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Benzoxazines as new human topoisomerase I inhibitors and potential poisons

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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.

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Abbreviations

EMSA:

Electrophoretic mobility shift assay

SAR:

Structure–activity relationship

BONC-001:

2-hydroxy-3,4-dihydro-2H-1,4-benzoxazin-3-one

BONC-013:

Ethyl 6-chloro-4-methyl-3-oxo-3,4-dihydro-2H-1,4-benzoxazin-2-acetate

IC50 :

50% Inhibitory concentrations

DIBOA:

1,4-benzoxazin-3-ones including 2,4-Dihydroxy-1,4-benzoxazin-3-one

DIMBOA:

6,7-dimethoxy-2-benzoxazolinone

MIC:

Minimum growth inhibition concentration

CPT:

Camptothecin

hTopo I:

Human DNA topoisomerase I

Sc:

Supercoiled bands

Nck:

Nicked bands

BONC-002:

Ethyl (3-oxo-3,4-dihydro-2H-1,4-benzoxazine-2-yl)acetate

Cappamensin A:

2H-1,4-benzoxazin-3(4H)-one, 6-methoxy-2-methyl-4-carbaldehyde

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Acknowledgments

We thank the Research Fund of Ankara University (Grant No: 12B3336002 and Grant No: 11A3336001) and The Scientific and Technological Research Council of Turkey (TÜBİTAK) (Grant No: TBAG-105 T081) for the financial support of this research. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Author information

Author notes

  1. Egemen Foto

    Present address: Department of Biotechnology, Faculty of Science, Necmettin Erbakan University, Konya, Turkey

Authors and Affiliations

  1. Izmir International Biomedicine and Genome Institute, iBG-izmir, Dokuz Eylül University, Izmir, Turkey

    Çigdem Özen

  2. Department of Molecular Biology, Faculty of Science, Hacettepe University, Ankara, Turkey

    Fatma Zilifdar & Nuran Diril

  3. Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Ankara University, Ankara, Turkey

    Betül Tekiner-Gülbaş, İlkay Yıldız, Esin Akı-Yalçın & İsmail Yalçın

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Contributions

EF, ÇÖ and FZ: Performing mechanistic studies and manuscript preparations. BTG: synthesis of the title compounds. İYi: SAR analysis and manuscript preparations. EAY: collaboration in the synthesis of the target compounds. ND: collaboration in manuscript preparations. İYa: supervision of the pharmacological part. All authors read and approved the final manuscript.

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Correspondence to Egemen Foto.

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Key-Points

• Benzoxazine derivatives inhibit human DNA topoisomerase I action.

• BONC-001 inhibited catalytic activity of hTopo I by interacting with DNA binding site of the enzyme.

• OH group at the 2nd position of benzoxazine ring was very important for catalytic inhibition.

• BONC-013, 77 times more effective than CPT, inhibited hTopo I activity by stabilizing covalent enzyme-DNA complex.

• The attachment of methyl group of the R1 position seemed to play a role for poison effect.

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Foto, E., Özen, Ç., Zilifdar, F. et al. Benzoxazines as new human topoisomerase I inhibitors and potential poisons. DARU J Pharm Sci 28, 65–73 (2020). https://doi.org/10.1007/s40199-019-00315-x

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  • DOI: https://doi.org/10.1007/s40199-019-00315-x

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