The Reactions of Mitomycin C with Dithiols II. Formation of Dithiol Cross-Links

We report that the clinically used antitumor drug mitomycin C reacts with excess dithiols to give dithiol cross-links as major products. Mechanistic studies reveal that three dithiol molecules participate at different stages of the reaction: in the reductive activation of mitomyicin C, in an alkylation at C1, and in an additional reduction that activates C10 for the second-arm alkylation by the dithiol. We hypothesize that the reactions reported here indicate that mitomycin C could act as a mechanism-based inhibitor of enzymes containing a dithiol active site. Introduction Mitomycin C (MC, 1) is an antitumor antibiotic used clinically in cancer chemotherapyand in ophthalmologic procedures. MC is a “smart” prodrug, inert towards nucleophiles in its original structure, but transformed into a highly reactive bis-electrophile after reduction. The mechanism of activation of MC is initiated by the reduction of the quinone ring of MC to form hydroquinone 2, that spontaneously eliminates methanol to form leucoaziridinomitosene 3 (Scheme 1), a bis-electrophile that alkylates cellular nucleophiles such as phosphate, bicarbonate, glutathione and DNA. 7 Sequential alkylation of DNA at the 1 and 10 positions of 3 generates a cytotoxic interstrand crosslink that specifically links N2 of opposite deoxyguanosine residues at CpG sites of double stranded DNA. Recent reports indicate that glucose regulatory protein (GRP58) plays a significant role in the cellular activation of MC, and that the activity of this protein resides in its two thioredoxin-like domains. The involvement of proteins containing a dithiol active site in the biological activity of MC is not restricted to the above mentioned activation by GRP58. We recently reported a successful activation of MC using simple dithiols as chemical models for the dithiol functional group present in the active site of proteins of the thioredoxin family. 10 Continuing with our investigation of simple thiols as a chemical model for dithiol-containing proteins in their reactions with MC we report here that dithiols, in addition to perform the reductive activation of MC, are alkylated by activated MC to form S,S’-crosslinks as the almost exclusive end products. Results and discussion The initial reactions were performed using DTT, and the product profile observed in the HPLC trace (Figure 1) consisted of four mitosenes with identical UV and MS, that we identified as the four possible diastereomeric mitosenes resulting from alkylation of DTT by a single MC molecule at its 1and 10positions. The HPLC analysis of reactions between the oxidoreductase cofactor DHLA and MC (Figure 1) revealed the formation of at least seven of the eight expected isomers, resulting from bis-alkylation of MC with one molecule of racemic DHLA. Figure 1. HPLC traces for the reaction of MC with dithiols: (a) 1,3-propanedithiol; (b) DTT; (c) DHLA. Reactions contained a molar ratio dithiol/MC = 5, and were performed at 20 oC for 8 hours, quenched by acidification and analyzed by LC/MS (λ = 310 nm). LC-HRMS gave exact masses that confirmed the molecular formula of the proposed DTT crosslinks 7 and DHLA crosslinks 8 (Chart 1). For a full characterization of the crosslinks of MC with dithiols, we used 1,3-propanedithiol as a means to simplify the composition of the reaction mixture. As expected, the reaction of MC with 1,3-propanedithiol (Figure 1) gave two isomers with UV and MS supporting structure 6. We should note here that the simple chromatograms obtained after the reaction of MC with dithiols (Figure 1) do not imply that all MC is converted to MC-dithiol crosslinks. Side reactions do occur but, as we will discuss later, they result in the formation of unidentified insoluble mitosenes that do not appear on the HPLC trace. The two isomers of 6 could be separated and fully characterized using NMR spectroscopy. The main evidence for the stereochemistry was obtained using circular dichroism, a well established technique to assign the stereochemical configuration at C-1 of 2-aminomitosenes. In our case, the isomers from the reaction of MC and 1,3-propanedithiol gave CD spectra that were almost symmetrical (Figure 3), and confirmed the expected stereochemistry at C-1, with the major isomer bearing a cis-configuration.