Discovery of Novel Bromophenol Hybrids as Potential Anticancer Agents through the Ros-Mediated Apoptotic Pathway: Design, Synthesis and Biological Evaluation

A series of bromophenol hybrids with N-containing heterocyclic moieties were designed, and their anticancer activities against a panel of five human cancer cell lines (A549, Bel7402, HepG2, HCT116 and Caco2) using MTT assay in vitro were explored. Among them, thirteen compounds (17a, 17b, 18a, 19a, 19b, 20a, 20b, 21a, 21b, 22a, 22b, 23a, and 23b) exhibited significant inhibitory activity against the tested cancer cell lines. The structure-activity relationships (SARs) of bromophenol derivatives were discussed. The promising candidate compound 17a could induce cell cycle arrest at G0/G1 phase and induce apoptosis in A549 cells, as well as caused DNA fragmentations, morphological changes and ROS generation by the mechanism studies. Furthermore, compound 17a suppression of Bcl-2 levels (decrease in the expression of the anti-apoptotic proteins Bcl-2 and down-regulation in the expression levels of Bcl-2) in A549 cells were observed, along with activation caspase-3 and PARP, which indicated that compound 17a induced A549 cells apoptosis in vitro through the ROS-mediated apoptotic pathway. These results might be useful for bromophenol derivatives to be explored and developed as novel anticancer drugs.


Introduction
The development of anticancer agents is the hot topic in medicinal chemistry based on the high incidence and lethality of cancer today. Although many effective drugs have been approved to treat cancer, therapies for it are less satisfactory due to side effects, thus there is need for novel therapeutic agents. Certain new targeted agents can block the growth of cancer cells by interfering with specific targeted molecules and have less toxicity than chemotherapy drugs, which offer a promise of treating cancer. Reactive oxygen species (ROS) play vital roles in cell growth, which associate with multiple changes in cellular functions (cell proliferation, migration, differentiation, apoptosis, etc.) in cancer cells [1]. Agents targeting ROS-mediated apoptotic pathway have proven to be attractive for cancer therapy [2]. Some natural and synthetic effective anticancer agents targeting ROS-mediated apoptotic pathway were reported [3][4][5].
Bromophenols, a class of natural marine products, and their derivatives possessed various potent activities, including antioxidative, anticancer, antithrombotic, antimicrobial, and antiinflammatory activities, which have attracted much attention [6]. A series of bromophenol hybrids with indolin-2-one moiety were designed and synthesized as potential anticancer agents in our previous work [7]. Among of them, compound WLJ18 ( Figure 1) displayed excellent antitumor activities against various human cancer cell lines and could inactivate invasion and metastasis. These encouraging results prompted us to further design and synthesize a new class of bromophenol derivatives as potential anticancer agents.
N-containing heterocyclic compounds are important compounds with excellent biological activities, such as antiviral, anti-inflammatory and antitumor activities [8]. Especially in new drug discovery, the N-containing heterocyclic moieties were widely introduced into pharmaceutical molecules. For example, the rings of piperidine, morpholine and piperazine were introduced into anticancer drugs (Gefitinib, Vandetanib, SU11274, Sunitinib, etc. (Figure 1)) and had positive effects on activities of drugs along with several properties of molecules, including drug-target interactions, metabolic stability and toxicity [9]. Usually, the combination of different biological units leads to synergistic activity, which is an efficient strategy for the design of novel antitumor agents [10][11][12][13]. Based on the above, we designed and synthesized a series novel of bromophenol hybrids with heterocyclic molecules containing N-containing heterocyclic compounds are important compounds with excellent biological activities, such as antiviral, anti-inflammatory and antitumor activities [8]. Especially in new drug discovery, the N-containing heterocyclic moieties were widely introduced into pharmaceutical molecules. For example, the rings of piperidine, morpholine and piperazine were introduced into anticancer drugs (Gefitinib, Vandetanib, SU11274, Sunitinib, etc. (Figure 1)) and had positive effects on activities of drugs along with several properties of molecules, including drug-target interactions, metabolic stability and toxicity [9]. nitrogen in our continuing research for novel anticancer agents. The antitumor activities of bromophenol hybrids were screened against a series of cancer cell lines in vitro using MTT assay, and the structure-activity relationships (SARs) of these analogs are also discussed. The mechanism studies of the promising candidate compound 17a were further investigated.

Chemistry
The general synthetic methods of compounds are shown in Scheme 1. Firstly, oxindole (1) was reacted with ClSO3H to yield compound 2. Then, compound 2 and 4-bromoaniline were heated for 3 h in THF at 80 °C to afford N-(4-bromophenyl)-2-oxoindoline-5-sulfonamide (3) [7]. The aldehyde 4 was reacted with alkyl dibromide under K2CO3 in dimethylformamide (DMF) to yield intermediates 5-7 [14]. Then compounds 5-7 were treated with the appropriate amine under Et3N in DMF to give compounds 8-16 [15]. Finally, the reactions between intermediates (8-16) and compound 3wereperformed under the condition of Knoevenagel condensation in ethanol with a catalytic amount of piperidine to give the desired derivatives 17-25 in good yields. The structures of compounds 8-25 are shown in Table 1. All of the synthesized derivatives were purified and their structures were characterized by spectroscopic means ( 1 H, 13 C-nuclear magnetic resonance (NMR), and high-resolution mass spectrometer (HRMS)). The configuration of the double bond in compounds 7-25 was assigned to E based on the spectra of 1 H NMR [16,17]. Physicochemical properties (including calculated logarithm of partition coefficient between n-octanol and H2O (cLogP), H2O solubility in mol/L (cLogS), polar surface area (TPSA), hydrogen bond acceptor (Ha), and hydrogen bond donor (Hd)), toxicity profiles (including mutagenic effect, tumorigenic effect, irritating effect and reproductive effect) and drug-likeness scores of these compounds were calculated and predicted using OSIRIS Property Explorer software at URL http://www.organicchemistry.org/prog/peo/ [18]. The calculation of physicochemical properties and prediction of toxicity risks are shown in Table 2

Cytotoxicity
All of the target compounds (17-25) were investigated for their in vitro anti-cancer activity against five human cancer cell lines, A549 (human lung cancer cell line), HepG2 (human hepatocellular carcinoma cell line), Bel7402 (human hepatocellular carcinoma cell line), HCT116 (human colorectal cancer cell line), and Caco2 (Human colonic epithelial cell line), using MTT method with sunitinib as a positive control. The IC 50 values of these compounds are listed in Table 3. As shown in Table 3, the rings of piperidine were introduced to bromophenol derivatives 17a and 17b, which exhibited excellent anticancer activities against the test cancer cell lines. Compounds 18a and 18b with ring of morpholine showed better activities against HCT116, Caco2 and A549 cell lines than that of compound WLJ18. On the contrary, the activity of compound 18c displayed weak activities against Bel7402, HepG2, HCT116 and Caco2, except A549 with the IC 50 value of 4.49 ± 0.73 µg/mL. When 1,4 -bipiperidine unit was introduced to bromophenol, the activities of compound 19a and 19b were increased. The introduction of N,N-diethylpiperidine moiety with two and three carbon chains (20a and 20b) could increase the activities against the test cancer cell lines with excellent IC 50 values. The anticancer activity of compound 21a containing ring of 4-methylpiperazine with two-carbon chain slightly increased comparing to compound WLJ18. When the number of the carbon chain was two atoms, compound 21b exhibited excellent anticancer activities, inhibiting the five cancer cell lines with IC 50 values of 5.20 ± 0.76 µg/mL, 3.25 ± 0.32 µg/mL, 5.83 ± 1.11 µg/mL, 4.43 ± 0.53 µg/mL and 7.52 ± 0.99 µg/mL, respectively. Compound 22a with 4-(pyrimidin-2-yl)piperazin-1-yl ring and two carbon chain showed potent activities against cancer cell lines of HCT116 and Caco2 with the IC 50 values of 3.59 ± 0.25 µg/mL and4.09 ± 0.76 µg/mL. However, its analogs (22b) with three carbon chains showed weak anticancer activities. The chain length could affect the anticancer activities of these hybrids based on the above results. In an effort to gain more potent bromophenol hybrids and their information of the SARs, we probed additional structural changes. The 4-(pyrazin-2-yl) piperazin-1-yl, 4-(2-(dimethylamino)ethyl) piperazin-1-yl and bis (2-hydroxyethyl) amino groups were incorporated with WLJ18. Disappointingly, the anticancer activities of compounds 24 and 25 obviously decreased, which indicated that the moieties of incorporation could affect the steric clash, electron density, or hydrogen-bonding capacity, resulting different anticancer activities of bromophenol hybrids.

Compound 17a Induce Morphological Changes in A549 Cells
Morphological changes of cancer cells are always associated with the growth inhibition induced by cytotoxic agents. We also took photos for the cells after treating compound 17a for 48 h. As shown in Figure 2A, compound 17a treated A549 cells showed morphological changes such as cell shrinkage, deformation and reduced number of viable cells. and 19b were increased. The introduction of N,N-diethylpiperidine moiety with two and three carbon chains (20a and 20b) could increase the activities against the test cancer cell lines with excellent IC50 values. The anticancer activity of compound 21a containing ring of 4-methylpiperazine with twocarbon chain slightly increased comparing to compound WLJ18. When the number of the carbon chain was two atoms, compound 21b exhibited excellent anticancer activities, inhibiting the five cancer cell lines with IC50 values of 5.20 ± 0.76 μg/mL, 3.25 ± 0.32 μg/mL, 5.83 ± 1.11 μg/mL, 4.43 ± 0.53 μg/mL and 7.52 ± 0.99 μg/mL, respectively. Compound 22a with 4-(pyrimidin-2-yl)piperazin-1-yl ring and two carbon chain showed potent activities against cancer cell lines of HCT116 and Caco2 with the IC50 values of 3.59 ± 0.25 μg/mL and4.09 ± 0.76 μg/mL. However, its analogs (22b) with three carbon chains showed weak anticancer activities. The chain length could affect the anticancer activities of these hybrids based on the above results. In an effort to gain more potent bromophenol hybrids and their information of the SARs, we probed additional structural changes. The 4-(pyrazin-2-yl) piperazin-1-yl, 4-(2-(dimethylamino)ethyl) piperazin-1-yl and bis (2-hydroxyethyl) amino groups were incorporated with WLJ18. Disappointingly, the anticancer activities of compounds 24 and 25 obviously decreased, which indicated that the moieties of incorporation could affect the steric clash, electron density, or hydrogen-bonding capacity, resulting different anticancer activities of bromophenol hybrids.

Compound 17a Induce Morphological Changes in A549 Cells
Morphological changes of cancer cells are always associated with the growth inhibition induced by cytotoxic agents. We also took photos for the cells after treating compound 17a for 48 h. As shown in Figure 2A, compound 17a treated A549 cells showed morphological changes such as cell shrinkage, deformation and reduced number of viable cells.

Compound 17a Inhibits Colony Formation Ability of A549 Cells
The colony formation experiment was performed to determine the long-term impact of compound 17a on A549 cells growth. A 10-day colony formation assay was performed in this study. A549 cells were seeded in six-well plates (500 cells/well). Cells were treated with various concentrations of compound 17a (0, 5, 10, 20 µg/mL), and incubated for 10 days to allow colony formation. The results revealed that the colony-forming ability of A549 cells was significantly and dose-dependently suppressed after compound 17a treatment ( Figure 2B,C). As shown in Figure 2C, 464.5 ± 10 of colonies were present in the control panel, whereas after treatment with 5 µg/mL compound 17a the number decreased to 263 ± 37. Further decrease to 133 ± 5 and 55 ± 17 occurred after treatment for 10 and 20 µg/mL, respectively. The results indicated that compound 17a had a significant inhibitory effect on the colony formation of A549 cells.

Compound 17a Induce Apoptosis in A549 Cells
To determine whether the compound 17a-induced reduction in cell viability was responsible for the induction of apoptosis, A549 cells were co-stained with PI and Annexin-V FITC, and the number of apoptotic cells was estimated by flow cytometry. The flow cytometric detection of phosphatidylserine (PS) expression in early apoptosis was employed (using fluorescence-conjugated annexin-V). This combination allows the differentiation among viable cells (AV−/PI−), early-phase apoptotic cells (AV+/PI−), late-phase apoptotic cells (AV+/PI+), and necrotic cells (AV−/PI+). A dose-dependent increase in the percentage of apoptotic cells was noted after the cells were treated for 48 h with compound 17a (0, 5, 10, 20 µg/mL). As shown in Figure 3A,B, 11.45 ± 1.20% of apoptotic cells were present in the control panel, whereas, after treatment with 5 µg/mL compound 17a, the population rose to 14.65 ± 1.06%. Further increase to 24.3 ± 6.36% and 63.8 ± 7.21% occurred after treatments of 10 and 20 µg/mL, respectively. The colony formation experiment was performed to determine the long-term impact of compound 17a on A549 cells growth. A 10-day colony formation assay was performed in this study. A549 cells were seeded in six-well plates (500 cells/well). Cells were treated with various concentrations of compound 17a (0, 5, 10, 20 μg/mL), and incubated for 10 days to allow colony formation. The results revealed that the colony-forming ability of A549 cells was significantly and dose-dependently suppressed after compound 17a treatment ( Figure 2B,C). As shown in Figure 2C, 464.5 ± 10 of colonies were present in the control panel, whereas after treatment with 5 μg/mL compound 17a the number decreased to 263 ± 37. Further decrease to 133 ± 5 and 55 ± 17 occurred after treatment for 10 and 20 μg/mL, respectively. The results indicated that compound 17a had a significant inhibitory effect on the colony formation of A549 cells.

Compound 17a Induce Apoptosis in A549 Cells
To determine whether the compound 17a-induced reduction in cell viability was responsible for the induction of apoptosis, A549 cells were co-stained with PI and Annexin-V FITC, and the number of apoptotic cells was estimated by flow cytometry. The flow cytometric detection of phosphatidylserine (PS) expression in early apoptosis was employed (using fluorescence-conjugated annexin-V). This combination allows the differentiation among viable cells (AV−/PI−), early-phase apoptotic cells (AV+/PI−), late-phase apoptotic cells (AV+/PI+), and necrotic cells (AV−/PI+). A dosedependent increase in the percentage of apoptotic cells was noted after the cells were treated for 48 h with compound 17a (0, 5, 10, 20 μg/mL). As shown in Figure 3A,B, 11.45 ± 1.20% of apoptotic cells were present in the control panel, whereas, after treatment with 5 μg/mL compound 17a, the population rose to 14.65 ± 1.06%. Further increase to 24.3 ± 6.36% and 63.8 ± 7.21% occurred after treatments of 10 and 20 μg/mL, respectively.

Compound 17a Causes DNA Fragmentations and Morphological Changes
An essential hallmark of apoptosis is DNA fragmentation and morphological changes. Morphological changes of apoptotic cells such as nuclear apoptotic bodies were analyzed by fluorescence microscopy with Hoechst 33258 staining. A549 cells were treated with 5, 10 and 20 µg/mL compound 17a for 48 h. As shown in Figure 3C, the treatment of the A549 cells with compound 17a resulted in the induction of chromatin condensation, fragmentation and clear apoptotic bodies that were visualized in fluorescence microscopy.

Compound 17a Induce G0/G1 Cell Cycle Arrest in A549 Cells
To elucidate whether the cytotoxicity induced by the derivatives was due to cell cycle arrest, A549 cells were treated with compound 17a (0, 5, 10, 20 µg/mL) for 48 h. Flow cytometry analysis showed A549 cells, which were treated with compound 17a, arrested in G0/G1 phase in a dose-dependent manner (Figure 4). When compared with control, compound 17a increased the population in the G1 phase from 57.85% to 80.63% at concentration of 20 µg/mL, while the G2/M phase was decreased. These findings denote that compound 17a can induce cell cycle arrest in G0/G1 phase.

Compound 17a Causes DNA Fragmentations and Morphological Changes
An essential hallmark of apoptosis is DNA fragmentation and morphological changes. Morphological changes of apoptotic cells such as nuclear apoptotic bodies were analyzed by fluorescence microscopy with Hoechst 33258 staining. A549 cells were treated with 5, 10 and 20 μg/mL compound 17a for 48 h. As shown in Figure 3C, the treatment of the A549 cells with compound 17a resulted in the induction of chromatin condensation, fragmentation and clear apoptotic bodies that were visualized in fluorescence microscopy.

Compound 17a Induce G0/G1 Cell Cycle Arrest in A549 Cells
To elucidate whether the cytotoxicity induced by the derivatives was due to cell cycle arrest, A549 cells were treated with compound 17a (0, 5, 10, 20 μg/mL) for 48 h. Flow cytometry analysis showed A549 cells, which were treated with compound 17a, arrested in G0/G1 phase in a dosedependent manner (Figure 4). When compared with control, compound 17a increased the population in the G1 phase from 57.85% to 80.63% at concentration of 20 μg/mL, while the G2/M phase was decreased. These findings denote that compound 17a can induce cell cycle arrest in G0/G1 phase. Cell cycle is regulated by a family of protein kinase complexes, including CDKs and cyclins, in eukaryotic cells [19]. The previous study has reported that the reduced activities of CDK 4 and cyclin D1 are the hallmarks of cell cycle arrest at the G1/S phase [20,21]. In this study, Western blot analysis showed that treatment of A549 cells with compound 17a at 20 μg/mL significantly decreased the level of cyclin D1 and CDK4.

Compound 17a Triggers ROS Generation
ROS are highly harmful elements to cells as they initiate oxidative stress and ultimately cause cellular damage. Excessive ROS generation renders cells vulnerable to apoptosis. To determine whether compound 17a triggers ROS generation in A549 cells to induce apoptosis, the ROS level was measured using 2′,7′-dichlorodihydrofluoresce in diacetate (DCFH-DA) as fluorescent probe. DCFH- Cell cycle is regulated by a family of protein kinase complexes, including CDKs and cyclins, in eukaryotic cells [19]. The previous study has reported that the reduced activities of CDK 4 and cyclin D1 are the hallmarks of cell cycle arrest at the G1/S phase [20,21]. In this study, Western blot analysis showed that treatment of A549 cells with compound 17a at 20 µg/mL significantly decreased the level of cyclin D1 and CDK4.

Compound 17a Triggers ROS Generation
ROS are highly harmful elements to cells as they initiate oxidative stress and ultimately cause cellular damage. Excessive ROS generation renders cells vulnerable to apoptosis. To determine whether compound 17a triggers ROS generation in A549 cells to induce apoptosis, the ROS level was measured using 2 ,7 -dichlorodihydrofluoresce in diacetate (DCFH-DA) as fluorescent probe. DCFH-DA is cleaved by intracellular esterases into its non-fluorescent form (DCFH), which is converted to a green fluorescent product, carboxy-DCF, via oxidation. A rapid production of ROS occurred after the exposure of A549 cells to compound 17a ( Figure 5A,B). When compared with control (100%), the mean DCF fluorescence increased by 150.10 ± 22.60%, 177.32 ± 15.60% and 214.60 ± 18.90%, when treated with 17a for 48 h. Treatment of compound 17a also increased the green fluorescence intensity in A549 cells ( Figure 5C).
Mar. Drugs 2017, 15, 343 9 of 18 DA is cleaved by intracellular esterases into its non-fluorescent form (DCFH), which is converted to a green fluorescent product, carboxy-DCF, via oxidation. A rapid production of ROS occurred after the exposure of A549 cells to compound 17a ( Figure 5A,B). When compared with control (100%), the mean DCF fluorescence increased by 150.10 ± 22.60%, 177.32 ± 15.60% and 214.60 ± 18.90%, when treated with 17a for 48 h. Treatment of compound 17a also increased the green fluorescence intensity in A549 cells ( Figure 5C). Cells were harvested and analyzed using FACS. (C) Cells were washed twice with PBS and analyzed using fluorescence microscopy (bar = 50 μm). All data were representative of three independent experiments. * p < 0.05; ** p < 0.01 vs. control group.

Effect of Compound 17a on the Expression of Apoptosis-Related Proteins
The Bcl-2 family members are important regulators of mitochondrial function during apoptosis. It has been recognized that accumulation of ROS does not kill cells directly, it triggers an apoptotic signing pathway that leads to cell death, such as increase the Bax/Bcl-2 ratio and activate caspase-3 and PARP [22]. In our study, the treatment of A549 cells induces decrease in the expression of the anti-apoptotic proteins Bcl-2 and down-regulation in the expression levels of Bcl-2 ( Figure 6). Treatment with compound 17a also activated caspase-3 and PARP in A549 cells ( Figure 6). All these data demonstrated that compound 17a induced A549 cells apoptosis in vitro, probably through the ROS-mediated apoptotic pathway.

Effect of Compound 17a on the Expression of Apoptosis-Related Proteins
The Bcl-2 family members are important regulators of mitochondrial function during apoptosis. It has been recognized that accumulation of ROS does not kill cells directly, it triggers an apoptotic signing pathway that leads to cell death, such as increase the Bax/Bcl-2 ratio and activate caspase-3 and PARP [22]. In our study, the treatment of A549 cells induces decrease in the expression of the anti-apoptotic proteins Bcl-2 and down-regulation in the expression levels of Bcl-2 ( Figure 6). Treatment with compound 17a also activated caspase-3 and PARP in A549 cells ( Figure 6). All these data demonstrated that compound 17a induced A549 cells apoptosis in vitro, probably through the ROS-mediated apoptotic pathway.

Chemistry
Reaction reagents were purchased from J&K Scientific Ltd. (Beijing, China). Organic solvents were analytical reagent grade and purchased from Tianjin Chemical Reagent Co., Ltd. (Tianjin, China) Column chromatography (CC): silica gel (200-300 mesh; Qingdao Makall Group Co., Ltd.; Qingdao, China). All reactions were monitored using thin-layer chromatography (TLC) on silica gel plates. 1 H and 13 C-NMR spectra were recorded on Bruker DRX 500 MHz spectrometers with tetramethylsilane (TMS) as the internal standard (Bruker, Bremerhaven, Germany). MS and HRMS spectra were determined on a LCMS-IT-TOF mass spectrometer (Shimadzu, Kyoto, Japan). Melting points were determined on a SGW X-4 Melting Point Apparatus (Shanghai Precision Science Instrument Co., Ltd.; Shanghai, China). The synthesized compounds were named using ChemBioDraw Ultra software (v 12.0, PerkinElmer, Waltham, MA, USA).

General Procedures for the Preparation of Compounds 5-7
The mixture of anhydrous K2CO3 (1.00 eq.) and the 3-bromo-4-hydroxy-5methoxybenzaldehyde 4 (1.00 eq.) were suspended in dry DMF. Then, an alkyl dibromide (1.10 eq.) was added in several portions (neat) during 0.5 h. This reaction mixture was stirred at 60-80 °C overnight. After complete turnover, H2O was added and the aqueous solution was extracted three times with chloroform. The combined organic layers washed with H2O, HCl and brine. Then, the organic layer was dried with Na2SO4 and removed in vacuo. The resulting residue was purified by

Chemistry
Reaction reagents were purchased from J&K Scientific Ltd. (Beijing, China). Organic solvents were analytical reagent grade and purchased from Tianjin Chemical Reagent Co., Ltd. (Tianjin, China) Column chromatography (CC): silica gel (200-300 mesh; Qingdao Makall Group Co., Ltd., Qingdao, China). All reactions were monitored using thin-layer chromatography (TLC) on silica gel plates. 1 H and 13 C-NMR spectra were recorded on Bruker DRX 500 MHz spectrometers with tetramethylsilane (TMS) as the internal standard (Bruker, Bremerhaven, Germany). MS and HRMS spectra were determined on a LCMS-IT-TOF mass spectrometer (Shimadzu, Kyoto, Japan). Melting points were determined on a SGW X-4 Melting Point Apparatus (Shanghai Precision Science Instrument Co., Ltd., Shanghai, China). The synthesized compounds were named using ChemBioDraw Ultra software (v 12.0, PerkinElmer, Waltham, MA, USA).

General Procedures for the Preparation of Compounds 5-7
The mixture of anhydrous K 2 CO 3 (1.00 eq.) and the 3-bromo-4-hydroxy-5-methoxybenzaldehyde The combined organic layers washed with H 2 O, HCl and brine. Then, the organic layer was dried with Na 2 SO 4 and removed in vacuo. The resulting residue was purified by column chromatography on silica gel to provide compounds 5-7. Details of synthesis of compounds 5-7 are given in the Supplementary Data.

General Procedure for the Preparation of Compounds 8-16
To a solution of compounds 5-7 (0.1 mmol) in DMF, the appropriate amine (0.3 mmol) and Et 3 N (160 µL, 1.17 mmol) was added. After stirring at room temperature (unless otherwise indicated) overnight, the mixture was poured into H 2 O. Then, the organic phase was extracted three times with EtOAc (4 × 30 mL) and washed with H 2 O (2 × 30 mL) and with brine (2 × 30 mL). The solvent was evaporated, and the residue was purified by column chromatography on silica gel to provide the compounds 8-16. Details of synthesis of compounds 8-16 are given in the Supplementary Data.

General Procedures for the Preparation of Compounds 17-25
The synthesis of compounds 2 and 3 were performed as our previous report [7]. Piperidine (50 µL) was added to a mixture of compound 3 (0.5 mmol) and appropriate aldehydes 8-16 (0.55 mmol) in ethanol (5 mL). The reaction mixture was heated to refluxing and stirred for 2 h, and TLC analysis indicated when the reaction was complete. The crude product was filtered, washed with ethanol and dried in a vacuum (if no solid precipitated, the crude product was chromatographed using a silica gel column) to afford the title compounds 17-25 as yellow solid.

MTT Assay
The cell viability was assessed by MTT assay as described previously [7]. Briefly, the cells were plated at a density of 3 × 10 3 cells/well in 96-well plates and incubated at 37 • C overnight before drug exposure. Cells were incubated with the tested compounds to achieve final concentrations (0, 2.5, 5, 10, 20, 40, 80 µg/mL) for 48 h. Twenty microliters of MTT (5 mg/mL) was added to each well and allowed to react for another 4 h. After removing the supernatant, 150 µL of DMSO was added to dissolve the formazan and the plates were read at 490 nm. All experiments were carried out in triplicate and the viability of the control cells was taken as 100% cell survival. The IC 50 values were analyzed by GraphPad Prime 5.0.

Colony Forming Assay
A549 cells (500 cells/well) were seeded in six-well plates. After 24 h, cells were treated with various concentrations of compound 17a (0, 5, 10, and 20 µg/mL) and incubated for 15 days to allow colony formation. The cells were then washed with PBS, fixed with 4% paraformalclehyde for 10 min. Next, colonies were stained with 0.1% crystal violet for 15 min at room temperature. The excel crystal violet solution was washed away with distilled H 2 O, colonies containing more than 50 cells were counted and evaluated [23].

Cell Cycle Analysis
The cell cycle was determined by flow cytometry with DNA staining to reveal the total amount of DNA. Briefly, A549 cells were seeded into six-well plates at a density of 5 × 10 5 cells per well. The cells were treated with 5, 10 and 20 µg/mL compound 17a for 48 h. Then the cells were harvested, washed twice with cold PBS, and fixed in cold 75% ethanol at −20 • C overnight. The next day, the cells were washed twice with cold PBS, re-suspended with cold PBS and stained with 20 µg/mL RnaseA (Sigma, St. Louis, MO, USA) for 30 min at 37 • C. The cells were washed and re-suspended with cold PBS containing 50 µg/mL propidium iodide (PI, Sigma, St. Louis, MO, USA) for 30 min at room temperature in the dark. The cell cycle phase distribution was analyzed in three different experiments using flow cytometry (Becton Dickinson, Franklin Lakes, NJ, USA).

Cell Apoptosis Analysis
A549 cells (2 × 10 5 /well) were plated in six-well plates and incubated for 24 h, then treated with test compounds with various concentration compound 17a (0, 5, 10, 20 µg/mL) for 48 h. Then 1~5 × 10 5 cells were harvested, washed twice with cold PBS, re-suspended in 500 µL Annexin V Binding Buffer, and 5 µL Annexin V-FITC and 5 µL PI were added. After being stained in the dark for 10min at room temperature, the cells were analyzed in three different experiments using flow cytometry (Becton Dickinson, Franklin Lakes, NJ, USA).