Synthesis and enzymatic activation of N-[N(alpha)-(4-amino-4-deoxypteroyl)-N(delta)-hemiphthaloyl-L-ornithiny]-L-phenylalanine, a candidate for antibody-directed enzyme prodrug therapy (ADEPT).
N-[N(alpha)-(4-amino-4-deoxypteroyl)-N(delta)-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 N(alpha)-(4-amino-4-deoxypteroyl)-N(delta)-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 degrees C. In a spectrophotometric kinetic assay with 50 microM dihydrofolate as the competing substrate in the presence of 65 microM NADPH, 1+bCPA stoichiometrically inhibited recombinant human DHFR (rhDHFR) with a K(i) of 0.35 pM. In contrast, 1 without bCPA was a poor inhibitor of rhDHFR (K(i)>10 microM). 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 (IC(50) 1.3-1.4 nM), whereas 1 in the absence of bCPA was >100-fold less potent (IC(50) 155 nM).
Wright
Joel E
JE
Dana-Farber Cancer Institute and Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, 44 Binney Street, Boston, MA 02115, USA. joel_wright@dfci.harvard.edu
Rosowsky
Andre
A
D000429:Alcohol Oxidoreductases / Q000037:antagonists & inhibitors; D000818:Animals; D000970:Antineoplastic Agents / Q000138:chemical synthesis / Q000378:metabolism / Q000494:pharmacology; D002268:Carboxypeptidases / Q000378:metabolism*; D043422:Carboxypeptidases A; D002417:Cattle; D002455:Cell Division / Q000187:drug effects; D004151:Dipeptides / Q000138:chemical synthesis* / Q000378:metabolism / Q000494:pharmacology; D004791:Enzyme Inhibitors / Q000138:chemical synthesis / Q000378:metabolism / Q000494:pharmacology; D006801:Humans; D020128:Inhibitory Concentration 50; D011355:Prodrugs / Q000138:chemical synthesis* / Q000378:metabolism / Q000494:pharmacology; D013329:Structure-Activity Relationship; D014407:Tumor Cells, Cultured / Q000187:drug effects
D000970:Antineoplastic Agents; D004151:Dipeptides; D004791:Enzyme Inhibitors; C473414:N-(N-(4-amino-4-deoxypteroyl)-N-hemiphthaloyl-ornithiny)-phenylalanine; D011355:Prodrugs; D000429:Alcohol Oxidoreductases; C056570:dihydroflavanol 4-reductase; D002268:Carboxypeptidases; D043422:Carboxypeptidases A