Cucurbalsaminones A–C, Rearranged ... - ACS Publications

Article Cite This: J. Nat. Prod. XXXX, XXX, XXX−XXX

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Cucurbalsaminones A−C, Rearranged Triterpenoids with a 5/6/3/6/ 5-Fused Pentacyclic Carbon Skeleton from Momordica balsamina, as Multidrug Resistance Reversers Andreia Mónico,† Cátia Ramalhete,†,‡ Vânia André,§ Gabriella Spengler,⊥ Silva Mulhovo,∥ M. Teresa Duarte,§ and Maria-José U. Ferreira*,†

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†

Research Institute for Medicines (iMed.ULisboa), Faculty of Pharmacy, Universidade de Lisboa, Avenida Prof. Gama Pinto, 1649-003 Lisbon, Portugal ‡ ATLÂ NTICA − Escola Universitária de Ciências Empresariais, Saúde, Tecnologias e Engenharia, Fábrica da Pólvora de Barcarena, 2730-036 Barcarena, Oeiras, Portugal § Centro de Química Estrutural, Instituto Superior Técnico, Universidade de Lisboa, Avenida Rovisco Pais, 1049-001 Lisbon, Portugal ⊥ Department of Medical Microbiology and Immunobiology, Faculty of Medicine, University of Szeged, Dóm tér 10, H-6720 Szeged, Hungary ∥ Centro de Estudos Moçambicanos e de Etnociências, Faculdade de Ciências e Matemática, Universidade Pedagógica, 21402161 Maputo, Mozambique S Supporting Information *

ABSTRACT: Three new triterpenoids, cucurbalsaminones A−C (1−3), featuring a unique 5/6/3/6/5-fused pentacyclic carbon skeleton, named cucurbalsaminane, were isolated from a methanol extract of Momordica balsamina. Their structures were elucidated by spectroscopic methods and corroborated, for 1, by structure solution using single-crystal X-ray diffraction analysis. A hypothetical biogenetic pathway for these compounds is proposed. Compounds 1−3 were evaluated for their Pglycoprotein (P-gp/ABCB1) modulation ability, using a mouse T-lymphoma MDR1-transfected cell model by the rhodamine123 accumulation assay, and displayed potent multidrug resistance (MDR)-reversing activity.

being highly oxidized, cucurbitacins differ also from more common tetracyclic triterpenes, due to the methyl group being located at C-9 rather than at C-10.5 Previously, chemical investigation of a methanol extract of the aerial parts of M. balsamina led to the isolation of several cucurbitane triterpenoids.7−11 Most of these compounds and semisynthetic derivatives were shown to be potent multidrug resistance (MDR) reversers by inhibiting P-glycoprotein (Pgp) efflux-pump activity.7,12 Additionally, some of these compounds showed selective antiproliferative activity against resistant cancer cells.13 As part of a continuing investigation for new anti-MDR triterpenoids from M. balsamina, herein are described the

Momordica balsamina L. (Cucurbitaceae) is a slender trailing herb native to some tropical and subtropical regions of Africa and Asia. Also known as African pumpkin, M. balsamina has been used for centuries due to its nutritional properties and medicinal value.1 Among its multiple biological activities are the anti-HIV activity of the fruit pulp extract2 and the antiplasmodial activity of the leaf and fruit extracts used as antimalarials in African traditional medicine.3 Its biological activities are attributed to the abundance of unique secondary metabolites biosynthesized by this plant, namely, the high content of tetracyclic triterpenoids with a cucurbitane-type skeleton that characterize the Momordica genus.4−6 The cucurbitane triterpene basic structure contains a 19-(10→ 9β)-abeo-10α-lanost-5-ene tetracyclic nucleus, with the double bond at C-5 and various oxygen-containing substituents being common characteristics of most of these compounds. Besides © XXXX American Chemical Society and American Society of Pharmacognosy

Received: January 7, 2019

A

DOI: 10.1021/acs.jnatprod.9b00019 J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products

Article

Figure 1. Structures of compounds 1−3.

isolation, structure elucidation, plausible biogenetic formation pathway, and MDR-reversing activity of three new compounds (1−3, Figure 1) with a unique pentacyclic carbon skeleton, which has been named cucurbalsaminane.

Table 1. 1H NMR Spectroscopic Data for Cucurbalsaminones A−C (1−3) (CDCl3, δ in ppm, J in Hz, 300 MHz)a

RESULTS AND DISCUSSION The air-dried powdered aerial parts of the African medicinal plant M. balsamina were extracted, exhaustively, with methanol. Fractionation of the EtOAc-soluble part of the methanol extract, using various chromatographic techniques, led to the purification of the new curcurbitanes 1−3. Compound 1, named cucurbalsaminone A, [α]23D −134.2 (c 0.1, CHCl3), was isolated as white crystals (mp 158−159 °C). Its molecular formula was determined to be C27H40O3, from the protonated molecular ion at m/z 413.30638 [M + H]+ in the HRESITOFMS (calcd for C 27H 41 O 3 , 413.30502), consistent with eight degrees of unsaturation. Its IR spectrum showed an absorption band for a carbonyl group at 1718 cm−1. The 1H NMR spectrum showed signals for one tertiary methyl group particularly deshielded (δH 2.08), along with five tertiary methyls (δH 0.83, 0.85, 1.06, 1.18, and 1.20) and one secondary methyl group (δH 0.86, d, J = 6.9 Hz) (Table 1). The 13C NMR spectrum displayed 27 carbon resonances, discriminated by a DEPT experiment as seven methyl groups, seven methylenes, five methines, and eight quaternary carbons (including three carbonyl signals at δC 216.2, 210.9, and 209.4). The 1H−1H COSY and HMQC spectra revealed two main fragments (A and B) (Figure 2). The connection of these spin systems was determined through long-range HMBC correlations of H-8 (δH 2.18) with C-6 (δC 39.4), C-9 (δC 36.9), C-10 (δC 42.5), C-11 (δC 32.3), C-14 (δC 49.9), C-19 (δC 29.6), and C-30 (δC 19.8). The ketone group at C-3 was assigned through a 2JC−H correlation between the carbonyl signal at δC 216.2 and H-2a (δH 2.31−2.41). Moreover, the 2JC−H correlation observed between the carbonyl signal at δC 210.9 and H-8 (δH 2.18) was used to determine its position at C-7. Additionally, the presence of the cyclopropane ring at positions C-5 and C-6 was indicated through observation of cross-peaks of H-6 (δH 1.99) with C-4 (δC 44.4) and C-5 (δC 39.8). The establishment of a carbonyl group at C-23 was corroborated by the 2JC−H correlation between C-23 (δC 209.4) and Me-24 (δH 2.08) and the 3JC−H correlation between C-22 (δC 51.2) and Me-24. A detailed analysis of the 2D NMR data (COSY, HMQC, and HMBC), along with the 1H and 13C NMR spectra, allowed the unambiguous assignment of all carbon signals (Table 2).

position

■

1a 1b 2a 2b 6 8 10 17 18 19 20 21 22a 22b 23 24 26 27 28 29 30

1

2

3

2.12−2.19, m 1.11−1.17, m 2.31−2.41, m 1.97−2.03, m 1.99, s 2.18, s 2.30−2.40, m 1.36−1.47, m 0.83, s 1.06, s 1.89−1.92, m 0.86, d (6.9) 2.47, dd (16.0, 2.8) 2.03−2.13, m

2.12−2.19, m 1.14−1.17, m 2.34−2.42, m 1.98−2.04, m 2.02, s 2.22, s 2.36−2.40, m 1.41−1.50, m 0.86, s 1.09, s 2.01−2.07, m 0.89, d (6.7) 2.49, dd (14.8, 2.6) 1.99−2.06, m

2.08, s

6.04, 2.13, 1.88, 1.21, 1.24, 0.88,

δH (J in Hz)

1. 18, s 1.20, s 0.85, s

m s s s s s

2.12−2.19, m 1.14−1.18, m 2.34−2.42, m 1.98−2.04, m 2.02, s 2.21, s 2.35−2.40, m 1.41−1.49, m 0.82, s 1.09, s 1.38−1.44, m 0.89, d (6.7) 2.20−2.23, m 1.70−1.74, m 5.61, dt (15.7, 7.3) 6.11, bd (15.7) 4.85, (2H, br s) 1.83, s 1.22, s 1.24, s 0.88, s

a

Assignments are based on HMQC, COSY, and HMBC experiments. Proton signals of H-11, H-12, H-15, and H-16 were overlapped and were not assigned.

The aforementioned data suggested that compound 1 is a 25,26,27-trinor-triterpene, with an unprecedented 5/6/3/6/5fused pentacyclic scaffold, differing from the characteristic tetracyclic skeleton usually found in cucurbitacins.4,5 This new skeleton has been named cucurbalsaminane. The relative configuration of compound 1 was determined from a NOESY experiment, assuming an α-orientation for the angular H-10 proton, characteristic of cucurbitacins.14 In this way, crosspeaks observed between H-10/Me-28, H-10/Me-30, Me-30/ H-17, and H-17/Me-21 supported their α-orientation. Conversely, the NOE correlations between H-8/Me-19, H8/Me-18, Me-19/H-6, and H6/Me-29 indicated the βorientation of these protons (Figure 2). The crystal structure of 1 established by X-ray crystallography (Figure 3) corroborated the assignments made by the NMR spectroscopic studies. Bond distances and angles were B



Article

Figure 2. Key (a) 1H−1H, HMBC and (b) NOESY correlations of cucurbalsaminone A (1). Energy minimization of the 3D structure of 1 was carried out in MOE (Molecular Operating Environment)15 through the MOPAC semiempirical quantum chemistry program, using the PM3 Hamiltonian and a root-mean-square gradient at 0.01 kcal/mol/A2. The pictures of the referred models of 1 were visualized using PyMOL.16

Table 2. 13C NMR Spectroscopic Data for Cucurbalsaminones A−C (1−3) (CDCl3, δ in ppm, 75 MHz)a 1

2

3

position

δC

DEPT

δC

DEPT

δC

DEPT

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30

20.5 37.9 216.2 44.4 39.8 39.4 210.9 63.3 36.9 42.5 32.3 30.7 46.3 49.9 36.5 28.4 49.7 16.8 29.6 33.1 20.1 51.2 209.4 30.8

CH2 CH2 C C C CH C CH C CH CH2 CH2 C C CH2 CH2 CH CH3 CH3 CH CH3 CH2 C CH3

19.5 21.4 19.8

CH3 CH3 CH3

20.6 37.9 216.2 44.5 39.9 39.5 210.9 63.4 36.5 42.5 32.4 30.7 46.4 50.0 37.0 28.5 49.7 16.8 29.7 33.6 20.1 51.8 201.5 124.4 155.1 20.8 27.8 19.6 21.5 19.9

CH2 CH2 C C C CH C CH C CH CH2 CH2 C C CH2 CH2 CH CH3 CH3 CH CH3 CH2 C CH C CH3 CH3 CH3 CH3 CH3

20.6 37.9 216.3 44.5 39.9 39.5 211.0 63.4 36.6 42.5 32.4 30.7 46.3 49.9 37.0 28.3 49.7 16.8 29.7 37.0 20.1 39.9 129.4 134.3 142.3 114.3 18.9 19.5 21.5 19.0

CH2 CH2 C C C CH C CH C CH CH2 CH2 C C CH2 CH2 CH CH3 CH3 CH CH3 CH2 CH CH C CH2 CH3 CH3 CH3 CH3

Figure 3. ORTEP diagram of the molecular structure of 1, with ellipsoids at the 50% probability level (color code: carbon, gray; oxygen, red; hydrogen, white).

Compound 2, cucurbalsaminone B, [α]23D −123.2 (c 0.1, CHCl3), was obtained as an amorphous, white powder. Its molecular formula was determined as C30H44O3, based on the protonated molecular ion peak at m/z 453.33802 [M + H]+ in the HRESITOFMS (calcd for C30H45O3, 453.33632), corresponding to nine degrees of unsaturation. The IR spectrum displayed absorption bands at 1723 and 1688 cm−1, indicating isolated and conjugated carbonyl groups. When comparing the 1 H and 13C NMR data of compound 1 with those of 2, it was deduced that both compounds share the same pentacyclic nucleus, differing only in the side chain. Thus, the 13C NMR and DEPT spectra of compound 2 revealed 30 carbon signals, corresponding to eight methyl groups, eight methylenes, five methines (one sp2 carbon at δC 124.4), and eight quaternary carbons, with three of these corresponding to carbonyl signals (δC 216.2, 210.9, and 201.5), and one sp2 carbon resonating at δC 155.1. The presence of one relatively high-field carbonyl signal (δC 201.5), along with one sp2 carbon signal at δC 155.1 and a deshielded olefinic proton of a trisubstituted double bond at δH 6.04, provided evidence for an α,β-unsaturated carbonyl group in the side chain. The 1H NMR spectrum also

a

Assignments are based on DEPT, HMQC, COSY, and HMBC experiments.

within the expected values for similar compounds (Table S2, Supporting Information). C



Article

skeleton, with an α,β-unsaturated carbonyl carbon at C-7, such as balsaminol E (cucurbita-5,24-dien-7-one-3β,23(R)-diol), previously isolated from M. balsamina,9 the key biochemical reactions would involve a 1,2-alkyl shift through C-3/C-4 Cbond cleavage and generation of a five-membered A ring through a Michael-type addition reaction. Intramolecular alkylation of the resulting carbocation at C-4 would give rise to the new cyclopropane ring by connecting C-4 with C-6. Through photochemical reactions, a photorearrangement of the α,β-unsaturated δ-diketone 4,4-dimethyl-17β-acetoxy-androst-5-ene-3,7-dione to two diastereoisomeric cyclopropyl diketones was previously reported.17−19 Previously, in a search for plant-derived compounds as MDR reversers, some cucurbitane-type triterpenes, isolated from M. balsamina, or acyl derivatives were found to be strong P-gp modulators.7,12 Thus, aiming at finding new active compounds for structure−activity relationship studies, the ability of compounds 1−3 as P-glycoprotein modulators was evaluated on a parental mouse T-lymphoma cell line and its corresponding MDR subline, overexpressing this transporter, by the rhodamine-123 accumulation assay, as assessed by flow cytometry. In this assay, the fluorescence activity ratio (FAR) was measured, which represents the intracellular accumulation of rhodamine-123 in MDR (L5178Y-MDR) and parental cells (L5178Y-PAR). Verapamil was used as positive control (20 μM). The compounds were tested at 0.2, 2, and 20 μM. Compounds with FAR values higher than 1 were considered as active P-gp modulators, and those with FAR values greater than 10 as strong modulators.20 At 2 μM, the three compounds (1−3) were found to be strong P-gp modulators (FAR = 14.2, 76.7, and 62.2, respectively; verapamil FAR = 6.6 at 20 μM). Even at the lowest concentration (0.2 μM), compounds 2 and 3 exhibited a significant FAR value (FAR > 3). Conversely, compound 1 was considered inactive at this concentration, suggesting the influence of side chain on the resultant antiMDR activity (Table 3). When comparing these results with

displayed signals for five tertiary methyl groups (δH 0.86, 0.88, 1.09, 1.21, and 1.24), two vinylic methyls (δH 1.88 and 2.13), and one secondary methyl group at δH 0.89 (d, J = 6.7 Hz), corroborating the above data. In the HMBC spectrum of 2, the heterocorrelations between both vinylic methyls (δC 20.8 and 27.8) and the olefinic proton H-24 (δH 6.04), together with the presence of HMBC crosspeaks from the carbonyl group at δC 201.5 to the methylene protons H-22 (δH 2.49, 1.99−2.06) and the olefinic proton, substantiated the presence of an enone system at the side chain. The relative configuration of 2, established by a NOESY experiment, was found to be identical to that of compound 1. Compound 3, named cucurbalsaminone C, [α]23D −80.4 (c 0.1, CHCl3), was also isolated as an amorphous, white powder. Its molecular formula of C30H44O2, deduced from the HRESITOFMS, showed a protonated molecular ion at m/z 437.34229 [M + H]+ (calcd for C30H45O2, 437.34141). The IR spectrum of 3 showed an absorption band at 1723 cm−1, providing evidence for the presence of a carbonyl group. Comparison of the NMR data with those of 1 and 2 revealed that compound 3 is based on the same cucurbalsaminane-type triterpenoid scaffold, but having a different side chain. Besides the signals of the nucleus, the 1H NMR spectrum showed resonances for two methyls, a secondary (δH 0.89) and a vinylic methyl group (δH 1.83). Moreover, a terminal double bond (δH 4.85, br s, 2H) and vinylic NMR signals of a transdisubstituted double bond (δH 6.11, br d, J = 15.7 Hz; 5.61, dt, J = 15.7, 7.3 Hz) were also observed. The 13C NMR spectrum revealed 30 carbon resonances, exhibiting signals for two sp2 methines (δC 129.4 and 134.3) and for a terminal double bond (δC 114.3, methylene group; δC 142.3 a quaternary carbon), thus corroborating the C-8 side chain proposed. Analysis of the HMBC spectrum revealed 3JC−H correlations between the olifinic carbon at C-24 (δC 134.3) and the terminal methylene (δH 4.85, br s), along with long-range correlations between the methine C-20 (δC 37.0) with the diastereotopic methylene protons at C-22 (δH 2.20−2.23, 1.70−1.749), indicating a (23E)-Δ23,25-conjugated diene. A plausible biosynthetic pathway for the carbon skeleton of cucurbalsaminones A−C is proposed (Scheme 1). In this way, starting from a precursor having a tetracyclic cucurbitane-type

Table 3. Effects of Compounds 1−3 on P-gp-Mediated R123 Efflux, in ABCB1-Transfected Mouse T-Lymphoma (L5178Y-MDR) Cells

Scheme 1. Proposed Biogenetic Pathway for Cucurbalsaminones 1−3

compound

concentration, μM

FARa

verapamil cucurbalsaminone A (1)

20.0 0.2 2.0 20.0 0.2 2.0 20.0 0.2 2.0 20.0 2.0%

6.6 1.4 14.2 87.5 5.9 76.7 105.5 3.1 62.2 117.7 1.1

cucurbalsaminone B (2)

cucurbalsaminone C (3)

DMSO a

FAR = (MDRtreated/MDR

control)/(Parentaltreated/

Parentalcontrol).

those found for cucurbitanes in the earlier studies mentioned above,7,12 it was found that compounds 1−3 exhibited stronger MDR-reversing activity than most of those compounds.7,12 When compounds 1−3 were assessed for their antiproliferative activity on the same cell lines by the MTT assay, no significant effects were found (Table S3, Supporting Information). In conclusion, the new structures 1−3 provided a new rearranged triterpenoid skeleton with an unprecedented 5/6/ D



Article

least-squares refinement was used for the non-H atoms with anisotropic thermal parameters. All the H atoms were inserted in idealized positions and allowed to refine in their parent carbon atom. The absolute configuration has not been established by anomalousdispersion effects in diffraction measurements on the crystal. The enantiomer (C5-S; C6-R; C8-R; C9-S; C10-S; C13-R; C14-S; C17-R; C20-R) has been assigned by reference to unchanging chiral centers in the biosynthetic procedure. Crystal data and details of data collection for 1 are summarized in Table S1 (Supporting Information). Crystallographic data have been deposited with the Cambridge Crystallographic Data Centre (CCDC 1878164). Copies of the data can be obtained, free of charge, from the CCDC, 12 Union Road, Cambridge CB2 1EZ, UK [fax: +44-(0)1223-336033 or e-mail: [email protected]]. Cell Lines and Cell Culture. L5178 mouse T-lymphoma cells were transfected with pHa MDR1/A retrovirus, as previously described.25,26 The ABCB1-expressing cell line was selected by culturing the infected cells with colchicine.27 Both parent and resistant cells were cultured and then incubated as previously reported.7,12 Antiproliferative Assay. The antiproliferative effects of compounds 1−3, in a range of increasing concentrations (0.26−133 μM), were tested on the L5178 mouse T-cell lymphoma cells and the human ABCB1-transfected subline, as previously reported.7,12 Rhodamine-123 Accumulation Assay. The rhodamine-123 accumulation assay was performed as previously described. The compounds were evaluated at final concentrations between 0.2 and 20 μM.7,12

3/6/5-fused pentacyclic scaffold. Moreover, preliminary data obtained on P-glycoprotein-mediated MDR reversing activity pointed to their potential as MDR reversers.

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EXPERIMENTAL SECTION

General Experimental Procedures. Optical rotations were obtained using a PerkinElmer 241 polarimeter. Infrared spectra were taken on an Affinity-1 (Shimadzu) FTIR infrared spectrophotometer. NMR spectra were recorded on a Bruker Advance 300 NMR spectrometer (1H 300 MHz; 13C 75 MHz), using CDCl3 as solvent. 1 H and 13C chemical shifts are expressed in δ (ppm) referenced to the solvent used, and the proton coupling constants J are in hertz (Hz). High-resolution mass spectra (HRMS) were recorded on an FTICRMS Apex Ultra (Brüker Daltonics) Tesla mass spectrometer. Column chromatography (CC) was carried out on SiO2 (Merck 9385) or a Combiflash system (Teledyne-Isco; Lincoln, NE, USA) using SiO2 or C18 prepacked columns. TLC was performed on precoated SiO2 F254 plates (Merck 5554 and 5744) and visualized under UV light and by spraying with sulfuric acid−methanol (1:1) followed by heating. Plant Material. The aerial parts of Momordica balsamina were collected in August 2014 at Gaza, Mozambique. The plant material identification was determined by Dr. Silva Mulhovo, a botanist, and a voucher specimen (30 SM) has been deposited at the herbarium (LMA) of the Instituto de Investigação Agronómica, Maputo, Mozambique. Extraction and Isolation. Dried aerial parts of M. balsamina (4.5 kg) were powdered and extracted exhaustively with methanol (8 × 15 L) at room temperature. Evaporation of the solvent (under vacuum, 40 °C) from the methanol extract afforded a residue of 865 g, which was suspended in a MeOH−H2O solution (1:2) and extracted with EtOAc. The resulting EtOAc extract was dried (Na2SO4) and evaporated, yielding a residue of 535 g, which was chromatographed over silica gel (3 kg) with mixtures of n-hexane−EtOAc and EtOAc− MeOH of increasing polarity. Eight fractions (fractions A−H) were obtained through TLC analysis. Fraction D (12.4 g), obtained with nhexane−EtOAc (8:2−7:3), was chromatographed using a reversedphase C18 column (100 g; MeOH−H2O, 1:1 to 1:0), yielding seven fractions (FD1−FD7). From the mother liquors of fraction FD2 (129 mg), after a silica gel column chromatography with mixtures of nhexane−EtOAc (1:0−7:3), was obtained 51 mg of compound 1. Compounds 2 (11 mg) and 3 (30 mg) were obtained by chromatography, over silica gel, of the mother liquors of fraction FD4, using, as eluents, mixtures of n-hexane−EtOAc (1:0−3:2). Cucurbalsaminone A (25,26,27-trinor-cucurbalsamina-3,7,23trione) (1): white crystals; mp 158−159 °C (n-hexane−EtOAc); [α]23D −134.2 (c 0.1, CHCl3); IR (KBr) νmax 2957, 2962, 1718, 1464, 1379 cm−1; 1H and 13C NMR, see Tables 1 and 2; HRESITOFMS m/ z 413.30638 [M + H]+ (calcd for C27H41O3, 413.30502). Cucurbalsaminone B (cucurbalsamin-24-ene-3,7,23-trione) (2): amorphous, white powder; [α]23D −123.2 (c 0.1, CHCl3); IR (KBr) νmax 2957, 2923, 1723, 1688 cm−1; 1H and 13C NMR, see Tables 1 and 2; HRESITOFMS m/z 453.33802 [M + H]+ (calcd for C30H45O3, 453.33632). Cucurbalsaminone C [(23E)-cucurbalsamina-23,25-diene-3,7dione] (3): amorphous, white powder; [α]23D −80.4 (c 0.1, CHCl3); IR (KBr) vmax 2957, 2926, 1723, 1458, 1379 cm−1; 1H and 13C NMR, see Tables 1 and 2; HRESITOFMS m/z 437.34229 (calcd for C30H45O2, 437.34141). X-ray Crystallography of Compound 1. A single crystal of 1 was mounted with Fomblin in a cryoloop, and the crystal data were collected on a Bruker AXS-KAPPA APEX II diffractometer with graphite-monochromated radiation (Mo Kα, λ = 0.710 73 Å) and the X-ray generator operating at 50 kV and 30 mA. Data collection was monitored by the APEX2 program.21 A correction for Lorentzian, polarization, and absorption effects was made using the SAINT and SADABS software.21 The SIR97 program22 was applied for structure solution, and SHELXL-9723 was used for full-matrix least-squares refinement on F2; these programs are in the WINGX version 2014.1 package.24 Non-H atoms were refined anisotropically. A full-matrix

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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.9b00019. Crystallographic data of 1 (CCDC 1878164), antiproliferative activity of compounds 1−3, HRESITOFMS, IR, and NMR data of 1−3 (PDF)

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AUTHOR INFORMATION

Corresponding Author

*Tel: +351 217 946 475. Fax: +351 217 946 470. E-mail: mjuferreira@ff.ulisboa.pt. ORCID

Vânia André: 0000-0001-5599-8355 M. Teresa Duarte: 0000-0003-0994-1352 Maria-José U. Ferreira: 0000-0002-8742-1486 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This study was financially supported by European Structural & Investment Funds through the COMPETE Programme and from National Funds through FCT, Fundaçaõ para a Ciência e a Tecnologia, projects PTDC/MED-QUI/30591/2017 and SAICTPAC/0019/2015. We also acknowledge Prof. Carlos Cordeiro, Faculty of Sciences, University of Lisbon, for the high-resolution mass spectrometric data (FCT, REDE/1501/ REM/2005).

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REFERENCES

(1) Thakur, G. S.; Bag, M.; Sanodiya, B. S.; Bhadouriya, P.; Debnath, M.; Prasad, G. B.; Bisen, P. S. Curr. Pharm. Biotechnol. 2009, 10, 667− 682. (2) Bot, Y. S.; Mgbojikwe, L. O.; Nwosu, C.; Ambiku, A.; Damshak, D. Afr. J. Biotechnol. 2007, 6, 47−52.

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