Benzoxazines as new human topoisomerase I inhibitors and potential poisons

Abstract

Background: The numbers of topoisomerase I targeted drugs on the market are very limited although they are used clinically for treatment of solid tumors. Hence, studies about finding new chemical structures which specifically target topoisomerase I are still remarkable.

Objectives: In this present study, we tested previously synthesized 3,4-dihydro-2H-1,4-benzoxazin-3-one derivatives to reveal their human DNA topoisomerase I inhibitory potentials.

Methods: We investigated inhibitory activities of 3,4-dihydro-2H-1,4-benzoxazin-3-one derivatives on human topoisomerase I by relaxation assay to clarify inhibition mechanisms of effective derivatives with EMSA and T4 DNA ligase based intercalation assay. With SAR study, it was tried to find out effective groups in the ring system.

Results: While 10 compounds showed catalytic inhibitory activity, 8 compounds were found to be potential topoisomerase poisons. 4 of them also exhibited both activities. 2-hydroxy-3,4-dihydro-2H-1,4-benzoxazin-3-one (BONC-001) was the most effective catalytic inhibitor (IC₅₀:8.34 mM) and ethyl 6-chloro-4-methyl-3-oxo-3,4-dihydro-2H-1,4-benzoxazin-2-acetate (BONC-013) was the strongest potential poison (IC₅₀:0.0006 mM). BONC-013 was much more poisonous than camptothecin (IC₅₀:0.034 mM). Intercalation assay showed that BONC-013 was not an intercalator and BONC-001 most probably prevented enzyme-substrate binding in an unknown way. Another important result of this study was that OH group instead of ethoxycarbonylmethyl group at R position of benzoxazine ring was important for hTopo I catalytic inhibition while the attachment of a methyl group of R1 position at R₂ position were play a role for increasing of its poisonous effect.

Conclusion: As a result, we presented new DNA topoisomerase I inhibitors which might serve novel constructs for future anticancer agent designs. Graphical abstract.

Keywords: Anticancer; Benzoxazine; Catalytic inhibitor; Topoisomerase I; Topoisomerase I poison.

Graphical abstract

Fig. 1

Biologically active 1,4-benzoxazines

Fig. 2

Effect of increasing concentrations of BONC-001 on the relaxation of plasmid DNA by topoisomerase I. 0.2 μg supercoiled DNA (pBR322) was incubated with or without hTopo I in the absence and presence of the test drug at the indicated concentration (mM). CPT was used at 0.12 mM. DNA samples were separated on 1% agarose gel without ethidium bromide. The gel was photographed under UV light. Rlx relaxed, Sc supercoiled

Fig. 3

Effect of increasing concentrations of BONC-013 on the relaxation of Sc plasmid DNA by topoisomerase I. Supercoiled DNA (pBR322) was incubated with or without hTopo I in the absence and presence of the test drug at six different concentrations (4–12 mM). CPT was used at 0.12 mM. DNA samples were separated by agarose gel electrophoresis without ethidium bromide. Nck nicked, Rlx relaxed, Sc supercoiled

Fig. 4

A graphical demonstration of IC₅₀ values for different hTopo I inhibitions of benzoxazine derivatives. (a) IC₅₀ values for catalytic inhibitors, (b) IC₅₀ values for poisons

Fig. 5

BONC-001 (green) prevents binding of hTopo I (orange) to substrate DNA (blue). Protein bound DNA bands of hTopo I (20 U) incubated with 0,2 μg supercoiled pBR322 DNA (Sc DNA) are shown. Controls were DNA alone (lane 1) and DNA with htopo I (lane 2). To the samples of lane 7 DNA was added together with BONC-001 (10 mM) to show that BONC-001 had no influence on the pBR322 mobility. Different concentrations (6, 8, 10 mM) of BONC-001 were added to the samples in lane 4, 5, 6. Binding of topoisomerase I to DNA in the presence of CPT (1 mM) was used as positive control (lane 3). The reactions were started with the addition of DNA and incubated for 6 min at 37 °C. Samples were separated on 1% TAE-agarose gel electrophoresis in the presence of ethidium bromide for 16 h

Fig. 6

Intercalation ability of BONC-013. T4 DNA ligase unwinding assay was carried out with BONC-013 and ethidium bromide (EtBr). 0.2 μg linear pBR322 plasmid DNA was religated by 400 units of T4 DNA ligase in the presence of increasing concentration of BONC-013 for 1 h at 37 °C. Gel was migrated for about 16 h at room temperature. Ln DNA Linear DNA, Sc DNA Supercoiled DNA