Nitrogen Mustards

Gillman and Philips' initial disclosure of 1 was accompanied by a mechanistic rationale that the sulfur mustard exerted its antiproliferative effects by the irreversible alkylation of 'phosphokinases.' The electrophilicity of 1 and the related nitrogen-based mustards react, via the intermediacy of either a thiarinium or an aziridinium cation, with the following exogenous nucleophiles: sulfides, alcohols, amines, phosphates, imidazoles, and carboxylates. In doing so, these agents possess the ability to alkylate proteins; however, their antiproliferative effects appear to be a function of their ability to covalently modify DNA and subsequent formation of DNA interstrand cross-links.4 The order of the relative nucleophilicity of positions on DNA for electrophiles has been determined to be N-7 of guanine >N-3 of adenine >N-7 of adenine >N-3 of guanine >N-1 of adenine >N-1 of cytosine5; see Figure 2 for numbering of the purines (A and G) and pyrimidines (Tand C).

Mustards covalently modify DNA by the mechanism shown in Figure 3. Ionization of one of the chlorides of the beta-chloroethylamine-substituent of 3, via an SN1 pathway, yields the highly reactive aziridinium cation 4 (Figure 3). Nucleophilic capture of 4 by one of the aforementioned sites on DNA yields mono-adduct 5. At this point, reformation of an aziridinium cation generates intermediate 6, which has the potential to be attacked by exogenous nucleophiles (X), for example, water, proteins, or other nucleic acids yielding 7. In addition, 6 can undergo a second alkylation by the same piece of DNA generating either the intrastrand cross-link 8 or the interstrand cross-links 9.6

The ability of a mustard agent, for example 2, to alkylate DNA begins a series of events that have a profound cumulative effect on the cellular machinery, ultimately leading to cell death. Treating cells with 2 results in formation of several alkylated moieties. Of particular interest are the interstrand cross-links formed between the N-7 of two different guanines. The resulting imidazolium species increases the enolic character of the residue, emulating the hydrogen bond accepting/donating array of adenine. During DNA replication, the alkylated base pairs with thymine resulting in a net GC to AT substitution.7'73 The increased imidazolium character of the modified guanine allows for possible hydrolysis of the heterocycle, or depurination of the guanine residue, both of which lead to DNA lesions,8 and ultimately strand scission.

The reactivity of the beta-chloroethylamine group is highly dependent on the nucleophilicity of the mustard nitrogen. For example, mechlorethamine reacts within minutes in the body upon intravenous dosing, thereby limiting its clinical utility. In fact, the chemical instability of 2 translates to a rather nonselective alkylating agent in vivo, with a variety of toxicities elicited in rapidly dividing tissues and systems. In an effort to find analogs that have increased chemical stability, as well as reduced toxicity, a variety of substituted arenes have been conjugated to the nitrogen to moderate the chemical reactivity of the resulting mustard. The most successful analogs have employed conjugation of the mustard group to an appropriately substituted arene, yielding chlorambucil (Leukeran, 10; Figure 4) and melphalan (Alkeran, 11). The conjugation of the arene onto the mustard nitrogen significantly retards the rate of aziridinium cation formation allowing for increased chemical stability, yielding orally active drugs. Unfortunately, the increased chemical stability of 10 and 11 does not translate into greater selectivity for malignant cells versus nontransformed cells. As a result, the chemical modifications of 2 to afford 10 and 11 have greatly impacted the physical properties of this class of compounds, while yielding only marginally decreased toxicity to normal tissue.

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