## 3 Results and Discussion
### In Vitro Biological Activity Characterization
Conversely, the introduction of a 2-chloro-4-nitrophenyl substituent in compound **18** led to a reduction in A549 viability to 44.1% (Figure 2). Following the exploration of the anticancer activity of pyrrolidone derivatives with aromatic substitutions, we aimed to investigate whether alkylation at the nitrogen position would enhance anticancer efficacy. To this end, ethyl iodide was reacted with pyrrolidone derivatives, yielding compounds **19**-**22** with \(N\)-ethyl substitutions. The alkylated compound **19**, which contained an aromatic substituent (R = phenyl), resulted in a reduction in A549 viability to 72.5%, comparable to that of the non-alkylated derivative (compound **9**). The alkylation of the para-chlorine substituted derivative produced compound **20**, which reduced A549 viability to 67.9%. Interestingly, the alkylation of the bromine-containing  
Figure 2: The _in vitro_ anticancer activity of pyrrolidone derivatives **2**-**26** against A549 human non-small cell lung cancer cells. The cells were exposed to a fixed 100 \(\upmu\)M concentration of compounds, or doxorubicin (DOX), cisplatin (CP), and citarabin (AraC), for 24 h, and then post-treatment viability was measured using an MTT assay. The line on the graph indicates a 50% inhibition threshold. The data are shown as mean \(\pm\) SD of three experimental replicas. *—\(p\) < 0.05.  
derivative yielded compound **21**, exhibiting moderate anticancer activity with a viability of 51.3%. Lastly, the \(N\)-alkylated derivative containing a 4-fluorophenyl substituent resulted in a reduced anticancer activity, maintaining A549 viability at 69.8% (Figure 2). After investigating the effects of heterocyclic and aromatic substitutions, we further aimed to characterize the impact of incorporating nitrogen-containing heterocycles on anticancer activity. Compound **23**, featuring a 3,5-dimethylpyrazole substituent, reduced A549 viability to 44.1%, while the 2,5-dimethylpyrrole derivative (compound **24**) demonstrated weaker activity, resulting in a viability of 51.0%. Notably, derivatives containing oxadiazole thione (compound **25**) and triazole thione (compound **26**) cores exhibited the strongest anticancer activity, reducing A549 viability to 28.0% and 29.6%, respectively. The anticancer activity of compounds **25** and **26** was significantly higher than that of cytarabine (\(p<0.05\)) (Figure 2). After characterizing the structure-dependent anticancer activity of synthesized compounds, we then moved forward and selected the most promising candidates with significantly (\(p<0.05\)) stronger anticancer activity than AraC and evaluated their dose-response kinetics in A549 cells. A549 cells were treated with increasing concentrations of the selected compounds (**6**, **25**, and **26**) or AraC, and cell viability was assessed post-treatment (Figure 3). All tested compounds demonstrated significant cytotoxic activity (\(p<0.05\)) relative to the untreated control. Notably, the halogenated derivative (compound **6**) exhibited cytotoxicity comparable to that of AraC (Figure 3). Additionally, the oxadiazole thione (compound **25**) and triazole thione (compound **26**) showed more potent cytotoxic effects than those induced by AraC. Collectively, these data demonstrate that the pyrrolidone derivatives demonstrate structure-dependent anticancer activity against A549 cells _in vitro_. The most promising compounds demonstrated comparable or stronger anticancer activity than AraC, a standard FDA-approved chemotherapeutic agent. These pyrrolidone derivatives could serve as a starting scaffold for the future hit-to-lead optimization to enhance the _in vitro_ anticancer activity.