Synthesis of trifluoromethyl-containing spiro-epoxyindole-pyrrolidines via a DABCO-catalyzed [3 + 2] cycloaddition and their antitumor activities.

We report a concise and general DABCO-catalyzed [3 + 2] cycloaddition that enables rapid assembly of a library of trifluoromethyl-containing spiroepoxyindole-pyrrolidine derivatives (3a-3x) under mild, operationally simple conditions, providing the target scaffolds in good to excellent yields (53-99 %, except 3e: 36 %) with high diastereoselectivity. Structural assignments were supported by multinuclear NMR, HRMS and single-crystal X-ray analysis (3a). A subset of the library was screened for antiproliferative activity in HeLa and K562 cells; compound 3p emerged as the most active hit (K562, IC50 = 7.02 ± 0.89 μM) with low toxicity to normal NCM460 cells. Integrative network pharmacology and molecular docking nominated STAT3, PIK3CA and JAK2 as putative targets of 3p, and detailed docking predicted favorable binding modes for both stereoisomers. Target prioritization for experimental follow-up focused on JAK2: cell-based assays showed a concentration-dependent decrease in phosphorylated JAK2 (p-JAK2) with no change in total JAK2, and surface plasmon resonance (SPR) experiments with JAK2 immobilized on a CM5 chip confirmed direct binding of 3p. Kinetic analysis (1,1 L fit) returned ka = 46.60 × 103 M-1·s-1, kd = 5.19 × 10-2 s-1 and KD = 1.11 × 10-6 M, indicating micromolar affinity and appreciable residence time consistent with functionally relevant engagement. Together, the synthetic chemistry, in silico target nomination and orthogonal target-engagement data identify 3p as a promising lead for further optimization and mechanistic study.