The reported diketopiperazine calpain inhibitor, cis-L-L-3,6-bis-(4-hydroxybenzyl)-1,4-dimethylpiperazine-2,5-dione 1, and its analogues 3 and 4 were synthesized from the corresponding amino acids. The previously assigned structure of 1 is confirmed but neither synthetic 1 nor its N-methylphenylalanine analogues 3 and 4 inhibit porcine erythrocyte calpain I.