Novel omega-N-amino analogs of B13 (Class E) were designed, synthesized and tested as inhibitors of acid ceramidase (ACDase) and potential anticancer agents deprived of unwanted lysosomal destabilization and ACDase proteolytic degradation properties of LCL204 [Szulc, Z. M.; Mayroo, N.; Bai, A.; Bielawski, J.; Liu, X.; Norris, J. S.; Hannun, Y. A.; Bielawska, A. Bioorg. Med. Chem. 2008, 16, 1015]. Representative analog LCL464, (1R,2R)-2-N-(12'-N,N-dimethylaminododecanoyl amino)-1-(4''-nitrophenyl)-1,3-propandiol, inhibited ACDase activity in vitro, with a similar potency as B13 but higher than LCL204. LCL464 caused an early inhibition of this enzyme at a cellular level corresponding to decrease of sphingosine and specific increase of C(14)- and C(16)-ceramide. LCL464 did not induce lysosomal destabilization nor degradation of ACDase, showed increased cell death demonstrating inherent anticancer activity in a wide range of different cancer cell lines, and induction of apoptosis via executioner caspases activation. LCL464 represents a novel structural lead as chemotherapeutic agent acting via the inhibition of ACDase.