The goal of this work was to synthesize new compounds for anticancer evaluation as a trial to obtain new antitumor agents with higher activity and fewer side effects. Therefore, the precursor 2,2'-(1,4-phenylenebis (thiazole-4,2-diyl))bis (3-(dimethylamino)acrylonitrile) was used to synthesize various azolopyrimidine derivatives connected to the thiazole moiety. Compounds -, including pyrazolopyrimidine, triazolopyrimidine, and others, were produced by reacting enaminonitrile with different -nucleophiles. Additionally, compounds -, such as isoxazole and pyrimidinethione derivatives, were obtained by reacting compound with guanidine, hydrazine hydrate, hydroxylamine hydrochloride, and thiourea. Enaminonitrile was also treated with barbituric acid, isoxazolone, and pyrazolone to yield pyranopyrimidine derivatives -. Moreover, enaminonitrile reacts with -nucleophiles namely ''acetylacetone, dimedone, 2-cyanomethylbenzothiazole, and 2-cyanomethylbenzimidazole'' to give pyrano derivatives , and fused pyridone derivatives and , respectively. The cytotoxic activity of novel compounds against HSV-1, HIV-1, and various cancer cell lines was assessed, with compounds , , and showing the strongest effects. Molecular docking studies further evaluated the binding affinity of these derivatives, with docking scores ranging from -7.8679 to -8.3013 kcal/mol. Several new azolopyrimidine derivatives linked to the thiazole moiety were effectively synthesized and assessed in the study, and they showed notable cytotoxic activity against HSV-1, HIV-1, and several cancer cell lines.