DNA binding, photocleavage, antimicrobial and cytotoxic properties of Ru(II) polypyridyl complexes containing BOPIP ligand, (BOPIP = {2-(4-(benzyloxy) phenyl)-1H-imidazo [4,5-f] [1,2]phenanthroline})

Article abstract of DOI:10.1080/15257770.2018.1549329

A novel ligand BOPIP (BOPIP = {2-(4-(benzyloxy)phenyl)-1H-imidazo[4,5-f][1,10]phenanthroline}) and its mononuclear Ru(II) polypyridyl complexes [Ru(phen)₂ BOPIP]²⁺(1) (phen = 1,10-Phenanthrolene), [Ru(bpy)₂ BOPIP]²⁺

Full text of DOI:10.1080/15257770.2018.1549329

Nucleosides, Nucleotides and Nucleic Acids
ISSN: 1525-7770 (Print) 1532-2335 (Online) Journal homepage: https://www.tandfonline.com/loi/lncn20
DNA binding, photocleavage, antimicrobial
and cytotoxic properties of Ru(II) polypyridyl
complexes containing BOPIP ligand, (BOPIPꢀ=ꢀ{2-
(4-(benzyloxy) phenyl)-1H-imidazo [4,5-f]
[1,2]phenanthroline})
Srinivas Gopu, Vuradi Ravi kumar, Kotha Laxma Reddy, Putta Venkat Reddy
& Satyanarayana Sirasani
To cite this article: Srinivas Gopu, Vuradi Ravi kumar, Kotha Laxma Reddy, Putta Venkat Reddy
& Satyanarayana Sirasani (2019): DNA binding, photocleavage, antimicrobial and cytotoxic
properties of Ru(II) polypyridyl complexes containing BOPIP ligand, (BOPIPꢀ=ꢀ{2-(4-(benzyloxy)
phenyl)-1H-imidazo [4,5-f] [1,2]phenanthroline}), Nucleosides, Nucleotides and Nucleic Acids, DOI:
10.1080/15257770.2018.1549329
To link to this article: https://doi.org/10.1080/15257770.2018.1549329
Published online: 19 Mar 2019.
Submit your article to this journal
Article views: 11
View Crossmark data
Full Terms & Conditions of access and use can be found at
https://www.tandfonline.com/action/journalInformation?journalCode=lncn20

NUCLEOSIDES, NUCLEOTIDES AND NUCLEIC ACIDS
https://doi.org/10.1080/15257770.2018.1549329
DNA binding, photocleavage, antimicrobial and
cytotoxic properties of Ru(II) polypyridyl complexes
containing BOPIP ligand, (BOPIP ¼ {2-(4-(benzyloxy)
phenyl)-1H-imidazo [4,5-f] [1,2]phenanthroline})
Srinivas Gopua,b, Vuradi Ravi kumara, Kotha Laxma Reddya,
Putta Venkat Reddya, and Satyanarayana Sirasania
aDepartment of Chemistry, University College of Science, Osmania University, Hyderabad,
b
Telangana State, India; Department of Chemistry, Government Degree College Manthani,
Peddapalli District, Telangana State, India
ABSTRACT
ARTICLE HISTORY
Received 13 August 2018
Accepted 9 November 2018
A novel ligand BOPIP (BOPIP ¼ {2-(4-(benzyloxy)phenyl)-1H-
imidazo[4,5-f][1,10]phenanthroline}) and its mononuclear Ru(II)
polypyridyl complexes [Ru(phen)2 BOPIP]2þ(1) (phen ¼ 1,10-
Phenanthrolene), [Ru(bpy)2 BOPIP]2þ(2) (bpy ¼ 2,20 bipyridyl),
[Ru(dmb)2 BOPIP]2þ(3) (dmb ¼ 4, 40 -dimethyl 2, 20 -bipyridine),
[Ru(Hdpa)2 BOPIP]2þ(4) (Hdpa ¼ 2,20dipyridylamine) have been
synthesized successfully and characterized by elemental ana-
lysis, UV-vis, IR, 1H, 13C-NMR, and ESI-MS Spectroscopy. The
interaction of these complexes with CT-DNA was studied using
absorption, emission techniques, viscosity measurements and
molecular docking studies. The docking study also supports
the binding ability of complexes obtained through the absorp-
tion and emission techniques. These studies reveal that the
Four Ru(II) polypyridyl complexes bind to DNA predominantly
by intercalation. The Antimicrobial activity and cytotoxicity of
these complexes are also reported.
KEYWORDS
Antimicrobial activity; DNA
binding; MTT Assay;
Molecular Docking;
Photocleavage; Viscosity
1. Introduction
Cancer is mostly considered as a group of dreadful diseases, characterized
by uncontrolled cell growth. Cancer, still proven to be one of the unruliest
diseases to which humans are subjected, and as yet no practical and com-
pletely effective drugs or methods to control are available. Hence, identifi-
cation of new effective, selective, and less cytotoxic anticancer agents is still
one of the most pressing health issues.[1–4] DNA, the carrier of genetic
information, has been identified as the primary target for a variety of
anticancer drugs because of their ability to interfere DNA transcription
CONTACT Satyanarayana Sirasani
ssnsirasani@gmail.com
Department of Chemistry, University College of
Science, Osmania University, Hyderabad 500007, Telangana State, India.
Color versions of one or more of the figures in the article can be found online at www.tandfonline.com/lncn.
ß 2019 Taylor & Francis Group, LLC

2
S. GOPU ET AL.
and replication, which are major steps of cell growth and division.[5] Thus,
knowing and understanding drug-DNA interactions is important to com-
prehend the mode of action of any anticancer drug targeting DNA. DNA
offers a number of sites for different covalent and noncovalent interactions
with the drugs.
The field of anticancer metallodrugs is dominated by platinum-based
compounds and the so-called “DNA paradigm”, which presumes that the
mechanism of action of metallodrugs relies on direct DNA damage.[6] The
quest for alternative drugs to the well-known cisplatin and its derivatives,
which are still used in more than 50% of the treatment regimes for patients
suffering from cancer, is highly desirable.[7,8] The development of more
efficient anticancer drugs with improved selectivity and diminished toxic
side effects is currently an area of intense research. With the objective of
developing compounds with a new mode of action in comparison to the
established anticancer drugs cisplatin, carboplatin, and oxaliplatin for treat-
ment of a broader range of tumors and with fewer side effects, many metal
complexes were investigated in recent years for their tumour inhibiting
properties.[9] New metal-based anticancer drugs may be able to widen the
spectrum of treatable cancers, reduce toxic side effects, and overcome plat-
inum resistance.
Ruthenium is the most attractive metal owing to its chemical and air sta-
bility, structural diversity, low toxicity and ability to mimic iron binding in
biological system, which finally supported them as highly potent anticancer
agents rather than platinum-based drugs.[10–12] Due to unique photophysical
properties, ruthenium complexes have been widely applied in DNA probing,
cellular imaging, protein monitoring, and anticancer activity.[13–20] Presently,
ruthenium complex NKP-1339 (trans-[tetrachloridobis (1H–indazole)
ruthenate(III)]) has successfully entered into the clinical trials.[21,22]
Changes in the structure of main ligand could be used to attain diverse
DNA binding ability of ruthenium(II) complexes. Therefore, extensive stud-
ies on different structured ligands are necessary to further elucidate the
DNA binding ability and its mechanism of Ru(II) complexes and discover
some new potential anticancer reagents. In this article, we report the syn-
thesis, characterization, DNA binding, light switching, photocleavage, cyto-
toxicity, and antimicrobial activity studies of the ligand 2-(4-(benzyloxy)
phenyl)-1H-imidazo[4,5-f][1, 10]phenanthroline (BOPIP) and four of its
ruthenium(II) complexes. [Ru(phen)2(BOPIP)]2þ (1), [Ru(bpy)2(BOPIP)]2þ
(2), [Ru(dmb)2(BOPIP)]2þ (3), [Ru(Hdpa)2(BOPIP)]2þ (4) (Scheme 1) The
absorption & emission studies, viscosity measurements, and photocleavage
studies show that the four complexes predominantly interact with DNA by
an intercalative mode. The cytotoxicity of these compounds evaluated by 3-
(4,5-dimethylthiazol-2-yl)2,5-diphenyltetrazolium bromide (MTT) assay.

NUCLEOSIDES, NUCLEOTIDES AND NUCLEIC ACIDS
3
Scheme 1. Schematic synthetic route for the preparation of complexes 1, 2, 3 and 4, Where
1¼ [Ru(Phen)2BOPIP]2þ, 2¼ [Ru(bpy)2 BOPIP]2þ, 3¼ [Ru(dmb)2 BOPIP]2þ, 4¼ [Ru(Hdpa)2 BOPIP]2þ
.
The cytotoxicity studies show that these compounds exhibit efficient activ-
ity against HeLa (human cervical cancer cell line) cell lines in a dose-
dependent manner. The antimicrobial activity experiments show that these
compounds exhibit decent antimicrobial activity.
2. Materials and methods
2.1. Materials
All reagents and solvents of analytical grade were commercial products and
were used as received unless otherwise stated. 1,10-Phenanthroline-5,6-dione,[23]
cis-[Ru(phen)2Cl2]. 2H2O, cis-[Ru(bpy)2Cl2].2H2O, cis-[Ru(dmb)2Cl2].2H2O,[24]
and cis-[Ru(Hdpa)2Cl2].2H2O[25] were synthesized according to literature

4
S. GOPU ET AL.
procedures. 4-(benzyloxy) benzaldehyde, RuCl3.3H2O, and MTT were procured
from Sigma-Aldrich. 1,10-Phenanthroline monohydrate, 2,20-bipyridine (bpy),
4,40-dimethyl-2,20-bipyridine (dmb), and 2,20-dipyridyl amine (Hdpa) were
acquired from Merck. Calf thymus DNA (CT-DNA) was bought from Aldrich,
Supercoiled pBR322 plasmid DNA (stored at ꢀ20 ꢁC) was obtained from
Fermentas Life Sciences and was used as received. Agarose was purchased from
Genei. Ultrapure Milli-Q water (18.2 mX) was used in all experiments and for
preparing various buffers double-distilled water was used. The HeLa human
cervical carcinoma cell line was obtained from NCCS, Pune, and was main-
tained in RPMI 1640 standard (Sigma Aldrich) supplemented with 10% (v/v)
fetal bovine serum, 2 m.mol L-glutamine, 4.5 g L-1 glucose, 19 nonessential
amino acids, and 19 antibiotics consisting of penicillin/streptomycin, gentami-
cin, amphotericin B, and nystatin (basal growth medium). Binding of the com-
plexes with CT-DNA was studied in tris(hydroxymethyl)aminomethane
(Tris)–HCl buffer (5 m.mol Tris–HCl, 50 m.mol NaCl, pH 7.2). A solution of
CT-DNA in Tris–HCl buffer gave a ratio of UV absorbance at 260 and 280 nm
of 1.8:1 to 1.9:1, indicating the DNA was sufficiently free of protein.[26] The
concentration of DNA per nucleotide was determined spectrophotometrically
using a molar absorptivity of 6,600 Mꢀ1 cmꢀ1 (260 nm).[27] Concentrated stock
solutions of CT-DNA were prepared in buffer and were determined by the UV
absorbance at 260 nm after 1:100 dilutions. Stock solutions were stored at 4 ꢁC
and used after not more than 4 days. Concentrated stock solutions of metal
complexes were prepared by dissolving calculated amounts of metal complexes
in DMSO and diluted suitably with the corresponding buffer to the concentra-
tions required for all the experiments.
2.2. Physical measurements
The UV-Vis spectra was recorded on Shimadzu UV-2600 spectrophotom-
eter. Cary Eclipse instrument serial number (MY12400004) Spectro fluor-
ometer was used to record the luminescence spectral data for determining
the binding constant values. IR spectra were recorded on a PerkinElmer
1
1605 Fourier transform IR spectrometer by means of KBr disks. H and
13C NMR spectra were recorded with a Bruker 400-MHz spectrometer with
dimethyl-d6 sulfoxide (DMSO-d6) as the solvent and tetramethylsilane as
the internal standard at room temperature. Elemental microanalysis (C, H,
and N) was conducted by using PerkinElmer 240 elemental analyser.
Electrospray ionization mass spectrometry (ESI–MS) mass spectra were
recorded with a Quattro LC triple quadrupole mass spectrometer fortified
with the MassLynx software program (Micromass, Manchester, UK).

NUCLEOSIDES, NUCLEOTIDES AND NUCLEIC ACIDS
5
2.3. Synthesis and characterization of ligand and complexes
The 1,10-phenanthroline-5,6-dione (Phendione),[23] cis-[Ru(phen)2Cl2], cis-
[Ru(bpy)2Cl2], cis-[Ru(dmb)2Cl2], and cis-[Ru(Hdpa)2Cl2][24,25] were syn-
thesized according to reported literature methods. Schematic diagram of
Ru(II) complexes were shown in Scheme 1.
2.4. Synthesis of ligand [BOPIP]
The ligand was synthesized according to the procedure in the literature.[28]
A mixture of phendione (0.53 g, 2.50 m.mol), 4-(benzyloxy) benzaldehyde
(0.743 g, 3.50 m.mol), ammonium acetate (3.88 g, 50.0 m.mol) is liquified in
glacial acetic acid (25 ml) and the ensuing solution was refluxed for 5h. A
clear wine-red colour solution attained. The above solution was cooled to
room temperature and transferred into distilled water, drop wise addition
of Conc. NH3 form a yellow precipitate, which was collected, washed with
H2O and dried. The crude product recrystallized with C5H5N.H2O and
dried (Yield: 81.04%). Anal. Data for C26H18N4O: Calcd(%): C, 77.59; H,
4.51; N, 13.9; found(%): C, 77.64; H, 4.45; N, 13.76. ES-MS(m/z) Calc: 402;
found: 403 [M þ H]þ. 1H–NMR (DMSO-d6, 400 MHz): d 8.93(d,2H),
8.26(d, 2H), 7.88(m, 5H), 7.44(t, 2H),7.27(d, 2H), 7.1(d,2H), 5.22(s,2H).
13C[1H] NMR (400 MHz, DMSO-d6, ppm): d153.8, 153.1, 140.4, 137, 128.2,
122.6, 115.4, 114.8, 69.8. IR (KBr, cmꢀ1): 3641 (m, N-H), 1118 (m, C-N),
1240 (m, C-O-C).
2.5. Synthesis of complexes
2.5.1. [Ru(phen)2(BOPIP)](ClO4)2.2H2O(1)
Cis-[Ru(Phen)2Cl2].2H2O (0.284 g, 0.5 m.mol), BOPIP (0.201 g, 0.5 m.mol)
dissolved in ethanol (25 ml) plus water (15ml) mixture and refluxed for 8h
at 120 ꢁC under N2 atmosphere. When the light purple colour solution was
obtained, it was cooled to room temperature and an equal volume of
saturated aqueous NaClO4 solution was added under vigorous stirring. The
yellow precipitate was collected and washed with small amounts of water,
ethanol and diethyl ether, then dried under vacuum (yield: 78%). Anal.
data for RuC50H34N8O: calcd (%): C, 69.51; H, 3.97; N, 12.97; found: þC2,
69.62; H, 3.88; N, 12.81. ES-MS(m/z) cal: 864; found: 866 [M þ H]
.
1H–NMR (DMSO-d6, 400 MHz): d 9.06(d,6H), 8.79(d, 6H), 8.21(d, 4H),
8.09(d, 2H),7.79(m, 6H), 7.2(d,2H), 5.25(s,2H). 13C[1H] NMR (400 MHz,
DMSO-d6, ppm): d 160.5, 153.2, 147.7, 137.2, 132.2, 130.9, 128.9, 128.2,
126.8, 122.5, 116.0, 115.7, 69.9. IR (KBr, cmꢀ1): 3475 (m, N-H), 1116 (m, C-N),
1143 (m, C-O-C), and 626 (m, Ru-N).

6
S. GOPU ET AL.
2.5.2. [Ru(bpy)2(BOPIP)](ClO4)2.2H2O(2)
This complex was synthesized by adopting the same procedure as described
above for Complex 1. taking a mixture of cis-[Ru(bpy)2Cl2].2H O (0.260 g,
2
0.5 m.mol), BOPIP (0.201 g, 0.5 m.mol) (yield: 78%). Anal. data for
RuC46H34N8O: calcd(%): C, 67.72; H, 4.20; N, 13.73; found(%): C, 67.82;
1
H, 4.23; N, 13.63. ES-MS(m/z) calc: 816; found: 817 [M þ H] þ1. H–NMR
(DMSO-d6, 400 MHz): d 9.10(d,2H), 8.9(d, 4H), 8.84(d, 2H), 8.11(t,
4H),8.28(t, 4H), 7.86(d, 2H), 7.61(d,4H), 7.44(t, 2H), 7.34(m, 5H),
7.22(d,2H), 5.25(s,2H). 13C[1H] NMR (400 MHz, DMSO-d6, ppm): d 160.6,
157.2, 153.2, 151.8, 138.4, 137.1, 128.9, 128.3, 124.9, 122.5, 116.0, 115.7,
69.9. IR (KBr, cmꢀ1): 3444 (m, N-H), 1078 (m, C-N), 1143 (m, C-O-C), and
626 (m, Ru-N).
2.5.3. [Ru(dmb)2(BOPIP)](ClO4)2.2H2O(3)
This complex was synthesized as described above by taking a mixture of
cis-[Ru(dmb)2Cl2].2H O (0.288 g, 0.5 m.mol), BOPIP (0.201 g, 0.5 m.mol)
2
(yield: 72.71%). Anal. data for RuC50H42NO9: calc. C, 53.82; H, 4.04; Cl,
6.76; N, 12.02; O, 13.73; Ru, 9.64; found: C, 54.01; H, 4.32; Cl, 6.60; þN2,
11.94; O, 13.82; Ru, 9.50. ES-MS(m/z) calc: 1048; found: 1050 [M þ H]
.
1H–NMR (DMSO-d6, 400 MHz): d 8.75(d, 6H), 8.28(d, 2H), 8.07(s, 4H),
7.66(d, 4H), 7.44(t, 2H),7.17(m, 5H), 5.24(s,2H), 2.46(s, 12H). 13C[1H]
NMR (400 MHz, DMSO-d6, ppm): d 160.5, 156.7, 150.0, 137.1, 132.2,
128.9, 128.2, 127.2, 122.6, 115.7, 69.9, 51.0. IR (KBr, cmꢀ1): 3444 (m, N-H),
1133 (m, C-N), 1141 (m, C-O-C), and 624 (m, Ru-N).
2.5.4. [Ru(Hdpa)2(BOPIP)](ClO4)2.2H2O(4)
This complex was synthesized as described above by taking a mixture of
cis-[Ru(Hdpa)2Cl2].2H2O (0.19 g, 0.5 m.mol), BOPIP (0.201 g, 0.5 m.mol)
(yield: 52.71%). Anal. data for RuC47H42Cl2N9O9: calc. C, 53.82; H, 4.04;
Cl, 6.76; N, 12.02; O, 13.73; Ru, 9.64; found: C, 54.01; H, 4.32; Cl, 6.60; þN2,
11.94; O, 13.82; Ru, 9.50. ES-MS(m/z) calc: 1048; found: 1050 [M þ H]
.
1H–NMR (DMSO-d6, 400 MHz): d 8.96(d, 4H), 8.25(d, 2H), 8.05(d, 2H),
7.74(t, 6H), 7.43(d, 2H), 6.97(m, 5H), 6.9(d, 6H), 5.24(s,2H), 4.2(s, 2H).
13C[1H] NMR (100 MHz, DMSO-d6, ppm): d 160.5, 153.8, 153.1, 137.1,
128.9, 128.2, 122.6, 115.9, 69.9. IR (KBr, cmꢀ1): 3444 (m, N-H), 1133 (m, C-
N), 1141 (m, C-O-C), and 624 (m, Ru-N).
2.6. DNA-binding and photocleavage experiments
2.6.1. UV-Visible absorption spectral studies
The DNA-binding studies were conducted at room temperature.
Concentrated stock solutions of metal complexes were prepared by

NUCLEOSIDES, NUCLEOTIDES AND NUCLEIC ACIDS
7
dissolving calculated amounts of metal complexes in DMSO and diluted
accordingly with the corresponding buffer to the concentrations required
for all the experiments. The absorption titrations were performed in
Tris–HCl buffer. The absorption titrations of the complex in buffer were
performed using a fixed complex concentration (20ml), to which increments
of the DNA stock solution was added. Ru–DNA solutions were incubated
for 5 min before the absorption spectra were recorded. The intrinsic bind-
ing constants Kb of these complexes with regard to DNA were calculated
by using the following equation.[29]
½
ꢂ
½
ꢂ
ð
DNA = e ꢀ e ¼ DNA = e ꢀ e þ 1=K e ꢀ e
f Þ fÞ bð fÞ
(1)
ð
a
b
b
where [DNA] is the concentration of DNA, ea, eb and ef correspond to the
apparent absorption extinction coefficient (Aobsd/[complex]), the extinction
coefficient for the complex in the fully bound form and the extinction coef-
ficient for the free complex respectively. The graph was plotted between
[DNA]/(ea-ef) versus [DNA] gave the intrinsic binding constant Kb. The Kb
value obtained from the ratio of slope to the intercept.
2.6.2. Florescence (Luminescence) spectral studies
The luminescence titrations were performed similarly to the absorption
titrations using Tris-HCl buffer. To the fixed metal concentration (10 ml),
various concentrations (10–200 ml) of DNA were added. The binding con-
stant was calculated using Scatchard equation.[30]
Â
Ã
Cb ¼ Ct ðF ꢀ F0Þ=ðFmax ꢀ F0Þ
(2)
where Ct is the total complex concentration, F is the observed fluores-
cence emission intensity at a given DNA concentration, F0 is the inten-
sity in the absence of DNA, and Fmax is when the complex is maximum
bound to DNA. From the Scatchard plot of r/Cf versus r, where r is the
Cb/[DNA] and Cf is the concentration of the free complex, the negative
slope gives the intrinsic binding constant Kb of the complexes based on
the relation
ð
Þ
r=Cf ¼ Kb 1 ꢀ nr
(3)
Quenching studies with [Fe(CN)6]4ꢀ were extended under this lumines-
cence experiment for further understanding the binding ability of these
complexes with DNA. We also observed an interesting thing that these
complexes are exhibiting the light switch on/off effect by taking the same
concentrations of Co2þ and Na2EDTA solutions in ideal concentrations of
complex in fluorescence titrations.

8
S. GOPU ET AL.
2.6.3. Viscosity studies
Ostwald viscometer was used for the viscosity studies, Ostwald viscometer
was immersed in thermo stated water bath maintained a constant tempera-
ture (30 0.1 ꢁC) by using BPE buffer (6 m.mol Na2HPO4, 2 m.mol
NaH2PO4, 1 m.mol Na2EDTA, pH ¼ 7.0). The used CT-DNA samples
approximately 200 base pairs in average length were prepared by sonication
to minimize the complexes arising from DNA flexibility.[31] Using the
digital stopwatch, the flow time was recorded and each sample was
repeated thrice. The recorded data were presented as (g/g0)1/3 versus con-
centration of [Ru(II)]/[DNA], where g is the viscosity of DNA in the pres-
ence of the complex, and g0 is the viscosity of DNA alone. Viscosity values
were calculated from the observed flow time of DNA-containing solutions
(t) corrected for the flow time of the buffer alone (t0).
2.6.4. Photocleavage experiment
For the gel electrophoresis experiments pH 8.0 buffer of 40 m.mol Tris
base, 20 m.mol acetic acid, and 1 m.mol EDTA was used. A buffer of
10 m.mol Tris–HCl and 1 m.mol Na2EDTA was used for dilution of
pBR322 DNA. Supercoiled pBR322 DNA (0.1 mg/mL) was treated with
ruthenium(II) complexes with concentrations of 20, 40, 80 ml, and the mix-
tures were irradiated at room temperature with a UV lamp (365 nm, 10 W)
for 60 min. A loading buffer containing 25% bromophenol blue, 0.25%
xylene cyanole, and 30% glycerol (2 mL) was added. The samples were then
analysed by 0.8% agarose gel electrophoresis at 50 V for 2 h. The gel was
stained with 2 mL (from 1 mg/100 mL) ethidium bromide and photographed
under UV light.[32] The gels were viewed with a gel documentation system
and photographed using a CCD camera (Alpha Innotech).
[CAUTION: Ethidium bromide is a mutagen and potential carcinogen.
Gloves should be worn and care should be taken when handling. UV light is
damaging to eyes and exposed skin. Protective eyewear and apron should be
worn at all times.]
The photocleavage experiments were also performed with singlet oxygen
(1O2) inhibitor Histidine and Hydroxyl free radical (ꢃOH) inhibitor
Mannitol to establish the reactive species responsible for the photoactivated
cleavage of the plasmid.
2.7. Antimicrobial studies
Antimicrobial studies were performed using standard disk diffusion
method.[33] The antibacterial activity of the complexes was studied against
Escherichia coli and Staphylococcus aureus. Each of the ruthenium(II) com-
plex was dissolved in DMSO at different concentrations of 10, 20, and

NUCLEOSIDES, NUCLEOTIDES AND NUCLEIC ACIDS
9
40 mg. Paper disks of Whatman filter paper no. 1 were sterilized in an auto-
clave. The paper disks saturated with 10 mL of the ruthenium(II) complex
were placed aseptically in Petri dishes containing LB agar medium inocu-
lated separately with E. coli and S. aureus. The Petri dishes were incubated
at 37 ꢁC, and the inhibition zones were recorded after 24 h of incubation.
The experiments were repeated twice and the average value was taken. The
results were also compared with the results for the standard antibacterial
drug Ampicillin.
2.8. Molecular docking studies
Accelry’s Discovery Studio (version 2.1) was used to design lead molecules,
estimate the docking interactions of a complex of drug and protein bind-
ing, and number of bonds formed by ligand with the target. The molecular
docking of ruthenium complexes 1, 2, 3 and 4 was performed using
LibDock.[34] LibDock is a high-throughput algorithm for docking ligands
into an active binding site on the receptor, which is also a site-features
docking algorithm. Accelry’s CHARMm force field was used throughout
the simulation before running LibDock. The crystal structure of human
DNA topoisomerase 1 (TOP1) receptor was downloaded from RCSB PDB
(PDB ID-1T8I), after downloading the PDB format of the protein, all the
water molecules of the protein were removed by using Discovery Studio
and stabilizing the charges, filling the missing residues, and generating the
side chains, according to the parameters available. The receptor should be
in a biologically active and stable state. After the receptor is constructed,
the active site within the receptor should be recognized. The receptor may
have many active sites but the one of the interest should be selected.
Ruthenium complexes were sketched using the tools Chemsketch and used
to dock into the target binding site. Ruthenium complex conformations
aligned to receptor interaction sites and the best poses were reported at the
end of docking simulations. The scoring functions have been used to esti-
mate binding affinity to screen out active and inactive compounds during
the process of virtual screening.[35]
2.9. Cytotoxicity assay in vitro (MTT Assay)
Standard MTT assay was conducted as described in the literature.[36] Cells
were placed in 96-well microassay culture plates (8 ꢄ 103 per well) in
200 mL and were grown overnight at 37 ꢁC in a 5% CO2 incubator.
Complexes 1–4, in the concentration range 1–100 mM, dissolved in DMSO
(Sigma-Aldrich), were added to the wells. Control wells were prepared by
addition of culture medium (200 mL). Wells containing culture medium

10
S. GOPU ET AL.
without cells were used as a negative control and cisplatin was used as a
positive control. DMSO was used as the vehicle control. A stock solution
of cisplatin (10 m.mol in DMSO) was freshly prepared for every experi-
ment. After 48 h, 20 mL of MTT solution [5 mg/mL in phosphate-buffered
saline (PBS)] was added to each well and the plates were wrapped in alu-
minium foil and incubated for 4 h at 37 ꢁC. The purple formazan product
was dissolved by addition of 100 mL of 100% DMSO to each well. The
absorbance was monitored at 620 nm using a 96-well plate reader. The
stock solutions of the metal complexes were prepared in DMSO, and in
all experiments, the percentage of DMSO was maintained in the range of
0.1–2%. DMSO by itself was found to be nontoxic to the cells until a con-
centration of 2%. Data were collected for three replicates each to obtain
the mean values. The IC50 values were determined by plotting the per-
centage viability versus concentration on a logarithmic graph and reading
the concentration at which 50% of cells remained viable relative to
the control.
3. Results and discussion
3.1. Electronic absorption titrations
Electronic absorption spectroscopy is the common means to study the
interaction between metal complexes and DNA.[37] For metallointercalators,
DNA binding is associated with hypochromism and a redshift in the metal
to ligand charge transfer (MLCT) and ligand bands.[38] This is primarily
due to the intercalation, involving strong stacking interactions between an
aromatic chromophore and the base pairs of DNA. The extent of the hypo-
chromism in a UV–visible band is consistent with the strength of the inter-
action.[39] Thus, to provide evidence for the possibility of binding of each
complex to CT-DNA, spectroscopic titrations of solutions of each of the
complexes with several concentrations of CT-DNA were examined. A char-
acteristic spectral curve of the complex at different DNA concentrations is
shown in Figure 1. As the DNA concentration is increased, the MLCT
bands of 1 at 453 nm, 2 at 462 nm, 3 at 467 nm, and 4 at 468 nm exhibit
hypochromism of about 14.46, 13.74, 11.64, and 15.01%, respectively, and
bathochromism of about 2–5 nm. To further elucidate the binding strength
of the complexes with regard to DNA, the intrinsic binding constant Kb
was determined in each case by monitoring the changes in their absorbance
in the MLCT band with increasing concentration of CT-DNA. The Kb val-
ues of 1, 2, 3, and 4 are 7.1 ꢄ 104 Mꢀ1, 3.4 ꢄ 104 Mꢀ1, 2.5 ꢄ 104 Mꢀ1, and
8.3 ꢄ 104 Mꢀ1, respectively. The values are smaller than that of those DNA
metallointercalators, such as [Ru(bpy)2(PPIP)]2þ Kb ¼ (4.3 ( 0.40) ꢄ 104
Mꢀ1), [Ru(phen)2(PPIP)]2þ Kb ¼ (1.13 ( 0.30)
ꢄ
105 Mꢀ1) and

NUCLEOSIDES, NUCLEOTIDES AND NUCLEIC ACIDS
11
Figure 1. Absorption spectra of complexes 1–4 in absence and presence of CT-DNA in Tris-HCl
buffer. Arrow shows hypochromism and bathochromism upon the increase of DNA concentra-
tion. Inserted plot,[DNA]/(ea-ef) versus [DNA] for the titration of DNA with Ru(II) complexes,
which gives intrinsic binding constant (Kb).
[Ru(bpy)2(dppz)]2þ (dppz ¼ dipyrido-[3,2-a:20,30-c]phenazine, Kb > 106
Mꢀ1), but bigger than that of the parent complex [Ru(phen)3]2þ
Kb ¼ (5.5 ꢄ 103 Mꢀ1).[38,40,41] Since the intercalator is common in all the
four complexes, the different DNA binding properties of the four com-
plexes are due to their diverse ancillary ligands. Going from bpy to phen,
the planar area and hydrophobicity increases, which may lead to a greater
binding affinity for DNA. The four additional methyl groups in complex 3
relative to complex 2 employ some steric hindrance, thus averting the com-
plex from intercalating as effectively, and so instigating a decrease in the
binding constant. The flexible nonplanar hdpa ligands approach more
closely and coordinate to ruthenium(II) more strongly than the rigid phen
ligands[42] and the NH group in Hdpa may employ some added interac-
tions such as hydrogen bonding with functional groups present on the
edge of the DNA.[43] This would contribute significantly to the greater
binding constant in contrast to the other three complexes. The Kb values of
all the complexes studied are in the order 4 > 1 > 2 > 3.

12
S. GOPU ET AL.
3.2. Luminescence titrations
To further understand the exact nature of the complex binding to DNA, lumi-
nescence titration experiments were performed at a fixed metal complex con-
centration (5 mM) in Tris buffer (pH 7.2) at ambient temperature. The change of
emission intensity is related to the extent to which the complex enters into the
hydrophobic environment inside the DNA. Figure 2 shows the fluorescence exci-
tation and emission spectra for the free and bound complexes 1–4 in the pres-
ence of different amounts of CT-DNA. Excitation wavelengths of 453, 462, 467,
and 468 nm were used for fluorescence measurements of complexes 1, 2, 3, and
4, respectively and emission wavelength found to be 602, 610, 618, and 627 nm.
When the CT-DNA was added to the solution of the complexes 1-4, the fluores-
cence intensity was found to increase. The fluorescence intensities of complexes
1, 2, 3, and 4 increased by 3.26, 3.18, 3.11, and 3.83 times, respectively, compared
with the intensities in the absence of CT-DNA. The emission enhancement of
the complexes 1-4 in the presence of CT-DNA is much smaller than that
observed for complexes [Ru(phen)2(PPIP)]2þ, [Ru(bpy)2(PPIP)]2þ and
[Ru(dmb)2(PPIP)]2þ.[41]
This
implies
that
[Ru(phen)2(PPIP)]2þ,
[Ru(bpy)2(PPIP)]2þ and [Ru(dmb)2(PPIP)]2þ may interact with CT-DNA more
strongly and when the complex intercalates between the DNA base pairs, the
mobility of the complex is restricted at the binding site and the hydrophobic
Figure 2. Emission spectra of complexes 1–4 in Tris-HCl buffer upon addition of CT-DNA. The
arrow shows the intensity change upon the increase of DNA concentration. Inset: Scatchard
plot of above complex, which gives binding constant (Kb).

NUCLEOSIDES, NUCLEOTIDES AND NUCLEIC ACIDS
13
environment inside the DNA helix reduces the accessibility of solvent water mol-
ecules to the complex, leading to a decrease of the vibrational modes of relax-
ation. The intrinsic binding constant from the fluorescence data was obtained
from a modified Scatchard equation[30] through a plot of r/Cf versus r, where r is
the binding ratio Cb/[DNA] and Cf is the free ligand concentration. Scatchard
plots for the complexes were constructed from luminescence spectra, and the
binding constants (Kb) were 7.29ꢄ 104 Mꢀ1, 3.61ꢄ 104 Mꢀ1, 2.57ꢄ 104 Mꢀ1,
and 9.8 ꢄ 104 Mꢀ1 for 1, 2, 3, and 4, respectively. The binding constants calcu-
lated are in comparable with the absorption spectra.
3.2.1. Quenching studies
Steady-state emission quenching experiments using [Fe(CN)6]4- as a quencher
may provide further information about complexes binding to DNA, but cannot
be used to determine the mode of binding. In quenching experiments, to main-
tain the ionic strength so that the quenching curves remain nonlinear, KCl was
added along with K4[Fe(CN)6] such that the final and total concentration was
constant at 4 ꢄ 10ꢀ3 M.[44] The Stern–Volmer quenching constant (Ksv) can
be determined using the Stern-Volmer equation,[45]
Figure 3. Quenching studies of complexes 1–4 in Tris-HCl with [Fe(CN)6]4ꢀ in the absence of
DNA (a), presence of DNA 1:20 (b) and 1:100 (c).

14
S. GOPU ET AL.
Table 1. DNA binding and Ksv data for Ruthenium(II) complexes.
Ksv values
Complex þ DNA
Absorbance
Absorption
kmax (nm)
(MLCT)
Hypo
chromism
(%)
binding
constant
(Kb)
Emission
binding
constant
Only
Complex
Complex
1:50
1:100
[Ru(Phen)2BOPIP]þ2 (1)
[Ru(bpy)2 BOPIP]þ2 (2)
[Ru(dmb)2 BOPIP]þ2 (3)
[Ru(Hdpa)2BOPIP]þ2 (4)
453
462
467
468
14.46
13.74
11.64
15.01
7.1 ꢄ 104
3.4 ꢄ 104
2.5 ꢄ 104
8.3 ꢄ 104
7.29 ꢄ 104
3.61 ꢄ 104
2.57 ꢄ 104
9.8 ꢄ 104
25279
17881
14026
28541
16585
12063
9997
5542
3053
2804
5847
17441
phen: 1,10-phenanthroline, bpy: 2,20-bipyridine, dmb: 4,40-dimethyl-2,20-bipyridine, bpip: 2-(4-(benzyloxy)phenyl)-
1H-imidazo [4,5-f][1,10]phenanthroline, hdpa: 2,20,-dipyridylamine, MLCT: metal-to-ligand charge transfer.
I0=I ¼ 1 þ KSV½Qꢂ
where I0 and I are the intensities of the fluorophore in the absence and
presence of the quencher, respectively, [Q] is the concentration of the
quencher, and Ksv is the linear Stern–Volmer quenching constant. In gen-
eral, positively charged free complex ions may be readily quenched by
[Fe(CN)6]4-, whereas the complex bound to DNA can be protected from
the quencher as the negative charge of [Fe(CN)6]4- will be repelled by the
negatively charged phosphate backbone of DNA, resulting in less quench-
ing of the bound complex compared with the free complex. Figure 3. shows
the Stern–Volmer plots for the free complexes in solution and the com-
plexes in the presence of increasing amounts of DNA. The Ksv values for
all four complexes are given in Table 1. From the quenching studies it is
clear that the DNA binding affinity of complexes follows the order
4 > 1>2 > 3, which is consistent with other results.[38,40,46]
3.2.2. On–off–On light switching behaviour
As shown in Figure 4 the emission spectral profile of DNA bound complex 1
elucidates the switching of emission on and off when Co2þ and EDTA are
added, respectively. The experiments were conducted using a method similar to
that developed by our research group earlier.[38,46] When the complex binds to
DNA (switch on), the emission intensity is high, but when we add Co2þ
(0.03 m.mol), the emission of DNA-bound complex 1 is quenched by Co2þ,
thus turning the light switch off,[47,48] owing to the formation of the
Co2þ–complex 1 heterometallic complex. When EDTA (0.03 m.mol) was added
to the buffer system containing Co2þ–complex 1, the emission intensity recov-
ered again (light switch on), based on the strong coordination of Co2þ to
EDTA (EDTA– Co2þ) and the complex becomes free. A similar observation
was made for other three complexes. The change in luminescence of the DNA-
bound complex in the presence of Co2þ and EDTA reveals its use in the
modulation of drug therapy.

NUCLEOSIDES, NUCLEOTIDES AND NUCLEIC ACIDS
15
Figure 4. DNA light switch on and off experimentally showing the luminescence changes upon
addition of Co2þ, EDTA and DNA to complex 1.
3.3. Viscosity studies
The DNA binding modes of complexes were further investigated by viscosity
measurement. The viscosity measurements of DNA is regarded as the least
uncertain and the critical test of a DNA binding model in solution in the
absence of crystallographic data and provides strong evidence for intercala-
tive DNA binding mode.[31,49] A classical intercalation model results in
lengthening the DNA helix as base pairs are detached to accommodate the
binding ligand, leading to the increase of DNA viscosity. In contrast, a par-
tial non-classical intercalation of ligand could bend (or kink) the DNA helix
and reduce its effective length.[50] For example, under suitable conditions,
intercalation of dye like EtBr roots a significant increase in the overall DNA
length. The effects of the complexes on the viscosity of rod-like DNA com-
paring with EtBr are shown in Figure 5. Though the intercalating ligand is
same in all complexes, there is a small difference in the viscosity, this is due
to the difference in the ancillary ligands. These further suggest that four
Ru(II) complexes show an intercalative binding mode to CT-DNA, which
parallel the absorption titration results. The increased degree of viscosity also
supports the order of binding of the complexes to DNA as determined by
other methods which follow the order EB >4 > 1>2 > 3 (Figure 5).
3.4. Photocleavage of pBR322 DNA
The cleavage reactions on plasmid DNA induced by ruthenium(II) com-
plexes were performed and monitored by agarose gel electrophoresis. When
circular plasmid DNA is subjected to electrophoresis, comparatively fast
migration is observed for the intact supercoiled form (form I). If scission

16
S. GOPU ET AL.
Figure 5. Viscosity studies of four complexes in BPE buffer with increasing amounts of com-
plexes 1-4 and Ethidium bromide (EtBr) on the relative viscosity of calf thymus DNA at room
temperature, a ¼ EtBr, 1 ¼ [Ru(Hdpa)2BOPIP]2þ, 2 ¼ [Ru(Phen)2BOPIP]2þ, 3 ¼ [Ru(bpy)2 BOPIP]2þ
,
4 ¼ [Ru(dmb)2 BOPIP]2þ
.
Figure 6. Photoactivated cleavage of pBR322 DNA in the absence (control) and presence of dif-
ferent concentrations (20, 40 and 80 mM) 0f ruthenium complexes (1–4) after irradiation under
UV light for 30 minutes.
occurs on one strand (nicking), the supercoiled form will relax to generate a
slower-moving open circular form (form II). If both strands are cleaved, a
linear form (form III) that migrates between form I and form II will be gen-
erated.[32] Figure 6 shows gel electrophoresis separation of pBR322DNA after
incubation with different concentrations of ruthenium(II) complexes and
irradiation at 365nm for 60 min. No DNA cleavage was observed for the
control, in which the metal complex was absent. When the concentration of
the ruthenium(II) complexes was increased, the amount of form I gradually

NUCLEOSIDES, NUCLEOTIDES AND NUCLEIC ACIDS
17
decreased, whereas the amount of form II increased. Under comparable
experimental conditions, all complexes showed photocleavage activity. The
pBR322 DNA photocleavage results for these complexes are consistent with
the results obtained for other ruthenium(II) polypyridyl complexes.[51,52] To
establish the reactive species responsible for the photoactivated cleavage of
the plasmid, we further investigated the influence of potentially inhibitive
agents. Histidine, a naturally occurring amino acid, has been widely used as
a scavenger of singlet molecular oxygen (1O2) especially during biological
1
photooxidation processes.[53] As reported O2 reacts with histidine to form a
transannular peroxide in its imidazole ring and thus loses its ability to react
with other species. Histidine is also one of the most reactive biomolecules
1
with regard to O2 and exists in the muscle of animal tissues. In the presence
of histidine (10 m.mol) (Figure 9), cleavage was absent (form II is not
observed) or very much reduced compared what was observed for the com-
1
plexes with DNA (absence of histidine). This indicates that O2 plays an
important role in the photocleavage mechanism. A photocleavage experiment
was also conducted in the presence of mannitol, an OH radical inhibitor
(Figure 7). In the presence of mannitol, form II is formed; hence, there is no
change in the cleavage pattern, which indicates that the OH, radical is not
1
responsible for cleavage and only O2 is responsible for photocleavage of
pBR322 in presence of the ruthenium(II) complexes.
3.5. Antimicrobial activity
Complexes 1–4 were screened in vitro for their microbial activity against E.
coli and S. aureus at 1 mg mL-1 concentration by the standard disk
Figure 7. Photoactivated cleavage of pBR322 DNA in the presence of [Ru(Phen)2BOPIP]2þ com-
plex after irradiation at 365 nm for 30 min in the presence of histidine and mannitol.

18
S. GOPU ET AL.
Table 2. Antibacterial activity of ruthenium(II) complexes.
Escherichia coli
(Gram negative)
Staphylococcus aureus
(Gram positive)
Compound
10mg
20mg
40mg
10mg
20mg
40mg
BOPIP
3
4
10
9
8
7
[Ru(Phen)2BOPIP]þ2 (1)
[Ru(bpy)2 BOPIP]þ2 (2)
[Ru(dmb)2 BOPIP]þ2 (3)
[Ru(Hdpa)2BOPIP]þ2 (4)
Ampicillin
6.5
6.0
5.5
5.0
10.0
8.0
9.2
12
10
11.5
10.4
18.0
12
11
11.7
11.2
14.0
12.2
13.5
12.5
21
8.7
Inhibition zone diameter in millimetres.
method. The results are expressed as inhibition zone diameter (in milli-
metres) versus the control (DMSO). The DMSO control showed negligible
activity as compared with the metal complexes. The antimicrobial activity
increased as the concentration of the compounds increased. The antibacter-
ial activity data for the complexes at various concentrations (Table 2) indi-
cate that the complexes exhibited appreciable activity against E. coli and S.
aureus. The activity increased with the increase in the concentrations of the
complexes. The complexes were more effective against E. coli than against
S. aureus but were less effective than the standard drug ampicillin. As an
increase in the lipophilic character of the complex favors its permeation
through the lipid layer of the bacterial membrane, it shows more activity.
These results are consistent with results from earlier studies.[54,55]
3.6. Molecular docking studies
Molecular docking studies The LibDock module from Discovery Studio was
used to perform the molecular docking of ruthenium complexes 1, 2, 3 and 4
with the active site pocket residues of human DNA TOP1. Human DNA
TOP1 is an essential enzyme that relaxes DNA supercoiling during replication
and transcription. The topoisomerase enzymes have been researched as targets
for the generation of new cancer treatments because when they are inhibited
in a cell, cell death results. Therefore, inhibitors of the topoisomerase enzymes
have the ability to kill all cells undergoing DNA replication, reading of the
DNA for protein production, or experiencing repair of DNA damage.
Subsequently, cancer cells divide much more rapidly than normal cells, the
cancer cells will be slaughtered by the topoisomerase inhibitors, however,
some normal cells with topoisomerase activity will also be killed. DNA TOP1
is overexpressed in tumor cells and is an important target in cancer chemo-
therapy. All the ruthenium complexes were docked into the active site pocket
of DNA TOP1, using LibDock. According to the results obtained from
LibDock simulation, all ruthenium complexes were ranked by the LibDock
scores. From the results, complex 4 exhibited the highest docking scores of
137.942 kcal/mol (Figure 8). The interactions and Dock scores of the

NUCLEOSIDES, NUCLEOTIDES AND NUCLEIC ACIDS
19
Figure 8. Molecular docking models illustrating the interaction between complexes with active
site pocket residues of human DNA topoisomerase 1 (PDB ID: 1T8I) target and showing inter-
molecular hydrogen bonds.
Table 3. The LibDock scores and docking interactions of the ruthenium complexes (1–4) with
human DNA TOP 1.
Libdock Score
Complex
(K.Cal/Mole)
Interacting Residues
Interacting atoms
H-Distance
2.2600
1
121.159
DC8,DT9,DG10, Gly478, Asp479,
Met782, Arg503, Gln778
5:H67 - F:DA12:O4’ complex
2
115.942
116.893
DC8, DT9, DG10, Gly478, Asp479,
Met782, Arg503, Gln778
complex:H63 - F:DA12:N3
complex:H63 - F:DA12:C2
DC8:H42 - O49 complex
complex:H68 - F:DA12:N3
complex:H62 - F:DA12:C1’
complex:H62 - F:DA12:C2’
complex:C7 - A:ARG503:HH11
B:DT10:H3 – complex: O49
1.764
2.205
2.392
1.967
2.154
1.9070
2.0590
2.061
3
DC8, DT9, DG10, Gly478, Asp479,
Met782, Arg503, Gln778
4
137.942
DT10, DC112, DA113, TGP11,
Asn722, Asp533,

20
S. GOPU ET AL.
Figure 9. Cell viability of HeLa cell lines invitro treatment with complexes 1, 2, 3 and 4. Each
data point is the mean standard error obtained from at least three independent experiment.
Table 4. The IC50 values for complexes 1–4 against HeLa cell lines.
S.No.
Compound
IC50 (mM)
1
2
3
4
5
[Ru(Phen)2BOPIP]þ2 (1)
[Ru(bpy)2 BOPIP]þ2 (2)
[Ru(dmb)2 BOPIP]þ2 (3)
[Ru(Hdpa)2BOPIP]þ2(4)
Cisplatin
27.76
31.59
36.42
24.38
4.81
ruthenium complexes with the active site pocket residues of human DNA TOP1
were tabulated in Table 3. The active site pocket residues of human DNA TOP1
were involved in hydrogen bonding formation with ruthenium complexes. A
higher score indicates a stronger receptor–ligand-binding affinity.
3.7. In vitro cytotoxicity
The cytotoxicity activity of all four complexes and the corresponding ligand
against the HeLa (human cervical cancer cell line) cell lines was evaluated by
MTT assay. Cisplatin was used as a positive control and DMSO as negative
control. The IC50 values obtained for four complexes are shown in Table 4.
The tumor cells in the presence of complexes 1–4 were incubated for 48 h.
The IC50 values for all the complexes ranged from 1 to 100mM, suggesting
that the ligand and the complexes exhibited antitumor activity against HeLa
cell lines to different degrees. These compounds all exhibit relatively lower in
vitro cytotoxicity against the selected HeLa cell line than cisplatin. Figure 9
showed that the cell viability decreased with increasing concentrations of
complexes 1, 2, 3 and 4. Among all these, complex 4 exhibited higher in
vitro cytotoxicity, with IC50 values of 24.38. This is may be due to the pres-
ence of an amine group (–NH–) between two pyridine moieties in Hdpa.[25]

NUCLEOSIDES, NUCLEOTIDES AND NUCLEIC ACIDS
21
The cytotoxicity activity of the complexes is consistent with their DNA bind-
ing abilities i.e. 4 > 1>2 > 3. The obtained IC50 values are also comparable
with the reported ruthenium (II) polypyridyl complexes.[56]
Conclusion
Four Ru(II) complexes [Ru(phen)2 BOPIP]2þ(1), [Ru(bpy)2 BOPIP]2þ(2)
[Ru(dmb)2 BOPIP]2þ(3), [Ru(Hdpa)2 BOPIP]2þ(4) were synthesized and
characterized. The absorption spectral studies, Luminescence titrations, and
viscosity measurements suggest that all the four complexes bind to CT-
DNA through intercalation. The intrinsic binding constants calculated
through absorption studies and fluorescence spectral studies are good in
agreement and complex 4 exhibits slightly higher intrinsic binding constant
among four complexes. Upon irradiation, under UV light all the four com-
plexes can cleave pBR322 DNA and proved that singlet oxygen(1O2) is
responsible for the cleavage of pBR322 DNA. All the four complexes
exhibit the Antimicrobial activity and showed cytotoxicity against A549
(human lung tumor cell line), Du145 (human prostate cancer cell line),
and HeLa (human cervical cancer cell line) cell lines. These complexes
exhibit relatively lower in vitro cytotoxicity against the selected cell lines
than cisplatin. Molecular docking studies support the Hydrogen bonding
and Vander Wall’s interactions play a major role in binding to DNA.
Acknowledgments
The University Grants Commission, New Delhi, India, is gratefully acknowledged for the
support in the form of Teacher Fellowship under Faculty Development programme to one
of the authors. We also extend our sincere thanks to CFRD Osmania University for provid-
ing instrumentation facilities.
References
[1] Wu, J.; Wu, S.; Shi, L.; Zhang, S.; Ren, J.; Yao, S.; Yun, D.; Huang, L.; Wang, J.; Li,
W.; et al. Design, Synthesis, and Evaluation of Asymmetric EF24 Analogues as
Potential Anti-cancer Agents for Lung Cancer. Eur. J. Med. Chem. 2017, 125,
1321–1331. DOI: 10.1016/j.ejmech.2016.10.027.
[2] Komor, A. C.; Barton, J. K. The Path for Metal Complexes to a DNA Target. Chem.
Commun. (Camb.) 2013, 49, 3617–3630.
[3] Lin, R.; Johnson, S. G.; Connolly, P. J.; Wetter, S. K.; Binnun, E.; Hughes, T. V.;
Murray, W. V.; Pandey, N. B.; Moreno-Mazza, S. J.; Adams, M.; et al. Synthesis and
Evaluation of 2,7-diamino-thiazolo[4,5-d] Pyrimidine Analogues as Anti-tumor
Epidermal Growth Factor Receptor (EGFR) tyrosine Kinase Inhibitors. Bioorg. Med.
Chem. Lett. 2009, 19, 2333–2337. DOI: 10.1016/j.bmcl.2009.02.067.
[4] Zheng, Y.; Zhu, L.; Fan, L.; Zhao, W.; Wang, J.; Hao, X.; Zhu, Y.; Hu, X.; Yuan, Y.;
Shao, J.; Wang, W. Synthesis, SAR and Pharmacological Characterization of Novel

22
S. GOPU ET AL.
Anthraquinone Cation Compounds as Potential Anticancer Agents. Eur. J. Med.
Chem. 2017, 125, 902–913. DOI: 10.1016/j.ejmech.2016.10.012.
[5] Syam, S.; Abdelwahab, S. I.; Al-Mamary, M. A.; Mohan, S. Synthesis of Chalcones
with Anticancer Activities. Molecules 2012, 17, 6179–6195.
[6] Hartley, J. A.; Hochhauser, D. Small Molecule Drugs - Optimizing DNA Damaging
Agent-Based Therapeutics. Curr. Opin. Pharmacol. 2012, 12, 398–402.
[7] Lippert, B. Cisplatin Chemistry and Biochemistry of a Leading Anticancer Drug
Wiley; Weinheim: Germany, 1999.
[8] Jakupec, M. A.; Galanski, M.; Arion, V. B.; Hartinger, C. G.; Keppler, B. K.
Antitumour Metal Compounds: More than Theme and Variations. Dalton Trans
2008, 2, 183–194. DOI: 10.1039/B712656P.
[9] Dyson, P. J.; Sava, G. Metal-Based Antitumour Drugs in the Post Genomic Era.
Dalton Trans. 2006, 16, 1929–1933. DOI: 10.1039/b601840h.
[10] Mendoza-Ferri, M. G.; Hartinger, C. G.; Mendoza, M. A.; Groessl, M.; Egger, A. E.;
Eichinger, R. E.; Mangrum, J. B.; Farrell, N. P.; Maruszak, M.; Bednarski, P. J.; et al.
Transfering the Concept of Multinuclearity to Ruthenium Complexes for
Improvement of Anticancer Activity. J. Med. Chem. 2009, 52, 916–925. DOI:
10.1021/jm8013234.
[11] Hartinger, C. G.; Metzler-Nolte, N.; Dyson, P. J. Challenges and Opportunities in
the Development of Organometallic Anticancer Drugs. Organometallics 2012, 31,
5677–5685. DOI: 10.1021/om300373t.
[12] Gasser, G.; Ott, I.; Metzler-Nolte, N. Organometallic Anticancer Compounds. J.
Med. Chem. 2011, 54, 3–25.
[13] Gill, M. R.; Garcia-Lara, J.; Foster, S. J.; Smythe, C.; Battaglia, G.; Thomas, J. A. A
Ruthenium(II) polypyridyl Complex for Direct Imaging of DNA Structure in Living
Cells. Nat. Chem. 2009, 1, 662–667.
[14] Vyas, N. A.; Ramteke, S. N.; Kumbhar, A. S.; Kulkarni, P. P.; Jani, V.; Sonawane,
U. B.; Joshi, R. R.; Joshi, B.; Erxleben, A. Ruthenium(II) polypyridyl Complexes with
Hydrophobic Ancillary Ligand as Ab Aggregation Inhibitors. Eur. J. Med. Chem.
2016, 121, 793–802. DOI: 10.1016/j.ejmech.2016.06.038.
[15] Puckett, C. A.; Ernst, R. J.; Barton, J. K. Exploring the Cellular Accumulation of
Metal Complexes. Dalton Trans. 2010, 39, 1159–1170.
ꢀ
[16] Cook, N. P.; Torres, V.; Jain, D.; Martı, A. A. Sensing Amyloid-b Aggregation Using
Luminescent Dipyridophenazine Ruthenium(II) Complexes. J. Am. Chem. Soc. 2011,
133, 11121–11123. DOI: 10.1021/ja204656r.
[17] Chen, Y.; Qin, M. Y.; Wu, J. H.; Wang, L.; Chao, H.; Ji, L. N.; Xu, A. L. Synthesis,
Characterization, and Anticancer Activity of Ruthenium(II)-b-Carboline
complexEur. Eur. J. Med. Chem. 2013, 70, 120–129.
[18] Liu, P.; Jia, J.; Zhao, Y.; Wang, K. Z. Recent Advances on Dark and Light-Activated
Cytotoxity of Imidazole-Containing Ruthenium Complexes. Mini Rev. Med. Chem.
2016, 16, 272–289.
[19] Poynton, F. E.; Bright, S. A.; Blasco, S.; Williams, D. C.; Kelly, J. M.; Gunnlaugsson,
T. The Development of Ruthenium(ii) Polypyridyl Complexes and Conjugates for in
vitro Cellular and in vivo Applications. Chem. Soc. Rev. 2017, 46, 7706.
[20] Vuradi, R. K.; Dandu, K.; Kumar, Y. P.; Vinoda Rani, M.; Rajender Reddy, M.;
Chintakuntla, N.; Ravi, C.; Thakur, S. S.; Rao, C. M.; Satyanarayana, S. Studies on
the DNA Binding and Anticancer Activity of Ru(II) polypyridyl Complexes by
Using
a
(2-(4-(Diethoxymethyl)-1H-imidazo[4,5-f][1,10]
phenanthroline))
Intercalative Ligand. New J. Chem. 2018, 42, 846. DOI: 10.1039/C7NJ03819D.

NUCLEOSIDES, NUCLEOTIDES AND NUCLEIC ACIDS
23
[21] Muhammad, N.; Guo, Z. Metal-based Anticancer Chemotherapeutic Agents. Curr.
Opin. Chem. Biol. 2014, 19, 144–153. DOI: 10.1016/j.cbpa.2014.02.003.
[22] Sava, G.; Bergamo, A.; Bonetti, A.; Leone, R.; Muggia, F.M.; Howell, S.B. (Eds.).
Synthesis and in Vitro (Anticancer) Evaluation of g 6 -Arene Ruthenium Complexes
Bearing Stannyl Ligands; Humana Press: Totowa, 2009,57–66.
[23] Yamada, M.; Nakamura, Y.; Hasegawa, T.; Itoh, A.; Kuroda, S.; Shimao, I. Synthesis
and Properties of Diamino-Substituted Dipyrido [3,2-a: 20,30-c]Phenazine. Bull.
Chem. Soc. Japan 1992, 65, 2007–2009.
[24] Sullivan, B. P.; Sullivan, D. J.; Salmon, B. P.; Mayer, T. J. Mixed Phosphine 2,2’-
Bipyridine Complexes of Ruthenium. Inorg. Chem. 1978, 17, 3334–3341. DOI:
10.1021/ic50190a006.
[25] Rajendran, V.; Murali, M.; Suresh, E.; Sinha, S.; Somasundaram, K.; Palaniandavar,
M. Mixed Ligand Ruthenium (II) Complexes of bis(pyrid-2-yl)-/bis(benzimidazol-2-
yl)-Dithioether and Diimines: Study of Non-covalent DNA Binding and
Cytotoxicity. Dalton Trans. 2008, 7(1),148–163. DOI: 10.1039/B710578A.
[26] Marmur, J. A Procedure for the Isolation of Deoxyribonucleic Acid from Micro-
Organisms. J. Mol. Bio. 1961, 3, 208–218. DOI: 10.1016/S0022-2836(61)80047-8.
[27] Reichmann, M. E.; Rice, S. A.; Thomas, C. A.; Doty, P. A Further Examination of
the Molecular Weight and Size of Desoxypentose Nucleic Acid. J. Am. Chem. Soc.
1954, 76, 3047–3053. DOI: 10.1021/ja01640a067.
[28] Steck, E. A.; Day, A. R. Reactions of Phenanthraquinone and Retenequinone with
Aldehydes and Ammonium Acetate in Acetic Acid Solution. J. Am. Chem. Soc.
1943, 65, 452–456.
[29] Wolfe, A.; Shimer, G. H.; Meehan, T. Polycyclic Aromatic Hydrocarbons Physically
Intercalate into Duplex Regions of Denatured DNA. Biochemistry 1987, 26,
6392–6396. DOI: 10.1021/bi00394a013.
[30] Mc Ghee, J. D.; Von Hippel, P. H. 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. 1974, 86, 469–489. DOI: 10.1016/0022-
2836(74)90031-X.
[31] Chaires, J. B.; Dattagupta, N.; Crothers, D. M. Selfassociation of Daunomycin.
Biochemistry 1982, 21, 3927–3932. DOI: 10.1021/bi00260a004.
[32] Barton, J. K.; Raphael, A. L. Photoactivated Stereospecific Cleavage of Double-helical
DNA by Cobalt(III) Complexes. J. Am. Chem. Soc. 1984, 106, 2466–2468. DOI:
10.1021/ja00320a058.
[33] Drew, W. L.; Barry, A. L.; Toole, R. O.; Sherris, J. C. Reliability of the Kirby-Bauer
Disc Diffusion Method for Detecting Methicillin-Resistant Strains of Staphylococcus
aureus. Appl. Microbiol. 1972, 24, 240–247.
[34] Diller, D. J.; Merz , K. M. Jr. High Throughput Docking for Library Design and
Library Prioritization. Proteins 2001, 43, 113–124.
[35] Jones, G.; Willet, P.; Glen, R. C. Molecular Recognition of Receptor Sites Using a
Genetic Algorithm with a Description of Desolvation. J. Mol. Biol. 1995, 245, 43–53.
DOI: 10.1016/S0022-2836(95)80037-9.
[36] Mosmann, T. Rapid Colorimetric Assay for Cellular Growth and Survival:
Application to Proliferation and Cytotoxicity Assays. J. Immunol. Methods 1983, 65,
45–55. DOI: 10.1016/0022-1759(83)90303-4.
[37] Barton, J. K.; Danishefsky, A.; Goldberg, J. Tris(phenanthroline)ruthenium(II):
Stereoselectivity in Binding to DNA. J. Am. Chem. Soc. 1984, 106, 2172–2176. DOI:
10.1021/ja00319a043.

24
S. GOPU ET AL.
[38] Pyle, A. M.; Rehmann, J. P.; Meshoyrer, R.; Kumar, C. V.; Turro, N. J.; Barton, J. K.
Mixed-ligand Complexes of Ruthenium(II): Factors Governing Binding to DNA. J.
Am. Chem. Soc. 1989, 111, 3051–3058. DOI: 10.1021/ja00190a046.
[39] Moucheron, C.; Mesmaeker, A. K. D.; Choua, S. Photophysics of
Ru(phen)2(PHEHAT)2þ: A Novel “Light Switch” for DNA and Photo-oxidant for
Mononucleotides. Inorg. Chem. 1997, 36, 584–592. DOI: 10.1021/ic9609315.
[40] Deepika, N.; Praveen Kumar, Y.; Shobha Devi, Ch.; Venkat Reddy, P.; Srishailam,
A.; Satyanarayana, S. Synthesis, characterization, and DNA binding, photocleavage,
cytotoxicity, cellular uptake, apoptosis, and on-off light switching studies of Ru(II)
mixed-ligand complexes containing 7-fluorodipyrido[3,2-a:20,30c]phenazine. J Biol
Inorg Chem, 2013, 18, 751–766.
[41] Tan, L.-F.; Chao, H.; Li, H.; Liu, Y.-J.; Sun, B.; Wei, W.; Ji, L.-N. Synthesis,
Characterization, DNA-Binding and Photocleavage Studies of [Ru(bpy)2(PPIP)]2þ
and [Ru(phen)2(PPIP)]2þ. J. Inorg. Biochem. 2005, 99, 513–528. DOI: 10.1016/
j.jinorgbio.2004.10.028.
[42] Ravi Kumar, V.; Nagababu, P.; Srinivas, G.; Reddy, M. R.; Rani, M. V.; Ravi, M.;
Satyanarayana, S. Investigation of DNA/BSA Binding of Three Ru(II) complexes by
Various Spectroscopic Methods, molecular Docking and Their Antimicrobial Activity.
J. Coord. Chem. 2017, 70, 3790–3809. DOI: 10.1080/00958972.2017.1407410.
[43] Tu, L. C.; Chen, C. S.; Hsiao, I. C.; Chern, J. W.; Lin, C. H.; Shen, Y. C.; Yeh, S. F.
Synthesis of 1-Substituted Carbazolyl-1,2,3,4-tetrahydro- and Carbazolyl-3,4-dihydro-
b-carboline Analogs as Potential Antitumor Agents. Chem. Biol. 2005, 12, 1317–1324.
[44] Ang, W. H.; Dyson, P. J. Classical and Non-Classical Ruthenium-Based Anticancer
Drugs: Towards Targeted Chemotherapy. Eur. J. Inorg. Chem. 2006, 4003–4018.
DOI: 10.1002/ejic.200600723.
[45] Satyanarayana, S.; Dabrowiak, J. C.; Chaires, J. B. Tris(phenanthroline)ruthenium(II)
enantiomer Interactions with DNA: mode and Specificity of Binding. Biochemistry
1993, 32, 2573–2584. DOI: 10.1021/bi00061a015.
[46] Lakowicz, J. R.; Weber, G. Quenching of Fluorescence by Oxygen. Probe for
Structural Fluctuations in Macromolecules. Biochemistry 1973, 12, 4161–4170. DOI:
10.1021/bi00745a020.
[47] Shobhadevi, C.; Satyanarayana, S. Review: Synthesis, characterization, and DNA-
binding Properties of Ru(II) molecular “light Switch” Complexes. J. Coord. Chem.
2012, 65, 474–486. DOI: 10.1080/00958972.2011.649736.
[48] Liu, X. W.; Shen, Y. M.; Lu, J. L.; Chen, Y. D.; Li, L.; Zhang, D. S. Synthesis, DNA-
binding and Photocleavage of “light Switch” complexes [Ru(bpy)2(pyip)]2þand
[Ru(phen)2(pyip)]2þ. Spectrochim. Acta. Part A. 2010, 77, 522–527.
[49] Chen, M.; Li, H.; Li, Q.; Xu, Z. Luminescence Properties of [Ru(bpy)(2)
MDHIP](2þ) modulated by the introduction of DNA, copper(II) ion and EDTA.
Spectrochim. Acta A Mol. Biomol. Spectrosc. 2010, 75, 1566–1570.
[50] Satyanarayana, S.; Dabrowiak, J. C.; Chaires, J. B. Neither DELTA.- nor.LAMBDA.-
Tris(phenanthroline)Ruthenium(II) Binds to DNA by Classical Intercalation.
Biochemistry 1992, 31, 9319–9324. DOI: 10.1021/bi00154a001.
[51] Satyanarayana, S.; Suh, D.; Fokt, I.; Przewloka, T.; Priebe, W.; Chaires, J. B. Parsing
the Free Energy of Anthracycline Antibiotic Binding to DNA. Biochemistry 1996,
35, 2047–2053.
[52] Liu, Y. J.; Zeng, C. H.; Huang, H. L.; He, L. X.; Wu, F. H. Synthesis, DNA-binding,
photocleavage, cytotoxicity and Antioxidant Activity of Ruthenium (II) polypyridyl
Complexes. Eur. J. Med. Chem. 2010, 45, 564–571.

NUCLEOSIDES, NUCLEOTIDES AND NUCLEIC ACIDS
25
[53] Huang, H. L.; Li, Z. Z.; Liang, Z. H.; Liu, Y. J. Cell Cycle Arrest, Cytotoxicity,
Apoptosis, DNA-Binding, Photocleavage, and Antioxidant Activity of Octahedral
Ruthenium(II) Complexes. Eur. J. Inorg. Chem. 2011, 2011, 5538–5547. DOI:
10.1002/ejic.201100848.
[54] Tan, L. F.; Liang, X. L.; Liu, X. H. Synthesis, double Stranded DNA-binding and
Photocleavage Studies of a Functionalized Ruthenium(II) complex with 7,70-methyle-
nedioxyphenyldipyrido[3,2-a:20,30-c]-Phenazine. J. Inorg. Biochem. 2009, 103, 441–447.
[55] Islam, M. S.; Hossain, M. B.; Reza, M. Y. Synthesis, Characterization and
Antibacterial Activity of Mixed Ligand Complexes of Pd(II) Ions with Oxalic Acid
and Heterocyclic Amines. J. Med. Sci. 2003, 3, 289–293.
[56] Morad, F. M.; Ajaily, M. M. E. L.; Gweirif, S. B. Preparation, Physical
Characterization and Antibacterial Activity of Ni (II) Schiff Base Complex. J. Sci.
Appl. 2007, 1, 72–78.
[57] Venkat Reddy, P.; Nagamani, C.; Reddy M, R.; Srishailam, A.; Nagasuryaprasad, K.;
Deepika, N.; Yashvanth, V. V. N.; Prakasham, R. S.; Satyanrayana, S. S.; Satyanarayana,
S. Synthesis and Evaluation of Invitro DNA/Protein Binding Affinity, Antimicrobial,
Antioxidant and Antitumor Activity of Mononuclear Ru (II) Moxed Polypyridyl
Complexes. J. Fluoresc. 2016, 26, 225–240. DOI: 10.1007/s10895-015-1705-z.