Automated Solid‐Phase Flow Synthesis for Scaffold Diversification of a High‐Throughput Screening Hit

ABSTRACT

High‐throughput screening (HTS) enables rapid hit identification, yet subsequent analogue synthesis and structure–activity relationship exploration remain constrained by labor‐intensive multistep workflows. Here, we describe an automated solid‐phase flow synthesis (SPS‐flow) platform for diversity‐oriented analogue generation of an HTS‐derived, non‐peptidic small‐molecule hit scaffold. A benzodiazepine hit against cyclin‐dependent kinase 11B (CDK11B) was selected as a proof‐of‐concept and translated into a modular 10–11‐step synthetic sequence through fragment‐guided retrosynthetic design. A sterically tuned triazene linker on 2‐chlorotrityl resin was developed to tolerate diverse reaction conditions while enabling traceless cleavage and post‐synthetic diazonium diversification, and Chemical Recipe File (CRF)‐based control coordinated continuous‐, circulation‐, and stop‐flow operations within a unified automated workflow. The platform enabled the synthesis of 42 benzodiazepine analogues in 5%–33% overall yields, with each sequence completed within approximately 45–48 h from a common resin‐bound precursor. Within the explored first‐generation design space, the library rapidly defined the synthetic accessibility and preliminary structure–activity landscape of the scaffold under SPS‐flow conditions, supporting early triage of the series and demonstrating that SPS‐flow can be used as a programmable platform for automated multistep diversification and early‐stage drug optimization.