title: Design, synthesis, and biological evaluation of cyclic and acyclic nitrobenzylphosphoramide mustards for E. coli nitroreductase activation.
abstract: In efforts to obtain anticancer prodrugs for antibody-directed or gene-directed enzyme prodrug therapy using E. coli nitroreductase, a series of nitrobenzylphosphoramide mustards were designed and synthesized incorporating a strategically placed nitro group in a position para to the benzylic carbon for reductive activation. All analogues were good substrates of E. coli nitroreductase with half-lives between 2.9 and 11.9 min at pH 7.0 and 37 degrees C. Isomers of the 4-nitrophenylcyclophosphamide analogues 3 and 5 with a benzylic oxygen para to the nitro group showed potent selective cytotoxicity in nitroreductase (NTR) expressing cells, while analogues 4 and 6 with a benzylic nitrogen para to the nitro group showed little selective cytotoxicity despite their good substrate activity. These results suggest that good substrate activity and the benzylic oxygen are both required for reductive activation of 4-nitrophenylcyclophosphamide analogues by E. coli nitroreductase. Isomers of analogue 3 showed 23,000-29,000x selective cytotoxicity toward NTR-expressing V79 cells with an IC(50) as low as 27 nM. They are about as active as and 3-4x more selective than 5-aziridinyl-2,4-dinitrobenzamide (CB1954). The acyclic 4-nitrobenzylphosphoramide mustard ((+/-)-7) was found to be the most active and most selective compound for activation by NTR with 170,000x selective cytotoxicity toward NTR-expressing V79 cells and an IC(50) of 0.4 nM. Compound (+/-)-7also exhibited good bystander effect compared to 5-aziridinyl-2,4-dinitrobenzamide. The low IC(50), high selectivity, and good bystander effects of nitrobenzylphosphoramide mustards in NTR-expressing cells suggest that they could be used in combination with E. coli nitroreductase in enzyme prodrug therapy.
authors.affiliation: Department of Pharmaceutical Chemistry, Ernest Mario School of Pharmacy, Rutgers, The State University of New Jersey, 160 Frelinghuysen Road, Piscataway, 08854, USA.
authors.forename: Yongying
authors.initials: Y
authors.lastname: Jiang
authors.forename: Jiye
authors.initials: J
authors.lastname: Han
authors.forename: Chengzhi
authors.initials: C
authors.lastname: Yu
authors.forename: Simon O
authors.initials: SO
authors.lastname: Vass
authors.forename: Peter F
authors.initials: PF
authors.lastname: Searle
authors.forename: Patrick
authors.initials: P
authors.lastname: Browne
authors.forename: Richard J
authors.initials: RJ
authors.lastname: Knox
authors.forename: Longqin
authors.initials: L
authors.lastname: Hu
mesh_terms: D000818:Animals; D000970:Antineoplastic Agents / Q000138:chemical synthesis* / Q000737:chemistry / Q000494:pharmacology; D002460:Cell Line; D045744:Cell Line, Tumor; D006224:Cricetinae; D003412:Cricetulus; D015195:Drug Design; D004789:Enzyme Activation; D029968:Escherichia coli Proteins / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D009601:Nitroreductases / Q000235:genetics / Q000378:metabolism*; D010752:Phosphoramide Mustards / Q000138:chemical synthesis* / Q000737:chemistry / Q000494:pharmacology; D011355:Prodrugs / Q000138:chemical synthesis* / Q000737:chemistry / Q000494:pharmacology; D013237:Stereoisomerism; D013329:Structure-Activity Relationship; D014162:Transfection
chemical_list: D000970:Antineoplastic Agents; D029968:Escherichia coli Proteins; D010752:Phosphoramide Mustards; D011355:Prodrugs; D009601:Nitroreductases