Article pubs.acs.org/jnp
C‑17 Lactam-Bearing Limonoids from the Twigs and Leaves of Amoora tsangii Guo-Yuan Zhu,† Guangying Chen,‡ Liang Liu,† Li-Ping Bai,*,† and Zhi-Hong Jiang*,† †
State Key Laboratory of Quality Research in Chinese Medicine, Macau Institute for Applied Research in Medicine and Health, Macau University of Science and Technology, Taipa, Macau, People’s Republic of China ‡ Key Laboratory of Tropical Medicinal Plant Chemistry of Ministry of Education, College of Chemistry & Chemical Engineering, Hainan Normal University, Hainan, People’s Republic of China S Supporting Information *
ABSTRACT: Twelve new lactam-bearing limonoids, amooramides A−L (1−12), were isolated from the twigs and leaves of Amoora tsangii, and their structures fully elucidated by spectroscopic analysis. These compounds represent the first examples of rings A,B-seco-limonoids bearing unusual lactam side chains at C-17, including a 3-substituted 1,5-dihydro-2Hpyrrol-2-one moiety in 1−7 and a 4-substituted 1,5-dihydro-2H-pyrrol-2-one unit in 8−12. Compound 9 inhibited TNFαinduced NF-κB activity by 64% at a concentration of 10 μM.
L
imonoids are a large class of nortriterpenoids that are mainly found in plants belonging to the Meliaceae family. To date, more than a thousand limonoids have been reported. They can be divided into four major groups, i.e., ring intact limonoids, seco-limonoids, rearranged limonoids, and limonoid derivatives.1 Nitrogen-bearing limonoids, which are also known as limonoid-based alkaloids, are a minor class of limonoids. These compounds consist of five lactam-bearing,2−5 four pyridine-bearing,6,7 and two maleimide-bearing limonoids.8,9 Amoora tsangii (Merr.) X. M. Chen is a meliaceous timber tree native to Hainan Island in China. The stem bark of this plant is used as a folk medicine to kill lice.10 A previous phytochemical investigation of the twigs and leaves of A. tsangii reported nine limonoids and four sesquiterpenoids.11 Our preliminary LC-MS study of the ethanolic extract of A. tsangii revealed the presence of several N-bearing limonoids, which prompted us to conduct an extensive study of limonoids in this plant. Herein, we describe the isolation and structural elucidation of 12 new Nbearing limonoids and the inhibitory activity of a selection of these compounds toward NF-κB.
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RESULTS AND DISCUSSION The twigs and leaves of A. tsangii were extracted twice with 80% EtOH, and the combined extracts were concentrated under reduced pressure, suspended in H2O, and extracted sequentially with petroleum ether and EtOAc. The EtOAc-soluble fraction was repeatedly purified by column chromatography over silica gel and ODS and by preparative HPLC to afford the N-bearing limonoids 1−12. Amooramide A (1) was obtained as a white, amorphous powder. Its molecular formula was determined to be C36H41NO10 by the 13C NMR data and an HRESIMS ion at m/z 648.2811 [M + H]+ (calcd for [M + H]+ 648.2803), indicating that 1 had 17 degrees of unsaturation. The IR © 2014 American Chemical Society and American Society of Pharmacognosy
spectrum of 1 showed strong absorptions at 1730 and 1690 cm−1, which indicated the presence of carbonyl groups. The 1H NMR data of 1 (Table 1) displayed resonances characteristic of a benzoyloxy group [δH 7.84 (2H, d, J = 7.4 Hz), 7.35 (2H, t, J = 7.4 Hz), and 7.49 (1H, t, J = 7.4 Hz)], a methoxy group (δH 3.70, 3H, s), an acetoxy methyl group (δH 1.93, 3H, s), and four tertiary methyls (δH 1.03, 1.17, 1.29, and 1.57, each 3H, s). The 13 C NMR (Table 3) data of 1 displayed 34 resonances (Table 3), which were classified by DEPT and HSQC experiments as a monosubstituted benzoyl, five ester carbonyls, an exocyclic Received: December 27, 2013 Published: March 12, 2014 983
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Table 1. 1H NMR Data of Compounds 1−6 (600 MHz in CDCl3, δ in ppm, J in Hz)a position 1 2 5 6a 6b 9 11 12 15 16a 16b 17 18 19 22 23a 23b 28 29 30a 30b 7-OMe 11-acyl 12-acyl-2′ 3′ (7′) 4′ (6′) 5′ N−H
1 6.99, d (13.0) 6.36, d (13.0) 3.36 (d, 9.4) 2.32 (d, 17.0) 2.21, dd (17.0, 3.13, d (7.4) 5.78, dd (10.7, 6.06, d (10.7) 3.92, s 2.68, dd (14.0, 2.07, dd (14.0, 3.02, dd (10.8, 1.17, s 1.03, s 6.60, brs 3.49, d (20.0) 3.05, d (20.0) 1.29, s 1.57, s 5.39, s 5.26, s 3.70, s 1.93, s 7.84, 7.35, 7.49, 6.79,
d (7.4) t (7.4) t (7.4) brs
2
9.4) 7.4)
10.8) 7.2) 7.2)
6.99, d (13.0) 6.36, d (13.0) 3.36 (d, 9.6) 2.31 (d, 17.0) 2.20, dd (17.0, 3.12, d (7.4) 5.79, dd (10.7, 6.04, d (10.7) 3.92, s 2.71, dd (14.0, 2.03, dd (14.0, 3.01, dd (10.5, 1.16, s 1.03, s 6.50, s 3.38, d (19.5) 2.95, d (19.5) 1.28, s 1.56, s 5.40, s 5.25, s 3.70, s 1.93, s 7.84, d (7.4) 7.36, t (7.4) 7.50, t (7.4)
3
9.6) 7.4)
10.5) 7.3) 7.3)
4
6.98, d (13.0) 6.38, d (13.0) 3.37 (d, 9.6) 2.32 (d, 17.0) 2.21, dd (17.0, 3.13, d (7.4) 5.78, dd (10.7, 6.06, d (10.7) 3.92, s 2.61, dd (14.0, 2.08, dd (14.0, 3.04, dd (10.6, 1.16, s 1.04, s 6.56, t (1.6) 3.55, dd (19.8, 3.12, dd (19.8, 1.29, s 1.57, s 5.40, s 5.27, s 3.71, s 1.94, s
9.6) 7.4)
10.6) 7.2) 7.2)
1.6) 1.6)
7.88, d (7.4) 7.37, t (7.4) 7.51, t (7.4)
5
6.93, d (13.0) 6.28, d (13.0) 3.26 (d, 9.4) 2.29 (d, 17.0) 2.19, dd (17.0, 9.4) 3.05, d (7.4) 5.63, dd (10.8, 7.4) 5.82, d (10.8) 3.88, s 2.52, dd (13.7, 10.5) 2.09, dd (13.7, 7.3) 3.01, dd (10.5, 7.3) 1.08, s 1.00, s 6.78, brs 3.88, d (20.3) 3.81, d (20.3) 1.29, s 1.55, s 5.35, s 5.22, s 3.72, s 2.11, s 2.06, m 1.58, m; 1.31, m 0.85, t (7.5) 0.95, d (7.1) 6.85, brs
6.93, d (13.0) 6.28, d (13.0) 3.27 (d, 9.3) 2.29 (d, 17.0) 2.19, dd (17.0, 3.06, d (7.4) 5.63, dd (10.7, 5.82, d (10.7) 3.88, s 2.59, dd (13.8, 2.07, dd (13.8, 3.00, dd (10.5, 1.07, s 1.00, s 6.76, s 3.87, d (20.1) 3.80, d (20.1) 1.29, s 1.55, s 5.35, s 5.22, s 3.72, s 2.11, s 2.26, m 1.03, d (7.0) 1.00, d (7.0)
6
9.3) 7.4)
10.5) 7.2) 7.2)
6.97, d (13.0) 6.27, d (13.0) 3.28 (d, 9.6) 2.30 (d, 17.0) 2.20, dd (17.0, 9.6) 3.06, d (7.3) 5.69, dd (10.7, 7.3) 5.88, d (10.7) 3.90, s 2.90, dd (13.7, 10.6) 2.13, m 3.04, dd (10.6, 7.3) 1.17, s 1.02, s 6.86, s 4.05, d (19.2) 3.86, d (19.2) 1.30, s 1.56, s 5.37, s 5.22, s 3.73, s 2.14, s 2.11, m 1.59, m; 1.33, m 0.87, t (7.5) 1.01, d (7.0)
5.96, brs
a
N-CH3 (2.67, s) in 2, N-CH2CH2OH (3.26 dt, 14.7, 5.0, H-1″a; 3.20 dt, 14.7, 5.0, H-1″b; 3.54 m, H-2″) in 3, N-4-hydroxy-2,6-dimethoxyphenyl (5.84 d, 2.1, H-3″; 5.86 d, 2.1, H-5″; 3.59 s, 2″-OMe; 3.64 s, 6″-OMe) in 6.
The relative configuration of 1 was determined by a NOESY experiment (Figure 2). The NOESY correlations from H-11 to H3-18α and H-9 allowed for the assignment of the 11α-H and 11β-acetoxy group. Similarly, the NOE interaction between H12 and H-17β suggested the β-orientation of H-12 and the 12αbenzoyloxy group. Correlations of H-5/H3-28α, H-5/H-9, and H-15/H3-18α indicated that H-5, H-9, and H-15 were αoriented. Thus, the structure of 1 was established as shown. Compounds 2 and 3 had molecular formulas C37H43NO10 and C38H45NO11, respectively, based on their HRESIMS and 13 C NMR data analyses. The 1D NMR data of 2 and 3 (Tables 1 and 3) were similar to those of 1, except that the amino proton in 1 was replaced by alkyl groups. A methyl group [δC 29.0; δH 2.67 (s)] was linked to the nitrogen in 2, as deduced from the HMBC correlations from N-CH3 to C-21 and C-23. In contrast, a −CH2CH2OH unit was connected to the nitrogen in 3, as indicated by the HMBC correlations from the hydrogens at δH 3.26 (dt, J = 14.7, 5.0 Hz, H-1″a) and 3.20 (dt, J = 14.7, 5.0 Hz, H-1″b) to C-21 and the COSY correlations from H-1″a and H-1″b to the hydrogen at δH 3.54 (2H, m, H2″). Compounds 2 and 3 were named amooramides B (2) and C (3), respectively. Amooramides D (4) and E (5) were isolated as amorphous, white solids, and their molecular formulas were determined by 13 C NMR and HRESIMS data to be C34H45NO10 and C33H43NO10, respectively. A comparison of the NMR data of 4 and 5 (Tables 1 and 3) with those of 1 indicated that these three compounds were structurally similar except for the acyloxy groups at C-12. The spectroscopic data of compound 4
double bond, a disubstituted double bond, one trisubstituted double bond, and six methyl, three methylene, six methine, and four quaternary carbons. NMR resonances typical for A,B-secolimonoids11 were also observed, including several resonances that were assigned to an α,β-unsaturated ε-lactone moiety [δC 148.8 (C-1), 122.1 (C-2), 166.9 (C-3), 83.7 (C-4); δH 6.99 (d, J = 13.0 Hz, H-1), 6.36 (d, J = 13.0 Hz, H-2)] and an exocyclic Δ8(30) double bond [δC 136.2 (C-8), 121.4 (C-30); δH 5.39 (s, H-30a), 5.26 (s, H-30b)]. Comparison of the 1H and 13C NMR spectroscopic data of 1 (Tables 1 and 3) with those of amotsangin A11 and mulavanin A12 indicated that these compounds were structurally related, except for the acyloxy group at C-12 and the C-17 side chain. The NMR resonances associated with the C-17 substituent [δC 135.5 (C-20), 174.3 (C-21), 142.7 (C-22), 45.9 (C-23); δH 6.60 (brs, H-22), 3.49 (d, J = 20.0 Hz, H-23a), 3.05 (d, J = 20.0 Hz, H-23b)] were similar to that of the 3-substituted 1,5-dihydro-2H-pyrrol-2-one unit in salannolactam-(21)2 and munronin D.4 Furthermore, the 1H NMR spectrum of 1 exhibited a resonance characteristic of an amino proton at δH 6.79 (brs), which was correlated with C-20, C-21, C-22, and C-23 in the HMBC spectrum. These data suggested a 3-substituted 1,5-dihydro-2H-pyrrol-2-one unit connected to C-17 in 1, which was confirmed by HMBC correlations from H-17 to C-20, C-21, and C-22. The benzoyloxy group was located at C-12 via an HMBC correlation from H-12 to C-1′. The proposed structure of 1 was further confirmed by 1H−1H COSY and HMBC analyses (Figure 1). 984
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Table 2. 1H NMR Data of Compounds 7−12 (600 MHz in CDCl3, δ in ppm, J in Hz) position 1 2 5 6a 6b 9 11 12 15 16a 16b 17 18 19 21a 21b 22 23a 23b 28 29 30a 30b OMe 11-acyl 12-acyl-2′ 3′ (7′) 4′ (6′) 5′ N-H(CH3)
7 6.96, d (12.9) 6.35, d (12.9) 3.33 (d, 9.1) 2.32 (d, 16.7) 2.21, dd (16.9, 3.19, d (7.4) 5.92, dd (10.6, 6.12, d (10.6) 3.93, s 2.81, dd (14.0, 2.05, dd (14.0, 3.03, dd (10.6, 1.21, s 1.06, s
6.60, 3.45, 2.89, 1.29, 1.57, 5.42, 5.29, 3.71, 8.01,
brs d (20.0) d (20.0) s s s s s s
7.87, 7.38, 7.51, 5.59,
d (7.4) t (7.4) t (7.4) brs
8
9.1) 7.4)
10.6) 7.4) 7.4)
6.94, d (13.0) 6.31, d (13.0) 3.33 (d, 9.4) 2.31 (d, 16.9) 2.21, dd (16.9, 3.12, d (7.4) 5.79, dd (10.7, 6.05, d (10.7) 3.96, s 2.37, dd (14.0, 1.93, dd (14.0, 2.86, dd (10.5, 1.04, s 1.02, s 3.71, d (19.5) 3.63, d (19.5) 5.82, brs
1.28, 1.57, 5.42, 5.30, 3.71, 1.82,
s s s s s s
7.91, d (7.4) 7.42, t (7.4) 7.55, t (7.4)
9
9.4) 7.4)
6.9) 10.5) 6.9)
10
6.85, d (13.0) 6.23, d (13.0) 3.27 (d, 9.3) 2.28 (d, 16.9) 2.20, dd (16.9, 3.07, d (7.4) 5.65, dd (10.8, 5.81, d (10.8) 3.91, s 2.32, dd (13.9, 1.80, dd (13.9, 2.83, dd (10.6, 1.05, s 1.00, s 3.87, d (19.6) 3.82, d (19.6) 5.91, s
1.29, 1.56, 5.37, 5.27, 3.72, 2.14, 2.15, 1.57, 0.85, 1.01,
9.3) 7.4)
6.9) 10.6) 6.9)
s s s s s s m m; 1.31, m t (7.4) d (7.1)
6.85, d (13.0) 6.23, d (13.0) 3.29 (d, 9.2) 2.28 (d, 17.0) 2.20, dd (17.0, 3.07, d (7.3) 5.64, dd (10.8, 5.80, d (10.8) 3.91, s 2.32, dd (13.9, 1.81, dd (13.9, 2.80, dd (10.7, 1.04, s 1.00, s 3.85, d (19.5) 3.79, d (19.5) 5.89, brs
1.30, 1.55, 5.37, 5.27, 3.73, 2.13, 2.36, 1.04, 1.03,
s s s s s s h (7.0) d (7.0) d (7.0)
5.74, brs
11
9.2) 7.3)
6.9) 10.7) 6.9)
6.94, d (13.0) 6.33, d (13.0) 3.36 (d, 9.5) 2.31 (d, 17.0) 2.21, dd (17.0, 3.13, d (7.4) 5.79, dd (10.7, 6.06, d (10.7) 3.95, s 2.34, dd (14.1, 1.93, dd (14.1, 2.78, dd (10.5, 1.01, s 1.02, s 3.54, d (19.5) 3.46, d (19.5) 5.82, s
1.29, 1.57, 5.42, 5.30, 3.71, 1.89,
s s s s s s
7.93, 7.46, 7.57, 2.43,
d (7.4) t (7.4) t (7.4) s
12
9.5) 7.4)
7.0) 10.5) 7.0)
6.85, d (13.0) 6.23, d (13.0) 3.27 (d, 9.3) 2.28 (d, 17.0) 2.19, dd (17.0, 3.06, d (7.2) 5.64, dd (10.8, 5.80, d (10.8) 3.90, s 2.31, dd (13.8, 1.80, dd (13.8, 2.77, dd (10.6, 1.02, s 1.00, s 3.74, brs
9.3) 7.2)
6.8) 10.6) 6.8)
5.91, s
1.29, 1.55, 5.37, 5.27, 3.73, 2.13, 2.15, 1.57, 0.85, 1.01, 2.94,
s s s s s s m m; 1.31, m t (7.4) d (7.1) s
NMR data of 8 (Tables 2 and 3) were similar to those of 1 except for resonances derived from the C-17 side chain. The 1H and 13C NMR spectra of 8 displayed signals for the C-17 side chain [δC 159.9 (C-20), 50.9 (C-21), 124.9 (C-22), 173.9 (C23); δH 3.71 (d, J = 19.5 Hz, H-21a), 3.63 (d, J = 19.5 Hz, H21b), 5.82 (brs, H-22)], which were identical to those of salamolactam-(23),2 a limonoid containing a 4-substituted 1,5dihydro-2H-pyrrol-2-one unit at C-17. The HMBC correlations from H-17 (δH 2.86, dd, J = 10.5, 6.9 Hz) to C-20, C-21, and C22 and from H-21 to C-23 further confirmed that the carbonyl carbon of the lactam in 8 was located at C-23. Thus, compound 8 is a 4-substituted 1,5-dihydro-2H-pyrrol-2-one isomer of 1. The proposed structure and relative configuration of 8 were confirmed by 2D NMR analyses (Figures 1 and 2). Compound 8 is the second example of a limonoid bearing a 4-substituted 1,5-dihydro-2H-pyrrol-2-one unit at C-172 and was named amooramide H (8). The molecular formulas of amooramides I (9) and J (10) were established on the basis of their HRESIMS and 13C NMR data analyses as C34H45NO10 and C33H43NO10, the same of 4 and 5, respectively. Consideration of the NMR data for 9 and 10 (Tables 2 and 3) revealed that they possessed the same C17 side chain (4-substituted 1,5-dihydro-2H-pyrrol-2-one unit) as 8 and were the 4-substituted 1,5-dihydro-2H-pyrrol-2-one isomers of 4 and 5, respectively. The HRESIMS spectrum (m/z 662.2961; calcd 662.2960 [M + H]+) of amooramide K (11) showed that it has the same molecular formula, C37H43NO10, as 2. The NMR spectroscopic data of 11 (Tables 2 and 3) were similar to those of 8, except
demonstrated the presence of a 2-methylbutanoyloxy moiety at C-12 (Tables 1 and 3), which was confirmed by an HMBC correlation between H-12 (δH 5.82, d, J = 10.8 Hz) and C-1′. The absolute configuration at C-2′ of the 2-methylbutanoyloxy moiety remains to be determined.11 In a similar manner, the NMR data for compound 5 indicated that it has a methylpropanoyloxy group at C-12. Compound 6 was assigned a molecular formula of C42H53NO13 based on its 13C NMR and HRESIMS data. Comparison of the 1D NMR data of 6 (Tables 1 and 3) with those of 4 showed that they have similar structures, except for the presence of an extra 4-hydroxy-2,6-dimethoxyphenyl group linked to nitrogen in 6. This deduction was confirmed by the observations that the carbon resonances for C-21 (δC 171.8) and C-23 (δC 52.5) in 6 were shifted upfield and downfield by 2.5 and 6.3 ppm, respectively, compared to those of 4, but were similar to those of 2 and 3. Therefore, the structure of 6 was established as shown and named amooramide F. The molecular formula of amooramide G (7) was determined to be C35H39NO10 by its 13C NMR and HRESIMS data. The NMR data of 7 (Tables 2 and 3) revealed that it possessed a similar structure to 1, except that the acetoxy group attached to C-11 in 1 is replaced with a formyloxy group (δH 8.01; δC 160.1) in 7. This assignment was supported by the HMBC correlations between the resonances of the formyloxy group and C-11 and H-11. The NOESY spectrum showed that the relative configuration of 7 is identical to that of 1. HRESIMS analysis revealed that compound 8 has the same molecular formula (C36H41NO10) as amooramide A (1). The 985
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Table 3. 13C NMR Data of Compounds 1−12 (in CDCl3, 150 MHz, δ in ppm)a position 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 28 29 30 7-OMe 11-acyl 12-acyl-1′ 2′ 3′ (7′) 4′ (6′) 5′
1
2
3
4
5
6
7
8
9
10
11
12
148.7 122.1 166.8 83.7 50.0 35.0 173.6 136.4 53.4 46.3 71.0 75.3 46.3 71.0 60.3 29.2 40.4 13.6 22.7 135.5 174.3 142.7 45.9 30.2 22.4 121.4 52.4 170.5 20.3 165.2 129.7 129.5 128.4 133.0
148.8 122.1 166.9 83.7 50.0 35.0 173.6 136.2 53.4 46.2 70.9 75.4 46.2 71.0 60.3 29.0 40.7 13.6 22.7 136.4 170.9 139.3 52.0 30.2 22.4 121.4 52.4 170.5 20.3 165.2 129.8 129.6 128.3 132.9
148.5 122.2 166.7 83.6 50.0 35.0 173.6 136.2 53.4 46.3 71.1 75.2 46.3 71.0 60.1 29.4 40.4 13.7 22.7 136.5 172.2 140.1 51.9 30.3 22.4 121.4 52.4 170.5 20.3 164.9 129.9 129.6 128.4 132.9
148.6 122.0 166.7 83.6 50.1 35.0 173.6 136.6 53.2 46.2 71.2 74.2 46.1 71.2 60.1 30.2 40.0 13.6 22.6 136.5 174.3 142.3 46.2 30.2 22.4 121.1 52.4 170.5 20.7 174.6 40.9 26.0 11.8 15.7
148.4 122.1 166.6 83.5 50.1 35.0 173.5 136.6 53.2 46.2 71.1 74.2 46.1 71.2 60.1 29.9 40.2 13.5 22.6 136.5 173.7 142.2 45.9 30.2 22.4 121.1 52.4 170.4 20.6 175.0 34.0 18.6 18.9
148.7 122.0 166.7 83.6 50.1 35.0 173.6 136.5 53.3 46.6 71.2 74.4 46.2 71.1 60.4 29.0 41.8 13.5 22.6 136.3 171.8 142.0 52.5 30.3 22.4 121.1 52.4 170.5 20.7 174.7 41.2 26.0 11.9 15.5
148.3 122.6 166.8 83.6 50.6 35.0 173.5 136.2 53.4 46.2 70.4 75.0 46.6 71.0 60.4 28.9 40.9 13.6 22.9 135.2 173.8 143.2 45.5 30.2 22.4 121.6 52.4 160.1
148.1 122.3 166.5 83.6 50.0 34.9 173.5 135.9 52.8 46.2 70.9 75.2 46.2 71.4 59.5 32.8 42.4 13.3 22.8 159.9 50.9 124.9 173.9 30.2 22.3 122.0 52.4 170.4 20.1 165.4 128.9 129.7 128.8 132.7
148.1 122.2 166.5 83.6 50.0 34.9 173.5 135.9 53.0 46.4 70.7 73.8 46.2 71.0 59.1 33.6 41.9 13.5 22.7 160.2 51.2 124.4 174.4 30.2 22.4 121.8 52.4 170.4 20.7 175.1 41.1 26.0 11.7 16.1
148.1 122.3 166.5 83.6 50.0 34.9 173.5 135.9 53.0 46.2 70.7 73.9 46.4 70.9 59.1 33.5 41.9 13.4 22.7 159.9 50.8 124.6 174.0 30.2 22.4 121.8 52.4 170.3 20.6 175.6 34.2 18.5 19.0
148.1 122.4 166.6 83.7 50.0 35.0 173.5 136.0 53.0 46.2 70.9 75.1 46.2 71.5 59.6 32.4 42.1 13.4 22.8 155.9 57.3 125.5 170.9 30.2 22.4 122.0 52.4 170.4 20.2 165.3 129.2 129.6 128.8 133.7
148.1 122.2 166.5 83.6 50.0 34.9 173.5 136.0 53.0 46.3 70.8 73.8 46.2 71.0 59.2 33.2 41.6 13.4 22.7 156.1 57.3 125.1 171.2 30.2 22.4 121.8 52.4 170.4 20.7 175.1 41.1 26.0 11.7 16.2
165.1 129.6 129.7 128.4 133.1
a N-CH3 (29.0) in 2, N-CH2CH2OH (46.2, C-1″; 62.0, C-2″) in 3, N-4-hydroxy-2,6-dimethoxyphenyl (106.1, C-1″; 156.9, C-2″; 92.9, C-3″; 158.4, C-4″; 93.0, C-5″;157.3, C-6″; 55.4, 2″-OMe; 55.6, 6″-OMe) in 6, N-CH3 (28.2) in 11, N-CH3 (28.7) in 12.
for the presence of an additional methyl group [δC 28.2; δH 2.43 (3H, s)]. The extra N-methyl group in 11 was defined by HMBC correlations from the N-CH3 group to C-21 (δC 57.3) and C-23 (δC 170.9). The structure of amooramide K (11) was thus determined to be N-methylamooramide H. Amooramide L (12) has the molecular formula C35H47NO10 based on its 13C NMR and HRESIMS data. Comparison of the NMR data of 12 (Tables 2 and 3) with those of 9 revealed that 12 contained an additional methyl group [δC 28.7; δH 2.94 (3H, s)]. The HMBC correlations from N-CH3 (δH 2.94, s) to C-21 (δC 57.3) and C-23 (δC 171.2) suggested that the methyl group was located at the nitrogen. On the basis of these results, the structure of amooramide L (12) was determined to be Nmethylamooramide I. Although four limonoids carrying a 3-substituted 1,5dihydro-2H-pyrrol-2-one moiety at C-17 and a limonoid bearing a 4-substituted 1,5-dihydro-2H-pyrrol-2-one unit have previously been reported from plants belonging to the Meliaceae family,2−5 amooramides A−L represent the first examples of A,B-seco-limonoids bearing unusual lactam side chains at C-17, including the 3-substituted 1,5-dihydro-2Hpyrrol-2-one in 1−7 and the 4-substituted 1,5-dihydro-2Hpyrrol-2-one in 8−12. These unusual C-17 lactam-bearing
limonoids further demonstrated the structural diversity of plant limonoids. Since these N-bearing A,B-seco-limonoids were isolated from the genus Amoora for the first time, they may be used as chemotaxonomic markers for this genus. Previous studies indicated that limonoids possess a wide range of biological activities, including insect antifeeding, antimicrobial, antiprotozoal, anti-inflammatory, antioxidant, and anticancer activities.13−17 Nuclear factor kappaB (NF-κB) is a key transcription factor that is responsible for regulating inflammation, innate immune responses, and cancer development.18,19 Abnormal NF-κB signaling is associated with a variety of different diseases, and NF-κB inhibitors from natural products could be used as potential anti-inflammatory and anticancer drug candidates. Prompted by continuing interest in the development of NF-κB inhibitors from natural products,20 we evaluated the NF-κB inhibitory activities of the major compounds 1, 2, 4, 8, and 9 using a luciferase-based NF-κB reporter gene assay.21 As shown in Figure 3, amooramide I (9) significantly inhibited the TNFα-induced NF-κB luciferase activity by 64% at a concentration of 10 μM, whereas the remaining compounds (1, 2, 4, and 8) were inactive. Furthermore, none of the tested compounds 1, 2, 4, 8, and 9 exhibited considerable cytotoxic activity toward the HepG2 cell 986
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Figure 1. Key 1H−1H COSY and HMBC correlations of compounds 1 and 8.
Figure 2. Selected NOESY correlations of compounds 1 and 8.
line at 10 μM (data not shown). These results suggest that compound 9 is a potential NF-κB inhibitor. It is conceivable that additional biological activities of amooramides will be found by using other bioassay models, and therefore these amooramides will be useful for investigating the structure− activity relationship of limonoids in future studies.
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measured on an Agilent 6230 Accurate-Mass TOF-LC/MS system. UHPLC analyses were carried out on an Agilent 1290 Infinity LC system using an Extend-C18 column (1.8 μm, 50 × 2.1 mm, i.d., Agilent). Semipreparative and preparative HPLC were conducted on the LabAlliance system using YMC-Pack ODS-A (10 μm, 250 × 10 mm, i.d., YMC) and VisionHT C18 Polar (5 μm, 250 × 22 mm, i.d., Grace) columns, with a gradient solvent system composed of H2O and CH3CN and flow rates of 4.0 and 12.0 mL/min, respectively. MPLC was conducted on the Sepacore Flash Chromatography System (Buchi, Switzerland) using a Siliabond C18 ODS column (40−63 μm, 460 × 36 mm, i.d., Silicycle, Canada). TLC was performed on plates precoated with silica gel 60 F254 (Merck) and reversed-phase RP-18 F254 (Merck), and the spots were visualized by spraying the plates with a 10% (v/v) solution of H2SO4 in EtOH, followed by heating. Column chromatography (CC) was carried out with silica gel (40−63 μm, Grace, USA) as packing material. All solvents were spectroscopic grade and purchased from Labscan Asia (Bangkok, Thailand) or distilled prior to use.
EXPERIMENTAL SECTION
General Experimental Procedures. Optical rotations were obtained on a Rudolph Research Analytical Autopol I automatic polarimeter (Na 589 nm). UV spectra were recorded on a Beckman Coulter DU800 spectrophotometer. IR spectra were obtained on an Agilent Cary 600 series FT-IR spectrometer (KBr). NMR spectra were recorded on a Bruker Ascend 600 NMR spectrometer (600 MHz for 1 H and 150 MHz for 13C) using standard Bruker pulse programs. The chemical shift (δ) values are presented in ppm relative to TMS, and coupling constants (J) are presented in Hz. HRESIMS spectra were 987
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1251, 1223, 1076, 1040, 928, 824, 637, 598; 1H and 13C NMR spectroscopic data, see Tables 1 and 3; HRESIMS m/z 628.3114 [M + H]+ (calcd for C34H46NO10, 628.3116), 650.2934 [M + Na]+ (calcd for C34H45NO10Na, 650.2936). Amooramide E (5): white, amorphous powder; [α]25D +140 (c 0.2, MeOH); UV (MeOH) λmax (log ε) 223 (4.24) nm; IR (KBr) νmax 3440, 2963, 2931, 1745, 1692, 1636, 1454, 1371, 1252, 1223, 1126, 1075, 1041, 931, 834, 751, 598 cm−1; 1H and 13C NMR spectroscopic data, see Tables 1 and 3; HRESIMS m/z 614.2957 [M + H]+ (calcd for C33H44NO10, 614.2960), 636.2776 [M + Na]+ (calcd for C33H43NO10Na, 636.2779). Amooramide F (6): white, amorphous powder; [α]25D +73 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 218 (4.32) nm; IR (KBr) νmax 3443, 2959, 2927, 2855, 1740, 1691, 1600, 1516, 1454, 1384, 1260, 1221, 1128, 1040, 1004, 929, 821, 716, 638 cm−1; 1H and 13C NMR spectroscopic data, see Tables 1 and 3; HRESIMS m/z 780.3590 [M + H]+ (calcd for C42H54NO13, 780.3590), 802.3410 [M + Na]+ (calcd for C42H53NO13Na, 802.3409). Amooramide G (7): white, amorphous powder; [α]25D +89 (c 0.2, MeOH); UV (MeOH) λmax (log ε) 222 (4.26), 273 (2.75) nm; IR (KBr) νmax 3433, 2956, 2926, 2857, 1732, 1690, 1632, 1602, 1451, 1385, 1273, 1167, 1126, 1071, 1027, 823, 712 cm−1; 1H and 13C NMR spectroscopic data, see Tables 2 and 3; HRESIMS m/z 634.2645 [M + H]+ (calcd for C35H40NO10, 634.2647), 656.2466 [M + Na]+ (calcd for C35H39NO10Na, 656.2466). Amooramide H (8): white, amorphous powder; [α]25D +82 (c 1, MeOH); UV (MeOH) λmax (log ε) 218 (4.21), 280 (2.42) nm; IR (KBr) νmax 3420, 2989, 2956, 1727, 1695, 1451, 1371, 1311, 1273, 1220, 1126, 1072, 1041, 1000, 926, 822, 713, 598 cm−1; 1H and 13C NMR spectroscopic data, see Tables 2 and 3; HRESIMS m/z 648.2812 [M + H]+ (calcd for C36H42NO10, 648.2803), 670.2626 [M + Na]+ (calcd for C36H41NO10Na, 670.2623). Amooramide I (9): white, amorphous powder; [α]25D +73 (c 1, MeOH); UV (MeOH) λmax (log ε) 213 (4.43) nm; IR (KBr) νmax 3405, 2970, 1735, 1696, 1458, 1414, 1371, 1252, 1220, 1126, 1078, 1041, 927, 864, 822, 682, 598 cm−1; 1H and 13C NMR spectroscopic data, see Tables 2 and 3; HRESIMS m/z 628.3123 [M + H]+ (calcd for C34H46NO10, 628.3116), 650.2937 [M + Na]+ (calcd for C34H45NO10Na, 650.2936). Amooramide J (10): white, amorphous powder; [α]25D +81 (c 0.2, MeOH); UV (MeOH) λmax (log ε) 214 (4.33) nm; IR (KBr) νmax 3422, 2976, 2930, 1738, 1693, 1637, 1438, 1372, 1252, 1221, 1126, 1075, 1040, 930, 821, 681, 597 cm−1; 1H and 13C NMR spectroscopic data, see Tables 2 and 3; HRESIMS m/z 614.2954 [M + H]+ (calcd for C33H44NO10, 614.2960), 636.2774 [M + Na]+ (calcd for C33H43NO10Na, 636.2779). Amooramide K (11): white, amorphous powder; [α]25D +111 (c 0.6, MeOH); UV (MeOH) λmax (log ε) 227 (4.28) nm; IR (KBr) νmax 3445, 2988, 2954, 1730, 1697, 1451, 1371, 1271, 1221, 1126, 1041, 926, 832, 714, 597, 458 cm−1; 1H and 13C NMR spectroscopic data, see Tables 2 and 3; HRESIMS m/z 662.2961 [M + H]+ (calcd for C 37 H 44 NO 10 , 662.2960), 684.2774 [M + Na] + (calcd for C37H43NO10Na, 684.2779). Amooramide L (12): white, amorphous powder; [α]25D +56 (c 0.5, MeOH); UV (MeOH) λmax (log ε) 224 (4.30) nm; IR (KBr) νmax 3444, 2971, 1736, 1707, 1439, 1373, 1255, 1222, 1127, 1079, 1042, 823, 679, 600 cm−1; 1H and 13C NMR spectroscopic data, see Tables 2 and 3; HRESIMS m/z 642.3287 [M + H]+ (calcd for C35H48NO10, 642.3273), 664.3101 [M + Na]+ (calcd for C35H47NO10Na, 664.3092). Cell Line and NF-κB Luciferase Assay. HepG2-NF-κB-Luc cells, which had been transfected with the NF-kB-luciferase gene, were kindly provided by Dr. C. H. Leung (University of Macau). The cells were cultured in DMEM medium (Invitrogen) supplemented with 100 U/mL penicillin, 100 μg/mL streptomycin, and 10% fetal bovine serum at 37 °C in a humidified atmosphere of 5% CO2 and 95% air. Inhibition of NF-κB activation was determined using a NF-κB luciferase assay according to a modified version of a previously described method.21 Briefly, HepG2-NF-κB-Luc cells were seeded overnight at 5 × 104 cells/well in a 24-well plate. The cells were preincubated with test compounds at 10 μM for 1 h and cotreated
Figure 3. NF-κB luciferase assay of selected compounds. HepG2-NFκB-Luc cells were pretreated with selected compounds at 10 μM for 1 h and then simulated with TNFα (10 ng/mL) for 4 h. The cell lysates were analyzed for luciferase activity to determine the extent of NF-κB inhibition. Data are expressed as mean ± SEM (n = 3). **p < 0.01, compared with TNFα treatment. Plant Material. The twigs and leaves of A. tsangii were collected from Hainan Island, Hainan Province, China, in May 2012. The species was authenticated by Qiong-Xin Zhong, who is an associate professor at the College of Life Sciences, Hainan Normal University. A voucher specimen (AT-201205) was deposited at the State Key Laboratory of Quality Research in Chinese Medicine, Macau University of Science and Technology. Extraction and Isolation. Air-dried twigs and leaves of A. tsangii (4.0 kg) were extracted with 80% EtOH (2 × 40 L) under reflux. The combined extracts were concentrated under reduced pressure to afford a brown residue (∼780 g), which was suspended in H2O (4 L) and sequentially extracted with petroleum ether (3 × 4 L) and EtOAc (3 × 4 L) to give the petroleum ether-soluble (50 g) and EtOAc-soluble (90 g) fractions. The EtOAc extract (80 g) was subjected to silica gel CC eluting with petroleum ether−acetone (100:0 → 50:50, v/v) to obtain fractions A−G. Fraction F (10 g) was repeatedly chromatographed on a reversed-phase RP-18 column eluting with MeOH−H2O (20:80 → 100:0, v/v) and further purified by semipreparative and preparative HPLC eluting with MeCN−H2O (50:50 → 60:40, v/v) to yield 1 (30.1 mg), 2 (8.7 mg), 3 (1.5 mg), 4 (13.8 mg), 6 (0.5 mg), 7 (0.5 mg), 10 (0.8 mg), 11 (1.1 mg), and 12 (1.2 mg). Fraction G (6.5 g) was separated in a similar manner to fraction F to afford 5 (0.6 mg), 8 (10.5 mg), and 9 (9.0 mg). Amooramide A (1): white, amorphous powder; [α]25D +81 (c 0.5, MeOH); UV (MeOH) λmax (log ε) 222 (4.16) nm; IR (KBr) νmax 3424, 2987, 2956, 1730, 1690, 1450, 1372, 1314, 1273, 1221, 1174, 1125, 1042, 926, 824, 713, 600 cm−1; 1H and 13C NMR spectroscopic data, see Tables 1 and 3; HRESIMS m/z 648.2811 [M + H]+ (calcd for C36H42NO10, 648.2803), 670.2633 [M + Na]+ (calcd for C36H41NO10Na, 670.2623). Amooramide B (2): white, amorphous powder; [α]25D +122 (c 1, MeOH); UV (MeOH) λmax (log ε) 231 (4.26) nm; IR (KBr) νmax 3457, 2989, 2953, 1746, 1690, 1451, 1372, 1272, 1221, 1175, 1125, 1070, 1041, 1000, 924, 822, 712, 579 cm−1; 1H and 13C NMR spectroscopic data, see Tables 1 and 3; HRESIMS m/z 662.2969 [M + H]+ (calcd for C37H44NO10, 662.296), 679.3229 [M + NH3]+ (calcd for C37H47N2O10, 679.3225), 684.2790 [M + Na]+ (calcd for C37H43NO10Na, 684.2779). Amooramide C (3): white, amorphous powder; [α]25D +127 (c 0.4, MeOH); UV (MeOH) λmax (log ε) 229 (4.26) nm; IR (KBr) νmax 3442, 2987, 2954, 2932, 1745, 1686, 1637, 1454, 1411, 1372, 1315, 1274, 1219, 1175, 1125, 1070, 1041, 924, 822, 713, 589 cm−1; 1H and 13 C NMR spectroscopic data, see Tables 1 and 3; HRESIMS m/z 692.3057 [M + H]+ (calcd for C38H46NO11, 692.3065), 714.2884 [M + Na]+ (calcd for C38H45NO11Na, 714.2885). Amooramide D (4): white, amorphous powder; [α]25D +83 (c 1, MeOH); UV (MeOH) λmax (log ε) 220 (4.24) nm; IR (KBr) νmax (cm−1) 3407, 2971, 2882, 1736, 1695, 1642, 1456, 1414, 1371, 1309, 988
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with TNFα (10 ng/mL) for 4 h. The firefly luciferase activities were measured using a Bright-Glo Luciferase Reporter Assay System (Promega) according to the manufacturer’s protocol using a multimode reader (Infinite 200 PRO, Tecan).
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(21) Leung, C. H.; Chan, D. S.; Kwan, M. H.; Cheng, Z.; Wong, C. Y.; Zhu, G. Y.; Fong, W. F.; Ma, D. L. ChemMedChem. 2011, 6, 765− 768.
ASSOCIATED CONTENT
S Supporting Information *
This material (HRESIMS, 1D and 2D NMR spectra of compounds 1−12) is available free of charge via the Internet at http://pubs.acs.org.
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AUTHOR INFORMATION
Corresponding Authors
*Phone: +853-88972403. Fax: +853-28825886. E-mail: lpbai@ must.edu.mo. *Phone: +853-88972777. Fax: +853-28825886. E-mail:
[email protected]. Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS We are grateful to Macao Science and Technology Development Fund, MSAR (063/2011/A3 and 039/2011/A2), for financial support.
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REFERENCES
(1) Tan, Q. G.; Luo, X. D. Chem. Rev. 2011, 111, 7437−522. (2) Kraus, W.; Klenk, A.; Bokel, M.; Vogler, B. Liebigs Ann. Chem. 1987, 4, 337−340. (3) Cheplogoi, P. K.; Mulholland, D. A. Phytochemistry 2003, 62, 1173−1178. (4) Qi, S. H.; Chen, L.; Wu, D. G.; Ma, Y. B.; Luo, X. D. Tetrahedron 2003, 59, 4193−4199. (5) Qi, S. H.; Wu, D. G.; Chen, L.; Ma, Y. B.; Luo, X. D. J. Agric. Food Chem. 2003, 51, 6949−6952. (6) Wu, J.; Zhang, S.; Bruhn, T.; Xiao, Q.; Ding, H.; Bringmann, G. Chem.−Eur. J. 2008, 14, 1129−1144. (7) Cui, J.; Ouyang, J.; Deng, Z.; Lin, W. Magn. Reson. Chem. 2008, 46, 894−897. (8) Wang, X. N.; Yin, S.; Fan, C. Q.; Wang, F. D.; Lin, L. P.; Ding, J.; Yue, J. M. Org. Lett. 2006, 8, 3845−3848. (9) Zhang, X. Y.; Li, Y.; Wang, Y. Y.; Cai, X. H.; Feng, T.; Luo, X. D. J. Nat. Prod. 2010, 73, 1385−1388. (10) Chen, W. Y.; Chang, C. C.; Chen, F. H. In Flora Hainanica (Hainan Zhiwu Zhi); Science Press: Beijing, 1974; Vol. 3, pp 66−67. (11) Chen, H. D.; Yang, S. P.; Liao, S. G.; Zhang, B.; Wu, Y.; Yue, J. M. J. Nat. Prod. 2008, 71, 93−97. (12) Lin, B. D.; Chen, H. D.; Liu, J.; Zhang, S.; Wu, Y.; Dong, L.; Yue, J. M. Phytochemistry 2010, 71, 1596−1601. (13) Fowles, R. G.; Mootoo, B. S.; Ramsewak, R. S.; Reynolds, W. F.; Nair, M. G. Nat. Prod. Commun. 2011, 6, 785−787. (14) Alam, A.; Haldar, S.; Thulasiram, H. V.; Kumar, R.; Goyal, M.; Iqbal, M. S.; Pal, C.; Dey, S.; Bindu, S.; Sarkar, S.; Pal, U.; Maiti, N. C.; Bandyopadhyay, U. J. Biol. Chem. 2012, 287, 24844−24861. (15) Ge, Y. H.; Liu, K. X.; Zhang, J. X.; Mu, S. Z.; Hao, X. J. J. Agric. Food Chem. 2012, 60, 4289−4295. (16) Obbo, C. J.; Makanga, B.; Mulholland, D. A.; Coombes, P. H.; Brun, R. J. Ethnopharmacol. 2013, 147, 220−223. (17) Patwardhan, C. A.; Fauq, A.; Peterson, L. B.; Miller, C.; Blagg, B. S.; Chadli, A. J. Biol. Chem. 2013, 288, 7313−7325. (18) Perkins, N. D. Nat. Rev. Cancer 2012, 12, 121−132. (19) Shih, V. F.; Tsui, R.; Caldwell, A.; Hoffmann, A. Cell Res. 2011, 21, 86−102. (20) Tse, A. K.; Wan, C. K.; Zhu, G. Y.; Shen, X. L.; Cheung, H. Y.; Yang, M.; Fong, W. F. Mol. Immunol. 2007, 44, 2647−2658. 989
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