Design, Synthesis, and Anticancer Activity Studies of Novel Quinoline-Chalcone Derivatives

Abstract

The chalcone and quinoline scaffolds are frequently utilized to design novel anticancer agents. As the continuation of our work on effective anticancer agents, we assumed that linking chalcone fragment to the quinoline scaffold through the principle of molecular hybridization strategy could produce novel compounds with potential anticancer activity. Therefore, quinoline-chalcone derivatives were designed and synthesized, and we explored their antiproliferative activity against MGC-803, HCT-116, and MCF-7 cells. Among these compounds, compound 12e exhibited a most excellent inhibitory potency against MGC-803, HCT-116, and MCF-7 cells with IC₅₀ values of 1.38, 5.34, and 5.21 µM, respectively. The structure–activity relationship of quinoline-chalcone derivatives was preliminarily explored in this report. Further mechanism studies suggested that compound 12e inhibited MGC-803 cells in a dose-dependent manner and the cell colony formation activity of MGC-803 cells, arrested MGC-803 cells at the G2/M phase and significantly upregulated the levels of apoptosis-related proteins (Caspase3/9 and cleaved-PARP) in MGC-803 cells. In addition, compound 12e could significantly induce ROS generation, and was dependent on ROS production to exert inhibitory effects on gastric cancer cells. Taken together, all the results suggested that directly linking chalcone fragment to the quinoline scaffold could produce novel anticancer molecules, and compound 12e might be a valuable lead compound for the development of anticancer agents.

1. Introduction

Chalcone is a natural product template which shows many versatile pharmacological activities especially anticancer activities [1,2,3]. Due to its simple chemistry and ease of synthesis, a large number of chalcone derivatives was discovered with variety of promising biological activity [4]. In fact, chalcone compounds have shown good therapeutic effects and clinical application potential as anticancer drugs for the treatment of human cancers [5,6,7]. In addition, chalcone fragment was also frequently utilized to design novel agents with other anticancer moieties to enhance the biological efficacy by the molecular hybridization strategy [8,9,10,11,12,13,14,15]. 4-Aminochalcone derivative 1 [12] displayed excellent inhibitory activity against NCI-H460, A549, and H1975 cells with IC₅₀ values of 2.3, 3.2, and 5.7 μM, respectively. Compound 1 was able to trigger ROS-mediated apoptosis in time- and concentration-dependent manners in NCI-H460 cells. In addition, compound 1 also displayed a better safety profile in animal models. Chalcone dithiocarbamate derivative 2 [13] was reported as a LSD1 inhibitor with an IC₅₀ value of 0.14 μM. Compound 2 exhibited potent anticancer activity against MOLT-4 cells (IC₅₀ = 0.87 μM) and was significantly effective in suppressing the growth of MOLT-4 xenograft tumor mouse model. Compound 3 [14] was successfully designed by the structural combination of the 1,3,5-triazine and chalcone fragments via a diether linker with potent antiproliferative activity against the MCF-7 and HCT116 cells (GI₅₀ = 0.127 and 0.116 μM, respectively). Additionally, compound 3 could potently inhibit the activity of cellular DHFR and TrxR in HCT-116 cells. [1,2,4] Triazolo [1,5-a] pyrimidine–chalcone derivative 4 [15] inhibited MGC-803 cells at the nanomolar level (GI₅₀ = 0.64 μM) and exhibited its anti-proliferative potency via inducing autophagy and increasing ROS level (Figure 1). Therefore, chalcone fragment might be a valuable and core moiety to design anticancer agents. In this work, we continued our efforts on the discovery of chalcone derivatives as potential anticancer activity.

Quinolines, as one class of N-containing heterocycles, have numerous advantages over other non-nitrogenous, which are widely used as “parental” compounds to synthesize molecules with variety of promising biological activity [16,17,18,19]. The quinoline motifs are frequently found in many compounds that show potent anticancer activity with different mechanisms [20,21,22,23,24,25,26,27]. Anlotinib [23] (multi-kinase inhibitor) and Bosutinib [24] (Src-Abl inhibitor), which are quinoline-based protein kinase inhibitors, have been approved for the treatment of human cancers. Quinoline–chalcone derivative 5 [25] as a potent tubulin inhibitor showed excellent anticancer potency with IC₅₀ values at nanomolar levels. In addition, compound 5 could arrest the cell cycle at the G2/M phase, induce apoptosis, depolarize mitochondria, and induce ROS generation in K562 cells. Quinoline chalcone 6 [26] is effective in exhibiting potent activity against HL60 cells with an IC₅₀ values of 0.59 μM. Novel phenylsulfonylurea derivative 7 [27] as an anticancer agent exhibited potent cytotoxicity activity against HepG-2, A549, and MCF-7 cells (IC₅₀ = 2.71, 7.47, and 6.55 μM, respectively) as well as moderate PI3K/mTOR dual inhibitory activity (Figure 2). Therefore, in this work, we use the quinoline moiety as the core scaffold of molecules to discover novel quinoline-based anticancer agents.

Molecular hybridization strategy is extensively used in drug design and discovery based on the combination of different bioactive moieties to produce new hybrids with the improved activities [28]. These interesting findings about chalcones and quinolines as anticancer agents led to molecular hybridization strategy of chalcone and quinoline scaffolds to generate novel anticancer agents. In this work, as the continuation of our work on the development of anticancer agents, we designed and synthesized novel quinoline-chalcone derivatives as anticancer activity (Figure 3).

2. Results and Discussion

2.1. Chemistry

Target quinoline-chalcone derivatives were synthesized by outlined procedures in Scheme 1. Commercially available 4-aminoacetophenone (8) reacted with aromatic aldehydes 9a–9j to afford compounds 10a–10j in the presence of NaOH in EtOH at 25 °C. Substitution reaction between compounds 10a–10j with commercially available 4-chloro-2-methylquinoline (11) gave target compounds 12a–12j in the presence of HCl in EtOH at 80 °C. In addition, Compounds 12a, 12b, 12e, and 12f then reacted with iodomethane or iodoethane in the presence of KOH in acetonitrile at 80 °C to give compounds 13a–13f. All the compounds were characterized by means of NMR and HREI-mass spectra which are showed in the Supplementary Materials.

2.2. Antiproliferative Activity and Structure Activity Relationship Analysis

According to the latest statistics from the International Agency for Research on Cancer (IARC), in 2020, the number of newly diagnosed patients of colorectal cancer, gastric cancer, and breast cancer ranked second, third, and fourth in China, respectively [29]. Therefore, the in vitro antiproliferative activities of new target compounds 12a–12j and 13a–13f were evaluated against MGC-803 cell line (human gastric cancer cells), HCT-116 cell line (human colon cancer cells), and MCF-7 (human breast cancer cells), with the 5-fluorouracil (5-Fu) as a positive control. The following Table 1 depicted the results of antiproliferative activity.

As shown in Table 1, most of quinoline-chalcone derivatives displayed potent antiproliferative activity against MGC-803, HCT-116 and MCF-7 cells with IC₅₀ values <20 µM. Particularly, compound 12e exhibited most excellent inhibitory potency against MGC-803, HCT-116, and MCF-7 cells with IC₅₀ values of 1.38, 5.34, and 5.21 µM, respectively, which were much lower than that of 5-Fu (IC₅₀ values = 6.22 µM, 10.4 μM, and 11.1 μM, respectively), which indicated that compound 12e was effective in inhibiting the activity of three kinds of tumor cells. In addition, most of compounds was more sensitive to MGC-803 cells than that of HCT-116 and MCF-7 cells. Therefore, the structure–activity relationships were discussed according to the results of antiproliferative activity of MGC-803 cells. As shown in Table 1, the types and positions of substituents (R₁) on chalcone group (A ring) have an important influence on its antiproliferative activity. Compared with 12f, compounds 12a–12e with electron-donating groups of A ring exhibited enhanced activity than compounds without substitution groups of A ring, but compounds 12g and 12i, with electron-withdrawing groups of A ring, despaired the antiproliferative activity more potent than that of compound 12f. In addition, the position of substituents (R₁) is also important. The inhibitory activity of compounds was less potent when the substituents (R₁) were at the 3-position of chalcone group (A ring) than that of the substituents (R₁) were at the 3-position of A ring (compounds 12b vs. 12c, 12g vs. 12i, and 12h vs. 12j). However, compound 12e with a 3,4,5-triOCH₃ substituent of chalcone group (A ring) exhibited better activity. The relationships between the electron-donating groups and electron-withdrawing groups of chalcone group (A ring) and the inhibitory potency on MGC-803 cells were 3,4,5-triOCH₃ > 3,4-diOCH₃ > 4-CH₃ > 4-Br > 4-OCH₃ > 3-OCH₃ > 3-Br > H > 4-Cl > 3-Cl. Next, the influence of R₂ was further explored. As shown in Table 1, the inhibitory activity of compounds 13a–13f was decreased when the H group was replaced by CH₃ or CH₃CH₂ substituent (compounds 13a vs. 12f, 13b vs. 12a, 13c vs. 12d, 13d vs. 12g, 13e vs. 12e, and 13f vs. 12e), indicating that the substituents of R₂ could not improve the inhibitory potency.

Based on above the antiproliferative activity results of compounds, we can conclude that linking quinoline fragment to the chalcone scaffold produces new hybrids with potential anticancer activity. The types and positions of substituents (R₁) on chalcone group (A ring) make a great influence on the inhibitory potency of compounds. Substituents of R₂ impaired the inhibitory potency of compounds (Figure 4).

2.3. Compound 12e Inhibited Gastric Cancer Cells

Compound 12e in this series of compounds showed the most excellent inhibitory activity against MGC-803 cells. The cell viability of the MGC-803 cells was significantly decreased after the treatment with different concentrations of compound 12e for 48 h (Figure 5D). The cell inhibition rate of the high concentration treatment group increased by more than 60%. Cell proliferation inhibition could be caused by cell cycle arrestment. As shown in Figure 5A,C, the results of cell cycle analysis showed that gastric cancer cells MGC-803 could arrest the cells in G2/M phase. The percentage of cells was upregulated by 20%. Compound 12e also showed significant activity on inhibiting the activity of cell colony formation (Figure 5B). With long-term low concentration treatment of compound 12e, the formatted colony was significantly decreased from the concentration of 900 nM. The detection of apoptosis-related proteins showed that the levels of cleaved-Caspase3/9 and cleaved-PARP, the markers of apoptosis, were significantly upregulated (Figure 5E). In conclusion, compound 12e could inhibit gastric cancer cells by arresting cells in G2/M phase and inducing apoptosis of MGC-803 cells.

2.4. Compound 12e Inhibited Gastric Cancer Cells through the Generation of Reactive Oxygen Species (ROS)

It has been reported in the literature that the molecular skeleton of chalcone can induce the production of ROS [12,15]. The assay labeling ROS using DCFH-DA probe revealed that compound 12e could effectively induce ROS generation at concentrations as low as 500 nM (Figure 6A). To investigate the direct relationship between the induction of ROS generation and tumor cell inhibition by compound 12e, gastric cancer cells (MGC-803 and SGC-7901 cells) were treated with the ROS inhibitor NAC in combination with compound 12e. As shown in Figure 6B,C, the results showed that the presence of NAC significantly reversed the cytostatic effect caused by compound 12e. The above results suggested that compound 12e could significantly induce ROS generation and was dependent on ROS production to exert inhibitory effects on gastric cancer cells.

3. Materials and Methods

All the chemical reagents were purchased from commercial suppliers (Energy chemical Company, Shanghai, China). Melting points were determined on an X-5 micromelting apparatus (Fukai Instrument, Beijing, China). NMR spectra data was recorded with a Bruker DPX 400 MHz spectrometer (Bruker, Billerica, MA, USA). High-resolution mass spectra (HRMS) data was obtained using a Waters Micromass Q-TOF Micromass spectrometer (Waters, Manchester, UK) by electrospray ionization (ESI).

3.1. Synthesis of Compounds 10a–10j

A solution of commercially available 4-aminoacetophenone 8 (1.0 mmol, 1.0 eq), aromatic aldehyde 9a–9j (1.0 mmol, 1.0 eq), NaOH (2.0 mmol, 2.0 eq) and were added into 20 mL EtOH at 25 °C. After 6 h, 20 mL water was added to the reaction mixture, giving yellow solid. Then, the crude product was obtained by filtration without further purification.

3.2. Synthesis of Compounds 12a–12j and 13a–13f

A solution of compounds 10a–10j (1.0 mmol, 1.0 eq), 4-chloro-2-methylquinoline 11 (1.0 mmol, 1.0 eq) were added into 10 mL EtOH at 80 °C in the presence of HCl. After 8 h, organic phases were evaporated to obtain crude products, and then were purified to give compounds 12a–12j by column chromatography. A solution of compounds 12a, 12b, 12e, 12f, and 12e (1.0 mmol, 1.0 eq), iodomethane or iodoethane (2.0 mmol, 2.0 eq), and KOH (2.0 mmol, 2.0 eq) were added into 10 mL acetonitrile at 80 °C. After 6 h, the reaction was quenched with water and the crude product extracted with ethyl acetate three times, organic phases were evaporated to obtain crude products, then were purified to give compounds 13a–13f by column chromatography (PE:EA = 5:1).

(E)-1-(4-((2-methylquinolin-4-yl)amino)phenyl)-3-(p-tolyl)prop-2-en-1-one (12a). Yellow powder, Yield, 68%, m.p.124–126 °C. ¹H NMR (400 MHz, DMSO-d₆) δ 9.31 (s, 1H), 8.30 (dd, J = 8.5, 1.4 Hz, 1H), 8.24–8.18 (m, 2H), 7.94 (d, J = 15.6 Hz, 1H), 7.87 (d, J = 8.3 Hz, 1H), 7.82–7.76 (m, 2H), 7.73 (d, J = 7.3 Hz, 1H), 7.71–7.67 (m, 1H), 7.56–7.51 (m, 1H), 7.50 (s, 1H), 7.48 (s, 1H), 7.33–7.23 (m, 3H), 2.56 (s, 3H), 2.36 (s, 3H). ¹³C NMR (100 MHz, DMSO-d₆) δ 187.52, 159.42, 149.29, 147.00, 146.09, 143.55, 140.90, 132.64, 131.52, 130.93, 130.01, 129.96, 129.26, 129.08, 124.85, 122.68, 121.47, 119.93, 119.06, 106.02, 25.60, 21.55. HRMS m/z calcd. for C₂₆H₂₃N₂O, [M + H]⁺: 379.1805, found: 379.1812.

(E)-3-(4-methoxyphenyl)-1-(4-((2-methylquinolin-4-yl)amino)phenyl)prop-2-en-1-one (12b). Yellow powder, Yield, 64%, m.p.111–113 °C. ¹H NMR (400 MHz, DMSO-d₆) δ 9.29 (s, 1H), 8.29 (dd, J = 8.5, 1.4 Hz, 1H), 8.24–8.16 (m, 2H), 7.89–7.82 (m, 4H), 7.74–7.66 (m, 2H), 7.50 (dd, J = 10.1, 7.1 Hz, 3H), 7.24 (s, 1H), 7.07–6.99 (m, 2H), 3.83 (s, 3H), 2.55 (d, J = 2.3 Hz, 3H). ¹³C NMR (100 MHz, DMSO-d₆) δ 187.46, 159.42, 149.38, 146.23, 144.70, 138.42, 131.92, 130.65, 129.96, 129.05, 128.04, 124.84, 122.69, 119.90, 119.56, 119.24, 119.17, 113.09, 110.95, 105.81, 55.95, 25.57. HRMS m/z calcd. for C₂₆H₂₃N₂O₂, [M + H]⁺: 395.1754, found: 395.1758.

(E)-3-(3-methoxyphenyl)-1-(4-((2-methylquinolin-4-yl)amino)phenyl)prop-2-en-1-one (12c). Yellow powder, Yield, 66%, m.p.173–175 °C. ¹H NMR (400 MHz, DMSO-d₆) δ 9.32 (s, 1H), 8.29 (dd, J = 8.5, 1.4 Hz, 1H), 8.26–8.19 (m, 2H), 8.00 (d, J = 15.6 Hz, 1H), 7.87 (dd, J = 8.5, 1.3 Hz, 1H), 7.75–7.68 (m, 2H), 7.57–7.48 (m, 4H), 7.44 (dt, J = 7.8, 1.4 Hz, 1H), 7.38 (t, J = 7.8 Hz, 1H), 7.26 (s, 1H), 7.03 (ddd, J = 8.1, 2.6, 1.0 Hz, 1H), 3.85 (s, 3H), 2.56 (s, 3H). ¹³C NMR (100 MHz, DMSO-d₆) δ 193.40, 160.16, 159.44, 143.53, 137.15, 137.08, 131.12, 131.06, 130.37, 130.04, 129.86, 128.97, 128.70, 124.94, 122.68, 122.03, 119.97, 119.08, 118.93, 114.81, 114.69, 113.64, 55.42, 25.49. HRMS m/z calcd. for C₂₆H₂₃N₂O₂, [M + H]⁺: 395.1754, found: 395.1760.

(E)-3-(3,4-dimethoxyphenyl)-1-(4-((2-methylquinolin-4-yl)amino)phenyl)prop-2-en-1-one (12d). Yellow powder, Yield, 69%, m.p.153–155 °C. ¹H NMR (400 MHz, DMSO-d₆) δ 9.30 (s, 1H), 8.30 (dd, J = 8.5, 1.4 Hz, 1H), 8.25–8.18 (m, 2H), 7.92–7.83 (m, 2H), 7.75–7.66 (m, 2H), 7.57 (d, J = 2.0 Hz, 1H), 7.54–7.46 (m, 3H), 7.39 (dd, J = 8.4, 2.0 Hz, 1H), 7.25 (s, 1H), 7.03 (d, J = 8.4 Hz, 1H), 3.88 (s, 3H), 3.83 (s, 3H), 2.55 (s, 3H).¹³C NMR (100 MHz, DMSO-d₆) δ 187.40, 159.40, 151.61, 149.53, 149.26, 146.84, 146.16, 144.02, 131.73, 130.88, 129.97, 129.08, 128.20, 124.84, 124.32, 122.68, 120.02, 119.90, 119.11, 112.04, 111.05, 105.87, 56.22, 56.07, 25.59. HRMS m/z calcd. for C₂₇H₂₅N₂O₃, [M + H]⁺: 425.1860, found: 425.1869.

(E)-1-(4-((2-methylquinolin-4-yl)amino)phenyl)-3-(3,4,5-trimethoxyphenyl)prop-2-en-1-one (12e). Brown power, Yield, 60%, m.p.122–124 °C. ¹H NMR (400 MHz, DMSO-d₆) δ 9.36 (s, 1H), 8.30 (dd, J = 8.5, 1.4 Hz, 1H), 8.26–8.20 (m, 2H), 7.95 (d, J = 15.5 Hz, 1H), 7.86 (d, J = 8.3 Hz, 1H), 7.75–7.65 (m, 2H), 7.54–7.48 (m, 3H), 7.25 (d, J = 3.5 Hz, 3H), 3.88 (s, 6H), 3.73 (s, 3H), 2.55 (s, 3H). ¹³C NMR (100 MHz, DMSO-d₆) δ 187.46, 153.60, 152.85, 144.00, 140.09, 131.49, 131.01, 130.91, 130.01, 129.03, 124.90, 122.68, 121.65, 119.92, 119.05, 106.89, 105.21, 60.63, 56.62, 56.19, 25.56. HRMS m/z calcd. for C₂₈H₂₇N₂O₄, [M + H]⁺: 455.1965, found: 455.1975.

(E)-1-(4-((2-methylquinolin-4-yl)amino)phenyl)-3-phenylprop-2-en-1-one (12f). Yellow powder, Yield, 58%, m.p.265-267 °C. ¹H NMR (400 MHz, DMSO-d₆) δ 11.10 (s, 1H), 8.89 (d, J = 8.5 Hz, 1H), 8.34 (d, J = 8.6 Hz, 2H), 8.17 (d, J = 8.4 Hz, 1H), 8.05–7.98 (m, 2H), 7.92 (dd, J = 6.7, 3.0 Hz, 2H), 7.83–7.76 (m, 2H), 7.74 (dd, J = 6.7, 4.8 Hz, 2H), 7.52–7.44 (m, 3H), 7.04 (s, 1H), 2.69 (s, 3H). ¹³C NMR (100 MHz, DMSO-d₆) δ 192.85, 160.84, 153.04, 151.68, 149.78, 136.85, 135.87, 131.12, 130.30, 129.59, 129.45, 128.90, 128.72, 128.54, 127.31, 126.55, 123.61, 123.54, 120.28, 114.25, 25.33. HRMS m/z calcd. for C₂₅H₂₁N₂O, [M + H]⁺: 365.1648, found: 365.1655.

(E)-3-(4-chlorophenyl)-1-(4-((2-methylquinolin-4-yl)amino)phenyl)prop-2-en-1-one (12g). Yellow powder, Yield, 58%, m.p.203–205 °C. ¹H NMR (400 MHz, DMSO-d₆) δ 11.07 (s, 1H), 8.86 (d, J = 8.6 Hz, 1H), 8.34 (d, J = 8.2 Hz, 2H), 8.15 (d, J = 8.5 Hz, 1H), 7.99 (dd, J = 22.2, 7.4 Hz, 4H), 7.79 (s, 1H), 7.74 (d, J = 6.3 Hz, 2H), 7.71 (s, 1H), 7.54 (d, J = 8.2 Hz, 2H), 7.04 (s, 1H), 2.68 (s, 3H). ¹³C NMR (100 MHz, DMSO-d₆) δ 188.29, 155.64, 153.94, 143.14, 142.67, 138.98, 135.67, 135.61, 134.27, 134.14, 131.13, 130.85, 129.48, 127.25, 124.78, 124.20, 123.10, 120.26, 117.24, 101.84, 20.32. HRMS m/z calcd. for C₂₅H₂₀ClN₂O, [M + H]⁺: 399.1259, found: 399.1261.

(E)-3-(4-bromophenyl)-1-(4-((2-methylquinolin-4-yl)amino)phenyl)prop-2-en-1-one (12h). Yellow powder, Yield, 67%, m.p.133–135 °C. ¹H NMR (400 MHz, DMSO-d₆) δ 9.32 (s, 1H), 8.29 (dd, J = 8.5, 1.4 Hz, 1H), 8.24–8.18 (m, 2H), 8.03 (d, J = 15.6 Hz, 1H), 7.87 (d, J = 8.5 Hz, 3H), 7.73–7.65 (m, 4H), 7.53 (d, J = 7.4 Hz, 1H), 7.51–7.47 (m, 2H), 7.26 (s, 1H), 2.56 (s, 3H). ¹³C NMR (100 MHz, DMSO-d₆) δ 192.55, 187.43, 142.10, 136.81, 135.12, 134.69, 132.35, 131.71, 131.59, 131.15, 131.07, 130.26, 130.02, 129.03, 128.92, 124.93, 122.70, 122.34, 118.83, 106.50, 25.56. HRMS m/z calcd. for C₂₅H₂₀BrN₂O, [M + H]⁺: 443.0754, found: 443.0756.

(E)-3-(3-chlorophenyl)-1-(4-((2-methylquinolin-4-yl)amino)phenyl)prop-2-en-1-one (12i). Yellow powder, Yield, 63%, m.p.212–214 °C. ¹H NMR (400 MHz, DMSO-d₆) δ 9.55 (s, 1H), 8.35 (dd, J = 16.5, 8.3 Hz, 1H), 8.26–8.06 (m, 3H), 7.86 (ddd, J = 11.1, 6.2, 2.6 Hz, 2H), 7.77–7.61 (m, 2H), 7.56–7.46 (m, 5H), 7.35 (t, J = 2.2 Hz, 1H), 7.23 (d, J = 23.6 Hz, 1H), 2.55 (d, J = 5.1 Hz, 3H). ¹³C NMR (100 MHz, DMSO-d₆) δ 187.37, 159.37, 149.33, 147.46, 146.10, 141.69, 137.67, 134.28, 131.15, 131.11, 131.07, 130.39, 129.94, 129.02, 128.31, 128.27, 124.82, 124.17, 123.02, 120.06, 118.91, 106.32, 25.60. HRMS m/z calcd. for C₂₅H₂₀ClN₂O, [M + H]⁺: 399.1259, found: 399.1266.

(E)-3-(3-bromophenyl)-1-(4-((2-methylquinolin-4-yl)amino)phenyl)prop-2-en-1-one (12j). Yellow powder, Yield, 64%, m.p.219–221 °C. ¹H NMR (400 MHz, DMSO-d₆) δ 9.33 (d, J = 9.0 Hz, 1H), 8.29–8.22 (m, 3H), 8.08 (d, J = 15.6 Hz, 1H), 7.87 (dd, J = 8.2, 3.8 Hz, 2H), 7.74–7.62 (m, 3H), 7.55–7.47 (m, 3H), 7.41 (q, J = 7.6 Hz, 2H), 7.27 (s, 1H), 2.55 (d, J = 4.8 Hz, 3H). ¹³C NMR (100 MHz, DMSO-d₆) δ 187.34, 159.43, 149.31, 147.30, 145.99, 141.69, 137.92, 133.30, 131.40, 131.17, 131.13, 129.98, 129.11, 128.68, 124.90, 124.10, 122.90, 122.69, 119.98, 118.91, 106.28, 25.61. HRMS m/z calcd. for C₂₅H₂₀BrN₂O, [M + H]⁺: 443.0754, found: 443.0761.

(E)-1-(4-(methyl(2-methylquinolin-4-yl)amino)phenyl)-3-phenylprop-2-en-1-one (13a). Brown power, Yield, 57%, m.p.160–162 °C. ¹H NMR (400 MHz, DMSO-d₆) δ 8.07–7.99 (m, 3H), 7.92–7.82 (m, 3H), 7.73 (ddd, J = 8.4, 6.8, 1.5 Hz, 1H), 7.70–7.61 (m, 2H), 7.49–7.41 (m, 5H), 6.78–6.71 (m, 2H), 3.52 (s, 3H), 2.69 (s, 3H). ¹³C NMR (100 MHz, DMSO-d₆) δ 188.47, 155.66, 153.97, 144.68, 142.52, 138.93, 135.74, 135.10, 134.31, 131.24, 130.82, 129.46, 129.41, 127.30, 124.81, 124.11, 122.33, 120.24, 117.19, 101.79, 25.92, 20.34. HRMS m/z calcd. for C₂₆H₂₃N₂O, [M + H]⁺: 379.1805, found: 379.1809.

(E)-1-(4-(methyl(2-methylquinolin-4-yl)amino)phenyl)-3-(p-tolyl)prop-2-en-1-one (13b). Yellow powder, Yield, 55%, m.p. 86–88 °C. ¹H NMR (400 MHz, DMSO-d₆) δ 8.06–7.99 (m, 3H), 7.83 (d, J = 15.6 Hz, 1H), 7.76–7.70 (m, 3H), 7.68–7.60 (m, 2H), 7.50–7.42 (m, 2H), 7.25 (d, J = 7.9 Hz, 2H), 6.78–6.71 (m, 2H), 3.52 (s, 3H), 2.69 (s, 3H), 2.34 (s, 3H). ¹³C NMR (100 MHz, DMSO-d₆) δ 192.74, 160.79, 152.99, 151.74, 138.61, 137.05, 133.07, 131.08, 130.30, 129.99, 129.62, 129.54, 129.31, 127.51, 127.36, 126.51, 123.64, 123.52, 120.20, 114.33, 25.31, 21.52, 21.30. HRMS m/z calcd. for C₂₇H₂₅N₂O, [M + H]⁺: 392.1889, found: 393.1964.

(E)-3-(4-methoxyphenyl)-1-(4-(methyl(2-methylquinolin-4-yl)amino)phenyl)prop-2-en-1-one (13c). Yellow powder, Yield, 63%, m.p.114–116 °C. ¹H NMR (400 MHz, DMSO-d₆) δ 8.05–7.98 (m, 3H), 7.80 (d, J = 8.8 Hz, 2H), 7.76–7.69 (m, 2H), 7.68–7.61 (m, 2H), 7.48–7.42 (m, 2H), 7.00 (d, J = 8.8 Hz, 2H), 6.78–6.70 (m, 2H), 3.81 (s, 3H), 3.52 (s, 3H), 2.68 (s, 3H). ¹³C NMR (100 MHz, DMSO-d₆) δ 192.63, 187.44, 161.58, 160.94, 149.48, 143.10, 137.74, 131.60, 130.95, 130.84, 130.43, 129.28, 126.53, 123.74, 123.45, 119.91, 114.88, 114.68, 114.44, 114.06, 55.78, 55.60, 25.14. HRMS m/z calcd. for C₂₇H₂₅N₂O₂, [M + H]⁺: 409.1911, found: 409.1919.

(E)-3-(4-chlorophenyl)-1-(4-(methyl(2-methylquinolin-4-yl)amino)phenyl)prop-2-en-1-one (13 d). Yellow powder, Yield, 67%, m.p.159–161 °C. ¹H NMR (400 MHz, DMSO-d₆) δ 8.03 (dd, J = 8.2, 5.8 Hz, 3H), 7.94–7.87 (m, 3H), 7.73 (ddd, J = 8.4, 6.8, 1.5 Hz, 1H), 7.68–7.62 (m, 2H), 7.54–7.49 (m, 2H), 7.48–7.43 (m, 2H), 6.74 (d, J = 9.0 Hz, 2H), 3.53 (s, 3H), 2.69 (s, 3H). ¹³C NMR (100 MHz, DMSO-d₆) δ 192.30, 160.83, 153.10, 151.63, 149.80, 135.96, 134.80, 133.47, 131.27, 131.15, 130.29, 129.62, 129.06, 128.74, 127.22, 126.53, 123.60, 123.53, 120.31, 114.22, 40.74. HRMS m/z calcd. for C₂₆H₂₂ClN₂O, [M + H]⁺: 413.1415, found: 413.1420.

(E)-1-(4-(methyl(2-methylquinolin-4-yl)amino)phenyl)-3-(3,4,5-trimethoxyphenyl)prop-2-en-1-one (13e). Yellow powder, Yield, 61%, m.p.162–164 °C. ¹H NMR (400 MHz, DMSO-d₆) δ 8.10–7.98 (m, 3H), 7.84 (d, J = 15.5 Hz, 1H), 7.73 (ddd, J = 8.4, 6.8, 1.5 Hz, 1H), 7.67–7.58 (m, 2H), 7.51–7.43 (m, 2H), 7.18 (s, 2H), 6.79–6.71 (m, 2H), 3.85 (s, 6H), 3.71 (s, 3H), 3.53 (s, 3H), 2.69 (s, 3H). ¹³C NMR (100 MHz, DMSO-d₆) δ 187.17, 160.78, 153.57, 152.79, 149.70, 143.36, 131.07, 130.99, 130.28, 129.55, 126.46, 123.77, 123.48, 121.84, 120.14, 119.91, 114.57, 114.42, 107.48, 106.75, 60.61, 60.53, 56.58, 56.14, 25.32. HRMS m/z calcd. for C₂₉H₂₉N₂O₄, [M + H]⁺: 469.2122, found: 469.213.

(E)-1-(4-(ethyl(2-methylquinolin-4-yl)amino)phenyl)-3-(3,4,5-trimethoxyphenyl)prop-2-en-1-one (13f). Yellow powder, Yield, 62%, m.p.148–150 °C. ¹H NMR (400 MHz, DMSO-d₆) δ 8.01 (dd, J = 8.3, 6.4 Hz, 2H), 7.85–7.70 (m, 2H), 7.63–7.57 (m, 1H), 7.55–7.46 (m, 1H), 7.45 (d, J = 8.6 Hz, 1H), 7.17 (s, 1H), 6.84–6.76 (m, 1H), 6.68 (d, J = 8.8 Hz, 1H), 6.63–6.57 (m, 1H), 4.00 (q, J = 7.0 Hz, 1H), 3.94 (q, J = 7.0 Hz, 1H), 3.84 (s, 3H), 3.70 (s, 2H), 3.63 (d, J = 5.6 Hz, 3H), 3.41 (d, J = 5.2 Hz, 3H), 2.70 (d, J = 7.4 Hz, 3H), 1.23 (dt, J = 21.2, 7.0 Hz, 3H). ¹³C NMR (100 MHz, DMSO-d₆) δ 186.97, 160.87, 153.57, 151.83, 150.24, 149.91, 143.17, 139.92, 131.16, 131.01, 130.21, 129.64, 128.09, 126.53, 124.09, 123.73, 121.89, 121.14, 114.07, 106.73, 60.60, 56.57, 47.26, 25.38, 13.32. HRMS m/z calcd. for C₃₀H₃₁N₂O₄, [M + H]⁺: 483.2278, found: 483.2290.

3.3. Cell Culture

Cell lines used were cultured in humidified incubator at 37 °C and 5% CO₂. The RPMI-1640 medium was supplemented with 10% fetal bovine serum, penicillin (100 U/mL), and streptomycin (0.1 mg/mL). All the cells were obtained from the Cell Bank of Type Culture Collection of Chinese Academy of Sciences (Shanghai, China).

3.4. MTT Assay

Cell lines were seeded into 126-well plates and incubated for 24 h. Then, cells were treated with different concentrations of compounds. Additionally, after another 48 h, MTT reagent (20 μL per well) was added and then incubated at 37 °C for 4 h. Formazan was then dissolved with DMSO. Absorbencies of formazan solution were measured at 4120 nm. The IC₅₀ values of tested compounds were calculated by SPSS version 17.0.

3.5. Western Blotting Analysis

Gastric cancer cells were seeded in dishes and treated with 12e or DMSO. After 48 h, MGC-803 cells were collected and then lysed. The denatured lysates of each group were electrophoretic separated in SDS-PAGE. Proteins were then transferred onto PVDF membranes from gels. After blocking for 2 h, membranes were incubated with primary antibodies conjugation. Then, the membranes were washed and incubated with 2nd antibodies. At last, specific proteins were detected.

3.6. General Methods

In this work, some other assays including colony formation assay were referred to our previous work [30,31].

4. Conclusions

Chalcone and quinoline are common scaffolds found in many compounds with many versatile pharmacological activities, especially anticancer activities. In this work, we assumed that linking chalcone fragment to the quinoline scaffold through the principle of molecular hybridization strategy could produce novel compounds with potential anticancer activity. Therefore, quinoline-chalcone derivatives were designed and synthesized, and we explored their antiproliferative activity against MGC-803, HCT-116, and MCF-7 cells. Among these compounds, compound 12e exhibited a most excellent inhibitory potency against MGC-803, HCT-116, and MCF-7 cells, with IC₅₀ values of 1.38, 5.34, and 5.21 µM, respectively, which were much lower than that of 5-FU (IC₅₀ values = 6.22, 10.4, and 11.1 μM, respectively). Further mechanism studies suggested that compound 12e inhibited the cell colony formation activity of MGC-803 cells in a dose-dependent manner. Meanwhile, compound 12e could arrest MGC-803 cells at the G2/M phase and significantly upregulate levels of apoptosis-related proteins (Caspase3/9 and cleaved-PARP) in MGC-803 cells. In addition, compound 12e could significantly induce ROS generation, and was dependent on ROS production to exert inhibitory effects on gastric cancer cells. Taken together, all the results suggested that compound 12e might be a valuable lead compound for the development of anticancer agents.

Supplementary Materials

The supplementary materials are available online.

Author Contributions

S.-Y.Z., W.L., and J.S. designed the research and contributed to revision of manuscript; Y.-F.G., X.-J.L., W.-B.L., and G.-X.Y. performed the synthetic work; X.-Y.Y., Y.-R.L., Y.-B.Z. and X.-Y.T. were responsible for the direction of the biological research; and Y.-F.G. contributed to writing of the manuscript. All authors have read and agreed to the published version of the manuscript.

Funding

This work was supported by the National Natural Sciences Foundations of China (U2004123 for Sai-Yang Zhang) and China Postdoctoral Science Foundation (No. 2019M632812 for Sai-Yang Zhang) and the Henan Scientific Innovation Talent Team, Department for Education (No. 19ITSTHN001 for Wen Zhao, China). Henan Association of Science and Technology (No. 2020HYTP056 for Sai-Yang Zhang, China) and Science and Technology Department of Henan Province (No. 20202310144, for Sai-Yang Zhang, China). The open fund of state key laboratory of Pharmaceutical Biotechnology, Nan-jing University, China (Grant no. KF-GN-202101).

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

Data of the compounds is available from the authors.

Conflicts of Interest

The authors declare no conflict of interest.

Sample Availability

Samples of the compounds are available from the authors.

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Figures, Scheme and Table

Figure 1. Structures of chalcone derivatives as anticancer agents previously reported.

Figure 2. Structures of quinoline derivatives as anticancer agents previously reported.

Figure 3. Design of novel quinoline-chalcone derivatives as anticancer agents.

Scheme 1. Synthesis of compounds 12a–12j and 13a–13f. Reagents and conditions: (a) EtOH, NaOH, 25 °C, 6 h; (b) EtOH, HCl, 80 °C, 8 h; and (c) iodomethane or iodoethane, KOH, acetonitrile, 80 °C, 8 h.

Figure 4. Summary of the structure-activity relationships.

Figure 5. Compound 12e inhibited gastric cancer cells. (A,C) Cell-cycle distribution detection, MGC-803 cells were treated with indicated concentrations of compound 12e for 48 h; (B) Cell colonies formatted, MGC-803 cells were treated indicated concentrations of compound 12e for 7 d; (D) Cell viability of MGC-803 cells, cells were treated indicated concentrations of compound 12e for 48 h; and (E) Apoptosis related proteins, MGC-803 cells were treated indicated concentrations of compound 12e for 48 h.

Figure 6. Compound 12e induced ROS generation. (A) The levels of ROS in MGC-803 cells, cells were treated indicated concentrations of compound 12e for 48 h, then ROS was labeled by DCFH-DA. (B,C) Cell viabilities of two gastric cancer cells, cells were treated with indicated concentrations of compound 12e and/or NAC for 48 h. Date were expressed as mean ± SD, n = 3. Three individual experiments were implemented for per group. ** p < 0.01, *** p < 0.001 vs. control.

Table 1

In vitro antiproliferative activity of compounds 12a–12j and 13a–13f against MGC-803, HCT-116, and MCF-7 cells.

Figure 6. Compound 12e induced ROS generation. (A) The levels of ROS in MGC-803 cells, cells were treated indicated concentrations of compound 12e for 48 h, then ROS was labeled by DCFH-DA. (B,C) Cell viabilities of two gastric cancer cells, cells were treated with indicated concentrations of compound 12e and/or NAC for 48 h. Date were expressed as mean ± SD, n = 3. Three individual experiments were implemented for per group. ** p < 0.01, *** p < 0.001 vs. control.

Compounds	R1	R2	IC50 (μM) a
			MGC-803	HCT-116	MCF-7
12a	4-CH3	H	1.86 ± 0.23	7.22 ± 1.28	6.86 ± 0.41
12b	4-OCH3	H	2.39 ± 0.36	8.61 ± 1.02	6.32 ± 0.47
12c	3-OCH3	H	2.63 ± 0.18	6.41 ± 0.78	4.43 ± 0.22
12d	3,4-diOCH3	H	1.62 ± 0.11	5.62 ± 0.41	7.86 ± 1.02
12e	3,4,5-triOCH3	H	1.38 ± 0.21	5.34 ± 0.39	5.21 ± 0.51
12f	H	H	3.24 ± 0.43	7.25 ± 0.82	8.23 ± 1.07
12g	4-Cl	H	3.82 ± 0.18	13.2 ± 1.39	7.41 ± 0.82
12h	4-Br	H	2.28 ± 0. 32	9.22 ± 0.82	7.82 ± 1.03
12i	3-Cl	H	4.25 ± 0.41	8.77 ± 0.74	10.21 ± 1.24
12j	3-Br	H	3.22 ± 0.19	16.2 ± 1.55	8.23 ± 1.06
13a	H	CH3	8.26 ± 1.48	>20	>20
13b	4-CH3	CH3	4.63 ± 0.52	10.2 ± 1.06	7.41 ± 0.56
13c	4-OCH3	CH3	4.54 ± 0.44	11.7 ± 1.12	7.82 ± 0.48
13d	4-Cl	CH3	8.25 ± 1.03	18.4 ± 1.47	15.2 ± 1.44
13e	3,4,5-triOCH3	CH3	3.73 ± 0.28	>20	10.7 ± 1.01
13f	3,4,5-triOCH3	CH3CH2	10.21 ± 1.21	>20	>20
5-Fu	-	-	6.22 ± 1.12	10.4 ± 1.23	11.1 ± 1.28

^a In vitro antiproliferative activity was assayed by exposure for 48 h.