The development process for syringolin A analogues having improved proteasome inhibitory and antitumor activity is described. The strategy was to first establish a convergent synthesis of syringolin A using a rare intramolecular Ugi three-component reaction in the last stage of the synthesis, so as to gain access toa set of structure-based analogues. The inhibitory activity of chymotrypsin-like activity of 20S proteasome was largely improved by targeting the S3 subsite of the β5 subunit. Cytotoxic activity was also improved by installing the membrane-permeable substituent. These biological properties are comparable to those of bortezomib, a clinically used first-line proteasome inhibitor.