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

Article abstract of DOI:10.1016/j.bioorg.2019.103393

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

Full text of DOI:10.1016/j.bioorg.2019.103393

BioorganicChemistryxxx(xxxx)xxxx
Contents lists available at ScienceDirect
Bioorganic Chemistry
journal homepage: www.elsevier.com/locate/bioorg
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
b 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
A R T I C L E I N F O
A B S T R A C T
Keywords:
A series of new 3,6,9-trisubstituted acridine derivatives with fluorine substituents on phenyl ring were syn-
thesized 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 to-
poisomerase 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).
3,6,9-Trisubstituted acridine derivatives
Fluorine
calf thymus DNA
Spectroscopic techniques
topoisomerases I/II
Cytotoxicity
Lung carcinoma cells
1. Introduction
with some molecules and the damage which this induces may lead to a
wide range of pathological changes. Studying the interaction of phar-
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, anti-
bacterial, 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 combi-
nation of acridine and fluorine in one molecule may lead to the de-
velopment of new molecules with more active and effective qualities.
DNA are prone to alterations under various conditions; interaction
maceutical 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 inter-
action [13–21].
3,6-Diaminoacridines are able to intercalate between the base of
DNA and inhibit topoisomerase II [22]. Further research has also re-
vealed 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 in-
tegrity 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: maria.kozurkova@upjs.sk (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/©2019ElsevierInc.Allrightsreserved.
Pleasecitethisarticleas:PatrikNunhart,etal.,BioorganicChemistry,https://doi.org/10.1016/j.bioorg.2019.103393

P. Nunhart, et al.
BioorganicChemistryxxx(xxxx)xxxx
́
́
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].
2.1.2. Synthesis of 2,2,4,4–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.
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 in-
corporated fluorine substituents (derivatives 8b and 8d), while deri-
vatives 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.
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).
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.
2.1.4. 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].
2. Experimental
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).
2.1. 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
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].
(ctDNA)
were
obtained
from
Sigma-Aldrich
(Germany).
Ethylenediaminetetraacetic acid (EDTA), ribonuclease (RNase A), pro-
teinase K and sodium lauryl sulfate (SDS) were purchased from Serva
(Germany), plasmid pUC 19 (2761 bp, DH 5α), and agarose (type II No-
A-6877) (Sigma), Topoisomerase I, Topoisomerase II, tris(hydro-
xymethyl) 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.
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
1H (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 chro-
matography (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).
(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 pro-
duct 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 decomposi-
tion; 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 2,2,4,4–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

P. Nunhart, et al.
BioorganicChemistryxxx(xxxx)xxxx
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-9-
chloroacridine (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 decom-
position; 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, H-
3′,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 decomposi-
tion; 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, H-
3′,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)
2.1.13. 3,6–Diamino–9–[(4–fluorophenyl)amino]acridine hydrochloride
(8b)
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%.
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, H-
2′,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 decomposi-
tion; 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, H-
3′,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,
2.1.11. General procedure for the preparation of 3,6-diamino-9-[(aryl)
amino]acridine 8a–8c
C5), 50.0 (NHCH2Ph). 19F NMR (375 MHz, DMSO–d6)
δ 117.27
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.
(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%.
2.1.16. 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 eva-
poration of 2-propanol under reduced pressure, and finally distilled
3

P. Nunhart, et al.
BioorganicChemistryxxx(xxxx)xxxx
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.
containing a freshly prepared 5 × complete assay buffer, 0.2 μg super-
coiled 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].
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%.
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) supple-
mented with 10% fetal bovine serum (FBS; Biosera, Boussens, France)
2.2. DNA binding experiments
and antibiotics (1% Antibiotic-Antimycotic 100 × and 50 × 10−3
g
Stock solutions of all derivatives at a concentration of 1 × 10−2
M
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.
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.
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).
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
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 ex-
periments 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 sig-
moidal fit to the metabolic activity data using OriginPro 8.5.0 SR1
(OriginLab Corp., Northampton, MA, USA).
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 pro-
cessed using GraphPad Prism 6 software.
The binding constants K of the ligand-DNA complexes were calcu-
lated 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 nu-
cleotides. The binding data were fitted using Gnu Octave 2.1.73 soft-
ware [33].
For the assessment of total cell numbers and viability within in-
dividual experimental groups, floating and adherent cells were har-
vested 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, re-
suspended in a staining solution (0.1% Triton X-100, 0.137 g L−1 ri-
bonuclease 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 argon-
ion 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 Soft-
ware 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.
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 dec-
atenation 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
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 12-
well μ-Chamber slides (Ibidi GmbH, Martinsried, Germany) and left to
settle down for 24 h. The studied derivatives 8a–8d were added to the
Table 1
Spectral absorption characteristic of acridine compounds 8a–8d.
Compound
λfree
λbound
Δλ
Hypochromicity
K × 106
[nm]
[nm]
[nm]
[%]
[M−1
]
8a
8b
8c
8d
405
380
384
385
407
397
394
393
2
40.5
53.8
51.7
54.1
1.22
2.32
2.22
2.28
17
10
8
4

P. Nunhart, et al.
BioorganicChemistryxxx(xxxx)xxxx
Scheme 1. Synthesis of 3,6–diamino–9(10H)acridone (4).
Scheme 2. Synthesis of trisubstituted acridine derivatives 8a–d.
5

P. Nunhart, et al.
BioorganicChemistryxxx(xxxx)xxxx
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 ana-
lysis. 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 ana-
lysis 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).
6

P. Nunhart, et al.
BioorganicChemistryxxx(xxxx)xxxx
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 ac-
cordance with a previously described procedure based on diphe-
nylmethane (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

P. Nunhart, et al.
BioorganicChemistryxxx(xxxx)xxxx
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
S
G2
Control
0
55.82
51.04
34.69
54.81
58.60
63.52
54.34
59.69
55.89
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
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
8a
5
1.20***
1.51***
0.79
50
5
8b
8c
8d
50
5
0.66
0.92
50
5
2.86
0.43
50
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 cy-
clization of compound 3 provided the main synthon, 3,6–diaminoacri-
done 4.
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 hy-
drolysis of chlorine derivate 6 (Scheme 2).
The key intermediate, 6, was prepared through the reaction of
POCl3 with precursor 5, which was in turn obtained through the reac-
tion 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
3.2. UV–Vis absorption spectroscopy
The UV–Vis absorption spectra of the novel derivatives 8a–8d
8

P. Nunhart, et al.
BioorganicChemistryxxx(xxxx)xxxx
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 con-
centrations of ctDNA resulting in a tendency toward hypochromism. A
significant hypochromic shift (40.5–54.1%) was observed for deriva-
tives 8a–8d, a feature which is typical for intercalators. The extent of
the hypochromism is usually consistent with the strength of inter-
calative 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 va-
lues of binding constants are comparable to those of classical inter-
calators such as ethidium bromide (K = 1.4 × 106 M−1), which sug-
gests that the mode of interaction is intercalation. Spectral absorption
characteristics of the studied acridine compounds are presented in
Table 1 and Fig. 1.
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 cata-
lyzed 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 in-
hibitory effect was observed for any of the compounds, with the ex-
ception of compound 8d at 30 and 50 × 10−6 M.
The compounds with fluorine substituents were found to exhibit
increased levels of binding affinity. If we compare the values of hypo-
chromism 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 deri-
vatives 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.
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].
3.3. Topoisomerase I inhibition
Since most acridine derivatives generally also have good inhibitory
activity against TopoI/II, we also aimed to verified whether re-
presentative 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].
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 dose-
and time-dependent manner, with derivative 8d displaying the highest
The supercoil relaxation activity of pBR322 topoisomerase I in the
presence of varying concentrations (5, 10 and 30 × 10−6 M) of com-
pounds 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 con-
centration-dependent extents following incubation with derivatives
9

P. Nunhart, et al.
BioorganicChemistryxxx(xxxx)xxxx
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.
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 de-
rivative 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 deriva-
tives 8a, 8b and 8c. For these derivatives, fluorescence was detected in
all three channels (Fig. 7). The maximal level of fluorescence was de-
tected 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
The results from the cell cycle distribution assay showed a con-
centration-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 de-
rivatives 8b, 8c and 8d (Table 3).
The derivatives containing fluorine (compounds 8b and 8d) de-
monstrated 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
10

P. Nunhart, et al.
BioorganicChemistryxxx(xxxx)xxxx
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 fluores-
cence intensity decreased a little bit in ratio to control. In the case of
derivative 8d, no significant changes in fluorescence were observed
(Fig. 7).
Declaration of Competing Interest
The authors declare that there is no conflict of interest in this work.
Acknowledgements
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/fibro-
blastic cell shape (approx. 93–95% of all cells, depending on cell via-
bility) (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 fluores-
cence 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 sphe-
rical 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.
This study was supported by Vedecká grantová agentúra MŠVVaŠ
SR a SAV (VEGA) Grant No. 1/0016/18, UHHK 00179906 and Medical
University Science Park in Košice (MediPark, Košice - Phase II)
ITMS2014+: 313011D103.
References
[1] D. Sabolová, P. Kristian, M. Kožurková, Proflavine/acriflavine derivatives with
versatile biological activities, J. Appl. Toxicol. 39 (2019), https://doi.org/10.1002/
jat.3818.
[2] J.W. Pridgeon, P.H. Klesius, M. Yildirim-Aksoy, Attempt to develop live attenuated
bacterial vaccines by selecting resistance to gossypol, proflavine hemisulfate, no-
vobiocin, or ciprofloxacin, Vaccine 31 (2013) 2222–2230, https://doi.org/doi:10.
1016/j.vaccine. 2013.03.004.
[3] P. Prasher, M. Sharma, Medicinal chemistry of acridine and its analogues,
MedChemComm. 9 (2018) 1589–1618, https://doi.org/10.1039/c8md00384j.
[4] L. Janovec, D. Sabolova, M. Kozurkova, H. Paulikova, P. Kristian, J. Ungvarsky, E.
Moravcikova, M. Bajdichova, D. Podhradsky, J. Imrich, Synthesis, DNA interaction,
and cytotoxic activity of a novel proflavine-dithiazolidinone pharmacophore,
Bioconjug. Chem. 18 (2007) 93–100, https://doi.org/ 10.1021/bc060168v.
[5] M. Kozurkova, D. Sabolova, L. Janovec, J. Mikes, J. Koval’, J. Ungvarsky,
M. Stefanisinova, P. Fedorocko, P. Kristian, J. Imrich, Cytotoxic activity of pro-
flavine diureas: synthesis, antitumor, evaluation and DNA binding properties of
1′,1″-(acridin-3,6-diyl)-3′,3″-dialkyldiureas, Bioorg. Med. Chem. 16 (2008)
3976–3984, https://doi.org/10.1016/j.bmc.2008.01.026.
Based on the results of our densitometric analysis, we found that
autofluorescence of control cells (cell line A549) is very low
[6] M.K. Gatasheh, S. Kannan, K. Hemalatha, N. Imrana, Proflavine an acridine DNA
intercalating agent and strong antimicrobial possessing potential properties of
carcinogen, Karbala Int. J. Mod. Sci. 3 (2017) 272–278, https://doi.org/10.1016/j.
kijoms.2017.07.003.
(MFI = 1.00
0.09;
measured
in
range:
0 = background,
100 = overexposed area). All the samples incubated with studied de-
rivatives exhibited significantly higher fluorescence intensity (MFI
[7] J. Ježek, J. Hlaváček, J. Šebestík, Biomedical Applications of Acridines: Derivatives,
Syntheses, Properties and Biological Activities with a Focus on Neurodegenerative
Diseases, Springer, 2017.
8a = 23.05
8c = 50.91
1.69;
MFI
8b = 34.49
5.39;
MFI
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).
[8] K. Haranahalli, T. Honda, I. Ojima, Recent progress in the strategic incorporation of
fluorine into medicinally active compounds, J. Fluor. Chem. 217 (2019) 29–40,
https://doi.org/10.1016/j.jfluchem.2018.11.002.
[9] I. Ojima, Fluorine in Medicinal Chemistry and Chemical Biology, John Wiley &
Sons, 2009.
[10] K. Müller, C. Faeh, F. Diederich, Fluorine in pharmaceuticals: looking beyond in-
tuition, Science 317 (2007) 1881–1886, https://doi.org/10.1126/science.1131943.
[11] J.-P. Begue, D. Bonnet-Delpon, Recent advances (1995–2005) in fluorinated phar-
maceuticals based on natural products, J. Fluor. Chem. 127 (2006) 992–1012,
https://doi.org/10.1016/j.jfluchem.2006.05.006.
4. Conclusion
[12] J. Wang, M. Sánchez-Roselló, J.L. Aceña, C. del Pozo, A.E. Sorochinsky, S. Fustero,
V.A. Soloshonok, H. Liu, Fluorine in pharmaceutical industry: fluorine-containing
drugs introduced to the market in the last decade (2001–2011), Chem. Rev. 114
(2013) 2432–2506, https://doi.org/10.1021/cr4002879.
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 spectro-
scopy 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.
[13] A. Rescifina, C. Zagni, M.G. Varrica, V. Pistara, A. Corsaro, Recent advances in small
organic molecules as DNA intercalating agents: synthesis, activity, and modeling,
Eur. J. Med. Chem. 74 (2014) 95–115, https://doi.org/ 0.1016/j.ejmech.2013.11.
029.
[14] M. Sirajuddin, S. Ali, A. Badshah, Drug-DNA interactions and their study by
UV–visible, fluorescence spectroscopies and cyclic voltametry, J. Photochem.
Photobiol. B Biol. 124 (2013) 1–19, https://doi.org/10.1016/j.jphotobiol.2013.03.
013.
[15] J. Plsikova, L. Janovec, J. Koval, J. Ungvarsky, J. Mikes, R. Jendzelovsky,
P. Fedorocko, J. Imrich, P. Kristian, J. Kasparkova, V. Brabec, M. Kozurkova, 3,6-bis
(3-alkylguanidino) acridines as DNA-intercalating antitumor agents, Eur. J. Med.
Chem. 57 (2012) 283–295, https://doi.org/10.1016/j.ejmech.2012.09.020.
[16] M. Misiak, F. Mantegazza, G. L Beretta, Methods for elucidation of DNA-anticancer
drug interactions and their applications in the development of new drugs, Curr.
Pharm. Des. 22 (2016) 6596–6611, https://doi.org/10.2174/
1381612822666160831114622.
[17] M. Tertis, M. Bucsa, G. Mesesan, A. Pirnau, C. Floare, R. Sandulescu, C. Cristea,
DNA intercalating ability of four acridine-N-oxides derivatives investigated by
spectral and electrochemical techniques, Farmacia 66 (2018) 688–692, https://doi.
org/10.34925/ farmacia.2018.4.19.
[18] J. Ježek, J. Hlaváček, J. Sebestík, Biomedical application of acridines, Prog. Drug
Res. (2017) ISBN 978-3-319-63953-6.
Contemporary strategies for introducing fluorine atoms into mole-
cules are centered on attempts to increase binding affinity, alter phy-
siochemical properties and improve anticancer activity. Compounds 8b
and 8d increased binding affinity (K = 2.32 and 2.28 × 106 M−1, re-
spectively), altered physiochemical properties and improved anticancer
activity.
[19] E.A. Lafayette, S.M. Vitalino de Almeida, M.G. da R. Pitta, E.I. Carneiro Beltrao, T.
G. da Silva, R. Olimpio de Moura, I. da R. Pitta, L.B.J. de Carvalho, M. do C.A. de
Lima, Synthesis, DNA binding and topoisomerase I inhibition activity of thiazacri-
dine and imidazacridine derivatives, Molecules 18 (2013) 15035–15050, https://
doi.org/ 10.3390/ molecules181215035.
[20] M. de M Silva, T.S. Macedo, H.M.P. Teixeira, D.R.M. Moreira, M.B.P. Soares, A.L. da
C Pereira, V. de L Serafim, F.J.B. Mendonca-Junior, M. do Carmo A de Lima, R.O. de
Moura, E.F. da Silva-Junior, J.X. de Araujo-Junior, M.D. de A Dantas, E. de O.O
11

P. Nunhart, et al.
BioorganicChemistryxxx(xxxx)xxxx
Nascimento, T.M.S. Maciel, T.M. de Aquino, I.M. Figueiredo, J.C.C. Santos,
topoisomerase inhibition: how can these mechanisms be explored to design more
specific anticancer agents? Biomed. Pharmacother. 96 (2017) 1538–1556 https://
doi.org/0.1016/j.biopha. 2017.11.054.
Correlation between DNA/HSA-interactions and antimalarial activity of acridine
derivatives: proposing a possible mechanism of action, J. Photochem. Photobiol. B
189 (2018) 165–175, https://doi.org/10.1016/j.jphotobiol.2018.10.016.
[21] M. Sakthi, A. Ramu, Synthesis, structure, DNA/BSA binding and antibacterial stu-
dies of NNO tridentate Schiff base metal complexes, J. Mol. Struct. 1149 (2017)
727–735, https://doi.org/10.1016/j.molstruc.2017.08.040.
[28] G. Capranico, J. Marinello, G. Chillemi, Type I DNA topoisomerases, J. Med. Chem.
60 (2017) 2169–2192, https://doi.org/10.1021/acs.jmedchem.6b00966.
[29] M.K. Kathiravan, M.M. Khilare, K. Nikoomanesh, A.S. Chothe, K.S. Jain,
Topoisomerase as target for antibacterial and anticancer drug discovery, J. Enzyme
Inhib. Med. Chem. 28 (2013) 419–435, https://doi.org/10.3109/14756366.2012.
658785.
[22] A. Mansouri, D. Haouzi, V. Descatoire, C. Demeilliers, A. Sutton, N. Vadrot, B.
Fromenty, G. Feldmann, D. Pessayre, A. Berson, Tacrine inhibits topoisomerases
and DNA synthesis to cause mitochondrial DNA depletion and apoptosis in mouse
liver, Hepatology. 38 (2003) 715–725, https://doi.org/10.1053/jhep.2003.50353.
[23] J. Janockova, J. Korabecny, J. Plsikova, K. Babkova, E. Konkolova, D. Kucerova, J.
Vargova, J. Koval, R. Jendzelovsky, P. Fedorocko, In vitro investigating of antic-
ancer activity of new 7-MEOTA-tacrine heterodimers, J. Enzyme Inhib. Med. Chem.
34 (2019) 877–897, https://doi.org/10.1080/14756366.2019.1593159.
[24] J. Janočková, J. Plšíková, R. Jendželovský, J. Mikeš, J. Kašpárková, V. Brabec,
S. Hamuľaková, P. Fedoročko, M. Kožurková, Tacrine derivatives as dual topoi-
somerase I and II catalytic inhibitors, Bioorg. Chem. 59 (2015) 168–176, https://
doi.org/10.1016/j.bioorg.2015.03.002.
[30] Mayur Y.C., Zaheeruddin, Peters G.J., Lemos C., Kathmann I., Prasad V.V. Synthesis
of 2-fluoro N(10)-substituted acridones and their cytotoxicity studies in sensitive
and resistant cancer cell lines and their DNA intercalation studies. Arch. Pharm. 342
(2009) 640–650, https://doi.org/10.1002/ardp.200900046.
[31] M. Matejova, L. Janovec, J. Imrich, Study of the synthesis of novel trisubstituted
acridines, Arkivoc (2015) 134–152, https://doi.org/10.3998/ark.5550190.p009.
033.
[32] J.D. McGhee, P.H. von Hippel, Theoretical aspects of DNA-protein interactions: co-
operative and non-co-operative binding of large ligands to a one-dimensional
homogeneous lattice, J. Mol. Biol. 86 (1974) 469–489, https://doi.org/10.1016/
0022-2836(74)90031-x.
[25] R.G. Gouveia, A.G. Ribeiro, M.A.S.P. Segundo, J.F. de Oliveira, M. do C.A. de Lima,
T.R.C. de Lima Souza, S.M.V. de Almeida, R.O. de Moura, Synthesis, DNA and
protein interactions and human topoisomerase inhibition of novel spiroacridine
derivatives, Bioorg. Med. Chem. 26 (2018) 5911–5921, https://doi.org/ 10.1016/j.
bmc.2018.10.038.
[33] J. Buša, GnuOctave, FEI Technical University in Košice, Košice, 2006.
[34] M. Los, C.J. Burek, C. Stroh, K. Benedyk, H. Hug, A. Mackiewicz, Anticancer drugs
of tomorrow: apoptotic pathways as targets for drug design, Drug Discov. Today 8
(2003) 67–77, https://doi.org/10.1016/S1359-6446(02)02563-1.
[35] K. Buch, T. Peters, T. Nawroth, M. Sänger, H. Schmidberger, P. Langguth,
Determination of cell survival after irradiation via clonogenic assay versus multiple
MTT assay. A comparative study, Rad. Oncol. 7 (2012) 1–6, https://doi.org/10.
1186/1748-717X-7-1.
[26] W. Zhang, B. Zhang, W. Zhang, T. Yang, N. Wang, C. Gao, C. Tan, H. Liu, Y. Jiang,
Synthesis and antiproliferative activity of 9-benzylamino-6-chloro-2-methoxy-acri-
dine derivatives as potent DNA-binding ligands and topoisomerase II inhibitors,
Eur. J. Med. Chem. 116 (2016) 59–70, https://doi.org/ 10.1016/j.ejmech.2016.03.
066.
[36] A. Kaplan, G.A. Ciftci, H.M. Kutlu, The apoptotic and genomic studies on A549 cell
line induced by silver nitrate, Tumor Biol. 39 (2017) 1–12, https://doi.org/10.
1177/1010428317695033.
[27] S.M.V. de Almeida, A.G. Ribeiro, G.C. de Lima Silva, J.E.F. Alves, E.I.C. Beltrão,
J.F. de Oliveira, L.B. de Carvalho, M. do Carmo Alves de Lima, DNA binding and
12