Mitochondria-Targeting Click-Derived Pyridinyltriazolylmethylquinoxaline-Based Y-Shaped Binuclear Luminescent Ruthenium(II) and Iridium(III) Complexes as Cancer Theranostic Agents

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Mitochondria-Targeting Click-Derived

Pyridinyltriazolylmethylquinoxaline-Based Y‑Shaped Binuclear

Luminescent Ruthenium(II) and Iridium(III) Complexes as Cancer

Theranostic Agents

Nilmadhab Roy,^⊥Utsav Sen,^⊥Yukti Madaan, Venkatesan Muthukumar, Seshu Varddhan,

Suban K. Sahoo, Debashis Panda, Bipasha Bose,* and Priyankar Paira *

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ABSTRACT: Due to several negative issues, market available drugs have been gradually losing their importance in the treatment of

cancer. With a view to discover suitable drugs capable of diagnosing as well as inhibiting the growth of cancer cells, we have aspired

to develop a group of theranostic metal complexes which will be (i) target speciﬁc, (ii) cytoselective, thus rendering the normal cell

unaﬀected, (iii) water-soluble, (iv) cancer cell permeable, and (v) luminescent, being beneﬁcial for healing the cancer eternally.

Therefore, to reach our goal, we have prepared novel Ru(II)- and Ir(III)-based bimetallic and hetero bimetallic scaﬀolds using click-

derived pyridinyltriazolylmethylquinoxaline ligands followed by metal coordination. Most of the compounds have displayed

signiﬁcant cytoselectivity against colorectal adenocarcinoma (Caco-2) and epithiloid cervical carcinoma (HeLa) cells with respect to

normal human embryonic kidney cells (HEK-293) compared to cisplatin [cis-diamminedichloroplatinum(II)] along with excellent

binding eﬃcacy with DNA as well as serum albumin. Complex [(η⁶-p-cymene)(η⁵-Cp*)Ru^IIIr^IIICl₂(K²-N,N-L)](PF₆)₂[RuIrL]

exhibited the best cytoselectivity against all the human cancer cells and was identiﬁed as the most signiﬁcant cancer theranostic agent

in terms of potency, selectivity, and ﬂuorescence quantum yield. Investigation of the localization of complex [Ir₂L] and [RuIrL] in

the more aggressive colorectal adenocarcinoma cell HT-29 indicates that mitochondria are the key cellular target for destroying

cancer cells. Mitochondrial dysfunction and G2/M phase cell cycle arrest in HT-29 cell were found to be involved in the apoptotic

cell death pathway induced by the test complexes [Ir₂L] and [RuIrL]. These results validate the concept that these types of

complexes will be reasonably able to exert great potential for tumor diagnosis as well as therapy in the near future.

INTRODUCTION

■

The great menace of cancer as the second most lethal disease

in developing countries has created a perilous situation in the

living world where medical science is still incapable of ﬁnding

out appropriate medicines to abate its rampant proliferation.^1,2

The urgent development of an eﬃcient anticancer drug is,

therefore, long awaited, which could be accomplished by the

means of advanced technology and improved conception in

Received: October 1, 2020

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Figure 1. Design of pyridinyltriazolylmethylquinoxaline-based binuclear luminescent Ru(II) and Ir(III) complexes.

cancer biology, the consequences of which led to the discovery

of cis-diamminedichloroplatinum(II) (cisplatin) by Rosenberg

in 1965, inaugurating the new era of anticancer research based

on metallopharmaceuticals.³The major concerns with

cisplatin, aside from its restricted scope of action, are its high

toxicity as well as its proclivity in creating tumor resistance in

patients.^4,5Eventually, all these short comings compelled

researchers to think of a new way for designing an outstanding

drug alternative to cisplatin having lower toxicity and less

issues with tumor resistance along with a broader spectrum of

activity.^6,7In this regard, ruthenium(II) and iridium(III)

complexes are undeniably the most superb candidates among

potential anticancer metallodrugs other than platinum-based

complexes. Their inherent properties like activity against

cisplatin resistant various cell lines, lower side eﬀects, less

toxicity to healthy cells for being highly selective toward cancer

cells,⁸greater aﬃnity to subcellular target (a particular

organelle), the ability of ruthenium to mimic iron in binding

to various biological molecules,⁸redox accessible oxidation

states, water tolerance,⁹and relatively low reduction

potential¹⁰have wonderfully boosted their capability to act

as anticancer metallodrugs. Keen interest in designing

ruthenium-based anticancer drugs has been mounting rapidly

since KP1019 and after that its sodium salt, KP1339, have

eﬀectively completed phase I clinical trials and are in the

process to enter phase-2 clinical trials in the near future.¹¹⁻¹⁵

Other remarkable success were observed for Ru(II) metallo

therapeutics such as TLD1433, which has entered phase-1 and

phase-2a clinical trials with photodynamic therapy (PDT) for

nonmuscle invasive bladder cancer treatment.¹⁶Many

researchers have successfully discovered various types of

novel Ru(II) scaﬀolds which have been investigated up to

preclinical studies. As an example, the Ru-PTA (1,3,5-triaza-7-

phosphaadamantane) complex RAPTA-C,¹⁷along with

Erlotinib, has exhibited proﬁcient anticancer activity. In

addition to this, more advancement on patents of ruthenium

complexes with various scaﬀolds has also been reported.¹⁸⁻²⁵

RAED44, a ruthenium-based compound with a 1,2-ethylenedi-

amine ligand was reported to have the ability of binding to

DNA in vitro neoplastic cells²⁶and to form adducts with

guanine. DW1/2²⁷is the ﬁrst ruthenium based antitumor

agent targeting through a signal transduction pathway. In 2004,

a new class of RAPTAs bearing phosphoadamantane and arene

ligands were reported as anticancer agents. For example,

[Ru(η⁶-p-cymene)Cl(2)(pta)], (RAPTA-C)²⁸and Ru(η⁶-

C₆H₅Me)-(pta)Cl₂(RAPTA-T) are well-known.²⁹A group

of researchers have been able to develop a number of cationic

biscyclometaleted Ir(III)-based metal complexes as marvelous

cellular imaging agents which becomes possible only by the

means of some fantastic features of Ir(III) organometallic

compounds. These are (i) large Stokes shifts (>100 nm) to

avoid inner ﬁlter eﬀects, (ii) rapid transmembrane activity

(short incubation time and less potential toxicity), (iii) long

luminescence lifetimes (100 ns) for time-resolved detection,

and (iv) enhanced photostabilities (less photobleaching) when

used in the imaging process. Moreover, a number of Ir(III)

complexes have recently been explored as anticancer as well as

cancer theranostic agents, and Ir(III) cyclometaleted com-

plexes have also appeared as eﬀective photodynamic

therapeutic agents. Recently, Peter J. Sadler, an eminent

inventor of various organometallic compounds from the

University of Warwick,³⁰obtained patents on the application

of organometallic iridium-based anticancer complexes with the

aid of the most modern concepts in the ﬁeld of metallodrugs.

The aforementioned complexes displayed dual properties of

killing cancer cells via DNA damage and simultaneously by

mitochondrial dysfunction by ROS production along with

detection of cancer cells in the human body. Mitochondria

have been deliberated as potential cellular O₂sensors and also

play a signiﬁcant role in cellular metabolism by activating the

B

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Scheme 1. Click-Derived Pyridinyltriazolylmethylquinoxaline ligand (L/LBr) Synthesis⁴⁹

extrinsic and intrinsic apoptotic pathways.³¹Therefore,

mitochondria have been taken into consideration as a prime

target for cancer cell annihilation. The intrinsic phosphor-

escence property of Ir(III) complexes can be used to track

drug delivery in the body and monitor the accumulation of a

drug in subcellular organelles, which assists with the

optimization therapy and ﬁnding the mechanism of cell

death.³²In the extracellular medium, the metabolic pathways

take part in competition among the aquation, reduction, and

hydrolysis after binding to transport proteins and cell-surface

biomolecules and after that the ability of diﬀusion into the

cells. The extent to which each drug participates in these steps

along with the focused pathways is very crucial in determining

whether the drug is mainly antimetastatic or cytotoxic.

Quinoxaline derivatives have already been recognized for

their biological activities and are signiﬁcant in the pharma-

ceutical industry having antimicrobial,³³antiviral,³⁴antituber-

culosis,³⁵anti-inﬂammatory,^36,37and anticancer activities.³⁸

Furthermore, “click chemistry” is being used to have a

pronounced imprint in material science,³⁹⁻⁴¹especially in

building up the novel structures of polymers^42,43and

dendrimers.⁴⁴The unique template “triazole” has also been

concomitant with quinoxaline moieties being associated with

antifungal, antibacterial, antiviral, antitumor, antimicrobial,

antitubercular, anti-inﬂammatory, antimalarial, anti-HIV, car-

diovascular, and also CNS activities.⁴⁵⁻⁴⁸Therefore, our prime

objective of this project is to synthesize a group of ligands

possessing bistriazole and quinoxaline moieties in a single

domain and their further exploration into multinuclear

complexes. Since both the ligand and the metal centers are

acquaintance with their selectivity as well as speciﬁcity in

anticancer action, the idea of developing signiﬁcant cytose-

lectivity and targeting speciﬁc binuclear Ru(II) and Ir(III)

complexes were on our mind to bring forth novel anticancer

metallo drugs that will be competent in showing the combined

properties of two diﬀerent metals in the same scaﬀold (Figure

1).

RESULTS AND DISCUSSION

■

Chemistry. Synthesis and Characterization. Syntheses of

ligands L and LBr and their respective complexes with Ru(II)

and Ir(III) [Ru₂L], [Ru₂LBr], [Ir₂L], [Ir₂LBr], [RuL], [IrL],

and [RuIrL] have been summarized in Schemes 1, 2, and 3. At

the outset, o-phenylenediamine derivatives and 1,4 dibromo-

2,3-butanedione were supported on silica and then irradiated

under microwave for 10 min at 100 °C (Scheme 1).⁴⁹After

completion of the reaction, silica gel was thoroughly washed

with ethanol followed by slow evaporation to get the gray

crystals of bis(bromomethyl)quinoxaline. The coversion of

bromide to azide was accomplished by the reaction of silica

supported mixture of bis(bromomethyl)quinoxaline and

sodium azide under microwave condition. Then ligands L

and LBr were prepared following the click chemistry protocol

brieﬂy by the treatment of silica supported 2,3-bis-

(azidomethyl)quinoxaline with 2-ethenylpyridine for 10−20

min. The crude triazoles were further recrystallized with

hexane-ethyl acetate (1:1) mixture and brown crystalline

ligands (L or LBr) were obtained with high yields. The ligands

1

L and LBr were then analyzed by H and ¹³C NMR, IR, and

mass spectroscopy.⁴⁹

1

In H NMR spectrum of 2,3-bis(bromomethyl)quinoxaline,

the most downﬁeld two protons near to the nitrogen atoms

appeared as double doublet (dd) at δ 8.07 ppm. The other two

aromatic protons were observed at δ 7.79 ppm as a double

doublet and last four aliphatic CH₂protons were observed as

singlet peak at δ 4.93 ppm. In ¹³C NMR spectrum, the most

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Scheme 2. One Pot Synthesis of Binuclear Ru(II) and Ir(III) Pyridinyltriazolylmethylquinoxaline Complexes

characteristic peaks of CH₂were found at δ 30.49 ppm. There

were no signiﬁcant diﬀerences observed in ¹H NMR spectra of

azidomethylquinoxaline and bromomethyquinoxaline. In the

¹³C NMR spectra, most characteristic peaks of CH₂of

compounds 2,3-bis(azidomethyl)quinoxaline and 2,3-bis-

(azidomethyl)-6-bromoquinoxaline were observed at δ 52−

53 ppm because of the presence of the azide group. The

characteristic peaks of NNN stretching of these

compounds were displayed at 2083 and 2094 cm⁻¹in the IR

precursors were treated with ligands (L, LBr) under sonication

for 2 h at ambient temperature. The progress of the reactions

was monitored by TLC. In order to facilitate anion exchange,

solid NH₄PF₆was added to the reaction mixture and again

sonicated for 2 h at ambient temperature. The solvent was

evaporated after completion of the reactions, and brown

powderlike crude products were obtained. Then the crude

products were washed thoroughly with hexane and recrystal-

lized from diethyl ether/methanol mixture to remove the

impurities. At last, brown needle-shaped crystals of Ru(II)

complexes [Ru₂L], [Ru₂LBr] and dark yellow ﬁne crystals of

Ir(III)complexes [Ir₂L], [Ir₂LBr] were obtained with high

yields (90−95%). The structures of all these complexes were

1

spectra. In the H NMR spectrum of compound L, the most

downﬁeld two protons adjacent to the two nitrogen atoms of

pyridine appeared as a broad singlet at δ 8.56 ppm. The

characteristic singlet peak of aromatic proton present in

triazole was observed at δ 8.38 ppm. The remaining aromatic

protons of pyridine rings and quinoxaline ring were observed

in the range of δ 7.76−8.18 ppm. Lastly, the characteristic four

aliphatic CH₂protons were recorded as singlet at δ 6.16 ppm.

In ESI-MS, the characteristic molecular ion peak of compound

L was observed as [M + H]⁺, 447.4. The complexes [Ru₂L],

[Ru₂LBr] and [Ir₂L], [Ir₂LBr] were formed by chloro bridge

cleavage of [(η⁶-p-cymene)RuCl(μ-Cl)]₂and [(η⁵-Cp*)IrCl-

(μ-Cl)]₂respectively with the reaction of ligands L, LBr

(Scheme 2). Herein, the alcoholic solutions of metal

1

analyzed by H, ¹³C, ¹⁹F, and ³¹P NMR, FT-IR, and HRMS.

1

In the H NMR spectrum, the protons of the ligand were

shifted to more downﬁeld incase of Ru(II) binuclear complex,

[Ru₂L]. For example, the peaks of L in the region of δ 7.26−

8.56 ppm were moved to δ 7.71−9.53 ppm in the complex,

[Ru₂L]. The four aliphatic CH₂protons which were displayed

in L at δ 6.15 ppm as a singlet were changed to two doublets in

the region of 6.61−6.75 ppm in the complex. The two sets of

methyl protons of the p-cymene ring were observed in the

range of δ 1.00−1.09 ppm, as two distinct doublets. The six

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Scheme 3. Synthesis of Ru(II) and Ir(III) Pyridinyltriazolylmethylquinoxaline Monometallic and Mixed Metallic Complexes

protons of another two methyl groups appeared as singlet at δ

2.17 ppm. Similarly, CH protons of the isopropyl groups were

found as multiplet in the region of δ 2.62−2.69 ppm. The eight

aromatic protons of p-cymene rings were appeared in the

region of δ 5.86−6.18 ppm. The characteristic phosphorus and

ﬂuorine peaks in ³¹P and ¹⁹F NMR spectra were observed in

the region of δ −153.04 to −135.47 ppm and δ −71.01 ppm,

respectively. The P−F stretching at 829 cm⁻¹, sp³C−H

stretching at 2970 cm⁻¹and sp³C−H bending at 1442 and

1404 cm⁻¹were observed in the IR spectrum which indicated

the existence of PF₆in Ru(II) complex. The HRMS peak at m/

z: 494.0699 [M − 2PF₆]²⁺conﬁrmed the formation of the

complex [Ru₂L]. The Ir(III) binuclear complex [Ir₂L] was

were observed in the range of δ 8.7−9.0 ppm and 89.4−92.6

ppm, respectively. The appearance of P−F stretching at 827

cm⁻¹, sp³C−H stretching at 3037 cm⁻¹, and sp³C−H bending

at 1423 cm⁻¹in the IR spectrum indicated the existence of PF₆

in Ir(III) complex. The HRMS peak at m/z: 586.1349 [M −

2PF₆]²⁺, conﬁrmed the formation of complex [Ir₂L].

The Ru(II) mononuclear complex [RuL] was prepared by

the treatment of [(η⁶-p-cymene)RuCl(μ-Cl)]₂in methanol

with the ligand L (1:2 equiv) under sonication for 2 h. After a

change in color from deep yellow to brown, 2.1 equiv of

NH₄PF₆was added to the reaction medium and sonicated for

another 2 h. The Ir(III) mononuclear complex [IrL] was also

prepared in a similar fashion. Furthermore, the Ru(II) and

Ir(III) mixed metal complex [RuIrL] was prepared by treating

[(η⁵-Cp*)IrCl(μ-Cl)]₂with complex [RuL] in (1:2 equiv)

methanol under sonication for 2 h. After complete conversion

to the product, ligand exchange was performed with the

addition of NH₄PF₆to the reaction medium in a similar

manner (Scheme 3). The complexes [RuL], [IrL], and

1

fully characterized by H, ¹³C, ¹⁹F, and ³¹P NMR, FT-IR, and

HRMS. Similarly, in complex [Ir₂L], the splitting patterns of

all the protons of ligands were moved toward the downﬁeld

region. The four aliphatic CH₂protons were displayed in L as a

singlet at δ 6.16 ppm changed to δ 6.69 ppm in the complex.

The ligand protons present in the complex were exhibited in

the range of δ 7.76−9.45 ppm. The most characteristic 30 Cp*

protons appeared as a singlet at δ 1.73 ppm. The characteristic

phosphorus and ﬂuorine peaks of PF₆were observed in the

region of δ −153.00 to −135.44 ppm and −71.06 to −69.17

ppm, respectively. In the ¹³C NMR spectrum, the characteristic

methyl carbon peaks and singlet carbons of the Cp* moiety

1

[RuIrL] were fully characterized by H and ¹³C NMR, IR,

and HRMS. The characteristic doublet peaks of two methyl

protons of p-cymene in complex [RuL] were observed at δ

1.01−1.09 ppm whereas other methyl protons appeared as a

singlet at δ 2.18 ppm. The characteristic multiplet peaks of CH

protons were observed in the range of δ 2.63−2.67 ppm.

E

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Table 1. Photophysical Characterization, Solubility, Lipophilicity, and Conductivity Study of the Complexes [Ru₂L],

[Ru₂LBr], [Ir₂L], [Ir₂LBr], and [RuIrL]

h

Λ_M(S cm²mol^‑1)

solubility

10%

a

b

c

d

e

f

g

complexes

[Ru₂L]

[Ru₂LBr]

[Ir₂L]

[Ir₂LBr]

[RuIrL]

cisplatin

λ_max(nm)

260, 300, 335 380, 450

260, 300,335 378, 450

λ_f(nm)

Stoke’s shift

OD

ε (M⁻¹cm⁻¹

)

(φ_f)

(M)

log P

DMSO

60, 130

53, 125

0.48

0.51

0.32

0.56

0.35

0.26

0.08

16000

17000

10667

18667

11667

8667

0.28

0.16

0.43

0.21

0.46

0.032

0.029

0.030

0.026

0.019

0.017

−

0.20 0.01

0.45 0.03

0.27 0.05

0.11 0.01

0.96 0.02

−

132

−

140

300

−

420

−

350

213

260, 300, 340 415, 450, 500 90, 125, 175

260, 300,345 384, 450, 496 54, 120, 166

260, 290, 330 380, 515

−

55, 190

−

102

136

34

−

200, 300

−

quinine sulfate 350

a

452

2667

c

0.546

−

b

d

e

f

Absorption maxima. Maximum emission wavelength (λ_exc325−330 nm). Optical density. Extinction coeﬃcient. Quantum yield. DMSO−

g

h

10% DMEM medium (1:99 v/v, comparable to cell media). n-Octanol/water partition coeﬃcients. Conductance in DMSO and 10% aqueous

DMSO (complex concentration 3 × 10⁻⁵M).

Likewise, the characteristic 15 Cp* protons of complex [IrL]

were appeared as singlet at δ 1.73 ppm. The HRMS peaks of

complex [RuL] and complex [IrL] appeared at m/z: 717.1552

[M − PF₆]⁺and 809.2205 [M − PF₆]⁺, respectively. In the

case of mixed metallic complex [RuIrL], the characteristic 15

Cp* protons were recorded as a singlet at δ 1.73 ppm, and the

methyl protons of p-cymene appeared as singlet at δ 2.18 ppm

and two doublets in the region of δ 1.00−1.09 ppm. The

characteristic CH protons of the isopropyl group were

observed as multiplet at δ 2.62−2.67 ppm. The HRMS peak

at m/z 540.1012 [M − 2PF₆]²⁺conﬁrmed the formation of the

complex [RuIrL].

Electronic Absorption (UV−Visible) and Fluorescence

Study. To emphasize the cellular imaging properties of the

synthesized complexes [Ru₂L], [Ru₂LBr], [Ir₂L], [Ir₂LBr],

and [RuIrL], UV and ﬂuorescence studies were conducted in

(1:9, v/v) dimethyl sulfoxide (DMSO): water mixture (Figure

S1 and S2). These complexes exhibited strong absorption

peaks in the range of 260−300 nm due to characteristic

intraligand (N∧N ligands) transitions (LLCT) and lower

energy broad absorption bands in the range of 320−450 nm

due to metal to ligand charge transfer (MLCT).^50,51The

absorption peak at the lower wavelength region appeared due

to the charge transfer between the π-bonding molecular orbital

of ligand (HOMO) to the π*-antibonding molecular orbital

(LUMO), i.e., due to π−π* transition, and the appearance of a

less intense absorption band at a higher wavelength region was

due to charge transfer from the metal’s low lying ﬁlled d-orbital

(t_2g) to the vacant higher energy antibonding π* molecular

orbitals of the ligands which was regarded as a MLCT

transition. The complexes showed emission in the range of

350−600 nm for MLCT by the electronic transition from

ligand π* molecular orbital to metal t_2gorbital. The

corresponding quantum yields (Φ_f) of these complexes were

calculated from the emission spectral data, which could be

used for photocytotoxicity and cellular imaging purposes

(equation ii, Table 1). It was evident that all the compounds

were signiﬁcantly ﬂuorescent, and compound [RuIrL] showed

the highest quantum yield (Φ_f), about 0.46 in 10% DMSO

medium, among them.

in hydrocarbon solvents. It is noteworthy to mention that these

complexes are soluble in the range of 5−8 mg per mL of

DMSO−10% DMEM medium (1:99 v/v, comparable to cell

media) at 25 °C. To know the lipophilic properties or drug-like

behavior of these complexes [Ru₂L], [Ru₂LBr], [Ir₂L],

[Ir₂LBr], and [RuIrL], we had estimated the n-octanol/

water partition coeﬃcient (log P_o/w, where P_o/w= the octanol/

water partition coeﬃcient), using the shake ﬂask method

(Figure S3 and Table 1).⁵²⁻⁵⁴The experimental log P_o/wvalues

of these complexes were obtained in the range 0.11−0.96.

Complex, [RuIrL] exhibited highest order of log P_o/wdue to

the lipophilic character of p-cymene, Cp* and iridium metal

which supported the signiﬁcant mitochondrial accumulation.

The bimetallic [Ru₂L], [Ir₂L], and heterobimetallic [RuIrL]

complexes displayed the molar conductance values of 132, 140,

and 136 S cm²mol⁻¹respectively in pure DMSO suggesting

their 1:2 electrolytic nature.⁵⁵Agreeably, the molar con-

ductance of all these complexes were signiﬁcantly increased in

10% aqueous DMSO (∼300−420 S cm²mol⁻¹,Table 1),

proposing their 1:4 electrolytic nature. Thus, the obtained

conductivity of these complexes attributed to the dissociation

of the M−Cl bond in the tested system and conﬁrmed the

ionization of complexes in aqueous medium which ensured

signiﬁcant binding properties of complexes with biomolecules.

Moreover, the conductance values of these complexes were

increased gradually with time up to 6 h after incubation in 10%

DMSO suggesting the easiness of departure of the labile −Cl

group from the complexes (Figure S4). On the other hand,

cisplatin exhibited poor conductance (Λ_M= 34 S cm²mol⁻¹)

in DMSO and showed 1:2 electrolytic property (Λ_M= 213 S

cm²mol⁻¹) in 10% DMSO.

Stability Study of the Complexes by UV−Vis Spec-

troscopy. The stability study of the three complexes, namely

[Ru₂L], [Ir₂L], and [RuIrL], was accomplished in two

mediums, 1% DMSO in water and GSH (1 mM) medium.

This study was needed to determine the stability of complexes

in the biological environment inside the cell so as to be a

potent therapeutic agent. A reasonable change in the UV−vis

spectral proﬁle with the λ_maxvalue over 48 h in 1% DMSO was

observed for each complex, suggesting that M−Cl bond was

moderately stable in less water content (Figure S5). Such

change in absorption bands indicates that these complexes can

easily form aqua complexes in bulk water medium, which can

also be quantitatively justiﬁed by the observed molar

conductivity of the respective complexes in aqueous

Solubility, Lipophilicity, and Conductivity Study. The

tumor-inhibiting potential of metal complexes is strongly

dependent on the equilibrium between hydrophilicity and

lipophilicity. These bimetallic or mixed metallic complexes are

highly soluble in DMSO and DMF and had moderate to good

solublity in H₂O, MeOH, EtOH, CH₃CN and poor solubility

DMSO.⁵⁶Moreover, the H NMR study in 10% DMSO/

1

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D₂O mixture did not show any change in spectral pattern

suggesting no dissociation of the ligands from the complexes

(Figure S6). It is well-known that glutathione (GSH) is a

crucial detoxifying agent in the presence of glutathione S-

transferase (GST) in cells.^57,58Henceforth, to see the activity

of GSH on these complexes, [Ru₂L], [Ir₂L], and [RuIrL], the

stability study was performed in the presence of excess (10

equiv) glutathione by time dependent UV−vis spectroscopy

which displayed minor changes in absorbance with time

(Figure S7), i.e., complexes were not being deactivated by

GSH. Biomolecules can exhibit the binding aﬃnity with the

metal complexes and therefore a competitive binding between

GSH and serum albumin with the complex may be started to

prevail when drug enters into the cell.^56,59,60But, in our case,

as GSH was incompetent to bind with these complexes,

binding with serum albumin will prevail when the drug will

enter into the cell, ruling out the probability of competitive

binding, which has been further justiﬁed from the binding

study with the serum albumin in the later section.

Theoretical Study. Computational Study. The density

functional theory (DFT) method was applied to compute the

probable 3D structure of the metal complexes. All calculations

were performed in the gas phase by using the B3LYP

exchange-correlation functional and the basis sets LANL2DZ

for Ru and Ir atoms whereas 6-31G** for the remaining atoms.

The computational programs Gaussian 09W and Gauss View

were used for the calculations.⁶²The computed structures of

the Y-shaped half-sandwich organometallic complexes were

shown in Figure 3. The quinoxaline unit separated the two

metal centers forming the half-sandwich complex by a

methylene group that provided suﬃcient ﬂexibility to form a

perfect Y-shaped organometallics. Each metal center in the

complexes formed a pseudo-octahedral coordination geometry,

where the metal atom (Ir or Ru) was coordinating to all six

carbon atoms of p-cymene ring in the case of Ru complexes

whereas all ﬁve carbon atoms of pentamethylcyclopentadiene

were coordinated in the case of Ir, along with a chloride ion

and the two nitrogen atoms of the pyridine-N and triazole-N.

The bond lengths and bond angles of the Ru complexes in

[Ru₂L], [Ru₂LBr] and [RuIrL] and Ir complexes in [Ir₂L],

[Ir₂LBr], and [RuIrL] are similar. Each metal center mimics

the well-known familiar “three leg piano-stool” structure, where

the seat is formed by the η⁵-/η⁶-arene and the three legs of the

stool are constituted by the pyridine-N and triazole-N atoms

and the chloride ion. In complex [RuIrL], the observed bond

angles for the triazole-N−Ru−Cl (83.79°) and pyridine-N−

Ru−Cl (84.33°) in the Ru center and the bond angles for the

triazole-N−Ir−Cl (84.31°) and pyridine-N−Ir−Cl (83.59°) in

the Ir center are close to 90° supporting the “piano-stool”

structure. The distance from the Ru center to the six carbons

of the arene has almost identical bond lengths (2.252−2.337

Å) with an average distance of 2.292 Å. The triazole-N and

pyridine-N atoms coordinated respectively to the Ru with the

bond distance of 2.077 and 2.139 Å, where the triazole-N is

coordinated more strongly than the pyridine-N. The calculated

Ru−Cl bond length was obtained as 2.423 Å. Similar to Ru

center, the distance from the Ir center to the ﬁve carbons of the

arene have almost similar bond length (2.199−2.238 Å) with

an average distance of 2.230 Å.

Electrochemical Properties. In order to get some insights

into the stability of the Ru(II) and Ir(III) state in these

complexes, the electrochemical behavior of the reported

complexes were studied with the aid of cyclic voltammetry at

a ﬁxed scan rate. Figure 2 represents a cyclic voltammetric

The triazole-N and pyridine-N atoms coordinated to the Ir

with the bond distance of 2.104 and 2.147 Å, respectively,

where the triazole-N is coordinated more strongly than the

pyridine-N. The Ir−Cl bond length was obtained as 2.438 Å.

Furthermore, to examine the changes in the electron densities

distribution, the highest occupied molecular orbital (HOMO)

and lowest unoccupied molecular orbital (LUMO) of L,

[Ru₂L] and [Ir₂L] were drawn and compared (Figure 4). In

case of L, the HOMO electron densities were distributed

mainly over the pyridinyl−triazolyl unit whereas the LUMO

electron densities were located over quinoxaline unit. Upon

complexation with the Ir/Ru atom, the HOMO electron

densities were mainly distributed around the metal center

whereas the LUMO was over the quinoxaline unit. The band

gap of L was decreased upon forming the complexes [Ru₂L]

and [Ir₂L]. These results indicate that internal charge transfer

occurred between the quinoxaline unit and the metal center.

Similar distributions were observed in other organometallics.

Molecular Docking Study. To identify possible binding

sites and mode of interactions of the complexes with the BSA

(bovine serum albumin) and DNA, molecular docking

experiments were performed to estimate the free energy of

binding using the Autodock 4.2 by applying the Lamarckian

Figure 2. Cyclic voltammetry response of the samples at the potential

window of −0.2 to −1.2 V vs Ag/AgCl in 0.2 M of sodium sulfate

solution at scan rate of 20 mV s⁻¹.

response of various Ru and Ir complexes in 0.2 M Na₂SO₄

medium (10 mL) with 0.5 mL of ethanol at a scan rate, 20 mV

s⁻¹. On the basis of the magnitude of anodic (i_pa) and cathodic

peak (i_pc) currents, it had been found that complex [Ir₂LBr]

showed an irreversible electrochemical behavior (i_pa≪ i_pc),

whereas, other complexes displayed well-deﬁned reversible

redox peak (i_pa≅i_pc) with an apparent standard electrode

potential, E^o′ = 0.600 0.50 V vs Ag/AgCl, which was closer

to the literature value, 0.55 V vs Ag/AgCl in neutral pH

solution.⁶¹The detailed information for the E^o′ values of all the

complexes have been provided in Table S1. On the basis of

obtained electrode potential values, we can depict that the

possible redox-state of these metal complexes will be Ru(III)/

Ru(II) and Ir(III)/Ir(II). This observation nicely described the

electroactive functionality of the synthesized complexes in

aqueous medium.

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Figure 3. DFT (B3LYP/6-31G**, LANL2DZ) computed structure of the complexes (a) [Ru₂L], (b) [Ru₂LBr], (c) [Ir₂L], (d) [Ir₂LBr], and (e)

[RuIrL].

genetic algorithm (LGA) docking parameters.⁶³The BSA

crystal structure (PDB ID: 45FS) is modeled using the

Swissmodel online tool, whereas the DNA 3D structure is

retrieved from the Protein Data Bank with reference PDB ID

1BNA. The docking complexes were analyzed by using the

BIOVia discovery studio.

BSA consists of three major domains (I, II, and III), where

the most electrostatic surfaces are found in between the

domain-II residues. The complexes were docked randomly

with BSA by keeping the coordinates of central grid point of

maps to x = 8.376, y = 21.661, and z = 106.639. The best pose

of the complexes were located between the domain II and III

(Figure 5a). The complexes were bound ﬁrmly at the

hydrophobic cavity of BSA with the dock score ranging from

−3.73 to −6.97 kcal/mol (Table S2). Analyses of the [Ru₂L]-

BSA docked structure indicated that the best pose is stabilized

by multiple noncovalent interactions of [Ru₂L] with the amino

acid residues such as ILE297, LEU301, PRO303, LEU304,

ARG336, HIS337, and LYS377 (Figure 5c). The higher

binding aﬃnity of [Ru₂L] complex toward BSA resulted

smaller inhibition constant (K_i) of 7.73 μM. Similarly, other

complexes like [Ir₂LBr] and [RuIrL] also showed eﬀective

binding at the hydrophobic cavity of BSA with estimated

inhibition constants of 23.36 μM and 101.51 μM, respectively.

The docking of metal complexes with DNA were performed

by adjusting the coordinates of central grid point of maps to x

= 13.358, y = 20.325, and z = 10.766. The metal complexes

docking with the DNA dodecamer posed strong electrostatic

interactions and a poor hydrophobic cavity. As shown in

Figure 4b, the best posed of the complexes [Ru₂L], [Ru₂LBr],

[Ir₂LBr], and [RuIrL] were stacked at one place via

interaction by binding to the major groove, whereas the

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Figure 4. DFT computed electron densities distributions of HOMOs and LUMOs in the ligand L (a) and the complexes [Ru₂L] (b) and [Ir₂L]

(c).

Figure 5. 3D views of the best poses of the [Ru₂L], [Ru₂LBr], [Ir₂L], [Ir₂LBr], and [RuIrL] complexes within the hydrophobic cavity of (a)BSA

and (b) DNA. Two views of the docked structures of [Ru₂L] with (c) BSA and (d) DNA showing the important interactions with the residues.

complex [Ir₂L] bound electrostatically to the outside edge of

the minor groove (Figure 5b). The binding energy of the

complexes with DNA was observed in the range of −5.53 to

−8.21 kcal/mol. Because of the higher aﬃnity of the complexes

toward DNA, the inhibition constants are also smaller in

comparison to BSA. Complexes like [Ru₂L], [RuIrL], and

[Ir₂LBr] showed the higher binding aﬃnity with the smaller

inhibition constant. With high dock score, the complex [Ru₂L]

was binding to most favorable sites of DNA base pairs

consisting of an electrostatic surface (Figure 5d).

oxaliplatin, and carboplatin and organic drugs (doxorubicin,

gemcitabine, 5-ﬂuorouracil, etc.).^64,53Therefore, the binding

eﬃcacy of metal complexes with DNA is a modern approach

for designing eﬀective chemotherapeutic drugs. To investigate

experimentally the binding aﬃnity of these complexes with

DNA, the reliable electronic absorption titration tool was

employed.⁶⁵There are two diﬀerent modes of binding of the

metal complexes with DNA, i.e., covalent and noncovalent

interactions (intercalation, groove binding, and electrostatic

interactions).⁶⁵The UV−vis spectral array of the metal

complexes can assuredly unveil the style of interaction of the

complexes with DNA. In order to evaluate the equilibrium

binding constant (K_b), binding site size (s), and binding mode,

DNA Binding Studies. UV and Fluorescence Spectro-

scopic Method. DNA is the vital pharmacological target for

various FDA approved anticancer metallodrugs like cisplatin,

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Figure 6. Absorption spectral traces for complexes (a) [Ru₂L], (b) [Ir₂L], and (c) [RuIrL] with increasing concentration of ct-DNA in DMSO

medium. (d) Plot associated with the titration of [Ru₂L], [Ir₂L], [RuIrL], and ct-DNA at 298 K to ﬁt the model of Bard and Thorp.

Table 2. Binding Parameters for Interaction of Complex [Ru₂L], [Ir₂L], and [RuIrL] with ct-DNA

a

b

c

d

complex

K_b(M^‑1)

S

K_SV(M^‑1)

K_app(M^‑1)

hypochromism

[Ru₂L]

[Ir₂L]

[RuIrL]

(0.46 0.99) × 10⁴

(0.75 1.30) × 10⁴

(0.44 0.67) × 10⁴

0.11 0.02

0.21 0.03

0.14 0.06

0.0044 × 10⁶

0.001 × 10⁶

0.01 × 10⁶

2.7 × 10⁶

2.46 × 10⁶

2.8 × 10⁶

26

12

43

a

K_b, equilibrium DNA binding constant from UV−visible absorption titration followed by nonlinear curve ﬁtting (MVH model, equation i.

b

c

d

Binding site size (per base pair). K_SV, Stern−Volmer quenching constant. K_app, apparent DNA binding constant from competitive displacement

from ﬂuorescence spectroscopy.

the complexes [Ru₂L], [Ir₂L], and [RuIrL] (concentration 5 ×

10⁻⁵M) were titrated against increasing concentrations of ct-

DNA (5−60 μM). In all cases, we observed a hypochromism

(Δε ∼ 10 × 10³M⁻¹cm⁻¹) at λ_abs∼ 300 nm with respect to

an increase in DNA concentration (Figure 6). Bard’s equation

was employed based on the McGhee−von Hippel (MvH)

model to evaluate the equilibrium binding constant (K_b) values

and binding site sizes (s, per base pair).^65gK_band s for

complexes [Ru₂L], [Ir₂L], and [RuIrL] were calculated from

equation i, 0.46 0.99 × 10⁴M⁻¹, 0.11 0.02 for [Ru₂L];

0.75 1.30 × 10⁴M⁻¹, 0.21 0.03 for [Ir₂L]; and 0.44 0.67

× 10⁴M^‑1, 0.14 0.06 for [RuIrL] in DMSO (Figure 6 and

Table 2).

M⁻¹)⁶⁶and lower than the classical DNA intercalator, EtBr (7

× 10⁵M⁻¹).⁶⁷Therefore, moderate binding constants with

binding site size (s) and signiﬁcant hypochromism in

absorbance on addition of ct-DNA ensured the intercalative

mode of binding which had been further justiﬁed by an

ethidium bromide (EtBr) binding assay.

The competitive binding studies of complexes [Ru₂L],

[Ir₂L], and [RuIrL] with EtBr-ct-DNA showed a decrease in

ﬂuorescence intensity (Figure S8). These complexes displaced

EtBr from ct-DNA grooves, and it got bound to the DNA base

pairs. Thus, the decrease in ﬂuorescence intensity was observed

as EtBr appeared in its free form and thereby free EtBr

concentration started to increase gradually in solution. The

excitation wavelength used for EtBr-bound DNA and

complexes [Ru₂L], [Ir₂L], and [RuIrL] was ﬁxed at 485 nm,

while the recorded emission wavelength was 600 nm. The

Ethidium Bromide (EtBr) Binding Study. The values of

these binding constants are comparable to some reported

DNA intercalative Ru(II) complexes (1.1 × 10⁴−4.8 × 10⁴

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Table 3. Binding Parameters for the Interaction of Complexes [Ru₂L], [Ir₂L], and [RuIrL] with BSA and HSA

a

b

c

d

e

f

complex

K (M^‑1)

n_BSA

K_HSA(M^‑1)

K_q

K (M^‑1)

n_HSA

[Ru₂L]

[Ir₂L]

[RuIrL]

0.72 × 10⁴

1.4 × 10⁴

2.435

2.508

2.423

0.11 × 10⁶

0.14 × 10⁶

1.065 × 10¹³

1.38 × 10¹³

0.46 × 10¹³

0.62 × 10⁴

0.18 × 10⁴

1.37

1.71

1.27

1.33 × 10⁴

0.05 × 10⁶

0.75 × 10⁴

a

b

c

d

K, binding constant with BSA.

n

_BSA, number of binding sites (BSA). K_HSA, Stern−Volmer quenching constant. K_q, quenching rate constant

e

f

(HSA). K, binding constant with HSA. n, number of binding sites (HSA).

concentration of DNA and EtBr used was 120 μM and 8 μM

respectively.The K_appvalues for the complexes [Ru₂L], [Ir₂L],

and [RuIrL] calculated from equation iii were 2.7 × 10⁶M⁻¹,

2.46 × 10⁶M⁻¹, and 2.8 × 10⁶M⁻¹respectively (Table 2). The

value of K_EtBrfound from literature is 1 × 10⁷M⁻¹. The Stern−

Volmer quenching constant (K_SV) calculated from equation iii

for the complexes [Ru₂L], [Ir₂L], and [RuIrL] were 0.0044 ×

10⁶M⁻¹, 0.001 × 10⁶M⁻¹, and 0.01 × 10⁶M⁻¹respectively

(Table 2). Complex [RuIrL] exhibited highest K_appvalue

among all the complexes as the combined property of both

Ru(II) and Ir(III) metals were prevailed in heterobinuclear

metal complex rather than homobinuclear ruthenium [Ru₂L]

and homobinuclear iridium [Ir₂L] complexes.

Viscosity Measurement. In order to ﬁnd out the binding

modes of these complexes with DNA, a hydrodynamic method

type viscosity study had been conducted. Binding via

intercalation causes the adjacent base pairs separation to

yield binding of drug molecules into the DNA double helix,

which leads to an increase of the length of DNA as well as their

viscosity. In the relative viscosity analysis, a faster increase in

the viscosity with the gradual increase in the ratio of drug to ct-

DNA in case of complex [RuIrL] was observed, which

indicated strong intercalative binding action (Figure S9).

BSA and HSA Binding Study. The ﬂuorescence emission

spectra of BSA in the absence and presence of complexes

[Ru₂L], [Ir₂L], and [RuIrL] were recorded with excitation at

280 nm while the emission was obtained at 350 nm (Figure

S10)^65e,fThe concentrations of complexes used for emission

spectra were 0−100 μM in distilled water, and the BSA

concentration was ﬁxed at 5 μM. The nonlinear and upward

curvature of the Stern−Volmer plot suggested that more than

one processes were involved in the overall quenching of BSA

(Figure S11). The binding aﬃnity (K) of these complexes was

calculated from Scatchard plot analysis showing strong binding

propensity of the tested complexes with BSA, which was

required for transport of protein-bound complexes in biological

systems (equation v, Figure S12, Table 3).

result, the nonlinearity of the Stern−Volmer plot for the

complexes with upward curvature can vividly demonstrate the

key role of both static and dynamic quenching in the overall

ﬂorescence quenching of HSA.

Biology. Cytotoxic Activity. The in vitro cytotoxicity of all

these complexes [Ru₂L], [Ru₂LBr], [Ir₂L], [Ir₂LBr], and

[RuIr L] was studied using standard 3-(4,5-dimethylthiazol-2-

yl)-2,5-diphenyltetrazolium bromide (MTT) assay protocol

beside a panel of cancer cell lines, i.e., human epitheloid cervix

carcinoma (HeLa) and colorectal adenocarcinoma cells (Caco-

2), and one normal cell line, i.e., human embryonic kidney cells

(HEK-293) in triplicates. Cisplatin had been used as standard

positive control drug. All the complexes exhibited signiﬁcant

cytotoxicity (5−23 μM) in the Caco-2 cell line, but in HeLa

cells, compounds [Ir₂L] (IC₅₀= 23.4 0.9 μM) and [RuIrL]

(IC₅₀= 14.2 1.2 μM) showed considerable potency. Most of

the complexes represented higher cytoselectivity than cisplatin

against all the human cancer cell lines tested. Among them,

complex [RuIrL] displayed the highest potency and selectivity

in both the cell lines (HeLa, IC₅₀= 14.2 1.2 μM, selectivity

>14; Caco-2, IC₅₀= 2.2 0.4 μM, selectivity >90) over the

noncancerous HEK-293 cell line (Table 4) as it acquired the

Table 4. Preliminary MTT Cytotoxicity Screening of

Synthesized Multinuclear Ru(II) and Ir(III) Complexes at

48 h of Drug Exposure

a

b

IC (μM)

SF

50

compound

Caco-2

HeLa

HEK 293 Caco-2

HeLa

[Ru₂L]

[Ru₂LBr]

[Ir₂L]

[Ir₂LBr]

[RuL]

5

0.8

50.5 1.1

>100

23.4 0.9

>100

65.9 1.0

70.5 0.8

14.2 1.2

−

>200

−

>40

>17

>18

>9

>12

>25

>90

−

>4

2

>9

2

>3

>14

−

12 1.1

11 1.0

23 2.1

17 1.2

[IrL]

8

0.8

[RuIrL]

DMSO

cisplatin

2.2 0.4

−

19.8 1.2

14.5 0.9

>50

>2.5

>3.4

In order to ﬁnd out the applicability of these complexes in

a

the human body, we emphasized the binding study with

human serum albumin (HSA) following a similar ﬂuorescence

quenching method like BSA (Figure S13). This study revealed

the strong binding aﬃnity of the complexes with HSA, which

was supported by the Stern−Volmer quenching constant

(K_HSA), quenching rate constant (K_q), and binding constant

(K) (equations iv and v, Figure S14, Figure S15, and Table 3).

The values of K_HSAfor all the complexes were observed in the

range of 0.05−0.14 × 10⁶M⁻¹. Complex [RuIrL] showed the

highest binding constant (K) (0.75 × 10⁴) among the others.

On the other hand, the biomolecular quenching rate constant

(K_q) for these complexes was obtained on the order of 10¹³.

This higher order of quenching rate constant surpassed the

maximum value for dynamic quenching (2 × 10¹⁰L mol⁻¹s⁻¹)

due to molecular collision, suggesting the successful

bimolecular quenching along with complex formation.^65fAs a

IC₅₀is the concentration of the synthesized complexes and cisplatin

at which 50% of cells undergo cytotoxic cell death under treatment.

b

SF (selectivity factor) = ratio of IC₅₀for HEK-293 to IC₅₀for all the

cancer cell lines.

highest lipophilicity, good solubility, and stability in the cancer

cell environment along with the synergistic eﬀect of both

ruthenium(II) and iridium(III) metals.⁶⁸

The in vitro cytotoxicity of the complexes [Ir₂L] and

[RuIrL] was further determined by MTT assay with the most

aggressive colorectal carcinoma cell line, HT-29. The tumor

cells were incubated with the test compounds at diﬀerent

concentrations (1−30 μM for [Ir₂L] and 2.5−20 μM for

[RuIrL] for 48 h in vitro. It was found that both the complexes

exhibited cytotoxicity on the colon cancer cell line (HT29) in a

dose dependent manner. Morphologically, cell shrinkage and

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Figure 7. MTT assay and EC₅₀value determination of HT-29 cells up on treatment with (a) [Ir₂L] and (c) [RuIrL] drugs. (b and d) Dose

dependent morphological changes of the HT-29 cell line, upon treatment with various doses of drug [Ir₂L] and [RuIrL]. Cells were costained with

Hoechst 33342 to visualize the nucleus. An arrow mark represents the cell damage. Scale bar 50 μm.

Figure 8. Co-localization of the drug [Ir₂L] in colon cancer cell line HT-29. (a) Control, (b) 10 μM, and (c) 25 μM of [Ir₂L] treatment for 48 h.

(d) Representative image of the drug localization of [Ir₂L] in the highest dose (25 μM), merged with the phase contrast ﬁeld. Scale bar 50 μm.

stress granules were observed upon treatment with 10 and 25

μM of [Ir₂L] and 10 and 15 μM of [RuIrL] complex (Figure

7). In HT-29 cells, the compound [Ir₂L] showed an EC₅₀

Morphological Analysis. To explore the action of these

complexes on the cancer cell morphology, we treated the

cancer cells with the test compounds at two diﬀerent

concentrations ([Ir₂L] at 10 and 25 μM and [RuIrL] at 10

and 15 μM) along with a control experiment. After 48 h of

treatment, costaining was performed with the live cell nuclear

stain Hoechst 33342.⁶⁹Signiﬁcant cell damage including cell

shrinkage and stress granules was observed in the HT-29 cell

line (Figure 7, parts b and d), and the changes were compared

with the control cell. Cell damage had started to manifest at 10

value of 27 μM (Figure 7a) while [RuIrL] showed an EC

̃

50

value of 18 μM (Figure 7c). Both complexes were localized in

̃

the cytoplasm of the HT-29 cancer cell line upon treatment.

Since most of the morphological changes and cellular changes

were observed within a 10−25 μM range for [Ir₂L] and a 10−

15 μM range for [RuIrL] complex, we chose the

aforementioned doses to proceed with further studies.

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Figure 9. Co-localization of the drug [RuIrL] in colon cancer cell line HT-29. (a) Control, (b) 10 μM, and (c) 15 μM of [RuIrL] treatment for 48

h. (d) Representative image of the drug localization of [RuIrL] in the highest dose (15 μM), merged with the phase contrast ﬁeld. Scale bar 50 μm.

μM of [Ir₂L] treatment, and the damages were increased with

a complex concentration of 25 μM (Figure 7b). On the other

hand, complex [RuIrL] exhibited the eﬀect from 10 to 15 μM

(Figure 7d). Maximum cell damages as compared to intact

control cells were observed at 25 μM and 15 μM for the

complexes [Ir₂L] and [RuIrL] respectively (Figure 7, parts b

and d).

Cell Localization Study. To obtain the information on

subcellular localization of the complexes [Ir₂L] and [RuIrL],

colocalization experiments were performed. As shown in

Figure 8, there is a good superposition of green ﬂuorescence

emission by the compound [Ir₂L] at 25 μM concentration

suggesting the penetration of the drug inside the cancer cell

membrane and then conﬁrming the subcellular localization of

Figure 10. Comparison of the level of penetration of two

the complex in cytoplasm but not in nucleus. Congruently, the

organometallic complexes [RuIrL] and [Ir₂L] in colon cancer cell

complex [RuIrL] had also shown a green ﬂuorescence with

line HT-29 cells. Graph represents the mean ﬂuorescence intensity

lesser intensity than that for the complex [Ir₂L] in spite of

being a potent compound for the HT-29 cell line (Figure 9).

Moreover, we had compared the penetration of both the

complexes by dint of measuring the mean ﬂuorescence

intensity which suggested greater penetration of the complex

[Ir₂L] in colon cancer cells in comparison to the [RuIrL]

complex (Figure 10). Therefore, this study helped us to

presume the subcellular localization of the complexes [Ir₂L]

and [RuIrL] in cytoplasmic mitochondria, which were further

corroborated by advanced analysis.

Cell-Cycle Analysis. With reference to the results of the

MTT assay, where there was an inhibition of cell proliferation,

it was further validated by determination of the cellular

morphology and localization of the drug to cancer cells. So, we

went ahead to investigate the DNA content by ﬂow cytometry.

As shown in the Figure 11, the colon cancer cells exhibited

high G0/G1 phase (∼50.82%) under control conditions, as

expected, and the S phase was ∼39.77% with ∼9.41% G2/M

phase (Figure 11d). The treatment of complex [Ir₂L] resulted

in diminishing of the S phase to ∼27.58 in 25 μM in HT-29

colorectal carcinoma cells along with signiﬁcant gradual

increment of the G2/M phase (from ∼9.41 to ∼31.36).

(MFI) of the drugs [Ir₂L] and [RuIrL] at their highest

concentrations. The diﬀerences between two groups were measured

by an unpaired Student t test. The P values of >0.12 (ns), 0.033 (*),

0.002 (**), and <0.0002 (***) were considered as signiﬁcant. Error

bar represents the standard error of mean (SEM).

Likewise, the complex [RuIrL], delivered an eﬀect on cell-

cycle in the HT-29 colorectal cancer cell line. As illustrated in

Figure 12, the colon cancer cells exhibited high G0/G1 phase

(∼46.45%) under control conditions and high S phase

(∼38.30%) with ∼25.25% G2/M phase (Figure 12d). The

treatment of [RuIrL] resulted in a decrease of the S phase to

∼26.38% in 25 μM in HT-29 colorectal carcinoma cells along

with a signiﬁcant increment of the G2/M phase (from ∼15.26

to ∼32.04%). In summary, both the organometallic complexes

were potent for the colon cancer cell line HT-29 and were able

to cause G2/M phase cell cycle arrest. Excessive stalling of the

cells at G2/M was a prelude to either cellular damage or

apoptosis that demanded further validation (Figure 11 and

12).

Mitotracker RED CMX ROS Staining Assay and

Colocalization Assay. Cellular uptake and localization of

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Figure 11. Cell cycle analysis of (a) control HT-29 cells, (b) 10 μM of drug [Ir₂L] treatment, (c) 25 μM of [Ir₂L] drug treatment. (d) Tabular and

(e) graphical representation of the diﬀerent phases of the cell-cycle up on diﬀerent treatment of the drug [Ir₂L]. The P values of >0.12 (ns), 0.033

(*), 0.002 (**), <0.0002 (***) were considered as signiﬁcant. Error bar represents the standard error of mean (SEM).

an anticancer agent is of major importance to enable it to

target key organelles of the cell. As mitochondria plays an

important role in detoxifying ROS in the cell, we performed

Mitotracker RED CMX ROS staining assay for validating the

eﬀect of complexes [Ir₂L] (concentrations 10 and 25 μM) and

[RuIrL] (concentrations 10 and 15 μM) on mitochondria

along with control experiment. We observed the complex

[RuIrL] was being localized in mitochondria causing the

mitochondrial damage (Figure 13). The complex [RuIrL]

increased the red ﬂuorescence intensity indicated the

increasing mass of the mitochondria (Figure 13). Moreover,

Mitotracker colocalization assay also revealed the signiﬁcant

mitochondrial localization of these complexes within 48 h of

incubation with good Pearson correlation coeﬃcient (0.66−

0.67) (Figure 14).

Mitochondrial Dysfunction. Mitochondrial dysfunction

acts as a hallmark of cell apoptosis by releasing various

proapoptotic proteins. Mitochondrial membrane potential

(MMP, ΔΨm) has been recognized as a key indicator of

mitochondrial function.³¹Consequently, JC-1, a MMP-

sensitive probe with red ﬂuorescence in normal mitochondria

and green ﬂuorescence in dysfunctional mitochondria was used

to identify the variation of MMP in HT-29 cells. CCCP

(carbonyl cyanide m-chlorophenylhydrazone) was used as a

positive control for this type of investigation. In Figures 15a

and 16a, it has been shown that the HT-29 cells exposed a

uniform red ﬂuorescence without the treatment of complex

indicating the normal mitochondrial function. After the

treatment of [Ir₂L] and [RuIrL] with HT-29 cells, bright

green ﬂuorescence was observed indicating the loss of MMP

along with mitochondrial dysfunction which is comparable

with the result of CCCP (Figure 15 and 16). Complex [Ir₂L]

reduced the mitochondrial membrane potential from 7 to 0.28

(Figure 15b) whereas complex [RuIrL] also decreased the

mitochondrial membrane potential to 0.3 (Figure 16b) after 48

h of treatment. Hence, both the potent complexes were

capable of aﬀecting mitochondria and thereby their eﬃciency

can be considered as comparable with the positive control

CCCP (Figure 15 and 16). In summary, the complexes

[RuIrL] and [Ir₂L] were competent to make the mitochon-

drial membrane “leaky”, leading to damage of mitochondria

which resulted in a cell apoptosis pathway.

Structure Activity Relationship Study (SAR). Struc-

ture−activity relationship (SAR) study revealed that the

potency of pyridinyltriazolylmethylquinoxaline-based Ru(II)

and Ir(III) complexes [Ru₂L], [Ir₂L], [Ru₂LBr], [Ir₂LBr],

[RuL], [IrL], and [RuIrL] in cancer cells varied with the

combination of the metals and substitution of bromine group

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Figure 12. Cell-cycle analysis of (a) control HT-29 cells, (b) 10 μM of drug [Ir₂L] treatment, and (c) 15 μM of [RuIrL] drug treatment. (d)

Tabular and (e) graphical representation of the diﬀerent phases of the cell-cycle up on diﬀerent treatment of the drug [RuIrL]. The p values of

>0.12 (ns), 0.033 (*), 0.00 2(**), and <0.0002 (***) were considered as signiﬁcant. The error bar represents the standard error of mean

(SEM).

Figure 13. Mitotracker staining assay in HT-29 cell line with the drug [RuIrL] for 48 h (a) untreated control, (b) treated with 10 μM [RuIrL], (c)

graphical representation of the increased MFI in treated colon cancer cells. The diﬀerences between the two groups were measured by an unpaired

Student t test. The p values of >0.12 (ns), 0.033 (*), 0.002 (**), and <0.0002 (***) were considered as signiﬁcant. The error bar represents the

standard error of mean (SEM). Scale bar 100 μm.

in the quinoxaline ring. Monoruthenium(II) complex [RuL]

showed moderate potency in both the cancer cell lines.

However, diruthenium(II) complex [Ru₂L] displayed much

better potency in both the cancer cell lines. The dipositive

charge of ruthenium triggered the reaction to follow

mitochondrial pathway by reducing the mitochondrial

membrane potential (MMP, ΔΨm), but the potency of the

complex was reduced signiﬁcantly after the introduction of a

bromo group in the quinoxaline moiety. Similar trends were

also observed in case of iridium(III) complexes [IrL], [Ir₂L],

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Figure 14. Mitotracker staining assay in HT-29 cell line with the colocalized drug [RuIrL] and [Ir₂L] for 48 h (a) untreated control, (b) treated

with 15 μM RuIrL, and (c) treated with 25 μM [Ir₂L] drug. The last image of every panel represents the Pearson correlation coeﬃcient of

colocalization. R-values between −1, considered as a strong negative relationship, and +1, considered as a strong positive relationship are observed.

Scale bar 100 μm.

Figure 15. (a) JC-1 staining and the determination of the mitochondrial membrane potential in HT-29 cell line by the drug [Ir₂L]. (b) Graph of

JC-1 staining in HT-29 cells upon treatment with drug [Ir₂L] in diﬀerent concentrations (10 and 25 μM) and positive control CCCP (5 μM). The

P values of >0.12 (ns), 0.033 (*), 0.002 (**), and <0.0002(***) were considered as signiﬁcant. The error bar represents the standard error of

mean (SEM). Scale bar 100 μm.

and [Ir₂LBr]. Bis(iridium) complexes [Ir₂L] and [Ir₂LBr]

were more potent and selective than bis(ruthenium)

complexes [Ru₂L] and [Ru₂LBr] because of their good

subcellular (mitochondria) accumulation. The mixed metallic

complex [RuIrL] showed highest the cytoselectivity (Caco-2 >

90; HeLa > 14) among all these complexes owing to its high

lipophilicity and strong mitochondria targeting ability.

complexes inside the cell, (ii) labile chlorine attached to

ruthenium and iridium was essential for several biomolecular

interactions, (iii) Ru(II) and Ir(III) bicationic complexes

enhanced the compound solubility and accelerated the

mitochondrial targeting aptitude by reducing the MMP, (iv)

triazolylmethylquinoxaline ligand acted as a main protagonist

for the DNA intercalating agent, (v) the luminescent property

of these complexes helped in the bioimaging application, and

(vi) complex [RuIrL] reigned over all the complexes by

The overall results showed us that (i) hydrophobic p-

cymene and Cp* ring facilitated the passive diﬀusion of these

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Figure 16. (a) JC-1 staining and the determination of the mitochondrial membrane potential in HT-29 cell line by the drug [RuIrL]. (b) JC-1

staining in HT-29 cells upon treatment with drug LRuIr in diﬀerent concentrations (10 and 25 μM) and positive control CCCP (5 μM). The P

values of >0.12 (ns), 0.033 (*), 0.002 (**), and <0.0002(***) were considered as signiﬁcant. The error bar represents the standard error of

mean (SEM). Scale bar 100 μm.

Figure 17. SAR study of pyridinyltriazolylmethylquinoxaline-based Ru(II) and Ir(III) complexes [Ru₂L], [Ir₂L], [Ru₂LBr], [Ir₂LBr], [RuL],

[IrL], and [RuIrL].

conveying remarkable cytotoselectivity because of it attaining

the highest lipophilicity, solubility, and stability along with

signiﬁcant subcellular (mitochondria) accumulation reinforced

by the synergistic eﬀect of both Ru(II) and Ir(II) metals

(Figure 17).

apoptotic power on cancer cells can be demonstrated by their

keen targeting aﬃnity toward mitochondria, the power house

of the cell triggering mitochondrial dysfunction associated with

the reduction of mitochondrial membrane potential making

the mitochondrial membrane “leaky” along with the cell cycle

arrest. Casting our vision to a DNA binding study also revealed

their good DNA intercalative inclination, and their high

binding proﬁciency to human serum albumin (HSA)

conﬁrmed their facile transportation to the cellular medium

through the bloodstream. In addition to this, the higher

quantum yield values attributed the good luminescent nature

of the complexes, gifting them an opulent imaging quality.

From all the aspects our prepared scaﬀolds were most active as

well as selective in destroying the cancer cells through the

detection of cancer cells exploiting their luminescence nature.

In a nut shell, it can be depicted that complex [RuIrL]

achieved paramount importance over all the complexes on the

basis of its excellent potency in association with the highest

percent of selectivity to cancer cell along with its superb

CONCLUSION

■

From these experiments, it can be nicely portrayed that we

have been able to develop mitochondria targeting novel

ruthenium- and iridium-based homo and hetero bimetallic

pyridinyltriazolylmethylquinoxaline complexes as successful

luminescent anticancer agents with appreciably high yields

and much greater stability to the biological environment. All

the prepared complexes displayed their eﬃciency toward HeLa

and Caco-2 cancer cell lines, rendering the normal cell

unaﬀected. However, the heterobimetallic complex [RuIrL]

had been able to leave an imprint of its superiority among all of

them by exposing its eﬃciency toward most aggressive

colorectal carcinoma cell line, HT-29. The exposition of their

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H stretching), 2962 (C−H Ar stretching), 1485 (C−H bending),

1425 (CH₂bending), 1209(C−N stretching), 765 (C−H stretching

of o-disubstituted group), 628 (C−Br stretching). ESI-MS (CH₃OH):

m/z 317 [M + H]⁺.

cellular imaging quality which will enable it to be explored as

an outstanding anticancer theranostic drug in the near future.

6-Bromo-2,3-bis(bromomethyl)quinoxaline. ¹H NMR (400

MHz, CDCl₃): Yield: 98%. Mp: 165−168 °C. R_f: 0.79 (1:3 ethyl

EXPERIMENTAL SECTION

■

Materials and Methods. The highest commercial quality

reagents and solvents were used. All analytical grade organic solvents

used for the chemical synthesis and chromatography were picked up

from E. Merck (India). Sodium ascorbate, copper sulfate, 1,4

dibromo-2,3-butanedione, sodium azide, 2-ethynyl pyridine, o-phenyl-

ene diamine, [(η⁶-p-cymene)RuCl(μ-Cl)]₂and [(η⁵-Cp*)IrCl(μ-

Cl)]₂were purchased from SPECTROCHEM and Sigma-Aldrich

Chemical Ltd., Merck. ct-DNA, bovine serum albumin (BSA), and

human serum albumin (HSA) were purchased from Sigma-Aldrich

Chemical Limited. HeLa, Caco-2, HT-29, and HEK-293 cell lines

were purchased from NCCS, Pune, and ATCC. DMEM medium, 1%

penicillin and streptomycin, and 1% Glutmax were purchased from

Gibco. Also, 10% fetal bovine serum and 0.25% trypsin−EDTA were

procured from Himidia and Thermo Fisher Scientiﬁc, USA

respectively. Hoechst 33342 and carbonyl cyanide m-chlorophenylhy-

drazone (CCCP) were purchased from Sigma-Aldrich Chemical

Limited. JC-1 stain was procured from Invitrogen. ¹H NMR, ¹³C

NMR, ¹⁹F NMR, and ³¹P NMR spectra were recorded on a 400 MHz

Advance Bruker DPX spectrometer with tetramethylsilane (TMS) as

internal standard. The chemical shifts were reported in ppm units.

Abbreviations are as follows: s, singlet; d, doublet; dd, double doublet;

t, triplet; m, multiplet. The melting points of the complexes were

measured on an Elchem Microprocessor based DT apparatus using an

open capillary tubes. TLC was performed on silica gel 60 F₂₅₄

precoated aluminum sheets (E. Merck, Germany) using the solvent

system hexane, ethyl acetate, and methanol, and solvents and spots

were visualized using a UV lamp. Infrared spectra (IR) were recorded

on a Shimadzu Aﬃnity FT-IR spectrometer in the range 4000−400

cm⁻¹. The mass spectra of the synthesized compounds were recorded

on Applied Biosystems (API-4000 ESI-mode), using methanol as

solvent. UV−visible spectra were recorded on a JASCO V-760

spectrometer using a 1 cm quartz cell and ﬂuorescence spectra on a

Hitachi F7000 ﬂuorescence spectrophotometer equipped with a

xenon lamp. Cyclic voltammetry study was performed by using an

electrochemical instrument, FRA2 μAutolab TypeIII, Potentiostat/

galvanostat, Metrohm/Autolab, Netherlands. Conductivity and

viscosity were measured using a TDS Conductometer and an

Ostwald Viscometer, respectively. For cytotoxicity (MTT assay),

Elisa reader and 96-well plate were used. Live cell morphology was

observed under an inverted microscope (Primovert, Zeiss), and the

live cell images were captured using Evos-M5000 ﬂuorescent imager

(Invitrogen, Thermo Fisher Scientiﬁc).

1

acetate: hexane). H NMR (CDCl₃, 400 MHz): δ 8.25 (s, 1H, H-4),

7.92 (d, J = 8.8 Hz, 1H, H-2), 7.85 (d, J = 8.8 Hz, 1H, H-1), 4.89 (s,

4H, CH₂, H-9, H-10). ¹³C NMR (400 MHz, CDCl₃): δ 151.8 (CH,

C-7), 151.2 (CH, C-8), 142.1 (CH, C-5), 140.3 (CH, C-6), 134.5

(CH, C-4), 131.40 (CH, C-1), 130.3 (CH, C-2), 125.1 (CH, C-3),

30.21 (CH₂, C-9), 30.11 (CH₂, C-10). IR (KBr, cm⁻¹): 3028 (sp³C−

H stretching), 2972 (C−H Ar stretching), 1595 (CC), 1473, 1415

(CH₂bending), 1209.37 (C−N stretching) 721.38 (C−H stretching

of o-disubstituted group). 634, 567 (C−Br stretching). ESI-MS

(CH₃OH): m/z 395 [M + H]⁺.

Synthesis of the 2,3-Bis(azidomethyl)quinoxaline Series.⁴⁹A 50

mg sample of 2,3-bromomethyl quinoxalines and sodium azide was

dissolved in ethanol. Then silica was added to the solution and mixed

uniformly to obtain a slurry. After complete drying, the reaction

mixture was kept in a microwave for 10 min at 40 W (50 °C). TLC

was monitored in 3:1 hexane/ethyl acetate solvent system, after every

2 min interval, to observe the change in the reaction. As soon as the

reaction was completed, the product from the solid supported mixture

was extracted using ethanol. After evaporation of ethanol, blackish

crystals of azidomethylquinoxalines were obtained with high yield

(∼94%).

2,3-Bis(azidomethyl)quinoxaline. Yield: 95%. Mp: 145−148 °C.

1

R_f: 0.68 (1:3 ethyl acetate:hexane). H NMR (CDCl₃, 400 MHz): δ

8.10 (dd, 2H, J₁= 6.4 Hz, J₂= 3.6 Hz, ArH, H-1, H-4), 7.78 (dd, 2H,

J₁= 6.4 Hz, J₂= 3.6 Hz, ArH, H-2, H-3), 4.70 (s, 4H, CH₂, H-9, H-

10). ¹³C NMR (400 MHz, CDCl₃): δ 148.3 (2 × CH, C-7, C-8),

140.9 (2 × CH, C-5, C-6), 130.3 (2 × CH, C-2, C-3), 128.7 (2 × CH,

C-1, C-4), 52.9(2 × CH₂, C-9, C-10). IR(KBr, cm⁻¹): 3053 (sp³C−

H stretching), 2129, 2083 (NNN stretching), 1566 (C−H

bending), 1452 (CH₂bending), 1240 (C−N stretching), 761 (C−H

stretching of o-disubstituted group). ESI-MS (CH₃OH): m/z 241 [M

+ H]⁺.

2,3-Bis(azidomethyl)-6-bromoquinoxaline. Yield: 95%. Mp:

1

145−148 °C. R_f: 0.68 (1:3 ethyl acetate:hexane). H NMR (CDCl₃,

400 MHz): δ 8.32 (s, 1H,ArH, H-4), 8.0 (d, 1H, J = 9.2 Hz, ArH, H-

2), 7.90 (d, 1H, J = 10.8 Hz,ArH, H-1), 4.71 (s, 4H, CH₂). ¹³C NMR

(400 MHz, CDCl₃): δ 150.4 (CH, C-7), 149.7 (CH, C-8), 141.9

(CH, C-5), 140.1 (CH, C-6), 134.3 (CH, C-1), 131.5 (CH, C-4),

130.4 (CH, C-2), 124.5 (CH, C-3), 53.3 (CH₂, C-9), 53.2 (CH₂, C-

10),IR(KBr, cm¹): 3045 (sp³C−H stretching), 2943 (C−H Ar

stretching), 2094, 2069 (NNN stretching), 1597 (C−H

bending), 1477 (CH₂bending), 1427 (CH₂bending), 1143 (C−N

stretching), 740 (C−H stretching of o-disubstituted group). ESI-MS

(CH₃OH): m/z 319 [M + H]⁺.

Synthetic Procedures. Synthesis of 2,3-Bis(bromomethyl)-

quinoxaline series.⁴⁹First, 100 mg of o-phenylene diamine

derivatives were taken in 50 mL beakers followed by addition of

1,4-dibromobutane-2,3-dione (1.1 equiv). The reagents were

dissolved in 10 mL of ethanol followed by the addition of silica

(100−200 mesh) so as to obtain a slurry. After dryingthe silica in air,

the solid supported reaction mixture was kept in a microwave at 60 W

(100 °C) for 10 min. TLC was monitored after every 2 min interval to

observe the progress of the reaction using a 3:1 hexane/ethyl acetate

solvent system. After the reaction was completed, the solid silica was

washed with ethanol (3 times) and ﬁltered oﬀ. The obtained ﬁltrate

was then evaporated to dryness and recrystallized from hexane/ethyl

acetate. Gray crystals of 2, 3-bis(bromomethyl)quinoxaline and 6-

bromo-2,3-bis(bromomethyl)quinoxalinewere obtained with 95−98%

yield. These compounds were characterized by ¹H and ¹³C NMR, IR,

and mass spectroscopy.

Synthesis of the 2,3-Bis[{4-(pyridin-2-yl)-1H-1,2,3-triazol-1-yl}-

methyl]quinoxaline Series (L, LBr).⁴⁹First, 50 mg of 2,3-bis-

(azidomethyl) quinoxaline derivatives were taken in 50 mL beakers

followed by addition of 2.5 equiv of 2-ethynylpyridine and an aqueous

solution of 5 mol % of CuSO₄and 5 mol % of sodium ascorbate. All

the reagents were dissolved properly, and then the required amount of

silica was added gradually to make a slurry. After drying in air, the

solid supported mixture was kept under microwave at 60 W (100 °C)

for 20 min. TLC was monitored every 5 min interval to keep on

checking the progression of the reaction. After completion of the

reaction, the product was extracted by ethanol washing and then

ﬁltered oﬀ. The solvent was evaporated to yield a crude triazole

product. The ﬁne brown crystals of 2,3-bis[{4-(pyridin-2-yl)-1H-

1,2,3-triazol-1-yl}methyl]quinoxalines (L, LBr) were obtained from

hexane−ethyl acetate (1:1) with a high yield (∼95%).

2,3-Bis(bromomethyl)quinoxaline. Yield: 95%. Mp: 150−155 °C.

1

R_f: 0.75 (1:3 ethyl acetate:hexane). H NMR (CDCl₃, 400 MHz): δ

8.07 (dd, 2H, J₁= 6.4 Hz, J₂= 3.6 Hz, ArH, H-1, H-4), 7.79 (dd, 2H,

J₁= 6.4 Hz, J₂= 3.6 Hz, ArH, H-2, H-3), 4.93 (s, 4H, CH₂, H-9, H-

10). ¹³C NMR (400 MHz, CDCl₃): δ 30.5 (2 × CH₂, C-9, C-10),

129.7 (2 × CH, C-7, C-8), 130.9 (2 × CH, C-5, C-6), 143.6 (2 × CH,

C-1, C-4), 150.9 (2 × CH, C-2, C-3). IR (KBr, cm⁻¹): 3018 (sp3 C−

2,3-Bis[{4-(pyridin-2-yl)-1H-1,2,3-triazol-1-yl}methyl]quinoxaline

1

(L). Yield: 95%. Mp: 172−177 °C. R_f: 0.26 (ethyl acetate). H NMR

(400 MHz, CDCl₃): δ 8.56 (s, 2H, CH, H-19, H-24), 8.38 (s, 2H,

CH, H-12, H-14), 8.16 (d, 2H, J = 6.4 Hz, CH, H-16, H-21), 8.06−

8.09 (m, 2H, CH, H-17, H-22), 7.80−7.83 (m, 2H, CH, H-18, H-23),

R

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7.78 (d, 2H, J = 8.0 Hz, CH, H-1, H-4),7.32(brs, 2H, CH, H-2, H-3)

6.16 (s, 4H, CH). IR (cm⁻¹, KBr): 3082 (sp³C−H stretching), 2970

(C−H Ar stretching), 2833 (CH₂asymmetric stretching), 1598 (C

C), 1570 (C−H bending), 1450 (CH₂bending), 1290 (C−N

stretching), 783 (CH bending), 756 (C−H stretching of o-

disubstituted group). ESI-MS (CH₃OH): m/z 447.1 [M + H]⁺.

6-Bromo-2,3-bis[{(4-(pyridin-2-yl)-1H-1,2,3-triazol-1-yl}methyl]-

quinoxaline (LBr). Yield: 95%. Mp: 295−297 °C. R_f: 0.31 (ethyl

acetate). ¹H NMR (400 MHz, DMSO-d₆): δ 8.69−8.73 (m, 2H, CH,

H-17, H-23), 8.62 (brs, 2H, CH, H-19, H-24), 8.16 (brs, 1H, CH, H-

4), 8.09 (d, J = 8 Hz, 2H, CH, H-16, H-21), 7.86−7.94 (m, 3H, CH,

H-2, H-18, H-24), 7.37(brs, 3H, CH, H-1, H-12, H-14), 6.33 (s, 4H,

CH₂, H-9, H-10). IR (KBr, cm⁻¹): 3535 (N−H symmetric), 3361

(asymmetric stretching), 2956 (C−H Ar stretching), 1600 (CC

stretching), 1421 (CH₂bending), 1228 (C−N stretching), 808 (C−H

bending), 617 (C−Br stretching). ESI-MS (CH₃OH): m/z 524[M +

H]⁺, 526 [M + H]⁺.

N, 10.32. Found: C, 38.46; H, 3.39; N, 10.02. Yield: 95%. Mp: 185−

187 °C. R_f(100% methanol): 0.34. ¹H NMR (DMSO-d₆, 400 MHz):

δ 9.49 (brs, 2H, H-19, H-24), 9.35 (brs, 2H, H-12, H-14), 8.33 (s,

2H, H-16, H-21), 8.25 (s, 2H, H-17, H-22), 7.97 (brs, 2H, H-1, H-4),

7.72 (brs, 3H, H-2, H-18, H-23), 6.66 (brs, 4H, H-9, H-10), 5.97−

6.00 (m, J = 5.60 Hz, 3H, p-cymene, H-3a, H-3b, H-4a), 5.83 (d, 2H,

J = 6.0 Hz, p-cymene, H-5a, H-6a), 5.78 (d, J = 6.0 Hz, 2H, p-cymene,

H-4b, H-5b), 5.74 (d, J = 6.0 Hz, 1H, p-cymene, H-6b), 2.74−2.85

(sept, 2H, p-cymene, H-8a, H-8b), 2.17 (s, 3H, p-cymene, H-1a), 2.16

(s, 3H, p-cymene, H-1b), 1.08 (d, J = 6.8 Hz, 6H, p-cymene, H-9a, H-

9b), 1.0 (d, J = 6.0 Hz, H-10a, H-10b). ¹³C(DMSO-d₆, 100 MHz): δ

{156.9, 156.3} (2C, C-15, C-20), 149.5 (2C, C-7, C-8), 148.4 (CH,

C-19), 146.7 (CH, C-24), 141.4 (C, C-5), 140.7 (C, C-6), 139.4

(2CH, C-17, C-22), 134.7 (CH, C-4), 131.4 (2C, C-11, C-13), 130.7

(2CH, C-12, C-14), 128.1 (CH, C-1), 126.5 (CH, C-2), 124.4 (CH,

C-16, C-21), 123.1 (CH, C-18, C-23), 106.8 (C, C-3), 104.1 (p-

cymene, C, C-2a, C-2b), 100.6 (p-cymene, C, C-8a, C-8b), {86.8,

85.9, 85.6, 84.8} (p-cymene, Ar−CH, C-3a, 3b, 4a, 4b, 5a, 5b, 6a, 6b),

54.1 (CH₂, C-9, C-10), {30.9, 30.4} (p-cymene, isopropyl-CH, C-8a,

C-8b), {22.4, 22.3, 21.9, 21.8} (p-cymene, isopropylCH₃, C-9a, C-9b,

C-10a, C-10b), {18.7, 18.3} (p-cymene, CH₃, C-1a, C-1b). ¹⁹F NMR

(DMSO-d₆, 376 MHz): δ −71.02 (PF₆), −69.13 (PF₆). ³¹P NMR

(DMSO-d₆, 162 MHz): δ −157.42 (PF₆), −153.03 (PF₆), −148.64

(PF₆), −144.25 (PF₆), −139.86 (PF₆), −135.47 (PF₆), −131.08

(PF₆). IR (cm⁻¹, KBr): 3622 (C−H stretching), 2966 (Sp³C−H

stretching), 1624 (N−H bending), 1440 (arm CC stretching),

1278 (C−N stretching), 827 (P−F stretching). ESI-MS (MeOH): m/

z: 533.0 [M − 2PF₆]²⁺. HRMS (MeOH): m/z 533.4242. HRMS

(MeOH): C₄₄H₄₅N₁₀BrCl₂F₁₂P₂Ru₂(M) calculated m/z, 535.0233

[M − 2PF₆]²⁺, 1380.9657 [M + Na]⁺; observed m/z, 535.0247 [M −

2PF₆]²⁺, 1380.9643 [M + Na]⁺.

II

Synthesis of [(η⁶-p-cymene)₂Ru₂Cl₂(K²-N,N-L/LBr)](PF₆)₂([Ru₂L],

[Ru₂LBr]). First, 25 mg (0.0725 mmol) of [(η⁶-p-cymene)RuCl(μ-

Cl)]₂was dissolved in 5 mL of methanol and stirred for 10 min to

dissolve the compound completely in methanol. Then 1.1 equiv of

previously prepared ligand (L, LBr) was added to the reaction

mixture. The mixture was sonicated for 2 h at ambient temperature. A

change in color from deep yellow to deep brown was observed. Then

2.5 equiv of NH₄PF₆was added to the reaction mixture, which was

stirred for another 2 h. The reaction was monitored by TLC using

100% methanol as solvent system. After the completion of the

reaction, methanol was evaporated to get the solid product. To

remove the impurities, crude product was washed thoroughly with

hexane followed by diethyl ether. The puriﬁed product was further

crystallized from methanol/diethyl ether system, and brown colored

ﬁne crystals were obtained with 90−95% yield. The structures of

Synthesis of [(η⁵-Cp*)₂Ir₂^IIICl₂(K²-N,N-L/LBr)](PF₆)₂([Ir₂L, Ir₂LBr]).

First 25 mg (0.017 mmol) of [(η⁵-Cp*)IrCl(μ-Cl)]₂was dissolved in

5 mL of methanol and stirred for 10 min to dissolve the compound

completely in methanol. Then 1.1 equiv of the previously prepared

ligand (L/LBr) was added to the reaction mixture. The mixture was

sonicated for 2 h at ambient temperature. A change in color from

orange to light yellow was observed. Then 2.5 equiv of NH₄PF₆was

added to the reaction mixture, which was stirred for another 2 h. The

reaction was monitored by TLC using 100% methanol as solvent

system. After the completion of the reaction, methanol was

evaporated to obtain the solid product. To remove the impurities,

crude product was washed thoroughly with hexane followed by

diethyl ether. The puriﬁed product was further crystallized from

methanol−dietheyl ether mixture to get yellow colored ﬁne crystals

with 90−95% yield. The structures of [Ir₂L] and [Ir₂LBr] were

analyzed by ¹H, ¹³C, ¹⁹F, and ³¹P NMR, FT-IR, and ESI-MS. Purity of

the complex was determined by C, H, N, and HRMS analysis.

[(η⁵-Cp*)₂Ir₂^IIICl₂(K²-N,N-L)](PF₆)₂([Ir₂L]). Yield: 45 mg (0.031

mmol, 98%). M_r(C₄₄H₄₈N₁₀Cl₂F₁₂P₂Ir₂) = 1462.19 g/mol. Anal.

Calcd for C₄₄H₄₈N₁₀Cl₂F₁₂P₂Ir₂: C, 36.14; H, 3.31; N, 9.58. Found:

C, 36.73; H 3.67; N, 9.73. Yield: 98%. Mp: 178−180^οC. R_f(100%

methanol): 0.30. ¹H NMR (DMSO-d₆, 400 MHz): δ 9.45 (s, 2H, H-

12, H-14), 8.99 (d, J = 5.6 Hz, 2H, H-19, H-24), 8.54 (s, 2H, H-16,

H-21), 8.31 (d, J = 4.8 Hz, 2H, H-17, H-22), 7.82 (s, 4H, H-2, H-3,

H-18, H-23), 7.78 (t, J = 6.4 Hz, 2H, H-1, H-4), 6.69 (s, 4H, H-9, H-

10), 1.73 (s, 30H, Cp*, H-6a, H-6b, H-7a, H-7b, H-8a, H-8b, H-9a,

H-9b, H-10a, H-10b). ¹³C NMR (DMSO-d₆, 100 MHz): δ 157.2 (C,

C-15), 156.5 (C, C-20), 152.9 (2C, C-7, C-8), 149.5 (2CH, C-19, C-

24), 148.4 (2C, C-5, C-6), 147.9 (2CH, C-17, C-22), 141.2 (2C, C-

11, C-13), 140.9 (CH, C-12), 140.6 (CH, C-14), 131.2 (CH, C-2, C-

3), 129.1 (2CH, C-1, C-4), 128.9 (CH, C-16), 127.9 (CH, C-21),

122.9 (2CH, C-18, C-23),{91.8, 91.3, 89.8, 89.4} (Cp*, 10C, C-1a,

1b, 2a, 2b, 3a, 3b, 4a, 4b, 5a, 5b), 54.2 (CH₂, C-9, C-10), (8.8, Cp*,

CH₃, C-6a, 6b, 7a, 7b, 8a, 8b, 9a, 9b, 10a, 10b). ¹⁹F NMR (DMSO-d₆,

400 MHz): δ −71.06 (PF₆), −69.17 (PF₆). ³¹P NMR (DMSO-d₆, 400

MHz): δ −152.98 (PF₆), −153.00 (PF₆), −148.61 (PF₆), −144.22

(PF₆), −139.83 (PF₆), −135.44 (PF₆). IR (cm⁻¹, KBr): 3319 (arm

C−H stretching), 3037 (Sp³C−H stretching), 1423 (arm CC

stretching), 1282 (C−N stretching), 1124 (C−O stretching), 827

1

[Ru₂L] and [Ru₂LBr] were analyzed by H, ¹³C, ¹⁹F, and ³¹P NMR,

FT-IR, and ESI-MS. Purity of these complexes were determined by C,

H, N, and HRMS analysis.

II

[(η⁶-p-cymene)₂Ru₂Cl₂(K²-N,N-L)](PF₆)₂([Ru₂L]). Yield: 50.4 mg

(0.0394 mmol, 97%). M_r(C₄₄H₄₆N₁₀Cl₂F₁₂P₂Ru₂) = 1277.88 g/mol.

Anal. Calcd for C₄₄H₄₆N₁₀Cl₂F₁₂P₂Ru₂: C, 41.36; H, 3.63; N, 10.96.

Found: C, 41.05; H, 3.55; N, 10.46. Yield: 97%. Mp: 178−180 °C. R_f

(100% methanol): 0.30. ¹H NMR (DMSO-d₆, 400 MHz): δ 9.52 (d, J

= 5.2 Hz, 2H, H-19, H-24), 9.39 (s, 2H, H-12, H-14), 8.38 (d, J = 7.6

Hz, 2H, H-16, H-21), 8.27 (t, J = 7.6 Hz, 2H, H-17, H-22), 7.80−7.87

(m, 4H, H-2, H-3, H-18, H-23), 7.73 (t, J = 6.8 Hz, 2H, H-1, H-4),

6.62−6.76 (m, 4H, H-9, H-10), 6.14−6.18 (m, 4H, p-cymene H-3a,

H-4a, H-5a, H-6a), 6.03 (d, J = 6.0 Hz, 2H, p-cymene H-3b, H-4b),

5.87 (d, J = 5.6 Hz, 2H, p-cymene H-5b, H-6b), 2.62−2.69 (m, 2H,

CH, H-8a, H-8b), 2.18 (s, 6H, Me, H-1a, H-1b), 1.08 (d, J = 6.8 Hz,

6H, p-cymene, H-9a, H-9b), 1.01 (d, J = 6.8 Hz, 6H, p-cymene, H-

10a, H-10b).¹³C (DMSO-d₆, 100 MHz): δ 156.3 (C, C-15), 154.0 (C,

C-20), 148.4 (2C, C-7, C-8), 146.6 (2CH, C-19, C-24), 140.6 (2C,

C-5, C-6), 138.5 (2C, C-11, C-13), 131.6 (2CH, C-17, C-22), 128.9

(CH, C-12), 127.90 (CH, C-14), 126.6 (CH, C-2), 125.9 (CH, C-3),

123.1 (CH, C-1), 122.2 (CH, C-4), 112.1 (2CH, C-16, C-21), 107.0

(2CH, C-18, C-23), 104.2 (p-cymene, C, C-2a, C-2b), 103.1 (p-

cymene, C, C-7a, C-7b), {86.1, 85.6, 83.9, 82.9} (p-cymene, 8CH, C-

3a, 3b, 4a, 4b, 5a, 5b, 6a, 6b), 54.2 (CH₂, C-9, C-10), 30.9 (p-cymene,

CH, C-8a, C-8b), {22.3, 21.9} (p-cymene, isopropyl CH₃, C-9a, C-

10a, C-9b, C-10b), 18.7 (p-cymene, CH₃, C-1a, C-1b). ¹⁹F NMR

(DMSO-d₆, 376 MHz): δ −71.01 (PF₆), −69.12 (PF₆). ³¹P NMR

(DMSO-d₆, 162 MHz): δ −153.04 (PF₆), −148.65 (PF₆), −144.26

(PF₆), −139.87 (PF₆), −135.47 (PF₆). IR (cm⁻¹, KBr): 3122 (C−H

stretching), 2970 (sp³C−H stretching), 1622 (CC), 1442 (CC

stretching), 1280 (C−N stretching), 829 (P−F stretching), 773 (C−

H bending). ESI-MS (MeOH): m/z 493.9 [M − 2PF₆]²⁺. HRMS

(MeOH): C₄₄H₄₆N₁₀Cl₂F₁₂P₂Ru₂(M) calculated m/z, 494.0686 [M

− 2PF₆]²⁺, 1301.0552 [M + Na]⁺; observed m/z, 494.0699 [M −

2PF₆]²⁺, 1301.0549 [M + Na]⁺.

II

[(η⁶-p-cymene)₂Ru₂Cl₂(K²-N,N-LBr)](PF₆)₂([Ru₂LBr]). Yield: 52.5

mg (0.0386 mmol, 95%). M_r(C₄₄H₄₅N₁₀BrCl₂F₁₂P₂Ru₂) = 1356.77

g/mol. Anal. Calcd for C₄₄H₄₅N₁₀BrCl₂F₁₂P₂Ru₂: C, 38.95; H, 3.34;

S

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(P−F stretching), 779 (C−H bending). ESI-MS (MeOH): m/z:

586.2 [M − 2PF₆]²⁺. HRMS (MeOH): C₄₄H₄₈N₁₀Cl₂F₁₂P₂Ir₂(M)

calculated m/z, 586.1349 [M − 2PF₆]²⁺, 1485.1880 [M + Na]⁺;

observed m/z, 586.1329 [M − 2PF₆]²⁺, 1485.1843 [M + Na]⁺.

[(η⁵-Cp*)₂Ir₂^IIICl₂(K²-N,N-LBr)](PF₆)₂([Ir₂LBr]). Yield: 46.0 mg

(0.0298 mmol, 95%). M_r(C₄₄H₄₇N₁₀BrCl₂F₁₂P₂Ir₂) = 1541.08 g/

mol. Anal. Calcd for C₄₄H₄₇N₁₀BrCl₂F₁₂P₂Ir₂: C, 34.29; H, 3.07; N,

9.09. Found: C, 33.80; H, 3.25; N, 8.34. Yield: 95%. Mp: 175−177^οC.

mL of methanol and stirred for 10 min to dissolve the compound

completely in methanol. Then 1.1 equiv (29.4 mg) of previously

prepared ligand (L) was added to the reaction mixture. The mixture

was sonicated for 2 h at ambient temperature. When a change in color

from orange to light yellow was observed, 1.1 equiv of NH₄PF₆was

added to the reaction mixture and stirred for another 2 h. The

reaction was monitored by TLC using 100% methanol as solvent

system. After the complete conversion, methanol was evaporated to

dryness in air. To remove the impurities, crude product was washed

thoroughly with 5 mL of hexane and dried. The puriﬁed product was

further crystallized from methanol/diethyl ether mixture. The brown

ﬁne crystals were obtained with 90−95% yield. The structure of [IrL]

1

R_f(100% methanol): 0.46. H NMR (DMSO-d₆, 400 MHz): δ 9.44

(s, 2H, H-12, H-14), 8.99 (d, J = 5.2 Hz, 2H, H-19, H-24), 8.56 (s,

2H, H-16, H-21), 8.33 (t, J = 7.2 Hz, 2H, H-17, H-22), 8.05 (s, 1H,

H-4), 7.96 (d, J = 8.4 Hz, 1H, H-2), 7.76−7.79 (m, 3H, H-1, H-18,

H-23), 6.70 (d, J = 9.2 Hz, 4H, H-9, H-10), 1.74 (s, 15H, Cp*, H-6a,

H-7a, H-8a, H-9a, H-10a). 1.73 (s, 15H, Cp*, H-6b, H-7b, H-8b, H-

9b, H-10b).¹³C NMR (DMSO-d₆, 400 MHz): δ 152.9 (2C, C-15, C-

20), 148.4 (C, C-7), 147.8 (C, C-8), 145.1 (CH, C-19), 144.5 (CH,

C-24), 141.2 (2C, C-5, C-6), 139.4 (2CH, C-17, C-22), 135.7 (C, C-

11), 134.5 (C, C-13), 132.8 (CH, C-4), 131.0 (CH, C-1), 128.7 (CH,

C-2), 127.9 (CH, C-12), 123.1 (CH, C-14), 120.9 (CH, C-16), 120.2

(CH, C-21), 117.2 (CH, C-18), 112.5 (CH, C-23), 107.4 (C, C-3),

{92.6, 91.3, 89.8, 89.4} (Cp*, C, C-1a, 1b, 2a, 2b, 3a, 3b, 4a, 4b, 5a,

5b), 54.2 (CH₂, C-9, C-10), {9.0, 8.9, 8.8, 8.7,8.1} (Cp*, CH₃, C-6a,

6b, 7a, 7b, 8a, 8b, 9a, 9b, 10a, 10b).¹⁹F NMR (DMSO-d₆, 376 MHz):

δ −71.05 (PF₆), −69.16 (PF₆). ³¹P NMR (DMSO-d₆, 162 MHz): δ

−157.38 (PF₆), −152.99 (PF₆), −148.60 (PF₆), −144.21 (PF₆),

−139.82 (PF₆), −135.43 (PF₆). IR (cm⁻¹, KBr): 3327 (arm C−H

stretching), 1427 (arm CC stretching), 1222 (C−N stretching),

825 (P−F stretching), 777 (C−H bending). ESI-MS (MeOH): m/z:

626.8 [M − 2PF₆]²⁺. HRMS (MeOH): C₄₄H₄₇N₁₀BrCl₂F₁₂P₂Ir₂(M)

calculated m/z, 625.0902 [M − 2PF₆]²⁺, 1563.0985 [M + Na]⁺;

observed m/z, 625.0889 [M − 2PF₆]²⁺, 1563.0984 [M + Na]⁺.

Synthesis of [(η⁶-p-cymene)Ru^IICl(K²-N,N-L)]PF₆([RuL]). A 25 mg

(0.041 mmol, 0.5 equiv) sample of [(η⁶-p-cymene)RuCl(μ-Cl)]₂was

dissolved in 5 mL of methanol and stirred for 10 min to dissolve the

compound completely in methanol. Then 38.2 mg (1.1 equiv) of

previously prepared ligand (L) was added to the reaction mixture.

The mixture was sonicated for 2 h at ambient temperature. As soon as

a change in color from deep yellow to deep brown was observed, 1.1

equiv of NH₄PF₆was added to the reaction mixture which was stirred

for another 2 h. The reaction was monitored by TLC using 100%

methanol as solvent system. After the complete conversion, methanol

was evaporated in order to have the solid product. Then solid crude

product was washed thoroughly with 5 mL of hexane and dried to

expel the impurities. The puriﬁed product was crystallized from

methanol−diethyl ether mixture and pure brown colored ﬁne crystals

were obtained with 90−95% yield. The structure of [RuL] was

examined by ¹H, ¹⁹F, and ³¹P NMR, FT-IR. and ESI-MS. Purity of the

sample was determined by HRMS analysis.

1

was conﬁrmed by H, ¹⁹F, and ³¹P NMR, FT-IR, and ESI-MS. Purity

of the complex was determined by HRMS analysis.

[(η⁵-Cp*)Ir^IIICl(K²-N,N-L)]PF₆([IrL]). Yield: 58 mg (0.060 mmol,

97%). M_r(C₃₄H₃₃N₁₀ClF₆PIr) = 954.33 g/mol. Anal. Calcd for

C₃₄H₃₃N₁₀ClF₆PIr: C, 42.79; H, 3.49; N, 14.68. Found: C, 42.97; H,

3.25; N, 14.34. Yield: 97%. Mp: 170−172^οC. R_f(100% methanol):

0.30. ¹H NMR (DMSO-d₆, 400 MHz): δ 9.47 (d, J = 6.4 Hz, 1H, H-

19), 9.4 (d, J = 10.4 Hz, 1H, H-24), 8.97−9.01 (m, 2H, H-12, H-21),

8.56 (d, J = 7.6 Hz, 1H, H-22), 8.47 (d, J = 6.4 Hz, 1H, H-16), 8.27−

8.35 (m, 2H, H-1, H-17), 7.81 (brs, 4H, H-2, H-3, H-4, H-14), 7.73−

7.78 (m, 2H, H-18, H-23), 6.67−6.72 (m, 2H, H-9), 6.38 (s, 2H, H-

10), 1.73 (s, 15H, H-1′, H-2′, H-3′, H-4′, H-5′). ¹⁹F NMR (DMSO-

d₆, 376 MHz): δ −71.05 (PF₆), −69.16 (PF₆). ³¹P NMR (DMSO-d₆,

162 MHz): δ −152.98 (PF₆), −152.99 (PF₆), −148.60 (PF₆),

−144.21 (PF₆), −139.82 (PF₆), −135.43 (PF₆), −131.04 (PF₆). IR

(cm⁻¹, KBr): 3128 (arm C−H stretching), 1450 (arm CC

stretching), 1261 (C−N stretching), 835 (P−F stretching), 777(C−

H bending). ESI-MS (MeOH): m/z: 810.1 [M − PF₆]⁺. HRMS

(MeOH): C₃₄H₃₃N₁₀ClF₆PIr (M) calculated m/z, 809.2207 [M −

PF₆]⁺; observed m/z, 809.2205 [M]⁺.

Synthesis of [(η⁶-p-cymene)(η⁵-Cp*)Ru^IIIr^IIICl₂(K²-N,N-L)](PF₆)₂

([RuIrL]). First 25 mg (0.031 mmol, 0.5 equiv) of [(η⁵-Cp*)IrCl(μ-

Cl)]₂was dissolved in 5 mL of methanol and stirred for 10 min to

dissolve the compound completely in methanol. Then 1.1 equiv of

previously prepared complex [RuL] was added to the reaction

mixture. The mixture was sonicated for 2 h at ambient temperature.

As soon as a change in color from orange to deep red was observed,

1.1 equiv of NH₄PF₆was added to the reaction mixture and stirred for

another 2 h to complete the reaction. The reaction was monitored by

TLC using 100% methanol as solvent system. After the completion of

reaction, methanol was evaporated. The impurities were removed by

washing the crude product thoroughly with 5 mL of hexane followed

by diethyl ether. The puriﬁed product was further crystallized from

methanol/diethyl ether and the brown colored ﬁne crystals of

compound [RuIrL] was obtained with 95%yield. The structure of

1

[RuIrL] were analyzed by H, ¹⁹F, ¹³C, and ³¹P NMR, FT-IR, and

[(η⁶-p-cymene)Ru^IICl(K²-N,N-L)]PF₆([RuL]). Yield: 67 mg (0.078

mmol, 95%). M_r(C₃₄H₃₂N₁₀ClF₆PRu) = 862.17 g/mol. Anal. Calcd

for C₃₄H₃₂N₁₀ClF₆PRu: C, 47.36; H, 3.74; N, 16.25 . Found: C,

47.78; H, 3.32; N, 16.34.Yield: 97%. Mp: 174−176^οC. R_f(100%

methanol): 0.36. ¹H NMR (DMSO-d₆, 400 MHz): δ 9.53 (d, J = 5.2

Hz, 1H, H-19), 9.42 (s, 1H, H-24), 8.75 (s, 1H, H-21), 8.40 (d, J =

5.6 Hz, 1H, H-12), 8.28 (t, J = 7.2 Hz, 2H, H-12, H16), 8.09 (s, 1H,

H-17), 7.92−7.94 (m, 2H, H-1, H-4), 7.87 (s, 2H, H-2, H-3), 7.79−

7.88 (m, 2H, H-23, H-14), 7.74 (t, J = 6.8 Hz, 1H, H-18), 6.63−6.77

(m, 2H, H-9), 6.33 (s, 2H, H-10), 6.15−6.19 (m, 2H, H-c, H-d), 6.05

(d, J = 5.6 Hz, 1H, H-e), 5.88 (d, J = 5.6 Hz, 1H, H-j), 2.64−2.67 (m,

1H, H-8), 2.18 (s, 3H, H-a), 1.08 (d, J = 6.8 Hz, 3H, H-i), 1.01 (d, J =

6.8 Hz, 3H, H-j). ¹⁹F NMR (DMSO-d₆, 376 MHz): δ −71.04 (PF₆),

−69.15 (PF₆). ³¹P NMR (DMSO-d₆, 162 MHz): δ −152.98 (PF₆),

−153.00 (PF₆), −148.61 (PF₆), −144.22 (PF₆), −139.83 (PF₆),

−135.44 (PF₆). IR (cm⁻¹, KBr): 3082 (arm C−H stretching), 1450

(arm CC stretching), 1280 (C−N stretching), 837 (P−F

stretching), 781(C−H bending). ESI-MS (MeOH): m/z: 717.6

[M]⁺. HRMS (MeOH): C₃₄H₃₂N₁₀ClF₆PRu(M) calculated m/z,

717.1543[M − PF₆]⁺; observed m/z, 717.1552.

ESI-MS. Purity of the complex was determined by C, H, N analysis

and HRMS.

[(η⁶-p-cymene)(η⁵-Cp*)Ru^IIIr^IIICl₂(K²-N,N-L)](PF₆)₂[RuIrL]. Yield:

82 mg (0.059 mmol, 95%). M_r(C₄₄H₄₇N₁₀Cl₂F₁₂P₂IrRu) = 1370.03

g/mol. Anal. Calcd for C₄₄H₄₇N₁₀Cl₂F₁₂P₂IrRu: C, 38.57; H, 3.46; N,

10.22. Found: C, 38.16; H, 3.31; N, 9.89.Yield: 98%. Mp: 185−

1

187^οC. R_f(100% methanol): 0.45. H NMR (DMSO-d₆, 400 MHz):

δ 9.53 (d, J = 5.2 Hz, 1H, H-19), 9.41−9.45 (m, 2H, H-1, H-4), 9.01

(d, J = 5.6 Hz, 1H, H-24), 8.55 (brs, 1H, H-12), 8.39 (brs, 1H, H-14),

8.26−8.34 (m, 2H, H-18, H-23), 7.78−7.83 (m, 4H, H-16, H-17, H-

21, H-22), 7.72−7.76 (m, 2H, H-2, H-3), 6.70 (s, 4H, H-9, H-10),

6.15−6.19 (m, 2H, H-3, H-4), 6.04 (d, J = 6.0 Hz, 1H, H-5), 5.87 (d,

J = 5.6 Hz, 1H, H-6), 2.62−2.67 (m, 1H, p-cymene, H-8), 2.18 (s,

3H, p-cymene, H-1), 1.73 (s, 15H, Cp*, H-6, H-7, H-8, H-9, H-10),

1.08 (d, J = 6.8 Hz, 3H p-cymene, H-9), 1.01 (d, J = 6.8 Hz, 3H, p-

cymene, H-10). ¹³C NMR (DMSO-d₆, 100 MHz): δ168.5 (2C, C-7,

C-8), 162.9 (2C, C-15, C-20), 152.9 (2CH, C-19, C-24), 148.0 (2C,

C-5, C-6), 140.6 (2CH, C-17, C-22), 131.6 (2C, C-11, C-13), 129.0

(2CH, C-12, C-14), 128.2 (2CH, C-1, C-4), 120.4 (2CH, C-2, C-3),

119.3 (2CH, C-16, C-21), 112.2 (2CH, C-18, C-23), {98.6, 92.6}

(Cp*, 5C, C-1, C-2, C-3, C-4, C-5), {89.8, 89.4, 85.9, 81.3} (p-

cymene, Ar−CH, C-3, C-4, C-5, C-6), 65.4 (CH₂, C-9, C-10), {22.9,

Synthesis of [(η⁵-Cp*)Ir^IIICl(K²-N,N-L)]PF₆([IrL]). First, 25 mg

(0.031 mmol, 0.5 equiv) of [(η⁵-Cp*)IrCl(μ-Cl)]₂was dissolved in 5

T

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20.9} (p-cymene, isopropyl-CH₃, C-9, C-10), 19.2 (p-cymene, CH₃,

C-1), {9.1, 8.8, 8.7} (Cp*, CH₃, C-6, C-7, C-8, C-9, C-10).¹⁹F NMR

(DMSO-d₆, 376 MHz): δ −71.06 (PF₆), −69.17 (PF₆). ³¹P NMR

(DMSO-d₆, 162 MHz): δ −152.98 (PF₆), −153.00 (PF₆), −148.61

(PF₆), −144.22 (PF₆), −139.83 (PF₆), −135.44 (PF₆). IR (cm⁻¹,

KBr): 3151 (arm C−H stretching), 1409 (arm CC stretching), 833

(P−F stretching), 775.38 (C−H bending). ESI-MS (MeOH): m/z:

539.8 [M − 2PF₆]²⁺. HRMS (MeOH): C₄₄H₄₇N₁₀Cl₂F₁₂P₂IrRu (M)

calculated m/z, 540.1017 [M − 2PF₆]²⁺, 1371.1397 [M + H]⁺;

observed m/z, 540.1012[M − 2PF₆]²⁺, 1371.1398 [M + H]⁺.

ACKNOWLEDGMENTS

■

The authors are grateful to the Department of Science and

Technology, Government of India, for supporting the work

through a DST-EMR project grant (EMR/2017/000816). The

authors are grateful to VIT University for providing VIT SEED

funding. We also acknowledge Professor A. Senthil Kumar and

his group for helping in the cyclic voltammetry study. We

acknowledge DST, New Delhi, India, for the DST-FIST

Project.

ASSOCIATED CONTENT

LIST OF ABBREVIATIONS

■

sı

* Supporting Information

The Supporting Information is available free of charge at

https://pubs.acs.org/doi/10.1021/acs.inorgchem.0c02928.

NMR:nuclear magnetic resonance

LCMS:liquid chromatography mass spectrometry

HRMS:high resolution mass spectrometry

ppm:parts per million

MLCT:metal ligand charge transfer

MTT:3-(4, 5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium

bromide

¹H, ³¹P, and ¹⁹F NMR, LCMS, IR, UV, and ﬂuorescence

spectra of all compounds and additional experimental

information (PDF)

GSH:glutathione

TLC:thin layer chromatography

s:singlet

d:doublet

brs:broad singlet

t:triplet

m:multiplet

OD:optical density

CT-DNA:calf-thymus DNA

BSA:bovine serum albumin

HSA:human serum albumin

EtBr:ethidium bromide

AUTHOR INFORMATION

■

Corresponding Authors

Priyankar Paira − Department of Chemistry, School of

advanced sciences, Vellore Institute of Technology, Vellore

632014, Tamilnadu, India; orcid.org/0000-0003-1698-

4895; Email: priyankar.paira@vit.ac.in

Bipasha Bose − Department Stem Cells and Regenerative

Medicine Centre, Institution Yenepoya Research Centre,

Yenepoya University, Derlakatte, Mangalore 575018,

Karnataka, India; Email: bipasha.bose@gmail.com

Authors

DFT:density functional theory

K_b:intrinsic binding constant

K_sv:Stern−Volmer quenching constant

Nilmadhab Roy − Department of Chemistry, School of

advanced sciences, Vellore Institute of Technology, Vellore

632014, Tamilnadu, India

K_app:apparent binding constant

Utsav Sen − Department Stem Cells and Regenerative

Medicine Centre, Institution Yenepoya Research Centre,

Yenepoya University, Derlakatte, Mangalore 575018,

Karnataka, India

Yukti Madaan − Department of Chemistry, School of advanced

sciences, Vellore Institute of Technology, Vellore 632014,

Tamilnadu, India

Venkatesan Muthukumar − Department of Chemistry, School

of advanced sciences, Vellore Institute of Technology, Vellore

632014, Tamilnadu, India

Seshu Varddhan − Department of Applied Chemistry, S. V.

National Institute of Technology (SVNIT) Ichchanath,

Surat, Gujrat 395007, India

Suban K. Sahoo − Department of Applied Chemistry, S. V.

National Institute of Technology (SVNIT) Ichchanath,

Surat, Gujrat 395007, India; orcid.org/0000-0003-

1751-5310

Debashis Panda − Department of Basic Sciences and

Humanities, Rajiv Gandhi Institute of Petroleum Technology,

An Institution of National Importance, Jais, Amethi 229304,

Uttar Pradesh, India; orcid.org/0000-0002-2333-3735

K:overall binding constant

REFERENCES

■

(1) Global Cancer - Facts and Figures; American Cancer Society:

2007; p 1.

(2) Thun, M. J.; DeLancey, J. O.; Center, M. M.; Jemal, A.; Ward, E.

M. The global burden of cancer: priorities for prevention. Carcino-

genesis 2010, 31, 100−110.

(3) Rosenberg, B.; Vancamp, L.; Trosko, J. E.; Mansour, V. H.

Platinum Compounds: a New Class of Potent Antitumour Agents.

Nature 1969, 222, 385.

(4) Reedijk, J. Improved understanding in platiniumantitumour

chemistry. Chem. Commun. 1996, 801−806.

(5) Ohndorf, U. M.; Rould, M. A.; He, Q.; Pabo, C. O.; Lippard, S. J.

Basis for Recognition of Cisplatin-Modified DNA by High-Mobility-

Group Proteins. Nature 1999, 399, 708−712.

(6) Borst, P.; Rottenberg, S.; Jonkers, J. How do real tumors become

resistant to cisplatin? Cell Cycle 2008, 7, 1353−1359.

(7) Barabas, K.; Milner, R.; Lurie, D.; Adin, C. Cisplatin: A review of

toxicities and therapeutic applications. Vet. Comp. Oncol. 2008, 6, 1−

18.

(8) Allardyce, C. S.; Dyson, P. J. Ruthenium in Medicine: Current

Clinical Uses and Future prospects. Platinum Met. Rev. 2001, 45, 62−

69.

Complete contact information is available at:

https://pubs.acs.org/10.1021/acs.inorgchem.0c02928

(9) Podlech, J.; Maier, T. C. Indium in Organic Synthesis. Synthesis

2003, 0633−0655.

(10) Li, C. J.; Chan, T. H. Organometallic reactions in aqueous

media with indium. Tetrahedron Lett. 1991, 32, 7017−7020.

(11) Hartinger, C. G.; Zorbas-Seifried, S.; Jakupec, M. A.; Kynast, B.;

Zorbas, H.; Keppler, B. K. From bench to bedside-preclinical and

early clinical development of the anticancer agent indazolium trans-

Author Contributions

^⊥N.R. and U.S. made an equal contribution.

Notes

The authors declare no competing ﬁnancial interest.

U

https://dx.doi.org/10.1021/acs.inorgchem.0c02928

Inorg. Chem. XXXX, XXX, XXX−XXX

Inorganic Chemistry

pubs.acs.org/IC

Article

[tetrachlorobis(1H-indazole)ruthenate(III)] (KP1019 or FFC14A). J.

Inorg. Biochem. 2006, 100, 891−904.

(12) Rademaker-Lakhai, J. M.; Van Den Bongard, D.; Pluim, D.;

Beijnen, J. H.; Schellens, J. H. M. A Phase I and Pharmacological

Study with Imidazolium-trans-DMSO-imidazole-tetrachlororuthenate,

a Novel Ruthenium Anticancer Agent. Clin. Cancer Res. 2004, 10,

3717−3727.

(13) Jakupec, M. A.; Arion, V. B.; Kapitza, S.; Reisner, E.; Eichinger,

A.; Pongratz, M.; Marian, B.; Graf v. Keyserlingk, N.; Keppler, B. K.

KP1019 (FFC14A) from bench to bedside: preclinical and early

clinical development –an overview. Int. J. Clin. Pharmacol. Ther. 2005,

43, 595−596.

(14) Levina, A.; Mitra, A.; Lay, P. A. Recent developments in

ruthenium anticancer drugs. Metallomics 2009, 1, 458−470.

(15) Zeng, L.; Gupta, P.; Chen, Y.; Wang, E.; Ji, L.; Chao, H.; Chen,

Z.-S. The development of anticancer ruthenium(II) complexes: from

single molecule compounds to nanomaterials. Chem. Soc. Rev. 2017,

46, 5771−5804.

(28) Chatterjee, S.; Kundu, S.; Bhattacharyya, A.; Hartinger, C. G.;

Dyson, P. J. The ruthenium(II)-arene compound RAPTA-C induces

apoptosis in EAC cells through mitochondrial and p53-JNK pathways.

JBIC, J. Biol. Inorg. Chem. 2008, 13, 1149−1155.

(29) Lee, R. F. S.; Escrig, S.; Maclachlan, C.; Knott, G. W.; Meibom,

A.; Sava, G.; Dyson, P. J. The Differential Distribution of RAPTA-T in

Non-Invasive and Invasive Breast Cancer Cells Correlates with Its

Anti-Invasive and Anti-Metastatic Effects. Int. J. Mol. Sci. 2017, 18,

1869.

(30) Novohradsky, V.; Zerzankova, L.; Stepankova, J.; Kisova, A.;

Kostrhunova, H.; Liu, Z.; Sadler, P. J.; Kasparkova, J.; Brabec, V. A

dual-targeting apoptosis inducing organometallic half-sandwich

iridium anticancer complex. Metallomics 2014, 6, 1491−1501.

(31) Rangasamy, S.; Ju, H.; Um, S.; Oh, D.-C.; Song, J. M.

Mitochondria and DNA Targeting of 5,10,15,20-Tetrakis(7-

sulfonatobenzo[b]thiophene) Porphyrin-Induced Photodynamic

Therapy via Intrinsic and Extrinsic Apoptotic Cell Death. J. Med.

Chem. 2015, 58, 6864−6874.

(16) (a) Smithen, D. A.; Yin, H.; Beh, M. H. R.; Hetu, M.; Cameron,

T. S.; McFarland, S. A.; Thompson, A. Synthesis and Photobiological

Activity of Ru(II) Dyads Derived from Pyrrole-2-carboxylate

Thionoesters. Inorg. Chem. 2017, 56, 4121−4132. (b) Monro, S.;

(32) Zhang, C.; Qiu, K.; Liu, C.; Huang, H.; Rees, T. W.; Ji, L.;

Zhang, Q.; Chao, H. Tracking mitochondrial dynamics during

apoptosis with phosphorescent fluorinated iridium(iii) complexes.

Dalton Trans. 2018, 47, 12907−12913.

́

Colon, K. L.; Yin, H.; Roque, J., 3rd; Konda, P.; Gujar, S.; Thummel,

(33) Singh, D. P.; Deivedi, S. K.; Hashim, S. R.; Singhal, R. G.

Synthesis and Antimicrobial Activity of some New Quinoxaline

Derivatives. Pharmaceuticals 2010, 3, 2416−2425.

R. P.; Lilge, L.; Cameron, C. G.; McFarland, S. A. Transition Metal

Complexes and Photodynamic Therapy from a Tumor-Centered

Approach: Challenges, Opportunities, and Highlights from the

Development of TLD1433. Chem. Rev. 2019, 119, 797−828.

(17) Berndsen, R. H.; Weiss, A.; Abdul, U. K.; Wong, T. J.; Meraldi,

P.; Griffioen, A. W.; Dyson, P. J.; Nowak-Sliwinska, P. Combination

of ruthenium(II)-arene complex [Ru(η⁶-p -cymene)Cl₂(pta)]

(RAPTA-C) and the epidermal growth factor receptor inhibitor

erlotinib results in efficient angiostatic and antitumor activity. Sci. Rep.

2017, 7, 43005.

(18) Sadler, P. J.; Lainez, R. F.; Abraba, H.; Michael, M.; Duncan, J.

M.; Ruthenium Anticancer Complexes. U.S. Patent US 20060058270

A1, Mar 16, 2006.

(19) Wenjie, M. Chair Ruthenium Complexes and Their Use as

Anticancer Agents. U.S. Patent US 8,357,678 B2, Jan 22, 2013.

(20) Mestroni, G.; Alessio, E.; Sava, G.; New Salts of Anionic

Complexes of Ru(III) as Antimetastatic and Antineoplastic Agents

WO Patent WO1998000431A1, Jan 8, 1998.

(21) Gamble, A. J.; Lynam, J. M.; Walton, P. H.; Synthesis an

Anticancer activity of Ruthenium(II) cis-cis-1,3,5-Triaminocyclohex-

ane Complexes. WO Patent WO2013038134A1, Mar 21, 2013.

(22) Morris, R. E.; Sadler, P. J.; Chen, H.; Jodrell, D.; Half-Sandwich

Ruthenium(II) Compounds Comprising Nitrogen Containing

Ligands for Treatment of Cancer. U.S. Patent US 20050239765 A1,

Dec 27, 2005.

(23) Morris, R. E.; Sadler, P. J.; Jodrell, D.; Chen, H.; Ruthenium(II)

Compounds for Use in the Therapy of Cancer. U.S. Patent US

6,936,634 B2, Aug 30, 2005.

(24) Keppler, B. K.; Medicament Preparations Containing Tumour-

Inhibiting Ruthenium(III) Complexes. WO Patent

WO1997036595A3, Nov 6, 1997.

(25) Mestroni, G.; Alessio, E.; Sava, G.; Lengo, E.; Zorzet, S.;

Bergamo, A. Ruthenium Dimeric Compounds Suitable as Antimeta-

static and Antineoplastic Agents. U.S. Patent US 6,921,824 B1, Jul 26,

2005.

(34) Rong, F.; Chow, S.; Yan, S.; Larson, G.; Hong, Z.; Wu, J.

Structure-activity relationship(SAR) studies of quinoxalines as novel

HCV NSSB RNA-dependent RNA polymerase inhibitors. Bioorg.

Med. Chem. Lett. 2007, 17, 1663−1666.

(35) Jaso, A.; Zarranz, B.; Aldana, I.; Monge, A. Synthesis of New

Quinoxaline-2-carboxylate 1,4-Dioxide derivatives as Anti-Mycobac-

terium tuberculosis Agents. J. Med. Chem. 2005, 48, 2019−2025.

(36) Smits, R. A.; Lim, H. D.; Hanzer, A.; Zuiderveld, O. P.; Guaita,

E.; Adami, M.; Coruzzi, G.; Leurs, R.; de Esch, I. J. P. Discovery of

Quinazolines as Histamine H₄Receptor Inverse Agonists Using a

Scaffold Hopping Approach. J. Med. Chem. 2008, 51, 2457−2467.

(37) Burguete, A.; Pontiki, E.; Hadjipavlou-Litina, D.; Villar, R.;

Vicente, E.; Solano, B.; Ancizu, S.; Perez-Silanes, S.; Aldana, I.;

Monge, A. Synthesis and anti-inflammatory/antioxidant activities of

some new ring substituted 3-phenyl-1-(1,4-di-N-oxide quinoxalin-2-

yl)-2-propen-1-one derivatives and of their 4,5-dihydro-(1H)-pyrazole

analogues. Bioorg. Med. Chem. Lett. 2007, 17, 6439−6443.

(38) Naylor, M. A.; Stephen, M. A.; Nolan, J.; Sutton, B.; Tochcer, J.

H.; Fielden, E. M.; Adams, G. E.; Strafford, I. J. Heterocyclic mono-N-

oxides with potential applications as bioreductive anti-tumour drugs:

Part 1. 8-Alkylamino-substituted phenylimidazo [1, 2-a] quinoxalines.

Anticancer Drug Res. 1993, 8, 439−461.

(39) Gil, M. V.; Arevalo, M. J.; Lopez, O. Click Chemistry what ’s in

a Name? Triazole Synthesis and Beyond. Synthesis 2007, 2007, 1589.

(40) Binder, W. H.; Kluger, C. Azide/Alkyne “Click ” Reactions:

Applications in Material Science and Organic Synthesis. Curr. Org.

Chem. 2006, 10, 1791−1815.

(41) Lutz, J. F. 1, 3-Dipolar Cycloadditions of Azides and Alkynes: A

Universal Ligation Tool in Polymer and Material Science. Angew.

Chem., Int. Ed. 2007, 46, 1018−1025.

(42) Binder, W. H.; Sachsenhofer, R. ‘Click ’ Chemistry in Polymer

and Materials Science. Macromol. Rapid Commun. 2007, 28, 15−54.

(43) Hawker, C. J.; Wooley, K. L. The convergence of synthetic

organic and polymer chemistries. Science 2005, 309, 1200−1205.

(44) Read, E. S.; Armes, S. P. Recent advances in shell cross-linked

micelles, Recent advances in shell cross-linked micelles. Chem.

Commun. 2007, 3021−3035.

(45) Singhal, N.; Sharma, P. K.; Dudhe, R.; Kumar, N. Recent

advancement of triazole derivatives and their biological significance. J.

Chem. Pharm. Res. 2011, 3, 120−126.

(46) Amr, A. E.; Sabry, N. M.; Abdulla, M. M. Synthesis, Reactions,

and Anti-inflammatory Activity of Heterocyclic Systems Fused to a

Thiophene Moiety Using Citrazinic Acid As Synthon. Monatsh. Chem.

2007, 138, 699−707.

(26) Adhireksan, Z.; Davey, G. E.; Campomanes, P.; Groessl, M.;

Clavel, C. M.; Yu, H.; Nazarov, A. A.; Yeo, C. H.; Ang, W. H.; Droge,

P.; Rothlisberger, U.; Dyson, P. J.; Davey, C. A. Combination of

ruthenium(II)-arene complex [Ru(η⁶-p -cymene)Cl₂(pta)] (RAPTA-

C) and the epidermal growth factor receptor inhibitor erlotinib results

in efficient angiostatic and antitumor activity. Nat. Commun. 2014, 5,

3462.

́

(27) Debreczeni, J. E.; Bullock, A. N.; Atilla, G. E.; Williams, D. S.;

Bregman, H.; Knapp, S.; Meggers, E. Ruthenium half-sandwich

complexes bound to protein kinase Pim-1. Angew. Chem., Int. Ed.

2006, 45, 1580−1585.

V

https://dx.doi.org/10.1021/acs.inorgchem.0c02928

Inorg. Chem. XXXX, XXX, XXX−XXX

Inorganic Chemistry

pubs.acs.org/IC

Article

(47) Fujiwara, N.; Nakajima, T.; Ueda, Y.; Fujita, H.; Kawakami, H.

Novel piperidinylpyrimidine derivatives as inhibitors of HIV-1 LTR

activation. Bioorg. Med. Chem. 2008, 16, 9804−9816.

(63) Morris, G. M.; Huey, R.; Lindstrom, W.; Sanner, M. F.; Belew,

R. K.; Goodsell, D. S.; Olson, A. J. AutoDock4 and AutoDockTools4:

Automated docking with selective receptor flexibility. J. Comput.

Chem. 2009, 30 (16), 2785−2791.

(64) Liu, P.; Wu, B.; Liu, J.; Dai, Y.; Wang, Y.; Wang, K. DNA

Binding and Photocleavage Properties, Cellular Uptake and Local-

ization, and in-Vitro Cytotoxicity of Dinuclear Ruthenium(II)

Complexes with Varying Lengths in Bridging Alkyl Linkers. Inorg.

Chem. 2016, 55, 1412−1422.

(48) Thirumurugan, P.; Matosiuk, D.; Jozwiak, K. Click Chemistry

for Drug Development and Diverse Chemical −Biology Applications.

Chem. Rev. 2013, 113, 4905−4979.

(49) Maiti, S.; Roy, N.; Babu, L. T.; Moharana, P.; Athira, C. C.;

Darsana Sreedhar, E.; De, S.; Ashok Kumar, S. K.; Paira, P. Cu(ii),

Ir(i) and CuOnanocatalyzed mild synthesis of luminescent sym-

metrical and unsymmetrical bis(triazolylmethyl)quinoxalines: bio-

compatibility, cytotoxicity, live cell imaging and biomolecular

interaction. New J. Chem. 2020, 44, 920−931.

(50) Chen, Y.; Qiao, L.; Ji, L.; Chao, H. Phosphorescent iridium(III)

complexes as multicolor probes for specific mitochondrial imaging

and tracking. Biomaterials 2014, 35, 2−13.

(65) (a) Liu, Z.-C.; Wang, B.-D.; Yang, Z.-Y.; Li, Y.; Qin, D.-D.; Li,

T.-R. Synthesis, crystal structure, DNA interaction and antioxidant

activities of two novel water-soluble Cu²⁺complexes derivated from 2-

oxo-quinoline-3-carbaldehyde Schiff-bases. Eur. J. Med. Chem. 2009,

44, 4477−4484. (b) Herebian, D.; Sheldrick, W. S. Synthesis and

DNA binding properties of bioorganometallic (η⁵-

pentamethylcyclopentadienyl)iridium(iii) complexes of the type

[(η⁵-C₅Me₅)Ir(Aa)(dppz)]ⁿ⁺(dppz = dipyrido[3,2-a :2 ′,3 ′-c ]-

phenazine, n = 1 −3), with S-coordinated amino acids (Aa) or

peptides. J. Chem. Soc., Dalton Trans. 2002, 966−974. (c) Grover, N.;

Welch, T. W.; Fairley, T. A.; Cory, M.; Thorp, H. H. Oxidation of

DNA by trans-dioxoruthenium(VI) complexes: self-inhibition of

DNA cleavage by metal complexes. Inorg. Chem. 1994, 33, 3544−

3548. (d) Sarkar, B.; Mondal, A.; Madaan, Y.; Roy, N.; Moorthy, A.;

Kuo, Y.−C.; Paira, P. Luminescent anticancer ruthenium(ii)-p-

cymene complexes of extended imidazophenanthroline ligands:

synthesis, structure, reactivity, biomolecular interactions and live cell

imaging. Dalton Transaction 2019, 48, 12257−12271. (e) P.K., A.;

Paira, P. Luminescent anticancer Ru(II)-arenebipyridine and

phenanthroline complexes: Synthesis, characterization, DFT studies,

biological interactions and cellular imaging application. J. Inorg.

Biochem. 2020, 208, 111099. (f) Mondal, A.; Paira, P. Hypoxia

efficient and glutathione-resistant cytoselective ruthenium (II)-p-

cymene-arylimidazophenanthroline complexes: biomolecular interac-

tion and live cell imaging. Dalton Transactions 2020, 49, 12865−

12878. (g) Khan, T. A.; Bhar, K.; Thirumoorthi, R.; Roy, T. K.;

Sharma, A. K. Design, synthesis, characterization and evaluation of the

anticancer activity of water-soluble halfsandwichruthenium(II)

arenehalido complexes. New J. Chem. 2020, 44, 239.

(66) (a) Zhen, Q. X.; Ye, B. H.; Zhang, Q. L.; Liu, J. G.; Li, H.; Ji, L.

N.; Wang, L. Synthesis, characterization and the effect of ligand

planarity of [Ru(bpy)₂L]²⁺on DNA binding affinity. J. Inorg. Biochem.

1999, 76, 47−53. (b) Pyle, A. M.; Rehmann, J. P.; Meshoyrer, R.;

Kumar, C. V.; Turro, N. J.; Barton, J. K. Mixed-ligand complexes of

ruthenium(II): factors governing binding to DNA. J. Am. Chem. Soc.

1989, 111, 3051−3058.

(67) (a) Chen, H.; Parkinson, J. A.; Morris, R. E.; Sadler, P. J. Highly

Selective Binding of Organometallic Ruthenium Ethylenediamine

Complexes to Nucleic Acids: Novel Recognition Mechanims. J. Am.

Chem. Soc. 2003, 125, 173−186. (b) Dimiza, F.; Perdih, F.; Tangoulis,

V.; Turel, I.; Kessissoglou, D. P.; Psomas, G. Interaction of copper(II)

with the non-steroidal anti-inflammatory drugs naproxen and

diclofenac: synthesis, structure, DNA- and albumin-binding. J. Inorg.

Biochem. 2011, 105, 476−489.

(51) Jin, C.; Liu, J.; Chen, Y.; Zeng, L.; Guan, R.; Ouyang, C.; Ji, L.;

Chao, H. Cyclometalated Iridium(III) Complexes as Two-Photon

Phosphorescent Probes for Specific Mitochondrial Dynamics

Tracking in Living Cells. Chem. - Eur. J. 2015, 21, 12000−12010.

̈

(52) Kubanik, M.; Holtkamp, H.; Sohnel, T.; Jamieson, S. M. F.;

Hartinger, C. G. Impact of the Halogen Substitution Pattern on the

Biological Activity of Organoruthenium 8-Hydroxyquinoline Anti-

cancer Agents. Organometallics 2015, 34, 5658−5668.

(53) Gilewska, A.; Barszcz, B.; Masternak, J.; Kazimierczuk, K.;

Sitkowski, J.; Wietrzyk, J.; Turlej, E. Similarities and differences in d⁶

low-spin ruthenium, rhodium and iridium half-sandwich complexes:

synthesis, structure, cytotoxicity and interaction with biological targets

JBIC. JBIC, J. Biol. Inorg. Chem. 2019, 24, 591−606.

(54) Aman, F.; Hanif, M.; Siddiqui, W. A.; Ashraf, A.; Filak, L. K.;

̈

Reynisson, J.; Sohnel, T.; Jamieson, S. M.; Hartinger, C. G. Anticancer

Ruthenium(η⁶-p -cymene) Complexes of Nonsteroidal Anti-inflamma-

tory Drug Derivatives. Organometallics 2014, 33, 5546−5553.

(55) (a) Geary, W. J. The use of conductivity measurements in

organic solvents for the characterization of coordination compounds.

Coord. Chem. Rev. 1971, 7, 81−122. (b) Ali, I.; Wani, W. A.; Saleem,

K. Empirical Formulae to Molecular Structures of Metal Complexes

by Molar Conductance, Synthesis and Reactivity in Inorganic. Synth.

React. Inorg., Met.-Org., Nano-Met. Chem. 2013, 43, 1162−1170.

(c) Banerjee, S.; Pant, I.; Khan, I.; Prasad, P.; Hussain, A.; Kondaiah,

P.; Chakravarty, A. R. Remarkable enhancement in photocytotoxicity

and hydrolytic stability of curcumin on binding to an oxovanadium-

(IV) moiety. Dalton Trans. 2015, 44, 4108−4122.

́

(56) (a) Díez, J.; Gimeno, J.; Lledos, A.; Suarez, F. J.; Vicent, C.

Imidazole Based Ruthenium(IV) Complexes as Highly Efficient

Bifunctional Catalysts for the Redox Isomerization of Allylic Alcohols

in Aqueous Medium: Water as Cooperating Ligand. ACS Catal. 2012,

́

2, 2087−2099. (b) Romero-Canelon, I.; Sadler, P. J. Next-Generation

Metal Anticancer Complexes: Multitargeting via Redox Modulation.

Inorg. Chem. 2013, 52, 12276−12291.

(57) Townsend, D. M.; Tew, K. D. The role of glutathione-S-

transferase in anti-cancer drug resistance. Oncogene 2003, 22, 7369−

7375.

(58) Campling, B. G.; Baer, K.; Baker, H. M.; Lam, Y. M.; Cole, S. P.

Do glutathione and related enzymes play a role in drug resistance in

small cell lung cancer cell lines? Br. J. Cancer 1993, 68, 327−335.

(68) Van Niekerk, A.; Chellan, P.; Mapolie, S. F. Heterometallic

Multinuclear Complexes as Anti-Cancer Agents-An Overview of

Recent Developments. Eur. J. Inorg. Chem. 2019, 2019, 3432−3455.

(69) Sen, U.; Shenoy P, S.; Bose, B. Opposing effects of low versus

high concentrations of water soluble vitamins/dietary ingredients

Vitamin C and niacin on colon cancer stem cells (CSCs). Cell Biol.

Int. 2017, 41, 1127−1145.

̌

̈

(59) Sersen, S.; Kljun, J.; Kryeziu, K.; Panchuk, R.; Alte, B.; Korner,

W.; Heffeter, P.; Berger, W.; Turel, I. Structure-Related Mode-of-

Action Differences of Anticancer Organoruthenium Complexes with

β -Diketones. J. Med. Chem. 2015, 58, 3984−3996.

(60) Gamcsik, M. P.; Kasibhatla, M. S.; Teeter, S. D.; Colvin, O. M.

Glutathione levels in human tumors. Biomarkers 2012, 17, 671−691.

(61) Saravanan, N.; Gandhi, M.; Senthil Kumar, A. High-valent

ruthenium(IV)-oxo complex stabilized mesoporous carbon (graphi-

tized)/nafion modified electrocatalyst for methanol oxidation reaction

in neutral pH. J. Electroanal. Chem. 2020, 874, 114457.

(62) Frisch, M. J.; Trucks, G. W.; Schlegel, H. B.; Scuseria, G. E.;

Robb, M. A.; Cheeseman, J. R.; Farkas, A. D.; Foresman, J. B.; Ortiz, J.

V.; Cioslowski, J.; Fox, D. J. Gaussian 09W; Gaussian, Inc.,

Wallingford, CT, 2009.

W

https://dx.doi.org/10.1021/acs.inorgchem.0c02928

Inorg. Chem. XXXX, XXX, XXX−XXX

Article Doi

Mitochondria-Targeting Click-Derived Pyridinyltriazolylmethylquinoxaline-Based Y-Shaped Binuclear Luminescent Ruthenium(II) and Iridium(III) Complexes as Cancer Theranostic Agents

DOI: 10.1021/acs.inorgchem.0c02928

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Full text of DOI:10.1021/acs.inorgchem.0c02928

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