Synthesis and enzymatic activation of N-[Nα-(4-amino-4-deoxypteroyl)-Nδ-hemiphthaloyl-l-ornithiny]-l-phenylalanine, a candidate for antibody-directed enzyme prodrug therapy (ADEPT) Original Research Article

N-[Nα-(4-Amino-4-deoxypteroyl)-Nδ-hemiphthaloyl-l-ornithinyl]-l-phenylalanine (1), a carboxypeptidase A (CPA) cleavable prodrug was synthesized for use in an antibody directed strategy to improve the therapeutic selectivity of Nα-(4-amino-4-deoxypteroyl)-Nδ-hemiphthaloyl-l-ornithine (2), an extremely potent nonpoly-glutamatable DHFR inhibitor which is also highly cytotoxic. Compound 1 was shown by HPLC analysis to give a >99% yield of 2 upon incubation with bovine CPA (bCPA) for 20 min at 25 °C. In a spectrophotometric kinetic assay with 50 μM dihydrofolate as the competing substrate in the presence of 65 μM NADPH, 1+bCPA stoichiometrically inhibited recombinant human DHFR (rhDHFR) with a Ki of 0.35 pM. In contrast, 1 without bCPA was a poor inhibitor of rhDHFR (Ki>10 μM). In a 72 h growth inhibition assay against cultured CCRF-CEM human leukemic lymphoblasts, the growth inhibitory activities of 1+bCPA, 2+bCPA, and 2 alone were the same (IC50 1.3–1.4 nM), whereas 1 in the absence of bCPA was >100-fold less potent (IC50 155 nM).