European Journal of Medicinal Chemistry 95 (2015) 400e415

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European Journal of Medicinal Chemistry journal homepage: http://www.elsevier.com/locate/ejmech

Original article

Synthesis and pharmacological evaluation of novel bisindole derivatives bearing oximes moiety: Identification of novel proapoptotic agents Hong-En Qu a, 1, Ri-Zhen Huang a, 1, Gui-Yang Yao c, 1, Jiu-Ling Li a, Man-Yi Ye a, Heng-Shan Wang a, *, Liangxian Liu b, ** a

State Key Laboratory for the Chemistry and Molecular Engineering of Medicinal Resources (Ministry of Education of China), School of Chemistry and Pharmaceutical Sciences of Guangxi Normal University, Guilin 541004, PR China Department of Chemistry and Chemical Engineering, Gannan Normal University, Ganzhou, Jiangxi 341000, PR China c Department of Pharmaceutical Engineering, School of Chemistry and Chemical Engineering, Southeast University, Nanjing 211189, PR China b

a r t i c l e i n f o

a b s t r a c t

Article history: Received 15 January 2015 Received in revised form 24 March 2015 Accepted 25 March 2015 Available online 27 March 2015

In an effort to develop potent anti-cancer chemopreventive agents, a novel series of bisindole derivatives bearing oxime moiety were synthesized. Structures of all compounds were characterized by NMR and HRMS. Anti-proliferative activities for all of these compounds were investigated by the method of MTT assay on 7 human cancer lines and the normal cell lines (HUVEC). Most of them showed a noteworthy anti-cancer activity in vitro, the half maximal inhibitory concentration (IC50) value is 4.31 mM of 4e against T24. The results from Hoechst 33258 and acridine orange/propidium iodide staining as well as annexinV-FITC assays provided evidence for an apoptotic cell death. The further mechanisms of compound 4e-induced apoptosis in T24 cells demonstrated that compound 4e induced the productions of ROS, and altered anti- and pro-apoptotic proteins, leading to mitochondrial dysfunction and activations of caspase-9 and caspase-3 for causing cell apoptosis. Moreover, the cell cycle analysis and western-blot analysis indicated that compound 4e effectively arrested T24 cells in G1 stage and possibly has an effect on cell cycle regulatory proteins particularly cyclin D1. © 2015 Elsevier Masson SAS. All rights reserved.

Keywords: Bisindole Oxime Apoptosis Anti-cancer Cell cycle arrest

1. Introduction Cancer is characterized by uncontrolled cell proliferation and has become the second cause of mortality in the world. Traditionally prescribed chemotherapeutic agents have serious problems with toxicity, instability and poor water solubility. Even more unfortunately, narrow therapeutic index of anticancer compounds and the problem of multidrug resistance [1] are some of the major hurdles in the successful practice of chemotherapy. Therefore, development of potent and specific anti-cancer agents is urgently needed.

* Corresponding author. State Key Laboratory Cultivation Base for the Chemistry and Molecular Engineering of Medicinal Resources, School of Chemistry & Pharmaceutical Science of Guangxi Normal University, Yucai Road 15, Guilin 541004, Guangxi, PR China. ** Corresponding author. E-mail addresses: [email protected] (H.-S. Wang), [email protected] (L. Liu). 1 Co-first author: These authors contributed equally to this work. http://dx.doi.org/10.1016/j.ejmech.2015.03.058 0223-5234/© 2015 Elsevier Masson SAS. All rights reserved.

Indole, the most pharmacodynamic nucleus in nature, has been a major constituent of number of clinically effective agents and incorporated in various naturally occurring indole alkaloids, such as indirubin and serotonin [2,3]. Indole derivatives, especially the 3substituted indoles [9e13], have been found to possess a wide range of pharmaceutical properties, such as anti-cancer [4], antibacterial [5], antiviral [6], anti-topoisomerase II [7] and antiinflammatory [8] activities. Additionally, biindole scaffolds are important motifs in many natural products and the medicinal chemistry world [14]. Modern studies had reviewed bis-indole (Fig. 1) alkaloids show significant bioactivities comparing to monoindole alkaloids [15,16]. Among them, several bis-indoles are in clinical trials, such as indirubin which have been approved as anticancer agent for children with acute promyelocytic leukemia. In addition, indirubin and meisoindigo have been identified as a potent inhibitor for cyclindependent kinases (CDKs) and GSK-3b [17,18]. However, the poor aqueous solubility and bioavailability preclude the extensive clinical application of indirubin and its derivatives. Recently, flexibility

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silica gel (eluant: EtOAc/PE ¼ 1:4) to yield the corresponding product 5 and 6 (Scheme 1). All the target compounds were fully characterized by 1H NMR, 13C NMR and HRMS. 2.2. Biological evaluation

Fig. 1. A structural comparison of the designed 2, 30 -Bi (3H-indol)-3-one oximes with indirubin, indirubine-30 -oxime, bis-isatins schiffbase and meisoindigo.

and aqueous solubility bis-isatins schiffbase which was directly connected via a bis-Schiff base linker (Fig. 1) were reported [19]. In mechanism study, this bis-isatins schiffbase arrested the cell cycle at the G2/M phase in HepG2 cells by down-regulating the expression of cyclin B1 and CDC 2. Despite the fact that the bis-isatins are well studied compounds, new bis-indole derivatives are continually being discovered in terms of their biological activity. In our previous works [20], we reported the synthesis of bisindole which directly connected via a single bond by an efficient one-pot procedure. The results from the MTT assay for these compounds showed a noteworthy anti-cancer activity in vitro. Meanwhile, the introduction of an oxime function into an appropriate skeleton is a reasonable approach to the preparation of potent cytotoxic agents and many oxime derivatives have exhibited potent inhibition activities against human tumors [21,22]. In continuation of our search for pharmacologically active bis-indole derivatives, it seemed to be reasonable to prepare and evaluate 2, 30 -Bi (3H-indol)-3-one oximes (Fig. 1) for their biological properties. It must be noted that a multitude of study was focused on the double bond coplanar skeleton. However, pharmacological activities of the single bond connecting bisindole were not reported. More over, in most previous studies, the oxime-containing bisindole derivatives have not been thoroughly explored for their regulation of cellular signaling apoptosis inducing and growth arrest effects. In this paper, we further conducted the synthesis and antiproliferative evaluation of the oxime-containing bis-indole derivatives. Furthermore, the mechanism of apoptotic effects induced by the representative compound 4e was also investigated. 2. Results and discussion 2.1. Chemistry 2.1.1. General procedure for the preparation of compounds To a solution of indole (1, 0.50 mmol) and NaNO2 (0.60 mmol) in CH3CN (1 mL) was added FeCl3$6H2O (0.125 mmol) under atmosphere and the mixture was stirred at room temperature for 15e72 h (monitored by TLC). The reaction mitxure was concentrated under reduced pressure. The residue was purified by flash chromatography on silica gel (eluant: EtOAc/PE ¼ 1:1) to yield the corresponding product 4 (Scheme 1). To a solution of indole (1, 0.50 mmol), alkyl bromide (0.7 mmol) and NaNO2 (1.0 mmol) in DMF (1 mL) was added RuCl3$3H2O (0.075 mmol) under atmosphere and the mixture was stirred at 40  C for 8e26 h (monitored by TLC). The reaction mixture was concentrated under reduced pressure. The residue was purified by flash chromatography on

2.2.1. Cytotoxicity test The inhibitory effect of these object compounds was evaluated by MTT assay against A549, MGC-803, HepG2, T24, SPCA-2, NCIH460, SK-OV-3, HUVEC normal cells line, and compared the data with 2,30 -Bi(3H-indol)-3-one oxime (4a) and the positive control 5fluorouracil (5-FU). The results were shown in Table 1 and Table 1S (Supplementary). In all human cancer cell lines, almost all of the compounds showed an increased cytotoxic activity as compared to 4a, and most of them even showed preferable cytotoxic activities than 5-FU. Compounds 4e, 4i and 5j showed good inhibitory activity for all the tested carcinoma cell lines. Compound 4c showed strong inhibitory activity selectivity for SK-OV-3 as well as HepG2, 4l selectivity for SPCA-2 and 5g selectivity for SK-OV-3. The others also showed good inhibitory except 4n for SK-OV-3, 4o for A549, NCI-H460 and SPCA2, 6c for NCI-H460 and SK-OV-3, 6d for NCI-H460. In particular, the 5, 50 -Dimethoxy-2, 30 -bi (3H-indol)-3-one oxime (4e) showed a noteworthy anti-cancer activity in vitro, IC50 value is 4.31 mM of 4e against T24. The results from the MTT assay for compounds 4, 5 and 6 using the human bladder carcinoma cell line T24 were presented in more detail in Fig. 2. The results revealed that the analogs 4d, 4n and 4o, obtained by inserting the methyl moiety in the positions 5 of indole showed quite different antiproliferative potencies: compound 4d, with IC50 value of 8.78 mM, was about 3-fold more active than derivatives 4n and 4o, and undoubtedly emerged as one of the most active compounds within this subset, thus suggesting that the C-5 insertion position plays a pivotal role. A beneficial effect was also observed with the modifications of the C-5 position: the derivative 4e was 4-fold more active than 4j. The presence of a weakly electron-withdrawing in position C5 seemed to be associated with a general increase in activity. Moreover, the modifications of oxime moiety caused a loss of potency: compounds 4 showed an even better cytotoxicity. In addition, the inhibition activities of compounds 4, 5 and 6 against HUVEC normal cell lines were also estimated in Table 1S (Supplementary). The results indicated that the cytotoxicity of most of compounds against cancer cells was much higher than HUVEC normal cells, making them good candidates as anti-cancer drugs. In summary, these modifications yield some small increase in cytotoxicity and a low but significant improvement for the selectivity of the compounds. 2.2.2. Induction of apoptosis Apoptosis is characterized as programmed cell death which leads to characteristic changes including blebbing, cell shrinkage, nuclear fragmentation, chromatin condensation, and chromosomal DNA fragmentation. Inhibition of apoptosis is considered an essential step in tumorigenesis and is one of the hallmarks of cancer, allowing the survival of cells that accumulate oncogenic events that otherwise would have been removed by apoptosis [23]. It is therefore necessary to consider cell apoptosis as another effective approach in cancer treatment. Thus, in order to address the cell death caused by compounds 4e, the mechanism of growth inhibition of T24 cell lines was evaluated. The morphological character changing of T24 cells were investigated under fluorescence microscopy using acridine orange (AO)/ ethidium bromide (EB), Hoechst 33258, JC-1 mitochondrial membrane potential staining to evaluate whether the growth inhibitory activity of the selected compound was related to the induction of

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Scheme 1. The synthesis of the compounds. Reactants and conditions: (i) NaNO2, CH3CN, FeCl3$6H2O, 25  C, 15e72 h; (ii, iii) alkyl bromide, NaNO2, DMF, RuCl3$3H2O, 40  C, 8e26 h.

Table 1 Effects of bisindole derivatives 4, 5 and 6 against different tumor cell lines. Compound

4b 4c 4d 4e 4f 4g 4h 4i 4j 4k 4l 4m 4n 4o 4p 5a 5b 5c 5d 5e 5f 5g 5h 5i 5j 5k 5l 5m 5n 5o 6a 6b 6c 6d 4a 5-Fu a

IC50 (mM)a R1

R2

A549

5-Br 5-F 5-CH3 5-OCH3 5-OBn 5-NH2 5-NHAc 5-NO2 5-CN 5-CO2CH3 6-F 6-CO2CH3 7-CH3 4-CH3 4-CN H 5-F 5-CN 6-F H 5-F 5-Br H H 5-Br 7-CH3 5-Br 7-CH3 H 5-OCH3 H 5-CH3 H H H e

e e e e e e e e e e e e e e e H H H H 4-CH3 4-CH3 4-CH3 4-F 4-CF3 4-CF3 3-F 3-Cl 3-Cl 2-Cl-4-F 2-Cl-4-F

16.17 11.94 11.18 6.09 19.49 14.82 21.91 10.44 14.91 20.47 21.26 25.94 17.52 44.86 33.59 19.34 10.53 14.18 15.69 22.44 13.58 13.27 26.99 21.94 8.45 23.28 12.94 31.86 21.40 19.07 30.95 25.49 36.99 36.71 21.81 38.23

e e

MGC-803 ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

0.96 1.34 1.27 1.11 0.87 0.63 0.96 0.84 1.57 1.80 0.88 0.49 0.87 1.25 0.93 0.79 0.35 1.55 0.67 2.58 0.59 0.04 0.77 0.56 1.62 1.95 0.25 0.94 0.54 0.14 1.18 0.86 0.77 0.64 1.25 0.62

13.18 12.26 9.47 7.66 14.51 13.17 17.85 10.78 18.38 22.43 22.05 29.76 16.90 24.96 35.37 15.42 21.31 15.49 21.94 22.78 20.79 31.18 33.04 30.60 11.07 31.94 7.60 26.43 18.52 16.14 24.31 22.25 23.32 29.86 20.58 47.39

± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

0.54 0.84 0.62 0.97 0.83 1.01 1.64 1.14 1.65 0.90 0.21 0.56 0.83 0.71 0.58 77 0.04 0.86 0.56 0.60 0.41 0.57 0.97 0.91 1.09 0.56 0.45 1.48 0.34 0.09 0.53 0.72 0.56 0.64 0.67 1.36

HepG2 14.35 9.31 10.68 9.92 19.03 18.47 28.19 8.25 30.11 15.26 13.82 20.75 15.58 21.29 40.49 19.52 17.34 25.08 24.11 19.07 19.62 20.39 35.49 22.30 11.07 34.14 13.30 22.81 25.45 24.11 31.30 34.30 33.61 32.12 20.43 31.98

± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

T24 0.37 0.83 0.93 0.34 0.95 0.76 0.60 1.35 1.68 0.53 0.65 1.42 1.81 0.43 1.63 0.31 1.21 0.86 0.39 0.64 1.37 1.32 0.27 0.45 1.09 0.95 0.56 0.15 0.56 0.39 0.99 1.15 1.15 0.90 0.32 0.78

12.48 10.13 8.78 4.31 11.82 9.38 16.76 13.15 28.35 11.47 11.68 14.35 23.52 15.02 19.38 12.15 12.52 18.63 13.31 23.47 20.04 21.37 23.05 18.87 9.88 21.20 9.34 17.55 20.03 12.43 32.14 25.12 35.17 35.37 27.43 40.57

± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

0.95 1.73 0.93 0.29 0.63 0.75 0.69 0.37 0.23 0.32 0.14 0.58 0.62 0.53 1.21 0.93 0.25 0.42 0.82 0.08 0.74 0.53 0.21 0.32 0.85 0.14 0.98 0.81 0.79 0.58 0.47 0.56 0.32 0.53 0.88 1.47

Each data represents mean ± S.D. from three different experiments performed in triplicate.

apoptosis. 2.2.2.1. Hoechst 33258 staining. Hoechst 33258 is a membrane permeable blue fluorescent dye which stains the cell nucleus. Live cells with uniformly light blue nuclei were observed under fluorescence microscope after treatment with Hoechst 33258, while apoptotic cells had bright blue nuclei because of karyopyknosis and chromatin condensation, and the nuclei of dead cells could not be

stained. T24 cells treated with compound 4e at 5 mM, 10 mM, 20 mM for 24 h were stained with Hoechst 33258, whereas the cells not dealt with the 4e was used as control at for 24 h. The results were given in Fig. 3. As shown in Fig. 3, cells not treated with the compound 4e were normally blue (in the web version), but most cell nuclei appeared to be highly condensed (brightly stained). On the other hand, for 4e treatment, apoptotic cells increased gradually in a dose-dependent

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(in the web version), whereas late apoptotic cells will be thickly stained as orange or display orange fragments and necrotic cells will be stained as orange with no condensed chromatin. The cytotoxicity of compound 4e at 5 mM, 10 mM, and 20 mM for 24 h against T24 cells was analyzed by AO/EB staining, and T24 cells not dealt with 4e was used as control. The results were shown in Fig. 4. Fig. 4 showed that the T24 cells treated with 4e at different concentration for 24 h had obviously changed. The nuclei stained as yellow green or orange, and the morphology showed pycnosis, membrane blebbing and cell budding. These phenomena are associated with cell apoptosis. In summary, the cells presented with apoptotic morphology. The nearly complete absence of red cells in compound 4e showed that it was associated with very low cytotoxicity. The results demonstrated that compound 4e could induce apoptosis with low cytotoxicity, which consistent with the results for Hoechst 33258 staining. Fig. 2. Cytotoxicity (IC50 values in mM with SD, from MTT assays) for compounds 4, 5 and 6 using the human bladder cancer cell line T24.

manner and exhibited typical changes including reduction of cellular volume, bright staining and condensed or fragmented nuclei. The results suggested that compound 4e was able to induce apoptotic cell morphology in T24 cell line. 2.2.2.2. AO/EB staining. AO is a vital dye which can stain nuclear DNA across an intact cell membrane, while EB can only stains cells that had lost their membrane integrity. Therefore, after concurrently treating with AO and EB, live cells will be uniformly stained as green (in the web version) and early apoptotic cells will be turbidly stained as green yellow or show green yellow fragments

2.2.2.3. Compound 4e-induced loss of mitochondrial membrane potential (DJm) in T24 cells. Mitochondria play a critical role in the life and death of cells, and they are known to be a major source and target of oxidative stress. Mitochondria maintain homeostasis in the cell by interacting with ROS and responding adequately to different stimuli. An abnormal cellular ROS balance can be activated by the structural injury of mitochondria. Furthermore, excess ROS production can induce mitochondrial damage. The damage of mitochondrial integrity and the consequent loss of mitochondrial membrane potential are the early events in the initiation and activation of apoptotic cascades [24]. To further determine the apoptosis-inducing effect of target compound 4e, the fluorescent probe JC-1 was used to design and detect the changes of mitochondrial membrane potential. JC-1, which is a kind of lipophilic cationic dye, can easily pass through

Fig. 3. Effects of compound 4e on morphological changes of T24 cells after staining with Hoechst 33258 dye. (a) The cells not treated with 4e were used as control, (b, c, d) Compound 4e treated T24 cells at concentrations of 5, 10 and 20 mM, respectively.

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Fig. 4. Compound 4e induced apoptotic in T24 cells were determined by AO/EB staining and were photographed via fluorescence microscopy. (a) Not dealt with compound 4e was used as control at for 24 h, (b, c, d) dealt with compound 4e for 24 h at concentrations of 5, 10 and 20 mM, respectively.

the plasma membrane into cells and accumulates in mitochondria [25]. The maximum excitation wavelength of JC-1monomers and Jaggregates were excited at 514 nm and 585 nm, respectively, and then light emissions were collected at 515e545 nm (green) and 570e600 nm (red). The effect of compound 4e on mitochondrial membrane potential loss at 5 mM, 10 mM and 20 mM for 24 h in T24 cells stained with JC-1 was investigated. The results were shown in Fig. 5. As shown in Fig. 5, cells were treated with the compound 4e showed strong green fluorescence and typical apoptotic morphology at different concentration for 24 h, while cells not treated with the compound 4e were normally red (in the web version). From the above discussions we may safely draw the conclusion that compound 4e induced apoptosis against T24 cell line. The experimental results were identical with that of previous, once again demonstrated that the 4e induced apoptosis against T24 cell line. 2.2.2.4. Compound 4e-induced apoptosis in T24 cells determined by Annexin V-FITC/PI. Annexin V binds to phosphatidylserine, which is exposed on the cell membrane and is the latest stages of cell death resulting from either apoptotic or necrotic processes. Hence, Annexin V-FITC staining is typically accompanied by a vital dye staining such as PI to identify the formation of apoptotic cells in the early stages (Annexin V-FITC positive, PI negative). Viable cells with intact membranes exclude PI, whereas the membranes of dead and damaged cells are permeable to PI. After this procedure, cells can be classified from FITC Annexin V and PI negative (viable, or no measurable apoptosis), to FITC Annexin V positive and PI negative (early apoptosis, membrane integrity is present) and finally to FITC Annexin V and PI positive (end stage apoptosis and death). In Fig. 6, flow cytometric analysis revealed apoptosis ratios

(including the early and late apoptosis ratios) for compound 4e were obtained after 24 h of treatment at concentrations of 5 mM, 10 mM, 20 mM with the highest apoptosis ratio being 32.7%. Moreover, the apoptosis of T24 cells treated with compound 4e increased gradually with concentration; Synchronously, the apoptosis ratios of compound 4e evaluated at different concentration points were found to 6.89% (5 mM), 11.9% (10 mM) and 32.7% (20 mM), respectively, were higher than that of control (2.74%). Apoptosis determined by Annexin V-FITC-PI Detection Kit confirmed the fact that compound 4e could induce apoptosis in T24 cells. 2.2.2.5. Increase of intracellular ROS level in T24 cells induced by compound 4e. Reactive oxygen species (ROS) are a byproduct of normal metabolism, including free radicals such as the superoxide anion, hydroxyl and lipid radicals, as well as oxidizing nonradical species such as hydrogen peroxide, peroxynitrite, and singlet oxygen. They often cause cellular damage and lead to cell death and tissue injury, especially at high concentrations. The generation of intracellular ROS may be responsible for the induction of apoptosis [26,27]. Therefore, we investigated the ROS levels by means of fluorescence of DCFH-DA. ROS generation in T24 cells was visualized by fluorescence microscopy, T24 cells were dealt with compound 4e with increasing concentrations (5 mM, 10 mM, 20 mM) for 24 h, and T24 cells not dealt with 4e was used as control at for 24 h. As indicated in Fig. 7, cells not treated with the compound 4e were normally green (in the web version). For 4e treatment, the cells showed strong green fluorescence. Hence, it could be summarized that compounds 4e significantly increased the intracellular level of ROS. 2.2.2.6. Induces caspase-dependent apoptosis in T24 cells. To further investigate the effect of compound 4e on apoptosis in T24 cells,

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Fig. 5. Effects of compound 4e on morphological changes and mitochondrial membrane potential (Djm) in T24 cells. (a) Not dealt with compound 4e was used as control at for 24 h, (b, c, d) dealt with compound 4e for 24 h at concentrations of 5, 10 and 20 mM, respectively.

expression of the apoptosis-related genes was detected by realtime quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) after treatment of compound 4e for 24 h at 5 mM, 10 mM, 20 mM. The levels of gene expression were normalized to control cells and calibrated with reference to b-actin. Up-regulation of apoptosis-related genes was observed and this effect was dosedependent (Fig. 8). When the cells were exposed to antiproliferative concentrations (IC50) for 24 h, the levels of caspase-8, AIF and Cyt c were up-regulated 8.03-fold, 4.47-fold and 8.09-fold (p < 0.05), respectively. Strong up-regulations were noticed in caspase-3, -9 (24.79-fold and 27.11-fold, respectively, p < 0.05). 2.2.2.7. Release of cytochrome c and activation of caspases were involved in the apoptosis induced by compound 4e. To confirm the molecular mechanisms involved in the observed apoptosis alterations, we investigated the effects of compound 4e on the expression of proteins important for mitochondria mediated apoptosis. Apoptosis occurs through two different pathways, that is the intrinsic and extrinsic pathways. Intrinsic pathway involves mitochondria that play a pivotal role in apoptosis, whereas extrinsic pathway is mediated by cell death receptors such as Fas, TNF a after receiving the death signal. It is well-known that intrinsic pathway is regulated by the Bcl-2 family of pro- and anti-apoptotic proteins, which stimulate the permeabilization of the mitochondrial outer membrane and cytochrome c released into the cytosol, promoting in the activation of the caspase cascade and the induction of apoptotic cell death [28,29]. The effects of compound 4e on the constitutive levels of Bax, Bcl-2 and cytochrome c in T24 cells are given in Fig. 9a. In comparison with the control cells, 4e induced a significant increase in the levels of Bax and a decrease in the expression of Bcl-2, in a dose-dependent manner. Compound 4e treatment caused an accumulation of cytochrome c in the cytosol,

most probably due to the release of mitochondrial cytochrome c. These results indicated an involvement of caspases in the apoptotic process downstream of mitochondria. Then, the roles of important caspases (caspase-9, caspase-3 and caspase-7) were investigated. As Fig. 9b shown, treatment of T24 cells with 4e caused a marked increase in the levels of caspase-9, caspase-3 and caspase-7 proteins compared to the control. These results revealed an involvement of caspases in the intrinsic apoptotic process downstream of mitochondria. Thus, compound 4e induced T24 cells apoptosis might decrease the activation of Bcl-2 and stimulate its downstream proteins which are associated with the mitochondriadependent apoptotic pathway. 2.2.3. Cell cycle perturbations To further examine how compound 4e suppressed the growth of T24 cancer cells, the effect of 4e on cell cycle distribution was investigated and the results of a typical experiment are shown in Fig. 10. As determined by flow cytometry, the exposure of T24 cells to compound 4e after 48 h, resulting in an obvious increase of the percentage of cells in G1 phase when compared with the control. 4e treatment caused 66.92% cells in G1 phase as compared to control showing 34.43%. Inversely, S phase cell population was decreased to 28.41% as compared to control having 62.00%. These results suggested the role of cell cycle arrest in compound 4e-induced growth inhibition of T24 cancer cells. Moreover, the molecular events involved in cellular response to the effective compound, were investigated and to this end, the levels of regulatory proteins implicated in G1 arrest, including p21, cyclin A, p53, cyclin E were evaluated. The cell cycle regulatory proteins which control the G1 to S phase transition are cyclins and cyclin-dependent kinases (CDKs). The activities of cyclin Ddependent CDK4 and CDK6 are detected first in mid-G1 phase and

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Fig. 6. Flow cytometric analysis of cells stained with Annexin V-FITC and PI. The T24 cells not treated with 4e were used as control, (a) Compound 4e treated T24 cells at concentrations of 5, 10 and 20 mM, respectively. (b, c, d) The numbers of living cells and early apoptotic cells were expressed as a percentage of the total cell number. Each bar represents the mean ± SD (n ¼ 5).

then increase as cells approach the G1/S boundary. Cyclin E is periodically expressed at maximum levels near the G1/S transition, binding to a different catalytic subunit, CDK2. The CDK inhibitors

are tumor suppressor proteins that interact with distinct cyclineCDK complexes and thereby inhibit the activity of these enzymes, such as p16, p21 and p27, which regulate the G1 to S

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Fig. 7. Compound 4e affected the levels of intracellular ROS in T24 cells. The T24 cells not treated with 4e were used as control, (b, c, d) Compound 4e treated T24 cells at concentrations of 5, 10 and 20 mM, respectively.

phase transition of the cell cycle [30,31]. In the present investigation the FACS data clearly suggested that cell cycle arrest occurs at G1 phase and possibly has an effect on cell cycle regulatory proteins. The levels of cyclins (cyclin A, cyclin D1 and cyclin E), CDKs (CDK2, CDK4) and CDK inhibitors (p53, p16, p21 and p27) have been determined by western blot analysis for the compound 4e in T24 cells for 24 h at 5 mM, 10 mM, 20 mM concentrations and compared to the control (Fig. 11). Western blot analysis of the T24 cells lysates revealed that 4e reduced the levels of CDK2, CDK4,

cyclin E and cyclin D1 and simultaneously increased the levels of p53, p16, p21 and p27 in a concentration-dependent manner thus indicating that the cells are effectively arrested at G1 phase of the cell cycle. However, 4e treatment did not alter the levels of cyclin A in T24 cells. 3. Experimental section 3.1. General Melting points were determined on a digital melting point apparatus and temperatures were uncorrected. Infrared spectra were measured with a Nicolet Avatar 360 FT-IR spectrometer using film KBr pellet techniques. 1H and 13C NMR spectra were recorded on Bruker spectrometers at 400 and 100 MHz, respectively. Chemical shifts were reported in ppm relative to TMS for 1H and 13C NMR spectra. CDCl3 or DMSO-d6 was used as the NMR solvent. Mass spectra were recorded with Bruker Dalton Esquire 3000 plus LC-MS apparatus. Elemental analysis was carried out on a PerkineElmer 240B instrument. Silica gel (300e400 mesh) was used for flash column chromatography, eluting (unless otherwise stated) with ethyl acetate/petroleum ether (PE) (60e90  C) mixture. 3.2. General procedure for the preparation of 4

Fig. 8. Effects of 4e after dealt for 24 h at different concentrations on the apoptosis cascade by qRT-PCR.

To a solution of indole (0.50 mmol) and NaNO2 (0.60 mmol) in CH3CN (1 mL) was added FeCl3$6H2O (0.125 mmol) under atmosphere and the mixture was stirred at room temperature for 15e72 h (monitored by TLC). The reaction mitxure was concentrated under reduced pressure. The residue was purified by flash chromatography on silica gel (eluant: EtOAc/PE ¼ 1:1) to yield the corresponding product.

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Fig. 9. Effect of compound 4e on cytochrome c release and levels of Bcl-2, Bax, Apaf-1, Caspase-9, Caspase-3, Caspase-7, PARP. T24 cells were treated with at 20 mM of compound 4e for 6, 12 and 24 h, respectively. Equal amount of protein was loaded on SDS-PAGE gel for western blot analysis as described in experimental section. b-actin was used as an internal control.

3.2.1. (E)-2,30 -Bi(3H-indol)-3-one oxime (4a) Red-brown solid, mp: 243e244  C (from EtOAc/PE ¼ 1:1). IR (KBr) nmax: 3172, 3050, 1548, 1438, 1008, 753 cm1. 1H NMR (400 MHz, DMSO-d6): d 13.34 (s, 1H), 11.82 (s, 1H), 8.51 (d, J ¼ 8.0 Hz, 1H, AreH), 8.32 (d, J ¼ 4.0 Hz, 1H, AreH), 8.04 (d, J ¼ 4.0 Hz, 1H, AreH), 7.49 (d, J ¼ 8.0 Hz, 1H, AreH), 7.43 (m, 2H, AreH), 7.22 (m, 3H, AreH). 13C NMR (100 MHz, DMSO-d6): d 162.6, 157.2, 155.4, 136.9, 132.2, 132.1, 127.2, 126.5, 125.8, 123.1, 122.9, 121.6, 121.5, 119.9, 112.5, 109.0. MS (ESI): 262 (MþHþ, 100), 284 (MþNaþ, 5). Anal calcd for C16H11N3O: C, 73.55; H, 4.24; N, 16.08. Found C, 73.76; H, 4.35; N, 15.89. 3.2.2. (E)-5,50 -Dibromo-2,30 -bi(3H-indol)-3-one oxime (4b) Brown solid, mp: 238e239  C (from EtOAc/PE ¼ 1:1). IR (KBr) nmax: 3413, 3138, 1539, 1438, 1357, 1025 cm1. 1H NMR (400 MHz, DMSO-d6): d 13.76 (s, 1H), 12.03 (s, 1H), 8.61 (d, J ¼ 1.8 Hz, 1H, AreH), 8.33 (d, J ¼ 2.8 Hz, 1H, AreH), 8.11 (d, J ¼ 1.8 Hz, 1H, AreH), 7.57 (dd, J ¼ 8.2, 1.8 Hz, 1H, AreH), 7.47 (d, J ¼ 8.6 Hz, 1H, AreH), 7.44 (d, J ¼ 8.2 Hz, 1H, AreH), 7.34 (dd, J ¼ 8.6, 1.8 Hz, 1H, AreH). 13C NMR (100 MHz, DMSO-d6): d 162.4, 155.9, 154.3, 135.7, 134.6, 133.6, 129.4, 128.1, 125.9, 124.8, 123.2, 121.8, 117.9, 114.7, 114.5, 108.3. MS (ESI): 418 (MþHþ, 50), 420 (MþHþ, 100), 422 (MþHþ, 50). Anal calcd for C16H9Br2N3O: C, 45.83; H, 2.16; N, 10.03. Found C, 45.91; H, 2.36; N, 9.92. 3.2.3. (E)-5,50 -Difluoro-2,30 -bi(3H-indol)-3-one oxime (4c) Red-brown solid, mp: 260e261  C (from EtOAc/PE ¼ 1:1). IR (KBr) nmax: 3353, 3144, 1552, 1464, 1010 cm1. 1H NMR (400 MHz, DMSO-d6): d 13.70 (s, 1H), 11.95 (s, 1H), 8.35 (d, J ¼ 2.7 Hz, 1H, AreH), 8.16 (dd, J ¼ 10.1, 2.6 Hz, 1H, AreH), 7.77 (dd, J ¼ 8.1, 2.6 Hz, 1H, AreH), 7.53 (dd, J ¼ 8.8, 4.6 Hz, 1H, AreH), 7.47 (dd, J ¼ 8.4, 4.6 Hz, 1H, AreH), 7.25 (dt, J ¼ 2.7, 9.5 Hz, 1H, AreH), 7.08 (dt, J ¼ 2.6, 9.1 Hz, 1H, AreH). 13C NMR (100 MHz, DMSO-d6): d 162.4, 159.8, 157.5, 154.7, 153.2, 133.6, 133.5, 133.4, 126.9 (d, J ¼ 11.1 Hz), 120.8 (d, J ¼ 8.6 Hz), 118.3 (d, J ¼ 24.5 Hz), 114.3 (d, J ¼ 26.3 Hz), 113.8 (d, J ¼ 8.6 Hz), 111.4 (d, J ¼ 26.3 Hz), 109.0, 107.5 (d, J ¼ 24.5 Hz). MS (ESI): 298 (MþHþ, 100). Anal calcd for C16H9F2N3O: C, 64.65; H, 3.05; N, 14.14. Found C, 64.90; H, 3.32; N, 13.78. 3.2.4. (E)-5,50 -Dimethyl-2,30 -bi(3H-indol)-3-one oxime (4d) Waxy solid. IR (KBr) nmax: 3292, 3161, 2926, 1550, 1458, 1012 cm1. 1H NMR (400 MHz, DMSO-d6): d 13.70e13.10 (s, 1H), 11.82 (s, 1H), 8.31 (d, J ¼ 2.4 Hz, 1H, AreH), 8.28 (s, 1H, AreH), 7.89 (s, 1H, AreH), 7.38 (d, J ¼ 8.2 Hz, 1H, AreH), 7.33 (d, J ¼ 7.8 Hz, 1H, AreH), 7.22 (d, J ¼ 8.2 Hz, 1H, AreH), 7.06 (d, J ¼ 7.8 Hz, 1H, AreH), 2.44 (s, 3H, CH3), 2.33 (s, 3H, CH3). 13C NMR (100 MHz, DMSO-d6):

d 161.3, 155.1, 135.3, 135.1, 132.8, 132.4, 132.1, 130.5, 127.8, 126.5, 124.8, 122.4, 121.3, 118.9, 112.3, 108.2, 21.9, 21.4. MS (ESI): 290 (M þ Hþ, 100), 312 (M þ Naþ, 10). Anal calcd for C16H11N3O: C, 73.55; H, 4.24; N, 16.08. Found C, 73.76; H, 4.35; N, 15.89. 3.2.5. (E)-5,50 -Dimethoxy-2,30 -bi(3H-indol)-3-one oxime (4e) Waxy solid. IR (KBr) nmax: 3286, 3125, 2932, 1551, 1475, 1273, 1216, 1011 cm1. 1H NMR (400 MHz, DMSO-d6): d 13.32 (s, 1H), 11.61 (s, 1H), 8.22 (d, J ¼ 2.7 Hz, 1H, AreH), 8.01 (d, J ¼ 2.6 Hz, 1H, AreH), 7.61 (d, J ¼ 2.6 Hz, 1H, AreH), 7.37 (d, J ¼ 8.8 Hz, 1H, AreH), 7.32 (d, J ¼ 8.4 Hz, 1H, AreH), 6.95 (dd, J ¼ 8.4, 2.7 Hz, 1H, AreH), 6.85 (dd, J ¼ 8.8, 2.6 Hz, 1H, AreH), 3.81 (s, 3H, CH3), 3.78 (s, 3H, CH3). 13C NMR (100 MHz, DMSO-d6): d 161.1, 157.9, 155.5, 155.3, 150.8, 131.8, 131.7, 127.1, 122.5, 120.2, 116.5, 113.7, 113.0, 112.6, 108.9, 105.1, 56.1, 55.9. MS (ESI): 322 (MþHþ, 100). Anal calcd for C18H15N3O3: C, 67.28; H, 4.71; N, 13.08. Found C, 67.53; H, 4.49; N, 12.81. 3.2.6. (E)-5,50 -Bis(benzyloxy)- 2,30 -bi(3H-indol)-3-one oxime (4f) Red-brown solid, mp: 237e239  C (from EtOAc/PE ¼ 1:1). IR (KBr) nmax: 3298 (s), 1549, 1451, 1212, 1130, 1007 cm1. 1H NMR (400 MHz, DMSO-d6): d 13.34 (s, 1H), 11.63 (s, 1H), 8.20 (d, J ¼ 2.5 Hz, 1H, AreH), 8.09 (d, J ¼ 2.0 Hz, 1H, AreH), 7.67 (d, J ¼ 2.2 Hz, 1H, AreH), 7.48 (d, J ¼ 7.5 Hz, 2H, AreH), 7.44 (d, J ¼ 7.5 Hz, 2H, AreH), 7.40e7.35 (m, 5H, AreH), 7.35e7.28 (m, 3H, AreH), 7.02 (dd, J ¼ 8.6, 2.2 Hz, 1H, AreH), 6.91 (dd, J ¼ 8.6, 2.2 Hz, 1H, AreH), 5.11 (s, 2H, CH2), 5.09 (s, 2H, CH2). 13C NMR (100 MHz, DMSO-d6): d 161.2, 156.9, 155.4, 154.4, 151.0, 138.1, 137.6, 132.0, 131.8, 128.9, 128.8, 128.3, 128.2, 128.1, 128.0, 127.1, 122.5, 120.2, 117.5, 114.8, 113.1, 113.0, 108.9, 106.9, 70.4, 70.3. MS (ESI): 474 (MþHþ, 100), 496 (MþNaþ, 10). Anal calcd for C30H23N3O3: C, 76.09; H, 4.90; N, 8.87. Found C, 76.33; H, 5.29; N, 9.05. 3.2.7. (E)-5,50 -Diamino-2,30 -bi(3H-indol)-3-one oxime (4g) Red-brown solid, mp: 165e167  C (from EtOAc/PE ¼ 2:1). IR (KBr) nmax: 3225 (br s), 1541, 1451, 1370, 1022 cm1. 1H NMR (400 MHz, DMSO-d6): d 11.25 (s, 1H), 10.96 (s, 1H), 8.10 (d, J ¼ 1.8 Hz, 1H, AreH), 7.77 (dd, J ¼ 8.7, 1.8 Hz, 1H, AreH), 7.46 (d, J ¼ 8.7 Hz, 1H, AreH), 7.39 (t, J ¼ 2.7 Hz, 1H, AreH), 7.28 (d, J ¼ 8.7 Hz, 1H, AreH), 7.24 (t, J ¼ 2.7 Hz, 1H, AreH), 7.12 (t, J ¼ 2.1 Hz, 1H), 6.65 (d, J ¼ 2.1 Hz, 1H), 6.56 (s, 1H), 6.29 (s, 2H). 13C NMR (100 MHz, DMSO-d6): d 148.0, 142.4, 136.9, 129.9, 128.4, 128.1, 127.0, 126.3, 121.5, 117.7, 116.2, 115.0, 112.6, 112.2, 103.1, 102.6. MS (ESI): 292 (MþHþ, 100). Anal calcd for C16H13N5O: C, 65.97; H, 4.50; N, 24.04. Found C, 66.32; H, 4.88; N, 23.69.

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Fig. 10. Cell cycle analysis of compound 4e treated T24 cells. T24 cells were treated with different concentrations (0, 5, 10 and 20 mM) of compound 4e for 48 h to determine DNA fluorescence and cell cycle phase distribution. (e) The numbers of G1, S and G2 phase cells were expressed as a percentage of the total cell number. The values represent mean ± S.D of three independent experiments (p < 0.01).

3.2.8. (E)-N,N0 -(3-(Hydroxyimino)-2,30 -bi(3H-indole)-5,50 -diyl) diacetamide (4h) Red-brown solid, mp: 228e229.5  C (from EtOAc/PE ¼ 2:1). IR (KBr) nmax: 3197 (br s), 1667, 1544, 1467, 1371, 1023 cm1. 1H NMR (400 MHz, DMSO-d6): d 13.35 (s, 1H), 11.66 (s, 1H), 9.97 (s, 1H), 9.91 (s, 1H), 8.42 (s, 1H, AreH), 8.27 (d, J ¼ 1.9 Hz, 1H, AreH), 8.23 (d,

J ¼ 2.9 Hz, 1H, AreH), 7.64 (dd, J ¼ 8.3, 1.9 Hz, 1H, AreH), 7.57 (dd, J ¼ 8.8, 1.9 Hz, 1H, AreH), 7.36 (d, J ¼ 8.8 Hz, 1H, AreH), 7.26 (d, J ¼ 8.3 Hz, 1H, AreH), 1.95 (s, 6H, 2CH3). 13C NMR (100 MHz, DMSOd6): d 168.5, 168.2, 161.6, 155.5, 152.6, 137.4, 133.8, 133.5, 132.2, 126.3, 122.3, 121.7, 119.5, 119.0, 117.0, 113.6, 112.1, 109.1, 24.4, 24.3. MS (ESI): 376 (MþHþ, 100), 398 (MþNaþ, 15). Anal calcd for

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Fig. 11. Effect of compound 4e on the levels of CDK2, CDK4, Cyclin A, Cyclin D1, Cyclin E, p53, p27, p21 and p16. T24 cells were treated with of compound 4e at 0, 5, 10, 20 mM for 24 h, respectively. Equal amount of protein was loaded on SDS-PAGE gel for western blot analysis as described in experimental section. b-actin was used as an internal control.

C20H17N5O3: C, 63.99; H, 4.56; N, 18.66. Found C, 64.26; H, 4.74; N, 18.72. 3.2.9. (E)-5,50 -Dinitro-2,30 -bi(3H-indol)-3-one oxime (4i) Red-brown solid, mp: 176e178  C (from EtOAc/PE ¼ 1:1). IR (KBr) nmax: 3350, 3189, 1551, 1439, 1330, 1092 cm1. 1H NMR (400 MHz, DMSO-d6): d 14.24 (s, 1H), 12.61 (s, 1H), 9.31 (d, J ¼ 2.2 Hz, 1H, AreH), 8.68 (d, J ¼ 2.2 Hz, 1H, AreH), 8.57 (s, 1H, AreH), 8.30 (dd, J ¼ 8.5, 2.4 Hz, 1H, AreH), 8.12 (dd, J ¼ 8.5, 2.4 Hz, 1H, AreH), 7.69 (d, J ¼ 8.0 Hz, 1H, AreH), 7.67 (d, J ¼ 8.0 Hz, 1H, AreH). 13C NMR (100 MHz, DMSO-d6): d 153.8, 145.6, 143.8, 143.3, 140.3, 136.8, 127.9, 127.8, 125.7, 121.4, 121.1, 120.0, 119.3, 118.8, 113.4, 110.5. MS (ESI): 352 (MþHþ, 100), 374 (MþNaþ, 20). Anal calcd for C16H9N5O5: C, 54.71; H, 2.58; N, 19.94. Found C, 55.03; H, 2.91; N, 19.89. 3.2.10. (E)-3-(Hydroxyimino)-2,30 -bi(3H-indole)-5,50 -dicarbonitrile (4j) Red-brown solid, mp: 56e58  C (from EtOAc/PE ¼ 1:2). IR (KBr) nmax: 3254, 3212, 3075, 2222, 1610, 1448, 1159, 991 cm1. 1H NMR (400 MHz, DMSO-d6): d 12.22 (s, 1H), 11.73 (s, 1H), 8.22 (d, J ¼ 8.3 Hz, 1H, AreH), 8.15 (dd, J ¼ 8.5, 1.5 Hz, 1H, AreH), 7.77 (s, 1H, AreH), 7.50 (d, J ¼ 8.3 Hz, 1H, AreH), 7.47 (d, J ¼ 2.6 Hz, 1H, AreH), 7.41 (dd, J ¼ 8.5, 1.5 Hz, 1H, AreH), 6.63 (s, 1H, AreH). 13C NMR (100 MHz, DMSO-d6): d 149.7, 146.7, 138.3, 137.5, 133.4, 127.3, 125.1, 124.7, 124.6, 124.5, 121.6, 121.0, 118.7, 113.7, 113.6, 111.4, 110.0, 101.7. MS (ESI): 301 (MþH2OþHþ, 100). Anal calcd for C18H9N5O: C, 69.45; H, 2.91; N, 22.50. Found C, 69.22; H, 2.83; N, 22.67. 3.2.11. (E)-Dimethyl 3-(hydroxyimino)-2,30 -bi(3H-indole)-5,50 dicarboxylate (4k) Red-brown solid, mp: 275e277  C (from EtOAc/PE ¼ 1:1). IR (KBr) nmax: 3284, 3138, 3050, 1687, 1548, 1435, 1286 cm1 1H NMR (400 MHz, DMSO-d6): d 13.84 (s, 1H), 12.28 (s, 1H), 9.15 (s, 1H, AreH), 8.56 (s, 1H, AreH), 8.46 (s, 1H, AreH), 8.03 (d, J ¼ 8.1 Hz, 1H,

AreH), 7.84 (d, J ¼ 8.5 Hz, 1H, AreH), 7.58 (d, J ¼ 8.1 Hz, 2H, AreH), 3.86 (s, 3H, CH3), 3.83 (s, 3H, CH3). 13C NMR (100 MHz, DMSO-d6): d 167.6, 166.4, 164.7, 160.6, 154.4, 139.7, 134.9, 134.0, 127.7, 127.0, 126.0, 125.2, 124.4, 123.4, 121.6, 120.0, 112.8, 109.8, 52.6, 52.3. MS (ESI): 378 (MþHþ, 100), 400 (MþNaþ, 30). Anal calcd for C20H15N3O5: C, 63.66; H, 4.01; N, 11.14. Found C, 63.91; H, 3.85; N, 10.78. 3.2.12. (E)-6,60 -Difluoro-2,30 -bi(3H-indol)-3-one oxime (4l) Red-brown solid, mp: 285e287  C (from EtOAc/PE ¼ 1:1). IR (KBr) nmax: 3144 (br s), 1545, 1424, 1373, 1022 cm1. 1H NMR (400 MHz, DMSO-d6): d 13.45 (s, 1H), 11.93 (s, 1H), 8.42 (m, 1H, AreH), 8.30 (m, 1H, AreH), 7.99 (m, 1H, AreH), 7.25 (t, J ¼ 10.0 Hz, 2H, AreH), 7.03 (m, 1H, AreH), 6.92 (m, 1H, AreH). 13C NMR (100 MHz, DMSO-d6): d 164.2, 161.0, 159.3 (d, J ¼ 12.7 Hz), 158.6, 154.1, 137.0 (d, J ¼ 12.7 Hz), 133.4, 128.4 (d, J ¼ 9.8 Hz), 123.8 (d, J ¼ 9.8 Hz), 123.1, 118.2, 112.0 (d, J ¼ 24.1 Hz), 110.0 (d, J ¼ 24.1 Hz), 108.8, 107.7 (d, J ¼ 24.1 Hz), 98.9 (d, J ¼ 24.1 Hz). MS (ESI): 298 (MþHþ, 100). Anal calcd for C16H9F2N3O: C, 64.65; H, 3.05; N, 14.14. Found C, 65.02; H, 3.17; N, 14.05. 3.2.13. (E)-Dimethyl 3-(hydroxyimino)-2,30 -bi(3H-indole)-6,60 dicarboxylate (4m) 1 H NMR (400 MHz, DMSO-d6): d 11.78 (s, 1H), 11.56 (s, 1H), 8.41 (d, J ¼ 0.8 Hz, 1H, AreH), 8.06 (dd, J ¼ 8.4, 1.4 Hz, 1H, AreH), 8.04 (dd, J ¼ 8.0, 1.4 Hz, 1H, AreH), 8.00 (d, J ¼ 0.8 Hz, 1H, AreH), 7.96 (d, J ¼ 8.1 Hz, 1H, AreH), 7.53 (dd, J ¼ 8.4, 1.4 Hz, 1H, AreH), 7.29 (d, J ¼ 8.0 Hz, 1H, AreH), 3.82 (s, 6H, 2CH3). 13C NMR (100 MHz, DMSOd6): d 167.5, 165.7, 150.8, 143.5, 135.4, 133.0, 129.7, 129.2, 129.0, 128.7, 128.5, 122.9, 122.6, 120.1, 118.5, 114.3, 112.5, 108.2, 57.0, 52.2. MS (ESI): 378 (MþHþ, 100), 400 (MþNaþ, 60). 3.2.14. (E)-7,70 -Dimethyl-2,30 -bi(3H-indol)-3-one oxime (4n) Red-brown solid, mp: 235e236  C (from EtOAc/PE ¼ 1:1). IR (KBr) nmax: 3340, 3225, 2922, 1548, 1438, 1377 cm1. 1H NMR

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(400 MHz, DMSO-d6): d 13.25 (s, 1H), 11.78 (s, 1H), 8.40 (d, J ¼ 7.5 Hz, 1H, AreH), 8.29 (d, J ¼ 3.0 Hz, 1H, AreH), 7.86 (d, J ¼ 7.5 Hz, 1H, AreH), 7.24 (d, J ¼ 7.6 Hz, 1H, AreH), 7.11 (t, J ¼ 7.6 Hz, 1H, AreH), 7.06 (d, J ¼ 7.5 Hz, 1H, AreH), 7.02 (d, J ¼ 7.5 Hz, 1H, AreH), 2.49 (s, 6H, 2CH3). 13C NMR (100 MHz, DMSO-d6): d 161.5, 155.8, 155.4, 136.4, 133.5, 131.4, 128.8, 126.3, 125.7, 124.8, 123.7, 121.7, 121.5, 121.2, 120.6, 109.7, 17.1, 16.3. MS (ESI): 290 (MþHþ, 100), 312 (MþNaþ, 15). Anal calcd for C18H15N3O: C, 74.72; H, 5.23; N, 14.52. Found C, 74.61; H, 5.45; N, 14.13. 0

0

3.2.15. 4, 4 -Dimethyl-2,3 -bi(3H-indol)-3-one oxime (4o) Red-brown solid, mp: 237e239  C (from EtOAc/PE ¼ 1:1). IR (KBr) nmax: 3376, 3137, 2921, 1543, 1463, 1438, 1017 cm1. 1H NMR (400 MHz, DMSO-d6): d 13.25 (s, 1H), 11.66 (s, 1H), 8.23 (dd, J ¼ 7.2, 2.4 Hz, 2H, AreH), 7.84 (s, 1H, AreH), 7.33 (d, J ¼ 8.3 Hz, 1H, AreH), 7.28 (d, J ¼ 7.3 Hz, 1H, AreH), 7.16 (t, J ¼ 7.8 Hz, 1H, AreH), 7.00 (d, J ¼ 8.3 Hz, 1H, AreH), 2.40 (s, 3H, CH3), 2.30 (s, 3H, CH3). 13C NMR (100 MHz, DMSO-d6): d 162.5, 155.5, 135.2, 134.9, 132.4, 131.9, 130.1, 129.7, 127.9, 126.7, 124.6, 122.5, 119.4, 118.5, 112.1, 111.9, 21.9, 21.3. MS (ESI): 290 (MþHþ, 100), 312 (MþNaþ, 10). Anal calcd for C18H15N3O: C, 74.72; H, 5.23; N, 14.52. Found C, 75.10; H, 4.97; N, 14.36. 0

0

3.2.16. 2,3 -Bi(1H-indole)-4,4 -dicarbonitrile (4p) Red-brown solid, mp: 168e169  C (from EtOAc/PE ¼ 1:1). IR (KBr) nmax: 3241 (br s), 2861, 2224, 1426, 974 cm1. 1H NMR (400 MHz, DMSO-d6): d 12.03 (s, 1H, NH), 11.75 (s, 1H, NH), 8.28 (d, J ¼ 8.1 Hz, 1H, AreH), 7.89 (d, J ¼ 7.7 Hz, 1H, AreH), 7.75 (t, J ¼ 7.8 Hz, 1H, AreH), 7.67 (d, J ¼ 8.1 Hz, 1H, AreH), 7.59 (d, J ¼ 7.8 Hz, 1H, AreH), 7.36 (d, J ¼ 2.6 Hz, 1H, AreH), 7.24 (t, J ¼ 7.7 Hz, 1H, AreH), 7.01 (s, 1H, AreH). 13C NMR (100 MHz, DMSOd6): d 151.1, 144.3, 135.9, 132.8, 132.7, 127.7, 126.9, 126.4, 125.9, 125.6, 120.1, 117.7, 117.6, 117.2, 117.0, 109.2, 105.6, 101.4. MS (ESI): 301 (MþH2OþHþ, 100). Anal calcd for C18H10N4: C, 76.58; H, 3.57; N, 19.85. Found C, 76.41; H, 3.53; N, 20.06. 3.3. General procedure for the preparation of 5 and 6 To a solution of indole (0.50 mmol), alkyl bromide (0.7 mmol) and NaNO2 (1.0 mmol) in DMF (1 mL) was added RuCl3$3H2O (0.075 mmol) under atmosphere and the mixture was stirred at 40  C for 8e26 h (monitored by TLC). The reaction mixture was concentrated under reduced pressure. The residue was purified by flash chromatography on silica gel (eluant: EtOAc/PE ¼ 1:4) to yield the corresponding product. 3.3.1. (E)-2,30 -Bi(3H-indol)-3-one O-benzyl oxime (5a) Red solid, mp: 201e203  C (from EtOAc/PE ¼ 1:4). IR (KBr) nmax: 3453, 3206, 1542, 1437, 1131. 1H NMR (400 MHz, DMSO-d6): d 11.91 (s, 1H), 8.51 (d, J ¼ 8.4 Hz, 1H), 8.38 (d, J ¼ 2.7 Hz, 1H), 7.91 (d, J ¼ 7.2 Hz, 1H), 7.54 (d, J ¼ 7.2 Hz, 2H), 7.51 (d, J ¼ 8.4 Hz, 1H), 7.46e7.39 (m, 4H), 7.36 (t, J ¼ 7.2 Hz, 1H), 7.25e7.18 (m, 2H), 7.17e7.12 (m, 1H), 5.59 (s, 2H). 13C NMR (100 MHz, DMSO-d6): d 162.0, 157.7, 155.1, 137.3, 137.0, 133.2, 132.6, 129.1, 129.0, 128.8, 127.5, 126.4, 126.0, 123.3, 122.9, 121.7, 121.4, 120.2, 112.6, 108.7, 78.6. HRESIMS calcd for [C23H17N3O þ H]þ 352.1450 (100%), found 352.1439 (100%). 3.3.2. (E)-5,50 -Difluoro-2,30 -bi(3H-indol)-3-one O-benzyl oxime (5b) Red-brown solid, mp: 250e252  C (from EtOAc/PE ¼ 1:4). IR (KBr) nmax: 3410, 2928, 1552, 1468, 1132. 1H NMR (400 MHz, DMSOd6): d 11.98 (s, 1H), 8.37 (d, J ¼ 3.0 Hz, 1H), 8.14 (dd, J ¼ 10.1, 2.6 Hz, 1H), 7.65 (dd, J ¼ 8.0, 2.6 Hz, 1H), 7.54 (d, J ¼ 7.2 Hz, 2H), 7.50 (dd, J ¼ 8.9, 4.6 Hz, 1H), 7.48e7.41 (m, 3H), 7.40e7.36 (m, 1H), 7.26 (dt,

411

J ¼ 2.6, 8.9 Hz, 1H), 7.08 (dt, J ¼ 2.6, 8.9 Hz, 1H), 5.60 (s, 2H). 13C NMR (100 MHz, DMSO-d6): d 161.8 (d, J ¼ 3.2 Hz), 160.5 (d, J ¼ 250.8 Hz), 158.7 (d, J ¼ 234.0 Hz), 154.4, 153.8, 137.1, 133.9, 133.6, 129.1 (d, J ¼ 2.6 Hz), 128.9, 126.8 (d, J ¼ 11.0 Hz), 122.1 (d, J ¼ 9.9 Hz), 121.2 (d, J ¼ 8.4 Hz), 119.1 (d, J ¼ 23.5 Hz), 114.6 (d, J ¼ 26.4 Hz), 113.8 (d, J ¼ 9.9 Hz), 111.4 (d, J ¼ 26.4 Hz), 108.7 (d, J ¼ 4.4 Hz), 107.7, 107.4, 78.9. HRESIMS calcd for [C23H15F2N3O þ H]þ 388.1261 (100%), found 388.1250 (100%). 3.3.3. (E)-3-(Benzyloxyimino)-2,30 -bi(3H-indol)-5,50 -dicarbonitrile (5c) Red solid, mp: 298e300  C (from EtOAc/PE ¼ 1:4). IR (KBr) nmax: 3432, 2923, 1543, 1449, 1128. 1H NMR (400 MHz, DMSO-d6): d 12.59 (s, 1H), 8.89 (s, 1H), 8.62 (s, 1H), 8.29 (s, 1H), 7.98 (dd, J ¼ 8.0, 1.6 Hz, 1H), 7.74 (dd, J ¼ 10.9, 8.3 Hz, 2H), 7.68 (dd, J ¼ 8.5, 1.6 Hz, 1H), 7.66e7.61 (m, 2H), 7.52e7.46 (m, 2H), 7.46e7.42 (m, 1H), 5.72 (s, 2H). 13C NMR (100 MHz, DMSO-d6): d 164.0, 160.4, 153.3, 139.0, 137.8, 137.0, 136.0, 130.3, 129.3, 129.2, 129.1, 129.0, 127.7, 126.5, 126.1, 121.7, 121.3, 120.7, 119.3, 114.4, 108.8, 108.3, 104.3, 79.4. HRESIMS calcd for [C25H15N5O þ H]þ 402.1355 (100%), found 402.1346 (100%). 3.3.4. (E)-6,60 -Difluoro-2,30 -bi(3H-indol)-3-one O-benzyl oxime (5d) Orange solid, mp: 202e203  C (from EtOAc/PE ¼ 1:4). IR (KBr) nmax: 3443, 3204, 2926, 1548, 1463, 1130. 1H NMR (400 MHz, DMSO-d6): d 12.07 (s, 1H), 8.48 (dd, J ¼ 8.8, 5.8 Hz, 1H), 8.44 (d, J ¼ 2.9 Hz, 1H), 7.96 (dd, J ¼ 8.2, 5.8 Hz, 1H), 7.61e7.55 (m, 2H), 7.51e7.45 (m, 2H), 7.45e7.40 (m, 1H), 7.35 (dd, J ¼ 6.3, 2.3 Hz, 1H), 7.33 (dd, J ¼ 6.3, 2.3 Hz, 1H), 7.12 (dt J ¼ 2.3, 9.6 Hz, 1H), 7.03 (dq J ¼ 2.3, 9.6 Hz, 1H), 5.64 (s, 2H). 13C NMR (100 MHz, DMSO-d6): d 165.4 (d, J ¼ 248.5 Hz), 163.6, 160.0 (d, J ¼ 236.6 Hz), 159.9 (d, J ¼ 7.9 Hz), 153.8, 137.2, 133.9, 129.1, 129.0, 128.9, 128.8, 128.7 (d, J ¼ 10.3 Hz), 123.9 (d, J ¼ 9.9 Hz), 123.0, 118.0, 112.2 (d, J ¼ 23.1 Hz), 110.1 (d, J ¼ 23.8 Hz), 108.6, 108.2 (d, J ¼ 24.6 Hz), 99.0 (d, J ¼ 25.7 Hz), 78.8. HRESIMS calcd for [C23H15F2N3O þ H]þ 388.1261 (100%), found 388.1253 (100%). 3.3.5. (E)-2,30 -Bi(3H-indol)-3-one O-(4-methylbenzyl) oxime (5e) Red solid, mp: 193e195  C (from EtOAc/PE ¼ 1:4). IR (KBr) nmax: 3411, 2925, 1542, 1441, 1133. 1H NMR (400 MHz, DMSO-d6): d 11.91 (s, 1H), 8.49 (dd, J ¼ 8.5, 1.6 Hz, 1H), 8.38 (d, J ¼ 2.9 Hz, 1H), 7.89 (d, J ¼ 7.4 Hz, 1H), 7.50 (dd, J ¼ 7.4, 1.6 Hz, 1H), 7.43 (d, J ¼ 7.7 Hz, 2H), 7.41 (d, J ¼ 3.6 Hz, 2H), 7.25e7.17 (m, 4H), 7.17e7.12 (m, 1H), 5.53 (s, 2H), 2.30 (s, 3H). 13C NMR (100 MHz, DMSO-d6): d 162.0, 157.7, 155.0, 138.2, 137.0, 134.2, 133.1, 132.5, 129.6, 129.2, 127.4, 126.4, 126.0, 123.3, 122.9, 121.7, 121.4, 120.1, 112.6, 108.7, 78.6, 21.3. HRESIMS calcd for [C24H19N3O þ H]þ 366.1606 (100%), found 366.1594 (100%). 3.3.6. (E)-5,50 -Difluoro-2,30 -bi(3H-indol)-3-one O-(4methylbenzyl) oxime (5f) Red solid, mp: 246e248  C (from EtOAc/PE ¼ 1:4). IR (KBr) nmax: 3404, 2922, 1548, 1460, 1128. 1H NMR (400 MHz, DMSO-d6): d 12.01 (s, 1H), 8.39 (d, J ¼ 3.0 Hz, 1H), 8.14 (dd, J ¼ 9.8, 2.6 Hz, 1H), 7.63 (dd, J ¼ 8.0, 2.6 Hz, 1H), 7.51 (dd, J ¼ 8.8, 4.6 Hz, 1H), 7.45 (d, J ¼ 8.0 Hz, 1H), 7.43 (d, J ¼ 8.0 Hz, 2H), 7.28 (dt, J ¼ 2.6, 9.8 Hz, 1H), 7.23 (d, J ¼ 8.0 Hz, 2H), 7.08 (dt, J ¼ 2.6, 8.8 Hz, 1H), 5.55 (s, 2H), 2.30 (s, 3H). 13 C NMR (100 MHz, DMSO-d6): d 161.8 (d, J ¼ 2.2 Hz), 160.6 (d, J ¼ 248.7 Hz), 158.7 (d, J ¼ 234.0 Hz), 154.3 (d, J ¼ 2.4 Hz), 153.7 (d, J ¼ 2.1 Hz), 138.4, 134.0, 133.9, 133.6, 129.7, 129.3, 126.8 (d, J ¼ 10.9 Hz), 122.1 (d, J ¼ 10.0 Hz), 121.2 (d, J ¼ 8.8 Hz), 119.1 (d, J ¼ 23.1 Hz), 114.6 (d, J ¼ 25.7 Hz), 113.9 (d, J ¼ 9.9 Hz), 111.5 (d, J ¼ 25.7 Hz), 108.7 (d, J ¼ 4.5 Hz), 107.6 (d, J ¼ 25.3 Hz), 78.9, 21.3. HRESIMS calcd for [C24H17F2N3O þ H]þ 402.1418 (100%), found

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402.1407 (100%). 3.3.7. (E)-5,50 -Dibromo-2,30 -bi(3H-indol)-3-one O-(4methylbenzyl) oxime (5g) Red-brown solid, mp: 195e197  C (from EtOAc/PE ¼ 1:8). IR (KBr) nmax: 3417, 3249, 2925, 1548, 1438, 1133. 1H NMR (400 MHz, DMSO-d6): d 12.14 (d, J ¼ 2.3 Hz, 1H), 8.60 (d, J ¼ 2.0 Hz, 1H), 8.39 (d, J ¼ 3.0 Hz, 1H), 7.99 (d, J ¼ 2.0 Hz, 1H), 7.62 (dd, J ¼ 8.2, 2.0 Hz, 1H), 7.47 (t, J ¼ 8.5 Hz, 2H), 7.44 (dd, J ¼ 8.2, 2.0 Hz, 2H), 7.36 (dd, J ¼ 8.5, 2.0 Hz, 1H), 7.24 (d, J ¼ 8.2 Hz, 2H), 5.57 (s, 2H), 2.31 (s, 3H). 13C NMR (100 MHz, DMSO-d6): d 161.8, 156.4, 153.8, 138.4, 135.8, 135.5, 134.1, 131.4, 129.7, 129.3, 128.0, 126.0, 124.8, 123.0, 122.1, 118.0, 114.8, 114.6, 108.0, 100.0, 79.0, 21.3. HRESIMS calcd for [C24H17Br2N3O  H]þ 519.9660 (50%), 521.9640 (100%), found 519.9669 (50%), 521.9648 (100%). 3.3.8. (E)-2,30 -Bi(3H-indol)-3-one O-(4-fluorobenzyl) oxime (5h) Red solid, mp: 169e171  C (from EtOAc/PE ¼ 1:8). IR (KBr) nmax: 3412, 3144, 2924, 1544, 1440, 1219. 1H NMR (400 MHz, DMSO-d6): d 11.92 (d, J ¼ 2.0 Hz, 1H), 8.50 (dd, J ¼ 6.7, 1.8 Hz, 1H), 8.38 (d, J ¼ 3.1 Hz, 1H), 7.91 (d, J ¼ 7.3 Hz, 1H), 7.64e7.59 (m, 2H), 7.51 (dd, J ¼ 6.7, 1.8 Hz, 1H), 7.43 (dd, J ¼ 4.7, 1.1 Hz, 2H), 7.29e7.21 (m, 4H), 7.18e7.13 (m, 1H), 5.59 (s, 2H). 13C NMR (100 MHz, DMSO-d6): d 162.6 (d, J ¼ 244.3 Hz), 162.0, 157.7, 155.1, 137.0, 133.6 (d, J ¼ 2.9 Hz), 133.2, 132.5, 131.3 (d, J ¼ 8.4 Hz), 127.5, 126.4, 126.0, 123.3, 122.9, 121.7, 121.4, 120.2, 115.9 (d, J ¼ 21.3 Hz), 112.6, 108.7, 77.8. HRESIMS calcd for [C23H16FN3O þ H]þ 370.1356 (100%), found 370.1347 (100%). 3.3.9. (E)-Bi(3H-indol)-3-one O-(4-(trifluoromethyl)benzyl) oxime (5i) Red solid, mp: 214e216  C (from EtOAc/PE ¼ 1:4). IR (KBr) nmax: 3419, 3216, 2925, 1547, 1328, 1122. 1H NMR (400 MHz, DMSO-d6): d 11.90 (d, J ¼ 2.0 Hz, 1H), 8.50 (dd, J ¼ 6.8, 2.0 Hz, 1H), 8.36 (d, J ¼ 2.9 Hz, 1H), 7.97 (d, J ¼ 7.3 Hz, 1H), 7.81 (d, J ¼ 8.2 Hz, 2H), 7.75 (d, J ¼ 8.2 Hz, 2H), 7.51 (dd, J ¼ 6.8, 2.0 Hz, 1H), 7.46e7.42 (m, 2H), 7.25e7.16 (m, 3H), 5.72 (s, 2H). 13C NMR (100 MHz, DMSO-d6): d 161.9, 157.8, 151.5, 142.3, 137.0, 133.3, 132.6, 129.3 (q, J ¼ 31.9 Hz), 129.2, 129.1, 127.7, 126.4, 126.1, 126.0 (q, J ¼ 3.6 Hz), 123.3, 122.9, 121.7, 121.4, 120.2, 112.6, 108.7, 77.5. HRESIMS calcd for [C24H16F3N3O þ H]þ 420.1324 (100%), found 420.1314 (100%). 3.3.10. (E)-5,50 -Dibromo-bi(3H-indol)-3-one O-(4-(trifluoromethyl) benzyl) oxime (5j) Red solid, mp: 235e237  C (from EtOAc/PE ¼ 1:4). IR (KBr) nmax: 3423, 3205, 2927, 1548, 1436, 1117. 1H NMR (400 MHz, DMSO-d6): d 12.08 (s, 1H), 8.59 (d, J ¼ 2.0 Hz, 1H), 8.31 (s, 1H), 8.03 (d, J ¼ 2.0 Hz, 1H), 7.75 (q, J ¼ 8.4 Hz, 4H), 7.60 (dd, J ¼ 8.4, 2.0 Hz, 1H), 7.46 (d, J ¼ 8.4 Hz, 1H), 7.41 (d, J ¼ 8.4 Hz, 1H), 7.34 (dd, J ¼ 8.4, 2.0 Hz, 1H), 5.71 (s, 2H). 13C NMR (100 MHz, DMSO-d6): d 161.7, 156.5, 154.4, 142.1, 135.8, 135.6, 134.0, 129.8, 129.4, 129.3, 129.2 (q, J ¼ 31.6 Hz), 128.0, 126.0, 125.9 (q, J ¼ 3.8 Hz), 124.8, 123.0, 122.1, 118.1, 114.7, 114.6, 108.0, 77.8. HRESIMS calcd for [C24H14Br2F3N3O  H]þ 573.9377 (50%), 575.9357 (100%), 577.9377 (50%), found 573.9386 (50%), 575.9366 (100%), 577.9345 (50%). 3.3.11. (E)-7,70 -Dimethyl-bi(3H-indol)-3-one O-(3-fluorobenzyl) oxime (5k) Brown solid, mp: 134e135  C (from EtOAc/PE ¼ 1:4). IR (KBr) nmax: 3431, 2925, 1550, 1447, 1132. 1H NMR (400 MHz, DMSO-d6): d 11.87 (d, J ¼ 2.0 Hz, 1H), 8.39 (d, J ¼ 7.8 Hz, 1H), 8.29 (d, J ¼ 2.9 Hz, 1H), 7.78 (d, J ¼ 7.2 Hz, 1H), 7.48 (q, J ¼ 7.8 Hz, 1H), 7.40e7.33 (m, 2H), 7.26 (d, J ¼ 7.6 Hz, 1H), 7.21 (t, J ¼ 8.3 Hz, 1H), 7.13 (t, J ¼ 7.6 Hz, 1H), 7.06 (t, J ¼ 7.2 Hz, 1H), 7.04 (t, J ¼ 7.2 Hz, 1H), 5.59 (s, 2H), 2.52 (s, 3H), 2.50 (s, 3H). 13C NMR (100 MHz, DMSO-d6): d 162.6 (d,

J ¼ 243.9 Hz), 160.8, 155.9, 140.4 (d, J ¼ 7.3 Hz), 136.4, 134.5, 131.7, 131.1 (d, J ¼ 8.4 Hz), 129.2, 126.2, 125.9, 125.3, 124.8, 124.7, 123.9, 121.9, 121.7, 121.1, 120.6, 115.6 (d, J ¼ 11.0 Hz), 115.4 (d, J ¼ 11.4 Hz), 109.3, 77.4, 17.2, 16.3. HRESIMS calcd for [C25H20FN3O þ H]þ 398.1669 (100%), found 398.1659 (100%). 3.3.12. (E)-5,50 -Dibromo-bi(3H-indol)-3-one O-(3-chlorobenzyl) oxime (5l) Red solid, mp: 234e235  C (from EtOAc/PE ¼ 1:4). IR (KBr) nmax: 3431, 2927, 1541, 1435, 1133. 1H NMR (400 MHz, DMSO-d6): d 12.15 (d, J ¼ 2.4 Hz, 1H), 8.61 (d, J ¼ 2.0 Hz, 1H), 8.37 (d, J ¼ 3.1 Hz, 1H), 8.04 (d, J ¼ 2.0 Hz, 1H), 7.65e7.61 (m, 2H), 7.55e7.51 (m, 1H), 7.50e7.43 (m, 4H), 7.37 (dd, J ¼ 8.5, 2.0 Hz, 1H), 5.63 (s, 2H). 13C NMR (100 MHz, DMSO-d6): d 161.7, 156.5, 154.2, 139.7, 135.8, 135.7, 135.6, 134.0, 133.7, 131.0, 129.8, 128.8, 128.0, 127.6, 126.0, 124.8, 123.0, 122.1, 118.1, 114.8, 114.6, 108.0, 77.9. HRESIMS calcd for [C23H14Br2ClN3O  H]þ 539.9114 (50%), 541.9093 (100%), 543.9073 (50%), found 539.9127 (50%), 541.9103 (100%), 543.9081 (50%). 3.3.13. (E)-7,70 -Dimethyl-bi(3H-indol)-3-one O-(3-chlorobenzyl) oxime (5m) Red solid, mp: 225e227  C (from EtOAc/PE ¼ 1:4). IR (KBr) nmax: 3415, 2924, 1552, 1434, 1129. 1H NMR (400 MHz, DMSO-d6): d 11.85 (s, 1H), 8.38 (d, J ¼ 7.9 Hz, 1H), 8.28 (d, J ¼ 3.1 Hz, 1H), 7.78 (d, J ¼ 7.0 Hz, 1H), 7.60 (s, 1H), 7.53e7.44 (m, 3H), 7.28 (d, J ¼ 7.7 Hz, 1H), 7.12 (t, J ¼ 7.5 Hz, 1H), 7.04 (t, J ¼ 7.5 Hz, 1H), 7.03 (d, J ¼ 7.0 Hz, 1H), 5.60 (s, 2H), 2.52 (s, 3H), 2.50 (s, 3H). 13C NMR (100 MHz, DMSO-d6): d 160.8, 155.9, 155.9, 140.0, 136.4, 134.6, 133.7, 131.7, 131.0, 129.3, 128.7, 128.6, 127.4, 126.2, 125.9, 125.3, 123.9, 121.9, 121.7, 121.1, 120.6, 109.3, 77.4, 17.2, 16.3. HRESIMS calcd for [C25H20ClN3O þ H]þ 414.1373 (100%), 416.1344 (32%), found 414.1364 (100%), 416.1328 (32%). 3.3.14. (E)-Bi(3H-indol)-3-one O-(2-chloro-4-fluorobenzyl) oxime (5n) Red solid, mp: 232e234  C (from EtOAc/PE ¼ 1:8). IR (KBr) nmax: 3409, 3204, 2923, 1543, 1441, 1130. 1H NMR (400 MHz, DMSO-d6): d 11.93 (s, 1H), 8.49 (d, J ¼ 7.3 Hz, 1H), 8.38 (d, J ¼ 2.3 Hz, 1H), 7.93 (d, J ¼ 7.3 Hz, 1H), 7.75 (t, J ¼ 7.3 Hz, 1H), 7.54 (d, J ¼ 8.8 Hz, 1H), 7.49 (d, J ¼ 7.7 Hz, 1H), 7.47e7.42 (m, 2H), 7.29 (t, J ¼ 8.5 Hz, 1H), 7.21 (d, J ¼ 6.6 Hz, 2H), 7.18e7.12 (m, 1H), 5.65 (s, 2H). 13C NMR (100 MHz, DMSO-d6): d 162.5 (d, J ¼ 248.2 Hz), 161.8, 157.7, 155.5, 137.0, 134.9 (d, J ¼ 10.8 Hz), 133.5 (d, J ¼ 9.2 Hz), 133.3, 132.7, 131.4 (d, J ¼ 3.5 Hz), 127.6, 126.4, 126.1, 123.3, 122.9, 121.7, 121.4, 120.2, 117.4 (d, J ¼ 25.2 Hz), 115.1 (d, J ¼ 21.1 Hz), 112.6, 108.6, 75.2. HRESIMS calcd for [C23H15ClFN3O þ H]þ 404.0966 (100%), 406.0936 (32%), found 404.0953 (100%), 406.0917 (32%). 3.3.15. (E)-5,50 -Dimethoxy-2,30 -bi(3H-indol)-3-one O-(2-chloro-4fluorobenzyl) oxime (5o) Brown solid, mp: 164e165  C (from EtOAc/PE ¼ 1:8). IR (KBr) nmax: 3323, 2865, 1503, 1383. 1H NMR (400 MHz, DMSO-d6): d 8.52 (s, 1H), 8.26 (d, J ¼ 2.8 Hz, 1H), 7.99 (d, J ¼ 2.1 Hz, 1H), 7.75 (t, J ¼ 7.9 Hz, 1H), 7.54 (d, J ¼ 8.8 Hz, 1H), 7.51 (d, J ¼ 2.1 Hz, 1H), 7.37 (d, J ¼ 8.8 Hz, 1H), 7.31 (d, J ¼ 8.4 Hz, 1H), 7.29 (t, J ¼ 6.7 Hz, 1H), 7.00 (dd, J ¼ 8.4, 2.1 Hz, 1H), 6.86 (dd, J ¼ 8.4, 2.1 Hz, 1H), 5.60 (s, 2H), 3.96 (s, 3H), 3.85 (s, 3H). 13C NMR (100 MHz, DMSO-d6): d 162.5 (d, J ¼ 248.8 Hz), 160.4, 157.9, 155.7, 155.4, 151.2, 134.9 (d, J ¼ 10.8 Hz), 133.5 (d, J ¼ 9.2 Hz), 132.1, 131.9, 131.4 (d, J ¼ 3.4 Hz), 127.0, 122.3, 120.5, 117.5 (d, J ¼ 25.2 Hz), 117.0, 115.1 (d, J ¼ 21.1 Hz), 114.6, 113.1, 112.7, 108.5, 105.2, 75.2, 56.0, 55.9. HRESIMS calcd for [C25H19ClFN3O3 þ H]þ 464.1177 (100%), 466.1148 (32%), found 464.1167 (100%), 466.1133 (32%).

H.-E. Qu et al. / European Journal of Medicinal Chemistry 95 (2015) 400e415

3.3.16. (E)-2,30 -Bi(3H-indol)-3-one O-allyl oxime (6a) Red solid, mp: 122e123  C (from EtOAc/PE ¼ 1:8). IR (KBr) nmax: 3371, 2921, 1549, 1431. 1H NMR (400 MHz, DMSO-d6): d 11.91 (s, 1H), 8.51 (d, J ¼ 7.0 Hz, 1H), 8.40 (d, J ¼ 2.9 Hz, 1H), 7.95 (d, J ¼ 7.2 Hz, 1H), 7.51 (dd, J ¼ 7.0, 1.5 Hz, 1H), 7.49e7.41 (m, 2H), 7.31e7.13 (m, 3H), 6.26e6.15 (m, 1H), 5.50 (dd, J ¼ 17.3, 1.5 Hz, 1H), 5.37 (d, J ¼ 10.4 Hz, 1H), 5.06 (d, J ¼ 5.8 Hz, 2H). 13C NMR (100 MHz, DMSO-d6): d 162.0, 157.7, 155.0, 137.0, 134.2, 133.1, 132.5, 127.5, 126.4, 126.0, 123.3, 122.9, 121.7, 121.4, 120.2, 119.3, 112.6, 108.7, 77.6. HRAPCIMS calcd for [C19H15N3O þ H]þ 302.1293 (100%), found 302.1284 (100%). 3.3.17. (E)-7,70 -Dimethyl-2,30 -bi(3H-indol)-3-one O-allyl oxime (6b) Red solid, mp: 168e169  C (from EtOAc/PE ¼ 1:4). IR (KBr) nmax: 3394, 2923, 1554, 1434, 1124. 1H NMR (400 MHz, CDCl3): d 8.60 (d, J ¼ 8.0 Hz, 1H), 8.42 (s, 1H), 8.32 (d, J ¼ 2.4 Hz, 1H), 7.85 (d, J ¼ 6.8 Hz, 1H), 7.27e7.21 (m, 2H), 7.10 (d, J ¼ 7.2 Hz, 1H), 7.04 (t, J ¼ 7.5 Hz, 1H), 6.24e6.12 (m, 1H), 5.47 (dq, J ¼ 17.3, 1.5 Hz, 1H), 5.36 (dq, J ¼ 1.2, 10.5 Hz, 1H), 4.98 (dt, J ¼ 1.2, 5.8 Hz, 2H), 2.63 (s, 3H), 2.52 (s, 3H). 13C NMR (100 MHz, CDCl3): d 161.0, 155.8, 155.7, 135.7, 133.8, 133.6, 129.9, 129.7, 126.1, 125.4, 124.9, 124.0, 122.0, 121.4, 121.0, 120.1, 118.5, 111.0, 77.3, 16.5, 16.2. HRESIMS calcd for [C21H19N3O þ H]þ 330.1606 (100%), found 330.1596 (100%). 3.3.18. (E)-2,30 -Bi(3H-indol)-3-one O-butyl oxime (6c) Red solid, mp: 191e193  C (from EtOAc/PE ¼ 1:8). IR (KBr) nmax: 3363, 2910, 1540, 1421. 1H NMR (400 MHz, DMSO-d6): d 11.89 (s, 1H), 8.52 (dd, J ¼ 6.7, 2.2 Hz, 1H), 8.41 (d, J ¼ 2.9 Hz, 1H), 7.93 (d, J ¼ 7.2 Hz, 1H), 7.52 (dd, J ¼ 6.7, 2.2 Hz, 1H), 7.47e7.41 (m, 2H), 7.28e7.21 (m, 2H), 7.20e7.14 (m, 1H), 4.53 (t, J ¼ 6.5 Hz, 2H), 1.86e1.77 (m, 2H), 1.52e1.43 (m, 2H), 0.96 (t, J ¼ 7.4 Hz, 3H). 13C NMR (100 MHz, DMSO-d6): d 162.0, 157.6, 154.7, 137.0, 133.0, 132.5, 127.3, 126.4, 126.0, 123.3, 122.9, 121.6, 121.5, 120.1, 112.6, 108.8, 76.8, 31.3, 19.1, 14.2. HRESIMS calcd for [C20H19N3O þ H]þ 318.1606 (100%), found 318.1592 (100%). 3.3.19. (E)-2,30 -Bi(3H-indol)-3-one O-cyclopropylmethyl oxime (6d) Red solid, mp: 183e185  C (from EtOAc/PE ¼ 1:8). IR (KBr) nmax: 3461, 3215, 1551, 1443, 1139. 1H NMR (400 MHz, DMSO-d6): d 11.86 (s, 1H), 8.52 (dd, J ¼ 6.5, 2.1 Hz, 1H), 8.40 (d, J ¼ 2.9 Hz, 1H), 7.95 (d, J ¼ 7.3 Hz, 1H), 7.51 (dd, J ¼ 7.0, 1.3 Hz, 1H), 7.43 (d, J ¼ 3.8 Hz, 2H), 7.26e7.14 (m, 3H), 4.36 (d, J ¼ 7.3 Hz, 2H), 1.39e1.30 (m, 1H), 0.66e0.58 (m, 2H), 0.46e0.40 (m, 2H). 13C NMR (100 MHz, DMSOd6): d 162.0, 157.6, 154.6, 137.0, 132.9, 132.5, 127.4, 126.4, 125.9, 123.2, 122.9, 121.6, 121.5, 120.1, 112.6, 108.8, 81. 5, 10.8, 3.5. HRAPCIMS calcd for [C20H17N3O þ H]þ 316.1450 (100%), found 316.1436 (100%). 3.4. In vitro cytotoxicity The NCI-H460, T24, HepG2, SPCA-2, MGC-803, A549, SK-OV-3 and HUVEC cell lines used in this study were all obtained from the Institute of Biochemistry and Cell Biology, China Academy of Sciences. All were supplemented with 10% heat-inactivated fetal bovine serum in a humidified atmosphere of 5% CO2/95% air at 37  C. In order to investigate the potential of compounds 4, 5, 6 and 5-FU, a commercial classical anticancer drug was used as a reference organic drug. Assays of cytotoxicity were determined in 96well, flat bottomed microtiter plates. The supplemented culture medium with cell lines was added to the wells. Compounds 4, 5, 6 and 5-FU were dissolved in the culture medium with 1% DMSO to give various concentrations. The resulted solutions were subsequently added to a set of wells. Control wells contained

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supplemented media with 1% DMSO. The microtiter plates were incubated at 37  C in a humidified atmosphere of 5% CO2/95% air for a further 2 day. Cytotoxic screening by 3-(4, 5-dimethylthiazol-2yl)-2, 5- di-phenyltetrazolium bromide (MTT) assay was conducted. At the end of each incubation period, the MTT solution (10 mL, 10 mg/mL) was added into each well and the cultures were incubated further for 4 h at 37  C in a humidified atmosphere of 5% CO2/ 95% air. After removal of the supernatant, DMSO (100 mL) was added to dissolve the formazan crystals. The absorbance was read by enzyme labeling instrument with 570/630 nm double wavelength measurement. The cytotoxicity was estimated based on the percentage cell survival in a dose dependent manner relative to the negative control. The final IC50 values were calculated by the Bliss method (n ¼ 5). All the tests were repeated in at least three independent experiments. 3.5. Hoechst 333258 staining Cells grown on a sterile cover slip in six-well plates were treated with compounds for a certain range of time. The culture medium containing compounds was removed, and the cells were fixed in 4% paraformaldehyde for 10 min. After being washed twice with PBS, the cells were stained with 0.5 mL of Hoechst 33258 (Beyotime, Haimen, China) for 5 min and then again washed twice with PBS. The stained nuclei were observed under a Nikon ECLIPSETE2000-S fluorescence microscope using 350 nm excitation and 460 nm emissions. 3.6. AO/EB staining Cells were seeded at a concentration of 5  104 cell/mL in a volume of 2 mL on a sterile cover slip in six-well tissue culture plates. Following incubation, the medium was removed and replaced with fresh medium plus 10% fetal bovine serum and supplemented with concentrations of compound 4e. After the treatment period, the cover slip with monolayer cells was inverted on a glass slide with 10 mL of AO/EB stain (100 mg/mL). Fluorescence was read on a Nikon ECLIPSETE2000-S fluorescence microscope (OLYMPUS Co., Japan). 3.7. Mitochondrial membrane potential staining JC-1 (Beyotime, Haimen, China) probe was employed to measure mitochondrial depolarization in T24 cells. Briefly, cells cultured in six-well plates after indicated treatments were incubated with an equal volume of JC-1 staining solution (5 mg/mL) at 37  C for 20 min and rinsed twice with PBS. Mitochondrial membrane potentials were monitored by determining the relative amounts of dual emissions from mitochondrial JC-1 monomers or aggregates using a Nikon ECLIPSETE2000-Sfluorescent microscope. Mitochondrial depolarization is indicated by an increase in the green/ redfluorescence intensity ratio. 3.8. Flow cytometry 3.8.1. Apoptosis analysis Apoptosis was discriminated with the annexin V-FITC/propidium iodide test. Cells were seeded at 2  106/well in 10% FBS DMEM into 6-well plates, and treated with compounds for 24 h. The cells were washed twice with cold Phosphate Buffered Saline (PBS) and then resuspend cells in 1  Binding Buffer (0.1 M Hepes/ NaOH (pH 7.4), 1.4 M NaCl, 25 mM CaCl2) at a concentration of 1  106 cells/mL. Transfer 100 mL of the solution (1  105 cells) to a 5 mL culture tube, and add 5 mL of FITC Annexin V (BD, Pharmingen) and 5 mL propidium iodide (PI) to each tube. Gently vortex the cells

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and incubate for 30 min at RT (25  C) in the dark. Add 200 mL PBS to each tube. Analysis was performed with the system software (Cell Quest; BD Biosciences). Lower left quadrant, viable cells (annexin V_/PI_); lower right quadrant, early apoptotic cells (annexin Vþ/PI_); upper right quadrant, late apoptotic cells (annexin Vþ/PIþ); upper left quadrant, necrotic cells (annexin V_/PIþ). The percentage of cells positive for PI and/or Annexin V-FITC was reported inside the quadrants. 3.8.2. Cell cycle analysis The cells lines were treated with indicated concentrations of compound 4e. After incubated for 48 h, cells were washed twice with ice-cold PBS, fixed and permeabilized with ice-cold 70% ethanol at 20  C overnight. The cells were treated with 100 mg/mL RNase A at 37  C for 30 min after washed with ice-cold PBS, and finally stained with 1 mg/mL propidium iodide (PI) in the dark at 4  C for 30 min. Analysis was performed with the system software (Cell Quest; BD Biosciences). 3.9. ROS assay T24 cells were seeded into six-well plates and subjected to various treatments. Following treatment, cells were incubated with 10 mM DCFH-DA (Beyotime, Haimen, China) dissolved in cell free medium at 37  C for 30 min in dark, and then washed three times with PBS. Cellular fluorescence was quantified using Nikon ECLIPSETE2000-S fluorescence microscope at an excitation of 485 nm and an emission of 538 nm. 3.10. Immunoblotting and real-time quantitative reversetranscription polymerase chain reaction (qRT-PCR) Total RNA was extracted from the T24 cells after treatment with 5 mM, 10 mM, 20 mM oxime-derived bisindole derivatives for 24 h using the RNA pure Kit (Aidlab, RN0302, China) as described previously. RNA samples were reverse-transcribed for 30 min at 42  C with the High Capacity cDNA Reverse Transcription Kit (TaKaRa, Biotechnology, Dalian). The SYBR® Green PCR Master Mix (Fermentas, K0251, Lithuania) and specific primer pairs were used for selected genes, and the primer pair for actin was used as the reference gene. RT-PCR was performed according to the following conditions: 2 min at 50  C, 10 min at 95  C, and 40 cycles of 15 s at 95  C and 1 min at 60  C using 0.5 mL of complementary (c) DNA, 2  SYBR Green PCR Master Mix, and 500 nM of the forward and reverse primers on a 7500 real-time PCR System (Applied Biosystems). The threshold cycle number (Ct) was calculated with the 7500 ABI software. Relative transcript quantities were calculated using the △Ct method with actin as the reference gene amplified from the same samples. Ct is the difference in the threshold cycles of messenger (m)RNA for selected genes relative to those of actin mRNA. The real-time RT-PCR was performed in triplicate for each experimental group. The PCR primers used here are given in Table 2S (See supporting information). 3.11. Western blot Total cell lysates from cultured T24 cells after compound treatments as mentioned earlier were obtained by lysing the cells in icecold RIPA buffer (1  PBS, 1% NP-40, 0.5% sodium deoxycholate and 0.1% SDS) and containing 100 mg/mL PMSF, 5 mg/mL Aprotinin, 5 mg/ mL Leupeptin, 5 mg/mL Pepstatin and 100 mg/mL NaF. After centrifugation at 12,000 rpm for 10 min, the protein in supernatant was quantified by Bradford method (BIO-RAD) using Multimode varioscan instrument (Thermo Fischer Scientifics). Thirty micrograms of protein per lane was applied in 12% SDS polyacrylamide

gel. After electrophoresis, the protein was transferred to polyvinylidine difluoride (PVDF) membrane (Amersham Biosciences). The membrane was blocked at room temperature for 2 h in TBST containing 5% blocking powder (Santacruz). The membrane was washed with TBST for 5 min, and primary antibody was added and incubated at 4  C overnight (O/N). Bax, Bcl-2, cytochrome c, Apaf-1, caspase-9, -7, -3, PARP, cyclin A, cyclin D1, cyclin E, CDK2, CDK4, p16, p21, p27 and p53 antibodies were purchased from Imgenex, USA. After three TBST washes, the membrane was incubated with corresponding horseradish peroxidase-labeled secondary antibody (1:2000) (Santa Cruz) at room temperature for 1 h. Membranes were washed with TBST three times for 15 min and the protein blots were visualized with chemiluminescence reagent (Thermo Fischer Scientifics Ltd.). The X-ray films were developed with developer and fixed with fixer solution. 3.12. Statistics The data were processed by the Student'st-test with the significance level P  0.05 using SPSS. 4. Conclusions In summary, the presence of an oxime group at position 2 seems essential for obtaining good cytotoxic effects of bisindoles, and introduction the polar substituent at C-5, C-50 significantly improved the anti-cancer bioactivities of the compounds. The results from Hoechst 33258 and acridine orange/propidium iodide (AO/EB) staining as well as annexinV-FITC assays provided evidence for an apoptotic cell death, which may be through increasing the intracellular ROS level, up-regulating the caspase-3, caspase-9 and cytochrome c signaling pathway. Flow cytometric data of these compounds showed increased G1 peak, which is suggestive of G1 cell cycle arrest. Furthermore, T24 cells have been found to be dependent on cyclin D1/CDK4 pathway for their continued proliferation. Interestingly, these compounds are effective as cyclin E and have shown a pronounced increase in p53, p21 and p16 activity. Therefore, the promising anti-proliferative and apoptosis-inducing activity of compound 4e can be further utilized as medicinal chemistry lead for future drug discovery. Acknowledgments This study was supported by the National Natural Science Foundation of China (No. 81260472 21362002 and 21431001), “BAGUI Scholar” Project and the Innovation Program for Graduate Students in Jiangsu Province (KYLX_0162) and the Foundation of Ministry of Education Innovation Team (NO. IRT1225). Appendix A. Supplementary data Supplementary data related to this article can be found at http:// dx.doi.org/10.1016/j.ejmech.2015.03.058. References [1] (a) B. Lallemand, F. Chaix, M. Bury, J. Dubois, R. Kiss, J. Med. Chem. 54 (2011) 6501e6513; (b) R. Vennila, S. Kamalraj, J. Muthumary, Biomed. Aging Pathol. 2 (2012) 16e18; (c) S.B. Bharate, J.B. Bharate, S.I. Khan, B.L. Tekwani, B. Singh, R.A. Vishwakarma, Eur. J. Med. Chem. 63 (2013) 435e443; (d) Q.Q. Yu, Y.N. Liu, L. Xu, C.P. Zheng, F.L. Le, X.Y. Qin, Y.Y. Liu, J. Liu, Eur. J. Med. Chem. 82 (2014) 82e95. [2] A. Ahmad, W.A. Sakr, K.M. Wahidur Rahman, Curr. Dr. Targ. 11 (2010) 652e666. [3] P. Singh, P. Kaur, V. Luxami, S. Kaur, S. Kumar, Bioorg. Med. Chem. 15 (2007)

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Synthesis and pharmacological evaluation of novel bisindole derivatives bearing oximes moiety: identification of novel proapoptotic agents.

In an effort to develop potent anti-cancer chemopreventive agents, a novel series of bisindole derivatives bearing oxime moiety were synthesized. Stru...
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