Abstract
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 IC50‐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.
Highlights
Introduction termed auristatinsThe second generation of analogues is characterized by the replacement of the N-terminal dimethylamino group with a nucleophilic secondary[11,12,13] or primary amine.[14]
Three novel auristatins were synthesized with the goal of capturing the inherent potency of dolastatin 10 (1) and decreased efflux permeability of monomethyl auristatin F (6)
The synthesis of the two central γ-amino acids of the pentapeptides was achieved by a chiral-pool approach starting from isoleucine and hydroxyproline, respectively
Summary
This hypothesis explains the striking differences between MMAE and MMAF on both counts. MMAE has been found to be slightly less cytotoxic in vitro than its auristatin analogue,[29] highlighting the importance of the N-terminal dimethylamino-moiety as found in dolastatin for inherent potency. As the C-terminal methyl ester of MMAF (6-OMe) has been found to display a pronounced apparent cytotoxicity and is frequently used as a benchmark in auristatin-SAR,[14,26] we hypothesized that this analogous modification would not distort the inherent potency of azastatin. Their apparent cytotoxicity towards three tumour cell lines was subsequently assessed and the differences were used to extrapolate the hypothetical, inherent potency of the azastatin core to gain insight into its potential utility in ADC research
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