Resin Glycosides from Ipomoea alba Seeds as Potential

Article pubs.acs.org/jnp

Resin Glycosides from Ipomoea alba Seeds as Potential Chemosensitizers in Breast Carcinoma Cells Sara Cruz-Morales,† Jhon Castañeda-Gómez,†,‡ Daniel Rosas-Ramírez,†,§ Mabel Fragoso-Serrano,† Gabriela Figueroa-González,†,⊥ Argelia Lorence,∥ and Rogelio Pereda-Miranda*,† †

Departamento de Farmacia, Facultad de Química, and §Departamento de Química de Biomacromoléculas, Instituto de Química, Universidad Nacional Autónoma de México, Ciudad Universitaria, 04510, Mexico City, Mexico ‡ Grupo Químico de Investigación y Desarrollo Ambiental, Programa de Licenciatura en Ciencias, Facultad de Educación, Universidad Surcolombiana, Neiva, Colombia ⊥ Laboratorio de Genómica, Unidad de Investigación Básica, Instituto Nacional de Cancerología, 14080, Mexico City, Mexico ∥ Arkansas Biosciences Institute and Department of Chemistry and Physics, Arkansas State University, P.O. Box 639, Jonesboro, Arkansas 72467, United States S Supporting Information *

ABSTRACT: Multidrug resistance is the expression of one or more efflux pumps, such as P-glycoprotein, and is a major obstacle in cancer therapy. The use of new potent and noncytotoxic efflux pump modulators, coadministered with antineoplastic agents, is an alternative approach for increasing the success rate of therapy regimes with different drug combinations. This report describes the isolation and structure elucidation of six new resin glycosides from moon vine seeds (Ipomoea alba) as potential mammalian multidrug-resistance-modifying agents. Albinosides IV−IX (1−6), along with the known albinosides I−III (7−9), were purified from the CHCl3-soluble extract. Degradative chemical reactions in combination with NMR spectroscopy and mass spectrometry were used for their structural elucidation. Four new glycosidic acids, albinosinic acids D−G (10−13), were released by saponification of natural products 3−6. They were characterized as tetrasaccharides of either convolvulinolic (11Shydroxytetradecanoic) or jalapinolic (11S-hydroxyhexadecanoic) acids. The potentiation of vinblastine susceptibility in multidrug-resistant human breast carcinoma cells of albinosides 1−6 was evaluated by modulation assays. The noncytotoxic albinosides VII (4) and VIII (5), at a concentration of 25 μg/mL, exerted the strongest potentiation of vinblastine susceptibility, with a reversal factor (RFMCF‑7/Vin+) of 201- and >2517-fold, respectively.

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plants and their main active secondary metabolites have been shown to modulate MDR in cancer cells, and some crude drugs have been investigated as anticancer agents in order to introduce new therapeutic alternatives.3 There is growing evidence that resin glycosides4a are modulators of efflux pumps that produce the multidrug-resistant phenotype in prokaryotic5 and eukaryotic6,7 cells. These resins are complex mixtures of an extensive family of secondary metabolites known as glycolipids and represent unique compounds in the plant kingdom confined to the Convolvulaceae (the morning glory family)4a and the Scrophulariaceae.4b Previous results from our research program have shown that murucoidin V (isolated from Ipomoea murucoides)8 and purgin II (from the officinal jalap root, I. purga)7b significantly lowered the efflux rate of rhodamine 123, a fluorescent P-gp substrate used to determine its accumulation in monolayer efflux assays

ancer cells are resistant when they are not susceptible to the concentration of a clinically used drug by developing a variety of mechanisms that result in the loss of their initial hypersensitivity to anticancer agents. It is common for cancer cells to express mechanisms that confer simultaneous resistance to various drugs that are structurally and functionally different, a phenomenon known as multidrug resistance (MDR).1 These mechanisms complicate treatment and dramatically increase both morbility and mortality as well as the financial costs for cancer therapy in health care systems worldwide. The most common reason to acquire cross resistance to a wide range of anticancer drugs is the expression of one or more ATPdependent efflux pumps, such as P-glycoprotein (MDR protein1/P-gp) and the breast cancer resistance protein (BCRP/ABCG2), which have the task of detecting and expelling drugs or any hydrophobic xenobiotic outside the cell.2 The use of efflux pump modulators coadministered with cytotoxic drugs results in a susceptibility equivalent to that of a cell without transporter expression. Commonly used medicinal © XXXX American Chemical Society and American Society of Pharmacognosy

Received: August 26, 2016

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DOI: 10.1021/acs.jnatprod.6b00782 J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products

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with resistant cells.6,7b Decreased expression of the P-gp by murucoidin V was also detected by immunofluorescence flow cytometry after treatment with an anti-P-gp monoclonal antibody.6 Incubation of vinblastine-resistant human breast carcinoma cells (MCF-7/Vin) with these resin glycosides also enhanced vinblastine susceptibility.6,7b On the basis of these results, we have employed this cytotoxicity assay for the facile identification of the capacity of each isolated noncytotoxic resin glycoside to modulate the resistance phenotype in the MCF-7 cell line as potential vinblastine chemosensitizers.6,7 Thus, our long-term efforts have been mainly focused on the chemical investigation of this type of glycolipids4 to identify new

potential chemosensitizers to further explore their underlying mechanism of action as MDR-modifying agents.5−7 Moon vine (I. alba L., formerly known as Ipomoea bona-nox and Calonyction aculeatum) is a cultivated ornamental and medicinal species of the night-blooming morning glory group that is native to tropical and subtropical Americas from Mexico and Florida to northern Argentina.9 In Mexico, a decoction of the aerial parts and flowers is used to treat paralysis and soft tissue swelling.7a As with many New World plants, the initial transport to the Old World was a result of their medical applications, learned from the indigenous people of the Americas.10 Laxatives were of prime interest to Europeans,10b and I. alba was one of the early recorded species from the B



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from those observed for albinosinic acid A, the glycosidic acid of albinoside I (7), suggested that the ester linkage for the macrolactone was also placed at the terminal pentose unit as in the case of model compound 7 (Figure S1, Supporting Information).7a Saponification of 1 liberated an H2O-soluble glycosidic acid and an organic solvent-soluble fraction, from which the released 2-methyl-2-butenoic (tga) acid was identified by GC-MS. The glycosidic acid was methylated and further acetylated to yield the peracetylated derivative of albinosinic acid A methyl ester. Comparison of the melting point, optical rotation, and 13C NMR data with published values confirmed its structure,7a which was also identified by HPLC comparison with an authentic sample. All protons of each saccharide unit for the natural product 1 were assigned by a combination of COSY (Figure S8, Supporting Information) and TOCSY NMR techniques (Table 1). Then, all carbons were sequentially assigned by HSQC studies (Table 2).4 In the low-field region of the HSQC spectrum, five anomeric signals were identified: Qui-1 (δH 4.78, δC 103.5), Glc-1 (δH 5.99, δC 101.2), Qui′-1 (δH 5.29, δC 104.4), Rha-1 (δH 5.64, δC 100.2), Rha′-1 (δH 5.82, δC 101.1) (Figures S6 and S7, Supporting Information). The interglycosidic connectivities were confirmed by HMBC experiments.4 For example, the following key correlations were observed: H-2 (δH 4.44) of Qui with C-1 (δC 101.2) of Glc; H-2 (δH 4.18) of Glc with C-1 (δC 101.1) of Rha′; H-6 (δH 4.20) of Glc with C-1 (δC 104.4) of Qui′; H-1 (δH 5.64) of Rha with C-2 (δC 81.3) of Qui′; and H-1 (δH 4.78) of Qui with C-11 (δC 81.5) of convolvulinolic acid (Figure S9, Supporting Information). The locations of the ester substituents were also recognized by HMBC experiments12 through 3JCH correlations between a specific carbonyl group and the pyranose ring proton: the lactonization site at C-2 of the terminal rhamnose unit (Rha) was established by the observed correlation of H-2 (δH 5.81) of Rha with C-1 (δC 173.5) of convolvulinolic acid, and the position of esterification by the tigloyl residue was confirmed by the correlation of H-2 (δH 5.98) of Rha′ with the carbonyl at δC 167.7. For albinoside V (2), the HRESIMS showed a [M − H]− peak at m/z 1137.5712, indicating a molecular formula of C54H89O25 (calcd error: δ = +1.2 ppm), which was 16 mass units higher than albinoside II.7a The initial loss of a niloyl residue afforded a peak at m/z 1037 [M − H − 100 (C5H8O2)]−, in addition to the peak at m/z 809 [1037 − C6H10O4 − 82 (C5H6O)]−, which indicated the consecutive loss of a methylpentose unit and a tigloyl residue for 2. The rest of the fragment peaks were produced by glycosidic cleavage at m/z 663 [809 − C6H10O4]−, 517 [663 − C6H10O4]−, 389 [517 + H2O − C6H10O4)]−, and 243 [389 − C6H10O4]−) (Figure S10, Supporting Information); this fragmentation pattern was similar to that previously reported for albinoside II (8).7a Saponification of this natural product 2 liberated tiglic and nilic acids, which were identified by GC-MS of the organic solventsoluble fraction. The H2O-soluble glycosidic acid was methylated and acetylated to yield the peracetylated derivative of albinosinic acid B methyl ester. Comparison of the melting point, optical rotation, and 13C NMR data with published values confirmed its structure.7a A similar NMR experimental approach to that described above was used for the structure elucidation of 2 in order to assign the 1H and 13C NMR spectra (Tables 1 and 2; Figures S11 and S12, Supporting Information) through COSY (Figure S13, Supporting Information) and HSQC experiments.4 The following key 3JCH correlations were observed to confirm the interglycosidic connectivities in the

Convolvulaceae used by the extinct Taino Amerindians of the Caribbean.10c The present in-depth chemical investigation of the moon vine resin glycoside profile from commercial seed samples was undertaken to unravel the structural diversity of this class of MDR reversal agents by isolating novel glycolipids. Six new resin glycosides, albinosides IV−IX (1−6), as well as the known albinosides I−III (7−9),7a were isolated from the CHCl3soluble extract of moon vine seeds, and their structures were established through chemical degradation, NMR spectroscopy, and mass spectrometry. Reversal of multidrug resistance by compounds 1−6 was also evaluated in vinblastine-resistant MCF-7/Vin cells in modulation assays.

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RESULTS AND DISCUSSION The resin glycoside fraction from the CHCl3-soluble moon vine seed extract was obtained by precipitation with MeOH. Then, individual major constituents were separated into nine fractions through preparative reversed-phase HPLC using peak-shaving and heart-cutting11 techniques. Previously isolated resin glycosides from this species,7a albinosides I−III (7−9) (Figures S1 and S2, Supporting Information), were used as standards to identify eluates containing new constituents. Co-elution experiments were done on an RP-18 column with an isocratic elution of MeOH−CH3CN−H2O, and six peaks were selected for further HPLC separation in the recycling mode.11 To achieve chromatographic homogeneity, each peak was recycled until overlapped components were separated. A refractive index detector was used to monitor this purification process. These approaches allowed the purification of six new compounds, named albinosides IV−IX (1−6). The main approach followed for the structure elucidation of the isolated resin glycosides involved the use of degradative chemical reactions in combination with spectroscopic and spectrometric methods.4a Saponification of the crude material fragmented the macrocyclic lactone and liberated the fatty acids that esterify the oligosaccharide core, which were further identified by GC-MS. Thus, their H2O-soluble glycosidic acids were used to generate 13C NMR profiles7d,10b for 1 and 2, containing known glycosidic acids, since their anomeric signals were readily distinguishable and used as fingerprints for pattern recognition and structural dereplication.7a For the minor compounds 3−6, four new glycosidic acids were identified as the saponification products and were named albinosinic acids D−G (10−13), respectively. Their acid-catalyzed hydrolysis released a mixture of monosaccharides, which underwent derivatization with L-cysteine to form thiazolidines. These derivatives allowed the identification of the constitutive monosaccharides for each oligosaccharide core by GC-MS as their trimethylsilane (TMS) ethers.7a Negative-ion FABMS of albinoside IV (1) afforded a peak at m/z 1053.5139 [M − H]− corresponding to the molecular formula C49H81O24 (calcd error: δ = +1.5 ppm) (Figure S5, Supporting Information). The elimination of 82 amu confirmed the presence of a tigloyl residue at m/z 971 [M − H − C5H6O]−. The other peaks were produced by consecutive glycosidic cleavages along the oligosaccharide core at m/z 825 [971 − C6H10O4 (methylpentose unit)]−, 679 [825 − C6H10O4]−, 533 [679 − C6H10O4]−, 389 [533 + H2O − C6H10O5 (hexose unit)]−, and 243 [389 − C6H10O4]−. This fragmentation pattern was similar to that previously reported for albinoside I,7a confirming a branched pentasaccharide core. Furthermore, the difference of 18 mass units for all these peaks C



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Table 1. 1H NMR Spectroscopic Data for Albinosides IV (1) and V (2) (Measured at 500 MHz in C5D5N, δ in ppm, J in Hz) position Qui-1 2 3 4 5 6 Glc-1 2 3 4 5 6a 6b Rha-1 2 3 4 5 6 Rha′-1 2 3 4 5 6 Qui′-1 2 3 4 5 6 Rha″-1 2 3 4 5 6 Conv 2a 2b 11 14 nla-2 3 4 5 tga-3 4 5

1 4.78, 4.44, 4.26, 3.69, 3.57, 1.49, 5.99, 4.18, 4.07, 3.68, 3.81, 4.20, 4.20, 5.64, 5.81, 4.07, 3.99, 5.10, 1.59, 5.82, 5.98, 4.65, 4.44, 4.37, 1.82, 5.29, 3.96, 4.19, 3.68, 3.88, 1.59,

d (8.0) dd (9.0, 8.0) m m m d (6.0) d (7.6) dd (9.0, 7.8) dd (9.0, 7.8) m ddd (9.0, 6.0, 3.0) m m brs brs dd (9.0, 3.0) dd (9.9, 9.9) dq (9.9, 6.5) d (6.5) brs dd (3.0, 2.0) dd (9.0, 3.0) dd (9.9, 9.9) m d (6.5) d (8.0) dd (9.0, 8.0) dd (9.0, 9.0) m dq (9.0, 6.0) d (6.0)

2.64, m 3.72−3.80, m 0.91, t (7.4)

7.12, dq (7.2, 1.2) 1.40, d (7.2) 1.91, brs

Table 2. 13C NMR Spectroscopic Data for Albinosides IV (1) and V (2) (Measured at 125 MHz in C5D5N, δ in ppm)

2 4.77, 4.42, 4.27, 4.27, 3.66, 1.59,

d (8.0) dd (9.0, dd (9.0, dd (9.0, dq (9.0, d (6.0)

8.0) 9.0) 9.0) 6.0)

5.61, brs 5.77, brs 4.07, dd (9.0, 3.0) 3.53, dd (9.9, 9.9) 3.64, m 1.50, d (6.5) 5.85, brs 4.13, m 5.50, dd (9.0, 3.0) 4.36, m 4.32, m 1.95, d (6.5) 5.33, d (8.0) 4.05, dd (9.0, 8.0) 3.79, dd (9.0, 9.0) 3.79, dd (9.0, 9.0) 3.71, dq (9.0, 6.0) 1.61, d (6.0) 5.73, brs 5.85, m 4.54, dd (9.0, 3.0) 4.30, m 4.25, m 1.87, d (6.5) 2.90, ddd (16.0, 9.6, 7.6) 2.43, ddd (16.0, 9.6, 7.6) 3.83−3.88, m 0.90, t (7.4) 2.82, dq (7.2, 6.8) 4.26−4.30, m 1.32, d (6.4) 1.25, d (7.2) 7.12, dq (7.2, 1.2) 1.49, d (7.2) 1.85, brs

HMBC spectrum (Figure S14, Supporting Information): H-2 (δH 4.42) of Qui with C-1 (δC 99.9) of Rha; H-4 (δH 3.53) of Rha with C-1 (δC 106.6) of Qui′; H-2 (δH 4.05) of Qui′ with C-1 (δC 99.7) of Rha″; H-1 (δH 5.85) of Rha′ with C-3 (δC 81.5) of Qui′; and H-1 (δH 4.77) of Qui with C-11 (δC 81.6) of convolvulinolic acid. The observed HMBC long-range correlations (2,3JCH) were used to support the macrolactonization site and the positions of esterification (Figure S14, Supporting Information). The following key correlations were

position

1

2

Qui-1 2 3 4 5 6 Glc-1 2 3 4 5 6 Rha-1 2 3 4 5 6 Rha′-1 2 3 4 5 6 Qui′-1 2 3 4 5 6 Rha″-1 2 3 4 5 6 Conv-1 2 11 14 nla-1 2 3 4 5 tga-1 2 3 4 5

103.5 78.7 78.9 76.6 72.3 18.2 101.2 77.9 75.4 70.0 76.5 69.7 100.2 71.7 74.2 77.6 67.3 17.4 101.1 71.9 70.1 76.1 70.0 18.1 104.4 81.3 77.7 76.7 72.5 18.4

102.9 77.4 80.8 79.3 77.7 18.6

173.5 33.2 81.5 14.3

167.7 129.5 138.4 14.6 12.6

99.9 72.9 74.2 75.7 72.3 18.7 100.1 73.4 72.2 85.5 68.6 19.8 106.6 70.5 81.5 73.2 72.9 18.6 99.7 73.6 72.4 79.4 70.5 18.2 172.9 34.1 81.6 14.6 174.5 48.9 69.3 19.6 12.7 167.9 129.3 138.4 14.7 12.7

observed: between H-3 (δH 5.50) of Rha′ and the carbonyl resonance at δc 172.9, assigned to the lactone functionality due to its 2JCH coupling with the diastereotopic C-2 methylene protons (δH 2.90 and 2.43); H-2 of the terminal branched rhamnose (δH 5.85, Rha″) and the carbonyl group of the niloyl moiety (δC 174.5); H-2 of the second saccharide unit (δH 5.77, Rha) and the carbonyl carbon for the tigloyl residue at δC 167.9. D



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Table 3. 1H NMR Spectroscopic Data for Albinosides VI−IX (3−6) (Measured at 500 MHz in C5D5N, δ in ppm, J in Hz) position Qui-1 2 3 4 5 6 Glc-1 2 3 4 5 6a 6b Qui′-1 2 3 4 5 6 Qui″-1 2 3 4 5 6 Rha-1 2 3 4 5 6 Fuc-1 2 3 4 5 6 Conv 2a 2b 11 14 Jal-2a 2b 11 16 ace-2 nla-2 3 4 5 tga-3 4 5 tga′-3 4 5

3 4.77, 4.31, 4.13, 3.76, 3.68, 1.46, 5.75, 4.11, 3.87, 3.67, 3.78, 4.14, 4.36, 5.07, 5.66, 5.66, 4.33, 3.64, 1.59,

5.80, 4.64, 4.78, 5.87, 5.21, 1.62,

3.33, 2.76, 3.77, 0.92,

d (8.0) dd (9.0, 8.0) dd (8.8, 8.0) dd (8.8, 8.0) dq (8.8, 5.6) d (6.0) d (7.6) t (8.0) m dd (9.6, 9.2) ddd (9.2, 6.0, 3.0) m dd (9.2, 3.0) d (8.0) dd (9.0, 8.0) dd (9.0, 9.0) dd (9.0, 8.0) dq (9.0, 6.0) d (6.0)

d (1.5) brs dd (9.0, 3.2) t (9.6) dq (10.0, 6.0) d (6.4)

ddd (16.4, 12.4, 2.8) ddd (16.4, 12.4, 2.8) m t (7.2)

4 4.78, 4.28, 4.28, 3.62, 3.66, 1.62,

5

d (8.0) dd (8.8, 8.0) dd (8.8, 8.0) m dq (9.0, 6.0) d (6.0)

5.64, d (8.0) 4.05, dd (9.0, 4.03, dd (9.0, 3.49, m 3.69, dq (9.2, 1.44, d (5.8) 5.04, d (8.0) 5.66, dd (9.0, 5.66, dd (9.0, 3.67, m 3.49, dq (9.0, 1.47, d (6.0) 5.83, brs 4.62, brs 4.75, dd (9.0, 5.86, t (8.0) 5.19−5.23, m 1.60, d (6.4)

6

4.75, 4.45, 4.27, 3.70, 3.54, 1.58, 5.98, 4.21, 5.82, 4.44, 3.84, 4.25, 4.25,

d (8.0) dd (9.2, m dd (9.6, dq (9.0, d (6.0) d (7.6) dd (9.2, dd (9.2, dd (9.2, dd (9.2, dd (9.2, dd (9.2,

5.67, 5.88, 4.81, 4.36, 4.78, 1.92, 5.28, 4.34, 4.23, 5.65, 3.95, 1.31, 2.83, 2.44, 3.85, 0.88,

brs m dd (9.0, 3.0) dd (9.6, 8.8) dq (8.8, 6.4) d (6.4) d (7.6) dd (9.2, 7.6) dd (9.2, 3.0) d (3.0) q (6.0) d (6.4) ddd (10.4, 7.6, 2.0) ddd (10.4, 7.6, 2.0) m t (7.2)

8.0) 9.0) 6.0)

7.6) 8.8) 9.2) 6.0) 6.0) 3.0)

4.76, 4.44, 4.28, 3.85, 3.63, 1.59, 5.99, 4.21, 5.84, 4.50, 3.86, 4.26, 4.26,

d (8.0) dd (9.2, dd (9.6, dd (9.6, dq (9.0, d (6.0) d (7.6) dd (9.2, dd (9.2, dd (9.2, dd (9.2, dd (9.2, dd (9.2,

5.67, 5.88, 4.23, 4.34, 4.78, 1.92, 5.29, 4.32, 4.31, 5.64, 3.92, 1.31,

brs d (2.0) dd (9.0, dd (9.6, dq (8.8, d (6.4) d (7.6) dd (9.2, dd (9.2, d (3.0) q (6.0) d (6.4)

8.0) 8.0) 9.0) 6.0)

7.6) 8.8) 9.2) 6.0) 6.0) 3.0)

8.0) 9.0) 5.8)

8.0) 9.0) 6.0)

2.4)

3.35, ddd (16.0, 12.4, 2.8) 2.76, ddd (16.0, 12.4, 2.8) 3.78−3.80, m 0.93, t (7.2)

3.0) 8.8) 6.4)

7.6) 3.0)

2.84, ddd (16.4, 9.0, 8.0) 2.43, ddd (16.4, 9.0, 8.0) 3.80−3.86, m 0.86, t (6.8) 2.05, 2.83, 4.14, 1.34, 1.24,

s dq (7.2, 7.1) m d (6.4) d (7.2)

2.06, s 2.79, dq (7.2, 7.1) 4.26−4.30, m 1.33, d (6.4) 1.24, d (7.2) 6.97, 1.46, 1.79, 7.12, 1.49, 1.82,

Albinoside VI (3) gave a sodium adduct ion at m/z 991.4726 [M + Na]+ in the HRMALDITOFMS corresponding to the molecular formula C45H76O22Na (calcd error: δ = +0.6 ppm).

dq (7.2, 1.6) d (7.2) brs dq (7.2, 1.2) d (7.2) brs

6.99, 1.49, 1.81, 7.12, 1.46, 1.86,

dq (8.0, 1.6) d (7.2) brs dq (7.2, 1.2) d (7.2) brs

The saponification of 3 released a mixture of acids, which was collected in Et2O. This mixture was analyzed by GC-MS and allowed the identification of acetic and nilic acids. The H2OE



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Table 4. 13C NMR Spectroscopic Data for Albinosides VI−IX (3−6) (Measured at 125 MHz in C5D5N, δ in ppm) position

3

4

5

6

position

Qui-1 2 3 4 5 6 Glc-1 2 3 4 5 6 Qui′-1 2 3 4 5 6 Qui″-1 2 3 4 5 6 Rha-1 2 3 4 5 6 Fuc-1

101.4 76.5 85.7 78.2 77.9 18.5 102.6 79.1 70.6 75.2 77.5 63.2 104.4 72.3 76.5 80.3 73.9 19.2

103.7 76.8 79.6 77.5 72.7 18.9

103.2 77.5 79.3 77.8 73.4 19.5 100.6 79.6 80.1 70.4 77.9 62.5

103.2 77.5 79.6 77.7 73.4 19.2 100.6 79.6 79.9 70.5 77.8 62.6

2 3 4 5 6 Conv-1 2a 11 14 Jal-1 2 11 16 ace-1 2 nla-1 2 3 4 5 tga-1 2 3 4 5 tga′-1 2 3 4 5

102.3 72.9 71.1 77.0 67.6 19.0

101.8 78.9 77.7 74.8 74.9 18.8 100.9 72.4 76.1 72.6 72.3 18.0 101.7 72.5 70.7 76.8 67.2 18.7

99.8 74.3 78.1 86.2 68.9 19.9 107.1

99.8 73.4 81.6 86.2 68.8 19.5 107.1

3

174.6 34.3 82.1 15.3

4

5

6 74.2 79.9 75.1 70.4 17.7

174.3 34.2 81.3 14.3

74.5 79.9 75.1 71.0 17.9 173.3 35.4 81.1 15.2

173.3 35.4 81.6 14.7 171.7 21.9 175.7 49.5 69.1 21.4 13.9

171.3 21.3 175.2 49.0 69.3 21.7 14.8 168.2 129.7 138.4 14.8 13.1 168.8 129.6 138.7 14.9 13.1

168.1 129.7 138.4 14.9 13.1 168.8 129.6 138.8 14.8 13.1

spectra of compound 3, four anomeric signals were confirmed at δH 4.77 (1H, d, J = 7.0 Hz; δC 101.4, Qui-1); 5.75 (1H, d, J = 7.6 Hz; δC 102.6, Glc-1); 5.07 (1H, d, J = 7.6 Hz; δC 104.4, Qui′-1); and 5.80 (1H, d, J = 1.2 Hz; δC 102.3, Rha-1). Therefore, four separate spin systems for the sugar skeletons were readily distinguished in the 1H−1H COSY and TOCSY spectra. The following key 3JCH correlations in the HMBC spectrum confirmed the glycosylation sequence (Figure 1): between C-1 (δC 101.4) of Qui and H-11 (δH 3.77) of the

soluble residue, albinosinic acid D (10), was identified as a tetrasaccharide of 11-hydroxytetradecanoic acid by ESIMS in the positive mode with a potassium adduct ion of high abundance at m/z 883 [M + K]+ and FABMS in the negative mode with a deprotonated molecule ion at m/z 843 [M − H]− and diagnostic ions at m/z 697 [843 − 146]−, 551 [697 − 146]−, 389 [551 − 162]−, and 243 [389 − 146]−.7a The liberated water-soluble sugars from 10 by an acid-catalyzed procedure underwent derivatization with L-cysteine to form thiazolidines, which were identified by GC-MS as their TMS ethers as quinovose, rhamnose, and glucose in a ratio of 2:1:1.7a This sugar analysis also confirmed the absolute configuration for the monosaccharides as the L-series for rhamnose and the Dseries for quinovose and glucose. For compound 3, the negative-ion FABMS allowed the identification of a deprotonated molecule at m/z 967 [M − H]− (Figure S15, Supporting Information). The initial loss of 100 mass units at m/z 867 [M − H − C5H8O2]− confirmed the presence of a niloyl residue. The consecutive loss of a methylpentose unit and a ketene (42 amu) generated the peak at m/z 679 [867 − C2H2O (acetyl) − C6H10O4]−. The rest of the fragmentation pattern at m/z 533 [679 − C6H10O4]−, 551 [679 + H2O − C6H10O4]−, 389 [551 − C6H 10O 5 (hexose unit)]−, and 243 [389 − C6H 10O 4]− confirmed the additional loss of two methylpentoses and one hexose unit. All proton and carbon signals (Tables 3 and 4; Figures S16 and S17, Supporting Information) were assigned sequentially by COSY (Figure S18, Supporting Information) and HSQC NMR studies. In the low-field region of the HSQC

Figure 1. Key HMBC correlations for compound 3 showing connectivities (3JCH) for anomeric carbons: (A) C-1 Qui/H-11 Conv; (B) C-1 Glc/H-2 Qui; (C) C-1 Rha/H-2 Glc; (D) C-1 Qui′/H-6 Glc (unlabeled connectivity: C-1/H-5 Qui′). F



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ppm) in the HRESIMS, in contrast to the ion [M − H]− at m/z 1017.5265 (C50H81O21, calcd error: δ = −1.08 ppm) for albinoside IX (6), indicating a difference of two methylene groups (28 atomic mass units) between these two compounds. Thus, these results suggested the presence of convolvulinolic acid (11S-hydroxytetradecanoic acid) as the aglycone for 5 and jalapinolic acid (11S-hydroxyhexadecanoic acid) as the aglycone for 6.7a,11 In the negative FABMS, peaks from the consecutive elimination for two tigloyl residues at m/z 907 [M − H − 82 (C5H6O)]− and 825 [907 − C5H6O]− were also observed for 5 (Figure S25, Supporting Information). The consecutive elimination of each sugar unit at m/z 679 [825 − C6H10O4 (methylpentose unit)]−, 533 [679 − C6H10O4 (methylpentose unit)]−, 389 [533 + H2O − C6H10O5 (hexose unit)]−, and 243 [389 − C6H10O4 (methylpentose unit)]− confirmed a tetrasaccharide of a convolvulinolic acid moiety for albinoside VIII (5). The difference of 28 mass units (two methylene groups) between compounds 5 and 6, as well as the production of the same general fragmentation pattern by glycosidic cleavage of each sugar moiety at m/z 935, 853, 707, 561, 417, and 271, confirmed the similar linear tetrasaccharide core in both acids and the presence of jalapinolic acid as the aglycone for compound 6 (Figure S30, Supporting Information).11 Pure compounds 5 and 6 were each saponified, and their Et2O-soluble fraction was analyzed by GC-MS. Both compounds released 2-methyl-2-butenoic acid. The H2Osoluble residue from compound 5 gave albinosinic acid F (12), which was identified as a tetrasaccharide of 11Shydroxytetradecanoic acid by positive ESIMS at m/z 883 [M + K]+ and in the negative mode with a deprotonated molecule at m/z 843 [M − H]− and diagnostic ions at m/z 697, 551, 389, and 243; compound 6 gave albinosinic acid G (13), which was characterized as a tetrasaccharide of 11S-hydroxyhexadecanoic acid by ESIMS and FABMS in the negative mode: m/z 871 [M − H]−, 725 [871 − 146]−, 579 [725 − 146]−, 417 [579 − 162] − , and 271 [417 − 146] −. The analysis of the monosaccharide mixtures obtained by acid hydrolysis was performed by GC-MS with their thiazolidine derivatives of Lcysteine as TMS ethers. The same equimolecular sugar composition for both compounds 12 and 13 was recognized: D-quinovose, L-rhammnose, D-fucose, and D-glucose. In the lowfield region of the HSQC spectrum of compound 5, four anomeric signals were observed at δH 4.75 (1H, d, J = 8.0 Hz; δC 103.2, Qui-1); 5.98 (1H, d, J = 7.6 Hz; δC 100.6, Glc-1); 5.67 (1H, brs; δC 99.8, Rha-1), 5.28 (1H, d, J = 7.6 Hz; δC 107.1, Fuc-1) (Tables 3 and 4; Figures S26 and S27, Supporting Information). An almost identical HSQC spectrum was registered for compound 6 with four anomeric signals centered at δH 4.76 (1H, d, J = 7.6 Hz; δC 103.2, Qui-1); 5.99 (1H, d, J = 7.6 Hz; δC 100.6, Glc-1); 5.67 (1H, brs; δC 99.8, Rha-1); and 5.29 (1H, d, J = 7.6 Hz; δC 107.1, Fuc-1) (Tables 3 and 4; Figures S31 and S32, Supporting Information). The interglycosidic connectivities were established on the basis of detailed long-range heteronuclear coupling correlations (3JCH) by HMBC studies. For example, the following key correlations were observed in compounds 5 and 6 (Figures S29 and S34, Supporting Information): (a) the connectivity between H-1 of Qui (5, δH 4.75; 6, δH 4.76) and C-11 of the fatty acid (5, δC 81.1; 6, δC 81.6); (b) H-2 of Qui (5, δH 4.45; 6, δH 4.44) with C-1 of Glc (δC 100.6); (c) H-2 of Glc (δH 4.21) with C-1 of Rha (δC 99.8); and (d) H-4 of Rha (5, δH 4.36; 6, δH 4.34) with C-1 of Fuc (δC 107.1). Accordingly, albinosinic acid F (12) corresponded to (11S)-hydroxytetradecanoic acid 11-O-β-D-

aglycone; H-1 (δH 4.77) of Qui and C-11 of the aglycone (δC 82.1, Conv); H-2 (δH 4.31) of Qui and C-1 (δC 102.6) of Glc; H-6 (δH 4.36) of Glc and C-1 (δC 104.4) of Qui′; C-1 (δC 102.3) of Rha and H-2 (δH 4.11) of Glc (Figure S19, Supporting Information). The lactonization and acylation positions for 3 were established by the following HMBC correlations: between H-2 (δH 5.66) of Qui′ and the carbonyl resonance at δc 174.6, assigned to the lactone functionality due to its 2JCH coupling with the diastereotopic C-2 methylene protons (δH 3.33 and 2.76); H-3 (δH 5.66) of Qui′ and the carbonyl group of the niloyl moiety (δC 175.7); and H-4 of the terminal branched rhamnose (δH 5.87, Rha) and the carbonyl group of the acetyl moiety (δC 171.7). Consequently, the structure of albinosinic acid D (10) corresponded to (11S)convolvulinolic acid 11-O-α-L-rhamnopyranosyl-(1→2)-O-[6deoxy-β-D-glucopyranosyl-(1→6)]-O-β-D-glucopyranosyl-(1→ 2)-O-6-deoxy-β-D-glucopyranoside. Albinoside VII (4) showed a sodium adduct at m/z 975.4777 [M + Na]+ in the HRMALDITOFMS, consistent with a molecular formula of C45H75O21Na (calcd error: δ = +0.6 ppm), indicating the difference of one oxygen (16 atomic mass units) between this compound and albinoside VI (3). In the negative FABMS (m/z 951 [M − H]−) (Figure S20, Supporting Information), the consecutive losses of one niloyl residue at m/z 851 [M − H − C5H8O2]−, one acetyl residue at m/z 809 [851 − C2H2O]−, and four methylpentoses at m/z 663, 517, 371, and 243 indicated that compound 4 is a tetraglycoside of convolvulinolic acid. Saponification of 4 liberated acetic and nilic acids, which were identified by GCMS of the organic solvent-soluble fraction. The H2O-soluble residue from compound 4 gave albinosinic acid E (11), which was identified as a tetrasaccharide of 11-hydroxytetradecanoic acid by positive FABMS at m/z 851 [M + Na]+; in negative mode ESIMS, a deprotonated molecule at m/z 827 [M − H]− with diagnostic ions at m/z 681, 535, 439, and 243 was observed. In the low-field region of the HSQC spectrum of compound 4, four anomeric signals were observed at δH 4.78 (1H, d, J = 8.0 Hz; δC 103.7, Qui-1); 5.64 (1H, d, J = 8.0 Hz; δC 101.8, Qui′-1); 5.04 (1H, d, J = 8.0 Hz; δC 100.9, Qui″-1); and 5.83 (1H, brs; δC 101.7, Rha-1). COSY (Figure S23, Supporting Information), and HSQC NMR studies were used to assign the 1H and 13C NMR spectra (Tables 3 and 4; Figures S21 and S22, Supporting Information). The following key 3JCH correlations in the HMBC spectrum confirmed the glycosylation sequence: between H-1 (δH 4.78) of Qui and C-11 of the aglycone (δC 81.3, Conv); H-2 (δH 4.28) of Qui and C-1 (δC 101.8) of Qui′; H-3 (δH 4.03) of Qui′ and C-1 (δC 100.9) of Qui″; and H-1 (δH 5.83) of Rha and C-2 (δC 78.9) of Qui′ (Figure S24, Supporting Information). The locations for the lactonization and the esterifications were established by the following HMBC correlations: between H-2 (δH 5.66) of Qui″ and the carbonyl resonance at δc 174.3, assigned to the lactone functionality due to its 2JCH coupling with the diastereotopic C2 methylene protons (δH 3.35 and 2.76); H-3 (δH 5.66) of Qui″ and the carbonyl group of the niloyl moiety (δC 175.2); and H4 of the terminal branched rhamnose (δH 5.86, Rha) and the carbonyl group of the acetyl moiety (δC 171.3). Therefore, the structure of albinosinic acid E (11) corresponded to (11S)convolvulinolic acid 11-O-α-L-rhamnopyranosyl-(1→2)-O-[6deoxy-β-D-glucopyranosyl-(1→3)]-O-6-deoxy-β-D-glucopyranosyl-(1→2)-O-6-deoxy-β-D-glucopyranoside. Albinoside VIII (5) yielded a deprotonated molecule peak at m/z 989.4998 [M − H]− (C48H77O21, calcd error: δ = +0.35 G



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Table 5. Cytotoxicity of Albinosides IV−IX (1−6)a IC50 (μM) compound

MCF-7 sens

MCF-7/Vin−

MCF-7/Vin+

MDA

HeLa

HCT15

HCT-116

1 2 3 4 5 6 vinblastine adriamycin camptothecin colchicine ellipticine podophyllotoxin

>20 >20 >20 >20 15.1 8.0 0.06 1.3 20 >20 >20 >20 >20 16.3 1.1 9.2 20 >20 >20 >20 >20 17.0 1.3 5.3 20 >20 >20 >20 >20 11.6 0.01 NT NT NT NT NT

>20 >20 >20 >20 17.4 9.6 0.002 NT NT NT NT NT

>20 >20 >20 >20 14.3 9.8 0.06 NT NT NT NT NT

>20 >20 >20 >20 15.1 15.9 0.05 NT NT NT NT NT

a

MCF-7 = breast carcinoma (sensitive MCF-7 cells and multidrug-resistant MCF-7/Vin cells, MCF-7/Vin+ cells were cultured in medium containing 0.192 μg/mL vinblastine; at the same time, a stock of MCF-7/Vin− cells was maintained in vinblastine-free medium); MDA-MB-231 = triple-negative breast cancer; HeLa = cervix carcinoma, HCT-15 = colon carcinoma; HCT-116 = colon carcinoma; NT = not tested.

Table 6. Modulation of Vinblastine Cytotoxicity in Drug-Sensitive MCF-7 and Multidrug-Resistant MCF-7/Vin by Albinosides IV−IX (1−6) reversal foldc

IC50 (μM) compounda

MCF-7/Vin−

MCF-7/Vin+

MCF-7 sens

RFMCF‑7/Vin−

RFMCF‑7/Vin+

RFMCF‑7 sens

vinblastine 1 2 3 4 5 6 reserpineb

1.2 ± 0.1 0.0007 ± 0.0002 0.0042 ± 0.001 1.32 ± 0.15 0.0037 ± 0.01 0.25 ± 0.03 87 11

Serial dilutions from 0.0007 to 15 μM vinblastine in the presence or absence of glycolipid (25 μg/mL). bReserpine = 5 μg/mL as positive control. RF = IC50 vinblastine/IC50 vinblastine in the presence of glycolipid. Each value represents the mean ± SD from three independent experiments.

a c

(BCRP/ABCG2), and comprised anthracyclines (adriamycin), podophyllotoxin derivatives, tropolone-like alkaloids (colchicine), indole alkaloids (reserpine), and other polyaromatic small-molecule DNA intercalators (ellipticine).2b The lack of cytotoxicity (IC50 > 20 μM) of the tested samples is an essential requirement to distinguish between a possible synergism and the real cytotoxic potentiation in the monolayer modulation assay through their reversal effects. On the basis of our previous results of drug uptake inhibition by resin glycosides with an anti-P-gp monoclonal antibody and rhodamine 123 in MDR MCF-7/Vin cells,6,7b the modulation assay was used to identify chemosensitizers through the potentiation of vinblastine susceptibility in MDR cells. This assay employed parental or vinblastine-sensitive (MCF-7 sens) and vinblastine-resistant (MCF-7/Vin− and MCF-7/Vin+) human breast cancer cells (Table 6). The reversal fold value (RFMCF‑7/Vin+), as a parameter of potency, was calculated from dividing the IC50 of vinblastine alone by the IC50 of vinblastine in the presence of test compounds.6 This simple bioassay has allowed us to identify murucoidin V (RFMCF‑7/Vin+ 255),6 albinoside III,7a purgin II,7b and jalapinosides I and II7c,d (RFMCF‑7/Vin+ > 2140), which proved to be substrates of P-gp in effluxing experiments.6,7b The noncytotoxic albinosides VII (4) and VIII (5) exerted the strongest potentiation of vinblastine susceptibility with a reversal factor (RFMCF‑7/Vin+) over 201- and >2517-fold, respectively. This potency in the reversal of the susceptibility to vinblastine was even better than the activity of

fucopyranosyl-(1→4)-O-α-L-rhamnopyranosyl-(1→2)-O-β-Dglucopyranosyl-(1→2)-O-6-deoxy-β-D-glucopyranoside, while the structure for albinosinic acid G (13) was characterized as (11S)-hydroxyhexadecanoic acid 11-O-β-D-fucopyranosyl-(1→ 4)-O-α-L-rhamnopyranosyl-(1→2)-O-β-D-glucopyranosyl-(1→ 2)-O-6-deoxy-β-D-glucopyranoside. For the natural products, the location of the macrolactonization by the aglycone, i.e, convolvulinolic acid for 5 and jalapinolic acid for 6, on the oligosaccharide core was determined by the observed 3JCH correlations between H-3 (5, δH 5.82; 6, δH 5.84) of glucose with C-1 (δC 173.3) of the fatty acid in the HMBC spectrum. The positions of acylation were also determined by 3JCH correlations between the carbonyl carbons and their corresponding geminal proton on the oligosaccharide core, indicating the same position for the two tigloyl residues in 5 and 6. Thus, one of the tigloyl residues (5, δC 168.2; 6, δC 168.1) was attached at C-2 (δH 5.88) of Rha, and the second one (δC 168.8) was linked at C-4 (5, δH 5.65; 6, δH 5.64) of the terminal fucose. Cytotoxicity screening using the sulforhodamine B method14 was evaluated, and data are presented in Table 5. All glycolipids were tested as chemosensitizers in vinblastine-resistant MCF-7/ Vin+ cells by the same method (Table 6). The cross-resistance profile displayed by the vinblastine MCF-7/Vin cells (Table 5) was consistent with the MDR expressed by the P-glycoprotein (MDR protein1/P-gp, ABCB1) and other transmembrane efflux pumps, such as the breast cancer resistance protein H



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at 37 °C in an atmosphere of 5% CO2 in air (100% humidity). To maintain drug resistance, MCF-7/Vin+ cells were cultured in medium containing 0.192 μg/mL vinblastine. At the same time, a stock of MCF-7/Vin cells was maintained in vinblastine-free medium (MCF-7/ Vin−). Plant Material. Seeds of moon vine (Ipomoea alba; item #01052PK-P1) were acquired from Park Seed (Greenwood, SC, USA) in March 2011. For authentication purposes, 10 seeds were germinated in soil, and five seedlings were grown to maturity in an environmental growth chamber under the following conditions: 25 °C, 65% humidity, 16:8 h photoperiod, and 150 μmol/m2/s light intensity. Voucher specimens were identified by Dr. Travis D. Marsico and deposited at the Arkansas State University Herbarium (STAR 027009). Extraction and Isolation. Dried seeds were milled (400 g) and exhaustively extracted by maceration at room temperature with hexane and then with CHCl3 to yield two extracts after removal of the solvents: an oily residue (11 g) and a dark syrup (8.2 g). The resin glycoside crude mixture of the CHCl3-soluble extract was obtained as a white solid (6.8 g) by precipitation with MeOH. Then, this crude was analyzed by reversed-phase C18 (Waters; 7 μm, 19 × 300 mm) HPLC using an isocratic elution with MeOH−CH3CN−H2O (5:4:1), at a flow rate of 4 mL/min, sample injection of 500 μL, concentration of 0.1 mg/μL. Comparison with reference compounds confirmed the presence of previously reported resin glycosides: albinoside I (7) (23 min, peak I), albinoside II (8) (99.5 min, peak VIII), albinoside III (9) (128 min, peak IX). Eluates across the peaks with tR values of 30.0 min (peak II), 38.2 min (peak III), 44.6 min (peak IV), 49.6 min (peak V), 63.1 min (peak VI), and 109.1 min (peak VII) were collected by the heart-cutting technique.10 Each subfraction was independently reinjected (sample injection, 500 μL; concentration, 0.1 mg/μL) and purified by preparative-scale recycling HPLC10 to achieve total homogeneity between 10 and 20 consecutive cycles employing a Symmetry C18 column (Waters; 7 μm, 19 × 300 mm), isocratic elution with MeOH−CH3CN−H2O (10:7:3), and a flow rate of 8 mL/min for the first subfraction. For the remaining fractions, isocratic elution with MeOH−CH3CN (7:3) with a flow rate of 8.5 mL/min was used. These procedures afforded pure compounds 1 (10 mg; tR 28.2 min) from peak III, 2 (10 mg; tR 7.9 min) from peak V, 3 (16.3 mg; tR 16.3 min) from peak II, 4 (28 mg; tR 20.0 min) from peak IV, 5 (23.4 mg; tR 9.05 min) from peak VI, and 6 (39.1 mg; tR 9.7 min) from peak VII. Albinoside IV (1): white powder; mp 143−146 °C; [α]589 −31.4, [α]578 −32.9, [α]546 −36.4, [α]436 −60.0, [α]365 −90.0 (c 1.0, MeOH); 1 H and 13C NMR, see Tables 1 and 2; negative FABMS m/z 1053 [M − H]−, 971 [M − H − C5H6O (tigloyl)]−, 907 [971 − H − H2O − CO2H]−, 825 [971 − C6H10O4 (methylpentose unit)]−, 679 [825 − C6H10O4 (methylpentose unit)]−, 533 [679 − C6H10O4 (methylpentose unit)]−, 389 [533 + H2O − C6H10O5 (hexose unit)]−, 243 [389 − C6H10O4 (methylpentose unit)]−; HRESIMS m/z 1053.5139 [M − H]− (calcd for C49H81O24 requires 1053.5123). Albinoside V (2): white powder; mp 136−140 °C; [α]589 −17.5, [α]578 −18.3, [α]546 −20.8, [α]436 −32.5, [α]365 −47.5 (c 1.0, MeOH); 1 H and 13C NMR, see Tables 1 and 2; negative FABMS m/z 1137 [M − H]−, 1037 [M − H − C5H8O2 (niloyl)]−, 891 [1037 − C6H10O4 (methylpentose unit)]−, 809 [891 − C5H6O (tigloyl)]−, 791 [809 − H 2 O] − , 745 [791 − H − CO 2 H] − , 663 [809 − C 6 H 10 O 4 (methylpentose unit)]−, 517 [663 − C6H10O4 (methylpentose unit)]−, 389 [517 + H2O − C6H10O4 (methylpentose unit)]−, 243 [389 − C6H10O4 (methylpentose unit)]−; HRESIMS 1137.5712 m/z [M − H]− (calcd for C54H89O25 requires 1137.5698). Albinoside VI (3): white powder; mp 148−152 °C; [α]589 −84.0, [α]578 −78.0, [α]546 −87.0, [α]436 −131.0, [α]365 −183.0 (c 1.0, MeOH); 1H and 13C NMR, see Tables 3 and 4; negative FABMS m/z 967 [M − H]−, 867 [M − H − C5H8O2 (niloyl)]−, 679 [867 − C2H2O (acetyl) − C6H10O4 (methylpentose unit)]−, 551 [679 + H2O − C 6 H 10 O 4 (methylpentose unit)] − , 533 [679 − C 6 H 10 O 4 (methylpentose unit)]−, 389 [551 − C6H10O5 (hexose unit)]−, 243 [389 − C6H10O4 (methylpentose unit)]−; HRMALDITOFMS m/z 991.4726 [M + Na]+ (calcd for C45H76O22Na requires 991.4720). Albinoside VII (4): white powder; mp 142−145 °C; [α]589 −103.8, [α]578 −86.9, [α]546 −99.2, [α]436 −147.7, [α]365 − 206.2 (c 1.0,

reserpine, a cytotoxic positive efflux pump control (Table 6), and displayed the same potency as the previously reported value for albinoside III (9, RF > 2140).7a Moderate activities were observed for albinosides V (2) (RF 3) and VI (3) (RF 2), which were similar to those reported for albinosides I (7, RF 3) and II (8, RF 3).7a Albinoside IV (1) did not exhibit a strong modulation of cytotoxicity in vinblastine-resistant MCF-7/Vin+ cells (RF 3). However, it modulated the MDR phenotype in MCF-7/Vin− cells with a reversal factor of 1700. Albinoside IX (6) was the only active compound against a variety of tumor cell lines (Table 5). Therefore, its reversal activity (RF > 2517) is the result of an additive synergism that could be substantial from a therapeutic perspective. As in previous reports,5−7 there is no evidence of the relationship between chemical structure and modulatory activity since minor variations in the acylation pattern of the oligosaccharide cores could affect their MDRreversal activities. The cross activity displayed among members of the albinoside series could represent an example of synergy between related components in crude extracts, with inactive cytotoxic compounds disabling a resistance mechanism, e.g., efflux pump-expressing cells, therefore, potentiating the activity of cytotoxic substances by the modulation of transporters that confer MDR through competing with toxins for binding to the efflux pump active site. Resin glycosides represent a new class of amphipathic relatively high molecular weight MDR modulators that deserve further biochemical investigations of their mechanism of action as chemosensitizers for their low cytotoxicity and potent selective behavior that could be used to identify effective therapeutic drug combination and lower their current doses, thereby decreasing toxic side effects in refractory malignancies. Our results suggest that convolvulaceous plants elaborate an array of amphipathic bioactive oligosaccharides, of which many have evolved to confer selective advantage to plants. This evolutionary process may have potential in the discovery of new MDR-modifying leads from plant sources.

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EXPERIMENTAL SECTION

General Experimental Procedures. Melting points were determined on a Fisher-Johns apparatus and are uncorrected. Optical rotations were measured with a PerkinElmer model 341 polarimeter. 1 H (500 and 400 MHz) and 13C (125.7 and 100 MHz) NMR experiments were conducted on a Varian Inova instrument. Negativeion LRFABMS were recorded using a matrix of triethanolamine on a Thermo DFS spectrometer. Negative-ion HRESIMS experiments were performed on a Bruker MicrOTOF-Q high-resolution quadruple-timeof-flight mass spectrometer according to the procedure previously described.6a The instrumentation used for HPLC analysis consisted of a Waters (Millipore Corp., Waters Chromatography Division, Milford, MA, USA) 600E multisolvent delivery system equipped with a refractive index detector (Waters 410). Control of the equipment, data acquisition, processing, and management of the chromatographic information were performed by the Empower 2 software (Waters). GC-MS was performed on a Thermo-Electron instrument coupled to a Thermo-Electron spectrometer using the conditions previously described in the preceding article on the chemistry of moon vine seeds.7a RPMI 1640 medium and fetal bovine serum were purchased from Gibco (Life Technologies, Carlsbad, CA, USA), and sulforhodamine B, reserpine, and vinblastine from Sigma-Aldrich (St. Louis, MO, USA). Colon (HCT-15 and HCT-116), cervix (HeLa), and breast (MCF-7 and MDA-MB-231) carcinoma cell lines were acquired from the American Type Culture Collection. The resistant counterpart MCF-7/Vin was developed and subcultured during five consecutive years, as previously reported.6 All cell lines were maintained in RPMI 1640 medium supplemented with 10% fetal bovine serum and cultured I



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MeOH); 1H and 13C NMR, see Tables 3 and 4; negative FABMS m/z 951 [M − H]−, 851 [M − H − C5H8O2 (niloyl)]−, 809 [851 − C2H2O (acetyl)]−, 663 [809 − C6H10O4 (methylpentose unit)]−, 535 [663 + H2O − C6H10O4 (methylpentose unit)]−, 517 [663 − C6H10O4 (methylpentose unit)]−, 389 [535 − C6H10O4 (methylpentose unit)]−, 371 [517 − C6H10O4 (methylpentose unit)]−, 243 [389 − C6H10O4 (methylpentose unit)]−; HRMALDITOFMS m/z 975.4777 [M + Na]+ (calcd for C45H76O21Na requires 975.4771). Albinoside VIII (5): white powder; mp 132−135 °C; [α]589 −20.0, [α]578 −21.6, [α]546 −23.2, [α]436 −36.8, [α]365 −53.7 (c 1.0, MeOH); 1 H and 13C NMR, see Tables 3 and 4; negative FABMS m/z 989 [M − H]−, 907 [M − H − C5H6O (tigloyl)]−, 825 [907 − C5H6O (tigloyl)]−, 679 [825 − C6H10O4 (methylpentose unit)]−, 533 [679 − C6H10O4 (methylpentose unit)]−, 389 [533 + H2O − C6H10O5 (hexose unit)]−, 243 [389 − C6H10O4 (methylpentose unit)]−; HRESIMS m/z 989.4998 [M − H]− (calcd for C48H77O21 requires 989.4963). Albinoside IX (6): white powder; mp 132−135 °C; [α]589 −9.0, [α]578 −9.5, [α]546 −11.3, [α]436 −15.9, [α]365 −19.5 (c 1.0, MeOH); 1 H and 13C NMR, see Tables 3 and 4; negative FABMS m/z 1017 [M − H]−, 935 [M − C5H6O (tigloyl)]−, 853 [935 − C5H6O (tigloyl)]−, 707 [853 − C6H10O4 (methylpentose unit)]−, 561 [707 − C6H10O4 (methylpentose unit)]−, 417 [561 + H2O − C6H10O5 (hexose unit)]−, 271 [417 − C6H10O4 (methylpentose unit)]−; HRFABMS m/z 1017.5265 [M − H]− (calcd for C50H81O21 requires 1017.5276). Alkaline Hydrolysis of Compounds 1−6. Individual solutions of compounds 1−6 (10 mg for each one) in 5% KOH−H2O (1 mL) were refluxed at 95 °C for 3 h. Then, the reaction mixtures were acidified to pH 5.0 and extracted with CHCl3 (2 × 5 mL) and Et2O (2 × 5 mL). The organic layer was washed with H2O, dried over anhydrous Na2SO4, evaporated under reduced pressure, and directly analyzed by CG-MS and comparison of their spectra and retention times with those of authentic samples.11 All analytical standards were purchased with a purity of >97%: acetic acid (240168, Aldrich); 3hydroxy-2-methylbutanoic acid (209603, Santa Cruz Biotechnology); tiglic acid (89450, Sigma-Aldrich). For compounds 3 and 4 acetic acid (tR 2.81 min) was detected: m/z [M]+ 60 (65), 45 (80), 43 (100), 29 (19), 15 (25); for compounds 1, 2, 5, and 6, tiglic acid (tR 6.95 min) was detected: m/z [M]+ 100 (30), 83 (18), 79 (38), 77 (40), 73 (100), 65 (9), 55 (22); and for compounds 2−4, 3-hydroxy-2-methylbutyric acid (tR 7.95 min) was detected: m/z [M]+ 118 (2.0), 115 (10), 101 (20), 84 (12), 73 (70), 60 (100). Preparation and identification of 4bromophenacyl (2R,3R)-3-hydroxy-2-methylbutyrate were performed according to a previously reported procedure: mp 56−59 °C; [α]D −6.0 (c 1.0 CHCl3); GC-MS m/z 118 (2.0), 115 (10), 101 (20), 84 (12), 73 (70), 60 (100). This transesterification procedure has been used to confirm the absolute configuration for 3-hydroxy-2methylbutyrate residue (nla).7a,10 The aqueous phases were extracted with n-BuOH (2 × 10 mL) and concentrated to give colorless solids. Saponification of compound 1 yielded albinosinic acid A (14; 6.2 mg): white powder; mp 148−150 °C; [α]D −27.6 (c 1.0, MeOH); HRFABMS m/z 989.4799 [M − H]−. Compound 2 afforded albinosinic acid B (15; 6.4 mg): white powder; mp 146−148 °C; [α]D −25.0 (c 1.0, MeOH); HRFABMS m/z 973.4850 [M − H]−. These two known glycosidic acids were identified by comparison of their physical and spectroscopic constants with published values.7a,13 Saponification of compound 3 produced 10 (7.2 mg), compound 4 formed 11 (7.3 mg), compound 5 afforded 12 (8.5 mg), and compound 6 yielded 13 (7.7 mg). Albinosinic acid D (10): white powder; mp 108−110 °C; [α]D −25 (c 0.4, MeOH); 1H NMR (C5D5N, 500 MHz) δ 4.82 (1H, d, J = 8.0 Hz, Qui-1), 4.44 (1H, dd, J = 9.0, 8.0 Hz, Qui-2), 4.33 (1H, dd, J = 9.0, 9.0 Hz, Qui-3), 3.72 (1H, dd, J = 9.0, 9.0 Hz, Qui-4), 3.54 (1H, dq, J = 9.0, 6.0 Hz, Qui- 5), 1.55 (3H, d, J = 6.0 Hz, Qui-6), 5.87 (1H, d, J = 7.5 Hz, Glc-1), 4.21 (1H, dd, J = 9.0, 8.0 Hz, Glc-2), 4.08 (1H, dd, J = 9.0, 8.0 Hz, Glc-3), 3.97 (1H, dd, J = 9.0, 9.0 Hz, Glc-4), 3.87 (1H, ddd, J = 9.0, 6.0, 3.0 Hz, Glc-5), 2.24 (1H, m, Glc-6a), 4.43 (1H, m, Glc-6b), 6.32 (1H, brs, Rha-1), 4.77 (1H, m, Rha-2), 5.03 (1H, dd, J = 9.0, 3.0 Hz, Rha-3), 4.45 (1H, m, Rha-4), 4.83 (1H, m, Rha-5), 1.80 (3H, d, J = 6.5 Hz, Rha-6), 5.86 (1H, d, J = 8.0 Hz, Qui′-1), 4.18 (1H,

dd, J = 9.0, 8.0 Hz, Qui′-2), 3.89 (1H, dd, J = 9.0, 9.0 Hz, Qui′-3), 3.57 (1H, dd, J = 9.0, 9.0 Hz, Qui′-4), 3.68 (1H, dq, J = 9.0, 6.0 Hz, Qui′5), 1.56 (3H, d, J = 6.0 Hz, Qui′-6); 2.50 (2H, m, Conv-2b), 2.35 (2H, t, J = 7.4 Hz, Conv-2b), 3.80 (1H, m, Conv-11), 0.92 (3H, t, J = 7.2 Hz, Conv-14); 13C NMR (125 MHz, C5D5N) δ 103.4 (CH, Qui-1), 70.2 (CH, Qui-2), 74.4 (CH, Qui-3), 78.3 (CH, Qui-4), 73.5 (CH, Qui-5), 19.6 (CH3, Qui- 6), 102.8 (CH, Glc-1), 80.4 (CH, Glc-2), 79.4 (CH, Glc-3), 76.3 (CH, Glc-4), 79.3 (CH, Glc-5), 64.3 (CH2, Glc-6), 102.6 (CH, Rha-1), 75.4 (CH, Rha-2), 80.3 (CH, Rha-3), 74.6 (CH, Rha-4), 80.0 (CH, Rha-5), 19.0 (CH3, Rha-6), 101.6 (CH, Qui′1), 78.1 (CH, Qui′-2), 75.3 (CH, Qui′-3), 74.5 (CH, Qui′-4), 74.4 (CH, Qui′-5), 17.4 (CH3, Qui′-6); 174.1 (CO, Conv-1), 34.5 (CH2, Conv-2), 73.5 (CH, Conv-11), 14.8 (CH3, Conv-14); positive ESIMS m/z 883 [M + K]+. Albinosinic acid E (11): white powder; mp 102−105 °C; [α]D −33.6 (c 1.1, MeOH); 1H NMR (C5D5N, 500 MHz) δ 4.85 (1H, d, J = 8.0 Hz, Qui-1), 4.44 (1H, dd, J = 9.0, 8.0 Hz, Qui-2), 4.33 (1H, dd, J = 9.0, 9.0 Hz, Qui-3), 3.72 (1H, dd, J = 9.0, 9.0 Hz, Qui-4), 3.54 (1H, dq, J = 9.0, 6.0 Hz, Qui-5), 1.48 (3H, d, J = 6.0 Hz, Qui-6); 5.87 (1H, d, J = 8.0 Hz, Qui′-1), 4.18 (1H, dd, J = 9.0, 8.0 Hz, Qui′-2), 3.89 (1H, dd, J = 9.0, 9.0 Hz, Qui′-3), 3.57 (1H, dd, J = 9.0, 9.0 Hz, Qui′-4), 3.68 (1H, dq, J = 9.0, 6.0 Hz, Qui′-5), 1.56 (3H, d, J = 6.0 Hz, Qui′-6); 6.31 (1H, brs, Rha-1), 4.77 (1H, m, Rha-2), 5.03 (1H, dd, J = 9.0, 3.0 Hz, Rha-3), 4.45 (1H, m, Rha-4), 4.83 (1H, m, Rha-5), 1.79 (3H, d, J = 6.5 Hz, Rha-6); 5.18 (1H, d, J = 8.0 Hz, Qui″-1), 4.18 (1H, dd, J = 9.0, 8.0 Hz, Qui″-2), 3.87 (1H, dd, J = 9.0, 9.0 Hz, Qui″-3), 3.55 (1H, dd, J = 9.0, 9.0 Hz, Qui″-4), 3.68 (1H, dq, J = 9.0, 6.0 Hz, Qui″- 5), 1.55 (3H, d, J = 6.0 Hz, Qui″-6); 2.53 (2H, m, Conv-2b), 2.38 (2H, t, J = 7.4 Hz, Conv-2b), 3.74 (1H, m, Conv-11), 0.88 (3H, t, J = 7.2 Hz, Conv-14); 13 C NMR (125 MHz, C5D5N) δ 102.6 (CH, Qui-1), 70.2 (CH, Qui2), 74.4 (CH, Qui-3), 78.3 (CH, Qui-4), 73.5 (CH, Qui-5), 17.4 (CH3, Qui- 6); 102.1 (CH, Qui′-1), 78.1 (CH, Qui′-2), 75.3 (CH, Qui′-3), 74.5 (CH, Qui′-4), 74.4 (CH, Qui′-5), 18.7 (CH3, Qui′- 6); 101.6 (CH, Rha-1), 70.3 (CH, Rha-2), 79.1 (CH, Rha-3), 77.0 (CH, Rha-4), 70.0 (CH, Rha-5), 19.2 (CH3, Rha-6); 106.3 (CH, Qui″-1), 78.1 (CH, Qui″-2), 75.3 (CH, Qui″-3), 74.5 (CH, Qui″-4), 74.4 (CH, Qui″-5), 18.7 (CH3, Qui″-6); 174.1 (CO, Conv-1), 34.6 (CH2, Conv2), 72.6 (CH, Conv-11), 14.7 (CH3, Conv-14); positive FABMS m/z 851 [M + Na]+; negative ESIMS m/z 827 [M − H]−. Albinosinic acid F (12): white powder; mp 104−106 °C; [α]D −27.3 (c 1.1, MeOH); 1H NMR (C5D5N, 500 MHz) δ 4.83 (1H, d, J = 8.0 Hz, Qui-1), 4.44 (1H, dd, J = 9.0, 8.0 Hz, Qui-2), 4.33 (1H, dd, J = 9.0, 9.0 Hz, Qui-3), 3.72 (1H, dd, J = 9.0, 9.0 Hz, Qui-4), 3.54 (1H, dq, J = 9.0, 6.0 Hz, Qui- 5), 1.52 (3H, d, J = 6.0 Hz, Qui-6), 5.88 (1H, d, J = 7.5 Hz, Glc-1), 4.21 (1H, dd, J = 9.0, 8.0 Hz, Glc-2), 4.08 (1H, dd, J = 9.0, 8.0 Hz, Glc-3), 3.97 (1H, dd, J = 9.0, 9.0 Hz, Glc-4), 3.87 (1H, ddd, J = 9.0, 6.0, 3.0, Hz, Glc-5), 2.24 (1H, m, Glc-6a), 4.43 (1H, m, Glc-6b), 6.31 (1H, brs, Rha-1), 4.77 (1H, m, Rha-2), 5.03 (1H, dd, J = 9.0, 3.0 Hz, Rha-3), 4.45 (1H, m, Rha-4), 4.83 (1H, m, Rha-5), 1.79 (3H, d, J = 6.5 Hz, Rha-6), 5.14 (1H, d, J = 7.5 Hz, Fuc-1), 4.40 (1H, dd, J = 9.0, 7.5 Hz, Fuc-2), 4.08 (1H, dd, J = 9.0, 3.0 Hz, Fuc-3), 3.97 (1H, d, J = 3.5 Hz, Fuc-4), 3.89 (1H, m, Fuc-5), 1.44 (3H, d, J = 6.5 Hz, Fuc-6); 2.56−2.50 (2H, m, Conv-2), 3.67 (1H, m, Conv-11), 0.90 (3H, t, J = 7.0 Hz, Conv-14); 13C NMR (125 MHz, C5D5N) δ 103.4 (CH, Qui-1), 70.2 (CH, Qui-2), 74.4 (CH, Qui-3), 78.3 (CH, Qui-4), 73.5 (CH, Qui-5), 19.6 (CH3, Qui- 6), 102.8 (CH, Glc-1), 80.4 (CH, Glc-2), 79.4 (CH, Glc-3), 76.3 (CH, Glc-4), 79.3 (CH, Glc5), 64.3 (CH2, Glc-6), 102.6 (CH, Rha-1), 75.4 (CH, Rha-2), 80.3 (CH, Rha-3), 74.6 (CH, Rha-4), 80.0 (CH, Rha-5), 19.0 (CH3, Rha6), 107.2 (CH, Fuc-1), 80.2 (CH, Fuc-2), 79.2 (CH, Fuc-3), 75.9 (CH, Fuc-4), 71.2 (CH, Fuc-5), 18.3 (CH3, Fuc-6); 177.7 (CO, Conv1), 36.4 (CH2, Conv-2), 89.9 (CH, Conv-11), 15.7 (CH3, Conv-14); positive ESIMS m/z 883 [M + K]+; negative ESIMS m/z 843 [M − H]−. Albinosinic acid G (13): white powder; mp 104−106 °C; [α]D −24 (c 1.0, MeOH); 1H NMR (C5D5N, 500 MHz) δ 4.86 (1H, d, J = 8.0 Hz, Qui-1), 4.44 (1H, dd, J = 9.0, 8.0 Hz, Qui-2), 4.34 (1H, dd, J = 9.0, 9.0 Hz, Qui-3), 3.72 (1H, dd, J = 9.0, 9.0 Hz, Qui-4), 3.54 (1H, dq, J = 9.0, 6.0 Hz, Qui- 5), 1.55 (3H, d, J = 6.0 Hz, Qui-6), 5.88 (1H, d, J = 7.5 Hz, Glc-1), 4.22 (1H, dd, J = 9.0, 8.0 Hz, Glc-2), 4.10 (1H, dd, J = J



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experiments, reserpine (5 μg/mL) was used as a positive control. The reversal fold (RF) value, as a parameter of potency, was calculated from dividing the IC50 of vinblastine alone by the IC50 of vinblastine in the presence of test compounds.

9.0, 8.0 Hz, Glc-3), 3.97 (1H, dd, J = 9.0, 9.0 Hz, Glc-4), 3.87 (1H, ddd, J = 9.0, 6.0, 3.0 Hz, Glc-5), 2.24 (1H, m, Glc-6a), 4.43 (1H, m, Glc-6b), 6.31 (1H, brs, Rha-1), 4.77 (1H, m, Rha-2), 5.03 (1H, dd, J = 9.0, 3.0 Hz, Rha-3), 4.45 (1H, m, Rha-4), 4.83 (1H, m, Rha-5), 1.92 (3H, d, J = 6.5 Hz, Rha-6), 5.18 (1H, d, J = 7.5 Hz, Fuc-1), 4.38 (1H, dd, J = 9.0, 7.5 Hz, Fuc-2), 4.08 (1H, dd, J = 9.0, 3.0 Hz, Fuc-3), 3.97 (1H, d, J = 3.5 Hz, Fuc-4), 3.89 (1H, m, Fuc-5), 1.48 (3H, d, J = 6.5 Hz, Fuc-6); 2.55−2.50 (2H, m, Jal-2), 3.91 (1H, m, Jal-11), 0.88 (3H, t, J = 7.0 Hz, Jal-16); 13C NMR (125 MHz, C5D5N) δ 102.9 (CH, Qui-1), 70.2 (CH, Qui-2), 74.4 (CH, Qui-3), 78.3 (CH, Qui-4), 73.5 (CH, Qui-5), 19.2 (CH3, Qui- 6), 102.8 (CH, Glc-1), 80.4 (CH, Glc2), 79.4 (CH, Glc-3), 76.3 (CH, Glc-4), 79.3 (CH, Glc-5), 63.7 (CH2, Glc-6), 102.6 (CH, Rha-1), 72.7 (CH, Rha-2), 68.4 (CH, Rha-3), 73.0 (CH, Rha-4), 64.0 (CH, Rha-5), 19.0 (CH3, Rha-6), 106.6 (CH, Fuc1), 80.2 (CH, Fuc-2), 79.2 (CH, Fuc-3), 75.9 (CH, Fuc-4), 71.2 (CH, Fuc-5), 17.5 (CH3, Fuc-6), 176.5 (CO, Jal-1), 35.4 (CH2, Jal-2), 80.7 (CH, Jal-11), 14.4 (CH3, Jal-16); negative ESIMS m/z 871 [M − H]−. Sugar Analysis. Compounds 10−13 (5 mg of each) in 10 mL of 4 N HCl were independently refluxed at 90 °C for 1 h. Then, each reaction mixture was diluted with 5 mL of H2O and extracted with ether (3 × 10 mL). The organic layer was evaporated to dryness, dissolved in CHCl3 (3 mL), and treated with CH2N2. The aqueous phase was neutralized with 1 N KOH and extracted with n-BuOH (10 mL), then washed with H2O (2 × 5 mL), and concentrated to give a solid. The thiazolidine derivatives of each sugar mixture were prepared according to previously described procedures,15 converted into volatile derivatives by treatment with chlorotrimethylsilane (Sigma Sil-A), and analyzed by CG-MS by applying the following conditions: DB-5MS (10 m × 0.18 mm, film thickness 0.18 μm); He, 2 mL/min; 100 °C isothermal for 3 min, linear gradient to 300 °C at 20 °C/min. Retention times for TMS derivatives of common sugar thiazolidines were used as standards for GC identification through coelution experiments with L-rhamnose tR 4.53 min, D-fucose tR 4.56 min, Dquinovose tR 4.59 min, and D-glucose tR 4.73 min. Identification of Aglycones. The derivatized (CH2N2) organic layer residues obtained from acid-catalyzed hydrolysis of albinosinic acids D−G (10−13) were individually submitted to normal-phase HPLC (ISCO, 21.2 × 250 mm, 10 μm) using isocratic elution [nhexane−CHCl3−Me2CO (6:3:1)] and a flow rate of 6 mL/min to give 2.0−3.5 mg from compounds 3−5 of methyl (11S)-hydroxytetradecanoate:11 tR 18.6 min; mp 27−29 °C; [α]D +1.5 (c 2, CHCl3); 13C NMR 174.4, 71.7, 51.5, 39.6, 37.5, 34.1, 29.6, 29.5, 29.3, 29.2, 29.1, 25.6, 24.9, 18.8, 14.1. An aliquot of this pure sample (2 mg) obtained from compounds 10−12 was derivatized with Sigma Sil-A for 5 min at 70 °C. GC-MS analysis gave one peak (tR 7 min): m/z [M]+ 330 (0.3), 315 (3.5), 287 (66.8), 145 (100), 73 (35.4). Treatment of derivatized organic layer residues from acid hydrolysis of compound 13 as described above afforded 2.0 mg of methyl (11S)-hydroxyhexadecanoate:11 tR 16.4 min; mp 42−44 °C; [α]D +7.3 (c 2, CHCl3); 13C NMR 174.4, 72.0, 51.4, 37.5, 37.4, 34.1, 31.9, 29.6, 29.5, 29.4, 29.2, 29.1, 25.6, 25.3, 24.9, 22.6, 14.1. This aglycone (2 mg) was derivatized with Sigma Sil-A and subjected to GC-MS analysis, which gave one peak (tR 12.8 min): m/z [M]+ 358 (0.3), 343 (0.5), 311 (10.5), 287 (59.7), 173 (100), 73 (46.3). Cytotoxicity and Modulation of Multidrug-Resistance Assays. Cytotoxicity of the resin glycosides (1−6) was determined using an SRB assay.14 Cells were harvested at log phase, treated in triplicate with various concentrations of the test samples (0.2−25 μg/ mL), and incubated for 72 h at 37 °C in a humidified atmosphere of 5% CO2. Results are expressed as the concentration that inhibits 50% of the growth of the control cells after the incubation period (IC50). The values were calculated from a semilog plot of the drug concentration (μg/mL) against the percentage of growth inhibition.6 Vinblastine was included as a positive control. The reversal effects as modulators were further investigated with the same method. MCF-7 and MDR MCF-7/Vin cells were seeded into 96-well plates and treated with various concentrations of vinblastine (0.00064−10 μg/ mL) in the presence or absence of glycolipids at 25 and 5 μg/mL for 72 h. The ability of glycolipids to potentiate vinblastine cytotoxicity was measured by calculating the IC50 as described above. In these

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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.6b00782. Structures for albinosides I−III (Figures S1 and S2) and albinosinic acids A−C (Figures S3 and S4); FABMS and NMR (1H, 13C, COSY, and HMBC) spectra of albinosides IV−IX (1−6; Figures S5−S34); modulation assay of vinblastine with compounds 5 and 6 (Figures S35 and S36) (PDF)

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AUTHOR INFORMATION

Corresponding Author

*Tel: +52 55 5622-5288. Fax: +52 55 5622-5329. E-mail: [email protected]. ORCID

Rogelio Pereda-Miranda: 0000-0002-0542-0085 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This research was supported by grants from Dirección General de Asuntos del Personal Académico (UNAM, IN215016) and Consejo Nacional de Ciencia y Tecnologı ́a (CB220535). A.L. thanks the Arkansas Biosciences Institute for funds in support of this project. S.C.-M. and J.C.-G. are grateful to CONACyT for graduate student scholarships. Thanks are due to G. Duarte ́ (USAI, Facultad de Quimica, UNAM) for the recording of mass spectra.

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REFERENCES

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