β,8β,9α,15βtetracetoxy-3β-benzoyloxy-6β-hydroxy-14-oxo-2βH,13αH-jatropha-14E,11E-diene (5),10 20-deoxyingenol (7),21,22 cycloartane triterpenes (8−10),23,24 p-hydroxybenzaldehyde (11),25 astragalin (12),26 and juglalin (13).27 The structures of these compounds are given in the Supporting Information (Figure S3). Jatrophane diterpenes 1−3 and 5 were investigated for their potential ability as MDR modulators. They were first tested on MDR1-gene transfected mouse lymphoma cells (L5178Y MDR) that specifically overexpress P-gp. In order to test the potential clinical application of the compounds, they were also assayed on multidrug-resistant human colon adenocarcinoma cells (COLO 320). Reversion of MDR phenotype was followed by flow cytometry, using a standard functional assay that measures intracellular accumulation of rhodamine-123, a fluorescent substrate analogue of epirubicine. The fluorescence activity ratio (FAR) values were used to evaluate the P-gp modulating potential. In this way, compounds with FAR values higher than 1 were considered to be active as P-gp modulators, and those with FAR values higher than 10 were regarded as strong modulators. 28 The compounds did not exhibit cytotoxicity at concentrations similar to or higher than the highest concentration used in the MDR reversal experiments (data not shown). Verapamil, a calcium channel blocker and chemosensitizer, was applied as a positive control.29 The results for MDR-reversal activity on the two cell lines are summarized in Table 2. On MDR mouse lymphoma cells, compounds were tested in four concentrations (2, 6, 20, and 60 μM). At the highest concentrations 1 and 2 were strong modulators of the efflux-pump activity (FAR = 12.1 and 23.1 at 20 μM, and FAR = 72.9 and 82.2 at 60 μM, respectively) and had a greater activity when compared to that of the positive control verapamil (FAR = 6.6 at 22 μM). Diterpene 5, previously isolated from Euphorbia serrulata and tested on L5178Y MDR cells with similar results,10 also showed good MDR modulating activity at 20 and 60 μM (FAR = 10.1 and 43.4, respectively). On the other hand, compound 3 revealed weak MDR modulating activity at 2, 6, and 20 μM and, however, a strong effect at 60 μM (FAR = 36.1). The results also showed a dose-

Table 2. Effect of Compounds 1−3 and 5 on Reversal of Multidrug Resistance on Human MDR1 Gene-Transfected Mouse Lymphoma Cells (L 5178 Y) and on Human Colon Adenocarcinoma Cells (COLO 320) fluorescence activity ratio (FAR) at different concentrations (μM)a L5178 Y compound 1 2 3 5

2 1.1 1.2 1.1 0.9

6 2.9 12.4 1.4 1.4

20 12.1 23.1 1.6 10.1

COLO 320 60 72.9 82.2 36.1 43.4

2 1.1 2.0 0.8 0.7

20 5.1 5.5 2.8 3.0

The FAR values of the positive control verapamil (22 μM) were 6.6 and 4.0 for L 5178 Y and COLO320 cells, respectively. a

dependent inhibitory effect of P-gp-mediated efflux for diterpenes 1, 2, and 5 (Table 2). On the COLO 320 cell line, the compounds were tested at two concentrations (2 and 20 μM). Compounds 1−3 and 5 exhibited FAR values of 1.1, 2.0, 0.8, and 0.7, respectively, at 2 μM. When tested at 20 μM, compounds 1 (FAR = 5.1), 2 (FAR = 5.5), and 3 (FAR = 3.0) showed P-gp modulatory activity similar to that of the positive control (Table 2). It is interesting to note that 1 and 2 were the most effective in elevating rhodamine-123 accumulation in both cell lines; however, the FAR values observed in COLO320 cells were lower than those obtained for the mouse lymphoma cells. The differences in the observed modulating effects between the two MDR cell lines may be associated with different levels of P-gp expression, which are lower in COLO 320 cells according to immunohistological studies.30 The tested diterpenes have several structural differences, namely, in the type of substitution at C-6, C-14, and C-15, the position of the double bonds, and the stereochemistry at C-2. The presence of two acetyl groups at C-14 and C-15 appears to play an important role in the MDR reversal activity, since 2 showed the highest P-gp modulating activity on both cell lines at 20 μM. In fact, a marked decrease of activity was observed in 3 that has an OH group at C-15. However, when comparing the effects of 1 and 3, which differ in the type of function at C14 and in the configuration at C-2, it was interesting to note that the presence of a carbonyl group at this position improves the activity, as was observed for 1. The different location of one of the double bonds and the substitution at C-6 also influenced the efflux pump activity, as demonstrated by the FAR values of 1 and 5 on the two cell lines. In this experiment, the configuration at C-2 did not seem to play a significant role in MDR modulatory activity, as was reported earlier for some jatrophane diterpenes.16 Another way to overcome MDR could be to discover new drugs able to modulate the expression of molecules involved in the apoptotic pathway and capable of inducing specific apoptosis for malignant tumor cells. Thus, it was considered important to clarify the role of the new isolated compounds as apoptosis inducers. Diterpenes 1−3 and 5 were evaluated on the COLO 320 cell line, using the annexin-V/propidium iodide assay described in the Experimental Section. An apoptosis inducer, 12H-benzo[α]phenothiazine, was used as the positive control. The compounds were tested at 20 μM, and the results are summarized in Table 3. None of the tested compounds were able to induce significant apoptosis and cell death. 1918



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Table 3. Effect of Compounds 1−3 and 5 (20 μM) on Apoptosis Induction on Human Colon Adenocarcinoma Cells (COLO 320)a A−PI− A−PI+ A+PI− A+PI+ M-627b 1 2 3 5

% early apoptosis

% total apoptosis

% cell death

0 0.01 11.65 2.24 17.55 6.72 5.39 3.00 4.40

0 0.01 0.18 1.71 58.56 4.97 7.70 4.93 4.60

0.40 8.17 0.02 0.19 3.24 0.74 1.11 1.11 0.90

as eluents. According to TLC differences, 12 crude fractions were obtained (A−L). Fraction C (3.65 g, n-hexane/EtOAc, 17:3 to 3:1) was subjected to CC (SiO2, 230 g) using n-hexane/EtOAc (1:0 to 0:1); after TLC monitoring, the fractions with similar profiles were combined. Fraction C.3, eluted with n-hexane/EtOAc (4:1), was repeatedly fractionated by CC using mixtures of n-hexane/CH2Cl2/EtOAc of increasing polarity to afford 8 (34 mg). Further purification by HPLC (MeOH/H2O, 9:1; 254 nm, 4 mL/min) yielded 6 (11 mg) and 10 (8 mg). Fraction C.4, eluted with n-hexane/EtOAc (3:1), was subjected to CC and recrystallized (EtOAc/n-hexane) to give 9 (16 mg). Mother liquors were further purified by CC, yielding 1 (108 mg). Fraction D (4.21 g, n-hexane/EtOAc, 3:1 to 7:3) was chromatographed on SiO2 (230 g) using n-hexane/EtOAc (1:0 to 0:1) to give seven subfractions. Fraction D.3, eluted with n-hexane/EtOAc (3:1), was separated by CC and preparative TLC (CHCl3/MeOH, 19:1), affording 11 (13 mg). Fraction D.4, eluted with n-hexane/EtOAc (7:3), was subjected to repeated CC and preparative TLC to yield 7 (10 mg), and a residue with a strong absorption at 254 nm was further purified by HPLC (MeOH/H2O, 7:3; 254 nm, 4 mL/min), affording 4 (20 mg), 2 (7 mg), and 3 (12 mg). Fraction D.5 was purified by CC and preparative TLC (CHCl3/MeOH, 19:1) to give 5 (15.4 mg). Fraction I (2.87 g) was chromatographed using mixtures of CH2Cl2/EtOAc and EtOAc/MeOH of increasing polarities. Fractions eluted with CH2Cl2/EtOAc (1:9) were recrystallized from MeOH/ EtOAc to give 12 (1.38 g). Fraction H (5.3 g) was subject to CC, and fractions eluted with CH2Cl2/EtOAc (1:9) were purified by recrystallization (EtOAc) to afford 13 (207 mg). Euphomelliferine (1), 3β-benzoyloxy-15β-hydroxy-7β,8β,9α-triacetoxy-14-oxo-2βH,13αH-jatropha-5E,11E-diene: white, amorphous solid; [α]20 D +7.0 (c 0.10, CHCl3); IR (CH2Cl2) νmax 3532, 2966, 1741, 1450, 1270, 716 cm−1; 1H NMR and 13C NMR (see Table 1); ESIMS m/z 621 [M + Na]+; HRLSIMS m/z 621.2663 [M + Na]+ (calcd for C33H42O10Na: 621.2675). Euphomelliferene A (2), 3β-benzoyloxy-7β,8β,9α,14β,15β-pentacetoxy-2αH,13αH-jatropha-5E,11E-diene): white, amorphous solid; [α]20 D −7.0 (c 0.10, CHCl3); IR (CH2Cl2) νmax 2972, 1740, 1449, 1237, 736, 712 cm−1; 1H NMR and 13C NMR (see Table 1); ESIMS m/z 707 [M + Na]+; HRLSIMS m/z 707.3021 [M + Na]+ (calcd for C37H48O12Na: 707.3043). Euphomelliferene B (3), 3β-benzoyloxy-15β-hydroxy7β,8β,9α,14β-tetracetoxy-2αH,13αH-jatropha-5E,11E-diene: white, amorphous solid; [α]20 D −18.0 (c 0.12, MeOH); IR (CH2Cl2) νmax 3373, 3045, 1746, 1595, 810, 752 cm−1; 1H NMR and 13C NMR (see Table 1); ESIMS m/z 665 [M + Na]+; HRLSIMS m/z 665.2928 [M + Na]+ (calcd for C35H46O11Na: 665.2937). 19(10→9)-abeo-8α,9β,10α-tirucalla-5,25-diene-3β,24-diol (6): white, amorphous solid; [α]20 D −2.0 (c 0.17, CHCl3); IR (CH2Cl2) νmax 3380, 2936, 2870, 1451, 1371, 1027, 897, 755 cm−1; 1H NMR (CDCl3, 400 MHz) δ 5.63 (1 H, d, J = 5.6 Hz, H-6), 4.92 (1 H, br s, H-26a), 4.84 (1 H, br s, H-26b), 4.01 (1 H, t, J = 6,4 Hz, H-24), 3.48 (1 H, br s, H-3), 1.72 (3H, s, H-27), 1.14 (3H, s, H-30), 1.05 (3H, s, H-29), 0.91 (3H, d, J = 6,0 Hz, H-21), 0.86 (3H, s, H-28), 0.83 (3H, s, H-19), 0.80 (3H, s, H-18); 13C NMR (CDCl3, 100.61 MHz) δ 147.8/ 147.5 (C, C-25), 142.0 (C, C-5), 121.8 (CH, C-6), 111.5/111.0 (CH2, C-26), 76.7 (CH, C-3), 76.4/76.3 (CH, C-24), 50.4 (CH, C-17), 49.4 (CH, C-10), 47.4 (C, C-14), 46.1 (C, C-13), 44.5 (CH, C-8), 40.9 (C, C-4), 35.8 (CH, C-20), 35.7 (CH2, C-11), 35.1 (C, C-9), 34.2 (CH2, C-15), 32.0/31.9 (CH2, C-23), 31.4 (CH2, C-22), 30.2 (CH2, C-12), 29.0 (CH3, C-29), 28.2 (CH2, C-16), 27.9 (CH2, C-2), 25.6 (CH3,C30), 25.2 (CH2, C-7), 18.9 (CH3, C-28), 18.8 (CH2, C-1), 18.7 (CH3, C-21), 17.6/17.2 (CH3,C-27), 16.5 (CH3, C-19), 15.2 (CH3, C-18); EIMS m/z 442 [M]+ (2), 290 (27), 275 (10), 163 (59), 161 (32), 152 (14), 134 (79); FABMS m/z 465 [M + Na]+; HRLSIMS m/z 465.3696 (calcd for C30H50O2Na: 465.3708). Cells and Cell Culture. The L5178Y mouse T-lymphoma cells (ECACC cat. no. 87111908, U.S. FDA, Silver Spring, MD, USA) were transfected with the pHa MDR1/A. The MDR1-expressing cell line was selected by culturing the infected cells with 60 ng/mL of colchicine to maintain the expression of the MDR phenotype. L5178Y

a

Control: A+: annexin V-FITC staining; A−: without annexin VFITC; PI+: propidium iodide staining; PI−: without propidium iodide. b Positive control: 12H-benzo(α)-phenothiazine (200 μM).

In conclusion, multidrug resistance mediated by P-gp has been extensively associated with its drug efflux activity. According to the results presented herein, the new jatrophane diterpenes 1 and 2 were able to successfully modulate P-gp efflux activity by reversing the MDR phenotype on both multidrug-resistant cell lines that overexpress this efflux pump. The induction of apoptosis through the modulation of this efflux pump could not be observed. Nevertheless, these macrocyclic diterpenes are promising lead compounds for the development of effective P-gp modulators.

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EXPERIMENTAL SECTION

General Experimental Procedures. Melting points were determined on a Köpffler apparatus and are uncorrected. Optical rotations were obtained using a Perkin-Elmer 241 polarimeter, with quartz cells of 1 dm path length; the samples were solubilized in CHCl3 or MeOH. IR spectra were determined on a Nicolet Impact 400 FTIR, and NMR spectra recorded on a Bruker ARX-400 NMR spectrometer (1H 400 MHz; 13C 100.61 MHz), using CDCl3, MeOD, or DMSO as solvents. Low-resolution mass spectra were taken on a Kratos MS25RF spectrometer (70 eV) and on a Micromass Quattro micro API (ESIMS); high-resolution mass spectra were recorded on a Micromass Autospec spectrometer. Column chromatography (CC) was carried out on SiO2 (Merck 9385). TLC was performed on precoated SiO2 F254 plates (Merck 5554 and 5744) and visualized under UV light and by spraying with H2SO4−AcOH−H2O (1:20:4) or H2SO4−H2O (1:1) followed by heating. HPLC was carried out on a Merck-Hitachi instrument, with UV detection, and a Merck-Hitachi D7500 integrator. Analytical HPLC was performed using a Merck LiChrospher 100 RP-18 (5 μm, 125 × 4 mm) column. Semipreparative HPLC was performed on a Merck LiChrospher 100 RP-18 (10 μm, 250 × 10 mm) column. Mixtures of MeOH/H2O and MeCN/H2O were used as eluents. Plant Material. Euphorbia mellifera (Euphorbiaceae) was collected in the Garcia d’Orta Garden, Lisbon, Portugal, and identified by Dr. Teresa Vasconcelos of Instituto Superior de Agronomia (ISA), University of Lisbon. A voucher specimen (no. 281) has been deposited at the herbarium of ISA. Extraction and Isolation. The air-dried aerial parts (2.5 kg) were repeatedly extracted with MeOH (11 × 11.5 L) at room temperature. The pooled extracts were evaporated under vacuum, and the concentrated extract was suspended in EtOH (2.7 L) and treated with an equal volume of 3% Pb(OAc)2. After 4 h, the suspension was filtered twice on a bed of Celite; the clear filtrate was concentrated under vacuum to remove most of the EtOH and then exhaustively extracted with EtOAc (16 L). After washing with brine, drying with Na2SO4, and evaporation to dryness, 44 g of a crude extract was obtained. This extract was chromatographed over SiO2 (750 g) using mixtures of n-hexane/EtOAc and EtOAc/MeOH of increasing polarity 1919



Article

(parental, PAR) mouse T-cell lymphoma cells and the human MDR1transfected subline (MDR) were cultured in McCoy’s 5A medium supplemented with 10% heat-inactivated horse serum, L-glutamine, and antibiotics. The cell lines were incubated in a humidified atmosphere (5% CO2, 95% air) at 37 °C. The human colon adenocarcinoma COLO 205 and COLO 320 MDR cell lines were cultured in RPMI 1640 medium supplemented with 10% heatinactivated fetal bovine serum, 2 mM L-glutamine, 1 mM Na-pyruvate, and 100 mM HEPES. The cell lines were incubated in a humidified atmosphere (5% CO2, 95% air) at 37 °C. The semiadherent human colon cancer cells were detached with 0.25% trypsin and 0.02% EDTA for 5 min at 37 °C. Antiproliferative Assay. Antiproliferative effects of the compounds were tested in a range of increasing concentrations (0.26−133 μM), using L5178Y mouse T-lymphoma and COLO 320 MDR cell lines as experimental models. The cells were distributed into 96-well flat bottom microtiter plates at a concentration of 1 × 105/mL with a final volume of 100 μL of medium per well. The different concentrations of each compound were added into duplicate wells. The plates were incubated at 37 °C for 72 h; at the end of the incubation period, 15 μL of MTT solution (from a 5 mg/mL stock) was added to each well and incubated for 4 h. Then, 100 μL of SDS (10%) solution was measured into each well, and the plates were further incubated overnight at 37 °C. The cell growth was determined by measuring the optical density (OD) at 550 nm (ref 630 nm) with a Dynatech MRX vertical beam ELISA reader. The percentage of inhibition of cell growth was determined according to the following equation:

temperature, the supernatant was removed, and the cells were resuspended in PBS. After this procedure, the apoptosis assay was carried out according to the rapid protocol of the kit. The fluorescence was analyzed immediately using a Becton Dickinson Facstar flow cytometer.

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S Supporting Information *

1 H−1H-COSY and selected HMBC correlations for 1−3; relevant NOESY correlations for 1−3; chemical structures of known compounds (7−13); 1D and 2D NMR spectra of compounds 1−3 and 6; flow cytometry data for 2 on human colon adenocarcinoma cells (COLO 320) and MDR L5178Y mouse T-lymphoma cells, at 2 and 20 μM concentrations, respectively. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*Tel: +351217946475. Fax: +351217946470. E-mail: mjuferreira@ff.ul.pt. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This study was supported by FCT (Fundaçaõ para a Ciência e a Tecnologia, Portugal), project PTDC/QUI-QUI/099815/ 2008. M.R. acknowledges FCT for her Ph.D. grant (SFRH/ BD/72915/2010).

⎡ ODsample − ODmedium control ⎤ 100 − ⎢ ⎥ × 100 ⎣ ODcells control − ODmedium control ⎦

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Assay for Rhodamine-123 Accumulation. The COLO 205 and COLO 320 MDR cells were adjusted to a density of 1 × 106/mL, resuspended in serum-free RPMI 1640 medium, and distributed in 500 μL aliquots. The PAR and MDR mouse lymphoma cells were adjusted to a density of 2 × 106/mL, resuspended in serum-free McCoy’s 5A medium, and distributed in 500 μL aliquots. The test compounds were added at 2 and 20 μM, verapamil (positive control) was added at 22 μM, and DMSO was added at 2% as solvent control. The samples were incubated for 10 min at room temperature, and then 10 μL (5.2 μM final concentration) of rhodamine-123 was added to the samples, followed by an incubation of 20 min at 37 °C. Finally, the samples were washed twice, resuspended in 500 μL of phosphate-buffered saline (PBS), and analyzed on a Becton Dickinson and Company FACScan flow cytometer (Franklin Lakes, USA). The histograms were evaluated regarding the mean fluorescence intensity, the standard deviation, and the peak channel of 10 000 individual cells belonging to the total and the gated populations. The fluorescence activity ratio was calculated on the basis of the measured fluorescence values (FL-1) of treated/untreated resistant cell line (MDR) and the sensitive cell line (PAR). FAR =

ASSOCIATED CONTENT

REFERENCES

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FL‐1MD treated /FL‐1MD untreated FL‐1PA treated /FL‐1PA untreated

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Jatrophane Diterpenes from Euphorbia mellifera and Their Activity as

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