This step is a classic reaction of the demethylation of dimethyl aldehyde under acidic conditions. 1-(2,2-dimethoxylethyl)-1,4-dihydro-3-methoxy-4-oxo-2,5-pyridinedicarboxylic acid-2-methyl ester (compound 1) (310 g, 1.0 mol) was combined with anhydrous formic acid (1,000 mL). The reaction mixture was heated to 65°C and allowed to react for 3 h under an argon atmosphere. The progress of the reaction was monitored using TLC (V(Petroleum ether):V(ethyl acetate) = 3:1, RF = 0.3–0.6). Afterward, the reaction mixture was concentrated under vacuum at 45°C to eliminate the formic acid, resulting in the formation of a crude oil compound. To remove any residual formic acid, acetonitrile (300 mL) was added, and the solvent was subsequently evaporated under vacuum. This process led to the isolation of compound 2 in the form of an oil.

The mechanism of this step involves the condensation of compound 2 with S-amino-propanol, leading to the formation of an intermediate compound, denoted as compound a. Compound a undergoes intramolecular cyclization to form intermediate compound b. The imine group within compound b then undergoes condensation with the ester group to yield compound 3. The mechanism of this step is documented in the previous article (Johns et al., 2013).

Compound 2 was dissolved in acetonitrile (1,000 mL), and then S-2-aminopropanol (125 g, 1.4 mol) was added to the solution. The reaction mixture was heated to 80°C–82°C and stirred for 2 h. The progress of the reaction was monitored using TLC (V(Petroleum ether):V(ethyl acetate) = 3:1, RF = 0.3–0.6). The mixture was then concentrated under 45°C, and dichloromethane (2000 mL) was added. After complete mixing, water (1,000 mL) was added, followed by the addition of 2 N hydrochloric acid to adjust the PH to the range of 1–2. The lower organic phase was separated and the upper aqueous phase was extracted three times with methylene chloride (500 mL). Combined with the organic phase, washed these using saturated salt water (400 mL) for three times. The resulting crude product, compound 3 (165 g), was further purified using methyl alcohol. ¹H NMR (400 MHz, DMSO) δ 15.46 (d, J = 8.1 Hz, 1H), 8.75 (d, J = 3.2 Hz, 1H), 5.43 (d, J = 82.2 Hz, 1H), 4.87 (d, J = 80.4 Hz, 1H), 4.41 (d, J = 47.8 Hz, 1H), 4.14 (d, J = 53.8 Hz, 2H), 3.89 (d, J = 4.2 Hz, 3H), 3.72 (dd, J = 10.8, 5.9 Hz, 1H), 3.14 (d, J = 85.1 Hz, 1H), 1.33 (d, J = 29.1 Hz, 3H).

This step is a classic reaction involving the condensation of a carboxyl group and an amino group. Compound 3 (30 g), HATU (38 g), DIPEA (25 g), aminophenylacetylene (23 g), and N,N-dimethylformamide (1500 mL) was mixed at room temperature, and reacted for 12 h. Water (500 mL) was added, and the mixture was stirred, filtered, and dried overnight to obtain compound 4 (34 g), yield 86.5%. ¹H NMR (400 MHz, DMSO) δ 12.53 (s, 1H), 8.69 (s, 1H), 7.95 (s, 1H), 7.58 (s, 1H), 7.38 (s, 1H), 7.22 (s, 1H), 5.42 (d, J = 78.6 Hz, 1H), 4.87 (d, J = 76.4 Hz, 1H), 4.40 (d, J = 25.2 Hz, 1H), 4.14 (d, J = 68.8 Hz, 2H), 3.80 (d, J = 72.6 Hz, 3H), 3.13 (d, J = 67.9 Hz, 1H), 2.81 (d, J = 63.8 Hz, 1H), 1.29 (d, J = 48.7 Hz, 3H).

This step is the classic of a click compound reaction, which has been previously reported in articles (Asemanipoor et al., 2020). Azide compounds (0.5 g), compound 4 (0.5 g), TBA (10 mL), water (10 mL), tetrahydrofuran (10 mL), chalcanthite (0.25 g), and sodium ascorbate (0.5 g) were mixed and reacted at 70°C. After reacted completely, dichloromethane (20 mL) was added, and the reaction was then filtered. The aqueous phase was extracted twice with dichloromethane, and the organic phase was combined, dried using anhydrous magnesium sulfate, followed by evaporated. The obtained residues was subsequently recrystallized using methanol, resulting in the final isolation of the target compounds 5a-5s, 6a-6i.

Human lung cancer cell line H460 and H1299, human liver normal cell line LO2 were bought from ATCC. DMEM or RPMI 1640 was used as the cell culture medium with 10% FBS and 1% penicillin/streptomycin. Cells were maintained in an incubator with 37°C humidified atmosphere and 5% CO₂.

Cell viability and IC₅₀ values were determined using the CCK8 assay kit. Cells were seeded at a density of 5 × 10³ cells per well in a 96-well plate. After overnight adhesion, 25 μM of compounds or vehicle control DMSO which all dissolved in the culture medium were added to cells and cultured for 48 h or 72 h. CCK8 reagent, dissolved in the medium, was then added to cells and incubated for 1 h. The absorbance at 450 nm was measured using a microplate spectrophotometer (Thermo). Cell viability was calculated, with the untreated group taken as 100%. For IC₅₀ values determination, different concentrations (0, 0.4 μM, 2 μM, 10 μM, 20 μM, 50 μM) of compounds were added to cells for 72 h. Subsequent CCK8 measurement allowed the calculation of inhibition percentage based on cell viability. The IC₅₀ values were computed by the prism statistical software. The CCK-8 assay was conducted three times and the repetitions in each time were at least three. All the values were expressed as Mean ± SD, p < 0.05 was considered statistically significant.

Cells with the density of 500 cells per well were seeded in a 6-well plate. Different concentrations (0, 2 μM, 4 μM, 8 μM, 16 μM and 32 μM) of compounds were added to cells and maintained for a 72- hour duration. And cells were cultured until the monoclonal appeared. Afterward, cells were fixed with 4% paraformaldehyde and stained by Giemsa dye. The plate clone numbers were photographed and counted using a microscope. Values were expressed as Mean ± SEM, p < 0.05 was considered statistically significant. Data were performed using Graph Prim 7.0.

Cells with a density of 5 × 10³ cells per well were seeded in a 96-well plate. After overnight incubation, cells were treated with different concentrations (0, 5 μM, 10 μM, 20 μM) of compounds for 24 h. The LIVE/DEAD Assay Kit was used to dye cells, and cells were observed and photographed under a fluorescent microscope. Live and Dead cell numbers were counted and the ratio of dead to living cells were calculated. Values were expressed as Mean ± SEM, p < 0.05 was considered statistically significant. Data were performed using Graph Prim 7.0.

Cells with a density of 3 × 10⁵ cells per well were seeded in 6-well plates. After overnight adhesion, cells were treated with different concentrations of compounds for 72 h. Cells were harvested and stained with Annexin V-FITC and propidium iodide. Apoptotic ratio was quantified using the flow cytometry (BD Biosciences) and analyzed using the FlowJo software v10. Values were expressed as Mean ± SD, p < 0.05 was considered statistically significant. Data were performed using Graph Prim 7.0.

Cells with a density of 5 × 10³ cells/well were cultured in a 96-well plate. The cells were treated with different concentrations (0, 5, 10, 20 μM) of compounds for 24 h. Then the medium was replaced with 10 μM DCFH-DA dissolved in culture medium and incubated for 30min at 37°C in dark. The fluorescent cells were observed and photographed using a fluorescent microscope.

Western blot was used to analyze protein expressions. Cells were cultured in 12-well plates and treated with different concentrations (0, 2, 4, 8 μM) of compounds for 72 h. Cell proteins were extracted using RIPA buffer with protease/phosphatase inhibitor cocktail (CST). 10%–15% sodium dodecyl sulfate polyacrylamide gel electrophoresis was used to separate proteins and nitrocellulose membranes (Millipore) was used to collected proteins. Antibodies used in western bolt were LC3 (3868s, CST), caspase 3 (9662s, CST), cyclin D (2922s, CST), cyclin E (20808s, CST), β-Catenin (9562s, CST), γH2AX (9718s, CST), PARP (46D11, CST), and β-actin (4967s, CST). Values were expressed as Mean ± SEM, p < 0.05 was considered statistically significant. Data were performed using Graph Prim 7.0.

In order to assess the potential cytotoxicity of the synthesized compounds, we selected two types of human non-small-cell lung cancer cell line, H460 and H1299. Cell viability and cytotoxicity were evaluated using the CCK8 assay (Xu et al., 2018). The cells were treated with 25 μM of compounds 5a-5s and 6a-6i for 48h and 72 h. The results, presented in Table 2, indicated that after 48 h of treatment, the compounds showed limited anti-proliferative effects against H460 and H1299 cells. Among them, compounds 5c, 5i, and 6h exhibited cell viability rates below 50% in H460 cells. However, against H1299 cells, all compounds exhibited cell viability rates exceeding 90%, indicating minor effects on this cell line. Upon extending the treatment period to 72 h, some compounds exhibited heightened cytotoxicity. Specifically, in H460 cells, compounds 5c, 5d, 5i, 5s, 6b, and 6h showed cell viability rates below 50%. In contrast, no compounds managed to reduce H1299 cell viability to less than 50%, yet compounds 5i, 5m, 6h and 6i achieved cell viability rates below 70%. These findings suggested that the synthesized compounds exhibited more pronounced cytotoxic effects against H460 cells compared to H1299 cells. Notably, compound 5i displayed the most significant cytotoxicity against both cell lines. It is worth noting that minimal effects on the cell viability of the normal cell line LO2 were observed when exposed to these compounds. Further, we determined the half-maximal inhibitory concentration (IC₅₀) of selected compounds against H460 cells, the results of which were summarized in Table 3. Remarkably, compound 5i exhibited the most potent cytotoxicity with an IC₅₀ value of 6.06 μM, followed by compounds 5c and 5s with IC₅₀ values of 9.65 μM, and 13.38 μM, respectively.

To assess the anti-proliferative effects of compound 5i, live/dead cell staining was performed using Calcein Acetoxymethyl Ester (Calcein-AM) and Propidium Iodide (PI) dyes (Xie et al., 2023). Calcein-AM highlights live cells through green fluorescence emission, while PI identifies dead cells with damaged membrane integrity, producing red fluorescence. H460 and H1299 cells were treated with different concentrations (0, 5, 10 and 20 μM) of compound 5i for 24 h. After 24 h treatment, cells were stained with Calcein-AM and PI, diluted in cultured medium, and incubated for an hour in dark. Then the fluorescence was detected and photographed using a fluorescent microscope. The results demonstrated that compound 5i significantly decreased the number of live cells and increased the number of dead cells in H460 cells (Figure 3A). In the 10 and 20 μM concentration groups, the number of live cells was dramatically reduced compared to the untreated group, and it showed less dead cells because of the largely decreased live cells in 20 μM concentration group (Figure 3A). Similarly, the same trends were observed in H1299 cells, as treatment with compound 5i led to a decrease in live cell numbers alongside a substantial rise in dead cells (Figure 3B). In addition, we also performed plate clone formation assay (Xu et al., 2018) to further investigate the anti-proliferation ability of compound 5i. The number of cell clones was decreased when treated with compound 5i, particularly in the 16 μM and 32 μM concentration groups in H460 cells (Figure 3C). Similarly, a reduction in the number of cell clones was also observed in H1299 cells following treatment with compound 5i (Figure 3D). These results indicated that compound 5i effectively inhibited cancer cell proliferation, thus underscoring its anticancer activity.

Apoptosis is a programmed-controlled process of cell self-destruction, and increased cell apoptosis can lead to cell death (Wang et al., 2023). To explore the preliminary molecular mechanisms of the synthesized compounds action, the apoptotic activity of compound 5i was evaluated by comparing the percentage of apoptotic cells with untreated cells using flow cytometry after staining with Annexin V-FITC and PI (Chen et al., 2022a). This assay facilitates the detection of necrotic cells (Q1, AV-/PI+), late apoptotic cells (Q2, AV+/PI+), early apoptotic cells (Q3, AV+/PI-), and live cells (Q4, AV-/PI-). H460 and H1299 cells were treated with different doses of compound 5i for 72h, and cells were collected, stained, and analyzed. The proportion of early and late apoptotic cells was added and their ratio relative to the total cells was calculated. The results demonstrated a significant increase in apoptotic cells in H460 cells treated with 2, 4 and 8 μM of compound 5i (Figure 4A). However, compound 5i exhibited weaker impact on cell apoptosis in H1299 cells, as the apoptotic cell population showed no significant change or even a decrease (Figure 4B). This was consistent with the results of cell viability measurement that compound 5i showed better antitumor effect on H460 cells rather than H1299 cells.

Reactive oxygen species (ROS) levels were determined quantitatively using the probe 2′-7′-dichlorodihydrofluorescin diacetate (H2DCFDA) (Kim and Xue, 2020). Under normal condition, ROS participated in mitochondrial electron transport, signal transduction, gene expression, and enzyme reactions. But excess or deficiency of ROS levels can result in the development of diseases including cancers, diabetes, atherosclerosis, neurodegenerative diseases and others (Wang et al., 2023). ROS induction is associated with mitochondrial membrane damage, cell apoptosis, and ultimately cell death (Dixon and Stockwell, 2014). To evaluate the effect of compound 5i on ROS generation, H460 cells were treated with 5 μM, 10 μM or 20 μM of compound 5i for 24 h. Subsequently, the cells were stained with peroxide sensitive fluorescent dye, 2′-7′-dichlorodihydro-flurescien diacetate (DCFH-DA), and visualized using a fluorescent microscope. DCFH-DA has no fluorescence and it can pass through cell membrane freely. After entry the cell, it could hydrolyze by intracellular esterase to DCHF and intracellular ROS can further oxidize DCFH to produce green fluorescent DCF. Results showed a significant augmentation in ROS generation within H460 cells treated with compound 5i (Figure 5). It is important to acknowledge that in the 10 μM and 20 μM groups, the ROS levels seemed less pronounced compared to the 5 μM group. This phenomenon can be attributed to the reduction in the number of live cells; however, the observed fluorescence appeared more intense (Figure 5). Previous studies have demonstrated that ROS production triggered apoptosis, autophagy, and cell death (Pellegrini et al., 2023). Our findings also showed that compound 5i could increase ROS levels in H460 cells which may lead to cancer cell apoptosis and cell death.

Western blot analysis was performed to assess the protein expressions involved in cell growth and cell death processes. H460 and H1299 cells were treated with different concentrations of compound 5i, and whole cell proteins were collected for analysis. Results demonstrated a significant increase in the expression of ubiquitin like molecule light chain 3 (LC3) (Tanida et al., 2004), an important marker in autophagy, in H460 cells treated with compound 5i (Figure 6A). This suggested that compound 5i could induce autophagy. However, there were no significant differences observed in the expression of other proteins involved in cell cycle, DNA damage or DNA repair, such as caspase3, Cyclin D, Cyclin E, cadherin-associated protein beta 1 (β-catenin), γ-H2AX (Sharma et al., 2012), and Poly (ADP-Ribose) Polymerase (PARP) (Hanzlikova et al., 2018), after compound 5i treatment in H460 cells (Figure 6A). In H1299 cells, no significant differences were observed in LC3 and caspase 3 expression levels (Figure 6B). Additionally, the levels of proteins related to cell cycle, including Cyclin D and Cyclin E, exhibited a decrease following treatment with compound 5i (Figure 6B). Notably, β-catenin expression remained unaltered in response to the treatment (Figure 6B). Treatment with compound 5i prompted DNA damage, as demonstrated by the increased expression of γ-H2AX, although PARP expression remained unaffected (Figure 6B). These results were consistent with the above. Autophagy has both tumor suppression and growth promotion effects in cancers, depending on the cellular context and cancer stage. On the one hand, autophagy inhibits cancer development via eliminating dysfunctional proteins and damaged mitochondria. On the other hand, autophagy can promote tumor growth by providing recycled metabolites to support cancer cell survival and growth (Wang et al., 2023). A number of studies have suggested that ROS induced autophagy as a regulator (Pellegrini et al., 2023). In our study, we found that compound 5i significantly induced autophagy which was consistent with the stimulated ROS levels shown above. Combined with the above results, we suggested that compound 5i promoted autophagy process and finally induced cell death in H460 cells. But in H1299 cells, compound 5i exerted its anticancer ability by suppressing cell division and proliferation.