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. 2022 Apr 30;27(9):2883.
doi: 10.3390/molecules27092883.

Acridine Based N-Acylhydrazone Derivatives as Potential Anticancer Agents: Synthesis, Characterization and ctDNA/HSA Spectroscopic Binding Properties

Mária Vilková  1 , Monika Hudáčová  2 , Nikola Palušeková  2 , Rastislav Jendželovský  3 , Miroslav Almáši  4 , Tibor Béres  5 , Peter Fedoročko  3 , Mária Kožurková  2
Affiliations

Acridine Based N-Acylhydrazone Derivatives as Potential Anticancer Agents: Synthesis, Characterization and ctDNA/HSA Spectroscopic Binding Properties

Mária Vilková et al. Molecules. .

Abstract

A series of novel acridine N-acylhydrazone derivatives have been synthesized as potential topoisomerase I/II inhibitors, and their binding (calf thymus DNA—ctDNA and human serum albumin—HSA) and biological activities as potential anticancer agents on proliferation of A549 and CCD-18Co have been evaluated. The acridine-DNA complex 3b (-F) displayed the highest Kb value (Kb = 3.18 × 103 M−1). The HSA-derivatives interactions were studied by fluorescence quenching spectra. This method was used for the calculation of characteristic binding parameters. In the presence of warfarin, the binding constant values were found to decrease (KSV = 2.26 M−1, Kb = 2.54 M−1), suggesting that derivative 3a could bind to HSA at Sudlow site I. The effect of tested derivatives on metabolic activity of A549 cells evaluated by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide or MTT assay decreased as follows 3b(-F) > 3a(-H) > 3c(-Cl) > 3d(-Br). The derivatives 3c and 3d in vitro act as potential dual inhibitors of hTopo I and II with a partial effect on the metabolic activity of cancer cells A594. The acridine-benzohydrazides 3a and 3c reduced the clonogenic ability of A549 cells by 72% or 74%, respectively. The general results of the study suggest that the novel compounds show potential for future development as anticancer agents.

Keywords: HSA binding; acridine; benzohydrazide; ctDNA; spectroscopic study.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Scheme 1
Scheme 1
Synthesis of N′-[(E)-(acridin-4-yl)methylidene]benzohydrazides 3a–e.
Scheme 2
Scheme 2
Synthesis of N′-[(E)-(acridin-4-yl)methylidene]benzohydrazides 7b–d.
Figure 1
Figure 1
Selected 1H, 13C and 15N NMR chemical shifts, and coupling constants for derivatives 3a and 7c.
Figure 2
Figure 2
Fluorescence quenching spectra of HSA at different concentrations of compound 3a with λex = 280 nm in a Tris−HCl/NaCl buffer, pH = 7.4, at 25 °C. Plain black line [HSA] = 4 μM; plain coloured line [3a] = 0–6.2 μM and dashed line [3a] alone = 3.7 μM. The arrows show the intensity change upon increasing concentrations of the quencher.
Figure 3
Figure 3
Synchronous fluorescence spectra of HSA + compound 3a and F/F0 plots at room temperature. ([HSA] = 4 μM; [3a] = 0–6.2 μM; dashed line for Δλ = 60 nm and plain line for Δλ = 15 nm; the arrows show the intensity change upon increasing concentrations of the quencher).
Figure 4
Figure 4
Three-dimensional fluorescence spectra and corresponding contour diagrams of free HSA and HSA:3a systems in a Tris—HCl/NaCl buffer, pH = 7.4 at 25 °C. ([HSA], [3a] = 4 μM).
Figure 5
Figure 5
Emission spectrum HSA:3a (1:1, black line) in the presence of increasing concentration site markers (A) warfarin and (B) ibuprofen (coloured line), and (C) graphical analysis of the competitive experiment, percentage of the initial fluorescence plot molar ratio (HSA/marker). The molar ratio of the HSA:marker is: 1:0.5, 1:1, 1:1.5, 1:2, 1:2.5, 1:3, 1:3.5, 1:4, 1:4.5 and 1:5, [warfarin/ibuprofen] = 0–20 μM.
Figure 6
Figure 6
UV-Vis absorption spectra of acridine derivatives (A–D) 3a–3d at different concentrations of ctDNA in a Tris-HCl buffer, pH = 7.4, at room temperature. The dashed line for free derivatives [3a–3d] = 25 μM and plain colour line for [ctDNA] = 74–680 μM. The arrows show the absorbance change upon increasing concentration of ctDNA. The inserts correspond to Benesi–Hildebrand plots.
Figure 7
Figure 7
Effects of derivatives 3a–3d on the metabolic activity of adenocarcinomic human alveolar basal epithelial cells (A549 cells) evaluated by MTT assay. The cells were treated with the indicated concentrations of the compounds for 24 (A) and 48 h (B). Statistical significance ** p < 0.01; *** p < 0.001 for each experimental group compared to the untreated cells.
Figure 8
Figure 8
Effects of derivatives 3a–3d on the metabolic activity of CCD-18Co cells evaluated by MTT assay. The cells were treated with the indicated concentrations of the compounds for 48 h. Statistical significance ** p < 0.01; *** p < 0.001 for each experimental group compared to the untreated cells.
Figure 9
Figure 9
Effect of derivatives 3a and 3c on viability (A) and total cell number (B) of A549 cells. The viability and total cell number were evaluated 48 h after derivatives addition and are expressed as percentage of the viable, eosin negative cells or as percentage of the total cell number, respectively. Statistical significance ** p < 0.01; *** p < 0.001 for each experimental group compared to the untreated cells.
Figure 10
Figure 10
Effect of derivatives 3a and 3c on A549 cells’ ability to form new colonies. (A) Representative image of colonies in both the presence and absence of compounds treatment. (B) Data represents the percentage of colony forming ability presented as the mean values ± S.D. of three independent experiments. Statistical significance *** p ˂ 0.001 for the experimental group compared to the untreated cells.
Figure 11
Figure 11
Effect of 3a and 3c compounds on cell cycle distribution in A549 cells. The cells were treated with the indicated concentrations of compounds for 48 h. Statistical significance * p < 0.05; ** p < 0.01 for each experimental group compared to the untreated cells.
Figure 12
Figure 12
The result of Topo I/IIα activity tests in the presence of derivatives 3a–3d. (A) Electrophoretogram for inhibition relaxation activity of hTopo I (2.5 U) by derivatives 3a–3d in concentrations of 25, 50 and 100 μM (lane 5–16), substrate-free SC pBR322 (0.5 μg) (lane 4), camptothecin (CPT, lane 2) and plasmid+hTopo I control (lane 3). (NOC—nicked-opened DNA form; R—relaxed DNA form; SC—supercoiled DNA form). (B) Electrophoretogram for inhibition decatenation activity of hTopo IIα (0.5 U) by derivatives 3a–3d in concentrations of 25, 50 and 100 μM (lane 5–16), substrate-free catenated kinetoplast DNA (cat kDNA) (0.5 μg) (lane 4), m-Amsacrine (AMSA, lane 2) and plasmid +hTopo IIα control (lane 3). (decat DNA—decatenated DNA; NC—nicked circular DNA form; L—linear DNA form).
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