Jatrophane Diterpenoids as Modulators of P-Glycoprotein-Dependent

Article pubs.acs.org/jmc

Jatrophane Diterpenoids as Modulators of P‑GlycoproteinDependent Multidrug Resistance (MDR): Advances of Structure− Activity Relationships and Discovery of Promising MDR Reversal Agents Jianyong Zhu,† Ruimin Wang,† Lanlan Lou, Wei Li, Guihua Tang, Xianzhang Bu,* and Sheng Yin* School of Pharmaceutical Sciences, Sun Yat-sen University, Guangzhou 510006, People’s Republic of China S Supporting Information *

ABSTRACT: The phytochemical study of Pedilanthus tithymaloides led to the isolation of 13 jatrophane diterpenoids (1−13), of which eight (1−8) are new. Subsequent structural modification of the major components by esterification, hydrolysis, hydrogenation, or epoxidation yielded 22 new derivatives (14−35). Thus, a jatrophane library containing two series of compounds was established to screen for P-glycoprotein (Pgp)-dependent MDR modulators. The activity was evaluated through a combination of Rho123 efflux and chemoreversal assays on adriamycin resistant human hepatocellular carcinoma cell line HepG2 (HepG2/ADR) and adriamycin resistant human breast adenocarcinoma cell line MCF-7 (MCF-7/ADR). Compounds 19, 25, and 26 were identified as potent MDR modulators with greater chemoreversal ability and less cytotoxicity than the thirdgeneration drug tariquidar. The structure−activity relationship (SAR) was discussed, which showed that modifications beyond just increasing the lipophilicity of this class of Pgp inhibitors are beneficial to the activity. Compound 26, which exhibited a remarkable metabolic stability in vitro and a favorable antitumor effect in vivo, would serve as a promising lead for the development of new MDR reversal agents.

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INTRODUCTION

One of the most accepted strategies to overcome Pgpmediated MDR is based on the development of reversal agents that when coadministered with an anticancer drug will restore their chemotherapeutic efficacy without directly killing MDR cells.4 In this manner, a considerable number of MDR reversal agents targeting Pgp have been reported. However, despite encouraging results in in vitro assays, to date, there are no reversal agents clinically available because of their intrinsic toxicity, low affinity, and unfavorable pharmacokinetic properties. For instance, first-generation modulators such as verapamil and cyclosporine A,5,6 besides revealing low affinity for Pgp, had distinct pharmacological actions on the cardiovascular (calcium channel blocker) and immune (immunosuppressant) systems. Second-generation modulators (e.g., valspodar, elacridar, biricodar, and dexverapamil) have reduced side

MDR designates a phenomenon where resistance to one drug is accompanied by resistance to drugs that are structurally and functionally unrelated. The development of MDR is one of the leading causes of treatment failure in the chemotherapy of malignant tumors. A primary mechanism of MDR is the overproduction of Pgp in the plasma membranes of resistant cells, where the Pgp acts as an energy-dependent efflux pump, reducing the intracellular accumulation of anticancer drugs.1 Pgp is the product of the human multidrug resistance gene (MDR1). It is classified as a pseudosymmetrical heterodimer, where each monomer is composed of six membrane spanning segments (i.e., transmembrane domains, TMDs) and one nucleotide-binding domain (NBD). The TMDs mediate the recognition and transport of substrates, while the NBDs are responsible for the ATP-binding and hydrolysis and consequently for conformational changes of the protein.2,3 © XXXX American Chemical Society

Received: April 20, 2016

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DOI: 10.1021/acs.jmedchem.6b00605 J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

Article

Figure 1. Structures of compounds 1−35.

and possess 5/12- and 5/11/3-membered carbon ring systems, respectively. The reported SARs suggested that the hydrophobicity was the key factor for their Pgp inhibition activities, and the detailed SARs of lathyranes were well investigated by esterification and epoxidation of the major isolates.10,12 For jatrophane diterpenes, the substitutions of the “southwestern” fragment (C-2, C-3, and C-5) were thought to be critical factors for the activity.8 The activity would be collapsed by the acylation of the free hydroxyl at C-3, and the hydroxylation or acylation of C-2 also led to a decrease of the activity. Moreover, analogues with a carbonyl at C-14, an acetoxyl at C-9, or a free hydroxyl at C-15 were beneficial to the activity.9 However,

effects, but showed increased toxicity and pharmacokinetic alterations of cytotoxic drugs.5,6 Third-generation MDR modulators, such as tariquidar (XR9576) and zosuquidar (LY335979),5,6 inhibit Pgp at the nanomolar level but still suffer the unexpected toxicity in phase III clinical trials. In recent years, the search for MDR modulators from natural products has attracted considerable interest of medicinal chemists. As a promising example, macrocyclic diterpenes isolated from the family Euphorbia, especially for jatrophanes and lathyranes, were found to be potential MDR modulators targeting Pgp.7−12 These two structurally related types of diterpenes are usually substituted with a variety of acyl groups B



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Figure 2. ORTEP diagram of compounds 1, 3, 6, 9, 12, and 33.

Among them, compounds 1 and 9−13, with good yield, gave us a good starting point to engage the structural modification for constructing a jatrophane library for this SAR study. As a result, a total of 22 new derivatives (14−35) were prepared through esterification, hydrolysis, or epoxidation modifications. The library containing 35 compounds represents two groups of jatrophanes (I and II) with the presence of 8-OAc or 8methylene (Figure 1). Rhodamine 123 (Rho123) efflux and chemoreversal assays indicated that a series of compounds were very effective in reversing the Pgp-dependent MDR on cell lines HepG2/ADR and MCF-7/ADR. The in vivo effect of compound 26 was also validated on a HepG2/ADR xenografts

these reported SARs of jatrophanes only based on the investigations of limited natural molecules within certain subtypes, lacking structurally diverse samples to support these conclusions. Besides, the pharmaceutical properties of these jatrophane Pgp inhibitors remain uninvestigated. In this regard, there is an urgent need for an illustration of detailed SARs based on the improved diversity of jatrophane categories, and subsequent pharmaceutical evaluation of potent compounds for development of jatrophane-based MDR reversal leads. In our continuing study toward understanding the chemistry of the plants from the Euphorbiaceae Family,13,14 13 jatrophane diterpenoids (1−13) were isolated from P. tithymaloides. C



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confirmed by a single crystal X-ray crystallographic analysis using anomalous scattering of Cu Kα radiation [the flack parameter, 0.03 (9)] (Figure 2). Therefore, compound 2 was determined to be (1S,2R,3S,4S,5S,8R,9S,14R,15S)1,7,8,9,14,15-hexaacetoxy-3-nicotinoyloxy-5-hydroxyjatropha6E,12E-diene and was given the trivial name peditithin B. Compound 3 had the molecular formula C39H50O16, as determined by HRESIMS and 13C NMR data. The NMR spectra of 3 revealed similar structural features to those found in 9, with the major differences being the presence of a terminal double bond (δC 152.3 and 115.7) and an oxymethine carbon signal (δC 66.4) in 3 instead of the Δ12 in 9, indicating that 3 was a Δ12-rearranged derivative of 9. HBMC correlations from H2-20 (δH 5.47 and 5.01) to C-13 (δC 152.3), C-14 (δC 71.2), and C-12 (δC 66.4) suggested that the exomethylene group was located at C-13, while HMBC correlations from H-12 (δH 4.86) to C-13, C-14, C-20, and C-10 suggested that a hydroxyl group was located at C-12. The relative configuration of 3 was assigned to be the same as that of 9 by NOESY experiment and by comparison of their 13C NMR data. In particular, the βorientation of 12-OH was assigned by NOE correlations of H12/H-4, H-8, and H-18. The structure including the absolute configuration of 3 was finally confirmed by a single crystal X-ray crystallographic analysis using anomalous scattering of Cu Kα radiation [the flack parameter, 0.0 (3)] (Figure 2). Therefore, compound 3 was determined to be (1S,2R,3S,4S,5S,8R,9S,12S,14R,15S)-1,7,8,9,14,15-hexaacetoxy3-benzoyloxy-5,12-dihydroxyjatropha-6E,13(20)-diene and was given the trivial name peditithin C. Compound 4 had the molecular formula C39H50O14, as determined by HRESIMS and 13C NMR data. The 1D NMR spectra of 4 exhibited most of the structural features found in 11, with the major difference being the presence of an acetoxy group (δC 169.0 and 19.5) in 4 instead of the ketone group (C7, δC 197.5) in 11. The acetyl group was located at C-7 by HMBC correlation from H-7 (δH 4.85) to the carbonyl group at δC 169.0. This was supported by the upfield-shifted C-8 signal in 4 (ca. 7 ppm) as compared to 11, bearing a 7-ketone. The structure of 4 was further established by detailed interpretation of its 2D NMR data. In particular, the 7β-OAc was assigned by NOE correlations of H-7/H-17, H-17/H-4, and H-8. Therefore, compound 4 was determined to be (1S,2R,3S,4S,7S,8R,9S,14R,15S)-1,7,8,9,14,15-hexaacetoxy-3benzoyloxyjatropha-5E,12E-diene and was given the trivial name peditithin D. Compound 5 had the molecular formula C39H48O16, as determined by HRESIMS and 13C NMR data. The NMR spectra of 5 revealed similar structural features to those of known analogue 1α,7,8β,9β,14α,15β-hexaacetoxy-3β-nicotinoyloxy-5,13β,11,12β-diepoxyjatropha-6(7)-ene, isolated from P. tithymaloides,15 with the major difference being the presence of a benzoyl group in 5 instead of the nicotinoyl group in the known analogue. The benzoyl group was located at C-3 by HMBC correlation from H-3 (δH 5.61) to the benzoyl carbonyl group at δC 165.3. 5 was the second example of jatrophane diterpenoids featuring a rare epoxide ring between C-5 and C13, which was verified by HMBC correlation of H-5 to C-13. The relative configuration of 5 was established on the basis of NOESY experiment and by analysis of its 1H−1H coupling constants. In particular, the NOE correlations of H-8/H-4, H12, and H3-18 and H-12/H3-18 indicated that these protons were cofacial and designated as α-oriented. Consequently, the NOE correlations of H-9/H3-19 and H-11, H-11/H3-20 and

mouse model. Herein, the structure elucidation, SAR, and chemoreversal ability of these compounds are described.

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RESULTS AND DISCUSSION Isolation and Structure Elucidation. The air-dried powder of the leaves and stems of P. tithymaloides (5 kg) was extracted with 95% EtOH at room temperature (rt) to give a crude extract, which was suspended in H2O and successively partitioned with petroleum ether (PE), EtOAc, and n-BuOH. Various column chromatographic separations of the PE extract afforded compounds 1−13 (Figure 1), of which 1−8 were new. Compound 1, a colorless crystal, had the molecular formula C30H40O11, as established by a HRESIMS ion at m/z 599.2470 [M + Na]+ (calcd for C30H40O11Na, 599.2463), corresponding to 11 degrees of unsaturation. The 1H NMR (Supporting Information, Table S1) and HMQC spectra exhibited signals for five acetyl methyl groups [δH 2.18 (3H, s), 2.15 (3H, s), 2.06 (3H × 2, s), and 2.02 (3H, s)], five methyl groups [1.78 (3H, s), 1.71 (3H, s), 1.21 (3H, d, J = 7.1 Hz), 1.05 (3H, s), and 1.01 (3H, s)], four oxymethine protons [δH 5.86 (1H, s), 5.82 (1H, s), 5.76 (1H, d, J = 7.1 Hz), and 5.15 (1H, s)], and three olefinic protons [δH 6.99 (1H, s), 5.83 (1H, dd, J = 1.8, 1.7 Hz), and 5.79 (1H, m)]. The 13C NMR (Supporting Information, Table S2) spectrum, in combination with DEPT experiments, resolved 30 carbon resonances attributable to one α,β-unsaturated ketone (δC 194.9), five acetyl carbonyl groups (δC 170.7, 170.4, 170.0, 169.7, and 169.5), three trisubstituted double bonds (δC 137.9, 134.6, 136.6, 135.2, 134.2, and 124.5), ten methyls, five sp3 methines (four oxygenated), and one sp3 methylene and two sp3 quaternary carbons (one oxygenated). As 9 of the 11 degrees of unsaturation were accounted for by one ketone, five carbonyls, and three double bonds, the remaining degree of unsaturation required that 1 was bicyclic. The above-mentioned information was quite similar to that of the coisolated known compound 11, except for the absence of one benzoyl group and the presence of an additional trisubstituted double bond (δC 136.6 and 135.2) in 1, indicating that 1 was a derivative of 11 with the elimination of a molecule of benzoic acid. The location of the additional double bond was assigned at Δ3 by HMBC correlations from H-3 (δH 5.83) to C4, C-5, C-2, and C-16 and from H-1 (δH 5.76) to C-3 and C-4. The relative configuration of 1 was assigned to be the same as that of 11 based on their similar 1D NMR data and the NOESY correlations. The structure including the absolute configuration of 1 was finally confirmed by a single crystal X-ray crystallographic analysis using anomalous scattering of Cu Kα radiation [the flack parameter, 0.01(12)] (Figure 2). Therefore, compound 1 was determined to be (1S,2R,8R,9S,14R,15S)1,8,9,14,15-pentaacetoxy-7-oxojatropha-3Z,5E,12E-triene and was given the trivial name peditithin A. Compound 2 had the molecular formula C38H49NO15, as determined by HRESIMS and 13C NMR data. The 1D NMR spectra of 2 exhibited most of the structural features found in 9, with the major difference being the presence of a nicotinoyl group in 2 instead of the benzoyl group in 9. The nicotinoyl group was located at C-3 by HMBC correlation from H-3 (δH 5.48) to the nicotinoyl carbonyl group at δC 165.0. The relative configuration of 2 was assigned to be the same as that of 9 by NOESY experiment and by comparison of their 13C NMR data. The absolute configuration of 2 was assigned to be the same as that of 9 by comparison of their CD spectra, which showed a similar Cotton effect at 209 nm (Δε−64.1) (Supporting Information, Figure S229). The structure of 9 was finally D



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Scheme 1. Synthesis of Jatrophane Derivativesa

a

Reagents and reaction conditions: (a) 1% NaOH in MeOH (m/v), rt, 1 h; (b) benzoyl chlorides, Pyr, rt, 12 h; (c) Dess−Martin periodinane, CH2Cl2, 3 h; (d) m-CPBA, CH2Cl2, rt, 3 h; (e) Ac2O, Pyr, rt, 12 h; (f) tosyl/p-bromobenzoyl/2-furoyl/2-thiophenecarbonyl/benzoyl chlorides, Pyr, rt, 12 h; (g) 10% Pd/C, CH2Cl2, H2, 10 h; (h) 1% HCl, rt, 1 h.

H3-19, and H3-20/H-14 assigned these protons to be βoriented (Supporting Information, Figure S230). The βorientation of H-5 was established by the large coupling constant between H-4 and H-5 (J = 12.4 Hz), while the small coupling constant between H-3 and H-4 (J = 5.4 Hz) suggested H-3 to be α-orientated. On consideration of the biosynthetic origin, 5 should share the same absolute configuration with those above-assigned analogues. Thus, 5 was determined to be (1S,2R,3S,4R,5S,8R,9S,11R,12R,13R,14R,15S)-1,7,8,9,14,15hexaacetoxy-3-benzoyloxy-5,13,11,12-diepoxyjatropha-6E-ene and was given the trivial name peditithin E. Compound 6, a colorless crystal, had the molecular formula C33H44O11, as established by HRESIMS and 13C NMR data. The 1D NMR spectra of 6 exhibited most of the structural

features found in 13, except for the presence of an additional acetyl group [δH 1.07 (3H, s); δC 19.7, 169.3] in 6, indicating that 6 was an acetylated derivative of 13. The location of the additional acetyl group was assigned at OH-7 by HMBC correlation from H-7 to the carbonyl group at δC 169.3. This was supported by the downfield-shifted H-7 signal in 6 with respect to that in 13 [δH 4.91 in 6; δH 4.05 in 13]. The structure including the absolute configuration of 6 was finally confirmed by a single crystal X-ray analysis [flack parameter, − 0.05 (15)] (Figure 2). Therefore, compound 6 was determined to be (1S,2S,3S,4S,7R,9R,13R,14R,15S)-7,9,15-triacetoxy-3-benzoyloxy-1,13,14-trihydroxyjatropha-5E,11E-diene and was given the trivial name peditithin F. E



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Figure 3. Jatrophanes inhibit Pgp-mediated Rho123 efflux. Flow cytometery is used to measure the retention of Rho 123 in P-glycoproteinexpressing HepG2/ADR cells. (A) Western bolt analysis of Pgp expression in HepG2/ADR and MCF-7/ADR cells. (B) (C) Accumulation fold of Rho123 expressed as the ratio of the fluorescent intensity of the compound treated cells to that of untreated cells. Fold of control = (the fluorescence of compound treated cell − background)/(the fluorescence of Rho123-only cell − background).

same hydrolysis product (14) as that of 6. Therefore, compound 8 was determined to be (1S,2S,3S,4S,7R,9R,13R,14R,15S)-9-acetoxy-3,7-dibenzoyloxy1,13,14,15-tetrahydroxyjatropha-5E,11E-diene and was given the trivial name peditithin H. The known compounds 9−13 were identified by comparison of their spectroscopic data with those in the literature.15 It is worth noting that in the current study the absolute configurations of the C-9 and C-13 in 12 and 13 were revised to be R, while the absolute configurations of C-9 in 9, 10, and 11 were revised as S. Structural modification. Compounds 1 and 9−13 isolated in good amounts were selected as the initial structures for the design of various derivatives to investigate the SAR related to the MDR (Scheme 1). Alkaline hydrolysis of 12, 9, and 1 for increase of their hydrophobicity afforded 14−16, 30, 27, and 28. The acylation of the free hydroxyls at C-1 or C-14 in 12 with acetic anhydride or tosyl/p-bromobenzoyl/2-furoyl/2thiophenecarbonyl/benzoyl chlorides in dry pyridine under nitrogen atmosphere to afford 17−23 in 50−97% yields and treatment of 10 with benzoyl chloride under the same conditions gives 29. Oxidation of 9, 13, and 17 with the Dess−Martin periodinane reagent afforded 32, 24, and 25, respectively. Direct oxidation of 11 and 9 with m-CPBA gave 12,13-epoxide derivatives 31 and 33, respectively. The intramolecular epoxy-ring opening of 33 with the presence of hydrochloric acid afforded 34 with a rare C-5−O−C-13 linkage. 34 was further acetylized by acetic anhydride in dry pyridine to

Compound 7 had the molecular formula C38H46O11, as determined by HRESIMS and 13C NMR data. The NMR data of 7 were similar to those of 13 except for the presence of an additional benzoyl group [δH 8.11 (2H, dd J = 7.8, 1.2 Hz), 7.51 (2H, dd J = 7.8, 7.2 Hz), 7.61 (1H, ddd J = 7.8, 7.2, 1.2 Hz); δC 165.0, 129.8, 129.7 × 2, 128.7 × 2, and 133.3] in 7, indicating that 7 was an benzoylated derivative of 13. The location of the benzoyl group was assigned at OH-14 by an HMBC correlation from H-14 to the benzoyl carbonyl group at δC 165.0. This was supported by the downfield-shifted H-14 signal in 7 with respect to that in 13 [δH 5.99 in 7; δH 4.27 in 13]. The relative configuration of 7 was assigned to be the same as that of 6 by NOESY experiment and by comparison of their 13 C NMR data. The structure of 7 was finally confirmed by alkaline hydrolysis of 7, which yielded the same hydrolysis product (14) as that of 6. Therefore, compound 7 was determined to be (1S,2S,3S,4S,7R,9R,13R,14R,15S)-9,15-diacetoxy-3,4-dibenzoyloxy-1,7,13-trihydroxyjatropha-5E,11E-diene and was given the trivial name peditithin G. Compound 8 had the molecular formula C36H44O10, as determined by HRESIMS and 13C NMR data, 43 mass units less than that of 12. The NMR spectra of 8 showed high similarity to those of 12 except for the absence of an acetyl group and the upfield-shifted carbon signal at C-15 (δC 82.9 in 8, δC 91.2 in 12), indicating that 8 was the 15-O-deacetylated derivative of 12. The relative configuration of 8 was assigned to be the same as that of 12 by NOESY experiment and by comparison of their 13C NMR data. The structure of 8 was finally confirmed by alkaline hydrolysis of 8, which yielded the F



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at 3-OH improved the activity (2 vs 9). This result was inconsistent with the previous studies,8 in which the free hydroxyls at C-3 and C-15 were considered to be the positive factors for the activity. The acylation of 14-OH was benefical to the activity (13 vs 7), while the presence of the carbonyl at C14 instead of this hydroxyl had little influence on the activity (17 vs 25). In contrast to Δ11, the epoxidation of Δ12 led to a dramatic decrease in activity (9 vs 33), while hydrogenation of Δ11 had little influence on the activity (26 vs 12). The oxidative (4 vs 11) or substituted (6 vs 12) patterns of C-7 showed little influence on the activity unless with the free hydroxyl on C-7 as mentioned above. The presence of 8-OAc or 8-methylene seemed indifferent to the activity, as compounds with pronounced activity were found in both groups (I and II). The presence of the rare C-5−O−C-13 bridge in jatrophanes was investigated for the first time, which seemed beneficial to the activity, as 35 with such a rare linkage and all of abovementioned favorable features (saturated ring A, 3-OBz, and the minimum of the free hydroxyls) turned out to be a potent Pgp inhibitor. The above-mentioned SAR information is summarized in Figure 4.

generate 35. Compound 26 was produced by palladiumcatalyzed hydrogenation of 12. Jatrophanes Inhibit Pgp-Mediated Rhodamine 123 Efflux. To assess the inhibitory potential of obtained compounds against Pgp, a flow cytometry-based Rho123 effluxion assay was performed. The high expressions of Pgp in HepG2/ADR and MCF-7/ADR were first validated by Western blot in the current work (Figure 3A). The efficiency of Rho123 accumulation was denoted by the relative fluorescence enhancement fold versus the control in the HepG-2/ADR cell line. The third-generation Pgp modulator, tariquidar, was used as positive control. In the first round screening at the concentration of 10 μM, 20 compounds (4−9, 11, 12, 17− 23, 25, 26, 29, 31, and 32) showed more potent inhibitory activity than that of tariquidar (Figure 3B), in which compound 5 with a rare oxygen bridge between C-5 and C-13 represented the most active compound (3.65 fold). Inspired by this result, 34 and 35 with such fragment were synthesized and were included in the second round screening performed at a lower concentration of 1.0 μM (Figure 3B). The inhibitory activity of tariquidar slightly decreased at this concentration, while several compounds (5, 12, 23, 26, 29, 32, and 35) were still better than or comparable to that of tariquidar. Based on the combined results, 18 compounds (4−7, 11, 12, 17, 19−23, 25, 26, 29, 31, 32, and 35) were chosen for a dose−effect dependence study at concentrations of 0.125, 0.25, 0.5, 1.0, and 2.0 μM respectively. Verapamil, the first-generation Pgp modulator, was also included as positive control. As shown in Figure 3C, most of the selected compounds exhibited a good dose-dependent manner and greater activity than that of verapamil at equal concentrations. Compounds 12, 26, 29, and 35, which showed comparable activity (2.80, 2.74, 2.78, 3.07 fold, respectively) to tariquidar (2.97 fold) at 2.0 μM while exhibiting greater potency (1.32, 1.34, 1.37, 1.81 fold, respectively) than tariquidar (1.04 fold) at 0.125 μM, were considered as potent Pgp inhibitors. To the best of our knowledge, compounds of this class represented the most effective jatrophane-type Pgp inhibitors reported so far. Structure−Activity Relationships. In general, compounds with the presence of double bonds at Δ2 or Δ3, such as 1, 24, 27, and 28, showed a dramatic decrease of the activity, indicating that the saturated five-membered ring was important for the activity. Most compounds with more than 3 hydroxyls (13−16 and 30) showed poor activity, which is in accordance with the general role of lipophilicity for Pgp inhibitors.7−12 However, within this group of compounds, the presence of free hydroxyls at the C-1−C-15−C-14−C-13 fragment was not harmful to their activity (8), while the hydroxylation of C-5 (30), C-7 (13−15), or C-9 (14−16) dramatically decreased the potency. The presence of 5-OH was unfavorable to activity, as the elimination or oxidation of 5-OH increased the potency, especially at a low concentration (4 vs 9 and 32 vs 9, respectively), though the 5-OH bearing compounds with fewer hydroxyls (9 and 10) could maintain moderate to high activity. Similarly, the presence of 12-OH was detrimental to activity, as 3 and 34 with this hydroxyl showed poor activity, and the epoxidation (between C-11 and C-12) or acetylation of them enhanced the activity (34 vs 5 and 35). The acylation effects of 1-OH were investigated for the first time, and they showed little influence on the activity (12 vs 17 and 19−23). The acylation of OH-15 did not significantly improve the activity, as shown by 12 vs 8, while the replacement of the nicotinoyl by benzoyl

Figure 4. SARs of jatrophane diterpenes on Pgp inhibition.

Jatrophane Reversal ADR Resistant Cancer Cell Lines HepG2 and MCF-7. As Pgp-mediated efflux is one of the most significant mechanisms of MDR, compounds with good Pgp inhibitory activities would possess the potential to reverse the Pgp-mediated drug resistance. In this regards, compounds 5, 12, 17, 19−23, 25, 26, 29, and 35 with high potency and good dose-dependent effects on Pgp inhibition were further investigated in a drug combination assay on HepG2/ADR and MCF-7/ADR cancer cell lines. First, the cytotoxicity of selected compounds toward both cell lines was evaluated by MTT assay. As shown in Table 1, all jatrophanes show no apparent cytotoxicity toward both ADR resistant cancer cell lines, while the positive control tariquidar was more impressive toward both cell lines, especially in MCF-7/ADR (IC50 = 13.1 μM). Nevertheless, considering the effective concentrations of these compounds in chemoreversal assays was much lower than their cytotoxicity, the intrinsic cytotoxicity of all these compounds was neglected in following evaluations. The MDR chemoreversal assay was initially performed by combination of 200 nM of tested compounds with various concentration of ADR. As shown in Table 1, the cytotoxity of ADR against HepG2/ADR (IC50 = 76.8 μM) and MCF-7/ ADR (IC50 = 72.2 μM) were dramatically increased by the addition of tested jatrophanes, with 29−95 and 4−55 reversal fold, respectively. This reversal ability was generally greater than verapamil (11 and 6 reversal fold, respectively) and weaker than tariquidar (82 and 54 reversal fold, respectively). Compounds 19, 25, and 26 which worked well toward both cell lines, were further tested at the lower (100 nM) and higher (500 nM) concentrations. Although no apparent dose-dependG



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Table 1. Cytotoxicity and Reversal Effects of Selected Compounds toward ADR-Resistant Cancer Cell Lines HepG2/ADR

MCF-7/ADR

Sensitivity Reversal Cmpd (nM)

IC50 (μM) 200 100 200 500 200 100 200 500 200 200 200 200 100 200 500 100 200 500 200 200 100 200 500 100 200 500

5 12

17 19

20 21 22 23 25

26

29 35 Tariquidar

VRP

Control (ADR)d

1.72 1.33 1.36 1.05 2.65 0.82 0.97 0.75 2.45 2.64 2.39 2.02 0.51 0.81 0.60 1.09 1.25 0.59 1.29 1.38 0.80 0.94 0.89 28.9 6.85 4.26 nae

± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

a

Cytotoxicity

Reversal Fold

0.38 0.31 0.16 0.12 0.16 0.10 0.09 0.05 0.42 0.55 0.72 0.73 0.16 0.21 0.04 0.24 0.00 0.13 0.33 0.83 0.31 0.13 0.10 0.45 2.01 1.16

b

IC50 (μM)

45 58 57 73 29 93 79 102 31 29 32 38 150 95 128 71 61 131 60 56 96 82 86 3 11 18 na

c

129.86 >150

93.26 >150

81.31 >150 >150 >150 >150

>150

>150 >150 37.2

138.42

76.8 ± 4.23

Sensitivity Reversal

Cytotoxicity

IC50 (μM)

Reversal fold

10.3 ± 2.58 /f 2.27 ± 0.81 / 3.27 ± 0.64 / 1.32 ± 0.33 / 3.16 ± 0.82 2.25 ± 0.67 1.77 ± 0.28 2.62 ± 1.15 / 2.49 ± 0.98 / / 1.98 ± 0.52 / 3.19 ± 1.35 19.7 ± 6.45 / 1.34 ± 0.41 / / 11.30 ± 5.15 / na

7 / 32 / 22 / 55 / 23 32 41 28 / 29 / / 36 / 23 4 / 54 / / 6 / na

IC50 (μM) 55.6 >150 >150 >150 >150 >150 >150 64.2 >150

>150 >150 >150 13.1

>150 72.2 ± 16.8

Values of IC50 (Pgp inhibitor+ADR) are the mean ± standard error (SE) of at least two independent experiments. bThe reversal fold is calculated as a ratio of IC50 (ADR) to IC50 (Pgp inhibitor+ADR). cIntrinsic cytotoxicity effects of compounds on cells. Values of IC50 of a single experiment. dValues of IC50 (ADR) are the mean ± standard error (SE) of three independent experiments. eNot available. fNot determined. a

ent relationship was observed after at least three repeated tests, substantial activities of these compounds were found at all concentrations, being much greater than verapamil and comparable to tariquidar. These observations suggested that these compounds could serve as promising leads in chemoreversal drug discovery of MDR. Compounds 19, 25, and 26 Showed Low Cytotoxicity on Normal Cell Line Human Lung Fibroblasts Cells. As some of the tested jatrophanes showed a pronounced chemoreversal effect and low cytotoxicity toward the HepG2/ ADR and MCF-7/ADR cell lines, their safety in anticancer therapy was further evaluated in a normal cell line, human lung fibroblasts (HLF). As shown in Table 2, no obvious toxic effects were observed for 19, 25, and 26 at a high concentration, up to 100 μM. In contrast, significant inhibition of HLF cell growth was observed for tariquidar. These results might suggest a better therapeutic index for these compounds than for tariquidar.

Molecular Modeling. To further understand and characterize the interaction between the jatrophanes and Pgp, the most promising compound (26) was selected and subjected to in silico analysis with a homology model of Pgp (PDB code: 3G5U). As shown in Figure 5, 26 was docked well in the TMD regions of Pgp. Four hydrogen bonds formed between OH-1,

Table 2. Cytotoxicity of Selected Compounds toward the HLF Cell Line

Figure 5. Binding pose of 26 with human Pgp. The Pgp model was generated based on the PDB structure of murine Pgp (Code: 3G5U) and was portrayed as a cartoon (gray). Residues involved in the interaction were colored yellow, while the surfaces (yellow) of Phe331, Phe978, and Ile335 were shown to indicate the hydrophobic pocket packing with the phenyl ring in 3-OBz (dotted, dark cyan). The hydrogen bonds were shown as red dashed lines. The structural figures were drawn in PyMOL.

Cmpd

19

25

26

Tariquidar

Verapamil

IC50 (μM)a

>100

>100

>100

16.69 ± 3.13

27.18 ± 1.46

Values of IC50 are the mean ± standard error (SE) of two independent experiments. a

H



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also revealed a superiority of coadministration of 26 over ADR alone. These results validated that as a potent Pgp inhibitor 26 could efficiently restore the sensitivity of MDR human tumors to chemotherapeutic ADR at well-tolerated doses.

OH-13, OH-14, and 3-OBz with the Gln720, Gln985, Phe978, and Tyr305 observed, respectively; the phenyl ring in 3-OBz packed into the hydrophobic pocket formed by Phe331, Phe978, and Ile335, and favored the binding. This model is generally consistent with the summarized SARs. The presence of Δ2 and Δ3 in ring A, which led to the elimination of 3-OBz, caused a dramatic decrease of the activity, possibly due to the disruption of the hydrogen bond of OH-1/Gln720 and the hydrophobic packing. Moreover, the replacement of 3-OBz by less hydrophobic 3-ONic led to a decrease in activity. Interestingly, the C-7−C-8−C-9 fragment pointed into a vacant space in the predicted model, which rationalized that the subsititutional variations on these positions had little influence on the activity. However, the presence of free hydroxyls at these positions would increase the polarity of the molecule, which prevented the entry of jatrophanes into the cell memberance, and led to a sharp decrease of inhibitory activity. Metabolic Stability. The metabolic stability of compound 26 in rat liver microsomes was examined and compared with tariquidar and verapamil, using testosterone as a positive control. As shown in Table 3, tariquidar, verapamil, and

Testosteroneb Tariquidar Verapamil 26

κ (min−1)

T1/2 (min)a

± ± ± ±

2.5 ± 0.15 210 ± 9.29 25 ± 2.52 866 ± 24.5

0.2665 0.0033 0.0235 0.0008

0.01665 0.00063 0.0038 0.00006

CONCLUSIONS

■

EXPERIMENTAL SECTION

Jatrophanes have been reported to be potential Pgp inhibitors.7−9 However, due to the restricted structural diversity and limited amount of these natural products, only very little SAR and biological property information was revealed. The jatrophane library constructed by phytochemical investigation and subsequent chemical modification in the current study allowed not only extensive biological evaluations but also indepth SAR investigation. The results clearly demonstrated that several of these compounds are promising Pgp-mediated MDR modulators, with potency stronger than previously reported analogues. Besides the general role of lipophilicity, the current SAR studies revealed for the first time that the saturated ring A was essential for the activity, while the free hydroxyls at the C1−C-15−C-14−C-13 fragment had little influence on the activity. In addition, the formation of a rare C-5−O−C-13 bridge would increase the activity, while epoxidation of Δ12 is detrimental to the activity. Negligible effects were also observed for acylation of 1-OH, hydrogenation of Δ11, and the oxidation pattern of C-8. Compounds 19, 25, and 26, which exhibited greater chemoreversal ability and less cytotoxicity than the third-generation MDR modulator tariquidar, were identified to be potent modulators. Among them, compound 26 exhibits a remarkable metabolic stability in vitro and a good antitumor effect in vivo, which makes it a lead for the development of new MDR reversal agents. Nevertheless, before becoming clinically useful MDR modulators, several pharmaceutical issues, including the in vivo pharmacokinetics, the drug safety, and the drug−drug interactions in combination therapy, should be addressed for 26 and its analogues in future studies.

Table 3. Metabolic Stability of 26 in Liver Microsomes of SD Rat Cmpd

■

Results are expressed as the mean ± SD of at least three independent experiments performed in triplicate. bThe positive control (testosterone) exhibited metabolic stability that was consistent with the literature and internal validation data. a

testosterone had a T1/2 of 210, 25, and 2.5 min, respectively. The T1/2 value of testosterone was consistent with previous reports and internal validation data.16 Comparatively, 26 demonstrated significant stability after incubation with rat liver microsomes (T1/2 = 866 min) under the same conditions. These data suggest that compound 26 possesses considerably greater metabolic stability in vitro than the reference compounds. In vivo evaluation. To assess the in vivo efficiency of the active compounds, a HepG2/ADR xenografts mice model was set up. Compound 26 was selected for evaluation based on the consideration of in vitro activity, metabolic stability, and availability. A 5 mg/kg dose of ADR was administrated intraperitoneally (ip) with or without the combination of 10 mg/kg dose of 26 in two groups of HepG2/ADR xenografts nude mice, respectively (Figure 6). As shown in Figure 6A, coadministration of 26 significantly reduced the final tumor volume of the nude mice (mean, 44.88 mm3) as compared to the control group (mean, 144.48 mm3). Correspondingly, the weight of excised tumors in the coadministration group (mean, 0.0209 g) was decreased significantly (P < 0.05) in contrast to the control group (mean, 0.0563 g) (Figures 6D−E), with the inhibiton rate (IR) of 62.89%. Remarkably, although 6 out of the 7 mice in the ADR-only group died within 12 days due to the serious side effects (Figures 6B−C), 5 mice in coadministration group survived more than 3 weeks, indicating that coadministration of 26 greatly extended the overall survival. Furthermore, the tumor volume curve (Figure 6A)

General. X-ray data were collected using an Angilent Xcalibur Nova X-ray diffractometer. Melting point was measured on an X-4 melting instrument and uncorrected. Optical rotations were measured on a Rudolph Autopol I automatic polarimeter. IR spectra were determined on a Bruker Tensor 37 infrared spectrophotometer. NMR spectra were measured on a Bruker AM-400 spectrometer at 25 °C. HRESIMS was performed on a Waters Micromass Q-TOF spectrometer. A Shimadzu LC-20 AT equipped with an SPD-M20A PDA detector was used for HPLC. A YMC-pack ODS-A column (250 × 10 mm, S-5 μm, 12 nm) were used for semipreparative HPLC separation. Silica gel (300−400 mesh, Qingdao Haiyang Chemical Co., Ltd.), C18 reversed-phase silica gel (12 nm, S-50 μm, YMC Co., Ltd.), and Sephadex LH-20 gel (Amersham Biosciences) were used for column chromatography (CC). All solvents were of analytical grade (Guangzhou Chemical Reagents Company, Ltd.). Doxorubicin hydrochloride (ADR) was purchased from Shenzhen Main Luck Pharmaceuticals Inc. (Guangzhou, CHN). Tariquidar was obtained from SelleckChem Inc. (Shanghai, CHN). The purity of all compounds were determined by HPLC, conducted on a Shimadzu LC-20AT series system, TC-C18 column (4.6 × 250 mm, 5 μm). The mobile phase consisted of water (A) and MeOH (B). A gradient program was used as follows: 0−9 min, 60%−100% B; 9−11 min, 100% B; 11−13 min, 100−60% B; 13−15 min, 60% B, at a flow rate of 1 mL/min. All compounds exhibited greater than 95% purity (Supporting Information, Figure S231−265). Pharmaceutical-grade dimethyl sulfoxide (DMSO) was acquired from Aladdin Inc. (Shanghai, CHN). (±)-Verapamil hydrochloride, rhodamin 123 (Rho123) and 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazoI



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Figure 6. Antitumor effect of the combination of ADR and 26 on the HepG2/ADR xenografts. (A) Tumor volume of xenografts nude mice. (B) Body weight of xenografts nude mice. (C) Survival rate of xenografts nude mice. (D) Mean tumor weight of surviving nude mice from the control group and the combination-schedule group. * Statistically significant difference in mean tumor weight compared with the control, P < 0.05. (E) Photograph of surviving nude mice and corresponding excised tumor. lium bromide (MTT) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Human hepatocellular carcinoma multidrug resistant HepG2/ADR cell line were generously provided by Prof. Kwok-Pui Fung (The Chinese University of Hong Kong, Hong Kong).17 Human breast adenocarcinoma multidrug resistant cell line MCF-7/ADR (ADR-selected ABCB1-overexpressing cell line) were kindly provided by Prof. Li-Wu Fu (Sun Yat-Sen University, P. R. China).18 The normal HLF cells were purchased from the Experimental Animal Center at Sun Yat-Sen University. Plant Material. Leaves and stems of P. tithymaloides were collected from the campus of Guangzhou University of Traditional Chinese Medicine, Guangzhou city, P. R. China, in August 2013, and were authenticated by Associate Professor Lin Jiang of Sun Yat-sen University. A voucher specimen (accession number: YPTZ-201309) has been deposited at the School of Pharmaceutical Sciences, Sun Yatsen University. Extraction and Isolation. The air-dried powder of the leaves and stems of P. tithymaloides (5 kg) was extracted with 95% EtOH (3 × 5 L) at rt to give 210 g of crude extract. The extract was suspended in H2O (1 L) and successively partitioned with PE (3 × 1 L), EtOAc (3 × 1 L), and n-BuOH (3 × 1 L) to yield three corresponding portions. The PE extract (64 g) was subjected to MCI gel CC eluted with a MeOH/H2O gradient (2:8 → 10:0) to afford Fractions I−VI. Separation of the Fraction I (6.5 g) by Sephadex LH-20 eluted with MeOH led to Fractions Ia−Id. Fraction Ia (1.7 g) was subjected to silica gel chromatography using PE/acetone mixtures (v/v 1:0 → 0:1) to afford Fractions Ia1−Ia3. Fraction Ia2 was loaded onto a Sephadex

LH-20 column eluted with MeOH to give 8 (10 mg). Fraction Ib (2.8 g) was chromatographed over a C18 reversed-phase (RP-C18) column eluted with MeOH/H2O (6:4 → 10:0) to afford Fractions Ib1−Ib4. Colorless crystals of 12 (1.5 g) and 13 (52.0 mg) were recrystallized from Fractions Ib2 and Ib3, respectively, in PE-acetone (5:1). Fraction Ic was purified using RP-HPLC (MeOH/H2O, 7:3, 3 mL/min) to give Fractions Ic1−Ic2. Colorless crystal of 6 (13.3 mg) was recrystallized from Fraction Ic1 in PE-acetone (5:1). Fraction Ic2 was loaded onto a Sephadex LH-20 column eluted with MeOH to give 7 (10 mg). Fraction II (6.3 g) was subjected to Sephadex LH-20 column eluted with EtOH to yield Fractions IIa−IId. Fraction IIa (1.5 g) was chromatographed over a C18 reversed-phase (RP-C18) column eluted with MeOH/H2O (7:3 → 10:0) to afford Fractions IIa1−IIa4. Colorless crystal of 1 (122.3 mg) was recrystallized from Fraction IIa2 in PE-EtOAc (6:1). Fraction IIa3 was loaded onto a Sephadex LH-20 column eluted with CH2Cl2-MeOH (1:1) to give 11 (69.2 mg) and 4 (10.2 mg). Fraction IIa4 was purified using RP-HPLC (MeOH/H2O, 8:2, 3 mL/min) to give 2 (11 mg, tR 11 min) and 10 (63 mg, tR 13 min). Fraction IIb (3.0 g) was subjected to silica gel CC (PE/EtOAc, 8:1 → 1:2) to give Frations IIb1−IIb3. Colorless crystal of 9 (1.0 g) was recrystallized from Fraction IIb2 in PE-acetone (5:1). Fraction IIb3 was purified using RP-HPLC (CH3OH/H2O, 8:2, 3 mL/min) to give 5 (20.7 mg, tR 14 min). Fraction III (1.1 g) was chromatographed over a C18 reversed-phase (RP-C18) column eluted with MeOH/H2O (7:3 → 10:0) to afford Fractions IIIa−IIIb. Fraction IIIb was loaded onto a Sephadex LH-20 column eluted with CH2Cl2-MeOH (1:1) to give Fractions IIIb1−IIIb2. Colorless crystal of 3 (10.3 mg) was J



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recrystallized from Fraction IIIb2 in PE-EtOAc (3:1). 1H and 13C NMR data of 1−8 were summarized in Supporting Information Tables S1 and S2, respectively. Compound 1. Colorless crystals, mp 185−186 °C; [α]25D − 18.9 (c 0.11, CHCl3); UV (MeOH) λmax (log ε) 251(3.57), 206 (3.88) nm; IR (KBr) νmax 2970, 1744, 1694, 1371, 1220, 1029, 765 cm−1; HREIMS m/z 599.2470 [M + Na]+ (calcd for C30H40O11Na, 599.2463). Compound 2. White powder; [α]25D − 58.4 (c 0.13, CHCl3); UV (MeOH) λmax (log ε) 263(3.61), 226 (3.85) nm; CD (c 1.32 × 10−4 M, MeOH) λmax (Δε) 209 (−64.1) nm; IR (KBr) νmax 3476, 2933, 1745, 1429, 1373, 1234, 754, 611 cm−1; HREIMS m/z 782.3013 [M + Na]+ (calcd for C38H49NO15Na, 782.2994). Compound 3. Colorless crystals, mp 215−219 °C; [α]25D − 34.2 (c 0.19, CHCl3); UV (MeOH) λmax 280(2.85), 232 (3.95) nm; IR (KBr) νmax 3567, 3520, 2973, 2937, 1761, 1370, 1222, 771, 715 cm−1; HREIMS m/z 797.3004 [M + Na]+ (calcd for C39H50O16Na, 797.2991). Compound 4. White powder; [α]25D + 53.3 (c 0.12, CHCl3); UV (MeOH) λmax (log ε) 273(3.06), 239 (3.57) nm; IR (KBr) νmax 2973, 1737, 1369, 1219, 1033, 713 cm−1; HREIMS m/z 675.3137 [M + Na]+ (calcd for C39H50O14Na, 675.3093). Compound 5. White powder; [α]25D − 43.3 (c 0.06, CHCl3); UV (MeOH) λmax (log ε) 273(2.67), 231 (3.87) nm; IR (KBr) νmax 2982, 1742, 1369, 1240, 1067, 1036, 712 cm−1; HREIMS m/z 795.2867 [M + Na]+ (calcd for C39H48O16Na, 795.2835). Compound 6. Colorless crystals, mp 160−163 °C; [α]25D + 11.9 (c 0.12, CHCl3); UV (MeOH) λmax (log ε) 264(3.05), 234 (3.87) nm; IR (KBr) νmax 3514, 2974, 2932, 1732, 1447, 1374, 1271, 1248, 1110, 757, 712 cm −1; HREIMS m/z 639.2789 [M + Na]+ (calcd for C33H44O11Na, 639.2776). Compound 7. White powder; [α]25D − 70.0 (c 0.10, CHCl3); UV (MeOH) λmax 273(3.20), 236 (3.93); IR (KBr) νmax 3633, 3534, 2981, 1711, 1350, 1268, 1253, 1110, 1025, 712 cm−1; HREIMS m/z 701.2968 [M + Na]+ (calcd for C38H46O11Na, 701.2932). Compound 8. White powder; [α]25D − 20.0 (c 0.07, CHCl3); UV (MeOH) λmax 273(3.28), 233 (3.81); IR (KBr) νmax 3471, 2940, 1714, 1452, 1366, 1262, 1244, 708 cm−1; HREIMS m/z 659.2853 [M + Na]+ (calcd for C36H44O10Na, 659.2827). Preparation of 14−16 by Alkaline Hydrolysis of 12. Compound 12 (50 mg) was treated with NaOH (1% in MeOH, 1 mL) at rt for 1 h. The mixture was then diluted with 5 mL of H2O, followed by the extraction of EtOAc (3 × 5 mL). The organic layer was dried and evaporated to give a residue, which was purified by RPHPLC (CH3OH/H2O, 7.5:2.5, 3 mL/min) to afford 14 (8.1 mg, tR 10 min), 15 (13.5 mg, tR 11 min), and 16 (5.5 mg, tR 12 min). (1S,2R,3S,4S,7R,9R,13R,14R,15S)-1,3,7,9,13,14,15-heptahydroxyjatropha-5E,11E-diene (14). White powder; [α]25D − 10.5 (c 0.17, MeOH); UV (MeOH) λmax (log ε) 210 (3.51) nm; IR (KBr) νmax 3419, 2965, 2930, 1657, 1081, 1027, 870 cm−1; 1H NMR (CD3OD, 400 MHz) δH 5.57 (1H, d, J = 10.6 Hz, H-5), 5.55 (1H, d, J = 15.7 Hz, H-12), 5.44 (1H, d, J = 15.7 Hz, H-11), 4.28 (1H, d, J = 6.5 Hz, H-7), 3.82 (1H, d, J = 11.7 Hz, H-1), 3.74 (1H, dd, J = 4.9, 4.7 Hz, H-3), 3.64 (1H, s, H-14), 3.54 (1H, dd, J = 10.6, 4.9 Hz, H-4), 3.47 (1H, dd, J = 3.4, 3.4 Hz, H-9), 2.07 (1H, dd, J = 15.3, 3.4 Hz, H-8a), 1.94 (1H, m, H-2), 1.77 (1H, m, H-8b), 1.71 (3H, s, H3-17), 1.28 (3H, s, H3-20), 1.08 (3H, s, H3-18), 1.06 (3H, d, J = 6.8 Hz, H3-16), 0.89 (3H, s, H319); 13C NMR (CD3OD, 100 MHz) δC 138.9 (C-6), 135.9 (C-11), 131.0 (C-12), 121.2 (C-5), 89.8 (C-1), 84.2 (C-15), 79.7 (C-14), 77.1 (C-3), 75.7 (C-13), 73.9 (C-7), 72.9 (C-9), 45.7 (C-2), 43.8 (C-4), 41.0 (C-10), 38.0 (C-8), 30.6 (C-20), 24.1 (C-19), 20.1 (C-18), 17.1 (C-17), 11.9 (C-16); ESIMS m/z 771.4 [2 M − H]−; HREIMS m/z 421.2003 [M + Cl]− (calcd for C20H34O7Cl, 421.1999). (1S,2S,3S,4S,7R,9R,13R,14R,15S)-3-benzoyloxy-1,7,9,13,14,15hexahydroxyjatropha-5E,11E-diene (15). White powder; [α]25D − 56.0 (c 0.26, MeOH); UV (MeOH) λmax (log ε) 272 (2.98), 229 (4.07) nm; IR (KBr) νmax 3470, 2965, 2932, 1706, 1452, 1287, 1085, 1032 cm−1; 1H NMR (CD3OD, 400 MHz) δH 5.62 (1H, d, J = 15.7 Hz, H-12), 5.58 (1H, d, J = 10.0 Hz, H-5), 5.52 (1H, d, J = 15.7 Hz, H-11), 5.38 (1H, dd, J = 4.3, 4.3 Hz, H-3), 4.15 (1H, d, J = 6.4 Hz, H7), 3.90 (1H, d, J = 11.9 Hz, H-1), 3.89 (1H, dd, J = 10.0, 4.3 Hz, H-

4), 3.76 (1H, s, H-14), 3.38 (1H, dd, J = 3.2, 3.0 Hz, H-9), 2.27 (1H, m, H-2), 2.06 (1H, dd, J = 15.2, 3.2 Hz, H-8a), 1.74 (3H, s, H3-17), 1.70 (1H, m, H-8b), 1.33 (3H, s, H3-20), 1.11 (3H, s, H3-19), 0.98 (3H, d, J = 6.7 Hz, H3-16), 0.90 (3H, s, H3-18). 3-OBz: 8.03 (2H, dd, J = 7.2, 1.5 Hz), 7.58 (1H, ddd, J = 7.5, 7.2, 1.5 Hz), 7.47 (2H, dd, J = 7.5, 7.2 Hz); 13C NMR (CD3OD, 100 MHz) δC 140.7 (C-6), 136.6 (C-11), 130.8 (C-12), 119.5 (C-5), 89.5 (C-1), 84.0 (C-15), 80.3 (C3), 79.6 (C-14), 75.7 (C-13), 73.3 (C-7), 73.0 (C-9), 45.0 (C-2), 42.1 (C-4), 41.0 (C-10), 38.0 (C-8), 30.4 (C-20), 24.0 (C-19), 19.9 (C18), 17.0 (C-17), 12.2 (C-16). 3-OBz: 168.0, 134.0, 131.8, 130.6 × 2, 129.4 × 2; ESIMS m/z 525.2 [M + Cl]−; HREIMS m/z 525.2271 [M + Cl]− (calcd for C27H38O8Cl, 525.2261). (1S,2S,3S,4S,7R,9R,13R,14R,15S)-3,7-dibenzoyloxy-1,9,13,14,15pentahydroxyjatropha-5E,11E-diene (16). White powder; [α]25D − 45.0 (c 0.20, CHCl3); UV (MeOH) λmax (log ε) 273 (3.25), 230 (4.12) nm; IR (KBr) νmax 3499, 2963, 2928, 1719, 1453, 1282, 1120, 1068 cm−1; 1H NMR (CDCl3, 400 MHz) δH 5.64 (1H, d, J = 15.7 Hz, H-12), 5.58 (1H, d, J = 15.7 Hz, H-11), 5.49 (1H, d, J = 10.2 Hz, H-5), 5.45 (1H, dd, J = 5.4, 4.0 Hz, H-3), 5.31 (1H, d, J = 6.3 Hz, H-7), 4.01 (1H, d, J = 11.6 Hz, H-1), 3.83 (1H, s, H-14), 3.77 (1H, dd, J = 10.2, 5.4 Hz, H-4), 3.66 (1H, d, J = 5.9 Hz, HO-14), 3.50 (1H, s, H-9), 2.51 (1H, s, HO-13), 2.17 (1H, m, H-2), 2.13 (1H, m, H-8a), 2.08 (1H, s, HO-1), 2.00 (1H, m, H-8b), 1.86 (3H, s, H3-17), 1.39 (3H, s, H3-20), 1.14 (3H, s, H3-18), 0.96 (3H, d, J = 6.7 Hz, H3-16), 0.93 (3H, s, H319). 3-OBz: 7.77 (2H, dd, J = 7.5, 1.0 Hz), 7.30 (1H, ddd, J = 7.5, 7.3, 1.0 Hz), 7.11 (2H, dd, J = 7.5, 7.3 Hz). 7-OBz: 7.54 (2H, dd, J = 7.1, 1.0 Hz), 7.28 (1H, ddd, J = 7.5, 7.1, 1.0 Hz), 6.98 (2H, dd, J = 7.5, 7.1 Hz); 13C NMR (CDCl3, 100 MHz) δC 136.7 (C-11), 136.3 (C-6), 128.0 (C-12), 118.5 (C-5), 88.8 (C-1), 82.9 (C-15), 78.2 (C-14), 77.7 (C-3), 75.0 (C-13), 74.6 (C-7), 73.4 (C-9), 44.1 (C-2), 41.0 (C-4), 40.2 (C-10), 35.1 (C-8), 30.6 (C-20), 23.1 (C-19), 19.0 (C-18), 16.6 (C-17), 11.7 (C-16). 3-OBz: 165.4, 132.6, 129.9, 129.3 × 2, 128.1 × 2. 7-OBz: 165.0, 132.3, 129.7, 129.0 × 2, 128.0 × 2; ESIMS m/z 629.2 [M + Cl]−; HREIMS m/z 629.2532 [M + Cl]− (calcd for C34H42O9Cl, 629.2523). Preparation of 17 and 18 by Acetylation of 12. Acetic anhydride (200 μL) was added to a stirred solution of 12 (15 mg) in freshly distilled pyridine (2 mL). The reaction mixture was stirred at rt for 12 h and quenched by adding 1 mL of H2O. After removal of solvent under vacuum, the residue was purified on a flash silica gel column eluted with CH2Cl2 to afford 17 (8.1 mg) and 18 (3.5 mg). (1S,2R,3S,4S,7R,9R,13R,14R,15R)-1,9,15-triacetoxy-3,7-dibenzoyloxy-13,14-dihydroxyjatropha-5E,11E-diene (17). White powder; [α]25D − 27.8 (c 0.18, CHCl3); UV (MeOH) λmax (log ε) 274 (3.11), 228 (4.13), 204 (3.92) nm; IR (KBr) νmax 3518, 2972, 2933, 1730, 1453, 1371, 1280, 1248, 713 cm−1; 1H NMR (CDCl3, 400 MHz) δH 5.82 (1H, d, J = 9.8 Hz, H-5), 5.60 (1H, d, J = 12.0 Hz, H1), 5.52 (1H, d, J = 15.9 Hz, H-12), 5.45 (1H, dd, J = 5.4, 4.0 Hz, H3), 5.28 (1H, d, J = 15.9 Hz, H-11), 5.25 (1H, d, J = 6.1 Hz, H-7), 5.13 (1H, dd, J = 3.5, 3.2 Hz, H-9), 4.72 (1H, d, J = 5.8 Hz, H-14), 4.05 (1H, dd, J = 9.8, 4.6 Hz, H-4), 2.36 (1H, m, H-2), 2.05 (2H, m, H2-8), 1.82 (3H, s, H3-17), 1.33 (3H, s, H3-20), 0.86 (3H, s, H3-18), 0.86 (3H, d, J = 6.7 Hz, H3-16), 0.85 (3H, s, H3-19). 3-OBz: 7.69 (2H, dd, J = 8.2, 1.2 Hz), 7.27 (1H, ddd, J = 8.2, 7.6, 1.2 Hz), 7.07 (2H, dd, J = 8.2, 7.6 Hz). 7-OBz: 7.55 (2H, dd, J = 8.4, 1.2 Hz), 7.24 (1H, ddd, J = 8.4, 7.6, 1.2 Hz), 6.97 (2H, dd, J = 8.4, 7.6 Hz). 15-OAc: 2.35 (3H, s). 1-OAc: 2.20 (3H, s). 9-OAc: 1.67 (3H, s); 13C NMR (CDCl3, 100 MHz) δC 134.3 (C-6), 132.0 (C-11), 129.7 (C-12), 119.1 (C-5), 89.8 (C-15), 86.5 (C-1), 77.1 (C-3), 74.5 (C-13), 74.2 (C-7), 73.9 (C-9), 71.8 (C-14), 43.8 (C-2), 41.6 (C-4), 39.5 (C-10), 32.3 (C-8), 31.4 (C20), 22.8 (C-19), 20.5 (C-18), 16.4 (C-17), 11.2 (C-16). 3-OBz: 165.2, 132.6, 129.4, 129.0 × 2, 128.1 × 2. 7-OBz: 165.0, 132.3, 129.2, 129.2 × 2, 127.7 × 2. 15-OAc: 170.9, 22.1. 9-OAc: 169.9, 20.5. 1-OAc: 169.2, 21.2; ESIMS m/z 755.1 [M + Cl]−; HREIMS m/z 755.2839 [M + Cl]− (calcd for C40H48O12Cl, 755.2840). (1S,2S,3S,4S,7R,9R,13R,14R,15S)-9,14,15-triacetoxy-3,7-dibenzoyloxy-1,13-dihydroxyjatropha-5E,11E-diene (18). White powder; [α]25D − 41.0 (c 0.06, CHCl3); UV (MeOH) λmax (log ε) 274 (3.42), 229 (4.31) nm; IR (KBr) νmax 3528, 2959, 2919, 1730, 1456, 1374, 1280, 1113, 1027, 709 cm−1; 1H NMR (CDCl3, 400 MHz) δH K



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5.79 (1H, d, J = 9.6 Hz, H-5), 5.67 (1H, s, H-14), 5.55 (1H, d, J = 15.5 Hz, H-12), 5.47 (1H, dd, J = 4.5, 4.2 Hz, H-3), 5.35 (1H, d, J = 15.5 Hz, H-11), 5.28 (1H, d, J = 3.2 Hz, H-7), 5.16 (1H, dd, J = 3.2, 3.0 Hz, H-9), 4.25 (1H, dd, J = 9.5, 4.5 Hz, H-4), 4.11 (1H, dd, J = 12.0, 2.4 Hz, H-1), 3.79 (1H, d, J = 2.4 Hz, HO-1), 2.25 (1H, m, H-2), 2.07 (2H, m, H2-8), 1.85 (3H, s, H3-17), 1.20 (1H, s, H-20), 0.97 (3H, s, H3-19), 0.96 (3H, s, H3-18), 0.94 (3H, d, J = 6.7 Hz, H3-16). 3-OBz: 7.65 (2H, dd, J = 7.5, 1.4 Hz), 7.28 (1H, ddd, J = 8.0, 7.2, 1.2 Hz), 7.05 (2H, dd, J = 8.0, 7.2 Hz). 7-OBz: 7.55 (2H, dd, J = 7.3, 1.1 Hz), 7.24 (1H, ddd, J = 8.0, 7.3, 1.1 Hz), 7.02 (2H, dd, J = 8.0, 7.3 Hz). 15-OAc: 2.45 (3H, s). 9-OAc: 2.18 (3H, s). 14-OAc: 1.70 (3H, s); 13C NMR (CDCl3, 100 MHz) δC 134.1 (C-6), 132.5 (C-11), 129.2 (C-12), 119.2 (C-5), 90.8 (C-15), 86.6 (C-1), 77.0 (C-3), 75.4 (C-13), 74.2 (C-7), 73.8 (C-9), 73.0 (C-14), 42.6 (C-2), 42.4 (C-4), 39.7 (C-10), 32.4 (C8), 31.5 (C-20), 22.8 (C-19), 20.6 (C-18), 16.4 (C-17), 11.6 (C-16). 3-OBz: 165.2, 132.6, 129.5, 128.9 × 2, 128.1 × 2. 7-OBz: 165.0, 132.2, 129.4, 129.2 × 2, 127.8 × 2. 15-OAc: 173.1, 22.2. 14-OAc: 170.1, 20.9. 9-OAc 170.0, 21.6; ESIMS m/z 743.3 [M + Na]+; HREIMS m/z 743.3062 [M + Na]+ (calcd for C40H48O12Na, 743.3038). General Preparation of 19−23 by Esterification of 12. Suitable acyl chloride (200 μL) was added to a stirred solution of 12 (15 mg) in freshly distilled pyridine (2 mL). The reaction mixture was stirred at rt for 12 h and quenched by adding 1 mL of H2O. After removal of solvent under vacuum, the residue was purified on a flash silica gel column eluted with CH2Cl2. (1S,2R,3S,4S,7R,9R,13R,14R,15R)-9,15-diacetoxy-1-Tosyl-3,7-dibenzoyloxy-13,14-dihydroxyjatropha-5E,11E-diene (19) was obtained from reaction with Tosyl chloride. Six mg, white powder; [α]25D − 20.6 (c 0.05, CHCl3); UV (MeOH) λmax (log ε) 273 (3.33), 227 (4.36), 208 (4.15) nm; IR (KBr) νmax 3448, 2965, 2927, 1723, 1454, 1369, 1280, 1180, 1116, 711 cm−1; 1H NMR (CDCl3, 400 MHz) δH 6.21 (1H, d, J = 11.6 Hz, H-1), 5.66 (1H, d, J = 15.7 Hz, H12), 5.49 (1H, d, J = 9.3 Hz, H-5), 5.48 (1H, dd, J = 6.5, 3.5 Hz, H-3), 5.42 (1H, d, J = 15.7 Hz, H-11), 5.21 (1H, dd, J = 3.6, 3.5 Hz, H-7), 4.95 (1H, dd, J = 3.5, 3.3 Hz, H-9), 4.23 (1H, dd, J = 9.3, 6.5 Hz, H-4), 4.17 (1H, s, H-14), 2.55 (1H, m, H-2), 2.04 (2H, m, H2-8), 1.78 (3H, s, H3-17), 1.38 (1H, s, H-20), 0.99 (3H, s, H3-19), 0.95 (3H, s, H3-18), 0.40 (3H, d, J = 6.8 Hz, H3-16). 3-OBz: 7.92 (2H, dd, J = 7.3, 1.0 Hz), 7.38 (1H, ddd, J = 7.7, 7.3, 1.0 Hz), 7.19 (2H, dd, J = 7.7, 7.3 Hz), 7OBz: 7.59 (2H, dd, J = 7.3, 1.0 Hz), 7.20 (1H, ddd, J = 7.7, 7.3, 1.0 Hz), 7.02 (2H, dd, J = 7.7, 7.3 Hz), 1-O-tosyl: 7.83 (2H, d, J = 8.3, Hz), 7.31 (2H, d, J = 8.3 Hz), 2.38 (3H, s). 15-OAc: 2.47 (3H, s). 9OAc: 1.72 (3H, s); 13C NMR (CDCl3, 100 MHz) δC 135.7 (C-6), 131.0 (C-12), 130.5 (C-11), 118.6 (C-5), 90.3 (C-15), 88.4 (C-1), 75.8 (C-14), 75.3 (C-13), 74.6 (C-3), 73.9 (C-9), 73.7 (C-7), 41.1 (C2), 40.3 (C-4), 39.7 (C-10), 32.4 (C-8), 31.8 (C-20), 23.3 (C-19), 20.9 (C-18), 16.0 (C-17), 10.7 (C-16). 3-OBz: 165.9, 132.4, 129.6 × 2, 129.5, 127.8 × 2. 7-OBz: 164.8, 132.2, 129.5 × 2, 129.4, 127.8 × 2. 1O-tosyl: 145.4, 132.9, 129.8 × 2, 128.3 × 2, 21.7. 15-OAc: 170.7, 22.8. 9-OAc: 169.7, 20.9; ESIMS m/z 855.1 [M + Na]+; HREIMS m/z 855.3057 [M + Na]+ (calcd for C45H52O13SNa, 855.3021). (1S,2R,3S,4S,7R,9R,13R,14R,15R)-9,15-diacetoxy-1-(p-bromobenzoyl)-3,7-dibenzoyloxy-13,14-dihydroxyjatropha-5E,11E-diene (20) was obtained from reaction with p-bromobenzoyl chloride. Eight mg, white powder; [α]25D − 51.9 (c 0.12, CHCl3); UV (MeOH) λmax (log ε) 239 (4.49), nm; IR (KBr) νmax 3565, 2974, 1729, 1588, 1278, 1098, 710 cm−1; 1H NMR (CDCl3, 400 MHz) δH 5.87 (1H, d, J = 9.8 Hz, H5), 5.83 (1H, d J = 11.7 Hz, H-1), 5.52 (1H, dd, J = 5.3, 4.3 Hz, H-3), 5.51 (1H, d, J = 15.8 Hz, H-11), 5.30 (1H, d, J = 15.8 Hz, H-12), 5.29 (1H, d, J = 3.8 Hz, H-7), 5.14 (1H, dd, J = 3.5, 2.9 Hz, H-9), 4.81 (1H, d, J = 5.8 Hz, H-14), 4.09 (1H, dd, J = 9.8, 5.3 Hz, H-4), 2.53 (1H, m, H-2), 2.07 (2H, m, H2-8), 1.86 (3H, s, H3-17), 1.38 (1H, s, H-20), 0.94 (3H, s, H3-19), 0.94 (3H, s, H3-18), 0.93 (3H, d, J = 6.7 Hz, H316). 1-O-bromobenzoyl: 7.89 (2H, d, J = 8.5 Hz), 7.64 (2H, d, J = 8.5 Hz). 3-OBz: 7.74 (2H, dd, J = 7.3, 1.5 Hz), 7.30 (1H, ddd, J = 7.7, 7.3, 1.5 Hz), 7.12 (2H, dd, J = 7.7, 7.3 Hz). 7-OBz: 7.57 (2H, dd, J = 7.3, 1.4 Hz), 7.28 (1H, ddd, J = 7.7, 7.3, 1.4 Hz), 6.99 (2H, dd, J = 7.7, 7.3 Hz). 15-OAc: 2.37 (3H, s). 9-OAc: 1.68 (3H, s); 13C NMR (CDCl3, 100 MHz) δC 134.5 (C-6), 132.1 (C-11), 129.9 (C-12), 119.2 (C-5), 90.1 (C-15), 87.3 (C-1), 77.1 (C-3), 74.8 (C-13), 74.2 (C-7), 73.8 (C-

9), 72.2 (C-14), 44.0 (C-2), 41.8 (C-4), 39.6 (C-10), 32.3 (C-8), 31.5 (C-20), 22.8 (C-19), 20.6 (C-18), 16.5 (C-17), 11.4 (C-16). 1-Obromobenzoyl: 164.9, 132.1 × 2, 130.9 × 2, 129.5, 128.4. 3-OBz: 165.3, 132.7, 129.5, 129.0 × 2, 128.2 × 2. 7-OBz: 165.0, 132.3, 129.2 × 2, 129.0, 127.8 × 2. 15-OAc: 171.0, 22.1. 9-OAc: 170.0, 20.9; ESIMS m/z 883.1 [M + Na]+; HREIMS m/z 883.2329 [M + Na]+ (calcd for C45H49BrO12Na, 883.2300). (1S,2R,3S,4S,7R,9R,13R,14R,15R)-9,15-diacetoxy-1-(2-furoyl)-3,7dibenzoyloxy-13,14-dihydroxyjatropha-5E,11E-diene (21) was obtained from reaction with 2-furoyl chloride. 7.5 mg, white powder; [α]25D − 16.0 (c 0.30, CHCl3); UV (MeOH) λmax (log ε) 230 (4.38), 208 (4.17) nm; IR (KBr) νmax 3511, 2970, 2933, 1730, 1583, 1280, 1113, 710 cm−1; 1H NMR (CDCl3, 400 MHz) δH 5.88 (1H, d, J = 9.8 Hz, H-5), 5.75 (1H, d J = 11.5 Hz, H-1), 5.57 (1H, d, J = 11.5 Hz, H11), 5.52 (1H, dd, J = 5.0, 4.3 Hz, H-3), 5.32 (1H, d, J = 15.5 Hz, H12), 5.31 (1H, d, J = 3.5 Hz, H-7), 5.17 (1H, dd, J = 3.3, 3.1 Hz, H-9), 4.81 (1H, d, J = 5.9 Hz, H-14), 4.13 (1H, dd, J = 9.8, 5.0 Hz, H-4), 2.52 (1H, m, H-2), 2.09 (2H, dd, J = 3.5, 3.3, H2-8), 1.87 (3H, s, H317), 1.37 (1H, s, H-20), 0.95 (3H, s, H3-19), 0.94 (3H, s, H3-18), 0.94 (3H, d, J = 6.8 Hz, H3-16). 3-OBz: 7.75 (2H, dd, J = 8.3, 1.2 Hz), 7.29 (1H, ddd, J = 8.3, 7.8, 1.2 Hz), 7.13 (2H, dd, J = 8.3, 7.8 Hz). 7-OBz: 7.60 (2H, dd, J = 8.2, 1.3 Hz), 7.27 (1H, ddd, J = 8.2, 7.6, 1.3 Hz), 6.99 (2H, dd, J = 8.2, 7.6 Hz). 1-O-2-furoyl: 7.59 (1H, dd, J = 3.5, 1.3 Hz), 7.33 (1H, dd, J = 3.5, 0.7 Hz), 6.56 (1H, dd, J = 3.5, 1.7 Hz). 15-OAc: 2.39 (3H, s). 9-OAc: 1.68 (3H, s); 13C NMR (CDCl3, 100 MHz) δC 134.3 (C-6), 132.0 (C-11), 129.7 (C-12), 119.1 (C-5), 90.0 (C-15), 86.9 (C-1), 77.4 (C-3), 74.5 (C-13), 74.2 (C-7), 73.8 (C-9), 72.0 (C14), 44.0 (C-2), 41.8 (C-4), 39.5 (C-10), 32.2 (C-8), 31.4 (C-20), 22.7 (C-19), 20.5 (C-18), 16.4 (C-17), 11.2 (C-16). 1-O-2-furoyl: 156.8, 146.2, 144.3, 118.8, 112.2. 3-OBz: 165.1, 132.6, 129.4, 129.0 × 2, 128.1 × 2. 7-OBz: 165.0, 132.1, 129.2, 129.1 × 2, 127.7 × 2. 15OAc: 170.8, 22.0. 9-OAc: 169.9, 20.7; ESIMS m/z 795.3 [M + Na]+; HREIMS m/z 795.3011 [M + Na]+ (calcd for C43H48O13Na, 795.2987). (1S,2R,3S,4S,7R,9R,13R,14R,15R)-9,15-diacetoxy-1-(thiophene-2carbonyl)-3,7-dibenzoyloxy-13,14-dihydroxyjatropha-5E,11E-diene (22) was obtained from reaction with 2-thiophenecarbonyl chloride. Seven mg, white powder; [α]25D − 12.8 (c 0.41, CHCl3); UV (MeOH) λmax (log ε) 274 (4.01), 233 (4.30) 208 (4.08) nm; IR (KBr) νmax 3479, 2971, 2933, 1730, 1453, 1280, 1096, 710 cm−1; 1H NMR (CDCl3, 400 MHz) δH 5.86 (1H, d, J = 9.8 Hz, H-5), 5.73 (1H, d J = 11.6 Hz, H-1), 5.55 (1H, d, J = 15.6 Hz, H-11), 5.51 (1H, dd, J = 5.0, 4.5 Hz, H-3), 5.30 (1H, d, J = 15.6 Hz, H-12), 5.29 (1H, d, J = 3.0 Hz, H-7), 5.14 (1H, dd, J = 3.1, 3.1 Hz, H-9), 4.84 (1H, d, J = 5.8 Hz, H14), 4.10 (1H, dd, J = 9.8, 5.0 Hz, H-4), 3.43 (1H, d, J = 5.8 Hz, HO14), 2.83 (1H, s, HO-13), 2.49 (1H, m, H-2), 2.07 (2H, dd, J = 3.5, 3.1 Hz, H2-8), 1.86 (3H, s, H3-17), 1.38 (1H, s, H-20), 0.96 (3H, d, J = 6.8 Hz, H3-16), 0.94 (3H, s, H3-18), 0.93 (3H, s, H3-19). 1-O-thiophene2-carbonyl: 7.89 (1H, d, J = 3.7 Hz), 7.60 (1H, d, J = 4.9 Hz), 7.17 (1H, dd, J = 4.9, 3.7 Hz). 3-OBz: 7.73 (2H, dd, J = 7.3, 1.0 Hz), 7.31 (1H, ddd, J = 7.7, 7.3, 1.0 Hz), 7.12 (2H, dd, J = 7.7, 7.3 Hz). 7-OBz: 7.57 (2H, dd, J = 7.3, 1.2 Hz), 7.26 (1H, ddd, J = 7.8, 7.3, 1.2 Hz), 6.98 (2H, dd, J = 7.8, 7.3 Hz). 15-OAc: 2.38 (3H, s). 9-OAc:1.67 (3H, s); 13 C NMR (CDCl3, 100 MHz) δC 134.5 (C-6), 132.1 (C-11), 129.6 (C-12), 119.1 (C-5), 90.1 (C-15), 87.4 (C-1), 77.4 (C-3), 74.6 (C13), 74.2 (C-7), 73.9 (C-9), 72.1 (C-14), 44.1 (C-2), 41.8 (C-4), 39.6 (C-10), 32.3 (C-8), 31.5 (C-20), 22.8 (C-19), 20.6 (C-18), 16.5 (C17), 11.4 (C-16). 1-O-thiophene-2-carbonyl: 160.6, 134.1, 132.9, 132.2, 128.4. 3-OBz: 165.2, 132.7, 129.5, 129.1 × 2, 128.2 × 2. 7-OBz: 165.0, 132.2, 129.3, 129.2 × 2, 127.8 × 2. 15-OAc: 171.0, 22.1. 9-OAc: 170.0, 20.8; ESIMS m/z 811.3 [M + Na]+; HREIMS m/z 811.2783 [M + Na]+ (calcd for C43H48O12SNa, 811.2759). (1S,2R,3S,4S,7R,9R,13R,14R,15R)-9,15-diacetoxy-1,3,7-tribenzoyloxy-13,14-dihydroxyjatropha-5E,11E-diene (23) was obtained from reaction with benzoyl chloride. Eight mg, white powder; [α]25D − 13.2 (c 0.50, CHCl3); UV (MeOH) λmax (log ε) 274 (3.50), 231 (4.43) nm; IR (KBr) νmax 3500, 2971, 2935, 1730, 1452, 1280, 1113, 1027, 982, 710 cm−1; 1H NMR (CDCl3, 400 MHz) δH 5.89 (1H, d, J = 9.7 Hz, H5), 5.88 (1H, d J = 11.8, H-1), 5.54 (1H, dd, J = 5.3, 4.3 Hz, H-3), 5.54 (1H, d, J = 15.5 Hz, H-11), 5.33 (1H, d, J = 15.5 Hz, H-12), 5.30 (1H, L



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(1S,2S,3S,4S,7R,9R,13R,14R,15S)-9,15-Fiacetoxy-3,7-dibenzoyloxy-1,13,14-trihydroxyjatropha-5E-ene (26). A solution of 12 (15 mg) in CH2Cl2 (5 mL) was treated with 10% Pd/C under an atmosphere of hydrogen for 10 h at rt. The reaction mixture was filtered and evaporated to dryness. The obtained residue was purified on a flash silica gel (CH2Cl2/MeOH, 300:1) to afford the 26 (9 mg). White powder; [α]25D +28.0 (c 0.17, CHCl3); UV (MeOH) λmax (log ε) 273 (3.34), 232 (4.24) nm; IR (KBr) νmax 3468, 2961, 2931, 1727, 1713, 1265, 1025, 711 cm−1; 1H NMR (CDCl3, 400 MHz) δH 6.15 (1H, d, J = 9.8 Hz, H-5), 5.66 (1H, d, J = 6.5 Hz, H-9), 5.47 (1H, dd, J = 4.6, 4.0 Hz, H-3), 5.37 (1H, d, J = 6.4 Hz, H-7), 4.73 (1H, d, J = 5.1 Hz, HO-13), 4.52 (1H, dd, J = 3.7 Hz, HO-14), 4.30 (1H, dd, J = 11.7, 3.4 Hz, H-1), 4.24 (1H, d, J = 3.7 Hz, H-14), 4.20 (1H, d, J = 3.5 Hz, HO-1), 4.07 (1H, dd, J = 9.8, 4.6 Hz, H-4), 2.50 (1H, dd, J = 12.8, 5.2 Hz, H-12b), 2.25 (1H, m, H-2), 2.15 (1H, m, H-8a), 1.91 (1H, m, H8b), 1.83 (3H, s, H3-17), 1.57 (1H, d, J = 12.8 Hz, H-11a), 1.23 (3H, s, H3-20), 1.14 (1H, ddd, J = 5.6, 5.5, 5.2 Hz, H-11b), 1.02 (3H, d, J = 6.6 Hz, H3-16), 0.84 (3H, s, H3-18), 0.75 (3H, s, H3-19). 3-OBz: 7.79 (2H, dd, J = 8.3, 1.2 Hz), 7.43 (1H, ddd, J = 8.3, 7.6, 1.2 Hz), 7.24 (2H, dd, J = 8.3, 7.6 Hz). 7-OBz: 7.61 (2H, dd, J = 8.3, 1.2 Hz), 7.29 (1H, ddd, J = 8.3, 7.6, 1.2 Hz), 6.96 (2H, dd, J = 8.3, 7.6 Hz). 15-OAc: 2.42 (3H, s). 9-OAc: 1.41 (3H, s); 13C NMR (CDCl3, 100 MHz) δC 137.9 (C-6), 118.5 (C-5), 91.3 (C-15), 87.8 (C-1), 78.1 (C-3), 74.7 (C-7), 73.4 (C-13), 72.8 (C-14), 72.0 (C-9), 43.5 (C-2), 41.9 (C-4), 37.4 (C-10), 32.0 (C-11), 31.5 (C-8), 29.1 (C-12), 27.2 (C-20), 24.1 (C-19), 23.0 (C-18), 17.2 (C-17), 11.8 (C-16). 3-OBz: 165.2, 132.7, 129.8, 129.4 × 2, 128.3 × 2. 7-OBz: 165.2, 132.3, 129.6, 129.1 × 2, 127.8 × 2. 15-OAc: 173.1, 22.0. 9-OAc: 170.4, 20.7; ESIMS m/z 679.0 [M − H]−; HREIMS m/z 703.3110 [M + Na]+ (calcd for C38H48O11Na, 703.3089). Preparation of 27 and 28 by Alkaline Hydrolysis of 1. Starting from 1 (20 mg), compounds 27 and 28 were prepared following the same procedures used for synthesis of 14−16. The reaction mixture was purified by RP-HPLC (CH3OH/H2O, 7.5:2.5, 3 mL/min) to afford 27 (4.1 mg, tR 12 min) and 28 (3.5 mg, tR 13 min). (1S,2R,8R,9S,14R,15S)-9,15-diacetoxy-1,8,14-trihydroxy-7-oxojatropha-3Z,5E,12E-triene (27). White powder; [α]25D − 58.8 (c 0.03, CDCl3); UV (MeOH) λmax (log ε) 255 (3.80), 209 (4.02) nm; IR (KBr) νmax 3476, 2959, 2918, 1736, 1711, 1676, 1242, 1107, 808 cm−1; 1H NMR (CDCl3, 400 MHz) δH 6.89 (1H, s, H-5), 5.83 (1H, s, H-3), 5.38 (1H, m, H-12), 5.02 (1H, s, H-9), 4.93 (1H, m, H-8), 4.92 (1H, s, H-14), 4.71 (1H, d, J = 2.3 Hz, HO-1), 4.38 (1H, d, J = 2.2 Hz, HO-14), 4.12 (1H, dd J = 7.5, 2.3 Hz, H-1), 3.56 (1H, d, J = 8.1 Hz, HO-8), 3.02, (1H, m, H-2), 2.73 (1H, dd, J = 15.8, 11.8 Hz, H-11a), 2.06 (1H, m, H-11b), 1.83 (3H, s, H3-20), 1.80 (3H, s, H3-17), 1.24 (3H, d, J = 7.0 Hz, H3-16), 1.20 (3H, s, H3-18), 0.98 (3H, s, H3-19). 15-OAc: 2.17 (3H, s). 9-OAc: 2.01 (3H, s); 13C NMR (CDCl3, 100 MHz) δC 201.5 (C-7), 138.3 (C-3), 137.2 (C-5), 137.1 (C-13), 136.3 (C-6), 134.3 (C-4), 122.8 (C-12), 97.8 (C-15), 87.6 (C-1), 77.7 (C9), 75.4 (C-14), 70.7 (C-8), 45.2 (C-2), 40.0 (C-10), 39.2 (C-11), 25.3 (C-19), 24.8 (C-18) 18.5 (C-16), 14.0 (C-20), 12.0 (C-17). 15OAc: 173.4, 22.0. 9-OAc:170.1, 20.5; EIMS m/z 473.1 [M + Na]+; HREIMS m/z 451.1023 [M + H]+ (calcd for C24H35O8, 451.2326). (1S,2R,8R,9S,14R,15S)-1,9,14,15-tetraacetoxy-8-hydroxy-7-oxojatropha-3Z,5E,12E-triene (28). White powder; [α]25D − 68.6 (c 0.07, CDCl3); UV (MeOH) λmax (log ε) 255 (3.75), 209 (3.93) nm; IR (KBr) νmax 3469, 2959, 2922, 1743, 1676, 1246, 1217, 1043, 1024, 882 cm−1; 1H NMR (CDCl3, 400 MHz) δH 6.97 (1H, s, H-5), 5.84 (1H, dd, J = 1.8, 1.7 Hz, H-3), 5.80 (1H, m, H-12), 5.80 (1H, s, H-14), 5.79 (1H, d, J = 7.0 Hz, H-1), 4.88 (1H, s, H-8), 4.87 (1H, s, H-9), 3.59 (1H, d, J = 8.0 Hz, HO-8), 2.98 (1H, m, H-2), 2.72 (1H, dd, J = 15.5, 12.1 Hz, H-11a), 1.82 (3H, s, H3-17), 1.68 (3H, s, H3-20), 1.22 (3H, d, J = 7.1 Hz, H3-16), 1.19 (3H, s, H3-18), 0.99 (3H, s, H3-19). 14-OAc: 2.15 (3H, s). 1-OAc: 2.06 (3H, s). 15-OAc: 2.04 (3H, s). 9OAc: 1.98 (3H, s); 13C NMR (CDCl3, 100 MHz) δC 201.1 (C-7), 137.0 (C-6), 136.7 (C-3), 136.3 (C-5), 135.4 (C-4), 133.5 (C-13), 125.3 (C-12), 94.0 (C-15), 83.0 (C-1), 78.3 (C-9), 74.8 (C-14), 70.8 (C-8), 44.1 (C-2), 40.2 (C-10), 39.7 (C-11), 25.5 (C-19), 24.7 (C18), 17.9 (C-16), 14.9 (C-20), 11.5 (C-17). 1-OAc:170.7, 21.0. 9-OAc: 169.9, 22.1. 15-OAc: 169.7, 20.3. 14-OAc: 169.4, 20.6; EIMS m/z

s, Hz, H-7), 5.15 (1H, dd, J = 3.5, 2.9 Hz, H-9), 4.86 (1H, d, J = 5.6 Hz, H-14), 4.11 (1H, dd, J = 9.7, 5.1 Hz, H-4), 2.52 (1H, m, H-2), 2.08 (2H, m, H2-8), 1.87 (3H, s, H3-17), 1.39 (1H, s, H-20), 0.95 (3H, s, H3-19), 0.94 (3H, s, H3-18), 0.93 (3H, d, J = 6.7 Hz, H3-16). 1-OBz: 8.02 (2H, dd, J = 7.3, 1.2 Hz), 7.61 (1H, ddd, J = 7.7, 7.3, 1.2 Hz), 7.50 (2H, dd, J = 7.7, 7.3 Hz). 3-OBz: 7.60 (2H, dd, J = 7.3, 1.1 Hz), 7.31 (1H, ddd, J = 7.8, 7.3, 1.1 Hz), 7.13 (2H, dd, J = 7.8, 7.3 Hz). 7-OBz: 7.58 (2H, dd, J = 7.2, 1.0 Hz), 7.28 (1H, ddd, J = 7.8, 7.2, 1.0 Hz), 7.00 (2H, dd, J = 7.8, 7.2 Hz). 15-OAc: 2.38 (3H, s). 9-OAc: 1.67 (3H, s); 13 C NMR (CDCl3, 100 MHz) δC 134.4 (C-6), 132.1 (C-11), 129.9 (C-12), 119.3 (C-5), 90.1 (C-15), 87.1 (C-1), 77.2 (C-3), 74.7 (C-13), 74.2 (C-7), 73.9 (C-9), 72.3 (C-14), 44.2 (C-2), 41.9 (C-4), 39.6 (C10), 32.4 (C-8), 31.5 (C-20), 22.8 (C-19), 20.6 (C-18), 16.5 (C-17), 11.4 (C-16). 1-OBz: 165.4, 133.2, 130.1, 129.3 × 2, 128.7 × 2. 3-OBz: 165.3, 132.7, 129.5, 129.1 × 2, 128.2 × 2. 7-OBz: 165.0, 132.2, 129.3, 129.3 × 2, 127.8 × 2. 15-OAc: 170.9, 22.1. 9-OAc: 170.0, 20.8; ESIMS m/z 805.3 [M + Na]+; HREIMS m/z 805.3217 [M + Na]+ (calcd for C45H50O12Na, 805.3194). (4S,9R,13R,14R,15R)-9,15-Diacetoxy-13,14-dihydroxyjatropha-2Z,5E,11E-triene (24). A solution of 13 (15 mg) in dried CH2Cl2 (5 mL) was added Dess-Martin periodinane reagent (15 mg). The reaction mixture was stirred for 3 h at rt, then filtered and evaporated to dryness. The obtained residue was purified on a flash silica gel (CH2Cl2/MeOH, 200:1) to afford the 24 (10 mg). White powder; [α]25D +6.7 (c 0.09, CHCl3); UV (MeOH) λmax (log ε) 246 (4.09) nm; IR (KBr) νmax 3450, 2968, 2931, 1741, 1723, 1372, 1238, 1026 cm−1; 1H NMR (CDCl3, 400 MHz) δH 7.34 (1H, s, H-3), 6.87 (1H, d, J = 7.2 Hz, H-5), 6.08 (1H, d, J = 15.9 Hz, H-11), 5.86 (1H, d, J = 15.9 Hz, H-12), 4.73 (1H, d, J = 7.2 Hz, H-9), 4.51 (1H, m, H-4), 4.13 (1H, s, H-14), 3.48 (1H, d, J = 14.3 Hz, H-8a), 2.29 (1H, dd, J = 14.3, 6.5 Hz, H-8b), 1.43 (1H, s, H-20), 1.87 (1H, d, J = 1.1 H3-16), 1.66 (3H, s, H3-17), 1.10 (3H, s, H3-18), 1.10 (3H, s, H3-19). 15-OAc: 2.06 (3H, s). 9-OAc: 2.03 (3H, s); 13C NMR (CDCl3, 100 MHz) δC 204.6 (C-1), 202.3 (C-7), 155.2 (C-3), 140.6 (C-2), 138.6 (C-5), 137.5 (C-6), 133.0 (C-11), 132.7 (C-12), 83.3 (C-15), 78.8 (C-9), 75.7 (C-14), 75.5 (C-13), 43.1 (C-4), 41.7 (C-8), 39.0 (C-10), 29.9 (C-20), 23.3 (C-19), 21.5 (C-18), 12.2(C-17), 10.6 (C-16). 9-OAc: 170.4, 20.9. 15-OAc: 168.2, 21.0; ESIMS m/z 471.3 [M + Na]+; HREIMS m/z 471.2008 [M + Na]+ (calcd for C24H32O8Na, 471.1989). (1S,2R,3S,4S,7R,9R,13R,15S)-1,9,15-Triacetoxy-3,7-dibenzoyloxy-13-hydroxy-14-oxojatropha-5E,11E-diene (25). A solution of 17 (15 mg) in dried CH2Cl2 (5 mL) was added Dess-Martin periodinane reagent (15 mg). The mixture was stirred for 3 h at rt, then filtered and evaporated to dryness. The obtained residue was purified by RP-HPLC (CH3OH/H2O, 7.5:2.5, 3 mL/min) to afford 25 (4.5 mg, tR 13 min). White powder; [α]25D − 41.3 (c 0.14, CHCl3); UV (MeOH) λmax (log ε) 273 (3.45), 232 (4.27) nm; IR (KBr) νmax 3533, 2961, 2917, 1753, 1729, 1455, 1375, 1278, 1263, 1071, 803, 712 cm−1; 1H NMR (CDCl3, 400 MHz) δH 5.84 (1H, d, J = 10.5 Hz, H-5), 5.72 (1H, d, J = 15.6 Hz, H-11), 5.62 (1H, d, J = 8.0 Hz, H-1), 5.59 (1H, d, J = 15.6 Hz, H-12), 5.50 (1H, dd, J = 3.8, 3.8 Hz, H-3), 5.35 (1H, d, J = 6.1 Hz, H-7), 5.10 (1H, d, J = 4.4 Hz, H-9), 3.84 (1H, s, HO-13), 3.69 (1H, dd, J = 10.5, 4.0 Hz, H-4), 2.60 (1H, m, H-2), 2.18 (1H, m, H-8a), 1.86 (1H, d, J = 15.6 Hz, H-8b), 1.59 (3H, s, H3-17), 1.41 (3H, s, H3-20), 1.03 (3H, d, J = 6.8 Hz, H3-16), 0.98 (3H, s, H318), 0.98 (3H, s, H3-19). 3-OBz: 7.92 (2H, dd, J = 7.4, 1.2 Hz), 7.62 (1H, ddd, J = 7.5, 7.4, 1.2 Hz), 7.45 (2H, dd, J = 7.5, 7.4 Hz). 7-OBz: 7.66 (2H, dd, J = 7.2, 1.0 Hz), 7.29 (1H, ddd, J = 7.5, 7.2, 1.0 Hz), 6.91 (2H, dd, J = 7.5, 7.2 Hz). 15-OAc: 2.51 (3H, s). 1-OAc: 2.08 (3H, s). 9-OAc: 1.47 (3H, s); 13C NMR (CDCl3, 100 MHz) δC 213.1 (C-14), 136.9 (C-11), 136.6 (C-6), 116.8 (C-5), 95.3 (C-15), 84.7 (C-1), 79.7 (C-13), 78.1 (C-3), 74.3 (C-9), 73.8 (C-7), 54.3 (C-4), 45.4 (C-2), 39.7 (C-10), 31.5 (C-8), 27.8 (C-20), 23.8 (C-19), 20.9 (C-18), 16.0 (C-17), 11.6 (C-16). 3-OBz: 165.5, 133.1, 129.54 × 2, 129.51, 128.6 × 2. 7-OBz: 164.9, 132.6, 129.4 × 2, 129.3, 127.9 × 2, 15-OAc: 170.9, 22.0. 9-OAc: 170.2, 20.6. 1-OAc: 169.6, 21.0; ESIMS m/z 741.3 [M + Na]+; HREIMS m/z 741.2902 [M + Na]+ (calcd for C40H46O12Na, 741.2881). M



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557.1 [M + Na]+; HREIMS m/z 557.2374 [M + Na]+ (calcd for C28H38O10Na, 557.2357). (1S,2R,3S,4S,5S,8R,9S,14R,15S)-7,8,9,14,15-Pentaacetoxy1,3-dibenzoyloxy-5-hydroxyjatropha-6E,12E-diene (29). Starting from 10 (15 mg), the title compound was esterified by benzoyl chloride (200 μL) following the same procedure used for the synthesis of 19−23 and was obtained as a white powder solid (10.5 mg). White powder; [α]25D − 104.1 (c 0.17, CHCl3); UV (MeOH) λmax (log ε) 273 (3.32), 232 (4.24) nm; IR (KBr) νmax 3483, 2966, 2926, 1732, 1373, 1274, 1229, 1112, 1027, 714 cm−1; 1H NMR (CDCl3, 400 MHz) δH 6.58 (1H, s, H-8), 6.17 (1H, s, H-14), 5.99 (1H, d, J = 8.8 Hz, H-12), 5.88 (1H, d, J = 11.7 Hz, H-1), 5.55 (1H, d, J = 4.0, 3.9 Hz, H-3), 5.28 (1H, s, H-9), 4.62 (1H, dd, J = 11.0, 2.8 Hz, H-5), 4.43 (1H, dd, J = 11.0, 4.6 Hz, H-4), 2.69 (1H, m, H-2), 2.39 (1H, dd, J = 16.4, 9.5 Hz, H-11a), 2.00 (1H, m, H-11b), 1.65 (1H, s, H-20), 1.35 (3H, s, H3-17), 1.01 (3H, s, H3-18), 0.96 (3H, d, J = 6.8 Hz, H3-16), 0.90 (3H, s, H3-19). 1-OBz: 8.04 (2H, dd, J = 7.2, 1.0 Hz), 7.57 (1H, ddd, J = 7.5, 7.2, 1.0 Hz), 7.46 (1H, dd, J = 7.5, 7.2 Hz). 3-OBz: 8.01 (2H, dd, J = 8.2, 1.0 Hz), 7.56 (1H, ddd, J = 8.2, 7.5, 1.1 Hz), 7.45 (1H, dd, J = 8.2, 7.5 Hz). 14-OAc: 2.26 (3H, s). 7-OAc: 2.16 (3H, s). 8-OAc: 2.14 (3H, s). 15-OAc: 2.10 (3H, s). 9-OAc: 2.04 (3H, s); 13C NMR (CDCl3, 100 MHz) δC 143.8 (C-7), 124.8 (C-12), 91.4 (C-15), 84.5 (C-1), 78.5 (C-9), 75.9 (C-3), 72.8 (C-14), 71.4 (C-5), 69.5 (C8), 47.4 (C-4), 44.2 (C-2), 41.2 (C-11), 38.7 (C-10), 29.3 (C-19), 20.5 (C-18), 17.7 (C-17), 17.3 (C-20), 11.9 (C-16). 1-OBz: 166.4, 133.0, 130.1, 129.8 × 2, 128.4 × 2. 3-OBz: 165.4, 133.2, 129.9, 129.5 × 2, 128.6 × 2. 8-OAc: 170.3, 21.2. 9-OAc: 170.3, 21.8. 14-OAc: 169.2, 21.0. 7-OAc: 169.1, 20.1. 15-OAc: 167.8, 20.9; ESIMS m/z 843.3 [M + Na]+; HREIMS m/z 843.3225 [M + Na]+ (calcd for C44H52O15Na, 843.3198). (1S,2S,3S,4S,5S,8R,9S,14R,15S)-7,8-Diacetoxy-3-benzoyloxy1,5,9,14,15-pentahydroxyjatropha-6E,12E-diene (30). Starting from 9 (40 mg), the title compound was prepared following the same procedures used for the synthesis of 14−16. The reaction mixture was purified by RP-HPLC (CH3OH/H2O, 7:3, 3 mL/min) to afford 30 (20.1 mg, tR 10 min). White powder; [α]25D − 12.0 (c 0.13, CHCl3); UV (MeOH) λmax (log ε) 273 (3.04), 229 (4.12) 211 (3.97) nm; IR (KBr) νmax 3409, 2972, 2920, 1752, 1726, 1372, 1260, 1228, 1083, 1025, 713 cm−1; 1H NMR (CDCl3, 400 MHz) δH 6.70 (1H, s, H-8), 5.85 (1H, s, H-12), 5.38 (1H, d, J = 3.7, 3.6 Hz, H-3), 5.08 (1H, s, H-9), 4.52 (1H, s, H-14), 4.47 (1H, d, J = 11.1 Hz, H-5), 4.05 (1H, d, J = 9.3 Hz, H-1), 3.78 (1H, dd, J = 11.1, 3.6 Hz, H-4), 2.40 (1H, m, H-11a), 2.23 (1H, m H-2), 1.94 (1H, m, H-11b), 1.76 (1H, s, H-20), 1.27 (3H, s, H3-17), 1.01 (3H, s, H3-18), 0.97 (3H, d, J = 6.5 Hz, H316), 0.86 (3H, s, H3-19). 1-OBz: 7.98 (2H, dd, J = 7.5, 1.0 Hz), 7.47 (1H, ddd, J = 7.5, 7.2, 1.0 Hz), 7.34 (1H, dd, J = 7.5, 7.4 Hz). 9-OAc: 2.10 (3H, s). 7-OAc:2.05 (3H, s). 8-OAc: 2.01 (3H, s); 13C NMR (CDCl3, 100 MHz) δC 143.1 (C-7), 136.9 (C-13), 121.7 (C-12), 88.8 (C-1), 85.1 (C-15), 78.9 (C-9), 77.7 (C-14), 76.0 (C-3), 71.0 (C-5), 69.9 (C-8), 45.4 (C-2), 44.6 (C-4), 41.2 (C-11), 38.7 (C-10), 29.3 (C19), 20.7 (C-18), 18.0 (C-17), 18.9 (C-20), 11.9 (C-16). 3-OBz: 165.5, 132.9, 129.9, 129.5 × 2, 128.4 × 2. 8-OAc: 170.9, 21.0. 9-OAc: 170.9, 21.0. 7-OAc: 167.8, 20.1; ESIMS m/z 655.3 [M + Na]+; HREIMS m/z 655.2748 [M + Na]+ (calcd for C31H42O11Na, 655.2619). (1S,2R,3S,4S,8R,9S,12S,13R,14R,15S)-1,8,9,14,15-Pentaacetoxy-3-benzoyloxy-12,13-epoxy-7-oxojatropha-5E-ene (31). A solution of 11 (15 mg) in dried CH2Cl2 (5 mL) was added m-CPBA (15 mg). The mixture was stirred for 3 h at rt, then concentrated to dryness. The obtained residue was purified on a flash silica gel eluted with CH2Cl2 to afford the 31 (10 mg). White powder; [α]25D +62.7 (c 0.26, CHCl3); UV (MeOH) λmax (log ε) 231 (3.93), 205 (3.76) nm; IR (KBr) νmax 2963, 2913, 1752, 1727, 1668, 1261, 1098, 1020, 800 cm−1; 1H NMR (CDCl3, 400 MHz) δH7.20 (1H, dd, J = 9.4, 1.3 Hz, H-5), 5.98 (1H, s, H-14), 5.84 (1H, d, J = 8.6 Hz, H-9), 5.66 (1H, dd, J = 4.0, 3.7 Hz, H-3), 5.49 (1H, d, J = 10.7 Hz, H-1), 5.36 (1H, d, J = 4.0 Hz, H-8), 3.79 (1H, dd, J = 9.4, 4.0 Hz, H-4), 3.73 (1H, dd, J = 5.5, 2.0 Hz, H-12), 2.25 (1H, m, H-2), 1.92 (3H, s, H-17), 1.47 (1H, d, J = 16.8 Hz, Ha-11), 1.11 (1H, d, J = 5.5 Hz, Hb-11), 1.06 (3H, s, H3-18), 1.05 (3H, d, J = 6.8 Hz, H3-16) 1.00 (3H, s, H3-19). 3-OBz: 8.07 (2H,

dd, J = 8.3, 1.3 Hz), 7.60 (1H, ddd, J = 8.3, 7.8, 1.3 Hz), 7.51 (1H, dd, J = 8.3, 7.8 Hz). 15-OAc: 2.37 (3H, s). 14-OAc: 2.21 (3H, s). 9-OAc: 2.08 (3H, s). 1-OAc: 2.03 (3H, s). 8-OAc: 1.20 (3H, s); 13C NMR (CDCl3, 100 MHz) δC 194.1 (C-7), 138.4 (C-6), 138.4 (C-5), 89.7 (C-15), 85.2 (C-1), 80.5 (C-8), 77.3 (C-3), 76.5 (C-9), 69.2 (C-14), 59.8 (C-13), 56.2 (C-12), 46.9 (C-4), 45.8 (C-2), 37.2 (C-11), 37.1 (C-10), 25.8 (C-19), 22.4 (C-18), 16.3 (C-20), 13.2 (C-17), 11.9 (C16). 3-OBz: 165.8, 133.6, 129.9 × 2, 129.4, 128.8 × 2. 9-OAc: 169.8, 21.1. 1-OAc: 169.8, 20.6. 8-OAc: 169.5, 19.3. 15-OAc: 169.2, 22.4. 14OAc: 168.9, 21.8; ESIMS m/z 737.3 [M + Na]+; HREIMS m/z 737.2809 [M + Na]+ (calcd for C37H46O14Na, 737.2780). (1S,2R,3S,4S,8R,9S,14R,15S)-1,7,8,9,14,15-Hexaacetoxy-3benzoyloxy-5-oxojatropha-6E,12E-diene (32). Starting from 9 (15 mg), the title compound was prepared following the same procedure used for the synthesis of 24 and was obtained as a white powder solid (10 mg). White powder; [α]25D − 115.9 (c 0.12, CHCl3); UV (MeOH) λmax (log ε) 273 (2.90), 230 (3.98) 206 (3.86) nm; IR (KBr) νmax 3646, 3558, 2974, 1744, 1374, 1247, 1226, 1095, 1058, 718 cm−1; 1H NMR (CDCl3, 400 MHz) δH 6.20 (1H, s, H-8), 6.09 (1H, s, H-14), 6.00 (1H, d, J = 4.1, 3.8 Hz, H-3), 5.71 (1H, d, J = 9.4 Hz, H-12), 5.48 (1H, d, J = 11.1 Hz, H-1), 4.82 (1H, d, J = 4.1 Hz, H-4), 4.75 (1H, s, H-9), 2.46 (1H, m, H-2), 2.26 (1H, m, H-11a), 1.97 (H-11b), 1.82 (1H, s, H-20), 1.33 (3H, s, H3-17), 0.97 (3H, d, J = 6.8 Hz, H3-16), 0.95 (3H, s, H3-19), 0.85 (3H, s, H3-18). 3-OBz: 7.98 (2H, dd, J = 8.3, 1.2 Hz), 7.55 (1H, ddd, J = 8.3, 7.7, 1.2 Hz), 7.42 (1H, dd, J = 8.3, 7.7 Hz). 1-OAc: 2.21 (3H, s). 14-OAc: 2.18 (3H, s). 9-OAc: 2.12 (3H, s). 15-OAc: 2.11 (3H, s). 7-OAc: 2.08 (3H, s). 8OAc: 2.06 (3H, s); 13C NMR (CDCl3, 100 MHz) δC 198.4 (C-5), 142.1 (C-7) 133.4 (C-13), 130.4 (C-6), 122.1 (C-12), 89.4 (C-15), 84.9 (C-1), 78.8 (C-9), 76.4 (C-3), 71.8 (C-14), 68.7 (C-8), 56.5 (C4), 44.2 (C-2), 40.2 (C-11), 38.4 (C-10), 28.5 (C-19), 20.1(C-18), 16.1 (C-20), 13.7 (C-17), 12.1 (C-16). 3-OBz: 165.5, 129.7 × 2, 129.5, 128.5 × 2. 8-OAc: 170.4, 20.9. 7-OAc: 170.0, 20.8. 1-OAc: 169.9, 20.7. 9-OAc: 169.6, 22.0. 14-OAc: 168.9, 20.7. 15-OAc: 167.6, 20.1; ESIMS m/z 779.2 [M + Na]+; HREIMS m/z 779.2917 [M + Na]+ (calcd for C39H48O15Na, 779.2885). (1S,2R,3S,4S,8R,9S,12S,13R,14R,15S)-1,7,8,9,14,15-Hexaacetoxy-3-benzoyloxy-12,13-epoxyjatropha-6E-ene (33). Starting from 9 (15 mg), the title compound was prepared following the same procedure used for the synthesis of 31 and was obtained as a colorless crystals in PE/acetone (10:1). Twelve mg, mp 180−182 °C; [α]25D − 38.7 (c 0.31, CHCl3); UV (MeOH) λmax (log ε) 273 (2.94), 229 (4.05) 207 (3.91) nm; IR (KBr) νmax 33474, 2976, 2939, 1759, 1728, 1375, 1228, 1073, 1030, 717 cm−1; 1H NMR (CDCl3, 400 MHz) δH 6.31 (1H, s, H-8), 6.18 (1H, s, H-14), 5.44 (1H, d, J = 4.5, 4.2 Hz, H3), 5.43 (1H, d, J = 11.4 Hz H-1), 5.19 (1H, s, H-9), 4.66 (1H, dd, J = 11.3, 3.7 Hz, H-5), 4.20 (1H, dd, J = 4.1 Hz, H-4), 3.57 (1H, d, J = 3.4 Hz, H-12), 2.30 (1H, m, H-2), 1.33 (1H, s, H-20), 1.32 (3H, s, H3-17), 1.05 (3H, s, H3-19), 0.87 (3H, d, J = 6.8 Hz, H3-16), 0.85 (3H, s, H318). 3-OBz: 7.96 (2H, dd, J = 8.0, 1.0 Hz), 7.57 (1H, ddd, J = 8.0, 7.7, 1.0 Hz), 7.71 (1H, dd, J = 8.0, 7.7 Hz). 7-OAc: 2.16 (3H, s). 9-OAc: 2.15 (3H, s). 1-OAc: 2.10 (3H, s). 14-OAc: 2.09 (3H, s). 15-OAc: 2.08 (3H, s), 8-OAc: 2.02 (3H, s); 13C NMR (CDCl3, 100 MHz) δC 144.1 (C-7), 129.8 (C-6), 88.4 (C-15), 84.9 (C-1), 79.5 (C-9), 76.2 (C-3), 71.0 (C-5), 68.7 (C-14), 68.1 (C-8), 60.1 (C-13), 54.9 (C-12), 47.9 (C-4), 44.2 (C-2), 40.5 (C-11), 38.0 (C-10), 29.4 (C-18), 19.3 (C19), 17.6 (C-20), 17.0 (C-17), 11.8 (C-16). 3-OBz: 165.4, 133.3, 130.1, 129.5 × 2, 128.6 × 2. 9-OAc: 170.7, 20.9. 1-OAc: 170.3, 20.9. 8OAc: 170.1, 20.7. 7-OAc: 169.3, 21.9. 14-OAc: 168.6, 20.8. 15-OAc: 167.7, 20.0; ESIMS m/z 797.3 [M + Na]+; HREIMS m/z 797.3024 [M + Na]+ (calcd for C39H50O16Na, 797.2991). (1S,2R,3S,4R,5S,8R,9S,12S,13S,14R,15S)-1,7,8,9,14,15-Hexaacetoxy-3-benzoyloxy-12-hydroxy-5,13-epoxyjatropha-6E-ene (34). Compound 33 (11 mg) was treated with HCl (1% in MeOH, 5 mL) at rt for 1 h. After removal of the solvent, the crude mixture was purified on a flash silica gel columneluted with CH2Cl2 to afford the 34 (9 mg). White powder; [α]25D − 59.3 (c 0.49, CHCl3); UV (MeOH) λmax (log ε) 273 (2.77), 230 (3.94) 205 (3.83) nm; IR (KBr) νmax 3522, 2966, 2936, 1747, 1247, 1081, 715 cm−1; 1H NMR (CDCl3, 400 MHz) δH 6.53 (1H, s, H-8), 5.91 (1H, s, H-14), 5.65 (1H, d, J = 3.6, N



Article

0.1200, Rsigma = 0.0625) which were used in all calculations. The final R1 was 0.0795 (>2sigma(I)) and wR2 was 0.2495 (all data). Flack parameter = 0.0 (3). Crystal Data for peditithin F (6). C33H44O11, (M = 616.68 g/mol): monoclinic, space group C2 (no. 5), a = 33.6576(3) Å, b = 9.29614(7) Å, c = 11.57569(12) Å, β = 98.0893(9)°, V = 3585.83(5) Å3, Z = 4, T = 103 (2) K, μ(Cu Kα) = 0.707 mm−1, Dc = 1.142 g/cm3, 35102 reflections measured (5.3° ≤ 2θ ≤ 143.58°), 6970 unique (Rint = 0.0333, Rsigma = 0.0188) which were used in all calculations. The final R1 was 0.0516 (>2sigma(I)) and wR2 was 0.1620 (all data). Flack parameter = −0.05(14). Crystal Data for (1S,2S,3S,4S,5S,8R,9S,14R,15S)-1,7,8,9,14,15hexaacetoxy-3-benzoyloxy-5-hydroxyjatropha-6E,12E-diene (9). Colorless crystals, mp 230−232 °C; C39H50O15·MeOH (M = 790.83 g/mol): orthorhombic, space group P212121 (no. 19), a = 9.42038(6) Å, b = 17.88633(13) Å, c = 24.9535(2) Å, V = 4204.56(6) Å3, Z = 4, T = 103(2) K, μ(Cu Kα) = 0.808 mm−1, Dc = 1.249 g/cm3, 41413 reflections measured (6.08° ≤ 2θ ≤ 143.4°), 8195 unique (Rint = 0.0413, Rsigma = 0.0259) which were used in all calculations. The final R1 was 0.0299 (>2sigma(I)) and wR2 was 0.0755 (all data). Flack parameter = 0.03 (9). Crystal Data for (1S,2S,3S,4S,7R,9R,13R,14R,15S)-9,15-diacetoxy3,7-dibenzoyloxy-1,13,14-trihydroxyjatropha-5E,11E-diene (12). Colorless crystals, mp 84−86 °C; C38H46O11, (M = 678.75 g/mol): orthorhombic, space group P212121 (no. 19), a = 10.40656(15) Å, b = 15.8771(2) Å, c = 21.6213(3) Å, V = 3572.40(9) Å3, Z = 4, T = 103 (2) K, μ(Cu Kα) = 0.760 mm−1, Dc = 1.262 g/cm3, 22849 reflections measured (6.908° ≤ 2θ ≤ 134.076°), 6300 unique (Rint = 0.0293, Rsigma = 0.0235) which were used in all calculations. The final R1 was 0.0299 (I > 2σ(I)) and wR2 was 0.0742 (all data). Flack parameter = −0.07 (6). Crystal Data for (1S,2S,3S,4S,8R,9S,12S,13R,14R,15S)1,7,8,9,14,15-hexaacetoxy-3-benzoyloxy-12,13-epoxyjatropha-6Eene (33). Colorless crystals, mp 112−114 °C; C39H50O16·MeOH, (M = 806.83 g/mol): orthorhombic, space group P212121 (no. 19), a = 9.42109(9) Å, b = 18.09352(15) Å, c = 24.80785(18) Å, V = 4228.76(6) Å3, Z = 4, T = 103 (2) K, μ(Cu Kα) = 0.832 mm−1, Dc = 1.267 g/cm3, 41995 reflections measured (6.04° ≤ 2θ ≤ 143.72°), 8276 unique (Rint = 0.0377, Rsigma = 0.0253) which were used in all calculations. The final R1 was 0.0313 (>2sigma(I)) and wR2 was 0.0796 (all data). Flack parameter = −0.10 (9). Cell Culture. HepG2/ADR, MCF-7/ADR and HLF were cultured in tissue culture flasks in RPMI1640 complete growth medium (Gibco BRL, USA) supplemented with 10% fetal bovine serum (FBS) (Gibco BRL, USA), 100 IU/mL penicillin, and 100 μg/mL streptomycin (Beyotime, CHN). All cells were maintained in a carbon dioxide incubator containing (37 °C, 5% CO2, 90% relative humidity (RH)). To maintain their MDR, HepG2/ADR, and MCF-7/ADR cells were cultured in medium containing 1.2 μM of Adriamycin. Western Blot Analysis. The total cellular samples were harvested and rinsed twice with ice-cold PBS buffer. Cell extracts were lysed in RIPA buffer (50 mM Tris (pH 7.4), 150 mM NaCl, 1% Triton X-100, 1% sodium deoxycholate, 0.1% SDS, sodium orthovanadate, sodium fluoride and EDTA) containing protease inhibitor cocktails (Roche Life Science, USA). Protein concentration was determined using the BCA protein assay kit (Thermo Fisher Scientific, Rockford, Illinois, USA). Cell lysates containing identical amounts of total protein (25 μg) were resolved by sodium dodecyl sulfate polycrylamide gelelectrophoresis (SDS-PAGE) and subsequently electrophoretically transferred onto PVDF membranes (Millipore, Darmstadt, Germany). After blocking in tris-buffered saline containing 0.1% of Tween 20 (TBST) with 5% (w/v) skim milk (Nestle Carnation, New Zealand) for 2 h at room temperature, the membranes were incubated with primary and secondary antibodies (Cell Signaling Technology, Beverly, MA, USA) and subsequently visualized with an enhanced chemiluminescence detection kit (Advansta WesternBright ECL, USA). GADPH was used as the loading control for the experimental data analysis. Intracellular Accumulation of Rho123. HepG2/ADR cells were seeded into 6-well plates at a density of 5 × 105/well and were

3.5 Hz, H-3), 5.40 (1H, d, J = 9.4 Hz, H-1), 5.09 (1H, s, H-9), 4.79 (1H, d, J = 10.5 Hz, H-5), 4.15 (1H, d, J = 3.7 Hz, H-12), 3.31 (1H, dd, J = 10.5, 3.5 Hz, H-4), 2.23 (1H, m, H-2), 2.20 (1H, dd, J = 15.9, 6.9 Hz, H-11a), 1.36 (1H, m, H-11b), 1.34 (1H, s, H-20), 1.31 (3H, s, H3-17), 1.01 (3H, d, J = 6.9 Hz, H3-16), 0.92 (3H, s, H3-18), 0.85 (3H, s, H3-19). 3-OBz: 8.00 (2H, dd, J = 7.2, 1.2 Hz), 7.59 (1H, ddd, J = 7.8, 7.2, 1.2 Hz), 7.45 (1H, dd, J = 7.8, 7.2 Hz). 14-OAc: 2.36 (3H, s). 15-OAc: 2.27 (3H, s), 9-OAc: 2.11 (3H, s). 7-OAc: 2.08 (3H, s). 1OAc: 2.06 (3H, s). 8-OAc: 1.99 (3H, s); 13C NMR (CDCl3, 100 MHz) δC 144.5 (C-7), 124.2 (C-6), 85.0 (C-1), 84.5 (C-15), 82.0 (C9), 80.2 (C-13), 75.5 (C-3), 72.1 (C-5), 69.8 (C-14), 69.0 (C-12), 66.6 (C-8), 48.3 (C-4), 47.6 (C-2), 46.6 (C-11), 37.6 (C-10), 29.0 (C19), 18.3 (C-18), 17.8 (C-20), 14.4 (C-17), 12.2 (C-16). 3-OBz: 165.6, 133.4, 129.7, 129.5 × 2, 128.5 × 2. 14-OAc: 173.2, 21.2. 9-OAc: 170.1, 20.8. 8-OAc: 170.0, 20.5. 15-OAc: 169.8, 22.2. 1-OAc: 169.4, 20.0. 7-OAc: 168.3, 20.9; ESIMS m/z 797.3 [M + Na]+; HREIMS m/z 797.3027 [M + Na]+ (calcd for C39H50O16Na, 797.2991). (1S,2R,3S,4R,5S,8R,9S,12S,13S,14R,15S)-1,7,8,9,12,14,15Heptaacetoxy-3-benzoyloxy-5,13-epoxyjatropha-6E-ene (35). Starting from 34 (8 mg), the title compound was prepared following the same procedure used for the sysnthesis of 17 and 18. The reaction mixture was purified by RP-HPLC (CH3OH/H2O, 8.5:1.5, 3 mL/ min) to afford 35 (4.6 mg, tR 12 min). White powder; [α]25D − 100 (c 0.04, CHCl3); UV (MeOH) λmax (log ε) 273 (3.26), 228 (4.22) 208 (4.18) nm; IR (KBr) νmax 2958, 2917, 1747, 1460, 1377, 1247, 1229, 1085, 1022 cm−1; 1H NMR (CDCl3, 400 MHz) δH 6.58 (1H, s, H-8), 5.93 (1H, s, H-14), 5.64 (1H, d, J = 3.5, 3.1 Hz, H-3), 5.44 (1H, d, J = 9.3 Hz, H-1), 5.43 (1H, m, H-12), 5.12 (1H, s, H-9), 4.82 (1H, d, J = 10.4 Hz, H-5), 3.32 (1H, dd, J = 10.4, 3.1 Hz, H-4), 2.31 (1H, m, H11a), 1.42 (1H, s, H-20), 1.32 (3H, s, H3-17), 1.05 (3H, s, H3-18), 0.98 (3H, d, J = 6.9 Hz, H3-16), 0.82 (3H, s, H3-19). 3-OBz: 8.00 (2H, dd, J = 7.2, 1.2 Hz), 7.59 (1H, ddd, J = 7.7, 7.2, 1.2 Hz), 7.45 (1H, dd, J = 7.7, 7.2 Hz). 1-OAc: 2.29 (3H, s). 8-OAc: 2.19 (3H, s). 9-OAc: 2.12 (3H, s). 7-OAc: 2.08 (3H, s). 12-OAc: 2.05 (3H, s). 14-OAc: 2.00 (3H, s). 15-OAc: 1.95 (3H, s); 13C NMR (CDCl3, 100 MHz) δC 144.7 (C-7), 123.7 (C-6), 84.9 (C-1), 84.7 (C-15), 81.9 (C-9), 79.5 (C-13), 75.4 (C-3), 72.0 (C-5), 70.6 (C-12), 67.5 (C-14), 66.7 (C-8), 48.2 (C4), 47.5 (C-2), 46.1 (C-11), 38.2 (C-10), 29.0 (C-20). 19.1 (C-18), 18.9 (C-20), 14.5 (C-17), 12.2 (C-16). 3-OBz: 165.6, 133.4, 129.7, 129.5 × 2, 128.5 × 2. 9-OAc: 170.3, 20.8. 15-OAc: 170.1, 21.5. 14OAc: 169.9, 20.9. 8-OAc: 169.8, 21.2. 12-OAc: 169.7, 20.5. 1-OAc: 169.6, 22.3. 7-OAc: 168.1, 20.0; ESIMS m/z 839.3 [M + Na]+; HREIMS m/z 839.3140 [M + Na]+ (calcd for C41H52O17Na, 839.3097). X-ray Crystal Structure Analysis. Single crystals of 1, 3, 6, 9, 12, and 33 were collected on an Xcalibur, Onyx, Nova diffractometer equipped with Cu Kα radiation (λ = 1.54184 Å). These structures were determined using direct methods and refined using olex2.19 All non-hydrogen atoms were refined using anisotropic thermal parameters. Hydrogen atoms were located by geometrical calculations. The absolute configurations of 1, 3, 6, 9, 12, and 33 were confirmed by refinement of the Flack parameters. Crystallographic data have been deposited at the Cambridge Crystallographic Data Centre, CCDC Numbers 1454874 (1), 1454875 (3), 1454876 (6), 1454877 (9), 1454878 (12), and 1454879 (33). These data are available free of charge from The Cambridge Crystallographic Data Centre via www. ccdc.cam.ac.uk/data_request/cif. Crystal Data for peditithin A (1). C30H40O11, (M = 576.62 g/mol): orthorhombic, space group P212121 (no. 19), a = 8.57949 (10) Å, b = 14.11566 (14) Å, c = 23.8181 (2) Å, V = 2884.49 (5) Å3, Z = 4, T = 103 (2) K, μ (Cu Kα) = 0.841 mm−1, Dc = 1.328 g/cm3, 28182 reflections measured (7.28° ≤ 2θ ≤ 143.46°), 5635 unique (Rint = 0.0648, Rsigma = 0.0437) which were used in all calculations. The final R1 was 0.0343 (>2sigma(I)) and wR2 was 0.0870 (all data). Flack parameter = 0.01(12). Crystal Data for peditithin C (3). C39H50O16, (M = 797.29 g/mol): monoclinic, space group P21 (no. 4), a = 13.3336(4) Å, b = 9.6931(4) Å, c = 17.6305(7) Å, β = 110.243(4)°, V = 2137.89(14) Å3, Z = 2,T = 103(2) K, μ(Cu Kα) = 0.823 mm−1, Dc = 1.250 g/cm3, 36670 reflections measured (5.34° ≤ 2θ ≤ 145.06°), 8231 unique (Rint = O



Article

incubated with given concentration of compounds and 10 μM Rho123 in the dark at 37 °C for 90 min. The cells were washed twice with icecold PBS after centrifugation, resuspended in PBS buffer, and analyzed by flow cytometry. Tariquidar and verapamil were evaluated at parallel concentration and time as a positive control. The fluorescence intensity of intracellular Rho123 was measured with a flow cytometer in FL1 channel (Em = 525 nm). Fold of control = (The fluorescence of compound treated cell−Background)/(The fluorescence of Rho123-only cell − Background). Cytotoxicity Analysis. Cytotoxicity was determined using the MTT colorimetric assay with minor modifications from that previously described.20 Cells (5 × 103 cells/well) were seeded into 96-well plates filled with culture medium containing various concentrations of test samples. The negative control was added DMSO (equal volume), and they were allowed to grow in carbon dioxide incubator (37 °C, 5% CO2, 90% RH). After 48 h of incubation, 20 μL of MTT solution (5 mg/mL) was added to each well, and the plate was further incubated for another 4 h, allowing viable cells to convert the yellow-colored MTT into dark blue formazan crystals. Subsequently, the medium was aspirated, and 150 μL DMSO was added to each well to dissolve the formazan crystals. The optical density (OD) was recorded on a TECAN Infinite M200 Pro multimode reader at 570 nm with a reference wavelength lecture at 630 nm. The IC50 values were expressed as concentration required to inhibit tumor cell proliferation by 50% and calculated by nonlinear regression of the survival curves in GraphPad Prism 5. All data are average values from triplicate samples. MDR Reversal Activity. HepG2/ADR and MCF-7/ADR cells were seeded into 96-well plates at a density of 5 × 103/well and were incubated with premeditated concentration of Pgp inhibitors (100 nM, 200 nM, or 500 nM) at a carbon dioxide incubator for 1 h. Then the cells were incubated with ADR (8 concentration gradients) together for an additional 48 h. The cytotoxicities of ADR with or without Pgp inhibitors were analyzed using the previously described MTT assay. Tariquidar and verapamil was used as a positive control, and DMSO was used as a negative control. The reversal fold is calculated as a ratio of IC50(ADR) to IC50(Pgp inhibitor+ADR). All experiments were performed at least two times. Molecular Modeling. The complex model of 26 with Pgp was generated by the Sybyl 7.3.5 software. The protein structure was extracted from the PDB file (PBD code 3G5U) and the ligand conformer was generated by MOE using the MMFF94x force field. Binding analysis were performed with Surflex-Dock in with threshold 0.5 and bloat 0, additional starting conformation per molecular was set to 5, and other parameters were set as default. The structural figures were drawn in PyMOL. Metabolic Stability. Rat liver microsomes were prepared according to previously described methods.21 The protein concentration was 31 mg/mL as determined by the method of Bradford.22 Compounds 26, tariquidar, verapamil, and testosterone were dissolved in CH3CN as a 10 mM stock solution and incubated with rat liver microsomes (1 mg of protein per mL, final concentration) at a final concentration of 100 μM in a final volume of 0.5 mL buffer solution (100.0 mM PBS; 3.0 mM MgCl2; 1.0 mM NADPH; pH = 7.4). The samples were incubated for various time intervals (0, 5, 10, 30, 60, 90, 120, 150, 180 min) at 37 °C in a water bath. The incubations were terminated at different time points by adding 0.5 mL of ice-cold CH3CN. A parallel incubation was performed in the absence of a NADPH-regenerating system and using microsomes as the negative control, and these reactions were terminated after the corresponding incubation times. After centrifugation with 15000 rpm at 4 °C for 10 min, the supernatants were directly analyzed by a HPLC-UV system (Agilent HPLC 1200 instrument, Supporting Information, Figure S266). Three independent experiments were performed in triplicate (Supporting Information, Figure S267). Tumor Xenografts Growth Inhibition Assay. All animal experiments complied with the Zhongshan School of Medicine Policy on the Care and Use of Laboratory Animals. Female BALB/c nude mice (5 weeks old) were purchased from the Experimental Animal Center at Sun Yat-Sen University and maintained in pathogen-free conditions to establish the model of xenografts of HepG2/ADR.23

HepG2/ADR cells were harvested during log-phase growth and resuspended in PBS at 6.67 × 107 cells/mL. Each mouse was injected subcutaneously in the right flank with 1 × 107 cells. When the tumor volume reached approximately 60 mm3, the mice were randomly divided into three groups of seven animals and treated ip with various regimens on days 0 (start of treatment), 3, and 6. The ADR-alone group was treated with ADR (5 mg/kg). The combination-schedule group, 26 (10 mg/kg) was administered ip 2 h before ADR (5 mg/kg) ip, whereas the control group was treated with an equivalent volume of normal saline (including 0.5% DMSO, 5% Kolliphor HS 15). Tumor size and body weight were measured every day. The tumor volume (V) was calculated using the formula V = (larger diameter) × (smaller diameter)2 /2, and growth curves were plotted using average tumor volume within each experimental group at the set time points. At the end of treatment, the animals were sacrificed, and the tumors were removed and weighed. The rate of inhibition (IR) was calculated according to the formula: IR = (1−Mean tumor weight of the experimental group/Mean tumor weight of the control group) × 100%. Differences were determined using the Student’s t test. Significance was determined at P < 0.05.

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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.jmedchem.6b00605. IR, HRESIMS, 1D and 2D NMR spectra of 1−8 and 14−35, 1H and 13C NMR spectra of known compounds 9−13, and HPLC chromatograms of compounds 1−35 (PDF) Complex model of 26 with Pgp (PDB ID code 3G5U) (PDB) Molecular formula strings for 1−35 (CSV)

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AUTHOR INFORMATION

Corresponding Authors

*(X.-Z. Bu) Phone and fax: +86-20-39943054. E-mail: [email protected]. *(S. Yin) Tel: +86-20-39943090 or +86-20-39943031. Fax: +86-20-39943090. E-mail: [email protected]. Author Contributions †

J.-Y. Zhu and R.-M. Wang contributed equally.

Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The authors thank the National High Technology Research and Development Program of China (863 Projects, no. 2015AA020928), the Guangdong Natural Science Funds for Distinguished Young Scholar (no. 2014A030306047), and the National Natural Science Foundation of China (No. 81573302, 81473083, and 81402813) for providing financial support to this work.

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ABBREVIATIONS USED MDR, multidrug resistance; Pgp, P-glycoprotein; Rho123, rhodamine 123; SAR, structure−activity relationship; TMD, transmembrane domain; PE, petroleum ether; NBD, nucleotide-binding domain; ADR, adriamycin; HLF, human lung fibroblasts; m-CPBA, meta-chloroperoxybenzoic acid; IC50, halfmaximal inhibitory concentration; ip, intraperitoneally; SDSPAGE, sodium dodecyl sulfate polyacrylamide gel electrophoresis; GADPH, glyceraldehyde-3-phosphate dehydrogenase; P



Article

(17) Chan, J. Y. W.; Chu, A. C. Y.; Fung, K. P. Inhibition of Pglycoprotein expression and reversal of drug resistance of human hepatoma HepG2 cells by multidrug resistance gene (mdr1) antisense RNA. Life Sci. 2000, 67, 2117−2124. (18) Fu, L. W.; Zhang, Y. M.; Liang, Y. J.; Yang, X. P.; Pan, Q. C. The multidrug resistance of tumor cells was reversed by tetrandrine in vitro and in xenografts derived from human breast adenocarcinoma MCF7/adr cells. Eur. J. Cancer 2002, 38, 418−426. (19) Dolomanov, O. V.; Bourhis, L. J.; Gildea, R. J.; Howard, J. A. K.; Puschmann, H. OLEX2: a complete structure solution, refinement and analysis program. J. Appl. Crystallogr. 2009, 42, 339−341. (20) Carmichael, J.; DeGraff, W. G.; Gazdar, A. F.; Minna, J. D.; Mitchell, J. B. Evaluation of a tetrazolium-based semiautomated colorimetric assay: assessment of chemosensitivity testing. Cancer Res. 1987, 47, 936−942. (21) Brunschweiger, A.; Iqbal, J.; Umbach, F.; Scheiff, A. B.; Munkonda, M. N.; Sevigny, J.; Knowles, A. F.; Muller, C. E. Selective nucleoside triphosphate diphosphohydrolase-2 (NTPDase2) inhibitors: Nucleotide mimeties derived from uridine-5′-carboxamide. J. Med. Chem. 2008, 51, 4518−4528. (22) Bradford, M. M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 1976, 72, 248−254. (23) Xiang, Q. F.; Zhang, D. M.; Wang, J. N.; Zhang, H. W.; Zheng, Z. Y.; Yu, D. C.; Li, Y. J.; Xu, J.; Chen, Y. J.; Shang, C. Z. Cabozantinib reverses multidrug resistance of human hepatoma HepG2/adr cells by modulating the function of P-glycoprotein. Liver Int. 2015, 35, 1010− 1023.

NADPH, reduced nicotinamide adenine dinucleotide phosphate

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REFERENCES

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Jatrophane Diterpenoids as Modulators of P-Glycoprotein-Dependent

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