Structure-based design, synthesis and biological testing of etoposide analog epipodophyllotoxin–N-mustard hybrid compounds designed to covalently bind to topoisomerase II and DNA
Graphical abstract
Introduction
Hybrid drug molecules that contain two distinct pharmacophores and that can simultaneously act at two pharmacological targets have the potential to exceed the potency, spectrum of activity, and efficacy of either of the constituent moieties from which they were derived. Several recent reviews have described the various approaches taken in designing hybrid drugs.1, 2, 3, 4, 5 In the anticancer drug development area these have included such molecules as an epipodophyllotoxin–lexitropsin hybrids; enediyne–lexitropsin hybrids;5 an epipodophyllotoxin–amsacrine hybrid;6 various N-mustard- and cisplatin–steroid hybrids;4 epipodophyllotoxin–camptothecin hybrids;7 and chlorambucil–distamycin A hybrids.3
The X-ray structure of two molecules of etoposide complexed in a ternary complex with topoisomerase IIβ (PDB ID: 3QX3) has recently been determined.8, 9 In this structure the glycosidic moiety of etoposide occupies a spacious binding pocket in the protein. The bound epipodophyllotoxin moiety of etoposide, unlike planar DNA intercalators that stack with two bases, stacks only with a single +5 guanine base in a DNA deformed structure.9 Etoposide is an effective anticancer agent10 and interfacial topoisomerase II inhibitor11, 12 which stabilizes a covalent topoisomerase II–DNA cleavable complex intermediate10, 11, 12, 13, 14 resulting in single- and double-strand DNA breaks that are cytotoxic. Compounds in which the glycosidic moiety of etoposide is modified (e.g., teniposide) or even replaced retain excellent activity.15, 16, 17, 18 In previous studies we described the synthesis and biological and topoisomerase IIα inhibitory activities of photoaffinity etoposide probes with a substitution on the glycosidic moiety18 or with the glycoside moiety replaced with a substituted phenyl group.17 Here, guided by molecular modeling and docking into the etoposide binding site in the X-ray structure of topoisomerase IIβ, we designed and synthesized a series of epipodophyllotoxin–N-mustard hybrid compounds. These hybrids were designed to contain a bifunctional N-mustard moiety that could alkylate protein residues on topoisomerase II or alkylate DNA bases when DNA and topoisomerase II are in a ternary complex with inhibitor. These N-mustard hybrids could also alkylate uncomplexed DNA.19 It was hypothesized that binding of an epipodophyllotoxin–N-mustard hybrid in the cleaved DNA–topoisomerase II complex would result in an N-mustard-derived covalent adduct being produced, and that this covalent adduct would irreversibly inactivate topoisomerase II. Hence, the hybrid molecules would have an advantage over etoposide which is bound in a thermodynamic equilibrium within an established complex between topoisomerase II and DNA and is free to dissociate. In addition, since these hybrid compounds may exert their antitumor effects by two independent mechanisms (targeting topoisomerase II and alkylating DNA), cancer cells should be much less able to develop resistance. Due to the known clinical utility of the N-mustards19 and of etoposide,10 a series of epipodophyllotoxin–N-mustard hybrids based on chlorambucil, melphalan and bendamustine moieties were designed, synthesized and tested for their biological activity in vitro and in cell-based assays.
Several of the newly synthesized hybrids incorporated a piperazine linker to extend the chain and to improve pharmacological properties. The most potent of the series had a National Cancer Institute (http://dtp.nci.nih.gov) mean NCI-60 cell line screen GI50 that was 17-fold more potent than etoposide and was 41-fold more potent than the most potent N-mustard melphalan. Using a variety of assays, all of the hybrids showed strong evidence for targeting topoisomerase II. In addition, the hybrids showed evidence consistent with the ability to function as alkylating agents as expected.
Section snippets
Chemistry
Two different strategies were used for the synthesis of the hybrid molecules containing both the epipodophyllotoxin and the N-mustard anticancer drug motifs. In the first approach the N-mustard compounds were directly linked to the epipodophyllotoxin as depicted in Scheme 1, Scheme 2. In the second approach the N-mustard compounds were linked to the epipodophyllotoxin analog via a piperazine linker as shown in Scheme 3, Scheme 4. In the first step (Scheme 1), the 4β-OH group in 1 (4′-O
Cell growth inhibitory effects of the hybrids and piperazine intermediates on human leukemia K562 cells and K/VP.5 cells with decreased levels of topoisomerase IIα and topoisomerase IIβ
The cell growth inhibitory effects of the synthesized hybrids and the piperazine advanced intermediates on human leukemia K562 cells and K/VP.5 cells25, 26 were compared to etoposide and the N-mustards chlorambucil, melphalan and bendamustine (Table 1). Most of the hybrids displayed sub-micromolar IC50 values comparable to that of etoposide. All of the hybrids were much more growth inhibitory than the three N-mustards tested. Cancer cells can acquire resistance to topoisomerase II poisons by
Conclusions
Etoposide and the N-mustards from which our hybrids are derived are thought to exert their anticancer activity by distinctively different mechanisms. Etoposide acts as a topoisomerase IIα interfacial inhibitor/poison10, 11, 12, 13 that results in stabilization of cleaved DNA and frank double strand breaks that lead to cell death. The N-mustards are thought to act through the formation of cross-links with DNA that results in DNA damage that is difficult to repair and also leads to cell death.
Chemistry
1H and 13C NMR spectra were recorded on a Bruker Avance NMR spectrometer operating at 300 MHz or 500 MHz, as indicated, for 1H NMR and 75 MHz (or 125 MHz as indicated) for 13C NMR, respectively, in CDCl3 or DMSO-d6. The chemical shifts are expressed as δ units with Si(CH3)4 as the internal standard. Melting points were uncorrected. The high resolution mass spectra were run on a Bruker microTOF Focus mass spectrometer (Fremont, CA) using electron spray ionization. TLC was performed on plastic-backed
Acknowledgments
Supported by Grants from the Canadian Institutes of Health Research, the Canada Research Chairs Program, and a Canada Research Chair in Drug Development to Brian Hasinoff and a United States NIH Grant CA090787 to Jack Yalowich. The authors declare no competing financial interest. The funding source(s) had no involvement in the study design; in the collection, analysis and interpretation of data; in the writing of the report; and in the decision to submit the article for publication.
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