Organometallic binuclear Ru(II) complexes: Design, synthesis, DNA/BSA binding interactions and in-vitro cytotoxicity against HeLa cell line

Article abstract of DOI:10.1016/j.inoche.2019.02.023

Synthesis, characterization, DNA/BSA binding and cytotoxicity of organometallic binuclear Ru (II) complexes [(p-cym)(Fc-AA)Ru(μ-Im)Ru(Fc-AA)(p-cym)]Cl (C1–C4; where: p-cym = p-cymene MeC₆H₄Prⁱ, Fc-AA = N-ferrocenyl amino acid conjugates (Fc-tyr1, Fc-phe2, Fc-leu3, Fc-trp4) and Im = Imidazole) have been described. The complexes C1–C4 exhibited strong DNA/BSA binding affinity and cytotoxicity against human cervical cancer HeLa cell line. Their binding with calf thymus CT-DNA and bovine serum albumin BSA have been examined by absorption and emission spectral studies. Intercalative interactions between complexes and CT-DNA have been affirmed by EB displacement studies and DNA viscosity measurements, while interaction with BSA via static quenching was explored by fluorescence titration. The anti-cancer activity of C1–C4 was studied using MTT assay on HeLa cell line and the corresponding IC₅₀ values were calculated. The IC₅₀ value (30.0 μM–120.0 μM) obtained were further compared with the well-known ruthenium complex NAMI-A, currently under phase II clinical trials which has IC₅₀ value 608.5 μM.

Full text of DOI:10.1016/j.inoche.2019.02.023

Accepted Manuscript
Organometallic binuclear Ru(II) complexes: Design, synthesis,
DNA/BSA binding interactions and in-vitro cytotoxicity against
HeLa cell line
Priyanka Khanvilkar, Ramadevi Pulipaka, Kavita Shirsath,
Ranjitsinh Devkar, Debjani Chakraborty
PII:
S1387-7003(18)31133-X
DOI:
https://doi.org/10.1016/j.inoche.2019.02.023
INOCHE 7281
Reference:
To appear in:
Inorganic Chemistry Communications
Received date:
Revised date:
Accepted date:
24 December 2018
5 February 2019
15 February 2019
Please cite this article as: P. Khanvilkar, R. Pulipaka, K. Shirsath, et al., Organometallic
binuclear Ru(II) complexes: Design, synthesis, DNA/BSA binding interactions and in-
vitro cytotoxicity against HeLa cell line, Inorganic Chemistry Communications,
https://doi.org/10.1016/j.inoche.2019.02.023
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ACCEPTED MANUSCRIPT
Organometallic Binuclear Ru(II) Complexes :Design, Synthesis, DNA/BSA binding
interactions and InVitro Cytotoxicity against HeLa cell line
a
a
b
b
a*
Priyanka Khanvilkar , Ramadevi Pulipaka , Kavita Shirsath , Ranjitsinh Devkar , Debjani Chakraborty
a
Department of Chemistry, The Maharaja Sayajirao University of Baroda, Vadodara, India
Department of Zoology, The Maharaja Sayajirao University of Baroda, Vadodara, India.
b
Corresponding author : Debjani Chakraborty*
Email: debchak23@gmail.com
Synthesis, Characterization, DNA/BSA binding and cytotoxicity of organometallic binuclear Ru (II)
complexes [(p-cym)(Fc-AA) Ru (μ-Im) Ru (Fc-AA) (p-cym)]Cl(C1-C4; where: p-cym = p-cymene
i
MeC H Pr , Fc-AA=N-ferrocenyl amino acidconjugates (Fc-tyr1, Fc-phe2, Fc-leu3, Fc-trp4) and Im =
6
4
Imidazole) have been described. The complexes C1-C4 exhibited strong DNA/BSA binding affinity and
cytotoxiciy against human cervical cancer HeLa cell line. Their binding with calf thymus CT-DNA and
bovine serum albumin BSAhave been examined by absorption and emission spectral studies.Intercalative
interactions between complexes and CT-DNA have been affirmed by EBdisplacement studies and DNA
viscosity measurements, while interaction with BSA via static quenching was explored by
fluorescencetitration. The anti-canceractivity of C1-C4was studied using MTT assay on HeLa cell line and
the corresponding IC values were calculated. The IC value (30.0μM-120.0μM) obtained were further
50
50
compared with the well-known ruthenium complex NAMI-A, currently under phase II clinical trials which
has IC value 608.5μM.
5
0
N-ferrocenyl amino acids, organometallic binuclear ruthenium (II) complexes, Imidazole, DNA/BSA
binding interactions, cytotoxicity, HeLa cell.
1

ACCEPTED MANUSCRIPT
Development and evaluation of effective anticancer agents exhibiting strong DNA and protein binding
capabilities, high cytotoxicity and low side effects are highly in-demand in medicinal and pharmaceutical
research. Organometallic compounds are an emerging category of therapeutics in anticancer research
offering advantages where regular drugs are not efficient due to resistance. These compounds provide
something alternative to inorganic therapeutics and organic drugs. Organometallic conjugation of
biological molecules like amino acids, peptides, nucleobases or natural products is the most common and
effective method for developing organometallic compounds which are medicinally promising candidates
[
1]. Our pursuit to design ferrocene based cytotoxic agent’s stems from the fact that the ferrocenyl group
can get reversibly oxidized to ferrocinium radical cation which can generate ROS under physiological
conditions. This makes the ferrocenyl moiety redox active and cytotoxic with potential bioapplications.
The first ruthenium anticancer compound to be studied on human beings was NAMI-A, a Ru (III)
coordination compound of formula [ImH][trans-RuCl4(dmso-S)(Im)] (dmso-S = sulfur-bonded dimethyl
sulfoxide,Im = imidazole) [2]. In addition Ru(II)–polypyridyl compounds have shown excellent
advantages as photosensitizers for photodynamic therapy [3]. Furthermore organometallic ruthenium-arene
complexes are being considered as alternative to platinum based drugs owing to their superior anticancer
activity, lower side effects and photocytotoxicity [4]. Since interaction between metal complexes and DNA
can cause DNA damage in cancer cells, study of the binding mechanism of metal complexes with DNA is
of importance. Cytotoxicity of the organometallic ruthenium compounds have been attributed to their
interaction with proteins, besides DNA [5]. Further, serum albumin is one of the most essential proteins
involved in the transportation,distribution, accumulation and excretion of the drugs in tumour cells. Bovine
serum albumin (BSA) is used as a model protein to evaluate binding behaviour and cytotoxicity of these
compounds [6].
The organometallic binuclear ruthenium (II) arene complexes (C1-C4) with ferrocene amino acid
conjugates as ligands have been synthesised and investigated for their interaction with CT-DNA and BSA.
Results obtained from the binding studies prompted the in-vitro cytotoxicity evaluation of the complexes
on human cervical cancer (HeLa) cell line using MTT assay, and the corresponding IC50 values were
obtained and compared with other well-known anti-cancer agent
All the chemicals and solvents used for synthesis and characterization of ligands and complexes were of
analytical grade and used as purchased. α-terpinene was purchased from Sigma-Aldrich, RuCl . 3H O and
3
2
BSA from Hi-media and CT-DNA, Tri-sodium citrate and EB (ethidium bromide) were purchased from
SRL (Sisco research laboratory, Mumbai, India.). The solvents used in the present study were purchased
from Merck and are of analytical grade. HeLa cell line was obtained from National centre for cell science
(
NCCS) Pune, India.
Electrospray ionization (ESI) mass spectral measurement was done using Applied Biosystem API 2000
-1
Mass spectrometer. Infrared spectra in the range 4000-400 cm were recorded on Perkin Elmer RX-1 FTIR
1
using KBr pellets. H NMR spectra were recorded on a Bruker AR X 400 Spectrometer at 400 MHz using
2

ACCEPTED MANUSCRIPT
DMSO as solvent.UV spectra of all the complexes were recorded in CH OH solution on Perkin Elmer
3
Lambda-35 dual beam UV-Vis spectrophotometer. Fluorescence spectra of the complexes were recorded
using DMSO solution on JASCO FP-6300 fluorescence spectrophotometer.
The ferrocenyl amino acids (Fc-AA)were synthesized according to the procedure reported [7].The
binuclear metal complexes Cl-C4 were synthesized in two steps. Firstly, the mononuclear complexes
6
6
[
Ru(η -p-cym)(Fc-AA)Cl]were synthesized by a typical µ-chlorido-bridge splitting reaction of [Ru(η -p-
cymene)Cl ] as described in the literature [8]. In the second step two equivalent molesof the mononuclear
2
2
complex was dissolved in a mixture of 5ml of CH Cl and 3ml of CH OH followed by addition of one
2
2
3
equivalent mole of imidazole. The reaction mixture was stirred for 10 min to form a homogeneous solution
and a solution of 1.0M NaOH was added to raise the pH upto ~11 and to deprotonate the –NH present in
the imidazole ring [9]. The solution was kept stirring for 24hr at r.t. under N atmosphere. The resultant
2
reddish brown crystalline solid was then filtered, washed with pet ether and dried in oven at 40°C for 1 h.
The complexes so obtained were recrystallized from dichloromethane and ether. The general procedure for
the synthesis of C1-C4 is given in fig-1.
Where R = -CH (4-hydroxyphenyl) Fc-Tyr (C1); -CH Ph Fc-Phe (C2); -CH CH (CH ) Fc-Leu (C3);
2
2
2
3 2
-
CH (3-indolyl)Fc-Trp (C4)
2
Fig.1.General Synthetic scheme of complexesC1-C4
6
C1was synthesized by the reaction of the mononuclear complex [Ru(η -p-cymene)(Fc-tyr)Cl](0.092 mmol,
6
0.0 mg) and the bridging ligand imidazole(0.046 mmol, 3.1mg). Solubility: DMSO, MeOH, CH Cl .
2 2
Yield: 76.4%; Molecular Weight:1330.32 g/mole; Molecular Formula: C H ClFe N O Ru ; Anal.
63
73
2
4
6
2
+
Found: C, 56.74; H, 5.51; N, 4.35. Calc.: C, 57.34; H, 5.85; N, 4.11. ESI-MS m/z: 1294.82 (M ) ;δ (400
H
6
MHz, DMSO-d ): 10.4 (s, 1H, -OH of tyrosine),7.3-8.5 (m, 4H, Ar-H oftyrosine), 5.81-5.75 (m, 4H, p-
cym Ar-H), 4.7 (t, 2H, substituted cyclopentadiene), 4.45 (t, 2H, substituted cyclopentadiene), 4.27 (s, 5H,
cyclopentadiene), 2.7 (q, 1H, p-cym-iso-prop-CH), 2.03 (s, 3H, p-cym Ar-CH ), 1.27 (d, 6H, p-cym- iso-
3
3

ACCEPTED MANUSCRIPT
-1
prop-(CH ) ),7.5 (d, 2H, imidazole CH=CH); FTIR (KBr/cm ):ν(O-H) 3452,ν(Ar)C-
3
2
H2963,νCOOassym1512, νCOOsym1326, ΔνCOO186.
6
C2 was synthesized by the reaction of [Ru(η -p-cymene)(Fc-phe)Cl] (0.094 mmol, 60.0 mg) and imidazole
(
0.047 mmol, 3.2 mg). Solubility: DMSO, MeOH, CH Cl .Yield: 64.3%; Molecular Weight 1299.44
2 2
g/mole; Molecular FormulaC H ClFe N O Ru ;Anal. Found: C, 58.41; H, 5.68; N, 4.10. Calc.: C,
63
73
2
4
4
2
+
6
5
8.72; H, 5.99; N, 4.21. ESI-MS m/z: 1263.94 (M ) ; δ (400 MHz, DMSO-d ): 7.10-7.45(m, 5H, Ar-H
H
of phenylalanine), 5.14-5.63 (m, 4H, p-cym Ar-H), 4.23 (t, 2H, substituted cyclopentadiene),4.64 (t, 2H,
substituted cyclopentadiene), 4.15 (s, 5H, cyclopentadiene), 2.93(q, 1H, p-cym-iso-prop-CH), 2.12 (s,
-1
3
H, p-cymAr-CH ), 1.25 (d, 6H, p-cym-iso-prop-(CH ) ); FTIR (KBr/cm ):ν(Ar)C-H2959,
3 3 2
νCOOassym1626, νCOOsym1367, ΔνCOO259.
6
C3 was synthesized by the reaction of[Ru(η -p-cymene)(Fc-leu)Cl](0.1 mmol, 60.0 mg) and
imidazole(0.05, 3.40 mg). Solubility: DMSO, MeOH, CH Cl . Yield: 67.9%; Molecular Weight 1231.42
2
2
g/mole; Molecular FormulaC H ClFe N O Ru ; Anal. Found: C, 54.15; H, 6.50; N, 5.35. Calc.: C,
57
77
2
4
4
2
+
6
5
4.54; H, 6.80; N, 5.72. ESI-MS m/z: 1195.92 (M ); δ (400 MHz, DMSO-d ): 5.45-5.90 (m, 4H, p-cym
H
Ar-H), 5.46 (t, 2H,substituted cyclopentadiene), 4.15 (t, 2H, substituted cyclopentadiene),4.25 (s, 5H,
cyclopentadiene), 2.89 (q, 1H, p-cym-isoprop-CH), 2.03 (s, 3H, p-cym Ar-CH ), 1.19 (d, 6H, p-cym-iso-
3
-1
prop-(CH ) ), 1.56 (d, 6H, leucine-iso-prop-(CH ) ); FTIR(KBr/cm ):ν(Ar)C-H2957, νCOOassym1603,
3
2
3 2
νCOOsym1326, ΔνCOO = 277.
6
C4 was synthesized by the reaction of [Ru(η -p-cymene)(Fc-trp)Cl](0.089 mmol, 60.0 mg) and
imidazole(0.04 mmol, 3.03 mg). Solubility:DMSO, MeOH, CH Cl .Yield: 62.8%; Molecular Weight:
2
2
1
377.52 g/mole; Molecular Formula:C H ClFe N O Ru ; Anal. Found: C, 59.29; H, 5.30; N, 5.12.
67 75 2 6 4 2
+
Calc.: C, 58.91; H, 5.60; N, 5.51. ESI-MS m/z: 1342.02 (M -Cl); δ (400 MHz, DMSO-d6): 12.6 (s, 1H,
H
indolinic N-H of tryptophan),5.94-5.35 (m, 4H, p-cym Ar-H), 4.72 (t, 2H, substituted
cyclopentadiene),4.59 (t, 2H, substituted cyclopentadiene), 4.78 (s, 5H,cyclopentadiene), 2.93 (q, 1H, p-
-1
cym-iso-prop-CH), 2.16(s, 3H, p-cymAr-CH3), 1.25(d, 6H,p-cym-iso-prop-(CH ) );FTIR (KBr/cm ):
3
2
ν(N-H) 3082, ν(Ar)C-H2961, νCOOassym1637, νCOOsym1370, ΔνCOO= 267.
The absorption spectral experiments to evaluate the interaction of the complexes with calf thymus DNA
were conducted in Tris-HCl/NaCl buffer (5mM Tris-HCl, 5mM NaCl) having pH 7.2 and using stock
solution of the complexes C1-C4(1mM) in DMSO at an ambient temperature. The stock solution of CT-
DNAwas prepared in buffer and stored at 4˚C. Absorption studies were carried out by keeping fixed
concentrations of complexes and varying the concentration of CT-DNA. Solution of the complexes were
prepared by diluting with tris buffer to obtain the desired concentration. While measuring the absorption of
the complexes, equal increments of CT-DNA solution were added to both the sample cell and to the
reference cell in order to eliminate the absorbance of CT-DNA itself. The binding constant (K ) of the
b
complexes to DNA were calculated using the McGhee-von Hippel (MvH) method [10].
4

ACCEPTED MANUSCRIPT
Fluorescence spectroscopy was employed to examine whether the complexes have the ability to displace
EB from DNA–EB complex and to confirm the intercalative mode of binding of the complexes.The
titration experiment was carried out by keeping the DNA-EB complex concentration constant and varying
the complex concentration. The variation in the fluorescence emission spectrum of the DNA–EB complex
was recorded by addition of aliquot of the complex. The fluorescence emission (λex = 520nm) was
measured from 530 nm to 700nm using 5nm slits.
Cannon–Ubbelohde viscometer was used to measure the relative viscosity of DNA. The viscosity
measurements of DNA (200 µM) solutions were carried out at a constant temperature of 32.0 ± 0.1 ºC in
the presence of complexes C1-C4 at [complex]/[DNA] ratio of 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
1/3
calculated. Data are presented as (η/η ) versus [complex]/[DNA], where η is the viscosity of DNA in the
0
presence of complex and η is the viscosity of DNA alone. Viscosity values were calculated from the
0
observed flow time of DNA-containing solutions (t) corrected for that of the buffer alone (t ), η = (t-t )/t
0
0
0
[
11].
The protein-binding study of C1-C4 was performed employing steady state fluorescence spectroscopy. In
the fluorescence quenching experiments, a 3.0 ml tris buffer solution of BSA (16.6 μM) was placed in a
quartz cuvette and titrated with increasing concentrations of the complexes. The effect of increasing
concentrations of the complexes on the emission of the tryptophan residues of BSA was monitored in the
range 300-500nm at an excitation wavelength of 296 nm.[12]
Cytotoxicityof the synthesised complexes was assessed by standard MTT colorimetric assay [13].HeLa
3
-1
cells (5.0 × 10 cells well ) were placed in 96-well culture plates (Tarson India Pvt. Ltd.) and grown
overnight at 37˚C in a 5% CO incubator. C1-C4 were added to the wells in the concentration range 0.5-
2
150 mg/ml. Control wells were prepared by addition of culture medium without the compounds. The plates
were incubated at 37˚C in a 5% CO incubator for 48 h. Standard 3-(4,5-dimethylthiazole)-2,5-
2
diphenyltetraazolium bromide MTT dye solution was then added to each well. After 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 addition of DMSO to dissolve the formazan crystals formed and further
incubation for 30 min. The optical density of each well was measured spectrophotometrically at 563 nm
using Biotek- ELX800MS universal ELISA reader (Bio-Tek instruments, Inc., Winooski, VT). The IC50
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.
5

ACCEPTED MANUSCRIPT
+
The ESI-Mass spectra of C1-C4 show m/z peaks corresponding to the molecular ion M . The m/z values
indicate that a p-cymene and an amino acid conjugated ferrocenyl ligand are coordinated to each of the
metal centres with a deprotonated imidazole as a bridging ligand, thereby resulting in the formation of a
binuclear complex. The molecular ion peaks have been provided in table -1.The mass spectra have been
provided in the supplementary file (Fig. S1).
Table 1m/z values of complexes C1-C4
Code
C1
C2
C3
C4
Calculated Mass
1294.82
1263.94
1195.92
1342.02
(
g/mol)
+
+
+
+
Observed Mass
1293.6(M -1)
1262.4(M -1)
1196.3(M )
1342.02(M )
(
g/mol)
-1
The IR spectra of C1-C4 (fig.S2) displayed characteristic strong stretching bands at1530-1660 cm due to
-1
-
asymmetric and weaker bands at 1325-1373 cm due to symmetric COO stretch of the conjugated amino
-1
acids respectively which were found as strong bands in the fingerprint region at 1580-1610 cm in the
spectra of free ferrocenyl ligands [8]. A monodentate coordination mode of the carboxylate group of the
ligands to the metal ion is indicated by the separation frequency(Δν)values which fall in the range 185-295
-
1
cm [14,15]. The broad O-H stretching band of free carboxylic acid group found in the ligands at ~3546
-
1
cm is completely lost in the IR spectra of the complexes indicating complexation via the carboxylate
-1
oxygen. Furthermore the N-H stretching bands at 2900-3000 cm observed in the spectra of the ferrocenyl
-1
amino acids were found to blue shifted to 3050-3200 cm in C1-C4 indicating complexation of the ligands
via the nitrogen of the secondary amine. The presence of weak to medium bands in the fingerprint regions
-1
owing to aromatic νC-H stretch (2900-3000 cm ) and strong bands due to the aromatic ν
in-plane
C=C
-1
vibrations (~1430-1667cm ) is indicative of presence of p-cymene in the complexes.The free ν
N-H
-
1
stretching of the imidazole around 3300-3364 cm was absent in the spectra of C1-C4 indicating
deprotonation of the imidazole N-H on co-ordination to the two Ru(II) metal centres. The overall changes
in the IR spectra suggest that the ferrocenyl-amino acid conjugates act as monoanionic bidentate ligands
interacting with the metal centres via nitrogen of the secondary amine and carboxylate oxygen whereas
disappearance of the imidazole N-H frequency suggested that two Ru(II) metal centres are bridged via an
imidazole anion resulting in binuclear complex formation.
The peaks arising due to carboxylic O-H and N-H of secondary amine in the free ferrocenyl ligands are no
longer seen in the NMR spectra of the complexes indicating coordination of the carboxylate oxygen and
nitrogen of secondary amine to the two ruthenium metal centres. Moreover the peaks owing to the aromatic
protons in amino acid nucleus are observed in the region δ = 7-9. The methyl and substituted methyl
6

ACCEPTED MANUSCRIPT
protons appear at around δ = 1-2 ppm. The N-H proton of free imidazole observed at δ = 11-13 as a sharp
singlet was absent in the NMR spectra of the complexes suggesting co-ordination of the deprotonated
1
imidazole N with one of the metal centre.The H NMR spectra of C1-C4 also show the following distinct
peaks corresponding to p-cymene; 6 proton doublet at δ= 1.19 ppm owing to two methyl protons of iso-
propyl group [CH(CH ) ], 3 proton singlet at δ= 2.08 ppm due to the Ar-methyl para to the iso-propyl
3
2
group, 1 proton quartet at δ=2.85 ppm attributed to –CH of the iso-propyl group and two 2 proton doublets
at δ=5.8 ppm which can be assigned to the Ar-protons of p-cymene. The mass spectra have been provided
in the supplementary file (Fig. S3).
The electronic absorption spectra of the complexes C1-C4 were recorded in CH OH solution in the region
3
200-450 nm. The electronic spectra of free ligands displayed intense absorption bands at 203-206 nm [8]
ascribable to intra ligand π→π* transition of the cyclopentadienyl rings of ferrocene [16] which were
observed in the longer wavelength region at 210-220nm (Fig. 2) in the complexes due to coordination with
Ru(II) metal centre. Absorption peaks in the region 275-277 nm corresponding to n→π* transitions of the
ferrocenyl ligands were also observed.
Fig. 2UV-vis. spectra of complexesC1-C4 recorded in CH OH with path length 1 cm
3
Table 2 UV-Vis peak assignments of C1-C4
Intra-ligand transitions
Code
(nm)
π-π*
216
211
216
218
n-π*
275
276
277
275
C1
C2
C3
C4
7

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The electronic absorption spectra of complexes C1-C4 consist of a well-resolved absorption band in the
range of 210-220 nm assigned to intraligand π→π* transitions. Upon the addition of DNA, the band
showed significant hypochromism accompanied with a red shift. These results indicate intercalative
binding mode of the complexes to DNA and are similar to various metallo-intercalators reported earlier
[
(
17]. Intercalation of small molecules to DNA generally results in hypochromism and red shift
bathochromism) of the absorption band due to strong stacking interaction between the aromatic
chromophore of the ligands and the base pair of the DNA [18,19].The UV spectra of complexes C1-C4
have been recorded in absence and presence of varying CT-DNA concentration (0-100 mM) within (Fig.
3
2
).The intrinsic binding constants K were obtained by monitoring the changes in the energy band at 210-
b
20nm with increasing concentration of DNA and using the equation:
[
DNA]/(ε -ε )=[DNA]/(ε -ε )+1/K (ε -ε )
A f b f b b f
where [DNA] is the concentration of DNA in base pairs, ε = Aobsd/ [compound], ε is the extinction
A
f
coefficient for the unbound compound and ε is the extinction coefficient for the compound in the fully
b
bound form. A plot of [DNA]/(ε -ε ) vs [DNA] (Fig. 4) gave a slope 1/(ε -ε )and an intercept(1/K )(ε -ε ).
A
f
b
f
b
A
f
The binding constant K is the ratio of the slope to the intercept. The observed values of the binding
b
3
constants K (table 3) follow the order C2>C4>C1>C3. Higher binding strength of C2 (9.3 x 10 ),C4 (8.6 x
b
3
3
1
0 ) and C1(5.5 x 10 ) may be attributed to the presence of additional planar aromatic moieties in the
amino acid conjugates which facilitates intercalation.The titration curves for complexes C2–C4 have been
provided as Supplementary material (Fig. S4).
Fig.3UV absorption spectra of C1 at increasing concentrations of CT-DNA, the arrow shows decrease in
intensity upon increasing concentration of the DNA.
8

ACCEPTED MANUSCRIPT
Fig.4: Plot of [DNA]/(ε -ε ) versus [DNA] for C1-C4. The slope to intercept ratio gave K values
A
f
b
The changes observed in the fluorescence spectra of EB on its binding to CT DNA are often used for the
interaction study between DNA and other compounds, such as metal complexes. The emission spectra of
DNA-EB (λ = 546 nm, λ = 610) complex in the absence and presence of increasing amounts of C1-C4
ex
em
have been recorded. Addition of complexes resulted in a significant decrease of the intensity of the
emission band at 609 nm with slight red shift of thebands indicating the competition of the complexes with
EB in binding to DNA (Fig.5). The observed quenching of DNA-EB fluorescence suggeststhat they
displace EB from the DNA-EB complex and interact with DNA probably by intercalation[20].The
quenching constants (K ) determined from the slopes of the plots I / I versus [Q]were used to evaluate
SV
0
the quenching efficiency of the complexes according to the Stern-Volmer equation [21].The Stern-Volmer
quenching plots (Fig. 6) illustrate that the quenching of fluorescence of the DNA-EB complex is in good
agreement (R = 0.93-0.99) with the linear Stern Volmer equation. The KSV values obtained are shown in
table 3.The titration curves for complexes C2–C4 have been provided as Supplementary material (Fig. S5).
Fig.5 Fluorescence emission spectra of DNA-EB complex at increasing concentrations of C1 the arrow
shows decrease in intensity (quenching of DNA-EB fluorescence) upon increasing concentration of the
complex.
9

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Fig.6Stern-Volmer quenching plot Io/I versus [Q] of DNA-EB for C1-C4
Table 3 K and K values of complexesC1-C4
b
SV
Code
C1
C2
C3
C4
-
1
3
4
3
3
3
3
3
3
K M
5.5 x 10
1.4 x 10
2nm
9.3 x 10
6.3 x 10
1nm
3.5 x 10
8.6 x 10
3nm
8.6 x 10
6.3 x 10
1nm
b
-
1
K M
sv
λ shift
In order to further confirm the modes of binding of complexes C1–C4 to CT-DNA, viscosity
measurements of DNA solutions were carried out in presence and absence of the complexes. Intercalation
elongates the double helix to accommodate the complex molecule in between the base pairs, leading to an
increase in the viscosity of DNA. In contrast, DNA groove binding typically causes less pronounced
(
positive or negative) or no changes in DNA solution viscosity [22-24]. The effects of C1–C4 and classical
intercalator EB on the viscosities of CT-DNA solution are shown in Fig. 7. With increasing
complex]/[DNA] concentration ratios, the relative viscosities of CT-DNA increased gradually indicative
of characteristic intercalative mode of binding.
[
1
0

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Fig. 7.Effect of increasing amounts of the complexes C1–C4 and ethidium bromide (EB) on the relative
viscosity of CT-DNA (200 mM) in Tris–HCl buffer at 32 ˚C. [Complex]/[DNA] = 0, 0.04, 0.08, 0.12, 0.16
and 0.20.
BSA solutions exhibit a strong fluorescence emission with a peak at 343 nm, due to the tryptophan
residues, when excited at 296 nm [25,26]. Addition of increasing concentrations of C1-C4 to a solution of
BSA (at a ratio of 0-7.5) results in a significant decrease of the protein fluorescence intensity (Fig.8)
suggesting that the complexes interact with BSA and causes quenching of fluorescence .The fluorescence
quenching is quantified by the Stern-Volmer equation : I / I= 1+ KSV [Q]. The KSV values obtained from
0
the plot of [Q] versus I / I (Fig. 9) are given in table 4. Binding of small molecules to a set of equivalent
0
sites on the protein results into an equilibrium between the free and bound molecules represented by the
double logarithm equation : log (Io-I)/I = log K + n log [Q] [27].The plot of log [(Io-I)/I] versus log [Q]
b
-1
for all the systems is linear (Fig.10) and the values of K (M ) and n (table 4) have been obtained from the
a
3
intercept and slope, respectively. The association binding constant K values for C1-C4 are 2.9 x 10 , 3.4
a
4
4
3
-1
3
6
-
x 10 , 4.8 x 10 and 8.4 x 10 M respectively. Values with a magnitude order in the range of 10 -10 M
1
for K are indicative of an efficient interaction with the protein [28-30].The number of binding sites n
a
indicates the number of independent class of binding sites for the complexes on BSA and generally n ≈
1[31].The n valuesfor the complexes average out to be 1 which suggests that there is one binding site
available on the protein. Moreover the linear nature of the double logarithm plots (Fig. 10) indicates that
only one of the tryptophan residues on BSA protein is interacting with the compounds[32].The titration
curves for complexes C2–C4 have been provided as Supplementary material in (Fig. S6).
1
1

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Fig.8:Fluorescence emission spectra of BSA at increasing concentrations of C1 the arrow shows decrease
in intensity (quenching of BSA fluorescence) upon increasing concentration of the complex.
Fig.9: Stern-Volmer quenching plot Io/I versus [Q] of BSA for C1-4
Fig.10: Double logarithmic plot from the quenching of BSA fluorescence by C1-C4 to determine the
binding constant K and number of binding sites n.
a
1
2

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Table 4 K , K and n values for complexes C1-C4
SV
a
Code
C1
C2
C3
C4
-
1
3
4
4
4
4
4
3
4
K M
a
2
3
.9 x 10
3.4 x 10
4.8 x 10
8.4 x 10
-
1
K M
sv
.3 x 10
0.8
3.7 x 10
1.01
2.9 x 10
1.04
4.5 x 10
0.8
n
In-vitro cytotoxicity tests of the binuclear complexes were performed on the human cervical cancer cell
line (HeLa). The selection of this cell line is purely based on their higher occurrence in human population
worldwide which is of prime concern. In-vitro cytotoxicity of the complexes C1-C4 in the HeLa cell line
was evaluated using MTT assay. The cell viabilities (%), obtained for HeLa cells with exposure to the
complexes for 48 h is depicted in Fig. 11. The antiproliferative activity of the complexes was found to be
dose dependent,that is, the cell viability decreased with increasing concentrations.The IC values of the
5
0
complexes have been tabulated in table 5. Although the synthesized ruthenium complexes are found to be
less active compared to cis platin (IC = 18.8 ± 3.4 μM), they are much more active compared to NAMI-A
50
(
ruthenium complex under phase II clinical trials) whose IC values have been found to be in the range of
50
608.5 ± 55.4 µM for HeLa cell lines[33,34] and those of RAPTA-C >1600 μM on treatment for 72 h [35].
Fig. 11. % Cell viability of complexes C1-C4towards HeLa human cervical cancer cell lines. Each point is
the mean ± standard error obtained from three independent experiments.
Table 5 :IC values for complexes C1-C4.
5
0
Code
µM
C1
C2
C3
C4
NAMI A
RAPTA-C
75.5± 5.77
31.1± 6.17
115.5±4.65
32.3±8.51
608.5 ± 55.4
>1600 μM
In summary, homo-binuclear Ru(II) complexes of p-cymene and ferrocenyl amino acid ligands with
imidazole as a bridging ligand were synthesized and characterized using various spectral techniques. DNA
and BSA binding studies reveal that the binuclear complexes have good binding affinities towards the
1
3

ACCEPTED MANUSCRIPT
macromolecules compared to the mononuclear complexes [8] due to more available binding sites, larger
cationic charge and a greater number of hydrophobic ligands than their mononuclear counterparts.Such
impressive binding efficacies broaden their scope for in-vitro investigations as potent anticancer agents
following which we carried out the cytotoxicity studies employing MTTassay. Lower IC values of the
5
0
complexes can be attributed to the presence of a hydrophobic arene moiety which facilitates cellular uptake
of the complexes.
The authors PK and PR gratefully acknowledge the University Grants Commission, MRP fellowship [UGC
F.No. 43-238/2014(SR)] and RFSMS-BSR fellowship [Letter no. F.5-71/2007(BSR)], respectively, for the
financial assistance. The authors would like to acknowledge the DST-FIST program for funding NMR
facility at the Department of Chemistry, Faculty of Science, The Maharaja Sayajirao University of Baroda.
The authors are thankful to the Heads, Department of Chemistry and Zoology,Faculty of Science, The
Maharaja Sayajirao University of Baroda for providing the necessary lab facilities and required
instrumentation facilities to carry out the research work.
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Organometallic Binuclear Ru(II) Complexes :Design, Synthesis, DNA/BSA binding
interactions and InVitro Cytotoxicity against HeLa cell line
a
a
b
b
Priyanka Khanvilkar , Pulipaka Ramadevi , Kavita Shirsath , Ranjitsinh Devkar ,
a*
Debjani Chakraborty
a
Department of Chemistry, The Maharaja Sayajirao University of Baroda, Vadodara, India
Department of Zoology, The Maharaja Sayajirao University of Baroda, Vadodara, India.
b
Corresponding author : Debjani Chakraborty*
Email: debchak23@gmail.com
Graphical Abstract
1
8

ACCEPTED MANUSCRIPT
Organometallic Binuclear Ru(II) Complexes :Design, Synthesis, DNA/BSA binding
interactions and Cytotoxicity against HeLa cell line.**
a
a
b
b
Priyanka Khanvilkar , Pulipaka Ramadevi , Kavita Shirsath , Ranjitsinh Devkar and Debjani
Chakraborty
a*
a
Department of Chemistry, The Maharaja Sayajirao University of Baroda, Vadodara, India
b
Department of Zoology, The Maharaja Sayajirao University of Baroda, Vadodara, India
*
Corresponding author: Debjani Chakraborty
Email: debchak23@gmail.com
Highlights:




Binuclear organometallic ruthenium complexes have been synthesized and
characterized.
DNA and BSA binding interactions have been established by UV-Vis, Viscosity
measurements and fluorescence studies respectively.
The binuclear complexes have good binding affinities towards the macromolecules
compared to the mononuclear complexes.
Complexes show better cytotoxic activity than NAMI-A and RAPTA
1
9