Identification, synthesis and properties of 5-(aziridin-1-YL)-2-nitro-4-nitrosobenzamide, A novel DNA crosslinking agent derived from CB1954

doi:10.1016/0006-2952(93)90487-H

Biochemical Pharmacology

Volume 46, Issue 5, 1 September 1993, Pages 797-803

https://doi.org/10.1016/0006-2952(93)90487-H Get rights and content

Abstract

5-(Aziridin-1-yl)-4-hydroxylamino-2-nitrobenzamide, the active form of 5-(aziridin-1-yl)-2,4-dinitrobenzamide (CB1954), can react spontaneously with oxygen, and in aqueous solution yields 5-(aziridin-1-yl)-2-nitro-4-nitrosobenzamide and hydrogen peroxide. Mild biological reducing agents such as NAD(P)H, reduced thiols and ascorbic acid rapidly re-reduced the nitroso compound to the hydroxylamine. Both compounds were equally efficient at inducing cytotoxicity and DNA interstrand crosslinking in cells when exposed in phosphate-buffered saline (PBS). Neither agent was capable of inducing cross-links in isolated DNA. When acetyl coenzyme A was included in the incubation, crosslink formation was seen with the hydroxylamine, but not with the nitroso compound. Thus, the nitroso compound is acting as a prodrug for the hydroxylamine, and needs to be reduced to this compound to exert its cytotoxic effects. In vivo anti-tumour tests showed that neither compound was effective in its own right. This may be due to the rapid reduction of the nitroso to the hydroxylamine, and the reaction of the hydroxylamine with serum proteins. The chemical synthesis of the 5-(aziridin-1-yl)-2-nitro-4-nitrosobenzamide, and an improved synthesis of 5-(aziridin-1-yl)-4-hydroxylamino-2-nitrobenzamide is described. These results emphasize the potential efficacy of the in situ activation of prodrugs such as CB1954 either by endogenous enzymes such as DT diaphorase, or by antibody directed enzyme prodrug therapy (ADEPT).

References (22)

LM Cobb et al.
2,4-Dinitro-5-ethyleneiminobenzamide (CB1954): a potent and selective inhibitor of the growth of the Walker carcinoma 256
Biochem Pharmacol
(1969)
JJ Roberts et al.
CB1954 (2,4-dinitro-5-aziridinyl benzamide) becomes a DNA interstrand crosslinking agent in Walker tumour cells
Biochem Biophys Res Commun
(1986)
RJ Knox et al.
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 (CB1954) by a nitroreductase enzyme in Walker carcinoma cells
Biochem Pharmacol
(1988)
RJ Knox et al.
The nitroreductase enzyme in Walker cells that activates 5-(aziridin-1-yl)-2,4-dinitro-benzamide (CB1954) to 5-(aziridin-1-yl)-4-hydroxyl-amino-2-nitrobenzamide is a form of NAD(P)H dehydrogenase (quinone) (EC 1.6.99.2)
Biochem Pharmacol
(1988)
T Cresteil et al.
High levels of expression of the NAD(P)H: quinone oxidoreductase (NQO1) gene in tumor cells compared to normal cells of the same origin
Biochem Pharmacol
(1991)
F Friedlos et al.
Potentiation of CB1954 cytotoxicity by reduced pyridine nucleotides in human tumour cells by stimulation of DT diaphorase activity
Biochem Pharmacol
(1992)
RJ Knox et al.
Bioactivation of CB1954: reaction of the active 4-hydroxylamino derivative with thioesters to form the ultimate DNA-DNA interstrand crosslinking species
Biochem Pharmacol
(1991)
F Friedlos et al.
The properties of total adducts and interstrand crosslinks in the DNA of cells treated with CB1954. Exceptional frequency and stability of the crosslink
Biochem Pharmacol
(1992)
MP Boland et al.
The differences in kinetics of rat and human DT diaphorase result in a differential sensitivity of derived cell lines to CB1954 (5-(aziridin-1-yl)-2,4-dinitrobenzamide)
Biochem Pharmacol
(1991)
RJ Riley et al.
DT-diaphorase and cancer chemotherapy
Biochem Pharmacol
(1992)

GM Anlezark et al.

The bioactivation of CB1954. I. Purification and properties of a nitroreductase enzyme from Escherichia coli—a potential enzyme for antibody directed enzyme prodrug therapy (ADEPT)

Biochem Pharmacol

(1992)

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It transpired that Walker 256 tumour cells produced a unique DT-diaphorase activity, not present in the other rodent and human cell lines tested, that activated CB1954 by reduction of its auxochromic nitro groups. The hydroxylamine produced is cancerostatic because it promotes the formation of interstrand C8-O6 DNA cross-links, which are ineffectively repaired by the cell [34,35]. The rat ‘DT-diaphorase’ is systematically named NQO1 (NAD(P)H:quinine oxidoreductase).
Despite substantial investment in prevention, treatment and aftercare, cancer remains a leading cause of death worldwide. More effective and accessible therapies are required. A potential solution is the use of endospore forming Clostridium species, either on their own, or as a tumour delivery vehicle for anti-cancer drugs. This is because intravenously injected spores of these obligate anaerobes can exclusively germinate in the hypoxic/necrotic regions present in solid tumours and nowhere else in the body. Research aimed at exploiting this unique phenomenon in anti-tumour strategies has been ongoing since the early part of the 20th century. Only in the last decade, however, has there been significant progress in the development and refinement of strategies based on spore-mediated tumour colonisation using a range of clostridial species. Much of this progress has been due to advances in genomics and our ability to modify strains using more sophisticated gene tools.
Understanding the broad substrate repertoire of nitroreductase based on its kinetic mechanism
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The hydroxylamine product is a basis for the toxicity of metronidazole and related antibiotics, which are activated in vivo by pathogen NRs (15, 16) (in anaerobes, other mechanisms reductively eliminate the nitro group producing nitro radicals that initiate oxidative stress (17, 18)). Thus chemistry of NR is being developed for clinical implementation against cancer via GDEPT (gene-directed enzyme prodrug therapy) and ADEPT (antibody-directed enzyme pro-drug therapy) wherein an NfsB-antibody fusion would be targeted to tumors where it would then reductively activate the prodrug CB1954 in ADEPT (8, 9, 19). Thus it is important to know what product NR produces.
The oxygen-insensitive nitroreductase from Enterobacter cloacae (NR) catalyzes two-electron reduction of nitroaromatics to the corresponding nitroso compounds and, subsequently, to hydroxylamine products. NR has an unusually broad substrate repertoire, which may be related to protein dynamics (flexibility) and/or a simple non-selective kinetic mechanism. To investigate the possible role of mechanism in the broad substrate repertoire of NR, the kinetics of oxidation of NR by para-nitrobenzoic acid (p-NBA) were investigated using stopped-flow techniques at 4 °C. The results revealed a hyperbolic dependence on the p-NBA concentration with a limiting rate of 1.90 ± 0.09 s⁻¹, indicating one-step binding before the flavin oxidation step. There is no evidence for a distinct binding step in which specificity might be enforced. The reduction of p-NBA is rate-limiting in steady-state turnover (1.7 ± 0.3 s⁻¹). The pre-steady-state reduction kinetics of NR by NADH indicate that NADH reduces the enzyme with a rate constant of 700 ± 20 s⁻¹ and a dissociation constant of 0.51 ± 0.04 mm. Thus, we demonstrate simple transient kinetics in both the reductive and oxidative half-reactions that help to explain the broad substrate repertoire of NR. Finally, we tested the ability of NR to reduce para-hydroxylaminobenzoic acid, demonstrating that the corresponding amine does not accumulate to significant levels even under anaerobic conditions. Thus E. cloacae NR is not a good candidate for enzymatic production of aromatic amines.
An unusually cold active nitroreductase for prodrug activations
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Citation Excerpt :
with flow rate of 1 ml/min. For the quantification, the extinction coefficients were used as follows: ε260 = 7880 M−1 cm−1 for the 4-hydroxylamine and ε260 = 5420 M−1 cm−1 for the 2-hydroxylamine.45 Using BLAST search46 with the sequence of E. coli nitroreductases (nfsB), a potential nitroreductase gene was identified from the genome sequence of S. saprophyticus.
A set of PCR primers based on the genome sequence were used to clone a gene encoding a hypothetical nitroreductases (named as Ssap-NtrB) from uropathogenic staphylococcus, Staphylococcus saprophyticus strain ATCC 15305, an oxygen insensitive flavoenzyme. Activity studies of the translation product revealed that the nitroreductase catalyses two electron reduction of a nitroaromatic drug of nitrofurazone (NFZ), cancer prodrugs of CB1954 and SN23862 at optimum temperature of 20 °C together with retaining its maximum activity considerably at 3 °C. The required electrons for such reduction could be supplied by either NADH or NADPH with a small preference for the latter. The gene was engineered for heterologous expression in Escherichia coli, and conditions were found in which the enzyme was produced in a mostly soluble form. The recombinant enzyme was purified to homogeneity and physical, spectral and catalytical properties were determined. The findings lead us to propose that Ssap-NtrB represents a novel nitro reductase with an unusual cold active property, which has not been described previously for prodrug activating enzymes of nitroreductases.
Kinetic and Structural Characterisation of Escherichia coli Nitroreductase Mutants Showing Improved Efficacy for the Prodrug Substrate CB1954
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Escherichia coli nitroreductase (NTR) is a flavoprotein that reduces a variety of quinone and nitroaromatic substrates. Among these substrates is the prodrug 5-[aziridin-1-yl]-2,4-dinitrobenzamide (CB1954) that is activated by NTR to form two products, one of which is highly cytotoxic. NTR in combination with CB1954 has entered clinical trials for virus-directed enzyme–prodrug therapy of cancer. Enhancing the catalytic efficiency of NTR for CB1954 is likely to improve the therapeutic potential of this system. We previously identified a number of mutants at six positions around the active site of NTR that showed enhanced sensitisation to CB1954 in an E. coli cell-killing assay. In this study we have purified improved mutants at each of these positions and determined their steady-state kinetic parameters for CB1954 and for the antibiotic nitrofurazone. We have also made a double mutant, combining two of the most beneficial single mutations. All the mutants show enhanced specificity constants for CB1954, and, apart from N71S, the enhancement is selective for CB1954 over nitrofurazone. One mutant, T41L, also shows an increase in selectivity for reducing the 4-nitro group of CB1954 rather than the 2-nitro group. We have determined the three-dimensional structures of selected mutants bound to the substrate analogue nicotinic acid, using X-ray crystallography. The N71S mutation affects interactions of the FMN cofactor, while mutations at T41 and F124 affect the interactions with nicotinic acid. The structure of double mutant N71S/F124K combines the effects of the two individual single mutations, but it gives a greater selective enhancement of activity with CB1954 over nitrofurazone than either of these, and the highest specificity constant for CB1954 of all the mutations studied.
Quinone Reductase-Mediated Nitro-Reduction: Clinical Applications
2004, Methods in Enzymology
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However the 4-nitroso compound does not react with thioesters. The nitroso compound is acting as a prodrug for the hydroxylamine and needs to be reduced to this compound to exert its cytotoxic effects.46 Importantly, in vivo, neither compound shows any anti-tumor activity.46
This chapter discusses the clinical applications of quinone reductase–mediated nitro-reduction. The quinone reductase enzyme NQO1 catalyzes the reduction of quinines to their hydroquinone form. NQO1 has a number of unusual properties. It can use either NADPH or NADH with equal activity as a cofactor, and it has been shown that simple reduced nicotinamide derivatives can also act as cofactors for this enzyme. This chapter examines the clinical applications of the nitroreductase activity of three quinone reductase enzymes, NQO1, E. coli B nitroreductase and, in particular, NQO2. The nitroreductase properties of these enzymes are particularly relevant to the treatment of cancer, and the rationale for this approach is described in the chapter. With NQO2, an endogenous human enzyme has been discovered with significant nitroreductase activity that can bioactivate a proven anti-tumor prodrug designated CB 1954. Prodrugs activated by nitro-reduction in cancer therapy are described also described in the chapter. The chapter discusses the use of NQO1 as a nitroreductase. The chapter describes the functional characterization of truncated NQO1 and NQO1/NQO2 chimeric enzyme preparations. Bioactivation of CB 1954 by NQO2, and biodistribution of NQO2 in humans is also explained in the chapter.
Quantitative structure-activity relationships in two-electron reduction of nitroaromatic compounds by Enterobacter cloacae NAD(P)H: Nitroreductase
2001, Archives of Biochemistry and Biophysics
Enterobacter cloacae NAD(P)H:nitroreductase (NR; EC 1.6.99.7) catalyzes the reduction of a series of nitroaromatic compounds with steady-state bimolecular rate constants (k_cat/K_m) ranging from 10⁴ to 10⁷ M⁻¹ s⁻¹. In agreement with a previously proposed scheme of two-step four-electron reduction of nitroaromatics by NR (Koder, R. L., and Miller, A.-F. (1998) Biochim. Biophys. Acta 1387, 395–405), 2 mol NADH per mole mononitrocompound were oxidized. An oxidation of excess NADH by polinitrobenzenes, including explosives 2,4,6-trinitrotoluene (TNT) and 2,4,6-trinitrophenyl-N-methylnitramine (tetryl), has been observed as a slower secondary process, accompanied by O₂ consumption. This type of “redox cycling” was not related to reactions of nitroaromatic anion-radicals, but was caused by the autoxidation of relatively stable reaction products. The initial reduction of tetryl and other polinitrophenyl-N-nitramines by E. cloacae NR was analogous to a two-step four-electron reduction mechanism of TNT and other nitroaromatics. The logs k_cat/K_m of all the compounds examined exhibited parabolic dependence on their enthalpies of single-electron or two-electron (hydride) reduction, obtained by quantum mechanical calculations. This type of quantitative structure–activity relationship shows that the reactivity of nitroaromatics towards E. cloacae nitroreductase depends mainly on their hydride accepting properties, but not on their particular structure, and does not exclude the possibility of multistep hydride transfer.

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Identification, synthesis and properties of 5-(aziridin-1-YL)-2-nitro-4-nitrosobenzamide, A novel DNA crosslinking agent derived from CB1954

Abstract

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