J. Nat. Prod. 2004, 67, 1483-1487
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Constituents of Senecio chionophilus with Potential Antitubercular Activity Jian-Qiao Gu,† Yuehong Wang,‡ Scott G. Franzblau,‡ Gloria Montenegro,§ and Barbara N. Timmermann*,† Department of Pharmacology and Toxicology, Division of Medicinal and Natural Products Chemistry, College of Pharmacy, University of Arizona, Tucson, Arizona 85721, Institute for Tuberculosis Research (M/C 964), College of Pharmacy, University of Illinois at Chicago, Chicago, Illinois 60612, and Departamento de Ciencias Vegetales, Facultad de Agronomı´a e Ingenierı´a Forestal, Pontificia Universidad Cato´ lica de Chile, Avenida Vicun˜ a Mackenna 4860, Santiago, Chile Received May 17, 2004
Two new sesquiterpenoids, (1S,4S,5R,10R)-1-hydroxy-6-isobutyryloxy-10H-9-oxofuranoeremophilane (1) and 1R-hydroxy-6β-(2ξ-methylbutyryloxy)-10RH-9-oxofuranoeremophilane (2), along with 21 known constituents, were isolated from the n-hexane and dichloromethane extracts of the above-ground biomass and roots of Senecio chionophilus. The structures of 1 and 2 were elucidated on the basis of spectroscopic evidence and chemical transformation methods. The absolute configuration of 1 was determined by Mosher ester methodology. All of the isolates were evaluated for their antitubercular potential against Mycobacterium tuberculosis in a microplate Alamar Blue assay. Compound 2, 6β-angeloyloxy-1R-hydroxy10RH-9-oxofuranoeremophilane (3), and 4′-hydroxyacetophenone (6) exhibited mild antitubercular activity at minimum inhibitory concentrations of 119, 114, and 121 µg/mL, respectively. Senecio chionophilus Phil. (Asteraceae) is a small woody shrub with erect stems, hairy leaves, and yellow flowers, indigenous to Argentina and Chile, where it grows in the Andes Mountains above 1500 m.1 Infusions of its aerial parts and roots are traditionally used in Chile for treating heavy colds and runny noses (Montenegro, personal communication). Up to the present, no phytochemical or biological activity information has been reported on this plant. As part of a collaborative search for novel antitubercular principles of plant and microbial organisms from dryland biodiversity of Latin America,2,3 n-hexane- and dichloromethane-soluble extracts of the above-ground biomass and roots of S. chionophilus exhibited inhibitory effects on the growth of Mycobacterium tuberculosis H37Rv in a microplate Alamar Blue assay (MABA) system with minimum inhibitory concentrations (MICs) of 50 and 100 µg/mL, respectively. Fractionation of these two extracts led to the isolation of two new furanoeremophilanes, 1 and 2, along with 21 known compounds. The structures of 1 and 2 were elucidated by spectroscopic evidence and chemical transformation methods. The absolute configuration of 1 was determined by Mosher ester methodology in a modified procedure in an NMR tube.4,5 All of the isolates were evaluated for their antitubercular potential as pure compounds, and the results are described herein. Results and Discussion Compound 1 was obtained as a white amorphous powder and gave a protonated molecular ion ([M + H]+) at m/z 335.1861 by high-resolution FABMS, consistent with an elemental formula of C19H26O5. This compound exhibited UV maxima at 205 and 285 nm, suggestive of the presence of considerable conjugation in the molecule. In the IR spectrum, absorption bands at 3490 cm-1 (OH), 1739 cm-1 (an ester carbonyl), and 1658 cm-1 (a conjugated keto) were apparent.6 Comparison of the 1H NMR data (Table 1) of 1 with those of 1R-hydroxy-6β-isobutyryloxy-10RH-9-oxofuranoeremophilane (5),7 which was also obtained in the * To whom correspondence should be addressed. Tel: +1-520-626-2481. Fax: +1-520-626-2515. E-mail:
[email protected]. † University of Arizona. ‡ University of Illinois at Chicago. § Pontificia Universidad Cato´lica de Chile.
10.1021/np049831z CCC: $27.50
present investigation, indicated that they are a pair of isomers, with the only difference being in the configuration at C-10. The furanoeremophilane skeleton of 1 was confirmed by the observed HMBC correlations (H-12/C-7, C-8, C-11; H3-13/C-7, C-11, C-12; H3-14/C-3, C-4, C-5; and H315/C-4, C-5, C-6, C-10) (Table 3). The hydroxyl group in 1 was located at C-1 rather than C-6 as a result of the observed 1H-1H COSY cross-peaks in CDCl3 between OH-1 (δH ) 4.95, brs) and H-1 (δH ) 3.91, m), which was further correlated with H-10 (δH ) 3.16, brs) and H2-2 (δH ) 1.59 and 1.96). The relative configuration of 1 was determined by a ROESY experiment (Figure 1), which gave diagnostic correlations from H-1β to H-2β, H-10β, and CH3-5β, as well as H-6R to CH3-4β and CH3-5β, leading to the assignment of H-1β, H-4R, H-6R, H-10β, and CH3-5β, respectively. A cis-fused A/B ring junction in 1 was established by the observed ROESY correlations between H-10 and H3-15 as well as the chemical shift of H3-15 at δH ) 1.07 in CDCl3, rather than at δH < 1.00 in the trans-fused series.8,9 The placement of the 6β-isobutyryloxy group in 1 was confirmed by the observation of a relatively high-field shift of H3-14 at δH ) 0.82 due to its quasi 1,3-diaxial interaction with H-6R in the cis-fused A/B ring series.8 In addition, the broad singlet splitting pattern for H-10 of 1 confirmed the assignment of a cis-disposition for H-1β/H-10β in the cisfused ring series, instead of a doublet (J ) 10 Hz) indicating an anti-configuration for H-1β/H-10R in the trans-fused series.7 Furthermore, a steroidal-like conformation of 1 was established on the basis of the observed spatial ROESY correlations between H-1β and CH3-5, along with H-6R and CH3-4 (Figure 1).8 The absolute configuration of the stereogenic center at C-1 in 1 was determined by a convenient Mosher ester procedure,4,5 which was performed directly in NMR tubes using deuterated pyridine as a solvent. Compound 1 was treated with (R)-(-)- and (S)-(+)-Rmethoxy-R-(trifluoromethyl)phenylacetyl chloride to obtain its (S)- (1s) and (R)-esters (1r), respectively (see Experimental Section). Analysis of ∆δΗ ) δΗ (1s) - δΗ (1r) data in C5D5N (Table 1) showed positive differences in chemical shifts for the protons at C-10 and C-15 and negative effects for the protons at C-2 and C-3. These effects indicated that the absolute configuration at C-1 of 1 is S. Accordingly, the absolute configurations at C-4, C-5, and C-10 were
© 2004 American Chemical Society and American Society of Pharmacognosy Published on Web 07/27/2004
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Journal of Natural Products, 2004, Vol. 67, No. 9
Gu et al.
Table 1. 1H NMR Data of Compounds 1, 1s, 1r, 2, and 2aa position 1 2 3 4 6 10 12 13 14 15 OH-1 2′ 3′ 4′ 5′ COCH3-1
1b 3.91 m 1.59 o 1.96 d-like (11) 1.39-1.50 o 1.39-1.50 o 1.54 o 6.00 brs 3.16 brs 7.43 d (1.0) 2.04 d (1.0) 0.82 d (6.2) 1.07 s 4.95 brs 2.56 sep (7.0) 1.17 d (7.0) 1.13 d (7.0)
1c 4.57 brs 1.80 m 1.80 m 1.31 m (4.3, 11) 2.84 o 1.90 o 7.66 o 2.83 brs 7.60 o 1.90 s 0.96 d (7.0) 1.13 s 6.81 br s 2.72 sep (7.0) 1.24 d (7.0) 1.22 d (7.0)
1sc
1rc
∆δΗ(1s-1r)
2b
2ab
5.75 br s 1.99 m 1.80 m 1.24 o 2.17 t-like (15) 1.86 o 6.76 s 3.07 brs 7.68 brs 1.86 s 0.92 d (7.0) 1.07 s
5.77 br s 1.99 m 1.98 m 1.38 d-like (14) 2.50 t-like (14) 1.86 o 7.14 brs 3.01 brs 7.28 brs 1.86 s 0.95 d (7.0) 1.05 s
Sd 0 -0.18 -0.14 -0.33 0 -0.38 +0.06 +0.40 0 -0.03 +0.02
5.29 m 1.39 o 2.12 t-like (11) 1.44 o 1.44 o 1.86 o 6.38 s 2.67 d (10) 7.33 br s 1.87 br s 0.90 (6.8) 0.99 s
2.66 sep (7.0) 1.19 d (7.0) 1.19 d (7.0)
2.72 sep (7.0) 1.24 d (7.0) 1.23 d (7.0)
-0.06 -0.05 -0.04
4.17 m (5.1) 1.39 o 2.05 ddd (5.3, 11) 1.46 o 1.46 o 1.84 o 6.35 s 2.39 d (9.6) 7.38 q (1.1) 1.89 d (1.1) 0.89 (6.8) 0.99 s 4.50 br s 2.43 m (7.0) 1.89 o, 1.55 o 0.99 t (7.4) 1.23 d (7.2)
2.43 sep (7.0) 1.87 o, 1.50 o 0.99 o 1.21 d (7.2) 2.02 s
a Spectra were recorded at 300.1 MHz, and values are reported in parts per million relative to TMS. Coupling constants in parentheses are reported in hertz. Multiplicities are as follows: brs, broad singlet; d, doublet; ddd, doublet of double doublets; m, multiplet; o, unclear due to overlapping signals; q, quartet; s, singlet; sep, septet; t, triplet. Assignments are based on 1H-1H COSY, HMQC, HMBC, and ROESY experiments. b Data in CDCl3. c Data in C5D5N. d Absolute configuration at C-1 of 1.
Table 2.
Figure 1. Selected ROESY correlations observed for 1.
13C
NMR Data of Compounds 1, 2, 2a, 4, 4a, and 5a
position
1
2
2a
4
4a
5
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 1′ 2′ 3′ 4′ 5′ COCH3 COCH3
69.3 d 31.8 t 29.7 t 33.0 d 47.1 s 67.9 d 135.2 s 147.5 s 190.3 s 53.6 d 121.5 s 145.9 d 7.8 q 15.4 q 15.8 q 176.3 s 34.2 d 19.1 q 18.9 q
66.4 d 32.8 t 30.1 t 42.0 d 50.9 s 74.6 d 135.9 s 146.6 s 189.2 s 60.8 d 121.1 s 145.9 d 8.9 q 17.9 q 8.9 q 175.8 s 41.1 d 25.7 t 11.7 q 15.5 q
67.3 d 31.2 t 29.7 t 41.8 d 51.1 s 74.6 d 133.5 s 147.2 s 184.7 s 57.9 d 120.7 s 144.9 d 9.0 q 17.7 q 8.8 q 175.8 s 41.1 d 25.7 t 11.7 q 15.5 q 170.5 s 21.2 q
66.4 d 32.8 t 30.2 t 41.6 d 51.0 s 74.6 d 136.2 s 146.4 s 189.2 s 60.7 d 121.1 s 146.0 d 8.9 q 17.7 q 8.7 q 172.7 s 43.4 t 24.6 d 22.7 q 22.7 q
67.3 d 31.2 t 29.7 t 41.4 d 51.3 s 74.7 d 133.8 s 147.1 s 184.7 s 57.9 d 120.6 s 144.9 d 8.8 q 17.5 q 8.7 q 172.7 s 43.4 t 24.6 d 22.7 q 22.7 q 170.5 s 21.2 q
66.3 d 32.8 t 30.1 t 41.9 d 50.9 s 74.7 d 136.0 s 146.5 s 189.2 s 60.7 d 121.1 s 146.0 d 8.9 q 17.8 q 8.8 q 176.3 s 34.5 d 18.7 q 18.6 q
Figure 2. Selected ROESY correlations observed for 2.
a Spectra were recorded in CDCl at 75.5 MHz, and values are 3 reported in parts per million relative to TMS. Multiplicities were 13 determined by C NMR and DEPT experiments and are designated as follows: s, singlet; d, doublet; t, triplet; q, quartet. Assignments are based on 1H-1H COSY, HMQC, HMBC, and ROESY experiments.
deduced as S, R, and R, respectively, on the basis of their relative configuration. All the 1H and 13C NMR signals were completely assigned by interpretation of the 1H-1H COSY, HSQC, and HMBC spectra. Thus, the structure of 1 was determined as (1S,4S,5R,10R)-1-hydroxy-6-isobutyryloxy10H-9-oxofuranoeremophilane. Compound 2 was obtained as a colorless amorphous solid, and its molecular formula was deduced as C20H28O5 from the HRFABMS data at m/z 349.2010 [M + H]+ (C20H29O5, calcd for 349.2015). As documented in the Experimental Section, 2 also exhibited UV, IR, and 1H NMR (Table 1) features similar to those of 5 and was therefore recognized as an analogue of the latter compound, with the only difference occurring in the ester group at C-6. A 2-methylbutyryloxy group of 2 was identified by observation of its characteristic 1H NMR signals within one spin system for two methyls at δH ) 0.99 (t, J ) 7.4 Hz, H3-4′)
and 1.23 (d, J ) 7.2 Hz, H3-5′), one methylene at δH ) 1.55 and 1.89 (overlapping, H2-3′), and one methine at δH ) 2.43 (m, J ) 7.0 Hz, H-2′). The presence of this ester side chain was confirmed by the 13C and DEPT NMR data of 2 (Table 2) indicating an ester carbonyl at δC ) 175.8 (C-1′), two methyls at δC ) 11.7 (C-4′) and 15.5 (C-5′), a methylene at δC ) 25.7 (C-3′), and a methine at δC ) 41.1 (C-2′). The 2-methylbutyryloxyl group was located at C-6 as a result of observed HMBC correlations between H-6 and C-4, C-5, C-7, C-8, C-15, and C-1′ (Table 3). The configuration of the 2-methylbutyryloxy unit could not be determined by the present NMR experiments. The relative configuration of 2 including the trans-fused A/B ring system was determined by a ROESY experiment (Figure 2), which gave diagnostic spatial correlations from H-1β to H-2β and CH3-5β, as well as H-10R to H-2R, H-4R, and H-6R, leading to the assignment of H-1β, CH3-5β, H-4R, H-6R, and H-10R, respectively.
Antitubercular Activity of Senecio chionophilus
Journal of Natural Products, 2004, Vol. 67, No. 9 1485
Table 3. Observed HMBC Correlations of 1, 2, and 2a (300.1 MHz, CDCl3) H
C of 1
C of 2
C of 2a
2 6 10 12 13 14 15 2′ 3′ 4′ 5′ acetate CH3
NOa NOa NOa 7, 8, 11 7, 11, 12 3, 4, 5 4, 5, 6, 10 1′, 3′, 4′ 1′, 2′ 1′, 2′
NOa 4, 5, 7, 8, 15, 1′ 1, 5, 6, 9, 15 7, 8, 11 7, 11, 12 3, 4, 5 5, 6, 10 3′, 4′, 5′ 1′, 2′, 4′, 5′ 2′, 3′ 1′, 2′, 3′
4, 10 4, 5, 7, 8, 15, 1′ 1, 5, 6, 9, 15 7, 8, 11 7, 11, 12 3, 4, 5 5, 6, 10 NOa NOa 2′, 3′ 1′, 2′, 3′ acetate CO
a
HMBC correlations were not observed in the spectra.
Furthermore, a coupling constant of 9.6 Hz between H-1 and H-10 in 2 indicated that these two neighboring protons existed in a diaxial orientation,7 and this required a steroidal-like conformation, which was further confirmed by the above-mentioned ROESY correlations between H-1β and CH3-5, as well as H-4R and H-10R (Figure 2).8 Moreover, an acetate derivative (2a) was generated after compound 2 was treated with pyridine and acetic anhydride by a routine procedure to confirm the presence of a free hydroxy group in 2. Therefore, 2 was identified as 1Rhydroxy-6β-(2ξ-methylbutyryloxy)-10RH-9-oxofuranoeremophilane, and all the NMR signals of 2 and 2a were unambiguously assigned in Tables 1-3.
(6),12 1R-acetoxy-6β-angeloyloxy-10RH-9-oxofuranoeremophilane,6 1R-acetoxy-6β-isobutyryloxy-10RH-9-oxofuranoeremophilane,13 R-amyrenone,14 β-amyrenone,14 R-amyrin,14 β-amyrin,14 1R-angeloyloxy-6β-isobutyryloxy-10RH-9-oxofuranoeremophilane,13 6β-angeloyloxy-10RH-9-oxofuranoeremophilane,6 caryophyllene 4β,5R-epoxide,15 daucosterol,16 1R-hydroxy-6β-propionyloxy-10RH-9-oxofuranoeremophilane,17 6β-isobutyryloxy-10RH-9-oxofuranoeremophilane,6 lupenone,14 lupeol,14 β-sitosterol,18 taraxasterol,14 and taraxasterone,14 by comparison of their physical and spectroscopic data (mp, [R]D, 1H NMR, 13C NMR, DEPT, 2D NMR, and MS) with reported data. The 13C NMR data of 4 and its acetate (4a) along with 5 have not been published; thus the complete assignments of their 13C NMR data were performed and are reported in Table 2. The occurrence of these isolates of S. chionophilus is in agreement with the fact that terpenoids, especially furanoeremophilanes, are widespread in the genus Senecio.19 However, it is noteworthy that in the present investigation we did not obtain any pyrrolizidine alkaloids (PA), which are reported to be one of the most characteristic secondary metabolites in other species in the same genus.20 The absence of PA in S. chionophilus was further confirmed by their negative detection in the methanolic extract when TLC plates were sprayed with Dragendorff’s reagent. It is well known that pyrrolizidine alkaloids in Senecio species cause hepatotoxicity to livestock feeding on this plant.21-23 The lack of PA in S. chionophilus may contribute to its ethnobotanical use to treat heavy colds by the local population in southern Chile. All of the 23 compounds obtained from S. chionophilus along with two O-acetyl derivatives (2a and 4a) were evaluated for their antitubercular potential against M. tuberculosis, according to established protocols.3 The results showed that compounds 2, 3, and 6 exhibited mild inhibitory activity with MIC values of 119, 114, and 121 µg/mL, respectively, when compared with the MIC of 0.03 µg/mL for rifampin, a positive control used for the microplate Alamar Blue assay.3 In contrast, the other 22 compounds were not regarded as active in this assay, since they did not meet the threshold MIC value of
