Kinetically labile ruthenium(ii) complexes of terpyridines and saccharin: effect of substituents on photoactivity, solvation kinetics, and photocytotoxicity

Article abstract of DOI:10.1039/d1dt00246e

Herein, we designed six kinetically labile ruthenium(ii) complexes containing saccharin (sac) and 4′-substituted-2,2′:6′,2′′-terpyridines (R-tpy),viz. trans-[Ru(sac)₂(H₂O)₃(dmso-S)] (1) and [Ru^II(R-tpy)(sac)₂(X)] [X = solvent molecule] (2-6). We intentionally kept the labile hydrolysable Ru-X bonds that were potentially activatedviasolvent-exchange reactions. This strategy generates a coordinative vacancy that allows further binding with potential biological targets. To gain insight into the electronic effects of ancillary ligands on Ru-X ligand-exchange kinetics or photoreactions, we have used a series of substituted terpyridines (R-tpy) and studied their solvation kinetics. The ternary complexes were also studied for their potential utility in Ru-assisted photoactivated chemotherapy (PACT) synergized with release of saccharin as a highly selective carbonic anhydrase IX (CA-IX) inhibitor, over-expressed in hypoxic tumors. The ternary complexes exhibit distorted octahedral geometry around Ru(ii) from two monodentatetransoidalsaccharin in the axial position, and tridentate terpyridines and labile solvent molecules at the basal plane (2-6). We studied their speciation, solvation kinetics, and photoreactivity in the presence of green LED light (λ_irr= 530 nm). All the complexes are relatively labile and undergo solvation in coordinating solvents (e.g.DMSO/DMF). The complexes undergo the ligand-substitution reaction, and their speciation and kinetics were studied by UV-Vis, ESI-MS,¹H-NMR, and structural analysis. We also attempted to assess the effect of various substituents on the ancillary terpyridine ligand (R-tpy) in photo-reactivity and ligand-exchange reactions. The photo-induced absorption and emission measurements suggested dissociation of the saccharin from the Ru-center supporting PACT pathways. The complexes display a significant binding affinity with CT-DNA (K_b～ 10⁴-10⁵M^?1) and bovine serum albumin (BSA) (K_BSA～ 10⁵M^?1). Cytotoxicity was studied in the dark and the presence of low energy UV-A light (365 nm) in cervical cancer cells (HeLa) and breast cancer cells (MCF7). Photoirradiation of the complexes induces the generation of reactive oxygen species (ROS) assessed using 1,3-diphenylisobenzofuran (DPBF) and intracellular DCFDA assays. The complexes are sufficiently internalized in cancer cells throughout the cytoplasm and nucleus and induce apoptosis as studied by staining with dual dyes using confocal microscopy.

Full text of DOI:10.1039/d1dt00246e

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Page 1 of 43
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DOI: 10.1039/D1DT00246E
Kinetically Labile Ruthenium(II) Complexes of Terpyridines and
Saccharin: Effect of Substituents on Photoactivity, Solvation Kinetics, and
Photocytotoxicity
a
bc
a
a
b
Priyaranjan Kumar , Prerana Singh , Sanjoy Saren , Sandip Pakira , Sri Sivakumar , Ashis K.
Patra*a
a
Author address: Department of Chemistry, Indian Institute of Technology Kanpur, Kanpur 208016,
Uttar Pradesh, India. E-mail: akpatra@iitk.ac.in
bDepartment of Chemical Engineering, DST Thematic Unit of Excellence on Soft Nanofabrication,
Indian Institute of Technology Kanpur, Kanpur 208016, Uttar Pradesh, India.
cDepartment of Biological Sciences & Bioengineering, Indian Institute of Technology Kanpur,
Uttar Pradesh 208016, India
1

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DOI: 10.1039/D1DT00246E
Abstract
Herein, we designed six kinetically labile ruthenium(II) complexes containing saccharin (sac) and
II
4
′-substituted-2,2:6,2-terpyridines (R-tpy), viz. trans-[Ru(sac) (H O) (dmso-S)] (1) and [Ru (R-
2
2
3
tpy)(sac) (X)] [X = solvent molecule] (2−6). We intentionally kept the labile hydrolysable Ru-X
2
bonds that were potentially activated via solvent-exchange reactions. This strategy generates the
coordinative vacancy that allows further binding with potential biological targets. To gain insight
of the electronic effects of ancillary ligands on Ru-X ligand-exchange kinetics or photoreactions,
we have used a series of substituted terpyridines (R-tpy) and studied their solvation kinetics. The
ternary complexes were also aimed for their potential utility in the Ru-assisted photoactivated
chemotherapy (PACT) synergized with release of saccharin as a highly selective carbonic anhydrase
IX (CA-IX) inhibitor, over-expressed in hypoxic tumors. The ternary complexes exhibit distorted
octahedral geometry around Ru(II) from two monodentate transoidal saccharin in axial position,
and tridentate terpyridines and labile solvent molecule at basal plane (2−6). We studied their
speciation, solvation kinetics, and photoreactivity in the presence of green LED light ( = 530
irr
nm). All the complexes are relatively labile and undergo solvation in coordinating solvents (e.g.
DMSO/DMF). The complexes undergo ligand-substitution reaction, their speciation and kinetics
1
were studied by UV-Vis, ESI-MS, H-NMR, and structural analysis. We also attempted to access
the effect of various substituents on ancillary terpyridine ligand (R-tpy) in photo-reactivity and
ligand-exchange reactions. The photo-induced absorption and emission measurements suggested
dissociation of the saccharin from Ru-center supporting PACT pathways. Complexes display a
4
6
-1
significant binding affinity with CT-DNA (K ~10 −10 M ) and bovine serum albumin (BSA)
b
5
-1
(
KBSA ~ 10 M ). The cytotoxicity studied in the dark and the presence of low energy UV-A light
(365 nm) in cervical cancer cells (HeLa) and breast cancer cells (MCF7). Photoirradiation of the
complexes induces the generation of reactive oxygen species (ROS) accessed using 1,3-
diphenylisobenzofuran (DPBF) and intracellular DCFDA assays. The complexes are sufficiently
internalized in cancer cells throughout the cytoplasm and nucleus and induce apoptosis studied by
staining with dual dyes using confocal microscopy.
2

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Introduction
Ruthenium complexes offer several desirable opportunities to the synthetic inorganic chemist for
designing targeted chemotherapeutics. Ruthenium has the flexibility to form covalent bonds with
various hard and soft donors (O, N, P, S and halogens, arenes) and wide structural diversity for
pharmacological targets. Therefore, the scope and flexibility for structural modulation and
peripheral ligand modification are far greater than the platinum drugs. Moreover, ruthenium is more
apt for multimodal therapeutic approaches due to its remarkable catalytic activity, redox modulation
II
III
via physiologically accessible oxidation-states (Ru , Ru ), tunable ligand-exchange kinetics, and
excellent photochemistry among d-block elements [1−6]. These multifaceted characters make Ru-
complexes at the forefront of medicinal inorganic chemistry for the last two decades [711]. The
medicinal inorganic chemists can satisfy their keen intuition, and creative designs for studying
multi-targeted metallodrugs due to the immense opportunity and diversity Ru-complexes offers in
multiple directions. Ruthenium complexes can exhibit their anticancer activity via activation of a
wide range of cellular pathways, offering to reinforce many cell-death mechanisms. Such a strategy
gives the prospect to tackle acquired drug resistance by Pt-drugs and their harmful side effects
[1215]. Ru-compounds' biological targets include DNA, proteins, enzymes, genes, and lipids
having a significant role in tumor growth, survival, and metabolism [16]. The high uptake of Ru-
complexes to cancerous tissue and lowered systematic toxicities are often correlated with its
advantageous periodic similarity with iron. The iron-transporter protein transferrin (Tf) meets the
high demand of Fe to cancer cells for their faster proliferation and metabolism. The Ru-agents
exploit Tf to facilitate their cellular internalization and exhibit tumor selectivity and high dose
tolerance compared to the platinum drugs [3].
The inorganic photosensitizers in combination with light and oxygen provide significant
opportunities for newer chemotherapeutic modalities-photodynamic therapy (PDT) or
photoactivated chemotherapy. The electronically excited states of certain metal complexes possess
easily accessible long-lived triplet excited states triggered by visible light activation. These excited
states undergo a series of photochemical reactions before deactivation to ground states, e.g.,
3
photorelease of bioactive ligands, energy transfer to O redox reactions, etc. [17]. The Ru-
2
,
photochemistry has been extensively utilized for developing non-invasive and clinically approved
PDT agents for cancer which provide selectivity via spatiotemporal control [18]. The PDT modality
is a strategy of generating cytotoxic reactive oxygen species (ROS) in situ from the photosensitizers
3

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3
upon excitation of preferably low energy light in the presence of O . Even with the low energy
2
photo-illumination in visible to near-infrared (NIR) can generate excited-state of the metal
3
-
2-
complexes, which can either transfer electrons to O to form superoxide (O ), peroxide (O ), and
2
2
2

1
hydroxyl radicals ( OH) (Type 1) or transfer energy to generate O (Type II) as major cytotoxic
2
species [1921]. Ru-polypyridyl complexes are a suitable choice as photosensitizers over the
organic chromophores because of superior absorption in the phototherapeutic window, low-lying
excited triplet states, and facile electron or energy transfer reactions to the biological substrates.
Moreover, the luminescent Ru(II) complexes are apt for cellular imaging probes, understanding
mechanism of actions, and theranostic applications [22]. Recently, McFarland and coworkers
reported the first Ru(II)-based PDT agent, TLD1433 containing α-terthienyl conjugated
imidazo[4,5-f][1,10]-phenanthroline photosensitizer and two 4,4′-dimethyl-2,2′ bipyridine ligands.
TLD1433 has entered phase 1b human clinical trials to treat non-muscle-invasive bladder cancer
(
NMIBC) and showed promising results [23, 24]. The solely O -dependence is a major limitation
2
of PDT modality, limiting its applicability for the treatment of solid and aggressive tumors
considering their hypoxic nature. Therefore, the treatment of solid hypoxic tumors remains
challenging for both classical chemotherapy and PDT. The combination of light to Ru-photocaged
3
complexes can activate them to dissociative MLCT triplet excited electronic states, which
3
interconvert further to MC electronic states. These processes end up forming new cytotoxic
chemical entities, known as photoactivated chemotherapy (PACT), providing spatiotemporal
control of the treatment modality. Interestingly, Ru-complexes with PACT applications do not
primarily require oxygen and have the potential to generate vacant sites on Ru by cleavage and
photosubstitution of usually monodentate or bidentate ligands selectively. Both the activated metal
center and/or photo-substituted ligand can impart biological activity in synergy [2529]. The metal-
solvated photoproduct can express binding and damage to the targets such as DNA like platins [29].
Therefore, the PACT strategy can be elegantly utilized for targeting solid hypoxic tumors. S. Bonnet
and coworkers recently reported nicotinamide phosphoribosyltransferase (NAMPT) inhibitor-
2
+
containing red-light-activated ( = 625 nm) Ru-caged complex namely [Ru(tpy)(biq)(L)] (biq =
ex
2
,2-biquinoline,
tpy
=
2,2;6-2-terpyridine
and
L
=
4-[({[4-(2-methyl-2-
propanyl)phenyl]sulfonyl}amino)methyl]-N-(3-pyridinyl)benzamide) capable of triggering the
cytotoxic ligand in oxygen-independent manner for skin and lung cancers [30]. Hypoxia is a major
obstacle in chemotherapy and photochemotherapy resulting in poor prognosis and drug resistance.
4

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To date, efficient hypoxia targeting agents are very limited in the literature. Thus, developing such
complexes is worthwhile to treat clinically challenging tumors with characteristics of poor prognosis
and resistance to multimodal chemotherapy and phototherapy [31−33].
In the context of exploiting various cancer hallmarks, the altered metabolic pathways of cancer cells
(Warburg effect) produce a considerably high amount of lactic acid, making tumour
microenvironment acidic [34]. Despite this adverse condition, cancer cells survive by local
upregulation of pH mediated from overexpressed carbonic anhydrase (CA)-IX and XII. Saccharin
(ortho-sulfobenzimide) is a potential inhibitor for CA-IX in nanomolar affinity, having more than
1
000-fold selectivity over the rest of the CA-isoforms. The inhibitory action of saccharin (sac) may
negatively affect tumor cells' survival by lowering intracellular pH [3537]. Therefore, the saccharin
releasing complexes could be a potential strategy to target hypoxic tumors [38].
II
Scheme 1. The rationale for design of [Ru (R-tpy)(sac) (X)] (2-6) complexes with substituted 4′-{(2-
2
pyrrolyl (2)/furyl (3)/thienyl (4)/pyridyl (5) and 3-pyridyl (6)}-2,2′:6,2-terpyridines (R-tpy) and X = solvents
(
CH CN, dmso, and H O) studied in this work.
3 2
Here, we have thoughtfully designed six Ru(II) complexes of bioactive saccharin (sac) ligands and
4
-substituted terpyridines (R-tpy) (2−6) with the intended labile exchangeable solvent coordination
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site to form activated Ru(II) complexes. These activated complexes are expected to interact with
biological targets such as nucleobases, oligonucleotides, amino acids, DNA, or proteins during their
intracellular passage (Scheme 1). Therefore, it is important to understand the equilibrium kinetics
of the formation of various ligand-exchange species and their fate in physiological conditions. To
gain insight into the electronic effects of peripheral ligands on photoreactions' kinetics or ligand-
exchange kinetics, we have used a series of substituted terpyridine (R-tpy). Moreover, R-tpy also
acts as effective photosensitizers and DNA-binder with varying 4-substituents for modulation of
photophysical and electronic properties. The choice of prognostic biomarker sac was based on its
potentials to inhibit carbonic anhydrase-IX (CA-IX) in possible synergism with photoactivated
chemotherapy (PACT) of Ru-polypyridyl complexes. Recently, we have reported the photo-induced
saccharin dissociation and DNA damage activity of octahedral Ru(II) complexes of saccharin
containing dipyridoqunoxaline (dpq) and dipyridophenazine (dppz) following ROS generation and
PACT pathways [39].
We report here the synthesis, spectral characterizations, solid-state structures, solution chemistry
II
and photoreactivity, and binding studies with DNA and serum proteins of a series of ternary [Ru (R-
tpy)(sac) (X)] (2-6) complexes containing accessible labile site (Ru-X) for feasible bioreactivity.
2
The complexes undergo solvent-exchange reactions in H O, DMF, CH CN, and DMSO,
2
3
demonstrating their kinetic lability. Interestingly, the complexes showed unique photo-induced
responses upon exposure of low energy green LED light (irr = 530 nm), corresponding to
photoactivation and probable release of saccharin. The cytotoxicity and photocytotoxicity of
complexes 1, 2, and 4 were evaluated from MTT assay in HeLa and MCF7 cancer cell lines.
Formation of reactive oxygen species (ROS) upon photoirradiation was studied from the
degradation of 1,3-diphenylisobenzofuran (DPBF) and staining of cancer cells with ROS-sensitve
2
',7'-dichlorodihydrofluorescein diacetate (H DCFDA) dye. The emissive complexes displayed
2
significant permeability, uptake, and induce apoptosis as determined by acridine orange/ethidium
bromide (AO/EB) dual staining. The complexes are envisioned as potential ROS-mediated
photoactivated chemotherapeutic agents.
6

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Results and Discussion
Synthesis, Characterization and Physiochemical aspects
All the Ru(II) complexes were synthesized in good yield from cis-[Ru(dmso-S) (dmso-O)Cl ],
3
2
+
-
sodium saccharinate dihydrate (Na sac 2H O) and 4′-substituted-2,2′:6′,2″-terpyridines (R-tpy) at
2
+
ambient conditions (Scheme 2). The reaction of Na Sac with a warm solution of cis-[Ru(dmso-
S) (dmso-O)Cl ] in methanol resulted in the formation of trans-[Ru(sac) (H O) (dmso-S)] (1). The
3
2
2
2
3
II
series of ternary complexes [Ru (R-tpy)(sac) (X)] (2−6) were synthesized from the binary complex
2
1
upon reaction with 4-{(2-pyrrolyl (2)/furyl (3)/thienyl (4)/pyridyl (5) and 3-pyridyl (6)}-
terpyridines (R-tpy). All the complexes were isolated as crystalline solids after work-up and remain
stable in ambient conditions. The complexes were characterized by ESI-MS, UV-Vis, FT-IR, NMR,
electrochemical analysis, fluorescence, and molecular structure was confirmed by single-crystal X-
ray diffraction studies. The selected results obtained from these physicochemical measurements are
shown in Table 1. The ESI-MS(+) analyses showed the presence of molecular ion peaks (M+)
corresponding to the complexes with the expected isotopic distribution pattern of Ru (Figs. S1S6).
1
The structural identity and purity of the complexes in solution were further evaluated from H NMR
(
Figs. S7S12). Noticeably, a slight deshielded peak corresponding to DMSO-d in addition to
6
referenced solvent (DMSO-d ) was observed, which originated from the coordination to the labile
6
site present in the complexes. All the complexes display characteristic chemical shift ( ppm) values
1
corresponding to substituted R-tpy and sac in the aromatic region of the H-NMR spectrum. Both
ESI-MS and NMR analyses suggest the structural integrity of ternary speciation in solution as
determined in solid-state. The FT-IR of complexes 1−6 showed the strong carbonyl (C=O) stretching
-1
frequencies (sac) ranging 1630−1643 cm (Fig. S13). The strong symmetric ( ) and asymmetric
sym
(
asym) stretching frequencies corresponding to the sulphonyl (SO2
)
group of sac ligand vary within
-1
-1,
1
146−1157 cm and 1286−1292 cm respectively [40]. Complexes 2−6 showed a lowered 
C=O
-1
than binary complex 1 with the minimum value for complex 4 (C=O = 1630 cm ). The ternary
complexes 2−6 with varying 4-terpyridine substitution are interesting in their distinct electronic
properties. Trans-[Ru(sac) (H O) (dmso-S)] (1) showed two weak absorption bands at 275 nm ( =
2
2
3
-1
-1
-1
-1
2
640 M cm ) and 365 nm ( = 370 M cm ) in DMF representing the ligand-based ππ*
1
transition and metal-to-ligand charge transfer ( MLCT) from Ru(dπ)  sac(π*) [41]. The
absorptions in the UV-region (284−350 nm) for the complexes 2−6 in DMF arise from the ligand-
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1
centered ( LC) ππ* transitions (Fig. 1(a) and Table 1). All the complexes displayed an intense
1
absorption band in the visible region of 496−560 nm assignable as MLCT originating from spin-
allowed Ru(dπ)tpy(π*) electronic transition [42, 43]. Interestingly, 4-(2-pyrrolyl/furyl/thienyl)-
1
R-tpy substitutions of the complexes 2−4 showed a red-shift of ~ 8−20 nm in their MLCT bands
than the 4-(2-pyridyl/3-pyridyl)-tpy indicating underlying electronic effects of the substituents. The
complexes with 2-furyl (3) and 2-thienyl (4) substituents showed similar MLCT bands (~505 nm)
with a higher molar absorptivity of the later complex. The 2-pyrrolyl substituent in complex 2
showed red-shift ~ 10-20 nm compared to the rest of the ternary complexes suggesting a relatively
1
low-energy MLCT state. The 3-pyridyl-tpy in 6 leads to an additional MLCT shoulder at 560 nm.
The UV-Vis absorption spectra of the complexes 1−6 in 1% (v/v) DMF−5 mM Tris-HCl/NaCl
buffer (pH = 7.2) showed small blue shifts of both the ππ* and Ru(dπ)tpy(π*) bands compared
with DMF solvent (Fig. S14 and Table S1). The complexes display multiple irreversible redox
responses in their cyclic voltammograms assignable to RuII/III and tpy ligands (Fig. 1(b), Fig. S15
II
III
and Table 1). The Ru Ru potential ranges from 1.01−1.12 V vs. Ag/AgCl, while saccharin
remained redox- inactive in these potential ranges [42, 44, 45].
8

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Scheme 2. General route for the synthesis of Ru(II) complexes: trans-[Ru(sac) (H O) (dmso-S)] (1) and
2
2
3
+
-
[
Ru(R-tpy)(sac) (X)] (X = solvent) (2−6). The reaction reagents and conditions: (a) 2 equiv. Na sac .2H O
2 2
(
sac = saccharin), MeOH, 3 h reflux; (b) KOH, NH (aq), EtOH, overnight stirring; (c) 1 (MeOH), 4′-
3
substituted-2,2′:6′,2″-terpyridine (CHCl ), 12 h reflux.
3
The lower redox potentials in case complexes 2−6 than 1 indicate the increased electron density at
Ru(II)-centre implies that the R-tpy ligands are more likely better -donors. The complexes 3 and
II
III
4
with 2-furyl and 2-thienyl substituents showed similar Ru Ru potential but the isomeric
complexes 6 showed the higher E (V) for the couple RuII/III than complex 5.
The luminescence spectra of the ligands in DMF showed R-tpy ligands are highly emissive and
showed fluorescence ranging 360395 nm upon excitation at 280 nm (Fig. S16 (b)). The saccharin
(
sac) was found only weakly emissive at  = 440 nm along with a weak shoulder at 345 nm. The
em
1
complexes 1−6 showed a ligand-centered ( LC) broad emissions ranging 430−460 nm ( = 360
ex
nm), and another emission peak at 407 nm (2−6) and 412 nm (1) respectively (Fig.1(d), Table 1)
[46]. Upon excitation at 280 nm, the complexes show two emission bands in the range of 345451
nm with em ~ 345 nm and ~450 nm respectively (Fig. S17 and Table S1). The resemblance of
emission spectral profile of sac and complexes suggested the fluorescence emission of the
complexes originating mainly from sac ligands for complexes 15 while both the 3-pytpy and sac
centered emission was observed for complex 6. The excitation of MLCT bands of the complexes
3
3
showed very poor emission, possibly due to closer MLCT and metal-centered excited ( MC) states
which favor radiationless thermal deactivation (Fig. S18) [47]. Moreover, the presence of labile Ru-
X (solv.) bond will facilitate the non-radiative decay of triplet Ru-centered excited states via
vibrational energy transfer (VET). The excitation spectra of the ligands are shown in Fig. 16 (a).
The excitation spectra of complexes at  = 450 nm in DMF showed a peak at ~ 285 nm and broad
em
peaks ranging 330−430 nm (Fig. 1(c)).
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Fig. 1. (a) Absorption spectra of the complexes 16 in DMF at 298 K. (b) The cyclovoltametric responses of
complexes 16 (1 mM) in DMF showing the Ru /RuIII redox couple [supporting electrolyte: 0.1 M
II
n
-1
Bu NClO ; working electrode: glassy carbon; counter electrode: Pt-wire, scan rate: 50 mVs ]. (c) Overlay
4
4
of the excitation spectra of the complexes 16 (10 μM) with em = 450 nm. (d) The emission spectra of the
complexes 16 (10 μM) in DMF. [  = 360 nm, ex. slit width = 10 nm, em. slit width = 10 nm, T = 298 K].
ex
Table 1. Selected photophysical and electrochemical data of complexes 1−6.
Complex λmaxa/nm, (ε/ ×10 M cm )
3
−1
−1
λemb/nm
IR /cm
C=O
c
-1
E (RuII/II)
d
(SO2
sym/asym
1148/1292
)
pc
(
)
`1
275 (2.64), 365 (0.37)
412, 435
407, 460
407, 435
407, 435
407, 433
1643
1642
1642
1630
1631
1.12
1.05
1.02
1.02
1.01
2
288 (18.33), 310 (17.77), 350 (11.86), 515 (5.02)
288 (18.81), 322 (24.67), 505 (6.73)
288 (20.53), 322 (24.60), 504 (7.59)
288 (25.18), 316 (21.39), 496 (7.79)
1148/1290
1146/1287
1157/1296
1147/1288
3
4
5
1
0

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6
284 (29.56), 320 (20.25), 497 (6.89), 560 (3.32)
407, 450
1636
1148/1286
1.05
a
b
UV–visible determined absorption peak maximum and molar extinction coefficient in DMF. Fluorescence
c
d
II/III
emission maxima with λexc = 360 nm. In KBr pellets. Redox potential (V) for the couple Ru vs. Ag/AgCl in the
presence of 0.1 M tetrabutylammonium perchlorate (TBAP) as supporting electrolyte in DMF.
Single crystal X-ray structures
The trans-[Ru(sac) (H O) (dmso-S)] (1) and [Ru(R-tpy)(sac) (X)] (X = solvent) (35) were
2
2
3
2
crystallized using slow evaporation under ambient conditions. Our efforts to crystallize complexes
and 6 remained unsuccessful. All the complexes crystallized in the triclinic P-1 space group with
2
two molecules in the unit cell. The ORTEP views of the molecular structures with labeled
heteroatoms are shown in Fig. 2, and the unit cell packing diagrams are shown in Fig. S19 in ESI.
The detailed crystallographic parameters are given in Table S2 and the selected bond lengths are
II
shown in Table S3 and S4 in ESI. The crystal structure of trans-[Ru (sac) (H O) (dmso-S)] (1)
2
2
3
showed distorted octahedral geometry coordinating of three H O, one dmso (S-donor) in equatorial
2
II
plane, and two axial saccharinates as N-donor in trans-conformation. The [Ru (R-tpy)(sac) (X)] (X
2
=
solvent) (35) showed distorted octahedral {RuN (solvent)} geometry with three basal sites
5
coordinated to N -donor terpyridine ligands (R-tpy), two axial sites bound to the N-donor
3
saccharinate (trans). The sixth equatorial site is coordinated to a solvent molecule (X), [X = CH CN
3
(
3); H O (4, 5)]. The Ru-X site is expected to be kinetically labile and can facilitate further ligand-
2
exchange reactions.
1
1

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Fig. 2. The ORTEP view of the molecular structure of trans-[Ru(sac) (H O) (dmso-S)] (1) and [Ru(R-
2
2
3
tpy)(sac) (X)] (1, 3, 5) with labeled heteroatoms. The thermal ellipsoids are drawn at its 50% probability
2
level. The hydrogen atoms were omitted for clarity.
1
2

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Table 2. Selected Ru(II)-sac/tpy bond lengths and the dihedral angle between the sac ligands
Complex
1
2.128(4)

3
4
5
RuN(sac)a (Å)
RuN4(tpy)b (Å)
Dihedral anglec
2.124(3)
1.944(3)
71.55
2.131(8)
1.927(7)
40.39
2.114(3)
1.921(3)
7.30
9.75
aSmallest bond length for Rusac. RuN bond length trans to solvent coordination site. The dihedral
b
c
angle between the [Ru1N1S1C7] and [Ru1N2S2C14] planes containing sac ligands.
The longest and shortest bond lengths in complex 1 are Ru−S(dmso) and Ru−O3(OH ), respectively.
2
A gradual decrease of the average Ru-(N)sac and Ru-Ntpy bond lengths were observed in 3−5 than
complex 1 reveals the substituents' associated electronic effects at 4′-tpy. The Ru-N bond distances
in complexes were in the range of reported analogous Ru(II) complexes [39, 4851]. The shortest
Ru-N bonds correspond to the Ru-(N) (1) and Ru-N4 (35) are shown in Table 2, suggesting a
sac
tpy
better π-acceptor ability of pytpy (5) than ftpy (3) and ttpy (4) [52]. The longest bond for complex
is Ru−N1(sac) at 2.134(3) Å and for complexes 4 and 5 is Ru1−O7 (OH ) at a distance of 2.191(6)
3
2
Å and 2.174(2) Å, respectively. Both the saccharinates are a little away from perfectly trans-
configuration as evidenced from N1-Ru1-N2: 173.27(16) (1), 176.11(13) (3), 172.2(3) (4)
1
73.30(12) (5). A strong intermolecular long-range π−π interaction was observed between the
saccharin rings at 3.376 Å for 1 and between the R-tpy rings at 3.359 Å (3), 3.376 Å (4), and 3.356
Å (5) and shown in Fig. S20. A significantly higher dihedral angle for complexes 3 (71.55) and 4
(40.39) are observed between the saccharin planes compared to complex 1 (7.30) and 5 (9.75)
(Table 2 and Fig. S21).
Solvation of the Complexes
Ruthenium(II) complexes with a labile site like the Pt(II) drugs show hydrolysis and are believed to
be a key step before interacting with the biological targets to exhibit its therapeutic efficacy. The
presence of labile Ru-Cl bond(s) is a crucial design criterion as evident from the molecular structures
of NAMI-A, KP1019, or half-sandwich Ru-arene complexes (e.g. RAPTA-C) and many recent
examples originally inspired form platins. However, the factors that influence Ru-X bond's lability
(X = monodentate ligands) or its variation on therapeutic efficacy are not investigated systematically
in the literature. Any metallodrug inside the cells obviously encounters a vast array of potential low
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3

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-
or high M.W. biologically relevant species as ligands ranging from H O, Cl , nucleotides, amino
2
acids, thiols, amino acids, peptides, nucleic acids or proteins, etc. Some of these reactions are
desirable for activation of the drug, while rest may lead to significant inactivation, efflux, or create
multiple roadblocks to reach the desirable pharmacological targets. Therefore, a sound
understanding of the modulation of such reaction kinetics and speciation in the physiological
condition is essential. Such pharmacokinetics information is vital in designing a truly effective drug
candidate that will survive the complex and competitive cellular environment and able to make its
way to the desired site at an effective dose. A deeper understanding of these reactions is also
essential to study the mechanism of action of multi-drug resistance (MDR) and how to overcome it.
The comparable rates of hydrolysis of Ru(II)-complexes with Pt(II), make them a suitable and
alternative choice of metal for Pt(II) in chemotherapy [53]. The rates of solvation/anation reactions
can be fine-tuned based on the ancillary ligands' electronic effects and substituents present on it.
The ligand substitution kinetics is directly associated with the pharmacokinetic profile of the Ru(II)-
drugs; higher ligand-exchange rates usually showcase higher cytotoxicity [54]. The labile Ru(II)-X
II
bonds in [Ru (R-tpy)(sac) (X)] (X = solvent) (2−6) are expected to undergo a facile hydrolysis to
2
form aqua-complexes followed by anticipated coordination with intracellular biological targets. The
solvolysis reaction kinetics of the complexes were studied in DMF and aqueous buffer using UV-
1
vis spectroscopy and in DMSO-d using H NMR to understand their lability and in situ speciation
6
in solution.
Fig. 3. The electronic absorption changes of the complexes 1 and 2 upon solvation for 240 mins in the dark.
(a) The solvation of complex 2 (58 μM) in the presence of DMF. Inset: The pseudo-first-order kinetic fit
measured from the changes in A308 nm for complex 2 upon solvation with DMF. (b) The hydrolysis kinetics of
1
4

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complex 1 (222 μM) in the presence of 11% (v/v) DMF−5 mM Tris-HCl/NaCl buffer (pH = 7.2) mixture.
Inset: The pseudo first-order kinetic fit of the changes in A350 nm upon hydrolysis.
Table 3. The solvation kinetic parameter for the complexes 1−6.
a
-1
b
c
-1
d
Complex
k (s )
t
1/2 (min)
k (s )
t1/2 (min)
1
2
3
4
5
(8.900.90) × 10-4
(1.300.04) × 10-4
(2.310.16) × 10-4
(2.370.10) × 10-4
(2.440.29) × 10-4
13.00
90.12
49.93
48.77
47.40
(2.60.14) × 10-4
(8.91.50) × 10-5
(5.621.80) × 10-5
(1.990.16) × 10-4
-
44.0
129.78
205.64
58.09
-
aSolvation rate constant in DMF at 298 K calculated from abs = 350 nm (1), 308 nm (2), 308 nm (3), 322
b
c
nm (4) and 318 nm (5). Half –lifetime from first-order kinetics of solvation (DMF). Hydrolysis rate
constant at 298 K calculated from abs = 350 nm (1), 286 nm (2), 322 nm (3), and 322 nm (4). dHalf –
lifetime from first-order kinetics of hydrolysis.
The solvation kinetics of the complexes 1−6 were studied in DMF and DMF−5 mM Tris-HCl/NaCl
buffer (pH = 7.2) in the dark condition from time-dependent absorption measurement (Fig. 3 and
Figs. S22−S24). The UV-Vis spectra of the complexes in DMF or aq. buffer showed
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5

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1
Fig. 4. Time-dependent concomitant evolution of H-NMR spectra of the complexes (DMSO-d , 500 MHz,
6
II
II
2
98 K) for 3 h at various ranges: (a, b) [Ru (furyl-tpy)(sac) (X)] (3), and (c, d) [Ru (thienyl-tpy)(sac) (X)]
2 2
(4).
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6

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Fig. 5. Proposed solvation reaction of the complexes in various coordinating solvents as identified from their
spectral measurements and single-crystal X-ray structures. Solvation of complex 1 in the buffer (top) and
II
generalized representation of complexes [Ru (R-tpy)(sac) (X)] (2-6) (bottom) in CH CN, buffer, and DMF.
2
3
concomitant changes in their absorbances. The pseudo first-order kinetic data for the rate of
solvation reaction (k) and half-life time (t ) are measured from the fitting of absorbance changes
1
/2
to the rate equation are shown in Table 3. As expected, the binary complex [Ru(sac) (H O) (dmso-
2
2
3
S)] (1) showed a higher rate of solvation amongst all complexes with multiple easily accessible
labile coordinating ligands. The rates of solvation in DMF were found in order of 10-4 s-1 and
-4
-5 -1
hydrolysis rates in the range of 10 −10 s . We observed comparatively higher rates of solvation
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7

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in the case of DMF than the aqueous buffer, possibly driven by stronger Ru-DMF bonds. The
complex 6 seems relatively stable and does not show any noticeable changes in absorbance for 4 h.
1
The solvation and lability of the complexes 16 were monitored using time-dependent H-NMR in
DMSO-d for 3 h (Fig. 4 and Figs. S25S28). The results showed the ternary speciation of the
6
complexes retained in solution and do not show changes in ligand-centered chemical shifts in
1
aromatic regions. Interestingly, we observed a gradually emerging H peak at ~ 2.50 ppm in addition
1
to the residual H-peak from the reference solvent (DMSO-d ) (2.46 ppm), which corresponds to
6
the coordination of solvent (DMSO-d ) with Ru-center. Upon increasing the time from 10 min to 3
6
1
h, the peak at ~ 2.50 ppm showed gradual enhancement in intensity. The time-dependent H-NMR
data suggests facile solvation and kinetic lability of Ru-X bond, resulting in a dynamic equilibrium
between Ru-bound DMSO and free DMSO. The relatively faster solvation for complex 1 and slower
solvation for complexes 5 and 6 support the kinetic rates from UV-vis studies (ESI†).
1
The ligand-exchange reactions with coordinating solvents are evidenced from UV-Vis, ESI-MS, H
NMR, and the presence of Ru-solvent bond during crystallization from such solvents. Such solvation
reactions are shown in Fig. 5. These labile complexes are anticipated to coordinate with biologically
relevant molecules as ligands in cancer cells.
Photoreactivity
The photoactivated Ru(II) complexes have been widely explored for PDT/ PACT applications due
to activation of a variety of photochemical reactions from easily accessible triplet excited states,
facile intersystem crossing, generation of ROS, and presence of intense low-energy absorption
bands available in the PDT window [1722]. The photoexcitation of Ru-polypyridyl complexes
having a strong MLCT band in the visible region undergoes photosubstitution of the monodentate
II
ligands by a solvent molecule [5557]. The presence of a series of R-tpy chromophores in [Ru (R-
tpy)(sac) (X)] (X = solvent) is anticipated to show photosubstitution of the monodentate saccharin
2
3
(
sac) ligands having therapeutically relevant hypoxia targeting properties due to accessible MC-
excited states.
The irradiation of white light (WL) (λ > 400 nm, 5V, 0.3 W) to the 2% (v/v) DMF-buffer mixture
ex
solution (pH 7.2) of complexes 1−6 showed different absorption profiles in comparison to dark (Fig.
S29). The complexes 1 and 6 on photo-illumination showed increased absorbance, while 2−5
showed an observable decrease of absorbance. The formation of the isosbestic points in UV-region
indicating generation of new photoproducts under visible-light irradiation.
1
8

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Fig. 6. The green light (λ = 530 nm) LED (3V, 158 lm @ 700mA) induced spectral changes observed for
ex
the complexes in DMF. (a) Absorption spectral traces of complex 2 (58 μM) for the 0−100 min. Inset:
Changes in absorbance at 308 nm of complex 2 with monoexponential curve fitting. (b) Absorption spectral
traces of complex 3 (39 μM) for 0−100 min. Inset: Changes in absorbance at 322 nm of complex 3 with
monoexponential curve fitting. (c) The unique enhancement of fluorescence emission (λ = 280 nm) intensity
ex
upon photoexposure of the complex 6 (20 μM) for 0−60 min, (d) Relative changes in the fluorescence
intensity of the complexes with the function of exposure time. Ex. slit width = 10 nm, em. slit width = 10
nm, T = 298 K. The direction of the arrow represents the concomitant changes.
We further studied the effect of green LED light (λ = 530 nm, 3V, 158 lm @ 700mA, from
irr
Luxeonstar LEDs, Canada) on the complexes in DMF from UV-Vis and fluorescence emission
studies. The green light (GL) induced significant changes in the UV-vis spectral traces (Figs. 6(a),
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9

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(b) and Fig. S30) with alteration in absorbance of both the ππ* and MLCT bands with the
formation of new peaks and formation of multiple isosbestic points..
The steady-state emission of complexes 1−5 showed a decrease of emission intensity; however,
complex 6 displayed a unique emission profile with significant enhancement of emission intensity
centered at 359 nm and bands ranging 430540 nm (Fig. 6(c), (d) and Fig. S31). The emission band
at 359 nm corresponds to 3-pytpy, while  at 430540 nm assigned to saccharin. The complexes
em
do not show any changes in their emission intensities in dark (Fig. S32).
1
Further, GL-induced photoactivity of complex 3 was studied using H-NMR spectroscopy.
Photoirradiation (060 min) resulted in decreased peak intensity at 6.78 ppm and ~ 8.73 ppm with
1
slightly deshielded chemical shifts (Fig. S33). We didn’t observe any significant changes in H-
II
NMR spectra possibly due to low photosubstitution yield as reported for [Ru (tpy)LCl ] type
2
complexes, (where L = monodentate ligands: pyridines and nitriles) and higher concentrations (mM)
used for NMR studies [58].
The above spectral results suggest distinct green-light induced photoactivity of complexes 26 due
to underlying photo-substitution reactions. The spectral changes occur probably through the
photorelease of sac ligands followed by the solvation as reported for several Ru(II)-complexes [27,
2
8].
DNA binding studies
DNA is the most explored target for many clinically successful chemotherapeutic drugs (e.g.
doxorubicins, 5-fluorouracil, gemcitabine, and platins) [59]. Ruthenium complexes with suitable
ligands choice can bind DNA in various modes including covalent coordination from electronically
rich nucleic acid (Guanine-N7), intercalation in nucleobases, major and minor groove depending on
their overall structure [60, 61]. The bound Ru-complexes potentially damage DNA or alter the
structural conformations of DNA, leading to inhibition in transcription and translation of cells. The
6
II
III
d -polypyridyl metal complexes (Ru /Rh ) have been reported for their advantageous molecular
light switch, charge-transfer reagents, metallointercaltors, metalloinsertors of DNA due to their
unique luminescence. These properties make these reagents a suitable diagnostic molecular probe
and therapeutic agent for cancer [6265]. The binding of metal complexes to DNA usually shows
both hypochromic and bathochromic shifts of their electronic spectra indicating π−π interactions
between base pairs and planar organic chromophore.
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The addition of CT-DNA to the complexes in 5 mM Tris-HCl/NaCl buffer (pH = 7.2) resulted in a
significant decrease in ππ* and MLCT bands for complexes 2−6 of its UV-Vis spectra
corresponding to terpyridine (Figs. 7(a) and S34). However, complex 1 without R-tpy ligand showed
a hyperchromic shift of ππ* band upon DNA addition and can be accounted from duplex
stabilization and tight binding with CT-DNA (Fig. S34(a)) [66, 67]. The intrinsic DNA binding
4
-1
6
-1
constant (K ) of the complexes found in the order of 10 M (1)−10 M (6) (Table 4). The K values
b
b
are comparable to reported [Ru(Cl-tpy)(en/dach)Cl]Cl complexes (en= ethylenediamine, dach =
,2-diaminocyclohexane) [67].
1
Fig. 7. The interaction of the complexes with CT-DNA in 5 mM Tris-HCl/NaCl buffer (pH = 7.2). (a) The
binding of complex 3 (49 μM) with CT-DNA (0−35 μM) in 2.1% (v/v) DMF-buffer mixture leads to the
hypochromic shift of their absorption bands. Inset: Determination of binding constant of complex 3 with
DNA from the slope to the intercept ratio of the linear fit plot [DNA]/Δ vs. [DNA]. (b) Ethidium bromide
af
(EthB) displacement assay for the complex 3. The gradual addition of complex (0−39 μM) to the multi-step
pretreated EthB (12 μM) with CT-DNA (14.4 μM) addition leads to significant quenching of fluorescence
intensity of the adduct at 605 nm. The bottom trace (black) corresponds to EthB emission and the top trace
(red) corresponds to EthB-DNA adduct. Inset: EthB displacement profile of complexes 2−6 from the
quenching of intensity I/I vs [complex] in ~ 2% (v/v) DMF-buffer.
0
The hypothesis that extended polypyridyls can show intercalation was further studied from ethidium
bromide (EthB) displacement assay. EthB is an emissive intercalator for DNA and used as
competitive probe for evaluation of intercaltive binding of other compounds. The EtBr alone in
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buffer media is poorly emissive due to quenching from H O molecule. However, it showed
2
enhanced red emission ~ 605 nm (λexc = 546 nm) upon binding to DNA [68]. The emissive EthB-
DNA adduct upon gradual addition of complexes 2−6 showed a significant fluorescence quenching
due to the displacement of EthB from DNA and binding of complexes via intercalation (Figs. 7(b)
6
and S35). The competitive apparent binding constant (K ) of complexes are in order of 10 derived
app
from their concentration corresponding to 50% emission quenching (C ) (Table 4) [69]. Complex
5
0
1
could not quench the emission of EthB-DNA adduct (Fig. S35(a)) signifying the presence of planar
R-tpy ligands is necessary for effective intercalation.
Table 4. DNA binding constants for the complexes 1−6.
Complex
1
2
3
4
5
6
a
-1
1.07  105 3.80  104 2.65  105 3.34  104 1.61  105
9.50  105
7.00 106
16.80
K /M
b
Kappb/M-1
-
-
5.23 106
3.74 106
3.97 106
3.18 106
c
C (M)
58.47
31.50
29.67
36.95
5
0
a
b
c
Intrinsic DNA binding constant. Apparent binding constant with DNA. The complex concentration
corresponding to 50% quenching of emission intensity of EthB-DNA adduct.
Protein binding studies
Serum proteins play an active role in the binding, distribution, metabolism, and pharmacokinetics
of drugs. The clinically tested anticancer Ruthenium complexes (NAMI-A and KP1019) show less
toxicity and tumor selectivity and are believed to act through HSA-mediated transport.
2
2

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Fig. 8. The BSA (2 μM) binding studies of the complexes (0−3.5 μM) in 0.7% (v/v) DMF−5 mM Tris-
HCl/NaCl buffer (pH = 7.2) mixture at 298 K, λexc/em = 295/345 nm and slit width ex/em = 10/5. (a) The
tryptophan fluorescence emission quenching of BSA upon increasing complex 5. Inset: The Stern-Volmer
plot for the complexes. (b) The modified Stern-Volmer plot for the determination of static equilibrium
binding constant from intercept and number of binding sites available (n) from the slope of the plot.
Table 5. BSA binding parameters for the complexes 1−6.
Complex
1
2
3
4
5
6
KBSAa (M )  10
-1
5
1.14
1.14
1.16
0.81
1.55
1.55
7.62
0.94
1.87
1.87
1.36
0.80
1.34
1.34
258
1.22
1.14
1.14
1.38
0.83
1.34
1.34
3.33
0.89
b
-1 -1
13
K (M s )  10
q
c
-1
4
K (M )  10
nd
a
c
b
Stern-Volmer constant for tryptophan-214 fluorescence emission quenching of BSA. Quenching rate constant.
d
Protein binding constant. The number of binding sites per molecule of BSA.
These Ru-compounds binds to the most abundant serum protein accumulate in tumor cells via leaky
blood vessels by the enhanced permeability retention (EPR) effect [70, 71]. The presence of two
tryptophan residues (Trp-134 and Trp-212) contributes to its significant intrinsic fluorescence in
BSA and is used as a spectral probe for drug binding studies. The alteration of fluorescence arises
from the conformational changes, ligand binding, subunit association, or protein denaturation [72,
7
3].
A significant quenching of fluorescence intensity of BSA (2 μM) at 346 nm (λexc = 295 nm) was
observed upon the gradual addition of complexes 1−6 (0−3.5 μM) (Fig. 8 and Fig. S36). The Stern-
Volmer quenching constant (KBSA), Stern-Volmer quenching rate constant (K ), the binding constant
q
(K), and the number of binding sites available per molecule (n) were determined [74] and shown in
4
6
-1
Table 5. The binding constants vary in the range 10 −10 M , suggesting an optimum affinity
towards BSA with lowest affinity for complex 1 and highest for complex 4. The n values are closer
to unity (0.80−1.22), suggesting a single binding site available per molecule of the protein. The
protein binding parameters KBSA, K , K, and n values agree with the reported Ru-terpyridine ternary
q
complexes [75].
Synchronous fluorescence spectral studies
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Lloyd introduced that the simultaneous scanning of both the excitation and emission
monochromators and monitoring fluorescence provides a valuable synchronous fluorescence
spectrum (SFS) [76]. The constant-wavelength or constant-energy or variable-angle synchronous
luminescence measurement can serve various analytical needs. The SFS provides several valuable
information such as spectral simplifications, reduced spectral bandwidth, and maintained
fluorescence sensitivity of multichromophoric residues with different spectral properties [77]. The
information on drug interactions with protein is crucially essential in the proteomics era, and the
SFS technique provides salient information at the molecular level.
Fig. 9. The synchronous fluorescence binding studies of BSA (2 μM) of the complexes 1−6 in 0.7% (v/v) 5
mM Tris-HCl/NaCl buffer (pH = 7.2) mixture at 298 K slit width ex/em = 10/5. (a) An overlay of I/I vs.
0
[Complex] for the decrease of intensity for Δλ = 15 nm. Inset: The spectral traces showing an intensity
decrease of complex 6. (b) An overlay of I/I vs. [Complex] for the decrease of intensity for Δλ = 60 nm.
0
Inset: The spectral traces show an intensity decrease of complex 6.
The fixed wavelength scanning of BSA with Δλ = 15 nm and Δλ = 60 nm exploits the information
of tryptophan and tyrosine chromophores present in the microenvironment of the protein. The
addition of aliquots of the complexes 1−6 to buffer solution of BSA (2 μM) resulted in significant
quenching of intensity for both the Δλ = 15 nm (10−32%) and Δλ = 60 nm (33−44%) measurements
(Fig. 9 and Figs. S37−S38). The pronounced changes in the intensity for Δλ = 60 nm suggests the
preferential binding with tryptophan residue over tyrosine. However, we did not observe many
changes in the hydrophobicity of the microenvironment around tryptophan residue with no apparent
shift of band position at Δλ = 60 nm [78].
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4

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Photoinduced singlet oxygen generation
1
The ability of the complexes to induce singlet oxygen ( O ) generation upon photoexposure of green
2
light LED (GL, λ = 530 nm) (3V, 158 lm @ 700mA) was performed from the absorption spectral
irr
1
measurement of 1,3-diphenylisobenzofuran (DPBF). DPBF in the presence of O undergoes [4+ 2]
2
cycloaddition reaction resulting in the formation of highly unstable endoperoxide, which
subsequently decomposes to 1, 2-dibenzoylbenzene (DBB) [79, 80]. We performed DPBF assay to
1
investigate O generation from complexes 2 and 4 upon GL exposure, and results are shown in Fig.
2
1
0 and Fig. S39. Interestingly, the DPBF (50 M) remains stable in the presence of the complexes
(10 M) in dark. However, GL illumination (0600 s) showed a remarkable decrease in A415 nm
corresponding to * electronic transition of DPBF. The control experiment with DPBF alone
upon GL exposure only showed a slight decline of A415 nm, suggesting the photoinduced in situ
1
generations of O from the complexes 2 and 4. The significant decrease of A
for complex 4
415 nm
2
1
(
96%) than 2 (54%) indicates efficient O generation from the thienyl substituent of R-tpy. The
2
presence of three thienyl moieties in imidazophenantroline ligand design in TLD1443- a Ru-based
1
PDT agent under clinical trial for bladder cancer is critical for higher O quantum yield [24].
2
Fig. 10. (a) Absorption spectral profile of DPBF (50 M) with 2 (10 M) upon irradiation with green light
1
LED ( =530 nm) for 0600 s signifying the formation of O . (b) Time-dependent relative absorbance
2
changes for complexes 2 (10 M) and 4 (10 M) with DPBF (50 M) in dark and upon green light irradiation.
Spectra was recorded at 298 K in DMF solution.
Cytotoxicity Studies
Photocytotoxicity
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The cytotoxicity profile of the selected complexes (1, 2, and 4) were tested in both the HeLa and
MCF7 cancer cells in the dark and low energy UV-A light (365 nm, 6 W) irradiation using MTT
assay (Fig. 11 and Figs. S40, S42 in ESI). The tested complexes showed relatively higher toxicity
towards HeLa over MCF7 cells in the dark. Complex 4 with 2-thienyl-tpy group found more
cytotoxic than the binary Ru(II) compound (1) and ternary complex 2 having 2-pyrrolyl-tpy ligand.
Fig. 11. The dose-dependent toxicity profile of complex 4 from the MTT assay was determined after 24 h
incubation. (a) Cytotoxicity profile in the dark for HeLa cells. (b) The overlay of dark toxicity and UV-A
light (365 nm, 6 W) induced phototoxicity in MCF7 cells.
Further, the photocytotoxicity of the complexes in MCF7 cells were also studied in the presence of
white light (λ > 400 nm, 5V, 0.3 W) (Fig. S41). We observed enhanced phototoxicity of complex
ex
4
1
in MCF7 cells at 365 nm UV-A light compared to white light irradiation. In contrast, complexes
and 2 do not differ in their phototoxicities to the MCF7 cells in UV-A light. This greater
phototoxicity for complex 4 can be accounted from its efficient ROS generation due to presence of
-thienyl substituted terpyridine (ttpy) as observed from DPBF assay compared to complex 2. The
2
presence of labile Ru-solvent bonds and associated ligand-exchange reactions are also expected to
1
deactivate the triplet Ru-excited states essential to generate O or other ROS involved in
2
photocytotoxicity.
Cellular uptake in MCF7 and HeLa cancer cells
Once the cytotoxicity of the complexes was studied, we attempted to study their uptake to both the
HeLa and MCF7 cell lines from the fluorescence microscopic imaging studies. As the complexes
are fluorescent and showed blue emission, the compounds' uptake can be visualized using confocal
laser scanning microscopy (CLSM).
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6

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Fig. 12. Cellular uptake of complexes 1, 2 and 4 (10 μM) in MCF7 cells after 4 h of treatment visualized by
confocal laser scanning microscopy with λ = 350 nm and λ = 460 nm. Top: complex 1, middle: complex
ex
em
2
and bottom: complex 4, scale bar = 50 μm.
The cellular uptake studies of complexes in MCF7 cells are shown in Fig. 12 and for HeLa cells in
Fig. S43 in ESI. Both the cells showed efficient localization of the complexes throughout the
cytoplasm and nucleus. Good permeability of the complexes to the cancer cells is advantageous in
targeting aggressive and hypoxic tumors.
Photoinduced intracellular ROS detection
To convince ourselves on the photocytotoxicity mechanism, we studied the intracellular ROS
generation in the presence of the tested Ru(II) complexes. 2',7'-dichlorodihydrofluorescein diacetate
2
7

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DOI: 10.1039/D1DT00246E
(
H DCFDA), a cell-permeable probe, which upon cleavage of the acetate groups by esterases and
2
oxidation is converted to highly fluorescent 2',7'-dichlorofluorescein (DCF) and utilized as an
indicator for ROS detection [81]. To affirm the role of the photosensitizing complexes 1, 2, 4 as
reactive oxygen species (ROS) generating agents, we performed DCFH-DA staining in MCF7 cells.
The control experiments were performed in the dark as negative control and hydrogen peroxide
(
H O ) as a conventional positive control. After 4 h treatment of complexes and photoirradiation (1
2 2
h) of low energy UV-A light (365 nm, 6 W), we observed significant ROS generation capability of
the complexes. Complex 4 showed higher ROS generation ability than 1 and 2 (Fig. 13). The
efficient ROS generation ability from complex 4 was consistent with the results obtained from
photodegradation of 1,3-diphenylisobenzofuran (DPBF) (Fig. 10) and higher phototoxicity in MCF7
cells (Fig. 11). In the dark control experiment, very feeble ROS was detected in the presence the
nd
complexes (Fig. S44). The positive control H O elicit significant ROS generation (Fig. 13 (a), 2
2
2
panel). The results are quantitated by mean fluorescence intensity (MFI) analysis using Image J
software suggesting higher efficiency of complex 4 (Fig. 13(b)).
Fig. 13. Detection of intracellular reactive oxygen species (ROS) from H DCFDA assay. (a) The confocal
2
laser scanning microscopy (CLSM) images of MCF7 cells, for untreated cells (negative control), H O
2
2
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treated (positive control) and after 4 h treatment with complexes 1, 2, 4 (10 M) followed by photoexposure
1 h) with UV-A light (365 nm). λ = 492 nm, λ = 527 nm, scale bar = 100 m. (b)
(
ex
em
Quantitative analysis comparing the levels of ROS intermediates in untreated and treated MCF7 cells on
exposure to UV-A light (365 nm). Values are mean ± S.D from three independent experiments (n = 3).
Apoptosis assessment
The apoptotic potential of complexes 1, 2, and 4 was substantiated on MCF7 cells in the dark (Fig.
1
4). The viable cells, early apoptotic cells, and the late apoptotic cells were examined by acridine
orange/ethidium bromide (AO/EB) dual staining which efficiently binds to nucleic acids by
intercalation [82]. These fluorescent dyes exactly aid in distinguishing cells in definite stages of
apoptosis. Acridine orange (AO), a vital dye, stains both the viable (green colour) and apoptotic
cells (yellow-green). Ethidium bromide (EtBr) stains necrotic cells red due to loss of membrane
integrity. Early apoptotic cells show characteristic features of nuclear condensation and apoptotic
bodies in yellow color. Late apoptotic cells are marked orange-red as they incorporate EtBr
demonstrating aberrant nuclear morphology. The confocal laser scanning microscopy images of
AO/EB staining indicate that complex 4 showed enhanced apoptosis compared to complexes 1 and
2
. Most of the cells appeared orange-red in color, representative of late apoptosis. Quantification of
apoptosis affirms complex 4 causes efficient apoptosis as compared to 1 and 2 (Fig. 14 (b)).
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