Synthesis and Anticancer Evaluation of 4-Anilinoquinolinylchalcone Derivatives

A series of 4-anilinoquinolinylchalcone derivatives were synthesized and evaluated for antiproliferative activities against the growth of human cancer cell lines (Huh-7 and MDA-MB-231) and normal lung cells (MRC-5). The results exhibited low cytotoxicity against human lung cells (MRC-5). Among them, (E)-3-{4-{[4-(benzyloxy)phenyl]amino}quinolin-2-yl}-1-(4-methoxyphenyl) prop-2-en-1-one (4a) was found to have the highest cytotoxicity in breast cancer cells and low cytotoxicity in normal cells. Compound 4a causes ATP depletion and apoptosis of breast cancer MDA-MB-231 cells and triggers reactive oxygen species (ROS)-dependent caspase 3/7 activation. In conclusion, it is worth studying 4-anilinoquinolinylchalcone derivatives further as new potential anticancer agents for the treatment of human cancers.


Introduction
Cancer is a disease that occurs when changes in a group of normal cells within the body lead to uncontrolled, abnormal growth forming a lump called a tumor [1]; this is true of all cancers except leukemia (cancer of the blood). If left untreated, tumors can grow and spread into the surrounding normal tissue or to other parts of the body via the bloodstream and lymphatic systems and can affect the digestive, nervous, and circulatory systems or release hormones that may affect body function. Liver and breast cancers are common malignancies worldwide and the leading causes of cancer-induced death [2].
Hybrid molecules not only have more favorable properties such as enhanced activity and improved specificity but also could overcome drug resistance, so hybridization of chalcone moiety with other anticancer pharmacophores represents a promising strategy to develop novel anticancer agents with high efficacy [19]. In recent years, numerous chalcone-quinoline hybrids have been prepared and evaluated for their anticancer activities. Some exhibited excellent in vitro and in vivo potency, revealing their potential as putative anticancer drugs [10]. In our previous study, we have synthesized a series of chalcone derivatives in which an aryl moiety was replaced with a quinoline nucleus and evaluated for their biological activities [20][21][22]. Among them, (E)-3-[3-(4-methoxyphenyl)quinolin-2-yl]-1-phenylprop-2-en-1-one (1) was active against the growth of H1299 and SKBR-3 with IC50 values of 1.41 and 0.70 µM, respectively [21].
Lapatinib, a tyrosine kinase dual inhibitor targeting epidermal growth factor receptors (EGFR) and Her2/Neu, has been widely accepted in ongoing preclinical or clinical applications for the treatment of various solid tumors, including those of the breast, lung, liver, head and neck, colon, prostate, gastric and ovarian cancers [23][24][25][26]. Abo-Zeid et al. demonstrated that lapatinib exhibited cytotoxicity on the MDA-MB-231 cell line with an IC50 value of 32.5 µM after 24 h of treatment [27]. Chen et al. have also revealed IC50 values of 2.11, 3.42, and 4.85 µM, respectively, for the lapatinib-treated Huh-7, HepG2, and HA22T cancer cell lines [27]. To further explore 4-anilinoquinolinylchalcone derivatives as potential anticancer agents, a series of novel target compounds ( Figure 1) were designed and synthesized. We considered the hybrid approach utilizing the chalcone structure of compound-1 and Phenylether amine substructure from lapatinib. The target structures are more drug-like with a shape similar to the compact cis-form of Combretastatin A4. They also possessed suitable molecular weights and the number of hydrogen-bond donating/accepting groups according to Lipinski's rule of five. These target compounds were evaluated for their antiproliferative activities against the growth of Huh-7 and MDA-MB-231 cancer cell lines and MRC-5 (normal human lung cells), and the results are outlined in Table 1.     [21]. d lapatinib was used as a reference drug.

Downregulation of ATP by Compound 4a against Breast Cancer Cells
ATP levels are another cell viability indicator [31][32][33]. To further confirm the inhibition of the cell proliferative ability of compound 4a, the cellular ATP levels of breast cancer MDA-MB-231 cells were assessed ( Figure 2). Like the XTT assay, 4a showed dose-responsive downregulation of ATP in breast cancer cells.

Upregulation of Apoptosis by Compound 4a against Breast Cancer Cells
To examine the apoptosis-inducing ability of compound 4a, the annexin V/7aminoactinomycin D (7-AAD) change in breast cancer MDA-MB-231 cells was assessed. Compound 4a showed more significant apoptosis of breast cancer cells at 5 and 10 µM than control and 1 µM (Figure 3). Similarly, some quinoline and chalcone derivatives, such as BPIQ [28] and S17 [30], show apoptosis induction. Compound 4a, consisting of both versatile quinoline and chalcone moieties, was also active in inducing cancer cell apoptosis.
ATP levels are another cell viability indicator [31][32][33]. To further confirm th tion of the cell proliferative ability of compound 4a, the cellular ATP levels o cancer MDA-MB-231 cells were assessed ( Figure 2). Like the XTT assay, 4a dose-responsive downregulation of ATP in breast cancer cells. . Data containing non-ove notes exhibited significant differences (p < 0.05). The letters (a, b, c) were provided by the JMP software for determine significance. "a" is the biggest value and others follow the ord

Upregulation of Apoptosis by Compound 4a against Breast Cancer Cells
To examine the apoptosis-inducing ability of compound 4a, the V/7-aminoactinomycin D (7-AAD) change in breast cancer MDA-MB-231 cells sessed. Compound 4a showed more significant apoptosis of breast cancer cells 10 µM than control and 1 µM ( Figure 3). Similarly, some quinoline and chalcone tives, such as BPIQ [28] and S17 [30], show apoptosis induction. Compound 4a, ing of both versatile quinoline and chalcone moieties, was also active in inducin cell apoptosis.  . Data containing non-overlapping notes exhibited significant differences (p < 0.05). The letters (a, b, c) were provided by the statistical JMP software for determine significance. "a" is the biggest value and others follow the order.
dose-responsive downregulation of ATP in breast cancer cells. . Data containing non-overlapping notes exhibited significant differences (p < 0.05). The letters (a, b, c) were provided by the statistical JMP software for determine significance. "a" is the biggest value and others follow the order.

Upregulation of Apoptosis by Compound 4a against Breast Cancer Cells
To examine the apoptosis-inducing ability of compound 4a, the annexin V/7-aminoactinomycin D (7-AAD) change in breast cancer MDA-MB-231 cells was assessed. Compound 4a showed more significant apoptosis of breast cancer cells at 5 and 10 µM than control and 1 µM (Figure 3). Similarly, some quinoline and chalcone derivatives, such as BPIQ [28] and S17 [30], show apoptosis induction. Compound 4a, consisting of both versatile quinoline and chalcone moieties, was also active in inducing cancer cell apoptosis. MDA-MB-231 cells were treated with 4a for 24 h, and their apoptosis status was determined by flow cytometry. Populations of annexin V (+)/7AAD were assigned as apoptosis (+) (%). Data, means ± SDs (n = 3). Data containing non-overlapping notes exhibited significant differences (p < 0.05). The letters (a, b, c) were provided by the statistical JMP software for determine significance. "a" is the biggest value and others follow the order. Populations of annexin V (+)/7AAD were assigned as apoptosis (+) (%). Data, means ± SDs (n = 3). Data containing non-overlapping notes exhibited significant differences (p < 0.05). The letters (a, b, c) were provided by the statistical JMP software for determine significance. "a" is the biggest value and others follow the order.

Reactive Oxygen Species (ROS) Were Induced by Compound 4a in Breast Cancer Cells
To evaluate the ROS-inducing ability of compound 4a, the flow cytometry-based ROS detection of breast cancer MDA-MB-231 cells was conducted. Compound 4a showed dose-responsive induction of ROS of breast cancer cells (Figure 4).

Upregulation of Caspase 3/7 Activity by Compound 4a against Breast Cancer Cells
Since apoptosis and ROS induction was observed, the role of ROS in apoptosis signaling is worth investigating its ability to trigger apoptosis. This potential role of ROS was examined by the pretreatment of N-acetylcysteine (NAC), a ROS inhibitor [35,36]. Several anticancer studies, such as psoralidin [37], nitrated [6,6,6]tricycles compound SK1 [38], and Aaptos suberitoid extract [39], have demonstrated that NAC can reverse the ROS-induced apoptosis in the example of caspase 3/7 activation.
Using the caspase 3/7 luminescence assay, the modulating ability of breast cancer MDA-MB-231 cells on caspase 3/7 signaling with compound 4a was evaluated. Like annexin V-detected apoptosis (Figure 3), compound 4a was inactive at the concentration of 1 µM. However, it exhibited a significant caspase 3/7 activation of breast cancer cells at 5 and 10 µM ( Figure 5). Moreover, this caspase 3/7 activation with compound 4a was suppressed by NAC pretreatment. These data suggest that compound 4a activates caspase 3/7 of breast cancer cells in an ROS-dependent manner. . Data containing non-overlapping notes exhibited significant differences (p < 0.05). The letters (a, b, c, d) were provided by the statistical JMP software for determine significance. "a" is the biggest value and others follow the order.

Upregulation of Caspase 3/7 Activity by Compound 4a against Breast Cancer Cells
Since apoptosis and ROS induction was observed, the role of ROS in apoptosis signaling is worth investigating its ability to trigger apoptosis. This potential role of ROS was examined by the pretreatment of N-acetylcysteine (NAC), a ROS inhibitor [35,36]. Several anticancer studies, such as psoralidin [37], nitrated [6,6,6]tricycles compound SK1 [38], and Aaptos suberitoid extract [39], have demonstrated that NAC can reverse the ROS-induced apoptosis in the example of caspase 3/7 activation.
Using the caspase 3/7 luminescence assay, the modulating ability of breast cancer MDA-MB-231 cells on caspase 3/7 signaling with compound 4a was evaluated. Like annexin V-detected apoptosis (Figure 3), compound 4a was inactive at the concentration of 1 µM. However, it exhibited a significant caspase 3/7 activation of breast cancer cells at 5 and 10 µM ( Figure 5). Moreover, this caspase 3/7 activation with compound 4a was suppressed by NAC pretreatment. These data suggest that compound 4a activates caspase 3/7 of breast cancer cells in an ROS-dependent manner.

Chemistry
Melting points were determined on an Electrothermal IA9100 (Electrothermal, . Data containing non-overlapping notes exhibited significant differences (p < 0.05). The letters (a, b, c, d) were provided by the statistical JMP software for determine significance. "a" is the biggest value and others follow the order.

Chemistry
Melting points were determined on an Electrothermal IA9100 (Electrothermal, Staffordshire, UK) melting point apparatus and were uncorrected. Nuclear magnetic resonance ( 1 H and 13 C) spectra were recorded on a Varian-Unity-400 spectrometer (Varian, Palo Alto, CA, USA). Chemical shifts were expressed in parts per million (δ) with tetramethylsilane (TMS) as an internal standard. Thin-layer chromatography was performed on silica gel 60 F-254 plates purchased from E. Merck and Co (Darmstadt, Germany). The elemental analyses were performed in the Instrument Center of the National Science Council at National Cheng-Kung University or National Taiwan University using Heraeus CHN-O Rapid EA (Heraeus, Waltham, MA, USA), and all values are within ± 0.4% of the theoretical compositions. (

Antiproliferative Assay
Cancer cells (Huh-7, MDA-MB-231) and normal lung cells (MRC-5) were purchased from Bioresources Collection and Research Center, Taiwan. Cells were maintained in the same standard medium, grown as a monolayer in DMEM (Gibco, Miami, FL, USA), and supplemented with 10% fetal bovine serum (FBS) and antibiotics, i.e., 100 IU/mL penicillin, 0.1 mg/mL streptomycin and 0.25 µg/mL amphotericin. The culture was maintained at 37 • C with 5% CO 2 in a humidified atmosphere. Cells (5 × 10 3 cells/well) were treated with tested compounds for 72 h in the medium containing 10% FBS. Cell viability was quantitated with the use of sodium 3 -[1-(phenylamino-carbonyl)-3,4-tetrazolium}bis(4-methoxy-6-nitro)benzene sulfonic acid hydrate (XTT) colorimetric assay (Biological Industries, Beit-Haemek, Israel). XTT labeling reagent (1 mg/mL) was mixed with electroncoupling reagent, following the manufacturer's instructions, and 50 µL of the mixture was added directly to the cells. The plates were further incubated at 37 • C for 4 h. Color was measured spectrophotometrically in a microtiter plate reader at 492 nm and used as a relative measurement of viable cell numbers. After drug treatment, the viable cell number was compared to the solvent and untreated control cell number. This information was used to determine the percent of control growth as (Ab treated /Ab control ) × 100, where Ab represents the mean absorbance (n = 3). The concentration that killed 50% of cells (IC 50 ) was determined from the linear portion of the curve by calculating the concentration of the agent that reduced the absorbance in treated cells, compared to control cells, by 50%.

Cellular ATP Assay
Cellular ATP was determined using the ATPlite luminescence reagent (PerkinElmer Life Sciences, Boston, MA, USA) [40,41]. According to the user instruction, the cell lysate of treated cells was reacted with ATPlite substrate for 5 min in the dark. Finally, the luminescence was detected by a luminometer (Berthold Technologies GmbH & Co., Bad Wildbad, Germany).

ROS Assay
The oxidative stress indicator, such as ROS, was selected to assess the impact of drug treatment on the change in oxidative stress. According to the manufacturer's instructions, the ROS reacting probe 2 ,7 -dichlorodihydrofluorescein diacetate (H 2 DCFDA) (Sigma-Aldrich) was applied to drug-treated cells [44,45]. Finally, these stained cells were conducted with flow cytometry.

Statistical Analysis
The significance was determined using JMP software 12 (SAS Institute Inc., Cary, NC, USA). Data containing non-overlapping notes exhibited significant differences.

Conclusions
In this study, we synthesized 4-anilinoquinolinylchalcone derivatives and evaluated them in vitro for their anticancer activities. These compounds were more potent in inhibiting Huh-7 and MDA-MB-231 cancer cell proliferation than the reference drug lapatinib.