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Biological evaluation of new organoruthenium(II) metallates containing 3-acetyl-8-methoxy-2H-chromen-2-one appended CNS donor Schiff bases
Accepted Manuscript
Biological evaluation of new organoruthenium(II) metallates containing 3-acetyl-8methoxy-2H-chromen-2-one appended CNS donor Schiff bases
G. Kalaiarasi, S. Rex Jeya Rajkumar, S. Dharani, Frank R. Fronczek, R. Prabhakaran
PII:
S0022-328X(18)30281-X
DOI:
10.1016/j.jorganchem.2018.04.030
Reference:
JOM 20422
To appear in:
Journal of Organometallic Chemistry
Received Date: 5 January 2018
Revised Date:
16 April 2018
Accepted Date: 21 April 2018
Please cite this article as: G. Kalaiarasi, S.R. Jeya Rajkumar, S. Dharani, F.R. Fronczek, R.
Prabhakaran, Biological evaluation of new organoruthenium(II) metallates containing 3-acetyl-8methoxy-2H-chromen-2-one appended CNS donor Schiff bases, Journal of Organometallic Chemistry
(2018), doi: 10.1016/j.jorganchem.2018.04.030.
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ACCEPTED MANUSCRIPT
Biological evaluation of new organoruthenium(II) metallates containing
3-acetyl-8-methoxy-2H-chromen-2-one appended CNS donor Schiff bases
G. Kalaiarasi,a S. Rex Jeya Rajkumar,b S. Dharani,a Frank R. Fronczek,c and
R. Prabhakaran a*
Department of Chemistry, Bharathiar University, Coimbatore 641 046, India.
b
Department of Biosciences and Technology, Karunya University, Coimbatore 641 114,
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a
India.
c
Department of Chemistry, Louisiana State University, Baton Rouge, LA 70803, USA
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Abstract
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A series of four new, cyclometallated ruthenium(II) complexes was synthesized from
3-acetyl-8-methoxy-2H-chromen-2-one functionalized 4(N)- substituted thiosemicarbazones
and characterized through various spectral and analytical methods. The molecular structures
of the complexes 1, 2 and 4 were determined by single-crystal X-ray diffraction analysis,
which confirmed that the complexes possess a distorted octahedral geometry with the ligands
coordinating in a dibasic tridentate fashion via C, N and S atoms. DNA [Calf Thymus DNA
(CT-DNA)] and protein [Bovine Serum Albumin (BSA) and Human Serum Albumin (HSA)]
binding studies indicated an intercalative mode of binding with DNA and static quenching
mechanism with proteins. The compounds cleaved plasmid DNA (pBR322) without
application of any external agent and acted well as free radical scavengers. A good spectrum
of antimicrobial activity was observed against four bacterial and five fungal pathogens. Two
cancerous cell lines, MCF-7 (human breast cancer) and A549 (human lung carcinoma) were
employed to test their in vitro cytotoxic activity using MTT assay. The complexes (1-4)
showed better activity with lower IC50 values over cisplatin. Further non toxic nature of the
complexes have been examined with human normal keratinocyte cell line HaCaT. Further,
the results of Lactate dehydrogenase release (LDH) and Nitric Oxide (NO) release supported
the cytotoxic nature of the compounds. The results of all the biological studies carried out
implied that the complex 3 bearing an ethyl substituent was observed to be the best.
Key words: Cyclometallated Ru(II) complexes; DNA/Protein binding; Antimicrobial studies;
Cytotoxicity, LDH/NO release.
Corresponding
author
Tel.:
+91-422-2428319;
rpnchemist@gmail.com (R. Prabhakaran)
Fax:
+91-422-2422387;
E-Mail:
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1. Introduction
Development of structurally novel and biologically applicable transition metal complexes is
of paramount importance in the field of inorganic research. Increasing attention is on
producing metal mediated drugs, primarily with minimal side effects and of highly targeted
delivery [1]. Thiosemicarbazide is one such ligand system whose diverse applications viz.
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antitumor, antifungal, antibacterial, antiviral, antiparasitic, etc., have attracted numerous
chemists and their activities were found to improve upon complexation with metals [1-6].
They usually have two coordination sites (N and S) and a possibility of an additional donor
site greatly enhances their biological and catalytic activities [7]. These Schiff base ligands act
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as chelating agents and can offer a variety of coordination modes [8]. Coumarin derivatives
condensed with thiosemicarbazides provide the above said additional donor site and exhibit
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noticeable activities. It is a naturally occurring compound widespread in plants. Their
anticoagulant and antithrombotic actions were clinically proven. Investigation on coumarin
compounds also revealed that they possess antibacterial, antifungal and anticancer
properties [9,10]. Coumarin derivatives target a number of pathways in cancer cells such as
cell cycle arrest, antimitotic activity, inhibition of kinase, angiogenesis, heat shock protein
(HSP90), telomerase, carbonic anhydrase, etc [9]. Novobiocin, clorobiocin and coumermycin
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A1 are some of the commercially available coumarin based antibiotics [11,12]. Since
coumarins and thiosemicarbazides are individually active in biological processes [2,3,13], the
complexation of coumarin thiosemicarbazones with a metal would certainly end up in
increased activity. The drawbacks observed with cisplatin made the chemists to search for
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new metal drugs and ruthenium complexes are found to be a better alternative for platinum
since it can avail different coordination modes [14], stable oxidation states and mimic iron in
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binding with biomolecules [15,16]. The synthesis of NAMI-A [17], KP1019 [18], NKP-1339
and their clinical trials in anticancer treatments depicts the effect of ruthenium complexes in
chemotherapy [19,20]. Although the potential of ruthenium complexes in therapy is assured,
a thorough understanding of their unique modes of action, identification of their biological
targets and improving their selectivity are need to be focused still [21]. Demoro et al
evidenced the anti-trypanosomal [22] and anti-tumour activities [21] of organoruthenium
thiosemicarbazone complexes in two separate works. A number of articles are available in
displaying the cytotoxic nature of ruthenium(II) complexes, including those reported by
Garza-Ortiz et al [23], Juinn Chow et al [24] and Chang et al [25].
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The potentiality of reported ruthenium compounds in chemotherapy triggered us to
develop new ruthenium based complexes. Our search resulted in the synthesis and evaluation
of biological activities of 3-acetyl-8-methoxy coumarin thiosemicarbazones appended Ru(II)
complexes.
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2. Results and Discussion
2.1. Synthesis and Characterization
The cyclometallated ruthenium(II) complexes were obtained by the direct reaction of the
3-acetyl-8-methoxy-2[H]-chromene-2-one-4(N)-substituted thiosemicarbazones (H2L1-4) with
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[RuHCl(CO)(PPh3)3] in benzene under reflux conditions, as mentioned in Scheme 1. The
complexes were characterized by IR, elemental analyses, UV−Visible, 1H and 13C NMR
spectroscopic techniques. The structures of the complexes (1, 2 and 4) were confirmed by X-
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ray crystallography. The ligands were observed to undergo C–H activation at the ortho
position of H3C-C=N, leading to the formation of a five-member metallacycle and behaved as
a dibasic tridentate donor. The new complexes (1–4) were stable to air and light, nonhygroscopic in nature and were soluble in common organic solvents such as
dichloromethane, chloroform, benzene, acetonitrile, ethanol, methanol, dimethylformamide
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and dimethylsulfoxide and the complexes were stable in aqueous solutions such as 1%
aqueous DMSO, phosphate buffer–DMSO (99:1) and Tris-HCl–DMSO (99:1), which was
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confirmed by UV-Vis spectroscopic techniques (Fig. S1).
H
N
H3CO
N
C
AC
C
O
H
S
O
[RuHCl(CO)(PPh3)3]
N
R
BENZENE, 7 h
H
N
C
P
Ru
S
O
N
H3CO
P
N
R
CO
P= PPh3
R=H
R=CH3
R=C2H5
R=C6H5
(1)
(2)
(3)
(4)
Scheme 1. Synthesis of new Ruthenium(II) Complexes
The IR spectral bands of the new ruthenium(II) complexes (1-4) were observed in the
ranges 1590-1598 cm−1 for C=N stretching vibrations [26] and 729-752 cm−1 for C−S stretch
[26,27]. The strong band that appeared around 1914-1938 cm−1 was assigned to the terminally
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coordinated carbonyl group of the complexes 1-4 [26]. In addition, vibrations corresponding
to the presence of triphenylphosphine also present in the region 1407-1434, 1089-1090, 694696 cm-1 [20,27].
The electronic spectra of the complexes displayed broad and weak absorption bands
in the region around 244−397 nm. The absorption observed at 244-279 nm is most likely due
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to a transition involving only ligand orbitals (π/π*and n/π*) [28]. The bands appearing at
around 318–324 nm were assigned to ligand to metal charge transfer transitions [7]. The
absorption at 397 nm is probably due to metal to ligand charge transfer transitions [27,28].
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The 1H-NMR spectra of the complexes recorded in DMSO showed all the expected
signals (Fig.S2–S5). In the spectra of complexes (1-4), a singlet appeared in the range
of δ 1.88-2.09 ppm has been assigned to the N=C-CH3 group protons [29]. The 1H-NMR
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spectra of the complexes confirmed the deprotonation at the C4-H and hydrazinic N(2)H protons
of the ligands H2L1-4 signifying the coordination of ligands in the anionic form after
deprotonation at C4 carbon atom of the pyrone ring and enolization and deprotonation of the
-NH-C=S group prior to coordination of thiolate sulphur atom [26,27] respectively. All the
aromatic protons resonated in the expected range of δ 7.15-7.59 ppm [7] and a singlet
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corresponding to the –OCH3 group occurred around δ 3.79-3.91 ppm [30]. A broad singlet
appeared at δ 5.74 ppm owing to the NH2 protons for 1. In the spectra of the complexes 2, 3
and 4, a quatret, triplet and singlet were observed at δ 6.09-6.29, δ 6.63-6.68 and δ 8.49 ppm
respectively due to the terminal –NH protons of the substituted thiosemicarbazone ligands
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H2L2-4 respectively [26]. In the spectra of the complexes (2 and 3), a doublet and a triplet was
observed around δ 2.10–3.11 and δ 0.73-1.15 ppm due to the methyl group of protons [30]. In
addition, a multiplet at δ 2.23–3.60 ppm corresponding to the methylene group of protons
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was observed in the spectrum of 3 [26].
From the 13C-NMR spectra of the complexes 1-4 (Fig. S6-S9), the carbon resonance
signals of C=N group appeared at
161.9–166.0 ppm [31]. Peaks at
162.9-166.4 ppm were
assigned to C-S carbon atom [27,32] and the methoxy carbon appeared at
Complexes 1-4 exhibited resonance at
56.1 ppm [30].
127.4-133.6 ppm due to the carbon atoms of the
triphenylphosphine groups [7]. A singlet at
193.6-208.6 ppm is assigned to the terminal
carbonyl carbon atom [7,33]. In complexes 2 and 3, the methyl carbon peak appeared at
22.0 and
22.1 ppm, respectively, while in complex 3, the methylene carbon was observed at
31.1 ppm [30].
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2.2. X-Ray crystallography
The molecular structures of the complexes (1, 2 and 4) have been determined by
single crystal X-ray diffraction method and their ORTEP drawings along with the atom
numbering scheme are shown in Fig. 1-3. A summary of the structure refinement of the
complexes 1, 2 and 4 are given in Table 2.
2.2.1. Crystal structure description of new Ru(II) complexes
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complexes are given in Table 1 and selected bond distances and bond angles of the
Though attempts were made to obtain crystals of all the four synthesized complexes,
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crystals suitable for X-ray diffraction studies could be obtained only for complexes 1, 2 and
4, and their structures have been solved by X-ray diffraction techniques. From the symmetry
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of the reflections and the solution of the structures, it is clear that the crystals belong to the
orthorhombic system with the P212121 space group (complex 1) and triclinic system with the
P-1 space group (complex 2 and 4). The ORTEP diagrams showed that the
thiosemicarbazone ligand is coordinated to the metal ion through the pyrone carbon (C3), N1
hydrazinic nitrogen, and thiolate sulfur atoms with the formation of two five-member ring
with a bite angle N(1)−Ru(1)−S(1) of 79.03(9)° for complex 1, 79.56(5)° for complex 2,
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79.75(3)° for complex 4, and a bite angle of C(3)-Ru(1)-N(1) of 78.10(14)° for complex 1,
78.16(8)° for complex 2, 78.24(4)° for complex 4. The Ru(1)−N(1) bond distance is 2.091(3)
Å for complex 1, 2.094(2) Å for complex 2, 2.0844(10) Å for complex 4, the Ru(1)−S(1)
distance is 2.4534(1) Å for complex 1, 2.4433(7) Å for complex 2, 2.4334(3) Å for complex
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4 and the Ru(1)−C(3) distance is 2.072(4) Å for complex 1, 2.058(2) Å for complex 2,
2.061(1) Å for complex 4. The remaining three sites are occupied by phosphorus atoms of
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two triphenylphosphine ligands which are mutually trans to each other with Ru(1)−P(1) and
Ru(1)−P(2) distances of 2.3759(11) Å and 2.3844(12) Å for complex 1, 2.3780(7) Å and
2.3582(6) Å for complex 2, 2.3664(3) Å and 2.3717(3) Å for complex 4, and a carbonyl
group with Ru(1)−C(14) distances of 1.851(4) Å for complex 1, Ru(1)−C(15) distances of
1.846(2) Å for complex 2, Ru(1)−C(20) distances of 1.8564(12) Å for complex 4. The trans
arrangement of bulky triphenylphosphine ligands may be due to the presence of CO, a
stronger π-acidic ligand that completes the hexa coordination, might have forced the bulky
triphenylphosphine to take trans position for steric reasons. The observed bond distances are
comparable with those found in other reported ruthenium complexes containing
triphenylphosphine [7,16,22,26,34,35]. This bond lengthening could be attributed to the
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strong trans influence of the bulkier triphenylphosphine ligands. The two triphenylphosphine
ligands which are mutually trans to each other and are slightly bent towards the carbonyl
group due to the steric requirements of somewhat bulky chelating ligand, causing a slight
deviation from a linear trans arrangement, which is evident from the bond angle of P(1)–
Ru(1)–C(14)= 87.4(1)° for complex 1, P(1)–Ru(1)–C(15)= 89.46(8)° for complex 2, P(1)–
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Ru(1)–C(20)= 89.12(4)° for complex 4, are smaller than bond angle of P(1)–Ru(1)–N(1)=
89.92(9)° for complex 1, P(1)–Ru(1)–N(1)= 89.62(6)° for complex 2, P(1)–Ru(1)–N(1)=
90.07(3)° for complex 4, and P(2)–Ru(1)–N(1)= 92.01(9)° for complex 1, P(2)–Ru(1)–N(1)=
90.56(6)° for complex 2, P(2)–Ru(1)–N(1)= 92.01(3)° for complex 4. The carbonyl group
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occupies the site trans to N(1). The cis bond angles found in the complexes agree well with
those reported for similar ruthenium complexes [7,16,26,34,35]. The trans angles
C(14)−Ru(1)−N(1) = 176.84(15)° for complex 1, C(15)−Ru(1)−N(1) = 178.39(9)° for
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complex 2, C(20)−Ru(1)−N(1) = 177.76(4)° for complex 4, P(2)−Ru−P(1) = 174.82(4)° for
1, 176.46(2)° for 2, 174.65(11)° for 3 and S(1)−Ru(1)−C(3) = 156.83(12)° for 1, 157.61(7)°
for 2 and 157.97(3)° for 4, deviated from linearity. The distorted octahedron is evidenced by
the longer Ru–P bonds when compared to the equatorial bonds and also the deviation from
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the corresponding cis and trans bond angles of 90° and 180°.
Fig. 1. ORTEP diagram of [Ru(8MAC-tsc)CO(PPh3)2] (1)
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Fig. 2. ORTEP diagram of [Ru(8MAC-mtsc)CO(PPh3)2] (2)
Fig. 3. ORTEP diagram of [Ru(8MAC-ptsc)CO(PPh3)2] (4)
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Table 1. Crystallographic data of the complexes (1, 2 and 4)
[Ru(8MACptsc)CO(PPh3)2
]
C56H45N3O4P2RuS.
(0.5)CH2Cl2
[Ru(8MACtsc)CO(PPh3)2]
[Ru(8MACmtsc)CO(PPh3)2]
Empirical formula
C50H41N3O4P2RuS
Formula weight
Temperature
Wavelength
Crystal system
Space group
Unit cell dimensions
a
b
c
942.93
90.0(5) K
0.71073 Å
Orthorhombic
P212121
C51H43N3O4P2RuS.
(0.34)H2O
962.99
90.0(5) K
0.71073 Å
Triclinic
P -1
1061.48
90.0(5) K
0.71073 Å
Triclinic
P-1
10.9779(3) Å
11.5073(3) Å
17.6704(5) Å
13.2638(6) Å
13.3525(6) Å
14.2108(7) Å
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16.648(3) Å
10.358(2)Å
24.080(5) Å
α
90°
β
90°
γ
90°
Volume
4152.4(14) Å3
Z
4
Density
1.508 Mg/m3
Absorption coefficient 0.557 mm-1
F(000)
1936
Crystal size
0.18 × 0.13 × 0.05 mm
Crystal shape
plate
θ range for data 1.487 to 26.799 °
collection
Limiting indices
-20≤h≤ 21, -13 ≤k≤ 12,
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Identification code
86.438(2) °
86.217(2) °
71.830(2) °
2383.80(19) Å3
2
1.479 Mg/m3
0.548 mm-1
1090
0.21 × 0.20 × 0.05 mm
Plate
1.868 to 30.029 °
0.27 × 0.23 × 0.08 mm
Lath
1.437 to 34.996 °
-15 ≤h≤ 15, -16 ≤k≤ 16,
-24 ≤l≤ 24
-30 ≤l≤ 27
12872 (R(int)=0.0300)
63045 (R(int)=0.0699)
multi-scan
multi-scan
leastFull-matrix
least- Full-matrix
2
2
squares on F
squares on F
-21 ≤h≤ 21, -21≤k≤
20, -22 ≤l≤ 22
71724 (R(int)=0.0220)
multi-scan
Full-matrix
least2
squares on F
63045/2/565
71724/2/636
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86.8063 °
83.3108 °
88.4161 °
2213.09(10) Å3
2
1.445 Mg/m3
0.525 mm-1
991
Independent reflections
Absorption correction
Refinement method
Data/Restraints/
Parameters
Goodness-of-fit on F2
Final
R
indices
[I>2σ(I)]
R indices (all data)
12872/3/588
1.029
1.006
R1 = 0.0323, wR2= R1 = 0.0429, wR2= R1 = 0.0315, wR2=
0.0890
0.0837
0.0700
R1 = 0.0418, wR2 = R1 = 0.0706, wR2 = R1 = 0.0384, wR2 =
0.1008
0.0881
0.0663
1.026
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Table 2. Selected bond lengths (Å) and bond angles (°) of the complexes (1, 2 and 4)
BOND LENGTHS
2
Ru1 C14
1.851(4)
Ru1 C3
2.072(4)
Ru1 N1
2.091(3)
Ru1 P1
2.376(1)
Ru1 P2
2.385(1)
Ru1 S1
2.453(1)
BOND ANGLES
Ru1 C15
Ru1 C3
Ru1 N1
Ru1 P1
Ru1 P2
Ru1 S1
2
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Ru1 C20
Ru1 C3
Ru1 N1
Ru1 P1
Ru1 P2
Ru1 S1
1.8564(12)
2.0616(11)
2.0844(10)
2.3664(3)
2.3717(3)
2.4334(3)
4
102.04(7)
157.61(7)
79.56(5)
88.00(2)
88.55(2)
89.59(7)
89.62(6)
176.45(2)
89.46(7)
90.46(7)
93.91(7)
90.56(6)
178.39(9)
78.16(8)
100.2(1)
95.44(8)
117.82(8)
115.99(8)
111.11(8)
113.25(8)
117.45(8)
111.78(8)
124.5(1)
118.1(2)
113.0(2)
130.7(2)
178.5(2)
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S1 Ru1 C15
S1 Ru1 C3
S1 Ru1 N1
S1 Ru1 P1
S1 Ru1 P2
P1 Ru1 C3
P1 Ru1 N1
P1 Ru1 P2
P1 Ru1 C15
P2 Ru1 C15
P2 Ru1 C3
P2 Ru1 N1
N1 Ru1 C15
N1 Ru1 C3
C3 Ru1 C15
C11 S1 Ru1
C16 P1 Ru1
C22 P1 Ru1
C28 P1 Ru1
C34 P2 Ru1
C40 P2 Ru1
C46 P2 Ru1
N2 N1 Ru1
C10 N1 Ru1
C2 C3 Ru1
C4 C3 Ru1
O4 C15 Ru1
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102.51(12)
156.83(1)
79.03(9)
89.25(4)
86.40(4)
94.6(1)
89.92(9)
174.82(4)
87.4(1)
90.85(12)
90.55(11)
92.01(9)
176.8(1)
78.1(1)
100.5(2)
94.9(2)
117.2(1)
115.7(1)
113.4(1)
116.8(1)
106.5(2)
118.1(2)
125.0(2)
118.3(3)
112.1(3)
132.6(3)
177.5(3)
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S1 Ru1 C14
S1 Ru1 C3
S1 Ru1 N1
S1 Ru1 P1
S1 Ru1 P2
P1 Ru1 C3
P1 Ru1 N1
P1 Ru1 P2
P1 Ru1 C14
P2 Ru1 C14
P2 Ru1 C3
P2 Ru1 N1
N1 Ru1 C14
N1 Ru1 C3
C3 Ru1 C14
C11 S1 Ru1
C15 P1 Ru1
C21 P1 Ru1
C27 P1 Ru1
C33 P2 Ru1
C39 P2 Ru1
C45 P2 Ru1
N2 N1 Ru1
C10 N1 Ru1
C2 C3 Ru1
C4 C3 Ru1
O4 C14 Ru1
1.846(2)
2.058(2)
2.094(2)
2.3780(7)
2.3582(6)
2.4433(7)
S1 Ru1 C20
S1 Ru1 C3
S1 Ru1 N1
S1 Ru1 P1
S1 Ru1 P2
P1 Ru1 C3
P1 Ru1 N1
P1 Ru1 P2
P1 Ru1 C20
P2 Ru1 C20
P2 Ru1 C3
P2 Ru1 N1
N1 Ru1 C20
N1 Ru1 C3
C3 Ru1 C20
C11 S1 Ru1
C33 P1 Ru1
C21 P1 Ru1
C27 P1 Ru1
C39 P2 Ru1
C45 P2 Ru1
C51 P2 Ru1
N2 N1 Ru1
C10 N1 Ru1
C2 C3 Ru1
C4 C3 Ru1
O4 C20 Ru1
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1
4
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101.52(4)
157.97(4)
79.75(3)
88.38(1)
86.66(1)
92.42(3)
90.07(3)
174.66(2)
88.12(4)
90.90(4)
92.92(3)
91.02(3)
177.76(5)
78.24(4)
100.51(5)
95.20(4)
112.18(4)
116.06(4)
117.07(4)
114.18(4)
112.12(4)
116.47(4)
124.55(8)
118.28(8)
112.85(8)
130.99(9)
176.78(1)
While dealing with the hydrogen-bonding interaction, in complex (2), we found the
donor–acceptor interactions between the O(2) oxygen atom of the molecule with O(5) oxygen
atom of the water came from solvent of crystallisation O(2)----O(5)= 3.015 Å (Fig. 4; Table
S1). In complex (4) we found the donor–acceptor distance (3.006 Å) corresponding to the
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O(4)-O(4) bond between the carbonyl oxygen atom of the first molecule and the carbonyl
oxygen atom of the second molecule. This interaction gave a pseudo binuclear structural
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appearance to the complex 4 (Fig. 5; Table S1).
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Fig. 4. ORTEP diagram of [Ru(8MAC-mtsc)CO(PPh3)2] (2) with hydrogen bonding
Fig. 5. ORTEP diagram of [Ru(8MAC-ptsc)CO(PPh3)2] (4) with hydrogen bonding
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2.3. DNA binding studies
2.3.1. UV–Vis absorption spectral titrations
The electronic absorption spectra of the complexes 1–4 (25 µM) in the absence and
presence of CT-DNA (2.5–25 µM) are depicted in Fig. 6. By using absorption spectral
technique, the intrinsic binding constant (Kb) value of the compounds was determined from
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the absorptivity changes of the respective compounds upon the incremental addition of CTDNA. The LMCT absorption band of the complex 1 at 322 nm exhibited a hypochromism of
about 31.91 % with a red shift of 3 nm. The absorption bands of complex 2 and 3 showed
hypochromism of about 39.06 % and 44.04 % with a bathochromic shift of 3 nm and 4 nm at
SC
322 and 320 nm respectively. Whereas, the absorption bands of the complex 4 at 283 nm and
393 nm exhibited the same phenomenon of hypochromism of about 31.42 % and 34.74 %,
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with a red shift of about 3 nm. The decrease in absorbance with increase in concentration of
CT-DNA may be due to the decrease in transition probabilities as a result of partial transfer
of electrons from the π orbital of the DNA base pairs to the coupled π* orbital of the
coordinated Schiff base to metal due to overlapping [36]. The extent of hypochromism in the
charge transfer band is an indication of the strength of intercalative interaction [36]. Above
spectral results obviously inferred that the complexes interact with CT-DNA via intercalation
TE
D
mode. The intrinsic binding constant Kbin was calculated from the ratio of slope to the y
intercept in plots of [DNA]/[εa −εf] versus [DNA] (Inset of Fig. 6) and are given in Table 3.
The obtained binding constant values showed that the cyclometallated Ru(II) complexes
behaved as potent binders to DNA through intercalation and the order of binding affinity of
EP
the complexes with CT-DNA is 3 > 2 > 1 > 4. This may be due to the presence of different
substitutents in the terminal nitrogen atom of the ligands. These results are comparable with
AC
C
earlier reports describing the intercalative mode of various ruthenium intercalators
[20,26,34,37-39].
ACCEPTED MANUSCRIPT
1.0
1.0
0.9
0.005
0.9
0.004
0.0010
0.8
0.0008
0.0006
0.003
0.002
0.7
0.8
0.0004
0.7
ABSORBANCE
ABSORBANCE
2
0.0012
[DNA]/( ε a-ε f)
1
0.0014
[DNA]/( ε a-ε f)
1.1
0.0002
0.0000
0.6
0
5
10
15
20
25
[DNA]
0.5
0.4
0.001
0.6
0.000
0
5
10
15
20
25
[DNA]
0.5
0.4
0.3
0.2
0.2
0.1
0.1
0.0
0.0
250
300
350
400
450
250
500
300
0.9
0.0010
0.8
0.0005
0.6
0.0000
0
5
10
0.5
450
500
0.0020
1.0
0.0015
15
20
[DNA]
0.4
0.3
0.2
25
ABSORBANCE
ABSORBANCE
0.7
1.1
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0.8
4
[DNA]/( ε a-ε f)
0.0020
400
SC
0.0025
0.9
[DNA]/(ε a-ε f)
1.0
350
WAVELENGTH (nm)
WAVELENGTH(nm)
3
RI
PT
0.3
0.0015
0.0010
0.0005
0.7
0.6
0.0000
0
5
10
15
20
25
[DNA]
0.5
0.4
0.3
0.2
0.1
0.1
0.0
250
300
350
400
450
0.0
250
300
350
400
450
500
WAVELENGTH (nm)
TE
D
WAVELENGTH(nm)
500
Fig. 6. Absorption titration spectra of complexes (1-4) (25 µM) with increasing
concentrations (2.5-25 µM) of CT-DNA (Tris HCl buffer, pH 7.2). Inset: Binding isotherms
EP
of the complexes 1-4 with CT-DNA
AC
C
2.3.2. EB-DNA quenching studies
The result obtained from the above experiments suggested that all the complexes can
bind with CT-DNA. Further, the binding mode of the complexes with CT-DNA has been
confirmed by ethidium bromide (EB) displacement studies. Upon each successive addition of
complexes to CT-DNA pretreated with EB ([DNA]/[EB] = 1), a gradual quenching of the
fluorescence intensity was observed (Fig. 7). The reduction of the fluorescence emission
intensity gives criteria to analyze the DNA binding propensity of the complexes and stacking
interaction (intercalation) between the adjacent DNA base pairs [40]. As the concentration of
the complexes increased from 10-100 µM, the emission band of DNA-bound EB exhibited
quenching up to 26.17, 30.88, 33.98 and 22.99 % of the initial fluorescence intensity together
ACCEPTED MANUSCRIPT
with a red shift of 2-4 nm for complex 1, 2, 3 and 4 respectively. This gives a direct evidence
for the intercalative binding mode of the complexes with DNA. The quenching data were
calculated by using Stern-Volmer plot and quenching constant is obtained as a slope from the
plot of Io/Icorr versus [Q] and are given in Table 3. The data showed that DNA-bound EB can
be more readily replaced by the complexes in the order 3 > 2 > 1 > 4, among the complexes
RI
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1-4, the ligand having terminal ethyl group showed better affinity towards the DNA when
compared to the phenyl and methyl group and hydrogen atom in the N-terminal position [26].
Further, the calculated KSV values of the compounds are significant when compared to the
1.35
400
1.30
350
2
IΟΟ/I Corr
1.25
1.6
1.5
350
1.20
IΟΟ/ICorr
400
1
SC
reported values [26,27,34].
1.4
250
1.05
0
20
40
60
80
[Q]
200
150
100
50
1.2
100
1.1
250
0
20
40
60
80
100
[Q]
200
150
100
50
0
0
550
600
650
700
750
WAVELENGTH (nm)
550
1.5
20
40
60
80
100
EP
[Q]
150
AC
C
100
50
600
1.25
650
WAVELENGTH (nm)
300
1.20
1.15
1.10
1.05
250
1.00
0
20
40
60
80
100
[Q]
200
150
100
50
0
550
750
1.30
IΟΟ/ICorr
IΟΟ/ICorr
1.0
0
700
1.35
350
1.3
1.1
200
4
1.4
1.2
250
650
400
1.6
300
600
WAVELENGTH (nm)
TE
D
350
3
EMISSION INTENSITY
300
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1.10
EMISSION INTENSITY
1.15
EMISSION INTENSITY
EMISSION INTENSITY
1.3
300
0
700
750
550
600
650
700
750
WAVELENGTH (nm)
Fig. 7. The emission spectra of the DNA–EB system (λexc= 515 nm, λem= 530–750 nm), in
the presence of complexs 1-4. [DNA] = 10 µM, [complex] = 10–100 µM, [EB] = 10 µM.
The arrow shows the emission intensity changes upon increasing complex concentration.
Inset: Stern−Volmer plot of the fluorescence titration of the complexes (1-4) (10-100 µM)
with DNA-EB (10 µM)
ACCEPTED MANUSCRIPT
Table 3: The binding constant (Kbin) and quenching constant (Ksv) values for the interactions
of the complexes (1–4) with CT-DNA
Binding constant
Kbin M-1
Quenching constant
KSV M-1
Complex 1
7.5280±0.321 x105
2.79±0.003 x103
Complex 2
9.2713±0.306 x105
3.43±0.006 x103
Complex 3
13.8736±0.320 x105
6.19±0.008 x103
Complex 4
6.2265±0.286 x105
2.75±0.002 x103
SC
2.3.3. Viscosity measurements
RI
PT
Compounds
In order to confirm the binding mode of the complexes with CT-DNA, viscosity
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measurements were carried out by keeping the DNA concentration (100 µM) as constant and
varying the concentrations of the compounds (20-100 µM). From Fig. 8, it is obvious that the
relative viscosity of DNA solutions increased upon increasing the concentration of the
complexes. Viscosity of DNA will increase while the complex intercalates between adjacent
DNA base pairs, which leads to an increase in the separation of base pairs at the intercalation
TE
D
site, resulting an increase in the overall DNA length [41,42] and the above results concluded
that compounds interacted with CT-DNA through an intercalative mode. In addition, from the
plot we concluded that the ability of the complexes to increase the viscosity of CT-DNA
depends upon the substitution on the N-terminal nitrogen of the ligand and the increasing
EP
order of viscosity of CT-DNA by the complexes is (NH-Ethyl) 3 > (NH-methyl) 2 > (NH-
(η /ηο)
1/3
AC
C
Hydrogen) 1> (NH-Phenyl) 4 which is consistent with their DNA binding results.
DNA VISCOSITY STUDIES
1.8
1.7
Complex 1
Complex 2
Complex 3
Complex 4
1.6
1.5
1.4
1.3
1.2
1.1
1.0
0.9
0.0
0.2
0.4
0.6
0.8
1.0
(1/R=[Complex]/[DNA])
Fig. 8. Effect of the complexes (1-4) on viscosity of CT-DNA
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2.4. pBR322 DNA cleavage studies
The newly synthesized organoruthenium(II) complexes (1-4) were studied for their
chemical nuclease activities by the agarose gel electrophoresis method with supercoiled
pBR322 DNA as the substrate, in the absence of external agents in a medium of 5 mM TrisHCl/50 mM NaCl buffer (pH 7.2). The DNA cleavage efficiency was measured by
RI
PT
determining the ability of the compound to convert the supercoiled DNA (SC Form) to Linear
circular form or nicked form (NC Form). All the four cyclometallated ruthenium(II)
complexes efficiently cleaved the supercoiled pBR322 DNA to nicked form and linear
circular form (Fig. 9). From Fig. 9 we inferred that the complexes to influence nuclease
accepting ability pertaining to the
SC
activity in the order 3 > 2 > 1≈ 4, which may be due to the change in the electron donating or
N-terminal substituent of the ligand and subsequent
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change in the polarisability on metal. Among the complexes, complex 3 with more electron
donating ethyl substitution on terminal nitrogen atom causes stronger distortion on DNA
strand showing more efficient DNA cleavage. The observed result pattern is parallel to their
AC
C
EP
TE
D
DNA binding affinity.
Fig. 9. Gel electrophoresis diagram showing the cleavage of supercoiled pBR322 DNA by
complexes 1-4 in 5% DMSO and 95% 5 mM Tris−HCl/50 mM NaCl buffer at pH 7.2 and 37
°C with an incubation time of 2 h. Lanes M: Marker; Lane C 1: Complex 1 (50 µM). Lane C
2: Complex 2 (50 µM); Lane C 3: Complex 3 (50 µM); Lane C 4: Complex 4 (50 µM); Lane
S: Metal precursor (50 µM); Forms SC, NC, and LC are supercoiled, nicked circular, and
linear circular DNA, respectively.
ACCEPTED MANUSCRIPT
2.5. Protein Binding Studies
The intrinsic fluorescence of BSA and HSA is mainly due to tryptophan residue
[43,44]. The binding of new Ru(II) complexes 1–4 with BSA and its homologue HSA was
investigated by fluorescence emission spectroscopy, since the albumin solution exhibits an
intense emission band (λex = 290 nm) at λem,max = 345 nm (for HSA) and 346 nm (for BSA)
RI
PT
which is assigned to the existence of tryptophans. The emission titration studies have been
performed at room temperature using BSA (10 µM)/ HSA (10 µM) with increasing
concentrations of complexes 1–4 (0−100 µM) in the range 290−500 nm. However, no
emission peak appeared around 340-345 nm when these ruthenium complexes were excited
SC
with the same excitation wavelength, suggesting that the ruthenium complexes 1-4 would not
induce fluorescence interference to serum albumin within the investigated excitation
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wavelength range. Addition of the above test compounds to BSA solution resulted in a
significant decrease in the fluorescence intensity of BSA at 346 nm, up to 63.39, 74.89, 78.71
and 72.34 % of the initial fluorescence intensity of BSA with 2-6 nm blue shift for complexes
1, 2, 3 and 4 respectively (Fig. 10). Addition of the compounds to HSA solution resulted in a
high quenching of the emission band of HSA at 345 nm up to 51.00, 57.37, 65.91 and 45.62
% of the initial fluorescence intensity together with a blue shift of 2-4 nm for complex 1, 2, 3
TE
D
and 4 respectively (Fig. 11). From the above observations, we may conclude that definite
interaction is taking place between the complexes and serum albumins.
Fig. S10 in the supporting information shows the absorption spectra of BSA and HSA
EP
in the absence and presence of complexes. The absorbance intensity of serum albumins
showed hypochromism with a small blue shift (∼ 2-5 nm) in the presence of complexes 1-4,
AC
C
indicating a static quenching mechanism of the serum albumins by complexes. By using
Stern Volmer quenching equation, the values of the Stern Volmer quenching constant (KSV)
and the quenching constant (Kq) for complexes interacting with serum albumins (BSA/HSA)
were calculated. The Stern Volmer quenching constant obtained as a slope of the plot of [Q]
verses the ratio of the fluorescence intensity in the absence (Iₒ) and in the presence of the
quencher (I) (Fig. 12; Table 4) and the results follow the order 3 > 2 > 1 > 4. The observed
Ksv values are comparable to those reported for other ruthenium complexes [20,26,34,38,39].
The quenching constant values (kq ≈ 1012 M-1 s-1) suggested the good binding affinity of the
complexes with serum albumins through static quenching mechanism [45].
ACCEPTED MANUSCRIPT
The binding constant Kbin and number of binding site (n) can be calculated from the
Scatchard equation and are given in Table 4 (Fig. 13), from these values we knew that the
complexes (1-4) showed strong binding affinity with serum albumins. Further, the obtained
results suggested that complexes 1-4 act as potent binders with serum albumins. From the
results, we confirmed that the Ru(II) complexes having a large hydrophobic area can interact
RI
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more efficiently with serum albumins via a static pathway. The obtained binding constant and
quenching constant values revealed that the ruthenium(II) complexes bind to both the
albumins in the following order 3 > 2 > 1 > 4. This observed trend can be explained on the
basis of electron donating ability and hydrophobicity of the compounds. Increase in the
SC
electron-donating ability of the N-terminal substituent of the coordinated ligand increases the
electron density on the electron deficient metal center [46]. As seen from the results, complex
3 with the enhanced hydrophobicity showed the best binding ability [34]. The obtained
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quenching constant and binding constant values of these new cyclometallated ruthenium(II)
complexes agree well with those reported for other ruthenium(II) complexes [20,47,48].
900
1
700
EMISSION INTENSITY
700
600
500
TE
D
EMISSION INTENSITY
2
800
800
400
300
200
100
0
300
350
400
450
600
500
400
300
200
100
0
300
EP
.
3
AC
C
900
350
400
900
4
800
800
700
EMISSION INTENSITY
EMISSION INTENSITY
450
WAVELENGTH (nm)
WAVELENGTH (nm)
600
500
400
300
200
100
700
600
500
400
300
200
100
0
0
300
320
340
360
380
400
WAVELENGTH (nm)
420
440
300
350
400
450
WAVELENGTH (nm)
Fig. 10. The emission spectra of BSA (10 µM; λexc= 280 nm; λemi= 346 nm) in the presence
of increasing amounts of complexes 1-4 (10–100 µM). The arrow shows the emission
intensity changes upon increasing complex concentration
ACCEPTED MANUSCRIPT
350
1
350
2
300
200
150
100
50
250
200
150
100
50
0
0
300
320
340
360
380
400
420
440
320
340
WAVELENGTH (nm)
360
380
400
420
440
SC
WAVELENGTH (nm)
350
350
3
300
EMISSION INTENSITY
300
250
250
M
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EMISSION INTENSITY
RI
PT
250
EMISSION INTENSITY
EMISSION INTENSITY
300
200
150
100
50
4
200
150
100
50
0
0
320
340
360
380
400
420
320
340
360
380
400
420
440
WAVELENGTH (nm)
TE
D
WAVELENGTH (nm)
440
EP
Fig. 11. The emission spectra of HSA (10 µM; λexc= 280 nm; λemi= 346 nm) in the presence
of increasing amounts of complexes 1-4 (10–100 µM). The arrow shows the emission
intensity changes upon increasing complex concentration
STERN VOLMER PLOT
5.0
A
4.5
Complex 1
Complex 2
Complex 3
Complex 4
4.0
B
Complex 1
Complex 2
Complex 3
Complex 4
2.8
2.6
2.4
IΟ/ICorr
AC
C
3.5
IΟ/ICorr
STERN VOLMER PLOT
3.0
3.0
2.5
2.2
2.0
1.8
1.6
2.0
1.4
1.5
1.2
1.0
0
20
40
60
[Q]
1.0
80
100
0
20
40
60
80
100
[Q]
Fig. 12. A) Stern−Volmer plot of the fluorescence titration of the complexes (1-4) (10-100 µM)
with BSA (10 µM). B) Stern−Volmer plot of the fluorescence titration of the complexes (1-4)
(10-100 µM) with HSA (10 µM)
ACCEPTED MANUSCRIPT
0.4
B
Complex 1
Complex 2
Complex 3
Complex 4
0.2
log ((FΟ-F)/F)
0.4
SCATCHARD PLOT
0.6
0.0
-0.2
-0.4
SCATCHARD PLOT
0.2
0.0
log ((FΟ-F)/F)
A
Complex 1
Complex 2
Complex 3
Complex 4
-0.2
-0.4
-0.6
-0.6
-0.8
-0.8
-1.2
-1.2
-1.4
-1.4
-5.0
-4.8
-4.6
-4.4
-4.2
-5.0
-4.0
log [Q]
-4.8
RI
PT
-1.0
-1.0
-4.6
-4.4
-4.2
-4.0
log [Q]
SC
Fig. 13. A) Scatchard plot of the fluorescence titration of the complexes (1-4) (10-100 µM)
with BSA (10 µM). B) Scatchard plot of the fluorescence titration of the complexes (1-4)
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(10-100 µM) with HSA (10 µM)
Table 4: Stern volmer quenching constant (Ksv), Quenching constant (kq), binding
constant (Kbin) and number of binding sites (n) for the interactions of ligands and
complexes (1-4) with BSA/HSA
BSA
Stern-Volmer
Ksv/M-1
Quenching
Constant (kq)
M-1s-1
Binding constant
Kbin/M-1
n
TE
D
Compounds
1.784±0.090x104
1.784±0.090x1012
4.363±0.182x105
1.3569±0.041
Complex 2
3.147±0.012x104
3.147±0.012x1012
6.914±0.088x105
1.3384±0.020
3.904±0.033x104
3.904±0.033x1012
1.361±0.105x106
1.3996±0.024
2.552±0.002x104
2.552±0.002x1012
3.490±0.043x105
1.2991±0.045
Complex 1
1.100±0.012x104
1.100±0.012x1012
3.808±0.039x104
1.1371±0.009
Complex 2
1.367±0.021x104
1.367±0.021x1012
4.460±0.014x104
1.1306±0.015
Complex 3
2.091±0.067x104
2.091±0.067x1012
9.131±0.041x104
1.1716±0.043
Complex 4
0.830±0.015x104
0.830±0.015x1012
3.212±0.015x104
1.1493±0.015
Complex 3
HSA
AC
C
Complex 4
EP
Complex 1
ACCEPTED MANUSCRIPT
2.5.1. Conformational Investigation
Synchronous fluorescence experiments have been performed to determine the
conformational changes in serum albumins such as BSA and HSA in the presence of
compounds. In the synchronous fluorescence spectra of the tyrosine residue of both the serum
albumins, the addition of the compounds to the solution of BSA/HSA showed hypochromism
RI
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with negligible shift in the emission wavelength (Fig. S11-S12). Synchronous fluorescence
spectra of BSA at ∆λ = 60 nm exhibited a decrease in fluorescence intensity up to 70.6978.04 % at 340 nm with significant blue shift for complexes (1-4) (Fig. S13), whereas the
spectra corresponding to the tryptophan residue of HSA, the addition of the compounds to the
SC
solution of HSA showed hypochromism upto 43.96-62.51 % for complexes (1-4) (Fig. S14).
The obtained results clearly revealed that compounds effectively bind with serum albumins
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and affect the conformation of the tryptophan and tyrosine micro regions.
2.5.2. Three-dimensional fluorescence spectra analysis
Three dimensional fluorescence spectroscopic studies have been performed to
investigate the microenvironmental changes in BSA/HSA during interaction with the
compounds. Fig. 14-15 shows the three dimensional emission spectra and contour ones of
TE
D
serum albumins in the absence and presence of ruthenium(II) complexes and their
corresponding characteristic parameters are provided in Table S2. The emission intensity of
peak ‘A’ corresponding to the Rayleigh first order scattering peak increased upon adding the
complexes to serum albumins. This is due to the compound formation of serum albumins
EP
with our ruthenium(II) complexes leading to an increase in the diameter of the
macromolecule which in turn resulted in the enhancement of scattering effect [49]. The
AC
C
fluorescence intensity of peak ‘B’ corresponding to the tryptophan and tyrosine residues
decreased with slight blue shift. The obtained results inferred that the molecular
conformational and microenvironment changes of protein occurred after interaction with the
complexes.
AC
C
EP
TE
D
M
AN
U
SC
RI
PT
ACCEPTED MANUSCRIPT
Fig. 14. Three-dimensional fluorescence spectra of BSA in the absence and presence of
ruthenium(II) complexes 1-4 (pH 7.4, 298 K, [BSA] =10 µM, [Complex] =10 µM)
AC
C
EP
TE
D
M
AN
U
SC
RI
PT
ACCEPTED MANUSCRIPT
Fig. 15. Three-dimensional fluorescence spectra of HSA in the absence and presence of
ruthenium(II) complexes 1-4 (pH 7.4, 298 K, [HSA] =10 µM, [Complex] =10 µM)
ACCEPTED MANUSCRIPT
2.6. In vitro Antioxidant activity
The compounds which exhibit radical scavenging activity are receiving much
attention because they possess interesting anticancer, anti-inflammatory and anti-ageing
activities [50]. The analysis of the DPPH radical scavenging ability of cyclometallated Ru(II)
complexes have been carried out along with the standard Vitamin C. The results showed that
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the activity of the compounds is higher than that of the standard, the DPPH scavenging
activity of the Ru(II) complexes (8.23-9.85 µM) follow the order of 3 > 2 > 1 > 4 (Fig. 16
and Fig. S15). Complex 3 shows the best DPPH scavenging activity among the
complexes. The phosphomolybdenum assay is quantitative, since the antioxidant activity is
SC
expressed as the number of equivalents of ascorbic acid (Table 5). Four new cyclometallated
Ru(II) complexes exhibited higher activity than their parent ligands among them complex 3
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contains the more electron donating ethyl substitution at terminal nitrogen showed higher
activity. A comparison of the radical scavenging activity of Ru(II) complexes 1–4 with that
of the reported Ru(II) Schiff base complexes may reveal that our new Ru(II) complexes 1–
4 act as potent radical scavengers [51].
EP
60
40
AC
C
IC50 VALUE (µM)
80
TE
D
DPPH RADICAL SCAVENGING ASSAY
100
20
0
STD
RU
Complex 1
Complex 2
Complex 3
Complex 4
Fig.16. The DPPH radical scavenging activity of the [RuHCl(CO)(PPh3)3] and new Ru(II)
complexes (1-4)
ACCEPTED MANUSCRIPT
Table 5: Estimation of Total antioxidant capacity of [RuHCl(CO)(PPh3)3] and new Ru(II)
complexes (1-4)
µg Ascorbic acid equivalents/ml
07.02±0.08
50.03±0.73
59.16±0.65
63.38±0.89
49.33±0.54
RI
PT
Compounds
[RuHClCO(PPh3)3]
Complex 1
Complex 2
Complex 3
Complex 4
2.7. Antimicrobial activity
SC
In vitro antimicrobial activities of four new complexes 1–4 were investigated with
few pathogenic bacteria such as Pseudomonas aeruginosa, Streptococcus pneumonie,
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Staphylococcus aureus, Salmonella paratyphi and with few fungi Candida albicans,
Aspergillus niger, Trichophyton rubrumi, Candida tropicalis and Aspergillus fumigatus. The
results are expressed as the zone of inhibition and minimum inhibitory concentration (MIC)
and the effect of the compounds was susceptible to their concentration used for inhibition
(Table S3-S6). For comparison, MIC values for positive control such as Gentamicin for
bacteria and Ketaconazole for fungi are also given. On comparing the activity of the
TE
D
complexes, complex 3 was more active on bacteria namely S. aureus, S. pneumonie and P.
aeruginosa. When tested against S. paratyphi, both the complexes 1 and 3 showed
comparable activity. Complex 3 was much more efficient in inhibiting the growth of fungal
species like T. rubrum (IC50 = 16.93±0.31 µM), A. fumigatus (IC50 = 15.78±0.18 µM) and C.
EP
albicans (IC50 = 14.43±0.17 µM), whereas complex 1 was the most active against A. niger.
Complexes 1 and 3 were approximately equally active over C. tropicalis. All the compounds
AC
C
showed lower activity than the controls used. Overall, complex 3 was the better candidate for
the growth inhibition of the microorganisms. The complexes showed different degrees of
antimicrobial activity due to the structural variations of themselves and variation on the group
of microorganisms [52]. Tested complexes had better antimicrobial activity than the ligands
against all pathogens. This may be explained by Tweedy’s chelation theory [53], which stated
that, upon complexation the polarity of metal ion gets reduced which increases the
lipophilicity of the metal complexes facilitating them to cross the cell membrane easily
[53,54]. The antimicrobial activity against the control disc with 10% aqueous DMSO showed
no zone of inhibition. In addition, antimicrobial activity of the complexes was compared with
ACCEPTED MANUSCRIPT
already reported ruthenium complexes, showing that the new Ru(II) complexes exhibited
significant activity [26,55].
ANTIBACTERIAL STUDIES
S. aureus
S. pneumonie
P. aeruginosa
S. paratyphi
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30
25
20
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15
10
5
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Minimum Inhibitory Concentration (µM)
35
0
RU
C1
C2
C3
C4
STD
Fig. 17. Anti bacterial activity of [RuHClCO(PPh3)3] and new Ru(II) complexes (1-4). Error
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bars represent the standard deviation of the mean (n=3)
50
35
30
T. rubrum
A. niger
A. fumigatus
C. tropicalis
C. albicans
EP
40
25
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C
Minimum Inhibitory Concentration (µM)
ANTIFUNCAL STUDIES
45
20
15
10
5
0
RU
C1
C2
C3
C4
STD
Fig. 18. Anti fungal activity of [RuHClCO(PPh3)3] and new Ru(II) complexes (1-4). Error
bars represent the standard deviation of the mean (n=3)
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2.8. Cytotoxicity studies
2.8.1. Antiproliferative Studies - Cancer Cell Growth Inhibition
Significant results obtained from DNA/protein binding, antioxidant and antimicrobial
studies motivated us to further explore their antiproliferative activities. The compounds were
subjected to study the in vitro cytotoxicity by MTT assay towards following three cell lines:
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human lung cancer cells (A549), human breast cancer cells (MCF-7) and human normal
keratinocyte cell lines (HaCaT). Cytotoxic activity of cisplatin against all the above cell lines
was investigated under the same experimental conditions for comparison purposes. The
anticancer activity was assessed on the basis of IC50 values (the concentration of a drug
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required to inhibit the growth of 50% of the cells) and the obtained values of the complexes
against selected three tumor cell lines are shown in Table 7. The results revealed that Ru(II)
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complexes exhibited potent antitumor activities than their parent ligands and standard drug
cisplatin against the selected cell lines in different concentrations and the antitumor activities
are concentration-dependent (Fig. S16 - S18). In human breast cancer cell lines (MCF-7), the
anticancer activity of the complexes follows the order cisplatin (16.79±0.08) < complex 4
(3.93±0.08) < complex 1 (3.86±0.11) < complex 2 (3.77±0.09) < complex 3 (3.72±0.11). The
cytotoxic nature of the compounds against human lung cancer cell lines (A549) is in the order
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cisplatin (15.10±0.05) < complex 4 (4.31±0.08) < complex 1 (4.12±0.11) < complex 2
(3.93±0.13) < complex 3 (3.81±0.10).
From the results we knew that cyclometallated Ru(II) complexes (1-4) appeared to be
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more cytotoxic against A549 and MCF-7 cells over their parent ligands. On the basis of the
results, the antiproliferative activity of the complexes has been arranged in the order
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3 > 2 > 1 > 4. The observed trend may be due to the presence of different substituents in the
terminal nitrogen atom of the ligands. The anticancer activity of the complexes increased
with increase in the electron donating ability of the substituent on the terminal nitrogen of the
coordinated thiosemicarbazones. It is notable that complex 3 having more electron donating
ethyl group at terminal nitrogen has a high antiproliferative activity, as compared with
remaining complexes against both the cell lines (MCF-7 and A549) and its IC50 value which
is almost five times greater than that of cisplatin indicated its high cytotoxic effects against
human cancer cells. Interestingly, this observed trend is in agreement with their previous
biological studies, suggesting that the anticancer activities of the tested compounds against
cancer cell lines may be related to their ability to intercalate the base pairs of the DNA and/or
their free radical scavenging activity. The compounds were also screened for their activity on
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the human normal keratinocyte cells (HaCaT) to examine the selectivity of the compounds
for cancer cells rather than normal cell lines and the results inferred that the complexes are
significantly non toxic to normal cells. The anticancer activity of our new cyclometallated
Ru(II) complexess are superior to those reported some other Ru(II) Schiff base complexes
against A549 cell lines and MCF-7 cells [14,20,21,24,27,56-59].
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Table 7. The IC50 values for the human breast cancer cell line MCF-7, human lung
carcinoma cancer cell line A549 and human normal keratinocyte cells (HaCaT) with new
organometallic Ru(II) complexes for 48h
IC50 values (µM)
A549
15.10±0.05
[RuHClCO(PPh3)3]
20.10±0.18
15.96±0.21
Complex 1
3.96±0.11
4.12±0.11
>40
Complex 2
3.74±0.09
3.93±0.13
>40
Complex 3
3.72±0.11
3.81±0.10
>40
Complex 4
3.85±0.08
4.31±0.08
>40
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HaCaT
>40
>40
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2.8.2. LDH assay
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Cisplatin
MCF-7
16.79±0.08
Compounds
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To further evaluate the toxicity of the compounds, activity of LDH was measured.
LDH is a stable cytoplasmic enzyme released into the culture medium due to the loss of
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membrane integrity resulting from apoptosis of cells. Hence, LDH release is used to analyse
drug induced cytotoxicity of cancer cells [60]. When cancer cell lines A549 and MCF-7 were
treated with the IC50 concentration of the new cyclometallated ruthenium(II) complexes 1-4
for a period of 48 h, a significant increase of LDH release in the culture medium was
observed (Fig. 19). These results indicated the efficiency of the complexes in inducing cell
death by collapsing the membrane integrity. The complexes showed good level of LDH
leakage in A549 and MCF-7 cells compared to cisplatin. This study authenticate that
complex 3 was more effective followed by complex 2, 1 and 4. The ruthenium(II) complexes
showed significant activity when compared with the earlier reports [34].
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LACTATE DEHYDROGENASE (%)
100
A549
MCF-7
90
70
60
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% of LDH release
80
50
40
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30
20
0
Control
Cisplatin
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10
C1
C2
C3
C4
Fig. 19. Percentage of lactate dehydrogenase released by the human cancer cell lines A549
and MCF-7 after an incubation period of 48 h with complexes 1-4. Error bars represent the
2.8.3. Nitric oxide assay
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standard mean error (n= 6).
The nitric oxide (NO) assay is also an important measure of cytotoxicity, as NO has
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been shown to directly inhibit methionine adenosyl transferase, leading to glutathione
depletion, and its reaction with superoxide generates the strong oxidant peroxynitrite, which
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can initiate lipid peroxidation or cause a direct inhibition of the mitochondrial respiratory
chain [61]. In the present study NO release by the new ruthenium(II) complexes 1-4 was
evaluated using A549 and MCF-7 cells. The quantification of the nitrite produced in the cell
media by the Griess assay is an indirect but cost-effective measurement of the amount of NO
produced by the cells. It is interesting to note that the complexes were found to release more
NO than the cisplatin and control (Fig. 20). In addition, complex 3 was more effective in
enhancing the level of NO in the culture medium and the activity follows the order 3 > 2 > 1
> 4 and hence the results confirmed the cytotoxic potential of the studied compounds.
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100
80
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70
60
50
40
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nmoles of nitrite released
A549
MCF-7
Nitric Oxide Release
90
30
10
0
Control
Cisplatin
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20
C1
C2
C3
C4
Fig. 20. Nitrite released (nmoles) by the human cancer cell lines A549 and MCF-7 after an
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incubation period of 48 h with complexes 1-4. Error bars represent the standard mean error
(n= 6).
3. Conclusion
synthesized
by
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New potential anticancer active cyclometallated Ru(II) complexes (1-4) were
the
complexation
of
3-acetyl-8-methoxycoumarin-4N-substituted
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thiosemicarbazones with [RuHCl(CO)(PPh3)3]. Elemental analyses and spectroscopic
characterizations (IR, UV-Vis, 1H NMR and 13C NMR) supported the formation of the
complexes. The crystal structures of the complexes 1, 2 and 4 have been solved by X-ray
crystallographic analysis. Intercalative binding mode of the compounds with CT-DNA was
suggested by the spectral, EB displacement assay and DNA viscosity measurements. The
compounds were able to bind well with BSA/HSA and a static quenching mechanism was
observed.
3D
fluorescence
experiments
indicated
the
changes
in
the
protein
microenvironment. The compounds have good radical scavenging property and antimicrobial
studies revealed the significant activity of the compounds. The complexes were better in
damaging cancerous cell lines than the cisplatin. They were found to be non toxic against
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human normal keratinocyte cell line HaCaT. Combining the overall results, it is evident that
the biological activity of the complexes follows the pattern 3 > 2 > 1 > 4 and the promising
results obtained from the biological studies suggested that the complexes can act as good
probes for further exposure in pharmaceutical applications..
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4. Experimental section
4.1. Materials and methods
All the reagents used were of analytical or chemically pure grade. Solvents were
purified and dried according to standard procedures [62]. 3-acetyl-8-methoxy-2H-chromen-2-
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one [63] and the metal precursor [RuHCl(CO)(PPh3)3] were prepared according to the
literature procedures [64]. The 3-acetyl-8-methoxy-2H-chromen-2-one-4(N)-substituted
thiosemicarbazones H2L1-4 were prepared with a slight modification of the reported method
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[37,63]. Buffers were prepared from doubly distilled water. Ethidium bromide (EB), bovine
serum albumin (BSA), calf thymus DNA (CT-DNA) and 3-(4,5-dimethylthiazol-2-yl)-2,5diphenyltetrazolium bromide (MTT) were purchased from HiMedia and used as received.
Melting points were measured in a Lab India apparatus. Infrared spectra were measured as
KBr pellets on a JASCO FT-IR 4100 instrument between 400–4000 cm−1. Elemental analyses
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of carbon, hydrogen, nitrogen and sulfur was determined by using Vario EL-III CHNS
analyser. The electronic spectra of the complexes were recorded with a JASCO V-630
spectrophotometer using DMSO as the solvent in the 800–200 nm range. Emission spectra
were recorded by using JASCO FP 6600 Spectrofluorimeter. 1H and 13C NMR spectra of the
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compounds were recorded in DMSO with a Bruker instrument with 400 MHz and 100 MHz
respectively, chemical shift relative to tetramethylsilane.
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4.2. X-ray Crystallography
Suitable single crystals for the complexes (1, 2 and 4) were obtained from
dichloromethane/methanol medium. Single crystal data collections and corrections for the
new Ru(II) complexes (1, 2 and 4) were carried out with a Bruker kappa APEX-II DUO 1000
CCD diffractometer using graphite monochromated Mo Kα (λ= 0.71073 Å) radiation at 90.05
K. All the calculations were done by using SHELXS-97 [65] and SHELXL-2014/7 programs
[66].
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4.3. General procedure for the synthesis of new ruthenium(II) complexes
[Ru(8MAC-Rtsc)(CO)(PPh3)2]
A solution of 3-acetyl-8-methoxy-coumarin thiosemicarbazone/ 3-acetyl-8-methoxycoumarin-4(N)-methylthiosemicarbazone/3-acetyl-8-methoxy-coumarin-4(N)-ethylthiosemi
carbazone / 3-acetyl-8-methoxy-coumarin-4(N)-phenylthiosemicarbazone (0.105 mmol) in 10
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cm3 of benzene was added dropwise to a boiling solution of [RuHCl(CO)(PPh3)3] (0.105
mmol) in benzene and refluxed for 7 h and allowed to stand for 4 days at room temperature.
Reddish orange solid formed was filtered, washed with petroleum ether (60–80 °C) and
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recrystallized from dichloromethane and methanol mixture (1:1 v/v).
4.3.1. Synthesis of [Ru(8MAC-tsc)(CO)(PPh3)2] (1)
Yield: 68 %. Mp. 163 °C. Anal. calcd. for C50H41N3O4P2RuS: C, 63.67; H, 4.39; N, 4.45; S,
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3.39. Found: C, 63.59; H, 4.30; N, 4.40; S, 3.35 %. FT-IR (ν, cm−1) in KBr: ν(C=O lactone)
1687, ν(C=N) 1590, ν(C-S) 729, ν(C≡O) 1914,
1434, 1089, 695 (for PPh3). UV-Vis
(DMSO), λmax (ε): 267 (15,252) nm (dm3mol−1cm−1) (Intraligand transition); 324 (16,786) nm
(dm3mol−1cm−1) (LMCT s→d). 1H NMR (400 MHz, DMSO-d6, δ ppm, J Hz): δ 7.15-7.31
(m, 31H, Ar-H), δ 6.62-6.66 (t, 1H, J=7.2, C6-H), δ 6.91-6.93 (d, 1H, J=8, C7-H), δ 3.79 (s,
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3H,-OCH3), δ 2.09 (s, 3H,-CH3), δ 5.74 (br s, 2H, -NH2). 13C NMR (100 MHz, DMSO-d6, δ
ppm): δ 201.4 (C≡O), δ 162.9 (C-S), δ 161.9 (C=N), δ 151.2 (C=O), δ 112.6 (C2), δ 145.7
(C3), δ 126.3 (C4), δ 121.4 (C5), δ 125.5 (C6), δ 121.1 (C7), δ 139.1 (C8), δ 127.9 (C9), δ
18.2 (-CH3), δ 56.1 (OMe), δ 128.6-133.3 (PPh3). Complex 1 was recrystallized from
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dichloromethane and methanol mixture (1:1 v/v) to yield red transparent, needle like crystals
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suitable for X-ray analysis.
4.3.2. Synthesis of [Ru(8MAC-mtsc)(CO)(PPh3)2] (2)
Yield: 62%. Mp. 206 °C. Anal. calcd. for C51H43N3O4P2RuS: C, 64.00; H, 4.45; N, 4.39; S,
3.34. Found: C, 63.63; H, 4.49; N, 4.34; S, 3.30 %. FT-IR (ν, cm−1) in KBr: ν(C=O lactone)
1682, ν(C=N) 1592, ν(C-S) 745, ν(C≡O) 1930,
3
−1
1407, 1089, 696 (for PPh3). UV-Vis
−1
(DMSO), λmax (ε): 244 (86,054) nm (dm mol cm ) (Intraligand transition); 318 (47,292) nm
(dm3mol−1cm−1) (LMCT s→d), 531 (1057) nm (dm3mol−1cm−1) (forbidden d→d transition).
1
H NMR (400 MHz, DMSO-d6, δ ppm, J Hz): δ 7.13-7.30 (m, 31H, Ar-H), δ 6.74-6.78 (t,
1H, J=7.6, C6-H), δ 6.85-6.87 (d, 1H, J=7.2, C7-H), δ 3.89 (s, 3H,-OCH3), δ 2.09 (s, 3H,CH3), δ 6.09-6.29 (q, 1H, terminal -NH), δ 2.10-2.11 (d, 3H, J=4.8,-NH-CH3). 13C NMR (100
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MHz, DMSO-d6, δ ppm): δ 193.6 (C≡O), δ 163.2 (C-S), δ 162.6 (C=N), δ 150.1 (C=O), δ
112.5 (C2), δ 145.7 (C3), δ 126.3 (C4), δ 121.6 (C5), δ 126.3 (C6), δ 121.1 (C7), δ 139.1
(C8), δ 137.6 (C9), δ 18.1 (-CH3), δ 56.1 (OMe), δ 22.0 (-NH-CH3), δ 127.4-133.6 (PPh3).
Needle shaped, transparent red colour crystals were obtained by recrystallization of
4.3.3. Synthesis of [Ru(8MAC-etsc)(CO)(PPh3)2] (3)
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complex 2 in dichloromethane and methanol mixture.
Yield: 66 %. Mp.146 °C. Anal. calcd. for C52H45N3O4P2RuS: C, 64.31; H, 4.68; N, 4.32; S,
3.30. Found: C, 63.25; H, 4.60; N, 4.27; S, 3.26 %. FT-IR (ν, cm−1) in KBr: ν(C=O lactone)
3
−1
1434, 1090, 696 (for PPh3). UV-Vis
−1
SC
1687, ν(C=N) 1590, ν(C-S) 752, ν(C≡O) 1938,
(DMSO), λmax (ε): 245 (77,054) nm (dm mol cm ) (Intraligand transition); 318 (43,524) nm
(dm3mol−1cm−1) (LMCT s→d). 1H NMR (400 MHz, DMSO-d6, δ ppm, J Hz): δ 7.13-7.63
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(m, 31H, Ar-H), δ 6.72-6.75 (t, 1H, J=7.2, C6-H), δ 6.89-6.91 (d, 1H, J=7.6, C7-H), δ 3.77 (s,
3H,-OCH3), δ 1.88 (s, 3H,-CH3), δ 6.39-6.68 (t, 1H, terminal -NH), δ 2.21-2.36 (m, 2H,
J=4.8,-NH-CH2), δ 0.70-0.73 (t, 3H, J=7.2,-CH2-CH3). 13C NMR (100 MHz, DMSO-d6, δ
ppm): δ 203.4 (C≡O), δ 165.8 (C-S), δ 163.2 (C=N), δ 153.1 (C=O), δ 112.4 (C2), δ 145.7
(C3), δ 129.5 (C4), δ 127.4 (C5), δ 127.9 (C6), δ 121.1 (C7), δ 139.5 (C8), δ 130.5 (C9), δ
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18.0 (-CH3), δ 56.1 (OMe), δ 31.1 (-NH-CH2), 22.0 (terminal -CH3), δ 128.3-133.6 (PPh3).
4.3.4. Synthesis of [Ru(8MAC-ptsc)(CO)(PPh3)2] (4)
Yield: 67%. Mp. 193 °C. Anal. calcd. for C56H45N3O4P2RuS: C, 65.99; H, 4.45; N, 4.12; S,
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3.14. Found: C, 65.01; H, 4.37; N, 4.05; S, 3.09 %. FT-IR (ν, cm−1) in KBr: ν(C=O lactone)
1682, ν(C=N) 1598, ν(C-S) 743, ν(C≡O) 1921, 1432, 1089, 694 (for PPh3). UV-Vis (DMSO),
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λmax (ε): 250 (99,515) nm (dm3mol−1cm−1) (Intraligand transition); 279 (80,102) nm
(dm3mol−1cm−1) (Intraligand transition); 397 (37,552) nm (dm3mol−1cm−1) (LMCT s→d). 1H
NMR (400 MHz, DMSO-d6, δ ppm, J Hz): δ 6.96-7.34 (m, 36H, Ar-H), δ 6.69-6.78 (m, 2H,
C6-H and C7-H), δ 3.80 (s, 3H,-OCH3), δ 1.99 (s, 3H,-CH3), δ 8.49 (s, 1H, terminal -NH).
13
C NMR (100 MHz, DMSO-d6, δ ppm): δ 208.6 (C≡O), δ 166.4 (C-S), δ 166.0 (C=N), δ
153.1 (C=O), δ 112.9 (C2), δ 145.8 (C3), δ 1296.4 (C4), δ 120.2 (C5), δ 121.3 (C6), δ 118.5
(C7), δ 141.3 (C8), δ 139.2 (C9), δ 18.6 (-CH3), δ 56.1 (OMe), δ 127.5-133.4 (PPh3). Single
crystals suitable for X-ray diffraction studies were obtained by recrystallisation of complex 4
in dichloromethane and methanol solution.
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4.4. Biomolecular interaction studies
The stability of the complexes was performed in 1 % aqueous DMSO, phosphate
buffer–DMSO (99:1) and Tris-HCl–DMSO (99:1). The stability was analyzed by monitoring
the electronic spectra over 24 h at room temperature on a JASCO 4100 spectrophotometer.
DNA binding studies, DNA viscosity studies, DNA cleavage experiments and protein
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binding studies have been carried out according to the method described in our earlier reports
[26,34,59].
4.5. In vitro antioxidant assays
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The DPPH radical scavenging assay of the compounds has been done according to the
reported method [67]. In this study, various concentrations of the experimental standard
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ascorbic acid, [RuHCl(CO)(PPh3)3] (20-100 µM) and complexes (2-10 µM) in methanol were
taken. Total antioxidant activity of the compounds was determined by the phosphomolybdate
method [68].
4.6. In vitro antimicrobial assay
Antimicrobial activities of the compounds were evaluated by agar well diffusion
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method [69] as reported, by taking Staphylococcus aureus, Streptococcus pneumonie,
Pseudomonas aeruginosa, Salmonella paratyphi and fungus such as Candida albicans,
Trichophyton rubrum, Aspergillus niger, Aspergillus fumigatus and Candida tropicalis.
Gentamicin and Ketaconazole were used as positive controls to study the antibacterial and
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antifungal activities respectively. The antimicrobial activity of the test compounds was
checked with various concentrations (25 µg/ml, 50 µg/ml and 100 µg/ml) against all the test
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pathogens. Each experiment was performed in triplicate and the results are represented as
average zone of inhibition and minimum inhibitory concentration of all the test pathogens.
4.7. Cytotoxicity studies
Cytotoxic activity of the compounds was tested with human lung cancer cell lines
A549, human breast cancer cell lines MCF-7 and human normal keratinocyte cells (HaCaT)
by using MTT assay, which was done according to the earlier literature methods [70] and IC50
values obtained from nonlinear regression using GraphPad Prism 5 [71]. The LDH release
[72] and NO release [73] assays of the compounds was evaluated by the earlier reported
methods.
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ACKNOWLEDGEMENT
The author G.K. greatly acknowledge DST, New Delhi, India for INSPIRE fellowship
(IF140225 dated 23.01.2014). The author S.D greatly acknowledged UGC, New Delhi, India
for UGC-BSR fellowship (F.25-1/2014-15(BSR)/7-26/2007(BSR) dated 05.11.2015).
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Supporting information
Crystallographic data for the complex 1, 2 and 4 have been deposited at the Cambridge
Crystallographic centre as supplementary publication [1570688 (1), 1570689 (2) and
(4)].
The
data
can
be
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w.w.w.ccdc.cam.ac.uk/conts/retrieving.html/
obtained
free
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1570690
of
charge
at
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HIGHLIGHTS
Four new organoruthenium(II) complexes have been synthesized and characterized
The DNA/protein interactions of ligands and complexes were studied by a variety of
techniques
The antimicrobial activity against four different bacteria and five fungi have also been
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examined
The antiproliferative activity was evaluated against MCF-7 and HeLa cell lines
The complexes 1-4 showed potent anticancer activity over the standard drug,
Cisplatin
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Assay on HaCaT cell lines showed that the compounds were non-toxic to those cells.