A new cytotoxic, DNA interstrand crosslinking agent, 5-(aziridin-1-yl)-4-hydroxylamino-2-nitrobenzamide, is formed from 5-(aziridin-1-yl)-2,4-dinitrobenzamide (CB 1954) by a nitroreductase enzyme in Walker carcinoma cells.

The properties of catenated nitrogen molecules, molecules containing internal chains of bonded nitrogen atoms, is of fundamental scientific interest in chemical structure and bonding, as nitrogen i...

Properties and Promise of Catenated Nitrogen Systems As High-Energy-Density Materials

Walker cells in vivo or in vitro are exceptionally sensitive to the monofunctional alkylating agent CB 1954 (5-(aziridin-1-yl)-2,4-dinitrobenzamide). The basis of the sensitivity is that CB 1954 forms DNA interstrand crosslinks in Walker cells but not in insensitive cells. Crosslink formation is due to the aerobic reduction of CB 1954 to form 5-(aziridin-1-yl)-4-hydroxylamino-2-nitrobenzamide by the enzyme DT diaphorase. The 4-hydroxylamine can not crosslink DNA directly but requires further activation by a non-enzymatic reaction with a thioester (such as acetyl coenzyme A). As predicted from their measured DT diaphorase activities, a number of rat hepatoma and hepatocyte cell lines are also sensitive to CB 1954. However, no CB 1954-sensitive tumours or cell lines of human origin have been found. This is because the rate of reduction of CB 1954 by the human form of DT diaphorase is much lower than that of the Walker enzyme (ratio of kcat = 6.4). To overcome this intrinsic resistance of human cells towards CB 1954 a number of strategies have been developed. First, analogues have been developed that are more rapidly reduced by the human form of CB 1954. Second, the cytotoxicity of CB 1954 can be potentiated by reduced pyridinium compounds. Third, a CB 1954 activating enzyme can be targeted to human tumours by conjugating it to an antibody (ADEPT). A nitroreductase enzyme has been isolated from E. coli that can bioactivate CB 1954 much more rapidly than Walker DT diaphorase and is very suitable for ADEPT. Thus CB 1954 may have a role in the therapy of human tumours.

The bioactivation of CB 1954 and its use as a prodrug in antibody-directed enzyme prodrug therapy (ADEPT)

The present study tested the hypothesis that the experimental antineoplastic imidazotetrazinone temozolomide degrades in the biophase to 3-methyl-(triazen-1-yl)imidazole-4-carboxamide (MTIC) and exerts its cytotoxicity via this species. MTIC is a metabolite of the antimelanoma agent dacarbazine and is thought to be responsible for the antineoplastic activity of the latter. Cytotoxicity in vitro was investigated in TLX5 murine lymphoma cells. MTIC and temozolomide were cytotoxic in the absence of mouse-liver microsomes, whereas dacarbazine required metabolic activation. The generation of MTIC from either dacarbazine, its primary metabolite 5-[3-(hydroxymethyl)-3-methyl-triazen-1-yl]-imidazole-4-carboxamid e (HMMTIC) or temozolomide was studied by reversed-phase high-performance liquid chromatography in incubation mixtures under the conditions of the cytotoxicity assay. MTIC was found in incubations of temozolomide with or without microsomes. Dacarbazine yielded MTIC (and HMMTIC) only when microsomes were included in the incubation mixture. Although the mode of action of temozolomide seems to be similar to that of dacarbazine, the results obtained in this study show that these agents differ markedly in their ability to generate the active species MTIC.

Comparison of the cytotoxicity in vitro of temozolomide and dacarbazine, prodrugs of 3-methyl-(triazen-1-yl)imidazole-4-carboxamide

Bioactivation of CB 1954: reaction of the active 4-hydroxylamino derivative with thioesters to form the ultimate DNA-DNA interstrand crosslinking species.

Walter C.J. Ross

Papers

Tumour inhibitory triazenes: structural requirements for an active metabolite.

The metabolism of the anti-tumour agent 5-(1-aziridinyl)-2,4-dinitrobenzamide (CB 1954)

N-(2-chloroethyl)-N-(2-hydroxyethyl)arylamines. Possible intermediates to potential carcinolytic agents bearing dissimilar reactive functions.