Article pubs.acs.org/jnp
Isolation, Structure Elucidation, and Absolute Configuration of Highly Oxygenated Germacranolides from Carpesium cernuum Qing-Xin Liu,†,‡ Yong-Xun Yang,‡ Jian-Ping Zhang,‡ Li-Ping Chen,‡ Yun-Heng Shen,‡ Hui-Liang Li,*,‡ and Wei-Dong Zhang*,†,‡,§ †
School of Traditional Chinese Medicine, China Pharmaceutical University, Nanjing 210009, People’s Republic of China Department of Natural Product Chemistry, School of Pharmacy, Second Military Medical University, Shanghai 200433, People’s Republic of China § Shanghai Institute of Pharmaceutical Industry, Shanghai 200400, People’s Republic of China ‡
S Supporting Information *
ABSTRACT: The new highly oxygenated germacranolides cernuumolides A−J (1−10) and the known compounds 11−20 were isolated from Carpesium cernuum. Among these compounds, 1−4 are 11-methoxymethylgermacranolides and 5−7 as well as 11−17 are 2,9-hemiacetal-linked germacranolides. Their structures were elucidated using NMR and HRESIMS analyses, and X-ray diffraction studies were used to confirm the absolute configurations of 1, 2, 5, 6, 8, and 9. Cernuumolides A−J were evaluated for their in vitro cytotoxicity against the A549, HCT116, MDAMB-231, and BEL7404 cell lines, and 8 exhibited moderate cytotoxicity with IC50 values in the 0.87−2.02 μM range.
■
T
he genus Carpesium (Compositae) consists of approximately 21 plant species in central Asia. In this genus, approximately 17 species and three varieties grow in mainland China.1 The plant Carpesium cernuum, as a Chinese folk medicine, has been used as an anti-inflammatory, analgesic, and detoxifying agent.2 In previous investigations, a series of structurally diverse compounds were isolated, including sesquiterpenoid lactones, sterols, aromatic compounds, and glycosides,3−8 with the sesquiterpenoid lactones being the major constituents. The new germacranolides cernuumolides A−J (1−10) and the known compounds 11−20 were indentified in the current investigation. Notably, compounds 5−7 and 11−17 are 2,9-hemiacetal-linked germacranolides that possess two possible diastereomers.9−11 Although the relative configurations of the 2,9-hemiacetal-linked germacranolides had been confirmed by NOESY analysis and comparison of their observed and reported 1H NMR data, their absolute configurations remain undefined.12−14 Herein, the isolation and structure elucidation of cernuumolides A−J are reported. Structurally, these highly oxygenated germacranolides contain as many as nine stereogenic centers. NOESY spectra and X-ray data analysis were used to confirm the relative and absolute configurations, respectively. Finally, their cytotoxic activities were also assessed. © 2016 American Chemical Society and American Society of Pharmacognosy
RESULTS AND DISCUSSION
Cernuumolide A (1), colorless monoclinic crystals, had a molecular formula of C25H38O10 based on a sodium adduct HRESIMS ion at m/z 521.2386 (calcd 521.2363). The 1H NMR data indicated the presence of an angeloyloxy group at δH 6.15, 2.01, and 1.96 as well as a 2-methylpropanoyloxy group at δH 2.68, 1.23, and 1.22 (Table 1). The spectrum contained a methoxy group at δH 3.38 and two additional methyl groups at δH 0.92 and 1.21. The 13C NMR and DEPT spectra showed the presence of 25 carbon resonances (Table 1), which consisted of six methyl groups, a methoxy group, three methylene groups, nine methine groups, an sp2 quaternary carbon, four carbonyl carbons, and an oxygenated tertiary carbon. On the basis of the 1 H−1H COSY data, five coupled spin systems corresponding to H3-14/H-10/H-1/H-2; H-5/H-6/H-7/H-8/H-9; H-7/H-11/ H-13; H-3′/H3-4′; and H3-3″/H-2″/H3-4″ were unambiguously established (Figure 1). HMBC correlations from H3-14 (δH 0.92) to C-1 (δC 27.3), C-9 (δC 80.9), and C-10 (δC 31.0) as well as from H-2α (δH 3.95) to C-3 (δC 219.0) were observed. The C-15 methyl protons (δH 1.21) exhibited HMBC correlations with C-3, C-4 (δC 81.9), and C-5 (δC Received: April 8, 2016 Published: September 26, 2016 2479
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Chart 1
Table 1. 1H and 13C NMR Spectroscopic Data for Compounds 1−4 in Methanol-d4 1a
a
2a
position
δC
1
27.3
δH (J in Hz)
2
33.5
3 4 5 6 7 8 9 10 11 12 13
219.0 81.9 80.6 82.0 40.9 68.9 80.9 31.0 41.8 178.6 71.3
14 15 16 1′ 2′
21.6 25.2 59.6 169.6 129.6
3′ 4′ 5′ 1″ 2″ 3″
139.0 16.2 21.1 177.9 35.5 19.6
6.15, m 2.01, m 1.96, m
4″ 5″
19.5
1.23, m
α 1.89, m β 1.59, m α 3.95, m β 2.23, m
5.47, 4.55, 2.63, 4.56, 4.97, 2.18, 3.33,
d (9.3) m m m d (10.5) m m
3.46, 3.71, 0.92, 1.21, 3.38,
dd (9.7, 3.8) dd (9.7, 4.1) d (6.6) s s
2.68, m 1.22, m
δC 27.0 33.2 219.3 81.9 80.5 82.0 40.9 68.7 81.1 30.9 41.8 178.7 71.3 21.6 25.3 59.6 175.1 44.7 26.9 23.0 23.0 168.7 129.1 139.6 16.2 20.8
3a
δH (J in Hz) α 1.86, m β 1.60, m α 3.92, m β 2.21, m
δC 27.2 33.5
5.57, 4.60, 2.62, 4.53, 4.88, 2.16, 3.34,
d (9.3) m m m d (10.7) m m
3.45, 3.70, 0.92, 1.22, 3.38,
dd (9.7, 3.8) dd (9.7, 4.2) d (6.9) s s
2.31, 2.30, 2.11, 1.00, 1.01,
m m m d (6.6) d (6.6)
219.3 81.9 80.5 81.9 40.9 68.9 80.9 31.0 41.8 178.6 71.3 21.6 25.2 59.6 169.6 129.1
4a
δH (J in Hz) α 1.90, m β 1.61, m α 3.97, m β 2.22, m
5.58, 4.62, 2.66, 4.60, 4.98, 2.21, 3.35,
d (9.2) m m m d (10.5) m m
3.47, 3.72, 0.92, 1.23, 3.39,
dd (9.8, 3.7) dd (9.8, 4.1) d (6.6) s s
δC
δH (J in Hz)
27.2 33.2 219.2 82.0 80.6 81.7 40.9 68.9 80.9 31.0 41.8 178.6 71.3 21.5 25.3 59.6 169.6 129.6
6.14, m 2.01, m 1.95, m
6.17, m
139.0 16.2 20.8 168.7 129.6 139.6
6.17, m
139.1 16.2 21.1 177.5 43.0 27.8
1.99, m 1.96, m
16.2 21.1
2.00, m 1.96, m
12.3 17.6
α 1.89, m β 1.61, m α 3.93, m β 2.22, m
5.48, 4.56, 2.62, 4.58, 4.97, 2.19, 3.35,
d (9.3) m m m d (10.3) m m
3.46, 3.71, 0.92, 1.22, 3.38,
dd (9.8, 3.7) dd (9.8, 4.1) d (6.8) s s
6.16, m 2.01, m 1.95, m 2.49, 1.77, 1.51, 0.96, 1.23,
m m m m m
Recorded at 500 and 125 MHz.
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difference in the NMR spectra of 2 and 1 was the presence of an extra methylene group in the spectrum of 2 (Table 1). These results implied the presence of a 3-methylbutyryloxy rather than a 2-methylpropanoyloxy group in 2, which were confirmed by the HMBC correlations of H3-4′ (δH 1.00) and H3-5′ (δH 1.01)/C-2′ (δC 44.7) and C-3′ (δC 26.9) as well as H-2′a (δH 2.31)/C-1′ (δC 175.1) (Figure S98, Supporting Information). The HMBC correlations of H-5 (δH 5.57)/C-1″ (δC 168.7) and H-9 (δH 4.88)/C-1′ revealed that the angeloyloxy and 3-methylbutyryloxy groups were attached to C-5 and C-9, respectively. A single crystal of 2 was obtained and subjected to X-ray crystallographic analysis using Cu Kα radiation (Figure 2). The structure of cernuumolide B (2) was thus defined as (4R,5R,6S,8S,9R,11R)-5-angeloyloxy-4,8-dihydroxy-11-methoxymethyl-9-(3-methylbutyryloxy)-3-oxogermacran-6,12-olide. Cernuumolide C (3) had a molecular formula of C26H38O10 via a sodium adduct HRESIMS ion at m/z 533.2384 (calcd 533.2363). The NMR spectra of 3 resembled those of 2 except for the chemical shifts around C-2′ and C-3′. An sp2 methine (δH 6.14, δC 139.0) and an sp2 quaternary carbon (δC 129.1) were observed in 3, indicating that the 3-methylbutyryloxy group in 2 was replaced by an angeloyloxy group in 3 (Table 1). This was confirmed by the HMBC correlations (Figure S98, Supporting Information): H3-5′ (δH 1.95)/C-2′ (δC 129.1) and C-1′ (δC 169.6), as well as H3-4′ (δH 2.01)/C-3′ (δC 139.0), and H-9 (δH 4.98)/C-1′. The structure of cernuumolide C (3) was thus defined as (4R*,5R*,6S*,8S*,9R*,11R*)-5,9-diangeloyloxy-4,8-dihydroxy-11-methoxymethyl-3-oxogermacran6,12-olide. The HRESIMS data of compound 4 suggested a molecular formula of C26H40O10. Analysis of the NMR spectra of 4 suggested that it was an isomer of 2 with a difference in the locations of two of the methyl groups. The HMBC data exhibited correlations of H3-5″ (δH 1.23)/C-1″ (δC 177.5) and C-2″ (δC 43.0) as well as H3-4″ (δH 0.96)/C-3″ (δC 27.8), implying that the 3-methylbutyryloxy group in 2 was substituted by a 2-methylbutanoyloxy group in 4 (Figure S98, Supporting Information). The 2-methylbutanoyloxy and angeloyloxy groups were located at C-5 and C-9, respectively, as determined by the HMBC correlations of H-5 (δH 5.48)/C1″ and H-9 (δH 4.97)/C-1′ (δC 169.6). Therefore, compound 4, cernuumolide D, was identified as (4R*,5R*,6S*,8S*,9R*,11R*)-9-angeloyloxy-4,8-dihydroxy-11-
Figure 1. Key COSY (blue −), HMBC (red →), and NOESY (black ↔) correlations for 1.
80.6). Therefore, a 10-membered ring comprising C-1 to C-10 was present. In addition, Me-14 and Me-15 were located at C10 and C-4, respectively. The presence of a lactone moiety was established from the following HMBC correlations: H-13 (δH 3.46, 3.71) with C-11 (δC 41.8)/C-12 (δC 178.6) and H-6 (δH 4.55) with C-12. The HMBC correlation of H3-16 (δH 3.38)/ C-13 (δC 71.3) revealed the C-13 location of a methoxy group. The C-13 methylene signal (δC 71.3) represents the characteristic carbon signal of the 11-methoxymethylgermacranolides. The locations of the 2-methylpropanoyloxy group at C-5 and the angeloyloxy group at C-9 were based on the HMBC correlations of H-5 (δH 5.47)/C-1″ (δC 177.9) and H-9 (δH 4.97)/C-1′ (δC 169.6). The NOE associations of H-5/H-7α, H8/H-7α, and H-10/H-7α revealed that these protons were αoriented. The NOESY correlations from H-9 to H-10 and H315 to H-5 suggested the α-orientations of H-9 and Me-15. In addition, H-6 and H-11 had β-orientations based on the NOE correlation of H-11/H-6β. These orientations were confirmed by Cu Kα X-ray crystallographic analysis (Figure 2). Therefore, the structure of cernuumolide A (1) was defined as (4R,5R,6S,8S,9R,11R)-9-angeloyloxy-4,8-dihydroxy-11-methoxymethyl-5-(2-methylpropanoyloxy)-3-oxogermacran-6,12-olide. In order to confirm that cernuumolides A−D (1−4) are natural products, these compounds and the acetonitrile extract of the plant were subjected to LCESIMS analysis (Supporting Information). The results showed that compounds 1−4 are indeed natural products and not artifacts due to Michael addition of MeOH to the α-methylene-γ-lactone functionality. The HRESIMS data indicated that the molecular formula of compound 2 (colorless prisms) was C26H40O10 via a sodium adduct ion at m/z 535.2529 (calcd 535.2519). The main
Figure 2. ORTEP representations of 1 and 2. 2481
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deduced from the NOE associations between H-3α (δH 2.00)/ H-5 and H-3α/H-10 (δH 2.54). The NOE correlation between H-5/H3-15 supported Me-15 being α-oriented (Figure 3). These results were confirmed by Cu Kα X-ray diffraction analysis (Figure 4). The X-ray diffraction study also indicated the β-orientations of H-2 and HO-9. Therefore, the structure of cernuumolide E (5) was defined as (2S,4S,5R,6S,8R,9R)-2,9epoxy-4,8,9-trihydroxy-5-(2-methylpropanoyloxy)germacran6,12-olide. The molecular formula of cernuumolide F (colorless prisms) was assigned as C20H28O8 via a sodium adduct HRESIMS ion (m/z 419.1684, calcd 419.1682). The NMR spectroscopic data (Table 2) of 6 resembled those of 5 except that the 2methylpropanoyloxy group in 5 was substituted by an angeloyloxy group in 6. It was supported by the HMBC correlations of H3-4′ (δH 1.96)/C-3′ (δC 138.8), H3-5′ (δH 1.97)/C-2′ (δC 129.6), and H3-5′/C-1′ (δC 169.5). The HMBC correlation of H-5 (δH 4.75)/C-1′ revealed the C-5 location of the angeloyloxy group (Figure S98, Supporting Information). The NOE association of H-8 (δH 3.68) to H-7α indicated that HO-8 was β-oriented. Orthorhombic crystals of 6 were obtained and subjected to Cu Kα radiation (Figure 4). The structure of 6, cernuumolide F, was thus defined as (2S,4S,5R,6S,8S,9R)-5-angeloyloxy-2,9-epoxy-4,8,9-trihydroxygermacran-6,12-olide. The HRESIMS data of cernuumolide G (7) suggested a molecular formula of C20H28O8. A comparison of the NMR spectra of 7 with those of 6 revealed that they possessed similar structures. The only difference in the structures was the orientation of HO-8. The α-orientation of HO-8 was proposed based on the NOE association between H-8 (δH 3.61)/H-6 (δH 5.27) (Figure S98, Supporting Information). Therefore, the structure of cernuumolide G (7) was defined as (2S*,4S*,5R*,6S*,8R*,9R*)-5-angeloyloxy-2,9-epoxy-4,8,9trihydroxygermacran-6,12-olide. Cernuumolide H (8), colorless orthorhombic crystals, had a molecular formula of C24H34O9 based on HRESIMS data. The 1 H NMR spectrum of 8 exhibited characteristic signals corresponding to an angeloyloxy group and a 2-methylpropanoyloxy group. The 13C NMR (DEPT) spectrum of 8 revealed the presence of an oxygenated tertiary carbon, a ketocarbonyl group, and an α-methylene-γ-lactone moiety. The remaining
methoxymethyl-5-(2-methylbutanoyloxy)-3-oxogermacran6,12-olide. Cernuumolide E (5), colorless tetragonal crystals, had a molecular formula of C19H28O8 via a sodium adduct HRESIMS ion at m/z 407.1684 (calcd 407.1682). The NMR data suggested the presence of an exocyclic methylene group and a 2-methylpropanoyloxy group. Two partial structure sequences [i.e., CH2(3)CH(2)CH2(1)CH(10)CH3(14) and CH(5)CH(6)CH(7)CH(8)] were evident in the 1H−1H COSY spectrum (Figure 3). The C−C interconnectivity of the
Figure 3. Key COSY (blue −), HMBC (red →), and NOESY (black ↔) correlations for 5.
fragments was based on the HMBC correlations of H3-14 (δH 1.17) with C-1 (δC 38.5), C-9 (δC 108.7), and C-10 (δC 37.3); H3-15 (δH 1.21) with C-3 (δC 45.5), C-4 (δC 72.8), and C-5 (δC 80.1); and H-8 (δH 3.59) with C-9 and C-10. The above information supported the presence of a 10-membered ring from C-1 to C-10 with two methyl groups located at C-4 and C-10. The HMBC correlations of H2-13 (δH 5.92, 6.34) with C-7 (δC 48.2)/C-12 (δC 172.5) and H-6 (δH 5.25) with C12 supported the presence of an α-methylene-γ-lactone group involving C-6, C-7, C-11, C-12, and C-13. The HMBC correlations of H-2 (δH 4.55) with C-4, C-9, and C-10 confirmed that the hemiacetal linkage involves C-2 and C-9. The C-9 signal (δC 108.7) represents the characteristic carbon signal of the 2,9-hemiacetal-linked germacranolides. The 2methylpropanoyloxy residue was attached to C-5 based on the HMBC correlation of H-5 (δH 4.63) with C-1′ (δC 178.7). The NOESY correlations between H-5/H-7 (δH 3.06) and H-6/H-8 suggested that these two pairs of hydrogens were cis-oriented. These four hydrogens were placed trans to their neighboring hydrogens. Therefore, H-5 and H-7 were α-oriented, while H-6 and H-8 were β-oriented. Me-14 had a β-orientation as
Figure 4. ORTEP representations of 5 and 6. 2482
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Table 2. 1H and 13C NMR Spectroscopic Data for Compounds 5−7 in Methanol-d4 5a
a
6b
7a
position
δC
δH (J in Hz)
δC
δH (J in Hz)
δC
δH (J in Hz)
1
38.5
37.8
74.9 45.5
α 1.96, m β 1.76, m 4.59, m α 2.04, dd (15.3, 12.1) β 1.62, dd (15.5, 4.0)
38.5
2 3
α 1.91, m β 1.72, m 4.55, m α 2.00, dd (15.6, 12.0) β 1.60, dd (15.7, 3.9)
α 1.92, m β 1.73, m 4.57, m α 2.04, dd (15.5, 12.1) β 1.62, dd (15.5, 4.0)
4 5 6 7 8 9 10 11 12 13
72.8 80.1 79.7 48.2 76.7 108.7 37.3 135.5 172.5 128.5
14 15 1′ 2′ 3′ 4′ 5′
15.8 30.9 178.7 35.4 19.6 19.4
4.63, 5.25, 3.06, 3.59,
d (9.9) dd (9.9, 1.5) dd (10.7, 1.8) d (10.7)
2.54, m
5.92, 6.34, 1.17, 1.21,
m m d (6.5) s
2.66, m 1.23, d (7.0) 1.19, d (7.0)
75.1 45.1 73.1 80.9 77.7 47.1 78.4 107.0 38.6 140.2 172.6 124.1
4.75, 5.52, 3.31, 3.68,
d (10.3) dd (10.2, 2.2) m br s
2.42, m
5.86, 6.30, 1.08, 1.24,
14.1 31.1 169.5 129.6 138.8 16.1 20.9
d (2.1) d (2.3) d (6.6) m
6.10, m 1.96, m 1.97, s
75.0 45.6 72.8 80.1 79.9 48.2 76.8 108.7 37.3 135.5 172.6 128.6 15.8 31.0 169.5 129.5 138.8 16.1 20.8
4.72, 5.27, 3.10, 3.61,
d (10.0) dd (9.9, 1.5) dd (10.7, 1.8) d (10.7)
2.56, m
5.93, 6.36, 1.18, 1.24,
m m d (6.6) s
6.10, m 1.96, m 1.96, s
Recorded at 600 and 150 MHz. bRecorded at 500 and 125 MHz.
Figure 5. ORTEP representations of 8 and 9.
data indicated the presence of two methylenes, six methines including two methyl groups (i.e., one oxygenated secondary and one secondary), and four oxymethine groups. These observations revealed that 8 was a germacranolide derivative similar to those from Allagopappus viscosissimus15 except that the ester residue at C-5 was a 2-methylpropanoyloxy group and at C-9 there was an angeloyloxy group in 8. The assignments were based on the HMBC correlations of H-5/C-1″ (δC 177.9) and H-9/C-1′ (δC 169.3) (Figure S98, Supporting Information). In addition, the NOE association between H-8 (δH 4.46)/H-7 (δH 3.05) indicated that HO-8 was β-oriented,
which was supported by the Cu Kα X-ray diffraction analysis (Figure 5). Therefore, the structure of cernuumolide H (8) was defined as (4R,5R,6S,8S,9R)-9-angeloyloxy-4,8-dihydroxy-5-(2methylpropanoyloxy)-3-oxogermacran-6,12-olide. Compound 9, colorless orthorhombic crystals, had a molecular formula of C23H34O8 established by HRESIMS data. Its NMR data (Table 3) revealed a secondary methyl group, an oxygenated secondary methyl group, an oxygenated tertiary carbon, three oxymethines, and an exocyclic methylene, and its data closely resembled those of incaspitolide A.16 The major difference was the orientation of the 8-(2-methylpropa2483
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Table 3. 1H and 13C NMR Spectroscopic Data for Compound 9 in CDCl3 and Compounds 8 and 10 in Methanol-d4 8a
a
position
δC
1
27.2
2
34.8
3
219.1
4 5 6 7 8 9 10 11 12 13
81.9 79.7 81.6 43.3 72.1 80.1 31.6 134.3 171.2 125.5
14 15 1′ 2′ 3′ 4′ 5′ 1″ 2″ 3″ 4″
21.5 24.9 169.3 129.4 139.2 16.2 21.0 177.9 35.5 19.5 19.6
9a δH (J in Hz)
δC
α 1.89, m β 1.74, m α 3.87, m β 2.27, m
δH (J in Hz)
d (9.6) dd (9.5, 6.5) m d (10.3) d (10.3) m
5.71, 6.34, 0.99, 1.24,
d (2.6) d (2.9) d (6.6) s
23.1
73.1 77.6 71.4 44.2 80.8 208.4 43.7 135.0 168.6 124.1
6.16, m 2.00, m 1.95, s 2.71, m 1.24, m 1.26, m
δC
α 1.70, m β 1.79, m α 1.03, m β 1.78, m α 1.57, m β 1.34, m
32.9
34.8
5.41, 4.69, 3.05, 4.46, 5.27, 2.24,
10a
4.59, 4.74, 3.79, 5.25,
d (1.6) d (2.1) d (6.4) s
20.0 24.6 175.8 33.7 18.6 18.7
2.64, m 1.20, m 1.20, m
177.0 33.9 18.8 18.8
2.68, m 1.20, m 1.20, m
1.70, m
23.6
α 1.25, m β 1.73, m α 1.49, m β 1.33, m
73.8 79.3 73.7 45.5 83.3 211.7 44.3 137.6 171.5 125.2
2.81, m
5.87, 6.44, 1.17, 1.15,
34.3
37.6
d (6.9) dd (6.8, 2.0) m d (1.8)
δH (J in Hz)
20.6 24.3 167.7 128.2 141.0 16.1 20.6 179.1 35.1 19.3 19.4
4.65, 4.71, 4.17, 5.49,
d (6.5) dd (6.8, 2.0) m d (2.0)
3.15, m
6.13, 6.34, 1.84, 1.15,
d (1.8) d (2.1) m s
6.18, m 1.92, m 1.14, s 2.66, m 1.18, m 1.19, m
Recorded at 500 and 125 MHz.
Table 4. Cytotoxic Activities of Compounds 1−10 against the A549, HCT116, MDA-MB-231, and BEL7404 Tumor Cell Lines (Mean ± SD, n = 3) compound 1 2 3 4 5 6 7 8 9 10 DOX
A549 IC50 (μM) >100 >100 >100 90.42 >100 >100 >100 12.14 29.91 18.36 0.015
± 5.19
± ± ± ±
1.09 2.59 0.62 0.00
HCT116 IC50 (μM) 49.59 71.53 36.37 57.95 >100 37.51 >100 0.87 2.65 5.51 0.007
± ± ± ±
3.41 4.98 2.86 3.91
>100 >100 >100 >100 >100 >100 >100 2.02 7.48 7.53 0.13
± 2.03 ± ± ± ±
MDA-MB-231 IC50 (μM)
0.05 0.21 0.29 0.00
noyloxy) group. The NOE correlation from H-8 (δH 5.25) to H-7 (δH 3.79) indicated the α-orientation of H-8 (Figure S98, Supporting Information). Finally, crystals of 9 were available from a mixture of MeOH and H2O, and the Cu Kα X-ray diffraction analysis was used to determine its absolute configuration (Figure 5). The structure of 9, cernuumolide I, was thus defined as (4S,5R,6S,8S)-5,8-di(2-methylpropanoyloxy)-4-hydroxy-9-oxogermacran-6,12-olide. The HRESIMS data of cernuumolide J (10) showed a molecular formula of C24H34O8. The NMR data (Table 3) of 10 resembled those of 9, except that the 2-methylpropanoyloxy signals of 9 were replaced by signals corresponding to an angeloyloxy group in 10. The HMBC correlations of H-8 (δH
± ± ± ±
0.12 0.63 0.51 0.01
BEL7047 IC50 (μM) 71.71 79.00 88.08 62.87 81.85 69.57 56.38 1.80 7.32 7.35 0.13
± ± ± ± ± ± ± ± ± ± ±
3.64 4.12 3.44 4.73 5.13 2.65 3.71 0.09 0.49 0.55 0.01
5.49) with C-1′ (δC 167.7) indicated the C-8 location of the angeloyloxy group (Figure S98, Supporting Information). Therefore, the structure of cernuumolide J (10) was defined as (4S*,5R*,6S*,8S*)-8-angeloyloxy-4-hydroxy-5-(2-methylpropanoyloxy)-9-oxogermacran-6,12-olide. Seven 2,9-hemiacetal-linked germacranolides including (2S,4S,5R,6S,8R,9R,2″S)-8-angeloyloxy-4,9-dihydroxy-2,9epoxy-5-(2-methylbutanoyloxy)germacran-6,12-olide (11),12 (2S,4S,5R,6S,8R,9R,2″R)-8-angeloyloxy-4,9-dihydroxy-2,9epoxy-5-(2-methylbutanoyloxy)germacran-6,12-olide (12),12 (2S,4S,5R,6S,8R,9R)-5,8-diangeloyloxy-4,9-dihydroxy-2,9epoxygermacran-6,12-olide (13),12 (2S*,4S*,5R*,6S*,8R*,9R*)-8-angeloyloxy-4,9-dihydroxy-2,92484
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epoxy-5-(2-methylpropanoyloxy)germacran-6,12-olide (14),14 (2S*,4S*,5R*,6S*,8R*,9R*)-8-angeloyloxy-4,9-dihydroxy-2,9epoxy-5-(3-methylbutyryloxy)germacran-6,12-olide (15), 17 (2R,4S,5R,6S,8R,9S,2″S)-8-angeloyloxy-4,9-dihydroxy-2,9epoxy-5-(2-methylbutanoyloxy)germacran-6,12-olide (16),12 and (2R*,4S*,5R*,6S*,8R*,9S*)-8-angeloyloxy-4,9-dihydroxy2,9-epoxy-5-(2-methylpropanoyloxy)germacran-6,12-olide (17)14 were established by analysis of their NMR data and comparison with literature values. These results were confirmed by Cu Kα X-ray diffraction analysis of compounds 11−13 and 16 (Figure S97, Supporting Information). The essential structural difference between compounds 11−15 and compounds 16 and 17 lies in the configuration of the hemiacetal linkage across the germacrane ring. The ether oxygen atom was α-oriented in compounds 11−15 and β-oriented in compounds 16 and 17. Three known compounds, namely, incaspitolide D (18),18 (4R*,5R*,6S*,8R*,9R*)-5-angeloyloxy-4,8-dihydroxy-9-(2methylpropanoyloxy)-3-oxogermacran-6,12-olide (19),15 and (4R*,5R*,6S*,8R*,9R*)-4,8-dihydroxy-5-(2-methylpropanoyloxy)-9-(3-methylbutyryloxy)-3-oxogermacran-6,12-olide (20),18 were also isolated. Their structures were established by comparison with their observed and reported NMR and MS data. The cytotoxic activities of compounds 1−10 against A549, HCT116, MDA-MB-231, and BEL7404 cell lines were evaluated using the MTT assay, and the positive control was doxorubicin (Table 4). Among the tested compounds, 8 exhibited moderate cytotoxicity against the HCT116, MDAMB-231, and BEL7404 cells with IC50 values of 0.87, 2.02, and 1.80 μM, respectively.
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g) was separated into 20 additional fractions (3A−3T) by a silica gel column eluted with mixtures of petroleum ether and EtOAc (from 5:1 to 1:1). Fr. 3K (8 g) was chromatographed using a C18 silica gel column with gradient elution using MeOH−H2O (from 30:70 to 100:0) to afford seven subfractions (3K1−3K7). Fr. 3K4 (1 g) was chromatographed on a Zorbax SB-C18 column (MeOH−H2O, 55:45) to yield compounds 1 (13 mg), 2 (9 mg), 3 (4 mg), and 4 (12 mg). Fr. 3K7 (0.6 g) was separated using the same method to give compounds 5 (10 mg), 6 (7 mg), and 7 (6 mg). Fr. 3K1 (0.9 g) was separated using the same method to afford compounds 8 (22 mg), 9 (9 mg), and 10 (10 mg). Cernuumolide A (1): colorless, monoclinic crystals (MeOH−H2O); mp 270−272 °C; [α]20D −106 (c 0.3, MeOH); UV (MeOH) λmax (log ε) 203.0 (4.26) nm; IR νmax (KBr) cm−1 3454, 2976, 2931, 1770, 1716, 1460, 1383, 1188, 1161; NMR data see Table 1; HRESIMS m/z 521.2386 [M + Na]+ (calcd C25H38O10Na, 521.2363). Cernuumolide B (2): colorless, hexagonal crystals (MeOH−H2O); mp 275−277 °C; [α]20D −151 (c 0.3, MeOH); UV (MeOH) λmax (log ε) 203.0 (4.22) nm; IR νmax (KBr) cm−1: 3446, 2968, 2935, 1755, 1716, 1458, 1369, 1184, 1151, 1038; NMR data see Table 1; HRESIMS m/z 535.2529 [M + Na]+ (calcd C26H40O10Na, 535.2519). Cernuumolide C (3): white, amorphous powder; [α]20D −83 (c 0.3, MeOH); UV (MeOH) λmax (log ε) 203.0 (4.27) nm; IR νmax (KBr) cm−1 3446, 2929, 1788, 1720, 1674, 1456, 1385, 1273, 1140; NMR data see Table 1; HRESIMS m/z 533.2384 [M + Na]+ (calcd C26H38O10Na, 533.2363). Cernuumolide D (4): white, amorphous powder; [α]20D −61 (c 0.3, MeOH); UV (MeOH) λmax (log ε) 203.0 (4.25) nm; IR νmax (KBr) cm−1 3452, 2924, 1770, 1716, 1633, 1468, 1383, 1234, 1186, 1074, 1034; NMR data see Table 1; HRESIMS m/z 535.2537 [M + Na]+ (calcd C26H40O10Na, 535.2519). Cernuumolide E (5): colorless, tetragonal crystals (MeOH); mp 226−228 °C; [α]20D +24 (c 0.3, MeOH); UV (MeOH) λmax (log ε) 209.0 (4.12) nm; IR vmax (KBr) cm−1 3450, 2924, 2548, 1743, 1660, 1468, 1344, 1236, 1142, 1039; NMR data see Table 2; HRESIMS m/z 407.1684 [M + Na]+ (calcd C19H28O8Na, 407.1682). Cernuumolide F (6): colorless, orthorhombic crystals (MeOH); mp 228−230 °C; [α]20D −27 (c 0.3, MeOH); UV (MeOH) λmax (log ε) 217.0 (4.50) nm; IR νmax (KBr) cm−1 3504, 2929, 1774, 1714, 1649, 1456, 1396, 1232, 1132, 1051; NMR data see Table 2; HRESIMS m/z 419.1684 [M + Na]+ (calcd C20H28O8Na, 419.1682). Cernuumolide G (7): white, amorphous powder; [α]20D +21 (c 0.3, MeOH); UV (MeOH) λmax (log ε) 216.0 (4.51) nm; IR νmax (KBr) cm−1 3446, 2974, 2935, 1766, 1647, 1460, 1385, 1232, 1155, 1041; NMR data see Table 2; HRESIMS m/z 419.1685 [M + Na]+ (calcd C20H28O8Na, 419.1682). Cernuumolide H (8): colorless, orthorhombic crystals (MeOH− H2O); mp 254−256 °C; [α]20D −57 (c 0.3, MeOH); UV (MeOH) λmax (log ε) 203.0 (4.22) nm; IR νmax (KBr) cm−1 3467, 2978, 2877, 1753, 1714, 1458, 1356, 1228, 1157, 1078; NMR data see Table 3; HRESIMS m/z 489.2111 [M + Na]+ (calcd C24H34O9Na, 489.2101). Cernuumolide I (9): colorless, orthorhombic crystals (MeOH); mp 239−241 °C; [α]20D −31 (c 0.3, MeOH); UV (MeOH) λmax (log ε) 203.0 (4.21) nm; IR νmax (KBr) cm−1 3516, 2979, 2937, 1776, 1749, 1722, 1469, 1356, 1275, 1203, 1124, 1061; NMR data see Table 3; HRESIMS m/z 461.2164 [M + Na]+ (calcd C23H34O8Na, 461.2151). Cernuumolide J (10): white, amorphous powder; [α]20D −19 (c 0.3, MeOH); UV (MeOH) λmax (log ε) 203.0 (4.23) nm; IR νmax (KBr) cm−1 3479, 2974, 2927, 1776, 1720, 1460, 1385, 1275, 1230, 1149, 1032; NMR data see Table 3; HRESIMS m/z 473.2170 [M + Na]+ (calcd C24H34O8Na, 473.2151). The crystallographic data for compounds 1, 2, 5, 6, 8, and 9 were deposited at the Cambridge Crystallographic Data Center with deposition numbers CCDC 1420441, 1424496, 1046396, 1052088, 1420617, and 1413200, respectively. Crystallographic data are given in the Supporting Information. Cytotoxicity Assays. Human cancer cells (A549, HCT116, MDAMB-231, and BEL7404) were cultured in 96-well plates for 72 h with 5% CO2 at 37 °C. The MTT assay was used to evaluate the survival
EXPERIMENTAL SECTION
General Experimental Procedures. Optical rotations were measured with a PerkinElmer model 341 polarimeter. UV spectra were recorded on the diode array detector on the HLPC (Agilent 1200). IR spectra were measured with a Bruker FTIR Vector 22 spectrometer. NMR spectra were recorded on Bruker Ascend-500 and Ascend-600 spectrometers. HRESIMS data were measured with an Agilent-6520Q-TOF mass spectrometer. LCESIMS analysis was done on an Agilent 6410B triple-stage quadrupole mass spectrometer equipped with an ESI ion source and an Agilent 1290 HPLC system. The following chromatographic substrates were utilized: Sephadex LH-20 (40−70 μm; Pharmacia Co., Ltd.); silica gel (200−300 mesh; Huiyou Silica Gel Development Co., Ltd.); macroporous resin HP-20 and YMC-gel ODS-A (50 μm; YMC, Milford, MA); DMSO (Merck, Sharp & Dohme, Ltd.); MTT (Sigma Chemical Co.); and cell lines (i.e., A549, HCT116, HL-60, and SK-Hep-1) (Shanghai Institute of Pharmaceutical Industry). The X-ray data were acquired on a Bruker SMART APEX II DUO instrument. Semipreparative HPLC was performed on an Agilent series 1260 HPLC instrument using a Zorbax SB-C18 column (5 μm particle size, 9.4 mm × 150 mm). An isocratic elution program employed MeOH−H2O (55:45, v/v). The detection wavelengths were 210 and 254 nm, and the flow rate was 2.0 mL/min. Plant Material. The whole Carpesium cernuum L. plant was collected in July 2014 from Longli County, Guizhou Province, People’s Republic of China, and identified by Prof. Bao-Kang Huang of the Second Military Medical University. A voucher specimen (GZzqsy470) was deposited at the Second Military Medical University. Extraction and Isolation. The dried, powdered whole C. cernuum plant (20.0 kg) was extracted with 95% EtOH (3 × 60 L) to afford a crude residue (2.5 kg), which was suspended in H2O and partitioned with EtOAc. The EtOAc portion (1 kg) was subjected to a macroporous resin column with gradient elution using MeOH−H2O (from 30:70 to 100:0) to yield five major fractions (1−5). Fr. 3 (170 2485
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rates of the cancer cells,19 and results were expressed as the mean value of triplicate determinations. The positive control was doxorubicin.
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(15) Gonzalez, A. G.; Bermejo, B. J.; Triana, M. J.; Eiroa, M. J.; Lopez, S. M. Phytochemistry 1992, 31, 330−331. (16) Gonzalez, A. G.; Bermejo, J.; Triana, J.; Eiroa, J. L.; Lopez, M. J. Nat. Prod. 1995, 58, 432−437. (17) Kim, M. R.; Suh, B. R.; Kim, J. G.; Kim, Y. H.; Kim, D. K.; Moon, D. C. Phytochemistry 1999, 52, 113−115. (18) Gao, X.; Lin, C. J.; Jia, Z. J. J. Nat. Prod. 2007, 70, 830−834. (19) Alley, M. C.; Scudiero, D. A.; Monks, A.; Hursey, M. L.; Czerwinski, M. J.; Fine, D. L.; Abbott, B. J.; Mayo, J. G.; Shoemaker, R. H.; Boyd, M. R. Cancer Res. 1988, 48, 589−601.
ASSOCIATED CONTENT
S Supporting Information *
The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jnatprod.6b00315. Crystallographic data (CIF) Crystallographic data (CIF) Crystallographic data (CIF) Crystallographic data (CIF) Crystallographic data (CIF) Crystallographic data (CIF) Crystallographic data (CIF) Crystallographic data (CIF) Crystallographic data (CIF) Crystallographic data (CIF) Physicochemical data and original spectra (PDF)
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AUTHOR INFORMATION
Corresponding Authors
*Fax (H.-L. Li): +86 (0)21 81871244. Tel: +86 (0)21 81871244. E-mail:
[email protected]. *Fax (W.-D. Zhang): +86 (0)21 81871244. Tel: +86 (0)21 81871244. E-mail:
[email protected]. Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS The work was supported by the National Nature Science Foundation of China (81102335, 81230090, 81473109, 81520108030).
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