Biological activity of quinazoline analogues and molecular modeling of their interactions with G-quadruplexes

https://doi.org/10.1016/j.bbagen.2020.129773Get rights and content

Highlights

  • 2-vinyl-quinazolin-4-ones (V2Qs) are moderate MDA breast cancer cell suppressors.

  • Probable mechanism of V2Qs' action is stacking to telomeric G-quadruplexes.

  • Computed DFT and RI-MP2 stacking energies reproduce observed trends in genotoxicity.

  • Heterocycle-quadruplex stabilization energy may be used for prediction of activity.

Abstract

Background

Quinazolines 1 to 6, with an aromatic or aryl-vinyl substituent in position 2 are selected with the aim to compare their structures and biological activity. The selection includes a natural alkaloid, schizocommunin, and the synthetic 2-(2′-quinolyl)-3H-quinazolin-4-one, known to interact with guanine-quadruplex dependent enzymes, respectively telomerase and topoisomerase.

Methods

Breast cancer cells of the MDA cell line have been used to study the bioactivity of the tested compounds by the method of Comet Assay and FACS analyses. We model observed effects assuming stacking interactions of studied heterocycles with a naked skeleton of G-quadruplex, consisting of guanine quartet layers and potassium ions. Interaction energies are computed using a dispersion corrected density functional theory method, and an electron-correlated molecular orbital theory method.

Results

Selected compounds do not remarkably delay nor change the dynamics of cellular progression through the cell cycle phases, while changing significantly cell morphology. Our computational models quantify structural effects on heterocyclic G4-complex stabilization energies, which directly correlate with observed biological activity.

Conclusion

Our computational model of G-quadruplexes is an acceptable tool for the study of interaction energies of G-quadruplexes and heterocyclic ligands, predicting, and allowing design of novel structures.

General significance

Genotoxicity of quinazolin-4-one analogues on human breast cancer cells is not related to molecular metabolism but rather to their interference with G-quadruplex regulatory mechanisms. Computed stabilization energies of heterocyclic ligand complexes of G-quadruplexes might be useful in the prediction of novel telomerase / helicase, topoisomerase and NA polymerase dependent drugs.

Introduction

Numerous plant and marine medicines with quinazoline components have been known to Asian folk practitioners for time immemorial. Presently, more than 220 natural alkaloids possess a quinazoline or quinazolinone fragment, and the interest in this class of compounds is steadily growing due to their broad and diverse biological activity and medicinal applicability [[1], [2], [3]]. Quinazoline heterocycles are structurally closely similar to a number of biologically active compounds, extending to the basic carriers of biological hereditary information, namely purines and pyrimidines as nucleic acid (NA) bases [1,2]. Quinazolin-4-ones are among the conveniently accessible heterocycles, frequently using the long known Niementowski synthesis [[1], [2], [3], [4]]. The latter heterocyclic molecules provide sufficient variability in the search for biologically potent compounds of prospective medicinal interest [[1], [2], [3], [4], [5]]. We have used aldol type conversions of 2-alkylsubstituted quinazolin-4-ones [6] looking for structural and topological similarities with nucleic acid base pairs [[5], [6], [7], [8], [9]], so that enhanced activity of potential novel derivatives might be expected beforehand.

There are a couple of important details of structural requirements to potentially biologically active molecules related to NA base pairs. First, it is the capability to form intra- and inter- molecular hydrogen bonds like the in-plane interactions of NA base pairs. The second requirement is the capability to participate in dispersion interactions of the type of stacking interactions between NA base pairs within the NA helix. With heterocyclic 2-substituted quinazolin-4-one analogues, both requirements are easy to satisfy, as is obvious with planar heterocyclic compounds 1–6 shown in the Scheme 1 below, possessing at least one proton donor – acceptor pair of atoms including nitrogen.

The naturally occurring schizocommunin 3 has been extensively studied aiming first at the clarification of its structure as a quinazoline derivative [7]. More recently, 3 and its derivatives have been found to react specifically with DNA in telomeres and thus to affect a deepest mechanism of cell proliferation and apoptosis by stabilization of guanine quadruplexes, G4, and thus inhibiting telomerase [8]. Pyridine and quinoline substituted quinazolin-4-ones 5 and 6 are known [5], and have also been reported as physiologically active, interacting with another guanine-quadruplex related enzyme, topoisomerase [[9], [10], [11]]. In this study, we design and synthesize the novel molecules 1 and 2, as well as 4, which is an aza-derivative of 3. Then we use the opportunity to investigate the biological activities of these four as well as of the two relatively well-known compounds, 5, and 6, in relation to their primary structural characteristics. Our results indicate that all listed compounds induce DNA damage of different extent depending on their structure on human breast cancer cells. In addition, the studied quinazoline analogues bring changes in the overall cellular morphology of the tested cells. We further use computational modeling of shown quinazolin-4-one derivatives 1–6, and attempt to find connections of theoretical quantities to the biological activity of the compounds. As far as mechanisms of the observed biological effects of 3, 5, and 6 have already been discussed, [[7], [8], [9], [10], [11], [12]] this might open possibilities to develop potential drugs based on heterocyclic compounds designed preliminarily. On the other hand, correspondence of model expectations for chosen heterocycles and experimentally registered effects may in turn contribute to understanding of biochemical mechanisms, and possibly direct further design of promising heterocyclic structures.

Our chosen selection logic for molecules, capable of hydrogen bonding and NA stacking, limits the range of eligible structures to aromatic or quasi-aromatic heterocycles while evidently including the growing set of natural aromatic alkaloids, known for many useful biological effects. [12]

Section snippets

2-Substituted analogues of quinazolin-4-one induce genotoxic stress on MDA cells

Human breast cancer cells of the MDA cell line have been used as a model to study the bioactivity of the tested compounds. The cells have been treated with compounds 1 to 6, Scheme 1, for 4 h at 37 °C. To test the genotoxic potential of the six tested substances we have performed the method of Comet assay, also called single-cell gel electrophoresis (SCGE). SCGE sensitively and precisely detects all kinds of damages in DNA including single-strand DNA breaks, double-strand DNA breaks and

Mechanism of action

So far, we have considered effects of structural variations of quinazoline analogues on their biological activity. To understand the reasons for the observed differences, we need to consider the interactions of studied molecules with their presumed biochemical counterparts. This is another more complicated level of molecular organization, which must include both studied heterocyclic molecules, and biological components. For this purpose, we attempt molecular dynamics simulation, MD, for the

Conclusions and outlook

Anticancer activity exhibited by natural alkaloids and synthetic heterocycles, including quinazoline analogues, may in a number of cases be due to their contribution to the stabilization of four-stranded G-quadruplexes, observed in guanine-rich NA sequences. This work uses a model of G-quadruplex stabilization by heterocyclic molecules stacking to its bottom. On the basis of this model, and without preliminary expectations of possible activity and/or biochemical mechanism, we find correlations

Chemistry of 2-substituted quinazolin-4-one analogues

An efficient two step aldol type synthesis of 2-substituted-quinazolinones involves double lithiation of 2-methyl-4(3H)-quinazolin-4-one and subsequent in situ trapping with variety of electrophiles.3 The same approach is applied for the preparation of 2-((3-oxoisoindolin-1-ylidene)methyl)quinazolin-4(3H)-one 1 (Scheme 3). Thus, lithiation of 2-methyl-quinazolin-4-one with 3 equivalents of LDA in THF at −78 °C followed by addition of phthalimide furnished the key intermediate 1a in 66% yield.

Author statement

Jose Kaneti: The G-quadruplex model; Computational modeling; Writing and editing of the computational part.

Milena Georgieva: Conceptualization, Methodology and Data analysis and Curation for genotoxicity results of the studied materials, Writing and Editing of the biological part.

Miroslav Rangelov: MD simulations.

Irena Philipova: Synthesis and characterization of the studied compounds.

Ivan Angelov: Fluorescence experiments.

Bela Vasileva: Methodology and Experimental design of genotoxicity

Declaration of Competing Interest

None declared.

Acknowledgements

This work has been supported by the Bulgarian Science Fund, via Grant Number DN 19/11 of Dec. 10, 2017.The funding organization, www.fni.bg, has not been otherwise involved in the study design, data collection and report writing. Most reported computations have been carried out on the AVITOHOL HPC cluster facility, https://www.top500.org/system/178609; last accessed Sep. 01, 2020.

References (60)

  • A. Hameed et al.

    Quinazoline and quinazolinone as important medicinal scaffolds: a comparative patent review (2011–2016)

    Expert Opin. Ther. Pat.

    (2018)
  • S. von Niementowski

    Synthesen von Chinazolinverbindungen

    J. Prakt. Chem.

    (1895)
  • E.S. Lee et al.

    Synthesis and Biological Properties of Selected 2-aryl-4(3H)-quinazolinones

    Heterocycl. Commun.

    (2004)
  • I. Philipova et al.

    Convenient synthesis of some 2-substituted 4(3H)-quinazolinone derivatives

    J. Heterocyclic Chem.

    (2006)
  • K. Uehata et al.

    Total synthesis of schizocommunin and revision of its structure

    J. Nat. Prod.

    (2013)
  • T. Che et al.

    Discovery of novel schizocommunin derivatives as telomeric G-quadruplex ligands that trigger telomere dysfunction and the deoxyribonucleic acid (DNA) damage response

    J. Med. Chem.

    (2018)
  • R.O. Dempsey et al.

    Rational design of quinazoline-based reversible inhibitors of human erythrocyte purine nucleoside phosphorylase

    Biochemistry

    (1991)
  • J. Davoll et al.

    Quinazoline analogues of folic acid

    J. Chem. Soc. C

    (1970)
  • K.J. Scanlon et al.

    Quinazoline analogues of folic acid as inhibitors of thymidylate synthetase from bacterial and mammalian sources

    Mol. Pharmacol.

    (1979)
  • T. Che et al.

    Natural Alcaloids and heterocycles as G-quadruplex ligands and potential anticancer agents

    Molecules

    (2018)
  • M. Bajpayee et al.

    The comet assay: assessment of in vitro and in vivo DNA damage

    Methods Mol. Biol.

    (2013)
  • A.R. Collins

    The comet assay: a heavenly method!

    Mutagenesis

    (2015)
  • M. Georgieva et al.

    Random, double- and single-strand DNA breaks can be differentiated in the method of comet assay by the shape of the comet image

    Electrophoresis

    (2015)
  • A. Hartmann et al.

    Recommendations for conducting the in vivo alkaline comet assay, 4th international comet assay workshop

    Mutagenesis

    (2003)
  • R.R. Tice et al.

    Single cell gel/comet assay: guidelines for in vitro and in vivo genetic toxicology testing

    Environ. Mol. Mutagen.

    (2000)
  • E. Peycheva et al.

    Comparison between alkaline and neutral variants of yeast comet assay

    Biotechnol. Biotechnol. Equip.

    (2009)
  • P.L. Olive et al.

    The comet assay: a method to measure DNA damage in individual cells

    Nat. Protoc.

    (2006)
  • D.F. Alvarez et al.

    Publishing flow cytometry data

    Am. J. Physiol. Lung Cell. Mol. Physiol.

    (2010)
  • L. Gay et al.

    Tumour cell heterogeneity

    F1000Research

    (2016)
  • S.B. Mhaske et al.

    Regioselective quinazolinone-directed ortho lithiation of quinazolinoylquinoline: practical synthesis of naturally occurring human DNA topoisomerase I poison luotonin A and luotonins B and E

    J. Organomet. Chem.

    (2004)
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