The comparative study of the DNA binding and biological activities of the quaternized dicnq as a dicationic form and its platinum(II) heteroleptic cationic complex

Article abstract of DOI:10.1016/j.bioorg.2019.03.009

The square-planar heteroleptic Pt(II) coordination compound [Pt(bpy)(dicnq)](NO₃)₂ (1) and the quaternized dicnq ligand, namely 12,13-dicyano-5,6-dihydrodipyrazino[2,3-f:1′,2′,3′,4′-lmn][1,10]phenanthroline-4,7-diium dibromide (2) (Fig. 1) were synthesized and fully characterized by means of FTIR, NMR, MALDI-TOF MS and the purity was confirmed by CHN analyses. The DNA binding profiles of 1 and 2 were identified in an identical condition. The biological activities of these compounds were investigated by the assays of transcription and replication inhibition, cytotoxic and antimicrobial activity. The result of this study indicates that, both compounds strongly bind to DNA via intercalation but only 1 has a strong nuclease activity. The coordination compound of dicnq (1) binds to the DNA only slightly stronger than the quaternized form of dicnq (2), and is more potent as an inhibitor of transcription and replication and therefore, 1 has more potential as an anticancer agent but the compounds did not show cytotoxic activity against MCF-7 and MDA-MB-231 breast cancer, and DLD-1 colon cancer cell lines it was found that they only had activities against HepG2 liver cancer cell line with following IC₅₀ values; 94.75 and 159.60 μM for 1 and 2, respectively. In addition, tested bacteria are more susceptible to compound 1. These biological activities of 1 may strongly be due to its ability to digest DNA as a chemical nuclease. According to this study, the quaternization of the ligand does not make biologically more active than the coordination compound of the same ligand in this case. The compound (1) is worth further investigation for its antitumor activities.

Full text of DOI:10.1016/j.bioorg.2019.03.009

Accepted Manuscript
The comparative study of the DNA binding and biological activities of the qua-
ternized dicnq as a dicationic form and its platinum(II) heteroleptic cationic
complex
Ufuk Yıldız, Abdurrahman Şengül, Irfan Kandemir, Füsun Cömert, Senem
Akkoç, Burak Coban
PII:
S0045-2068(18)31394-4
DOI:
https://doi.org/10.1016/j.bioorg.2019.03.009
YBIOO 2835
Reference:
To appear in:
Bioorganic Chemistry
Received Date:
Revised Date:
Accepted Date:
30 November 2018
1 March 2019
4 March 2019
Please cite this article as: U. Yıldız, A. Şengül, I. Kandemir, F. Cömert, S. Akkoç, B. Coban, The comparative study
of the DNA binding and biological activities of the quaternized dicnq as a dicationic form and its platinum(II)
heteroleptic cationic complex, Bioorganic Chemistry (2019), doi: https://doi.org/10.1016/j.bioorg.2019.03.009
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The comparative study of the DNA binding and biological activities of the
quaternized dicnq as a dicationic form and its platinum(II) heteroleptic
cationic complex
1
1
2
3
4
*1
Ufuk Yıldız , Abdurrahman Şengül , Irfan Kandemir , Füsun Cömert , Senem Akkoç , Burak Coban
*1
Department of Chemistry, Faculty of Arts and Sciences, Zonguldak Bulent Ecevit University, Zonguldak
6
2
7100, Turkey
Department of Biology, Faculty of Science, Ankara University, Ankara 06560, Turkey
3
Department of Microbiology, Faculty of Medicine, Zonguldak Bulent Ecevit University, Zonguldak 67600,
Turkey
4
Faculty of Pharmacy, Suleyman Demirel University, Isparta 32260, Turkey
*
Corresponding Author: e-mail: burakcoban@yahoo.com

The comparative study of the DNA binding and biological activities of the quaternized dicnq as
a dicationic form and its platinum(II) heteroleptic cationic complex [Pt(bpy)(dicnq)](NO
Abstract
)
3 2
The square-planar heteroleptic Pt(II) coordination compound [Pt(bpy)(dicnq)](NO
3
) (1) and the
2
quaternized dicnq ligand, namely 12,13-dicyano-5,6-dihydrodipyrazino[2,3-f:1',2',3',4'-
lmn][1,10]phenanthroline-4,7-diium dibromide (2) (Figure 1) were synthesized and fully characterized
by means of FTIR, NMR, MALDI-TOF MS and the purity was confirmed by CHN analyses. The
DNA binding profiles of 1 and 2 were identified in an identical condition. The biological activities of
these compounds were investigated by the assays of transcription and replication inhibition, cytotoxic
and antimicrobial activity.
The result of this study indicates that, both compounds strongly bind to DNA via intercalation but only
1
has a strong nuclease activity. The coordination compound of dicnq (1) binds to the DNA only
slightly stronger than the quaternized form of dicnq (2), and is more potent as an inhibitor of
transcription and replication and therefore, 1 has more potential as an anticancer agent but the
compounds did not show cytotoxic activity against MCF-7 and MDA-MB-231 breast cancer, and
DLD-1 colon cancer cell lines it was found that they only had activities against HepG2 liver cancer
cell line with following IC50 values; 94.75 and 159.60 µM for 1 and 2, respectively. In addition, tested
bacteria are more susceptible to compound 1. These biological activities of 1 may strongly be due to
its ability to digest DNA as a chemical nuclease. According to this study, the quaternization of the
ligand does not make biologically more active than the coordination compound of the same ligand in
this case. The compound (1) is worth further investigation for its antitumor activities.
Keywords: DNA binding, intercalation, dicnq, transcription inhibition, PCR inhibition, cytotoxicity,
antibacterial susceptibility.
Introduction

Cellular DNA is the major target of most antitumor and antibacterial drugs. Therefore, the study of the
binding of a small molecule to DNA has been a very attractive field of research in recent years. It is
extremely important to determine the binding mode of these molecules for the design of new and more
selective DNA-targeted drugs. Molecules can bind to DNA as intercalators, groove binders and
electrostatically external binders. After the success of cisplatin, many platinum complexes have been
synthesized to study their DNA interactions. On the other hand, cationic nitrogen containing
polycyclic aromatic compounds have been known as DNA intercalators such as natural plant alkaloids
or synthetic compounds showing antitumor and antibacterial activities (1, 2). A variety of transition
metal complexes of a known phenanthroline based ligand, dipyridophenazine (dppz) (3, 4) and one of
its important derivatives, 6,7-dicyanodipyrido[2,2-d:2′,3′-f]quinoxaline (dicnq) have been extensively
studied including the crystal structures of Cu(II) and Ag(I) complexes (5), spectroscopic properties of
Au(III) and Pt(II) complexes (6), DNA affinities of Ru(II), Co(II), Ni(II), Mn(II) complexes (7-11). In
the search for new potential DNA binders, a new square-planar Pt(II) coordination compound
[Pt(bpy)(dicnq)](NO
3
2
) (1) and a new cationic derivative obtained by the quaternization of dicnq
ligand, namely 12,13-dicyano-5,6-dihydrodipyrazino[2,3-f:1′,2′,3′,4′-lmn][1,10]phenanthroline-4,7-
diium dibromide (2) (Figure 1) have been synthesized, characterized, their DNA binding profiles were
identified and finally biological activities including inhibition of transcription and replication,
cytotoxicity against MCF-5 and MDA-MB-231 breast, HepG2 liver and DLD-1 colon cancer cell lines
and antimicrobial activities against selected bacteria were investigated.
Material and Methods
The synthesis of 6,7-dicyanodipyrido[2,2-d:2′,3′-f]quinoxaline (dicnq).
An ethanolic solution of 1,10-phenanthroline-5,6-dione (5.0 g, 23.8 mmol) and diaminomaleonitrile
(2.57 g, 23.8 mmol) was heated in the presence of a few drops of acetic acid for 2 h under reflux. The
mixture was allowed to cool to r.t. and the precipitate was filtered and washed with ethyl alcohol to
give an orange-brown solid (3.07 g, 42 %). M.p. 335 °C; FT-IR: 3068 (Ar-CH), 2241 (CN), 1584,

−
1 1
1
570, 1505, 1461, 1448, 1387, 1372, 1332, 1262, 1222, 1141, 1074, 1207, 828, 812, 741, 688 cm ; H
+
NMR (300 MHz, DMSO-d
6
) 9.42 (dd, 2H), 9.36 (dd, 2H), 8.05 (dd, 2H); EI-MS m/z 282 (M ).
Synthesis of Pt(bpy)(dicnq)(NO
3
2
) (1).
A solution of dicnq (0.03 g, 0.10 mmol) in ethanol was added into the aqueous solution of
Pt(bpy)(H O) ](NO (0.11 mmol) which was prepared in situ (12). Then, the mixture was heated
under reflux for 2 h. After cooling down to r.t., the solution was diluted with water (60 mL) and then
filtered out. To this, a saturated aqueous NaNO solution was added at r.t. to allow a yellow precipitate
after a few days (0.048 g, 63 %) [Pt(bpy)(dicnq)](NO : M.p. 375 °C (decomp.). FT-IR: 3057w (OH),
240 (CN), 1584vs, 1504vs, 1440vs, 1286vs, 1271vs, 1252vs, 1140vs, 929, 812, 745 cm ; H NMR:
[
2
2
)
3 2
3
)
3 2
−1 1
2
(200 MHz, DMSO-d
6
, δ): 9.52 (d, 2H), 9.40 (d, 2H), 8.62 (d, 2H), 8.55 (d, 2H), 8.49 (t, 2H), 8.16 (m,
+
2
H), 7.91 (t, 2H). MALDI-TOF MS m/z 758 [M+H] (Calculated for C26
H
14
N
10
O Pt 758.08).
6
16
Calculated for C26H N
10
O Pt (%): C, 40.27; H, 2.08; N, 18.06. Found (%): C, 40.14; H, 2. 4; N, 17.9.
7
Synthesis of 12,13-dicyano-5,6-dihydrodipyrazino[2,3-f:1′,2′,3′,4′-lmn][1,10]-phenanthroline-4,7-
diium dibromide (2).
6,7-dicyanodipyridoquinoxaline (dicnq) (0.03 g, 0.1 mmol) in excess of ethylene bromide (5 mL) was
heated under reflux for 12 h. The mixture was left to cool down to r.t. and the precipitate was collected
by filtration after washing with plenty of acetone and ether to give quarternized dicnq (2) (0.014 g,
46%) as gray-green needles: M.p. 380 °C (decomp.). FT-IR: 3432-3376 (OH), 3035 (Ar-CH), 2936
(Alkyl C-H stretch), 2240 (CN), 1587, 1537, 1497, 1414, 1340, 1310, 1242, 1231, 1160, 1110, 1035,
-
1 1
1
2
1
3
008, 928, 845, 742, 709 cm ; H NMR: (300 MHz, CHCl
3
, δ): 10.3 (d, 2H); 9.79 (d, 2H); 8.81 (dd,
, δ): 156.6, 151.6, 144.0, 137.9, 135.6, 134.4, 133.7,
33.2, 130.1, 128.4, 127.2, 115.7, 53.0. MALDI-TOF MS m/z 311.3 [M+H]; calculated for [M+H],
11.10. Anal. Calc. for C18 : C, 42.71; H, 2.79; N, 16.60 %. Found: C, 42.7; H, 2.9; N, 16.5
13
H); 5.72 (s 4H); C NMR: (300 MHz, CHCl
3
H
14Br
2
N
6
O
2
%.
Spectroscopic measurements. The absorption spectra were recorded by using Varian Cary 100
spectrophotometer. Perkin Elmer LS 55 fluorescence spectrometer was used to record the emission

spectra. For the measurements, 0.5 mL DMSO solutions of the compounds (about 1 mmol) were
prepared and they were diluted to a final concentration of 20 μM by using 5 mM ammonium acetate
buffer (pH 7.5) and used for titrations. The CT-DNA stock solution was added in portions of 1 μL and
the mixture was incubated for 10 min before the spectra were recorded and repeated until the end of
○
the decrease at the spectrum. The temperature was set to 25 ± 0.1 C.
The competitive assays for each compound against ethidium bromide (EtBr) were investigated by
fluorescence spectrophotometry. The EtBr-DNA complex solution (50:1) was titrated with the
compound solutions and the emission peak at 640 nm (ex = 453 nm) was recorded.
Viscosity. The ubbelohde viscometer was used to run the viscosity measurements at a constant
temperature of 30.0 ±0.1 °C. The flow times of the DNA solutions (150 µM) in the presence of
compounds or DNA alone were measured with a stopwatch with three repeats. Data were presented as
1/3
(η/η
0
)
vs binding ratio, where η is the viscosity of DNA in the presence of the compound and η is
0
the viscosity of DNA alone.
Agarose gel electrophoresis. Plasmid DNA (pBR322) was used in the gel electrophoresis
experiments run on 1 % agarose gel in a tris(hydroxymethyl)aminomethane-borate-EDTA (TAE)
running buffer solution. The reaction mixtures were prepared (10 mL) in 50 mM ammonium acetate
○
buffer, pH 7.5 at 0 C containing pBR322 (0.1 μg) and the compounds (0-200 μM) and then incubated
○
at 36 C for 1 h with and without the addition of 2 µL of 30 % hydrogen peroxide solution. A potential
-1
of 35 V for 4 h was applied to the gel at room temperature. The EtBr solution (0.5 μg mL ) for 30 min
was used to stain the gel, soaked in water for 20 min and then visualized under UV light.
Transcription inhibition. RNA synthesis kit and the recommended DNA template from Promega
were used for this experiment. The DNA template was incubated with the compounds before it was
added to the reaction mixture containing rNTPs (ATP, UTP, CTP, and GTP; 25 mM), T7 transcription
buffer, T7 enzyme mix. The mixtures were reacted for 1 h at 37 °C. The products were analyzed by
electrophoresis (30 V, 2 h) using 1 % agarose gels and TAE running buffer solution.

Inhibition of PCR amplification. This procedure is a modified method of PCR stop assay carried out
in invertebrate (honeybee) mitochondrial DNA (mtDNA) region amplifications (13). The PCR
inhibition in invertebrate mtDNA was performed through amplification of two mitochondrial gene
regions (COI- Carboxyl Oxidase I- approx. 1000bp, CytB-Cytochrome B- approx. 500bp) in honeybee
mtDNA separately. The PCR reactions were performed in 1 × PCR mix containing Dream Taq Green
PCR Master Mix (Thermo Scientific, #K1082), 10pmol of each primer pairs (COI-F 5′-
TTAAGATCCCCAGGATCATG-3′, COI-R 5′-TGCAAATACTGCACCTATTG-3′ and CytB-F 5′-
TATGTACTACCATGAGGACAAATATC-3′,
CytB-R
5′-
ATTACACCTCCTAATTTATTAGGAAT-3′) and the total volume completed to 25 µL with PCR
grade pure water. PCR reactions for both honeybee mtDNA regions were incubated in a thermocycler
with the following cycling conditions: 94 °C for 1 min, followed by 35 cycles of 94 °C for 1 min, 50
°C for 2 min and 72 °C for 3 min. Amplified PCR products were resolved on 1 % agarose gel, EtBr
stained and photographed under UV light.
Cell culture. Human breast epithelial adenocarcinoma cancer cell line (MCF-7), human liver
epithelial hepatocellular carcinoma cell line (HepG2), human breast epithelial adenocarcinoma cell
line (MDA-MB-231) and human epithelial colorectal adenocarcinoma cell line (DLD-1) were cultured
in DMEM medium supplemented with 10% FBS and 1% Glutamax. The cells were seeded into sterile
96-well plates at a density of 4 × 103 cells/well and maintained at 37 °C under a humidified
atmosphere containing 5% CO for 24 h. After 24 h and 48 h incubation time, seeding cells were
2
exposed to compounds at nine different concentration following 200, 100, 50, 20, 10, 5, and 5 µM.
Then, 96-well plates were again incubated for 24 h. Then, MTT stock solution (50 µL, 5 mg/mL) was
added to each well and was incubated in an incubator for 4 h. Then, medium was removed and 200 µL
of DMSO was added to each well. The plates were placed on a plate rocker for 30 minutes. The
absorbance values were measured at 560 nm using Promega reader device. IC50 values were
calculated with the GraphPad Prism 7 software program.
Antimicrobial activity of the compounds was determined by the micro-broth-dilution method, as
recommended by the European Committee on Antimicrobial Susceptibility Testing (EUCAST). The

stock solutions were prepared in DMSO at 500 mg/µl and serial two-fold dilutions of each chemical
totally 10 dilutions between 250-0.48 µg/ml) were prepared with the cation-adjusted Mueller-Hinton
(
broth (CAMHB) in sterile 96-well polystyrene U-bottom broth micro-dilution trays. Staphylococcus
aureus ATCC 29213, Enterococcus faecalis ATCC 29212, Escherichia coli ATCC 25922 and
4
Pseudomonas aeruginosa ATCC 27853 were used (10 CFU/well) as test microorganisms.
Cefotaxime was used as the control antimicrobial. The lowest concentration of the compounds and
antimicrobial demonstrating complete inhibition of growth was determined as minimal inhibitor
concentration (MIC). Tests were repeated twice.
Results
Chemistry
The ligand dicnq was synthesized according to the method explained elsewhere (7, 14). The product
1
was obtained in good yield. The FTIR and H NMR data are identical to the literature data. The
-
1
characteristic CN stretching appears at around 2240 cm in the IR spectra of all compounds. The IR
spectra of the cationic derivative 2 and the Pt(II) complex (1) show the OH stretching vibrations at
-
1
around 3400-3600 cm due to the presence of lattice water in both molecules. The observed doublet at
-1
3
432-3376 cm in the spectrum of 2 can be assigned to the hydrogen bonded water molecules in the
lattice (11). The IR spectrum of 2 shows the characteristic stretching of the aliphatic protons at 2936
-1
cm that undoubtedly confirms the attachment of the ethylene group to the tertiary N atoms of the
ligand (dicnq) that is an evidence of the completion of the quaternization. The remaining peaks are
very similar with only slightly shifting of the corresponding CN and CC stretching vibrations as
1
1
expected. In the H NMR, the observed three doublet of doublets at the aromatic region of the H
NMR spectra confirm the symmetrical 1,10-phenanthroline moiety (6, 7, 14). The precursor,
Pt(bpy)Cl
remove the fine powder of AgCl precipitate, the solution was repeatedly filtered and finally
centrifuged. The resulting pale yellow solution of [Pt(bpy)(H O) ](NO was treated with dicnq in
2
(13) was treated with an equimolar amount of AgNO
3
in aqueous solution. In order to
2
2
)
3 2

ethanol, then the mixture was set to reflux for three hours and finally followed by the addition of a
saturated NaNO solution to give [Pt(bpy)(dicnq)](NO (1). The MALDI-TOF MS, the observed
3
)
3 2
molecular mass at m/z 757, confirms the purity and the integrity of 1. The CHN analysis showed that
the complex is monohydrated in the solid state as revealed by the IR which shows the characteristic
-
1
1
vibration bands between 3200 and 3500 cm . In the H NMR spectrum, the aromatic protons (α)
(Table 1) adjacent to imine nitrogen were shifted because of the coordinating platinum atom. The
additional four peaks belonging to the bipyridine ligand further confirms the structure of the complex.
Dicnq in excess of ethylene bromide was heated at reflux under dry nitrogen atmosphere for 3 hours to
-
1
yield the quaternized dicnq (2) as a bromide salt. In the IR spectrum, the peaks at 2240 and 2931 cm
which are attributable to the nitrile and the alkyl groups, respectively, support the structure. The
protonated nitrogen atoms in 2 caused deshielding of the aromatic protons as listed in Table 1. The
1
aliphatic protons which appear at 5.72 ppm in the H NMR spectrum and the aliphatic carbon atoms at
13
5
3 ppm in the C NMR spectrum clearly confirm the completion of the quaternization of the 1,10-
phenanthroline tertiary N atoms in 2. The MALDI-TOF MS further confirms the molecular structure
at m/z 311.3. The CHN analysis confirms that this highly charged compound has two molecules of
water of crystallization in the solid state which is well agreed with the FTIR showing a characteristic
-
1
water stretching band at 3432 cm .
Figure 1
1
Table 1. Comparison of the H NMR chemical shifts of aromatic protons of the dicnq
compounds.
Protons (ppm)
Compounds
solvent
α
[6] 9.38
9.47
β
γ
dicnq
dicnq
q-dicnq
8.03
7.97
8.80
9.38 DMSO-d6
9.52 CDCl
3
(2) 9.80
10.30 CDCl
3

2
+
[
Pt(bpy)dicnq]
(1) 8.55
8.49
9.52 DMSO-d6
DNA binding studies
UV titration experiment is a valuable method to study the interactions of compounds with DNA. An
apparent hypochromism and a bathochromism in the spectrum can be attributed to intercalation mode
of binding to DNA. Figure 2 shows the absorption spectra of the compounds in the absence and
presence of CT-DNA. With the addition of DNA to 1 or 2, the apparent hypochromism with 1-2 nm
red shift in the UV spectrum was observed. The hypochromism was found to be 18 % for 1 and 14 %
for 2 at around 1:1 DNA-to-drug concentration ratio. For a better evaluation and the comparison
between two compounds, the binding constants K were calculated:
b
[DNA]/(ε
A
-ε
f
)= [DNA]/(ε
B
-ε
f
) + 1/K
b
B
(ε -ε
f
)
(1)
where [DNA] is in base pairs, 
and  are absorptivity values of free and the fully bound form of the compound. The binding
constant K is given by the ratio of the slope to the intercept in the [DNA]/( - ) vs [DNA] plot. The

is absorption coefficient of the compound for each measurement, and

f
B
b
A
f
5
−1
calculated binding constants were found 8.2 (± 0.5) and 8.0 (± 0.4) × 10 M . Both compounds have
higher binding affinities against ds-DNA in comparison to the original ligand (11). The platinum(II)
2
+
complex (1) binds stronger than the analogous [Pt(bpy)en] indicating that the addition of dicnq
ligand significantly improves the binding of the complex (15) (Table 2). The quaternized dicnq (2) can
bind to DNA stronger than the free ligand itself (Table 2), probably because of the additional positive
charges on the ligand having potential electrostatic interaction ability with the anionic phosphate
+
groups of the DNA structure. In comparison, the octahedral [Mn(dicnq)(H
2
O)
3
(NO
3
)] complex ion
has a similar binding affinity while 1,10-phenanthroline complexes of Ru(II), Ni(II), Co(III) with
dicnq have lower binding values (Table 1) (7, 10, 11). This may be due to the steric effect of the two
additional phenanthrene rings of these octahedral complexes. The results are also compatible to those
5
−1
of other cationic compounds and the Pt(II) complexes such as q-dppz (K
b
= 3.4 × 10 M ), q-pip (K =
b
5
−1
5
−1
2+
4
-1
2+
4
.4 × 10 M ), and q-hpip (K
b
= 7.5 × 10 M ), [Pt(bpy)(pip)] (K = 2.9 × 10 M ), [Pt(bpy)(hpip)]
b

4
-1
2+
4
-1
2+
4
-1
(K
b
= 5.4 × 10 M ), [Pt(bpy)(iip)] (K
b
= 3.6 × 10 M ), and [Pt(bpy)(miip)] (K = 7.0 × 10 M ) (4,
b
16-19).
Figure 2
Table 2. A comparison of 1 and 2 with dicnq and its other analogous metal complexes in the
literature according to the binding parameters by the UV titration method.
UV-max.
nm)
K
b
Red shift
(nm)
Hypochromism
(%)
Compounds
References
4
-1
(
(×10 M )
dicnq
Pt(dicnq)(bpy)
q-dicnq]
Mn(dicnq)(H
Co(dicnq)(phen)
Ni(dicnq)(phen)
Ru(dicnq)(phen)
Pt(bpy)(en)]
[10]
-
-
[10]
[9]
[9]
[6]
[14]
305
310
451
305
409
369
351
317
17.0
82.0
80.0
50.0
0.6
2.1
3.3
15.6
5
1
2
6
3
5
4
5
18
18
14
24
28
35
16
49
(1)
2
+
[
(2)
+
[
[
[
[
[
2
O)
2
NO
3
]
3
+
2
]
2
+
2
]
2
+
2
]
2
+
Competitive fluorescence studies have been extensively used for the identification of the binding
mode based on the competition between the unknown drug and a known fluorescence dye with a
known binding mode. This is very useful if a dye with high fluorescence emission at the binding site
of DNA reduces its fluorescence intensity when it is subjected to the addition of the unknown drug,
this means that both unknown drug and known dye have the same binding mode to DNA. Figure 3
shows the changes in the fluorescence intensity of ethidium bromide (EtBr) a known intercalator (20)
bonded to DNA with the additions of 1 and 2. Both compounds reduce the fluorescence intensity of
EtBr emission indicating that they bonded to DNA via intercalation or in some cases as groove binders
(21).
The binding constants can be calculated by the classical Stern–Volmer;
0
I /I= 1 + Kapp
(2)

where I
0
and I represent the fluorescence intensities in the absence and presence of a compound; Kapp
/I vs.
compound]/[DNA] (Figure 3). The binding constant of EtBr was used as 4.5×10 M (22), the
is a linear Stern–Volmer quenching constant dependent on the quenching plot of I
0
5
−1
[
5
5
−1
calculated DNA binding constants (Kapp) were 8.8 × 10 and 3.8 × 10 M for 1 and 2, respectively.
These binding values well correlate with those of UV–vis titration.
Figure 3
Viscosity measurements are reliable and precise to length change and an indication of a classical
intercalation (23). Figure 4 shows the effects of EtBr, 1 and 2 on the viscosity of DNA. The classical
intercalator, EtBr, increased the viscosity of DNA. A concentration-depended slight viscosity increase
compares to EtBr indicates that 1 and 2 are intercalating between the DNA base pairs (4).
Figure 4
The gel electrophoresis was used to study DNA cleavage activities of the compounds. Supercoiled
plasmid DNA, pBR322 in ammonium acetate buffer (pH 7.5) was used in this study. Plasmid DNA
pBR322 in its form I resembles a relatively long run on the gel and cleavage on one strand generates
an open circular form (form II) creating a short move on the gel (24). Figure 5 shows agarose gel
electrophoresis assay of pBR322 incubated with each of compounds. With increasing concentration of
1
(Figure 5A, lane 2-3), the amount of form I of pBR322 diminished gradually. Multiple scissions in
the DNA were occurred by 1 and small fragments of DNA cannot be visualized in the gel as indicated
by the previous studies (15, 25, 26). On the other hand, 2 showed no effect on the DNA structure even
at high concentration (Figure 5B). However, in the presence of hydrogen peroxide, single strand DNA
cleavage was observed for pBR322 treated with 2, the form I gradually diminished, while form II

increased. According to these results, 1 exerts extensive damage to the DNA but 2 generates a
moderate cleavage on the DNA backbone when an oxidizing agent is used.
Figure 5
Biological activities
Transcription inhibition: A variety of anticancer agents act as inhibitors of DNA transcription(27-
30). The inhibitors of transcription usually stabilize the double-stranded DNA template by non-
covalent binding and therefore prevent the strand separation. Some transition metal complexes of
planar polypyridyl ligands were shown to inhibit transcription by the degree of their binding to
dsDNA (31, 32). Figure 6 shows a varying concentration of the compounds affecting the amount of
mRNA produced by transcription. The control lanes indicate the maximum amount of the production
with no compounds added. Increasing concentration of both compounds decreased the amount of the
mRNA produced in a concentration depended manner (lanes 3–5). The inhibition of transcription was
more effective by 1, when the concentration of both compounds was 400 μM and over (lanes 5-6)
transcription was completely inhibited, another word, there is no mRNA synthesized. This result is
coherent with the DNA binding studies.
Figure 6
Inhibition of PCR amplification: PCR (Polymerase Chain Reaction) is a revolutionary method
developed by Kary Mullis in the 1980s (33). PCR is based on using the ability of DNA polymerase to
synthesize new strand of DNA complementary to the offered template strand. It is an in vitro DNA
replication reaction. The compounds 1 and 2 were further evaluated by using a PCR assay if they can

stabilize the DNA duplex template, and therefore inhibit DNA polymerase. Figure 7 shows the PCR
final products of the assay mixtures containing compounds. The DNA polymerase inhibition was very
effective for inhibiting the enzyme at a low concentration of 1 for the 500 bp long cyt-B template but
at higher concentration for the 1000 bp long CO-I template. Whereas, 2 is almost inactive for DNA
polymerase even at very high concentrations for both templates.
Figure 7
Cytotoxic activity: Compounds were screened for learning their cytotoxic activity against MCF-7,
HepG2, MDA-MB-231 and DLD-1 cell lines using the MTT assay method for a period of 24 h and 48
h. The IC50 results are given in Table 3. In this study, we firstly screened our samples for 24 h against
breast cancerous cell line (MCF-7). However, IC50 values were obtained higher than 200 µM.
Therefore, the screening time was increased from 24 h to 48 h. However, the values obtained were
again greater than 200 µM. Likewise; they were also tested in MDA-MB-231, HepG2 and DLD-1 cell
lines for 48 h. However, according to above MTT results, compounds 1 and 2 had not efficiency in
two mentioned breast cancerous cell lines and colon cancer cell line, it was found that they only had
activities in the liver cell line (HepG2) with following IC50 values; 94.75 and 159.60 µM for 1 and 2,
respectively. On the other hand, the positive control drugs cisplatin and ploxal-S gave lower IC50
values as 30.38 and 22.25 µM, respectively.
Table 3. IC50 values of the compounds 1 and 2 against cancerous cell lines.
Compounds
IC50 (µM)
MCF-7
4 h
˃200
˃200
9.72
HepG2 MDA-MB-231
DLD-1
2
48 h
˃200
˃200
N.T.*
27.60
48 h
94.75
159.60
30.38
22.25
48 h
˃200
˃200
2.78
4.30
48 h
˃200
˃200
60.79
N.T.*
1
2
Cisplatin
Ploxal-S
N.T.* Not tested
N.T.*

Figure 8
Antimicrobial activity: Identified MIC values of the compounds are given in Table 4. MIC of the
control microorganism (Escherichia coli ATCC 25922) was 0.03 µg/ml for cefotaxime (expected
value; 0.03-0.125 µg/ml).
The platinum(II) complex (1) showed a moderate antimicrobial activity against all microorganisms
tested but 2 has a very weak or no activity. This result is consistent with the nuclease activities of the
compounds. A number of platinum(II) complexes have been also shown to have similar antibacterial
activities (34-36).
Table 4. Identified MIC values of the compounds 1 and 2.
S. aureus
E. faecalis
E. coli
P. aeruginosa
ATCC 27853
Compounds
ATCC 29213 ATCC 29212 ATCC 25922
1
2
31.2
125
15.6
250
31.2
125
31.2
125
Conclusions
Two compounds of dicnq: a square-planar heteroleptic Pt(II) coordination compound
Pt(bpy)(dicnq)(NO (1) and a quaternized dicnq 2,13-dicyano-5,6-dihydrodipyrazino[2,3-f:1',2',3',4'-
)
3 2
lmn][1,10]phenanthroline-4,7-diium dibromide (2) are reported. A number of physicochemical
methods suggested that these compounds strongly bind to DNA via intercalative binding interactions
with different binding affinities. The nuclease activities of the compounds against pBR322 DNA
indicate that 1 severely digests the nucleic acid in low concentrations. On the other hand, 2 itself is
inactive but induces single strand cleavage only in the presence of peroxide. The DNA functions:
transcription and replication are inhibited by 1 and therefore it can be considered as a potential
antitumor agent, both compounds were found to be moderately active against HepG2 liver cancer cell
line and 1 is more active but no activities found against other cell lines tested. On the other hand, 1

also showed weak antibacterial activities against both gram-positive and gram-negative bacteria
whereas 2 showed a very weak or no activity. These biological activities of 1 may be related to the
nuclease activities of this compound. Further studies are required to assess the cytotoxic properties of
1
in vivo against different cell lines to elucidate the actual mechanism of its biological activity.
Acknowledgements
We are grateful for the financial support of Zonguldak Bulent Ecevit University with grant # 2015-
72118496-13. We thank to Ms. Tuğba Demiray and Ms. Sena (Apaydın) Hasanoğlu for the technical
contribution to the project.
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Figure Captions
Figure 1. Synthesis of 1 and 2.
Figure 2. UV-Vis spectra of 1 and 2 upon the addition of CT-DNA in Tris-HCl buffer (5
mM), [Compounds] = 20 μM, [DNA]= 0-20 μM. The change in absorbance was shown by
arrows. Inset: plots of [DNA]/(ε
A
-ε ) vs [DNA].
f
Figure 3. The change in fluorescence spectra of EtBr-DNA complex upon addition of 1 (left)
and 2 (right) (0-22 and 24 μM, respectively). The arrows indicate the changes while the
concentration increasing. Inset: emission-quenching curve of EtBr-DNA complex.
Figure 4. Plot of relative viscosity of CT-DNA vs [compound]/[DNA] in Tris buffer (5 mM
Tris HCl, 25 mM NaCl) at pH 7. Showing EtBr ■, [1] ♦, [2] ▲.
Figure 5. Agarose gel electrophoresis results of pBR322 DNA after treatment with increasing
concentrations of 1 (a), 2 (b). Lane 1, DNA alone; lanes 2–6, DNA was incubated for 1 h with
increasing concentrations (20, 40, 60, 80, 100, 200 µM) of the compounds and (c) Lane 1,
DNA alone; lane 2 DNA+H
2
2
O , lanes 3–9, DNA was incubated for 1 h in the presence of
H
2
O
2
incubated for 1 h with increasing concentrations (20, 40, 60, 80, 100, 200, 400 µM) of
2
.
Figure 6. Transcribed mRNA on the gel electrophoresis in the presence of 1 for A) and 2 for
B). Lane 1: positive control (max. amount of mRNA); lanes 2–6: increasing concentrations of
(
100, 200, 300, 400, and 600 µM).
Figure 7. Effect of 1 and 2 on the hybridization of oligomers (COI-F 5′-TTAAGATCCCCAGGATCATG-3′,
COI-R
5′-TGCAAATACTGCACCTATTG-3′
and
CytB-F
5′-TATGTACTACCATGAGGACAAATATC-3′,
CytB-R
5′-
ATTACACCTCCTAATTTATTAGGAAT-3′) in the PCR assay. The reactions of oligomers with various
concentrations of compounds were assessed. The C lines are controls (no compounds used

and the maximum amount of DNA synthesized) and increasing concentrations of the
compounds (5, 50, 500 μM) were added.
Figure 8. Cell viability ratios of HepG2 depending on concentrations of 1 and 2.

Highlights




[Pt(bpy)(dicnq)](NO
3
) (1) and quaternized dicnq (2) were synthesized and characterized
2
1 and 2 are both good DNA intercalators
1 has strong nuclease, transcriptase and Taq-polimerase inhibitory activities
1 is a potent antimicrobial
1
is cytotoxic against HepG2 Liver cancer cell line

Graphical abstract