Resin Glycosides from Ipomoea Alba Seeds As Potential

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Resin Glycosides from Ipomoea alba Seeds as Potential Chemosensitizers in Breast Carcinoma Cells † † ‡ † § † Sara Cruz-Morales, Jhon Castañeda-Gomez,́ , Daniel Rosas-Ramírez, , Mabel Fragoso-Serrano, † ⊥ ∥ † Gabriela Figueroa-Gonzalez,́ , Argelia Lorence, and Rogelio Pereda-Miranda*, † § Departamento de Farmacia, Facultad de Química, and Departamento de Química de Biomacromoleculas,́ Instituto de Química, Universidad Nacional Autonomá de Mexico,́ Ciudad Universitaria, 04510, Mexico City, Mexico ‡ Grupo Químico de Investigacioń y Desarrollo Ambiental, Programa de Licenciatura en Ciencias, Facultad de Educacion,́ Universidad Surcolombiana, Neiva, Colombia ⊥ Laboratorio de Genomica,́ Unidad de InvestigacionBá sica,́ 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 (11S- hydroxytetradecanoic) 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.

ancer cells are resistant when they are not susceptible to plants and their main active secondary metabolites have been C the concentration of a clinically used drug by developing a shown to modulate MDR in cancer cells, and some crude drugs variety of mechanisms that result in the loss of their initial have been investigated as anticancer agents in order to hypersensitivity to anticancer agents. It is common for cancer introduce new therapeutic alternatives.3 There is growing cells to express mechanisms that confer simultaneous resistance evidence that resin glycosides4a are modulators of efflux pumps to various drugs that are structurally and functionally different, that produce the multidrug-resistant phenotype in prokaryotic5 a phenomenon known as multidrug resistance (MDR).1 These and eukaryotic6,7 cells. These resins are complex mixtures of an mechanisms complicate treatment and dramatically increase extensive family of secondary metabolites known as glycolipids both morbility and mortality as well as the financial costs for and represent unique compounds in the plant kingdom cancer therapy in health care systems worldwide. The most confined to the Convolvulaceae (the morning glory family)4a common reason to acquire cross resistance to a wide range of and the Scrophulariaceae.4b anticancer drugs is the expression of one or more ATP- Previous results from our research program have shown that 8 dependent efflux pumps, such as P-glycoprotein (MDR murucoidin V (isolated from Ipomoea murucoides) and purgin 7b protein1/P-gp) and the breast cancer resistance protein II (from the officinal jalap root, I. purga) significantly lowered (BCRP/ABCG2), which have the task of detecting and the efflux rate of rhodamine 123, a fluorescent P-gp substrate expelling drugs or any hydrophobic xenobiotic outside the used to determine its accumulation in monolayer efflux assays cell.2 The use of efflux pump modulators coadministered with cytotoxic drugs results in a susceptibility equivalent to that of a Received: August 26, 2016 cell without transporter expression. Commonly used medicinal Published: November 23, 2016

Chart 1

with resistant cells.6,7b Decreased expression of the P-gp by potential chemosensitizers to further explore their underlying − murucoidin V was also detected by immunofluorescence flow mechanism of action as MDR-modifying agents.5 7 cytometry after treatment with an anti-P-gp monoclonal Moon vine (I. alba L., formerly known as Ipomoea bona-nox antibody.6 Incubation of vinblastine-resistant human breast and Calonyction aculeatum) is a cultivated ornamental and carcinoma cells (MCF-7/Vin) with these resin glycosides also medicinal species of the night-blooming morning glory group 6,7b that is native to tropical and subtropical Americas from Mexico enhanced vinblastine susceptibility. On the basis of these and Florida to northern Argentina.9 In Mexico, a decoction of results, we have employed this cytotoxicity assay for the facile fl fi the aerial parts and owers is used to treat paralysis and soft identi cation of the capacity of each isolated noncytotoxic resin tissue swelling.7a As with many New World plants, the initial glycoside to modulate the resistance phenotype in the MCF-7 transport to the Old World was a result of their medical 6,7 cell line as potential vinblastine chemosensitizers. Thus, our applications, learned from the indigenous people of the long-term efforts have been mainly focused on the chemical Americas.10 Laxatives were of prime interest to Europeans,10b investigation of this type of glycolipids4 to identify new and I. alba was one of the early recorded species from the

3094 DOI: 10.1021/acs.jnatprod.6b00782 J. Nat. Prod. 2016, 79, 3093−3104 Journal of Natural Products Article

Convolvulaceae used by the extinct Taino Amerindians of the from those observed for albinosinic acid A, the glycosidic acid Caribbean.10c of albinoside I (7), suggested that the ester linkage for the The present in-depth chemical investigation of the moon macrolactone was also placed at the terminal pentose unit as in vine resin glycoside profile from commercial seed samples was the case of model compound 7 (Figure S1, Supporting 7a fi undertaken to unravel the structural diversity of this class of Information). Saponi cation of 1 liberated an H2O-soluble MDR reversal agents by isolating novel glycolipids. Six new glycosidic acid and an organic solvent-soluble fraction, from resin glycosides, albinosides IV−IX (1−6), as well as the known which the released 2-methyl-2-butenoic (tga) acid was − − 7a fi albinosides I III (7 9), were isolated from the CHCl3- identi ed by GC-MS. The glycosidic acid was methylated and soluble extract of moon vine seeds, and their structures were further acetylated to yield the peracetylated derivative of established through chemical degradation, NMR spectroscopy, albinosinic acid A methyl ester. Comparison of the melting and mass spectrometry. Reversal of multidrug resistance by point, optical rotation, and 13C NMR data with published compounds 1−6 was also evaluated in vinblastine-resistant values confirmed its structure,7a which was also identified by MCF-7/Vin cells in modulation assays. HPLC comparison with an authentic sample. All protons of each saccharide unit for the natural product 1 were assigned by ■ RESULTS AND DISCUSSION a combination of COSY (Figure S8, Supporting Information) The resin glycoside fraction from the CHCl -soluble moon vine and TOCSY NMR techniques (Table 1). Then, all carbons 3 4 seed extract was obtained by precipitation with MeOH. Then, were sequentially assigned by HSQC studies (Table 2). In the individual major constituents were separated into nine fractions low-field region of the HSQC spectrum, five anomeric signals fi δ δ δ δ through preparative reversed-phase HPLC using peak-shaving were identi ed: Qui-1 ( H 4.78, C 103.5), Glc-1 ( H 5.99, C 11 ′ δ δ δ δ and heart-cutting techniques. Previously isolated resin 101.2), Qui -1 ( H 5.29, C 104.4), Rha-1 ( H 5.64, C 100.2), 7a ′ δ δ glycosides from this species, albinosides I−III (7−9) (Figures Rha -1 ( H 5.82, C 101.1) (Figures S6 and S7, Supporting S1 and S2, Supporting Information), were used as standards to Information). The interglycosidic connectivities were con- 4 identify eluates containing new constituents. Co-elution firmed by HMBC experiments. For example, the following key δ δ experiments were done on an RP-18 column with an isocratic correlations were observed: H-2 ( H 4.44) of Qui with C-1 ( C − − δ δ elution of MeOH CH3CN H2O, and six peaks were selected 101.2) of Glc; H-2 ( H 4.18) of Glc with C-1 ( C 101.1) of 11 ′ δ δ ′ for further HPLC separation in the recycling mode. To Rha ; H-6 ( H 4.20) of Glc with C-1 ( C 104.4) of Qui ; H-1 δ δ ′ δ achieve chromatographic homogeneity, each peak was recycled ( H 5.64) of Rha with C-2 ( C 81.3) of Qui ; and H-1 ( H 4.78) δ until overlapped components were separated. A refractive index of Qui with C-11 ( C 81.5) of convolvulinolic acid (Figure S9, detector was used to monitor this purification process. These Supporting Information). The locations of the ester sub- 12 approaches allowed the purification of six new compounds, stituents were also recognized by HMBC experiments 3 fi named albinosides IV−IX (1−6). through JCH correlations between a speci c carbonyl group The main approach followed for the structure elucidation of and the pyranose ring proton: the lactonization site at C-2 of the isolated resin glycosides involved the use of degradative the terminal rhamnose unit (Rha) was established by the δ δ chemical reactions in combination with spectroscopic and observed correlation of H-2 ( H 5.81) of Rha with C-1 ( C spectrometric methods.4a Saponification of the crude material 173.5) of convolvulinolic acid, and the position of esterification fragmented the macrocyclic lactone and liberated the fatty acids by the tigloyl residue was confirmed by the correlation of H-2 δ ′ δ that esterify the oligosaccharide core, which were further ( H 5.98) of Rha with the carbonyl at C 167.7. fi − − identi ed by GC-MS. Thus, their H2O-soluble glycosidic acids For albinoside V (2), the HRESIMS showed a [M H] were used to generate 13C NMR profiles7d,10b for 1 and 2, peak at m/z 1137.5712, indicating a molecular formula of δ containing known glycosidic acids, since their anomeric signals C54H89O25 (calcd error: = +1.2 ppm), which was 16 mass were readily distinguishable and used as fingerprints for pattern units higher than albinoside II.7a The initial loss of a niloyl recognition and structural dereplication.7a For the minor residue afforded a peak at m/z 1037 [M − H − 100 − − compounds 3−6, four new glycosidic acids were identified as (C5H8O2)] , in addition to the peak at m/z 809 [1037 − − the saponification products and were named albinosinic acids C6H10O4 82 (C5H6O)] , which indicated the consecutive D−G(10−13), respectively. Their acid-catalyzed hydrolysis loss of a methylpentose unit and a tigloyl residue for 2. The rest released a mixture of monosaccharides, which underwent of the fragment peaks were produced by glycosidic cleavage at − − − − derivatization with L-cysteine to form thiazolidines. These m/z 663 [809 C6H10O4] , 517 [663 C6H10O4] , 389 [517 − − − − derivatives allowed the identification of the constitutive +H2O C6H10O4)] , and 243 [389 C6H10O4] ) (Figure monosaccharides for each oligosaccharide core by GC-MS as S10, Supporting Information); this fragmentation pattern was their trimethylsilane (TMS) ethers.7a similar to that previously reported for albinoside II (8).7a Negative-ion FABMS of albinoside IV (1)afforded a peak at Saponification of this natural product 2 liberated tiglic and nilic m/z 1053.5139 [M − H]− corresponding to the molecular acids, which were identified by GC-MS of the organic solvent- δ formula C49H81O24 (calcd error: = +1.5 ppm) (Figure S5, soluble fraction. The H2O-soluble glycosidic acid was Supporting Information). The elimination of 82 amu confirmed methylated and acetylated to yield the peracetylated derivative the presence of a tigloyl residue at m/z 971 [M − H − of albinosinic acid B methyl ester. Comparison of the melting − 13 C5H6O] . The other peaks were produced by consecutive point, optical rotation, and C NMR data with published glycosidic cleavages along the oligosaccharide core at m/z 825 values confirmed its structure.7a A similar NMR experimental − − − [971 C6H10O4 (methylpentose unit)] , 679 [825 approach to that described above was used for the structure − − − − 1 13 C6H10O4] , 533 [679 C6H10O4] , 389 [533 + H2O elucidation of 2 in order to assign the H and C NMR spectra − − − C6H10O5 (hexose unit)] , and 243 [389 C6H10O4] . This (Tables 1 and 2; Figures S11 and S12, Supporting Information) fragmentation pattern was similar to that previously reported through COSY (Figure S13, Supporting Information) and 7a fi 4 3 for albinoside I, con rming a branched pentasaccharide core. HSQC experiments. The following key JCH correlations were Furthermore, the difference of 18 mass units for all these peaks observed to confirm the interglycosidic connectivities in the

3095 DOI: 10.1021/acs.jnatprod.6b00782 J. Nat. Prod. 2016, 79, 3093−3104 Journal of Natural Products Article

Table 1. 1H NMR Spectroscopic Data for Albinosides IV (1) Table 2. 13C NMR Spectroscopic Data for Albinosides IV (1) δ J δ and V (2) (Measured at 500 MHz in C5D5N, in ppm, in and V (2) (Measured at 125 MHz in C5D5N, in ppm) Hz) position 12 position 12 Qui-1 103.5 102.9 Qui-1 4.78, d (8.0) 4.77, d (8.0) 2 78.7 77.4 2 4.44, dd (9.0, 8.0) 4.42, dd (9.0, 8.0) 3 78.9 80.8 3 4.26, m 4.27, dd (9.0, 9.0) 4 76.6 79.3 4 3.69, m 4.27, dd (9.0, 9.0) 5 72.3 77.7 5 3.57, m 3.66, dq (9.0, 6.0) 6 18.2 18.6 6 1.49, d (6.0) 1.59, d (6.0) Glc-1 101.2 Glc-1 5.99, d (7.6) 2 77.9 2 4.18, dd (9.0, 7.8) 3 75.4 3 4.07, dd (9.0, 7.8) 4 70.0 4 3.68, m 5 76.5 5 3.81, ddd (9.0, 6.0, 3.0) 6 69.7 6a 4.20, m Rha-1 100.2 99.9 6b 4.20, m 2 71.7 72.9 Rha-1 5.64, brs 5.61, brs 3 74.2 74.2 2 5.81, brs 5.77, brs 4 77.6 75.7 3 4.07, dd (9.0, 3.0) 4.07, dd (9.0, 3.0) 5 67.3 72.3 4 3.99, dd (9.9, 9.9) 3.53, dd (9.9, 9.9) 6 17.4 18.7 5 5.10, dq (9.9, 6.5) 3.64, m Rha′-1 101.1 100.1 6 1.59, d (6.5) 1.50, d (6.5) 2 71.9 73.4 Rha′-1 5.82, brs 5.85, brs 3 70.1 72.2 2 5.98, dd (3.0, 2.0) 4.13, m 4 76.1 85.5 3 4.65, dd (9.0, 3.0) 5.50, dd (9.0, 3.0) 5 70.0 68.6 4 4.44, dd (9.9, 9.9) 4.36, m 6 18.1 19.8 5 4.37, m 4.32, m Qui′-1 104.4 106.6 6 1.82, d (6.5) 1.95, d (6.5) 2 81.3 70.5 Qui′-1 5.29, d (8.0) 5.33, d (8.0) 3 77.7 81.5 2 3.96, dd (9.0, 8.0) 4.05, dd (9.0, 8.0) 4 76.7 73.2 3 4.19, dd (9.0, 9.0) 3.79, dd (9.0, 9.0) 5 72.5 72.9 4 3.68, m 3.79, dd (9.0, 9.0) 6 18.4 18.6 5 3.88, dq (9.0, 6.0) 3.71, dq (9.0, 6.0) Rha″-1 99.7 6 1.59, d (6.0) 1.61, d (6.0) 2 73.6 Rha″-1 5.73, brs 3 72.4 2 5.85, m 4 79.4 3 4.54, dd (9.0, 3.0) 5 70.5 4 4.30, m 6 18.2 5 4.25, m Conv-1 173.5 172.9 6 1.87, d (6.5) 2 33.2 34.1 Conv 2a 2.64, m 2.90, ddd (16.0, 9.6, 7.6) 11 81.5 81.6 2b 2.43, ddd (16.0, 9.6, 7.6) 14 14.3 14.6 11 3.72−3.80, m 3.83−3.88, m nla-1 174.5 14 0.91, t (7.4) 0.90, t (7.4) 2 48.9 nla-2 2.82, dq (7.2, 6.8) 3 69.3 3 4.26−4.30, m 4 19.6 4 1.32, d (6.4) 5 12.7 5 1.25, d (7.2) tga-1 167.7 167.9 tga-3 7.12, dq (7.2, 1.2) 7.12, dq (7.2, 1.2) 2 129.5 129.3 4 1.40, d (7.2) 1.49, d (7.2) 3 138.4 138.4 5 1.91, brs 1.85, brs 4 14.6 14.7 5 12.6 12.7

HMBC spectrum (Figure S14, Supporting Information): H-2 δ δ δ δ ′ ( H 4.42) of Qui with C-1 ( C 99.9) of Rha; H-4 ( H 3.53) of observed: between H-3 ( H 5.50) of Rha and the carbonyl δ ′ δ ′ δ Rha with C-1 ( C 106.6) of Qui ; H-2 ( H 4.05) of Qui with resonance at c 172.9, assigned to the lactone functionality due δ ″ δ ′ δ 2 C-1 ( C 99.7) of Rha ; H-1 ( H 5.85) of Rha with C-3 ( C to its JCH coupling with the diastereotopic C-2 methylene ′ δ δ δ 81.5) of Qui ; and H-1 ( H 4.77) of Qui with C-11 ( C 81.6) of protons ( H 2.90 and 2.43); H-2 of the terminal branched δ ″ convolvulinolic acid. The observed HMBC long-range rhamnose ( H 5.85, Rha ) and the carbonyl group of the niloyl 2,3 δ δ correlations ( JCH) were used to support the macrolactoniza- moiety ( C 174.5); H-2 of the second saccharide unit ( H 5.77, ﬁ δ tion site and the positions of esteri cation (Figure S14, Rha) and the carbonyl carbon for the tigloyl residue at C Supporting Information). The following key correlations were 167.9.

3096 DOI: 10.1021/acs.jnatprod.6b00782 J. Nat. Prod. 2016, 79, 3093−3104 Journal of Natural Products Article

1 − − δ J Table 3. H NMR Spectroscopic Data for Albinosides VI IX (3 6) (Measured at 500 MHz in C5D5N, in ppm, in Hz) position 3456 Qui-1 4.77, d (8.0) 4.78, d (8.0) 4.75, d (8.0) 4.76, d (8.0) 2 4.31, dd (9.0, 8.0) 4.28, dd (8.8, 8.0) 4.45, dd (9.2, 8.0) 4.44, dd (9.2, 8.0) 3 4.13, dd (8.8, 8.0) 4.28, dd (8.8, 8.0) 4.27, m 4.28, dd (9.6, 8.0) 4 3.76, dd (8.8, 8.0) 3.62, m 3.70, dd (9.6, 9.0) 3.85, dd (9.6, 9.0) 5 3.68, dq (8.8, 5.6) 3.66, dq (9.0, 6.0) 3.54, dq (9.0, 6.0) 3.63, dq (9.0, 6.0) 6 1.46, d (6.0) 1.62, d (6.0) 1.58, d (6.0) 1.59, d (6.0) Glc-1 5.75, d (7.6) 5.98, d (7.6) 5.99, d (7.6) 2 4.11, t (8.0) 4.21, dd (9.2, 7.6) 4.21, dd (9.2, 7.6) 3 3.87, m 5.82, dd (9.2, 8.8) 5.84, dd (9.2, 8.8) 4 3.67, dd (9.6, 9.2) 4.44, dd (9.2, 9.2) 4.50, dd (9.2, 9.2) 5 3.78, ddd (9.2, 6.0, 3.0) 3.84, dd (9.2, 6.0) 3.86, dd (9.2, 6.0) 6a 4.14, m 4.25, dd (9.2, 6.0) 4.26, dd (9.2, 6.0) 6b 4.36, dd (9.2, 3.0) 4.25, dd (9.2, 3.0) 4.26, dd (9.2, 3.0) Qui′-1 5.07, d (8.0) 5.64, d (8.0) 2 5.66, dd (9.0, 8.0) 4.05, dd (9.0, 8.0) 3 5.66, dd (9.0, 9.0) 4.03, dd (9.0, 9.0) 4 4.33, dd (9.0, 8.0) 3.49, m 5 3.64, dq (9.0, 6.0) 3.69, dq (9.2, 5.8) 6 1.59, d (6.0) 1.44, d (5.8) Qui″-1 5.04, d (8.0) 2 5.66, dd (9.0, 8.0) 3 5.66, dd (9.0, 9.0) 4 3.67, m 5 3.49, dq (9.0, 6.0) 6 1.47, d (6.0) Rha-1 5.80, d (1.5) 5.83, brs 5.67, brs 5.67, brs 2 4.64, brs 4.62, brs 5.88, m 5.88, d (2.0) 3 4.78, dd (9.0, 3.2) 4.75, dd (9.0, 2.4) 4.81, dd (9.0, 3.0) 4.23, dd (9.0, 3.0) 4 5.87, t (9.6) 5.86, t (8.0) 4.36, dd (9.6, 8.8) 4.34, dd (9.6, 8.8) 5 5.21, dq (10.0, 6.0) 5.19−5.23, m 4.78, dq (8.8, 6.4) 4.78, dq (8.8, 6.4) 6 1.62, d (6.4) 1.60, d (6.4) 1.92, d (6.4) 1.92, d (6.4) Fuc-1 5.28, d (7.6) 5.29, d (7.6) 2 4.34, dd (9.2, 7.6) 4.32, dd (9.2, 7.6) 3 4.23, dd (9.2, 3.0) 4.31, dd (9.2, 3.0) 4 5.65, d (3.0) 5.64, d (3.0) 5 3.95, q (6.0) 3.92, q (6.0) 6 1.31, d (6.4) 1.31, d (6.4) Conv 2a 3.33, ddd (16.4, 12.4, 2.8) 3.35, ddd (16.0, 12.4, 2.8) 2.83, ddd (10.4, 7.6, 2.0) 2b 2.76, ddd (16.4, 12.4, 2.8) 2.76, ddd (16.0, 12.4, 2.8) 2.44, ddd (10.4, 7.6, 2.0) 11 3.77, m 3.78−3.80, m 3.85, m 14 0.92, t (7.2) 0.93, t (7.2) 0.88, t (7.2) Jal-2a 2.84, ddd (16.4, 9.0, 8.0) 2b 2.43, ddd (16.4, 9.0, 8.0) 11 3.80−3.86, m 16 0.86, t (6.8) ace-2 2.05, s 2.06, s nla-2 2.83, dq (7.2, 7.1) 2.79, dq (7.2, 7.1) 3 4.14, m 4.26−4.30, m 4 1.34, d (6.4) 1.33, d (6.4) 5 1.24, d (7.2) 1.24, d (7.2) tga-3 6.97, dq (7.2, 1.6) 6.99, dq (8.0, 1.6) 4 1.46, d (7.2) 1.49, d (7.2) 5 1.79, brs 1.81, brs tga′-3 7.12, dq (7.2, 1.2) 7.12, dq (7.2, 1.2) 4 1.49, d (7.2) 1.46, d (7.2) 5 1.82, brs 1.86, brs

Albinoside VI (3) gave a sodium adduct ion at m/z 991.4726 The saponiﬁcation of 3 released a mixture of acids, which was + [M + Na] in the HRMALDITOFMS corresponding to the collected in Et2O. This mixture was analyzed by GC-MS and δ ﬁ molecular formula C45H76O22Na (calcd error: = +0.6 ppm). allowed the identi cation of acetic and nilic acids. The H2O-

3097 DOI: 10.1021/acs.jnatprod.6b00782 J. Nat. Prod. 2016, 79, 3093−3104 Journal of Natural Products Article 13 − − δ Table 4. C NMR Spectroscopic Data for Albinosides VI IX (3 6) (Measured at 125 MHz in C5D5N, in ppm) position 3456 position 3456 Qui-1 101.4 103.7 103.2 103.2 2 74.5 74.2 2 76.5 76.8 77.5 77.5 3 79.9 79.9 3 85.7 79.6 79.3 79.6 4 75.1 75.1 4 78.2 77.5 77.8 77.7 5 71.0 70.4 5 77.9 72.7 73.4 73.4 6 17.9 17.7 6 18.5 18.9 19.5 19.2 Conv-1 174.6 174.3 173.3 Glc-1 102.6 100.6 100.6 2a 34.3 34.2 35.4 2 79.1 79.6 79.6 11 82.1 81.3 81.1 3 70.6 80.1 79.9 14 15.3 14.3 15.2 4 75.2 70.4 70.5 Jal-1 173.3 5 77.5 77.9 77.8 2 35.4 6 63.2 62.5 62.6 11 81.6 Qui′-1 104.4 101.8 16 14.7 2 72.3 78.9 ace-1 171.7 171.3 3 76.5 77.7 2 21.9 21.3 4 80.3 74.8 nla-1 175.7 175.2 5 73.9 74.9 2 49.5 49.0 6 19.2 18.8 3 69.1 69.3 Qui″-1 100.9 4 21.4 21.7 2 72.4 5 13.9 14.8 3 76.1 tga-1 168.2 168.1 4 72.6 2 129.7 129.7 5 72.3 3 138.4 138.4 6 18.0 4 14.8 14.9 Rha-1 102.3 101.7 99.8 99.8 5 13.1 13.1 2 72.9 72.5 74.3 73.4 tga′-1 168.8 168.8 3 71.1 70.7 78.1 81.6 2 129.6 129.6 4 77.0 76.8 86.2 86.2 3 138.7 138.8 5 67.6 67.2 68.9 68.8 4 14.9 14.8 6 19.0 18.7 19.9 19.5 5 13.1 13.1 Fuc-1 107.1 107.1 soluble residue, albinosinic acid D (10), was identified as a spectra of compound 3, four anomeric signals were confirmed δ δ tetrasaccharide of 11-hydroxytetradecanoic acid by ESIMS in at H 4.77 (1H, d, J = 7.0 Hz; C 101.4, Qui-1); 5.75 (1H, d, J = δ δ the positive mode with a potassium adduct ion of high 7.6 Hz; C 102.6, Glc-1); 5.07 (1H, d, J = 7.6 Hz; C 104.4, abundance at m/z 883 [M + K]+ and FABMS in the negative ′ δ − Qui -1); and 5.80 (1H, d, J = 1.2 Hz; C 102.3, Rha-1). mode with a deprotonated molecule ion at m/z 843 [M − H] Therefore, four separate spin systems for the sugar skeletons − − − and diagnostic ions at m/z 697 [843 146] , 551 [697 were readily distinguished in the 1H−1H COSY and TOCSY − − − 7a 146] , 389 [551 − 162] , and 243 [389 − 146] . The 3 spectra. The following key JCH correlations in the HMBC liberated water-soluble sugars from 10 by an acid-catalyzed spectrum confirmed the glycosylation sequence (Figure 1): procedure underwent derivatization with L-cysteine to form between C-1 (δ 101.4) of Qui and H-11 (δ 3.77) of the thiazolidines, which were identified by GC-MS as their TMS C H 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 D- series 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 − − − fi H C5H8O2] con rmed 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 − − − C6H10O5 (hexose unit)] , and 243 [389 C6H10O4] confirmed the additional loss of two methylpentoses and one hexose unit. All proton and carbon signals (Tables 3 and 4; Figure 1. Key HMBC correlations for compound 3 showing Figures S16 and S17, Supporting Information) were assigned 3 connectivities ( JCH) for anomeric carbons: (A) C-1 Qui/H-11 sequentially by COSY (Figure S18, Supporting Information) Conv; (B) C-1 Glc/H-2 Qui; (C) C-1 Rha/H-2 Glc; (D) C-1 and HSQC NMR studies. In the low-field region of the HSQC Qui′/H-6 Glc (unlabeled connectivity: C-1/H-5 Qui′).

3098 DOI: 10.1021/acs.jnatprod.6b00782 J. Nat. Prod. 2016, 79, 3093−3104 Journal of Natural Products Article δ δ − − aglycone; H-1 ( H 4.77) of Qui and C-11 of the aglycone ( C ppm) in the HRESIMS, in contrast to the ion [M H] at m/z δ δ δ − 82.1, Conv); H-2 ( H 4.31) of Qui and C-1 ( C 102.6) of Glc; 1017.5265 (C50H81O21, calcd error: = 1.08 ppm) for δ δ ′ δ ff H-6 ( H 4.36) of Glc and C-1 ( C 104.4) of Qui ; C-1 ( C albinoside IX (6), indicating a di erence of two methylene δ 102.3) of Rha and H-2 ( H 4.11) of Glc (Figure S19, groups (28 atomic mass units) between these two compounds. Supporting Information). The lactonization and acylation Thus, these results suggested the presence of convolvulinolic positions for 3 were established by the following HMBC acid (11S-hydroxytetradecanoic acid) as the aglycone for 5 and δ ′ correlations: between H-2 ( H 5.66) of Qui and the carbonyl jalapinolic acid (11S-hydroxyhexadecanoic acid) as the aglycone resonance at δc 174.6, assigned to the lactone functionality due for 6.7a,11 In the negative FABMS, peaks from the consecutive 2 − − to its JCH coupling with the diastereotopic C-2 methylene elimination for two tigloyl residues at m/z 907 [M H 82 δ δ ′ − − − protons ( H 3.33 and 2.76); H-3 ( H 5.66) of Qui and the (C5H6O)] and 825 [907 C5H6O] were also observed for 5 δ carbonyl group of the niloyl moiety ( C 175.7); and H-4 of the (Figure S25, Supporting Information). The consecutive δ − terminal branched rhamnose ( H 5.87, Rha) and the carbonyl elimination of each sugar unit at m/z 679 [825 C6H10O4 δ − − group of the acetyl moiety ( C 171.7). Consequently, the (methylpentose unit)] , 533 [679 C6H10O4 (methylpentose − − − structure of albinosinic acid D (10) corresponded to (11S)- unit)] , 389 [533 + H2O C6H10O5 (hexose unit)] , and 243 α → − − fi convolvulinolic acid 11-O- -L-rhamnopyranosyl-(1 2)-O-[6- [389 C6H10O4 (methylpentose unit)] con rmed a deoxy-β-D-glucopyranosyl-(1→6)]-O-β-D-glucopyranosyl-(1→ tetrasaccharide of a convolvulinolic acid moiety for albinoside 2)-O-6-deoxy-β-D-glucopyranoside. VIII (5). The difference of 28 mass units (two methylene Albinoside VII (4) showed a sodium adduct at m/z 975.4777 groups) between compounds 5 and 6, as well as the production [M + Na]+ in the HRMALDITOFMS, consistent with a of the same general fragmentation pattern by glycosidic δ molecular formula of C45H75O21Na (calcd error: = +0.6 cleavage of each sugar moiety at m/z 935, 853, 707, 561, ppm), indicating the difference of one oxygen (16 atomic mass 417, and 271, confirmed the similar linear tetrasaccharide core units) between this compound and albinoside VI (3). In the in both acids and the presence of jalapinolic acid as the negative FABMS (m/z 951 [M − H]−) (Figure S20, aglycone for compound 6 (Figure S30, Supporting Informa- Supporting Information), the consecutive losses of one niloyl tion).11 Pure compounds 5 and 6 were each saponified, and − − − residue at m/z 851 [M H C5H8O2] , one acetyl residue at their Et2O-soluble fraction was analyzed by GC-MS. Both − − m/z 809 [851 C2H2O] , and four methylpentoses at m/z compounds released 2-methyl-2-butenoic acid. The H2O- 663, 517, 371, and 243 indicated that compound 4 is a soluble residue from compound 5 gave albinosinic acid F tetraglycoside of convolvulinolic acid. Saponification of 4 (12), which was identified as a tetrasaccharide of 11S- liberated acetic and nilic acids, which were identified by GC- hydroxytetradecanoic acid by positive ESIMS at m/z 883 [M + MS of the organic solvent-soluble fraction. The H2O-soluble +K] and in the negative mode with a deprotonated molecule residue from compound 4 gave albinosinic acid E (11), which at m/z 843 [M − H]− and diagnostic ions at m/z 697, 551, 389, was identified as a tetrasaccharide of 11-hydroxytetradecanoic and 243; compound 6 gave albinosinic acid G (13), which was acid by positive FABMS at m/z 851 [M + Na]+; in negative characterized as a tetrasaccharide of 11S-hydroxyhexadecanoic mode ESIMS, a deprotonated molecule at m/z 827 [M − H]− acid by ESIMS and FABMS in the negative mode: m/z 871 [M with diagnostic ions at m/z 681, 535, 439, and 243 was − H]−, 725 [871 − 146]−, 579 [725 − 146]−, 417 [579 − observed. In the low-field region of the HSQC spectrum of 162]−,and271[417− 146]−. The analysis of the δ compound 4, four anomeric signals were observed at H 4.78 monosaccharide mixtures obtained by acid hydrolysis was δ δ (1H, d, J = 8.0 Hz; C 103.7, Qui-1); 5.64 (1H, d, J = 8.0 Hz; C performed by GC-MS with their thiazolidine derivatives of L- ′ δ ″ 101.8, Qui -1); 5.04 (1H, d, J = 8.0 Hz; C 100.9, Qui -1); and cysteine as TMS ethers. The same equimolecular sugar δ 5.83 (1H, brs; C 101.7, Rha-1). COSY (Figure S23, composition for both compounds 12 and 13 was recognized: Supporting Information), and HSQC NMR studies were used D-quinovose, L-rhammnose, D-fucose, and D-glucose. In the low- to assign the 1H and 13C NMR spectra (Tables 3 and 4; Figures field region of the HSQC spectrum of compound 5, four 3 δ S21 and S22, Supporting Information). The following key JCH anomeric signals were observed at H 4.75 (1H, d, J = 8.0 Hz; fi δ δ correlations in the HMBC spectrum con rmed the glyco- C 103.2, Qui-1); 5.98 (1H, d, J = 7.6 Hz; C 100.6, Glc-1); δ δ δ sylation sequence: between H-1 ( H 4.78) of Qui and C-11 of 5.67 (1H, brs; C 99.8, Rha-1), 5.28 (1H, d, J = 7.6 Hz; C δ δ the aglycone ( C 81.3, Conv); H-2 ( H 4.28) of Qui and C-1 107.1, Fuc-1) (Tables 3 and 4; Figures S26 and S27, δ ′ δ ′ δ ( C 101.8) of Qui ; H-3 ( H 4.03) of Qui and C-1 ( C 100.9) Supporting Information). An almost identical HSQC spectrum ″ δ δ ′ of Qui ; and H-1 ( H 5.83) of Rha and C-2 ( C 78.9) of Qui was registered for compound 6 with four anomeric signals δ δ (Figure S24, Supporting Information). The locations for the centered at H 4.76 (1H, d, J = 7.6 Hz; C 103.2, Qui-1); 5.99 fi δ δ lactonization and the esteri cations were established by the (1H, d, J = 7.6 Hz; C 100.6, Glc-1); 5.67 (1H, brs; C 99.8, δ ″ δ following HMBC correlations: between H-2 ( H 5.66) of Qui Rha-1); and 5.29 (1H, d, J = 7.6 Hz; C 107.1, Fuc-1) (Tables 3 and the carbonyl resonance at δc 174.3, assigned to the lactone and 4; Figures S31 and S32, Supporting Information). The 2 functionality due to its JCH coupling with the diastereotopic C- interglycosidic connectivities were established on the basis of δ δ ″ 3 2 methylene protons ( H 3.35 and 2.76); H-3 ( H 5.66) of Qui detailed long-range heteronuclear coupling correlations ( JCH) δ and the carbonyl group of the niloyl moiety ( C 175.2); and H- by HMBC studies. For example, the following key correlations δ 4 of the terminal branched rhamnose ( H 5.86, Rha) and the were observed in compounds 5 and 6 (Figures S29 and S34, δ carbonyl group of the acetyl moiety ( C 171.3). Therefore, the Supporting Information): (a) the connectivity between H-1 of δ δ δ structure of albinosinic acid E (11) corresponded to (11S)- Qui (5, H 4.75; 6, H 4.76) and C-11 of the fatty acid (5, C α → δ δ δ convolvulinolic acid 11-O- -L-rhamnopyranosyl-(1 2)-O-[6- 81.1; 6, C 81.6); (b) H-2 of Qui (5, H 4.45; 6, H 4.44) with β → β δ δ deoxy- -D-glucopyranosyl-(1 3)]-O-6-deoxy- -D-glucopyrano- C-1 of Glc ( C 100.6); (c) H-2 of Glc ( H 4.21) with C-1 of → β δ δ δ syl-(1 2)-O-6-deoxy- -D-glucopyranoside. Rha ( C 99.8); and (d) H-4 of Rha (5, H 4.36; 6, H 4.34) with δ Albinoside VIII (5) yielded a deprotonated molecule peak at C-1 of Fuc ( C 107.1). Accordingly, albinosinic acid F (12) − − δ β m/z 989.4998 [M H] (C48H77O21, calcd error: = +0.35 corresponded to (11S)-hydroxytetradecanoic acid 11-O- -D-

3099 DOI: 10.1021/acs.jnatprod.6b00782 J. Nat. Prod. 2016, 79, 3093−3104 Journal of Natural Products Article

a Table 5. Cytotoxicity of Albinosides IV−IX (1−6) μ IC50 ( M) compound MCF-7 sens MCF-7/Vin− MCF-7/Vin+ MDA HeLa HCT15 HCT-116 1 >20 >20 >20 >20 >20 >20 >20 2 >20 >20 >20 >20 >20 >20 >20 3 >20 >20 >20 >20 >20 >20 >20 4 >20 >20 >20 >20 >20 >20 >20 5 15.1 >20 >20 >20 17.4 14.3 15.1 6 8.0 16.3 17.0 11.6 9.6 9.8 15.9 vinblastine 0.06 1.1 1.3 0.01 0.002 0.06 0.05 adriamycin 1.3 9.2 5.3 NT NT NT NT camptothecin <0.0018 <0.0018 <0.0018 NT NT NT NT colchicine 0.05 2.8 2.8 NT NT NT NT ellipticine 1.6 1.6 2.0 NT NT NT NT podophyllotoxin 0.02 0.2 0.3 NT NT NT NT aMCF-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) μ c IC50 ( M) reversal fold − a + − + compound MCF-7/Vin MCF-7/Vin MCF-7 sens RFMCF‑7/Vin RFMCF‑7/Vin RFMCF‑7 sens vinblastine 1.2 ± 0.1 1.5 ± 0.2 0.05 ± 0.01 1 0.0007 ± 0.0002 0.5 ± 0.026 0.014 ± 0.11 1700 3 4 2 0.0042 ± 0.001 0.52 ± 0.38 0.0014 ± 0.001 283 3 37 3 1.32 ± 0.15 0.66 ± 0.3 0.012 ± 0.006 1 2 4 4 0.0037 ± 0.01 0.0075 ± 0.14 0.0015 ± 0.01 322 201 34 5 0.25 ± 0.03 <0.0006 <0.0006 5 >2517 >87 6 <0.0006 <0.0006 <0.0006 >1984 >2517 >87 reserpineb 0.06 ± 0.02 0.5 ± 0.3 0.005 ± 0.002 20 3 11 aSerial 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. c ± RF = IC50 vinblastine/IC50 vinblastine in the presence of glycolipid. Each value represents the mean SD from three independent experiments. fucopyranosyl-(1→4)-O-α-L-rhamnopyranosyl-(1→2)-O-β-D- (BCRP/ABCG2), and comprised anthracyclines (adriamycin), glucopyranosyl-(1→2)-O-6-deoxy-β-D-glucopyranoside, while podophyllotoxin derivatives, tropolone-like alkaloids (colchi- the structure for albinosinic acid G (13) was characterized as cine), indole alkaloids (reserpine), and other polyaromatic 2b (11S)-hydroxyhexadecanoic acid 11-O-β-D-fucopyranosyl-(1→ small-molecule DNA intercalators (ellipticine). The lack of α → β → μ 4)-O- -L-rhamnopyranosyl-(1 2)-O- -D-glucopyranosyl-(1 cytotoxicity (IC50 >20 M) of the tested samples is an essential 2)-O-6-deoxy-β-D-glucopyranoside. For the natural products, requirement to distinguish between a possible synergism and the location of the macrolactonization by the aglycone, i.e, the real cytotoxic potentiation in the monolayer modulation convolvulinolic acid for 5 and jalapinolic acid for 6, on the assay through their reversal effects. On the basis of our previous 3 oligosaccharide core was determined by the observed JCH results of drug uptake inhibition by resin glycosides with an δ δ correlations between H-3 (5, H 5.82; 6, H 5.84) of glucose anti-P-gp monoclonal antibody and rhodamine 123 in MDR δ 6,7b with C-1 ( C 173.3) of the fatty acid in the HMBC spectrum. MCF-7/Vin cells, the modulation assay was used to identify 3 The positions of acylation were also determined by JCH chemosensitizers through the potentiation of vinblastine correlations between the carbonyl carbons and their corre- susceptibility in MDR cells. This assay employed parental or sponding geminal proton on the oligosaccharide core, vinblastine-sensitive (MCF-7 sens) and vinblastine-resistant indicating the same position for the two tigloyl residues in 5 (MCF-7/Vin− and MCF-7/Vin+) human breast cancer cells δ δ and 6. Thus, one of the tigloyl residues (5, C 168.2; 6, C (Table 6). The reversal fold value (RFMCF‑7/Vin+), as a δ 168.1) was attached at C-2 ( H 5.88) of Rha, and the second parameter of potency, was calculated from dividing the IC50 δ δ δ one ( C 168.8) was linked at C-4 (5, H 5.65; 6, H 5.64) of the of vinblastine alone by the IC50 of vinblastine in the presence of terminal fucose. test compounds.6 This simple bioassay has allowed us to 14 6 7a Cytotoxicity screening using the sulforhodamine B method identify murucoidin V (RFMCF‑7/Vin+ 255), albinoside III, 7b 7c,d was evaluated, and data are presented in Table 5. All glycolipids purgin II, and jalapinosides I and II (RFMCF‑7/Vin+ > 2140), were tested as chemosensitizers in vinblastine-resistant MCF-7/ which proved to be substrates of P-gp in effluxing Vin+ cells by the same method (Table 6). The cross-resistance experiments.6,7b The noncytotoxic albinosides VII (4) and profile displayed by the vinblastine MCF-7/Vin cells (Table 5) VIII (5) exerted the strongest potentiation of vinblastine + was consistent with the MDR expressed by the P-glycoprotein susceptibility with a reversal factor (RFMCF‑7/Vin ) over 201- and (MDR protein1/P-gp, ABCB1) and other transmembrane >2517-fold, respectively. This potency in the reversal of the efflux pumps, such as the breast cancer resistance protein susceptibility to vinblastine was even better than the activity of

3100 DOI: 10.1021/acs.jnatprod.6b00782 J. Nat. Prod. 2016, 79, 3093−3104 Journal of Natural Products Article ° reserpine, a cytotoxic positive efflux pump control (Table 6), at 37 C in an atmosphere of 5% CO2 in air (100% humidity). To and displayed the same potency as the previously reported maintain drug resistance, MCF-7/Vin+ cells were cultured in medium μ value for albinoside III (9, RF > 2140).7a Moderate activities containing 0.192 g/mL vinblastine. At the same time, a stock of MCF-7/Vin cells was maintained in vinblastine-free medium (MCF-7/ were observed for albinosides V (2) (RF 3) and VI (3) (RF 2), − Vin ). which were similar to those reported for albinosides I (7,RF3) Plant Material. Seeds of moon vine (Ipomoea alba; item #01052- and II (8, RF 3).7a Albinoside IV (1) did not exhibit a strong + PK-P1) were acquired from Park Seed (Greenwood, SC, USA) in modulation of cytotoxicity in vinblastine-resistant MCF-7/Vin March 2011. For authentication purposes, 10 seeds were germinated in cells (RF 3). However, it modulated the MDR phenotype in soil, and five seedlings were grown to maturity in an environmental − MCF-7/Vin cells with a reversal factor of 1700. Albinoside IX growth chamber under the following conditions: 25 °C, 65% humidity, (6) was the only active compound against a variety of tumor 16:8 h photoperiod, and 150 μmol/m2/s light intensity. Voucher cell lines (Table 5). Therefore, its reversal activity (RF > 2517) specimens were identified by Dr. Travis D. Marsico and deposited at is the result of an additive synergism that could be substantial the Arkansas State University Herbarium (STAR 027009). − Extraction and Isolation. from a therapeutic perspective. As in previous reports,5 7 there Dried seeds were milled (400 g) and exhaustively extracted by maceration at room temperature with hexane is no evidence of the relationship between chemical structure and then with CHCl3 to yield two extracts after removal of the and modulatory activity since minor variations in the acylation solvents: an oily residue (11 g) and a dark syrup (8.2 g). The resin ff pattern of the oligosaccharide cores could a ect their MDR- glycoside crude mixture of the CHCl3-soluble extract was obtained as a reversal activities. The cross activity displayed among members white solid (6.8 g) by precipitation with MeOH. Then, this crude was μ × of the albinoside series could represent an example of synergy analyzed by reversed-phase C18 (Waters; 7 m, 19 300 mm) HPLC − − between related components in crude extracts, with inactive 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 cytotoxic compounds disabling a resistance mechanism, e.g., μ fi efflux pump-expressing cells, therefore, potentiating the activity 0.1 mg/ L. Comparison with reference compounds con rmed the of cytotoxic substances by the modulation of transporters that presence of previously reported resin glycosides: albinoside I (7) (23 min, peak I), albinoside II (8) (99.5 min, peak VIII), albinoside III (9) confer MDR through competing with toxins for binding to the ffl (128 min, peak IX). Eluates across the peaks with tR values of 30.0 min e ux pump active site. Resin glycosides represent a new class (peak II), 38.2 min (peak III), 44.6 min (peak IV), 49.6 min (peak V), of amphipathic relatively high molecular weight MDR 63.1 min (peak VI), and 109.1 min (peak VII) were collected by the modulators that deserve further biochemical investigations of heart-cutting technique.10 Each subfraction was independently their mechanism of action as chemosensitizers for their low reinjected (sample injection, 500 μL; concentration, 0.1 mg/μL) and cytotoxicity and potent selective behavior that could be used to purified by preparative-scale recycling HPLC10 to achieve total ff homogeneity between 10 and 20 consecutive cycles employing a identify e ective therapeutic drug combination and lower their μ × ff Symmetry C18 column (Waters; 7 m, 19 300 mm), isocratic elution current doses, thereby decreasing toxic side e ects in refractory − − fl with MeOH CH3CN H2O (10:7:3), and a ow rate of 8 mL/min malignancies. Our results suggest that convolvulaceous plants for the first subfraction. For the remaining fractions, isocratic elution elaborate an array of amphipathic bioactive oligosaccharides, of − fl with MeOH CH3CN (7:3) with a ow rate of 8.5 mL/min was used. ff which many have evolved to confer selective advantage to These procedures a orded pure compounds 1 (10 mg; tR 28.2 min) plants. This evolutionary process may have potential in the from peak III, 2 (10 mg; tR 7.9 min) from peak V, 3 (16.3 mg; tR 16.3 discovery of new MDR-modifying leads from plant sources. 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): − ° α − white powder; mp 143 146 C; [ ]589 31.4, ■ EXPERIMENTAL SECTION α − α − α − α − [ ]578 32.9, [ ]546 36.4, [ ]436 60.0, [ ]365 90.0 (c 1.0, MeOH); General Experimental Procedures. Melting points were 1H and 13C NMR, see Tables 1 and 2; negative FABMS m/z 1053 [M − − − − − − − − determined on a Fisher-Johns apparatus and are uncorrected. Optical H] , 971 [M H C5H6O (tigloyl)] , 907 [971 H H2O − − − − rotations were measured with a PerkinElmer model 341 polarimeter. CO2H] , 825 [971 C6H10O4 (methylpentose unit)] , 679 [825 1 13 − − H (500 and 400 MHz) and C (125.7 and 100 MHz) NMR C6H10O4 (methylpentose unit)] , 533 [679 C6H10O4 (methyl- − − − experiments were conducted on a Varian Inova instrument. Negative- pentose unit)] , 389 [533 + H2O C6H10O5 (hexose unit)] , 243 − − ion LRFABMS were recorded using a matrix of triethanolamine on a [389 C6H10O4 (methylpentose unit)] ; HRESIMS m/z 1053.5139 − − Thermo DFS spectrometer. Negative-ion HRESIMS experiments were [M H] (calcd for C49H81O24 requires 1053.5123). Albinoside V (2): − ° α − performed on a Bruker MicrOTOF-Q high-resolution quadruple-time- white powder; mp 136 140 C; [ ]589 17.5, fl α − α − α − α − of- ight mass spectrometer according to the procedure previously [ ]578 18.3, [ ]546 20.8, [ ]436 32.5, [ ]365 47.5 (c 1.0, MeOH); described.6a The instrumentation used for HPLC analysis consisted of 1H and 13C NMR, see Tables 1 and 2; negative FABMS m/z 1137 [M − − − − − − a Waters (Millipore Corp., Waters Chromatography Division, Milford, H] , 1037 [M H C5H8O2 (niloyl)] , 891 [1037 C6H10O4 − − − − MA, USA) 600E multisolvent delivery system equipped with a (methylpentose unit)] , 809 [891 C5H6O (tigloyl)] , 791 [809 − − − − − refractive index detector (Waters 410). Control of the equipment, data H2O] , 745 [791 H CO2H] , 663 [809 C6H10O4 − − acquisition, processing, and management of the chromatographic (methylpentose unit)] , 517 [663 C6H10O4 (methylpentose − − − information were performed by the Empower 2 software (Waters). unit)] , 389 [517 + H2O C6H10O4 (methylpentose unit)] , 243 − − GC-MS was performed on a Thermo-Electron instrument coupled to a [389 C6H10O4 (methylpentose unit)] ; HRESIMS 1137.5712 m/z − − Thermo-Electron spectrometer using the conditions previously [M H] (calcd for C54H89O25 requires 1137.5698). Albinoside VI (3): − ° α − described in the preceding article on the chemistry of moon vine white powder; mp 148 152 C; [ ]589 84.0, 7a α − α − α − α − seeds. [ ]578 78.0, [ ]546 87.0, [ ]436 131.0, [ ]365 183.0 (c 1.0, RPMI 1640 medium and fetal bovine serum were purchased from MeOH); 1H and 13C NMR, see Tables 3 and 4; negative FABMS m/z − − − − − − Gibco (Life Technologies, Carlsbad, CA, USA), and sulforhodamine 967 [M H] , 867 [M H C5H8O2 (niloyl)] , 679 [867 − − B, reserpine, and vinblastine from Sigma-Aldrich (St. Louis, MO, C2H2O (acetyl) C6H10O4 (methylpentose unit)] , 551 [679 + H2O − − − USA). Colon (HCT-15 and HCT-116), cervix (HeLa), and breast C6H10O4 (methylpentose unit)] ,533[679 C6H10O4 − − − (MCF-7 and MDA-MB-231) carcinoma cell lines were acquired from (methylpentose unit)] , 389 [551 C6H10O5 (hexose unit)] , 243 − − the American Type Culture Collection. The resistant counterpart [389 C6H10O4 (methylpentose unit)] ; HRMALDITOFMS m/z fi + MCF-7/Vin was developed and subcultured during ve consecutive 991.4726 [M + Na] (calcd for C45H76O22Na requires 991.4720). 6 Albinoside VII (4): − ° α − years, as previously reported. All cell lines were maintained in RPMI white powder; mp 142 145 C; [ ]589 103.8, α − α − α − α − 1640 medium supplemented with 10% fetal bovine serum and cultured [ ]578 86.9, [ ]546 99.2, [ ]436 147.7, [ ]365 206.2 (c 1.0,

3101 DOI: 10.1021/acs.jnatprod.6b00782 J. Nat. Prod. 2016, 79, 3093−3104 Journal of Natural Products Article

MeOH); 1H and 13C NMR, see Tables 3 and 4; negative FABMS m/z dd, J = 9.0, 8.0 Hz, Qui′-2), 3.89 (1H, dd, J = 9.0, 9.0 Hz, Qui′-3), 3.57 − − − − − − ′ ′ 951 [M H] , 851 [M H C5H8O2 (niloyl)] , 809 [851 (1H, dd, J = 9.0, 9.0 Hz, Qui -4), 3.68 (1H, dq, J = 9.0, 6.0 Hz, Qui - − − − ′ C2H2O (acetyl)] , 663 [809 C6H10O4 (methylpentose unit)] , 535 5), 1.56 (3H, d, J = 6.0 Hz, Qui -6); 2.50 (2H, m, Conv-2b), 2.35 (2H, − − − [663 + H2O C6H10O4 (methylpentose unit)] , 517 [663 t, J = 7.4 Hz, Conv-2b), 3.80 (1H, m, Conv-11), 0.92 (3H, t, J = 7.2 − − 13 δ C6H10O4 (methylpentose unit)] , 389 [535 C6H10O4 (methyl- Hz, Conv-14); C NMR (125 MHz, C5D5N) 103.4 (CH, Qui-1), − − − pentose unit)] , 371 [517 C6H10O4 (methylpentose unit)] , 243 70.2 (CH, Qui-2), 74.4 (CH, Qui-3), 78.3 (CH, Qui-4), 73.5 (CH, − − [389 C6H10O4 (methylpentose unit)] ; HRMALDITOFMS m/z Qui-5), 19.6 (CH3, Qui- 6), 102.8 (CH, Glc-1), 80.4 (CH, Glc-2), + 975.4777 [M + Na] (calcd for C45H76O21Na requires 975.4771). 79.4 (CH, Glc-3), 76.3 (CH, Glc-4), 79.3 (CH, Glc-5), 64.3 (CH2, Albinoside VIII (5): − ° α − white powder; mp 132 135 C; [ ]589 20.0, Glc-6), 102.6 (CH, Rha-1), 75.4 (CH, Rha-2), 80.3 (CH, Rha-3), 74.6 α − α − α − α − ′ [ ]578 21.6, [ ]546 23.2, [ ]436 36.8, [ ]365 53.7 (c 1.0, MeOH); (CH, Rha-4), 80.0 (CH, Rha-5), 19.0 (CH3, Rha-6), 101.6 (CH, Qui - 1H and 13C NMR, see Tables 3 and 4; negative FABMS m/z 989 [M 1), 78.1 (CH, Qui′-2), 75.3 (CH, Qui′-3), 74.5 (CH, Qui′-4), 74.4 − − − − − − ′ ′ H] , 907 [M H C5H6O (tigloyl)] , 825 [907 C5H6O (CH, Qui -5), 17.4 (CH3, Qui -6); 174.1 (CO, Conv-1), 34.5 (CH2, − − − − (tigloyl)] , 679 [825 C6H10O4 (methylpentose unit)] , 533 [679 Conv-2), 73.5 (CH, Conv-11), 14.8 (CH3, Conv-14); positive ESIMS − − + C6H10O4 (methylpentose unit)] , 389 [533 + H2O C6H10O5 m/z 883 [M + K] . − − − Albinosinic acid E (11): − ° α (hexose unit)] , 243 [389 C6H10O4 (methylpentose unit)] ; white powder; mp 102 105 C; [ ]D − − − 1 δ HRESIMS m/z 989.4998 [M H] (calcd for C48H77O21 requires 33.6 (c 1.1, MeOH); H NMR (C5D5N, 500 MHz) 4.85 (1H, d, J 989.4963). = 8.0 Hz, Qui-1), 4.44 (1H, dd, J = 9.0, 8.0 Hz, Qui-2), 4.33 (1H, dd, J Albinoside IX (6): − ° α − white powder; mp 132 135 C; [ ]589 9.0, = 9.0, 9.0 Hz, Qui-3), 3.72 (1H, dd, J = 9.0, 9.0 Hz, Qui-4), 3.54 (1H, α − α − α − α − [ ]578 9.5, [ ]546 11.3, [ ]436 15.9, [ ]365 19.5 (c 1.0, MeOH); dq, J = 9.0, 6.0 Hz, Qui-5), 1.48 (3H, d, J = 6.0 Hz, Qui-6); 5.87 (1H, 1H and 13C NMR, see Tables 3 and 4; negative FABMS m/z 1017 [M d, J = 8.0 Hz, Qui′-1), 4.18 (1H, dd, J = 9.0, 8.0 Hz, Qui′-2), 3.89 (1H, − − − − − − ′ ′ H] , 935 [M C5H6O (tigloyl)] , 853 [935 C5H6O (tigloyl)] , dd, J = 9.0, 9.0 Hz, Qui -3), 3.57 (1H, dd, J = 9.0, 9.0 Hz, Qui -4), 3.68 − − − ′ ′ 707 [853 C6H10O4 (methylpentose unit)] , 561 [707 C6H10O4 (1H, dq, J = 9.0, 6.0 Hz, Qui -5), 1.56 (3H, d, J = 6.0 Hz, Qui -6); 6.31 − − − (methylpentose unit)] , 417 [561 + H2O C6H10O5 (hexose unit)] , (1H, brs, Rha-1), 4.77 (1H, m, Rha-2), 5.03 (1H, dd, J = 9.0, 3.0 Hz, − − 271 [417 C6H10O4 (methylpentose unit)] ; HRFABMS m/z Rha-3), 4.45 (1H, m, Rha-4), 4.83 (1H, m, Rha-5), 1.79 (3H, d, J = 6.5 − − ″ 1017.5265 [M H] (calcd for C50H81O21 requires 1017.5276). Hz, Rha-6); 5.18 (1H, d, J = 8.0 Hz, Qui -1), 4.18 (1H, dd, J = 9.0, 8.0 Alkaline Hydrolysis of Compounds 1−6. Individual solutions of Hz, Qui″-2), 3.87 (1H, dd, J = 9.0, 9.0 Hz, Qui″-3), 3.55 (1H, dd, J = − − ″ ″ compounds 1 6 (10 mg for each one) in 5% KOH H2O (1 mL) 9.0, 9.0 Hz, Qui -4), 3.68 (1H, dq, J = 9.0, 6.0 Hz, Qui - 5), 1.55 (3H, were refluxed at 95 °C for 3 h. Then, the reaction mixtures were d, J = 6.0 Hz, Qui″-6); 2.53 (2H, m, Conv-2b), 2.38 (2H, t, J = 7.4 Hz, fi × acidi ed to pH 5.0 and extracted with CHCl3 (2 5 mL) and Et2O(2 Conv-2b), 3.74 (1H, m, Conv-11), 0.88 (3H, t, J = 7.2 Hz, Conv-14); × 13 δ 5 mL). The organic layer was washed with H2O, dried over C NMR (125 MHz, C5D5N) 102.6 (CH, Qui-1), 70.2 (CH, Qui- anhydrous Na2SO4, evaporated under reduced pressure, and directly 2), 74.4 (CH, Qui-3), 78.3 (CH, Qui-4), 73.5 (CH, Qui-5), 17.4 ′ ′ analyzed by CG-MS and comparison of their spectra and retention (CH3, Qui- 6); 102.1 (CH, Qui -1), 78.1 (CH, Qui -2), 75.3 (CH, 11 ′ ′ ′ ′ times with those of authentic samples. All analytical standards were Qui -3), 74.5 (CH, Qui -4), 74.4 (CH, Qui -5), 18.7 (CH3, Qui - 6); purchased with a purity of >97%: acetic acid (240168, Aldrich); 3- 101.6 (CH, Rha-1), 70.3 (CH, Rha-2), 79.1 (CH, Rha-3), 77.0 (CH, ″ hydroxy-2-methylbutanoic acid (209603, Santa Cruz Biotechnology); Rha-4), 70.0 (CH, Rha-5), 19.2 (CH3, Rha-6); 106.3 (CH, Qui -1), tiglic acid (89450, Sigma-Aldrich). For compounds 3 and 4 acetic acid 78.1 (CH, Qui″-2), 75.3 (CH, Qui″-3), 74.5 (CH, Qui″-4), 74.4 (CH, + ″ ″ (tR 2.81 min) was detected: m/z [M] 60 (65), 45 (80), 43 (100), 29 Qui -5), 18.7 (CH3, Qui -6); 174.1 (CO, Conv-1), 34.6 (CH2, Conv- (19), 15 (25); for compounds 1, 2, 5, and 6, tiglic acid (t 6.95 min) 2), 72.6 (CH, Conv-11), 14.7 (CH3, Conv-14); positive FABMS m/z R − was detected: m/z [M]+ 100 (30), 83 (18), 79 (38), 77 (40), 73 (100), 851 [M + Na]+; negative ESIMS m/z 827 [M − H] . Albinosinic acid F (12): − ° α 65 (9), 55 (22); and for compounds 2−4, 3-hydroxy-2-methylbutyric white powder; mp 104 106 C; [ ]D + − 1 δ acid (tR 7.95 min) was detected: m/z [M] 118 (2.0), 115 (10), 101 27.3 (c 1.1, MeOH); H NMR (C5D5N, 500 MHz) 4.83 (1H, d, J (20), 84 (12), 73 (70), 60 (100). Preparation and identification of 4- = 8.0 Hz, Qui-1), 4.44 (1H, dd, J = 9.0, 8.0 Hz, Qui-2), 4.33 (1H, dd, J bromophenacyl (2R,3R)-3-hydroxy-2-methylbutyrate were performed = 9.0, 9.0 Hz, Qui-3), 3.72 (1H, dd, J = 9.0, 9.0 Hz, Qui-4), 3.54 (1H, − ° α according to a previously reported procedure: mp 56 59 C; [ ]D dq, J = 9.0, 6.0 Hz, Qui- 5), 1.52 (3H, d, J = 6.0 Hz, Qui-6), 5.88 (1H, − 6.0 (c 1.0 CHCl3); GC-MS m/z 118 (2.0), 115 (10), 101 (20), 84 d, J = 7.5 Hz, Glc-1), 4.21 (1H, dd, J = 9.0, 8.0 Hz, Glc-2), 4.08 (1H, (12), 73 (70), 60 (100). This transesterification procedure has been dd, J = 9.0, 8.0 Hz, Glc-3), 3.97 (1H, dd, J = 9.0, 9.0 Hz, Glc-4), 3.87 used to confirm the absolute configuration for 3-hydroxy-2- (1H, ddd, J = 9.0, 6.0, 3.0, Hz, Glc-5), 2.24 (1H, m, Glc-6a), 4.43 (1H, methylbutyrate residue (nla).7a,10 m, Glc-6b), 6.31 (1H, brs, Rha-1), 4.77 (1H, m, Rha-2), 5.03 (1H, dd, The aqueous phases were extracted with n-BuOH (2 × 10 mL) and J = 9.0, 3.0 Hz, Rha-3), 4.45 (1H, m, Rha-4), 4.83 (1H, m, Rha-5), concentrated to give colorless solids. Saponification of compound 1 1.79 (3H, d, J = 6.5 Hz, Rha-6), 5.14 (1H, d, J = 7.5 Hz, Fuc-1), 4.40 yielded albinosinic acid A (14; 6.2 mg): white powder; mp 148−150 (1H, dd, J = 9.0, 7.5 Hz, Fuc-2), 4.08 (1H, dd, J = 9.0, 3.0 Hz, Fuc-3), ° α − − − C; [ ]D 27.6 (c 1.0, MeOH); HRFABMS m/z 989.4799 [M H] . 3.97 (1H, d, J = 3.5 Hz, Fuc-4), 3.89 (1H, m, Fuc-5), 1.44 (3H, d, J = Compound 2 afforded albinosinic acid B (15; 6.4 mg): white powder; 6.5 Hz, Fuc-6); 2.56−2.50 (2H, m, Conv-2), 3.67 (1H, m, Conv-11), − ° α − 13 δ mp 146 148 C; [ ]D 25.0 (c 1.0, MeOH); HRFABMS m/z 0.90 (3H, t, J = 7.0 Hz, Conv-14); C NMR (125 MHz, C5D5N) 973.4850 [M − H]−. These two known glycosidic acids were identified 103.4 (CH, Qui-1), 70.2 (CH, Qui-2), 74.4 (CH, Qui-3), 78.3 (CH, by comparison of their physical and spectroscopic constants with Qui-4), 73.5 (CH, Qui-5), 19.6 (CH3, Qui- 6), 102.8 (CH, Glc-1), published values.7a,13 Saponification of compound 3 produced 10 (7.2 80.4 (CH, Glc-2), 79.4 (CH, Glc-3), 76.3 (CH, Glc-4), 79.3 (CH, Glc- ff mg), compound 4 formed 11 (7.3 mg), compound 5 a orded 12 (8.5 5), 64.3 (CH2, Glc-6), 102.6 (CH, Rha-1), 75.4 (CH, Rha-2), 80.3 mg), and compound 6 yielded 13 (7.7 mg). (CH, Rha-3), 74.6 (CH, Rha-4), 80.0 (CH, Rha-5), 19.0 (CH3, Rha- Albinosinic acid D (10): − ° α − white powder; mp 108 110 C; [ ]D 25 6), 107.2 (CH, Fuc-1), 80.2 (CH, Fuc-2), 79.2 (CH, Fuc-3), 75.9 1 δ (c 0.4, MeOH); H NMR (C5D5N, 500 MHz) 4.82 (1H, d, J = 8.0 (CH, Fuc-4), 71.2 (CH, Fuc-5), 18.3 (CH3, Fuc-6); 177.7 (CO, Conv- Hz, Qui-1), 4.44 (1H, dd, J = 9.0, 8.0 Hz, Qui-2), 4.33 (1H, dd, J = 9.0, 1), 36.4 (CH2, Conv-2), 89.9 (CH, Conv-11), 15.7 (CH3, Conv-14); 9.0 Hz, Qui-3), 3.72 (1H, dd, J = 9.0, 9.0 Hz, Qui-4), 3.54 (1H, dq, J = positive ESIMS m/z 883 [M + K]+; negative ESIMS m/z 843 [M − 9.0, 6.0 Hz, Qui- 5), 1.55 (3H, d, J = 6.0 Hz, Qui-6), 5.87 (1H, d, J = H]−. Albinosinic acid G (13): − ° α − 7.5 Hz, Glc-1), 4.21 (1H, dd, J = 9.0, 8.0 Hz, Glc-2), 4.08 (1H, dd, J = white powder; mp 104 106 C; [ ]D 24 1 δ 9.0, 8.0 Hz, Glc-3), 3.97 (1H, dd, J = 9.0, 9.0 Hz, Glc-4), 3.87 (1H, (c 1.0, MeOH); H NMR (C5D5N, 500 MHz) 4.86 (1H, d, J = 8.0 ddd, J = 9.0, 6.0, 3.0 Hz, Glc-5), 2.24 (1H, m, Glc-6a), 4.43 (1H, m, Hz, Qui-1), 4.44 (1H, dd, J = 9.0, 8.0 Hz, Qui-2), 4.34 (1H, dd, J = 9.0, Glc-6b), 6.32 (1H, brs, Rha-1), 4.77 (1H, m, Rha-2), 5.03 (1H, dd, J = 9.0 Hz, Qui-3), 3.72 (1H, dd, J = 9.0, 9.0 Hz, Qui-4), 3.54 (1H, dq, J = 9.0, 3.0 Hz, Rha-3), 4.45 (1H, m, Rha-4), 4.83 (1H, m, Rha-5), 1.80 9.0, 6.0 Hz, Qui- 5), 1.55 (3H, d, J = 6.0 Hz, Qui-6), 5.88 (1H, d, J = (3H, d, J = 6.5 Hz, Rha-6), 5.86 (1H, d, J = 8.0 Hz, Qui′-1), 4.18 (1H, 7.5 Hz, Glc-1), 4.22 (1H, dd, J = 9.0, 8.0 Hz, Glc-2), 4.10 (1H, dd, J =

3102 DOI: 10.1021/acs.jnatprod.6b00782 J. Nat. Prod. 2016, 79, 3093−3104 Journal of Natural Products Article

9.0, 8.0 Hz, Glc-3), 3.97 (1H, dd, J = 9.0, 9.0 Hz, Glc-4), 3.87 (1H, experiments, reserpine (5 μg/mL) was used as a positive control. The ddd, J = 9.0, 6.0, 3.0 Hz, Glc-5), 2.24 (1H, m, Glc-6a), 4.43 (1H, m, reversal fold (RF) value, as a parameter of potency, was calculated Glc-6b), 6.31 (1H, brs, Rha-1), 4.77 (1H, m, Rha-2), 5.03 (1H, dd, J = from dividing the IC50 of vinblastine alone by the IC50 of vinblastine in 9.0, 3.0 Hz, Rha-3), 4.45 (1H, m, Rha-4), 4.83 (1H, m, Rha-5), 1.92 the presence of test compounds. (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 ■ ASSOCIATED CONTENT (1H, d, J = 3.5 Hz, Fuc-4), 3.89 (1H, m, Fuc-5), 1.48 (3H, d, J = 6.5 *S Supporting Information Hz, Fuc-6); 2.55−2.50 (2H, m, Jal-2), 3.91 (1H, m, Jal-11), 0.88 (3H, 13 δ The Supporting Information is available free of charge on the t, J = 7.0 Hz, Jal-16); C NMR (125 MHz, C5D5N) 102.9 (CH, Qui-1), 70.2 (CH, Qui-2), 74.4 (CH, Qui-3), 78.3 (CH, Qui-4), 73.5 ACS Publications website at DOI: 10.1021/acs.jnat- (CH, Qui-5), 19.2 (CH3, Qui- 6), 102.8 (CH, Glc-1), 80.4 (CH, Glc- prod.6b00782. 2), 79.4 (CH, Glc-3), 76.3 (CH, Glc-4), 79.3 (CH, Glc-5), 63.7 (CH2, Structures for albinosides I−III (Figures S1 and S2) and Glc-6), 102.6 (CH, Rha-1), 72.7 (CH, Rha-2), 68.4 (CH, Rha-3), 73.0 albinosinic acids A−C (Figures S3 and S4); FABMS and (CH, Rha-4), 64.0 (CH, Rha-5), 19.0 (CH3, Rha-6), 106.6 (CH, Fuc- NMR (1H, 13C, COSY, and HMBC) spectra of 1), 80.2 (CH, Fuc-2), 79.2 (CH, Fuc-3), 75.9 (CH, Fuc-4), 71.2 (CH, albinosides IV−IX (1−6; Figures S5−S34); modulation Fuc-5), 17.5 (CH3, Fuc-6), 176.5 (CO, Jal-1), 35.4 (CH2, Jal-2), 80.7 − − assay of vinblastine with compounds 5 and 6 (Figures (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 S35 and S36) (PDF) 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 ■ AUTHOR INFORMATION ether (3 × 10 mL). The organic layer was evaporated to dryness, Corresponding Author dissolved in CHCl3 (3 mL), and treated with CH2N2. The aqueous phase was neutralized with 1 N KOH and extracted with n-BuOH (10 *Tel: +52 55 5622-5288. Fax: +52 55 5622-5329. E-mail: × mL), then washed with H2O(2 5 mL), and concentrated to give a [email protected]. solid. The thiazolidine derivatives of each sugar mixture were prepared ORCID according to previously described procedures,15 converted into volatile 0000-0002-0542-0085 derivatives by treatment with chlorotrimethylsilane (Sigma Sil-A), and Rogelio Pereda-Miranda: analyzed by CG-MS by applying the following conditions: DB-5MS Notes (10 m × 0.18 mm, film thickness 0.18 μm); He, 2 mL/min; 100 °C The authors declare no competing financial interest. isothermal for 3 min, linear gradient to 300 °Cat20°C/min. Retention times for TMS derivatives of common sugar thiazolidines ■ ACKNOWLEDGMENTS were used as standards for GC identification through coelution This research was supported by grants from Direccioń General experiments with L-rhamnose t 4.53 min, D-fucose t 4.56 min, D- R R de Asuntos del Personal Academicó (UNAM, IN215016) and quinovose tR 4.59 min, and D-glucose tR 4.73 min. fi ı́ Identi cation of Aglycones. The derivatized (CH2N2) organic Consejo Nacional de Ciencia y Tecnolog a (CB220535). A.L. layer residues obtained from acid-catalyzed hydrolysis of albinosinic thanks the Arkansas Biosciences Institute for funds in support acids D−G(10−13) were individually submitted to normal-phase of this project. S.C.-M. and J.C.-G. are grateful to CONACyT HPLC (ISCO, 21.2 × 250 mm, 10 μm) using isocratic elution [n- for graduate student scholarships. Thanks are due to G. Duarte − − fl ́ hexane CHCl3 Me2CO (6:3:1)] and a ow rate of 6 mL/min to give (USAI, Facultad de Quimica, UNAM) for the recording of − − 2.0 3.5 mg from compounds 3 5 of methyl (11S)-hydroxytetrade- mass spectra. 11 − ° α 13 canoate: tR 18.6 min; mp 27 29 C; [ ]D +1.5 (c 2, CHCl3); C NMR 174.4, 71.7, 51.5, 39.6, 37.5, 34.1, 29.6, 29.5, 29.3, 29.2, 29.1, ■ REFERENCES 25.6, 24.9, 18.8, 14.1. 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3104 DOI: 10.1021/acs.jnatprod.6b00782 J. Nat. Prod. 2016, 79, 3093−3104