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.

Mechanism of Action and Preclinical Antitumor Activity of the Novel Hypoxia-Activated DNA Cross-Linking Agent PR-104

“…Compound 9 had a mass spectrum (m/z 373; 1 Br) consistent with cyclization via intramolecular alkylation of the ortho hydroxylamine by the mesylate leaving group of the mustard to form a tetrahydroquinoxaline [as reported following ortho nitroreduction of the dinitrobenzamide chloromustard SN 23862 (21,30)]. The corresponding hydrolysis product 10 (m/z 311, no halogens) showed an absorbance spectrum distinct from the para nitroreduction products (Supplementary Fig.…”

“…2A). The ortho nitroreduction pathway generates a monofunctional mustard and is unlikely to contribute to cytotoxicity (30), but its stable end product 10 may be a useful biomarker for hypoxic activation of PR-104A.…”

Agentes antineoplásicos biorredutíveis: uma nova alternativa para o tratamento de tumores sólidos

“…Em 1986, Denny e Wilson lançaram as bases teóricas do planejamento racional de mostardas nitroaromáticas como potenciais agentes citotóxicos seletivos para células em hipóxia 18 . A partir de então, várias mostardas nitroaromáticas, como as mostardas (16) e (17), vêm sendo extensivamente estudadas como pró-fármacos 18,20,[78][79][80][81][82][83][84] .…”

“…Em células em hipóxia, o grupo nitro é reduzido a hidroxilamino e/ou amino (doador de elétrons), aumentando a densidade eletrônica no anel aromático, com conseqüente ativação da mostarda nitrogenada 18 . 81 .…”

Studies relating to the synthesis, enzymatic reduction and cytotoxicity of a series of nitroaromatic prodrugs

“…18 Analogues of CB 1954 1 have also been prepared and studied as potential cytotoxic agents 19 as have the structurally related nitrogen-mustard derivatives SN 23862 5 and its analogues. [20][21][22][23][24][25][26] The 2-nitro-group in SN 23862 5 is reduced by E. coli NR producing the amine derivative 6 thus facilitating the formation of an aziridinium species 7 from the mustard moiety. In this Letter, we report the synthesis and evaluation (enzymatic and cytotoxicity) of a series of Nnitroarylated 1,2,3,4-tetrahydroisoquinoline derivatives with a core structure represented by formula 8 as potential nitroaromatic prodrugs.…”