Silver(i) complexes of 3-methoxy-4-hydroxybenzaldehyde thiosemicarbazones and triphenylphosphine: structural, cytotoxicity, and apoptotic studies

Article abstract of DOI:10.1039/d0dt01134g

Novel silver(i) complexes of the type [AgCl(PPh3)2(L)] {PPh3 = triphenylphosphine; L = VTSC = 3-methoxy-4-hydroxybenzaldehyde thiosemicarbazone (1); VMTSC = 3-methoxy-4-[2-(morpholine-1-yl)ethoxy]benzaldehyde thiosemicarbazone (2); VPTSC = 3-methoxy-4-[2-

Full text of DOI:10.1039/d0dt01134g

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A. Vieira de Godoy Netto, Dalton Trans., 2020, DOI: 10.1039/D0DT01134G.
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DOI: 10.1039/D0DT01134G
ARTICLE
Silver(I) complexes of 3-methoxy-4-hydroxybenzaldehyde
thiosemicarbazones and triphenylphosphine: structural, cytotoxic
and apoptotic studies
Received 00th January 20xx,
Accepted 00th January 20xx
Débora E. S. Silva,a  Amanda B. Becceneri,b João V. B. Santiago,a José A. Gomes Neto,a Javier Ellena,c
Márcia R. Cominetti,b José C. M. Pereira,a Michael J. Hannond and Adelino V. G. Nettoa 
DOI: 10.1039/x0xx00000x
Novel silver(I) complexes of the type [AgCl(PPh3)2(L)] {PPh3
hydroxybenzaldehyde thiosemicarbazone (1); VMTSC 3-methoxy-4-[2-(morpholine-1-yl)ethoxy)benzaldehyde
thiosemicarbazone (2); VPTSC 3-methoxy-4-[2-(piperidine-1-yl)ethoxy)benzaldehyde thiosemicarbazone (3)} were
= triphenylphosphine; L= VTSC = 3-methoxy-4-
=
=
synthesized and fully characterized by spectroscopic techniques. The molecular structures of complexes 2 and 3 were
determined by single crystal X-ray diffraction. The compounds 1-3 exhibited appreciable cytotoxic activity against human
tumor cells (lung A549, breast MDA-MB-231 and MCF-7) with IC50 values in 48 h of incubation ranging from 5.6 to 18 M.
Cellular uptake studies showed that complexes 1-3 were efficiently internalized after 3 hours of treatment in MDA-MB-231
cells. The effects of complex 1 in cell morphology, cell cycle, induction of apoptosis, mitochondrial potential membrane
(m) and reactive oxygen species (ROS) production have been evaluated in triple negative breast cancer (TNBC) cells MDA-
MB-231. Our results showed that complex 1 induced typical morphological alterations of cell death, an increase in cells at
sub-G1 phase, apoptosis and mitochondrial membrane depolarization. Furthermore, DNA binding studies evidenced that 1
can bind to ct-DNA and does so without modifying the B-structure of the DNA, but that the binding is weak compared to
Hoechst 33258.
Introduction
The anticancer potential of silver-based compounds have
Breast cancer affected more than 2 million women
worldwide in 2018 being the leading cause of cancer death.1
Triple negative breast cancer (TNBC) is the most aggressive
breast cancer subtype, comprises about 20% of all cases, and
has the shorter overall survival. TNBC is characterized by a lack
of expression of estrogen (ER) and progesterone receptors (PR),
and does not exhibit amplification of the human epidermal
growth factor receptor 2 (HER2).2 Since no specific molecular
targets has been identified for the TNBC, chemotherapy is the
main available therapeutic option. However, its limited efficacy
makes it crucial to develop new anticancer agents against
TNBC.3
attracted considerable interest in the last few decades.4,5
A
growing body of evidence suggests that these complexes
generally exert their cytotoxicity by altering the membrane
structure, interacting with nucleic acids and inducing oxidative
stress.6 However, their action against tumor cells is strictly
dependent on a number of parameters such as solubility and
stability in aqueous media/biological medium, rate of release of
AgI, and lipophilicity. Such parameters may be fine-tuned by
carefully selecting a suitable set of ligands.5,7 Among the
available ligands, phosphines have been systematically
employed in the design of new metal complexes for therapeutic
purposes due to its versatility and rich coordination chemistry.8
In addition, the lipophilic character of phosphines may assist in
crossing the cellular membrane, consequently, increasing the
cell internalization of the metal compound.9 Since the
pioneering work of Berners-Price and Sadler10, considerable
efforts have been devoted to the investigation of the antitumor
potential of silver phosphine complexes. There is a growing
number of silver compounds containing the “Ag(PR3)2(L)” core
that have gained increasing attention due to their promising
cytotoxic effects on a variety of tumor cells.11,12,13,14,15,16,17 For
instance, Engelbrecht et al.17 have described that
a. Department of General and Inorganic Chemistry, Department of Analytical
Chemistry, UNESP – São Paulo State University, Institute of Chemistry, CEP 14800-
060 Araraquara, SP, Brazil. E-mail: deboraeduarda.soares@gmail.com;
adelino.netto@unesp.br
b. Department de Gerontology, Federal University of São Carlos, CEP 13565-905 São
Carlos, SP, Brazil.
c. São Carlos Institute of Physics, University of São Paulo, CEP 13.566-590, São Carlos,
SP, Brazil.
d. School of Chemistry, University of Birmingham, Birmingham, B15 2TT, UK.
e. † Footnotes relating to the title and/or authors should appear here.
f. Electronic Supplementary Information (ESI) available: [details of any
supplementary information available should be included here]. See
DOI: 10.1039/x0xx00000x
[Ag(SCN)(PR3)2] (PR3
=
tris-4-methoxyphenylphosphine)
induced mitochondrial apoptosis in esophageal squamous
carcinoma (SNO) cell line. This silver complex was more active
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Journal Name
than cisplatin and showed reduced toxicity towards two non- vanillin with 1-(2-Chloroethyl)morpholine hydrochloride and 1-
View Article Online
24
malignant cells (dermal and kidney cell lines). Complexes of the (2-Chloroethyl)piperidine hydrochlorideD, OrI:e1s0p.1e0c3t9i/vDe0lyD.T011T34hGe
type [Ag(L)(PPh3)2] (L
triphenylphosphine) induced apoptosis in leiomyosarcoma microcrystalline solids in good yields (70-80%) by the
(LMS) and human breast adenocarcinoma (MCF-7) cell lines.16
condensation of thiosemicarbazide and aldehydes VM and VP,
=
benzoic acids; PPh3
=
new compounds VMTSC and VPTSC were obtained as
Recently, we have studied the cytotoxic and apoptotic respectively, in the presence of HCl as catalyst. Compounds
effects of compounds of the type [Ag(phen)(L)]NO3 {phen = VMTSC and VPTSC were further analyzed using elemental
1
1
1,10-phenantroline;
L
=
2-formylpyridine-N(4)-R- analyses, IR spectroscopy, H, COSY, H-13C HSQC and HMBC
thiosemicarbazones) on TNBC cell line MDA-MB-231.18 One of NMR spectroscopy. The details of the synthetic procedures and
these complexes was not only more active than cisplatin, but structural characterization are described in the Experimental
also was less toxic for the non-tumor breast cell line MCF-10A. Section. The purity of the compounds VMTSC and VPTSC was
These findings have prompted us to investigate the antitumor further confirmed by elemental analysis. The condensation of
potential of new silver compounds obtained by the thiosemicarbazide and functionalized aldehydes was confirmed
replacement of 1,10-phenanthroline by triphenylphosphine by the disappearance of the characteristic CO bands at ca.
(PPh3). However, as
a
result of the enhancement of 1700 cm-1 in the IR spectra of VM and VP together with the
hydrophobicity caused by PPh3, the metal phosphine complexes appearance of the typical absorptions from thiosemicarbazone
tend to be less soluble in polar solvents.5 Recently, Bisceglie et moiety at ca. 3170 cm-1 (NH), 1600 cm-1 (CN) and 758 – 780
al.19 have modified thiosemicarbazones with polar groups, such cm-1 (CS).25 The NMR spectra of the thiosemicarbazones
as N-ethylmorpholine, aiming the modulation of the VMTSC and VPTSC were in full agreement with their molecular
1
lipophilicity/hydrophilicity of metal complexes. In this work, we structure, being observed only one set of signals (see ESI). H
have used vanillin thiosemicarbazones (3-methoxy-4- NMR spectra of VMTSC and VPTSC clearly exhibited the typical
hydroxybenzaldehyde thiosemicarbazone) O-alkylated with N- signals of thiosemicarbazone moiety: a singlet at 11.31 ppm
ethylmorpholine and N-ethylpiperidine moieties to enhance the (=N-NH-), two broad signals at ca. 8.2 – 8.0 ppm (-NH2) and a
solubility of silver complexes in polar solvents.
singlet at 7.95 ppm (-HC=N).25 Besides the expected signals of
In the framework of our ongoing research on new bioactive piperidine and morpholine groups, 1H NMR spectra also showed
metal complexes,20,21,22,23 we report herein the synthesis, the characteristic signals for ethylene groups and vanillin over
cytotoxic and apoptotic studies of new tetrahedral silver(I) the spectral range of 4.1-2.6 and 7.5-7.0 ppm, respectively.
compounds of
the type [AgCl(PPh3)2(L)] {PPh3
=
Integration of the proton signals was in accord with the
triphenylphosphine;
L= VTSC 3-methoxy-4- requirements for VMTSC and VPTSC. The most important
=
hydroxybenzaldehyde thiosemicarbazone (1); VMTSC = 3- feature of DEPTQ spectra is the appearance of a low field
methoxy-4-[2-(morpholine-1-yl)ethoxy)benzaldehyde resonance at ca. 142.5 ppm which was assigned to the iminyl
thiosemicarbazones (2); VPTSC = 3-methoxy-4-[2-(piperidine-1- carbon [-HC=N-] together with the presence of the thiocarbonyl
yl)ethoxy)benzaldehyde thiosemicarbazone (3)}. The cytotoxic group signal (C=S) at 177.5 ppm.
activities of 1-3 were evaluated against breast cancer cell lines
MDA-MB-231 (TNBC) and MCF-7 (hormone-responsive) and
lung cancer cell line (A549). As a comparison, the toxic effects
of silver compounds on non-tumor breast cells (MCF-10A) have
also been determined. The effects of 1 on MDA-MB-231 cells
Silver(I) compounds
The complexes 1-3 were synthesized from the reaction of
AgCl with 3-methoxy-4-R-benzaldehyde thiosemicarbazone and
PPh3 = triphenylphosphine in a 1:1:2 molar ratio, respectively
regarding cell morphology, apoptosis, cell cycle, reactive oxygen
(Scheme 1). The compounds were characterized by IR and NMR
species (ROS) formation and mitochondrial potential
spectroscopy, elemental analysis and single-crystal X-ray
diffraction. Microanalysis data confirmed the expected
membrane (m) have also been investigated. The DNA
binding ability of 1 has also been investigated by means of
circular dichroism and competitive assays with Hoechst 33258.
formulation. The IR spectra of 1–3 showed characteristics bands
at 1600 (C=N) and 745 (C=S) cm-1 from thiosemicarbazones.
The shift of the C=S band from 758-780 cm-1 (free ligands) to
lower frequency suggested the coordination via sulfur atom.25
Results and discussion
In addition, no shift of the (C=N) absorption was noticed,
Synthesis and characterization
supporting the S-monodentate coordination in the solid state.
The presence of intense absorption at 1095 cm-1 evidenced
coordination of triphenylphosphine in the compounds 1-3.26
The molecular structures of complexes 2 and 3 were
determined by single-crystal X-ray diffraction (Fig. 1). The
crystallographic data and selected bond parameters are listed
in Tables 1 and S1, respectively. Both complexes crystalize in the
monoclinic space group I2/a with one chlorine, one sulfur atom
of the thiosemicarbazone and two phosphorous from two
triphenylphosphine molecules coordinated to the silver(I) atom.
Thiosemicarbazones
The synthesis of the new thiosemicarbazones was
accomplished using a two-step synthetic protocol: i) O-
alkylation of the vanillin followed by Schiff-base condensation
of the appropriate functionalized aldehyde with
thiosemicarbazide, as depicted in Scheme 1. Compounds 3-
methoxy-4-[2-(morpholine-1-yl)ethoxy)benzaldehyde (VM) and
3-methoxy-4-[2-(piperidine-1-yl)ethoxy)benzaldehyde
were prepared by the treatment of the commercially available
(VP)
2 | J. Name., 2012, 00, 1-3
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DOI: 10.1039/D0DT01134G
S
A)
O
N
O
N
H
NH2
H3CO
Cl
NH2CSNHNH2
H3CO
Acetone
K2CO3
55 ºC
H3CO
OH
+
CH3CH2OH + H2O
O
O
N
N
N
. HCl
R
R
R
a: R= CH2
b: R= O
VP: R= CH2
VM: R= O
VPTSC: R= CH2
VMTSC: R= O
B)
Cl
H3CO
RO
H3CO
N
N
HN
Ag
PPh3
CH3CN
HN
S
S
AgCl
2PPh3
+
+
PPh3
RO
NH2
NH2
R = H (1); N-ethylmorpholine (2) and N-ethylpiperidine (3)
Scheme 1 Reaction scheme of (A) thiosemicarbazones and (B) silver(I) compounds.
The metal coordination form a distorted tetrahedral with bond assignable to the vanillin aromatic protons (H2, H5, H6),
angles ranging from 99.8° to 124.21°. The Ag-S bond length are methoxyl group, and N-ethylmorpholinyl/piperidinyl moieties
2.6013 Å and 2.6325 Å in 2 and 3, respectively, which are close were not affected on coordination. The typical resonances of
to that reported ones for similar silver complexes [AgCl(κ1-S- triphenylphosphine protons were noticed in the range of 7.33 –
L)(PPh3)2] (L=pyridine-2-carbaldehyde thiosemicarbazone) and 7.43 ppm. The 31P NMR spectra of complexes 1-3 in DMSO-d6
[AgCl(η1-S-HIntsc)(Ph3P)2] (HIntsc
thiosemicarbazone) with
=
indole-N1-methyl-3- exhibited one singlet at δ 4.8 – 2.9 ppm at 298 K because of the
neutral monodentate rapid intermolecular exchange of the PPh3 ligands, which is a
thiosemicarbazones.27,28 The Ag-P bond lengths in complexes 2 well-documented behavior for phosphine based complexes of
and 3 are comparable to those observed for similar complexes coinage metals.30,31,32
in the literature.27,28 The chloride ligand is involved in
In order to evaluate the stability of 1-3 under biologically
intramolecular hydrogen bonding with the NH of relevant conditions, we have conducted some time-dependent
thiosemicarbazone, leading to the formation of six membered experiments in solution phase. Due to their low aqueous
ring.
solubility, 1-3 were dissolved in a mixture of DMSO-d6/D2O
(80:20%, v/v) and their 1H NMR spectra were collected at 0, 24
and 48 h after sample preparation (Fig. S13). At 0 h, the
aromatic region of the spectra of 1 and 2 (Figure S13) exhibited
a complex multiplet from PPh3 ligands centred at 7.24 ppm
which became relatively more resolved at 24 h and remained
unchanged at 48 h. This finding may be associated with a change
of the coordination mode of thiosemicarbazone in solution. No
significant spectral changes were noticed over this period for 3.
In all cases, no signals assignable to the free ligands were
detected over this period. UV-Vis spectra of the silver
complexes 1-3 in Tris-HCl buffer (pH 7.4, [Cl-] = 50 mM, 2%
DMSO, 298 K) were also registered at 0, 24 and 48 h (Fig. S14).
Over the 48-h period in Tris-HCl buffer, the CT band (ca. 321 nm)
decreased in intensity (15-37%), which may indicate that a
considerable amount of the silver complexes remained intact in
the presence of Cl-.
Solution studies
1H and DEPTQ NMR spectra of complexes 1-3 in DMSO-d6
showed all the expected signals and integration. Upon
coordination, the hydrazinic proton (=N-NH2) signal was shifted
ca. 0.3-0.4 ppm to downfield, suggesting that
thiosemicarbazones act as neutral ligands. The iminyl-HC=N
signal did not change significantly after complexation. In the
DEPTQ NMR spectra of 1-3, the C=S signal was displaced ca. 1.1-
1.5 ppm upfield compared to the free thiosemicarbazones,
indicating a weakening of the C=S bond as a result of the Ag-S
bond formation.29 A downfield shifted of ca. 1.0-1.3 ppm was
noticed for the azomethine carbon resonance (-HC=N) upon
coordination, which suggested the coordination of imine
nitrogen to silver(I) ion. This finding may indicate that
thiosemicarbazone acts as a N,S-ligand in solution. The signals
This journal is © The Royal Society of Chemistry 20xx
J. Name., 2013, 00, 1-3 | 3
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DOI: 10.1039/D0DT01134G
Table 1 Crystallographic data and experimental details for complexes 2 and 3.
2
3
CCD Number
Empirical formula
Formula weight
Temperature (K)
Crystal system
Space group
a (Å)
1992316
C51H52AgClN4O3P2S
1992321
C47H49AgCl2N6O2P20.13C2H3
1016.69
293(2)
monoclinic
I2/a
25.4270(9)
974.64
298
monoclinic
I2/a
25.5169(10)
b (Å)
12.1243(5)
10.2681(3)
c (Å)
33.2299(10)
43.6410(13)
α (°)
90
90
β (°)
100.785(3)
102.951(3)
γ (°)
90
90
Volume (Å3)
Z
10063.3(6)
11143.5(7)
8
1.162
8
ρcalc (g/cm3)
μ (mm )
1.342
0.621
‑1
0.557
F(000)
4200.0
4016.0
Crystal size (mm3)
0.35 × 0.28 × 0.2
Mo Kα (λ = 0.71073)
5.03 to 55
0.25 × 0.2 × 0.18
MoKα (λ = 0.71073)
5.098 to 68.974
-31 ≤ h ≤ 39, -14 ≤ k ≤ 15, -68 ≤ l ≤ 54
94501
Radiation
2Θ range for data collection (°)
Index ranges
Reflections collected
Independent reflections
Data/restraints/parameters
Goodness-of-fit on F2
Final R indexes [I>=2σ (I)]
Final R indexes [all data]
Largest diff. peak/hole / e Å-3
-32 ≤ h ≤ 33, -15 ≤ k ≤ 13, -42 ≤ l ≤ 43
23269
11513 [Rint = 0.0257, Rsigma = 0.0390]
11513/0/569
22366 [Rint = 0.0601, Rsigma = 0.0670]
22366/0/569
1.060
1.010
R1 = 0.0366, wR2 = 0.0722
R1 = 0.0628, wR2 = 0.0882
0.45/-0.40
R1 = 0.0530, wR2 = 0.1186
R1 = 0.1286, wR2 = 0.1488
0.83/-0.60
Cytotoxic activity
The cytotoxic activity of vanillin thiosemicarbazones and
compounds 1-3 in breast (MCF-7 and MDA-MB-231) and lung
(A549) human tumor cells has been evaluated by using the MTT
assay after 48 hours of incubation.33 The IC50 values are
summarized in Table 2. The assays were also performed with
cisplatin under same experimental conditions, as a reference
drug. The vanillin thiosemicarbazones VTSC, VMTSC and VPTSC
are not cytotoxic, no drug response was observed at
concentrations > 100 M in all the tested tumor cell lines. On
the other hand, the silver(I) compounds 1-3 were more
cytotoxic than cisplatin against MDA-MB-231, MCF-7 and A549
cells, with IC50 values ranging from 5.63 to 18.80 M. The
compounds 1-3 displayed comparable cytotoxicity, suggesting
(2)
that
the
incorporation
of
O-alkylated
vanillin
thiosemicarbazones did not increase their activity. Particularly,
the complex 1 was more cytotoxic in TNBC cells MDA-MB-231
(5.63 ± 0.13 M) than in hormone sensitive cells MCF-7 (16.63
± 1.08 M). As a comparison, the cytotoxic effects of
compounds 1-3 were also evaluated towards the non-tumor cell
line MCF-10A. The silver(I) compounds exhibited higher
selectivity index (SI) values (in the range of 1.3 - 1.7) compared
to cisplatin (0.9) (Table 2). It is important to emphasize that
silver compounds can take part in competing ligand exchanging
reactions with biological components during the cytotoxic
experiments, giving rise to new species which may be
responsible for the observed activity. Therefore, the IC50 values
may not reflect rigorously the activity of either the original silver
complexes, free ligands or Ag(I) ion.
(3)
Fig. 1 Ortep type diagram of the asymmetric unit of the complexes 2 and 3
showing the atomic labelling scheme around the silver(I) atom and ellipsoids
at the 50% probability level.
4 | J. Name., 2012, 00, 1-3
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Table 2 IC50 values (M) of compounds against human tumour cells A549, MCF-7 and MDA-MB-231 and non-tumour breast cells MVCiFew-1A0rAtic.le Online
DOI: 10.1039/D0DT01134G
Compound
A549
MCF-7
MDA-MB-231
5.63 ± 0.13
6.72 ± 0.46
MCF-10A
8.39 ± 0.11
11.61 ± 0.88
SI
1
7.48 ± 0.21
8.15 ± 1.21
16.63 ± 1.08
5.78 ± 0.57
1.5
1.7
2
3
6.46 ± 0.51
>100
18.80 + 0.63
>100
7.16 ± 0.10
>100
9.53 ± 0.64
1.3
-
-
VTSC
VMTSC
VPTSC
-
-
-
>100
>100
>100
>100
>100
>100
-
Cisplatin
23.36 ± 0.42
49.60 ± 3.69
43.85 ± 2.4
39.12 ± 4.87
0.9
a Selectivity index (SI): IC50 MCF-10A/IC50 MDA-MB-231
Cellular uptake
48 h, stained with propidium iodide (PI) and the DNA content
was determined using flow cytometry. The percentage of cell
population in each phase is shown in Fig. 3. It was observed a
notable increase of the sub-G1 cell population after treatment
with complex 1, evidencing the induction of internucleosomal
DNA fragmentation, which is a result of apoptosis.39 The
percentage of cells at sub-G1 phase in the control (5.5%)
increased to 23.3% after treatment. Concomitantly, a significant
decrease in G1 cell population from 70.4% (control) to 43% and
an increase in S cell population from 10.9% (control) to 15%,
were also observed in MDA-MB-231 cells treated with complex
1. The treatment with camptothecin (a pro-apoptotic drug)40
induced significant effects in the MDA-MB-231 cycle, the sub-
G1 cell population increased to 60.6%, whereas G1 and S cell
population decreased to 31.1 and 3.7%, respectively (Fig. 3).
The cellular uptake ability of metal complexes have been
related to their cytotoxic activity.34,35 In this sense, the cellular
uptake of complexes 1-3 was evaluated in TNBC cells MDA-MB-
231. The Ag uptake was determined in MDA-MB-231 cells
exposed to 12 M of compounds for 3 hours. The initial
concentration of Ag in the solutions of complexes 1-3 used to
the treatment was also quantified. Based on the initial
concentration of 1-3, the percentage of Ag uptake was
calculated. As shown in Fig 2, about 30% of 1-3 added to the cell
medium were internalized in MDA-MB-231 cells after 3 hours of
incubation. Consistently, the Ag uptake results are in agreement
with in vitro cytotoxic activity of compounds 1-3. These results
evidenced that the modification of vanillin thiosemicarbazones
with N-ethylmorpholine and N-ethylpiperidine moieties did not
improve their cytotoxic effects neither cellular uptake. For
comparison purposes, cellular uptake studies on compounds of Cell apoptosis
the type [Ag(phen)(L)]NO3 {phen = 1,10-phenantroline; L = 2-
To evaluate the apoptosis induced by the complex 1 in TNBC
formylpyridine-N(4)-R-thiosemicarbazones) revealed that only
8-12% of them were taken up by MDA-MB-231 cells after 6
hours of incubation.18 This finding agrees well with previous
observation that the lipophilic nature of PPh3 ligands plays an
important role in cellular membrane crossing, thus, enhancing
the cell internalization of the metal complexes.9
cells, the Annexin V/ 7-AAD double staining assay was
performed. MDA-MB-231 cells were exposed to 6 and 12 M of
1 for 48 h, stained with Annexin V and 7-AAD and analyzed by
flow cytometry. As shown in Fig. 4, the percentage of apoptotic
cells significantly increased, after treatment with complex 1,
compared to the control cells. In addition, the percentage of
necrosis was insignificant in treated cells. The total percentage
of apoptotic cells increased from 10.9% (control) to 69.8% after
treatment with 12 M of 1. The treatment with camptothecin
induced 43.4% of apoptosis in MDA-MB-231 cells (Fig. 4). These
results indicated that complex 1 potently induces late apoptosis
in MDA-MB-231 cells.
Morphological studies
In order to examine the cytotoxic effects of complex 1 in
MDA-MB-231 cells, the cell morphology was analyzed over a
period of 48 hours of incubation. As shown in Fig. S15, the
untreated cells exhibited normal morphology. On the other
hand, the MDA-MB-231 cells treated with 1 showed typical
morphological features of cell death. The morphological
changes were concentration and time-dependent. MDA-MB-
231 cells treated with 1 exhibited a significant decrease in cell
density. The treatment for 24 h at 12 and 24 M induced the
appearance of shrunk and round cells. After 48 h, these changes
were observed for all tested concentrations. These
morphological alterations have been ascribed to cell death via
apoptosis.36,37
Cell cycle
Considering that cell cycle arrest may be an effective
strategy to inhibit the proliferation of tumor cells,38 the effect
of complex 1 in the cell cycle distribution of MDA-MB-231 cells
was investigated. TNBC cells were incubated with 6 M of 1 for
Fig. 2 Cellular uptake of silver(I) compounds after 3 h of treatment in
MDA-MB-231 cells
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The mitochondrial membrane potential (m) assay
View Article Online
DOI: 10.1039/D0DT01134G
In order to verify whether apoptosis induced by the complex
1 in TNBC cells is mediated by the intrinsic pathway, the effects
of 1 on mitochondrial membrane potential (m) were
examined. MDA-MB-231 cells were treated with 1 (6, 12 and 24
M) for 4 h, stained with JC-1 and analyzed by flow cytometry.41
JC-1 is mitochondrial fluorescent dye that forms aggregates
with red fluorescence in cells with normal mitochondria. On the
other hand, in apoptotic cells with damaged mitochondria, the
membrane potential decreases, and monomers with green
fluorescence can be seen. As shown in Fig. 5, the control cells
exhibited red fluorescence. MDA-MB-231 cells treated with
complex 1 showed changes in mitochondrial m compared to
the control cells, indicating the mitochondrial membrane
depolarization in a concentration-dependent manner (Fig. 5). In
4 h of treatment at 24 M of 1, the percentage of cells emitting
green fluorescence increased to 47%, compared to 2.1% in
untreated control cells. Cisplatin did not affect the
mitochondrial membrane potential in TNBC cells under similar
experimental conditions (Fig. 5).
Fig. 3 A) Effects of complex 1 on MDA-MB-231 cell cycle. TNBC cells
were treated for 48 h with 1 (6 M) or camptothecin (Campto, 20 M).
Data are the mean of experiments in triplicate (*p ≤ 0.01, **p ≤ 0.001
and ***p ≤ 0.0001). B) Representative histograms of cell cycle.
ROS formation
The intrinsic apoptosis pathway can be triggered by
intracellular ROS generation.42 In addition, ROS generation has
been proposed as common mediator for apoptosis promoted
by many metal complexes.43,44,45 So, as a preliminary study, the
intracellular ROS formation was investigated by 2',7'-
dichlorodihydrofluorescein diacetate (H2DCFDA) staining. The
oxidation of non-fluorescent dye (H2DCFDA) by intracellular
ROS produce the fluorescent probe 2',7'-dichlorofluorescein
(DCF). The effect of complex 1 on intracellular ROS formation
was evaluated in TNBC cells (Fig. 6). MDA-MB-231 cells were
exposed to 1 (6 and 12 M) for 24 h, stained with H2DCFDA and
analyzed by confocal fluorescence microscopy. Control cells
exhibited no significant green fluorescence. After treatment
with 1, a remarkable increase of green fluorescence in the TNBC
cells was observed, demonstrating intracellular ROS formation
induced by complex 1. Cisplatin has also promoted the increase
of green fluorescence in the cells (Fig. 6). Our findings may
DNA interaction
The binding of metal complexes with DNA can be explored
using circular dichroism (CD) spectroscopy.47,48 The CD
spectrum of ct-DNA was recorded in the presence of increasing
concentrations of 1 (Fig. S16). The typical DNA signature CD
spectroscopy was unchanged on addition of complex 1,
confirming that a B-DNA structure is retained when the two
species interact. No induced peaks in the complex spectroscopy
were observed.
Competitive binding studies with Hoechst 33258 were also
performed. Hoechst 33258 binds to the minor groove of DNA
and displays a strong fluorescence when bound to DNA.49 Thus,
the quenching of the intrinsic fluorescence of Hoechst-bound
DNA upon addition a metal complex can be used to examine
whether the metal complex can bind to DNA and displace the
Hoechst 33258. Silver(I) complex 1 was titrated into a ct-DNA
solution pre-treated with Hoechst 33258, and the emission
intensity was recorded. As shown in Fig. S17, the emission
intensity of Hoechst-bound DNA at 458 nm decreased by >70%
upon addition of 1, confirming that 1 can bind to DNA and can
displace the Hoechst 33258 from the minor groove. Although
the displacement of Hoechst is sometimes ascribed to
competitive minor groove binding (and this would potentially
be feasible for this complex), Hoechst can also be displaced by
agents that bind elsewhere on the DNA.50 While complex 1 can
clearly displace Hoechst, it is a less strong binder, and even in
presence of a ten-fold excess of complex the Hoechst
fluorescence is not completely quenched.
suggest that compound
1 induces an enhancement of
intracellular ROS production in the MDA-MB-231 cells, which
would be related to apoptotic cell death pathway. However,
although H2DCFDA probe is widely employed for detecting
intracellular H2O2 and other oxidants, caution should be taken
when interpreting these results. According to Kalyanaraman et
46
al., several limitations are associated with the use of this
protocol due to the complex intracellular redox chemistry of
HDCF carboxylate anion.
At this point, it seems instructive to make some discussion
regarding possible mechanisms, which might account for the
cytotoxic effects of silver complexes on MDA-MB-231 cell line.
6 | J. Name., 2012, 00, 1-3
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In our study, the new thiosemicarbazones VTSC, VMTSC and
ARTICLE
Besides gold-based compounds, silver(I) complexes have
View Article Online
VPTSC are non cytotoxic against MDA-MB-231, MCF-7 and A549 been identified to trigger apoptosiDsOIb: 1y0.10t3a9r/gDe0tDinTg0113t4hGe
tumor cells. An enhancement of the cytotoxicity is observed in mitochondria and/or inhibiting TrxRs.55,63 The elegant Allison’s
all cases when thiosemicarbazones and triphenylphosphine are work on the anti-cancer activity of [Ag(NHC)2][AgBr2] (NHC = 1-
coordinated to silver ion, most likely due to the higher cellular benzyl-3-methylimidazolium) revealed that this compound is a
accumulation as a result of the increase of lipophilicity. In fact, powerful inhibitor of human TrxR (IC50 value of 2.39 ± 0.59 nM),
cellular uptake results clearly indicated the internalization of leading to an enhancement of ROS production and
silver complexes by the MDA-MB-231 cells, supporting the subsequently apoptotic cell death.67 In addition, it induced the
above conclusions. DNA binding studies have been carried out complete inhibition of Topoisomerase I at 0.16 M, resulting in
since the cytotoxic effect of metal complexes is frequently DNA damage together with the inhibition of PARP-1 and
attributed to their ability to induce DNA damage, resulting in glycolysis in tumor cells.67 A number of studies has also
cell death.51 Our results from DNA binding studies revealed that demonstrated the potential of silver(I) phosphine-based
1 displayed a poor DNA-binding affinity in cell free conditions, complexes to induce significant effects on mitochondria, which
which may suggesting distinct pathways to cytotoxicity from are associated with the mitochondriotropic properties of
that found for cisplatin. The cytotoxic effect of complex 1 on phosphines. For instance, triphenylphosphonium groups
+
+
MDA-MB-231 cells was also evaluated under an inverted light (HPPh3 /alkyl-PPh3 ) have been used as vehicles to delivery
microscope. Upon incubation with 1, MDA-MB-231 cells drugs to mitochondria within because of their cationic charge
exhibited the typical morphological changes associated with and high lipophilicity.37,55,68 Thus, the coordination of
apoptosis: cell shrinkage and rounding together with their phosphines is supposed to play an important role in targeting
detachment from the plate.36,37 Annexin V/ 7AAD flow mitochondria. For example, the complex [Ag(SCN)(PR3)] (PR3 =
cytometry analysis results confirm the apoptotic cell death in tris(4-methoxyphenyl)phosphine) induces apoptosis via
the MDA-MB-231 cells induced by compound 1. In order to intrinsic pathway by the mitochondrial membrane
examine the changes that take place in cell cycle progression depolarization, increase of ROS levels, caspase-9 cleavage and
from inhibition of MDA-MB-231 cell proliferation, cell cycle cytochrome c release.17 Complexes of the type [Ag(nim)(PR3)n]
distribution analysis using flow cytometry has been carried out. (nim = nimesulide; PR3 = triarylphosphines; n = 1, 2) induces the
Upon incubation with complex 1, an increase of the sub-G1 cell apoptotic pathway in MCF-7 cells via mitochondrion damage.37
population with a concomitant accumulation in S-phase cells In addition, these silver phosphine complexes showed
was noticed. The increase of the sub-G1 cell population inhibitory activity against the lipoxygenase (LOX), an enzyme
indicates the induction of internucleosomal DNA fragmentation mainly distributed to mitochondrion and responsible for the
as a result of apoptosis].39 In addition, complex 1 also induces S- oxidation of polyunsaturated fatty acids to leukotrienes or
phase cell cycle arrest, implying the inhibition of DNA synthesis prostaglandins.37
or S-phase specific DNA damage.52 Given its low reactivity However, reports on TrxR inhibition by silver phosphine
towards ct-DNA, complex 1 may interfere with important compounds are still scarce in literature. According to Santini et
enzymes in DNA replication, such as Topoisomerase I, a highly al.,69 the cytotoxic activity of [Ag(PTA)4]PF6 (PTA = 1,3,5-triaza-
active enzyme during S-phase.53 Another important difference 7-phosphaadamantane) against tumour cells has been
between complex 1 and cisplatin can be noticed in their effects proposed to be the result of its ability to inhibit cytosolic TrxR
on mitochondrial membrane potential of MDA-MB-231 cells. (IC50 value of 10.3 nM). Recently, Landini et al. have reported
Compound 1 induces a significant mitochondrial membrane the cytotoxicity and TrxR inhibition studies on complexes of the
depolarization in a concentration-dependent manner whilst type [MX(PEt3)] and [M(PEt3)2]+ (M = Ag, Au; X = halides; PEt3=
cisplatin does not cause any effect under similar experimental triethylphosphine).70 Although silver complexes have elicited
conditions. These results suggest that complex 1 induces the the TrxR inhibition at IC50 values ranging from 11-49 nM, they
apoptosis of MDA-MB-231 cells triggered by mitochondrial- were less potent than their corresponding gold analogues.
dependent pathway.54
According to the authors, the moderate effect induced by silver
The ability of metal compounds to target mitochondria and compounds in the TrxR activity in treated cells compared to that
induce the intrinsic (mitochondrial-dependent) apoptotic of gold species suggests that additional mechanism of cell
pathway has attracted considerable attention in the last few damage may be involved.
decades.55–59 Much of this work has been focused on the role of
Based on the results of the studies discussed above, we
gold compounds as “antimitochondrial agents”.59–62 Several suggest that mitochondria may be one of the cellular targets
mechanistic studies have demonstrated that the cytotoxic responsible for the cytotoxic effects of 1 in the MDA-MB-231
action of gold(I/III) compounds is linked with the inhibition of cells rather than DNA. However, further studies are required to
Thioredoxin reductases (TrxRs), a family of selenoproteins that evidence if these silver species or their products of biological
take part in redox control of the mitochondrial permeability reactions are able to reach nucleus so that DNA binding may be
transition.60,63–66 According to Bindoli et al.,62 inhibition of significant to the cytotoxicity. At this point, the mechanisms of
mitochondrial and cytosolic TrxRs leads to an enhancement of action associated with its mitochondrial pathway remain to be
H2O2 levels inside mitochondria and changes the permeability elucidated. Although we cannot confirm that 1 induces ROS
condition of the mitochondrial membranes, eventually leading production, its interaction with Thioredoxin reductases and
to cell apoptosis.62
other proteins cannot be discarded.
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View Article Online
DOI: 10.1039/D0DT01134G
Fig. 4 A) Complex 1 induce apoptosis in TNBC cells. MDA-MB-231 cells were exposed to 1 and analyzed by flow cytometry. B) Percentage of apoptotic cells
compared to the control cells. The percentage of cells in apoptosis was calculated as the sum of the cells in recent and late apoptosis. Camptothecin (Campto,
20 M) was used as positive control. A representative dot plot for each condition is shown. Data are the mean of experiments in triplicate (*p ≤ 0.05, **p ≤
0.01 and ***p ≤ 0.001).
Fig. 5 A) Effects of complex 1 on mitochondrial membrane potential (m) of MDA-MB-231 cells. Cells with normal mitochondrial membranes are
exhibited in red, whereas cells with loss of m are depicted in green. TNBC cells were incubated with 1 (6, 12 and 24 M) or 50 M of cisplatin
8 | J. Name., 2012, 00, 1-3
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(Cisp) for 4 h, and stained with JC-1. B) Percentage of cells emitting green fluorescence. A representative dot plot for each conditionViisewshAortwiclne.ODnalintae
are the mean of experiments in triplicate (*p ≤ 0.001 and **p ≤ 0.0001).
DOI: 10.1039/D0DT01134G
but the interaction with enzymatic targets, such as Thioredoxin
reductases, cannot be ruled out. These findings demonstrated
that silver compounds could be potentially useful for the
development of new metal-based drugs to treat triple negative
breast cancer proliferation.
Experimental section
Chemistry
Silver chloride and organic reagents were purchased from
Sigma. Literature procedures were followed for the synthesis of
3-methoxy-4-hydroxybenzaldehyde
thiosemicarbazone
(VTSC)71 and the functionalized aldehydes (3-methoxy-4-[2-
(piperidine-1-yl)ethoxy)benzaldehyde (VP) and 3-methoxy-4-[2-
(morpholine-1-yl)ethoxy)benzaldehyde (VM).24 The silver(I)
complexes were synthetized under protection of light.
Elemental analyses data were obtained with a Perkin Elmer
2400 series II. IR spectra were recorded on a Perkin Elmer
Spectrum 200 spectrometer in the range of 4000–400 cm-1
using KBr pellets. The 1H and DEPTQ NMR spectra were
recorded on a 14.1 T Bruker Avance III HD spectrometer at 298
K.
Fig. 6 Effects of complex 1 on intracellular ROS formation in MDA-MB-
231 cells. TNBC cells were incubated with 1 (6 and 12 M) or 50 M of
cisplatin (Cisp) for 24 h, and stained with H2DCFDA.
Crystal structure determination
Conclusions
Single crystal X-ray diffraction data for both complexes were
collected at room temperature (293 K) on a Rigaku XTALAB-
MINI diffractometer using MoKα radiation (0.71073 Å)
monochromated by graphite. The cell determination and the
final cell parameters were obtained on all reflections using the
software CrysAlisPro.72 Data integration and scaled was carried
out using the software CrysAlisPro. The structures were solved
by direct method using SHELXT73 and refined using full-matrix
least-squares method within SHELXL software,74 included in
Olex2.75 In all cases, non-hydrogen atoms were clearly resolved
in the Fourier maps and anisotropically refined. Hydrogen
atoms were stereochemically positioned and refined using the
riding model.74 The crystallographic data are summarized in
Table 1. The crystallographic illustrations were prepared
MERCURY program.76 The CIF file of structures 2 and 3 were
deposited in the Cambridge Structural Data Base under the
code CCDC 1992316 and 1992321, respectively. Copies of the
data can be obtained, free of charge, via www.ccdc.cam.ac.uk.
In this work, we have synthesized new tetrahedral silver(I)
compounds of the type [AgCl(PPh3)2(L)] {PPh3
=
triphenylphosphine; L= VTSC 3-methoxy-4-
=
hydroxybenzaldehyde thiosemicarbazone (1); VMTSC = 3-
methoxy-4-[2-(morpholine-1-yl)ethoxy)benzaldehyde
thiosemicarbazones (2); VPTSC = 3-methoxy-4-[2-(piperidine-1-
yl)ethoxy)benzaldehyde thiosemicarbazone (3)} and evaluated
their cytotoxic and apoptotic effects. The molecular structures
of 2 and 3 have been confirmed by single crystal X-ray
diffraction studies. The new vanillin thiosemicarbazones VMTSC
and VPTSC were inactive against human tumor breast (MDA-
MB-231 and MCF-7) and lung (A549) cells. The new silver
compounds are more potent than cisplatin against all tested
tumour cells. In general, silver complexes 1-3 showed
comparable cytotoxicity, indicating that the incorporation of O-
alkylated vanillin thiosemicarbazones did not result in an
increased cytotoxicity. After 3 hours of incubation, ca. 30% of 1-
3 were internalized in MDA-MB-231 cells, which suggest that a
significant amount of the complexes reach the cell's interior.
Compound 1 induced apoptosis and mitochondrial membrane
depolarization. DNA binding studies confirm that 1 can interact
with DNA (and will do so via a mechanism different to that of
cisplatin) though it does so weakly which implies that other
targets in the cell may be equally or more important. Although
the exact pharmacological targets associated with the
cytotoxicity of 1 remain unclear, our evidences indicated
mitochondria as a potential intracellular targets for these silver
compounds rather than DNA. The mechanisms of action
involved with its antimitochondrial activity are still unknown
Synthesis
Thiosemicarbazones
3-methoxy-4-hydroxybenzaldehyde thiosemicarbazone (VTSC)
Yield 81%. IR (KBr, cm-1): 3170 (NH); 1598 (C=N); 758 (C=S).
1H NMR (DMSO-d6, δ ppm): 11.26 (s, 1H, =N-NH-), 9.49 (s, 1H,
OH), 8.11 and 7.96 (s, 2H, NH2), 7.93 (s, 1H, HC=N), 7.47 (d, J =
1.75 Hz, 1H, H2van), 7.02 (dd, J = 8.20, 1.85 Hz, 1H, H6van), 6.78
(d, J = 8.20 Hz, 1H, H5van), 3.82 (s, 3H, OCH3). DEPTQ NMR
(DMSO-d6, δ ppm): 177.38 (C=S), 148.84 (C4), 148.14 (C3),
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142.97 (HC=N), 125.64 (C1), 122.44 (C6), 115.24 (C5), 109.27 NMR (DMSO-d6, δ ppm): 11.62 (s, 1H, =N-NH-), 9.53 (s, 1H, OH),
View Article Online
(C2), 55.81 (OCH3).
8.48 and 8.19 (s, 2H, NH2), 8.00 (s, 1H, HDCO=NI: 1)0, .710.5309/D(d0,DJT0=1113.47G5
Hz, 1H, H2van), 7.44 (m, 6H, PPh3), 7.34 (m, 24H, PPh3), 7.04 (dd,
J = 8.0, 1.75 Hz, 1H, H6van), 6.79 (d, J = 8.0 Hz, 1H, H5van), 3.83
(s, 3H, OCH3). 13C NMR (DMSO-d6, δ ppm): 175.97 (C=S), 149.06
3-methoxy-4-[2-(4-morpholine-1-yl)ethoxy]-benzaldehyde
thiosemicarbazone (VMTSC)
A round bottom flask was charged with 3-methoxy-4-[2- (C4), 148.15 (C3), 144.15 (HC=N), 133.57 (CH PPh3), 133.20 (Cq
(morpholine-1-yl)ethoxy)benzaldehyde (2.24 mmols), PPh3), 130.13 (CH PPh3), 128.91 (CH PPh3), 125.37 (C1), 122.68
thiosemicarbazide (2.24 mmols) and 20 mL of ethanol aqueous (C6), 115.23 (C5), 109.34 (C2), 55.79 (OCH3).
solution (Vwater :Vethanol = 3:1). After the addition of a catalytic
amount of diluted HCl, the reaction mixture was kept under
The compounds 2 and 3 were prepared similarly to 1.
magnetic stirring at room temperature. After 24 h, the pH of the [AgCl(PPh3)2(VMTSC)] (2)
reaction media was adjusted to 9 by adding K2CO3, affording a Yield 48%. Anal. Calc. for C51H52AgClN4O3P2S (%): C, 60.87; H,
microcrystalline solid. The solid was filtered off, washed 5.21; N, 5.57. Found (%): C, 58.08; H, 5.20; N, 5.38. IR (KBr, cm-
carefully with ice water and cool MeOH, and dried under 1): 1600 (C=N); 1095 (P-CPh); 745  (C=S). 1H NMR (DMSO-d6,
vacuum. Yield: 72 %. Anal. Calc. for C15H28N4O6S (%): C, 45.90; δ ppm): 11.60 (s, 1H, =N-NH-), 8.44 and 8.19 (s, 2H, NH2), 8.01
H, 7.19; N, 14.28. Found (%): C, 45.87; H, 6.88; N, 14.17. IR (KBr, (s, 1H, HC=N), 7.53 (d, J=1.75 Hz, 1H, H2van), 7.45 (m, 6H, PPh3),
cm-1): 3170 (NH); 1600 (C=N); 766 (C=S). 1H NMR (DMSO-d6, 7.36 (m, 24H, PPh3), 7.13 (dd, 8.40, 1.75 Hz, 1H, H6van) 6.99 (d,
δ ppm): 11.31 (s, 1H, =N-NH-), 8.16 and 8.02 (s, 2H, NH2), 7.96 J= 8.40 Hz, 1H, H5van), 4.10 (t, J= 6.0 Hz, 2H, OCH2CH2N), 3.82
(s, 1H, HC=N), 7.51 (d, J=1.85 Hz, 1H, H2van), 7.11 (dd, J= 8.40, (s, 3H, OCH3), 3.57 (t, J= 4.5 Hz, 4H, H1morf), 2.69 (t, J=5.7 Hz,
1.85 Hz, 1H, H6van), 6.99 (d, J=8.40 Hz, 1H, H5van), 4.09 (t, J= 2H, OCH2CH2N), 2.47 (b, 2H, H2morf). 13C NMR (DMSO-d6, δ
6.0 Hz, 2H, OCH2CH2N), 3.81 (s, 3H, OCH3), 3.57 (t, J= 4.5 Hz, 4H, ppm): 176.40 (C=S), 149.95 (C4), 149.30 (C3), 143.50 (HC=N),
H1morf), 2.69 (t, J=5.7 Hz, 2H, OCH2CH2N), 2.48 (b, 4H, H2morf). 133.41 (CH PPh3), 133.28 (Cq PPh3), 130.07 (CH PPh3), 128.84
DEPTQ NMR (DMSO-d6, δ ppm): 177.53 (C=S), 149.80 (C4), (CH PPh3), 126.87 (C1), 122.32 (C6), 112.53 (C5), 108.88 (C2),
149.32 (C3), 142.52 (HC=N), 127.10 (C1), 122.17 (C6), 112.53 66.19 (C1morf), 66.17 (OCH2CH2N), 56.95 (OCH2CH2N), 55.78
(C5), 108.79 (C2), 66.19 (C1morf), 66.13 (OCH2CH2N), 56.98 (OCH3), 53.68 (C2morf).
(OCH2CH2N), 55.80 (OCH3), 53.71 (C2morf).
[AgCl(PPh3)2(VPTSC)] (3)
3-methoxy-4-[2-(4-piperidine-1-yl)ethoxy]-benzaldehyde
thiosemicarbazone (VPTSC)
Yield 53%. Anal. Calc. for C54H57AgClN5O2P2S (%): C, 62.04; H,
5.50; N, 6.70. Found (%): C, 61.92; H, 5.48; N, 6.54. IR (KBr, cm-
The preparation was the same as that of VMTSC, except that 3- 1): 1600 (C=N); 1095 (P-CPh); 745  (C=S).1H NMR (DMSO-d6,
methoxy-4-[2-(piperidine-1-yl)ethoxy)benzaldehyde (VP) was δ ppm): 11.73 (s, 1H, =N-NH-), 8.54 and 8.26 (s, 2H, NH2), 8.03
used. Yield: 83 %. Anal. Calc. for C16H30N4O5S (%): C, 49.21; H, (s, 1H, HC=N), 7.53 (d, J= 1.85 Hz, 1H, H2van), 7.43 (m, 6H, PPh3),
7.74; N,14.35. Found (%): C, 49.05; H, 7.77; N, 14.14. IR (KBr, cm- 7.33 (m, 24H, PPh3), 7.13 (dd, J= 1.85, 8.35 Hz, 1H, H6van), 7.00
1
1): 3170 (NH); 1600 (C=N); 780 (C=S). H NMR (DMSO-d6, δ (d, J= 8.40 Hz, 1H, H5van), 4.08(t, J= 6 Hz, 2H, OCH2CH2N), 3.82
ppm): 11.31 (s, 1H, =N-NH-), 8.15 and 8.02 (s, 2H, NH2), 7.95 (s, (s, 3H, OCH3), 2.66(t, J= 6 Hz, 2H, OCH2CH2N), 2.44 (b, 4H,
1H, HC=N), 7.51 (d, J= 1.85 Hz, 1H, H2van), 7.11 (dd, J= 1.85, H1pip), 1.49 (q, J= 6 Hz, 4H, H2pip), 1.37 (b, 2H, H3pip). 13C NMR
8.40 Hz, 1H, H6van), 6.98 (d, J= 8.40 Hz, 1H, H5van), 4.07 (t, J= (DMSO-d6, δ ppm): 176.03 (C=S), 150.03 (C4), 149.28 (C3),
6 Hz, 2H, OCH2CH2N), 3.81 (s, 3H, OCH3), 2.65 (t, J= 6 Hz, 2H, 143.79 (HC=N), 133.44 (CH PPh3), 133.16 (Cq PPh3), 130.10 (CH
OCH2CH2N), 2.43 (b, 4H, H1pip), 1.48 (q, J= 6 Hz, 4H, H2pip), 1.36 PPh3), 128.81 (CH PPh3), 126.76 (C1), 122.42 (C6), 112.43 (C5),
(b, 2H, H3pip). DEPTQ NMR (DMSO-d6, δ ppm): 177.51 (C=S), 108.82 (C2), 66.30 (OCH2CH2N), 57.31 (OCH2CH2N), 55.77
149.85 (C4), 149.28 (C3), 142.53 (HC=N), 127.01 (C1), 122.18 (OCH3), 54.46 (C1pip), 25.56 (C2pip), 23.92 (C3pip).
(C6), 112.47 (C5), 108.77 (C2), 66.31 (OCH2CH2N), 57.32
(OCH2CH2N), 55.78 (OCH3), 54.48 (C1pip), 25.57 (C2pip), 23.93 Stability studies
(C3pip).
1
The H NMR spectra of compounds 1-3 in DMSO-d6/D2O (80:
20% v/v) were acquired on a 14.1 T Bruker Avance III HD
spectrometer at 0, 24 and 48 h after preparation of samples
Silver(I) compounds
(298 K).
[AgCl(PPh3)2(VTSC)] (1)
3-methoxy-4-hydroxybenzaldehyde thiosemicarbazone (80 mg,
The UV-VIS spectra of compound 1-3 (2x10-5 mol L-1) prepared
in Tris-HCl buffer (5 mM Tris-HCl and 50 mM NaCl, pH 7.4) with
0.355 mmol) was added to a suspension of AgCl (50.89 mg,
2% DMSO were recorded at 0, 24 and 48 h after preparation of
0.355 mmol) in 10 mL of acetonitrile and stirring for 24 hours at
solutions.
50 °C. After this period, triphenylphosphine (186.28 mg, 0.71
mmol) was added and stirred for 30 minutes leading to a clear
solution. The solution was kept to crystallization at room
Cell culture. The human lung tumor cell line A549 was obtained
from ATCC, and human breast tumor cell lines MCF-7 and MDA-
temperature. Yield: 40%. Anal. Calc. for C45H41AgClN3O2P2S
MB-231 were from Rio de Janeiro Cell Bank (BCRJ). The cells
(%): C, 60.51; H, 4.63; N, 4.70. Found (%): C, 59.94; H, 4.77; N,
were maintained at 37 °C in 5% CO2 in Dulbecco’s Modified
4.69. IR (KBr, cm-1): 1598 (C=N); 1095 (P-CPh); 745  (C=S). 1H
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Eagle´s medium (DMEM), supplemented with 10% of FBS, apoptosis. The MDA-MB-231 cells (0.4x105 cells pVeirewwAertlicl)lewOnelirnee
gentamicin sulfate (50 mg L-1) and amphotericin B (2 mg L-1). The seeded in 24-well plates and maintainedDaOtI3: 170.°1C03in9/D50%DTC0O1213f4oGr
non-tumor cells MCF-10A (breast) were obtained from Olivia 24 h and then, treated with 6 and 12 M of 1 for 48 h. The cells
Newton-John Institute Cancer Research Institute, maintained at were washed with PBS and incubated with 2.5 μL de 7AAD and
37 °C in 5% CO2 in Dulbecco's Modified Eagle´s Medium and 2.5 μL PE-Annexin-V (BD Biosciences) for 15 min. Next, the cells
Nutrient Mixture F-12 (DMEM/ F12), supplemented with 5% were collected with a scraper and analyzed in an Accuri C6 flow
horse serum, EGF (0.02 mg mL-1), hydrocortisone (0.05 mg mL- cytometer (BD Biosciences) using the CSampler software (BD
1), insulin (0.01 mg mL-1), L- glutamine (2 mM), penicillin (100 UI Biosciences).
mL-1) and streptomycin (100 mg mL-1).
Cell cycle assay. MDA-MB-231 cells (1x105 cells per well) were
Cell viability. The cytotoxic effects of compounds were seeded in 12-well plates and incubated at 37 °C in 5% CO2 for 24
determined
by
MTT
[3-(4,5-dimethylthiazol-2-yl)-2,5- h. After incubation, the cells were treated with 6 M of 1 for 48
diphenyltetrazolium bromide] assay. The cells (1.5x104 cells/ h. Then, the cells were harvested and fixed with 70% ethanol at
100L) were seeded in 96-well plates and incubated at 37 °C in -20 °C for 24 h, washed with PBS and incubated with a solution
5% CO2 for 24 h. Then, the cells were treated with different containing 0.02 g mL-1 RNase A (Sigma) and 10 g mL-1 PI
concentrations of compounds for 48 h. The silver compounds (Sigma) at 37 °C for 30 min. Next, the cells were analyzed in an
and ligands were solubilized in DMSO, whereas cisplatin was Accuri C6 flow cytometer (BD Biosciences) using the CSampler
prepared in DMF. The control cells were incubated with 0.5% of software (BD Biosciences).
DMSO (vehicle). After treatment, the medium was removed and
the cells were incubated with MTT (1 mg mL-1) for 4 h. Next, the The mitochondrial membrane potential (m) assay. Flow
formazan crystals were solubilized in isopropanol and cytometry assays using the BD MitoScreen kit (BD Biosciences)
absorbance measured on a micro plate reader (BioTek, Epoch) were performed to investigate the effects of compound 1 in the
at 570 nm. The IC50 values were calculated using the Hill’s mitochondrial m. MDA-MB-231 cells (1x105 cells per well)
equation in the Graph Pad Prism software 6.0.
were seeded in 12-well plates, incubated at 37 °C in 5% CO2 for
24 h and then, treated with 6, 12 and 24 M of 1 for 4 h. The
Cellular uptake. MDA-MB-231 cells (2x105 cells per well) were cells were harvested and incubated with 10 g mL-1 of JC-1
seeded in 12-well plates and incubated at 37 °C in 5% CO2. After (5,5′,6,6′-Tetrachloro-1,1′,3,3′-tetraethyl-imidacarbocyanine
24 h, the cells were treated with 12 M of compounds 1-3 for 3 iodide) at 37 °C for 15 min. Next, the cells were washed with
h. Then, the medium was removed and cells were washed with PBS and analyzed in an Accuri C6 flow cytometer (BD
PBS (phosphate buffered saline), trypsinized and the cell Biosciences) using CSampler software (BD Biosciences).
suspension counted. The total concentration of Ag was
determined using an Analytik Jena ContrAA 700 high-resolution
continuum source graphite furnace atomic absorption
ROS assay. MDA-MB-231 cells (0.4x105 cells per well) were
seeded in 24-well plates, incubated at 37 °C in 5% CO2 for 24 h
spectrometer. Analytical working solution containing 50 g L-1
and then, exposed to 6 and 12 M of 1 for 24 h. The cells were
Ag was prepared daily by dilution of the 1000 mg L-1 stock
washed with PBS and incubated with 10 M of H2DCFDA (2',7'-
standard solution (SpecSol®, QUIMLAB, Brazil) and acidified to
dichlorodihydrofluorescein diacetate, Invitrogen) at 37 °C for 20
1.0% (v/v) HNO3 solution. The blank solution was 1.0 % (v/v)
min. Then, the cells were analyzed using a confocal fluorescence
HNO3. External calibration was adopted. The calibration curve
microscope (Carl Zeiss LSM 800).
was performed by addition of appropriate aliquots of analytical
working solution into the atomizer in order to obtain 50, 100,
150, 250, 350, 500 and 750 pg of Ag. The samples consisting in
cell suspension with internalized compounds 1-3 were diluted
DNA binding assays
All experiments were carried out from a fresh solution of ct-DNA
(Sigma) prepared in Tris-HCl buffer (5 mM Tris-HCl and 50 mM
in deionized water and homogenized on a vortex mixer and
NaCl, pH 7.4). The compound 1 solution was initially prepared
aliquots of 5 L analyzed. Each sample was read in triplicate. To
in DMSO and then diluted to different concentrations in Tris-HCl
consider the matrix effects, the concentration of Ag was also
buffer, with the DMSO concentration kept below 3%.
determined in samples with initial concentration of 1-3 (12 M).
The CD spectra were recorded on a spectropolarimeter JASCO
The results are expressed as percentage of Ag uptake (See ESI).
J-815 in a continuous mode scanning (100 nm min-1) with
accumulations of 4 scans, from 235 to 300 nm. The spectra were
obtained for free ct-DNA (50 µM) and in the presence of
Morphology assay. The MDA-MB-231 cells (0.4x105 cells per
well) were seeded in 24-well plates, incubated at 37 °C in 5%
different concentrations of 1 (from 2.5 to 15 M). The samples
CO2 for 24 h and then, exposed to 6, 12 and 24 M of compound
were incubated for 24 h at 37 °C.
1 for 48 h. Cell morphology was examined under an inverted
microscope (LABOMED TCM 400) and the images were
The competitive binding studies were performed in a JASCO J-
815 equipped with fluorescence detector. The DNA and
recorded using a camera (LABOMED iVu5100).
Hoechst 33258 concentrations were kept fixed at 60 µM and 6
µM, respectively, while the concentration of the compound 1
range from 0 to 60 µM. The samples were maintained in rest for
Apoptosis assay. The Annexin V/ 7-AAD double staining assay
was performed to analyze the effects of compound 1 on
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J. Name., 2013, 00, 1-3 | 11
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Journal Name
5 min before the measurements and then, the spectra were 15
obtained from 360 to 600 nm with excitation wavelength at 350
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The authors declare that they have no conflict of interest.
Acknowledgements
The authors are grateful for the financial support provided by
FAPESP (proc. 2013/26203-5, 2015/12098 and 2016/177115),
CNPq (proc. 422105/2016-3, 407676/2016-3 and 305190/2017-
2) and CAPES.
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