Ru(II)-based complexes with N-(acyl)-N′,N′-(disubstituted)thiourea ligands: Synthesis, characterization, BSA- and DNA-binding studies of new cytotoxic agents against lung and prostate tumour cells

Article abstract of DOI:10.1016/j.jinorgbio.2015.04.008

Abstract Four ruthenium(II)-based complexes with N-(acyl)-N′,N′-(disubstituted)thiourea derivatives (Th) were obtained. The compounds, with the general formula trans-[Ru(PPh₃)₂(Th)(bipy)]PF₆, interact with bovine serum alb

Full text of DOI:10.1016/j.jinorgbio.2015.04.008

Ru(II)-based complexes with N-(acyl)-N’,N’-(disubstituted)thiourea ligands:
Synthesis, characterization, BSA- and DNA-binding studies of new cytotoxic
agents against lung and prostate tumour cells
Rodrigo S. Correa, Katia M. de Oliveira, Fa´bio G. Delolo, Anislay Al-
varez, Rau´l Mocelo, Ana M. Plutin, Marcia R. Cominetti, Eduardo E.
Castellano, Alzir A. Batista
PII:
DOI:
Reference:
S0162-0134(15)00102-6
doi: 10.1016/j.jinorgbio.2015.04.008
JIB 9708
To appear in:
Journal of Inorganic Biochemistry
Received date:
Revised date:
Accepted date:
14 January 2015
12 April 2015
13 April 2015
Please cite this article as: Rodrigo S. Correa, Katia M. de Oliveira, F´abio G. Delolo, Anis-
lay Alvarez, Rau´l Mocelo, Ana M. Plutin, Marcia R. Cominetti, Eduardo E. Castellano,
Alzir A. Batista, Ru(II)-based complexes with N-(acyl)-N’,N’-(disubstituted)thiourea
ligands: Synthesis, characterization, BSA- and DNA-binding studies of new cytotoxic
agents against lung and prostate tumour cells, Journal of Inorganic Biochemistry (2015),
doi: 10.1016/j.jinorgbio.2015.04.008
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ACCEPTED MANUSCRIPT
Ru(II)-based complexes with N-(acyl)-N’,N’-(disubstituted)thiourea
ligands: synthesis, characterization, BSA- and DNA-binding studies of new
cytotoxic agents against lung and prostate tumour cells
Rodrigo S. Correaa,*, Katia M. de Oliveiraa, Fábio G. Deloloa, Anislay Alvarezb, Raúl
Mocelob, Ana M. Plutinb, Marcia R. Cominettic, Eduardo E. Castellanod, Alzir A. Batistaa,*
a Departamento de Química, Universidade Federal de São Carlos – UFSCar, Rodovia
Washington Luís KM 235, CP 676, 13561-901, São Carlos, SP, Brazil; bLaboratório de
Síntesis Orgánica, Facultad de Química, Universidad de la Habana, Habana 10400, Cuba.
cDepartamento de Gerontologia, Universidade Federal de São Carlos, São Carlos, SP, Brazil;
dDepartamento de Física e Informática, Instituto de Física de São Carlos, Universidade de São
Paulo, CP 369, 13560-970, São Carlos, SP, Brazil.
Corresponding authors: Rodrigo S. Correa (rodricorrea@ufscar.br). Alzir A. Batista
(daab@ufscar.br). Tel.: +55 1633518285; Fax: +55 1633518350.
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Abstract
Four ruthenium(II)-based complexes with N-(acyl)-N’,N’-(disubstituted)thiourea
derivatives (Th) were obtained. The compounds, with the general formula trans-
[Ru(PPh3)2(Th)(bipy)]PF6, interact with bovine serum albumin (BSA) and DNA. BSA-
binding constants, which were in the range of 3.3–6.5 x 104 M-1, and the thermodynamic
parameters (G, H and S), suggest spontaneous interactions with this protein by
electrostatic forces due to the positive charge of the complexes. Also, binding constant by
spectrophotometric DNA titration (Kb = 0.8–1.8 x 104 M) and viscosity studies indicate
weak interactions between the complexes and DNA. Cytotoxicity assays against DU-145
(prostate cancer) and A549 (lung cancer) tumour cells revealed that the complexes are more
active in tumour cells than in normal (L929) cells, and that they present high cytotoxicity (low
IC50 values) compared with the reference metallodrug, cisplatin.
Keywords: Ruthenium/phosphine/diimine complexes, acylthiourea ligands, prostate cancer,
lung cancer, DNA binding; BSA binding.
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1. Introduction
In recent years, ruthenium(II) complexes have attracted attention due to their wide
variety of structures, reactivities and applications, in particular those compounds with
catalytic and anticancer activities [1-3]. Many research groups are exploring ruthenium
complexes containing specific molecular fragments in order to design new bioactive
anticancer metallodrugs, such as ruthenium/arene [4–5] and ruthenium/staurosporine
analogues [6], and some of these are in early preclinical trials [7]. In the last ten years, our
research group has investigated the reactivity and biological properties of ruthenium
complexes containing phosphine and diimine ligands, which exhibit antitubercular,
antitumour, antileishmanial and antiplasmodial activities [8-11]. Therefore, as part of our
ongoing effort to design new bioactive metallodrug candidates, in this paper we used O,S-
chelating acylthiourea ligands to synthesize a new phosphine/diimine/ruthenium(II) class of
compounds with biological properties.
Thiourea derivatives were employed as an ionophore in amperometric sensors for
Cd(II) [12], and they can be included in the field of coordination chemistry as versatile
ligands. Conformational isomerism, steric effects and the presence of donor sites in their
structures can provide several possible coordination modes [13, 14]. Usually, N-(acyl)-N’,N’-
(disubstituted)thiourea ligands form very stable complexes with a six-membered ring,
presenting different complex:ligand stoichiometry, such as 1:2 or 1:3, according to the
coordination number of the metal ion. Recently, we have explored the ability of N-acyl-N',N'-
disubstituted thiourea derivatives to form 1:3 and 1:2 stable complexes with transition metals,
specifically with Co(III), Ni(II) and Cu(II) [15-20]. In such metal complexes, the acylthiourea
derivatives acted as anionic O,S-chelating ligands with a cis configuration. In addition,
neutral acylthiourea derivatives can also bind the metal centre as monodentate ligands through
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the sulfur atom, such as observed in Cd(II) [21] and Cu(I) complexes [22]. Within the scope
of coordination chemistry, a wide range of d-block metals with different oxidation states were
synthesized with this type of ligand, presenting different modes of coordination [15-34].
Some of these complexes exhibited cytotoxicity against tumour cells, and were more
cytotoxic than the free ligands [27-32]. Furthermore, catalytic [33] and luminescent [34]
properties of this kind of complex were attributed to the presence of acylthiourea derivatives
as ligands. Despite the fact that acylthioureas have been used frequently as ligands for
syntheses of transition metal complexes, only two ruthenium(III) complexes [32, 35] have
been investigated so far, and just one has been studied by X-ray diffraction. Thus, here we
present the first report on ruthenium(II)/triphenylphosphine/2,2’-bipyridine complexes
containing acylthiourea derivatives as the ligand. We describe here the synthesis,
characterization, and X-ray crystallographic and biological studies of complexes containing
one acylthiourea ligand per ruthenium(II) ion, which is different to the previously reported
complexes that contained ruthenium(III) [32, 35].
The Figure 1 illustrates the structure of the four 1-(acyl)-3,3-(disubstituted)thiourea
derivatives used as ligands [N-benzoyl-N’,N’-diphenylthiourea (BzPh2Th), N-(2-furoyl)-
N’,N’-diphenylthiourea (FuPh2Th), N-benzoyl-N’,N’-dibenzylthiourea (BzBn2Th) and N-(2-
furoyl)-N’,N’-dibenzylthiourea (FuBn2Th)]. In this paper, the metal/DNA and metal/albumin
binding ability was also evaluated. Cytotoxic studies on a prostate tumour cell line [DU-145
(ATCC: HTB-81)], lung tumour cells [A549 (CCL-185)] and the L929 normal cell line were
carried out.
INSERT FIGURE 1
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2. Material and Methods
2.1
Materials, measurements and methods
All manipulations involving solutions of the complexes were performed under argon.
All solvents used in the work were purified by standard methods. Chemicals used were of
reagent grade or comparable purity. The RuCl3.3H2O, triphenylphosphine (PPh3), 2,2’-
bipyridine (bipy) and reagents used in the syntheses of acylthiourea derivatives (benzoyl
chloride, furoyl chloride, dibenzylamine, diphenylamine and KSCN) were used as received
from Aldrich. The cis-[RuCl2(bipy)(PPh3)2] complex was prepared according to the published
procedure [36].
The IR spectra were recorded on a FT-IR Bomem-Michelson 102 spectrometer in the
range 4000–250 cm-1 using CsI pellets. Conductivity data (presented as μS/cm) were obtained
in CH2Cl2 using a Micronal model B-330 connected to a Pt with constant cell equal to 0.089
cm-1; measurements were made at room temperature using 1 mM solutions of the complexes.
1H and 13C NMR spectra were recorded on a Bruker DRX 400 MHz, internally referenced to
TMS (tetramethylsilane), chemical shift (δ), multiplicity (m), spin-spin coupling constant (J),
and integral (I). CDCl3 was used as solvent. The 31P{1H} chemical shifts are reported in
relation to H3PO4 (85% v/v). The UV-Vis spectra of the complexes in CH2Cl2 were recorded
on a Hewlett Packard diode array–8452A. Cyclic voltammetry experiments were carried out
at 25 ºC in CH2Cl2 containing 0.10 M Bu4NClO4 (tetrabutylammonium perchlorate, TBAP)
(Fluka Purum), with a BAS-100B/W Bioanalytical Systems Inc. electrochemical analyser.
The working and auxiliary electrodes were stationary Pt foils; a Luggin capillary probe was
used and the reference electrode was Ag/AgCl. Under these conditions, the ferrocene (Fc) is
oxidized at 0.43 V (Fc+/Fc). The microanalyses were performed in the Microanalytical
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Laboratory of the Chemistry Department of the Federal University of São Carlos, with an EA
1108 CHNS microanalyser (Fisons Instruments).
2.2
Synthesis
The acylthiourea ligands were prepared using a standard procedure [13] by reacting
the benzoyl/furoyl chloride with KSCN in anhydrous acetone, followed by condensation with
dibenzylamine/diphenylamine. The reaction mixture was poured into cold water, resulting in
precipitation of a solid, which was purified by recrystallization from acetone–water solution
1
(1:1 v/v). The identity of the products was confirmed by comparing their infrared, H and 13C
NMR data with those reported in the literature [13–21].
To obtain the complexes (1–4), the acylthiourea derivative (0.12 mmol) was dissolved
in a Schlenk flask in 50 mL of a mixture of dichloromethane/methanol (2:1 v/v) with 20 μL of
triethylamine and KPF6 (0.12 mmol; 15.0 mg). Next, as the precursor, 100 mg (0.11 mmol) of
the cis-[RuCl2(PPh3)2(bipy)] (chlorines in the cis position to each other, and PPh3 in the trans
position to each other) was added. The solution was kept under reflux, under an inert
atmosphere and with stirring for 24 h. For each reaction, the final solution was concentrated
to ca. 2 mL, and 10 mL of water was added to precipitate an orange powder. The solids were
filtered off, washed with warm water, diethyl ether separately, and dried under vacuum.
N-benzoyl-N’,N’-diphenylthiourea (BzPh2Th) [C20H16N2OS]: exp. (cal) C, 72.26 (72.19); H,
4.85 (4.80); N, 8.43 (8.40); S, 9.64 (9.59). Yield: 81 %: m.p. (ºC): 205-206 . IR (cm-1): (νN-
H) 3150; (νC-H) 3000, 2910; (νC=O) 1680; (νC=C) 1610; (I, N-C=S) 1520; (II, N-C=S)
1350; (III, N-C=S) 1160; (IV, N-C=S) 930. 1H NMR (400 MHz, CDCl3, 298 K): (ppm) 8.73
(hydrogen atom of N-H), 7.60-6.92 (15 hydrogen atoms of Ph). 13C NMR (100 MHz, CDCl3,
298 K): (ppm) 182.49 (C=S); 162.34 (C=O); 145.73, 143.15, 132.84, 132.73-117.82 (C-Ph).
UV-Visible (UV-Vis) (CH2Cl2, 10-5 M): /nm (/M-1cm-1) 248 (18,252), 265 (17,475).
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N-(2-furoyl)-N’,N’-diphenylthiourea (FuPh2Th) [C18H14N2O2S]: exp. (cal) C, 67.06 (67.00);
H, 4.38 (4.33); N, 8.69 (8.65); S, 9.94 (9.90). Yield: 73 %: m.p. (ºC): 126-128. IR (cm-1): (νN-
H) 3120; (νC-H) 3005, 2910; (νC=O) 1690; (νC=C) 1615; (I, N-C=S) 1530; (II, N-C=S)
1
1325; (III, N-C=S) 1163; (IV, N-C=S) 1025. H NMR (400 MHz, CDCl3, 298 K): (ppm)
13
8.91 (hydrogen atom of N-H), 7.44 -6.49 (13 hydrogen atoms of Ph and Fu). C NMR (100
MHz, CDCl3, 298 K): (ppm) 183.06(C=S); 153.40 (C=O); 146.87, 145.78, 145.52, 144.96-
116.58 (C-Ph) and 113.55-111.73 (C-Fu). UV-Vis (CH2Cl2, 10-5 M): /nm (/M-1cm-1) 235
(13,750), 284 (23,000).
N-benzoyl-N’,N’-dibenzylthiourea (BzBn2Th) [C22H20N2OS]: exp. (cal) C, 73.30 (72.89); H,
5.59 (5.48); N, 7.77 (7.71); S, 8.89 (8.82). Yield: 83 %: m.p. (ºC): 136-138. IR (cm-1): (νN-H)
3330; (νC-H) 3000, 2900; (νC=O) 1690; (νC=C) 1583; (I, N-C=S) 1514; (II, N-C=S) 1355;
1
(III, N-C=S) 1192; (IV, N-C=S) 932. H NMR (400 MHz, CDCl3, 298 K): (ppm) 8.58 (N-
H), 7.80-7.11 (15H of Ph), 5.23 and 4.72 (4H of 2CH2). 13C NMR (100 MHz, CDCl3, 298 K):
(ppm) 181.94 (C=S); 163.95 (C=O); 135.38, 134.67, 133.10, 132.48-127.90 (C-Ph), 56.73
and 56.00 (2CH2). UV-Vis (CH2Cl2, 10-5 M): /nm (/M-1cm-1) 257 (12,484), 321 (12,725).
N-(2-furoyl)-N’,N’-dibenzylthiourea (FuBn2Th) [C20H18N2O2S]: exp. (cal) C, 68.55 (68.49);
H, 5.18 (5.04); N, 7.99 (7.93); S, 9.15 (9.08). Yield: 76 %: m.p. (ºC): 146-147. IR (cm-1): (νN-
H) 3320; (νC-H) 3020, 2905; (νC=O) 1693; (νC=C) 1608; (I, N-C=S) 1580; (II, N-C=S)
1
1425; (III, N-C=S) 1173; (IV, N-C=S) 1020. H NMR (400 MHz, CDCl3, 298 K): (ppm)
8.72 (N-H), 7.53-6.56 (13 H of Ph and Fu). 13C NMR (100 MHz, CDCl3, 298 K): (ppm)
180.83(CS); 153.95 (CO); 146.11, 145.59, 135.25, 134.59-127.79 (C-Ph); 117.66-113.00 (C-
Fu) and 56.22 (2CH2). UV-Vis (CH2Cl2, 10-5 M): /nm (/M-1cm-1) 237 (9,760), 275
(19,337).
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trans-[Ru(PPh3)2(BzPh2Th)(bipy)]PF6 (1): Yield: 120 mg (81%). Anal. Calc. for
[RuC66H53N4OP2S]PF6: exp. (calc) C, 63.05 (63.00); H, 4.27 (4.25); N, 4.31 (4.45); S, 2.62
(2.55) %. Molar conductance (μS/cm, CH2Cl2) 54.1. IR (cm-1): (νCHPPh3, bipy, Th) 3078, 3055,
2922, 2853; (νC=Nbipy) 1603; (νC=O) 1588; (νC=C and νC=N) 1500, 1489, 1489, 1479, 1435,
1402, 1356; (νC-S) 1294; ν-P) 1090; (νring) 1072, 1024, 1001; (νP-F) 841; (C=S) 764; (νRu-
31
P) 517, (νRu-S) 492; (νRu-N) 403; (νRu-O) 355. P{1H} NMR (162 MHz, CDCl3, 298 K):
1
(ppm) 21.12 (s). H NMR (400 MHz, CDCl3, 298 K): (ppm) 9.21 and 8.16 (2H, C-H of
bipy adjacent to the coordinated nitrogen atoms); 7.88-6.74 (30H atoms of PPh3, 15H
aromatic of BzPh2Th and 6H aromatic of bipy). 13C NMR (100 MHz, CDCl3, 298 K): (ppm)
177.36 (C=S); 172.59 (C=O); 158.74, 157.07, 153.56, 151.87, 151.18, 148.70, 145.11,
138.54, 136.13, 135.76, 133.28 – 123.37 (C-Ph; C-bipy, C-PPh3). UV-Vis (CH2Cl2, 10-5 M):
/nm (/M-1cm-1) 281 (4820), 407 (903), 476 (664).
trans-[Ru(PPh3)2(FuPh2Th)(bipy)]PF6 (2): Yield: 135 mg (89%). Anal. Calc. for
[RuC64H53N4O2P2S]PF6: exp. (calc) C, 61.50(61.49); H, 3.97(4.27); N, 4.24(4.48); S, 2.34
(2.56) %. Molar conductance (μS/cm, CH2Cl2) 56.1. IR (cm-1): (νCHPPh3, bipy, Th) 3080, 3059,
2922, 2852; (νC=Nbipy) 1603; (νC=O) 1578; (νC=Cfuroyl) 1512; (νC=N and νC=C) 1491,1481,
1471, 1456, 1435, 1418, 1391, 1358; (νCS) 1292; (νC-P) 1090; (νring) 1072, 1026, 999; (νP-F)
31
841; (CS) 766; (νRu-P) 520; (νRu-S) 496; (νRu-N) 403; (νRu-O) 357. P{1H} NMR (162
MHz, CDCl3, 298 K): (ppm) 21.95 (s). 1H NMR (400 MHz, CDCl3, 298 K): (ppm) 9.40
and 8.25 (2H, C-H of bipy adjacent to the coordinated nitrogen atoms); 7.94-6.28 (30H
aromatic hydrogen atoms of PPh3, 13H aromatic hydrogen atoms of FuPh2Th and 6H
aromatic hydrogen atoms of bipy). 13C NMR (100 MHz, CDCl3, 298 K): (ppm) 176.36 (CS);
165.05 (CO); 159.59, 158.95, 156.93, 153.30, 149.53, 148.92, 145.37, 144.93, 135.81-122.88
(C-Ph; C-bipy, C-PPh3), 117.81-111.84 (C-Furoyl). UV-Vis (CH2Cl2, 10-5 M): /nm (/M-
1cm-1) 282 (3540), 406 (900), 472 (706).
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trans-[Ru(PPh3)2(BzBn2Th)(bipy)]PF6 (3): Yield: 95 mg (65%). Anal. Calc. for
[RuC68H57N4OP2S]PF6: exp. (calc) C, 61.60(61.59); H, 4.37(4.12); N, 4.77(4.49); S, 2.38
(2.57) %. Molar conductance (μS/cm, CH2Cl2) 57.0. IR (cm-1): (νCHPPh3, bipy, Th) 3080, 3059,
3028, 2922, 2853; (νC=Nbipy) 1603; (νC=O) 1585; (νC=N and νC=C) 1508, 1495, 1481, 1470,
1450, 1433, 1412, 1400, 1354; (νCS) 1267; (δCH2) 1207; (νC-P) 1088; (νring) 1072, 1028,
1001; (νP-F) 839; (CS) 758; (νRu-P) 521; (νRu-S) 490; (νRu-N) 405; (νRu-O) 360. 31P{1H}
1
NMR (162 MHz, CDCl3, 298 K): (ppm) 22.07 (s). H NMR (400 MHz, CDCl3, 298 K):
(ppm) 9.26 and 8.48 (2H, C-H of bipy adjacent to the coordinated nitrogen atoms); 7.83-6.98
(30H aromatic hydrogen atoms of PPh3, 15H aromatic hydrogen atoms of BzBn2Th and 6H
aromatic hydrogen atoms of bipy); 4.95 and 4.74 (4H, CH2 of BzBn2Th). 13C NMR (100
MHz, CDCl3, 298 K): (ppm) 176.55 (CS); 173.02 (CO); 158.94, 157.18, 153.56, 148.59,
139.02-123.47 (C-Ph; C-bipy, C-PPh3), 53.41 and 51.60 (CH2). UV-Vis (CH2Cl2, 10-5 M):
/nm (/M-1cm-1) 279 (2676), 392 (546), 483 (367).
trans-[Ru(PPh3)2(FuBn2Th)(bipy)]PF6 (4): Yield: 110 mg (74%). Anal. Calc. for
[RuC66H57N4O2P2S]PF6: exp. (calc) C, 62.34(62.11); H, 4.70(4.34); N, 4.54(4.39); S, 2.28
(2.51) %. Molar conductance (μS/cm, CH2Cl2) 56.5. IR (cm-1): (νCHPPh3, bipy, Th) 3080, 3059,
3028, 2924, 2854; (νC=Nbipy) 1605; (νC=O) 1578; (νC=Cfuroyl) 1522; (νC=N and νC=C) 1495,
1481, 1466, 1452, 1433, 1416, 1383, 1356, 1309; (νC-S) 1267; (δCH2) 1211; (νC-P) 1090;
(νring) 1080, 1028, 1001; (νP-F) 843; (C=S) 762; (νRu-P) 518; (νRu-S) 494; (νRu-N) 401;
1
(νRu-O) 353. 31P{1H} NMR (162 MHz, CDCl3, 298 K): (ppm) 22.49 (s). H NMR (400
MHz, CDCl3, 298 K): (ppm) 9.41 and 8.54 (2H, C-H of bipy adjacent to the coordinated
nitrogen atoms); 7.42-6.45 (30H atoms of PPh3, 13H aromatic atoms of FuBn2Th and 6H
13
aromatic atoms of bipy); 4.90 and 4.56 (4H, CH2 of FuBn2Th). C NMR (100 MHz, CDCl3,
298 K): (ppm) 175.53 (CS); 164.70 (CO); 159.18, 157.07, 153.49, 148.56, 145.09, 137.24-
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123.59 (C-Ph; C-bipy, C-PPh3), 113.23-111.80 (C-Furoyl), 52.95 and 51.36 (CH2). UV-Vis
(CH2Cl2, 10-5 M): /nm (/M-1cm-1) 280 (2035), 392 (497), 480 (351).
2.3
X-ray structure determination
Orange single-crystals of the complexes 1–4 were grown from diethyl ether diffusion
into a dichloromethane solution of the complex. Room temperature (298 K) X-ray diffraction
experiments were carried out using a suitable crystal mounted on glass fibre and positioned on
the goniometer head. Intensity data were measured on an Enraf–Nonius Kappa-CCD
diffractometer with graphite monochromated MoKα radiation (λ = 0.71073 Å). The cell
refinements were performed using the software Collect [37] and Scalepack [38], and the final
cell parameters were obtained on all reflections. Data reduction was carried out using the
software Denzo-SMN and Scalepack [38]. The structures were solved by the direct method
using SHELXS-97 [39] and refined using the software SHELXL-97 [39]. The Gaussian
method was used for the absorption corrections [40]. Non-hydrogen atoms of the complexes
were unambiguously located, and a full-matrix, least-squares refinement of these atoms with
anisotropic thermal parameters was carried out. In all ligands of the complexes 1–4, the
aromatic C–H hydrogen atoms were positioned stereochemically and were refined with fixed
individual displacement parameters [Uiso(H) = 1.2 Ueq(Csp2)] using a riding model with
aromatic C–H bond lengths fixed at 0.93 Å. Methylene groups of the BzBn2Th and FuBn2Th
ligands in complexes 3 and 4 were also set as isotropic with a thermal parameter 20% greater
than the equivalent isotropic displacement parameter of the atom to which each one was
bonded, and C–H bond lengths were fixed at 0.97 Å. Tables and structure representations
were generated by WinGX [41] and MERCURY [42], respectively. The main crystal data
collections and structure refinement parameters for 1–4 are summarized in Table 1.
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INSERT TABLE 1
2.4 Bovine serum albumin binding experiments
The protein binding study was performed by a tryptophan fluorescence quenching
experiment using bovine serum albumin (BSA, 2.5 µM) in buffer (4.5 mM Tris-HCl, 0.5 mM
NaOH, 50 mM NaCl) at pH 7.4. The extinction of the emission intensity of the tryptophan
residue at 340 nm (excitation wavelength of 280 nm) was monitored using the complexes as
suppressors in different concentrations (0–100 µM) in DMSO. Fluorescence spectra were
recorded from 300 to 500 nm and performed in triplicate, using an opaque 96-well plate.
Fluorescence spectra were recorded on a SpectraMax M3 at different temperatures (295 and
310 K).
2.5 DNA titration and viscosity experiments
A standard solution of calf thymus DNA (ctDNA) from Sigma-Aldrich was prepared
in Tris-HCl buffer (5 mM Tris-HCl, pH 7.2). The concentration of this ctDNA solution was
measured from its absorption intensity at 260 nm using the molar absorption coefficient value
of 6600 M-1 cm-1. The ctDNA solution is protein-free, given that the ratio of UV absorbance
at 260 and 280 nm is about 1.8:1. The solution of ruthenium complexes 1–4 used in the
experiments was prepared in Tris-HCl buffer containing 2% DMSO. In the titration
experiments, different concentrations of the ctDNA were used while the ruthenium complex
was at 25 μM. Sample correction was made for the absorbance of ctDNA and the spectra were
recorded after solution equilibration for 1 min. The intrinsic equilibrium binding constant
(Kb) of the complexes to ctDNA was obtained by monitoring changes in the absorption
intensity with increasing concentration of ctDNA, and was analysed by regression analysis.
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Viscosity experiments were carried out using an Ostwald viscometer maintained at a
constant temperature of 25 °C in a thermostatic bath. The viscosity of the DNA solution was
measured in the presence of increasing amounts of the complexes (1–4).
The flow times were measured with an automated timer. Each sample was measured
three times, and an average flow time was calculated. The obtained data are presented as
(η/η0)1/3 versus r, where η is the viscosity of DNA in the presence of the complexes, and η0 is
the viscosity of DNA alone in buffer solution [43–46].
2.6 Cell culture assay
The cytotoxic activities of ruthenium complexes 1–4 were evaluated against the
human prostate tumour cell line DU-145 (ATCC: HTB-81) and against lung tumour A549
(CCL-185) cells. For initial screening of antitumour candidates, the in vitro assays were
performed using the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide)
method, a colorimetric assay in which the mitochondria of viable cell reduct the soluble
yellow tetrazolium salt to blue formazan crystals [47]. The A549 cells were maintained in
Dulbecco's modified Eagle's medium (DMEM) and the DU-145 and L929 (ATCC: CCL-1)
cells were maintained in RPMI-1640 medium. All cell lines were supplemented with 10% of
foetal bovine serum (FBS), at 37 ºC in a humidified 5% CO2 atmosphere. After reaching
confluence, cells were detached by trypsinization and 1.5 × 104 cells/well were seeded in 200
μL of complete medium in 96-well assay microplates. The plates were incubated at 310 K in
5% CO2 for at least 12 h to allow cell adhesion prior to compound testing. All tested
compounds were dissolved in sterile DMSO (stock solution with maximum concentration of
20 mM) and diluted to 100 to 0.05 µM (final concentration in each well), where 1 μL aliquots
were added to 200 μL of medium (final concentration of 0.5% DMSO/well). Cells were
incubated with compounds for 48 h at 310 K in 5% CO2.
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In order to verify the cytotoxic effect of the complexes, the cell viability was
measured after incubation with the complexes. Cells were twice washed with phosphate
buffered saline (PBS) and MTT solution (0.5 mg/mL, 50 µL/well) was added to cells and
incubated for 4 hours, after which 100 μL of isopropanol was added to dissolve the
precipitated formazan crystals. The conversion of MTT to formazan by metabolically viable
cells was measured in an automated microplate reader at 595 nm. To analyse the cell viability,
the control (cells with only DMSO) was taken as the reference (100%). All experiments were
carried out in triplicate. The percentage of viable cells was calculated as the mean with
respect to the DMSO control. The IC50 (drug concentration at which 50% of the cells are
viable relative to the control) values were obtained by non-linear fitting using GraphPad
Prism software.
3. Results and Discussion
3.1 Synthesis
The reactions of cis-[RuCl2(PPh3)2(bipy)] with N-Acyl-N',N'-disubstituted thiourea
derivatives (Th) produce complexes with the general formula [Ru(PPh3)2(Th)(bipy)]PF6, in
which the N-acyl-N',N'-disubstituted thiourea ligand is chelated and negatively charged. The
synthetic step used in this work was straightforward and with good yield, providing
satisfactory elemental analysis data. The molar conductance measurements for compounds
(1–4) were carried out in dichloromethane, and the results, ranging 54.1–57.0 μS/cm, are
consistent with 1:1 type compounds [48].
3.2 Crystal structure and molecular assembly analysis of complexes 1–4
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The X-ray studies confirm the structures proposed for the complexes: trans-
[Ru(PPh3)2(BzPh2Th)(bipy)]PF6 (1), trans-[Ru(PPh3)2(FuPh2Th)(bipy)] PF6 (2), trans-
[Ru(PPh3)2(BzBn2Th)(bipy)]PF6 (3), and trans-[Ru(PPh3)2(FuBn2Th)(bipy)]PF6 (4) (Fig. 2
and Fig. S4, supplementary information). The complexes present one acylthiourea derivative,
one 2,2’-bipyridine, and two triphenylphosphine ligands, forming slightly distorted octahedral
geometries, highlighted by the bond angles around the metal centres (Table 3). The N–Ru–N
bond angles on the equatorial position are far from the expected value of 90° due to the
tension of the five-membered chelate ring of the bipy, while the six-membered ring formed by
the acylthiourea derivatives ligands are less tensioned, presenting values close to 90o. The P–
Ru–N and P1–Ru–P2 (PPh3 ligands adopting a trans configuration) bond angles are close to
90o and 180°, respectively (Table 3). Ru–Nbipy and Ru–PPPh3 bond lengths found in complexes
1–4 are in agreement with values present in complexes obtained from the same Ru(II)
precursor [11, 49].
INSERT FIGURE 2
INSERT TABLE 3
The crystal structures of two free ligands, FuBn2Th [50] and BzBn2Th [51], were
previously published. In the free ligands, the C2=S1 (1.661–1.676 Å) and C1=O1 (1.214–
1.215 Å) bond lengths indicate a double-bond character, whereas the C1–N1 and C2–N1
bonds are single (1.373–1.412 Å). As a result of acylthiourea coordination, the bond lengths
present significant C2–S1 and C1–O1 lengthening and C–N shortening (Table 3).
The conformation between the thiocarbonyl and the carbonyl groups of metal-free
ligands is twisted [50, 51, 52]. After coordination, the conformation of the N’N’-dibenzyl
group changed significantly. In the BzBn2Th and FuBn2Th free ligands, the torsion angles
14

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between the two benzyl groups are 108º and 104o, respectively, whereas in the complexes 3
and 4, the torsion angles are similar with values close to -153o. The planarity of the
acylthiourea moiety is highlighted by the dihedral angle between the planes passing through
the N1–C1–O1–C3 atoms and N1–C2–S2–N2 atoms. For the complexes 1, 2, 3 and 4, the
dihedral angles are 1.51º, 7.84º, 5.44º and 10.16o, respectively, suggesting that the N-furoyl
groups, as well as the N’,N’-dibenzyl groups, contribute to distorting the coordinated
acylthiourea moiety. This result is also supported by analysing the torsion angle of the
acylthiourea moiety, in which the C1—N1—C2—S1 fragment tends to be more distorted than
the O1—C1—N1—C2 fragment, particularly in complexes 2–4 (Table 3).
In the crystal structure of complexes 2 and 4, the oxygen atom of the furan group
presents a syn conformation related to the oxygen of the coordinated carbonyl. The
conformation adopted by the furan is allowed due to the weak intramolecular interaction
between the C–H groups of the PPh3 or bipy ligands with the oxygen of furan (Fig. S8,
supplementary information).
The crystal structure of free ligands BzBn2Th and FuBn2Th exhibit patterns with
N−H…S and C−H…O interactions forming infinite chains [50, 51]. However, in the
complexes (1–4), both sulfur and oxygen atoms are coordinated and the N–H group is absent.
Therefore, to stabilize the crystal structure of the complexes 1–4, there are only weak C–
H…F–P intermolecular interactions, as well as van der Waals forces and π–π stacking, which
can be mapped using the Hirshfeld surface and the fingerprint plots analysis (Fig. S5 and S6,
supplementary information). It is well known that the non-classical interactions can provide
good structural motifs for the construction of extended architectures and can stabilize the
crystal structures of metal complexes with acylthiourea derivatives [15, 20, 53, 54]. These
intermolecular interactions may be responsible for binding the complex to biological targets.
15

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3.3 Infrared and ultraviolet-visible spectroscopy
In the IR spectra of complexes 1–4, the vN–H stretching band, at around 3200 cm–1, is
not present, suggesting that the coordinated ligand is deprotonated. The free ligands present
an intense band in the region between 1800 and 1610 cm–1, which is assigned to vC=O
stretching vibrations [28, 29, 55] and is absent in the spectra of the complexes, evidencing
that upon the ligand coordination, the carbonyl absorption shifts to lower frequencies around
1575–1590 cm-1. Strong bands in the region of 1550-1300 cm–1 are characteristic of vC=N
and vC=C stretching vibrations and are present in the spectra of the ligands and of the
complexes. In the complexes, the bands related to νC S and δC S absorptions occur in the
regions around 1260 and 760 cm-1, respectively. The complexes exhibit νRu-P stretching in
the range 521–511 cm-1. Also, the νRu-S, νRu-N and νRu-O stretching vibrations occur as
weak bands in a region of low intensity at about 500–350 cm-1 [55].
3.4 Multinuclear 31P{1H}, 1H and 13C NMR experiments
In the 1H NMR spectra of the free ligands, a shoulder band corresponding to a singlet
of the N–H group proton is observed in the region of 8.10–8.70 ppm [28, 29, 50]. This kind of
signal is absent in the spectra of the complexes, indicating the deprotonation of the
acylthioureide group by the coordination of the ligand, which acts as a monoanionic species,
1
to the metal. Additionally, the H NMR spectra of complexes 1–4 showed the characteristic
deshielded signal at 9.41 and 8.54 ppm, corresponding to the ortho hydrogen atoms of the
bipy ligand. Other aromatic hydrogen atoms resonances are in the range 6.28–7.94 ppm,
which are attributed to the protons present in the triphenylphosphine and bipy, as well as the
aromatic H atoms of the N-(acyl)-N’,N’-(disubstituted)thiourea ligands. Complexes 2 and 4
present two distinct signals in the regions of 6.55–6.39 and 6.45–6.29 ppm, which can be
attributed to the hydrogen atoms of the furan group. Additionally, complexes 3 and 4 showed
16

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two shielded signals at 4.95–4.90 and 4.74–4.56 ppm, which are assigned to the methylene
13
protons of the N’,N’-dibenzyl groups. The C NMR spectra of complexes 1–4 show signals
at around 177.4–175.5 and 172.6–164.7 ppm, which are assigned to the C S and C O
groups, respectively. For the free ligands, these signals occur at about 183 ppm for C=S, and
close to 163 ppm for the C=O group, evidencing the coordination of the metal to the ligands
through O,S atoms. In the complexes, the shifts of these two peaks to lower (CS) and higher
frequencies (CO), respectively, compared with the free ligands, may be attributed to the
-
delocalization of π electrons of the S Ru O chelate rings, accounting for the opposite
displacements with respect to the free ligand. The same effect was previously observed for
other transition metal complexes containing N-(acyl)-N’,N’-(disubstituted)thiourea ligands
[19, 23, 31].
Aromatic carbon atoms were also identified in the range 159–123 ppm. In the spectra
of complexes 2 and 4, the signals in the range 118–111 ppm belong to the carbon atoms of the
furan group, while the complexes 3 and 4 present signals at around 53–51 ppm, which are
assigned to carbon atoms of the CH2 groups.
The 31P{1H} experiments show the presence of only one singlet signal for coordinated
phosphorus of each complex around 22–21 ppm (Table 2) due to equivalence of the two
phosphorus atoms in trans configuration [11, 36, 51]. In the 31P{1H} spectra of complexes 1–
-
4, heptet signals of the PF6 anion around -144 ppm were observed.
INSERT TABLE 2
3.5 Electrochemical studies
The electrochemical behaviour of complexes 1–4 in cyclic voltammetric experiments
were similar to those found for other Ru(II) complexes that present monophosphine as ligands
17

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in a trans configuration [11]. These experiments were performed in the same conditions and it
was observed that complexes 1–4 exhibit a quasi-reversible redox process assigned to one-
electron Ru(II)/Ru(III), with Epa ranging from 920 to 958 mV (Table 2). The similar
electrochemical behaviour observed for all complexes may be due to the same
stereochemistry found for them. Recently, Barbosa et al. [11] reported the electrochemistry of
the complex trans-[Ru(PPh3)2(Lap)(bipy)]PF6 (Lap = lapachol), which presents an oxidation
process higher than 1.0 V. In this compound, the O–O-chelated lapachol presents two hard
oxygen atoms coordinated to the metal centre, whereas in the complexes with acylthiourea
ligands, there is one soft (sulfur) and one hard (oxygen) atom coordinated. The oxidation
process occurs at a potential lower than 1.0 V because the sulfur atom is a better electron
donor than the oxygen. The E½ values found for complexes 1–4 were considerably more
anodic than that observed for the precursor [RuCl2(PPh3)2(bipy)] (Table 2), indicating that the
ruthenium centre is more stable in the complexes containing the acylthiourea ligands
compared with the precursor. Such metal stabilization is assumed to be due to the replacement
of two chlorides by a negatively charged chelating ligand.
3.6. Fluorescence spectra of BSA–Ru(II) complexes
Albumin is the most abundant protein in plasma, thus its interaction with complexes 1-
4 was evaluated. Complex binding to this protein may lead to reduction or enhancement of
their biological properties because this protein presents the ability to transport drugs and
nutrients through the organism [56]. BSA solutions exhibit a strong fluorescence emission
with a peak at 340 nm when excited at 280 nm, which is provided mainly by two residues of
tryptophan: Trp-134, which is located on the surface of domain I, and Trp-213, which is
located within the hydrophobic pocket of domain II [56]. The interaction of ruthenium(II)
complexes with BSA was studied by a fluorescence quenching experiment. The experiments
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were carried out by adding the ruthenium(II) complexes in increasing concentrations (0–100
μM) to BSA (2.5 μM) at 295 and 310 K, then following the decrease in fluorescence intensity
(Table 4; Figure 3A).
INSERT TABLE 4
INSERT FIGURE 3
The fluorescence quenching can be illustrated by the well-known Stern–Volmer
equation [57]:
F0 F = 1 Ksv[Q = 1 [Q
q 0
where F0 and F represent the fluorescence intensities of BSA in the absence and presence of
the complexes, respectively, Ksv is the Stern–Volmer quenching constant, kq is the
bimolecular quenching rate constant (units M-1S-1). is the emission lifetime of the
0
fluorescent biomolecule in the absence of quencher ( = 6.2 ns) [58] and [Q] is the
0
concentration of the quencher (the Ru(II) complex). The value of Ksv is obtained as the slope
of the linear plot of F0/F vs. [Q]. In addition, according to kq = KSV/ 0, the quenching rate
constant kq can be calculated. The binding constant (Kb) and the number of binding sites (n)
can be determined using the following equation:
(F0 F)
log
= log Kb n log [Q
F
where Kb is the equilibrium constant to a site and n is the number of binding sites per BSA
molecule. Kb and n values were obtained from the intercept and slope through the double-
logarithm regression curve of log[(F0-F)/F] vs. log[Q]. The effect of complex 1 on BSA
fluorescence intensity is depicted in Figure 3A. Table 4 shows the constant values for the
complexes and the ligands BzBn2Th and FuBn2Th. The ligands BzPh2Th and FuPh2Th do not
19

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interact with BSA, highlighting the importance in BSA binding of the N’,N’-dibenzyl
substituent in the free ligand.
The different mechanisms of quenching are usually classified as dynamic quenching
and static quenching, and these can be distinguished by their different dependencies on
temperature and viscosity. Static quenching refers to the formation of a complex between
quencher and fluorophore in the ground state. When increasing the temperature, the BSA-
complex formation decreases leading to a decrease in the fluorescence quenching. Dynamic
quenching refers to contact between fluorophores and the quencher during the transient
existence of the excited state. In this case, the number of collisions depends upon diffusion,
and thus higher temperatures increase the fluorescence quenching, and as a result the
quenching constant increases [58]. Accordingly, in this paper, the Ksv values are reduced
when the temperature is increased. Therefore, the static mechanism is responsible for the
interactions between the complexes 1–4 and BSA. Furthermore, the maximum scatter
collision-quenching constant values, kq, occur around at 1012 M−1s−1 for the complexes 1–4,
which is higher than maximum kq value for dynamic quenching [kq of 2 × 1010 M−1s−1],
indicating the existence of static quenching mechanism [58]. The values of n are close to 1,
indicating that there is only one binding site in the BSA for each complex.
The thermodynamic parameters (H°, S° and G°) were analysed to evaluate the
intermolecular forces involving the molecules of the complex and BSA. As discussed in the
X-ray structure analysis, in the solid state, the complexes can present interactions that include
hydrogen bonds, van der Waals forces, electrostatic forces and hydrophobic interaction
forces. The types of interactions can occur between the BSA and the complexes, and are
indicated by the sign and magnitude of the thermodynamic parameters. The values for H° 
0 and S°  0 implies the involvement of hydrophobic forces in protein binding, H°  0 and
S°  0 correspond to van der Waals and hydrogen bonding interactions, and H°  0 and
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S°  0 suggests an electrostatic force [59]. As observed in Table 4, the positive S° and
negative H° values indicate that electrostatic forces occur in complexes 1–4, which are
allowed by the positive charge of the complexes with negative regions of the protein.
Furthermore, the negative value of G° indicates a spontaneous interaction between them.
The magnitude of the BSA-binding constant of the complexes 1–4, compared with other
Ru(II) complexes reported recently [60], suggests a moderate interaction with BSA molecule.
Thus, the molecules of the complexes 1–4 can be stored in protein and released at desired
targets.
3.7 DNA binding: UV-Vis spectrophotometric and viscosity studies
All complexes exhibit the same behaviour when ctDNA is added, in which absorption
spectra decrease at the rate of about 1–10%, suggesting a weak interaction between complex
and DNA. Binding constants, Kb, were calculated according to the equation [61]:
[ctDNA /(εa-εf) = [ctDNA /(εb-εf) + 1/Kb(εb-εf)
in which [ctDNA] is the concentration of ctDNA in base pairs, εa is the ratio of the
absorbance/[Ru , εf is the extinction coefficient of the free Ru(II) complex, and εo is the
extinction coefficient of the complex in the fully bound form. The ratio of the slope to the
intercept in the plot of [DNA]/(εa-εf) vs. [DNA] gives the value of Kb, which was calculated
from the metal to ligand charge transfer (MLCT) absorption band (λmax) at around 400 nm
(Table 5).
INSERT TABLE 5
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Slight changes were observed by the addition of ctDNA in the absorption spectra of
complexes 1–4 due to low hypochromism and also by the low Kb values compared with those
reported in the literature (Kb ranging from 10-3 to 10-4 M-1) [62–64]. Figure 3B depicts
electronic spectra obtained for complex 1, which is similar to those obtained for complexes 2–
4. Considering the molecular structure and positive charge of the complexes, electrostatic
interactions with ctDNA are expected, involving the negatively charged phosphate groups of
DNA. The Kb values found in this report for complexes 1–4 are comparable with those found
for [Ru(tpy)(PHBI)]2+ and [Ru(tpy)(PHNI)]2+ (PHBI
=
2-(2-benzimidazole)-1,10-
phenanthroline; PHNI = 2-(2-naphthoimidazole)-1,10-phenanthroline; tpy = 2,2′:6′,2″-
terpyridyl [65]) and other cationic Ru(II) complexes that bind to DNA through electrostatic
interaction [66]. Electrostatic interactions are weaker than the intercalative interactions
observed in complexes containing planar ligands (Kb = 10-5–106 M-1) [62], and those formed
by hydrogen bonds between complex and DNA [63]. In addition, Kb values of complexes 1–4
are lower than those observed for the classical intercalator ethidium bromide (Kb ≥ 106 M-1)
[64].
Complexes 2 and 4 have a slightly higher affinity to ctDNA than complexes 1 and 3,
suggesting that, in solution, the oxygen atom from the furan group of the complexes 2 and 4
could interact weakly with ctDNA by hydrogen bonds, increasing the affinity between them.
The interaction between the complexes and ctDNA was also evaluated by viscosity
experiments, which can provide a good idea of the mode of interaction between the complex
and the ctDNA. It is well known that classical intercalators, such as ethidium bromide, lead to
an increase in the viscosity of ctDNA because separation of the base pairs occurs to
accommodate the intercalator. A covalent DNA-binding mode may cause its fragmentation,
thus decreasing the DNA viscosity [43–46]. The ctDNA viscosity did not increase
significantly when the concentration of the complexes was increased (See the plot (η/ηo)1/3 vs.
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[complex]/[DNA] in supplementary information; Fig. S10). These small changes suggest the
existence of only weak interactions between the complexes and ctDNA.
3.8 Biological activity
Complexes 1–4 were evaluated against the DU-145 (human prostate carcinoma),
A549 (human lung carcinoma) and L929 (normal) cell lines. The IC50 values were calculated
from the dose-survival curves obtained after 48 h of metallodrug treatment with an MTT
assay (Table 6). The complexes reported here are more cytotoxic than the free ligands,
highlighting the importance of the metal to the antitumour biological activity. Complex 3 was
the most active against DU-145 cells, while complex 1 was the most potent against A549
cells. For comparison, the cytotoxicity of cisplatin was evaluated under the same experimental
conditions. The complexes were more active against human DU-145 and A549 cell lines than
cisplatin (Table 6). Interestingly, complex 3 was more selective than cisplatin in the human
DU-145 cell line.
INSERT TABLE 6
The FuPh2Th and FuBn2Th free ligands present activity against DU-145, while
BzPh2Th and BzBn2Th are inactive up to 200 µM (Table 6), suggesting that the furan group is
important for the activity of the free molecule against this tumour cell line. On the other hand,
the BzPh2Th and FuBn2Th metal-free ligands were active against the L929 cell line,
highlighting that the N,N’-dibenzyl improve the activity of these molecules considering
normal cells. Considering the A549 cell line, all free ligands were inactive up to 200 µM.
Recently, our research group reported bis-triphenylphosphine/Pd(II) complexes with
the ligands studied here [31]. The Pd(II) complexes with BzPh2Th and BzBn2Th ligands were
23

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inactive against DU-145, MDA-MB-231 and L-929 cell lines, while the complexes with
FuPh2Th and FuBn2Th were cytotoxic against MDA-MB-231 and L-929 cell lines only, with
IC50 values ranging from 35 to 70 μM. In these types of Pd(II) complexes, the activity can be
attributed to the steric hindrance of the ligands, as well as the presence of the non-coordinated
oxygen from the furan group in the molecule of the ligands [31]. In the present report,
complexes of ruthenium(II) were very active in the studied cell lines, presenting very low IC50
values. These results show that ruthenium(II) complexes with the same ligands are more
active than palladium(II) complexes, highlighting the importance of ruthenium(II) compounds
as potential anticancer drugs.
The four Ru(II)-based complexes with N-(acyl)-N’,N’-(disubstituted)thiourea ligands
were more cytotoxic against DU-145 and A549 tumour cells than L929 normal cells. It is
worth mentioning that to realise a structure-activity relationship by changing ligand
substituents is not possible, given that the IC50 values obtained are very close and the number
of complexes studied is too small for this purpose. The DNA-binding studies suggesting weak
interactions with the Ru(II) complexes cannot explain their cytotoxic potency. The
mechanism of action of the complexes studied in this work may involve targets other than
DNA, such as topoisomerase [67].
4. Conclusions
In summary, in this report four new ruthenium(II) complexes containing bis-
triphenylphosphines, bipyridine and N-(acyl)-N’,N’-(disubstituted)thiourea derivatives as
ligands were synthesized and characterized, and their cytotoxicity against A549 and DU-145
cancer cells was evaluated and compared with normal (L929) cells. The crystal structures of
the complexes were determined, and showed that the triphenylphosphine ligands were in the
trans configuration, as suggested by 31P{1H} NMR experiments. The studies on
24

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complex/BSA binding show a static fluorescence quenching mechanism. The thermodynamic
parameters of the complex/BSA binding indicate a spontaneous interaction between these two
species and the presence of electrostatic forces between them. This same kind of weak
interactions can be suggested by the spectrophotometric titrations and viscosity studies of
complex/DNA binding, with low DNA-binding constants and no significant changes in the
DNA viscosity. Furthermore, the IC50 values against cancer cells (A549 and DU-145) for the
complexes are lower than the IC50 values of cisplatin (reference drug) and the free ligands.
These results reveal that N-(acyl)-N’,N’-(disubstituted)thiourea derivatives coordinated to
ruthenium provide good cytotoxicity against tumour cells. In this series of ruthenium(II)
complexes, the complexes 2 and 4 interact more strongly with the DNA molecule than
complexes 1 and 3, supporting our previous suggestion that the non-coordinate oxygen atom
from the furoyl ring can interact directly with this biomolecule. Thus, the fact that the IC50
values for the A549 and DU-145 cancer cells for all four complexes were essentially the same
suggests that for this series of complexes the mechanism of action may be not only through
the DNA, but also through other biomolecules, such as topoisomerase. This study is ongoing
in our laboratory to test this hypothesis.
Acknowledgment
We thank the Brazilian Research Council CNPq and FAPESP. CAPES provide
financial support to this research by the project CAPES/MES-CUBA 123/11. R.S. Correa
thanks FAPESP (2013/26559-4) and CAPES (14267-13-0) for postdoctoral fellowships. Also,
K.M. Oliveira thanks FAPESP (2014/04147-9) for doctoral scholarship and A.Alvarez also
thanks Capes/MES-CUBA 022451/2013.
Appendix A. Supplementary data
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Coordinates and other crystallographic data have been deposited with the deposition
codes CCDC 1043557, CCDC 1043558, CCDC1043559 and CCDC 1043560, for 1, 2, 3 and
4, respectively. Copies of this information may be obtained from The Director, CCDC, 12
Union Road, Cambridge, CB2 1EZ, UK, Fax: +44 1233 336033, E-mail:
deposit@ccdc.cam.ac.uk or www.ccdc.cam.ac.uk.
References
[1] M. Bacac, A.C. G. Hotze, K. V. D. Schilden, J. G. Haasnoot, S. Pacor, E. Alessio, G.
Sava, J. Reedijk, J. Inorg. Biochem. 98 (2004) 402-412.
[2] M. Gras, B. Therrien, G. Süss-Fink, P. Stepnicka, A. K. Renfrew, P. J. Dyson, J. Org.
Chem. 693 (2008) 3419-3424.
[3] W. Kandioller, E. Balsano, S. M. Meier, U. Jungwirth, S. Göschl, A. Roller, M. A.
Jakupec, W. Berger, B. K. Keppler, C. G. Hartinger, Chem. Commun. 49 (2013) 3348-3350.
[4] H. Chen, J. A. Parkinson, R. E. Morris, P. J. Sadler, J. Am. Chem. Soc. 125 (2003) 173–
186.
[5] A. Bergamo, A. Masi, P.J. Dyson, G. Sava, Int. J. Oncol. 33 (2008) 1281–1289.
[6] J.E. Debreczeni, A.N. Bullock, G.E. Atilla, D.S. Williams, H. Bregman, S. Knapp, E.
Meggers, Angew. Chem. Int. Ed. 45 (2006) 1580–1585.
[7] A. Bergamo, C. Gaiddon, J.H.M. Schellens, J.H. Beijnen, G. Sava. J. Inorg. Biochem. 106
(2012) 90–99.
[8] F.R. Pavan, G. Von Poelhsitz, M.I.F. Barbosa, S.R.A. Leite, A.A. Batista, J. Ellena, L.S.
Sato, S.G. Franzblau, V. Moreno, D. Gambino, C.Q.F. Leite, Eur. J. Med. Chem. 46 (2011)
5099-5107.
26

ACCEPTED MANUSCRIPT
[9] F.B. Nascimento, G. Von Poelhsitz, F.R. Pavan, D.N. Sato, C.Q.F. Leite, H.S.S. Araujo, J.
Ellena, E.E. Castellano, V.M. Deflon, A.A. Batista, J. Inorg. Biochem. 102 (2008) 1783-1789.
[10] E.R. dos Santos, M.A. Mondelli, L.V. Pozzi, R.S. Corrêa, H.S. Salistre-de-Araújo, F.R.
Pavan, C.Q.F. Leite, J. Ellena, V.R.S. Malta, S.P. Machado, A.A. Batista, Polyhedron 51
(2013) 292–297
[11] M.I.F. Barbosa, R.S. Correa, K.M. de Oliveira, C. Rodrigues, J. Ellena, O.R.
Nascimento, V.P.C. Rocha, F.R. Nonato, T.S. Macedo, J.M. Barbosa-Filho, M.B.P. Soares,
A.A. Batista, J. Inorg. Biochem. 136 (2014) 33–39.
[12] O. Estevez-Hernandez, J.L. Hidalgo, E. Reguera, I. Naranjo, Sens. Actuators, B 120
(2007) 766–772.
[13] A. Plutin, H. Márquez, M. Morales, M. Sosa, L. Morán, Y. Rodríguez, M. Suárez, C.
Seoane, N. Martín, Tetrahedron 56 (2000) 1533 - 1594.
[14] K.R. Koch. Coord. Chem. Rev. 216–217 (2001) 473–488.
[15] H. Pérez, Y.P. Mascarenhas, A.M. Plutin, R.S. Corrêa, J. Duque. Acta Crystallogr. Sect.
E, 64 (2008) m503.
[16] H. Pérez, R.S. Corrêa, A.M. Plutin, B. O'Reilly, J. Duque, Acta Crystallogr. Sect. E, 64
(2008) m733-m734.
[17] H. Pérez, R.S. Corrêa, J. Duque, A.M. Plutin, B. O'Reilly, Acta Crystallogr. Sect. E, 64
(2008) m916.
[18] H. Pérez, R.S. Corrêa, A.M. Plutín, O. Calderon, J. Duque, Acta Crystallogr. Section E,
65 (2009) m242.
[19] H. Pérez, R.S. Corrêa, B. O’Reilly, A.M. Plutín, C.C.P. Silva, Y.P. Mascarenhas. J.
Struct. Chem. 53 (2012) 921–926.
27

ACCEPTED MANUSCRIPT
[20] B. O’Reilly, A. M. Plutín, H. Pérez, O. Calderón, R. Ramos, R. Martínez, R. A. Toscano,
J. Duque, H. Rodríguez-Solla, R. Martínez-Alvarez, M. Suárez, N. Martín, Polyhedron 36
(2012) 133–140.
[21] J. Duque, O. Estévez-Hernández, E. Reguera, J. Ellena, R.S. Correa. J. Coord. Chem. 62
(2009) 2804–2813.
[22] N. Gunasekaran, S.W. Ng, E.R.T. Tiekink, R. Karvembu. Polyhedron 34 (2012) 41–45.
[23] S.S. Tan, A.A. Al-abbasi, M.I.M. Tahir, M.B. Kassim, Polyhedron, 68 (2014) 287–294.
[24] B. Schmitt, T.I.A. Gerber, E. Hosten, R. Betz, Inorg. Chem. Commun. 24 (2012) 136–
139.
[25] J.G. Małecki, J. Nycz, Polyhedron, 55 (2013) 49–56.
[26] J. Schroer, U. Abram, Polyhedron 28 (2009) 2277–2283.
[27] N. Selvakumaran, S.W. Ng, E.R.T. Tiekink, R. Karvembu, Inorg. Chim. Acta. 376
(2011) 278–284.
[28] W. Hernández, E. Spodine, J. C. Muñoz, L. Beyer, U. Schroder, J. Ferreira, M. Pavani
Bioinorg. Chem. Appl., 1 (2003) 271-284.
[29] W. Hernández, E. Spodine, L. Beyer, U. Schröder, R. Richter, J. Ferreira, M. Pavani,
Bioinorg. Chem. Appl., 3 (2005) 299-316.
[30] A. Rodger, K.K. Patel, K.J. Sanders, M. Datt, C. Sacht, M.J. Hannon. J. Chem. Soc.,
Dalton Trans. (2002) 3656–3663
[31] A.M. Plutin, R. Mocelo, A. Alvarez, R. Ramos, E.E. Castellano, M.R. Cominetti, A.E.
Graminha, A.G. Ferreira, A.A. Batista, J. Inorg. Biochem. 134 (2014) 76-82.
[32] S. Mihai, M. Negoiu, Rev. Chim., 7 (2012) 63.
[33] N. Gunasekaran, P. Jerome, S.W. Ng, E.R.T Tiekink, R. Karvembu, J. Mol. Catal. A:
Chem. (2012) 353–354.
28

ACCEPTED MANUSCRIPT
[34] V. Cîrcu, M. Ilie, F. Dumitrascu, I. Neagoe, S. Pasculescu, Polyhedron, 28 (2009) 3739–
3746.
[35] J.Sieler, R.Richter, E.Hoyer, L.Beyer, O.Lindqvist, L. Anderson. Z. Anorg. Allg. Chem.
580 (1990) 167-174.
[36] A.A. Batista, M.O. Santiago, C.L. Donicci, I.S. Moreira, P.C. Healy, S.J. Berners-Price,
S.L. Queiroz, Polyhedron 20 (2001) 2123-2128.
[37] COLLECT Data Collection Software; Nonius: Delft, 1998.
[38] Z. Otwinowski, W. Minor, In Methods in Enzymology: Macromolecular
Crystallography, Part A; Carter, C. W., Jr., Sweet, R. M., Eds.; Academic Press: New York,
1997; Vol. 276, pp 307−326.
[39] G.M. Sheldrick, Acta Crystallogr. Sect. A. 64 (2008) 112−122.
[40] P. Coppens, L. Leiserowitz, D. Rabinovich. Acta Crystallogr. 18 (1965) 1035-1038.
[41] L. J. Farrugia. J. Appl. Crystallogr. 32 (1999) 837-838.
[42] C.F. Macrae, I.J. Bruno, J.A. Chisholm, P.R. Edgington, P. Mccabe, E. Pidcock, L.R.
Monge, R. Taylor, J. van de Streek, P.A. Wood, J. Appl. Crystallogr. 41 (2008) 466-470.
[43] G. Cohen, H. Eisenberg, Biopolymers 8 (1969) 45-55.
[44] A. Sellamuthu, R. Ravishankaran, A. A. Karande, M. Kandaswamy, Dalton Trans. 41
(2012) 12970- 12983.
[45] Z. Petra, P. Franc, T. Iztok, G. Gerald, P. Georges, J. Inorg. Biochem. 117 (2012) 35–47.
[46] N. Maribel, C. William, A. R. H-P., S. Anibal, A. M. Jesús, T. Peter, S-D. R. A., J.
Inorg. Biochem. 105 (2011) 1684–1691.
[47] T. Mosmann. J. Immun. Meth., 65 (1983) 55-63.
[48] W. J. Geary. Coord. Chem. Rev. 7 (1971) 81–122.
[49] F. T. Martins, R. S. Corrêa, A. A. Batista, J. Ellena. Cryst. Eng. Comm. 16 (2014) 7013–
7022.
29