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Design, synthesis and biological evaluation of E-ring modified evodiamine derivatives as novel antitumor agents
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CCLET-2929; No. of Pages 5 Chinese Chemical Letters xxx (2014) xxx–xxx
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Original article
Design, synthesis and biological evaluation of E-ring modified evodiamine derivatives as novel antitumor agents Kun Fang a,1, Guo-Qiang Dong a,1, Hai Gong b,1, Na Liu a, Zhen-Gang Li a, Shi-Ping Zhu a, Zhen-Yuan Miao a, Jian-Zhong Yao a, Wan-Nian Zhang a, Chun-Quan Sheng a,* a b
School of Pharmacy, Second Military Medical University, Shanghai 200433, China Department of Radiation Oncology, Ji’nan Military General Hospital, Jinan 250031, China
A R T I C L E I N F O
A B S T R A C T
Article history: Received 4 December 2013 Received in revised form 6 March 2014 Accepted 7 March 2014 Available online xxx
A series of novel E-ring modified evodiamine derivatives were designed and synthesized as antitumor agents. Their capacity to interfere with the catalytic activity of topoisomerase I and II was evaluated by the relaxation assay. In vitro antitumor activity results revealed that compound 12 showed good antitumor activity with a broad spectrum. Its binding modes with topoisomerase I and II were clarified by molecular docking. ß 2014 Chun-Quan Sheng. Published by Elsevier B.V. on behalf of Chinese Chemical Society. All rights reserved.
Keywords: Evodiamine derivatives Topoisomerase I Topoisomerase II Antitumor activity
1. Introduction The use of effective antitumor agents derived from natural products is a promising strategy for cancer chemotherapy [1]. It is reported that more than 50% of antitumor drugs have been developed from natural products or their synthetic derivatives [2,3]. Antitumor natural products also provide novel probes for chemical biology and target identification studies. For example, camptothecin (target: DNA topoisomerase I, Top1) and taxol (target: microtubule) represent two historic achievements in antitumor drug discovery [4]. Evodiamine is a quinazolinocarboline alkaloid with diverse biological activities which was isolated from the fruits of the traditional Chinese herb Evodiae fructus (Chinese name: Wu-ChuYu) [5]. In particular, evodiamine showed anti-proliferative activities against a wide variety of cancer cells by inducing apoptosis [6]. It is a good antitumor lead compound because of its broad-antitumor spectrum, low toxicity, and suitable physicochemical properties. In our previous studies, evodiamine was
* Corresponding author. E-mail address: [email protected] (C.-Q. Sheng). 1 These authors contributed equally to this article.
identified as a Top1 inhibitor by structure-based virtual screening [7]. Further structural optimization studies identified several highly active evodiamine derivatives which showed potent in vitro and in vivo antitumor activities [8]. In silico target identification and biological assays revealed that the evodiamine derivatives were dual inhibitors of Top1 and topoisomerase II (Top2) [8]. Inspired by these encouraging results, herein a series of E-ring modified evodiamine derivatives were designed and synthesized. Compound 12 showed good antitumor activity with a broad spectrum. Its binding modes with topoisomerase I and II were clarified by molecular docking. 2. Experimental Nuclear magnetic resonance (NMR) spectra were recorded on Bruker AVANCE300 and AVANCE500 spectrometers (Bruker Company, Germany), with TMS as an internal standard and DMSO-d6 as the solvent. Chemical shifts (d values) and coupling constants (J values) are expressed in ppm and Hz, respectively. ESI mass spectra were performed on an API-3000 LC–MS spectrometer. TLC analysis was carried out on silica gel plates GF254 (Qindao Haiyang Chemical, China). Silica gel column chromatography was performed with Silica gel 60G (Qindao Haiyang Chemical, China). Commercial solvents were used without any pretreatment.
http://dx.doi.org/10.1016/j.cclet.2014.03.043 1001-8417/ß 2014 Chun-Quan Sheng. Published by Elsevier B.V. on behalf of Chinese Chemical Society. All rights reserved.
Please cite this article in press as: K. Fang, et al., Design, synthesis and biological evaluation of E-ring modified evodiamine derivatives as novel antitumor agents, Chin. Chem. Lett. (2014), http://dx.doi.org/10.1016/j.cclet.2014.03.043
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Scheme 1. Synthetic routes of the target compounds. Reagents and conditions: (a) Ethyl formate, reflux, 6 h, yield 100%; (b) POCl3, CH2Cl2, 0 8C, 6 h, yield 85%; (c) Triphosgene, THF, reflux, 4 h, yield 90%-95%; (d) CH3I, KOH, DMF, rt, 2 h, yield 60–87%; (e) CH2Cl2, 45 8C, 6 h, yield 50%-62%; (f) 10% Pd/C, H2, DMF, r.t., 12 h, yield 11–73%; (g) 2-bromoacetyl bromide, Et3N, CH2Cl2, 0 8C, 1 h, yield 84%; (h) Na2S9H2O, DMF, r.t., 12 h, yield 70%; (i) 2-(1-piperazinyl)pyridine-4-boronic acid pinacol ester, Pd(PPh3)4, Na2CO3, H2O, reflux, 3.5 h, yield 51%.
2.1. Chemistry The synthetic route of E-ring modified evodiamine derivatives is described in Scheme 1. 3,4-Dihydro-b-carboline (4) was prepared by reacting tryptamine 2 with ethyl formate, followed by intramolecular ring closure in the presence of POCl3. Substituted 2aminobenzoic acid (5) was treated with triphosgene to provide isatoic anhydride, which was methylated by CH3I to afford compound 7 [8]. Condensation of 3,4-dihydro-b-carboline (4) with N-methyl isatoic anhydride 7 provided E-ring derivatives 8a–c. Reduction of the nitro group of compounds 8a and 8b using 10% Pd/C and H2 afforded the amino derivatives 9a and 9b. Compound 9a was further acylated to give compound 10, which was subsequently treated with Na2S to provide compound 11. The Suzuki-Miyaura reaction of compound 8c with 2-(1-piperazinyl)pyridine-4-boronic acid pinacol ester afforded the target compound 12. Synthesis of 3-nitroevodiamine (8a): A solution of 3,4-dihydrob-carboline [8] (4, 0.17 g, 1 mmol) and 1-methyl-6-nitro-1Hbenzo[d][1,3]oxazine-2,4-dione [8] (7a, 0.2 g, 1 mmol) in CH2Cl2 (15 mL) were heated to 45 8C and stirred for 6 h. Then the reaction mixture was concentrated to one-half of its original volume. The precipitate was collected by filtration and washed with alcohol to yield the product 8a (0.21 g, 62%) as yellow solid. 1H NMR (300 MHz, DMSO-d6): d 2.68–2.70 (m, 1 H), 2.95–3.01 (m, 2H), 3.45 (s, 3H), 4.60–4.64 (m, 1H), 6.50 (s, 1H), 6.96–7.11 (m, 4H), 7.32 (d, 1H, J = 7.8 Hz), 7.42 (d, 1H, J = 7.8 Hz), 8.24 (dd, 1H, J = 9.0 Hz, 2.7 Hz), 8.43 (d, 1H, J = 2.7 Hz), 10.89 (s, 1H). ESI-MS: Calcd. for C19H16N4O3 [M H] : m/z 347.12; found: 347.76. The synthetic method for target compounds 8b–c was similar to that of compound 8a. 2-Chloro-3-nitroevodiamine (8b): Yellow solid (0.33 g, yield: 50%). 1H NMR (300 MHz, DMSO-d6): d 2.68–2.73 (m, 1H), 2.95–3.01 (m, 2H), 3.46 (s, 3H), 4.61–4.67 (m, 1H), 6.52 (s, 1H), 7.00–7.16
(m, 3H), 7.35 (d, 1H, J = 7.5 Hz), 7.43 (d, 1H, J = 7.5 Hz), 8.37 (s, 1H), 10.90 (s, 1H). ESI-MS: Calcd. for C19H14ClN4O3 [M H] : m/z 381.79; found: 381.81. 3-Iodoevodiamine (8c): White solid (0.07 g, yield: 50%). 1 H NMR (500 MHz, DMSO-d6): d 2.75–2.78 (m, 1H), 2.91–2.92 (m, 1H), 2.96 (s, 3H), 3.19–3.21 (m, 1H), 4.58–4.62 (m, 1H), 6.17 (s, 1H), 6.87 (d, 1H, J = 8.6 Hz), 7.01 (t, 1H, J = 7.6 Hz), 7.11 (t, 1H, J = 7.6 Hz), 7.35 (d, 1H, J = 8.1 Hz), 7.46 (d, 1H, J = 8.1 Hz), 7.45 (dd, 1H, J = 8.6 Hz, 2.1 Hz), 7.98 (d, 1H, J = 2.1 Hz), 11.03 (s, 1H). ESI-MS: Calcd. for C19H17IN3O [M+H]+: m/z 430.26; found: 430.39. Synthesis of 3-aminoevodiamine (9a): To a stirring solution of 8a (0.12 g, 0.34 mmol) in DMF (10 mL), 10% Pd/C (0.01 g) was added. The reaction mixture was stirred under hydrogen atmosphere for 12 h at room temperature. Then the mixture was diluted with water (50 mL) and extracted with EtOAc (3 50 mL). The combined organic layers were washed with saturated sodium chloride solution (3 50 mL), dried over anhydrous Na2SO4 and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (CH2Cl2–MeOH, gradient 100:1, 100:2) to give compound 9a (0.08 g, 73%) as a yellow solid. 1H NMR (500 MHz, DMSO-d6): d 2.27 (s, 3H), 2.77–2.79 (m, 1H), 2.88–2.92 (m, 1H), 3.11–3.13 (m, 1H), 4.63–4.67 (m, 1H), 5.10 (s, 2H), 5.90 (s, 1H), 6.78 (dd, 1H, J = 8.5 Hz, 2.5 Hz), 6.94 (d, 1H, J = 8.5 Hz), 7.01 (t, 1H, J = 7.5 Hz), 7.10–7.14 (m, 2H), 7.36 (d, 1H, J = 8.0 Hz), 7.51 (d, 1H, J = 8.0 Hz), 11.26 (s, 1H). ESI-MS: Calcd. for C19H19N4O [M+H]+: m/z 319.15; found: 319.93. The synthetic method for target compounds 9b was similar to that of compound 9a. 2-Chloro-3-aminoevodiamine (9b): Red solid (0.09 g, yield: 11%). 1H NMR (500 MHz, DMSO-d6): d 2.27 (s, 3H), 2.73–2.77 (m, 2H), 3.11–3.13 (m, 1H), 4.63–4.66 (m, 1H), 5.10 (s, 2H), 5.90 (s, 1H), 6.78 (dd, 1H, J = 8.5 Hz, 2.5 Hz), 6.94 (d, 1H, J = 8.5 Hz), 7.01 (t, 1H, J = 7.5 Hz), 7.10–7.14 (m, 2H), 7.36 (d, 1H, J = 8.0 Hz), 11.26 (s, 1H).
Please cite this article in press as: K. Fang, et al., Design, synthesis and biological evaluation of E-ring modified evodiamine derivatives as novel antitumor agents, Chin. Chem. Lett. (2014), http://dx.doi.org/10.1016/j.cclet.2014.03.043
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ESI-MS: Calcd. for C19H18ClN4O [M+H]+: m/z 353.83; found: 353.49. Synthesis of 2-bromoacetamidoevodiamine (10): To a stirring solution of 9a (0.1 g, 0.3 mmol) and Et3N (0.04 mL, 0.3 mmol) in CH2Cl2 (20 mL), 2-bromoacetyl bromide (0.04 mL, 0.47 mmol) was added and stirred for 1 h at 0 8C. The solvent was removed under reduced pressure. The residue was purified by silica gel column chromatography (CH2Cl2:MeOH = 100:1, v/v) to afford 10 (0.11 g, yield 84%) as yellow solid. 1H NMR (300 MHz, DMSO-d6): d 2.69 (s, 3H), 2.85 (m, 2H), 3.15 (m, 1H), 4.01 (s, 2H), 4.62 (m, 1H), 6.07 (s, 1H), 7.00 (t, 1H, J = 7.8 Hz), 7.08–7.13 (m, 2H), 7.35 (d, 1H, J = 7.8 Hz), 7.48 (d, 1H, J = 7.8 Hz), 7.73 (dd, 1H, J = 9.0 Hz, 2.4 Hz), 8.03 (d, 1H, J = 2.7 Hz), 10.41 (s, 1H), 11.16 (s, 1H). ESI-MS: Calcd. for C21H19BrN4O2 [M+H]+: m/z 439.07; found: 439.75. Synthesis of 2-mercaptoacetamidoevodiamine (11): A solution of 10 (0.05 g, 0.11 mmol) and Na2S9H2O (0.27 g, 1.1 mmol) in DMF (5 mL) was stirred at room temperature for 12 h. The solvent was removed in vacuo and the residue was diluted with water and filtered from insolubles. The filtrate was acidified to pH 1.2 with 3 mol/L HCl. The precipitate was collected and purified by silica gel column chromatography (CH2Cl2–MeOH, gradient 100:1– 100:3) to give compound 11 (0.03 g, yield 70%) as a yellow solid. 1 H NMR (300 MHz, DMSO-d6): d 1.30 (s, 1 H), 2.66 (s, 3H), 2.86 (m, 2H), 3.20 (m, 1H), 3.48 (s, 2H), 4.64 (m, 1H), 6.06 (s, 1H), 7.01 (t, 1H, J = 7.2 Hz), 7.10–7.15 (m, 2H), 7.36 (d, 1H, J = 8.1 Hz), 7.50 (d, 1H, J = 8.1 Hz), 7.73 (d, 1H, J = 9.3 Hz), 8.07 (s, 1H), 10.19 (s, 1H), 11.19 (s, 1H). ESI-MS: Calcd. for C21H20N4O2S [M + H]+: m/z 393.14; found: 393.84. Synthesis of 3-(2-(piperazin-1-yl)pyridin-4-yl)evodiamine (12): Under an nitrogen atomosphere, 8c (0.1 g, 0.23 mmol), 2(1-piperazinyl)pyridine-4-boronic acid pinacol ester (0.1 g, 0.35 mmol) and tetrakistriphenylphosphine palladium (40 mg) were dissolved in DMF (5 mL). An aqueous sodium carbonate solution (2 mol/L, 1 mL) was added and the mixture was heated under reflux for 3.5 h. After cooled to room temperature, the reaction mixture was diluted with water (50 mL) and then extracted with EtOAc (3 50 mL). The combined organic layers were washed with saturated sodium chloride solution (3 50 mL), dried over anhydrous Na2SO4 and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (CH2Cl2:MeOH = 100:1, v/v) to give compound 12 (0.056 g, yield 51%) as a yellow solid. 1H NMR (300 MHz, DMSO-d6): d 2.15 (s, 3H), 2.82 (m, 1H), 3.01 (m, 1H), 3.16 (m, 1H), 3.74 (s, 3H), 3.81 (s, 3H), 4.64 (m, 1H), 6.25 (s, 1H), 6.85 (d, 1H, J = 9.3 Hz), 6.92 (s, 1H), 6.99 (t, 1H, J = 7.8 Hz), 7.05–7.11 (m, 2H), 7.34 (d, 1H, J = 7.8 Hz), 7.44 (d, 1H, J = 7.8 Hz), 7.85 (dd, 1H, J = 8.7 Hz, 2.1 Hz), 8.04 (d, 1H, J = 2.1 Hz), 8.09 (d, 1H, J = 5.1 Hz), 10.99 (s, 1H). ESI-MS: Calcd. for C28H28N6O [M+H]+: m/z 465.24; found: 465.84. 2.2. Biological assay Top1-mediated supercoiled pBR322 relaxation assay: Relaxation assays were carried out as previously described [7,8]. The reaction buffer contained 35 mmol/L Tris–HCl (pH 8.0), 72 mmol/L KCl, 5 mmol/L MgCl2, 5 mmol/L dithiothreitol, 5 mmol/L spermidine, 0.1% bovine serum albumin (BSA), 0.25 mg pBR322 plasmid DNA, and 1 unit of Top1 (TaKaRa Biotechnology Co., Ltd., Dalian) in a total volume of 20 mL. After incubation at 37 8C for 15 min, the reaction was stopped by the addition of 2 mL of 10 loading buffer (0.9% sodium dodecyl sulfate (SDS), 0.05% bromophenol blue, and 50% glycerol). Then, the DNA samples were subjected to electrophoresis in 0.8% agarose gel in TAE (Tris–acetate–EDTA) at 8 V/cm for 1 h. Gels were stained with ethidium bromide (0.5 mg/mL) for 60 min. The DNA band was visualized over UV light and photographed with Gel Doc Ez imager software (Bio-Rad Laboratories Ltd.).
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Top2a-mediated supercoiled pBR322 relaxation assay: Relaxation assays were performed using Topoisomerase II Drug Screening Kits (TopoGEN, Inc.) [8]. The reaction buffer contained 50 mmol/L Tris–HCl (pH 8.0), 150 mmol/L NaCl, 10 mmol/L MgCl2, 5 mmol/L dithiothreitol, 30 mg/mL bovine serum albumin (BSA), 2 mmol/L ATP, 0.25 mg pBR322 plasmid DNA, and 0.75 unit of Top2a (TopoGEN, Inc.) in a total volume of 20 mL. After incubation at 37 8C for 30 min, the reaction was terminated by the addition of 2 mL 10 gel loading buffer (0.25% bromophenol blue, 50% glycerol). The reaction products were analyzed on 1% agarose gel at 8 V/cm for 1 h with TAE (Tris–acetate–EDTA) as the running buffer. Gels were stained with ethidium bromide (0.5 mg/mL) for 60 min. The DNA band was visualized over UV light and photographed with Gel Doc Ez imager software (Bio-Rad Laboratories Ltd.). In vitro cytotoxicity assay: Cells were seeded in 96-well microtiter plates at a density of 5 103/well and treated in triplicate with gradient concentrations of compounds in a humidified atmosphere with 5% CO2 at 37 8C for 72 h. 20 mL of MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) solution (5 mg/mL) was added to each well and the plate was incubated for an additional 4 h. The formazan was dissolved in 100 mL of DMSO. The absorbance (OD) was read on a WellscanMK2 microplate reader (Labsystems) at 570 nm. The concentration causing 50% inhibition of cell growth (IC50) was determined by the Logit method [9,10]. All experiments were performed three times. 2.3. Molecular docking In silico docking was performed using GOLD 5.1 [11] on a Linux PC as described before [8]. The X-ray crystallographic structures of CPT-DNA-Top1 ternary complex (PDB code: 1T8I [12]) and ATPase domain of human Top2a (PDB code: 1ZXM [13]) were obtained from the Protein Data Bank. They were prepared for molecular docking analysis in Discovery Studio 3.0 [14] by removing the ligand from the binding pocket, merging nonpolar hydrogens, adding polar hydrogens, and rendering Gasteiger charges to each atom. 3. Results and discussion 3.1. In vitro antitumor activity The growth inhibitory activities of the target compounds toward human cancer cell lines A549 (lung cancer), MDA-MB-435 (breast cancer), and HCT116 (colon cancer) were determined using MTT assay. Evodiamine was used as the reference drug. When the 2bromoacetamide group was attached on the position 3 of evodiamine E-ring, compound 10 showed moderate in vitro antitumor activity against MDA-MB-435 (IC50 = 22.69 mmol/L), A549 (IC50 = 153.97 mmol/L) and HCT 116 (IC50 = 28 mmol/L) cell lines (Table 1). However, the loss of the antitumor activity was observed for the corresponding thio-derivative 11. 2-Chloro-3-nitro evodiamine (8b) also showed moderate antitumor activity against all three of the tested cancer cell lines. When its nitro group was reduced to the amino group, compound 9b showed significantly Table 1 In vitro antitumor activity of evodiamine derivatives (IC50, mmol/L). Compounds
8b 9b 10 11 12 Evodiamine
Structure
IC50 (mmol/L) MDA-MB-435
A549
HCT116
31.37 1.35 22.69 >250 2.78 20.20
204.15 >250 153.97 >250 6.80 100.00
35.97 75.19 28.00 >250 5.94 100.00
Please cite this article in press as: K. Fang, et al., Design, synthesis and biological evaluation of E-ring modified evodiamine derivatives as novel antitumor agents, Chin. Chem. Lett. (2014), http://dx.doi.org/10.1016/j.cclet.2014.03.043
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improved activity against the MDA-MB-435 cell line (IC50 = 1.35 mmol/L). Interestingly, the compound with a 2-(piperazin-1-yl)pyridyl group at the position 3 of evodiamine E-ring (compound 12) showed good antitumor activity with a broad spectrum. The IC50 values of compound 12 against MDA-MB-435, A549 and HCT 116 cell lines were 2.78 mmol/L, 6.80 mmol/L and 5.94 mmol/L, respectively. As compared with evodiamine, most of the target compounds showed better antitumor activity and broader spectra. 3.2. Top1/Top2 inhibitory activity Human Top2 can be classified into two subgroups, Top2a and Top2b. Top2a increases its expression during the cell cycle and mitosis, and its concentration is much higher in tumor cells. Top2b expression remains relatively constant and at low level throughout the cell cycle [15,16]. Thus, Top2a is a good target for antitumor drug discovery. Previously, evodiamine and its derivatives were identified as dual Top1/Top2 inhibitors [8]. Herein, Top1- and Top2amediated pBR322 DNA relaxation assays with purified Top1 and Top2a were used to investigate the inhibitory activity of compound 12 with its concentrations from 10 mmol/L to 100 mmol/L. Camptothecin (CPT) and etoposide (Eto) were employed as positive controls. As shown in Fig. 1A, compound 12 inhibited Top1mediated relaxation of pBR322 DNA in a concentration-dependent manner. It showed moderate inhibitory activity at 100 mmol/L and lower activity at 50 mmol/L. The inhibitory activity of compound 12 was much higher than evodiamine, which only showed marginal activity at 100 mmol/L [7]. Compound 12 was also proven to be a potent inhibitor of the catalytic activity of Top2a (Fig. 1B). Particularly, it showed comparable inhibitory activity against Top2-mediated DNA relaxation to that of Eto at 100 mmol/L and 50 mmol/L, respectively. 3.3. Binding mode with Top1/Top2 In an attempt to understand the binding mode of compound 12 to Top1-DNA complex and ATP-binding domain of Top2a, docking simulation was performed. As indicated in the interaction model between 12 and Top1-DNA complex (Fig. 2A), its E-ring and pyridyl group intercalates at the site of DNA cleavage and forms p-stacking interactions with both the 1 (upstream) and +1 (downstream) base pairs. The N atom of pyridine forms a hydrogen bond with the hydroxyl group of Thr718. AB-ring is faced into the major groove and its indole NH undergoes a strong hydrogen bond interaction with the oxygen atom of Guanine. Moreover, compound 12 is
Fig. 1. Top1 and Top2 inhibitory activity of compound 12. (A) Inhibition of Top1 relaxation activity ranging from 10 mmol/L to 100 mmol/L. Lane 1, supercoiled plasmid DNA; Lane 2, DNA + Top1; Lanes 3–5, DNA + Top1 + CPT (100, 50 and 10 mmol/L); Lanes 6–8, DNA + Top1 + 12 (100, 50 and 10 mmol/L). (B) Inhibition of Top2 relaxation activity ranging from 10 mmol/L to 100 mmol/L. Lane 1, supercoiled plasmid DNA; Lane 2, DNA + Top2; Lanes 3–5, DNA + Top1 + Eto (100, 50 and 10 mmol/L); Lanes 6–8, DNA + Top2 + 12 (100, 50 and 10 mmol/L). Lanes order from left to right.
observed to be well-fitted into the ATP-binding domain of Top2a (Fig. 2B). It is capable of making four hydrogen-bonds to enhance the stabilization of the 12-ATPase complex. The N atom of pyridine is shown to participate in hydrogen bonding interactions with Ala167 and Lys168, respectively. The amino group on the piperazine also can form two hydrogen bonds with backbone amide of Arg162 and Asn163. ChemPLP score was also calculated to evaluate the binding affinity. The most active compound 12 showed the highest binding affinity to Top1 and Top2a with ChemPLP sore 82.54 and 61.00, respectively. In contrast, removal of the piperazine group led to the decrease of the binding affinity (ChemPLP sore: 68.04 for Top1 and 54.48 for Top2a). Evodiamine showed the lowest ChemPLP scores (Top1: 64.45, Top2a: 53.34). The computational results were consistent with the biological activities and highlighted the importance of the pyridine and piperazine group for Top1 and Top2a binding.
Fig. 2. Binding mode with Top1/Top2. (A) Binding mode of 12 to Top1–DNA complex. (B) Binding mode of 12 to ATP-binding domain of Top2. The figure was generated using PyMol (http://www.pymol.org/).
Please cite this article in press as: K. Fang, et al., Design, synthesis and biological evaluation of E-ring modified evodiamine derivatives as novel antitumor agents, Chin. Chem. Lett. (2014), http://dx.doi.org/10.1016/j.cclet.2014.03.043
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4. Conclusion In summary, a series of novel E-ring modified evodiamine derivatives were designed and synthesized as antitumor agents. Top1/Top2 inhibitory assay and further molecular docking confirmed that the derivatives acted by dual inhibition of Top1 and Top2. As compared with evodiamine, most of the compounds showed better antitumor activity and broader spectra. Compound 12 showed good antitumor activity with an IC50 ranging from 2.78 mmol/L to 5.94 mmol/L. It can be used as a good starting point for further optimization. Acknowledgments This work was supported by National Natural Science Foundation of China (Nos. 81222044, 81373278), Key Project of Science and Technology of Shanghai (No. 11431920402), the National Basic Research Program of China (No. 2014CB541800), Shanghai RisingStar Program (No. 12QH1402600), and Shanghai Municipal Health Bureau (No. XYQ2011038). References [1] G.M. Cragg, P.G. Grothaus, D.J. Newman, Impact of natural products on developing new anti-cancer agents, Chem. Rev. 109 (2009) 3012–3043. [2] D.J. Newman, G.M. Cragg, Natural products as sources of new drugs over the 30 years from 1981 to 2010, J. Nat. Prod. 75 (2012) 311–335. [3] D.J. Newman, G.M. Cragg, Natural products as sources of new drugs over the last 25 years, J. Nat. Prod. 70 (2007) 461–477.
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Please cite this article in press as: K. Fang, et al., Design, synthesis and biological evaluation of E-ring modified evodiamine derivatives as novel antitumor agents, Chin. Chem. Lett. (2014), http://dx.doi.org/10.1016/j.cclet.2014.03.043
CCLET-2929; No. of Pages 5 Chinese Chemical Letters xxx (2014) xxx–xxx
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Chinese Chemical Letters journal homepage: www.elsevier.com/locate/cclet
Original article
Design, synthesis and biological evaluation of E-ring modified evodiamine derivatives as novel antitumor agents Kun Fang a,1, Guo-Qiang Dong a,1, Hai Gong b,1, Na Liu a, Zhen-Gang Li a, Shi-Ping Zhu a, Zhen-Yuan Miao a, Jian-Zhong Yao a, Wan-Nian Zhang a, Chun-Quan Sheng a,* a b
School of Pharmacy, Second Military Medical University, Shanghai 200433, China Department of Radiation Oncology, Ji’nan Military General Hospital, Jinan 250031, China
A R T I C L E I N F O
A B S T R A C T
Article history: Received 4 December 2013 Received in revised form 6 March 2014 Accepted 7 March 2014 Available online xxx
A series of novel E-ring modified evodiamine derivatives were designed and synthesized as antitumor agents. Their capacity to interfere with the catalytic activity of topoisomerase I and II was evaluated by the relaxation assay. In vitro antitumor activity results revealed that compound 12 showed good antitumor activity with a broad spectrum. Its binding modes with topoisomerase I and II were clarified by molecular docking. ß 2014 Chun-Quan Sheng. Published by Elsevier B.V. on behalf of Chinese Chemical Society. All rights reserved.
Keywords: Evodiamine derivatives Topoisomerase I Topoisomerase II Antitumor activity
1. Introduction The use of effective antitumor agents derived from natural products is a promising strategy for cancer chemotherapy [1]. It is reported that more than 50% of antitumor drugs have been developed from natural products or their synthetic derivatives [2,3]. Antitumor natural products also provide novel probes for chemical biology and target identification studies. For example, camptothecin (target: DNA topoisomerase I, Top1) and taxol (target: microtubule) represent two historic achievements in antitumor drug discovery [4]. Evodiamine is a quinazolinocarboline alkaloid with diverse biological activities which was isolated from the fruits of the traditional Chinese herb Evodiae fructus (Chinese name: Wu-ChuYu) [5]. In particular, evodiamine showed anti-proliferative activities against a wide variety of cancer cells by inducing apoptosis [6]. It is a good antitumor lead compound because of its broad-antitumor spectrum, low toxicity, and suitable physicochemical properties. In our previous studies, evodiamine was
* Corresponding author. E-mail address: [email protected] (C.-Q. Sheng). 1 These authors contributed equally to this article.
identified as a Top1 inhibitor by structure-based virtual screening [7]. Further structural optimization studies identified several highly active evodiamine derivatives which showed potent in vitro and in vivo antitumor activities [8]. In silico target identification and biological assays revealed that the evodiamine derivatives were dual inhibitors of Top1 and topoisomerase II (Top2) [8]. Inspired by these encouraging results, herein a series of E-ring modified evodiamine derivatives were designed and synthesized. Compound 12 showed good antitumor activity with a broad spectrum. Its binding modes with topoisomerase I and II were clarified by molecular docking. 2. Experimental Nuclear magnetic resonance (NMR) spectra were recorded on Bruker AVANCE300 and AVANCE500 spectrometers (Bruker Company, Germany), with TMS as an internal standard and DMSO-d6 as the solvent. Chemical shifts (d values) and coupling constants (J values) are expressed in ppm and Hz, respectively. ESI mass spectra were performed on an API-3000 LC–MS spectrometer. TLC analysis was carried out on silica gel plates GF254 (Qindao Haiyang Chemical, China). Silica gel column chromatography was performed with Silica gel 60G (Qindao Haiyang Chemical, China). Commercial solvents were used without any pretreatment.
http://dx.doi.org/10.1016/j.cclet.2014.03.043 1001-8417/ß 2014 Chun-Quan Sheng. Published by Elsevier B.V. on behalf of Chinese Chemical Society. All rights reserved.
Please cite this article in press as: K. Fang, et al., Design, synthesis and biological evaluation of E-ring modified evodiamine derivatives as novel antitumor agents, Chin. Chem. Lett. (2014), http://dx.doi.org/10.1016/j.cclet.2014.03.043
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Scheme 1. Synthetic routes of the target compounds. Reagents and conditions: (a) Ethyl formate, reflux, 6 h, yield 100%; (b) POCl3, CH2Cl2, 0 8C, 6 h, yield 85%; (c) Triphosgene, THF, reflux, 4 h, yield 90%-95%; (d) CH3I, KOH, DMF, rt, 2 h, yield 60–87%; (e) CH2Cl2, 45 8C, 6 h, yield 50%-62%; (f) 10% Pd/C, H2, DMF, r.t., 12 h, yield 11–73%; (g) 2-bromoacetyl bromide, Et3N, CH2Cl2, 0 8C, 1 h, yield 84%; (h) Na2S9H2O, DMF, r.t., 12 h, yield 70%; (i) 2-(1-piperazinyl)pyridine-4-boronic acid pinacol ester, Pd(PPh3)4, Na2CO3, H2O, reflux, 3.5 h, yield 51%.
2.1. Chemistry The synthetic route of E-ring modified evodiamine derivatives is described in Scheme 1. 3,4-Dihydro-b-carboline (4) was prepared by reacting tryptamine 2 with ethyl formate, followed by intramolecular ring closure in the presence of POCl3. Substituted 2aminobenzoic acid (5) was treated with triphosgene to provide isatoic anhydride, which was methylated by CH3I to afford compound 7 [8]. Condensation of 3,4-dihydro-b-carboline (4) with N-methyl isatoic anhydride 7 provided E-ring derivatives 8a–c. Reduction of the nitro group of compounds 8a and 8b using 10% Pd/C and H2 afforded the amino derivatives 9a and 9b. Compound 9a was further acylated to give compound 10, which was subsequently treated with Na2S to provide compound 11. The Suzuki-Miyaura reaction of compound 8c with 2-(1-piperazinyl)pyridine-4-boronic acid pinacol ester afforded the target compound 12. Synthesis of 3-nitroevodiamine (8a): A solution of 3,4-dihydrob-carboline [8] (4, 0.17 g, 1 mmol) and 1-methyl-6-nitro-1Hbenzo[d][1,3]oxazine-2,4-dione [8] (7a, 0.2 g, 1 mmol) in CH2Cl2 (15 mL) were heated to 45 8C and stirred for 6 h. Then the reaction mixture was concentrated to one-half of its original volume. The precipitate was collected by filtration and washed with alcohol to yield the product 8a (0.21 g, 62%) as yellow solid. 1H NMR (300 MHz, DMSO-d6): d 2.68–2.70 (m, 1 H), 2.95–3.01 (m, 2H), 3.45 (s, 3H), 4.60–4.64 (m, 1H), 6.50 (s, 1H), 6.96–7.11 (m, 4H), 7.32 (d, 1H, J = 7.8 Hz), 7.42 (d, 1H, J = 7.8 Hz), 8.24 (dd, 1H, J = 9.0 Hz, 2.7 Hz), 8.43 (d, 1H, J = 2.7 Hz), 10.89 (s, 1H). ESI-MS: Calcd. for C19H16N4O3 [M H] : m/z 347.12; found: 347.76. The synthetic method for target compounds 8b–c was similar to that of compound 8a. 2-Chloro-3-nitroevodiamine (8b): Yellow solid (0.33 g, yield: 50%). 1H NMR (300 MHz, DMSO-d6): d 2.68–2.73 (m, 1H), 2.95–3.01 (m, 2H), 3.46 (s, 3H), 4.61–4.67 (m, 1H), 6.52 (s, 1H), 7.00–7.16
(m, 3H), 7.35 (d, 1H, J = 7.5 Hz), 7.43 (d, 1H, J = 7.5 Hz), 8.37 (s, 1H), 10.90 (s, 1H). ESI-MS: Calcd. for C19H14ClN4O3 [M H] : m/z 381.79; found: 381.81. 3-Iodoevodiamine (8c): White solid (0.07 g, yield: 50%). 1 H NMR (500 MHz, DMSO-d6): d 2.75–2.78 (m, 1H), 2.91–2.92 (m, 1H), 2.96 (s, 3H), 3.19–3.21 (m, 1H), 4.58–4.62 (m, 1H), 6.17 (s, 1H), 6.87 (d, 1H, J = 8.6 Hz), 7.01 (t, 1H, J = 7.6 Hz), 7.11 (t, 1H, J = 7.6 Hz), 7.35 (d, 1H, J = 8.1 Hz), 7.46 (d, 1H, J = 8.1 Hz), 7.45 (dd, 1H, J = 8.6 Hz, 2.1 Hz), 7.98 (d, 1H, J = 2.1 Hz), 11.03 (s, 1H). ESI-MS: Calcd. for C19H17IN3O [M+H]+: m/z 430.26; found: 430.39. Synthesis of 3-aminoevodiamine (9a): To a stirring solution of 8a (0.12 g, 0.34 mmol) in DMF (10 mL), 10% Pd/C (0.01 g) was added. The reaction mixture was stirred under hydrogen atmosphere for 12 h at room temperature. Then the mixture was diluted with water (50 mL) and extracted with EtOAc (3 50 mL). The combined organic layers were washed with saturated sodium chloride solution (3 50 mL), dried over anhydrous Na2SO4 and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (CH2Cl2–MeOH, gradient 100:1, 100:2) to give compound 9a (0.08 g, 73%) as a yellow solid. 1H NMR (500 MHz, DMSO-d6): d 2.27 (s, 3H), 2.77–2.79 (m, 1H), 2.88–2.92 (m, 1H), 3.11–3.13 (m, 1H), 4.63–4.67 (m, 1H), 5.10 (s, 2H), 5.90 (s, 1H), 6.78 (dd, 1H, J = 8.5 Hz, 2.5 Hz), 6.94 (d, 1H, J = 8.5 Hz), 7.01 (t, 1H, J = 7.5 Hz), 7.10–7.14 (m, 2H), 7.36 (d, 1H, J = 8.0 Hz), 7.51 (d, 1H, J = 8.0 Hz), 11.26 (s, 1H). ESI-MS: Calcd. for C19H19N4O [M+H]+: m/z 319.15; found: 319.93. The synthetic method for target compounds 9b was similar to that of compound 9a. 2-Chloro-3-aminoevodiamine (9b): Red solid (0.09 g, yield: 11%). 1H NMR (500 MHz, DMSO-d6): d 2.27 (s, 3H), 2.73–2.77 (m, 2H), 3.11–3.13 (m, 1H), 4.63–4.66 (m, 1H), 5.10 (s, 2H), 5.90 (s, 1H), 6.78 (dd, 1H, J = 8.5 Hz, 2.5 Hz), 6.94 (d, 1H, J = 8.5 Hz), 7.01 (t, 1H, J = 7.5 Hz), 7.10–7.14 (m, 2H), 7.36 (d, 1H, J = 8.0 Hz), 11.26 (s, 1H).
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ESI-MS: Calcd. for C19H18ClN4O [M+H]+: m/z 353.83; found: 353.49. Synthesis of 2-bromoacetamidoevodiamine (10): To a stirring solution of 9a (0.1 g, 0.3 mmol) and Et3N (0.04 mL, 0.3 mmol) in CH2Cl2 (20 mL), 2-bromoacetyl bromide (0.04 mL, 0.47 mmol) was added and stirred for 1 h at 0 8C. The solvent was removed under reduced pressure. The residue was purified by silica gel column chromatography (CH2Cl2:MeOH = 100:1, v/v) to afford 10 (0.11 g, yield 84%) as yellow solid. 1H NMR (300 MHz, DMSO-d6): d 2.69 (s, 3H), 2.85 (m, 2H), 3.15 (m, 1H), 4.01 (s, 2H), 4.62 (m, 1H), 6.07 (s, 1H), 7.00 (t, 1H, J = 7.8 Hz), 7.08–7.13 (m, 2H), 7.35 (d, 1H, J = 7.8 Hz), 7.48 (d, 1H, J = 7.8 Hz), 7.73 (dd, 1H, J = 9.0 Hz, 2.4 Hz), 8.03 (d, 1H, J = 2.7 Hz), 10.41 (s, 1H), 11.16 (s, 1H). ESI-MS: Calcd. for C21H19BrN4O2 [M+H]+: m/z 439.07; found: 439.75. Synthesis of 2-mercaptoacetamidoevodiamine (11): A solution of 10 (0.05 g, 0.11 mmol) and Na2S9H2O (0.27 g, 1.1 mmol) in DMF (5 mL) was stirred at room temperature for 12 h. The solvent was removed in vacuo and the residue was diluted with water and filtered from insolubles. The filtrate was acidified to pH 1.2 with 3 mol/L HCl. The precipitate was collected and purified by silica gel column chromatography (CH2Cl2–MeOH, gradient 100:1– 100:3) to give compound 11 (0.03 g, yield 70%) as a yellow solid. 1 H NMR (300 MHz, DMSO-d6): d 1.30 (s, 1 H), 2.66 (s, 3H), 2.86 (m, 2H), 3.20 (m, 1H), 3.48 (s, 2H), 4.64 (m, 1H), 6.06 (s, 1H), 7.01 (t, 1H, J = 7.2 Hz), 7.10–7.15 (m, 2H), 7.36 (d, 1H, J = 8.1 Hz), 7.50 (d, 1H, J = 8.1 Hz), 7.73 (d, 1H, J = 9.3 Hz), 8.07 (s, 1H), 10.19 (s, 1H), 11.19 (s, 1H). ESI-MS: Calcd. for C21H20N4O2S [M + H]+: m/z 393.14; found: 393.84. Synthesis of 3-(2-(piperazin-1-yl)pyridin-4-yl)evodiamine (12): Under an nitrogen atomosphere, 8c (0.1 g, 0.23 mmol), 2(1-piperazinyl)pyridine-4-boronic acid pinacol ester (0.1 g, 0.35 mmol) and tetrakistriphenylphosphine palladium (40 mg) were dissolved in DMF (5 mL). An aqueous sodium carbonate solution (2 mol/L, 1 mL) was added and the mixture was heated under reflux for 3.5 h. After cooled to room temperature, the reaction mixture was diluted with water (50 mL) and then extracted with EtOAc (3 50 mL). The combined organic layers were washed with saturated sodium chloride solution (3 50 mL), dried over anhydrous Na2SO4 and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (CH2Cl2:MeOH = 100:1, v/v) to give compound 12 (0.056 g, yield 51%) as a yellow solid. 1H NMR (300 MHz, DMSO-d6): d 2.15 (s, 3H), 2.82 (m, 1H), 3.01 (m, 1H), 3.16 (m, 1H), 3.74 (s, 3H), 3.81 (s, 3H), 4.64 (m, 1H), 6.25 (s, 1H), 6.85 (d, 1H, J = 9.3 Hz), 6.92 (s, 1H), 6.99 (t, 1H, J = 7.8 Hz), 7.05–7.11 (m, 2H), 7.34 (d, 1H, J = 7.8 Hz), 7.44 (d, 1H, J = 7.8 Hz), 7.85 (dd, 1H, J = 8.7 Hz, 2.1 Hz), 8.04 (d, 1H, J = 2.1 Hz), 8.09 (d, 1H, J = 5.1 Hz), 10.99 (s, 1H). ESI-MS: Calcd. for C28H28N6O [M+H]+: m/z 465.24; found: 465.84. 2.2. Biological assay Top1-mediated supercoiled pBR322 relaxation assay: Relaxation assays were carried out as previously described [7,8]. The reaction buffer contained 35 mmol/L Tris–HCl (pH 8.0), 72 mmol/L KCl, 5 mmol/L MgCl2, 5 mmol/L dithiothreitol, 5 mmol/L spermidine, 0.1% bovine serum albumin (BSA), 0.25 mg pBR322 plasmid DNA, and 1 unit of Top1 (TaKaRa Biotechnology Co., Ltd., Dalian) in a total volume of 20 mL. After incubation at 37 8C for 15 min, the reaction was stopped by the addition of 2 mL of 10 loading buffer (0.9% sodium dodecyl sulfate (SDS), 0.05% bromophenol blue, and 50% glycerol). Then, the DNA samples were subjected to electrophoresis in 0.8% agarose gel in TAE (Tris–acetate–EDTA) at 8 V/cm for 1 h. Gels were stained with ethidium bromide (0.5 mg/mL) for 60 min. The DNA band was visualized over UV light and photographed with Gel Doc Ez imager software (Bio-Rad Laboratories Ltd.).
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Top2a-mediated supercoiled pBR322 relaxation assay: Relaxation assays were performed using Topoisomerase II Drug Screening Kits (TopoGEN, Inc.) [8]. The reaction buffer contained 50 mmol/L Tris–HCl (pH 8.0), 150 mmol/L NaCl, 10 mmol/L MgCl2, 5 mmol/L dithiothreitol, 30 mg/mL bovine serum albumin (BSA), 2 mmol/L ATP, 0.25 mg pBR322 plasmid DNA, and 0.75 unit of Top2a (TopoGEN, Inc.) in a total volume of 20 mL. After incubation at 37 8C for 30 min, the reaction was terminated by the addition of 2 mL 10 gel loading buffer (0.25% bromophenol blue, 50% glycerol). The reaction products were analyzed on 1% agarose gel at 8 V/cm for 1 h with TAE (Tris–acetate–EDTA) as the running buffer. Gels were stained with ethidium bromide (0.5 mg/mL) for 60 min. The DNA band was visualized over UV light and photographed with Gel Doc Ez imager software (Bio-Rad Laboratories Ltd.). In vitro cytotoxicity assay: Cells were seeded in 96-well microtiter plates at a density of 5 103/well and treated in triplicate with gradient concentrations of compounds in a humidified atmosphere with 5% CO2 at 37 8C for 72 h. 20 mL of MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) solution (5 mg/mL) was added to each well and the plate was incubated for an additional 4 h. The formazan was dissolved in 100 mL of DMSO. The absorbance (OD) was read on a WellscanMK2 microplate reader (Labsystems) at 570 nm. The concentration causing 50% inhibition of cell growth (IC50) was determined by the Logit method [9,10]. All experiments were performed three times. 2.3. Molecular docking In silico docking was performed using GOLD 5.1 [11] on a Linux PC as described before [8]. The X-ray crystallographic structures of CPT-DNA-Top1 ternary complex (PDB code: 1T8I [12]) and ATPase domain of human Top2a (PDB code: 1ZXM [13]) were obtained from the Protein Data Bank. They were prepared for molecular docking analysis in Discovery Studio 3.0 [14] by removing the ligand from the binding pocket, merging nonpolar hydrogens, adding polar hydrogens, and rendering Gasteiger charges to each atom. 3. Results and discussion 3.1. In vitro antitumor activity The growth inhibitory activities of the target compounds toward human cancer cell lines A549 (lung cancer), MDA-MB-435 (breast cancer), and HCT116 (colon cancer) were determined using MTT assay. Evodiamine was used as the reference drug. When the 2bromoacetamide group was attached on the position 3 of evodiamine E-ring, compound 10 showed moderate in vitro antitumor activity against MDA-MB-435 (IC50 = 22.69 mmol/L), A549 (IC50 = 153.97 mmol/L) and HCT 116 (IC50 = 28 mmol/L) cell lines (Table 1). However, the loss of the antitumor activity was observed for the corresponding thio-derivative 11. 2-Chloro-3-nitro evodiamine (8b) also showed moderate antitumor activity against all three of the tested cancer cell lines. When its nitro group was reduced to the amino group, compound 9b showed significantly Table 1 In vitro antitumor activity of evodiamine derivatives (IC50, mmol/L). Compounds
8b 9b 10 11 12 Evodiamine
Structure
IC50 (mmol/L) MDA-MB-435
A549
HCT116
31.37 1.35 22.69 >250 2.78 20.20
204.15 >250 153.97 >250 6.80 100.00
35.97 75.19 28.00 >250 5.94 100.00
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improved activity against the MDA-MB-435 cell line (IC50 = 1.35 mmol/L). Interestingly, the compound with a 2-(piperazin-1-yl)pyridyl group at the position 3 of evodiamine E-ring (compound 12) showed good antitumor activity with a broad spectrum. The IC50 values of compound 12 against MDA-MB-435, A549 and HCT 116 cell lines were 2.78 mmol/L, 6.80 mmol/L and 5.94 mmol/L, respectively. As compared with evodiamine, most of the target compounds showed better antitumor activity and broader spectra. 3.2. Top1/Top2 inhibitory activity Human Top2 can be classified into two subgroups, Top2a and Top2b. Top2a increases its expression during the cell cycle and mitosis, and its concentration is much higher in tumor cells. Top2b expression remains relatively constant and at low level throughout the cell cycle [15,16]. Thus, Top2a is a good target for antitumor drug discovery. Previously, evodiamine and its derivatives were identified as dual Top1/Top2 inhibitors [8]. Herein, Top1- and Top2amediated pBR322 DNA relaxation assays with purified Top1 and Top2a were used to investigate the inhibitory activity of compound 12 with its concentrations from 10 mmol/L to 100 mmol/L. Camptothecin (CPT) and etoposide (Eto) were employed as positive controls. As shown in Fig. 1A, compound 12 inhibited Top1mediated relaxation of pBR322 DNA in a concentration-dependent manner. It showed moderate inhibitory activity at 100 mmol/L and lower activity at 50 mmol/L. The inhibitory activity of compound 12 was much higher than evodiamine, which only showed marginal activity at 100 mmol/L [7]. Compound 12 was also proven to be a potent inhibitor of the catalytic activity of Top2a (Fig. 1B). Particularly, it showed comparable inhibitory activity against Top2-mediated DNA relaxation to that of Eto at 100 mmol/L and 50 mmol/L, respectively. 3.3. Binding mode with Top1/Top2 In an attempt to understand the binding mode of compound 12 to Top1-DNA complex and ATP-binding domain of Top2a, docking simulation was performed. As indicated in the interaction model between 12 and Top1-DNA complex (Fig. 2A), its E-ring and pyridyl group intercalates at the site of DNA cleavage and forms p-stacking interactions with both the 1 (upstream) and +1 (downstream) base pairs. The N atom of pyridine forms a hydrogen bond with the hydroxyl group of Thr718. AB-ring is faced into the major groove and its indole NH undergoes a strong hydrogen bond interaction with the oxygen atom of Guanine. Moreover, compound 12 is
Fig. 1. Top1 and Top2 inhibitory activity of compound 12. (A) Inhibition of Top1 relaxation activity ranging from 10 mmol/L to 100 mmol/L. Lane 1, supercoiled plasmid DNA; Lane 2, DNA + Top1; Lanes 3–5, DNA + Top1 + CPT (100, 50 and 10 mmol/L); Lanes 6–8, DNA + Top1 + 12 (100, 50 and 10 mmol/L). (B) Inhibition of Top2 relaxation activity ranging from 10 mmol/L to 100 mmol/L. Lane 1, supercoiled plasmid DNA; Lane 2, DNA + Top2; Lanes 3–5, DNA + Top1 + Eto (100, 50 and 10 mmol/L); Lanes 6–8, DNA + Top2 + 12 (100, 50 and 10 mmol/L). Lanes order from left to right.
observed to be well-fitted into the ATP-binding domain of Top2a (Fig. 2B). It is capable of making four hydrogen-bonds to enhance the stabilization of the 12-ATPase complex. The N atom of pyridine is shown to participate in hydrogen bonding interactions with Ala167 and Lys168, respectively. The amino group on the piperazine also can form two hydrogen bonds with backbone amide of Arg162 and Asn163. ChemPLP score was also calculated to evaluate the binding affinity. The most active compound 12 showed the highest binding affinity to Top1 and Top2a with ChemPLP sore 82.54 and 61.00, respectively. In contrast, removal of the piperazine group led to the decrease of the binding affinity (ChemPLP sore: 68.04 for Top1 and 54.48 for Top2a). Evodiamine showed the lowest ChemPLP scores (Top1: 64.45, Top2a: 53.34). The computational results were consistent with the biological activities and highlighted the importance of the pyridine and piperazine group for Top1 and Top2a binding.
Fig. 2. Binding mode with Top1/Top2. (A) Binding mode of 12 to Top1–DNA complex. (B) Binding mode of 12 to ATP-binding domain of Top2. The figure was generated using PyMol (http://www.pymol.org/).
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4. Conclusion In summary, a series of novel E-ring modified evodiamine derivatives were designed and synthesized as antitumor agents. Top1/Top2 inhibitory assay and further molecular docking confirmed that the derivatives acted by dual inhibition of Top1 and Top2. As compared with evodiamine, most of the compounds showed better antitumor activity and broader spectra. Compound 12 showed good antitumor activity with an IC50 ranging from 2.78 mmol/L to 5.94 mmol/L. It can be used as a good starting point for further optimization. Acknowledgments This work was supported by National Natural Science Foundation of China (Nos. 81222044, 81373278), Key Project of Science and Technology of Shanghai (No. 11431920402), the National Basic Research Program of China (No. 2014CB541800), Shanghai RisingStar Program (No. 12QH1402600), and Shanghai Municipal Health Bureau (No. XYQ2011038). References [1] G.M. Cragg, P.G. Grothaus, D.J. Newman, Impact of natural products on developing new anti-cancer agents, Chem. Rev. 109 (2009) 3012–3043. [2] D.J. Newman, G.M. Cragg, Natural products as sources of new drugs over the 30 years from 1981 to 2010, J. Nat. Prod. 75 (2012) 311–335. [3] D.J. Newman, G.M. Cragg, Natural products as sources of new drugs over the last 25 years, J. Nat. Prod. 70 (2007) 461–477.
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