Phenotypic screening, a widely utilized technique in drug research and development, has the advantage of not
relying on specific molecular targets, enabling a more comprehensive evaluation of the potential efficacy of
drugs. Our group developed and synthesized a series of novel 1H-pyrazole [3, 4-d]pyrimidine derivatives. We
used the phenotypic screening method to assess their activities. The results showed that compound 8r had
excellent inhibitory activity against the esophageal cancer cell line KYS-30 (IC50 = 3.53 ± 0.04 μM), significantly
better than the positive control compound 5-Fu. Further activity studies indicated that 8r could not only
considerably inhibit the colony formation and invasion of KYS-30 but also showed excellent safety both in vitro
and in vivo. Subsequently, we treated rat cardiac muscle cells H9C2 with high concentrations (10 μM and 20 μM)
of 8r. Investigations revealed that cardiac muscle cells maintained a survival rate exceeding 80 %, verifying the
minimal cardiotoxicity of compound 8r. The research confirmed the technical benefits of phenotypic screening in
maintaining a balance between drug effectiveness and safety, while also offering a novel approach for creating
anticancer drugs with high efficacy and low toxicity