Monitoring the DNA by ruthenium complexes of heterocyclic N,S-donor ligands and evaluation of biological activities

Article abstract of DOI:10.1007/s00706-016-1708-8

Neutral N,S-donor bidentate ligands have been synthesized and characterized by NMR and IR spectroscopic techniques. The ligands have been used to synthesized ruthenium(II) complexes ([Ru(L¹–L⁶)PPh₃)₂Cl₂

Full text of DOI:10.1007/s00706-016-1708-8

Monatsh Chem
DOI 10.1007/s00706-016-1708-8
ORIGINAL PAPER
Monitoring the DNA by ruthenium complexes of heterocyclic
N,S-donor ligands and evaluation of biological activities
Parag S. Karia1 Pankajkumar A. Vekariya1 Anshul P. Patidar1
•
•
•
Ravi R. Patel2 Mohan N. Patel1
•
Received: 28 February 2015 / Accepted: 15 February 2016
Ó Springer-Verlag Wien 2016
Abstract Neutral N,S-donor bidentate ligands have been
synthesized and characterized by NMR and IR spectroscopic
techniques. The ligands have been used to synthesized
ruthenium(II) complexes ([Ru(L1–L6)PPh3)2Cl2]) and
ruthenium(III) complexes ([Ru(L1–L6)PPh3)Cl3]). Synthe-
sized complexes have been characterized using elemental
analysis, UV–Vis spectroscopy, magnetic measurements,
LC–MS, and IR spectroscopy. Broth dilution technique was
used to investigate antibacterial activity against two Gram-
positive and three Gram-negative microorganisms and
results show that all complexes are more potent than the
respective ligands. UV–Vis absorption titration and viscos-
ity measurements have been carried out to investigate the
binding mode and binding strength of complexes with her-
ring sperm DNA. The quantitative binding strength (Kb) of
the complexes was found in the range of 0.521 3 105–
2.94 3 105 M-1 and indicate intercalative mode of binding.
Gel electrophoresis study was carried out to study the
cleavage of pUC19 supercoiled DNA. DNA nuclease
activity data for the complexes are found higher than
respective ligands and metal salt. Brine shrimp bioassay was
carried out to perform cytotoxicity study. IC50 values of the
complexes were found in the range of 5.69 1.06–14.62
Graphical abstract
Keywords N,S-donor ligands Á
Ru(II)/Ru(III) complexes Á Nucleic acid interaction Á
Cytotoxicity
Introduction
Current progress in the field of molecular biology has
recognized various bioactive compounds that may be log-
ically targeted via inorganic compounds as medication.
Metal complexes have come upon as feasible alternatives
to organic compounds as scaffolds for the design of
molecularly targeted agents due to number of significant
advantages [1, 2]. Among these metal compounds, transi-
tion metal complexes interacting to DNA have been centre
of bioinorganic investigations for at least three decades due
to their noteworthy applications in DNA structural probes
and molecular switches, disease resistance, development in
drug effectiveness, and new drug design and screening [3–
6]. Primary importance of using transition metals is owing
to variable oxidation states and geometries due to which
the coordination sphere of ligands can be rationally
0.87 lg cm-3
.
Electronic supplementary material The online version of this
article (doi:10.1007/s00706-016-1708-8) contains supplementary
material, which is available to authorized users.
& Mohan N. Patel
jeenen@gmail.com
1
Department of Chemistry, Sardar Patel University,
Vallabh Vidyanagar 388 120, Gujarat, India
2
Department of Bioscience, Sardar Patel University,
Vallabh Vidyanagar 388 120, Gujarat, India
123

P. S. Karia et al.
planned. This aspect can be exploited to prepare novel values are close to the 1.73 BM indicating that the com-
structural geometries (e.g. octahedral or square-planar plexes contain one unpaired electron, with ruthenium ion in
motifs) that are not usually accessible to organic mole- its ?3 oxidation state.
cules. Second point of selecting transition metal complexes
DMSO was used as a medium to obtain the electronic
is that the thermodynamic and kinetic properties of these spectra of the synthesized complexes. In case of ruthe-
metal complexes are sensitive toward the nature of ligands nium(II) complexes 1–6, three distinct bands are observed
attached and hence, metal-based therapeutics can be easily in the range of 240–800 nm. The band in the range of
optimized to achieve the desired biological applications 262–273 nm is corresponding to intra ligand charge
through alteration of the supporting ligands. In transition transfer band. The band around 420–440 nm is attributed to
metal compounds, platinum based drugs have drawn much metal to ligand charge transfer band. The peak around
attention after the clinical success of cisplatin. However, 550–568 nm is due to d–d transition. While in case of
such metal based drugs are hindered by several problems ruthenium(III) complexes 7–12, in the electronic spectra, a
such as serious toxicity, side effects and major problem band obtained around 270 nm corresponds to intraligand
with resistance [7]. These unsolved problems in platinum- charge transfer band. The band around 350 nm is due to
based anticancer therapy have inspired increased research metal-to-ligand charge transfer band. The peak around
efforts in the search for new non-platinum-based metal 540 nm is assigned due to d–d transition.
species as cytotoxic agents [8], and currently, there is much
interest in the design of ruthenium complexes as anticancer IR spectra
agents [9]. Many Ru(II) and Ru(III) complexes have been
reported to exhibit potentially useful cytotoxicity recently, Vibrations modes of the ligands are affected by coordina-
including mer-[Ru(III)(terpy)Cl3] [10], a-[Ru(II)(azpy)2- tion to ruthenium ion, corresponding to shifting in energy
Cl2] [11], and Ru(II) organometallic complexes of the type for bands in the IR spectra. These bands are also sensitive
[(g6-arene)Ru(II)(en)Cl][PF6] [12]. Most extraordinarily, to the substituent in the complexes. The IR spectral data
two other Ru(III) complexes, namely NAMI-A ([ImH][- obtained due to the chelation of ligands to the ruthenium
trans-RuCl4(DMSO)(Im)]) and KP1019 ([IndH][trans- metal ion are represented in the Supplementary Material.
RuCl4(Ind)2]) have already entered clinical trials. NAMI-A The m(C=N) band of ligands (1390–1397 cm-1) are shifted
is nontoxic in vitro while shows very good antimetastatic to higher frequency in complexes (1506–1522 cm-1) sug-
activity [13]. KP1019 has been introduced into phase I gesting the coordination of ligand through nitrogen atom of
clinical trials against colon carcinomas and their metastases pyridine ring [20]. The m(C=S) stretching bands of ligands
in 2003 [14].
(1162–1179 cm-1
) are shifted to higher frequency
In continuation of our earlier work [15], to understand (1234–1260 cm-1), which is attributed to the chelation of
the role of chelating effect of ligands with ruthenium(II)/ ruthenium ion to sulfur atom of thiophene ring. Bands
(III) in biological application, reaction of N,S-donor observed in the of range 1548–1578 cm-1 assigned as
ligands derived from pyridinium salt of 2-acetylthiophene m(C=C)ar stretching. The m(C–H)ar. Stretching bands are
and substituted enones, with ruthenium precursor [RuCl3- observed in the range of 3034–3067 cm-1. Bands observed
PPh3] has been carried out. Biological activity of the in the range of 545–572 and 454–475 cm-1 are assigned
complexes was carried out performing antibacterial, DNA due to m(Ru–N) and m(Ru–S) stretching vibration,
binding, DNA cleavage, and cytotoxicity study.
respectively.
Thermal analysis
Results and discussion
The TG analyses for complex 1 and complex 7 carried out
at a heating rate of 10 °C per minute from ambient tem-
perature to 900 °C under N2 atmosphere. Thermogram of
Magnetism and electronic spectra
Room temperature magnetic susceptibility of the synthe- the complex 1 and complex 7 are represented in the Sup-
sized complexes has been measured by Gouy’s magnetic plementary Material. In case of complex 1, no loss in
balance using mercury tetrathiocyanatocobaltate(II) as a weight is observed up to 170 °C indicating the absence of
calibrant. No change in the weight is observed in the water molecules. The first loss observed in the temperature
Gouy’s balance when the complexes 1–6 were kept in the range of 180–450 °C is due to the loss of chlorine atoms
external magnetic field, indicating the absence of unpaired and triphenylphosphine moiety. The second mass loss
electron. This also confirms ruthenium in the complexes is occurring in the temperature range of 530–780 °C is
in ?2 oxidation state. The magnetic moment of complexes attributed to loss of ligand moiety and leaving behind the
7–12 are found in the range 1.79–1.92 BM. The obtained residue of metal oxide. While in the TG curve of complex
123

Monitoring the DNA by ruthenium complexes of heterocyclic N,S-donor ligands and evaluation…
7, no loss in weight is observed up to 160 °C indicating the
absence of water molecules. The first mass loss occurs in
the temperature range of 170–250 °C, which is attributed to
loss of three chlorine atoms, second mass loss occurring in
the temperature range of 350–490 °C corresponding to
removal of triphenylphosphine moiety and the third mass
loss around 580–800 °C is due to decomposition of neutral
bidentate ligand, and leaving behind metal oxide as
residue.
the standard antibiotics like ciprofloxacin and ofloxacin.
The mechanism of toxicity of the complexes may be
ascribed to the increase of the lipophilic nature of the
complexes arising from chelation. The mode of action of
antimicrobials may involve different targets in pathogens,
e.g. interference with cell wall synthesis and damage to the
cytoplasmic membrane, as a result of which cell perme-
ability may be altered leading to cell death. Another
mechanism of toxicity of these complexes to microorgan-
isms may be due to the inhibition of energy production or
ATP production, by inhibiting respiration or by the
uncoupling of oxidative phosphorylation. The increase in
antimicrobial activity of metal complexes can be the result
of: (I) chelate effects, (II) nature of the ligands, (III) total
charge of the complex, (IV) nature of the ion neutralising
the complex, and (V) nuclearity of the metal centre in the
compounds. The inhibition activity seems to be governed
in certain degree by the facility of coordination at the metal
centre as well as electronic nature of the ligands (Figs. 1,
2).
Mass spectra
Mass spectrum of complex 1 and complex 7 are repre-
sented in the Supplementary Material. In the mass
spectrum of complex 1, the peaks at m/z = 1063.09,
1065.09, 1067.01, and 1069.06 are assigned as molecular
ion peak (M?), (M ? 2), (M ? 4), (M ? 6), respectively.
The peak observed at m/z = 1026.09 is due to loss of one
chlorine atom. The peak at m/z = 911.11 is due to the loss
of second chlorine atom. Several other fragments at m/
z = 898.00, 729.04, 696.05, 661.06, 626.02, 466.91,
365.02, and 262.08 are observed, which are attributed to
fragments associated with complex and proposed frag-
mentation pattern of complex 1 is shown in Supplementary
Material. In the mass spectrum of complex 7, molecular ion
peak at m/z = 837.96, 839.99, 841.93, 843.92, and 845.90
are assigned as (M?), (M ? 2), (M ? 4), (M ? 6), and
(M ? 8) peaks due to the presence of four chlorine atoms.
The peak observed at m/z = 799.00 is due to loss of one
chlorine atom. The peak at m/z = 470.89 is due to the loss
of neutral bidentate ligand. Several other fragments at m/
z = 764.09, 729.02, 466.93, 436.00, 398.90, 365.07,
364.01 and 262.10 are observed, which attributed to frag-
ments associated with complex. Proposed fragmentation
pattern for the complex 7 is shown in Supplementary
Material.
DNA binding mode investigation
Quantitative binding strength of the complex was carried
out by absorption titration method. In the intercalation
mode of binding of metal complex with DNA, there is an
interaction of p* orbital of the intercalated ligand which
may couple with the p orbital of the DNA base pairs,
resulting in the lowering of p–p* transition energy and thus
displays a red shift in the transition [16]. On the other hand,
hypochromism is observed when there is a coupling of a p
orbital with partially filled electrons [17]. The overlay
spectrum of complex 1 and 7 are represented in Figs. 3 and
4, respectively. In the overlay spectrum of complex 1 and
7, hypochromism and red shift are observed, which suggest
the intercalation mode of binding. The intrinsic binding
constant values (Kb) for the synthesized complexes are
represented in the Table 2. The Kb values for the synthe-
sized complexes are found in the range of 0.521 3 105–
2.94 3 105 M-1, which are higher than DD - [{Ru(Me2-
bpy)2}2(l-bpm)]4? (1 9 103) [18], while the obtained Kb
values are lower as compared to the [Ru(bpy)2(dppz)]2?
([106) [19]. The difference in the Kb value of the synthe-
sized complexes is attributed to their relative hydrophobic
character of the ligands which is affected by the substituent
present in the ligands [20]. To further confirm the binding
mode of the complexes, viscosity measurements were
carried out. The viscosity titration curve of the ruthenium
complexes 1–12 are represented in Fig. 3.
In vitro antibacterial activity
The interpretation of antimicrobial potency is carried out in
terms minimum inhibitory concentration (MIC) defined as
the lowest concentration, which inhibits the growth of
microorganism, referred by lack of turbidity in the tube. If
for a specific concentration of the test compounds no tur-
bidity is observed, then a whole experimental procedure is
repeated with the next dilution i.e. half the concentration of
test compound that was previously added. This procedure
is repeated until the faint turbidity by the inoculums itself
is observed and the said concentration is termed as MIC.
The result of MIC in terms of lM is represented in Table 1.
The result of antibacterial activity indicates that the syn-
thesized complexes are more potent than the reference
ligands but the complexes are less potent as compared to
The figure shows that successive addition of ruthenium
complexes to the solution of herring sperm (HS-) DNA
increases its relative viscosity, suggesting classical inter-
calation mode of binding as hypochromism with red shift is
123

P. S. Karia et al.
Table 1 MIC values of synthesized ligands L1–L6 and complexes 1–12
Sr. no.
MIC values
Gram-positive
S. Aureus
Gram-negative
B. subtilis
S. marcescens
P. aeruginosa
E. coli
L1
L2
L3
L4
L5
L6
1
265
245
280
235
270
250
155
190
200
170
210
235
125
160
175
135
165
170
255
280
275
265
255
240
210
210
260
230
235
245
130
140
190
160
165
210
230
260
290
280
265
325
160
180
210
180
180
220
120
140
175
150
150
180
300
350
450
345
390
470
170
165
225
185
200
225
120
145
160
155
170
185
190
255
215
210
240
225
145
165
190
175
200
210
115
125
150
120
140
190
2
3
4
5
6
7
8
9
10
11
12
Fig. 1 Electronic absorption spectra of complex 1 with increasing
concentration of herring sperm DNA (HS-DNA) in phosphate buffer.
Inset plots of [DNA]/(ea - ef) versus [DNA] for the titration of DNA
with ruthenium(III) complexes
Fig. 2 Electronic absorption spectra of complex 1 with increasing
concentration of herring sperm DNA (HS-DNA) in phosphate buffer.
Inset plots of [DNA]/(ea - ef) versus [DNA] for the titration of DNA
with ruthenium(III) complexes
leading to the increase of the relative specific viscosity of
DNA. The conclusion is in agreement with absorption
titration method. Similar results were reported for the
ethidium bromide (EB) and [Ru(phen)2(MIP)](ClO4)2-
2.5H2O [21].
observed in absorption titration measurement. If blue shift
was observed in absorption titration, the interaction mode
of complexes with DNA will be the partial intercalation.
Ruthenium complexes interact with DNA by incorporation
of planar aromatic ligand into DNA base pairs, hence
123

Monitoring the DNA by ruthenium complexes of heterocyclic N,S-donor ligands and evaluation…
Fig. 3 Effect on relative
viscosity of HS-DNA under the
influence of increasing amount
of complexes at 37 1 °C in
phosphate buffer (Na2HPO4/
NaH2PO4, pH 7.2)
Fig. 4 Average % mortality of
brine shrimp against increasing
amount of complex
concentration
Brine shrimp lethality bioassay: a cytotoxicity
profile
Table 2 Intrinsic binding constant (Kb) values of the complex
Complexes
Kb 9 105 M-1
Complexes
Kb 9 105 M-1
1
2
3
4
5
6
1.74
7
8
2.94
1.86
1.41
1.40
1.29
1.49
All test tubes were incubated for 24 h at room temperature.
Number of dead nauplii is counted after 24 and 48 h. With
the help of magnifying glass. The percentage mortality of
brine shrimp nauplii was determined from the number of
dead nauplii. And the LC50 was calculated from the plot of
log of concentration of samples plotted against percent of
mortality of nauplii. From the result represented in Figs. 4
1.23
1.42
9
0.759
0.521
0.585
10
11
12
123

P. S. Karia et al.
Fig. 5 LC50 value of the
complexes
Fig. 6 Cleavage of pUC19
plasmid DNA under the
influence of ruthenium(II)
complexes. Lane 1 DNA
control, lane 2 RuCl3Á3H2O,
lane 3 [Ru(L1)(PPh3)2Cl2] (1),
lane 4 [Ru(L2)(PPh3)2Cl2] (2),
lane 5 [Ru(L3)(PPh3)2Cl2] (3),
lane 6 [Ru(L4)(PPh3)2Cl2] (4),
lane 7 [Ru(L5)(PPh3)2Cl2] (5),
lane 8 [Ru(L6)(PPh3)2Cl2] (6)
and 5, it is inferred that % mortality of brine shrimp found
to increase with increase in the concentration of complexes
and the complexes of ruthenium(III) exhibit higher toxicity
at lower concentration than other synthesized ruthenium(II)
complexes, suggesting its low concentration effects the
growth of Artemia cysts. However, the LC50 of synthesized
(complexes ? DNA) shows three bands, an additional
linear form (form II) indicating a significant cleavage of
DNA by the test complexes in reference to parent
compound RuCl3Á3H2O. Quantitative measure of the
DNA cleavage in terms of % cleavage for the test
compounds are represented in the Table 3. As the
complexes are observed to cleave the DNA, it is con-
cluded that the complexes control the growth of the
microorganisms by cleaving the DNA.
complexes (LC50 = 5.69 1.06–14.62 0.87 lg cm-3
)
are comparable to standard anticancer agent cisplatin
(LC50 \ 4 lg cm-3).
Photochemical investigation of DNA nuclease
activity
Conclusion
Figures 6 and 7 show the cleavage of pUC19 DNA by
ruthenium(II) and ruthenium(III) complexes respectively,
along with reference standard RuCl3Á3H2O. The differ-
ence was observed in the bands of RuCl3Á3H2O (lane 2)
and complexes (lanes 3–8) compared with the control
DNA (lane 1). Control DNA shows only two bands
corresponding to supercoiled form (form I) and circular
form (form III). Lane 2 (RuCl3Á3H2O ? DNA) also
show two bands but intensity of supercoiled form is
Synthesized blackish-brown Ru(II) complexes are unsta-
ble in air and diamagnetic while reddish-brown Ru(III)
complexes are stable in air. Electronic spectra support the
ruthenium ion in an octahedron geometry. It is found that
experimental percentage for C, H, and N atom in the
complex matches well with the theoretical values for the
proposed structure. The proof for the proposed structure
has also been strengthened from molecular ion peak in the
mass spectrum analysis and from the decomposition of
fragments occurring in TGA analysis at different
different
from
that
of
control.
Lane
3–8
123

Monitoring the DNA by ruthenium complexes of heterocyclic N,S-donor ligands and evaluation…
Fig. 7 Cleavage of pUC19 plasmid DNA under the influence of
ruthenium(III) complexes. Lane 1 DNA control, lane 2 RuCl3Á3H2O,
lane 3 [Ru(L1)(PPh3)Cl3] (7), lane 4 [Ru(L2)(PPh3)Cl3] (8), lane 5
[Ru(L3)(PPh3)Cl3] (9), lane
6
[Ru(L4)(PPh3)Cl3] (10), lane
7
[Ru(L5)(PPh3)Cl3] (11), lane 8 [Ru(L6)(PPh3)Cl3] (12)
Table 3 Complexes mediated cleavage data of pUC19 DNA
Compounds
% SC
% OC
% LC
% Cleavage
Gel-1
Control
86.8
67.1
9.5
13.2
32.9
68.5
68.1
77.1
76.5
62.2
72.8
–
–
RuCl3Á3H2O
–
22.69
89.05
86.63
81.79
85.36
72.00
86.05
[Ru(PPh3)2(L1)Cl2] (1)
[Ru(PPh3)2(L2)Cl2] (2)
[Ru(PPh3)2(L3)Cl2] (3)
[Ru(PPh3)2(L4)Cl2] (4)
[Ru(PPh3)2(L5)Cl2] (5)
[Ru(PPh3)2(L6)Cl2] (6)
Gel-2
22.5
20.3
7.4
11.6
15.8
12.7
24.3
12.1
16.9
13.4
15.0
Control
83.1
66.1
11.6
12.4
19.6
21.6
29.4
26.3
16.9
33.9
60.1
66.9
64.2
63.9
57.9
60.1
–
–
RuCl3Á3H2O
–
20.45
86.04
85.07
76.41
74.00
64.62
68.35
[Ru(PPh3)(L1)Cl3] (7)
[Ru(PPh3)(L2)Cl3] (8)
[Ru(PPh3)(L3)Cl3] (9)
[Ru(PPh3)(L4)Cl3] (10)
[Ru(PPh3)(L5)Cl3] (11)
[Ru(PPh3)(L6)Cl3] (12)
28.3
18.4
16.1
14.5
12.7
18.6
temperature range supporting the theoretically proposed
structure of complex. The increase in the antimicrobial
potency of the complexes as compared to ligands is
attributed to the chelate effect which increases lipophilicity
of complexes. Complex 7 showed higher potency towards
all the bacterial strains compared to other test compounds.
All the synthesized ruthenium complexes bind in an
intercalative mode of binding action and the quantitative
binding strength of ruthenium(III) complexes are found
higher than ruthenium(II) complexes. Cytotoxicity studies
of metal complexes shows good potency against brine
shrimp and 100 % mortality is achieved after 48 h of
incubation. LC50 values of ruthenium(III) complexes are
found to lower compared to ruthenium(II) complexes with
similar ligands, indicating significantly high toxicity of
ruthenium(III) complexes as compared to ruthenium(II)
complexes. LC50 values of the complexes are found to be
lower as compared to potassium dichromate suggesting
that complexes are significantly toxic at low concentration
and their lower dose can inhibit the 50 % of population of
test species as compared to the higher dose required for the
potassium dichromate and can be further explored for
in vivo cytotoxicity studies against cell lines. Highly effi-
cient cleavage of supercoiled pUC19 DNA is observed for
all the complexes compared to the metal salt.
Experimental
All the chemicals purchased were of analytical grade and
used as such without further purification. Ruthenium
trichloride trihydrate, 2-acetylthiophene, 3-chloroben-
zaldehyde, 3-fluorobenzaldehyde, 3-bromobenzaldehyde,
4-chlorobenzaldehyde,
4-fluorobenzaldehyde,
123

P. S. Karia et al.
Scheme 1
H
O
O
CH3
O
C
C
R1
4''
NaOH
MeOH,
R1 R2
R2
Ligands
at
5''
6''
Stir
room
R1
R2
3''
2''
Cl
temp.
L1
L2
L3
L4
L5
L6
R2
F
H
H
H
F
R1
Cl
1''
Cl
Br
H
H
H
enones
Substituted
4
Substituted
aldehydes
-Chloro-
acetophenone
p
NH
4OAc,
MeOH
6 h
5
3
2
2'''
5'''
Reflux,
3'
6
1'''
2'
S
3'''
Cl
Br
N
4'
1
CH3
4'''
I
I
6'''
Pyridine,
2
1'
Cl
5'
S
2 h
Reflux,
N
S
O
S -donor bidentate
N,
ligands
O
2-Acetylthiopene
salt of
2-acetylthiopene
Pyridinium
000
CDCl3, Me4Si): d = 164.75 (C4 ), 156.16 (C2), 152.93
4-bromobenzaldehyde, and HS-DNA were purchased from
Sigma Chemical Co. (India). Ethidium bromide (EB),
bromophenol blue, agarose, and Luria Broth (LB) were
purchased from Himedia (India). Culture of pUC19 bac-
teria (MTCC 47) was purchased from Institute of Microbial
Technology (Chandigarh, India).
0
(C6), 149.32 (C2 ), 145.09 (C1 ), 137.34 (C1 ), 134.76
000
00
000
(C6 ), 134.73 (C2 ), 128.94 (C5 ), 128.91 (C3 ), 128.86
000
0
0
0
00
00
(C4 ), 128.30 (C5 ), 128.03 (C3 ), 127.93 (C6 ), 124.85
00
00
ꢀ
(C2 ), 116.07 ppm; IR: m = 3066 m(C–H)ar, 1545 m(C=C),
1397 m(C=N), 1179 m(C=S), 1229 m(C–F), 1092 m(C–Cl),
1381, 1362 pyridine skeleton band, 800 m(C–H), 1112, 826
A Perkin-Elmer 240 Elemental Analyser was used to
collect microanalytical data (C, H, N). Room temperature
magnetic susceptibility was measured by Gouy’s method
using mercury tetrathiocyanatocobaltate(II) as the calibrant
(vg = 16.44 9 10-6 cgs units at 20 °C), on Citizen Bal-
ance. The diamagnetic correction has been made using
Pascal’s constant. FT-IR data were collected with the help
of FT-IR ABB Bomen MB 3000 spectrophotometer. UV–
Vis spectra of the complex were recorded on UV-160A
UV–Vis spectrophotometer, Shimadzu (Japan) and cor-
rected for background due to solvent absorption. The
percentage of pUC19 DNA cleavage was quantified by
AlphaDigiDocTM RT. Version V.4.0.0 PC-Image software.
(p-substituted ring) cm-1
.
2,4-Bis(4-chlorophenyl)-6-(thiophen-2-yl)pyridine
(L2, C21H13Cl2NS)
It has been synthesized by the reaction between pyridinium
salt of 2-acetylthiophene and enone of p-chlorobenzalde-
hyde and p-chloroacetophenone. Yield 60 %; m.p.:
140 °C; 1H NMR (400 MHz, CDCl3, Me4Si): d = 8.13
0
000 000
, ), 7.68 (2H, d, H2
5
000,6000
),
(2H, d, H3,5), 7.75 (3H, d, H5 ,3
0
00 00 00 00
0
7.54–7.46 (5H, m, H3 ,2 ,3 ,5 ,6 ), 7.18 (1H, dd, H4 ) ppm;
13C NMR (100 MHz, CDCl3, Me4Si): d = 156.28 (C6),
0
153.03 (C2), 151.26(C4), 149.13 (C2 ), 145.02 (C1 ),
000
00
137.31 (C1 ), 137.09 (C4 ), 135.44 (C4 ), 129.38 (C3 ,5 ),
000
00
00 00
00 00
), 128.40 (C2 ,6 ), 128.29 (C2
000,5000
000,6000
), 128.06
128.94 (C3
2-(4-Chlorophenyl)-4-(4-fluorophenyl)-6-(thiophen-2-
yl)pyridine (L1, C21H13ClFNS)
0
(C5 ), 127.98 (C4 ), 124.89 (C3 ), 116.21 (C3), 115.26 (C5)
0
0
ꢀ
ppm; IR: m = 3061 m(C–H)ar, 1546 m(C=C), 1397 m(C=N),
Modified Krohnke pyridine synthesis was used for the
synthesis of ligands [22]. Enone synthesized by the
reaction between p-fluorobenzaldehyde and p-chloroace-
tophenone, was refluxed with pyridinium salt of 2-
acetylthiopene in presence of ammonium acetate for 6 h.
The reaction mixture was allowed to cool at room
temperature. The solid product was filtered and recrystal-
lized from hexane. General reaction scheme for the
synthesis of ligands is shown in Scheme 1. Yield 65 %;
m.p.: 138 °C; 1H NMR (400 MHz, CDCl3, Me4Si):
1176 m(C=S), 1082 m(C–Cl), 1383, 1304 pyridine skeleton
band, 778 m(C–H), 1123, 830 (p-substituted ring) cm-1
.
4-(4-Bromophenyl)-2-(4-chlorophenyl)-6-(thiophen-2-
yl)pyridine (L3, C21H13ClBrNS)
It has been synthesized by the reaction between pyridinium
salt of 2-acetylthiophene and enone of p-bromobenzalde-
hyde and p-chloroacetophenone. Yield 62 %; m.p.:
123 °C; 1H NMR (400 MHz, CDCl3, Me4Si): d = 8.12
0
000 000
, ), 7.68 (2H, d, H2
5
000,6000
),
(2H, d, H3,5), 7.73 (3H, t, H5 ,3
0
000
,
3-
00 00
0
00 00
d = 8.14–8.11 (2H, m, H3,5), 7.74–7.69 (5H, m, H5 ,2
7.60 (2H, d, H2 ,6 ), 7.48 (3H, dd, H3 ,3 ,5 ), 7.18 (1H, t,
H4 ) ppm; 13C NMR (100 MHz, CDCl3, Me4Si):
00
0
00 00
5
000,6000
0
,
), 7.51–7.46 (3H, m, H3 ,3 ,5 ), 7.26–7.22 (2H, m,
H2 , 6 ), 7.19–7.16 (1H, m, H4 ) ppm; 13C NMR (100 MHz,
00
00
0
0
d = 156.29 (C6), 153.92 (C2), 153.07 (C4), 149.19 (C2 ),
123

Monitoring the DNA by ruthenium complexes of heterocyclic N,S-donor ligands and evaluation…
000
145.00 (C1 ), 137.54 (C1 ), 137.26 (C4 ), 135.46 (C4 ),
00
000
00
0
000,6000
), 120.43 (C4 ), 118.14
000,5000
126.62 (C3
), 122.83 (C2
00 00
132.34 (C3 ,5 ), 128.93 (C3
00 00
), 128.68 (C2 ,6 ), 128.29
00 00 0 00 00
(C5 ,6 ), 116.50 (C3 ), 115.07 (C2 ,4 ), 112.52 (C3), 109.12
000,5000
0
0
0
), 128.04 (C5 ), 128.00 (C4 ), 124.92 (C3 ), 116.12
000,6000
ꢀ
(C2
(C5) ppm; IR: m = 3055 m(C–H)ar., 1548 m(C=C), 1393
(C3), 115.17 (C5) ppm; IR: mꢀ = 3052 m(C–H)ar., 1545
m(C=C), 1390 m(C=N), 1170 m(C=S), 1095 m(C–Cl), 1012
m(C–Br), 1380, 1360 pyridine skeleton band, 750 m(C–H),
m(C=N), 1154 m(C=S), 1180 m(C–Cl), 1008 m(C–Br) 1354
pyridine skeleton band, 789, 821(m-substituted ring)
cm-1
.
1150, 815 (p-substituted ring) cm-1
.
Synthesis of complexes
2-(4-Chlorophenyl)-4-(3-fluorophenyl)-6-(thiophen-2-
yl)pyridine (L4, C21H13ClFNS)
A ruthenium precursor [RuCl3(PPh3)3] was prepared by the
refluxing the methanolic solution of RuCl3Á3H2O and PPh3
(1:3) in presence of conc. HCl for 1 h. The reddish brown
precipitate obtained was filtered, dried and recrystallized
from hot methanol.
It has been synthesized using the chalcone prepared from
the reaction between m-fluorobenzaldehyde and p-
1
chloroacetophenone. Yield 69 %; m.p.: 118 °C; H NMR
(400 MHz, CDCl3, Me4Si): d = 8.35–8.32 (2H, dd,
000,6000
000,5000
), 8.14–8.09 (1H, dd,
H2
), 8.26–8.20 (2H, dd, H3
0
H3), 7.99–7.91 (2H, m, H3 ,5 ), 7.71–7.69 (1H, dd, H5),
0
Dichloro[2-(4-chlorophenyl)-4-(4-fluorophenyl)-6-(thio-
phen-2-yl)pyridine]bis(triphenylphosphine)ruthenium(II)
[RuCl2(PPh3)2L1] (1, C57H43Cl3FNP2RuS)
00 00 00
00
7.63–7.61 (3H, dd, H2 ,4 ,6 ), 7.42–7.34 (1H, t, H5 ), 7.30–
7.22 (1H, dd, H4 ) ppm; 13C NMR (100 MHz, CDCl3,
0
00
Me4Si): d = 158.68 (C3 ), 155.78 (C6), 149.83 (C2),
It has been synthesized by refluxing the solution of
ruthenium precursor [RuCl3(PPh3)3] in toluene (0.1 mmol)
with the methanolic solution of 2-(4-chlorophenyl)-4-(4-
fluorophenyl)-6-(thiophen-2-yl)pyridine (L1, 0.1 mmol) in
presence of Et3N as a reducing agent (0.1 mmol in
methanol) and LiCl (0.4 mmol in methanol) (to prevent
excessive loss of Cl from the synthesized precursor) for 4 h
(Scheme 2) [23, 24]. The resulting solution has been
concentrated to half of its volume and the product has been
separated by adding small amount of pet ether (60:80). The
obtained blackish brown product was washed with
methanol and dried under vacuum. Yield 15.0 %; m.p.:
244.1 °C; UV–Vis (DMSO): kmax = 550, 440, 273 nm.
00
0
148.23 (C4), 143.07 (C1 ), 141.85 (C2 ), 139.12 (C1 ),
000
000
136.52 (C4 ), 131.23 (C5 ), 129.91 (C3 , C5 ), 128.71
0
000
000
000 000 0 0 00 00
, ), 127.55 (C3 ,4 ,5 ), 123.5 (C6 ), 116.70 (C3),
6
(C2
00
00
115.11 (C4 ), 112.14 (C2 ), 108.90 (C5) ppm; IR:
ꢀ
m = 3066 m(C–H)ar, 1546 m(C=C), 1398 m(C=N), 1157
m(C=S), 1225 m(C–F), 1092 m(C–Cl), 1363 pyridine skele-
ton band, 788, 826 (m-substituted ring) cm-1
.
4-(3-Chlorophenyl)-2-(4-chlorophenyl)-6-(thiophen-2-
yl)pyridine (L5, C21H13Cl2NS)
It has been synthesized using the chalcone prepared from the
reaction between m-chlorobenzaldehyde and p-chloroace-
tophenone. Yield 58 %; m.p.: 109 °C; 1H NMR (400 MHz,
CDCl3, Me4Si): d = 8.16–8.14 (3H, d, H3,2 ’,6 ’ ), 7.84 (1H,
0
s, H5), 7.52–7.752 (2H, m, H3 ’ ,5 ’ ), 7.51–7.50 (1H, t, H5 ),
00
0 0
Dichloro[2,4-bis(4-chlorophenyl)-6-(thiophen-2-
yl)pyridine]bis(triphenylphosphine)ruthenium(II) [RuCl2(-
PPh3)2L2] (2, C57H43Cl4NP2RuS)
0
0
0
00
ppm; 13C NMR
0
0
0
00 00 00
7.50–7.48 (6H, m, H3 ,4 ,5 ,2 ,4 ,6 )
00
(100 MHz, CDCl3, Me4Si): d = 157.10 (C3 ), 154.20 (C6),
It has been synthesized using [RuCl3(PPh3)3] and 2,4-
bis(4-chlorophenyl)-6-(thiophen-2-yl)pyridine (L2). Yield
15.9 %; m.p.: 253.0 °C; UV–Vis (DMSO): kmax = 554,
440, 263 nm.
0
00
0
150.33 (C2), 143.44 (C4 ), 138.16 (C1 , C2 ), 136.52 (C1
000
,
4-
00), 132.56 (C3
), 130.13 (C2
0
), 128.25 (C3 ,4 ,5 ),
0
0
000,5000
000,6000
00 00
00
126.12 (C3,5 ,6 ), 123.57 (C4 ), 116.54 (C2 ), 115.45 (C5)
00
ꢀ
ppm; IR: m = 3067 m(C–H)ar, 1548 m(C=C), 1396 m(C=N),
Dichloro[4-(4-bromophenyl)-2-(4-chlorophenyl)-6-(thio-
phen-2-yl)pyridine]bis(triphenylphosphine)ruthenium(II)
[RuCl2(PPh3)2L3] (3, C57H43BrCl3NP2RuS)
1158 m(C=S), 1182 m(C–Cl), 1354 pyridine skeleton band,
785, 826 (m-substituted ring) cm-1
.
It has been synthesized using [RuCl3(PPh3)3] and 4-(4-
bromophenyl)-2-(4-chlorophenyl)-6-(thiophen-2-yl)pyri-
dine (L3). Yield 17.2 %; m.p.: 234.7 °C; UV–Vis
(DMSO): kmax = 557, 431, 262 nm.
4-(3-Bromophenyl)-2-(4-chlorophenyl)-6-(thiophen-2-
yl)pyridine (L6, C21H13BrClNS)
It has been synthesized using the chalcone prepared from
the reaction between m-bromobenzaldehyde and p-
1
chloroacetophenone. Yield 52 %; m.p.: 90 °C; H NMR
(400 MHz, CDCl3, Me4Si): d = 8.15–8.11 (2H, d,
Dichloro[2-(4-chlorophenyl)-4-(3-fluorophenyl)-6-(thio-
phen-2-yl)pyridine]bis(triphenylphosphine)ruthenium(II)
[RuCl2(PPh3)2L4] (4, C57H43Cl3FNP2RuS)
00
2 ’,6 ’ ), 7.96–7.97 (1H, d, H3), 7.80–7.60 (4H, m,
0 0
H
0
0
0
0
0
0
0
0
00 00
H
2 ’,4 ’,3 ’ ,5 ’ ), 7.58–7.41 (5H, m, H5,3 ,5 ,5 ,6 ), 7.18–7.16
It has been synthesized using [RuCl3(PPh3)3] and 2-(4-
chlorophenyl)-4-(3-fluorophenyl)-6-(thiophen-2-yl)pyri-
dine (L4). Yield 16.9 %; m.p.: 247.5 °C; UV–Vis
(DMSO): kmax = 560, 426, 269 nm.
(1H, d, H4 ) ppm; 13C NMR (100 MHz, CDCl3, Me4Si):
0
00
d = 158.12 (C3 ), 154.58 (C6), 149.03 (C2), 143.02 (C4),
00
139.19 (C1 ), 137.34 (C2 ), 130.73 (C1
000,4000
0
0
), 128.52 (C5 ),
123

P. S. Karia et al.
Scheme 2
R1
R2
PPh3
Conc. HCl
PPh3
R2
S
Cl
Ru
LiCl, Et3N
Toluene, reflux, 4 h
RuCl
N
R1
.
3(PPh3)3
RuCl3 3H2O
Cl
MeOH
PPh3
h
Reflux 1
Cl
N
S
R1
R2
Cl
Complex
1
2
3
4
5
6
F
H
H
H
F
Cl
Br
Cl
Br
H
H
H
Dichloro[4-(3-chlorophenyl)-2-(4-chlorophenyl)-6-(thio-
phen-2-yl)pyridine]bis(triphenylphosphine)ruthenium(II)
[RuCl2(PPh3)2L5] (5, C57H43Cl4NP2RuS)
Trichloro[4-(4-bromophenyl)-2-(4-chlorophenyl)-6-(thio-
phen-2-yl)pyridine](triphenylphosphine)ruthenium(III)
[RuCl3(PPh3)L3] (9, C39H28BrCl4NPRuS)
It has been synthesized using [RuCl3(PPh3)3] and 4-(3-
chlorophenyl)-2-(4-chlorophenyl)-6-(thiophen-2-yl)pyri-
dine (L5). Yield 15.2 %; m.p.: 256.8 °C; UV–Vis
(DMSO): kmax = 568, 422, 265 nm.
It has been synthesized using [RuCl3(PPh3)3] and 4-(4-
bromophenyl)-2-(4-chlorophenyl)-6-(thiophen-2-yl)pyri-
dine (L3). Yield 17 %; m.p.: 288 °C; leff = 1.84 B.M.;
UV–Vis (DMSO): kmax = 560, 369, 272 nm.
Dichloro[4-(3-bromophenyl)-2-(4-chlorophenyl)-6-(thio-
phen-2-yl)pyridine]bis(triphenylphosphine)ruthenium(II)
[RuCl2(PPh3)2L6] (6, C57H43BrCl3NP2RuS)
Trichloro[2-(4-chlorophenyl)-4-(3-fluorophenyl)-6-(thio-
phen-2-yl)pyridine](triphenylphosphine)ruthenium(III)
[RuCl3(PPh3)L4] (10, C39H28Cl4FNPRuS)
It has been synthesized using the [RuCl3(PPh3)3] and 4-(3-
bromophenyl)-2-(4-chlorophenyl)-6-(thiophen-2-yl)pyri-
dine (L6). Yield 14.8 %; m.p.: 272.0 °C; UV–Vis
(DMSO): kmax = 560, 420, 263 nm.
It has been synthesized using [RuCl3(PPh3)3] and 2-(4-
chlorophenyl)-4-(3-fluorophenyl)-6-(thiophen-2-yl)pyri-
dine (L4). Yield 18 %; m.p.: 275 °C; leff = 1.79 B.M.;
UV–Vis (DMSO): kmax = 544, 358, 270 nm.
Trichloro[2-(4-chlorophenyl)-4-(4-fluorophenyl)-6-(thio-
phen-2-yl)pyridine](triphenylphosphine)ruthenium(III)
[RuCl3(PPh3)L1] (7, C39H28Cl4FNPRuS)
Trichloro[4-(3-chlorophenyl)-2-(4-chlorophenyl)-6-(thio-
phen-2-yl)pyridine](triphenylphosphine)ruthenium(III)
[RuCl3(PPh3)L5] (11, C39H28Cl5NPRuS)
It has been synthesized by refluxing the solution of ruthenium
precursor [RuCl3(PPh3)3] in toluene (0.1 mmol) with the
methanolic solution of 2-(4-chlorophenyl)-4-(4-fluo-
rophenyl)-6-(thiophen-2-yl)pyridine (L1, 0.1 mmol) for 4 h
(Scheme 3). The resulting solution has been concentrated to
half of its volume and the product was separated by adding
small amount of pet ether (60:80). The blackish brown product
obtained was washed with methanol, toluene, and finally dried
under vacuum. Yield 17 %; m.p.: 249 °C; leff = 1.92 B.M.;
UV–Vis (DMSO): kmax = 540, 385, 270 nm.
It has been synthesized using [RuCl3(PPh3)3] and 4-(3-
chlorophenyl)-2-(4-chlorophenyl)-6-(thiophen-2-yl)pyri-
dine as ligand (L5). Yield 19 %; m.p.: 259 °C; leff = 1.91
B.M.; UV–Vis (DMSO): kmax = 551, 367, 271 nm.
Trichloro[4-(3-bromophenyl)-2-(4-chlorophenyl)-6-(thio-
phen-2-yl)pyridine](triphenylphosphine)ruthenium(III)
[RuCl3(PPh3)L6] (12, C39H28BrCl4NPRuS)
It has been synthesized using [RuCl3(PPh3)3] 4-(3-bro-
mophenyl)-2-(4-chlorophenyl)-6-(thiophen-2-yl)pyridine
(L6). Yield 18 %; m.p.: 278 °C; leff = 1.92 B.M.; UV–Vis
(DMSO): kmax = 558, 371, 270 nm.
Trichloro[2,4-bis(4-chlorophenyl)-6-(thiophen-2-
yl)pyridine](triphenylphosphine)ruthenium(III)
[RuCl3(PPh3)L2] (8, C39H28Cl5NPRuS)
In vitro antibacterial activity
It has been synthesized using [RuCl3(PPh3)3] and 2,4-
bis(4-chlorophenyl)-6-(thiophen-2-yl)pyridine (L2). Yield
20 %; m.p.: 255 °C; leff = 1.89 B.M.; UV–Vis (DMSO):
kmax = 562, 362, 272 nm.
The aim of this study was to evaluate in vitro bacterial
potency of the ruthenium complexes against two Gram-
positive (Bacillus subtilis and Serratia marcescens) and
123

Monitoring the DNA by ruthenium complexes of heterocyclic N,S-donor ligands and evaluation…
Scheme 3
R1
R2
PPh3
R2
Conc. HCl
PPh3
S
Cl
Ru
Cl
Toluene
4 h
.
N
R1
RuCl
3(PPh3)3
RuCl3 3H2O
Cl
MeOH
Reflux
1 h
Reflux
Cl
N
R1
R2
S
Complex
Cl
7
F
H
H
H
F
Cl
Br
8
Cl
Br
H
H
H
9
10
11
12
three Gram-negative (Escherichia coli, Pseudomonas
aeruginosa, and Staphylococcus aureus). A broth dilution
method was employed for screening antibacterial activity.
A pre-culture of bacteria was used to prepare bacterial
culture in Luria Broth. 50 cm3 of 2 % LB solution was
prepared and sterilized after transferring 10 cm3 of its
aliquots and definite concentration of test compounds into
five corning tubes one for each bacterial species. Then
20 9 10-3 cm3 of bacterial culture from previously pre-
pared control for each species was added into different
corning tubes and inoculated for 24 h at 37 °C. This
experiment is repeated until only a faint turbidity appears
in the tube. The lowest concentration, which showed no
visible growth after 24 h subculture was considered as MIC
for each compound.
½DNAŠ
½DNAŠ
1
¼
þ
ðea À ef Þ ðeb À ef Þ Kbðeb À ef Þ
where ea, ef, and eb correspond to Aobsd/[complex], the
extinction coefficient for the free complex and the extinc-
tion coefficient for the complex in the fully bound form,
respectively.
The binding of small molecules to DNA cannot be
determined only by absorption titration method. Visco-
metric measurement is a supporting tool to confirm the
binding mode. Viscosity measurements have been largely
used to investigate the conformational changes of DNA
induced by binding of complexes. In electrostatic and
groove binding viscosity of DNA solution remain unal-
tered, while in classical intercalative binding viscosity will
rise. Among these interactions, intercalation and groove
binding are the most important DNA-binding modes as
they invariably lead to cellular degradation. Herein, we
carried out viscosity measurement at 37.0 1 °C using a
constant thermometric bath. To a solution of sonicated HS-
DNA (approximately 200 bp) taken in Ubbelohde vis-
cometer, a predetermined volume of the synthesized
complex was supplemented. Subsequent to thorough agi-
tation, resulting mixture was subjected to the measurement
of relative viscosity. For each aliquot of the complex
solution added, measurement was done thrice and average
time was calculated. The relative viscosity ratio (g/g0) has
been plotted against the r-bound ([complex]/[DNA])
(where g and g0 were the relative viscosity for DNA in the
presence or absence of complexes, respectively). Viscosity
measurements can sensitively detect the lengthening and
unwinding of a DNA helix induced by the binding of
intercalators [28], and thus provides evidence of interca-
lation for small DNA-binding molecules.
DNA binding mode investigation
UV–visible spectral titration of ruthenium complexes (in
DMSO) with HS-DNA in phosphate buffer was carried out
to inspect the binding mode for the complexes. In an
experiment, fixed increment of DNA solution in phosphate
buffer (100 9 10-3 cm3) was added to sample cell holding
in definite concentration of complex solution (20 lM) and
to the reference cell containing buffer and DMSO as a
control to nullify the effect of DMSO in sample holding
cell, and allowed to incubate for 10 min prior to the spectra
being recorded [25–27]. The change in absorbance against
wavelength at metal-to-ligand charge transfer (MLCT)
band and intra-ligand transfer (ILT) band with increasing
concentration of DNA was monitored to examine the
binding strength in terms of intrinsic binding constant Kb.
The intrinsic binding constant values was calculated using
the following equation
123

P. S. Karia et al.
Brine shrimp lethality bioassay: a cytotoxicity
profile
References
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Researchers can look for cytotoxic compounds, if they are
interested in developing a therapeutic that targets rapidly
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16, and 20 lg cm-3 were prepared from a stock solution of
1000 lg cm-3 of the test compounds. Final volume in each
test tube was adjusted to 2.5 cm3 (1.0 cm3 sea salt solution,
0.450 cm3
double
distilled
water,
0.050 cm3
DMSO ? complex and remaining 1.0 cm3 was added as
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The impact of DNA cleavage in inorganic medicinal
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The samples were added to the isolated pUC19 DNA of
E. Coli and incubated for 24 h before subjected to elec-
trophoresis.
System
of
0.020 cm3
(0.010 cm3
DNA ? 0.005 cm3 test compound ? 0.005 cm3 quench-
ing dye) for each test compound along with standard DNA
marker containing TE buffer ware loaded into the gel
chamber wells and subjected to electrophoresis for 3–4 h
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visualized under UV illuminator and photographed to cal-
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Acknowledgments We are thankful to the Head of chemistry
department and UGC RFSMS Scheme for providing financial support.
123