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Norlignans from Pouzolzia occidentalis
Phytochemistry Letters 3 (2010) 29–32
Contents lists available at ScienceDirect
Phytochemistry Letters journal homepage: www.elsevier.com/locate/phytol
Norlignans from Pouzolzia occidentalis M. Mohammed a, A.R. Maxwell a,*, R. Ramsewak a, W.F. Reynolds b a b
Department of Chemistry, University of the West Indies, St. Augustine, Trinidad and Tobago Department of Chemistry, University of Toronto, Toronto, Ontario M5S1A1, Canada
A R T I C L E I N F O
A B S T R A C T
Article history: Received 3 October 2009 Received in revised form 26 October 2009 Accepted 27 October 2009 Available online 12 November 2009
Two structurally interesting new norlignans named 2-hydroxy-4-[4-hydroxyphenyl-(4-hydroxy-3methoxybenzyl)]-3-(3,5-dihydroxyphenyl)tetrahydrofuran (1) (pouzolignan A), and 1,4-dihydroxy-3[4-hydroxyphenyl-(4-hydroxy-3-methoxybenzyl)]-2-(3,5-dihydroxyphenyl)butane (2) (pouzolignan B), were isolated from the EtOAc fraction of the methanol extract of Pouzolzia occidentalis. Compound 3, the methyl ether of 1, most likely an artifact, was also isolated. The overall structures and relative stereochemistry were elucidated largely by analysis of 1D and 2D NMR spectral data. ß 2009 Phytochemical Society of Europe. Published by Elsevier B.V. All rights reserved.
Keywords: Pouzolzia occidentalis Urticaceae Norlignans Pouzolignans A (1) and B (2)
1. Introduction Recent reports on the biological activity of extracts of plants of the Urticaceae family have indicated that they have antidiabetic and antilipidaemic (Momo et al., 2006), antibacterial (Sayeed et al., 2003), anti-HIV (Piccinelli et al., 2005) and anticancer (Konrad et al., 2000; Luo et al., 2003) activities. As part of our continuing effort to discover the chemical constituents and medicinal properties of the flora of Trinidad and Tobago, we have studied the plant Pouzolzia occidentalis (syn. P. palmieri) Wedd. (Urticaceae), on which no previous studies have been reported. We report herein the isolation of two new norlignans, 2-hydroxy-4-[4hydroxyphenyl-(4-hydroxy-3-methoxybenzyl)]-3-(3,5-dihydroxyphenyl)tetrahydrofuran (1) and 1,4-dihydroxy-3-[4-hydroxyphenyl-(4-hydroxy-3-methoxybenzyl)]-2-(3,5-dihydroxyphenyl)butane (2). 2. Results and discussion The crude methanol extract of the aerial parts of the plant was suspended in 80% CH3OH/H2O and the mixture successively extracted with petrol, CHCl3 and EtOAc. Repeated chromatography on the EtOAc fraction yielded three unusual norlignans. Compounds 1 and 2 named pouzolignans A and B, respectively, and compound 3, the methyl ether of 1, presumably formed during the isolation of the latter. Compound 1 was isolated as a brown gum. It + gave an ½a25 D of 32.4 (c 0.1, MeOH) and an M ion in the HREIMS at
* Corresponding author. Tel.: +868 662 6013; fax: +868 645 3771. E-mail address: [email protected] (A.R. Maxwell).
m/z 424.1512, which indicated a molecular formula of C24H24O7 (calcd. for C24H24O7, 424.1522). The infra-red spectrum with absorptions at 3374 and 1602 cm1 indicated hydroxyl and aromatic groups, respectively. 1D and 2D NMR experiments enabled elucidation of the structure of 1. Signals at dH 7.00 (2H, d, J = 8.5 Hz) and dH 6.59 (2H, d, J = 8.5 Hz) in the 1H NMR spectrum, the corresponding signals at dC 130.4 (2C) and dC 115.9 (2C) in the HMQC spectrum and two quaternary carbon signals at dC 156.7 and dC 136.7 indicated a 1,4-disubstituted and monooxygenated aromatic ring (A). The presence of a second aromatic ring (B), which was 1,2,4-trisubstituted and dioxygenated, was deduced from the signals at dH 6.69 (1H, d, J = 8.0 Hz), dH 6.73 (1H, dd, J = 8.0, 2.0 Hz) and dH 6.80 (1H, d, J = 2.0 Hz), the corresponding carbon signals at dC 116.0, 121.0 and 112.6 along with quaternary carbon signals at dC 148.9, 145.9 and 137.8. The presence of a third aromatic ring (C) which was 1,3,5-trisubstituted and dioxygenated followed from the signals at dH 6.04 (2H, t, J = 2.0 Hz) and dH 5.91 (1H, d, J = 2.0 Hz), the related carbon signals at dC 107.0 (2C) and dC 101.8 and the quaternary carbon signals at dC 147.1 and dC 159.4 (2C). A tetrahydrofuran-2-ol moiety was evident from the following data: a 1H multiplet at dH 3.12 (dC 53.0, C-4) showed cross peaks in the COSY spectrum to a 1H multiplet at dH 2.78 (dC 59.6, C-3) and the signals at dH 4.04 (t, J = 8.5 Hz) and dH 3.83 (m) which were due to a pair of methylene protons attached to a carbon resonating at dC 72.8 (C-5). The signal at dH 2.78 (H-3) showed a COSY cross peak to a signal at dH 5.21 (dC 106.4, C-2) which in turn gave an HMBC correlation to the methylene carbon signal at dC 72.8, C-5. The attachment of the aromatic ring C to the carbon resonating at dC 59.6 (C-3) was deduced from the HMBC correlations of the attached proton (dH 2.78, H-3) to the carbon
1874-3900/$ – see front matter ß 2009 Phytochemical Society of Europe. Published by Elsevier B.V. All rights reserved. doi:10.1016/j.phytol.2009.10.008
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M. Mohammed et al. / Phytochemistry Letters 3 (2010) 29–32
signals at dC 147.1 (C-1000 ) and dC 107.0 (C-2000 /C-6000 ). The signal at dH 3.12 (H-4) in addition to the cross peaks at dH 2.78 (H-3) and at dH 4.04 and dH 3.83 (H2-5) in the COSY spectrum already mentioned, also gave a COSY cross peak to the signal at dH 3.95 (dC 56.8) which itself showed cross peaks in the HMBC spectrum at dC 72.8 (C-5) and dC 53.0 (C-4) as well as at dC 112.6 (C00 -2), 121.0 (C-600 ), 130.4 (C-20 /C-60 ), 136.7 (C-10 ) and 137.8 (C-100 ). These data revealed that the aromatic rings A and B were attached to the carbon resonating at dC 56.8 and that this carbon was bonded to the carbon of the tetrahydrofuran ring resonating at dC 53.0 (C-4). Finally, the methoxy signal at dH 3.78 (dC 56.5) gave a cross peak in the HMBC spectrum to the quaternary aromatic carbon signal at dC 148.9 (C-300 ), confirming the presence of the methoxy group on aromatic ring B. The structure of 1 was therefore assigned as shown. The relative stereochemistry around the tetrahydrofuran ring was determined from vicinal H–H coupling constants and from analysis of the NOESY correlations. Thus, the proton at C-2 which couples only with H-3 occurs as a doublet at dH 5.21 with a coupling constant of only 2.0 Hz. This indicates that H-2 must be trans to H-3 since in five membered rings only the trans vicinal couplings can have such low values (Wu et al., 2002; Wu and Cremer, 2003). The signal at dH 2.78 (H-3) shows a coupling constant of 5.4 Hz with the signal at dH 3.12 (H-4) but in the NOESY spectrum gives only a weak cross peak to this signal, while it gives a very strong cross peak to the signal at dH 3.97 (H-6). This pointed to the trans relationship of H-3 and H-4 and the cis relationship between H-3 and H-6 and hence the trans relationship between H-4 and H-6. The NOESY spectrum also enabled assignment of the signals for the protons at C-5 that are cis and trans to H-4. Interestingly, the large coupling constant (11.5 Hz) between H-4 and H-6 must mean that these two protons are essentially anti to each other. Indeed, the minimum energy conformation of 1 obtained from molecular mechanics calculations (MM2 force field) showed an angle of ca.1798 between these two protons. Compound 2 was also isolated as a brown gum with an ½a25 D of 29.6 (c 0.1, MeOH). 1D and 2D NMR data (Table 2) indicated a structure similar to that of compound 1. However, the hemiacetal carbon present in compound 1 was absent in compound 2, which now showed two oxymethylene carbons rather than one as in compound 1. Furthermore, correlation observed between the H-2 (dH 5.21) and C-5 (dC 72.8) signals in the HMBC spectrum of compound 1 was absent in the HMBC spectrum of 2. These data suggested that a tetrahydrofuran ring was not present in compound 2. The fact that both the positive ion ([M+Na]+ peak at m/z 449.1577) and the negative ion ([MH] at m/z 425.1600) HRESIMS led to a molecular formula of C24H26O7 for compound 2, which therefore contained two more hydrogen atoms than compound 1, confirmed this suggestion. It was concluded that the tetrahydrofuran ring of compound 1 had opened to give the two hydroxymethyl groups in compound 2 as shown. Compound 3 was also isolated and characterized by both NMR and mass spectral data. It gave an [a]D of 44.4 (c 0.1, MeOH) and an M+ ion at m/z 438.1678, corresponding to a molecular formula of C25H26O7. The IR as well as the 1D and 2D NMR spectral data of 3 closely matched those of compound 1 and analysis of the NMR spectral data in a manner analogous to that given above led to the same gross structure for 3 as for 1. The extra carbon indicated by the molecular formula of 3 was deduced from the NMR spectral data to be due to a methoxy carbon which resonated at d 54.9 (dH 3.30). This methoxy proton signal showed an HMBC correlation to the carbon signal at d 113.0 (dH 4.74, H-2) and the COSY spectrum indicated long range coupling between the methoxy proton and H2 (d 4.74). Compound 3 was therefore identified as the methyl ether of 1. While 3 is a new compound it is unlikely to be a natural
product and is most likely an artifact arising from the extraction/ isolation procedures. 3. Experimental 3.1. General Optical rotations were measured on a Polatronic D digital polarimeter. IR spectra were recorded on a PerkinElmer RX1 FT-IR spectrophotometer as Nujol1 mulls and UV spectra were recorded in MeOH solutions on a Cary 50 UV–vis instrument. NMR spectra were run in MeOH-d6 on a Varian Unity 600 NMR spectrometer. Mass spectral data were obtained from either a Micromass 70S250 mass spectrometer or a MDS Sciex QStar mass spectrometer. TLC was performed on silica gel 60 PF254+366 plates (Merck), 0.25 mm thick for analytical and 1.0 mm thick for preparative work. Column chromatography was performed using Merck silica gel 60 (70–230 mesh). Sephadex1 LH-20 used for size exclusion chromatography was supplied by Sigma–Aldrich. The solvents petrol (60–80 8C), CHCl3, and EtOAc were all distilled prior to use. 3.2. Plant material The aerial parts of the plant were collected in December 2005 along Riverside Rd., Curepe, Trinidad. A voucher specimen (TRIN 36472) is deposited at the National Herbarium of Trinidad and Tobago. 3.3. Extraction and isolation The dried, ground plant material (1.3 kg) was exhaustively extracted with methanol (16 L) in the cold. The filtered extract was evaporated under reduced pressure to yield 85 g of crude extract. This was suspended in 80% MeOH/H2O and extracted successively with petrol, CHCl3, and EtOAc to give fractions of masses 7.2, 6.8 and 1.4 g, respectively. The EtOAc extract (1.4 g) was subjected to Si gel column chromatography eluting with CHCl3 containing increasing amounts of EtOH to yield eight fractions (I–VIII) after combination based on TLC analysis of the total of one hundred fractions. Repeated Si gel column chromatography (CHCl3/MeOH, 85:15; CHCl3/EtOAc, 1:1) on fraction III followed by gel permeation chromatography (Sephadex1 LH-20, CHCl3/EtOAc, 1:1) yielded compound 1 (3 mg). Compound 2 (8 mg) was isolated from fraction IV after preparative layer chromatography followed by column chromatography (EtOAc/MeOH/H2O, 400:20:18) of the more polar band. Compound 3 (2.6 mg) was obtained after repeated Si gel column chromatography (petrol/EtOAc, 3:2) on fraction II. 3.4. 2-Hydroxy-4-[4-hydroxyphenyl-(4-hydroxy-3methoxybenzyl)]-3-(3,5-dihydroxyphenyl)-tetrahydrofuran (1) Brown gum. ½a25 D 32.4 (c 0.1, MeOH); UV, lmax MeOH nm (log e): 270 (3.48); IR, ymax Nujol1 (cm1): 3374 (–OH), 1602 (Ar C–C); 1H and 13C NMR data, see Table 1. HREIMS (positive ion): found m/z 424.1512 [M]+; calculated for C24H24O7 m/z 424.1522. 3.5. 1,4-Dihydroxy-3-[4-hydroxyphenyl-(4-hydroxy-3methoxybenzyl)]-2-(3,5-dihydroxyphenyl)butane (2) Brown gum. ½a25 D 29.6 (c 0.1, MeOH); UV, lmax MeOH nm (log e): 270 (3.94); IR, ymax Nujol1 (cm1): 3360 (–OH), 1608 (Ar C–C); 1H and 13C NMR data, see Table 2. HRESIMS (positive ion): found m/z 449.1577 [M+Na]+; calculated for C24H26O7Na m/z 449.1594; negative ion: found m/z 425.1600 [MH]; calculated for C24H25O7 m/z 425.1605.
M. Mohammed et al. / Phytochemistry Letters 3 (2010) 29–32
31
Table 1 NMR data of 1.a. Position
2 3 4 5b 5a 6 10 20 ,60 30 ,50 40 100 200 300 400 500 00 600 1000 2000 ,6000 3000 ,5000 4000 300 -OCH3 a
H–1H COSY
HMQC
dC
dH
106.4 59.6 53.0 72.8
5.21 2.78 3.12 3.83 4.04 3.95 – 7.00 6.59 – – 6.80 – – 6.69 6.73 – 5.91 – 6.04 3.78
56.8 136.7 130.4 115.9 156.7 137.8 112.6 148.9 145.9 116.0 121.0 147.1 107.0 159.4 101.8 56.5
(d, 2.0) (m) (m) (m) (t, 8.5) (d, 11.5) (d, 8.5) (d, 8.5)
(d, 2.0)
(d, 8.0) (dd, 2.0, 8.0) (d, 2.0) (t, 2.0)
1
HMBC
dH
dC
2.78 3.12, 5.21 2.78, 3.95, 4.04, 3.83 4.04 3.83 3.12 – 6.59 7.00
59.6, 56.8, 56.8, 56.8, 56.8, 53.0,
6.73 – – 6.73 6.69, 6.80 – 6.04 – 5.91 –
56.8, 121.0, 145.9,148.9
72.8, 147.1 106.4, 107.0, 147.1 59.6, 147.0 53.0 59.6, 106.4 72.8, 112.6, 130.4, 136.7, 137.8
56.8, 130.4, 156.7 116.0, 136.7, 156.7
137.8, 148.9 56.8, 112.6, 145.9 59.6, 101.8, 107.0, 159.4 107.0, 159.4 148.9
Data acquired at 600 MHz (1H) and 125 MHz (13C) in CD3OD with TMS as internal standard. Multiplicities and coupling constants (in Hz) are in parentheses.
Table 2 NMR data of 2.a. Position
dC 2
66.9
3 4 5
49.5 46.7 63.1
6 10 20 ,60 30 ,50 40 100 200 300 400 500 600 1000 2000 ,6000 3000 ,5000 4000 300 -OCH3 a
H–1H COSY
HMQC
53.1 136.8 130.3 116.6 157.0 138.1 116.8 149.0 145.8 116.3 121.1 143.3 109.5 159.5 102.2 56.3
dH 3.98 3.93 2.98 2.85 3.30 3.35 3.46 – 7.25 6.77 – – 6.75 – – 6.62 6.64 – 6.19 – 6.16 3.77
(dd, 11.4, 7.2) (dd, 11.4, 6.0) (m) (m) (m) (t, 9.0) (d, 12.0) (d, 8.5) (d, 8.5)
(d, 1.5)
(d, 8.0) (dd, 1.5, 8.0) (d, 2.0) (t, 2.0) (s)
1
HMBC
dH
dC
3.93 3.98 3.93, 3.98, 2.85 2.98, 3.35, 3.30, 3.46 2.85 2.85 2.85 – 6.77 7.25 – – 6.64 –
– 143.3 143.3, 109.5, 66.9, 63.1, 53.1, 46.7 – –
6.64 6.62, 6.75 – 6.16 – 6.19 –
138.1, – 157.0, 157.0, – – – – –– – 149.0, – 159.4, – – 149.0
136.8, 130.3, 121.1, 46.7 130.3, 53.1 136.8, 53.1, 116.6
145.8, 138.1, 121.1, 53.1 109.5, 102.2, 49.5
Data acquired at 600 MHz (1H) and 125 MHz (13C) in CD3OD with TMS as internal standard. Multiplicities and coupling constants (Hz) are in parentheses.
3.6. 4-[4-Hydroxyphenyl-(4-hydroxy-3-methoxybenzyl)]-3-(3,5dihydroxyphenyl)-2-methoxy-tetrahydrofuran (3) Brown gum. ½a25 D 44.4 (c 0.1, MeOH); UV, lmax MeOH nm (log e): 270 (3.89); IR, ymax Nujol1 (cm1): 3370 (–OH), 1602 (Ar C–C); 1H (600 MHz, CD3OD): d 6.98 (1H, d, J = 8.5 Hz, H-20 /H-60 ), 6.76 (1H, d, J = 1.5 Hz, H-200 ), 6.71 (2H, dd, J = 8.0, 1.5 Hz, H-600 ), 6.68 (1H, d, J = 8.0 Hz, H-500 ), 6.60 (2H, d, J = 8.5 Hz, H-30 /50 ), 6.05 (1H, t, J = 2.0 Hz, H-4000 ), 5.89 (2H, d, J = 2.0 Hz, H-2000 /6000 ), 4.74 (1H, br s, H2), 4.05 (1H, t, J = 8.5 Hz, H-5a), 3.87 (1H, d, J = 11.5 Hz, H-6), 3.78 (1H, s, OMe-300 ), 3.65 (1H, dd, J = 7.5, 8.5 Hz, H-5b), 3.30 (1H, s, OMe-2), 3.09 (1H, m, H-4), 2.82 (1H, d, J = 4.0 Hz, H-3); 13C NMR (125 MHz, CD3OD): d 159.5 (C-3000 /C-5000 ), 156.7 (C-40 ), 149.0 (C-300 ), 147.2 (C-1000 ), 146.0 (C-400 ), 137.8 (C-100 ), 136.7 (C-10 ), 130.3 (C-20 /C-
60 ), 121.0 (C-600 ), 116.3 (C-500 ), 115.8 (C-30 /C-50 ), 113.0 (C-2), 112.6 (C-200 ), 106.9 (C-2000 /C-6000 ), 101.5 (C-4000 ), 73.1 (C-5), 58.3 (C-3), 57.1 (C-6), 56.6 (OMe-300 ), 54.6 (OMe-2), 53.2 (C-4); HREIMS (positive ion): found m/z 438.1678 [M]+; calculated for C25H26O7 m/z 438.1679. Acknowledgements The authors wish to thank Dr. Alex Young of the University of Toronto for the MS analyses and Mr. W. Johnson for help in the collection and identification of the plant material. Financial support from the Department of Chemistry, University of the West Indies and NSERC of Canada is gratefully acknowledged.
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References Konrad, L., Muller, H.-H., Lenz, C., Laubinger, H., Aumuller, G., Lichius, J.J., 2000. Antiproliferative effect on human prostate cancer cells by a stinging nettle root (Urtica dioica) extract. Planta Med. 66, 44–47. Luo, Y., Liu, Y., Luo, X., Li, B., Zhang, G., 2003. Cytotoxic alkaloids from Boehmeria siamensis. Planta Med. 69, 842–845. Momo, C.E.N., Oben, J.E., Tazoo, D., Dongo, E., 2006. Antidiabetic and hypolipidaemic effects of a methanol/methylene-chloride extract of Laportea ovalifolia (Urticaceae), measured in rats with alloxan-induced diabetes. Ann. Trop. Med. Parasitol. 100, 69–74.
Piccinelli, A.L., Mahmood, N., Mora, G., Poveda, L., De Simone, F., Rastrelli, L., 2005. Anti-HIV activity of dibenzylbutyrolactone-type lignans from Phenax species endemic in Costa Rica. J. Pharm. Pharmacol. 57, 1109–1115. Sayeed, A., Sattar, M.A., Akteruzzaman, M., Islam, A., Sadik, G., Bhuiyan, M., 2003. A prenylated isoflavone from Pouzolzia indica: its in vitro antimicrobial activity and cytotoxin evaluation. Orient. J. Chem. 19, 35–40. Wu, A., Cremer, D., Auer, A.A., Gauss, J., 2002. Extension of the Karplus relationship for NMR spin–spin coupling constants to nonplanar ring systems: pseudorotation of cyclopentane. J. Phys. Chem. A 106, 657–667. Wu, A., Cremer, D., 2003. A new approach for determining the conformational features of pseudorotating ring molecules utilizing calculated and measured NMR spin–spin coupling constants. J. Phys. Chem. 107, 1797–1810.
Contents lists available at ScienceDirect
Phytochemistry Letters journal homepage: www.elsevier.com/locate/phytol
Norlignans from Pouzolzia occidentalis M. Mohammed a, A.R. Maxwell a,*, R. Ramsewak a, W.F. Reynolds b a b
Department of Chemistry, University of the West Indies, St. Augustine, Trinidad and Tobago Department of Chemistry, University of Toronto, Toronto, Ontario M5S1A1, Canada
A R T I C L E I N F O
A B S T R A C T
Article history: Received 3 October 2009 Received in revised form 26 October 2009 Accepted 27 October 2009 Available online 12 November 2009
Two structurally interesting new norlignans named 2-hydroxy-4-[4-hydroxyphenyl-(4-hydroxy-3methoxybenzyl)]-3-(3,5-dihydroxyphenyl)tetrahydrofuran (1) (pouzolignan A), and 1,4-dihydroxy-3[4-hydroxyphenyl-(4-hydroxy-3-methoxybenzyl)]-2-(3,5-dihydroxyphenyl)butane (2) (pouzolignan B), were isolated from the EtOAc fraction of the methanol extract of Pouzolzia occidentalis. Compound 3, the methyl ether of 1, most likely an artifact, was also isolated. The overall structures and relative stereochemistry were elucidated largely by analysis of 1D and 2D NMR spectral data. ß 2009 Phytochemical Society of Europe. Published by Elsevier B.V. All rights reserved.
Keywords: Pouzolzia occidentalis Urticaceae Norlignans Pouzolignans A (1) and B (2)
1. Introduction Recent reports on the biological activity of extracts of plants of the Urticaceae family have indicated that they have antidiabetic and antilipidaemic (Momo et al., 2006), antibacterial (Sayeed et al., 2003), anti-HIV (Piccinelli et al., 2005) and anticancer (Konrad et al., 2000; Luo et al., 2003) activities. As part of our continuing effort to discover the chemical constituents and medicinal properties of the flora of Trinidad and Tobago, we have studied the plant Pouzolzia occidentalis (syn. P. palmieri) Wedd. (Urticaceae), on which no previous studies have been reported. We report herein the isolation of two new norlignans, 2-hydroxy-4-[4hydroxyphenyl-(4-hydroxy-3-methoxybenzyl)]-3-(3,5-dihydroxyphenyl)tetrahydrofuran (1) and 1,4-dihydroxy-3-[4-hydroxyphenyl-(4-hydroxy-3-methoxybenzyl)]-2-(3,5-dihydroxyphenyl)butane (2). 2. Results and discussion The crude methanol extract of the aerial parts of the plant was suspended in 80% CH3OH/H2O and the mixture successively extracted with petrol, CHCl3 and EtOAc. Repeated chromatography on the EtOAc fraction yielded three unusual norlignans. Compounds 1 and 2 named pouzolignans A and B, respectively, and compound 3, the methyl ether of 1, presumably formed during the isolation of the latter. Compound 1 was isolated as a brown gum. It + gave an ½a25 D of 32.4 (c 0.1, MeOH) and an M ion in the HREIMS at
* Corresponding author. Tel.: +868 662 6013; fax: +868 645 3771. E-mail address: [email protected] (A.R. Maxwell).
m/z 424.1512, which indicated a molecular formula of C24H24O7 (calcd. for C24H24O7, 424.1522). The infra-red spectrum with absorptions at 3374 and 1602 cm1 indicated hydroxyl and aromatic groups, respectively. 1D and 2D NMR experiments enabled elucidation of the structure of 1. Signals at dH 7.00 (2H, d, J = 8.5 Hz) and dH 6.59 (2H, d, J = 8.5 Hz) in the 1H NMR spectrum, the corresponding signals at dC 130.4 (2C) and dC 115.9 (2C) in the HMQC spectrum and two quaternary carbon signals at dC 156.7 and dC 136.7 indicated a 1,4-disubstituted and monooxygenated aromatic ring (A). The presence of a second aromatic ring (B), which was 1,2,4-trisubstituted and dioxygenated, was deduced from the signals at dH 6.69 (1H, d, J = 8.0 Hz), dH 6.73 (1H, dd, J = 8.0, 2.0 Hz) and dH 6.80 (1H, d, J = 2.0 Hz), the corresponding carbon signals at dC 116.0, 121.0 and 112.6 along with quaternary carbon signals at dC 148.9, 145.9 and 137.8. The presence of a third aromatic ring (C) which was 1,3,5-trisubstituted and dioxygenated followed from the signals at dH 6.04 (2H, t, J = 2.0 Hz) and dH 5.91 (1H, d, J = 2.0 Hz), the related carbon signals at dC 107.0 (2C) and dC 101.8 and the quaternary carbon signals at dC 147.1 and dC 159.4 (2C). A tetrahydrofuran-2-ol moiety was evident from the following data: a 1H multiplet at dH 3.12 (dC 53.0, C-4) showed cross peaks in the COSY spectrum to a 1H multiplet at dH 2.78 (dC 59.6, C-3) and the signals at dH 4.04 (t, J = 8.5 Hz) and dH 3.83 (m) which were due to a pair of methylene protons attached to a carbon resonating at dC 72.8 (C-5). The signal at dH 2.78 (H-3) showed a COSY cross peak to a signal at dH 5.21 (dC 106.4, C-2) which in turn gave an HMBC correlation to the methylene carbon signal at dC 72.8, C-5. The attachment of the aromatic ring C to the carbon resonating at dC 59.6 (C-3) was deduced from the HMBC correlations of the attached proton (dH 2.78, H-3) to the carbon
1874-3900/$ – see front matter ß 2009 Phytochemical Society of Europe. Published by Elsevier B.V. All rights reserved. doi:10.1016/j.phytol.2009.10.008
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M. Mohammed et al. / Phytochemistry Letters 3 (2010) 29–32
signals at dC 147.1 (C-1000 ) and dC 107.0 (C-2000 /C-6000 ). The signal at dH 3.12 (H-4) in addition to the cross peaks at dH 2.78 (H-3) and at dH 4.04 and dH 3.83 (H2-5) in the COSY spectrum already mentioned, also gave a COSY cross peak to the signal at dH 3.95 (dC 56.8) which itself showed cross peaks in the HMBC spectrum at dC 72.8 (C-5) and dC 53.0 (C-4) as well as at dC 112.6 (C00 -2), 121.0 (C-600 ), 130.4 (C-20 /C-60 ), 136.7 (C-10 ) and 137.8 (C-100 ). These data revealed that the aromatic rings A and B were attached to the carbon resonating at dC 56.8 and that this carbon was bonded to the carbon of the tetrahydrofuran ring resonating at dC 53.0 (C-4). Finally, the methoxy signal at dH 3.78 (dC 56.5) gave a cross peak in the HMBC spectrum to the quaternary aromatic carbon signal at dC 148.9 (C-300 ), confirming the presence of the methoxy group on aromatic ring B. The structure of 1 was therefore assigned as shown. The relative stereochemistry around the tetrahydrofuran ring was determined from vicinal H–H coupling constants and from analysis of the NOESY correlations. Thus, the proton at C-2 which couples only with H-3 occurs as a doublet at dH 5.21 with a coupling constant of only 2.0 Hz. This indicates that H-2 must be trans to H-3 since in five membered rings only the trans vicinal couplings can have such low values (Wu et al., 2002; Wu and Cremer, 2003). The signal at dH 2.78 (H-3) shows a coupling constant of 5.4 Hz with the signal at dH 3.12 (H-4) but in the NOESY spectrum gives only a weak cross peak to this signal, while it gives a very strong cross peak to the signal at dH 3.97 (H-6). This pointed to the trans relationship of H-3 and H-4 and the cis relationship between H-3 and H-6 and hence the trans relationship between H-4 and H-6. The NOESY spectrum also enabled assignment of the signals for the protons at C-5 that are cis and trans to H-4. Interestingly, the large coupling constant (11.5 Hz) between H-4 and H-6 must mean that these two protons are essentially anti to each other. Indeed, the minimum energy conformation of 1 obtained from molecular mechanics calculations (MM2 force field) showed an angle of ca.1798 between these two protons. Compound 2 was also isolated as a brown gum with an ½a25 D of 29.6 (c 0.1, MeOH). 1D and 2D NMR data (Table 2) indicated a structure similar to that of compound 1. However, the hemiacetal carbon present in compound 1 was absent in compound 2, which now showed two oxymethylene carbons rather than one as in compound 1. Furthermore, correlation observed between the H-2 (dH 5.21) and C-5 (dC 72.8) signals in the HMBC spectrum of compound 1 was absent in the HMBC spectrum of 2. These data suggested that a tetrahydrofuran ring was not present in compound 2. The fact that both the positive ion ([M+Na]+ peak at m/z 449.1577) and the negative ion ([MH] at m/z 425.1600) HRESIMS led to a molecular formula of C24H26O7 for compound 2, which therefore contained two more hydrogen atoms than compound 1, confirmed this suggestion. It was concluded that the tetrahydrofuran ring of compound 1 had opened to give the two hydroxymethyl groups in compound 2 as shown. Compound 3 was also isolated and characterized by both NMR and mass spectral data. It gave an [a]D of 44.4 (c 0.1, MeOH) and an M+ ion at m/z 438.1678, corresponding to a molecular formula of C25H26O7. The IR as well as the 1D and 2D NMR spectral data of 3 closely matched those of compound 1 and analysis of the NMR spectral data in a manner analogous to that given above led to the same gross structure for 3 as for 1. The extra carbon indicated by the molecular formula of 3 was deduced from the NMR spectral data to be due to a methoxy carbon which resonated at d 54.9 (dH 3.30). This methoxy proton signal showed an HMBC correlation to the carbon signal at d 113.0 (dH 4.74, H-2) and the COSY spectrum indicated long range coupling between the methoxy proton and H2 (d 4.74). Compound 3 was therefore identified as the methyl ether of 1. While 3 is a new compound it is unlikely to be a natural
product and is most likely an artifact arising from the extraction/ isolation procedures. 3. Experimental 3.1. General Optical rotations were measured on a Polatronic D digital polarimeter. IR spectra were recorded on a PerkinElmer RX1 FT-IR spectrophotometer as Nujol1 mulls and UV spectra were recorded in MeOH solutions on a Cary 50 UV–vis instrument. NMR spectra were run in MeOH-d6 on a Varian Unity 600 NMR spectrometer. Mass spectral data were obtained from either a Micromass 70S250 mass spectrometer or a MDS Sciex QStar mass spectrometer. TLC was performed on silica gel 60 PF254+366 plates (Merck), 0.25 mm thick for analytical and 1.0 mm thick for preparative work. Column chromatography was performed using Merck silica gel 60 (70–230 mesh). Sephadex1 LH-20 used for size exclusion chromatography was supplied by Sigma–Aldrich. The solvents petrol (60–80 8C), CHCl3, and EtOAc were all distilled prior to use. 3.2. Plant material The aerial parts of the plant were collected in December 2005 along Riverside Rd., Curepe, Trinidad. A voucher specimen (TRIN 36472) is deposited at the National Herbarium of Trinidad and Tobago. 3.3. Extraction and isolation The dried, ground plant material (1.3 kg) was exhaustively extracted with methanol (16 L) in the cold. The filtered extract was evaporated under reduced pressure to yield 85 g of crude extract. This was suspended in 80% MeOH/H2O and extracted successively with petrol, CHCl3, and EtOAc to give fractions of masses 7.2, 6.8 and 1.4 g, respectively. The EtOAc extract (1.4 g) was subjected to Si gel column chromatography eluting with CHCl3 containing increasing amounts of EtOH to yield eight fractions (I–VIII) after combination based on TLC analysis of the total of one hundred fractions. Repeated Si gel column chromatography (CHCl3/MeOH, 85:15; CHCl3/EtOAc, 1:1) on fraction III followed by gel permeation chromatography (Sephadex1 LH-20, CHCl3/EtOAc, 1:1) yielded compound 1 (3 mg). Compound 2 (8 mg) was isolated from fraction IV after preparative layer chromatography followed by column chromatography (EtOAc/MeOH/H2O, 400:20:18) of the more polar band. Compound 3 (2.6 mg) was obtained after repeated Si gel column chromatography (petrol/EtOAc, 3:2) on fraction II. 3.4. 2-Hydroxy-4-[4-hydroxyphenyl-(4-hydroxy-3methoxybenzyl)]-3-(3,5-dihydroxyphenyl)-tetrahydrofuran (1) Brown gum. ½a25 D 32.4 (c 0.1, MeOH); UV, lmax MeOH nm (log e): 270 (3.48); IR, ymax Nujol1 (cm1): 3374 (–OH), 1602 (Ar C–C); 1H and 13C NMR data, see Table 1. HREIMS (positive ion): found m/z 424.1512 [M]+; calculated for C24H24O7 m/z 424.1522. 3.5. 1,4-Dihydroxy-3-[4-hydroxyphenyl-(4-hydroxy-3methoxybenzyl)]-2-(3,5-dihydroxyphenyl)butane (2) Brown gum. ½a25 D 29.6 (c 0.1, MeOH); UV, lmax MeOH nm (log e): 270 (3.94); IR, ymax Nujol1 (cm1): 3360 (–OH), 1608 (Ar C–C); 1H and 13C NMR data, see Table 2. HRESIMS (positive ion): found m/z 449.1577 [M+Na]+; calculated for C24H26O7Na m/z 449.1594; negative ion: found m/z 425.1600 [MH]; calculated for C24H25O7 m/z 425.1605.
M. Mohammed et al. / Phytochemistry Letters 3 (2010) 29–32
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Table 1 NMR data of 1.a. Position
2 3 4 5b 5a 6 10 20 ,60 30 ,50 40 100 200 300 400 500 00 600 1000 2000 ,6000 3000 ,5000 4000 300 -OCH3 a
H–1H COSY
HMQC
dC
dH
106.4 59.6 53.0 72.8
5.21 2.78 3.12 3.83 4.04 3.95 – 7.00 6.59 – – 6.80 – – 6.69 6.73 – 5.91 – 6.04 3.78
56.8 136.7 130.4 115.9 156.7 137.8 112.6 148.9 145.9 116.0 121.0 147.1 107.0 159.4 101.8 56.5
(d, 2.0) (m) (m) (m) (t, 8.5) (d, 11.5) (d, 8.5) (d, 8.5)
(d, 2.0)
(d, 8.0) (dd, 2.0, 8.0) (d, 2.0) (t, 2.0)
1
HMBC
dH
dC
2.78 3.12, 5.21 2.78, 3.95, 4.04, 3.83 4.04 3.83 3.12 – 6.59 7.00
59.6, 56.8, 56.8, 56.8, 56.8, 53.0,
6.73 – – 6.73 6.69, 6.80 – 6.04 – 5.91 –
56.8, 121.0, 145.9,148.9
72.8, 147.1 106.4, 107.0, 147.1 59.6, 147.0 53.0 59.6, 106.4 72.8, 112.6, 130.4, 136.7, 137.8
56.8, 130.4, 156.7 116.0, 136.7, 156.7
137.8, 148.9 56.8, 112.6, 145.9 59.6, 101.8, 107.0, 159.4 107.0, 159.4 148.9
Data acquired at 600 MHz (1H) and 125 MHz (13C) in CD3OD with TMS as internal standard. Multiplicities and coupling constants (in Hz) are in parentheses.
Table 2 NMR data of 2.a. Position
dC 2
66.9
3 4 5
49.5 46.7 63.1
6 10 20 ,60 30 ,50 40 100 200 300 400 500 600 1000 2000 ,6000 3000 ,5000 4000 300 -OCH3 a
H–1H COSY
HMQC
53.1 136.8 130.3 116.6 157.0 138.1 116.8 149.0 145.8 116.3 121.1 143.3 109.5 159.5 102.2 56.3
dH 3.98 3.93 2.98 2.85 3.30 3.35 3.46 – 7.25 6.77 – – 6.75 – – 6.62 6.64 – 6.19 – 6.16 3.77
(dd, 11.4, 7.2) (dd, 11.4, 6.0) (m) (m) (m) (t, 9.0) (d, 12.0) (d, 8.5) (d, 8.5)
(d, 1.5)
(d, 8.0) (dd, 1.5, 8.0) (d, 2.0) (t, 2.0) (s)
1
HMBC
dH
dC
3.93 3.98 3.93, 3.98, 2.85 2.98, 3.35, 3.30, 3.46 2.85 2.85 2.85 – 6.77 7.25 – – 6.64 –
– 143.3 143.3, 109.5, 66.9, 63.1, 53.1, 46.7 – –
6.64 6.62, 6.75 – 6.16 – 6.19 –
138.1, – 157.0, 157.0, – – – – –– – 149.0, – 159.4, – – 149.0
136.8, 130.3, 121.1, 46.7 130.3, 53.1 136.8, 53.1, 116.6
145.8, 138.1, 121.1, 53.1 109.5, 102.2, 49.5
Data acquired at 600 MHz (1H) and 125 MHz (13C) in CD3OD with TMS as internal standard. Multiplicities and coupling constants (Hz) are in parentheses.
3.6. 4-[4-Hydroxyphenyl-(4-hydroxy-3-methoxybenzyl)]-3-(3,5dihydroxyphenyl)-2-methoxy-tetrahydrofuran (3) Brown gum. ½a25 D 44.4 (c 0.1, MeOH); UV, lmax MeOH nm (log e): 270 (3.89); IR, ymax Nujol1 (cm1): 3370 (–OH), 1602 (Ar C–C); 1H (600 MHz, CD3OD): d 6.98 (1H, d, J = 8.5 Hz, H-20 /H-60 ), 6.76 (1H, d, J = 1.5 Hz, H-200 ), 6.71 (2H, dd, J = 8.0, 1.5 Hz, H-600 ), 6.68 (1H, d, J = 8.0 Hz, H-500 ), 6.60 (2H, d, J = 8.5 Hz, H-30 /50 ), 6.05 (1H, t, J = 2.0 Hz, H-4000 ), 5.89 (2H, d, J = 2.0 Hz, H-2000 /6000 ), 4.74 (1H, br s, H2), 4.05 (1H, t, J = 8.5 Hz, H-5a), 3.87 (1H, d, J = 11.5 Hz, H-6), 3.78 (1H, s, OMe-300 ), 3.65 (1H, dd, J = 7.5, 8.5 Hz, H-5b), 3.30 (1H, s, OMe-2), 3.09 (1H, m, H-4), 2.82 (1H, d, J = 4.0 Hz, H-3); 13C NMR (125 MHz, CD3OD): d 159.5 (C-3000 /C-5000 ), 156.7 (C-40 ), 149.0 (C-300 ), 147.2 (C-1000 ), 146.0 (C-400 ), 137.8 (C-100 ), 136.7 (C-10 ), 130.3 (C-20 /C-
60 ), 121.0 (C-600 ), 116.3 (C-500 ), 115.8 (C-30 /C-50 ), 113.0 (C-2), 112.6 (C-200 ), 106.9 (C-2000 /C-6000 ), 101.5 (C-4000 ), 73.1 (C-5), 58.3 (C-3), 57.1 (C-6), 56.6 (OMe-300 ), 54.6 (OMe-2), 53.2 (C-4); HREIMS (positive ion): found m/z 438.1678 [M]+; calculated for C25H26O7 m/z 438.1679. Acknowledgements The authors wish to thank Dr. Alex Young of the University of Toronto for the MS analyses and Mr. W. Johnson for help in the collection and identification of the plant material. Financial support from the Department of Chemistry, University of the West Indies and NSERC of Canada is gratefully acknowledged.
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