A series of cystargolide-based β-lactone analogues containing nitrogen atoms at the Pz portion of the scaffold were prepared and evaluated as proteasome inhibitors, and for their cytotoxicity profile toward several cancer cell lines. Inclusion of one, two or even three nitrogen atoms at the Pz portion of the cystargolide scaffold is well tolerated, producing analogues with low nanomolar proteasome inhibition activity, in many cases superior to carfilzomib. Additionally, analogue 8g , containing an ester and pyrazine group at Pz, was shown to possess significant activity toward RPMI 8226 cells (IC 50 = 21 nM) and to be less cytotoxic toward the normal tissue model MCF10A cells than carfilzomib.