Synthesis of N5-furoxan-functionalized pyrazolo[3,4-d]pyrimidinones as novel nonclassical DHFR/TS inhibitors with...

Dihydrofolate reductase (hDHFR) and thymidylate synthase (TS) are pivotal folate-cycle enzymes that synergistically regulate DNA biosynthesis and cancer cell proliferation. Herein, the design and synthesis of multifunctional N5-furoxan-based pyrazolo[3,4-d]pyrimidinones (MAHS-1-11) as putative first-in-class hDHFR and TS inhibitors are reported. Among the synthesized analogs, MAHS-3 and MAHS-4 emerged as the most potent broad-spectrum, dose-dependent antiproliferative agents against the NCI-USA 60 cell panel examined herein, displaying MGI% values of 62.79% and 63.37%, respectively, comparable to methotrexate (65.60%), with GI₅₀s ranging from 0.276 to 85.2 μM. Both compounds exhibited good-to-moderate cytostaticity, with TGIs ranging from 10.6 to 82.5 μM and predominantly non-lethal effects across most cancer types tested. Notably, MAHS-4 showed a consistent inhibitory pattern across the full NCI panel, affording subpanel GI₅₀ (MG-MID) values of 3.84-20.33 μM and an overall full-panel MG-MID of 13.76 μM, with pronounced activity against leukemia, NSCLC, renal, prostate, and breast cancer subpanels. Enzymatic assays revealed moderate hDHFR inhibition by MAHS-3 and MAHS-4 (IC₅₀ = 52.60 and 83.53 μM, respectively), whereas both compounds demonstrated superior TS inhibition relative to 5-FU, with approximately 1.5- and 2.5-fold enhanced potency, respectively. Mechanistically, both analogs induced G2/M-stage arrest, upregulated p21/p27, and activated the intrinsic Cas-dependent apoptotic pathway, as evidenced by increased Bax, Cas-7, and Cas-9, alongside reduced Bcl-2 and PARP-1 expression. Furthermore, both compounds exerted pronounced anti-metastatic effects, markedly elevated intracellular NO levels, and induced autophagy-related cell death. Molecular modeling studies showed that both compounds adopt a tortuous L-shaped conformation via the alkyl linker, enabling favorable spatial overlay with reference inhibitors within the target binding pocket, and identified the 5-membered heterocyclic moiety and the C6 region as key optimization hotspots. Furthermore, both compounds complied with Lipinski's and Pfizer's drug-likeness criteria. Overall, MAHS-3 and MAHS-4 represent promising next-generation TS-targeted antifolate preliminary hit compounds demonstrating moderate hDHFR inhibitory activity and requiring substantial optimization and pharmacokinetic evaluation.