title: Reductive chemistry of the novel hypoxia-selective cytotoxin 5-[N,N-bis(2-chloroethyl)amino]-2,4-dinitrobenzamide.
abstract: 5-[N,N-Bis(2-chloroethyl)amino]-2,4-dinitrobenzamide (1; SN 23862) is a novel bioreductive drug whose selective toxicity for hypoxic cells appears due to oxygen-inhibited enzymatic reduction of one of the nitro groups to the corresponding amine or hydroxylamine. Radiolytic reduction of 1 using up to four reducing equivalents in 1 N sodium formate was shown to proceed via electron addition to the 4-nitro group, thereby identifying this substituent as the most electron-affinic site in the molecule. The initially-formed 4-hydroxylamine and its N-hydroxytetrahydroquinoxaline half-mustard cyclization product (formed by intramolecular reaction with one arm of the adjacent mustard group) are reduced to the corresponding 4-amines upon further addition of electrons, although reduction of the 2-nitro group leading to 2,4-diamino products begins after addition of only six electron equivalents. Radiolytic reduction of the structurally similar 5-(aziridin-1-yl)-2,4-dinitrobenzamide (2; CB 1954) with six electron equivalents also occurs at the 4-nitro group to give the 4-hydroxylamine and 4-amine. The product mixture from reduction of 2 is less complex, largely because the corresponding 4-hydroxylamine and 4-amine are stable. The major reduction products of 1 were chemically synthesized by unequivocal routes to provide authentic samples for identification of the products of radiolytic reduction and to allow determination of their cytotoxicities. The 2- and 4-amino derivatives of 1 are significantly more cytotoxic than the parent drug, although the toxicity of the 4-amine is moderated by its facile conversion to the corresponding less toxic tetrahydroquinoxaline half-mustard. Although the 2- and 4-hydroxylamino derivatives were prepared by chemical reduction of 1, their toxicity could not be evaluated because of their instability. The 4-hydroxylamine reacts intramolecularly with the 5-mustard group somewhat more rapidly than does the 4-amine, while the 2-hydroxylamine is converted into a 2,2'-azoxy dimer following aerial oxidation to the 2-nitroso derivative. The fully reduced 2,4-diamino derivative of 1 is 10-fold more cytotoxic again than the 2-amine and, surprisingly, does not undergo spontaneous intramolecular alkylation. This elucidation of the reduction chemistry of 1 will facilitate further investigations of the toxic products generated from this compound both by hypoxic tumor cells and by ADEPT enzymes.
authors.affiliation: Cancer Research Laboratory, University of Auckland School of Medicine, New Zealand.
authors.forename: B D
authors.initials: BD
authors.lastname: Palmer
authors.forename: P
authors.initials: P
authors.lastname: van Zijl
authors.forename: W A
authors.initials: WA
authors.lastname: Denny
authors.forename: W R
authors.initials: WR
authors.lastname: Wilson
mesh_terms: D000816:Aniline Mustard / Q000031:analogs & derivatives* / Q000494:pharmacology; D000970:Antineoplastic Agents; D002455:Cell Division / Q000187:drug effects*; D002460:Cell Line; D055598:Chemical Phenomena; D002627:Chemistry, Physical; D002851:Chromatography, High Pressure Liquid; D000860:Hypoxia; D066298:In Vitro Techniques; D009588:Nitrogen Mustard Compounds / Q000138:chemical synthesis / Q000737:chemistry / Q000633:toxicity*; D010084:Oxidation-Reduction; D011355:Prodrugs / Q000737:chemistry
chemical_list: D000970:Antineoplastic Agents; D009588:Nitrogen Mustard Compounds; D011355:Prodrugs; C076205:5-(N,N-bis(2-chloroethyl)amino)-2,4-dinitrobenzamide; D000816:Aniline Mustard