Rational Design of Azastatin as a Potential ADC Payload with Reduced Bystander Killing

Auristatins are a class of ultrapotent microtubule inhibitors, whose growing clinical popularity in oncology is based upon their use as payloads in antibody‐drug conjugates (ADCs). The most widely utilized auristatin, MMAE, has however been shown to cause apoptosis in non‐pathological cells proximal to the tumour (“bystander killing”). Herein, we introduce azastatins, a new class of auristatin derivatives encompassing a side chain amine for antibody conjugation. The synthesis of Cbz‐azastatin methyl ester, which included the C2‐elongation and diastereoselective reduction of two proteinogenic amino acids as key transformations, was accomplished in 22 steps and 0.76 % overall yield. While Cbz‐protected azastatin methyl ester (0.13–3.0 nM) inhibited proliferation more potently than MMAE (0.47–6.5 nM), removal of the Cbz‐group yielded dramatically increased IC(50)‐values (9.8–170 nM). We attribute the reduced apparent cytotoxicity of the deprotected azastatin methyl esters to a lack of membrane permeability. These results clearly establish the azastatins as a novel class of cytotoxic payloads ideally suited for use in next‐generation ADC development.