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Minor alkamides from Heliopsis longipes S.F. Blake (Asteraceae) fresh roots
Phytochemistry Letters 4 (2011) 275–279
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Minor alkamides from Heliopsis longipes S.F. Blake (Asteraceae) fresh roots Sugey Lo´pez-Martı´nez, A. Berenice Aguilar-Guadarrama, Marı´a Yolanda Rios * Centro de Investigaciones Quı´micas, Universidad Auto´noma del Estado de Morelos, Avenida Universidad 1001, Col. Chamilpa, 62209 Cuernavaca, Morelos, Mexico
A R T I C L E I N F O
A B S T R A C T
Article history: Received 3 December 2010 Received in revised form 12 April 2011 Accepted 21 April 2011 Available online 13 May 2011
From the fresh roots of Heliopsis longipes three new minor alkamides: longipinamide A (N-isobutyl-8,10diynoic-3Z-undecenamide), longipenamide A (N-isobutyl-syn-8,9-dihydroxy-2E,6Z-decadienamide) and longipenamide B (N-isobutyl-syn-6,9-dihydroxy-2E,7E-decadienamide); three known alkamides: affinin (spilanthol, N-isobutyl-2E,6Z,8E-decatrienamide), N-isobutyl-2E,6Z-decadienamide and Nisobutyl-2E-decenamide; and 11 other known compounds were isolated. The structures of the three new minor alkamides were established by 1D and 2D NMR spectroscopy including 1H, 13C, DEPT, COSY, HSQC, and HMBC experiments, as well as by EI and FAB+ mass spectrometry. To our knowledge, this is the first report of the isolation of linear dihydroxyalkamides as natural products. ß 2011 Phytochemical Society of Europe. Published by Elsevier B.V. All rights reserved.
Keywords: Heliopsis longipes Asteraceae Alkamides Linear dihydroxyalkamides
1. Introduction Heliopsis longipes S.F. Blake is an annual climber that grows from July to October. It is an endemic plant of the central region of Mexico (Molina-Torres et al., 1999; Cilia-Lo´pez et al., 2008), where is popularly called ‘‘chilcuague’’ or ‘‘chilcuan’’ (Jacobson et al., 1947; Jacobson, 1954, 1955; Correa et al., 1971). The first chemical analysis of this plant was performed by Acree, Jacobson, and Haller in 1945 (Acree et al., 1945a,b), and in 1947, H. longipes Blake was identified as source of affinin, which previously had been thought to derive from Erigeron affinis D. C (Jacobson et al., 1947). Since its first analysis, this medicinal plant has been studied by several research groups, with the purpose of investigating the fungistatic and bacteriostatic (Molina-Torres et al., 2004), analgesic (Rios et al., 2007), anti-hyperalgesic (Acosta-Madrid et al., 2009), antinociceptive (Ogura et al., 1982; De´ciga-Campos et al., 2010; ˜ o-Corte´s et al., 2010), and anti-inflammatory properties Carin (Herna´ndez et al., 2009). The roots from H. longipes have been used in folk medicine to treat pain, particularly tooth and oral pain (Correa et al., 1971; Jacobson, 1954), and ulcerative conditions (Correa et al., 1971; Colvard et al., 2006; Gutie´rrez-Lugo et al., 1996). They have also been used as a mouth anesthetic and as an antiparasitic. Its local analgesic and anesthetic effects suggest the presence of compounds with corresponding activities. Until now, however, analgesic activity has been associated only with the presence of
* Corresponding author. Tel.: +52 777 329 70 00x6024; fax: +52 777 329 79 97. E-mail address: [email protected] (M.Y. Rios).
affinin [N-isobutyl-2E,6Z,8E-decatrienamide (1)], hereafter referred to as ‘‘spilanthol’’ the major chemical component found in the roots of this plant (Rios et al., 2007; De´ciga-Campos et al., 2010). The isolated yield of 1 from the roots is approximately 0.73% with respect to the dry weight from the roots and 45.23% with respect to the total dry extract weight. This alkamide is highly abundant, and it is very difficult to isolate the minor alkamides included in the extract. It is possible, however, that these minor alkamides contribute to the analgesic activity observed in the total extract. The present paper describes the isolation and the structural elucidation of three new minor alkamides, N-isobutyl8,10-diynoic-3Z-undecenamide (2), N-isobutyl-syn-8,9-dihydroxy-2E,6Z-decadienamide (3), and N-isobutyl-syn-6,9-dihydroxy-2E,7E-decadienamide (4) from H. longipes roots, together with affinin (1), and additional previously isolated chemical compounds such as bornyl ester of deca-2E,6Z,8E-trienoic acid (Molina-Torres et al., 1995), N-2-methylbutyl-deca-2E,6Z,8E-trienamide (Molina-Torres et al., 1996), undeca-2E-en-8,10-dyinoic acid isobutylamide (Bauer et al., 1989), N-isobutyl-2E,6Z-decenamide (Cilia-Lo´pez et al., 2008) N-isobutyl-2E-decenamide (CiliaLo´pez et al., 2008), squalene (Bauer et al., 1989), b-sitosteryl palmitate (Pogliani et al., 1994), stigmasteryl palmitate (Nielsen and Kofod, 1963), lupeyl acetate (Mahato and Kundu, 1994), lupeol (Burns et al., 2000), angelicoidenol (Mahmooda et al., 1983), bsitosterol (Aldrich library, 1992a), and stigmasterol (Aldrich library, 1992b). Affinin (1) increases the release of GABA in the temporal cerebral cortex (Rios et al., 2007). Furthermore, 1 inhibits NO production in murine macrophages, which efficiently downregulates the production of inflammatory mediators IL-1b, IL-6 and
1874-3900/$ – see front matter ß 2011 Phytochemical Society of Europe. Published by Elsevier B.V. All rights reserved. doi:10.1016/j.phytol.2011.04.014
S. Lo´pez-Martı´nez et al. / Phytochemistry Letters 4 (2011) 275–279
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Compound 2 has, on the basis of HREIMS [(M)+, m/z 231.1614] a molecular formula C15H21NO, indicating six degrees of unsaturation. Two of these degrees were due to the presence of one amide carbonyl group (1646 cm 1 in the IR spectrum and dC 171.1 ppm in the 13C NMR spectrum), and one double bond (dC 124.3 and
TNF-a, and attenuates the expression of COX-2 and iNOS (Matthias et al., 2008). Affinin also can permeates the skin (Boonen et al., 2010). The characterization of natural alkamide analogues of affinin contributes to the study of novel molecules that may have interesting activities that merit further evaluation.
O
H N
NH
O
H
2
1 R2
H N R1
O
R4
R3
3 R1 = R2 = H, R3 = R4 = OH, Δ6,7 4 R1 = R3 = H, R2 = R4 = OH, Δ7,8
2. Results and discussion The roots H. longipes biosynthesize alkamides and other interesting natural products. Alkamides have been associated with the analgesic and anesthetic properties found in this medicinal plant. These natural products are pale yellow oils that show a characteristic dark brown and blue absorption pattern at short- and long-wave, respectively, when visualized under UV light on TLC plates. They also develop characteristic brilliant yellow spots when they are visualized using (NH4)4Ce(SO4) in 2 N H2SO4. Making use of this identification criteria, and on the basis of NMR spectroscopic data, we isolated and characterized the three minor, previously undescribed alkamides, N-isobutyl-8,10-diynoic-3Zundecenamide (2), N-isobutyl-syn-8,9-dihydroxy-2E,6Z-decadienamide (3), and N-isobutyl-syn-6,9-dihydroxy-2E,7E-decadienamide (4). The structural elucidation of these molecules is described herein.
134.3 ppm). The other four degrees of unsaturation were attributed to the presence of two conjugate alkyne triple bonds, located at the terminal position of a chain, which is in accord with the observation of three signals at dC 77.9, 68.5 and 65.3 ppm, and one doublet signal at dC 65.0 ppm (dH 2.01 ppm) in the 13C NMR spectrum (Table 1). These data suggested a linear structure for this compound. An N-isobutyl structural fragment was deduced from the presence of one doublet of doublets signal with integration corresponding to two hydrogens at dH 3.07 ppm assigned to an Nmethylene group (dC 47.0 ppm, t), one heptuplet at dH 1.77 ppm (dC 28.6 ppm, d), and one doublet signal with an integration of six hydrogens at dH 0.90 ppm (dC 20.2 ppm, q) (Table 1). Consequently, this compound corresponds to an alkamide with a carboncontaining chain of eleven carbons at the fatty acid residue. The double bond is located at C3–C4 according to the observed doublet of doublets signal with an integration of two hydrogens at dH 2.96 ppm (dC 40.7 ppm, t) that corresponds to one methylene unit alpha to the amide carbonyl group (H2). One double signal centered
Table 1 1 H and 13C NMR data (J in Hz) of compounds 2–4 (d ppm, in CDCl3). H/C
2
3
H 1 2 3 4 5 6 7 8 9 10 11 NHa b c d,e
– 2.96 2.95 5.60 5.59 2.18 1.64 2.28 – – – 2.01 5.93 3.07 1.77 0.90
dd (4.8, 2.0) dd (4.8, 0.8) d (5.6) ddd (5.6, 2.0, 0.8) m q (6.8) td (6.8, 1.2)
t (1.2) (bs) dd (6.8,6.0) h (6.8) d (6.8)
4
C
H
C
H
C
171.1 s 40.7 t
– 5.85 d (15.6)
166.5 s 124.4 d
– 5.90 d (15.6)
166.8 s 124.1 d
124.3 d 134.3 d 31.6 t 18.6 t 27.5 t 77.9 s 68.5 s 65.3 s 65.0 d – 47.0 t 28.6 d 20.2 q
6.79 ddd (15.6,6.4,6.4) 2.25 (m)
143.8 d 31.7 t 26.9 t 129.4 d 132.2 d 71.3 d 70.5 d 17.7 q – – 47.2 t 28.8 d 20.4 q
6.77 2.22 1.64 4.06 5.61 5.68 4.26 1.23 – 6.94 3.08 1.78 0.90
144.0 d 28.1 t 35.6 t 71.1 d 131.8 d 135.0 d 67.7 d 23.3 q – – 47.1 t 28.7 d 20.4 q
5.55 5.54 4.34 3.82 1.30 – 6.04 3.13 1.79 0.92
dd (11.2,5.2) dd (11.2,8.4) dd (8.4,4.0) dq (6.4,4.0) d (6.4) (bs) dd (6.4,6.4) h (6.8) d (6.8)
dt (15.6,6.8) (m) (m) ddd (13.2,6.8,5.6) ddd (15.6, 12.4, 5.6) ddd (15.6, 12.4, 5.6) dq (6.4, 12.4) d (6.4) dd (14.0,6.4) dd (6.4,6.4) h (6.8) d (6.8)
S. Lo´pez-Martı´nez et al. / Phytochemistry Letters 4 (2011) 275–279
O b
3
4
1 2
5
H N
6
NH a
c
277
7
d e
H
8
9
10
2 1
4 3
5
7 8
O
11
6
HO
9
OH
3
2
OH H N OH
O
4 Fig. 1. Selected COSY and HMBC correlations for compounds 2–4
at dH 5.60 (J = 5.6 Hz), and one doublet of double doublets signal at dH 5.59 ppm (J = 5.6, 2.0, 0.8 Hz), with integration for one hydrogen each (dC 124.3 and 134.3 ppm, both d), corresponds to H3 and H4, respectively. The 5.6 Hz coupling constant value for these signals establish a Z configuration for this double bond. Carbons C5 to C7 are three methylene units, the first one is allylic in accordance with the multiple signal at dH 2.18 ppm (dC 31.6), and the last of which is propargylic as evidenced by the observed resonance signal at dH 2.28 ppm (dC 27.5 ppm, t). This natural product corresponds to Nisobutyl-8,10-diynoic-3Z-undecenamide (2) and the structure is in agreement with the observed COSY and HMBC correlations, shown in Fig. 1 and Table 2. Undeca-2E-en-8,10-dyinoic acid isobutylamide. Two isomers of compound 2 [undeca-2Z-en-8,10-dyinoic acid isobutylamide (N-isobutyl-8,10-diynoic-2Z-undecenamide) and undeca-2E-en-8,10-dyinoic acid isobutylamide (N-isobutyl8,10-diynoic-2E-undecenamide)] have been already isolated from Echinaceae angustifolia (Bauer et al., 1989). The last one was also isolated of the dried roots from H. longipes, and its analgesic activity, determined by means of GABA release in mice brain slices was evaluated, being inactive (Rios et al., 2007). Alkamide 3 showed a molecular weight of m/z 255.1834 in the HREIMS, corresponding to a molecular formula C14H25NO3 with
COSY;
HMBC correlations.
three degrees of unsaturation. In accordance with this molecular formula, this natural product includes an N-isobutyl fragment and 1 fatty acid residue containing 10 carbons. In addition, resonances characteristic of the N-isobutyl fragment were observed at dH 3.13 ppm (N-CH2, dC 47.2 ppm, t), dH 1.79 ppm and 0.92 ppm (dC 28.8 ppm, d; 20.4 ppm, q, respectively) in the 1H and 13C NMR spectra. The fatty acid residue includes one a,b-unsaturated carbonyl group, which corresponds to the observed signals at dH 6.79 ppm (H3, dC 143.8 ppm), 5.85 ppm (H2, dC 124.4 ppm, d), and dC 166.5 ppm (C1, s). In the HMBC spectrum (Fig. 1, Table 2), hydrogens H2 and H3 showed correlation cross peaks with C4 (dC 31.7 ppm), whereas H3 was correlated with C5 (dC 26.9 ppm). This observation indicated that the chain elongation is due to two methylene groups. One additional cis double bond showed resonance signals at dH 5.55 ppm and 5.54 ppm (both d, J = 11.2 Hz, dC 129.4 ppm and 132.2 ppm). This double bond is located at C6–C7 in accordance with the correlation cross peak between both H4 and H5 (dH 2.25 ppm) with C6 (dC 129.4 ppm) and of H6 (dH 5.55 ppm) with C5 (Fig. 1B). Two oxygenated carbons are located at C8 and C9 and showed resonances at dH 4.34 ppm (dC 71.3 ppm, d) and 3.82 ppm (dC 70.5 ppm, d), respectively. H7 (dH 5.54 ppm) and H9 (dH 3.82 ppm) showed correlation cross peaks
Table 2 Observed COSY and HMBC correlations for compounds 2–4. 2
1 2 3 4 5 6 7 8 9 10 11 NHa b c d e
3
4
COSY
HMBC
COSY
HMBC
COSY
HMBC
– H3, H2,
– C1, C3, C4 C2, C5
– H3 H2, H4 H3, H6
– C1, C1, C3, C4, C5 – C9 C8 – – – C1, – Cb, Cb,
– H3 H2, H4 H3, H5 H4, H6 H5, H7 H6, H8 H7, H9 H8, H10 H9 – Hb NH, Hc Hb, Hd, He Hc Hc
– C1, C1, – C4, C8 C6, C6, – C8, – C1 C1, Cb, Cb, Cb,
5 5
H2, 6 H5, H7 H6 – – – – Hb NH, Hc Hb, Hd, He Hc Hc
C3, C4, C6, – – – C9, C1 C1, Cb, Cb, Cb,
C4, C6 C5, C7, C8 C9, C8
C10 Cc, Cd, Ce Cd, Ce Cc, Ce Cc, Cd
H5 H8 H7, H9 H8, H10 H9 – Hb NH, Hc Hb, Hd, He Hc Hc
C4 C4, C5 C5 C6
Cc, Cd, Ce Cc, Ce Cc, Cd
C4 C4 C6 C8, C9 C9 C9
Cc, Cd, Ce Cd, Ce Cc, Ce Cc, Cd
278
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with C8 (dC 71.3 ppm), whereas H8 (dH 4.34 ppm) correlated with C9 (dC 70.5 ppm). H8 and H9 showed a 4.0 Hz coupling constant corresponding to a 608 dihedral angle and correlation cross peaks at the NOESY experiment, indicating that 8- and 9-hydroxyl groups have a syn relationship. This compound corresponds to N-isobutylsyn-8,9-dihydroxy-2E,6Z-decadienamide (3). N-Isobutyl-syn-6,9-dihydroxy-2E,7E-decadienamide (compound 4) is an isomer of compound 3. In compound 4, the hydroxyl groups are located at C6 and C9, and a trans double bond is at C7–C8 on the basis of the observed correlation cross peaks in the HMBC spectrum (Fig. 1, Table 2) and coupling constant values. In addition, H5 (dH 1.64 ppm) correlated with the oxygenated carbon C6 (dC 71.1 ppm), whereas H6 (dH 4.06 pm) showed a correlation cross peak with C8 (dC 135.0 ppm). Three correlation cross peaks were observed for H7 (dH 5.61 ppm), with C6, C8, and C9 (dc 67.7 ppm), while H10 (dH 1.23 ppm) correlated with C8 and C9. NOE difference experiment at 100 8C on the signal at dH 4.26 shown increases of those at dH 4.06 and irradiation on the signal at dH 4.06 shown increases of those at dH 4.26, corroborating an synrelationship between both 6- and 8-hydroxyl groups. Compounds 2–4 are minor chemical constituents of H. longipes roots. Their isolation and characterization from fresh, but not from stored roots, suggests a possible instability. These compounds may be important because alkamides constitute valuable molecules with possible antinociceptive, analgesic, and anesthetic properties. Actually, our group makes efforts to isolate compounds 2–4 in adequate quantities to assess their pharmacological properties as analgesic. 3. Experimental 3.1. General procedures Compounds were isolated by open column chromatography (CC). The isolation procedures and purity of compounds were monitored by thin layer chromatography (TLC), by visualizing with UV-light and spraying with (NH4)4Ce(SO4)4 in 2 N H2SO4. Optical rotations were measured on a PerkinElmer 341 MC digital polarimeter using CHCl3 as the solvent. IR spectra were obtained in KBr or as films (CHCl3) on a Bruker Vector 22 IR spectrometer. All 1 H, 13C, and 2D NMR experiments were performed in CDCl3 on a Varian Unity 400 spectrometer equipped with a 5 mm inverse detection pulse field gradient probe at 25 8C, at 400 MHz for 1H NMR and 100 MHz for 13C NMR. Chemical shifts were referenced to tetramethylsilane (TMS) as an internal standard. EI and FAB+MS spectra were recorded on a Jeol JMX-AX 505 HA mass spectrometer in a matrix of nitrobenzyl alcohol for FAB+MS. 3.2. Plant material The fresh roots of Heliopsis longipes (Asteraceae) were collected on 28 July 2007 at ‘‘Real de Xichu’’, Guanajuato and were identified by M.C. Ramiro Rios Go´mez of the Facultad de Estudios Superiores Zaragoza, UNAM, Me´xico. A voucher specimen (number 5904) was deposited at FEZA Herbarium, UNAM. 3.3. Extraction and isolation Fresh roots of H. longipes (4.725 kg) were extracted at room temperature with acetone. The extraction solvent was concentrated to dryness under vacuum to render 76.5 g of extract (1.2% yield). Fractionation of the acetone extract by means of open CC (silica gel, 100-230 mesh; Natland Int. Corp.; 11.5 cm 50 cm) was performed with a step gradient of n-hexane-acetone 100:0 to 40:60, collecting 143 250-mL fractions. On the basis of TLC analysis, these fractions were pooled into eight groups: G-1 (4.9 g, n-hexane
100%), G-2 (7.9 g, n-hexane:acetone 9:1), G-3 (4.5 g, n-hexane:acetone 9:1), G-4 (17.5 g, n-hexane:acetone 8:2), G-5 (10.3 g, nhexane:acetone 6:4), G-6 (8.7 g, n-hexane:acetone 6:4), G-7 (5.4 g, n-hexane:acetone 4:6, and G-8) (1.7 g, n-hexane:acetone 4:6). G-1 was an essential oil (4.9 g, 6.40% yield with respect to the total acetone extract). Additional groups were subjected to flash column chromatography: G-2 (silica gel, 230–400 mesh; Natland Int. Corp.; 4.5 cm i.d. 10 in., eluent n-hexane 100% to n-hexane:CH2Cl2 80:20) collecting 314 125-mL fractions, to yield squalene (314.0 mg, 0.41%), a mixture of b-sitosteryl palmitate and stigmasteryl palmitate (445.3 mg, 0.58%), lupeyl acetate (183.4 mg, 0.24%), a mixture of b-sitosterol and stigmasterol (866.2 mg, 1.13%), lupeol (230.7 mg, 0.30%) and the bornyl ester of deca-2E,6Z,8E-trienoic acid (174 mg, 0.23%); G-3 to G-6 (nhexane:CH2Cl2 70:30) to afford practically only affinin (34.6 g, 45.23%), accompanied by minor amounts of N-isobutyl-8,10diynoic-3Z-undecenamide (2, 82.0 mg, 0.10%), N-isobutyl-2Edecenamide (124.4 mg, 0.16%), and N-isobutyl-2E,6Z-decenamide (313.9 mg, 0.41%); G-7 (silica gel, 230–400 mesh; Natland Int. Corp.; 3 cm i.d. 10 in., eluent CH2Cl2:acetone 80:10 to 60:40) collecting 214 25-mL fractions, to yield angelicoidenol (187.2 mg, 0.24%) and N-isobutyl-syn-8,9-dihydroxy-2E,6Z-decadienamide (3, 73 mg, 0.10%); and finally fraction G-8 (silica gel, 230– 400 mesh; Natland Int. Corp.; 2.5 cm i.d. 10 in., eluent CH2Cl2:acetone 80:20 to 60:40) collecting 59 10-mL fractions, to afford Nisobutyl-syn-6,9-dihydroxy-2E,7E-decadienamide (4, 163 mg, 0.21%). 3.3.1. N-Isobutyl-8,10-diynoic-3Z-undecenamide (2) Pale yellow oil; UV (CHCl3, 1 10 3 M) lmax (log e) 242 (2.79), 251 (2.69), 277 (2.43), 350 (1.80) nm; IR (CHCl3): nmax = 3433 (NH), 3305 (C–H stretching), 2959, 2932, 2871 (CH, CH2, CH3), 2224 (CC), 1646 (C5 5O), 1552 (NH), 1266, 971, 739 cm 1; EIMS m/z 232 [M+H]+ (22), 230 [M H]+ (16), 216 [M-CH3]+ (5), 203 (8), 188 [MC3H7]+ (12), 174 [M-C4H9]+ (8), 159 [M-C4H10N]+ (10), 145 (13), 131 [C10H11]+ (48), 117 [C9H9]+ (60), 114 [C6H12NO]+ (28), 104 [C8H8]+ (23), 91 [C7H7]+ (53), 57 [C4H9]+ (100); HREIMS m/z 231.1614 [M]+ (calcd. for C15H21NO, 231.1623). See Table 1 for the 1 H and 13C NMR spectroscopic data. 3.3.2. N-Isobutyl-syn-8,9-dihydroxy-2E,6Z-decadienamide (3) Pale yellow oil; [a]D20 – 2.5 (c 1.7, CHCl3); UV (CHCl3) lmax (log e) 243 (4.11), 301 (2.99), 353 (1.30) nm; IR (CHCl3): nmax = 3315 (OH, NH), 2961, 2926, 2871 (CH, CH2, CH3), 1669 (C5 5O), 1627 (C5 5C), 1514, 1370, 1255, 979 cm 1; EIMS m/z 255 [M]+ (5), 238 [MOH]+ (4), 210 [M-C2H5O]+ (77), 184 [M-C4H9N]+ (20), 167 [MC4H8O2]+ (39), 149 [M-C4H10N-OH-OH]+ (100), 141 [C8H15NO]+ (68), 115 [C6H11O2]+ (16), 109 (12), 91 [C7H7]+ (11), 71 (14) 57 [C4H9]+ (100); HREIMS m/z 255.1834 [M]+ (calcd for C14H25NO3, 2551.1834). See Table 1 for the 1H and 13C NMR spectroscopic data. 3.3.3. N-Isobutyl-syn-6,9-dihydroxy-2E,7E-decadienamide (4) Pale yellow oil; [a]D20 – 1.2 (c 1.6, CHCl3); UV (CHCl3) lmax (log e) 243 (3.99), 346 (1.74) nm; IR (CHCl3): nmax = 3309 (OH, NH), 2960, 2925, 2869 (CH, CH2, CH3), 1669 (C5 5O), 1628 (C5 5C), 1555, 1370, 1255, 979 cm 1; FAB+MS m/z 256 [M+H]+ (22), 238 [M+HH2O]+ (92), 184 (9), 203 (8), 154 (24), 119 (30), 105 (34), 91 [C7H7]+ (53), 81 (67), 69 (73), 55 [C4H9]+ (100); HRFAB+MS m/z 256.1923 [M+H]+ (calcd for C14H26NO3, 256.1913). See Table 1 for the 1H and 13 C NMR spectroscopic data. Acknowledgements This work was supported financially by CONACyT (Grants number 79584-Q and 48358-Q). SLM thank PROMEP and ICYTDF for a postdoctoral fellowship. We are grateful to Biol. Enrique
S. Lo´pez-Martı´nez et al. / Phytochemistry Letters 4 (2011) 275–279
Salazar Leyva, M. C. Ramiro Rios-Gomez, Dr. Blanca Domı´nguez Mendoza, Ing. Victoria Labastida, and T.C. Marı´a Medina Pastor for technical assistance. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.phytol.2011.04.014. References ˜ eda-Herna´ndez, G., Cilia-Lo´pez, V.G., Carin ˜ o-Corte´s, R., Acosta-Madrid, I.I., Castan Pe´rez-Herna´ndez, N., Ferna´ndez-Martı´nez, E., Ortı´z, M.I., 2009. Interaction between Heliopsis longipes and diclofenac on the thermal hyperalgesia test. Phytomedicine 16, 336–341. Acree, F., Jacobson, M., Haller, H.L., 1945a. An amide possessing insecticidal properties from the roots of Erigeron affinis D. C. J. Org. Chem. 10, 236–242. Acree, F., Jacobson, M., Haller, H.L., 1945b. The structure of affinin, the insecticidal amide from Erigeron affinis D. C. J. Org. Chem. 10, 449–451. Aldrich Library of 13C and 1H FT NMR spectra, vol. 3. 1992, p. 569A. Aldrich Library of 13C and 1H FT NMR spectra, vol. 3. 1992, p. 569B. Bauer, R., Reminger, P., Wagner, H., 1989. Alkamides from the roots of Echinacea angustifolia. Phytochemistry 28, 505–508. Boonen, J., Baert, B., Roche, N., Burvenich, C., De Spiegeleer, B., 2010. Transdermal behaviour of the N-alkylamide spilanthol (affinin) from Spilanthes acmella (Compositae) extracts. J. Ethnopharmacol. 127, 77–84. Burns, D., Reynolds, W.F., Buchanan, G., Reese, P.B., Enriquez, R.G., 2000. Assignment of 1H and 13C spectra and investigation of hindered side-chain rotation in lupeol derivatives. Magn. Reson. Chem. 38, 488–493. ˜ o-Corte´s, R., Gayosso-De-Lucio, J.A., Ortiz, M.I., Sa´nchez-Gutie´rrez, M., Garcı´aCarin Reyna, P.B., Cilia-Lo´pez, V.G., Pe´rez-Herna´ndez, N., Moreno, E., Ponce-Monter, H., 2010. Antinociceptive, genotoxic and histopathological study of Heliopsis longipes S.F Blake in mice. J. Ethnopharmacol. 130, 216–221. Cilia-Lo´pez, V.G., Aguirre-Rivera, R.J., Reyes-Agu¨ero, J.A., Jua´rez-Flores, B., 2008. Etnobota´nica de Heliopsis longipes (Asteraceae: Heliantheae). Bol. Soc. Bot. Mex. 83, 83–89. Colvard, M.D., Cordell, G.A., Villalobos, R., Sancho, G., Soejarto, D.D., Pestle, W., Echeverri, T.L., Perkowitz, K.M., Michel, J., 2006. Survey of medical ethnobotanicals for dental and oral medicine conditions and pathologies. J. Ethnopharmacol. 107, 134–142. Correa, J., Roquet, S., Dı´az, E., 1971. Multiple NMR analysis of the affinin. Org. Magn. Reson. 1–5.
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De´ciga-Campos, M., Rios, M.Y., Aguilar-Guadarrama, A.B., 2010. Antinociceptive effect of Heliopsis longipes extract and affinin in mice. Planta Med. 76, 665–670. Gutie´rrez-Lugo, M., Barrientos-Benı´tez, T., Luna, B., Ramı´rez-Gama, R., Bye, R., Linares, E., Mata, R., 1996. Antimicrobial and cytotoxic activities of some crude drug extracts from Mexican medicinal plants. Phytomedicine 2, 341–347. Herna´ndez, I., Ma´rquez, L., Martı´nez, I., Dieguez, R., Delporte, C., Prieto, S., MolinaTorres, J., Garrido, G., 2009. Anti-inflammatory effects of ethanolic extract and alkamides-derived from Heliopsis longipes roots. J. Ethnopharmacol. 124, 649–652. Jacobson, M., Acree, F., Haller, H.L., 1947. Correction of the source of ‘‘affinin’’ (Nisobutyl-2,6,8-decatrienoamide). J. Org. Chem. 12, 731–732. Jacobson, M., 1954. Constituents of Heliopsis longipes species III. Cis-trans isomerism in affinin. J. Am. Chem. Soc. 76, 4606–4608. Jacobson, M., 1955. Constituents of Heliopsis longipes species IV. The total synthesis of trans-affinin. J. Am. Chem. Soc. 77, 2461–2463. Mahato, S.B., Kundu, A.P., 1994. 13C NMR Spectra of pentacyclic triterpenoids A compilation of some salient features. Phytochemistry 37, 1517–1575. Mahmooda, U., Singha, S.B., Thakur, R.S., 1983. Angelicoidenol, a bicyclic monoterpene from the seeds of Pleurospermum angelicoides. Phytochemistry 22, 774–776. Matthias, A, Connellan, P., Thompson, D., Bone, K.M., Lehmann, R.P., 2008. Acute immune modulatory effects of bioavailable Echinacea alkamides. Planta Med. 74, 1008–1009. Molina-Torres, J., Garcı´a-Cha´vez, A., Ramı´rez-Cha´vez, E., 1999. Antimicrobial properties of alkamides presents in flavoring plants traditionally used in Mesoamerica: affinin and capsaicin. J. Ethnopharmacol. 64, 241–248. Molina-Torres, J., Salgado, R., Ramı´rez, E., del Rı´o, R.E., 1995. Presence of the ornyl ester of deca-2E,6Z,8E-trienoic acid in Heliopsis longipes roots. J. Nat. Prod. 58, 1590–1591. Molina-Torres, J., Salgado, R., Ramı´rez, E., del Rı´o, R.E., 1996. Purely olefinic alkamides in Heliopsis longipes and Acmella (Spilanthes) oppositifolia. Biochem. Syst. Ecol. 24, 43–47. Molina-Torres, J., Salazar-Cabrera, C.J., Armenta-Salinas, C., Ramı´rez-Cha´vez, E., 2004. Fungistatic and bacteriostatic activities of alkamides from Heliopsis longipes roots: affinin and reduced amides. J. Agric. Food Chem. 52, 4700–4704. Nielsen, B.E., Kofod, H., 1963. Constituents of the Umbelliferous Plants I. Constituents of the root of Angelica archangelica L. Acta Chem. Scand. 17, 1161–1163. Ogura, M., Cordell, G.A., Quinn, M.L., Leon, C., Benoit, P.S., Soejarto, D.D., Farnsworth, N.R., 1982. Ethnopharmacologic studies I: rapid solution to a problem oral use of Heliopsis longipes by means of a multidisciplinary approach. J. Ethnopharmacol. 64, 215–219. Pogliani, L., Ceruti, M., Ricchiardi, G., Viterbo, D., 1994. An NMR and molecular mechanics study of squalene and squalene derivatives. Chem. Phys. Lipids 70, 21–34. Rios, M.Y., Aguilar-Guadarrama, A.B., Gutie´rrez, M.C., 2007. Analgesic activity of affinin, an alkamide from Heliopsis longipes (Compositae). J. Ethnopharmacol. 110, 364–367.
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Phytochemistry Letters journal homepage: www.elsevier.com/locate/phytol
Minor alkamides from Heliopsis longipes S.F. Blake (Asteraceae) fresh roots Sugey Lo´pez-Martı´nez, A. Berenice Aguilar-Guadarrama, Marı´a Yolanda Rios * Centro de Investigaciones Quı´micas, Universidad Auto´noma del Estado de Morelos, Avenida Universidad 1001, Col. Chamilpa, 62209 Cuernavaca, Morelos, Mexico
A R T I C L E I N F O
A B S T R A C T
Article history: Received 3 December 2010 Received in revised form 12 April 2011 Accepted 21 April 2011 Available online 13 May 2011
From the fresh roots of Heliopsis longipes three new minor alkamides: longipinamide A (N-isobutyl-8,10diynoic-3Z-undecenamide), longipenamide A (N-isobutyl-syn-8,9-dihydroxy-2E,6Z-decadienamide) and longipenamide B (N-isobutyl-syn-6,9-dihydroxy-2E,7E-decadienamide); three known alkamides: affinin (spilanthol, N-isobutyl-2E,6Z,8E-decatrienamide), N-isobutyl-2E,6Z-decadienamide and Nisobutyl-2E-decenamide; and 11 other known compounds were isolated. The structures of the three new minor alkamides were established by 1D and 2D NMR spectroscopy including 1H, 13C, DEPT, COSY, HSQC, and HMBC experiments, as well as by EI and FAB+ mass spectrometry. To our knowledge, this is the first report of the isolation of linear dihydroxyalkamides as natural products. ß 2011 Phytochemical Society of Europe. Published by Elsevier B.V. All rights reserved.
Keywords: Heliopsis longipes Asteraceae Alkamides Linear dihydroxyalkamides
1. Introduction Heliopsis longipes S.F. Blake is an annual climber that grows from July to October. It is an endemic plant of the central region of Mexico (Molina-Torres et al., 1999; Cilia-Lo´pez et al., 2008), where is popularly called ‘‘chilcuague’’ or ‘‘chilcuan’’ (Jacobson et al., 1947; Jacobson, 1954, 1955; Correa et al., 1971). The first chemical analysis of this plant was performed by Acree, Jacobson, and Haller in 1945 (Acree et al., 1945a,b), and in 1947, H. longipes Blake was identified as source of affinin, which previously had been thought to derive from Erigeron affinis D. C (Jacobson et al., 1947). Since its first analysis, this medicinal plant has been studied by several research groups, with the purpose of investigating the fungistatic and bacteriostatic (Molina-Torres et al., 2004), analgesic (Rios et al., 2007), anti-hyperalgesic (Acosta-Madrid et al., 2009), antinociceptive (Ogura et al., 1982; De´ciga-Campos et al., 2010; ˜ o-Corte´s et al., 2010), and anti-inflammatory properties Carin (Herna´ndez et al., 2009). The roots from H. longipes have been used in folk medicine to treat pain, particularly tooth and oral pain (Correa et al., 1971; Jacobson, 1954), and ulcerative conditions (Correa et al., 1971; Colvard et al., 2006; Gutie´rrez-Lugo et al., 1996). They have also been used as a mouth anesthetic and as an antiparasitic. Its local analgesic and anesthetic effects suggest the presence of compounds with corresponding activities. Until now, however, analgesic activity has been associated only with the presence of
* Corresponding author. Tel.: +52 777 329 70 00x6024; fax: +52 777 329 79 97. E-mail address: [email protected] (M.Y. Rios).
affinin [N-isobutyl-2E,6Z,8E-decatrienamide (1)], hereafter referred to as ‘‘spilanthol’’ the major chemical component found in the roots of this plant (Rios et al., 2007; De´ciga-Campos et al., 2010). The isolated yield of 1 from the roots is approximately 0.73% with respect to the dry weight from the roots and 45.23% with respect to the total dry extract weight. This alkamide is highly abundant, and it is very difficult to isolate the minor alkamides included in the extract. It is possible, however, that these minor alkamides contribute to the analgesic activity observed in the total extract. The present paper describes the isolation and the structural elucidation of three new minor alkamides, N-isobutyl8,10-diynoic-3Z-undecenamide (2), N-isobutyl-syn-8,9-dihydroxy-2E,6Z-decadienamide (3), and N-isobutyl-syn-6,9-dihydroxy-2E,7E-decadienamide (4) from H. longipes roots, together with affinin (1), and additional previously isolated chemical compounds such as bornyl ester of deca-2E,6Z,8E-trienoic acid (Molina-Torres et al., 1995), N-2-methylbutyl-deca-2E,6Z,8E-trienamide (Molina-Torres et al., 1996), undeca-2E-en-8,10-dyinoic acid isobutylamide (Bauer et al., 1989), N-isobutyl-2E,6Z-decenamide (Cilia-Lo´pez et al., 2008) N-isobutyl-2E-decenamide (CiliaLo´pez et al., 2008), squalene (Bauer et al., 1989), b-sitosteryl palmitate (Pogliani et al., 1994), stigmasteryl palmitate (Nielsen and Kofod, 1963), lupeyl acetate (Mahato and Kundu, 1994), lupeol (Burns et al., 2000), angelicoidenol (Mahmooda et al., 1983), bsitosterol (Aldrich library, 1992a), and stigmasterol (Aldrich library, 1992b). Affinin (1) increases the release of GABA in the temporal cerebral cortex (Rios et al., 2007). Furthermore, 1 inhibits NO production in murine macrophages, which efficiently downregulates the production of inflammatory mediators IL-1b, IL-6 and
1874-3900/$ – see front matter ß 2011 Phytochemical Society of Europe. Published by Elsevier B.V. All rights reserved. doi:10.1016/j.phytol.2011.04.014
S. Lo´pez-Martı´nez et al. / Phytochemistry Letters 4 (2011) 275–279
276
Compound 2 has, on the basis of HREIMS [(M)+, m/z 231.1614] a molecular formula C15H21NO, indicating six degrees of unsaturation. Two of these degrees were due to the presence of one amide carbonyl group (1646 cm 1 in the IR spectrum and dC 171.1 ppm in the 13C NMR spectrum), and one double bond (dC 124.3 and
TNF-a, and attenuates the expression of COX-2 and iNOS (Matthias et al., 2008). Affinin also can permeates the skin (Boonen et al., 2010). The characterization of natural alkamide analogues of affinin contributes to the study of novel molecules that may have interesting activities that merit further evaluation.
O
H N
NH
O
H
2
1 R2
H N R1
O
R4
R3
3 R1 = R2 = H, R3 = R4 = OH, Δ6,7 4 R1 = R3 = H, R2 = R4 = OH, Δ7,8
2. Results and discussion The roots H. longipes biosynthesize alkamides and other interesting natural products. Alkamides have been associated with the analgesic and anesthetic properties found in this medicinal plant. These natural products are pale yellow oils that show a characteristic dark brown and blue absorption pattern at short- and long-wave, respectively, when visualized under UV light on TLC plates. They also develop characteristic brilliant yellow spots when they are visualized using (NH4)4Ce(SO4) in 2 N H2SO4. Making use of this identification criteria, and on the basis of NMR spectroscopic data, we isolated and characterized the three minor, previously undescribed alkamides, N-isobutyl-8,10-diynoic-3Zundecenamide (2), N-isobutyl-syn-8,9-dihydroxy-2E,6Z-decadienamide (3), and N-isobutyl-syn-6,9-dihydroxy-2E,7E-decadienamide (4). The structural elucidation of these molecules is described herein.
134.3 ppm). The other four degrees of unsaturation were attributed to the presence of two conjugate alkyne triple bonds, located at the terminal position of a chain, which is in accord with the observation of three signals at dC 77.9, 68.5 and 65.3 ppm, and one doublet signal at dC 65.0 ppm (dH 2.01 ppm) in the 13C NMR spectrum (Table 1). These data suggested a linear structure for this compound. An N-isobutyl structural fragment was deduced from the presence of one doublet of doublets signal with integration corresponding to two hydrogens at dH 3.07 ppm assigned to an Nmethylene group (dC 47.0 ppm, t), one heptuplet at dH 1.77 ppm (dC 28.6 ppm, d), and one doublet signal with an integration of six hydrogens at dH 0.90 ppm (dC 20.2 ppm, q) (Table 1). Consequently, this compound corresponds to an alkamide with a carboncontaining chain of eleven carbons at the fatty acid residue. The double bond is located at C3–C4 according to the observed doublet of doublets signal with an integration of two hydrogens at dH 2.96 ppm (dC 40.7 ppm, t) that corresponds to one methylene unit alpha to the amide carbonyl group (H2). One double signal centered
Table 1 1 H and 13C NMR data (J in Hz) of compounds 2–4 (d ppm, in CDCl3). H/C
2
3
H 1 2 3 4 5 6 7 8 9 10 11 NHa b c d,e
– 2.96 2.95 5.60 5.59 2.18 1.64 2.28 – – – 2.01 5.93 3.07 1.77 0.90
dd (4.8, 2.0) dd (4.8, 0.8) d (5.6) ddd (5.6, 2.0, 0.8) m q (6.8) td (6.8, 1.2)
t (1.2) (bs) dd (6.8,6.0) h (6.8) d (6.8)
4
C
H
C
H
C
171.1 s 40.7 t
– 5.85 d (15.6)
166.5 s 124.4 d
– 5.90 d (15.6)
166.8 s 124.1 d
124.3 d 134.3 d 31.6 t 18.6 t 27.5 t 77.9 s 68.5 s 65.3 s 65.0 d – 47.0 t 28.6 d 20.2 q
6.79 ddd (15.6,6.4,6.4) 2.25 (m)
143.8 d 31.7 t 26.9 t 129.4 d 132.2 d 71.3 d 70.5 d 17.7 q – – 47.2 t 28.8 d 20.4 q
6.77 2.22 1.64 4.06 5.61 5.68 4.26 1.23 – 6.94 3.08 1.78 0.90
144.0 d 28.1 t 35.6 t 71.1 d 131.8 d 135.0 d 67.7 d 23.3 q – – 47.1 t 28.7 d 20.4 q
5.55 5.54 4.34 3.82 1.30 – 6.04 3.13 1.79 0.92
dd (11.2,5.2) dd (11.2,8.4) dd (8.4,4.0) dq (6.4,4.0) d (6.4) (bs) dd (6.4,6.4) h (6.8) d (6.8)
dt (15.6,6.8) (m) (m) ddd (13.2,6.8,5.6) ddd (15.6, 12.4, 5.6) ddd (15.6, 12.4, 5.6) dq (6.4, 12.4) d (6.4) dd (14.0,6.4) dd (6.4,6.4) h (6.8) d (6.8)
S. Lo´pez-Martı´nez et al. / Phytochemistry Letters 4 (2011) 275–279
O b
3
4
1 2
5
H N
6
NH a
c
277
7
d e
H
8
9
10
2 1
4 3
5
7 8
O
11
6
HO
9
OH
3
2
OH H N OH
O
4 Fig. 1. Selected COSY and HMBC correlations for compounds 2–4
at dH 5.60 (J = 5.6 Hz), and one doublet of double doublets signal at dH 5.59 ppm (J = 5.6, 2.0, 0.8 Hz), with integration for one hydrogen each (dC 124.3 and 134.3 ppm, both d), corresponds to H3 and H4, respectively. The 5.6 Hz coupling constant value for these signals establish a Z configuration for this double bond. Carbons C5 to C7 are three methylene units, the first one is allylic in accordance with the multiple signal at dH 2.18 ppm (dC 31.6), and the last of which is propargylic as evidenced by the observed resonance signal at dH 2.28 ppm (dC 27.5 ppm, t). This natural product corresponds to Nisobutyl-8,10-diynoic-3Z-undecenamide (2) and the structure is in agreement with the observed COSY and HMBC correlations, shown in Fig. 1 and Table 2. Undeca-2E-en-8,10-dyinoic acid isobutylamide. Two isomers of compound 2 [undeca-2Z-en-8,10-dyinoic acid isobutylamide (N-isobutyl-8,10-diynoic-2Z-undecenamide) and undeca-2E-en-8,10-dyinoic acid isobutylamide (N-isobutyl8,10-diynoic-2E-undecenamide)] have been already isolated from Echinaceae angustifolia (Bauer et al., 1989). The last one was also isolated of the dried roots from H. longipes, and its analgesic activity, determined by means of GABA release in mice brain slices was evaluated, being inactive (Rios et al., 2007). Alkamide 3 showed a molecular weight of m/z 255.1834 in the HREIMS, corresponding to a molecular formula C14H25NO3 with
COSY;
HMBC correlations.
three degrees of unsaturation. In accordance with this molecular formula, this natural product includes an N-isobutyl fragment and 1 fatty acid residue containing 10 carbons. In addition, resonances characteristic of the N-isobutyl fragment were observed at dH 3.13 ppm (N-CH2, dC 47.2 ppm, t), dH 1.79 ppm and 0.92 ppm (dC 28.8 ppm, d; 20.4 ppm, q, respectively) in the 1H and 13C NMR spectra. The fatty acid residue includes one a,b-unsaturated carbonyl group, which corresponds to the observed signals at dH 6.79 ppm (H3, dC 143.8 ppm), 5.85 ppm (H2, dC 124.4 ppm, d), and dC 166.5 ppm (C1, s). In the HMBC spectrum (Fig. 1, Table 2), hydrogens H2 and H3 showed correlation cross peaks with C4 (dC 31.7 ppm), whereas H3 was correlated with C5 (dC 26.9 ppm). This observation indicated that the chain elongation is due to two methylene groups. One additional cis double bond showed resonance signals at dH 5.55 ppm and 5.54 ppm (both d, J = 11.2 Hz, dC 129.4 ppm and 132.2 ppm). This double bond is located at C6–C7 in accordance with the correlation cross peak between both H4 and H5 (dH 2.25 ppm) with C6 (dC 129.4 ppm) and of H6 (dH 5.55 ppm) with C5 (Fig. 1B). Two oxygenated carbons are located at C8 and C9 and showed resonances at dH 4.34 ppm (dC 71.3 ppm, d) and 3.82 ppm (dC 70.5 ppm, d), respectively. H7 (dH 5.54 ppm) and H9 (dH 3.82 ppm) showed correlation cross peaks
Table 2 Observed COSY and HMBC correlations for compounds 2–4. 2
1 2 3 4 5 6 7 8 9 10 11 NHa b c d e
3
4
COSY
HMBC
COSY
HMBC
COSY
HMBC
– H3, H2,
– C1, C3, C4 C2, C5
– H3 H2, H4 H3, H6
– C1, C1, C3, C4, C5 – C9 C8 – – – C1, – Cb, Cb,
– H3 H2, H4 H3, H5 H4, H6 H5, H7 H6, H8 H7, H9 H8, H10 H9 – Hb NH, Hc Hb, Hd, He Hc Hc
– C1, C1, – C4, C8 C6, C6, – C8, – C1 C1, Cb, Cb, Cb,
5 5
H2, 6 H5, H7 H6 – – – – Hb NH, Hc Hb, Hd, He Hc Hc
C3, C4, C6, – – – C9, C1 C1, Cb, Cb, Cb,
C4, C6 C5, C7, C8 C9, C8
C10 Cc, Cd, Ce Cd, Ce Cc, Ce Cc, Cd
H5 H8 H7, H9 H8, H10 H9 – Hb NH, Hc Hb, Hd, He Hc Hc
C4 C4, C5 C5 C6
Cc, Cd, Ce Cc, Ce Cc, Cd
C4 C4 C6 C8, C9 C9 C9
Cc, Cd, Ce Cd, Ce Cc, Ce Cc, Cd
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with C8 (dC 71.3 ppm), whereas H8 (dH 4.34 ppm) correlated with C9 (dC 70.5 ppm). H8 and H9 showed a 4.0 Hz coupling constant corresponding to a 608 dihedral angle and correlation cross peaks at the NOESY experiment, indicating that 8- and 9-hydroxyl groups have a syn relationship. This compound corresponds to N-isobutylsyn-8,9-dihydroxy-2E,6Z-decadienamide (3). N-Isobutyl-syn-6,9-dihydroxy-2E,7E-decadienamide (compound 4) is an isomer of compound 3. In compound 4, the hydroxyl groups are located at C6 and C9, and a trans double bond is at C7–C8 on the basis of the observed correlation cross peaks in the HMBC spectrum (Fig. 1, Table 2) and coupling constant values. In addition, H5 (dH 1.64 ppm) correlated with the oxygenated carbon C6 (dC 71.1 ppm), whereas H6 (dH 4.06 pm) showed a correlation cross peak with C8 (dC 135.0 ppm). Three correlation cross peaks were observed for H7 (dH 5.61 ppm), with C6, C8, and C9 (dc 67.7 ppm), while H10 (dH 1.23 ppm) correlated with C8 and C9. NOE difference experiment at 100 8C on the signal at dH 4.26 shown increases of those at dH 4.06 and irradiation on the signal at dH 4.06 shown increases of those at dH 4.26, corroborating an synrelationship between both 6- and 8-hydroxyl groups. Compounds 2–4 are minor chemical constituents of H. longipes roots. Their isolation and characterization from fresh, but not from stored roots, suggests a possible instability. These compounds may be important because alkamides constitute valuable molecules with possible antinociceptive, analgesic, and anesthetic properties. Actually, our group makes efforts to isolate compounds 2–4 in adequate quantities to assess their pharmacological properties as analgesic. 3. Experimental 3.1. General procedures Compounds were isolated by open column chromatography (CC). The isolation procedures and purity of compounds were monitored by thin layer chromatography (TLC), by visualizing with UV-light and spraying with (NH4)4Ce(SO4)4 in 2 N H2SO4. Optical rotations were measured on a PerkinElmer 341 MC digital polarimeter using CHCl3 as the solvent. IR spectra were obtained in KBr or as films (CHCl3) on a Bruker Vector 22 IR spectrometer. All 1 H, 13C, and 2D NMR experiments were performed in CDCl3 on a Varian Unity 400 spectrometer equipped with a 5 mm inverse detection pulse field gradient probe at 25 8C, at 400 MHz for 1H NMR and 100 MHz for 13C NMR. Chemical shifts were referenced to tetramethylsilane (TMS) as an internal standard. EI and FAB+MS spectra were recorded on a Jeol JMX-AX 505 HA mass spectrometer in a matrix of nitrobenzyl alcohol for FAB+MS. 3.2. Plant material The fresh roots of Heliopsis longipes (Asteraceae) were collected on 28 July 2007 at ‘‘Real de Xichu’’, Guanajuato and were identified by M.C. Ramiro Rios Go´mez of the Facultad de Estudios Superiores Zaragoza, UNAM, Me´xico. A voucher specimen (number 5904) was deposited at FEZA Herbarium, UNAM. 3.3. Extraction and isolation Fresh roots of H. longipes (4.725 kg) were extracted at room temperature with acetone. The extraction solvent was concentrated to dryness under vacuum to render 76.5 g of extract (1.2% yield). Fractionation of the acetone extract by means of open CC (silica gel, 100-230 mesh; Natland Int. Corp.; 11.5 cm 50 cm) was performed with a step gradient of n-hexane-acetone 100:0 to 40:60, collecting 143 250-mL fractions. On the basis of TLC analysis, these fractions were pooled into eight groups: G-1 (4.9 g, n-hexane
100%), G-2 (7.9 g, n-hexane:acetone 9:1), G-3 (4.5 g, n-hexane:acetone 9:1), G-4 (17.5 g, n-hexane:acetone 8:2), G-5 (10.3 g, nhexane:acetone 6:4), G-6 (8.7 g, n-hexane:acetone 6:4), G-7 (5.4 g, n-hexane:acetone 4:6, and G-8) (1.7 g, n-hexane:acetone 4:6). G-1 was an essential oil (4.9 g, 6.40% yield with respect to the total acetone extract). Additional groups were subjected to flash column chromatography: G-2 (silica gel, 230–400 mesh; Natland Int. Corp.; 4.5 cm i.d. 10 in., eluent n-hexane 100% to n-hexane:CH2Cl2 80:20) collecting 314 125-mL fractions, to yield squalene (314.0 mg, 0.41%), a mixture of b-sitosteryl palmitate and stigmasteryl palmitate (445.3 mg, 0.58%), lupeyl acetate (183.4 mg, 0.24%), a mixture of b-sitosterol and stigmasterol (866.2 mg, 1.13%), lupeol (230.7 mg, 0.30%) and the bornyl ester of deca-2E,6Z,8E-trienoic acid (174 mg, 0.23%); G-3 to G-6 (nhexane:CH2Cl2 70:30) to afford practically only affinin (34.6 g, 45.23%), accompanied by minor amounts of N-isobutyl-8,10diynoic-3Z-undecenamide (2, 82.0 mg, 0.10%), N-isobutyl-2Edecenamide (124.4 mg, 0.16%), and N-isobutyl-2E,6Z-decenamide (313.9 mg, 0.41%); G-7 (silica gel, 230–400 mesh; Natland Int. Corp.; 3 cm i.d. 10 in., eluent CH2Cl2:acetone 80:10 to 60:40) collecting 214 25-mL fractions, to yield angelicoidenol (187.2 mg, 0.24%) and N-isobutyl-syn-8,9-dihydroxy-2E,6Z-decadienamide (3, 73 mg, 0.10%); and finally fraction G-8 (silica gel, 230– 400 mesh; Natland Int. Corp.; 2.5 cm i.d. 10 in., eluent CH2Cl2:acetone 80:20 to 60:40) collecting 59 10-mL fractions, to afford Nisobutyl-syn-6,9-dihydroxy-2E,7E-decadienamide (4, 163 mg, 0.21%). 3.3.1. N-Isobutyl-8,10-diynoic-3Z-undecenamide (2) Pale yellow oil; UV (CHCl3, 1 10 3 M) lmax (log e) 242 (2.79), 251 (2.69), 277 (2.43), 350 (1.80) nm; IR (CHCl3): nmax = 3433 (NH), 3305 (C–H stretching), 2959, 2932, 2871 (CH, CH2, CH3), 2224 (CC), 1646 (C5 5O), 1552 (NH), 1266, 971, 739 cm 1; EIMS m/z 232 [M+H]+ (22), 230 [M H]+ (16), 216 [M-CH3]+ (5), 203 (8), 188 [MC3H7]+ (12), 174 [M-C4H9]+ (8), 159 [M-C4H10N]+ (10), 145 (13), 131 [C10H11]+ (48), 117 [C9H9]+ (60), 114 [C6H12NO]+ (28), 104 [C8H8]+ (23), 91 [C7H7]+ (53), 57 [C4H9]+ (100); HREIMS m/z 231.1614 [M]+ (calcd. for C15H21NO, 231.1623). See Table 1 for the 1 H and 13C NMR spectroscopic data. 3.3.2. N-Isobutyl-syn-8,9-dihydroxy-2E,6Z-decadienamide (3) Pale yellow oil; [a]D20 – 2.5 (c 1.7, CHCl3); UV (CHCl3) lmax (log e) 243 (4.11), 301 (2.99), 353 (1.30) nm; IR (CHCl3): nmax = 3315 (OH, NH), 2961, 2926, 2871 (CH, CH2, CH3), 1669 (C5 5O), 1627 (C5 5C), 1514, 1370, 1255, 979 cm 1; EIMS m/z 255 [M]+ (5), 238 [MOH]+ (4), 210 [M-C2H5O]+ (77), 184 [M-C4H9N]+ (20), 167 [MC4H8O2]+ (39), 149 [M-C4H10N-OH-OH]+ (100), 141 [C8H15NO]+ (68), 115 [C6H11O2]+ (16), 109 (12), 91 [C7H7]+ (11), 71 (14) 57 [C4H9]+ (100); HREIMS m/z 255.1834 [M]+ (calcd for C14H25NO3, 2551.1834). See Table 1 for the 1H and 13C NMR spectroscopic data. 3.3.3. N-Isobutyl-syn-6,9-dihydroxy-2E,7E-decadienamide (4) Pale yellow oil; [a]D20 – 1.2 (c 1.6, CHCl3); UV (CHCl3) lmax (log e) 243 (3.99), 346 (1.74) nm; IR (CHCl3): nmax = 3309 (OH, NH), 2960, 2925, 2869 (CH, CH2, CH3), 1669 (C5 5O), 1628 (C5 5C), 1555, 1370, 1255, 979 cm 1; FAB+MS m/z 256 [M+H]+ (22), 238 [M+HH2O]+ (92), 184 (9), 203 (8), 154 (24), 119 (30), 105 (34), 91 [C7H7]+ (53), 81 (67), 69 (73), 55 [C4H9]+ (100); HRFAB+MS m/z 256.1923 [M+H]+ (calcd for C14H26NO3, 256.1913). See Table 1 for the 1H and 13 C NMR spectroscopic data. Acknowledgements This work was supported financially by CONACyT (Grants number 79584-Q and 48358-Q). SLM thank PROMEP and ICYTDF for a postdoctoral fellowship. We are grateful to Biol. Enrique
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