Abstract
Antiproliferative bioassay-guided fractionation of the ethanolic extract of the endemic Madagascan plant Metaporana sericosepala led to the first natural product isolation of a butenolide diterpene which was synthesized during an anti-inflammatory study in 1988. The structure of the compound was elucidated as 3-homofarnesyl-4-hydroxybutenolide (1) by analysis of its spectroscopic data, including 1D- and 2D-NMR data and chemical evidence. The once synthetic compound can now also be considered as a natural product. Compound 1 had modest antiproliferative activity towards the A2780 ovarian cancer cell line with an IC50 value of 8 μM.
Keywords: Convolvulaceae, Metaporana, Diterpene, Butenolide, Antiproliferative, A2780
As part of our ongoing search for anticancer drugs from Madagascan plants in accordance with the framework of the International Cooperative Biodiversity Group (ICBG) program [2], an ethanol extract of the stems of the endemic plant Metaporana sericosepala Verdc. (Convolvulaceae) exhibited weak antiproliferative activity (IC50 20 μg/mL) against the A2780 ovarian cancer cell line, and its extract was thus selected for chemical investigation. This investigation led to the isolation of the butenolide diterpene (1). Compound 1 was identified as the naturally-occurring counterpart of a compound synthesized in 1988 [3]. This investigation also updated the NMR spectroscopic data for 1 from a simple 1D proton spectrum to include 13C, DEPT, HMQC and HMBC spectra. Similar 3-akyl-4-hydroxybutenolide terpenes have been isolated with reported antiproliferative activities against L5178Y (lymphoma) and MDA-MB-231 (breast cancer) cell lines [4].
This is the first chemical investigation on the species Metaporana sericosepala and the genus Metaporana. Another similar genus from the Convolvulaceae family, Bonamia (not to be confused with the parasite) has been shown in the literature to produce monoterpenes and lignans [5]. The isolation, structure elucidation, and antiproliferative properties of 1 are reported herein.
Liquid-liquid partitioning of the ethanolic extract of Metaporana sericosepala between hexanes, dichloromethane, and water gave an active dichloromethane fraction (IC50 14 μg/mL). C18 solid phase extraction followed by C18 HPLC of the dichloromethane fraction afforded 1. Compound 1 was then identified as the γ-hydroxybutenolide, 3-homofarnesyl-4-hydroxybutenolide (Figure 1) [3]. This is the first report of the isolation of 1 from a natural source.
Figure 1.
Structure and numbering for compound 1.
Compound 1 had the chemical formula of C20H30O3 as determined by high resolution ESIMS (m/z 319.2273 [M+H+], calculated 319.2268 for C20H31O +). The infrared spectrum of 1 displayed characteristic hydroxyl (3385 cm−1) and carbonyl (1755 cm−1) stretching absorptions, and its UV/Vis spectrum displayed a strong absorbance at 228 nm, consistent with a γ-hydroxybutenolide [6].
The 1H-NMR spectroscopic data of 1 (Table 1) displayed two olefinic singlet proton resonances at δ 5.88 (s, 1H) and δ 6.02 (s, 1H) as well as characteristic resonances for a homofarnesyl group. There are three overlapping vinyl protons at δ 5.09 (m, 3H), two singlet signals equivalent to three methyls (δ 1.60, 6H; δ 1.63, 3H) and an allyl coupled doublet methyl at δ 1.68 (d, J= 0.9 Hz, 3H). Two well-resolved methylene units were coupled to one another with resonances at δ 2.32 (q, J = 7.1 Hz, 2H) and δ 2.46 (t, J = 7.1 Hz, 2H). An additional four overlapping methylene units at δ 2.11-1.95 (m, 9H) are believed to be stacked with a hydroxyl proton based on the total integration.
Table 1.
1H- and 13C-NMR data for Compound 1.
| Position | 13C | 1H | DEPT | HMBC (1H → 13C) |
|---|---|---|---|---|
| 1 | 170.7 | C | ||
| 2 | 118.1 | 5.88 s | CH | C-1, C-3, C-4 |
| 3 | 168.8 | C | ||
| 4 | 98.5 | 6.02 brs | CH | C-1, C-2 |
| 5 | 25.4 | 2.46 t (7.1) | CH2 | |
| 6 | 27.9 | 2.32 q (7.1) | CH2 | C-3, C-5, C-7, C-8 |
| 7 | 122.19 | 5.09 m | CH | C-5, C-6, C-20 |
| 8 | 137.7 | C | ||
| 9 | 39.8 | 2.02 m | CH2 | C-8, C-10, C-11, C-20 |
| 10 | 26.9 | 2.05 m | CH2 | C-9, C-11 |
| 11 | 124.0 | 5.09 m | CH | C-9, C-10, C-19 |
| 12 | 135.5 | C | ||
| 13 | 39.9 | 1.97 m | CH2 | C-12, C-14, C-15, C-19 |
| 14 | 26.6 | 2.08 m | CH2 | C-13, C-15, C-16 |
| 15 | 124.5 | 5.09 m | CH | C-13, C-14, C-17, C-18 |
| 16 | 131.5 | C | ||
| 17 | 25.9 | 1.68 d (0.9) | CH3 | C-15, C-16, C-18 |
| 18 | 17.7 | 1.60 brs | CH3 | C-14, C-15, C-16, C-17 |
| 19 | 16.4 | 1.60 brs | CH3 | C-11, C-12 |
| 20 | 16.2 | 1.63 s | CH3 | C-7, C-8, C-9 |
Measured in CDCl3 (1H-NMR at 600 MHz and 13C-NMR at 150 MHz).
The 13C-NMR spectroscopic data of 1 (Table 1) displayed twenty carbon resonances, which is consistent with the observed and calculated mass spectroscopic data. The carbon resonances revealed the presence of a lactone, a β-enone, and a hemiacetal with resonances at δ 170.7 (C-1), δ 168.8 (C-3), and δ 98.5 (C-4), respectively. Resonances for four vinyl methine carbons were observed at δ 124.5 (C-15), δ 124.0 (C-11), δ122.1 (C-7), and δ 118.1 (C-2). These observations are also consistent with thew presence of γ-hydroxybutenolide and homofarnesyl units, where the lactone, β-enone, hemiacetal, and one vinyl carbon would belong to the γ-hydroxybutenolide and the remaining three vinyl carbons belong to the homofarnesyl unit. Four less intense olefinic carbons were observed at δ 168.8 (C-3), 137.7 (C-8), δ 135.5 (C-12), and δ 131.5 (C-16). The remaining carbon signals must belong to the six methylene units and four methyls with resonances at δ 39.9 (C-13), δ 39.8 (C-9), δ 27.9 (C-6), δ 26.9 (C-10), δ 26.6 (C-14), δ 25.4 (C-5), δ 25.9 (C-17), δ 17.7 (C-18), δ 16.4 (C-19), and δ 16.2 (C-20).
The DEPT 135 spectrum distinguished C-1, C-3, C-8, C-12, and C-16 as quaternary carbons and C-2, C-7, C-11, and C-15 as methine carbons. It could also be seen that there were six methylene carbons corresponding to carbons C-5, C-6, C-9, C-10, C-13, and C-14. This experiment also revealed C-17 as a methyl or methine group and not a methylene unit. The remaining signals C-18, C-19, and C-20 were also revealed to be methyl or methine carbons.
Interpretation of the HMQC data revealed C-17, C-18, C-19, and C-20 as methyl groups with correlations from δH 1.68 (d, J = 0.9, 3H) to δC 25.9, δH 1.60 (brs, 6H) to δC 17.7, δH 1.60 (brs, 6H) to δC 16.4, and δH 1.63 (s, 3H) to δC 16.2, respectively. The downfield shift of C-17 (δ 25.9) is due to the local environment created by the vinyl proton and the trans methylene group. This observation also supports the double bonds on the homofarnesyl unit as all (E), since C-18, C-19, and C-20 are not shifted down field like C-17 [7]. The HMQC also revealed the chemical shifts of the four overlapping methylene proton units as δH 1.97 to δC 39.9 (C-13), δH 2.02 to δC 39.8 (C-9), δH 2.05 to δC 26.9 (C-10), and δH 2.08 to δC 26.6 (C-14). The two well-resolved methylene units displayed correlations from δH 2.46 to δC 25.4 (C-5) and δH 2.32 to δC 27.9 (C-6). The HMQC spectrum displayed the correlations of the three overlapping vinyl protons at δH 5.09 to C-15, C-11, and C-7. The proton at δH 5.88 (s, 1H) displayed a correlation to δC 118.1 (C-2), the last of the vinyl carbons. The final proton at δH 6.02 (brs, 1H) was correlated to the hemiacetal carbon δC 98.5 (C-4).
The end of the homofarnesyl unit was found by observing the HMBC correlations from H-17 to C-15, C-16, and C-18 (Figure 2). The second isoprene unit of the homofarnesyl chain is connected to the first isoprene unit between C-14 and C-13 and the third isoprene unit between C-10 and C-9, supported by HMBC correlations from H-13 to C-14 and C-12, and H-9 to C-10 and C-8, respectively. Correlations from H-2 to C-1, C-3, and C-4 add further support for the presence of the lactone hemiacetal butenolide. Finally, correlations from H-6 to C-3, C-5, and C-7 support the attachment of the homofarnesyl unit between C-3 and C-5. For other key HMBC correlations refer to Figure 2.
Figure 2.
Key HMBC correlations for compound 1.
The configurations of the double bonds of 1 were assigned by comparisons with the literature data. If a 2-alkyl-4-hydroxybutenolide had been observed then an upfield carbon resonance of around δ 130 would be expected for C-2, but this resonance was not observed. For a 3-alkyl-4-hydroxybutenolide a carbon resonance between δ 170 and δ 160 would be expected for C-3. A carbon resonance was observed at δ 168.8 and assigned to C-3 [8]. This observation supports the presence of a 3-alkyl-4-hydroxybutenolide.
The presumed equilibrium between the ring-opened and ring-closed forms of the hemiacetal at C-4 suggested that 1 should be racemic. In the event, a small [α]D of −1.18° was observed, consistent with a slight excess of one enantiomer.
Compound 1 was previously only known as a synthetic compound, but it can now be considered as a natural product. It exhibited modest antiproliferative activity (IC50 8 μM) in the A2780 ovarian cancer cell line assay.
Experimental
General experimental procedures
Silica gel and silica gel C18, both 40-63 μm, were used for TLC and the C18 solid phase extraction. HPLC isolations were conducted with a Shimadzu SCL-10A VP controller, SPD-M10A VP photodiode array detector and two LC-10AT VP pumps with a 250 × 4.60 mm, Luna 5 micron, C18 Phenomenex column. UV and IR spectra were measured on Shimadzu UV-1201 and Bruker Tensor 27 FTIR spectrometers. Optical rotation was recorded on a JASCO P-2000 polarimeter. The mass spectrum was obtained on an Agilent 6220 LC/MS TOF. 1H- and 13C-NMR data were recorded on a Bruker 600 spectrometer in CDCl3 with TMS as an internal standard.
Antiproliferative Bioassay
The A2780 ovarian cancer cell line assay was performed at Virginia Polytechnic Institute and State University as previously reported [9]. The A2780 cell line is a drug-sensitive ovarian cancer cell line [10].
Plant Material
Stem, leaf, and flower samples of Metaporana sericosepala were collected from the dry forests of Ambilobe, Marivorahona, Amboahangibe, and Mahamasina in the Diana region of the Antsiranana province, Madagascar, on August 9, 2007 at 12°58'18"S 049°09'46"E. The plant is a liana vine with white flowers. Voucher specimens with collection number Randrianaivo 1478 have been deposited in herbaria at the Parc Botanique and Zoologique de Tsimbazaza (TAN), at the Centre National d' Application des Recherches Pharmaceutiques in Antananarivo, Madagascar (CNARP), at the Missouri Botanical Garden in St. Louis, Missouri (MO), and at the Muséum National d' Histoire Naturelle in Paris, France (P).
Extraction
Dried stems of Metaporana sericosepala (278 g) were extracted with ethanol to give 14.8 g of extract designated MG4354, of which 4.0 g was made available to Virginia Tech.
Isolation of 1
One gram of the ethanol extract of Metaporana sericosepala was dissolved in 60 mL of MeOH and extracted with hexanes (3 × 80 mL) to provide 145 mg of a hexanes-soluble fraction. The MeOH fraction was dried and suspended in 70 mL of water and extracted with dichloromethane (3 × 80 mL) to give 631 mg of a water-soluble fraction and 47 mg for the dichloromethane fraction. The dichloromethane fraction was found to be active (IC50 14 μg/mL) and was then divided into three fractions by a C18 reverse-phase solid phase extraction (SPE) with 60% MeOH, 80% MeOH, and 100% MeOH. The 80% MeOH fraction (9.0 mg) was found to be the most active fraction with an IC50 value of 4 μg/mL. Further separation with reverse-phase C18 HPLC afforded eleven fractions. The most active fraction (peak at 18 min fr. 5, 2.1 mg, IC50 3 μg/mL) was determined to be compound 1.
3-Homofarnesyl-4-hydroxybutenolide (1)
[α]D: −1.18° (c 3.67×10−3, CH3OH).
IR (CHCl3): 3385, 2968, 2924, 2855, 1755, 1740 cm−1.
UV/Vis λmax (MeOH) nm (log ε): 228
1H NMR (600 MHz, CDCl3): See Table 1.
13C NMR (150 MHz CDCl3): See Table 1.
ESIMS: m/z [M + H+] calcd for C20H31O3+: 319.2268; found: 319.2273.
Supplementary Material
Acknowledgments
This International Cooperative Biodiversity Group project was supported by the Fogarty International Center, the National Cancer Institute, the National Institute of Allergy and Infectious Diseases, the National Institute of Mental Health, the National Institute on Drug Abuse, the National Heart Lung and Blood Institute, the National Center for Complementary and Alternative Medicine, the Office of Dietary Supplements, the National Institute of General Medical Sciences, the Biological Sciences Directorate of the National Science Foundation, and the Office of Biological and Environmental Research of the U.S. Department of Energy under Cooperative Agreement U01 TW00313 with the International Cooperative Biodiversity Groups. This project was also supported by the National Research Initiative of the Cooperative State Research, Education and Extension Service, USDA, Grant #2008-35621-04732. These supports are gratefully acknowledged. Work at Virginia Tech was supported by the National Science Foundation under Grant CHE-0619382 for the purchase of the Bruker Avance 600 NMR spectrometer and Grant CHE-0722638 for the purchase of the Agilent 6220 mass spectrometer. We thank Mr. B. Bebout for obtaining the mass spectra. Fieldwork essential for this project was conducted under a collaborative agreement between the Missouri Botanical Garden and the Parc Botanique et Zoologique de Tsimbazaza and a multilateral agreement between the ICBG partners, including the Centre National d’Application des Recherches Pharmaceutiques. We gratefully acknowledge courtesies extended by the Government of Madagascar (Ministére des EauxetForêts). We thank A. Rakotondrafara, S. Rakotonandrasana, A. S. Manasse, A. Razanakolona, and V. Benjara for assistance with the plant collection.
Footnotes
Supplementary data: Supplementary data, consisting of the 1H and 13C NMR spectra of 1, are available on the publisher’s website.
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