Article pubs.acs.org/jnp
Karwinaphthopyranones from the Fruits of Karwinskia parvifolia and Their Cytotoxic Activities Claudia Rojas-Flores,†,‡ María Yolanda Rios,†,‡ Rebeca López-Marure,§ and Horacio F. Olivo*,† †
Medicinal and Natural Products Chemistry, The University of Iowa, Iowa City, Iowa 52242, United States Centro de Investigaciones Químicas, Universidad Autónoma del Estado de Morelos, Avenida Universidad 1001, Col. Chamilpa, 62209 Cuernavaca, Morelos, Mexico § Departamento de Biología Celular, Instituto Nacional de Cardiología “Ignacio Chávez,”, Juan Badiano No. 1, Colonia Sección 16, Tlalpan, C. P. 14080, México D. F., Mexico ‡
S Supporting Information *
ABSTRACT: The new 2-acetyl-8-methoxynaphthol (1) and five new “dimeric” napthopyranones, karwinaphthopyranones A1 and A2 (2 and 3) and karwinaphthopyranones B1−B3 (4−6), possessing a methoxy group at C-5′, were isolated together with four other known compounds from the dried fruits of Karwinskia parvifolia. The structures of compounds 2−6 were determined by spectroscopic data interpretation. Cell culture assays showed that some of these compounds possess antiproliferative activities in representative human cancer cell lines, with half-maximal growth inhibitory concentrations in the micromolar range.
naphthol (1), two new dimeric dihydroxyanthracenones, karwinaphthopyranones A1 (2) and A2 (3), and three new dimeric dihydroxyanthracenones with different oxidation states, karwinaphthopyranones B1 (4), B2 (5), and B3 (6), along with four known compounds (7−10). The structures of the new compounds were assigned by detailed spectroscopic analysis.
Karwinskia humboldtiana (Schult.) Zucc. (buckthorn), commonly known as “coyotillo” or “tullidora”, is a poisonous shrub of the Rhamnaceae family, which grows extensively in Mexico, the southwestern United States, and in some regions of Central America. The toxicity of its fruit was known even before the Spanish came to America.1 Accidental ingestion of the poisonous fruit by humans produces a progressive, ascending flaccid paralysis a few days later.2 The fruit and the roots of K. humboldtiana have been the most studied in the past three decades.3 Cytotoxicity of one of the toxins isolated from the seeds of K. humboldtiana was compared to five clinically used chemotherapeutic agents.4 Thus, T-514 (later renamed peroxisomocine A1) showed high selective cytotoxicity toward lung, liver, and colon cancer cells when compared to healthy cells from the same tissues. Chemical screening of different species of the genus Karwinskia showed that K. parvifolia possesses the highest concentration levels of T-514.5 Interest in isolating T-514 for further biological studies prompted us to investigate K. parvifolia. In the present study, the chemical constituents of the fruits of Karwinskia parvifolia Rose (Rhamnaceae) collected near Tasquillo, Hidalgo, Mexico, were investigated. A large-scale extraction of the dried fruits of K. parvifolia yielded a new © XXXX American Chemical Society and American Society of Pharmacognosy
■
RESULTS AND DISCUSSION Air-dried and powdered K. parvifolia fruits were extracted initially with hexanes to remove fatty acids and waxes. The plant material was then extracted with dichloromethane, and this dried extract was fractionated chromatographically with benzene−acetone. These fractions were then purified again, eluting with hexanes−ethyl acetate mixtures to afford 10 compounds, 2-acetyl-8-methoxynaphthol (1), karwinaphthopyranones A1 and A2 (2 and 3), karwinaphthopyranones B1 to B3 (4−6), and four known compounds (7−10), as described below. Four known compounds (7−10), related to 1−6, were also isolated in this investigation. Of these, torachrysone monoReceived: May 27, 2014
A
dx.doi.org/10.1021/np500430q | J. Nat. Prod. XXXX, XXX, XXX−XXX
Journal of Natural Products
Article
(8), when treated with HCl in acetic acid.5a 1-O-Methylchrysophanol (10) has been isolated from Cassia obtusifolia.14 This anthraquinone was also obtained chemically by condensation of a vinyl ketene with a napthoquinone.15 The new products 1−6 were isolated and characterized. These compounds are analogous to monomers and dimers from other species of Karwinskia. The structure elucidation and assignment of signals of 1H and 13C NMR (Tables 1 and 2) were corroborated by DEPT-135 and 2D experiments (copies of COSY, HMQC, and HMBC). Copies of 1D and 2D spectra can be found in the Supporting Information. 2-Acetyl-8-methoxynaphthol (1) was isolated in the initial fractions, with the molecular formula deduced from the (+)-HRFABMS being C13H12O3. The 13C NMR signals for this compound showed a carbonyl ketone at δC 203.7 and 10 aromatic carbons, with two of them attached to oxygens (δC 164.4 and 159.7). The 1H NMR data indicated the presence of a phenolic proton forming a bridge bond with a carbonyl (δH 14.51), a methyl ether (δH 4.04), and an acetyl group (δH 2.69). All aromatic signals were well resolved in the 1H NMR spectrum. H-3 coupled with H-4 (δH 7.64 and 7.18, J = 8.7 Hz), and H-6 (δH 7.50) coupled with both H-5 and H-7 (δH 7.31 and 6.87). A COSY experiment was used to corroborate these couplings. In the HMBC spectrum, a correlation was observed between H-3 and the acetyl carbonyl, C-1, and C-4a. Additionally, H-6 correlated with C-4a and C-8. The molecular formula of karwinaphthopyranone A1 (2) was deduced to be C 33 H 34 O 9 by (+)-HRFABMS analysis, corresponding to 17 degrees of unsaturation. The 13C NMR data (Table 2) of this compound showed signals for a ketone at δC 203.0 and an α,β-unsaturated ketone at 183.1, confirmed by an IR strong absorption band at 1632 cm−1. Altogether, 18 signals were observed corresponding to aromatic and vinyl carbons, with five of these corresponding to methine carbons. The methine carbons corresponded to three aromatic rings
methyl ether (7) was isolated earlier from the roots of K. humboldtiana6 and in addition from the seeds of Cassia tora.7 Anthracenone T-544 was isolated from the fruits of K. humboldtiana6 and also from the roots of K. parvifolia. This anthracenone was isolated as an inseparable mixture of diastereomers.8,2 T-544 was renamed tullidinol, as it was found to be the agent responsible for the neurotoxicity found in the fruit.9 Anthracenone T-514 or peroxisomicine A1 (8) was isolated from K. humboldtiana and other species of Karwinskia.6 T-514 was renamed peroxisomicine A1 (PA1) because it was found to cause an irreversible and selective damage of yeast peroxisomes in vivo.10 PA1 was also found to inhibit catalase in vitro. 11 Minor diastereomers of PA1 have also been reported.12,13 Bis-anthracenone T-478 (9) was obtained previously chemically by dehydration of peroxisomicine A1
Table 1. 1H (300 MHz) NMR Spectroscopic Data for Compounds 2−6 (δ in ppm, J in Hz) 2a
3b
position
δH (J in Hz)
δH (J in Hz)
2 Me-3 4
2.14, 0.90, 2.53, 2.41, 7.18, 8.63, 6.63,
7.05, d (1.6) 2.44, s 7.60, d (1.6)
2.80, m 1.44, s 3.16, br s
6.50, s 2.32, s/2.18, s 6.85, s/6.67, s
6.65, s 2.36, s/2.23, s 7.60, d (1.2)/7.04, s
7.91, d (8.0) 8.49, d (8.0)
7.24, d (8.7) 8.32, d (8.7) 6.99, br s
6.73, d (7.6)/6.20, m 8.04, d (7.8)/7.97, d (7.8) 4.48, d (15.7)/4.43, d (15.7) 11.44, s/11.18, s 12.00, s/11.73, s
7.91, d (8.1)/6.98, d (8.1) 8.51, d (8.1)/8.29, d (8.0) 11.92, s/11.73, s 12.41, s/12.20, s
4.89, 1.58, 3.35, 1.13, 1.98, 3.06, 6.99, 7.55,
4.95, 1.55, 3.47, 1.19, 1.78, 3.07, 6.85, 7.37,
5 6 10 HO-1 HO-8 HO-9 1′ Me-1′ 3′ Me-3′ 4′ MeO-5′ 6′ 7′ MeO-7′ 8′ MeO-9′ a
m s d (16.0) d (16.0) d (8.2) d (8.2) br s
10.04, s 16.06, br s 5.38, d (6.5) 1.95, d (6.5) 3.48, m 1.06, d (6.2) 1.97, m 3.07, s 6.68, d (2.0) 3.09, s 6.28, d (2.0) 3.42, s
11.85, s 12.30, s 4.93, 1.55, 3.45, 1.18, 1.70, 3.09, 6.30,
m d (6.4) m d (6.2) m s d (2.3)
3.72, s 6.43, d (2.3) 3.94, s
4b
5/5′-epi-5b
δH (J in Hz)
6/5′-epi-6b
δH (J in Hz)
9.76, s 16.98, br s 4.93, m 1.55, d (6.5) 3.41, m 1.16, d (6.3) 1.98, m 3.06, s 6.87, d (8.1) 7.32, dd (8.1, 8.1) 6.88, d (8.1) 3.95, s
δH (J in Hz)
m d (6.5)/1.52, d (6.5) m d (6.4)/1.11, d (6.0) m s/3.04, s d (8.2) dd (8.2, 8.2)
7.26, d (8.0)/6.84, d (8.0) 4.00, s/3.93, s
m d (6.4) m d (6.0)/1.18, d (6.0) m s/3.05, s d (7.6)/6.82, d (7.6) dd (7.8, 7.8)/7.34, dd (7.9, 7.9)
6.92, d (8.6)/6.88, d (8.4) 3.97, s/3.95, s
Data measured in C6D6. bData measured in CDCl3. B
dx.doi.org/10.1021/np500430q | J. Nat. Prod. XXXX, XXX, XXX−XXX
Journal of Natural Products
Article
Table 2. 13C (75 MHz) NMR Spectroscopic Data for Compounds 2−6 (δ in ppm) position 1 2 3 Me-3 4 4a 5 6 7 8 8a 9 9a 10 10a 1′ Me-1′ 3′ Me-3′ 4′ 4′a 5′ MeO-5′ 5′a 6′ 7′ MeO-7′ 8′ 9′ MeO-9′ 9′a 10′ 10′a a
2a
3b
4b
5/5′-epi-5b
6/5′-epi-6b
δC, type
δC, type
δC, type
δC, type
δC, type
203.0, 50.9, 70.1, 28.5, 43.0, 135.6, 117.7, 131.1, 125.0, 154.8, 113.0, 166.4, 109.6, 118.0, 139.3, 71.7, 22.1, 68.9, 21.6, 33.1, 143.3, 77.0, 50.0, 148.0, 103.9, 163.5, 54.6, 98.7, 161.7, 55.5, 118.4, 183.1, 141.1,
C CH2 C CH3 CH2 C CH CH C C C C C CH C CH CH3 CH CH3 CH2 C C CH3 C CH C CH3 CH C CH3 C C C
162.6, 124.4, 149.3, 22.2, 121.2, 132.8, 119.2, 134.6, 138.5, 159.9, 115.5, 192.5, 113.7, 181.9, 133.1, 71.0, 21.4, 68.4, 21.2, 32.4, 142.4, 76.2, 50.0, 145.7, 103.7, 163.2, 55.3, 98.4, 161.2, 56.1, 116.9, 182.9, 140.6,
C CH C CH3 CH C CH CH C C C C C C C CH CH3 CH CH3 CH2 C C CH3 C CH C CH3 CH C CH3 C C C
202.8, 51.0, 76.3, 28.9, 43.0, 134.7, 117.6, 136.8, 123.9, 153.8, 112.4, 165.9, 109.2, 118.0, 138.8, 70.8, 21.3, 68.5, 21.1, 32.3, 145.4, 76.0, 50.0, 145.0, 110.6, 132.7,
C CH2 C CH3 CH2 C CH CH C C C C C CH C CH CH3 CH CH3 CH2 C C CH3 C CH CH
161.8/161.6, C 117.0/117.0, CH 147.6/147.3, C 22.1/21.9, CH3 121.1/120.7, CH ? 119.3/119.7, CH 133.0/132.3, CH 130.0/130.5, C 158.1/158.0, C 116.3/116.3, C 195.2/191.5, C 114.7/114.3, C 32.5/32.3, CH2 ? 70.9/71.0, CH 21.2/21.3, CH3 68.7/68.4, CH 21.6/22.0, CH3 32.1/31.9, CH2 144.0/143.6, C 75.7/75.6, C 50.0/49.9, CH3 144.4/144.4, C 110.8/111.0, CH 133.1/132.6, CH
162.5/162.4, C 124.2/115.7, CH 149.2/147.8, C 22.0/21.9, CH3 119.5/121.0, CH 141.2/132.8, C 119.3/119.1, CH 134.7/133.5, CH 137.8/128.2, C 158.8/158.6, C 115.3/115.2, C 193.2/192.2, C 113.4/113.2, C 181.5/?, C 141.1/132.8, C 70.9/70.9, CH 21.2/21.1, CH3 68.4/68.4, CH 21.1/21.1, CH3 32.2/32.2, CH2 143.5/143.2, C 75.9/75.6, C 49.9/49.8, CH3 144.7/144.2, C 111.2/110.7, CH 132.9/132.6, CH
119.6, 158.8, 55.9, 122.4, 184.3, 139.7,
CH C CH3 C C C
118.7/118.5, CH 158.9/158.9, C 56.1/56.2, CH3 122.6/122.4, C 191.3/184.1, C 140.4/140.3, C
119.3/118.0, CH 159.0/158.8, C 55.9/55.9, CH3 122.5/122.4, C 183.9/183.8, C 140.6/140.0, C
Data measured in C6D6. bData measured in CDCl3.
according to the aromatic signals observed in the 1H NMR spectrum (Table 1). The 1H NMR downfield signals at δH 16.06 and 10.04 corresponded to two hydroxy groups, and the two doublets (δH 8.63 and 7.18, J = 8.2 Hz) and a singlet (δH 6.63) to the aromatic protons in the dihydroxyanthracenone moiety. The two methylene carbons at δC 50.9 and 43.0, the methyl carbon at δC 28.5, and the quaternary carbon at δC 70.1 supported the presence of a dihydroxyanthracenone moiety. All the 1H and 13C NMR signals with the exception of those for C6 and C-7 for the dihydroxyanthracenone moiety are very similar to those previously reported for peroxisomicine A1 (8).13 The two remaining aromatic protons (δH 6.28 and 6.68, J = 2.0 Hz) and methines (δC 98.7 and 103.9) were assigned to ring C′ of the naphthopyranone moiety. Carbons 7′ and 9′ (δC 163.5 and 161.7, respectively) were found to bear methoxy groups. The α,β-unsaturated carbonyl was assigned to ring B′. The two methyl group signals in the 1H NMR spectrum appeared as doublets at δH 1.95 and 1.06. These two methyl group signals appeared in the 13C NMR spectrum at δC 22.1 and 21.6, and the two methyl groups attached to C-1′ (δC 71.7) and C-3′ (δC 68.9) were assigned to the 2,3-dihydropyran ring A′. The methylene C-4′ showed resonances at δC 33.1 and δH 1.97, while the C-5′ quaternary carbon was clearly observed in C6D6 at δC 77.0 (confirmed by DEPT-135 and HMBC). This
chemical shift suggests that the third methoxy group (δC 50.0) is attached to C-5′. The presence of this methoxy group is responsible for the chemical shift changes observed in H-6, C-6, and C-7 in the dihydroxyanthracenone moiety of this compound and PA1. The connection between the 3,4dihydronaphthopyrone and the dihydroxyanthracenone moieties was assigned between C-5′ and C-7, similar to several previous dimeric analogues isolated from Karwinskia, seedlings of Cassia torosa,16 and also in several toadstools.17
Figure 1. Selected HMBC correlations observed for 2 and 5. C
dx.doi.org/10.1021/np500430q | J. Nat. Prod. XXXX, XXX, XXX−XXX
Journal of Natural Products
Article
aromatic protons (δH 6.50 and 6.85). The 13C NMR data showed two pairs of carbonyl carbons assigned to C-9 and C10′. The nonaromatic methyl ether (δH 3.06, δC 49.9) was assigned at C-5′ (δC 75.6). Two upfield proton resonances (δH 12.00 and 11.44) suggested the presence of two phenolic protons forming a bridge head with the C-9 carbonyl group (δC 195.2). A methylene proton (δH 4.48) was assigned to C-10 (δC 32.5). Karwinaphthopyranone B2 (5) was determined as a dehydration product of karwinaphthopyrone B1 (4). The molecular formula of karwinaphthopyranone B3 (6) was determined as C32H28O8 by (+)-HRFABMS analysis. Both the 1 H and 13C NMR data were very similar to those of karwinaphthopyranone B2, indicating these compounds to be very closely related analogues. Similar to karwinaphthopyranone B2, karwinaphthopyranone B3 was obtained as a 5:4 mixture of diastereomers. The molecular formula indicated 19 degrees of unsaturation, one more than B1. The 13C NMR data showed the presence of three carbonyl groups (δC 183.9, 193.2, and 181.5). In turn, the 1H NMR data showed an ABX system (δH 6.85, 7.37, and 6.92), two methine protons (δH 4.95 and 3.47), methylene protons (δH 1.78, 2H), and two methyl groups (δH 1.55 and 1.19), corresponding to the naphthopyrone moiety. The 1H NMR data also showed an AB system (δH 7.91 and 8.51) and two meta-coupled protons (δH 6.65 and 7.60). Signals for an aromatic methyl ether (δH 3.97, δC 55.9) and also a nonaromatic methyl ether (δH 3.07, δC 49.9) were observed. The downfield signals present in the 1H NMR spectrum were considered as typical 1,8-dihydroxyanthraquinone resonances (δH 12.41 and 11.92). The carbonyl group (δC 181.5) was assigned at C-10. Karwinaphthopyranone B3 (6) was assigned as an oxidation product of karwinaphthopyranone B2 (5).
The molecular formula of karwinaphthopyranone A2 (3) was deduced as C33H30O9 by (+)-HRFABMS analysis, representative of 19 degrees of unsaturation. Interestingly, this compound was isolated as a pure diastereomer, compared to karwinaphthopyranones B2 and B3. The 13C NMR data of this compound showed signals for three carbonyl carbons at δC 192.5, 182.9, and 181.9 and 20 aromatic/vinyl carbons, with four of these being attached to oxygens (δC 163.2, 162.6, 161.2, and 159.9) and six methines (δC 134.6, 124.4, 121.2, 119.2, 103.7, and 98.4). Also observed were signals for two aromatic methyl ethers (δC 56.1 and 55.3), a nonaromatic methyl ether (δC 50.0), two methines attached to oxygen (δC 71.0 and 68.4), a methylene (δC 32.4), and three methyl carbons (δC 22.2, 21.4, and 21.2) (Table 2). The occurrence of two aromatic methyl ethers suggested this compound is an analogue of karwinaphthopyranone A1. The 19 degrees of unsaturation inherent in the molecular formula suggested three aromatic rings, three other rings, three carbonyl groups, and one olefin. The two oxygenated methines (δC 71.0 and 68.4) and the methylene carbon (δC 32.4) suggested the presence of a dihydropyran ring (ring C′). The 1H NMR data showed two downfield signals for the chelated phenols at δH 12.30 and 11.85, corresponding to a 1,8-dihydroxyanthaquinone, in addition to signals of six aromatic protons, all of them doublets, two at δH 8.49 and 7.91 (Jortho = 8 Hz), two at 7.60 and 7.05 (Jmeta = 1.6 Hz), and two at 6.43 and 6.30 (Jmeta = 2.3 Hz), with each pair of protons belonging to an aromatic ring, two methine protons at δH 4.93 and 3.45, two aromatic methyl ethers (δH 3.94 and 3.72), and one nonaromatic methyl ether (δH 3.09). The chelated protons OH-1 and OH-8 showed long-range correlations with the aromatic carbons C-2 and C-6 in the HMBC spectrum. The structure was assigned for this compound (karwinaphthopyranone B3), which is a dehydrated and oxidized analogue of karwinaphthopyranone A1 (2). The molecular formula of karwinaphthopyranone B1 (4) was deduced as C32H32O8 from its (+)-HRFABMS. This compound presented signals in its NMR spectra very similar to those of karwinaphthopyranone A1, but an aromatic proton replaced the methoxy group resonance at C-7′ (Tables 1 and 2). The 1H NMR spectrum showed three aromatic protons coupled to each other on ring C′ (δH 6.87, 7.32, and 6.88) and three protons corresponding to the dihydroxyanthracenone moiety in a similar manner to karwinaphthopyranone A1 (2), as an AB system (δH 7.24 and 8.32) and a singlet (δH 6.99). The aromatic methyl ether (δH 3.95, δC 55.9) was assigned at C-9′ (δC 158.8), and the nonaromatic methyl ether (δH 3.06, δC 50.0) attached to C-5′ (δC 76.0). The 13C NMR aromatic region showed six CH carbons, confirming the presence of only one aromatic methoxy group. This compound was assigned as the 7′-desmethoxy analogue of karwinaphthopyranone A1. The molecular formula for karwinaphthopyrone B2 (5) was deduced as C32H30O7 from its (+)-HRFABMS, corresponding to 18 degrees of unsaturation. The small amount of this compound and the presence of an inseparable diastereomer complicated its characterization. Karwinaphthopyrone B2 was obtained as a 5:4 mixture of diastereomers. Signals for an ABX system (δH 6.99, 7.55, and 7.26), an aromatic methyl ether (δH 4.00), two methine protons (δH 4.89 and 3.35), two methyl groups (δH 1.58 and 1.13), and the methylene protons (δH 1.98) suggested the presence of the same naphthopyranone moiety as in karwinaphthopyrone B1. However, the AB system proton signals present at H-5 and H-6 appeared at a slightly different region (δH 6.73 and 8.04), and there were two more
Compounds 1−10 were evaluated for cytotoxic activity against the A549 (human lung cancer), HeLa (human cervical cancer), ZR-7530 (human breast cancer), HCT-15 (human colon cancer), U373 (human brain cancer), and Hep G2 (human liver cancer) cell lines (Table 3).18 Three compounds showed activity against the Hep G2 cell line, tarachrysone monomethyl ether (7) (IC50 9.6 μM), 1-methylchrysophanol (10) (IC50 9.3 μM), and karwinaphthopyrone B1 (4) (IC50 4.6 μM). Three compounds showed activity against the ZR-7530 cell line, peroxisomicine A1 (8) (IC 50 5.8 μM), bisanthracenone 9 (IC50 9.6 μM), and karwinaphthopyranone B1 (4) (IC 50 5.5 μM). Peroxisomicine A1 (8) and karwinaphthopyranone B1 (4) showed activity against the HCT-15 cell line (IC50 4.9 and 4.6 μM, respectively). Peroxisomicine A1 (8) also showed activity against the HeLa cell line (IC50 4.9 μM). Interestingly, compounds possessing the 8,9-dihydroxyanthracenone showed more potent cytotoxic activities. D
dx.doi.org/10.1021/np500430q | J. Nat. Prod. XXXX, XXX, XXX−XXX
Journal of Natural Products
Article
fractionated on silica gel column chromatography (14.0 × 4.0 cm, hexane−AcOEt, 80:20, to hexane−AcOEt, 60:40) and yielded 1methylchrysophanol (10) (119.1 mg, 0.017% yield), torachrysone monomethyl ether (7) (34.0 mg, 0.048% yield), and karwinaphthopyranone B2 (5) (126.7 mg, 0.18% yield). Fractions B6 and B7 were combined (2.0 g) and fractionated on a silica gel column (14.0 × 4.5 cm, hexane−AcOEt, 70:30, to hexane−AcOEt, 60:40) and yielded karwinaphthopyranone B3 (6) (15.3 mg, 0.022% yield) and karwinaphthopyranone B1 (4) (9.5 mg, 0.013% yield). Fraction C1 (1.3 g) was purified with a silica gel chromatographic column (14.0 × 4.0 cm, hexane−AcOEt, 70:30, to hexane−AcOEt, 60:40), to afford karwinaphthopyranone B3 (6) (9.8 mg, 0.014% yield) and an impure subfraction. Subfraction C1-1 (257 mg) was purified by silica gel chromatography (14.0 × 2.0 cm, benzene 100% to benzene−acetone, 96:4), yielding tullidinol (20.2 mg, 0.028% yield). Fractions C2−D3 yielded anthracenone T-510 (2.7 g, 3.8% yield). Fractions D4 and D5 (2.6 g) were separated using a silica gel chromatographic column (14.0 × 4.0 cm, CH2Cl2−MeOH−NH4OH, 94:5:1) and yielded an impure subfraction (645 mg). Subfraction D5-1 was purified by a chromatographic column containing silica gel (14.0 × 3.5 cm, CH2Cl2−acetone, 90:10), yielding karwinaphthopyranone A2 (3) (39.9 mg, 0.057% yield), and fractions D6−E3 yielded karwinaphthopyranone A2 (3) (2.4 g, 3.4% yield). Fraction E4 (821 mg) was purified by silica gel column chromatography (12.0 × 2.0 cm, CH2Cl2−MeOH−NH4OH, 89:10:1) and yielded karwinaphthopyranone A1 (2) (16.1 mg, 0.023% yield). Fraction E5 (357 mg) was fractionated in a silica gel chromatographic column (16.0 × 2.0 cm, CH2Cl2−MeOH− NH4OH, 89:10:1, to CH2Cl2−MeOH−NH4OH, 78:20:2) and yielded PA-1 (8) (6.9 mg, 0.01% yield). Fraction E6 yielded a mixture of karwinaphthopyranone A1 (2) and PA-1 (8) (658 mg, 8:2 ratio, 0.93% yield). 2-Acetyl-8-methoxynaphthol (1): yellow needles; mp 118−119 °C; UV (CHCl3) λmax (log ε) 386 (2.99), 259 (3.70) nm; IR (film) νmax 3011, 2837, 1621, 1577, 1379, 1348, 1275, 1262, 1152, 1086, 968, 911, 818, 793 cm−1; 1H NMR (CDCl3, 300 MHz) δ 14.51 (1H, br s, OH-1), 7.64 (1H, d, J = 8.7 Hz, H-3), 7.50 (1H, dd, J = 8.1, 8.1 Hz, H6), 7.31 (1H, d, J = 8.1 Hz, H-5), 7.18 (1H, d, J = 8.7 Hz, H-4), 6.87 (1H, d, J = 8.1 Hz, H-7), 4.04 (3H, s, CH3O-8), 2.69 (3H, s, COCH3); 13 C NMR (CDCl3, 75 MHz) δ 203.7 (s, COCH3), 164.4 (s, C-1), 159.7 (s, C-8), 140.4 (s, C-4a), 130.7 (d, C-6), 126.2 (d, C-3), 120.6 (d, C-5), 118.5 (d, C-4), 116.4 (s, C-8a), 114.4 (s, C-2), 106.5 (d, C7), 56.4 (q, CH3O-8), 27.8 (q, COCH3); 1H NMR (C6D6, 300 MHz) δ 15.06 (1H, br s, OH-1), 7.43 (1H, d, J = 8.8 Hz, H-3), 7.39 (1H, dd, J = 8.0,7.2 Hz, H-6), 7.35 (1H, m, H-5), 7.15 (1H, d, J = 8.8 Hz, H-4), 6.58 (1H, dd, J = 7.2, 1.7 Hz, H-7), 3.61 (3H, s, CH3O-8), 2.28 (3H, s, COCH3); 13C NMR (C6D6, 75 MHz) δ 203.6 (s, COCH3), 165.9 (s, C-1), 161.0 (s, C-8), 141.2 (s, C-4a), 131.1 (d, C-6), 127.2 (d, C-3), 120.9 (d, C-5), 118.6 (d, C-4), 117.7 (s, C-8a), 115.3 (s, C-2), 107.0 (d, C-7), 56.2 (q, CH3O-8), 27.7 (q, COCH3); (+) FABMS m/z 217 [C13H13O3, M + H]+ (100), 216 [M]+ (65), 201 (28), 183 (8), 154 (9), 136 (13), 115 (13), 109 (12), 95 (18), 91 (17), 55 (15); (+)-HRFABMS m/z 217.0880 [M + H]+ (calcd for C13H13O3, 217.0865). Karwinaphthopyranone A1 (2): dark brown resin; [α]25D +147.3 (c 1.0, CHCl3); UV (CHCl3) λmax (log ε) 242 (3.93), 274 (3.99), 420 (3.34) nm; IR (film) νmax 3431, 2968, 2930, 2852, 1632, 1599, 1456, 1419, 1380, 1319, 1272, 1160, 1094, 1070 cm−1; 1H NMR (C6D6, 300 MHz), see Table 1; 13C NMR (C6D6, 75 MHz), see Table 2; (+) FABMS m/z 574 [C33H34O9, M]+ (24), 570 (21), 525 (18), 499 (17), 458 (17), 423 (16), 397 (100), 317 (16), 289 (22), 255 (47), 219 (28); (+)-HRFABMS m/z 574.2289 [M]+ (calcd for C33H34O9, 574.2203). Karwinaphthopyranone A2 (3): dark red resin; [α]25D +138.4 (c 1.0, CHCl3); UV (CHCl3) λmax (log ε) 236 (3.56), 244 (3.70), 438 (3.04) nm; IR (film) νmax 3280, 2929, 2853, 2357, 1735, 1600, 1455, 1424, 1287, 1262, 1219, 1147, 1074 cm−1; 1H NMR (CDCl3, 300 MHz), see Table 1; 13C NMR (CDCl3, 75 MHz), see Table 2; (+) FABMS m/z 570 [C33H30O9, M]+ (9), 538 (7), 494 (5), 460 (6), 391 (8), 346 (7), 307 (61), 289 (58), 260 (25), 219 (100); (+)-HRFABMS m/z 570.1940 [M]+ (calcd for C33H30O9, 570.1890).
Table 3. Cytotoxic Activity (IC50 μM) of Selected Compounds for Cancer Cell Lines
a
A549
HeLa
ZR7530
HCT15
U373
Hep G2
compound
IC50 (μM)
IC50 (μM)
IC50 (μM)
IC50 (μM)
IC50 (μM)
IC50 (μM)
4 7 8 9 10 etoposidea
>10 >10 >10 >10 >10 9.3
>10 >10 4.9 >10 >10 3.4
5.5 >10 5.8 9.6 >10 2.5
4.6 >10 4.9 >10 >10 4.6
>10 >10 >10 >10 >10 8.5
4.6 9.6 >10 >10 9.3 3.6
Etoposide was used as positive control.
In summary, six new compounds (1−6) were isolated from the fruits of K. parvifolia. Five of them (2−6) are dimeric molecules possessing a dihydronaphthopyranone attached to a dihydroxyanthracenone in different oxidation states. Interestingly, these five dimeric compounds possess a nonaromatic methyl ether attached to C-5′. Cytotoxicity studies revealed that some of these compounds exhibit activity against several cancer cell lines.
■
EXPERIMENTAL SECTION
General Experimental Procedures. Melting points were measured on a Thomas-Hoover melting point apparatus. Optical rotations were recorded on a JASCO P-1020 polarimeter. UV spectra were obtained using a Shimadzu UV-2550 spectrophotometer. IR spectra were obtained using a JASCO FT-IR-4100 spectrometer. NMR spectra were acquired using a Bruker AVANCE-300 NMR spectrometer. A JEOL JMS-700 high-resolution mass spectrometer was used to obtain mass spectrometry data by EIMS and (+)-HRFABMS. Silica gel (230−400 mesh, Sorbent Technologies, Atlanta, GA, USA) was used for CC. Fresh fruits of K. parvifolia were collected and identified by Prof. ́ Ramiro Rios-Gó mez (Facultad de Estudios Superiores Zaragoza, UNAM) at Carretera Tasquillo a Zimapan, Hidalgo (N 20°36′23″, W 99°20′16″), at 1899 m above sea level in May 2012. A specimen (voucher number 13235) was deposited at the Facultad de Estudios Superiores Zaragoza herbarium. Air-dried and powdered K. parvifolia fruits (1.37 kg) were extracted three times with hexane (3.5 L × 3 days) at room temperature to remove fatty acids and waxes. The plant material was covered with CH2Cl2 and extracted three times with this solvent (3.5 L × 3 days, each) to render an oily extract (70.3 g). The dichloromethane extract (70.3 g) was adsorbed on 40−63 μm mesh silica gel (55 g) and placed in a glass chromatography column containing silica gel (15.0 × 4.0 cm). Fractions (200 mL each) were collected during the chromatographic process. Initially, the column was eluted with 100% hexane to remove fatty acids and waxes (fractions A1−A5). Gradients were carried out by successive additions of benzene−acetone mixtures, 50:1 (fractions B1−B7), 20:1 (fractions C1−C7), 10:1 (fractions D1−D7), and 5:1 (fractions E1−E7). Precautions were taken in the use of benzene including working in a fumehood. Fraction B2 (7.7 g, 12.0 × 4.5 cm silica gel column chromatography, hexane 100% to hexane−AcOEt, 85:15) yielded anthracenone T-478 (9) (10.4 mg, 0.015% yield). Fraction B3 (2.4 g, 14.0 × 4.0 cm silica gel column chromatography, hexane 100% to hexane−AcOEt, 85:15) yielded torachrysone monomethyl ether (7) (51.2 mg, 0.073% yield). Fraction B4 (4.1 g, 14.0 × 4.0 cm silica gel column chromatography, hexane−AcOEt, 95:5, to hexane−AcOEt, 90:10) yielded torachrysone monomethyl ether (7) (13.2 mg, 0.019% yield), 2-acetyl-8methoxynaphthol (1) (34.0 mg, 0.048% yield), and an impure subfraction (237 mg). Subfraction B4-1 was purified by silica gel column chromatography (14.0 × 2.0 cm, benzene−acetone, 97:3), yielding tullidinol (26.0 mg, 0.037% yield). Fraction B5 (1.6 g) was E
dx.doi.org/10.1021/np500430q | J. Nat. Prod. XXXX, XXX, XXX−XXX
Journal of Natural Products
Article
Karwinaphthopyranone B1 (4): light brown resin; [α]25D −37.4 (c 0.6, CHCl3); UV (CHCl3) λmax (log ε) 242 (3.25), 273 (3.42), 415 (2.78) nm; IR (film) νmax 3381, 2990, 2971, 1631, 1585, 1469, 1410, 1383, 1329, 1268, 1202, 1096, 1077 cm−1; 1H NMR (CDCl3, 300 MHz), see Table 1; 13C NMR (CDCl3, 75 MHz), see Table 2; (+) FABMS m/z 545 [C32H33O8, M + H]+ (9), 544 [M]+ (8), 513 (9), 415 (8), 393 (13), 307 (100), 289 (65) 219 (38); (+)-HRFABMS m/z 545.2142 [M + H]+ (calcd for C32H33O8, 545.2175). Karwinaphthopyranone B2 (5): light brown resin; [α]25D +141.5 (c 0.8, CHCl3); UV (CHCl3) λmax (log ε) 243 (4.20), 338 (3.86), 368 (3.91) nm; IR (film) νmax 3360, 2975, 2932, 2837, 2367, 2357, 2340, 1635, 1603, 1428, 1372, 1287, 1269, 1079, 1064 cm−1; 1H NMR (CDCl3, 300 MHz), see Table 1; 13C NMR (CDCl3, 75 MHz), see Table 2; (+) FABMS m/z 527 [C32H31O7, M + H]+ (13), 525 (44), 493 (53), 452 (32), 423 (27), 408 (26), 393 (21), 373 (18), 298 (20), 252 (28), 219 (100); (+)-HRFABMS m/z 527.2096 [M]+ (calcd for C32H31O7, 527.2070). Karwinaphthopyranone B3 (6): dark brown resin; [α]25D +136.9 (c 1.0, CHCl3); UV (CHCl3) λmax (log ε) 260 (4.24), 337 (3.64), 438 (3.68) nm; IR (film) νmax 3300, 2972, 2932, 2853, 1663, 1622, 1595, 1469, 1424, 1370, 1267, 1077 cm−1; 1H NMR (CDCl3, 300 MHz), see Table 1; 13C NMR (CDCl3, 75 MHz), see Table 2; (+) FABMS m/z 541 [C32H29O8, M + H]+ (4), 307 (17), 289 (13) 219 (28), 154 (100), 136 (74), 89 (28); (+)-HRFABMS m/z 541.1877 [M + H]+ (calcd for C32H29O8, 541.1862). Cytotoxicity Testing. The cytotoxicities of all compounds against A549 (human lung carcinoma), HeLa (human cervical cancer), ZR7530 (human breast cancer), HCT-15 (human colon cancer), U373 (human brain cancer), and Hep G2 (human liver cancer) cells were tested by using the MTT method.18 Etoposide was used as a positive control.
■
Phytochemistry 1985, 24, 1681−1683. (c) Yussim, L. F.; Lara, O. R.; Benavides, A.; Hernandez, B.; Fernandez, R.; Tamariz, J.; Zepeda, G. Phytochemistry 1995, 40, 1429−1431. (4) Piñeyro-López, A.; Martinez de Villareal, L.; Gonzalez-Alanis, R. Toxicology 1994, 92, 217−227. (5) Waksman, N.; Martinez, L. Rev. Latinoam. Quim. 1989, 20, 27− 29. (6) Dreyer, D. L.; Arai, I.; Bachman, C. D.; Anderson, W. R.; Smith, R. G.; Daves, G. D. J. Am. Chem. Soc. 1975, 97, 4985−4990. (7) Shibata, S.; Murishita, E.; Kaneda, M.; Kimura, Y.; Takido, M.; Takahashi, S. Chem. Pharm. Bull. 1969, 17, 454−457. (8) (a) Arai, I.; Dreyer, D. L.; Anderson, W. R., Jr.; Daves, G. D., Jr. J. Org. Chem. 1978, 43, 1253−1254. (b) Waksman, N.; BenavidesCortez, G.; Rivas-Galindo, V. Phytochemistry 1999, 50, 1041−1046. (9) Bermudez, M. V.; Gonzalez-Spencer, D.; Guerrero, M.; Waksman, N.; Piñeyro, A. Toxicon 1986, 24, 1091−1097. (10) Sepulveda, J.; van der Klei, I. J.; Keizer, I.; Piñeyro-Lopez, A.; Harder, W.; Veenhuis, M. FEMS Microbiol. Lett. 1992, 91, 207−212. (11) Moreno-Sepulveda, M.; Vargas-Zapata, R.; Esquivel-Escobedo, D.; Waksman de Torres, N.; Piñeyro-Lopez, A. Planta Med. 1995, 61, 337−340. (12) Rivas Galindo, V.; Cuevas, G.; Garza, L.; Waksman, N. Arkivoc 2005, 224−233. (13) Rivas-Galindo, V.; Waksman, N. Nat. Prod. Lett. 2001, 15, 243− 251. (14) Guo, H.; Chang, Z.; Rujun, Y.; Guo, D.; Zheng, J. Phytochemistry 1998, 49, 1623−1625. (15) Savard, J.; Brassard, P. Tetrahedron 1984, 40, 3455−3464. (16) Takahashi, S.; Kitanaka, S.; Takaido, M.; Sankawa, U.; Shibata, S. Phytochemistry 1977, 16, 999−1002. (17) Muller, M.; Lamottke, K.; Steglich, W.; Busemann, S.; Reichert, M.; Bringmann, G.; Spiteller, P. Eur. J. Org. Chem. 2004, 4850−4855. (18) (a) Mosmann, T. J. Immunol. Methods 1983, 65, 55−63. (b) Tao, Z.; Zhou, Y.; Lu, J.; Duan, W.; He, X.; Lin, L.; Ding, J.; Qin, Y. J. Cancer Biol. Ther. 2007, 6, 691−696.
ASSOCIATED CONTENT
S Supporting Information *
Bioassays, additional data for compounds 1−6, and 1D and 2D NMR spectra for all compounds. This material is available free of charge via the Internet at http://pubs.acs.org.
■
AUTHOR INFORMATION
Corresponding Author
*Tel: 319-335-8849. Fax: 319-335-8766. E-mail:
[email protected]. Notes
The authors declare no competing financial interest.
■
ACKNOWLEDGMENTS This work was financially supported by a BSFP grant (University of Iowa). We thank the Universidad Autónoma del Estado de Morelos and CONACyT (grant number 178520) for a sabbatical stint to M.Y.R. and CONACyT for an international exchange fellowship to C.R.F. We thank Prof. ́ Ramiro Rios-Gó mez (Facultad de Estudios Superiores Zaragoza, UNAM) for the collection and identification of the plant material.
■
REFERENCES
(1) Piñeyro-López, A.;Waksman, N. In Studies in Natural Products Chemistry; Atta-ur-Rahman, Ed.; Elsevier: Oxford, UK, 2000; Vol. 22, pp 555−606. (2) (a) Ramos-Alvarez, M.; Bessudo, L.; Sabin, A. B. J. Am. Med. Assoc. 1969, 207, 1481−1492. (b) The toxic effect of the fruit has been attributed to a toxin (T-467) named tullidinol. See: Waksman, N.; Benavides-Cortez, G.; Rivas-Galindo, V. Phytochemistry 1999, 50, 1041−1046. (3) (a) Dominguez, X. A.; Garza, L. Phytochemistry 1972, 11, 1186. (b) Mitscher, L. A.; Gollapudi, S. R.; Oburn, D. S.; Drake, S. F
dx.doi.org/10.1021/np500430q | J. Nat. Prod. XXXX, XXX, XXX−XXX
