3-[(E)-(acridin-9′-ylmethylidene)amino]-1-substituted thioureas and their biological activity

Article abstract of DOI:10.1016/j.saa.2017.03.014

This paper describes the synthesis of a novel series of acridine thiosemicarbazones through a two-step reaction between various isothiocyanates and hydrazine followed by treatment with acridin-9-carbaldehyde. The properties of this series of seven new derivatives are studied using NMR and biochemical techniques, and the DNA-binding properties of the compounds are determined using spectrophotometric studies (UV–vis absorption, fluorescence, and circular/linear dichroism) and viscometry. The binding constants K are estimated as being in the range of 2.2 to 7.8?×?10⁴?M^??1 and the percentage of hypochromism was found to be 22.11–49.75% (from UV–vis spectral titration). Electrophoretic experiments prove that the novel compounds demonstrate moderate inhibitory effects against Topo I activity at a concentration of 60?×?10^??6?M.

Full text of DOI:10.1016/j.saa.2017.03.014

Accepted Manuscript
3
-[(E)-(acridin-9′-ylmethylidene)amino]-1-substituted thioureas
and their biological activity
Michal Bečka, Mária Vilková, Othman Salem, Jana Kašpárková,
Viktor Brabec, Mária Kožurková
PII:
S1386-1425(17)30182-8
doi: 10.1016/j.saa.2017.03.014
SAA 14993
DOI:
Reference:
Spectrochimica Acta Part A: Molecular and Biomolecular
Spectroscopy
To appear in:
Received date:
Revised date:
Accepted date:
18 November 2016
8 February 2017
4 March 2017
Please cite this article as: Michal Bečka, Mária Vilková, Othman Salem, Jana Kašpárková,
Viktor Brabec, Mária Kožurková , 3-[(E)-(acridin-9′-ylmethylidene)amino]-1-substituted
thioureas and their biological activity. The address for the corresponding author was
captured as affiliation for all authors. Please check if appropriate. Saa(2017), doi: 10.1016/
j.saa.2017.03.014
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ACCEPTED MANUSCRIPT
3
-[(E)-(acridin-9'-ylmethylidene)amino]-1-substituted thioureas and their
biological activity
1
1
2
3
3
Michal Bečka , Mária Vilková , Othman Salem , Jana Kašpárková , Viktor Brabec , Mária
2
,4,*
Koţurková
1
2
Department of Organic Chemistry, Department of Biochemistry, Institute of Chemistry, Faculty of Science,
P.J.Šafárik University, Moyzesova 11, Košice, Slovak Republic;
3
4
Department of Biophysics, Faculty of Science, Palacky University, Slechtitelu 27, 783 71 Olomouc; Biomedical
Research Center, University Hospital Hradec Kralove, Sokolovska 581, Hradec Kralove, Czech Republic
*
Correspondence: maria.kozurkova@upjs.sk; Tel.: +421-904-577-04
Keywords
Acridine, Thiosemicarbazone, DNA-binding, Topoisomerase I, Spectroscopic study
This paper describes the synthesis of a novel series of acridine thiosemicarbazones through a
two-step reaction between various isothiocyanates and hydrazine followed by treatment with
acridin-9-carbaldehyde. The properties of this series of seven new derivatives are studied
using NMR and biochemical techniques, and the DNA-binding properties of the compounds
are determined using spectrophotometric studies (UV-Vis absorption, fluorescence, and
circular/linear dichroism) and viscometry. The binding constants K are estimated as being in
4
-1
the range of 2.2 to 7.8 × 10 M and the percentage of hypochromism was found to be 22.11–
9.75% (from UV-Vis spectral titration). Electrophoretic experiments prove that the novel
compounds demonstrate moderate inhibitory effects against Topo I activity at a concentration
4
-6
of 60 × 10 M.
Introduction
Thiosemicarbazones have been studied due to the wide range of bioactivities: antibacterial,
antifungal, antineoplastic and antiviral [1-4]. Nowadays, thiosemicarbazones are extensively
studied as anticancer drugs. Several synthetic molecules have been reported to be anticancer
drugs and Triapine entered into clinical trials [5,6]. Antitumor activity of thiosemicarbazones
is extremely differentiated and it is very much depended on the typology of tumor cells [7].
Several mechanisms have been proposed so far. Topo II and ribonucleotide reductase have

ACCEPTED MANUSCRIPT
been proposed as the two primary targets of these molecules [8]. The addition of acridine is
known to have a marked effect on the chemical and biological properties of many types of
compounds [9,10]. In view of these literature reports, we have focused our work on the
synthesis of a new series of acridine thiosemicarbazones. The properties of these new
derivatives are studied using NMR and biochemical techniques, and the DNA-binding
properties of the compounds are determined using spectrophotometric studies (UV-Vis
absorption, fluorescence, and circular/linear dichroism) and viscometry. The study will also
examine the inhibitory effect of these compounds on topoisomerase activity I.
Materials and Methods
Materials
Agarose (type II No-A-6877), Ampicilin, Plasmid pBR322, Ethylenediaminetetraacetic
acid disodium salt (Na EDTA), Ethylenediaminetetraacetic acid (EDTA), Sodium chloride
2
(
NaCl), Potassium chloride (KCl), Calcium chloride (CaCl ), Propidium iodide (PI), Ethidium
2
bromide (EtBr), Triton X-100 (Sigma-Aldrich, Germany). Bovine serum albumin (BSA),
Magnesium chloride (MgCl ), Dithiothreitol (DTT) (TaKaRa, Japan). Chloroform, Glucose,
2
Glycerol, Glycerin, Hydrogen chloride (HCl), H BO KH PO , K HPO Na HPO Na H PO
4
3
3,
2
4
2
4,
2
4,
2
2
(
Lachema, Czech Republic). Trishydroxymethylaminomethane (Tris) (Roth, Germany).
Ethanol, Isopropyl alcohol (Lach-Ner, Czech Republic), NaHCO , Amphotericin, Penicillin,
3
Streptomycin (Invitrogen, USA) and Dimethyl sulfoxide (DMSO), sodium dodecyl sulphate
(SDS) (Serva (Germany). Calf thymus DNA (ctDNA) (Sigma-Aldrich, Germany), Calf
thymus topoisomerase I and 10 × DNA Topoisomerase I buffer (Takara, Japan).
Synthesis of 5a–5g
All of the reactions mentioned above were performed in an inert atmosphere of nitrogen.
All reagents were used as supplied without prior purification. The progression of the reaction
was monitored with analytical thin layer chromatography using TLC-sheets ALUGRAM-SIL
G/UV254 (Machereye Nagel, Germany). Purification by flash chromatography was
performed using silica gel (60 Å, 230400 mesh, Merck) with the indicated eluent. NMR
spectra were recorded at room temperature on a Varian VNMRS NMR spectrometer
1
13
15
operating at 599.87 MHz for H, 150.84 MHz for C, and 60.79 MHz for N. Spectra were
1
13
recorded at 25 °C in DMSO-d . The residual protic solvent of DMSO-d ( H,  2.50 ppm; C,
6
6

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1
13

39.0 ppm) was as the internal references in the H and C spectra (Fig. S1). Melting points
were determined on a Boetius hot-stage apparatus and are uncorrected. Elemental analysis
was performed on a Perkin Elmer CHN 2400 elemental analyser.
Synthesis of 3-[(E)-(acridin-9'-ylmethylidene)amino]-1-substituted thioureas 5a–g
Method I: Isothiocyanates 4a–g (0.483 mmol) and hydrazine hydrate (50%, 0.483 mmol)
were added to methanolic solutions (4 mL) of acridin-9-carbaldehyde (3, 100 mg, 0.483
mmol). The reaction mixtures were refluxed for 12–24 h (TLC monitoring), and the
precipitates were filtered off, dried and crystallized (DMF/ethanol).
Method II: Isothiocyanates 4a–g (0.483 mmol), hydrazine hydrate (50%, 0.483 mmol) and
molecular sieves 4 Å were added to methanolic solutions (4 mL) of acridin-9-carbaldehyde
(3, 100 mg, 0.483 mmol). The reaction mixtures were refluxed for 12–24 h (TLC monitoring),
and the precipitates were filtered off, dried and crystallized (DMF/ethanol).
Method III: Hydrazine hydrate (50%, 0.283 mmol) was added to ethanolic solutions (2 mL)
of isothiocyanates 4a–g (0.283 mmol). The reaction mixtures were stirred at room
temperature (TLC monitoring; n-Hex/ethylacetate = 4/1), and acridin-9-carbaldehyde (3, 50
mg, 0.283 mmol) and 2 drops of glacial acetic acid were then added. The reaction mixtures
were refluxed until aldehyde 3 disappeared (TLC monitoring; n-Hex/ethylacetate = 4/1), and
the precipitates were filtered off, dried and crystallized (DMF/ethanol).
Spectroscopic measurements
UV-vis spectra were obtained with a Varian Cary 100 UV-Vis spectrophotometer. UV-Vis
spectra titrations were performed using a 0.01 M Tris-HCl buffer, pH = 7.4. Calf thymus
DNA (ctDNA) was used without further purification. The DNA concentration was determined
-1
-1
by the UV absorbance at 260 nm (ε = 6600 M cm ) [11]. The stock solution of ctDNA gave
a ratio of UV absorbance at 260 and 280 nm (A /A ) of 1.82, indicating that the DNA was
2
60 280
sufficiently free of protein contamination [12]. Solutions of acridine derivatives 5a5g (10 ×
-
3
-3
1
7
0 M in 5 × 10 M DMSO) were transferred to the quartz cuvette at a final concentration of
-5
.5 × 10 M. All measurements were performed at 25 °C in a rectangular quartz cuvette with
a 1 cm path length. The binding affinities were calculated from the absorbance spectra
following the method of McGhee and von Hippel using data from a Scatchard plot [13,14].
The binding data were fitted using GNU Octave 2.1.73 software [15]. The standard Gibb´s

ACCEPTED MANUSCRIPT
free energy for DNA binding was calculated using the following equation: ΔG° = -RT.lnK,
where R is the general gas constant, T the absolute temperature and K the binding constant
[
16,17].
Competitive displacement assay
The competitive studies of each derivative with EB were investigated with fluorescence
spectroscopy in order to examine whether the derivative is capable of displacing EB from the
ctDNA-EB complex. Ethidium bromide (EB) emits intense fluorescence in the presence of
ctDNA, due to its strong intercalation between the adjacent ctDNA base pairs. Previous
studies have reported that the enhanced fluorescence can be quenched by replacement by a
second molecule [18,19]. The fluorescence spectra of EB were measured using an excitation
wavelength of 500 nm and the emission range was set between 520 and 800 nm. The widths
of both the excitation and emission slits were set at 10 nm. The intercalating effect of studied
acridine derivatives with the DNA–EB complex was studied by gradually adding specific
-4
amounts of studied acridine derivatives 5ag 0–5.8 × 10 M to quartz cuvettes contained
fixed concentrations of the ctDNA-EB complex solution. The influence of the addition of
each compound to the DNA-EB complex solution was obtained by recording the variation of
fluorescence emission spectra.
Circular/linear dichroism
Circular dichroism (CD) measurements were performed with a J-810 Jasco
spectropolarimeter using a 1 cm path-length quartz cuvette at 24 °C, 0.01 M Tris-HCl buffer
-4
(
pH 7.4). The CD spectra of ctDNA (c = 5.8 × 10 M) incubated with varying concentrations
of derivatives 5ag were investigated over a wavelength range of 230–350 nm in a nitrogen
atmosphere. The results are presented as a mean of three scans from which the buffer
background was electronically subtracted [20].
Flow linear dichroism (LD) spectra were collected using a flow Couette cell in a Jasco J-
7
20 spectropolarimeter adapted for LD measurements. Molecules of longer length such as
DNA (minimum length of ~250 bp) can be orientated in a flow Couette cell. The flow cell
consists of a fixed outer cylinder and a rotating solid quartz inner cylinder, separated by a gap
of 0.5 mm, giving a total pathlength of 1 mm. LD spectra of ctDNA at a concentration of 1.2
-3
-6
×
10 M modified by derivatives 5ag (8–40 × 10 M) were recorded at 25 °C in 0.01 M
Tris-HCl buffer, in the range of 220–600 nm.

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Viscometric measurements
Viscosity experiments were conducted with an AMV Automated Micro Viscometer (Anton
Paar GmbH, Austria) using a 1.6-mm capillary tube at a constant temperature of 25 ± 0.1 °C.
Titrations were performed by adding various concentrations of derivatives 5ag (2.5–10 ×
-5
-3
1/3
1
0 M) to a ctDNA solution (1.6 × 10 M). The results are presented as (η/η ) versus the
0
ratio of the concentration of the compound to ctDNA, where η is the viscosity of ctDNA in
the presence of the compound and η is the viscosity of ctDNA alone. Viscosity values were
0
calculated from the observed flow time of DNA-containing solutions (t) and corrected for the
buffer solution (t ), η = (t−t )/t .
0
0
0
Determination of the impact of acridine derivatives on plasmid DNA
Varying concentrations of the studied acridine derivatives were added to plasmid DNA
pUC19/ pBR322 (1.4 µg). The samples were immediately incubated in darkness at 37 °C for
-6
1
8 h with gentle shaking after which 3 × 10 L of electrophoresis loading buffer
(bromophenol blue) was added. Samples were analysed with gel electrophoresis using 1%
agarose gels at 7 V/cm for 2.5 h using 0.5 × TBE buffer followed by staining in 10 mg/mL
ethidium bromide. The gel was then visualized under UV-light and photographed.
DNA topoisomerase I relaxation assay
Topoisomerase I was added to solutions of plasmid supercoiled DNA pUC19 (1.4
µg/pBR322, 1.4 µg/pHOT-1, 0.25 µg) in a relaxation buffer. Once full relaxation had been
achieved, varying amounts of derivatives 5ag were added to the relaxation buffer and the
-3
-3
solutions were incubated at 37 °C for 45 min (3.5 × 10 M Tris-HCl (pH=8.0), 5 × 10 M
-3
-2
-2
MgCl , 7.2 × 10 M KCl, 0.1% bovine serum albumin, 5 × 10 M spermidine, 5 × 10 M
2
dithiothreitol). The DNA samples were then electrophoresed on a 1% agarose gel at 7 V/cm
for 2.5 h with a running buffer (5× TBE). EB was used as a staining agent to allow the
visualization of the gels with UV-light.
Results and discussion
The use of multicomponent reactions to synthesize novel compounds has become common
in modern organic chemistry as such reactions are an elegant method of maximizing the
structural complexity and convergence of compounds whilst minimizing the cost of synthesis
[
21]. As part of our research, we investigated the one-pot and catalyst-free synthesis of

ACCEPTED MANUSCRIPT
thiosemicarbazones via multicomponent coupling reactions which was reported by Cunha and
da Silva (Scheme 1, Table 1 - method I). We observed that a part of the starting material,
derivative 3, was recovered and that the final yield of the desired product was 44–79% (Table
1
). In order to increase the yield of derivative 5, we carried out the reaction in the presence of
molecular sieves (Table 1 - method II). The obtained yield of derivative 5 was moderately
higher, but the longer reaction time and the difficulty involved in separating the final product
from the starting material limited the potential application of the procedure. Therefore we
decided instead to use the method reported by Benmohammed et al which offers a simple and
efficient procedure for the synthesis of thiosemicarbazones (Scheme 2, Table 1 – method III)
[
22].
The synthesis of thiosemicarbazones 5a–g is shown in Scheme 2. An initial substrate of
acridin-9-carbaldehyde (3) [23] was prepared from 9-(bromomethyl)acridine (2) [24] which
was obtained from 9-methylacridine (1) [22,25] (Scheme 1).
Br
CH3
NBS/AIBN
Na/2-nitropropane
DMSO
rt, 2h
CCl4
N
N
8
0 °C, 6h
1
2
S
H
N
R
N
N
N
H
H
H
O
NH -NH x H O
RNCS 4ag
2
2
2
MeOH
reflux, 1224h
N
3
5ag
R = Me (a), Pr (b), Ph (c), 4-CH O-Ph (d),
3
3
-CH O-Ph (e), 4-F-Ph (f), 4-NO -Ph (g)
3
2
Scheme 1 Multicomponent synthesis of 3-[(E)-(acridin-9'-ylmethylidene)amino]-1-
substituted thioureas 5a–g.
Thiosemicarbazones 5a–g were synthesized in two steps. The first step was the preparation of
thiosemicarbazide 7a–g from commercially available isothiocyanates 4a–g and hydrazine
hydrate in ethanol at room temperature (Scheme 2). The reaction of thiosemicarbazides 7a–g
with acridine-9-carbaldehyde (3) in the presence of few drops of acetic acid led to favourable
yields of thiosemicarbazones 5a–g (80%–92%), as is shown in Table 1. The crude products
were further purified by recrystallization using DMF/ethanol to furnish analytically pure
compounds 5a–g.

ACCEPTED MANUSCRIPT
S
H
R
N
N
N
H
H
N
S
H
N
Akr-CHO (3)
N
R
R NH
S
CH COOH (cat)
DMSO-d6
NH NH x H O
3
2
2
2
R NCS
ag
NH
NH2
EtOH
rt, 313 h
EtOH
rt, 13 h
rt
N
N
H
4
7ag
5
ag
6
c,g
R = Me (a), Pr (b), Ph (c), 4-CH O-Ph (d),
3
3
-CH O-Ph (e), 4-F-Ph (f), 4-NO -Ph (g)
3
2
Scheme 2 Two-step synthesis of 3-[(E)-(acridin-9'-ylmethylidene)amino]-1-substituted
thioureas 5a–g and corresponding 1-substituted-5H,10'H-spiro[acridine-9',2-[1,4,5]triazine]-
6
-thiones 6c,g.
Table 1 Reaction of aldehyde 3 with isothiocyanates 4a–g.
a
b
c
Product
Method I
reaction time
h)
Method II
reaction time
Method III
reaction time
yield
(%)
46
yield
(%)
52
yield
(%)
91
R
(
(h)
26
24
20
21
22
20
16
(h)
3
5
a
Me
Pr
24
23
19
20
22
21
17
5
b
50
61
4
92
5
c
Ph
79
81
6
80
5
d
4-CH
3-CH
3
3
O-Ph
O-Ph
65
68
13
9
87
5
e
44
58
91
5
f
4-F-Ph
4-NO -Ph
71
79
8
91
5
g
78
85
12
90
2
a
b
c
Multicomponent reaction: MeOH, reflux, Multicomponent reaction: MeOH, MS, reflux, Step reaction: EtOH,
rt.
1
13
15
H, C and N NMR data (chemical shifts and coupling constants) for compounds 5ag
are shown in Experimental and the numbering of atoms is indicated in Fig. 2. Unambiguous
assignments for all protons, carbons and nitrogens were obtained by correlating the results of
1
D and 2D NMR analysis.
Acridine and phenyl rings protons were assigned according to the chemical shift and
multiplicity data and through sequential analysis of the results of the NOE and COSY
experiments. Protons H-1',8' were easily defined by considering the NOE effects which were
observed between them and H-9; the ortho protons of the phenyl ring displayed NOE effects
with an H-1 proton (Fig. 1). COSY and HSQC spectra were then examined in order to

ACCEPTED MANUSCRIPT
identify the remaining protons and carbons. The quaternary carbons were assigned on the
basis of the analysis of HMBC experiments.
1
13
1
13
The atoms of the thiosemicarbazide fragment were identified using H, C-HSQC, H, C-
1
15
HMBC and H, N-HMBC data (Fig. 2).
H
H
.69
H
S 4
2.28
H
N
N
N
4.64
0.68
2.72
H
H
H 1
.74
13.71
4
.13H
N
H
H
Fig. 1 Conformation of derivative 5c with key NOE enhancements (in %).
H
H
H
S
H
3
1
1''
N
N
4
H
N
H
H
8
'
8
'a
9
'a
H
H
9
'
1'
4'
4'a
1
0'a N
5'
10'
H
1
13
1
15
Fig. 2 The structure of compound 5c together with H, C-HMBC () and H, N-HMBC
() correlations assisting in the structural elucidation.
Thiosemicarbazones possess the unique capability of forming Z and E isomers [26,27]. A
number of authors have noted that compounds possessing a Z isomer generally exhibit a NH-3
signal in the 14–15 ppm range, whereas those compounds possessing an E form display a
signal in the 9-12 ppm range [28].
The NMR spectra for all of the compounds examined in our study showed only one set of
peaks, demonstrating the preferential formation of one isomer over the other. Based on the
chemical shifts values we propose that all of the compounds exist in an E isomer
configuration.
The stereochemistry of thiosemicarbazones 5a-g were also determined by using NOESY
experiments (Fig. 1). The saturation of the H-3 proton in derivative 5c resulted in NOE

ACCEPTED MANUSCRIPT
enhancement at the H-5 proton (2.72%). Furthermore, the saturation of H-5 showed NOE
enhancement at the H-3 (2.28%) and H-1',8' protons (13.71%). The saturation of the H-1
proton resulted in strong NOE enhancement at H-2'',6'' (4.69%), whereas weaker NOE was
observed at H-1',8' (1.74%). These observations are in agreement with the results of
compounds featuring an E-configuration.
1
The H NMR spectrum of compound 5c which had been freshly dissolved in DMSO-d
6
showed that the compound was formed exclusively of E isomers. However, the same NMR
sample of compound 5c was examined after the solution had been standing for some time, and
the amount of E isomer was found to have decreased and a new compound had developed as
shown in Figure S1. After 9 days it was possible to determine the structure of this minor form,
which was termed spiro derivative 6c. A similar form of spiro cyclization was also observed
in derivative 5g.
The distinct resonances of the spiro carbons C-9' allowed clear recognition of the structures
in each case (61.7 ppm for 6c and 62.1 ppm for 6g), but the findings were confirmed by
further 1D and 2D NMR experiments. Three distinct one-proton signals (11.58 ppm, 9.14
1
ppm and 7.35 ppm) were observed in the H NMR spectra of derivative 6c. The assignment of
1
13
1
the one-proton NH and CH signals was performed using the results of H, C-HSQC and H,
1
5
N-HSQC spectra. The unambiguous assignment of the proton at C-5 was provided by
NOESY correlations to H-1', 8', the proton at N-10' by NOESY correlation to H-4', 5', thus
permitting the assignment of H-3 by default. The assignment of acridine protons H-1', 8' were
obtained from NOESY correlations with H-5, followed by COSY correlations beginning at H-
1
3
1
', 8'. The assignment of the aromatic region of the C NMR spectrum was done using the
HSQC. The signals for C-8'a, 9'a were readily assigned from their HMBC correlations with
H-5, H-10', H-2', 7', H-4',5' and the signals for C-4'a,10'a from their HMBC correlations with
H-1',8', H-3',6'. The signal for the quaternary carbon C-9' showed a correlation with H-1', 8'
and H-5 in the HMBC spectrum (Fig. 3).
H
H
H
N
S
3
N
H
1''
N
H
H 5
1'
1
H
H
8
'
5'
10'
N
H
4'
H

ACCEPTED MANUSCRIPT
1
13
Fig. 3 The structure of spiro compound 6c together with selected H, C-HMBC () and
1
15
H, N-HMBC () correlations assisting in the structural elucidation.
The interaction of the newly synthesized acridine derivatives 5a–5g with ctDNA was
-3
monitored with spectrophotometric titrations in a Tris-HCl buffer (10 × 10 M, pH=7.4). In
order to evaluate the general affinity of derivatives 5a–5g towards ctDNA, the binding
interactions of these compounds with ctDNA were examined with spectrophotometric
titrations (in the region of 300–600 nm). When compounds bind to DNA, their UV-Vis curves
show a characteristic shift of the absorption maximum wavelength (bathochromic or
hypsochromic shift) and a decrease (hypochromicity) [29,30] or increase (hyperchromicity) in
absorbance [31]. In the case of our novel acridine derivatives, a significant decrease in
absorbance was recorded when the concentration of ctDNA was increased. In addition, a
bathochromic shift of the chromophore was also observed in the absorbance, which suggests
that acridine derivatives 5a–5g can interact with DNA. The absorption spectra of the studied
acridine derivatives 5a–5g in both the absence and presence of ctDNA are given in Figs. S2.
In general, the process of DNA binding places the molecule in an environment which is
different from that of the uncomplexed molecule in solution. Due to these different features
the electron distribution of the molecule is distorted by π-stacking with the bases, and this
causes significant differences between the absorption properties of molecules in complexed
and uncomplexed forms [32-34]. Reducing the distance between the intercalated compounds
and DNA bases allows hypochromism to occur [35]. Consequently, the energy level of the π-
π* electron transition decreases which thereby results in a red shift.
We examined the rate of hypochromism for each of the novel derivatives, and compound
5
g was found to show the greatest decrease in peak intensity (Table 2). Absorbance intensity
changes were used to calculate the DNA binding constants (K) of the acridine derivatives
using the method proposed by McGhee and von Hippel (Table 2). The binding constants K
4
-1
calculated for the novel derivatives ranged from 2.2 to 7.8 × 10 M , and suggest the high
affinity of the studied acridine derivatives towards ctDNA base pairs. Typical K values for
4
6
-1
intercalation complexes between organic dyes and DNA range from 1 × 10 to 1 × 10 M
5
and are usually significantly smaller than the binding constants of groove binders (1 × 10 to 1
9
−1
×
10 M ) [32].

ACCEPTED MANUSCRIPT
Table 2 UV-Vis absorption data of acridine derivatives 5a–5g.
Compound
λmax free
λmax bound
Δλ
Hypochromicity
K
n
ΔG
-
1
-1
(
nm)
(nm)
(nm)
(%)
(M )
(kJ mol )
4
5
5
5
a
b
c
366
370
364
375
371
418
367
372
364
375
372
419
363
1
2
30.62
20.36
24.4
3.4×10
2.8±0.2
3.7±0.3
2.5±0.2
2.8±0.5
3.1±0.3
2.8±05
2.7±0.4
-25.9
-28.0
-24.8
-25.6
- 26.4
-25.6
-25.6
4
4
4
4
4
4
7.8×10
2.2×10
3.0×10
4.1×10
3.0×10
2.7×10
0
5
d
0
22.11
37.6
5
5
5
e
f
1
1
28.9
g
367
-4
49.75
The K values increased in the following relationship: 5b > 5e > 5a > 5d, 5f > 5g > 5c. The
high value of K recorded for derivative 5b suggests a strong binding affinity towards ctDNA,
and supports the idea that intercalative π-stacking interactions of the ligands with DNA are
essential for their efficient electron transfer reaction. Moreover, the uniformity of the
calculated values of K for different substituted acridine rings validates the assumption that the
DNA binding mechanism is heavily dependent on a common structural component of all of
the studied systems; the acridine skeleton. The lateral substituents of the molecules appear to
play a much less important role in the binding process [33]. The absence of any clear isobestic
point in the compound-ctDNA spectra indicates that more than one type of binding may be
occurring [33]. The exact mode of interaction between the compounds and ctDNA could not
be established and hence further experiments were required to understand the binding mode
fully.
In order to determine the interaction of acridine derivatives 5a–5g with ctDNA in more
detail, a competitive EB binding study was performed using fluorescence experiments [36].
EB is a phenanthridine fluorescent dye forming soluble complexes with nucleic acids and
emitting intense fluorescence in the presence of ctDNA due to the intercalation of the planar
phenanthridinium ring between adjacent base pairs on the double helix [37]. However, not
only the DNA intercalators but also groove DNA binders could cause the reduction in the
emission intensities of DNA bound EB. For example, the non-intercalating DNA groove
binding agents of berenil, bisamidines, spermine and spermidine were reported to be capable
of displacing EB from DNA [38]. Thus, any change in fluorescent intensity of dye-DNA

ACCEPTED MANUSCRIPT
complex upon addition of a small molecule can offer valuable information regarding the mode
of interaction which has occurred [39,40].
The fluorescence spectra of EB with ctDNA in both the absence and presence of the studied
acridine derivatives were measured using an excitation wavelength of 500 nm and the
emission range was set between 520 and 800 nm. The The results are shown in Fig 4 and S3.
Interestingly, the results of the competitive DNA binding assay demonstrate that the addition
of derivatives 5a–5g, compounds which may bind to DNA more strongly than EB, induced a
rapid decrease in the fluorescent intensity of the emission band of the ctDNA-EB system. The
competitive binding interaction between acridine complexes 5a–5g and EB with ctDNA was
also found to have occurred. By comparing the obtained results, derivatives 5a, 5e and 5g
were found to be particularly effective in displacing EB from ctDNA. Fig. 5 and S3 show that
derivatives 5a–g had replaced EB in the DNA helix. Since EtBr intercalates DNA through
interactions with the minor groove, the displacement of EtBr (quantified by fluorescence) by
the titration of a compound is suggestive of an intercalative or minor groove binding [41].
-
5
-6
Fig. 4 Fluorescent displacement of EB (6.0 × 10 M) bound to DNA (1.0 × 10 M bp) at
-4
-3
increasing concentrations of derivative 5a (0–8 × 10 M), λ = 500 nm, in 10 × 10 M Tris-
ex
HCl (pH = 7.4, 24 C), corresponding to the curves from top to bottom, respectively.
The insertion of a planar molecule into B-DNA causes the extension and local unwinding
of the polynucleotide double helix [42]. This process causes substantial changes in the relative
positions of the bases in the complex region. In addition to affecting the hydrodynamic
properties of DNA, the modified structure containing the planar ligand also dramatically alters
the chiroptical response of the polynucleotide. The results of circular dichroism spectroscopy
assays demonstrate the perturbation in B-DNA conformation upon interaction with acridine
derivatives.

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The CD spectra of a series of ctDNA solutions were recorded in both the absence and
presence of acridine derivatives 5a–5g. Fig. 5 and S4 depict the changes which occurred in
these bands in the presence of acridine derivatives, and provide evidence of the nature of the
formation of the derivative-ctDNA complex. Acridine derivatives are not optically active in
solution, they could acquire chirality upon binding to DNA, and thus contribute to CD
spectrum. Obvious changes occurred in the CD spectra following the addition of derivatives
5
a–5g to the complex, and the conformational transition of DNA can be traced through the
changes in the positions and intensity of these spectral bands in the CD spectra. The results
show a considerable increase in the circular dichroism spectrum of DNA at 275 nm as a
consequence of the insertion of each of the studied compounds into the base-pairs. A notable
reduction in the intensity of the negative band at 245 nm was observed for all complexes.
Classical intercalation reactions tend to alter the intensity of the two bands due to the strong
stacking interactions of the nucleotide bases and more stable conformations (right-handed B
conformations of ctDNA), whereas simple groove binding and electrostatic interactions show
a lower perturbation or no perturbation affect whatsoever on the bases’ stacking and ellipticity
bands [43]. Here, it is pertinent to mention that these results are in agreement with the
findings of earlier fluorescence studies in which the acridine derivatives were found to be able
to replace the dye molecule in the DNA complex. It is therefore possible to suggest that the
CD findings provide more evidence of the likelihood of the intercalative binding mode of
interaction.
8
4
0
-
4
8
-
3
00
400
500
600
Wavelenght [nm]
Fig. 5 Effect of compound 5a on CD spectra of ctDNA. CD spectra of ctDNA (4.5 ×
-
3
-2
1
(
0 M bp) in 1 × 10 M Tris-HCl (pH = 7.4) at varying concentrations of compounds 5a
-5
8.6–43.5 × 10 M). Each spectrum was obtained at 25 °C. Arrow indicates the changes upon
increasing amounts of the compounds.

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LD spectroscopy measures the difference in absorption of light polarised both parallel and
perpendicular to the orientation axis of the molecule. In general, small molecules do not orient
in flow and therefore exhibit no LD signal, whereas molecules which are very long and thin
(e.g. natural DNA) orient very easily in flow and display strong LD signals. The helical axis
orients along the flow axis, with the base pairs orienting approximately perpendicular to this
axis [44]. The π-π* transitions in the DNA base pairs therefore result in a negative LD signal
at 260 nm.
In this experiment, multiple LD analyses of the same samples were undertaken by
-3
comparing first the signal intensity at 259 nm of samples of ctDNA (1.2×10 M). The LD
spectra of ctDNA and ctDNA in the presence of acridine derivatives 5a–5g are given in Fig. 6
and S5. Interestingly, ctDNA shows a characteristic negative peak with a maximum at 259 nm
due to π-π* transitions in the DNA bases. Clear changes were then observed in the spectra
when acridine derivatives 5a–5g were added to the solution; there was a contribution in this
region of the spectrum. At increasing concentrations of the derivatives, the intensity of the
signal was seen to increase in comparison to the ctDNA alone. An increase in the amplitude
of the negative 259 nm LD band of DNA is usually associated with DNA stiffening [45-50]
However, an unambiguous interpretation of the changes of LD signal at 259 nm is impossible
because acridine derivatives absorb in this region. In the region of above 300 nm, a broad
negative peak was observed for all of the studied acridine derivatives, these findings were
particularly marked in the cases of derivatives 5b, 5c and 5e. This observation is in
agreement with a coplanar arrangement of the DNA base pairs and the acridine derivatives
moiety either intercalated or located in a groove and oriented parallel with base pairs. Thus,
the LD results listed above suggest that the structural reformation of ctDNA occurs as a result
of interaction with the acridine compounds. This finding is further supported by the data from
other spectroscopic techniques, and allows us to determine the interaction stereochemistry in
more detail and offers us a deeper insight into the structure-activity relationships of the
complexes. As the results of our LD experiments do not unambiguously distinguish between
intercalative and groove DNA binding modes, it was necessary to further experiments.

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-3
Fig. 6 Linear dichroism spectra of ctDNA (1.2 × 10 M bp, blue line) in the presence of
-6
increasing amounts of derivative 5a (8–40 × 10 M ) in 1M Tris solution, pH = 7.4
In order to characterize the interactions between derivatives 5a–5g and DNA in more
detail, viscosity measurements were carried out on derivative-ctDNA complexes at varying
concentrations of the studied compounds. Viscometric measurements of DNA are considered
to be regarded as the least ambiguous and the most effective tool for determining the binding
modes of small molecules with DNA [47]. When a ligand inserts itself between adjacent base
pairs within the DNA duplex, the helix must unwind to create a gap in order to accommodate
the incoming ligand. This unwinding process increases the overall contour length of the helix
and, as a result, the DNA solution becomes more viscous. In contrast, groove binding
compounds do not require insertion between the base pairs, and therefore DNA lengthening
does not occur and the viscosity of the solution is not significantly altered. Viscometric
experiments can therefore be employed to distinguish between different non-covalent DNA
1
/3
binding modes [45]. Fig. 7 shows dependence of the relative specific viscosity (η/η ) against
0
the mole ratio r = [ligand]/[DNA]. It is shown that the relative specific viscosity of ctDNA
was not much affected as the concentration of studied derivatives increased, compared with
that for increasing concentration of EtBr in the ctDNA solution. Thus, these viscosity data are
consistent with the hypothesis that compounds bind to DNA in a specific orientation(s), via a
non-intercalative and possibly a groove-binding mode.

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5
5
5
5
5
5
5
a
b
c
d
e
f
2.3
2.1
1.9
1.7
1.5
1.3
1.1
0.9
g
EtBr
-0.02
0.03
0.08
0.13
0.18
0.23
r = [ligand]/[DNA]
-5
Fig. 7 Effect of increasing concentrations of derivatives 5a5g (2.5–10 × 10 M) on the
-3
viscosity of ctDNA. Concentration of ctDNA remained constant (1.6 × 10 M bp).
Topoisomerases (Topo) are key cellular enzymes which prevent DNA strands from
becoming tangled by cutting the DNA helix and causing it to wind and unwind and play an
important role in the transcription and replication of DNA [48,49]. The ligand which occupies
the topoisomerase binding site may suppress the association of topoisomerase with DNA.
DNA intercalators which inhibit topoisomerase activity or which form stabilized ternary
complexes with DNA and topoisomerase have high potential for development as DNA
targeting anticancer drugs [51,52]. The acridine derivatives presented in this paper show a
high level of interaction with DNA and therefore it would be of interest to examine whether
the compounds are also capable of inhibiting some of the nuclear enzymes involved in DNA
processing, such as Topo I.
In this assay, the ability of the studied derivatives to affect the Topo I-mediated relaxation of
supercoiled DNA was investigated. Plasmid pBR322 DNA was used as a substrate and was
incubated with appropriate amounts of Topo I in both the absence and presence of various
-6
concentrations (30-60×10 M) of derivatives 5a–5g. Fig. 8 shows the relative affinity of all of
the studied compounds on the relaxation of plasmid pBR322 DNA mediated by Topo I. As
was expected, the supercoiled pBR322 DNA was fully relaxed by the enzyme in the absence
of the studied samples (lane Top I, Fig. 8). Acridine derivatives 5a–5g failed to inhibit the
-6
relaxation of the supercoiled DNA substantially at a concentration of 30×10 M (lanes a), but
-6
at a higher concentration of 60 × 10 M, derivatives 5a–5g showed moderate levels of Topo

ACCEPTED MANUSCRIPT
I inhibitory activity (lanes b). These results suggest that the studied compounds are able to
affect the topology of DNA which may form a foundation for their future development as
Topo I inhibitors.
Fig. 8 Inhibition of topoisomerase I-induced DNA relaxation by acridine derivatives 5a–5g. Native
supercoiled pBR322 (1.4 µg, lane pBR322) was incubated for 45 min at 37 ºC with 2 units of
-6
topoisomerase I in the absence (lane Topo I) or presence of compounds (a = 30, b = 60 × 10 ). The
samples were run on agarose gel followed by ethidium bromide staining. sc= supercoiled; oc= open-
circular.
Conclusion
In this work seven new acridine derivatives (5a–5g) are synthesized and investigated. The
formation and properties of the acridine-DNA complex were studied using UV-Vis
absorbance fluorescence spectra. All of the studied acridine derivatives were found to exhibit
4
-1
binding constants with ctDNA in the range of 2.2 to 7.8 × 10 M , and the compound with the
most efficient rate of binding to ctDNA in vitro was found to be derivative 5b. The
derivatives were also found to demonstrate moderate inhibitory effects on Topo I activity at
-6
concentrations above 60 × 10 M. The findings presented in this paper offer significant
information about the interaction between acridines compounds and DNA, features which are
of great use in the potential development of drugs with enhanced or more selective activity
and greater clinical efficacy.
Acknowledgements
This study was supported by VEGA grant No. 1/0001/13, MH CZ-DRO (UHHK,
0
0179906). The authors from the Department of Biophysics, Palacky University acknowledge
the support from the project of Palacky University (IGAPrF 2016 013).

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H
H
H
N
S
3
N
H
1''
N
H
H 5
1'
1
H
H
8
'
5'
10'
N
H
4'
H
1
13
1
15
The structure of spiro compound 6c together with selected H, C-HMBC () and H, N-
HMBC () correlations assisting in the structural elucidation.
Graphical abstract

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Highlights
o The synthesis of a novel series of acridine thiosemicarbazones.
o The binding constants for derivatives 5a-5g with DNA were K = 2.2 to 7.8 × 10 M .
o The studied samples displayed an effect on topoisomerase I activity.
4
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