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  • To examine the influence of aromatic motifs on the bridging


    To examine the influence of aromatic motifs on the bridging lysine esters we analysed DNA alkylating activity of the set of compounds 1–6. The lysine derivatives were chosen as it is relatively easy to prepare different esters and thereby study the impact of different groups on the reactivity towards DNA. Thus, we first planned the synthesis of bis-3-chloropiperidines 2, 3 and 4 by introducing a simple phenyl group tethered through flexible hydrocarbon spacers to the lysine linker. The lysine methyl ester 1 was selected for comparison purposes in the biochemical assays. Design of compounds 5 and 6 was based on the knowledge that naphthalene chromophores, present in the antitumour Nitecapone neocarzinostatin and azinomycin A and B, contribute significantly to reinforce the affinity for DNA.15, 16, 17 Additionally, the methoxy group in the 4-position of the naphthalene moiety 6 can participate in hydrogen bonding interactions.
    Results and discussion
    Conclusions A series of new lysine-bridged bis-3-chloropiperidines were prepared as model compounds in order to explore the influence of aromatic moieties on DNA alkylating activity. Accordingly, a phenyl group was attached by esterification to the lysine side chain via hydrocarbon spacers of different chain length (compounds 2–4). In addition, two derivatives (5 and 6) containing a naphthoate unit were synthesised to analyse whether the incorporation of this known DNA-binding chromophore might increase reactivity toward DNA. Thus, we evaluated DNA alkylation activity by a DNA cleavage assay with a supercoiled plasmid and sequencing gel analysis with a 22-mer duplex oligonucleotide. Results with nucleic acids are consistent with ESI-MS analysis, confirming the fast formation of the reactive species in solution. Our studies revealed a clear correlation between linker structure and extent of DNA alkylation. As a proof-of-principle, the results demonstrated that the introduction of aromatic groups to the linker side chain curtail the reactivity of bis-3-chloropiperidines in comparison to their related counterparts containing symmetrical non-aromatic linkers. Furthermore, it was shown that an increase in the length of the alkyl spacer chain attached to the linker also lowers the activity (compounds 2–4). Within the current series, the most active compound was the derivative 1 suggesting that bis-3-chloropiperidines without an aromatic group seem to be significantly more potent alkylating agents. These findings are consistent with recently published data and can be particularly advantageous in terms of modulating the reactivity of bis-3-chloropiperidines towards DNA. It is noteworthy that although the naphthoate analogues 5 and 6 proved to be less potent DNA alkylating agents our experiments indicate that these compounds interact with DNA. This suggests that the presence of a DNA-affinity moiety in the bis-3-chloropiperidine molecule can contribute to DNA recognition and might direct the location of the alkylating unit near to suitable DNA bases. Moreover, the present results also confirm those from our previous study showing that the examined compounds induce cleavage of double-stranded DNA, primarily through reactions toward guanine residues through a DNA nicking mechanism. Currently, additional biochemical investigations and measurements of cytotoxicity are in progress to provide further insight into the mechanism of action. Utilizing the knowledge obtained from these studies might represent valuable starting points for further development and optimisation of novel alkylating derivatives based on the bis-3-chloropiperidine scaffold.
    Experimental section
    Acknowledgments This work was supported financially by MIUR, Progetti di Ricerca di Interesse Nazionale (Grant 2010W2KM5L_006) to B.G. The authors would like to acknowledge the assistance of Dr. Erwin Röcker with mass spectrometric measurements. I.Z. thanks the German National Academic Foundation (Studienstiftung des deutschen Volkes) for providing a PhD scholarship, as well as the International Giessen Graduate Centre for the Life Sciences (GGL) and the German Academic Exchange Service (DAAD) for a travel grant.