Pharmacophore-Based Structure Optimization of <i>S</i>-Trityl-<scp>l</scp>-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 <i>S</i>-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. <i>rac</i>-<b>1-C</b> and <b>2-C</b> were synthesized via a key allylation reaction of ethylene-bridged triarylmethanol mediated by XtalFluor-E. Compound <b>2-C</b> (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-<i>p</i>-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.