Also, a series of pyrazolo[3,4-d]pyrimidine derivatives was developed as multifunctional antifolate analogs targeting both TS and DHFR.123 Compound 26 was the most potent analog, displaying broad-spectrum antiproliferative activity across NSCLC, CNS, ovarian, prostate, colon, melanoma, breast, and renal cancer cell lines, with high selectivity and non-lethal cytostatic effects. The gene discussed is TYMS; the disease is non-small cell lung carcinoma.