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Additionally, the click chemistry requires three steps (amination, azidation and click reaction), which was the leading cause of the low synthetic yield of Abz-E2B. This should decrease the log P value to less than 4. 00, increasing the water solubility 22. All of the compounds were optically resolved by high-performance liquid chromatography (HPLC) on a chiral column before testing their effects in vitro and in vivo.