- Email: [email protected]
New coumarins from Pterocaulon virgatum (L.) DC
Biochemical Systematics and Ecology 34 (2006) 165e169 www.elsevier.com/locate/biochemsyseco
New coumarins from Pterocaulon virgatum (L.) DC Dominick Maes a, Silvia Debenedetti b, Norbert De Kimpe a,* a
Department of Organic Chemistry, Faculty of Bioscience Engineering, Ghent University, Coupure Links 653, B-9000 Ghent, Belgium b Department of Biological Sciences, National University of La Plata, Calle 115 and 47, 1900 La Plata, Argentina Received 16 December 2004; accepted 5 July 2005
Abstract Three new coumarins, 5-hydroxy-6,7-methylenedioxycoumarin, 5-(2-hydroxy-3-methoxy-3-methylbutoxy)-6,7-methylenedioxycoumarin and 5-(2-hydroxy-3-methyl-3-butenyloxy)coumarin, were isolated from the petrol ether extract of the aerial parts of Pterocaulon virgatum. Their structures were elucidated based on spectroscopic evidence. Ó 2005 Elsevier Ltd. All rights reserved. Keywords: Coumarins; Pterocaulon virgatum
1. Introduction The genus Pterocaulon (Asteraceae) is widely distributed in North and South America and Australia. From the 18 species of the genus, 12 have medicinal reports (Cabrera and Ragonese, 1978). Pterocaulon virgatum DC is endemic in northeastern Argentina, southern Brazil and Paraguay. Aerial parts of the plant are used in Argentine traditional medicine as digestive, emenagogue, insecticide and as an agent against snake bites (Debenedetti et al., 1999). In previous reports we described the isolation and identification of five flavonoids (Debenedetti et al., 1983), mono and dicaffeoylquinic derivatives (Debenedetti et al., 1993; Martino et al., 1979), and several new and known 6,7-dioxygenated coumarins (Debenedetti et al., 1998) and 5-oxy-6,7-methylenedioxycoumarins (Debenedetti et al., 1994, 1997). The present report deals with the isolation of three new trioxygenated coumarins from P. virgatum, namely 5-(2-hydroxy-3-methoxy-3-methylbutoxy)-6,7-methylenedioxycoumarin 1, 5-(2-hydroxy-3-methyl-3-butenyloxy)6,7-methylenedioxycoumarin 2 and 5-hydroxy-6,7-methylenedioxycoumarin 3. 4' 1' 2' 3'
O O
6
O
7
5
8
1
4a
4OH 3
8a
O 1
2
OMe O O O
O
O
2
* Corresponding author. Tel.: C32 9 264 59 51; fax: C32 9 264 62 43. E-mail address: [email protected] (N. De Kimpe). 0305-1978/$ - see front matter Ó 2005 Elsevier Ltd. All rights reserved. doi:10.1016/j.bse.2005.09.001
OH
OH
O O
O
O
3
O
D. Maes et al. / Biochemical Systematics and Ecology 34 (2006) 165e169
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2. Materials and methods 2.1. General experimental procedures Melting points are uncorrected. UV spectra were recorded in MeOH. IR: KBr pellet. EIMS spectra were obtained by direct probe insertion at 70 eV. 1H NMR and 13C NMR were recorded at 270 and 67.8 MHz, respectively, in CDCl3 and DMSO-d6 using TMS as internal standard. 2.2. Plant material Aerial parts of P. virgatum (L.) DC were collected from the Experimental Station INTA, Concepcio´n del Uruguay, Entre Rios, Argentina, during November 1989 and identified by Dr. B. Soraru´, ‘Juan A. Dominguez’ Museum, Faculty of Pharmacy and Biochemistry, University of Buenos Aires. A herbarium species No. SD-041 is preserved in the Department of Pharmacology of the same institution. 2.3. Isolation and identification The air dried and powdered aerial parts of P. virgatum (950 g) were extracted at room temperature with 25% aq. methanol. The methanol was removed under reduced pressure and the aqueous layer was partitioned with petrol ether, chloroform, diethyl ether and ethyl acetate. The concentrated petrol ether extract (1 g) was subjected to CC on silica gel and eluted with a gradient of petrol ether:ethyl acetate mixture (25e100%). Fractions were collected (15 ml) and monitored by silica gel TLC using toluene:ethyl acetate (7:3) as mobile phase. Similar fractions were combined into 20 fractions (IeXX). Fraction XI (140 mg) was further chromatographed by CPC model CCC-1000, Pharma-Tech Research Corp., using hexane:EtOAc:MeOH:H2O (1:1:1:1) upper phase as mobile phase. Flow rate, 1 ml/min; pressure, 105 psi; rotation speed, 1050 rpm; run time, 270 min and fraction volume, 5 ml. A total of 85 fractions was obtained. The fractions were combined, followed by TLC using silica gel, and the same mobile phase was used in the CPC. Fractions 32e37 and 39e43 were evaporated in vacuo and recrystallized from MeOH, yielding 17 mg of 5-(2hydroxy-3-methoxy-3-methylbutoxy)-6,7-methylenedioxycoumarin 1 and 11 mg of 5-(2-hydroxy-3-methyl-3-butenyloxy)-6,7-methylenedioxycoumarin 2, respectively. Fraction XV was further purified from Sephadex LH20 using MeOH as eluent to obtain 10 mg of 5-hydroxy-6,7methylenedioxycoumarin 3. 2.3.1. 5-(2-Hydroxy-3-methoxy-3-methylbutoxy)-6,7-methylenedioxycoumarin (1) ÿ1 UV lMeOH max nm: 335 sh, 315, 257 sh, 235 nm IR nmax cm : 3431 (broad, OH); 1722 (C]O), 1629 (C]CAr). EIMS C C m/z (rel. int.): 322 [M] (29), 206 [M ÿ R C H] (44), 73 [(CH3)2C]OCH3]C (100). 1H NMR: see Table 1. 13C NMR: see Table 2. Table 1 1 H NMR data of coumarins 1e3 H
1 (CDCl3)
2 (CDCl3)
3 (DMSO-d6)
3 4 8 6,7-OCH2O
6.22 d (J Z 9.8 Hz) 8.03 d (J Z 9.8 Hz) 6.55 s 6.019 d (J Z 1.4 Hz) 6.032 d (J Z 1.4 Hz) 4.32 dd (J Z 10.5, 8.3 Hz) 4.52 dd (J Z 10.5, 3.0 Hz) 3.86 dd (J Z 8.3, 3.0 Hz) 1.21 s 1.25 s 3.26 s 2.50 s e
6.22 d (J Z 9.8 Hz) 7.97 d (J Z 9.8 Hz) 6.56 s 6.019 d (J Z 1.4 Hz) 6.032 d (J Z 1.4 Hz) 4.31 dd (J Z 10.7, 8.1 Hz) 4.45 dd (J Z 10.7, 3.3 Hz) 4.45 dd (J Z 8.1, 3.3 Hz) 5.03 s and 5.14 s 1.82 s e e e
6.19 8.04 6.63 6.10
1# 2# 4# 3#-Me 3#-OMe 2#-OH 5#-OH
d (J Z 9.6 Hz) d (J Z 9.6 Hz) s s
e e e e e e 10.86 s
D. Maes et al. / Biochemical Systematics and Ecology 34 (2006) 165e169
167
Table 2 13 C NMR data of coumarins 1e3 C
1 (CDCl3)
2 (CDCl3)
3 (DMSO-d6)
2 3 4 4a 5 6 7 8 8a 6,7-OCH2O 1# 2# 3# 4# 3#-Me 3#-OMe
161.5 112.0 139.1 107.2 137.2 132.3 151.6 92.9 152.6 102.0 73.6 76.0 78.0 20.6 20.9 49.3
161.3 112.0 138.8 107.0 136.8 132.2 151.5 93.0 152.5 102.1 74.8 74.1 143.3 113.2 18.8 e
160.5 s 110.5 d 139.4 d 105.3 s 136.0 s 130.8 s 151.2 sa 90.05 d 151.7 sa 102.0 t e e e e e e
a
s d d s s s sa d sa t t d s q q q
s d d s s s sa d sa t t d s t q
May be interchanged.
2.3.2. 5-(2-Hydroxy-3-methyl-3-butenyloxy)-6,7-methylenedioxycoumarin (2) UV lMeOH nm: 335 sh, 315, 257 sh, 235 nm. IR nmax cmÿ1: 3410 (broad, OH); 1730 (br, C]O), 1630 (C]CAr). max EIMS m/z (rel. int.): 290 [M]C (57), 206 [M ÿ R C H]C (100), 178 [206 ÿ CO] (47). 1H NMR: see Table 1. 13C NMR: see Table 2. 2.3.3. 5-Hydroxy-6,7-methylenedioxycoumarin (3) nm: 245 sh, 322; lMeONa nm: 250, 340. IR nmax cmÿ1: 3447 (broad, OH); 1685 (br, C]O and C]C), UV lMeOH max max C 1591. EIMS m/z (rel. int.): 206 [M] (100), 178 [M ÿ CO]C (84), 149 (53). 1H NMR: see Table 1. 13C NMR: see Table 2. 3. Results and discussion The aqueous methanol extract of the aerial parts of P. virgatum was subjected to successive extraction with petrol, chloroform, diethyl ether and ethyl acetate. The concentrated petrol ether extract was submitted to CPC separation, yielding 5-(2-hydroxy-3-methoxy-3-methylbutoxy)-6,7-methylenedioxycoumarin 1, 5-(2-hydroxy-3-methyl-3-butenyloxy)-6,7-methylenedioxycoumarin 2 and 5-hydroxy-6,7-methylenedioxycoumarin 3. The IR spectra of compounds 1e3 showed the presence of a lactone carbonyl, characteristic of coumarins, at 1710 cmÿ1 together with the bands of an aromatic ring at 1620 and 1575 cmÿ1. The UV spectrum of the three compounds exhibited UV absorption typical for 5,6,7-trioxygenated coumarins (Debenedetti et al., 1994). Compound 3 showed a bathochromic shift when dissolved in NaOMe/MeOH, revealing the presence of a phenolic hydroxy group. The 1H NMR spectra of compounds 1e3 showed a pair of doublets at d 6.22 and 8.03 (J Z 9.8 Hz) (1), at d 6.22 and 7.97 (J Z 9.8 Hz) (2) and at d 6.19 and 8.04 (J Z 9.6 Hz) (3), for H-3 and H-4, respectively, which is typical of coumarins substituted at C-5. Singlets at 6.55 (1), 6.56 (2) and 6.63 (3), suggested the presence of only one aromatic proton, thus these coumarins had to be trisubstituted. The doublets at 6.02 and 6.03 (J Z 1.4 Hz) (1 and 2) or the singlet at 6.10 (3) revealed the presence of a methylenedioxy substituent. In the 1H NMR spectrum, a long range coupling was visible between the H-4 doublet and the aromatic proton H-8, suggesting a 5,6,7-trioxygenated substitution pattern for the three coumarins. This oxygenation pattern was confirmed by comparison of the spectroscopic data of coumarins 1e3 with those obtained for 5-(3-methyl-2-butenyloxy)-6,7-methylenedioxycoumarin 4, whose structure was unequivocally established by X-ray crystallographic analysis (Debenedetti et al., 1994). The newly isolated compounds also show structural analogy with 5-methoxy-6,7-methylenedioxycoumarin 5, 5-(2,3-dihydroxy-3-methylbutoxy)6,7-methylenedioxycoumarin 6 and 5-(3-methyl-2-oxobutoxy)-6,7-methylenedioxycoumarin 7, all isolated from the same plant (Debenedetti et al., 1994, 1997).
D. Maes et al. / Biochemical Systematics and Ecology 34 (2006) 165e169
168
OH
OCH3
O
O
O
O O
O
4
O
O
O
O
5
O
O
O
6
O
OH
O O
O
O O
O
7
Further information on the structure of the side chains at C5 was derived from the 1H and 13C NMR spectra (Tables 1 and 2). Compound 1 exhibited two pairs of double doublets at d 4.32 (J Z 10.5, 8.3 Hz) and 4.52 (J Z 10.5, 3.0 Hz) corresponding to the AB-part of an ABX system in a OeCH2eCHeOe moiety, and a doublet of doublets at d 3.86 (J Z 8.3, 3.0 Hz) showing the X-part of the same system. A singlet at d 3.26 (3H, OMe) and a singlet signal at d 1.25 (6H, Me2) suggested the presence of a (CH3)2CeOCH3 fragment. Together with the broad singlet at d 2.50 (1H, OH) it proves that compound 1 is in fact 5-(2-hydroxy-3-methoxy-3-methylbutoxy)-6,7-methylenedioxycoumarin. Compound 2 showed two pairs of doublets at d 4.31 (J Z 10.7, 8.1 Hz) and 4.45 (J Z 10.7, 3.3 Hz) corresponding to the AB-part of an ABX system in a OeCH2eCHeOe moiety, and a doublet of doublets at d 4.45 (J Z 8.1, 3.3 Hz) showing the X-part of the same system. In addition, the 1H NMR spectrum exhibited two broad singlets at d 5.03 and 5.14 (1H, each), typical of terminal vinylic protons, and a singlet signal at d 1.82 (3H) characteristic of a methyl group, suggesting the presence of a CH2]C(CH3) fragment. Compound 3 showed a singlet at d 10.86 and was, therefore, identified as 5-hydroxy-6,7-methylenedioxycoumarin. The Argentine Pterocaulon genus comprises 11 species (Zuloaga and Morrone, 1999) of which only six species (P. virgatum, Pterocaulon polystachyum, Pterocaulon balansae, Pterocaulon lanatum, Pterocaulon alopecuroides and Pterocaulon purpurascens) were phytochemically investigated. From these species several known and new coumarins, with different oxygenation patterns, were identified. Four of the six investigated Pterocaulon species (P. virgatum, P. polystachyum, P. balansae and P. lanatum) contained 5-oxy-6,7-methylenedioxycoumarins (Debenedetti et al., 1996, 1997, 1998, 1999; Magalhaes et al., 1981; Palacios et al., 1999; Vera et al., 2001; Vilegas et al., 1995). The three new coumarins 5-(2-hydroxy-3-methoxy-3-methylbutoxy)-6,7-methylenedioxycoumarin 1, 5-(2-hydroxy-3-methyl3-butenyloxy)-6,7-methylenedioxycoumarin 2 and 5-hydroxy-6,7-methylenedioxycoumarin 3, isolated from P. virgatum, show the same substitution pattern which is rare in nature but seems to be characteristic for the Pterocaulon genus. Acknowledgements The authors are indebted to SECyT (Argentina), the ‘Fund for Scientific Research (FWO) e Flanders’ (Belgium) and the ‘Special Research Fund (BOF) e Ghent University’ (Belgium) for financial support. References Cabrera, A.L., Ragonese, A.M., 1978. Revisio´n de ge´nero Pterocaulon (Compositae). Darviniana 21, 185e257. Debenedetti, S.L., Ferraro, G.E., Coussio, J.D., 1983. Flavonoides aislados de Pterocaulon virgatum. Acta Farm. Bonaer. 2 (1), 1. Debenedetti, S.L., Palacios, P.S., Wilson, E.G., Coussio, J.D., 1993. HPLC analysis of caffeoylquinic acid contents in argentine medicinal plants. Acta Hortic. 333, 191. Debenedetti, S.L., Palacios, P.S., Nadinic, E.L., Coussio, J.D., De Kimpe, N., Boeykens, M., Feneau-Dupont, J., Declercq, J.P., 1994. 5-(3-Methyl2-butenyloxy)-6,7-methylenedioxycoumarin, a 5,6,7-trioxygenated coumarin from Pterocaulon virgatum. J. Nat. Prod. 57, 1539e1542. Debenedetti, S.L., Nadinic, E.L., Gomez, M.A., Coussio, J.D., De Kimpe, N., Boeykens, M., 1996. Purpurasolol, a highly oxygenated coumarin from Pterocaulon purpurascens. Phytochemistry 42, 563e564. Debenedetti, S.L., De Kimpe, N., Boeykens, M., Coussio, J.D., Kesteleyn, B., 1997. Structural revision of four coumarins from Pterocaulon species. Phytochemistry 45, 1515e1517. Debenedetti, S.L., Nadinic, E.L., Coussio, J.D., De Kimpe, N., Boeykens, M., 1998. Two 6,7-dioxygenated coumarins from Pterocaulon virgatum. Phytochemistry 48, 707e710. Debenedetti, S.L., Abbaspour Tehrani, K., Van Puyvelde, L., De Kimpe, N., 1999. Isopurpurasol, a coumarin from Pterocaulon virgatum. Phytochemistry 51, 701e703. Magalhaes, A.F., Magalhaes, E.G., Leitao Filho, H.F., Frighetto, R.T.S., Barros, S.M.G., 1981. Coumarins from Pterocaulon balansae and P. lanatum. Phytochemistry 20, 1369e1371.
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Martino, V.S., Debenedetti, S.L., Coussio, J.D., 1979. Caffeoylquinic acids from Pterocaulon virgatum and Pluchea sagittalis. Phytochemistry 18, 2052. Palacios, P.S., Rojo, A.A., Coussio, J.D., De Kimpe, N., Debenedetti, S.L., 1999. 6,7-Dioxygenated and 5,6,7-trioxygenated coumarins from Pterocaulon polystachyum. Planta Med. 65, 294e295. Vera, N., Bardo´n, A., Catala´n, C., Gedris, T., Herz, W., 2001. New coumarins from Pterocaulon polystachyum. Planta Med. 67, 674e677. Vilegas, W., Boralle, N., Cabrera, A., Bernardo, A.C., Pozetti, G.L., Fontes Arantes, S., 1995. Coumarins and a flavonoid from Pterocaulon alopecuroides. Phytochemistry 38, 1017e1019. Zuloaga, F.O., Morrone, O., 1999. Cata´logo de las plantas vasculares de la Repu´blica Argentina, vol. II. Dicotyledoneae (Asteraceae), 179e180.
New coumarins from Pterocaulon virgatum (L.) DC Dominick Maes a, Silvia Debenedetti b, Norbert De Kimpe a,* a
Department of Organic Chemistry, Faculty of Bioscience Engineering, Ghent University, Coupure Links 653, B-9000 Ghent, Belgium b Department of Biological Sciences, National University of La Plata, Calle 115 and 47, 1900 La Plata, Argentina Received 16 December 2004; accepted 5 July 2005
Abstract Three new coumarins, 5-hydroxy-6,7-methylenedioxycoumarin, 5-(2-hydroxy-3-methoxy-3-methylbutoxy)-6,7-methylenedioxycoumarin and 5-(2-hydroxy-3-methyl-3-butenyloxy)coumarin, were isolated from the petrol ether extract of the aerial parts of Pterocaulon virgatum. Their structures were elucidated based on spectroscopic evidence. Ó 2005 Elsevier Ltd. All rights reserved. Keywords: Coumarins; Pterocaulon virgatum
1. Introduction The genus Pterocaulon (Asteraceae) is widely distributed in North and South America and Australia. From the 18 species of the genus, 12 have medicinal reports (Cabrera and Ragonese, 1978). Pterocaulon virgatum DC is endemic in northeastern Argentina, southern Brazil and Paraguay. Aerial parts of the plant are used in Argentine traditional medicine as digestive, emenagogue, insecticide and as an agent against snake bites (Debenedetti et al., 1999). In previous reports we described the isolation and identification of five flavonoids (Debenedetti et al., 1983), mono and dicaffeoylquinic derivatives (Debenedetti et al., 1993; Martino et al., 1979), and several new and known 6,7-dioxygenated coumarins (Debenedetti et al., 1998) and 5-oxy-6,7-methylenedioxycoumarins (Debenedetti et al., 1994, 1997). The present report deals with the isolation of three new trioxygenated coumarins from P. virgatum, namely 5-(2-hydroxy-3-methoxy-3-methylbutoxy)-6,7-methylenedioxycoumarin 1, 5-(2-hydroxy-3-methyl-3-butenyloxy)6,7-methylenedioxycoumarin 2 and 5-hydroxy-6,7-methylenedioxycoumarin 3. 4' 1' 2' 3'
O O
6
O
7
5
8
1
4a
4OH 3
8a
O 1
2
OMe O O O
O
O
2
* Corresponding author. Tel.: C32 9 264 59 51; fax: C32 9 264 62 43. E-mail address: [email protected] (N. De Kimpe). 0305-1978/$ - see front matter Ó 2005 Elsevier Ltd. All rights reserved. doi:10.1016/j.bse.2005.09.001
OH
OH
O O
O
O
3
O
D. Maes et al. / Biochemical Systematics and Ecology 34 (2006) 165e169
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2. Materials and methods 2.1. General experimental procedures Melting points are uncorrected. UV spectra were recorded in MeOH. IR: KBr pellet. EIMS spectra were obtained by direct probe insertion at 70 eV. 1H NMR and 13C NMR were recorded at 270 and 67.8 MHz, respectively, in CDCl3 and DMSO-d6 using TMS as internal standard. 2.2. Plant material Aerial parts of P. virgatum (L.) DC were collected from the Experimental Station INTA, Concepcio´n del Uruguay, Entre Rios, Argentina, during November 1989 and identified by Dr. B. Soraru´, ‘Juan A. Dominguez’ Museum, Faculty of Pharmacy and Biochemistry, University of Buenos Aires. A herbarium species No. SD-041 is preserved in the Department of Pharmacology of the same institution. 2.3. Isolation and identification The air dried and powdered aerial parts of P. virgatum (950 g) were extracted at room temperature with 25% aq. methanol. The methanol was removed under reduced pressure and the aqueous layer was partitioned with petrol ether, chloroform, diethyl ether and ethyl acetate. The concentrated petrol ether extract (1 g) was subjected to CC on silica gel and eluted with a gradient of petrol ether:ethyl acetate mixture (25e100%). Fractions were collected (15 ml) and monitored by silica gel TLC using toluene:ethyl acetate (7:3) as mobile phase. Similar fractions were combined into 20 fractions (IeXX). Fraction XI (140 mg) was further chromatographed by CPC model CCC-1000, Pharma-Tech Research Corp., using hexane:EtOAc:MeOH:H2O (1:1:1:1) upper phase as mobile phase. Flow rate, 1 ml/min; pressure, 105 psi; rotation speed, 1050 rpm; run time, 270 min and fraction volume, 5 ml. A total of 85 fractions was obtained. The fractions were combined, followed by TLC using silica gel, and the same mobile phase was used in the CPC. Fractions 32e37 and 39e43 were evaporated in vacuo and recrystallized from MeOH, yielding 17 mg of 5-(2hydroxy-3-methoxy-3-methylbutoxy)-6,7-methylenedioxycoumarin 1 and 11 mg of 5-(2-hydroxy-3-methyl-3-butenyloxy)-6,7-methylenedioxycoumarin 2, respectively. Fraction XV was further purified from Sephadex LH20 using MeOH as eluent to obtain 10 mg of 5-hydroxy-6,7methylenedioxycoumarin 3. 2.3.1. 5-(2-Hydroxy-3-methoxy-3-methylbutoxy)-6,7-methylenedioxycoumarin (1) ÿ1 UV lMeOH max nm: 335 sh, 315, 257 sh, 235 nm IR nmax cm : 3431 (broad, OH); 1722 (C]O), 1629 (C]CAr). EIMS C C m/z (rel. int.): 322 [M] (29), 206 [M ÿ R C H] (44), 73 [(CH3)2C]OCH3]C (100). 1H NMR: see Table 1. 13C NMR: see Table 2. Table 1 1 H NMR data of coumarins 1e3 H
1 (CDCl3)
2 (CDCl3)
3 (DMSO-d6)
3 4 8 6,7-OCH2O
6.22 d (J Z 9.8 Hz) 8.03 d (J Z 9.8 Hz) 6.55 s 6.019 d (J Z 1.4 Hz) 6.032 d (J Z 1.4 Hz) 4.32 dd (J Z 10.5, 8.3 Hz) 4.52 dd (J Z 10.5, 3.0 Hz) 3.86 dd (J Z 8.3, 3.0 Hz) 1.21 s 1.25 s 3.26 s 2.50 s e
6.22 d (J Z 9.8 Hz) 7.97 d (J Z 9.8 Hz) 6.56 s 6.019 d (J Z 1.4 Hz) 6.032 d (J Z 1.4 Hz) 4.31 dd (J Z 10.7, 8.1 Hz) 4.45 dd (J Z 10.7, 3.3 Hz) 4.45 dd (J Z 8.1, 3.3 Hz) 5.03 s and 5.14 s 1.82 s e e e
6.19 8.04 6.63 6.10
1# 2# 4# 3#-Me 3#-OMe 2#-OH 5#-OH
d (J Z 9.6 Hz) d (J Z 9.6 Hz) s s
e e e e e e 10.86 s
D. Maes et al. / Biochemical Systematics and Ecology 34 (2006) 165e169
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Table 2 13 C NMR data of coumarins 1e3 C
1 (CDCl3)
2 (CDCl3)
3 (DMSO-d6)
2 3 4 4a 5 6 7 8 8a 6,7-OCH2O 1# 2# 3# 4# 3#-Me 3#-OMe
161.5 112.0 139.1 107.2 137.2 132.3 151.6 92.9 152.6 102.0 73.6 76.0 78.0 20.6 20.9 49.3
161.3 112.0 138.8 107.0 136.8 132.2 151.5 93.0 152.5 102.1 74.8 74.1 143.3 113.2 18.8 e
160.5 s 110.5 d 139.4 d 105.3 s 136.0 s 130.8 s 151.2 sa 90.05 d 151.7 sa 102.0 t e e e e e e
a
s d d s s s sa d sa t t d s q q q
s d d s s s sa d sa t t d s t q
May be interchanged.
2.3.2. 5-(2-Hydroxy-3-methyl-3-butenyloxy)-6,7-methylenedioxycoumarin (2) UV lMeOH nm: 335 sh, 315, 257 sh, 235 nm. IR nmax cmÿ1: 3410 (broad, OH); 1730 (br, C]O), 1630 (C]CAr). max EIMS m/z (rel. int.): 290 [M]C (57), 206 [M ÿ R C H]C (100), 178 [206 ÿ CO] (47). 1H NMR: see Table 1. 13C NMR: see Table 2. 2.3.3. 5-Hydroxy-6,7-methylenedioxycoumarin (3) nm: 245 sh, 322; lMeONa nm: 250, 340. IR nmax cmÿ1: 3447 (broad, OH); 1685 (br, C]O and C]C), UV lMeOH max max C 1591. EIMS m/z (rel. int.): 206 [M] (100), 178 [M ÿ CO]C (84), 149 (53). 1H NMR: see Table 1. 13C NMR: see Table 2. 3. Results and discussion The aqueous methanol extract of the aerial parts of P. virgatum was subjected to successive extraction with petrol, chloroform, diethyl ether and ethyl acetate. The concentrated petrol ether extract was submitted to CPC separation, yielding 5-(2-hydroxy-3-methoxy-3-methylbutoxy)-6,7-methylenedioxycoumarin 1, 5-(2-hydroxy-3-methyl-3-butenyloxy)-6,7-methylenedioxycoumarin 2 and 5-hydroxy-6,7-methylenedioxycoumarin 3. The IR spectra of compounds 1e3 showed the presence of a lactone carbonyl, characteristic of coumarins, at 1710 cmÿ1 together with the bands of an aromatic ring at 1620 and 1575 cmÿ1. The UV spectrum of the three compounds exhibited UV absorption typical for 5,6,7-trioxygenated coumarins (Debenedetti et al., 1994). Compound 3 showed a bathochromic shift when dissolved in NaOMe/MeOH, revealing the presence of a phenolic hydroxy group. The 1H NMR spectra of compounds 1e3 showed a pair of doublets at d 6.22 and 8.03 (J Z 9.8 Hz) (1), at d 6.22 and 7.97 (J Z 9.8 Hz) (2) and at d 6.19 and 8.04 (J Z 9.6 Hz) (3), for H-3 and H-4, respectively, which is typical of coumarins substituted at C-5. Singlets at 6.55 (1), 6.56 (2) and 6.63 (3), suggested the presence of only one aromatic proton, thus these coumarins had to be trisubstituted. The doublets at 6.02 and 6.03 (J Z 1.4 Hz) (1 and 2) or the singlet at 6.10 (3) revealed the presence of a methylenedioxy substituent. In the 1H NMR spectrum, a long range coupling was visible between the H-4 doublet and the aromatic proton H-8, suggesting a 5,6,7-trioxygenated substitution pattern for the three coumarins. This oxygenation pattern was confirmed by comparison of the spectroscopic data of coumarins 1e3 with those obtained for 5-(3-methyl-2-butenyloxy)-6,7-methylenedioxycoumarin 4, whose structure was unequivocally established by X-ray crystallographic analysis (Debenedetti et al., 1994). The newly isolated compounds also show structural analogy with 5-methoxy-6,7-methylenedioxycoumarin 5, 5-(2,3-dihydroxy-3-methylbutoxy)6,7-methylenedioxycoumarin 6 and 5-(3-methyl-2-oxobutoxy)-6,7-methylenedioxycoumarin 7, all isolated from the same plant (Debenedetti et al., 1994, 1997).
D. Maes et al. / Biochemical Systematics and Ecology 34 (2006) 165e169
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OH
OCH3
O
O
O
O O
O
4
O
O
O
O
5
O
O
O
6
O
OH
O O
O
O O
O
7
Further information on the structure of the side chains at C5 was derived from the 1H and 13C NMR spectra (Tables 1 and 2). Compound 1 exhibited two pairs of double doublets at d 4.32 (J Z 10.5, 8.3 Hz) and 4.52 (J Z 10.5, 3.0 Hz) corresponding to the AB-part of an ABX system in a OeCH2eCHeOe moiety, and a doublet of doublets at d 3.86 (J Z 8.3, 3.0 Hz) showing the X-part of the same system. A singlet at d 3.26 (3H, OMe) and a singlet signal at d 1.25 (6H, Me2) suggested the presence of a (CH3)2CeOCH3 fragment. Together with the broad singlet at d 2.50 (1H, OH) it proves that compound 1 is in fact 5-(2-hydroxy-3-methoxy-3-methylbutoxy)-6,7-methylenedioxycoumarin. Compound 2 showed two pairs of doublets at d 4.31 (J Z 10.7, 8.1 Hz) and 4.45 (J Z 10.7, 3.3 Hz) corresponding to the AB-part of an ABX system in a OeCH2eCHeOe moiety, and a doublet of doublets at d 4.45 (J Z 8.1, 3.3 Hz) showing the X-part of the same system. In addition, the 1H NMR spectrum exhibited two broad singlets at d 5.03 and 5.14 (1H, each), typical of terminal vinylic protons, and a singlet signal at d 1.82 (3H) characteristic of a methyl group, suggesting the presence of a CH2]C(CH3) fragment. Compound 3 showed a singlet at d 10.86 and was, therefore, identified as 5-hydroxy-6,7-methylenedioxycoumarin. The Argentine Pterocaulon genus comprises 11 species (Zuloaga and Morrone, 1999) of which only six species (P. virgatum, Pterocaulon polystachyum, Pterocaulon balansae, Pterocaulon lanatum, Pterocaulon alopecuroides and Pterocaulon purpurascens) were phytochemically investigated. From these species several known and new coumarins, with different oxygenation patterns, were identified. Four of the six investigated Pterocaulon species (P. virgatum, P. polystachyum, P. balansae and P. lanatum) contained 5-oxy-6,7-methylenedioxycoumarins (Debenedetti et al., 1996, 1997, 1998, 1999; Magalhaes et al., 1981; Palacios et al., 1999; Vera et al., 2001; Vilegas et al., 1995). The three new coumarins 5-(2-hydroxy-3-methoxy-3-methylbutoxy)-6,7-methylenedioxycoumarin 1, 5-(2-hydroxy-3-methyl3-butenyloxy)-6,7-methylenedioxycoumarin 2 and 5-hydroxy-6,7-methylenedioxycoumarin 3, isolated from P. virgatum, show the same substitution pattern which is rare in nature but seems to be characteristic for the Pterocaulon genus. Acknowledgements The authors are indebted to SECyT (Argentina), the ‘Fund for Scientific Research (FWO) e Flanders’ (Belgium) and the ‘Special Research Fund (BOF) e Ghent University’ (Belgium) for financial support. References Cabrera, A.L., Ragonese, A.M., 1978. Revisio´n de ge´nero Pterocaulon (Compositae). Darviniana 21, 185e257. Debenedetti, S.L., Ferraro, G.E., Coussio, J.D., 1983. Flavonoides aislados de Pterocaulon virgatum. Acta Farm. Bonaer. 2 (1), 1. Debenedetti, S.L., Palacios, P.S., Wilson, E.G., Coussio, J.D., 1993. HPLC analysis of caffeoylquinic acid contents in argentine medicinal plants. Acta Hortic. 333, 191. Debenedetti, S.L., Palacios, P.S., Nadinic, E.L., Coussio, J.D., De Kimpe, N., Boeykens, M., Feneau-Dupont, J., Declercq, J.P., 1994. 5-(3-Methyl2-butenyloxy)-6,7-methylenedioxycoumarin, a 5,6,7-trioxygenated coumarin from Pterocaulon virgatum. J. Nat. Prod. 57, 1539e1542. Debenedetti, S.L., Nadinic, E.L., Gomez, M.A., Coussio, J.D., De Kimpe, N., Boeykens, M., 1996. Purpurasolol, a highly oxygenated coumarin from Pterocaulon purpurascens. Phytochemistry 42, 563e564. Debenedetti, S.L., De Kimpe, N., Boeykens, M., Coussio, J.D., Kesteleyn, B., 1997. Structural revision of four coumarins from Pterocaulon species. Phytochemistry 45, 1515e1517. Debenedetti, S.L., Nadinic, E.L., Coussio, J.D., De Kimpe, N., Boeykens, M., 1998. Two 6,7-dioxygenated coumarins from Pterocaulon virgatum. Phytochemistry 48, 707e710. Debenedetti, S.L., Abbaspour Tehrani, K., Van Puyvelde, L., De Kimpe, N., 1999. Isopurpurasol, a coumarin from Pterocaulon virgatum. Phytochemistry 51, 701e703. Magalhaes, A.F., Magalhaes, E.G., Leitao Filho, H.F., Frighetto, R.T.S., Barros, S.M.G., 1981. Coumarins from Pterocaulon balansae and P. lanatum. Phytochemistry 20, 1369e1371.
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