## 3 Results and Discussion
### In Vitro Biological Activity Characterization  
The Anticancer Activity Characterization of Pyrrolidone Derivatives **2**-**26**  
After synthesizing and characterizing the pyrrolidone derivatives (compounds **2**-**26**), we subsequently aimed to evaluate their _in vitro_ anticancer activity utilizing the A549 non-small cell lung cancer cell line as a model. To identify the most promising pyrrolidone derivatives exhibiting anticancer properties, we initially exposed the A549 cells to a fixed concentration of 100 \(\upmu\)M for each compound. The post-treatment cell viability was subsequently measured and compared to the cytotoxic effects induced by standard chemotherapy agents, including doxorubicin, cisplatin, and cytarabine (Figure 2). The following chemotherapy agents were chosen due to their use in clinical oncology alone or in combination to treat and manage various lung-derived cancers. The initial testing revealed that both starting materials, carboxylic acid (compound **2**) and methyl ester (compound **3**), exhibited no significant anticancer activity against A549 cells, maintaining viabilities of 92.7% and 95.4%, respectively, after a 24-hour exposure. However, the transformation of the methyl ester to hydrazide (compound **4**) resulted in a marked increase in anticancer activity, as evidenced by a reduced cell viability of 69.7%. The further incorporation of an unsubstituted or substituted isatin moiety into the hydrazide framework yielded compounds **5** and **6** (Scheme 1). Notably, compound **5** (R=H) demonstrated significant anticancer activity, decreasing A549 cell viability to 43.8%. In contrast, the substitution of the hydrogen radical with a bromine atom in compound **6** markedly enhanced the anticancer efficacy, reducing A549 viability to 27.5%. Furthermore, the anticancer activity of compound **6** was found to be significantly greater than that induced by cytarabine (AraC), which resulted in a cell viability of 53.4% (\(p<0.05\)) (Figure 2). The reaction of hydrazide **4** with various heterocyclic aldehydes yielded compounds that contained nitrofuran (compound **7**) and nitrothiophene (compound **8**) substitutions. Among these derivatives, the nitrofuran derivative (compound **7**) exhibited a reduction in A549 viability to 67.8%. Conversely, the nitrothiophene derivative (compound **8**) displayed particularly promising anticancer activity, with a notable decrease in A549 cell viability to 34.7% (Figure 2). The further reaction of hydrazide **4** with various aromatic aldehydes yielded a series of compounds featuring aromatic substitutions (compounds **9**-**18**). The incorporation of a phenyl group in compound **9** resulted in a reduction in A549 cell viability to 74.8%. Subsequent substitutions of halogen radicals at the para position of the phenyl ring led to the synthesis of compounds **10**-**12**, which exhibited moderate anticancer activity. Specifically, compound **10**, bearing a chlorine substituent at the para position, decreased A549 viability to 56.0%. Additionally, substitution of chlorine with bromine or fluorine in compounds **11** and **12** further reduced A549 viability to 52.5% and 38.4%, respectively. However, the introduction of a methoxy group at the para position in compound **13** diminished the anticancer activity, resulting in 74.1% viability. Furthermore, ortho- and para-halogen substitutions (R = 2,4-diFC\({}_{6}\)H\({}_{3}\)) did not enhance anticancer activity, as demonstrated by compound **14**, which maintained a viability of 66.5% (Figure 2). In contrast, the incorporation of a 2,4,6-trimethoxyphenyl substituent in compound **15** yielded favorable anticancer activity, reducing A549 viability to 37.8%. Subsequent substitutions of chlorines at the _ortho_ and _meta_ positions of the phenyl ring resulted in compounds **16** and **17**, both exhibiting similar anticancer activity with viabilities of 66.1% and 66.3%, respectively.