Correlation of in Situ Oxazolidine Formation with Highly Synergistic

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Correlation of in situ oxazolidine formation with highly synergistic cytotoxicity and DNA crosslinking in cancer cells from combinations of doxorubicin and formaldehyde Benjamin L. Barthel, Erin L Mooz, Laura Elizabeth Wiener, Gary G Koch, and Tad H Koch J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.5b01956 • Publication Date (Web): 16 Feb 2016 Downloaded from http://pubs.acs.org on February 21, 2016

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Journal of Medicinal Chemistry

Correlation of in situ oxazolidine formation with highly synergistic cytotoxicity and DNA crosslinking in cancer cells from combinations of doxorubicin and formaldehyde

Benjamin L. Barthel1, Erin L. Mooz1, Laura Elizabeth Wiener2, Gary G. Koch2, Tad H. Koch1,*

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Department of Chemistry and Biochemistry, University of Colorado Boulder, Boulder, Colorado 80309

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Department of Biostatistics, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina,

27599 Abstract The anthracyclines are a class of antitumor compounds that are successful and widely-used but suffer from cardiotoxicity and acquired tumor resistance. Formaldehyde interacts with anthracyclines to enhance antitumor efficacy, bypass resistance mechanisms, improve the therapeutic profile, and change the mechanism of action from a topoisomerase II poison to a DNA crosslinker. Contrary to current dogma, we show that both efficient DNA crosslinking and potent synergy in combination with formaldehyde correlate with the anthracycline’s ability to form cyclic formaldehyde conjugates as oxazolidines moieties and that the cyclic conjugates are better crosslinking agents and cytotoxins than acyclic conjugates. We also provide evidence that suggests that the oxazolidine forms in situ, since cotreatment with doxorubicin and formaldehyde is highly cytotoxic to dox-resistant tumor cell lines, and that this benefit is absent in combinations of formaldehyde and epirubicin, which cannot form stable oxazolidines. These results have potential clinical implications in the active field of anthracycline prodrug design and development.

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Introduction The anthracycline class of antineoplastic agents is represented in current chemotherapy protocols by four compounds: doxorubicin (dox, 1), daunorubicin (dau, 2), idarubicin (ida, 3), and epirubicin (epi, 4, Chart 1). The class shares the structural features of a tetracyclic anthraquinone ring system coupled to an amino sugar (daunosamine) via a glycosidic linkage. Structural differences between the agents are minimal; ida differs from dau by the modification on the 4 position, dox is equivalent to dau in all respects except for a hydroxyl moiety at the 14 position, and epi and dox only differ in the stereochemistry of the hydroxyl group at the 4’ location in the daunosamine. Clinically, the anthracyclines are some of the most successful anticancer drugs ever developed, with dox being the most commonly used. Applications are wide-ranging and the compounds are used across a broad spectrum of both solid (carcinomas and sarcomas) and non-solid tumors.1 The accepted mechanism of action derives from interaction with topoisomerase II (TopoII) through formation of a stable ternary complex of cut DNA-drug-TopoII. The re-ligation of the DNA is inhibited by the presence of the drug, resulting in double-strand DNA breaks.2-4 Other modes of action have been demonstrated, though their contributions to the clinical effectiveness of the drugs are unclear.5-8 Successful treatment with anthracyclines is hindered most significantly by both acute and chronic cardiotoxicity leading to the formation of congestive heart failure in the long term.9-11 Further, anthracyclines are susceptible to the development of resistance by a variety of mechanisms, the most relevant of which results from P170 glycoprotein (P170gp)-mediated export of the drug from the cell.12-14 A growing body of evidence points towards the conclusion that anthracyclines may be activated to greater potencies by either co-administration with formaldehyde-releasing compounds or by inclusion of formaldehyde in the structure. In recent years, we have synthesized and characterized several compounds falling into the latter category, the most relevant of which is doxazolidine (doxaz, 5).15 The oxazolidine moiety results from the reaction of the formaldehyde with the cis configuration of the vicinyl aminol on the daunosamine to form a geminyl aminol (6a), which then either ring-closes directly or via the Schiff base (6b, Scheme 1, top). Doxaz is between 1 and 4 orders of magnitude more potent than dox against a

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broad spectrum of human cancer cell lines, including those that are resistant to dox.15, 16 This gain in potency is achieved without a corresponding increase in toxicity towards cardiomyocytes, resulting in greatly increased therapeutic potential. Reaction with an additional equivalent of formaldehyde results in pseudo-dimerization of doxaz units to form doxoform (doxF, 7),17 which is also highly toxic, likely through rapid hydrolysis to free the two units of doxaz. Corresponding structures have been hypothesized or synthesized with dau.17 In contrast, consistent with literature precedent,18, 19 the trans-amino alcohol found in epi does not result in stable oxazolidines upon reaction with formaldehyde, but instead favors the formation of a bicyclic structure consisting of two epi units and three units of formaldehyde known as epiform (epiF, 10; Scheme 1, bottom). While epiF does display increased cytotoxicity over epi and overcomes resistance, it is far less potent that doxaz or doxF. Each of these anthracycline-formaldehyde conjugates is hydrolytically unstable to varying degrees. Doxaz and doxF are the most unstable, exhibiting half-lives of approximately 3 min.15, 17 EpiF is more stable with respect to hydrolysis to aminols 8 and 9 (Scheme 1). The half-life under physiological conditions is approximately 2 h and loss of the formaldehyde from 8 and 9 to form epi was not observed at pH 7.4.20 The presence of formaldehyde in anthracycline treatments results in a significant modification of the mechanism of action. As stated above, the primary mechanism of action for anthracyclines is inhibition of the re-ligation of DNA via TopoII. Deficiency in this enzyme is sufficient to reduce sensitivity to dox by approximately 10-fold, but results in very little change to doxaz sensitivity.16 Mass spectrometry and other experiments from our lab and others indicate that the presence of formaldehyde enables direct DNA alkylation at sequences of 5’-GC-3’ by dox,15, 17, 21, 22 dau,21, 23 and epi20, 24, 25 in sensitive and resistant cell lines, thus allowing function independent of TopoII. Coldwell, et al, have demonstrated that doxformaldehyde conjugates can form with clinically relevant concentrations and endogenous formaldehyde; although, the contribution of these crosslinks to the clinical effects of dox is currently unclear.26 In the current model for crosslink formation with anthracycline-formaldehyde conjugates, adducts are formed by attack of the formaldehyde carbon by the 2-amino group of a tautomer of a guanine base in the minor

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groove of DNA (Scheme 2).15 The “virtual crosslink” is completed by hydrogen bonding between the drug’s 9-OH and the guanine on the opposite strand.27 Here, we present evidence that the oxazolidine moiety is the critical structural feature that allows highly-efficient DNA crosslinking and that combinations of anthracyclines and formaldehyde are highly synergistic only when stable oxazolidine formation is possible. Our data suggest that in opposition to what has been dogmatic in anthracycline-formaldehyde interactions, the oxazolidine is capable of forming in situ and that the significant potentiation of anthracycline activity by formaldehyde is primarily a result of that formation. Our work suggests that significant clinical benefit may be gained by combining oxazolidine-capable anthracyclines with treatments that elevate tumor formaldehyde levels, particularly with drug-resistant tumors. Results and Discussion Doxazolidine (doxaz) induces sequence-dependent, functionally-relevant DNA crosslinks in living, cellular systems. Previous work from our lab has demonstrated that incubation of doxaz, via the dimer doxoform, with short sequences of GC-rich DNA results in alkylation of the DNA by the drug.17 Further, results from our lab and others have shown that under conditions in which oxidation-reduction processes allow for the production of formaldehyde, DNA-drug adducts form in a sequence-specific manner.27-29 Much of this work, along with the crystal structure of daunorubicin covalently bound to DNA,23 were produced in cell-free systems using isolated DNA and tightly controlled conditions. We now demonstrate widespread crosslinking activity from doxaz in a complex and dynamic cellular system utilizing the alkaline Single-Cell Gel Electrophoresis (SCGE) assay, commonly referred to as the comet assay (see Supporting Information for a cartoon). In this assay, cells are treated with the prospective genotoxic agent, then embedded in agarose, lysed and electrophoresed under alkaline conditions. The free ends and small fragments of DNA that result from strand breaks will migrate through the gel, while large, intact DNA does not. Thus, after staining, the structures resemble comets in which the density of the stain in the tail is indicative of the extent of DNA strand breaks.

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Initially, we treated cells as adherent cultures, followed by washing, trypsinizing, counting, and diluting prior to lysis in the agarose gels. However, in this form, we found that the assay was poorly reproducible; untreated cells varied significantly in the extent of DNA damage (data not shown). Subsequent tests indicated that the handling of the cells during trypsinization was of critical importance. Since we planned to extensively utilize the comet assay to test many different conditions of drug combinations, the protocol was modified to reduce experimental variability resulting from trypsinization of individual treatment groups. Instead, a large population of cells was trypsinized as a whole, then divided into treatments groups, which were treated in suspension. This had the added benefit of allowing the cells to be embedded into the gel and lysed more quickly after removal of the drugs, thereby preventing cellular repair machinery from masking any genotoxic effects. Using our modified comet assay protocol, we tested the genotoxic effects of dox, epi, and doxaz over 90 min of treatment, in both dox-sensitive MiaPaca-2 human pancreatic carcinoma cells and doxresistant NCI/ADR-RES (ADR cells) human ovarian carcinoma cells (Figure 1 and Table 1). As expected, 100 µM dox and 100 µM epi both induced significant increases in DNA damage content (p

Correlation of in Situ Oxazolidine Formation with Highly Synergistic

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