Note Cite This: J. Nat. Prod. 2017, 80, 3038-3042
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Lupane-Type Triterpenes of Phoradendron vernicosum Lía S. Valencia-Chan,†,‡ Isabel García-Cámara,†,‡ Luis W. Torres-Tapia,† Rosa E. Moo-Puc,§ and Sergio R. Peraza-Sánchez*,† †
Unidad de Biotecnología, Centro de Investigación Científica de Yucatán (CICY), Calle 43 No. 130, Col. Chuburná de Hidalgo, Mérida, Yucatán, México 97205 § Unidad de Investigación Médica Yucatán, Unidad Médica de Alta Especialidad, Centro Médico Ignacio García Téllez, Instituto Mexicano del Seguro Social (IMSS), Calle 41 No. 439, Col. Industrial, Mérida, Yucatán, México 97200 S Supporting Information *
ABSTRACT: Three new lupane-type triterpenes, 3α,24dihydroxylup-20(29)-en-28-oic acid (1), 3α,23-dihydroxy-30oxolup-20(29)-en-28-oic acid (2), and 3α,23-O-isopropylidenyl-3α,23-dihydroxylup-20(29)-en-28-oic acid (3), together with eight known compounds (4−11) were isolated from a methanol extract of Phoradendron vernicosum aerial parts. The chemical structures of 1−3 were determined on the basis of spectroscopic data interpretation. The isolated compounds were tested against seven human cancer cell lines and two normal cell lines.
flavonoids.6−10 However, little is known on the chemical composition of other Phoradendron species, with this being the first phytochemical report on P. vernicosum. After conducting extraction of P. vernicosum with methanol and further successive chromatographic procedures, 11 isolates were obtained. Three new triterpenes, 3α,24-dihydroxylup20(29)-en-28-oic acid (1), 3α,23-dihydroxy-30-oxolup-20(29)en-28-oic acid (2), and 3α,23-O-isopropylidenyl-3α,23-dihydroxylup-20(29)-en-28-oic acid (3), were isolated. Also obtained were eight known compounds, namely, lupenone (4),11 betulinic acid (5),12 3-epi-betulinic acid (6),13 3α,23dihydroxylup-20(29)-en-28-oic acid (7),14 betulone (8), betulonic acid (9),15 oleanolic acid (10),16,17 and betulin (11).18 Compounds 1−3 showed bands at 3500 to 3068, 2942 to 2870, 1731 to 1692, and 1650 to 1635 cm−1 in their FT-IR spectra, suggesting the presence of hydroxy and carbonyl groups and double bonds, respectively. Compound 1 was obtained as a white, amorphous solid. Its molecular formula C30H48O4 was determined by HRFABMS (m/z 472.3545). The 1 H NMR data (Table 1) indicated the presence of four methyl singlets (δH 0.78, 0.89, 0.99, 1.11), a vinylic methyl group at δH 1.79 (s), and two olefinic methylene protons at δH 4.95 (1H, br s) and 4.78 (1H, br s), for which the chemical shifts and small coupling constants were found typical of a vinylic methylene unit, confirming the presence of an isopropenyl moiety in the molecule, and a characteristic lupane Hβ-19 signal at δH 3.55 (1H, m). Also, an oxygenated methine proton at δH 3.95 (br s)
C
ancer is one of the principal causes of mortality globally according to the World Health Organization and led to 8.2 million deaths in 2012.1 This group of diseases is characterized by abnormal cell growth, and various drugs currently exist for its treatment. However, these therapies possess diverse disadvantages, such as high cost, undesired side effects, and low efficacy against metastasis and can lead to the development of multidrug resistance mechanisms in abnormal cells, among others.2 In consequence, current research is focused on finding and developing new anticancer drugs with minimal toxic effects. One approach used for this task is the isolation of compounds from plants, since many natural metabolites have proved to be good leads to be developed as effective drugs currently used for chemotherapy in cancer. Among the diverse criteria applied to choose appropriate plants for the discovery of bioactive metabolites, one is selection based on traditional medicine.3 Thus, as part of a search for new compounds with potential anticancer activity from medicinal plants of the Yucatan peninsula in Mexico, 21 species were selected according to their use in the treatment of cancer-like symptoms recorded in the ethnobotanical literature, and then they were evaluated for cytotoxic activity. After this initial step, a methanol extract of Phoradendron vernicosum Greenm. (Santalaceae) was found to show activity against a nasopharyngeal carcinoma cell line.4 In Mexico, the genus Phoradendron has a wide geographical distribution, and plants belonging to this taxon are referred to as mistletoe species. Phoradendron species have been used in different parts of the world in traditional medicine for the treatment of diverse illnesses, including some resembling cancer symptoms.5 Previous chemical analyses of some Phoradendron species have led to the isolation of triterpenes and © 2017 American Chemical Society and American Society of Pharmacognosy
Received: March 1, 2017 Published: November 9, 2017 3038
DOI: 10.1021/acs.jnatprod.7b00177 J. Nat. Prod. 2017, 80, 3038−3042
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alcohol groups. The 13C NMR spectrum (Table 1) indicated the presence of 30 carbons, including a signal at δC 195.8 corresponding to a formyl group, a signal at δC 179.2 of an sp2 carbon of a carboxylic acid group, two signals at δC 158.1 (C20) and 134.2 (C-29) of sp2 carbons of a typical olefinic group, and two oxygenated carbons at δC 76.2 (C-3) and δC 71.8 (C23). The HMBC experiment (Figure 2) showed correlations between H-16/C-28, supporting the location of the carboxylic acid group at C-28. A COSY experiment exhibited coupling of the formyl group proton (δH 9.73) with H-29a, H-29b, and H19 (Figure 2); the correlations observed in the HMBC spectrum between δH 9.73 and C-20 and C-29 proved useful to assign the formyl group at C-30. Protons of the hydroxymethylene group (H-23) showed correlations with C3 and C-24, confirming its location on the A-ring. Finally, the position of the primary hydroxy group was determined to be at C-23 by a NOESY-1D selective experiment, and, for this purpose, CH3-24 was irradiated at 463.95 Hz (0.77 ppm) to a mixing time of 500 ms (128 scans), observing correlations with H-3, H-23, and CH3-25 (Figure 3). In this manner, the new compound 2 was proposed structurally as 3α,23-dihydroxy-30oxolup-20(29)-en-28-oic acid. Compound 3 was obtained as a white, amorphous solid. Its molecular formula was established as C33H52O4 by HREIMS (m/z 512.3841 [M]+). The 1H NMR spectrum (Table 1) exhibited six signals from seven methyl groups (δH 0.68, 0.85, 0.93, 1.01, 1.41 × 2, 1.69). Of these, the methyl group at C-30 (δH 1.69) and the vinylic methylene group at C-29 (δH 4.74 and 4.61) of an isopropenyl moiety were consistent with compound 3 being based on a lupane skeleton. Two protons of an oxygenated methylene (δH 3.66 and 3.23, H-23) and one proton of an oxygenated methine (δH 3.61, H-3), having a βorientation since it was observed as a broad singlet, were also noted in this spectrum. When these signals and the signal of a carbonyl group (δC 182.0, C-28) in the 13C NMR spectrum were compared with those of 1, it was found that compound 3 has an additional ring formed by an isopropylidene moiety (δC 98.0, 29.2, 19.3) attached to the oxygens of the primary and secondary alcohols. The oxygenated methylene protons H-23 showed HMBC cross-linked correlations with the quaternary carbon at δC 98.0, along with C-2 (δC 23.6), C-3 (δC 73.0), C-4 (δC 35.1), C-5 (δC 43.0), and C-24 (δC 17.2). This quaternary carbon was determined to be bonded between C-3 and C-23 as an acetonide group forming a 1,3-dioxane moiety. Also, a ROESY determination indicated an interaction between H-23 and H-5, while a NOESY-1D selective experiment confirmed a spatial correlation among CH3-24 and CH3-25 (Figures S23 and S24, Supporting Information); for the latter, CH3-24 was irradiated at 396.91 Hz (0.68 ppm) to a mixing time of 250 ms (32 scans). These two 1H−1H NMR experiments assisted in the assignment of the acetonide group to a cis α-orientation. Moreover, the signals for the acetonide group were compared with those reported in the literature for such analogues by Tsuda and co-workers,19 helping to establish that the O,Oisopropylidene group and ring A have a cis-decalin conformation (type D). Based on this evidence, the structure of the new compound 3 was elucidated as 3α,23-Oisopropylidenyl-3α,23-dihydroxylup-20(29)-en-28-oic acid. Compounds 1−11 were evaluated for their cytotoxic activities against seven human cancer cell lines (MCF-7, MDA-MB-231, HeLa, SiHa, DU-145, KB, and Hep2) and two normal cell lines (Vero and Hek-293), using docetaxel as a positive control. Compounds 1−11 exhibited cytotoxic activity
and two oxygenated methylene protons at δH 3.89 (1H, d, J = 11.4 Hz) and 3.70 (1H, d, J = 11.4 Hz) were observed, corroborating the presence of the hydroxy groups detected in the IR spectrum. The 13C NMR spectrum supported by DEPT experiments revealed the presence of 30 carbons (Table 1): six methines, including an oxygenated methine carbon at δC 76.3 (C-3); 12 methylenes, one of them oxygenated at δC 71.9 (C24), and another vinylic methylene carbon at δC 110.4 (C-29) from the isopropenyl moiety; five methyl groups at δC 19.9 (C30), 18.5 (C-23), 17.1 (C-27), 17.0 (C-25), and 15.4 (C-26); and seven quaternary carbons, with one being a carbonyl group at δC 179.4 (C-28), characteristic of a carboxylic acid, and one olefinic quaternary carbon at δC 151.8 (C-20). Therefore, the core structure of 1 was determined to be a lupane-type triterpenoid. A comprehensive analysis of the HSQC, HMBC, and DEPT 135 spectra of 1 allowed the assignments of the proton and carbon NMR signals unambiguously. Location of the C-3 hydroxy group was determined via HMBC experiment, in which H-3 exhibited 3J correlations with C-5 and C-24. The α-orientation of OH-3 was assigned on the basis of the small J value of proton H-3 (δH 3.95, br s). In addition, long-range correlations between the oxygenated methylene protons H-24/ C-3, C-4, C-5, and C-23 proved the allocation of a primary hydroxy group at C-24. It is noteworthy that by comparing these signals with those previously reported for 3α,23dihydroxylup-20(29)-en-28-oic acid (sapogenol),14 no significant differences were found, so therefore the position of the hydroxy group was determined by a NOESY experiment (Figure 1), in which the spatial coupling between H-24/H-25 was observed, thus determining that compound 1 is 3α,24dihydroxylup-20(29)-en-28-oic acid. Compound 2 was isolated as white, crystal-like needles. The HRFABMS of 2 showed a molecular ion peak at m/z 486.3338 in accordance with a molecular formula of C30H46O5. The 1H NMR spectra (Table 1) exhibited signals corresponding to four methyl groups (δH 1.06, 0.96, 0.86, 0.77). The presence of an isopropenyl moiety was detected by the occurrence of two signals (δH 6.37, 6.00) belonging to a methylene group, with the vinylic methyl group replaced by a formyl group. This was inferred by a singlet at δH 9.73, which is a key difference compared to 3α,23-dihydroxylup-20(29)-en-28-oic acid (sapogenol).14 A multiplet at δH 3.97 corresponding to H-19 was likewise evident, and also two doublets (δH 3.88 and 3.69, J = 10.8 Hz) forming an AX system and a broad singlet at δH 3.94 suggested the occurrence of both primary and secondary 3039
DOI: 10.1021/acs.jnatprod.7b00177 J. Nat. Prod. 2017, 80, 3038−3042
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Table 1. NMR Spectroscopic Data (δ in ppm, J in Hz) for Compounds 1−3 1 position
δCa
1a 1b 2a 2b 3 4 5 6a 6b 7a 7b 8 9 10 11a 11b 12a 12b 13 14 15a 15b 16a 16b 17 18 19 20 21a 21b 22a 22b 23a 23b 24a 24b 25 26 27 28 29a 29b 30 (CH3)2C (CH3)2C (CH3)2C
34.2, CH2 27.2, CH2 76.3, 41.3, 44.2, 18.9,
CH C CH CH2
35.0, CH2 41.8, 51.4, 38.1, 21.7,
C CH C CH2
26.6, CH2 39.1, CH 43.4, C 30.7, CH2 33.3, CH2 57.1, 50.2, 48.3, 151.8, 31.7,
C CH CH C CH2
38.0, CH2 18.5, CH3 71.9, CH2 17.0, 15.4, 17.1, 179.4, 110.4,
CH3 CH3 CH3 C CH2
19.9, CH3
2
δH (mult., J in Hz)b 1.79, 1.37, 1.97, 1.76, 3.95,
m m m m br s
2.12, 1.59, 1.39, 1.63, 1.35,
d (12) m m m m
1.65, t (11.4) 1.51, 1.22, 1.95, 1.19, 2.74,
m m m m t (12)
1.86, 1.21, 2.61, 1.48,
t (13.2) d (13.2) d (12.6) m
1.70 3.55, m 2.25, 1.51, 2.25, 1.59, 0.78,
m m m m s
3.89, 3.70, 0.89, 0.99, 1.11,
d (11.4) d (11.4) s s s
4.95, br s 4.78, br s 1.79, s
δ Ca 34.2, CH2 27.2, CH2 76.2, 41.3, 44.2, 18.9,
CH C CH CH2
35.0, CH2 41.7, 51.3, 37.9, 21.6,
C CH C CH2
28.4, CH2 38.9, CH 43.3, C 30.7, CH2 33.1, CH2 57.3, 51.7, 51.2, 158.1, 32.7,
C CH CH C CH2
37.9, CH2 71.8, CH2 18.5, CH3 17.1, 16.9, 15.3, 179.2, 134.2,
CH3 CH3 CH3 C CH2
195.8, CH
3
δH (mult., J in Hz)b 1.73, 1.45, 1.93, 1.75, 3.94,
m m m m br s
2.10, 1.58, 1.37, 1.61, 1.35,
d (11.4) m m m m
1.60 d (10.8) 1.44, m 1.12, m 1.42, m 1.02, m 2.69 ddd (12.6, 12.6, 2.4) 1.85, 1.22, 2.63, 1.55,
m m m m
m m m m d (10.8) d (10.8) s
0.86, s 1.06, s 0.96, s 6.37, s 6.00, s 9.73, s
33.1, CH2 23.6, CH2 73.0, 35.1, 43.0, 17.7,
CH C CH CH2
34.0, CH2 40.8, 50.2, 36.9, 20.6,
C CH C CH2
25.4, CH2 38.3, CH 42.4, C 29.6, CH2 33.1, CH2
2.06 3.97, m 2.45, 1.56, 2.24, 1.81, 3.88, 3.69, 0.77,
δCc
56.3, 49.2, 46.9, 150.4, 30.5,
C CH CH C CH2
37.0, CH2 68.3, CH2 17.2, CH3 16.5, 16.1, 14.8, 182.0, 109.6,
CH3 CH3 CH3 C CH2
19.3, 29.2, 19.3, 98.0,
CH3 CH3 CH3 C
δH (mult., J in Hz)d 1.35, 1.28, 1.83, 1.43, 3.61,
m m t (13.5) m br s
1.78, 1.36, 1.29, 1.52, 1.33,
d (12.6) t (7.2) d (13.5) m m
1.46, d (11.4) 1.46, 1.23, 1.69, 1.06, 2.17,
m m m dd (12.6, 3.6) td (12.6, 1.8)
1.52, 1.19, 2.26, 1.41,
m d (12.6) m m
1.61, t (11.4) 3.01, ddd (10.8, 10.8, 4.8) 1.98, 1.40, 1.96, 1.47, 3.66, 3.23, 0.68,
m m m m d (11.4) d (11.4) s
0.85, s 0.93, s 1.01, s 4.74, 4.61, 1.69, 1.41, 1.41,
s s s s s
a13
C NMR data were measured in C5D5N at 125 MHz. b1H NMR data were measured in C5D5N at 600 MHz. c13C NMR data were measured in CDCl3 at 100 MHz. d1H NMR data were measured in CDCl3 at 400 MHz.
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(CC50) in the range 10.2−197.4 μM (Table S1, Supporting Information) and antiproliferative activity (IC50) in the range 12.5−204.5 μM (Table S2, Supporting Information). Although these results classify compounds 1−11 as nonactive according to the National Cancer Institute (CC50 or IC50 values ≤10 μM, active),2 the selectivity indices (SI) of compound 3 (41.0 for cytotoxicity assay and 39.9 for antiproliferative assay) against the nasopharyngeal carcinoma (KB) cell line seem worthy of mention.
EXPERIMENTAL SECTION
General Experimental Procedures. Melting points were determined on a Laboratory Devices, Inc., micromelting point apparatus, model Mel-Temp II, and are uncorrected. Optical rotations were measured on a Rudolph Research Analytical Autopol IV polarimeter. IR spectra were recorded on KBr disks on a Nicolet Protégé Fourier transform IR spectrometer. NMR spectra were recorded in CDCl3, C5D5N, or (CD3)2CO on a Bruker Avance 400 spectrometer and 600 Varian Direct Drive spectrometer. The chemical shifts are given in δ (ppm) with residual deuterated solvent as internal reference and coupling constants in Hz. HREIMS and HRFABMS 3040
DOI: 10.1021/acs.jnatprod.7b00177 J. Nat. Prod. 2017, 80, 3038−3042
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using silica gel 60 (63−200, 40−63, or 2−25 μm particle size) or Sephadex LH-20 from Sigma-Aldrich. Plant Material. Aerial parts of P. vernicosum were collected in October 2011 on Hunucmá- Sisal highway (21°05′38.0″ N, 89°58′21.4″ W), Yucatán (Mexico), and the plant was identified by Tech. Paulino Simá-Polanco (CICY). A voucher sample (PS 2999) was deposited at the U Najil Tikin Xiu herbarium of CICY. The aerial parts were dried under artificial light (50−60 °C) for 3 days and then ground. Extraction and Isolation. Dried aerial parts of P. vernicosum (6.2 kg) were extracted by maceration at room temperature with methanol (MeOH) and dried on a rotatory evaporator under vacuum to obtain 658 g of MeOH extract, which was then suspended in MeOH−H2O (1:3), and the resultant hydroalcoholic solution was successively partitioned with hexane, CH2Cl2, and EtOAc solvents to obtain after removal of the solvents in vacuo the hexane (A, 43.4 g), CH2Cl2 (B, 115.5 g), and EtOAc (C, 71.9 g) fractions, respectively. The active hexane fraction (A, 43.4 g) was chromatographed on a silica gel 60 (2−25 μm) column using hexane, mixtures of hexane− EtOAc (9:1−2:8), EtOAc, and mixtures of EtOAc−MeOH (7:3 and 1:1) as mobile phases, to yield 10 fractions (A1−A10). Fraction A3 (200 mg) was subjected to CC on a silica gel 60 (63−200 μm) column eluting with hexane−EtOAc (98:2) to afford lupenone (4, 87.1 mg). Fraction A6 (11.0 g) was chromatographed on a silica gel 60 column using CH2Cl2, mixtures of CH2Cl2−EtOAc (98:2−1:1), EtOAc, and a mixture of EtOAc−MeOH (7:3), providing 11 fractions (B1−B11). When B4 (3.5 g) was redissolved with CH2Cl2, a precipitate was collected to produce 3-epi-betulinic acid (6, 198.1 mg). The remainder of fraction B4 was separated on a chromatographic column on Sephadex LH-20 using a mixture of hexane−CHCl3−MeOH (2:1:1) as mobile phase, yielding seven fractions (C1−C7). Fraction C4 (1.29 g) was purified by silica gel preparative TLC eluted with hexane− acetone (9:1) to give betulinic acid (5, 16 mg) and a mixture (20 mg), which was submitted to silica gel preparative TLC eluted with hexane−acetone (9:1) to give betulone (8, 8.0 mg) and betulonic acid (9, 10.0 mg). Fraction B6 (318 mg) was purified by silica gel CC using CHCl3−EtOAc (2:8) to obtain four fractions (D1−D4). Fraction D2 (252.5 mg) was subjected to silica gel CC (63−200 μm) eluted with a mixture of CHCl3−EtOAc (9:1) to give 3α,23-dihydroxylup-20(29)en-28-oic acid (7, 11 mg). Fraction A7 (709.4 mg) was subjected to silica gel CC using CH2Cl2−EtOAc (9:1) and MeOH to yield 11 fractions (E1−E11); fraction E11 was found to be pure oleanolic acid (10, 88.5 mg). Fraction E9 (348.6 mg) was subjected to silica gel CC using hexane−EtOAc (8:2) to give betulin (11, 68.6 mg). Fraction A8 (2.9 g) was separated by silica gel CC with hexane−EtOAc (7:3) to obtain 13 subfractions (F1−F13). Fraction F10 (584.7 mg) produced a precipitate (F10A, 248.9 mg) and a filtrate (F10B, 335.8 mg); the filtrate F10B was subjected to silica gel CC with isocratic elution with hexane−acetone (8:2) to yield compound 1 (215.7 mg). Fraction F13 (380.2 mg) was subjected to silica gel CC using gradient elution with hexane, hexane−EtOAc (98:2−1:1), and acetone as mobile phases, obtaining 15 subfractions (G1−G15). Fraction G10 (66.2 mg) was chromatographed over a Sephadex LH-20 column, eluted with hexane−CHCl3−MeOH (2:1:1), to afford compound 2 (49.1 mg). Likewise, the CH2Cl2 fraction (B, 10 g) was subjected to vacuum liquid chromatography on silica gel with hexane−acetone mixtures of increasing polarity (9:1, 8:2, 7:3, 6:4, 1:1) to yield 10 fractions (H1− H10). Fraction H8 (8.3 g) was subjected to silica gel CC using hexane−acetone (9:1) as mobile phase to give 10 subfractions (I1− I10). Fraction I2 (615 mg) was chromatographed over silica gel with hexane−acetone (9:1) to afford 20 subfractions (J1−J20). Fraction J12 turned out to be pure betulinic acid (5, 13.2 mg). Fraction J11 (71.7 mg) was chromatographed over a Sephadex LH-20 column using MeOH as mobile phase to afford four fractions (K1−K4). Separation of fraction K2 (57.2 mg) on a silica gel column with hexane−acetone (8:2) as mobile phase yielded compound 3 (29.8 mg). Fraction J4 (244.3 mg) was purified using a silica gel column with hexane−acetone (20:1) to yield compound 1 (119.1 mg). 3α,24-Dihydroxylup-20(29)-en-28-oic acid (1): white, amorphous powder (C5H5N); mp 199−200 °C; [α]26D −35.0 (c 0.2, CHCl3); IR
Figure 1. Key 1H−1H NOESY correlations of compound 1.
Figure 2. Key 1H−1H COSY and HMBC correlations of compound 2.
Figure 3. Key 1H−1H NOESY correlations of compound 2.
Figure 4. Key HMBC correlations of compound 3.
were measured with a JEOL GCMate II. GC-MS analyses were performed on an Agilent-Technologies gas chromatograph (model 6890N) coupled to a mass detector (model 5975B). A 30-m-long capillary column (Ultra II, 0.25 mm internal diameter, 0.33 μm thick, 5% diphenyl−95% dimethylsiloxane stationary phase) was used; the ionization power was at 70 eV. Precoated TLC silica gel 60 F254 aluminum sheets from Sigma-Aldrich were used for thin-layer chromatography (0.25 and 0.5 mm layer thickness for analytical and preparative TLC, respectively) and visualized under short (254 nm) and long (366 nm) wavelength UV light or a spray reagent (H2SO4− AcOH−H2O, 1:20:4). Column chromatography (CC) was conducted 3041
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(KBr) νmax 3500, 3070, 2942, 1699, 1635 cm−1; 1H and 13C NMR data, see Table 1; HRFABMS m/z 472.3545 (calcd for C30H48O4, 472.3552). 3α,23-Dihydroxy-30-oxolup-20(29)-en-28-oic acid (2): white, crystal-like needles (C5H5N); mp 188−190 °C; [α]26D −30.9 (c 0.5, CHCl3−MeOH, 1:1); IR (KBr) νmax 3500, 2942, 1692 cm−1; 1H and 13 C NMR data, see Table 1; HRFABMS m/z 486.3338 (calcd for C30H46O5, 486.3345). 3α,23-O-Isopropylidenyl-3α,23-dihydroxylup-20(29)-en-28-oic acid (3): white, amorphous powder (CHCl3); mp 111−113 °C; [α]26D −14.8 (c 0.5, CHCl3−MeOH, 1:1); IR (KBr) νmax 3068, 2984, 2937, 2870, 1731, 1699, 1650, 1235, 1195 cm−1; 1H and 13C NMR data, see Table 1; HREIMS m/z 512.3841 (calcd for C33H52O4, 512.3865). Cell Culture. Cell lines of cervix adenocarcinoma (HeLa, ATCCCCL-2), cervix squamous carcinoma (SiHa, ATCC-HTB-35), breast adenocarcinoma (MCF-7, ATCC-HTB-22; MDA-MB-231, ATCCHTB-26), prostate adenocarcinoma (DU-145, ATCC-HTB-81), nasopharynx carcinoma (KB, ATCC-CL-17), laryngeal carcinoma (Hep-2, ATCC-CCL-23), green monkey kidney cells (Vero, ATCCCCL-81), and human cell embryonic kidney cell line (Hek-293, ATCC-CRL-1573), from the American Type Culture Collection (ATCC), were kindly provided by Veronica Vallejo-Ruiz from the East Biomedical Research Center (IMSS, Mexico). The cells were cultured in sterile Costar T25 flasks containing D-MEM medium (Gibco), supplemented with fetal bovine serum (FBS) (10%, v/v), 100 U/mL penicillin G, and 100 μg/mL streptomycin at 37 °C under a 5% CO2 atmosphere (95% humidity). Cytotoxicity and Antiproliferative Assays. Compounds 1−11 were evaluated for their cytotoxic and antiproliferative effects on different cancer cell lines, as reported by Skehan et al.20 Results are expressed as the concentration of agent that reduces cell growth by 50% (CC50 or IC50, respectively), calculated by GraphPad Prism 5 software. Docetaxel was used as positive control. All tests were performed in triplicate. In addition, the level of harmfulness on normal cells was evaluated by determining the selectivity index.21
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REFERENCES
(1) World Health Organization. http://www.who.int/topics/cancer/ en/ (accessed May 12, 2016). (2) National Cancer Institute. http://www.cancer.gov/ (accessed August 11, 2016). (3) Patwardhan, B. J. Ethnopharmacol. 2005, 100, 50−52. (4) Caamal-Fuentes, E.; Torres-Tapia, L. W.; Simá-Polanco, P.; Peraza-Sánchez, S. R.; Moo-Puc, R. J. Ethnopharmacol. 2011, 135, 719−724. (5) Waizel Bucay, J.; Herrera Santoyo, J.; Cortés, D. A.; Villarreal Ortega, M. L. Rev. Inst. Nac. Cancerol. (Mex.) 1994, 40, 133−137. (6) Dossaji, S. F.; Becker, H.; Exner, J. Phytochemistry 1983, 22, 311− 312. (7) Kashiwada, Y.; Wang, H.-K.; Nagao, T.; Kitanaka, S.; Yasuda, I.; Fujioka, T.; Yamagishi, T.; Cosentino, L. M.; Kozuka, M.; Okabe, H.; Ikeshiro, Y.; Hu, C.-Q.; Yeh, E.; Lee, K.-H. J. Nat. Prod. 1998, 61, 1090−1095. (8) López-Martínez, S.; Navarrete-Vázquez, G.; Estrada-Soto, S.; León-Rivera, I.; Rios, M. Y. Nat. Prod. Res. 2013, 27, 130−136. (9) Rios, M. Y.; Salinas, D.; Villarreal, M. L. Planta Med. 2001, 67, 443−446. (10) Varela, B. G.; Fernández, T.; Ricco, R. A.; Cerdá Zolezzi, P.; Hajos, S. E.; Gurni, A. A.; Alvarez, E.; Wagner, M. L. J. Ethnopharmacol. 2004, 94, 109−116. (11) Pereira, F. B. M.; Domingues, F. M. J.; Silva, A. M. S. Nat. Prod. Lett. 1996, 8, 97−103. (12) Peng, C.; Bodenhausen, G.; Qiu, S.; Fong, H. H. S.; Farnsworth, N. R.; Yuan, S.; Zheng, C. Magn. Reson. Chem. 1998, 36, 267−278. (13) Wang, X.-Y.; Zhang, S.-Y.; Li, J.; Liu, H.-N.; Xie, X.; Nan, F.-J. Acta Pharmacol. Sin. 2014, 35, 1463−1472. (14) Yook, C.-S.; Liu, X.-Q.; Chang, S.-Y.; Park, S.-Y.; Nohara, T. Chem. Pharm. Bull. 2002, 50, 1383−1385. (15) Satiraphan, M.; Pamonsinlapatham, P.; Sotanaphun, U.; Sittisombut, C.; Raynaud, F.; Garbay, C.; Michel, S.; Cachet, X. Biochem. Syst. Ecol. 2012, 44, 407−412. (16) Ayatollahi, A. M.; Ghanadian, M.; Afsharypour, S.; Abdella, O. M.; Mirzai, M.; Askari, G. Iran. J. Pharm. Res. 2011, 10, 287−294. (17) Caligiani, A.; Malvasi, G.; Palla, G.; Marseglia, A.; Tognolini, M.; Bruni, R. Food Chem. 2013, 136, 735−741. (18) Tijjani, A.; Ndukwe, I. G.; Ayo, R. G. Trop. J. Pharm. Res. 2012, 11, 259−262. (19) Tsuda, Y.; Sano, T.; Isobe, K.; Miyauchi, M. Chem. Pharm. Bull. 1974, 22, 2396−2401. (20) Skehan, P.; Storeng, R.; Scudiero, D.; Monks, A.; McMahon, J.; Vistica, D.; Warren, J. T.; Bokesch, H.; Kenney, S.; Boyd, M. R. J. Natl. Cancer Inst. 1990, 82, 1107−1112. (21) Mena-Rejón, G.; Caamal-Fuentes, E.; Cantillo-Ciau, Z.; CedilloRivera, R.; Flores-Guido, J.; Moo-Puc, R. J. Ethnopharmacol. 2009, 121, 462−465.
ASSOCIATED CONTENT
S Supporting Information *
The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jnatprod.7b00177. Selected 1H and 13C NMR, 1H−1H COSY, DEPT 135, NOESY, ROESY, HMBC, and HRMS spectra of compounds 1−3 (PDF)
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Note
AUTHOR INFORMATION
Corresponding Author
*E-mail:
[email protected]. Phone: 999-942-8330. ORCID
Sergio R. Peraza-Sánchez: 0000-0002-8161-501X Author Contributions
́ L. S. Valencia-Chan and I. Garcia-Cá mara contributed equally.
‡
Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS This work was supported by the National Council of Science and Technology of Mexico (CONACYT) under grant 156755; L.S.V.-C. received scholarship 423469/290915 from CONACYT; R.E.M.-P. obtained a research grant from the IMSS Foundation. The authors wish to thank P. Simá-Polanco ́ (CICY) for the plant authentication; the Facultad de Quimica at UADY in Mérida (Mexico), for the 400 MHz NMR and HRMS facilities; and LANNBIO at Cinvestav-Mérida (Mexico), for the 600 MHz NMR spectra. 3042
DOI: 10.1021/acs.jnatprod.7b00177 J. Nat. Prod. 2017, 80, 3038−3042
