A Fluorescent Silver(I) Carbene Complex with Anticancer Properties: Synthesis, Characterization, and Biological Studies

Article abstract of DOI:10.1002/cmdc.201800672

The silver(I) N-heterocyclic carbene (NHC) complex bis(1-(anthracen-9-ylmethyl)-3-ethylimidazol-2-ylidene) silver chloride ([Ag(EIA)₂]Cl), bearing two anthracenyl fluorescent probes, has been synthesized and characterized. [Ag(EIA)₂]Cl is stable in organic solvents and under physiological conditions, and shows potent cytotoxic effects in vitro toward human SH-SY5Y neuroblastoma cells. The interactions of [Ag(EIA)₂]Cl with a few model biological targets have been studied as well as its ability to be internalized in cells. The in vitro anticancer activity is apparently related to the level of drug internalization. Notably, [Ag(EIA)₂]Cl does not react with a few model proteins, but is capable of binding the C-terminal dodecapeptide of thioredoxin reductase hTrxR(488–499) and to strongly inhibit the activity of this enzyme. Binding occurs through an unconventional process leading to covalent binding of one or two carbene ligands to the C-terminal dodecapeptide with concomitant release of the silver cation. To the best of our knowledge, this mode of interaction is reported here for the first time for Ag(NHC)₂ complexes.

Full text of DOI:10.1002/cmdc.201800672

Accepted Article
Title: A Fluorescent Silver(I) Carbene Complex with Anticancer
Properties: Synthesis, Characterization and Biological Studies
Authors: Maria Giulia Fabbrini, Damiano Cirri, Alessandro Pratesi,
Lorenzo Ciofi, Tiziano Marzo, Annalisa Guerri, Silvia Nistri,
Alfonso Dell’Accio, Tania Gamberi, Mirko Severi, Andrea
Bencini, and Luigi Messori
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A Fluorescent Silver(I) Carbene Complex with Anticancer
Properties: Synthesis, Characterization and Biological Studies
Maria Giulia Fabbrini, #a Damiano Cirri, #b Alessandro Pratesi, b Lorenzo Ciofi, b Tiziano Marzo, c, b
*
Annalisa Guerri, a Silvia Nistri, d Alfonso Dell’Accio, d Tania Gamberi, e Mirko Severi, a Andrea Bencini a
and Luigi Messori b*
Abstract: The silver(I) NHC (N-heterocyclic carbene) complex bis(1-
(anthracen-9-ylmethyl)-3-ethylimidazol-2-ylidene) silver chloride,
([Ag(EIA)2]Cl) hereafter, bearing two anthracenyl fluorescent probes
has been synthesized and characterised. [Ag(EIA)2]Cl is stable in
organic solvents and under physiological-like conditions, and shows
potent cytotoxic effects in vitro toward human SH-SY5Y
neuroblastoma cells. The interactions of [Ag(EIA)2]Cl with a few model
biological targets have been studied here as well as its ability to be
internalized in cells. The in vitro anticancer activity is apparently
related to the level of drug internalisation. Notably, [Ag(EIA)2]Cl does
not react with a few model proteins, but is capable to bind the C-
terminal dodecapeptide of thioredoxin reductase hTrxR(488-499).
Binding occurs through an unconventional process leading to covalent
binding of one or two carbene ligands to the C-terminal dodecapeptide
with concomitant release of the silver cation. This mode of interaction,
to the best of our knowledge, is reported here for the first time for
Ag(NHC)2 complexes; consequently, a potent enzyme inhibition is
measured.
problems associated to the use of Pt drugs still remain unsolved.3
A family of metal-based compounds with promising features is
that of carbene complexes. Metal-N-heterocyclic carbenes (NHC)
have been extensively investigated in recent years for various
applications, including medical and pharmacological ones.
Particularly attractive for biomedical purpose appears the high
stability of these complexes being often greater than for the
corresponding metal complexes bearing phosphane ligands.4
This is one of the reasons why the synthesis and characterisation
of the carbene complexes of several transition metals has gained
so much attention. Many gold, copper, platinum, palladium,
iridium, ruthenium and silver carbenes have now been tested as
experimental drugs with promising results.5
Beyond the above motivations, the interest in anticancer
applications of carbene complexes is also driven by the
observation that gold or silver carbene compounds seem to have
non-genomic targets for their pharmacological activity. In this
frame, many research groups worldwide, reported on the ability of
gold-based carbenes to bind thioredoxin reductase, an important
enzyme involved in the redox homeostasis of cells; apparently
metal binding occurs at the level of the redox-active C terminal
motif bearing a selenocysteine.6
Introduction
In 2013, Rigobello and co-workers, published a paper on the
synthesis, characterisation and anticancer properties of two novel
silver- or gold-based carbene complexes bearing a 1-(anthracen-
Since cisplatin was first approved by Food and Drug
Administration (FDA) for cancer treatment in 1978, the
development of new and more effective metal-based anticancer
drugs has represented a primary challenge for scientists.1 Despite
the great efforts made in the last decades, only two other platinum
based drugs (i.e. carboplatin and oxaliplatin) and one As-based
drug (i.e. As2O3, trisenox)2 have received approval worldwide as
anticancer agents. In addition, even considering the important
improvements in the administration protocols, some of the major
9-ylmethyl)-3-methylimidazolium
ligand
with
fluorescent
properties. These compounds manifested pronounced in vitro
anticancer properties being also strong inhibitors of TrxR.
Remarkably, the silver complex was more efficient than its gold
counterpart in contrasting cancer cell proliferation as well as in
inhibiting thioredoxin reductase activity.7 We were intrigued in
evaluating whether small structural modifications of 1-(anthracen-
9-ylmethyl)-3-methylimidazol-2-ylidene silver chloride complex,
([AgCl(MIA)] hereafter), for instance the replacement of the
methyl substituent of imidazole with an ethyl one, might
appreciably affect the chemical and biological profiles of the
resulting compound. We soon realized that upon ligand
modification, when the product is slowly crystallized from the
reaction mixture, the structure of the silver center resulted to be
different. Indeed, at variance with the parent complex bearing the
methyl substituent in position 3 of imidazole, a structure of the
type [Ag(NHC)2]X, was the only one afforded both in the solid
state and in solution. Thus, we report here on the synthesis,
chemical and biological features of this fluorescent complex
characterized by the ability to inhibit thioredoxin reductase
apparently through direct binding to the active site redox motif.
Remarkably, this latter process has been conveniently
investigated by high resolution ESI-MS using the C-terminal
dodecapeptide of thioredoxin reductase hTrxR(488-499) bearing
the active site redox motif, with a method already established in
our laboratory.6a,b
[a]
[b]
M. G. Fabbrini, Dr. A. Guerri, Dr. M. Severi, Prof. A. Bencini
Department of Chemistry “U. Schiff”, University of Florence, Via
della Lastruccia 3, 50019 Sesto Fiorentino, Italy.
D. Cirri, Dr. A. Pratesi, Dr. L. Ciofi, Dr. T. Marzo, Prof. L Messori
Laboratory of Metals in Medicine (MetMed), Department of
Chemistry “U. Schiff”, University of Florence, Via della Lastruccia 3,
50019 Sesto Fiorentino, Italy. E-mail: luigi.messori@unifi.it
Dr. T. Marzo
Department of Pharmacy, University of Pisa. Via Bonanno Pisano 6,
56126, Pisa, Italy. E-mail: tiziano.marzo@unipi.it
Dr. S. Nistri, A. Dell’Accio
Department of Experimental and Clinical Medicine, Largo Brambilla
3, 50134 Florence, Italy.
[c]
[d]
[e]
Dr. M. Severi
Experimental and Clinical Biomedical Sciences “Mario Serio”,
University of Florence, Viale GB Morgagni 50, 50134, Florence,
Italy.
# These two authors equally contributed.
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Results and Discussion
Synthesis and characterisation of [Ag(EIA)2]Cl
We have first prepared the study complex according to the
synthetic procedure reported in the experimental section. The
complex was designed by modifying the ligand reported by
Rigobello and co-workers in their work (see figure 1).
Figure 2 X-ray structure of complex [Ag(EIA)2]Cl: C-Ag bond length 2.093(4) Å
and c-Ag-C’ angle 167.7° (C’ is reported by the symmetry operation -x+1,+y,-
z-1/2).
Absorption and emission spectra in the UV-Vis
To test the complex stability with respect to dissociation in solution,
we recorded UV-vis absorption spectra of [Ag(EIA)2]Cl in common
organic solvents (DMSO, DMF) as well as in buffered aqueous
solutions at physiological or slightly acidic buffered pH.
[Ag(EIA)2]Cl displays the typical structured absorption band of the
anthracene chromophore, with a maximum at 370 nm (figure 6).
This band does not show any change within 24 hours, testifying
the complex stability in an aqueous medium (Figure 3) and in
DMSO or DMF solutions (see supporting material).
Figure 1 Structures of the ligand previously reported (left)7 and of the modified
ligand used in this work (right, (5) in Scheme 1).
After the synthesis of the modified ligand (see supporting material
for NMR spectra and X-ray structure), we intended to prepare its
1:1 silver complex, i.e. [AgCl(EIA)]. In contrast to expectations,
through slow crystallisation of the product, as described in the
experimental part, we obtained selectively and in good yield a
product which turned out to correspond to the bifunctional silver
complex [Ag(EIA)2]Cl. Its NMR and ESI-MS spectra are reported
in the supporting material.
Crystal structure
The crystal structures of the free ligand (5) and of [Ag(EIA)2]Cl
show that the ligand in both molecules retains the same
conformation with respect to the mean planes containing the
anthracene ring and the ethylimidazole moiety (88.55 in (5)
compared to 82.25 in [Ag(EIA)2]Cl). On the contrary, the torsion
angle of the ethyl pendant varies from -44.15° in 5 to 77.17 in
[Ag(EIA)2]Cl. The solid-state crystal structure of [Ag(EIA)2]Cl
confirms the bifunctional structure already described in solution
(Figure 2 and supporting information for further data).
These results point out that, at variance with [Ag(MIA)]Cl, the
small modification made at the level of position 3 of imidazole,
strongly affected the nature of the resulting species leading to a
final product characterized by two carbene ligands bound to the
metal center both in the solid state and in solution.
Figure 3 Time course spectra of [Ag(EIA)2]Cl (10-5 M, phosphate buffer 50 mM
pH 7.4, 1% DMSO) for 24 h; see supporting material for UV-Vis spectrum in
pure DMSO and DMF.
At slightly acidic pH (pH=4.5, acetate buffer), a generalised
raising of the baseline was detected probably originating from a
slight precipitation of the complex. However, such precipitation
does not affect the shape of the absorption bands that remain
substantially unaltered supporting a rather high stability even
under these conditions (see supporting material). Next, we
performed fluorescence experiments. Spectra were recorded
using different excitation wavelengths ranging from 335 to 405 nm
(see supporting information). In figure 4 the emission spectrum
obtained for an excitation wavelength of 350 nm is reported. The
spectrum shows three main emission bands falling at 395, 420,
440 nm. Also, it is interesting to note that the presence of the silver
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atom does not affect significantly the spectrophotometric
properties of the ligand being these substantially unaltered when
compared to those of the free ligand (supporting material for
spectral details).
Complex
µg of metal/ µg of proteins
[Ag(EIA)2]Cl
cisplatin
1.4x10-3±0.2x10-3 (a)
1.6x10-4 ± 0.3x10-4 (b)
2.1x10-4 ± 0.7x10-4 (b)
Ag(MIA)Cl
Table 2. Metals level (per µg of proteins) measured after 30 minutes of exposure
of SH-SY5Y cells to 10 µM of [Ag(EIA)2]Cl, cisplatin and [AgCl(MIA)]. Values
with the same letter are not statistically different at 5 % significance level
according to the Tukey test.
It is observed that [Ag(EIA)2]Cl shows a far higher ability to enter
cells being its uptake about ten-fold greater than cisplatin and
about six-fold greater than [AgCl(MIA)]. The increased cell
internalization of [Ag(EIA)2]Cl, is probably correlated to its higher
lipophilicity. In turn, the greater cell internalization might account
for its larger cytotoxicity.
Figure 4 Emission spectra of [Ag(EIA)2]Cl (10-8 M in 50 mM phosphate buffer
pH 7.4 in presence of 0.001% DMSO) λexc=350 nm.
Thioredoxin reductase inhibition by [Ag(EIA)2]Cl
Being the cellular redox system and, more specifically,
thioredoxin reductase one of the likely targets for the cytotoxic
effects of gold and silver carbene complexes,6,7,9 we decided to
determine the inhibitory potency of [Ag(EIA)2]Cl toward this
enzyme (table 3).
Cytotoxic effects and cellular uptake
Next, the in vitro cytotoxic effects of [Ag(EIA)2]Cl toward human
SH-SY5Y neuroblastoma cells were measured in comparison
with cisplatin (the clinical established drug in the treatment of
neuroblastoma) and [AgCl(MIA)]. Results highlighted that
[Ag(EIA)2]Cl possesses cytotoxic properties greater than both
compounds. In particular, an IC50 value of 1.059 ± 0.042 µM was
determined being this value about 10 and 2-fold lower than
cisplatin and Ag(MIA)Cl respectively (table 1).
Complex
IC50 (µM)
[Ag(EIA)2]Cl
0.493±0.04
Table 3 Thioredoxin Reductase Activity Assay. IC50 value (µM) was determined
treating 1 U/L of TrxR with [Ag(EIA)2]Cl (from 0.1 nM to 1 mM). Result is reported
as average value for three independent experiments ± SD.
Complex
IC50
[Ag(EIA)2]Cl
cisplatin
1.059 ± 0.042
9.987 ± 0.506
2.093 ± 0.704
The experiments were done according to the procedure detailed
in materials and methods.10 The obtained IC50 value falling in the
submicromolar range points out that [Ag(EIA)2]Cl produces a
strong inhibition of TrxR, in nice agreement with its cytotoxic
effects toward the SH-SY5Y cancer cell line.
[AgCl(MIA)]
Table 1. IC50 Values (µM) in SH-SY5Y cells, determined for [Ag(EIA)2]Cl,
cisplatin and Ag(MIA)Cl. Result is reported as average value for three
independent experiments ± SD.
MS studies of the interaction of [Ag(EIA)2]Cl with the C-
terminal dodecapeptide of hTrxR
Afterward, to disclose the molecular basis for TrxR inhibition,
[Ag(EIA)2]Cl was incubated with the C-terminal dodecapeptide of
hTrxR typically used as a model for the interaction of metal
compounds with this enzyme (figure 5).6
Indeed, this latter dodecapeptide, with aminoacidic sequence Ac-
SGGDILQSGCUG-NH2, corresponds to the tryptic C-terminal
fragment (488-499) of hTrxR. In the peptide sequence the N-
terminal aminoacid has been acetylated and the C-terminal has
been amidated to avoid undesired interactions with the metal
compound. In figure 5 the mass spectrum of this peptide is
displayed showing an intense peak at m/z 1183.39 Da. In the inset
a zoom is reported with the detail of the characteristic isotopic
Uptake experiments were subsequently performed to assess
whether the different log P values of these compounds (that may
be tentatively associated to a different ability to permeate cell
membrane) do affect importantly cellular accumulation. Indeed,
the three compounds show rather different log P values being -
2.4 for cisplatin8 and 0.90 and 0.50 for [Ag(EIA)2]Cl and
[AgCl(MIA)] respectively. Results of the uptake experiments are
reported in table 2.
pattern due to the presence of
selenocysteine residue.
a
selenium-containing
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100%
80%
60%
40%
20%
0%
100%
1205.3792
1225.3611
100%
80%
60%
40%
20%
0%
1183.3950 100%
1184.3953
1198.6403
1469.5459
1205.3744
1244.3079
1755.6933
1321.6092
1400
Mass/Charge, Da
1600
1400
1600
Figure 6 ESI-MS spectrum of the C-terminal dodecapeptide hTrxR (Ac-
SGGDILQSGCUG-NH2) dissolved in MilliQ water (10-4 M) in presence of 5.0 eq.
of dithiothreitol (DTT) and 1.0 eq. of [Ag(EIA)2]Cl incubated for 2 h at 37 °C. The
inset shows the isotopic pattern for the bis-adduct.
Mass/Charge, Da
Figure 5 ESI-MS spectrum of the C-terminal dodecapeptide hTrxR (Ac-
SGGDILQSGCUG-NH2) dissolved in MilliQ water (10-4 M). The inset shows the
characteristic selenium isotopic pattern.
To confirm this unconventional reactivity, we have carried out the
same ESI-MS experiments using the complex bis(1-(anthracen-
The ESI-MS spectrum recorded for [Ag(EIA)2]Cl in the presence
of the dodecapeptide bearing the redox-active motif of thioredoxin
reductase revealed that the metal compound reacts rapidly with
this peptide. Indeed, after 2 h of incubation at 37 °C, the MS
spectrum (figure 6) clearly shows the presence of two main
signals, one at m/z 1469.54 Da corresponding to the mono-adduct
and one at m/z 1755.69 Da belonging to the bis-adduct where one
or two carbene ligands, deprived of the metal center, are bound
to the peptide. Assignments were consistent with theoretical
simulations of the isotopic pattern reported in the supplementary
information, that were in very good agreement with the
experimental ones depicted in the insets of figure 6. The peak at
m/z 1205.37 Da is assigned to the unreacted peptide associated
with sodium.
9-ylmethyl)-3-propylimidazol-2-ylidene)
silver
bromide,
([Ag(PIA)2]Br) i.e. the same complex with a propyl substituent in
the position 3 of the imidazole. This complex has been
synthesised following the same synthetic route as in the case of
[Ag(EIA)2]Cl and characterised accordingly. Its crystal structure
has been also determined (see supporting material). A reactivity
superimposable to that of [Ag(EIA)2]Cl emerged, indicating that
the uncommon mechanism of binding described in the case of
[Ag(EIA)2]Cl is conserved upon replacement of the ethyl
substituent in the position 3 of the imidazole with the propyl one.
Additionally, the same incubation was carried out also with
[AgCl(MIA)] i.e. the monofunctional silver complex already
reported in reference 7. At variance with the two difunctional silver
complexes i.e. ([Ag(EIA)2]Cl and ([Ag(PIA)2]Br) no adducts were
found.
Next, to gain further insight into the mechanistic aspects involved
in the pharmacological action of ([Ag(EIA)2]Cl, we reacted the
silver complex with three model proteins i.e. cytochrome c,
ribonuclease A and lysozyme and analyzed the incubated
solutions through a well-established protocol relying on high
resolution mass spectrometry.11 Indeed, binding and interaction
with non-genomic targets such as proteins is recognized to play a
key role for the activity of silver and gold carbene complexes.7, 10
Contrary to expectations, no evidence of adduct formation was
detected even for long incubation times (72 h, see supporting
material for spectra).
Fluorescence microscopy
Finally, to evaluate the intracellular distribution of [Ag(EIA)2]Cl,
SH-SY5Y cells were treated with this silver complex for different
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times and then observed under a fluorescence microscope. No
fluorescence signal is detectable in the control cells (Fig. 8A).
Conversely, a weak blue fluorescence signal appears in the
peripheral cytoplasm about 30 min after the treatment with
[Ag(EIA)2]Cl, reaching its maximum intensity at 60 min. By the
observation of the fluorescence pattern, the fluorescent complex
seems to accumulate within rounded bodies compatible with
endocytic vesicles as suggested by their distribution in the sub-
plasmalemmal cytoplasm (figure 7B).
cytotoxicity value, supporting the view of thioredoxin reductase
being one of the likely cellular targets. The reaction of [Ag(EIA)2]Cl
toward the C-terminal dodecapeptide of thioredoxin reductase
hTrxR(488-499) has been investigated by HRMS experiments.
Notably, [Ag(EIA)2]Cl is able to bind tightly the C-terminal
fragment, most probably at the level of the S-Se redox motif,
through an unconventional mechanism -involving a sort of
transmetalation- with concomitant loss of the silver center. This
finding is particularly important and, to the best of our knowledge,
such mode of binding is here reported for the first time. Finally, it
is notable that the presence of the anthracene fluorophore allows
the facile tracking of the complex through fluorescence
microscopy analysis; we found that [Ag(EIA)2]Cl accumulates
within rounded-shaped bodies that are compatible with endocytic
vesicles.
Experimental Section
Figure 7 Representative image of SH-SY5Y cells: controls (A); treated with 10
μM [Ag(EIA)2]Cl for 1 h (B).
Chemistry. Synthesis were carried out through modifications of
12
those reported in literature.7,
The overall synthetic route is
Conclusions
displayed in Scheme 1. Details on the synthesis of products 2-5
are reported in supporting material.
There is today a wide interest in the development of metal
carbene complexes combining cytotoxic and fluorescent
properties. Here, we have reported on the synthesis and chemical
characterisation of a novel monocationic silver(I) NHC (N-
heterocyclic carbene) complex, namely bis(1-(anthracen-9-
ylmethyl)-3-ethylimidazol-2-ylidene) silver chloride, bearing an
anthracenyl fluorescent probe. The complex has been fully
characterised by NMR, ESI mass spectrometry, elemental
analysis and X-ray crystallography. The chemical and biological
characterisation of this compound has revealed a few peculiar
and interesting features.
Quite surprisingly, replacement of the methyl group of [AgCl(MIA)]
with an ethyl substituent at position 3 of the imidazole ring leads
to the formation of a cationic silver complex bearing two carbene
ligands. The selective obtainment of the bis-carbene complex is
attributable to a number of factors including the steric hindrance
of the imidazole substituent, the nature of the counterion and the
reaction environment as already reported by Youngs and
coworkers.12
Scheme 1 Synthetic route for bis(1-(anthracen-9-ylmethyl)-3-
ethylimidazol-2-ylidene) silver chloride.
([Ag(EIA)2]Cl is highly stable in common organic solvents or in
buffered physiological-like solutions and is unreactive toward
small model proteins. These properties make it highly suitable for
biological and cellular studies.
Synthesis of bis(1-(anthracen-9-ylmethyl)-3-ethylimidazol-2-
ylidene) silver chloride ([Ag(EIA)2]Cl). (5) (101 mg, 0.31 mmol)
was stirred with silver(I) oxide (40 mg, 0.17 mmol) in methanol (7
ml) for 24 h. Then, 20 mL of dichloromethane were added to the
mixture and the suspension was filtered over celite in order to
remove both unreacted Ag2O and AgCl formed during reaction.
After concentration in vacuo crystallisation was allowed at -20 °C
by adding hexane. During the next four days the dark-grey
precipitate that slowly appeared was daily filter-off. Finally, the
Remarkably, ([Ag(EIA)2]Clmanifested a cytotoxic potency ten-fold
higher than cisplatin and two-fold higher than [AgCl(MIA)] when
assayed in a reference neuroblastoma cell line (the respective
IC50 values being 1.059, 9.987 and 2.093 µM). The in vitro
pharmacological activity of [Ag(EIA)2]Cl well correlates with its
ability to enter cancer cells that in turn is probably dependent on
its greater lipophilicity compared with [AgCl(MIA)] and cisplatin.
[Ag(EIA)2]Cl, inhibits potently TrxR with an IC50 value of 0.493 µM;
interestingly this value is in nice agreement with the determined
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plate-shaped crystals of complex were filtered and washed with
hexane. Yield: 61%.
complexes were also added in 1:3 metal complex/peptide molar
ratio and the mixture incubated for 2 h at 37 °C.
1H NMR (400.13 MHz, MeOD) δ: 8.59 (s, 2H, Ant H10), 8.18 (d,
4H, Ant H1, J = 8.1 Hz), 8.07 (d, 4H, Ant H4, J = 8.2 Hz), 7.53 -
7.45 (m, 8H, Ant H2,3), 7.31 (s, 2H, Im H4’), 7.23 (s, 2H, Im H5’),
5.91 (s, 4H, AntCH2Im), 3.58 (b, 4H, ImCH2CH3), 1.13 (b, 6H,
ImCH2CH3); 13C NMR (100.61 MHz, MeOD) δ: 183.57 (Im C2’),
133.05 (Ant C4a), 132.17 (Ant C8a), 130.83 (Ant C10), 130.57
(Ant C4), 128.51 (Ant C2), 126.48 (Ant C3), 124.59 (Ant C9),
123.67 (Ant C1), 123.22 (Im C5’), 121.53 (Im C4’), 48.25
(AntCH2Im), 47.82 (ImCH2CH3), 17.07 (ImCH2CH3); ESI-MS: m/z
Interaction between [Ag(EIA)2]Cl and model proteins was carried
out as described in our previous works:11 solutions of cytochrome
c, ribonuclease A and lysozyme (10-4 M) were incubated for 72 h
at 37 °C with [Ag(EIA)2]Cl (3:1 complex to protein ratio) in 20 mM
ammonium acetate buffer pH 6.8 with the presence of 3% of
organic solvent (DMSO or DMF). Aliquots were sampled after 24,
48 and 72 h. ESI-MS spectra were acquired through direct
infusion at 10 μl min-1 flow rate in a TripleTOF® 5600+ mass
spectrometer (Sciex, Framingham, MA, U.S.A.), equipped with a
DuoSpray® interface operating with an ESI probe. The ESI
source parameters were optimized as follows: positive polarity,
Ionspray Voltage Floating 5500 V, Temperature 400 °C, Ion
source Gas 1 (GS1) 40; Ion source Gas 2 (GS2) 30; Curtain Gas
(CUR) 25, Declustering Potential (DP) 100 V, Collision Energy
(CE) 10 V. For acquisition, Analyst TF software 1.7.1 (Sciex) was
used and deconvoluted masses were obtained by using the Bio
Tool Kit micro-application v.2.2 embedded in PeakView™
software v.2.2 (Sciex).
681.20. Elemental analysis of C,
N
and
H
for
C40H36AgClN4·3(CH2Cl2) [calculated C: 53.20%, H: 4.36%, N:
5.77%, experimental: C: 53.22%, H: 4.04%, N: 5.02%].
Spectrophotometric analysis. Solution behavior of [Ag(EIA)2]Cl
was assessed through spectrophotometric studies performed with
a Varian Cary 50 Bio UV-Vis spectrophotometer. For absorption
experiments solutions of complex (10-5 M) were prepared in
dimethylsulfoxide (DMSO), dimethylformamide (DMF) or 50 mM
phosphate buffer (pH=7.4) in presence of 1% of organic solvent.
Emission spectra of complex (10-8 M), in 50 mM phosphate buffer
(pH=7.4) with the presence of 0.001% of DMF, were recorded
with a Perkin Elmer LS55 Luminescence Spectrometer. All the
experiments were carried out at 25 °C.
X-ray crystallography. Single crystal suitable for X-ray
diffraction experiments for (5) were obtained by adding to the
reaction mixture acetonitrile and diethyl ether. This solution was
stored at -20 °C for four days. After this time, yellow needles-
shaped crystals were obtained. For [Ag(EIA)2]Cl, colourless plate-
shaped crystals were straightforwardly obtained as product of the
synthesis as already described above in the paragraph reporting
the synthesis of compound. Similarly, for bis(1-(anthracen-9-
ylmethyl)-3-propylimidazol-2-ylidene) silver chloride [(Ag(PIA)2]Br
hereafter, (see results and discussion section and supporting
material where this complex is labeled as (8)), suitable crystals
were obtained as product of the synthesis. Data collections were
performed on an Oxford Diffraction Xcalibur3 diffractometer at
100 K and with Mo Kα radiation (λ = 0.71073 Å). Data collection
and reduction were performed through the suite CrysAlis.14 The
absorption correction was applied with the program SCALE3
ABSPACK, also integrated in the CrysAlis suite. The structures
were solved by direct methods implemented in SIR9715 and
refined by full-matrix least-squares on F2 using the SHELXL
software16 All the non-hydrogen atoms were refined
anisotropically by full-matrix least-squares methods on F2. All the
H atoms (except those of H2O in (5)) were placed in calculated
positions with isotropic thermal parameters depending on that of
the atom to which they are bound and included in structure factor
calculations in the final stage of full-matrix least-squares
refinement. In the crystal structure of compound [Ag(EIA)2]Cl, the
silver and chlorine atom lie in special position and the occupancy
factor is 0.5 for both atoms. CCDC 1844077-1844078 and
1856791 contain the supplementary crystallographic data.
Log P determination. The octanol/water partition coefficient was
assessed through a modified shake flask method previously used
in our laboratories.8
NMR spectroscopy. NMR spectra were acquired on a Bruker
Avance III 400 console equipped with a Bruker Ultrashield 400
Plus superconducting magnet (resonating frequencies: 400.13
MHz and 100.61 MHz for 1H and 13C respectively) and a 5mm
PABBO BB-1H/D Z-GRD Z108618/0049 probe. All spectra were
recorded at room temperature (25 +/- 2 °C) in solvents with a
deuteration degree of 99.8% and calibrated on solvent residual
signals.13 Chloroform-d was purchased from Sigma-Aldrich, while
DMSO-d6 was purchased from Deutero.de. Signals of compound
(5) were attributed through additional 2D NMR experiments i.e.
COSY, NOESY, HSQC and 13C{1H} DEPT-135 (see supporting
information). Signal of [Ag(EIA)2]Cl were attributed through
comparison with compound (5).
ESI-MS. Interaction between the silver complex and the synthetic
dodecapeptide Ac-SGGDILQSGCUG-NH2, corresponding to the
C-terminal tryptic fragment of hTrxR, was assessed by high
resolution ESI-MS.6a,b
The dodecapeptide was dissolved in LC-MS grade water (final
concentration 10-4 M solution) and 5.0 eq. of dithiothreitol (DTT)
were added to reduce the S-Se bond, then, 1.0 eq. of the silver
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Cell Cultures. Human SH-SY5Y neuroblastoma cells were
cultured in Dulbecco’s modified Eagle’s medium: Ham’s Nutrient
Mixture F12 (1:1) (Sigma Aldrich), supplemented with 10% heat-
inactivated foetal bovine serum (FBS, Invitrogen, Carlsbad, CA,
USA), 2 mM glutamine, 250 U/ml penicillin G and 250 μg/mL
streptomycin (Sigma-Aldrich), in a humidified atmosphere with
5% CO2 at 37 °C.
coverslip and treated with 10 µM [Ag(EIA)2]Cl for 20 min, 30 min,
60 min and 90 min. Control cells, not treated with this silver
complex, were used. At end of treatment, the cells were fixed with
2% paraformaldehyde for 10 min at room temperature and then
observed under an epifluorescence Zeiss Axioskop microscope
(Mannheim, Germany) using 358 nm excitation wavelength, with
a 100X objective. The fluorescence images were captured using
a Leica DFC310 FX 1.4‐megapixel digital camera, equipped with
the Leica software application suite LAS V3.8 (Leica
Microsystems, Mannheim, Germany).
Cytotoxicity assay. Cell viability was measured using the
CellTiter-Blue® Reagent (Promega, Milan, Italy). SH-SY5Y cells
(1 x104/well) were seeded in 96-well plates and treated with
[Ag(EIA)2]Cl (0,5-2,5 µM), cisplatin (10-50 µM) and [AgCl(MIA)]
(2-10 µM) for 24h. At end treatments, CellTiter-Blue® Reagent
was added to each well and incubated for 2 h at 37 °C.
Fluorescence was measured in a multiplate reader (Infinite
M200PRO, Tecan, Switzerland) at 560/590 nm. The IC50 values
for each compound were determined from the specific dose-
response curves by non-linear analysis, using GraphPad Prism
2.0 statistical program (GraphPad Software, San Diego, CA,
USA) and expressed as means ± standard deviation of at least 4
independent experiments.
Thioredoxin Reductase activity Assay. The inhibitory effects of
complex toward thioredoxin reductase activity (from rat liver) were
determined by quantification of the ability of compound to directly
reduce 5,5′-Dithiobis(2-nitrobenzoic acid) (DTNB) in presence of
NADPH as already reported in literature.10
Acknowledgements
We gratefully acknowledge Beneficentia Stiftung, ITT (Istituto
Toscano Tumori), Ente Cassa Risparmio Firenze (ECR) and
AIRC for funding the projects (IG-16049) and “Advanced mass
spectrometry tools for cancer research: novel applications in
proteomics, metabolomics and nanomedicine” (Multi-user
Equipment Program 2016, Ref. code 19650). COST Action
CM1105 is also acknowledged. T.M. thanks AIRC-FIRC
(Fondazione Italiana per la Ricerca sul Cancro) for the 3-years
Fellowship for Italy, Project Code: 18044 and University of Pisa
(PRA_2017_25).
Drugs uptake. SH-SY5Y cells were seeded in 6-well plates
(5x105 cells/well) and allowed to adhere. The culture medium was
replaced with medium without phenol red and FBS, and the cells
were incubated with [Ag(EIA)2]Cl, cisplatin or [AgCl(MIA)] (10 µM)
for 30 min. At end of treatments, the cells were harvested in
distilled H2O to induce osmotic shock. The determination of
metals concentration in the cell lysates was performed as
previously reported8 in triplicate by a Varian 720-ES Inductively
Coupled Plasma Atomic Emission Spectrometer (ICP-AES)
equipped with a CETAC U5000 AT+ ultrasonic nebulizer, in order
to increase the method sensitivity. Before the analysis, fixed
volume of samples, were moved in vials and digested in a thermo-
reactor at 80 °C for 3 h with 1 mL of aqua regia (HCl suprapure
grade and HNO3 suprapure grade in 3:1 ratio) and 5 mL of
ultrapure water (≤18 MΩ). Next, sample were spiked with 1 ppm
of Ge used as an internal standard and analyzed. Calibration
standards were prepared by gravimetric serial dilution from a
commercial standard solution of Pt at 1000 mg L−1. The
wavelength used for Pt and Ag determination were 214.424 and
338.289 nm respectively whereas for Ge the line at 209.426 nm
was used. The operating conditions were optimized to obtain
maximum signal intensity, and between each sample, a rinse
solution of HCl suprapure grade and HNO3 suprapure grade in 3:1
ratio was used in order to avoid any “memory effect”. The values
of Ag or Pt were normalized to cellular proteins, determined by
the micro-bicinchoninic acid (BCA) method, and expressed as μg
of metal/μg of proteins.
Keywords: thioredoxin reductase; silver carbene complexes;
fluorescence; anticancer drugs; mass spectrometry.
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A
novel fluorescent Silver(I) NHC
Maria Giulia Fabbrini, #a Damiano Cirri,
#b Alessandro Pratesi, b Lorenzo Ciofi, b
Tiziano Marzo, c, b* Annalisa Guerri, a
Silvia Nistri, d Alfonso Dell’Accio, d
Tania Gamberi, e Mirko Severi, a
complex has been synthesized and
characterized. This complex is highly
stable and unreactive toward common
model proteins, but able to inhibit
thioredoxin reductases. It is highly
cytotoxic in neuroblastoma cancer
cells being its pharmacological action
likely related to an unconventional
mechanism of binding to thioredoxin
reductases.
Andrea Bencini a and Luigi Messori b*
Page No. – Page No.
A Fluorescent Silver(I) Carbene
Complex with Anticancer Properties:
Synthesis, Characterization and
Biological Studies
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