Epoxylathyrol Derivatives: Modulation of ABCB1-Mediated Multidrug

Article pubs.acs.org/jnp

Epoxylathyrol Derivatives: Modulation of ABCB1-Mediated Multidrug Resistance in Human Colon Adenocarcinoma and Mouse T‑Lymphoma Cells Ana M. Matos,† Mariana Reis,† Noélia Duarte,† Gabriella Spengler,‡ Joseph Molnár,‡ and Maria-José U. Ferreira*,† †

Downloaded by RUTGERS UNIV on September 3, 2015 | http://pubs.acs.org Publication Date (Web): September 2, 2015 | doi: 10.1021/acs.jnatprod.5b00370

Research Institute for Medicines (iMed.ULisboa), Faculdade de Farmácia, Universidade de Lisboa, 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: Epoxyboetirane A (1), a macrocyclic diterpene that was found to be inactive as an ABCB1 modulator, was submitted to several chemical transformations, aimed at generating a series of compounds with improved multidrug resistance (MDR)-modifying activity. Overall, 23 new derivatives were prepared, in addition to the already reported epoxylathyrol (2) and methoxyboetirol (3). Their anti-MDR potential was assessed through both functional and chemosensitivity assays on resistant human colon adenocarcinoma and human ABCB1-gene transfected L5178Y mouse lymphoma cells. Structure−activity relationship analysis showed that different substitution patterns led to distinct ABCB1 inhibitory activities, although intrinsic cellular characteristics seemed to influence the modulatory behavior. A considerable enhancement in MDR-modifying activity was observed for aromatic compounds in both cell lines, particularly in 3,17-disubstituted esters derived from 3, a Payne-rearranged Michael adduct of 2. All compounds tested were revealed to interact synergistically with doxorubicin, and ATPase inhibition by three representative MDR-modifying compounds was also investigated. On account of its outstanding ABCB1 inhibitory activity at 0.2 μM and overall remarkable bioactive profile, methoxyboetirane B (22) was found to be a new promising lead for MDR-reversing anticancer drug development.

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membrane efflux-pump proteins belonging to the ABC transporters.3,4 A promising strategy for MDR reversal is the concomitant administration of anticancer drugs with nontoxic molecules able to effectively inhibit ABCB1-mediated efflux, ultimately restoring the cytotoxic concentration of the active agents in cancer cells.5 Indeed, several compounds have been reported for their MDR-modifying ability. However, none of them have been approved for clinical use so far.2 Jatrophane and lathyrane-type macrocyclic diterpenes isolated from Euphorbia species have shown to be potent inhibitors of the ABCB1 efflux-pump activity.6−10 However, despite the already existing work on this topic, there is still a path to be followed for optimizing plant-derived macrocyclic diterpenes as ABCB1 modulators. Thus, the present investigation focused on the development of a small library of lathyrane diterpenes with an epoxylathyrol skeleton through

ultidrug resistance (MDR) to chemotherapy remains a major challenge in the treatment of infectious diseases and cancer. One of the most commonly found and best known MDR mechanisms in cancer is the overexpression of the ABCB1 gene-encoded product, P-glycoprotein (P-gp, ABCB1), which belongs to the family of energy-dependent membrane efflux pumps known as ATP-binding cassette (ABC) transporters. When overexpressed by cancer cells, P-gp enhances the efflux rate of anticancer drugs, thus decreasing their intracellular concentration to a level below which they are unable to kill tumor cells. Owing to its unusual broad substrate specificity, ABCB1 overexpression has evolved as one of the major MDR mechanisms, and, therefore, it is considered currently an important molecular target in the reversal of MDR in cancer patients.1,2 The existence of transporter proteins homologous to ABCB1 in organisms ranging from prokaryotes to eukaryotes supports drug efflux as a general mechanism of MDR. This is the case of fungal infections, where one of the most important mechanisms of antifungal MDR is due to the overexpression of © XXXX American Chemical Society and American Society of Pharmacognosy

Received: April 28, 2015

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DOI: 10.1021/acs.jnatprod.5b00370 J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products

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Scheme 1. Preparation of Epoxyboetirane A (1) Derivatives (2−6 and 21−26)a

a Reagents and conditions: (i) 5% KOH in MeOH (m/v), rt, 3 h, 65% (2) and 13% (3); (ii) 5% KOH in EtOH (m/v), rt, 6 h, 45% (2), 11% (4); (iii) LiAlH4, THF, 0 °C, 1 h, then 10% NaOH(aq) (m/v), 4% (5) and 44% (6); (iv) acyl chloride or anhydride, DMAP (cat.), TEA/CH2Cl2 (1:1), rt, 6−24 h; (v) acyl chloride or anhydride DMAP (cat.), TEA/CH2Cl2 (1:1), rt, 48 h.

besides epoxylathyrol (2), this reaction led to a new rearranged adduct, named ethoxyboetirol (4), which resulted from an αketol rearrangement that has also been found to occur naturally in other lathyrane- and jatrophane-modified diterpenes.13,14 In addition, the compound underwent again a base-catalyzed migration of the epoxy function, followed by opening at C-5 by the nucleophilic attack of one deprotonated molecule of ethanol. Ethoxyboetirol (4), reported herein for the first time, displayed an 1H NMR spectrum that was consistent with the newly added ethoxy group, which appeared as a triplet at δH 1.22 (J = 7.0 Hz) and two multiplets at δH 3.69 and 3.84 due to the diasteriotopic protons of the methylene group. Moreover, when comparing the spectrum to that of epoxylathyrol (2), H-4 was shifted downfield (from δH 1.80 in 2 to δH 3.70 in 4), which was indicative of an α-ketol rearrangement.13,14 Regarding the 13C NMR spectrum, the carbonyl function at C-15 was displayed at δC 210.8 and C-12 was shifted significantly upfield (δC 133.5), confirming the absence of the α,β-unsaturated carbonyl present in epoxylathyrol (2). The analysis of the NOESY spectrum confirmed the predicted relative configuration at C-6 (Figure S1, Supporting Informa-

molecular derivatization, directed toward the establishment of structure−activity relationships regarding their MDR-modifying activity. The effect of the introduced chemical transformations in the structure of the original scaffold of epoxyboetirane A (1) was evaluated in vitro through the rhodamine-123 accumulation assay on human colon adenocarcinoma and mouse lymphoma cells and corresponding MDR sublines, in addition to a series of cytotoxicity, drug combination, and ATPase activity assays.

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RESULTS AND DISCUSSION Chemistry. Epoxyboetirane A (1), isolated from Euphorbia boetica Boiss. (Euphorbiacae),11 initially was hydrolyzed in a methanolic solution of potassium hydroxide, affording epoxylathyrol (2) as the major reaction product and a modest yield of a Payne-rearranged Michael adduct, named methoxyboetirol (3) (Scheme 1, i). Both compounds were identified by comparison of their 1H NMR and 13C NMR spectra with those reported in the literature.12 In order to obtain a methoxyboetirol (3) analogue having an ethoxy group at C12, epoxyboetirane A (1) was hydrolyzed using an ethanolic solution of potassium hydroxide (Scheme 1, ii). However, B



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Scheme 2. Preparation of Epoxylathyrol (2) Derivatives (7−20)a

a Reagents and conditions: (i) acyl chloride, DMAP (cat.), TEA/CH2Cl2 (1:1), rt, 2 h or 50 °C, 4 d (12); (ii) acyl chloride, DMAP (cat.), TEA/ CH2Cl2 (1:1), 50 °C, 7 d; (iii) carbamoyl chloride, DMAP (cat.), TEA/CH2Cl2 (1:1), 50 °C, 5 h−3 d.

tion); however, the relative configuration at C-14 could not be attributed. Reduction of epoxyboetirane A (1) with LiAlH4 afforded two new epimeric products at C-14: pentoxyboetirol A (5), in a very small amount, and pentoxyboetirol B (6) as the major product (Scheme 1, iii), indicating that the α-face of the carbonyl is much more accessible to the reagent than the β-face. In both compounds, the 1H NMR spectra displayed, as expected, two new signals corresponding to the proton geminal to the hydroxy group at C-14 (δH 4.37 and 4.08, for 5 and 6, respectively) and to the C-17 methyl group (δH 1.25 and 1.22 for 5 and 6, respectively), and the 13C NMR spectra showed resonances at δC 85.4 and 78.1 for C-14. An upfield shift from δC 54.6 to δC 25.2/24.2 was also observed for C-17, corroborating the opening of the epoxy function. These two epimers were then distinguished in terms of their relative stereochemistry through NOESY experiments (Figures S2 and S3, Supporting Information). Having obtained sufficiently large amounts of epoxylathyrol (2), methoxyboetirol (3), and pentoxyboetirol B (6), several subsequent acylation reactions were performed using a set of different anhydrides and acyl chlorides (Schemes 1 and 2). Overall, 20 new ester and carbamate derivatives were prepared in this study.

Compounds 7−15 (epoxyboetiranes K−S) exhibited very similar 1H NMR data when compared with those of epoxylathyrol (2). The most significant differences were generally related to protons H-3 and H-5 and their corresponding carbons due to paramagnetic effects (max. ΔδH‑3 = +1.61 ppm, max. ΔδC‑3 = +3.1 ppm; max. ΔδH‑5 = +2.05 ppm, max. ΔδC‑5 = +6.2 ppm). Overall, the β-carbons C4 and C-6 also showed diamagnetic effects (max. ΔδC‑4 = −2.8 ppm; max. ΔδC‑6 = −1.7 ppm), and the γ-carbons C-1 and C-7 were shifted downfield (max. ΔδC‑1 = +0.9 ppm; max. ΔδC‑7 = +2.3 ppm), similarly to the α-carbons. In contrast, the 1H NMR spectra of boetiranes A (16) and B (17) showed a second-order AB system corresponding to both H-17 diasteriotopic protons at a much lower field when compared to the remaining epoxyboetiranes K−S (7−15). This refers to the hydrolysis of the 6,17-epoxy ring and was additionally supported by the lower field 13C NMR resonances of carbons C-5, C-6, and C17. Both compounds underwent the earlier mentioned Payne rearrangement, giving rise to a new primary alkoxy ion at position C-17, which was highly susceptible to further acylation. Moreover, the newly formed 5,6-epoxy function most likely suffered a nucleophilic attack by a hydroxide ion at C-5, resulting in inversion of the configuration at C-6. HMBC and NOESY experiments confirmed the proposed structure and the C



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Table 1. MDR-Reversal Activity of Compounds 2, 7−15, and 18−20 on ABCB1-Transfected L5178Y Mouse Lymphoma (L5178Y) and Multidrug-Resistant Human Colon Adenocarcinoma Cells (Colo 320)a

Verapamil 20 μM (positive control): FAR(ABCB1-transfected L5178Y cells) = 17.72, FAR(Colo 320 cells) = 8.95; DMSO 2% (negative control): FAR(ABCB1-transfected L5178Y cells) = 0.78, FAR(Colo 320 cells) = 0.62. a

The 1H and 13C NMR spectroscopic data of epoxycarbamoylboetiranes A−C (18−20) clearly resembled those of

relative stereochemistry of this type of compound, respectively (Figure S4, Supporting Information). D



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Table 2. MDR-Reversal Activity of Compounds 3, 5, 6, and 21−26 on ABCB1-Transfected L5178Y Mouse Lymphoma (L5178Y) and Multidrug-Resistant Human Colon Adenocarcinoma Cells (Colo 320)a

Verapamil 20 μM (positive control): FAR(ABCB1-transfected L5178Y cells) = 17.72, FAR(Colo 320 cells) = 8.95; DMSO 2% (negative control): FAR(ABCB1-transfected L5178Y cells) = 0.78, FAR(Colo 320 cells) = 0.62. a

also measured in these cancer cell lines, but they were not significant (Table S2, Supporting Information). The lowest IC50 value obtained was 22.5 ± 1.61 μM (12). Some of the most active compounds, which were also found to be strong ABCB1 modulators, were additionally tested on mouse embryonic fibroblasts, a noncancerous cell experimental model, showing no significant cytotoxicity. The rhodamine-123 accumulation assay was performed with compounds 2−26 on both the parental human colon adenocarcinoma and mouse T-lymphoma cell lines and corresponding MDR sublines in order to investigate their activity as ABCB1 modulators. The fluorescence activity ratio (FAR) values, used to evaluate the ABCB1 modulating potential, were calculated through the equation given in Section 4.2. For human ABCB1-gene-transfected mouse lymphoma cells, where ABCB1 is highly expressed, compounds with FAR values higher than 1.0 were considered active as ABCB1 modulators, and those with FAR values higher than 10 were regarded as strong modulators. Concerning Colo 320 MDR cells, it should be noted that ABCB1 expression is much

compounds 7−15. The structures of methoxyboetiranes A−C (21−23) and pentoxyboetiranes B1−B3 (24−26) were similarly elucidated based on the spectroscopic data of the original lathyrane diterpenesmethoxyboetirol (3) and pentoxyboetirol B (6), respectivelycomplemented by HMQC and HMBC spectra. Biological Assays. The antiproliferative activities of compounds 2−26 were evaluated on human colon adenocarcinoma cell lines (sensitive Colo 205 and Colo 320 MDR cells) and on sensitive (L5178Y PAR) and human ABCB1-genetransfected mouse lymphoma cells (L5178Y MDR cells), using the thiazolyl blue tetrazolium bromide (MTT) assay. When comparing the IC50 values presented in Table S1 (Supporting Information), it is clear that this set of derivatives possesses different cell growth inhibitory activities depending on the cell line evaluated. Significant effects were observed on human colon adenocarcinoma cells for derivatives 22 (IC50 = 5.2 ± 2.5 μM and 6.9 ± 2.7 μM on sensitive and resistant cells, respectively) and 23 (IC50 = 8.2 ± 3.6 μM and 9.3 ± 3.2 μM on sensitive and resistant cells, respectively). Cytotoxic effects were E



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Table 3. MDR-Reversal Activity of Compounds 4, 16, and 17 on ABCB1-Transfected L5178Y Mouse Lymphoma (L5178Y) and Multidrug-Resistant Human Colon Adenocarcinoma Cells (Colo 320)a

a Verapamil 20 μM (positive control): FAR(ABCB1-transfected L5178Y cells) = 17.72, FAR(Colo 320 cells) = 8.95; DMSO 2% (negative control): FAR(ABCB1-transfected L5178Y cells) = 0.78, FAR(Colo 320 cells) = 0.62.

In order to find a correlation between the ABCB1 modulatory activity and some general physicochemical properties, four molecular descriptors were calculated for compounds 2−26: molecular weight (MW), molecular volume (MV), topological polar surface area (TPSA), and logarithm of the octanol/water partition coefficient (log P). Structure−activity relationship studies were conducted considering FAR values obtained at 2 μM. The results illustrated the occurrence of a preferential MW (558.6−726.71 g/mol) and lipophilicity (c log P 5.30−6.59) for optimal activity (Table S3, Supporting Information). However, the effect of lipophilicity was clearly dependent upon the intrinsic cellular characteristics, with Colo 320 cells requiring slightly less lipophilic modulators than L5178Y mouse lymphoma cells in order to achieve full MDR phenotype reversion. Moreover, an increased TPSA lowered ABCB1-reversal activity, while a medium-range MV (496.78− 566.44 Å3) seemed to be beneficial. Some exceptions to all general tendencies, however, disclosed the importance of ideal structural features. As expected, different substituents and acylation patterns led to distinct ABCB1 inhibitory activities, although some divergences between the results obtained for the two cancer cell lines indicated that intrinsic cellular characteristics might also play a key role in the ability of these compounds to reverse the MDR phenotype. All acylated compounds were unquestionably more active than their corresponding parent compounds, thus corroborating previous reports.11,15 As referred to above, compounds bearing aromatic groups have shown to be extremely effective, contrasting with boetirane A (16), methoxyboetirane A (21), and epoxycarbamoylboetiranes A−C (18−20), which, indeed, clearly failed to deliver satisfactory results. When comparing the activities of epoxyboetiranes K (7) and L (8) (Table 1), a 2-fold decrease of activity was observed for compound 7 on both cell lines (FAR = 22.82 on L5178Y cells; FAR = 2.70 on Colo 320 cells) probably due to steric hindrance caused by both biphenyl ester groups. In fact, this compound was the one with the highest MV (658.1) in the set of

lower on human Colo 320 MDR cells, and thus the FAR values are not comparable. Compounds were tested at 20 μM, at 2 μM, and, in some cases (8−10, 12, 17, 22, 23, 26), at 0.2 μM. Verapamil was used as a positive control (20 μM). The results are summarized in the form of sets of related derivatives (Tables 1−3). Covered are (a) the epoxyboetirane and epoxycarbamoylboetirane groups (2, 7−15, and 18−20), which are aliphatic or aromatic esters and carbamates obtained through acylation of epoxylathyrol (2, Table 1); (b) the methoxyboetirane group (21−23), which are esters of methoxyboetirol (3) (Table 2); (c) the pentoxyboetirane group (24−26), esters of pentoxyboetirol B (6, Table 2); and (d) ethoxyboetirol (4) and boetiranes A (16) and B (17) (Table 3). In contrast to epoxyboetirane A (1; FAR 1.0 at 20 μM)11 and the parent polyhydroxylated diterpenes (2, 3, 5, 6) that were inactive or barely active, when tested at 20 μM most of the ester derivatives were shown to be very strong modulators, acting in a concentration-dependent manner (Tables 1 and 2). The most active derivatives were those with aromatic moieties (7−14, 17, 22, 23, 25, 26) that exhibited FAR values ranging from 30.97 (7) to 83.29 (9) for L5178Y MDR cells and from 2.64 (7) to 15.40 (10) for Colo 320 cells (verapamil, FAR = 17.72 and 8.95 at 20 μM on L5178Y and Colo 320 cells, respectively). Moreover, at 2 μM, compounds 7−11, 14, 22, 23, and 26 were still extremely active (FAR 22.82−86.39 for L5178Y cells; FAR 2.70−9.85 for Colo 320 cells). In particular, epoxyboetiranes L and N (8, 10) and methoxyboetirane B (22) exhibited a demonstrable MDR-modulating activity at 0.2 μM, notwithstanding the 100-fold decrease in their concentration level (FAR 13.67 and 11.9 on L5178Y cells and 2.54 and 6.34 on Colo 320 cells, respectively) (Tables 1 and 2; Figures S5−S16, Supporting Information). The carbamoyl derivatives (18−20) revealed weak MDRmodulating activity for both cell lines even at the highest concentration used (FAR 1.38−1.61 on L5178Y cells; FAR 1.42−1.55 on Colo 320 cells). F




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compounds tested (2−26). It also has the highest log P value (9.88). Comparison of the activity of compounds sharing benzoyl moieties within the set of epoxyboetirane derivatives (9, 10, 11, 12; Table 1) suggested that the substitution pattern of the benzene rings influences the activity. This effect, which depends on the cell line, seems to be mostly related with the position (meta/para) of the substituent, independently of its nature, suggesting it is due to steric hindrance. In fact, for mouse lymphoma cells, at 2 μM, compounds 11 (FAR = 38.72) and 12 (FAR = 8.03), bearing the strong electron-withdrawing trifluoromethyl (CF3−) group, and the electron-donating amino function, respectively, at the para position, were less active than compounds 9 (FAR = 86.39) and 10 (FAR = 60.62), which are both meta-substituted with electron-donating groups (CH 3 − and CH 3 O−, respectively). The parasubstituted benzoyl moieties are longer than the metasubstituted ones, which might cause a steric hindrance similar to that suggested for compound 7, bearing biphenyl groups. Comparable conclusions could be deduced for the methoxyboetirol derivatives, 23 (CF3− meta-substituted; FAR = 39.30) and 22 (without substituents; FAR = 67.06) (Table 2). Epoxyboetiranes Q (13) and R (14) are an unusual homologous pair of epoxylathyrol (2) derivatives, since they each result from a bioisosteric replacement of an oxygen with a sulfur atom at the five-membered aromatic ester function. This difference was enough for a decrease in the ability of epoxyboetirane Q (13) to modulate ABCB1-mediated efflux on both cell lines (the FAR values dropped from 31.06 to 6.09 for L5178Y cells and from 8.44 to 1.67 for Colo 320 cells). Pentoxyboetirane B1 (24, FAR 11.37 and 2.76) does not possess any aromatic group, and, still, it was not the least active compound within the set of pentoxyboetirol B (6) derivatives. In fact, this may be considered as a good ABCB1 modulator, and, since it is acylated at several key positions of the parental scaffold, this may be sufficient to elicit activity. When compared with pentoxyboetirane B2 (25), pentoxyboetirane B3 (26) was found to be more active. It has an additional benzoyl group at position C-14, thus suggesting that the acylation pattern at C-3 and C-14 may enhance ABCB1-binding interactions when the ester groups include aromatic rings. Altogether, this study points toward aromatic moieties as being fundamental features of the ABCB1 pharmacophore, leading to a relevant enhancement in MDR-modifying activity of these lathyrane diterpenes when compared to nonaromatic analogues, whether they are diacylated at positions C-3 and C5, C-3 and C-17, or C-3 and C-14. The establishment of electrostatic interactions and aromatic π−π electron stacking with phenylalanine and tyrosine amino acid residues present in the drug-binding site may be the main underlying reason, as predicted by previous computational studies on ABCB1 drugbinding affinity.16,17 In order to study the type of in vitro interactions between the test compounds and the anticancer drug doxorubicin, the most active substances in the rhodamine-123 accumulation assay at 2 μM, having two aromatic moieties (8−10, 12−14, 17, 22, 24), were evaluated in a combination chemotherapy model on MDR mouse T-lymphoma cells. Compounds 17 and 25, having only one benzoyl moiety, a carbamoyl derivative (18), and two acetylated derivatives (21, 24) were also evaluated. Several concentrations of the compounds and the doxorubicin were tested. The extent of interactions between doxorubicin and the tested compound was calculated and expressed using the combination index (CI) value.18

It was found that all compounds tested interacted synergistically with doxorubicin, and six of them (9, 10, 13, 17, 22, 23) displayed very strong interactions, being able to enhance the cytotoxicity of doxorubicin with CI values ranging from 0.053 to 0.097 (Table 4). A strong synergistic effect was found for Table 4. Type and Strength of the Interaction between Several Derivatives and Doxorubicin on Human ABCB1Transfected L5178Y Mouse Lymphoma Cells ratioa

CI ± SDb

epoxyboetirane L (8) epoxyboetirane M (9)

4:1 5:1

0.233 ± 0.008 0.066 ± 0.004

epoxyboetirane N (10)

5:1

0.097 ± 0.026

epoxyboetirane P (12) epoxyboetirane Q (13)

5:1 5:1

0.172 ± 0.067 0.053 ± 0.010

epoxyboetirane R (14) boetirane B (17)

5:1 9:1

0.196 ± 0.068 0.051 ± 0.013

epoxycarbamoylboetirane A (18) methoxyboetirane A (21) methoxyboetirane B (22)

7:1

0.861 ± 0.309

6:1 5:1

0.214 ± 0.090 0.057 ± 0.020

methoxyboetirane C (23)

5:1

0.054 ± 0.026

pentoxyboetirane B1 (24) pentoxyboetirane B2 (25)

5:1 6:1

0.205 ± 0.019 0.230 ± 0.150

compound

interaction strong synergism very strong synergism very strong synergism strong synergism very strong synergism strong synergism very strong synergism slight synergism strong synergism very strong synergism very strong synergism strong synergism strong synergism

a

Data are shown as the best combination ratio between the tested compounds and doxorubicin. bCombination index (CI) values are represented as the mean of three CI values calculated on the basis of different drug ratios ± standard deviation (SD) of the mean, for an inhibitory concentration of 50% (IC50). CI < 0.1 = very strong synergism; 0.1 < CI < 0.3 = strong synergism; 0.85 < CI < 0.9 = slight synergism.15

methoxyboetirane A (21), which exhibited a low CI value (0.214), even though it did not display a good ABCB1 modulation activity, suggesting that other mechanisms might be responsible for its activity (its FAR values were 1.16 and 2.07 at 2 and 20 μM, respectively). As ATP hydrolysis and substrate transport by ABCB1 are tightly coupled, the measurement of ATPase activity provides a convenient way of disclosing much valuable information on the type of interaction between MDR modulators and ABCB1. Owing to their high MDR-modifying activity at low concentrations, epoxyboetiranes N (10) and R (14) and methoxyboetirane B (22) were chosen to be further evaluated in a colorimetric ATPase activity assay as representatives of the present set of epoxylathyrol (2) and methoxyboetirol (3) derivatives. The assay consists of two complementary subsets of tests: (i) the activation assay, in which the compounds are evaluated for their stimulating or inhibitory effects on the basal vanadate-sensitive ATPase activity, leading to conclusions regarding their interaction with ABCB1, and (ii) the inhibition assay, which is performed in the presence of a known ABCB1 substrate, verapamil, and aims to characterize ABCB1 efflux inhibitors (or slowly transported substrates) through their effect on the verapamil-stimulated vanadate-sensitive ATPase activity. The results are displayed in Figure 1 as the relative ATPase activity of each compound, with 100% taken as the G



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compounds seem to inhibit verapamil transport by ABCB1 at high concentrations, either in a concentration-dependent manner (10 and 14) or not (22), indicating that they are all slowly transported by ABCB1, thus reducing the transport rate of other substrates such as verapamil. Additionally, there was a noncompetitive inhibitory behavior observed at high concentrations for epoxyboetirane R (14; Figure 1B), since it inhibited not only the stimulated ABCB1 ATPase activity but also the baseline vanadate-sensitive ATPase activity (especially above 25 μM).19 This suggests that an allosteric effect might be taking place, in which the compound has a relatively low affinity to the allosteric site of ABCB1, but, when present at sufficiently high concentrations, it may be able to induce conformational changes hampering intramolecular movements necessary for the cleavage of ATP. As for methoxyboetirane B (22; Figure 1C), ATPase activity was decreased to 50% at all tested concentrations in the presence of verapamil (100%). In light of these and previous results, it is fair to assume that the distinct modulatory behavior of methoxyboetirane B (22) is most likely due to its aromatic acylation pattern at C-3 and C-17, contrasting with other evaluated derivatives with ester groups at positions C-3 and C-5. In conclusion, by chemically modifying the basic scaffold of epoxyboetirane A (1), previously described as only a modest MDR modulator,11 a strong increase in ABCB1 reversal activity of lathyrane macrocyclic diterpenes could be obtained. This was accomplished with the introduction of three diaromatic acylation patterns in the original molecule, which furthermore provided a valuable source of data for structure−activity relationship studies on multidrug-resistant human colon adenocarcinoma and mouse T-lymphoma cell lines. Optimal ranges of MW, lipophilicity, TPSA, and MV for improved modulation ability of ABCB1 efflux were found, as well as additional evidence on structural key points for greater affinity to the drug-binding site. In particular, the present results suggested strongly that 3,17-dibenzoyl derivatives may be able to significantly optimize aromatic π−π electron stacking interactions and maintain activity at concentrations as low as 0.2 μM, as shown for methoxyboetirane B (22). The analysis of ATPase inhibitory profiles of three representative MDRmodifying compounds additionally revealed that they all act as ABCB1 slowly transported substrates and reinforced the distinct modulatory ability of 22. Thus, these studies highlight the potential of methoxyboetirane B (22) as a promising multitarget lead for natural-product-based anticancer drug development.

Figure 1. Effect of epoxyboetirane N (10, A), epoxyboetirane R (14, B), and methoxyboetirane B (22, C) on ABCB1 ATPase activity. In the inhibition assay, compounds were tested in the presence of verapamil (40 μM). The effects are presented as the relative ATPase activity, with 100% taken as the stimulated vanadate-sensitive ATPase activity and 0% assumed to be the baseline vanadate-sensitive ATPase activity. Results are expressed as the means ± SD from experiments performed in triplicate.

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EXPERIMENTAL SECTION

General Experimental Procedures. All solvents were dried according to published methods and distilled prior to use. All the other reagents were obtained from commercial suppliers and were used without further purification. Flash column chromatography (CC) was performed on silica gel (Merck 9385) or using CombiFlash Rf200 (Teledyne Isco). Merck silica gel 60 F254 plates were used for analytical TLC, with visualization under UV light (λ 254 and 366 nm) and by spraying with H2SO4/MeOH (1:1), followed by heating. For preparative TLC, 20 × 20 cm × 0.5 mm silica plates were used (Merck 1.05774). Melting points were determined on a Kofler apparatus. Specific optical rotations were obtained using a PerkinElmer 241-MC polarimeter, using quartz cells of 1 dm path length. IR spectra were determined on a Shimadzu IRAffinity-1 FTIR spectrophotometer using KBr disks. NMR spectra were recorded on a Bruker ARX-400 NMR spectrometer (1H 400 MHz; 13C 100.61 MHz), using CDCl3, MeOD, C5D5N, or DMSO-d6 as solvents; signal attribution follows the

stimulated vanadate-sensitive ATPase activity and 0% assumed to be the baseline vanadate-sensitive ATPase activity. From the analysis of the activation assays, it is clear that the three compounds interact with ABCB1, since they all stimulated the basal ABCB1 ATPase activity. The most pronounced stimulatory effect was observed for epoxyboetirane R (14; Figure 1A; FAR = 31.1 at 2 μM), which accordingly did not inhibit ABCB1 efflux-pump activity as robustly as the other two compounds, epoxyboetirane N (10) and methoxyboetirane B (22) (FAR = 60.6 and 67.0 at 2 μM). Moreover, all H




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Pentoxyboetirol B (6): amorphous, white powder; [α]25 D −120 (c 0.13, CHCl3); IR (KBr) νmax 3420 cm−1; 1H NMR (400 MHz, CDCl3) δ 0.75 (1H, t, J = 10.6 Hz, H-9), 0.97 (1H, m, H-8b), 1.03 (3H, s, CH3-18), 1.05 (3H, d, J = 9.3 Hz, CH3-16) 1.09 (1H, m, H-7b), 1.12 (3H, s, CH3-19), 1.20 (1H, m, H-11), 1.22 (3H, s, CH3-17), 1.50 (1H, d, J = 2.3 Hz, H-4), 1.60 (1H, m, H-8a), 1.68 (3H, s, CH3-20), 1.70 (1H, m, H-1β), 1.72 (1H, m, H-7b), 1.88 (1H, br s, H-2), 2.21 (1H, dd, J = 14.3 and 9.7 Hz, H-1α), 3.18 (1H, s, OH), 4.08 (1H, s, H-14), 4.10 (1H, s, OH) 4.25 (1H, d, J = 2.7 Hz, H-5), 4.38 (1H, s, H-3), 4.43 (1H, s, OH), 4.68 (1H, s, OH), 4.78 (1H, s, OH), 5.72 (1H, d, J = 10.5 Hz, H-12) ppm; 13C NMR (100 MHz, CDCl3) δ 14.2 (C-16), 15.6 (C-19), 16.6 (C-20), 18.9 (C-8), 21.9 (C-10), 24.2 (C-17), 25.4 (C-11), 29.0 (C-18), 31.7 (C-9), 36.9 (C-7), 37.0 (C-2), 46.7 (C-4), 47.2 (C-1), 74.4 (C-5), 75.1 (C-6), 78.1 (C-14), 79.6 (C-3), 85.2 (C15), 123.3 (C-12), 132.0 (C-13) ppm; ESIMS m/z 354.30 [M]+. General Procedure for Acylation Reactions. A solution of the corresponding parent compound 2, 3, and 6 in dry triethylamine and CH2Cl2 (1:1) was stirred for 5 min at room temperature after the addition of catalytic amounts of DMAP. Then, the suitable anhydride or acyl chloride was added, and the reaction mixture was stirred and monitored by TLC. The experimental conditions (time and temperature) were adjusted in each case to force the consumption of all starting material. The residue was concentrated under reduced pressure and purified by flash column chromatography. Epoxyboetirane K (7): obtained from the reaction of 2 (20 mg, 0.057 mmol) with biphenyl-4-carbonyl chloride (78 mg, 0.360 mmol, 6.3 equiv) for 24 h at room temperature. The residue was purified by CC (n-hexane/EtOAc, 1:0 to 17:3, and n-hexane/CH2Cl2, 1:1 to 0:1) to afford 14 mg (0.020 mmol, 35% yield) of an amorphous, white 1 powder: [α]25 D +323 (c 0.08, CHCl3); H NMR (400 MHz, DMSO-d6) δ 0.78 (3H, d, J = 5.9 Hz, CH3-16), 0.95 (1H, t, J = 12.0 Hz, H-7a), 1.17 (1H, m, H-9), 1.19 (3H, s, CH3-18), 1.21 (3H, s, CH3-19), 1.60 (1H, m, H-11), 1.63 (2H, m, H-1β and H-8b), 1.82 (3H, s, CH3-20), 1.82 (2H, m, H-7b and H-8a), 1.95 (1H, m, H-4), 2.17 (1H, br s, H2), 2.37 (1H, br s, H-17a), 2.62 (1H, br s, H-17b), 2.97 (1H, dd, J = 11.3 and 7.3 Hz, H-1α), 5.57 (1H, s, H-3), 6.32 (1H, s, OH-15), 6.54 (1H, d, J = 7.8 Hz, H-5), 7.43 (2H, d, J = 6.1 Hz, H-11′ and H-11″), 7.49 (4H, dd, J = 14.2 and 7.0 Hz, H-10′, H-10″, H-12′, and H-12″), 7.57 (4H, s, H-9′, H-9″, H-13′, and H-13″), 7.66 (4H, d, J = 5.3 Hz, H-4′, H-4″, H-6′, and H-6″), 7.72 (5H, d, J = 7.8 Hz, H-12, H-3′, H3″, H-7′, and H-7″) ppm; 13C NMR (100 MHz, DMSO-d6) δ 12.5 (C20), 14.3 (C-16), 16.4 (C-19), 20.0 (H-8), 25.5 (C-10), 28.6 (C-18), 28.9 (C-11), 33.3 (C-7), 34.7 (C-9), 38.1 (C-2), 48.0 (C-1), 49.8 (C4), 54.3 (C-17), 59.3 (C-6), 66.2 (C-5), 80.3 (C-3), 86.5 (C-15), 126.3 (C-4′ and C-4″), 127.0 (C-6′)*, 127.1 (C-6″)*, 128.0 (C-2′)*, 128.4 (C-9′ and C-9″), 128.5 (C-2″)*, 128.7 (C-13′ and C-13″), 129.2 (C-10′, C-10″, C-11′, C-11″, C-12′ and C-12″), 130.0 (C-3′, C3″, C-7′ and C-7″), 134.8 (C-13), 139.0 (C-8′ and C-8″), 139.1 (C-5′ and C-5″), 148.5 (C-12), 165.0 (C-1′), 167.0 (C-1″), 201.3 (C-14) ppm; ESIMS m/z 711.41 [M + H]+. Epoxyboetirane L (8): obtained from the reaction of 2 (24 mg, 0.069 mmol) with 2-naphthoyl chloride (78 mg, 0.409 mmol, 6.0 equiv) for 2 h at room temperature. The residue was purified by CC (n-hexane/EtOAc, 1:0 to 9:1) and preparative TLC (CH2Cl2) to afford 32 mg (0.049 mmol, 53% yield) of an amorphous, yellow 1 powder: [α]25 D +254 (c 0.09, CHCl3); H NMR (400 MHz, DMSO-d6) δ 0.75 (3H, d, J = 5.7 Hz, CH3-16), 0.94 (1H, t, J = 12.0 Hz, H-7a), 1.17 (3H, s, CH3-18), 1.18 (1H, m, H-9), 1.20 (3H, s, CH3-19), 1.63 (1H, m, H-11), 1.67 (1H, m, H-1β), 1.76 (1H, m, H-8b), 1.81 (3H, s, CH3-20), 1.94 (1H, m, H-7b), 1.97 (2H, m, H4 and H-8a), 2.19 (1H, br s, H-2), 2.36 (1H, s, H-17a), 2.61 (1H, s, H-17b), 2.98 (1H, dd, J = 12.6 and 7.2 Hz, H-1α), 5.61 (1H, s, H-3), 6.32 (1H, s, OH-15), 6.64 (1H, d, J = 7.7 Hz, H-5), 7.38 (1H, d, J = 8.2 Hz, H-11′)*, 7.54 (3H, m, H-7′, H-7″, and H-11″)*, 7.63 (2H, m, H-6′ and H-6″), 7.71 (3H, m, H-5′, H-8′, and H-8″)*, 7.81 (2H, m, H-5″ and H-12)*, 7.90 (1H, d, J = 8.1 Hz, H-10′)*, 7.94 (1H, d, J = 8.1 Hz, H-10″)*, 7.99 (1H, m, H-3′)*, 8.04 (1H, m, H-3″)* ppm; 13C NMR (100 MHz, DMSO-d6) δ 12.2 (C-20), 14.3 (C-16), 16.5 (C-19), 20.0 (C-8), 25.6 (C-10), 28.6 (C-18), 29.0 (C-11), 33.4 (C-7), 34.9 (C-9), 38.0 (C-2), 48.1 (C-1), 49.8 (C-4), 54.5 (C-17), 59.4 (C-6), 66.4 (C-5), 80.6 (C-3), 86.6 (C-

numbered structures provided as Supporting Information (Appendix). Chemical shifts are expressed in δ (ppm) referenced to the solvent used, and the proton coupling constants J in hertz (Hz). Spectra were assigned using appropriate COSY, DEPT, HMQC, and HMBC sequences. Permutable signals between the same position in other ester functions are marked by asterisks. ESIMS analysis was performed on a triple quadrupole (QT) Micromass Quattro Micro AP1 mass spectrometer, with an ion source set in a positive ESI ionization mode. All tested compounds were purified to ≥95% purity as determined by HPLC. Preparation of Epoxylathyrol (2) and Methoxyboetirol (3). Epoxyboetirane A (1, 471 mg, 0.989 mmol), previously isolated from Euphorbia boetica,11 was suspended in 5% KOH in methanol (7 mL). After stirring for 3 h at room temperature, the mixture was worked up by dilution with 10 mL of distilled water followed by extraction with ethyl acetate (7 × 10 mL). The organic layer was dried and evaporated to give a residue that was purified by CC (n-hexane/EtOAc, 1:0 to 2:1) to afford 255 mg (0.642 mmol, 65% yield) of 2 and 32 mg (0.084 mmol, 13% yield) of 3. Preparation of Ethoxyboetirol (4). Compound 1 (414 mg, 0.869 mmol) was suspended in 5% KOH in ethanol (11 mL). After stirring for 6 h at room temperature, the mixture was worked up by dilution with 10 mL of distilled water followed by extraction with ethyl acetate (7 × 10 mL). The organic layer was dried to give a residue that was further purified by CC (n-hexane/EtOAc, 1:0 to 2:3) to afford 125 mg (0.357 mmol, 45% yield) of 2 and 13 mg (0.033 mmol, 11% yield) of 4. Ethoxyboetirol (4): amorphous, white powder; [α]25 D −36 (c 0.1, CHCl3); IR (KBr) νmax 3414, 1718 cm−1; 1H NMR (400 MHz, CDCl3) δ 0.40 (1H, m, H-8b), 0.86 (1H, t, J = 12.0 Hz, H-9), 0.95 (3H, s, CH3-19), 1.03 (3H, s, CH3-18), 1.10 (3H, d, J = 6.3 Hz, CH316), 1.22 (3H, t, J = 7.0 Hz, O-CH2CH3), 1.32 (3H, m, H-1β, H7-b and H-11), 1.57 (1H, m, H-8a), 1.77 (1H, m, H-2), 1.94 (3H, s, CH320), 1.96 (1H, m, H-7a), 2.48 (1H, dd, J = 14.5 and 4.5 Hz, H-1α), 3.18 (1H, t, J = 9.8 Hz, H-3), 3.46 (1H, m, H-17a), 3.70 (2H, m, H-4 and O-CH2CH3), 3.86 (2H, m, H-17b and O-CH2CH3), 4.08 (1H, s, H-5), 4.68 (1H, d, J = 11.0 Hz, H-12) ppm; 13C NMR (100 MHz, CDCl3) δ 12.3 (C-20), 15.6 (O-CH2CH3), 15.7 (C-19), 18.1 (C-16), 18.5 (C-8), 22.8 (C-10), 25.1 (C-11), 28.9 (C-18), 33.8 (C-9), 36.4 (C-7), 38.4 (C-2), 41.1 (C-1), 54.8 (C-4), 66.8 (C-17), 67.6 (OCH2CH3), 75.2 (C-3), 76.9 (C-5), 76.9 (C-6), 82.4 (C-14), 133.5 (C12), 135.6 (C-13), 210.8 (C-15); ESIMS m/z 396.32 [M]+. Preparation of Pentoxyboetirol A (5) and Pentoxyboetirol B (6). Compound 1 (302 mg, 0.63 mmol) was dissolved in dry THF (5 mL), and the solution was cooled to 0 °C. LiAlH4 (240 mg, 10 equiv) was then added, and the mixture was stirred for 1 h at 0 °C. Aqueous 10% NaOH solution (1.5 mL) was added, and the mixture stirred for another 10 min to neutralize the excess of LiAlH4. The gray precipitate obtained was poured into 100 mL of a mixture of EtOAc/H2O (4:1) and filtered through a pad of Celite under reduced pressure. The aqueous layer was extracted with ethyl acetate (3 × 50 mL), the organic layer was then dried, and the solvent was evaporated. The obtained residue was purified by CC (n-hexane/EtOAc, 1:0 to 1:4) to afford 8 mg (0.023 mmol, 4% yield) of 5 and 99 mg (0.279 mmol, 44% yield) of 6. 1 Pentoxyboetirol A (5): yellow oil; [α]25 D −106 (c 0.1, CHCl3); H NMR (400 MHz, CDCl3) δ 0.79 (1H, m, H-9), 0.95 (1H, m, H-8b), 1.05 (3H, s, CH3-18), 1.08 (1H, m, H-7b), 1.10 (3H, d, J = 8.0 Hz, CH3-16), 1.11 (3H, s, CH3-19), 1.21 (1H, m, H-11), 1.25 (3H, s, CH3-17), 1.41 (1H, d, J = 2.3 Hz, H-4), 1.45 (1H, dd, J = 15.4 and 10.6 Hz, H-1β), 1.65 (3H, s, CH3-20), 1.67 (1H, m, H-8a), 1.76 (1H, m, H-7a), 1.90 (1H, br s, H-2), 2.78 (1H, dd, J = 15.4 and 10.6 Hz, H1α), 4.06 (1H, s, H-5), 4.37 (1H, s, H-14), 4.45 (1H, s, H-3), 5.27 (1H, d, J = 10.6 Hz, H-12) ppm; 13C NMR (100 MHz, CDCl3) δ 10.4 (C-20), 14.2 (C-16), 15.1 (C-19), 18.5 (C-8), 21.8 (C-10), 23.4 (C11), 25.2 (C-17), 25.6 (C-18), 31.8 (C-9), 36.2 (C-2), 36.3 (C-7), 42.0 (C-1), 47.9 (C-4), 73.7 (C-5), 74.7 (C-6), 79.6 (C-3), 85.4 (C14), 88.7 (C-15), 130.0 (C-12), 132.7 (C-13) ppm; ESIMS m/z 354.30 [M]+. I




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DMSO-d6) δ 12.7 (C-20), 14.7 (C-16), 16.6 (C-19), 20.1 (C-8), 25.7 (C-10), 28.8 (C-18), 28.8 (C-11), 33.3 (C-7), 34.9 (C-9), 38.0 (C-2), 47.9 (C-1), 49.6 (C-4), 54.4 (C-17), 59.4 (C-6), 67.3 (C-5), 81.4 (C3), 86.7 (C-15), 125.5 (C-4′ and C-6′)*, 125.6 (C-4″ and C-6″)*, 130.1 (C-3′ and C-7′)*, 132.7 (C-13), 138.6 (C-2′)*, 138.7 (C-2″)*, 148.5 (C-12), 164.1 (C-1′), 164.2 (C-1″), 201.2 (C-14) ppm; signals corresponding to C-5′, C-5″, CF3-5′, and CF3-5″ were not detected; ESIMS m/z 695.29 [M + H]+. Epoxyboetirane P (12): obtained from the reaction of 2 (23 mg, 0.066 mmol) with 4-(dimethylamino)benzoyl chloride (73 mg, 0.446 mmol, 6.8 equiv) for 96 h under reflux. The residue was purified by preparative TLC with n-hexane/EtOAc (3:2) to afford 15 mg (0.023 mmol, 35% yield) of an amorphous, white powder: [α]25 D +26.0 (c 0.08, CHCl3); 1H NMR (400 MHz, DMSO-d6) δ 0.85 (1H, m, H-7a), 0.87 (3H, d, J = 6.7 Hz, CH3-16), 1.17 (4H, s, H-9 and CH3-19), 1.18 (3H, s, CH3-18), 1.44 (1H, m, H-1β), 1.52 (1H, m, H-4), 1.58 (1H, m, H-11), 1.76 (4H, s, H-8b and CH3-20), 1.81 (1H, s, H-2), 1.98 (1H, m, H-8a), 2.00 (1H, m, H-7b), 2.14 (1H, s, H-17a), 2.49 (1H, s, H17b), 2.99 (13H, s, H-1α, NMe2-5′ and NMe2-5″), 3.93 (1H, s, H-3), 5.57 (1H, s, OH-15), 6.32 (1H, d, J = 9.2 Hz, H-5), 6.71 (4H, dd, J = 13.0 and 8.8 Hz, H-4′, H-4″, H-6′ and H-6″), 7.60 (1H, d, J = 11.8 Hz, H-12), 7.76 (4H, dd, J = 13.6 and 8.7 Hz, H-3′, H-3″, H-7′, and H-7″) ppm; 13C NMR (100 MHz, DMSO-d6) δ 12.4 (C-20), 14.4 (C-16), 16.3 (C-19), 20.0 (C-8), 25.5 (C-10), 28.6 (C-11), 29.0 (C-18), 33.4 (C-7), 35.0 (C-9), 37.8 (C-2), 39.7 (NMe2-5′ and NMe2-5″), 48.0 (C1), 52.0 (C-4), 54.1 (C-17), 59.2 (C-6), 65.5 (C-5), 78.7 (C-3), 88.5 (C-15), 110.8 (C-4′ and C-6′)*, 110.9 (C-4″ and C-6″)*, 116.7 (C-2′ and C-2″), 130.9 (C-3′ and C-7′)*, 131.0 (C-3″ and C-7″)*, 134.7 (C-13), 149.5 (C-12), 153.1 (C-5′)*, 153.3 (C-5″)*, 165.6 (C-1′), 167.6 (C-1″), 200.5 (C-14) ppm; ESIMS m/z 615.45 [M − C2H6 + H]+, 498.45 [M − C9H10NO + 2H]+. Epoxyboetirane Q (13): obtained from the reaction of 2 (19 mg, 0.054 mmol) with 2-furoyl chloride (70 μL, 0.710 mmol, 13.1 equiv) for 4 h at room temperature. The residue was purified by CC (nhexane/EtOAc, 1:0 to 4:1) to afford 25 mg (0.046 mmol, 86% yield) 1 of an amorphous, white powder: [α]25 D +162 (c 0.08, CHCl3); H NMR (400 MHz, DMSO-d6) δ 0.76 (3H, d, J = 6.4 Hz, CH3-16), 0.91 (1H, t, J = 12.0 Hz, H-7a), 1.17 (4H, s, H-11 and CH3-19), 1.18 (3H, s, CH3-18), 1.52 (1H, m, H-1β), 1.60 (2H, m, H-8b and H-9), 1.79 (3H, s, CH3-20), 1.81 (1H, m, H-4), 1.96 (2H, m, H-7b and H-8a), 2.11 (1H, br s, H-2), 2.30 (1H, s, H-17a), 2.57 (1H, s, H-17b), 2.90 (1H, dd, J = 12.4 and 7.9 Hz, H-1α), 5.40 (1H, s, H-3), 6.11 (1H, s, OH-15), 6.45 (1H, d, J = 7.6 Hz, H-5), 6.53 (1H, s, H-4′)*, 6.58 (1H, d, J = 1.6 Hz, H-4″)*, 6.86 (1H, d, J = 1.3 Hz, H-5′)*, 7.09 (1H, d, J = 3.4 Hz, H-5″)*, 7.68 (1H, d, J = 11.5 Hz, H-12), 7.77 (2H, d, J = 11.9 Hz, H-3′ and H-3″); 13C NMR (100 MHz, DMSO-d6) δ 12.6 (C-20), 14.4 (C-16), 16.5 (C-19), 20.1 (C-8), 25.7 (C-10), 28.8 (C-18), 29.2 (C-11), 33.4 (C-7), 35.0 (C-9), 38.1 (C-2), 47.9 (C-1), 49.6 (C-4), 54.5 (C-17), 59.4 (C-6), 66.1 (C-5), 80.3 (C-3), 86.6 (C-15), 112.0 (C-4′)*, 112.2 (C-4″)*, 118.5 (C-5′)*, 118.8 (C-5″)*, 134.7 (C-13), 140.3 (C-12), 143.3 (C-2′)*, 143.8 (C-2″)*, 147.5 (C-3′)*, 147.8 (C3″)*, 157.3 (C-1′), 157.4 (C-1″), 201.1 (C-14); ESIMS m/z 539.30 [M + H]+. Epoxyboetirane R (14): obtained from the reaction of 2 (19 mg, 0.054 mmol) with 2-tiophenecarbonyl chloride (70 μL, 0.654 mmol, 12.1 equiv) for 4 h at room temperature. The residue was purified by CC (n-hexane/EtOAc, 1:0 to 4:1) and preparative TLC with nhexane/EtOAc (4:1) to afford 33 mg (0.058 mmol, quantitative yield) 1 of an amorphous, white powder: [α]25 D +231 (c 0.08, CHCl3); H NMR (400 MHz, DMSO-d6) δ 0.77 (3H, d, J = 6.4 Hz, CH3-16), 0.89 (1H, t, J = 12.5, H7a), 1.17 (1H, m, H-11), 1.18 (3H, s, CH3-19), 1.19 (3H, s, CH3-18), 1.54 (1H, m, H-1β), 1.59 (2H, m, H-8b and H-9), 1.79 (3H, s, CH3-20), 1.80 (1H, m, H-4), 1.91 (1H, m, H-7a), 1.96 (1H, m, H-8a), 2.13 (1H, br s, H-2), 2.29 (1H, s, H-17a), 2.59 (1H, d, J = 2.2 Hz, H-17b), 2.93 (1H, dd, J = 12.5 and 7.9 Hz, H-1α), 5.41 (1H, s, H-3), 6.22 (1H, s, OH-15), 6.51 (1H, d, J = 8.2 Hz, H-5), 7.03 (1H, t, J = 4.1 Hz, H-4′)*, 7.10 (1H, d, J = 4.3 Hz, H-4″)*, 7.34 (1H, d, J = 2.3 Hz, H-5′)*, 7.57 (1H, d, J = 2.9 Hz, H-5″)*, 7.75 (1H, d, J = 11.8 Hz, H-12), 7.80 (2H, t, J = 4.3 Hz, H-3′ and H-3″); 13C NMR (100 MHz, DMSO-d6) δ 12.8 (C-20), 14.8 (C-16), 16.8 (C-19), 20.4

15), 124.7 (C-11′)*, 124.9 (C-11″)*, 126.4 (C-2′)*, 126.7 (C-6′ and C-6″)*, 126.9 (C-2″)*, 127.6 (C-7′, C-7″, C-10′, and C-10″)*, 128.3 (C-8′)*, 128.5 (C-8″)*, 129.0 (C-5′)*, 129.2 (C-5″)*, 130.5 (C-3′)*, 130.7 (C-3″)*, 131.6 (C-4′)*, 131.7 (C-4″)*, 134.6 (C-13), 134.9 (C9′ and C-9″)*, 149.4 (C-12), 165.1 (C-1′), 165.2 (C-1″), 201.2 (C14) ppm; ESIMS m/z 659.37 [M + H]+. Epoxyboetirane M (9): obtained from the reaction of 2 (32 mg, 0.091 mmol) with m-toluoyl chloride (70 μL, 0.531 mmol, 5.8 equiv) for 2 h at room temperature. The residue was purified by CC (nhexane/EtOAc, 1:0 to 4:1) and preparative TLC (CH2Cl2) to afford 39 mg (0.067 mmol, 73% yield) of white crystals: [α]25 D +235 (c 0.08, CHCl3); mp 181−182 °C (CH2Cl2/MeOH); IR (KBr) νmax 3414, 1710 cm−1; 1H NMR (400 MHz, DMSO-d6) δ 0.74 (3H, d, J = 6.2 Hz, CH3-16), 0.89 (1H, t, J = 11.5 Hz, H-7a), 1.17 (1H, m, H-9), 1.19 (6H, s, CH3-18 and CH3-19), 1.60 (1H, m, H-1β and H-11), 1.72 (1H, d, J = 12.6 Hz, H-8b), 1.81 (3H, s, CH3-20), 1.89 (2H, m, H-7b and H-8a), 1.93 (1H, s, H-4), 2.15 (1H, br s, H-2), 2.20 (3H, s, Me6′)*, 2.25 (3H, s, Me-6″)*, 2.34 (1H, d, J = 0.6 Hz, H-17a), 2.54 (1H, d, J = 0.8 Hz, H-17b), 2.94 (1H, dd, J = 12.1 and 7.8 Hz, H-1α), 5.49 (1H, s, H-3), 6.24 (1H, s, OH-15), 6.55 (1H, d, J = 8.5 Hz, H-5), 7.16 (1H, m, H-5′)*, 7.21 (2H, m, H-7′ and H-7″), 7.28 (2H, m, H-4′ and H-4″), 7.36 (2H, m, H-3′ and H-3″), 7.49 (1H, d, J = 7.1 Hz, H-5″)*, 7.74 (1H, d, J = 11.7 Hz, H-12) ppm; 13C NMR (100 MHz, DMSOd6) δ 12.4 (C-20), 14.3 (C-16), 16.4 (C-19), 20.8 (Me-6′)*, 20.9 (Me6″ and C-8)*, 25.6 (C-10), 28.7 (C-18), 29.0 (C-11), 33.4 (C-7), 34.8 (C-9), 37.7 (C-2), 48.1 (C-1), 49.6 (C-4), 54.6 (C-17), 59.5 (C-6), 66.1 (C-5), 80.3 (C-3), 88.5 (C-15), 126.4 (C-5′)*, 126.5 (C-5″)*, 127.9 (C-4′ and C-4″), 128.5 (C-6′)*, 129.0 (C-6″)*, 129.6 (C-7′)*, 129.8 (C-7″)*, 133.4 (C-3′)*, 133.5 (C-3″)*, 134.6 (C-13), 137.2 (C2′)*, 137.3 (C-2″)*, 149.7 (C-12), 165.3 (C-1′ and C-1″), 201.7 (C14) ppm; ESIMS m/z 587.36 [M + H]+. Epoxyboetirane N (10): obtained from the reaction of 2 (26 mg, 0.074 mmol) with 3-methoxybenzoyl chloride (70 μL, 0.498 mmol, 6.2 equiv) for 72 h at room temperature. The residue was purified by CC (n-hexane/EtOAc, 1:0 to 17:3) to afford 24 mg (0.039 mmol, 52% yield) of an amorphous, white powder: [α]25 D +146 (c 0.09, CHCl3); 1 H NMR (400 MHz, DMSO-d6) δ 0.74 (3H, d, J = 6.2 Hz, CH3-16), 0.89 (1H, m, H7-a), 1.17 (1H, m, H-9), 1.18 (3H, s, CH3-18 and CH319), 1.52 (1H, m, H-1β), 1.59 (1H, m, H-11), 1.70 (1H, m, H-8b), 1.80 (3H, s, CH3-20), 1.86 (1H, m, H-4), 1.93 (2H, m, H-7b and H8a), 2.15 (1H, br s, H-2), 2.31 (1H, s, H17-a), 2.58 (1H, s, H-17b), 2.96 (1H, dd, J = 12.2 and 8.1 Hz, H-1α), 3.64 (3H, s, OMe-6′)*, 3.71 (3H, s, OMe-6″)*, 5.46 (1H, s, H-3), 6.35 (1H, s, OH-15), 6.56 (1H, d, J = 7.9 Hz, H-5), 6.99 (1H, m, H-7′)*, 7.07 (1H, m, H-7″)*, 7.09 (2H, m, H-5′ and H-5″), 7.14 (1H, m, H-3′)*, 7.26 (2H, m, H-3″ and H-4′)*, 7.28 (1H, m, H-4″)*, 7.77 (1H, d, J = 11.6 Hz, H-12) ppm; 13 C NMR (100 MHz, DMSO-d6) δ 12.7 (C-20), 14.6 (C-16), 16.6 (C19), 20.2 (C-8), 25.8 (C-10), 28.9 (C-18), 29.3 (C-11), 33.6 (C-7), 35.1 (C-9), 38.2 (C-2), 48.4 (C-1), 49.9 (C-4), 54.7 (C-17), 55.3 (OMe-6′ and OMe-6″), 59.6 (C-6), 66.5 (C-5), 80.8 (C-3), 86.8 (C15), 113.9 (C-7′)*, 114.4 (C-7″)*, 119.1 (C-5′)*, 119.6 (C-5″), 121.7 (C-3′)*, 121.9 (C-3″), 129.4 (C-4′)*, 129.5 (C-4″)*, 130.6 (C-2′)*, 131.5 (C-2″), 134.8 (C-13), 149.6 (C-12), 158.9 (C-6′ and C-6″), 165.1 (C-1′ and C-1″), 201.6 (C-14) ppm; ESIMS m/z 619.36 [M + H]+. Epoxyboetirane O (11): obtained from the reaction of 2 (26 mg, 0.060 mmol) with 4-(trifluoromethyl)benzoyl chloride (70 μL, 0.470 mmol, 7.9 equiv) for 2 h at room temperature. The residue was purified by CC (n-hexane/EtOAc, 1:0 to 19:1) and preparative TLC with CH2Cl2 to afford 30 mg (0.043 mmol, 72% yield) of an 1 amorphous, white powder: [α]25 D +139 (c 0.09, CHCl3); H NMR (400 MHz, DMSO-d6) δ 0.75 (3H, d, J = 6.4 Hz, CH3-16), 0.96 (1H, t, J = 10.7 Hz, H-7a), 1.17 (1H, m, H-9), 1.19 (6H, s, CH3-18 and CH3-19), 1.58 (1H, m, H-1β), 1.64 (1H, m, H-11), 1.83 (4H, s, H-8b and CH3-20), 1.86 (1H, m, H-7b), 1.96 (1H, m, H-8a), 1.98 (1H, m, H-4), 2.20 (1H, br s, H-2), 2.40 (1H, d, J = 1.5 Hz, H-17a), 2.64 (1H, d, J = 0.5 Hz, H-17b), 2.96 (1H, dd, J = 12.1 and 7.8 Hz, H-1α), 5.57 (1H, s, H-3), 6.38 (1H, s, OH-15), 6.56 (1H, d, J = 7.4 Hz, H-5), 7.65 (4H, s, H-4′, H-4″, H-6′, and H-6″), 7.70 (1H, s, H-12), 7.75 (4H, q, J = 8.5 Hz, H-3′, H-3″, H-7′, and H-7″) ppm; 13C NMR (100 MHz, J




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(C-8), 26.0 (C-10), 29.0 (C-11), 29.4 (C-18), 33.8 (C-7), 35.2 (C-9), 38.4 (C-2), 48.4 (C-1), 49.8 (C-4), 54.8 (C-17), 59.6 (C-6), 66.8 (C5), 81.0 (C-3), 86.8 (C-15), 128.1 (C-4′ and C-4″), 132.8 (C-2′)*, 133.6 (C-2″)*, 133.9 (C-5′)*, 134.0 (C-5″)*, 134.7 (C-3′ and C-3″), 134.9 (C-13), 149.8 (C-12), 161.3 (C-1′ and C-1″), 201.4 (C-14); ESIMS m/z 571.24 [M + H]+. Epoxyboetirane S (15): obtained from the reaction of 2 (20 mg, 0.057 mmol) with 1-adamantanecarbonyl chloride (71 mg, 0.357 mmol, 6.3 equiv) for 2 h at room temperature. The residue was purified by CC (n-hexane/EtOAc, 1:0 to 49:1) and preparative TLC with n-hexane/EtOAc (17:3) to afford 27 mg (0.040 mmol, 70% yield) of an amorphous, white powder: [α]25 D +71 (c 0.09, CHCl3); IR (KBr) νmax 1726 cm−1; 1H NMR (400 MHz, CDCl3) δ 0.68 (1H, td, J = 13.1 and 7.1 Hz, H-7a), 0.96 (3H, d, J = 6.6 Hz, CH3-16), 1.10 (3H, s, CH3-18), 1.18 (3H, s, CH3-19), 1.20 (1H, m, H-11), 1.24 (1H, m, H7b), 1.48 (1H, m, H-8b), 2.09 (3H, s, CH3-20), 2.30 (1H, d, J = 4.7 Hz, H-17a), 2.54 (2H, dd, J = 14.8 and 9.3 Hz, H-1α and H-4), 2.71 (1H, d, J = 4.7 Hz, H-17b), 4.68 (1H, d, J = 10.7 Hz, H-5), 5.47 (1H, t, J = 3.6 Hz, H-3), 5.97 (1H, d, J = 11.1 Hz, H-12), signals corresponding to adamantane protons appear at δH 2.02−1.69 ppm and were overlapped with signals corresponding to protons H-1β, H-2, H-8a, and H-9; 13C NMR (100 MHz, CDCl3) δ 14.1 (C-20), 14.4 (C16), 15.6 (C-19), 20.3 (C-8), 23.5 (C-10), 28.8 (C-11 and C-18), 34.5 (C-7), 35.2 (C-9), 37.4 (C-2), 47.5 (C-1), 51.1 (C-17), 51.5 (C-4), 62.0 (C-6), 73.1 (C-5), 77.1 (C-3), 84.2 (C-15), 135.7 (C-13), 142.0 (C-12), 175.8 (C-1′), 177.2 (C-1″), 206.0 (C-14) ppm; signals corresponding to adamantane carbons appeared at δC 39.0−38.8, 36.7−36.5, and 28.1−28.0 ppm; ESIMS m/z 675.50 [M]+. Boetirane A (16): obtained from the reaction of 2 (29 mg, 0.083 mmol) with propionic anhydride (70 μL, 0.546 mmol, 6.6 equiv) for 7 days under reflux. The residue was purified by CC (n-hexane/EtOAc, 1:0 to 9:1) to afford 2 mg (0.004 mmol, 5% yield) of an amorphous, 1 white powder: [α]25 D −43 (c 0.1, CHCl3); H NMR (400 MHz, CDCl3) δ 0.96 (3H, d, J = 6.8 Hz, CH3-16), 1.05 (3H, t, J = 7.6 Hz, CH3-3′)*, 1.11 (3H, s, CH3-18), 1.14 (1H, m, H-8b), 1.15 (6H, t, J = 7.6 Hz, CH3-3″ and CH3-3‴)*, 1.16 (1H, m, H-9), 1.20 (3H, s, CH319), 1.36 (1H, dd, J = 10.8 and 9.2 Hz, H-11), 1.48 (1H, m, H-8a), 1.50 (1H, m, H-7a), 1.82 (1H, dd, J = 14.5 and 11.4 Hz, H-1β), 2.03 (3H, s, CH3-20), 2.17 (1H, m, H-7b), 2.24 (6H, m, H-2′, H-2″, and H2‴), 2.32 (1H, m, H-2), 2.48 (1H, dd, J = 14.6 and 9.3 Hz, H-1α), 2.65 (1H, s, OH), 2.80 (1H, dd, J = 10.8 and 4.0 Hz, H-4), 3.63 (2H, dd, J = 14.0 and 12.0 Hz, H-17a and H-17b), 4.55 (1H, s, OH), 5.44 (1H, t, J = 3.6 Hz, H-3), 5.56 (1H, d, J = 10.8 Hz, H-5), 5.96 (1H, d, J = 11.2 Hz, H-12); 13C NMR (100 MHz, CDCl3) δ 8.9 (C-3′)*, 9.0 (C-3″)*, 9.1 (C-3‴)*, 14.0 (C-20), 14.3 (C-16), 15.5 (C-19), 15.9 (C-8), 25.3 (C-10), 26.4 (C-11), 27.5 (C-2′)*, 27.5 (C-2″)*, 27.7 (C-2‴)*, 27.8 (C-7), 28.8 (C-18), 34.7 (C-9), 36.5 (C-2), 48.5 (C-1), 49.8 (C-4), 66.1 (C-17), 69.1 (C-5), 73.3 (C-6), 78.1 (C-3), 84.7 (C-15), 134.5 (C-13), 143.2 (C-12), 172.5 (C-1′), 174.3 (C-1″), 174.8 (C-1‴), 206.7 (C-14); ESIMS m/z 537.31 [M + H]+. Boetirane B (17): obtained from the reaction of 2 (50 mg, 0.143 mmol) with o-anisoyl chloride (159 mg, 1.506 mmol, 10.5 equiv) for 7 days under reflux. The residue was purified by CC [n-hexane/EtOAc (1:0 to 1:1)] and preparative TLC with CH2Cl2/MeOH (99:1) to afford 13 mg (0.004 mmol, 14% yield) of an amorphous, white 1 powder: [α]25 D −64 (c 0.09, CHCl3); H NMR (400 MHz, DMSO-d6) δ 0.86 (1H, m, H-8b), 0.95 (3H, d, J = 5.1 Hz, CH3-16), 1.09 (3H, s, CH3-18), 1.12 (1H, m, H-9), 1.15 (3H, s, CH3-19), 1.30 (1H, m, H7b), 1.37 (1H, m, H-11), 1.52 (1H, m, H-8a), 1.55 (1H, m, H-7a), 1.68 (1H, m, H-1β), 1.83 (1H, s, OH), 1.97 (3H, s, CH3-20), 2.38 (2H, m, H-1α and H-2), 2.63 (1H, d, J = 7.8 Hz, H-4), 3.69 (3H, s, OMe-7′)*, 3.75 (3H, s, OMe-7″)*, 3.94 (1H, d, J = 11.2 Hz, H-17a), 4.01 (1H, d, J = 11.0 Hz, H-17b), 4.13 (1H, s, H-5), 4.27 (1H, s, OH), 4.81 (1H, s, OH), 5.66 (1H, d, J = 11.1 Hz, H-12), 5.71 (1H, s, H-3), 6.71 (1H, t, J = 7.5 Hz, H-4′)*, 6.90 (1H, t, J = 7.4 Hz, H-4″)*, 7.08 (2H, m, H-6′ and H-6″)*, 7.46 (1H, t, J = 8.1 Hz, H-5′)*, 7.52 (1H, t, J = 8.0 Hz, H-5″)*, 7.76 (1H, d, J = 7.5 Hz, H-3′)*, 7.77 (1H, d, J = 7.6 Hz, H-3″)*; 13C NMR (100 MHz, DMSO-d6) δ 13.7 (C-20), 14.8 (C-16), 15.6 (C-19), 15.7 (C-8), 24.2 (C-10), 25.4 (C-11), 27.3 (C-7), 28.6 (C-18), 33.8 (C-9), 36.0 (C-2), 47.3 (C-1), 51.7 (C-4), 55.9

(OMe-7′ and OMe-7″), 67.8 (C-17), 68.1 (C-5), 74.1 (C-6), 80.1 (C3), 84.5 (C-15), 112.7 (C-6′ and C-6″), 119.9 (C-2′ and C-4′)*, 120.0 (C-4″)*, 120.8 (C-2″)*, 131.2 (C-3′)*, 131.4 (C-3″)*, 133.4 (C-5′)*, 133.9 (C-5″)*, 136.8 (C-13), 139.8 (C-12), 158.8 (C-7′ and C-7″), 164.8 (C-1′), 165.6 (C-1″), 207.2 (C-14); ESIMS m/z 636.29 [M]+. Epoxycarbamoylboetirane A (18): obtained from the reaction of 2 (20 mg, 0.057 mmol) with dimethylcarbamoyl chloride (70 μL, 0.760 mmol, 13.3 equiv) for 5 h under reflux. The residue was purified by CC (n-hexane/EtOAc, 1:0 to 7:3) and preparative TLC with nhexane/EtOAc (1:1) to afford 15 mg (0.036 mmol, 62% yield) of an 1 amorphous, yellow powder: [α]25 D +29 (c 0.09, CHCl3); H NMR (400 MHz, C5D5N) δ 1.08 (1H, m, H-7b), 1.09 (3H, s, CH3-18), 1.14 (3H, d, J = 4.9 Hz, CH3-16), 1.20 (3H, s, CH3-19), 1.59 (1H, t, J = 8.0 Hz, H-11), 1.98 (1H, m, H-1β), 2.03 (3H, s, CH3-20), 2.11 (4H, m, H-2, H-4, H-7a, and H-8b), 2.18 (1H, m, H-9), 2.32 (1H, m, H-8a), 2.58 (2H, s, H-17a and H-17b), 2.82 (6H, s, N′Me2-1′), 3.63 (1H, m, H1α), 4.41 (1H, s, H-3), 5.78 (1H, s, OH), 6.66 (1H, s, OH), 6.74 (1H, d, J = 9.4 Hz, H-5), 8.11 (1H, d, J = 11.4 Hz, H-12); 13C NMR (100 MHz, C5D5N) δ 13.8 (C-20), 15.7 (C-19), 17.4 (C-16), 21.9 (C-8), 26.9 (C-10), 29.6 (C-18), 30.7 (C-11), 35.5 (C-7), 36.4 (N′Mea-1′), 36.8 (C-9), 37.4 (N′Meb-1′), 39.7 (C-2), 50.5 (C-1), 54.2 (C-4), 56.1 (C-17), 60.9 (C-6), 69.6 (C-5), 81.1 (C-3), 90.8 (C-15), 136.6 (C13), 151.1 (C-12), 158.2 (C-1′), 202.1 (C-14); ESIMS m/z 422.25 [M + H]+. Epoxycarbamoylboetirane B (19): obtained from the reaction of 2 (25 mg, 0.071 mmol) with diethylcarbamoyl chloride (100 μL, 0.790 mmol, 11.1 equiv) for 72 h under reflux. The residue was purified by CC (n-hexane/EtOAc, 1:0 to 7:3) to afford 37 mg (0.067 mmol, 94% yield) of an amorphous, white powder: [α]25 D +85 (c 0.08, CHCl3); IR (KBr) νmax 1701, 1314 cm−1; 1H NMR (400 MHz, CDCl3) δ 0.97 (1H, m, H-7b), 1.00 (1H, m, H-9), 1.01 (3H, d, J = 6.6 Hz, CH3-16), 1.08 (3H, s, CH3-19), 1.15 (3H, s, CH3-18), 1.16 (4H, m, CH3-4′, CH3-4″, CH3-5′, and CH3-5″), 1.46 (1H, dd, J = 11.1 and 8.4 Hz, H11), 1.76 (1H, s, H-8b), 1.74 (1H, dd, J = 9.2 and 2.3 Hz, H-4), 1.86 (3H, s, CH3-20), 1.94 (1H, br s, H-2), 2.09 (1H, m, H-8a), 2.20 (1H, m, H-7a), 2.34 (1H, s, H-17a), 2.50 (1H, d, J = 11.4 Hz, H-17b), 3.01 (2H, ddq, J = 21.3, 14.0, and 7.0 Hz, H-2′a and H-2″a)*, 3.33 (5H, m, H-1α, H-3′ and H-3″)*, 3.47 (1H, td, J = 14.2 and 7.1 Hz, H-2′b)*, 3.91 (1H, dq, J = 13.9 and 6.1 Hz, H-2″b)*, 4.07 (1H, s, H-3), 6.16 (1H, d, J = 9.2 Hz, H-5), 6.59 (1H, d, J = 11.4 Hz); 13C NMR (100 MHz, CDCl3) δ 12.7 (C-20), 13.7 (C-16), 13.8 (C-4′)*, 13.8 (C-4″)*, 14.5 (C-5′)*, 14.6 (C-5″)*, 16.5 (C-19), 20.8 (C-8), 25.7 (C-10), 29.2 (C-18), 29.3 (C-11), 33.9 (C-7), 34.8 (C-9), 38.6 (C-2), 41.5 (C-2′)*, 41.9 (C-2″)*, 42.0 (C-3′)*, 42.7 (C-3″)*, 47.9 (C-1), 52.5 (C-4), 55.4 (C-17), 59.8 (C-6), 67.7 (C-5), 79.4 (C-3), 92.1 (C-15), 136.7 (C13), 143.2 (C-12), 155.9 (C-1′), 156.8 (C-1″), 197.5 (C-14); ESIMS m/z 549.03 [M + H]+. Epoxycarbamoylboetirane C (20): obtained from the reaction of 2 (23 mg, 0.066 mmol) with 4-morpholinocarbonyl chloride (100 μL, 0.872 mmol, 13.3 equiv) for 24 h under reflux. The residue was purified by CC (n-hexane/EtOAc, 1:0 to 1:1) to afford 31 mg (0.054 mmol, 82% yield) of white crystals: mp 125−126 °C (CH2Cl2/ 1 MeOH); [α]25 D +120 (c 0.1, CHCl3); H NMR (400 MHz, CDCl3) δ 0.95 (1H, t, J = 3.2 Hz, H-7b), 1.03 (4H, d, J = 6.4 Hz, H-9 and CH316), 1.14 (3H, s, CH3-19), 1.18 (3H, s, CH3-18), 1.29 (1H, dd, J = 11.2 and 8.4 Hz, H-11), 1.58 (1H, m, H-1β), 1.62 (1H, m, H-8b), 1.73 (1H, dd, J = 9.2 and 2.4 Hz, H-4), 1.87 (3H, s, CH3-20), 1.93 (1H, m, H-2), 2.10 (1H, m, H-8a), 2.22 (1H, dd, J = 14.0 and 5.6 Hz, H-7a), 2.32 (1H, s, H-17a), 2.52 (1H, d, J = 3.2 Hz, H-17b), 3.28 (1H, m, H2′a)*, 3.36 (1H, dd, J = 13.6 and 8.0 Hz, H-1α), 3.54 (5H, m, H-3′a, H-4′, H-4″, H-5′, and H-5″)*, 3.66 (4H, m, H-2′b, H-2″a, H-2″b, and H-3′b)*, 4.04 (1H, s, H-3), 6.12 (1H, d, J = 9.2 Hz, H-5), 6.65 (1H, d, J = 11.2 Hz, H-12); 13C NMR (100 MHz, CDCl3) δ 12.6 (C-20), 13.5 (C-16), 16.5 (C-19), 20.5 (C-8), 25.6 (C-10), 29.0 (C-11 and C-18), 33.6 (C-7), 34.5 (C-9), 38.4 (C-2), 44.0 (C-2′)*, 44.5 (C-2″ and C4′)*, 44.8 (C-4″)*, 47.5 (C-1), 52.0 (C-4), 55.1 (C-17), 59.3 (C-6), 66.5 (C-3′ and C-5′)*, 66.6 (C-3″ and C-5″)*, 67.9 (C-5), 79.1 (C-3), 92.4 (C-15), 136.3 (C-13), 143.3 (C-12), 150.2 (C-1′), 153.2 (C-1″), 196.6 (C-14); ESIMS m/z 577.40 [M + H]+. K




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Pentoxyboetirane B1 (24): obtained from the reaction of 6 (20 mg, 0.056 mmol) with acetic anhydride (1 mL) for 48 h at room temperature. The residue was purified by CC (n-hexane/EtOAc, 1:0 to 3:7) to afford 4 mg (0.007 mmol, 13% yield) of brown oil: [α]25 D −80 (c 0.06, CHCl3); 1H NMR (400 MHz, CDCl3) δ 0.85 (1H, m, H-9), 0.90 (3H, d, J = 6.6 Hz, CH3-16), 1.05 (3H, s, CH3-18), 1.10 (1H, m, H-8b), 1.19 (3H, s, CH3-19), 1.21 (2H, m, H-7b and H-11), 1.25 (3H, s, CH3-17), 1.77 (3H, s, CH3-20), 1.80 (2H, m, H-1β and H-8a), 1.96 (3H, s, CH3-2c)*, 1.97 (3H, s, CH3-2d)*, 2.03 (1H, d, J = 2.6 Hz, H4), 2.09 (3H, s, CH3-2e)*, 2.10 (6H, s, CH3-2a and CH3-2b)*, 2.17 (2H, m, H-2 and H-7a), 2.49 (1H, dd, J = 15.6 and 8.9 Hz, H-1α), 5.38 (1H, d, J = 10.9 Hz, H-12), 5.60 (1H, s, H-5), 5.74 (1H, t, J = 4.0 Hz, H-14), 5.89 (1H, s, H-3); 13C NMR (100 MHz, CDCl3) δ 14.8 (C-16), 15.7 (C-19), 16.1 (C-20), 19.5 (C-8), 21.2 (C-2a)*, 21.8 (C2b and C-2c)*, 22.6 (C-2d)*, 22.4 (C-2e)*, 22.9 (C-10), 24.3 (C-11), 26.4 (C-17), 29.3 (C-18), 38.0 (C-2), 38.8 (C-7), 45.7 (C-1), 48.4 (C4), 72.5 (C-5), 76.3 (C-6), 77.7 (C-14), 77.9 (C-3), 91.7 (C-15), 124.9 (C-12), 129.3 (C-13), 169.6 (C-1a)*, 170.6 (C-1b)*, 171.3 (C1c)*, 171.8 (C-1d)*, 172.5 (C-1e)*; ESIMS m/z 604.29 [M + H + K]+. Pentoxyboetirane B2 (25) and Pentoxyboetirane B3 (26): obtained from the reaction of 6 (24 mg, 0.068 mmol) with benzoyl chloride (100 μL, 0.861 mmol, 12.7 equiv) for 24 h at room temperature. The residue was purified by CC (n-hexane/EtOAc, 1:0 to 7:3) to afford 9 mg (0.02 mmol, 29% yield) of 25 (amorphous, white 1 powder): [α]25 D −42 (c 0.08, CHCl3); H NMR (400 MHz, CDCl3) δ 0.83 (1H, m, H-9), 0.97 (3H, d, J = 6.5 Hz, CH3-16), 1.08 (3H, s, CH3-18), 1.09 (1H, m, H-8b), 1.15 (3H, s, CH3-19), 1.20 (3H, s, CH3-17), 1.24 (1H, m, H-7b), 1.40 (1H, s, H-11), 1.64 (1H, m, H-8a), 1.73 (1H, m, H-1β), 1.75 (3H, s, CH3-20), 1.80 (1H, m, H-2), 1.97 (1H, d, J = 3.0 Hz, H-4), 2.04 (1H, s, OH), 2.17 (1H, m, H-7a), 2.29 (1H, m, H-1α), 3.00 (1H, s, OH), 3.85 (1H, s, OH), 4.25 (1H, s, H14), 4.28 (1H, s, H-5), 5.39 (1H, s, OH), 5.83 (1H, d, J = 10.9 Hz, H12), 5.94 (1H, s, H-3), 7.46 (2H, t, J = 7.4 Hz, H-4′ and H-6′), 7.58 (1H, t, J = 7.4 Hz, H-5′), 8.07 (2H, d, J = 8.2 Hz, H-3′ and H-7′); 13C NMR (100 MHz, CDCl3) δ 14.9 (C-16), 15.1 (C-19), 15.9 (C-20), 19.1 (C-8), 22.5 (C-10), 24.6 (C-17), 25.7 (C-11), 29.4 (C-18), 32.4 (C-9), 37.6 (C-7), 37.9 (C-2), 47.0 (C-4), 48.7 (C-1), 74.0 (C-5), 75.6 (C-6), 78.9 (C-14), 80.3 (C-3), 88.3 (C-15), 123.4 (C-12), 128.9 (C4′), 129.0 (C-6′), 129.9 (C-3′), 130.0 (C-7′), 130.5 (C-2′), 132.1 (C13), 133.4 (C-5′), 165.2 (C-1′); ESIMS m/z 458 [M]+. This reaction also afforded 10 mg (0.018 mmol, 26% yield) of 26 (amorphous, white 1 powder): [α]25 D +18 (c 0.080, CHCl3); H NMR (400 MHz, CDCl3) δ 0.85 (1H, m, H-9), 1.00 (3H, d, J = 6.7 Hz, CH3-16), 1.03 (3H, s, CH3-18), 1.12 (1H, m, H-8b), 1.19 (3H, s, CH3-17), 1.26 (2H, m, H7b and H-11), 1.74 (1H, m, H-8a), 1.87 (1H, m, H-1β), 1.88 (3H, s, CH3-20), 1.92 (1H, m, H-7a), 2.16 (1H, d, J = 4.2 Hz, H-4), 2.31 (1H, m, H-2), 2.43 (1H, dd, J = 13.9 and 9.7 Hz, H-1α), 3.36 (1H, s, OH), 3.85 (1H, s, OH), 4.45 (1H, s, H-5), 5.64 (1H, s, H-4), 5.69 (1H, d, J = 10.7 Hz, H-12), 6.00 (1H, t, J = 4.3 Hz, H-14), 7.46 (4H, dt, J = 19.4 and 7.6 Hz, H-4′, H-6′, H-4″, and H-6″), 7.58 (2H, m, H-5′ and H5″), 8.07 (4H, dd, J = 13.1 and 7.8 Hz, H-3′, H-7′, H-3″, and H-7″); 13 C NMR (100 MHz, CDCl3) δ 15.2 (C-16), 15.4 (C-19), 17.0 (C20), 19.2 (C-8), 22.5 (C-10), 24.7 (C-11), 25.6 (C-17), 29.1 (C-18), 32.5 (C-9), 37.0 (C-7), 37.5 (C-2), 46.9 (C-4), 47.5 (C-1), 74.1 (C-5), 74.9 (C-6), 80.2 (C-14), 80.5 (C-3), 84.9 (C-15), 125.2 (C-12), 129.0 (C-4′, C-4″, C-6′, and C-6″)*, 129.4 (C-13), 129.8 (C-3′ and C-7′)*, 129.9 (C-2′)*, 130.0 (C-3″ and C-7″)*, 130.4 (C-2″)*, 133.6 (C-5′)*, 133.7 (C-5″)*, 165.8 (C-1′), 165.8 (C-1″); ESIMS m/z 562.28 [M]+. Cell Lines and Cultures. L5178Y mouse T-lymphoma cells (ECACC catalog no. 87111908, U.S. FDA, Silver Spring, MD, USA) were transfected with the pHa MDR1/A retrovirus.20 The ABCB1expressing cell line was selected by culturing the infected cells with 60 ng/mL of colchicine (Sigma-Aldrich Chemie GmbH, Steinheim, Germany), to maintain the MDS phenotype expression. L5178Y (parental, PAR) mouse T-cell lymphoma cells and the human ABCB1transfected subline were cultured in McCoy’s 5A supplemented with 10% heat-inactivated horse serum, 100 U/L L-glutamine, and 100 mg/ L penicillin−streptomycin mixture, all obtained from Sigma-Aldrich. The human colon adenocarcinoma cell lines (Colo 205 parent and

Methoxyboetirane A (21): obtained from the reaction of 3 (20 mg, 0.052 mmol) with acetic anhydride (1 mL) for 12 h at room temperature. The residue was purified by CC (n-hexane/EtOAc, 1:0 to 7:3) to afford 26 mg (0.052 mmol, 98% yield) of a brown oil: [α]25 D −35 (c 0.1, CHCl3); 1H NMR (400 MHz, CDCl3) δ 0.29 (1H, t, J = 9.1 Hz, H-9), 0.85 (2H, m, H-8b and H-11), 0.92 (3H, s, CH3-19), 0.93 (3H, d, J = 5.7 Hz, CH3-16), 1.03 (3H, s, CH3-18), 1.11 (1H, m, H-7b), 1.19 (3H, d, J = 7.2 Hz, CH3-20), 1.60 (2H, m, H-7a and H8a), 1.65 (1H, m, H-1β), 1.86 (1H, m, H-2), 2.06 (1H, m, H-1α), 2.08 (3H, s, CH3-2′)*, 2.09 (3H, s, CH3-2″)*, 2.13 (3H, s, CH3-2‴)*, 2.79 (1H, d, J = 8.5 Hz, H-5), 3.06 (1H, dd, J = 8.3 and 4.5 Hz, H-4), 3.33 (3H, s, OMe-12), 3.50 (1H, dd, J = 10.7 and 4.6 Hz, H-12), 3.70 (1H, d, J = 12.0 Hz, H-17a), 3.88 (1H, m, H-13), 4.39 (1H, d, J = 12.1 Hz, H-17b), 5.52 (1H, br s, H-3); 13C NMR (100 MHz, CDCl3) δ 10.9 (C-20), 13.5 (C-16), 16.1 (C-19), 18.2 (C-10), 19.5 (C-8), 21.0 (C2′)*, 21.1 (C-2″)*, 21.6 (C-2‴)*, 25.6 (C-11), 27.2 (C-9), 27.5 (C7), 28.9 (C-18), 36.4 (C-2), 41.9 (C-13), 46.7 (C-4), 49.0 (C-1), 56.4 (12-OMe), 57.2 (C-5), 61.7 (C-6), 67.2 (C-17), 78.2 (C-3), 80.0 (C12), 88.2 (C-15), 170.1 (C-1′ and C-1″), 171.3 (C-1‴), 211.03 (C14); ESIMS m/z 483.96 [M − CH3CO + H2O]+, 435.28 [M − C3H5O2]+. Methoxyboetirane B (22): obtained from the reaction of 3 (17 mg, 0.044 mmol) with benzoyl chloride (70 μL, 0.603 mmol, 13.6 equiv) for 6 h at room temperature. The residue was purified by CC (nhexane/EtOAc, 1:0 to 8:1) to afford 27 mg (0.046 mmol, 97% yield) 1 of an amorphous, yellow powder: [α]25 D −31 (c 0.09, CHCl3); H NMR (400 MHz, CDCl3) δ 0.32 (1H, t, J = 9.2, H-9), 0.88 (4H, s, H11 and CH3-19), 1.00 (3H, d, J = 6.6 Hz, CH3-16), 1.02 (3H, s, CH318), 1.23 (3H, d, J = 6.7 Hz, CH3-20), 1.24 (1H, m, H-8b), 1.67 (1H, m, H-8a), 1.86 (1H, t, J = 13.1 Hz, H-1β), 2.00 (2H, m, H-2 and H7b), 2.18 (1H, m, H-7a), 2.20 (1H, m, H-1α), 3.02 (1H, d, J = 8.5 Hz, H-5), 3.24 (1H, dd, J = 8.6 and 4.3 Hz, H-4), 3.34 (3H, s, OMe-12), 3.53 (1H, dd, J = 10.7 and 4.6 Hz, H-12), 3.94 (1H, m, H-13), 4.02 (1H, d, J = 12.1 Hz, H-17a), 4.53 (1H, d, J = 12.1 Hz, H-17b), 5.81 (1H, s, H-3), 7.30 (2H, t, J = 7.8 Hz, H-4′ and H-6′)*, 7.41 (2H, t, J = 7.7 Hz, H-4″ and H-6″)*, 7.50 (1H, t, J = 7.4 Hz, H-5′)*, 7.56 (1H, t, J = 7.5 Hz, H-5″)*, 7.94 (2H, d, J = 7.3 Hz, H-3′ and H-7′)*, 8.01 (2H, d, J = 7.3 Hz, H-3″ and H-7″)*; 13C NMR (100 MHz, CDCl3) δ 10.7 (C-20), 13.3 (C-16), 15.8 (C-19), 17.9 (C-10), 19.5 (C-8), 25.4 (C-11), 27.0 (C-9), 27.8 (C-7), 28.6 (C-18), 36.9 (C-2), 41.7 (C-13), 49.0 (C-1), 56.2 (OMe-12), 56.7 (C-5), 61.7 (C-6), 67.0 (C-17), 78.2 (C-12), 80.0 (C-3), 88.1 (C-15), 128.4 (C-4′ and C-6′)*, 128.6 (C-4″ and C-6″)*, 129.5 (C-3′ and C-7′)*, 129.6 (C-3″ and C-7″)*, 133.2 (C-5′)*, 133.3 (C-5″)*, 133.4 (C-2′)*, 134.2 (C-2″)*, 165.5 (C-1′), 166.6 (C-1″), 210.9 (C-14); ESIMS m/z 608.38 [M + H2O]+, 591.35 [M + H]+. Methoxyboetirane C (23): obtained from the reaction of 3 (25 mg, 0.065 mmol) with 4-(trifluoromethyl)benzoyl chloride (75 μL, 0.505 mmol, 7.7 equiv) for 24 h at room temperature. The residue was purified by CC (n-hexane/EtOAc, 1:0 to 17:3) to afford 16 mg (0.022 mmol, 34% yield) of an amorphous, white powder: [α]25 D −17 (c 0.1, CHCl3); 1H NMR (400 MHz, CDCl3) δ 0.33 (1H, t, J = 9.0 Hz, H-9), 0.82 (1H, m, H-11), 1.00 (3H, d, J = 6.6 Hz, CH3-16), 1.03 (3H, s, CH3-18), 1.23 (4H, d, J = 6.0 Hz, H-8b and CH3-20), 1.63 (1H, m, H8a), 1.84 (1H, t, J = 13.1 Hz, H-1β), 2.03 (2H, m, H-2 and H-7b), 2.19 (2H, m, H-1α and H-7a), 2.96 (1H, d, J = 8.6 Hz, H-5), 3.26 (1H, dd, J = 8.4 and 4.4 Hz, H-4), 3.34 (3H, s, OMe-12), 3.49 (1H, m, H-12), 3.92 (1H, m, H-13), 4.10 (1H, d, J = 12.2 Hz, H-17a), 4.55 (1H, d, J = 12.1 Hz, H-17b), 5.83 (1H, t, J = 3.4 Hz, H-3), 7.54 (2H, d, J = 8.2 Hz, H-4′ and H-6′)*, 7.69 (2H, d, J = 8.1 Hz, H-4″ and H-6″)*, 8.02 (2H, d, J = 8.1 Hz, H-3′ and H-7′)*, 8.12 (2H, d, J = 8.1 Hz, H-3″ and H7″)*; 13C NMR (100 MHz, CDCl3) δ 10.7 (C-20), 13.3 (C-16), 15.8 (C-19), 17.9 (C-10), 19.5 (C-8), 25.4 (C-11), 27.0 (C-9), 27.8 (C-7), 28.6 (C-18), 36.8 (C-2), 41.7 (C-13), 48.9 (C-1), 56.2 (OMe-12), 56.8 (C-5), 61.6 (C-6), 67.0 (C-17), 78.0 (C-12), 88.0 (C-15), 122.0 (CF3-5′)*, 124.8 (CF3-5″)*, 125.4 (C-4′ and C-6′)*, 125.7 (C-4″ and C-6″)*, 130.0 (C-3′, C-3″, C-7′ and C-7″)*, 134.7 (C-5′ and C-5″)*, 164.3 (C-1′), 165.2 (C-1″), 210.8 (C-14); ESIMS m/z 744.35 [M + H2O]+, 695.29 [M − CH3O]+. L




Article

medium control wells, to a final volume of 200 μL per well. The plates were incubated for 48 h at 37 °C in a CO2 incubator, and at the end of the incubation period, the cell growth was determined by the MTT staining method, as described earlier. Drug interactions were evaluated using CompuSyn software.24 Each dose−response curve (for individual agents as well as combinations) was fit to a linear model using the median effect equation, in order to obtain the median effect value (corresponding to the IC50) and slope (m).18,25 The goodnessof-fit was assessed using the linear correlation coefficient, r, and only data from analysis with r > 0.90 were presented. The extent of interaction between drugs was expressed using the combination index, in which a CI value close to 1 indicates additivity, while a CI < 1 is defined as synergy and a CI > 1 as antagonism. ATPase Activity Assays. ABCB1 ATPase activity was determined using the SB-MDR1 PREADEASY ATPase kit (SOLVO Biotechnology, Szeged, Hungary) according to the manufacturer’s instructions. Briefly, the purified Sf9 insect membrane vesicles (4 μg protein/well), expressing high levels of human ABCB1, were incubated in 50 μL of ATPase assay buffer plus the assayed compounds and 2 mM MgATP for 10 min at 37 °C. Compounds were tested at 0.78, 1.56, 3.13, 6.25, 12.5, 25.0, 50.0, and 100.0 μM. The final concentration of DMSO in the experiment was 2%. The ATPase reaction was stopped, and the inorganic phosphate (Pi) produced was measured colorimetrically (optical density was read at 630 nm). The amount of Pi liberated by the transporter was proportional to its activity. Hence, ATPase activities were determined as the difference of the measured Pi liberation with and without the presence of 1.2 mM sodium orthovanadate (vanadate-sensitive ATPase activity). This ATPase kit comprises two different tests: the activation and inhibition assays. The activation assay detects compounds that are transported by ABCB1 and thus stimulate baseline vanadate-sensitive ATPase activity, such as verapamil (40 μM), which was used as a positive control. In the inhibition assay, the compounds were incubated in the presence of verapamil (40 μM), and thus, inhibitors may reduce the verapamilstimulated vanadate-sensitive ATPase activity. In some cases inhibitors may inhibit the baseline transporter ATPase activity as well, such as cyclosporine A (40 μM), also used as a positive control.

Colo 320/MDR-LRP expressing ABCB1), ATCC-CCL-220.1 (Colo 320), and CCL-222 (Colo 205) were purchased from LGC Promochem, Teddington, England. The cells were cultured in RPMI 1640 medium supplemented with 10% heat-inactivated fetal bovine serum, 2 mM L-glutamine, 1 mM Na pyruvate, and 100 mM HEPES. The semiadherent human colon cancer cells were detached with 0.25% trypsin and 0.02% EDTA for 5 min at 37 °C. Antiproliferative Assays. The antiproliferative effects of all compounds were tested in a range of decreasing concentrations using both mouse lymphoma and human colon adenocarcinoma cell lines as experimental models, in 96-well flat-bottomed microtiter plates. First, the compounds were diluted in 100 μL of medium. The maximum tested concentration of each compound was 100 μM, with the exception of compounds 9, 13, and 15, which was 50 μM due to DMSO solubility problems. Then, 6 × 103 cells in 100 μL of medium were added to each well, with the exception of the medium control wells. The culture plates were initially incubated at 37 °C for 72 h, and, at the end of the incubation period, 20 μL of MTT (Sigma-Aldrich Chemie GmbH, Steinheim) solution (5 mg/mL in PBS) was added to each well and incubated for another 4 h. Then, 100 μL of 10% SDS (sodium dodecyl sulfate, Sigma) solution (10% in 0.01 M HCl) was added into each well, and the plates were further incubated overnight at 37 °C. Cell growth was determined by measuring the optical density (OD) at 550 nm (ref 630 nm) with a Multiscan EX ELISA reader (Thermo Labsystems, Cheshire, WA, USA). The percentage of inhibition of cell growth was determined according to eq 1. All experiments were performed in triplicate. The results were expressed as the mean ± SD, and the IC50 values were obtained by best fitting the dose-dependent inhibition curves in GraphPad Prism 5 software.21,22 Only data from analysis with R2 > 0.90 were presented. ⎡ ODsample − ODmedium control ⎤ 100 − ⎢ ⎥ × 100 ⎣ ODcell control − ODmedium control ⎦

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Rhodamine-123 Accumulation Assay. Mouse PAR T-lymphoma cells or human Colo 320 colon adenocarcinoma cells were adjusted to a density of 2 × 106 cells/mL, resuspended in serum-free McCoy’s 5A medium or RPMI 1640, respectively, and distributed in 500 μL aliquots into Eppendorf centrifuge tubes. Aliquots (10 μL) of test compounds were added at various concentrations (0.2, 2, or 20 μM), and verapamil (positive control, EGIS Pharmaceuticals PLC, Budapest, Hungary) was added at 20 μM. DMSO at 4% was also added as solvent control. The samples were incubated for 10 min at room temperature, after which 10 μL of rhodamine-123 (5.2 μM final concentration) was added. After 20 min of incubation at 37 °C, the samples were washed twice, resuspended in 500 μL of phosphatebuffered saline (PBS), and analyzed by flow cytometry (Partec CyFlow Space Instrument, Partec GmbH, Münster, Germany). The resulting histograms were evaluated regarding mean fluorescence intensity (FL1), standard deviation, both forward scatter (FSC) and side scatter (SSC) parameters, and the peak channel of 20 000 individual cells belonging to the total and the gated populations (Figures S5−S16, Supporting Information). The fluorescence activity ratio was calculated on the basis of the quotient between FL-1 of treated/ untreated resistant cell line (ABCB1-transfected mouse lymphoma or Colo 320 human colon adenocarcinoma cells) over the respective treated/untreated sensitive cell line (PAR mouse lymphoma or Colo 205 human colon adenocarcinoma cells), according to eq 2.23

FAR =

FL‐1 MDR treated/FL‐1 MDR untreated FL‐1 PAR treated/FL‐1 PAR untreated

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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.5b00370. Additional figures and tables illustrating the structural identification of new compounds, cytotoxicity and antiproliferative assays, histograms related to the rhodamine-123 accumulation assays on human colon adenocarcinoma and mouse lymphoma cells, physicochemical properties of compounds, and numbered structures of new compounds (PDF)

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AUTHOR INFORMATION

Corresponding Author

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

The authors declare no competing financial interest.

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Drug Combination Assay. Doxorubicin (2 mg/mL, Teva Pharmaceuticals, Budapest, Hungary) was serially diluted in the horizontal direction as previously described, starting with 8 μg/mL. The resistance modifier was subsequently diluted in the vertical direction, starting with 20 μg/mL. The dilutions of doxorubicin were made in a horizontal direction in 100 μL, and the dilutions of the resistance modifiers vertically in the microtiter plate in 50 μL volume. The cells were resuspended in culture medium and distributed into each well in 50 μL containing 1 × 104 cells, with the exception of the

ACKNOWLEDGMENTS The authors would like to acknowledge FCT (Fundaçaõ para a Ciência e a Tecnologia) for financing this study (projects PTDC/QEQ-MED/0985/2012, Pest-OE/SAU/UI4013/2014, REDE/1518/REM2005, Ph.D. grant SFRH/BD/72915/2010) and Dr. J. Ferreira for providing ESIMS data at the Faculdade de Farmácia da Universidade de Lisboa. M




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REFERENCES

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Epoxylathyrol Derivatives: Modulation of ABCB1-Mediated Multidrug

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