Synthesis and evaluation of anticancer activity of new 9-acridinyl amino acid derivatives

Compounds 6, 7, 8 and 9 exhibit lower or similar IC₅₀ values compared to amsacrine and showed a lack of toxicity towards unstimulated normal human leucocytes.

Abstract

A series of eleven 9-acridinyl amino acid derivatives were synthesized using a two-step procedure. Cytotoxicity was tested on the K562 and A549 cancer cell lines and normal diploid cell line MRC5 using the MTT assay. Compounds 6, 7, 8 and 9 were the most active, with IC₅₀ values comparable to or lower than that of chemotherapeutic agent amsacrine. 8 and 9 were especially effective in the A549 cell line (IC₅₀ ≈ 6 μM), which is of special interest since amsacrine is not sufficiently active in lung cancer patients. Cell cycle analysis revealed that 7 and 9 caused G2/M block, amsacrine caused arrest in the S phase, while 6 and 8 induced apoptotic cell death independently of the cell cycle regulation. In comparison to amsacrine, 6, 7, 8, and 9 showed similar inhibitory potential towards topoisomerase II, whereas only 7 showed DNA intercalation properties. In contrast to amsacrine, 6, 7, 8 and 9 showed a lack of toxicity towards unstimulated normal human leucocytes.

Introduction

Acridine analogues constitute a class of compounds with anti-inflammatory, anticancer, antimicrobial, antitubercular, antiparasitic, antimalarial, antiviral and fungicidal activities. Two major considerations for the use of acridines as potential anticancer compounds are the effectiveness of their semi-planar heterocyclic structure to intercalate between base pairs in the double-stranded DNA structure and the redox properties of acridine.1–4 Additionally, acridine derivatives prevent proper functioning of tumor cells by inhibition of the enzymes that control the topology of DNA: topoisomerase, telomerase and cyclin-dependent kinases.5–7 Acridine derivatives are also widely considered as potent photosensitizers for photodynamic therapy in tumour treatment.8

To date, a variety of acridine derivatives have been synthesized and tested for antitumor activity. Amsacrine (m-AMSA) was the first synthetic topoisomerase II poison approved for clinical use. It is still used in the treatment of acute leukemia and Hodgkin's and non-Hodgkin's lymphoma, but without significant activity against solid tumors. However, the side effects, development of resistance and low bioavailability limit its use. Therefore, a lot of effort has been made by synthetic chemists to produce new derivatives with improved antitumor activity.9,10 Other acridine analogues, such as N-(2-(dimethylamino)ethyl)acridine-4-carboxamide (DACA) and triazoloacridone, showed good antitumor efficacy and entered clinical studies (Fig. 1).11–14

Fig. 1. Chemical structures of some reported acridines with antitumor activity.

Previously performed studies showed that various acridinyl amino acid derivatives displayed good antitumor activity. Gellerman described the synthesis of N-substituted 9-aminoacridine and bis-acridine derivatives containing electron-withdrawing or electron-donating groups, including amino acid residues.15 These derivatives showed good antitumor activity in comparison with commercial 9-aminoacridine and m-amsacrine, probably due to enhancement of biologically important chelating properties leading to formation of more powerful DNA damaging reactive species. Singh and co-workers presented the synthesis of amino acid derivatives of acridone as potential leads to anticancer drugs,16 while Gao and co-workers developed amino acid acridone analogues possessing a 3,5-dimethoxyphenyl moiety.17,18 According to these results, cell death can be caused by multiple mechanisms, such as inhibition of aerobic glycolysis, mitochondrial oxidative phosphorylation, DNA damage and oxidative stress. Lyakhov et al. synthesized a series of 9-acridine amino acid derivatives and showed that the antiproliferative activity of these compounds depends on the selected amino acid and its chain length (Fig. 1).19

The aims of this study were the synthesis of novel acridinyl amino acid derivatives with improved anticancer activity and lower toxicity in comparison to amsacrine, and investigation of underlying mechanisms of their action.

Results and discussion

Design and synthesis

Compounds tested in this study were designed in accordance with the results of antiproliferative activity of the 9-acridinyl amino acid derivatives synthesized by Lyakhov et al.19 These authors tested the activity on cell division and cell growth of five compounds containing aliphatic amino acids (linear side chains) using cucumber roots and observed a significant influence of the side chain length on inhibition of cell growth. Therefore, acridines containing amino acids as a side chain are promising candidates, but attention should be paid to the selection of amino acids. In our study, the following compounds were designed: compounds with side chain lengths most similar to those with the highest reported activity (compounds 6, 8 and 9, Scheme 1), compounds with longer side chains and higher lipophilicity (compounds 5, 7, 10 and 11, Scheme 1), and compounds with the methyl ester of aromatic amino acids as the side chain (compounds 1–4, Scheme 1, because the influence of this type of amino acid was not evaluated before).

Scheme 1. Chemical structures and synthesis of 9-acridinyl amino acid derivatives.

Compounds 1–11 were synthesized using a modified procedure presented by Lyakhov et al.19 In contrast to this procedure, a one-pot synthesis was performed, without the isolation of the expected 9-alkoxy intermediate. In the first step, a solution of 9-chloroacridine in alcohol was mixed with a previously prepared solution of alkoxide (a solution of sodium methoxide in methanol, sodium ethoxide in ethanol or sodium propoxide in propanol) and refluxed for 2.5 hours. In the second step, the corresponding amino acid was added into the reaction mixture and reflux was continued for the next 4 hours. Additionally, a solution of alkoxide in alcohol was prepared in accordance with the amino acid ester used in the second step in order to prevent transesterification. This procedure was applied because products were obtained in higher yields when compared to the direct reaction between an amino acid and 9-chloroacridine in a basic environment. Synthesis and chemical structures of the synthesized compounds are presented in Scheme 1.

In vitro anticancer activity

Cytotoxicity

Effects of compounds 1–11 on cell viability were examined and compared to those of amsacrine – an anilinoacridine with known antileukemic activity.20 Compounds were tested in the K562 (chronic myelogenous leukemia) and A549 (lung epithelial carcinoma) cancer cell lines, as well as in normal diploid cell line MRC5 (lung fetal fibroblasts). Cells were treated in the micromolar range for 72 h and viability was measured using a tetrazolium based MTT assay.21 The MTT assay is an indicator of cellular metabolic activity. IC₅₀ values (Table 1) were defined as a 50% decrease in the capability of reducing MTT dye compared to that of dimethyl sulfoxide (DMSO) treated cells. Compounds 6, 7, 8 and 9 were the most potent inhibitors in the MTT assay, with IC₅₀ values below 20 μM, comparable to or lower than that of amsacrine. Compounds 8 and 9 were especially effective in the A549 cell line (IC₅₀ ≈ 6 μM) (Fig. 2). This is of special interest, as amsacrine itself did not have sufficient antitumor activity in lung cancer patients.22 As with other non-targeted chemotherapeutics, the tested compounds were also toxic towards proliferating normal MRC5 cells, although with higher IC₅₀ values. Many cancer therapeutics are used despite their toxicity in the clinic, and amsacrine has been shown to decrease white blood cell count.22,23 Therefore, we tested the toxicity of compounds 6–9 towards unstimulated normal human leucocytes. Leucocytes prepared from human blood were treated with increasing concentrations of compounds 6, 7, 8 and 9 and compared to those with amsacrine in an MTT assay after 24 h or 72 h of treatment (Fig. 3). While amsacrine was toxic in a dose- and time-dependent manner, the tested compounds showed a lack of toxicity towards leucocytes. This was especially prominent for compounds 8 and 9. Given the low IC₅₀ values of compounds 8 and 9 in the A549 cell line, this finding makes them the most promising agents from the group.

Table 1. IC₅₀ values of amsacrine and compounds 1–11.

Compound	K562	A549	MRC5
Amsacrine	13.8 ± 8.0 a	22.2 ± 2.8	15.4 ± 2.6
1	40.6 ± 2.1	25.7 ± 4.9	47.6 ± 4.4
2	46.1 ± 2.0	34.3 ± 9.2	65.1 ± 11.2
3	46.6 ± 3.7	29.5 ± 1.6	56.1 ± 9.6
4	42.4 ± 1.4	34.1 ± 1.0	46.95 ± 14.3
5	62.9 ± 11.8	62.6 ± 1.1	49.8 ± 6.5
6	11.2 ± 0.4	9.5 ± 0.9	15.8 ± 3.2
7	21.8 ± 5.4	19.3 ± 4.0	16.9 ± 1.7
8	19.2 ± 2.4	6.15 ± 0.6	12.1 ± 1.6
9	16.4 ± 2.5	6.3 ± 0.2	11.6 ± 1.3
10	34.5 ± 4.8	36.3 ± 2.0	25.1 ± 3.2
11	75.0 ± 8.0	72.4 ± 1.8	58.8 ± 9.1

^aIC₅₀ ± SD [μM].

Fig. 2. Survival curves for A549, K562 and MRC5 cells treated with increasing concentrations of amsacrine or compounds 6, 7, 8 and 9 for 72 h.

Fig. 3. Survival curves for normal human leucocytes treated with increasing concentrations of amsacrine or compounds 6, 7, 8 and 9 for 24 h (A) or 72 h (B).

Cytotoxicity of the most potent compounds (6, 7, 8 and 9) was also confirmed in the long-term survival assay in the A549 cell line. At MTT-based IC₅₀ values, the tested compounds decreased colony growth by more than 95%, compared to that of DMSO treated cells (Fig. 4).

Fig. 4. Bright-field images of A549 cells ten days after the exposure to compounds 6, 7, 8 and 9 (A) and quantification of the colony growth from two independent experiments (B).

Derivatives with linear aliphatic side chains (5–11) showed either good (6–9) or poor (5 and 11) activity, which is in accordance with the results obtained by Lyakhov et al.19 for similar compounds. Differences in side chain length might affect both pharmacokinetic and pharmacodynamic properties and very long side chains (5 and 11) are unfavorable for antiproliferative activity.

Cell cycle analysis

In order to test whether the most potent compounds 6–9 induce the inhibition of cell viability through modulation of the cell cycle progression, flow cytometry analysis of the cell cycle distribution was performed with propidium iodide stain. Cell cycle checkpoints help ensure the accuracy of DNA replication and division.24 The cell-cycle DNA damage checkpoints occur late in the G1 phase, which prevents entry to the S phase, and late in the G2 phase, which prevents entry to mitosis, and can arrest the cell cycle in response to DNA damage to allow time for DNA repair.25 The A549 cell line was chosen as a model system, as it had higher sensitivity to compounds 6–9 than to amsacrine. Amsacrine was originally designed to intercalate into DNA through the acridine moiety, but is historically significant as the first topoisomerase II poison,26 inducing a block in the S phase of the cell cycle. The effects of compounds 6–9 were analyzed at MTT-based IC₅₀ values after 24 h and 48 h of treatment (Fig. 5). Amsacrine treatment, as expected, caused arrest in the S phase (at 24 h, Fig. 5A) and subsequent apoptosis, detectable as an increase in the sub G1 phase at 48 h (late apoptotic cells with fragmented DNA content, Fig. 5B). Compound 6 had no effect on the cell cycle distribution after 24 h or 48 h of treatment. Compound 7 caused accumulation in the G2/M phase, with 18% and 29% of cells compared to 9% and 11% of control cells, at 24 h and 48 h, respectively. G2/M accumulation was also present for cells treated with compound 9 although to a lesser extent (12% and 19% of cells, at 24 h and 48 h, respectively). Compound 8 did not induce significant changes in the cell cycle distribution. In conclusion, for compounds 7 and 9, the observed decrease in cell viability could be explained by the G2/M block in the cell cycle progression. Generally, G2/M block was observed for acridines or other classes of compounds acting either as topoisomerase inhibitors or topoisomerase poisons, which can also involve other targets and mechanisms.27–32

Fig. 5. Cell cycle analysis of A549 cells treated with amsacrine or compounds 6, 7, 8 and 9. Quantification of phase distribution after 24 and 48 hours of treatment from three independent experiments (A and B) and representative graphs at 48 h (C).

Cell death analysis

The potential of compounds 6, 7, 8, and 9 and amsacrine to induce apoptosis of A549 cells was analyzed after 24 h and 48 h of treatment with MTT-based IC₅₀ concentrations by flow cytometry with Annexin V-FITC/propidium iodide staining. All the tested compounds significantly increased the percentage of apoptotic cells (one-way ANOVA), with compounds 6 and 7 being comparable to amsacrine (Fig. 6). For compound 6, which did not induce changes in the cell cycle distribution, induction of apoptosis seems to be the main cause of the decrease of cell viability. Compounds 8 and 9 induced apoptosis, although to a lesser extent than the other tested compounds. Nevertheless, these results are of importance as compounds 8 and 9 were used at concentrations more than 3 times lower than that of amsacrine, and as described above, have the best toxicity profile.

Fig. 6. Cell death analysis of A549 cells treated with amsacrine or compounds 6, 7, 8 and 9. Quantification from three independent experiments: one-way ANOVA with Dunnett's multiple comparison test (a) and representative graphs after 48 h of treatment (B).

Electrophoretic mobility shift assay

To examine the potential of compounds 6, 7, 8, and 9 and amsacrine to directly interact with double stranded DNA, the electrophoretic mobility shift assay was performed. Whole genomic DNA was isolated from A549 cells and 0.5 μg of DNA was loaded for 1 h with 100 μM tested compounds in 5 mM Tris-HCl/50 mM NaCl buffer. While all the tested compounds showed some binding to DNA, amsacrine, a known intercalating agent, induced a strong shift in DNA mobility in the agarose gel, as well as compound 7, implying its DNA intercalating properties (Fig. 7).

Fig. 7. Electrophoretic mobility shift assay. A549 DNA mobility in 0.8% agarose gel in the presence of amsacrine (amsa), 6, 7, 8 and 9.

Determination of inhibitory activities on human DNA topoisomerase IIα

The results of activity of compounds 6, 7, 8 and 9 against human topoisomerase IIα are presented in Table 2 and compared to that of amsacrine.

Table 2. IC₅₀ values of amsacrine and compounds 6, 7, 8, and 9.

Compound	IC50 a
Amsacrine	16 ± 8
6	14 ± 5
7	16 ± 7
8	13 ± 9
9	14 ± 5

^aμM.

All tested compounds exhibit similar inhibitory effects on topoisomerase IIα (IC₅₀ values 13–16 μM) in comparison to amsacrine (IC₅₀ value 16 μM). Amsacrine intercalates into DNA due to the presence of an acridine ring, whereas its side chain is responsible for the inhibition of topoisomerase II. As a result, ternary complex amsacrine–DNA–topoisomerase II is formed. This was confirmed by Chourpa and Manfait using surface-enhanced Raman scattering (SERS) spectroscopy and the same authors also proved that amsacrine can interact with topoisomerase II alone.33 This could be used to explain the ability of compounds 6, 8 and 9 to inhibit topoisomerase II despite the lack of DNA intercalating properties.

Conclusions

A series of eleven 9-acridinyl amino acid derivatives were synthesized using a modification of a previously published two-step procedure.19 Compounds 6, 7, 8 and 9 were the most potent viability inhibitors in cancer cells, in particular compounds 8 and 9 in the A549 lung epithelial carcinoma cell line (IC₅₀ ≈ 6 μM). Importantly, in contrast to amsacrine, compounds 6, 7, 8 and 9 showed a lack of toxicity towards unstimulated normal human leucocytes. Cell cycle analysis revealed that compounds 7 and 9 caused G2/M block in A549 cells, while compounds 6 and 8 induced apoptotic cell death independent of the cell cycle regulation. In comparison to amsacrine, compounds 6, 7, 8, and 9 showed similar inhibitory potential towards topoisomerase IIα, whereas only compound 7 showed DNA intercalation properties. Finally, favourable pharmacokinetic and druglikeness properties (ESI†), lack of toxicity towards unstimulated normal human leucocytes and a better activity profile than amsacrine make compounds 6, 7, 8 and 9 the best candidates for further investigations.

Experimental section

Synthesis of acridine derivatives – chemicals, equipment and general procedure

9-Chloroacridine (97%), 1-methyl-l-tryptophan (95%), ethyl 4-aminobutyrate hydrochloride (98%), thionyl chloride (97%), sodium pieces and diethyl ether (≥99.7%) were purchased from Sigma Aldrich (Steinheim, Germany). l-Phenylalanine methyl ester hydrochloride (98%), β-alanine ethyl ester hydrochloride (98%) and l-histidine methyl ester dihydrochloride (98%) were purchased from Acros Organics (Geel, Belgium). Chloroform (stabilised with amylene, ≥99%), ethyl acetate (for analysis, ≥99.8%) and methanol (extra dry, for synthesis) were obtained from Fisher Scientific (Loughborough, UK), whereas silica gel 60 GF₂₅₄ for preparative thin layer chromatography, hexane (anhydrous, 95%) and propanol (anhydrous, 99.7%) were obtained from Merck (Darmstadt, Germany). Absolute ethanol was purchased from Carlo Erba (Rodano, Italy), whereas ethyl 6-aminohexanoate (98%) was purchased from Alfa Aesar (Karlsruhe, Germany). 1-Methyl-l-tryptophan methyl ester hydrochloride, 4-aminobutyric acid propyl ester hydrochloride, 6-aminohexanoic acid propyl ester hydrochloride, 8-aminooctanoic acid methyl ester hydrochloride and β-alanine propyl ester hydrochloride were synthesized and physicochemically characterized in our laboratory (unpublished results). The synthesized compounds were structurally characterized by determining melting points and by spectroscopic methods (IR, NMR, MS/MS, HRMS). Melting points were determined on a Boetius PHMK 05 apparatus (Radebeul, Germany). IR spectra were recorded on a Nicolet iS10 (Thermo Fisher Scientific Inc., Madison, WI, USA), whereas NMR spectra were recorded on a BRUKER AVANCE III 400 NMR spectrometer (Bruker Biospin GmbH, Rheinstetten, Germany). MS/MS analyses were performed on a TSQ Quantum Access MAX mass spectrometer (Thermo Fisher Scientific Inc., Madison, WI, USA). Accurate masses were determined on an Orbitrap UHPLC-HRMS (Thermo Fisher Scientific Inc., Madison, WI, USA).

Sodium alkoxide solutions (sodium methoxide in methanol, sodium ethoxide in ethanol and sodium propoxide in propanol) were prepared by dissolving sodium (150 mg) in 25 mL of the corresponding alcohol at room temperature. 9-Chloroacridine (0.3 mmol, 1 eq.) was dissolved in alcohol (methanol, ethanol or propanol; 3 mL) and the corresponding sodium alkoxide solution (3 mL) was added. The reaction mixture was refluxed for 2.5 h, amino acid (0.42 mmol, 1.4 eq.) was added and the reaction mixture was refluxed for an additional 4 h (Scheme 1). The reaction mixture was evaporated to dryness, the residue was dissolved in a mixture of chloroform and methanol and purified by column or preparative thin layer chromatography. Details on synthesis and physicochemical characterization data of the synthesized compounds are presented in the ESI.† Purity of synthesized compounds was evaluated using the HPLC method (experimental details and chromatograms are presented in the ESI†) and ranged from 96.2% (compound 4) to 99.8% (compound 5).

Evaluation of anticancer activity

MTT assay

Two human cancer cell lines K562 (ATCC-CCL-243, chronic myelogenous leukemia) and A549 (ATCC-CCL-185, lung epithelial carcinoma) and one normal diploid cell line MRC5 (ATCC-CCL-171, lung fetal fibroblast) were obtained from the ATCC and maintained in RPMI-1640 medium (Sigma Aldrich) supplemented with 10% fetal bovine serum, penicillin (192 U mL^–1) and streptomycin (200 μg mL^–1). Cells were grown at 37 °C in 5% CO₂ and a humidified air atmosphere. The cytotoxicity of the tested compounds was determined with the 3-(4,5-dimethylthiazol-yl)-2,5-diphenyltetrazolium bromide (MTT, Sigma-Aldrich) assay. Cells were seeded into 96-well cell culture plates at a cell density of 7000 cells per well. After 24 h, cells were exposed to serial dilutions of amsacrine or the tested compounds in the 6.25 to 100 μM range. After an incubation period of 72 h, MTT solution was added to each well; after 4 h incubation, formazan crystals were dissolved in 10% sodium dodecyl sulfate and absorbances were measured at 570 nm on a Multiskan EX reader (Thermo Labsystems). IC₅₀ values (μM) were determined from the dose response curves as the concentration of the compound causing a 50% decrease in MTT reduction compared to the control (DMSO) using Prism 7 (GraphPad).

Preparation and treatment of leucocytes from human blood

Leucocytes were separated from whole heparinized blood of healthy volunteers by Histopaque®-1077 (Sigma Aldrich) density gradient centrifugation. Leucocytes collect at the plasma/Histopaque interphase and erytrocytes and granulocytes sediment to the bottom of the tube. Interphase cells were washed three times, then counted and resuspended in supplemented RPMI-1640 medium. Such prepared cells (150 000 per well) were seeded in 96-well microtiter plates. After 2 h, cells were exposed to serial dilutions of amsacrine or the tested compounds in the 6.25 to 100 μM range. After 24 h and 72 h, cell viability was analyzed in the MTT assay.

Long-term cell survival assay

10³ A549 cells per well were seeded in a 6-well plate format and treated for 48 h with selected compounds with IC₅₀ concentrations derived from the MTT assay. Cells were then washed and colonies were allowed to grow for 10 days, after which the cells were fixed with 4% formaldehyde and stained with 0.05% crystal violet. After photographing, the dye was extracted with 10% acetic acid and quantified at 570 nM on a Multiskan EX reader (Thermo Labsystems).

Cell cycle analysis

Cells were treated for 24 h with MTT-based IC₅₀ concentrations of compounds and quantitative analysis of the cell cycle phase distribution was performed by flow-cytometric analysis (Calibur Becton Dickinson flow cytometer and Cell Quest computer software). Shortly, the DNA in fixed RNaseA (1 mg mL^–1) treated A549 cells was stained with 400 mg mL^–1 propidium iodide (PI) and 20 000 events were counted.

Annexin V-FITC apoptotic assay

Quantitative analysis of apoptotic and necrotic cell death induced by the investigated compounds and amsacrine, as a reference compound, was performed using an Annexin V-FITC apoptosis detection kit, according to the manufacturer's instructions (BD Biosciences). Shortly, after 24 h or 48 h treatment, A549 cells were trypsinized, washed twice with ice-cold PBS and resuspended in 200 mL binding buffer (10 mM HEPES/NaOH pH 7.4, 140 mM NaCl, 2.5 mM CaCl₂). 2 × 10⁵ cells were stained with Annexin V-FITC and PI. After 15 min incubation at room temperature in the dark, cells were analyzed using a FACS Calibur Becton Dickinson flow cytometer and Cell Quest computer software.

Electrophoretic mobility shift assay

500 000 A549 cells were trypsinized and centrifuged at 1500 rpm for 10 minutes. Cell pellets were resuspended in 300 μL lysis buffer with 10% SDS and 20 mg ml^–1 proteinase K. After 1 h incubation at 56 °C, lysates were mixed with 100 μL of 6 M NaCl and briefly vortexed. Solutions were centrifuged for 3 minutes at 14 000 rpm and the supernatant containing DNA was transferred into a new tube. DNA was precipitated with absolute ethanol, washed twice with 70% ethanol and air dried. DNA was resuspended in distilled water and the concentration was determined on a BioSpec-nano spectrophotometer (Shimadzu Biotech). 0.5 μg of A549 DNA was incubated for 1 h at room temperature in 100 μM solution of the tested compounds in 5 mM Tris-HCl/50 mM NaCl buffer. DNA was loaded onto 0.8% agarose gel and separated for 4 h at 30 V. To visualize DNA bands, the gel was stained with GelRed nucleic acid gel stain (Biotium) for 10 minutes at room temperature.

Determination of inhibitory activities on human DNA topoisomerase IIα

Inhibitory activities were determined in an assay from Inspiralis on streptavidin-coated 96-well microtitre plates from Thermo Scientific Pierce. First, the plates were rehydrated with a buffer (20 mM Tris·HCl, 0.01% w/v BSA, 0.05% v/v Tween 20, 137 mM NaCl, pH 7.6) and then the biotinylated oligonucleotide was immobilized. After washing off the unbound oligonucleotide, the enzyme assay was performed. A reaction volume of 30 μL in buffer (50 mM Tris·HCl, 10 mM MgCl₂, 125 mM NaCl, 5 mM DTT, 0.1 μg mL^–1 albumin, 1 mM ATP, pH 7.5) contained 1.5 U of human DNA topoisomerase II, 0.75 μg of supercoiled pNO1 plasmid, and 3 μL of an inhibitor solution containing 10% DMSO and 0.008% Tween 20. Reaction solutions were incubated at 37 °C for 30 min. After that, TF buffer (50 mM NaOAc, 50 mM NaCl and 50 mM MgCl₂, pH 5.0) was added to terminate the enzymatic reaction. After additional incubation for 30 min at RT, during which the biotin–oligonucleotide–plasmid triplex was formed, the unbound plasmid was washed off using TF buffer and Diamond dye in T10 buffer (10 mM Tris·HCl, 1 mM EDTA, pH 8.0) was added. The fluorescence was measured with a microplate reader (BioTek Synergy H4, excitation: 485 nm, emission: 537 nm). Initial screening was done at 100 or 10 μM concentrations of inhibitors followed by IC₅₀ determination using seven concentrations of the tested compounds. GraphPad Prism 6 software was used to calculate the IC₅₀ values. The results are reported as the average value of two independent measurements. As the positive control, etoposide (IC₅₀ = 71 μM) was used.

Ethical statement

All experiments were performed in accordance with the Guidelines of Good Laboratory Practice (Sl. glasnik RS, 28/2008), and experiments were approved by the Ethics Committee of the Institute for Oncology and Radiology of Serbia (decision number 5665-01, 2014). Informed consents were obtained from human participants of this study.

Conflicts of interest

The authors declare no conflict of interest.