To obtain a better tumor cell killing mediated by cytotoxic T lymphocytes (CTLs), several strategies were developed that might prime tumor-specific T-cell responses. One such strategy is combining the immune checkpoint inhibitors (CPI) blockade with radiotherapy (RT), considering the synergy between these two types of therapies. Here, we present an in vitro experimental model of cocultures of human tumor cell lines (A549 lung carcinoma and SK-Mel-1 melanoma) and immune cells (CTLs), in which CTLs activation is attempted by irradiation of the tumor cells with ionizing radiations and by the treatment of CTLs with an anti-programmed death-1 (anti-PD-1) drug. This model is suitable for studying cellular death, inflammatory responses, molecule expression, and immunotherapy.

Background/Objectives: Combining heterocyclic fragments provides a strategy for developing compounds for exploratory anticancer evaluation. This study aimed to synthesize and characterize five thiazolyl-methylthio-1,3,4-thiadiazolyl-quinazolin-4(3H)-one compounds (5a–e), assess their properties in silico, and investigate the selected ones for cytotoxicity and their effects on irradiation-associated DNA lesions. Methods: Structures were characterized by IR, HRMS, and ¹H- and ¹³C-NMR spectroscopy. Computational studies included physicochemical and ADMETox predictions, multi-platform target prediction, and molecular docking to A2A, Erβ, and CDK5. Compounds 5a and 5b were evaluated for cytotoxicity and cell-death profiles on one colorectal cancer (CRC) cell line (HT-29), two breast cancer (BC) cell lines (MCF-7 and MDA-MB-231), and one healthy fibroblast cell line (BJ), using cisplatin (Cis) as a reference. DNA lesions were assessed by the Comet assay in both breast cancer models before, immediately after, and 24 h after 4 Gy irradiation. Results: Compounds 5a–e were obtained in yields of 45.17–83.27%. Compounds 5a and 5b had lower 24-h IC₅₀ values than cisplatin in MCF-7 cells, but higher values in the other tested cell lines. Compound 5b showed higher selectivity indexes than both 5a and cisplatin across the cancer models, although its values of 1.14–1.34 indicated modest selectivity. Cell-death profiles differed between treatments, with larger necrotic fractions following 5a or 5b treatment in MCF-7 cells. Both compounds were associated with higher post-irradiation lesion scores than untreated irradiated controls. Scores increased further at 24 h in MCF-7 cells but decreased in MDA-MB-231 (triple-negative) cells. Pre-irradiation scores were also elevated in treated MCF-7 cells. Computational profiling identified comparatively more favorable predicted solubility for 5a and 5b. Cross-platform ADMET comparison consistently indicated low Caco-2 permeability, CYP3A4 inhibition, potential liver-injury risk, and high plasma–protein binding, while several absorption, transporter, and safety endpoints remained discordant. Target predictions showed weak cross-platform convergence. Docking provided the strongest, although moderate, structural support for A2A binding by 5a–d. Conclusions: The findings support further investigation of 5a and 5b, particularly their effects on irradiation-associated DNA lesions. Priorities include improving potency and selectivity, validating in silico predictions, and evaluating radiosensitizing potential through clonogenic survival studies.