Cytotoxic Plakortides from the Brazilian Marine Sponge Plakortis

Article pubs.acs.org/jnp

Cytotoxic Plakortides from the Brazilian Marine Sponge Plakortis angulospiculatus Evelyne A. Santos,† Amanda L. Quintela,‡ Elthon G. Ferreira,§ Thiciana S. Sousa,‡ Francisco das Chagas L. Pinto,‡ Eduardo Hajdu,⊥ Mariana S. Carvalho, Sula Salani,⊥ Danilo D. Rocha,† Diego V. Wilke,† Maria da Conceiçaõ M. Torres,‡ Paula C. Jimenez,§,▽ Edilberto R. Silveira,‡ James J. La Clair,∥ Otília Deusdênia L. Pessoa,*,‡ and Letícia V. Costa-Lotufo*,†,§,○ †

Departamento de Fisiologia e Farmacologia, Universidade Federal do Ceará, Fortaleza, 60.430-270, Brazil Departamento de Química Orgânica e Inorgânica, Universidade Federal do Ceará, Fortaleza, 60.021-970, Brazil § Instituto de Ciências do Mar, LABOMAR, Universidade Federal do Ceará, Fortaleza, 60.165-081, Brazil ⊥ Departamento de Invertebrados, Museu Nacional, Universidade Federal do Rio de Janeiro, Rio de Janeiro, Rio de Janeiro, 20.940-040, Brazil ∥ Xenobe Research Institute, P.O. Box 3052, San Diego, California 92163-1052, United States ‡

S Supporting Information *

ABSTRACT: Three new plakortides, 7,8-dihydroplakortide E (1), 2, and 10, along with known natural products 3, 4, spongosoritin A (5), 6−8, and plakortide P (9), were isolated from Brazilian specimens of Plakortis angulospiculatus. Compounds 2, 3, 5, and 7−9 displayed cytotoxic activities with IC50 values ranging from 0.2 to 10 μM. Compounds that contained a dihydrofuran ring were generally less active and displayed time dependence in their activity. The activities of compounds 2 and 7−9, carboxylic acids bearing a common sixmembered endoperoxide, were higher overall than for compounds 3 and 5. The modes underlying the cytotoxic actions of plakortides 2, 3, 5, 7, and 9 were further investigated using HCT-116 cells. While dihydrofurans 3 and 5 induce a G0/ G1 arrest, six-membered peroxides 2, 7, and 9 delivered a G2/M arrest and an accumulation of mitotic figures, indicating a distinctly different antimitotic response. Confocal analysis indicated that microtubules were not altered after treatment with 2, 7, or 9, therein suggesting that the mitotic arrest may be unrelated to cytoskeletal targets. Overall, we find that two related classes of natural products obtained from the same extract offer cytostatic activity, yet they do so through discrete pathways.

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have clinical status for drug development.7,8 More recently, eribulin mesylate, a synthetic derivative of halichondrin B obtained from Halichondria okadai, completed clinical trials and was added to the anticancer chemotherapy drug arsenal.9 While new structural motifs are becoming scarce, the reinvestigation and expansion of established natural product families still offer a key function for drug discovery. First, and perhaps most practically, data from large collections of natural products provide a comparative resource for the establishment and validation of structural assignments. Second, examination of a large set of analogues often provides strong support for bioactivity and mode of action studies, in part by offering positive and negative controls for comparative analyses. Finally, these activity analyses often provide a key first level of SAR data. Along these lines, we report on the discovery of three new plakortides, compounds 1, 2, and 10, along with seven known

ver the last four decades, the laboratory of William Fenical has revealed many key lessons for the natural products community. Of these, several play a fundamental role in expanding marine natural products research in Brazil. In particular, these include lessons on the (a) need to explore multiple methods for lead isolation;1,2 (b) importance of combining isolation and structure elucidation efforts with medicinal chemistry;3 and (c) vitality of understanding a natural product’s unique mode of action.4 Here we describe a study that combines aspects of compound isolation to further understand the structure−activity relationships (SAR) and associated biological activity in a complex panel of natural products isolated from marine sponges in the Plakortis genus (Supporting Information Figure S1). Marine sponges are one of the most relevant sources of bioactive compounds from the marine environment.5 Since the isolation of the arabinonucleosides from Tectitethya (formerly Cryptotethya) crypta, by Bergmann and Feeney in the early 1950s,6 these organisms have provided thousands of secondary metabolites, many of which are under preclinical evaluation or © XXXX American Chemical Society and American Society of Pharmacognosy

Received: November 10, 2014

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DOI: 10.1021/np5008944 J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products

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Figure 1. Structures of an additional seven known compounds also obtained from P. angulospiculatus extracts. Specific rotations are provided as observed (top) and reported values (bottom).

Specimens of P. angulospiculatus were obtained during an expedition to Flecheiras Beach, Ceara, Brazil, on March 30, 2014. A sample of P. angulospiculatus tissue (26.1 g, wet weight) was extracted with EtOH to yield 3.7 g of an organic extract. Samples of this extract were fractionated over HR-X resin (20 g) eluting sequentially with H2O, aqueous CH3CN, CH3CN, and CH2Cl2. We identified activity in the CH3CN fraction using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay as a guide.26 A sample of this fraction (866 mg) was subjected to a combination of flash chromatography and semipreparative normal-phase HPLC to deliver 10 compounds. Using a combination of specific rotation, HRESIMS, and 1H NMR and 13C NMR data, we identified the first seven compounds (Figure 1) as 6-desmethyl6-ethylspongosoritin A (3, 33.0 mg),10,23,26 6-desmethyl-6ethylspongosoritin-9,10-dihydrospongosoritin A (4, 4.4 mg),10,26 spongosoritin A (5, 29.4 mg),11,27 9,10-dihydrospongosoritin A (6, 6.2 mg),11 compound 7 (14.7 mg),28,29 compound 8 (5.2 mg), 28 and plakortide P (9, 4.6 mg),10,23,26,30,31 by comparison with the literature data (see Supporting Figure S2 for 13C NMR comparative plots). Spectroscopic data from the remaining three materials, compounds 1 (5.1 mg), 2 (5.8 mg), and 10 (25 mg), did not match the literature data, and hence they were subjected to a complete spectroscopic analysis. Compound 1 was isolated as a colorless resin with a specific rotation [α]20D of +26 (c 0.36, CHCl3). Its molecular formula, C21H36O4, which displays four degrees of unsaturation, was deduced by HRESIMS. This formula was confirmed by analysis of the 13C NMR and 13C NMR DEPT spectra (Table 1), which identified five methyl groups (CH3), seven methylene carbons (CH2), six methine carbons (CH), four of which were sp2

compounds (Figure 1) from specimens of Plakortis angulospiculatus collected off the northeast coast of Brazil. P. angulospiculatus sponges from the Brazilian coast were previously studied, leading to the isolation of a plakortenone and five plakortides, including 5 and 9.10,11

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RESULTS AND DISCUSSION As part of bioprospecting efforts directed at the identification of novel anticancer compounds from the Brazilian coastline, we investigated the chemistry and bioactivity of a cytotoxic extract derived from P. angulospiculatus. Plakortis (Homosclerophorida, Plakinidae) species occur largely within tropical regions, with equivalent numbers reported from the three major oceans. Recent reports list eight species known from the Tropical Western Atlantic (Brazil and the West Indies), seven from the Pacific, and six from the Indian Ocean including west Australia and the Red Sea.12 Sponges from this genus are typically recognized as sources of cyclic endoperoxides containing fiveor six-membered oxygenated rings,13 which are known to retain antiparasitic, antimicrobial, and anticancer activities.14,15 Plakortin, the first representative of this group, was isolated from a Caribbean specimen of Plakortis halichondrioides in 1978.16 Nevertheless, this class has since expanded, and related compounds, such as plakinic acids (plakinic acid A),17,18 plakortones (plakortone B),19,20 plakortolides (plakortolide E),21,22 and plakortides (plakortide E19−21 and plakortide O23−25), have enhanced the structural diversity and expanded the profile of their biological activity. B



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Table 1. NMR Spectroscopic Data (1H 300 MHz, 13C 75 MHz, CDCl3) for 7,8-Dihydroplakortide E (1), 2, and 10 7,8-dihydroplakortide E (1)a no.

δC, type

1 2

170.7, C 119.5, CH

3 4 5

152.6, CH 87.4, C 54.6, CH2

6 7 8 9

89.8, 43.3, 26.8, 44.0,

10 11 12 13 14 15 16 17

42.3, CH 133.3, CH 132.7, CH 25.8, CH2 14.5, CH3 30.9, CH2 9.0, CH3 29.3, CH2

18 19

8.84, CH3 20.6, CH2

20

29.2, CH3

21

11.9, CH3

C CH2 CH CH2

δH (J in Hz) 6.11, d (15.8) 6.97, d (15.8) 2.32, d (12.1) 2.26, d (12.1) 1.13, m 1.52, m 1.49, m 1.85, 4.95, 5.35, 2.00, 0.96, 1.83, 0.89, 1.75, 1.64, 0.92, 0.86,

m dd (15.2, 9.0) dt (15.2, 6.3) m t (7.4) m t (7.4) m m t (7.9) d (6.1)

1.33, m 1.13, m 0.82, t (7.4)

2 δC, type 177.3, C 31.5, CH2

3.06, 2.42, 4.44, 2.09, 1.72, 1.27,

78.7, CH 35.5, CH 35.6, CH2 84.5, C 127.3, CH 137.6, C 47.7, CH2

dd (15.9, 9.4) dd (15.9, 3.3) m m m m

5.12, s 2.10, 1.95, 2.05, 5.11, 5.37, 2.00, 0.98, 1.18, 0.93, 1.61, 1.53, 0.87, 1.71,

42.7, CH 133.2, CH 131.9, CH 25.8, CH2 14.2, CH3 25.2, CH2 11.2, CH3 32.7, CH2 7.8, CH3 17.1, CH3 28.1, CH3

m m m dd (15.3, 8,3) dt (15.3, 6.3) m t (7.2) m t (7.3) m m t (7.4) s

1.36, m 1.20, m 0.84 t (7.3)

11.8, CH3

22 a

10 δH (J in Hz)

δC, type 175.1, C 31.1, CH2 78.8, CH 35.7, CH 35.9, CH2 84.7, C 127.0, CH 143.4, C 41.3, CH2 36.9, 25.7, 23.4, 29.1, 14.4, 32.8, 10.7, 33.1,

CH CH2 CH2 CH2 CH3 CH2 CH3 CH2

7.9, CH3 22.7, CH2 12.5, CH3 25.2, CH2 11.2, CH3

δH (J in Hz) 3.05, 2.40, 4.44, 2.13, 1.72, 1.26,

dd (15.9, 9.7) dd (15.9, 3.4) m m dd (13.4, 3.8) m

5.17, s 2.01, 1.98, 1.41, 1.26, 1.20, 1.26, 0.90, 1.19, 0.91, 1.62,

dd (13.4, 7.9) dd (13.4, 8.1) m m m m m m m m

0.85, 2.21, 2.13, 0.96,

m m m t (7.2)

1.26, m 1.10, m 0.82, m

Traces of acetone were observed in the NMR data of this sample.

4S, 6S, and 10R based on the fact that the specific rotation for 1, [α]20D +26 (c 0.36, CHCl3), was of the same sign as plakortide E (4S, 6R, 10R), natural [α]20D +75 (c 2.23, CHCl3) and synthetic [α]20D +87 (c 0.87, CHCl3), and that only the 4S, 6S, and 10R configuration has been reported as naturally occurring.19−21 Further evidence was also obtained by comparing the 13C NMR data with literature values, as shown in Figure 2b; significant deviations (Δ > 1.5 ppm) were observed only at C-7 and C-8. The assignment of C-8 in 1 could not be unambiguously assigned by NMR data and, hence, will likely require chemical synthesis for its assignment, as was the case for plakinic acid A,18 plakortone B,20,21 plakortolide E,22 plakortide E,20,21 and compound 7.29 On the basis of our data, we named this compound 7,8-dihydroplakortide E (1), as the only difference between this compound and plakortide E is a double bond at C-7 and C-8.19−21 Compound 2 was obtained as a yellow resin with a specific rotation of [α]20D −145 (c 1.1, CHCl3). In contrast to 1, the 1H NMR and 13C NMR data of 2 were compatible with a planar structure containing a six-membered endoperoxide consistent with plakortide H, [α]20D +5.5 (c 2.9),32 and 4-epi-plakortide H, [α]20D +19 (c 0.13, CHCl3).33 Similarities in the NMR data indicated that 2 was most closely related to the corresponding carboxylic acid of plakortide H. The relative configuration of 2 was determined by NOE correlations obtained from a NOESY spectrum of 2 (Figure 2a). These data suggested that 2 differed from plakortide H acid in the configuration of C-6 relative to the configurations of C-3 and C-4. 13C NMR data analyses

hybridized as part of the two double bonds, and three nonprotonated carbons (C). The presence of two olefins was further confirmed by four signals in the 1H NMR spectrum of 1 (δH 6.97, 6.11, 5.35, and 4.95, Table 1). One carbon at δC 170.7 (C-1) was assigned as a carbonyl from a carboxylic acid, an observation that was consistent with IR spectroscopy (νmax at 3430 and 1689 cm−1). We then turned to examine the gCOSY NMR spectrum to elucidate the structural backbone of 1 (Figure 2) and identified a motif that bore a similarity to plakortide E. As anticipated, the HMBC spectrum exhibited long-range correlations for the methyl and methylene protons of both ethyl substituents with the oxygenated carbon atoms at δC 87.4 (C-4) and 89.8 (C-6), as well as correlations of both olefinic protons at δH 6.11 (H-2) and 6.97 (H-3) with carbon C-4 (Figure 2a). The second olefin was identified as a trans-olefin (J = 15.2 Hz) between C-11 and C-12 through HMBC correlations with δC 25.8 (C-13) and the carbon of a methyl group at δC 14.5 (C-14). The HMBC data also identified a methyl group attached to C-8 and an additional ethyl group at C-10. Next, the relative configuration was deduced by comparison of NOESY data collected from 1 with the published data for plakortide E.20,21 This included NOE interactions between the axial proton at C-5 and the protons on C-3 and C-7, as well as the equatorial proton at C-5 and protons on C-15 and C-17 (Figure 2). These data indicated that the side chain extending from C-7 was on the opposite side of the ethyl groups at C-4 and C-6. We then tentatively assigned the configuration of 1 as C



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the methyl ester, as 10 was obtained as the corresponding carboxylic acid. Considering the similarity to plakortide P (9) and 11 and the fact that the specific rotations of 10 and 11 were opposite in sign, we assigned 10 as the enantiomeric acid of methyl ester 11. As with 2 and 11,30 we could not assign the configuration at the remote C-10 stereocenter and, therefore, tentatively describe it as 11,12-dihydroplakortide P. Next, we evaluated the cytotoxicity of compounds 1−10 by comparing their activities in colon cancer (HCT-116) and metastatic prostate cancer (PC-3M) cells with nontumor lung fibroblast (MRC-5) cells. After 72 h of incubation, compounds 2, 3, 5, and 7−9 were considered cytotoxic, with IC50 values of 142 2.8 1.9−4.2 8.1 4.7−14 19 12−31 5.4 2.6−11 37 16−81 3.8 3.0−4.8 10 6.5−17 3.6 1.7−7.9 65 14−293 0.02 0.02−0.03

>142 12 9.3−15 >163

>142 51 17−155 >163

>162

92 35−244 3.3 1.2−9.6 52 9.0−303 2.0 1.1−3.6 0.5 0.2−0.9 0.2 0.0−0.3 31 15−64 2.0 0.7−5.0

N.T.b 14 9.3−23 59 25−138 >162

3 4 5 6

Figure 2. Selected NMR data. (a) gCOSY, HMBC, and NOESY correlations for compounds 1 and 2. Insets depict NOESY interactions. (b) 13C NMR analyses comparing chemical shifts between 7,8-dihydroplakortide E (1) and plakortide E.34 (c) 13C NMR analyses comparing chemical shifts between 2 and plakortide H.32 (d) 13C NMR analyses comparing chemical shifts between 10 and 11.30 M denotes the methyl group of the corresponding methyl esters. As we isolated both 2 and 10 as acids and compared them against known compounds that were methyl esters, the presented value for M was highly negative. *Denotes that data were not present in the literature.

24 h

compd

7 8 9 10 +c

25 4.2−149 >171 3.7 1.9−5.9 2.8 2.1−3.9 2.2 1.3−3.8 20 8.6−48 0.4 0.2−0.7

44 >171 N.T. N.T. 3.2 0.5−8.1 27 19−37 1.9 0.6−6.3

a

Data are presented as IC50 values and 95% confidence interval (CI 95%) for colon (HCT-116), metastatic prostate (PC-3M), and nontumor lung (MRC-5) cells. Experiments were performed in triplicate. bN.T.−not determined. c+ denotes the positive control doxorubicin.

provided further support for this assignment, as the major differences between 2 and plakortide H appeared proximal to C-6 and C-5 and C-17 and at the position of esterification, C-1 (Figure 2c). Compound 2, therefore, represents the second diastereomer of plakortide H that has been isolated from Plakortis specimens. The configuration at the remote C-10 stereocenter, as was the case for plakortide H32 or 4-epiplakorited H,33 could not be determined. However, analyses of other plakortides have suggested the R configuration.23,30,33 The third new material, compound 10, was isolated as a colorless resin with a specific rotation of [α]20D of −79 (c 0.28, CHCl3). Its molecular formula, C22H40O4, as determined by HRESIMS, indicated the presence of four degrees of unsaturation. On further comparison with the literature, we found that its NMR spectrum was comparable to that of plakortide P (9) (Figure 1),23 albeit without the olefin at C11−C-12. In addition, similarities were obtained with compound 11, [α]23D of +12.4 (c 0.4, CHCl3),30 bearing an absolute configuration of 3R,4R,6S.30 Comparison of the 1H NMR and 13C NMR data between 10 and 11 showed comparable chemical shifts (Figure 2d) with the exception of

compound containing a five-membered endoperoxide ring, was supported by the fact that plakortide E, a closely related compound, lacked activity (>100 μM) when screened in the macrophage cell line J744.1.34 The observed activity pattern was quite variable among tested compounds, especially when time dependence and selectivity are considered. Compounds 3−6, which contain a dihydrofuran ring, were generally less active, with significant activity observed only for compounds 3 and 5 against HCT-116 cells (IC50’s of 8.1 and 5.4 μM, respectively). These compounds also displayed time dependence in their cytotoxic activity, as their IC50 values increased up to 8.2-fold upon reduction of the time of incubation from 72 h to 24 h. When comparing the toxicity against nontumor cells, only compound 3 was selective, as it was 20 times more active against HCT-116 cells than against MRC-5 cells. The activity of the second group, compounds 2 and 7−10, which contained a D



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common six-membered endoperoxide, was higher overall than compounds 3−6, with IC50 values ranging from 0.2 to 10 μM. In general, they displayed poor selectivity, with the exception of compound 2, which was 18 times more toxic to HCT-116 cells when compared to nontumor MRC-5 cells. The cytotoxic activities of 2 and 7−9 were not affected by time, providing comparable activity after 24 or 72 h (Table 2). IC50 values for 9 were at least 10 times smaller than those obtained with 10, while these compounds only differ by a double bond at C-11/ C-12, indicating the importance of this structural difference to the biological activity. We then selected five active compounds from these groups, 2, 3, 5, 7, and 9, for further biological evaluation. We began by applying flow cytometry to examine the effects of 3 and 5 on HCT-116 cells. First, we determined that cell density was reduced in cultures treated with both compounds (Figure 3a).

Figure 4. Effects of select compounds on cell cycle progression. Effects of (a) a dihydrofuran ring containing 3 and 5 or (b) six-membered endoperoxides 2, 7, and 9 on HCT-116 cell cycle progression. HCT116 cells were untreated (C−), treated with 0.1 μM doxorubicin (C+), or treated with 2, 3, 5, 7, or 9 for 24 h and then analyzed by flow cytometry. Ten thousand events were acquired in each replicate. Representative histograms from one experiment are shown along with the average outcome in pie graphs. The percentages of G0/G1, S, and G2/M phases were analyzed using the ModFit LT 4.0 program.

visualized only at 15 μM, and 2 did not present significant effects at the tested concentrations (5 and 15 μM). In contrast to 3 and 5, six-membered endoperoxide compounds delivered a G2/M arrest (Figure 4b). Plakortide P (9) has previously displayed antineuroinflammatory and antiparasitic effects, emphasizing a highly selective activity against Leishmania chagasi.10 Nevertheless, screening through the NCI-60 human tumor cell line panel indicated a lack of selectivity, with IC50 values ranging from 0.01 to 1.9 μM.23 Our data also returned similar activities as well as a lack of cell selectivity (Table 2). A combination of microscopic methods provided an excellent means to characterize the cell cycle arrest. As shown in Figure 5, conventional staining with a Quick Panoptic Kit confirmed the increased presence of mitotic cells. Further analysis was conducted by confocal microscopy. HCT-116 cells were treated with 50 μM 3, 50 μM 5, 5 μM 2, 5 μM 7, or 2.5 μM 9, fixed, and stained for their nucleus and microtubules. As seen in Figure 6, select cells treated with 2 or 9 displayed induction of spindle formation but failed to complete the assembly process. This resulted in cells that display an apparent block during anaphase (white arrow, Figure 6). This was in contrast to the effects of paclitaxel, which resulted in a uniform blockage of spindle formation as given by accumulation of cells at metaphase (yellow arrows, Figure 6). While the mode by which 2 and 9 inhibited spindle assembly remains inconclusive, it appears to operate independent of microtubule polymerization. Interestingly, these effects were not observed with compound 3 or 5. Various plakortides, namely, F, G, and H, are known to increase Ca2+ uptake by activating cardiac sarcoplasmic/

Figure 3. Analysis of the activities of selected compounds on HCT116 cell density and viability. Effects of dihydrofurans 3 and 5 on (a) cellular growth and (b) membrane integrity. Effects of six-membered endoperoxides 2, 7, and 9 on (c) cellular growth and (d) membrane integrity. Cells were either untreated (C−), treated with 0.1 μM doxorubicin (C+), treated with 3 or 5, or treated with 2, 7, or 9 for 24 h and then analyzed by flow cytometry after staining with PI. Data are presented as mean values ± standard error of the mean (SEM) from three independent experiments each conducted in triplicate. Ten thousand events were acquired for each replicate. *p < 0.05 are given by control by ANOVA and followed by Dunnett’s comparison test.

This was accompanied by a reduction in membrane integrity at 100 μM (Figure 3b), providing evidence for cell death, albeit at a high dose. Further inspection through cell cycle analyses indicated that these compounds induced blockage at G0/G1 (Figure 4a). Prior studies indicated that spongosoritin A (5) displayed moderate antiparasitic and hemolytic activities and a low cytotoxicity against murine macrophages. Additionally, it was shown to be inactive at 86 μM against tumor cell lines.10 We used similar methods to examine the group of sixmembered endoperoxide compounds 2, 7, and 9. Plakortide P (9) was more active than 2 and 7, significantly diminishing cell density at 2.5 μM (Figure 3c), accompanied by cell death at the higher concentrations (Figure 3d), while the effects of 7 were E



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endoplasmic reticulum Ca2+-ATPases (SERCAs).19 Hence, some of plakortide biological properties have been related to the disruption of Ca2+ homeostasis, such as the antifungal activity mediated by plakortide F.35 Calcium plays a central function in many physiological processes, including regulation of the cell cycle.36,37 For instance, Whitaker and Patel revealed the roles played by cytosolic Ca2+ in controlling cell cycle progression in sea urchin embryos.38 Their studies indicated that a transient increase of intracellular Ca2+ is recorded during the G1 to S transition, the G2 to M transition, and the transition from metaphase to anaphase during mitosis. Therefore, compounds that induce Ca2+ influx such as amiodarone, an antiarrhythmic drug and antifungal agent, arrest yeast cells at G1/G0 and G2/M phases via a calcineurin-mediated mechanism, as also suggested to partake in the activity of plakortide F.35,39 The antimitotic effect induced by the plakortides studied herein, mainly that of plakortide P, may, in some instances, be related to increased Ca2+ levels, although this hypothesis requires further investigation.

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CONCLUSION We now report the identification of three new plakortides, 7,8dihydroplakortide E (1), 2, and 10, and seven known compounds that represent three families of natural products from Plakortis. While these compounds share a common biosynthetic origin, the branches within these pathways lead to natural products with different effects on mammalian cells. As illustrated in Figure 7, the isolated compounds cluster into different groups of cytostatic responses, including no observed effects (compounds 1, 4, 6, and 10), time-dependent cytotoxic activity for 3 and 5, and time-independent cytotoxic activity for 2 and 7−9. A more detailed investigation of active compounds 2, 3, 5, 7, and 9 suggested that these compounds presented a different activity profile, while dihydrofuran 3 and 5 arrested cells in G0/G1 and six-membered endoperoxide (2, 7, and 9) arrested cells in G2/M, leading to an accumulation of mitotic figures. The structural requirements involved in the cytotoxic activity of the plakotides are far from being fully elucidated. While

Figure 5. Morphological changes. Images depicting HCT-116 cells stained with hematological staining kit (see Experimental Section) after 24 h treatment with 5 μM 2, 50 μM 3, 50 μM 5, 5 μM 7, 2.5 μM 9, 0.4% DMSO (negative control, C−), or 100 nM doxorubicin (positive control, C+). Magnification was at 200×. Bars denote 20 μm. Arrows indicate mitotic figures.

Figure 6. Confocal fluorescent microscopic analyses. Images depicting the effects of 5 μM 2, 50 μM 3, 50 μM 5, 5 μM 7, and 2.5 μM 9 on HCT-116 cells after 24 h incubation. For each panel, cells were stained with DAPI (nucleus, blue) and Alexa Fluor 647−α-tubulin mAb (microtubule, red) and imaged on a 710 LSM confocal microscope (Zeiss) at a magnification of 630×. The negative control (C−) was 0.15% DMSO in PBS, and 250 nM doxorubicin (C+) and 50 nM paclitaxel were positive controls. White and yellow arrows depict mitotic cells from treatment with paclitaxel or or 2 or 9, respectively. Bars denote 10 μm. F



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Extraction. A sample of P. angulospiculatus (26.1 g, wet weight) was cut into 1 cm2 pieces, immersed in EtOH (250 mL), and ultrasonicated for 10 min. This procedure was repeated four times. The four extracts were collected and evaporated under reduced pressure to yield 3.7 g of an EtOH extract. Fractionation. The extract was fractionated using a combination of hydrophobic polystyrene resins and silica gel chromatography. This began by fractionating the entire extract (3.7 g) over a 20 g column of Chromabond HR-X resin (Machery-Nagel), eluting sequentially with H2O (500 mL), 7:3 CH3CN/H2O (300 mL), CH3CN (300 mL),and CH2Cl2 (300 mL), to deliver four fractions weighing 2.1 g (H2O), 617 mg (CH3CN/H2O), 866 mg (CH3CN), and 165 mg (CH2Cl2), respectively. Aliquots of each of these fractions were dissolved in DMSO at 10 or 1 mg/mL and screened against three cell lines for cytotoxicity. The CH3CN fraction demonstrated the highest activity, inducing 86% and 100% cell death at 5 and 50 μg/mL, respectively. The CH3CN fraction was then subjected to a combination of fractionation on silica gel followed by semipreparative normal-phase HPLC eluting with gradients of n-hexane/EtOAc, semipreparative normal-phase HPLC eluting with gradients of CH2Cl2/MeOH, or semipreparative C18 HPLC eluting with gradients of MeOH/H2O. Using TLC as a guide to select and pool fractions, the entire CH3CN fraction (866 mg) was mined to deliver 8,9-dihydroplakortide E (1) (5.1 mg), compound 2 (5.8 mg), 6-desmethyl-6-ethylspongosoritin A (3) (33.0 mg), 6-desmethyl-6-ethyl-9,10-dihydrospongosoritin A (4) (4.4 mg), spongosoritin A (5) (29.4 mg), 9,10-dihydrospongosoritin A (6) (6.2 mg), compound 7 (14.7 mg), compound 8 (5.2 mg), plakortide P (9) (4.6 mg), and compound 10 (25.4 mg). 7,8-Dihydroplakortide E (1): colorless resin; [α]20D +26 (c 0.36, CHCl3); UV (MeOH) λmax 201 nm; IR (film) νmax 3430, 1698, 1655, 1460, 1306, 1282 cm−1; 1H NMR (300 MHz, CDCl3) and 13C NMR (75 MHz, CDCl3) data, Table 1; HRESIMS m/z 375.2484 [M + Na]+ (calcd for C21H36O4Na, 375.2506). Compound 2: yellow resin; [α]20D −145 (c 1.1, CHCl3); UV (MeOH) λmax 202 nm; IR (film) νmax 3432, 1713, 1461, 1380, 967 cm−1; 1H NMR (300 MHz, CDCl3) and 13C NMR (75 MHz, CDCl3) data, Table 1; HRESIMS m/z 375.2524 [M + Na]+ (calcd for C21H36O4Na, 375.2506). Compound 10: colorless resin; [α]20D −79 (c 0.28, CHCl3); UV (MeOH) λmax 200 nm; 1H NMR (300 MHz, CDCl3) and 13C NMR (75 MHz, CDCl3) data, Table 1; HRESIMS m/z 391.2824 [M + Na]+ (calcd for C22H40O4Na, 391.2828). Cytotoxicity Assays. Cell lines were maintained in RPMI 1640 medium supplemented with 10% fetal bovine serum (v/v), 2 mM glutamine, 100 U/mL penicillin, and 100 μg/mL streptomycin. Cells were cultured at 37 °C under a 5% CO2 atmosphere and regularly split to maintain logarithmic growth phase. Cytotoxic activity was evaluated using HCT-116 (colon adenocarcinoma) and PC-3 M (metastatic prostate carcinoma) tumor cells along with a human nontumor cell line, MRC-5 (fetal lung fibroblast), by collecting IC50 values using the MTT assay.24 Briefly, cells were plated into 96-well plates (5 × 104 cells/mL) and cultured for 24 h prior to screening. Negative and positive controls were given by 0.15% DMSO (C−) or 5 μM doxorubicin (C+). Three hours before the end of the incubation periods, 150 μL of a 1 mM stock solution of MTT was added to each well. After 3 h, the colorimetric response was read using a Multiskan FC multiplate reader (Fisher Scientific). The effect was quantified as the percentage of the control absorbance at 595 nm. IC50 values along with 95% confidence intervals were calculated by nonlinear regression using GraphPad Prism 4.0 (Intuitive Software for Science). Cell Viability Analyses. HCT-116 cells were seeded in a 24-well plate (5 × 104 cells/mL), cultured for 24 h to initiate cell growth, and treated with media containing 5 μM 2, 15 μM 2, 50 μM 3, 100 μM 3, 5 μM 5, 100 μM 5, 5 μM 7, 15 μM 7, 2.5 μM 9, 5 μM 9, 0.15% DMSO (v/v) (negative control), or 100 nM doxorubicin (positive control). After incubation for 24 h, the cells were harvested with 0.05% trypsin-EDTA (Gibco), centrifuged, and resuspended in a 5 μg/mL propidium iodide (PI) solution (Sigma-Aldrich). After 5 min incubation in the dark, 10 000 events were acquired using an Accuri C6 flow cytometer (BD Biosciences) on a gated region to exclude

Figure 7. Summary of SAR data. Two different biological activities were observed as given by blockage of the cell cycle during G0/G1 or G2/M. While each of the natural products shared a common motif (orange), different activities were observed for molecules containing the dihydrofuran (green), five-membered endoperoxide (red), versus six-membered endoperoxide (blue) motifs.

cytotoxicity is a very common response in cells treated with either five-membered or six-membered endoperoxides,40,41 the literature fails to provide a detailed survey of the specific differences regarding their efficacy. Our results indicate that sixmembered endoperoxides, derived from the same biosynthetic origin as their five-membered counterparts, were indeed more active. Overall these data begin to suggest that while endoperoxides display cytotoxic activity, structural features such as relative and absolute configurations, as well as functionality, can provide selective activity by activation of different pathways. Overall, these studies indicate the importance of developing SARs between related members and families of endoperoxide-containing natural products. As learned through our interactions with Professor William Fenical, natural products offer far more information when viewed through a comparative eye.

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EXPERIMENTAL SECTION

General Experimental Procedures. Specific rotations were measured on a digital 341 polarimeter (PerkinElmer). IR spectra using KBr pellets were recorded using a FT-IR 1000 spectrometer (PerkinElmer). NMR spectra were collected on either an Avance DRX-500 (Bruker) or DPX-300 (Bruker) spectrometer. HRESIMS spectra were acquired using an LCMS-IT-TOF spectrometer (Shimadzu). HPLC analysis was carried out using a Class LC-10 system (Shimadzu) equipped with an SPD-M10Avp diode array UV− vis detector (Shimadzu), a 250 mm × 10 mm i.d. × 5 μm LC-Si analytical column (Supelco), and C18 semipreparative column (Supelco). Column chromatography was carried out on silica gel 60 (EM Biosciences) and Chromabond HR-X resin (Machery-Nagel). Thin-layer chromatography (TLC) was conducted using precoated 0.20 mm Kieselgel 60 F254 polyester sheets (EM Biosciences). Fractions and pure compounds were monitored by TLC analysis, and plates were developed by spraying with vanillin/perchloric acid/EtOH solution followed by heating. Collection and Identification of Biological Material. The sponge Plakortis angulospiculatus was collected on March 30, 2014, at Flecheiras Beach (3°13′7,29″ S−39°15′48,40″), Ceará state, northeastern Brazil. The material was transferred to ice shortly afterward and transported to the laboratory to be processed. A small portion was preserved in 96% EtOH for species identification. A voucher specimen (MNRJ 17753) was deposited in the Porifera collection of the Museu Nacional, Universidade Federal do Rio de Janeiro, Rio de Janeiro, Brazil. G



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Notes

debris and doublets from the analysis. The differences between the negative control and experimental groups were determined by analysis of variance (ANOVA) followed by Dunnett’s test on GraphPad Software 4.0 (Intuitive Software for Science). The minimal significance level was set at p < 0.05. Cell Cycle Analyses. HCT-116 cells were seeded in a 24-well plate (5 × 104 cells/mL), cultured for 24 h to initiate cell growth, and treated with media containing 5 μM 2, 15 μM 2, 50 μM 3, 100 μM 3, 5 μM 5, 100 μM 5, 5 μM 7, 15 μM 7, 2.5 μM 9, 5 μM 9, 0.15% DMSO (v/v) (negative control), or 100 nM doxorubicin (positive control). After incubation for 24 h, the cells were harvested with trypsin-0.05% EDTA (Gibco), centrifuged, and resuspended in a solution containing 5 μg/mL PI, 0.1% citrate, and 0.1% Triton X-100. After 30 min incubation in the dark, 10 000 events were acquired on an Accuri C6 flow cytometer (BD Biosciences) set with gated regions to exclude debris and doublets from the analysis. Cell cycle histograms were acquired using the FL3 channel (excitation at λmax 488 nM and emission λmax 620 nM) to capture the red fluorescence emitted from PI−DNA complexes. The cell cycle profile was analyzed in linear scale with the ModFit LT 4.0 software (Verity Software House). The histograms depicted in Figure 4 were created with FlowJo X (Tree Star). The differences between negative control and experimental groups were determined by analysis of variance (ANOVA) followed by Dunnett’s test using GraphPad Software 4.0 (Intuitive Software for Science). The minimal significance level was set at p < 0.05. Morphological Analysis. HCT-116 cells were seeded in a 24-well plate (5 × 104 cells/mL), cultured for 24 h to initiate cell growth, and treated with media containing 5 μM 2, 15 μM 2, 50 μM 3, 100 μM 3, 5 μM 5, 100 μM 5, 5 μM 7, 15 μM 7, 2.5 μM 9, 5 μM 9, 0.15% DMSO (v/v) (negative control), or 100 nM doxorubicin (positive control). After 24 h exposure, the cells adhered to coverslips were fixed, stained using a kit for fast staining of hematologic cells (Quick Panoptic Kit, Laboclin, Paraná), and mounted on glass slides. Cells were examined for morphological changes under light microscopy using a BX-41 microscope (Olympus). Confocal Microscopy. HCT-116 cells were seeded in a 24-well plate (5 × 104 cells/mL), cultured for 24 h to initiate cell growth, and treated with media containing 5 μM 2, 15 μM 2, 50 μM 3, 100 μM 3, 5 μM 5, 100 μM 5, 5 μM 7, 15 μM 7, 2.5 μM 9, 5 μM 9, 0.15% DMSO (v/v) (negative control), or 100 nM doxorubicin (positive control). After the incubation for 24 h, cells were fixed with 3.7% formaldehyde, permeabilized with 0.25% Triton-X, and counterstained. Counterstaining was conducted by staining the nucleus with 10.9 μM 4′,6-diamino-2-phenylindole dilactate (DAPI) (Life Technologies) and tubulin with 1:200 Alexa Fluor 647−anti-α-tubulin (Life Technologies). Cells were examined for morphological changes using an LSM 710 confocal microscope (Zeiss).

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The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by grants from the Conselho ́ Nacional de Desenvolvimento Cientifico e Tecnológico (CNPq) and the Fundaçaõ Cearense de Apoio ao Desenvolví mento Cientifico e Tecnológico (FUNCAP−Programa Á reas Estratégicas) to L.V.C.L. and O.D.L.P. L.V.C.L. is currently a researcher fellow from Fundaçaõ de Amparo à Pesquisa do Estado de São Paulo (FAPESP #2014/11721-3).

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ASSOCIATED CONTENT

S Supporting Information *

Copies of NMR spectroscopic data sets for compounds 1 and 2 are available free of charge via the Internet at http://pubs.acs. org.

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REFERENCES

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AUTHOR INFORMATION

Corresponding Authors

*Tel: +55-85-3366-9441. Fax: +55-85-3366-9782. E-mail: [email protected] (O. D. L. Pessoa). *Tel: +55-85-3366-7029. Fax: +55-85-3366-8333. E-mail: [email protected] (L. V. Costa-Lotufo). Present Addresses ▽

Departamento de Ciências do Mar, Universidade Federal de São Paulo, Santos, São Paulo, Brazil. ○ The Núcleo de Pesquisa em Produtos Naturais e Sintéticos (NPPNS), Faculdade de Ciências Farmacêuticas de Ribeirão Preto (FCFRP), Universidade de São Paulo (USP), Ribeirão Preto, São Paulo, Brazil. H



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Cytotoxic Plakortides from the Brazilian Marine Sponge Plakortis

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