Fitoterapia 100 (2015) 133–138
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Six new alkaloids from Melodinus henryi Ke Ma, Jun-Song Wang, Jun Luo, Ling-Yi Kong ⁎ State Key Laboratory of Natural Medicines, Department of Natural Medicinal Chemistry, China Pharmaceutical University, 24 Tong Jia Xiang, Nanjing 210009, People's Republic of China
a r t i c l e
i n f o
Article history: Received 28 September 2014 Accepted in revised form 21 November 2014 Available online 3 December 2014 Keywords: Melodinus henryi Bisindole alkaloids Quinolinic melodinus alkaloids Isolation Structural elucidation
a b s t r a c t A total of six new alkaloids, melodinhenines A–F (1–6), were isolated from Melodinus henryi. Melodinhenines A and B are new eburnan–vindolinine-type bisindole alkaloids and melodinhenines C–F are new quinolinic melodinus alkaloids. Their structures were elucidated through extensive spectroscopic methods including 2D NMR and HRESIMS analyses. The absolute configuration of 1 and 2 was determined using ECD exciton chirality method. To the best of our knowledge, this is the first report on the determination of the absolute configuration of eburnan–vindolinine-type bisindole alkaloid using this method. © 2014 Elsevier B.V. All rights reserved.
1. Introduction The genus Melodinus (Apocynaceae) includes 53 species which are distributed mainly in the tropical and subtropical areas of Asia and Australia. Many plants of this genus are used as folk medicine to treat hernia, indigestion, and abdominal pain in Yunnan, Guangdong, and Guangxi provinces of China [1]. Previous chemical investigations indicated that these plants were rich in various skeletal types of alkaloids and their dimeric forms, including scandine-type alkaloids such as melodines T and U [2], aspidosperma-type alkaloids such as melodinines M–S [2], rearranged aspidosperma-type alkaloids, such as melotenine A [3], ketolactam derivative, such as melohenine B [4], condylocarpan-type alkaloids such as 19S-methoxytubotaiwine N4-oxide [5], and melodines–aspidosperma-type bisindole alkaloids such as melosuavines A–C [6]. Pharmacological investigations on these alkaloids have demonstrated promising antitumor activity and antifertility activity [7,8]. As a part of our ongoing research on structurally diverse and potentially bioactive alkaloids [9,10], six new alkaloids, melodinhenines A–F (1–6), were isolated from Melodinus henryi. Of these new ⁎ Corresponding author. Tel./fax: +86 25 8327 1405. E-mail address: [email protected] (L.-Y. Kong).
http://dx.doi.org/10.1016/j.fitote.2014.11.021 0367-326X/© 2014 Elsevier B.V. All rights reserved.
alkaloids, melodinhenines A and B were rare eburnan– vindolinine-type bisindole alkaloids, and their structures were elucidated by spectroscopic methods. The absolute configuration of 1 and 2 was determined by ECD exciton chirality method, and this is the first report on the determination of the absolute configuration of eburnan–vindolinine-type bisindole alkaloid using this method. Herein, the isolation and structural elucidation of these isolates are described. 2. Experimental 2.1. General Optical rotations were recorded using a JASCO P-1020 polarimeter. UV spectra were recorded using a Shimadzu UV2450 spectrophotometer. ECD spectra were measured with a JASCO J-810 spectropolarimeter. IR spectra were recorded on KBr discs using a Bruker Tensor 27 spectrometer. 1D and 2D NMR spectra were acquired with a Bruker AV III-500 NMR instrument at 500 MHz (1H) and 125 MHz (13C). ESIMS and HRESIMS spectra were recorded using an Agilent 1100 series LC-MSD-Trap-SL mass analyzer and an Agilent 6520B Q-TOF mass instrument, respectively. Column chromatography (CC) was performed with ODS (40–63 μm, Fuji, Japan) and Sephadex
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LH-20 (Pharmacia, Sweden) columns. Preparative HPLC was performed on a Shimadzu LC-8A system (Shimadzu, Tokyo, Japan) equipped with a Shim-pack RP-C18 column (200 mm × 20 mm i.d., 10 μm, Shimadzu, Tokyo, Japan) with a flow rate of 10.0 mL/min and a column temperature of 25 °C, and detection was performed with a binary channel UV detector at 210 and 230 nm. 2.2. Plant material The stems of M. henryi were collected from Yunnan province of China in March 2013 and identified by Mr. Shuncheng Zhang, Xishuangbanna Tropical Botanical Garden, Chinese Academy of Sciences. A voucher specimen (No. 130312) was deposited in the Department of Natural Medicinal Chemistry, China Pharmaceutical University. 2.3. Extraction and isolation The air-dried stems of M. henryi (7 kg) were extracted with 95% EtOH (3 × 4 h). The crude extract was partitioned between EtOAc and 1% HCl. The aqueous phase was adjusted to pH 9–10 with ammonia water, and extracted with EtOAc to obtain crude alkaloids. The crude alkaloids were subjected to a D-101 macropous resion column, eluted with a gradient of EtOH–H2O (30:70 to 90:10, v/v), to afford four fractions (Fr.1–4). Fr.1 was subjected to a MCI gel column, eluted with a gradient of MeOH–H2O (30:70 to 90:10, v/v) to yield seven subfractions (Fr.1.1–1.7). Fr.1.3 was subjected to an RP-C18 column, eluted with a gradient of MeOH–H2O (30:70 to 50:50, v/v), finally purified by preparative HPLC using MeOH–H2O (30:70, containing 0.1% TFA, flow rate: 10 ml/min, retention time: 31 min, 35 min) to yield 3 (30 mg) and 4 (40 mg). Fr.2 was separated by an RP-C18 column, eluted with MeOH–H2O (30:70 to 70:30, v/v), to afford five subfractions (Fr.2.1–2.5), and Fr.2.2 was subjected to a Sephadex LH-20 column and eluted with MeOH, finally purified by preparative HPLC using MeOH–H2O (35:65, containing 0.1% TFA, flow rate: 10 ml/min, retention time: 36 min, 42 min) to yield 5 (6 mg) and 6 (8 mg). Fr.3 was subjected to a MCI gel column, eluted with a gradient of MeOH–H2O (30:70 to 90:10, v/v) to yield seven subfractions (Fr.3.1–3.7). Fr.3.2 was subjected to an RP-C18 column, eluted with a gradient of MeOH–H2O (30:70 to 90:10, v/v), finally purified by preparative HPLC using MeOH–H2O (68:32, containing 0.1% TFA, flow rate: 10 ml/min, retention time: 31 min, 35 min) to yield 1 (6 mg) and 2 (8 mg). Melodinhenine A (1): yellow solid; [α]24D +46 (c 1, CHCl3); UV (MeOH): λmax (log ε): 225 (4.53), 286 (3.41), and 316 (3.38) nm; ECD (c 3.4 × 10−4 M, MeOH) λmax (Δε): 233 nm (−0.04), and 255 nm (+5.44); IR (KBr) υmax 3324, 1681, 1462, and 1203 cm−1; ESI-MS (positive): m/z 613.6 [M + H]+; HRESIMS (positive): m/z 613.3538 [M + H]+, (calcd. 613.3537); and 1H and 13C NMR data see Table 1. Melodinhenine B (2): yellow solid; [α]24D −19 (c 1, CHCl3); UV (MeOH): λmax (log ε): 222 (4.53), 286 (3.41), and 318 (3.38) nm; ECD (c 3.4 × 10−4 M, MeOH) λmax (Δε): 233 nm (−0.05), and 255 nm (+10.65); IR (KBr) υmax 3386, 1721, 1462, and 1203 cm−1; ESI-MS (positive): m/z 629.5 [M + H]+ HRESIMS (positive): m/z 629.3487 [M + H]+ (calcd. 629.3486); and 1H and 13C NMR data see Table 2.
Table 1 1 H NMR (500 MHz) and methanol-d4. No. 2 3a 3b 5a 5b 6a 6b 7 8 9 10 11 12 13 14 15 16 17a 17b 18 19a 19b 20 21
13
C NMR (125 MHz) data of compound 1 in
δH (mult; J, Hz) 3.81, m 3.38, m 3.78, m 3.81, m 3.39, m 3.22, m
δC
No.
133.8 44.3
2′ 3′a 3′b 5′a 5′b 6′a 6′b 7′ 8′ 9′ 10′ 11′ 12′ 13′ 14′ 15′ 16′ 17′a 17′b 18′ 19′ 20′ 21′ OMe CO
52.7 17.1 105.3 127.7 119.5 121.2 122.2 113.5 139.1 122.2 129.9 57.7 43.3
7.45, d, (10.0) 7.03, t, (10.0) 6.88, t, (10.0) 6.49, d, (10.0) 5.62, m 6.03, m 4.78, dd, (11.6, 4.0) 2.41, m 1.98, m 1.07, t, (7.6) 1.96, m 1.73, m
7.9 33.8 38.3 59.6
5.08, s
δH (mult; J, Hz) 3.22, m 3.33, m 3.24, m 3.26, m 1.74, m 2.21, m 7.32, m 7.00, d, (10.0) 6.99, dd, (10.0, 2.0) 6.03, d, (2.0) 5.67, m 6.06, m 3.07, dd, (12.0, 6.4) 2.35, m 1.74, m 1.15, d, (6.8) 2.09, q, (6.8) 4.07, s 3.64, s
δC 83.2 50.1 52.8 33.5 60.3 139.1 123.1 128.7 134.1 124.4 151.1 129.9 124.4 39.9 28.9 7.1 50.2 45.7 59.6 51.6 175.1
Melodinhenine C (3): white amorphous powder; [α]24D +38 (c 1, CHCl3); UV (MeOH): λmax (log ε): 220 (4.53), 284 (3.41), and 293 (3.38) nm; IR (KBr) υmax 3426, 1681, 1462, and 1203 cm−1; ESI-MS (positive): m/z 367.3 [M + H]+; HRESIMS (positive): m/z 367.1651 [M + H]+ (calcd. 367.1652); and 1H and 13C NMR data see Table 3.
Table 2 1 H NMR (500 MHz) and methanol-d4. No. 2 3a 3b 5a 5b 6a 6b 7 8 9 10 11 12 13 14 15 16 17a 17b 18 19a 19b 20 21
δH (mult; J, Hz) 3.81, m 3.38, m 3.78, m 3.81, m 3.39, m 3.22, m
7.49, d, (10.0) 7.03, t, (10.0) 6.91, t, (10.0) 6.49, d, (10.0) 5.62, m 6.03, m 2.46, m 2.12, m 1.07, t, (7.6) 1.96, m 1.73, m 5.08, s
13
C NMR (125 MHz) data of compound 2 in
δC
No.
133.2 44.3
2′ 3′a 3′b 5′a 5′b 6′a 6′b 7′ 8′ 9′ 10′ 11′ 12′ 13′ 14′ 15′ 16′ 17′a 17′b 18′ 19′ 20′ 21′ OMe CO
52.7 17.1 105.3 127.7 119.5 121.3 123.1 113.5 139.1 122.2 128.7 80.4 43.3 7.9 33.8 38.3 59.6
δH (mult; J, Hz) 3.22, m 3.33, m 3.24, m 3.26, m 1.74, m 2.21, m 7.32, m 7.00, d, (10.0) 6.99, dd, (10.0, 2.0) 6.03, d, (2.0) 5.67, m 6.06, m 3.07, dd, (12.0, 6.4) 2.36, m 2.04, m 1.35, d, (6.8) 2.09, q, (6.8) 4.07, s 3.64, s
δC 85.2 50.1 50.2 33.8 59.2 139.1 123.1 128.7 134.1 124.7 150.9 131.0 125.3 43.3 26.2 9.9 51.6 44.2 59.8 51.6 175.1
K. Ma et al. / Fitoterapia 100 (2015) 133–138 Table 3 1 H NMR (500 MHz) and methanol-d4.
13
C NMR (125 MHz) data of compounds 3 and 4 in
3 Position 2 3a 3b 5a 5b 6a 6b 7 8 9 10 11 12 13 14 15 16 17a 17b 18a 18b 19 20 21 CO OMe
δH (J in Hz) 3.68, m 3.75, m 3.66, m 3.98, m 2.36, m 2.68, m
6.85, d, (9.0) 6.76, dd (9.0,2.0) 6.87, d, (2.0) 5.86, d, (10.0) 6.15, d, (10.0) 2.74, m 3.13, m 5.07, m 5.16, m 5.82, m 4.02, s 3.26, s
Table 4 1 H NMR (500 MHz) and methanol-d4.
4 δC 167.8 45.8 53.5 33.7 60.7 126.5 118.5 116.8 155.5 113.9 129.3 116.6 135.6 62.2 44.1 113.9 141.9 46.6 80.7 169.9 54.7
δC
Position
3.72, m
169.8 46.5
2 3a 3b 5a 5b 6a 6b 7 8 9 10 11 12 13 14 15 16 17a 17b 18 19 20 21 CO
7.45, d, (10.0) 7.18, t, (10.0) 7.34, t, (10.0) 7.00, d, (10.0) 5.88, d, (10.0) 6.16, d, (10.0) 2.15, m 2.78, m 5.06, m 5.18, m 5.85, m 4.08, s 3.56, s
13
C NMR (125 MHz) data of compounds 5 and 6 in
5
δH (J in Hz)
3.65, m 3.05, m 2.36, m 2.78, m
135
54.5 33.8 60.6 125.1 126.9 125.2 130.4 117.3 137.1 116.8 135.4 62.4 44.1 115.1 141.9 45.8 80.4 168.3 53.5
Melodinhenine D (4): white amorphous powder; [α]24D −21 (c 1, CHCl3); UV (MeOH): λmax (log ε): 220 (4.53), 284 (3.41), and 293 (3.38) nm; IR (KBr) υmax 3324, 1681, 1462, and 1203 cm−1; ESI-MS (positive): m/z 351.3 [M + H]+; HRESIMS (positive): m/z 351.1706 [M + H]+ (calcd. 351.1703); and 1H and 13C NMR data see Table 3. Melodinhenine E (5): white amorphous powder; [α]25D +8 (c 0.5, MeOH); UV (MeOH) λmax (log ε) 284 (3.62), and 215 (4.97) nm; IR (KBr) υmax 3401, 2658, 1716, 1204, and 1033 cm−1; ESI-MS (positive): m/z 321.3 [M + H]+; HRESIMS m/z 321.1595 (calcd. 321.1598); and 1H and 13C NMR data see Table 4. Melodinhenine F (6): white amorphous powder; [α]25D −12 (c 0.5, MeOH); UV (MeOH) λmax (log ε) 284 (3.62), and 215 (4.97) nm; IR (KBr) υmax 3407, 2654, 1724, 1204, and 1033 cm−1; ESI-MS (positive): m/z 321.3 [M + H]+; HRESIMS m/z 321.1595 (calcd. 321.1598); and 1H and 13C NMR data see Table 4. 3. Results and discussion Melodinhenine A (1) was obtained as a yellow solid, and its molecular formula was established as C40H45N4O2 by HRESIMS (m/z 613.3538 [M + H]+, calcd. 613.3537), requiring 20 indices of hydrogen deficiency. The IR absorption bands at 3386 and 1721 cm−1 resulted from the –NH and ester carbonyl functionalities. Its 1H NMR data (methanol-d4, 500 MHz) (Table 1) showed an indole moiety with an unsubstituted aryl moiety (δH 7.50 (1H, d, J = 10.0 Hz), 7.06 (1H, t, J = 10.0 Hz), 6.98 (1H, t, J = 10.0 Hz), and 6.76 (1H, d, J = 10.0 Hz)), a typical splitting pattern of a 1,2,4-trisubstitued aromatic ring (δH 6.05, d, J = 2.0 Hz; δH 6.49, dd, J = 2.0, 10.0 Hz; δH 5.68, d,
δH (J in Hz) 3.99, m 4.32, m 3.58, m 3.86, m 2.16, m 2.72, m
7.32, d, (10.0) 7.06, t, (10.0) 7.26, t, (10.0) 6.98, d, (10.0) 6.05, m 6.21, m 2.38, m 2.48, m 0.96, d, (5.0) 2.79, q, (5.0) 4.41, s
6 δC 168.7 47.6 56.2 37.4 58.3 130.0 124.6 125.0 130.2 118.1 138.6 122.6 127.2 68.3 40.3 8.5 53.4 48.1 62.9 207.9
δH (J in Hz) 3.96, m 4.32, m 3.53, m 3.86, m 2.26, m 2.49, m
7.38, d, (10.0) 7.04, t, (10.0) 7.26, t, (10.0) 6.98, d, (10.0) 6.02, m 6.25, m 2.18, m 2.68, m 0.89, d, (5.0) 2.12, q, (5.0) 4.29, s
δC 169.0 47.6 55.9 36.5 56.7 130.2 125.1 124.9 130.2 118.0 138.4 122.1 125.6 69.0 37.1 11.7 52.4 46.9 68.9 209.0
J = 10.0 Hz), a methoxyl resonance at δH 3.79 (3H, s), and two methyl resonances at δH 1.16 (1H, d, 5.0 Hz) and 1.07 (1H, t, 10.0 Hz). The 13C NMR (Table 1) and HSQC spectra suggested that 1 possessed 40 carbons, including three methyl, nine methylene, sixteen methine, and twelve quaternary carbons. Therefore, the gross structure of 1 was determined to be a bisindole alkaloid, consisting of eburnan and vindolinine units, similar to melodinine H [11]. The HMBC correlations of H-17 to C-16, H-16 to C-11′, and H-17 to C-12′ (Fig. 2) indicated that the two units were connected via C-16 and C-11′. Thus, the planar structure of 1 was determined as shown in Fig. 2. The relative configuration of 1 was confirmed by analyses of 1H–1H coupling constant and ROESY correlations (Fig. 3). The coupling constant of H-16 (dd, J = 11.6, 4.0 Hz) suggested H-16 to be β oriented by comparison with that of Omethylvincanol (dd, J = 9.5, 5.7 Hz) and celastromelidine (dd, J = 10.0, 5.0 Hz) [11,12]. The ROESY cross peaks of H-16/ H-15 and H-21/H-19 indicated that H-21 and the C-20 ethyl were α oriented. In vindolinine unit, the ROESY correlations of H-19′/H-16′, and H-21′/H-16′ indicated that H-16′, H-19′, and H-21′ were on the same side. The absolute configuration of 1 was determined using the ECD exciton chirality method. The ECD spectrum of 1 showed a positive Cotton effect at 255 nm (Δε + 5.44) and a negative Cotton effect at 233 nm (Δε − 0.04), which was indicative of P-helicity between the two indole chromophores, hence permitting assignment of 16R absolute configuration (Fig. 4). Thus, the absolute configuration of 1 was assigned as depicted. Melodinhenine B (2) was obtained as a yellow solid, and its molecular formula was established as C40H45N4O3 by HRESIMS (m/z 613.3487 [M + H]+, calcd. 613.3486), requiring 20 indices of hydrogen deficiency. The IR absorption bands at 3386 and 1721 cm−1 resulted from the –NH and ester carbonyl functionalities. The 1D NMR data of 2 resembled those of 1,
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K. Ma et al. / Fitoterapia 100 (2015) 133–138
Fig. 1. Structures of compounds 1–6.
indicating that 2 was also an eburnan–vindolinine-type bisindole alkaloid. The C-16 resonance of 2 was deshielded relative to that of 1, indicating that a hydroxy group, instead of a proton, was attached to C-16, which was further confirmed by the HMBC correlations of H-17 to C-16 and C-20. Thus, the planar structure of 2 was established. The relative configuration of 2 was confirmed by analysis of ROESY spectrum of 2. The ROESY cross peak of H-21/H-19 indicated that H-21 and the C-20 ethyl were in the same side. In vindolinine unit, the ROESY correlations of H-19′/H-16′, and H-21′/H-16′ indicated that H-16′, H-19′, and H-21′ were in the same side. The absolute configuration of 2 was also determined using the ECD exciton chirality method. The ECD spectrum of 2 showed a positive Cotton effect at 255 nm (Δε + 10.65) and a negative Cotton effect at 233 nm (Δε − 0.05), which was indicative of P-helicity between the two indole chromophores, hence permitting assignment of 16R absolute configuration. Thus, the absolute configuration of 2 was assigned as depicted. Melodinhenine C (3) was obtained as a white amorphous powder, and its molecular formula was established as C21H22N2O4 by HRESIMS (m/z 367.1651, calcd. 367.1652). The IR absorption bands at 3426 and 1681 cm−1 resulted
from the –NH and ester carbonyl groups. Its 1H NMR data (Table 3) showed a typical splitting pattern of a 1,2,4trisubstitued aromatic ring (δH 6.87, d, J = 2.0 Hz; δH 6.76, dd, J = 2.0, 9.0 Hz; δH 6.85, d, J = 9.0 Hz), a methoxy resonance at δH 3.58 (s). The 13C NMR (Table 3) and HSQC spectra suggested that 3 possessed 21 carbons, including one methyl, five methylenes, eight methines, and seven quaternary carbons. Therefore, the gross structure of 3 was established as scandinetype alkaloid. The 1H and 13C NMR data of 3 were similar to those of 10-hydroxyscandine except for two differences [2]. First, the aromatic moiety resonances at δH 6.85, d, J = 2.0 Hz, δH 6.76, dd, J = 2.0, 9.0 Hz, δH 6.87, d, J = 9.0 Hz, δC 126.5, δC 113.9, δC 114.9, δC 155.5, δC 116.8, and δC 129.3, were different from those of 10-hydroxyscandine, suggesting that the hydroxyl group was attached to C-11 rather than C-10, which was further confirmed by the HMBC correlations (Fig. 5) of H-10 (δH 6.76, dd, J = 2.0, 9.0 Hz) to C-8 (δC 126.5), C-11 (δC 155.5), and C-12 (δC 118.5), and H-12 to C-8. Second, the C-7 resonances at
19 16
15 21
21' 16'
Fig. 2. Selected HMBC correlations of 1.
19'
Fig. 3. Selected ROESY correlations of 1.
K. Ma et al. / Fitoterapia 100 (2015) 133–138
137
6 21
15
17
Fig. 4. The ECD spectra of 1. A positive first and a negative second Cotton effect indicated the positive chirality (P-helicity) between the two indole chromophores of 1.
(δC 60.7), C-20 (δC 46.6), and C-21 (δC 80.7), rather than C-7 (δC 62.7), C-20 (δC 43.9), and C-21 (δC 81.7) in 10hydroxyscandine, indicated that the configuration of C-21 was different from that of 10-hydroxyscandine [2]. The βorientation of H-21 could be confirmed by the ROESY cross peaks (Fig. 6) of H-21 (δH 4.05) to H-6b (δH 2.68), H-15 (δH 6.15), and H-17 (δH 2.74). According to the ROESY experiment of 3, the other asymmetric carbons had the same relative configuration as those of 10-hydroxyscandine. Finally, the structure of 3 was established as shown in the Fig. 1. Melodinhenine D (4) was obtained as a white amorphous powder, and its molecular formula was established as C21H22N2O4 by HRESIMS (m/z 351.1706, calcd. 351.1703). The 1D NMR data of 4 was similar to 3 with the exception of signals corresponding to an unsubstituted aromatic moiety [δH 7.45 (1H, d, J = 10.0 Hz), 7.34 (1H, t, J = 10.0 Hz), 7.18 (1H, t, J = 10.0 Hz), 7.00 (1H, d, J = 10.0 Hz)]. This observation indicated that the planar structure of 4 was similar to 3 except for the absence of C-11 hydroxyl group. According to the ROESY experiment, all asymmetric carbons of 4 had the same relative configuration as those of 3. Finally, the structure of 4 was established as shown in the Fig. 1. Melodinhenine E (5) was isolated as a light yellow solid, and its molecular formula was established as C20H20N2O2 by HRESIMS (m/z 321.1595, calcd. 321.1598). Its 1H NMR data (Table 4) showed an unsubstituted aromatic moiety with signals at δH 7.32 (1H, d, J = 10.0 Hz), δH 7.26 (1H, d, J = 10.0 Hz), δH 7.06 (1H, t, J = 10.0 Hz), and δH 6.98 (1H, t, J = 10.0 Hz). The 13C NMR (Table 4) and HSQC spectra suggested
Fig. 5. The key HMBC correlations of 3.
Fig. 6. Selected ROESY correlations of 3.
that 5 possessed 20 carbons, including one methyl, four methylene, eight methine, and seven quaternary carbons. Therefore, the gross structure of 5 was established as scandine-type alkaloid. The 1H and 13C NMR data of 5 were similar to those of 19-epimeloscandonine except for the resonances of C-7 (δC 58.3), C-20 (δC 48.1), and C-21 (δC 62.9) [8]. This observation indicated that 5 was C-21-epimer of 19epimeloscandonine. The β-orientation of H-21 could be confirmed by the ROESY cross peaks (Fig. 7) of H-21 (δH 4.05) to H-6b (δH 2.68), and H-17 (δH 2.74). The other asymmetric carbons had the same relative configuration as those of 19epimeloscandonine on the basis of the ROESY experiment of 5. Finally, the structure of 5 was established as shown in the Fig. 1. Melodinhenine F (6) was isolated as a light yellow solid, and its molecular formula was established as C20H20N2O2 by HRESIMS (m/z 321.1595, calcd. 321.1598). The similarity of the 1H and 13C NMR spectroscopic data of 6 (Table 4) to those of meloscandonine indicated that 6 was also C-21-epimer of
6
21
Fig. 7. Selected ROESY correlations of 5.
17
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K. Ma et al. / Fitoterapia 100 (2015) 133–138
Scheme 1. The plausible biogenetic pathway for 1 and 2.
meloscandonine, which was the same as the relationship between 5 and 19-epimeloscandonine [8]. To the best of our knowledge, 1 and 2 are new examples of the eburnan–vindolinine type bisindole alkaloid. They might be derived from eburnan-type and vindolinine-type alkaloids via a coupling reaction. In this reaction, a providing electrophillic center indole alkaloid attacks to a providing nucleophillic center indole alkaloid in the acidic medium [13]. Here, we made a plausible biosynthetic pathway of them, which was presented in Scheme 1. Acknowledgments The work was supported by the Priority Academic Program Development of Jiangsu Higher Education Institutions (PAPD) and the Program for Changjiang Scholars and Innovative Research Team in University (IRT1193). References [1] Tian Y, Li PY. Flora of China. vol. 63. Beijing: Science Press; 1977 17–63. [2] Liu YP, Li Y, Cai XH, Li XY, Kong LM, Cheng GG, et al. Melodinines M–U, cytotoxic alkaloids from Melodinus suaveolens. J Nat Prod 2012;75:220–4.
[3] Feng T, Li Y, Liu YP, Cai XH, Wang YY, Luo XD. Melotenine A, a cytotoxic monoterpenoid indole alkaloid from Melodinus tenuicaudatus. Org Lett 2010;5:968–71. [4] Feng T, Cai XH, Li Y, Wang YY, Liu YP, Xie MJ, et al. Melohenines A and B, two unprecedented alkaloids from Melodinus henryi. Org Lett 2009;21: 4834–7. [5] Cai XH, Li Y, Liu YP, Li XN, Bao MF, Luo XD. Phytochemistry 2012;83: 116–24. [6] Liu YP, Zhao YL, Feng T, Cheng GG, Zhang BH, Li Y, et al. Melosuavines A–H, cytotoxic bisindole alkaloid derivatives from Melodinus suaveolens. J Nat Prod 2013;76:2322–9. [7] Yan KX, Hong SL, Feng XZ. Demethyltenicausine, a new bisindole alkaloid from Melodinus hemsleyanus. Yaoxue Xuebao 1998;33:597–9. [8] Guo LW, Zhou YL. Alkaloids from Melodinus hemsleyanus. Phytochemistry 1993;34:563–6. [9] Liu Y, Wang JS, Wang XB, Kong LY. Two novel dimeric indole alkaloids from the leaves and twigs of Psychotria henryi. Fitoterapia 2013;86: 178–82. [10] Ma K, Wang JS, Luo J, Yang MH, Yao HQ, Sun HB, et al. Bistabercarpamines A and B, first vobasinyl–chippiine-type bisindole alkaloid from Tabernaemontana corymbosa. Tetrahedron Lett 2014;55:101–4. [11] Feng T, Li Y, Wang YY, Cai XH, Liu YP, Luo XD. Cytotoxic indole alkaloids from Melodinus tenuicaudatus. J Nat Prod 2010;73:1075–9. [12] Li CM, Zheng HL, Wu SG, Sun HD. Quinolinic melodinus alkaloids from stem bark Melodinus khasianus. Acta Bot Yunn 1994;16:315–7. [13] James PK, John B, Feike B, James C, Walter JC, Kaoru F, et al. Total synthesis of indole and dihydroindole alkaloids in the vinblastine-–vincristine series the chloroindolenine approach. Helv Chim Acta 1975;58:1690–719.
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Six new alkaloids from Melodinus henryi Ke Ma, Jun-Song Wang, Jun Luo, Ling-Yi Kong ⁎ State Key Laboratory of Natural Medicines, Department of Natural Medicinal Chemistry, China Pharmaceutical University, 24 Tong Jia Xiang, Nanjing 210009, People's Republic of China
a r t i c l e
i n f o
Article history: Received 28 September 2014 Accepted in revised form 21 November 2014 Available online 3 December 2014 Keywords: Melodinus henryi Bisindole alkaloids Quinolinic melodinus alkaloids Isolation Structural elucidation
a b s t r a c t A total of six new alkaloids, melodinhenines A–F (1–6), were isolated from Melodinus henryi. Melodinhenines A and B are new eburnan–vindolinine-type bisindole alkaloids and melodinhenines C–F are new quinolinic melodinus alkaloids. Their structures were elucidated through extensive spectroscopic methods including 2D NMR and HRESIMS analyses. The absolute configuration of 1 and 2 was determined using ECD exciton chirality method. To the best of our knowledge, this is the first report on the determination of the absolute configuration of eburnan–vindolinine-type bisindole alkaloid using this method. © 2014 Elsevier B.V. All rights reserved.
1. Introduction The genus Melodinus (Apocynaceae) includes 53 species which are distributed mainly in the tropical and subtropical areas of Asia and Australia. Many plants of this genus are used as folk medicine to treat hernia, indigestion, and abdominal pain in Yunnan, Guangdong, and Guangxi provinces of China [1]. Previous chemical investigations indicated that these plants were rich in various skeletal types of alkaloids and their dimeric forms, including scandine-type alkaloids such as melodines T and U [2], aspidosperma-type alkaloids such as melodinines M–S [2], rearranged aspidosperma-type alkaloids, such as melotenine A [3], ketolactam derivative, such as melohenine B [4], condylocarpan-type alkaloids such as 19S-methoxytubotaiwine N4-oxide [5], and melodines–aspidosperma-type bisindole alkaloids such as melosuavines A–C [6]. Pharmacological investigations on these alkaloids have demonstrated promising antitumor activity and antifertility activity [7,8]. As a part of our ongoing research on structurally diverse and potentially bioactive alkaloids [9,10], six new alkaloids, melodinhenines A–F (1–6), were isolated from Melodinus henryi. Of these new ⁎ Corresponding author. Tel./fax: +86 25 8327 1405. E-mail address: [email protected] (L.-Y. Kong).
http://dx.doi.org/10.1016/j.fitote.2014.11.021 0367-326X/© 2014 Elsevier B.V. All rights reserved.
alkaloids, melodinhenines A and B were rare eburnan– vindolinine-type bisindole alkaloids, and their structures were elucidated by spectroscopic methods. The absolute configuration of 1 and 2 was determined by ECD exciton chirality method, and this is the first report on the determination of the absolute configuration of eburnan–vindolinine-type bisindole alkaloid using this method. Herein, the isolation and structural elucidation of these isolates are described. 2. Experimental 2.1. General Optical rotations were recorded using a JASCO P-1020 polarimeter. UV spectra were recorded using a Shimadzu UV2450 spectrophotometer. ECD spectra were measured with a JASCO J-810 spectropolarimeter. IR spectra were recorded on KBr discs using a Bruker Tensor 27 spectrometer. 1D and 2D NMR spectra were acquired with a Bruker AV III-500 NMR instrument at 500 MHz (1H) and 125 MHz (13C). ESIMS and HRESIMS spectra were recorded using an Agilent 1100 series LC-MSD-Trap-SL mass analyzer and an Agilent 6520B Q-TOF mass instrument, respectively. Column chromatography (CC) was performed with ODS (40–63 μm, Fuji, Japan) and Sephadex
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K. Ma et al. / Fitoterapia 100 (2015) 133–138
LH-20 (Pharmacia, Sweden) columns. Preparative HPLC was performed on a Shimadzu LC-8A system (Shimadzu, Tokyo, Japan) equipped with a Shim-pack RP-C18 column (200 mm × 20 mm i.d., 10 μm, Shimadzu, Tokyo, Japan) with a flow rate of 10.0 mL/min and a column temperature of 25 °C, and detection was performed with a binary channel UV detector at 210 and 230 nm. 2.2. Plant material The stems of M. henryi were collected from Yunnan province of China in March 2013 and identified by Mr. Shuncheng Zhang, Xishuangbanna Tropical Botanical Garden, Chinese Academy of Sciences. A voucher specimen (No. 130312) was deposited in the Department of Natural Medicinal Chemistry, China Pharmaceutical University. 2.3. Extraction and isolation The air-dried stems of M. henryi (7 kg) were extracted with 95% EtOH (3 × 4 h). The crude extract was partitioned between EtOAc and 1% HCl. The aqueous phase was adjusted to pH 9–10 with ammonia water, and extracted with EtOAc to obtain crude alkaloids. The crude alkaloids were subjected to a D-101 macropous resion column, eluted with a gradient of EtOH–H2O (30:70 to 90:10, v/v), to afford four fractions (Fr.1–4). Fr.1 was subjected to a MCI gel column, eluted with a gradient of MeOH–H2O (30:70 to 90:10, v/v) to yield seven subfractions (Fr.1.1–1.7). Fr.1.3 was subjected to an RP-C18 column, eluted with a gradient of MeOH–H2O (30:70 to 50:50, v/v), finally purified by preparative HPLC using MeOH–H2O (30:70, containing 0.1% TFA, flow rate: 10 ml/min, retention time: 31 min, 35 min) to yield 3 (30 mg) and 4 (40 mg). Fr.2 was separated by an RP-C18 column, eluted with MeOH–H2O (30:70 to 70:30, v/v), to afford five subfractions (Fr.2.1–2.5), and Fr.2.2 was subjected to a Sephadex LH-20 column and eluted with MeOH, finally purified by preparative HPLC using MeOH–H2O (35:65, containing 0.1% TFA, flow rate: 10 ml/min, retention time: 36 min, 42 min) to yield 5 (6 mg) and 6 (8 mg). Fr.3 was subjected to a MCI gel column, eluted with a gradient of MeOH–H2O (30:70 to 90:10, v/v) to yield seven subfractions (Fr.3.1–3.7). Fr.3.2 was subjected to an RP-C18 column, eluted with a gradient of MeOH–H2O (30:70 to 90:10, v/v), finally purified by preparative HPLC using MeOH–H2O (68:32, containing 0.1% TFA, flow rate: 10 ml/min, retention time: 31 min, 35 min) to yield 1 (6 mg) and 2 (8 mg). Melodinhenine A (1): yellow solid; [α]24D +46 (c 1, CHCl3); UV (MeOH): λmax (log ε): 225 (4.53), 286 (3.41), and 316 (3.38) nm; ECD (c 3.4 × 10−4 M, MeOH) λmax (Δε): 233 nm (−0.04), and 255 nm (+5.44); IR (KBr) υmax 3324, 1681, 1462, and 1203 cm−1; ESI-MS (positive): m/z 613.6 [M + H]+; HRESIMS (positive): m/z 613.3538 [M + H]+, (calcd. 613.3537); and 1H and 13C NMR data see Table 1. Melodinhenine B (2): yellow solid; [α]24D −19 (c 1, CHCl3); UV (MeOH): λmax (log ε): 222 (4.53), 286 (3.41), and 318 (3.38) nm; ECD (c 3.4 × 10−4 M, MeOH) λmax (Δε): 233 nm (−0.05), and 255 nm (+10.65); IR (KBr) υmax 3386, 1721, 1462, and 1203 cm−1; ESI-MS (positive): m/z 629.5 [M + H]+ HRESIMS (positive): m/z 629.3487 [M + H]+ (calcd. 629.3486); and 1H and 13C NMR data see Table 2.
Table 1 1 H NMR (500 MHz) and methanol-d4. No. 2 3a 3b 5a 5b 6a 6b 7 8 9 10 11 12 13 14 15 16 17a 17b 18 19a 19b 20 21
13
C NMR (125 MHz) data of compound 1 in
δH (mult; J, Hz) 3.81, m 3.38, m 3.78, m 3.81, m 3.39, m 3.22, m
δC
No.
133.8 44.3
2′ 3′a 3′b 5′a 5′b 6′a 6′b 7′ 8′ 9′ 10′ 11′ 12′ 13′ 14′ 15′ 16′ 17′a 17′b 18′ 19′ 20′ 21′ OMe CO
52.7 17.1 105.3 127.7 119.5 121.2 122.2 113.5 139.1 122.2 129.9 57.7 43.3
7.45, d, (10.0) 7.03, t, (10.0) 6.88, t, (10.0) 6.49, d, (10.0) 5.62, m 6.03, m 4.78, dd, (11.6, 4.0) 2.41, m 1.98, m 1.07, t, (7.6) 1.96, m 1.73, m
7.9 33.8 38.3 59.6
5.08, s
δH (mult; J, Hz) 3.22, m 3.33, m 3.24, m 3.26, m 1.74, m 2.21, m 7.32, m 7.00, d, (10.0) 6.99, dd, (10.0, 2.0) 6.03, d, (2.0) 5.67, m 6.06, m 3.07, dd, (12.0, 6.4) 2.35, m 1.74, m 1.15, d, (6.8) 2.09, q, (6.8) 4.07, s 3.64, s
δC 83.2 50.1 52.8 33.5 60.3 139.1 123.1 128.7 134.1 124.4 151.1 129.9 124.4 39.9 28.9 7.1 50.2 45.7 59.6 51.6 175.1
Melodinhenine C (3): white amorphous powder; [α]24D +38 (c 1, CHCl3); UV (MeOH): λmax (log ε): 220 (4.53), 284 (3.41), and 293 (3.38) nm; IR (KBr) υmax 3426, 1681, 1462, and 1203 cm−1; ESI-MS (positive): m/z 367.3 [M + H]+; HRESIMS (positive): m/z 367.1651 [M + H]+ (calcd. 367.1652); and 1H and 13C NMR data see Table 3.
Table 2 1 H NMR (500 MHz) and methanol-d4. No. 2 3a 3b 5a 5b 6a 6b 7 8 9 10 11 12 13 14 15 16 17a 17b 18 19a 19b 20 21
δH (mult; J, Hz) 3.81, m 3.38, m 3.78, m 3.81, m 3.39, m 3.22, m
7.49, d, (10.0) 7.03, t, (10.0) 6.91, t, (10.0) 6.49, d, (10.0) 5.62, m 6.03, m 2.46, m 2.12, m 1.07, t, (7.6) 1.96, m 1.73, m 5.08, s
13
C NMR (125 MHz) data of compound 2 in
δC
No.
133.2 44.3
2′ 3′a 3′b 5′a 5′b 6′a 6′b 7′ 8′ 9′ 10′ 11′ 12′ 13′ 14′ 15′ 16′ 17′a 17′b 18′ 19′ 20′ 21′ OMe CO
52.7 17.1 105.3 127.7 119.5 121.3 123.1 113.5 139.1 122.2 128.7 80.4 43.3 7.9 33.8 38.3 59.6
δH (mult; J, Hz) 3.22, m 3.33, m 3.24, m 3.26, m 1.74, m 2.21, m 7.32, m 7.00, d, (10.0) 6.99, dd, (10.0, 2.0) 6.03, d, (2.0) 5.67, m 6.06, m 3.07, dd, (12.0, 6.4) 2.36, m 2.04, m 1.35, d, (6.8) 2.09, q, (6.8) 4.07, s 3.64, s
δC 85.2 50.1 50.2 33.8 59.2 139.1 123.1 128.7 134.1 124.7 150.9 131.0 125.3 43.3 26.2 9.9 51.6 44.2 59.8 51.6 175.1
K. Ma et al. / Fitoterapia 100 (2015) 133–138 Table 3 1 H NMR (500 MHz) and methanol-d4.
13
C NMR (125 MHz) data of compounds 3 and 4 in
3 Position 2 3a 3b 5a 5b 6a 6b 7 8 9 10 11 12 13 14 15 16 17a 17b 18a 18b 19 20 21 CO OMe
δH (J in Hz) 3.68, m 3.75, m 3.66, m 3.98, m 2.36, m 2.68, m
6.85, d, (9.0) 6.76, dd (9.0,2.0) 6.87, d, (2.0) 5.86, d, (10.0) 6.15, d, (10.0) 2.74, m 3.13, m 5.07, m 5.16, m 5.82, m 4.02, s 3.26, s
Table 4 1 H NMR (500 MHz) and methanol-d4.
4 δC 167.8 45.8 53.5 33.7 60.7 126.5 118.5 116.8 155.5 113.9 129.3 116.6 135.6 62.2 44.1 113.9 141.9 46.6 80.7 169.9 54.7
δC
Position
3.72, m
169.8 46.5
2 3a 3b 5a 5b 6a 6b 7 8 9 10 11 12 13 14 15 16 17a 17b 18 19 20 21 CO
7.45, d, (10.0) 7.18, t, (10.0) 7.34, t, (10.0) 7.00, d, (10.0) 5.88, d, (10.0) 6.16, d, (10.0) 2.15, m 2.78, m 5.06, m 5.18, m 5.85, m 4.08, s 3.56, s
13
C NMR (125 MHz) data of compounds 5 and 6 in
5
δH (J in Hz)
3.65, m 3.05, m 2.36, m 2.78, m
135
54.5 33.8 60.6 125.1 126.9 125.2 130.4 117.3 137.1 116.8 135.4 62.4 44.1 115.1 141.9 45.8 80.4 168.3 53.5
Melodinhenine D (4): white amorphous powder; [α]24D −21 (c 1, CHCl3); UV (MeOH): λmax (log ε): 220 (4.53), 284 (3.41), and 293 (3.38) nm; IR (KBr) υmax 3324, 1681, 1462, and 1203 cm−1; ESI-MS (positive): m/z 351.3 [M + H]+; HRESIMS (positive): m/z 351.1706 [M + H]+ (calcd. 351.1703); and 1H and 13C NMR data see Table 3. Melodinhenine E (5): white amorphous powder; [α]25D +8 (c 0.5, MeOH); UV (MeOH) λmax (log ε) 284 (3.62), and 215 (4.97) nm; IR (KBr) υmax 3401, 2658, 1716, 1204, and 1033 cm−1; ESI-MS (positive): m/z 321.3 [M + H]+; HRESIMS m/z 321.1595 (calcd. 321.1598); and 1H and 13C NMR data see Table 4. Melodinhenine F (6): white amorphous powder; [α]25D −12 (c 0.5, MeOH); UV (MeOH) λmax (log ε) 284 (3.62), and 215 (4.97) nm; IR (KBr) υmax 3407, 2654, 1724, 1204, and 1033 cm−1; ESI-MS (positive): m/z 321.3 [M + H]+; HRESIMS m/z 321.1595 (calcd. 321.1598); and 1H and 13C NMR data see Table 4. 3. Results and discussion Melodinhenine A (1) was obtained as a yellow solid, and its molecular formula was established as C40H45N4O2 by HRESIMS (m/z 613.3538 [M + H]+, calcd. 613.3537), requiring 20 indices of hydrogen deficiency. The IR absorption bands at 3386 and 1721 cm−1 resulted from the –NH and ester carbonyl functionalities. Its 1H NMR data (methanol-d4, 500 MHz) (Table 1) showed an indole moiety with an unsubstituted aryl moiety (δH 7.50 (1H, d, J = 10.0 Hz), 7.06 (1H, t, J = 10.0 Hz), 6.98 (1H, t, J = 10.0 Hz), and 6.76 (1H, d, J = 10.0 Hz)), a typical splitting pattern of a 1,2,4-trisubstitued aromatic ring (δH 6.05, d, J = 2.0 Hz; δH 6.49, dd, J = 2.0, 10.0 Hz; δH 5.68, d,
δH (J in Hz) 3.99, m 4.32, m 3.58, m 3.86, m 2.16, m 2.72, m
7.32, d, (10.0) 7.06, t, (10.0) 7.26, t, (10.0) 6.98, d, (10.0) 6.05, m 6.21, m 2.38, m 2.48, m 0.96, d, (5.0) 2.79, q, (5.0) 4.41, s
6 δC 168.7 47.6 56.2 37.4 58.3 130.0 124.6 125.0 130.2 118.1 138.6 122.6 127.2 68.3 40.3 8.5 53.4 48.1 62.9 207.9
δH (J in Hz) 3.96, m 4.32, m 3.53, m 3.86, m 2.26, m 2.49, m
7.38, d, (10.0) 7.04, t, (10.0) 7.26, t, (10.0) 6.98, d, (10.0) 6.02, m 6.25, m 2.18, m 2.68, m 0.89, d, (5.0) 2.12, q, (5.0) 4.29, s
δC 169.0 47.6 55.9 36.5 56.7 130.2 125.1 124.9 130.2 118.0 138.4 122.1 125.6 69.0 37.1 11.7 52.4 46.9 68.9 209.0
J = 10.0 Hz), a methoxyl resonance at δH 3.79 (3H, s), and two methyl resonances at δH 1.16 (1H, d, 5.0 Hz) and 1.07 (1H, t, 10.0 Hz). The 13C NMR (Table 1) and HSQC spectra suggested that 1 possessed 40 carbons, including three methyl, nine methylene, sixteen methine, and twelve quaternary carbons. Therefore, the gross structure of 1 was determined to be a bisindole alkaloid, consisting of eburnan and vindolinine units, similar to melodinine H [11]. The HMBC correlations of H-17 to C-16, H-16 to C-11′, and H-17 to C-12′ (Fig. 2) indicated that the two units were connected via C-16 and C-11′. Thus, the planar structure of 1 was determined as shown in Fig. 2. The relative configuration of 1 was confirmed by analyses of 1H–1H coupling constant and ROESY correlations (Fig. 3). The coupling constant of H-16 (dd, J = 11.6, 4.0 Hz) suggested H-16 to be β oriented by comparison with that of Omethylvincanol (dd, J = 9.5, 5.7 Hz) and celastromelidine (dd, J = 10.0, 5.0 Hz) [11,12]. The ROESY cross peaks of H-16/ H-15 and H-21/H-19 indicated that H-21 and the C-20 ethyl were α oriented. In vindolinine unit, the ROESY correlations of H-19′/H-16′, and H-21′/H-16′ indicated that H-16′, H-19′, and H-21′ were on the same side. The absolute configuration of 1 was determined using the ECD exciton chirality method. The ECD spectrum of 1 showed a positive Cotton effect at 255 nm (Δε + 5.44) and a negative Cotton effect at 233 nm (Δε − 0.04), which was indicative of P-helicity between the two indole chromophores, hence permitting assignment of 16R absolute configuration (Fig. 4). Thus, the absolute configuration of 1 was assigned as depicted. Melodinhenine B (2) was obtained as a yellow solid, and its molecular formula was established as C40H45N4O3 by HRESIMS (m/z 613.3487 [M + H]+, calcd. 613.3486), requiring 20 indices of hydrogen deficiency. The IR absorption bands at 3386 and 1721 cm−1 resulted from the –NH and ester carbonyl functionalities. The 1D NMR data of 2 resembled those of 1,
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K. Ma et al. / Fitoterapia 100 (2015) 133–138
Fig. 1. Structures of compounds 1–6.
indicating that 2 was also an eburnan–vindolinine-type bisindole alkaloid. The C-16 resonance of 2 was deshielded relative to that of 1, indicating that a hydroxy group, instead of a proton, was attached to C-16, which was further confirmed by the HMBC correlations of H-17 to C-16 and C-20. Thus, the planar structure of 2 was established. The relative configuration of 2 was confirmed by analysis of ROESY spectrum of 2. The ROESY cross peak of H-21/H-19 indicated that H-21 and the C-20 ethyl were in the same side. In vindolinine unit, the ROESY correlations of H-19′/H-16′, and H-21′/H-16′ indicated that H-16′, H-19′, and H-21′ were in the same side. The absolute configuration of 2 was also determined using the ECD exciton chirality method. The ECD spectrum of 2 showed a positive Cotton effect at 255 nm (Δε + 10.65) and a negative Cotton effect at 233 nm (Δε − 0.05), which was indicative of P-helicity between the two indole chromophores, hence permitting assignment of 16R absolute configuration. Thus, the absolute configuration of 2 was assigned as depicted. Melodinhenine C (3) was obtained as a white amorphous powder, and its molecular formula was established as C21H22N2O4 by HRESIMS (m/z 367.1651, calcd. 367.1652). The IR absorption bands at 3426 and 1681 cm−1 resulted
from the –NH and ester carbonyl groups. Its 1H NMR data (Table 3) showed a typical splitting pattern of a 1,2,4trisubstitued aromatic ring (δH 6.87, d, J = 2.0 Hz; δH 6.76, dd, J = 2.0, 9.0 Hz; δH 6.85, d, J = 9.0 Hz), a methoxy resonance at δH 3.58 (s). The 13C NMR (Table 3) and HSQC spectra suggested that 3 possessed 21 carbons, including one methyl, five methylenes, eight methines, and seven quaternary carbons. Therefore, the gross structure of 3 was established as scandinetype alkaloid. The 1H and 13C NMR data of 3 were similar to those of 10-hydroxyscandine except for two differences [2]. First, the aromatic moiety resonances at δH 6.85, d, J = 2.0 Hz, δH 6.76, dd, J = 2.0, 9.0 Hz, δH 6.87, d, J = 9.0 Hz, δC 126.5, δC 113.9, δC 114.9, δC 155.5, δC 116.8, and δC 129.3, were different from those of 10-hydroxyscandine, suggesting that the hydroxyl group was attached to C-11 rather than C-10, which was further confirmed by the HMBC correlations (Fig. 5) of H-10 (δH 6.76, dd, J = 2.0, 9.0 Hz) to C-8 (δC 126.5), C-11 (δC 155.5), and C-12 (δC 118.5), and H-12 to C-8. Second, the C-7 resonances at
19 16
15 21
21' 16'
Fig. 2. Selected HMBC correlations of 1.
19'
Fig. 3. Selected ROESY correlations of 1.
K. Ma et al. / Fitoterapia 100 (2015) 133–138
137
6 21
15
17
Fig. 4. The ECD spectra of 1. A positive first and a negative second Cotton effect indicated the positive chirality (P-helicity) between the two indole chromophores of 1.
(δC 60.7), C-20 (δC 46.6), and C-21 (δC 80.7), rather than C-7 (δC 62.7), C-20 (δC 43.9), and C-21 (δC 81.7) in 10hydroxyscandine, indicated that the configuration of C-21 was different from that of 10-hydroxyscandine [2]. The βorientation of H-21 could be confirmed by the ROESY cross peaks (Fig. 6) of H-21 (δH 4.05) to H-6b (δH 2.68), H-15 (δH 6.15), and H-17 (δH 2.74). According to the ROESY experiment of 3, the other asymmetric carbons had the same relative configuration as those of 10-hydroxyscandine. Finally, the structure of 3 was established as shown in the Fig. 1. Melodinhenine D (4) was obtained as a white amorphous powder, and its molecular formula was established as C21H22N2O4 by HRESIMS (m/z 351.1706, calcd. 351.1703). The 1D NMR data of 4 was similar to 3 with the exception of signals corresponding to an unsubstituted aromatic moiety [δH 7.45 (1H, d, J = 10.0 Hz), 7.34 (1H, t, J = 10.0 Hz), 7.18 (1H, t, J = 10.0 Hz), 7.00 (1H, d, J = 10.0 Hz)]. This observation indicated that the planar structure of 4 was similar to 3 except for the absence of C-11 hydroxyl group. According to the ROESY experiment, all asymmetric carbons of 4 had the same relative configuration as those of 3. Finally, the structure of 4 was established as shown in the Fig. 1. Melodinhenine E (5) was isolated as a light yellow solid, and its molecular formula was established as C20H20N2O2 by HRESIMS (m/z 321.1595, calcd. 321.1598). Its 1H NMR data (Table 4) showed an unsubstituted aromatic moiety with signals at δH 7.32 (1H, d, J = 10.0 Hz), δH 7.26 (1H, d, J = 10.0 Hz), δH 7.06 (1H, t, J = 10.0 Hz), and δH 6.98 (1H, t, J = 10.0 Hz). The 13C NMR (Table 4) and HSQC spectra suggested
Fig. 5. The key HMBC correlations of 3.
Fig. 6. Selected ROESY correlations of 3.
that 5 possessed 20 carbons, including one methyl, four methylene, eight methine, and seven quaternary carbons. Therefore, the gross structure of 5 was established as scandine-type alkaloid. The 1H and 13C NMR data of 5 were similar to those of 19-epimeloscandonine except for the resonances of C-7 (δC 58.3), C-20 (δC 48.1), and C-21 (δC 62.9) [8]. This observation indicated that 5 was C-21-epimer of 19epimeloscandonine. The β-orientation of H-21 could be confirmed by the ROESY cross peaks (Fig. 7) of H-21 (δH 4.05) to H-6b (δH 2.68), and H-17 (δH 2.74). The other asymmetric carbons had the same relative configuration as those of 19epimeloscandonine on the basis of the ROESY experiment of 5. Finally, the structure of 5 was established as shown in the Fig. 1. Melodinhenine F (6) was isolated as a light yellow solid, and its molecular formula was established as C20H20N2O2 by HRESIMS (m/z 321.1595, calcd. 321.1598). The similarity of the 1H and 13C NMR spectroscopic data of 6 (Table 4) to those of meloscandonine indicated that 6 was also C-21-epimer of
6
21
Fig. 7. Selected ROESY correlations of 5.
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Scheme 1. The plausible biogenetic pathway for 1 and 2.
meloscandonine, which was the same as the relationship between 5 and 19-epimeloscandonine [8]. To the best of our knowledge, 1 and 2 are new examples of the eburnan–vindolinine type bisindole alkaloid. They might be derived from eburnan-type and vindolinine-type alkaloids via a coupling reaction. In this reaction, a providing electrophillic center indole alkaloid attacks to a providing nucleophillic center indole alkaloid in the acidic medium [13]. Here, we made a plausible biosynthetic pathway of them, which was presented in Scheme 1. Acknowledgments The work was supported by the Priority Academic Program Development of Jiangsu Higher Education Institutions (PAPD) and the Program for Changjiang Scholars and Innovative Research Team in University (IRT1193). References [1] Tian Y, Li PY. Flora of China. vol. 63. Beijing: Science Press; 1977 17–63. [2] Liu YP, Li Y, Cai XH, Li XY, Kong LM, Cheng GG, et al. Melodinines M–U, cytotoxic alkaloids from Melodinus suaveolens. J Nat Prod 2012;75:220–4.
[3] Feng T, Li Y, Liu YP, Cai XH, Wang YY, Luo XD. Melotenine A, a cytotoxic monoterpenoid indole alkaloid from Melodinus tenuicaudatus. Org Lett 2010;5:968–71. [4] Feng T, Cai XH, Li Y, Wang YY, Liu YP, Xie MJ, et al. Melohenines A and B, two unprecedented alkaloids from Melodinus henryi. Org Lett 2009;21: 4834–7. [5] Cai XH, Li Y, Liu YP, Li XN, Bao MF, Luo XD. Phytochemistry 2012;83: 116–24. [6] Liu YP, Zhao YL, Feng T, Cheng GG, Zhang BH, Li Y, et al. Melosuavines A–H, cytotoxic bisindole alkaloid derivatives from Melodinus suaveolens. J Nat Prod 2013;76:2322–9. [7] Yan KX, Hong SL, Feng XZ. Demethyltenicausine, a new bisindole alkaloid from Melodinus hemsleyanus. Yaoxue Xuebao 1998;33:597–9. [8] Guo LW, Zhou YL. Alkaloids from Melodinus hemsleyanus. Phytochemistry 1993;34:563–6. [9] Liu Y, Wang JS, Wang XB, Kong LY. Two novel dimeric indole alkaloids from the leaves and twigs of Psychotria henryi. Fitoterapia 2013;86: 178–82. [10] Ma K, Wang JS, Luo J, Yang MH, Yao HQ, Sun HB, et al. Bistabercarpamines A and B, first vobasinyl–chippiine-type bisindole alkaloid from Tabernaemontana corymbosa. Tetrahedron Lett 2014;55:101–4. [11] Feng T, Li Y, Wang YY, Cai XH, Liu YP, Luo XD. Cytotoxic indole alkaloids from Melodinus tenuicaudatus. J Nat Prod 2010;73:1075–9. [12] Li CM, Zheng HL, Wu SG, Sun HD. Quinolinic melodinus alkaloids from stem bark Melodinus khasianus. Acta Bot Yunn 1994;16:315–7. [13] James PK, John B, Feike B, James C, Walter JC, Kaoru F, et al. Total synthesis of indole and dihydroindole alkaloids in the vinblastine-–vincristine series the chloroindolenine approach. Helv Chim Acta 1975;58:1690–719.
