Microwave-assisted synthesis and DNA-binding studies of half-sandwich ruthenium(II) arene complexes containing phenanthroimidazole-triarylamine hybrids

Article abstract of DOI:10.1080/00958972.2021.1885650

A new series of half-sandwich Ru(II)-arene complexes with phenanthroimidazole-triarylamine hybrid ligands were synthesized and scrutinized for DNA binding studies. The metal complexes were prepared via microwave-assisted synthesis with high yield and purity. The interaction of Ru(II)-arene complex with calf thymus DNA was studied by spectrophotometric methods. All six complexes exhibited significant interaction with CT-DNA. Methoxyphenyl-substituted metal complex showed the highest binding efficiency with a binding constant (K_b) of 5.053 × 10⁴ M^?1 due to its electron-rich nature. The methoxyphenyl group remarkably enhanced the interaction through stabilization of π-orbital of the metal complex which resulted in the efficient coupling. Phenyl- and thiophene-substituted ligands also showed good binding constants of 4.908 × 10⁴ M^?1 and 4.509 × 10⁴ M^?1, respectively. The absorption and ethidium bromide displacement studies declare that both the intercalation and groove binding is anticipated by these complexes. These Ru(II) complexes with phenanthroimidazole-triarylamine hybrid ligands can be realized as efficient DNA binding agents.

Full text of DOI:10.1080/00958972.2021.1885650

Journal of Coordination Chemistry
ISSN: (Print) (Online) Journal homepage: https://www.tandfonline.com/loi/gcoo20
Microwave-assisted synthesis and DNA-binding
studies of half-sandwich ruthenium(II) arene
complexes containing phenanthroimidazole-
triarylamine hybrids
Ramakrishnan Abhijna Krishna, Ramachandran Dheepika, Mathiyan
Muralisankar & Samuthira Nagarajan
To cite this article: Ramakrishnan Abhijna Krishna, Ramachandran Dheepika, Mathiyan
Muralisankar & Samuthira Nagarajan (2021) Microwave-assisted synthesis and DNA-binding
studies of half-sandwich ruthenium(II) arene complexes containing phenanthroimidazole-
triarylamine hybrids, Journal of Coordination Chemistry, 74:4-6, 838-849, DOI:
10.1080/00958972.2021.1885650
To link to this article: https://doi.org/10.1080/00958972.2021.1885650
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JOURNAL OF COORDINATION CHEMISTRY
2021, VOL. 74, NOS. 4–6, 838–849
https://doi.org/10.1080/00958972.2021.1885650
Microwave-assisted synthesis and DNA-binding studies
of half-sandwich ruthenium(II) arene complexes
containing phenanthroimidazole-triarylamine hybrids
Ramakrishnan Abhijna Krishna, Ramachandran Dheepika, Mathiyan Muralisankar
and Samuthira Nagarajan
Department of Chemistry, Central University of Tamil Nadu, Thiruvarur, India
ABSTRACT
ARTICLE HISTORY
Received 2 August 2020
Accepted 2 January 2021
A new series of half-sandwich Ru(II)-arene complexes with phenanthroi-
midazole-triarylamine hybrid ligands were synthesized and scrutinized
for DNA binding studies. The metal complexes were prepared via micro-
wave-assisted synthesis with high yield and purity. The interaction of
Ru(II)-arene complex with calf thymus DNA was studied by spectro-
photometric methods. All six complexes exhibited significant interaction
with CT-DNA. Methoxyphenyl-substituted metal complex showed the
highest binding efficiency with a binding constant (Kb) of 5.053ꢀ 104
KEYWORDS
Ruthenium(II) arene;
phenanthroimidazole; triar-
ylamine; DNA-binding
M
ꢁ1 due to its electron-rich nature. The methoxyphenyl group remark-
ably enhanced the interaction through stabilization of p-orbital of the
metal complex which resulted in the efficient coupling. Phenyl- and thio-
phene-substituted ligands also showed good binding constants of
4.908ꢀ 104 Mꢁ1 and 4.509ꢀ 104 Mꢁ1, respectively. The absorption and
ethidium bromide displacement studies declare that both the intercal-
ation and groove binding is anticipated by these complexes. These Ru(II)
complexes with phenanthroimidazole-triarylamine hybrid ligands can be
realized as efficient DNA binding agents.
CONTACT Samuthira Nagarajan
snagarajan@cutn.ac.in
Department of Chemistry, Central University of Tamil
Nadu, Thiruvarur, 610005, India
Supplemental data for this article is available online at https://doi.org/10.1080/00958972.2021.1885650.
ß 2021 Informa UK Limited, trading as Taylor & Francis Group

JOURNAL OF COORDINATION CHEMISTRY
839
1. Introduction
Revolution in cancer therapy was heralded by the discovery of cisplatin in 1965 [1, 2].
Cytotoxicity induced by platinum drugs is due to the cross-linking of DNA and thereby
inhibiting the replication and protein synthesis. However, severe side-effects like
neurotoxicity, renal toxicity, myelo suppression, ototoxicity, hair loss, and poor solubil-
ity of the drug in the liquid medium have restricted the applications [3–5]. As an alter-
native, owing to the excellent physicochemical properties, ruthenium complexes are
now receiving attention as potent anticancer agents [6]. Two of the complexes such
as sodium salt of indazolium [trans-[tetrachloridobis(1H-indazole)ruthenate(III)]
(KP1339)
and
imidazolium
[trans-tetrachlorido(dimethylsulfoxide)(1H-
imidazole)ruthenate(III)] (NAMI-A) have successfully reached the stage of human trials
[7]. Ru(II)-p-cymene-PTA complex is also progressing towards the clinical trials [8].
These complexes are beneficial over platinum complexes as they possess similar bind-
ing nature as iron and antimetastatic as well [9]. Also, ruthenium complexes have
become a promising class of metallodrugs because of their excellent DNA-binding
properties and their good interaction with the blood proteins. The solubility of com-
plexes with blood proteins provides an easier path for transportation and protects the
complexes against premature elimination or degradation [10]. Besides, modification of
Ru complexes with different ligand systems gives the effective change in treating a
diverse type of tumor. Hence, global research is focusing on different applications of
ruthenium complexes with various biologically significant ligands [11].
In the background of developing good DNA-binding agents containing Ru, the
‘piano-stool’ skeleton of half-sandwich Ru(II)-arene complexes offer many possibilities
to modify the structural characteristics and biological action of the complexes, which
leads to possible antiproliferative activity towards tumor cells [12, 13]. Additionally, the
piano-stool confirmation of Ru(II) arene complexes allows it to show a variety of cata-
lytic applications like closing olefin metathesis [14], C(sp2)-H activation of substituted
pyridines [15], oxidation of alcohols [16], and asymmetric catalysis for Diels–Alder reac-
tions [17]. Dyson and Aird et al. developed half-sandwich ruthenium complexes having
similar ligand exchange properties as that of platinum (II) anticancer drugs [18, 19].
According to them, the biological activities of complexes are decided by the nature of
chelating arene ligands. Gianferrara et al. [20] classified metal anticancer compounds
based on their mode of action and categorized them into groups of functional com-
pounds. The activity of complexes depended on the type of metal center, its electronic
configuration, oxidation state, and its hard-soft nature. Isostructural piano-stool com-
plexes with different metal ions have been studied for their biological activity [21, 22].
The Ru(II) compound (RM175) and its osmium(II) analog (AFAP51) displayed significant
variations in vitro and in vivo anticancer activities. Casini and co-workers demonstrated
that Rh(III) and Ir(III) complexes are inactive towards anticancer activity by comparing
the CpRh(III) and CpIr(III) derivatives and osmium complexes of RAPTA-C to the NAMI-
A Ru(III) complex [23]. The enhanced stability of half-sandwich Ru(II) complexes with
heterocycles due to hydrogen bonding plays an important role in the anticancer
mechanism [24]. In the absence of stable hydrogen bond interactions, dissociation of

840
R. ABHIJNA KRISHNA ET AL.
the ligand occurs in the solution, confirmed by NMR spectroscopic studies. Hydrogen-
bonding based structure-activity relationship has modified the drug action mechanism
at the cellular and molecular levels [25, 26].
The present work came from our continuous interests in studying the chemothera-
peutic activity of half sandwiched ruthenium(II) complexes. 1,10-Phenanthroline (phen)
is a typical chelating bidentate ligand that shows novel properties by coordinating to
different transition metals [27–29]. It is a heteroaromatic system having beautifully
placed nitrogen atoms acting cooperatively, with remarkable characters such as rigidity,
planarity, hydrophobicity, and electron deficiency [30, 31]. Triarylamine (TAA) derivatives
exhibit various pharmacological activities. Organometallic half-sandwich Ir(III) complexes
containing TAA substituted bipyridyl ligands were able to oxidize NADH to NADþ effi-
ciently and they showed good binding property to DNA and bovine serum albumin
[32]. They hold an exceptional mode of interaction with DNA and are exclusively bound
to quadruplex DNA [33, 34]. Imidazole is important in the field of medicinal chemistry
as it is a constituent in biologically important molecules like histidine, histamine, biotin,
alkaloids, nucleic acids, etc. and has shown anticancer activity [35, 36].
In this investigation, we have designed and synthesized six new half-sandwich
ruthenium(II) arene complexes containing phen-TAA hybrids. The metal complexes
were synthesized by focused microwave-assisted reaction with high purity and yield.
Interaction modes of the complexes with DNA have been investigated by UV-Vis
absorption and emission spectroscopic techniques. The binding and cleavage proper-
ties of phen-metal complexes were modulated by functionalization of the phen unit.
The introduction of electron-rich substituents on the TAA side influences the rate of
interaction with CT-DNA.
2. Experimental
2.1. Materials and methods
All reagents and solvents were purchased from commercial sources and used as
received without purification. [RuCl2(g6-p-cymene)]2 was synthesized as per the litera-
ture method [37]. Phen-TAA hybrids were newly synthesized in our research laboratory
[38]. 1H and 13C NMR spectra were collected on a Bruker 400 MHz spectrometer in
CDCl3. Mass spectral analysis was performed on a Thermo Scientific Exactive Plus
UHPLC MS spectrometer. FT-IR spectra were recorded on a Perkin Elmer spectrometer.
UV-Vis spectra were acquired on a Jasco spectrophotometer. Emission spectra were
obtained from the Perkin Elmer spectrophotometer. Thermogravimetric analyses were
done on a Perkin Elmer thermogravimetric analyzer with a ramp rate of 10 ꢂC per
minute from 30 to 800 ꢂC under an N2 atmosphere.
2.2. Synthesis of metal complexes
[Ru(g6-p-cymene)Cl2]2 was prepared by dissolving 0.5 g of ruthenium(III) chloride in
25 mL of ethanol and sonicated for 15 min in a round bottom flask. a-Pellandrene
(4 mL) was added to the reaction mixture, stirred to get a clear solution, and conse-
quently refluxed for four hours. The obtained clear red solution was kept in the deep

JOURNAL OF COORDINATION CHEMISTRY
841
freezer for 2 days. The obtained reddish-brown crystals were washed with methanol
and dried in vacuum to get a 76% yield of [Ru(g6-p-cymene)Cl2]2.
TAA aldehydes were initially synthesized as per the procedure reported [39]. The
phen-TAA hybrid ligands were prepared by heating a mixture of 1,10-phenanthroline-
5,6-dione (5 mmol), ammonium acetate (100 mmol), corresponding TAA aldehydes
(6 mmol), and glacial acetic acid (4 mL) at 100 ꢂC in 250 watt for about 20 min in a
CEM microwave synthesizer. The reaction mixture was poured into ice to get a yellow
precipitate and was collected by filtration using Whatman 40 filter paper. The precipi-
tate was washed with ammonia solution to neutralize and consequently, washed with
hot water. The precipitate was dried to get yellow solid in 98% yield.
[(C6H6)RuCl2]2 (0.1 mmol) and phen-TAA ligands (0.2 mmol) in 12 mL of dichlorome-
thane were allowed to react in a CEM microwave synthesizer for 30 min (pre-stirring:
2 min., temperature: 60 ꢂC, power: 250 watt, pressure: 275 PSI). The addition of hexane
to the reaction mixture gave an orange-yellow solid. The solid was collected by filtra-
tion, washed with hexane several times, and air-dried to get 91% of the product.
[(g6-p-cym)RuII(1)(Cl)]Cl, 7: Yield: 95%. ESI-MS Calcd for C41H35Cl2N5Ru [M-Cl]þ,
1
734.162; found, 734.144. H NMR (400 MHz, CDCl3) d (ppm): 13.731 (s, 1H), 13.1539 (d,
J ¼ 5.6 Hz, 1H), 12.192 (d, J ¼ 5.6, 1H), 11.985 (d, J ¼ 12.4 Hz, 1H), 11.868 (s, 1H), 11.267
(t, J ¼ 6.8 Hz, 3H), 11.0875 (m, 6H), 10.1265 (d, J ¼ 5.2 Hz, 1H), 9.345 (d, J ¼ 5.6 Hz, 1H),
6.500 (s, 6H), 6.214 (s, 3H), 5.180 (s, 3H), 5.790 (d, J ¼ 5.6 Hz, 6H), 3.930 (s, 1H). 13C NMR
(100 MHz, CDCl3) d (ppm): 145.81, 128.41, 126.87, 124.23, 122.84, 120.73, 102.99, 83.05,
38.79, 32.89, 29.55, 20.79, 17.93.
[(g6-p-cym)RuII(2)(Cl)]Cl, 8: Yield: 90%. ESI-MS Calcd for C55H47Cl2N5O2Ru [M–Cl]þ,
1
946.246; found, 946.242. H NMR (400 MHz, CDCl3) d (ppm): 14.78 (s, 1H), 9.505 (s, 2H),
8.958 (s, 2H), 8.376 (s, 3H), 7.798 (s, 1H), 7.695 (s, 1H), 7.492 (s, 1H), 7.216 (s, J ¼ 6.8 Hz,
4H), 6.952 (d, J ¼ 7.6 Hz, 4H), 5.902 (m, 3H), 2.616 (s, 1H), 2.229 (s, 4H), 1.862 (s, 6H),
1.256 (s, 3H), 0.932 (s, 6H).13C NMR (100 MHz, CDCl3) d (ppm): 158.95, 149.40, 145.54,
143.40, 136.18, 136.21, 133.21, 128.61, 128.02, 127.61, 126.01, 125.51, 125.41, 122.19,
114.19, 113.20, 86.47, 86.97, 83.97, 55.34, 31.07, 30.90, 29.69, 22.05, 18.83.
[(g6-p-cym)RuII(3)(Cl)]Cl, 9: Yield: 92%. ESI-MS Calcd for C49H39Cl2N5S2Ru [M-Cl]þ,
1
898.137; found, 898.135. H NMR (400 MHz, CDCl3) d (ppm): 15.026 (s, 1H), 10.169 (s,
1H), 9.501 (d, J ¼ 19.6 Hz, 2H), 9.108 (s, 1H), 8.476 (s, 2H), 7.868 (s, 1H), 7.744 (s, 1H),
7.511 (d, J ¼ 7.6 Hz, 4H), 7.386 (m, 6H), 7.11 (d, J ¼ 6.8 Hz, 3H), 5.909 (m, 2H), 2.935 (m,
1H), 2.636 (s, 1H), 2.227 (s, 2H), 1.725 (s, 2H), 1.256 (s, 3H), 0.954 (s, 6H). 13C NMR
(100 MHz, CDCl3) d (ppm): 145.96, 141.77, 131.30, 128.71, 127.50, 126.22, 125.41,
122.56, 119.66, 86.45, 84.24, 9931.17, 29.97, 21.97, 18.84.
[(g6-p-cym)RuII(4)(Cl)]Cl, 10: Yield: 91%. ESI-MS Calcd for C53H43Cl2N5Ru [M–Cl]þ,
1
886.225; found, 886.232. H NMR (400 MHz, CDCl3) d (ppm): 14.921 (s, 1H), 10.099 (s,
1H), 9.563 (d, J ¼ 10 Hz, 3H), 9.081 (s, 1H), 8.482 (s, 1H), 7.854 (s, 1H), 7.737 (s, 1H),
7.581 (d, J ¼ 6.8 Hz, 3H), 7.520 (d, J ¼ 7.2 Hz, 3H), 7.421 (t, J ¼ 6.4 Hz, 3H), 7.335 (m, 2H),
7.227 (d, J ¼ 6.4 Hz), 5.919 (m, 4H), 2.612 (s, 1H), 2.194 (s, 2H), 1.928 (s, 3H), 1.257 (s,
3H), 0.921 (s, 6H). 13C NMR (100 MHz, CDCl3) d (ppm): 145.96, 141.77, 131.30, 127.50,
126.22, 25.41, 122.56, 119.66, 86.45, 84.24, 31.17, 29.70, 21.97, 18.87.
[(g6-p-cym)RuII(5)(Cl)]Cl, 11: Yield: 89%. ESI-MS Calcd for C41H33Cl2I2N5Ru [M-Cl]þ,
1
985.955; found, 985.982. H NMR (400 MHz, CDCl3) d (ppm): 15.097 (s, 1H), 10.222 (s,

842
R. ABHIJNA KRISHNA ET AL.
1H), 9.561 (s, 1H), 9.483 (s, 1H), 9.174 (s, 1H), 8.513 (s, 1H), 7.910 (s, 1H), 7.780 (s, 1H),
7.564 (d, J ¼ 6.8 Hz, 4H), 7.138 (d, J ¼ 6.8 Hz, 1H), 6.881 (d, J ¼ 7.2 Hz, 2H), 5.913 (m, 3H),
2.675 (s, 1H), 2.246 (s, 2H), 1.675 (s, 6H), 1.255 (s, 3H), 0.982 (s, 3H). 13C NMR (100 MHz,
CDCl3) d (ppm): 148.38, 146.55, 143.75, 138.63, 128.90, 126.74, 123.28, 86.97, 31.16,
29.70, 22.19, 22.03.
[(g6-p-cym)RuII(6)(Cl)]Cl, 12: Yield: 92%. ESI-MS Calcd for C65H59Cl2N5Ru [M-Cl]þ,
1
1046.350; found, 1046.356. H NMR (400 MHz, CDCl3) d (ppm): 15.052 (s, 1H), 10.197 (s,
1H), 9.613 (s, 1H), 9.131 (s, 1H), 8.573 (s, 1H), 8.506 (s, 1H), 7.903 (m, 3H), 7.456 (t,
J ¼ 6.8 Hz, 5H), 7.456 (t, J ¼ 6.8 Hz, 5H), 7.366 (d, J ¼ 5.6 Hz, 4H), 7.222 (d, J ¼ 7.2 Hz, 2H),
7.099 (d, J ¼ 9.2 Hz, 3H), 5.904 (m, 3H), 4.199 (s, 1H), 2.658 (s, 1H), 2.236 (s, 3H), 1.803
(s, 3H), 1.324 (s, 18H), 0.960 (s, 6H). 13C NMR (100 MHz, CDCl3) d (ppm): 151.42, 132.84,
131.32, 129.12, 125.64, 124.39, 120.35, 34.80, 34.44, 31.37, 31.20, 29.70, 22.17,
22.04, 18.95.
2.3. DNA interaction studies
UV-Vis Absorption and fluorescence studies: The interaction mode of complexes with
CT-DNA was explored by UV-vis absorption and fluorescence emission studies. The
experiments were performed in 50 mMNaCl/5 mM trisHCl (pH 7.2) solution at room
temperature. The calf-thymus DNA (CT-DNA) was prepared by sonicating it in tris-HCl/
NaCl buffer for three days at 4 ꢂC and stored at 4 ꢂC. The DNA was free of proteins as
indicated by the absorption ratio 1.9 of the stock solution of CT-DNA at kmax 260 and
280 nm. Ten-fold dilution of the bulk DNA solution was done to show maximum
absorbance at 260 nm with the absorption coefficient value of 6600 Mꢁ1 cmꢁ1 per
nucleotide. The titration experiments were made by keeping the complex concentra-
tion constant (5 mM) and varying the CT-DNA concentration (20–50 mM). Samples were
equilibrated before recording each spectrum [40, 41] and a significant absorbance
change was noticed. The intrinsic binding constant (Kb) of the complexes with CT-DNA
was quantitatively calculated from the following equation to compare the effect of dif-
ferent substitutions [41]:
½
ꢃ
DNA
½
ꢃ
¼ DNA =ðeb ꢁ ef Þ þ 1=Kbðeb ꢁ ef Þ
e ꢁ e
a
ð
f Þ
where [DNA] ¼ concentration of DNA in base pairs, ea (apparent extinction coefficient) ¼
observed absorbance/[complex] and ef (extinction coefficient for free complex) ¼ absorb-
½
ꢃ
ance for blank solution/[complex]. Kb was calculated from the plot of DNA =ðea ꢁ ef )
versus [DNA] by taking the ratio of the slop and the intercept. The competitive binding
study of the metal complexes with a known intercalator, ethidium bromide (EB), was
conducted to further understand the binding mode of complexes with CT-DNA. EB solu-
tion was prepared in Tris-HCl/NaCl buffer. The change in fluorescence emission intensity
at 599 nm was recorded.
3. Results and discussion
The metal complexes were designed with the three coordination sites of ruthenium in
[(g6-arene)RuII(X)(Y)(Z)]þ is occupied by the arene moiety and the other ligands occupy

JOURNAL OF COORDINATION CHEMISTRY
843
the rest three coordination sites which resemble the piano stool. The arene group pro-
vides a hydrophobic nature to the complex that helps it to enter the cells by stabiliz-
ing þ2 oxidation state of the ruthenium. The phen unit of the ligand is highly capable
of making chelation with the metal ions. TAA substitution together with the formation
of imidazole moiety in the phen unit enhances the DNA binding activity of the
metal complex.
The phen-TAA hybrids (1–6) were newly synthesized (Scheme 1) and systematically
characterized. The complexes [(g6-p-cymene)Ru(g2-N,N-L)Cl]Cl (L ¼ 1–6) were synthe-
sized from phen-TAA hybrids (1–6) and [RuCl2(g6-p-cymene)]2 precursors in 2:1 molar
ratio in DCM at 60 ꢂC in a CEM microwave synthesizer. All the complexes are orange-
yellow solids, air-stable, non-hygroscopic, and highly soluble in chloroform, dichloro-
methane, dimethylformamide, dimethylsulphoxide, methanol, and acetonitrile.
3.1. FT-IR and NMR characterization
IR absorption observed in the range 1583–1617 cmꢁ1 and 1456–1569 cmꢁ1 corre-
sponds to the groups m(C@N) and m(C@C), respectively. A negative shift of ꢄ60 cmꢁ1
in m(C@N) in comparison to the ligand is suggestive of coordination of ruthenium ion
is through the nitrogen atoms. The broad band observed in the range
1
3013–3066 cmꢁ1 corresponds to N-H stretching frequency of the ligand. The H NMR
spectra of complexes were recorded in CDCl3. The aliphatic protons of p-cymene moi-
ety were shielded and gave sharp singlets in the range of 0.925–0.985 ppm. Another
set of singlets around 1.256–1.800 ppm correspond to the 3H of the p-cymene. The N-
H signal was observed in the highly deshielded region of 10–15 ppm as compared to
that of the ligand N-H protons also supports the coordination of the ligands to metal.
In the 13C NMR spectra of the complexes, the signals that appeared around
18–29 ppm and 84–86 ppm were assigned to the aliphatic and aromatic carbon atoms
in the p-cymene group, respectively. Peaks in the range of 145–158 ppm and
132–149 ppm correspond to the -C ¼ N and -C-N; these peaks appeared to be slightly
deshielded from the ligand peaks [11, 38]. The signals for all other protons and car-
bons were located in the appropriate regions. TGA was done at the ramp rate of 10 ꢂC
in the temperature range of 25 to 800 ꢂC. The compounds show a single step of
decomposition in the range of 330 to 370 ꢂC and the order of thermal stability was
observed to be 9 > 12 > 10 > 8 > 11 > 7. The high decomposition temperature of
Figure 1. (a) Absorption and (b) emission spectra of 7–12.

844
R. ABHIJNA KRISHNA ET AL.
Table 1. Photophysical properties and binding constant values of 7–12.
kabs
(nm)
Absorption coefficient
Binding constant
Complex
kem (nm)
Stokes’ shift (cmꢁ1
)
e (104 Mꢁ1 cmꢁ1
)
Kb (1 ꢀ 104 Mꢁ1
)
7
8
9
10
11
12
310, 362
298, 363
298, 364
304, 364
317, 362
304, 371
480
487
474
466
463
434
6790
7014
6375
6013
6026
3912
4.063
5.317
5.022
5.923
4.970
6.603
3.487
5.053
4.509
4.908
4.103
4.252
thiophene and tert-butylphenylacetylene substituted metal complexes 9 and 12 is due
to extended molecular architecture. Metal complexes without substitution 7 and with
iodine substitution have the lowest decomposition temperature (thermograms are
given in Figure S25).
3.2. Photophysical studies
UV-vis absorption and emission studies were carried out to understand the photophys-
ical behavior of metal complexes. Absorption behavior was investigated in DMF (10ꢁ5
M) (Figure 1(a)) and data are represented in Table 1. Complexes 7–12 exhibited intra-
ꢅ
ligand p-p transition bands around 306–317 nm with a bathochromic shift of
35–18 nm. The absorption band around 364–370 nm corresponds to n-p transition
ꢅ
and shows a hypsochromic shift of 2–8 nm as compared to the free ligand. This differ-
ence occurs because, during complex formation, the orbitals of the nitrogen atom in
ꢅ
ꢅ
the formation of molecular bonding levels have variation in energy for p-p and n-p
transitions [42]. A new absorption band around 515–518 nm corresponds to d-d
transition of the metal ion electrons. Complex 12 exhibited the highest absorption
coefficient (6.603 ꢀ 10ꢁ4 Mꢁ1 cmꢁ1) due to the efficient electron delocalization of
tert-butylphenyl substituent. Besides, the acetylene group improved electronic distri-
bution. Complexes 8, 9 and 10 also exhibited good absorbance due to the presence
of methoxyphenyl, thiophene, and phenyl groups, respectively, as the substituents
capable of enhancing conjugation. Complexes 7 and 11 having –H and –I, respect-
ively, exhibited poor absorbance due to the lower conjugation and absence of effi-
cient auxochromic groups. All the complexes exhibited a greater absorption
coefficient than that of the ligand, due to spin and Laporte allowed ligand to metal
charge transition. This explains the change of the transitions towards higher wave-
lengths [38]. Emission spectra were recorded in DMF (10ꢁ7 M) and the spectra are pre-
sented in Figure 1(b). All the complexes exhibited significant Stokes shifts, as shown in
Table 1. The shape of emission spectra is not mirrored images of the absorption spec-
tra, indicating the geometrical changes of the molecule in the excited state [43].
3.3. DNA-binding studies
3.3.1. Electronic absorption studies
UV spectra of the complexes in the presence and absence of CT-DNA are shown in
Figure 2. With increase in the concentration of CT-DNA, absorption bands exhibited
hypochromism along with a hypsochromic shift of 1–8 nm, suggesting an intimate

JOURNAL OF COORDINATION CHEMISTRY
845
Figure 2. Representative absorption spectra of complexes with CT-DNA in Tris-HCl buffer.
ꢅ
association via intercalation [44]. During this process, the p orbital of the metal com-
plex will couple with the p orbitals of the base pairs, thereby decreasing the energy of
ꢅ
p-p transition. Therefore, these interactions resulted in hypochromism [45]. The mag-
nitude of hypsochromic shift and hypochromism indicates the binding strength of
complexes [46].
Intrinsic binding constant values are calculated by putting each set of data into the
equation [DNA]/(eaꢁef) ¼ [DNA]/(ebꢁef) þ 1/[Kb(ebꢁef)]. It gave a straight line with a
slope of 1/(ebꢁef) and y-intercept of 1/[Kb(ebꢁef)] (Table 1, Figure S32). The calculated
values of binding constants were in a similar range of other reported complexes [46].
The binding affinity of complexes is the order of 8 > 10 > 9 > 12 > 11 > 7. The higher
binding affinity of methoxyphenyl-substituted TAA-phen complex is due to the ten-
ꢅ
ꢅ
dency to donate electrons to the p orbital [44]. This will also stabilize the p orbital
and reduces the energy gap between the p orbital of DNA and p orbital of the metal
ꢅ
complex, resulting in efficient coupling [45]. Complex 10 with phenyl substitution has
a higher binding constant than thiophene-substituted 9 due to effective delocalization.
The unsubstituted metal complex 7 has the lowest binding constant because of the
absence of any activating groups. The presence of azole moiety in the ligand can favor
the interaction through p–p interaction and H-bonding and thus responsible for the
good binding constant values. The higher affinity of imidazole moiety in DNA binding
and its anticancer activity has been reported [47].
3.3.2. Fluorescence spectroscopic studies
The studies were carried out in Tris-HCl/NaCl buffer (pH 7.3). The titrations were done
by keeping the complex concentration fixed (5 mM) and varying the concentration of
CT-DNA (5–45 mM). Since all the complexes were fluorescent in solution, EB was not
used for the binding studies [48]. A gradual increase in the emission intensity was
observed on the incremental addition of DNA to the complex solution. The enhance-
ment of fluorescence is due to the penetration of the complex into the hydrophobic
environment of the DNA, thereby avoiding the quenching effect of solvent water

846
R. ABHIJNA KRISHNA ET AL.
Figure 3. Representative emission spectra of complexes with varying addition of CT-DNA in Tris-
HCl buffer.
Figure 4. Representative emission spectra of EB bound CT-DNA with the addition of varying con-
centrations of a metal complex solution.
molecules [49]. This will allow greater electrostatic interaction between the metal com-
plex and DNA in a more rigid and hydrophobic environment with less access to solv-
ent molecules. The marked increase in the emission intensity by the binding of the
complex to the CT-DNA agrees with those observed for other intercalators [48]. The
hypsochromic shift of 60–67 nm observed for 7–10 may be due to the difference in
the mode of interaction of these complexes compared to 11 and 12. These results
show that the complex bound more strongly to the CT-DNA. The emission spectra of
complexes with CT-DNA in Tris-HCl buffer is shown in Figure 3.
3.3.3. Comparative binding study with EB
The comparative binding of the metal complex with CT-DNA was performed to under-
stand the binding mode through intercalation or groove binding in nature. The titra-
tion was performed by taking a 1:1 ratio of CT-DNA (2.5 mM) and EB (2.5 mM) solution

JOURNAL OF COORDINATION CHEMISTRY
847
Scheme 1. Synthetic route to phen-TAA hybrid ligands (1–6), half-sandwiched Ru(II) arene com-
plexes (7–12) and their molecular structure.
and varying concentrations of metal complex solution (2.5–20 mM). The emission inten-
sity was observed to increase gradually on the incremental addition of the metal com-
plex [50]. Thus the experiment shows that the addition of metal complex to the
solution does not affect the emission intensity of the EB. This observation confirms
that the two probes (EB and CT-DNA) bound independently and do not depend on
the other [50]. Thus it reveals that the metal complex also binds through groove-bind-
ing to CT-DNA since EB is a known intercalator [51, 52]. The emission spectra of the
comparative binding study of EB bound DNA with the metal complex is shown in
Figure 4.
4. Conclusion
Six new ruthenium(II) arene complexes containing phenanthroimidazole-TAA hybrid
ligands were synthesized using a microwave synthesizer in good yield with high

848
R. ABHIJNA KRISHNA ET AL.
purity. All the complexes were characterized by analytical and spectral techniques. The
metal complex substituted with thiophene is found to be the most thermally stable as
observed from TGA studies. Interaction of the complexes with CT-DNA was investi-
gated using UV-vis absorption and fluorescence spectroscopic techniques. UV-Vis
absorption studies give information about the intercalative binding of complexes. The
binding ability of the complexes was calculated and the methoxyphenyl-substituted
was possessing good binding efficiency with a Kb value of 5.053 ꢀ 104 Mꢁ1. The effi-
cient intercalative binding ability of this complex may be due to its good electron-
donating substituent. Emission spectra give information regarding the groove binding
nature of complexes. Thus, since our metal complex of interest has several functional
centers, it is found to interact with CT-DNA through intercalation as well as
groove binding.
Acknowledgement
The authors thank the Central University of Tamil Nadu, Thiruvarur, India for the
research facilities.
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