Phytochemistry xxx (2014) xxx–xxx
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Quinolone and indole alkaloids from the fruits of Euodia rutaecarpa and their cytotoxicity against two human cancer cell lines Nan Zhao a,b, Zhan-Lin Li a,b,⇑, Da-Hong Li a,b, Ya-Ting Sun a,b, Dong-Ting Shan a,b, Jiao Bai a,b, Yue-Hu Pei a,b, Yong-Kui Jing c, Hui-Ming Hua a,b,⇑ a b c
Key Laboratory of Structure-Based Drug Design & Discovery, Ministry of Education, Shenyang Pharmaceutical University, Shenyang 110016, PR China School of Traditional Chinese Materia Medica, Shenyang Pharmaceutical University, Shenyang 110016, PR China Department of Medicine, Mount Sinai School of Medicine, New York, NY 10029, USA
a r t i c l e
i n f o
Article history: Received 16 May 2014 Received in revised form 11 July 2014 Available online xxxx Keywords: Euodia rutaecarpa Rutaceae Quinolone alkaloid Indole alkaloid Growth inhibitory activity
a b s t r a c t Four quinolone alkaloids (1–4) and three indole alkaloids (20–22), together with 30 known alkaloids (5–19, 23–37), were isolated from the fruits of Euodia rutaecarpa. Their structures were established by spectroscopic analyses. The in vitro cytotoxic activities of these alkaloids against leukaemia HL-60 and prostate cancer PC-3 cell lines were evaluated. Ó 2014 Elsevier Ltd. All rights reserved.
1. Introduction
2. Results and discussion
The dried and nearly ripe fruits of Euodia rutaecarpa (Juss.) Benth. (Rutaceae) have been used as a traditional Chinese medicine for treatment of headache, abdominal pain, dysentery, postpartum haemorrhage, and amenorrhea (Chinese Pharmacopoeia Commission, 2010). A number of quinolone and indole alkaloids have also been reported (Yang and Teng, 2007; Noboru et al., 1989; Tang et al., 1996; Tohru et al., 1988) from E. rutaecarpa, and some of these alkaloids have exhibited promising antiinflammatory (Ko et al., 2007; Adams et al., 2004), antibacterial (Kazunari et al., 2002), and cytotoxic activities (Jiang and Hu, 2009). As an ongoing search for bioactive natural alkaloids, the isolation and structural elucidation of four new quinolone alkaloids (1–4) and three indole alkaloids (20–22), together with thirty known alkaloids, are reported herein. The growth inhibitory activities of the isolated compounds were evaluated against HL-60 and PC-3 cell lines.
The air-dried fruits of E. rutaecarpa were extracted with 75% ethanol. After concentration, the extract was successively partitioned with petroleum ether (P.E.), CHCl3, EtOAc, and n-butanol. The P.E., CHCl3, and EtOAc soluble fractions were submitted to separation and purification by column chromatography over silica gel as well as reversed-phase (ODS), Sephadex LH-20, and finally preparative HPLC yielding 18 quinolone alkaloids (Fig. 1) and 19 indole alkaloids (Fig. 2), of which seven were new compounds. The known compounds were identified as: 1-methyl-2-[(Z)-5undecenyl]-4(1H)-quinolone (5) (Tang et al., 1996), 1-methyl-2[(Z)-6-undecenyl]-4(1H)-quinolone (6) (Tang et al., 1996), 1-methyl-2-[(Z)-7-tridecenyl]-4(1H)-quinolone (7) (Tang et al., 1996), evocarpine (8) (Tang et al., 1996), 1-methyl-2-[7-carbonyl(E)-9-tridecenyl]-4(1H)-quinolone (9) (Huang et al., 2012), euocarpine D (10) (Wang et al., 2013), 1-methyl-2-[(4Z,7Z)-4,7-tridecadienyl]-4(1H)-quinolone (11) (Tohru et al., 1988), 1-methyl-2[(6Z,9Z)-6,9-pentadecadienyl]-4(1H)-quinolone (12) (Tohru et al., 1988), 1-methyl-2-octyl-4(1H)-quinolone (13) (Somanathan and Smith, 1981), 1-methyl-2-nonyl-4(1H)-quinolone (14) (Tang et al., 1996; Kostova et al., 1999), 1-methyl-2-decyl-4(1H)-quinolone (15) (Tang et al., 1998), 1-methyl-2-undecyl-4(1H)-quinolone (16) (Lee et al., 2003), dihydroevocarpine (17) (Tohru et al., 1988), 1-methyl-2-pentadecenyl-4(1H)-quinolone (18) (Tohru et al., 1988), evodiamine (19) (Zhang et al., 1999), wuzhuyurutine B
⇑ Corresponding authors at: Key Laboratory of Structure-Based Drug Design & Discovery, Ministry of Education, Shenyang Pharmaceutical University, Shenyang 110016, PR China. Tel./fax: +86 24 23986465. E-mail addresses: [email protected] (Z.-L. Li), [email protected] (H.-M. Hua). http://dx.doi.org/10.1016/j.phytochem.2014.10.020 0031-9422/Ó 2014 Elsevier Ltd. All rights reserved.
Please cite this article in press as: Zhao, N., et al. Quinolone and indole alkaloids from the fruits of Euodia rutaecarpa and their cytotoxicity against two human cancer cell lines. Phytochemistry (2014), http://dx.doi.org/10.1016/j.phytochem.2014.10.020
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(23) (Teng and Yang, 2006), bouchardatine (24) (Wattanapiromsakul et al., 2003), rutaecarpine (25) (Zhang et al., 1999), 1-hydroxyrutaecarpine (26) (Wattanapiromsakul et al., 2003; Ng et al., 1987), 3-hydoxyrutaecarpine (27) (Li et al., 2001), 7-hydroxyrutaecarpine (28) (Wu et al., 1995), hortiacine (29) (Yang et al., 1995), 7,8-dehydrorutaecarpine (30) (Ikuta et al., 1998), dehydroevodiamine (31) (Zhang et al., 1999), goshuyuamide-I (32) (Shoji et al., 1989), goshuyuamide-II (33) (Shoji et al., 1989), N-(2-methylaminobenzoyl) tryptamine (34) (Shoji et al., 1988), evodiamide (35) (Shoji et al., 1988), wuchuyuamideI (36) (Yang and Teng, 2007), and 1,2,3,4-tetrahydro-1-oxo-carboline (37) (Tang et al., 1996) by comparison of their spectroscopic data with reported data. Compound 1 was isolated as a colourless oil with a molecular formula of C21H27NO2, as determined by HRESIMS (m/z 326.2117 [M+H]+). Its N-methyl-4(1H)-quinolone skeleton was determined by its UV absorptions at 239, 321, and 334 nm (Huang and Yu, 1988). The 1H and 13C NMR spectra showed characteristic signals of a N-methyl-4(1H)-quinolone at dH 6.01 (1H, s, H-3), 8.14 (1H, d, J = 7.9 Hz, H-5), 7.36 (1H, dd, J = 7.9, 7.0 Hz, H-6), 7.70 (1H, dd, J = 8.6, 7.0 Hz, H-7), 7.76 (1H, d, J = 8.6 Hz, H-8) and dC 155.3 (C2), 109.9 (C-3), 175.9 (C-4), 126.0 (C-4a), 125.3 (C-5), 123.0 (C-6), 132.0 (C-7), 116.8 (C-8), 141.8 (C-8a). The resonances of an a,bunsaturated carbonyl group were observed at dH 6.82 (dt, J = 15.8, 6.5 Hz, H-40 ), 6.04 (d, J = 15.8 Hz, H-50 ), and dC 147.2 (C40 ), 130.5 (C-50 ), 200.0 (C-60 ). The characteristic fragment ions of quinolone alkaloids were given in the EIMS including the ones arising from McLafferty rearrangement at m/z 173, a displacement rearrangement at m/z 186, and an elimination of the carbonyl at m/z 144. The HMBC correlations (Fig. 3) supported the position of an a,b-unsaturated carbonyl group in the side-chain, in which the proton signals (dH 6.82, 6.04) correlated with C-30 (dC 31.5), suggesting that the olefinic carbons were assigned at C-40 and
5 6 7 8
1
O 4
4a
8a
3
10 11'
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6'
11
8' 13'
7'
OH
3
15'
OH
4 5 6 7 8
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12
4'
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13
15
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16 13'
8'
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14
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9'
O
O 2
8'
O
O 5'
7'
9
N 2 R CH 3 4'
1'
C-50 and that the carbonyl carbon was assigned at C-60 . Moreover, the double bond was determined to be in the E-form due to the coupling constant (JH-40 ,50 = 15.8 Hz). Thus, 1 was assigned as 1-methyl-2-[6-carbonyl-(E)-4-undecenyl]-4(1H)-quinolone. Compound 2 was obtained as a colourless oil and its molecular formula was determined to be C23H31NO2 from its molecular ion peak at m/z 354.2438 [M+H]+ (calcd. for C23H32NO2, 354.2428) in the HRESIMS, indicating compound 2 had two more methylene groups than 1. The UV spectrum and EIMS (m/z 173, 144, 186) suggested that 2 should be a quinolone alkaloid. The 1H and 13C NMR spectra closely resembled those of 1, except for the integral of the protons in the high field region. The assignment of the position of an a,b-unsaturated carbonyl group was determined by long-range correlations from H-90 b (2.16, 1H, m,) to C-70 (130.3) and C-80 (147.0) and the a-cleavage fragment ions at m/z 228, 296 in EIMS, indicating that the carbonyl was assigned at C-60 and that the olefinic carbons were assigned at C-70 and C-80 . Therefore, the structure of 2 was concluded to be 1-methyl-2-[6-carbonyl-(E)-7tridecenyl]-4(1H)-quinolone. Compound 3 was obtained as white needles. The molecular formula, C25H39NO2, was assigned by the quasi-molecular ion peak at m/z 386.3056 [M+H]+ (calcd. for C25H40NO2, 386.3054) in HRESIMS. Its IR spectrum showed the presence of hydroxyl (3419 cm1) and aromatic (1597 cm1) groups. The characteristic absorption peaks at 239, 321, and 334 nm in the UV spectrum indicated the presence of an N-methyl-4(1H)-quinolone skeleton (Huang and Yu, 1988), which was supported by the 1H and 13C NMR signals at dH 3.72 (3H, s, N–CH3), 6.01 (s, H-3), 8.14 (dd, J = 7.9, 1.3 Hz, H-5), 7.35 (td, J = 7.3, 1.2 Hz, H-6), 7.70 (ddd, J = 8.7, 6.6, 1.6 Hz, H-7), 7.75 (br d, J = 7.7 Hz, H-8); dC 34.2 (N–CH3), 155.3 (C-2), 109.8 (C-3), 175.8 (C-4). The 1H and 13C NMR spectra also displayed signals for one hydroxymethyl group [dH 4.28 (t, J = 5.1 Hz, –OH), 3.35 (td, J = 6.5, 5.1 Hz, H-150 ); dC 60.7 (C-150 )], which was located at
17 18
7'
9'
10' 15'
8'
9' 10'
11' 13' 15'
Fig. 1. Structures of compounds 1–18 from Euodia rutaecarpa.
Please cite this article in press as: Zhao, N., et al. Quinolone and indole alkaloids from the fruits of Euodia rutaecarpa and their cytotoxicity against two human cancer cell lines. Phytochemistry (2014), http://dx.doi.org/10.1016/j.phytochem.2014.10.020
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8b
11
12 a
12
8
8a
N H
R4
7
N
13 a
O
13 b 14 a
4
1
3
19 R = H
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R H C 3
R5 N
N H
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20 R = CH2 OH 9 10
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HN14
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R = COOCH3
22
R = COOH
23 24
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OH
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O N N H N H 3C
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R2
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N H H 3 CHN 32
OCH3 H
O HO
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O
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N H N H 3C
H N
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33
N O
O
N CH3
R N N H H3 CHN
36
N H 37
R = COOH R = CHO
NH O
34
R =H
35
R = CH3
O
Fig. 2. Structures of compounds 19–37 from Euodia rutaecarpa.
O
O O
O
N
N
CH 3
CH 3 1
2 Fig. 3. Key HMBC correlations of 1–4.
the terminus of the side-chain. The assigned structure was further verified by a detailed HMBC analysis (Fig. 3). Consequently, compound 3 was identified as 1-methyl-2-[15-hydroxyl-pentadecenyl]-4(1H)-quinolone. Compound 4 was obtained as white needles. Its HRESIMS exhibited a quasi-molecular ion peak at m/z 358.2744 [M+H]+ corresponding to a molecular formula of C23H35NO2, which was a C2H4 unit less than that of compound 3. The IR and UV spectra were similar to those of 3. A comparison of the 1H and 13C NMR spectroscopic data of 4 and 3 showed that they were closely related analogues differing only in the length of the side-chain. Hence, compound 4 was assigned as 1-methyl-2-[13-hydroxyl-tridecenyl]-4(1H)-quinolone. Compound 20 was isolated as a white powder. Its molecular formula was determined to be C20H19N3O2 by HRESIMS ([M+Na]+
at m/z = 356.1363). The IR spectrum showed the presence of O–H (3415 cm1), N–H (3228 cm1), and aromatic groups (1614, 1595, 1488, and 1471 cm1). The UV absorptions at 226, 268, and 326 nm showed the presence of an indole chromophore (Teng and Yang, 2006). The 1H NMR spectrum of 20 displayed signals of two spin systems for 1,2-disubstitued aromatic rings dH 7.11 (2H, m, H-1, 3), 7.49 (1H, dd, J = 8.0, 7.3 Hz, H-2), 7.87 (1H, d, J = 7.6 Hz, H-4), 7.52 (1H, d, J = 7.9 Hz, H-9), 7.03 (1H, dd, J = 7.9, 7.3 Hz, H-10), 7.14 (1H, m, H-11), 7.43 (1H, d, J = 8.1 Hz, H-12), one N–H group (dH 11.25), one hydroxymethyl group(dH 5.20, 3.92, 3.72, dC 64.9) and one N–CH3 group (dH 2.44, dC 38.7). The 13 C NMR spectrum showed resonances assigned to an indole ring and a quinazolone ring, as well as one hydroxymethyl group (dC 64.9) and two methylene groups at dC 40.1, 20.4, suggesting that compound 20 was an indoloquinazoline alkaloid. A comparison
Please cite this article in press as: Zhao, N., et al. Quinolone and indole alkaloids from the fruits of Euodia rutaecarpa and their cytotoxicity against two human cancer cell lines. Phytochemistry (2014), http://dx.doi.org/10.1016/j.phytochem.2014.10.020
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COOCH3 N
N H HOH2 C
O
N H
N
N
O
HN
COOH N
N H
O
HN
H 3C 20
22
21 Fig. 4. Key HMBC correlations of 20–22.
with the NMR spectroscopic data of evodiamine (19) indicated that compound 20 possessed one more hydroxymethyl group than 19. The hydroxymethyl group was assigned to C-13b based on the long-range correlations from the protons at dH 3.92 and 3.72 to the carbon signals at dC 130.2 (C-13a) and 76.2 (C-13b) in the HMBC spectrum (Fig. 4). Thus, compound 20 was identified as 13b-hydroxymethylevodiamine. Compound 21 was isolated as a white powder. Its HRESIMS gave an [M+H]+ ion at m/z 320.1030 (calcd. 320.1030) consistent with a molecular formula of C18H13N3O3. The UV absorptions of 21 at 221, 245sh, 354, and 373 nm were similar to those of wuzhuyurutine B (23) (Teng and Yang, 2006). The IR spectrum showed the absorptions of N–H (3447 cm1) and carbonyl (1686 and 1654 cm1) groups. The characteristic signals for a typical indole moiety at dH 12.86 (1H, br. s, 13-NH), 8.11 (1H, br. d, J = 8.2 Hz, H-9), 7.28 (1H, td, J = 7.6, 1.0 Hz, H-10), 7.36 (1H, td, J = 7.6, 1.0 Hz, H-11), 7.68 (1H, br. d, J = 8.0 Hz, H-12) were observed in the 1H NMR spectrum. The 1H NMR spectrum also gave resonances for a quinazolinone moiety at dH 7.83 (1H, d, J = 8.0 Hz, H-1), 7.90 (1H, td, J = 8.0, 1.2 Hz, H-2), 7.59 (1H, td, J = 8.0, 1.0 Hz, H-3), 8.20 (1H, dd, J = 8.0, 1.2 Hz, H-4). The NMR signals at dH 4.00 (3H, s, –COOCH3) and dC 167.4 (–COOCH3), 52.4 (–COOCH3) indicated the existence of a methyl carboxylate. A comparison of the 1H and 13C NMR spectroscopic data of compounds 21 and 23 suggested that they were similar compounds. An N–H group was located on the N-14 position instead of N-6 in 21 due to the 3J correlation of the proton at dH 13.79 (N–H) with C-1 (dC 127.5) in the HMBC spectrum. Correspondingly, N-6 and C-13b formed an imine moiety. Thus, the structure of 21 was established and named wuzhuyurutine C.
Compound 22 was isolated as a white powder. Its molecular formula was assigned as C17H11N3O3 by HRESIMS ([M+Na]+ m/z 328.0687, calcd. 328.0693). Its 1H and 13C NMR spectra exhibited 8 aromatic protons, two N–H protons at dH 13.86 and 12.73, a carboxylic proton at dH 14.52, and 17 sp2 carbons. 22 showed the same molecular formula and similar NMR data to 23, indicating that both were a pair of isomers. A comparison of the 1H and 13C NMR spectroscopic data of 22 with those of 21 indicated that they were similar, with a difference in the loss of a methoxyl, the presence of the active proton of a carboxylic acid at dH 14.52, and the down-field shift of the carboxylic carbon at dC 169.3 in 22, suggesting the existence of a free carboxyl instead of the methyl ester in 21. The above deduction was supported by the HMBC correlations (Fig. 4). Therefore, 22 was determined to be an isomer of wuzhuyurutine B and named wuzhuyurutine D. 2.1. Cytotoxicity assay The cytotoxicities of the isolated compounds were evaluated against HL-60 (human leukaemia) and PC-3 (human prostate cancer) cell lines. The results are provided in Table 1. Most of the tested compounds showed cytotoxic activity against HL-60 cell lines, among which compound 19 exhibited the highest activity, even when compared with the positive control (5-Fu). Among the quinolone alkaloids, compounds 4, 8, and 17, with a C13 side-chain, and 16, with a C11 chain, showed stronger cytotoxic activity against HL-60 cell lines than other analogues. Hydroxylation at the terminus of the side-chain (4 and 17) enhanced the activity. The position of a double bond in the C13 side-chain (7 and 8) was shown to affect the activity. It is noteworthy that the
Table 1 The in vitro cytotoxic activities of the isolated compounds against HL-60 and PC-3 cancer cell lines.
a b
Compounds
HL-60 GI50a ± SD (lM)
PC-3 GI50a ± SD (lM)
Compounds
HL-60 GI50a ± SD (lM)
PC-3 GI50a ± SD (lM)
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19
58.13 ± 1.45 >80 20.36 ± 3.44 12.07 ± 2.28 54.10 ± 0.27 54.24 ± 6.07 21.04 ± 0.50 10.44 ± 3.21 30.84 ± 2.62 73.14 ± 0.71 39.28 ± 3.79 41.31 ± 0.53 21.04 ± 0.50 >80 36.72 ± 0.31 10.73 ± 1.20 16.02 ± 1.65 >80 0.51 ± 0.14
>80 >80 31.99 ± 1.98 >80 >80 >80 >80 15.11 ± 2.87 >80 >80 >80 >80 >80 >80 >80 17.61 ± 3.31 >80 >80 14.35 ± 1.58
20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 5-Fub
>80 70.08 ± 1.56 24.88 ± 1.41 >80 71.88 ± 6.13 >80 8.34 ± 2.72 11.94 ± 2.00 >80 >80 >80 20.06 ± 1.67 13.62 ± 1.10 31.39 ± 3.21 57.43 ± 4.21 >80 >80 >80 1.84 ± 0.29
>80 80 46.50 ± 1.22 >80 >80 >80 58.52 ± 3.21 >80 >80 >80 >80 >80 >80 >80 >80 >80 >80 >80 >80
GI50 is the concentration that inhibited 50% of cell growth. The data shown are means ± SD of three independent experiments. Positive control.
Please cite this article in press as: Zhao, N., et al. Quinolone and indole alkaloids from the fruits of Euodia rutaecarpa and their cytotoxicity against two human cancer cell lines. Phytochemistry (2014), http://dx.doi.org/10.1016/j.phytochem.2014.10.020
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a,b-unsaturated carbonyl at the side-chain (1, 2, 9 and 10) decreased the activity. For indolopyridoquinazoline alkaloids (25–30), a phenolic hydroxyl at ring-E (26 and 27) increased activity. N-methylation (31) and the further reduction of the C–N double bond (19) of the quinazoline moiety (25) increased the activity. Further hydroxymethylation of C-13b resulted in loss of activity (19 and 20). Compounds 8, 16, and 19 showed moderate activities against PC-3 cells. 3. Concluding remarking Seven new alkaloids, including four quinolone, three indole and 30 known alkaloids were isolated from the fruit of E. rutaecarpa. Their structures were elucidated by comprehensive spectroscopic analyses. The a,b-unsaturated carbonyl group in the side-chain of compounds 1, 2, 9, and 10 was originally derived from a allylic hydroxyl, which was unstable in the natural environment and oxidized to a a,b-unsaturated carbonyl (Huang et al., 2012), and unfortunately was not obtained. The two indoloquinazoline alkaloids, 22 and 23, were isomers that showed different chromogenic reactions. Compound 22 showed black–blue colour visualised by Dragendorff’s reagent. Interestingly, compound 22 exhibited moderate inhibitory activity against HL-60 and PC-3 cell lines, while 23 did not show any effects. The cytotoxicities of quinolone alkaloids may be related to the length of the side-chain. The a,b-unsaturated carbonyl at the side-chain decreased the cytotoxicity. The phenolic hydroxyl at ring-E of the indolopyridoquinazoline alkaloids exhibited higher cytotoxicity than those with an unsubstituted ring-E. 4. Experimental 4.1. General experimental procedures Melting points were measured on an Electrothermal MEL-TEMP melting point apparatus without correction. UV spectra were recorded using a Shimadzu UV-2201 spectrometer. IR spectra were obtained on a Bruker IFS-55 spectrometer (using a KBr disk method). NMR spectra were recorded on Bruker ARX-300 and AV-600 NMR spectrometers with TMS as an internal standard. Mass spectra were acquired on a Bruker micrOTOF-Q mass spectrometer (for HRESIMS) and an Agilent 5975 GC mass spectrometer (for EIMS). Column chromatography (CC) was performed with silica gel (Qingdao Haiyang Chemical Co., Ltd., China), ODS (50 lm, YMC Co. Ltd., Kyoto, Japan) and Sephadex LH-20 (GE Healthcare, Sweden). Semi-preparative HPLC was conducted on a YMC ODS-A column (250 20 mm I.D., 5 lm) equipped with an LC-6AD pump and an SPD-20A UV–Vis detector (Shimadzu Co., Ltd., Japan). 4.2. Plant material Plant material was purchased from Yuntian Drugstore, Anguo, Hebei Province, China, in August 2011 and was identified as fruits of E. rutaecarpa (Juss.) Benth by Prof. Jincai Lu (School of Traditional Chinese Materia Medica, Shenyang Pharmaceutical University, Shenyang, China). A voucher sample (WZY-20110910) was deposited in the Department of Natural Products Chemistry, Shenyang Pharmaceutical University, Shenyang, China. 4.3. Extraction and isolation of new compounds The fruits of Euodia rutaecarpa (10.5 kg) were extracted with EtOH–H2O(75:25, v/v, 63 L 3 3 h) under conditions of reflux. After evaporation of the solvent under reduced pressure, the
5
residue was suspended in H2O and partitioned with P.E. (3 20 L), CHCl3 (3 20 L), EtOAc (3 20 L) and n-BuOH (3 20 L), successively. The P.E. extract (46 g) was fractionated by CC silica gel using a gradient system of P.E. (60–90 °C)–acetone (100:0 to 100:50, v/v) as an eluent to afford eight fractions (Fr.A– Fr.H). Fr.G was separated into five subfractions (Fr.G1–Fr.G5) by on silica gel CC with a system of P.E.-acetone (100:3, 100:5, 100:7, 100:10, 100:20). Fr.G4 was submitted to Sephadex LH-20 CC followed by semi-preparative RP-HPLC eluted with MeOH– H2O (80:20, v/v, 5.0 mL/min) to afford 14 (9.0 mg, tR 44.0 min), 6 (4.2 mg, tR 54.5 min), 15 (2.9 mg, tR 62.0 min), 11 (10.5 mg, tR 73.5 min), 16 (12.0 mg, tR 87.0 min), and mixture I (tR 107.0 min), which was purified by PTLC to obtain 8 (21.0 mg) and 2 (3.4 mg). Fr. G5 was subjected to Sephadex LH-20 CC eluted with MeOH followed by semi-preparative RP-HPLC eluted with MeOH–H2O (80:20, v/v, 5.0 mL/min) to afford mixture II (tR 56.0 min), which purified by PTLC to obtain 1 (2.1 mg) and 5 (4.6 mg), and 13 (2.3 mg, tR 87.5 min). Fr.F was separated into four fractions (Fr.F1–Fr.F4) by silica gel CC with a gradient solvent system of CH2Cl2–MeOH (100:0, 100:1, 100:3, 100:5, v/v). Fr.F2 was separated by semi-preparative RP-HPLC eluted with MeOH–H2O (80:20, v/v, 5.0 mL/min) to afford 18 (1.9 mg, tR 60.0 min), mixture III (tR 86.0 min), 10 (3.5 mg, tR 92.0 min), 12 (38.1 mg, tR 111.0 min) and 17 (45.0 mg, tR 148.0 min). Mixture III was purified by PTLC to obtain 7 (9.7 mg) and 9 (2.8 mg). Fr.E was subjected to D101 macroporous resin CC eluted with MeOH–H2O (30:70, 60:40, 100:0, v/v) to afford three fractions (Fr.E1–Fr.E3). Fr.E3 was subjected to semi-preparative RP-HPLC (MeOH–H2O 73:27, v/v, 2.0 mL/min) to obtain 28 (6.2 mg, tR 18.0 min). The CHCl3 extract (300 g) was subjected to Al2O3 CC using a gradient system of increasing polarity of CH2Cl2–MeOH (100:0–100:200, v/v). The collected fractions were combined based on their TLC characteristics to yield ten fractions (Fr.I–Fr.R). Fr.K was purified by ODS (MeOH:H2O, 70:30, v/v) to give 30 (5.9 mg). Fr.L was subjected to Sephadex LH-20 to afford 29 (5.8 mg). Fr.M was dissolved in MeOH and filtered to yield the filtrate, which was purified by PTLC to afford 25 (28.2 mg) and 19 (20.7 mg). Fr.N was subjected to Sephadex LH-20 to afford four subfractions (Fr.N1–Fr.N4). Fr.N2 was purified by ODS (MeOH:H2O, 30:70, v/v) to yield 31 (36.3 mg). Fr.N4 was purified by semi-preparative HPLC (MeOH:H2O, 70:30, v/v) to yield 34 (8.0 mg, tR 14.2 min). Fr.O (5.0 g) was separated by ODS CC to yield 3 subfractions (Fr.O1–Fr.O3). Fr.O1 was repeatedly recrystallised in acetone to afford mixture IV, which was further purified by HPLC on a semi-preparative YMC C-18 column using MeOH–H2O (70:30, v/v) as the mobile phase to yield 20 (2.0 mg, tR 20.8 min) and 35 (2.9 mg, tR 23.1 min). Fr.O2 was subjected to HPLC to yield 33 (10.8 mg, tR 12.0 min) and 32 (3.2 mg, tR 18.0 min). Compound 22 (9.8 mg) was obtained from Sephadex LH-20 CC of Fr.O3. Fr.P was separated by ODS CC and eluted with a MeOH–H2O gradient (from 20:80 to 80:20, v/v) to afford four subfractions (Fr.P1–Fr.P4). Fr.P2 was purified by Sephadex LH-20 CC to obtain 36 (12.1 mg) and 37 (6.8 mg). Fr.P3 was subjected to silica gel CC eluted with P.E.-acetone (7:1, v/v) to yield 26 (4.9 mg) and MeOH to yield 4 (4.6 mg). Compound 3 (18.0 mg) was obtained from Fr.P4 via Sephadex LH-20 CC eluted with CH2Cl2–MeOH (1:1, v/v). Fr.Q was separated by ODS CC and eluted with MeOH–H2O (35:65, v/v, Fr.Q1) and MeOH–H2O (40:60, v/v, Fr.Q2). Fr.Q1 was repeatedly recrystallised in acetone to afford 24 (3.9 mg). The remaining mother liquor of Fr.Q1 was purified by HPLC (MeOH:H2O, 75:25, v/v) to give 27 (3.4 mg, tR 29.0 min). The EtOAc extract (102 g) was fractionated by silica gel CC using a gradient system of increasing polarity of CH2Cl2–MeOH (100:0– 100:50, v/v). The collected fractions were combined based on their TLC characteristics to yield four fractions (Fr.S–Fr.V). Fr.T was repeatedly recrystallised in MeOH to yield 21 (15.6 mg) and the
Please cite this article in press as: Zhao, N., et al. Quinolone and indole alkaloids from the fruits of Euodia rutaecarpa and their cytotoxicity against two human cancer cell lines. Phytochemistry (2014), http://dx.doi.org/10.1016/j.phytochem.2014.10.020
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mother liquor was purified by semi-preparative HPLC (MeOH:H2O, 75:25, v/v) to obtain 23 (4.1 mg, tR 29.5 min). 4.3.1. (1) 1-methyl-2-[6-carbonyl-(E)-4-undecenyl]-4(1H)-quinolone Colourless oil; UV (MeOH) kmax (log e): 239 (4.49), 321 (4.15), 334 (4.16) nm; IR (KBr) tmax: 2923, 2851, 1621, 1597, 1567, 1468 cm1; 1H NMR (600 MHz, DMSO-d6): d 8.14 (1H, d, J = 7.9 Hz, H-5), 7.76 (1H, d, J = 8.6 Hz, H-8), 7.70 (1H, dd, J = 8.6, 7.0 Hz, H-7), 7.36 (1H, dd, J = 7.9, 7.0 Hz, H-6), 6,82 (1H, dt, J = 15.8, 6.5 Hz, H-40 ), 6.04 (1H, d, J = 15.8 Hz, H-50 ), 6.01 (1H, s, H-3), 3.72 (3H, s, –NCH3), 2.76 (2H, t, J = 6.8 Hz, H-10 ), 2.55 (2H, m, H-30 ), 2.52 (2H, t, J = 7.1 Hz, H-70 ), 2.26 (1H, m, H-20 b), 2.14 (1H, m, H-20 a), 1.54 (1H, m, H-80 b), 1.43 (1H, m, H-80 a), 1.35 (2H, m, H-90 ), 1.22 (2H, m, H-100 ), 0.83 (3H, t, J = 7.3 Hz, 110 –CH3); 13C NMR (150 MHz, DMSO-d6): d 200.0 (C-60 ), 175.9 (C-4), 155.3 (C2), 147.2 (C-40 ), 141.8 (C-8a), 132.0 (C-7), 130.5 (C-50 ), 126.0 (C4a), 125.3 (C-5), 123.0 (C-6), 116.8 (C-8), 109.9 (C-3), 38.9 (C-70 ), 34.4 (–NCH3), 33.8 (C-10 ), 31.5 (C-30 ), 27.9 (C-80 ), 27.8 (C-20 ), 25.9 (C-90 ), 21.7 (C-100 ), 13.8 (110 -CH3); HRESIMS m/z 326.2117 [M+H]+ (calcd. for C21H28NO2 326.2115). EI-MS m/z 254, 200, 186 (100), 173, 144. 4.3.2. (2) 1-methyl-2-[6-carbonyl-(E)-7-tridecenyl]-4(1H)-quinolone Colourless oil; UV (MeOH) kmax (log e): 239 (4.45), 321 (4.12), 334 (4.13) nm; IR (KBr) tmax: 2922, 2851, 1623, 1598, 1467 cm1; 1H NMR (600 MHz, DMSO-d6): d 8.14 (1H, br d, J = 7.7 Hz, H-5), 7.76 (1H, d, J = 8.6 Hz, H-8), 7.70 (1H, ddd, J = 7.0, 6.8, 1.5 Hz, H-7), 7.36 (1H, dd, J = 7.7, 7.0 Hz, H-6), 6,82 (1H, dt, J = 15.8, 8.4 Hz, H-80 ), 6.06 (1H, d, J = 15.8 Hz, H-70 ), 6.02 (1H, s, H-3), 3.72 (3H, s, –NCH3), 2.75 (2H, t, J = 7.4 Hz, H-10 ), 2.52 (2H, t, J = 6.9 Hz, H-50 ), 2.16 (1H, m, H-90 b), 1.95 (1H, m, H-90 a), 1.60 (2H, m, H-20 ), 1.59 (1H, m, H-40 b), 1.54 (1H, m, H-40 a), 1.42 (2H, m, H-100 ), 1.33 (2H, m, H-30 ), 1.25 (2H, m, H-110 ), 1.23 (2H, m, H-120 ), 0.84 (3H, t, J = 7.9 Hz, 130 -CH3); 13C NMR (150 MHz, DMSO-d6): d 200.0 (C-60 ), 175.9 (C-4), 155.4 (C-2), 147.0 (C-80 ), 141.9 (C-8a), 132.0 (C-7), 130.3 (C-70 ), 126.0 (C-4a), 125.2 (C-5), 123.1 (C-6), 116.8 (C-8), 109.8 (C-3), 39.3 (C-50 ), 34.4 (–NCH3), 33.8 (C-10 ), 31.6 (C-90 ), 28.7 (C-40 ), 28.1 (C-20 ), 28.0 (C-30 ), 27.1 (C-100 ), 26.1 (C-110 ), 21.8 (C-120 ), 13.4 (130 -CH3); HR-ESI-MS m/z 354.2438 [M+H]+ (calcd. for C23H32NO2 354.2438). EI-MS m/z 310, 282, 256, 228, 186 (100), 173, 144. 4.3.3. (3) 1-methyl-2-[15-hydroxyl-pentadecenyl]-4(1H)-quinolone White needles (MeOH); mp. 111–112 °C; UV (MeOH) kmax (log e): 239 (4.43), 321 (4.10), 334 (4.11) nm; IR (KBr) tmax: 3419, 2916, 1618, 1596, 1558, 1468 cm1; 1H NMR (300 MHz, DMSOd6): d 8.14 (1H, dd, J = 7.9, 1.3 Hz, H-5), 7.75 (1H, br d, J = 7.7 Hz, H-8), 7.70 (1H, ddd, J = 8.7, 6.6, 1.6 Hz, H-7), 7.35 (1H, td, J = 7.3, 1.2 Hz, H-6), 6.01 (1H, s, H-3), 4.28 (1H, t, J = 5.3 Hz, –OH), 3.72 (3H, s, –NCH3), 3.36 (2H, J = 6.5, 5.1 Hz, H-150 ), 2.75 (2H, t, J = 7.6 Hz, H-10 ), 1.60 (2H, m, H-20 ), 1.37 (2H, m, H-140 ), 1.32 (2H, m, H-30 ), 1.22 (10H, m, H-40 –H-130 ); 13C NMR (75 MHz, DMSOd6): d 175.8 (C-4), 155.3 (C-2), 141.8 (C-8a), 131.9 (C-7), 126.1 (C-4a), 125.2 (C-5), 122.9 (C-6), 116.9 (C-8), 109.8 (C-3), 60.7 (C150 ), 34.2 (–NCH3), 33.7 (C-10 ), 32.5 (C-140 ), 29.1–28.0 (C-20 –C130 ); HR-ESI-MS m/z 386.3056 [M+H]+ (calcd. for C25H40NO2 386.3054). 4.3.4. (4) 1-methyl-2-[13-hydroxyl-tridecenyl]-4(1H)-quinolone White needles; mp. 109–110 °C; UV (MeOH) kmax (log e): 239 (4.50), 321 (4.15), 334 (4.16) nm; IR (KBr) tmax: 3403, 2918, 2849, 1619, 1598, 1557, 1467 cm1; 1H NMR (600 MHz, CDCl3): d 8.44 (1H, dd, J = 8.0, 1.4 Hz, H-5), 7.67 (1H, td, J = 7.2, 1.5 Hz, H7), 7.52 (1H, d, J = 8.7 Hz, H-8), 7.38 (1H, t, J = 7.2 Hz, H-6), 6.30 (1H, s, H-3), 3.76 (3H, s, –NCH3), 3.63 (2H, t, J = 6.7 Hz, H-130 ), 2.72 (2H, t, J = 7.7 Hz, H-10 ), 1.68 (2H, m, H-20 ), 1.56 (2H, m,
H-120 ), 1.42 (2H, m, H-30 ), 1.331.22 (16H, m, H-40 –H-110 ); 13C NMR (150 MHz, CDCl3): d 177.7 (C-4), 155.2 (C-2), 142.0 (C-8a), 132.3 (C-7), 126.8 (C-5), 126.4 (C-4a), 123.6 (C-6), 115.5 (C-8), 111.2 (C-3), 63.1 (C-130 ), 34.9 (C-10 ), 34.5 (–NCH3), 32.9 (C-120 ), 28.6 (C-20 ), 29.1–28.0 (C-30 –C-100 ), 25.9 (C-110 ); HR-ESI-MS m/z 358.2744 [M+H]+ (calcd. for C23H36NO2 358.2741). 4.3.5. (20) 13b-hydroxymethylevodiamine White powder (MeOH); UV (MeOH) kmax (log e): 226 (4.63), 268 (3.96), 326 (2.79) nm; IR (KBr) tmax: 3415, 3228, 1614, 1595, 1488, 1471.cm1; 1H NMR (600 MHz, DMSO-d6): d 11.25 (1H, br. s, NH), 7.87 (1H, d, J = 7.6 Hz, H-4), 7.52 (1H, d, J = 7.9 Hz, H-9), 7.49 (1H, dd, J = 8.0, 7.3 Hz, H-2), 7.43 (1H, d, J = 8.1 Hz, H-12), 7.14 (1H, m, H-11), 7.11 (2H, m, H-1, 3), 7.03 (1H, dd, J = 7.9, 7.3 Hz, H-10), 5.20 (1H, br t, J = 5.5 Hz, –CH2OH), 4.94 (1H, dd, J = 12.4, 4.5 Hz, Hb-7), 3.92 (1H, dd, J = 11.2, 5.5 Hz, –CH2OH), 3.72 (1H, dd, J = 11.2, 5.5 Hz, –CH2OH), 3.29 (1H, dd, J = 12.4, 3.8 Hz, Ha-7), 2.90 (1H, dd, J = 14.8, 2.7 Hz, Hb-8), 2.70 (1H, m, Ha-8), 2.44 (3H, s, –NCH3); 13C NMR (150 MHz, DMSO-d6): d 161.3 (C-5), 148.9 (C-14a), 136.6 (C-12a), 133.1 (C-2), 130.2 (C-13a), 127.6 (C-4), 125.6 (C-8b), 122.3 (C-3), 122.0 (C-11), 121.9 (C-4a), 121.3 (C-1), 118.7 (C-10), 118.3 (C-9), 113.0 (C-8a), 111.7 (C-12), 76.2 (C13b), 64.9 (–CH2OH), 40.1 (C-7), 38.4 (–NCH3), 20.4 (C-8); HRESI-MS m/z 356.1363 [M+Na]+ (calcd. for C20H19N3NaO2 356.1369). 4.3.6. (21) wuzhuyurutine C White powder (MeOH); UV (MeOH) kmax (log e): 221 (4.29), 245sh (4.16), 354 (4.19), 373 (4.03) nm; IR (KBr) tmax: 3447, 1686, 1654, 1583, 1464, 1446 cm1; 1H NMR (600 MHz, DMSOd6): d 13.79 (1H, br. s, 14-NH), 12.86 (1H, br. s, 13-NH), 8.20 (1H, dd, J = 8.0, 1.2 Hz, H-4), 8.11 (1H, br. d, J = 8.2 Hz, H-9), 7.90 (1H, td, J = 8.0, 1.2 Hz, H-2), 7.83 (1H, d, J = 8.0 Hz, H-1), 7.68 (1H, br. d, J = 8.0 Hz, H-12), 7.59 (1H, td, J = 8.0, 1.0 Hz, H-3), 7.36 (1H, td, J = 7.6, 1.0 Hz, H-11), 7.28 (1H, td, J = 7.6, 1.0 Hz, H-10), 4.00 (3H, s, –COOCH3); 13C NMR (150 MHz, DMSO-d6): d 167.4 (C-7), 160.8 (C-5), 148.4 (C-14a), 146.0 (C-13b), 135.6 (C-12a), 134.8 (C-2), 134.6 (C-13a), 127.5 (C-1), 127.2 (C-3), 126.5 (C-8b), 126.1 (C-4), 124.9 (C-11), 122.6 (C-10), 122.4 (C-9), 121.6 (C-4a), 113.1 (C12), 106.3 (C-8a), 52.4 (–COOCH3); HR-ESI-MS m/z 319.1030 [M+H]+ (calcd. for C18H14N3O3 320.1030). 4.3.7. (22) wuzhuyurutine D White powder (MeOH); UV (MeOH) kmax (log e): 221 (4.46), 245sh (4.28), 347 (4.35), 363 (4.21) nm; IR (KBr) tmax: 3402, 3401, 1664, 1584 cm1; 1H NMR (300 MHz, DMSO-d6): d 14.52 (1H, br. s, –COOH), 13.86 (1H, br. s, 14-NH), 12.73 (1H, br. s, 13NH), 8.19 (2H, br d, J = 8.0 Hz, H-4, 9), 7.90 (1H, td, J = 8.0, 1.0 Hz, H-2), 7.84 (1H, d, J = 7.6 Hz, H-1), 7.69 (1H, d, J = 7.8 Hz, H-12), 7.57 (1H, td, J = 8.0, 1.0 Hz, H-3), 7.35 (1H, dd, J = 7.8, 7.1 Hz, H11), 7.26 (1H, dd, J = 8.2, 7.3 Hz, H-10); 13C NMR (150 MHz, DMSO-d6): d 169.3 (C-7), 161.0 (C-5), 148.5 (C-14a), 146.2 (C13b), 135.7 (C-12a), 134.8 (C-2), 134.4 (C-13a), 127.4 (C-18b), 127.3 (C-1), 127.1 (C-3), 126.1 (C-4), 124.9 (C-11), 122.5 (C-10), 122.7 (C-9), 121.7 (C-4a), 113.1 (C-12), 107.7 (C-8a); HR-ESI-MS m/z 328.0687 [M+Na]+ (calcd. 328.0693). 4.4. Cell culture and growth inhibition assay The human leukaemia cell line HL-60 and the human prostate cancer PC-3 cell line were purchased from the American Type Culture Collection (ATCC Rockville, MD, USA) and grown in RPMI-1640 medium (Gibco, New York, NY, USA) supplied with 100 IU/mL penicillin, 100 lg/mL streptomycin, 1 mM D-glutamine and 10% heat-inactivated foetal bovine serum (Gibco). Cells were incubated in standard conditions (37 °C under 5% CO2 and humidified atmosphere).
Please cite this article in press as: Zhao, N., et al. Quinolone and indole alkaloids from the fruits of Euodia rutaecarpa and their cytotoxicity against two human cancer cell lines. Phytochemistry (2014), http://dx.doi.org/10.1016/j.phytochem.2014.10.020
N. Zhao et al. / Phytochemistry xxx (2014) xxx–xxx
HL-60 cell viability was determined by the trypan blue exclusion test (Wang et al., 2006). Briefly, cells in logarithmic growth were dispensed into 24-well microplates at 4 104 cells/mL and incubated with various concentrations of the test compounds at 37 °C under 5% CO2 and 95% air for 3 days. The compounds were dissolved in DMSO and then diluted to the proper concentrations. Cell viability was determined after staining the cells, and the total cell number was determined using a hematocytometer. PC-3 cell viability was determined by an MTT assay (Plumb et al., 1989). Briefly, cells were dispensed into microtiter plates at 2 104 cells/mL in 1 mL of 1640 medium/well and incubated overnight at 37 °C in 5% CO2 and 95% air. The test compounds were diluted with fresh medium and placed into microtiter plates in a total volume of 200 ll. Untreated control cultures were maintained under the same conditions. A total of 50 lL of a 0.4% MTT solution (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide; Thiazolyl blue) was added to each well of the plate. After 3–4 h incubation, MTT was removed from the wells and the formazan crystals were dissolved in DMSO (200 lL) and added to each well. The plates were then shaken for 10 min. The OD was read using in a microplate reader (Bio-RAD) at 570 nm. The growth inhibition in cells after the treatment with different concentration (GI50) was obtained from a regression analysis of the concentration response data. 5-Fluorouracil (5-Fu) was used as the positive control, and 0.1% DMSO was used as the negative control. Three independent experiments were duplicated, and the data are presented as the mean ± SD. Acknowledgements The authors wish to thank the financial support from the National Natural Science Foundation of China (No. 81172958) and the National Key Technology R&D Program (2012BAI30B02). We are grateful to Mr. Yi Sha and Mrs. Wen Li (Analytical Testing Center, Shenyang Pharmaceutical University, Shenyang, People’s Republic of China) for their measurements of the NMR data. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.phytochem.2014. 10.020. References Adams, M., Kunert, O., Haslinger, E., Bauer, R., 2004. Inhibition of leukotriene biosynthesis by quinlone alkaloids from the fruits of Evodia rutaecarpa. Planta Med. 70, 904–908. Chinese Pharmacopoeia Commission, 2010. Pharmacopoeia of the People’s Republic of China, vol. I. China Medical Science and Technology Press, Beijing, p. 160. Huang, L., Yu, D.Q., 1988. Application of Ultraviolet Spectrum in Organic Chemistry (ii). Science Press, Beijing, p. 100. Huang, X., Li, W., Yang, X.W., 2012. New cytotoxic quinolone alkaloids from fruits of Evodia rutaecarpa. Fitoterapia 83, 709–714.
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Ikuta, A., Nakamura, T., Urabe, H., 1998. Indolopyridoquinazoline, furoquinoline and canthinone type alkaloids from Phellodendron amurense callus tissues. Phytochemistry 48, 285–291. Jiang, J.L., Hu, C.P., 2009. Evodiamine: a novel anti-cancer alkaloid from Evodia rutaecarpa. Molecules 14, 1852–1859. Kazunari, T., Kazuhide, H., Norio, H., Masaki, H., Takashi, T., Tetsuya, Y., Toshio, W., Yasuhiro, F., Yasuhiro, T., Takema, N., Shigetoshi, K., Eiji, I., Kazuo, K., Tetsuo, A., 2002. In vivo action of novel alkyl methyl quinolone alkaloids against Helicobacter pylori. J. Antimicrob. Chemother. 50, 547–552. Ko, H.C., Wang, Y.H., Liou, K.T., Chen, C.M., Chen, C.H., Wang, W.Y., Chang, S., Hou, Y.C., Chen, K.T., Chen, C.F., Shen, Y.C., 2007. Anti-inflammatory effects and mechanisms of the ethanol extract of Evodia rutaecarpa and its bioactive components on neutrophils and microglial cells. Eur. J. Pharmacol. 555, 211– 217. Kostova, I., Ivanova, A., Mikhova, B., Klaiber, I., 1999. Alkaloids and coumarins from Ruta graveolens. Monatsh. Chem. 130, 703–707. Lee, M.K., Hwang, B.Y., Lee, S.A., Oh, G.J., Choi, W.H., Hong, S.S., Lee, K.S., Ro, J.S., 2003. 1-Methyl-2-undecyl-4(1H)-quinolone as an irreversible and selective inhibitor of type B monoamine oxidase. Chem. Pharm. Bull. 51, 409–411. Li, X.C., Dunbar, D.C., ElSohly, H.N., Walker, L.A., Clark, A.M., 2001. Indolopyridoquinazoline alkaloid from Leptothyrsa sprucei. Phytochemistry 58, 627–629. Ng, K.M., Gray, A.I., Waterman, P.G., 1987. Limonoids, alkaloids, and a coumarin from the root and stem barks of Tetradium glabrifolium. J. Nat. Prod. 50, 1160– 1163. Noboru, S., Akemi, U., Akio, I., Nobuaki, S., Shigenobu, A., 1989. Two novel alkaloids from Evodia rutaecarpa. J. Nat. Prod. 52, 1160–1162. Plumb, J.A., Milroy, R., S.B., Kaye, 1989. Effects of the pH dependence of 3-(4,5dimethylthiazol-2-yl)-2,5-diphenylteterazolium bromide-formazan absorption on chemosensitivity determined by a novel tetrazolium-based assay. Cancer Res. 49, 4435–4440. Shoji, N., Umeyama, A., Iuchi, A., Saito, N., Arihara, S., 1989. Two novel alkaloids from Evodia rutaecarpa. J. Nat. Prod. 52, 1160–1162. Shoji, N., Umeyama, A., Iuchi, A., Saito, N., Takemoto, T., 1988a. Isolation of an amide, a possible key precursor to evodiamine, from Evodia rutaecarpa. J. Nat. Prod. 51, 161–162. Shoji, N., Umeyama, A., Iuchi, A., Saito, N., Takemoto, T., 1988b. Isolation of a new alkaloid from Evodia rutaecarpa. J. Nat. Prod. 51, 791–792. Somanathan, R., Smith, K.M., 1981. Synthesis of some 2-alkyl-4-quinolone and 2alkyl-4-methoxyquinolone alkaloids. J. Heterocyclic Chem. 18, 1077–1079. Tang, Y.Q., Feng, X.Z., Huang, L., 1996a. Quinolone alkaloids from Evodia rutaecarpa. Phytochemistry 43, 719–722. Tang, Y.Q., Feng, X.Z., Huang, L., 1996b. Studies on the chemical constituents of Evodia rutaecarpa [Juss] Benth. Acta Pharm. Sin. 31, 151–155. Tang, Y.Q., Wu, K.M., Feng, X.Z., Huang, L., 1998. Synthesis and bioaction of 2-alkyl4(1H)-quinolone. Acta Pharm. Sin. 33, 121–127. Teng, J., Yang, X.W., 2006. Two new indoloquinazoline alkaloids from the unripe fruits of Evodia rutaecarpa. Heterocycles 68, 1691–1698. Tohru, S., Toshio, M., Masanori, K., Akira, U., 1988. Limonoids and quinolone alkaloids from Evodia rutaecarpa Bentham. Chem. Pharm. Bull. 36, 4453–4461. Wang, F., Hua, H.M., Pei, Y.H., Chen, D., Jing, Y.K., 2006. Triterpenoids from the resin of Styrax tonkinensis and their antiproliferative and differentiation effects in human leukemia HL-60 cells. J. Nat. Prod. 69, 807–810. Wang, X.X., Zan, K., Shi, S.P., Zeng, K.W., Jiang, Y., Guan, Y., Xiao, C.L., Gao, H.Y., Wu, L.J., Tu, P.F., 2013. Quinolone alkaloids with antibacterial and cytotoxic activities from the fruits of Evodia rutaecarpa. Fitoterapia 89, 1–7. Wattanapiromsakul, C., Forster, P.I., Waterman, P.G., 2003. Alkaloids and limonoids from Bouchardatia neurococca: systematic significance. Phytochemistry 64, 609–615. Wu, T.S., Yeh, J.H., Wu, P.L., Chen, K.T., Lin, L.C., Chen, C.Fu., 1995. 7Hydroxyrutaecarpine from Tetradium glabrifolium and Tetradium ruticarpum. Heterocycles 41, 1071–1076. Yang, L.M., Chen, C.F., Lee, K.H., 1995. Synthesis of rutaecarpine and cytotoxic analogues. Bioorg. Med. Chem. Lett. 5, 465–468. Yang, X.W., Teng, J., 2007. Chemical constituents of the unripe fruits of Evodia rutaecarpa. J. Chin. Pharm. Sci. 16, 20–23. Zhang, H., Yang, X.W., Chui, Y.R., 1999. Complete assignment of 1H and 13C NMR chemical shifts of evodiamine, rutaecarpine and dehydroevodiamine. Chin. J. Magn. Reson. 16, 563–568.
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Quinolone and indole alkaloids from the fruits of Euodia rutaecarpa and their cytotoxicity against two human cancer cell lines Nan Zhao a,b, Zhan-Lin Li a,b,⇑, Da-Hong Li a,b, Ya-Ting Sun a,b, Dong-Ting Shan a,b, Jiao Bai a,b, Yue-Hu Pei a,b, Yong-Kui Jing c, Hui-Ming Hua a,b,⇑ a b c
Key Laboratory of Structure-Based Drug Design & Discovery, Ministry of Education, Shenyang Pharmaceutical University, Shenyang 110016, PR China School of Traditional Chinese Materia Medica, Shenyang Pharmaceutical University, Shenyang 110016, PR China Department of Medicine, Mount Sinai School of Medicine, New York, NY 10029, USA
a r t i c l e
i n f o
Article history: Received 16 May 2014 Received in revised form 11 July 2014 Available online xxxx Keywords: Euodia rutaecarpa Rutaceae Quinolone alkaloid Indole alkaloid Growth inhibitory activity
a b s t r a c t Four quinolone alkaloids (1–4) and three indole alkaloids (20–22), together with 30 known alkaloids (5–19, 23–37), were isolated from the fruits of Euodia rutaecarpa. Their structures were established by spectroscopic analyses. The in vitro cytotoxic activities of these alkaloids against leukaemia HL-60 and prostate cancer PC-3 cell lines were evaluated. Ó 2014 Elsevier Ltd. All rights reserved.
1. Introduction
2. Results and discussion
The dried and nearly ripe fruits of Euodia rutaecarpa (Juss.) Benth. (Rutaceae) have been used as a traditional Chinese medicine for treatment of headache, abdominal pain, dysentery, postpartum haemorrhage, and amenorrhea (Chinese Pharmacopoeia Commission, 2010). A number of quinolone and indole alkaloids have also been reported (Yang and Teng, 2007; Noboru et al., 1989; Tang et al., 1996; Tohru et al., 1988) from E. rutaecarpa, and some of these alkaloids have exhibited promising antiinflammatory (Ko et al., 2007; Adams et al., 2004), antibacterial (Kazunari et al., 2002), and cytotoxic activities (Jiang and Hu, 2009). As an ongoing search for bioactive natural alkaloids, the isolation and structural elucidation of four new quinolone alkaloids (1–4) and three indole alkaloids (20–22), together with thirty known alkaloids, are reported herein. The growth inhibitory activities of the isolated compounds were evaluated against HL-60 and PC-3 cell lines.
The air-dried fruits of E. rutaecarpa were extracted with 75% ethanol. After concentration, the extract was successively partitioned with petroleum ether (P.E.), CHCl3, EtOAc, and n-butanol. The P.E., CHCl3, and EtOAc soluble fractions were submitted to separation and purification by column chromatography over silica gel as well as reversed-phase (ODS), Sephadex LH-20, and finally preparative HPLC yielding 18 quinolone alkaloids (Fig. 1) and 19 indole alkaloids (Fig. 2), of which seven were new compounds. The known compounds were identified as: 1-methyl-2-[(Z)-5undecenyl]-4(1H)-quinolone (5) (Tang et al., 1996), 1-methyl-2[(Z)-6-undecenyl]-4(1H)-quinolone (6) (Tang et al., 1996), 1-methyl-2-[(Z)-7-tridecenyl]-4(1H)-quinolone (7) (Tang et al., 1996), evocarpine (8) (Tang et al., 1996), 1-methyl-2-[7-carbonyl(E)-9-tridecenyl]-4(1H)-quinolone (9) (Huang et al., 2012), euocarpine D (10) (Wang et al., 2013), 1-methyl-2-[(4Z,7Z)-4,7-tridecadienyl]-4(1H)-quinolone (11) (Tohru et al., 1988), 1-methyl-2[(6Z,9Z)-6,9-pentadecadienyl]-4(1H)-quinolone (12) (Tohru et al., 1988), 1-methyl-2-octyl-4(1H)-quinolone (13) (Somanathan and Smith, 1981), 1-methyl-2-nonyl-4(1H)-quinolone (14) (Tang et al., 1996; Kostova et al., 1999), 1-methyl-2-decyl-4(1H)-quinolone (15) (Tang et al., 1998), 1-methyl-2-undecyl-4(1H)-quinolone (16) (Lee et al., 2003), dihydroevocarpine (17) (Tohru et al., 1988), 1-methyl-2-pentadecenyl-4(1H)-quinolone (18) (Tohru et al., 1988), evodiamine (19) (Zhang et al., 1999), wuzhuyurutine B
⇑ Corresponding authors at: Key Laboratory of Structure-Based Drug Design & Discovery, Ministry of Education, Shenyang Pharmaceutical University, Shenyang 110016, PR China. Tel./fax: +86 24 23986465. E-mail addresses: [email protected] (Z.-L. Li), [email protected] (H.-M. Hua). http://dx.doi.org/10.1016/j.phytochem.2014.10.020 0031-9422/Ó 2014 Elsevier Ltd. All rights reserved.
Please cite this article in press as: Zhao, N., et al. Quinolone and indole alkaloids from the fruits of Euodia rutaecarpa and their cytotoxicity against two human cancer cell lines. Phytochemistry (2014), http://dx.doi.org/10.1016/j.phytochem.2014.10.020
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(23) (Teng and Yang, 2006), bouchardatine (24) (Wattanapiromsakul et al., 2003), rutaecarpine (25) (Zhang et al., 1999), 1-hydroxyrutaecarpine (26) (Wattanapiromsakul et al., 2003; Ng et al., 1987), 3-hydoxyrutaecarpine (27) (Li et al., 2001), 7-hydroxyrutaecarpine (28) (Wu et al., 1995), hortiacine (29) (Yang et al., 1995), 7,8-dehydrorutaecarpine (30) (Ikuta et al., 1998), dehydroevodiamine (31) (Zhang et al., 1999), goshuyuamide-I (32) (Shoji et al., 1989), goshuyuamide-II (33) (Shoji et al., 1989), N-(2-methylaminobenzoyl) tryptamine (34) (Shoji et al., 1988), evodiamide (35) (Shoji et al., 1988), wuchuyuamideI (36) (Yang and Teng, 2007), and 1,2,3,4-tetrahydro-1-oxo-carboline (37) (Tang et al., 1996) by comparison of their spectroscopic data with reported data. Compound 1 was isolated as a colourless oil with a molecular formula of C21H27NO2, as determined by HRESIMS (m/z 326.2117 [M+H]+). Its N-methyl-4(1H)-quinolone skeleton was determined by its UV absorptions at 239, 321, and 334 nm (Huang and Yu, 1988). The 1H and 13C NMR spectra showed characteristic signals of a N-methyl-4(1H)-quinolone at dH 6.01 (1H, s, H-3), 8.14 (1H, d, J = 7.9 Hz, H-5), 7.36 (1H, dd, J = 7.9, 7.0 Hz, H-6), 7.70 (1H, dd, J = 8.6, 7.0 Hz, H-7), 7.76 (1H, d, J = 8.6 Hz, H-8) and dC 155.3 (C2), 109.9 (C-3), 175.9 (C-4), 126.0 (C-4a), 125.3 (C-5), 123.0 (C-6), 132.0 (C-7), 116.8 (C-8), 141.8 (C-8a). The resonances of an a,bunsaturated carbonyl group were observed at dH 6.82 (dt, J = 15.8, 6.5 Hz, H-40 ), 6.04 (d, J = 15.8 Hz, H-50 ), and dC 147.2 (C40 ), 130.5 (C-50 ), 200.0 (C-60 ). The characteristic fragment ions of quinolone alkaloids were given in the EIMS including the ones arising from McLafferty rearrangement at m/z 173, a displacement rearrangement at m/z 186, and an elimination of the carbonyl at m/z 144. The HMBC correlations (Fig. 3) supported the position of an a,b-unsaturated carbonyl group in the side-chain, in which the proton signals (dH 6.82, 6.04) correlated with C-30 (dC 31.5), suggesting that the olefinic carbons were assigned at C-40 and
5 6 7 8
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O 4
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O
O 2
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N 2 R CH 3 4'
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C-50 and that the carbonyl carbon was assigned at C-60 . Moreover, the double bond was determined to be in the E-form due to the coupling constant (JH-40 ,50 = 15.8 Hz). Thus, 1 was assigned as 1-methyl-2-[6-carbonyl-(E)-4-undecenyl]-4(1H)-quinolone. Compound 2 was obtained as a colourless oil and its molecular formula was determined to be C23H31NO2 from its molecular ion peak at m/z 354.2438 [M+H]+ (calcd. for C23H32NO2, 354.2428) in the HRESIMS, indicating compound 2 had two more methylene groups than 1. The UV spectrum and EIMS (m/z 173, 144, 186) suggested that 2 should be a quinolone alkaloid. The 1H and 13C NMR spectra closely resembled those of 1, except for the integral of the protons in the high field region. The assignment of the position of an a,b-unsaturated carbonyl group was determined by long-range correlations from H-90 b (2.16, 1H, m,) to C-70 (130.3) and C-80 (147.0) and the a-cleavage fragment ions at m/z 228, 296 in EIMS, indicating that the carbonyl was assigned at C-60 and that the olefinic carbons were assigned at C-70 and C-80 . Therefore, the structure of 2 was concluded to be 1-methyl-2-[6-carbonyl-(E)-7tridecenyl]-4(1H)-quinolone. Compound 3 was obtained as white needles. The molecular formula, C25H39NO2, was assigned by the quasi-molecular ion peak at m/z 386.3056 [M+H]+ (calcd. for C25H40NO2, 386.3054) in HRESIMS. Its IR spectrum showed the presence of hydroxyl (3419 cm1) and aromatic (1597 cm1) groups. The characteristic absorption peaks at 239, 321, and 334 nm in the UV spectrum indicated the presence of an N-methyl-4(1H)-quinolone skeleton (Huang and Yu, 1988), which was supported by the 1H and 13C NMR signals at dH 3.72 (3H, s, N–CH3), 6.01 (s, H-3), 8.14 (dd, J = 7.9, 1.3 Hz, H-5), 7.35 (td, J = 7.3, 1.2 Hz, H-6), 7.70 (ddd, J = 8.7, 6.6, 1.6 Hz, H-7), 7.75 (br d, J = 7.7 Hz, H-8); dC 34.2 (N–CH3), 155.3 (C-2), 109.8 (C-3), 175.8 (C-4). The 1H and 13C NMR spectra also displayed signals for one hydroxymethyl group [dH 4.28 (t, J = 5.1 Hz, –OH), 3.35 (td, J = 6.5, 5.1 Hz, H-150 ); dC 60.7 (C-150 )], which was located at
17 18
7'
9'
10' 15'
8'
9' 10'
11' 13' 15'
Fig. 1. Structures of compounds 1–18 from Euodia rutaecarpa.
Please cite this article in press as: Zhao, N., et al. Quinolone and indole alkaloids from the fruits of Euodia rutaecarpa and their cytotoxicity against two human cancer cell lines. Phytochemistry (2014), http://dx.doi.org/10.1016/j.phytochem.2014.10.020
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8b
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R = COOH R = CHO
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R = CH3
O
Fig. 2. Structures of compounds 19–37 from Euodia rutaecarpa.
O
O O
O
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N
CH 3
CH 3 1
2 Fig. 3. Key HMBC correlations of 1–4.
the terminus of the side-chain. The assigned structure was further verified by a detailed HMBC analysis (Fig. 3). Consequently, compound 3 was identified as 1-methyl-2-[15-hydroxyl-pentadecenyl]-4(1H)-quinolone. Compound 4 was obtained as white needles. Its HRESIMS exhibited a quasi-molecular ion peak at m/z 358.2744 [M+H]+ corresponding to a molecular formula of C23H35NO2, which was a C2H4 unit less than that of compound 3. The IR and UV spectra were similar to those of 3. A comparison of the 1H and 13C NMR spectroscopic data of 4 and 3 showed that they were closely related analogues differing only in the length of the side-chain. Hence, compound 4 was assigned as 1-methyl-2-[13-hydroxyl-tridecenyl]-4(1H)-quinolone. Compound 20 was isolated as a white powder. Its molecular formula was determined to be C20H19N3O2 by HRESIMS ([M+Na]+
at m/z = 356.1363). The IR spectrum showed the presence of O–H (3415 cm1), N–H (3228 cm1), and aromatic groups (1614, 1595, 1488, and 1471 cm1). The UV absorptions at 226, 268, and 326 nm showed the presence of an indole chromophore (Teng and Yang, 2006). The 1H NMR spectrum of 20 displayed signals of two spin systems for 1,2-disubstitued aromatic rings dH 7.11 (2H, m, H-1, 3), 7.49 (1H, dd, J = 8.0, 7.3 Hz, H-2), 7.87 (1H, d, J = 7.6 Hz, H-4), 7.52 (1H, d, J = 7.9 Hz, H-9), 7.03 (1H, dd, J = 7.9, 7.3 Hz, H-10), 7.14 (1H, m, H-11), 7.43 (1H, d, J = 8.1 Hz, H-12), one N–H group (dH 11.25), one hydroxymethyl group(dH 5.20, 3.92, 3.72, dC 64.9) and one N–CH3 group (dH 2.44, dC 38.7). The 13 C NMR spectrum showed resonances assigned to an indole ring and a quinazolone ring, as well as one hydroxymethyl group (dC 64.9) and two methylene groups at dC 40.1, 20.4, suggesting that compound 20 was an indoloquinazoline alkaloid. A comparison
Please cite this article in press as: Zhao, N., et al. Quinolone and indole alkaloids from the fruits of Euodia rutaecarpa and their cytotoxicity against two human cancer cell lines. Phytochemistry (2014), http://dx.doi.org/10.1016/j.phytochem.2014.10.020
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COOCH3 N
N H HOH2 C
O
N H
N
N
O
HN
COOH N
N H
O
HN
H 3C 20
22
21 Fig. 4. Key HMBC correlations of 20–22.
with the NMR spectroscopic data of evodiamine (19) indicated that compound 20 possessed one more hydroxymethyl group than 19. The hydroxymethyl group was assigned to C-13b based on the long-range correlations from the protons at dH 3.92 and 3.72 to the carbon signals at dC 130.2 (C-13a) and 76.2 (C-13b) in the HMBC spectrum (Fig. 4). Thus, compound 20 was identified as 13b-hydroxymethylevodiamine. Compound 21 was isolated as a white powder. Its HRESIMS gave an [M+H]+ ion at m/z 320.1030 (calcd. 320.1030) consistent with a molecular formula of C18H13N3O3. The UV absorptions of 21 at 221, 245sh, 354, and 373 nm were similar to those of wuzhuyurutine B (23) (Teng and Yang, 2006). The IR spectrum showed the absorptions of N–H (3447 cm1) and carbonyl (1686 and 1654 cm1) groups. The characteristic signals for a typical indole moiety at dH 12.86 (1H, br. s, 13-NH), 8.11 (1H, br. d, J = 8.2 Hz, H-9), 7.28 (1H, td, J = 7.6, 1.0 Hz, H-10), 7.36 (1H, td, J = 7.6, 1.0 Hz, H-11), 7.68 (1H, br. d, J = 8.0 Hz, H-12) were observed in the 1H NMR spectrum. The 1H NMR spectrum also gave resonances for a quinazolinone moiety at dH 7.83 (1H, d, J = 8.0 Hz, H-1), 7.90 (1H, td, J = 8.0, 1.2 Hz, H-2), 7.59 (1H, td, J = 8.0, 1.0 Hz, H-3), 8.20 (1H, dd, J = 8.0, 1.2 Hz, H-4). The NMR signals at dH 4.00 (3H, s, –COOCH3) and dC 167.4 (–COOCH3), 52.4 (–COOCH3) indicated the existence of a methyl carboxylate. A comparison of the 1H and 13C NMR spectroscopic data of compounds 21 and 23 suggested that they were similar compounds. An N–H group was located on the N-14 position instead of N-6 in 21 due to the 3J correlation of the proton at dH 13.79 (N–H) with C-1 (dC 127.5) in the HMBC spectrum. Correspondingly, N-6 and C-13b formed an imine moiety. Thus, the structure of 21 was established and named wuzhuyurutine C.
Compound 22 was isolated as a white powder. Its molecular formula was assigned as C17H11N3O3 by HRESIMS ([M+Na]+ m/z 328.0687, calcd. 328.0693). Its 1H and 13C NMR spectra exhibited 8 aromatic protons, two N–H protons at dH 13.86 and 12.73, a carboxylic proton at dH 14.52, and 17 sp2 carbons. 22 showed the same molecular formula and similar NMR data to 23, indicating that both were a pair of isomers. A comparison of the 1H and 13C NMR spectroscopic data of 22 with those of 21 indicated that they were similar, with a difference in the loss of a methoxyl, the presence of the active proton of a carboxylic acid at dH 14.52, and the down-field shift of the carboxylic carbon at dC 169.3 in 22, suggesting the existence of a free carboxyl instead of the methyl ester in 21. The above deduction was supported by the HMBC correlations (Fig. 4). Therefore, 22 was determined to be an isomer of wuzhuyurutine B and named wuzhuyurutine D. 2.1. Cytotoxicity assay The cytotoxicities of the isolated compounds were evaluated against HL-60 (human leukaemia) and PC-3 (human prostate cancer) cell lines. The results are provided in Table 1. Most of the tested compounds showed cytotoxic activity against HL-60 cell lines, among which compound 19 exhibited the highest activity, even when compared with the positive control (5-Fu). Among the quinolone alkaloids, compounds 4, 8, and 17, with a C13 side-chain, and 16, with a C11 chain, showed stronger cytotoxic activity against HL-60 cell lines than other analogues. Hydroxylation at the terminus of the side-chain (4 and 17) enhanced the activity. The position of a double bond in the C13 side-chain (7 and 8) was shown to affect the activity. It is noteworthy that the
Table 1 The in vitro cytotoxic activities of the isolated compounds against HL-60 and PC-3 cancer cell lines.
a b
Compounds
HL-60 GI50a ± SD (lM)
PC-3 GI50a ± SD (lM)
Compounds
HL-60 GI50a ± SD (lM)
PC-3 GI50a ± SD (lM)
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19
58.13 ± 1.45 >80 20.36 ± 3.44 12.07 ± 2.28 54.10 ± 0.27 54.24 ± 6.07 21.04 ± 0.50 10.44 ± 3.21 30.84 ± 2.62 73.14 ± 0.71 39.28 ± 3.79 41.31 ± 0.53 21.04 ± 0.50 >80 36.72 ± 0.31 10.73 ± 1.20 16.02 ± 1.65 >80 0.51 ± 0.14
>80 >80 31.99 ± 1.98 >80 >80 >80 >80 15.11 ± 2.87 >80 >80 >80 >80 >80 >80 >80 17.61 ± 3.31 >80 >80 14.35 ± 1.58
20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 5-Fub
>80 70.08 ± 1.56 24.88 ± 1.41 >80 71.88 ± 6.13 >80 8.34 ± 2.72 11.94 ± 2.00 >80 >80 >80 20.06 ± 1.67 13.62 ± 1.10 31.39 ± 3.21 57.43 ± 4.21 >80 >80 >80 1.84 ± 0.29
>80 80 46.50 ± 1.22 >80 >80 >80 58.52 ± 3.21 >80 >80 >80 >80 >80 >80 >80 >80 >80 >80 >80 >80
GI50 is the concentration that inhibited 50% of cell growth. The data shown are means ± SD of three independent experiments. Positive control.
Please cite this article in press as: Zhao, N., et al. Quinolone and indole alkaloids from the fruits of Euodia rutaecarpa and their cytotoxicity against two human cancer cell lines. Phytochemistry (2014), http://dx.doi.org/10.1016/j.phytochem.2014.10.020
N. Zhao et al. / Phytochemistry xxx (2014) xxx–xxx
a,b-unsaturated carbonyl at the side-chain (1, 2, 9 and 10) decreased the activity. For indolopyridoquinazoline alkaloids (25–30), a phenolic hydroxyl at ring-E (26 and 27) increased activity. N-methylation (31) and the further reduction of the C–N double bond (19) of the quinazoline moiety (25) increased the activity. Further hydroxymethylation of C-13b resulted in loss of activity (19 and 20). Compounds 8, 16, and 19 showed moderate activities against PC-3 cells. 3. Concluding remarking Seven new alkaloids, including four quinolone, three indole and 30 known alkaloids were isolated from the fruit of E. rutaecarpa. Their structures were elucidated by comprehensive spectroscopic analyses. The a,b-unsaturated carbonyl group in the side-chain of compounds 1, 2, 9, and 10 was originally derived from a allylic hydroxyl, which was unstable in the natural environment and oxidized to a a,b-unsaturated carbonyl (Huang et al., 2012), and unfortunately was not obtained. The two indoloquinazoline alkaloids, 22 and 23, were isomers that showed different chromogenic reactions. Compound 22 showed black–blue colour visualised by Dragendorff’s reagent. Interestingly, compound 22 exhibited moderate inhibitory activity against HL-60 and PC-3 cell lines, while 23 did not show any effects. The cytotoxicities of quinolone alkaloids may be related to the length of the side-chain. The a,b-unsaturated carbonyl at the side-chain decreased the cytotoxicity. The phenolic hydroxyl at ring-E of the indolopyridoquinazoline alkaloids exhibited higher cytotoxicity than those with an unsubstituted ring-E. 4. Experimental 4.1. General experimental procedures Melting points were measured on an Electrothermal MEL-TEMP melting point apparatus without correction. UV spectra were recorded using a Shimadzu UV-2201 spectrometer. IR spectra were obtained on a Bruker IFS-55 spectrometer (using a KBr disk method). NMR spectra were recorded on Bruker ARX-300 and AV-600 NMR spectrometers with TMS as an internal standard. Mass spectra were acquired on a Bruker micrOTOF-Q mass spectrometer (for HRESIMS) and an Agilent 5975 GC mass spectrometer (for EIMS). Column chromatography (CC) was performed with silica gel (Qingdao Haiyang Chemical Co., Ltd., China), ODS (50 lm, YMC Co. Ltd., Kyoto, Japan) and Sephadex LH-20 (GE Healthcare, Sweden). Semi-preparative HPLC was conducted on a YMC ODS-A column (250 20 mm I.D., 5 lm) equipped with an LC-6AD pump and an SPD-20A UV–Vis detector (Shimadzu Co., Ltd., Japan). 4.2. Plant material Plant material was purchased from Yuntian Drugstore, Anguo, Hebei Province, China, in August 2011 and was identified as fruits of E. rutaecarpa (Juss.) Benth by Prof. Jincai Lu (School of Traditional Chinese Materia Medica, Shenyang Pharmaceutical University, Shenyang, China). A voucher sample (WZY-20110910) was deposited in the Department of Natural Products Chemistry, Shenyang Pharmaceutical University, Shenyang, China. 4.3. Extraction and isolation of new compounds The fruits of Euodia rutaecarpa (10.5 kg) were extracted with EtOH–H2O(75:25, v/v, 63 L 3 3 h) under conditions of reflux. After evaporation of the solvent under reduced pressure, the
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residue was suspended in H2O and partitioned with P.E. (3 20 L), CHCl3 (3 20 L), EtOAc (3 20 L) and n-BuOH (3 20 L), successively. The P.E. extract (46 g) was fractionated by CC silica gel using a gradient system of P.E. (60–90 °C)–acetone (100:0 to 100:50, v/v) as an eluent to afford eight fractions (Fr.A– Fr.H). Fr.G was separated into five subfractions (Fr.G1–Fr.G5) by on silica gel CC with a system of P.E.-acetone (100:3, 100:5, 100:7, 100:10, 100:20). Fr.G4 was submitted to Sephadex LH-20 CC followed by semi-preparative RP-HPLC eluted with MeOH– H2O (80:20, v/v, 5.0 mL/min) to afford 14 (9.0 mg, tR 44.0 min), 6 (4.2 mg, tR 54.5 min), 15 (2.9 mg, tR 62.0 min), 11 (10.5 mg, tR 73.5 min), 16 (12.0 mg, tR 87.0 min), and mixture I (tR 107.0 min), which was purified by PTLC to obtain 8 (21.0 mg) and 2 (3.4 mg). Fr. G5 was subjected to Sephadex LH-20 CC eluted with MeOH followed by semi-preparative RP-HPLC eluted with MeOH–H2O (80:20, v/v, 5.0 mL/min) to afford mixture II (tR 56.0 min), which purified by PTLC to obtain 1 (2.1 mg) and 5 (4.6 mg), and 13 (2.3 mg, tR 87.5 min). Fr.F was separated into four fractions (Fr.F1–Fr.F4) by silica gel CC with a gradient solvent system of CH2Cl2–MeOH (100:0, 100:1, 100:3, 100:5, v/v). Fr.F2 was separated by semi-preparative RP-HPLC eluted with MeOH–H2O (80:20, v/v, 5.0 mL/min) to afford 18 (1.9 mg, tR 60.0 min), mixture III (tR 86.0 min), 10 (3.5 mg, tR 92.0 min), 12 (38.1 mg, tR 111.0 min) and 17 (45.0 mg, tR 148.0 min). Mixture III was purified by PTLC to obtain 7 (9.7 mg) and 9 (2.8 mg). Fr.E was subjected to D101 macroporous resin CC eluted with MeOH–H2O (30:70, 60:40, 100:0, v/v) to afford three fractions (Fr.E1–Fr.E3). Fr.E3 was subjected to semi-preparative RP-HPLC (MeOH–H2O 73:27, v/v, 2.0 mL/min) to obtain 28 (6.2 mg, tR 18.0 min). The CHCl3 extract (300 g) was subjected to Al2O3 CC using a gradient system of increasing polarity of CH2Cl2–MeOH (100:0–100:200, v/v). The collected fractions were combined based on their TLC characteristics to yield ten fractions (Fr.I–Fr.R). Fr.K was purified by ODS (MeOH:H2O, 70:30, v/v) to give 30 (5.9 mg). Fr.L was subjected to Sephadex LH-20 to afford 29 (5.8 mg). Fr.M was dissolved in MeOH and filtered to yield the filtrate, which was purified by PTLC to afford 25 (28.2 mg) and 19 (20.7 mg). Fr.N was subjected to Sephadex LH-20 to afford four subfractions (Fr.N1–Fr.N4). Fr.N2 was purified by ODS (MeOH:H2O, 30:70, v/v) to yield 31 (36.3 mg). Fr.N4 was purified by semi-preparative HPLC (MeOH:H2O, 70:30, v/v) to yield 34 (8.0 mg, tR 14.2 min). Fr.O (5.0 g) was separated by ODS CC to yield 3 subfractions (Fr.O1–Fr.O3). Fr.O1 was repeatedly recrystallised in acetone to afford mixture IV, which was further purified by HPLC on a semi-preparative YMC C-18 column using MeOH–H2O (70:30, v/v) as the mobile phase to yield 20 (2.0 mg, tR 20.8 min) and 35 (2.9 mg, tR 23.1 min). Fr.O2 was subjected to HPLC to yield 33 (10.8 mg, tR 12.0 min) and 32 (3.2 mg, tR 18.0 min). Compound 22 (9.8 mg) was obtained from Sephadex LH-20 CC of Fr.O3. Fr.P was separated by ODS CC and eluted with a MeOH–H2O gradient (from 20:80 to 80:20, v/v) to afford four subfractions (Fr.P1–Fr.P4). Fr.P2 was purified by Sephadex LH-20 CC to obtain 36 (12.1 mg) and 37 (6.8 mg). Fr.P3 was subjected to silica gel CC eluted with P.E.-acetone (7:1, v/v) to yield 26 (4.9 mg) and MeOH to yield 4 (4.6 mg). Compound 3 (18.0 mg) was obtained from Fr.P4 via Sephadex LH-20 CC eluted with CH2Cl2–MeOH (1:1, v/v). Fr.Q was separated by ODS CC and eluted with MeOH–H2O (35:65, v/v, Fr.Q1) and MeOH–H2O (40:60, v/v, Fr.Q2). Fr.Q1 was repeatedly recrystallised in acetone to afford 24 (3.9 mg). The remaining mother liquor of Fr.Q1 was purified by HPLC (MeOH:H2O, 75:25, v/v) to give 27 (3.4 mg, tR 29.0 min). The EtOAc extract (102 g) was fractionated by silica gel CC using a gradient system of increasing polarity of CH2Cl2–MeOH (100:0– 100:50, v/v). The collected fractions were combined based on their TLC characteristics to yield four fractions (Fr.S–Fr.V). Fr.T was repeatedly recrystallised in MeOH to yield 21 (15.6 mg) and the
Please cite this article in press as: Zhao, N., et al. Quinolone and indole alkaloids from the fruits of Euodia rutaecarpa and their cytotoxicity against two human cancer cell lines. Phytochemistry (2014), http://dx.doi.org/10.1016/j.phytochem.2014.10.020
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mother liquor was purified by semi-preparative HPLC (MeOH:H2O, 75:25, v/v) to obtain 23 (4.1 mg, tR 29.5 min). 4.3.1. (1) 1-methyl-2-[6-carbonyl-(E)-4-undecenyl]-4(1H)-quinolone Colourless oil; UV (MeOH) kmax (log e): 239 (4.49), 321 (4.15), 334 (4.16) nm; IR (KBr) tmax: 2923, 2851, 1621, 1597, 1567, 1468 cm1; 1H NMR (600 MHz, DMSO-d6): d 8.14 (1H, d, J = 7.9 Hz, H-5), 7.76 (1H, d, J = 8.6 Hz, H-8), 7.70 (1H, dd, J = 8.6, 7.0 Hz, H-7), 7.36 (1H, dd, J = 7.9, 7.0 Hz, H-6), 6,82 (1H, dt, J = 15.8, 6.5 Hz, H-40 ), 6.04 (1H, d, J = 15.8 Hz, H-50 ), 6.01 (1H, s, H-3), 3.72 (3H, s, –NCH3), 2.76 (2H, t, J = 6.8 Hz, H-10 ), 2.55 (2H, m, H-30 ), 2.52 (2H, t, J = 7.1 Hz, H-70 ), 2.26 (1H, m, H-20 b), 2.14 (1H, m, H-20 a), 1.54 (1H, m, H-80 b), 1.43 (1H, m, H-80 a), 1.35 (2H, m, H-90 ), 1.22 (2H, m, H-100 ), 0.83 (3H, t, J = 7.3 Hz, 110 –CH3); 13C NMR (150 MHz, DMSO-d6): d 200.0 (C-60 ), 175.9 (C-4), 155.3 (C2), 147.2 (C-40 ), 141.8 (C-8a), 132.0 (C-7), 130.5 (C-50 ), 126.0 (C4a), 125.3 (C-5), 123.0 (C-6), 116.8 (C-8), 109.9 (C-3), 38.9 (C-70 ), 34.4 (–NCH3), 33.8 (C-10 ), 31.5 (C-30 ), 27.9 (C-80 ), 27.8 (C-20 ), 25.9 (C-90 ), 21.7 (C-100 ), 13.8 (110 -CH3); HRESIMS m/z 326.2117 [M+H]+ (calcd. for C21H28NO2 326.2115). EI-MS m/z 254, 200, 186 (100), 173, 144. 4.3.2. (2) 1-methyl-2-[6-carbonyl-(E)-7-tridecenyl]-4(1H)-quinolone Colourless oil; UV (MeOH) kmax (log e): 239 (4.45), 321 (4.12), 334 (4.13) nm; IR (KBr) tmax: 2922, 2851, 1623, 1598, 1467 cm1; 1H NMR (600 MHz, DMSO-d6): d 8.14 (1H, br d, J = 7.7 Hz, H-5), 7.76 (1H, d, J = 8.6 Hz, H-8), 7.70 (1H, ddd, J = 7.0, 6.8, 1.5 Hz, H-7), 7.36 (1H, dd, J = 7.7, 7.0 Hz, H-6), 6,82 (1H, dt, J = 15.8, 8.4 Hz, H-80 ), 6.06 (1H, d, J = 15.8 Hz, H-70 ), 6.02 (1H, s, H-3), 3.72 (3H, s, –NCH3), 2.75 (2H, t, J = 7.4 Hz, H-10 ), 2.52 (2H, t, J = 6.9 Hz, H-50 ), 2.16 (1H, m, H-90 b), 1.95 (1H, m, H-90 a), 1.60 (2H, m, H-20 ), 1.59 (1H, m, H-40 b), 1.54 (1H, m, H-40 a), 1.42 (2H, m, H-100 ), 1.33 (2H, m, H-30 ), 1.25 (2H, m, H-110 ), 1.23 (2H, m, H-120 ), 0.84 (3H, t, J = 7.9 Hz, 130 -CH3); 13C NMR (150 MHz, DMSO-d6): d 200.0 (C-60 ), 175.9 (C-4), 155.4 (C-2), 147.0 (C-80 ), 141.9 (C-8a), 132.0 (C-7), 130.3 (C-70 ), 126.0 (C-4a), 125.2 (C-5), 123.1 (C-6), 116.8 (C-8), 109.8 (C-3), 39.3 (C-50 ), 34.4 (–NCH3), 33.8 (C-10 ), 31.6 (C-90 ), 28.7 (C-40 ), 28.1 (C-20 ), 28.0 (C-30 ), 27.1 (C-100 ), 26.1 (C-110 ), 21.8 (C-120 ), 13.4 (130 -CH3); HR-ESI-MS m/z 354.2438 [M+H]+ (calcd. for C23H32NO2 354.2438). EI-MS m/z 310, 282, 256, 228, 186 (100), 173, 144. 4.3.3. (3) 1-methyl-2-[15-hydroxyl-pentadecenyl]-4(1H)-quinolone White needles (MeOH); mp. 111–112 °C; UV (MeOH) kmax (log e): 239 (4.43), 321 (4.10), 334 (4.11) nm; IR (KBr) tmax: 3419, 2916, 1618, 1596, 1558, 1468 cm1; 1H NMR (300 MHz, DMSOd6): d 8.14 (1H, dd, J = 7.9, 1.3 Hz, H-5), 7.75 (1H, br d, J = 7.7 Hz, H-8), 7.70 (1H, ddd, J = 8.7, 6.6, 1.6 Hz, H-7), 7.35 (1H, td, J = 7.3, 1.2 Hz, H-6), 6.01 (1H, s, H-3), 4.28 (1H, t, J = 5.3 Hz, –OH), 3.72 (3H, s, –NCH3), 3.36 (2H, J = 6.5, 5.1 Hz, H-150 ), 2.75 (2H, t, J = 7.6 Hz, H-10 ), 1.60 (2H, m, H-20 ), 1.37 (2H, m, H-140 ), 1.32 (2H, m, H-30 ), 1.22 (10H, m, H-40 –H-130 ); 13C NMR (75 MHz, DMSOd6): d 175.8 (C-4), 155.3 (C-2), 141.8 (C-8a), 131.9 (C-7), 126.1 (C-4a), 125.2 (C-5), 122.9 (C-6), 116.9 (C-8), 109.8 (C-3), 60.7 (C150 ), 34.2 (–NCH3), 33.7 (C-10 ), 32.5 (C-140 ), 29.1–28.0 (C-20 –C130 ); HR-ESI-MS m/z 386.3056 [M+H]+ (calcd. for C25H40NO2 386.3054). 4.3.4. (4) 1-methyl-2-[13-hydroxyl-tridecenyl]-4(1H)-quinolone White needles; mp. 109–110 °C; UV (MeOH) kmax (log e): 239 (4.50), 321 (4.15), 334 (4.16) nm; IR (KBr) tmax: 3403, 2918, 2849, 1619, 1598, 1557, 1467 cm1; 1H NMR (600 MHz, CDCl3): d 8.44 (1H, dd, J = 8.0, 1.4 Hz, H-5), 7.67 (1H, td, J = 7.2, 1.5 Hz, H7), 7.52 (1H, d, J = 8.7 Hz, H-8), 7.38 (1H, t, J = 7.2 Hz, H-6), 6.30 (1H, s, H-3), 3.76 (3H, s, –NCH3), 3.63 (2H, t, J = 6.7 Hz, H-130 ), 2.72 (2H, t, J = 7.7 Hz, H-10 ), 1.68 (2H, m, H-20 ), 1.56 (2H, m,
H-120 ), 1.42 (2H, m, H-30 ), 1.331.22 (16H, m, H-40 –H-110 ); 13C NMR (150 MHz, CDCl3): d 177.7 (C-4), 155.2 (C-2), 142.0 (C-8a), 132.3 (C-7), 126.8 (C-5), 126.4 (C-4a), 123.6 (C-6), 115.5 (C-8), 111.2 (C-3), 63.1 (C-130 ), 34.9 (C-10 ), 34.5 (–NCH3), 32.9 (C-120 ), 28.6 (C-20 ), 29.1–28.0 (C-30 –C-100 ), 25.9 (C-110 ); HR-ESI-MS m/z 358.2744 [M+H]+ (calcd. for C23H36NO2 358.2741). 4.3.5. (20) 13b-hydroxymethylevodiamine White powder (MeOH); UV (MeOH) kmax (log e): 226 (4.63), 268 (3.96), 326 (2.79) nm; IR (KBr) tmax: 3415, 3228, 1614, 1595, 1488, 1471.cm1; 1H NMR (600 MHz, DMSO-d6): d 11.25 (1H, br. s, NH), 7.87 (1H, d, J = 7.6 Hz, H-4), 7.52 (1H, d, J = 7.9 Hz, H-9), 7.49 (1H, dd, J = 8.0, 7.3 Hz, H-2), 7.43 (1H, d, J = 8.1 Hz, H-12), 7.14 (1H, m, H-11), 7.11 (2H, m, H-1, 3), 7.03 (1H, dd, J = 7.9, 7.3 Hz, H-10), 5.20 (1H, br t, J = 5.5 Hz, –CH2OH), 4.94 (1H, dd, J = 12.4, 4.5 Hz, Hb-7), 3.92 (1H, dd, J = 11.2, 5.5 Hz, –CH2OH), 3.72 (1H, dd, J = 11.2, 5.5 Hz, –CH2OH), 3.29 (1H, dd, J = 12.4, 3.8 Hz, Ha-7), 2.90 (1H, dd, J = 14.8, 2.7 Hz, Hb-8), 2.70 (1H, m, Ha-8), 2.44 (3H, s, –NCH3); 13C NMR (150 MHz, DMSO-d6): d 161.3 (C-5), 148.9 (C-14a), 136.6 (C-12a), 133.1 (C-2), 130.2 (C-13a), 127.6 (C-4), 125.6 (C-8b), 122.3 (C-3), 122.0 (C-11), 121.9 (C-4a), 121.3 (C-1), 118.7 (C-10), 118.3 (C-9), 113.0 (C-8a), 111.7 (C-12), 76.2 (C13b), 64.9 (–CH2OH), 40.1 (C-7), 38.4 (–NCH3), 20.4 (C-8); HRESI-MS m/z 356.1363 [M+Na]+ (calcd. for C20H19N3NaO2 356.1369). 4.3.6. (21) wuzhuyurutine C White powder (MeOH); UV (MeOH) kmax (log e): 221 (4.29), 245sh (4.16), 354 (4.19), 373 (4.03) nm; IR (KBr) tmax: 3447, 1686, 1654, 1583, 1464, 1446 cm1; 1H NMR (600 MHz, DMSOd6): d 13.79 (1H, br. s, 14-NH), 12.86 (1H, br. s, 13-NH), 8.20 (1H, dd, J = 8.0, 1.2 Hz, H-4), 8.11 (1H, br. d, J = 8.2 Hz, H-9), 7.90 (1H, td, J = 8.0, 1.2 Hz, H-2), 7.83 (1H, d, J = 8.0 Hz, H-1), 7.68 (1H, br. d, J = 8.0 Hz, H-12), 7.59 (1H, td, J = 8.0, 1.0 Hz, H-3), 7.36 (1H, td, J = 7.6, 1.0 Hz, H-11), 7.28 (1H, td, J = 7.6, 1.0 Hz, H-10), 4.00 (3H, s, –COOCH3); 13C NMR (150 MHz, DMSO-d6): d 167.4 (C-7), 160.8 (C-5), 148.4 (C-14a), 146.0 (C-13b), 135.6 (C-12a), 134.8 (C-2), 134.6 (C-13a), 127.5 (C-1), 127.2 (C-3), 126.5 (C-8b), 126.1 (C-4), 124.9 (C-11), 122.6 (C-10), 122.4 (C-9), 121.6 (C-4a), 113.1 (C12), 106.3 (C-8a), 52.4 (–COOCH3); HR-ESI-MS m/z 319.1030 [M+H]+ (calcd. for C18H14N3O3 320.1030). 4.3.7. (22) wuzhuyurutine D White powder (MeOH); UV (MeOH) kmax (log e): 221 (4.46), 245sh (4.28), 347 (4.35), 363 (4.21) nm; IR (KBr) tmax: 3402, 3401, 1664, 1584 cm1; 1H NMR (300 MHz, DMSO-d6): d 14.52 (1H, br. s, –COOH), 13.86 (1H, br. s, 14-NH), 12.73 (1H, br. s, 13NH), 8.19 (2H, br d, J = 8.0 Hz, H-4, 9), 7.90 (1H, td, J = 8.0, 1.0 Hz, H-2), 7.84 (1H, d, J = 7.6 Hz, H-1), 7.69 (1H, d, J = 7.8 Hz, H-12), 7.57 (1H, td, J = 8.0, 1.0 Hz, H-3), 7.35 (1H, dd, J = 7.8, 7.1 Hz, H11), 7.26 (1H, dd, J = 8.2, 7.3 Hz, H-10); 13C NMR (150 MHz, DMSO-d6): d 169.3 (C-7), 161.0 (C-5), 148.5 (C-14a), 146.2 (C13b), 135.7 (C-12a), 134.8 (C-2), 134.4 (C-13a), 127.4 (C-18b), 127.3 (C-1), 127.1 (C-3), 126.1 (C-4), 124.9 (C-11), 122.5 (C-10), 122.7 (C-9), 121.7 (C-4a), 113.1 (C-12), 107.7 (C-8a); HR-ESI-MS m/z 328.0687 [M+Na]+ (calcd. 328.0693). 4.4. Cell culture and growth inhibition assay The human leukaemia cell line HL-60 and the human prostate cancer PC-3 cell line were purchased from the American Type Culture Collection (ATCC Rockville, MD, USA) and grown in RPMI-1640 medium (Gibco, New York, NY, USA) supplied with 100 IU/mL penicillin, 100 lg/mL streptomycin, 1 mM D-glutamine and 10% heat-inactivated foetal bovine serum (Gibco). Cells were incubated in standard conditions (37 °C under 5% CO2 and humidified atmosphere).
Please cite this article in press as: Zhao, N., et al. Quinolone and indole alkaloids from the fruits of Euodia rutaecarpa and their cytotoxicity against two human cancer cell lines. Phytochemistry (2014), http://dx.doi.org/10.1016/j.phytochem.2014.10.020
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HL-60 cell viability was determined by the trypan blue exclusion test (Wang et al., 2006). Briefly, cells in logarithmic growth were dispensed into 24-well microplates at 4 104 cells/mL and incubated with various concentrations of the test compounds at 37 °C under 5% CO2 and 95% air for 3 days. The compounds were dissolved in DMSO and then diluted to the proper concentrations. Cell viability was determined after staining the cells, and the total cell number was determined using a hematocytometer. PC-3 cell viability was determined by an MTT assay (Plumb et al., 1989). Briefly, cells were dispensed into microtiter plates at 2 104 cells/mL in 1 mL of 1640 medium/well and incubated overnight at 37 °C in 5% CO2 and 95% air. The test compounds were diluted with fresh medium and placed into microtiter plates in a total volume of 200 ll. Untreated control cultures were maintained under the same conditions. A total of 50 lL of a 0.4% MTT solution (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide; Thiazolyl blue) was added to each well of the plate. After 3–4 h incubation, MTT was removed from the wells and the formazan crystals were dissolved in DMSO (200 lL) and added to each well. The plates were then shaken for 10 min. The OD was read using in a microplate reader (Bio-RAD) at 570 nm. The growth inhibition in cells after the treatment with different concentration (GI50) was obtained from a regression analysis of the concentration response data. 5-Fluorouracil (5-Fu) was used as the positive control, and 0.1% DMSO was used as the negative control. Three independent experiments were duplicated, and the data are presented as the mean ± SD. Acknowledgements The authors wish to thank the financial support from the National Natural Science Foundation of China (No. 81172958) and the National Key Technology R&D Program (2012BAI30B02). We are grateful to Mr. Yi Sha and Mrs. Wen Li (Analytical Testing Center, Shenyang Pharmaceutical University, Shenyang, People’s Republic of China) for their measurements of the NMR data. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.phytochem.2014. 10.020. References Adams, M., Kunert, O., Haslinger, E., Bauer, R., 2004. Inhibition of leukotriene biosynthesis by quinlone alkaloids from the fruits of Evodia rutaecarpa. Planta Med. 70, 904–908. Chinese Pharmacopoeia Commission, 2010. Pharmacopoeia of the People’s Republic of China, vol. I. China Medical Science and Technology Press, Beijing, p. 160. Huang, L., Yu, D.Q., 1988. Application of Ultraviolet Spectrum in Organic Chemistry (ii). Science Press, Beijing, p. 100. Huang, X., Li, W., Yang, X.W., 2012. New cytotoxic quinolone alkaloids from fruits of Evodia rutaecarpa. Fitoterapia 83, 709–714.
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Please cite this article in press as: Zhao, N., et al. Quinolone and indole alkaloids from the fruits of Euodia rutaecarpa and their cytotoxicity against two human cancer cell lines. Phytochemistry (2014), http://dx.doi.org/10.1016/j.phytochem.2014.10.020
