Synthesis of tetrahydroquinoline derivatives modified at position 3
We synthesized the new tetrahydroquinoline derivatives through the following three-step pathway (Fig. 2a). The starting compound was ethyl benzoylacetate (1). In the first step, ethyl benzoylacetate and a variety of benzyl-type halides were subjected to the SN2 reaction. The reaction was carried out in dimethylformamide (DMF) in the presence of K2CO3. Then, the resulting benzyl-type derivatives of ethyl benzoylacetate (2a–e) were subjected to ammonolysis in 24% ammonia water solution. The corresponding benzoylopropanamides (3a–e) were condensed with cyclohexanone in the presence of TsOH and anhydrous MgSO4 to obtain desirable tetrahydroquinolinones (4a–e) modified with benzyl-type substituents at position 3.21 After biological evaluation, the most active 3-(1-naphthylmethyl)-4-phenyl-5,6,7,8-tetrahydro-1H-quinolin-2-one (4a) was converted into chlor22 and methoxy23 derivatives (5, 6) (Fig. 2b). It is worth mentioning that in some cases tautomers and rotamers were observed, which is described in the supplementary information.
Biology Evaluation
Compound 4a significantly inhibits the viability of human nonsmall cell lung cancer and colon cancer cells with minimal toxicity to nonmalignant human kidney cells
All new tetrahydroquinoline derivatives were assessed for cytotoxicity on a broad panel of cancer and normal cell lines, using the MTT assay. The half-inhibitory growth inhibitory concentration (IC50) values of each compound were calculated after 72 h of treatment and presented in Table 1. The results of the biological evaluation showed that compounds 4b, 4c, 4d and 4e did not have any effect on the tested cell lines, whereas compounds 5, 4a, and 6 significantly decreased the viability of colon cancer cells (HCT-116) with an IC50 value of approximately 13 µM. Additionally, 4a and 6 (A549) had an IC50 value of 11.33 ± 0.67 and 40.18 ± 0.94 µM, respectively, and exhibited almost no suppressive effect on human normal HEK293 kidney cells compared to the effect of compounds on A549 and HCT-116 cells (Fig. 3). In this assay, the IC50 value of 5-FU and ETP was recorded as a positive control. Based on the observations, 4a was recognized as the most potent, and was therefore chosen for further analysis of the mechanisms underlying the antiproliferative action.
Table 1
Cytotoxic activity of tetrahydroquinoline derivatives against various cancer and normal cells presented as an IC50 ± SD (µM) value referring to the concentration that inhibits 50% of cell growth
Compound | Cell lines | |
HCT-116 | A549 | MCF-7 | HepG2 | Hela | HEK293 | |
IC50 (µM) | |
5 | 13.10 ± 0.96 | > 50 | > 50 | > 50 | > 50 | > 50 | |
4a | 12.18 ± 1.61 | 11.33 ± 0.67 | > 50 | > 50 | > 50 | 49.01 ± 2.21 | |
6 | 15.61 ± 1.29 | 40.18 ± 0.94 | > 50 | > 50 | 50 | > 50 | |
4b | > 50 | > 50 | > 50 | > 50 | > 50 | > 50 | |
4c | > 50 | > 50 | > 50 | > 50 | > 50 | > 50 | |
4d | > 50 | > 50 | > 50 | > 50 | > 50 | > 50 | |
4e | > 50 | > 50 | > 50 | > 50 | > 50 | > 50 | |
5-FU | 5.78 ± 0.38 | 8.12 ± 1.01 | 8.41 ± 0.98 | 13.12 ± 0.32 | 11.09 ± 1.53 | 16.82 ± 0.34 | |
ETP | 0.39 ± 0.01 | 0.54 ± 0.21 | 0.83 ± 0.15 | 5.01 ± 0.22 | 3.40 ± 0.51 | 1.91 ± 0.97 | |
Next, the effect of 4a on the number of cell-forming clones was examined by the colony formation assay. As shown in Fig. 3, the number of observed colonies was found to be significantly decreased after treatment in comparison to the DMSO-treated control, and this effect was dose-dependent.
Compound 4a induces sub-G1-phase cell cycle arrest in A549 cells
The antiproliferative effect of 4a was further assessed by examining the progression of the cell cycle (Fig. 4). The tested compound induced cell cycle arrest at the sub-G1 phase in A549 cells. The fraction of cells in this phase increased significantly from 3.2% (DMSO-treated cells) to 15.5% and 33.8% (cells treated with the compound for 24 and 48 h, respectively). The observed changes corresponded with a decrease in the fraction of cells at the G0/G1 phase and were time-dependent. In the case of HCT-116 cells, treatment with the tested compound led to a significant decrease in the fraction of cells in the S-phase and the effect was time-dependent. However, the pattern of cell distribution was similar in both treated and control cells.
Compound 4a induces caspase-dependent apoptosis in A549 cells
To further explain the mechanism by which 4a induced cell death in cancer cells, a flow cytometric analysis was performed by dual-staining the cells with 7-aminoactinomycin D (7-AAD) and Annexin V-fluorescein isothiocyanate (FITC). As shown in Fig. 5, exposure of A549 cells to 4a at its IC90 concentration for 6 h caused a significant decrease in the percentage of surviving cells (20.76 ± 1.24%, p = 0.0004), with a simultaneous increase in the fraction of early-apoptotic cells [7-AAD (–), Annexin V-FITC (+); 14.03 ± 1.74%, p = 0.0078] in comparison to DMSO-treated control cells (Fig. 5). Treatment with the tested compound for 24 h caused 3.9-fold augmentation in apoptosis [early: 7-AAD (–), Annexin V-FITC (+); late: 7-AAD (+), Annexin V-FITC (+)] in comparison to the vehicle. Further incubation of cells with 4a for 48 h led to a significant increase in the percentage of apoptotic cells (p < 0.0001), while the level of the necrotic fraction was similar to that in control.
Compound 4a induces apoptosis via intrinsic and extrinsic pathways
Apoptosis induction via both intrinsic and extrinsic pathways is mediated by caspase cascade events.24 Therefore, in this study, the ability of 4a to activate two effector caspases (caspase-3 and caspase-7) was evaluated by flow cytometry. As shown in Fig. 5, after 6 h of exposure to this compound, a slight increase (2.32 ± 0.05-fold) in the activity of caspase-3/7 was observed in A549-treated cells. After 24 and 48 h of incubation, remarkable activation of caspase-3/7 (3.9 ± 0.33-fold, p = 0.04) was observed in comparison to the vehicle.
Furthermore, the expression of several apoptosis-related proteins was measured by Western blotting. As shown in Fig. 6, treatment of A549 cells with 4a resulted in time-dependent modulation of expression of all investigated proteins. The expression of BCL-2 protein was significantly decreased, while the expression of BID was slightly activated and that of BAD and BAX protein was higher than that in the control group. Treatment with 4a also led to the disappearance of the band corresponding to intact PARP-1 resulting in its proteolytic cleavage and the formation of an 85-kDa fragment. In addition, all the examined caspases (caspase-3, caspase-8, and caspase-9) were cleaved into their corresponding catalytically active forms. Expression of AIF remained unchanged in comparison to DMSO-treated control, which confirmed that treatment with 4a induced caspase-dependent cell death.25