FITOTE-02857; No of Pages 8 Fitoterapia xxx (2014) xxx–xxx
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Fitoterapia
F
journal homepage: www.elsevier.com/locate/fitote
Cassane-type diterpenoids from the seed kernels of Caesalpinia bonduc
2Q1
Lin Wu, Jun Luo, Yangmei Zhang, Xiaobing Wang, Lei Yang ⁎, Lingyi Kong ⁎
3 4
State Key Laboratory of Natural Medicines, Department of Natural Medicinal Chemistry, China Pharmaceutical University, 24 Tong Jia Xiang, Nanjing 210009, People's Republic of China
R O
O
1
5
a b s t r a c t
9 14 10 15 11 16 12 17 13 18 19 21 20 22 23 24 26 25
Article history: Received 17 November 2013 Accepted in revised form 3 January 2014 Accepted 9 January 2014 Available online xxxx
Seven new cassane diterpenoids (1–7), along with three known compounds (8–10), were isolated from the seed kernels of Caesalpinia bonduc. The structures were elucidated by extensive 1D and 2D NMR (HSQC, HMBC and ROESY) and mass (HRESIMS) spectroscopic data analyses. The structure and absolute configuration of compound 1 were confirmed by a single-crystal X-ray diffraction experiment. All isolates were tested for their cytotoxicity against HepG-2, MCF-7 and MG-63 cells, and 8–10 showed weak inhibitory activities. © 2014 Published by Elsevier B.V.
C
T
Keywords: Caesalpinia bonduc Cassane diterpenoids Cytotoxicity
E
27
1. Introduction
29 30
Caesalpinia bonduc (Linn.) Roxb. (Fabaceae) is a famous medicinal plant widely distributed in tropical and subtropical regions of Southeast Asia, and the seeds of this plant have long been used as a traditional Chinese medicine (TCM) to treat common cold, fever and dysentery [1]. Phytochemical research indicated that cassane furanoditerpenoids are the most active constituents distributed in the genus Caesalpinia, which display significant antiproliferative [2], antimalarial [3], antibacterial [4], antihelmintic [5], and antineoplastic effects [6]. Following our research program of discovering novel terpenes from folk medicinal plants [7–9], seven new cassanetype diterpenoids, caesalls A–F (1–7), and three known compounds, norcaesalpinins MC (8) [10], caesalpinins D (9) [11], and bonducellpin D (10) [12], were isolated from the EtOH extract of seed kernels of title plant. Their structures were elucidated by extensive 1D and 2D NMR (HSQC, HMBC, and ROESY) and mass (HRESIMS) spectroscopic data analyses, and that of 1 was confirmed by a single-crystal X-ray diffraction experiment. Compounds 8–10 showed weak inhibitory activities against HepG-2 and MG-63 with IC50 values between 27.6
39 40 41 42 43 44 45 46 47 48
R
O
C
37 38
N
35 36
U
33 34
R
28
31 32
P
i n f o
D
a r t i c l e
E
7 8
⁎ Corresponding authors. Tel./fax: +86 25 83271405. E-mail addresses: [email protected] (L. Yang), [email protected] (L. Kong).
and 39.8 μM. From the observed cytotoxic activities, we hypothesize that the presence of the γ-lactone formed between the oxygen at C-7 and the C-17 carbonyl in the cassane diterpenoids plays an important role for the cytotoxic activities. Herein, the isolation, structural elucidation and cytotoxicity of them are reported.
49 50
2. Experimental
55
2.1. General experimental procedures
56
Melting points were measured on an X-4 digital display micro-melting apparatus, uncorrected. Optical rotations were determined with a JASCO P-1020 polarimeter. UV spectra were performed on a Shimadzu UV-2450 spectrophotometer. IR spectra were recorded in KBr-disk on a Bruker Tensor 27 spectrometer. 1D and 2D NMR spectra were acquired on a Bruker AV-500 NMR instrument at 500 MHz (1H) and 125 MHz (13C) in CDCl3, CD3OD and DMSO-d6. HRESIMS was carried out on an Agilent UPLC-Q-TOF (6520B). Column chromatography (CC) was done using silica gel (Qingdao Marine Chemical Co., Ltd., China), ODS (40–63 μm, Fuji, Japan), or Sephadex LH-20 (Pharmacia, Sweden). Preparative HPLC was carried out using a Shimadzu LC-6A instrument with a SPD-10A detector using a shim-pack RP-C18 column (20 × 200 mm). Analytical HPLC
57
0367-326X/$ – see front matter © 2014 Published by Elsevier B.V. http://dx.doi.org/10.1016/j.fitote.2014.01.011
Please cite this article as: Wu L, et al, Cassane-type diterpenoids from the seed kernels of Caesalpinia bonduc, Fitoterapia (2014), http://dx.doi.org/10.1016/j.fitote.2014.01.011
51 52 53 54
58 59 60 61 62 63 64 65 66 67 68 69 70
2 t1:1 t1:2
L. Wu et al. / Fitoterapia xxx (2014) xxx–xxx
Table 1 1 H NMR (500 Hz) and
13
C NMR (125 Hz) spectra data for compounds 1–3 (J in Hz, δ in ppm).
Position
1a δH (multi, J in Hz)
δC, type
δH (multi, J in Hz)
δC, type
δH (multi, J in Hz)
δC, type
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 t1:29 t1:30 t1:31 t1:32 t1:33 t1:34 t1:35 t1:36 t1:37 t1:38 t1:39 t1:40 t1:41
1 2
5.64, s 1.98, m 2.11, m 1.95, m 1.17, m
76.0, CH 22.8, CH2
4.67, s 1.87, m 1.45, m 1.85, m 0.94, m
73.5, CH 22.0, CH2
4.62, 1.51, 1.83, 0.88, 1.83,
74.5, CH 22.1, CH2
t1:42 t1:43
a
71
5.43, t (9.0)
73.5, CH
2.33, m 2.63, td (12.5, 2.0)
7.01, s
127.9, C 139.0, C 49.0, C 104.3, CH
44.5, 36.9, 43.9, 37.0,
6.72, d (2.0) 7.52, d (2.0) 2.39, s
153.8, C 126.1, C 128.3, C 105.0, CH 144.7, CH 16.0, CH3
18 19 20 1-OCOCH3 1-OCOCH3 6-OCOCH3 6-OCOCH3 7-OCOCH3 7-OCOCH3 5-OH 6-OH 7-OH 12-OCH3 16-OCH3
1.36, s 1.34, s 1.33, s
5.86, d (2.0) 5.37, d (2.0) 5.06, s 4.90, s 1.04, s 1.00, s 1.06, s
30.8, CH3 25.2, CH3 29.3, CH3 169.7, C 21.4, CH3
1.96, s
2.03, s
C
1.98, s
R
E
1.88, s
Recorded in CDCl3. Recorded in DMSO-d6.
3.00, s 3.38, s
was measured on an Agilent 1200 Series instrument with a DAD detector using a shim-pack VP-ODS column (250 × 4.6 mm).
73
2.2. Plant material
74 75
78 79
The seeds of C. bonduc (Linn.) Roxb. were purchased from Chengdu City, Sichuan Province of China in March 2012, and were authenticated by Professor Min-Jian Qin, Department of Medicinal Plants, China Pharmaceutical University. A voucher specimen (No. CC201203) was deposited in the Department of Natural Medicinal Chemistry, China Pharmaceutical University.
80
2.3. Extraction and isolation
81
The powdered air-dried seed kernels of C. bonduc (2.8 kg) were extracted with 95% EtOH (3 × 4 h). The EtOH extract was concentrated under reduced pressure. The crude extract (790 g) was suspended in water and successively partitioned with petroleum ether, CH2Cl2. Fractionation of the CH2Cl2 (142 g) extract was performed on a silica gel column using a gradient of petroleum ether–acetone (50:1–3:1) to yield four fractions (Fr. 1–4) by TLC analysis. Fr. 3 (12.6 g) was then run
82 83 84 85 86 87 88
N C
U
76 77
O
72
109.3, 144.4, 141.0, 121.8, 105.8, 109.8,
3.69, dd (8.5, 6.5)
C C C CH CH CH2
P
12 13 14 15 16 17
1.26, t (12.5) 1.71, dd (12.5, 2.0)
CH CH C CH2
30.1, CH3 23.8, CH3 16.4, CH3 169.6, C 20.7, CH3 169.8, C 21.2, CH3 169.9, C 20.7, CH3
32.0, CH2
F
36.6, CH2
s m m m m
R O O
5.33, d (9.0)
37.8, C 78.3, C 74.8, CH
2.78, dd (17.0,7.5) 3.31, dd (17.0,7.5)
8 9 10 11
b
39.0, C 77.4, C 70.0, CH
4.51, t (7.5)
7
31.7, CH2
D
4 5 6
33.0, CH2
E
3
3b
T
t1:4
2b
R
t1:3
38.1, C 77.5, C 75.4, CH
3.81, dd (15.5, 7.5)
73.0, CH
2.14, m 2.60, td (12.0,5.5)
43.2, 37.3, 43.6, 22.4,
2.15, m 2.48, m
6.56, 7.43, 5.33, 5.16, 1.22, 1.18, 1.10,
(1.5) (1.5) s s s s s
1.98, s
CH CH C CH2
150.9, 119.3, 140.1, 106.6, 141.8, 106.5,
C C C CH CH CH2
31.2, CH3 24.6, CH3 16.8, CH3 169.8, C 21.0, CH3
3.14, br s 4.27, d (6.5) 4.38, d (6.5) 48.9, CH3 55.7, CH3
on an ODS column using a step gradient of MeOH–H2O (40:60–90:10), to afford seven subfractions (Fr. 3.1–3.7). Fr. 3.3 was chromatographed over a Sephadex LH-20 column eluted with MeOH and further purified by preparative HPLC using MeOH–H2O (70:30, 10 ml/min) to yield 6 (77 mg, tR = 30 min). Fr. 3.6 was chromatographed over an ODS column with a continuous gradient of MeOH–H2O (50:50 to 100:0) to yield five fractions (Fr. 3.6.1–3.6.5). Fr. 3.6.2 was chromatographed over a Sephadex LH-20 column, eluted with MeOH to yield 1 (9 mg). Fr. 3.6.3 was eluted with MeOH–H2O (65:35) on an ODS column and further purified by preparative HPLC using MeOH–H2O (60:40, 10 ml/min) to yield 4 (5 mg, tR = 37 min). Fr. 3.4 was subjected to ODS column with gradient MeOH–H2O (50:50 to 100:0) to yield six fractions (Fr. 3.4.1–3.4.6). Fr. 3.4.4 was subjected to a silica gel column, eluted with petroleum ether–acetone (4:1), to obtain four subfractions (Fr. 3.4.4.1–3.4.4.4). Separation of Fr.3.4.4.3 was achieved by preparative HPLC with MeOH–H2O (55:45, 10 ml/min), yielding 7 (6 mg, tR = 40 min). Fr. 3.4.3 was eluted with MeOH–H2O (60:40) on an ODS column, and four subfractions (Fr. 3.4.3.1–3.4.3.4) were collected. Fr. 3.4.3.3 was further purified by preparative HPLC with
Please cite this article as: Wu L, et al, Cassane-type diterpenoids from the seed kernels of Caesalpinia bonduc, Fitoterapia (2014), http://dx.doi.org/10.1016/j.fitote.2014.01.011
89 90 91 92 93 94 95 96 97 98 99 100 101 102 103 104 105 106 107 108 109 110
L. Wu et al. / Fitoterapia xxx (2014) xxx–xxx
Table 2 1 H NMR (500 Hz) and
13
F
O
R O
P
D
130 131
C NMR (125 Hz) spectra data for compounds 4–7 (J in Hz, δ in ppm).
a
E
128 129
5a
6b
7a
Position
4
δH (multi, J in Hz)
δC, type
δH (multi, J in Hz)
δC, type
δH (multi, J in Hz)
δC, type
δH (multi, J in Hz)
δC, type
1 2
4.71, 1.92, 1.53, 1.81, 1.00,
74.2, CH 22.2, CH2
4.77, 1.78, 1.94, 5.06,
74.2, CH 27.1, CH2
4.85, 1.69, 1.99, 1.93, 1.06,
76.9, CH 23.3, CH2
4.72, 1.90, 2.38, 1.85, 1.57,
76.2, CH 25.8, CH2
3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 1-OCOCH3 1-OCOCH3 3-OCOCH3 3-OCOCH3 6-OCOCH3 6-OCOCH3 7-OCOCH3 7-OCOCH3 5-OH 7-OH 14-OCH3 a b c
1.92, 1.61, 4.07, 2.10, 2.55,
s m m m m
29.4, CH2 37.7, C 76.4, C 36.1, CH2
m m m m m
66.8, 43.7, 37.8, 42.6, 22.2,
2.14, m 2.55, m
6.55, 7.42, 5.43, 5.16, 0.98, 1.06, 1.12,
d (2.0) d (2.0) s s s s s
2.01, s
CH CH CH C CH2
150.7, 119.4, 140.3, 106.6, 141.7, 107.0,
s dt (14.0, 4.0) m dd (12.5, 4.0)
C C C CH CH CH2
5.49, s
c
5.49, sc 2.67, m 2.73, m
27.9, CH3 24.9, CH3 17.2, CH3 169.5, C 20.9, CH3
2.16, dd (11.0, 4.0) 2.68, m
6.58, 7.48, 5.09, 4.79, 1.08, 1.03, 1.25,
d (2.0) d (2.0) s s s s s
1.90, s 2.00, s 2.01, s
3.13, d (1.5) 4.34, d (7.0)
T
126 127
C
124 125
E
122 123
R
120 121
R
118 119
O
116 117
C
115
N
113 114
Caesall C (3): white powder; [α]25D +118° (c 0.10, MeOH); UV (MeOH) λmax (log ε) 208 (4.96), 227 (sh) (4.86) nm; IR (KBr) νmax 3459, 2310, 1640, 1399, 1244 cm−1; 1H and 13C NMR data, see Table 1; HRESIMS m/z 413.1932 [M+Na]+ (calcd for C22H30NaO6, 413.1935). Caesall D (4): white powder; [α]25D +35.5° (c 0.08, MeOH); UV (MeOH) λmax (log ε) 205 (5.03) nm; IR (KBr) νmax 3454, 2311, 1639, 1400, 1242 cm−1; 1H and 13C NMR data, see Table 2; HRESIMS m/z 397.1988 [M+Na]+ (calcd for C22H30NaO5, 397.1988). Caesall E (5): white powder; [α]25D + 101.6° (c 0.05, MeOH); UV (MeOH) λmax (log ε) 206 (4.58), 227 (sh) (4.43) nm; IR (KBr) νmax 3453, 2313, 1738, 1639, 1400, 1246, 1039 cm− 1; 1H and 13C NMR data, see Table 2; HRESIMS m/z 555.2198 [M+Na]+ (calcd for C28H36NaO10, 555.2201). Caesall F (6): white powder; [α]25D +57.3° (c 0.14, MeOH); UV (MeOH) λmax (log ε) 215 (3.80) nm; IR (KBr) νmax 3455, 2311, 1742, 1639, 1513, 1399, 1247, 1035 cm−1; 1H and 13C NMR data, see Table 2; HRESIMS m/z 529.2406 [M+Na]+ (calcd for C27H38NaO9, 529.2408).
MeOH–H2O (70:30, 10 ml/min) to obtain 5 (3.0 mg, tR = 28 min). Fr. 3.5 (2.7 g) was eluted with a gradient of increasing MeOH (50–100%) in water on an ODS column to yield six subfractions (Fr. 3.5.1–Fr. 3.5.6). Fr. 3.5.2 was subjected to Sephadex LH-20 eluted with MeOH to produce four subfractions (Fr. 3.5.2.1–3.5.2.4). Fr. 3.5.2.1 separated by preparative HPLC with MeOH–H2O (60:40, 10 ml/min) to give 3 (3.0 mg, tR = 32 min). Fr. 3.5.5 was purified by preparative HPLC with MeOH–H2O (70:30, 10 ml/min) to obtain 2 (2.8 mg, tR = 24 min). Caesall A (1): colorless crystals; mp 200–203 °C; [α]25D + 13.8° (c 0.10, MeOH); UV (MeOH) λmax (log ε) 205 (4.19), 250 (3.73), 281 (3.23), 291 (3.15) nm; IR (KBr) νmax 3455, 2973, 2310, 1726, 1639, 1383, 1252, 1080 cm−1; 1H and 13C NMR data, see Table 1; HRESIMS m/z 395.1830 [M+Na]+ (calcd for C22H28NaO5, 395.1829). Caesall B (2): white powder; [α]25D +53.2° (c 0.10, MeOH); UV (MeOH) λmax (log ε) 207 (5.09) nm; IR (KBr) νmax 3449, 1743, 1639, 1400, 1244 cm−1; 1H and 13C NMR data, see Table 1; HRESIMS m/z 559.2516 [M+Na]+ (calcd for C28H40NaO10, 559.2514).
U
111 112
3
2.03, s 4.47, br s
71.9, CH
42.5, C 79.2, C 75.2, CH 74.4, 41.1, 37.4, 44.1, 22.5,
CH CH CH C CH2
150.7, 119.0, 138.8, 106.6, 142.2, 104.6,
C C C CH CH CH2
24.2, CH3 18.8, CH3 16.9, CH3 170.0, C 20.8, CH3 169.8, C 20.7, CH3 169.9, C 21,2, CH3 169.9, C 20.9, CH3
s m m m m
5.50, s 5.86, s 1.92, td (14.5, 3.0) 3.11, td (11.5, 5.0) 2.30, dd (16.0, 5.0) 2.42, dd (16.0, 11.5)
6.44, d (2.0) 7.33, d (2.0) 1.39, s 1.13, s 1.14, s 1.25, s 2.07, s
2.04, s 1.95, s
33.5, CH2 39.6, C 80.0, C 77.8, CH 75.4, 49.7, 35.4, 45.7, 24.0,
CH CH CH C CH2
152.7, C 120.7, C 74.4, C 110.3, CH2 142.5, CH2 26.0, CH3 31.2, CH3 25.3, CH3 17.6, CH3 171.7, C 21.9, CH3
172.6, C 21.9, CH3 172.6, C 21.9, CH3
s m m m m
4.72, s 3.88, m 1.57, m 2.79, m 2.07, m 2.38, m
2.99, 6.28, 7.37, 0.99,
m d (2.0) d (2.0) d (7.0)
1.03, s 1.08, s 1.06, s
35.7, CH2 40.7, C 77.4, C 72.9, CH 65.0, 41.9, 31.1, 43.0, 21.2,
CH CH CH C CH2
148.0, C 122.1, C 26.7, CH 109.8, CH 140.6, CH 16.8, CH3
2.04, s
23.0, CH3 24.9, CH3 17.4, CH3 169.2, C 20.9, CH3
2.04, s
169.3, C 20.7, CH3
3.10, d (2.5) 4.31, d (7.0) 3.18, s
51.9, CH3
Recorded in DMSO-d6. Recorded in CD3OD. Overlapped signals.
Please cite this article as: Wu L, et al, Cassane-type diterpenoids from the seed kernels of Caesalpinia bonduc, Fitoterapia (2014), http://dx.doi.org/10.1016/j.fitote.2014.01.011
132 133 134 135 136 137 138 139 140 141 142 143 144 145 146 147 148 149 150 151 152
L. Wu et al. / Fitoterapia xxx (2014) xxx–xxx
E
D
P
R O O
F
4
C
R
R
E
187
Colorless crystals of 1 were obtained from CH2Cl2–MeOH. 188 Crystal data were obtained on a Bruker Smart-1000 CCD 189 with a graphite monochromator with Cu Kα radiation (λ = 190
O
185 186
2.4. X-ray crystallographic analysis
N C
183 184
Caesall G (7): white powder; [α]25D + 5.2° (c 0.10, MeOH); UV (MeOH) λmax (log ε) 216 (3.80) nm; IR (KBr) νmax 3450, 2311, 1729, 1639, 1399, 1266, 1234,1050 cm−1; 1H and 13C NMR data, see Table 2; HRESIMS m/z 457.2199 [M+Na]+ (calcd for C24H34NaO7, 457.2197).
U
182 181 180 179 178 177 176 175 174 173 172 171 170 169 168 167 166 165
T
Fig. 1. Compounds from the seed kernels of C. bonduc.
Fig. 2. Single-crystal X-ray of compound 1.
Please cite this article as: Wu L, et al, Cassane-type diterpenoids from the seed kernels of Caesalpinia bonduc, Fitoterapia (2014), http://dx.doi.org/10.1016/j.fitote.2014.01.011
5
P
R O
O
F
L. Wu et al. / Fitoterapia xxx (2014) xxx–xxx
203
210
The cytotoxicity of compounds 1–10 was assessed via the MTT method using the HepG-2, MG-63 and MCF-7 cancer cell lines. The cells were grown in DMEM supplemented with 10% fetal bovine serum and cultured at a density of 5000 cell/mL in
211
E
2.5. Cytotoxicity assay
T
C
E
201 202
R
199 200
R
197 198
204
O
195 196
Cambridge Crystallographic Data Centre (deposition number: CCDC 966727). Copies of the data can be obtained free of charge via www.ccdc.cam.ac.uk/conts/retrieving.html or from the Cambridge Crystallographic Data Centre, 12, Union Road, Cambridge CB2 1EZ, UK. [Fax: (+44) 1223-336-033 or e-mail: [email protected]].
C
194
1.54184 Å) at 290(2) K. The structure was solved by direct methods using the SHELXS-97 [13] and expanded using difference Fourier techniques, refined with the SHELXL-97 [14]. Crystal data of 1: C22H28O5 (M = 372.44); monoclinic crystal (0.32 × 0.24 × 0.19 mm3); space group P21; unit cell dimensions a = 8.2412(2) Å, b = 7.86130(10) Å, c = 15.8285(3) Å, β = 104.899(2)°, V = 991.00(3) Å3; Z = 2; Dcalcd. = 1.248 mg/m3; μ = 0.710 mm−1; 7892 reflections measured (11.1 ≤ 2Θ ≤ 138.86); 2584 unique (Rint = 0.0174) which were used in all calculations; the final refinement gave R1 = 0.0311 (N 2sigma(I)) and wR2 = 0.0757 (all data); flack parameter = − 0.06(18). Crystallographic data for compound 1 have been deposited in the
N
192 193
U
191
D
Fig. 3. Key HMBC and ROESY of compound 2.
Fig. 4. Key HMBC and ROESY of compound 3.
Please cite this article as: Wu L, et al, Cassane-type diterpenoids from the seed kernels of Caesalpinia bonduc, Fitoterapia (2014), http://dx.doi.org/10.1016/j.fitote.2014.01.011
205 206 207 208 209
212 213 214
6
Compound
Hep-G2
t3:4 Q2 t3:5 t3:6 t3:7 t3:8 t3:9
1–7 8 9 10 Cisplatinb
t3:10 t3:11
a
IC50 ± SD (μM)
IC50 ± SD (μM)
IC50 ± SD (μM)
N100 39.8 ± 0.4 34.2 ± 0.8 27.6 ± 1.1 8.4 ± 0.9
N100 N100 44.5 ± 1.0 35.4 ± 1.4 24.8 ± 1.2
N100 N100 N100 N100 N100
F
Values present mean ± SD of triplicate experiments. Positive control.
245
3. Results and discussion
246
Compound 1 was obtained as colorless crystals and its molecular formula, C22H28O5, was determined by HRESIMS (m/z 395.1829 [M+Na]+). The IR absorptions at 3455 and 1726 cm−1 indicated the presence of hydroxyl and ester carbonyl groups. The 1H NMR spectrum (Table 1) showed two mutually coupled olefinic protons at δH 6.72 (1H, d, J = 2.0 Hz) and δH 7.52 (1H, d, J = 2.0 Hz) suggested the presence of a fused furan ring. The 13C NMR spectrum showed 22 carbon signals, including 8 olefinic and aromatic carbons (δC 104.3, 105.0, 126.1, 127.9, 128.3, 139.0, 144.7 and 153.8). These data suggested that the basic carbon skeleton of 1 was cassane-type diterpene and similar to caesalpinin MD [10]. The conjugation of the benzene ring with the fused furan ring was confirmed by the HMBC correlations of H-11 (δH 7.01, s) with C-12 (δC 153.8) and C-13 (δC 126.1) and of H3-17 (δH 2.39, s) with C-8 (δC 127.9), C-9 (δC 139.0), C-11 (δC 104.3), C-13 (δC 126.1) and C-14 (δC 128.3). Two proton signals at δH 2.78 (1H, dd, J = 17.0, 7.5 Hz) and 3.31 (1H, dd, J = 17.0, 7.5 Hz) were attached to C-7 (δC 36.6), which was determined by the HSQC correlations between H2-7 and C-7 and the cross peaks of H2-7 with C-8 (δC 127.9) and C-9 (δC 139.0) in the HMBC spectrum. Furthermore, the HMBC correlations of \OCOCH3 (δH 1.96, s) and H-1 (δH 5.64, s) with \OCOCH3 (δC 169.7) and of H2-7 with C-6 (δC 70.0) indicated that the acetoxy group was located at C-1 and the hydroxyl group at C-6. Thus, the planar structure of compound 1 was established as shown in Fig. 1. The relative configuration of 1 was mainly established by ROESY correlations. Cross peaks of H3-20 with H-1 and H-6 indicated that the acetoxy group at C-1 and the hydroxyl group at C-6 were α-oriented. The absolute configuration of 1 was determined by X-ray diffraction study as shown in Fig. 2. Thus, the structure was confirmed and the absolute configuration of 1 was finally determined to be 1S,5R,6S,10S and named caesall A.
254 255 256 257 258 259 260 261 262 263 264 265 266 267 268 269 270 271 272 273 274 275 276 277 278 279 280
C
E
252 253
R
250 251
R
248 249
O
247
N C
241 242
U
239 240
Compound 2 was obtained as white amorphous powder, and its molecular formula was assigned as C28H40O10 on the basis of its HRESIMS. The 1H NMR spectrum (Table 1) displayed three methyl signals at δH 1.04 (3H, s), 1.00 (3H, s) and 1.06 (3H, s), two methoxy signals at δH 3.38 (3H, s) and 3.00 (3H, s), and three acetoxy methyl signals at δH 2.03 (3H, s), 1.98 (3H, s) and 1.88 (3H, s). The 13C NMR and HSQC spectra of 2 showed 28 carbon signals for eight methyl groups, four methylene (one olefinic carbon at δC 109.8), seven methine (one olefinic carbon at δC 121.8; six carbon atoms bearing oxygen at δC 73.5, 78.3, 73.5, 74.8, 109.3 and 105.8) and nine quaternary carbons (two olefinic carbon atoms at δC 141.0 and 144.4; three ester carbonyls at δC 169.6, 169.8 and 169.9). In the HSQC and HMBC spectra, the proton signals at δH 5.06 (1H, s) and 4.90 (1H, s) were directly linked to the carbon signals at δC 109.8 and showed long-distance correlations with C-8 (δC 44.5), C-13 (δC 144.4) and C-14 (δC 141.0), suggesting that the double bond was between C-14 and C-17. Furthermore, The HMBC correlations (Fig. 3) of H-1 (δH 4.67, s) with \OCOCH3 (δC 169.6), H-6 (δH 5.33, d, J = 9.0 Hz) with \OCOCH3 (δC 169.8), H-7 (δH 5.43, t, J = 9.0 Hz) with \OCOCH3 (δC 169.9), 12-OCH3 (δH 3.00, s) with C-12 (δC 109.3), and 16-OCH3 (δH 3.38, s) with C-16 (δC 105.8) indicated that the acetoxy groups were attached to C-1, C-6, and C-7 and that two methoxy groups were attached to C-12 and C-15. The olefinic proton signals at δH 5.86 (1H, d, J = 2.0 Hz) were located at C-15 (δC 121.8) on the basis of the correlations of H-15 with C-12, C-13 and C-14. These data suggested that 2 was a tetracyclic cassane diterpene with isomerized dihydrofuran ring moiety. The relative configuration of compound 2 was established through analysis of its ROESY spectrum (Fig. 3). H3-20 (δH 1.06, s) had cross peaks with H-1 (δH 4.67, s) and H-6 (δH 5.33, d, J = 9.0 Hz), while H-16 (δH 5.37, d, J = 2.0 Hz) had cross peaks with H-8 (δH 2.33, m), which indicated that they were β-oriented. The interaction of H-7 (δH 5.43, t, J = 9.0 Hz) with H3-18 (δH 1.04, s) and 12-OCH3 (δH 3.00, s) suggested that they were α-oriented. Compound 3, a white amorphous powder, exhibited the molecular formula C22H30O6 according to HRESIMS (m/z 413.1932 [M+Na]+). The 1H NMR spectrum showed the presence of three methyl groups at δH 1.22 (3H, s), 1.18 (3H, s) and 1.10 (3H, s), an acetoxy methyl signal at δH 1.98 (3H, s), and three oxymethine resonances at δH 4.62 (1H, s), 3.69 (1H, dd, J = 8.5, 6.5 Hz) and 3.81 (1H, dd, J = 15.5, 7.5 Hz). The 1,2-disubstituted furan ring was evident from low-field doublets at δH 6.56 (1H, d, J = 1.5 Hz) and 7.43 (1H, d, J = 1.5 Hz). The exo-methylene protons were revealed by the
D
243 244
a 96-well microtiter plate. Five different concentrations of each compound in DMSO were subsequently added to the wells. Each concentration was tested in triplicate. After incubation under 5% CO2 at 37 °C for 48 h, 20 μL of MTT (5 mg/mL) was added to each well, and the cells were incubated for another 4 h. Then, the liquid in each well was removed, and DMSO (150 μL) was added. The absorbance (OD values) at 570 nm with a 630 nm reference was measured on a Universal Microplate Reader.
237 238
R O O
b
MCF-7
P
236 235 234 233 232 231 230 229 228 227 226 225 224 223 222 221 220 219
MG-63 a
E
t3:3
Table 3 In vitro cytotoxic activities of compounds 1-10.
T
t3:1 t3:2
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Please cite this article as: Wu L, et al, Cassane-type diterpenoids from the seed kernels of Caesalpinia bonduc, Fitoterapia (2014), http://dx.doi.org/10.1016/j.fitote.2014.01.011
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L. Wu et al. / Fitoterapia xxx (2014) xxx–xxx
351 352 353
354 Q6 355 356 357 Q7 358 359 360 361 362 363 364 365 366 367 368 369 370 371 372 373 374 375 376 377 378 379 380 381 382 383 384 385 386 387 388
422 Q11
This research work was financially supported by the Youth Fund Project of Basic Research Program of Jiangsu Province (Natural Science Foundation, BK2012351), the National High Technology Research and Development Program of China (863 Program) (2013AA093001), the Fundamental Research Funds for the Central Universities (JKPZ2013011), the Program for Changjiang Scholars and Innovative Research Team in University (PCSIRT-IRT1193), and the Project Funded by the Priority Academic Program Development of Jiangsu Higher Education Institutions (PAPD).
423
References
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O
F
Acknowledgments
P
349 350 Q5
D
347 Q4 348
389 390
E
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difference being the relative configuration at C-14. The Rf value on HPLC (14.65 min for 6 and 7.43 min for 5α-hydroxy14β-methoxy-1α, 6α, 7β-triacetoxyvouacapane, MeOH: H2O =70:30) indicated that the polarity of 6 is smaller than 5α-hydroxy-14β-methoxy-1α, 6α, 7β-triacetoxyvouacapane. The structure of 6 was established as 5α-hydroxy-14αmethoxy-1α, 6α, 7β-triacetoxyvouacapane and named caesall F. Compound 7 exhibited [M+Na]+ ion peak at m/z 457.2199 using HRESIMS, corresponding to the molecular formula C24H34O7. The obtained 1H and 13C NMR signals (Table 2) were closely related to those of 6, except for a hydroxyl group replace an acetoxy group at C-7 (δC 65.0) and H3-17 (δH 0.99, d, J = 7.0 Hz) to be α-oriented in 7. The HMBC correlation of C-8 (δC 41.9) with 7-OH (δH 4.31, d, J = 7.0 Hz) suggested that the hydroxyl group was located at C-7. The relative stereochemistry of 7 was deduced by the analysis of the ROESY correlations. The cross peaks from H3-17 to H-9 and H-7 indicated that the H3-17 was to be α-oriented. Thus, the structure of 7 was described as shown (Fig. 1) and named caesall G. Compounds 1–10 were tested for their cytotoxicity against three human cancer cell lines (HepG-2, MCF-7 and MG-63) using the MTT method. The obtained IC50 values were between 27.6 and 39.8 μM, and cisplatin was used as a positive control (Table 3). Compounds 1–7 were inactive against all of the tested cell lines. Compounds 8–10 showed low activity against HepG-2 (IC50 27.66–39.83 μM). Compounds 9–10 also showed low activity against MG-63 (IC50 35.48–44.5 μM). None of the compounds showed activity against MCF-7. On the basis of these biological results, we hypothesize that the presence of the γ-lactone was formed between the oxygen at C-7 and the C-17 carbonyl in the cassane diterpenes that may enhance the cytotoxic activities of the compounds.
T
343 344
C
341 342
E
339 340
R
337 338
R
335 336
O
333 334
C
332
N
330 331 Q3
signals at δH 5.33 (1H, s) and 5.16 (1H, s). The NMR data for this compound (Table 1) were similar to those of 5α-hydroxy1α,6α,7β-triacetoxyvouacapan-14(17)-ene [15], except for two acetoxy groups that were replaced by two hydroxyl groups at C-6 and C-7. The full assignments were determined by analysis of the HSQC and HMBC data. In the HMBC spectrum (Fig. 4), H2-17 showed correlations to C-8 and C-13, which supported the location of the exocyclic double bond between C-14 and C-17 and conjugated with the furan ring. The locations of the acetoxy group at C-1 and two hydroxyl groups at C-6 and C-7 were determined by the correlations of H-1 (δH 4.62, s) and \OCOCH3 (δH 1.98, s) with \OCOCH3 (δC 169.8), of 6-OH (δH 4.27, d, J = 6.5 Hz) with C-6 (δC 75.4) and of 7-OH (δH 4.38, d, J = 6.5 Hz) with C-7 (δC 73.0) in the HMBC spectrum. The relative configuration of 3 was established by ROESY correlations. The correlations of H3-20 (δH 1.10, s) with H-1 (δH 4.62, s) and H-6 (δH 3.69, dd, J = 8.5, 6.5 Hz), of H3-19 (δH 1.18, s) with H-6 (δH 3.69, dd, J = 8.5, 6.5 Hz), of H3-18 (δH 1.22, s) with 6-OH (δH 4.27, d, J = 6.5 Hz) indicated H-1 (δH 4.62, s) and H-6 (δH 3.69, dd, J = 8.5, 6.5 Hz) to be β-oriented. On the other hand, ROESY correlations between 7-OH (δH 4.38, d, J = 6.5 Hz) and 8-H (δH 2.11, m) indicated H-7 (δH 3.81, dd, J = 15.5, 7.5 Hz) to be α-oriented. The molecular formula of 4 was assigned as C22H30O5 on the basis of HRESIMS (m/z 397.1988 [M+Na]+). The 1H and 13 C NMR spectra of 4 were similar to those of 3 except for the hydroxyl group at C-6 in 3 that was absent in 4. In the HMBC spectrum, the correlations of 5-OH (δH 3.13, d, J = 1.5 Hz) with C-6 (δC 36.1) and of 7-OH (4.34, d, J = 7.0 Hz) with C-8 (δC 43.7) suggested a hydroxyl group at C-7. The relative configuration of compound 4 was determined from the ROESY spectrum. The NOEs from H-1 to H3-20, H-8 and H-11ax (δH 2.55, m); from 5-OH to H3-18, H-7 and H-9 indicated that rings A and B are in chair conformations with a trans-fused ring junction, thus confirming the relative configurations at C-1, C-5 and C-7. Compound 5 was shown to possess a molecular formula of C28H36O10 by HRESIMS (m/z 555.2198 [M+Na]+). A comparison of the NMR data (Table 2) of 5 and 4 indicated the presence of three additional acetoxy groups at C-3, C-6 and C-7 in 5. The HMBC correlations of H-1 (δH 4.77, s) with \OCOCH3 (δC 20.8) and \OCOCH3 (δC 169.8), H-3 (δH 5.06, dd, J = 12.5, 4.0 Hz) with \OCOCH3 (δC 20.7) and \OCOCH3 (δC 169.9), H-6 (δH 5.49, s) with\OCOCH3 (δC 21.2) and H-7 (δH 5.49, s) with\OCOCH3 (δC 20.9), \OCOCH3 (δH 2.01) with C-6 (δC 75.2) and\OCOCH3 (δH 2.03) with C-7 (δC 74.4), together with the above molecular formula confirmed the above assumption. The relative configuration of 5 was deduced by the analysis of the ROESY correlations. The correlations of H3-20 with H-1, H-6 and H-7 indicated H-1, H-6 and H-7 to be β-oriented and of H3-18 with 5-OH and H-3 indicated H-3 to be α-oriented. Compound 6 gave [M+Na]+ peak at m/z 529.2406 (HRESIMS) for the molecular formula C27H38O9. The NMR spectra of 6 are similar to those of 5α-hydroxy-14β-methoxy-1α, 6α, 7β-triacetoxyvouacapane [15], except that the signal for H-9 (δH 3.11, td, J = 11.5, 5.0 Hz) in 6 is shifted to lower field. The ROESY correlation between H-8 (δH 1.92, td, J = 14.5, 3.0 Hz) and H3-17 (δH 1.39, s) indicated H3-17 to be β-oriented. The above data suggested that 6 is a stereoisomer of 5α-hydroxy14β-methoxy-1α, 6α, 7β-triacetoxyvouacapane, with the
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[1] Jiangsu New Medical College. Dictionary of Chinese traditional medicine. Shanghai People's Publishing House; 1977 1289. [2] Hou Y, Cao S, Brodie P, Miller JS, Birkinshaw C, Ratovoson F, et al. Antiproliferative cassane diterpenoids of Cordyla madagascariensis ssp. Madagascariensis from the Madagascar rainforest. J Nat Prod 2008; 71:150–2. [3] Kalauni SK, Awale S, Tezuka Y, Banskota AH, Linn TZ, Asih PBS, et al. Antimalarial activity of cassane- and norcassane-type diterpenes from Caesalpinia crista and their structure–activity relationship. Biol Pharm Bull 2006;29:1050–2. [4] Dickson RA, Houghton PJ, Hylands PJ. Antibacterial and antioxidant Cassane diterpenoids from Caesalpinia benthamiana. Phytochemistry 2007;68:1436–41.
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[10] Kalauni SK, Awale S, Tezuka Y, Banskota AH, Linn TZ, Kadota S. Cassaneand norcassane-type diterpenes of Caesalpinia crista from Myanmar. J Nat Prod 2004;67:1859. [11] Linn TZ, Awale S, Tezuka Y, Banskota AH, Kalauni SK, Attamimi F, et al. Cassane- and norcassane-type diterpenes from Caesalpinia crista of Indonesia and their antimalarial activity against the growth of Plasmodium falciparum. J Nat Prod 2005;68:706. [12] Peter SR, Tinto WF, Bonducellpins AD. New cassane furanoditerpenes of Caesalpinia bonduc. J Nat Prod 1997;60:1219. [13] Sheldrick GM. SHELXS-97, program for crystal structure resolution. Germany: University of Göttingen; 1997. [14] Sheldrick GM. SHELXL-97, program for crystal structure refinement. Germany: University of Göttingen; 1997. [15] Jiang RW, Ma SC, But PPH, Mak TCW. New antiviral cassane furanoditerpenes from Caesalpinia minax. J Nat Prod 2001;64:1266–72.
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[5] Jabbar A, Zaman MA, Iqbal Z, Yaseen M, Shamim A. Anthelmintic activity of Chenopodium album (L.) and Caesalpinia crista (L.) against trichostrongylid nematodes of sheep. J Ethnopharmacol 2007;114:86–91. [6] Yadav PP, Maurya R, Sarkar J, Arora A, Kanojiya S, Sinha S, et al. Cassane diterpenes from Caesalpinia bonduc. Phytochemistry 2009;70:256–61. [7] Luo J, Wang JS, Wang XB, Luo JG, Kong LY. Chuktabularins E-T, 16norphragmalin limonoids from Chukrasia tabularis var. Velutina. J Nat Prod 2010;73:835–43. [8] Zhang JY, Wu FH, Qu W, Liang JY. Two new cassane diterpenoids from seeds of Caesalpinia sappan Linn. Chin J Nat Med 2012;10(3):0218–21. [9] Zhang M, Iinuma M, Wang JS, Oyama M, Ito Tetsuro, Kong LY. Terpenoids from Chloranthus serratus and their anti-inflammatory activities. J Nat Prod 2012;75:694–8.
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Contents lists available at ScienceDirect
Fitoterapia
F
journal homepage: www.elsevier.com/locate/fitote
Cassane-type diterpenoids from the seed kernels of Caesalpinia bonduc
2Q1
Lin Wu, Jun Luo, Yangmei Zhang, Xiaobing Wang, Lei Yang ⁎, Lingyi Kong ⁎
3 4
State Key Laboratory of Natural Medicines, Department of Natural Medicinal Chemistry, China Pharmaceutical University, 24 Tong Jia Xiang, Nanjing 210009, People's Republic of China
R O
O
1
5
a b s t r a c t
9 14 10 15 11 16 12 17 13 18 19 21 20 22 23 24 26 25
Article history: Received 17 November 2013 Accepted in revised form 3 January 2014 Accepted 9 January 2014 Available online xxxx
Seven new cassane diterpenoids (1–7), along with three known compounds (8–10), were isolated from the seed kernels of Caesalpinia bonduc. The structures were elucidated by extensive 1D and 2D NMR (HSQC, HMBC and ROESY) and mass (HRESIMS) spectroscopic data analyses. The structure and absolute configuration of compound 1 were confirmed by a single-crystal X-ray diffraction experiment. All isolates were tested for their cytotoxicity against HepG-2, MCF-7 and MG-63 cells, and 8–10 showed weak inhibitory activities. © 2014 Published by Elsevier B.V.
C
T
Keywords: Caesalpinia bonduc Cassane diterpenoids Cytotoxicity
E
27
1. Introduction
29 30
Caesalpinia bonduc (Linn.) Roxb. (Fabaceae) is a famous medicinal plant widely distributed in tropical and subtropical regions of Southeast Asia, and the seeds of this plant have long been used as a traditional Chinese medicine (TCM) to treat common cold, fever and dysentery [1]. Phytochemical research indicated that cassane furanoditerpenoids are the most active constituents distributed in the genus Caesalpinia, which display significant antiproliferative [2], antimalarial [3], antibacterial [4], antihelmintic [5], and antineoplastic effects [6]. Following our research program of discovering novel terpenes from folk medicinal plants [7–9], seven new cassanetype diterpenoids, caesalls A–F (1–7), and three known compounds, norcaesalpinins MC (8) [10], caesalpinins D (9) [11], and bonducellpin D (10) [12], were isolated from the EtOH extract of seed kernels of title plant. Their structures were elucidated by extensive 1D and 2D NMR (HSQC, HMBC, and ROESY) and mass (HRESIMS) spectroscopic data analyses, and that of 1 was confirmed by a single-crystal X-ray diffraction experiment. Compounds 8–10 showed weak inhibitory activities against HepG-2 and MG-63 with IC50 values between 27.6
39 40 41 42 43 44 45 46 47 48
R
O
C
37 38
N
35 36
U
33 34
R
28
31 32
P
i n f o
D
a r t i c l e
E
7 8
⁎ Corresponding authors. Tel./fax: +86 25 83271405. E-mail addresses: [email protected] (L. Yang), [email protected] (L. Kong).
and 39.8 μM. From the observed cytotoxic activities, we hypothesize that the presence of the γ-lactone formed between the oxygen at C-7 and the C-17 carbonyl in the cassane diterpenoids plays an important role for the cytotoxic activities. Herein, the isolation, structural elucidation and cytotoxicity of them are reported.
49 50
2. Experimental
55
2.1. General experimental procedures
56
Melting points were measured on an X-4 digital display micro-melting apparatus, uncorrected. Optical rotations were determined with a JASCO P-1020 polarimeter. UV spectra were performed on a Shimadzu UV-2450 spectrophotometer. IR spectra were recorded in KBr-disk on a Bruker Tensor 27 spectrometer. 1D and 2D NMR spectra were acquired on a Bruker AV-500 NMR instrument at 500 MHz (1H) and 125 MHz (13C) in CDCl3, CD3OD and DMSO-d6. HRESIMS was carried out on an Agilent UPLC-Q-TOF (6520B). Column chromatography (CC) was done using silica gel (Qingdao Marine Chemical Co., Ltd., China), ODS (40–63 μm, Fuji, Japan), or Sephadex LH-20 (Pharmacia, Sweden). Preparative HPLC was carried out using a Shimadzu LC-6A instrument with a SPD-10A detector using a shim-pack RP-C18 column (20 × 200 mm). Analytical HPLC
57
0367-326X/$ – see front matter © 2014 Published by Elsevier B.V. http://dx.doi.org/10.1016/j.fitote.2014.01.011
Please cite this article as: Wu L, et al, Cassane-type diterpenoids from the seed kernels of Caesalpinia bonduc, Fitoterapia (2014), http://dx.doi.org/10.1016/j.fitote.2014.01.011
51 52 53 54
58 59 60 61 62 63 64 65 66 67 68 69 70
2 t1:1 t1:2
L. Wu et al. / Fitoterapia xxx (2014) xxx–xxx
Table 1 1 H NMR (500 Hz) and
13
C NMR (125 Hz) spectra data for compounds 1–3 (J in Hz, δ in ppm).
Position
1a δH (multi, J in Hz)
δC, type
δH (multi, J in Hz)
δC, type
δH (multi, J in Hz)
δC, type
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 t1:29 t1:30 t1:31 t1:32 t1:33 t1:34 t1:35 t1:36 t1:37 t1:38 t1:39 t1:40 t1:41
1 2
5.64, s 1.98, m 2.11, m 1.95, m 1.17, m
76.0, CH 22.8, CH2
4.67, s 1.87, m 1.45, m 1.85, m 0.94, m
73.5, CH 22.0, CH2
4.62, 1.51, 1.83, 0.88, 1.83,
74.5, CH 22.1, CH2
t1:42 t1:43
a
71
5.43, t (9.0)
73.5, CH
2.33, m 2.63, td (12.5, 2.0)
7.01, s
127.9, C 139.0, C 49.0, C 104.3, CH
44.5, 36.9, 43.9, 37.0,
6.72, d (2.0) 7.52, d (2.0) 2.39, s
153.8, C 126.1, C 128.3, C 105.0, CH 144.7, CH 16.0, CH3
18 19 20 1-OCOCH3 1-OCOCH3 6-OCOCH3 6-OCOCH3 7-OCOCH3 7-OCOCH3 5-OH 6-OH 7-OH 12-OCH3 16-OCH3
1.36, s 1.34, s 1.33, s
5.86, d (2.0) 5.37, d (2.0) 5.06, s 4.90, s 1.04, s 1.00, s 1.06, s
30.8, CH3 25.2, CH3 29.3, CH3 169.7, C 21.4, CH3
1.96, s
2.03, s
C
1.98, s
R
E
1.88, s
Recorded in CDCl3. Recorded in DMSO-d6.
3.00, s 3.38, s
was measured on an Agilent 1200 Series instrument with a DAD detector using a shim-pack VP-ODS column (250 × 4.6 mm).
73
2.2. Plant material
74 75
78 79
The seeds of C. bonduc (Linn.) Roxb. were purchased from Chengdu City, Sichuan Province of China in March 2012, and were authenticated by Professor Min-Jian Qin, Department of Medicinal Plants, China Pharmaceutical University. A voucher specimen (No. CC201203) was deposited in the Department of Natural Medicinal Chemistry, China Pharmaceutical University.
80
2.3. Extraction and isolation
81
The powdered air-dried seed kernels of C. bonduc (2.8 kg) were extracted with 95% EtOH (3 × 4 h). The EtOH extract was concentrated under reduced pressure. The crude extract (790 g) was suspended in water and successively partitioned with petroleum ether, CH2Cl2. Fractionation of the CH2Cl2 (142 g) extract was performed on a silica gel column using a gradient of petroleum ether–acetone (50:1–3:1) to yield four fractions (Fr. 1–4) by TLC analysis. Fr. 3 (12.6 g) was then run
82 83 84 85 86 87 88
N C
U
76 77
O
72
109.3, 144.4, 141.0, 121.8, 105.8, 109.8,
3.69, dd (8.5, 6.5)
C C C CH CH CH2
P
12 13 14 15 16 17
1.26, t (12.5) 1.71, dd (12.5, 2.0)
CH CH C CH2
30.1, CH3 23.8, CH3 16.4, CH3 169.6, C 20.7, CH3 169.8, C 21.2, CH3 169.9, C 20.7, CH3
32.0, CH2
F
36.6, CH2
s m m m m
R O O
5.33, d (9.0)
37.8, C 78.3, C 74.8, CH
2.78, dd (17.0,7.5) 3.31, dd (17.0,7.5)
8 9 10 11
b
39.0, C 77.4, C 70.0, CH
4.51, t (7.5)
7
31.7, CH2
D
4 5 6
33.0, CH2
E
3
3b
T
t1:4
2b
R
t1:3
38.1, C 77.5, C 75.4, CH
3.81, dd (15.5, 7.5)
73.0, CH
2.14, m 2.60, td (12.0,5.5)
43.2, 37.3, 43.6, 22.4,
2.15, m 2.48, m
6.56, 7.43, 5.33, 5.16, 1.22, 1.18, 1.10,
(1.5) (1.5) s s s s s
1.98, s
CH CH C CH2
150.9, 119.3, 140.1, 106.6, 141.8, 106.5,
C C C CH CH CH2
31.2, CH3 24.6, CH3 16.8, CH3 169.8, C 21.0, CH3
3.14, br s 4.27, d (6.5) 4.38, d (6.5) 48.9, CH3 55.7, CH3
on an ODS column using a step gradient of MeOH–H2O (40:60–90:10), to afford seven subfractions (Fr. 3.1–3.7). Fr. 3.3 was chromatographed over a Sephadex LH-20 column eluted with MeOH and further purified by preparative HPLC using MeOH–H2O (70:30, 10 ml/min) to yield 6 (77 mg, tR = 30 min). Fr. 3.6 was chromatographed over an ODS column with a continuous gradient of MeOH–H2O (50:50 to 100:0) to yield five fractions (Fr. 3.6.1–3.6.5). Fr. 3.6.2 was chromatographed over a Sephadex LH-20 column, eluted with MeOH to yield 1 (9 mg). Fr. 3.6.3 was eluted with MeOH–H2O (65:35) on an ODS column and further purified by preparative HPLC using MeOH–H2O (60:40, 10 ml/min) to yield 4 (5 mg, tR = 37 min). Fr. 3.4 was subjected to ODS column with gradient MeOH–H2O (50:50 to 100:0) to yield six fractions (Fr. 3.4.1–3.4.6). Fr. 3.4.4 was subjected to a silica gel column, eluted with petroleum ether–acetone (4:1), to obtain four subfractions (Fr. 3.4.4.1–3.4.4.4). Separation of Fr.3.4.4.3 was achieved by preparative HPLC with MeOH–H2O (55:45, 10 ml/min), yielding 7 (6 mg, tR = 40 min). Fr. 3.4.3 was eluted with MeOH–H2O (60:40) on an ODS column, and four subfractions (Fr. 3.4.3.1–3.4.3.4) were collected. Fr. 3.4.3.3 was further purified by preparative HPLC with
Please cite this article as: Wu L, et al, Cassane-type diterpenoids from the seed kernels of Caesalpinia bonduc, Fitoterapia (2014), http://dx.doi.org/10.1016/j.fitote.2014.01.011
89 90 91 92 93 94 95 96 97 98 99 100 101 102 103 104 105 106 107 108 109 110
L. Wu et al. / Fitoterapia xxx (2014) xxx–xxx
Table 2 1 H NMR (500 Hz) and
13
F
O
R O
P
D
130 131
C NMR (125 Hz) spectra data for compounds 4–7 (J in Hz, δ in ppm).
a
E
128 129
5a
6b
7a
Position
4
δH (multi, J in Hz)
δC, type
δH (multi, J in Hz)
δC, type
δH (multi, J in Hz)
δC, type
δH (multi, J in Hz)
δC, type
1 2
4.71, 1.92, 1.53, 1.81, 1.00,
74.2, CH 22.2, CH2
4.77, 1.78, 1.94, 5.06,
74.2, CH 27.1, CH2
4.85, 1.69, 1.99, 1.93, 1.06,
76.9, CH 23.3, CH2
4.72, 1.90, 2.38, 1.85, 1.57,
76.2, CH 25.8, CH2
3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 1-OCOCH3 1-OCOCH3 3-OCOCH3 3-OCOCH3 6-OCOCH3 6-OCOCH3 7-OCOCH3 7-OCOCH3 5-OH 7-OH 14-OCH3 a b c
1.92, 1.61, 4.07, 2.10, 2.55,
s m m m m
29.4, CH2 37.7, C 76.4, C 36.1, CH2
m m m m m
66.8, 43.7, 37.8, 42.6, 22.2,
2.14, m 2.55, m
6.55, 7.42, 5.43, 5.16, 0.98, 1.06, 1.12,
d (2.0) d (2.0) s s s s s
2.01, s
CH CH CH C CH2
150.7, 119.4, 140.3, 106.6, 141.7, 107.0,
s dt (14.0, 4.0) m dd (12.5, 4.0)
C C C CH CH CH2
5.49, s
c
5.49, sc 2.67, m 2.73, m
27.9, CH3 24.9, CH3 17.2, CH3 169.5, C 20.9, CH3
2.16, dd (11.0, 4.0) 2.68, m
6.58, 7.48, 5.09, 4.79, 1.08, 1.03, 1.25,
d (2.0) d (2.0) s s s s s
1.90, s 2.00, s 2.01, s
3.13, d (1.5) 4.34, d (7.0)
T
126 127
C
124 125
E
122 123
R
120 121
R
118 119
O
116 117
C
115
N
113 114
Caesall C (3): white powder; [α]25D +118° (c 0.10, MeOH); UV (MeOH) λmax (log ε) 208 (4.96), 227 (sh) (4.86) nm; IR (KBr) νmax 3459, 2310, 1640, 1399, 1244 cm−1; 1H and 13C NMR data, see Table 1; HRESIMS m/z 413.1932 [M+Na]+ (calcd for C22H30NaO6, 413.1935). Caesall D (4): white powder; [α]25D +35.5° (c 0.08, MeOH); UV (MeOH) λmax (log ε) 205 (5.03) nm; IR (KBr) νmax 3454, 2311, 1639, 1400, 1242 cm−1; 1H and 13C NMR data, see Table 2; HRESIMS m/z 397.1988 [M+Na]+ (calcd for C22H30NaO5, 397.1988). Caesall E (5): white powder; [α]25D + 101.6° (c 0.05, MeOH); UV (MeOH) λmax (log ε) 206 (4.58), 227 (sh) (4.43) nm; IR (KBr) νmax 3453, 2313, 1738, 1639, 1400, 1246, 1039 cm− 1; 1H and 13C NMR data, see Table 2; HRESIMS m/z 555.2198 [M+Na]+ (calcd for C28H36NaO10, 555.2201). Caesall F (6): white powder; [α]25D +57.3° (c 0.14, MeOH); UV (MeOH) λmax (log ε) 215 (3.80) nm; IR (KBr) νmax 3455, 2311, 1742, 1639, 1513, 1399, 1247, 1035 cm−1; 1H and 13C NMR data, see Table 2; HRESIMS m/z 529.2406 [M+Na]+ (calcd for C27H38NaO9, 529.2408).
MeOH–H2O (70:30, 10 ml/min) to obtain 5 (3.0 mg, tR = 28 min). Fr. 3.5 (2.7 g) was eluted with a gradient of increasing MeOH (50–100%) in water on an ODS column to yield six subfractions (Fr. 3.5.1–Fr. 3.5.6). Fr. 3.5.2 was subjected to Sephadex LH-20 eluted with MeOH to produce four subfractions (Fr. 3.5.2.1–3.5.2.4). Fr. 3.5.2.1 separated by preparative HPLC with MeOH–H2O (60:40, 10 ml/min) to give 3 (3.0 mg, tR = 32 min). Fr. 3.5.5 was purified by preparative HPLC with MeOH–H2O (70:30, 10 ml/min) to obtain 2 (2.8 mg, tR = 24 min). Caesall A (1): colorless crystals; mp 200–203 °C; [α]25D + 13.8° (c 0.10, MeOH); UV (MeOH) λmax (log ε) 205 (4.19), 250 (3.73), 281 (3.23), 291 (3.15) nm; IR (KBr) νmax 3455, 2973, 2310, 1726, 1639, 1383, 1252, 1080 cm−1; 1H and 13C NMR data, see Table 1; HRESIMS m/z 395.1830 [M+Na]+ (calcd for C22H28NaO5, 395.1829). Caesall B (2): white powder; [α]25D +53.2° (c 0.10, MeOH); UV (MeOH) λmax (log ε) 207 (5.09) nm; IR (KBr) νmax 3449, 1743, 1639, 1400, 1244 cm−1; 1H and 13C NMR data, see Table 1; HRESIMS m/z 559.2516 [M+Na]+ (calcd for C28H40NaO10, 559.2514).
U
111 112
3
2.03, s 4.47, br s
71.9, CH
42.5, C 79.2, C 75.2, CH 74.4, 41.1, 37.4, 44.1, 22.5,
CH CH CH C CH2
150.7, 119.0, 138.8, 106.6, 142.2, 104.6,
C C C CH CH CH2
24.2, CH3 18.8, CH3 16.9, CH3 170.0, C 20.8, CH3 169.8, C 20.7, CH3 169.9, C 21,2, CH3 169.9, C 20.9, CH3
s m m m m
5.50, s 5.86, s 1.92, td (14.5, 3.0) 3.11, td (11.5, 5.0) 2.30, dd (16.0, 5.0) 2.42, dd (16.0, 11.5)
6.44, d (2.0) 7.33, d (2.0) 1.39, s 1.13, s 1.14, s 1.25, s 2.07, s
2.04, s 1.95, s
33.5, CH2 39.6, C 80.0, C 77.8, CH 75.4, 49.7, 35.4, 45.7, 24.0,
CH CH CH C CH2
152.7, C 120.7, C 74.4, C 110.3, CH2 142.5, CH2 26.0, CH3 31.2, CH3 25.3, CH3 17.6, CH3 171.7, C 21.9, CH3
172.6, C 21.9, CH3 172.6, C 21.9, CH3
s m m m m
4.72, s 3.88, m 1.57, m 2.79, m 2.07, m 2.38, m
2.99, 6.28, 7.37, 0.99,
m d (2.0) d (2.0) d (7.0)
1.03, s 1.08, s 1.06, s
35.7, CH2 40.7, C 77.4, C 72.9, CH 65.0, 41.9, 31.1, 43.0, 21.2,
CH CH CH C CH2
148.0, C 122.1, C 26.7, CH 109.8, CH 140.6, CH 16.8, CH3
2.04, s
23.0, CH3 24.9, CH3 17.4, CH3 169.2, C 20.9, CH3
2.04, s
169.3, C 20.7, CH3
3.10, d (2.5) 4.31, d (7.0) 3.18, s
51.9, CH3
Recorded in DMSO-d6. Recorded in CD3OD. Overlapped signals.
Please cite this article as: Wu L, et al, Cassane-type diterpenoids from the seed kernels of Caesalpinia bonduc, Fitoterapia (2014), http://dx.doi.org/10.1016/j.fitote.2014.01.011
132 133 134 135 136 137 138 139 140 141 142 143 144 145 146 147 148 149 150 151 152
L. Wu et al. / Fitoterapia xxx (2014) xxx–xxx
E
D
P
R O O
F
4
C
R
R
E
187
Colorless crystals of 1 were obtained from CH2Cl2–MeOH. 188 Crystal data were obtained on a Bruker Smart-1000 CCD 189 with a graphite monochromator with Cu Kα radiation (λ = 190
O
185 186
2.4. X-ray crystallographic analysis
N C
183 184
Caesall G (7): white powder; [α]25D + 5.2° (c 0.10, MeOH); UV (MeOH) λmax (log ε) 216 (3.80) nm; IR (KBr) νmax 3450, 2311, 1729, 1639, 1399, 1266, 1234,1050 cm−1; 1H and 13C NMR data, see Table 2; HRESIMS m/z 457.2199 [M+Na]+ (calcd for C24H34NaO7, 457.2197).
U
182 181 180 179 178 177 176 175 174 173 172 171 170 169 168 167 166 165
T
Fig. 1. Compounds from the seed kernels of C. bonduc.
Fig. 2. Single-crystal X-ray of compound 1.
Please cite this article as: Wu L, et al, Cassane-type diterpenoids from the seed kernels of Caesalpinia bonduc, Fitoterapia (2014), http://dx.doi.org/10.1016/j.fitote.2014.01.011
5
P
R O
O
F
L. Wu et al. / Fitoterapia xxx (2014) xxx–xxx
203
210
The cytotoxicity of compounds 1–10 was assessed via the MTT method using the HepG-2, MG-63 and MCF-7 cancer cell lines. The cells were grown in DMEM supplemented with 10% fetal bovine serum and cultured at a density of 5000 cell/mL in
211
E
2.5. Cytotoxicity assay
T
C
E
201 202
R
199 200
R
197 198
204
O
195 196
Cambridge Crystallographic Data Centre (deposition number: CCDC 966727). Copies of the data can be obtained free of charge via www.ccdc.cam.ac.uk/conts/retrieving.html or from the Cambridge Crystallographic Data Centre, 12, Union Road, Cambridge CB2 1EZ, UK. [Fax: (+44) 1223-336-033 or e-mail: [email protected]].
C
194
1.54184 Å) at 290(2) K. The structure was solved by direct methods using the SHELXS-97 [13] and expanded using difference Fourier techniques, refined with the SHELXL-97 [14]. Crystal data of 1: C22H28O5 (M = 372.44); monoclinic crystal (0.32 × 0.24 × 0.19 mm3); space group P21; unit cell dimensions a = 8.2412(2) Å, b = 7.86130(10) Å, c = 15.8285(3) Å, β = 104.899(2)°, V = 991.00(3) Å3; Z = 2; Dcalcd. = 1.248 mg/m3; μ = 0.710 mm−1; 7892 reflections measured (11.1 ≤ 2Θ ≤ 138.86); 2584 unique (Rint = 0.0174) which were used in all calculations; the final refinement gave R1 = 0.0311 (N 2sigma(I)) and wR2 = 0.0757 (all data); flack parameter = − 0.06(18). Crystallographic data for compound 1 have been deposited in the
N
192 193
U
191
D
Fig. 3. Key HMBC and ROESY of compound 2.
Fig. 4. Key HMBC and ROESY of compound 3.
Please cite this article as: Wu L, et al, Cassane-type diterpenoids from the seed kernels of Caesalpinia bonduc, Fitoterapia (2014), http://dx.doi.org/10.1016/j.fitote.2014.01.011
205 206 207 208 209
212 213 214
6
Compound
Hep-G2
t3:4 Q2 t3:5 t3:6 t3:7 t3:8 t3:9
1–7 8 9 10 Cisplatinb
t3:10 t3:11
a
IC50 ± SD (μM)
IC50 ± SD (μM)
IC50 ± SD (μM)
N100 39.8 ± 0.4 34.2 ± 0.8 27.6 ± 1.1 8.4 ± 0.9
N100 N100 44.5 ± 1.0 35.4 ± 1.4 24.8 ± 1.2
N100 N100 N100 N100 N100
F
Values present mean ± SD of triplicate experiments. Positive control.
245
3. Results and discussion
246
Compound 1 was obtained as colorless crystals and its molecular formula, C22H28O5, was determined by HRESIMS (m/z 395.1829 [M+Na]+). The IR absorptions at 3455 and 1726 cm−1 indicated the presence of hydroxyl and ester carbonyl groups. The 1H NMR spectrum (Table 1) showed two mutually coupled olefinic protons at δH 6.72 (1H, d, J = 2.0 Hz) and δH 7.52 (1H, d, J = 2.0 Hz) suggested the presence of a fused furan ring. The 13C NMR spectrum showed 22 carbon signals, including 8 olefinic and aromatic carbons (δC 104.3, 105.0, 126.1, 127.9, 128.3, 139.0, 144.7 and 153.8). These data suggested that the basic carbon skeleton of 1 was cassane-type diterpene and similar to caesalpinin MD [10]. The conjugation of the benzene ring with the fused furan ring was confirmed by the HMBC correlations of H-11 (δH 7.01, s) with C-12 (δC 153.8) and C-13 (δC 126.1) and of H3-17 (δH 2.39, s) with C-8 (δC 127.9), C-9 (δC 139.0), C-11 (δC 104.3), C-13 (δC 126.1) and C-14 (δC 128.3). Two proton signals at δH 2.78 (1H, dd, J = 17.0, 7.5 Hz) and 3.31 (1H, dd, J = 17.0, 7.5 Hz) were attached to C-7 (δC 36.6), which was determined by the HSQC correlations between H2-7 and C-7 and the cross peaks of H2-7 with C-8 (δC 127.9) and C-9 (δC 139.0) in the HMBC spectrum. Furthermore, the HMBC correlations of \OCOCH3 (δH 1.96, s) and H-1 (δH 5.64, s) with \OCOCH3 (δC 169.7) and of H2-7 with C-6 (δC 70.0) indicated that the acetoxy group was located at C-1 and the hydroxyl group at C-6. Thus, the planar structure of compound 1 was established as shown in Fig. 1. The relative configuration of 1 was mainly established by ROESY correlations. Cross peaks of H3-20 with H-1 and H-6 indicated that the acetoxy group at C-1 and the hydroxyl group at C-6 were α-oriented. The absolute configuration of 1 was determined by X-ray diffraction study as shown in Fig. 2. Thus, the structure was confirmed and the absolute configuration of 1 was finally determined to be 1S,5R,6S,10S and named caesall A.
254 255 256 257 258 259 260 261 262 263 264 265 266 267 268 269 270 271 272 273 274 275 276 277 278 279 280
C
E
252 253
R
250 251
R
248 249
O
247
N C
241 242
U
239 240
Compound 2 was obtained as white amorphous powder, and its molecular formula was assigned as C28H40O10 on the basis of its HRESIMS. The 1H NMR spectrum (Table 1) displayed three methyl signals at δH 1.04 (3H, s), 1.00 (3H, s) and 1.06 (3H, s), two methoxy signals at δH 3.38 (3H, s) and 3.00 (3H, s), and three acetoxy methyl signals at δH 2.03 (3H, s), 1.98 (3H, s) and 1.88 (3H, s). The 13C NMR and HSQC spectra of 2 showed 28 carbon signals for eight methyl groups, four methylene (one olefinic carbon at δC 109.8), seven methine (one olefinic carbon at δC 121.8; six carbon atoms bearing oxygen at δC 73.5, 78.3, 73.5, 74.8, 109.3 and 105.8) and nine quaternary carbons (two olefinic carbon atoms at δC 141.0 and 144.4; three ester carbonyls at δC 169.6, 169.8 and 169.9). In the HSQC and HMBC spectra, the proton signals at δH 5.06 (1H, s) and 4.90 (1H, s) were directly linked to the carbon signals at δC 109.8 and showed long-distance correlations with C-8 (δC 44.5), C-13 (δC 144.4) and C-14 (δC 141.0), suggesting that the double bond was between C-14 and C-17. Furthermore, The HMBC correlations (Fig. 3) of H-1 (δH 4.67, s) with \OCOCH3 (δC 169.6), H-6 (δH 5.33, d, J = 9.0 Hz) with \OCOCH3 (δC 169.8), H-7 (δH 5.43, t, J = 9.0 Hz) with \OCOCH3 (δC 169.9), 12-OCH3 (δH 3.00, s) with C-12 (δC 109.3), and 16-OCH3 (δH 3.38, s) with C-16 (δC 105.8) indicated that the acetoxy groups were attached to C-1, C-6, and C-7 and that two methoxy groups were attached to C-12 and C-15. The olefinic proton signals at δH 5.86 (1H, d, J = 2.0 Hz) were located at C-15 (δC 121.8) on the basis of the correlations of H-15 with C-12, C-13 and C-14. These data suggested that 2 was a tetracyclic cassane diterpene with isomerized dihydrofuran ring moiety. The relative configuration of compound 2 was established through analysis of its ROESY spectrum (Fig. 3). H3-20 (δH 1.06, s) had cross peaks with H-1 (δH 4.67, s) and H-6 (δH 5.33, d, J = 9.0 Hz), while H-16 (δH 5.37, d, J = 2.0 Hz) had cross peaks with H-8 (δH 2.33, m), which indicated that they were β-oriented. The interaction of H-7 (δH 5.43, t, J = 9.0 Hz) with H3-18 (δH 1.04, s) and 12-OCH3 (δH 3.00, s) suggested that they were α-oriented. Compound 3, a white amorphous powder, exhibited the molecular formula C22H30O6 according to HRESIMS (m/z 413.1932 [M+Na]+). The 1H NMR spectrum showed the presence of three methyl groups at δH 1.22 (3H, s), 1.18 (3H, s) and 1.10 (3H, s), an acetoxy methyl signal at δH 1.98 (3H, s), and three oxymethine resonances at δH 4.62 (1H, s), 3.69 (1H, dd, J = 8.5, 6.5 Hz) and 3.81 (1H, dd, J = 15.5, 7.5 Hz). The 1,2-disubstituted furan ring was evident from low-field doublets at δH 6.56 (1H, d, J = 1.5 Hz) and 7.43 (1H, d, J = 1.5 Hz). The exo-methylene protons were revealed by the
D
243 244
a 96-well microtiter plate. Five different concentrations of each compound in DMSO were subsequently added to the wells. Each concentration was tested in triplicate. After incubation under 5% CO2 at 37 °C for 48 h, 20 μL of MTT (5 mg/mL) was added to each well, and the cells were incubated for another 4 h. Then, the liquid in each well was removed, and DMSO (150 μL) was added. The absorbance (OD values) at 570 nm with a 630 nm reference was measured on a Universal Microplate Reader.
237 238
R O O
b
MCF-7
P
236 235 234 233 232 231 230 229 228 227 226 225 224 223 222 221 220 219
MG-63 a
E
t3:3
Table 3 In vitro cytotoxic activities of compounds 1-10.
T
t3:1 t3:2
L. Wu et al. / Fitoterapia xxx (2014) xxx–xxx
Please cite this article as: Wu L, et al, Cassane-type diterpenoids from the seed kernels of Caesalpinia bonduc, Fitoterapia (2014), http://dx.doi.org/10.1016/j.fitote.2014.01.011
281 282 283 284 285 286 287 288 289 290 291 292 293 294 295 296 297 298 299 300 301 302 303 304 305 306 307 308 309 310 311 312 313 314 315 316 317 318 319 320 321 322 323 324 325 326 327
L. Wu et al. / Fitoterapia xxx (2014) xxx–xxx
351 352 353
354 Q6 355 356 357 Q7 358 359 360 361 362 363 364 365 366 367 368 369 370 371 372 373 374 375 376 377 378 379 380 381 382 383 384 385 386 387 388
422 Q11
This research work was financially supported by the Youth Fund Project of Basic Research Program of Jiangsu Province (Natural Science Foundation, BK2012351), the National High Technology Research and Development Program of China (863 Program) (2013AA093001), the Fundamental Research Funds for the Central Universities (JKPZ2013011), the Program for Changjiang Scholars and Innovative Research Team in University (PCSIRT-IRT1193), and the Project Funded by the Priority Academic Program Development of Jiangsu Higher Education Institutions (PAPD).
423
References
433
R O
O
F
Acknowledgments
P
349 350 Q5
D
347 Q4 348
389 390
E
345 346
difference being the relative configuration at C-14. The Rf value on HPLC (14.65 min for 6 and 7.43 min for 5α-hydroxy14β-methoxy-1α, 6α, 7β-triacetoxyvouacapane, MeOH: H2O =70:30) indicated that the polarity of 6 is smaller than 5α-hydroxy-14β-methoxy-1α, 6α, 7β-triacetoxyvouacapane. The structure of 6 was established as 5α-hydroxy-14αmethoxy-1α, 6α, 7β-triacetoxyvouacapane and named caesall F. Compound 7 exhibited [M+Na]+ ion peak at m/z 457.2199 using HRESIMS, corresponding to the molecular formula C24H34O7. The obtained 1H and 13C NMR signals (Table 2) were closely related to those of 6, except for a hydroxyl group replace an acetoxy group at C-7 (δC 65.0) and H3-17 (δH 0.99, d, J = 7.0 Hz) to be α-oriented in 7. The HMBC correlation of C-8 (δC 41.9) with 7-OH (δH 4.31, d, J = 7.0 Hz) suggested that the hydroxyl group was located at C-7. The relative stereochemistry of 7 was deduced by the analysis of the ROESY correlations. The cross peaks from H3-17 to H-9 and H-7 indicated that the H3-17 was to be α-oriented. Thus, the structure of 7 was described as shown (Fig. 1) and named caesall G. Compounds 1–10 were tested for their cytotoxicity against three human cancer cell lines (HepG-2, MCF-7 and MG-63) using the MTT method. The obtained IC50 values were between 27.6 and 39.8 μM, and cisplatin was used as a positive control (Table 3). Compounds 1–7 were inactive against all of the tested cell lines. Compounds 8–10 showed low activity against HepG-2 (IC50 27.66–39.83 μM). Compounds 9–10 also showed low activity against MG-63 (IC50 35.48–44.5 μM). None of the compounds showed activity against MCF-7. On the basis of these biological results, we hypothesize that the presence of the γ-lactone was formed between the oxygen at C-7 and the C-17 carbonyl in the cassane diterpenes that may enhance the cytotoxic activities of the compounds.
T
343 344
C
341 342
E
339 340
R
337 338
R
335 336
O
333 334
C
332
N
330 331 Q3
signals at δH 5.33 (1H, s) and 5.16 (1H, s). The NMR data for this compound (Table 1) were similar to those of 5α-hydroxy1α,6α,7β-triacetoxyvouacapan-14(17)-ene [15], except for two acetoxy groups that were replaced by two hydroxyl groups at C-6 and C-7. The full assignments were determined by analysis of the HSQC and HMBC data. In the HMBC spectrum (Fig. 4), H2-17 showed correlations to C-8 and C-13, which supported the location of the exocyclic double bond between C-14 and C-17 and conjugated with the furan ring. The locations of the acetoxy group at C-1 and two hydroxyl groups at C-6 and C-7 were determined by the correlations of H-1 (δH 4.62, s) and \OCOCH3 (δH 1.98, s) with \OCOCH3 (δC 169.8), of 6-OH (δH 4.27, d, J = 6.5 Hz) with C-6 (δC 75.4) and of 7-OH (δH 4.38, d, J = 6.5 Hz) with C-7 (δC 73.0) in the HMBC spectrum. The relative configuration of 3 was established by ROESY correlations. The correlations of H3-20 (δH 1.10, s) with H-1 (δH 4.62, s) and H-6 (δH 3.69, dd, J = 8.5, 6.5 Hz), of H3-19 (δH 1.18, s) with H-6 (δH 3.69, dd, J = 8.5, 6.5 Hz), of H3-18 (δH 1.22, s) with 6-OH (δH 4.27, d, J = 6.5 Hz) indicated H-1 (δH 4.62, s) and H-6 (δH 3.69, dd, J = 8.5, 6.5 Hz) to be β-oriented. On the other hand, ROESY correlations between 7-OH (δH 4.38, d, J = 6.5 Hz) and 8-H (δH 2.11, m) indicated H-7 (δH 3.81, dd, J = 15.5, 7.5 Hz) to be α-oriented. The molecular formula of 4 was assigned as C22H30O5 on the basis of HRESIMS (m/z 397.1988 [M+Na]+). The 1H and 13 C NMR spectra of 4 were similar to those of 3 except for the hydroxyl group at C-6 in 3 that was absent in 4. In the HMBC spectrum, the correlations of 5-OH (δH 3.13, d, J = 1.5 Hz) with C-6 (δC 36.1) and of 7-OH (4.34, d, J = 7.0 Hz) with C-8 (δC 43.7) suggested a hydroxyl group at C-7. The relative configuration of compound 4 was determined from the ROESY spectrum. The NOEs from H-1 to H3-20, H-8 and H-11ax (δH 2.55, m); from 5-OH to H3-18, H-7 and H-9 indicated that rings A and B are in chair conformations with a trans-fused ring junction, thus confirming the relative configurations at C-1, C-5 and C-7. Compound 5 was shown to possess a molecular formula of C28H36O10 by HRESIMS (m/z 555.2198 [M+Na]+). A comparison of the NMR data (Table 2) of 5 and 4 indicated the presence of three additional acetoxy groups at C-3, C-6 and C-7 in 5. The HMBC correlations of H-1 (δH 4.77, s) with \OCOCH3 (δC 20.8) and \OCOCH3 (δC 169.8), H-3 (δH 5.06, dd, J = 12.5, 4.0 Hz) with \OCOCH3 (δC 20.7) and \OCOCH3 (δC 169.9), H-6 (δH 5.49, s) with\OCOCH3 (δC 21.2) and H-7 (δH 5.49, s) with\OCOCH3 (δC 20.9), \OCOCH3 (δH 2.01) with C-6 (δC 75.2) and\OCOCH3 (δH 2.03) with C-7 (δC 74.4), together with the above molecular formula confirmed the above assumption. The relative configuration of 5 was deduced by the analysis of the ROESY correlations. The correlations of H3-20 with H-1, H-6 and H-7 indicated H-1, H-6 and H-7 to be β-oriented and of H3-18 with 5-OH and H-3 indicated H-3 to be α-oriented. Compound 6 gave [M+Na]+ peak at m/z 529.2406 (HRESIMS) for the molecular formula C27H38O9. The NMR spectra of 6 are similar to those of 5α-hydroxy-14β-methoxy-1α, 6α, 7β-triacetoxyvouacapane [15], except that the signal for H-9 (δH 3.11, td, J = 11.5, 5.0 Hz) in 6 is shifted to lower field. The ROESY correlation between H-8 (δH 1.92, td, J = 14.5, 3.0 Hz) and H3-17 (δH 1.39, s) indicated H3-17 to be β-oriented. The above data suggested that 6 is a stereoisomer of 5α-hydroxy14β-methoxy-1α, 6α, 7β-triacetoxyvouacapane, with the
U
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