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Antitumor and anti-Mycobacterium tuberculosis agents based on cationic ruthenium complexes with amino acids
Inorganica Chimica Acta 463 (2017) 1–6
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Inorganica Chimica Acta
journal homepage: www.elsevier.com/locate/ica
Research paper
Antitumor and anti-Mycobacterium tuberculosis agents based on cationic
ruthenium complexes with amino acids
Edjane R. dos Santos a,⇑, Rodrigo S. Corrêa b, Lucas V. Pozzi a, Angelica E. Graminha a,
Heloisa S. Selistre-de-Araújo c, Fernando R. Pavan d, Alzir A. Batista a,⇑
a
Departamento de Química, Universidade Federal de São Carlos, C.P. 676, CEP 13565-905 São Carlos, SP, Brazil
Departamento de Química, ICEB, Universidade Federal de Ouro Preto, CEP 35400-000 Ouro Preto, MG, Brazil
Departamento de Ciências Fisiológicas, Universidade Federal de São Carlos, C.P. 676, CEP 13565-905 São Carlos, SP, Brazil
d
Departamento de Ciências Biológicas, Faculdade de Ciências Farmacêuticas, UNESP, CEP 14800-900 Araraquara, SP, Brazil
b
c
a r t i c l e
i n f o
Article history:
Received 25 March 2017
Accepted 6 April 2017
Available online 8 April 2017
Keywords:
Ruthenium complexes
Amino acid
Diastereoisomers
Cytotoxicity
Anti-Mycobacterial tuberculosis
a b s t r a c t
Six new complexes of Ru(II)/phenanthroline/1,4-bis(diphenylphosphino)butane containing amino acids
(Glycine, L-Alanine, L-Valine, L-Tyrosine, L-Methionine or L-Tryptophan) were synthesized and characterized by IR, 31P{1H}, 13C and 1H NMR spectroscopies and cyclic voltammetry experiments. These data suggest the presence of diastereoisomers, except for the complex with glycine, amino acid that does not
exhibit chiral carbon. The compounds are active against the MDA-MB-231 tumor cells and against
Mycobacterium tuberculosis. The cationic ruthenium complexes with amino acids, reported here, show
similar cytotoxicity against the MDA-MB-231 tumor cells. When compared with analogs complexes containing 2,20 -bipyridine as ligands, instead of 1,10-phenatroline, the new complexes studied here are, in
general, roughly twice more active than the 2,20 -bipyridine ones and their IC50 values comparable with
the cisplatin. In addition, low MICs values were obtained against Mycobacterium tuberculosis compared
with the reference drugs, cycloserine and ethambutol.
Ó 2017 Elsevier B.V. All rights reserved.
1. Introduction
Transition metal complexes represent a very important class of
promising chemotherapeutics. Many researchers have proposed a
large number of metal complexes as potential anticancer agents,
and/or with antibacterial properties [1–21]. Among then, ruthenium compounds have been widely investigated, showing interesting biological effects [22–26].
In addition, metal complexes with amino acids have been
explored due to their notable potential role in cancer therapy
[1,17–20,27–30], in which octahedral complexes containing amino
acids tend to form diastereoisomers due to their chiral carbon,
forming complexes with D and K configurations around the metal
[31–37].
Recently, our research group has published interesting biological results of ruthenium/biphosphine complexes exhibiting good
cytotoxicity against tumor cells and/or good activity anti-Mycobacterial tuberculosis [1,2,15,17,19–20,38,39]. Thus, continuing the
search for antitumor agents with better cytotoxicity against tumor
⇑ Corresponding authors.
E-mail addresses: edjanemrocha@gmail.com (E.R. dos Santos), daab@ufscar.br
(A.A. Batista).
http://dx.doi.org/10.1016/j.ica.2017.04.012
0020-1693/Ó 2017 Elsevier B.V. All rights reserved.
cells and/or also with good anti-Mycobacterial tuberculosis properties, we present here the synthesis, characterization and investigation of a series of ruthenium complexes with amino acids. Also, the
cytotoxic activity against the MDA-MB-231 tumor cells and against
Mycobacterium tuberculosis were carried out.
2. Experimental
2.1. Materials and measurements
All reactions were carried out under an argon atmosphere using
standard Schlenk techniques. Reagent grade solvents were appropriately dried and distilled before use. All chemicals used were of
reagent grade or of comparable purity. All chemicals were purchased from Aldrich or Fluka and were used as received. The precursor cis-[RuCl2(dppb)(phen)] was prepared using published
procedures [19], where dppb means 1,4-bis(diphenylphosphino)
butane and phen is phenanthroline.
The IR spectra of the complexes were recorded on a FT-IR
Bomem-Michelson 102 spectrometer in the 4000–200 cm1
region, using solid samples pressed in CsI pellets. All nuclear magnetic resonance (NMR) experiments were recorded on a BRUKER
2
E.R. dos Santos et al. / Inorganica Chimica Acta 463 (2017) 1–6
spectrometer, in a BBO 5 mm probe at room temperature, observing 1H at 400.13 MHz using deuterated chloroform (CDCl3) as solvent and 31P{1H} at 161.98 MHz in CH2Cl2 using a capillary of D2O
to get the lock. In the 31P{1H} experiments chemical shifts are with
respect 85% H3PO4 signal as external reference and 1H and 13C{1H}
experiments were calibrated by TMS signal as internal references.
The molar conductivity measurements (Km) were taken in dichloromethane at 25 °C, using concentrations of 1.0 103 M for the
complexes. Cyclic voltammetry (CV) experiments were carried
out at room temperature in dichloromethane (CH2Cl2) containing
0.100 mol L1 Bu4NClO4 (TBAP) (FlukaPurum) using a BAS-100B/
W Bioanalytical Systems Instrument. The working and auxiliary
electrodes were stationary Pt foils; the reference electrode was of
Ag/AgCl, in a Luggin capillary probe. Ultraviolet–visible (UV–vis)
spectra were recorded on a HP 8452A diode array spectrophotometer. CHNS elemental analyses were carried out on EA 1108 of the
FISONS.
2.2. Synthesis
2.2.1. Synthesis of complexes [Ru(AA-H)(dppb)(phen)]PF6,
AA = glycine; L-Alanine; L-Valine; L-Tyrosine; L-Methionine; LTryptophan
The synthetic procedure used in this work was similar to that
used in the literature [1,20]. As an example, 0.011 g (0.15 mmol)
of glycine was added to a Schlenk flask, and was dissolved in
50 mL of methanol previously deoxygenated. Once the glycine
was fully solubilized, 0.08 g (0.10 mmol) of the precursor, cis[RuCl2(dppb)(phen)], was added to the flask followed by an addition of 0.025 g (0.16 mmol) of NH4PF6. The mixture was allowed
to reflux for 48 h. After completing the reaction, the solution volume was reduced to about 1.0 mL, and deoxygenated diethyl ether
was added promoting the precipitation of a solid, which was collected on a fritted funnel, washed with water and ethyl ether,
and dried under vacuum. Yield 0.086 g (90%). This synthetic route
was used for the other amino acid complexes synthesized in this
work. The yields for the other complexes were also in the order
of 90%. The characterization of the synthesized complexes are
described above:
2.2.1.1. [Ru(Gly-H)(dppb)(phen)]PF6. UV–vis (CH2Cl2) k/nm 266,
374; IR (CsI) m/cm1 3439, 1616, 1385, 852, 557, 509, 420; 1H
NMR (400.21 MHz, CDCl3) d 1.37 (m, 1H, CH2), 2.20–2.00 (m, 2H,
CH2), 2.54–2.25 (m, 3H, CH2), 2.64 (m, 1H, CH2), 3.52 (m, 1H,
CH2), 3.57 (t, 4H, Gly), 5.99 (t, 2H, J 8.8, Ph), 6.65 (t, 1H, J 6.8, phen),
7.12 (dt, 1H, J 6.8, 1.6, phen), 7.45–7.35 (m, 4H, Ph), 7.66–7.48 (m,
5H, Ph), 7.82–7.72 (m, 3H, Ph), 7.87 (t, 2H, J 8.0, Ph), 8.05–7.96 (m,
1H, phen; 2H, Ph), 8.14 (d, 1H, J 9.2, phen), 8.52–8.49 (m, 1H, phen;
2H, Ph), 8.74 (d, 1H, J 8.4, phen), 9.20 (d, 1H, J 5.2, phen), 9.39 (m,
1H, phen); 13C NMR (100.05 MHz, CDCl3) d 52.4, 183.6; 31P{1H}
NMR (162 MHz, CH2Cl2/D2O) d (d, 47.2 and 40.7, 2JPP 33.4); KM/
(X1 cm2 mol1) 30.03; calcd for C42H40F6N3O2P3Ru: C, 54.43; H,
4.35; N, 4.53%; found: C, 54.43; H, 4.49; N, 4.51%.
2.2.1.2. [Ru(L-Ala-H)(dppb)(phen)]PF6. UV–vis (CH2Cl2) k/nm 270,
408; IR (CsI) m/cm1 3439, 1622, 1385, 854, 557, 509, 420; 1H
NMR (400.21 MHz, CDCl3) d 1.37 (m, 1H, CH2), 1.48 (s, 3H, CH3,
Ala), 2.20–2.00 (m, 2H, CH2), 2.54–2.25 (m, 3H, CH2), 2.64 (m,
1H, CH2), 3.52 (m, 1H, CH2), 3.79 (t, 3H, Ala), 5.99 (t, 2H, J 8.8,
Ph), 6.65 (t, 1H, J 6.8, phen), 7.12 (dt, 1H, J 6.8, 1.6, phen), 7.45–
7.35 (m, 4H, Ph), 7.66–7.48 (m, 5H, Ph), 7.82–7.72 (m, 3H, Ph),
7.87 (t, 2H, J 8.0, Ph), 8.05–7.96 (m, 1H, phen; 2H, Ph), 8.14 (d,
1H, J 9.2, phen), 8.52–8.49 (m, 1H, phen; 2H, Ph), 8.74 (d, 1H, J
8.4, phen), 9.20 (d, 1H, J 5.2, phen), 9.39 (m, 1H, phen); 13C NMR
(100.05 MHz, CDCl3) d 19.2, 48.3, 185.3 and 182.9; 31P{1H} NMR
(162 MHz, CH2Cl2/D2O) d (d, 46.9 and 38.4, 2JPP 33.2) and (d, 44.7
and 38.1, 2JPP 33.2); KM/(X1 cm2 mol1) 27.29; calcd for C43H42F6N3O2P3Ru: C, 54.90; H, 4.50; N, 4.47%; found: C, 54.81; H, 4.46; N,
4.13%.
2.2.1.3. [Ru(L-Val-H)(dppb)(phen)]PF6. UV–vis (CH2Cl2) k/nm 270,
408; IR (CsI) m/cm1 3443, 1624, 1385, 847, 557, 509, 420; 1H
NMR (400.21 MHz, CDCl3) d 1.37 (m, 1H, CH2), 1.7 (t, 6H, CH3,
Val), 2.20–2.00 (m, 2H, CH2), 2.3 (t, 1H, CH3,Val), 2.54–2.25 (m,
3H, CH2), 2.64 (m, 1H, CH2), 3.52 (m, 1H, CH2), 3.6 (t, 3H, Val),
5.99 (t, 2H, J 8.8, Ph), 6.65 (t, 1H, J 6.8, phen), 7.12 (dt, 1H, J 6.8,
1.6, phen), 7.45–7.35 (m, 4H, Ph), 7.66–7.48 (m, 5H, Ph), 7.82–
7.72 (m, 3H, Ph), 7.87 (t, 2H, J 8.0, Ph), 8.05–7.96 (m, 1H, phen;
2H, Ph), 8.14 (d, 1H, J 9.2, phen), 8.52–8.49 (m, 1H, phen; 2H,
Ph), 8.74 (d, 1H, J 8.4 Hz; phen), 9.20 (d, 1H, J 5.2 Hz; phen), 9.39
(m, 1H, phen); 13C NMR (100.05 MHz, CDCl3) d 30.0, 58.4, 182.2
and 180.9; 31P{1H} NMR (162 MHz, CH2Cl2/D2O) d (d, 47.0 and
38.9, 2JPP 33.0) and (d, 42.9 and 38.5, 2JPP 32.8); KM/(X1 cm2 mol1) 34.17; calcd for C45H46F6N3O2P3Ru: C, 55.79; H, 4.79; N,
4.34%; found: C, 55.42; H, 4.80; N, 4.26%.
2.2.1.4. [Ru(L-Tyr-H)(dppb)(phen)]PF6. UV–vis (CH2Cl2) k/nm 270,
408; IR (CsI) m/cm1 3443, 1616, 1385, 860, 557, 509, 420; 31P
{1H} NMR (162 MHz, CH2Cl2/D2O) d (d, 47.8 and 38.9, 2JPP 34.4)
and (d, 42.9 and 38.1, 2JPP 32.9); KM/(X1 cm2 mol1) 30.99; calcd
for C49H46F6N3O3P3Ru: C, 56.98; H, 4.49; N, 4.07%; C, 57.00; H,
4.48; N, 4.03%.
2.2.1.5. [Ru(L-Met-H)(dppb)(phen)]PF6. UV–vis (CH2Cl2) k/nm 270,
406; IR (CsI) m/cm1 3441, 1624, 1385, 843, 557, 509, 420; 1H
NMR (400.21 MHz, CDCl3) d 1.82 (m, 4H, dppb, CH2(CH2)2CH2),
2.0 (s, 3H, CH3, Met), 2.3 (t, 4H, Met), 2.77 (m, 4H, dppb, CH2(CH2)2(CH2), 3.8 (t, 3H, Met), 5.99 (t, 2H, J 8.8; Ph), 6.65 (t, 1H, J
6.8, phen), 7.12 (dt, 1H, J 6.8, 1.6, phen); 7.45–7.35 (m, 4H, Ph);
7.66–7.48 (m, 5H, Ph); 7.82–7.72 (m, 3H, Ph), 7.87 (t, 2H, J 8.0,
Ph), 8.05–7.96 (m, 1H, phen; 2H, Ph), 8.14 (d, 1H, J 9.2, phen),
8.52–8.49 (m, 1H, phen; 2H, Ph), 8.74 (d, 1H, J 8.4, phen), 9.20 (d,
1H, J 5.2, phen), 9.39 (m, 1H, phen); 31P{1H} NMR (162 MHz, CH2Cl2/D2O) d (d, 48.0 and 40.1, 2JPP 32.8) and (d, 45.1 and 39.7, 2JPP
32.3); KM/(X1 cm2 mol1) 32.85; calcd for C45H46F6N3O2P3SRu:
exptl (calc) C, 54.00; H, 4.63; N, 4.20; S, 3.20%; C, 53.15; H, 4.22;
N, 4.10; S, 3.15%.
2.2.1.6. [Ru(L-Trp-H)(dppb)(phen)]PF6. UV–vis (CH2Cl2) k/nm 266,
408; IR (CsI) m/cm1 3441, 1624, 1385, 854, 557, 509, 426; 1H
NMR (400.21 MHz, CDCl3) d 1.37 (m, 1H, CH2), 2.20–2.00 (m, 2H,
CH2), 2.54–2.25 (m, 3H, CH2), 2.64 (m, 1H, CH2), 3.52 (m, 1H,
CH2), 3.55–3.06 (t, 5H, Trp), 5.99 (t, 2H, J 8.8, Ph), 6.65 (t, 1H, J
6.8 Hz, phen), 7.57–6.7 (s, 5H, Trp), 7.12 (dt, 1H, J 6.8, 1.6 phen),
7.45–7.35 (m, 4H, Ph), 7.66–7.48 (m, 5H, Ph), 7.82–7.72 (m, 3H,
Ph), 7.87 (t, 2H, J 8.0, Ph), 8.05–7.96 (m, 1H, phen; 2H, Ph), 8.14
(d, 1H, J 9.2, phen), 8.52–8.49 (m, 1H, phen; 2H, Ph), 8.74 (d, 1H,
J 8.4, phen), 9.20 (d, 1H, J 5.2, phen), 9.39 (m, 1H, phen), 10.96 (s,
1H, Trp); 13C NMR (100.05 MHz, CDCl3) d 27.7, 53.4, 183.0 and
181.0; 31P{1H} NMR (162 MHz, CH2Cl2/D2O) d (d, 49.4 and 41.0,
2
Jpp 34.0) and (d, 44.8 and 40.7, 2Jpp 33.0); KM/(X1 cm2 mol1)
28.70; calcd for C51H47F6N4O2P3Ru: C, 58.01; H, 4.49; N, 5.31%;
found: C, 57.79; H, 4.45; N, 5.47%.
All these spectra are showed in Supplementary information
(Figs. S1–S28).
2.3. Cell culture assay in MDA-MB-231
The in vitro cytotoxicity assays on cultured human tumor cell
lines still represent the standard method for the initial screening
of potential anticancer agents. Thus, as a first step in assessing
their pharmacological properties, the new ruthenium complexes
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E.R. dos Santos et al. / Inorganica Chimica Acta 463 (2017) 1–6
were assayed against a human breast tumor cell line MDA-MB-231
(ATCC No. HTB-26). The cells were routinely maintained in Dulbecco’s Modified Eagle’s medium (DMEM) supplemented with
10% fetal bovine serum (FBS), at 37 °C in a humidified 5% CO2
atmosphere. After reaching confluence, the cells were detached
by trypsinization and counted. For the cytotoxicity assay, 5 104
cells were seeded in 200 lL of complete medium in 96-well assay
microplates (Corning Costar). The plates were incubated at 37 °C in
5% CO2 for 24 h to allow cell adhesion, prior to drug testing. All
tested compounds were diluted in sterile DMSO (stock solution
with maximum concentration of 20 mmol L1) to 5, 2.5, 0.2, and
0.02 mmol L1. From each of these dilute samples, 2 lL aliquots
were added to 200 lL medium (without FBS) giving a final concentration of DMSO of approximately 1% and a final concentration of
the complex diluted about 100 times. Cells were exposed to the
compounds for a 48 h period. Cell respiration, as an indicator of cell
viability, was determined by the mitochondrial-dependent reduction of MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromide] to formazan [40]. MTT solution (0.5 mg mL1) was
added to cell cultures and incubated for 3 h, after which 100 lL
of isopropanol were added to dissolve the precipitated formazan
crystals. The conversion of MTT to formazan by metabolically
viable cells was monitored in an automated microplate reader at
570 nm. The percent cell viability was calculated by dividing the
average absorbance of the cells treated with the test compounds
by that of the control; and it was plotted against drug concentration (logarithmic scale) to determine the IC50 (drug concentration
at which 50% of the cells are viable relative to the control), with
the error estimated from the average of 3 trials.
2.4. Anti-M. tuberculosis activity assay
Antimycobacterial activities of each tested compound and the
standard drugs cycloserine and ethambutol were determined in
triplicate in 96 sterilized flat bottomed micro-plates (Falcon
3072; Becton Dickinson, Lincoln Park, NJ, USA) and Middlebrook
7H9 Broth (Difco) supplemented with oleic acid–albumin–dextrose–catalase (OADC) enrichment (BBL/Becton Dickinson, Sparks,
MD, USA). The concentrations of the tested compound ranged from
0.15 to 250 lg mL1. The anti-M. tuberculosis activity of the compounds was determined by the REMA (Resazurin Microtiter Assay)
method and was used to measure the minimal inhibitory concentration (MIC) for the tested compounds (minimum concentration
necessary to inhibit 90% growth of M. tuberculosis H37Rv ATCC
27294) [41]. The development of the color pink in the wells was
taken as an indicator of bacterial growth and the maintenance of
the color blue as the contrary. Thus, the MIC was assumed to be
the lowest concentration able to inhibit the change of color from
blue to pink. MIC values were determined by fluorescence measured on a SPECTRAfluor Plus microfluorimeter (TecanÒ), with
excitation at 530 nm and emission at 590 nm.
3. Results and discussion
3.1. Synthesis of the complexes
Six complexes of general formula [Ru(AA-H)(dppb)(phen)]PF6
(AA-H = deprotonated amino acids; dppb = 1,4-bis(diphenylphosphino) butane and phen = 1,10-phenanthroline) were synthesized
and characterized. The elemental analysis data for the synthesized
complexes and their molar conductivity measurements (Km), carried out in dichloromethane at 25 °C, using concentrations of
1.0 103 mol L1 are described in the experimental section.
These data are in agreement with the suggested formulation,
where the complexes are positively charged.
3.1.1. 31P{ 1H}, 13C{ 1H} and 1H NMR studies
Diastereoisomers were formed for these complexes having chiral amino acids in their structures, which were emphasized by the
presence of four doublets in their 31P{1H} NMR spectra (See Table 1,
Fig. 1B, S12–S16). In this case, the 31P{1H} NMR spectrum of the
complex with glycine, a non-chiral amino acid, exhibits only two
doublets (Fig. 1C). This behavior is in agreement with other Ru
(II)/amino acid complexes previously reported by us, and by others
[1,20,35,42].
In the 13C NMR spectrum of the complex with glycine (Fig. S6),
the C1 atom of the COO- group is shifted to lower field (183.6 ppm),
when compared with the spectrum of metal-free glycine
(175.3 ppm). Moreover, the same is observed for the complexes
with the chiral amino acids, however in this case all spectra
(Figs. S7–S10) show duplication for the signal corresponding the
C1 atom, due to the presence of diastereoisomers [1,20,42–44].
3.1.2. Infrared, UV–visible and electrochemical experiments
The IR spectra of this series of complexes are very similar for all
the complexes (Figs. S17–S22), confirming the presence of the
coordinated amino acid ligand in their structures. Thus, deformation bands of the NH2 group, at around 3400 cm1 and vibrational
modes of the carboxylate group occur in the region of 1620 cm1
for the masCOO- and at about 1384 cm1, for the related msCOO–. In
the IR spectra of the complexes, the mM–O bands at about
509 cm1, and mM–N at about 420 cm1 were also observed, confirming the coordination of the amino acids by nitrogen and oxy
gen atoms. In addition, the D (m
asCOO msCOO) values at about
1
236 cm confirms that the carboxylates of the amino acids are
mono coordinated to the metal center [45].
Concerning the UV–vis experiments, the phenanthroline ligand
absorbs in the same region of the dppb and of the amino acids, thus
it is difficult to distinguish and assign each transition band in the
spectra of the complexes (Table 2, Figs. S23–S28). Therefore, it
could be suggested that at about 250 nm, there is a mixture of
p ? p⁄ bands of these ligands. The band around 400 nm was
assigned as a charge transfer metal–ligand (MLCT) of orbitals
dpRu ? 3pr⁄dp(diphosphine) and dpRu ? p⁄(phen, amino acids) [46], which
is shifted to higher energies of approximately 20 nm compared with
the starting complex cis-[RuCl2(dppb)(phen)].
The results of the cyclic voltammetry measurements for the
complexes with general formula [Ru(AA-H)(dppb)(phen)]PF6 are
presented in Table 3, Fig. 2 and S29–S34. The complexes showed
a quasi-reversible process, corresponding to an oxidation redox
process of one-electron (RuII/RuIII), and another oxidation process
belonging to the amino acid ligand [47].
3.1.3. In vitro antitumor and anti-M. tuberculosis assays
The MDA-MB-231 tumor cells (human breast carcinoma) were
exposed to complexes for a period of 48 h. It is worth mentioning
Table 1
Chemical shifts (d) for the 31P{1H} NMR spectra of [Ru(AA-H)(dppb)(phen)]PF6
complexes.
Complex
(d) ppm
2
[Ru(Gly-H)(dppb)(phen)]PF6
[Ru(L-Ala-H)(dppb)(phen)]PF6
47.2 and 40.7
46.9 and 38.4
44.7 and 38.1
47.0 and 38.9
42.9 and 38.5
47.8 and 38.9
42.9 and 38.1
48.0 and 40.1
45.1and 39.7
49.4and 41.0
44.8 and 40.7
33.4
33.2
33.2
33.0
32.8
34.4
32.9
32.8
32.3
34.0
33.0
[Ru(L-Val-H)(dppb)(phen)]PF6
[Ru(L-Tyr-H)(dppb)(phen)]PF6
[Ru(L-Met-H)(dppb)(phen)]PF6
[Ru(L-Trp-H)(dppb)(phen)]PF6
Jp-p(Hz)
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E.R. dos Santos et al. / Inorganica Chimica Acta 463 (2017) 1–6
Fig. 1. Diastereoisomers structures (A). 31P{1H} NMR spectra for the complexes: (B) [Ru(L-Ala-H)(dppb)(phen)]PF6 and (C) [Ru(Gly-H)(dppb)(phen)]PF6.
Table 2
Data of the UV–vis spectra of the complexes [Ru(AA-H)(dppb)(phen)]PF6, in CH2Cl2
solutions [5.0 105 L mol1].
Complex
k (nm)
(e) Molar
absorptivity
L mol1 cm1
Transition
[Ru(Gly-H)(dppb)(phen)]PF6
266
294(sh)*
374
270
290(sh)*
408
270
292(sh)*
408
270
292(sh)*
408
270
292(sh)*
406
270
290(sh)*
408
26460
–
3494
28354
–
4616
30833
–
4870
31626
–
4891
30911
–
4666
30251
–
4330
p ? p*
–
dp ? p*
p ? p*
–
dp ? p*
p ? p*
–
dp ? p*
p ? p*
–
dp ? p*
p ? p*
–
dp ? p*
p ? p*
–
dp ? p*
[Ru(L-Ala-H)(dppb)(phen)]PF6
[Ru(L-Val-H)(dppb)(phen)]PF6
[Ru(L-Tyr-H)(dppb)(phen)]PF6
[Ru(L-Met-H)(dppb)(phen)]PF6
[Ru(L-Trp-H)(dppb)(phen)]PF6
*
Fig. 2. The Cyclic voltammogram of the [Ru(L-Val-H)(dppb)(phen)]PF6 complex:
TBAP 0.1 mol L1; CH2Cl2; Ag/AgCl; scan rate of 100 mV s1.
Table 4
IC50 values for the MDA-MB-231 cell line and MIC values for the M. tuberculosis of the
[Ru(AA-H)(dppb)(phen)]PF6 complexes, the ligands and the reference drugs.
sh = sholder.
Table 3
Cyclic Voltammetry data (mV) for complexes [Ru(AA-H)(dppb)(phen)]PF6 in CH2Cl2,
0.1 mol L1 PTBA, 100 mV s1. Work and auxiliary electrodes of Pt, and Ag/AgCl as a
reference electrode.
Complex
RuII/RuIII
(Epa)
RuIII/RuII
(Epc)
E1/2
ipa/ipc
[Ru(Gly-H)(dppb)(phen)]PF6
[Ru(L-Ala-H)(dppb)(phen)]PF6
[Ru(L-Val-H)(dppb)(phen)]PF6
[Ru(L-Tyr-H)(dppb)(phen)]PF6
[Ru(L-Met-H)(dppb)(phen)]PF6
[Ru(L-Trp-H)(dppb)(phen)]PF6
1112
1075
1079
1096
1113
1053
1027
999
979
–
1003
–
1069
1044
1029
–
1058
–
1.40
0.90
0.98
–
0.95
–
that the complexes are stable in the conditions of the in vitro studies (1% DMSO + DMEM, supplemented with 10% fetal bovine
serum), for at least five days, as showed 31P{1H} NMR experiments.
In this case the 31P{1H} NMR data of the complexes of fresh
Complex
IC50 (lmol L1)
MIC (lg mL1)
[Ru(Gly-H)(dppb)(phen)]PF6
Ru(L-Ala-H)(dppb)(phen)]PF6
[Ru(L-Val-H)(dppb)(phen)]PF6
[Ru(L-Tyr-H)(dppb)(phen)]PF6
[Ru(L-Met-H)(dppb)(phen)]PF6
[Ru(L-Trp-H)(dppb)(phen)]PF6
cis-[RuCl2(dppb)(phen)]
cis-[PtCl2(NH3)2]
Amino acids
dppb
phen
Cycloserine
Ethambutol
4.17 ± 0.16
6.68 ± 0.02
7.44 ± 0.86
3.04 ± 0.60
5.84 ± 0.28
4.72 ± 0.07
23.86 ± 0.65
2.44 ± 0.24
>200
>200
>200
–
1.02
1.53
1.56
1.51
1.59
3.10
0.78
7.80
–
>25
>25
>25
12.5–50.0
5.62
solutions and after 5 days were the same, and comparable with
those one obtained in CH2Cl2 solutions. The IC50 (drug concentration at which 50% of the cells are viable relative to the control)
E.R. dos Santos et al. / Inorganica Chimica Acta 463 (2017) 1–6
5
Fig. 3. Graph of cell viability against MDA-MB-231 breast cancer cell lines for the series of complexes [Ru(AA-H)(dppb)(phen)]PF6, MTT assays.
values, calculated from the dose-survival curves as shown in Fig. 3
and Table 4. For comparison purposes, the cytotoxicity of cisplatin
was also evaluated under the same experimental conditions.
The complexes with amino acids are all positively charged compounds, whereas the precursor, cis-[RuCl2(dppb)(phen)], is neutral.
This may be one of the factors contributing to the lower IC50 values
for the complexes with amino acids. Indeed, the IC50 values of the
complexes with amino acids are similar, with values in the range of
3.04–7.44 lmol L1 (Table 4). This indicates that structural variation of the amino acid ligands does not affect the cytotoxicity of
the compounds; activity of the amino acid complexes could be
similarity to those reported in the literature [30,48–49] and studies
performed by our research group [1], revealed weak interaction
between the complexes and DNA. Therefore, these results allow
us to suggest that the complexes studied in this work also present
weak interaction with DNA [50].
Comparing the cytotoxicity results obtained for [Ru(AA-H)
(dppb)(phen)]PF6 complexes with the IC50 values of analogous
complexes, it can be observed that the complexes reported here
present more active, when compared with complexes with 2,20 bipyridine,
with
formulae
[Ru(AA-H)(dppb)(bipy)]PF6
(IC50 = 5.0 ± 1.5 to 28.5 ± 5.2 lmol L1) [1], suggesting the importance of the planarity e rigidity of the phenanthroline ligand, to
increase the biological activity of the complexes here studied.
The in vitro anti-M. tuberculosis properties of the complexes
with amino acids were evaluated against M. tuberculosis H37Rv
ATCC 27294. The MIC values of the complexes summarized here
are presented in Table 4. These values are comparable to ethambutol (MIC 5.61 lg mL1) and cycloserine (MIC 12.5–50.0 lg mL1),
which are first-line and second-line drugs, respectively [51,52].
These results confirm the anti-Mycobacterium tuberculosis activities
of the [Ru(AA-H)(dppb)(phen)]PF6 complexes. Furthermore, the
MIC results show that complexation of amino acids improved
activity of the complexes compared with the precursor cis-[RuCl2(dppb)(phen)].
The biphosphinic ligand (1, 4-bis-(diphenylphosphino)butane)
is slightly active against the Mycobacterium tuberculosis
(MIC > 50 lg mL1) [39]. Therefore, when this molecule is coordinated to the ruthenium, the inhibitory activity of the new species
is improved greatly as shown by the data presented in Table 4.
4. Conclusion
Six new ruthenium complexes with general formulae [Ru(AAH)(dppb)(phen)]PF6 were synthesized, characterized and their biological activity was evaluated, against MDA-MB-231 tumor cells
and as anti-Mycobacterium tuberculosis.
The 31P{1H} NMR data show the presence of diastereoisomers
for those complexes containing amino acid ligand, which have chiral carbon in their structures. The IC50 values for the MDA-MB-231
tumor cells obtained for Ru(II) complexes showed better activity
than the cisplatin (reference drug). The anti-Mycobacterium tuberculosis activity assays for the new complexes provided evidence
that the complexes show higher activity against M. tuberculosis
H37Rv, than the ethambutol, a first-line drug in several schemes
of conventional tuberculosis treatment, and also higher than the
cycloserine, a second-line drug used in the tuberculosis treatment.
Thus, these good biological results suggest that the title complexes are potential metallodrugs to be used against the MDAMB-231 tumor cells and anti-mycobacterium tuberculosis.
Acknowledgements
We would like to thank CNPq, CAPES and FAPESP for the financial support.
Appendix A. Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.ica.2017.04.012.
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