Article pubs.acs.org/jnp
Cembrane-Type Diterpenoids from the Chinese Liverworts Chandonanthus hirtellus and C. birmensis Rui-Juan Li,†,§ Zhao-Min Lin,†,§ Ya-Qi Kang,† Yan-Xia Guo,‡ Xin Lv,† Jin-Chuan Zhou,† Song Wang,† and Hong-Xiang Lou*,† †
Department of Natural Products Chemistry, Key Lab of Chemical Biology of the Ministry of Education, School of Pharmaceutical Science, Shandong University, Jinan 250012, People’s Republic of China ‡ Department of Biochemistry and Molecular Biology, School of Medicine, Shandong University, Jinan 250012, People’s Republic of China S Supporting Information *
ABSTRACT: Six new cembrane-type diterpenoids (1−6) were isolated from two species of Chandonanthus: Chandonanones A, B, and D−F (1, 2, and 4−6) were isolated from C. hirtellus, and chandonanones B, C, E, and F (2, 3, 5, and 6) from C. birmensis. Five known diterpenoids, (8E)4α-acetoxy-12α,13α-epoxycembra-1(15),8-diene (7), isochandonanthone (8), chandonanthone (9), anadensin (10), and 2,10,14triacetoxy-7,8,18,19-diepoxydolabell-3(E)-ene (11), were also obtained. The structures of the new metabolites were established by analyses of their spectroscopic data (1D NMR, 2D NMR, HRESIMS, and IR). The absolute configurations of compounds 1 and 2 were unequivocally confirmed using single-crystal X-ray diffraction analysis with Cu Kα radiation. Cytotoxicity tests of the isolated diterpenoids against seven cancer cell lines (DU145, PC3, A549, PC12, NCI-H292, NCI-H1299, and A172) revealed that some of the diterpenoids had weak activity.
L
2,10,14-triacetoxy-7,8,18,19-diepoxydolabell-3(E)-ene (11),6 were also obtained. Herein, we report the isolation, structure elucidation, and cytotoxicity testing of all of the compounds.
iverworts are distinguished from other phyla (mosses and hornworts) by characteristic cellular oil bodies that consist of hydrophobic terpenoids and aromatic compounds, many of which possess intriguing biological properties, such as antifungal, cytotoxic, antimicrobial, insect antifeedant, and multidrug resistance (MDR) reversal activities.1−4 Previous chemical investigations showed that the majority of the constituents of the genus Chandonanthus (Lophoziaceae), such as cembrane- and dolabellane-type diterpenoids, are structurally similar to those of marine organisms.1,5−11 Furthermore, cembrane-type diterpenoids are rare in liverworts and have been reported only from this genus so far.7 Thus, cembrane-type diterpenoids are considered to be chemotaxonomic markers of Chandonanthus in liverworts.9 In the course of our ongoing search for new bioactive metabolites from Chinese liverworts, we investigated the chemical constituents of the leafy liverworts C. hirtellus and C. birmensis, which are distributed throughout the southern and eastern regions of Asia12 and were collected in the Mount Mao’er, Guangxi Zhuang Autonomous Region, People’s Republic of China, in April 2011. Six new cembrane-type diterpenoids (1−6) were isolated from these two species: chandonanones A, B, and D−F (1, 2, and 4−6) were isolated from C. hirtellus, and chandonanones B, C, E, and F (2, 3, 5, and 6) from C. birmensis. Five known diterpenoids, (8E)-4αacetoxy-12α,13α-epoxycembra-1(15),8-diene (7),7 isochandonanthone (8),5 chandonanthone (9),5 anadensin (10),7 and © 2014 American Chemical Society and American Society of Pharmacognosy
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RESULTS AND DISCUSSION Chandonanone A (1), which was isolated as colorless needles, was assigned the molecular formula C22H34O5 based on 13C NMR data and a quasi-molecular ion at m/z 396.2746 [M + NH4]+ (calcd 396.2745) by HRESIMS, requiring six indices of hydrogen deficiency. The IR spectrum revealed absorption bands for ester carbonyl (1728 cm−1), keto carbonyl (1702 cm−1), and unsaturated carbonyl (1676 cm−1) groups, which were supported by the presence of resonances at δC 170.3 (4OCOCH3), 214.0 (C-7), and 196.4 (C-14), respectively, in the 13 C NMR spectrum. The remaining oxygen atom was determined to be part of an oxirane moiety, which was confirmed by the appearance of resonances at δH 3.49 (s, H-13) and δC 64.6 (C-13) and 64.8 (C-12) in the 1D NMR spectra. Further analyses of the 1D NMR data (Tables 1 and 2) of 1 displayed two vinyl methyls [δH 1.86 (s, H3-16) and 2.06 (s, H3-17)], one secondary methyl [δH 1.04 (d, J = 6.7 Hz, H319)], three tertiary methyls [δH 1.54 (s, H3-18), 1.23 (s, H3-20), and 1.99 (s, 4-OCOCH3)], seven methylenes, two methines, of Received: November 11, 2013 Published: February 3, 2014 339
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9. Thus, the gross structure of 1 was determined to be that depicted. In the NOESY spectrum of 1, the correlations between H13/H-11β/H-8 suggested that H-8 and H-13 adopted βorientations. The NOE cross-peak between H-11α/H3-20 established that Me-20 was α-oriented (Figure 1). To confirm the structure and determine its absolute configuration, compound 1 was crystallized from MeOH to afford a crystal of the monoclinic space group P21, which was analyzed using Xray crystallography (Figure 2). Single-crystal X-ray diffraction analysis using the anomalous scattering of Cu Kα radiation yielded a Flack parameter of 0.09(16) (CCDC 969045), which permitted the unambiguous assignment of the absolute configuration as 4R, 8S, 12S, and 13R. Chandonanone B (2) had the same molecular formula, C22H34O5, as 1 by 13C NMR data and HRESIMS (m/z 401.2296 [M + Na]+). The 1D NMR data (Tables 1 and 2) of 2 and 1 were closely comparable. Analyses of 2D NMR data revealed that compounds 2 and 1 had the same molecular structures and were likely a pair of diastereomers. X-ray crystallographic analysis using a single crystal of compound 2 and Cu Kα radiation (CCDC 969046) confirmed the structure and revealed that compound 2 was the C-8 epimer of compound 1. Thus, the absolute configuration of 2 is 4R, 8R, 12S, and 13R (Figure 2). The HRESIMS of chandonanone C (3) revealed an [M + H]+ ion peak at m/z 377.2322 (calcd 377.2323, C22H33O5), indicating one additional index of hydrogen deficiency compared to 1. The NMR data (Tables 1 and 2) of 3 were similar to those of 1, with a noticeable difference being the presence of trisubstituted double-bond resonances [δH 6.72 (br s, H-9); δC 139.0 (C-8) and 143.0 (C-9)], which were
which one was oxygenated (CH-13), two oxygenated quaternary carbons [C-4 (δC 83.7) and C-12], one conjugated double bond [δC 131.9 (C-1) and 150.5 (C-15)], one ester carbonyl carbon (4-OCOCH3), and two keto carbonyl carbons (C-7 and C-14). These data suggested a cembrane skeleton similar to that of (8E)-4α-acetoxy-12α,13α-epoxycembra-1(15),8-diene (7), which was isolated by Komala et al. from C. hirtellus.7 The 1H−1H COSY correlations (Figure 1) of 1 established three segments: C-2(H2)−C-3(H2), C-5(H2)−C6(H 2 ), and C-19(H 3 )−C-8(H)−C-9(H 2 )−C-10(H 2 )−C11(H2). The keto carbonyl at C-7 was confirmed using HMBC correlations (Figure 1) from H3-19 to C-7, C-8, and CTable 1. 1H NMR Spectroscopic Data for Compounds 1−6a 1b
position 2a 2b 3a 3b 5a 5b 6a 6b 7 8 9a 9b 10a 10b 11a 11b 13 16 17 18 19 20 4-OAc 7-OH a
2b
2.49 2.10 2.00 1.77 2.02 2.33 2.36 2.46
m td (13.0, 4.7) md m td (13.0, 4.7) m m m
2.35 2.15 1.67 1.61 2.33 2.10 2.55 2.41
m dd (13.2, 4.2) m m m m dt (18.6, 6.6) dt (18.6, 6.6)
2.74 1.78 1.43 1.62 1.10 2.00 1.39 3.49 1.86 2.06 1.54 1.04 1.23 1.99
m m m m m md (α-H) m (β-H) s s s s d (6.7) s s
2.62 1.65 1.51 1.44 1.35 2.11 1.32 3.53 1.80 1.89 1.54 1.06 1.31 1.97
s (6.6) m (α-H) m (β-H) m m m (α-H) m (β-H) s s s s d (6.6) s s
3b 2.37 2.18 1.83 1.44 2.58 2.09 2.82 2.35
4b
m m m m m m m md
2.35 m 2.20 1.50 1.84 1.48 1.70 1.46 4.02
m m md m m md d (9.2)
6.72 br s
5.40 br s
2.45 2.35 2.21 1.89 3.46 1.80 1.92 1.55 1.81 1.34 2.03
2.30 2.06 2.08 1.88 3.60 1.85 1.99 1.45 1.65 1.22 2.01
m md m (α-H) m (β-H) s s s s s s s
m m m (α-H) m (β-H) s s s s s s s
5c
6c
2.79 2.57 2.06 1.73 2.66 2.56 4.08
m m md m t like (11.5) m dd (8.6, 7.5)
2.98 ddd (14.0, 12.8, 6.0) 2.48 t (13.2) 2.10 md
2.89 1.96 1.76 4.63
m m m dd (10.6, 9.4)
2.08 2.11 1.73 4.38
md md (α-H) m (β-H) m
2.10 1.47 3.53 1.91 1.94 1.50 1.13 1.29 1.98
d (13.5) (α-H) d (13.5) (β-H) br s s s s d (4.8) s s
2.01 1.40 3.67 1.94 2.05 1.52 1.05 1.25 2.02 4.28
m (α-H) t (13.0) (β-H) s s s s d (6.2) s s s
1.99 m 1.37 m 2.18 m
Chemical shifts (δ) are expressed in ppm, and J values are presented in Hz. bRecorded at 600 MHz in CDCl3. cRecorded at 600 MHz in acetone-d6. Signals overlapped.
d
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Table 2. 13C NMR Spectroscopic Data (δ) for Compounds 1−6a
a
position
1b
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 4-OAc
131.9 23.1 37.9 83.7 29.5 39.0 214.0 43.9 34.4 23.4 37.6 64.8 64.6 196.4 150.5 22.8 23.8 23.9 17.2 15.5 170.3 22.5
2b s t t s t t s d t t t s d s s q q q q q s q
134.2 23.8 38.1 83.6 29.9 36.1 213.8 46.0 33.9 23.9 37.3 65.3 63.7 201.3 142.6 21.3 22.9 23.9 16.3 16.4 170.3 22.4
3b s t t s t t s d t t t s d s s q q q q q s q
133.4 23.3 38.5 83.8 35.0 31.7 203.2 139.0 143.0 23.6 35.7 63.7 63.5 199.7 144.9 21.7 23.1 23.6 12.0 16.5 170.0 22.5
4b s t t s t t s s d t t s d s s q q q q q s q
133.0 23.4 37.0 83.7 33.9 29.7 77.6 137.8 127.5 21.7 35.2 63.2 63.6 198.2 147.4 22.8 23.6 23.7 12.1 18.3 170.5 22.5
5c s t t s t t d s d t t s d s s q q q q q s q
134.9 22.3 39.2 86.0 31.0 89.0 163.2 34.0 38.9 77.0 45.6 62.7 65.5 197.6 147.3 22.3 23.3 24.5 17.4 14.7 170.6 22.5
6c s t t s t d s d t d t s d s s q q q q q s q
133.8 22.1 38.0 85.7 24.1 33.8 107.7 41.3 38.3 73.7 47.5 63.1 66.0 196.9 149.4 22.5 22.9 23.7 13.5 14.7 170.6 22.1
s t t s t t s d t d t s d s s q q q q q s q
Chemical shifts (δ) are expressed in ppm. bRecorded at 150 MHz in CDCl3. cRecorded at 150 MHz in acetone-d6.
Figure 1. Key HMBC (solid arrows), 1H−1H COSY (bold lines), and NOESY (dashed arrows) correlations for compound 1.
supported by HMBC correlations of H3-19 with C-7, C-8, and C-9 and 1H−1H COSY correlations of H-9/H2-10/H2-11 (Figure S21). NOE correlations of H-13/H-11β and H-11α/ H3-20 suggested β- and α-orientations for H-13 and Me-20, respectively. The β-orientation of Me-18 was determined by the NOE correlations of H-13/Ha-3/H3-18. Moreover, the NOE cross-peak between H-9 and H-13 established the E configuration of the Δ8(9) double bond (Figure S22). Thus, the structure of 3 was determined to be that shown. The molecular formula of chandonanone D (4) was deduced to be C22H34O5 by 13C NMR data and HRESIMS, suggesting one fewer index of hydrogen deficiency than 3. Accordingly, in the 1D NMR spectra of 4, the resonances of an oxygenated methine [δH 4.02 (d, J = 9.2 Hz, H-7); δC 77.6 (C-7)] provided evidence that the C-7 carbonyl group in 3 was replaced by a hydroxy methine group in 4, which was confirmed by 1H−1H COSY correlations of H2-5/H2-6/H-7 and HMBC correlations between H-7 and C-5 (δC 33.9), C-9 (δC 127.5), and C-19 (δC
12.1) (Figure S32). NOE cross-peaks of H3-17/H-13/H-11β/ H-9/H-7 and H3-16/Ha-3/H3-18 suggested that H-7, H-13, and Me-18 were β-oriented. The α-orientation of Me-20 was determined by the NOE correlation of H3-20/H-11α (Figure S33). The HRESIMS and 13C NMR data of chandonanone E (5) revealed a molecular formula of C22H32O5, indicating that 5 possessed seven indices of hydrogen deficiency. The 1H and 13 C NMR resonance patterns (Tables 1 and 2) of 5 were similar to those of isochandonanthone (8) and chandonanthone (9), which were isolated by Shy et al. from C. hirtellus.5 Analyses of the NMR data of 5 indicated that an oxygen linkage between C-7 (δC 163.2) and C-4 (δC 86.0) was absent. In contrast, an acetoxy group [δH 1.98 (s, H3); δC 22.5 and 170.6] was present at C-4. The HMBC correlation between H-10 (δH 4.63, dd, J = 10.6, 9.4 Hz) and C-7 established the oxygen linkage between C-7 and C-10 (δC 77.0). Furthermore, a trisubstituted double bond Δ6(7) was shown by the HMBC 341
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and 2) of 6 were similar to those of 5, apart from the absence of resonances for the Δ6(7) double bond, the appearance of an exchangeable proton resonance at δH 4.28 (s, 7-OH), and an additional methylene at δH 2.18 (m, H2-6) in 6. These observations suggested that the Δ6(7) double bond in 5 was hydrated in 6, which was further confirmed by HMBC correlations between 7-OH and C-6 (δC 33.8), C-7 (δC 107.7), and C-8 (δC 41.3) and 1H−1H COSY correlations of H2-5/H2-6 (Figure S52). Therefore, C-7 was determined to be a hemiketal carbon. The similar NOE correlation patterns of 6 and 5 indicated that they have the same relative configurations, with the exception that 7-OH had an α-orientation, as determined by the NOE correlations of H-9α/7-OH/H3-19 (Figure S53). Compounds 5 and 6 were obtained as a mixture from C. birmensis. They were also isolated as a mixture from C. hirtellus and then further separated using HPLC. However, they interconverted quickly as a result of the reversible hydration and dehydration processes. The proposed hydration and dehydration reactions are shown in Figure S65.13,14 The known diterpenoids 7, 8, and 11 from C. hirtellus and 8, 9, and 10 from C. birmensis were identified as (8E)-4α-acetoxy12α,13α-epoxycembra-1(15),8-diene (7),7 isochandonanthone (8),5 chandonanthone (9),5 anadensin (10),7 and 2,10,14triacetoxy-7,8,18,19-diepoxydolabell-3(E)-ene (11)6 by comparing their observed and reported spectroscopic data. Cembrane-type diterpenoids are also widespread in marine organisms.11,15 Such chemical similarities suggest that the genus Chandonanthus and some families of marine organisms may have an evolutionary relationship. Meanwhile, this research provides further evidence for cembrane-type diterpenoids as the chemotaxonomic markers of Chandonanthus in liverworts. The cytotoxicities of compounds 1−11 were tested against the DU145 and PC3 human prostate carcinoma cell lines, A549, NCI-H292, and NCI-H1299 human lung carcinoma cell lines, PC12 rat pheochromocytoma cell line, and A172 human astrocyte cell line using the MTT method;16 cisplatin was used as the positive control (Table 3). However, the majority of the compounds were inactive (IC50 > 50 μM) or showed weak activity (IC50 20−50 μM). Compound 1 exhibited weak cytotoxic activities, with IC50 values of 24.5 ± 1.1 and 17.2 ± 0.6 μM against PC12 and NCI-H1299 cells, respectively, and
Figure 2. X-ray crystallographic structures of 1 and 2.
correlations of H-6 (δH 4.08, d, J = 8.6, 7.5 Hz) with C-8 (δC 34.0) and H3-19 (δH 1.13, d, J = 4.8 Hz) with C-7 as well as the 1 H−1H COSY correlations of H2-5/H-6 (Figure 3). The observed NOE correlations between H-13/H-11β/H-8/H-6/ H3-18 showed that H-8, H-13, and Me-18 were β-oriented, while the NOE correlations of H3-20 with H-11α and H-10 revealed α-orientations for H-10 and Me-20 (Figure 3). Chandonanone F (6) was assigned the molecular formula C22H34O6 based on 13C NMR data and HRESIMS, which showed 18 mass units more than 5. The NMR data (Tables 1
Figure 3. Key HMBC (solid arrows), 1H−1H COSY (bold lines), and NOESY (dashed arrows) correlations for compound 5. 342
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Table 3. Cytotoxicity of Compounds 1−11 against Seven Cell Linesa IC50 (μM)
a
compound
DU145
PC3
A549
1 2 3 4 5 6 7 8 9 10 11 cisplatinc
43.7 ± 0.1 b 29.2 ± 0.6
24.3 ± 1.0 44.9 ± 0.7 19.9 ± 0.4
31.1 ± 0.7 21.5 ± 1.1 25.2 ± 1.1
PC12 24.5 ± 43.8 ± 40.6 ± 30.2 ± 41.1 ± 37.1 ± 47.1 ± 36.4 ± 31.1 ± 31.9 ±
1.1 2.2 0.2 0.8 3.7 1.4 3.1 0.4 0.1 1.0
NCI-H292
NCI-H1299
21.4 ± 38.9 ± 48.7 ± 48.1 ± 18.7 ±
17.2 ± 19.5 ± 40.3 ± 20.9 ± 28.3 ± 23.5 ± 41.6 ± 34.7 ± 21.9 ±
0.5 0.8 0.7
1.6
0.5
0.6 1.0 1.2 0.9 0.7 0.4 1.1
0.3 0.9
A172 23.1 ± 31.9 ± 37.3 ± 20.7 ±
0.9 1.0 1.8
1.0
The values presented are the means ± SD of triplicate experiments. b“” not active, IC50 values >50 μM. cPositive control substance. 60:40 to 100:0), to give four subfractions (2AA−2AD). Fraction 2AA (27 mg) was purified using semipreparative HPLC (MeOH−H2O, 68:32, 1.8 mL/min) to yield 2 (10.0 mg, tR = 22.5 min). Fraction 2AB (84 mg) was separated using silica gel CC (800 mg, 1.5 cm × 5 cm, 200−300 mesh, petroleum ether−acetone, 80:1) to give 8 (12.0 mg) and a mixture (23 mg), which was further separated using HPLC (MeCN−H2O, 55:45, 1.0 mL/min) to yield 6 (6.5 mg, tR = 5.1 min) and 5 (11.6 mg, tR = 25.3 min). Fraction 2AD was subjected to semipreparative HPLC (MeOH−H2O, 85:15, 1.8 mL/min) to give 11 (2.3 mg, tR = 25.1 min) and 7 (4.0 mg, tR = 17.7 min). Fraction 2B (20 mg) was purified using Sephadex LH-20 CC (2 cm × 120 cm, MeOH) to yield 1 (13.0 mg). Fraction 2C (246 mg) was separated using Sephadex LH-20 CC (2 cm × 120 cm, MeOH), followed by RP C18 Lobar CC (MeOH−H2O, 60:40 to 100:0), to give three subfractions (2CA−2CC). Fraction 2CC (85 mg) was further purified using semipreparative HPLC (MeOH−H2O, 70:30, 1.8 mL/min) to yield 4 (8.0 mg, tR = 31.2 min). The air-dried powder of the whole plant material of C. birmensis (300 g) was extracted using 95% EtOH at room temperature (1.8 L × 3 each for one week). The crude extract (25.6 g) was chromatographed using MCI gel CC (25 g, 2.5 cm × 25 cm, MeOH−H2O, 3:7 to 100:0) to give four fractions, 1−4. Fraction 2 (650 mg) was subjected to silica gel CC (4 g, 1.5 cm × 10 cm, 200−300 mesh, petroleum ether−acetone, 250:1 to 1:1) to give six subfractions (2A− 2F). Fraction 2B (511 mg) was separated using RP C18 Lobar CC (MeOH−H2O, 60:40 to 100:0) to give six subfractions (2BA−2BF). Fraction 2BB (80.3 mg) was purified using semipreparative HPLC (MeOH−H2O, 68:32, 1.6 mL/min) to yield 2 (18.0 mg, tR = 26.0 min). Fraction 2BD (49 mg) was purified using Sephadex LH-20 CC (2 cm × 120 cm, MeOH) to yield a mixture of 5 and 6 (6 mg). Fraction 2BE (138.9 mg) was separated using semipreparative HPLC (MeOH−H2O, 76:24, 1.6 mL/min) to yield 9 (3.43 mg, tR = 35.0 min), while fraction 2BF (27.9 mg) was purified using semipreparative HPLC (MeOH−H2O, 72:28, 1.6 mL/min) to yield 8 (0.51 mg, tR = 27.5 min). Fraction 2C (226 mg) was separated using Sephadex LH20 CC (2 cm × 120 cm, MeOH), followed by RP C18 Lobar CC (MeOH−H2O, 60:40 to 100:0), to give seven subfractions (2CA− 2CG). Fraction 2CA (25.5 mg) was further purified using semipreparative HPLC (MeCN−H2O, 52:48, 1.6 mL/min) to yield 10 (2.45 mg, tR = 22.5 min) and 3 (2.26 mg, tR = 26.5 min). Chandonanone A (1): colorless needles (MeOH); mp 184−186 °C; [α]25D −16 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 259 (3.12) nm; ECD (MeOH) λmax (Δε) 288 (+0.17), 217 (−0.70) nm; IR νmax 2937, 1728, 1702, 1676, 1371, 1247 cm−1; for 1H and 13C NMR data, see Tables 1 and 2; HRESIMS (positive mode) m/z 396.2746 [M + NH4]+ (calcd for C22H38O5N, 396.2745). Chandonanone B (2): colorless needles (MeOH); mp 164−165 °C; [α]25D −60 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 258 (3.30) nm; ECD (MeOH) λmax (Δε) 321 (+0.34), 266 (−0.68), 207 (−1.67) nm; IR νmax 2936, 1729, 1678, 1372, 1249 cm−1; for 1H and 13C NMR
compound 2 showed weak cytotoxicity against NCI-1299 cells, with an IC50 value of 19.5 ± 1.0 μM.
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EXPERIMENTAL SECTION
General Experimental Procedures. Melting points were measured using an X-6 micromelting point apparatus (Bejing TECH Instrument Co. Ltd., China) and are uncorrected. Optical rotations were obtained using a GYROMAT-HP polarimeter. UV spectra were acquired with an Agilent 8453E UV−visible spectroscopy system, with ECD spectra being obtained on a Chirascan spectropolarimeter (Applied Photophysics Ltd., Leatherhead, UK). IR spectra were recorded using a Nicolet iN 10 Micro FTIR spectrometer. NMR spectra were recorded on a Bruker Avance DRX-600 spectrometer operating at 600 (1H) and 150 (13C) MHz, with TMS as the internal standard. HRESIMS was performed on an LTQ-Orbitrap XL. HPLC separations were performed on an Agilent 1200 G1311A quaternary pump equipped with an Agilent 1200 G1322A degasser, an Agilent 1200 G1329B 1260ALS, an Agilent 1200 G1315D DAD detector, and ZORBAX SB-C18 5 μm columns (9.4 mm × 250 mm and 4.6 mm × 250 mm). Silica gel (200−300 mesh; Qingdao Haiyang Chemical Co. Ltd., Qingdao, P. R. China), RP C18 silica gel (40−63 μm, FuJi), Sephadex LH-20 (25−100 μm; Pharmacia Biotek, Denmark), and MCI gel (CHP20P, 75−150 μm, Mitsubishi Chemical Industries Ltd.) were used for CC. TLC was carried out with glass precoated silica gel GF254 plates (Qingdao Haiyang Chemical Co. Ltd.). The compounds were visualized under UV light and by spraying with H2SO4−EtOH (1:9, v/v) followed by heating. Implementation of the MM2 force field in ChemBio3D Ultra software from CambridgeSoft Corporation (Cambridge, MA, USA, ver. 11.0) was used to calculate the molecular models. Plant Material. C. hirtellus and C. birmensis were collected in April 2011 from Mount Mao’er, Guangxi Zhuang Autonomous Region, P. R. China, and authenticated by Prof. Yuan-Xin Xiong, College of Life Sciences, Guizhou University, P. R. China. Voucher specimens (No. 20110410-20 and 20110414-15) were deposited at the Department of Natural Products Chemistry, School of Pharmaceutical Sciences, Shandong University, P. R. China. Extraction and Isolation. The air-dried powder of the whole plant material of C. hirtellus (180 g) was extracted using 95% EtOH at room temperature (1.0 L × 3 each for one week). The crude extract (9.5 g) was suspended in H2O (80 mL) and partitioned successively with Et2O (4 × 80 mL) and n-BuOH (3 × 60 mL). After removal of the organic solvent, the Et2O fraction (6.6 g) was chromatographed using MCI gel CC (15 g, 2.5 cm × 15 cm, MeOH−H2O, 3:7 to 100:0) to give five fractions, 1−5. Fraction 2 (0.8 g) was separated using silica gel CC (8 g, 1.5 cm × 20 cm, 200−300 mesh, petroleum ether− acetone, 250:1 to 1:1) to give three subfractions (2A−2C). Fraction 2A (277 mg) was further separated using Sephadex LH-20 CC (2 cm × 120 cm, MeOH), followed by RP C18 Lobar CC (MeOH−H2O, 343
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at 37 °C under 5% CO2 for 4 h. The absorbance of the solution was measured at 570 nm on a plate reader (Biorad, USA). IC50 values (the concentration resulting in 50% inhibition of cell growth) for compounds 1−11 were calculated from the plotted results, with untreated cells being set at 100%. The experiments were conducted a minimum of three times.
data, see Tables 1 and 2; HRESIMS (positive mode) m/z 401.2296 [M + Na]+ (calcd for C22H34O5Na, 401.2298). Chandonanone C (3): colorless oil; [α]25D −13 (c 1.5, MeOH); UV (MeOH) λmax (log ε) 220 (3.61), 261 (3.18) nm; ECD (MeOH) λmax (Δε) 217 (−0.75) nm; IR νmax 2936, 1724, 1373, 1248 cm−1; for 1 H and 13C NMR data, see Tables 1 and 2; HRESIMS (positive mode) m/z 377.2322 [M + H]+ (calcd for C22H33O5, 377.2323). Chandonanone D (4): colorless needles (MeOH); mp 146−147 °C; [α]25D −102 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 259 (3.87) nm; ECD (MeOH) λmax (Δε) 256 (−3.80), 219 (−2.71), 202 (+1.55) nm; IR νmax 3209, 2928, 1723, 1681, 1374, 1252 cm−1; for 1H and 13C NMR data, see Tables 1 and 2; HRESIMS (positive mode) m/z 401.2299 [M + Na]+ (calcd for C22H34O5Na, 401.2298). Chandonanone E (5): colorless needles (MeOH); mp 176−178 °C; [α]25D −12 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 261 (3.91) nm; ECD (MeOH) λmax (Δε) 315 (+0.50), 271 (+0.99), 205 (−5.88) nm; IR νmax 2936, 1728, 1676, 1373, 1251 cm−1; for 1H and 13C NMR data, see Tables 1 and 2; HRESIMS (positive mode) m/z 377.2322 [M + H]+ (calcd for C22H33O5, 377.2323). Chandonanone F (6): colorless needles (MeOH); mp 182−184 °C; [α]25D −53 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 261 (3.87) nm; ECD (MeOH) λmax (Δε) 315 (+0.45), 271 (+0.92), 204 (−5.49) nm; IR νmax 3451, 2966, 1730, 1677, 1374, 1250 cm−1; for 1H and 13C NMR data, see Tables 1 and 2; HRESIMS (positive mode) m/z 395.2429 [M + H]+ (calcd for C22H35O6, 395.2428). X-ray Crystal Structure Analysis. Colorless needles of chandonanones A (1) and B (2) were obtained from a MeOH solution. Intensity data were collected on a Bruker APEX DUO diffractometer equipped with an APEX II CCD using Cu Kα radiation. Cell refinement and data reduction were performed using Bruker SAINT. The structures were solved by direct methods using SHELXS97.17 Refinements were performed with SHELXL-97 using full-matrix least-squares with anisotropic displacement parameters for all of the non-hydrogen atoms. The H atoms were placed in calculated positions and refined using a riding model. Molecular graphics were computed using SHELXS-97. Crystallographic data (excluding structure factor tables) for the structures of chandonanones A (1) and B (2) have been deposited with the Cambridge Crystallographic Data Center as supplementary publications no. CCDC 969045 for 1 and CCDC 969046 for 2. Copies of the data can be obtained free of charge upon application to CCDC, 12 Union Road, Cambridge CB 1EZ, UK [fax: Int. +44(0) (1223) 336 033; e-mail:
[email protected]]. Chandonanone A (1): C22H34O5, M = 378.49, monoclinic, a = 11.7016(5) Å, b = 13.6677(6) Å, c = 13.7418(6) Å, α = 90.00°, β = 108.5910(10)°, γ = 90.00°, V = 2083.10(16) Å3, T = 100(2) K, space group P21, Z = 4, μ(Cu Kα) = 0.676 mm−1, 21 081 reflections measured, 7124 independent reflections (Rint = 0.0432). The final R1 values were 0.0481 (I > 2σ(I)). The final wR(F2) values were 0.1320 (I > 2σ(I)). The final R1 values were 0.0485 (all data). The final wR(F2) values were 0.1327 (all data). The goodness of fit on F2 was 1.107. The Flack parameter is 0.09(16).18 The Hooft parameter is 0.01(5) for 3103 Bijvoet pairs.19 Chandonanone B (2): C22H34O5, M = 378.49, monoclinic, a = 5.6773(2) Å, b = 13.9911(5) Å, c = 13.0148(5) Å, α = 90.00°, β = 95.410(2)°, γ = 90.00°, V = 1029.18(7) Å3, T = 100(2) K, space group P21, Z = 2, μ(Cu Kα) = 0.684 mm−1, 8221 reflections measured, 3232 independent reflections (Rint = 0.0460). The final R1 values were 0.0471 (I > 2σ(I)). The final wR(F2) values were 0.1185 (I > 2σ(I)). The final R1 values were 0.0472 (all data). The final wR(F2) values were 0.1187 (all data). The goodness of fit on F2 was 1.097. The Flack parameter is 0.21(17). The Hooft parameter is 0.07(8) for 1353 Bijvoet pairs. Cytotoxic Activity Assay. The MTT [3-(4,5-dimethylthiazol-2yl)-2,5-diphenyl-2H-tetrazolium bromide, Sigma] assay was used for the in vitro evaluations of cytotoxicity in 96-well plates.16 The cells were plated in 96-well plates for 24 h prior to treatment and were continuously exposed to different concentrations of the compounds for 24 h. All of the samples for the cytotoxicity assays were dissolved in DMSO. Subsequently, 10 μL of MTT (5 mg/mL in phosphatebuffered saline) was added to each well, and the plates were incubated
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ASSOCIATED CONTENT
S Supporting Information *
1D NMR, 2D NMR, HRESIMS, IR, UV, and ECD spectra of the new compounds 1−6; key HMBC (solid arrows), 1H−1H COSY (bold lines), and NOESY (dashed arrows) correlations for compounds 2−4 and 6; and X-ray data of compounds 1 and 2 are available free of charge via the Internet at http://pubs.acs. org.
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AUTHOR INFORMATION
Corresponding Author
*Tel: +86-531-8838-2012. Fax: +86-531-8838-2019. E-mail:
[email protected]. Author Contributions §
R.-J. Li and Z.-M. Lin contributed equally to this paper.
Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS This work was financially supported by the National Natural Science Foundation of China (Nos. 30925038 and 30730109). The authors are grateful to Mrs. J. Ren, Mr. B. Ma, and Mr. S.Q. Wang for the NMR measurements, Mrs. Y.-H. Gao for the MS measurements, and Mrs. J. Xing for the HRESIMS measurements. We kindly acknowledge Dr. X.-N. Li (Kunming Institute of Botany) for the single-crystal X-ray diffraction determinations.
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REFERENCES
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