An investigation on 3-acetyl-7-methoxy-coumarin Schiff bases and their Ru(ii) metallates with potent antiproliferative activity and enhanced LDH and NO release

Article abstract of DOI:10.1039/c7ra12104k

New cyclometallated ruthenium(ii) complexes of 3-acetyl-7-methoxycoumarin-4N-substituted thiosemicarbazones were synthesized and characterized by analytical and spectral techniques. The crystal structures of the ligands H₂L^1-3 and complexes (1, 2 and 4) were confirmed by X-ray crystallography. The analysis showed that the ligands have undergone C-H activation at the C(4) carbon of the pyrone ring and acted in a tridentate fashion by binding through C, N and S atoms. CT-DNA and protein (BSA/HSA) binding studies were carried out to analyze their interaction with biomolecules. Good binding affinity with DNA was observed with intercalative binding mode, which was further confirmed by EB displacement and viscosity measurement studies. The quenching mechanism with BSA/HSA was found to be static. Three dimensional (3D) fluorescence measurements were carried out to validate the micro environmental changes in the serum albumins. Their antioxidant propensity and antimicrobial study insisted that the compounds displayed good spectrum of activity. Evaluation of their anticancer potential against MCF-7 (human breast cancer) and A549 (human lung carcinoma) cell lines revealed that the complexes exhibited better activity than the ligands and cisplatin. Further, the results of LDH and NO release assays supported the cytotoxic nature of the compounds. The non-toxic nature of the compounds was established by testing against the non-cancerous cell line HaCaT (human normal keratinocyte).

Full text of DOI:10.1039/c7ra12104k

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An investigation on 3-acetyl-7-methoxy-coumarin
Schiff bases and their Ru(II) metallates with potent
antiproliferative activity and enhanced LDH and NO
release†
Cite this: RSC Adv., 2018, 8, 1539
G. Kalaiarasi,a S. Rex Jeya Rajkumar,b S. Dharani,a J. G. Małeckic
a
*
and R. Prabhakaran
New cyclometallated ruthenium(II) complexes of 3-acetyl-7-methoxycoumarin-4N-substituted
thiosemicarbazones were synthesized and characterized by analytical and spectral techniques. The
crystal structures of the ligands H2L1–3 and complexes (1,
2 and 4) were confirmed by X-ray
crystallography. The analysis showed that the ligands have undergone C–H activation at the C(4) carbon
of the pyrone ring and acted in a tridentate fashion by binding through C, N and S atoms. CT-DNA and
protein (BSA/HSA) binding studies were carried out to analyze their interaction with biomolecules. Good
binding affinity with DNA was observed with intercalative binding mode, which was further confirmed by
EB displacement and viscosity measurement studies. The quenching mechanism with BSA/HSA was
found to be static. Three dimensional (3D) fluorescence measurements were carried out to validate the
micro environmental changes in the serum albumins. Their antioxidant propensity and antimicrobial
study insisted that the compounds displayed good spectrum of activity. Evaluation of their anticancer
potential against MCF-7 (human breast cancer) and A549 (human lung carcinoma) cell lines revealed that
the complexes exhibited better activity than the ligands and cisplatin. Further, the results of LDH and NO
release assays supported the cytotoxic nature of the compounds. The non-toxic nature of the
compounds was established by testing against the non-cancerous cell line HaCaT (human normal
keratinocyte).
Received 3rd November 2017
Accepted 17th December 2017
DOI: 10.1039/c7ra12104k
rsc.li/rsc-advances
already reported that the cytotoxicity of thiosemicarbazones is
mostly related with their parent aldehyde or ketone, metal
chelation efficacy and terminal amino substitution.11,12
Introduction
Thiosemicarbazides are a versatile class of compounds with
indispensable properties such as antitumor, antifungal, anti-
bacterial, antiviral, antiparasitic, etc.1–6 They are N, S donor
ligands whose activity can be greatly enhanced by the presence
of additional donor sites and a variety of coordination modes
can be shown by these systems.7–9 Potential ligands are formed
by attaching thiosemicarbazides with carbonyl compounds
having a heterocyclic moiety. A good deal of research can be
possible on such ligands and their metal complexes.10 It is
Coumarin is one among the natural products found extensively
in plants, which exhibits various pharmacological activities.13,14
Coumarin derived antibiotics such as novobiocin, clorobiocin
and coumermycin A1 are commercialized.15,16 Their ability to
inhibit human immunodeciency virus integrase made them to
be analysed in the treatment of HIV.17 Reports are available for
testing coumarin derivatives against various tumor18 and
neuronal cell lines.19 Generation of satisfactory clinical
compounds is one of the promising approaches to attain
effective and less toxic chemodrugs. The present scenario of
inorganic research faces difficulties in raising highly active
metal based drugs, the main criteria of which being less toxic
and target specic. Among others, cancer stands as the most
threatening disease and consistent attempts are made to
develop appropriate chemotherapeutic drugs. Successful
application of cisplatin as an anticancer agent provoked the
chemists to search for other active metal complexes.20,21
Recently, ruthenium complexes have come into the lime light
since two such complexes namely NAMI-A22 and KP1019 23
aDepartment of Chemistry, Bharathiar University, Coimbatore 641 046, India. E-mail:
rpnchemist@gmail.com; Fax: +91-422-2422387; Tel: +91-422-2428319
bDepartment of Biosciences and Technology, Karunya University, Coimbatore 641 114,
India
cDepartment of Crystallography, Silsian University, Szkolna 9, 40-006 Katowice,
Poland
† Electronic supplementary information (ESI) available: Crystallographic data for
the ligands H2L1–3 and complex 1, 2 and 4. CCDC 1580118 (H2L1), 1580119 (H2L2),
1580120 (H2L3), 1580121 (1), 1580122 (2) and 1580123 (4). For ESI and
crystallographic data in CIF or other electronic format see DOI:
10.1039/c7ra12104k
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entered into clinical trials, attesting the efficiency of ruthenium dichloromethane, toluene, methanol, DMSO and DMF. By
in the treatment of cancer. Moreover, exhibition of diverse using UV-visible spectroscopic techniques, the stability of the
coordination modes, stable oxidation states (from ꢀ2 to 8) and compounds in aqueous solutions was conrmed (Fig. S1 in the
mimicking iron in binding biomolecules makes ruthenium ESI†). The spectra recorded immediately and aer 24 h did not
a better alternative for platinum.24 Sufficient number of articles show any appreciable change in the intensity and the position
are available in the literature reporting the anticancer efficiency of the bands.
of ruthenium complexes including those by Garza-Ortiz et al.,25
and Juinn Chow et al.26
Assignments of selected characteristic IR band positions
provided signicant indication for the formation of 3-acetyl-7-
Considering the foregoing facts and our continuous inves- methoxycoumarin-4(N)-substituted thiosemicarbazone ligands
tigation on thiosemicarbazone complexes of various transition and their ruthenium complexes (Fig. S2–S10†). A band in the
metals, herein we report the synthesis, spectral characteriza- region 1600–1644 cmꢀ1 is assigned to the n(C]N),27 whereas the
tion, X-ray diffraction analysis, DNA/protein binding, antioxi- shiing of this band to 1606–1607 cmꢀ1 revealed the coordina-
dant, antimicrobial and anticancer studies of 3-acetyl-7- tion of azomethine nitrogen atom to the metal ion.28,29 A band
methoxycoumarin-4N-substituted thiosemicarbazones and appeared at 826–835 cmꢀ1 in the ligands due to vibration of the
their cyclometallated ruthenium(II) complexes.
C]S group, which disappeared in the spectra of the complexes
and a new band corresponding to the C–S group appeared at 744–
745 cmꢀ1 indicating that the other coordination is through thi-
olate sulphur aer enolization followed by deprotonation.30 In all
the complexes, terminally coordinated carbonyl group appeared
Results and discussion
Synthesis and characterization
.
as a strong band in the region 1918–1925 cmꢀ1 29,30 The stretch-
The ligands H2L1–4 were synthesized by the reaction of 3-acetyl-
7-methoxy-2H-chromene-2-one with 4(N)-substituted thio-
semicarbazides in methanol, which was precipitated as yellow
solid from the reaction mixture. Reacting an equimolar mixture
of these Schiff base ligands with [RuHClCO(PPh3)3] in benzene
under reux for 7 h afforded a reddish orange solution
(Scheme 1). Characterization of the ligands and complexes were
done by using elemental analyses, infrared spectroscopy, UV-Vis
and NMR spectroscopy. The structures of the ligands H2L1–3
and complexes (1, 2 and 4) were conrmed by X-ray crystallo-
graphic study. X-ray crystal structural determination clearly
showed that the ligands have undergone C–H activation at the
ortho position of H3C–C]N and acted in a tridentate, bianionic
manner binding through CNS atoms. Both the ligands and
complexes are air stable and soluble in ethanol, chloroform,
ing frequencies of triphenylphosphine group were observed
around 1404–1434, 1087–1090, 694–696 cmꢀ1
.
30,31
The electronic spectra of the ligands H2L1–4 and complexes
1–4 were recorded in DMSO. In the spectra of the free ligands,
the higher energy absorption bands appeared around 275–
282 nm have been assigned to (p / p*) transitions and the
bands observed at 352–356 nm have been assigned to (n / p*)
transitions.32 In the complexes 1–4, the high energy absorption
band observed in the region 268–337 nm has been assigned to
intra ligand transitions, the lowest energy band in the region
357–380 nm has been attributed to the ligand to metal charge
transfer (LMCT) transitions.7
The 1H NMR spectra of 3-acetyl-7-methoxy-coumarin, 3-
acetyl-7-methoxy-coumarin-4(N)-substituted
Scheme 1 Synthesis of the ligands H2L1–4 and their new ruthenium(II) complexes.
1540 | RSC Adv., 2018, 8, 1539–1561
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thiosemicarbazone ligands H2L1–4 and their Ru(II) complexes renement of the ligands is listed in Table 1 and selected inter
(1–4) showed the signals in the expected regions (Fig. S11–S19†). atomic distances and bond angles are summarized in Table S1.†
The singlets that appeared at d 10.26–10.77 ppm were assigned The structure of the ligand H2L1 together with the atom label-
to the hydrazinic proton of the free ligands, these signals were ling scheme is shown in Fig. 1. The ligand H2L1 crystallized in
ꢀ
absent in the complexes (1–4), supporting the enolization and the triclinic system with P1 space group. The monomeric units
deprotonation of the NH–C]S group upon coordination of the of the ligand H2L1 were arranged in a dimeric manner by the
thiolate sulfur to the Ru(II) ion.7,30 The singlet due to the N]C– weaker N(1)–H(1)/O(1) hydrogen bonds (Fig. S20, Table S2†).
2
ꢀ
CH3 proton appeared at d 2.24–2.33 ppm in the spectra of free The ligand H2L crystallized in the trigonal system with P3 space
ligand, which underwent an upeld shi in the spectra of the group. Six crystallographically independent molecules were
complexes to d 1.88–2.09 ppm, suggesting that the coordination present in the unit cell. In ligand H2L2, we found the donor–
occurred via the nitrogen atom of N]C–CH3 group.29,33,34 The acceptor distance as 3.013 A corresponding to the N(1)–O(1) and
˚
spectra of the ligands H2L1–4 displayed a singlet at d 7.98– O(1)–N(1) bond between the two molecules (Fig. 2). This inter-
8.45 ppm, which is corresponding to the hydrogen atom at C(4) action gave a pseudo bimolecular appearance to the ligand H2L2
carbon, however, for complexes 1–4 no resonance could be (Fig. S21†). The X-ray single crystal structure of the ligand H2L3
attributed to C4(H), which indicated that the C(4) carbon atom is shown in Fig. 3. From the symmetry of the reections and
of the pyrone ring is coordinated to the metal aer deprotona- solution of the structures, it is clear that the crystals belong to
ꢀ
tion. In addition, the spectra of all the ligands and complexes the triclinic P1 space group. H-Bonding in this structure is
exhibited a series of signals for aromatic protons at d 6.29– distinctly different from that observed in H2L1 and H2L2. The
7.74 ppm 7,30 and a singlet for –OCH3 protons around d 3.74– ligand H2L3 involved an intramolecular dipolar interaction
3.93 ppm.35
between coumarin oxygen (O2) and azomethine nitrogen (N3)
atoms as shown in Fig. S22 (Table S2†).
The existence of thione form in the ligands is conrmed by
X-ray crystallography
˚
˚
the C]S bond lengths, which are of 1.696 (3) A, 1.677 (3) A and
Crystal structure of the ligands H2L1–3. The molecular
structures of the ligands H2L1–3 have been determined by single-
crystal X-ray diffraction studies. A summary of the structure
1
2
3
˚
1.671 (4) A for H2L , H2L and H2L respectively and the bond
1
˚
length of C]N group (C(2)–N(3) ¼ 1.289(3) A for H2L , C(3)–
Table 1 Crystallographic data of ligands H2L1, H2L2 and H2L3
Identication code
Empirical formula
Formula weight
[H2-7MAC-tsc]
C13H13N3O3S$CH3OH
323.36
[H2-7MAC-mtsc]
C14H15N3O3S
305.35
[H2-7MAC-etsc]
C15H17N3O3S
319.38
Temperature
Wavelength
295(2) K
0.7107 A
295(2) K
0.7107 A
295(2) K
0.7107 A
˚
˚
˚
Crystal system
Space group
Triclinic
P1
Trigonal
P3
Triclinic
P1
ꢀ
ꢀ
ꢀ
Unit cell dimensions
a
B
C
˚
˚
˚
8.3702 (6) A
17.5595 (9) A
7.5260 (6) A
˚
˚
˚
8.8979(8) A
17.5595 (9) A
8.0281 (6) A
˚
˚
˚
12.4669 (9) A
97.411 (7)ꢁ
8.6657 (7) A
13.5383 (10) A
a
90ꢁ
89.670 (6)ꢁ
87.437 (6)ꢁ
67.782 (7)ꢁ
b
106.008 (6)ꢁ
90ꢁ
g
113.670 (8)ꢁ
120ꢁ
3
3
˚
3
˚
˚
Volume
786.53 (12) A
2314.0 (3) A
756.43 (11) A
Z
2
6
2
Density
Absorption coefficient
F(000)
1.365 mg mꢀ3
1.315 mg mꢀ3
1.402 mg mꢀ3
0.227 mmꢀ1
340
0.223 mmꢀ1
960
0.230 mmꢀ1
336
Crystal size
0.02 ꢂ 0.08 ꢂ 0.34 mm
0.06 ꢂ 0.08 ꢂ 0.31 mm
0.13 ꢂ 0.14 ꢂ 0.38 mm
Crystal shape
q range for data collection
Limiting indices
Needle
Needle
Prism
4.223 to 27.424ꢁ
ꢀ11 # h # 11, ꢀ12 # k # 12,
ꢀ16 # l # 17
4.003 to 25.505ꢁ
ꢀ22 # h # 24, ꢀ24 # k # 23,
ꢀ7 # l # 11
4.100 to 28.715ꢁ
ꢀ10 # h # 10, ꢀ11 # k # 10,
ꢀ17 # l # 17
2619
Reections collected
Independent reections
Completeness to q
2480
2007
3726 (R(int) ¼ 0.0307)
3576 (R(int) ¼ 0.0321)
3626 (R(int) ¼ 0.0427)
26.32ꢁ
26.32ꢁ
26.32ꢁ
Absorption correction
Renement method
Data/restraints/parameters
Goodness-of-t on F2
Final R indices [I > 2s(I)]
R indices (all data)
Multi-scan
Multi-scan
Multi-scan
Full-matrix least-squares on F2
3726/0/215
Full-matrix least-squares on F2
3576/0/201
Full-matrix least-squares on F2
3626/0/203
1.042
1.007
1.055
R1 ¼ 0.0841, wR2 ¼ 0.1760
R1 ¼ 0.0553, wR2 ¼ 0.1094
R1 ¼ 0.0889, wR2 ¼ 0.2249
R1 ¼ 0.0984, wR2 ¼ 0.2032
R1 ¼ 0.1141, wR2 ¼ 0.1298
R1 ¼ 0.1151, wR2 ¼ 0.2422
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Fig. 1 ORTEP diagram of [H2-7MAC-tsc] (H2L1).
Fig. 2 ORTEP diagram of [H2-7MAC-mtsc] (H2L2).
2
3
˚
˚
N(3) ¼ 1.285(3) A for H2L , C(4)–N(3) ¼ 1.273(5) A for H2L ) is in
agreement with a formal C]N bond length.36 In the ligands
H2L1–3, the thione sulfur atom S(1) is trans to the N(3) nitrogen
atom of C]N group about C(1)–N(2) bond, this structural
arrangement corresponds to E-isomer, which is conrmed by
a torsion angle of S(1)–C(1)–N(2)–N(3) bond, 179.6(2) for H2L1,
178.1(2) for H2L2 and 176.6(3) for H2L3. The bond distances in
the 3-acetyl-7-methoxy-4(N)-thiosemicarbazones H2L1–3 agree
Crystal structure description of new Ru(II) complexes. The
molecular structures of the complexes (1, 2 and 4) have been
determined by single crystal X-ray crystallographic studies to
conrm the coordination modes of the 3-acetyl-7-methoxy-2H-
chromene-2-one 4(N)-substituted thiosemicarbazones in the
complexes. The summary of the data collection and the
renement parameters have been given in Table 2 whereas
selected bond lengths and bond angles are given in Table S3.†
The ORTEP view along with the atomic numbering scheme of
complexes 1, 2 and 4 are given in Fig. 4–6. The X-ray crystal
structures revealed that the complexes 1, 2 and 4 crystallized in
well
with
the
values
observed
for
other
thiosemicarbazones.27,37,38
Fig. 3 ORTEP diagram of [H2-7MAC-etsc] (H2L3).
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Table 2 Crystallographic data of complexes 1, 2 and 4
Identication code
Empirical formula
Formula weight
[Ru(7-MAC-tsc)(CO)(PPh3)2] (1)
C50H41N3O4P2RuS$CH3OH
974.97
[Ru(7-MAC-mtsc)(CO)(PPh3)2] (2)
C51H43N3O4P2RuS
956.95
[Ru(7-MAC-ptsc)(CO)(PPh3)2] (4)
C56H45N3O4P2RuS
1019.02
Temperature
Wavelength
295(2) K
0.7107 A
295(2) K
0.7107 A
295(2) K
0.7107 A
˚
˚
˚
Crystal system
Space group
Triclinic
P1
Triclinic
P1
Triclinic
P1
ꢀ
ꢀ
ꢀ
Unit cell dimensions
a
B
C
˚
˚
˚
13.0569 (5) A
12.2016 (7) A
13.3038 (5) A
˚
˚
˚
13.7232 (5) A
13.4519 (7) A
13.4870 (5) A
˚
˚
˚
13.8479 (5) A
15.1541 (9) A
14.3074 (5) A
a
89.094 (3)ꢁ
75.301 (3)ꢁ
70.576 (3)ꢁ
82.857 (4)ꢁ
79.550 (5)ꢁ
65.934 (5)ꢁ
85.881 (5)ꢁ
85.582 (5)ꢁ
69.949 (3)ꢁ
b
g
3
˚
3
˚
3
˚
Volume
2257.10 (15) A
2229.9 (2) A
2401.56 (16) A
Z
2
2
2
Density
Absorption coefficient
F(000)
1.435 mg mꢀ3
1.425 mg mꢀ3
1.409 mg mꢀ3
0.516 mmꢀ1
1004
0.520 mmꢀ1
984
0.487 mmꢀ1
1048
Crystal size
0.02 ꢂ 0.15 ꢂ 0.24 mm
0.08 ꢂ 0.22 ꢂ 0.26 mm
0.01 ꢂ 0.13 ꢂ 0.31 mm
Crystal shape
q range for data collection
Limiting indices
Plate
Plate
Plate
3.793 to 28.999ꢁ
ꢀ15 # h # 17, ꢀ17 # k # 18,
ꢀ14 # l # 18
8354
3.605 to 28.757ꢁ
ꢀ15 # h # 16, ꢀ17 # k # 18,
ꢀ18 # l # 19
7740
3.857 to 29.163ꢁ
ꢀ17 # h # 17, ꢀ18 # k # 18,
ꢀ19 # l # 15
8210
Reections collected
Independent reections
Completeness to q
10 636 (R(int) ¼ 0.0334)
10 577 (R(int) ¼ 0.067)
11 129 (R(int) ¼ 0.0376)
26.32ꢁ
26.32ꢁ
26.32ꢁ
Absorption correction
Renement method
Data/restraints/parameters
Goodness-of-t on F2
Final R indices [I > 2s(I)]
R indices (all data)
Multi-scan
Multi-scan
Multi-scan
Full-matrix least-squares on F2
10 636/0/580
Full-matrix least-squares on F2
10 577/0/562
Full-matrix least-squares on F2
11 129/0/610
1.034
1.092
1.016
R1 ¼ 0.0390, wR2 ¼ 0.0833
R1 ¼ 0.0839, wR2 ¼ 0.2024
R1 ¼ 0.0425, wR2 ¼ 0.0824
R1 ¼ 0.0582, wR2 ¼ 0.0924
R1 ¼ 0.1085, wR2 ¼ 0.2330
R1 ¼ 0.0689, wR2 ¼ 0.0916
the triclinic P1 space group. In the complexes, the ligands P(1)–Ru(1)–C(1) 89.41(8)ꢁ for complex 1, 88.3(1)ꢁ for complex 2,
coordinated to the ruthenium ion through the N(1) nitrogen, 90.17(8)ꢁ for complex 4, are smaller than bond angle of P(1)–
pyrone carbon C(3) and thiolate sulphur atoms, forming two Ru(1)–N(1) ¼ 90.25(6)ꢁ for complex 1, P(1)–Ru(1)–N(1) ¼
ve member chelate rings with a bite angle N(1)–Ru(1)–S(1) of 93.3(1)ꢁ for complex 2, P(1)–Ru(1)–N(1) ¼ 90.94(6)ꢁ for
78.39(6)ꢁ for complex 1, 78.90(1)ꢁ for complex 2, 79.38(6)ꢁ for complex 4, and P(2)–Ru(1)–N(1) ¼ 93.40(6)ꢁ for complex 1,
complex 4, and a bite angle of C(5)–Ru(1)–N(1) of 78.58(8)ꢁ for P(2)–Ru(1)–N(1) ¼ 89.2(1)ꢁ for complex 2, P(2)–Ru(1)–N(1) ¼
complex 1, 78.10(2)ꢁ for complex 2, C(13)–Ru(1)–N(1) of 89.94(6)ꢁ for complex 4. The observed bond distances of Ru–P
78.18(9)ꢁ for complex 4. The fourth site is occupied by the are comparable with those found in other reported ruthenium
carbon atom of the carbonyl group to form a CNSC square- complexes containing triphenylphosphine.7,29,30,39–41 The bond
plane. The carbonyl group occupied the site trans to the N1 distances of Ru(1)–P(1) and Ru(1)–P(2) are comparatively
nitrogen, which is conrmed from the bond angle N(1)–Ru(1)– longer than those observed for basal planar bonds, such as
ꢀ
C(1) of 179.0(1)ꢁ for complex 1, 177.5(2)ꢁ for complex 2, and Ru(1)–N(1) [2.077–2.097 A], Ru(1)–Cpyrone [2.062–2.077 A] and
˚
˚
178.10(2)ꢁ for complex 4 and bond length of Ru(1)–C(1) Ru(1)–C(1) [1.851–1.87 A]. Due to the variation in the bond
˚
˚
˚
distances of 1.846(2) A for complex 1, 1.853(4) A for complex 2 length and bond angles, ruthenium(II) ion sitting in a CNOSP2
˚
and 1.848(2) A for complex 4 found similar to the reported coordination environment and adopted a distorted octahedral
complexes.29 The remaining axial coordination sites are lled geometry. The selected bond distances of complexes (1, 2 and
up by phosphorous atoms of two triphenylphosphine ligands, 4) such as Ru–P, Ru–O, Ru–S, Ru–N and Ru–C and bond angles
which are mutually trans to each other with Ru(1)–P(1) and agree very well with the similar reported ruthenium(II)
Ru(1)–P(2) distances of 2.379(8) A and 2.376(8) A for complex 1, complexes.7,29–31,37,39–41
˚
˚
˚
˚
˚
2.365(1) A and 2.383(1) A for complex 2 and 2.376(8) A and
While dealing with the hydrogen-bonding interactions, in
˚
2.376(8) A for complex 4 and are slightly bent towards the the complexes 2 and 4, we found the donor–acceptor distance
˚
carbonyl group due to the steric requirements of somewhat (2.954 for (2), 3.054 A for (4)) corresponding to the O(4)–O(4)
bulky chelating ligand, causing a slight deviation from a linear bond between the carbonyl oxygen atom of the rst molecule
trans arrangement, which is evident from the bond angle of and with the second molecule. This interaction gave a pseudo
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Fig. 4 ORTEP diagram of [Ru(7MAC-tsc)CO(PPh3)2] (1).
Fig. 5 ORTEP diagram of [Ru(7MAC-mtsc)CO(PPh3)2] (2).
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Fig. 6 ORTEP diagram of [Ru(7MAC-ptsc)CO(PPh3)2] (4).
binuclear structural appearance to the complex (2) and (4) binding constants Kbin were obtained according to eqn (S1).†
(Fig. S23–S24; Table S2†).
The binding constant value was calculated from the plot of
[DNA]/(3a ꢀ 3f) versus [DNA] and the data were given in Table 3
(Fig. 8). From the results, the intrinsic DNA-binding constants
Kbin were found to be in the order Complex 3 > Complex 2 >
DNA binding studies
UV-Vis absorption spectral titrations. DNA-binding studies Complex 1 > Complex 4 > H2L3 > H2L2 > H2L1 > H2L4. This may
are important for the rational design and construction of new be due to the presence of different substituents in the terminal
and more efficient drugs targeted to DNA.42 The binding affinity nitrogen atom of the ligands. These results are comparable with
of the ligands and their organoruthenium(II) complexes with CT- earlier reports describing the intercalative mode of various
DNA can be measured by using UV-Vis spectroscopy. The UV-Vis ruthenium intercalators.30,31,44
absorption spectra of the free ligands and their complexes in the
EB-DNA quenching studies. The competitive binding exper-
absence and presence of CT DNA are given in Fig. S25† and 7. In iments were carried out on the EB–CT-DNA system by varying
the presence of DNA, the absorption bands at about 345–349 nm the concentrations of the ligands and complexes to get further
for the ligands H2L1–4 exhibited hypochromism of about 41.46– information about the binding mode of compounds with DNA.
48.41% accompanied by a small red shi. When the concentra- In our studies, it was noted that upon concomitant addition of
tion of DNA is increased, all the new organoruthenium(II) the compounds to the EB-DNA system, emission intensity
complexes (1–4) showed a decrease in absorbance in the charge decreased progressively, indicative of competition between EB
transfer band at 330–348 nm to the extent of about 48.91–53.07% and compounds towards CT-DNA in binding/chelation
with a red-shi (bathochromic shi) of 2–8 nm. The decrease in (Fig. S26†). The reduction of the uorescence emission inten-
absorbance with increase in concentration of CT-DNA may be sity gives criteria to investigate the DNA binding propensity of
due to the decrease in transition probabilities as a result of the compounds and stacking interaction (intercalation)
partial transfer of electrons from the p orbital of the DNA base between the adjacent DNA base pairs.45 As shown in Fig. S26,†
pairs to the coupled p* orbital of the coordinated Schiff base to the uorescence intensity of EB-DNA gradually reduced with
metal due to overlapping.43 The extent of hypochromism in the increasing concentrations of compounds indicating that the
charge transfer band is an indication of the strength of inter- metal complexes bound to DNA by competing with EB. As the
calative interaction.43 The spectral characteristics obviously concentration of the compounds increased from 10–100 mM,
indicated that both the ligands and metal complexes interacted the emission band of DNA-bound EB exhibited quenching upto
with DNA most likely through an intercalation mode involving 19.98, 24.07, 28.36, 23.38, 27.83, 36.67, 36.99 and 28.92% of the
a stacking interaction between the aromatic chromophore and initial uorescence intensity together with a red shi of 2–4 nm
base pairs of DNA.
for H2L1, H2L2, H2L3, H2L4, complexes 1, 2, 3 and 4 respectively.
In order to compare the DNA-binding affinities of the ligands This provides a direct evidence for the intercalative binding
and their metal complexes quantitatively, their intrinsic mode of the compounds with DNA.
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Fig. 7 Absorption titration spectra of complexes (1–4) with increasing concentrations (2.5–25 mM) of CT-DNA (Tris–HCl buffer, pH 7.2).
Further quantitative measurement of the magnitude of versus [Q] (Fig. 9) and given in Table 3. The experimental results
interaction was ascertained by the classical Stern–Volmer showed that all the Ru(II) complexes bind to DNA more strongly
equation. Quenching constant KSV is used to evaluate the than their free ligands and the quenching constant value
quenching efficiency and is obtained from the slope of Io/I increased in the order 3 > 2 > 1 > 4 which also validated the
electronic absorption spectral results. Further, the calculated
KSV values of the compounds are signicant when compared to
the reported values.30,31
Table 3 The binding constant (Kbin) and quenching constant (KSV
)
Viscosity measurements. Viscosity measurements are sensi-
tive to changes of DNA length and are regarded as one of the
most effective test for the binding mode of the compounds with
DNA.46,47 In order to conrm the binding modes of the free
ligands and their Ru(II) complexes with CT-DNA, viscosity
measurement study was carried out. The effects of the ligands
and metal complexes on the relative viscosity of CT DNA are
given in Fig. 10. From Fig. 10, it is obvious that the specic
viscosity of the DNA sample increases with the addition of the
compounds. 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 site,
resulting an increase in the overall DNA length48,49 and the
values for the interaction of the ligands H2L1–4 and complexes (1–4)
with CT-DNA
Binding constant
Kbin (Mꢀ1
Quenching constant
KSV (Mꢀ1
Compounds
)
)
H2L1
2.1246 ꢃ 0.308 ꢂ 105
3.4346 ꢃ 0.306 ꢂ 105
7.1852 ꢃ 0.304 ꢂ 105
2.1058 ꢃ 0.288 ꢂ 105
1.2544 ꢃ 0.304 ꢂ 106
1.3958 ꢃ 0.320 ꢂ 106
1.4428 ꢃ 0.279 ꢂ 106
1.0636 ꢃ 0.334 ꢂ 106
2.79 ꢃ 0.005 ꢂ 103
3.14 ꢃ 0.002 ꢂ 103
3.95 ꢃ 0.009 ꢂ 103
2.91 ꢃ 0.005 ꢂ 103
4.16 ꢃ 0.002 ꢂ 103
5.52 ꢃ 0.009 ꢂ 103
5.62 ꢃ 0.003 ꢂ 103
4.00 ꢃ 0.002 ꢂ 103
H2L2
H2L3
H2L4
Complex 1
Complex 2
Complex 3
Complex 4
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degree of viscosity may be depending upon the substitution on
N-terminal nitrogen of the ligands and the increasing order of
viscosity of CT-DNA by the compounds is complex 3 > complex 2
> complex 1 > complex 4 > H2L3 > H2L2 > H2L1 > H2L4, which is
consistent with the above experimental results.
On the basis of above spectroscopic studies along with the
viscosity measurements, it is revealed that the ligands and their
organoruthenium(II) complexes can bind to CT DNA via an
intercalative mode and the new Ru(II) complexes bind to CT
DNA strongly than their free ligands alone.
DNA cleavage activity. The newly synthesized Schiff base
ligands H2L1–4 and their cyclometallated ruthenium(II)
complexes (1–4) were studied for their DNA cleavage activity by
the method of agarose gel electrophoresis against supercoiled
pBR322 DNA as the substrate, in the absence of external addi-
tives in a medium of 5 mM Tris–HCl/50 mM NaCl buffer (pH
7.2). The change in the DNA structure from supercoiled form to
nicked or linear form produces change in the extent of migra-
tion in the gel. Moreover, one strand cleavage occurring in SC
form will reduce to produce a nicked circular form (NC), which
is a slower-moving form. If both strands are cleaved, linear
circular (LC) form will be generated which migrates between SC
and NC forms.50 For comparison purposes, plasmid DNA was
incubated in presence of the representative ligands (H2L1–4) and
Fig. 8 Binding isotherms of the ligands H2L1–4 and complexes 1–4
with CT-DNA.
ꢁ
their corresponding complexes (1ꢀ4) for 3 h at 37 C. All the
compounds efficiently cleaved the supercoiled pBR322 DNA to
nicked form and linear circular form (Fig. 11). Obviously, the
DNA cleaving efficacy of the ruthenium complexes are higher
than that of the ligands, which correlates quite well with their
DNA binding affinity. From Fig. 11, we knew that the complexes
showed potent nuclease activity without any external reagent
and complex 3 with more electron donating ethyl substitution
on terminal nitrogen atom causes stronger distortion on DNA
strand leading to more efficient DNA cleavage followed by
complex 2, complex 1 and complex 4. This resulted pattern is
consistent with the DNA binding studies results.
Fig. 9 Stern–Volmer plot of the fluorescence titration of the ligands
H2L1–4 and complexes (1–4) (10–100 mM) with DNA-EB (10 mM).
Fig. 10 Effect of the ligands H2L1–4 and complexes (1–4) on the
viscosity of CT-DNA.
Fig. 11 Gel electrophoresis diagram showing the cleavage of super-
coiled pBR322 DNA by ligands H2L1–4 and 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 L1: ligand 1 (50 mM).
Lane L2: ligand 2 (50 mM); Lane L3: ligand 3 (50 mM); Lane L4: ligand 4
(50 mM); Lane 1: complex 1 (50 mM). Lane 2: complex 2 (50 mM); Lane 3:
complex 3 (50 mM); Lane 4: complex 4 (50 mM); Lane S: metal
precursor (50 mM); forms SC, NC, and LC are supercoiled, nicked
above results concluded that compounds interacted with CT-
DNA through an intercalative mode. The results showed that
the increasing rate of viscosity was different for ligands and
complexes and the complexes exhibited a higher increasing rate
due to the chelation of the ligands with Ru(II) ion. The increased circular, and linear circular DNA, respectively.
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Fig. 12 (A) Plot of % relative fluorescence intensity (% I/Io) vs. r (r ¼ [compound]/[BSA]) (B) plot of % relative fluorescence intensity (% I/Io) vs. r (r ¼
[compound]/[HSA]).
4 respectively with a 2–4 nm of hypsochromic shi (Fig. 12A).
The uorescence intensities of HSA decreased upto 53.86%,
46.41%, 51.00%, 28.61%, 41.01%, 50.56%, 53.45% and 39.80%
for H2L1, H2L2, H2L3, H2L4, complex 1, complex 2, complex 3
and complex 4 respectively with an increase in the concentra-
tion of the compounds, accompanied by a blue shi of 2–5 nm
(Fig. 12B). The obtained results conrmed the interaction of the
ligands and complexes with serum albumins. The absorption
spectra of the serum albumins in the absence and presence of
ligands H2L1–4 and the complexes 1–4 are given in Fig. S29 in
the ESI.† On adding ligands and complexes 1–4 to albumins,
Protein binding studies. In order to investigate the binding
of BSA and its homologue HSA with the ligands and new Ru(II)
complexes, the quenching of its uorescence emission spectra
upon addition of compounds has been studied (Fig. S27 and
S28†), since the albumin solution exhibits an intense emission
band (lex ¼ 290 nm) at lem,max ¼ 345 nm (for HSA) and 346 nm
(for BSA) which is assigned to the existence of tryptophans.51,52
Addition of the compounds to BSA resulted in the quenching of
its uorescence intensity at 346 nm upto 55.22%, 43.73%,
43.92%, 39.87%, 62.61%, 71.15%, 63.19% and 58.68% for H2L1,
H2L2, H2L3, H2L4, complex 1, complex 2, complex 3 and complex
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
Quenching constant kq/Mꢀ1
Compounds
Stern–Volmer KSV/Mꢀ1
sꢀ1
Binding constant Kbin/Mꢀ1
n
BSA
(H2L1)
1.193 ꢃ 0.024 ꢂ 104
7.730 ꢃ 0.009 ꢂ 103
1.464 ꢃ 0.018 ꢂ 104
6.660 ꢃ 0.012 ꢂ 103
1.674 ꢃ 0.040 ꢂ 104
2.382 ꢃ 0.011 ꢂ 104
1.756 ꢃ 0.080 ꢂ 104
1.454 ꢃ 0.030 ꢂ 104
1.193 ꢃ 0.024 ꢂ 1012
0.773 ꢃ 0.009 ꢂ 1012
1.464 ꢃ 0.018 ꢂ 1012
0.666 ꢃ 0.012 ꢂ 1012
1.674 ꢃ 0.040 ꢂ 1012
2.382 ꢃ 0.011 ꢂ 1012
1.756 ꢃ 0.080 ꢂ 1012
1.454 ꢃ 0.030 ꢂ 1012
1.971 ꢃ 0.042 ꢂ 103
3.003 ꢃ 0.010 ꢂ 103
14.11 ꢃ 0.030 ꢂ 103
1.111 ꢃ 0.018 ꢂ 103
2.251 ꢃ 0.029 ꢂ 104
3.323 ꢃ 0.027 ꢂ 104
17.49 ꢃ 0.030 ꢂ 104
2.178 ꢃ 0.019 ꢂ 104
0.8075 ꢃ 0.029
0.8915 ꢃ 0.011
0.9947 ꢃ 0.010
0.8039 ꢃ 0.021
1.0408 ꢃ 0.030
1.0478 ꢃ 0.031
1.266 ꢃ 0.0290
1.0486 ꢃ 0.019
(H2L2)
(H2L3)
(H2L4)
Complex 1
Complex 2
Complex 3
Complex 4
HSA
(H2L1)
1.073 ꢃ 0.014 ꢂ 104
0.909 ꢃ 0.010 ꢂ 104
1.424 ꢃ 0.016 ꢂ 104
0.377 ꢃ 0.010 ꢂ 104
0.741 ꢃ 0.010 ꢂ 104
1.088 ꢃ 0.030 ꢂ 104
1.189 ꢃ 0.030 ꢂ 104
0.675 ꢃ 0.008 ꢂ 104
1.073 ꢃ 0.014 ꢂ 1012
0.909 ꢃ 0.010 ꢂ 1012
1.424 ꢃ 0.016 ꢂ 1012
0.377 ꢃ 0.010 ꢂ 1012
0.741 ꢃ 0.010 ꢂ 1012
1.088 ꢃ 0.030 ꢂ 1012
1.189 ꢃ 0.030 ꢂ 1012
0.675 ꢃ 0.008 ꢂ 1012
2.095 ꢃ 0.036 ꢂ 103
2.549 ꢃ 0.016 ꢂ 103
2.615 ꢃ 0.019 ꢂ 103
1.165 ꢃ 0.018 ꢂ 103
2.273 ꢃ 0.017 ꢂ 104
6.341 ꢃ 0.039 ꢂ 104
7.194 ꢃ 0.041 ꢂ 104
0.297 ꢃ 0.024 ꢂ 104
0.8166 ꢃ 0.010
0.8599 ꢃ 0.017
0.8130 ꢃ 0.021
0.8702 ꢃ 0.018
1.1329 ꢃ 0.018
1.1859 ꢃ 0.004
1.2027 ꢃ 0.030
0.9118 ꢃ 0.025
(H2L2)
(H2L3)
(H2L4)
Complex 1
Complex 2
Complex 3
Complex 4
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Fig. 13 (A) Stern–Volmer plot of the fluorescence titration of the ligands H2L1–4 and complexes (1–4) (10–100 mM) with BSA (10 mM). (B) Stern–
Volmer plot of the fluorescence titration of the ligands H2L1–4 and complexes (1–4) (10–100 mM) with HSA (10 mM).
the absorbance intensity of serum albumins was decreased with and this result is consistent with the pattern in DNA binding
a red shi of 2 nm. The observed changes indicated a static studies.30,44 The quenching constant values for the quenching of
quenching mechanism of serum albumins by the ligands and serum albumins by the compounds (kq z 1012 Mꢀ1 sꢀ1) sug-
complexes (1–4). To obtain a quantitative insight into the gested a good binding affinity through static quenching
quenching progression, the Stern–Volmer quenching constant mechanism.53
(KSV) and the quenching constant (Kq) were calculated from the
Furthermore, the equilibrium binding constant and
Stern–Volmer equation using the Io/ICorr versus [Q] plot number of binding sites were evaluated by using the Scatchard
(Table 4).53 The observed linearity in the plots (Fig. 13) indicated equation. The Kbin values were derived from the graph between
the ability of the compounds to quench the emission intensity log[(Fo ꢀ F)/F] and log[Q] (Fig. 14) and are given in Table 4.
of serum albumins and the order of quenching constant of the From the results, we conrmed that the Ru(II) complexes
compounds is complex 3 > complex 2 > complex 1 > complex 4 > having a large hydrophobic area can interact more efficiently
H2L3 > H2L2 > H2L1 > H2L4. The observed KSV values are than the ligands with serum albumins via a static pathway.
comparable to those reported for other ruthenium complexes The higher binding affinity of the complexes over the ligands
Fig. 14 (A) Scatchard plot of the fluorescence titration of the ligands H2L1–4 and complexes (1–4) (10–100 mM) with BSA (10 mM). (B) Scatchard
plot of the fluorescence titration of the ligands H2L1–4 and complexes (1–4) (10–100 mM) with HSA (10 mM).
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may be due to the efficient binding of protein moiety with the those with DNA binding studies, complex 3 > complex 2 >
complexed metal ions. Here, the binding affinities of the complex 1 > complex 4 > H2L3 > H2L2 > H2L1 > H2L4. Variation
complexes with serum albumins followed the same order as in the binding affinity of the compounds with serum albumins
Fig. 15 Three-dimensional fluorescence spectra of BSA in the absence and presence of ruthenium(II) complexes 1–4 (pH 7.4, 298 K, [BSA] ¼ 10
mM, [complex] ¼ 10 mM).
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depends on the electron-donating ability of the ligand, i.e. the with the increase in electron-donating ability of the substit-
substitution on the N-terminal nitrogen atom. The binding uent on the terminal nitrogen of the coordinated thio-
capability of the compounds to serum albumins increased semicarbazone ligand. The obtained quenching constant and
Fig. 16 Three-dimensional fluorescence spectra of HSA in the absence and presence of ruthenium(II) complexes 1–4 (pH 7.4, 298 K, [HSA] ¼ 10
mM, [complex] ¼ 10 mM).
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binding constant values of these new cyclometallated ruth- ruthenium(II) complexes leading to an increase in the diameter
enium(II) complexes agree well with those reported for other of the macromolecule which in turn resulted in the enhance-
ruthenium(II) complexes.31,44,54–56
ment of scattering effect.58 The uorescence intensity of peak ‘B’
Conformational investigation. The conformational changes corresponding to the tryptophan and tyrosine residues
of the protein molecular environment in the vicinity of the decreased with slight blue shi. From the results, it is inferred
uorophore functional groups have been investigated by that the molecular microenvironment and conformational
synchronous uorescence spectroscopy. Synchronous uores- changes of protein occurred aer interaction with the
cence spectra show Trp residues of serum albumins only at the complexes and ligands.
wavelength interval (Dl) of 60 nm and Tyr residues only at Dl of
Antioxidant studies. Since the synthesized ligands and Ru(II)
15 nm. For both Dl ¼ 15 and 60 nm, uorescence intensities complexes showed good DNA binding affinity, it is considered
have been decreased with an increasing amount of compounds worthwhile to investigate their antioxidant activity. It has been
(Fig. S30–S33†). However, the magnitude of quenching and shi reported that free radical species such as reactive oxygen species
of wavelength are greater at Dl ¼ 60 nm. This result showed that (ROS), are involved in the pathogenesis of various diseases
compounds interacted with both Trp and Tyr residues but in through effects on DNA directly and by acting as a tumour
greater magnitude with Trp residues.
promoter.59,60 The DPPH free radical scavenging activity of the
Three-dimensional uorescence spectra analysis. To inves- ligands and complexes were studied and quantitatively antiox-
tigate the micro environmental changes in BSA/HSA during idant properties were determined by using phosphomolybdate
interaction with the compounds, three dimensional uores- method. We knew that the inhibitory effects of the tested
cence spectroscopic studies have been performed. The changes compounds on DPPH radical are concentration dependent and
observed in 3D emission spectra and contour lines of serum the suppression ratio increases with increasing sample
albumins in the absence and presence of ligands and complexes concentrations (Fig. S36†). As seen from Fig. 17, the IC50 values
are given in Fig. 15, 16, S34 and S35 in the ESI† and their cor- of the ligands H2L1, H2L2, H2L3, H2L4, complex 1, complex 2,
responding characteristic parameters are provided in Table 5. complex 3, complex 4 and vitamin C (standard) are 83.17 ꢃ 1.50,
The emission spectra of serum albumins such as BSA and HSA 80.75 ꢃ 1.34, 67.28 ꢃ 1.44, 91.21 ꢃ 1.54, 7.13 ꢃ 0.23, 6.75 ꢃ
have shown three characteristic peaks – peaks A and C corre- 0.18, 5.28 ꢃ 0.24, 7.39 ꢃ 0.14 and 98.72 ꢃ 1.50 respectively.
sponds to rst and second order Rayleigh scattering and peak B From the results, it is revealed that the complexes exhibited
corresponds to the spectral characteristics of Trp and Tyr resi- good radical scavenging activity over the standard ascorbic acid
dues of proteins.57 The emission intensity of Rayleigh rst order and ligands. In the phosphomolybdenum assay, the antioxidant
scattering peak increased upon adding the compounds to activity is expressed as the number of equivalents of ascorbic
serum albumins. This is due to the uorophore-quencher acid (Table 6). The total antioxidant activity of the compounds is
complex formation of serum albumins with our ligands/ in the following order complex 3 > complex 2 > complex 1 >
Table 5 Three-dimensional fluorescence spectral characteristics of BSA/HSA and BSA/HSA-complexes systems
Rayleigh scattering peaks
Peak position
Fluorescence peaks
Peak position
Stokes
Dl (nm)
Compounds
lex/lem (nm nmꢀ1
)
Intensity (F)
lex/lem (nm nmꢀ1
)
Stokes Dl (nm)
Intensity (F)
BSA
BSA
280/280
280/280
280/280
280/280
280/280
280/280
280/280
280/280
280/280
0
0
0
0
0
0
0
0
0
637.73
681.98
704.90
764.34
751.37
867.32
837.83
958.23
741.59
280/343
280/340
280/342
280/342
280/342
280/338
280/339
280/339
280/340
63
60
62
62
62
58
59
59
60
514.37
436.40
437.11
415.28
486.41
395.56
372.76
304.43
368.76
BSA + H2L1
BSA + H2L2
BSA + H2L3
BSA + H2L4
BSA + complex 1
BSA + complex 2
BSA + complex 3
BSA + complex 4
HSA
HSA
280/280
280/280
280/280
280/280
280/280
280/280
280/280
280/280
280/280
0
0
0
0
0
0
0
0
0
480.02
563.09
647.85
582.94
659.33
652.04
790.59
612.58
830.46
280/333
280/335
280/332
280/336
280/334
280/335
280/331
280/337
280/332
53
55
52
56
54
55
51
57
52
289.67
281.73
278.16
275.92
281.01
268.14
269.53
266.21
276.12
HSA + H2L1
HSA + H2L2
HSA + H2L3
HSA + H2L4
HSA + complex 1
HSA + complex 2
HSA + complex 3
HSA + complex 4
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complex 4 > H2L3 > H2L2 > H2L1 > H2L4 > ascorbic acid >
[RuHClCO(PPh3)3]. The results concluded that the metal
complexes are better antioxidants than their parent ligands
which may be due to the chelation of the ligands to metal ion.35
The radical scavenging ability of the new complexes is greater
than that of few other reported ruthenium complexes, con-
taining Schiff base ligands.9
Antimicrobial studies. The free ligands and their cyclo-
metallated ruthenium(II) carbonyl complexes were screened for
their in vitro antimicrobial activity against certain pathogenic
bacterial and fungal species at three different concentrations
using disc diffusion method. The test solutions were prepared
in 10% aqueous DMSO and the results of the antimicrobial
activities are expressed as the zone of inhibition and minimum
inhibitory concentration (MIC) and are given in Tables S4–S7 in
the ESI†, Fig. 18 and 19. From the results, we concluded that the
ligands and complexes exhibited signicant activity, but they
did not reach the effectiveness of the conventional bacteriocide
gentamicin and fungicide ketoconazole. Tested complexes had
better antimicrobial activity than the ligands against all path-
ogens. This may be explained by Tweedy's chelation theory.61
Coordination of ligands reduce the polarity of the metal ion
essentially by partial sharing of its positive charge with the
donor groups within the chelate ring system formed during the
coordination and leading to the increase in lipophilic nature of
the central metal atom, which favours the effective permeation
through the lipid layer of microorganism.61,62 On comparing the
antifungal activity of the complexes, complex 3 was more active
on four fungi namely Aspergillus niger, Aspergillus fumigatus,
Candida tropicalis and Candida albicans followed by complex 2,
complex 4 and complex 1. When tested against T. rubrum, the
activity of the complexes in the order of 3 > 2 > 1 > 4. In anti-
bacterial studies, the complex 3 was effective against S. aureus
followed by complex 2, 1 and 4 and in the case of S. pneumonie
the activity of the complexes follows the order of 3 > 1 > 4 > 2. On
comparing the activity of the complexes, complexes 2 and 3
were more active on bacteria namely P. aeruginosa. When tested
Fig. 17 The DPPH radical scavenging activity of the ligands,
[RuHClCO(PPh3)3] and new Ru(II) complexes.
Table
6 Estimation of Total antioxidant capacity of ligands,
[RuHClCO(PPh3)3] and new Ru(II) complexes (1–4)
mg ascorbic
Compounds
acid equivalents/ml
H2L1
33.98 ꢃ 0.29
37.10 ꢃ 0.43
40.01 ꢃ 0.27
32.90 ꢃ 0.57
07.02 ꢃ 0.08
54.13 ꢃ 0.28
58.27 ꢃ 0.46
62.71 ꢃ 0.37
54.99 ꢃ 0.65
H2L2
H2L3
H2L4
[RuHClCO(PPh3)3]
Complex 1
Complex 2
Complex 3
Complex 4
Fig. 18 Antibacterial activity of ligands H2L1–4, [RuHClCO(PPh3)3] and new Ru(II) complexes (1–4). Error bars represent the standard deviation of
the mean (n ¼ 3).
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Fig. 19 Antifungal activity of ligands H2L1–4, [RuHClCO(PPh3)3] and new Ru(II) complexes (1–4). Error bars represent the standard deviation of the
mean (n ¼ 3).
against S. paratyphi complex 1 stood out as good followed by the number of coumarin derivatives have shown considerable anti-
complexes 4, 3 and 2 respectively. The compounds showed cancer activity against a number of cell lines. All the 3-acetyl-7-
different degrees of antimicrobial activity due to the structural methoxy-coumarin-4(N)-substituted thiosemicarbazone ligands,
variations of themselves and variation on the group of micro- metal precursor [RuHClCO(PPh3)3] and their new cyclo-
organisms.63 In addition, antimicrobial activity of the metallated Ru(II) complexes were assessed for their cytotoxicity
complexes was compared with already reported ruthenium with two human derived cell lines namely human lung carci-
complexes, showing that the new Ru(II) complexes exhibited noma (A549) and human breast cancer cells (MCF-7) by using
better activity.64,65
MTT assay. For comparison purpose, cisplatin was used as
Anticancer studies. The DNA/protein binding studies, anti- a positive control under identical conditions. The dose–response
oxidant and antimicrobial studies had shown that the ligands curves are given in Fig. 20–22 and the results are shown in
and ruthenium(II) complexes studied here have therapeutic Table 7. The IC50 values for the ligands and their complexes for
potentials and were subjected to study their anticancer activity. A
Fig. 20 The newly synthesized ligands, [RuHClCO(PPh3)3] and cisplatin inhibit MCF-7 and A549 cells proliferation in a dose dependent manner.
MCF-7 and A549 cells were treated with different concentrations of ligands for 48 h, the cell viability was determined and the results were
expressed as percentage cell viability with control. Results shown are mean, which are three separate experiments performed in triplicate.
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Fig. 21 The newly synthesized ruthenium(II) complexes (1–4) inhibit MCF-7 and A549 cells proliferation in a dose dependent manner. MCF-7 and
A549 cells were treated with different concentrations of complexes for 48 h, the cell viability was determined and the results were expressed as
percentage cell viability with control. Results shown are mean, which are three separate experiments performed in triplicate.
Fig. 22 The newly synthesized ligands H2L1–4, Ru(II) complexes, [RuHClCO(PPh3)3] and cisplatin inhibit HaCaT cells proliferation in a dose
dependent manner. HaCaT cells were treated with different concentrations of compounds for 48 h, the cell viability was determined and the
results were expressed as percentage cell viability with control. Results shown are mean, which are three separate experiments performed in
triplicate.
the MCF-7 and A549 showed that the ligands and their Ru(II) complex 1, 2.62 ꢃ 0.07 for complex 2, 2.53 ꢃ 0.10 for complex 3
complexes were cytotoxic to these cells.
and 3.02 ꢃ 0.05 for complex 4.
Whilst most of the Schiff bases did not display good anti-
The ligands H2L1–4 and complexes exhibited high cytotoxic
cancer activity, the coumarin appended thiosemicarbazones effects on lung cancer cells with low IC50 values indicating their
H2L1–4 against MCF-7 cell line exhibited IC50 values of 13.06 ꢃ efficiency in killing cancer cells even at low concentrations. In
0.29 mM, 12.12 ꢃ 0.32 mM, 11.27 ꢃ 0.21 mM and 13.11 ꢃ 0.25 mM A549, the anticancer activity of the compounds follows the order
respectively, which were lower than that of cisplatin, indicating ruthenium precursor (15.96 ꢃ 0.21) < cisplatin (15.10 ꢃ 0.05) <
ligands showed good activity over cisplatin. Against the human H2L4 (13.83 ꢃ 0.18) < H2L1 (12.64 ꢃ 0.24) < H2L2 (12.12 ꢃ 0.16) <
breast cancer cell line MCF-7, the antiproliferative activity of H2L3 (11.63 ꢃ 0.15) < complex 4 (3.05 ꢃ 0.12) < complex 1 (2.96
Ru(II) complexes 1–4 was higher than that of their parent ꢃ 0.07) < complex 2 (2.93 ꢃ 0.07) < complex 3 (2.37 ꢃ 0.04).
ligands and cisplatin with lower IC50 values of 2.86 ꢃ 0.17 for
From this study, we concluded that the coumarin-appended
Schiff base derivatives had potent activity than cisplatin used to
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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 the ligands H2L1–4, [RuHClCO(PPh3)3]
and new organometallic Ru(II) complexes for 48 h
In order to investigate the selectivity of the compounds for
cancer cells rather than normal cell lines, the compounds were
also screened for their anticancer activity on the human normal
keratinocyte cells (HaCaT). In the noncancerous cell line, all the
compounds showed their nontoxic nature. Furthermore, the
IC50 values exhibited by the complexes showed a higher cyto-
toxic effect when compared to the other reported Ru(II)
complexes.24,26,30,31,44,66,67
IC50 values (mM)
Compounds
MCF-7
A549
HaCaT
Cisplatin
H2L1
16.79 ꢃ 0.08
13.06 ꢃ 0.29
12.12 ꢃ 0.32
11.27 ꢃ 0.21
13.11 ꢃ 0.25
20.10 ꢃ 0.18
2.86 ꢃ 0.17
2.62 ꢃ 0.07
2.53 ꢃ 0.10
3.02 ꢃ 0.05
15.10 ꢃ 0.05
12.64 ꢃ 0.17
12.12 ꢃ 0.16
11.63 ꢃ 0.15
13.83 ꢃ 0.18
15.96 ꢃ 0.21
2.96 ꢃ 0.07
2.93 ꢃ 0.07
2.37 ꢃ 0.04
3.05 ꢃ 0.12
>40
>40
>40
>40
>40
>40
>40
>40
>40
>40
Lactate dehydrogenase release. LDH is a stable cytoplasmic
enzyme that is released into the culture medium following loss
of membrane integrity and serves as a general mean to assess
cytotoxicity resulting from chemical compounds or environ-
mental toxic factors.68 In the present study, LDH leakage into
the culture medium of the compounds treated A549 and MCF-7
cells was analyzed. It was observed that the new ligands and
their Ru(II) complexes could potently induce the release of LDH
into the culture medium of A549 and MCF-7 cells when they are
treated with their respective IC50 concentrations for 48 h,
indicating that the compounds could rupture the plasma
membrane (Fig. 23). The results conrmed the cytotoxic effect
of the ligands and complexes on lung and breast cancer cell
lines. The compounds could induce LDH leakage as high as that
of cisplatin. The induction of LDH release was found to be
higher for complexes than their parent ligands when comparing
among them and with the control. Among the compounds
examined, complex 3 was found to be more potent in inducing
LDH leakage into the culture than the rest. These results are
comparable with the earlier reports.30
Nitric oxide release. Nitrite is the stable product of the nitric
oxide released in response to oxidative stress. The amount of
nitrite in the culture medium corresponds to the level of nitric
oxide. Hence the level of nitrite is estimated to measure the NO
produced aer complex treatment. The level of nitrite was
found to increase signicantly in the ligands and Ru(II)
complexes treated A549 and MCF-7 cells compared to the
control. The increased level of nitrite in the cell culture medium
further conrms the cytotoxic effects of the presently studied
H2L2
H2L3
H2L4
[RuHClCO(PPh3)3]
Complex 1
Complex 2
Complex 3
Complex 4
treat human breast cancer and human lung cancer. In addition,
the coordination of the ligands to the Ru(II) ion increases the
anticancer activity of the complexes to six times greater than the
ligands and eight times greater than the cisplatin against both
the cell lines. Thus, the presence of the substituent at N-
terminal nitrogen seems to be important for varying the order
of activity of the compounds. In both MCF-7 and A549 cell lines,
ruthenium(II) complex 3 containing more electron donating
ethyl group at N-terminal nitrogen exhibited high activity fol-
lowed by complex 2 (NH–Me), complex 1 (NH–H) and complex 4,
which has electron withdrawing phenyl group at terminal
nitrogen atom of the ligand. On the basis of the results, the
antiproliferative activity of these compounds has been arranged
in the order 3 > 2 > 1 > 4 > H2L3 > H2L2 > H2L1 > H2L4. Inter-
estingly, this observation 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.
Fig. 23 Percentage of lactate dehydrogenase released by the human
cancer cell lines A549 and MCF-7 after an incubation period of 48 h Fig. 24 Nitrite released (nmoles) by the human cancer cell lines A549
with ligands H2L1–4 and complexes 1–4. Error bars represent the and MCF-7 after an incubation period of 48 h with ligands H2L1–4 and
standard mean error (n ¼ 6).
complexes 1–4. Error bars represent the standard mean error (n ¼ 6).
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compounds. The induction of cytotoxicity in terms of NO and human normal keratinocyte cells (HaCaT) were obtained
release in A549 and MCF-7 cells follows the order of 3 > 2 > 1 > 4 from the National Center for Cell Science (NCCS), Pune, India.
> H2L3 > H2L2 > H2L1 > H2L4 > cisplatin. These results authen- Melting points were measured in a Lab India apparatus.
ticated the results obtained by MTT and LDH leakage assays Infrared spectra were measured as KBr pellets on a JASCO FT-
indicating that complex 3 is more effective than the remaining IR 4100 instrument between 400–4000 cmꢀ1. Elemental anal-
three complexes (Fig. 24). The nitric oxide release by the ysis of carbon, hydrogen, nitrogen and sulfur was determined
compounds is higher than that of cisplatin, and it is better than by using Vario EL III CHNS at the Department of Chemistry,
those reported for other ruthenium(II) complexes containing Bharathiar University, Coimbatore, India. The electronic
triphenylphosphines.30 The results of the nitric oxide assay spectra of the compounds were recorded with a JASCO V-630
support the concept that the complex-induced cell death is spectrophotometer using DMSO as the solvent in 800–
mediated by reactive oxygen species generation.
200 nm range. Emission spectra were recorded by using JASCO
FP 6600 Spectrouorimeter. 1H NMR spectra were recorded in
DMSO at room temperature with a Bruker 400 MHz instru-
ment, chemical shi relative to tetramethylsilane. The
Conclusion
A series of ligands 3-acetyl-7-methoxycoumarin-4N-substituted stability of the compounds was performed in 1% aqueous
thiosemicarbazones were prepared and consecutively DMSO and phosphate buffer–DMSO (99 : 1). The stability was
made to undergo complexation with ruthenium precursor analyzed by monitoring the electronic spectra over 24 h at
[RuHCl(CO)(PPh3)3]. The reactions ended up in cyclometallated room temperature on a JASCO 4100 spectrophotometer.
organometallic Ru(II) complexes. Analytical and spectral studies
accounted for the formation of the complexes. The ligands X-ray crystallography
acted in a tridendate manner by bonding through C, N and S
atoms. A systematic study on their DNA/protein binding prop-
Suitable single crystals for the ligands H2L1–3 and complexes
(1,
2
and 4) were obtained from methanol and
erties and antioxidant activities was carried out. Experimental
results suggest that the ligands and complexes can bind to DNA
via an intercalation mode and a static quenching with proteins.
Evaluation of their inhibitory potency against bacterial and
fungal pathogens revealed that the compounds possess a good
spectrum of antimicrobial activity. The in vitro cytotoxic activity
of the complexes was ascertained via MTT assay and the IC50
values were found in the range of 2.53 ꢃ 0.10–3.02 ꢃ 0.05 mM
and 2.37 ꢃ 0.04–3.05 ꢃ 0.12 mM for MCF-7 and A549 cancerous
cell lines, respectively. Moreover, LDH and NO release assays
conrm the anticancer potential of the tested compounds. The
results validated that complex 3 is a potent chemotherapeutic
drug among others. This may be attributed to the more electron
donating ability of the N-terminal ethyl group. In addition, the
present investigation lights up the potent antiproliferative effect
of ruthenium complexes by inhibiting the viability of A549 and
MCF-7 cells. Hence, further studies on animal models to
elucidate the clear mechanism of action of the complexes are
highly warranted to unveil the ruthenium complexes as anti-
cancer drugs.
dichloromethane/methanol medium respectively. Single crystal
data collections and corrections for the ligands (H2L1–3) and
new Ru(II) complexes (1, 2 and 4) were carried out with a Gemini
Xcaliber Atlas four circle diffractometer using graphite mono-
˚
chromated Mo Ka (l ¼ 0.71073 A) radiation at 295 K. All the
calculations were done by using SHELXS-200, SHELXL-2015/7
and Olex-2 programs.72
Preparation of 3-acetyl-7-methoxy-2H-chromen-2-one71
An ethanolic solution (10 cm3) of 4-methoxysalicylaldehyde
(1.22 g, 1 mmol) was taken along with the catalytic amount of
piperidine and ethylacetoacetate (1.95 g, mmol) and was
reuxed for 5 h with continuous stirring. The reaction mixture
was then cooled to room temperature, which afforded yellow
precipitate. The crude product was ltered, washed with
ethanol (3 ꢂ 10 cm3) and recrystallized from ethanol to yield an
ꢁ
yellow crystalline product. Yield ¼ 89%. Mp 118–120 C; anal.
calcd for C12H10O4: C, 66.11; H, 4.63; found: C, 66.08; H, 4.61;
UV-Vis (DMSO), lmax (3): 354 (26 868) nm (dm3 molꢀ1 cmꢀ1); IR
1
(n, cmꢀ1): n(C]O lactone) 1731, n(C]O acetyl group) 1681. H
NMR (400 MHz, DMSO-d6, d ppm, J Hz): d 8.628 (s, 1H, C4–H),
d 7.857–7.877 (d, J ¼ 8, 1H, C8–H), d 6.996–7.057 (m, 2H, C5–H
and C6–H), d 3.894 (s, 3H, ꢀOCH3), d 2.573 (s, 3H, ꢀCH3).
Experimental section
Materials and methods
All the reagents used were of analytically or chemically pure
grade. Solvents were puried and dried according to standard
procedures.69 The metal precursor [RuHCl(CO)(PPh3)3]70
Synthesis of ((1E)-1-(1-(7-methoxy-2-oxo-2H-chromen-3-yl)
ethylidene) thiosemicarbazone) [H2-7MAC-tsc] (H2L1)71
and 3-acetyl-7-methoxy-chromene-2-one (3-acetyl-7-methoxy Thiosemicarbazide (0.417 g, 4.58 mmol) was dissolved in 30
coumarin)71 were prepared according to the literature proce- cm3 of methanol with continuous stirring and gently heated for
dure. Doubly distilled water was used to prepare buffers. a period of 30 min. This was added to a methanolic solution (20
Ethidium bromide (EB), serum albumins (BSA/HSA), calf cm3) of 3-acetyl-7-methoxy-2H-chromen-2-one (1 g, 4.58 mmol).
thymus DNA (CT-DNA) and 3-(4,5-dimethyl thiazol-2-yl)-2,5- To this, few drops of glacial acetic acid were added and the
diphenyltetrazolium bromide (MTT) were purchased from mixture was reuxed for 2 h with continuous stirring. The
HiMedia (Mumbai, India) and used as received. Human lung mixture was then cooled to room temperature whereby a yellow
cancer cell lines A549, human breast cancer cell lines MCF-7 crystalline compound precipitated. This was collected by
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ltration, washed well with cold methanol and dried under
vacuum. The compound was recrystallized from DMF–meth-
anol (1 : 9 v/v). Yellow colored ne single crystals suitable for X-
ray analysis were collected. Yield: 72%. Mp: 213 ꢁC. Anal. calcd
for C13H13N3O3S: C, 53.58; H, 4.50; N, 14.42; S, 11.00. Found: C,
53.56; H, 4.47; N, 14.41; S, 11.00%. FT-IR (n, cmꢀ1) in KBr: n(C]
O lactone) 1728, n(C]N) 1644, n(–NH2) 3279, n(–NH) 3136, n(C]
S) 831. UV-Vis (DMSO), lmax (3): 276 (20 943) nm (dm3
molꢀ1 cmꢀ1); 354 (30 488) nm (dm3 molꢀ1 cmꢀ1). 1H NMR (400
MHz, DMSO-d6, d ppm, J Hz): d 7.986 (s, 1H, C4–H), d 7.719–
7.740 (d, 1H, J ¼ 8.4, C5–H), 7.039–7.094 (m, 2H, Ar–H), d 3.931
(s, 3H, –OCH3), d 2.302 (s, 3H, –CH3), d 10.424 (s, 1H, NH–C]S),
d 8.477 & 8.409 (2 br s, 2H, –NH2).
Synthesis of ((1E)-1-(1-(7-methoxy-2-oxo-2H-chromen-3-yl)
ethylidene) 4(N)-phenyl thiosemicarbazone) [H2-7MAC-ptsc]
(H2L4)
The ligand [H2-7MAC-ptsc] was prepared from 4-(N)-phenyl-
thiosemicarbazide (0.766 g, 4.58 mmol) and 3-acetyl-7-methoxy-
2H-chromen-2-one (1 g, 4.58 mmol) in the presence of glacial
acetic acid. The compound was recrystallised by using meth-
ꢁ
anol. Yield: 77%. Mp: 186 C. Anal. calcd for C19H17N3O3S: C,
62.10; H, 4.67; N, 11.43; S, 8.72. Found: C, 62.07; H, 4.64; N, 11.4;
S, 8.69%. FT-IR (n, cmꢀ1) in KBr: n(C]O lactone) 1700, n(C]N)
1600, n(terminal –NH) 3241, n(–NH) 3114, n(C]S) 826. UV-Vis
(DMSO), lmax (3): 282 (27 646) nm (dm3 molꢀ1 cmꢀ1); 356
1
(41 701) nm (dm3 molꢀ1 cmꢀ1). H NMR (400 MHz, DMSO-d6,
The very similar method was followed to synthesize the
following compounds.
d ppm, J Hz): d 8.458 (s, 1H, C4–H), d 6.691–7.524 (m, 8H, Ar–H),
d 3.885 (s, 3H, –OCH3), d 2.332 (s, 3H, –CH3), d 10.777 (s, 1H,
NH–C]S), d 10.126 (s, 1H, terminal –NH).
Synthesis of ((1E)-1-(1-(7-methoxy-2-oxo-2H-chromen-3-yl)
ethylidene) 4(N)-methyl thiosemicarbazone) [H2-7MAC-mtsc]
(H2L2)
Synthesis of new ruthenium(II) complexes
Synthesis of [Ru(7MAC-tsc)(CO)(PPh3)2] (1). A solution of
The ligand [H2-7MAC-mtsc] was prepared from 4-(N)-methyl- [H2-7MAC-tsc] (0.021 g; 0.105 mmol) in 10 cm3 of benzene was
thiosemicarbazide (0.481 g, 4.58 mmol) and 3-acetyl-7-methoxy- added dropwise to a boiling solution of [RuHCl(CO)(PPh3)3]
2H-chromen-2-one (1 g, 4.58 mmol) in the presence of glacial (0.100 g, 0.105 mmol) in benzene and reuxed for 7 h and
acetic acid. Single crystals suitable for X-ray diffraction studies allowed to stand for 4 days at room temperature. Reddish
were obtained by recrystallisation of ligand H2L2 in methanol. orange solid formed was ltered, washed with petroleum ether
Yield: 73%. Mp: 117 ꢁC anal. calcd for C14H15N3O3S: C, 55.05; H, (60–80 ꢁC) and crystallized from dichloromethane and meth-
4.96; N, 13.76; S, 10.49. Found: C, 55.03; H, 4.93; N, 13.71; S, anol mixture (1 : 1 v/v) to yield red transparent needle like
10.48%. FT-IR (n, cmꢀ1) in KBr: n(C]O lactone) 1714, n(C]N) crystals suitable for X-ray analysis. Yield: 66%. Mp: 247 ꢁC. Anal.
1608, n(terminal –NH) 3262, n(–NH) 3210, n(C]S) 834. UV-Vis calcd for C50H41N3O4P2RuS: C, 63.67; H, 4.39; N, 4.45; S, 3.39.
(DMSO), lmax (3): 275 (24 094) nm (dm3 molꢀ1 cmꢀ1); 352 Found: C, 63.64; H, 4.37; N, 4.42; S, 3.37%. FT-IR (n, cmꢀ1) in
1
(33 647) nm (dm3 molꢀ1 cmꢀ1). H NMR (400 MHz, DMSO-d6, KBr: n(C]O lactone) 1685, n(C]N) 1607, n(C–S) 745, n(C^O)
d ppm, J Hz): d 8.314 (s, 1H, C4–H), d 7.684–7.705 (d, J ¼ 8.4, 1H, 1918, 1436, 1090, 696 (for PPh3). UV-Vis (DMSO), lmax (3): 337
1
C5–H), 6.985–7.029 (m, 2H, Ar–H), d 3.879 (s, 3H, –OCH3), (33 654) nm (dm3 molꢀ1 cmꢀ1) (LMCT s / d). H NMR (400
d 2.244 (s, 3H, –CH3), d 10.377 (s, 1H, NH–C]S), d 8.465–8.490 MHz, DMSO-d6, d ppm, J Hz): d 7.150–7.547 (m, 31H, Ar–H),
(q, 1H, terminal –NH), d 3.027–3.037 (d, J ¼ 4, 1H, terminal d 6.290–6.444 (m, 2H, C6–H and C8–H), d 3.842 (s, 3H, –OCH3),
–NH–CH3).
d 1.977 (s, 3H, –CH3), d 5.651 (br s, 2H, –NH2).
A similar method was followed to synthesize other ruth-
enium(II) complexes.
Synthesis of [Ru(7MAC-mtsc)(CO)(PPh3)2] (2). Complex 2
was prepared by the same procedure as described for 1, with
H2L2 (0.105 mmol) as a ligand. Needle shaped, transparent pink
Synthesis of ((1E)-1-(1-(7-methoxy-2-oxo-2H-chromen-3-yl)
ethylidene) 4(N)-ethyl thiosemicarbazone) [H2-7MAC-etsc]
(H2L3)
The ligand [H2-7MAC-etsc] was prepared from 4-(N)-ethyl- colour crystals were obtained on slow evaporation of the reac-
ꢁ
thiosemicarbazide (0.546 g, 4.58 mmol) and 3-acetyl-7-methoxy- tion mixture. Yield: 62%. Mp >300 C. Anal. calcd for C51H43
-
2H-chromen-2-one (1 g, 4.58 mmol) in the presence of glacial N3O4P2RuS: C, 64.00; H, 4.45; N, 4.39; S, 3.34. Found: C, 63.97;
acetic acid. The compound was recrystallised by using meth- H, 4.43; N, 4.36; S, 3.30%. FT-IR (n, cmꢀ1) in KBr: n(C]O
anol to yield suitable yellow crystals for X-ray analysis. Yield: lactone) 1680, n(C]N) 1606, n(C–S) 744, n(C^O) 1925, 1404,
76%. Mp: 174 ꢁC. Anal. calcd for C15H17N3O3S: C, 56.40; H, 1087, 695 (for PPh3). UV-Vis (DMSO), lmax (3): 268 (45 067) nm
5.37; N, 13.15; S, 10.05. Found: C, 56.37; H, 5.34; N, 13.11; S, (dm3 molꢀ1 cmꢀ1) (intraligand transition); 363 (20 485) nm
10.01%. FT-IR (n, cmꢀ1) in KBr: n(C]O lactone) 1725, n(C]N) (dm3 molꢀ1 cmꢀ1) (LMCT s / d). 1H NMR (400 MHz, DMSO-d6,
1606, n(terminal –NH) 3241, n(–NH) 3155, n(C]S) 835. UV-Vis d ppm, J Hz): d 7.139–7.637 (m, 31H, Ar–H), d 6.62–6.63 (m, 1H,
(DMSO), lmax (3): 276 (14 245) nm (dm3 molꢀ1 cmꢀ1); 353 C8–H), d 6.87–6.90 (dd, 1H, J ¼ 2.4, 8.8, C6–H), d 3.842 (s, 3H,
1
(21 388) nm (dm3 molꢀ1 cmꢀ1). H NMR (400 MHz, DMSO-d6, –OCH3), d 2.098 (s, 3H, –CH3), d 5.972–6.098 (q, 1H, terminal
d ppm, J Hz): d 8.291 (s, 1H, C4–H), d 7.694–7.715 (d, J ¼ 8.4, 1H, –NH), d 1.221 (s, 3H, –NH–CH3).
C5–H), d 7.679–7.034 (m, 2H, Ar–H), d 3.881 (s, 3H, –OCH3),
Synthesis of [Ru(7MAC-etsc)(CO)(PPh3)2] (3). Complex 3 was
d 2.247 (s, 3H, –CH3), d 10.269 (s, 1H, NH–C]S), d 8.472–8.499 prepared by the same procedure as described for 1 with H2L3
(t, J ¼ 5.6, 1H, terminal –NH), d 3.593–3.622 (p, 2H, terminal (0.105 mmol) as a ligand. Red solid formed was ltered, washed
–NH–CH2), d 1.136–1.172 (t, J ¼ 7.2, 3H, –CH3).
with petroleum ether (60–80 ꢁC) and crystallized from
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dichloromethane and methanol to yield orange crystals. Yield: Candida tropicalis. The above said all test organisms were ob-
62%. Mp 237 ꢁC. Anal. calcd for C52H45N3O4P2RuS: C, 64.31; H, tained from the MTCC, Chandigarh, India and Microbiological
4.68; N, 4.32; S, 3.30. Found: C, 63.29; H, 4.64; N, 4.29; S, 3.28%. laboratory, Coimbatore, Tamil Nadu, India. The antimicrobial
FT-IR (n, cmꢀ1) in KBr: n(C]O lactone) 1681, n(C]N) 1606, activity of the test compounds was checked with various
n(C–S) 745, n(C^O) 1923, 1432, 1087, 695 (for PPh3). UV-Vis concentrations (25 mg mlꢀ1, 50 mg mlꢀ1 and 100 mg mlꢀ1
)
(DMSO), lmax (3): 268 (16 367) nm (dm3 molꢀ1 cmꢀ1) (intra- against all the test pathogens. Gentamicin and ketoconazole
ligand transition); 357 (38 703) nm (dm3 molꢀ1 cmꢀ1) (LMCT s were used as positive controls to study the antibacterial and
/ d). 1H NMR (400 MHz, DMSO-d6, d ppm, J Hz): d 7.216–7.634 antifungal activities respectively. Each experiment was per-
(m, 31H, Ar–H), d 6.644–6.921 (m, 2H, C6–H and C8–H), d 3.774 formed in triplicate and the results are represented as average
(s, 3H, –OCH3), d 1.887 (s, 3H, –CH3), d 6.18–6.23 (t, 1H, terminal zone of inhibition and minimum inhibitory concentration of all
–NH), d 1.126–1.288 (m, 2H, J ¼ 4.8, –NH–CH2), d 0.701–0.737 (t, the test pathogens.
3H, J ¼ 7.2, –CH2–CH3).
Synthesis of [Ru(7MAC-ptsc)(CO)(PPh3)2] (4). Complex 4 was
Cytotoxicity studies
prepared by the same procedure as described for 1 with H2L4
Cytotoxic activity of the compounds was tested with human
(0.105 mmol) as a ligand and [RuHCl(CO)(PPh3)3] (0.105 mmol).
lung cancer cell lines A549, human breast cancer cell lines MCF-
7 and human normal keratinocyte cells (HaCaT) by using MTT
Red solid formed was ltered, washed with petroleum ether
(60–80 ꢁC) and crystallized from dichloromethane and meth-
assay, which was done according to the earlier literature
anol to yield pink crystals. Yield: 60%. Mp 230 ꢁC. Anal. calcd for
methods77 and IC50 values obtained from nonlinear regression
C
56H45N3O4P2RuS: C, 65.99; H, 4.45; N, 4.12; S, 3.14. Found: C,
using GraphPad Prism 5.78 The LDH release79 and NO release80
assays of the compounds was evaluated by the earlier reported
methods.
65.96; H, 4.42; N, 4.09; S, 3.12%. FT-IR (n, cmꢀ1) in KBr: n(C]O
lactone) 1681, n(C]N) 1606, n(C–S) 744, n(C^O) 1920, 1431,
1089, 694 (for PPh3). UV-Vis (DMSO), lmax (3): 278 (19 763) nm
(dm3 molꢀ1 cmꢀ1) (intraligand transition); 365 (10 531) nm
(dm3 molꢀ1 cmꢀ1) (LMCT s / d), 380 (10 424) nm (dm3
molꢀ1 cmꢀ1) (LMCT s / d). 1H NMR (400 MHz, DMSO-d6,
d ppm, J Hz): d 6.909–7.324 (m, 36H, Ar–H), d 6.635–6.757 (m,
2H, C6–H & C8–H), d 3.793 (s, 3H, –OCH3), d 1.957 (s, 3H, –CH3),
d 8.488 (s, 1H, terminal –NH).
Conflicts of interest
There are no conicts to declare.
Acknowledgements
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).
Biomolecular interaction studies
The stability of the compounds was performed in 1% aqueous
DMSO and phosphate buffer : DMSO (99 : 1). The stability was
analyzed by monitoring the electronic spectra for the period of
24 h at room temperature on a JASCO 4100 spectrophotometer.
DNA binding studies, EB-displacement assays, DNA viscosity
studies, DNA cleavage experiments and protein binding studies References
have been done according to the reported methods.30,31,35,44,73
The detailed procedures for these experiments are provided in
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