Similarities and differences in d6 low-spin ruthenium, rhodium and iridium half-sandwich complexes: synthesis, structure, cytotoxicity and interaction with biological targets

JBIC Journal of Biological Inorganic Chemistry

https://doi.org/10.1007/s00775-019-01665-2

ORIGINAL PAPER

Similarities and diferences in d⁶low‑spin ruthenium, rhodium

and iridium half‑sandwich complexes: synthesis, structure,

cytotoxicity and interaction with biological targets

Agnieszka Gilewska¹· Barbara Barszcz¹· Joanna Masternak¹· Katarzyna Kazimierczuk²· Jerzy Sitkowski^3,4

Joanna Wietrzyk⁵· Eliza Turlej⁵

·

Received: 5 February 2019 / Accepted: 8 May 2019

Abstract

In this paper, we discussed the similarities and diferences in d⁶low-spin half-sandwich ruthenium, rhodium and iridium

complexes containing 2,2′-biimidazole (H₂biim). Three new complexes, {[RuCl(H₂biim)(η⁶-p-cymene)]PF₆}₂·H₂O (1), [(η⁵-

Cp)RhCl(H₂biim)]PF₆(2), and [(η⁵-Cp)IrCl(H₂biim)]PF₆(3), were fully characterized by CHN, X-ray difraction analysis,

UV–Vis, FTIR, and ¹H, ¹³C and ¹⁵N NMR spectroscopies. The complexes exhibit a typical pseudooctahedral piano-stool

geometry, in which the aromatic arene ring (p-cymene or Cp) forms the seat, while the bidentate 2,2′-biimidazole and chloride

ion form the three legs of the piano stool. Moreover, the cytotoxic activities of the compounds were examined in the LoVo,

HL-60, MV-4-11, MCF-7 human cancer cell lines and BALB/3T3 normal mouse fbroblasts. Notably, the investigated com-

plexes showed no cytotoxic efects towards the normal BALB/3T3 cell line compared to cisplatin, which has an IC₅₀value of

2.20 µg. Importantly, 1 displayed the highest activity against HL-60 (IC₅₀4.35 µg). To predict a binding mode, we explored

the potential interactions of the metal complexes with CT-DNA and protein using UV absorption and circular dichroism.

The obtained data suggest that the complexes could interact with CT-DNA via an outside binding mode. Moreover, binding

of the complexes with the GSH via UV–Vis and ESI mass spectra was determined. Comparative studies have shown that the

rhodium complex (2) is the most GSH reactive, which is probably responsible for its deactivation towards LoVo and MCF-7

tumour cells. The infuence of the metal ion on the biological activity of isostructural Rh(III) and Ir(III) complexes was an

important goal of the presented investigation.

Keywords Organoruthenium(II) · Organorhodium(III) · Organoiridium(III) · 2,2′-Biimidazole · Antitumor activity

Electronic supplementary material The online version of this

article (https://doi.org/10.1007/s00775-019-01665-2) contains

supplementary material, which is available to authorized users.

Introduction

By screening the literature connected to the efort of sci-

entists attempting to resolve the problems associated with

cancer treatment, it should be noted that organometal-

lic, bioinorganic and bioorganic chemistry are important

branches of this subject. Contemporary medicine, includ-

ing chemotherapy, is mainly based on Pt-anticancer drugs

to overcome the following important problems: (1) drug

resistance, (2) toxic side efects, and (3) lack of activity

against several types of cancer [1]. Therefore, scientists

have been paying special attention to organometallic com-

plexes in recent years when looking for new chemothera-

peutics. Special eforts have been focused on ruthenium

complexes, such as [ImH][trans-Ru(Im)₂Cl₄] (KP1019,

Im=imidazole) [2, 3] and [ImH][trans-Ru(DMSO)(Im)Cl₄]

(NAMI-A, Im=imidazole, DMSO=dimethylsulfoxide) [4,

* Joanna Masternak

joanna.masternak@ujk.edu.pl

1

Institute of Chemistry, Jan Kochanowski University, 15G

Świętokrzyska Str., 25-406 Kielce, Poland

2

Department of Inorganic Chemistry, Faculty of Chemistry,

Gdańsk University of Technology, 11/12 G. Narutowicza Str.,

80-233 Gdańsk, Poland

3

Institute of Organic Chemistry, Polish Academic of Science,

Kasprzaka 44/52, 01-224 Warsaw, Poland

4

National Medicines Institute, Chełmska 30/34,

00-725 Warsaw, Poland

5

Institute of Immunology and Experimental Therapy, Polish

Academy of Sciences, 12 Weigla Str., 53-114 Wrocław,

Poland

Vol.:(0123456789)

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JBIC Journal of Biological Inorganic Chemistry

5], as anti-tumour and antimetastatic agents, respectively.

This research has inspired the search for metallodrugs

based on an increasing range of transition metals, such as

gold, rhodium, iridium, and osmium. Going in this direc-

tion, the group of Sadler and Dyson [6, 7] developed ruthe-

nium half-sandwich complexes that ar e characterized by

similar ligand exchange kinetics to platinum(II) anticancer

drug candidates and whose biological activities are mainly

afected by the nature of the chelating ligands (two legs of

the piano-stool) and arene ligands that form the piano-stool

complexes. Alessio et al. [8] worked on a categorization

of metal anticancer compounds based on their modes of

action and classifed these complexes into groups of func-

tional compounds. Additionally, the anticancer activities of

functional compounds will depend on their metal centres,

electron confguration (depending on the place in which the

metal is situated in the periodic table), hard-soft nature and

oxidation state. Interestingly, a study from Sadler’s group

[9–11] focused on the biological activity of isostructural

piano-stool complexes that only difered in the metal centre

showed that the ruthenium(II) compound RM175 and its

osmium(II) analogue AFAP51 exhibited signifcant difer-

ences in their anticancer activities in vitro and in vivo. In

contrast, Dyson et al. [12] compared the osmium analogue

of RAPTA-C, as well as the CpRh(III) and CpIr(III) deriva-

tives to the NAMI-A Ru(III) complex, and demonstrated

that both the Rh(III) and Ir(III) compounds are inactive.

Moreover, Samuelson et al. [13] demonstrated that the

hydrogen bond stability of half-sandwich Ru(II) complexes

with heterocycles plays an important role in the anticancer

mechanism. The presence of strong hydrogen bonding was

better at stabilizing the complexes. However, in the absence

of stabilizing interactions, the ligand dissociates in solu-

tion, which was confrmed by NMR spectroscopy studies.

These structure–activity relationships based on hydrogen

bonding may have modulated the drug action mechanism

at both the cellular and molecular levels [14, 15]. During

past several years, we have been working to wards Ru(II)

piano-stool complexes, including {[RuCl(L¹)(η⁶-p-cymene)]

PF₆}₂·H₂O (L¹=2,2′-bis(4,5-dimethylimidazole)) [16], [(η⁶-

p-cymene)RuCl(2,2′-PyBIm)]PF₆[17 ] and Rh(II) dimeric

complex (Et₃NH)₂[Rh₂(µ₂-L)₄Cl₂] (L⁻= thiophene-2-car-

boxylate) [18], as well as focusing on their cytotoxic activ-

ity and DNA/protein binding. The main goal of our eforts

was to obtain a potential chemotherapeutic that does not

afect normal cell lines but possesses better cytotoxic activ-

ity against cancer cell lines. In continuation of our work,

which was inspired by Sadler and Dyson [9–12], we focused

on determining the infuence of complexes t hat difer only

in their central metal ion [Rh(III) and Ir(III)] on cytotoxic

activity. Additionally, we attempted to estimate whether the

presence of hydrogen bonds and noncovalent interactions

(HS analysis) infuenced the stability and cytotoxic activity

of the obtained complexes. Therefore, the main purposes of

the present work were as follows: (1) synthesis and full phys-

icochemical characterization of d⁶low-spin arene ruthenium

and isostructural pentamethylcyclopentadienyl rhodium

and iridium half-sandwich complexes with 2,2′-biimida-

zole, (2) determination and comparison of lipophilicity and

in vitro cytotoxic properties, and (3) investigation of the

types of interactions with biological targets (DNA, HSA,

and GSH). To achieve these goals, elemental analysis; sin-

1

gle-crystal X-ray difraction; H, ¹³C, and ¹⁵N NMR; and

infrared and UV–Vis spectroscopy methods were success-

fully applied. Moreover, the potential cytotoxic efects of

the {[RuCl(H₂biim)(η⁶-p-cymene)]PF₆}₂·H₂O (1), [(η⁵-Cp)

RhCl(H₂biim)]PF₆(2), and [(η⁵-Cp)IrCl(H₂biim)]PF₆(3)

complexes were examined towards the LoVo (colorectal

adenoma), MV-4-11 (myelomonocytic leukaemia), MCF-7

(breast adenocarcinoma), HL-60 (promyelocytic leukaemia)

and normal BALB/3T3 (mouse fbroblast) cell lines. With

the aim of gaining deeper insights into the mechanisms of

action, we investigated the interactions with DNA, HSA via

UV–Vis and CD methods. Moreover, binding of the com-

plexes with the GSH via UV–Vis and ESI mass spectra was

determined.

Experimental section

Materials and physical measurements

[(η⁶-p-Cymene)Ru(µ-Cl)Cl]₂(97%), [CpRh(μ-Cl)Cl]₂

(99%), [CpIr(μ-Cl)Cl]₂(99%), 2,2′-biimidazole (H₂biim),

NH₄PF₆(99%), cisplatin, 5,5′-dithio-bis(2-nitrobenzoic acid)

(DTNB) (99%), human serum albumin (HSA) (lyophilized

powder, < 0.007% fatty acid), l-glutathione reduced form

(GSH) (lyophilized powder, > 98%) were purchased from

Sigma-Aldrich. Analytical grade solvents were purchased

from Chempur. Tris(hydroxymethyl)aminomethane-HCl

(5 mM Tris–HCl/50 mM NaCl, pH 7.2) and PBS (10 mM,

pH=7.4) bufer were prepared using MiliQ water.

Elemental analysis was run on an Elementar Vario

Micro Cube analyzer. The FTIR spectra were recorded

on a Nicolet 380 spectrophotometer in the spectral range

4000–500 cm⁻¹using the ATR-difusive refection method.

UV–Vis measurements in methanolic solutions were per-

formed on UV–Vis spectrophotometer (V-630 Jasco) using

1 cm cuvettes against methanol as reference solutions. All

absorbance measurements were recorded at room tempera-

ture and the concentrations 1.0×10⁻⁴for ligand (H₂biim)

and complex 1, 2, 3. Circular dichroism spectra were

recorded with a Jasco J-815 spectropolarimeter (Jasco Inc.)

equipped with a Jasco Peltier-type temperature controller.

The ¹H, ¹³C and ¹⁵N NMR spectra were recorded at 25 °C in

DMSO-d₆solutions on a Varian VNMRS-600 (¹H 600 MHz,

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JBIC Journal of Biological Inorganic Chemistry

¹³C 150 MHz, ¹⁵N 43.5 MHz) spectrometer equipped with a

5-mm PFG AutoXID (¹H/X¹⁵N-³¹P) probe. Standard pulse

sequences were used except the ¹H-{¹⁵N} correlation. Gra-

dient-enhanced IMPACT-HMBC [19] ¹H-{¹⁵N} correlation

spectra were optimized for a coupling constant of 6 Hz with

the following experimental conditions: an acquisition time

of 0.3 s, spectral windows of 8000 (F2) and 18,000 (F1)

Hz, 2700 complex data points in t2, 256 or 512 complex

data points in t1, 4 scans per increment, 30 ms WURST-2

mixing sequence centered within the 60 ms preparation

interval (ASAP2) and a 150 Ernst angle as the excitation

pulse [20]. The data were processed with linear prediction

in t1 followed by zero flling in both dimensions. Gauss-

ian weighting functions were applied in both domains prior

to Fourier transformation. All the spectra were referenced

according to IUPAC recommendations [21]. The ESI mass

spectra of free GSH, GSH-cisplatin, GSH-complexes (1–3)

were recorded on a micrOTOF-Q II (Brucker) equipped with

syringe pump. The dry gas fow rate was at 4.0 l/min; the

dry heater was operated at 180 °C; the capillary voltage was

set at 4.500 V and collision energy was variable and set

between 2 and 10 eV. The sample solutions were prepared

in acetonitrile–methanol mixture (1:1).

Union Road, Cambridge CB2 1EZ, UK (Fax: (+44) 1223-

336-033; Email: deposit@ccdc.cam.ac.uk).

Synthesis of complexes 1–3

Preparation of {[RuCl(H₂biim)(η⁶‑p‑cymene)]PF₆}₂·H₂O (1)

A solution of the precursor compound [(η⁶-p-cymene)

Ru(µ-Cl)Cl]₂(0.25 mmol, 0.153 g), ligand 2,2′-biimida-

zole (0.5 mmol, 0.067 g) and NH₄PF₆(0.5 mmol, 0.0815 g)

was dissolved in a 20 ml mixed solution (CH₃OH:CH₂Cl₂;

3 V:1 V) with one drop of H₂O. The reaction mixture

was refuxed for 12 h (~65 °C). After 2 weeks, the result-

ing orange crystals suitable for single-crystal X-ray dif-

fraction were filtered and dried in a vacuum box. The

crystals were collected at 75% yield. Anal. Calc. (%) for

C₃₂H₄₂Cl₂F₁₂N₈OP₂Ru₂: C, 33.95; H, 3.67; N, 10.19. Found:

1

C, 33.42; H, 3.50; N, 10.22; H NMR (600 MHz, DMSO-

d₆, δ ppm): 1.00 [6H, d, (C_28/29H₃)₂C₂₇H)]; 2.61 [1H, m,

(C_28/29H₃)₂C₂₇H)]; 6.00 [2H, C₂₃H, C₂₅H, (p-cymene)];

5.80 [2H, C₂₂H, C₂₆H, (p-cymene)]; 2.07 (3H, s, C₃₀H₃);

7.84 (2H, s, C_4/4′H); 7.50 (2H, s, C_5/5′H); 12.91 (2H, s,

N_1/1′H). ¹³C NMR (150 MHz, DMSO-d₆, δ ppm): 22.1 (C₂₈,

C₂₉); 30.9 (C₂₇); 102.6 (C₂₄); 83.3 (C₂₃, C₂₅); 81.7 (C₂₂,

C₂₆); 99.8 (C₂₁); 18.7 (C₃₀); 138.2 [C_2/2′(H₂biim)]; 131.8

[C_4/4′(H₂biim)]; 121.1 [C_5/5′(H₂biim)]. ¹⁵N NMR (43.5 MHz,

DMSO-d₆, δ ppm): − 218.5 (N_1/1′H); − 181.7 (N_3/3′). IR

(cm⁻¹): 3260(br), 3145(w), 1527(ms), 1468(ms), 1321(ms),

1188(ms), 1093(ms), 849(vs), 754(ms), 679(ms), 557(s).

Crystal structure determination and refnement

Single-crystal X-ray difraction data of compounds 1–3 were

collected at 120(2) K on a Stoe IPDS-2T difractometer

with graphite-monochromated Mo-Kα radiation. Data col-

lection and image processing were performed with X-Area

1.75 (STOE & Cie GmbH, 2015) [22]. Intensity data were

scaled with LANA (part of X-Area) to minimize diferences

of intensities of symmetry-equivalent refections (multi-scan

method). The crystal was thermostated in nitrogen stream at

120 K using CryoStream-800 device (Oxford CryoSystem,

UK) during the entire experiment. The structure was solved

with direct methods and w as refined with the SHELX-

2016/6 program package [23, 24] with the f ull-matrix least

squares procedure based on F2. The Olex [25] and WingX

[26] program suites were used to prepare the fnal version

o f CIF fles. Diamond [27] was used to prepare the fgures.

All C–H type hydrogen atoms were attached at their geo-

metrically expected positions and refned as riding on heav-

ier atoms with the usual constraints. In the all compounds,

residual electron density is somewhat high and localizes near

the heavier Ru/Ir atoms, and is not attributable to an addi-

tional atom. A summary of crystallographic data is shown

in Table S1. Crystallographic data for structure of 1, 2 and

3 reported in this paper have been deposited with the Cam-

bridge Crystallographic Data Center as supplementary pub-

lications No. CCDC 1881107–1881109. Copies of the data

can be obtained free of charge on application to CCDC, 12

Preparation of [(η⁵‑Cp)RhCl(H₂biim)]PF₆(2)

A mixture of the precursor compound [CpRh(μ-Cl)Cl]₂

(0.1 mmol, 0.0618 g) in 5 ml of methanol was added drop-

wise to a 15 ml mixed solution of the ligand 2,2′-biimidazole

(CH₃OH:CH₂Cl₂; 3 V:1 V) (0.2 mmol, 0.026 g) and NH₄PF₆

(0.25 mmol, 0.0407 g). The reaction mixture was stirred and

refuxed for 10 h. After that step, the solution was allowed to

stand at room temperature to crystallize. The resulting com-

plex was fltered of and dried in a vacuum box after a week.

The crystals were collected at 62% yield. Anal. Calc. (%) for

C₁₆H₂₁ClF₆N₄PRh: C, 34.77; H, 3.83; N, 10.13. Found: C,

34.20; H, 3.51; N, 9.64; ¹H NMR (600 MHz, DMSO-d₆, δ

ppm): 1.68 [15H, (Cp–C_11–15H₃)]; 7.56 (2H, s, C_4/4′H); 7.54

(2H, s, C_5/5′H); 12.90 (2H, s, N_1/1′H). ¹³C NMR (150 MHz,

DMSO-d₆, δ ppm): 9.3 (Cp–C_11–15H₃); 95.4 (Cp–C_6–10

)

(J=8.2 Hz); 138.2 [C_2/2′(H₂biim)]; 128.2 [C_4/4′(H₂biim)];

121.4 [C_5/5′(H₂biim)]. ¹⁵N NMR (43.5 MHz, DMSO-d₆, δ

ppm): − 218.7 (N_1/1′H); − 173.8 (N_3/3′) (J = 18.6 Hz). IR

(cm⁻¹): 3169(w), 1531(ms), 1433(ms), 1179(ms), 1095(ms),

1021(ms), 836(vs), 746(ms), 608(ms), 557(s).

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JBIC Journal of Biological Inorganic Chemistry

Preparation of [(η⁵‑Cp)IrCl(H₂biim)]PF₆(3)

additional 72 h. Cisplatin, a platinum-based chemotherapy

drug, was used as a standard comparison treatment. The

ruthenium complex stock solution was always freshly pre-

pared by dissolving 1 mg of compound in 100 µl DMSO.

After that step, the obtained solution was diluted in culture

medium to reach the required concentrations (ranging from

0.1 to 100 µg/ml). The fnal concentration of DMSO in the

cell culture medium did not exceed 0.1% (v/v), which was

shown to have no efect on cell growth. The in vitro cyto-

toxic activity results were expressed as IC₅₀values, which

is the concentration of compound (in µg) that inhibits the

tumour cell proliferation rate by 50% compared with the

untreated control cells. The results were calculated from

2 to 3 independent experiments.

Complex 3 was prepared similar to complex (2) using

[CpIr(μ-Cl)Cl]₂(0.1 mmol, 0.0797 g) as the metal source.

The yellow crystals appeared after a week and were col-

lected at 49% yield. Anal. Calc. (%) for C₁₆H₂₁ClF₆N₄PIr:

C, 29.93; H, 3.29; N, 8.73. Found: C, 30.17; H, 3.03;

1

N, 8.78; H NMR (600 MHz, DMSO-d₆, δ ppm): 1.69

[15H, (Cp–C_11–15H₃)]; 7.56 (2H, s, C_4/4′H); 7.54 (2H,

s, C_5/5′H); 13.15 (2H, s, N_1/1′H). ¹³C NMR (150 MHz,

DMSO-d₆, δ ppm): 9.1 (Cp–C_11–15H₃); 87.1 (Cp–C_6–10);

139.9 [C_2/2′(H₂biim)]; 127.8 [C_4/4′(H₂biim)]; 121.8

[C_5/5′(H₂biim)]. ¹⁵N NMR (43.5 MHz, DMSO-d₆, δ ppm):

− 218.1 (N_1/1′H); − 189.7 (N_3/3′). IR (cm⁻¹): 3172(w),

1533(ms), 1458(ms), 1181(ms), 1093(ms), 1025(ms),

835(vs), 740(ms), 652(ms), 565(s).

Determination of stability

The stability of complexes 1, 2 and 3 was examined in a mix-

ture of one drop of DMSO and 5 mM Tris–HCl/50 mM NaCl

bufer at pH 7.2 and determined by UV–Vis spectroscopy.

The solution was maintained at room temperature (rt). After

0 and 24 h, electronic spectra were recorded with a Jasco 630

UV–Vis spectrophotometer. To confrm the stability of the

complexes, ¹³C NMR spectra were recorded in DMSO at

room temperature at two diferent time points: immediately

after dissolution and after 24 h.

Cells and cytotoxicity assay

The cytotoxicity of the investigated complexes (1–3) was

determined by microculture with sulforhodamine B SRB

(all adherent cells) and MTT ([3-(4,5-dimethyl(thiazol-

2-yl)-2,5-diphenyltetrazoliumbromide)]) [for cells in sus-

pension (leukaemia cells)] assays [28, 29] with the fol-

lowing four tumour cell lines: colorectal adenoma (LoVo),

promyelocytic leukaemia (HL-60), myelomonocytic leu-

kaemia (MV-4-11), and breast adenocarcinoma (MCF-7),

as well as a healthy mouse fbroblast (BALB/3T3) cell

line. All tested cell lines were obtained from the Ameri-

can Type Culture Collection (Rockville, MD, USA) and

maintained at the Cell Culture Collection of the Institute

of Immunology and Experimental Therapy (Wrocław,

Poland). The colorectal adenoma cells were cultured in a

mixture of F12K-ATCC medium. The breast cancer cells

were bred in Eagle and MEM medium supplemented with

glutamine and insulin (Sigma-Aldrich). The myelomono-

cytic leukaemia carcinoma cells were grown in a mixture

of RPMI 1640 w/GLUTAMAX-I medium (Gibco) supple-

mented with pyruvate (Sigma-Aldrich). The promyelocytic

leukaemia carcinoma cells were grown in a mixture of

RPMI 1640 w/GLUTAMAX-I medium (Gibco) supple-

mented with glucose (Sigma-Aldrich). The fbroblast cells

were cultured in DMEM medium (Gibco) supplemented

with glutamine (Sigma-Aldrich). Moreover, all the tested

media were supplemented with 10% HealthCare foetal

bovine serum (Sigma-Aldrich), streptomycin (Sigma-

Aldrich) and penicillin (Polfa Tarchomin). All cell lines

were grown at 37 °C in a humidifed atmosphere with 5%

CO₂. The cells were plated in 96-well plates (Sarstedt,

Germany) at a density of 10⁴cells per well and were cul-

tured in 100 µl of culture medium. After a 24 h incuba-

tion period, the cells were treated with compound for an

Partition coefcient

The lipophilicity of 1, 2, 3 was determined using the shake-

fask method [30]. The compounds (ca. 10⁻⁴M) were dis-

solved in aqueous phase (5 mM Tris–HCl/50 mM NaCl

bufer pH 7.2), and this solution was added to n-octan-1-ol.

The mixtures were mechanically mixed in tubes for 1 h,

12 h, 24 h, 30 h and 48 h until reaching solubility equilib-

rium followed by centrifugation (5000 rpm, 15 min). After

separation, the phases were analysed by UV–Vis spectros-

copy to determine the amount of the compound in the water

phase. The absorption values before and after shaking were

compared. The phase water–octanol equilibrium was estab-

lished after 30 h for complexes 2 and 3 and after 24 h for

complex 1. The partition coefcient (log P) for each com-

pound was calculated based on the Lambert–Beer Law to

determine the log P values. The procedures were repeated

six times for each complex, and the values of the log P are

the mean standard deviation.

Interaction with biomolecules

Calf thymus DNA

Binding of {[RuCl(H₂biim)(η⁶-p-cymene)]PF₆}₂·H₂O (1),

[(η⁵-Cp)RhCl(H₂biim)]PF₆(2) and [(η⁵-Cp)IrCl(H₂biim)]

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JBIC Journal of Biological Inorganic Chemistry

PF₆(3) with polymeric CT-DNA was evaluated via UV–Vis

absorption titrations and CD spectroscopy. The stock solu-

tions of the complexes and CT-DNA were prepared using

5 mM Tris–HCl/50 mM NaCl bufer at pH 7.2. The DNA

concentration per nucleotide was determined by UV absorp-

tion measurements using the molar absorption coefcient

ε₂₅₈=6 600 M⁻¹cm⁻¹[31]. Stock solutions were stored at

4 °C and used within 7 days. All experiments were per-

formed by maintaining the concentration of complexes con-

stant (300 µM) while varying the CT-DNA concentration

from 0 to 300 µM. The CD spectra of the solutions were

measured after 24 h and 48 h of incubation at 37 °C in the

range from 200 to 350 nm under a dry nitrogen atmosphere.

In contrast, the UV–Vis spectra were recorded after 40 min

and 24 h of incubation at room temperature in the range

from 200 to 600 nm. From the absorption titration data, the

intrinsic binding constant (K_b) of the metal complexes with

CT-DNA was determined using the equation given as:

absorbance of free HSA at 278 nm and ε_ais the absorbance

of HSA in the presence of diferent concentrations of the

complex) versus [complex] is a linear curve, and the binding

constant (K_b) can be obtained from the ratio of the intercept

to the slope. The results of HSA interaction with complexes

via CD spectroscopy were expressed as mean residue ellip-

ticity (MRE) in deg cm²dmol⁻¹. The value of MRE can be

obtained using the equation MRE=[Θ]/(nlC × 10), where

[Θ] is the CD in millidegrees obtained from the spectra (at

208 nm), n is the number of amino acid residues (585 for

HSA), l is the path length of the cell (0.2 cm), and C is

the mol fraction of the protein. Then, the helical content

of HSA can be calculated from the [Θ] value at 208 nm

according to the equation % helix={(− MRE₂₀₈− 4000)/

(33000 − 4000)} × 100 as described by Liu et al. [33].

Glutathione

(

)

(

)

(

)

The reactivity towar ds glutathione was determined using Ell-

man’s method [34–36] to measure the unreacted GSH con-

centration after incubation with the complexes. The proce-

dure is based on the reaction of the thiol with DTNB to give

the mixed disulfde and 2-nitro-5-thiobenzoic acid which

was quantitatively determined by the UV–Vis spectroscopy

at 412 nm. Mixtures of 1.0 ml of GSH (sample concentra-

tion 0.5–3.0 mM) 1 ml of 1–3 complexes (0.5 mM) and 8 ml

of H₂O were incubated at 25 °C in the dark for 30 h. Next

2.5 ml of the reaction mixture was treated with 2.5 ml of

DTNB in a phosphate bufer (PBS, 10 mM, pH 7.4) and the

absorbance was determined. A comparison with the refer-

ence GSH concentration/absorbance relationship allowed the

determination of unreacted thiol concentration. The obtained

concentrations are the average of three independent meas-

urements. Moreover, to examined products of the reaction

GSH with the compounds the ESI mass spectra of free GSH,

GSH cisplatin, GSH complexes (1–3) were recorded.

[DNA]∕ ꢀ_a− ꢀ_f= [DNA]∕ ꢀ_b− ꢀ_f+ 1∕K_bꢀ_b− ꢀ_f,

where [DNA] is the concentration of DNA in base pairs, ε_a

is the extinction coefcient of the complex at a given DNA

concentration, ε_fis the extinction coefcient of the com-

plex in free solution, and ε_bis the extinction coefcient of

the complex when fully bound to DNA. A plot of [DNA]/

(ε_a− ε_f) versus [DNA] gave a slope and an intercept equal

to 1/(ε_b− ε_f) and 1/K_b/(ε_b− ε_f), respectively. The intrinsic

binding constant K_bis the ratio of the slope to the intercept.

Human serum albumin

The interaction of {[RuCl(H₂biim)(η⁶-p-cymene)]

PF₆}₂·H₂O (1), [(η⁵-Cp)RhCl (H₂biim)]PF₆(2) and [(η⁵-

Cp)IrCl(H₂biim)]PF₆(3) with HSA was studied by UV–Vis

absorption titrations at 210–320 nm wavelengths; λ_maxwas

recorded at 278 nm (1 cm cuvette). The CD spectroscopy

was performed from 220 to 300 nm (0.2 cm cuvette) in

5 mM Tris–HCl/50 mM NaCl bufer at pH 7.2. Solid HSA

was dissolved in Tris–HCl/NaCl bufer (5/50 mM at pH 7.2)

and the resulting stock HSA solution was kept at 4 °C for a

maximum of 1 day. All the experiments involved 16.5 µM

HSA (UV–Vis spectroscopy) or 10 µM HSA (CD spectros-

copy) and increasing amounts of the complexes dissolved in

Tris–HCl/NaCl bufer (pH=7.2). The CD spectra of these

solutions were measured after 24 h of incubation at 37 °C,

while the UV–Vis spectra were measured after 40 min and

24 h of incubation at room temperature. The concentrations

of the stock solution of complexes 1, 2, and 3 were 1.5×10⁻⁴

M (UV–Vis spectroscopy) and 3 × 10⁻⁴M (CD spectros-

copy). The HSA concentration was determined spectropho-

tometrically using an extinction coefcient ε₂₈₀= 36 600

M⁻¹cm⁻¹[32].The plot of (ε_a− ε_f) (where ε_fis the initial

Results and discussion

The reaction of appropriate metal precursors: dichloro(p-

cymene)ruthenium(II) dimer [(η⁶-p-cymene)Ru(µ-Cl)

Cl]₂, pentamethylcyclopentadienylrhodium(III) dimer

[CpRh(μ-Cl)Cl]₂, pentamethylcyclopentadienyliridium-

(III) dimer [CpIr(μ-Cl)Cl]₂, with 2,2-biimidazole ligand

(H₂biim) with appropriate ratio in dry mixture of metha-

nol/dichloromethane resulted in the formation of d⁶low-

spin half-sandwich ruthenium, rhodium and iridium com-

plexes: {[RuCl(H₂biim)(η⁶-p-cymene)]PF₆}₂·H₂O (1),

[(η⁵-Cp)RhCl(H₂biim)]PF₆(2) and [(η⁵-Cp)IrCl(H₂biim)]

PF₆(3) (Fig. 1). All the complexes were isolated as orange

to yellow solids and were found to be stable in air and

1 3

JBIC Journal of Biological Inorganic Chemistry

Cl

Ir

Rh

Ru

Ir

Rh

Ru

Cl

2,2’-biimidazole (H₂biim)

MeOH/CH₂Cl₂, 10 h/65^oC, NH₄PF₆

13

26

25

29

+

8

21

24

9

14

7

6

12

27

28

30

22

23

10

Cl

11

15

Cl

-

Ru

PF₆

-

Rh

PF₆

³N

N^3'

³N

N₁

N^3'

4

5

4'

5'

2

2'

4

5

4'

5'

N₁

H

N_1'

H

N_1'

H

H O

2

H

Fig. 1 Schematic presentation of the syntheses of complexes 1–3

non-hydroscopic. These complexes are soluble in hot water,

methanol, DMSO but insoluble in hexane and diethyl ether.

The structure can be considered as a distorted octahedron

because the angles at the ruthenium(II) ions are close to 90°

[76.7(3)–86.9(2)°]. The distance observed between the two

ruthenium centres in the dimer [7.9687(11) Å] excludes any

possible metal–metal interactions.

Description of the structures

The crystal structure determination data are summarized in

Table S1. The selected bond distances and bond angles are

tabulated in Table 1. The perspective views of the crystal

structures of the presented complexes are depicted in Fig. 2a.

The asymmetric units of 1 contain two cationic complex

molecules, two PF^¯₆counterions and one molecule of water.

The asymmetric units of complexes 2 and 3 contain a cati-

onic complex molecule and a PF^¯₆counterion. The coordi-

nation sphere of each Ru(1) and Ru(2) is built up by an η⁶-

p-cymene, a chloride and an N^∩N-κ²N,N′ 2,2′-biimidazole

ligand. The distance from ruthenium(II) to the centroid of

the p-cymene ring is similar to that observed in the literature

[37]. The Ru–Cl (2.433(2) and 2.424(3) Å) and Ru–N bond

lengths (from 2.093 to 2.115(8) Å) are in the expected range.

The crystal packing of complex 1 shows some inter-

molecular interactions, including N–H···O, N/C/–H···F,

C–H···π and π···π, that contribute to the stability of the

compound. Moreover, two symmetrically independent

[RuCl(H₂biim)(η⁶-p-cymene)]⁺cations are held together

via π···π interactions between the parallel-displaced imida-

zole rings [3.7835(2) Å] and an N–H···Cl interaction [N(11)/

(11′)–H(11)/(11′)···Cl(1), 2.37 and 2.45 Å, respectively]

(Fig. 2b). Consequently, a chain of hydrogen bonds is cre-

ated along the crystallographic direction (Fig. S2a), and the

crystal structure of the complex exhibits tetrameric units that

are separated by water molecules (Fig. S2b).

The next two metal complexes with the general formula

[(η⁵-Cp)M(H₂biim)Cl][PF₆], in which M = Rh(III) (2) or

1 3

JBIC Journal of Biological Inorganic Chemistry

Table 1 Bond lengths and

1

2

3

angles for complexes 1–3

Distances (Å)

Ru(1)···Cg1

Ru(1)–Cl(1)

Ru(1)–N(3)

Ru(1)–N(3′)

Ru(2)···Cg2

Ru(2)–Cl(2)

Ru(2)–N(13)

Ru(2)–N(13′)

1.6692(7) Rh(1)···Cp

1.7609(2) Ir(1)···Cp

2.4419(6) Ir(1)–Cl(1)

1.7734(5)

2.4408(13)

2.109(4)

2.433(2)

2.103(8)

2.081(8)

1.6680(8)

2.424(3)

2.115(8)

2.093(8)

Rh(1)–Cl(1)

Rh(1)–N(1)

Rh(1)–N(3)

2.114(2)

2.117(2)

Ir(1)–N(1)

Ir(1)–N(3)

2.105(4)

Angles (°)

N(3′)–Ru(1)–N(3)

N(3′)–Ru(1)–Cl(1)

N(3)–Ru(1)–Cl(1)

N(13′)–Ru(2)–N(13)

N(13′)–Ru(2)–Cl(2)

N(13)–Ru(2)–Cl(2)

76.7(3)

86.9(2)

85.4(2)

76.2(3)

83.0(2)

86.0(2)

N(3)–Rh(1)–N(1)

N(1)–Rh(1)–Cl(1)

N(3)–Rh(1)–Cl(1)

77.01(9)

87.42(6)

87.04(6)

N(3)–Ir(1)–N(1)

N(1)–Ir(1)–Cl(1)

N(3)–Ir(1)–Cl(1)

76.06(14)

85.15(10)

85.94(10)

Fig. 2 Molecular structure of asymmetric unit of complex 1 (a) with marked N–H···O, N/C/O–H···F, N–H···Cl, C–H···π and π···π interaction (b)

Ir(III) (3) and H₂biim represents 2,2′-biimidazole, display

the standard “three-leg piano-stool” geometry (Fig. 3).

The half-sandwich rhodium(III) complex (2) consists of a

π-bonded η⁵-pentamethylcyclopentadienyl ligand, a doubly

σ-bonded 2,2′-biimidazole, as well as a chloride ligand. The

Rh–C bond distances in complex 2 [ranging from 2.1311(32)

to 2.1632(22) Å] are consistent with previous reports [37,

38]. The structure of the analogous iridium(III) complex

(3 ) shows slightly longer distances between the metal ion

and the Cp ring [2.1445(47)–2.1794(44) Å]. Accordingly,

replacement of the rhodium with iridium in compound 3 sur-

prisingly compresses the M–N bond lengths of the chelating

κ²N,N′–H₂biim ligand (see Table 1 ) and is similar to those

found in other Rh and Ir-η⁵-Cp compounds [37, 38].

Thus, the M(III)···Cg and M(III)-N,N-H₂biim bonds are

similar in complexes 2 and 3; however, careful inspection of

Table 1 shows that there are some diferences. The slightly

longer Ir···Cg distance [1.7734(5) Å] compared with the

Rh···Cg distance [1.7609(2) Å] and the slightly shorter Ir–N

and Rh–N distances suggest stronger interactions between

the iridium(III) centre and the N^∩N-2,2′-biimidazole

ligand compared to the stronger interactions between the

rhodium(III) ion and the Cp ring. Additionally, the M–Cl

bond distance in both complexes 2 and 3 [equal to 2.4419(6)

1 3

JBIC Journal of Biological Inorganic Chemistry

Fig. 3 Molecular structures of 2

(left) and 3 (right)

Fig. 4 2D fngerprint plots of the most signifcant intermolecular interactions for 1 (a), 2 (b) and 3 (c) complexes with percentage of interaction

and 2.4408(13) Å, respectively] is relativ ely longer than that

in previously reported complexes [37, 38]. In the crystal

packing of the rhodium and iridium complexes, we can

observe similar π···π interactions between the imidazole

rings and a relatively short C–H···F or N–H···Cl noncovalent

(C–H···F according to Fig. 4) strongly contribute to the crys-

tal packing. The smaller shares (see Fig. 4 for details) are

attributed to the H···Cl, C···H, C···C and N···H interactions,

respectively.

The above-mentioned data pointed out that the presence

of N–H/C–H hydrogen bond donors in complexes favours

multiple noncovalent interactions (especially for 1), and

probably infuenced solution stability. It should be under-

lined that compounds stability in solution is signifcantly

afected by strong bonding chelating ligand (H₂biim) and

the inertness of metal ions [Rh(III), Ir(III)].

−

interaction between the ligands and the anions (PF₆, Cl⁻)

(Fig. S3, Table S2).

Hirshfeld surface analysis

The purpose of examining the interactions in the crystal

structures is the opportunity to form noncovalent interac-

tions in biological systems; these interactions not only pro-

vide detailed information regarding close contacts but also

regarding more distant interactions for 1, 2, and 3 through

Hirshfeld surface (HS) analysis and the obtained associ-

ated 2D fngerprint plots. For the Ru(II) (1), Rh(III) (2),

and Ir(III) (3) complexes, the H···F and H···H interactions

Spectral studies of the complexes

NMR, UV–Vis and FTIR spectroscopy techniques as well as

elemental analysis were used to characterize the complexes.

The analytical data from these complexes are consistent with

the formulations.

1 3

JBIC Journal of Biological Inorganic Chemistry

NMR spectroscopy

UV–visible spectroscopy

Formation of 1–3 has been deduced from ¹H, ¹³C and ¹⁵

N

The UV–visible spectra of metal complexes 1–3 and H₂biim

(ligand) were determined in 1 × 10⁻⁴M dry methanol at

room temperature, and these spectra are presented in Figure

S9. The Ru(II), Rh(III) and Ir(III) complexes are d⁶low-spin

metal complexes containing proper geometry-flled orbitals

at the metal centres, which can interact with the ligand’s low

lying π* orbitals, possibly resulting in metal-to-ligand charge

transfer (MLCT) transitions (Table S3). The low energy

absorption bands observed in the range from 330 to 410 nm

are assigned to metal-to-ligand charge transition (MLCT)

dπ(M) to π*(L) transitions, while the high energy absorp-

tion bands observed in the range from 208 to 285 nm may

be attributed to ligand-centre π–π*/n–π* transitions [39–42].

The pentamethylcyclopentadienyl (Cp) mainly shows lo w-

intensity π→π* transition bands in the UV region overlap-

ping with the biimidazole-based π→π*/n→π* transitions

[43, 44].

NMR spectral studies. The resulting spectra are depicted

in Figs. S5–S8 in the Electronic Supplementary Materials

(ESM); the ensuing data are summarized in the experimental

section, and some of the data are presented in Table 2.

¹³C and ¹⁵N NMR are interesting to compare the strength

of the coordination (by Lewis acids) of the rhodium and

iridium central metal ions in the obtained complexes (2

and 3, respectively). Both the metal ions are coordinated

by the same η⁵-pentamethylcyclopentadienyl ligand (Cp).

Considering the δCp–C, we noticed that the π-donating

efect is much more efective in the case of the rhodium

complex (shift δ=95.4 ppm) compared to the iridium com-

plex (shift δ=87.1 ppm). This was confrmed by the X-ray

data [Rh(1)···Cp, 1.7609(2) Å and Ir(1)···Cp, 1.7734(5) Å].

Consequently, the efective positive charge of the metal ion

is decreasing, which is refected in the Rh–N and Ir–N bond

lengths with H₂biim [Rh(1)–N(1), 2.114(2) Å; Rh(1)–N(3),

2.117(2) Å; Ir(1)–N(1), 2.109(4) Å; and Ir(1)–N(3), 2.105(4)

Å]. This weaker coordination interaction of H₂biim with the

rhodium ion compared to the iridium ion was confrmed

by the ¹⁵N NMR results (shift δ_Rh=− 173.8 ppm and shift

δ_Ir=− 189.7 ppm). The chelating ligand H₂biim is the same

in both complexes; thus, Δ_coord.=δ_complex− δ_ligandcan be esti-

mated according to the δ_Rh,Irshift (the same is correct for the

Cp ligand shift). It should be noted that the ligand H₂biim is

a tautomer under the experimental conditions, which efec-

tively makes it impossible to measure the nitrogen spec-

trum and to determine the NH shift in the proton spectrum.

Interestingly, by analysing the ¹³C NMR spectra of the rho-

dium complex, we observed a doublet around δ 95.4 ppm

(J_Rh–C= 8.2 Hz). Moreover, the ¹⁵N NMR spectra of the

same complex display a doublet centred ca. − 173.8 ppm,

with a J_Rh–Nvalue of 18.6 Hz, which is in the range typical

for related compounds.

FTIR spectroscopic study

The FTIR spectra of all the compounds exhibited strong

bands in the region at 1527, 1531, and 1533 cm⁻¹and 1468,

1433, and 1458 cm⁻¹, which corresponds to C=C and C=N

stretching frequencies of the imidazole moieties in 1, 2 and

3, respectively. The N–H stretching frequency in the ligand

(3144 cm⁻¹) is also present in the same region in all the

complexes (ν=3145–3172 cm⁻¹), indicating that deproto-

nation does not occur. Additionally, the metal complexes

−

show sharp, characteristic peaks for the PF₆counter ion

at 849 cm⁻¹(1), 836 cm⁻¹(2) and 835 cm⁻¹(3) [45, 46].

Moreover, the IR spectra (Fig. S10) of the ruthenium com-

plex show a broad absorption band at 3260 cm⁻¹for ν(O–H),

which confrmed the presence of a water molecule in the

complex structure. Additionally, vibrations appearing in the

2980–2700 cm⁻¹region are associated with aliphatic C–H

stretching vibrations for the methyl and methine groups from

the p-cymene moiety.

Table 2 ¹H, ¹³C and ¹⁵

N

Compound δH4

δH5

δC4

δC5

δCp–C/p-

cymene-

C_(Ru)

δN3

δN1

NMR chemical shifts of 1–3

complexes in DMSO-d₆

1

7.84(+ 0.73) 7.50(+ 0.52) 131.8(+ 3.1) 121.1(+ 3.2) 102.6

7.56(+ 0.45) 7.54(+ 0.56) 128.2(− 0.5) 121.4(+ 3.5) 95.4

7.56(+ 0.45) 7.54(+ 0.56) 127.8(− 0.5) 121.8(+ 3.5) 87.1

− 181.7 − 218.5

− 173.8 − 218.7

− 189.7 − 218.1

2

3

H₂biim

7.11

6.98

128.7

117.9

–

The coordination shifts (∆_coord) are shown in parentheses

Δ_coord.=δ_complex− δ_ligand; Cp_{(Rh, Ir)}-η⁵-pentamethylcyclopentadienyl

1 3

JBIC Journal of Biological Inorganic Chemistry

Stability studies in solution

in diferent cell components should infuence on t heir bio-

logical activity. According to Horobin et al. [48, 49], a log

P in the range from − 5 to 0 facilitates compounds crossing

both cellular and nuclear membranes, so for rhodium com-

pound 2 (log P = − 0.105) accumulation probably occurs

mainly in the nuclei and lysosomes, whereas a log P > 0

suggests that the ruthenium (1) and iridium (3) compounds

will specifcally accumulate in the mitochondria and endo-

plasmic reticulum, but this requires further more detailed

research. Taking this into consideration, the obtained com-

plexes should possess diferent cytotoxic activities, which

are mainly infuenced by the type of metal centre ion in 1, 2,

and 3 and the types of DNA/HSA interactions. The obtained

unexpected results for the lipophilicity of the rhodium and

iridium isostructural complexes were confrmed by multiple

repetitions of the experiment.

Following the characterization of the complexes using stand-

ard spectroscopic methods before performing the biological

studies, the stability of obtained complexes 1–3 was stud-

ied by UV–Vis and ¹³C NMR spectroscopy. To assess the

stability of the complexes in aqueous solution, we selected

Tris–HCl/NaCl bufer solution (pH 7.2, 298 K). The pres-

ence of a drop of DMSO ensured the solubility of the

complexes. The spectra were unaltered over time, and no

absorbance changes were observed in the UV–Vis spectra

(Fig. 5), which indicated that the complexes were stable.

Furthermore, stability experiments were performed on the

complexes in DMSO-d₆by ¹³C NMR spectroscopy. The ¹³C

NMR spectra of the analysed complexes showed no change

over 24 h (Fig. S11). As a result, the stability studies suggest

that the compounds have sufcient stability for the prepara-

tion of samples for biological assays. The signifcant sta-

bility of the complexes is probably due to the presence of

strong hydrogen bonds and multiple noncovalent interac-

tions (Fig. 4). The obtained compounds are inert towards

substitution reactions due to the presence of strong bond-

ing chelating ligand (H₂biim) and inert metal ions [Rh(III),

Ir(III)] which infuenced compounds’ stability in solution

signifcantly.

The cell growth inhibition in the presence of complexes

1–3 was determined using MTT and SRB assays after a 96 h

incubation period against a panel of four human tumour cell

lines, including LoVo, HL-60, MV-4-11, and MCF-7 cells,

as well as the BALB/3T3 healthy mouse cell line (Table 3).

Notably, the obtained complexes show no cytotoxic efects

towards the normal BALB/3T3 cell line compared to cis-

platin (IC₅₀= 2.20 µg). Importantly, {[RuCl(H₂biim)(η⁶-

p-cymene)]PF₆}₂·H₂O (1) displayed the highest activity

against HL-60 cells (IC₅₀4.35 µg), while the IC₅₀values of

Rh(III) and Ir(III) were 36.2 µg and 30.86 µg, respectively.

According to the log P values (log P > 0), the ruthenium

and iridium complexes are active against the same cancer

cell lines (LoVo, HL-60, and MV-4-11), whereas the rho-

dium complex (log P<0) was only active against HL-60 and

MV-4-11. All the compounds were not only inactive against

the normal cell line at the tested concentration range but also

against the MCF-7 cell line. These results suggest the cyto-

toxic selectivity of the obtained organometallic complexes.

It should be underlined that the biological potencies of two

isostructural half-sandwich rhodium and iridium complexes

(metals from the same group) in the same overall ligand set

are not the same. Studying similarities and diferences in

biological activity of investigated analogues, it should be

pointed out that the rhodium complex is not active against

Antiproliferative activity and lipophilicity

Lipophilicity is a very important physicochemical fea-

ture related to the pharmacokinetic behaviour of drug-

like compounds [47]. Using the shake-flask technique,

we obtained the log P value for 1 at +0.22 0.01 and 2

at − 0.54 0.02, whereas for 3, the log P = + 1.55 0.04,

indicating that [(η⁵-Cp)RhCl(H₂biim)]⁺is more hydro-

philic than {[RuCl(H₂biim)(η⁶-p-cymene)]⁺and [(η⁵-Cp)

IrCl(H₂biim)]⁺. Interestingly, in this case, the replacement

of rhodium with a heavier homolog from the same group,

iridium, in the obtained isostructural complex (2 and 3) led

to signifcant diferences in the lipophilicity (Fig. 6). Due

to, the absorption and distribution of drug-like complexes

with metal centres from the same group which accumulate

1,5

1,0

0,5

0,0

2,5

2,0

1,5

1,0

0,5

0,0

1,5

Ru(II) complex ( ) 0h

Ru(II) complex (1

1

Ir(III) complex (3) 0h

Ir(III) complex (3) 24h

Rh(III) complex (2) 0h

Rh(III) complex (2) 24h

1,0

0,5

0,0

) 24h

300

400

500

600

300

400

500

600

300

400

500

600

Wavelenght (nm)

Fig. 5 UV–Vis spectra of complexes at rt in bufer

1 3

JBIC Journal of Biological Inorganic Chemistry

Fig. 6 Comparison of lipophi-

licity, probably location in the

cell and cytotoxic activity of

analysed complexes

Table 3 IC₅₀values

Compound

IC₅₀(μg)

MV-4-11

representing the antiproliferative

activity of analysed complexes

in panel of four human cancer

cell lines and normal mice

HL-60

MCF-7

> 100

LoVo

BALB/3T3

> 100

{[RuCl(H₂biim)(η⁶-p-

53 9.9

4.35 0.15

90 11

cymene)]PF₆}₂·H₂O

fbroblasts compared to cisplatin

[(η⁵-Cp)RhCl(H₂biim)]PF₆

[(η⁵-Cp)IrCl(H₂biim)]PF₆

H₂biim

50 13

81.5 9.4

> 100

36.2 7.7

30.86 0.75

> 100

2.17 0.55

> 100

2.20 0.43

61

0

> 100

1.96 0.68

Cisplatin

0.55 0.15

0.25 0.12

MCF-7 and LoVo cells, while the iridium compound is only

not active against the MCF-7 cell line. Moreover, the rho-

dium complex is approximately twofold more active against

MV-4-11 but slightly less active against the HL-60 cell line

compared with the iridium complex. The diferences in the

biological activity of potential drugs, such as discussed com-

plexes, are most likely derived from their absorption and

distribution into diferent cell components, depending on

their lipophilicity (log P_Rh=− 0.54, log P_Ir=+1.55). Simi-

lar diferences in anticancer activities in vitro and in vivo

have been discussed by Sadler et al. [9–11] for ruthenium(II)

(RM175) and its osmium(II) analogue (AFAP51).

Interactions with biomolecules

Organometallic complexes act as potential drugs by interact-

ing with various targets including DNA and proteins, which

is pr obably the explanation for their anticancer activities

[50, 51]. Taking this into account and our previously pub-

lished anticancer results [16–18], we evaluated the poten-

tial interactions between CT-DNA, HSA and GSH with the

obtained ruthenium(II) (1), rhodium(III) (2) and iridium(III)

(3) complexes.

The interactions of 1, 2, and 3 with CT-DNA were studied

by UV–Vis and CD spectroscopy to investigate the possi-

ble DNA-binding modes and to determine the binding con-

stants (K_b). The UV–Vis spectra shown in Fig. 7a display

the maximum absorption of {[RuCl(H₂biim)(η⁶-p-cymene)]

1 3

JBIC Journal of Biological Inorganic Chemistry

(a)

(b)

(c)

0,20

0,15

0,10

0,05

0,00

0

1:1

20000

15000

10000

5000

0

0.43

1.0

1.86

2.43

0.1

0.2

0.3

0.4

0.8

1

2

1

0

1:5

1:10

1:15

1:25

1

0

-5000

-10000

-15000

-20000

220

240

260

280

300

400

300

400

500

600

Wavelenght (nm)

0,20

0,15

0,10

0,05

0,09

0,08

0,07

0,06

1:1

1:5

1:10

1:15

1:25

1:35

0

20000

10000

0

2

0.1

0.2

0.3

0.4

0.8

1

0,05

400

2

Wavelenght (nm)

0

1

0

0.43

1.0

-10000

-20000

1.43

1.86

220

240

260

280

300

400

500

300

400

Wavelenght (nm)

2,5

2,0

1,5

1,0

0,5

0,0

1:1

1:5

1:10

1:15

1:25

0

20000

0

2

1

0

0,01

0.1

0.2

0.3

0.4

0.8

1

15000

10000

5000

0.43

1.0

1.86

2.43

3

0

-5000

-10000

-15000

-20000

0,00

400

450

Wavelenght (nm)

220

240

260

280

300

200

300

400

300

400

Wavelenght (nm)

Fig. 7 Spectra of 1, 2 and 3 complexes interactions with: a CT-DNA–

UV–Vis spectra (7 µM complex: 0.0, 3, 7, 13, 17 µM of CT-DNA)

(zoom portion of fgure shows clearly the existence of changes); b

CT-DNA–CD spectra (CT-DNA 300 µM) after 24 h incubation at dif-

ferent [complex]/[DNA] ratios at 37 °C; c HSA in the presence of dif-

ferent concentrations of complex

PF₆}₂·H₂O at 401 nm and [(η⁵-Cp)RhCl(H₂biim)]PF₆

at 398 nm; moreover, a hyperchromic effect with a red

shift of approximately 1 nm was observed upon the addi-

tion of CT-DNA. The [(η⁵-Cp)IrCl(H₂biim)]PF₆complex

exhibited hyperchromism at 401 nm but with a blue shift

of approximately 3 nm. These spectral characteristics

(hyperchromic efect) suggest that the complexes interact

with CT-DNA through non-intercalative binding modes

such as outside binding. This may be due to electrostatic

interactions between the positively charged [RuCl(H₂biim)

(η⁶-p-cymene)]⁺, [(η⁵-Cp)IrCl(H₂biim)]⁺and [(η⁵-Cp)

IrCl(H₂biim)]⁺complex units and the negatively charged

phosphate backbone at the periphery of the double-helix CT-

DNA [16, 52 ]. These data are in agreement with previous

reports in t he literature [53] and with previous data obtained

by us [16]. The magnitude of the binding strengths of the

compounds with CT-DNA can be evaluated via their intrin-

sic binding constant K_b. The K_bvalues are equal 2.5 × 10⁵

M⁻¹, 5 × 10⁴M⁻¹, and 5 × 10⁴M⁻¹for the ruthenium (1),

rhodium (2) and iridium (3) complexes, respectively. The

presented data demonstrated that all the complexes distinctly

interact with CT-DNA and that complex 1 binds the most

efciently.

The circular dichroism spectra were adopted to confrm

our results obtained by UV–Vis. The changes in the CD

signals of the DNA observed upon interaction with drugs

may often be assigned to corresponding changes in DNA

structure. The observed CD spectrum of calf thymus DNA

consists of a positive band at 275 nm (UV: λ_max, 260 nm)

due to base stacking and a negative band at 245 nm due to

helicity, whic h is characteristic of DNA in the right-handed

B form [54, 55]. The CD spectra obtained for CT-DNA and

1, 2, and 3 in diferent DNA:complex ratios (1:1, 5:1, 10:1,

15:1, 25:1) after 24 h (Fig. 7b) of incubation imply a non-

intercalative mode of interaction between the DNA and

complexes. The DNA region of the spectrum from 210 to

300 nm revealed changes in the DNA conformation with a

positive peak at approximately 278 nm showing increased

ellipticity and a negative band (~ 248 nm) showing a ten-

dency for continuously decreased ellipticity with increasing

1 3

JBIC Journal of Biological Inorganic Chemistry

Table 4 Concentration of unreacted GSH after 30 h incubation with

concentrations of the 1, 2, and 3 complexes (Fig. 7). Addi-

tionally, the maximum absorption peak for Ru(II) at 268 nm

was accompanied by a red shift (ca. 8 nm), while that the

peak intensity for Ir(III) at 278 nm was changed with a

blue shift of approximately 2 nm, which suggests that the

ruthenium complex interacts with CT-DNA much more

extensively. CD spectral analysis of the Rh(III) complex

upon the addition of CT-DNA did not suggest signifcant

changes in the band at 278 nm and was not accompanied by

a wavelength shift. According to the literature data, little or

no perturbation in the base stacking and helicity bands sug-

gests simple groove binding and electrostatic interactions

with the complex [56].

complexes

Complex

Concentration of unreacted

GSH (mM) GSH₀=0.05 mM

1

0.035 0.001

0.018 0.001

0.027 0.001

0.034 0.001

2

3

Cisplatin

GSSG, which simultaneously causes an increase in ROS lev-

els, pointing to the cytotoxic activities of complexes. Simul-

taneously, GSH can coordinate with the metals in anticancer

drugs to form less toxic GSH conjugates, thereby detoxify-

ing anticancer drugs. According to our UV–Vis studies, rho-

dium complex (2) is the most GSH reactive while ruthenium

complex 1 is less active and its activity is similar to cisplatin

(Table 4). Therefore, low afnity of the complex to GSH

might have a beneft in potential therapy.

Due to albumin’s remarkable binding properties and high

abundance in blood plasma, serum plays a crucial role in

the drug delivery system [57]. To understand the nature of

its binding with the complexes, the absorption intensity of

human serum albumin (HSA) has been investigated in the

presence of the complexes under study. Absorbance titration

studies have been performed at room temperature using HSA

(0.5 μM) with varying concentrations of 1–3 (0–50 μM) in

the range of 290–500 nm (λ_max278 nm). From the spectra

(Fig. 7c), it is clear that the absorption intensity of HSA

band increases without any shift in the presence of the ruthe-

nium, rhodium and iridium complexes indicating a static

interaction between HSA and the complexes. Using the

graph of (ε_a− ε_f) versus [complex], the binding constants

were calculated as 1.5 × 10⁴(1), 5 × 10⁴(2), and 1 × 10⁴

(3). These values are within the range of 10⁴to 10⁶M⁻¹, as

expected for good HSA carrier activity in vivo [58]. To gain

a better understanding in the conformational behaviour of

HSA in the albumin: ruthenium/rhodium or iridium com-

plex, CD spectroscopic measurements were performed. The

CD spectra of HSA at pH 7.2 exhibited two negative minima

at 208 and 222 nm, which is characteristic of an α-helical

protein structure. Since the α-helix is just one secondary

structure element, the structural changes in albumin could be

evaluated by the α-helical structure content [58]. Figure S12

shows the CD spectra of native HSA and HSA with increas-

ing concentrations of complexes. As shown, the ellipticity

values were found to increase in the presence of complexes

1–3, which indicated the loss of protein α-helical structure.

The changes in the helicity of HSA indicate that the chains

keep unfolding in the presence of diferent concentrations of

the complexes (Fig. S12).

Moreover, to examined products of the GSH reaction with

the compounds the ESI mass spectra of free GSH, GSH-

cisplatin, GSH-complexes (1–3) were recorded (Fig. S13).

The detailed information of several interaction products ions

extracted from reaction solution is given in Table 5. The

results show that ruthenium (1), rhodium (2) and iridium

(3) complexes formed adducts with GSH, with fragments of

GSH or fragments of GSSG (Table 5, Fig. S13a–c).

Conclusion

In conclusion, new d⁶organometallic ruthenium (1), rhodium

(2) and iridium (3) complexes containing 2,2′-biimidazole

(H₂biim) as a chelating ligand were prepared and characterized.

Crystallographic studies showed that the piano-stool complex

1 possesses a dimeric structure due to strong hydrogen bonds

between the cationic species. In contrast, the asymmetric units

in complexes 2 and 3 consist of cationic complexes in a mono-

meric form. The obtained compounds possess signifcant solu-

tion stability, which is probably achieved due to the presence of

strong hydrogen bonds and multiple noncovalent interactions

(especially for 1). It should be underlined that the chelating

ligand (H₂biim) and the inertness of the metal ions [Rh(III),

Ir(III)] signifcantly infuenced compounds’ stability in solu-

tion. Similar to the osmium and ruthenium complex analogues

investigated by Sadler’s group, the biological potencies of the

two obtained isostructural half-sandwich rhodium and iridium

complexes (metals from the same group) are not the same. The

diferences in complexes 2 and 3 are potentially derived from

their absorption and distribution into diferent cell compo-

nents, which is based on their lipophilicity (log P_Rh=− 0.54,

log P_Ir=+1.55). A comparison of the cytotoxicity showed

The literature data demonstrated that glutathione

(γ-glutamylcysteinylglycine, GSH) containing (SH) groups

act as enzyme cofactors, antioxidants and antitoxins. There-

fore, GSH has various physiological functions and is the

most abundant nonprotein thiol in mammalian cells [59].

It was presented that the cytotoxicity of some organometal-

lic anticancer compounds [60] is derived from the fact that

the complex acts as a catalyst for the oxidation of GSH to

1 3

JBIC Journal of Biological Inorganic Chemistry

Table 5 Assignment of (+) mass ions in the ESI mass spectra shown in Fig. S13 compared to calculated mass ions

m/z

Assignment

Calcd.

m/z

Assignment

Calcd.

{[RuCl(H₂biim)(η⁶-p-cymene)]PF₆}₂·H₂O

[(η⁵-Cp)RhCl(H₂biim)]PF₆

288.23

368.99

404.97

676.02

808.92

[RuCl(H₂biim)H₂O]⁺

288.93

368.06

404.03

676.19

808.18

371.00

308.04

406.97

544.00

678.03

698.01

Cisplatin

288.23

308.03

353.19

381.22

554.45

711.44

[(η⁵-Cp)Rh(Hbiim)]⁺

371.07

308.09

407.05

544.03

678.16

698.15

[Ru(Hbiim)(η⁶-p-cymene)]⁺

[GSH+H]⁺

[RuCl(H₂biim)(η⁶-p-cymene)]⁺

[Ru(H₂biim)(η⁶-p-cymene)+GS]⁺

[Ru(H₂biim)(η⁶-p-cymene)+GSSG^a]⁺

[(η⁵-Cp)RhCl(H₂biim)]⁺

[(η⁵-Cp)RhCl(H₂biim)PF₆+Na]⁺

[(η⁵-Cp)Rh(H₂biim)+GS]⁺

[(η⁵-Cp)Rh(H₂biim)+GSSG^b]⁺

[(η⁵-Cp)IrCl(H₂biim)]PF₆

308.04

461.05

497.01

549.66

[GSH+H]⁺

308.09

461.13

497.10

549.13

563.13

768.21

[PtClNH₃CH₃CN]⁺

[GSH+H]⁺

287.99

308.09

353.06

381.01

554.04

711.03

[(η⁵-Cp)Ir(Hbiim)]⁺

[(η⁵-Cp)IrCl(H₂biim)]⁺

[(η⁵-Cp)Ir(H₂biim)+GS^a]⁺

[(η⁵-Cp)Ir(H₂biim)+GS^b]⁺

[(η⁵-Cp)Ir(H₂biim)+GS]⁺

[GSH+2Na]⁺

[Pt+GS^c]⁺

563.68

768.08

[PtClNH₃+GSH]⁺

[PtCl+GSSG^c]⁺

GSH

201.94

409.10

531.31

[GSH^d+K]⁺

201.99

409.05

531.11

553.14

569.29

585.23

779.60

[GSSG-CO₂+H]⁺

569.17

585.16

779.30

[GSSG^d-H]⁺

[GSSG-CO+H]⁺

[GSSG^e+CH₃CN+Na]⁺

[GSSG^f+CH₃CN+H]⁺

[3GSH-2CO₂-2CO+2H]⁺

553.29

GSH (glutathione)=C₁₀H₁₇N₃O₆S; GSH (glutathione fragments)=^aC₃H₃OS; ^bC₃H₄NOS; ^cC₇H₁₀N₂O₂S; ^dC₅H₉NO₃S; GSSG (glutathione

b

c

d

e

disulfde)=C₂₀H₃₂N₆O₁₂S₂; GSSG (glutathione disulfde fragments)=^aC₁₄H₂₅N₅O₇S₂; C₉H₁₈N₄O₅S₂; C₁₅H₂₃N₅O₉S₂; C₁₂H₁₆N₄O₈S₂; C₁₅H-

₂₃N₄O₉S₂; ^fC₁₆H₂₆N₅O₁₀S₂

Acknowledgements The authors gratefully acknowledge dr hab.

Mariusz Urbaniak for help with ESI experiments. Additionally, the

authors thank the fnancial support of the Polish Ministry of Science

and Higher Education (Projects nos. 612074 and 612415).

that the rhodium complex is not active against MCF-7 and

LoVo cells, while the iridium compound is only not active

against the MCF-7 cell line. Moreover, the rhodium complex

is approximately twofold more active against MV-4-11 cells

but slightly less active against the HL-60 cell line compared

with the iridium complex. Importantly, {[RuCl(H₂biim)(η⁶-p-

cymene)]PF₆}₂·H₂O (1) displayed the highest activity against

HL-60 cells (IC₅₀4.35 µg). Additionally, it should be empha-

sized that the successfully obtained complexes were inactive

against the normal cell line. UV–Vis and CD data suggest that

the compounds can efciently bind to protein targets (HSA)

and interact with calf thymus DNA. The spectral characteris-

tics (hyperchromic efect) suggest that the complexes interact

with CT-DNA through electrostatic interactions between the

positively charged [RuCl(H₂biim)(η⁶-p-cymene)]⁺, [(η⁵-Cp)

IrCl(H₂biim)]⁺and [(η⁵-Cp)IrCl(H₂biim)]⁺complex units and

the negatively charged phosphate backbone at the periphery of

the DNA double-helix. Moreover, we studied the interactions

of the analysed complexes with GSH. The ESI mass spectra

results confrmed that 1, 2 and 3 obtained complexes bound to

GSH and formed adducts with GSH, with fragments of GSH

or GSSG. Additionally, the UV–Vis results demonstrated that

ruthenium complex 1 is considerably less active in the reaction

with GSH than the rhodium (2) and iridium (3) complexes.

Simultaneously, the ruthenium(II) dimer displays better cyto-

toxicity compared to complexes 2 and 3.

Open Access This article is distributed under the terms o f the Crea-

tive Commons Attribution 4.0 International License (http://creativeco

mmons.org/licenses/by/4.0/), which permits unrestricted use, distribu-

tion, and reproduction in any medium, provided you give appropriate

credit to the original author(s) and the source, provide a link to the

Creative Commons license, and indicate if changes were made.

References

1. Yan YK, Melchart M, Habtemariam A, Sadler PJ (2005) Orga-

nometallic chemistry, biology and medicine: ruthenium arene

anticancer complexes. Chem Commun 38:4764–4776

2. Lentz F, Drescher A, Lindauer A, Henke M, Hilger RA,

Hartinger CG, Scheulen ME, Dittrich C, Keppler BK, Jaehde

U (2009) Pharmacokinetics of a novel anticancer ruthenium

complex (KP1019, FFC14A) in a phase I dose-escalation study.

Anticancer Drugs 20:97–103

3. Hartinger ChG, Jakupec MA, Zorbas-Seifried S, Groessl M,

Egger A, Berger W, Zorbas H, Dyson PJ, Keppler BK (2008)

KP1019, a new redox-active anticancer agent—preclinical

development and results of a clinical phase I study in tumor

patients. Chem Biodiversity 5:2140–2155

4. Dömötör O, Hartinger ChG, Bytzek AK, Kiss T, Keppler BK,

Enyedy EA (2013) Characterization of the binding sites of the

anticancer ruthenium(III) complexes KP1019 and KP1339 on

1 3

JBIC Journal of Biological Inorganic Chemistry

22. STOE & Cie GmbH (2015) X-Area 1.75, software package for col-

lecting single-crystal data on STOE area-detector difractometers,

for image processing, scaling refection intensities and for outlier

rejection. STOE & Cie GmbH, Darmstadt

human serum albumin via competition studies. J Biol Inorg

Chem 18:9–17

5. Sava G, Capozzi I, Clerici K, Gagliardi G, Alessio E, Mestroni

G (1998) Pharmacological control of lung metastases of solid

tumours by a novel ruthenium complex. Clin Exp Metastasis

16:371–379

23. Sheldrick GM (2014) Acta Crystallogr Sect A 64:112

24. Sheldrick GM (2014) SHELXL-2014. University of Göttingen

and Bruker AXS, Karlsruhe

6. Dougan SJ, Sadler PJ (2007) The design of organometallic

ruthenium arene anticancer agents. Chimia 61:704–715

7. Dyson PJ (2007) Systematic design of a targeted organometallic

antitumour drug in pre-clinical development. Chimia 61:698

8. Gianferrara T, Bratsos I, Alessio E (2009) A categorization of

metal anticancer compounds based on their mode of action.

Dalton Trans 37:7588–7598

25. Dolomanov OV, Bourhis LJ, Gildea RJ, Howard JAK, Puschmann

H (2009) OLEX2: a complete structure solution, refnement and

analysis program. J Appl Cryst 42:339

26. Farrugia LJ (2012) WinGX and ORTEP for Windows: an update.

J Appl Crystallogr 45:849

27. Brandenburg K, Putz H, Diamond-crystal and molecular struc-

ture visualisation crystal impact, Rathausgasse 30, Bonn: GbR,

1997–2000, Version 3.1 G

9. Bergamo A, Masi A, Peacock AF, Habtemariam A, Sadler PJ,

Sava G (2010) In vivo tumour and metastasis reduction and

in vitro efects on invasion assays of the ruthenium RM175

and osmium AFAP51 organometallics in the mammary cancer

model. J Inorg Biochem 104:79–86

28. Skehan P, Storeng R, Scudiero D, Monks A, McMahon J, Vistica

D, Warren JT, Bokesch H, Kenney S, Boyd MR (1990) New col-

orimetric cytotoxicity assay for anticancer-drug screening. J Natl

Cancer Inst 82:1107

10. Aird RE, Cummings J, Ritchie AA, Muir M, Morris RE, Chen

H, Sadler PJ, Jodrell DI (2002) In vitro and in vivo activity

and cross resistance profles of novel ruthenium(II) organome-

tallic arene complexes in human ovarian cancer. Br J Cancer

86(10):1652–1657

29. Łakomska I, Szłyk E, Sitkowski J, Kozerski L, Nasulewicz A,

Pełczyńska M, Wietrzyk J, Opolski A (2004) Multinuclear NMR

spectroscopy and antitumor activity of novel platinum(II) com-

plexes with 5,7-disubstituted-1,2,4-triazolo[1,5-a]pyrimidines. J

Inorg Biochem 98(1):167–172

11. Guichard SM, Else R, Reid E, Zeitlin B, Aird R, Muir M, Dodds

M, Fiebig H, Sadler PJ, Jodrell DI (2006) Anti-tumour activity in

non-small cell lung cancer models and toxicity profles for novel

ruthenium(II) based organo-metallic compounds. Biochem Phar-

macol 71:408–415

30. Albert A (1979) Selective toxicity. The physico-chemical basis of

therapy. Chapman and Hall, London, pp 662–664

31. Srishailam A, Kumar YP, Gabra NMD, Reddy PV, Deepika N,

Veerababu N, Satyanarayana S (2013) Synthesis, DNA-binding,

cytotoxicity, photo cleavage, antimicrobial and docking studies of

Ru(II) polypyridyl complexes. J Fluoresc 23:897–908

32. Zhang GJ, Keita B, Craescu CT, Miron S, Oliveira PD, Nadjo

L (2007) Polyoxometalate binding to human serum albumin:

athermodynamic and spectroscopic approach. J Phys Chem B

111:11253–11259

12. Casini A, Edafe F, Erlandsson M, Gonsalvi L, Ciancetta A, Re N,

Ienco A, Messori L, Peruzzini M, Dyson PJ (2010) Rationaliza-

tion of the inhibition activity of structurally related organometallic

compounds against the drug target cathepsin B by DFT. Dalton

Trans 39:5556–5563

13. Mitra R, Das S, Shinde SV, Sinha S, Somasundaram K, Samuel-

son AG (2012) Anticancer activity of hydrogen-bond-stabilized

half-sandwich RuII complexes with heterocycles. Chem Eur J

18:12278–12291

33. Ge YSh, Jin Ch, Song Z, Zhang JQ, Jiang FL, Liu Y (2014) Multi-

spectroscopic analysis and molecular modeling on the interaction

of curcumin and its derivatives with human serum albumin: a

comparative study. Spectrochim Acta Part A Mol Biomol Spec-

trosc 124:265–276

14. Das S, Sinha S, Britto R, Somasundaram K, Samuelson AG

(2010) Cytotoxicity of half sandwich ruthenium(II) complexes

with strong hydrogen bond acceptor ligands and their mechanism

of action. J Inorg Biochem 104(2):93–104

34. Ellman GL (1959) Tissue sulfhydryl groups. Archiv Biochem

Biophys 82:70–77

35. Kuduk-Jaworska J, Waszkiewicz K (2000) Malatoplatinum(II)

complexes—carboplatin analogs. Transit Met Chem 25:443–449

36. Jakubowski M, Łakomska I, Sitkowski J, Wiśniewska J (2018)

Dicarboxylato platinum(II) complexes containing dimethyl sul-

foxide and triazolopyrimidine as potential anticancer agents: syn-

thesis, structural and biological studies in solution. New J Chem

42:8113–8122

15. Morris RE, Aird RE, Murdch PS, Chen H, Cummings J, Hughes

ND, Parsons S, Parkin A, Boyd G, Jodrell DI, Sadler PJ (2001)

Inhibition of cancer cell growth by ruthenium(II) arene com-

plexes. J Med Chem 44:3616–3621

16. Gilewska A, Masternak J, Kazimierczuk K, Trynda J, Wietrzyk J,

Barszcz B (2018) Synthesis, structure, DNA binding and antican-

cer activity of mixed ligand ruthenium(II) complex. J Mol Struct

1155:288–296

37. Kumar A, Gupta RK, Paitandi RP, Singh KB, Trigun SK, Hundal

MS, Pandey DS (2016) Cationic Ru(II), Rh(III) and Ir(III) com-

plexes containing cyclic p-perimeter and 2-aminophenyl benzimi-

dazole ligands: synthesis, molecular structure, DNA and protein

binding, cytotoxicity and anticancer activity. J Organomet Chem

801:68–79

17. Rogala P, Jabłońska-Wawrzycka A, Kazimierczuk K, Borek A,

Błażejczyk A, Wietrzyk J, Barszcz B (2016) Synthesis, crystal

structure and cytotoxic activity of ruthenium(II) piano-stool com-

plex with N,N-chelating ligand. J Mol Struct 1126:74–82

18. Masternak J, Gilewska A, Kazimierczuk K, Khavryuchenko OV,

Wietrzyk J, Trynda J, Barszcz B (2018) Synthesis, physicochemi-

cal and theoretical studies on new rhodium and ruthenium dimers.

Relationship between structure and cytotoxic activity. Polyhedron

154:263–274

38. Yadav M, Singh AK, Pandey DS (2009) First examples of het-

eroleptic dipyrrin/η5-pentamethylcyclopentadienyl rhodium/

iridium(III) complexes and their catalytic activity. Organometal-

lics 28(16):4713–4723

39. Palepu NR, Adhikari S, Premkumar R, Verma AK, Shepherd SL,

Phillips RM, Kaminsky W, Kollipara MR (2016) Half-sandwich

ruthenium, rhodium and iridium complexes featuring oxime

ligands: structural studies and preliminary investigation of in vitro

and in vivo anti-tumour activities. Appl Organomet Chem. https

://doi.org/10.1002/aoc.3640

19. Furrer J (2010) A robust, sensitive, and versatile HMBC experi-

ment for rapid structure elucidation by NMR: IMPACT-HMBC.

Chem Commun 46:3396–3398

20. Kupce E, Freeman R (2007) Fast multidimensional NMR by

polarization sharing. Magn Reson Chem 45:2–4

21. Wishart DS, Bigam CG, Yao J, Abildgaard F, Dyson HJ, Oldfeld

E, Markley JL, Sykes BD (1995) 1H, 13C and 15N chemical shift

referencing in biomolecular NMR. J Biomol NMR 6:135–140

1 3

JBIC Journal of Biological Inorganic Chemistry

40. Shadap L, Joshi N, Poluri KM, Kollipara MR, Kaminsky W

(2018) Synthesis and structural characterization of arene d⁶metal

complexes of sulfonohydrazone and triazolo ligands: high potency

o f triazolo derivatives towards DNA binding. Polyhedron. https://

doi.org/10.1016/j.poly.2018.08.051

51. Wu B, Ong MS, Groessl M, Adhireksan Z, Hartinger CG, Dyson

PJ, Davey CA (2011) A ruthenium antimetastasis agent forms

specifc histone protein adducts in the nucleosome core. Chem

Eur J 17(13):3562–3566

52. Shahabadi N, Kashanian S, Mahdavi M, Sourinejad N (2011)

DNA interaction and DNA cleavage studies of a new platinum(II)

complex containing aliphatic and aromatic dinitrogen ligands.

Bioinorg Chem Appl 2011:1–10

41. Gerloch M, Constable EC (1994) Transition metal chemistry: the

valence shell in d-block chemistry. Wiley Online Library, Oxford

42. Govindaswamy P, Mozharivskyj YA, Kollipara MR (2005) Syn-

theses, spectral and structural studies of Schif base complexes of

η5-pentamethylcyclopentadienyl rhodium and iridium. Polyhe-

dron 24:1710–1716

53. Shahabadi N, Hadidi S (2012) Spectroscopic studies on the inter-

action of calf thymus DNA with the drug levetiracetam. Spectro-

chim Acta A Mol Biomol Spectrosc 96:278–283

43. Didier P, Ortmans I, Kirsch-De Mesmaeker A, Watts R (1993)

Electrochemistry and absorption and emission spectros-

copy of new orthometalated complexes of rhodium(III) and

iridium(III) with the ligands 1,4,5,8-tetraazaphenanthrene and

1,4,5,8,9,12-hexaazatriphenylene. Inorg Chem 32(23):5239–5245

44. Sullivan B, Salmon D, Meyer T (1978) Mixed phosphine 2,2′-bipy-

ridine complexes of ruthenium. Inorg Chem 17(12):3334–3341

45. Bellamy L (1954) Infrared spectra of complex molecules.

Methuen, London, p 175

54. Palaniappan K, Prabhakaran R, Dallemer F, Natarajan K (2014)

Phtophysical properties and in vitro cytotoxicity studies of new

Ru(II) carbonyl complexes and mixed geometrical Ru(II)–Ni(II)

complex in HSDNA/BSA protein and human lung (A549) and

liver (HepG2) cells. RSC Adv 4:51850–51864

55. Ivanov VI, Minchenkova LE, Schyolkina AK, Polytayer AI (1973)

Diferent conformations of double-stranded nucleic acid in solu-

tion as revealed by circular dichroism. Biopolymers 12(1):89–110

56. Shahabadi N, Kashanian S, Darabi F (2010) DNA binding and

DNA cleavage studies of a water soluble cobalt(II) complex con-

taining dinitrogen Schif base ligand: the efect of metal on the

mode of binding. J Med Chem 45(9):4239–4245

46. Nakamoto K (1986) Infrared and Raman spectroscopy of inor-

ganic and coordination compounds. Wiley, New York

47. Aman F, Hanif M, Siddiqui WA, Ashraf A, Filak LK, Reynisson

J, Söhnel T, Jamieson SMF, Hartinger ChG (2014) Anticancer

ruthenium(η⁶-p-cymene) complexes of nonsteroidal anti-infam-

matory drug derivatives. Organometallics 33:5546–5553

48. Horobin RW, Stockert JC, Rashid-Doubell F (2006) Fluorescent

cationic probes for nuclei of living cells: why are they selective? A

quantitative structure–activity relations analysis. Histochem Cell

Biol 126:165–175

57. Pan X, Qin P, Liu R, Wang J (2011) Characterizing the interaction

between tartrazine and two serum albumins by a hybrid spectro-

scopic approach. J Agri Food Chem 58:6650–6656

58. Huang S, Peng S, Zhu F, Lei X, Xiao Q, Su W, Liu Y, Huang

CH, Zhang L (2016) Multispectroscopic investigation of the

interaction between two ruthenium(II) arene complexes of cur-

cumin analogs and human serum albumin. Biol Trace Elem Res

169:189–203

49. Mazuryk O, Magiera K, Rys B, Suzenet F, Kieda C, Brindell

M (2014) Multifaceted interplay between lipophilicity, protein

interaction and luminescence parameters of non-intercalative

ruthenium(II) polypyridyl complexes controlling cellular imag-

ing and cytotoxic properties. J Biol Chem 19(8):1305–1316

50. Colina-Vegas L, Villarreal W, Navarro M, de Oliveira CR,

Graminha AI, Maia PI, Defon VM, Ferreira AG, Cominetti MR,

Batista AA (2015) Cytotoxicity of Ru(II) piano–stool complexes

with chloroquine and chelating ligands against breast and lung

tumor cells: interactions with DNA and BSA. J Inorg Biochem

153:150–161

59. Berg JM, Tymoczko JL, Stryer L (2002) Biochemistry. Freeman

Company, New York

60. Dougan SJ, Habtemariam A, McHale SE, Parsons S, Sadler PJ

(2008) Catalytic organometallic anticancer complexes. Proc Natl

Acad Sci USA 105(33):11628–11633

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jurisdictional claims in published maps and institutional afliations.

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Similarities and differences in d6 low-spin ruthenium, rhodium and iridium half-sandwich complexes: synthesis, structure, cytotoxicity and interaction with biological targets

DOI: 10.1007/s00775-019-01665-2

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Article abstract of DOI:10.1007/s00775-019-01665-2

Full text of DOI:10.1007/s00775-019-01665-2

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