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First Enantiospecific Syntheses of Marine Merosesquiterpenes Neopetrosiquinones A and B: Evaluation of Biological Activity Ikram Chayboun,† Ettahir Boulifa,† Ahmed Ibn Mansour,† Fernando Rodriguez-Serrano,‡ Esther Carrasco,‡ Pablo Juan Alvarez,‡ Rachid Chahboun,*,§ and Enrique Alvarez-Manzaneda*,§ †

Laboratoire de Chimie Organique Appliquée, Département de Chimie, Faculté des Sciences, Université Abdelmalek Essaâdi, Tetouan, Morocco ‡ Instituto de Biopatología y Medicina Regenerativa (IBIMER) and §Departamento de Química Orgánica, Facultad de Ciencias, Instituto de Biotecnología, Universidad de Granada, 18071 Granada, Spain S Supporting Information *

ABSTRACT: The first enantiospecific syntheses of neopetrosiquinones A (6) and B (7), two merosesquiterpenes isolated from the deep-water sponge Neopetrosia cf. proxima, from the labdane diterpene trans-communic acid (10) have been achieved. A key step of the synthetic sequence is the simultaneous aromatization of the C ring and the benzylic oxidation on C-7 of an advanced intermediate, mediated by the oxygen−DDQ system. The in vitro antiproliferative activities of neopetrosiquinone B (7) and of the synthetic intermediates 8 and 9 against human breast (MCF-7), lung (A-549), and colon (T-84) tumor cell lines have been assayed. The most potent was compound 9 (IC50 = 4.1 μM), which was twice as active as natural compound 7 (IC50 = 8.3 μM) against A549 cells. In addition, the treatment with these compounds resulted in an induction of apoptosis. These findings indicate that the terpene benzoquinones reported here might be potentially useful as anticancer agents.

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(8,12),(9,11)-diene).10 The (+)-diene precursor of natural (−)-cyclozonarone (1) has been prepared starting from the drimanic alcohol (+)-albicanol,10a whereas the (−)-diene has been synthesized from the drimanic dialdehyde polygodial10b and from the labdane diterpene (+)-manool.11 Several total syntheses of xestoquinone (2) have also been reported.12 Syntheses for the alisiaquinones (3−5) and the neopetrosiquinones (6, 7) have not been reported hitherto. Herein we report the first enantiospecific syntheses of neopetrosiquinones A (6) and B (7), starting from natural diterpene trans-communic acid (10). The processes take place in short synthetic sequences utilizing mild reaction conditions. The in vitro antiproliferative and cytotoxic activities of neopetrosiquinone B (7) and of the synthetic intermediates 8 and 9 against human breast (MCF-7), lung (A-549), and colon (T-84) tumor cell lines have been assessed.

uring the past few years a series of structurally related sesquiterpene quinones, bearing a wide range of potent biological activities, have been isolated from a variety of marine organisms. Representative examples of this type of metabolite are cyclozonarone (1), obtained from the brown alga Dictyoperis undulate,1 xestoquinone (2), from the sponge Neopetrosia sapra,2 and alisiaquinones A (3) and B (4) and alisiaquinol (5), isolated from an unidentified deep-water sponge.3 Cyclozonarone (1) shows feeding-deterrent activity against young abalones and is also active against Trypanosoma cruzi (Chagas disease) with an IC50 value of 700 nM.4 Xestoquinone (2) inhibits the oncogenic protein kinase pp60v‑src 5 and the Ca2+ ATPase from the skeletal muscle myosin;6 this compound also showed cytotoxic effects in human breast tumor T47D cell lines7 and moderate in vivo activity in mice infected with Plasmodium berghei NK65.8 Alisiaquinones were reported to have activity against the enzyme targets plasmodial kinase PFnek-1 and protein farnesyl transferase and against Plasmodium falciparum both in vitro and in vivo.3 More recently, two related metabolites, neopetrosiquinones A (6) and B (7), have been isolated from the deep-water sponge Neopetrosia cf. proxima. These compounds show in vitro inhibitory activity against the PANC-1 pancreatic carcinoma and the DLD-1 colon carcinoma tumor cell lines.9 The enantiospecific synthesis of cyclozonarone (1) has been achieved after the Diels−Alder cycloaddition of 1,4-benzoquinone and the corresponding drimanic exocyclic diene (drima© XXXX American Chemical Society and American Society of Pharmacognosy

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RESULTS AND DISCUSSION In the context of our research on the synthesis of bioactive marine metabolites from abundant natural terpenoids,13 we have tackled the syntheses of neopetrosiquinones A (6) and B (7) starting from trans-communic acid (10), a labdane diterpene very abundant in some species of Juniperus14 and Cupressus.15 Received: December 4, 2014

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DOI: 10.1021/np500975b J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products

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which was then converted into amine 19, after refluxing with potassium hydroxide in MeOH for 2 h. This amine was also directly obtained from isocyanate 17, after prolongued refluxing with KOH in MeOH. The diene 11 was finally obtained when amine 19 was methylated and the resulting tetraalkyl ammonium salt was treated with potassium tert-butoxide. Next, the elaboration of the tetracyclic skeleton of the target compounds was undertaken (Scheme 3). When diene 11 was successively refluxed with 1,4-benzoquinone (12) in toluene for 3 h and then with methyl iodide and sodium hydride in tetrahydrofuran for 4 h, the tetracyclic ester 13 was obtained in high yield. A first approach to ketone 8 involved the aromatization of the C ring of ester 13. This was smoothly converted into compound 20 after treatment with manganese dioxide, an oxidant rarely utilized to achieve the ring aromatization. However, all attempts at oxidizing the benzylic C-7 position of the later compound, utilizing a variety of reagents, were unsuccesful. Interestingly, this objective was achieved when acetyl derivative 22 was treated with 2,3dichloro-5,6-dicyano-1,4-benzoquinone (DDQ), which caused the simultaneous aromatization of the C ring and the benzylic oxidation, affording the acetoxy ketone 23, which was then converted into compound 8. A possible mechanism for the transformation of compound 22 into ketone 23, an unprecedented oxidation for this type of compound mediated by the oxygen−DDQ system, is depicted in Scheme 4. This involves the dehydrogenation of compound 22, leading to diene I, which then undergoes the abstraction of a hydrogen atom at C-18, with subsequent capture of the hydroperoxy radical at C-7, to afford hydroperoxide II, which after dehydration is transformed into ketone 23. Finally, hydroxyketone 8 was transformed into the target compounds 6 and 7 (Scheme 5). The treatment of hydroquinone dimethyl ether 8 with AgO and nitric acid gave quinone 9, which was then converted into neopetrosiquinone A (6), after oxidation with pyridinium chlorochromate (PCC). When hydroxy ketone 8 was treated with sodium borohydride, the alkene 24 was obtained. Oxidation of this compound with AgO and nitric acid led finally to neopetrosiquinone B (7). Biological Assays. In Vitro Cytotoxicity Assay. We assessed the antiproliferative actions of compounds 7, 8, and 9 against human breast (MCF-7), lung (A-549), and colon (T84) tumor cell lines. Activity (IC50 < 10 μM) was observed for compounds 7 and 9 (Table 1). The most active compound was 9. Thus, the mean IC50 was 6.2 μM for compound 9 versus 9.2 μM for compound 7, considering the three human cell lines together. The optimal result was observed for compound 9 (IC50 = 4.1 μM), which was twice as active as compound 7 (IC50 = 8.3 μM) against A-549 cells. Figure 1 shows the dose− response curves of selected compounds. Recently, it was found that compounds 6 and 7 inhibit the in vitro proliferation of the DLD-1 human colorectal adenocarcinoma cell line with IC50 values of 3.7 and 9.8 μM, respectively, and the PANC-1 human pancreatic carcinoma cell line with IC50 values of 6.1 and 14 μM, respectively.9 In addition, compound 6 also inhibited the proliferation of the AsPC-1 human pancreatic carcinoma cell line (IC50 = 6.1 μM). The authors reported an IC50 above 15 μM of compound 7 against A-549 cells.9 However, in this study we found an IC50 of 8.3 μM. The different results may be due to the purity of the compounds and/or due to the initial cell density of the cultures, which was 5 × 103 cells/cm2 in our assays and above 5 × 104 cells/cm2 in the earlier study.9,18

Scheme 1 shows the syntheses we planned of neopetrosiquinones A (6) and B (7) from acid 10. The C ring Scheme 1. Syntheses of Neopetrosiquinones A (6) and B (7)

of the target compounds will be elaborated through a Diels− Alder cycloaddition of diene 11, obtained after the oxidative degradation of the side chain of diterpene 10, with 1,4benzoquinone (12). The successive reduction of the methyl ester to a primary alcohol and oxidation of the B and C rings will lead to hydroxy ketone 8, an intermediate precursor of target compounds 6 and 7 (Scheme 1). Scheme 2 shows the synthesis of diene 11 from transcommunic acid (10). The reductive ozonolysis of methyl ester 1416 gave in good yield aldehyde 15,17 which after treatment with the Jones reagent afforded acid 16.17 When this was treated with diphenylphosphoryl azide (DPPA), in the presence of triethylamine, the isocyanate 17 was formed. The reduction of the latter with sodium borohydride gave formamide 18, B



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Scheme 2. Synthesis of Diene 11 from Acid 10

Scheme 3. Synthesis of Tetracyclic Intermediate 8 from Diene 11

influence intracellular bioavailability.19 The log P of the studied molecules was determined by using Marvin 6.0.5 software (ChemAxon Ltd., 2013). Log P values are 3.3, 3.6, and 2.4 for compounds 7, 8, and 9, respectively. A proportional relationship was found between the IC50 in the three tumor lines and the log P values of the compounds (Spearman’s correlation coefficient 1, p = 0). Therefore, the lipophilicity may justify the differences found in the activity of the compounds. Cell Cycle and Apoptosis Analysis. The cell cycle is an important chemotherapeutic target. However, inhibiting the cell cycle may not be sufficient to cause antitumor effects.20 For that reason, we carried out a global evaluation of the effects of compounds on cell cycle and apoptosis. Cultures of A-549, MCF-7, and T-84 were first maintained in a serum-free medium for 24 h and then were induced at low (IC50) or high (2 × IC50) concentration of compound 7, 8, or 9 for 24 h in complete medium. We next determined the percentage of cells in G0−G1, S, and G2−M phases of the cell cycle and the sub-G1 fraction, which represents cells undergoing apoptosis.21−24

Compounds 6 and 7 have a very similar structure. The major differences are the replacement of the C-19 aldehyde functionality and C-7 ketone functionality, present in compound 6, with a primary alcohol and a C-6/C-7 double bond in compound 7, respectively. The greater in vitro activity of compound 6 and its ability to block the interaction between β-catenin and Tcf4 have been attributed to the aldehyde functionality.9 However, we have found that compound 9, which differs from compound 7 in the presence of a ketone group on C-7, shows a 1.2−2-fold improvement in the activity with respect to the natural compound. Therefore, it seems that the C-7 ketone functionality plays an important role in the activity of these terpene benzoquinones. Furthermore, as expected, compound 8, the precursor of compounds 7 and 9, with a di-O-methyl hydroquinone D ring, is inactive (IC50 > 10 μM) . Lipophilicity (log P) is a key parameter of the biological effect developed by many compounds, because it can determine their capacity to penetrate biological membranes and therefore C



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Scheme 4. Possible Mechanism for the Transformation of Compound 22 into Ketone 23, Mediated by the Oxygen−DDQ System

Scheme 5. Syntheses of Neopetrosiquinones A (6) and B (7) from Tetracyclic Intermediate 8

lation in G0−G1. In the other cell lines, where only a little accumulation of cells at sub-G1 was observed, compounds 7 and 8 appear to cause a slight accumulation of cells in the G0− G1 phase of the cell cycle. Although compound 9 also leads to apoptosis in the MCF-7 cells, there is no indication of G0−G1 accumulation in the other cell lines, which suggests that it may have a distinct mechanism compared to the other two compounds. Taken together, these results suggest that compound 7 produces an accumulation of A-549 and T-84 cells in G0−G1, whereas compound 8 seems to produce G0−G1 accumulation in cultures of the three cell lines. However, further studies will be necessary to demonstrate the effects of the compounds on the cell cycle, especially at the molecular level. Compounds 7, 8, and 9 increased the sub-G1 population in the three cell lines, indicating induction of apoptosis. Apoptosis is an ordered cellular process that occurs under physiological and pathological conditions that leads to a programmed cell

Table 1. Antiproliferative Activities of Compounds 7, 8, and 9 against A-549, MCF-7, and T-84 Cells IC50 (μM)a

a

compound

A-549

MCF-7

T-84

7 8 9

8.3 ± 0.5 21 ± 1 4.1 ± 0.5

7.7 ± 0.5 33 ± 3 6.6 ± 0.8

11.5 ± 0.9 42 ± 2 8.0 ± 1.0

Data are means ± SEM of four independent determinations.

In the MCF-7 breast cancer cell line, all three compounds led to accumulation of cells in the sub-G1 peak at 2 × IC50 concentration, indicating induction of apoptosis. Interestingly, compound 8 is less potent in its ability to initiate apoptosis in this cell line, as indicated by only 6.6% accumulation in the subG1 peak at the IC50 concentration as compared to 32% and 34% for compounds 7 and 9, respectively. However, at that concentration, compound 8 appears to produce an accumuD



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Figure 1. Dose−response curves of compounds 7, 8, and 9 against A-549, MCF-7, and T-84 cells. Each point represents the mean of four independent determinations, and error bars indicate SEM.

cause a slight increase in the G 0 /G 1 population at concentrations lower than those that cause apoptosis. We hypothesize that the cells might trigger apoptosis in response to cell cycle arrest, resulting in elevated levels of apoptosis found after the induction of cells with compound 8 at 2 × IC50. In the same way, compounds 7 and 8 appear to produce accumulation of A-549 and T-84 cells in G0−G1, associated with a slightly greater percentage of apoptotic cells than control. Although this hypothesis has to be confirmed, this mechanism of action is similar to that reported for various other compounds, including acetylsalicylic acid against hepatocellular carcinoma cells,30 chromenopyrazolediones against hormone-sensitive prostate tumor cells,31 natural extracts of Garcina epunctata against promyelocytic leukemia,32 the synergic action of quercetin and 2-methoxyestradiol against human hepatocellular carcinoma lines,33 and novel merosesquiterpenes, as reported recently.34,35 In summary, the first enantiospecific syntheses of marine sponge metabolites neopetrosiquinones A (6) and B (7) from the labdane diterpene trans-communic acid (10) have been achieved. The spectroscopic properties of synthetic compounds were similar to those reported for the natural products.9,36 Unfortunately, the specific rotations of the natural quinones were not obtained, which prevents confirmation of the absolute configuration of the isolated marine metabolites.37 Compounds 7 and 9 demonstrated antiproliferative activity in vitro against human breast, colon, and lung tumor cells (IC50 < 10 μM).38 The most active compound was 9, with a mean IC50 of 6.2 μM versus 9.2 μM for 7 (natural compound; neopetrosiquinone B). The most potent was compound 9 (IC50 = 4.1 μM), which was twice as active as the natural compound (IC50 = 8.3 μM) against A-549 cells. These results suggest that the C-7 ketone group plays an important role in the activity of these terpene benzoquinones. The treatment with compounds resulted in an induction of apoptosis. Moreover, compound 7 appears to cause a slight accumulation of A-549 and T-84 cells in G0−G1, whereas compound 8 seems to produce G0−G1 accumulation in cultures of the three cell lines. However, additional analysis will be necessary to demonstrate the effects of the compounds on the cell cycle. These findings indicate that the terpene benzoquinones reported here might be potentially useful as

death. In cancer, different molecular mechanisms can prevent the induction of apoptosis, resulting in the survival of malignant cells. Therefore, compounds able to induce or restore apoptotic pathways are potentially useful against tumoral cells.21 In fact, numerous novel and commercial drugs have a mechanism of action mediated by apoptosis.22,23 During apoptosis, cells experience a cascade of events that result in chromatin condensation, nuclear and DNA fragmentation, rounding up, and cell volume reduction. Finally, apoptosis involves membrane blebbing, alterations of organelles, loss of membrane integrity, and the formation of apoptotic bodies. Usually several hours are required for the process, although the time depends on the cell type and the stimulus.21 One valuable method to detect apoptosis is the quantification of the sub-G1 fraction by flow cytometry, which represents the percentage of cells undergoing DNA fragmentation.24 As discussed above, after 24 h of induction of MCF-7 cells with compounds there was a significant increase in the sub-G1 population. In A-549 and T-84 lines, although compounds increased the sub-G1 population, only compound 7 in T-84 (14%) and compound 8 in A-549 (18%) surpassed 10% after induction with 2 × IC50 for 24 h. Consequently, in A-549 and T-84 cells, either compounds act through a mechanism that does not involve apoptosis or a longer incubation time is required. To determine whether the increased fraction of MCF7 cells in sub-G1 after the treatment with compounds was due to apoptosis, induced cultures were processed for the TUNEL assay (terminal deoxynucleotidyl transferase-mediated nick end labeling).25,26 After 6 h of treatment, we found that compounds are capable of inducing apoptosis in MCF-7 cells (Figure 3). The above findings indicate that the action of compounds 7, 8, and 9 on MCF-7 cells is mediated by the induction of apoptosis independently of changes in cell cycle distribution. Similarly, Eucheuma cottonii polyphenol-rich extract (ECME) has antiproliferative activity against MCF-7 cells and induces apoptosis without cell cycle blockage.27 This effect has also been found in other cell types, such as human leukemic cells treated with an allosteric inhibitor of group I PAK activation (IPA-3)28 or HeLa cells treated with ethanolic extracts of Corallina pilulifera.29 However, compounds 7 and 8 seem to E


1.3 0.5b 0.7b 0.5b 0.7 1.0b ± ± ± ± ± ± 18 7.1 12 6.1 17 15 1.0b 0.9b 0.9 0.3b 0.7 1.4b

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1.0b 0.4b 0.8b 0.3b 0.5 1.2b ± ± ± ± ± ± 59 71 68 76 63 62 0.9b 1.4b 0.5b 0.5b 0.6b 0.3b ± ± ± ± ± ± 6.2 14 5.7 8.2 5.6 6.3 0.3b 0.1b 0.3b 0.1b 0.6b 1.0b ± ± ± ± ± ± a

9

8

The data are means ± SEM of three independent determinations. bSignificant difference with control group, p < 0.05.

17 1.7 17 1 19 9.7 0.6b 0.1b 0.5b 0,1b 0.9b 0.5b ± ± ± ± ± ± 12 0.7 11 0.7 9.4 6.4 0.9b 0.1b 0.3b 0.2b 1.2b 0.7b ± ± ± ± ± ± 39 5 66 6.9 37 24 1.5b 3.1b 0.4b 8.8b 4.1b 5.4b ± ± ± ± ± ± 32 93 6.6 91 34 60 1.2b 0.8b 1.1b 0.2b 2.3 2.2b ± ± ± ± ± ± 17 15 14 8 20 17 1.5b 0.9 0.7 0.1b 2.3 2.1b ± ± ± ± ± ± 7

IC50 2 × IC50 IC50 2 × IC50 IC50 2 × IC50

4 6.1 5.7 18 3.9 8.1

0.7 0.6b 0.5b 0.4b 0.7 0.6b

62 67 68 67 63 59

0.5 0.3b 1.1b 0.3b 1.7 0.3b ± ± ± ± ± ± ± ± ± ± ± ±

16 12 12 7.5 14 16

65 ± 1.1

G0−G1 (%) sub-G1 (%)

4 ± 0.3 28 ± 0.3

G2−M (%) S (%)

17 ± 0.8 51 ± 0.9

G0−G1 (%) sub-G1 (%)

4 ± 1.1 22 ± 1.8

G2−M (%)

62 ± 1.0 3.5 ± 1.1 control:

S (%) G0−G1 (%) sub-G1 (%) treatment:

12 ± 0.9

MCF-7 A-549

Table 2. Effect of Compounds 7, 8, and 9 on A-549, MCF-7, and T-84 Cell Cyclea

EXPERIMENTAL SECTION

General Experimental Procedures. Optical rotation measurements were carried out on a PerkinElmer 341 polarimeter, utilizing a 1 dm length cell and CHCl3 as a solvent. Infrared spectra (IR) were recorded as thin films on a Mattson Satellite FTIR spectrophotometer with samples between sodium chloride plates. NMR spectra were recorded at 500 MHz, for 1H, and at 125 MHz, for 13C, as indicated in each case, on a Varian Direct Drive spectrometer. Chemical shifts are expressed in parts per million (δ scale) downfield from tetramethylsilane. The signals of the 13C NMR were assigned utilizing DEPT experiments and on the basis of heteronuclear correlations. Highresolution mass spectra (HRMS) were carried out on a Q-TOF Waters Synap G2 spectrometer, utilizing the electrospray ionization (ESI) technique. Thin-layer chromatography (TLC) was performed using silica gel 60 F254 precoated plates (0.25 mm) and visualized by UV fluorescence quenching and phosphomolybdic acid solution staining. Chromatography separations were carried out by flash column on silica gel 60 (230−400 Mesh), utilizing hexane−methyl tert-butyl ether mixtures as eluent. CH2Cl2 was dried over calcium hydride. Benzene and tetrahydrofuran were dried over sodium benzophenone. Syntheses of Compounds 6−24. Synthesis of Methyl transCommunate (14).16 Diazomethane (obtained by addition of a solution of 6 g of Diazald in 35 mL of diethyl ether to 25 mL of 10% KOH in MeOH) was bubbled through a solution of acid 10 (3 g, 9.92 mmol) in 15 mL of diethyl ether until the consumption of starting material. After evaporation, ester 14 (3.1 g, 96%) was obtained. Synthesis of (1S,4aR,5S,8aR)-Methyl 1,4a-Dimethyl-6-methylene5-(2-oxoethyl)decahydronaphthalene-1-carboxylate (15).17 A solution of ester 14 (2.0 g, 6.33 mmol) in CH2Cl2 (60 mL) was slowly bubbled with an O3/O2 mixture at −78 °C for 2 h. The solution was flushed with argon, and triphenylphosphine (2.82 g, 10.76 mmol) was added, and the mixture was kept under stirring at rt for 6 h. After evaporation under vacuum, the crude residue was fractionated in methyl tert-butyl ether (30 mL)−H2O (30 mL) and extracted with ether (3 × 30 mL). The combined organic phase was dried over anhydrous Na2SO4 and evaporated to give a crude residue, which after column chromatography on silica gel (98% hexanes−MTBE) afforded aldehyde 15 (1.32 g, 75%), as a colorless oil: IR (film) νmax 1720, 1644, 1227, 1156, 893 cm−1; 1H NMR (CDCl3, 400 MHz) δ 0.51 (s, 3H), 1.18 (s, 3H), 1.4 (d, J = 12.6 Hz, 1H), 1.51 (d, J = 12.0 Hz, 1H), 1.65 (d, J = 12.3 Hz, 1H), 2.0 (m, 2H), 2.19 (d, J = 13.3 Hz, 1H), 3.60 (s, 3H), 4.37 (s, 1H), 4.82 (s, 1H), 9.62 (s, 1H); 13C NMR (CDCl3, 75 MHz) δ 12.9 (CH3), 19.8 (CH2), 25.7 (CH2), 28.7 (CH3), 31.4 (C), 37.9 (CH2), 38.1 (CH2), 39.4 (CH2), 39.9 (CH2), 44.2 (C), 50.2 (OCH3), 51.3 (CH), 56.4 (CH), 108.1 (CH2),148.0 (C), 177.5 (C O), 203.2 (CO); HRESIMS m/z 301.1768 [M + Na]+ (calcd for C17H26O3Na, 301.1780). Synthesis of 2-((1S,4aR,5S,8aR)-5-(Methoxycarbonyl)-5,8a-dimethyl-2-methylenedecahydronaphthalen-1-yl)acetic Acid (16).17 Jones reagent (1.5 mL) was added dropwise to a solution of aldehyde 15 (2 g, 7.19 mmol) in acetone (20 mL) at 0 °C until the TLC revealed the consumption of starting material, and the mixture was kept under stirring at rt for 30 min. After evaporation of the solvent, methyl tert-butyl ether (40 mL) was added and the mixture was washed with H2O (5 × 10 mL). Then the organic phase was washed with 5% aqueous NaOH (3 × 10 mL) and 5% HCl (35 mL), methyl tert-butyl ether (60 mL) was added to the aqueous phase, and the phases were shaken and separated. The organic phase was washed with H2O (3 × 20 mL) and brine (2 × 20 mL), dried over anhydrous Na2SO4, and evaporated to give compound 16 (1.7 g, 82%), as a white solid: [α]20D +12 (c 0.09, CHCl3); IR (film) νmax 3400, 1721, 1710, 1646, 1300, 1265, 1229, 1154 cm−1; 1H NMR (CDCl3, 500 MHz) δ 0.51 (s, 3H), 1.10 (ddd, J = 13.2, 13.2, 3.6 Hz, 1H), 1.18 (s, 3H), 1.21

± ± ± ± ± ±

18 ± 1.0

anticancer agents. The different effects of compounds must reflect the molecular differences among the cell lines. Studies are under way to further explore their mechanism of action and to gather key data for structure-based drug design.

17 9 15 9.4 15 17

G2−M (%) S (%)

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T-84

13 ± 0.6


F



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Figure 2. Representative panels of flow cytometry analysis of the effects of compounds 7, 8, and 9 on the fraction of MCF-7 cells in sub-G1. Cultures were incubated with complete medium for 24 h without (control) or with compounds at low (IC50) or high (2 × IC50) concentration. (m, 1H), 1.39 (dd, J = 12.9, 2.4 Hz, 1H), 1.53 (m, 1H), 1.65 (br d, J = 12.4 Hz, 1H), 1.70−1.90 (m, 2H), 1.91−2.08 (m, 2H), 2.17 (br d, J = 13.1 Hz, 1H), 2.25−2.60 (m, 4H), 3.60 (s, 3H), 4.53 (br s, 1H), 4.79 (br s, 1H); 13C NMR (CDCl3, 125 MHz) δ 12.7 (CH3), 19.8 (CH2), 25.7 (CH2), 28.7 (CH3), 30.8 (CH3), 37.9 (CH2), 38.1 (CH2), 39.0 (CH2), 39.4 (C), 44,2 (C), 51.2 (CH), 51.6 (CH3), 55.8 (CH), 106.5 (CH2), 148.2 (C), 177.6 (C), 179.9 (C); HRESIMS m/z 317.1734 [M + Na]+ (calcd for C17H26O4Na, 317.1729). Synthesis of (1S,4aS,5S,8aR)-Methyl 5-(Formamidomethyl)-1,4adimethyl-6-methylenedecahydronaphthalene-1-carboxylate (18). Diphenyl phosphoryl azide (2.0 g, 7.48 mmol) was added to a mixture of acid 16 (2 g, 6.68 mmol) and triethylamine (757 mg, 7.48 mmol) in dioxane (10 mL) at 0 °C, under an argon atmosphere, and the mixture was kept under stirring at rt for 45 min. After evaporation of the solvent, methyl tert-butyl ether (30 mL) was added and the solution was washed with H2O (3 × 10 mL) and brine (2 × 10 mL). The organic phase was dried over anhydrous Na2SO4 and evaporated to give a crude residue, which was dissolved in benzene (10 mL) and then refluxed for 15 min until TLC revealed the disappearance of starting material. Then, the solvent was evaporated under vacuum and the resulting crude residue (1.8 g, 91%), consisting of isocyanate 17, was utilized in the next step without purification. Compound 17: IR (film) νmax 3080, 2250, 1729, 1644, 1400, 1300, 1230, 1165 cm−1; HRESIMS m/z 314.1726 [M + Na]+ (calcd for C17H25NO3Na, 314.1732).

Sodium borohydride (1.6 g, 30.9 mmol) was added to a solution of the above crude residue in EtOH (25 mL), and the mixture was stirred at rt for 30 min. After evaporation of the solvent, H2O (20 mL) was added and the mixture was extracted with methyl tert-butyl ether (3 × 30 mL). The combined organic phases were washed with H2O (3 × 15 mL) and brine (2 × 15 mL), dried over anhydrous Na2SO4, and evaporated to give a crude residue, which after column chromatography on silica gel (40% MTBE−hexanes) afforded formamide 18 (1.5 g, 83%), as a white solid: [α]25D +51 (c 0.08, CHCl3); IR (film) νmax 3400, 2925, 2850, 1721, 1651, 1522, 1449, 1384, 1227, 1156, 1094, 982, 897, 758 cm−1; 1H NMR (CDCl3, 500 MHz) δ 0.55 (s, 3H), 1.05 (ddd, J = 13.2, 13.2, 4.1 Hz, 1H), 1.17 (s, 3H), 1.25 (ddd, J = 14.0, 14.0, 4.2 Hz, 1H), 1.32 (dd, J = 12.5, 3.1 Hz, 1H), 1,55 (m, 1H), 1.76−2.10 (m, 6H), 2.17 (br d, J = 11.2 Hz, 1H), 2.42 (ddd, J = 11.5, 3.5, 3.5 Hz, 1H), 3.02 (ddd, J = 14.0, 11.4, 3.4 Hz, 1H), 3.60 (s, 3H), 3.90 (ddd, J = 14.0, 7.5, 2.8 Hz, 1H), 4.53 (s, 1H), 4.93 (s, 1H), 5.59 (br s, 1H), 8.09 (s, 1H); 13C NMR (CDCl3, 125 MHz) δ 12.9 (CH3), 19.8 (CH2), 25.9 (CH2), 28.8 (CH3), 33.6 (CH2), 38.0 (CH2), 38.1 (CH2), 39.0 (CH2), 39.8 (C), 44.1 (C), 51.2 (OCH3), 55.9 (CH), 56.0 (CH), 106.9 (CH2), 147.1 (C), 160.9 (CO), 177.4 (CO); HRESIMS m/z 316.1897 [M + Na]+ (calcd for C17H27NO3Na, 316.1889). Synthesis of (1S,4aS,5S,8aR)-Methyl 5-(Aminomethyl)-1,4a-dimethyl-6-methylenedecahydronaphthalene-1-carboxylate (19) from Formamide 18. KOH (2 N, 10 mL) was added to a solution G



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3H), 1.01 (ddd, J = 13.4, 13.4, 3.9 Hz, 1H), 1.15 (s, 3H), 1.30 (dd, J = 12.1, 2.8 Hz, 1H), 1.46 (ddd, J = 13.3, 13.3, 4.0 Hz, 1H), 1.54 (m, 1H), 1.70 (br d, J = 12.7 Hz, 1H), 1.83−2.02 (m, 4H), 2.16 (br d, 13.4 Hz, 1H), 2.43 (ddd, J = 12.6, 2.8, 2.8 Hz, 1H), 3.60 (s, 3H), 4.57 (d, J = 1.7 Hz, 1H), 4.61 (br t, J = 2.1 Hz, 1H), 4.75 (br t, J = 2.1 Hz, 1H), 4.8 (d, J = 1.7 Hz, 1H); 13C NMR (CDCl3, 125 MHz) δ 19.2 (CH3), 19.8 (CH2), 24.3 (CH2), 28.7 (CH3), 36.2 (CH2), 37.6 (CH2), 38.2 (CH2), 40.5 (C), 44.4 (C), 51.1 (OCH3), 53.9 (CH), 104.9 (CH2), 108.8 (CH2), 149.8 (C), 160.5 (C), 177.5 (CO); HRESIMS m/z 271.1668 [M + Na]+ (calcd for C16H24O2Na, 271.1674). Procedure b: Iodomethane (2.67 g, 18.8 mmol) was added to a solution of amine 19 (1 g, 3.77 mmol) in ether (20 mL), and the mixture was stirred at reflux for 24 h. The solid that separated was filtered and then dissolved in anhydrous THF (20 mL). To this solution was added potassium tert-butoxide (545 mg, 4.86 mmol), and the mixture was stirred at rt for 20 h. Then, the solvent was evaporated under vacuum, the resulting residue was dissolved in methyl tert-butyl ether (50 mL), and the organic phase was washed with H2O (3 × 15 mL) and brine (2 × 15 mL), dried over anhydrous Na2SO4, and evaporated to give diene 11 (820 mg, 87%), as a colorless oil. Synthesis of (4S,4aR,12bS)-Methyl 8,11-Dimethoxy-4,12b-dimethyl-1,2,3,4,4a,5,6,7,12,12b-decahydrotetraphene-4-carboxylate (13). 1,4-Benzoquinone (12) (383 mg, 3.54 mmol) was added to a solution of diene 11 (800 mg, 3.22 mmol) in toluene (10 mL), and the mixture was refluxed for 3 h. After evaporation of the solvent, a crude residue (1.2 g) was obtained, which was utilized in the next step without further purification. To a solution of this crude residue (1.2 g) in anhydrous THF (20 mL) were added successively sodium hydride (350 mg, 14.58 mmol) and iodomethane (1.4 g, 9.86 mmol) at 0 °C under an argon atmosphere. The mixture was refluxed for 4 h, then poured into ice, and extracted with methyl tert-butyl ether (3 × 30 mL). The combined organic phases were washed with H2O (3 × 15 mL) and brine (2 × 15 mL), dried over anhydrous Na2SO4, and evaporated to give a crude residue, which after column chromatography on silica gel (10% MTBE−hexanes) afforded compound 13 (1.14 g, 92%), as a white solid: [α]25D +46.7 (c 0.16, CHCl3); IR (film) νmax 2933, 2882, 1721, 1603, 1464, 1376, 1255, 1091, 792, 759 cm−1; 1H NMR (CDCl3, 500 MHz) δ 0.87 (s, 3H), 1.05 (ddd, J = 13.5, 13.5, 4.2 Hz, 1H), 1.19 (ddd, J = 13.8, 13.8, 4.2 Hz, 1H), 1.23 (s, 3H), 1.45 (dd, J = 12.6, 1.7 Hz, 1H), 1.56 (m, 1H), 1.80−1.96 (m, 3H), 2.00−2.18 (m, 3H), 2.22 (br d, J = 13.3 Hz, 1H), 3.08−3.18 (m, 4H), 3.64 (s, 3H), 3.78 (s, 6H), 6.61 (s, 2H); 13C NMR (CDCl3, 125 MHz) δ 17.3 (CH3), 19.7 (CH2), 20.7 (CH2), 23.1 (CH2), 28.5 (CH3), 30.0 (CH2), 32.7 (CH2), 37.0 (CH2), 37.8 (CH2), 38.1 (C), 44.0 (C), 51.1 (OCH3), 53.5 (CH), 55.6 (OCH3), 55.7 (OCH3), 106.71 (CH), 106.76 (CH), 124.4 (C), 124.8 (C), 125.7 (C), 133.8 (C), 150.6 (C), 150.1 (C), 178.1 (CO); HRESIMS m/z 407.2203 [M + Na]+ (calcd for C24H32O4Na, 407.2198). Synthesis of (4S,4aR,12bS)-Methyl 8,11-Dimethoxy-4,12b-dimethyl-1,2,3,4,4a,5,6,12b-octahydrotetraphene-4-carboxylate (20). Manganese dioxide (1.26 g, 14.5 mmmol) was added to a solution of ester 13 (286 mg, 0.75 mmol) in CHCl3 (20 mL), and the mixture was stirred at rt for 14 h. After filtration and evaporation, ester 20 (229 mg, 80%) was obtained as a white solid: [α]25D +39.7 (c 0.11, CHCl3); IR (film) νmax 2947, 2834, 1724, 1597, 1459, 1435, 1251, 1110, 975, 897, 799, 759, 723 cm−1; 1H NMR (CDCl3, 500 MHz) δ 1.02 (ddd, J = 13.0, 13.0, 4.0 Hz, 1H), 1.04 (s, 3H), 1.23 (s, 3H), 1.20−1.26 (m, 1H), 1.46 (ddd, J = 13.4, 13.4, 4.0 Hz, 1H), 1.55−1.64 (m, 1H), 1.98−2.10 (m, 1H), 2.12−2.32 (m, 3H), 2.47 (d, J = 12.9 Hz, 1H), 2.82 (ddd, J = 17.2, 13.0, 6.0 Hz, 1H), 3.01 (dd, J = 16.6, 4.7 Hz, 1H), 3.60 (s, 3H), 3.86 (s, 6H), 6.55 (d, J = 8.3 Hz, 1H), 6.58 (d, J = 8.3 Hz, 1H), 7.8 (s, 1H), 8.02 (s, 1H); 13C NMR (CDCl3, 125 MHz) δ 20.1 (CH2), 21.3 (CH2), 23.8 (CH3), 28.6 (CH3), 32.5 (CH2), 37.7 (CH2), 38.1 (C), 40.0 (CH2), 44.2 (C), 51.3 (OCH3), 53.0 (CH), 55.6 (OCH3), 55.7 (OCH3), 101.7 (CH), 102.2 (CH), 124.6 (C), 125.2 (C), 134.5 (C), 147.4 (C), 148.9 (C), 149.4 (C), 178.0 (CO); HRESIMS m/z 405.2036 [M + Na]+ (calcd for C24H30O4Na, 405.2042). Synthesis of ((4S,4aR,12bS)-8,11-Dimethoxy-4,12b-dimethyl1,2,3,4,4a,5,6,7,12,12b-decahydrotetraphen-4-yl)methanol (21).

Figure 3. TUNEL DNA fragmentation assay. Cultures of MCF-7 cells were incubated with complete medium for 6 h without (control) or with compounds 7 (IC50), 8 (2 × IC50), or 9 (IC50). The nuclei of cells with fragmented DNA showed a bright green color. Few apoptotic cells appeared in the control cell culture (a). In contrast, cultures exposed to compound 7 (b), 8 (c), or 9 (d) have significantly more apoptotic cells than controls. Bars = 50 μm. of formamide 18 (1.5 g, 5.11 mmol) in MeOH (10 mL), and the mixture was refluxed for 2 h. After evaporation of the solvent, methyl tert-butyl ether (30 mL) was added and the resulting solution was washed with H2O (3 × 10 mL) and brine (2 × 10 mL), dried over anhydrous Na2SO4, and evaporated to give amine 19 (1.18 g, 87%) as a colorless syrup: [α]25D +39.0 (c 0.11, CHCl3); IR (film) νmax 3409, 2935, 2850, 1721, 1644, 1449, 1229, 1154, 891, 755, 666 cm−1; 1H NMR (CDCl3, 500 MHz) δ 0.47 (s, 3H), 1.01 (ddd, J = 13.4, 13.4, 4.0 Hz, 1H), 1.13 (s, 3H), 1.16 (ddd, J = 13.1, 13.1, 4.7 Hz, 1H), 1.26 (br s, 2H), 1.28 (dd, J = 12.5, 2.9 Hz, 1H), 1.47 (m, 1H), 1.69 (br d, J = 10.5 Hz, 1H), 1.72−1.2.00 (m, 5H), 2.13 (br d, J = 13.3 Hz, 1H), 2.38 (ddd, J = 6.6, 4.7, 2.6 Hz, 1H), 2.71 (dd, J = 12.8, 10.3 Hz, 1H), 2.83 (dd, J = 12.8, 2.4 Hz, 1H), 3.56 (s, 3H), 4.42 (s, 1H), 4.89 (s, 1H); 13C NMR (CDCl3, 125 MHz) δ 13.1 (CH3), 19.8 (CH2), 26.1 (CH2), 28.7 (CH3), 36.8 (CH2), 38.1 (CH2), 38.5 (CH2), 39.0 (NCH2), 39.7 (C), 44.1 (C), 51.0 (OCH3), 56.1 (CH), 60.1 (CH), 106.0 (CH2), 147.4 (C), 177.5 (CO); HRESIMS m/z 288.1934 [M + Na]+ (calcd for C16H27NO2Na, 288.1939). Synthesis of Amine 19 from Isocyanate 17. KOH (2 N, 5 mL) was added to a solution of isocyanate 17 (100 mg, 0.34 mmol) in MeOH (5 mL), and the mixture was refluxed for 48 h, after which TLC revealed the consumption of starting material. The mixture was evaporated, and the resulting residue was acidified by addition of 2 N HCl and extracted with methyl tert-butyl ether (3 × 20 mL). The aqueous phase was basified with 2 N NaOH and extracted with methyl tert-butyl ether (3 × 20 mL). The combined organic phase was washed with H2O (3 × 10 mL) and brine (2 × 10 mL), dried over anhydrous Na2SO4, and evaporated to give amine 19 (30 mg, 33%). Synthesis of (1S,4aS,8aR)-Methyl 1,4a-Dimethyl-5,6dimethylenedecahydronaphthalene-1-carboxylate (11). Procedure a: To a solution of amine 19 (200 mg, 0.75 mmol) in ether (15 mL) was added iodomethane (321 mg, 2.26 mmol), and the mixture was stirred at rt for 60 min. The solid that separated was filtered and then dissolved in anhydrous THF (15 mL). Sodium hydride (58 mg, 2.43 mmol) was added to this solution, at 0 °C under an argon atmosphere, and the mixture was refluxed for 20 h. Then, the mixture was poured into ice and extracted with methyl tert-butyl ether (3 × 30 mL). The combined organic phase was washed with H2O (3 × 15 mL) and brine (2 × 15 mL), dried over anhydrous Na2SO4, and evaporated to give a crude residue, which after column chromatography on silica gel (5% MTBE−hexanes) afforded diene 11 (56 mg, 33%), as a colorless oil: [α]25D −81 (c 0.09, CHCl3); IR (film) νmax 2935, 2852, 1726, 1635, 1458, 1225, 1163, 894 cm−1; 1H NMR (CDCl3, 500 MHz) δ 0.74 (s, H



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LiAlH4 (163 mg, 4.2 mmol) was added to a solution of ester 13 (1.5 g, 3.9 mmol) in dry THF at 0 °C under an argon atmosphere, and the mixture was refluxed for 2 h, at which time TLC showed no starting material. Then, the mixture was poured into ice, and 5% aqueous HCl was added slowly until the separated solid was dissolved. The mixture was extracted with methyl tert-butyl ether (3 × 30 mL), and the combined phases were washed with H2O (3 × 15 mL) and brine (2 × 15 mL), dried over anhydrous Na2SO4, and evaporated to give alcohol 21 (1.24 g, 90%) as a colorless syrup: [α]25D +39.0 (c 0.11, CHCl3); IR (film) νmax 3429, 2925, 1638, 1481, 1255, 1092, 1021, 758 cm−1; 1H NMR (CDCl3, 500 MHz) δ 0.98 (ddd, J = 13.4, 13.4, 3.4 Hz, 1H), 1.02 (s, 3H), 1.03 (s, 3H), 1.20−1.27 (m, 3H), 1.36 (dd, J = 12.3, 4.3 Hz, 1H), 1.42−1.52 (m, 3H), 1.55 (ddd, J = 10.2, 3.4, 3.4 Hz, 1H), 1.72−1.86 (m, 2H), 1.90−2.12 (m, 2H), 3.09−3.31 (m, 4H), 3.51 (d, J = 10.9 Hz, 1H), 3.78 (s, 3H), 3.79 (s, 3H), 3.83 (d, J = 10.9, 1H), 6.60 (s, 2H); 13C NMR (CDCl3, 125 MHz) δ 18.8 (CH2), 19.1 (CH2), 20.1 (CH3), 23.1 (CH2), 26.9 (CH3), 30.3 (CH2), 32.2 (CH2), 35.3 (CH2), 36.8 (CH2), 37.4 (C), 38.7 (C), 52.4 (CH), 55.5 (OCH3), 55.6 (OCH3), 65.4 (CH2), 106.5 (CH), 106.5 (CH), 123.3 (C), 124.6 (C), 125.5 (C), 134.7 (C), 150.5 (C), 150.9 (C); HRESIMS m/z 379.2257 [M + Na]+ (calcd for C23H32O3Na, 379.2249). Synthesis of ((4S,4aR,12bS)-8,11-Dimethoxy-4,12b-dimethyl1,2,3,4,4a,5,6,7,12,12b-decahydrotetraphen-4-yl)methyl Acetate (22). Acetic anhydride (4 mL) was added to a solution of alcohol 21 (1.3 g, 3.65 mmol) in pyridine (8 mL), and the mixture was kept at rt for 30 min. Then, H2O (5 mL) was added at 0 °C, and the reaction mixture was stirred for 5 min. The mixture was then extracted with methyl tert-butyl ether (3 × 30 mL). The combined organic phases were washed with 2 N HCl (3 × 10 mL), saturated aqueous NaHCO3 (3 × 10 mL), and brine (2 × 10 mL), dried over anhydrous Na2SO4, and evaporated to give acetate 22 (1.33 g, 92%) as a colorless syrup: [α]25D +47.8 (c 0.15, CHCl3); IR (film) νmax 2932, 1736, 1481, 1373, 1255, 1093, 1031, 756 cm−1; 1H NMR (CDCl3, 500 MHz) δ 1.01 (s, 3H), 1.05 (m, 1H), 1.06 (s, 3H), 1.22 (ddd, J = 13.0, 13.0, 3.9 Hz, 1H), 1.28 (d, J = 4.6 Hz, 1H), 1.42 (dd, J = 12.9, 1.5 Hz, 1H), 1.50− 1.70 (m, 3H), 1.75 (br d, J = 13.8 Hz, 1H), 1.88 (dd, J = 13.0, 6.9 Hz, 1H), 2.00−2.20 (m, 2H), 2.06 (s, 3H), 3.11 (s, 2H), 3.10−3.34 (m, 2H), 3.781 (s, 3H), 3.785 (s, 3H), 3.96 (d, J = 11.1 Hz, 1H), 4.30 (d, J = 11.1 Hz, 1H), 6.61 (s, 2H); 13C NMR (CDCl3, 125 MHz) δ 18.6 (CH2), 19.0 (CH2), 20.1 (CH3), 20.9 (CH3), 23.0 (CH2), 27.2 (CH3), 30.3 (CH2), 32.2 (CH2), 36.1 (CH2), 36.7 (CH2), 37.1 (C), 37.7 (C), 52.4 (CH), 55.5 (OCH3), 55.6 (OCH3), 67.2 (CH2), 106.6 (CH), 106.6 (CH), 123.4 (C), 124.6 (C), 125.5 (C), 134.6 (C), 150.7 (C), 150.9 (C), 171.4 (CO); HRESIMS m/z 421.2360 [M + Na]+ (calcd for C25H34O4Na, 421.2355). Synthesis of ((4S,4aR,12bS)-8,11-Dimethoxy-4,12b-dimethyl-6oxo-1,2,3,4,4a,5,6,12b-octahydrotetraphen-4-yl)methyl Acetate (23). To a solution of acetate 22 (500 mg, 1.25 mmol) in dioxane (10 mL) was added 2,3-dichloro-5,6-dicyano-1,4-benzoquinone (1.41 g, 6.25 mmol), and the mixture was kept at rt for 24 h. The solvent was evaporated under vacuum, and the resulting crude residue was purified by column chromatography on silica gel (20% MTBE−hexanes) to give compound 23 (500 mg, 97%) as a colorless syrup: [α]25D +2.0 (c 0.14, CHCl3); IR (film) νmax 2931, 2855, 1733, 1681, 1627, 1589, 1462, 1435, 1269, 1224, 1114, 1034, 972, 804, 757 cm−1; 1H NMR (CDCl3, 500 MHz) δ 1.06 (s, 3H), 1.20 (ddd, J = 14.9, 14.9, 3.8 Hz, 1H), 1.33 (s, 3H), 1.71−1.78 (m, 4H), 2.08 (dd, J = 14.4, 3.7 Hz, 1H), 2.10 (s, 3H), 2.63 (br d, J = 1.1 Hz, 1H), 2.80 (dd, J = 18.1, 14.4 Hz, 1H), 2.93 (dd, J = 18.1, 3.6 Hz, 1H), 3.94 (s, 3H), 3.95 (s, 3H), 4.09 (d, J = 11.2 Hz, 1H), 4.42 (d, J = 11.2 Hz, 1H), 6.64 (d, J = 8.3 Hz, 1H), 6.77 (d, J = 8.3 Hz, 1H), 8.14 (s, 1H), 8.95 (s, 1H); 13C NMR (CDCl3, 125 MHz) δ 18.7 (CH2), 21.0 (CH3), 24.6 (CH3), 27.0 (CH3), 36.3 (CH2), 36.5 (CH2), 37.0 (C), 38.3 (C), 38.6 (CH2), 49.5 (CH), 55.7 (OCH3), 55.8 (OCH3), 66.9 (OCH2), 103.1 (CH), 106.1 (CH), 116.5 (CH), 123.7 (CH), 124.5 (C), 128.6 (C), 129.1 (C), 149.0 (C), 151.0 (C), 151.1 (C), 171.0 (C), 198.8 (CO); HRESIMS m/z 433.1996 [M + Na]+ (calcd for C25H30O5Na, 433.1991). Synthesis of (4S,4aR,12bS)-4-(Hydroxymethyl)-8,11-dimethoxy4,12b-dimethyl-1,2,3,4,4a,5-hexahydrotetraphen-6(12bH)-one (8).

HCl (1 N, 0.3 mL) was added to a solution of acetate 23 (213 mg, 0.519 mmol) in MeOH (7 mL). After 20 h of stirring at rt, the mixture was extracted with methyl tert-butyl ether (20 mL). The organic phase was washed with H2O (3 × 10 mL), dried over anhydrous Na2SO4, and evaporated to give a crude residue, which after column chromatography on silica gel (40% MTBE−hexanes) afforded alcohol 8 (180 mg, 94%): [α]25D −0.99 (c 1.11, CHCl3); IR (film) νmax 3448, 2927, 2854, 1677, 1626, 1462, 1269, 758 cm−1; 1H NMR (CDCl3, 500 MHz) δ 1.06 (s, 3H), 1.12 (ddd, J = 13.3, 13.3, 4.0 Hz, 1H), 1.30 (s, 3H), 1.72−1.87 (m, 3H), 1.98 (dt, J = 13.8, 3.0 Hz, 1H), 2.08 (dd, J = 14.2, 4.0 Hz, 1H), 2.61 (dd, J = 10.6, 2.8 Hz, 1H), 2.76 (dd, J = 18.0, 14.3 Hz, 1H), 2.82 (dd, J = 18.0, 3.6 Hz, 1H), 3.67 (d, J = 10.8 Hz, 1H), 3.94 (s, 3H), 3.95 (d, J = 10.8 Hz, 1H), 3.96 (s, 3H), 6.64 (d, J = 8.4 Hz, 1H), 6.77 (d, J = 8.4 Hz, 1H), 8.14 (s, 1H), 8.95 (s, 1H); 13C NMR (CDCl3, 125 MHz) δ 18.75 (CH2), 24.7 (CH3), 26.4 (CH3), 35.3 (CH2), 36.4 (CH2), 38.3 (C), 38.4 (C), 38.6 (CH2), 49.5 (CH), 55.7 (CH3), 55.8 (CH3), 65.1 (CH2), 103.0 (CH), 106.1 (CH), 116.3 (CH), 123.7 (CH), 124.5 (C), 128.8 (C), 129.1 (C), 149.0 (C), 151.1 (C), 151.4 (C), 199.2 (C); HRESIMS m/z 391.1892 [M + Na]+ (calcd for C23H28O4Na, 391.1885). Synthesis of (4S,4aR,12bS)-4-(Hydroxymethyl)-4,12b-dimethyl1,2,3,4,4a,5-hexahydrotetraphene-6,8,11(12bH)-trione (9). To a solution of alcohol 8 (25 mg, 0.068 mmol) in dioxane (3 mL) were added at 0 °C 6 N HNO3 (0.5 mL) and AgO (17 mg, 0.138 mmol). After 6 min under stirring at rt, the mixture was evaporated and the crude residue was dissolved in methyl tert-butyl ether (20 mL). The organic phase was washed with H2O (3 × 10 mL), dried over anhydrous Na2SO4, and evaporated to give a crude residue, which after column chromatography on silica gel (40% MTBE−hexanes) afforded quinone 9 (18 mg, 78%) as a yellow syrup: [α]25D +42.6 (c 0.5, CHCl3); IR (film) νmax 3389, 2928, 1672, 1258, 1029, 843, 666 cm−1; 1 H NMR (500 MHz, CDCl3) δ 1.07 (s, 3H), 1.12 (ddd, J = 13.5, 13.5, 4.8 Hz, 1H), 1.31 (s, 3H),1.64 (m, 1H), 1.72−1.87 (m, 2H), 1.94 (br d, J = 13.9 Hz, 1H), 2.02 (d, J = 14.5, 3.5 Hz, 1H), 2.56 (br d, J = 12.6 Hz, 1H), 2.84 (dd, J = 18.2, 14.5 Hz, 1H), 2.93 (dd, J = 18.2, 3.5 Hz, 1H), 3.71 (d, J = 10.9 Hz, 1H), 3.86 (d, J = 10.9 Hz, 1H), 7.02 (d, J = 10.7 Hz, 1H), 7.01 (d, J = 10.7 Hz, 1H), 8.14 (s, 1H), 8.70 (s, 1H); 13 C NMR (125 MHz, CDCl3) δ 21.0 (CH2), 26.3 (CH3), 29.0 (CH3), 37.8 (CH2), 38.7 (CH2), 40.6(CH2), 41.0 (C), 41.6 (C), 51.7 (CH), 67.8 (CH2), 125.5 (CH), 129.2 (CH), 132.6 (C), 136.9 (C), 137.3 (C), 141.4 (CH), 142.0 (CH), 163.63 (C), 187.3 (C), 186.4 (C), 200.0 (C); HRESIMS m/z 361.1409 [M + Na]+ (calcd for C21H22O4Na, 361.1416). Synthesis of Neopetrosiquinone A (6). Pyridinium chlorochromate (19 mg, 0.088 mmol) was added to a solution of compound 9 (20 mg, 0.059 mmol) in CH2Cl2 (3 mL) under an argon atmosphere, and the mixture was stirred at room temperature for 15 min. Then it was filtered through a silica gel pad and washed with a mixture of CH2Cl2− methyl tert-butyl ether (20:10 mL). The solvent was evaporated to give compound 6 (15 mg, 75%) as a yellow syrup: [α]25D +40.4 (c 0.5, CHCl3); IR (film) νmax 2924, 1672, 1413, 841 cm−1; 1H NMR (500 MHz, CDCl3) δ 1.14 (s, 3H), 1.19 (s, 3H), 1.26 (m, 1H), 1.64 (m, 1H), 1.80−1.90 (m, 2H), 2.19 (dd, J = 14.2, 4.1 Hz, 1H), 2.28 (br d, J = 14.1 Hz, 1H), 2.57 (br d, J = 12.5 Hz, 1H), 3.11 (dd, J = 18.0, 4.1 Hz, 1H), 3.19 (dd, J = 18.0, 14.2 Hz, 1H), 7.02 (d, J = 10.6 Hz, 1H), 7.04 (d, J = 10.6 Hz, 1H), 8.16 (s, 1H), 8.75 (s, 1H), 9.88 (s, 1H); 13C NMR (126 MHz, CDCl3) δ 18.8 (CH2), 22.8 (CH3), 23.6 (CH3), 34.4 (CH2), 35.3 (CH2), 37.3 (CH2), 39.0 (C), 47.6 (C), 49.0 (CH), 123.3 (CH), 126.7 (CH), 130.1 (C), 134.3 (C), 134.7 (C), 138.7 (CH), 139.3 (CH), 159.2 (C), 183.7 (C), 184.5 (C), 195.9 (C), 204.2 (CH); HRESIMS m/z 359.1260 [M + Na]+ (calcd for C21H20O4Na, 359.1259). Synthesis of ((4S,4aR,12bS)-8,11-Dimethoxy-4,12b-dimethyl1,2,3,4,4a,12b-hexahydrotetraphen-4-yl)methanol (24). To a solution of compound 8 (40 mg, 0.108 mmol) in EtOH (3 mL) was added sodium borohydride (8 mg, 0.2 mmol) under an argon atmosphere, and the mixture was stirred at rt for 40 min. Then H2O (1 mL) was added, and the mixture was further stirred for 5 min. The mixture was then extracted with methyl tert-butyl ether (15 mL), and the organic phase was washed with H2O (3 × 10 mL), dried over I



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anhydrous Na2SO4, and evaporated to give 292 mg of a crude residue. This was dissolved in CH2Cl2 (5 mL), Amberlyst A-15 (75 mg) was added, and the resulting mixture was stirred at rt for 15 min. After filtration, the solution was dried over anhydrous Na2SO4 and evaporated to give compound 24 (35 mg, 97%): [α]25D −181.4 (c 1.52, CHCl3); IR (film) νmax 2925, 1458, 1076, 772, 666 cm−1; 1H NMR (500 MHz, CDCl3) δ 1.11 (s, 6H), 1.13 (m, 1H), 1.46 (br s, 1H), 1.74−1. 89 (m, 3H), 1.92 (br d, J = 14.4 Hz, 1H), 2.36 (dd, J = 3.1, 2.7 Hz, 1H), 2.44 (br d, J = 10.3 Hz, 1H), 3.78 (d, J = 11.2 Hz, 1H), 3.90 (d, J = 11.2 Hz, 1H), 3.95 (s, 3H), 3.96 (s, 3H), 6.21 (dd, J = 9.7, 2.7 Hz, 1H), 6.63 (d, J = 8.6 Hz, 1H), 6.67 (d, J = 8.6 Hz, 1H), 6.75 (dd, J = 9.7, 3.1 Hz, 1H), 7.86 (s, 1H), 7.96 (s, 1H); 13C NMR (125 MHz, CDCl3) δ 18.7 (CH2), 21.8 (CH3), 26.1 (CH3), 35.2 (CH2), 36.2 (CH2), 38.3 (C), 51.0 (CH), 55.7 (CH3), 66.0 (CH2), 102.4 (CH), 103.1 (CH), 114.2 (CH), 124.8 (C), 125.8 (C), 128.3 (CH), 129.8 (CH), 131.3 (C), 146.8 (C), 149.6 (C), 149.6 (C); HRESIMS m/z 375.1933 [M + Na]+ (calcd for C23H28O3Na, 375.1936). Synthesis of Neopetrosiquinone B (7). HNO3 (6 N, 0.5 mL) and AgO (14 mg, 0.113 mmol) were added to a solution of compound 24 (20 mg, 0.056 mmol) in dioxane (3 mL), and the mixture was stirred at rt for 5 min. After evaporation under vacuum, the resulting residue was dissolved in methyl tert-butyl ether (20 mL) and the organic phase was washed with H2O (3 × 10 mL), dried over anhydrous Na2SO4, and evaporated to give a crude residue, which after column chromatography on silica gel (50% hexanes−MTBE) afforded compound 7 (12 mg, 66%) as a yellow syrup: [α]25D −115 (c 0.1, CHCl3); IR (film) νmax 3426, 2924, 1667, 1311, 756, 666 cm−1; 1H NMR (500 MHz, CDCl3) δ 1.08 (s, 3H), 1.11 (s, 3H), 1.26 (m, 1H), 1.73−1.81 (m, 3H), 1.90 (br d, J = 14.2 Hz, 1H), 2.31 (dd, J = 3.2, 2.8 Hz, 1H), 2.35 (m, 1H), 3.75 (d, J = 11.2 Hz, 1H), 3.83 (d, J = 11.2 Hz, 1H), 6.43 (dd, J = 9.7, 2.8 Hz, 1H), 6.68 (dd, J = 9.7, 3.2 Hz, 1H), 6.91 (d, J = 10.3 Hz, 1H), 6.93 (d, J = 10.3 Hz, 1H), 7.72 (s, 1H), 7.88 (s, 1H); 13C NMR (151 MHz, CDCl3) δ 18.7 (CH2), 20.7 (CH3), 26.2 (CH3), 35.3 (CH2), 36.1 (CH2), 38.5 (C), 38.5 (C), 50.5 (CH), 66.3 (CH2), 120.8 (CH), 124.3 (CH), 126.8 (CH), 130.5 (C), 131.1 (C), 134.5 (CH), 138.2 (C), 138.6 (CH), 139.1 (CH), 153.8 (C), 185.1 (C), 185.3 (C); HRESIMS m/z 345.1473 [M + Na]+ (calcd for C21H22O3Na, 345.1467). Biological Assays. Cell Lines and Culture. Human lung tumor line A-549, human breast adenocarcinoma line MCF-7, and human colorectal carcinoma line T-84 were supplied by the Department of Cell Cultures of the Granada University Scientific Instrumentation Center. All lines were cultured at 37 °C in 5% CO2 and 90% humidity with Dulbecco’s modified Eagle medium, supplemented with 10% heat-inactivated fetal bovine serum, 10 mL/L penicillin−streptomycin 100×, and 2 mM L-glutamine. Culture media and respective supplements were supplied by Sigma-Aldrich. In Vitro Antiproliferative Assay. In order to calculate the IC50 of compounds, 5 × 103 cells/cm2 were seeded in quadruplicate. At 24 h, cells were induced with increasing compound concentrations for 3 days. Subsequently, cells were fixed with 10% cold trichloroacetic acid (4 °C) and stained with 0.4% sulforhodamine in 1% acetic acid. The colorant was solubilized with 10 mM Tris-base pH 10.5, and optical density values were determined by colorimeter at 492 nm (Multiskan EX, Thermo Electron Corporation). IC50 values were calculated from the semilogarithmic dose−response curve by linear interpolation. Cell Cycle and Sub-G1 Analysis. Cells were seeded (5 × 104/cm2), and the culture medium was replaced with a serum-free medium for 24 h to synchronize their cell cycles, as described elsewhere.39−41 Twentyfour hours later, they were again placed in culture medium with serum and induced with compounds for 24 h. After this time, cultures were washed with PBS, fixed with 70% cold EtOH, and incubated with a DNA extraction solution (0.2 M Na2HPO4, 0.1 M citric acid, pH 7.8) for 15 min at 37 °C. Cells were then centrifuged, washed with PBS, and resuspended in 250 μL of a solution of propidium iodide (40 μg/ mL) and RNase (100 μg/mL) for 30 min at 37 °C in the dark. Finally, samples were analyzed in a FACScan flow cytometer, using a linear scale for the cell cycle and a logarithmic scale to determine the sub-G1

fraction. Results were analyzed with FlowJo software (v 7.6.5, Tree Star, Inc.). TUNEL Assay. The TUNEL assay was used to detect DNA fragmentation. MCF-7 cells were plated on chamber slides. Following induction with compounds 7 (IC50), 8 (2 × IC50), or 9 (IC50) for 6 h, cells were fixed in MeOH for 30 min at rt and then slides were washed in phosphate-buffered saline (PBS), pH 7.4. Permeabilization was performed using 0.1% Triton X-100 (Merck, Germany) and 0.1% sodium citrate for 2 min on ice. After washing once with PBS, 30 μL of TUNEL mixture (Roche) was added, and samples were left to incubate 60 min at 37 °C in a moist chamber in the dark. Finally, slides were washed three times with PBS and analyzed by fluorescence microscopy (Leica DM IL LED Fluo). The nuclei of cells with fragmented DNA showed bright green color.

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ASSOCIATED CONTENT

S Supporting Information *

Copies of 1H and 13C NMR spectra. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected] (R. Chahboun). *Tel: (+34) 958 248 089. E-mail: [email protected] (E. AlvarezManzaneda). Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We thank the Spanish Ministry of Science and Innovation (Project CTQ 2009-09932) and the Regional Government of Andalucia (Project P11-CTS-7651 and assistance for the FQM348 and CTS-107 groups) for financial support. I.C. thanks the Spanish Agency of International Cooperation and Development (AECID) for the predoctoral grant provided. E.C. received a FPU predoctoral fellowship (Ministry of Education, Culture and Sport).

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REFERENCES

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