Reactions of 1,3-bis(2-chloroethyl)-1-nitrosourea and 1-(2-chloroethyl)-3-cyclohexyl-1-nitrosourea in aqueous solution

Weinkam, Robert J.; Lin, Huey-Shin

doi:10.1021/jm00196a009

Cited by 34 publications

(28 citation statements)

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“…However, deuterium substitution in some of these structures, such as the antitumor agent 1,3-bis(2-chloroethyl)-1-nitrosourea, has revealed multiple mechanisms of elimination and hydrolysis in generating reactive metabolites (Brundrett et al, 1976;Weinkam and Lin, 1979).…”

Section: Use Of Deuterium Isotope Effects To Probe P450 Reactionsmentioning

confidence: 99%

The Use of Deuterium Isotope Effects to Probe the Active Site Properties, Mechanism of Cytochrome P450-Catalyzed Reactions, and Mechanisms of Metabolically Dependent Toxicity

Nelson¹,

Trager²

2003

Drug Metab Dispos

143

121

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This article is available online at http://dmd.aspetjournals.org ABSTRACT:Critical elements from studies that have led to our current understanding of the factors that cause the observed primary deuterium isotope effect, (k H /k D )obs, of most enzymatically mediated reactions to be much smaller than the "true" or intrinsic primary deuterium isotope effect, k H /k D , for the reaction are presented. This new understanding has provided a unique and powerful tool for probing the catalytic and active site properties of enzymes, particularly the cytochromes P450 (P450). Examples are presented that illustrate how the technique has been used to determine k H /k D , and properties such as the catalytic nature of the reactive oxenoid intermediate, prochiral selectivity, the chemical and enzymatic mechanisms of cytochrome P450-catalyzed reactions, and the relative active site size of different P450 isoforms. Examples are also presented of how deuterium isotope effects have been used to probe mechanisms of the formation of reactive metabolites that can cause toxic effects.Historically, the determination of deuterium isotope effects has been a powerful tool to help unravel the intricacies of carbon-hydrogen bond cleavage and define the mechanism of specific chemical reactions. The intrinsic primary isotope effect, k H /k D , for a reaction is the magnitude of the isotope effect on the rate constant for the bond-breaking step (C-H versus C-D) of the reaction and is related to the symmetry of the transition state for that step. The larger the isotope effect, the more symmetrical the transition state, with the theoretical limit being 9 at 37°C in the absence of tunneling effects (Bell, 1974). The chemical events leading to the transition state and subsequent formation of products are the descriptors of a chemical mechanism. It is the relationship of k H /k D to transition state that provides mechanistic insight. Thus, k H /k D is the quantity that needs to be known, and for homogenous chemical reactions, the experimentally observed deuterium isotope effect, (k H /k D )obs, where (k H /k D )obs is defined as the ratio of a kinetic parameter such as V max or V max /K m obtained from a nondeuterated substrate to a deuterated substrate, is identical to k H /k D . This is generally not true of enzymatically mediated reactions and is the reason for the much less successful application of deuterium isotope effects to such reactions until the system was better understood. The experimentally observed isotope effect, (k H /k D )obs, was invariably found to be much smaller than k H /k D , the intrinsic isotope effect for that reaction, thereby obscuring both meaning and mechanism. Even more perplexing was the observation that (k H /k D

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Section: Use Of Deuterium Isotope Effects To Probe P450 Reactionsmentioning

confidence: 99%

The Use of Deuterium Isotope Effects to Probe the Active Site Properties, Mechanism of Cytochrome P450-Catalyzed Reactions, and Mechanisms of Metabolically Dependent Toxicity

Nelson¹,

Trager²

2003

Drug Metab Dispos

143

121

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“…The anti-tumour activity of both compounds is dependent upon formation of a reactive alkyldiazonium ion (Weinkam and Lin, 1979;Denny et al, 1994) and subsequent alkylation of the accessible nucleophilic atoms of DNA, predominantly N7-guanine, followed by N3-adenine and o6-guanine (Roberts, 1978). Temozolomide cytotoxicity can largely be accredited to methylation of 06-guanine (Domoradzki et al, 1984;Margison and O'Connor, 1990) and N3-adenine (Karran et al, 1982); the cytotoxicity/mutagenicity of 06-methylguanine being attributed to the induction of futile cycling in the long patch mismatch repair pathway, which results in prolonged DNA strand interruptions and the inhibition of subsequent replication Karran et al, 1993).…”

mentioning

confidence: 99%

3-aminobenzamide and/or O6-benzylguanine evaluated as an adjuvant to temozolomide or BCNU treatment in cell lines of variable mismatch repair status and O6-alkylguanine-DNA alkyltransferase activity

Wedge

Porteous²,

Newlands³

1996

Br J Cancer

106

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Summary 06-benzylguanine (06-BG) and 3-aminobenzamide (3-AB) inhibit the DNA repair proteins o6_ alkylguanine-DNA alkyltransferase (AGT) and poly(ADP-ribose) polymerase (PARP) respectively. The effect of 06-BG and/or 3-AB on temozolomide and 1,3-bis(2-chloroethyl)-nitrosourea (BCNU) cytotoxicity, was assessed in seven human tumour cell lines: six with an AGT activity of >80 fmol mg-' protein (Mer+) and one with an AGT activity of <3 fmol mg protein (Mer-). Three of the Mer+ cell lines (LS174T, DLD1 and HCT1 16) were considered to exhibit resistance to methylation by a mismatch repair deficiency (MMR-), each being known to exhibit microsatellite instability, and DLD1 and HCT1 16 having well-characterised defects in DNA mismatch binding. Potentiation was defined as the ratio between an IC50 achieved without and with a particular inhibitor treatment. Temozolomide or BCNU cytotoxicity was not potentiated by either inhibitor in the Mer-cell line. Preincubation with 06-BG (100 /M for 1 h) was found to potentiate the cytotoxicity of temozolomide by 1.35-to 1.57-fold in Mer+/MMR+ cells, but had no significant effect in Mer+/MMR-cells.In comparison, 06-BG pretreatment enhanced BCNU cytotoxicity by 1.94-to 2.57-fold in all Mer+ cell lines. Post-incubation with 3-AB (2 mm, 48 h) potentiated temozolomide by 1.35-to 1.59-fold in Mer+/MMR+ cells, and when combined with 06-BG pretreatment produced an effect which was at least additive, enhancing cytotoxicity by 1.97-to 2.16-fold. 3-AB treatment also produced marked potentiation (2.20-to 3.12-fold) of temozolomide cytotoxicity in Mer+/MMR-cells. In contrast, 3-AB produced marginal potentiation of BCNU cytotoxicity in only three cell lines (1.19-to 1.35-fold), and did not enhance the cytotoxicity of BCNU with o6_ BG treatment in any cell line. These data suggest that the combination of an AGT and PARP inhibitor may have a therapeutic role in potentiating temozolomide activity, but that the inhibition of poly(ADP-ribosyl)ation has little effect on the cytotoxicity of BCNU.

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“…BCNU has proven clinically useful in the management of brain tumours and lymphomas (Young et al, 1971;Edwards et al, 1980) but has limited therapeutic efficacy because of intrinsic or acquired tumour resistance (Walker and Hurwitz, 1970;Carter and Wasserman, 1976). This compound rapidly decomposes at physiological pH to yield an alkylating chloroethyldiazonium ion and a carbamoylating isocyanate (Montgomery et al, 1967;Weinkam and Lin, 1979 (Tong et al, 1982). It is this ability to cross-link DNA that correlates with chloroethylnitrosourea cytotoxicity (Lown et al, 1978;Bodell et al, 1985;Jiang et al, 1989).…”

mentioning

confidence: 99%

Potentiation of temozolomide and BCNU cytotoxicity by O6-benzylguanine: a comparative study in vitro

Wedge

Porteus²,

May³

et al. 1996

Br J Cancer

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Depletion of the DNA repair protein O(6)-alkylguanine-DNA alkyltransferase (AGT) with O(6)-benzylguanine (O(6)-BG) has been widely shown to enhance 1,3-bis(2-chloroethyl)-nitrosourea (BCNU) activity. This study aimed to determine whether temozolomide, a methylating imidazotetrazinone, would similarly benefit from combination with O(6)-BG. Seven human cell lines were examined with AGT activities ranging from <6 fmol mg-1 protein to >700 fmol mg-1 protein. Comparisons with BCNU were made on both single and multiple dosing schedules, since temozolomide cytotoxicity is highly schedule dependent. In single-dose potentiation studies, cells were preincubated with 100 microM O(6)-BG for 1 h, a treatment found to deplete AGT activity by >90% for 24 h. No potentiation of either temozolomide or BCNU cytotoxicity was observed in two glioblastoma cell lines with <6 fmol mg-1 protein AGT. In all other cell lines studied potentiation of BCNU toxicity by O(6)-BG was between 1.6- and 2.3-fold and exceeded that of temozolomide (1.1- to 1.7-fold). The magnitude of this potentiation was unrelated to AGT activity and the relative potentiation of temozolomide and BCNU cytotoxicity was found to be highly variable between cell lines. In multiple dosing studies two colorectal cell lines (Mawi and LS174T) were treated with temozolomide or BCNU at 24 h intervals for up to 5 days, with or without either 100 microM O(6)-BG for 1 h or 1 microM O(6)-BG for 24 h, commencing 1 h before alkylating treatment. Extended treatment with 1 microM O(6)-BG produced greater potentiation than intermittent treatment with 100 microM O(6)-BG. Potentiation of temozolomide cytotoxicity increased linearly in Mawi with each subsequent dosing: from 1.4-fold (day 1) to 4.2-fold (day 5) with continuous 1 microM O(6)-BG. In contrast, no potentiation was observed in LS174T, a cell line that would appear to be 'tolerant' of methylation. Potentiation of BNCU cytotoxicity increased in both cell lines with repeat dosing, although the rate of increase was less than that observed with temozolomide and continuous 1 microM O(6)-BG in Mawi. These results suggest that repeat dosing of an AGT inhibitor and temozolomide may have a clinical role in the treatment of tumours that exhibit AGT-mediated resistance.

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Reactions of 1,3-bis(2-chloroethyl)-1-nitrosourea and 1-(2-chloroethyl)-3-cyclohexyl-1-nitrosourea in aqueous solution

Cited by 34 publications

References 1 publication

The Use of Deuterium Isotope Effects to Probe the Active Site Properties, Mechanism of Cytochrome P450-Catalyzed Reactions, and Mechanisms of Metabolically Dependent Toxicity

The Use of Deuterium Isotope Effects to Probe the Active Site Properties, Mechanism of Cytochrome P450-Catalyzed Reactions, and Mechanisms of Metabolically Dependent Toxicity

3-aminobenzamide and/or O6-benzylguanine evaluated as an adjuvant to temozolomide or BCNU treatment in cell lines of variable mismatch repair status and O6-alkylguanine-DNA alkyltransferase activity

Potentiation of temozolomide and BCNU cytotoxicity by O6-benzylguanine: a comparative study in vitro

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