Article pubs.acs.org/jnp
Cytotoxic Dammarane-Type Triterpenoids from the Stem Bark of Dysoxylum binecteriferum Hui-Jiao Yan, Jun-Song Wang, and Ling-Yi Kong* State Key Laboratory of Natural Medicines, Department of Natural Medicinal Chemistry, China Pharmaceutical University, 24 Tong Jia Xiang, Nanjing 210009, People’s Republic of China S Supporting Information *
ABSTRACT: Fourteen new dammarane-type triterpenoids (1−14) and 11 known analogues were isolated from the stem bark of Dysoxylum binecteriferum. The absolute configurations were established by comparison with the literature or by Mo2(OAc)4induced electronic circular dichroism data. All isolates were evaluated for their cytotoxicities against three human cancer cell lines as well as their inhibitory effects on lipopolysaccharide-induced nitric oxide production in RAW264.7 cells. Compounds 4 and 8 displayed moderate cytotoxicities against HepG2 with IC50 values of 6.5 and 8.0 μM, respectively.
T
he genus Dysoxylum (Meliaceae) comprises approximately 75 species that are mainly distributed in India, Southeast Asia, Australia, and New Zealand.1 Many of these species have been used in folk medicine for the treatment of fever, convulsions, hemorrhage, rigid limbs, or facial distortion in children.2,3 Previous investigations had led to the isolation of a diverse range of bioactive secondary metabolites, such as antifeeding limonoids,4 cytotoxic alkaloids,5 and antibacterial and cytotoxic triterpenoids.6 Dysoxylum binecteriferum, a timber species, primarily found in the tropical areas of southern mainland China,1 has rarely been investigated. To continue our research on bioactive constituents, 14 new and 11 known dammarane-type triterpenoids were isolated from the stem bark of D. binecteriferum. Herein, the isolation and structural elucidation of these isolates, as well as their cytotoxic and anti-inflammatory properties, are described.
methyl groups (δH 0.70, 0.89, 0.91, 0.99, 1.11, 1.15, and 1.19, each 3H, s) were observed in the 1H NMR spectrum. These data corresponded to a dammarane triterpene skeleton similar to that of cabraleadiol,7 which was also isolated in this study. The gross structure of 1 was deduced from the HMBC spectrum (Figure 1a). The HMBC correlations between the tertiary methyl protons and their neighboring carbons were used to establish the dammarane backbone. All expected correlations (Me-18→C-8, 7, 9, 14; Me-19→C-10, 1, 5, 9; and Me-30→C-14, 8, 13, 15) displayed strong cross-peaks. Furthermore, the HMBC crosspeaks from the oxygenated methine protons at δH 3.68 (brs, W1/2 = 6.5 Hz, H-3) to carbons at δC 33.7 (C-1), 43.9 (C-5), and 18.0 (C-29), from the geminal protons at δH 3.40 and 3.54 (each d, J = 11.5 Hz, H2-28) to carbons at δC 40.9 (C-4), 43.9 (C-5), 77.3 (C-3), and 18.0 (C-29), and from the remaining methyl protons at δH 0.70 (H3-29) to carbons at δC 40.9 (C-4), 43.9 (C-5), 71.7 (C-28), and 77.3 (C-3) located two hydroxy groups at C-3 and C-28. The characteristic NMR data [δC 86.8 (C-20, s), δC 86.6 (C24, d); δH 3.64 (1H, dd, J = 5.5, 10.0 Hz, H-24)] suggested the presence of a 20,24-epoxy moiety and hence a tetrahydrofuran ring in the side chain. The gem-dimethyl protons at δH 1.19 (s,
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RESULTS AND DISCUSSION Compound 1 was assigned a molecular formula of C30H52O4, as deduced from its positive HRESIMS (found [M + Na]+ m/z 499.3757, calcd 499.3758) and 13C NMR data, indicative of five indices of hydrogen deficiency. The 13C NMR (Table 1) data showed 30 carbon resonances, which were classified by the HSQC spectrum as seven methyl, 11 methylene (one oxygenated), six methine (two oxygenated), and six quaternary carbons (two oxygenated). The five indices of hydrogen deficiency suggested that 1 was pentacyclic. Seven tertiary © 2014 American Chemical Society and American Society of Pharmacognosy
Received: August 27, 2013 Published: February 18, 2014 234
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Table 1. 1H (500 MHz) and 13C NMR (125 MHz) Data of Compounds 1−5 in CDCl3 1 position 1a
1.46, m 1.33 1.95, m
2b
1.47, m
3
3.68, brs (W1/2 = 6.5)
25 26 27 28a 28b 29a 29b 30
2 δC
1.67a 1.39, m 1.64, m 1.27, m 1.53, m 1.55, 1.22, 1.25, 1.63,
m m m m
1.48, m 1.08, m 1.78, m 1.33a 1.86, m 0.99, s 0.89, s 1.15, s 1.88, m 1.67a 1.81, m 3.64, dd (10.0, 5.5) 1.19, s 1.11, s 3.54, d (11.5) 3.40 d (11.5) 0.70, s 0.91, s
δH (J in Hz) a
33.7
1.45
26.1
1.30, m 1.94, m
a
1b 2a
4 5 6 7a 7b 8 9 10 11a 11b 12 13 14 15a 15b 16a 16b 17 18 19 20 21 22a 22b 23a 23b 24
a
δH (J in Hz)
3 δH (J in Hz)
δC
33.7
1.99, ddd (13.5, 7.0, 3.0) 1.39, m 2.62, ddd (16.0, 12.0, 7.5) 2.30, ddd (16.0, 6.0, 3.0)
39.8
26.0
1.45a 77.3 40.9 43.9 18.3 35.2 40.9 51.0 37.4 22.0 27.3 43.1 50.5 31.7 26.7 50.1 15.8 16.6 86.8 27.4 35.1 26.8 86.6 70.6 28.1 24.3 71.7
3.68, brs (W1/2 = 6.4) 1.66, m 1.38, m 1.63a 1.27, m 1.51, m 1.55, 1.21, 1.26, 1.58,
m m m m
1.46, m 1.08, m 1.80a 1.45a 1.81, m 0.97, s 0.88, s 1.13, s 1.70, m 1.63a 1.85, m 1.80a 3.73, dd (7.0, 7.0)
18.0
1.21, 1.12, 3.54, 3.40, 0.70,
s s d (11.5) d (11.5) s
16.8
0.91, s
4
δC
77.3 40.9 43.9 18.3 35.2 40.9 51.0 37.4 21.8 27.7 43.2 50.5 31.7 26.4 49.8 15.7 16.6 86.7 23.9 35.9
1.65, 1.41, 1.60, 1.30,
52.8 49.8 19.4 34.9
m m m m
40.7 50.4 37.1 22.2
1.49, m 1.55, 1.28, 1.27, 1.61,
m m m m
1.47a 1.11, m 1.78, m 1.47a 1.81, m 1.02, s 1.06, s
18.0
s s d (11.5) d (11.5) s
16.9
0.88, s
1.66, 1.40, 1.62, 1.32,
26.4
1.15, 1.87, 1.67, 1.84,
s m m m
83.6
3.64, dd (10.0, 5.5)
16.7
1.19, 1.11, 3.64, 3.42, 1.02,
39.9
35.6
1.58, m 2.59, m,
34.5
2.38, m
16.8
0.88, s
27.4 43.2 50.4 31.7 26.1 50.1 16.3 15.7 86.8 27.2 35.1 26.6
s s d (11.5) d (11.5) s
δC
1.93, m
40.7 50.9 37.1 22.4
1.48, m
m m m m m s s
71.7 27.7 24.6 67.2
δH (J in Hz)
39.8
52.8 49.9 19.5 34.9
m m m m
1.49, 1.09, 1.78, 1.33, 1.88, 1.03, 1.07,
49.9 15.7 16.2 86.6 23.9 35.9
5 δC
219.3
m m m m
26.0
1.21, 1.12, 3.65, 3.42, 1.02,
71.7 27.7 24.6 71.7
1.41, m 2.63, ddd (15.5, 12.0, 7.0) 2.30, ddd (15.5, 6.0, 3.0)
1.56, 1.28, 1.26, 1.68,
27.6 43.3 50.4 31.7
s m m m m dd (7.0, 7.0)
83.6
2.00, m
219.2
1.14, 1.70, 1.63, 1.85, 1.80, 3.73,
26.8
35.6
δH (J in Hz)
86.7 70.6 28.0 24.3 67.2 16.9 16.3
221.6
1.63, 1.50, 1.53, 1.34,
m m m m
1.45, m 1.52, 1.24, 1.25, 1.68,
m m m m
1.47, 1.08, 1.77, 1.33, 1.89, 0.98, 0.89,
m m m m m s s
1.15, 1.87, 1.69, 1.84,
s m m m
3.64, dd (10.0, 5.5) 1.19, s 1.11, s 1.27, s 3.98, d (11.5) 3.45, d (11.5) 0.89, s
50.0 56.1 19.5 35.0 40.5 51.1 36.9 22.9 27.4 43.3 50.2 31.7 26.1 50.2 15.2 16.5 86.7 27.3 35.1 26.6 86.7 70.6 28.0 24.3 22.4 66.1 17.3
Signal pattern unclear due to overlapping.
H3-26) and 1.11 (s, H3-27) displayed cross-peaks with carbons at δC 86.6 (C-24) in the HMBC spectrum, placing the 2hydroxyisopropyl group [δC 70.6 (C-25), 28.1 (C-26), 24.3 (C27)] at C-24. Other HMBC correlations (Figure 1a) further confirmed the side chain structure. Thus, the gross structure of 1 was established as shown. With regard to the relative configurations, the characteristic broad H-3 singlet suggested that it was in an equatorial position and β-oriented. Other relative configurations of 1 were elucidated mainly by the ROESY spectrum (Figure 1b). The typically observed correlations of Me-30 (14α-Me)/H-17α, H9α/Me-30, H-5α/H-9, Me-19/Me-18 (8β-Me), Me-29 (4βMe)/Me-19 (10β-Me), Me-18/H-13β, and H-13β/Me-21
Figure 1. Selected HMBC (H→C) (a) and ROESY (dashed ↔) correlations (b) of 1.
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Chart 1
each d, J = 11.5 Hz) and Me-29 (δH 1.02, s) to C-3 (δC 219.2), from H-28a (δH 3.65) to C-5, and from H-28b (δH 3.42) to Me-29. The relative configuration of 3 was the same as that of 2 based on their similar ROESY correlations. The ROESY crosspeaks of H-28b/H-5α, H-2a (δH 2.62)/Me-19β, and H-2a/Me29 (4β-Me) suggested that the hydroxy group was located at C28 (4α-Me). Thus, the structure of 3 was defined as (20S,24R)epoxy-25, 28-dihydroxydammar-3-one. Compound 4 differed from 3 only in the configuration of C-24. The characteristic proton resonance at δH 3.64 (dd, J = 10.0, 5.5 Hz) and carbon resonances at δC 86.8 (s, C-20), 86.7 (d, C-24), 27.2 (q, C-21), 34.9 (t, C-22), and 70.6 (s, C-25) resulted in the assignment of the (20S, 24S) absolute configuration for 4.8,9 Accordingly, the structure of 4 was assigned as (20S,24S)-epoxy-25,28dihydroxydammar-3-one. The NMR data of 5 (Table 1) were generally similar to those of 4 except for the resonances corresponding to ring A. The similar HMBC correlations suggested that they shared the same gross structure. The ROESY cross-peaks between the oxygenated methylene proton H-29a (δH 3.98, d, J = 11.5 Hz) and Me-19β and between H-9α and Me-28 in 5 indicated the β-orientation of the hydroxymethyl group at C-4. Thus, the structure of 5 was defined as (20S,24S)-epoxy-25,29-dihydroxydammar-3-one. Compound 6 was assigned a molecular formula of C30H50O4 by its 13C NMR data and positive HRESIMS ion at m/z 497.3603 [M + Na]+ (calcd for C30H50O4Na, 497.3601). The 1 H and 13C NMR data of 6 (Table 2) displayed resonances typical of dammarane triterpenoids. The HMBC correlations from the proton at δH 3.83 (dd, J = 7.0, 7.0 Hz) to C-18 (δC 9.49 q, γ-gauche effect), C-6 (δC 29.0), and C-9 (δC 49.8) and from Me-18 (δH 1.02, s) to C-7 (δC 74.5) suggested the presence of a hydroxy group at C-7. The ROESY cross-peaks of Me-30 (δH 0.95)/H-7, H-5 (δH 1.48)/H-7, and H-9 (δH 1.34)/ H-7 revealed the α-orientation of H-7. These data indicated that the structure of 6 was similar to that of salvilymitone.11 The 13C NMR data of the epoxy ring, especially the resonances of C-20 (δC 86.5), C-21 (δC 26.9), C-22 (δC 34.8), and C-24 (δC 86.5), were highly similar to those of the (20S,24S)-epimer.
revealed that 1 had the same configurations as other reported dammarane triterpenoids,7 and the cross-peak of H-5α/H2-28 confirmed the α-orientation of the C-4 hydroxymethyl group. The stereochemistry of the substituted tetrahydrofuran moiety at C-17 of the triterpenoid skeleton has been thoroughly studied.7−9 The chemical shifts for C-21, C-22, and C-24 in compounds with the 20S and 24R configurations were approximately δC 23.6−23.9, 35.5−35.7, and 83.2, whereas those for C-21, C-22, and C-24 in compounds with 20S and 24S configurations were δC 27.0−27.2, 34.6−34.8, and 86.5.9 Thus, the C-20 and C-24 absolute configurations of 1 were both assigned as S according to the characteristic 13C NMR resonances of C-21 (δC 27.4), C-22 (δC 35.1), and C-24 (δC 86.6). Thus, the structure of compound 1 was assigned as (20S,24S)-epoxydammarane-3α,25,28-triol. Compound 2 had a molecular formula of C30H52O4, the same as that of 1, as determined by the 13C NMR data and the observed pseudomolecular ion at [M + Na]+ m/z 499.3751 (calcd for C30H52O4Na, 499.3758). Comparison of the NMR data of 1 and 2 (Table 1) revealed a considerable degree of similarity except for the side chain resonances. The large chemical shift discrepancy (Δδ −3.0) at C-24 suggested that 2 is a C-24 epimer of 1, which was supported by the ROESY correlation between Me-21 and H-24 in 2, not observed in 1. Moreover, the characteristic 13C NMR resonances of C-21 (δC 23.9), C-22 (δC 35.9), and C-24 (δC 83.6) indicated the 20S and 24R configurations. Thus, the structure of 2 was defined as (20S,24R)-epoxydammarane-3α,25,28-triol. Compounds 3−5 had the same molecular formula of C30H50O4, as indicated by their 13C NMR data and positive HRESIMS ion at m/z 475.3780, 475.3783, and 475.3785, respectively ([M + H]+, calcd for C30H51O4, 475.3782), 2 mass units less than those of 1 and 2. Comparison of their 1H and 13 C NMR data (Table 1) indicated that 3−5 were analogues of 1 and 2. The major difference between 3 and 2 was the presence of a carbonyl group (δC 219.2) at C-3 in 3 instead of the oxymethine group (δH 3.68, brs; δC 77.3) in 2. The proposed structure was confirmed by the HMBC correlations from H2-2 (2.62, ddd; 2.30, ddd), H2-28 (δH 3.42 and 3.65, 236
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Table 2. 1H (500 MHz) and 13C NMR (125 MHz) Data of Compounds 6−10 in CDCl3 6 position 1a 1b 2a 2b 3 4 5 6a 6b 7a 7b 8 9 10 11a 11b 12a 12b 13 14 15a 15b 16a 16b 17 18 19 20 21 22a 22b 23a 23b 24 25 26 27 28a 28b 29a 29b 30 COOMe a
δH (J in Hz) 1.91, m 1.43, m 2.46, m
1.48, m 1.64a 1.25, m 3.83, dd (7.0, 7.0)
7 δC 39.5
δH (J in Hz)
1.56, m 1.35, m 1.79, m 1.37, m 1.64a 1.70, m 1.33, m 1.86, m
1.45, m
40.1
25.7
2.50, m 2.42, ddd (10.5, 7.5, 4.5)
34.3
3.40, t (3.0) 1.25, m
76.5 37.5 49.8
29.0
1.41a
18.5
74.5
1.57, m
35.4
217.3 47.1 52.7
45.9 49.8 36.7 22.3 26.2 43.8 49.4 34.9 27.3
1.41a 1.52, 1.16, 1.84, 1.35, 1.46,
m m m m m
1.45, m 1.11, m 1.71, m
40.8 50.7 37.9 21.5 25.5 43.7 50.6 31.4
19.9 34.8
40.6 50.1 37.1 22.2
1.40, m 1.53, 1.26, 1.84, 1.37, 1.51,
m m m m m
25.6 43.9 50.5 31.3
1.98, m
49.3
0.99, s 0.94, s
16.2 15.5 89.2 27.7 39.7
1.45, s 7.38, d (5.5)
16.6 16.3 92.6 23.9 159.8
26.4
6.05, d (5.5)
121.4 172.7
1.47, 2.56, 1.93, 4.52,
27.0
s m, m dd (9.0, 8.0)
s
δH (J in Hz) a
1.54 1.43a 2.36, m 2.17, m
2.02, dd (13.0, 3.0) 1.86a 1.44, m 1.54a
10 δC
69.4 176.9
1.19, s 1.12, s 1.10, s
70.3 27.8 24.1 28.8
0.93, s
28.6
1.08, s
27.1
1.05, s
21.0
0.83, s
22.4
1.04, s
21.3
0.95, s
16.2
0.88, s
15.7
0.89, s
16.4
δH (J in Hz)
δC
35.2
1.60, m
34.8
28.8
2.34, m 2.18, m
28.7
174.9 151.7 46.6 26.2
1.97, dd (12.5, 3.0) 1.35, m
34.5
1.51, m
1.24a
47.7
9.5 16.0 86.5 26.9 34.8
86.5
1.56, m 1.49, m 1.58, m
27.3
1.02, s 0.93, s
3.64, dd (10.0, 5.5)
1.36, m
1.52, m 1.16, m 1.65, m
48.7
s m m m
2.05, td (10.5, 6.0) 0.90, s 0.83, s
217.9 47.7 55.6
1.33, m
1.84, m
1.16, 1.88, 1.70, 1.85,
9 δC
33.9
1.24, m 1.34, m
δH (J in Hz)
m m m m
33.9
1.40, 1.28, 1.94, 1.54,
8 δC
174.9 147.9 51.1 25.0 34.2
1.22, m
1.53, m 1.43a 1.24a 1.80a 1.12, m 1.60, m 1.47a 1.10, m 1.75, m 1.47a 1.80, m 1.00, s 0.86, s 1.13, s 1.69, m 1.63, m 1.86a 1.80a 3.72, dd (7.0, 7.0)
1.21, s 1.12, s 5.19, brs 4.92, brs 4.07, dd (21.5, 12.0) 0.88, s 3.66, s
40.3 41.5 39.4 22.5 27.5 43.3 50.7 31.7 25.9 49.8 15.6 20.0 86.6 23.8
1.52, m 1.40a 1.27, m 1.90, m 1.71, m 1.40a 1.04, m 1.83, m 1.50, m 1.76, m 1.01, s 0.85, s 1.17, s
40.4 41.5 39.4 22.6 28.0 42.6 51.1 31.7 25.6 51.5 15.6 20.4 76.5 26.3 26.4
36.1 26.4 83.6 71.7 27.7 24.6 112.4
1.99, m 1.38, m 3.43, dd (5.0, 2.5)
67.5
1.25, 1.16, 4.84, 4.66, 1.73,
s s brs brs s
16.6 51.9
0.88, s 3.67, s
23.7 71.3 74.3 28.5 26.9 113.6 23.5
16.8 51.8
Signal pattern unclear due to overlapping.
Thus, the structure of 6 was defined as (20S,24S)-epoxy-7β,25dihydroxydammar-3-one. Compound 7 was obtained as a white powder. The HRESIMS showed an [M + H]+ ion at m/z 415.3209, and together with the 13C NMR data, this indicated a molecular formula of C27H42O3. Resonances for two olefinic protons (δH 6.05, 7.38, each d, J = 5.5 Hz) and six methyl singlets (δH 0.90, 0.834, 0.93, 0.830, 0.88, and 1.45) in the 1H NMR spectrum and 27 carbon signals in the 13C NMR spectrum (Table 2)
suggested that 7 possessed a trinortriterpenoid skeleton similar to that of cabraleahydroxylactone (3α-hydroxy-25,26,27trinordammar-24,20α-olide),12 which was also isolated in this investigation. However, the main differences occurred through the formation of a double bond between C-22 and C-23 in 7. The HMBC cross-peaks (Figure 2) from Me-21 (δH 1.45) to the oxygenated quaternary C-20 (δC 92.6) and the olefinic C-22 (δC 159.8) and from the olefinic proton H-23 (δH 6.05) to the lactone carbon (δC 172.7) indicated the presence of an α,β237
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deficiency indicated that 9 is tetracyclic, suggesting the cleavage of ring A. The NMR spectroscopic data of 9 are similar to those of methyl shoreate [methyl (20S,24S)-epoxy-25-hydroxy-3,4secodammar-4(28)-en-3-oate],9,14 which was also isolated in this investigation. The difference between the two compounds is the presence of an oxymethylene in 9 instead of a tertiary methyl group in methyl shoreate. The HMBC correlations (Figure 3a) from H2-28 to C-5 (δC 46.6, d) and C-29 (δC 67.5, t) and from H2-29 to C-4 (δC 151.7, s) and C-28 (δC 112.4, t) confirmed hydroxylation at C-29. Compound 9 shared the same relative configurations as those of other shoreic acid-type C-23 compounds (Figure 3b). The (20S,24R)-epoxide configurations of 9 were assigned on the basis of the characteristic chemical shifts of the carbon resonances of C-21 (δC 23.8 ppm), C-22 (δC 36.1 ppm), and C-24 (δC 83.6 ppm).9 Hence, the structure of 9 was defined as methyl (20S,24S)-epoxy-25,29-dihydroxy-3,4-secodammar4(28)-en-3-oate. Compound 10 had a molecular formula of C31H52O4, as deduced from its positive HRESIMS (found [M + H]+ m/z 489.3939, calcd 489.3938) and 13C NMR data, which indicated five indices of hydrogen deficiency. The 1H and 13C NMR data of 10 (Table 2) resembled those of 3,4-secodammarane methyl shoreate,9,14 especially by the presence of resonances for a typical exocyclic double bond [δH 4.66 (brs), 4.84 (brs); δC 147.9 (C-4), 113.6 (C-28)]; an ester carbonyl carbon (δC 174.9, C-3); a methoxy group [δH 3.67 (s); δC 51.8]; and six tertiary methyl groups (δH 1.01, 0.85, 1.17, 1.25, 1.16, and 0.88). The NMR spectra of 10 displayed an oxygenated methine [δH 3.43 (dd, J = 5.0, 2.5 Hz), δC 71.3 (C-24)] and two oxygenated quaternary carbons [δC 76.5 (C-20), 74.3 (C25)], which suggested the presence of a six-membered pyran instead of a tetrahydrofuran ring at C-17. The HMBC correlations from Me-21 (δH 1.17, s) to C-17 (δC 51.5), C20 (δC 76.5), and C-22 (δC 26.4) and from H-24 to C-22 (δC 26.4), C-23 (δC 23.7), and C-27 (δC 26.9) confirmed the presence of a 3-hydroxytetrahydropyranyl group at C-17.15 The ROESY correlations of H-24 with Me-26 and Me-27, together with the small coupling constant of H-24, suggested that it was β-equatorially oriented. The S configuration was assigned to C20, as is common in dammarane triterpenoids, especially those isolated in this investigation. Thus, the structure of 10 was defined as methyl (20S,25)-epoxy-24α-hydroxy-3,4-secodammar-4(28)-en-3-oate. Compound 11, an oil, had a molecular formula of C32H56O6 based on its positive HRESIMS ion at m/z 559.3972 [M + Na]+ and the 13C NMR data, which together indicated five indices of hydrogen deficiency. Comparison of its NMR data with those of 9 revealed similar features in rings B−D and the side chain. The HMBC correlations from the methyl singlet H329 (δH 1.17, s) to δC 71.7 (t, C-28), 77.6 (s, C-4), and 46.2 (d, C-5) and from the geminal protons at δH 3.52 and 3.31 (2H, each d, J = 11.0 Hz, H-28) to C-4, C-5, and C-29 (δH 22.2, q) revealed the presence of a 1,2-dihydroxyisopropyl moiety at C5. The presence of a propionic ester moiety was supported by the HMBC correlations from H-1a (δH 2.53) to C-2 (δC 29.3, t) and from H2-1 (δH 2.53, ddd; 1.74, m) and H2-2 (δH 2.46 ddd; 2.16, ddd) to a carbonyl carbon at δC 175.8 (C-3). Additional proton resonances at δH 1.26, (t, 7.0) and 4.12, (q, 7.0) and carbon resonances at δC 14.5 and 60.7 (O-Et) suggested that 11 was an ethyl ester. The absolute configuration of the vicinal diol (4-OH and 29-OH) moiety in 11 was
Figure 2. Selected HMBC (H→C) correlations of 7 and 8.
unsaturated-γ-lactone chain. Compound 7 shared the same relative configuration as those of other dammarane-type compounds based on their similar ROESY correlations. Thus, the structure of 7 was defined as 3α-hydroxy-25,26,27trinordammar-22(23)-en-24,20α-olide. Compound 8 was assigned a molecular formula of C27H42O4 from the [M + Na]+ ion at m/z 453.2976 and the 13C NMR data. Comparison of the NMR data of 8 (Table 2) with those of cabralealactone (3-oxo-25,26,27-trinordammar-24,20αolide)13 revealed that 8 carried a C-23 hydroxy group. This was confirmed by the HMBC correlations (Figure 2) from Me21 (δH 1.47, s) to C-22 (δC 39.7, t), C-17 (δC 39.7, d), and C20 (δC 39.7, s), from H-22a (δH 2.56, m) to C-23 (δC 69.4, d) and C-24 (δC 176.9, s), and from H-22b (δH 1.98, m) to C-20 (δC 39.7, s), C-21 (δC 27.7, q), and C-23 (δC 69.4, d). The relative configurations of 8 were determined by the ROESY spectrum in the same manner as for cabralealactone. The ROESY correlation of H-23/Me-21β indicated the αorientation for 23-OH. Thus, the structure of 8 was defined as 23α-hydroxy-3-oxo-25,26,27-trinordammar-24,20-α-olide. Compound 9 was obtained as a white powder, and its molecular formula was determined to be C31H52O5 from the positive HRESIMS and 13C NMR data, which together indicated six indices of hydrogen deficiency. The 1H and 13C NMR spectra (Table 2) showed a typical exocyclic double bond [δH 4.92 (brs), 5.19 (brs); δC 151.7 (C-4), 112.4 (C-28)], an ester carbonyl carbon (δC 174.9, C-3), an oxymethylene [δH 4.07, (dd, J = 21.5, 12.0, H2-29); δC 67.5 (C-29)], an oxymethine [δH 3.72, (dd, J = 7.0, 7.0, H-24); δC 83.6 (C-24)], two oxygenated quaternary carbons [δC 86.6 (C-20), 71.7 (C25)], a methoxy group [δH 3.66 (s); δC 51.9], and six other tertiary methyl groups (δH 1.00, 0.86, 1.13, 1.21, 1.12, and 0.88). Except for one carbon belonging to the methyl ester, the remaining 30 carbons and the strong cross-peaks in the HMBC spectrum correlating the tertiary methyl protons with their neighboring carbons (Figure 3a) suggested that 9 is a 3,4secodammarane derivative with a 20,24-epoxy-type tetrahydrofuran side chain. In addition to the two double bonds and the carbonyl group, the remaining four indices of hydrogen
Figure 3. Selected HMBC (H → C) (a) and ROESY (dashed ↔) correlations (b) of 9. 238
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(22),14 ethyl shoreate (23),22 methyl eichlerianate (24),10 and (24S)-dammar-20-ene-3α,24,25-triol (25)23 (Supporting Information, Figure S1) by comparing their observed and reported spectroscopic data. All compounds were evaluated for their cytotoxicity using the A549 (non-small-cell lung cancer), MCF-7 (breast cancer), and HepG2 (hepatocellular carcinoma) human cell lines (Table 4). Compounds 4 and 8 displayed moderate activities against HepG2 with IC50 values of 6.5 ± 1.1 and 8.0 ± 0.6 μM, respectively. All isolates were further tested for their inhibitory effect on LPS-stimulated NO production in RAW 264.7 cells. Compounds 1, 11, and methyl eichlerialactone displayed inhibition with IC50 values of 64.9 ± 5.5, 57.7 ± 3.8, and 31.8 ± 2.5 μM, respectively, as compared with the positive control Nmonomethyl-L-arginine at 39.2 ± 1.5 μM. The other compounds showed IC50 values greater that 100 μM and were considered inactive. Meliaceous plants have attracted broad interest in the field of natural products. Limonoids, a class of highly oxygenated, modified nortriterpenoids, are characteristic components of the Meliaceae family and are well known for their structural diversity and potential biological significance. Structurally, limonoids are derived from tetracyclic triterpenoids similar to euphol or tirucallol by a series of oxidative changes interspersed with molecular rearrangements.24a The presence of the protolimonoids and limonoids had been used for taxonomic purposes.24b Members of the genus Dysoxylum, however, characteristically produce nondegraded triterpenoids.25 There are only eight reports concerning limonoids from Dysoxylum.26 Our study revealed that D. binectariferum is rich in dammaranes but poor in limonoids, which might be of some chemotaxonomical significance to the position of the genus Dysoxylum in the Meliaceae family.
determined by the induced electronic circular dichroism (ECD) spectra of its in situ complex with Mo2(OAc)4 in DMSO solution (Snatzke’s method).16,17 On the basis of the empirical helicity rule relating the sign of the Cotton effect of the diagnostic O−C−C−O moiety,18 the negative Cotton effect at 310 nm indicated a 4R configuration (Figure 4A). Accordingly, the structure of 11 was defined as ethyl (4R,20S,24R)-epoxy4,25,28-trihydroxy-3,4-secodammar-3-oate.
Figure 4. ECD curves of the Mo2(OAc)4 complexes of 11 (A) and 13 (B).
Compounds 12−14 displayed quasimolecular ions of [M + Na]+ at m/z 559.3973, 559.3971, and 559.3970 in the HRESIMS, respectively. Their NMR spectra were similar to those of 11. The major chemical shift discrepancies corresponded to regions near the C-4 and C-24 stereogenic centers. The NMR data of the tetrahydrofuran ring in 12, especially the characteristic proton resonance at δH 3.64 (dd, J = 10.0, 5.0 Hz) and carbon resonances at δC 86.7 (s, C-20), 86.6 (d, C-24), 27.3 (q, C-21), 35.3 (t, C-22), and 70.5 (s, C25), suggested the 20S and 24S configurations and indicated that 12 was the C-24 isomer of 11. The structure of compound 12 was thus defined as ethyl (4R,20S,24S)-epoxy-4,25,28trihydroxy-3,4-secodammar-3-oate. Compound 13 exhibited some differences with regard to the vicinal diol moiety (Table 3). The NMR spectra showed an oxymethylene [δH 3.83, 3.42, (d, J = 10.5 Hz, H2-29); δC 67.9, (C-29)]; a methyl [δH 1.27 (s, H3-28); δC 26.2, (C-28)]; and a methine [δH 1.49, (m, H-5); δC 50.7, (C-5)], suggesting that 13 was the C-4 epimer of 12. This assertion was confirmed using Snatzke’s method: the positive Cotton effect at 310 nm in the induced ECD spectrum of the complex of 13 with Mo2(OAc)4 indicated a 4S configuration (Figure 4B). Accordingly, the structure of 13 was established as ethyl (4S,20S,24S)-epoxy-4,25,29-trihydroxy3,4-secodammar-3-oate. Similarly, 14 was assigned 20S and 24R configurations according to the NMR data of the tetrahydrofuran ring, especially the characteristic proton resonance at δH 3.72 (dd, J = 7.5, 7.5 Hz) and carbon resonances at δC 86.5 (s, C-20), 83.6 (d, C-24), 23.7 (q, C-21), 36.1 (t, C-22), and 71.7 ppm (s, C-25). Therefore, the structure of 14 was defined as ethyl (4S,20S,24R)-epoxy-4,25,29-trihydroxy-3,4-secodammar-3-oate. The known compounds were identified as cabraleadiol (15),7 3-epiocotillol (16),8,19 carbraleahydroxylactone (17),12 cabralealactone (18),13 eichlerialactone (19),20 methyl eichlerialactone (20),21 ethyl eichlerialactone (21),21 methyl shoreate
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EXPERIMENTAL SECTION
General Experimental Procedures. Optical rotations were performed on a JASCO P-1020 polarimeter. IR spectra (KBr disks, in cm−1) were recorded on a Bruker Tensor 27 spectrometer. ECD spectra were carried out using a JASCO 810 spectropolarimeter. NMR spectra were obtained on a Bruker AV-500 NMR instrument at 500 MHz (1H) and 125 MHz (13C) with TMS as an internal standard. HRESI mass spectra were acquired on an Agilent 6520B Q-TOF mass instrument. Silica gel (Qingdao Marine Chemical Co., Ltd., China), ODS (40−63 μm, Fuji, Japan), and Sephadex LH-20 (Pharmacia, Sweden) were used for open column chromatography. Preparative HPLC was carried out using an Agilent 1100 Series instrument equipped with a 1100 Series multiple wavelength detector, an Alltech ELSD 3300 detector (Grace), and a Shim-pak RP-C18 column (200 × 20 mm). Plant Material. The air-dried stem bark of D. binecteriferum was collected from Xishuangbanna, Yunnan Province, People’s Republic of China, in August 2011, and was authenticated by Professor ShunCheng Zhang, Xishuangbanna Botanical Garden, Chinese Academy of Sciences, People’s Republic of China. A voucher specimen (accession number HG201108) was deposited in the Department of Natural Medicinal Chemistry, China Pharmaceutical University. Extraction and Isolation. The air-dried stem bark of D. binecteriferum (10 kg) was refluxed three times with 95% ethanol. After removal of the solvent under reduced pressure, the crude extract (1500 g) was suspended in H2O (1 L) and partitioned with CH2Cl2 (3 × 1 L). The CH2Cl2 extract (300 g) was crudely separated by column chromatography (CC) eluting with a gradient of petroleum ether (PE)−EtOAc (1:0 to 1:2), and the material was eluted with PE− EtOAc (2:1 to 1:1) to afford four fractions (A−D), as monitored by TLC. Fraction A was subjected to an ODS column using a step 239
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Table 3. 1H (500 MHz) and 13C NMR (125 MHz) Data of Compounds 11−14 in CDCl3 11 position 1a 1b 2a 2b 3 4 5 6a 6b 7a 7b 8 9 10 11a 11b 12a 12b 13 14 15a 15b 16a 16b 17 18 19 20 21 22a 22b 23a 23b 24 25 26 27 28a 28b 29a 29b 30 COOEt a
δH (J in Hz) 2.53, 1.48, 2.46, 2.16,
1.60, 1.55, 1.50, 1.74, 1.20,
m m m m
m m m m m
1.54, m 1.59, 1.28, 1.78, 1.15, 1.61,
m m m m m
1.44, 1.07, 1.72, 1.46, 1.81, 0.98, 1.05,
m m m m m s s
1.13, 1.70, 1.63, 1.86, 1.80, 3.72,
s m m m m dd (7.5, 7.5)
1.21, 1.12, 3.52, 3.31, 1.17,
s s d (11.0) d (11.0) s
0.86, s 1.26, t (7.0) 4.12, q (7.0)
12 δC 35.0 29.3 175.8 77.6 46.2 21.6 34.7 40.2 42.7 41.4 22.4 27.7 43.4 50.7 31.7 25.9 49.8 15.5 21.1 86.6 23.7 36.2 26.4 83.6 71.7 27.6 24.6 71.7
δH (J in Hz) 2.53, 1.48, 2.47, 2.18,
1.60, 1.61, 1.27, 1.74, 1.20,
m m m m
m m m m m
1.54, m 1.55, 1.51, 1.80, 1.23, 1.67,
m m m m m
1.44, 1.08, 1.75, 1.31, 1.87, 1.00, 1.06,
m m m m m s s
1.15, 1.88, 1.66, 1.85,
s m m m
3.64, dd (10.0, 5.0)
22.2
1.19, 1.11, 3.52, 3.31, 1.18,
s s d (11.0) d (11.0) s
16.5 14.5 60.7
0.86, s 1.26, t (7.0) 4.12, q (7.0)
13 δC
δH (J in Hz)
34.7 29.3 175.9 77.6 46.2 22.4 34.9 40.2 42.8 41.4 21.9 27.3 43.3 50.6 31.7 26.1 50.0 15.6 21.1 86.7 27.3 35.3 26.6
2.32, m 1.49a 2.44, m 2.20, m
1.49a 1.51, m 1.21, m 1.74a 1.24, m 1.52, m 1.55, m 1.50, m 1.81, m 1.24a 1.67a 1.44, m 1.08, m 1.76, m 1.33, m 1.87a 0.98, s 0.98, s 1.14, s 1.86, m 1.67a 1.87a
86.6 70.5 28.1 24.4 71.7
3.63, dd (9.5, 5.0)
22.2
3.83, 3.42, 0.86, 1.26, 4.13,
16.5 14.5 60.8
1.19, s 1.11, s 1.27, s d (10.5) d (10.5) s t (7.0) q (7.0)
14 δC 34.9 29.3 175.8 77.4 50.7 22.0 34.9 40.3 42.6 41.1 21.2 27.3 43.3 50.6 31.7 26.1 50.0 15.5 20.8 86.7 27.3 35.2 26.6 86.6 70.5 28.1 24.3 26.2 67.9 16.5 14.5 60.9
δH (J in Hz) 2.30, m 1.46a 2.43, m 2.18, m
1.49, 1.56, 1.48, 1.72, 1.23,
m m m m m
1.51, m 1.59, m 1.21, m 1.79a 1.15, m 1.60, m 1.43, m 1.08, m 1.77, m 1.46a 1.80, m 0.96, s 0.96, s 1.12, s 1.68, m 1.62, m 1.86, m 1.79a 3.72, dd (7.5, 7.5) 1.20, s 1.11, s 1.26, s 3.82, 3.42, 0,85, 1.26, 4.12,
d (10.5) d (10.5) s t (7.0) q (7.0)
δC 34.9 29.3 175.8 77.5 50.6 21.2 34.8 40.2 42.6 41.1 21.8 27.6 43.4 50.7 31.7 25.9 49.7 15.5 20.8 86.5 23.7 36.1 26.2 83.6 71.7 27.6 24.6 26.4 67.8 16.5 14.5 60.84
Signal pattern unclear due to overlapping. mg), 3 (2.5 mg), 4 (5.0 mg), 5 (4.6 mg), and 9 (2.3 mg). (For detailed procedures on extraction and isolation, see Supporting Information.) Compound 1: colorless powder; [α]25D +45 (c 0.1, CH2Cl2); IR (KBr) νmax 3445, 2968, 1639, 1400 cm−1; 1H and 13C NMR data, see Table 1; HRESIMS m/z 499.3757 [M + Na]+ (calcd for C30H52O4Na, 499.3758). Compound 2: colorless powder; [α]25D +24 (c 0.1, CH2Cl2); IR (KBr) νmax 3442, 2966, 1640, 1400, 1384, 1124, 1081, 657, 602 cm−1; 1 H and 13C NMR data, see Table 1; HRESIMS m/z 499.3751 [M + Na]+ (calcd for C30H52O4Na, 499.3758). Compound 3: colorless powder; [α]25D +26 (c 0.1, CH2Cl2); IR (KBr) νmax 3424, 2964, 2928, 2882, 1687, 1630, 1462, 1400, 1379, 1170, 1112, 1042 cm−1; 1H and 13C NMR data, see Table 1; HRESIMS m/z 475.3780 [M + H]+ (calcd for C30H51O4, 475.3782).
gradient of MeOH−H2O (from 30% to 90% MeOH) to afford eichlerialactone (30.0 mg), methyl eichlerialactone (10.0 mg), ethyl eichlerialactone (4.0 mg), 1 (8.0 mg), 2 (2.0 mg), 10 (5.0 mg), 7 (5.2 mg), and 8 (3.3 mg). Fraction B was chromatographed on silica gel with MeOH−CH2Cl2 (from 1:40 to 1:20) to afford compounds 11 (27.2 mg), 12 (3.3 mg), 13 (5.0 mg), and 14 (4.8 mg). Fraction C was chromatographed on reversed-phase C18 silica gel eluting with MeOH−H2O (from 30% to 90% MeOH) to give methyl eichlerianate (5.0 mg), methyl shoreate (8.0 mg), ethyl shoreate (9.0 mg), 3epiocotillol (5.0 mg), cabraleadiol (7.2 mg), cabralealactone (4.3 mg), and carbraleahydroxylactone (3.4 mg). Fraction D was purified by ODS gel CC with MeOH−H2O (from 30% to 90% MeOH) to afford compounds 6 (2.0 mg), (24S)-dammar-20-ene-3α,24,25-triol (3.2 240
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first ECD was recorded immediately, and the time evolution was surveyed until stationary (approximately 0−30 min). The inherent ECD spectrum was subtracted. The absolute configuration was determined by the sign at around 310 nm in the observed ECD spectra.16,17 Cytotoxic Bioassays. All cmpounds were evaluated for cytotoxic activity by an MTT assay as described in the literature.27 Cells were obtained from the Cell Bank of the Shanghai Institute of Cell Biology. Cisplatin was used as a positive control, and the experiments were conducted in three independent replicates. NO Production Bioassays. The protocol for NO production bioassays was provided in previously published papers.28,29 Nmonomethyl-L-arginine was used as the positive control. All experiments were performed in three independent replicates.
Table 4. Cytotoxicity of Isolates against Three Cancer Cell Linesa,b sample 1 2 4 8 9 (24S)-dammar-20-ene3α,24,25-triol methyl shoreate ethyl shoreate ethyl eichlerialactone cisplatinc
A549
MCF-7
>50 >50 28.2 ± 2.0 12.4 ± 1.3 >50 35.0 ± 2.6
30.4 23.0 18.3 26.7 >50 16.7
12.9 ± 1.2 12.9 ± 1.1 >50 13.6 ± 2.1
± ± ± ±
1.3 1.7 1.6 2.4
HepG2 ± 1.1
± 1.8
20.2 >50 6.5 8.0 28.0 5.9
± ± ± ±
1.1 0.6 1.4 0.5
5.6 ± 0.6 18.4 ± 1.5 >50 10.6 ± 2.0
6.3 14.9 38.4 9.7
± ± ± ±
0.7 0.9 2.4 1.3
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ASSOCIATED CONTENT
S Supporting Information *
Results are expressed as IC50 values in μM. bThe remaining 14 compounds were inactive for all cell lines (IC50 > 50 μM). cPositive controls. a
Details for extraction and isolation; copies of HRESIMS, 1H and13C NMR, and 2D NMR spectra of compounds 1−14; chemical structural information of the known compounds 15− 25. This material is available free of charge via the Internet at http://pubs.acs.org.
Compound 4: colorless powder; [α]25D −8 (c 0.1, CH2Cl2); IR (KBr) νmax 3441, 2988, 1654, 1637, 1459, 1403, 1127, 1102 cm−1; 1H and 13C NMR data, see Table 1; HRESIMS m/z 475.3783 [M + H]+ (calcd for C30H51O4, 475.3782). Compound 5: colorless powder; [α]25D +39 (c 0.2, CH2Cl2); IR (KBr) νmax 3445, 1638, 1400, 1124, 1098, 660, 603 cm−1; 1H and 13C NMR data, see Table 1; HRESIMS m/z 475.3785 [M + H]+ (calcd for C30H51O4, 475.3782). Compound 6: colorless powder; [α]25D +62 (c 0.03, CH2Cl2); IR (KBr) νmax 3441, 2957, 2922, 2851, 1638, 1401 cm−1; 1H and 13C NMR data, see Table 2; HRESIMS m/z 497.3603 [M + Na]+ (calcd for C30H50O4Na 497.3601). Compound 7: colorless powder; [α]25D −10 (c 0.2, CH2Cl2); IR (KBr) νmax 3428, 2927, 1755, 1633, 1454, 1400, 1207, 1108, 820, 722, 663 cm−1; 1H and 13C NMR data, see Table 2; HRESIMS m/z 415.3209 [M + H]+ (calcd for C27H43O3, 415.3207). Compound 8: colorless powder; [α]25D +98 (c 0.03, CH2Cl2); IR (KBr) νmax 3441, 1768, 1702, 1636, 1449, 1400, 1129, 1100,657, 601 cm−1; 1H and 13C NMR data, see Table 2; HRESIMS m/z 453.2976 [M + Na]+ (calcd for C27H42O4Na, 453.2975). Compound 9: colorless powder; [α]25D +25 (c 0.1, CH2Cl2); IR (KBr) νmax 3430, 2970, 2924, 1628, 1459, 1400, 1261, 1168, 1050, 883, 671, 617 cm−1; 1H and 13C NMR data, see Table 2; HRESIMS m/z 527.3704 [M + Na]+ (calcd for C31H52O5Na, 527.3707). Compound 10: colorless powder; [α]25D +90 (c 0.1, CH2Cl2); IR (KBr) νmax 3428, 2962, 2922, 1633, 1455, 1400, 1378, 1166, 1114, 1051 cm−1; 1H and 13C NMR data, see Table 2; HRESIMS m/z 489.3939 [M + H]+ (calcd for C31H53O4, 489.3938). Compound 11: colorless oil; [α]25D +42 (c 0.03, CH2Cl2); IR (KBr) νmax 3442, 2959, 2925, 1641, 1462, 1400, 1122, 1098, 657, 601 cm−1; 1H and 13C NMR data, see Table 3; HRESIMS m/z 559.3972 [M + Na]+ (calcd for C32H56O6Na, 559.3969). Compound 12: colorless oil; [α]25D +89 (c 0.1, CH2Cl2); IR (KBr) νmax 3440, 2962, 2925, 1639, 1400, 1261, 1094, 1034, 802 cm−1; 1H and 13C NMR data, see Table 3; HRESIMS m/z 559.3973 [M + Na]+ (calcd for C32H56O6Na, 559.3969). Compound 13: colorless oil; [α]25D +25 (c 0.3, CH2Cl2); IR (KBr) νmax 3442, 2966, 1710, 1635, 1457, 1384, 1163, 1124, 1095, 1037, 657, 602 cm−1; 1H and 13C NMR data, see Table 3; HRESIMS m/z 559.3971 [M + Na]+ (calcd for C32H56O6Na, 559.3969). Compound 14: colorless oil; [α]25D +28 (c 0.4, CH2Cl2); IR (KBr) νmax 3448, 2973, 1637, 1399, 1384, 1124, 1097, 657, 603 cm−1; 1H and 13 C NMR data, see Table 3; HRESIMS m/z 559.3970 [M + Na]+ (calcd for C32H56O6Na, 559.3969). C-4 Absolute Configuration of Compounds 11 and 13. Snatzke’s method was used according to the published literature.16,17 Spectroscopic grade DMSO was dried with 4 Å molecular sieves, and mixtures of 1:1.2 diol−Mo2(OAc)4 were subjected to ECD measurement at a compound concentration of 0.8 mg/mL. After mixing, the
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AUTHOR INFORMATION
Corresponding Author
*E-mail: [email protected]. Tel: +86-25-83271405. Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS The research was financially supported by the Program for Changjiang Scholars and Innovative Research Team in University (IRT1193) and the Priority Academic Program Development of Jiangsu Higher Education Institutions (PAPD).
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REFERENCES
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Cytotoxic Dammarane-Type Triterpenoids from the Stem Bark of Dysoxylum binecteriferum Hui-Jiao Yan, Jun-Song Wang, and Ling-Yi Kong* State Key Laboratory of Natural Medicines, Department of Natural Medicinal Chemistry, China Pharmaceutical University, 24 Tong Jia Xiang, Nanjing 210009, People’s Republic of China S Supporting Information *
ABSTRACT: Fourteen new dammarane-type triterpenoids (1−14) and 11 known analogues were isolated from the stem bark of Dysoxylum binecteriferum. The absolute configurations were established by comparison with the literature or by Mo2(OAc)4induced electronic circular dichroism data. All isolates were evaluated for their cytotoxicities against three human cancer cell lines as well as their inhibitory effects on lipopolysaccharide-induced nitric oxide production in RAW264.7 cells. Compounds 4 and 8 displayed moderate cytotoxicities against HepG2 with IC50 values of 6.5 and 8.0 μM, respectively.
T
he genus Dysoxylum (Meliaceae) comprises approximately 75 species that are mainly distributed in India, Southeast Asia, Australia, and New Zealand.1 Many of these species have been used in folk medicine for the treatment of fever, convulsions, hemorrhage, rigid limbs, or facial distortion in children.2,3 Previous investigations had led to the isolation of a diverse range of bioactive secondary metabolites, such as antifeeding limonoids,4 cytotoxic alkaloids,5 and antibacterial and cytotoxic triterpenoids.6 Dysoxylum binecteriferum, a timber species, primarily found in the tropical areas of southern mainland China,1 has rarely been investigated. To continue our research on bioactive constituents, 14 new and 11 known dammarane-type triterpenoids were isolated from the stem bark of D. binecteriferum. Herein, the isolation and structural elucidation of these isolates, as well as their cytotoxic and anti-inflammatory properties, are described.
methyl groups (δH 0.70, 0.89, 0.91, 0.99, 1.11, 1.15, and 1.19, each 3H, s) were observed in the 1H NMR spectrum. These data corresponded to a dammarane triterpene skeleton similar to that of cabraleadiol,7 which was also isolated in this study. The gross structure of 1 was deduced from the HMBC spectrum (Figure 1a). The HMBC correlations between the tertiary methyl protons and their neighboring carbons were used to establish the dammarane backbone. All expected correlations (Me-18→C-8, 7, 9, 14; Me-19→C-10, 1, 5, 9; and Me-30→C-14, 8, 13, 15) displayed strong cross-peaks. Furthermore, the HMBC crosspeaks from the oxygenated methine protons at δH 3.68 (brs, W1/2 = 6.5 Hz, H-3) to carbons at δC 33.7 (C-1), 43.9 (C-5), and 18.0 (C-29), from the geminal protons at δH 3.40 and 3.54 (each d, J = 11.5 Hz, H2-28) to carbons at δC 40.9 (C-4), 43.9 (C-5), 77.3 (C-3), and 18.0 (C-29), and from the remaining methyl protons at δH 0.70 (H3-29) to carbons at δC 40.9 (C-4), 43.9 (C-5), 71.7 (C-28), and 77.3 (C-3) located two hydroxy groups at C-3 and C-28. The characteristic NMR data [δC 86.8 (C-20, s), δC 86.6 (C24, d); δH 3.64 (1H, dd, J = 5.5, 10.0 Hz, H-24)] suggested the presence of a 20,24-epoxy moiety and hence a tetrahydrofuran ring in the side chain. The gem-dimethyl protons at δH 1.19 (s,
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RESULTS AND DISCUSSION Compound 1 was assigned a molecular formula of C30H52O4, as deduced from its positive HRESIMS (found [M + Na]+ m/z 499.3757, calcd 499.3758) and 13C NMR data, indicative of five indices of hydrogen deficiency. The 13C NMR (Table 1) data showed 30 carbon resonances, which were classified by the HSQC spectrum as seven methyl, 11 methylene (one oxygenated), six methine (two oxygenated), and six quaternary carbons (two oxygenated). The five indices of hydrogen deficiency suggested that 1 was pentacyclic. Seven tertiary © 2014 American Chemical Society and American Society of Pharmacognosy
Received: August 27, 2013 Published: February 18, 2014 234
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Table 1. 1H (500 MHz) and 13C NMR (125 MHz) Data of Compounds 1−5 in CDCl3 1 position 1a
1.46, m 1.33 1.95, m
2b
1.47, m
3
3.68, brs (W1/2 = 6.5)
25 26 27 28a 28b 29a 29b 30
2 δC
1.67a 1.39, m 1.64, m 1.27, m 1.53, m 1.55, 1.22, 1.25, 1.63,
m m m m
1.48, m 1.08, m 1.78, m 1.33a 1.86, m 0.99, s 0.89, s 1.15, s 1.88, m 1.67a 1.81, m 3.64, dd (10.0, 5.5) 1.19, s 1.11, s 3.54, d (11.5) 3.40 d (11.5) 0.70, s 0.91, s
δH (J in Hz) a
33.7
1.45
26.1
1.30, m 1.94, m
a
1b 2a
4 5 6 7a 7b 8 9 10 11a 11b 12 13 14 15a 15b 16a 16b 17 18 19 20 21 22a 22b 23a 23b 24
a
δH (J in Hz)
3 δH (J in Hz)
δC
33.7
1.99, ddd (13.5, 7.0, 3.0) 1.39, m 2.62, ddd (16.0, 12.0, 7.5) 2.30, ddd (16.0, 6.0, 3.0)
39.8
26.0
1.45a 77.3 40.9 43.9 18.3 35.2 40.9 51.0 37.4 22.0 27.3 43.1 50.5 31.7 26.7 50.1 15.8 16.6 86.8 27.4 35.1 26.8 86.6 70.6 28.1 24.3 71.7
3.68, brs (W1/2 = 6.4) 1.66, m 1.38, m 1.63a 1.27, m 1.51, m 1.55, 1.21, 1.26, 1.58,
m m m m
1.46, m 1.08, m 1.80a 1.45a 1.81, m 0.97, s 0.88, s 1.13, s 1.70, m 1.63a 1.85, m 1.80a 3.73, dd (7.0, 7.0)
18.0
1.21, 1.12, 3.54, 3.40, 0.70,
s s d (11.5) d (11.5) s
16.8
0.91, s
4
δC
77.3 40.9 43.9 18.3 35.2 40.9 51.0 37.4 21.8 27.7 43.2 50.5 31.7 26.4 49.8 15.7 16.6 86.7 23.9 35.9
1.65, 1.41, 1.60, 1.30,
52.8 49.8 19.4 34.9
m m m m
40.7 50.4 37.1 22.2
1.49, m 1.55, 1.28, 1.27, 1.61,
m m m m
1.47a 1.11, m 1.78, m 1.47a 1.81, m 1.02, s 1.06, s
18.0
s s d (11.5) d (11.5) s
16.9
0.88, s
1.66, 1.40, 1.62, 1.32,
26.4
1.15, 1.87, 1.67, 1.84,
s m m m
83.6
3.64, dd (10.0, 5.5)
16.7
1.19, 1.11, 3.64, 3.42, 1.02,
39.9
35.6
1.58, m 2.59, m,
34.5
2.38, m
16.8
0.88, s
27.4 43.2 50.4 31.7 26.1 50.1 16.3 15.7 86.8 27.2 35.1 26.6
s s d (11.5) d (11.5) s
δC
1.93, m
40.7 50.9 37.1 22.4
1.48, m
m m m m m s s
71.7 27.7 24.6 67.2
δH (J in Hz)
39.8
52.8 49.9 19.5 34.9
m m m m
1.49, 1.09, 1.78, 1.33, 1.88, 1.03, 1.07,
49.9 15.7 16.2 86.6 23.9 35.9
5 δC
219.3
m m m m
26.0
1.21, 1.12, 3.65, 3.42, 1.02,
71.7 27.7 24.6 71.7
1.41, m 2.63, ddd (15.5, 12.0, 7.0) 2.30, ddd (15.5, 6.0, 3.0)
1.56, 1.28, 1.26, 1.68,
27.6 43.3 50.4 31.7
s m m m m dd (7.0, 7.0)
83.6
2.00, m
219.2
1.14, 1.70, 1.63, 1.85, 1.80, 3.73,
26.8
35.6
δH (J in Hz)
86.7 70.6 28.0 24.3 67.2 16.9 16.3
221.6
1.63, 1.50, 1.53, 1.34,
m m m m
1.45, m 1.52, 1.24, 1.25, 1.68,
m m m m
1.47, 1.08, 1.77, 1.33, 1.89, 0.98, 0.89,
m m m m m s s
1.15, 1.87, 1.69, 1.84,
s m m m
3.64, dd (10.0, 5.5) 1.19, s 1.11, s 1.27, s 3.98, d (11.5) 3.45, d (11.5) 0.89, s
50.0 56.1 19.5 35.0 40.5 51.1 36.9 22.9 27.4 43.3 50.2 31.7 26.1 50.2 15.2 16.5 86.7 27.3 35.1 26.6 86.7 70.6 28.0 24.3 22.4 66.1 17.3
Signal pattern unclear due to overlapping.
H3-26) and 1.11 (s, H3-27) displayed cross-peaks with carbons at δC 86.6 (C-24) in the HMBC spectrum, placing the 2hydroxyisopropyl group [δC 70.6 (C-25), 28.1 (C-26), 24.3 (C27)] at C-24. Other HMBC correlations (Figure 1a) further confirmed the side chain structure. Thus, the gross structure of 1 was established as shown. With regard to the relative configurations, the characteristic broad H-3 singlet suggested that it was in an equatorial position and β-oriented. Other relative configurations of 1 were elucidated mainly by the ROESY spectrum (Figure 1b). The typically observed correlations of Me-30 (14α-Me)/H-17α, H9α/Me-30, H-5α/H-9, Me-19/Me-18 (8β-Me), Me-29 (4βMe)/Me-19 (10β-Me), Me-18/H-13β, and H-13β/Me-21
Figure 1. Selected HMBC (H→C) (a) and ROESY (dashed ↔) correlations (b) of 1.
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Chart 1
each d, J = 11.5 Hz) and Me-29 (δH 1.02, s) to C-3 (δC 219.2), from H-28a (δH 3.65) to C-5, and from H-28b (δH 3.42) to Me-29. The relative configuration of 3 was the same as that of 2 based on their similar ROESY correlations. The ROESY crosspeaks of H-28b/H-5α, H-2a (δH 2.62)/Me-19β, and H-2a/Me29 (4β-Me) suggested that the hydroxy group was located at C28 (4α-Me). Thus, the structure of 3 was defined as (20S,24R)epoxy-25, 28-dihydroxydammar-3-one. Compound 4 differed from 3 only in the configuration of C-24. The characteristic proton resonance at δH 3.64 (dd, J = 10.0, 5.5 Hz) and carbon resonances at δC 86.8 (s, C-20), 86.7 (d, C-24), 27.2 (q, C-21), 34.9 (t, C-22), and 70.6 (s, C-25) resulted in the assignment of the (20S, 24S) absolute configuration for 4.8,9 Accordingly, the structure of 4 was assigned as (20S,24S)-epoxy-25,28dihydroxydammar-3-one. The NMR data of 5 (Table 1) were generally similar to those of 4 except for the resonances corresponding to ring A. The similar HMBC correlations suggested that they shared the same gross structure. The ROESY cross-peaks between the oxygenated methylene proton H-29a (δH 3.98, d, J = 11.5 Hz) and Me-19β and between H-9α and Me-28 in 5 indicated the β-orientation of the hydroxymethyl group at C-4. Thus, the structure of 5 was defined as (20S,24S)-epoxy-25,29-dihydroxydammar-3-one. Compound 6 was assigned a molecular formula of C30H50O4 by its 13C NMR data and positive HRESIMS ion at m/z 497.3603 [M + Na]+ (calcd for C30H50O4Na, 497.3601). The 1 H and 13C NMR data of 6 (Table 2) displayed resonances typical of dammarane triterpenoids. The HMBC correlations from the proton at δH 3.83 (dd, J = 7.0, 7.0 Hz) to C-18 (δC 9.49 q, γ-gauche effect), C-6 (δC 29.0), and C-9 (δC 49.8) and from Me-18 (δH 1.02, s) to C-7 (δC 74.5) suggested the presence of a hydroxy group at C-7. The ROESY cross-peaks of Me-30 (δH 0.95)/H-7, H-5 (δH 1.48)/H-7, and H-9 (δH 1.34)/ H-7 revealed the α-orientation of H-7. These data indicated that the structure of 6 was similar to that of salvilymitone.11 The 13C NMR data of the epoxy ring, especially the resonances of C-20 (δC 86.5), C-21 (δC 26.9), C-22 (δC 34.8), and C-24 (δC 86.5), were highly similar to those of the (20S,24S)-epimer.
revealed that 1 had the same configurations as other reported dammarane triterpenoids,7 and the cross-peak of H-5α/H2-28 confirmed the α-orientation of the C-4 hydroxymethyl group. The stereochemistry of the substituted tetrahydrofuran moiety at C-17 of the triterpenoid skeleton has been thoroughly studied.7−9 The chemical shifts for C-21, C-22, and C-24 in compounds with the 20S and 24R configurations were approximately δC 23.6−23.9, 35.5−35.7, and 83.2, whereas those for C-21, C-22, and C-24 in compounds with 20S and 24S configurations were δC 27.0−27.2, 34.6−34.8, and 86.5.9 Thus, the C-20 and C-24 absolute configurations of 1 were both assigned as S according to the characteristic 13C NMR resonances of C-21 (δC 27.4), C-22 (δC 35.1), and C-24 (δC 86.6). Thus, the structure of compound 1 was assigned as (20S,24S)-epoxydammarane-3α,25,28-triol. Compound 2 had a molecular formula of C30H52O4, the same as that of 1, as determined by the 13C NMR data and the observed pseudomolecular ion at [M + Na]+ m/z 499.3751 (calcd for C30H52O4Na, 499.3758). Comparison of the NMR data of 1 and 2 (Table 1) revealed a considerable degree of similarity except for the side chain resonances. The large chemical shift discrepancy (Δδ −3.0) at C-24 suggested that 2 is a C-24 epimer of 1, which was supported by the ROESY correlation between Me-21 and H-24 in 2, not observed in 1. Moreover, the characteristic 13C NMR resonances of C-21 (δC 23.9), C-22 (δC 35.9), and C-24 (δC 83.6) indicated the 20S and 24R configurations. Thus, the structure of 2 was defined as (20S,24R)-epoxydammarane-3α,25,28-triol. Compounds 3−5 had the same molecular formula of C30H50O4, as indicated by their 13C NMR data and positive HRESIMS ion at m/z 475.3780, 475.3783, and 475.3785, respectively ([M + H]+, calcd for C30H51O4, 475.3782), 2 mass units less than those of 1 and 2. Comparison of their 1H and 13 C NMR data (Table 1) indicated that 3−5 were analogues of 1 and 2. The major difference between 3 and 2 was the presence of a carbonyl group (δC 219.2) at C-3 in 3 instead of the oxymethine group (δH 3.68, brs; δC 77.3) in 2. The proposed structure was confirmed by the HMBC correlations from H2-2 (2.62, ddd; 2.30, ddd), H2-28 (δH 3.42 and 3.65, 236
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Table 2. 1H (500 MHz) and 13C NMR (125 MHz) Data of Compounds 6−10 in CDCl3 6 position 1a 1b 2a 2b 3 4 5 6a 6b 7a 7b 8 9 10 11a 11b 12a 12b 13 14 15a 15b 16a 16b 17 18 19 20 21 22a 22b 23a 23b 24 25 26 27 28a 28b 29a 29b 30 COOMe a
δH (J in Hz) 1.91, m 1.43, m 2.46, m
1.48, m 1.64a 1.25, m 3.83, dd (7.0, 7.0)
7 δC 39.5
δH (J in Hz)
1.56, m 1.35, m 1.79, m 1.37, m 1.64a 1.70, m 1.33, m 1.86, m
1.45, m
40.1
25.7
2.50, m 2.42, ddd (10.5, 7.5, 4.5)
34.3
3.40, t (3.0) 1.25, m
76.5 37.5 49.8
29.0
1.41a
18.5
74.5
1.57, m
35.4
217.3 47.1 52.7
45.9 49.8 36.7 22.3 26.2 43.8 49.4 34.9 27.3
1.41a 1.52, 1.16, 1.84, 1.35, 1.46,
m m m m m
1.45, m 1.11, m 1.71, m
40.8 50.7 37.9 21.5 25.5 43.7 50.6 31.4
19.9 34.8
40.6 50.1 37.1 22.2
1.40, m 1.53, 1.26, 1.84, 1.37, 1.51,
m m m m m
25.6 43.9 50.5 31.3
1.98, m
49.3
0.99, s 0.94, s
16.2 15.5 89.2 27.7 39.7
1.45, s 7.38, d (5.5)
16.6 16.3 92.6 23.9 159.8
26.4
6.05, d (5.5)
121.4 172.7
1.47, 2.56, 1.93, 4.52,
27.0
s m, m dd (9.0, 8.0)
s
δH (J in Hz) a
1.54 1.43a 2.36, m 2.17, m
2.02, dd (13.0, 3.0) 1.86a 1.44, m 1.54a
10 δC
69.4 176.9
1.19, s 1.12, s 1.10, s
70.3 27.8 24.1 28.8
0.93, s
28.6
1.08, s
27.1
1.05, s
21.0
0.83, s
22.4
1.04, s
21.3
0.95, s
16.2
0.88, s
15.7
0.89, s
16.4
δH (J in Hz)
δC
35.2
1.60, m
34.8
28.8
2.34, m 2.18, m
28.7
174.9 151.7 46.6 26.2
1.97, dd (12.5, 3.0) 1.35, m
34.5
1.51, m
1.24a
47.7
9.5 16.0 86.5 26.9 34.8
86.5
1.56, m 1.49, m 1.58, m
27.3
1.02, s 0.93, s
3.64, dd (10.0, 5.5)
1.36, m
1.52, m 1.16, m 1.65, m
48.7
s m m m
2.05, td (10.5, 6.0) 0.90, s 0.83, s
217.9 47.7 55.6
1.33, m
1.84, m
1.16, 1.88, 1.70, 1.85,
9 δC
33.9
1.24, m 1.34, m
δH (J in Hz)
m m m m
33.9
1.40, 1.28, 1.94, 1.54,
8 δC
174.9 147.9 51.1 25.0 34.2
1.22, m
1.53, m 1.43a 1.24a 1.80a 1.12, m 1.60, m 1.47a 1.10, m 1.75, m 1.47a 1.80, m 1.00, s 0.86, s 1.13, s 1.69, m 1.63, m 1.86a 1.80a 3.72, dd (7.0, 7.0)
1.21, s 1.12, s 5.19, brs 4.92, brs 4.07, dd (21.5, 12.0) 0.88, s 3.66, s
40.3 41.5 39.4 22.5 27.5 43.3 50.7 31.7 25.9 49.8 15.6 20.0 86.6 23.8
1.52, m 1.40a 1.27, m 1.90, m 1.71, m 1.40a 1.04, m 1.83, m 1.50, m 1.76, m 1.01, s 0.85, s 1.17, s
40.4 41.5 39.4 22.6 28.0 42.6 51.1 31.7 25.6 51.5 15.6 20.4 76.5 26.3 26.4
36.1 26.4 83.6 71.7 27.7 24.6 112.4
1.99, m 1.38, m 3.43, dd (5.0, 2.5)
67.5
1.25, 1.16, 4.84, 4.66, 1.73,
s s brs brs s
16.6 51.9
0.88, s 3.67, s
23.7 71.3 74.3 28.5 26.9 113.6 23.5
16.8 51.8
Signal pattern unclear due to overlapping.
Thus, the structure of 6 was defined as (20S,24S)-epoxy-7β,25dihydroxydammar-3-one. Compound 7 was obtained as a white powder. The HRESIMS showed an [M + H]+ ion at m/z 415.3209, and together with the 13C NMR data, this indicated a molecular formula of C27H42O3. Resonances for two olefinic protons (δH 6.05, 7.38, each d, J = 5.5 Hz) and six methyl singlets (δH 0.90, 0.834, 0.93, 0.830, 0.88, and 1.45) in the 1H NMR spectrum and 27 carbon signals in the 13C NMR spectrum (Table 2)
suggested that 7 possessed a trinortriterpenoid skeleton similar to that of cabraleahydroxylactone (3α-hydroxy-25,26,27trinordammar-24,20α-olide),12 which was also isolated in this investigation. However, the main differences occurred through the formation of a double bond between C-22 and C-23 in 7. The HMBC cross-peaks (Figure 2) from Me-21 (δH 1.45) to the oxygenated quaternary C-20 (δC 92.6) and the olefinic C-22 (δC 159.8) and from the olefinic proton H-23 (δH 6.05) to the lactone carbon (δC 172.7) indicated the presence of an α,β237
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deficiency indicated that 9 is tetracyclic, suggesting the cleavage of ring A. The NMR spectroscopic data of 9 are similar to those of methyl shoreate [methyl (20S,24S)-epoxy-25-hydroxy-3,4secodammar-4(28)-en-3-oate],9,14 which was also isolated in this investigation. The difference between the two compounds is the presence of an oxymethylene in 9 instead of a tertiary methyl group in methyl shoreate. The HMBC correlations (Figure 3a) from H2-28 to C-5 (δC 46.6, d) and C-29 (δC 67.5, t) and from H2-29 to C-4 (δC 151.7, s) and C-28 (δC 112.4, t) confirmed hydroxylation at C-29. Compound 9 shared the same relative configurations as those of other shoreic acid-type C-23 compounds (Figure 3b). The (20S,24R)-epoxide configurations of 9 were assigned on the basis of the characteristic chemical shifts of the carbon resonances of C-21 (δC 23.8 ppm), C-22 (δC 36.1 ppm), and C-24 (δC 83.6 ppm).9 Hence, the structure of 9 was defined as methyl (20S,24S)-epoxy-25,29-dihydroxy-3,4-secodammar4(28)-en-3-oate. Compound 10 had a molecular formula of C31H52O4, as deduced from its positive HRESIMS (found [M + H]+ m/z 489.3939, calcd 489.3938) and 13C NMR data, which indicated five indices of hydrogen deficiency. The 1H and 13C NMR data of 10 (Table 2) resembled those of 3,4-secodammarane methyl shoreate,9,14 especially by the presence of resonances for a typical exocyclic double bond [δH 4.66 (brs), 4.84 (brs); δC 147.9 (C-4), 113.6 (C-28)]; an ester carbonyl carbon (δC 174.9, C-3); a methoxy group [δH 3.67 (s); δC 51.8]; and six tertiary methyl groups (δH 1.01, 0.85, 1.17, 1.25, 1.16, and 0.88). The NMR spectra of 10 displayed an oxygenated methine [δH 3.43 (dd, J = 5.0, 2.5 Hz), δC 71.3 (C-24)] and two oxygenated quaternary carbons [δC 76.5 (C-20), 74.3 (C25)], which suggested the presence of a six-membered pyran instead of a tetrahydrofuran ring at C-17. The HMBC correlations from Me-21 (δH 1.17, s) to C-17 (δC 51.5), C20 (δC 76.5), and C-22 (δC 26.4) and from H-24 to C-22 (δC 26.4), C-23 (δC 23.7), and C-27 (δC 26.9) confirmed the presence of a 3-hydroxytetrahydropyranyl group at C-17.15 The ROESY correlations of H-24 with Me-26 and Me-27, together with the small coupling constant of H-24, suggested that it was β-equatorially oriented. The S configuration was assigned to C20, as is common in dammarane triterpenoids, especially those isolated in this investigation. Thus, the structure of 10 was defined as methyl (20S,25)-epoxy-24α-hydroxy-3,4-secodammar-4(28)-en-3-oate. Compound 11, an oil, had a molecular formula of C32H56O6 based on its positive HRESIMS ion at m/z 559.3972 [M + Na]+ and the 13C NMR data, which together indicated five indices of hydrogen deficiency. Comparison of its NMR data with those of 9 revealed similar features in rings B−D and the side chain. The HMBC correlations from the methyl singlet H329 (δH 1.17, s) to δC 71.7 (t, C-28), 77.6 (s, C-4), and 46.2 (d, C-5) and from the geminal protons at δH 3.52 and 3.31 (2H, each d, J = 11.0 Hz, H-28) to C-4, C-5, and C-29 (δH 22.2, q) revealed the presence of a 1,2-dihydroxyisopropyl moiety at C5. The presence of a propionic ester moiety was supported by the HMBC correlations from H-1a (δH 2.53) to C-2 (δC 29.3, t) and from H2-1 (δH 2.53, ddd; 1.74, m) and H2-2 (δH 2.46 ddd; 2.16, ddd) to a carbonyl carbon at δC 175.8 (C-3). Additional proton resonances at δH 1.26, (t, 7.0) and 4.12, (q, 7.0) and carbon resonances at δC 14.5 and 60.7 (O-Et) suggested that 11 was an ethyl ester. The absolute configuration of the vicinal diol (4-OH and 29-OH) moiety in 11 was
Figure 2. Selected HMBC (H→C) correlations of 7 and 8.
unsaturated-γ-lactone chain. Compound 7 shared the same relative configuration as those of other dammarane-type compounds based on their similar ROESY correlations. Thus, the structure of 7 was defined as 3α-hydroxy-25,26,27trinordammar-22(23)-en-24,20α-olide. Compound 8 was assigned a molecular formula of C27H42O4 from the [M + Na]+ ion at m/z 453.2976 and the 13C NMR data. Comparison of the NMR data of 8 (Table 2) with those of cabralealactone (3-oxo-25,26,27-trinordammar-24,20αolide)13 revealed that 8 carried a C-23 hydroxy group. This was confirmed by the HMBC correlations (Figure 2) from Me21 (δH 1.47, s) to C-22 (δC 39.7, t), C-17 (δC 39.7, d), and C20 (δC 39.7, s), from H-22a (δH 2.56, m) to C-23 (δC 69.4, d) and C-24 (δC 176.9, s), and from H-22b (δH 1.98, m) to C-20 (δC 39.7, s), C-21 (δC 27.7, q), and C-23 (δC 69.4, d). The relative configurations of 8 were determined by the ROESY spectrum in the same manner as for cabralealactone. The ROESY correlation of H-23/Me-21β indicated the αorientation for 23-OH. Thus, the structure of 8 was defined as 23α-hydroxy-3-oxo-25,26,27-trinordammar-24,20-α-olide. Compound 9 was obtained as a white powder, and its molecular formula was determined to be C31H52O5 from the positive HRESIMS and 13C NMR data, which together indicated six indices of hydrogen deficiency. The 1H and 13C NMR spectra (Table 2) showed a typical exocyclic double bond [δH 4.92 (brs), 5.19 (brs); δC 151.7 (C-4), 112.4 (C-28)], an ester carbonyl carbon (δC 174.9, C-3), an oxymethylene [δH 4.07, (dd, J = 21.5, 12.0, H2-29); δC 67.5 (C-29)], an oxymethine [δH 3.72, (dd, J = 7.0, 7.0, H-24); δC 83.6 (C-24)], two oxygenated quaternary carbons [δC 86.6 (C-20), 71.7 (C25)], a methoxy group [δH 3.66 (s); δC 51.9], and six other tertiary methyl groups (δH 1.00, 0.86, 1.13, 1.21, 1.12, and 0.88). Except for one carbon belonging to the methyl ester, the remaining 30 carbons and the strong cross-peaks in the HMBC spectrum correlating the tertiary methyl protons with their neighboring carbons (Figure 3a) suggested that 9 is a 3,4secodammarane derivative with a 20,24-epoxy-type tetrahydrofuran side chain. In addition to the two double bonds and the carbonyl group, the remaining four indices of hydrogen
Figure 3. Selected HMBC (H → C) (a) and ROESY (dashed ↔) correlations (b) of 9. 238
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(22),14 ethyl shoreate (23),22 methyl eichlerianate (24),10 and (24S)-dammar-20-ene-3α,24,25-triol (25)23 (Supporting Information, Figure S1) by comparing their observed and reported spectroscopic data. All compounds were evaluated for their cytotoxicity using the A549 (non-small-cell lung cancer), MCF-7 (breast cancer), and HepG2 (hepatocellular carcinoma) human cell lines (Table 4). Compounds 4 and 8 displayed moderate activities against HepG2 with IC50 values of 6.5 ± 1.1 and 8.0 ± 0.6 μM, respectively. All isolates were further tested for their inhibitory effect on LPS-stimulated NO production in RAW 264.7 cells. Compounds 1, 11, and methyl eichlerialactone displayed inhibition with IC50 values of 64.9 ± 5.5, 57.7 ± 3.8, and 31.8 ± 2.5 μM, respectively, as compared with the positive control Nmonomethyl-L-arginine at 39.2 ± 1.5 μM. The other compounds showed IC50 values greater that 100 μM and were considered inactive. Meliaceous plants have attracted broad interest in the field of natural products. Limonoids, a class of highly oxygenated, modified nortriterpenoids, are characteristic components of the Meliaceae family and are well known for their structural diversity and potential biological significance. Structurally, limonoids are derived from tetracyclic triterpenoids similar to euphol or tirucallol by a series of oxidative changes interspersed with molecular rearrangements.24a The presence of the protolimonoids and limonoids had been used for taxonomic purposes.24b Members of the genus Dysoxylum, however, characteristically produce nondegraded triterpenoids.25 There are only eight reports concerning limonoids from Dysoxylum.26 Our study revealed that D. binectariferum is rich in dammaranes but poor in limonoids, which might be of some chemotaxonomical significance to the position of the genus Dysoxylum in the Meliaceae family.
determined by the induced electronic circular dichroism (ECD) spectra of its in situ complex with Mo2(OAc)4 in DMSO solution (Snatzke’s method).16,17 On the basis of the empirical helicity rule relating the sign of the Cotton effect of the diagnostic O−C−C−O moiety,18 the negative Cotton effect at 310 nm indicated a 4R configuration (Figure 4A). Accordingly, the structure of 11 was defined as ethyl (4R,20S,24R)-epoxy4,25,28-trihydroxy-3,4-secodammar-3-oate.
Figure 4. ECD curves of the Mo2(OAc)4 complexes of 11 (A) and 13 (B).
Compounds 12−14 displayed quasimolecular ions of [M + Na]+ at m/z 559.3973, 559.3971, and 559.3970 in the HRESIMS, respectively. Their NMR spectra were similar to those of 11. The major chemical shift discrepancies corresponded to regions near the C-4 and C-24 stereogenic centers. The NMR data of the tetrahydrofuran ring in 12, especially the characteristic proton resonance at δH 3.64 (dd, J = 10.0, 5.0 Hz) and carbon resonances at δC 86.7 (s, C-20), 86.6 (d, C-24), 27.3 (q, C-21), 35.3 (t, C-22), and 70.5 (s, C25), suggested the 20S and 24S configurations and indicated that 12 was the C-24 isomer of 11. The structure of compound 12 was thus defined as ethyl (4R,20S,24S)-epoxy-4,25,28trihydroxy-3,4-secodammar-3-oate. Compound 13 exhibited some differences with regard to the vicinal diol moiety (Table 3). The NMR spectra showed an oxymethylene [δH 3.83, 3.42, (d, J = 10.5 Hz, H2-29); δC 67.9, (C-29)]; a methyl [δH 1.27 (s, H3-28); δC 26.2, (C-28)]; and a methine [δH 1.49, (m, H-5); δC 50.7, (C-5)], suggesting that 13 was the C-4 epimer of 12. This assertion was confirmed using Snatzke’s method: the positive Cotton effect at 310 nm in the induced ECD spectrum of the complex of 13 with Mo2(OAc)4 indicated a 4S configuration (Figure 4B). Accordingly, the structure of 13 was established as ethyl (4S,20S,24S)-epoxy-4,25,29-trihydroxy3,4-secodammar-3-oate. Similarly, 14 was assigned 20S and 24R configurations according to the NMR data of the tetrahydrofuran ring, especially the characteristic proton resonance at δH 3.72 (dd, J = 7.5, 7.5 Hz) and carbon resonances at δC 86.5 (s, C-20), 83.6 (d, C-24), 23.7 (q, C-21), 36.1 (t, C-22), and 71.7 ppm (s, C-25). Therefore, the structure of 14 was defined as ethyl (4S,20S,24R)-epoxy-4,25,29-trihydroxy-3,4-secodammar-3-oate. The known compounds were identified as cabraleadiol (15),7 3-epiocotillol (16),8,19 carbraleahydroxylactone (17),12 cabralealactone (18),13 eichlerialactone (19),20 methyl eichlerialactone (20),21 ethyl eichlerialactone (21),21 methyl shoreate
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EXPERIMENTAL SECTION
General Experimental Procedures. Optical rotations were performed on a JASCO P-1020 polarimeter. IR spectra (KBr disks, in cm−1) were recorded on a Bruker Tensor 27 spectrometer. ECD spectra were carried out using a JASCO 810 spectropolarimeter. NMR spectra were obtained on a Bruker AV-500 NMR instrument at 500 MHz (1H) and 125 MHz (13C) with TMS as an internal standard. HRESI mass spectra were acquired on an Agilent 6520B Q-TOF mass instrument. Silica gel (Qingdao Marine Chemical Co., Ltd., China), ODS (40−63 μm, Fuji, Japan), and Sephadex LH-20 (Pharmacia, Sweden) were used for open column chromatography. Preparative HPLC was carried out using an Agilent 1100 Series instrument equipped with a 1100 Series multiple wavelength detector, an Alltech ELSD 3300 detector (Grace), and a Shim-pak RP-C18 column (200 × 20 mm). Plant Material. The air-dried stem bark of D. binecteriferum was collected from Xishuangbanna, Yunnan Province, People’s Republic of China, in August 2011, and was authenticated by Professor ShunCheng Zhang, Xishuangbanna Botanical Garden, Chinese Academy of Sciences, People’s Republic of China. A voucher specimen (accession number HG201108) was deposited in the Department of Natural Medicinal Chemistry, China Pharmaceutical University. Extraction and Isolation. The air-dried stem bark of D. binecteriferum (10 kg) was refluxed three times with 95% ethanol. After removal of the solvent under reduced pressure, the crude extract (1500 g) was suspended in H2O (1 L) and partitioned with CH2Cl2 (3 × 1 L). The CH2Cl2 extract (300 g) was crudely separated by column chromatography (CC) eluting with a gradient of petroleum ether (PE)−EtOAc (1:0 to 1:2), and the material was eluted with PE− EtOAc (2:1 to 1:1) to afford four fractions (A−D), as monitored by TLC. Fraction A was subjected to an ODS column using a step 239
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Table 3. 1H (500 MHz) and 13C NMR (125 MHz) Data of Compounds 11−14 in CDCl3 11 position 1a 1b 2a 2b 3 4 5 6a 6b 7a 7b 8 9 10 11a 11b 12a 12b 13 14 15a 15b 16a 16b 17 18 19 20 21 22a 22b 23a 23b 24 25 26 27 28a 28b 29a 29b 30 COOEt a
δH (J in Hz) 2.53, 1.48, 2.46, 2.16,
1.60, 1.55, 1.50, 1.74, 1.20,
m m m m
m m m m m
1.54, m 1.59, 1.28, 1.78, 1.15, 1.61,
m m m m m
1.44, 1.07, 1.72, 1.46, 1.81, 0.98, 1.05,
m m m m m s s
1.13, 1.70, 1.63, 1.86, 1.80, 3.72,
s m m m m dd (7.5, 7.5)
1.21, 1.12, 3.52, 3.31, 1.17,
s s d (11.0) d (11.0) s
0.86, s 1.26, t (7.0) 4.12, q (7.0)
12 δC 35.0 29.3 175.8 77.6 46.2 21.6 34.7 40.2 42.7 41.4 22.4 27.7 43.4 50.7 31.7 25.9 49.8 15.5 21.1 86.6 23.7 36.2 26.4 83.6 71.7 27.6 24.6 71.7
δH (J in Hz) 2.53, 1.48, 2.47, 2.18,
1.60, 1.61, 1.27, 1.74, 1.20,
m m m m
m m m m m
1.54, m 1.55, 1.51, 1.80, 1.23, 1.67,
m m m m m
1.44, 1.08, 1.75, 1.31, 1.87, 1.00, 1.06,
m m m m m s s
1.15, 1.88, 1.66, 1.85,
s m m m
3.64, dd (10.0, 5.0)
22.2
1.19, 1.11, 3.52, 3.31, 1.18,
s s d (11.0) d (11.0) s
16.5 14.5 60.7
0.86, s 1.26, t (7.0) 4.12, q (7.0)
13 δC
δH (J in Hz)
34.7 29.3 175.9 77.6 46.2 22.4 34.9 40.2 42.8 41.4 21.9 27.3 43.3 50.6 31.7 26.1 50.0 15.6 21.1 86.7 27.3 35.3 26.6
2.32, m 1.49a 2.44, m 2.20, m
1.49a 1.51, m 1.21, m 1.74a 1.24, m 1.52, m 1.55, m 1.50, m 1.81, m 1.24a 1.67a 1.44, m 1.08, m 1.76, m 1.33, m 1.87a 0.98, s 0.98, s 1.14, s 1.86, m 1.67a 1.87a
86.6 70.5 28.1 24.4 71.7
3.63, dd (9.5, 5.0)
22.2
3.83, 3.42, 0.86, 1.26, 4.13,
16.5 14.5 60.8
1.19, s 1.11, s 1.27, s d (10.5) d (10.5) s t (7.0) q (7.0)
14 δC 34.9 29.3 175.8 77.4 50.7 22.0 34.9 40.3 42.6 41.1 21.2 27.3 43.3 50.6 31.7 26.1 50.0 15.5 20.8 86.7 27.3 35.2 26.6 86.6 70.5 28.1 24.3 26.2 67.9 16.5 14.5 60.9
δH (J in Hz) 2.30, m 1.46a 2.43, m 2.18, m
1.49, 1.56, 1.48, 1.72, 1.23,
m m m m m
1.51, m 1.59, m 1.21, m 1.79a 1.15, m 1.60, m 1.43, m 1.08, m 1.77, m 1.46a 1.80, m 0.96, s 0.96, s 1.12, s 1.68, m 1.62, m 1.86, m 1.79a 3.72, dd (7.5, 7.5) 1.20, s 1.11, s 1.26, s 3.82, 3.42, 0,85, 1.26, 4.12,
d (10.5) d (10.5) s t (7.0) q (7.0)
δC 34.9 29.3 175.8 77.5 50.6 21.2 34.8 40.2 42.6 41.1 21.8 27.6 43.4 50.7 31.7 25.9 49.7 15.5 20.8 86.5 23.7 36.1 26.2 83.6 71.7 27.6 24.6 26.4 67.8 16.5 14.5 60.84
Signal pattern unclear due to overlapping. mg), 3 (2.5 mg), 4 (5.0 mg), 5 (4.6 mg), and 9 (2.3 mg). (For detailed procedures on extraction and isolation, see Supporting Information.) Compound 1: colorless powder; [α]25D +45 (c 0.1, CH2Cl2); IR (KBr) νmax 3445, 2968, 1639, 1400 cm−1; 1H and 13C NMR data, see Table 1; HRESIMS m/z 499.3757 [M + Na]+ (calcd for C30H52O4Na, 499.3758). Compound 2: colorless powder; [α]25D +24 (c 0.1, CH2Cl2); IR (KBr) νmax 3442, 2966, 1640, 1400, 1384, 1124, 1081, 657, 602 cm−1; 1 H and 13C NMR data, see Table 1; HRESIMS m/z 499.3751 [M + Na]+ (calcd for C30H52O4Na, 499.3758). Compound 3: colorless powder; [α]25D +26 (c 0.1, CH2Cl2); IR (KBr) νmax 3424, 2964, 2928, 2882, 1687, 1630, 1462, 1400, 1379, 1170, 1112, 1042 cm−1; 1H and 13C NMR data, see Table 1; HRESIMS m/z 475.3780 [M + H]+ (calcd for C30H51O4, 475.3782).
gradient of MeOH−H2O (from 30% to 90% MeOH) to afford eichlerialactone (30.0 mg), methyl eichlerialactone (10.0 mg), ethyl eichlerialactone (4.0 mg), 1 (8.0 mg), 2 (2.0 mg), 10 (5.0 mg), 7 (5.2 mg), and 8 (3.3 mg). Fraction B was chromatographed on silica gel with MeOH−CH2Cl2 (from 1:40 to 1:20) to afford compounds 11 (27.2 mg), 12 (3.3 mg), 13 (5.0 mg), and 14 (4.8 mg). Fraction C was chromatographed on reversed-phase C18 silica gel eluting with MeOH−H2O (from 30% to 90% MeOH) to give methyl eichlerianate (5.0 mg), methyl shoreate (8.0 mg), ethyl shoreate (9.0 mg), 3epiocotillol (5.0 mg), cabraleadiol (7.2 mg), cabralealactone (4.3 mg), and carbraleahydroxylactone (3.4 mg). Fraction D was purified by ODS gel CC with MeOH−H2O (from 30% to 90% MeOH) to afford compounds 6 (2.0 mg), (24S)-dammar-20-ene-3α,24,25-triol (3.2 240
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first ECD was recorded immediately, and the time evolution was surveyed until stationary (approximately 0−30 min). The inherent ECD spectrum was subtracted. The absolute configuration was determined by the sign at around 310 nm in the observed ECD spectra.16,17 Cytotoxic Bioassays. All cmpounds were evaluated for cytotoxic activity by an MTT assay as described in the literature.27 Cells were obtained from the Cell Bank of the Shanghai Institute of Cell Biology. Cisplatin was used as a positive control, and the experiments were conducted in three independent replicates. NO Production Bioassays. The protocol for NO production bioassays was provided in previously published papers.28,29 Nmonomethyl-L-arginine was used as the positive control. All experiments were performed in three independent replicates.
Table 4. Cytotoxicity of Isolates against Three Cancer Cell Linesa,b sample 1 2 4 8 9 (24S)-dammar-20-ene3α,24,25-triol methyl shoreate ethyl shoreate ethyl eichlerialactone cisplatinc
A549
MCF-7
>50 >50 28.2 ± 2.0 12.4 ± 1.3 >50 35.0 ± 2.6
30.4 23.0 18.3 26.7 >50 16.7
12.9 ± 1.2 12.9 ± 1.1 >50 13.6 ± 2.1
± ± ± ±
1.3 1.7 1.6 2.4
HepG2 ± 1.1
± 1.8
20.2 >50 6.5 8.0 28.0 5.9
± ± ± ±
1.1 0.6 1.4 0.5
5.6 ± 0.6 18.4 ± 1.5 >50 10.6 ± 2.0
6.3 14.9 38.4 9.7
± ± ± ±
0.7 0.9 2.4 1.3
■
ASSOCIATED CONTENT
S Supporting Information *
Results are expressed as IC50 values in μM. bThe remaining 14 compounds were inactive for all cell lines (IC50 > 50 μM). cPositive controls. a
Details for extraction and isolation; copies of HRESIMS, 1H and13C NMR, and 2D NMR spectra of compounds 1−14; chemical structural information of the known compounds 15− 25. This material is available free of charge via the Internet at http://pubs.acs.org.
Compound 4: colorless powder; [α]25D −8 (c 0.1, CH2Cl2); IR (KBr) νmax 3441, 2988, 1654, 1637, 1459, 1403, 1127, 1102 cm−1; 1H and 13C NMR data, see Table 1; HRESIMS m/z 475.3783 [M + H]+ (calcd for C30H51O4, 475.3782). Compound 5: colorless powder; [α]25D +39 (c 0.2, CH2Cl2); IR (KBr) νmax 3445, 1638, 1400, 1124, 1098, 660, 603 cm−1; 1H and 13C NMR data, see Table 1; HRESIMS m/z 475.3785 [M + H]+ (calcd for C30H51O4, 475.3782). Compound 6: colorless powder; [α]25D +62 (c 0.03, CH2Cl2); IR (KBr) νmax 3441, 2957, 2922, 2851, 1638, 1401 cm−1; 1H and 13C NMR data, see Table 2; HRESIMS m/z 497.3603 [M + Na]+ (calcd for C30H50O4Na 497.3601). Compound 7: colorless powder; [α]25D −10 (c 0.2, CH2Cl2); IR (KBr) νmax 3428, 2927, 1755, 1633, 1454, 1400, 1207, 1108, 820, 722, 663 cm−1; 1H and 13C NMR data, see Table 2; HRESIMS m/z 415.3209 [M + H]+ (calcd for C27H43O3, 415.3207). Compound 8: colorless powder; [α]25D +98 (c 0.03, CH2Cl2); IR (KBr) νmax 3441, 1768, 1702, 1636, 1449, 1400, 1129, 1100,657, 601 cm−1; 1H and 13C NMR data, see Table 2; HRESIMS m/z 453.2976 [M + Na]+ (calcd for C27H42O4Na, 453.2975). Compound 9: colorless powder; [α]25D +25 (c 0.1, CH2Cl2); IR (KBr) νmax 3430, 2970, 2924, 1628, 1459, 1400, 1261, 1168, 1050, 883, 671, 617 cm−1; 1H and 13C NMR data, see Table 2; HRESIMS m/z 527.3704 [M + Na]+ (calcd for C31H52O5Na, 527.3707). Compound 10: colorless powder; [α]25D +90 (c 0.1, CH2Cl2); IR (KBr) νmax 3428, 2962, 2922, 1633, 1455, 1400, 1378, 1166, 1114, 1051 cm−1; 1H and 13C NMR data, see Table 2; HRESIMS m/z 489.3939 [M + H]+ (calcd for C31H53O4, 489.3938). Compound 11: colorless oil; [α]25D +42 (c 0.03, CH2Cl2); IR (KBr) νmax 3442, 2959, 2925, 1641, 1462, 1400, 1122, 1098, 657, 601 cm−1; 1H and 13C NMR data, see Table 3; HRESIMS m/z 559.3972 [M + Na]+ (calcd for C32H56O6Na, 559.3969). Compound 12: colorless oil; [α]25D +89 (c 0.1, CH2Cl2); IR (KBr) νmax 3440, 2962, 2925, 1639, 1400, 1261, 1094, 1034, 802 cm−1; 1H and 13C NMR data, see Table 3; HRESIMS m/z 559.3973 [M + Na]+ (calcd for C32H56O6Na, 559.3969). Compound 13: colorless oil; [α]25D +25 (c 0.3, CH2Cl2); IR (KBr) νmax 3442, 2966, 1710, 1635, 1457, 1384, 1163, 1124, 1095, 1037, 657, 602 cm−1; 1H and 13C NMR data, see Table 3; HRESIMS m/z 559.3971 [M + Na]+ (calcd for C32H56O6Na, 559.3969). Compound 14: colorless oil; [α]25D +28 (c 0.4, CH2Cl2); IR (KBr) νmax 3448, 2973, 1637, 1399, 1384, 1124, 1097, 657, 603 cm−1; 1H and 13 C NMR data, see Table 3; HRESIMS m/z 559.3970 [M + Na]+ (calcd for C32H56O6Na, 559.3969). C-4 Absolute Configuration of Compounds 11 and 13. Snatzke’s method was used according to the published literature.16,17 Spectroscopic grade DMSO was dried with 4 Å molecular sieves, and mixtures of 1:1.2 diol−Mo2(OAc)4 were subjected to ECD measurement at a compound concentration of 0.8 mg/mL. After mixing, the
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AUTHOR INFORMATION
Corresponding Author
*E-mail: [email protected]. Tel: +86-25-83271405. Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS The research was financially supported by the Program for Changjiang Scholars and Innovative Research Team in University (IRT1193) and the Priority Academic Program Development of Jiangsu Higher Education Institutions (PAPD).
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REFERENCES
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