Fluorinated 3,6,9-trisubstituted acridine derivatives as DNA interacting agents and topoisomerase inhibitors with A549 antiproliferative activity

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Fluorinated 3,6,9-trisubstituted acridine derivatives as DNA interacting agents and topoisomerase inhibitors with A549 antiproliferative activity Patrik Nunharta, Eva Konkoľováa, Ladislav Janovecb, Rastislav Jendželovskýc, Jana Vargovác, ⁎ Juraj Ševcc, Mária Matejováb, Beata Miltákováb, Peter Fedoročkoc, Mária Kozurkovaa,d, a

Department of Biochemistry, Institute of Chemistry, Faculty of Science, P. J. Šafárik University in Košice, Šrobárova 2, 041 54 Košice, Slovak Republic Department of Organic Chemistry, Institute of Chemistry, Faculty of Science, P. J. Šafárik University in Košice, Šrobárova 2, 041 54 Košice, Slovak Republic c Department of Cellular Biology, Institute of Biology and Ecology, Faculty of Science, P. J. Šafárik University in Košice, Šrobárova 2, 041 54 Košice, Slovak Republic d Biomedical Research Center, University Hospital Hradec Kralove, Sokolska 581, 500 05 Hradec Kralove, Czech Republic b

ARTICLE INFO

ABSTRACT

Keywords: 3,6,9-Trisubstituted acridine derivatives Fluorine calf thymus DNA Spectroscopic techniques topoisomerases I/II Cytotoxicity Lung carcinoma cells

A series of new 3,6,9-trisubstituted acridine derivatives with fluorine substituents on phenyl ring were synthesized and their interaction with calf thymus DNA was investigated. Analysis using UV–Vis absorbance spectra provided valuable information about the formation of the acridine-DNA complex. In addition, compounds 8b and 8d were found to display an increased binding affinity (K = 2.32 and 2.28 × 106 M−1, respectively). Topo I/II inhibition mode assays were also performed, and the results verify that the novel compounds display topoisomerase I and II inhibitory activity; compounds 8a, 8b and 8c completely inhibited topoisomerase I activity at a concentration of 60 × 10−6 M, but only compound 8d showed partial ability to inhibit topoisomerase II at concentrations of 30 and 50 × 10−6 M. The ability of the derivatives to impair cell proliferation was tested through an analysis of cell cycle distribution, quantification of cell number, viability studies, metabolic activity measurement and clonogenic assay. The content and localization of the derivatives in cells were analyzed using flow cytometry and fluorescence microscopy. The compounds 8b and 8d altered the physiochemical properties and improved antiproliferative activity in A549 human lung carcinoma cells (compound 8d displayed the highest level of activity, 4.25 × 10−6 M, after 48 h).

1. Introduction The 3,6-diaminoacridines and their derivatives are currently used as a versatile scaffold in medicinal chemistry for designing novel hybrid compounds. These new agents may offer improved pharmacological and toxicological profiles against several pathological conditions such as cancer and microbial diseases [1]. They are among the most studied compounds and are also widely used as antimalarial, antiviral, antibacterial, antiprotozoal and anti-tubercular agents [2–7]. Haranahalli et al. [8] have shown that fluorine plays a significant role in chemical biology and drug discovery, as evidenced by the large number of fluorine-containing drugs, which have been approved by the FDA. It is widely accepted that incorporating fluorine into molecules plays an important role in medicinal chemistry [9–12]. Therefore, the combination of acridine and fluorine in one molecule may lead to the development of new molecules with more active and effective qualities. DNA are prone to alterations under various conditions; interaction

with some molecules and the damage which this induces may lead to a wide range of pathological changes. Studying the interaction of pharmaceutical agents with DNA is essential in understanding their mode of interaction. Most ligands bind to DNA through a number of different modes, primarily groove binding, intercalation or electrostatic interaction [13–21]. 3,6-Diaminoacridines are able to intercalate between the base of DNA and inhibit topoisomerase II [22]. Further research has also revealed that these derivatives could also engage in interaction with Topo I [23,24]. Beyond their normal functions, topoisomerases are important cellular targets in the treatment of human cancers [25–29]. Some of the most powerful clinically used anticancer drugs act by causing DNA disorders. Topoisomerase inhibitors block the ligation step of the cell cycle, generating single and double stranded breaks that harm the integrity of the genome, thereby leading to apoptosis in proliferating cells and ultimately cell death. DNA topoisomerase directly regulates the topological structure of DNA and induces DNA damages by enhancing

⁎ Corresponding author at: Department of Biochemistry, Institute of Chemistry, Faculty of Science, P. J. Šafárik University in Košice, Šrobárova 2, 041 54 Košice, Slovak Republic. E-mail address: [email protected] (M. Kozurkova).

https://doi.org/10.1016/j.bioorg.2019.103393 Received 9 September 2019; Received in revised form 10 October 2019; Accepted 22 October 2019 0045-2068/ © 2019 Elsevier Inc. All rights reserved.

Please cite this article as: Patrik Nunhart, et al., Bioorganic Chemistry, https://doi.org/10.1016/j.bioorg.2019.103393

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2.1.2. Synthesis of 2,2́,4,4́–tetranitrodibenzophenone (3) The synthesis of this material was carried out according to the previously published method [31]. Yield 94%. mp 234–236 °C; 1H NMR (600 MHz, DMSO–d6): δ 8.95 (2H, d, H-3′,3″, J = 2.2 Hz), 8.64 (2H, dd, H-5′,5″, J1 = 8.5 Hz, J2 = 2.2 Hz), 8.06–8.01 (2H, d, H-6′,6″, J = 2.0 Hz). 13C NMR (150 MHz, DMSO–d6) δ 187.8 (CO), 149.7, 147.6, 134.4, 132.4, 128.3, 120.21.

the formation of TopoI/II-DNA cleavage complexes or suppressing DNA religation. Topoisomerases are therapeutic targets for antibacterial and also anticancer drugs. Several studies have shown the important role played by acridines as inhibitors of topoisomerases [15,29]. A series of 2-fluoro N-substituted acridones with varying alkyl side chain length have been reported, exhibiting promising cytotoxic effects against sensitive and resistant cancer cell lines while their DNA-binding properties, based on their affinity or intercalation with DNA, were evaluated [30]. As part of our ongoing studies into the DNA binding properties of acridines [4,5,15], a series of new derivatives with varying side chain length were synthesized and analyzed. Two of these novel agents incorporated fluorine substituents (derivatives 8b and 8d), while derivatives 8a and 8c were used as a control. Our research focuses on the capability of both compounds to interfere with human topoisomerase I and II and on the in vitro antiproliferative activity of the derivatives against the human lung carcinoma cell line A549. Contemporary approaches to the incorporation of fluorine atoms into molecules are centered on increasing the binding affinity, altering the physiochemical properties and improving the anticancer activity of the resultant compounds.

2.1.3. Synthesis of 3,6–diaminoacridine–9(10H)–one (4) The synthesis of this material was carried out according to the previously published method [31]. Yield 80%, mp > 300 °C; 1H NMR (400 MHz, DMSO–d6) δ 10.61 (1H, s, NH), 7.78 (2H, d, H-1,8, J = 8.4 Hz), 6.41 (2H, dd, H-2,7, J1 = 8.4 Hz, J2 = 2.0 Hz), 6.34 (2H, d, H-4,5, J = 2.0 Hz), 5.86 (4H, s, 2 × NH2). 13C NMR (100 MHz, DMSO–d6) δ 174.7 (CO), 153.1 (C3, C6), 143.6 (C4a, C10a), 127.7 (C1, C8), 112.3 (C8a, C9a), 110.5 (C2, C7), 96.3 (C4, C5). 2.1.4. Synthesis of 3,6–di(butanoylamino)acridine–9(10H)–one (5) The synthesis of this material was carried out according to the previously published method [31]. Light brown solid, yield 49%, mp > 300 °C; 1H NMR (400 MHz, DMSO–d6) δ 11.63 (1H, s, NH–10), 10.25 (2H, s, 2×(NHCO)), 8.15 (2H, s, H-4,5), 8.09 (2H, d, H-1,8, J = 8.4 Hz), 7.20 (2H, d, H-2,7, J = 8.4 Hz), 2.37 (4H, t, 2×(COCH2), J = 7.2 Hz), 1.65 (4H, m, 2x (CH2CH3)), 0.95 (t, 6H, 2×(CH2CH3), J = 7.2 Hz). 13C NMR (100 MHz, DMSO–d6) δ 174.89 (CO), 171.84 (NH–CO), 143.14 (C3, C6), 142.02 (C4a, C10a), 126.7 (C1, C8), 116.3 (C8a, C9a), 113.1 (C2, C7), 104.6 (C4, C5), 38.4 (COCH2), 18.5 (CH2CH3), 13.6 (CH2CH3).

2. Experimental 2.1. Materials and methods The reagents which were commercially obtained for the synthesis of new compounds were of the highest commercial quality and were used without further purification. All solvents were distilled and dried using standard methods. All other chemicals and reagents were purchased at reagent grade and were used without further purification. Propidium iodide (PI), Amsacrine (AMSA), Camphtothecin (CPT), Triton X-100, dimethyl sulfoxide (DMSO) and calf thymus DNA (ctDNA) were obtained from Sigma-Aldrich (Germany). Ethylenediaminetetraacetic acid (EDTA), ribonuclease (RNase A), proteinase K and sodium lauryl sulfate (SDS) were purchased from Serva (Germany), plasmid pUC 19 (2761 bp, DH 5α), and agarose (type II NoA-6877) (Sigma), Topoisomerase I, Topoisomerase II, tris(hydroxymethyl) aminomethane (Tris), Hefe extract, Tryptone/Peptone, and ampicillin were purchased from Roth (Germany) and isopropyl alcohol. All other chemicals were purchased from Lach-Ner (Czech Republic): isopropyl alcohol, bromophenol blue (BFM), ethanol, glucose, glycerol, hydrochloric acid (HCl), chloroform, isoamyl alcohol, boric acid, acetic acid, sodium chloride, and potassium acetate. 1 H (400 MHz, 600 MHz) and 13C (100 MHz, 150 MHz) NMR spectra were measured on a Varian Mercury Plus or a Varian VNMRS NMR spectrometer at room temperature in DMSO–d6. Chemical shifts (δ in ppm) are given from the internal solvent, DMSO–d6 2.50 ppm for 1H and 39.5 ppm for 13C. 19F (375 MHz) NMR spectra were measured on a Varian Mercury Plus spectrometers at room temperature in DMSO–d6 with trifluoroacetic acid (76.55 ppm) as the internal standard. Melting points were determined with a Koffler hot-stage apparatus and are uncorrected. Elemental analyses were performed on a Perkin–Elmer analyzer CHN 2400. Reactions were monitored using thin–layer chromatography (TLC) with Silufol plates and detection at 254 nm. Preparative column chromatography was performed using a Kieselgel Merck 60 column, type 9385 (grain size 250 nm).

2.1.5. Synthesis of 3,6–di(butanoylamino)–9–chloroacridine (6) The synthesis of this material was carried out according to the previously published method [31]. Yield 47%, mp 153–155 °C; 1H NMR (400 MHz, DMSO–d6) δ 10.45 (2H, s, 2×(NHCO)), 8.58 (2H, d, H-4,5, J = 2.0 Hz), 8.27 (2H, d, H-1,8, J = 9.6 Hz), 7.76 (2H, dd, H-2,7, J1 = 9.6 Hz, J2 = 2.0 Hz,), 2.43 (4H, t, 2×(COCH2), J = 7.6 Hz), 1.70 (4H, m, 2×(CH2CH3)), 0.98 (6H, t, 2×(CH2CH3), J = 7.6 Hz). 13C NMR (100 MHz, DMSO–d6) δ 172.1 (2 × CO), 149.5 (C3, C6), 141.2 (C4a, C10a), 139.1 (C9), 124.8 (C1, C8), 121.7 (C2, C7), 119.3 (C8a, C9a), 113.6 (C4, C5), 38.5 (COCH2), 18.3 (CH2CH3), 13.6 (CH2CH3). 2.1.6. General procedure for the preparation of 3,6–di (butanoylamino)–9–[(aryl)amino] acridine 7a–7i [31] Chloroacridine 6 (1.0 g, 2.6 mmol) was dissolved in hot acetonitrile (60 mL) and the corresponding amine (8.0 mmol) was then added to the solution. The mixture was refluxed for 1 hr. and the precipitated product was filtered off and washed with acetonitrile. In order to remove the remaining amine, the product was suspended in acetone, and the mixture was stirred vigorously under reflux for 2 hr. Upon cooling, the product was filtered off and crystallized from ethanol to produce yellow crystals. 2.1.7. 3,6–Bis(butanoylamino)–9–[phenyl(amino)]acridine hydrochloride (7a) Yellow crystalline solid, Yield 85%, mp: > 300 °C with decomposition; 1H NMR (400 MHz, DMSO–d6) δ 13.91 (1H, s, NH + –10), 11.13 (1H, s, 9–(NH)), 10.93 (2H, s, 2×(NHCO)), 8.53 (2H, d, H-4,5, J = 2.2 Hz), 8.06 (2H, d, H-1,8, J = 9.4 Hz), 7.50–7.43 (2H, m, H-3′,5′), 7.41–7.32 (5H, m, H2,H7,H2′,H6′,H4′), 2.43 (4H, t, 2x(COCH2), J = 7.2 Hz), 1.72–1.58 (4H, m, 2x(CH2CH3)), 0.93 (6H, t, 2x(CH2CH3), J = 7.2 Hz). 13C NMR (100 MHz, DMSO–d6) δ 172.8 (CO), 152.9 (C9), 144.6 (C3, C6), 141.9 (C4a, C10a), 141.4 (C1′), 129.8 (C3′, C5′), 126.8 (C1, C8), 126.6 (C4′), 124.1 (C2′, C6′), 116.7 (C2, C7), 109.7 (C8a, C9a), 104.4 (C4, C5), 38.5 (COCH2), 18.4 (CH2CH3), 13.6 (CH2CH3).

2.1.1. Synthesis of 2,2́,4,4́–tetranitrodiphenylmethane (2) The synthesis of this material was carried out according to the previously published method [31]. Cream solid, yield 84%, mp 168–170 °C; 1H NMR (600 MHz, DMSO–d6): δ 8.82 (2H, d, H-3′,3″, J = 2.5 Hz), 8.49 (2H, dd, H-5′,5″, J1 = 8.6 Hz, J2 = 2.5 Hz), 7.60 (2H, d, H-6′,6″, J = 8.6 Hz), 4.79 (2H, s, CH2). 13C NMR (150 MHz, DMSO–d6) δ 148.6, 146.6, 139.7, 133.7, 127. 8, 120.4 35.1. 2

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Anal. Calcd for C27H28N4O2 × HCl (476.99): C, 67.99; H, 6.13; N, 11.75%. Found: C, 64.31; H, 6.59; N, 12.50%.

2.1.11.2. Reaction path utilizing chloro derivate 9. 3,6-Diamino-9chloroacridine (9) (100 mg, 0.4 mmol) was dissolved in anhydrous dimethylformamide to which 0.6 mmol of amine was then added. The reaction mixture was stirred at 110 °C for 1 h under an inert atmosphere of nitrogen. The reaction was monitored using TLC and eluting with a methanol–ammonia mixture (10: 1 by volume). Once the reaction had finished, the products were precipitated from the solution through the addition of diethyl ether. After filtration and drying, the products were purified using column chromatography on a silica gel using ethyl acetate and crystallized from an ethanol–tetrahydrofuran mixture.

2.1.8. 3,6–Bis(butanoylamino)–9–[(4–fluorophenyl)amino]acridine hydrochloride (7b) Yellow crystalline solid, Yield 90%, mp : > 300 °C with decomposition; 1H NMR (600 MHz, DMSO–d6) δ 13.89 (1H, s, NH + –10), 11.11 (1H, s, 9–(NH)), 10.90 (2H, s, 2×(NHCO)), 8.53 (2H, d, H-4,5, J = 2.2 Hz), 8.06 (2H, d, H-1,8, J = 9.4 Hz), 7.44–7.40 (2H, m, H-2′,6′), 7.39 (2H, dd, H-2,7, J1 = 9.4 Hz, J2 = 2.2 Hz), 7.33–7.29 (2H, m, H3′,5′), 2.43 (4H, t, 2x(COCH2), J = 7.2 Hz), 1.69–1.61 (4H, m, 2x (CH2CH3)), 0.93 (6H, t, 2x(CH2CH3), J = 7.2 Hz). 13C NMR (150 MHz, DMSO–d6) δ 172.8 (CO), 160.3 (d, C4′, J = 244.2 Hz), 153.1 (C9), 144.6 (C3, C6), 141.9 (C4a, C10a), 137.6 (C1′), 126.7 (C1, C8), 126.3 (d, C2′, C6′, J = 8.4 Hz), 116.7 (C2, C7), 116.6 (d, C3′, C5′, J = 22.8 Hz), 109.5 (C8a, C9a), 104.4 (C4, C5), 38.5 (COCH2), 18.4 (CH2CH3), 13.6 (CH2CH3). 19F NMR (375 MHz, DMSO–d6) δ 117.20 (4–F– Ph). Anal. Calcd for C27H27FN4O2 × HCl (494.98): C, 65.51; H, 5.70; N, 11.32%. Found: C, 65.49; H, 6.11; N, 12.00%.

2.1.12. 3,6–Diamino–9–[(phenyl)amino]acridine hydrochloride (8a) Yellow crystalline solid, yield 80%, mp > 265 °C with decomposition; 1H NMR (400 MHz, DMSO–d6) δ 13.34 (1H, s, NH + –10), 10.13 (1H, s, 9–(NH)), 7.81 (2H, d, H-1,8, J = 9.4 Hz), 7.35–7.31 (2H, m, H3′,5′), 7.15–7.11 (3H, m, H-2′,6′, H4′), 6.85–6.81 (2H, m, H-4,5), 6.68 (2H, d, H-2,7, J = 9.4 Hz). 13C NMR (100 MHz, DMSO–d6) δ 153.9 (C9), 150.8 (C3, C6), 143.2 (C4a, C10a), 143.0 (C1′), 129.4 (C3′, C5′), 127.5 (C4′), 124.0 (C2′, C6′), 121.7 (C1, C8), 114.9 (C2, C7), 106.7 (C8a, C9a), 95.2 (C4, C5). Anal. Calcd for C19H16N4 × HCl (336.81): C, 67.75; H, 5.09; N, 16.63%. Found: C, 66.33; H, 5.75; N, 14.99%.

2.1.9. 3,6–Di(butanoylamino)–9–(benzylamino)acridine hydrochloride (7c) Performed according to the literature protocol, reference 31. Yellow crystalline solid, yield 77%, mp > 300 °C; 1H NMR (600 MHz, DMSO–d6) δ 13.31 (1H, bs, NH–10), 10.84 (2H, s, 2x (NHCO)), 10.12 (1H, bs, 9–(NHCH2)), 8.44–8.41(4H, m, H-1,8, H-4,5), 7.46–7.43 (4H, m, H-2′,6′, H-2,7), 7.40–7.37 (2H, m, H-3′,5′), 7.31–7.28 (1H, m, H-4′), 5.27 (2H, s, NHCH2Ph), 2.42 (4H, t, 2x (COCH2), J = 7.2 Hz), 1.67–1.61 (4H, m, 2x(CH2CH3)), 0.92 (6H, t, 2x (CH2CH3), J = 7.2 Hz). 13C NMR (150 MHz, DMSO–d6) δ 172.7 (CO), 155.4 (C9), 144.4 (C3, C6), 141.4 (C4a, C10a), 137.4 (C1′), 128.9 (C3′, C5′), 127.6 (C4′), 126.8 (C1, C8), 126.7 (C2′, C6′), 116.0 (C2, C7), 108.1 (C8a, C9a), 104.4 (C4, C5), 50.5 (NHCH2Ph), 38.5 (COCH2), 18.4 (CH2CH3), 13.6 (CH2CH3). Anal. Calcd for C28H30N4O2 × HCl (491.02): C, 68.49; H, 6.36; N, 11.41% N. Found: C, 68.70; H, 6.45; N, 11.25%.

2.1.13. 3,6–Diamino–9–[(4–fluorophenyl)amino]acridine hydrochloride (8b) Yellow crystalline solid, yield 80%, mp 195–197 °C; 1H NMR (400 MHz, DMSO–d6) δ 13.41 (1H, s, NH + –10), 10.30 (1H, s, 9–(NH)), 7.83 (2H, d, H-1,8, J = 9.0 Hz), 7.28–7.08 (4H, m, H-4,5, H2′,6′), 6.90 (2H, s, H-3′,5′), 6.73 (2H, d, H-2,7, J = 9.0 Hz). 13C NMR (100 MHz, DMSO–d6) δ 159.0 (d, C4′, J = 241.6 Hz), 152.9 (C9), 151.2 (C3, C6), 142.9 (C4a, C10a), 139.4 (C1′), 127.5 (C1, C8), 124.1 (d, C5′, C6′, J = 8.3 Hz), 116.1 (d, C3′, C5′, J = 22.6 Hz), 115.3 (C2, C7), 106.8 (C8a, C10a), 96.3 (C4, C5). 19F NMR (375 MHz, DMSO–d6) δ 122.35 (4–F–Ph). Anal. Calcd for C19H15FN4 × HCl (354.80): C, 64.32; H, 4.55; N, 15.79%. Found: C, 63.81; H, 4.69; N, 15.61%. 2.1.14. 3,6–Diamino–9–(benzylamino)acridine (8c) Yellow crystalline solid, yield 80%, mp 170–172 °C; 1H NMR (600 MHz, DMSO–d6) δ 8.04 (2H, d, H-1,8, J = 9.0 Hz), 7.40–7.39 (2H, m, H-2′,6′), 7.37–7.34 (2H, m, H-3′,5′), 7.27–7.25 (1H, m, H-4′), 6.61–6.58 (4H, m, H-2,7, H-4,5), 6.39 (4H, bs, 3,6–(2xNH2)) 5.02 (2H, s, NHCH2Ph). 13C NMR (150 MHz, DMSO–d6) δ 153.5 (C9), 152.9 (C3, C6), 144.1 (C4a, C10a), 139.1 (C1′), 128.6 (C3′, C5′), 127.1 (C4′), 126.7 (C1, C8, C2′, C6′), 113.4 (C2, C7), 104.9 (C8a, C9a), 96.7 (C4, C5), 51.1 (NHCH2Ph). Anal. Calcd for C20H18N4 (314.39): C, 76.41; H, 5.77; N, 17.82%. Found: C, 76.61; H, 5.84; N, 17.96%.

2.1.10. 3,6–Di(butanoylamino)–9–[(4–fluorobenzyl)amino]acridine hydrochloride (7d) Yellow crystalline solid, yield 70%, mp: 203–205 °C; 1H NMR (400 MHz, DMSO–d6) δ 13.34 (1H, bs, NH + –10), 10.87 (2H, s, 2x (NHCO), 10.11 (1H, bs, 9–(NHCH2)), 8.45–8.41 (4H, m, H-1,8, H-4,5), 7.53–7.48 (2H, m, H-2′,6′), 7.45 (2H, d, H-2,7, J = 9.6 Hz), 7.27–7.17 (2H, m, H-3′,5′), 5.26 (2H, s, NHCH2Ph), 2.42 (4H, t, 2x(COCH2), J = 7.2 Hz), 1.77–1 0.53 (4H, m, 2x(CH2CH3)), 0.92 (6H, t, 2x (CH2CH3), J = 7.2 Hz). 13C NMR (100 MHz, DMSO–d6) δ 172.7 (CO), 161.5 (d, C4′, J = 240.0 Hz), 155.3 (C9), 144.4 (C3, C6), 141.4 (C4a, C10a), 133.5 (C1′, J = 3.0 Hz), 128.9 (d, C2′, C6′, J = 8.1 Hz), 126.9 (C1, C8), 116.0 (C2, C7), 115.5 (d, C3′, C5′, J = 21.4 Hz), 108.1 (C8a, C9a), 104.4 (C4, C5), 49.8 (NHCH2Ph), 38.5 (COCH2), 18.4 (CH2CH3), 13.6 (CH2CH3).19F NMR (375 MHz, DMSO–d6) δ 116.64 (4–F–Bn). Anal. Calcd for C28H29FN4O2 × HCl (509.01): C, 66.07; H, 5.94; N, 11.01% N. Found: C, 65.82; H, 5.59; N, 11.21%.

2.1.15. 3,6–Diamino–9–[(4–fluorobenzyl)amino]acridine (8d) Yellow crystalline solid, yield 65%, mp > 260 °C with decomposition; 1H NMR (600 MHz, DMSO–d6) δ 8.07 (2H, d, H-1,8, J = 9.0 Hz), 7.43 (2H, dd, H-2′,6′, J1 = 8.4 Hz, J2 = 5.4 Hz), 7.20–7.17 (2H, m, H3′,5′), 6.65–6.60 (4H, m, H-4,5, H-2,7), 6.58 (4H, bs, 3,6–(2xNH2)), 5.04 (2H, s, NHCH2Ph). 13C NMR (150 MHz, DMSO–d6) δ 161.4 (d, C4′, J = 240.0 Hz), 153.9 (C9), 153.6 (C3, C6), 143.1 (C4a, C10a),134.9 (d, C1′, J = 3.0 Hz), 128.7 (d, C2′, C6′, J = 8.1 Hz), 127.0 (C1, C8), 115.5 (d, C3′, C5′, J = 21.4 Hz), 113.6 (C2, C7), 104.1 (C8a, C9a), 95.6 (C4, C5), 50.0 (NHCH2Ph). 19F NMR (375 MHz, DMSO–d6) δ 117.27 (4–F–Bn). Anal. Calcd for C20H17FN4 (332.37): C, 72.27; H, 5.16; N, 16.86%. Found: C, 71.83; H, 5.18; N, 17.03%.

2.1.11. General procedure for the preparation of 3,6-diamino-9-[(aryl) amino]acridine 8a–8c 2.1.11.1. Reaction path using the hydrolysis of derivatives 8a–8d [31]. Derivatives 7a–7i (1.0 mmol) were dissolved in 2–propanol (25 mL) and 0.05 mL of concentrated sodium hydroxide solution was then added to the mixture. The reaction mixture was stirred for 5 hr at 80 °C. The process of the reaction was monitored using TLC (ethyl acetate). The resulting mixture was then diluted dropwise with water until the precipitate had formed. The obtained mixture was cooled slowly to room temperature and then left overnight to crystallize. Products 8a–8c were crystallized in the form of hydrochlorides from a mixture of MeOH and THF.

2.1.16. Preparation of 3,6–diamino–9–chloracridine (9) Derivative 7 (0.5 g, 2.0 mmol) was added to 2-propanol (25 mL) and 0.05 mL of concentrated sodium hydroxide solution was then added to the solution of reactant 7. The reaction mixture was stirred at 85 °C for 5 h, then cooled to room temperature, concentrated through the evaporation of 2-propanol under reduced pressure, and finally distilled 3

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water (25 mL) was added to give a red precipitate. Product 9 was then filtered off, washed well with distilled water to neutral pH on the filter. After drying, product 9 was crystallized from ethanol. Yellow red solid, yield 70%, mp > 300 °C with decomposition; 1H NMR (600 MHz, DMSO–d6) δ 7.92 (2H, d, H-1,8, J = 9.6 Hz), 6.99 (2H, dd, H-2,7, J1 = 9.6 Hz, J2 = 2.4 Hz), 6.78 (2H, d, H-4,5, J = 2.4 Hz), 6,03 (4H, s, 2xNH2). 13C NMR (150 MHz, DMSO–d6) δ 151.4 (C3, C6), 150.9 (C4a, C10a), 138.8 (C9), 125.3 (C1, C8), 119.7 (C2, C7), 115.4 (C8a, C9a), 103.1 (C4, C5). Anal. Calcd for C13H10ClN3 (243.69): C, 64.07; H, 4.14; N, 17.24%. Found: C, 62.77; H, 4.37; N, 17.14%.

containing a freshly prepared 5 × complete assay buffer, 0.2 μg supercoiled pHOT-1 and 5U human Topo IIα. Reactions were incubated in the absence or in the presence of the studied derivatives (5, 10, 30 and 50 × 10−6 M) at 37 °C for 30 min. The method used to perform the experiments has been published previously [24,27]. 2.5. Biological studies Cell culture conditions. Human lung carcinoma cell line A549 was purchased from the American Type Culture Collection (ATCC, Rockville, MD, USA). Cells were grown in Kaighn’s modification of F-12 Ham Nutrient Mixture (Sigma-Aldrich, St. Louis, MO, USA) supplemented with 10% fetal bovine serum (FBS; Biosera, Boussens, France) and antibiotics (1% Antibiotic-Antimycotic 100 × and 50 × 10−3 g L−1 gentamicin; Biosera) at 37 °C, 95% humidity and 5% CO2. Cells were seeded on 12-well μ-Chamber slides (ibidi GmbH, Martinsried, Germany), 6 and 96-well plates (TPP, Trasadingen, Switzerland) and left to settle for 24 h. For flow cytometric analysis of derivatives content, floating and adherent cells were harvested together 6 and 24 h after treatment with the derivatives, washed in PBS and resuspended in Hank’s balanced salt solution (HBSS). Intracellular levels of derivatives were detected using a BD FACSCalibur flow cytometer (Becton Dickinson, San Jose, CA, USA) and established based on fluorescence excitation at 488 nm. The fluorescence was detected via a 530/30 nm band-pass filter (FL-1), 585/ 42 band-pass filter (FL-2) and 670 nm long-pass filter (FL-3). The results were analyzed using FlowJo software (TreeStar Inc., Ashland, OR, USA). MTT assays were performed in order to evaluate changes in the metabolic activity of cells that had occurred as a consequence of treatment with the derivatives. The method used to perform the experiments has been described in detail previously [15,23]. The results were evaluated as percentages of the absorbance of the untreated control. IC50 values for the derivatives were extrapolated from a sigmoidal fit to the metabolic activity data using OriginPro 8.5.0 SR1 (OriginLab Corp., Northampton, MA, USA). For the assessment of total cell numbers and viability within individual experimental groups, floating and adherent cells were harvested 24 h after derivatives treatment and evaluated using a Bürker chamber with eosin staining. The total cell number was expressed as a percentage of the untreated control of the total cell number. Viability was expressed as a percentage of viable, eosin negative cells. For flow cytometric analysis of the cell cycle, floating and adherent cells were harvested together 24 h after treatment with the derivatives, washed in cold PBS, fixed in cold 70% ethanol and kept at −20 °C overnight. Prior to analysis, the cells were washed twice in PBS, resuspended in a staining solution (0.1% Triton X-100, 0.137 g L−1 ribonuclease A and 0.02 g L−1 propidium iodide – PI) and incubated in the dark at RT for 30 min. The DNA content was analysed using a BD FACSCalibur flow cytometer (Becton Dickinson) with a 488 nm argonion excitation laser, and fluorescence was detected via a 585/42 nm band-pass filter (FL-2). The method used to perform the experiments has been described previously [5]. ModFit 3.0 software (Verity Software House, Topsham, ME, USA) was used to generate DNA content frequency histograms and to quantify the percentage of cells in the individual cell cycle phases. For the clonogenic assay, floating and adherent cells were harvested together 24 h after derivatives treatment, counted using a Bürker chamber with eosin staining and 800 viable cells per well were seeded in 6-well plates. After 7 days of cultivation under standard conditions, the cells in the plates were fixed and stained with 1% methylene blue dye in methanol. Visualized colonies were scanned, counted and the results were evaluated as percentages of the untreated control. A549 cells (1.5 × 104 cm−2/3.0 × 104 cm−2) were seeded on 12well μ-Chamber slides (Ibidi GmbH, Martinsried, Germany) and left to settle down for 24 h. The studied derivatives 8a–8d were added to the

2.2. DNA binding experiments Stock solutions of all derivatives at a concentration of 1 × 10−2 M were prepared by dilution using DMSO. Calf thymus DNA (ctDNA) stock solution was prepared by dissolving 10 mg/mL in a Tris-EDTA buffer (TE buffer, pH 8.0 contains: 1 × 10−2 M Tris-HCl, and 1 × 10−3 M EDTA). The concentration of the DNA solution was calculated spectrophotometrically at 260 nm using a molar extinction coefficient of 6600 cm−1 M−1. The ratio of A260/A280 was found to be 1.82, which is indicative of the purity of the DNA. The absorption spectra of free acridine derivatives and its complexes with ctDNA (calf thymus DNA) were measured on a Varian Cary 100 UV–vis spectrophotometer in a 2 × 10−3 dm3 0.01 M Tris-HCl buffer (pH 7.4) in a quartz cuvette (1 cm path length). The concentration of ctDNA in the solution was gradually increased (ranging from 0 to 3.7 × 10−5 M) until the saturation state of the ctDNA by the studied compound had been reached for use in the binding experiments. Each absorption spectrum was scanned at a wavelength range of 200–600 nm. All measurements were performed at 25 °C. The measured UV–Vis data for compounds 8a–8d (Table 1) were graphically processed using GraphPad Prism 6 software. The binding constants K of the ligand-DNA complexes were calculated using the absorption titration data and the McGhee and von Hippel equation [32], where K is the binding constant, n is the binding site size in the base pairs, Cf is the molar concentration of free ligands and r is the number of ligand molecules that bind to one mol of nucleotides. The binding data were fitted using Gnu Octave 2.1.73 software [33]. 2.3. Topoisomerase I relaxation assay The effect of derivatives 8a–8d on the relaxation of plasmid DNA with human topoisomerase I (hTOPI) was estimated using a negatively supercoiled plasmid pBR322 (0.5 × 10−6 g) which was incubated for 30 min at 37 °C with 2 units of hTOPI (Inspiralis, Ltd., UK) in both the absence and presence of the studied acridine derivatives (5, 10, 30 and 50 × 10−6 M, respectively [24]. 2.4. Topoisomerase II inhibition assay A freshly prepared 5 × complete assay buffer for Topo II decatenation containing 0.16 μg catenated kinetoplast DNA (kDNA) and 2 U of human Topo IIα in the absence or presence of derivatives 8a–8d (5, 10, 30 and 50 × 10−6 M) was incubated for 45 min at 37 °C. DNA cleavage reactions were carried out in a 20 × 10−6 L final volume Table 1 Spectral absorption characteristic of acridine compounds 8a–8d. Compound

λfree [nm]

λbound [nm]

Δλ [nm]

Hypochromicity [%]

K × 106 [M−1]

8a 8b 8c 8d

405 380 384 385

407 397 394 393

2 17 10 8

40.5 53.8 51.7 54.1

1.22 2.32 2.22 2.28

4

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Scheme 1. Synthesis of 3,6–diamino–9(10H)acridone (4).

Scheme 2. Synthesis of trisubstituted acridine derivatives 8a–d.

5

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Fig. 1. Absorption spectra of compounds 8a–8d in Tris-HCl (pH 7.4) in the presence and absence of ctDNA. Arrows indicate changes in absorption spectra at increasing ctDNA concentrations.

Fig. 2. Electrophoresis agarose gel showing inhibition of human topoisomerase I (hTOPI) activity induced DNA relaxation by derivatives 8a–8d.

cells for another 24 h. After cultivation with derivates, samples (control without derivatives and experimental groups) were washed with PBS solution buffer to remove non-cell-absorbed derivative residues and fixed with 4% paraformaldehyde for 20 min at room temperature. Samples were washed with PBS, mounted and coverslipped with Prolong Gold Antifade media (Thermo Fisher Scientific). Samples were visualized using an inverted fluorescence microscope Leica DMI6000 B (Leica Microsystems, Mannheim, Germany), using the same objective and parameters: objective–HCX PL APO CS 10.0 × 0.40 DRY UV; camera–DFC420 C; channels - transmitted light channel, fluorescent channel (filter cube: I3; excitation range: blue; excitation filter: BP 450–490; dichromatic mirror = 510; suppression filter: LP 515). The fluorescent channel was combined with the transmitted light channel to

allow all cells to be observed, including control. Microphotographs were captured and evaluated using Leica LAS AF Lite (Leica Microsystems, Mannheim, Germany). Densitometric analysis was performed using the Ellipse 2 software (ViDiTo, Slovak Republic). The images were desaturated prior to analysis. For calibration purposes, the signal from background in each sample (area without cells) was set as 0 and signal from overexposed area (R = 255; G = 255; B = 255) was set as 100. Densitometic analysis of signal from perinuclear space was performed in 20 cells per sample (cells incubated with particular derivates or control) and was expressed as median fluorescence intensity (MFI).

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Fig. 3. Topoisomerase II inhibition assays – effect of compounds 8a–8d on the decatenation of kDNA by (hTOPII).

Fig. 4. Effect of 8a–8d derivatives on metabolic activity in A549 cancer cell lines evaluated by an MTT assay. The results are presented as the mean values ± SD of three independent experiments; statistical significance (**): p < 0.01, (***): p < 0.001 for each experimental group compared to untreated control.

samples were analyzed using one-way analysis of variance (ANOVA) followed by the Tukey-Kramer post hoc tests for multiple comparisons. The experimental groups treated with derivatives were compared with the control group: (*): p < 0.05, (**): p < 0.01, (***): p < 0.001.

Table 2 IC50 values of derivatives 8a–8d in the A549 cancer cell line. Incubation time

8a

8b

8c

8d

Cisplatin*

24 h 48 h

51.48 18.66

n.d. n.d.

61.13 7.50

62.47 4.25

31.25 5.1

3. Results and discussion

The results are presented in µM. * Data were acquired from publication: Kaplan et al. [36].

3.1. Synthetic study

2.6. Statistical analysis

The synthetic pathway described herein begins with the preparation of 3,6–diaminoacridone 4 as an access to the trisubstituted acridines. The preparation of 3,6–diaminoacridone 4 was carried out in accordance with a previously described procedure based on diphenylmethane (1) (Scheme 1). The subsequent steps of the process, the

Data were analyzed using a one-way ANOVA with Tukeýs post-test and are expressed as the mean ± standard deviation (SD) of at least three independent experiments. The differences between MFI of 7

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Table 3 Effect of derivatives 8a–8d on cell cycle distribution. Results were evaluated 24 h after treatment with derivatives. Treatment group

Concentration [× 10−6 M]

G1

Control 8a

0 5 50 5 50 5 50 5 50

55.82 51.04 34.69 54.81 58.60 63.52 54.34 59.69 55.89

8b 8c 8d

S ± ± ± ± ± ± ± ± ±

1.87 0.48 1.05*** 3.47 1.65 3.72 5.01 1.32 3.81

33.94 26.48 40.04 34.82 31.09 24.00 33.87 26.91 31.24

G2 ± ± ± ± ± ± ± ± ±

1.66 1.00 0.50 2.45 1.82 3.81 5.56 0.90 6.93

10.24 22.47 25.26 10.37 10.31 12.48 11.79 13.04 12.86

± ± ± ± ± ± ± ± ±

0.62 1.20*** 1.51*** 0.79 0.66 0.92 2.86 0.43 3.14

The results are presented as a mean ± SD (n = 3), statistical significance (***): p < 0.001 for the particular experimental group compared to untreated control.

Fig. 5. Effect of derivatives 8a–8d on changes in viability and cellularity evaluated 48 h after treatment. The results are presented as a mean ± SD, statistical significance: (*): p < 0.05 for each experimental group compared to untreated control (C – control).

Fig. 6. Effect of derivatives 8a–8d on colony forming ability of A549 cancer cells evaluated using clonogenic assay, (A) The results are presented as the mean values ± SD of three independent experiments; statistical significance (**): p < 0.01, (***): p < 0.001 for each experimental group compared to untreated control, (B) Picture of one representative experiment from at least three independent experiments is presented.

nitration of diphenylmethane (1) and oxidation of 2, proceeded in yields similar to those obtained in the published results [31]. The cyclization of compound 3 provided the main synthon, 3,6–diaminoacridone 4. The key intermediate, 6, was prepared through the reaction of POCl3 with precursor 5, which was in turn obtained through the reaction of acridone 4 with butyric anhydride at increased temperature. The synthesis of acridines 7a–7c involved the reaction of 6 with the related amines (Scheme 2). The alkali hydrolysis of derivatives 7a–7c was then performed in order to remove the protecting groups and obtain the final products, derivatives 8a–8c. Although the previously described

reaction pathway is applicable for the synthesis of derivatives 7a–7c, we realized that the preparation of derivatives 8a–8c could also be achieved by utilizing 3,6-diamino-9-chloracridine (9) (Scheme 2). The decision to utilize this method was taken because of the problematic separation and purification of the products prepared using the first reaction pathway. Derivative 9 was prepared through the alkali hydrolysis of chlorine derivate 6 (Scheme 2). 3.2. UV–Vis absorption spectroscopy The UV–Vis absorption spectra of the novel derivatives 8a–8d 8

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Fig. 7. Flow cytometric analysis of intracellular level of studied derivatives 8a–8d. The results are presented as the mean values ± SD of three independent experiments; statistical significance (*): p < 0.05, (**): p < 0.01, (***): p < 0.001 for each experimental group compared to untreated control.

(Table 1) in the presence of ctDNA show significant absorption bands in the region of 300–550 nm. Fig. 1 describes the interaction of derivatives 8a–8d with increasing amounts of ctDNA and shows that the absorption bands of the ligand-DNA complex were affected by increasing concentrations of ctDNA resulting in a tendency toward hypochromism. A significant hypochromic shift (40.5–54.1%) was observed for derivatives 8a–8d, a feature which is typical for intercalators. The extent of the hypochromism is usually consistent with the strength of intercalative interaction. For this reason, we calculated binding constant using McGhee von Hippel Equation [32]. The binding constants of complexes 8a–8d with ctDNA were in the range of K = 1.22–2.28 × 106 M−1. The results show that derivative 8d, the compound in which the phenyl ring and acridine core are separated by a methylene group, displayed the highest constant. The estimated values of binding constants are comparable to those of classical intercalators such as ethidium bromide (K = 1.4 × 106 M−1), which suggests that the mode of interaction is intercalation. Spectral absorption characteristics of the studied acridine compounds are presented in Table 1 and Fig. 1. The compounds with fluorine substituents were found to exhibit increased levels of binding affinity. If we compare the values of hypochromism reported in this work with those of our previous works [4,23], we can observe a closer proximity of the acridine chromophore to DNA in both of the compounds containing fluorine. The absence of tight isosbestic points obtained during titration experiments for derivatives 8a, 8c and 8d indicates that more than one type of DNA-ligand complex may have been formed, a finding which is in contrast to the results obtained for derivative 8b.

8a–8d. Incubation with compound 8d resulted in no inhibitory activity at all of the tested concentrations. The derivatives were found to have fully inhibited the relaxation of plasmid DNA at concentration 30 × 10−6 M and higher. Camptothecin (CPT), a quinoline alkaloid which inhibits the DNA enzyme Topo I, was used as a positive control. CPT has shown effective inhibitory action on nucleic acid synthesis in mammalian cells [27,28]. 3.4. Decatenation of topoisomerase II The specific reaction of Topo II with DNA is a decatenation reaction as it involves the simultaneous cleavage of two strands of the DNA-helix [27,28]. The ability of derivatives 8a–8d to inhibit the Topo II catalyzed decatenation of kinetoplast DNA (kDNA) was also studied. The appearance of kDNA monomers was monitored in the reaction in order to determine whether an open circular decatenated (nicked) kDNA form or a closed circular decatenated kDNA form had developed. If Topo II retains its normal function, catenated kDNA (the top band with the lowest mobility) disappears and bands for open circular intermediate and closed circular decatenated kDNA (which migrates further) appear. In contrast, the absence of these bands in the results indicates that inhibition of the enzyme had occurred. As is shown in Fig. 3 no inhibitory effect was observed for any of the compounds, with the exception of compound 8d at 30 and 50 × 10−6 M. 3.5. Biological studies For biological effect analysis of novel compounds 8a–8d, we prefer to use multiple assays that reflect real physiological/pathological changes in more detail and on single cell level. It is generally known that most of the chemotherapeutic agents exert their cytotoxic effect either by induction of cancer cell apoptosis or by cell cycle arrest at a specific point [34]. MTT colorimetric assay is based on the reduction of the yellow water-soluble tetrazolium salt (MTT) into an insoluble crystalline blue formazan product by cellular oxidoreductases of viable cells. This transformation is not possible on dead cells, therefore, the resulting formazan crystal formation is proportional to the number of viable cells with an active metabolism [35]. The ability of the studied compounds to inhibit the metabolic activity of the human lung carcinoma cell line A549 was determined using this method. Derivatives were applied at a concentration range of from 2.5 to 50.0 × 10−6 M. As is clear from Fig. 4, compounds 8a, 8c and 8d inhibited metabolic activity in a doseand time-dependent manner, with derivative 8d displaying the highest

3.3. Topoisomerase I inhibition Since most acridine derivatives generally also have good inhibitory activity against TopoI/II, we also aimed to verified whether representative compounds have inhibitory effect on these targets. Given that the studied compounds 8a–8d are able to interact with ctDNA, it is important to consider whether they are also able to inhibit some of the cell nucleus enzymes involved in DNA operations [25–27]. The supercoil relaxation activity of pBR322 topoisomerase I in the presence of varying concentrations (5, 10 and 30 × 10−6 M) of compounds 8a–8d is shown in Fig. 2. The results demonstrate that the supercoiled DNA (line pBR) was fully relaxed by the enzyme in the absence of a compound (lane pBR + Topo), and that relaxation was inhibited to varying and concentration-dependent extents following incubation with derivatives 9

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Fig. 8. Accumulation of studied derivatives 8a–8d in A549 cancer cells after 24 h co-cultivation. Representative microphotographs of A549 cancer cells cultivated only in medium (control) (A–C) or in medium containing derivative 8a (D–F), 8b (G–I), 8c (J–L) or 8d (M–O). Scale bar = 100 µm.

level of efficacy. The results obtained from the MTT assays were also used to determine the IC50 values of the tested derivatives and these values are listed in Table 2. Compound 8d (4.25 × 10−6 M, after 48 h) was found to be the most effective agent against the A549 cancer cell line, while compound 8b was found to be ineffective (Table 2). The cisplatin was used as control. The results from the cell cycle distribution assay showed a concentration-dependent effect of derivative 8a in inducing an increase in the accumulation of cells in the G2/M phase. The onset of accumulation was accompanied with a decrease in the G0/G1 phase of cell cycle. No significant changes were observed in the case of cells treated with derivatives 8b, 8c and 8d (Table 3). The derivatives containing fluorine (compounds 8b and 8d) demonstrated no effect on cell viability at all concentrations of less than 50 × 10−6 M. On the other hand, the total cell number was decreased in the case of cells treated with derivatives 8a, 8c and 8d. The results from these two different types of analyses together with the findings of the metabolic activity assay (Fig. 5) indicate that while the treated cells

remain alive, they are no longer able to metabolize and proliferate. Clonogenic assay is a simple technique which identifies biological alteration leading to irreversible losses of proliferative capacity and thus the loss of a cells ability to form new colonies [35]. In order to test the effect of the studied compounds on colony formation or clonogenic ability, A549 cell line was treated with two different concentrations of these derivatives. As is shown in Fig. 6, a significant decrease in colony formation was observed in the presence of higher concentrations of all of the tested compounds. However, a similar effect in the case of derivative 8c and 8d at a concentration of 5 × 10−6 M was also detected. Flow cytometric analysis of the content of the derivatives in A549 cancer cells revealed an increased fluorescence in the case of derivatives 8a, 8b and 8c. For these derivatives, fluorescence was detected in all three channels (Fig. 7). The maximal level of fluorescence was detected in the case of derivative 8a 6 h after its addition to the cells. Later analyses performed 24 h after treatment with the derivative revealed that the fluorescence was lower, but still significant. A contrary trend was observed in the case of derivative 8b, in which maximal 10

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fluorescence was detected 24 h after its addition to the cells. The second highest level of fluorescence was observed in the case of derivative 8c 6 h after treatment of cells, however 24 h after treatment the fluorescence intensity decreased a little bit in ratio to control. In the case of derivative 8d, no significant changes in fluorescence were observed (Fig. 7). Based on morphological analysis of cells incubated with studied derivatives, we noticed altered morphology in cells incubated with 8c and 8d derivatives compared to control. Under normal conditions, human lung adenocarcinoma cells A549 possessed multipolar/fibroblastic cell shape (approx. 93–95% of all cells, depending on cell viability) (Fig. 8 - control A) and very low autofluorescence after excitation with blue light (Fig. 8 – control B). In cells incubated with 8a, cells retained their multipolar shape (approx. 92% cells) and the fluorescence signal was apparent in perinuclear space, indicating the more or less uniform accumulation of the derivate 8a in cell cytoplasm (Fig. 8a (D–F)). However, cells incubated with 8d tended to achieve the spherical shape (approx. 25% cells) (Fig. 8d (J-L)) and this tendency was even more obvious in cells incubated with 8b (approx. 64% cells) (Fig. 8b (G-I)) and 8c (approx. 76% cells) (Fig. 8c (J-L), indicating potential adverse effect of these derivatives to cellular metabolism and/ or functions. In 8b, 8c and 8d samples, we observed bright fluorescence signal in cell cytoplasm with uneven distribution predominantly at one pole of the cell body. This indicated the accumulation of the chemical compound in particular part of the cytoplasm. Based on the results of our densitometric analysis, we found that autofluorescence of control cells (cell line A549) is very low (MFI = 1.00 ± 0.09; measured in range: 0 = background, 100 = overexposed area). All the samples incubated with studied derivatives exhibited significantly higher fluorescence intensity (MFI 8a = 23.05 ± 1.69; MFI 8b = 34.49 ± 5.39; MFI 8c = 50.91 ± 3.13; MFI 8d = 36.82 ± 4.21) compared to control cells (P < 0.001; ANOVA followed by Tukey-Kramer post hoc test). Between experimental groups, differences in MFI were considered to be significant as follows: 8a vs 8d (P < 0.05); 8a vs 8c (P < 0.001); 8b vs 8c (P < 0.01) and 8c vs 8d (P < 0.05).

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4. Conclusion In summary, this study offers a deeper insight into the mode of action of a novel series compounds incorporating fluorine substituents with important macromolecule (DNA). Our study has described the antiproliferative effect of the new compounds against human lung carcinoma cell line A549 and its inhibition of topoisomerase action. The interaction of the new fluorinated acridine derivatives with ctDNA has also been studied and analyzed using a UV–Vis technique. The results of the binding studies suggest that these small molecules bind to DNA through intercalation. The results of our UV–Vis absorption spectroscopy prove that the binding of derivatives 8a–8d has occurred with a binding constant value K = 1.22–2.28 × 106 M−1. Our results indicate that derivatives 8a, 8b and 8c act as an effective Topo I inhibitors and compound 8d as a partial Topo II inhibitor at concentrations 30 × 10−6 M and higher. From a biological point of view, derivative 8d was found to be the most effective against human lung carcinoma cell line A549. Even at the relatively low concentration of 5 × 10−6 M, the derivatives were found to have decreased cell metabolism and reduced the ability of the cancer cells to form new colonies. Contemporary strategies for introducing fluorine atoms into molecules are centered on attempts to increase binding affinity, alter physiochemical properties and improve anticancer activity. Compounds 8b and 8d increased binding affinity (K = 2.32 and 2.28 × 106 M−1, respectively), altered physiochemical properties and improved anticancer activity.

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