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Selective Coordination Mode of Acylthiourea Ligands in Half-Sandwich Ru(II) Complexes and Their Cytotoxic Evaluation.
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Article
Selective Coordination Mode of Acylthiourea Ligands in HalfSandwich Ru(II) Complexes and Their Cytotoxic Evaluation
Beatriz N. Cunha,* Liany Luna-Dulcey, Ana M. Plutin, Rafael G. Silveira, João Honorato, Raúl R. Cairo,
Tamires D. de Oliveira, Marcia R. Cominetti, Eduardo E. Castellano, and Alzir A. Batista*
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ABSTRACT: In this study, half-sandwich Ru(II) complexes containing acylthiourea ligands of the general type [Ru(η6-p-cymene)(PPh3)(S)Cl]PF6 (1m−6m) and [Ru(η6-p-cymene)(PPh3)(S−O)]PF6 (1b−
6b) where S/S−O = N’,N’-disubstituted acylthiourea were synthesized
and characterized (via elemental analyses, IR spectroscopy, 1H NMR
spectroscopy, 13C{1H} NMR spectroscopy, and X-ray diffractometry),
and their cytotoxic activity was evaluated. The different coordination
modes of the acylthiourea ligands, monodentately via S (1m−6m) and
bidentately via S,O (1b−6b), to ruthenium were modulated from
different synthetic routes. The cytotoxicity of the complexes was
evaluated in five human cell lines (DU-145, A549, MDA-MB-231,
MRC-5, and MCF-10A) by MTT assay. The IC50 values for prostate
cancer cells (2.89−7.47 μM) indicated that the complexes inhibited
cell growth, but that they were less cytotoxic than cisplatin (2.00 μM). Unlike for breast cancer cells (IC50 = 0.28−0.74 μM) and
lung cancer cells (IC50 = 0.51−1.83 μM), the complexes were notably more active than the reference drug, and a remarkable
selectivity index (SI 4.66−19.34) was observed for breast cancer cells. Based on both the activity and selectivity, complexes 5b and
6b, as well as their respective analogous complexes in the monodentate coordination 5m and 6m, were chosen for further
investigation in the MDA-MB-231 cell line. These complexes not only induced morphology changes but also were able to inhibit
colony formation and migration. In addition, the complexes promoted cell cycle arrest at the sub-G1 phase inducing apoptosis.
Interaction studies by viscosity measurements, gel electrophoresis, and fluorescence spectroscopy indicated that the complexes
interact with the DNA minor groove and exhibit an HSA binding affinity.
■
patients use cisplatin in their treatment.5 However, more and
more, cisplatin treatment is becoming limited by the
development of tumor cell resistance and its side effects.6,7
The discovery of other potential drugs has mobilized the
scientific community, and metallodrugs have received special
attention.8,9 After cisplatin, a second generation of platinum
drugs, such as carboplatin and oxaliplatin, was approved and
many others are in clinical trials, which makes platinum the
most investigated metal in the development of new metallodrugs.10 Nonetheless, the search for less toxic drugs also
stimulated investigations focused on other metal centers.
Ruthenium complexes have been highlighted mainly due to
Ru(III) imidazole and indazole compounds, NAMI-A and
INTRODUCTION
The incidence rate of new cases of cancer has increased
significantly worldwide. Breast cancer is the most common
cancer for women and is the second leading cause of cancer
death among women, while for men, lung and prostate cancers
are the main types diagnosed. Considering both sexes, lung,
breast, and prostate cancers are responsible for about 33% of
the total number of new detected cases.1 The discovery of
cisplatin anticancer properties revolutionized cancer therapy
since the approval for the clinical use of cisplatin as a
chemotherapeutic agent in 1978, and it is currently widely
used.2,3
Cisplatin has been used alone or combined with other drugs
as a first-line treatment, as an adjuvant, or even as neoadjuvant
therapy and is associated with the treatments of bladder,
cervical, lung, ovarian, and testicular cancer as well as
malignant mesothelioma and squamous carcinoma of the
head and neck. In addition, cisplatin is also related to the
treatment of other types of cancers where the first-line
treatment failed, or in specific situations that preclude the
standard treatment.4 It is estimated that 50% of all cancer
© XXXX American Chemical Society
Received: January 31, 2020
A
https://dx.doi.org/10.1021/acs.inorgchem.0c00319
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Article
Scheme 1. Synthesis of Complexes 1m−6m and 1b−6b with the Respective Acylthiourea Ligands
breast (MCF-10A) and lung (MRC-5) human non-tumor cell
lines. Four selected complexes were investigated not only
regarding their effects on cell morphology, colony formation,
and migration on MDA-MB-231 but also regarding cell cycle
and cellular apoptosis. Additionally, interaction studies with
DNA and human serum albumin (HSA) were carried out.
KP1019/KP1339, which showed activities against primary
cancer and metastasis.11,12
More recently, a new perspective of ruthenium complexes
was marked by the investigation of organometallic ruthenium(II)-arene complexes, which were observed mainly in the
studies conducted by Dyson and Sadler. In this field, the most
promising complexes are [Ru(η6-p-cymene)(PTA)Cl2] (PTA
= 1,3,5-triaza-7-phosphaadamantane), termed RAPTA-C, and
[Ru(η6-p-cymene)(en)Cl]+ (en = ethylenediamine). The first
is recognized for its activity in vivo as an antimetastatic agent,
and the second for its cytotoxicity to a range of cancer cell
lines, as well as for its metastatic property.13 Thus,
investigations focused on ruthenium(II)-arene complexes
with different ligand classes have received great attention in
the biological field.14−17
Acylthiourea derivatives are established compounds which
have been known for more than a century, and their extensive
variety of applications makes them interesting molecules.15 In
coordination chemistry, the acylthioureas act as versatile
ligands due to the presence of S,N,O donor atoms in their
structures, which allows for several coordination modes with
different metal centers: (1) O,S bonded to a metal (anionicbidentate),18 (2) S bonded to a metal (neutral-monodentate),19 (3) N bonded to a metal (neutral-monodentate),20 (4)
O,N,N bonded to a metal (anionic-bidentate),21 (5) O,S
bonded to a metal and N bonded to a metal (anionicbriged),22 and (6) S,N bonded to a metal (anionicbidentate).23 The cytotoxicity in the different cell lines of
acylthiourea complexes with Ni(II), Co(III), Pd(II), Pt(II),
Ru(II), and Au(I) has demonstrated the potential for these
compounds as anticancer agents.24−29
In the present paper, we focus our study on the investigation
of the cytotoxic properties of half-sandwich Ru(II) complexes
with acylthioureas acting as monodentate and bidentate
ligands. Thus, 12 new complexes of types [Ru(η6-p-cymene)(PPh3)(S)Cl]PF6] and [Ru(η6-p-cymene)(PPh3)(S−O)]PF6]
were obtained, where PPh3 is triphenylphosphine and S/S−O
(1m and 1b) is N-(benzoyl)-N′,N′-dimethyl thiourea, S/S-O
(2m and 2b) is N-(benzoyl)-N′,N′-diethyl thiourea, S/S−O
(3m and 3b) is N-(2-furoyl)-N′,N′-dimethyl thiourea, S/S−O
(4m and 4b) is N-(2-furoyl)-N′,N′-diethyl thiourea, S/S−O
(5m and 5b) is N-(2-thiophenyl)-N′,N′-dimethyl thiourea,
and S/S−O (6m and 6b) is N-(2-thiophenyl)-N′,N′-diethyl
thiourea. The in vitro antiproliferative activity of the complexes
was evaluated against breast (MDA-MB-231), lung (A549),
and prostate (DU-145) human tumor cell lines, as well as
■
RESULTS AND DISCUSSION
Synthesis and Characterization. The reactions of the
precursor complex [Ru(η6-p-cymene)(PPh3)Cl2] with N-acylN′,N′-disubstituted thiourea produced 12 new complexes with
different coordination modes in this ligand class, as can be seen
in Scheme 1. By performing the synthesis in methanol only, the
monodentate coordination (via the S atom) of acylthiourea to
ruthenium is favored (1m−6m). However, by adding sodium
bicarbonate salt to the reaction medium another coordination
mode of the ligand, such as chelate and negatively charged
(1b−6b), is obtained. Additionally, if the reaction is conducted
in a methanol/H2O (1:1) mixture the bidentate complexes are
also formed (1b−6b). For the latter, such an effect is possible
due to the hydrolysis of complexes 1m−6m as reported in our
previous study, where similar behavior was observed for the
ruthenium complexes with monosubstituted acylthioureas.30
All complexes were isolated as hexafluorophosphate salts of the
type 1:1 electrolytes, as supported by molar conductance,
whose measurements were carried out in acetone (128.7−
139.8 S cm2 mol−1).
The single crystal structures of the complexes were obtained
by ether diffusion into an acetone solution of complexes 1m−
6m and by the slow evaporation of a dichlomethane/methanol
solution for complexes 1b, 2b, and 4b−6b. The coordination
of the acylthiourea ligands to the metal, monodentate via the S
atom (1m−6m) and bidentate via the S,O atoms (1b−6b),
was unambiguously confirmed by X-ray techniques. Crystal
data are shown in the Supporting Information (Tables S1−
S3). The selected bond distances and angles are summarized in
Table 1 as well as in Tables S4 and S5. All of the crystallized
complexes present a half-sandwich three-legged “piano-stool”
structure in which the ruthenium center has a pseudooctahedral geometry, as can be seen in Figure 1 and Figure
S39. The six carbon atoms of the η6-p-cymene ligand form the
seat, while the legs of the “piano-stool” structure are
constituted by phosphorus (triphenylphosphine), sulfur
(acylthiourea), and chlorine atoms (1m−6m) or phosphorus
(triphenylphosphine), sulfur, and oxygen (acylthiourea) atoms
B
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and single bond, showing evidence of the existence of πelectron delocalization in a six-membered chelate ring formed
upon the bidentate coordination of acylthiourea.34
Acylthiourea molecules can assume different conformations,
the most commonly known of which are S, M, Z, and U, where
these letters refer to the position of the CO and CS bonds
in relation to the central N−H bond.35 In our case, upon
coordination the acylthiourea moiety assumed different
conformations, as shown in Figure 1. In the monodentate
complexes (1m−6m), the acylthioureas adopted a nonplanar
distorted S-shaped conformation where the CO and CS
groups are located at opposite positions, favoring this
coordination mode. On the other hand, for the bidentate
complexes (1b, 2b, and 4b−6b) the U conformation was
observed, in which it plays an important role in the π-electron
delocalization for the bidentate S,O coordination mode of the
acylthiourea.
Complexes 1m−6m exhibit a band in their IR spectra
ranging from 3194 to 3116 cm−1, assigned as NH stretching
vibrations, which is also present in the spectra of free
acylthiourea. However, upon coordination to the metal this
band was shifted to lower frequencies, showing evidence that
the coordinate form of the ligand is protonated. As expected,
this band is not observed for complexes 1b−6b, which
indicates the anionic nature of acylthiourea upon coordination
to metal. The νCO, around 1680 cm−1, in complexes 1m−
6m did not change significantly when compared with the free
ligand. On the other hand, for complexes 1m−6m the νCO
showed a significant displacement to negative regions, by
around 90−183 cm−1, as expected for the S,O coordination
mode of the ligands.24 For all complexes, the νCS band, at
around 1260 cm−1, shifted for lower energy regions due to the
weakening of the CS bond upon coordination of the ligand
to the ruthenium metal. An intense band near 840 cm−1 in the
complexes refers to νP−F and shows evidence of the presence
of PF6−, as supported by the molar conductance values. Weak
vibrations were observed at around 495−501 cm−1 and around
336−377 cm−1, which are characteristic of Ru−S and Ru−O
stretching, respectively. For complexes 1m−6m, only the
νRu−S was observed due to the monodentate coordination of
Table 1. Selected Interatomic Distances and Bond Angles
for Complexes 5m, 6m, 5b, and 6b
bond lengths (Å)
Ru−P
Ru−S
Ru−Cl
Ru−O
S(1)−C(1)
O(1)−C(2)
N(1)−C(1)
N(2)−C(1)
N(1)−C(2)
P−Ru−Cl
S−Ru−Cl
S−Ru−P
S−Ru−O
P−Ru−O
5m
6m
5b
6b
2.3799(11)
2.3843(11)
2.4201(11)
2.3799(15)
2.3696(15)
2.4208(16)
2.3670(12)
2.3564(13)
2.3459(13)
2.3507(13)
1.698(4)
1.217(6)
1.392(6)
1.303(6)
1.382(6)
2.073(3)
1.709(6)
1.716(5)
1.204(8)
1.276(6)
1.388(8)
1.347(6)
1.307(8)
1.324(7)
1.398(8)
1.313(7)
bond angles (deg)
5m
6m
92.42(4)
90.51(4)
82.77(4)
91.36(6)
91.18(6)
82.81(5)
2.091(4)
1.720(5)
1.269(7)
1.345(7)
1.345(7)
1.320(7)
5b
6b
88.36(5)
89.21(10)
87.49(10)
87.05(5)
89.14(11)
88.67(11)
Article
(1b−6b), as confirmed by bond angles of S(1)−Ru−Cl, S(1)−
Ru−P, P−Ru−Cl, S(1)−Ru−O(1), and O(1)−Ru−P around
90°.
The crystal structures of the five free ligands 1,31 2 (CCDC
14315), 3,32 4,33 and 5 (CCDC 1029544) were previously
published. The average bond lengths of the thiocarbonyl group
(S(1)−C(1) = 1.697−1.709 Å) in complexes 1m−6m are
longer than the respective bond lengths of the free ligand (S−
C = 1.666−1.688 Å), but the double-bond character is
maintained; CO and all C−N bond lengths remained
practically unchanged. In the bidentate coordination (1b−6b),
both thiocarbonyl and carbonyl bond lengths are longer than
the corresponding free ligand as well as the analogous
monodentate complexes, but the C−N distances (C(1)−
N(1) = 1.341−1.357 Å; C(2)N(2) = 1.308−1.328 Å) are
significantly shorter than both the free ligands and the
monodentate complexes. The length of C−S, C−O, and C−
N in the thiocarbonyl fragment (SCNCO) is between a double
Figure 1. Crystal structures of complexes 5m, 6m, 5b, and 6b. For the sake of clarity, the PF6− counterions are not included.
C
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Table 2. Characteristic 13C{1H} NMR Signals in Experimental and Theoretical Data (ppm) For Acylthiourea Ligands and
Complexes
13
13
C NMR experimental
Ligands
C NMR theoretical
Complexes
CO
CS
1
164.5
181.6
2
164.9
181.2
3
154.6
180.5
4
155.1
180.0
5
159.0
181.0
6
159.4
180.5
1m
1b
2m
2b
3m
3b
4m
4b
5m
5b
6m
6b
CO
CS
165.2
172.4
165.7
171.7
155.1
164.1
155.8
164.4
159.6
168.0
160.1
168.3
181.0
174.9
180.7
173.7
180.1
174.0
179.8
172.6
180.3
173.5
180.0
172.5
Ligands
Complexes
CO
CS
1
163.7
179.4
2
164.0
177.8
3
155.7
185.4
4
153.1
177.3
5
157.8
178.6
6
158.7
178.5
1m
1b
2m
2b
3m
3b
4m
4b
5m
5b
6m
6b
CO
CS
164.6
164.8
166.0
168.0
152.8
156.6
152.9
158.7
156.8
159.0
157.2
161.0
178.2
167.6
177.0
169.2
176.3
167.6
175.0
168.9
177.1
166.5
177.2
162.4
Figure 2. 13C{1H} NMR spectra of complexes 4m, 4b, and 1a (* reported early)29 and the respective free ligands in acetone-d6.
behaviors in their 13C{1H} NMR spectra, mainly in the CO
and CS carbon signals, when compared with the free ligands,
as shown in Table 2. For complexes 1m−6m, these signals did
not change significantly, shifting less than 1 ppm for higher and
lower frequencies. Complexes 1b−6b presented the same
trend, but prominent shifts were observed, close to 9 and 8
ppm for CO and CS carbon signals, respectively.
These results are in agreement with the literature, where for
the monodentate (S) coordination of acylthiourea to different
metal centers the CO and CS carbon signals remain
virtually unchanged.36−38 In the bidentate (S,O) coordination
mode, a considerable displacement is observed, around 10
ppm, for higher and lower frequencies in relation to the CS
and CO carbon signals of free acylthiourea, respectively.39−41 This considerable displacement is due to the πelectron delocalization of the chelate ring formed upon
coordination of the acylthiourea molecule to the metal center,
as also supported by X-ray diffraction.
Additionally, for an unusual coordination mode of the
acylthiourea ligand to the metal (bidentate through the S,N
amidic), both the CS and CO signals are shifted to lower
frequencies by around 9 ppm compared to the free ligand,20,30
suggesting that in this case there is not a resonance of the πelectrons in the four-member chelate. The same trend was
observed regarding the shifts of the CS and CO signals in
the 13C{1H} NMR from theoretical data obtained by density
functional theory (DFT), which are in good agreement with
the acylthiourea ligand, while for the bidentate form (1b−6b),
both νRu−S and νRu−O were present.
All complexes were characterized by not only multinuclear
31 1
P{ H}, 1H, and 13C{1H} NMR experiments but also twodimensional correlation spectroscopy, COSY, HSQC, and
HMBC for the correct assignment of signals (Figures S1−
S38). With regard to 1H and 13C{1H} NMR spectra, the
signals of all synthesized complexes show the expected number
of peaks, splitting, and intensities corresponding with pcymene, triphenylphosphine, and acylthiourea ligands in a
1:1:1 ratio. In the 31P{1H} spectra of the complexes, two sets
of signals were observed: a singlet for coordinated phosphorus
around 28−29 ppm for the 1m−6m complexes and one
around 33−35 ppm for the 1b−6b complexes, which shifted to
lower frequencies when compared with the precursor (24
ppm) as a consequence of the Cl− labilization. Another set
refers to a septet signal of the PF6− anion at −144 ppm.
In the 1H NMR spectra of the free acylthiourea ligands there
is a singlet referring to the N−H hydrogen in the range of
9.19−9.65 ppm. For complexes 1m−6m, this signal was shifted
to 11.05−11.37 ppm due to the proximity of N−H to the
coordination center, but for complexes 1b−6b the N−H
hydrogen is absent. This result confirms the neutral and
anionic forms of the acylthiourea ligands upon their
coordination to the metal center for the monodentate and
bidentate complexes, respectively, as supported by the IR
technique. Complexes 1m−6m and 1b−6b showed different
D
https://dx.doi.org/10.1021/acs.inorgchem.0c00319
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Table 3. IC50 Values Obtained from in Vitro Cytotoxic Assays against DU-145, A549, MDA-MB-231, and MCF-10A Cell Lines
with 48 h of Incubation for the Complexes Compared with the Reference Drug (Cisplatin), Precursor, and Acylthiourea
Ligands
IC50 ± SD (μM)
complex
DU-145
A549
MDA-MB-231
MRC-5
MCF-10A
SI1a
SI2b
1m
2m
3m
4m
5m
6m
1b
2b
3b
4b
5b
6b
ligands
precursor
cisplatin
5.60 ± 0.33
3.87 ± 0.09
6.01 ± 0.26
5.73 ± 0.27
4.45 ± 0.62
2.89 ± 0.05
7.47 ± 0.41
3.27 ± 0.17
7.37 ± 0.94
6.22 ± 0.08
3.19 ± 0.75
4.08 ± 0.19
>100
49.68 ± 1.79
2.00 ± 0.47
1.39 ± 0.22
0.62 ± 0.12
1.83 ± 0.01
0.69 ± 0.18
0.71 ± 0.02
0.51 ± 0.02
2.41 ± 0.03
0.87 ± 0.02
1.41 ± 0.11
0.56 ± 0.08
1.32 ± 0.14
0.59 ± 0.07
>100
12.73 ± 0.17
11.84 ± 1.19
0.73 ± 0.07
0.74 ± 0.02
0.50 ± 0.02
0.56 ± 0.03
0.67 ± 0.09
0.38 ± 0.05
0.61 ± 0.13
0.39 ± 0.18
0.46 ± 0.08
0.35 ± 0.09
0.34 ± 0.05
0.28 ± 0.02
>100
21.63 ± 1.31
2. 44 ± 0.20
1.61 ± 0.54
0.94 ± 0.05
2.47 ± 0.27
1.12 ± 0.18
1.23 ± 0.33
1.22 ± 0.50
1.83 ± 0.43
0.87 ± 0.04
1.85 ± 0.27
1.29 ± 0.01
1.11 ± 0.06
1.03 ± 0.02
>100
50.65 ± 0.21
29.09 ± 0.78
4.25 ± 0.55
3.45 ± 0.22
5.72 ± 0.44
4.55 ± 0.22
6.67 ± 0.95
6.61 ± 0.51
3.14 ± 0.58
5.50 ± 0.31
6.17 ± 0.13
3.46 ± 0.20
6.19 ± 0.25
4.26 ± 0.38
>100
19.09 ± 1.03
29.45 ± 0.85
1.16
1.52
1.35
1.62
1.73
2.39
0.76
1.00
1.31
2.30
0.84
1.75
5.82
4.66
11.44
8.12
9.95
17.39
5.15
14.10
13.41
9.89
19.34
15.21
3.98
2.46
0.88
12.07
a
SI1 = IC50 MRC-5/IC50 A549. bSI2 = IC50 MCF-10A/IC50 MDA-MB-231.
Figure 3. Effects of complex 6b at IC50, 2 × IC50, and 5 × IC50 concentrations in the breast cells. (A) Morphology of MDA-MB-231 and MCF-10A
at 0, 24, and 48 h. (B) Colony formation and quantification of colony number and size. Significant at the *p < 0.05, **p < 0.01, ***p < 0.001, and
****p < 0.0001 levels using ANOVA.
breast (MDA-MB-231), and two nontumorigenic human cell
lines, lung (MRC-5) and breast (MCF-10A), by colorimetric
MTT assay. The IC50 values and selective index (SI) are
summarized in Table 3. All complexes were significantly more
cytotoxic than the respective precursor and acylthiourea
ligands for all evaluated cell lines, indicating that the
coordination of acylthiourea to the ruthenium enhanced the
cytotoxicity of both the precursor and the ligands. Moreover,
the antiproliferative activity of the complexes was notably
greater than that of the reference drug, cisplatin, in MDA-MB231 and A549 cells, while for DU-145 cells the complexes were
slightly less active than cisplatin.
the experimental data for different coordination modes that
include the S,N mode, as can be seen in Table 2 and Table S6.
Thus, we suggest that the 13C{1H} NMR technique may be
very useful, not only for allowing the prediction of the
coordination mode of the acylthiourea ligands to the
ruthenium center but also for the coordination to the other
diamagnetic metal center, based only on CS and CO
carbon shifts of complexes when compared to the free ligand,
as shown in Figure 2.
Cytotoxic Activity. The in vitro toxicity of the complexes
and the free acylthiourea molecules was evaluated against three
human tumor cell lines, prostate (DU-145), lung (A549), and
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Figure 4. Effects of complexes 5m, 6m, 5b, and 6b on MDA-MB-231 cells. (A) Boyden chamber assay of complex 6b at different concentrations
and control with FBS (FBS+) and without FBS (FBS−). Significant at the ***p < 0.0001 level using ANOVA. (B) Effects of complex 6b at
different concentrations on cell cycle distribution. (C) Apoptosis by PE-Annexin V with 7-AAD assay. Control corresponds to the untreated cells,
and camptothecin was used as a positive control. Significant at the *p < 0.05 and ***p < 0.0001 levels using ANOVA. Cell cycle distribution.
(Figure 3A and Figures S40−S42). The evaluated complexes
showed a similar effect on MDA-MB-231 cells, inducing
shrinkage, membrane blebbing, and cell detachment, which are
characteristic of apoptotic cell death.45,46 These effects were
intensified when both the concentration of complexes and the
time of exposure were increased. For nontumorigenic breast
cells, the complexes did not promote considerable morphological changes; only at the highest concentrations studied was
a slight inhibition for cell proliferation observed. Thus, the
selectivity of the complexes toward cancer cells is clear.
Colony formation is a useful assay in determining the
effectiveness of cytotoxic agents based on cell reproduction
after treatment with the compound of interest.47 The
complexes, at different concentrations, considerably inhibited
the colony formation and colony size in MDA-MB-231 cells
when compared with the control (Figure 3B and Figure S43),
indicating not only the cytotoxic but also the cytostatic
properties of these complexes.
Since migration is an important step of angiogenesis, which
is related to tumor metastasis, the ability of the complexes to
inhibit the migration cell in the MDA-MB-231 cell line was
investigated by both wound healing and Boyden chamber
migration assays (Figure 4A and Figures S44−S47). Unlike the
control, where the cells spontaneously migrated until complete
wound closure, the complexes decreased the migration
capacity of breast tumor cells, and the existence of the
wound after 24 h of cell treatment with the complexes was
observed. This result was also supported by Boyden chamber
assay, in which the complexes drastically reduced the migration
in a dose-dependent manner. Considering the concentration of
0.75 μM after 24 h, the number of migrated cells corresponded
to 25, 60, 79, and 81 for complexes 6b, 6m, 5b, and 5m,
respectively, which are markedly lower values than for the
positive control (323 cells), confirming the antimigratory
properties of the studied complexes. Despite the fact that
complexes 5m, 6m, and 5b showed a similar ability to inhibit
the migration, complex 6b was notably the most inhibitory.
The therapeutic window was investigated for lung and breast
cell lines, where the complexes were more active. The
complexes displayed a slight selectivity toward lung cancer
cells, showing SI values lower than for the cisplatin. However,
for the MDA-MB-231 cell line, the selectivity of all complexes
toward cancer cells is clear, with SI around 20 for complexes
5b and 6b, which are higher than for cisplatin.
Despite the similar effect of the complexes for specific cell
lines, a trend was generally observed: complexes with a
monodentate coordination mode of the acylthiourea were
more cytotoxic than the analogous complexes in a bidentate
form for DU-145 and A549 cells. The opposite occurs for the
MDA-MB-231 cell line, where the complexes in bidentate form
showed lower IC50 values to the detriment of the monodentate
complexes. In addition, complexes with an R1 = thiophene
group led to the most cytotoxicity in all studied cancer cell
lines. Furthermore, the relationship of the R2 chain length and
activity indicates that the cytotoxicity increases with the chain
length of the alkane. In almost all complexes and cell lines,
complexes where R2 = ethyl group were more cytotoxic than
the others. Thus, the most active complex for prostate and lung
cancer cells was 6m; for breast cancer cells, complex 6b was
the most active while 6m was the most selective.
Many publications have shown that increasing the length of
the carbon chain increases the lipophilicity of complexes as
well as the cytotoxicity.42,43 It is known that lipophilicity has an
influence on the lipid solubility and tissue and cellular
uptake.44 From these results, considering that complexes 5b,
6b, 5m, and 6m were the most active, with remarkable
selectivity on the MDA-MB-231 cell line, they were selected
for more detailed biological studies that included an
investigation of the mechanism of cell death in this cell line.
Therefore, studies were performed with these complexes on
cell morphology, clonogenic and antimigration assays,
apoptosis analysis, and their interaction with DNA and
human serum albumin (HSA).
After exposure of the complexes to breast cancer cells for 24
and 48 h, morphological changes and cell death were observed
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Article
Figure 5. (A) Effects of the concentration of complexes 5m, 6m, 5b, 6b, thiazole orange (TO), and cisplatin (CP) on the relative viscosity of CTDNA at 25 °C. (B) Effects of the concentration of complexes 5m, 6m, 5b, and 6b on the electrophoresis of plasmid pBR322 DNA in different Ri
(0.25, 0.5, 1.0, and 1.5), molecular weight marker (MW), and DNA in DMSO (DNA). (C) Emission spectra of Hoechst 33258 (5.0 μM)-CTDNA (175 μM) (λex = 343 nm) at different concentrations (0−50 μM) of complexes 6m and 6b at 25 °C. (D) Emission spectra of thiazole orange
(5.0 μM)-CT-DNA (175 μM) (λex = 480 nm) at different concentrations (0−50 μM) of complexes 6m and 6b at 25 °C.
The effects of the complexes on cell cycle regulation in
MDA-MB-231 cells were evaluated by propidium iodide
staining/flow cytometry. All of the studied complexes had
similar behavior on cell cycle progression, promoting a
significantly increased accumulation at the sub-G1 phase cells
in a dose-dependent manner, as shown in Figure 4B and Figure
S48. However, complex 6b was the most effective, since it
promoted a greater accumulation in the sub-G1 phase cells in
lower concentrations than the other complexes. It is known
that an increase in the sub-G1 portion is associated with
apoptosis as a cell death pathway.45
In order to confirm that the complexes induce apoptosis in
the MDA-MB-231 cells, as indicated by both cell morphology
assay and cell cycle analysis, a quantitative analysis was
performed by flow cytometry. A marked increase in the
apoptotic population (Figure 4C and Figure S49) was
observed after treatment with the complexes in a dosedependent manner. The evaluated complexes similarly induced
apoptosis in breast cells, where the percentage of apoptotic
cells was nearly 80% following treatment with complexes 5m,
6m, and 5b at 10 μM; in the case of complex 6b, this
percentage was around 70, 73, and 75% at 1, 2, and 3 μM,
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respectively. The degree of apoptosis in the MDA-MB-231
cells induced by complex 6b was greater and in lower
concentrations than those in complexes 5m, 6m, and 5b, which
is in agreement with the cytotoxic activity and cell cycle
investigation. Commonly, apoptosis is the death mechanism
used for Ru(II) arene complexes with anticancer activity, and
in many cases it is associated with an increase in the sub-G1
phase of the cell cycle.14,15,48,49
The viscosity experiment has proven to be a useful method
for investigating the mode of binding of the complexes to DNA
by the relationship between viscosity and DNA fragment
lengths.50 The relative viscosity of DNA did not change
significantly when increasing the concentration of the
complexes, as seen in Figure 5A. This profile is clearly different
from thiazole orange (TO), an intercalating molecule, and
cisplatin, a covalent binder, which are used as reference
compounds. No changes or less-significant changes in the
viscosity of DNA can be indicative of weak interactions or a
lack of interaction. Electrostatic and groove binding are
recognized as weak interactions where the viscosity of DNA
remains unaltered.
The interaction of the complexes with DNA was also
investigated by electrophoretic mobility of plasmid pBR322
DNA on agarose in gel after an incubation for 18 h at 37 °C.
As shown in Figure 5B, an unusual behavior was observed:
with the increase of Ri, there was a decrease in the intensity of
all bands. However, this effect was more prominent for the
monodentate complexes with R2 = ethyl; for complexes 5m
and 6m, no band was visible in Ri = 1.5. In some
circumstances, groove binders can replace intercalates with
ethidium bromide, a marker used in this assay, resulting in the
absence of visible bands.51
Based on viscosity and electrophoretic mobility experimental
data, the possibility of the interaction of the complexes with
the DNA minor groove was investigated by the displacement
of the Hoechst 33258 assay. Hoechst is a fluorescent dye, wellknown for its ability to bind DNA via the minor groove.52
Thus, compounds capable of interacting with DNA via the
minor groove can replace Hoechst, resulting in the quenching
of the fluorescence intensity of the Hoechst-DNA complex.
Successive additions of the complexes lead to a reasonable
decrease in the fluorescence intensity of the Hoechst-DNA
system (Figure 5C and Figure S50). Additionally, a thiazole
orange displacement assay was carried out in order to evaluate
if the complexes are also able to displace an intercalative
compound. This assay was performed in the same conditions
evaluated for the Hoechst assay. In the presence of increasing
amounts of complexes, a moderate decrease in the fluorescence
of the TO-DNA complex was observed (Figure 5D and Figure
S50). However, it can be observed that the monodentate
complexes (5m and 6m) promoted a greater quenching of
fluorescence when compared to the the bidentate complexes
(5b and 6b).
This result is in agreement with that observed in the DNA
electrophoresis, supporting that the absence of visible bands in
the agarose gel was related to the displacement of the DNA
intercalator ethidium bromide, which is a strong indicator that
the complexes interact with DNA via the minor groove since
the quenching of the fluorescence of the TO-DNA system is
insignificant when compared with the quenching exhibited for
the H33258-DNA system. This kind of interaction has already
been reported for other cytotoxic Ru(II) arene complexes.53,54
Article
Fluorescence spectroscopy is a widely disseminated method
for studying interactions between small molecules and HSA, in
which tryptophan is the main residue contributing to the
intrinsic fluorescence of this protein (HSA).55 Under the
studied conditions, the complexes were nonfluorescent. As
shown in Figure 6 and Figure S51, the fluorescence emission of
HSA was quenched regularly with additions of the complexes,
which confirms the interaction between the complexes and
HSA.
Typically, two mechanisms are associated with fluorescence
quenching: static and dynamic, which can be expressed by the
Stern−Volmer equation and are distinguished from a depend-
Figure 6. Fluorescence quenching spectra of HSA at different
concentrations of complex 6b at 298 K and fluorescence spectra of
HSA-dansylglycine (DG) titrated with complex 6b at 298 K.
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Article
Table 4. Stern−Volmer Quenching Constants (Ksv), Binding Constants (Kb), Numbers of Binding Sites (n), and
Thermodynamic Parameters for Ru(II) Complexes at Different Temperatures
5m
6m
5b
6b
t* (°C)
Ksv* (104)
Kb* (105)
n
ΔH°*
ΔS°*
ΔG°*
298
310
298
310
298
310
298
310
(4.59 ± 0.09)
(4.30 ± 0.12)
(5.66 ± 0.13)
(5.36 ± 0.13)
(5.15 ± 0.01)
(4.81 ± 0.01)
(6.51 ± 0.07)
(6.21 ± 0.06)
(0.40 ± 0.07)
(0.74 ± 0.02)
(0.61 ± 0.03)
(1.19 ± 0.29)
(2.83 ± 0.09)
(25.10 ± 2.25)
(16.27 ± 1.74)
(39.62 ± 1.75)
0.96
1.01
1.05
1.1
1.14
1.33
1.31
1.39
38.97
218.9
43.68
267.63
139.53
572.58
56.94
308.27
−26.26
−28.89
−27.28
−36.07
−31.1
−37.98
−35.43
−39.15
prostate cancer cells. Moreover, this cytotoxicity was
significantly higher than cisplatin for breast and lung cancer
cells. The structure−activity relation suggested that the
complexes, where R1 = thiophene and R2 = ethyl, promoted
a greater activity in all studied cell lines. In general,
monodentate complexes were more cytotoxic in A549 and
DU-145 cells, while bidentate complexes were more efficient
for MDA-MB-231 cells. Complexes 5m, 6m, 5b, and 6b inhibit
both colony formation and cell migration in breast cancer cells
and induce morphological changes. These complexes additionally promote cell cycle arrest at the sub-G1 phase, and
apoptosis cell death. The interaction studies with biomolecules
indicate that complexes 5m, 6m, 5b, and 6b bind to DNA via
the minor groove and show a considerable affinity for HSA site
II. These results highlight the potential of the half-sandwich
Ru(II)/acylthiourea complexes as anticancer agents, encouraging us to explore their chemical properties in more detail in
order to better understand this class of compound for greater
modulation.
ence on temperature and viscosity by the Stern−Volmer
constant (Ksv). For all complexes, Ksv values decreased with an
increase temperatures (Table 4), indicating a static mechanism, since in the case of the dynamic mechanism the opposite
is true and Ksv increases with an increase in temperatures.
The binding constant (Kb) values increased with the
temperature (Table 4), implying that temperature favors the
complex-HSA binding. The Kb values suggest that bidentate
complexes (105−106) exhibit a higher HSA binding affinity
than monodentate complexes (104−105), and complexes with
R1 = ethyl which also showed a higher protein affinity when
compared to complexes with R1 = methyl. Essentially,
hydrogen bonds, van der Waals forces, and electrostatic and
hydrophobic interactions are involved in protein-molecule
binding, and the thermodynamic parameters (ΔG° = free
energy, ΔH° = enthalpy, and ΔS° = entropy) can predict the
binding mode.55 As summarized in Table 4, the negative values
of ΔG° for the complexes reveal that the protein binding
process is spontaneous. Additionally, both positive ΔH° and
ΔS° values show evidence of the involvement of hydrophobic
forces in the interaction of the complexes with HSA.
The major drug binding sites on HSA are described as sites I
and II, which are located in subdomains IIA and IIIA of the
protein, respectively. It is established that dansylated amino
acids are useful as fluorescent probes in order to distinguish the
binging sites.56,57 In order to indicate the HSA binding site for
the complexes, a competitive assay was carried out using
dansylglycine (DG) as a marker, since this compound binds to
HSA via site II. The HSA-DG complex resulted in increased
fluorescence that was blue-shifted (to 470 nm) compared to
dansylglycine, as expected. However, a considerable decrease
in the fluorescence intensity was observed with successive
additions of complexes, as can be seen in Figure 6 and Figure
S52. All studied complexes showed similar behavior. This
result indicates the affinity of the complexes for the HSA site
II.
■
■
EXPERIMENTAL SECTION
Synthesis of [Ru(η6-p-cymene)(PPh3)(S)Cl]PF6 Complexes.
The precursors and acylthiourea ligands were synthesized according
to previous reports.32,58−61 The [Ru(η6-p-cymene)(PPh3)Cl2] (0.180
mmol, 0.100 g) was dissolved in 20 mL of deaerated methanol, with a
subsequent addition of the respective acylthiourea ligand (0.198
mmol). The mixture was kept under an inert atmosphere and was
stirred for 0.5 h. Later on, NH4PF6 (0.300 mmol, 0.050 g) was added.
One hour later, the reaction mixture was concentrated under reduced
pressure until the precipitation of an orange solid, which was filtered
off, washed with water (3 × 5 mL) and cold methanol (3 × 5 mL),
and dried under vacuum.
Synthesis of [Ru(η6-p-cymene)(PPh3)(S−O)]PF6 Complexes.
The acylthiourea ligand (0.198 mmol) and NaHCO3 (0.198 mmol,
0.017 g) were dissolved in 20 mL of deaerated methanol. Afterward,
[Ru(η6-p-cymene)(PPh3)Cl2] (0.180 mmol, 0.100 g) was added and
the reaction mixture was stirred for 2.5 h under an inert atmosphere.
For precipitation of the yellow solid, NH4PF6 (0. 300 mmol, 0.050 g)
was added. The solid was filtered off, washed with water (3 × 5 mL)
and diethyl ether (3 × 5 mL), and dried under vacuum.
These complexes were also obtained by another synthetic route, in
which the acylthiourea ligand (0.190 mmol) was dissolved in 20 mL
of a deaerated methanolic solution of [Ru(η6-p-cymene)(PPh3)Cl2]
(0.180 mmol, 0.100 g). After stirring for 0.5 h, under an inert
atmosphere, 20 mL of deaerated water was added. The mixture was
stirred for 15 h, and the product, which was obtained after the
addition of NH4PF6 (0. 300 mmol, 0.050 g), was filtered off, washed
with water (3 × 5 mL) and diethyl ether (3 × 5 mL), and dried under
vacuum.
X-ray Structure Determination. The monodentate complexes
were crystallized from a diethyl ether diffusion into an acetone
solution of the complexes, while for the bidentate complexes the
crystallization occurred by a process of low evaporation of the
CONCLUSIONS
Different conditions in the synthetic route modulated distinct
coordination modes of acylthiourea ligands in the Ru(II)
complexes: monodentately only through S (1m−6m) and
bidentately via S,O (1b−6b). This ligand class showed
different conformations upon coordination, “S” and “U”
forms, showing that the type of conformation is associated
with specific coordination modes. Interestingly, the 13C{1H}
NMR technique may be an effective tool to predict the
coordination mode among the many possibilities of acylthiourea ligands in different metal centers. Acylthiourea ligand
insertion in the half-sandwich Ru(II) complexes contributed
considerably to the greater cytotoxicity in breast, lung, and
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complexes in a dichloromethane/methanol solution. The single
crystal measurements by X-ray diffraction were performed on
Enraf−Nonius Kappa-CCD and Apex II Duo diffractometers with
graphite monochromated MoKα radiation (λ = 0.71073 Å). Cell
refinements were carried out using the Collect software,62 and the
structures were obtained by the direct method using SHELXS-97
software.63 The Gaussian method was used for the absorption
corrections.64 The table and structure representations were generated
by WinGX software65 and MERCURY software,66 respectively. The
main crystal data collections and structure refinement parameters for
all complexes are summarized in the Supporting Information.
Theoretical Calculations. The Gaussian 09 revision E.01
electronic structure program suite was used for all DFT calculations.67
The local density approximation, characterized by the Vosko-WilkNusair (SVWN5) parametrization, was used for the gas phase
geometry optimizations.68 Crystallographic coordinates were used for
the optimization (when available) without any restriction of
symmetry, and the harmonic frequencies were calculated using the
second analytical derivatives and verified for the absence of imaginary
frequencies.69 The ruthenium was described by LANL2DZ relativistic
effective core potentials (ECP) that replace 28 core electrons with a
nonlocal effective potential and an associated basis set for the
remaining electrons, while the 6-31G+(d,p) basis set was used for all
other atoms.70,71 In order to determine the chemical shifts of 13C, the
GIAO method was used and the data were compared to the
experimental data using eq 1.72
δiso,calc = σref − σiso
Article
violet for 30 min. The colonies formed were analyzed in both number
and size using ImageJ software.
Migration. Cell migration was investigated by two methods:
wound healing and transwell using Boyden chambers. In the wound
healing assay, MDA-MB-231 cells (1.5 × 105 cells/well) were plated
into 12-well plates and incubated properly until the culture reached
100% confluence. Afterward, a scratch was made in the central portion
of every well using a micropipette tip. The cells were incubated with
the complexes in the corresponding concentration of IC50 (48 h) for
24 h. After this time, 40× images were captured with an inverted
optical microscope (Nikon, TS100) coupled to a system for image
capture in two different fields by well. For the transwell assay, a
density of 0.5 × 105 cells/well of MDA-MB-231 was incubated with
the complexes at different concentrations and seeded on the upper
chamber in a DMEM medium without FBS. In the lower chamber, a
DMEM medium with 10% FBS was added. The eventual migration
process occurred for 22 h at 37 °C in 5% CO2. Cells that remained in
the upper chamber were removed with a cotton swab, and cells that
migrated through the upper chamber membrane were fixed with
methanol and stained with 1% toluidine blue. Migrated cells were
quantified by manual counting.
Cell Cycle Analysis. MDA-MB-231 cells (4 × 105 cells/well)
were placed in a 6-well plate and incubated with different
concentrations of Ru(II) complexes and 32 μM campthothecin
(positive control) for 24 h. Afterward, the cells were collected and
washed three times with PBS and fixed overnight in 70% ethanol. The
cells were washed again with PBS, incubated with RNaseA (0.2 mg/
mL) for 30 min at 37 °C, and stained with a hypotonic fluorochrome
solution (5 μg/mL PI, 0.1% sodium citrate, and 0.1% Triton-X-100) 1
h before flow-cytometric analysis. Cell cycle phase distribution was
analyzed using Cell Quest (BD Biosciences) computer software with
an Accuri C6 flow cytometer (BD Biosciences).
Apoptosis Assay. Apoptotic activity of the evaluated complexes
on MDA-MB-231 cells was analyzed by flow cytometry using a PEAnnexin-V Apoptosis Detection Kit. The cells (7.0 × 104 cells/well)
were plated into 12-well plates and after 24 h of incubation at 37 °C
were exposed to different concentrations of complexes for 24 h.
Treated cells were washed with cold PBS and then resuspended in a
200 μL of binding buffer (10 mM HEPES/NaOH, pH 7.4, 140 mM
NaCl, 2.5 mM CaCl2). PE-Annexin V (2.5 mL) and 7-AAD (2.5 mL)
were added in each well and incubated for 20 min at room
temperature in the dark. Next, 200 μL of binding buffer was added for
analysis using an Accuri C6 flow cytometer. The fluorescence was
quantified by Cell Quest software (BD Biosciences).
DNA Binding Experiments. DNA interaction studies with the
complexes were performed by viscosity, electrophoresis, and
fluorescence quenching experiments using calf thymus CT-DNA or
plasmid pBR322. The complexes were dissolved in sterile DMSO, and
the assays were carried out based on the dilution, with a final
concentration of 10% DMSO for all experiments. Viscosity experiments were carried out using the Ostwald viscometer at 25 °C in a
thermostatic water bath. The concentration of the CT-DNA solution
in a Tris-HCl buffer (5 mM Tris-HCl, 50 mM NaCl, pH 7.2) was
fixed (250 μM), while the concentration of the complexes varied from
0 to 100 μM. The flow time was measured using a digital stopwatch.
The relative specific viscosity (η/η0)1/3 values, where η is the relative
viscosity of DNA in the presence of the complexes and η0 is the
relative viscosity of DNA, were plotted versus [complex]/[DNA]
ratios. Equation 2 was used to determine the relative viscosity of DNA
(η0) values from the flow time of the DNA solution (t) corrected for
the flow time of the buffer (t0).
(1)
where σref = 182.4254 ppm was used for C (TMS). The TMS crystal
structure (CCDC 678366) was optimized at the same level of
complex molecule theory.
Cell Culture. MDA-MB-231 (ATCC no. HTB-26) human breast
tumor cells, A549 (ATCC no. CCL-185) human lung tumor cells, and
MRC-5 (ATCC no.CCL-171) nontumor human lung cells were
maintained in a DMEM medium containing 10% fetal bovine serum
(FBS). DU-145 (ATCC no. HTB-81) human prostate tumor cells
were maintained in an RPMI-1640 medium also supplemented with
10% FBS. The nontumor human breast cell line, MCF-10A (ATCC
no. CRL-10317), was cultivated in a DMEM/F12 medium containing
5% horse serum, EGF (0.02 mg/mL), hydrocortisone (0.05 mg/mL),
cholera toxin (0.001 mg/mL), insulin (0.01 mg/mL), penicillin (100
IU/mL), streptomycin (100 mg/mL), and L-glutamine (2 mM). All
cell lines were maintained at 37 °C in a humidified 5% CO2
atmosphere.
Proliferation Assay. The effects of the Ru(II) complexes on the
viability of cells were determined by colorimetric assay using MTT [3(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide]. A density of 1.5 × 104 cells/well was seeded in 150 μL of supplemented
medium into 96-well plates. After 24 h, the cells were treated with
different concentrations of the complexes (dissolved in sterile
DMSO) and incubated for 48 h. MTT (1 mg/mL) was added (30
μL/well), and the plates were incubated again for 4 h. After removal
of the medium, the formazan crystals were dissolved in isopropanol.
The optical density was measured at 540 nm using a 96-well
multiscanner autoreader (ELISA).
Cell Morphology. MDA-MB-231 and MCF-10A cells were
seeded (1.0 × 105 cells/well) in a 12-well plate and incubated in a
supplemented medium at 37 °C in 5% CO2 for 24 h. After treatment
of the cells with different concentrations of complexes (dissolved in
sterile DMSO, and for the control only sterile DMSO) the cell
morphology was examined in an inverted microscope (Nikon, T5100)
with 40× magnification.
Colony Formation. MDA-MB-231 cells (300 cells/well) were
seeded in a 6-well plate and maintained in a supplemented medium at
37 °C in 5% CO2 for 24 h. Cells were treated with different
concentrations of the complexes for 48 h; for the control, only sterile
DMSO (0.5%) was added at same conditions. The medium was
replaced by a fresh medium without any complex, and the plates were
incubated for 10 days. The cells were washed with PBS, fixed with
methanol and acid acetic (3:1) for 5 min, and stained with 5% crystal
13
η0 =
t − t0
t0
(2)
DNA electrophoresis experiments were performed using agarose
gel. Complexes at different ratios between 0.25 and 1.5 were
incubated with pBR322 DNA (38 μM) at 37 °C for 18 h. The
samples were electrophoresed for 1.5 h at 80 V on 1% agarose gel in a
Tris-acetate-EDTA (TAE) buffer (0.45 M Tris-HCl, 0.45 M acetic
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acid, 10 mM EDTA) and stained with ethidium bromide (2 μL/50
mL). After the electrophoretic run, the bands were visualized using a
UV-light transilluminator (ChemiDoc equipment).
The Hoechst 33258 and thiazole orange (TO) displacement assay
was carried out by the fluorescence quenching experiment with the
Hoechst/TO (5.0 μM)-CT DNA (175 μM) complex 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 Hoechst CT DNA at 495
nm (excitation wavelength of 343 nm) or of the TO at 530 nm
(excitation wavelength of 480 nm) was monitored using the
complexes as suppressors at different concentrations (0−50 μM) in
DMSO. Fluorescence spectra were recorded in triplicate using an
opaque 96-well plate.
HSA Binding Experiments. HSA interaction studies with the
complexes were performed by a fluorescence quenching experiment,
where the HSA concentration in buffer (4.5 mM Tris-HCl, 0.5 mM
NaOH, 50 mM NaCl) at pH 7.4 was kept constant (5 μM), while the
concentration of the complexes was increased from 5 to 50 μM. The
extinction of the emission intensity of the HSA tryptophan residues at
305 nm (excitation wavelength 270 nm) was monitored at 25 and 37
°C. For data analysis, the classical Stern−Volmer equation was used
(eq 3):
F0
= 1 + Kqτo[Q] = 1 + K sv[Q]
F
Cambridge Crystallographic Data Centre, 12 Union Road,
Cambridge CB2 1EZ, UK; fax: +44 1223 336033.
■
F0 − F
= log Kb + n log[Q]
F
́
Beatriz N. Cunha − Departamento de Quimica,
Universidade
Federal de São CarlosUFSCar, 13561-901 São Carlos, SP,
Brazil; Instituto Federal GoianoIFGoiano, 76300-000 Ceres,
GO, Brazil; orcid.org/0000-0003-3079-1854;
Email: beatriznc_@hotmail.com
́
Alzir A. Batista − Departamento de Quimica,
Universidade
Federal de São CarlosUFSCar, 13561-901 São Carlos, SP,
Brazil; Email: daab@ufscar.br
Authors
Liany Luna-Dulcey − Departamento de Gerontologia,
Universidade Federal de São CarlosUFSCar, 13561-901 São
Carlos, SP, Brazil
́
Ana M. Plutin − Laboratório de Sintesis
Orgánica, Facultad de
́
Quimica,
Universidad dela HabanaUH, Habana 10400,
Cuba
́
Rafael G. Silveira − Departamento de Quimica,
Universidade
Federal de São CarlosUFSCar, 13561-901 São Carlos, SP,
Brazil; Instituto Federal GoianoIFGoiano, 76300-000 Ceres,
GO, Brazil
́
João Honorato − Departamento de Quimica,
Universidade
Federal de São CarlosUFSCar, 13561-901 São Carlos, SP,
Brazil; orcid.org/0000-0002-1127-6083
́
Raúl R. Cairo − Laboratório de Sintesis
Orgánica, Facultad de
́
Quimica,
Universidad dela HabanaUH, Habana 10400,
Cuba
́
Tamires D. de Oliveira − Departamento de Quimica,
Universidade Federal de São CarlosUFSCar, 13561-901 São
Carlos, SP, Brazil
Marcia R. Cominetti − Departamento de Gerontologia,
Universidade Federal de São CarlosUFSCar, 13561-901 São
Carlos, SP, Brazil; orcid.org/0000-0001-6385-7392
́ e Informática,
Eduardo E. Castellano − Departamento de Fisica
́ de São Carlos, Universidade de São Paulo
Instituto de Fisica
USP, 13560-970 São Carlos, SP, Brazil
Complete contact information is available at:
https://pubs.acs.org/10.1021/acs.inorgchem.0c00319
(3)
(4)
The thermodynamic parameters ΔH, ΔS, and ΔG were obtained
by eqs 5 and 6
ln
K 2 ijj 1
1 yz ΔH
= jj − zzz
j
K1
T2 z{ R
k T1
(5)
where K1 and K2 are the binding constants at temperatures T1 and T2,
respectively; R is the gas constant.
ΔG = − RT ln K = ΔH − T ΔS
(6)
To evaluate the interaction of the complexes with HSA site II, a
displacement assay was performed using the compound dansylglycine
as a fluorescent probe. The experiments were performed by adding
varying amounts of the complexes (0−50 μM, final concentrations) to
a mixture of 5 μM HSA and 5 μM dansylglycine in buffer (4.5 mM
Tris-HCl, 0.5 mM NaOH, 50 mM NaCl) at pH 7.4. After adding the
complexes, the solutions were incubated for 5 min at 25 °C before
measurements were taken. The extinction of the emission intensity of
the dansylglycine at 470 nm (excitation wavelength 340 nm) was
monitored at 25 °C.
■
AUTHOR INFORMATION
Corresponding Authors
where F0 and F correspond to the fluorescence intensities in the
absence and presence of the quencher, respectively; [Q] is the
quencher concentration; and Ksv is the Stern−Volmer quenching
constant.
The binding constant (Kb) as well as the number of binding sites
(n) was determined by plotting the double log graph of the
fluorescence data using eq 4.
log
Article
Notes
The authors declare no competing financial interest.
■
ACKNOWLEDGMENTS
This study was financed in part by the Coordenaçaõ de
́
Aperfeiçoamento de Pessoal de Nivel
Superior - Brazil
(CAPES) - Finance Code 001, CNPq, and FAPESP.
■
ASSOCIATED CONTENT
sı Supporting Information
*
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Measurements and characterization of all complexes
(PDF)
Accession Codes
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free of charge via www.ccdc.cam.ac.uk/data_request/cif, or by
emailing data_request@ccdc.cam.ac.uk, or by contacting The
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