Pharmacophore-Based Structure Optimization of S‑Trityl‑l‑Cysteine Derivatives for Cytotoxic Payload Applications

Kinesin spindle protein (KSP) represents a promising target for cancer therapy with a mechanism of action distinct from conventional microtubule agents. We previously identified S-trityl-_L-cysteine (STLC) derivatives as potent and selective KSP inhibitors. In this study, we designed STLC derivatives with diverse physicochemical properties for further structural optimization using in silico analyses. rac-1-C and 2-C were synthesized via a key allylation reaction of ethylene-bridged triarylmethanol mediated by XtalFluor-E. Compound 2-C (HCT116 half growth inhibition (GI₅₀): 1.2 nM) was identified as the minimal pharmacophore for KSP inhibition. Additionally, we explored the feasibility of using these inhibitors as linker-payloads in antibody-drug conjugates (ADCs). Guided by structure-activity relationships, we synthesized enzyme-cleavable valine-citrulline-p-aminobenzyloxycarbonyl linker STLC derivatives (VC-linker-STLCs) capable of cathepsin B-mediated payload release. Slight structural differences in the payload affected the cleavage reaction rate. These results suggest that VC-linker-STLCs have potential uses as linker-payloads for ADCs.