Abstract
An aqueous acetone 4-(4'-nitrobenzyl)pyridine (NBP) assay for alkylating activity was used at neutral pH to reproduce the Swain-Scott-Ogston reactivity sequence of nucleophile n constants, based on bimolecular competition between NBP and added nucleophiles for alkylation by nitrogen mustards, methanesulfonate esters, methyl iodide, epichlorohydrin, or 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU). Nucleophile concentration-dependent decreases in the 540-nm NBP product(s) permitted determination of the second-order rate constant ratio, kx/knbp, for alkylation of the competing nucleophile/NBP. The slope, s*, of several log(kx/knbp) values versus n constants closely paralleled literature Swain-Scott s constants and could be derived from the value for thiosulfate competition, 1og(kss2oo3/knbp). Alkylating activity, defined by the maximal 540-nm absorbance found for reactions up to 1 hr, was clearly related to an alkylating agent’s nucleophilic sensitivity [s, s*, or log(kss2oo3/knbp) value]. Values of log(kSS2oo3/knbp) corresponding to s constants 0.78-1.39 for clinical alkylating agents increased in the order 1-chloroethyl-3-(4-methylcyclohexyl)-1-nitrosourea ∼ streptozotocin < 1,4-bis(methanesulfonoxy)butane ∼ BCNU < 1,1',l ''-phosphinothioylidynetrisaziridine ∼ methyl-bis(2-chloroethyl)amine < 4-[bis(2-chloroethyl)amino]-L-phenylalanine. NBP competition methods for determination of nucleophilic selectivity ratios should be highly useful for rapid identification of protective nucleophile-clinical alkylating agent combinations, and for classification of alkylating agent biological mechanism of action according to recent interpretations of alkylating agent kinetic behavior.
ACKNOWLEDGMENTS I am most grateful to Drs. Charles Heidelberger and George Olah for encouragement and helpful advice, and to Dr. Michael Colvin for his stimulating review of the manuscript. I am indebted to Margaret Soh for her patient typing.
- Copyright © 1981 by The American Society for Pharmacology and Experimental Therapeutics
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