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Chemical constituents from Aphanamixis grandifolia
FITOTE-02803; No of Pages 5 Fitoterapia xxx (2013) xxx–xxx
Contents lists available at ScienceDirect
Fitoterapia
2Q1 3 4 5 6 7
Chemical constituents from Aphanamixis grandifolia
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Rong Zhang a,b, Hong-Ping He a, Ying-Tong Di a, Shun-Lin Li a, Guo-Ying Zuo c, Yu Zhang, a,⁎, Xiao-Jiang Hao a,⁎ a
State Key Laboratory of Phytochemistry and Plant Resources in West China, Kunming Institute of Botany, Chinese Academy of Sciences, Kunming 650201 Yunnan, PR China b University of Chinese Academy of Sciences, Beijing 100039, PR China c Research Center of Natural Medicine, Clinical School of Kunming General Hospital of Chengdu Military Command, Kunming 650032, PR China
a b s t r a c t
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Article history: Received 11 September 2013 Accepted in revised form 25 October 2013 Accepted 28 October 2013 Available online xxxx
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Four new terpenoids, nemoralisins D–G (1–4), were isolated from the leaves and stems of Aphanamixis grandifolia, along with two known diterpenoids, nemoralisin C and nemoralisin. Among them, compound 1 is the first example of norsesquiterpenoid with δ-lactone moiety, and nemoralisins E–G (2–4), are a class of acyclic diterpenoids, which are structurally related nemoralisin C and nemoralisin. These structures were established on the basis of spectroscopic methods and the absolute configuration of 1 was determined by comparison of quantum chemical TDDFT calculated and experimental ECD spectra. Nemoralisins D–G (1–4) were tested for their cytotoxicities on HL-60, SMMC-7721, A-549, MCF-7, and SW480 human tumor cell lines (IC50 N 40 μM), as well as the antimicrobial activities on Staphylococcus aureus, Pseudomonas aeruginosa, MRSA92# and MRSA98# (MIC N 50 μg/mL). © 2013 Elsevier B.V. All rights reserved.
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Keywords: Aphanamixis grandifolia Diterpenoid TDDFT Nemoralisins D–G
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1. Introduction
37 38
The species of Aphanamixis grandifolia (Meliaceae) is a wild timber tree mainly distributed in the tropical areas of the south of China, Indo China Peninsula, Malaysia and Republic Indonesia [1]. A series of compounds including limonoids [2,3], triterpenoids [4–6], diterpenoids [7], sesquiterpenoids and ligans have been isolated and identified from this species and the genus Aphanamixis in previous study. Those compounds have a range of biological activities such as insect antifeedant [8,9], antibacterial, anti-inflammatory [10], antimalarial [11], and anticancer [12,13]. As an ongoing program to explore structural diversity and to screen bioactive natural products from the family Meliaceae, four new terpenoids, nemoralisins D–G (1–4) with two known diterpenoids, nemoralisin C [14] and nemoralisin [15], were isolated from the leaves and stems of A. grandifolia. Compound 1 is the first example of
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journal homepage: www.elsevier.com/locate/fitote
⁎ Corresponding authors. Tel.: +86 871 65223263; fax: +86 871 65223070. E-mail addresses: [email protected] (Y. Zhang,), [email protected] (X.-J. Hao).
norsesquiterpenoid with δ-lactone moiety and nemoralisins E–G (2–4) were derivatives of nemoralisin C, and nemoralisin. In this paper, we report the isolation, structural elucidation and bioactivity of these new compounds.
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2. Experimental
56
2.1. General
57
General experiment procedure. Optical rotations were measured with a Jasco P-1020poplar meter. UV spectra were obtained using a Shimadu UV-2401A spectrophotometer. A Tenor 27 spectrophotometer was used for IR spectra as KBr pellets. 1D and 2D NMR spectra were recorded on Bruker AM-400, Bruker DRX-500 spectrometers with TMS as internal standard. HREIMS were performed on Waters Autospec Premier P776 spectrometer. ECD spectra were recorded with an Applied Photophysics Chirascan spectrometer. Semipreparative HPLC was performed on an Agilent 1100 liquid chromatography with a Waters X-bridge C18 (4.6 × 250 mm, 5 μm) column. Column chromatography (CC) was performed over silica gel (200–300 and 300–400 mesh, Qingdao Marine Chemical, Inc., Qingdao, PR
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0367-326X/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.fitote.2013.10.014
Please cite this article as: Zhang R, et al, Chemical constituents from Aphanamixis grandifolia, Fitoterapia (2013), http://dx.doi.org/ 10.1016/j.fitote.2013.10.014
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China) and Sephadex LH-20 (40–70 μm, Amersham Pharmacia Biotech AB, Uppsala, Sweden).
X-bridge C18 (4.6 × 250 mm, 5 μm) column with 40% MeOH/ 100 H2O to afford compound 2 (27.1 mg). 101
73
2.2. Plant material
2.4. Nemoralisin D (1)
102
74 75
Colorless oil; [α]20 D − 15.7 (c 0.27, MeOH); IR (KBr) νmax 3413, 2939, 2931, 1715, and 1247 cm−1; UV (MeOH) λmax 203 (1.67), CD (0.00081 M, MeOH) λmax (Δε) 195 (+ 0.35), 213 (− 0.25), 252 (− 0.27); 1H and 13C NMR data (see Tables 1 and 2); ESIMS m/z 259 [M + Na]+; HREIMS m/z 236.1408 [M]+. (calc. C14H20O3, 236.1412).
103 104
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The leaves and stems of A. grandifolia were collected in Jinping, Yunnan Province of China in September, 2010. The plant was identified by Prof. Xun Gong, Kunming Institute of Botany, Chinese Academy of Sciences. A specimen of this plant (NO. KIB 0596224) was deposited in the Kunming Institute of Botany.
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2.3. Extraction and isolation
2.5. Nemoralisin E (2)
81
The powdered stem and leaves of A. grandifolia Bl. (9.0 Kg) were refluxed for three times with methanol. The solvent was evaporated in vacuo to obtain a crude extract. After suspending in water, the crude extract was extracted successively with petroleum ether and ethyl acetate. The petroleum ether extract (200 g) was subjected to silica gel column chromatograph, eluted with petroleum ether/ethyl acetate (from 1:0 to 1:1) and chloroform/methanol (from 15:1 to 0:1) yielding five fractions (Fr.1–Fr.5). Fr.2 (28 g) was subjected to RP-18 eluting with MeOH/H2O (from 40% to 100%) to afford seven fractions (Fr.B1–Fr.B7). Fraction B3 (500 mg) was purified by Sephadex LH-20 eluted with MeOH and a series of silica gel column chromatography to afford compounds nemoralisin C (13.6 mg) and 1 (9.4 mg), respectively. Fraction B4 (3.0 g) was purified using Sephadex LH-20 eluted with MeOH, then by a silica gel column chromatography eluted with petroleum ether/acetone 4:1 to yield compound 4 (19.1 mg), 3 (103 mg), nemoralisin (15.6 mg) and fraction Fr. B4-4, respectively. Fr. B4-4 was further purified by Semipreparative HPLC using a Waters
Colorless oil; [α]20 D − 1.7 (c 0.20, MeOH); IR (KBr) νmax 3428, 2931, 1702, 1556, and 1383 cm−1; UV (MeOH) λmax 289 (1.75), 204 (1.95), 1H and 13C NMR data (see Tables 1 and 2); ESIMS m/z 355 [M + Na]+; HREIMS m/z 332.1983 [M]+. (calc. C20H28O4, 332.1988).
92 93 94 95 96 97 98 99
t1:1 t1:2
Table 1 1 H NMR data of compounds 1–4. Position
1a
t1:4 t1:5 t1:6 t1:7 t1:8 t1:9 t1:10 t1:11 t1:12 t1:13 t1:14 t1:15 t1:16 t1:17 t1:18 t1:19 t1:20 t1:21 t1:22 t1:23 t1:24 t1:25 t1:26 t1:27 t1:28
2a 2b 3 4a 4b 5a 5b 6 8a 8b 9a 9b 10a 10b 11 12 13 14 16 17 18a 18b 19 20 21
5.80 (s)
t1:29 t1:30
a b
O
t1:3
C
2.20 (dd, 16.4, 3.6) 2.38 (m) 5.10 (ddd,12.0, 8.1, 4.0) (d, 8.5) (m) (m) (m) (m) (t, 8.0) (t, 8.0)
U
N
5.31 2.02 2.02 1.73 1.73 2.41 2.41
119 120
O
R O
P
D
Colorless oil; [α]20 D − 20.1 (c 0.24, MeOH); IR (KBr) νmax 122 3426, 2978, 2931, 1702, 1699, 1559, and 1380 cm−1; UV 123 (MeOH) λmax 294 (1.27), 203 (1.35), 1H and 13C NMR data 124
4a
5.54 (s)
1.98 (dd, 16.0, 8.0) 2.18 (dd, 16.0, 8.0) 1.83(m) 1.14 (m) 1.32 (m) 1.85 (m) 1.85 (m) 4.96 (t, 6.5) 2.06 (m) 2.06 (m) 1.35 (m) 1.35 (m) 1.39 (m)
5.82 (s)
(t, 7.8) (t, 7.8) (m) (m) (t, 7.0) (t, 7.5) (t, 7.5) (m) (m) (t, 7.8) (t, 7.8)
5.47 (s) (3H, (3H, (s) (s) (3H, (3H,
117 118
121
2.7. Nemoralisin G (4)
3a
1.29 1.29 5.36 5.91 1.45 1.78
113 114
116
2.15 (dd, 16.4, 3.6) 2.32 (m) 5.12 (ddd, 12.0, 8.1, 4.0) 5.36 2.18 2.18 2.38 2.38 6.55
(d, 8.5) (t, 7.0) (t, 7.0) (m) (m) (t, 7.0)
2.48 (m) 2.13 (3H, s) 1.69 (3H, s) 1.98 (3H, s)
111 112
Colorless oil; [α]20 D − 7.6 (c 0.46, MeOH); IR (KBr) νmax 3424, 2977, 2931, 1702, 1632, and 1589 cm−1; UV (MeOH) λmax 262 (1.09), 201 (0.77);1H and 13C NMR data (see Tables 1 and 2); ESIMS m/z 373 [M + Na]+; HREIMS m/z 350.2457 [M]+. (calc. C21H34O4, 350.2457).
2b
2.53 2.53 2.05 2.05 5.06 1.91 1.91 1.49 1.49 2.16 2.16
110
115
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90 91
107 108
2.6. Nemoralisin F (3)
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88 89
C
86 87
E
84 85
105 106
109
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82 83
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76 77
F
71 72
5.20 (s)
5.43 (s)
s) s)
1.23 (3H, s) 1.23 (3H, s) 1.07 (3H, d, 7.0)
1.39 (3H, s) 1.39 (3H, s) 1.89 (3H, s)
s) s)
1.43 (3H, s) 0.80 (3H, d, 6.5) 3.50 (3H, s)
1.75 (3H, s) 1.98 (3H, s)
Data measured in CDCl3 at 400 MHz. Data measured in CDCl3/CD3OD (2:1) at 400 MHz.
Please cite this article as: Zhang R, et al, Chemical constituents from Aphanamixis grandifolia, Fitoterapia (2013), http://dx.doi.org/ 10.1016/j.fitote.2013.10.014
R. Zhang et al. / Fitoterapia xxx (2013) xxx–xxx
2b
3a
4a
t2:4 t2:5 t2:6 t2:7 t2:8 t2:9 t2:10 t2:11 t2:12 t2:13 t2:14 t2:15 t2:16 t2:17 t2:18 t2:19 t2:20 t2:21 t2:22 t2:23 t2:24
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21
165.3 116.7 156.8 34.8 74.4 122.7 141.7 38.6 21.0 42.3 208.8 29.6 16.4 22.9
168.6 116.2 160.8 33.1 26.3 124.2 134.7 38.8 26.4 31.5 138.3 184.4 99.4 208.8 88.5 22.7 22.7 121.3 15.5 25.1
173.4 41.3 29.8 36.5 24.9 124.5 134.3 39.2 25.1 33.4 35.3 195.8 99.6 207.2 88.0 22.7 22.6 17.6 15.6 19.5 51.1
165.1 116.6 156.9 35.0 73.9 122.6 141.3 38.0 26.7 137.0 126.1 184.3 98.9 207.3 88.2 23.0 23.0 13.1 16.8 23.1
t2:25 t2:26
a
Data measured in CDCl3 at 100 MHz. Data measured in CDCl3/CD3OD (2:1) at 100 MHz.
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2.8. Bioassay
153 154
The cytotoxicities of all the compounds were evaluated by MTT on MCF-7 (human breast cancer), HL-60 (human cervical cancer), A-549 (Human lung carcinoma), SMMC-7721 (human hepatocellular carcinoma), and SW-480 cell lines (human colon carcinoma) with taxol as positive control. These compounds were also screened for their antimicrobial activities against four microorganisms, Staphylococcus aureus, Pseudomonas aeruginosa, MRSA92# and MRSA98#. The minimum inhibitory concentrations (MICs) of these compounds were determined
E
R
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161
N C
159 160
Nemoralisin D (1) was obtained as a colorless oil, and its molecular formula was determined as C14H20O3 by HR-EI-MS with m/z 236.1408 [M]+ (calcd. 236.1412) and five degrees of unsaturation. The IR spectrum showed the presence of carbonyl group (1715 cm−1). The 13C NMR and DEPT spectra displayed 14 carbon signals, including two carbonyls, two double bonds, one sp3 methines, four sp3 methylenes, and three sp3 methyls. Since two carbonyl groups and two double bonds accounted for four out of five unsaturation, the remaining one degree of unsaturation was assumed for the presence of a ring system in 1. The structure of compound 1 was elucidated by analysis of 2D NMR data including 1H–1H COSY, HMBC, and HSQC. 1H–1H COSY spectrum of 1 revealed two partial structures a (C-4 to C-6) and b (C-8 to C-10) (Fig. 2). Connection of the partial structures of a and b through C-7 was established by HMBC correlations from H-6 (δH 5.31), H3-13 (1.69) to C-7 (δC 141.7). HMBC connections of H3-12 (δH 2.13) with C-11 (δC 208.8), C-10 (42.3), from H2-9 (δH 1.72) to C-11 indicated the linkage of partial structure b and C-12 through the ketone carbonyl C-11. The presence of α, β-unsaturated δ-lactone was established by HMBC correlation of H2-4 (δH 2.20, 3.38) with C-2 (δC 116.7), C-3 (δC 156.8), and H-5 (δH 5.10) with C-1 (δC 165.3). Moreover, C-14 linked to C-3, which can be proved by HMBC correlation of H3-14 (δH 1.10) with C-2, C-4 (δC 34.8). Hence the structure of 1 was established as shown in Fig. 1. The ROESY correlations of H-5/Me-13 and H-6/H-4b indicated an E-geometry of the Δ6(7) double bond. The absolute configuration of the only chiral center at C-5 was finally established by ECD calculation based on two possible isomers (5R)-1, and (5S)-1 [17]. The calculated ECD spectrum for (5S)-1 matched well with the experimental ECD spectrum of 1 (Fig. 3), thus establishing the absolute configuration of 1 as depicted. Nemoralisin E (2) was shown to have molecular formula C20H28O4 on the basis of HR-EI-MS with m/z 332.1983 [M]+ (calcd. 332.1988) and 7° of unsaturation. The NMR data of 2 showed high similarity to those of nemoralisin [15], except
166
U
157 158
C
150 151
(see Tables 1 and 2); CD (0.00071 M, MeOH) λmax (Δε) 198 (+0.37), 211 (−0.86), 253 (−0.97); ESIMS m/z 369 [M + Na]+; HREIMS m/z 346.1784 [M]+. (calcd for C20H26O5, 346.1780).
155 156
165
D
b
3. Results and discussion
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1a
R O O
Position
P
t2:3
131 148 147 146 145 144 143 142 141 140 139 138 137 136 135 134 133 132 149
by the 2-fold dilution method. Cytotoxicity evaluations and 162 antimicrobial tests were performed according to the previously 163 described protocol [16]. 164
Table 2 13 C NMR data of compounds 1–4.
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t2:2
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t2:1
3
Fig. 1. Molecular structures of 1–4.
Please cite this article as: Zhang R, et al, Chemical constituents from Aphanamixis grandifolia, Fitoterapia (2013), http://dx.doi.org/ 10.1016/j.fitote.2013.10.014
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R O
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Fig. 2. HMBC (arrow) and 1H–1H COSY (bold) correlations of 1–4.
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Acknowledgments
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D
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indicated an E-geometry of the Δ6(7) double bond. However, the configurations at C-3 and C-11 could not be assigned from the available data. Nemoralisin G (4) was obtained as colorless oil. Its molecular formula was determined as C20H26O5 by HR-EI-MS with m/z at 346.1784 [M]+ (calcd. 346.1780) which had one more oxygen atom than nemoralisin [15]. The NMR data of compound 4 were very similar to those of nemoralisin except that the presence of a trisubstituted double bond in 4. The assignment of Δ10(11) double bond was established by HMBC correlations of H3-18 (δH 1.89) with C-10 (δC 137.0) and C-11 (δC 126.1), as well as H-13 (δH 5.43) with C-11. The molecular formula inferred by HREIMS spectrum showed five oxygen atoms which is one more than that of structure we had established. The chemical shift of C-12 (δC 184.3) was significantly shifted upfield ca. Δδ + 11.2 compared to the known nemoralisin, this observation and the unsaturation requirement for 4 indicated a peroxide bond between C-12 and C-15 [18], which was further confirmed by the fragment peak at m/z 287 [M + H − CO − O2]+ contributed by the cleavage of a peroxyl group [19–21]. The E-geometries of Δ6(7) and Δ10(11) double bonds were assigned on the basis of ROESY correlations of Me-19/H-5, and Me-18/ CH2-9. In addition, the absolute configuration of 4 was identical to that of 1, as revealed by their similar ECD curves (Supplementary data, Fig. S34). Biogenetically, the precursor of compounds 1–4 could be traced back to the known nemoralisin and nemoralisin C (Scheme 1). After oxidation, nemoralisin C would yield the key intermediate i which undergo a rearrangement to afford 1 [22]. Dehydration and oxidation of nemoralisin C would readily produce 4. Compounds 2 and 3 might be derived from the key intermediate iii which involves oxidation and reduction as the key steps, respectively. Only nemoralisin C showed weak cytotoxicity to SMMC-7721 with IC50 22.67 μM. Moreover, these compounds were inactive against all the tested strains (MIC N 50 μg/mL).
T
C
215 216
E
213 214
R
211 212
R
209 210
O
208
C
206 207
N
204 205
that the presence of a Δ11(18) terminal double bond (δH 5.36, 5.91; δC 121.3, 138.3) and the absence of the α, β-unsaturated δ-lactone ring in 2. This assignment was confirmed by 1H–1H COSY and HMBC spectra, especially by the HMBC correlations of H2-10 (δH 2.16) to C-11 (δC 138.3), C-18 (121.3). Therefore, the structure of 2 was established as shown in Fig. 2. In the ROESY spectrum, the correlations of H-2/Me-20, H-6/CH2-8, and Me-19/CH2-5 indicated Δ2(3) and Δ6(7) double bonds took Z- and E-geometries, respectively. Nemoralisin F (3) was shown to have molecular formula C21H34O4 on the basis of HR-EI-MS with m/z 350.2457 [M]+ (calcd. 350.2457). The 1H and 13C NMR data of 3 suggested that 3 was also an analog of nemoralisin [15]. The differences were the existence of a methoxy group and the absence of Δ2(3) double bond in 3, which could be demonstrated by HMBC correlations of H3-20 (δH 0.80) with C-2 (δC 41.3), C-3 (δC 29.8), as well as 1H, 1H-COSY cross-peaks of H2-2/H-3/ H2-4 (H3-20)/H2-5/H-6 in 3. Moreover, the methoxy group was attached to C-1 (δC 173.4) by HMBC correlations of H3-21 (δH 3.50) to C-1. The planar structure of 3 was further verified by a combination analysis of the HSQC, HMBC, and 1H–1H COSY data (Fig. 2). The ROESY correlations of Me-19/CH2-5
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203
Fig. 3. Experimental and calculated ECD spectra of nemoralisin D (1).
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This work was financially supported by the National Basic 281 Research Program of China (2009CB522303). The authors 282 thank Prof. Yan Li, State Key Laboratory of Phytochemistry 283
Please cite this article as: Zhang R, et al, Chemical constituents from Aphanamixis grandifolia, Fitoterapia (2013), http://dx.doi.org/ 10.1016/j.fitote.2013.10.014
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Appendix A. Supplementary data
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Supplementary data to this article can be found online at http://dx.doi.org/10.1016/j.fitote.2013.10.014.
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Contents lists available at ScienceDirect
Fitoterapia
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Chemical constituents from Aphanamixis grandifolia
R O O
1
Rong Zhang a,b, Hong-Ping He a, Ying-Tong Di a, Shun-Lin Li a, Guo-Ying Zuo c, Yu Zhang, a,⁎, Xiao-Jiang Hao a,⁎ a
State Key Laboratory of Phytochemistry and Plant Resources in West China, Kunming Institute of Botany, Chinese Academy of Sciences, Kunming 650201 Yunnan, PR China b University of Chinese Academy of Sciences, Beijing 100039, PR China c Research Center of Natural Medicine, Clinical School of Kunming General Hospital of Chengdu Military Command, Kunming 650032, PR China
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Article history: Received 11 September 2013 Accepted in revised form 25 October 2013 Accepted 28 October 2013 Available online xxxx
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Four new terpenoids, nemoralisins D–G (1–4), were isolated from the leaves and stems of Aphanamixis grandifolia, along with two known diterpenoids, nemoralisin C and nemoralisin. Among them, compound 1 is the first example of norsesquiterpenoid with δ-lactone moiety, and nemoralisins E–G (2–4), are a class of acyclic diterpenoids, which are structurally related nemoralisin C and nemoralisin. These structures were established on the basis of spectroscopic methods and the absolute configuration of 1 was determined by comparison of quantum chemical TDDFT calculated and experimental ECD spectra. Nemoralisins D–G (1–4) were tested for their cytotoxicities on HL-60, SMMC-7721, A-549, MCF-7, and SW480 human tumor cell lines (IC50 N 40 μM), as well as the antimicrobial activities on Staphylococcus aureus, Pseudomonas aeruginosa, MRSA92# and MRSA98# (MIC N 50 μg/mL). © 2013 Elsevier B.V. All rights reserved.
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Keywords: Aphanamixis grandifolia Diterpenoid TDDFT Nemoralisins D–G
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1. Introduction
37 38
The species of Aphanamixis grandifolia (Meliaceae) is a wild timber tree mainly distributed in the tropical areas of the south of China, Indo China Peninsula, Malaysia and Republic Indonesia [1]. A series of compounds including limonoids [2,3], triterpenoids [4–6], diterpenoids [7], sesquiterpenoids and ligans have been isolated and identified from this species and the genus Aphanamixis in previous study. Those compounds have a range of biological activities such as insect antifeedant [8,9], antibacterial, anti-inflammatory [10], antimalarial [11], and anticancer [12,13]. As an ongoing program to explore structural diversity and to screen bioactive natural products from the family Meliaceae, four new terpenoids, nemoralisins D–G (1–4) with two known diterpenoids, nemoralisin C [14] and nemoralisin [15], were isolated from the leaves and stems of A. grandifolia. Compound 1 is the first example of
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⁎ Corresponding authors. Tel.: +86 871 65223263; fax: +86 871 65223070. E-mail addresses: [email protected] (Y. Zhang,), [email protected] (X.-J. Hao).
norsesquiterpenoid with δ-lactone moiety and nemoralisins E–G (2–4) were derivatives of nemoralisin C, and nemoralisin. In this paper, we report the isolation, structural elucidation and bioactivity of these new compounds.
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2. Experimental
56
2.1. General
57
General experiment procedure. Optical rotations were measured with a Jasco P-1020poplar meter. UV spectra were obtained using a Shimadu UV-2401A spectrophotometer. A Tenor 27 spectrophotometer was used for IR spectra as KBr pellets. 1D and 2D NMR spectra were recorded on Bruker AM-400, Bruker DRX-500 spectrometers with TMS as internal standard. HREIMS were performed on Waters Autospec Premier P776 spectrometer. ECD spectra were recorded with an Applied Photophysics Chirascan spectrometer. Semipreparative HPLC was performed on an Agilent 1100 liquid chromatography with a Waters X-bridge C18 (4.6 × 250 mm, 5 μm) column. Column chromatography (CC) was performed over silica gel (200–300 and 300–400 mesh, Qingdao Marine Chemical, Inc., Qingdao, PR
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0367-326X/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.fitote.2013.10.014
Please cite this article as: Zhang R, et al, Chemical constituents from Aphanamixis grandifolia, Fitoterapia (2013), http://dx.doi.org/ 10.1016/j.fitote.2013.10.014
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China) and Sephadex LH-20 (40–70 μm, Amersham Pharmacia Biotech AB, Uppsala, Sweden).
X-bridge C18 (4.6 × 250 mm, 5 μm) column with 40% MeOH/ 100 H2O to afford compound 2 (27.1 mg). 101
73
2.2. Plant material
2.4. Nemoralisin D (1)
102
74 75
Colorless oil; [α]20 D − 15.7 (c 0.27, MeOH); IR (KBr) νmax 3413, 2939, 2931, 1715, and 1247 cm−1; UV (MeOH) λmax 203 (1.67), CD (0.00081 M, MeOH) λmax (Δε) 195 (+ 0.35), 213 (− 0.25), 252 (− 0.27); 1H and 13C NMR data (see Tables 1 and 2); ESIMS m/z 259 [M + Na]+; HREIMS m/z 236.1408 [M]+. (calc. C14H20O3, 236.1412).
103 104
78 79
The leaves and stems of A. grandifolia were collected in Jinping, Yunnan Province of China in September, 2010. The plant was identified by Prof. Xun Gong, Kunming Institute of Botany, Chinese Academy of Sciences. A specimen of this plant (NO. KIB 0596224) was deposited in the Kunming Institute of Botany.
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2.3. Extraction and isolation
2.5. Nemoralisin E (2)
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The powdered stem and leaves of A. grandifolia Bl. (9.0 Kg) were refluxed for three times with methanol. The solvent was evaporated in vacuo to obtain a crude extract. After suspending in water, the crude extract was extracted successively with petroleum ether and ethyl acetate. The petroleum ether extract (200 g) was subjected to silica gel column chromatograph, eluted with petroleum ether/ethyl acetate (from 1:0 to 1:1) and chloroform/methanol (from 15:1 to 0:1) yielding five fractions (Fr.1–Fr.5). Fr.2 (28 g) was subjected to RP-18 eluting with MeOH/H2O (from 40% to 100%) to afford seven fractions (Fr.B1–Fr.B7). Fraction B3 (500 mg) was purified by Sephadex LH-20 eluted with MeOH and a series of silica gel column chromatography to afford compounds nemoralisin C (13.6 mg) and 1 (9.4 mg), respectively. Fraction B4 (3.0 g) was purified using Sephadex LH-20 eluted with MeOH, then by a silica gel column chromatography eluted with petroleum ether/acetone 4:1 to yield compound 4 (19.1 mg), 3 (103 mg), nemoralisin (15.6 mg) and fraction Fr. B4-4, respectively. Fr. B4-4 was further purified by Semipreparative HPLC using a Waters
Colorless oil; [α]20 D − 1.7 (c 0.20, MeOH); IR (KBr) νmax 3428, 2931, 1702, 1556, and 1383 cm−1; UV (MeOH) λmax 289 (1.75), 204 (1.95), 1H and 13C NMR data (see Tables 1 and 2); ESIMS m/z 355 [M + Na]+; HREIMS m/z 332.1983 [M]+. (calc. C20H28O4, 332.1988).
92 93 94 95 96 97 98 99
t1:1 t1:2
Table 1 1 H NMR data of compounds 1–4. Position
1a
t1:4 t1:5 t1:6 t1:7 t1:8 t1:9 t1:10 t1:11 t1:12 t1:13 t1:14 t1:15 t1:16 t1:17 t1:18 t1:19 t1:20 t1:21 t1:22 t1:23 t1:24 t1:25 t1:26 t1:27 t1:28
2a 2b 3 4a 4b 5a 5b 6 8a 8b 9a 9b 10a 10b 11 12 13 14 16 17 18a 18b 19 20 21
5.80 (s)
t1:29 t1:30
a b
O
t1:3
C
2.20 (dd, 16.4, 3.6) 2.38 (m) 5.10 (ddd,12.0, 8.1, 4.0) (d, 8.5) (m) (m) (m) (m) (t, 8.0) (t, 8.0)
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N
5.31 2.02 2.02 1.73 1.73 2.41 2.41
119 120
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R O
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D
Colorless oil; [α]20 D − 20.1 (c 0.24, MeOH); IR (KBr) νmax 122 3426, 2978, 2931, 1702, 1699, 1559, and 1380 cm−1; UV 123 (MeOH) λmax 294 (1.27), 203 (1.35), 1H and 13C NMR data 124
4a
5.54 (s)
1.98 (dd, 16.0, 8.0) 2.18 (dd, 16.0, 8.0) 1.83(m) 1.14 (m) 1.32 (m) 1.85 (m) 1.85 (m) 4.96 (t, 6.5) 2.06 (m) 2.06 (m) 1.35 (m) 1.35 (m) 1.39 (m)
5.82 (s)
(t, 7.8) (t, 7.8) (m) (m) (t, 7.0) (t, 7.5) (t, 7.5) (m) (m) (t, 7.8) (t, 7.8)
5.47 (s) (3H, (3H, (s) (s) (3H, (3H,
117 118
121
2.7. Nemoralisin G (4)
3a
1.29 1.29 5.36 5.91 1.45 1.78
113 114
116
2.15 (dd, 16.4, 3.6) 2.32 (m) 5.12 (ddd, 12.0, 8.1, 4.0) 5.36 2.18 2.18 2.38 2.38 6.55
(d, 8.5) (t, 7.0) (t, 7.0) (m) (m) (t, 7.0)
2.48 (m) 2.13 (3H, s) 1.69 (3H, s) 1.98 (3H, s)
111 112
Colorless oil; [α]20 D − 7.6 (c 0.46, MeOH); IR (KBr) νmax 3424, 2977, 2931, 1702, 1632, and 1589 cm−1; UV (MeOH) λmax 262 (1.09), 201 (0.77);1H and 13C NMR data (see Tables 1 and 2); ESIMS m/z 373 [M + Na]+; HREIMS m/z 350.2457 [M]+. (calc. C21H34O4, 350.2457).
2b
2.53 2.53 2.05 2.05 5.06 1.91 1.91 1.49 1.49 2.16 2.16
110
115
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90 91
107 108
2.6. Nemoralisin F (3)
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88 89
C
86 87
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84 85
105 106
109
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82 83
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76 77
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71 72
5.20 (s)
5.43 (s)
s) s)
1.23 (3H, s) 1.23 (3H, s) 1.07 (3H, d, 7.0)
1.39 (3H, s) 1.39 (3H, s) 1.89 (3H, s)
s) s)
1.43 (3H, s) 0.80 (3H, d, 6.5) 3.50 (3H, s)
1.75 (3H, s) 1.98 (3H, s)
Data measured in CDCl3 at 400 MHz. Data measured in CDCl3/CD3OD (2:1) at 400 MHz.
Please cite this article as: Zhang R, et al, Chemical constituents from Aphanamixis grandifolia, Fitoterapia (2013), http://dx.doi.org/ 10.1016/j.fitote.2013.10.014
R. Zhang et al. / Fitoterapia xxx (2013) xxx–xxx
2b
3a
4a
t2:4 t2:5 t2:6 t2:7 t2:8 t2:9 t2:10 t2:11 t2:12 t2:13 t2:14 t2:15 t2:16 t2:17 t2:18 t2:19 t2:20 t2:21 t2:22 t2:23 t2:24
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21
165.3 116.7 156.8 34.8 74.4 122.7 141.7 38.6 21.0 42.3 208.8 29.6 16.4 22.9
168.6 116.2 160.8 33.1 26.3 124.2 134.7 38.8 26.4 31.5 138.3 184.4 99.4 208.8 88.5 22.7 22.7 121.3 15.5 25.1
173.4 41.3 29.8 36.5 24.9 124.5 134.3 39.2 25.1 33.4 35.3 195.8 99.6 207.2 88.0 22.7 22.6 17.6 15.6 19.5 51.1
165.1 116.6 156.9 35.0 73.9 122.6 141.3 38.0 26.7 137.0 126.1 184.3 98.9 207.3 88.2 23.0 23.0 13.1 16.8 23.1
t2:25 t2:26
a
Data measured in CDCl3 at 100 MHz. Data measured in CDCl3/CD3OD (2:1) at 100 MHz.
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2.8. Bioassay
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The cytotoxicities of all the compounds were evaluated by MTT on MCF-7 (human breast cancer), HL-60 (human cervical cancer), A-549 (Human lung carcinoma), SMMC-7721 (human hepatocellular carcinoma), and SW-480 cell lines (human colon carcinoma) with taxol as positive control. These compounds were also screened for their antimicrobial activities against four microorganisms, Staphylococcus aureus, Pseudomonas aeruginosa, MRSA92# and MRSA98#. The minimum inhibitory concentrations (MICs) of these compounds were determined
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159 160
Nemoralisin D (1) was obtained as a colorless oil, and its molecular formula was determined as C14H20O3 by HR-EI-MS with m/z 236.1408 [M]+ (calcd. 236.1412) and five degrees of unsaturation. The IR spectrum showed the presence of carbonyl group (1715 cm−1). The 13C NMR and DEPT spectra displayed 14 carbon signals, including two carbonyls, two double bonds, one sp3 methines, four sp3 methylenes, and three sp3 methyls. Since two carbonyl groups and two double bonds accounted for four out of five unsaturation, the remaining one degree of unsaturation was assumed for the presence of a ring system in 1. The structure of compound 1 was elucidated by analysis of 2D NMR data including 1H–1H COSY, HMBC, and HSQC. 1H–1H COSY spectrum of 1 revealed two partial structures a (C-4 to C-6) and b (C-8 to C-10) (Fig. 2). Connection of the partial structures of a and b through C-7 was established by HMBC correlations from H-6 (δH 5.31), H3-13 (1.69) to C-7 (δC 141.7). HMBC connections of H3-12 (δH 2.13) with C-11 (δC 208.8), C-10 (42.3), from H2-9 (δH 1.72) to C-11 indicated the linkage of partial structure b and C-12 through the ketone carbonyl C-11. The presence of α, β-unsaturated δ-lactone was established by HMBC correlation of H2-4 (δH 2.20, 3.38) with C-2 (δC 116.7), C-3 (δC 156.8), and H-5 (δH 5.10) with C-1 (δC 165.3). Moreover, C-14 linked to C-3, which can be proved by HMBC correlation of H3-14 (δH 1.10) with C-2, C-4 (δC 34.8). Hence the structure of 1 was established as shown in Fig. 1. The ROESY correlations of H-5/Me-13 and H-6/H-4b indicated an E-geometry of the Δ6(7) double bond. The absolute configuration of the only chiral center at C-5 was finally established by ECD calculation based on two possible isomers (5R)-1, and (5S)-1 [17]. The calculated ECD spectrum for (5S)-1 matched well with the experimental ECD spectrum of 1 (Fig. 3), thus establishing the absolute configuration of 1 as depicted. Nemoralisin E (2) was shown to have molecular formula C20H28O4 on the basis of HR-EI-MS with m/z 332.1983 [M]+ (calcd. 332.1988) and 7° of unsaturation. The NMR data of 2 showed high similarity to those of nemoralisin [15], except
166
U
157 158
C
150 151
(see Tables 1 and 2); CD (0.00071 M, MeOH) λmax (Δε) 198 (+0.37), 211 (−0.86), 253 (−0.97); ESIMS m/z 369 [M + Na]+; HREIMS m/z 346.1784 [M]+. (calcd for C20H26O5, 346.1780).
155 156
165
D
b
3. Results and discussion
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Position
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t2:3
131 148 147 146 145 144 143 142 141 140 139 138 137 136 135 134 133 132 149
by the 2-fold dilution method. Cytotoxicity evaluations and 162 antimicrobial tests were performed according to the previously 163 described protocol [16]. 164
Table 2 13 C NMR data of compounds 1–4.
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t2:2
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t2:1
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Fig. 1. Molecular structures of 1–4.
Please cite this article as: Zhang R, et al, Chemical constituents from Aphanamixis grandifolia, Fitoterapia (2013), http://dx.doi.org/ 10.1016/j.fitote.2013.10.014
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Fig. 2. HMBC (arrow) and 1H–1H COSY (bold) correlations of 1–4.
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Acknowledgments
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indicated an E-geometry of the Δ6(7) double bond. However, the configurations at C-3 and C-11 could not be assigned from the available data. Nemoralisin G (4) was obtained as colorless oil. Its molecular formula was determined as C20H26O5 by HR-EI-MS with m/z at 346.1784 [M]+ (calcd. 346.1780) which had one more oxygen atom than nemoralisin [15]. The NMR data of compound 4 were very similar to those of nemoralisin except that the presence of a trisubstituted double bond in 4. The assignment of Δ10(11) double bond was established by HMBC correlations of H3-18 (δH 1.89) with C-10 (δC 137.0) and C-11 (δC 126.1), as well as H-13 (δH 5.43) with C-11. The molecular formula inferred by HREIMS spectrum showed five oxygen atoms which is one more than that of structure we had established. The chemical shift of C-12 (δC 184.3) was significantly shifted upfield ca. Δδ + 11.2 compared to the known nemoralisin, this observation and the unsaturation requirement for 4 indicated a peroxide bond between C-12 and C-15 [18], which was further confirmed by the fragment peak at m/z 287 [M + H − CO − O2]+ contributed by the cleavage of a peroxyl group [19–21]. The E-geometries of Δ6(7) and Δ10(11) double bonds were assigned on the basis of ROESY correlations of Me-19/H-5, and Me-18/ CH2-9. In addition, the absolute configuration of 4 was identical to that of 1, as revealed by their similar ECD curves (Supplementary data, Fig. S34). Biogenetically, the precursor of compounds 1–4 could be traced back to the known nemoralisin and nemoralisin C (Scheme 1). After oxidation, nemoralisin C would yield the key intermediate i which undergo a rearrangement to afford 1 [22]. Dehydration and oxidation of nemoralisin C would readily produce 4. Compounds 2 and 3 might be derived from the key intermediate iii which involves oxidation and reduction as the key steps, respectively. Only nemoralisin C showed weak cytotoxicity to SMMC-7721 with IC50 22.67 μM. Moreover, these compounds were inactive against all the tested strains (MIC N 50 μg/mL).
T
C
215 216
E
213 214
R
211 212
R
209 210
O
208
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206 207
N
204 205
that the presence of a Δ11(18) terminal double bond (δH 5.36, 5.91; δC 121.3, 138.3) and the absence of the α, β-unsaturated δ-lactone ring in 2. This assignment was confirmed by 1H–1H COSY and HMBC spectra, especially by the HMBC correlations of H2-10 (δH 2.16) to C-11 (δC 138.3), C-18 (121.3). Therefore, the structure of 2 was established as shown in Fig. 2. In the ROESY spectrum, the correlations of H-2/Me-20, H-6/CH2-8, and Me-19/CH2-5 indicated Δ2(3) and Δ6(7) double bonds took Z- and E-geometries, respectively. Nemoralisin F (3) was shown to have molecular formula C21H34O4 on the basis of HR-EI-MS with m/z 350.2457 [M]+ (calcd. 350.2457). The 1H and 13C NMR data of 3 suggested that 3 was also an analog of nemoralisin [15]. The differences were the existence of a methoxy group and the absence of Δ2(3) double bond in 3, which could be demonstrated by HMBC correlations of H3-20 (δH 0.80) with C-2 (δC 41.3), C-3 (δC 29.8), as well as 1H, 1H-COSY cross-peaks of H2-2/H-3/ H2-4 (H3-20)/H2-5/H-6 in 3. Moreover, the methoxy group was attached to C-1 (δC 173.4) by HMBC correlations of H3-21 (δH 3.50) to C-1. The planar structure of 3 was further verified by a combination analysis of the HSQC, HMBC, and 1H–1H COSY data (Fig. 2). The ROESY correlations of Me-19/CH2-5
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Fig. 3. Experimental and calculated ECD spectra of nemoralisin D (1).
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This work was financially supported by the National Basic 281 Research Program of China (2009CB522303). The authors 282 thank Prof. Yan Li, State Key Laboratory of Phytochemistry 283
Please cite this article as: Zhang R, et al, Chemical constituents from Aphanamixis grandifolia, Fitoterapia (2013), http://dx.doi.org/ 10.1016/j.fitote.2013.10.014
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Appendix A. Supplementary data
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Supplementary data to this article can be found online at http://dx.doi.org/10.1016/j.fitote.2013.10.014.
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Scheme 1. Biogenetic pathway proposed for compounds 1–4.
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