Abstract
A new lignan, 3,4,5′-trimethoxy-3′,4′-methylenedioxy-7,9′:7′,9 diepoxylignan (1) (6-[4-(3,4-dimethoxy-phenyl)-tetrahydro-furo[3,4-c.]furan-1-yl]-4-methoxy-benzo[CitationCitation] dioxole) together with two known lignans, 7′-epi.-sesartemin (2) and diayangambin (3), and a known flavonoid, 5-hydroxy-7,4′-dimethoxyflavone (4), were isolated from the leaves of Piper fimbriulatum. C. DC. Their structures were assigned by a combination of one- and two-dimensional NMR techniques. 7′-epi.-Sesartemin (2) showed the highest larvicidal activity against Aedes aegypti. (LC100 17.6 µg/ml) and weak antiplasmodial (IC50 7.0 µg/ml) and antitrypanosomal (IC50 39.0 µg/ml) activities. None of the compounds was active against Leishmania mexicana..
Introduction
Piper fimbriulatum. (Piperaceae) C. DC. is a slender shrub of 5 m height commonly known as canotillo. in Central America and is distributed in shaded sites in moist forests and along streams and roadsides of Colombia, Costa Rica, and Panama (Tebbs, Citation1989). In our ongoing search for larvicidal principles, the chloroform extract of the leaves of Piper fimbriulatum. showed larvicidal activity against Aedes aegypti. (LC100 37.5 µg/ml). Bioassay-guided fractionation of the chloroform extract of the leaves of P. fimbriulatum., using larvae of Aedes aegypti., resulted in the isolation of one new compound 3,4,5′-trimethoxy-3′,4′-methylenedioxy-7,9′:7′,9 diepoxylignan (1) () in addition to two known lignans (2, 3) and a known flavonoid (4). Compounds 1, 2, and 3 were also tested for their antiparasitic activity against Plasmodium falciparum., Leishmania mexicana., and Trypanosoma cruzi.. The structure elucidation of the new natural product 1 and the larvicidal and antiparasitic activities of compounds 1–3 are discussed herein.
Figure 1 Structures of compounds 1–4.
Materials and Methods
Melting points are uncorrected. Optical rotations were measured with a Perkin-Elmer 141 polarimeter. IR spectra were recorded on a Perkin Elmer 1310 spectrophotometer. NMR spectra were recorded on a Varian UNITY 500 spectrometer in CDCl3 at 500 MHz and 125 MHz for 13C NMR. Mass spectra were obtained on a Jeol JMS-AX 505 HAD mass spectrometer at 70 eV. Silica gel 60 (Merck 70-230 mesh) (Darmstadt, Germany) and Sephadex LH = 20 SIGMA (St. Louis, MO, USA) were used for column chromatography. Silica gel plates 60 F254 O. 25 mm thickness, Merck (Darmstadt, Germany) were used for TLC.
Plant material
Piper fimbriulatum. C. DC. was collected in Altos de Campana National Park, Dichapetalaceae trail, on April 11, 1997. Prof. Mireya D. Correa, Director of the Herbarium of the University of Panama (PMA), where voucher specimens (FLORPAN 2790) are deposited, established its taxonomic identification.
Extraction and isolation
Air-dried powdered leaves of P. fimbriulatum. (619.0 g) were extracted with CHCl3 by percolation at room temperature and the extract concentrated to obtain a residue (63.0 g; 10.0%). The chloroform extract was tested against larvae of Aedes aegypti.. The chloroform extract (8.0 g) was subjected to bioactivity-guided fractionation by repeated column chromatography using silica gel and gradient of CHCl3─MeOH (99:1) → MeOH 100% as eluent. Sixteen fractions (100 ml each one) were obtained and submitted to larvicidal assay. Two active fractions (PF-A; PF-B) were combined and submitted to column chromatography with CH2Cl2─EtOAc (95:5) → CH2Cl2─EtOAc (85:15). Four major compounds were eluted and purified by crystallization in MeOH from fractions 5, 6, 7, and 8, respectively: 3,4,5′-trimethoxy-3′,4′-methylenedioxy-7,9′:7′,9 diepoxylignan (1) (116 mg, 0.018%), 7′-epi.-sesartemin (2) (158 mg, 0.025%), diayangambin (3) (125 mg, 0.020%), and 5-hydroxy-7,4′-dimethoxyflavone (4) (82 mg, 0.013%).
3,4,5′-Trimethoxy-3′,4′-methylenedioxy-7,9′:7′,9 diepoxylignan (1)
Colorless needles from MeOH. mp (uncorr.): 134–136; [α]D + 115.2 (c. 0.241, CHCl3); UV (EtOH) λmax (log ε) 204 (5.39), 233 (4.54), 278 (4.16) nm; IR (KBr) νmax 2920, 2850, 1630, 1520, 1460, 1270, 1140, 1090, 730 cm−1; 1H NMR (CDCl3, 500 MHz) δ 6.93 (1H, d., J. = 1.9 Hz, H-2), 6.89 (1H, dd., J. = 1.9, 8.1, H-6), 6.84 (1H, d., J. = 8.1, H-5), 6.60 (1H, s., H-6′), 6.52 (1H, s., H-2′), 5.98 (2H, s., H-3′a), 4.83 (1H, d., J. = 5.3, H-7′), 4.42 (1H, d., J. = 7.3, H-7), 4.12 (1H, d., J. = 9.6, H-9α), 3.93 (3H, s., H-5′a), 3.90 (3H, s., H-3a), 3.88 (3H, s., H-4a), 3.84 (1H, d., J. = 9.4, H-9′β), 3.83 (1H, d., J. = 9.6, H-9β), 3.34 (1H, m., H-9′α), 3.32 (1H, m., H-8′), 2.91 (1H, m., H-8); 13C NMR (CDCl3, 125 MHz) δ 149.2 (C, C-4), 148.8 (C, C-3′), 148.7 (C, C-3), 143.5 (C, C-5′), 134.1 (C, C-4′), 133.5 (C, C-1), 132.9 (C, C-1′), 118.5 (CH, C-6), 110.9 (CH, C-5), 109.0 (CH, C-2), 104.8 (CH, C-6′), 101.4 (OCH2O, C-3′a), 99.9 (CH, C-2′), 87.6 (CH, C-7), 82.0 (CH, C-7′), 70.9 (CH2, C-9), 69.6 (CH2, C-9′), 56.6 (OCH3, C-5′a), 55.9 (OCH3, C-4a), 55.9 (OCH3, C-3a), 54.4 (CH, C-8), 50.1 (CH, C-8′). HSQC, HMBC, and NOESY were used to confirm these assignments. EIMS m./z. 400 (100), 219 (18), 191 (20), 181 (10), 180 (10), 179 (15), 177 (15), 166 (10), 165 (80), 152 (8), 151 (50). HREIMS m./z. 400.1550 (calcd for C22H24O7, 400.1522).
7-epi-Sesartemin (2)
UV (EtOH) λmax (log ε) 208 (4.94), 238 (4.00), 275 (3.46); 1H NMR (CDCl3, 500 MHz) δ 6.59 (1H, d., J. = Hz, H-6′), 6.52 (1H, d., J. = 0.85 Hz, H-2′), 6.51 (2H, s., H-2, H-6), 5.98 (2H, s., H-3′a), 4.83 (1H, d., J. = 5.12 Hz, H-7′), 4.41 (1H, d., J. = 7.26, H-7), 4.14 (1H, d., J. = 9.61 Hz, H-9b), 3.93 (3H, s., H-5′a), 3.91–3.85 (2H, m., H-9a, H-9′a), 3.89 (6H, s., H-3a, H-5a), 3.84 (3H, s., H-4a); 13C NMR (CDCl3, 500 MHz) δ 153.4 (C, C-3, C-5), 148.80 (C, C-3′), 143.5 (C, C-5′), 137.4 (C, C-4), 136.70 (C, C-1), 134.1 (C, C-4′), 132.9 (C, C-1′), 104.8 (CH, C-6′), 102.8 (CH, C-2), 101.4 (CH2, C-3′a), 99.8 (CH, C-2′), 87.8 (CH, C-7′), 81.9 (CH, C-7), 70.9 (CH2, C-9), 69.7 (CH2, C-9′), 60.8 (CH3, C-4a), 56.6 (CH3, C-5′a), 56.1 (CH3, C-3a, C-5a), 54.5 (CH, C-8), 50.0 (CH, C-8′); EIMS m./z. 430 [M]+ (100), 400 (15), 249 (12), 224 (12), 207 (13), 195 (23), 181 (30), 165 (27).
Diayangambin (3)
UV (EtOH) λmax (log ε) 208 (5.38), 240 (4.29), 274 (3.63); 1H NMR (CDCl3, 500 MHz) δ 6.61 (4H, s., H-2, H-6, H-2′, H-6′), 4.92 (2H, d., J. = 4.9 Hz, H-7, H-7′), 3.89 (12H, s., CH3O-4a, CH3O-4′a), 3.85 (6H, s., CH3O-3a, CH3O-5a, CH3O-3′a, CH3O-5′a), 3.74 (2H, dd., J. = 1.3, 9.6 Hz, H-9b, H-9′b), 3.59 (2H, dd., H-9a, H-9′a), 3.21 (2H, m., H-8, H-8′); 13C NMR (CDCl3, 500 MHz) δ 153.2 (C, C-3, C-5, C-3′, C-5′), 136.9 (C, C-4, C4′), 134.5 (C, C-1, C-1′), 103.0 (C, C-2, C-6, C-2′, C-6′), 84.0 (CH, C-7, C-7′), 68.8 (CH2, C-9, C-9′), 60.8 (CH3O, C-4a, C-4′a), 56.0 (CH3O, C-3a, C-5a, C-3′a, C-5′a), 49.4 (CH, C-8, C-8′). EIMS m./z. 446 [M]+ (100), 250 (25), 197 (38), 181 (62), 169 (25).
5-Hydroxy-7,4′-dimethoxyflavone (4)
1H NMR (CDCl3, 500 MHz) δ 10.50 (1H, s., OH-5a), 7.82 (2H, d., J. = 9.1, H-3′, H-5′), 7.00 (2H, d., J. = 9.1, H-2′, H-6′), 6.55 (1H, s., H-3), 6.45 (1H, d., J. = 2.1, H-8), 6.34 (1H, d., J. = 2.1, H-6), 3.88 (3H, s., CH3O-7a), 3.87 (3H, s., CH3O-4a); 13C NMR (, 500 MHz) δ 182.5 CDCI3(C, C-4), 165.4 (C, C-7), 164.0 (C, C-5), 162.6 (C, C-2), 162.2 (C, C-4′), 157.7 (C, C-4a, C-8a), 128.0 (CH, C-2′, C-6′), 123.5 (C, C-1′), 114.5 (CH, C-3′, C-5′), 104.33 (CH, C-3), 98.1 (CH, C-6), 92.6 (CH, C-8), 55.8 (C, CH3O-7a), 55.6 (C, CH3O-4′a); EIMS m./z. 298 [M]+ (100).
Larvicidal activity
The larvicidal activity was determined on larvae of Aedes aegypti. in a 96-well plate according to the method of Ceplenau (Citation1993), modified by Solís et al. (Citation1996). Tetrametrin was used as a standard larvicidal agent.
Antimalarial activity
The antimalarial activity was determined with a chloroquine-resistant (Indochina clone W2) strain of Plasmodium falciparum. according to the novel DNA-based microfluorimetric method of Corbett et al. (Citation2004). Chloroquine was used as a standard antimalarial agent.
Antileishmanial activity
The antileishmanial activity was determined on WHO reference strain of Leishmania mexicana. MOHM/B2/82/BELZ promastigotes according to the protocol of Cornelly et al. (Citation2003). Amphotericin B was used as a standard antileishmanial agent.
Antitrypanosomal activity
The intracellular assay was carried out according to Buckner et al.(Citation1996) on the recombinant Tulahuen clone C4 of Trypanosoma cruzi. (trypomastigotes), which expresses β-galactosidase (βGal) as a reporter enzyme, provided by F. Buckner (University of Washington, Seattle, WA, USA). Nifurtimox was tested as a standard antitrypanosomal agent.
Results and Discussion
The isolation of the three lignans (1–3) and one flavonoid (4) was carried out according to the procedures described in “Material and Methods.” Three known compounds 7′-epi.-sesartemin (2) (MacRae & Towers, 1985), diayangambin (3) (Russell & Fenemore, Citation1973), and 5-hydroxy-7,4′-dimethoxyflavone (4) (Yang et al., Citation1995) were identified by comparison of their spectral data with those published in the literature.
The HREIMS spectrum showed a [M]+ peak at m./z. 400.1550 corresponding to the molecular formula C22H24O7. The 1H NMR, 13C NMR and HMQC data of 1 showed five aromatic protons [δH/δC 6.93 (H-2)/109.0 (C-2), 6.89 (H-6)/118.5 (C-6), 6.84/110.9 (C-5), 6.60 (H-6′)/104.8 (C-6′), 6.52 (H-2′)/99.9 (C-2′)]; one methylenedioxy group [δH/δC5.98 (H-3′a)/101.4 (C-3′a)]; four methine protons [δH/δC 4.83 (H-7′)/82.0 (C-7′); 4.42 (H-7)/87.6 (C-7); 3.32 (H-8′)/50.1 (CH, C-8′), 2.91(H-8)/54.4 (C-8)]; three methoxy groups [δH/δC 3.93 (H-5′a)/56.6 (C-5′a), 3.90 (H-3a)/55.9 (OCH3, C-3a), 3.88 (H-4a)/55.9 (C-4a)]; two methylene groups [δH/δC 4.12 (H-9α), 3.83 (H-9β)/70.9 (C-9), 3.84 (H-9′ β), 3.34 (H-9′α)/69.6 (C-9′)]; and quaternary carbons [δC 149.2 (C-4), 148.8 (C-3′), 148.7 (C-3), 143.5 (C-5′), 134.1 (C-4′), 133.5 (C-1), 132.9 (C-1′)]. The key HMBC correlations of H-7β with C-1, C-8, C-9, H-7′α with C-1′, C-2′, C-6′, C-8′, C-9′, H-9β with C-8, and H-9′α with C-8′ strongly supported the presence of a bis-tetrahydrofuran ring. Additional HMBC correlations are shown in Figure 2. The spatial relationships in the molecule were then deduced from the NOESY spectrum of 1. In particular, the cross-peaks implicating H-8/H-8′, H-8/H-9α, H-8′/H-9′α, H8′/H-7′α indicated that these protons were all on the same face of the molecule and had α orientation, whereas those observed with H-7β/H-9β, H-7β/H-9′β established that these other substituents were on the other side of the molecule and had β orientation (Figure 3). The linkage between C-8 and C-8′ was confirmed to be cis., as is typical of naturally occurring bis-tetrahydrofuran lignans. From the NOESY data and the optical rotation, the compound 1 was concluded to be an unsymmetrically substituted bis-tetrahydrofuran lignan, possessing an axial aromatic ring at C-7 and an equatorial aromatic ring at C-7′ (Russell & Fenemore, Citation1973). On the basis of the above spectroscopic data, the structure of 1 was assigned as 3,4,5′-trimethoxy-3′,4′-methylenedioxy-7,9′:7′,9 diepoxylignan (6-[4-(3,4-dimethoxy-phenyl)-tetrahydro-furo[3,4-c.]furan-1-yl]-4-methoxy-benzo [Citation1Citation3]dioxole) (1), a new natural product.
All spectral and optical rotation data obtained for 2, 3, and 4 are in close agreement with those reported for 7′-epi.-sesartemin (MacRae & Towers, Citation1985), diayangambin (Russell & Fenemore, Citation1973), and 5-hydroxy-7,4′-dimethoxyflavone (Yang et al., Citation19955). Compound 2 was isolated from the bark of Virola elongata. (MacRae & Towers, Citation1985), compound 3 was first reported in Macropiper excelsum. (Russell & Fenemore, Citation1973), and compound 4 was isolated from Biota orientalis. (Yang et al., Citation1995). De Leo´n et al. (Citation2002) reported in vitro. and in vivo. immunomodulatory and anti-inflammatory activities of diayangambin (3).
shows the larvicidal activity and in vitro. antiparasitic activity against Plasmodium falciparum., Leishmania donovani., and Trypanosoma cruzi. of compounds 1–3. Compound 2 was the most active of the three tested compounds against P. falciparum. (W2: chloroquine-resistant strain) and T. cruzi. with IC50 values of 7.0 and 39 µg/ml, respectively, and Aedes aegypti. larvae (LC100 17.6 µg/ml).
Table 1.. Larvicidal and antiparasitic activities of isolated compounds.
The larvicidal activity against Aedes aegypti. has been previously reported in Piper acutisleginum. (Olsen et al., Citation1993), while Piper rusbyi. showed antileishmanial activity against L. amazonensis., L. braziliensis., and L. donovani. (Fournet et al., Citation1994), and antitrypanosomal activity against T. cruzi. (Fournet et al., Citation1994).
Acknowledgments
We thank the International Foundation for Science for funding a project (grant no. F/1081-2) (PNS) and the International Matsumae Foundation of Japan for a postdoctoral fellowship grant to P.N.S. This investigation also received financial support from the UNICEF/UNDP/World Bank/WHO Special Programme for Research and Training in Tropical Diseases (TDR) (Project ID A2076) the Organization of American States, and Fundación Natura. We are grateful to Dr. Eduardo Ortega-Barría (INDICASAT, Panama) for the antiparasitic screening of compounds.
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