Synthesis of BZR‐Cotoxin IV Analog and Evaluation of Its Cytotoxic Properties

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ABSTRACTOur research team has successfully synthesized BZR‐cotoxin IV, despite encountering considerable difficulties stemming from its unique structural features. The molecule's ester bond is particularly susceptible to hydrolysis, which leads to instability during the synthesis process. Moreover, the tendency of its linear precursor to form diketopiperazine (DKP) adds another layer of complexity to the synthesis. To overcome these obstacles, we adopted a strategy centered on developing an analog compound. This approach was guided by structure–activity relationship (SAR) principles. The key modification involved substituting the hydroxy acid residue (Hiv) found in the original BZR‐cotoxin IV with its corresponding amino acid, valine. This strategic alteration was intended to not only potentially improve the compound's characteristics but also to streamline the synthesis procedure. The synthesis of [Val]7‐BZR‐cotoxin IV was accomplished using a hybrid approach that combined solid‐phase and solution‐phase peptide synthesis techniques. The linear precursor of [Val]7‐BZR‐cotoxin IV was produced using Fmoc chemistry on CTC resin, with HATU/HOAt serving as the coupling reagent for amide bond formation. This process yielded a 9.25% pure product as a white powder. The cyclization step was performed using HATU in a dilute solution, which produced cyclic [Val]7‐BZR‐cotoxin IV with a yield of 12.9% as a white powder. Cytotoxicity test was conducted on both the linear and cyclic forms of [Val]7‐BZR‐cotoxin IV together with its natural BZR‐cotoxin IV against HeLa cancer cells. The result showed moderate activity, with IC50 values of 297.60 μM for the linear precursor and 161.58 μM for the cyclic analog. Notably, the cyclic [Val]7‐BZR‐cotoxin IV demonstrated higher cytotoxic activity compared with its natural counterpart, BZR‐cotoxin IV, which had an IC50 value of 214.01 μM.