Luminescent ruthenium(II)-para-cymene complexes of aryl substituted imidazo-1,10-phenanthroline as anticancer agents and the effect of remote substituents on cytotoxic activities

Article abstract of DOI:10.1016/j.ica.2020.120066

Ruthenium complexes are currently significant attention in medicinal chemistry as they offer various properties which make them an appropriate choice for drug development. Herein, a series of ruthenium(II)-p-cymene-2-aryl-imidazo-1,10-phenanthroline derivatives have been prepared and characterised by elemental analysis, infrared, LC-mass and NMR techniques. The structural and chemical properties shows that Ru(II) complexes have got rigidity, planarity, aromaticity, hydrogen donating and accepting capability which aids both solubility and interaction with biomolecules. The binding strength of these complexes with DNA and BSA were found to be 10⁴–10⁶ M^?1. The competitive displacement of ethidium bromide (EtBr) from DNA in the presence of complex reveals an intercalation or groove binding further this was supported by viscosity and in-silico studies. The cytotoxicity study of these Ru(II) complexes were conducted with two cancer cell lines (MDA-MB-231 and HeLa) and one human embryonic kidney cells (HEK-293). The study revealed that [(η⁶-p-cymene)RuCl (κ²-N,N-2-(4-fluorophenyl)-1H-imidazo[4,5-f][1,10]Phenanthroline].PF₆ (4e), [(η⁶-p-cymene)RuCl(κ²-N,N-2-(4-bromophenyl)-1H-imidazo[4,5-f][1,10]Phenanthroline].PF₆ (4f) and [(η⁶-p-cymene)RuCl(κ²-N,N-2-(4-nitrophenyl)-1H-imidazo[4,5-f][1,10]Phenanthro line].PF₆ (4g) were found exhibit least inhibitory concentration (IC₅₀) and high selectivity with respect to HeLa and MDA-MB-231. The activity of the Ru(II) complexes were position and substituents dependent.

Full text of DOI:10.1016/j.ica.2020.120066

InorganicaChimicaActa515(2021)120066
Contents lists available at ScienceDirect
Inorganica Chimica Acta
journal homepage: www.elsevier.com/locate/ica
Research paper
Luminescent ruthenium(II)-para-cymene complexes of aryl substituted
imidazo-1,10-phenanthroline as anticancer agents and the effect of remote
substituents on cytotoxic activities
⁎
Sourav Dea, R. Selva Kumara, Ashna Gauthamanb, S.K. Ashok Kumara, , Priyankar Pairaa,
Anbalagan Moorthyb, Subhasis Banerjeec
a Department of Chemistry, School of Advance Sciences, Vellore Institute of Technology, Vellore-632014, Tamil Nadu, India
b Department of Biotechnology, School of Bioscience & Technology, Vellore Institute of Technology, Vellore 632014, Tamil Nadu, India
c Department of Pharmaceutical Chemistry, Gupta College of Technological Sciences, Asansol 713301, West Bengal, India
A R T I C L E I N F O
A B S T R A C T
Keywords:
DNA
Ruthenium complexes are currently significant attention in medicinal chemistry as they offer various properties
which make them an appropriate choice for drug development. Herein, a series of ruthenium(II)-p-cymene-2-
aryl-imidazo-1,10-phenanthroline derivatives have been prepared and characterised by elemental analysis, in-
frared, LC-mass and NMR techniques. The structural and chemical properties shows that Ru(II) complexes have
got rigidity, planarity, aromaticity, hydrogen donating and accepting capability which aids both solubility and
interaction with biomolecules. The binding strength of these complexes with DNA and BSA were found to be
104–106 M−1. The competitive displacement of ethidium bromide (EtBr) from DNA in the presence of complex
reveals an intercalation or groove binding further this was supported by viscosity and in-silico studies. The
cytotoxicity study of these Ru(II) complexes were conducted with two cancer cell lines (MDA-MB-231 and HeLa)
and one human embryonic kidney cells (HEK-293). The study revealed that [(η6-p-cymene)RuCl (κ2-N,N-2-(4-
BSA
Gel electrophoresis
MTT
Intercalation
In-silico study
fluorophenyl)-1H-imidazo[4,5-f][1,10]Phenanthroline].PF6
(4e),
[(η6-p-cymene)RuCl(κ2-N,N-2-(4-bromo-
phenyl)-1H-imidazo[4,5-f][1,10]Phenanthroline].PF6 (4f) and [(η6-p-cymene)RuCl(κ2-N,N-2-(4-nitrophenyl)-
1H-imidazo[4,5-f][1,10]Phenanthro line].PF6 (4g) were found exhibit least inhibitory concentration (IC50) and
high selectivity with respect to HeLa and MDA-MB-231. The activity of the Ru(II) complexes were position and
substituents dependent.
1. Introduction
wide spectrum of tumours and this has opened door for development
several platinum drugs, i.e., carboplatin, oxaloplatin worldwide [5-7].
Cancer is the second life-threatening diseases after cardiovascular
and has a major impact on society. It is caused by mutation in genes,
which leads to atypical and unrestricted cell growth. Treatment of any
cancer aims to remove or destroy the cancerous cells without killing
normal cells. The most common types of treatment for cancer include
surgery, radiation, and chemotherapy which can be used either alone or
in combination with each other or other therapies. In chemotherapy,
the use of drugs to kill cancer cells by inhibiting cell division process
but their ultimate drawback is non-specificity towards normal cells,
which results in unwanted adverse drug reaction [1]. In this context,
organometallic compounds have recently been found to be promising
anticancer drug candidates [2-4]. The serendipitous discovery of cis-
platin as the first FDA (Food Drug Administration) approved Pt(II)
metal-based complex by Rosenberg in 1965 was useful clinically for
However, their therapeutics applications are being strongly restricted
due to poor aqueous solubility, toxicity to normal cells and drug re-
sistant problems [8-11]. The new strategies for the design of new an-
ticancer drugs is finding an alternative metal centre to platinum that
possesses new structures and modes of action to overcome the draw-
backs associated with cisplatin therapy [12-19]. It exhibits similar li-
gand exchange kinetics to platinum under physiological conditions, and
are less toxic than platinum drugs. This low toxicity is supposed to
relate to the redox potential of Ru(II) complexes under physiological
conditions. The rate of ligand exchange (10−2–10−3 s−1) related with
labile chlorine with water. Further, an ability to mimic iron in binding
to specific biomolecules such as albumin and transferrin in the blood
stream, which is beneficial for its delivery to cells with negligible side
effects, making ruthenium complexes a proper choice as efficient
⁎ Corresponding author.
https://doi.org/10.1016/j.ica.2020.120066
Received 13 August 2020; Received in revised form 1 October 2020; Accepted 1 October 2020
Availableonline07October2020
0020-1693/©2020ElsevierB.V.Allrightsreserved.

S. De, et al.
InorganicaChimicaActa515(2021)120066
Fig. 1. Molecular structures of (a) clinically approved (b) previously reported Ru(II)- imidazo-1,10-phenanthroline compounds.
anticancer drugs [20-22]. In this perspective, a recent advances in the
discovery of Ru(III) complexes such as NAMI-A, (N)KP1019 and
KP1339 and Ru(II)-based therapeutic TLD1433 with proven anti-pro-
liferative activity (Fig. 1). But, the poor solubility of KP1019 and low
therapeutic index of NAMI-A restrict their entry in phase II clinical
trials [23-26]. Besides, KP1339 clinically approved Ru(II)-arene com-
plexes (AH54 and AH63), which are active in the radio sensitization of
human colorectal cancer cells and RAPTA-C in human ovarian carci-
noma cells (A2780) [27-29].
purchased from Thermo-Fisher Scientific. Thin layer chromatography
was performed on pre-coated silica gel 60 F254 aluminium sheets (E.
Merck, Germany) using ethyl acetate and methanol (2:1, v/v) mixture
and spots were recognized under both normal light and UV light. The
melting point of all the compounds were measured on an Elchem
Microprocessor based DT apparatus using open capillary tube and are
uncorrected. NMR spectra (1H, 13C, 31P) were recorded on a Bruker 400
Variant at 400 MHz spectra and 31P NMR chemical shift was recorded
in ppm using 30% H3PO4 as an internal standard. Tetramethylsilane
(TMS) was used as internal reference and the chemical shifts are re-
ported in ppm unit. Mass data was carried out on a Schimadzu LC-MS-
4000 instrument having 4000 triple quadrupole MS using methanol as
the solvent. The reported m/z peak values stand for the major peaks
with an isotopic distribution. Infrared spectra (IR) were recorded on a
The search for more biologically active complexes related to imi-
dazole derivatives encompassing a diverse range of biological activities
due to its electron-rich and behaves as a strong σ-donor to surrounding
environment. A small change in electronic properties can significantly
change the hydrogen-bond donating or accepting properties [22,30-
32]. Further, previously reported, Ru(II) complexes of 2-aryl sub-
stituted imidazophenanthroline ligands as best DNA intercalator by
affording planarity [33-39] shown in Fig. 1. However, the development
of novel Ru(II)-arene complexes with extended π-conjugation is still a
challenge for target-oriented cancer therapy and cellular imaging
[40,41].
Shimadzu Affinity FT-IR spectrometer in the range of 4000–400 cm−1
.
UV–Visible spectra were recorded on a JASCO V-760 spectrometer and
fluorescence measurements were carried out using JASCO fluorescence
spectrophotometer (FP8200) equipped with a xenon and helium lamp
using 1 cm quartz cell. The ELISA reader and 96-well plates were used
for the MTT assay.
An ample literature survey show that there is no study on synergic
effect of Ru(II) para-cymene and position of variety of substituent
groups on phenyl core fused with phenanthroline imidazole. In con-
tinuation of our ongoing work on anticancer organo-ruthenium [42-
46], we have designed complexes comprising ligand (aryl substituted
imidazo-1,10-phenanthroline) and co-ligand [Ru(II)-p-cymene] having
some characteristics functional units such as (i) a labile chlorine atom
which helps the breakage of Ru-Cl bond by nucleophilic attack and
generates a reactive site on ruthenium, subsequent binding with other
biomolecules like proteins, thiols and DNA bases under physiological
conditions [47] (ii) η6-arene moieties which stabilize the oxidation state
of central metal, bind to receptor surfaces and assist their transporta-
tion through cell membrane [48,49] (Fig. 2).
2.2. Synthesis of Ru(II) based Imidazo-1,10-phenanthroline derivatives
(4a-4j)
Initially, 100 mg (0.475 mmol) of 1,10-phenanthroline-5,6-dione
(1) was taken in a round-bottom flask and 1.1 equivalent of benzal-
dehyde derivatives (2) was added followed by 294 mg (3.81 mmol, 8
equiv.) of ammonium acetate. The contents were dissolved in a mixture
of glacial acetic acid (7 mL) and water (3 mL) and sonicated the reac-
tion mixture for about 7–8 h at 100 °C in the presence of 2–3 drops of
sulphuric acid (Scheme 1). The progress of the reaction was monitored
by TLC using ethyl acetate: methanol (2:1, v/v) solvent mixture. Later,
it was transfer to ice-cold water followed by addition of aqueous am-
monia and kept aside for 1 h. The obtained precipitate was separated by
common filtration and kept for air drying for about 1 h. Further, the
compound was washed with hexane and ethyl acetate mixture (3:1, v/
v) to remove associated impurities. The pure product was obtained by
recrystallization using diethyl ether offer a solid powder in good yield
(~85–95%). In order to prepare Ru(II) complexes (4a-4j), one
equivalent of dichloro(p-cymene)Ru(II)dimer (50 mg, 0.081 mmol) was
dissolved in 10 mL of water in round bottom flask kept for sonication
for 15 min. Subsequently, 2.1 equivalent of previously prepared ligand
2. Experimental
2.1. Material and methods
All analytical grade solvents and reagents were purchased from SD
fine chemicals, India. Various benzaldehyde derivatives were obtained
from Alfa Aesar while ammonium acetate, ammonia, 1,10-phenan-
throline-5,6-dione and dichloro-p-cymene ruthenium(II)chloride were
2

S. De, et al.
InorganicaChimicaActa515(2021)120066
Fig. 2. Structural features of ruthenium (II)-p-cymene-2-aryl-imidazo-1,10-phenanthroline derivatives.
3a-3j (3a: 50.8 mg, 3b: 50.3 mg, 3c: 50.6 mg, 3d: 50.9 mg, 3e:
50.8 mg, 3f: 50.6 mg, 3g: 50.5 mg, 3h: 50.3 mg, 3i: 50.7 mg, 3j:
50.8 mg) was added to the reaction mixture followed by sonication for
about 2 h at room temperature. A significant color changes occurred in
the reaction mixture (yellow solution to brown precipitate). In order to
get crystalline product, ammonium hexafluorophosphate (32.5 mg
(0.199 mmol, 2.5 equiv.) was added to the reaction mixture and al-
lowed reaction to complete in 1 h. The progress of the reaction was
observed by TLC using pure methanol as eluent. After completion of the
reaction, water was evaporated by rotary evaporator. The obtained
crude product was washed with hexane and recrystallized by using
ether and methanol in solvent mixture (95:5, v/v) to get the desired
product with good yield (~88–91%). The possible mechanism for final
product formation has been depicted in Scheme S1.
J = 8 Hz, CH), 8.99 (d, 2H, CH) (Fig. S8); LC-MS: 313.1 [M + 1]+ (Fig.
S9).
2.2.4
4-(1H-imidazo[4,5-f][1,10]Phenanthroline-2-yl)-N,N-di-
methylaniline (3d): 4-dimethyl amino benzaldehyde (72 mg,
0.483 mmol); yield: 92%; Mw (C21H17N5): 339.40 g mol−1; m.p:
305–307 °C; Rf: 0.39 (2% ethyl acetate in methanol); FT-IR spectra
(cm−1): 3186 (CeH stretching), 2810 (alkyl CeH stretching), 1608
(NeH bending), 1485 (C]C stretching), 1192 (CeN stretching) (Fig.
S10); 1H NMR (400 MHz, DMSO‑d6): δ 6.51 (d, 1H, J = 8 Hz, CH), 6.81
(d, 1H, J = 8 Hz, CH), 7.47 (s, 1H, CH), 7.72 (m, 1H, CH), 7.94 (d, 1H,
CH), 8.20 (d, 1H, J = 2 Hz, CH), 8.67 (d, 2H, CH), 8.95 (d, 2H, CH),
9.13 (m, 1H, CH), 2.91, 3.10 (s, 6H, CH3) (Fig. S11); LC-MS: 340.0
[M]+ (Fig. S12).
2.2.5
2-(4-flurophenyl)-1H-imidazo[4,5-f][1,10]Phenanthroline
2.2.1
2-phenyl-(1H-imidazo[4,5-f][1,10]Phenanthroline
(3a):
(3e): 4-fluro benzaldehyde (50 μl, 0.466 mmol); yield: 93%; Mw
(C19H11FN4): 314.32 g mol−1; m.p: 304–306 °C; Rf: 0.51 (2:1 v/v, ethyl
acetate:methanol); FT-IR spectra (cm−1): 3055 (CeH stretching), 1606
(NeH bending), 1450 (C]C stretching), 1396 (CeN stretching), 1219
(C-F stretching) (Fig. S13); 1H NMR (400 MHz, DMSO‑d6): δ 7.45 (t, 2H,
J = 8 Hz, CH), 7.82 (m, 2H, CH), 8.32 (m, 2H, CH), 8.90 (d, 2H, CH),
9.02 (d, 2H, CH) (Fig. S14); LC-MS: 314.9 [M]+ (Fig. S15).
Benzaldehyde (48 μl, 0.470 mmol); yield: 95%; Mw (C19H12N4):
296.33 g mol−1; m.p: 307–309 °C; Rf: 0.49 (2% ethyl acetate in me-
thanol); FT-IR spectra (cm−1): 3159 (CeH stretching), 1552 (NeH
bending), 1458 (C]C stretching), 1394 (CeN stretching) (Fig. S1); 1H
NMR (400 MHz, DMSO‑d6): δ 7.40 (d, 1H, J = 8 Hz, ArH), 7.45 (d, 3H,
CH), 8.22 (d, 3H, J = 8 Hz, CH), 9.03 (s, 4H, CH) (Fig. S2); LC-MS:
297.3 [M + 1]+ (Fig. S3).
2.2.6 2-(4-bromophenyl)-(1H-imidazo[4,5-f][1,10]Phenanthroline
(3f):4-bromo benzaldehyde (90 mg, 0.486 mmol); yield: 85%; Mw
(C19H11BrN4): 375.23 g mol−1; m.p: 301–303 °C; Rf: 0.59 (2:1 v/v,
ethyl acetate: methanol); FT-IR spectra (cm−1): 2927 (CeH stretching),
1548 (NeH bending), 1398 (C]C stretching), 1273 (CeN stretching),
621 (C-Br stretching) (Fig. S16); 1H NMR (400 MHz, DMSO‑d6): δ 7.45
(t, 2H, CH), 7.82 (m, 2H, CH), 8.32 (t, 2H, CH), 8.90 (d, 2H, J = 4 Hz,
CH), 9.02 (d, 2H, CH) (Fig. S17); LC-MS: 374.8 [M]+ (Fig. S18).
2.2.2 2-(4-methoxyphenyl)-1H-imidazo[4,5-f][1,10]Phenanthroline
(3b): 4-methoxy benzaldehyde (58 μl, 0.477 mmol); yield: 93%; Mw
(C20H14N4O): 326.36 g mol−1; m.p: 309–311 °C; Rf: 0.48 (2:1 v/v, ethyl
acetate:methanol); FT-IR spectra (cm−1): 3014 (CeH stretching), 1610
(NeH bending), 1481 (C]C stretching), 1398 (CeN stretching) (Fig.
S4); 1H NMR (400 MHz, DMSO‑d6): δ 7.13 (d, 2H, J = 8 Hz, CH), 7.79
(m, 2H, CH), 8.25 (d, 2H, CH), 8.94 (d, 4H, CH), 3.84 (s, CH3O) (Fig.
S5); LC-MS: 327.2 [M + 1]+ (Fig. S6).
2.2.7
2-(4-nitrophenyl)-1H-imidazo[4,5-f][1,10]Phenanthroline
2.2.3 4-(1H-imidazo[4,5-f][1,10]Phenanthrolin-2-yl)phenol (3c): 4-
hydroxy benzaldehyde (48 μl, 0.481 mmol); yield: 92%; Mw
(C19H12N4O): 312.33 g mol−1; m.p: 303–304 °C. Rf: 0.49 (2:1 v/v, ethyl
acetate:methanol); FT-IR spectra (cm−1): 3196 (OH stretching), 3070
(CeH stretching), 1612 (NeH bending), 1481 (C]C stretching), 1182
(CeN stretching) (Fig. S7); 1H NMR (400 MHz, DMSO‑d6): δ 6.96 (t, 2H,
CH), 7.80 (m, 2H, CH), 8.10 (d, 2H, J = 8 Hz, CH), 8.89 (d, 2H,
(3g): 4-nitro benzaldehyde (72 mg, 0.476 mmol); yield: 89%; Mw
(C19H11N5O2): 341.33 g mol−1; m.p: 302–304 °C; Rf: 0.53 (2:1 v/v,
ethyl acetate:methanol); FT-IR spectra (cm−1): 3093 (CeH stretching),
1598 (NeH bending), 1450 (C]C stretching), 1330 (CeN stretching),
1504 and 1330 (–NO2 group) (Fig. S19); 1H NMR (400 MHz, DMSO‑d6):
δ 7.84 (m, 2H, CH), 8.48 (m, 4H, CH), 8.90 (d, 2H, CH), 8.90 (d, 2H,
J = 4 Hz, CH), 9.04 (d, 2H, CH) (Fig. S20); LC-MS: 341.7 [M]+ (Fig.
Scheme 1. Reagent and conditions to synthesis complexes (4a-4j).
3

S. De, et al.
InorganicaChimicaActa515(2021)120066
S21).
8.19 (s, 2H, ArH), 8.34 (d, 2H, J = 8 Hz, ArH), 9.33 (s, 1H, ArH), 9.84
(s, 2H, ArH) (Fig. S42); 31P NMR (DMSO‑d6, 162 MHz): δ −135.43 to
−152.98 (m, PF6) (Fig. S43); 19F NMR (DMSO‑d6, 376 MHz): δ −70.11
(6F, PF6) (Fig. S44); LC-MS (MeOH): m/z: 583.4 [M]+ (Fig. S45).
2.2.8 2-(1H-imidazo[4,5-f][1,10]Phenanthroline-2-yl)phenol (3h):
2-hydroxybenzaldehyde (50 μl, 0.479 mmol); yield: 94%; Mw
(C19H12N4O): 312.33 g mol−1; m.p: 300–302 °C; Rf: 0.48 (2:1 v/v, ethyl
acetate:methanol); FT-IR spectra (cm−1): 3062 (OH stretching), 2922
(CeH stretching), 1624 (NeH bending), 1433 (C]C stretching), 1256
(CeN stretching) (Fig. S22); 1H NMR (400 MHz, DMSO‑d6): δ 6.97 (m,
1H, OH), 7.15 (m, 1H, CH), 7.26 (t, 1H, CH), 7.54 (t, 1H, CH), 7.75 (t,
2H, J = 4 Hz, CH), 7.91 (m, 1H, CH), 8.21 (t, 1H, CH), 8.84 (d, 1H,
CH), 8.94 (s, 2H, CH), 9.15 (s, 1H, NH) (Fig. S23); LC-MS: 313.0 [M]+
(Fig. S24).
2.3.4
[(η6-p-cymene)RuCl(κ2-N,N-4-(1H-imidazo[4,5-f][1,10]
Phenanthroline-2-yl)-N,N- dimethylaniline].PF6 (4d): Yield: 91%; Mw
(C31H31N5ClF6PRu):
755.10
g
mol−1
;
Anal.
Calcd
for
C
31H31N5ClF6PRu: C 49.26, H 4.10, N 9.27, observed: C 49.36, H 4.13,
N 9.30; m.p: 191–193 °C; Rf (1% methanol in ethyl acetate): 0.42; FT-IR
spectra (cm−1): ʋ 1654 (NeH bending), 1053, 1024 (CeN stretching),
819 (P-F stretching) (Fig. S46); 1H NMR (DMSO‑d6, 400 MHz): δ 0.88
(s, 6H, cymene isopropyl-CH3), 2.19 (s, 3H, cymene-CH3), 2.49–2.61
(m, 1H, cymene-CH), 3.04 (s, 6H, CH3, ArH), 6.10 (d, 2H, J = 6 Hz,
cymene ArH), 6.32 (d, 2H, J = 6 Hz, cymene ArH), 6.92 (s, 2H, ArH),
8.11–8.24 (m, 5H, ArH), 9.12–9.17 (m, 1H, ArH), 9.81 (s, 2H, ArH)
(Fig. S47); 19F NMR (DMSO‑d6, 376 MHz): δ −70.10 (6F, PF6) (Fig.
S48); 31P NMR (DMSO‑d6, 162 MHz): δ −135.43 to −152.98 (m, PF6)
(Fig. S49); LC-MS (MeOH): m/z: 613.6 [M]+ (Fig. S50).
2.2.9 2-(2-chlorophenyl)-(1H-imidazo[4,5-f][1,10]Phenanthroline
(3i): 2-chloro benzaldehyde (54 μl, 0.480 mmol); yield: 94%; Mw
(C19H11 ClN4): 330.78 g mol−1; m.p: 307–308 °C; Rf: 0.52 (2:1 v/v,
ethyl acetate:methanol); FT-IR spectra (cm−1): 3167 (CeH stretching),
1670 (NeH bending), 1400 (C]C stretching), 1350 (CeN stretching),
734 (C-Cl stretching) (Fig. S25); 1H NMR (400 MHz, CDCl3): δ 7.42 (m,
4H, CH), 7.66 (m, 3H, CH), 8.46 (d, 1H, CH), 9.11 (d, 3H, CH) (Fig.
S26); LC-MS: 331.0 [M]+ (Fig. S27).
2.3.5
[(η6-p-cymene)RuCl(κ2-N,N-2-(4-fluorophenyl)-1H-imidazo
2.2.10
2-(1H-imidazo[4,5-f][1,10]Phenanthroline-2-yl)-4-ni-
[4,5-f][1,10]Phenanthroline].PF6
(4e):
Yield:
mol−1
; Anal.
93%;
Calcd
Mw
for
trophenol (3j): 2-hydroxy-5-nitro benzaldehyde (80 mg, 0.478 mmol);
yield: 94%; Mw (C19H11N5O3): 358.33 g mol−1; m.p: 300–302 °C; Rf:
0.43 (2:1 v/v, ethyl acetate:methanol); FT-IR spectra (cm−1): 3317 (OH
stretching), 2945 (CeH stretching), 1656, 1413 (–NO2 group), 1018
(CeN stretching) (Fig. S28); 1H NMR (400 MHz, DMSO‑d6): δ 6.91 (d,
1H, J = 8 Hz, OH), 7.80 (m, 2H, CH), 8.08 (d, 1H, CH), 8.86 (d, 2H,
CH), 9.01 (m, 3H, CH) (Fig. S29); LC-MS: 359.7 [M + 1]+ (Fig. S30).
(C29H25N4ClF7PRu):
730.03
g
C
29H25N4ClF7PRu: C 47.66, H 3.42, N 7.67, observed: C 47.79, H 3.42,
N 7.34; m.p: 187–190 °C; Rf (1% methanol in ethyl acetate): 0.45; FT-IR
spectra (cm−1): ʋ 2978 (CeH stretching), 1647 (NeH bending), 1053,
1024 (CeN stretching), 819 (P-F stretching) (Fig. S51); 1H NMR
(DMSO‑d6, 400 MHz): δ 0.90 (s, 6H, cymene isopropyl-CH3), 2.19 (s,
3H, cymene CH3), 2.49–2.63 (m, 1H, cymene CH), 6.10 (d, 2H,
J = 6 Hz, cymene ArH), 6.33 (d, 2H, J = 6.4 Hz, cymene ArH), 7.47 (t,
2H, ArH), 8.17–8.21 (m, 2H, ArH), 8.44 (t, 2H, J = 5.6, ArH), 9.33 (s,
2H, ArH), 9.85 (d, 2H, J = 5.2 Hz, ArH) (Fig. S52); 19F NMR (DMSO‑d6,
376 MHz): δ −70.10 (6F, PF6) (Fig. S53); 31P NMR (DMSO‑d6,
162 MHz): δ −135.43 to −152.98 (m, PF6) (Fig. S54); LC-MS (MeOH):
m/z: 585.2 [M]+ (Fig. S55).
2.3.1
[(η6-p-cymene)RuCl(κ2-N,N-2-phenyl-1H-imidazo[4,5-f]
[1,10]Phenanthroline)].PF6 (4a): Yield: 90%; Mw (C29H26N4ClF6PRu):
712.024 g mol−1; Anal. Calcd for C29H26N4ClF6PRu: C 48.87, H 3.65, N
7.86, observed: C 48.98, H 3.69, N 7.90; m.p: 178–180 °C; Rf (1%
methanol in ethyl acetate): 0.41; FT-IR spectra (cm−1): ʋ 1656 (NeH
bending), 1053, 1024 (CeN stretching), 821 (P-F stretching) (Fig. S31);
1H NMR (DMSO‑d6, 400 MHz): δ 0.90 (s, 6H, cymene isopropyl-CH3),
2.19 (s, 3H, cymene-CH3), 2.61–2.66 (m, 1H, cymene-CH), 6.11 (d, 2H,
J = 6 Hz, cymene ArH), 6.34 (d, 2H, J = 6 Hz, cymene ArH), 7.58–7.67
(m, 3H, ArH), 8.21 (s, 1H, ArH), 8.35 (d, 2H, J = 7.6 Hz, ArH), 9.30 (s,
2H, ArH), 9.86 (s, 2H, ArH) (Fig. S32); 19F NMR (DMSO‑d6, 376 MHz):
δ −70.11 (6F, PF6) (Fig. S33); 31P NMR (DMSO‑d6, 162 MHz): δ
−135.43 to −152.98 (m, PF6) (Fig. S34); LC-MS (MeOH): m/z: 567.7
[M]+ (Fig. S35)
2.3.6 [(η6-p-cymene)RuCl(κ2-N,N-2-(4-bromophenyl)-1H-imidazo
[4,5-f][1,10]Phenanthroline].PF6
(4f):
Yield:
95%;
Mw
(C29H25N4BrClF6PRu): 790.93
g
mol−1
;
Anal. Calcd for
C29H25N4BrClF6PRu: C 43.99, H 3.16, N 7.08, observed: C 44.04, H
3.19, N 7.10; m.p: 142–144 °C, Rf (1% methanol in ethyl acetate): 0.35;
FT-IR spectra (cm−1): ʋ 1658 (NeH bending), 1053, 1024 (CeN
stretching), 819 (P-F stretching) (Fig. S56); 1H NMR (DMSO‑d6,
400 MHz): δ 0.90 (s, 6H, cymene isopropyl-CH3), 2.20 (s, 3H, cymene
CH3), 2.50–2.62 (m, 1H, cymene CH), 6.11 (d, 2H, J = 6.4 Hz, cymene
ArH), 6.34 (d, 2H, J = 6 Hz, cymene ArH), 7.83 (d, 2H, J = 8.4 Hz,
ArH), 8.19 (t, 2H, J = 5.6 Hz, ArH), 8.33 (d, 2H, J = 8.4 Hz, ArH), 9.29
(d, 2H, J = 8 Hz, ArH), 9.84 (d, 2H, J = 5.2 Hz, ArH) (Fig. S57); 19F
NMR (DMSO‑d6, 376 MHz): δ −70.11 (6F, PF6) (Fig. S58); 31P NMR
(DMSO‑d6, 162 MHz): δ −135.43 to −157.38 (m, PF6) (Fig. S59); LC-
MS (MeOH): m/z: 645.6 [M]+ (Fig. S60).
2.3.2 [(η6-p-cymene)RuCl(κ2-N,N-2-(4-methoxyphenyl)-1H-imidazo
[4,5-f][1,10]Phenanthroline].PF6
(4b): Yield:
mol−1
; Anal.
92%;
Mw
(C30H28N4OClF6PRu):
742.06
g
Calcd for
C
30H28N4OClF6PRu: C 48.51, H 3.77, N 7.54, observed: C 48.62, H
3.90, N 7.62; m.p: 183–185 °C; Rf (1% methanol in ethyl acetate): 0.38;
FT-IR spectra (cm−1): ʋ 3014 (CeH stretching), 1656 (NeH bending),
1053, 1024 (CeN stretching), 819 (P-F stretching) (Fig. S36); 1H NMR
(DMSO‑d6, 400 MHz): δ 0.90 (s, 6H, cymene isopropyl-CH3), 2.20 (s,
3H, cymene-CH3), 2.50–2.67 (m, 1H, cymene-CH), 3.87 (s, CH3), 6.11
(d, 2H, J = 6 Hz, cymene ArH), 6.34 (d, 6H, J = 5.6 Hz, cymene ArH),
7.18 (d, 3H, J = 8.4, ArH), 8.19 (s, 2H, ArH), 8.34 (d, 2H, J = 8 Hz,
ArH), 9.33 (s, 1H, ArH), 9.84 (s, 2H, ArH) (Fig. S37); 19F NMR
(DMSO‑d6, 376 MHz): δ −70.11 (6F, PF6) (Fig. S38); 31P NMR
(DMSO‑d6, 162 MHz): δ −135.43 to −152.98 (m, PF6) (Fig. S39); LC-
MS (MeOH): m/z: 599.6 [M]+ (Fig. S40).
2.3.7
[(η6-p-cymene)RuCl(κ2-N,N-2-(4-nitrophenyl)-1H-imidazo
[4,5-f][1,10]Phenanthroline].
PF6
(4g):
Yield:
90%;
Mw
(C29H25N5O2ClF6PRu): 757.03
g
mol−1
;
Anal. Calcd for
C
29H25N5O2ClF6PRu: C 45.96, H 3.30, N 9.24, observed: C 46.02, H
3.33, N 9.32; m.p: 176–178 °C; Rf (1% methanol in ethyl acetate): 0.38;
FT-IR spectra (cm−1): ʋ 1656 (NeH bending), 1053, 1024 (CeN
stretching), 819 (P-F stretching) (Fig. 5.61); 1H NMR (DMSO‑d6,
400 MHz): δ 0.92 (s, 6H, cymene isopropyl-CH3), 2.21 (s, 3H, cymene
CH3), 2.51–2.65 (m, 1H, cymene CH), 6.13 (d, 2H, J = 6.4 Hz, cymene
ArH), 6.35 (d, 2H, J = 6 Hz, cymene ArH), 8.19 (t, 2H, J = 5.2 Hz,
ArH), 8.45 (d, 2H, J = 4.8 Hz, ArH), 8.64 (d, 2H, J = 8.4 Hz, ArH),
9.32 (d, 2H, J = 6.8 Hz, ArH), 9.86 (d, 2H, J = 5.2 Hz, ArH) (Fig. S62);
19F NMR (DMSO‑d6, 376 MHz): δ −70.1 (6F, PF6) (Fig. S63); 31P NMR
(DMSO‑d6, 162 MHz): δ −131.03 to −157.37 (m, PF6) (Fig. S64); LC-
MS (MeOH): m/z: 612.2 [M]+ (Fig. S65).
2.3.3
[(η6-p-cymene)RuCl(κ2-N,N-4-(1H-imidazo[4,5-f][1,10]
Phenanthroline-2-yl)phenol].
PF6
g
(4c): Yield:
mol−1
; Anal.
89%;
Mw
(C29H26N4OClF6PRu):
728.03
Calcd for
C
29H26N4OClF6PRu: C 47.80, H 3.57, N 7.69, observed: C 47.19, H
3.31, N 7.11; m.p: 138–140 °C; Rf (1% methanol in ethyl acetate): 0.42;
FT-IR spectra (cm−1): ʋ 1656 (NeH bending), 1053, 1024 (CeN
stretching), 819 (P-F stretching) (Fig. S41); 1H NMR (DMSO‑d6,
400 MHz): δ 0.90 (s, 6H, cymene isopropyl-CH3), 2.20 (s, 3H, cymene-
CH3), 2.50–2.63 (m, 1H, cymene-CH), 6.10 (d, 2H, J = 6 Hz, cymene
ArH), 6.34 (d, 2H, J = 6 Hz, cymene ArH), 7.18 (d, 3H, J = 8.4, ArH),
2.3.8 PF6 (4h): Yield: 85%; Mw (C29H26N4OClF6PRu):
728.03 g mol−1; Anal. Calcd for C29H26N4OClF6PRu: C 47.80, H 3.57, N
7.69, observed: C 47.02, H 3.84, N 7.73; m.p: 171–173 °C; Rf (1%
4

S. De, et al.
InorganicaChimicaActa515(2021)120066
methanol in ethyl acetate): 0.46; FT-IR spectra (cm−1): ʋ 2978 (CeH
stretching), 1629 (NeH bending), 1053, 1024 (CeN stretching), 819 (P-
F stretching) (Fig. S66); 1H NMR (DMSO‑d6, 400 MHz): δ 0.90 (s, 6H,
cymene isopropyl-CH3), 2.19 (s, 3H, cymene CH3), 2.49–2.63 (m, 1H,
cymene CH), 6.12 (d, 2H, J = 6.4 Hz, cymene ArH), 6.34 (d, 2H,
J = 6.4 Hz, cymene ArH), 7.10 (q, 3H, ArH), 7.51 (t, 1H, J = 5.2 Hz,
ArH), 8.30 (d, 1H, J = 6.8 Hz, ArH), 9.37 (d, 1H, J = 8.4 Hz, ArH),
9.88 (d, 2H, J = 4.8 Hz, ArH), 9.98 (d, 1H, J = 10.2 Hz, ArH) (Fig.
where, φ, I, OD and η related to quantum yield, peak area, absorbance
at λmax, refractive index of solvent.
2.5. Electrolytic study
To know the ionic nature of the complex, molar conductivity of each
Ru(II) complex was carried out in pure solvent (DMF/DMSO) and
partially aqueous. The conductivity of each solution was measured
using a conductivity-TDS meter-307 (Systronics, India) with cell con-
stant 1.0 cm−1 [52] and the molar conductivity (ΛM) was calculated
using the formula (ii).
S67); 19F NMR (DMSO‑d6, 376 MHz): δ −70.12 (6F, PF6) (Fig. S68); 31
P
NMR (DMSO‑d6, 162 MHz): δ −131.03 to −152.98 (m, PF6) (Fig. S69);
LC-MS (MeOH): m/z: 583.3 [M]+ (Fig. S70).
2.3.9 [(η6-p-cymene)RuCl(κ2-N,N-2-(2-chlorophenyl)-1H-imidazo
K × 1000
=
(ii)
[4,5-f][1,10]Phenanthroline].PF6
(4i):
Yield:
mol−1
; Anal.
95%;
Calcd
Mw
for
M
C
(C29H25N4Cl2F6PRu):
746.48
g
where, K and C are the specific conductivity and concentration of the
solute respectively.
C
29H25N4Cl2F6PRu: C 46.61, H 3.34, N 7.50, observed: C 46.71, H 3.81,
N 7.86; m.p: 143–145 °C; Rf (1% methanol in ethyl acetate): 0.39; FT-IR
spectra (cm−1): ʋ 2980 (CeH stretching), 1656 (NeH bending), 1053,
1024 (CeN stretching), 821 (P-F stretching) (Fig. S71); 1H NMR
(DMSO‑d6, 400 MHz): δ 0.91 (s, 6H, cymene isopropyl-CH3), 2.20 (s,
3H, cymene CH3), 2.50–2.64 (m, 1H, cymene CH), 6.12 (d, 2H,
J = 6.4 Hz, cymene ArH), 6.36 (d, 2H, J = 6 Hz, cymene ArH), 7.63 (q,
2H, ArH), 7.75 (d, 1H, J = 7.6 Hz, ArH), 7.93 (d, 1H, J = 6 Hz, ArH),
8.21 (t, 2H, 5.2 Hz, ArH), 9.22 (d, 2H, J = 8.4 Hz, ArH), 9.88 (d, 2H,
J = 5.2 Hz, ArH) (Fig. S72); 19F NMR (DMSO‑d6, 376 MHz): δ −70.11
(6F, PF6) (Fig. S73); 31P NMR (DMSO‑d6, 162 MHz): δ −135.43 to
−152.99 (m, PF6) (Fig. S74); LC-MS (MeOH): m/z: 601.2 [M]+ (Fig.
S75).
2.6. DNA binding study
2.6.1. Electronic absorption spectra
The DNA binding assay was carried out by using ligands (3a, 3e and
3g, 1 × 10−5 M) and Ru(II) complexes (4a, 4e and 4g, 1 × 10−5 M) in
Tris-HCl buffer (pH 7.4) in phosphate buffer media [53]. Initially, an
equal amount of DNA (1 mL, 2.87 × 10−4 M) transferred to both
cuvettes and sequentially added ligand or Ru(II)-complex to get ab-
sorption spectra of DNA-Ru(II)-complex interaction. Before each mea-
surement, sample was equilibrated with CT-DNA for about 5 min. The
intrinsic DNA binding constant (Kb) was calculated using the equation
(iii). Also, the UV–visible absorbance spectra of ligands and Ru(II)
complex were taken in aqueous medium.
2.3.10
[(η6-p-cymene)RuCl(κ2-N,N-2-(1H-imidazo[4,5f][1,10]
Phenanthroline-2-yl)-4-nitro phenol].PF6 (4j): Yield: 89%; Mw
(C29H25N5O3ClF6PRu): 773.03
g
mol−1
;
Anal. Calcd for
C
29H25N5O3ClF6PRu: C 45.01, H 3.23, N 9.05, observed: C 45.06, H
DNA
DNA
1
a
=
+
(iii)
3.26, N 9.13; m.p: 193–195 °C; Rf (1% methanol in ethyl acetate): 0.43;
FT-IR spectrum (cm−1): ʋ 1658 (NeH bending), 1051, 1024 (CeN
stretching), 821 (P-F stretching) (Fig. S76); 1H NMR (DMSO‑d6,
400 MHz): δ 0.91 (s, 6H, cymene isopropyl-CH3), 2.20 (s, 3H, cymene
CH3), 2.49–2.64 (m, 1H, cymene CH), 5.76–5.82 (m, 1H, ArH), 6.11 (d,
2H, J = 6.4 Hz, cymene ArH), 6.36 (d, 2H, J = 6.4 Hz, cymene ArH),
7.09 (d, 2H, J = 8.8 Hz, ArH), 8.13–8.17 (m, 3H, ArH), 9.15 (s, 1H,
ArH), 9.25 (d, 2H, J = 5.2 Hz, ArH) (Fig. S77); 19F NMR (DMSO‑d6,
376 MHz): δ −70.14 (6F, PF6) (Fig. S78); 31P NMR (DMSO‑d6,
162 MHz): δ −135.41 to −152.97 (m, PF6) (Fig. S79); LC-MS (MeOH):
m/z: 628.5 [M]+ (Fig. S80).
(
)
(
)
Kb (
)
f
a
f
b
f
where [DNA] is the concentration of DNA, εa, εf and εb are the apparent
extinction coefficient for the complex, extinction coefficient of the
complex in its free form and extinction coefficient of the complex when
fully bound to DNA respectively [54]. The linear plot obtained by
plotting [DNA]/(εa-εf) vs. [DNA] using Origin Lab, version 8.5. The
ratio of the slope to intercept from the linear fit gives the value of the
intrinsic binding constant (Kb).
2.6.2. Relative viscosity study
To investigate the binding interaction of the Ru(II) complex with
DNA was studied by viscosity measurements using Ostwald’s visc-
ometer [55-57]. Each experiment was performed for three times, and
the average flow time was calculated. The data was plotted as (η/η0)1/3
vs. [complex]/[DNA], where η and η0 corresponds to viscosity of DNA
in the presence of the ligand, and viscosity of DNA alone respectively.
The viscosity of DNA was calculated using the formula η0 = (t-t0)/t0
where t and t0 represents the efflux time of DNA and PBS buffer solution
respectively.
2.3. Theoretical study
All computational calculations were performed in the gas phase
using density functional theory (DFT). The Becke three-parameter Lee-
Yang-Parr (B3LYP) exchange correlation functional using Gaussian 09
computational codes [50]. The standard basic set 6-31G(d,p) was used
for lighter elements such as C, H, N, O, F, Cl, and Br atoms and LanL2DZ
effective core potential for Ru atom. All the optimizations were done
with zero negative vibrational frequency in gas phase.
2.6.3. EtBr displacement assay
The EtBr displacement assay was carried out to explain the mode of
binding between the ligands and Ru(II)-complexes with DNA [58]. The
intercalation of EtBr to DNA is accompanied by intense fluorescence
emission due to the formation of the EtBr-DNA adduct. The apparent
binding constant (Kapp) of the complex to CT DNA was determined from
the emission intensity of EtBr taken in 5 mM Tris-HCl buffer (pH 7.4).
The relative binding tendency of the complex to DNA was calculated
from the reduction of the emission intensity. The value of apparent
binding constant (Kapp) was obtained by using the equation (iv)
2.4. Fluorescence study
Fluorescence quantum yield (φ) was calculated by using the com-
parative William's method which involves the use of well-characterized
standard with the known quantum yield value using water and MTT
condition [51]. Quinine sulphate was used as a reference (0.546 in
0.5 M NaOH), 350 nm excitation energy and emission recorded at
450–515 nm. The gradients of the plots are proportional to the quantum
yield (φ) of the studied system. The data obtained and quantum yield
value calculated according to the equation (i):
Kapp×[complex]50 = KEtBr × [EtBr](iv)
IS
IR
ODR
ODS
=
×
×
×
S (i)
where [complex]50 is the concentration of the complex at 50%
R
quenching of DNA-bound EtBr emission intensity, KEtBr = 1.0x107
R
5

S. De, et al.
InorganicaChimicaActa515(2021)120066
M−1, binding constant of EtBr and concentration of EtBr is used 8 μM.
KSV is Stern-Volmer quenching constant [59]. The value of KSV was
calculated from the following equation (v).
I0
I
= 1 + KBSA [Q] = 1 + Kq 0 [Q](vi)
K
Kq
=
BSA (vii)
I0
I
0
= 1 + KSV [Q](v)
I0
I
log
= logK + nlog [Q](viii)
where I0 and I are emission intensities of EtBr-DNA in the absence and
I
in the presence of complex of concentration [Q].
where, I0 and I are emission intensities of BSA in the absence and in the
presence of quencher of concentration [Q] while KSV, kq and τ0 are
related to quenching constant, quenching rate constant and average
lifetime of the tryptophan (1 × 10−8 s), while K and n signifies binding
constant and number of binding sites calculated by using Scatchard
equation (viii) [67].
2.6.4. In-silico study
The synthesized phenanthroline ligands and their Ru(II) complexes
were subjected to molecular docking study using Autodock vina [60],
encompassing Lamarckian genetic algorithm (LGA) to predict binding
affinities of several conformers and AutoDock Tools (ADT) to execute
the operation and subsequent calculations. With the current computa-
tional resources, such a huge docking calculation with the large ex-
perimental HS-DNA prompted the process to opt a smaller section of
DNA with the sequence d(CCGTCGACGG) (PDB entry:423D, a sequence
commonly used in oligodeoxynucleotide study) [61] procured from
Protein Data Bank [62] with resolution of 1.60 Å was built using Au-
todock4 package to expedite over DNA-binding properties of all the
ligands and their respective Ru-complexes considered for the present
study. It is the 2D structures of (3a-3j) and (4a-4j) all the synthesized
compounds, developed from ACD ChemSketch Freeware, from which
all the corresponding coordinates were obtained and subsequently
transformed into PDB form through a toolbox that can speak several
languages of chemical data [63]. Separate files for both DNA and li-
gands were made using AutoDock Tools. Each atom in both target and
ligand was fed with Gasteiger charges. Prior docking, the binding site
was assigned developing a grid box with a spacing of 1 Å and
26 × 26 × 26 number of points was used in x, y and z directions. The
target was further refined to pdbqt for the final operation. With an
exhaustiveness of 8, Autodock generated nine significant conformers
for each ligand and their respective Ru-complexes. The necessary cal-
culations were done in a Dell system (3.4 GHz processor, 4 GB RAM,
1 TB Hard disk operating system. The scoring functions obtained out of
the process were screened to fix the conformer lying close to the active
site residues and subsequently analysed for its binding pattern. PyMOL
(The PyMOL Molecular Graphics System, Version 1.3, Schrodinger,
LLC) molecular graphics program was used to study the orientation of
each conformer within the active site.
2.8. In-vitro cytotoxic study
It is based on the reduction of the yellow tetrazolium salt (3-[4, 5-
dimethyl thiazol-2-yl]-2, 5 diphenyl tetrazolium bromide) by mi-
tochondrial dehydrogenases to form a blue MTT formazan in viable
cells [68,69]. Each Ru(II) complex was dissolved in 0.1% DMSO and
then serial dilution with cell medium. Two different cancer cell lines
such as HeLa, MDA-MB-231 and normal cell lines HEK-293 were used
in this assay. The cisplatin was used as a positive control. The entire
cells were cultured in 100 μl of a growth medium in 96-well plates and
incubated at 37 °C under 5% CO2 overnight. After 24 h of incubation
time, the cultured cells were exposed to different concentrations of li-
gands (9–300 µM). The effect DMSO on cells was studied by interacting
cells with 0.1% DMSO. After 24 h of incubation time, the medium was
superfluous and cell cultured plate was incubated with 100 μl of MTT
reagent (1 mg/mL) for 3 h at 37 °C. Then the suspension was kept on
micro vibrator for 10 min and subsequently the absorbance was re-
corded using ELISA reader at 620 nm. The experiment was also con-
ducted in triplicate. The growth inhibition percentage was calculated
using the formula: percentage growth inhibition = 100-[(AD × 100)/
AB], where AD represents measured absorbance in wells which consists
samples and AB represents absorbance of the blank wells.
3. Results and discussion
3.1. Structural studies of 3a-3j and 4a-4j
In order to rationalize the experimental protein binding study,
molecular docking study was performed. The crystallographic structure
of BSA with the PDB ID: 4F5S [64] was collected from fetched from the
protein data bank. The additional thing done during the protein pre-
paration was the exclusion of water molecule in order to avoid the
unwanted interaction with the docked conformers. The grid size con-
sidered for the protein is 30, 26 and 24 along the X, Y and Z axes with a
spacing of 1 Å encircling all the putative active site residues of which
the most prominent are Trp213 and Trp134 [65]. The working principle
and the output parameters were as similar as the above mentioned DNA
docking.
The structural analysis of ligands (3a-3j) and Ru(II) complexes (4a-
4j) were analysed by NMR (1H, 19F and 31P), FT-IR and LC-MS (Fig. S1-
S80). A brief spectroscopic characterization of 3e and 4e compounds
were discussed here. The NMR spectral studies of 3e (Fig. S14) con-
taining ten aromatic protons present in the phenanthroline backbone
was observed at δ 7.45–9.02 ppm. The most de-shielded proton, ad-
jacent to nitrogen in phenanthroline ring show a doublet at 9.02 ppm.
From FT-IR spectra (Fig. S13), the vibrational bands at 3055, 1606,
1396 and 1219 cm−1 were due to CeH stretching, NeH bending, CeN
stretching and C-F stretching respectively. The LC-MS chromatogram
show a molecular ion peak at m/z 314.90 [M]+ this is well matched
with calculated mass of 314.32 and it confirms the formation of 3e (Fig.
S15). In the case of 4e complex, the NMR spectral studies show a
characteristics peak: (i) the para cymene six methyl protons as singlet at
δ 0.89–0.91, one single methyl peak at δ 2.19 and four aromatic protons
gave distinct peaks in the range of δ 6.10–6.34, (ii) ten aromatic protons
of 1,10-phenanthroimidazole were observed at δ 7.47–9.85 ppm (iii) in
19F NMR show characteristics peak at −70.14 ppm (iv) 31P show seven
characteristics peaks in the region of −131.02 to −157.36 ppm (Fig.
S52-S54). A characteristic change in the splitting pattern of the protons
in arene unit and downfield effects were observed after binding to Ru
(II) metal centre. The 1H NMR resonances for the arene protons of the
Ru(II)-arene complex was shifted downfield relative to the corre-
sponding starting Ru(II) dimer. From FT-IR spectra (Fig. S51), the
2.7. Protein binding study
Serum albumin proteins found a major component in blood plasma
proteins and plays significant role in drug transport and metabolism
[66]. An interaction of the drug with bovine serum albumin (BSA) has
been studied from tryptophan emission quenching. Emission intensity
of BSA at 340 nm decreases gradually with increasing the concentration
of Ru(II) complex which confirms the interaction has occurred. The Ru
(II) complex solution was slowly added to the solution of BSA (2 μM) in
5 mM Tris-HCl/NaCl buffer (pH 7.2) and the quenching of the emission
at 340 nm (λex, 295 nm) was recorded. The quenching constant (KBSA
)
was determined by using Stern-Volmer equation (iv and vii).
6

S. De, et al.
InorganicaChimicaActa515(2021)120066
Fig. 3. Absorption and emission spectral responses of 3e and 4e (1 × 10−5 M) in DMSO and DMSO-water (1:1, v/v) media.
complex 4e show characteristics peak at 3442 (NeH stretching), 2978
(CeH stretching), 2250 (CeN nitrile), 1647 (C]C stretching), 1053
and 1024 (CeH bending), 819 (C-F stretching), 758 (CeH bending).
The LC-MS spectrum showed a significant peak at m/z 585.20 (calcu-
lated 585.07) for [M+] which is characterized by the unique Ru isotope
pattern (Fig. S55). The rest of the ligands and their Ru(II) complexes
were also characterized in a similar approach.
3.2. Absorption and emission study
Absorbance and emission abilities of ligands (3a-3j, 1 × 10−5 M)
and Ru(II) complexes of ligands (4a-4j, 1 × 10−5 M) were recorded in
pure DMSO as well as in DMSO:water (1:1,v/v) media in the range of
200–800 nm (Fig. 3, Fig. S81). These derivatives showed two major
electronic bands in the region 280–300 and 325–350 nm. These elec-
tronic bands are due to intra-ligand π-π* transitions from phenan-
throline and imidazole moieties. The molar absorptivity (ε) va-
lues ~ 3.0 × 104 L.mol−1 cm−1 and ~ 2.2 × 104 L.mol−1 cm−1
respectively. The electronic spectra of the Ru(II) complexes (4a-4j)
showed two absorption bands at 290–300 nm and 340–360 nm. These
transitions are due to π-π* transitions and metal-to-ligand charge
transitions (MLCT, Ru (dπ)- > (π*) phen) respectively. The molar ab-
sorptivity (ε) values ~ 4.0 × 104 L.mol−1 cm−1 and ~ 1.9 × 104
L.mol−1 cm−1 respectively.
Fig. 4. Optimized molecular geometry of 3a and 4a by DFT/B3LYP method.
The emission spectra of ligands (3a-3j) and their Ru(II) complexes
(4a-4j) were studied by using wavelength maximum (280 nm) in pure
DMSO and aqueous DMSO (1:1, v/v). The emission spectral response of
ligand and Ru(II) complex of ligands were observed at 400–550 nm and
320–450 nm respectively. The quantum yield of Ru(II) complex has
higher than the corresponding ligand. However, the quantum yield of
Ru(II) complex was found to be highest in pure DMSO compare to
DMSO:water (1:1, v/v). Out of ten compound studied, 3g and 4j ex-
hibits highest quantum yield of 0.38 and 0.49 respectively (Fig. S82 and
Table S1). This shows that both ligands and Ru(II) complexes can be
used for bio-imaging applications.
Fig. 5. FMO’s of 3a and 4a by DFT/B3LYP method.
imidazole phenanthroline and on Ru metal centre while LUMO’s are
present on Ru atom with the energy gap of 0.72 eV which indicates the
possible electron transfer from MLCT. The energies of frontier mole-
cular orbitals (EHOMO and ELUMO), energy band gap (ΔE) electro-
negativity (χ), chemical potential (μ), global hardness (ɳ), global soft-
ness (S) and global electrophilicity index (ω) [71-74]. As seen from
Table S2, the stability of Ru(II) complexes are more compare to cor-
responding ligands. The energy gap (ΔE) is an important parameter to
characterize the chemical reactivity and kinetic stability of the mole-
cule. The small energy gap indicates the charge transfer easily occurs in
it which further influence the biological activity of the compound.
In order to support experimental findings, computational studies of
(3a-3j) and their Ru(II) complexes (4a-4j) were carried out by using
combined DFT-B3LYP method. Different quantum-chemical parameters
were calculated by applying B3LYP/6-31G**/LanL2DZ ECP methods
such as total molecular energy, ESP charges, energies of HOMO and
LUMO orbitals. The highly pre-organized planar geometries of 3a-3j
and distorted tetrahedral geometry of 4a-4j were optimized as shown in
Fig. 4. The electrostatic potential mapped onto the constant electron
density surface [70]. The maximum negative region which preferred
site for electrophilic attack indicated as red color and the maximum
positive region which preferred site for nucleophilic attack as shown
blue color (Fig. 4). The frontier molecular orbitals and energy gap be-
tween HOMO and LUMO of ligands (3a-3j) and complexes (4a-4j) were
calculated and displayed in Fig. 5. In the case of 3a, HOMO and LUMO
orbitals are located on imidazole phenanthroline with an energy gap of
4.11 eV whereas in the case of 4a, the HOMOs mainly located on
3.3. Stability study
For unique therapeutics purpose, a drug has to be stable within the
cells in an internal physiological conditions. In order to meet this
7

S. De, et al.
InorganicaChimicaActa515(2021)120066
Fig. 6. UV–Visible spectral stability studies of 3e and 4e (1 × 10−5 M) in (a) water (b) 0.1 mM GSH (c) MTT condition (5% DMSO in phosphate buffer).
requirement, an experiment was carried out to check the stability of the
prepared compounds (3a, 3e, 4a and 4e) in water, 0.1 mM GSH and 5%
DMSO in phosphate buffer media. The stability of the compound was
measured over a period of 24 h at regular time interval for selected
derivatives using UV–visible spectral response (Fig. 6). Results show
that stability of 3a and 3e in water and MTT media remains same in
terms of their absorbance and wavelength maximum even after contact
of 24 h. But, there is a small decrease in absorbance was observed after
24 h contact of 3a and 3e with 0.1 mM GSH. A similar procedure was
adopted for 4a and 4e to check their stability. Results shows that in case
of water, both 4a and 4e were showing moderately decrease in absor-
bance in water and GSH media due to formation of aqua complex and
ligand exchange reaction with various endogenous nucleophiles present
in GSH phase respectively. In the presence of MTT there is no change in
absorbance and wavelength shift were observed. To gain more insight
view of stability, further stability test was proved by using 1H NMR
studies in DMSO‑d6 and DMSO‑d6:D2O (6:4, v/v). Results show that
there is no significant change in NMR pattern has been observed and
hence 4e complex was found to be stable in both conditions (Fig. S83-
S84).
like purine and pyrimidine. On interacting with DNA with 3e and 4g
revealed hyperchromism effect results electrostatic interaction between
the ligand and DNA owing to unstacking of base pairs followed by
denaturation of DNA. It leads to ligand-DNA well intact and allowing
more light to be absorbed. But 3a and 4e with DNA interaction displays
hypochromic effect because ligand intercalates in between the base
pairs of DNA, the π* orbital of the intercalating ligand can pair with the
π- orbital of the base pairs, and the coupling π* orbital is moderately
occupied by electrons, therefore decreasing the transition possibilities
and resulting in hypochromism effect [75,76]. In order to quantify
these interactions, the intrinsic binding constant (Kb) for all selected
ligand and their Ru(II) complex were calculated from the plot [DNA]/
(εa-εf) vs. [DNA] (Fig. S85). The Kb value for ligands and corresponding
Ru(II) complex with DNA was found to be in the order of 3e > 3g >
3a and 4e > 4g > 4a respectively (Table 1). Further, high Kb value
reveals that 3e (8.3 × 105 M−1) and 4e (2.4 × 106 M−1) were ex-
pected to be a good DNA cleaving properties.
3.5.2. EtBr displacement assay
The competitive binding studies of the ligands (3a, 3e and 3g) and
corresponding Ru(II)-complexes (4a, 4e and 4g) with CT-DNA
(120 μM) were calculated using emission study in the presence of EtBr
(8 μM) [77] by sequential addition of Ru complex (1 mM). During
spectral response, both ligand and Ru(II)-complex have been excited at
485 nm and emission was recorded at 590–598 nm. The EtBr dis-
placement assay is a proficient fluorescence spectral method for the
analysis of DNA intercalative binding of complex with CT-DNA. As EtBr
is a planar structure and it is a very sensitive fluorescent probe which
interacts with DNA via π-π* intercalation. It is a feebly emissive in
phosphate buffer, but in the presence of CT-DNA, it displays enhanced
fluorescence due to intercalation in DNA double helix. The addition of
the ligand or complex in the EtBr-DNA binary complex results in re-
duction of fluorescence intensity due to displacement of EtBr from the
DNA double helix. The obtained results reveals that the substantial
decrease of fluorescence intensity of EtBr-bound DNA in presence of
ligand or complex signifying noticeable intercalative binding mode
(Fig. 8 and Fig. S86). The degree of fluorescence quenching of EtBr pre-
treated DNA might be used to conclude the apparent binding constant
(Kapp) of the ligands or complexes with CT-DNA [78]. Besides, The 50%
quenching of DNA-EtBr occurred at concentration of 55–35 μM for both
ligands and Ru(II) complexes. Besides, the SV quenching constant
shown in Table 1 follows trend 3e > 3a > 3g and 4g > 4e > 4a
with EtBr-DNA respectively due to more electrostatic and groove
binding with DNA.
3.4. Molar conductivity study
To work as drug, given compound has to exhibit good lipophilicity
and electrolytic nature. In order to confirm ionic nature of the com-
pounds, the solubility of Ru(II) complex was performed from non-polar
to protic solvents. The solubility data reveals that ligands (3a-3j) and
their Ru(II) complex (4a-4j) were insoluble in chloroform, moderately
soluble in water but highly soluble in DMSO and DMF medium. Further,
these compounds are exhibiting solubility in the range of 6–10 mg/mL
in DMSO:water (9:1, v/v) media. The ionic nature of Ru(II) complex
was well understand by knowing their molar conductivity. It is obvious
that all complexes exhibit electrolytic conductivity in pure and partially
aqueous DMSO and DMF media (Table S3). This may be due to high
dielectric constant of the selected solvent and good solubility together
leads to ionization of the complex. A similar trend was observed with
rest of the Ru(II) complexes but 4e exhibits highest molar conductivity
compare to other complexes. Due to this high ionization, the complex
behave more cationic character and it is expected that the binding ca-
pacity of this complex with DNA could be more in physiological con-
ditions.
3.5. DNA binding study
3.5.1. Electronic absorption titration studies
The binding interaction of selected ligands (3a, 3e and 3g,
1 × 10−5 M) and corresponding Ru(II)-complexes (4a, 4e and 4g,
1 × 10−5 M) with CT-DNA (2.87 × 10−4 M) were studied by using
UV–Visible spectroscopic technique. As seen from Fig. 7, alone CT-DNA
exhibits an absorption band (260 nm −290 nm) with a maximum ab-
sorption at 258 nm which is due to π-π* transitions of DNA base pairs
3.5.3. Viscosity study
To investigate the binding interaction of the selected complexes (4a,
4e and 4g) with DNA, viscosity measurements were carried out on CT-
DNA by different the concentrations of the complexes. Due to various
types of interaction of complex with DNA, there is a change in relative
viscosity of DNA in presence of complex. Results reveals that relative
8

S. De, et al.
InorganicaChimicaActa515(2021)120066
Fig. 7. UV–visible spectral responses of 3e and 4e (1 × 10−5 M) in 5 mM Tris-HCl-NaCl buffer solution (pH = 7.2) with incremental addition of CT-DNA
(2.87 × 10−4 M).
viscosity (η/η0)1/3 vs. Ru(II) complex to CT-DNA in mole ratio is shown
in Fig. 9 and Table S4. The obtained results reveals that there is a
gradual increase in relative viscosity with increasing in concentration of
4g. However, in the case of 4a and 4e there is sudden increase up to
ri = 0.2, beyond this value there is gradual increase was observed. The
increasing in viscosity follows this could be due to effective intercala-
tion compare to other Ru(II) complexes. Also, we have compared the
performance Ru(II) complex with EtBr, results show that 4e exhibits
higher intercalation tendency compare to rest of the Ru(II) complexes.
derivative such as ligands (3a-3j) and their respective Ru(II) complexes
(4a-4j) were carried out with the DNA duplex comprising of d(CGCG
AATTCGCG) dodecamer sequence. The assessment of each docked
compounds were made by screening the scoring functions/binding
energy, as shown in Table S5. The negative values as obtained in each
compound clearly indicate a fair binding with the DNA. Compounds 3e
and 4e were selected to be highly active owing to their highest binding
energy, i.e., −9.4 kcal/mol and −9.3 kcal/mol respectively. A similar
interaction was observed in the other compounds like 3f, 3g, 4f and 4g.
Binding pose of best conformer of highly active compounds are shown
in Fig. 11a. The orientation of ligands 3e and 3g within the DNA could
be of minor groove binding, whereas the most stable conformers of the
complexes, 4e and 4g were found to be lying in between the base pairs,
thus intercalates DNA. The increase in binding energy can be attributed
to the presence of electronegative groups. Compounds 3e and 4e pos-
sess fluorine atom, despite acting as an electron withdrawing group,
occupying less area within the target site as its van der waals radii is
close to hydrogen, whereas in case of 3g and 4g containing electron
withdrawing group as nitro, occupying large area compare to fluorine,
thus differs from the former.
3.5.4. DNA cleaving study
An ability of the Ru(II) complex to destroy DNA was tested using
agarose gel electrophoresis. Initially, 10 mL (200 mg) of plasmid DNA
(≈10 kb) was mixed with an equal volume of Ru(II) complex with
different concentration (0.1, 0.01 or 0.05 mg/mL) in a total volume of
20 mL. Then mixture was incubated for 1 h at 37 0C and loaded on
agarose gel containing 1.0 mg/mL EtBr after the addition of 2 mL buffer
solution containing 25% bromophenol blue, 0.25% xylene cyanol, and
30% glycerol. Plasmid DNA was used as a positive control. The elec-
trophoresis was carried out at 50 V for 1 h in Tris–HCl buffer. The gel
plate was visualized using a gel documentation instrument. Results
reveals that plasmid DNA (≈10 kb) was totally degraded within 1.5 h
(Fig. 10) with all Ru(II) complexes. This may be due to breaking of the
double and triple hydrogen bonds present in the nucleotide bases.
In developing an understanding between the protein–ligand inter-
actions, molecular docking of all the compounds, both the ligands and
their respective complexes was conducted procuring the crystal-
lographic structure of BSA. It is clearly stated in literature that BSA
comprised of two essential binding sites, where Trp213 is a part of
hydrophobic site and Trp134, the other essential residue lying on the
surface of hydrophilic region. The scoring functions as documented in
Table S6, indicate compounds 3e, 3g, 4e and 4g were among the highly
interactive. Both the ligand and its respective complex (Fig. 11b) were
occupying the putative active sites. Almost all the compounds are or-
iented close to the hydrophobic regions which is well supported with
the visibility of of Trp 213. In a nutshell, the insilico study stands well
3.5.5. In-silico study
Binding mode analysis is an essential step in understanding the ar-
rangement of several atoms of target active site and their respective
ligand while they are in their interactive mode. When the target is
found to be DNA, the usual way by which most of the conventional
complementary drugs are working is through intercalation. In the
present study, molecular docking of two sets of phenanthroline
Table 1
Binding parameters of ligand and Ru(II) complexes with (a) DNA, (b) DNA-EtBr.
Ligand
(a)
(b)
Kb (M−1
)
% hypochromism
% hyperchromism
Ksv (M−1
)
Kapp (M−1
)
3a
3e
3g
4a
4e
4g
5.6 × 103
8.3 × 105
3.7 × 105
1.7 × 104
2.4 × 106
1.1 × 106
41.60
–
–
2.3 × 104
4.0 × 104
1.7 × 104
2.8 × 104
3.2 × 105
4.6 × 105
2.5 × 106
2.7 × 106
2.3 × 106
3.2 × 106
2.9 × 106
4.8 × 106
86.26
–
62.47
45.46
85.92
–
–
57.72
Kb, intrinsic DNA binding constant; Ksv, Stern-Volmer quenching constant; Kapp, apparent DNA binding constant.
9

S. De, et al.
InorganicaChimicaActa515(2021)120066
Fig. 8. Fluorescence spectral responses of the EtBr bound DNA in the presence of 3e and 4e at pH 7.2 (λex = 485; λem = 598 nm).
DMSO‑d6:D2O) and (iii) 4e with BSA (1 mM, 3:2, DMSO‑d6:D2O) were
recorded. Alone 4e exhibits seven 1H peaks, upon addition of CT-DNA
and BSA to 4e resulted in downfield shift of all the seven peaks (Fig.
S87). This shift clearly show electrostatic interaction between 4e and
DNA and BSA (Table S7). These results are well matched with experi-
mental findings obtained from conductivity and viscosity.
3.6. Protein binding assay
Serum albumin proteins found ~55% of total plasma proteins and
play a vital role in drug transport and metabolism. The binding inter-
action of ligands (3a, 3e and 3g) and Ru(II) complexes (4a, 4e and 4g)
with BSA was studied using intrinsic tryptophan emission quenching of
BSA in the presence of the ligand and Ru(II) complex (Fig. 12 and Fig.
S88). Upon a steady increase in the concentration of the ligand or
complex, the emission intensity of BSA at around 350 nm decreases
steadily. But in case of ligand 3e a minor blue shift of 345 nm to 360 nm
of emission maximum can be indicative of an increase in hydro-
phobicity of the microenvironment around the tryptophan residues.
These variations happened owing to ligand and protein interaction and
further suggested that the protein tertiary structure was disrupted and
finally losses its activity [79]. The quenching of emission resultant from
numerous molecular interactions arises due to changes in the secondary
structure of the protein upon binding of ligand or complex. In order to
quantify these interactions, the SV quenching constant (KBSA), binding
affinity (K) and number of binding sites (n) have been calculated from
slope of the linear plot of I0/I vs. [complex or ligand] using the SV
Fig. 9. Effect of increasing amounts of compounds on the viscosity of CT-DNA
at 298 K ([EtBr] = 1 × 10−6 mol/L; [DNA] = 1 × 10−6 mol/L; [com-
plex] = 1 × 10−3 mol/L).
with the experimental data.
3.5.6. NMR pattern of complex 4e with interaction of CT-DNA and BSA
The binding mechanism of 4e with DNA and BSA were studied NMR
spectral response using deuterated solvents. Accordingly, the 1H NMR
spectral response of (i) alone 4e (1 mM) (ii) 4e with DNA (1 kb, 3:2
Fig. 10. DNA degradation study of complexes (Lane 1–10 kb Plasmid DNA marker, Lane 2 –plasmid DNA, Lane 3 to 8 Plasmid DNA with compounds).
10

S. De, et al.
InorganicaChimicaActa515(2021)120066
Fig. 11. Molecular docking interaction of (a) DNA and (b) BSA with compounds (3e, 3g, 4e, and 4g).
equation. As seen from Table 2, the rate of bimolecular quenching
constant (kq) calculated from KSV and τ0 was observed to be 6.1 × 1014
and 3.7 × 1014 L. M−1 s−1 for 4e and 3e. These values are higher than
the maximum possible value for dynamic quenching (2.0 × 1010 L.
M−1 s−1), signifying the participation of static quenching mechanism
in the presence of Ru(II) arene complexes. Herein, the high quenching
rate constant, kq (1014 L. M−1 s−1), specifies an active bimolecular
quenching together with binding. Further, the SV quenching constant
and binding affinity of complex is more compare to ligand while
number of binding site is remain same in both cases. This may be due to
more electrostatic interaction is expected in Ru(II)complex compare to
corresponding ligand.
Table 2
Binding parameters of ligand and Ru(II) complexes with BSA.
Ligand
KBSA (M−1
)
kq (M−1 s−1
)
K(M−1
)
n
3a
3e
3g
4a
4e
4g
4.4 × 104
3.7 × 106
3.8 × 104
4.8 × 104
6.1 × 106
4.8 × 106
4.4 × 1012
3.7 × 1014
3.8 × 1012
4.8 × 1012
6.1 × 1014
4.8 × 1014
1.7 × 104
2.5 × 104
2.4 × 103
3.6 × 104
4.5 × 104
5.6 × 104
0.8
2.0
1.6
0.9
1.9
1.8
KBSA, Stern Volmer quenching constant; Kq, quenching rate constant; K, binding
constant with BSA; n, number of binding sites.
different effects on each cell line. The IC50 value was perceived in the
range of 4.25–36.56 μM in HeLa cells and 2.37–78.41 μM in MDA-MB-
231 cells shown in Table 3. Among the synthesized compounds, com-
plex 4e exhibits most potency and selectivity in HeLa cell line while 4f
and 4g in MDA-MB-231 cell line (Fig. S89). DMSO was used as control
and it didn’t show any inhibition of cancer cell growth. The cytotoxicity
performance in terms of IC50 and selectivity factor of present com-
pounds were compared with previously reported anticancer agents
[80,81] (Table S8). The results shows that 4e, 4f and 4g were found to
be highly potent and selective against HeLa and MDA-MB-231 cell lines
as compare to reported anti-cancer agents.
3.7. Cytotoxicity and structure activity relationship (SAR) studies
The cytotoxicity study of synthesized complexes (4a-4j) were as-
sessed by using MTT assay protocol as a panel of cell lines that are
MDA-MB-231, HeLa and HEK-293 in triplicates. Cells were well main-
tained with complexes along with cisplatin as a standard positive
control with increase in concentration from 9 to 300 μM for 24 h. As
seen from Table 3, the study revealed that in case of HeLa cell, the
selectivity factor order found to be 4e > 4g > 4i > 4f > 4b > 4h >
cisplatin > 4a > 4j > 4d while with MDA-MB-231 the order found
tobe4g > 4f > 4c > 4e > cisplatin > 4d > 4a > 4j > 4b > 4h >
4i. The majority of the complex showed higher potency and selectivity
in HeLa cells than the standard drug. Tested complexes showed
The MTT assay results were used to establish Structure Activity
Relationship (SAR) exists in the studied compounds. Accordingly, the
Fig. 12. Fluorescence quenching of BSA in the absence and presence of increasing concentration of 3e and 4e 298 K (Tris HCl/NaCl buffer- 5 mM, pH = 7.2,
λex = 295 nm and λem = 350 nm).
11

S. De, et al.
InorganicaChimicaActa515(2021)120066
Table 3
CRediT authorship contribution statement
MTT cytotoxicity screening of 4a-4j complexes at 24 h of exposure.
Sourav De: Conceptualization, Methodology. R. Selva Kumar: .
Ashna Gauthaman: . S.K. Ashok Kumar: Investigation, Writing -
original draft. Priyankar Paira: . Anbalagan Moorthy: . Subhasis
Banerjee: .
Ligand
*Cell Line (IC50 μM)
**Selectivity factor
HeLa
MDA-MB-231 HEK 293
HeLa
MDA-MB-
231
4a
4b
4c
4d
4e
4f
32.79
3.63
1.2 28.03
1.7 203.37
2.1 53.40
2.1
6.20
7.25
1.55
Declaration of Competing Interest
1.5
34.33
1.7 3.99
2.1 17.59
1.2
14.71
12.33
36.56
3.25
1.3
88.89
4.3
3.2
3.8
4.1
7.21 22.27
1.1 71.85
1.96
4.08
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influ-
ence the work reported in this paper.
1.8
8.23
2.47
2.37
1.9
85.43
93.38
178.22
26.28 10.38
17.42 37.80
5.36
1.7
1.2
2.2
1.8
4g
4h
4i
8.38
2.4 21.65 75.19
11.69
5.39
1.3 52.74
1.5 102.77
1.6 96.26
1.3 63.06
1.9
8.79
17.86
5.31
1.94
1.22
1.96
7.74
3.2
78.41
2.3
Acknowledgements
4j
11.87
1.9 32.08
1.5
1.8
Cisplatin 9.7
1.5
8.3
1.1
64.21
6.62
Authors are grateful to Department of Science and Technology,
Government of India, for supporting the work through the project grant
(EMR/2017/000816). Authors are also thankful to DST-VIT-FIST for
NMR and GC-MS for research facilities.
*IC50 Concentration at which 50% of cells undergo cytotoxic cell death. **SF
(selectivity factor) = ratio of IC50 of HEK-293 and IC50 for all the cancer cell
lines. HEK-293 fibroblasts are generally selected as the model for healthy cells
in the evaluation of chemotherapeutic drug selectivity. 24 h incubation time for
HeLa and MDA-MB-231cell line.
Appendix A. Supplementary data
position of substituent on the phenyl ring and nature of functional
group influence the cytotoxicity effect. The potency of the complex
increases when the –OH group is present at Y position while its pre-
sence at X and Z positions, the IC50 value was decreased. The presence
of electron withdrawing at Y position certainly the cytotoxicity effect
increases and hence complex 4e, 4f and 4g were found to be most
potent complexes. Hence, substituting -H by different functional groups
such as -F, -Br, -OH, OCH3, -N(CH3)2 and -NO2 provides various deri-
vatives with characteristic biological activity. However, when the -NO2
and -F groups are present at Y position the selectivity and cytotoxicity
was observed in MDA-MB-231 and HeLa respectively. This could be due
to following reasons: (a) hydrophobic arene moieties enrich the cellular
accumulation of the complex (b) labile –Cl group enhance the prob-
ability of covalent interaction of the complex with DNA (c) strong
electrostatic bonding of phosphate group present in DNA with Ru(II)
ions (d) phenanthroline core will provide tunable metal binding site (e)
the presence of imidazole moiety responsible for interaction with DNA
and protein through supramolecular interaction (f) presence of lipo-
philic halide group with extended conjugation can enhance rigid con-
firmation and increase binding affinity towards the binding site.
Supporting information includes detailed characterization data of
NMR, FT-IR and LC-MS, emission spectra, quantum yield of compounds
with the active site of DNA and BSA. Supplementary data to this article
can be found online at https://doi.org/10.1016/j.ica.2020.120066.
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