Interactions of aromatic nitrogen mustards with DNA

Abstract

Bis(2-chloroethyl)amines or nitrogen mustards are amongst the most useful drugs used in cancer chemotherapy. Nitrogen mustards may alkylate any accessible nucleophilic atom in nucleic acids, proteins or other biomolecules. The most effective nitrogen mustards almost universally possess two functional groups, suggesting that the anticancer action involves the formation of crosslinks between macromolecular sites. Our initial studies were designed to elucidate the general action of nitrogen mustards. Rate constants of hydrolysis of a series of aromatic nitrogen mustards Ar-X-pC6H4-N(CH2CH2Cl)2 (X = O, CH2, CONH, S, CO, and SO2) in buffered aqueous acetone mixtures have been obtained using an HPLC technique which allows evaluation of the rate constants for hydrolytic displacement of both chlorines in consecutive reactions. Hydrolysis has a general base-catalysed component and is accompanied by external chloride ion return. An aziridinium ion intermediate is implicated in at least one pathway. The mustards also alkylate the nucleophiles thiourea and 4-(4'-nitrobenzyl)pyridine, and again the aziridinium ion intermediate is involved since the kinetic behaviour rules out a direct SN2 pathway. The rate constants of hydrolysis of a series of 4-substituted aniline mustards Ar-X-pC6H4-N(CH2CH2Cl)2, where Ar is 4-anilinoquinolinium and X = O, CH2, CONH and CO, have been measured in water and in 0.02 M imidazole buffer at 37°C and in 50% aqueous acetone at 66°C. The equilibrium binding constants of the compounds and their hydrolytic products to nucleic acids of differing base composition have been determined at varying ionic strengths, and the results are consistent with the compounds binding, as expected, in the DNA minor groove. The alkylating reactivity of the mustards towards these nucleic acids has been measured in water at 37°C and in 0.01 M HEPES buffer over a range of temperatures from 25°C to 60°C. Evaluation of the thermodynamic parameters for these kinetic and equilibrium studies suggests that the interaction with nucleic acids is via an internal SN2 mechanism involving an aziridinium ion. The rate constants of hydrolysis of, and of alkylation of calf thymus DNA by, a series of 4-substituted aniline mustards Ar-X-pC6H4-N(CH2CH2Cl)2, where Ar is an acridine, benzisoquinolinodione or bisbenzimidazole chromophore and X is a link group containing -(CH2)n- or -O(CH2)n- where n varies from 0 to 6, have been measured in 0.01 M HEPES buffer at 37°C. Comparison of these results with those for equilibrium binding constants of the compounds to calf thymus DNA shows that with increasing length of the polymethylene linker the rate of alkylation increases but non-covalent binding becomes weaker. A relationship was also sought between chain length and biological properties but this was only apparent for the bisbenzimidazole mustards, with potency maximizing at a carbon-linker chain unit containing two methylene groups. The in vivo antitumour activities depend to some extent on the nature of the DNA-directed chromophore. There appears to be some relationship between biological activity and the extent of DNA alkylation, with the least active compounds having the smallest binding constants. However, DNA-targeting appears to allow the use of less reactive mustards. The binding of fifteen 4-substituted aniline mustards to DNA has been examined by agarose-gel electrophoresis. The 4-substituents included a quinoline, acridine, benzisoquinolinodione or bisbenzimidazole chromophore attached by linker chains of varying length. The quinoline mustard and bisbenzimidazole mustards have been shown to be minor groove binding agents, while the 9-aminoacridine mustards and the benzisoquinolinodione- mustards are intercalating agents. A simple agarose-gel electrophoretic method has been developed for determining the superhelical density of closed supercoiled DNA. The gel pattern depends on both the nature and the concentration of the mustard and suggests that noncovalent interactions occur between the non-alkylating moieties of these drugs and DNA, prior to covalent reaction. The evidence from the isolation of the DNA alkylated products suggests that the primary alkylation site by a quinoline monofunctional mustard appears to be mostly in the DNA major groove.