Organoruthenium (II) complexes featuring pyrazole-linked thiosemicarbazone ligands: Synthesis, DNA/BSA interactions, molecular docking, and cytotoxicity studies

Article abstract of DOI:10.1002/aoc.6343

A series of pyrazol-derived thiosemicarbazone ligands (L1–L4) were synthesized and reacted with [Ru(p-cymene)(μ-Cl)Cl]₂ to yield a series of “piano-stool”-type binuclear ruthenium (II)–arene-thiosemicarbazone complexes (C1–C8) of the general type [(Ru(η⁶-p-cym)L)₂(μ-im/azpy)] Cl_1–2 (L = diphenylpyrazole thiosemicarbazone; cym = p-cymene; im = imidazole; azpy = 4,4′-azopyridine). The thiosemicarbazone ligands act as N and S donors binding to the Ru(II) center via the imine nitrogen and the thione sulfur atoms. The complexes were characterized by NMR, FTIR, UV–Vis spectroscopy, and ESI⁺ mass spectrometry. The binding of the complexes to calf thymus deoxyribonucleic acid (CT-DNA) and bovine serum albumin (BSA) was evaluated, and it has been established that the binuclear complexes have good binding efficacies with DNA (K_b?= 10⁴–10⁵?M^?1) and BSA (K_a?= 10⁵–10⁶?M^?1). This is attributed to the arene moieties present in the ligands of the complexes that can have hydrophobic interactions with DNA/BSA. Ethidium bromide (EB) displacement studies and DNA viscosity measurements revealed intercalative interaction of the complexes with DNA. Static interaction of the complexes with BSA was revealed by fluorescence quenching studies. Molecular docking studies confirmed base stacking, H-bonding, and hydrophobic interactions with the biomolecules. In vitro antiproliferative studies of the complexes affirmed that the complexes are cytotoxic towards the HeLa (human cervical cancer) cell line with IC₅₀ values in range of 17.3–41.3?μM.

Full text of DOI:10.1002/aoc.6343

Received: 17 February 2021
DOI: 10.1002/aoc.6343
Revised: 27 May 2021
Accepted: 31 May 2021
F U L L P A P E R
Organoruthenium (II) complexes featuring pyrazole-linked
thiosemicarbazone ligands: Synthesis, DNA/BSA
interactions, molecular docking, and cytotoxicity studies
1
2
3
Priyanka Khanvilkar
| Soumya R. Dash
| Devjani Banerjee
|
4
4
1
Aliasgar Vohra
| Ranjitsinh Devkar
| Debjani Chakraborty
1
Department of Chemistry, The Maharaja
Sayajirao University of Baroda, Vadodara,
India
Abstract
A series of pyrazol-derived thiosemicarbazone ligands (L1–L4) were
2
Physical and Material Chemistry
synthesized and reacted with [Ru(p-cymene)(μ-Cl)Cl] to yield a series of
2
Division, CSIR-NCL Pune, Pune, India
3
“
piano-stool”-type
binuclear
ruthenium
(II)–arene-thiosemicarbazone
Cell and Molecular Biology Division, The
6
complexes (C1–C8) of the general type [(Ru(η -p-cym)L) (μ-im/azpy)] Cl
2
1–2
Maharaja Sayajirao University of Baroda,
Vadodara, India
(L = diphenylpyrazole thiosemicarbazone; cym = p-cymene; im = imidazole;
4
0
Department of Zoology, The Maharaja
azpy = 4,4 -azopyridine). The thiosemicarbazone ligands act as N and S donors
Sayajirao University of Baroda, Vadodara,
India
binding to the Ru(II) center via the imine nitrogen and the thione sulfur
atoms. The complexes were characterized by NMR, FTIR, UV–Vis spectros-
+
Correspondence
copy, and ESI mass spectrometry. The binding of the complexes to calf
Debjani Chakraborty, Department of
Chemistry, Faculty of Science, The M S
University of Baroda, Vadodara, India.
Email: debjani.chakraborty-chem@
msubaroda.ac.in
thymus deoxyribonucleic acid (CT-DNA) and bovine serum albumin (BSA)
was evaluated, and it has been established that the binuclear complexes
4
5
ꢀ1
have good binding efficacies with DNA (Kb = 10 –10
M ) and BSA
5
6
ꢀ1
(
K = 10 –10 M ). This is attributed to the arene moieties present in the
a
Funding information
ligands of the complexes that can have hydrophobic interactions with
DNA/BSA. Ethidium bromide (EB) displacement studies and DNA viscosity
measurements revealed intercalative interaction of the complexes with DNA.
Static interaction of the complexes with BSA was revealed by fluorescence
quenching studies. Molecular docking studies confirmed base stacking,
H-bonding, and hydrophobic interactions with the biomolecules. In vitro
antiproliferative studies of the complexes affirmed that the complexes are
cytotoxic towards the HeLa (human cervical cancer) cell line with IC50 values
in range of 17.3–41.3 μM.
DST-PURSE, Grant/Award Number:
FS/206/19-20; RFSMS-BSR, Grant/Award
Number: F.5-71/2007(BSR); University
Grants Commission, Grant/Award
Number: UGC F.No. 43-238/2014(SR)
K E Y W O R D S
binuclear ruthenium (II) complexes, DNA/BSA binding interactions, HeLa human cervical
carcinoma, pyrazole-derived thiosemicarbazone
1
| INTRODUCTION
already in use as drugs for therapeutic purposes. Therefore,
their design and synthesis is an important field of research.
Pyrazoles represent an important pharmacophore with
diverse biological activities, and some derivatives are
Modification of the substituents at positions 1, 3, and 5 has
resulted into new pyrazole derivatives with wide spectrum
Appl Organomet Chem. 2021;e6343.
wileyonlinelibrary.com/journal/aoc
© 2021 John Wiley & Sons, Ltd.
1 of 17
https://doi.org/10.1002/aoc.6343

2
of 17
KHANVILKAR ET AL.
biological activities. Structural changes at the different posi-
tions of the basic molecule results in better pharmacologi-
cal activities, providing it with antimicrobial,
anticonvulsant, analgesic, anti-inflammatory, antiviral,
antimalarial, and anticancer properties. Pyrazole-
pyrimidine derivatives have shown antiproliferative activity
against NCl-H226 (human lung carcinoma), NPC-TW01
family,
triapine
(3-aminopyridine-2-carboxaldehyde
thiosemicarbazone), was extensively investigated in
numerous clinical phase I and II trials in mono or com-
bination therapies. Ribonucleotide reductases are the
0
primary cellular target of Triapine. The TSCs, N -(6,7-
dihydroquinolin-8(5H)-ylidene)-4-(pyridin-2-yl)piperazine-
1-carbothiohydrazide (COTI-2), and di-2-pyridylketone-
4-cyclohexyl-4-methyl-3-thiosemicarbazone (DpC) have
entered the human clinical trials, renewing interest in
this class of therapeutically useful compounds.
It was observed that tethering two different biologi-
cally active moieties can bestow the resulting compound
(
nasopharyngeal cancer), T-cell leukemia (Jurkat) cells,
[1–3]
and a group of other human cancer cell lines.
A series
[12,13]
of pyrazole–sulfonamide conjugates with antiproliferative
activity against HeLa and C6 cell lines was reported
[4]
by Mert et al.
Via et al. reported antiproliferative
[
14]
activities of benzothiopyranopyrazole derivatives exerted
with new biological properties.
A conjugation of TSC
[5]
on HeLa and HL-60 cells. A phenyl, p-chlorophenyl, or
p-methoxyphenyl substituent at 1-position and a methoxy
substituent at the 7-position of the heterocyclic moiety was
found to be responsible for the activities.
and pyrazol derivatives can result into a potent compo-
nent for anticancer and other therapeutic drugs. In
addition, biological properties of thiosemicarbazones
have been found to be augmented by coordination to the
[
15,16]
In vitro antiproliferative activities of a series of
functionally substituted pyrazoles, on a panel of 60 cell
metal ions.
This approach was utilized by combin-
[17]
ing the Ru–arene fragment to thiosemicarbazones.
[6]
0
lines was investigated by Nitulescu et al. N-benzoyl-N -
The piano-stool-type Ru(II)-arene-based compounds
(
3-(4-bromophenyl)-1H-pyrazol-5-yl)-thiourea
showed
are envisaged as potential and compelling candidates for
[7]
[18,19]
promising results. Prasad et al.
investigated novel
cancer treatment.
The arene moiety provides signifi-
4
,5-dihydropyrazole derivatives that were found to be
cant pharmacological properties to the complexes,
namely, impact in cellular uptake, enhanced hydropho-
bicity, and biomolecular recognition process leading to
improved passive transport across the cell membrane. It
is evident that the interaction of metal complexes with
DNA or protein is enhanced due to the π-stacking of the
arene ligand that explains their biological activity and
more effective than cis-platin. The derivative with an
electron-withdrawing chloro group in the 4-position of
the lipophilic heterocycle exhibited high anticancer activ-
ity (IC50 = 4.94 mM) against HeLa cell lines. Rai et al.
reported the synthesis and anticancer activity of a series
of pyrazole chalcones against MCF-7 and HeLa cell
lines. The 4-fluoro-phenyl and 5-fluoro-pyridine deriva-
tives showed the highest inhibition in human MCF-7 and
HeLa cell lines. Two comprehensive reviews by Ansari
[8]
[20]
various pharmacological properties.
The
dicyanamidobenzene-bridged
diruthenium
complex
[{Ru(tpy)(thd)} (μ-dicyd)][PF ]{dicyd = 1,4
2
6
[9]
[10]
0
0
00
et al. and Karrouchi et al.
highlight the different
-dicyanamidobenzene, tpy = 2,2 :6 ,2 -terpyridine, thd = 2,
synthesis methods and the pharmacological properties of
pyrazole derivatives developed by many researchers
around the globe.
2,6,6-tetramethyl-3,5-heptanedione} was synthesized and
[
21]
characterized by X-ray crystallography.
XRD studies
of the binuclear Ru(II)-arene-triphenylphosphine com-
plex with terephthalic acid as bridging ligand were
carried by Honorato et al. The bidentate O–O coordina-
tion mode of the carboxylate groups was unambiguously
Thiosemicarbazones (TSCs) are well known for their
versatility and various pharmacological properties includ-
ing anticancer activity. Heterocyclic thiosemicarbazones
have gained considerable interest in medicinal chemistry
due to their ability to diffuse through the semipermeable
membrane of the cell lines. They show a range of biological
activities including antitubercular, antibacterial, antimalar-
ial, antileprosy, antiparasitic, antineoplastic, antiviral,
[
22]
confirmed by the X-ray technique.
This paper reports the design, synthesis, and biologi-
cal investigation of novel binuclear ruthenium (II)-arene
complexes (C1–C8) containing pyrazole substituted
thiosemicarbazone ligands (L1–L4).
[11]
antiproliferative, antioxidant, and antitumor activities.
Thiosemicarbazones (TSCs) are inhibitors of the
iron containing enzyme ribonucleotide reductase,
2 | EXPERIMENTAL SECTION
2.1 | Materials and instrumentation
which catalyzes the conversion of ribonucleotides to
0
2
-deoxyribonucleotides and hence is essential for DNA
[12]
synthesis and repair.
The first discovered representa-
tive of this class of compounds was 2-formylpyridine
thiosemicarbazone that exhibited potent anticancer
activity. Another well-known representative of this
The chemicals and solvents used for synthesis and
characterization of the complexes were of analytical
grade. Pyrazole carboxaldehyde was purchased from

KHANVILKAR ET AL.
3 of 17
Sigma-Aldrich; Thiosemicarbazide was purchased from
SRL (Sisco Research Laboratory, Mumbai, India). The
(0.01 M) in methanol with a small amount of glacial
acetic acid added was refluxed for 10 h. The progress of
the reaction was monitored by TLC. After completion
of the reaction, the mixture was poured into crushed ice;
the separated product was filtered off, washed with cold
methanol, and dried under vacuum. The ligands were
recrystallized in methanol, and pure white crystalline
product was obtained. The yield and spectral data of
ligands are discussed below.
6
precursor [Ru(η -p-cymene)Cl ] was prepared according
2
2
[23,24]
to the procedure cited in literature.
CT-DNA, tri-
sodium citrate, and EB (ethidium bromide) were pur-
chased from SRL (Sisco Research Laboratory, Mumbai,
India). RuCl ꢁ3H O and BSA (bovine serum albumin)
3
2
were purchased from Hi-media. HeLa cell line was
obtained from National Centre for Cell Science (NCCS),
Pune, India. ESI mass spectra of the complexes were
recorded on Applied Biosystem API 2000 Mass spectrom-
eter. Infrared spectra (400–4000 cmꢀ1) were recorded on
2.2.1 | 1-((1,3-Diphenyl-1H-pyrazol-4-yl)
methylene)thiosemicarbazone (L1)
1
α-Bruker FTIR with samples prepared as KBr pellets. H
NMR spectra were recorded on a Bruker AR X 400 Spec-
trometer at 400 MHz using DMSO as solvent. C, H, and
N elemental analysis were performed on a PerkinElmer
L1 was synthesized by condensation reaction of
thiosemicarbazide (2 mmol, 183 mg) and 1,3-diphenyl-
1H-pyrazole-4-carboxaldehyde (2 mmol, 498 mg). Solubil-
ity: MeOH, DMSO, DMF; yield 79.6%; molecular weight
321.4 g/mol; molecular formula C H N S; color: white;
2
40B elemental analyzer. UV spectra were recorded in
ꢀ
6
DMSO solution at concentrations in the range 10
–
ꢀ
3
1
0
M on Perkin Elmer Lambda-35 dual beam UV–Vis
17
15 5
spectrophotometer. Fluorescence spectra were recorded
in solution on JASCO FP-6300 fluorescence spectropho-
tometer. To evaluate the data obtained from DNA/BSA
interaction (titration) experiments, the OriginPro 8 soft-
ware was employed. ORCA program package (version
anal.: found: C, 63.27; H, 4.32; N, 21.39. Calc.: C,
63.53; H, 4.70; N, 21.79; MS m/z: obs (calc): 322.2 (321.4)
+
1
(M + 1); H-NMR (DMSO-d ): δppm 7.37, (tri, 1H, Ar–H);
6
7.42–7.51, (m, 5H, Ar–H); 7.77, (d, 2H, J = 6.8 Hz, Ar–H);
7.28, (s, 1H, Ar–H); 7.79, (d, 2H, J = 7.6 Hz, Ar–H); 8.03,
4
.0.1.2) was used for geometry optimization.
7.79 (s, 2H, NH ); 8.36 (s, 1H, HC=N); 10.07, (s, 1H,
2
ꢀ
1
N–NH); IR (KBr, cm ): ν
356, 3280; ν(C=N) 1599; ν(N–N) 1053; ν(C=S)assym 1291;
2883, ν(NNH) 3434; ν(NH2)
Ar)C–H
(
3
2
.2 | General procedure for the
preparation of pyrazol thiosemicarbazones
L1–L4)
ν(C=S)sym 816.
(
2.2.2 | 1-((1,3-Diphenyl-1H-pyrazol-4-yl)
The ligands were synthesized and characterized
methylene)-4-methyl-3-thiosemicarbazone (L2)
[25]
according to the literature (Scheme 1).
equimolar amount of a substituted thiosemicarbazide
0.01 M) and 1,3-diphenyl-1H-pyrazole-4-carboxaldehyde
In brief, an
Prepared by condensation of 4-methyl-3-thiosemicarbazide
(1.9 mmol, 200 mg) and 1,3-diphenyl-1H-pyrazole-
(
SCHEME 1 General synthetic route to diphenyl pyrazol thiosemicarbazones L1–L4

4
of 17
KHANVILKAR ET AL.
4
-carboxaldehyde (1.9 mmol, 472 mg). Solubility: MeOH,
2.3 | General synthetic procedure of
6
DMSO, DMF; yield 80.4%; molecular weight 335.4 g/mol;
molecular formula C H N S; color: pale yellow; anal.:
found: C, 64.11; H, 4.72; N, 20.51. Calc.: C, 64.45; H,
[(Ru(η -p-cym)L) (μ-im/azpy)]Cl
2
1–2
complexes: (C1–C8)
18
17 5
+
5
.11; N, 20.88; MS m/z: obs (calc): 337.1 (335.4) (M + 2);
The ligand L in 2.5-ml methanol was added to a solution
1
6
H-NMR (DMSO-d ): δppm 7.47, (tri, 1H, Ar–H);
of [Ru(η -p-cymene)Cl ] in 2.5-ml CH Cl in the molar
6
2 2
2
2
7
.49–7.59, (m, 5H, Ar–H); 7.69, (d, 2H, J = 6.8 Hz, Ar–H);
ratio 2:1, and the reaction mixture was stirred overnight
at room temperature. Slow evaporation of the solution
yielded the reddish brown mononuclear complex [Ru(η -
p-cym)(L)]Cl, which was filtered, washed with pet ether
8
.22, (s, 1H, Ar–H); 7.91, (d, 2H, J = 7.6 Hz, Ar–H); 8.28,
6
(s, 1H, NH–CH ); 3.03, (s, 3H, N–CH ); 9.08 (s, 1H,
3
3
ꢀ
1
HC=N); 11.41 (s, 1H, N–NH); IR (KBr, cm ): ν
(Ar)C–H
2932, ν(NN–H) 3389; ν(NH–CH3) 3325; ν(C=N) 1598; ν(N–N)
and CH Cl , and dried. The bridging ligand imidazole or
2
2
0
1054; ν(C=S)assym 1266; ν(C=S)sym 823.
4,4 -azopyridine was added to the mononuclear complex
dissolved in a mixture of 5 ml of CH Cl and 3 ml of
2
2
CH OH in 1:2 ratios. To deprotonate the ring –NH, 1.0-M
3
2
.2.3 | 1-((1,3-Diphenyl-1H-pyrazol-4-yl)
NaOH solution was added after addition of the imidazole
[
26]
methylene)-4-phenyl-3-thiosemicarbazone (L3)
ligand.
The solution was stirred for 24 h at room tem-
perature under N atmosphere. The reddish brown solid
2
Condensation of 4-phenyl-3-thiosemicarbazide (2.9 mmol,
obtained was filtered, washed with pet ether, and dried
in oven at 40 C for 1 h. The complexes so obtained were
ꢂ
5
00 mg) and 1,3-diphenyl-1H-pyrazole-4-carboxaldehyde
(
2.9 mmol, 742 mg) yielded L3. Solubility: MeOH, DMSO,
recrystallized from dichloromethane and ether. Scheme 2
shows the general synthetic route for preparation of the
complexes.
DMF; yield 81.06%; molecular weight 397.5 g/mol; molec-
ular formula C H N S; color: whitish yellow; anal.:
23
19 5
found: C, 69.10; H, 4.47; N, 17.39. Calc.: C, 69.50; H,
+
4
.82; N, 17.62; MS m/z: obs (calc): 398.1 (397.5) (M + 1);
1
H-NMR (DMSO-d ): δppm 7.38, (tri, 1H, Ar–H);
2.4 | Chemical characterization data of
the complexes
6
7
8
7
.50–7.58, (m, 5H, Ar–H); 7.72, (d, 2H, J = 6.7 Hz, Ar–H);
.34, (s, 1H, Ar–H); 7.92, (d, 2H, J = 7.4 Hz, Ar–H);
.21–7.49, (5H, m, N–C H ); 9.86, (s, 1H, NH–C H ); 9.27,
6
2.4.1 | [(Ru(η -p-cym)L1) (μ-im)]Cl (C1)
6
5
6
5
2
ꢀ
1
(
s, 1H, HC=N); 11.77, (s, 1H, N–NH); IR (KBr, cm ): ν
(Ar)
2
964; ν(NN–H) 3335; ν(NH–C6H5) 3290; ν(C=N) 1597; ν(N–N)
Yield: 62.9%; molecular weight 1214.9 g/mol; molecular
formula C H ClN Ru S ; anal.: found: C, 55.99; H,
C–H
1
065; ν(C=S)assym 1268; ν(C=S)sym 824.
57
60
12
2 2
4
.78; N, 13.77. Calc.: C, 56.36; H, 4.98; N, 13.84. ESI-MS
+
m/z: obs (calc): 1179.27 (1179.5) (M ); δ (400 MHz,
H
2
.2.4 | 1-((1,3-Diphenyl-1H-pyrazol-4-yl)
methylene)-4-(naphthalen-1-yl)-
-thiosemicarbazone (L4)
DMSO-d ) 7.38, (tri, 1H, Ar–H); 7.55, (m, 5H, Ar–H);
6
7.69, (d, 2H, J = 6.8 Hz, Ar–H); 7.77, (s, 1H, Ar–H); 7.83,
3
(d, 2H, J = 7.6 Hz, Ar–H); 8.22, (s, 2H, NH ); 8.69, (s, 1H,
2
HC=N); 5.82–5.77; (m, 4H, p-cym Ar–H); 2.84–2.50,
(q, 1H, p-cym-iso-prop-CH); 1.97, (s, 3H, p-cym Ar–CH3);
1.05, (d, 6H, p-cym-iso-prop-(CH ) ); 7.93 (d, 2H, imidaz-
The synthesis of L4 was carried out by condensation
of 4-(1-naphthyl)-3-thiosemicarbazide (1.6 mmol,
3
1
2
ꢀ
3
50 mg) and 1,3-diphenyl-1H-pyrazole-4-carboxaldehyde
ole CH=CH); FTIR (KBr/cm ): ν(
2959, ν
Ar)C–H
(NH2)
ꢀ
1
2
ꢀ1
(1.6 mmol, 399 mg). Solubility: MeOH, DMSO, DMF;
3050, ν(C=N) 1588, ν(C–S)sym 770; Ʌ (Ω ꢁm ꢁM ) 75.
M
yield 85.4%; molecular weight 447.2 g/mol; molecular
formula C H N S; color: yellow; anal.: found: C,
27
21 5
6
7
1
2.22; H, 4.38; N, 15.41. Calc.: C, 72.46; H, 4.73; N,
5.65; MS m/z: obs (calc): 448.1 (447.2) (M + 1);
2.4.2 | [(Ru(η -p-cym)L2) (μ-im)]Cl (C2)
2
+
1
H-NMR (DMSO-d ): δppm 7.34, (tri, 1H, Ar–H);
Yield: 77.5%; molecular weight 1243.0 g/mol; molecular
formula C H ClN Ru S ; anal. found: C, 56.61; H,
6
7
.56–7.58, (m, 5H, Ar–H); 7.75, (d, 2H, J = 6.8 Hz,
Ar–H); 7.75, (s, 1H, Ar–H); 7.88, (d, 2H, J = 8.6 Hz,
Ar–H); 7.46–7.56, (7H, m, N–C H ); 10.17,
59
64
12
2 2
4.89; N, 13.17. Calc.: C, 57.01; H, 5.19; N, 13.52. ESI-MS
+
m/z: obs (calc): 1207.7 (1207.5) (M ); δ (400 MHz,
10
7
H
(
(
3
s, 1H, NH–C H ); 9.26, (s, 1H, HC=N); 11.88,
DMSO-d ) 7.50, (tri, 1H, Ar–H); 7.59, (m, 5H, Ar–H);
10
7
6
s, 1H, N–NH); IR (KBr, cmꢀ ): ν(Ar)C–H 2971; ν(NN–H)
1
7.68, (d, 2H, J = 6.8 Hz, Ar–H); 7.79, (s, 1H, Ar–H); 7.80,
334; ν(NH–C10H7) 3123; ν(C=N) 1597; ν(N–N) 1047; ν(C=S)
(d, 2H, J = 7.6 Hz, Ar–H); 7.92, (s, 1H, NH–CH ); 8.22,
3
1276; ν(C=S)sym 807.
(s, 1H, HC=N); 5.82–5.73, (m, 4H, p-cym Ar–H);
assym

KHANVILKAR ET AL.
5 of 17
3 6 5 7
SCHEME 2 General synthetic route to complexes C1–C8, n = 1, 2 and R = H, CH , C H , C10H
2
.95–2.49, (q, 1H, p-cym-iso-prop-CH); 1.98, (s, 3H,
3.79; N, 11.86. Calc.: C, 63.03; H, 4.95; N, 11.46. ESI-MS
m/z: obs (calc): 1433.2 (1431.4) (M + 1); δH (400 MHz,
+
p-cym Ar–CH ); 1.02, (d, 6H, p-cym-iso-prop-(CH ) );
3
3 2
ꢀ
1
7
.92, (d, 2H, imidazole CH=CH); FTIR (KBr/cm ): ν(Ar)
DMSO-d ) 7.37, (tri, 1H, Ar–H); 7.55, (m, 5H, Ar–H);
6
2
962, ν
3124, ν(C=N) 1597, ν(C–S)sym 748; ɅM
7.63, (d, 2H, J = 6.8 Hz, Ar–H); 7.65, (s, 1H, Ar–H); 7.81,
(d, 2H, J = 7.6 Hz, Ar–H); 7.75, (s, 1H, NH–C H ); 8.32,
C–H
(NH–CH3)
ꢀ1
ꢀ
1
2
(
Ω
ꢁm ꢁM ) 72.
10
7
(
3
s, 1H, HC=N); 6.68–5.92, (m, 4H, p-cym Ar–H),
.93–3.35, (q, 1H, p-cym-iso-prop-CH); 2.49, (s, 3H,
6
2
.4.3 | [(Ru(η -p-cym)L3) (μ-im)]Cl (C3)
p-cym Ar–CH ); 1.93, (d, 6H, p-cym-iso-prop-(CH ) );
7
2
3
3 2
ꢀ
1
.75, (d, 2H, imidazole CH=CH); FTIR (KBr/cm ): ν(Ar)
Yield: 48.4%; molecular weight 1367.1 g/mol; molecular
formula C H ClN Ru S ; anal.: found: C, 60.12; H,
2959, ν
(Ω ꢁm ꢁM ) 70.
3138, ν(C=N) 1595, ν(C–S)sym 775; ɅM
C–H
(NH–C10H7)
ꢀ1
ꢀ
1
2
69
68
12
2 2
4
.89; N, 11.98. Calc.: C, 60.62; H, 5.01; N, 12.29. ESI-MS
+
m/z: obs (calc): 1330.3 (1331.1) (M ꢀ 1); δ (400 MHz,
H
6
DMSO-d ) 7.38, (tri, 1H, Ar–H); 7.53, (m, 5H, Ar–H);
2.4.5 | [(Ru(η -p-cym)L1) (μ-azpy)]Cl (C5)
6
2
2
7
.64, (d, 2H, J = 6.8 Hz, Ar–H); 7.79, (s, 1H, Ar–H); 7.82,
(
(
2
d, 2H, J = 7.6 Hz, Ar–H); 8.10, (s, 1H, NH–C H ); 8.34,
s, 1H, HC=N); 5.81–5.50, (m, 4H, p-cym Ar–H);
.50–2.24, (q, 1H, p-cym-iso-prop-CH); 2.15, (s, 3H,
Yield: 61.7%; molecular weight 1366.6 g/mol; molecular
formula C H Cl N Ru S ; anal.: C, 58.99; H,
4.73; N, 15.07. Calc.: C, 59.33; H, 4.98; N, 15.14.
6
5
64
64
2
14
2 2
2
+
p-cym Ar–CH ); 1.15, (d, 6H, p-cym-iso-prop-(CH ) );
7
ESI-MS m/z: obs (calc): 644.8 (645.7) (M ꢀ 1); δH
3
3 2
ꢀ
1
.82, (d, 2H, imidazole CH=CH); FTIR (KBr/cm ): ν(Ar)
(400 MHz, DMSO-d ) 7.06, (tri, 1H, Ar–H); 7.10, (m,
6
2
970, ν
3163, ν(C=N) 1589, ν(C–S)sym 756; ɅM
5H, Ar–H); 7.12, (d, 2H, J = 6.8 Hz, Ar–H); 7.81,
(s, 1H, Ar–H); 7.84, (d, 2H, J = 7.6 Hz, Ar–H); 8.18,
C–H
(NH–C6H5)
ꢀ1
ꢀ
1
2
(
Ω
ꢁm ꢁM ) 76.
(
s, 2H, NH ); 8.21, (s, 1H, HC=N); 6.84–6.79, (m, 4H,
2
p-cym Ar–H); 3.16–2.24, (q, 1H, p-cym-iso-prop-CH);
6
2
.4.4 | [(Ru(η -p-cym)L4) (μ-im)]Cl (C4)
1.75, (s, 3H, p-cym Ar–CH ); 1.15, (d, 6H, p-cym-
2
3
ꢀ
1
iso-prop-(CH ) ); FTIR (KBr/cm ): ν(Ar)C–H 2961,
3
2
Yield: 58.7%; molecular weight 1467.3 g/mol; molecular
formula C H ClN Ru S ; anal.: found: C, 62.98; H,
ν(NH2) 3159, ν
ɅM (Ω ꢁm ꢁM ) 134.
1589, ν(C–S)sym 761, νN=N 1408;
(C=N)
ꢀ1
ꢀ
1
2
77
72
12
2 2

6
of 17
KHANVILKAR ET AL.
[31]
6
[30]
2
.4.6 | [(Ru(η -p-cym)L2) (μ-azpy)]Cl (C6)
programs.
The resolution of Identity (ri)
and multi-
2
2
[
32]
pole accelerated resolution of Identity (marij)
mations with dispersion correction (disp3)
approxi-
[
33]
Yield: 78.8%; molecular weight 1394.6 g/mol; molecular
formula C H Cl N Ru S ; anal.: C, 59.52; H, 4.87; N,
have been
used for all the calculations. Solvent corrections were
66
68
2
14
2 2
1
4.56. Calc.: C, 59.89; H, 5.18; N, 14.82, ESI-MS m/z: obs
incorporated in all calculations using the COSMO
2
+
[34]
(
7
calc): 660.3 (661.2) (M ꢀ 1); δH (400 MHz, DMSO-d6)
model,
Mercury 4.0.0
with methanol (ε = 32.7) as the solvent.
[
35]
.33, (tri, 1H, Ar–H); 7.36, (m, 5H, Ar–H); 7.77, (d, 2H,
software was used for visualization.
J = 6.8 Hz, Ar–H); 7.79, (s, 1H, Ar–H); 7.81, (d, 2H,
Time-dependent density functional theory (TD-
[
36]
J = 7.6 Hz, Ar–H); 8.44, (s, 1H, NH–CH ); 8.46, (s, 1H,
DFT)
calculations were carried out by employing the
3
[
37]
HC=N); 5.82–5.77, (m, 4H, p-cym Ar–H); 3.35–2.86, (q,
Gaussian 09 program
of theory.
tinuum solvation model (CPCM)
with CAM-B3LYP/def2SVP level
An implicit conductor-like polarizable con-
[
38]
1
H, p-cym-iso-prop-CH); 1.97, (s, 3H, p-cym Ar–CH );
3
1
ꢀ
[39]
1
.17, (d, 6H, p-cym-iso-prop-(CH ) ); FTIR (KBr/cm ):
was used to consider
3
2
ν(Ar)C–H 2960, ν
3162, ν
1588, ν(C–S)sym
the solvent effect of methanol. Previously obtained
optimized geometries for C2 and C6 were taken as input
for the calculations, and the data for the corresponding
UV–Vis spectra were obtained.
(NH–CH3)
(C=N)
ꢀ
1
2
ꢀ1
7
64, νN=N 1402; Ʌ (Ω ꢁm ꢁM ) 135.
M
6
2.4.7 | [(Ru(η -p-cym)L3) (μ-azpy)]Cl (C7)
2
2
Yield: 89.7%; molecular weight 1518.8 g/mol; molecular
formula C H Cl N Ru S ; anal.: C, 62.73; H, 4.67; N,
2.6 | DNA binding experiments
76
72
2
14
2 2
1
3.16. Calc.: C, 63.05; H, 5.01; N, 13.54, ESI-MS m/z: obs
2.6.1 | Binding study using absorption
spectroscopy
2
+
(calc): 724.2 (723.9) (M ); δ (400 MHz, DMSO-d ) 7.36,
H
6
(tri, 1H, Ar-H); 7.63, (m, 5H, Ar–H); 7.72, (d, 2H,
J = 6.8 Hz, Ar–H); 7.42, (s, 1H, Ar–H); 7.64, (d, 2H,
J = 7.6 Hz, Ar–H); 8.24, (s, 1H, NH–C H ); 8.45, (s, 1H,
UV spectroscopy was employed to study the interaction
of compounds with CT DNA and to investigate the possi-
ble binding modes of CT DNA. Absorption titrations
were carried out with constant compound concentrations
while changing the CT-DNA concentration within. Stock
solution of the complexes was diluted with tris buffer to
get the desired concentration (30 μM). Equal increments
of CT-DNA were added at different ratios to the com-
pound solution and the reference solution to eliminate
the absorbance of CT-DNA itself, while measuring the
absorption.
6
5
HC=N); 5.49–5.44, (m, 4H, p-cym Ar–H); 2.24–2.17, (q,
1
H, p-cym-iso-prop-CH); 1.91, (s, 3H, p-cym Ar–CH );
3
1
ꢀ
1
.03, (d, 6H, p-cym-iso-prop-(CH ) ); FTIR (KBr/cm ):
3
2
ν(Ar)C–H 2960, ν
3045, ν
1590, ν(C=S)sym
(NH–C6H5)
(C=N)
ꢀ
1
2
ꢀ1
7
53, νN=N 1404; Ʌ (Ω ꢁm ꢁM ) 133.
M
6
2
.4.8 | [(Ru(η -p-cym)L4) (μ-azpy)]Cl (C8)
2
2
Yield: 89.3%; molecular weight 1618.9 g/mol; molecular
formula C H Cl N Ru S ; anal.: C, 64.79; H, 4.55; N,
84
76
2
14
2 2
1
2.42. Calc.: C, 65.18; H, 4.95; N, 12.67. ESI-MS m/z: obs
2.6.2 | DNA-EB competitive binding studies
2
+
(
7
calc): 772.2 (773.2) (M ꢀ1); δH (400 MHz, DMSO-d6)
.38, (tri, 1H, Ar–H); 7.44, (m, 5H, Ar–H); 7.70, (d, 2H,
The competitive binding and the mode of binding
with DNA have been investigated with fluorescence
spectroscopy in order to examine whether the
complexes can displace EB from the DNA–EB complex
and bind to DNA through intercalation. The DNA–EB
complex was prepared by adding EB and DNA in
tris buffer. A certain amount of a solution of each
complex was added step by step into the solution of
the DNA–EB complex. The influence of the addition of
each complex has been obtained by recording the
variation in the fluorescence emission spectra of the
DNA–EB complex. The fluorescence intensities were
measured at 609 nm (524 nm excitation) after addition
of different concentrations of the complexes at
different ratios.
J = 6.8 Hz, Ar–H); 7.78, (s, 1H, Ar–H); 7.99, (d, 2H,
J = 7.6 Hz, Ar–H); 8.23, (s, 1H, NH–C H ); 8.57, (s, 1H,
HC=N); 7.01–6.99, (m, 4H, p-cym Ar–H); 2.51–2.49, (q,
1
10
7
H, p-cym-iso-prop-CH); 1.97, (s, 3H, p-cym Ar–CH );
3
1
ꢀ
1
.02, (d, 6H, p-cym-iso-prop-(CH ) ); FTIR (KBr/cm ):
3 2
ν(Ar)C–H 2959, ν
7
3161, ν
1593, ν(C=S)sym
(NH–C10H7)
(C=N)
ꢀ
1
2
ꢀ1
75, νN=N 1431; Ʌ (Ω ꢁm ꢁM ) 131.
M
2
.5 | Geometry optimization
All DFT calculations were performed by using the b-p
functional
basis set by employing Turbo mole 6.4 suite of
[27]
[28]
[29]
and def2-SVP
(def2-ecp for “Ru”)

KHANVILKAR ET AL.
7 of 17
2.6.3 | Viscosity measurement
563 nm using Biotek-ELX800MS universal ELISA reader
(
Bio-Tek instruments, Inc., Winooski, VT). The IC50
The relative viscosity of DNA (200 μM) solutions was
values were determined by plotting the percentage
viability versus concentration on a logarithmic graph and
reading off the concentration at which 50% of cells
remained viable relative to the control. Each experiment
was repeated at least three times to obtain mean values.
measured by Cannon–Ubbelohde viscometer at
a
ꢂ
constant temperature of 32.0 ± 0.1 C in the presence of
complexes C1–C8. The [complex]/[DNA] ratios were
fixed at 0, 0.04, 0.08, 0.12, 0.16, and 0.20 in Tris–HCl
buffer (pH 7.2). Digital stopwatch with least count of
0
.01 s was used for flow time measurement with accuracy
of ±0.1 s. The flow time of each sample was measured
three times, and an average flow time was calculated.
The data are plotted as (η/η0) versus [complex]/[DNA],
3 | RESULTS AND DISCUSSION
1/3
6
3.1 | Synthesis of the [(Ru(η -p-
where η and η is the viscosity of the DNA solution in the
cym) L) (μ-im/azpy)]Cl complexes
0
2
2
1–2
absence and presence of complex, respectively. Viscosity
values were calculated from the observed flow time of
DNA-containing solutions (t) corrected for that of the
A new series of binuclear ruthenium (II) arene
6
complexes of the type [(Ru(η -p-cym)L) (μ-im/azpy)]Cl
2
1–2
[40]
buffer alone (t ), η = (t ꢀ t )/t .
(C1–C8) were achieved by reacting the mononuclear com-
0
0
0
6
plexes [Ru(η -p-cym)(L)Cl] with the bridging ligand
0
imidazole/4,4 -azopyridine in the ratio 2:1, in CH Cl/
2
2
.7 | BSA binding experiments
CH OH mixture, as shown in Figure 2. The synthesized
3
complexes were found to be stable in air at room tempera-
ture and non-hygroscopic. The elemental analysis data for
the complexes matched well with the calculated values,
thus confirming their proposed composition.
The BSA-binding study was performed employing steady
state fluorescence spectroscopy. A solution of bovine
serum albumin (BSA, 16.6 μM) in buffer (containing
1
5-mM tri-sodium citrate and 150-mM NaCl at pH 7.0)
was used to carry out the tryptophan fluorescence
quenching experiments. The quenching of emission
intensity of the tryptophan residues of BSA at 343 nm
was monitored in the presence of increasing concentra-
3.2 | Characterization
The ESI-mass spectra (Figure S1) show m/z peaks
corresponding to the molecular ions that give evidence
of the formation of binuclear complexes with the bridg-
ing ligand. Due to the large size of the complexes, vari-
ous fragments may be formed during ionization.
Fragmentation of the complexes by removal of the
bridging and/or the terminal ligand(s) can occur.
Fragmentation of the ligand backbone while the ligand
is still bound to the metal ion often occurs. The forma-
tion of the peaks (Table S1) with their assigned m/z
values may be rationalized in the following way. Loss
of a hydrogen atom H from the organic ligand
framework tends to yield a (M ꢀ 1) peak whereas the
(M + 1) peak is due to protonated molecular ion
[41]
tions of the complexes.
Fluorescence spectra were
recorded from 300 to 500 nm at an excitation wavelength
of 296 nm.
2.8 | Evaluation of anticancer activity
The synthesized complexes were assessed for their cyto-
[
42]
toxic activity by standard MTT colorimetric assay.
3
ꢀ1
HeLa cells (5.0 ꢃ 10 cells well ) were placed in 96-well
culture plates (Tarson India Pvt. Ltd.) and grown over-
ꢂ
night at 37 C in a 5% CO incubator. C1–C8 were added
2
to the wells in the concentration range 0.5–150 mg/ml;
+
control wells were prepared by addition of culture
(M + H) . The molecular ion peak values for the com-
medium without the compounds. The plates were incu-
plexes indicate that a p-cymene and a bidentate
ꢂ
bated at 37 C in a 5% CO incubator for 48 h and stan-
pyrazole-thiosemicarbazone ligand is coordinated to
2
0
dard 3-(4,5-dimethylthiazole)-2,5-diphenyltetraazolium
bromide MTT dye solution was added to each well. After
each of the two Ru(II) ions with an imidazole or a 4,4 -
azopyridine ligand bound simultaneously to both the
metal centers. Thus, each of the Ru(II) ions is six coor-
dinated with a distorted octahedral geometry. The com-
plexes C1–C4 and C5–C8 have molar conductance
4
h of incubation, the culture media was thrown out, and
the wells were washed with phosphate buffer saline
Hi-Media, India Pvt. Ltd.), which was followed by addi-
(
ꢀ
3
ꢀ1
2
ꢀ1
tion of DMSO to dissolve the formazan crystals formed
and further incubation for 30 min. The optical density of
each well was measured spectrophotometrically at
(10 M in DMSO) in the range of 72–75 (Ω m M
)
ꢀ
1
2
ꢀ1
ꢂ
and 131–135 (Ω
suggesting 1:1 and 1:2 electrolytic behavior.
m
M
), respectively, at 38 C
[
43]

8
of 17
KHANVILKAR ET AL.
In the IR spectra of the complexes C1–C8, the free
ligand ν(C=S) asymmetric and symmetric absorption in the
wavelength range 200–700 nm. The red shift of the
intense absorption bands at 223–227 nm assigned to
intra-ligand π ! π* transition of the ligand aromatic
rings to 226–230 nm in the complexes is attributed to
their coordination with Ru(II) metal center. Another
medium intensity peak owing to the intraligand N
and S centered n ! π* transitions, in the range of
358–380 nm have blue shifted to 313–365 nm on com-
plexation. This shift in the wavelength indicates N and
S coordination of the thiosemicarbazone ligand to the
metal center. For complexes C5–C8, one additional
peak was observed within 264–278 nm, due to the
ꢀ
1
region 1260–1290 and 807–824 cm is shifted to a single
ν(C–S) absorption at 748–775 cmꢀ , due to enolization of –
NH–C=S and subsequent co-ordination via deprotonated
sulfur. The strong band in the range 1597–1599 cmꢀ char-
acteristic of the azomethine group ν(C=N) is shifted to
1
1
ꢀ
1[[17]]
1
590–1588 cm
due to coordination through the
azomethine nitrogen atom. The bands in the fingerprint
regions 2959–2970 cmꢀ and 1457–1638 cm owing to
aromatic νC–H stretch and aromatic νC=C in plane vibra-
tions, respectively, are indicative of presence of p-cymene
in all the complexes. Characteristic band around
1
ꢀ1
intraligand n ! π* transitions of the azopyridine
ꢀ
1
[46]
3
300–3364 cm due to the ν(N–H) stretching of the free
moiety.
The λ
values of all the transitions taking
max
imidazole was found to be absent in the spectra of C1–C4,
place in the complexes have been tabulated in
Table S2.
0
while the ν
stretching of the 4,4 -azopyridine ligand is
N=N
observed at 1402–1487 cmꢀ in the spectra of C5–C8. The
analysis of the IR spectra suggests that all the pyrozlyl
thiosemicarbazones act as monoanionic bidentate ligands
and interact with the metal center via the azomethine
group and deprotonated sulfur. The IR spectra of all the
complexes have been provided as supporting information
1
Full geometry optimizations of compounds were
carried out using the DFT method at b-p functional and
def2-SVP (def2-ecp for “Ru”) basis set by employing
Turbomole 6.4 suite of programs as mention above in
Section 2.5. This functional has been shown to give more
accurate results for organometallic complexes. All calcu-
lations were performed using the approximations with
dispersion correction (disp3) program. The DFT calcula-
tions for geometry optimization provide some insight into
the structure of the complexes. Optimized structures of
the complexes C1 and C5 are shown in Figure 1. Piano-
stool type geometry is seen around each metal center of
the binuclear complexes with the Ru(II) ions π-bonded to
the arene ring. The average Ru–Ru distance in the
binuclear complexes with imidazole as bridging ligands is
(
Figure S2).
1
The H NMR spectra of C1–C8 (Figure S3) show
distinct peaks corresponding to p-cymene. The pres-
ence of a p-cymene ligand was confirmed by the
presence of 6-proton doublet at δ = 1.93–1.02 ppm
owing to two methyl protons of iso-propyl group [CH
(
CH ) ], 3-proton singlet at δ = 2.49–1.91 ppm due to
3
2
the Ar-methyl group para to the iso-propyl group,
1
–
-proton quartet at δ = 3.95–2.24 ppm attributed to
CH of the iso-propyl group, and two 2-proton dou-
0
6.14 Å, whereas in complexes with 4,4 –azopyridine as
blets at δ = 7.01–5.44 ppm assigned to the 4 Ar-protons
of p-cymene.
bridging ligands, the average distance is 13.26 Å due to
[44]
0
The imine proton observed in the
the presence of longer 4,4 -azopyridine ligand. The bond
range δ = 8.36–10.17 ppm in free ligands is shifted
upfield at δ = 8.22–8.69 ppm in the complexes indicat-
ing coordination via HC=N. All the aromatic protons
present in pyrozole ring are observed in the excepted
region. The absence of a singlet at δ = 11–13 ppm
angle values reveal a pseudo octahedral coordination of
the ruthenium centers. The metal–ligand bond lengths
and bond angles tabulated in Table 1 are in well agree-
[
47,48]
ment with the values in the literature.
Optimized
structures of the rest of the complexes are given in
Figure S5.
[34]
owing to the N–H proton of free imidazole,
in the
NMR spectra of C1–C4, suggests co-ordination of the
deprotonated imidazole N with the metal center. Apart
from this, CH=CH proton of imidazole at δ = 7.75–
In the absence of XRD data, time-dependent density
functional theory (TD-DFT) calculations were done to
provide further evidence for the bridging structure by
correlating the experimentally observed UV–Vis spectra
with the corresponding computationally obtained data.
Two complexes C2 and C6, one each from the imidazole
and azopyridine bridged series, were selected for
the calculations. The previously obtained optimized
structures were used as input geometries for the TD-DFT
calculations, and the UV–Vis spectral data generated
(Table S3) was found to be in close agreement with the
experimentally observed UV–Vis spectra.
7
.93 ppm is observed in spectra of C1–C4 indicating
the presence of imidazole ring. In case of C5–C8, a
slight upfield shift of the doublet peak due to four pro-
0
tons in the pyridine ring of 4,4 -azopyridine is observed
1
at δ = 7.28–8.48 ppm compared with H NMR of pure
0
4
,4 - azopyridine at δ = 7.75–8.89 ppm, indicating its
[45]
co-ordination to the metal center(s).
The electronic absorption spectra of the complexes
C1–C8 (Figure S4) show two major bands in the

KHANVILKAR ET AL.
9 of 17
FIGURE 1 Optimized structures of the complexes C1 and C5
3.3 | Stability studies
3.4 | DNA binding studies
Due to the less aqueous solubility of the metal complexes,
DNA/BSA binding and cytotoxicity studies were carried
out using stock solutions of the complexes prepared in
DMSO. The solutions were then diluted with tris buffer
so that the test solutions contained no more than 2%
DMSO. To check the stability of the complexes in DMSO,
UV–Visible spectra of the complexes C1–C8 were
recorded at room temperature in the range 200–700 nm
at time intervals up to 48 h. No changes in the UV–Vis
spectra were observed, implying that the complexes are
stable in DMSO solution.
One of the primary targets for many metallotherapeutics
(e.g., cisplatin, carboplatin, and oxaliplatin) and
organic anticancer drugs (doxorubicin, gemcitabine,
5-fluorouracil, etc.) is the nucleus DNA of the cancer
[49]
cells. Significant emphases have been given to the design
of complexes with ligand scaffolds that bind to DNA with
site selectivity. Several Ru(II) complexes have shown signifi-
[50]
cant DNA binding affinity.
The binding mode and
affinity between complexes under study and CT-DNA
were evaluated by the viscosity measurements, UV–Vis
absorption, and fluorescence emission spectroscopy.

10 of 17
KHANVILKAR ET AL.
TABLE 1 Metal ligand bond
L1
C1
L2
C2
L3
C3
L4
C4
lengths and bond angles of complexes
under study obtained from geometry
optimization
Bond Length (in Å)
Ru1 … … … … Ru2
Ru1–N1
C5
C6
C7
C8
6.15
13.29
2.10
2.36
2.10
2.10
2.36
2.10
6.17
13.26
2.09
2.36
2.10
2.09
2.36
2.10
6.13
13.26
2.09
2.36
2.10
2.09
2.36
2.10
6.11
13.26
2.09
2.37
2.09
2.09
2.37
2.09
2.09
2.36
2.09
2.09
2.39
2.09
2.09
2.37
2.09
2.08
2.42
2.10
2.09
2.36
2.09
2.08
2.40
2.10
2.09
2.36
2.08
2.08
2.40
2.10
Ru1–S1
Ru1–N2
0
Ru2–N1
0
Ru2–S1
Ru2–N3
Bond angle (in degree)
S1–Ru1–N1
80.9
81.1
90.1
84.6
81.0
90.1
84.6
80.7
81.2
89.2
84.3
81.1
89.2
84.3
81.0
81.1
89.0
84.6
81.2
89.2
84.7
81.1
81.0
88.7
84.4
81.0
88.5
84.3
S1–Ru1–N2
89.3
81.2
79.0
93.5
83.4
90.2
84.2
78.2
93.8
83.4
88.2
83.0
78.8
93.3
83.2
88.4
83.4
78.8
93.4
82.7
N1–Ru1–N2
0
0
S1 –Ru2–N1
0
S1 –Ru2–N3
0
N1 –Ru2–N3
FIGURE 2 UV absorption spectra of C1 at
increasing concentrations of CT-DNA, the arrow
shows decrease in intensity upon increasing
concentration of the complex
3
.4.1 | Binding study using absorption
constants K , for the complexes were calculated from the
b
Mehan's equation.
[
51]
spectroscopy
Absorption titration method was employed to evaluate
the binding ability of the complexes to CT-DNA. The UV
½DNAꢄ=ðεA ꢀεf Þ ¼ ½DNAꢄ=ðεb ꢀεf Þþ1=Kb ðεb ꢀεf Þ,
ꢀ
6
spectra of all the complexes (10 M) have been recorded
in the absence and presence of varying CT-DNA concen-
tration (at [DNA]/[C] ratio of 0–3.01) (Figure 2). C1–C8
showed hypochromism (50.5%–58.6%) accompanied with
a blue shift (1–3 nm) indicating an intercalative interac-
tion with DNA. The titration plots of remaining
complexes have been provided in Figure S6. The binding
where [DNA] is the concentration of DNA in base pairs,
εA = Aobsd/[compound], ε is the extinction coefficient for
f
the unbound compound, and ε is the extinction coeffi-
b
cient for the compound in the fully bound form. The plot
of [DNA]/(ε ꢀ ε ) versus [DNA] (Figure S7) gave a slope
A
f
1/(ε ꢀ ε ) and an intercept (1/K )(ε ꢀ ε ). K , the
b
f
b
A
f
b
intrinsic binding constant, is the ratio of the slope to the

KHANVILKAR ET AL.
11 of 17
intercept. The binding constants values, calculated for
suggested by absorption titration study. The emission
spectra of the DNA-EB adduct (λex = 546 nm, λem = 610)
in the absence and presence of increasing amounts of
complexes (at a [C]/[DNA–EB] ratio of 0–0.62) have been
recorded. Addition of complexes resulted in decrease in
the intensity of the emission band at 610 nm up to
35.02%–42.67% indicating that the complexes compete
with EB in binding to DNA (Figure 3). The observed
quenching of DNA–EB fluorescence on addition of the
complexes suggests that they intercalate DNA by dis-
placing EB from the DNA–EB complex. The titration
plots of remaining complexes have been shown in
Figure S8.
4
5
ꢀ1
C1–C8 (Table 2) in the range of 1.6 ꢃ 10 –1.2 ꢃ 10 M
reveal strong binding of the complexes with the biomole-
cule. Slightly higher K values in the range of
b
5
ꢀ1
(
1.60–2.17) ꢃ 10 M were obtained for half-sandwich
6
Ru(η -p-cymene) complexes with the pyrazole-based
ligands [2-(1H-pyrazol-3-yl)pyridine (L1), 3-(furan-2-yl)-
1
H-pyrazole (L2), and 3-(thiophen-2-yl)-1H-pyrazole
[52]
5
5
ꢀ1
(L3)]
and 2.47 ꢃ 10 and 3.73 ꢃ 10 M , respectively,
6
for the mononuclear and binuclear Ru(η -p-cymene)
complexes containing the indole thiosemicarbazone
[53]
ligand.
However, the binding constant values are
comparable with those observed in the case of Ni(II) bis
ꢀ
4
ꢀ1
(
thiosemicarbazone) complexes (1.40–2.90 ꢃ 10
M
)
The quenching constant (KSV) values were calculated
from the slopes of straight lines obtained from the Stern-
4
ꢀ1 [54]
and binuclear Ni(II) complexes (1.17–11.6 ꢃ 10 M ).
The effect of size, shape, hydrophobicity, and electrolytic
behavior of the metal complexes play significant roles in
[
58]
Volmer equation
:
[55]
binding of the complexes to DNA.
It is observed that
Io=I ¼ 1þKSV ½Qꢄ,
the complexes C3, C7, C4, and C8 with 4-phenyl/napthyl
substituted thiosemicarbazone ligand show high binding
affinities due to the additional planar aromatic rings pre-
sent in their ligand scaffolds which facilitate hydrophobic
where Io and I are the emission intensities in the absence
and the presence of the quencher C1-C8 respectively,
[Q] is the concentration of the quencher, and KSV is the
Stern-Volmer constant which can be obtained from the
slope of the plot of Io/I versus [Q] (Figure S9). The plots
illustrate that the quenching of DNA–EB fluorescence by
the complexes is in good agreement (R = 0.98) with the
linear Stern-Volmer equation. The Stern-Volmer
quenching constant KSV values with standard deviations
[56]
interactions with the nucleobases.
C1, C5, C2, and C6
with 4-hydrogen/methyl substituted thiosemicarbazone
ligand display low binding affinities due to no additional
planar aromatic rings. The standard Gibb's free energy
for DNA binding was calculated using the following
[57]
relation.
(
SD) given in Table 2 are in the range of (1.1–9.2) ꢃ
ꢂ
4 ꢀ1
ΔG b ¼ ꢀRT ln Kb,
10 M .
where R is universal gas constant and T is temperature.
ꢂ
All the complexes are showing negative value of ΔG b
indicating spontaneity of reaction.
3.4.3 | DNA viscosity measurement
To further confirm the proposed intercalative mode of
interaction between the complexes and DNA, viscosity
measurements were carried out in the absence and in the
presence of the complexes added gradually. A steady
increase in the relative viscosity of CT-DNA was observed
on increasing the concentrations of the complexes
(0–200 μM) in Tris–HCl/NaCl buffer (200 μM). The
3
.4.2 | EB displacement study using
emission spectroscopy
EB displacement experiment was carried out to confirm
the intercalative mode of binding of the complexes as
TABLE 2
K
b
and KSV values of
ꢀ1
ꢀ1
ꢂ
ꢀ1
Code
C1
K
b
M
K
SV
M
λ shift
2 nm
1 nm
1 nm
1 nm
2 nm
3 nm
2 nm
1 nm
ΔG kJmol
complexes
4
4
2.8 ꢃ 10 ± 0.007
4.6 ꢃ 10 ± 0.009
7.2 ꢃ 10 ± 0.017
6.8 ꢃ 10 ± 0.041
2.6 ꢃ 10 ± 0.026
1.6 ꢃ 10 ± 0.012
1.1 ꢃ 10 ± 0.032
1.2 ꢃ 10 ± 0.120
1.1 ꢃ 10 ± 0.035
ꢀ25.37
ꢀ26.60
ꢀ27.71
ꢀ27.57
ꢀ25.19
ꢀ23.98
ꢀ28.76
ꢀ28.98
4
4
C2
5.5 ꢃ 10 ± 0.027
4
4
C3
1.7 ꢃ 10 ± 0.036
4
4
C4
1.3 ꢃ 10 ± 0.042
4
4
C5
9.2 ꢃ 10 ± 0.036
4
4
C6
3.1 ꢃ 10 ± 0.028
5
4
C7
6.0 ꢃ 10 ± 0.036
5
4
C8
4.3 ꢃ 10 ± 0.042

12 of 17
KHANVILKAR ET AL.
relative viscosity slopes of the DNA solutions in the pres-
ence of the complexes were calculated (slope values
3.5 | BSA binding studies
0
.44–0.49) and compared with the classical intercalator
Literature have revealed that there can be electrostatic,
hydrophobic and van der Waals interactions between
BSA and metal complexes and also and hydrogen bond
formation. Fluorescence spectrophotometry is a common
technique to study molecular interaction with BSA. The
fluorescence emissions of the serum protein are due to
tyrosine, tryptophan, and phenylalanine residues. The
effect of addition of the complexes on the fluorescence
emission of BSA was monitored to evaluate the interac-
tion of the complexes with the protein. Addition of
ethidium bromide (slope value of 0.94). Thus the inser-
tion of complexes in between the base pairs elongates the
double helix, leading to an increase in the viscosity of
DNA. Complexes binding with DNA grooves cause less
[52]
noticeable or no variation in the viscosity.
The effects
of C1–C8 on the viscosity of CT-DNA solution are shown
in Figure 4. With increasing [complex]/[DNA] concentra-
tion ratios, the relative viscosity of CT-DNA increased
[
54]
gradually speculative of intercalative mode of binding.
FIGURE 3 Plot of fluorescence emission
intensity I versus wavelength λ for CT DNA-EB
complex at different concentrations of C1, the
arrow shows decrease in fluorescence intensity
on increasing concentration of the complex
FIGURE 4 Effect of increasing amounts of
the complexes and ethidium bromide on the
relative viscosity of CT-DNA (200 mM) in
ꢂ
Tris–HCl buffer at 32 C. [Complex]/[DNA] = 0,
0.04, 0.08, 0.12, 0.16 and 0.20

KHANVILKAR ET AL.
13 of 17
increasing concentrations of the complexes to a solution
of BSA (at [C]/[BSA] ratio of 0–2.8) results in a signifi-
cant decrease of the protein fluorescence intensity at
3.6 | Docking studies
Molecular docking study was employed to identify the
principle binding site of both the complexes with DNA
and the BSA protein. Crystal structure of DNA and BSA
were obtained from the Brookhaven Protein Database
(PDB) (http://www.rcsb.org/pdb). Structures of the com-
plexes C3 and C7 were created in ACD/Chemsketch
3
44 nm (Figure 5). The observed hypochromism with a
small blue shift (ꢅ2 nm) in the presence of C1–C8
indicates a static quenching of BSA fluorescence by the
[59]
complexes.
The complexes interact hydrophobically
with proteins, which is evident from the observed hyp-
ochromism. The titration plots of remaining complexes
are shown in Figure S10. The values of the Stern-Volmer
quenching constant (KSV) obtained from the plot of [Io/I]
versus [Q] (Figure S11), for the complexes interacting
[
62]
12.01 software
(Figure S13) and converted into .mol
file format to .pdb by Argus Lab. Molecular docking of
the selected complexes with DNA (PDB: 1BNA) and BSA
(PDB ID: 4F5S) were analyzed and visualized using
4
5
ꢀ1
[63]
with BSA are in the order of 10 –10 M (Table 3). This
indicates strong interaction with the protein. The KSV
values reveal that the complexes are more efficient in
quenching the fluorescence of BSA compared with the
Argus Lab 4.0.1.
Argus Lab is a docking tool based on
Lamarckian genetic algorithm having exact grid resolu-
tion along with precise predictions of the ligand binding
sites.
“AScore” scoring function was selected for the docking
studies. The docking algorithm was set as Argus dock with
exhaustive search and the grid resolution was fixed to
3
4
ꢀ1
ligands (10 –10 M ) due to increased hydrophobicity.
The plot of log [(Io ꢀ I)/I] versus log [Q] for all the
complexes (Figure S12) is linear; the association binding
ꢀ
1
constant (K M ), and the number of binding sites per
a
albumin (n) have been obtained by double logarithm
[
60]
equation.
The Ka values for the complexes are within
TABLE 3
a
KSV, K , and n values for complexes
5
6
ꢀ1
61]
the range of 10 –10 M as expected from a good BSA
carrier activity in vivo.
ꢀ1
ꢀ1
Code
C1
K
a
M
K
SV
M
n
[
5
5
1.5 ꢃ 10 ± 0.187
1.4 ꢃ 10 ± 0.235
1.0
1.2
1.1
1.2
1.3
1.4
1.2
1.3
6
4
C2
1.1 ꢃ 10 ± 0.191
9.9 ꢃ 10 ± 0.173
log ðIoꢀIÞ=I ¼ log Ka þnlog ½Qꢄ
5
5
C3
7.1 ꢃ 10 ± 0.186
1.5 ꢃ 10 ± 0.208
5
6
ꢀ1
6
5
The K values in the order of 10 –10 M also indicate
C4
1.6 ꢃ 10 ± 0.184
1.6 ꢃ 10 ± 0.164
a
5
5
strong binding of the complexes to BSA in agreement to
the Ksv values. The linear nature of the double logarithm
plots and the n values calculated for C1-C8, indicate that
only one of the tryptophan residues on BSA protein is
interacting with the complexes.
C5
3.6 ꢃ 10 ± 0.185
8.1 ꢃ 10 ± 0.154
5
4
C6
1.6 ꢃ 10 ± 0.185
1.0 ꢃ 10 ± 0.148
6
5
C7
1.2 ꢃ 10 ± 0.183
8.1 ꢃ 10 ± 0.180
6
5
C8
4.3 ꢃ 10 ± 0.187
5.6 ꢃ 10 ± 0.152
FIGURE 5 Plot of Fluorescence emission
intensity versus wavelength for BSA at
increasing concentrations of C1, the arrow
shows decrease in the fluorescence intensity
with increasing concentration of the complex

14 of 17
KHANVILKAR ET AL.
4
2
Å. The size for binding box was set to
2.48 Å ꢃ 26.74 Å ꢃ 43.40 Å for C3 and 22.48 Å ꢃ 26.16 Å
3.7 | In silico ADME toxicity profile
ꢃ 42.48 Å for C7 with DNA, and 17.08 Å ꢃ 27.99 Å
ꢃ 15.50 Å for C3 with BSA, respectively. The ligand dock-
ing method used for following studies was set to “Flexible
mode” and the precision factor was set to “Regular.”
ADME explains the absorption, distribution, metabolism
and excretion of the ligands designed, particularly drugs.
SwissADME (http://www.swissadme.ch/) is one of the
online tools which elucidate the pharmacological and
toxicological properties of a drug/complex. In the present
study, the simplified molecular input line entry specifica-
tions (SMILES) were uploaded into the database. This
leads to the generation of result in the form of Boiled
The stability of docked poses was decided by
ArgusLab energy functions and the hydrogen bonds
[64]
formed between receptor and ligands.
figure for BSA-C3 interactions, was also generated by
Protein-Ligand Interaction Profiler.” The tool gave us
the idea regarding hydrophobic interaction and
Further 3D,
[
69]
“
egg.
The tool gives all the data related to physicochem-
ical properties, lipophilicity, water solubility, pharmaco-
[65]
[70]
π-stacking interaction.
kinetics, druglikeness, and medicinal chemistry.
Validation of the docking interactions for the selected
complexes C3 and C7 with DNA (PDB: 1BNA) are shown
in Figures S14–S16. It was observed that both the com-
plexes showed complete base stacking interaction with
DNA, where the nitrogen base sequence in one strand is
C G C G A A T T C G C G and for the complemen-
ADME properties of the complexes C3 and C7 were
evaluated. The results (Table S9) reveal that the ruthe-
nium complexes were showing low gastrointestinal
(GI) absorption along with no blood brain barrier perme-
ation. Even though they showed interaction with P-gp
substrate but no inhibitory action was observed with
cytochrome P450 enzymes. This explains that the drugs
can be properly metabolized and can be eliminated from
the system without damaging cytochrome P450 enzymes.
1
2 3 4 5 6 7 8 9 10 11 12
tary sequence is G C G C T T A A G C G C .
24 23 22 21 20 19 18 17 16 15 14 13
The most significant binding interaction was based on
the hydrogen bond formed between complex-DNA inter-
faced to the substrate binding site. The hydrogen bond
less than 3 Å was considered. C3 was found to be inter-
acting with thymine nitrogen base at positions 7 and
3.8 | Antiproliferative studies
8
(Table S4) whereas C7 was found to be interacting both
with adenine at position 6 and 18 and thymine at posi-
tions 7, 8, and 19 (Table S5).
Since the DNA and BSA binding studies indicated a rea-
sonably strong interaction of the complexes with the bio-
molecules, MTT assay was carried out in a dose
dependent fashion to analyze the antiproliferative activity
of the binuclear complexes C1–C8 with HeLa (human
cervical cancer) for an incubation period of 48 h. This cell
line was selected due to higher occurrence of cervical
cancer in human population worldwide, which is of
prime concern. After the incubation period, the % of via-
ble cancer cells was reduced. The plot of percentage of
cell viability versus complex concentration (Figure 6)
depicted the dose dependent cell death induced by the
tested compounds. The corresponding IC50 values are
given in Table 4. The IC50 values of the synthesized
ruthenium complexes in the range of 17.3–41.3 μM reveal
that they show moderate cytotoxicity in HeLa cells and
are less active compared with cis-platin (IC50 = 18.8
± 3.4 μM) except C2 (IC50 = 17.3 ± 1.82 μM). The
Preferable binding site of C3 with BSA is given in
Table S6. In order to explain the interaction, hydrogen
bond less than 4 Å was considered. C3 showed significant
hydrogen bond interaction with Ser219, Tyr331, Trp213,
and Arg217 of chain A. Protein–Ligand Interaction Pro-
filer tool postulated the hydrophobic and π-stacking
interactions explaining the hydrophobic interaction
playing a major role in the binding of the ruthenium
complex with BSA (Figure S17; Tables S7 and S8). The
amino acid found to involve in hydrophobic interactions
were Ala212, 216, 225; Trp213; Val215, 230, 292, 342;
Leu218, 330, 346; Lys221; Phe222, 227; Glu226; Tyr331.
Drugs usually bind to BSA at two major sites Sudlow's
site 1 and site II having specialized cavity of subdomain
[66]
IIA and IIIA.
The important ligand binding site on
BSA is located in the subdomains IIA and IIIA, which
[67]
are the Sudlow sites.
Above interactions suggest that
anticancer
activities
are
in
the
order
of
C3 binds to BSA in the hydrophobic cavity of Site I in
C2 > C8 > C1 > C3 > C4 > C5 > C6 > C7. C2 with a
IC50 value lower than that of cisplatin shows the highest
cytotoxicity followed by C8 (IC50 = 23.7 ± 8.90 μM). The
high DNA binding affinity and enhanced cytoxicity indi-
cate DNA to be the target molecule for C8. However, the
order of cytotoxicity of the complexes C1–C7 is not in
line with their DNA binding capabilities which suggest
that DNA may not be the target for the complexes
[68]
subdomain IIA.
C3 is capable of quenching the trypto-
phan fluorescence by binding to the hydrophobic region
of protein facilitated by hydrophobic interaction of
methyl and isopropyl groups of p-cymene and the aro-
matic groups of the pyrazolyl thiosemicarbazone ligand.
Thus the ligand hydrophobicity contributes to the BSA
protein binding affinity of the complex.

KHANVILKAR ET AL.
15 of 17
FIGURE 6 Percent cell viability verses
concentration plots of C1–C8 on HeLa Human
cervical cancer cell lines. Each point is the
mean ± standard error obtained from three
independent experiments
TABLE 4 The IC50 values for the human cervical cancer cell line HeLa with C1–C8 for 48 h
Code
μM
C1
C2
C3
C4
24.4 ± 2.96
C5
17.3 ± 1.82
C6
29.6 ± 5.14
C7
31.7 ± 3.24
C8
Code
μM
32.6 ± 2.04
35.9 ± 5.71
41.3 ± 11.44
23.7 ± 8.90
in vitro. Also, high positive charge (+2) on C8 may result
residues Ser219, Tyr331, Arg217, and Trp213. Hydropho-
bic and stacking interactions with the protein were also
observed. C1-C8 were found to be more active than some
of the Ru(II)-arene complexes with hybrid TSC ligands.
The complex C2 showed more cytotoxicity compared to
cis platin towards HeLa cancer cells.
in electrostatic interactions between the positively
2
+
charged [(Ru(p-cym)L) (μ-azpy)] complex and the neg-
2
atively charged polar head groups of the lipid bilayer of
the cell membrane, hence an enhanced cellular uptake.
In contrast, the complexes C5–C7 with +2 charges
exhibit lower antitumor activity, which suggests that
enhanced cellular uptake may not always lead to
enhanced cytotoxicity. The complexes were found to be
more active against HeLa cells compared to Ru(II)-arene
complexes with triarylamine-thiosemicarbazone hybrid
ACKNOWLEDGMENTS
P.K. greatly acknowledge the University Grants
Commission, New Delhi, for the financial assistance,
MRP fellowship (UGC F.No. 43-238/2014(SR)),
RFSMS-BSR fellowship (Letter no. F.5-71/2007(BSR)),
and DST-PURSE fellowship (Letter no. FS/206/19-20),
respectively. The author would like to acknowledge the
DST-FIST program for funding NMR facility at the
Department of Chemistry, Faculty of Science, and The
Maharaja Sayajirao University of Baroda. The author is
thankful to the Heads, Department of Chemistry and
Zoology, Faculty of Science, The Maharaja Sayajirao Uni-
versity of Baroda, for providing the necessary laboratory
and instrumentation facilities to carry out the research
work. The author is thankful to the Centre of Excellence
in Scientific Computing (COESC), CSIR-NCL Pune, for
providing computational facilities.
[20]
ligands except for one complex as reported.
4
| CONCLUSION
Binuclear Ru(II) complexes of p-cymene and the pyrazol-
thiosemicarbazone hybrid ligands were synthesized and
characterized using various spectral techniques. The
complexes showed good binding propensities towards
4
5
ꢀ1
5
6
ꢀ1
DNA (K = 10 –10 M ) and BSA (Ka = 10 –10 M
)
b
as revealed by their binding studies. The binuclear com-
plexes were found to have better binding affinities
towards DNA/BSA due to their enhanced hydrophobicity
compared to their mononuclear analogues. DNA docking
studies of C3 and C7 revealed base stacking interactions
and formation of H-bonds with thyamine and adenine
bases. In addition BSA docking studies of complex C3
indicated H- bond formation with the amino acid
AUTHOR CONTRIBUTIONS
Priyanka Khanvilkar: Formal analysis; methodology;
software. Soumya Dash: Software. Devjani Banerjee:
Software. Aliasgar Vohra: Formal analysis. Ranjitsinh

16 of 17
KHANVILKAR ET AL.
Devkar: Formal analysis; resources; supervision.
Debjani Chakraborty: Investigation; supervision.
[16] D. X. West, A. E. Liberta, S. B. Padhye, R. C. Chikate, P. B.
Sonawae, A. S. Kumbhar, R. G. Yerande, Coord. Chem. Rev.
1993, 123, 49.
[
[
17] F. A. Bechford, G. Leblanc, J. Thessing, M. Shaloski Jr., B. J.
CONFLICT OF INTEREST
The authors declare that there is no conflict of
interest regarding the publication of this article.
Frost, L. Li, N. P. Seeram, Inorg. Chem. Commun. 2009, 12,
1094.
18] R. Pettinari, F. Marchetti, C. Di Nicola, C. Pettinari, A.
Galindo, R. Petrelli, L. Cappellacci, M. Cuccioloni, L. Bonfili,
A. M. Eleuteri, M. F. C. Guedes da Silva, A. J. L. Pombeiro,
Inorg. Chem. 2018, 57, 14123.
19] E. P ꢀa unescu, M. Soudani, P. Martin, R. Scopelliti, M. Lo Bello,
P. J. Dyson, Organometallics 2017, 36, 3313.
DATA AVAILABILITY STATEMENT
The data that supports the findings of this study are
available in the supporting information of this article.
[
[
20] M. Muralisankar, R. Dheepika, J. Haribabu, C. Balachandran,
S. Aoki, N. S. P. Bhuvanesh, S. Nagarajan, ACS Omega 2019, 4,
ORCID
Priyanka Khanvilkar https://orcid.org/0000-0001-5938-
11712.
9
725
[
21] M. Fabre, J. Jaud, M. Hliwa, J. P. Launay, J. Bonvoisin, Inorg.
Soumya R. Dash https://orcid.org/0000-0001-7267-6104
Devjani Banerjee https://orcid.org/0000-0002-4287-
Chem. 2006, 45, 9332.
[22] J. Honorato, K. M. Oliveira, C. M. Leite, L. Colina-Vegas, J. A.
N oꢁ brega, E. E. Castellano, J. Ellena, R. S. Correa, A. A.
Batista, J. Braz. Chem. Soc. 2020, 31, 2237.
2
398
Aliasgar Vohra https://orcid.org/0000-0002-4845-084X
Ranjitsinh Devkar https://orcid.org/0000-0002-9863-
[
[
[
23] M. A. Bennett, A. K. Smith, J. Chem. Soc. Dalton Trans.
1974, 233.
7
418
Debjani Chakraborty https://orcid.org/0000-0001-5871-
784
24] M. A. Bennett, T. N. Huang, T. W. Matheson, A. K. Smith, S.
Ittel, W. Nickerson, Inorg. Synth. 1982, 21, 74.
25] H. D. Patel, S. M. Divatia, E. D. Clercq, Indian J. Chem. 2013,
4
52, 535.
REFERENCES
[26] R. N. Patel, N. Singh, K. K. Shukla, U. K. Chauhan, Spe-
ctrochim. Acta A 2005, 61, 287.
[
[
1] C. Roder, M. Thomson, Drugs R&D 2015, 15, 13.
2] B. Bosco, A. Defant, A. Messina, T. Incitti, D. Sighel, A. Bozza,
Y. Ciribilli, A. Inga, S. Casarosa, I. Mancini, Molecules 2018,
[
[
[
[
[
[
[
27] a) A. D. Becke, Phys. Re. A 1988, 38, 3098; b) J. P. Perdew,
Phys. Re. B 1986, 33, 8822.
28] A. Schäfer, H. Horn, R. J. Ahlrichs, Chem. Phys. 1992, 97,
2
3, 1996.
2
571.
29] C. Hättig, G. Schmitz, J. Koßmann, Phys. Chem. Chem. Phys.
012, 14, 6549.
[
[
[
[
[
[
[
3] Y. Y. Huang, L. Y. Wang, C. H. Chang, Y. H. Kuo, F. F. Wong,
Tetrahedron 2012, 68, 9658.
4] S. Mert, A. S. Ya ꢀg lıo ꢀg lu, I. Demirtas, R. Kasımo ꢀg ulları, Med.
Chem. Res. 2014, 23, 1278.
5] L. D. Via, A. M. Marini, S. Salerno, C. L. Motta, A. Toninello,
Bioorg. Med. Chem. 2009, 17, 326.
6] G. M. Nitulescu, C. Draghici, A. V. Missir, Eur. J. Med. Chem.
2
30] R. Ahlrichs, M. Bar, M. Häser, H. Horn, C. Kölmel, Chem.
Phys. Lett. 1989, 162, 165.
31] K. Eichkorn, O. Treutler, H. Öhm, M. Haser, R. Ahlrichs,
Chem. Phys. Lett. 1995, 240, 283.
32] M. Sierka, A. Hogekamp, R. Ahlrichs, J. Chem. Phys. 2003,
118, 9136.
33] a) J. Hepburn, G. Scoles, R. Penco, Chem. Phys. Lett. 1975, 36,
2
010, 45, 4914.
7] S. Alyar, N. Özbek, N. O. Iskeleli, N. Karacan, Med. Chem. Res.
013, 22, 2061.
_
2
451; b) S. Grimme, J. Antony, S. Ehrlich, H. Krieg, J. Chem.
8] S. U. Rai, A. M. Isloor, P. Shetty, K. S. R. Pai, H. K. Fun,
Arabian J. Chem. 2015, 8, 317.
9] A. Ansari, A. Ali, A. M. Shamsuzzaman, New J. Chem. 2017,
Phys. 2010, 132, 154104.
[34] A. Klamt, G. Schuurmann, J. Chem. Soc. Perkin Trans. 1993,
2
, 799.
4
1, 16.
[
35] C. F. Macrae, P. R. Edgington, P. McCabe, E. Pidcock, G. P.
[
10] K. Karrouchi, S. Radi, Y. Ramli, J. Taoufik, Y. N. Mabkhot,
F. A. Al-aizari, M. Ansar, Molecules 2018, 23, 134.
Shields, R. Taylor, M. Towler, J. Van de Streek, J. Appl. Cryst.
2
006, 39, 453.
[
[
11] N. P. Prajapati, H. D. Patel, Synth. Commun. 2019, 49, 2767.
12] O. Dömötör, M. A. Kiss, G. T. G ꢁa l, N. V. May, G. Spengler, M.
Nové, A. C. Gašparovi ꢁc , E. Frank, E. A. Enyedy, J. Inorg. Bio-
chem. 2020, 202, 110883.
13] A. Sirbu, O. Palamarciuc, M. V. Babak, J. M. Lim, K. Ohui,
E. A. Enyedy, D. S. Shova, D. Darvasiov ꢁa , P. Rapta, W. H.
Ang, V. B. Arion, Dalton Trans. 2017, 46, 3833.
[
36] a) E. Runge, E. K. U. Gross, Phys. Rev. Lett. 1984, 52, 997; b) C.
Adamo, D. Jacquemin, Chem. Soc. Rev. 2013, 42, 845.
37] M. J. Frisch, G. W. Trucks, H. B. Schlegel, G. E. Scuseria,
M. A. Robb, J. R. Cheeseman, Gaussian 09, Rev D.1, Gaussian,
Inc, Wallingford CT 2013.
[
[
[
38] a) T. Yanai, D. P. Tew, N. C. Handy, Chem. Phys. Lett. 2004,
3
93, 51; b) J. O. F. Thompson, L. B. Klein, T. I. Sølling,
[
14] C. S. Allardyce, A. Dorcier, C. Scolaro, P. J. Dyson, Appl.
Organomet. Chem. 2005, 19, 1.
15] J. García-Tojal, A. García-Orad, J. L. Serra, J. L. Pizarro, L.
Lezama, M. I. Arriortua, T. Rojo, J. Inorg, Biochemistry 1999,
M. J. Paterson, D. Townsend, Chem. Sci. 2016, 7, 1826; c)
M. D. J. Waters, A. B. Skov, M. A. B. Larsen, C. M. Clausen,
P. M. Weber, T. I. Sølling, Phys. Chem. Chem. Phys. 2019, 21,
[
2283.
7
5, 45.

KHANVILKAR ET AL.
17 of 17
[
[
[
39] M. Cossi, N. Rega, G. Scalmani, V. Barone, J. Comput. Chem.
003, 24, 669.
40] M. N. Patel, C. R. Patel, H. N. Joshi, K. P. Thakor, Spectrochim.
Acta A 2014, 127, 261.
41] A. Tarushi, K. Lafazanis, J. Kljun, I. Turel, A. A. Pantazaki, G.
Psomas, D. P. Kessissoglou, J. Inorg. Biochem. 2013, 121, 53.
42] T. Mosmann, J. Immunol. Methods 1983, 65, 55.
43] I. Ali, W. A. Wani, K. Saleem, Synth. React. Inorg., Met.-Org.,
Nano-Met. Chem. 2013, 43, 1162.
[58] J. R. Lakowicz, G. Weber, Biochemistry 1973, 12, 4161.
2
[59] K. N. A. Rahman, J. Haribabu, C. Balachandran, N. S. P.
Bhuvanesh, R. Karvembu, A. Sreekanth, Polyhedron 2017,
135, 35.
[60] T. K. Goswami, B. V. S. K. Chakravarthi, M. Roy, A. A.
Karande, A. R. Chakravarty, Inorg. Chem. 2011, 50, 8452.
[61] M. Anjomshoa, S. J. Fatemi, M. Torkzadeh-Mahani, H.
Hadadzadeh, Spectrochim. Acta Part. A. 2014, 127, 511.
[62] ACD/ChemSketch, version 2020.1.2, Advanced Chemistry
Development, Inc, Toronto, ON, Canada, http://www.acdlabs.
com 2020.
[
[
[
[
[
44] R. Pulipaka, S. R. Dash, P. Khanvilkar, S. S. Jana, R. V.
Devkar, D. Chakraborty, Trans. Met. Chem. 2019, 38, 603.
45] X. M. Liu, L. H. Xie, J. B. Lin, R. B. Lin, J. P. Zhang, X. M.
Chen, Dalton Trans. 2011, 40, 8549.
46] L. N. Zhu, L. Yi, W. Dong, W. Z. Wang, Z. Q. Liu, Q. M.
Wang, D. Z. Liao, Z. H. Jiang, S. P. Yan, J. Coord. Chem. 2006,
[63] M. Thompson, ArgusLab 4.0.1., Planaria software LLC, Seattle,
Wash, USA, 2004.
[64] A. Hafeez, Z. S. Saify, A. Naz, F. Yasmin, N. Akhtar,
J. Comput. Med. 2013, 312183.
5
9, 457.
[65] S. Salentin, S. Schreiber, V. J. Haupt, M. F. Adasme, M.
Schroeder, Nucleic Acids Res. 2015, 43, W443.
[66] N. Zaidi, E. Ahmad, M. Rehan, G. Rabbani, M. R. Ajmal, Y.
Zaidi, J. Phys. Chem. B 2013, 117, 2595.
[67] G. Sudlow, D. J. Birkett, D. N. Wade, Mol. Pharmacol. 1976,
12, 1052.
[
47] T. Ya-Wen, C. Yun-Fan, L. Yong-Jie, C. Kuan-Hung, C. H.
Lin, H. Jui-Hsien, Molecules 2018, 23, 59.
48] A. A. Adebayo, P. A. Ajibade, J. Chem. 2016, 15, 3672062.
49] P. Kumar, S. Dasari, A. K. Patra, Eur. J. Med. Chem. 2017,
[
[
1
36, 52.
[
[
[
50] S. Thota, D. A. Rodrigues, D. C. Crans, E. J. Barreir, J. Med.
[68] K. Zheng, F. Liu, X. M. Xu, Y. T. Li, Z. Y. Wu, W. Yan, New
J. Chem. 2014, 38, 2964.
[69] A. Daina, V. Zoete, Chem Med Chem. 2016, 11, 1117.
[70] A. Daina, O. Michirlin, V. Zoete, Sci. Rep. 2017, 7, 42717.
Chem. 2018, 61, 5805.
51] A. M. Pyle, J. P. Rehmann, R. Meshoyrer, C. V. Kumar, N. J.
Turro, J. K. Barton, J. Am. Chem. Soc. 1989, 111, 3051.
52] Y. C. Huang, J. Haribabu, C. M. Chien, G. Sabapathi, C. K.
Chou, R. Karvembu, P. Venuvanalingam, W. M. Ching, M. L.
Tsai, S. C. N. Hsu, J. Inorg. Biochem. 2019, 194, 74.
53] J. Haribabu, G. Sabapathi, M. M. Tamizh, C. Balachandran,
N. S. P. Bhuvanesh, P. Venuvanalingam, R. Karvembu, Organ-
ometallics 2018, 37, 1242.
SUPPORTING INFORMATION
Additional supporting information may be found online
in the Supporting Information section at the end of this
article.
[
[
[
[
54] G. Kalaiarasi, S. Dharani, S. R. J. Rajkumar, V. M. Lynch, R.
Prabhakaran, J. Inorg. Biochem. 2020, 211, 111176.
55] A. Bouskila, E. Amouyal, C. Verchere-Beaur, I. Sasaki, A.
Gaudemer, J. Photochem. Photobiol. B. Biol. 2004, 76, 69.
56] R. Mudavath, B. Ushaiah, C. K. Prasad, K. Sudeepa, P.
Ravindar, S. N. T. Sunitha, C. S. Devi, J. Biomol. Struct. Dyn.
How to cite this article: P. Khanvilkar, S.
R. Dash, D. Banerjee, A. Vohra, R. Devkar,
D. Chakraborty, Appl Organomet Chem 2021,
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2
020, 38, 2849.
[
57] H. Laila, A. Rahman, M. Rafat, E. I. Khatib, L. A. B. Nassr,
A. M. Abu-Dief, M. Ismael, A. A. Seleem, Spectrochim. Acta A
2
014, 117, 366.