Ru(II)/clotrimazole/diphenylphosphine/bipyridine complexes: Interaction with DNA, BSA and biological potential against tumor cell lines and Mycobacterium tuberculosis

Article abstract of DOI:10.1016/j.jinorgbio.2016.06.023

Three ruthenium complexes [RuCl(CTZ)(bipy)(P-P)]PF₆ [P-P?=?1,2-bis(diphenylphosphino)ethane (dppe-1), 1,4-bis(diphenylphosphino)butane (dppb-2) and 1,1′-bis(diphenylphosphino)ferrocene (dppf-3), bipy?=?2,2′-bipiridine and clotrimazole (CTZ) 1-[(2-chlorophenyl)diphenylmethyl]-1H-imidazole] were synthesized. These complexes were characterized by a combination of elemental analysis, molar conductivity, infrared and UV–vis spectroscopy, ¹H, ¹³C{¹H} and ³¹P{¹H} nuclear magnetic resonance techniques, cyclic voltammetry and mass spectroscopy. Bovine serum albumin binding constants, which were in the range of 1.30–36.00?×?10⁴?M^??1, and thermodynamic parameters suggest spontaneous interactions with this protein by electrostatic forces due to the positive charge of the complexes. DNA interactions studied by spectroscopic titration, viscosity measurements, gel electrophoresis, circular dichroism, ethidium bromide displacement and reactions with guanosine and guanosine monophosphate indicated the DNA binding affinity primarily through non-covalent interactions. All complexes 1–3 were tested against the human carcinoma cell lines MCF-7 (breast), A549 (lung) and DU-145 (prostate) presenting promising IC₅₀ values, between 0.50 and 14.00?μM, in some cases lower than the IC₅₀ for the reference drug (cisplatin). The antimicrobial activity assays of the complexes provided evidence that they are potential agents against mycobacterial infections, specifically against Mycobacterium tuberculosis H37Rv.

Full text of DOI:10.1016/j.jinorgbio.2016.06.023

Ru(II)/clotrimazole/diphenylphosphine/bipyridine complexes: Interaction
with DNA, BSA and biological potencial against tumor cell lines and My-
cobacterium tuberculosis
Legna Colina-Vegas, Jocely Lucena Dutra, Wilmer Villarreal, Joa˜o Honorato
de A. Neto, Marcia Regina Cominetti, Fernando Pavan, Maribel Navarro,
Alzir A. Batista
PII:
DOI:
Reference:
S0162-0134(16)30187-8
doi: 10.1016/j.jinorgbio.2016.06.023
JIB 10025
To appear in:
Journal of Inorganic Biochemistry
Received date:
Revised date:
Accepted date:
7 March 2016
17 June 2016
23 June 2016
Please cite this article as: Legna Colina-Vegas, Jocely Lucena Dutra, Wilmer Villarreal,
Jo˜ao Honorato de A. Neto, Marcia Regina Cominetti, Fernando Pavan, Maribel Navarro,
Alzir A. Batista, Ru(II)/clotrimazole/diphenylphosphine/bipyridine complexes: Interac-
tion with DNA, BSA and biological potencial against tumor cell lines and Mycobacterium
tuberculosis, Journal of Inorganic Biochemistry (2016), doi: 10.1016/j.jinorgbio.2016.06.023
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ACCEPTED MANUSCRIPT
Ru(II)/clotrimazole/diphenylphosphine/bipyridine complexes: Interaction with DNA, BSA and
biological potencial against tumor cell lines and Mycobacterium tuberculosis
Legna Colina-Vegas1*, Jocely Lucena Dutra1, Wilmer Villarreal1, João Honorato de A. Neto1, Marcia
Regina Cominetti2, Fernando Pavan3, Maribel Navarro4, Alzir A. Batista1*
1Departamento de Química, Universidade Federal de São Carlos-SP, CEP 13565-905, Brazil.
2Departamento de Gerontologia, Universidade Federal de São Carlos-SP, CEP 13565-905, Brazil.
3Departamento de Ciências Biológicas, Faculdade de Ciências Farmacêuticas, UNESP, CEP 14800-900,
Araraquara, SP, Brazil.
4Directoria de Metrologia Aplicada a Ciências da Vida, Instituto Nacional de Metrologia, Qualidade e
Tecnologia, INMETRO, RJ, Brazil.
*Corresponding authors: Alzir A. Batista daab@ufscar.br. Legna Colina-Vegas esleg_24@hotmail.com
Abstract
Three ruthenium complexes [RuCl(CTZ)(bipy)(P-P)]PF6 [P-P =1,2-bis(diphenylphosphino)ethane
(dppe-1), 1,4-bis(diphenylphosphino)butane (dppb-2) and 1,1’-bis(diphenylphosphino)ferrocene (dppf-
3), bipy= 2,2’-bipiridine and clotrimazole (CTZ) 1-[(2-chlorophenyl)diphenylmethyl]-1H-imidazole]
were synthesized. These complexes were characterized by a combination of elemental analysis, molar
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31
conductivity, infrared and UV–vis spectroscopy, H, C{1H} and P{1H} nuclear magnetic resonance
techniques, cyclic voltammetry and mass spectroscopy. Bovine serum albumin binding constants, which
were in the range of 1.30–36.00 x 104 M-1, and thermodynamic parameters suggest spontaneous
interactions with this protein by electrostatic forces due to the positive charge of the complexes. DNA
interactions studied by spectroscopic titration, viscosity measurements, gel electrophoresis, circular
dichroism, ethidium bromide displacement and reactions with guanosine and guanosine monophosphate
indicated the DNA binding affinity primarily through non-covalent interactions. All complexes 1–3 were
tested against the human carcinoma cell lines MCF-7 (breast), A549 (lung) and DU-145 (prostate)
presenting promising IC50 values, between 0.50 to 14.00 μM, in some cases lower than the IC50 for the
reference drug (cisplatin). The antimicrobial activity assays of the complexes provided evidence that they
are potential agents against mycobacterial infections, specifically against M. tuberculosis H37Rv.
Keywords: Ruthenium-bisdiphenylphosphine, Clotrimazole, antitumor and anti-Mycobacterium
tuberculosis
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1. Introduction
Clotrimazole (CTZ), first synthesized by Karl Hienz Büchel in the late 1960s, was originally
developed as an antifungal agent [1]. In addition there was promising research concerning the use of CTZ
against other diseases, such as sickle cell anemia, malaria, beriberi, tineapedis, Chagas disease and cancer
[2]. Thus, this organic compound has a broad spectrum of biological activity, high efficacy and minimal
side effects. It was shown that clotrimazole is able to inhibit the proliferation of cancer cells in vitro and
in vivo, as it inhibits enzyme activity involved in glycolysis stopping the cell cycle [3]. In 1993, the first
metal complex containing clotrimazole was reported, [RuCl2(CTZ)2], which showed high trypanocidal
activity against Chagas disease, combined with low toxicity [4]. Furthermore, various authors have
demonstrated that the CTZ coordination for Ru(II)-η6-p-cymene, Au(I) and Cu(II) enhances its activity
against the epimastigote form of Trypanosoma cruzi, indicating that all of them had their activity on
parasite proliferation improved, when compared with the activity of the free CTZ [5,6,7]. CTZ metal
complexes have shown potential applications in medicinal chemistry and synergistic effects were studied,
thus, its metal complexes with ions such as Cu(II), Co(II), Ni(II), Zn(II) [8] and Pt(II) [9] had their
cytotoxic activity improved (IC50 values) in cell growth inhibition of human carcinoma, when compared
to the activity of the free ligand.
Biological activity of various ruthenium complexes stands out due to the ability of having action, in
vitro and in vivo, against some tumor cell lines, as well as lower cytotoxicity, compared with platinum
complexes. The structure of some of the most successful ruthenium complexes can be seen in Figure 1,
three Ru(III) anticancer agents: indazolium [trans-RuCl4(1H-indazole)2] (KP1019), sodium [trans-
RuCl4(1H-indazole)2] (NKP-1339) and imidazolium [trans-RuCl4(1H-imidazole)(DMSO-S)] (NAMI-A)
are currently undergoing clinical trials [10], and one Ru(II) complex: [Ru(p-cymene)Cl2(1,3,5-Triaza-7-
phosphaadamantane)] (RAPTA-C) also shows promising activities [11].
The potential use of ruthenium complexes for cancer treatment has motivated our research group to
synthesize new Ru(II)-diphenylphosphine [12] and Ru(II)-triphenylphosphine [13] complexes and to test
them against a range of tumor cells, yielding encouraging results. Thus, over the last seven years we have
synthesized some ruthenium/phosphine/diimine complexes (Figure 2), which showed promising activity
against tumor cell lines, and also against Mycobacterium tuberculosis [14,15,16,17,18]. M. tuberculosis
is the causative agent of tuberculosis, and in 2014 an estimated 9.6 million of new cases and 1.5 million
died from this disease (1.1 million HIV-negative cases and 0.4 million HIV-positive cases) [19].
Treatment for M. tuberculosis (MTB) is expensive, long, it causes some side effects and encounters
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resistance. Therefore many researchers are looking for new alternative drugs to treat this illness. Thus,
some ruthenium and gold complexes have been shown to be very propitious, with minimum inhibitory
concentrations (MICs) comparable to, or even better than some reference drugs used to treat tuberculosis.
This paper describes the synthesis, spectroscopic and electrochemical characterization of three new
complexes with the general formula [RuCl(CTZ)(bipy)(P-P)]PF6, where P–P
=
1,2-
bis(diphenylphosphino)ethane (dppe, 1), 1,4-bis(diphenylphosphino)butane (dppb, 2) and 1,1’-
bis(diphenylphosphino)ferrocene (dppf, 3) and bipy= 2,2’-bipiridine. The diphenylphosphines were
chosen as co-ligands in these complexes in order to stabilize the complexes due to their good π acceptor
properties [20]. Here, we also present the preliminary in vitro tests of antimycobacterial and antitumor
activities (A549, DU-145 and MCF-7) of the complexes. Finally, the interaction study of the synthesized
ruthenium complexes with DNA by spectroscopic titration, viscosity measurements, gel electrophoresis,
circular dichroism, ethidium bromide (EB) displacement, guanosine and guanosine monophosphate, and
with bovine serum albumin (BSA), by fluorescence quenching were carried out.
2. Experimental section
2.1 Materials and methods
All the syntheses of the complexes were performed under argon atmosphere. Solvents were
purified by standard methods. All chemicals used were of reagent grade or comparable purity. The
RuCl3·3H2O and the bis-(diphenylphosphine) ligands were purchased from Sigma-Aldrich. The
precursors cis-[RuCl2(P-P)(bipy)] were prepared according to the literature [21].
The electrochemical experiments were carried out using a BAS-100B/W MF-9063 Bioanalytical
Systems Instrument under argon atmosphere at room temperature with tetrabutylammonium perchlorate
(TBAP, FlukaPurum) as a supporting electrolyte. The electrochemical cell was equipped with platinum
working and auxiliary electrodes and Ag/AgCl as the reference electrode in a Luggin capillary probe, a
medium in which ferrocene is oxidized at 0.43 V (Fc+/Fc). Voltammograms were performed at a scan
rate of 0.100 V s−1. The infrared spectra (IR) were recorded on a FTIR Bomem-Michelson 102
spectrometer in the range of 4000–200 cm-1 using CsI pellets. Conductivity data were obtained in acetone
using a MeterLab CDM2300, measurements were made at room temperature using 1 mM solutions of the
complexes. The ultraviolet-visible (UV-Vis) spectra of the complexes in CH2Cl2 were recorded on a
Hewlett Packard diode array–8452A.
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31
1
1
1
All the 1D and 2D NMR experiments (1H, 13C{1H}, P{1H}, H-1H gCOSY, H-13C gHSQC, H-13C
gHMBC) were recorded on a 9.4 T Bruker Avance III spectrometer with a 5 mm internal diameter
indirect probe with ATMATM (Automatic Tuning Matching), holding the temperature stable at 300 K. In
general, 20 mg samples of CTZ and metal-CTZ complexes were dissolved in deuterated
dimethylsulfoxide (DMSO-d6, Cambridge Isotope Laboratories, Inc., USA). Mass spectra were obtained
by direct infusion in a Waters Synapt Mass Spectrometer in positive ion mode, utilizing CH3OCH3
(LC/MS grade from Honeywell; B & J Brand) as the solvent.
2.2 Synthesis
General procedure (Scheme 1-reaction 6). A solution of cis-[RuCl2(P-P)(bipy)] (0.100 mmol) in
dichloromethane (25 mL) was stirred until complete dissolution was achieved, an excess of KPF6 was
added; after 1 h the CTZ (0.15 mmol) dissolved in dichloromethane was added. The resultant mixture
was stirred and refluxed for 6 hours under argon atmosphere. Afterwards, the final orange solutions were
concentrated to ca. 2 mL and the solids were precipitated by adding n-hexane. The solids obtained were
filtrated and washed with water (3 x 5 mL) to remove KPF6 and KCl salts, n-hexane (3 x 5 mL) to
remove CTZ in excess, and dried under vacuum.
[RuCl(CTZ)(dppe)(bipy)]PF6 (1). Yield 92%; elemental analysis (%) Calc. for C58H49Cl2F6N4P3Ru calc.
(found) C 58.99 (58.98) H 4.18 (4.19) N 4.74 (4.69). Molecular weigth (MW) 1180.92; IR: υ(C=C) 1485
cm-1; υ(C=N) 1434 cm-1; υ(P-C) 1000 cm-1; υ(P-F) 842 cm-1; δ(P-F) 557 cm-1; υ(Ru-P) 503-526 cm-1;
υ(Ru-Cl) 316 cm-1. UV–vis [(CH2Cl2) λ/nm (ε, M−1 L cm−1)]: 236 (42.090), 296 (17.175), 438 (2.098).
1H NMR [400 MHz, dmso-d6, 298 K: ppm (m, I, attribution)]: 9.52, 8.54 and 8.25 (m, 4H, aromatic
protons of bipy ligand), 7.70-6.40 (m, 41H, overlap of aromatic protons of dppe (20H), bipy (4H) and
CTZ (17H) ligand), 3.08, 1.24 and 0.94 (m, 4H, CH2 aliphatic of dppe ligand).13C{1H} NMR [100.62
MHz, dmso-d6, 298 K: ppm (attribution)]: due to an overlap of the aromatic hydrogen, it was only
possible to identify a set of signals at carbons, 158.10-122.91 (Cq and CH of dppe, bipy and CTZ
31
ligands), 75.43 (C24-CTZ), 22.04 (CH2-P of dppe ligand). P{1H} NMR [162 MHz, dmso-d6, 298 K:
ppm (m, attribution)]: 65.70, 63.50 (d, P of dppe), -144.50 (h, PF6). High resolution ESI(+)-MS
(acetone): [M+H]+ (1081.1761 m/z, 7.15 %), [M –PF6]+ (1037.0835 m/z, 100.00 %), [M –CTZ –PF6]+
(691.0082 m/z, 92.85 %), [CTZ –Imidazol group]+ (277.0487, 67.20%).
[RuCl(CTZ)(dppb)(bipy)]PF6 (2).Yield 90%; elemental analysis (%) Calc. for C60H53Cl2F6N4P3Ru·H2O
calc. (found) C 58.73 (58.95) H 4.52 (4.76) N 4.57 (4.67), MW: 1226.99; IR: υ(C=C) 1483 cm-1; υ(C=N)
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1434 cm-1; υ(P-C) 1000 cm-1; υ(P-F) 840 cm-1; δ(P-F) 557 cm-1; υ(Ru-P) 507-518 cm-1; υ(Ru-Cl) 318 cm-
1
1. UV–vis [(CH2Cl2) λ/nm (ε, M−1 L cm−1)]: 236 (41.093), 298 (17.160), 458 (2.490). H NMR [400
MHz, dmso-d6, 298 K: ppm (m, I, attribution)]: 8.73, 8.37, 7.90 (d, 6H, aromatic protons of bipy
ligand),7.48-6.22 (m, 39H, overlap of aromatic protons of dppb (20H), bipy (2H) and CTZ (17H) ligand),
3.80, 3.20, 2.38 (m, 4H, aliphatics-CH2-P of dppb ligand), 1.91, 1.45, 1.18 (m, 4H, aliphatics C-(CH2)2-C
13
of dppb ligand). C{1H} NMR [100.62 MHz, dmso-d6, 298 K: ppm (attribution)]:due to an overlap of
the aromatic hydrogen, it was only possible to identify a set of signals at carbons, 159.07-121.62 (Cq and
CH of dppb, bipy and CTZ ligands), 74.95 (C24-CTZ), 27.88:25.83 (-CH2-P of dppb ligand), 23.09:20.85
(C-(CH2)2-C of dppb ligand).31P{1H}NMR [162 MHz, dmso-d6, 298 K: ppm (m, attribution)]: 39.10,
37.40 (d, P of dppb), -144.50 (h, PF6). High resolution ESI(+)-MS (acetone): [M –PF6]+ (1063.1721 m/z,
7.15 %), [M –CTZ –PF6]+ (719.0262 m/z, 100 %), [CTZ –Imidazol group]+ (277.0487, 75.85%).
[RuCl(CTZ)(dppf)(bipy)]PF6 (3). Yield 86%; elemental analysis (%) Calc. for C66H53Cl2F6FeN4P3Ru
calc. (found) C 59.30 (59.10) H 4.00 (4.39) N 4.19 (4.22), MW 1336.88; IR: υ(C=C) 1483 cm-1; υ(C=N)
1433 cm-1; υ(P-C) 1000 cm-1; υ(P-F) 840 cm-1;δ(P-F) 547 cm-1; υ(Ru-P) 511-520 cm-1; υ(Ru-Cl) 349 cm-
1
1. UV–vis (CH2Cl2) λ/nm (ε,M−1 L cm−1) 241 (46.275), 299 (20.148), 463 (3.773). H NMR [400 MHz,
dmso-d6, 298 K: ppm (m, I, attribution)]: 8.21, 6.52 (m, 45H, an overlap of aromatic protons of phenyl
groups of dppf (20H), bipy (8H) and CTZ (17H) ligand), 4.99, 4.52, 3.58 (m, 8H, aromatic C-H ferrocene
13
group of dppf). C{1H} NMR [100.62 MHz, dmso-d6, 298 K: ppm (attribution)]: due to an overlap of
the aromatic hydrogen, it was only possible to identify a set of signals at carbons, 158.97-122.71 (Cq and
CH of dppf, bipy and CTZ ligands), 75.37 (C24-CTZ), 73.92-72.97(CH of dppf ligand).31P{1H} NMR
[162 MHz, dmso-d6, 298 K: ppm (m, attribution)]: 38.40, 35.40 (d, P of dppf), -144.50 (h, PF6). High
resolution ESI(+)-MS (acetone): [M –PF6]+ (1191.0986 m/z, 7.15 %), [M –CTZ –PF6]+ (846.9580 m/z,
83.33 %), [CTZ-Imidazol group]+ (277.0487, 75.85%).
2.3 Interaction studies
Spectroscopic Measurements. All measurements with CT-DNA (calf thymus DNA from Sigma-
Aldrich) were carried out in a Tris–HCl buffer (5 mM Tris–HCl and 50 mM NaCl, pH 7.4). The DNA
concentration per nucleotide was determined by absorption spectrophotometric analysis using a molar
absorption coefficient of 6.600 mol-1.L.cm-1 at 260 nm [22]. The spectroscopic titrations were carried out
by adding increasing amounts of DNA to a solution of the complex, at a fixed concentration, in a quartz
cell and recording the UV–vis spectrum after each addition. The intrinsic binding constant Kb was
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determined from the plot of [DNA]/( εa-εf) vs [DNA], where [DNA] is the concentration of DNA in base
pairs, and the apparent absorption coefficients, εa, εf, εb correspond to Aobs/[Ru] the extinction coefficient
for the free ruthenium complex and ruthenium complex in the totally bound form, respectively. The data
were fitted to Eq. 1, with a slope equal to 1/(εb - εf) and the intercept equal to 1/ [Kb(εb - εf)] and Kb was
obtained from the ratio of the slope to the intercept [23].
[DNA]/(εa-εf) = [DNA]/(εb-εf) + 1/[Kb(εb-εf)]
(Eq. 1)
Viscosity experiments. Viscosity measurements were carried out using an Ostwald viscometer
immersed in a water bath maintained at 25 °C. The DNA concentration in a Tris–HCl buffer was kept
constant in all the samples, while the complex concentration was increased from 0 to 30 μM. The flow
time was measured at least 5 times with a digital stopwatch and the mean value was calculated. Data are
presented as (η/η0)1/3 versus the [complex]/[DNA] ratio, where η and η0 are the specific viscosities of
DNA in the presence and absence of the complex, respectively. The values of η and η0 were calculated
using the expression (t - tb)/tb, where t is the observed flow time and tb is the flow time of the buffer
alone.
Circular Dichroism (CD) experiments. CD spectra were recorded on a spectropolarimeter JASCO
J720 between 600 and 200 nm in a continuous scanning mode (200 nm/min). The final data are expressed
in molar ellipticity (millidegrees). All of the CD spectra were generated and represented averages of three
scans. Stock solutions (1.5 mM) of each complex were freshly prepared in DMSO prior to use. An
appropriate volume of each solution was added to the samples of a freshly prepared solution of CT-DNA
(100 μM) in a Tris–HCl buffer to achieve molar ratios ranging from 0.1 to 0.3 drug/DNA. The samples
were incubated at 37 °C for 18 h.
Agarose gel electrophoresis studies. 10 μL of pBR322 plasmid DNA in a Tris-HCl buffer were
incubated at 37 °C for 20 h with molar ratios (Ri) of the Ru(II) compounds between 0.5 and 4.0. After
incubation, 5 µl of each sample was separated by electrophoresis in a 1% agarose gel for 90 min at 100 V
using a Tris borate–EDTA buffer (TBE) and stained with ethidium bromide (5 μL ethidium bromide per
50 mL agarose gel mixture). Samples of free DNA and DNA with DMSO were used as controls. The
DNA bands were visualized as an image using a UV light transilluminator (ChemiDoc MP, Bio-Rad).
Ethidium bromide displacement. Assays were recorded on a spectrofluorimeter Spectra Max M3
in the 540−680 nm range with an excitation wavelength of 520 nm to solutions of CT-DNA (200 μM),
EB (40 μM) and complex (100 μM), inTris-HCl buffer at 25 °C.
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Reaction with guanosine and 5'-guanosine monophosphate. The interaction between the Ru-CTZ
1
complexes and DNA components was also studied and monitored by NMR spectroscopy making the H
and 31P{1H} NMR spectra at 25 °C in different time intervals. The study between complex 2 and
guanosine monophosphate was carried out in DMSO:D2O solutions. The interaction between complex 2
and the nucleotide guanosine was performed in DMSO.
Protein interaction. BSA (~2.5 μM) was prepared by dissolving the protein in Tris–HCl buffer
and the complexes were dissolved in DMSO. The BSA concentration was determined by absorption
spectrophotometric analysis using a molar absorption coefficient of 43.824 mol-1.L.cm-1 at 279 nm [24].
For fluorescence measurements, the BSA concentration in Tris–HCl buffer was kept constant in all the
samples, while the complex concentration was increased from 0.78 to 100 μM, and quenching of the
emission intensity of the BSA tryptophan residues at 340 nm (excitation wavelength 280 nm) was
monitored at different temperatures (295 and 310 K). The experiments were carried out in triplicate and
analyzed using the classical Stern–Volmer equation:
F0/F = 1 + Kqηo[Q] = 1 + Ksv[Q] (Eq. 2)
where F0 and F are the fluorescence intensities in the absence and presence of the quencher, respectively,
[Q] the quencher concentration, and Ksv Stern–Volmer the quenching constant, which can be written as
Kq= Ksv/ηo (Eq. 3)
where Kq is the biomolecular quenching rate constant and ηo is the average lifetime of the fluorophore in
the absence of the quencher (~10-9 s) [25]. The binding constant (Kb) and number of complexes bound to
BSA (n) were determined by plotting the double log graph of the fluorescence data using:
log [(F0-F)/F] = log Kb + nlog[Q] (Eq. 4)
The thermodynamic parameter (ΔH) was calculated from equation:
ln (K2/K1) = [(1/T1)-(1/T2)]ΔH/R (Eq. 5);
where K1 and K2 are the binding constants at temperatures T1 and T2, respectively, and R is the gas
constant. Furthermore, the change in free energy (ΔG) and entropy (ΔS) were calculated from the
following equation:
ΔG = -RT lnK = ΔH – TΔS (Eq. 6)
Determination of octanol-water distribution coefficients (Log Dow). Water–octanol partition
coefficients were determined using the shake flask method [26]. An UV-visible (UV-vis) calibration
curve was prepared in the range 10–80 μM in n-octanol with 2% of dimethylsulfoxide for solubilization
of the complexes. The determination was carried out at pH 7.4 in a mixture of equal volumes of water
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and n-octanol with 2% of dimethylsulfoxide and continuous shaking for 18 h at room temperature. The
concentration of complex in n-octanol was measured spectrophotometrically in order to determine values
of P=[compound](in octanol)/[compouns](in water).
2.4 Biological experiments
Cell Viability Assays. In vitro cytotoxicity assays on cultured human tumor cell lines still represent
the standard method for initial screening of antitumor agents. Before performing the biological screening,
the stability of the complexes was tested using the 31P{1H} NMR technique in DMSO solution containing
30% of Tris-HCl buffer. After 48 h, the spectra of the complexes were the same, when compared with
those recorded using fresh solutions. Thus, as a first step to assess their pharmacological properties, the
ruthenium complexes were assayed against human lung tumor line A549 (ATCC No. CCL-185), human
prostate tumor line DU-145 (ATCC No. HTB-81), human breast tumor line MCF-7 (ATCC No. HTB-
22), and the normal cell line L929 (ATCC No. CCL-1). The cells were routinely maintained with
Dulbecco’s Modified Eagle’s medium (DMEM-for L929 and A549) or RPMI 1640 (for MCF-7 and DU-
145) supplemented with 10% of fetal bovine serum (FBS), at 37 °C in a humidified 5% CO2 atmosphere.
For the cytotoxicity assay, 1.5 x 104 cells/well were seeded in 200 µL of complete medium in 96-well
plates (Corning Costar). Each complex was dissolved in DMSO from 40 to 0.01 mM, one microliter of
each complex sample was added to 200 µL medium. Cells were exposed to the complex for a 48 h
period. The conversion of MTT, 3-(4,5-dimethylthiazol-2-yl-2,5-diphenyltetrazolium bromide), to
formazan by metabolically viable cells was monitored by an automated microplate reader at 540 nm.
Morphological observations. For the morphological study DU-145 prostate tumor cells were seeded
at a density of 0.8 × 105 cells/well into 24-well plates. After allowing 24 h to adhere, images of cells
treated, with or without complex 2, were taken at 0, 2, 8, 24 and 48 h.
Antimycobacterial activity assay. The anti-M. tuberculosis activity of the ligands and of the
ruthenium complexes was determined using the REMA (Resazurin Microtiter Assay) method [27]. Stock
solutions of the compounds were prepared in DMSO and diluted in Middlebrook 7H9 broth (Difco,
Detroit, MI, USA) supplemented with oleic acid, albumin, dextrose and catalase (OADC enrichment
BBL/Becton-Dickinson, Sparks, MD, USA) so that final drug concentration ranges from 0.1 to 25 g.mL-1
could be obtained. The serial dilutions were performed in a Precision XS Microplate Sample Processor
(Biotek™). The rifampicin was dissolved in distilled water, and used as a standard drug. A suspension of
the MTB H37Rv ATCC 27294 was cultured in Middlebrook 7H9 broth supplemented with OADC and
0.05% Tween 80. The culture was frozen at -80⁰C in aliquots. After two days the colony forming unit
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(CFU)/mL of an aliquot was carried out. The concentration was adjusted to 5.105 CFU/mL and 100 µL of
the inoculum was added to each well of a 96-well microtiter plate together with 100µL of the compound.
Samples were set up in triplicate. The plate was incubated for 7 days at 37⁰C. After 24 h, 30 μL of 0.01%
resazurin (solubilized in water) was added. The fluorescence of the wells was read after 24 h using a
TECAN Spectrafluor. The MIC was defined as the lowest concentration resulting in 90% inhibition of
growth of MTB.
3. Results and discussion
3.1 Spectroscopic and analytical characterization
The reduction of ruthenium(III) chloride with excess of triphenylphosphine conducted in refluxing
methanol, afforded the brown crystalline solid of neutral complex [RuIICl2(PPh3)3], the
triphenylphosphine serves as the reducing agent yielding triphenylphosphine oxide. As presented in
scheme 1, [RuIICl2(PPh3)3] reacts with bidentate phosphine and bypiridine ligand in a sequence of
substitution reactions to give the precursor cis-[RuIICl2(P-P)(N-N)] that upon stirring and reflux, in
dichloromethane solution, and the presence of CTZ result in the new cationic compounds 1-3.
All three complexes were prepared in good yields (more than 85%), their general structures are
proposed based on spectroscopical data, as well as elemental analyses, which were in good agreement
1
31
with proposed formulations, molar conductivity measurements, and H, 13C{1H} and P{1H} NMR
spectra. Molar conductivities of 1, 2 and 3 in acetone, were in the range of 1:1 electrolytes [28]. The
electronic spectra for the complexes showed two bands in the UV region (240-300 nm) presents in the
free ligands and assigned to π→π* transitions from the aromatic rings are also present in the spectra of
the complexes, probably involving the phosphine, bipyridine and clotrimazole ligands [29,30], and one
band in the visible region (~ 450 nm), which can be assigned as metal to ligand charge transfer
transitions from Ru(dπ) to the ligand (π*), similar assignments have been proposed for other Ru(II)
complexes [31,32] and previous work by this group [33]. The solid state FT-IR spectra of the complexes
presented the characteristic band of Ru-P (502-521 cm-1), PF6 (841 and 557 cm-1), Ru-N (425-430 cm-1),
Ru-Cl (316-349 cm-1) and the imidazole ring (CH stretch) in the range of 3057-3064 cm-1.
The electrochemical behavior of the redox-active compounds were studied at room temperature using
the cyclic voltammetry technique, at a platinum disk electrode, in dichloromethane with TBAP as a
supporting electrolyte. The obtained voltammograms of the complexes are shown in Figure 3. Complexes
1 and 2 showed a similar electrochemical behavior, a quasi-reversible one-electron Ru(III)/Ru(II) redox
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process, with a potential at about 1000mV. For complex 3, this process is around ~ 800mV and other
redox process ~ 1570mV were also found, characteristics of the Fe(III)/Fe(II) couple from the ferrocene
molecule in the dppf ligand. In order to confirm this, an electrolysis at 1570 mV was carried out and the
product was reacted with NH4SCN. The red solution obtained is a characteristic of the formation of
[Fe(SCN)6]3- species. As expected, the E1/2 values found for the new complexes were considerably more
anodic than those observed for the respective precursors, between 177-480 mV, indicating a more
stabilized Ru(II) center in the new derivatives than in their precursors. This stabilization is suggested to
be due to the substitution of chlorido (ζ and π-donor) by an imidazole ring (ζ-donor and π-acceptor). The
proposed composition for the complexes were also confirmed by mass spectrometry analyses. The
ESI(+)-MS spectra for complex 1-3 showed a signal corresponding to a protonated species [M +H]+(only
for the complex 1) and its fragmentations generating the product ion corresponding to the loss of
hexafluorophosphate ion [M –PF6]+, followed by the loss of the clotrimazole ligand, forming the [M –PF6
–CTZ]+ and a peak corresponding to the free ligand [CTZ –Imidazole group]+.
31
The P{1H} NMR spectra of all complexes in CH2Cl2/D2O (Figure 4) present a typical AX spin
system indicating the magnetic non-equivalence of the two phosphorus atoms, corresponding to the P
trans N (bipy) and P trans N (CTZ). The chemical shifts and coupling constants (2JP-P) are shown in
Table 1. In the precursors, cis-[Ru(bipy)(P-P)Cl2], the high-field doublet corresponds to the P trans Cl, as
previously described [34], the shift to higher energy is indicative of the coordination of the metal ions to
the N3, from the CTZ ligand. The 1H NMR spectra for all complexes showed signals for CTZ, bipyridine
and the phosphine phenyl groups as a series of overlapped multiplets between the 9.20-6.20 ppm region,
corresponding to 45 hydrogen atoms. Furthermore, additional multiplets for complexes 1 and 2 are
observed corresponding to CH2 groups of dppe (3.08, 1.24, 0.94 ppm) and dppb (3.80, 3.20, 2.38, 1.91,
1.45, 1.18 ppm), and for 3 attributed to ferrocene of dppf ligand (4.99, 4.52 and 3.58 ppm).
3.2 Biological targets: DNA and BSA binding studies
DNA binding studies
To explore the possibility of DNA as a potential target for the complexes, spectroscopic studies were
carried out. UV-visible absorption spectroscopy is a versatile and normally engaged method to determine
the binding characteristics of metal complexes with DNA. The intense absorption spectral band around
300 nm, observed for all complexes, were used to evaluate their possible interaction with CT-DNA, in
Tris-HCl buffer at pH 7.4. The experiments were carried out keeping the concentration of the Ru(II)
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complexes constant, and varying the concentration of the CT-DNA. When adding the solution of CT-
DNA to each complex 1-3, a strong decrease in absorption intensity (hypochromism, ∼36%) was
observed. In order to compare the binding strength of the three complexes quantitatively, the intrinsic
binding constant Kb was calculated using the neighbor exclusion equation (Eq. 1). The Kb values are in
the trend 3 > 1 ≈ 2> CTZ. The Kb value for 3 (1.65x104 M-1) is two-fold higher than for 1 and 2 (1: 0.90;
2: 0.84x104 M-1) and ten-fold higher than for the CTZ (0.16 x104 M-1), indicating higher binding affinities
of 3 to CT-DNA, when compared with the other species. From the results obtained, it has been found that
all Ru(II) complexes show a good binding affinity to DNA and the values lie within the interval for
which a compound is considered to be interacting with DNA [35].
Viscosity measurements are successfully used to determine if there are intercalation or non-
intercalation binding modes of the complexes to DNA [36]. It is well known that classical intercalators,
such as ethidium bromide, lead to an increase in the viscosity of CT-DNA, when in the presence of
complexes, because separation of the base pairs occurs to accommodate the intercalator [37]. A covalent
DNA-binding mode may cause its fragmentation, thus decreasing the DNA viscosity [38]. Thus, in this
study it can be observed that the viscosity of the solution of CT-DNA did not change significantly when
the concentration of the complexes 1-3 was increased, indicating that ruthenium complexes bind with
DNA in a non-intercalative manner.
The CD spectral technique is very sensitive for diagnosing changes in the secondary structure of
DNA, resulting from drug–DNA interactions. A typical CD spectrum of calf thymus CT-DNA shows a
maximum at 275 nm, due to the base stacking and a minimum at 248 nm attributed to the right-handed
helicity, characteristic of the B conformation. To determine if the Ru(II)-CTZ complexes cause changes
in the helical structure of DNA, CD spectra of CT-DNA with increasing concentrations of compounds 1–
3 and free CTZ were acquired, up to molar ratio drug/DNA (Ri):0.3, and a significant change was not
observed in the CD magnitude (see Figure S7 in supplementary information).
Ethidium bromide (3,8-diamino-5-ethyl-6-phenyl phenanthridium bromide, EB) is a known DNA
intercalator agent, widely used as a sensitive fluorescent probe for DNA due to its high fluorescence
when bound to the nucleic acid. The free EB molecule shows reduced emission intensity as a
consequence of the solvent quenching or of a photoelectron transfer mechanism [39], when bound to
DNA, EB shows a remarkable enhancement in fluorescence, due to a steric protection that the
nucleobases provide to the dye molecule [40]. The presence of another species with affinity towards
DNA may result in a decrease in the emission intensity of the EB-DNA adduct, caused by either a
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competition for binding sites, a change in DNA conformation or through a photoelectron transfer
mechanism. EB displacement assays were performed to extract more information about the binding DNA
and Ru(II)-CTZ complexes by monitoring the emission intensity of DNA-bound EB fluorescence. In
Figure 5, it can be observed that after adding the complexes (100 μM) to CT-DNA solution (200 μM)
pretreated with EB, (40 μM) ([DNA]/[EB] : 5) in 5 mM of Tris-HCl, the emission intensity of DNA-
bound EB decreases 9.90% (for 1, 2) and 17.30% (for 3), which is consistent with the order of the above
Kb values obtained by UV-Vis absorption spectral titration. This result indicates that 3 at higher
concentrations would compete with DNA-bound EB more efficiently than 1 and 2, and indicate that the
complexes can partially displace the DNA-bound EB suggesting low to moderate competition with EB,
this DNA binding behavior was observed previously for non-intercalative complexes containing CTZ
like as Cu(II), Co(II), Ni(II), Zn(II) [8] and also complexes containing antimicrobial drugs ofloxacin
(ofloH) and norfloxacin (nfH) with general formula: [Cu(nfH)(phen)Cl]Cl, [Cu(nfH)2]Cl2 and
[Cu(ofloH)2][(CuCl2)2] [41].
The interaction of complexes 1–3 with pBR322 DNA was studied by agarose gel electrophoresis
(Figure 6) by incubating the Ru(II) complexes or free clotrimazole, with pBR322 DNA (40 μM) in Tris-
HCl buffer at pH 7.4 for 18 h, at 37 °C, with Ri 0.5, 2.0 and 4.0. The control experiment in lane 2 shows
the corresponding open circular, linear and super coiled form for untreated plasmid DNA. Lines 3-5
correspond to the plasmid incubated with clotrimazole, while lines 4–14 correspond to the plasmid
incubated with different concentrations of complexes 1, 2 and 3. The CTZ ligand does not induce a
noticeable alteration in the mobility of the plasmid, however it is clear that the increase in the
concentration of each Ru(II)-CTZ complex caused remarkable changes in the mobility of the plasmid.
Only two bands are evident for all complexes at Ri 0.5, representing both the linear and open circular
forms. At Ri 2.0 and Ri 4.0, no bands (either relaxed or linear band) are visible for the DNA. The first
possible explanation for this observation is that EB was completely expelled out of the plasmid due to
DNA binding of the complex at higher concentrations, leading to quenching of the EB emission. Another
alternative for this observed effect could be due to changes in the DNA structures, suggesting a strong
interaction of the complexes with the plasmid, inhibiting the EB binding. In all likelihood, a combination
of both effects probably occur: EB expels and changes in the DNA structure. Similar behavior was
observed previously for the [Ru(Hdpa)2(diimine)]ClO4 [42] and [Ru(bpy)(L)]PF6 complexes [43].
In conclusion, from all the data obtained it can be suggested that the Ru(II)-CTZ compounds present
DNA binding affinity, primarily through weak interactions, such as hydrogen bonding by major or minor
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groove and/or electrostatic interactions involving the negatively charged phosphate groups of DNA,
considering the positive charge of the complexes. Thus, in order to verify these observations, the reaction
between complex 2 and guanosine or guanosine monophosphate was studied to check the possible
1
lability of the chlorido ligand present in the complex. In Figure S8 (supplementary information), the H
and 31P{1H} NMR spectrum of complex 2 and the mixture of complex 2: guanosine are shown. As can be
31
seen, after mixing the reagents, no changes were observed in the P{1H} NMR spectrum for complex 2
at different times, indicating that there was not a strong association with the DNA components.
BSA binding studies
Fluorescence spectroscopy is an effective method to explore the interaction between small
molecules and macromolecules. To understand the mechanism of interaction between 1-3 and BSA,
fluorescence quenching experiments were carried out. The fluorescence of BSA comes from its
tryptophan, tyrosine and phenylalanine residues, where the latter two contribute to its fluorescence in
only a small part [44]. The fluorescence intensity of a compound can be decreased by a variety of
molecular interactions, such as excited-state reactions, molecular rearrangements, energy transfer,
ground-state complex formation and collision quenching [45]. Static quenching refers to fluorophore-
quencher complex formation. Thus, when the temperature is increased, this process occurs less efficiently
since the fluorophore-quencher complex is likely to be less stable under the conditions, leading to a
decrease in the fluorescence quenching. Moreover, dynamic quenching refers to a process whereby the
fluorophore and the quencher come into contact during the lifetime of the excited-state and higher
temperatures increase the fluorescence quenching, and as a result the quenching constant increases [46].
In order to ascertain the fluorescence quenching mechanism, the fluorescence quenching data were
measured at different temperatures (295 and 310 K), and the results are shown in Table 2.
These results show that KSV for 1-3 are directly related to the increase in temperature, indicating
that the probable quenching mechanism reaction is initiated by compound formation rather than by
dynamic collision. Moreover, the values of Kq were in the range of (9.18-27.37) x1013M-1s-1 for all
complexes, far higher than 2.0×1010 M-1s-1, the maximum possible value for dynamic quenching [47],
indicating the existence of a static quenching mechanism. The number of binding sites is approximately
equal to 1, indicating that there is only one binding site in the BSA for each complex, similar or equal to
those reported before for other metal complexes [48].
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The interaction forces between drugs and biomolecules may include electrostatic interactions,
multiple hydrogen bonds, van der Waals interactions, hydrophobic and steric contacts within the
antibody-binding site, etc [49]. The thermodynamic parameters, enthalpy change (ΔH), entropy change
(ΔS) and free energy change (ΔG) are the main means used to confirm the binding modes. From the
thermodynamic standpoint, ΔH> 0 and ΔS> 0 imply a hydrophobic interaction; ΔH< 0 and ΔS< 0 reflects
van der Waals force or hydrogen bond formation; and ΔH< 0 and ΔS> 0 suggests an electrostatic force.
As observed in Table 2, the positive ΔH and ΔS values reveal the predominance of hydrophobic
interactions of the compounds with BSA. Furthermore, the negative ΔG values reveal that the interaction
process is spontaneous. The magnitude of the BSA-binding constant of complexes 1–3, compared with
other Ru(II) complexes reported recently [60], suggests a moderate interaction with BSA molecule.
3.3 In vitro biological experiments
Growth inhibition assay and morphological observations
Since the 1990s, M-CTZ complexes have been described, where M = Pt(II), Pd(II), Au(I), Ru(II),
Cu(II) among other ions, showing that these complexes present a biological potential, mainly against
parasites such as T. cruzi, T brucei and Leishmania and pathogenic agents of neglected diseases such as
Trypanosomiasis, Chagas diseases and Leishmaniasis. Recently, a new family of organometallic
complexes with formula [Ru(η6-p-cymene)(X)(CTZ)], where X: Cl2, bipyridine, ethylenediamine or
acetylacetonate, have shown high in vitro activity against Leishmania major and Trypanosoma cruzi and
low toxicity toward normal mammalian cells. Therefore, few reports of metal complexes with CTZ have
been evaluated against tumor cells. However, free clotrimazole showed a significant decrease in the
MCF-7 cell viability, altering its morphological and inhibiting the glycolysis. A comparison with
previously reported CTZ metal complexes is significant: in 2006, Pd–(CTZ)2 complex showed very poor
cytotoxicity against four human tumor cell lines: MDA-MB-231, PANC-1, SKBR-3 and HT-29, values
in the 89-159 µM range [50]. By exchanging the metal center from Pd to Pt, two Pt-(CTZ)2 complexes
were reported and both showed inhibitory effects on five cells lines: HT-29, LoVo, MCF-7, SKBR-3 and
PC-3 with IC50 values in the 5–25 µM range [9]. Current reports (the first in 2012) showed a synergistic
effect in the binding of the CTZ to metal ions such as Cu(II), Co(II), Ni(II), and Zn(II), toward three
human carcinoma cells: Hela, PC-3 and HCT-15, with IC50 values in the 3.5-29.8 µM range, suggesting
that selected Cu(II)-CTZ complexes induce cell death via an apoptotic pathway [8]. Furthermore, more
recently, in 2013, a series of six Ru–CTZ compounds were studied, showing their activities against three
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prostate tumor, cervical cancer and lymphoblastic lymphoma cell lines, with IC50 values in the 2-546 µM
range [7].
Thus, having this in mind, complexes 1–3 were evaluated against the A549 (human lung
carcinoma), DU-145 (human prostate carcinoma), MCF-7 (human breast carcinoma) and L929 (normal)
cell line. The IC50 values were calculated from the dose-survival curves obtained after 48 h of Ru(II)-
CTZ complexes treatment with an MTT assay (Table 3). The results obtained using this assay show that
all three Ru(II)-CTZ complexes presented growth inhibitory effects on three tumor cells lines at doses
appreciably lower in comparison with free CTZ, under the same conditions, highlighting the importance
of the Ru(II)/bipyridine/phosphine moiety to the antitumor biological activity. For the sake of
comparison, the cytotoxicity of cisplatin was evaluated under the same experimental conditions and the
complexes were more active against human A549 and complexes 1-2 were more active against MCF-7
cell lines than cisplatin. The most remarkable effect was observed in the multidrug-resistant lung cancer
cell A549, in which the complexes exert the strongest activity, with IC50 values between 0.47 to 4.79 µM.
In this cell line, the free ligand shows a much weaker cytotoxicity, yielding an IC50 value of 14.47 µM.
Under an inverted microscope, cell shape and changes in it can be observed clearly. As shown in Figure
7, the DU-145 prostate tumor cells show epithelial cell morphology, while in the control group there
were very few round cells. Cells treated with complex 2 showed obvious morphological changes after the
first 24 h; cells treated for 48 h showed, in addition to morphological changes, a loss of adhesion, a
epithelial form and confluence, indicating the possibility of apoptosis.
The values of log D (Table 3) are between the interval 0.48-0.84, so that the biphosphine moiety
does not modify significantly lipophilicity for complexes 1-3 and no quantitative correlation between
lipophilicity and biological activity was observed.
Anti-Mycobacterium tuberculosis activity. The literature describes several cases in which a
connection can be made between the structure and activity of coordination compounds, where structural
properties can directly interfere with the mechanisms of action of the compounds with anti-
mycobacterium tuberculosis activity. Our research group reported Ru(II) complexes containing
byphosphines and bipyridines as binders that were extremely promising, showing minimum inhibitory
concentration values similar or better than the current drugs used to treat this disease. Generally, Ru(II)
compounds containing bidentate as binders (N-O, N-S or O-O) and two diphosphines or
diphosphines/bipyridine ligands show MIC values better than the precursor compound containing
chlorido atoms in place of the bidentate ligands. For these reasons, the in vitro anti-MTB activities of the
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free ligand and the Ru(II)-CTZ complexes were tested against MTB H37Rv ATCC 27294 (see Figure 8).
The MIC values are reported in Table 4. The activity found for the complexes is comparable or better
than those of some commonly used anti-M. tuberculosis agents, such as cycloserine (MIC = 12.50-50
μM), gentamicin (MIC = 4.20-8.40 μM), tobramycin (MIC = 8.56-17.10 μM) and clarithromycin (MIC =
10.70-21.40 μM) [51]. Antimicrobial activity assays of the new complexes provided evidence that
complexes 1, 2 and 3 are potential agents against mycobacterial infections, specifically against M.
tuberculosis H37Rv.
4. Conclusions
Three complexes combining clotrimazole with different Ru(II)-diphosphine/bipyridine complexes, with
general formula [RuCl(CTZ)(bipy)(P-P)]PF6, were synthesized and characterized by a variety of
techniques in a solid state and in solution. The complexes binding to BSA with moderate affinity
through a static quenching mechanism and the thermodynamic parameters reveal the predominance of
hydrophobic interactions with the protein. The activity of the new compounds was evaluated against
three human tumor cell lines and very promising results were obtained, yet all of them had poor
selectivity indices (~1) versus healthy cells, worse that even cisplatin. Indeed the IC50 values of Ru(II)-
CTZ complexes on A549 (lung) and MCF-7 (breast) tumor cells lines were lower than those obtained for
the free clotrimazole and for the cisplatin (reference drug). The MIC values of anti-M. tuberculosis
activity obtained for the complexes showed an activity comparable or better than that shown for various
second-line drugs used to treat illnesses. They also provided us with evidence that they are potential
agents against mycobacterial infections, encouraging the continuation of studies of these types of
compounds.
5. Abbreviations
CTZ
bipy
Clotrimazole
2,2`-bypiridine
BSA
dppe
dppb
dppf
Bovine serum albumin
1,2-bis(diphenylphosphino)ethane
1,4-bis(diphenylphosphino)butane
1,1’-bis(diphenylphosphino)ferrocene
Mycobacterum tuberculosis
MTB
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DMSO
TBAP
ATCC
CT
Dimethyl sulfoxide
Tetrabutylammonium perchlorate
America type culture collection
Calf thymus
EB
Ethidium bromide
CD
Circular dichroism
Ri
Molar ratios
MTT
CFU
MIC
3-(4,5-dimethylthiazol-2-yl-2,5-diphenyltetrazolium bromide
Colony forming unit
Minimum inhibitory concentration
Ksv
Kq
ηo
Stern-Volmer constant
Biomolecular quenching rate constant
Average lifetime of the fluorophore
ACKNOWLEDGMENTS
We would like to thank CNPq, CAPES, FAPESP for the financial support. We also thank Prof. Dr.
Otaciro Nascimento for the CD spectra at IFSC-USP and Dra. Soraya Ochs for the ESI-MS spectra at
INMETRO.
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Figure 1. Structure of the most promising ruthenium complexes in medicinal chemistry
Figure 2. Selected Ru(II)-Bisdiphenylphosphine metal complexes with significant antimycobacterial
properties prepared in our group [14-18]
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Scheme 1. Synthesis of the Ru(II)/clotrimazole/diphenylphosphine/bipyridine complexes
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Figure 3. Cyclic voltammogram for complexes [RuCl(CTZ)(bipy)(P-P)]PF6 in CH2Cl2, (TBAP 0.1 M;
Ag/AgCl; work electrode Pt; 100 mV.s−1)
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Table 1. Electrochemical, NMR 31P{1H} and molar conductivity data for complexes 1-3
Epa (mV)
1164
Epc (mV)
1011
E1/2
Ipa/Ipc
1.14
1.22
31P{1H} (ppm)
63.5; 65.7
2J(P-P)(Hz)
16.4
ΛM**
132.0
133.3
1087
1083
1
2
1135
1031
37.4; 39.1
36.0
862/1570*
716/1375*
---
----
35.4; 38.4
28.5
123.2
3
* FeII/FeIII potencial values. **ΛM: ohm-1cm2mol-1
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Figure 4. 31P{1H} NMR spectra of complexes [RuCl(CTZ)(bipy)(P-P)]PF6, in CH2Cl2/D2O
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Figure 5. Fluorescence spectra obtained from EB displacement assays. [CT-DNA]= 200 μM, [EB] = 40
μM and [Complex]= 100 μM, inTris-HCl buffer at 25 °C.
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Figure 6. Electrophoresis of plasmid pBR322 DNA incubated in 5 mM Tris-HCl buffer for 18 h at 37 °C
treated with 0.5, 2.0 and 4.0 eq. of CTZ and complexes 1, 2 and 3. MM: molecular marker, C: control,
pBR322 with DMSO.
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Table 2. Stern-Volmer quenching constant (Ksv, M-1), biomolecular quenching rate constant (Kq, M-1s-1),
binding constant (Kb,M-1), the number of binding sites (n), ΔG0(KJ.mol-1), ΔH0 (KJ.mol-1) and ΔS0
(J.mol-1K) values for the complex-BSA system at different temperatures
Ksv
Kq
Kb
n
ΔG
ΔH
ΔS
(x104)
(x1013)
(x104)
295
310
295
310
295
310
295
310
0.22
0.22
1.48
1.52
1.77
1.90
3.74
3.93
1.39
0.06
0.05
1.36
1.43
1.49
1.49
3.30
3.36
1.20
-1.22
60.47 246.50
CTZ
1.39
1.77
0.85
1.03
0.75
0.63
0.87
0.90
-1.59
-2.34
-2.48
-2.34
-2.48
-0.49
-0.54
9.18
4.31
2.86
3.82
93.84
89.15
29.65
1
2
3
9.43
10.91
11.77
23.19
27.37
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Table 3. Biological activity of the ruthenium compounds against four tumor cell lines for 48h incubation
and distribution coefficient (log D) at pH 7.4
IC50 (μM)
IS1
IS2
IS3
log D
A549
DU-145
MCF-7
L929
CTZ
14.47 ± 0.95 15.82 ± 0.23 16.80 ± 2.52
9.74 ± 2.05
0.67
0.61
0.58
--
2.11 ± 0.15
0.47 ± 0.05
4.79 ± 0.40
3.64 ± 0.50
3.32 ± 0.45
7.40 ± 0.35
2.33 ± 0.40
3.83 ± 0.12
1.68 ± 0.32
13.28 ± 1.00
2.61 ± 0.13
0.48 ± 0.05
5.89 ± 0.80
1.23
1.02
1.22
1.14
0.71
0.14
0.80
7.09
0.68
0.28
0.44
1.18
0.60 ± 0.06
0.48 ± 0.02
0.84 ± 0.10
--
1
2
3
Cisplatin* 14.42± 1.45
13.98 ± 2.02 16.53 ± 2.38
* Reference drug. IS1=IC50L929/IC50A549; IS2=IC50L929/IC50DU-145, IS3=IC50L929/IC50MCF-7
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