DNA targeting half sandwich Ru(ii)-: P-cymene-N^N complexes as cancer cell imaging and terminating agents: Influence of regioisomers in cytotoxicity

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DNA targeting half sandwich Ru(II)-p-cymene-N^N

complexes as cancer cell imaging and terminating

agents: inﬂuence of regioisomers in cytotoxicity†

Cite this: DOI: 10.1039/d0dt03107k

a

b

a

Ashaparna Mondal,‡ Utsav Sen,‡ Nilmadhab Roy, Venkatesan Muthukumar,

c

b

a

Suban Kumar Sahoo,

Bipasha Bose* and Priyankar Paira

*

For diagnosing and annihilating cancer in the human body, herein, we have adopted a one pot con-

venient synthetic protocol to synthesize a library of half sandwich Ru(II)-p-cymene-N^N complexes

under continuous sonication and isolated their regioisomers by preparative thin layer chromatography fol-

lowed by justiﬁcation of stability using DFT. The present work has resulted in a library of ruthenium arene

complexes and their isolated regioisomers following environmentally benign green processes and their

6

screening of anticancer activity in terms of cytotoxicity and selectivity against cancer cell lines where [(η -

p-cymene)RuCl{2-(5,6-dichloro-1H-benzo[d]imidazole-2-yl)quinolone}] (11j) has been elicited to be sig-

niﬁcantly more potent as well as selective in Caco-2 and HeLa cell lines than the normal HEK-293 cell

line compared to cisplatin and it has even shown marked cytotoxicity against the more aggressive HT-29

colorectal cancer cell line being capable of producing oxidative stress or arresting the cell cycle.

Moreover, these types of Ru(II)–arene complexes exhibited excellent binding eﬃcacy with DNA and the

6

Received 5th September 2020,

Accepted 5th December 2020

compounds

[(η -p-cymene)RuCl{5-chloro-2-(6-(4-chlorophenyl)pyridin-2-yl)benzo[d]thiazole}]PF

6

(

8l4), [(η -p-cymene)Ru-2-(6-(benzofuran-2-yl)pyridin-2-yl)-5-chlorobenzo[d]thiazole (8l9) and [(η -p-

cymene)RuCl{2-(6-nitro-1H-benzo[d]imidazol-2-yl)quinolone}]Cl (11f’) and might be applied for cancer

theranostic treatment due to their good ﬂuorescence properties and remarkable potency.

DOI: 10.1039/d0dt03107k

rsc.li/dalton

7

–9

platin were found to be potent anticancer agents.

These

Introduction

complexes were eﬃcacious against a majority of cancers, but

Metal drugs have been employed over centuries to palliate several drawbacks such as poor aqueous solubility, low selecti-

1

–5

diﬀerent kinds of fatal diseases especially cancer. However, vity, drug resistance and high levels of in vivo toxicity (neuro-

the boundary line between toxic and therapeutic doses caused toxicity, nephrotoxicity, thrombocytopenia, myelosuppression,

1

0–12

the scientists to deeply research drug discovery. As a conse- neutropenia and neuropathy) have limited their use.

The

quence, the door to the modern area of research on metal challenge ahead was, therefore, to develop new and more

drugs was opened when Michele Peyrone discovered cisplatin eﬀective metal-based anticancer agents. Nowadays, ruthenium

in 1844 and its anticancer activity came into the limelight in based complexes have attracted interest as potential cytotoxic

1

965 when Rosenburg reported its eﬃcacy for the treatment of agents against a number of cancer cell lines and are regarded

various cancers such as lung, head, neck, esophageal, testicu- as promising candidates as alternatives to cisplatin and its

6

13

lar and ovarian cancer. Alongside cisplatin, many other plati- derivatives. This is because of their variable oxidation states,

num(II) based drugs such as carboplatin, oxaliplatin and neda- faster rate of ligand exchange and good bioavailability, low

general toxicity and highly selective cytotoxicity towards prolif-

erative cells, activity against some cisplatin resistant cell lines,

imitating the binding nature of iron to some biological mole-

a

Department of Chemistry, School of advanced sciences, Vellore Institute of

Technology Vellore, 632014 Tamilnadu, India. E-mail: priyankar.paira@vit.ac.in

b

cules, water solubility and octahedral geometry and their elec-

Department Stem Cells and Regenerative Medicine Centre, Institution Yenepoya

Research Centre, Yenepoya University, University Road, Derlakatte, Mangalore

75018, Karnataka, India. E-mail: bipasha.bose@gmail.com

1

4–19

tronic and steric properties.

Some of the Ru-based com-

5

plexes such as [imiH]trans-[Ru(N-imi)(S-DMSO)Cl ] (NAMI-A),

4

c

Department of Applied Chemistry, S.V. National Institute of Technology (SVNIT),

[

indH]trans-[Ru(Nind)

2

Cl

4

]

(KP1019) and [Na]trans-[Ru(N-

Ichchanath, Surat, Gujrat-395 007, India. E-mail: suban_sahoo@rediﬀmail.com

20–22

ind) Cl (IT-139) have reached clinical trial stage.

2

4

]

†

Electronic supplementary information (ESI) available. CCDC 1991248. For ESI

However, NAMI-A and KP1019 have not been used for phase II

clinical studies due to low therapeutic index and poor solubi-

and crystallographic data in CIF or other electronic format see DOI: 10.1039/

D0DT03107K

2

3

‡

Equal contribution.

lity, respectively. The antitumor properties of the kinetically

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and thermodynamically unstable Ru(III) complexes are revealed study of a few ruthenium(II) arene complexes of the 2-aryldia-

when they are reduced to their corresponding stable Ru(II) zole derivative to be eﬀective on A2780, A2780 cis, and MCF-7

6

8

counterparts in vivo under biological circumstances (hypoxia, cell lines. Stefan Nikolić et al. reported p-cymene ruthenium

2

4–26

acidic pH (6.7) and high levels of glutathione).

Of late, chlorido complexes of 7-chloroquinoline that showed cytose-

6

9

various organometallic Ru(II), inorganic Ru(II) and nano- lective toxicity against the K562 neoplastic cell line. The iso-

material Ru(II) complexes have been recognised as anticancer meric construction of Ru(II)–arene complexes can be achieved

2

7–31

70

agents, with powerful therapeutic properties.

Some Ru(II) by altering the position of substituents in N^N ligands.

complexes bearing 1,10-phenanthroline and 2,2′-bipyridine Therefore, the isolation of a series of regioisomers, further

3

2–34

have also been employed as potent anticancer agents.

A

extension of π-conjugation and enhancement of cytotoxic ele-

new remarkable Ru(II) therapeutic TLD1433 entered phase I gance with the introduction of various aromatic moieties by

and phase IIa clinical trials for the treatment of nonmuscle replacing the –Br group through the Suzuki coupling reaction

3

5

invasive bladder cancer with photodynamic therapy (PDT).

and after that the selection of the best one among them in

Ruthenium arene complexes identified as piano-stool or half- terms of potency by dint of biological evaluation was a main

sandwich complexes have been recognised as promising anti- point of interest for establishing some novel Ru(II)–arene N^N

cancer drugs towards a wide spectrum of cancer types (breast, complexes as distinct anticancer agents which have not been

2

7,36–40

colon, lung, and pancreatic cancer cells).

reported. Convinced by the aforementioned objective and con-

The design of Ru(II)–arene complexes can achieve amphi- tinuous interest in organoruthenium compounds, we have

6

philic properties imparted by the hydrophilic metal centre and designed and synthesized a series of η -p-cymene Ru(II) com-

1

5,41,42

the hydrophobic arene moiety.

Therefore, in the design plexes of 2-(6-bromopyridinyl), 2-(6-arylpyridinyl) and 2-quino-

of Ru(II)–arene anticancer drugs, the potency of the drug is linyl substituted benzimidazole (BIZ), benzothiazole (BTZ) and

governed by the nature of the arene part, the labile halide benzoxazole (BOZ) scaﬀolds which can be more cytoselective

4

3–48

group and the bidentate chelating ligand.

The potency, than previously reported platinum or ruthenium drugs against

7

1

target specificity and bioimaging properties of such complexes cancer cells (Fig. 2). These molecules are classically gifted

can be altered by varying the nature of the bidentate chelating with four essential elements in the coordination sphere of the

ligand. In this framework, derivatives of benzimidazole, ben- Ru(II) centre: (i) an η -arene moiety, which stabilizes the oxi-

6

zothiazole and benzoxazole moieties possess a wide spectrum dation state of the metal cation to facilitate the cellular mem-

of pharmacological activities including antifungal, antiplas- brane transportation, (ii) a leaving group (X), which endures

moidal, antiviral, antiinflammatory, antiproliferative, antitu- easy dissociation to accelerate the coordination of the metal

mor, and antimicrobial activity, and are used as in vivo ion with target biomolecules through covalent interaction, (iii)

4

9–57

2

imaging agents (Fig. 1).

In addition, quinoline has also an ancillary bidentate ligand (κ -N^N), which may control the

been explored as antiinflammatory, antimicrobial and antitu- reactivity toward diﬀerent biomolecules (DNA, enzymes)

5

8–61

mor agents during the 20th century.

A large number of through noncovalent interaction, (iv) the overall charge and

quinoline derivatives, therefore, exhibited a broad spectrum of counter ion identity, which can aﬀect the solubility and per-

anticancer activity including potency against breast, prostate, meability, and (v) substitution in the N^N ligand for maintain-

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2–65

liver and colon cancer.

Moreover, a few drugs such as the ing the lipophilicity and construction of the regioisomer to

camptothecin containing quinoline moiety have been used identify the more potent isomer.

6

clinically for the treatment of cancer (Fig. 1).

Gumus et al. reported a platinum complex of 2-phenylben- been considered here as the safest solvent with several advan-

zimidazole that was potent against human RD tages such as easy availability, nontoxicity, less energy con-

To circumvent the harsh reaction conditions, water has

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7

72

Rhabdomyosarcoma cell line. Marta Martínez-Alonso et al. sumption, ease of isolation, etc. With an attempt to reduce

reported the synthesis and structure activity relationship (SAR) the negative impact on the environment created by organo-

metallic synthetic processes, we were specifically interested in

6

Fig. 1 Anticancer drugs containing benzimidazole, benzothiazole, and

quinoline moieties.

Fig. 2 Design of Ru(II)-η -p-cymene benzimidazole (BIZ), benzothia-

zole (BTZ) and benzoxazole (BOZ) scaﬀolds.

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using water as a green solvent and accomplished the reaction

by employing a sonication technique for the synthesis of the

designed complexes.

Results and discussion

Synthesis and preliminary characterization

Initially, we have introduced a convenient one-pot protocol for

6

the synthesis of (η -p-cymene)Ru(II)-2-(6-bromopyridinyl)benzi-

6

midazole (BIZ) and (η -p-cymene)Ru(II)-2-(6-bromopyridinyl)

benzothiazole (BTZ) (Scheme 1). The reactions were carried

out using water as the solvent under sonication without using

any catalyst, a procedure which was completely free from the

conventional use of hazardous chemicals. At the outset, o-phe-

nylene diamine (1a) was treated with 6-bromopyridine-2-car-

boxaldehyde (2) under sonication at 60 °C for 1 h to synthesize

2

-(6-bromopyridin-2-yl)-1H-benzo[d]imidazole ligand (3a).

Complete conversion of ligand 3a was confirmed by scrutiniz-

ing the TLC results (thin layer chromatography) (3 : 1 hexane/

6

ethyl acetate). Soon after this, 0.5 equivalents of [RuCl(μ-Cl)(η -

2

p-cymene)] (4) was added in the same reaction mixture and

was sonicated continuously for another 15 min. Formation of

the new complex was confirmed by TLC (100% ethyl acetate).

At the end, solvent water was evaporated using a rotary evapor-

6

ator and the crude product of (η -p-cymene)ruthenium(II)-2-(6-

bromopyridin-2-yl)-benzimidazole complex (5a) was collected.

To remove possible impurities, the collected crude product

was subjected to repeated washing with ether followed by

recrystallization from a ethyl acetate/hexane mixture (1 : 1). Fig. 3 Synthesized (η -p-cymene)ruthenium(II)-2-(6-bromopyridinyl)

The structure of the complex 5a was confirmed by nuclear

6

BIZ, BTZ complexes.

magnetic resonance (NMR), Fourier transform infrared (FT-IR)

and electro spray ionization mass spectrometry (ESI-MS) tech-

niques. After confirming the structure of 5a, we have extended

our scope by synthesizing other complexes (5b–5h) following

the same protocol (Scheme 1 and Fig. 3).

In the next step, we selected 2-(6-bromopyridinyl)benzothia-

zole (BTZ) scaﬀolds (3g–h) for achieving further extension in

π-conjugation by substituting the bromo group with an aro-

matic moiety via the Suzuki coupling reaction using aryl

boronic acids (Scheme 2). These newly synthesized ligands [7

(

g1–g3; l1–l9)] then underwent complexation with 0.5 equiva-

6

lents of [RuCl(μ-Cl)(η -p-cymene)]

2

(4) to produce another

series of ruthenium complexes [8(g1–g3; l1–l9)] (Scheme 2 and

Fig. 4).

6

Scheme 1 One pot synthesis of (η -p-cymene)ruthenium(II)-2-(6-bro-

Scheme 2 Synthesis of (η -p-cymene)ruthenium(II)-2-(6-phenylpyridi-

mopyridinyl) BIZ and BTZ complexes.

nyl) BTZ complexes.

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6

Fig. 4 Synthesized

(η -p-cymene)ruthenium(II)-2-(6-bromopyridinyl)

BIZ, BTZ complexes. Following the same protocol as Scheme 1 another

series of ruthenium(II)arene-2-quinolinyl benzimidazole complexes

(

11a–b, 11d–f, 11h–n) were also prepared using quinoline-2-carboxal-

dehyde instead of 6-bromopyridine-2-carboxaldehyde (Scheme 3 and

Fig. 5).

6

Fig. 5 Synthesized (η -p-cymene)ruthenium(II)-2-quinolinyl BIZ, BTZ

and BOZ complexes.

nance peaks for arene protons of the complexes 5a and 5g

were shifted downfield and there was a change in splitting

pattern as the C₂_vsymmetry of the complex was interrupted

upon ligand coordination. The aromatic protons of the

cymene ring in the ruthenium(II)-6-bromopyridinyl benzimida-

zole complex (5a) displayed four distinct doublets at δ

1

6

.05–6.45 ppm in the H spectrum due to its diastereotopic

6

nature. The protons of one methyl group of p-cymene gave one

singlet peak at δ 2.24 ppm. The other six methyl protons of the

isopropyl group in the p-cymene ring were observed in the

range of δ 0.82–0.88 ppm. The characteristic peaks of the CH

Scheme 3 One pot synthesis of (η -p-cymene)ruthenium(II)-2-quinoli-

nyl BIZ, BTZ and BOZ complexes.

To know the reaction pattern, a few ligands (3a, 3g, 10a, proton in the p-cymene ring were detected in the range of δ

0h) were isolated before adding the ruthenium precursor and 2.79–2.88 ppm as multiplets. The characteristic seven aromatic

were characterized by H NMR. In the H NMR spectra of protons of the ligand were found in a more deshielded region

-bromopyridinyl benzimidazole ligand (3a), characteristic (δ 7.55–8.57 ppm) compared to the free ligand (δ

1

6

peaks of aromatic protons were observed in the downfield 7.30–8.38 ppm). The complex 5a exhibited a characteristic

+

region of the spectrum (δ 7.30–8.38 ppm). The most molecular ion peak with m/z 543.3 [M] . In the case of the

deshielded –NH proton of the benzimidazole ring of com- ruthenium(II)-6-bromopyridinyl benzothiazole complex (5g),

pound 3a exhibited a broad singlet peak at δ 10.56 ppm. Six similar changes were noticed in which characteristic four aro-

aromatic protons of 6-bromopyridinyl benzothiazole (3g) matic protons present in the p-cymene ring exhibited two dis-

exhibited characteristic peaks in the same aromatic region (δ tinct doublets at δ 5.33 and 5.46 ppm. The peaks for six aro-

7

.36–8.27 ppm). The spectral pattern of the ligand changed matic protons of the 6-bromopyridinyl benzothiazole ligand

significantly after complex formation compared to the spec- were observed at δ 7.42–8.33 ppm. The complex exhibited a

+

trum of the corresponding ruthenium dimer where the reso- molecular ion peak at m/z 560.9 [M] with a characteristic

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Fig. 6 ORTEP diagram of ligand 7l1 with (a) thermal ellipsoids are

drawn at the 50% probability level and (b) crystal packing.

ruthenium isotopic pattern which confirmed the formation of

1

5

g. Significant spectral changes were observed when the

H

Fig. 7 Schematic diagram for the preparation of ruthenium(II) benzimi-

NMR spectra of 2-quinolinyl ligands (10a and 10h) and their dazole (BIZ), benzothiazole (BTZ) and benzoxazole (BOZ) scaﬀolds.

respective ruthenium(II) complexes (11a and 11h) were com-

pared. To establish the generality and scope of this particular

synthetic approach, various Ru(II)-p-cymene quinolinyl

scaﬀolds (11a–b, 11d–f, 11h–n) were synthesized and charac-

terized spectroscopically and analytically with significant

yields (see the ESI†).

Likewise, isolated 2-(6-arylpyridinyl) ligands [7(g1–g3; l1–l9)]

were characterized by NMR and mass spectroscopy. The struc-

ture of ligand 7l1 was further confirmed by single crystal XRD

spectra, the aromatic protons of the p-cymene ring of the

complex 11d showed two doublets at δ 5.57 and 5.67 and one

broad singlet at δ 5.73 while the other isomer 11d′ showed two

doublets at δ 5.61 and δ 5.66 and one multiplet at δ 5.72–5.76.

The methyl protons in the p-cymene ring of complex 11d

exhibited two doublets at δ 0.71 and 0.74 ppm whereas com-

pound 11d′ displayed the cymene methyl peak at δ 0.74 as a

doublet (Fig. S2†). The isolated yield, detailed spectral data

and pictures for all the isomers have been described in the

Experimental section and the ESI†. The plausible mechanism

for the formation of these two isomers as well as their yield

variation has been elucidated below. In general imidazole

exists in two isomeric forms (L and L ) in the medium. When

(Fig. 6). Ligand 7l1 crystallizes in the monoclinic space group,

P21/n (see the ESI, CCDC 1991248, Table S2–S8†). Each unit cell

contains four complexes. Atomic coordinates, bond lengths,

anisotropic displacement parameters, hydrogen coordinates,

torsion angles and hydrogen bonds are presented in Table S2

(see the ESI†). Their corresponding ruthenium complexes

A

B

[8(g1–g3; l1–l9)] were also characterized by NMR and mass spec-

the Ru(II)-p-cymene dimer was added to the reaction medium

troscopy. The diﬀerence in the NMR splitting pattern and

δ values in complexes [8(g1–g3; l1–l9)] compared to ligands

both the isomers were intended to react with it. Subsequently,

1

80° free rotation of the aryl group in isomer L_Bchanges its

conformation to bind with ruthenium. Thus two isomers of

ruthenium complexes (RuL and RuL ) have been isolated in

the case of mono substituted (3b, 3d–f, 3k–l) and unsymmetri-

[7(g1–g3; l1–l9)] evidently confirmed the complex formation.

A

B

Proposed mechanism for the formation of the regioisomer

The formation of ligands might involve intermolecular con- cal 4,5 di-substituted benzimidazole ligands (3c)(Fig. 8). On

densation, intramolecular condensation and subsequent aerial the other hand, unsubstituted (3a, 10a) and symmetrical 4,5

oxidation influenced by water molecules which facilitate the di-substituted benzimidazole ligands (10i, 10j) remained

rate of nucleophilic substitution as well as cyclization (Fig. 7). intact as only one Ru(II) isomer (5a, 11a, 11i, 11j). Isomer RuL

When mono substituted (1b, 1d–f, 1k–l) or unsymmetrical di- containing electron withdrawing groups (–Cl, –F, –CF , –NO

substituted-1,2-diamino benzene (1c) was subjected to react at 5-position of the benzimidazole ring cannot pull the

with the aldehydes followed by complexation with the ruthe- π-electrons so easily to their vacant orbitals as the p orbital of

nium(II)–arene dimer, two regioisomers with slight R

variation the ring carbon atom and respective vacant orbitals of electron

A

3

2

)

z

f

were formed (Fig. 3 and 5) due to the free rotation of the aryl withdrawing groups become non-parallel to each other owing

group through the C–C single bond. We separated those two to strong steric crowding between methyl groups of p-cymene

regioisomers by the preparative thin layer chromatographic and the concerned electron withdrawing group which is why

method. The compounds were eluted in 100% ethyl acetate electron density on the donor nitrogen atom of the benzimida-

and then separated (Fig. S1†). The crude products were recrys- zole ring remains more reserved for better co-ordination with

tallized from ethyl acetate in hexane (1 : 1). Those separated ruthenium than in RuL

B

. For this reason, the extent of positive

isomers were characterized by H NMR which revealed that character of ruthenium is more diminished in RuL compared

both the isolated compounds gave almost the same splitting to RuL giving rise to slightly more polarity in RuL than in

pattern and had the same number of protons with very slight RuL . Also, the simultaneous existence of one –Cl and one –F

diﬀerence in the δ values (see the ESI†). As estimated from the group in the benzimidazole ring follows the same trend

1

A

B

A

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Fig. 9 DFT computed structure of the complex 5d and its regioisomer

5d’.

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

of the arene has shown a similar bond length (2.237 Å–

Fig. 8 Mechanistic pathway for the formation of two Ru(II)

regioisomers.

2

.312 Å) with an average distance of 2.271 Å. The N1 and N2

atoms coordinated to the Ru centre with the bond distance

.215 Å (Ru–N1) and 2.087 Å (Ru–N2), where the benzimida-

zole–N is coordinated more strongly than the pyridine–N. The

2

depending on the position of the –F group. The isomers pos- bond length of 2.393 Å was obtained for Ru–Cl. The bond para-

sessing an electron withdrawing group at the 5-position meters discussed for the first complex are almost the same as

A f

(isomer RuL ) which acquired higher R were formed in greater those for the other six complexes and also comparable with

yield than their corresponding polar regioisomers (isomer those for the reported similar Ru(II) arene complexes. As tabu-

RuL ) due to the ardent aﬃnity towards the formation of RuL_Alated in Table S1,† most of the complexes preferred the first

B

−

1

−1

having an electron dense N-atom for stronger coordination isomer (5d: −0.62 kcal mol ; 5f: −1.19 kcal mol ; 11b:

with ruthenium metal. However, the substitution of the elec- −0.11 kcal mol ; 11d: −0.55 kcal mol ; 11f: −1.07 kcal

tron donating group (–OMe) will reverse the polarity of mol ; 11l: −0.14 kcal mol ) over the second regioisomer (5d′,

isomers which was substituted with electron withdrawing 5f′, 11b′, 11d′, 11f′, 11l′), except the complex 11e′ (1.27 kcal

groups and produced a similar trend of yield. The stability of mol ) that energetically preferred the second regioisomer.

−

1

−1

−

1

−1

−

1

both the regioisomers was further justified by the DFT study.

The calculated bond distance (Ru–N2) obtained from DFT

reinforces the former explanation regarding the polarity and

ease of formation of stable isomers. It is noteworthy to

DFT analysis

The structure of the seven half-sandwich organometallic com- mention that quinolinyl analogues were obtained in a greater

6

plexes of the type [Ru(η -arene)(L)Cl]Cl and their respective yield than pyridinyl analogues which can be well justified by

regioisomers was optimized by using density functional theory the calculated bond distance (Ru–N1) in DFT analysis. These

(

DFT) in the gas phase. All calculations were performed by results coincide with the nature of spots found in TLC as well

using the exchange–correlation functional B3LYP and the as the isolated yields of the isomers. However, the relative

basis sets LANL2DZ for the Ru atom and 6-31G** for the energy diﬀerence between two regioisomers of the complexes

remaining atoms coded in the computational program is much less and hence both the structures can exist. The

Gaussian 09 W. The optimized structure of the complexes 5d, energy gap between the HOMO–LUMO of the complexes was

5

f, 11b, 11d, 11e, 11f, and 11l and their corresponding regio- analysed because of the direct relationship with the chemical

isomers 5d′, 5f′, 11b′, 11d′, 11e′, 11f′, and 11l′ is shown in reactivity and kinetic stability: the lower HOMO–LUMO gap is a

Fig. 9, Fig. S3 and S4.† The calculated structure has the suitable condition for increased reactivity and decreased kinetic

pseudo-octahedral coordination geometry, where the Ru(II) stability whereas the reverse is true for a higher energy gap.

atom is coordinated to all six carbon atoms of the p-cymene

ring, the two nitrogen atoms of the bidentate benzimidazole

Photophysical study

derivatives and a chloride ion. The regioisomers of the com- To get insights into the quantum yield of the synthesized com-

plexes can be diﬀerentiated from the position of the functional plexes, UV-visible spectroscopy was performed. Compounds

group present in the benzimidazole unit. Also, the familiar were dissolved in 5% DMSO in PBS buﬀer and the concen-

5

−1

“

three leg piano-stool” structure is well suited to explain the tration of about 1 × 10⁻mol L was maintained during all

6

[

Ru(η -arene)(L)Cl]Cl complexes, where the seat is formed by the spectroscopic studies. The characteristic intraligand (π–π*)

the η -arene and the three legs of the stool are constituted by transitions (N^N ligands) appeared at 250–300 nm and

300–360 nm and weak metal to ligand charge transfer ( MLCT)

The calculated bond angles i.e. N1–Ru–Cl (82.17°) and N2– at 370–470 nm for (η -p-cymene)ruthenium(II)-2-(6-bromopyri-

Ru–Cl (84.57°) are nearly 90° supporting the “piano-stool” dinyl) complexes (Fig. S5†).

6

1

the two nitrogen atoms and the chloride ion.

6

7

3,74

Similar trends were observed

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6

for the extended π-electronic series i.e. (η -p-cymene)ruthe- showed moderate fluorescence quantum yield (0.024, Table 1),

6

nium(II)-2-(6-arylpyridinyl) scaﬀolds. The UV-Vis spectra of (η - while, 11f′ exhibited the best fluorescence quantum yield 0.22

p-cymene)ruthenium(II)-2-quinolinyl complexes showed good among all the members of the quinoline series (Table 1).

6

splitting in MLCT absorption bands in the range of However, all the (η -p-cymene)ruthenium(II)-2-(6-arylpyridinyl)

3

50–470 nm which was much more prominent than MLCT scaﬀolds displayed significant fluorescence quantum yield in

6

absorption bands of (η -p-cymene)ruthenium(II)-2-(6-bromo- the range of 0.17–0.51, (Table 1) because of their extended π

pyridinyl) complexes (Fig. S6†).

7

3,74

After that, quinine sulfate electronic structure. Among them, compounds 8l1, 8l4, and

in 0.5 M H SO solution was used as the reference standard 8l5–8l9 exhibited remarkable fluorescence having quantum

2

4

for the calculation of the respective quantum yields from the yields in the range of 0.41–0.51 (Table 1).

fluorescence study. Quantum yields of the complexes were cal-

culated from equation (ii) (Table 1). Among the (η -p-cymene)

6

Solubility, lipophilicity and conductivity

ruthenium(II)-2-(6-bromopyridinyl) series compound 5d′ Both hydrophilicity and lipophilicity were studied to uncover

the tumour inhibiting potential of these metal complexes.

These complexes are highly soluble in aqueous DMSO, H O,

2

MeOH, EtOH and CH

in the range of 5–8 mg per ml of DMSO-10% DMEM medium

1% DMSO in DMEM, 1 : 99 v/v, comparable to cell media) at

3

CN. These Ru(II) complexes were soluble

Table 1 Photophysical characterization, solubility, lipophilicity and

conductivity study of the synthesized ruthenium complexes

(

a

b

λ

max

,

λ

f

,

Stokes

shift

Quantum 25° C (Table S2†). The lipophilicity of these complexes was

c

d

Samples

[nm]

OD

yield, φ

evaluated using the n-octanol/aqueous buﬀer partition coeﬃ-

5

a

b

b′

c

c′

d

d’

e

330

400

324

330

345

390

365

250

404

450

455

402

390

434

400

470

475

444

452

376

—

74

50

55

78

60

89

55

80

85

79

87

126

1.19

0.65

0.60

1.29

1.12

0.83

0.60

1.08

0.016

—

0.011

—

0.012

0.024

0.007

—

cient (log P_o_/_w, where P_o_/_w= the octanol/water partition coeﬃ-

cient, PH 7.4) study by means of the shake flask method

75

(

Table S2†). The experimental log Po/w values of these com-

plexes were obtained in the range of 0.6–1.4 (Table S2†).

Among the pyridine series, complex 5d and it’s regioisomer

5d′ exhibited the highest log Po/w (∼1.3) due to the presence of

e′

f

f’

0.9

the lipophilic –CF group. In the extended π electronic series

3

0.959

0.812

2.69

—

(

8g1–8g3, 8l1–8l9) all the compounds showed significant lipo-

philicity (0.9–1.20) because of the presence of a hydrophobic

aryl group. Among the quinoline series, compounds 11d, 11d′,

g (less soluble)

3

70

—

5

8

h

335

343

334

341

328

325

260

444

477

450

455

400

397

350

400

404

399

401

488

500

490

394

384

390

356

352

370

444

354

356

414

398

394

392

400

432

386

394

378

452

109

134

116

114

72

90

60

81

82

173

185

177

92

79

84

54

50

61

135

65

66

109

89

84

89

94

57

79

66

1.022

0.255

0.353

0.128

0.345

0.525

—

1

1j, 11k and 11k′ exhibited the best lipophilicity (∼1.3–1.4)

due to –Cl, –F and –CF substitution. These ruthenium com-

plexes exhibited molar conductance values of ∼28–35 S

g1

g2

g3

l1

l2

l3

0.21

0.30

0.25

0.43

0.17

—

0.33

0.51

0.49

0.41

0.51

0.49

0.04

0.02

0.016

0.023

0.04

—

0.03

0.22

0.019

0.06

—

0.027

—

3

2

−1

cm M in pure DMSO. However, the molar conductance of all

these complexes did not increase significantly in 10% DMSO

2

−1

3

40

0.270

0.229

0.217

0.322

0.505

0.175

0.128

0.456

0.340

0.701

0.983

0.668

0.304

0.674

0.530

0.229

0.727

0.340

0.835

0.307

0.129

0.484

0.714

0.687

1.048

0.29

(∼30–40 S cm M , Table S2†), suggesting their 1 : 1 electro-

8

1

l4

l5

l6

l7

l8

l9

1a

1b

1b’

1d

1d’

1e

1e’

1f

323

318

319

315

313

302

305

306

302

309

289

290

305

309

310

303

306

375

307

328

330

350

lytic nature in pure DMSO as well as in 10% DMSO respectively

76

as the aqua complex was not formed certainly. These results

satisfactorily supported the complex-DNA non-covalent inter-

action in a cancer environment (low pH, high GSH).

Cytotoxic activity

The in vitro cytotoxicity of all the synthesized Ru(II)-p-cymene-

pyridinyl (5a–h, 5b′–f′, 8g1–g3, 8l–l9) and quinolinyl (11a–b,

11d–f, 11h–n, 11b′, 11d′–f′, 11k′–l′) complexes (Table 2) was

evaluated using standard 3-(4,5-dimethylthiazol-2-yl)-2,5-

diphenyltetrazolium bromide (MTT) bioassay besides a panel

of cancer cell lines such as human epitheloid cervix carcinoma

1f’

1h

1i

(

HeLa), human colorectal adenocarcinoma cell line (Caco-2)

1j

and one normal human embryonic kidney cells (HEK-293) in

triplicate over 48 h incubation at a concentration from 1 to

1k

1k′

1l

1l’

1m

1n

200 µM. As a contrast, the toxicity of cisplatin was tested as

well. There are diﬀerences between normal cells and cancer

cells, such as cell proliferation, physiological activities and

microenvironment, which could present diverse sensitivity to

drug treatment. Most of the organoruthenium scaﬀolds exhibi-

ted higher potency and selectivity than cisplatin in all the cell

48

102

—

0.57

Quinine sulfate

a

b

c

Absorption maxima.

Quantum yield.

Emission wavelength. Optical density.

d

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Table 2 MTT cytotoxicity screening of Ru(II)–arene complexes and (Table 2). Whereas, in Caco-2 cells, the IC50 values of these

their regioisomers

derivatives were found to be in the range of 6–68 µM. Herein,

we have also noticed that two regioisomers showed diﬀerent

levels of cytoselectivity in cancer cells with respect to the

Caco-2 HeLa normal cell. In general, more polar isomers (5b′–d′, 5f′, 11b′,

a

b

IC50 (µM)

SF

c

Compound Caco-2

HeLa

HEK 293

1

1d′, 11f′, and 11k′–l′) exhibited higher potency than less polar

5

8

1

a

b

b′

c

c′

d

d′

e

e′

f

f′

g

h

g1

g2

g3

l1

l2

l3

l4

l5

l6

l7

l8

l9

1a

1b

1b′

1d

1d′

1e

1e′

1f

1f′

1h

1i

1j

1k

1k′

1l

1l′

1m

1n

17.18 ± 1.4

29.24 ± 1.6

8.68 ± 1.2

29.76 ± 0.9

23.12 ± 0.8

68.89 ± 1.3

11.8 ± 1.2

25.50 ± 1.9

24.26 ± 1.4

20.33 ± 1.6

12.16 ± 1.4

48.38 ± 1.9

9.01 ± 0.9

76.89 ± 2.6

62.44 ± 2.6

46.20 ± 0.6

74.88 ± 1.6

34.82 ± 1.6

11.2 ± 1.3

11.16 ± 1.5

10.51 ± 0.9

7.8 ± 1.4

7.41 ± 1.5

7.73 ± 1.7

5.8 ± 1.6

15.17 ± 2.2

11.99 ± 2.5

13.22 ± 1.6

11.4 ± 1.9

7.07 ± 1.4

9.19 ± 1.7

240 ± 1.5

239 ± 1.8

219 ± 1.6

14.11

8.17

25.23

7.82

9.39

2.98

17.30

7.65

8.08

9.38

16.85

5.23

22.4

21.41

isomers (5b–d, 5f, 11b, 11d, 11f, 11k–l) towards the cancer

20.83 cells except methoxy substituted isomers (5e, 11e, 5e′ and

233.9 ± 1.6

217.25 ± 1.4

205.37 ± 1.4

204.2 ± 1.4

195.21 ± 1.4

196.12 ± 1.5

190.70 ± 1.4

205 ± 1.7

29.87

29.31

26.56

1

1e′).

Among all the complexes, 11j showed the best potency and

35.20 selectivity (31–55 fold) in both the cancer cell lines with

12.86

16.35

14.42

respect to normal cells (Fig. S7†). IC50 values of this complex

against HeLa and Caco-2 were observed to be 3.52 ± 1.7 μM (p

17.98 < 0.001) and 6.16 ± 0.8 μM (p < 0.05) respectively. Remarkably,

IC50 values of this complex in all the cancer cells were lower

than those of cisplatin by approximately 3–5 fold. In terms of

2.80 selectivity, complex 11j was 11–18 fold more selective than cis-

platin towards cancer cells compared to normal kidney cells.

However, the (η -p-cymene)ruthenium(II) complexes having

4.73 extended π-conjugated ligands (8g1–g3, 8l–l9) exhibited less

potency than pyridinyl and quinolinyl complexes. Among

them, complexes 8l4 and 8l9 showed the best potency and

2.44 selectivity in HeLa and Caco-2 cell lines, respectively. Herein,

most of the compounds displayed 2–55 fold selectivity in

cancer cells than the normal kidney cell. Whereas, the stan-

25.36 dard cisplatin exhibited only 2–3 fold selectivity. Once we

intended our attention towards the quantum yield of pyridinyl

and quinolinyl complexes in water, complex 11f displayed the

13.85 best quantum yield (0.22) with moderate IC₅₀in both the

cancer cell lines (39.9 µM and 11.7 µM in Caco-2 and HeLa cell

line respectively). This complex also showed many fold higher

18.74 selectivity in cancer cells. Among the π-conjugated ruthenium

complexes, compounds 8l1, 8l4, 8l5–8l9 exhibited remarkable

fluorescence having quantum yields in the range of 0.41–0.51,

37.30 while, the compounds 8l1, 8l5–8l8 having remarkable fluo-

253.20 ± 1.9

259.23 ± 1.4 28.77

35.81

28.20

1.35

154.32 ± 2.6 209.11 ± 1.6

67.32 ± 1.1 189.14 ± 1.6

157.99 ± 3.2 236.93 ± 1.4

247.65 ± 1.4 242.19 ± 1.7

2.71

3.02

5.12

3.23

5.41

0.94

1.49

0.97

2.23

6

84.45 ± 3.7

188.60 ± 1.7

189.63 ± 1.9

280.81 ± 1.8 18.64

200.12 ± 1.4 40.03 ± 1.2

15.06 ± 2.7

57.03 ± 2.4

7.97 ± 1.7

128.02 ± 2.1 164.92 ± 1.9

35.23

1.28

1.09

2.89

1.53

2.99

2.49

140.03 ± 2.4 197.52 ± 1.4 215.36 ± 1.9

66.48 ± 2.4

72.36 ± 1.9

3.53 ± 1.4

8.69 ± 0.6

17.23 ± 1.3

9.74 ± 0.7

30.92 ± 1.8

13.27 ± 1.

54.94 ± 1.2

7.4 ± 0.9

67.5 ± 2.4

39.9 ± 2.1

43.5 ± 2.2

64.34 ± 2.6

6.16 ± 0.8

13.39 ± 1.7

12.58 ± 1.4

46.33 ± 1.8

17.4 ± 1.2

11.89 ± 1.2

17.07 ± 0.7

17.9 ± 1.2

81.55 ± 2.0

119.13 ± 1.8 180.51 ± 1.9

80.60 ± 2.7

7.33 ± 1.9

10.25 ± 1.8

9.73 ± 1.2

7.6 ± 1.2

199.35 ± 1.8

1.51

2.10

24.45

169.96 ± 1.4 48.14

179.23 ± 2.4 20.62

259.97 ± 1.8 15.08

232.70 ± 1.6 23.89

228.37 ± 1.8

172.30 ± 1.4 12.98

187.16 ± 1.7 3.40

172.18 ± 1.9 23.26

171.61 ± 2.9

170.39 ± 1.4

181.84 ± 1.7

153.29 ± 1.8

192.3 ± 1.9

243.16 ± 1.9 18.15

293.96 ± 1.8 23.36

23.91

30.04

27.34

7.38

′

6.3 ± 1.4

13.51 ± 1.8

12.17 ± 1.4

13.8 ± 1.4

11.7 ± 2.4

9.7 ± 1.7

25.69 ± 1.2

3.52 ± 1.7

8.17 ± 1.8

7.88 ± 1.8

11.91 ± 17

10.69 ± 0.9

10.47 ± 1.8

14.31 ± 2.7

13.26 ± 0.6

14.14

12.43

14.56

2.54

4.27

4.18

2.38

31.21

5.96

54.63

29.76

187.10 ± 1.5

255.16 ± 1.9 14.66

262.00 ± 13 22.03

149.77 ± 1.7

50.22

4.03

15.70

23.86

25.02

rescence quantum yields, have been ruled out from cancer

theranostic application due to their non-potency in cancer

10.46 cells. However, these complexes might be suitable as bio-

8.77

2.80

Cisplatin

3.80

imaging agents. Compounds 8l4 and 8l9 exhibited high fluo-

IC50 is the concentration at which 50% of cells undergo cytotoxic cell rescence quantum yields and remarkable cytoselectivity in

a

death due to organoruthenium and cisplatin treatment in triplicate. It

HeLa and Caco-2 cell lines, respectively. In this aspect, com-

was calculated by non-linear sigmoidal curve fitting in the dose

pounds 8l4, 8l9 and 11f′ might be appropriate for cancer thera-

response model using Origin 8.5. Each value represents the mean ± SD

b

(

standard deviation) of three independent experiments. SF (selectivity nostic application.

factor) = ratio of IC50 for HEK-293 and IC50 for all the cancer cell lines.

HEK-293 fibroblasts are generally selected as the model for healthy Stability studies

c

cells in the evaluation of chemotherapeutic drug selectivity. 48 h

incubation time. Statistical significance is indicated as P < 0.05 based

on student’s t-test using Origin 8.5 software (the statistical significance

UV-vis spectroscopic method. A chemical entity should have

proper stability profile to become a potential therapeutic

agent. To accomplish the stability study, complexes 5h and 11j

were selected as two model compounds based on their potency

and selectivity in both the cancer cells. Two diﬀerent media

(

p) of the data is <0.05 or better).

lines with respect to the normal kidney cell. Herein, we used such as 5% DMSO in phosphate buﬀer and water were used

DMSO as a control which did not show any inhibition of cells. for this study. The absorbance was recorded at a regular time

6

In HeLa cells, the IC₅₀values for the (η -p-cymene)ruthenium interval over a period of 24 h and the results obtained have

6

(

II)-2-(6-arylpyridinyl) (5a–h, 5b′–f′) and (η -p-cymene)ruthe- been presented in the ESI.† It was evident that the drug

nium(II)-2-quinolinyl complexes (11a–b, 11d–f, 11h–n, 11b′, showing insignificant changes in absorption bands along with

1

1d′–f′, 11k′–l′) were found to be in the range of 3–25 µM time would be more stable in the corresponding medium.

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According to our investigation, compounds 5h and 11j showed transition was noticed with increasing DNA concentration

insignificant changes in their absorption bands in 5% DMSO/ from 0–100 μM (Tris-HCl buﬀer, pH 7.8, t = 25 °C) for the

PBS medium (PH 7.4) which was recorded over a 24 h time complex 5h and 11j (Fig. 10). This phenomenon was clear evi-

period (Fig. S8†). These results indicated that these complexes dence for the complex to interact with DNA in an intercalative

1

were stable in the existing medium and could be a good drug manner (in MLCT) as well as in electrostatic mode (in π–π*).

candidate for bioassays. To exhaust cancer cell proliferation, Similar to other DNA intercalators, such as ethidium bromide

DNA of such cells can be regarded as a potential therapeutic (EtBr), the complex is supposed to interact through π–π stack-

target. Usually compounds which are able to form aqua com- ing between DNA base pairs and the aromatic heterocyclic

7

9,80

plexes in the cellular environment are considered to interact group.

Intrinsic binding constant, K

b

for these entire com-

for complex

with DNA base pairs via covalent mode of action which result plexes, was calculated by using the equation (i). K

in disruption of the DNA structural motif. To know the aquatic 5h and 11j was found to be 0.54 × 10 M and 1.04 × 10 M

stability of both the complexes (5h and 11j), UV-vis study was respectively which were similar in magnitude with that of

performed which showed a slight hyperchromic shift in their EtBr–DNA (7 × 10 M ).

absorption bands over a time period of 48 h (Fig. S8†). Such the lower order of intrinsic binding constant (0.1 × 10 M

b

5

−1

5

−1

5

−1 81,82

However, complex 8l7 exhibited

3

−1

,

minor changes in absorption bands of these compounds indi- Fig. S12† and Table 3) and these results were correlated with

cated the slow dissociation of the –Cl ligand from the Ru(II)– the cytotoxicity data.

arene complexes. The steric bulkiness of the N^N ligand might

7

Ethidium bromide displacement assay

slow down the hydrolysis of Ru(II) complexes.

NMR spectroscopy method. In order to know the stability of The same compounds were used for steady-state ethidium

such complexes in DMSO, NMR spectra of compounds 5h and bromide (EtBr) displacement assay by fluorescence spec-

1

6

1j were recorded at 0th, 6th and 24th h in DMSO-d (Fig. S9 troscopy to investigate the way of drug–DNA interaction.

and S10, see ESI†). Pattern and δ values of characteristic peaks Ethidium bromide is a very strong DNA intercalator and struc-

for protons in all the spectra remained intact which indicated

that the complexes were stable in DMSO. To explain the

aquatic stability of complex 11j, the NMR spectrum was

recorded at 0th, 6th and 24th h after dissolving in D O at PH

2

7

.4 (Fig. S11, see the ESI†). However, no significant change in

the δ values was observed after 24 h which indicated that

complex 11j is fairly stable in water.

Biomolecular interaction

DNA binding studies. DNA is one of the important biological

targets for cancer therapy. Small molecules possessing a

charged centre can eﬃciently interact with DNA either in a

covalent or non-covalent (binding to major or minor groove,

intercalation etc.) manner. If any aromatic heterocyclic moiety

partially interferes with the hydrogen bonds between nitrogen

base pairs leading to disruption of the DNA structure, the way

7

8

of interaction is known as intercalation. The study involving

interaction of drug with DNA is important to get a clear under-

standing of the mechanism which can be performed following

spectroscopic method.

UV-vis and fluorescence spectroscopy methods. The behav-

iour of compounds in the presence of DNA was studied by UV

and fluorescence spectroscopy methods. As usual the ruthe-

nium complex 5h, under observation, possessed two distinct

absorption bands at 250–300 nm and 340–500 nm in the UV-

vis absorption spectrum which attributed to the intraligand

1

(

π–π*) transitions (N^N ligands) and MLCT transition respect-

ively, whereas, compound 11j displayed two π–π* bands at

50–300 nm and 300–340 nm, and MLCT band at 350–500 nm.

2

However, complex 8l7 did not show any significant MLCT

band except one π–π* band at 300–350 nm. After treating these

complexes (10 μM in Tris-HCl buﬀer, pH 7.8) with Calf

Thymus DNA (ct-DNA) a significant hyperchromic shift of

Fig. 10 UV-vis absorption spectrum of complex (a) 5h and (b) 11j in the

absence and in presence of 10–100 μM of ct-DNA in TrisHCl buﬀer (pH

7.8, T = 25 °C). Change in absorbance with increasing DNA concen-

tration in the direction of the arrow. Inset: plot of [DNA]/(ε − ε ) vs.

a

f

absorbance in π–π* transition and hypochromic shift in MLCT [DNA] for (c) 5h and (d) 11j.

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Table 3 The Stern–Volmer quenching constant (Ksv), number of binding sites (n), apparent binding constant (Kapp), intrinsic binding constant (K

and overall binding constant (K) for the complexes 5h, 11j and 8l7

b

),

a

sv [M⁻¹

app [M⁻¹

b

c

[M⁻¹

K^d[M⁻¹]

e

Complex

K

]

K

]

K

b

]

n

4

6

5

3

5

1

8

h

1j

l7

3.2 × 10₄

2.7 × 10

2 × 10

0.5 × 10

3.9 × 10

1.5 × 10

1.6 × 10

0.91 ± 0.02

1.2 ± 0.01

0.9 ± 0.04

6

5

3

1.04 × 10

4

6

3

4.0 × 10

0.8 × 10

0.1 × 10

a

b

c

K

sv, Stern–Volmer quenching constant.

b

Kapp, constant from competitive displacement. K , intrinsic DNA binding constant from UV-vis

e

d

absorption titration. K, overall binding constant. n, number of binding sites.

tural probe that gives a strong fluorescence while intercalating strong noncovalent binding (especially intercalating) between

DNA base pairs. The enhanced fluorescence can be quenched the complex and DNA. However, an extended π complex like

6

by other chemical moiety which can competitively replace EtBr 8l7 exhibited K_a_p_p= 0.8 × 10 .

8

3

and binds to the same site. Solution containing DNA bound

ethidium bromide showed intense fluorescence emission peak GSH and BSA protein interaction study

at 608 nm when excited at 485 nm. Fluorescence intensity was

GSH is the most prevalent non-protein thiol in mammalian

cells which acts as a detoxifying agent. Most of the cancer

cells become resistant to diﬀerent drugs by increasing their

quenched every time after incubating with increasing concen-

tration of compounds 5h, 11j (Fig. 11) and 8l7 (Fig. S13†).

Both the spectra of compounds 5h and 11j gave evidence of

intercalation. The extent of hypochromism for complexes 5h

and 11j was more than 50% accompanying a small blue shift

of approximately 8 nm. However, hypochromism for complex

84

cellular GSH level as it expedites the rate of deactivation

85,86

through the GSH–drug covalent interaction.

Thus, the

interaction of GSH with metal–drugs is an important para-

meter for their eﬃciency in the mammalian cancer cell.

Generally, organoruthenium complexes showed good stability

8

l7 is lower than 40%. The Stern–Volmer quenching constant

sv), number of binding sites (n), apparent binding constant

K_a_p_p) and overall binding constant (K) for the complexes were

(

K

87

in physiological conditions at high chloride concentrations.

Thus, the stabilities of the synthesized Ru(II) complexes were

studied in the presence of excess (10 eq.) glutathione (GSH)

using UV-vis spectroscopy. When a drug enters a biological

system it needs to be taken up in the cell environment to exert

its therapeutic activity. However, upon cell entry, where the

chloride level is lower, metal–drugs react with the intracellular

nucleophiles. Serum albumins are a major part of plasma pro-

teins and facilitate transportation and metabolism of various

exogenous and endogenous compounds associated with cell

tabulated (Table 3, equation (iii), (iv), (vi) which indicated that

both pyridinyl and quinolinyl complexes were able to bind

DNA in an intercalative manner. The observed values of K_b,

K

sv, Kapp and K are lower in magnitude than literature values

7

of classical intercalators, such as EtBr–DNA (KEtBr = 1.0 × 10

M

−

1

83

) (Table 3). Nevertheless, high K_a_p_pand K_s_vindicated the

8

metabolism. Human serum albumin (HSA), a structural ana-

logue of bovine serum albumin (BSA) which is present predo-

minantly in the cellular environment helps the drug to invade

8

9,91

the cell by forming a more active coordinate complex.

This

protein protects the Ru(II)–arene complex from deactivation by

7

7,90,91

GSH.

In general Ru(II)–arene complexes express strong

aﬃnity to BSA than GSH as the quantity of serum albumin in

7

7,90,91

cell is much higher than that of GSH.

In this study it

6

was found that the (η -p-cymene)ruthenium(II)-2-(6-bromopyri-

dinyl) complex (5h) made a strong interaction with GSH within

3

h of incubation. A strong hypochromic shift in absorbance

and blue shift in wavelength was observed after 48 h of inter-

action (Fig. S14†). Therefore, Ru(II)-p-cymene-bromopyridinyl

complexes might lose their activity in the cancer cell due to

the presence of excess GSH. However, these complexes exhibi-

ted decent potency and selectivity in cancer cells. Hence, the

albumin impasses the Ru(II) complexes to protect them from

GSH facilitating their delivery to the target site. In comparison

6

to this, the (η -p-cymene)ruthenium(II)-2-quinolinyl complex

(

11j) showed insignificant changes in absorption bands

Fig. 11 Ethidium bromide ﬂuorescence quenching study with com-

(

Fig. S14†). More steric quinoline analogues helped to slow

0 0

pounds (a) 5h and (b) 11j. Inset: plot of I /I vs. [Q] and plot of log [(I − I)/

I] vs. log [Q] for both the compounds.

down the hydrolysis rate and also the reaction with GSH which

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prevented deactivation by the coordination of GSH with the Ru Table 4 The Stern–Volmer quenching constant (Ksv), number of

9

2

binding sites (n), apparent binding constant (K_a_p_p) and overall binding

constant (K) for the complexes 5l, 11c and 8lh for BSA binding assay

(

II)–arene complex. In our study, bovine serum albumin

BSA) was chosen as a target protein for drug interaction

because of its excellent ligand binding abilities, good bio-

availability and a structural homology of human serum

albumin (HSA). BSA molecule shows intrinsic fluorescence

due to the presence of aromatic amino acids like tyrosine,

phenylalanine, mainly tryptophan in its quaternary structure.

A strong fluorescence quenching of BSA can be observed upon

interaction with the drug molecule although this quenching

method is sensitive when the binding takes place nearby the

tyrosine and tryptophan amino acids, so it will not provide

BSA a _[_M−1

b

_[_M−1 _S−1

_Kc _[_M₋₁_]

d

Complexes

K

]

K

q

]

n

4

12

6

5

1

8

h

1j

l7

7.5 × 10

3.4 × 10

1.8 × 10

7.5 × 10

3.4 × 10

0.2 × 10

1.6 × 10

0.4 × 10

1.2 × 10

1.4 ± 0.03

1.3 ± 0.02

1.5 ± 0.06

4

5

12

6

7

a

b

K

BSA, Stern–Volmer quenching constant.

stant. K, binding constant with BSA. n, number of binding sites.

q

K , quenching rate con-

c

d

8

3,85

information about the overall binding of the complex.

red shift of 4 nm was observed when complex 11j interacted

Fig. 12 shows the eﬀect of drug (5h, 8l7 and 11j) addition in with BSA. The Stern–Volmer quenching constant (Ksv) and

BSA stock solution (3.0 μM in phosphate buﬀer, pH 7.2) which bimolecular quenching rate constant (K ) were calculated

depicted that fluorescence of BSA was massively quenched using the classical Stern–Volmer equation (v). For the 5h-BSA

q

4

−1

(

more than 50%) with increase of drug concentration. A small complex, Ksv and K

q

were found to be 7.5 × 10 M and 7.5 ×

1

2

−1

s⁻¹respectively. Similarly for the 11h-BSA complex

was 3.4 × 10 M and K was found to be 3.4 × 10

1

0

M

4

−1

12

−1

K

M

sv

1

q

−

s

. Compound 8l7 also showed higher order of Ksv and K

Table 4). The K

were in the order of 10

q

(

q

values of the newly synthesized complexes

1

2

−1 −1

M

s

which were higher enough

than the maximum scatter collision quenching constant (2.0 ×

1

0

−1 −1

93

10

M

s ) of BSA quenchers. Binding constants of these

complexes and number of binding sites were calculated using

Scatchard equation (vi) and tabulated (Table 4). The high

potency of these complexes in cancer cells may be attributed

to the strong binding with serum albumin which overcomes

the drug resistance by GSH.

Structure activity relationship

Structure–activity relationship (SAR) study revealed that the

potency of Ru(II)-p-cymene-pyridinyl (5a–h, 5b′–f′, 8g1–g3,

8

1

l–l9) and ruthenium(II)-p-cymene-2-quinolinyl (11a–b, 11d–f,

1h–n, 11b′, 11d′–f′, 11k′–l′) complexes in cancer cells varies

with the substitution in the benzimidazole ring. For example,

unsubstituted Ru(II)-p-cymene-2-pyridinyl benzimidazole (5a)

showed significant potency and selectivity in both the cancer

cells. However, after introducing an electronegative halogen

group in the benzene ring, the potency of the complexes (5b,

5c, 5c′, and 5d) in the Caco-2 cell line dropped drastically and

increased significantly in the HeLa cell line. However, steri-

cally less hindered two isomers 5b′ and 5d′ exhibited signifi-

cant potency and selectivity in the Caco-2 cell line with

respect to normal cells. From this consequence it is evident

that the position of lipophilic –Cl and –CF

core moiety is essential to maintain Caco-2 permeability and

potency. The presence of lipophilic –F, –Cl and –CF groups

3

groups in the

3

increases the membrane permeability in the HeLa cell. This

is why compounds 5c and 5d and their regiosiomers exhibi-

ted the highest potency in HeLa cells among all other Ru(II)-

p-cyemene-2-pyridyl complexes. The introduction of an elec-

tron donating –OMe group in the benzene ring (5e and 5e′)

clearly displayed a moderate potency in both the cancer cells.

Fig. 12 Interaction of complexes (a) 5h, (b) 8l7 and (c) 11j with BSA.

0

Inset: Stern–Volmer plot of I /I vs. concentration of the complexes.

2

Electron withdrawing –NO substituted Ru(II) complexes (5f

Scatchard plot of log([I

0

− I]/I) vs. log[complex] for BSA in the presence

of the complexes.

and 5f′) also showed decent potency in both the cell lines.

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When the benzothiazole group was introduced in place of (iii) labile chlorine attached to ruthenium was essential for

benzimidazole, cytotoxicity of complex 5h in both the cell several biological interactions including DNA and protein, (iv)

lines was improved to a greater extent. Although compound more polar regioisomers except methoxy substituted com-

5

g exhibited high potency in HeLa cells, its cytotoxicity sig- plexes displayed more potency and selectivity than corres-

6

nificantly deteriorated in Caco-2 cells. In the case of (η -p- ponding less polar regioisomers in both the cancer cells, (v)

cymene)ruthenium(II)-2-quinolinyl complexes, the complex Ru(II) cationic complexes improved the compound solubility

having unsubstituted imidazole (11a) exhibited excellent and facilitated the electrostatic interaction between the nega-

potency in Caco-2 (IC₅₀= 8.69 ± 0.6 µM) and HeLa cell line tively charged DNA phosphate backbone and cationic ruthe-

(IC50 = 7.33 ± 1.9 µM). The potency of the complexes in CaCo- nium(VI) 2-aryl benzimidazoles acted as good DNA intercala-

2

cells has been reduced (IC50 = 13–67 µM) after introducing tors and also as a pharmacophore, (vii) the –NH group

the electron withdrawing or donating substituents excluding present in the benzimidazole moiety was crucial for hydrogen

complex 11j. For example, IC50 of complex 11i having an elec- bonding interaction with DNA base pair, (viii) ligands having

tron donating methyl group in the benzimidazole ring extended π electronic systems enhanced the quantum yield of

increased significantly in both the cancer cell lines (Caco-2: the complex which helped in cellular imaging and (ix) steri-

6

4.34 ± 2.6, HeLa: 25.69 ± 1.2). Mono chloro substituted ben- cally hindered quinoline analogues helped to slow down the

zimidazole ruthenium complexes (11b and 11b′) exhibited drug–GSH interaction and prevented the deactivation

much better potency than compound 11i in both the cancer (Fig. 13).

cells. Dichloro substituted benzimidazole ruthenium complex

(

11j) presented the best cytotoxicity profile in both the cell In vitro cytotoxicity of selected complex 11j against most

lines (CaCo2: IC₅₀= 6.16 ± 0.8 µM, HeLa: IC₅₀= 3.52 ± 1.7) aggressive colorectal carcinoma cell line HT-29

among all the synthesized organoruthenium complexes

because of its significant cell permeability (log p = 1.32 ±

Herein, complex 11j exhibited the most promising eﬀect on

both the cancer cells (Caco-2 and HeLa). For this very cause,

we focused our attention on the potency of complex 11j

against the mostaggressive colorectal carcinoma cell line

HT-29. MTT assay result showed a dose dependent drop of pro-

liferation of colon cancer cells as well as cervical cancer cells

0

×

.05) as well as high overall DNA binding constant (K = 1.04

10 ). These results signify the importance of electronegative

b

5

chloro substitution in the 2-quinolyl benzimidazole ligand for

acquiring potency. similar trend of cytotoxicity was

A

observed in the case of complex 11k and 11k′ while both the

complexes showed less cytotoxicity than complex 11b′ against

the Caco-2 cell line. After introducing the less electronegative

bromine atom in the benzimidazole ring, potency of the com-

plexes (11l and 11l′) was reduced in both the cell lines.

Although complex 11d and its regioisomer 11d′ exhibited sig-

nificant cytotoxicity like complex 5d and 5d′ in HeLa cells

because of the lipophilic –CF substituent, satisfactory results

3

were not observed in the Caco-2 cell line. Moderate to high

potency of the complexes 11e/11e′ (–OMe substitution) and

(

HeLa). The in vitro cytotoxicity of the compound 11j was

assessed against HeLa and HT-29 cell lines performing MTT

experiment. The tumor cells were incubated with the test com-

pound (11j) at diﬀerent concentrations (1–15 µM) for 24 h

in vitro. It was found that the compound 11j exhibited signifi-

cant cytotoxicity on both the commercially available cell lines

in a dose dependent manner.

In HT-29 and HeLa cell lines, the compound exhibited an

IC50 value of 6.8 µM and 4.4 µM respectively (Fig. 14A and B).

The aforementioned aggressiveness of HT-29 cells can be well-

justified by an IC₅₀value of 6.8 µM which was higher than that

of comparatively non-aggressive HeLa cell line (Fig. 14A).

Furthermore, we had carried out all the experiments using the

drug concentrations of 6.8 µM and 5 µM against tested cell

lines. In this study we had further quantified the eﬀective dose

1

2

1f/11f′ (–NO substitution) was observed in both the cell

lines. Similar results were observed in complexes 11h, 11m

and 11n. It was also observed that after introducing sterically

congested aryl groups to the pyridinyl ligands of the ruthe-

nium complexes, potency was reduced drastically which can

be correlated with the DNA binding data. However, complex

8

l4 in both the cell lines and complex 8l9 against the Caco-2

cell line exhibited significant potency. In general it has been

observed that more polar regioisomers exhibited much better

eﬃcacy than their corresponding regioisomers except

methoxy substituted complexes. The polar regioisomer

increased the positive character of ruthenium which in turn

enhanced the electrostatic binding to DNA. On the other

hand, release of steric hindrance in polar regioisomer helps

in intercalation of DNA but enhancement in lability of –Cl

influences the scaﬀold to bind with DNA covalently. Overall

results revealed that (i) the hydrophobic p-cymene ring facili-

tated the passive diﬀusion of these complexes inside the cell,

(ii) lipophilic fluorine and chlorine substitution in the ligand

was important for cell membrane permeability and potency, Fig. 13 SAR study of Ru(II)-p-cymene-2-arylbenzimidazole complexes.

Dalton Trans.

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Fig. 14 MTT assay and EC50 value determination of (A) HT-29 cells and (B) HeLa cell line. (C) Dose dependent morphological changes of HT-29 cell

line, upon treatment with various doses of drug 11j. Cells were co-stained with Hoechst 33342 to visualise nucleus. Arrow mark represents the cell

damage.

by determining their corresponding EC50 value on the HT-29 tration, % of dead cells was also increased. This data also cor-

cell line. In fact, in all our studies, we have used IC50 and EC50 roborated with our morphology data and further studies on

as synonyms.

oxidative damage and ROS generation were carried out.

Morphological analysis

ROS generation by complex 11j in HT-29 cells

Subsequently, we wanted to explore the action of such a DCFDA (2′,7′-dichlorofluorescin diacetate) is a colourless dye

complex on the cancer cell morphology. Hence, we treated the that binds with the ROS produced in the cell and gives a green

9

7

cancer cells with the test compound at diﬀerent concen- fluorescent signal. Consequently, the treatment of Ru(II) com-

trations (1–6.8 µM) and after 24 h the cells were co-stained plexes on HT-29 cells for 24 h led to ROS production, which

9

4

with live cell nuclear stain Hoechst 33342. As a result, signifi- resulted in oxidative stress on cancer cells and facilitated the

7

7,98

cant cell damage including cell shrinkage and membrane oxidation GSH to GSSG.

blebbing were clearly visualized in the HT-29 cell line production was detected in the HT-29 cells upon treatment of

Fig. 14C). The manifestation of cell damage started to prevail 6.8 µM of 11j as compared to untreated control. The fluo-

As shown in Fig. 15A, highest ROS

(

at 5 µM concentration of compound treated cells (Fig. 14C(ii)) rescence intensity of cell containing ROS was quantified and

and the damage was increased prominently at the complex represented in the form of bar graph (Fig. 15C). The formula

concentration of 6.8 µM (Fig. 14C(iii)) as compared to intact used to determine the % of DCFDA positive cell is % of ROS

control cells. In conclusion, the ruthenium scaﬀold 11j had producing cells = number of green cells/number of Blue or

+

ve

been promoted to cellular damage with the destruction of the Hoechst cells ×100. Hence, the drug 11j proved to be indu-

cell membrane.

cing cellular damage through ROS generation in colon cancer

cells. The deactivation of these complexes by GSH was low

because (i) they displayed higher aﬃnity to serum albumin; (ii)

Compound 11j causes DNA damage to colorectal cancer cells

7

GSH reacted competitively with ROS and converted to GSSG.

As DNA is the primary target of an anticancer drug, we

observed the DNA damage of cancer cells by using compound

Cell cycle analysis

1

1j which can be justified by dead cell counting assay with the

help of well-known DNA intercalating agent propidium As per the results of MTT assay, there was an inhibition of cell

9

5

iodide. Upon treatment of complex 11j to the HT-29 cells, we proliferation that was further validated by determining the cel-

observed the PI positive cells at 6.8 µM concentration. lular damage, DNA damage and oxidative stress to cancer cells.

Thereafter, PI positive cells were enumerated as well as the % So, we went forward to investigate the DNA content by flow

of dead cells were quantified by the following formula and has cytometry. As shown in the Fig. 16A, the colon cancer cells

been represented in the form of a bar graph (Fig. 15B). % of exhibited high S phase (∼37.15%) in control conditions, as

+

ve

dead cells = number of PI

cells/number of Blue or expected and G /G1 phase was ∼37.15% with ∼26.25% G2/M

0

+

ve

Hoechst cells × 100 which corresponds to the DNA damage, phase (Fig. 16). The treatment of complex 11j resulted in an

due to oxidative stress to the cells through ROS production.

9

6

increase in G0/G1 phase to ∼71.51% in 5 µM and ∼66% in

It was also observed that with increasing the drug concen- 6.8 µM which indicated G0/G1 cell cycle was arrested.

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Fig. 15 The drug 11j caused genomic DNA damage and also ROS production in HT-29 cells: (A) representative images of live cell vs. dead cell enu-

meration along with DCFDA co-staining and (B) graphical representation of % of dead cells upon treatment with diﬀerent concentration of drug 11j.

+ve

(

C) Graphical representation of % of DCFDA

cells upon treatment with diﬀerent concentrations of drug 11j. (D) Representative merged image of

an apoptotic cells upon treatment of 6.8 µM 11j, with Hoechst 33342, DCFDA and PI. Scale bar is 100 µm. Error bars indicate mean ± SD, and rep-

resent at least three independent experiments. Statistical signiﬁcance was determined using two tailed unpaired Student’s t-test. ***P < 0.001 con-

sidered as highly signiﬁcant.

tic, late apoptotic and necrotic/dead cells. The values were nor-

malized with a control value and represented as a bar graph. In

summary, the apoptotic event observed in the HT-29 colorectal

cancer cell line upon treatment with compound 11j was due to

excess ROS production or cell cycle arrest.

Cellular imaging study

A bio-imaging study was performed with the decently potent

Fig. 16 Cell cycle analysis with (A) control HT-29 cells, (B) the treat-

ment of 5 µM 11j and (C) the treatment of 6.8 µM 11j.

and fluorescent complexes among all the synthesized com-

6

pounds, i.e., [(η -p-cymene)RuCl{2-(6-nitro-1H-benzo[d]imida-

6

zol-2 yl)quinolone}]Cl (11f′), and [(η -p-cymene)RuCl{5-chloro-

2

-(6-(4-chlorophenyl)pyridin-2-yl)benzo[d]thiazole}]PF₆

(8l4)

Congruently, the S phase decreased to 19% in 11j drug treated

cells (Fig. 16). Excessive stalling of the cells at G0/G1 is pre-

luded to either cellular quiescence or apoptosis that needed

further validation. Similarly in our study, we had observed the

HT-29 cells undergoing G0/G1 phase arrest upon treatment

with ruthenium–drug 11j.

using the HeLa cell line.

After incubating the cancer cells with compound 11f′

20 μM) for 2 h a strong red fluorescence was visualized in live

(

cells under a fluorescence microscope using a green filter (exci-

tation filter 480–550 nm) (Fig. 18). Similarly, significant cellular

uptake of compound 8l4 in HeLa cells was further confirmed by

time dependent fluorescence imaging study (Fig. 19). As this

fluorescent complex (8l4) exhibited high cytoselectivity (18–35

fold) in cancer cells, this one could be a candid choice for

cancer theranostics. Co-localization of the complex with DAPI

showed that intracellular distribution of complex 8l4 was

mainly in the nucleus, rather than other organelles (Fig. 20).

Apoptosis assay

The process of tumorigenesis is closely correlated with sus-

tained proliferation of the cancer cells. Until now our experi-

ments suggested that HT-29 colon cancer cells were compelled

to stop proliferation upon treatment of 11j. Moreover, irrevers-

ible DNA damage held to produce ROS in the cells which paved

9

the way of apoptosis. Hence, we assessed the detection of

apoptotic protein/marker, Annexin-V. As shown in Fig. 17, the

maximum number of apoptotic and dead cells were observed in

HT-29 cells upon treatment of 10 µM and 6.8 µM of complex

Experimental

Materials and methods

1

1j. We observed a significant diﬀerence in control as well as in All reagents and solvents were purchased of highest commer-

treated cells, with regards to the percentage of live, early apopto- cial grade without any further purification. Dichloro(p-

Dalton Trans.

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Fig. 17 Annexin-V-FITC assay on HT-29 cell line before and after treatment with 11j: (A) typical scatter plot for representation of Annexin-V-FITC

assay where X axis is log of green ﬂuorescence (Annexin-V) and Y axis is log of red ﬂuorescence (PI). The four quadrants represent lower left-live

cells (Q1); lower right (Q2)-early apoptotic; upper left-necrotic (Q4); upper right (Q3)-late apoptotic. (B) Graphical representation of the simpliﬁed

values of the same scatter plot, normalized with the control cells (the percentage of late apoptotic cells of the control was subtracted from the per-

centage of late apoptotic cells of each of the treatment for normalization). Error bars indicate mean ± SD, and represent at least three independent

experiments.

cymene)ruthenium(II) dimer and o-phenylene diamine deriva-

tives were purchased from Sigma Aldrich Chemical Ltd.

Aminothiophenol, aminophenol, pyridine-2-carboxaldehyde,

6-bromo-pyridine-2-carboxaldehyde, quinoline 2-carboxalde-

hyde, and aryl boronic acids were obtained from Alfa Aesar

1

13

chemicals Ltd. H and C NMR spectra were taken on a

Bruker DPX spectrometer at 400 MHz and 100 MHz respect-

Fig. 18 Fluorescence and bright-ﬁeld images of live cells: (a) bright-

ﬁeld image of HeLa cell with compound 11f’ (20 µM in PBS buﬀer); incu- ively with tetramethylsilane as internal standard and the

bation time 2 h. (b) Fluorescence image of HeLa cell with compound 11f’ chemical shifts are reported in ppm units. Thin layer chrom-

(

20 µM in PBS buﬀer); incubation time 2 hours and (c) merged images of

atography was performed on pre-coated silica gel 60 F₂₅₄

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

system in methanol/ethyl acetate mixture and spots were

identified by UV light. The melting points were measured on

Elchem Microprocessor based DT apparatus using open capil-

lary tubes and are uncorrected. Molar conductivities of potent

compounds were checked using TDS-Conductometer. Mass

data were obtained using a Shimadzu ESI-MS-4000 instrument

having 4000 triple quadruple MS, using methanol as the

solvent. UV-Visible spectra were recorded on a JASCO V-730

spectrometer and fluorescence measurements were carried out

using a HITACHI fluorescence spectrophotometer (F-7000)

equipped with a xenon lamp. Fluorescence imaging study was

performed using an Olympus model CKX41 microscope.

Human colon carcinoma cell line (HT-29, Caco-2), cervical

cancer cell line (HeLa) and normal kidney cell line HEK-293

were purchased from the National centre for cell science

a and b. Scale bar 100 µm.

(

NCCS), Pune. Two cell lines were maintained in DMEM

Gibco) supplemented with 10% fetal bovine serum (Himedia,

Fig. 19 Fluorescence images of live cells with compound 8l4 (20 µM in

PBS buﬀer); for diﬀerent incubation times: (a) incubation time 4 h, (b)

incubation time 8 h, (c) incubation time 12 h and (d) incubation time

India), 1% penicillin and streptomycin and 1% Glutmax

2

Gibco, Thermo Scientific, USA) at 37 °C in 5% CO .

(

24 h. Scale bar 400 µm.

Additional 1% sodium pyruvate and 1% non-essential amino

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product was washed with hexane followed by recrystallization

from ethyl acetate. Finally, dark brown needle shaped crystals

of compound 7(g11–g3, l1–l9) were obtained with high yields

(

∼90–95%).

0 mg (0.049 mmol) of [Ru(p-cymene)Cl ] (4) was dissolved

3

2

in 10 ml of water in 50 ml round bottom flask by stirring for

0 min. Thereafter, 2.1 equivalents of previously prepared

1

ligands [7(g1–g3, l1–l3)] were added to the reaction mixture

followed by sonicating for another 15 min. A change in colour

occurred from deep yellow to brown. Then 20 mg (0.121 mmol,

2.5 eqv.) of NH PF was added to the reaction mixture and

Fig. 20 Co-localization images of complex 8l4 (20 μM) in live HeLa

cells after 4 h of incubation with nucleus dye DAPI. Excitation wave-

length is 480 nm. Scale bar 50 μm.

4

6

stirred for another 15 min. The formation of ruthenium

complex was confirmed by TLC using a 100% ethylacetate

solvent system. Finally the resulting solution was evaporated to

acid (NEAA) were added to the media for HeLa cells. The cells dryness. The crude product was further recrystallized from

were trypsinized upon reaching 70–80% confluency using diethyl ether/methanol mixture via the vapour diﬀusion

0

.25% trypsin-EDTA (Thermo Fisher Scientific, USA). For MTT method. The brown fine crystals of complexes [8(g1–g3, l1–l9)]

assay an Elisa reader and 96-well plate have been used.

were obtained with high yield (∼90–95%).

Synthetic procedures

General procedure for one pot synthesis of ruthenium(II)-p-

cymene complexes (5a–h, 5b′–f′, 11a–b, 11d–f, 11h–n, 11b′,

Conclusions

1

1d′–f′, 11k′–l′). A 5 ml aqueous solution of 1,2-diaminoben- In summary, we have established a convenient “on water” pro-

zene/2-aminothiophenol/2-aminophenol (1a–n) and 6-bromo- tocol for the library synthesis of Ru(II)-p-cymene-2-aryl benzi-

pyridine-2-carboxaldehyde (2)/quinoline-2-carboxaldehyde (9) midazole (BIZ), benzothiazole (BTZ) and benzoxazole (BOZ)

(1 : 1 equivalent) was suspended and homogenized. The reac- scaﬀolds under sonication along with the isolation of the

tion mixture was subjected to sonication at 60° C for 1 h. corresponding regioisomers by preparative thin layer chrom-

Formation of the ligand was confirmed by TLC using 25% atography and stability has been supported by density func-

ethylacetate in hexane as the solvent system. Once the product tional theory (DFT). Therefore this technique will be the

2 2

was formed, [Ru(p-cymene)Cl ] (4, 0.5 equivalents w.r.t. start- pioneer to presage the bright prospect of synthesizing a diver-

ing material) was added to the reaction mixture without isolat- sity of oriented newer organoruthenium compounds for anti-

ing the ligands. The reaction was continued under the same cancer screening. Here, most of the reported complexes have

conditions for another 15 min. After that the formation of the displayed significantly more potency as well as marked selecti-

ruthenium complex was confirmed by TLC using 100% ethyla- vity against HeLa and Caco-2 cell lines than the normal kidney

cetate as the solvent system. Finally the resulting solution was cell line (HEK-293) compared to the frequently used cisplatin.

evaporated to dryness. The crude product underwent repeated Again, it is a matter of great interest that more polar regio-

ether washing followed by recrystallization from ethyl acetate isomers except methoxy substituted complexes have conveyed

in hexane (1 : 1) with a significant overall yield (90–97%). more potency and selectivity than the corresponding less polar

Regioisomers were isolated by plate layer chromatography regioisomers in both the cancer cells. A meticulous structure

using various ratios of solvent systems (hexane : ethylactetate).

activity relationship (SAR) study has clearly revealed the substi-

General procedure for synthesis of Suzuki coupled ligand [7 tuent eﬀect of those isomers on cytotoxicity. By virtue of this,

g1–g3, l1–9)] and their corresponding ruthenium(II)-p-cymene complex 11j has emerged as the most potent and selective anti-

(

complexes [8(g1–g3, l1–l9)]. 50 mg of compound 3k/3l, 1.3 cancer agent in Caco-2 and HeLa cancer cell lines with respect

equivalents of aryl boronic acid (6a–j), 0.5 mol% of (PPh ) Pd to the normal cell line. Furthermore, the best anticancer

3

4

and 20 mol% of K

two necked round bottom flask. The reaction was performed at ing the more aggressive HT-29 colorectal cancer cells either by

20 °C in inert atmosphere for 12 h. The completion of reac- creating oxidative stress or following the way of cell cycle

2 3

CO with 5 mL of water were placed in a activity of 11j has been more prominent because of demolish-

1

tion was marked by colour change of the reaction mixture arrest. Beside this, the strong binding eﬃcacy of all the com-

from orange to dark brown. TLC was monitored to confirm the plexes with DNA and BSA has proved that they will be capable

completion of reaction. After that the reaction mixture was of being eﬀectively transported to the cancer cell so that they

poured into crushed ice medium followed by extraction with can easily target the DNA in order to terminate the uncon-

ethyl acetate using a separating funnel. The organic layer (i.e., trolled proliferation of cancer cells. Also, the solubility, stabi-

ethyl acetate) was separated and water layer was drained oﬀ. lity and lipophilicity of these complexes have been accurately

After 4–5 times washing, the organic layers were collected and found to be correlated with the eﬀectiveness of these com-

dried with anhydrous sodium sulphate and the solvent was plexes as successful drug candidates. In line with this, cellular

evaporated to dryness by using a rotary evaporator. The dried imaging study under a fluorescence microscope with the com-

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plexes 11f′ and 8l4 in the HeLa cell line produced red fluo-

rescent images of the cancer cell due to their high quantum

9 M. Shimada, H. Itamochi and J. Kigawa, Cancer Manage.

Res., 2013, 5, 67.

yields whereas the most fluorescent 8l4 exhibits the highest 10 M. Frezza, S. Hindo, D. Chen, A. Davenport, S. Schmitt,

quantum yield and best cytoselectivity in the HeLa cancer cell

line proving itself as a remarkable cancer theranostic agent

D. Tomco and Q. P. Dou, Curr. Pharm. Des., 2010, 16, 1813–

1825.

among all the complexes. At last in light of this work it can be 11 V. Milacic, D. Fregona and Q. P. Dou, Histol. Histopathol.,

foretold that all the complexes may appear as eﬀective anti-

2008, 23, 101–108.

cancer agents in the near future.

12 G. Gasser, I. Ottand and N. Metzler-Nolte, Organometallic

Anticancer Compounds, J. Med. Chem., 2011, 54, 3–25.

1

3 E. Alessio, Eur. J. Inorg. Chem., 2017, 2017, 1549–1560.

14 C. S. Allardyce and P. J. Dyson, Platinum Met. Rev., 2001, 45,

2–69.

Conﬂicts of interest

6

There are no conflicts to declare.

15 A. K. Singh, D. S. Pandey, Q. Xu and P. Braunstein, Coord.

Chem. Rev., 2014, 270, 31–56.

1

6 F. Chen, I. Romero-Canelón, J. J. Soldevila-Barreda,

J.-I. Song, J. P. Coverdale, G. J. Clarkson, J. Kasparkova,

A. Habtemariam, M. Wills, V. Brabec and P. J. Sadler,

Organometallics, 2018, 37, 1555–1566.

Acknowledgements

The authors are grateful to the Department of Science and

Technology, Government of India for supporting the work

through the DST-EMR project grant (EMR/2017/000816). The

authors are grateful to VIT University for providing VIT SEED

funding. We acknowledge DST, New Delhi, India for the

DST-FIST project.

1

7 K. Irena, Curr. Med. Chem., 2006, 13, 1085–1107.

8 R. P. Paitandi, S. Mukhopadhyay, R. S. Singh, V. Sharma,

S. M. Mobin and D. S. Pandey, Inorg. Chem., 2017, 56,

12232–12247.

1

9 Y.-J. Liu, C.-H. Zeng, Z.-H. Liang, J.-H. Yao, H.-L. Huang,

Z.-Z. Li and F.-H. Wu, Eur. J. Med. Chem., 2010, 45, 3087–

3

095.

Notes and references

2

0 A. Bergamo, C. Gaiddon, J. Schellens, J. Beijnen and

1

U. Sliwinska, F. P. Pruchnik, I. Pelinska, S. Ułaszewski,

G. Sava, J. Inorg. Biochem., 2012, 106, 90–99.

A. Wilczok and A. Zajdel, J. Inorg. Biochem., 2008, 102, 21 P.-S. Kuhn, V. Pichler, A. Roller, M. Hejl, M. Jakupec,

1

947–1951.

S. Banerjee and A. R. Chakravarty, Acc. Chem. Res., 2015,

8, 2075–2083.

W. Kandioller and B. Keppler, Dalton Trans., 2015, 44, 659–

668.

22 S. W. Chang, A. R. Lewis, K. E. Prosser, J. R. Thompson,

M. Gladkikh, M. B. Bally, J. J. Warren and C. J. Walsby,

Inorg. Chem., 2016, 55, 4850–4863.

2

3

4

A. Gatti, A. Habtemariam, I. Romero-Canelón, J.-I. Song,

B. Heer, G. J. Clarkson, D. Rogolino, P. J. Sadler and

M. Carcelli, Organometallics, 2018, 37, 891–899.

23 S. Thota, D. A. Rodrigues, D. C. Crans and E. J. Barreiro,

J. Med. Chem., 2018, 61, 5805–5821.

4

5

6

P. J. Jarman, F. Noakes, S. Fairbanks, K. Smitten,

I. K. Griﬃths, H. K. Saeed, J. A. Thomas and C. Smythe, 24 C. G. Hartinger, M. Groessl, S. M. Meier, A. Casini and

J. Am. Chem. Soc., 2019, 141(7), 2925–2937. P. J. Dyson, Chem. Soc. Rev., 2013, 42, 6186–6199.

A. P. Basto, N. Anghel, R. Rubbiani, J. Müller, D. Stibal, 25 E. S. Antonarakis and A. Emadi, Cancer Chemother.

F. Giannini, G. Süss-Fink, V. Balmer, G. Gasser and

J. Furrer, Metallomics, 2019, 11, 462–474.

Pharmacol., 2010, 66, 1–9.

26 A. I. Minchinton and I. F. Tannock, Nat. Rev. Cancer, 2006,

6, 583–592.

(a) A. Alama, B. Tasso, F. Novelli and F. Sparatore,

Organometallic compounds in oncology: implications of 27 F. Wang, A. Habtemariam, E. P. L. van der Geer,

novel organotins as antitumor agents, Drug Discovery

Today, 2009, 14, 500–508; (b) M. J. Piccart, H. Lamb and

J. B. Vermorken, Current and future potential roles of the

platinum drugs in the treatment of ovarian cancer, Ann.

R. Fernández, M. Melchart, R. J. Deeth, R. Aird,

S. Guichard, F. P. A. Fabbiani, P. Lozano-Casal,

I. D. H. Oswald, D. I. Jodrell, S. Parsons and P. J. Sadler,

Proc. Natl. Acad. Sci. U. S. A., 2005, 102, 18269–18274.

Oncol., 2001, 12, 1195–1203; (c) M. Peyrone, Ueber die ein- 28 R. E. Morris, R. E. Aird, P. del Socorro Murdoch, H. Chen,

wirkung des ammoniaks auf platinchlorür, Ann. Chem.

Pharm., 1844, 51, 1–29; (d) B. Rosenberg, L. Vancamp,

J. Cummings, N. D. Hughes, S. Parsons, A. Parkin, G. Boyd

and D. I. Jodrell, J. Med. Chem., 2001, 44, 3616–3621.

J. E. Trosko and V. H. Mansour, Platinum Compounds: a 29 F. P. Rodrigues, Z. A. Carneiro, P. Mascharak, C. Curti and

New Class of Potent Antitumour Agents, Nature, 1969, 222,

85–386.

A. J. Wagstaﬀ, A. Ward, P. Benfield and R. C. Heel, Drugs,

989, 37, 162–190.

E. Raymond, S. Chaney, A. Taamma and E. Cvitkovic, Ann.

Oncol., 1998, 9, 1053–1071.

R. S. da Silva, Coord. Chem. Rev., 2016, 306, 701–707.

30 W. L. Kwong, K. Y. Lam, C. N. Lok, Y. T. Lai, P. Y. Lee and

C. M. Che, Angew. Chem., 2016, 128, 13722–13726.

31 M. Montani, G. V. B. Pazmay, A. Hysi, G. Lupidi,

R. Pettinari, V. Gambini, M. Tilio, F. Marchetti, C. Pettinari

and S. Ferraro, Pharmacol. Res., 2016, 107, 282–290.

3

7

8

1

Dalton Trans.

View Article Online

Paper

Dalton Transactions

32 L. Zeng, Y. Chen, J. Liu, H. Huang, R. Guan, L. Ji and 53 M. Gaba, S. Singh and C. Mohan, Eur. J. Med. Chem., 2014,

H. Chao, Sci. Rep., 2016, 6, 19449. 76, 494–505.

33 S. S. Karki, S. Thota, S. Y. Darj, J. Balzarini and 54 L. Garuti, M. Roberti and G. Bottegoni, Curr. Med. Chem.,

E. DeClercq, Bioorg. Med. Chem., 2007, 15, 6632–6641. 2014, 21, 2284–2298.

34 C. M. Clavel, E. Paunescu, P. Nowak-Sliwinska, 55 C. S. Ra, B. Y. Jung and G. Park, Heterocycles, 2004, 62, 793–

A. W. Griﬃoen, R. Scopelliti and P. J. Dyson, J. Med. Chem.,

015, 58, 3356–3365.

5 D. A. Smithen, H. Yin, M. H. R. Beh, M. Hetu,

T. S. Cameron, S. A. McFarland and A. Thompson, Inorg.

Chem., 2017, 56, 4121–4132.

6 Y. K. Yan, M. Melchart, A. Habtemariam and P. J. Sadler,

Chem. Commun., 2005, 4764–4776.

7 P. C. Bruijnincx and P. J. Sadler, Curr. Opin. Chem. Biol.,

802.

2

56 S. Aiello, G. Wells, E. L. Stone, H. Kadri, R. Bazzi,

D. R. Bell, M. F. G. Stevens, C. S. Matthews, T. D. Bradshaw

and A. D. Westwell, J. Med. Chem., 2008, 51, 5135–5139.

57 D. Alagille, R. M. Baldwin and G. D. Tamagnan,

Tetrahedron Lett., 2005, 46, 1349–1351.

58 T. Eicher and S. Hauptmann, in The Chemistry of

Heterocycles, Wiley-VCH, Weinheim, Germany, 2nd edn,

2003, pp. 316.

3

2

008, 12, 197–206.

8 S. W. Magennis, A. Habtemariam, O. Novakova, J. B. Henry, 59 Y. L. Chen, C. J. Huang, Z. Y. Huang, C. H. Tseng,

S. Meier, S. Parsons, I. D. Oswald, V. Brabec and

P. J. Sadler, Inorg. Chem., 2007, 46, 5059–5068.

F. S. Chang, S. H. Yang, S. R. Lin and C. C. Tzeng, Bioorg.

Med. Chem., 2006, 14, 3098–3105.

3

9 T. Bugarcic, O. Nováková, L. Zerzánková, O. Vrána, 60 Y. L. Chen, Y. L. Zhao, C. M. Lu, C. C. Tzeng and

J. Kašpárková, A. Habtemariam, S. Parsons, P. J. Sadler and

V. Brabec, J. Med. Chem., 2008, 51, 5310–5319.

0 F. Wang, J. Xu, A. Habtemariam, J. Bella and P. J. Sadler,

J. Am. Chem. Soc., 2005, 127, 17734–17743.

J. P. Wang, Bioorg. Med. Chem., 2006, 14, 4373–4378.

61 Y. Feng, E. Lau, M. Scortegagna, C. Ruller, S. K. De,

E. Barile, S. Krajewski, P. Aza-Blanc, R. Williams,

A. B. Pinkerton, M. Jackson, L. Chin, M. Pellecchia,

M. Bosenberg and Z. A. Ronai, Pigm. Cell Melanoma Res.,

2013, 26, 136–142.

4

1 J. Furrerand and G. Süss-Fink, Coord. Chem. Rev., 2016,

3

09, 36–50.

2 A. F. Peacock and P. J. Sadler, Chem. – Asian J., 2008, 3, 62 S. Adsule, V. Barve, D. Chen, F. Ahmed, Q. P. Dou,

1

890–1899.

S. Padhye and F. H. Sarkar, J. Med. Chem., 2006, 49, 7242–

7246.

63 T. Nakamura, M. Oka, K. Aizawa, H. Soda, M. Fukuda,

K. Terashi, K. Ikeda, Y. Mizuta, Y. Noguchi, Y. Kimura,

T. Tsuruo and S. Kohno, Biochem. Biophys. Res. Commun.,

1999, 255, 618–624.

3 K. D. Upadhyay, N. M. Dodia, R. C. Khunt, R. S. Chaniara

and A. K. Shah, ACS Med. Chem. Lett., 2018, 9, 283–288.

4 O. Afzal, S. Kumar, M. R. Haider, M. R. Ali, R. Kumar,

M. Jaggi and S. Bawa, Eur. J. Med. Chem., 2015, 97, 871–

9

10.

4

5 J. Camacho, A. Barazarte, N. Gamboa, J. Rodrigues, 64 B. S. Reddy and A. Rivenson, Cancer Res., 1993, 53, 3914–

R. Rojas, A. Vaisberg, R. Gilman and J. Charris, Bioorg.

Med. Chem., 2011, 19, 2023–2029.

6 M. L. Richards, S. C. Lio, A. Sinha, K. K. Tieu and

3918.

65 A. Shi, T. A. Nguyen, S. K. Battina, S. Rana, D. J. Takemoto,

P. K. Chiang and D. H. Hua, Bioorg. Med. Chem. Lett., 2008,

18, 3364–3368.

4

J. C. Sircar, J. Med. Chem., 2004, 47, 6451–6454.

7 A. Sharma, V. Luxami and K. Paul, Bioorg. Med. Chem. Lett., 66 M. E. Wall, M. C. Wani, C. E. Cook, K. H. Palmer,

2

013, 23, 3288–3294.

A. T. McPhail and G. A. Sim, J. Am. Chem. Soc., 1966, 88,

3888–3890.

8 Y. Tong, J. J. Bouska, P. A. Ellis, E. F. Johnson, J. Leverson,

X. Liu, P. A. Marcotte, A. M. Olson, D. J. Osterling and 67 F. Gumus, I. Pamuk, T. Ozden, S. Yildiz, N. N. Dirl,

M. Przytulinska, J. Med. Chem., 2009, 52, 6803–6813.

9 B. Narasimhan, D. Sharma and P. Kumar, Med. Chem. Res.,

E. Oksuzoglu, S. Gur and A. Ozkul, J. Inorg. Biochem., 2003,

94, 255–262.

4

2

012, 21, 269–283.

68 M. M. Alonso, N. Busto, F. A. Jalón, B. R. Manzano,

J. M. Leal, A. M. Rodríguez, B. García and G. Espino, Inorg.

Chem., 2014, 53, 11274–11288.

5

0 K. Kawasaki, M. Masubachi, K. Morikami, S. Sogebe,

T. Aoyama, H. Ebiike, S. Niizuma, M. Hayase,

T. Fujii, K. Sakata, H. Shindoh, Y. Shiratori, Y. Aoki, 69 S. Nikolić, D. M. Opsenica, V. Filipović, B. Dojčinović,

T. Ohtsuka and N. Shimma, Bioorg. Med. Chem. Lett., 2003,

3, 87–91.

S. Aranđelović, S. Radulović and S. G. Šipka,

Organometallics, 2015, 34, 3464–3473.

1

5

1 R. S. Ferreira, M. A. Dessoy, I. Pauli, M. L. Souza, R. Krogh, 70 I. N. Stepanenko, A. Casini, F. Edafe, M. S. Novak,

A. I. L. Sales, G. Oliva, L. C. Dias and A. D. Andricopulo,

V. B. Arion, P. J. Dyson, M. A. Jakupec and B. K. Keppler,

J. Med. Chem., 2014, 57, 2380–2392.

Inorg. Chem., 2011, 50, 12669–12679.

2 S. R. LaPlante, M. Boes, C. Brochu, C. Chabot, 71 (a) G. R. Jadhav, S. Sinha, M. Chhabra and P. Paira, Bioorg.

R. Coulombe, J. R. Gillard, A. Jakalian, M. Poirier,

J. Rancourt, T. Stammers, B. Thavonekham, P. L. Beaulieu,

G. Kukolj and Y. S. Tsantrizos, J. Med. Chem., 2014, 57,

Med. Chem. Lett., 2016, 26, 2695–2700; (b) S. K. Subran,

S. Banerjee, A. Mondal and P. Paira, New J. Chem., 2016, 40,

10333–10343; (c) A. Mondal, S. De, S. Maiti, B. Sarkar,

A. K. Sk, R. Jacob, A. Moorthy and P. Paira, J. Photochem.

1

845–1854.

Dalton Trans.

View Article Online

Dalton Transactions

Paper

Photobiol., B, 2018, 178, 380–394; (d) S. De, S. Ray 83 A. Chandra, K. Singh, S. Singh, S. Sivakumar and

Chaudhuri, A. Panda, G. R. Jadhav, R. S. Kumar, A. K. A. Patra, Dalton Trans., 2016, 45, 494–497.

P. Manohar, N. Ramesh, A. Mondal, A. Moorthy, 84 D. M. Townsend and K. D. Tew, Oncogene, 2003, 22, 7369–

S. Banerjee, P. Paira and A. K. Sk, New J. Chem., 2019, 43, 7375.

291–3302; (e) B. Sarkar, A. Mondal, Y. Madaan, N. Roy, 85 A. L. Ortega, S. Mena and J. M. Estrela, Cancers, 2011, 3,

3

A. Moorthy, Y.-C. Kuo and P. Paira, Dalton Trans., 2019, 48,

1285–13210.

1

2257–12271.

86 B. G. Campling, K. Baer, H. M. Baker, Y. M. Lam and

S. P. Cole, Br. J. Cancer, 1993, 68, 327–335.

7

2 P. Tundo, P. Anastas, D. S. Black, J. Breen, T. Collins,

S. Memoli, J. Miyamoto, M. Polyakoﬀ and W. Tumas, Pure 87 J. X. Ong, C. W. Yap and W. H. Ang, Inorg. Chem., 2012, 51,

Appl. Chem., 2000, 72, 1207–1228. 12483–12492.

3 Y. Chen, L. Qiao, L. Ji and H. Chao, Biomaterials, 2014, 35, 88 S. Bi, Y. Sun, C. Qiao, H. Zhang and C. Liu, J. Lumin., 2009,

–13. 129, 541–547.

4 C. Jin, J. Liu, Y. Chen, L. Zeng, R. Guan, C. Ouyang, 89 M. T. Larsen, M. Kuhlmann, M. L. Hvam and K. A. Howard,

7

2

L. Jiand and H. Chao, Chem. – Eur. J., 2015, 21, 12000–

2010.

Mol. Cell. Ther., 2016, 4, 3.

90 J. C. Pessoa and I. Tomaz, Curr. Med. Chem., 2010, 17,

1

7

5 M. Kubanik, H. Holtkamp, T. Söhnel, S. M. F. Jamieson

and C. G. Hartinger, Organometallics, 2015, 34, 5658–5668.

6 (a) M. Sunita, B. Anupama, B. Ushaiah and

3701–3738.

91 A. Levina, A. Mitra and P. A. Lay, Metallomics, 2009, 1, 458–

7

470.

C. GyanaKumari, Arabian J. Chem., 2017, 10, S3367–S3374; 92 K. Purkait, S. Karmakar, S. Bhattacharyya, S. Chatterjee,

(

b) W. J. Geary, Coord. Chem. Rev., 1971, 7, 81–122; (c) I. Ali,

S. K. Dey and A. A. Mukherjee, Dalton Trans., 2015, 44,

5969–5973.

93 N. Shahabadi and M. Mohammadpour, Spectrochim. Acta,

Part A, 2012, 86, 191–195.

W. A. Wani and K. Saleem, Synth. React. Inorg., Met.-Org.,

Nano-Met. Chem., 2013, 43, 1162–1170.

7 S. Bhattacharyya, K. Purkait and A. Mukherjee, Dalton

7

8

Trans., 2017, 46, 8539.

94 U. Sen, P. Shenoy and B. Bose, Cell Biol. Int., 2017, 41(10),

1127–1145.

95 G. Cosa, K.-S. Focsaneanu, J. R. N. McLean, J. P. McNamee

and J. C. Scaiano, Photochem. Photobiol., 2001, 73, 585–599.

96 M. A. Kang, E.-Y. So, A. L. Simons, D. R. Spitz and T. Ouchi,

Cell Death Dis., 2012, 3(1), e249.

97 B. Bose, A. R. Najwa and S. S. P, Mol. Biotechnol., 2019,

61(2), 145–152.

98 U. S. Srinivasa, B. W. Q. Tana, B. A. Vellayappan and

A. D. Jeyasekharanac, Redox Biol., 2019, 25, 101084.

8 A. A. R. Despaigne, J. G. da Silva, P. R. da Costa, R. G. dos

Santos and H. Beraldo, Molecules, 2014, 19, 17202–17220.

9 O. Novakova, H. Chen, O. Vrana, A. Rodger, P. J. Sadler and

V. Brabec, Biochemistry, 2003, 42, 11544–11554.

0 H. Chen, J. A. Parkinson, R. E. Morris and P. J. Sadler,

J. Am. Chem. Soc., 2003, 125, 173–186.

1 F. Dimiza, F. Perdih, V. Tangoulis, I. Turel, D. P. Kessissoglou

and G. Psomas, J. Inorg. Biochem., 2011, 105, 476–489.

2 M. Ganeshpandian, R. Loganathan, E. Suresh,

A. Riyasdeen, M. A. Akbarsha and M. Palaniandavar, Dalton 99 D. D. Zio, V. Cianfanelli and F. Cecconi, Antioxid. Redox

Trans., 2014, 43, 1203–1219. Signaling, 2013, 19(6), 559–571.

Dalton Trans.

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