VobasinylâIboga Alkaloids from Tabernaemontana elegans: Cell

Article pubs.acs.org/jnp

Vobasinyl−Iboga Alkaloids from Tabernaemontana elegans: Cell Cycle Arrest and Apoptosis-Inducing Activity in HCT116 Colon Cancer Cells Angela Paterna,† Sofia E. Gomes,† Pedro M. Borralho,† Silva Mulhovo,‡ Cecília M. P. Rodrigues,† and Maria-José U. Ferreira*,† †

Research Institute for Medicines (iMed.ULisboa), Faculty of Pharmacy, Universidade de Lisboa, 1649-003 Lisbon, Portugal Centro de Estudos Moçambicanos e de Etnociências (CEMEC), Faculty of Natural Sciences and Mathematics, Pedagogical University, 21402161 Maputo, Mozambique

‡

S Supporting Information *

ABSTRACT: Phytochemical investigation of the roots of the African medicinal plant Tabernaemontana elegans led to the isolation of three new (1−3) and two known (4 and 5) bisindole alkaloids of the vobasinyl−iboga type. The structures of 1−3 were assigned by spectroscopic methods, mainly using 1D and 2D NMR experiments. All of the isolated compounds were evaluated for their cytotoxicity against HCT116 colon and HepG2 liver carcinoma cells by the MTS metabolism assay. Compounds 1−3 and 5 were found to be cytotoxic to HCT116 colon cancer cells, displaying IC50 values in the range 8.4 to >10 μM. However, the compounds did not display significant cytotoxicity against HepG2 cancer cells. The cytotoxicity of compounds 1−3 and 5 was corroborated using a lactate dehydrogenase assay. Hoechst staining and nuclear morphology assessment and caspase-3/7 activity assays were also performed for investigating the activity of compounds 1−3 and 5 as apoptosis inducers. The induced inhibition of proliferation of HCT116 cells by compounds 1 and 2 was associated with G1 phase arrest, while compounds 3 and 5 induced G2/M cell cycle arrest. These results showed that the new vobasinyl−iboga alkaloids 1−3 and compound 5 are strong inducers of apoptosis and cell cycle arrest in HCT116 colon cancer cells.

N

regulators, which control the correct entry and progression through the cell cycle, are commonly altered in tumors. Tumor cells frequently lose cell cycle checkpoint controls, which contributes to tumor development. Therefore, targeting the impaired checkpoints provides new approaches to improve current therapeutic strategies.14 Our ongoing search for anticancer agents from medicinal plants7,15−20 has led us to study the African medicinal plant Tabernemonatana elegans Stapf., which in the past has afforded several monoterpene indole and bisindole alkaloids.7,21 Aiming to increase the set of bioactive indole alkaloids on hand, in this paper are reported the isolation and structure elucidation of three new (1−3) and two known (4, 5) bisindole alkaloids of the vobasinyl−iboga type and the investigation of their ability to induce apoptosis and arrest cell cycle in human HCT116 colon and HepG2 liver carcinoma cells.

atural products and their derivatives have been playing a key role in drug discovery and development for the treatment of human diseases, with many current drugs having been obtained from plant sources.1,2 Plants belonging to the genus Tabernaemontana (Apocynaceae), which occur in tropical and subtropical regions of the world, including Africa, are recognized as rich sources of indole alkaloids.3 They are able to biosynthesize a large variety of monoterpene indole alkaloids of different skeletal types, where the biogenetic precursors are tryptamine and secologanin, which constitute the indole and terpenoid portions, respectively.4 Many studies have confirmed the therapeutic potential of indole alkaloids, and these are known to exert an effect on a wide range of biological targets such as cytotoxic activity for different cell lines5−8 and anticholinesterase,9 vasorelaxant,10 and antiplasmodial activities against Plasmodium falciparum.8 As an example, the clinically active anticancer drugs vinblastine and vincristine are indole alkaloids obtained from plants of the family Apocynaceae.11 Deregulation of apoptotic pathways can be considered a pivotal phenomenon for cancer development and also to confer drug resistance, which in turn is one of the main causes of anticancer treatment failure.12,13 In addition, several cell cycle © 2016 American Chemical Society and American Society of Pharmacognosy

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RESULTS AND DISCUSSION

The roots of T. elegans were extracted with methanol.7 Subsequent acid−base partitioning of the crude methanol Received: June 16, 2016 Published: October 5, 2016 2624

DOI: 10.1021/acs.jnatprod.6b00552 J. Nat. Prod. 2016, 79, 2624−2634

Journal of Natural Products

Article

Figure 1. Bisindole alkaloids (1−5) isolated from the roots of T. elegans.

NH/OH and ester carbonyl functions, respectively. The ESIMS data of 1 showed a protonated molecule at m/z 723 [M + H]+. The molecular formula, C43H54N4O6, corresponding to 19 degrees of unsaturation, was deduced from the HRTOFESIMS, which exhibited a protonated molecular ion at m/z 723.4117 [M + H] + (calcd for C43H55N4O6, 723.4116). Analysis of the NMR data, including those obtained from 2D experiments (1H−1H COSY, HMQC, and HMBC), indicated compound 1 to be a bisindole alkaloid constituted by vobasinyl and iboga moieties. Thus, these structural features were corroborated by the presence of two broad singlets in the 1H NMR spectrum, at δH 7.68 and 7.53, without correlation in the HMQC spectrum,

extract led to an alkaloid fraction. Fractionation of the dichloromethane-soluble part of the latter yielded three new vobasinyl−iboga-type bisinsole alkaloids (1−3). The known bisinsole alkaloids tabernaelegantine A (4) and tabernaelegantine D (5) (Figure 1) were also isolated and identified by comparison of their spectroscopic data (Supporting Information) to those reported in the literature.22 Compound 1, named (19′S)-hydroxytabernaelegantine A, was isolated as a light yellow, amorphous powder, [α]20D −1.3. The UV spectrum (285, 294, and 305 nm) exhibited absorption bands corresponding to the indole chromophore. The IR absorptions at 3445 and 1722 cm−1 suggested the presence of 2625



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and 1.00 (d, J = 6.3 Hz). A signal at δH 4.03 and the presence of a broad singlet at δH 3.47, without correlation in the HMQC spectrum, corroborated the presence of a hydroxy group (iboga unit). Furthermore, a double doublet at δH 5.23 (J = 12.8, 2.9 Hz), with a corresponding carbon at δ 35.1, was assigned to H3. The 13C NMR and DEPT spectra confirmed the presence of two methyl esters and a hydroxy group. They revealed the presence of 43 carbon signals, comprising 13 quaternary carbons (two ester carbonyl groups at δC 172.0 and 174.2), 15 methines (six sp2 and nine sp3, one bearing an oxygen at δC 71.4), nine sp3 methylenes, and six methyls (three methoxy groups at δC 50.1, 52.8, and 56.9 and a N-Me group at δC 42.6) (Table 1). Further structural features in compound 1 were obtained by two-dimensional NMR experiments (1H−1H COSY, HMQC, and HMBC), which, together with literature data, allowed unambiguous assignments of all proton and carbon resonances (Table 1). The 1H−1H COSY (3J and 4J couplings) and HMQC experiments revealed the key fragments −N−CH− CH2− and −N−CH2−CH−CH−CH2−CH, which are characteristics of the vobasinyl unit, and the spin systems −NCH2CH2− and − N−CH2−CH−CH2−CH−CH− belonging to the iboga unit (Figure 2). The heteronuclear 2JC−H and 3 JC−H correlations displayed in the HMBC spectrum of 1 established the connection of the referred spin systems and the location of the functional groups. Thus, the linkage of the two indole units at C-3/C-12′ was indicated by the key HMBC correlations from H-3 (δH 5.23) to C-12′ (2J) and C-11′ (3J). This connection was corroborated by the upfield proton resonance at δH 0.68, typical of one of the diastereotopic protons at C-17′ (iboga unit), which is exposed to the aromatic ring current effect. Moreover, the HMBC cross-peaks between H-19′ (δH 4.03) and Me-18′ (δC 20.3) and C-21′ supported the placement of the OH group at C-19′ (iboga unit). The location of the methoxy group at C-11′ was confirmed by the nuclear Overhauser effect observed between H-10′ and OMe-11′ in the NOESY spectrum. The relative configurations of the tetrahedral stereocenters of 1 were determined by a NOESY experiment (Figure 3). The αoriented H-15 and H-14′, due to the occurrence of the C-5/C16/C-15 and the C-14′/C-3′/N-4′ bridges in the vobasinyl and

corresponding to the indole NH of both units. Furthermore, the 1H NMR spectrum displayed resonances for six aromatic protons, of which four, at δH 7.01, 7.06, 7.12, and 7.66, were assigned to an unsubstituted indole moiety (vobasinyl unit). Two doublets at δH 7.26 and 6.84, with ortho-coupling, indicated the presence of an indole unit substituted at C-11′ and C-12′ (iboga unit). Moreover, three singlets, corresponding to methoxy groups, were observed at δH 3.96, 3.72, and 2.52, with the latter showing a diamagnetic shift due to the anisotropic effect of the aromatic ring (vobasinyl unit). As could be observed in an energy-minimized 3D structure23,24 of 1 (Figure 3), the ester at C-16 was positioned over the

Figure 2. Key 1H−1H COSY and HMBC correlations of compound 1.

shielding zone of the benzene ring and thus shifted upfield. A characteristic singlet at δH 2.62, indicative of an N-Me group, was also observed besides several aliphatic protons between δH 0.68 and 5.23 and two methyl groups at δH 0.92 (t, J = 7.3 Hz)

Figure 3. Energy-minimized 3D structure and NOESY correlations of compound 1. Energy minimization was carried out with the MMFF94x force field and a root-mean-square gradient of 0.1, using MOE (Molecular Operating Environment).23 Mercury CSD 2.0 was used to visualize the preferred conformation.24 2626



Article

Table 1. 1H and 13C NMR Spectroscopic Data (δ) for Compound 1 (300 and 75 MHz, Respectively) δH (J in Hz)a

δCa

5.23, dd (2.9, 12.8)

136.9 35.1

position 2 3

a

5 6 7 8 9 10 11 12 13 14a 14b 15

4.12, m 3.30, m

1.90, m 2.44, m 2.94, m

16 18

2.76, bt (2.7) 0.92, t (7.3)

19 20 21 N-Me −COOMe −COOMe N−H

1.24, 1.84, 2.55, 2.62,

7.66, 7.12, 7.06, 7.01,

bd (8.2) m m m

m m m s

2.52, s 7.68, bs

position 2′ 3′a 3′b 5′ 6′ 7′ 8′ 9′ 10′ 11′ 12′ 13′ 14′

59.2 19.6 110.2 129.5 118.1 119.6 122.3 109.9 136.2 29.2 33.1

15′a 15′b 16′ 17′a 17′b 18′ 19′ 20′ 21′ OMe′-11 −COOMe′ −COOMe′ N′−H 19′-OH

49.8 11.5 23.6 43.9 49.6 42.6 172.0 50.1

δH (J in Hz)a 2.74, 2.44, 3.30, 2.94,

m m m m

7.26, d (8.7) 6.84, d (8.7)

1.24, m 1.24, m 1.69, m 0.68, 1.69, 1.00, 4.03, 1.56, 3.65, 3.96,

m m d (6.3) m bs bs s

3.72, s 7.53, bs 3.47, bs

δ Ca 135.4 51.0 52.1 21.5 108.7 124.1 117.2 105.4 152.3 115.0 135.3 26.6 22.8 53.6 35.2 20.3 71.4 39.4 59.7 56.9 174.2 52.8

Spectrum recorded in CDCl3.

Table 2. 1H and 13C NMR Spectroscopic Data (δ) for Compound 2 (300 and 75 MHz, Respectively) δH (J in Hz)a

position 2 3 5 6a 6b 7 8 9 10 11 12 13 14a 14b 15 16 18 19 20 21 N-Me −COOMe −COOMe N−H

a

5.16, 4.11, 3.04, 3.30,

7.62, 7.10, 7.10, 7.10,

dd (3.0, 12.7) td (3.1, 8.0) m m

d (7.4) m m m

1.96, m 2.40,m 2.05, m 2.78, m 0.94, m 1.26, m 1.85, m 2.39 m 2.61, s 2.54, bs 7.86, bs

δCa

position

137.0 35.3 59.2 19.5

2′ 3′ 5′ 6′

110.3 129.2 117.7 119.6 122.4 110.5 136.1 29.4

7′ 8′ 9′ 10′ 11′ 12′ 13′ 14′

38.0 49.6 11.5

15′ 16′ 17′a 17′b 18′ 19′ 20′ 21′ OMe′-11 −COOMe′ −COOMe′ N′−H

23.6 43.7 49.7 42.7 172.0 50.2

δH (J in Hz)a

2.54, bs 3.04, m

7.25, d (8.1) 6.82, d (8.7)

1.71, m 2.94, m 0.76, 1.64, 0.88, 1.94, 1.64, 4.31, 3.94,

m m t (7.3) m m bs s

3.69, s 7.48, bs

δ Ca 133.0 175.9 42.6 20.8 108.0 123.5 117.1 105.4 152.3 114.8 135.3 30.9 33.1 54.9 33.8 11.3 27.6 35.4 55.9 56.8 172.5 52.8

Spectrum recorded in CDCl3. 2627



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Table 3. 1H and 13C NMR Spectroscopic Data (δ) for Compound 3 (300 and 75 MHz, Respectively) position 2 3 5 6a 6b 7 8 9 10 11 12 13 14a 14b 15 16 18 19 20 21 N-Me −COOMe −COOMe N−H a

δH (J in Hz)a 5.03, 4.04, 3.19, 3.32,

7.53, 7.03, 7.03, 7.03,

bd (11.4) td (2.7, 7.8) m m

m m m m

1.96, m 2.33, m 2.80, m 3.08, 0.90, 1.41, 1.78, 2.54, 3.68,

bd (12.6) m m m m s

2.64, bs 7.59, bs

δ Ca

position

138.0 36.9 59.4 19.1

2′ 3′ 5′ 6′

111.0 130.1 117.5 118.8 121.4 110.1 136.1 31.1

7′ 8′ 9′ 10′ 11′ 12′ 13′ 14′

33.1

15′a 15′b 16′ 17′ 18′ 19′ 20′ 21′ OMe′-11 −COOMe′ −COOMe′ N′−H

47.2 11.6 23.7 43.9 49.5 42.5 173.2 53.0

δH (J in Hz)a

δ Ca

2.71, m 2.90, m

132.4 175.9 50.9 21.7

6.77, s

6.85, s 2.54, m 1.20, m 1.96, m 2.21, 0.90, 1.41, 1.41, 4.40, 3.92,

m m m m bs s

2.42, s 7.91, bs

109.3 121.5 118.0 128.7 153.7 92.8 135.0 38.2 32.8 55.6 36.2 11.5 27.7 29.9 56.2 55.9 171.9 49.9

Spectrum recorded in CDCl3.

the new structure of the bisindole alkaloid 1 was established as (19′S)-hydroxytabernaelegantine A. Compound 2 was isolated as a yellow, amorphous powder with [α]20D −3.6. The indole chromophore was detected from the UV spectrum (284, 295, and 306 nm). The IR absorptions at 3375 and 1732 cm−1 suggested the presence of an NH and a carbonyl group. The ESIMS data showed a protonated molecular ion at m/z 721 [M + H]+. The molecular formula, C43H52N4O6, corresponding to 20 degrees of unsaturation, was deduced from the HRTOFESIMS peak at m/z 721.3961 [M + H]+ (calcd for C43H53N4O6, 721.3959). Comparison of NMR data of compound 2 with those of compound 1 indicated that the two bisindole alkaloids share the same vobasinyl unit linked with the iboga moiety at C-3/C-12′, which was supported by two doublets with ortho-coupling values at δH 7.25 (H-9′, J = 8.1 Hz) and 6.82 (H-10′, J = 8.7 Hz) in the 1H NMR spectrum and by the heteronuclear 3JH‑10′‑C‑12′ correlation found in the HMBC spectrum. The 13C NMR spectrum of 2 showed the presence of a new signal at δC 175.9 and the absence of the methylene at δC 51.0 (C-3′), indicating the oxidation of the methylene at C-3′ to a carbonyl group (iboga unit) (Table 2). The placement of the new carbonyl function at C-3′ was confirmed by the heteronuclear 2JC−H and 3JC−H HMBC correlation of H-14′ (δH 1.71) and H-21′ (δH 4.31) to C-3′. Furthermore, compound 2 was found to contain a β-ethyl chain at C-20′ instead of the hydroxyethyl group found in compound 1 (Table 2). The presence of an ethyl chain at C-20′, evidenced by the 1 H−1H COSY spectrum (−CH−CH2−CH3), was further supported by the HMBC correlations H-18′/C-20′ and H19′/C-21′. The relative configuration of 2, determined by a NOESY experiment, taking into account the coupling constant

iboga moieties, respectively, were taken as references on a biogenetic basis.3 A β-orientation was assigned to H-3 due to the large coupling values J3−14 (12.8 Hz). The NOE correlation between H-3 and N−H, indicating the proximity between both protons, corroborated the previously published stereochemical details.25 Nuclear Overhauser cross-peaks observed between H15/H-16 and H-15/H-18, indicating the same α-orientation for these three protons, supported the α-ethyl chain at C-20 and the stereochemistry at C-16. Moreover, the configuration at C20 was corroborated by the upfield resonance values of C-14 (δC14 29.2) and downfield chemical shifts of C-16 and C-21 (δC16 49.8 and δC21 49.6), when compared to those found for the known compound 4 (δC14 36.8, δC16 44.0, and δC21 47.0), which bears a β-oriented ethyl group at C-20.5 The remarkable shielding of the methyl ester group at C-16 (δH 2.52) (Figure 3) was in agreement with the S-configuration at this carbon. In the iboga unit, the configurations at C-14′, C-19′, C-20′, and C21′ were indicated by NOESY correlations of H-14′/H-20′, H14′/H-21′, and H-21′/H-19′ (Figure 3), which provided evidence for the α-orientation of these protons. The stereochemistry at C-19′ (S) was further assigned based on the chemical shift values of C-15′ (δC 22.8) and C-21′ (δC 59.7), which were identical with those reported for the corresponding carbons of the monomeric iboga alkaloid heyneanine, which bears a hydroxyethyl group at C-2026,27 (19 S series of iboga alkaloids). On the contrary, when compared to 19-epiheyneanine27 (19 R series of iboga alkaloids), the carbons C15′ and C-21′ showed diamagnetic (ΔδC‑15 = −5.8 ppm) and paramagnetic effects (ΔδC‑21 = +5 ppm), respectively. The chemical shift values assigned to C-15′ (δC 22.8) and C21′ (δC 59.7) supported the configuration at C-19′ (S).26 Thus, 2628



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In further studies, HCT116 cells were exposed to IC50 and 2fold IC50 values of compounds 1−3 and 5 or 5-FU, for 72 h. Cells exposed to vehicle (dimethyl sulfoxide, DMSO) and cells without exposure to either compound or vehicle (no addition) were used as controls. To explore the association of test compound-induced growth inhibition with regulation of cell cycle progression, cells were collected and stained with propidium iodide (PI), and the cellular DNA content was determined by flow cytometry. The percentage of cells in different phases of the cell cycle was analyzed after a 24 h exposure. As shown in Figure 4, compounds 1 and 2 induced significant accumulation of cells in the G1 phase at the IC50 (49% and 46%, respectively, p < 0.05) and 2-fold IC50 concentrations (55% and 62%, respectively, p < 0.05), compared to vehicle exposure (31%), and a concomitant decrease in the S phase. Treatment with compounds 3 and 5 also impacted on the cell cycle progression of HCT116 cells. At the doses tested, these compounds did not induce G1 phase arrest, but rather a significant accumulation of cells in the G2-M phase. Treatment of HCT116 cells with compound 3 at its IC50 concentration resulted in a nonsignificant induction of cell cycle arrest in the G2-M phase (32%), although at the 2-fold IC50 concentration a significant accumulation of cells in G2-M phase (44%, p < 0.05) was detected, compared to vehicle control cells. Compound 5 also induced significant accumulation of cells in the G2-M phase, corresponding to 77% and 47% for the IC50 and the 2-fold IC50, respectively (p < 0.05). According to the literature, exposure to IC50 and 2-fold IC50 of 5-FU in HCT116 cells results in G1-S phase arrest.28 Then, a lactate dehydrogenase (LDH) assay was used to assess general cell death, measuring the enzymatic activity of LDH release from damaged cells. The presence of the LDH in the cell culture supernatant is a consequence of damaged cytoplasmic membrane generally associated with cell death.29 The results obtained showed that exposure of compounds 1−3 and 5 at the IC50 concentration resulted in 2.5- to 2.8-fold increased LDH release in HCT116 cells compared to vehicle (DMSO) exposures, compared with 2.2- to 2.5-fold increased LDH release on 5-FU exposure (p < 0.01, Figure 5). Subsequently, the induction of apoptosis by compounds (1− 3 and 5) was assessed by evaluation of nuclear morphology after a Hoechst staining assay. Compound treatment induced higher levels of apoptosis in HCT116 cells, as shown in Figure 6. Cell exposure to the IC50 concentration of the four compounds (1−3 and 5) for 24 h led to 11%, 16%, 16%, and 31% apoptotic cells, respectively, compared to vehicle control cells, which in turn displayed less than 2% apoptotic cells (p < 0.01). Moreover, exposure of the test compounds at their 2-fold IC50 concentration led to 13%, 25%, 37%, and 40% apoptotic cells, respectively. To corroborate the apoptosis induction by the test compounds (1−3 and 5), a caspase-3/7 activity assay was performed. All compounds, at their IC50 concentrations, increased caspase-3/7 activation by values ranging from 1.6to 5.2-fold, compared with the DMSO vehicle control, and by values ranging from 1.5- to 3.7-fold, compared with the positive control 5-FU (Figure 7).

values and some characteristic NMR signals referred to above, was found to be the same as compound 1 for both units. In particular, the β-orientation of the ethyl chain at C-20′ of the iboga unit was indicated by the NOE interactions between H20′ (δH 1.64) and the α-oriented protons H-14′ (δH 1.71) and H-21′ (δH 4.31). When comparing compound 2 with similar compounds, such as 3′-oxotabernaelegantine A,8 bearing a βethyl chain at C-20 (vobasinyl unit), the presence of an α-ethyl chain at this carbon in compound 2 was indicated by the upfield chemical shifts of C-18 (ΔδC‑18 = −1.4 ppm) and C-19 (ΔδC‑19 = −2.1 ppm), along with the downfield shift of C-16 (ΔδC‑16 = +7.1 ppm). Thus, the structure of compound 2 was established as 3′-oxotabernaelegantine C. Compound 3 was isolated as a yellow, amorphous powder, [α]20D +15.6. The UV spectrum exhibited absorption bands at 283, 294, and 305 nm, corroborating the presence of the indole chromophore. The presence of NH and carbonyl functions was suggested by the IR absorptions at 3406 and 1724 cm−1. The low-resolution ESIMS exhibited a protonated molecular ion at m/z 721 [M + H]+. The HRTOFESIMS indicated the same molecular formula, C43H52 N4O6, as compound 2, through the protonated molecular ion at m/z 721.3963 [M + H]+ (calcd for C43H53N4O6, 721.39596), thus indicating 20 degrees of unsaturation. Careful analysis of the NMR data of compounds 2 and 3 showed that the only difference between these compounds was in the linkage of the monomeric units. The main differences could be recognized in the substitution pattern of the aromatic ring of the iboga unit. Thus, in compound 3, the 1H NMR spectrum showed two singlets at δH 6.77 and 6.85, indicating a linkage at C-10′ and C-3 (Table 3), which was supported by the chemical shift values found for the diastereotopic protons at C-17′ (δH 2.21).25 The relative configuration of all stereocenters of compound 3 was found to be the same as that found for compound 2. Hence, the structure of 3 was determined as 3′-oxotabernaelegantine D. The growth inhibitory properties of the five bisindole alkaloids (1−5) and 5-fluorouracil (5-FU) were investigated by the MTS metabolism assay, calculating half-maximal inhibitory concentration (IC50) after 72 h of treatment. The IC50 values in HCT116 colon cancer cells were 8.4 (1), >10 (2), 8.8 (3), 8.5 (5), and 2.5 μM (5-FU). In HepG2 cancer cells, the compounds did not show relevant cytotoxicity, suggesting selectivity for human HCT116 colon cancer cells. Compound 4 did not display cytotoxic activity (IC50 > 10 μM) in both cancer cell lines (Table 4). Table 4. IC50 Values (μM) of 1−5 As Assessed by the MTS Assay HCT116 cells

HepG2 cells

compound

IC50 (μM)a

95% CI

IC50 (μM)a

1 2 3 4 5 5-FU

8.4 >10 8.8 >10 8.5 2.5

7.83 to 9.06

>10 >10 >10 >10 >10 >10

8.24 to 9.45 7.91 to 9.11 2.46 to 5.84

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a

EXPERIMENTAL SECTION

General Experimental Procedures. Optical rotations were obtained using a PerkinElmer 241 polarimeter, with quartz cells of 1 dm path length. The UV spectra were performed using a UV-1603 (Shimadzu) UV−visible spectrophotometer. The infrared spectra were

After exposure of HCT116 and HepG2 cancer cells to compounds 1−5, 5-FU, and DMSO solvent (control) for 72 h. Results are mean values from at least three independent experiments, with 95% confidence intervals. 2629



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Figure 4. Effects of compounds 1−3 and 5 on cell cycle distribution. HCT116 human colon cancer cells were treated with compounds (1−3 and 5), 5-FU, or DMSO vehicle control for 24 h. Cellular DNA was stained with PI, and flow cytometry analysis was performed to determine cell cycle distribution. Following flow cytometry analysis, frequencies of cells in each phase of the cell cycle were calculated using Mod Fit LT 4.1 software. Histograms show one representative example from three independent experiments (upper panel). Results are expressed in the graph bar as means ± SEM of three different experiments (lower panel). aIC50, b2-fold IC50; †p < 0.01 and *p < 0.05 from DMSO vehicle control, §p < 0.01 and ‡p < 0.05 from 5-FU. MHz) and a Bruker 300 Ultra-Shield instrument (1H 300 MHz, 13C 75 MHz). 1H and 13C NMR chemical shifts are expressed in δ (ppm), referenced to the solvent used, and the proton coupling constants (J) in hertz (Hz). Spectra were assigned using appropriate 1H−1H COSY, DEPT, HMQC, and HMBC sequences. Column chromatography was performed on silica gel (Merck 9385), aluminum oxide 90 active

collected on an Affinity-1 (Shimadzu) FTIR spectrophotometer. Lowresolution mass spectrometry was conducted with a Triple Quadrupole mass spectrometer (Micromass Quattro Micro API, Waters), and high-resolution mass spectra were recorded on a FTICR-MS Apex Ultra (Bruker Daltonics) 7 T instrument. NMR spectra were recorded on a Bruker 400 Ultra-Shield instrument (1H 400 MHz, 13C 100.61 2630



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1232 cm−1; 1H and 13C NMR spectroscopic data (CDCl3), see Table 2; ESIMS (positive mode) m/z 721 [M + H]+; HRESITOFMS m/z 721.3961 [M + H]+ (calcd for C43H53N4O6, 721.3959). 3′-Oxotabernaelegantine D (3): yellow, amorphous powder; [α]20D +15.6 (c 0.1, CHCl3); UV (MeOH) λmax (log ε) 283 (3.45), 294 (3.46), 305 (3.48) nm; IR (NaCl) νmax 3406, 1724, 1637 cm−1; 1H and 13C NMR spectroscopic data (CDCl3), see Table 3; ESIMS (positive mode) m/z 721 [M + H]+; HRESITOFMS m/z 721.3963 [M + H]+ (calcd for C43H53N4O6, 721.3959). Cell Culture. Human colon carcinoma cells HCT116 (ATCC, CCL-247) were grown in McCoy’s 5A medium supplemented with 10% fetal bovine serum and 1% antibiotic/antimycotic (Gibco, Life Technologies, Paisley, UK) and maintained at 37 °C in a humidified atmosphere of 5% CO2. Human liver carcinoma cells HepG2 (ATCC, HB-8065) were grown in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal bovine serum, 1% nonessential amino acids, and 1% antibiotic/antimycotic (Invitrogen). Cells were seeded in 96-well plates at 5000 cells/well for MTS and caspase-3/7 activity assays; in 35 mm dishes at 150 000 cells/dish for morphological evaluation of apoptosis; and in six-well plates at 300 000 cells/well for cell cycle analysis. Test Compounds. Stock solutions of 0.5−50 mM of the test compounds (1−5) and the positive control 5-fluorouracil (Sigma Chemical Co., St. Louis, MO, USA) were prepared in sterile DMSO (Sigma Chemical Co.). Twenty-four hours after cell plating, the medium was removed and replaced with fresh medium containing 1−5 and 5-FU at the indicated final concentrations, or DMSO vehicle control, for the indicated exposure times. MTS Assay. Cell viability was evaluated by the CellTiter 96 Aqueous Non-Radioactive Cell Proliferation Assay (Promega, Madison, WI, USA), using 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium inner salt (MTS), after 72 h of cell treatment. This method is used to screen cancer cell sensitivity to commercially available, chemical synthesized, or extracted test compounds.30−32 In brief, it is a homogeneous, colorimetric method for determining the number of viable cells in proliferation, cytotoxicity, or chemosensitivity assays. The CellTiter96 Aqueous Assay is composed of solutions of MTS and an electron coupling reagent, phenazine methosulfate (PMS). MTS is bioreduced by cells into a formazan product that is soluble in a tissue culture medium. The absorbance of the formazan product at 490 nm can be measured directly from 96-well assay plates without additional processing. The conversion of MTS into the aqueous soluble formazan product is accomplished by dehydrogenase enzymes found in metabolically active cells. The quantity of formazan product was measured in a GloMaxMulti+ detection system (Promega) at 490 nm, as absorbance is directly proportional to the number of viable cells in culture. IC50 and 95% CI (confidence interval) values were determined using GraphPad Prism v.5.00 (GraphPad Software, San Diego, CA, USA), where IC50 is the half-maximal inhibitory concentration for a particular compound, and 95% CI represents a probability of 95% that the confidence interval produced will contain the true parameter value (IC50). Cell Cycle Analysis. After treatment of HCT116 cells with vehicle (DMSO), test compounds 1−3 and 5, or 5-FU at IC50 and 2-fold IC50 concentrations for 24 h, floating and trypsinized adherent cells were collected, centrifuged, resuspended in ice-cold PBS, and fixed under gentle vortexing by dropwise addition of an equal volume of ice-cold 80% (v/v) ethanol (−20 °C). Samples were then stored at 4 °C, for at least 18 h, followed by incubation with PI (25 μg/mL, in PBS), 30 min at 37 °C, before flow cytometry analysis. Since PI also stains doublestranded RNA, RNase A (10 μg/mL) was added to the staining solution. Subsequently, the DNA content was measured using a Guava EasyCyte 5HT flow cytometer (GuavaTechnologies, Inc., Hayward, CA, USA) and analyzed using Mod Fit LT 4.1 software (Verity Software House, Inc., Topsham, ME, USA). Lactate Dehydrogenase Assay. Cell viability was measured by the LDH assay using the cytotoxicity detection kit (Roche Applied Science, USA). In brief, after exposure of cells to vehicle (DMSO), test compounds (1, 2, 3, and 5), or 5-FU at IC50 and 2-fold IC50 concentrations for 72 h, cell culture supernatants were centrifuged at

Figure 5. General cell death as assessed by the LDH assay after exposure of HCT116 human colon cancer cells to compounds 1−3 and 5, 5-FU, or DMSO vehicle control for 72 h. Results are expressed as the means ± SEM of at least three different experiments. aIC50, b2fold IC50; *p < 0.05 from DMSO vehicle control, ‡p < 0.05 from 5-FU. neutral (Merck 1077), and RP18 gel (YMC-GEL ODS-A). Merck silica gel 60 F254 and Merck aluminum oxide 60 F254 neutral plates were used in analytical TLC, with visualization under UV light and by spraying with either Dragendorff’s reagent or a solution of H2SO4− MeOH (1:1), followed by heating. Plant Material. The roots of Tabernaemontana elegans were collected at Maputo, Mozambique, in February 2011. Taxonomic identification was performed by the botanist Dr. Silva Mulhovo, Centro de Estudos Moçambicanos e de Etnociências, Universidade Pedagógica, Maputo, Mozambique. A voucher specimen (23/SM) has been deposited at the herbarium (LMA) of the Instituto de Investigaçaõ Agrária de Moçambique (IIAM), Maputo, Mozambique. Extraction and Isolation. The air-dried, powdered roots (3.5 kg) of T. elegans were extracted with MeOH (4 × 15 L) as previously described.7 Briefly, the MeOH residue (500 g) was dissolved in Et2O and extracted with 10% CH3COOH. The pH of the acid layer was adjusted to 9 by the addition of dilute NH4OH. The basic layer was successively extracted with CH2Cl2 and EtOAc, yielding the CH2Cl2(90 g) and EtOAc-soluble (2 g) fractions. The dichloromethanesoluble fraction was subjected to silica gel column chromatography, using mixtures of solvents of increasing polarity (n-hexane−EtOAc and EtOAc−MeOH), to yield fractions A−O. The crude fraction B (2.5 g) was fractionated by silica gel column chromatography (CH2Cl2− MeOH, from 10:0 to 9:1, v/v), affording 12 subfractions (B1−B12). Fraction B2 (50 mg) was subjected to preparative TLC (n-hexane− CH2Cl2, 3:2 plus 1% trimethylamine), leading to tabernaelegantine D (5, 35 mg). Crude fraction M (3.9 g) was subjected to RP-18 column chromatography (H2O−MeOH, from 10:0 to 0:10, v/v), affording 18 subfractions (M1−M18). Fraction M11 (603 mg) was subjected to silica gel column chromatography (CH2Cl2−MeOH, from 10:0 to 3:2, v/v) to afford five fractions (M11a−M11e). Subfraction M11a (200 mg) was subjected to preparative TLC (n-hexane−CH2Cl2, 1:1 plus 1% trimethylamine, v/v), affording compounds 1 (23 mg), 2 (21 mg), and 3 (27 mg). Subsequently, fraction M14 (360 mg) was subjected to aluminum oxide column chromatography (n-hexane−EtOAc, from 10:0 to 1:9 plus 1% trimethylamine, v/v), giving six subfractions (M14a−M14f). Fraction M14c (90 mg) was subjected to preparative TLC (n-hexane−CH2Cl2, 1:1 plus 1% trimethylamine), affording tabernaelegantine A (4, 7 mg). (19′S)-Hydroxytabernaelegantine A (1): light yellow, amorphous powder; [α]20D −1.3 (c 0.1, CHCl3); UV (MeOH) λmax (log ε) 285 (3.45), 294 (3.47), 305 (3.48) nm; IR (NaCl) νmax 3445, 1722, 1635, 1234 cm−1; 1H and 13C NMR spectroscopic data (CDCl3), see Table 1; ESIMS (positive mode) m/z 723 [M + H]+; HRESITOFMS m/z 723.4117 [M + H]+ (calcd for C43H55N4O6, 723.4116). 3′-Oxotabernaelegantine C (2): yellow, amorphous powder; [α]20D −3.6 (c 0.1, CHCl3); UV (MeOH) λmax (log ε) 284 (3.45), 295 (3.47), 306 (3.48) nm; IR (NaCl) νmax 3375, 1732, 1660, 1446, 2631



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Figure 6. Evaluation of effects on apoptosis of compounds 1−3 and 5. Evaluation of changes in nuclear morphology by fluorescence microscopy of Hoechst-stained nuclei after 24 h incubation of HCT116 human colon cancer cells with compounds 1−3 and 5, 5-FU, or DMSO vehicle control. Representative images of compound effect on apoptosis (400× magnification), with white arrows highlighting apoptotic cells (upper panels). Quantification of apoptosis (lower panels). Results are expressed as means ± SEM of at least three different experiments. aIC50, b2-fold IC50; †p < 0.01 from DMSO vehicle control, ‡p < 0.05 and §p < 0.01 from 5-FU. 250g for 2 min and 50 μL of clear supernatant was transferred to a new flat-bottomed, 96-well plate. Cells attached in the well were lysed by adding 50 μL of lysis solution diluted in culture medium to obtain a cell lysate. Subsequently, 50 μL of LDH reagent mix was added to each well containing either cell culture supernatant or cell lysate, followed by a 15 min incubation at room temperature, protected from light. Absorbance was read at 490 nm, with 620 nm reference wavelengths using a model 680 microplate reader (Bio-Rad). The percentage of LDH release was determined as the ratio between released LDH (supernatant) and the total LDH (supernatant + cell lysate), in the same well, as previously described.33 Hoechst Staining. Hoechst labeling of cells was used to detect apoptotic nuclei by evaluation of nuclear morphology under fluorescence microscopy. Cells were treated with vehicle (DMSO), test compounds (1, 2, 3, and 5), or 5-FU at IC50 and 2-fold IC50 concentrations, and, 24 h after exposure, cells were fixed in 4% paraformaldehyde in phosphate-buffered saline (PBS), pH 7.4, for 10 min at 25 °C and then incubated with Hoechst dye 33258 (Sigma) at 5 μg/mL in PBS for 5 min. Cells were then washed with PBS and

mounted using PBS−glycerol (3:1, v/v). Fluorescent nuclei were scored and categorized according to the condensation and staining characteristics of chromatin. Normal nuclei showed noncondensed chromatin dispersed over the entire nucleus. Apoptotic nuclei were identified by condensed chromatin, contiguous to the nuclear membrane, as well as nuclear fragmentation of condensed chromatin. Three random microscopic fields per sample of approximately 100 nuclei were counted, and mean values were expressed as the percentage of apoptotic nuclei. Caspase 3/7 Activity Assay. Activity of effector caspase-3 and -7 was measured using the Caspase-Glo 3/7 assay (Promega). This assay is based on the cleavage of a pro-luminescent substrate containing the specific DEVD sequence recognized by caspase-3 and -7 to release aminoluciferin in cell lysates. The subsequent luciferase cleavage of the unconjugated aminoluciferin generates a luminescent signal directly proportional to the amount of caspase activity present in the sample. For this purpose, 75 μL of Caspase-Glo 3/7 reagent was added to each well, and the mixture was incubated at room temperature for 30 min, leading to complete cell lysis, stabilization of substrate cleavage by 2632



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(6) Mansoor, T. A.; Ramalho, R. M.; Mulhovo, S.; Rodrigues, C. M. P.; Ferreira, M. J. U. Bioorg. Med. Chem. Lett. 2009, 19, 4255−4258. (7) Mansoor, T. A.; Borralho, P. M.; Dewanjee, S.; Mulhovo, S.; Rodrigues, C. M. P.; Ferreira, M. J. U. J. Ethnopharmacol. 2013, 149, 463−470. (8) Girardot, M.; Deregnaucourt, C.; Deville, A.; Dubost, L.; Joyeau, R.; Allorge, L.; Rasoanaivo, P.; Mambu, L. Phytochemistry 2012, 73, 65−73. (9) Ingkaninan, K.; Changwijit, K.; Suwanborirux, K. J. Pharm. Pharmacol. 2006, 58, 847−852. (10) Zaima, K.; Koga, I.; Iwasawa, N.; Hosoya, T.; Hirasawa, Y.; Kaneda, T.; Ismail, I. S.; Lajis, N. H.; Morita, H. J. Nat. Med. 2013, 67, 9−16. (11) Cragg, G. M.; Newman, D. J. J. Ethnopharmacol. 2005, 100, 72− 79. (12) Lage, H. Cell. Mol. Life Sci. 2008, 65, 3145−3167. (13) Baird, R. D.; Kaye, S. B. Eur. J. Cancer 2003, 39, 2450−2461. (14) Stewart, Z. A.; Westfall, M. D.; Pietenpol, J. A. Trends Pharmacol. Sci. 2003, 24, 139−145. (15) Mansoor, T. A.; Ramalhete, C.; Molnár, J.; Mulhovo, S.; Ferreira, M. J. U. J. Nat. Prod. 2009, 72, 1147−1150. (16) Mansoor, T. A.; Ramalho, R. M.; Mulhovo, S.; Rodrigues, C. M. P.; Ferreira, M. J. U. Bioorg. Med. Chem. Lett. 2009, 19, 4255−4258. (17) Mansoor, T. A.; Ramalho, R. M.; Rodrigues, C. M. P.; Ferreira, M. J. U. Phytother. Res. 2012, 26, 692−696. (18) Duarte, N.; Varga, A.; Cherepnev, G.; Radics, R.; Molnár, J.; Ferreira, M. J. U. Bioorg. Med. Chem. 2007, 15, 546−554. (19) Vieira, C.; Duarte, N.; Reis, M. A.; Spengler, G.; Madureira, A. M.; Molnár, J.; Ferreira, M. J. U. Bioorg. Med. Chem. 2014, 22, 6392− 6400. (20) Ferreira, M. J. U.; Duarte, N.; Reis, M.; Madureira, A. M.; Molnár, J. Phytochem. Rev. 2014, 13, 915−935. (21) Paterna, A.; Borralho, P. M.; Gomes, S. E.; Mulhovo, S.; Rodrigues, C. M. P.; Ferreira, M. J. U. Bioorg. Med. Chem. Lett. 2015, 25, 3556−3559. (22) Bombardelli, E.; Bonati, A.; Gabetta, B.; Martinelli, E. M.; Mustich, G.; Danieli, B. J. J. Chem. Soc., Perkin Trans. 1 1976, 1432− 1438. (23) MOE v2008.10; Chemical Computing Group Inc.: Montreal, Quebec, Canada, 2008. (24) Macrae, C. F.; Bruno, I. J.; Chisholm, J. A.; Edgington, P. R.; McCabe, P.; Pidcock, E.; Rodriguez-Monge, L.; Taylor, R.; van de Streek, J.; Wood, P. A. Mercury CSD 2.0 - New Features for the Visualization and Investigation of Crystal Structures. http://www.ccdc. cam.ac.uk. (25) Kam, T. S.; Sim, K. M.; Pang, H. S. J. Nat. Prod. 2003, 66, 11− 16. (26) Takayama, H.; Suda, S.; Chen, I. S.; Kitajima, M.; Aimi, N.; Sakai, S. Chem. Pharm. Bull. 1994, 42, 280−284. (27) Wenkert, E.; Cochran, D. W.; Gottlieb, H. E.; Hagaman, E. W.; Filho, R. B.; Matos, F. J. d. A.; Madruga, M. I. L. M. Helv. Chim. Acta 1976, 59, 2437−2442. (28) Yoshikawa, R.; Kusunoki, M.; Yanagi, H.; Noda, M.; Furuyama, J.; Yamamura, T.; Hashimoto-Tamaoki, T. Cancer Res. 2001, 61, 1029−1037. (29) Goergen, J. L.; Marc, A.; Engasser, J. M. Cytotechnology 1993, 11, 189−195. (30) Borralho, P. M.; Kren, B. T.; Castro, R. E.; da Silva, I. B.; Steer, C. J.; Rodrigues, C. M. P. FEBS J. 2009, 276, 6689−6700. (31) Silva, T. F. S.; Martins, L. M. D. R. S.; da Silva, M. F. C.; Fernandes, A. R.; Silva, A.; Borralho, P. M.; Santos, S.; Rodrigues, C. M. P.; Pombeiro, A. J. L. J. Chem. Soc., Dalton Trans. 2012, 41, 12888− 12897. (32) Galluzzi, L.; Aaronson, S. A.; Abrams, J.; Alnemri, E. S.; Andrews, D. W.; Baehrecke, E. H.; Bazan, N. G.; Blagosklonny, M. V.; Blomgren, K.; Borner, C.; Bredesen, D. E.; Brenner, C.; Castedo, M.; Cidlowski, J. A.; Ciechanover, A.; Cohen, G. M.; De Laurenzi, V.; De Maria, R.; Deshmukh, M.; Dynlacht, B. D.; El-Deiry, W. S.; Flavell, R. A.; Fulda, S.; Garrido, C.; Golstein, P.; Gougeon, M. L.; Green, D. R.;

Figure 7. Evaluation of test compounds’ effect on caspase-3/7 activity. Caspase-3/7 activity after exposure of HCT116 human colon cancer cells to compounds 1−3 and 5, 5-FU, or DMSO vehicle control for 24 h. Results are expressed as the means ± SEM of at least three different experiments. aIC50, b2-fold IC50; *p < 0.05 and †p < 0.01 from DMSO vehicle control, ‡p < 0.05 and §p < 0.01 from 5-FU. caspases, and accumulation of luminescent signal. The resulting luminescence was measured using the GloMax-Multi+ detection system (Promega). Statistical Analysis. All data were expressed as means ± standard error (SEM) from at least three independent experiments. Statistical analysis was performed using Student’s t test. Values of p < 0.05 were considered significant.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jnatprod.6b00552. Spectroscopic data of 1−5 (PDF)

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AUTHOR INFORMATION

Corresponding Author

*Tel (M. J. U. Ferreira): 351 21 7946470. Fax: 351 21 7946470. E-mail: mjuferreira@ff.ulisboa.pt. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This study was supported financially by Fundaçaõ para a Ciência e a Tecnologia (FCT), Portugal (projects: PTDC/ QEQ-MED/0905/2012; Pest-OE/SAU/UI4013/2014; Ph.D. grant SFRH/BD/77612/2011). The authors thank the Portuguese Embassy in Mozambique, as well as the Portuguese Office of International Affairs for plant transport. We also acknowledge Prof. C. Cordeiro, Faculdade de Ciências, Universidade de Lisboa, for high-resolution mass spectrometric data (FCT, REDE/1501/REM/2005).

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REFERENCES

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