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. 2022 Dec;37(1):299-314.
doi: 10.1080/14756366.2021.2007905.

Topo II inhibition and DNA intercalation by new phthalazine-based derivatives as potent anticancer agents: design, synthesis, anti-proliferative, docking, and in vivo studies

Mohamed M Khalifa  1 , Ahmed A Al-Karmalawy  2 , Eslam B Elkaeed  3 , Mohamed S Nafie  4 , Mohamed A Tantawy  5   6 , Ibrahim H Eissa  1 , Hazem A Mahdy  1
Affiliations

Topo II inhibition and DNA intercalation by new phthalazine-based derivatives as potent anticancer agents: design, synthesis, anti-proliferative, docking, and in vivo studies

Mohamed M Khalifa et al. J Enzyme Inhib Med Chem. 2022 Dec.

Abstract

This research presents the design and synthesis of a novel series of phthalazine derivatives as Topo II inhibitors, DNA intercalators, and cytotoxic agents. In vitro testing of the new compounds against HepG-2, MCF-7, and HCT-116 cell lines confirmed their potent cytotoxic activity with low IC50 values. Topo II inhibition and DNA intercalating activities were evaluated for the most cytotoxic members. IC50 values determination demonstrated Topo II inhibitory activities and DNA intercalating affinities of the tested compounds at a micromolar level. Amongst, compound 9d was the most potent member. It inhibited Topo II enzyme at IC50 value of 7.02 ± 0.54 µM with DNA intercalating IC50 of 26.19 ± 1.14 µM. Compound 9d was then subjected to an in vivo antitumor examination. It inhibited tumour proliferation reducing solid tumour volume and mass. Additionally, it restored liver enzymes, proteins, and CBC parameters near-normal, indicating a remarkable amelioration in their functions along with histopathological examinations.

Keywords: DNA; Topo II; antitumer; intercalators; phthalazine.

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Conflict of interest statement

No potential conflict of interest was reported by the author(s).

Figures

Figure 1.
Figure 1.
Some reported DNA intercalators and topoisomerase II inhibitors showing their common pharmacophoric features.
Figure 2.
Figure 2.
Molecular hybridisation of triazolo phthalazine moieties with different recommended anticancer moieties based on the basic pharmacophoric features of doxorubicin as DNA intercalator and topoisomerase II inhibitors.
Scheme 1.
Scheme 1.
General procedure for synthesis of target compounds 7a,b, 8a,b and 9a-d; Reagents and conditions: (i) NH2NH2.H2O / EtOH/reflux/5 h, (ii) POCl3/heating/1 h, (iii) NH2NH2.H2O / EtOH/reflux/0.5 h, (iv) Butyric anhydride/reflux/1 h, (v) NH2NH2.H2O / EtOH/reflux/0.5 h, (vi) The appropriate Isocyanates/EtOH/reflux/3 h, (vii) The appropriate Isothiocyanate/EtOH/reflux/3h, (viii) The appropriate Aromatic aldehydes / EtOH / gl. acetic acid / reflux/4h.
Scheme 2.
Scheme 2.
General procedure for synthesis of intermediates 11a-c, Reagents and conditions: (i) chloroacetyl chloride/DMF/stirring/1.5h.
Scheme 3.
Scheme 3.
General procedure for synthesis of target compounds 14a-d and 15; Reagents and conditions: (i) 1) CS2/KOH/EtOH/reflux/3 h, 2) HCl, (ii) KOH/absolute EtOH/reflux/0.5 h, (iii) N-Aryl-2-chloroacetamide derivatives 11a-c/DMF/heating/KI/heating/3 h. (iv) Chloroacetamide/DMF/heating over water bath/KI/heating/3 h.
Figure 3.
Figure 3.
Methodology and Experimental design of the in vivo study.
Figure 4.
Figure 4.
Left panel: Bar chart representation of the effect of compound 9d treatment on the proliferation of solid tumour mass in the SEC-bearing mice. Right panel: Morphological representation for the tumour mass volume of A: SEC-group, B: SEC+ 9d, and C: SEC + DOX. Values are expressed as Mean ± SEM values of mice in each group (n = 7). Signs of * and # are values with significant differences in tumour weight and tumour volume, respectively compared to SEC control using an unpaired t-test (P ≤ 0.05) using GraphPad prism.
Figure 5.
Figure 5.
Histopathological examinations of liver tissues of SEC-bearing mice in different treatments (A) Normal control group that shows the normal structure of central vein surrounded with hepatocytes. (B) SEC control group shows pyknosis (arrows) & karyolysis (arrowhead), hydropic degeneration of hepatocytes, and loss of cell boundaries. (C) SEC group treated with 9d (5 mg/Kg BW) that shows hepatic cells are near normal and show tissue improvement as compared with a little hydropic degeneration. (D) SEC group treated with DOX shows tissue enhancement like normal group, but still, some hydropic degeneration, pyknosis (arrows) and karyolysis (arrowhead) were shown. (H&E stain, magnification ×200).
Figure 6.
Figure 6.
Superimposition of the co-crystallised ligand (light green) and the docking pose (light yellow) of the same molecule.
Figure 7.
Figure 7.
Binding of etoposide with DNA-Topo II, the hydrogen bonds are represented in red dashed lines.
Figure 8.
Figure 8.
Binding of doxorubicin with DNA-Topo II, the hydrogen bonds are represented in red dashed lines.
Figure 9.
Figure 9.
3D and 2D illustration of compound 9d in the Topo II active site.
Figure 10.
Figure 10.
3D and 2D illustration of compound 14a in the Topo II active site.
Figure 11.
Figure 11.
The expected ADMET study of the target compounds.
See this image and copyright information in PMC

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