Journal of Inorganic Biochemistry 156 (2016) 133144

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Journal of Inorganic Biochemistry

journal homepage: www.elsevier.com/locate/jinorgbio

Group 11 complexes with amino acid derivatives: Synthesis and

antitumoral studies
Lourdes Ortego a, Margarida Meireles b, Cornelia Kasper c, Antonio Laguna a,
M. Dolores Villacampa a,, M. Concepcin Gimeno a,
a
Departamento de Qumica Inorgnica, Instituto de Sntesis Qumica y Catlisis Homognea (ISQCH), CSIC-Universidad de Zaragoza, E-50009 Zaragoza, Spain
b
Universidade de Lisboa, Faculdade de Cincias, Departamento de Qumica e Bioqumica e Centro de Qumica e Bioqumica, Campo Grande, 1749-016, Lisboa, Portugal
c
Department of Biotechnology, Institute for Applied Microbiology, University of Material Resource and Life Science, Muthgasse 18, 1180, Vienna, Austria

a r t i c l e i n f o a b s t r a c t

Article history: Gold(I), gold(III), silver(I) and copper(I) complexes with modied amino acid esters and phosphine ligands have
Received 22 September 2015 been prepared in order to test their cytotoxic activity. Two different phosphine fragments, PPh3 and PPh2py
Received in revised form 15 December 2015 (py = pyridine), have been used. The amino acid esters have been modied by introducing an aromatic amine
Accepted 28 December 2015
as pyridine that coordinates metal fragments through the nitrogen atom, giving complexes of the type
Available online 30 December 2015
[M(L)(PR3)]+ or [AuCl3(L)] (L = L-valine-N-(4-pyridylcarbonyl) methyl ester (L1), L-alanine-N-(4-pyridylcarbonyl)
Keywords:
methyl ester (L2), L-phenylalanine-N-(4-pyridylcarbonyl) methyl-ester) (L3); M = Au(I), Ag(I), Cu(I), PR3 = PPh3,
Antitumor properties PPh2py). The in vitro cytotoxic activity of metal complexes was tested against four tumor human cell lines and one
Gold(I) tumor mouse cell line. A metabolic activity test (3-(4,5-dimethyl-thiazol-2-yl)-2,5-diphenyl tetrazolium bromide,
Silver(I) MTT) was used and IC50 values were compared with those obtained for cisplatin. Several complexes displayed
Copper(I) signicant cytotoxic activities. In order to determine whether antiproliferation and cell death are associated with
Amino acid derivatives apoptosis, NIH-3T3 cells were exposed to ve selected complexes (Annexin V+ FITC, PI) and analyzed by ow
Apoptosis cytometry. These experiments showed that the mechanism by which the complexes inhibit cell proliferation induc-
ing cell death in NIH-3T3 cells is mainly apoptotic.
2015 Elsevier Inc. All rights reserved.

1. Introduction
In the last decades chemotherapeutic research based on metal
complexes has increased considerably, since the good results with
platinum(II) compounds as cisplatin, the most widely used metal-
based antitumor drug. Gold complexes have been largely studied and
have gained growing interest because of their high cell growth inhibi-
tion [14]. Gold(I) complexes with several ligands such as phosphines,
thiolates, N-heterocyclic carbenes [59], or gold(III) species [1014]
have been studied for their cytotoxic activities. Many of these deriva-
tives have shown promising cytotoxic activities, in some cases overcom-
ing cisplatin resistance to specic cancer cells. In addition they have
shown a mechanism of action clearly different from that of platinum
drugs. Several targets have been identied for gold complexes; the inhi-
bition of thioredoxine reductase being one of the more important to
date [2,1521]. In spite of the great number of reported gold complexes
which show activity in the biological systems, not many gold derivatives
Corresponding authors.
E-mail addresses: dvilla@unizar.es (M.D. Villacampa), gimeno@unizar.es
(M.C. Gimeno).
with amino acids have been reported [22,23]. We have previously
reported on the synthesis and functionalization of gold(I) phosphine
nicotinic acid thiolate with amino acids providing complexes with
very good cytotoxic activity [24,25].
Silver(I) complexes were used as therapeutic compounds from
the middle ages mainly as antimicrobial agents. They have recently
regained attention as promising antitumor drugs, as shown in recent re-
views [26,27]. Several silver(I) derivatives with O-, N-, P- or S- donor li-
gands or N-heterocyclic carbene ligands have shown high cytotoxic
behavior against diverse human tumor cells [2834]. Mechanisms of
action of silver(I) derivatives are not yet clear. However interactions of
the complexes with DNA and thiol groups of the proteins have been
observed [26,27].
Copper is an essential trace metal for organisms. This plays an im-
portant role to catalyze metabolic oxidations and transfer electrons in
some enzymes as superoxide dismutase or cytochrome-c oxidase. The
international agency for research on cancer has not classied copper
as a human carcinogen. Copper complexes have been investigated as
potential antitumor agents because they may be less toxic for normal
cells than for cancerous ones. However, as all essential trace metals,
copper become toxic at slightly higher concentrations than the optimal
level. Principal toxicity may be the result of its redox activity (Cu(I)/
Cu(II)) and its afnity for heteroatoms of proteins or other

http://dx.doi.org/10.1016/j.jinorgbio.2015.12.018
0162-0134/ 2015 Elsevier Inc. All rights reserved.
134 L. Ortego et al. / Journal of Inorganic Biochemistry 156 (2016) 133144
biomolecules. An important number of copper complexes, specially
copper(II), have been screened for their antitumor properties. They
have shown encouraging perspectives and their mechanism of action
has been investigated [35]. Although copper(I) complexes have been
less studied compared to copper(II), an important number of them
have showed potent cytotoxic activity. Copper(I) complexes with P-
donor ligands [3641] or N-heterocyclic carbene ligands [42] have
shown strong cytotoxic activities. Research has indicated possible dif-
ferences (in the mechanisms of action) between copper complexes
and antitumor platinum derivatives.
Synthesis of gold, silver and copper complexes with modied amino
acid groups could be interesting in view of the possible biological prop-
erties. The amino acids can serve as good carriers to deliver the metallic
atoms to the biological target. In cancerous cells the amino acid recep-
tors are overexpressed and consequently the complexes could be
more selective to abnormal cells. Therefore, it was thought that it
would be a good idea to try to introduce metallic ions into the tumor
cells by amino acid derivatives as ligands, to facilitate the cell perme-
ation. In addition, it was considered that these ligands would be more
biocompatible and therefore reduce side effects in vivo.
L1L3 ligands and the corresponding meta piridyl-aminoacid deriv-
atives (Scheme 1) were previously prepared to synthesize a series of
fac tricarbonyl rhenium diimine complexes [43]. The amino acid methyl
esters derivatives were modied by introducing a pyridine group which
enhances the ability to coordinate metal atoms. Their applications in
uorescent microscopy cell imaging in the MCF-7 cell line were report-
ed. These derivatives showed a good uptake but with a markedly
different effect on the cells. Interestingly, after irradiation, rhenium
complexes with para amino acid derivatives induced damage in the
cell structure and cell death was observed. The cells treated with the
complexes with meta amino acid derivatives remained healthy.
This caused us to consider the hypothesis that coinage metal com-
plexes synthesized with the para and meta substituted pyridine amino
acid derivatives could have interesting cytotoxic properties. Here, we
report on the synthesis, structural characterization and on the study of
the biological activity of several group 11 metal complexes with the
para derivatives. Unfortunately, it was not possible to measure the
cytotoxic activity of gold(I) derivatives with the meta pyridyl-amino
acid ligands, due to their poor stability in solution at room temperature.
The complexes decomposed to metallic gold in a few hours. The in vitro
cytotoxic activities of metal complexes were tested against four tumor
human cell lines, A-549 (human lung), Hep-G2 (human liver cancer),
HeLa (human cervix epithelial carcinoma), MCF-7 (breast cancer) and
one tumor mouse cell line, NIH-3T3 (mouse broblast) showing strong
antitumor activity in vitro. Further experiments have been carried out in
order to determine the mechanism for which the complexes inhibit cell
proliferation and cause cell death.
Scheme 1. Depiction of selected ligands and their precursor.
2. Results and discussion
2.1. Synthesis and spectroscopic characterization
Amino acid derivatives L1L3 were synthesized by reaction of
commercially isonicotinic acid chloride with the corresponding
amino acid methyl ester compounds, in the presence of
triethylamine to allow the condensation reaction (Scheme 1), by
following literature precedents [43,44]. Spectroscopic information
is shown in the Experimental section.
The coordination chemistry of these ligands towards group 11
metal complexes has been studied. Gold(I) compounds 16 have
been prepared by reaction of L1L3 with an equimolecular amount
of the corresponding metal phosphine complexes [Au(OTf)(PPh3)]
or [Au(OTf)(PPh2py)] (OTf = CF3SO3, triuoromethanesulfonate),
whereas the gold(III) species 79 have been obtained by the reaction
of the ligands with AuCl3nH2O (Scheme 2).
In the same manner, the reaction of L1L3 with the silver or copper
complexes [Ag(OTf)(PR3)n] (n = 1, 2) or [Cu(NO3)(PPh3)2] has afforded
complexes 1018 and 1921, respectively (Scheme 3).
They have been characterized by means of IR, elemental analysis and
NMR spectroscopy. Assignments of the 1H NMR and 13C NMR signals
were made on the basis of 2D 1H COSY and HSQC spectra. Complete
spectroscopic information of the complexes has been collected in the
Experimental section.
The 1H NMR spectra of the compounds in acetone-d6 presented the
expected resonances for all the protons (see Experimental section).
They showed, in Au(I), Au(III) and Ag(I), a low-eld shift of the pyridine
protons H(2) and H(3), the nearest to the nitrogen atom, compared to
those found in the free ligands, that conrms the coordination through
the pyridine nitrogen atom to the metal. The 13C{1H} NMR also showed
a low-eld shift of the pyridine carbons C(2) and C(3). 1H NMR spectra
of Cu(I) complexes 19 (L1), 20 (L2) and 21 (L3) showed the resonances
for the corresponding amino ester derivative without no signicant
changes to those observed in the free amino acid ester ligands. The
phenyl resonances indicated that only one phosphine group is coordi-
nated to the copper metal in each compound.
The 31P{1H} NMR spectra of gold(I) and copper(I) compounds con-
sist on one singlet from the phosphorus atom coordinated to the
metal. The silver complexes with PPh3 are uxional in solution. Their
31 1
P{ H} NMR spectra at room temperature display one broad resonance
for complexes 11, 14, 16 and 17 or two broad resonances for complexes
10 and 13. This kind of uxionality is typical in the coordination chem-
istry of silver and can be attributed to exchange phenomena involving
the neutral ligands. In the low temperature spectra the expected com-
plex in each reaction was identied as the major product. At 203 K the
31 1
P{ H} NMR spectra of the mono-phosphine complexes 10, 13 and 16
 L. Ortego et al. / Journal of Inorganic Biochemistry 156 (2016) 133144
Scheme 2. Synthesis of the gold(I) and gold(III) complexes with L1L3: i) [Au(OTf)(PR3)] and ii) AuCl3nH2O.
consist of two doublets due to coupling with 107Ag and 109Ag nuclei
(J107AgP around 600 Hz, J109AgP around 700 Hz) that correspond to
the main derivative, [Ag(L)(PPh3)](OTf), and two doublets (J107AgP
around 450 Hz, J109AgP around 550 Hz) assigned to the corresponding
bis(phosphine) species [Ag(L)(PPh3)2](OTf) (Fig. 1).
At 203 K the 31P{1 H} NMR spectra of the bis(phosphine) com-
plexes 11, 14 and 17 show two doublets for the main derivative
[Ag(L)(PPh3 )2](OTf), as the most intense resonances, and also as
minor components two doublets for the mono(phosphine),
[Ag(L)(PPh 3 )](OTf), and two doublets (J 107 AgP around 250 Hz,
J109AgP around 300 Hz) that can be assigned to the tris(phosphine),
[Ag(L)(PPh3 ) 3 ](OTf), derivatives. An equilibrium between phos-
phine derivatives can be proposed as has been observed in other sil-
ver complexes [4550]. The 31P{1H} NMR spectra of complexes 12,
15 and 18 at r.t. show a broad resonance, that splits into a slightly
broad 6 symmetric lines at 203 K (Fig. 2). These spectra do not
match with a phosphorus atom coupled to both silver isotopomers,
were only two doublets would appear. It resembles that of the
starting material, which has a dinuclear structure, [Ag2 (OTf) 2(-
PPh2py)2], with bridging P,N units forming an eight membered ring
[51], and corresponds to an AB system for the phosphorus atoms
coupled to both silver nuclei with different coupling constants. For
this reason we proposed a similar structure for the PPh 2 py com-
plexes, in which the modied pyridil amino ester ligand would coor-
dinate to each silver atom of the dinuclear unit (Fig. 2).
2.2. Crystal structure determination
The crystal structures of gold(III) complexes 7 and 8 have been de-
termined by X-ray diffraction studies. They crystallized in the
Scheme 3. Synthesis of the silver(I) and copper(I) complexes with L1L3: i) [Ag(OTf)(PR3)n] and ii) [Cu(NO3)(PPh3)2].
135
monoclinic space group P21/c with two and one molecules by asymmet-
ric unit, respectively (Figs. 3 and 4). The gold atoms in these complexes
show a slightly distorted square-planar AuCl3N coordination. The angles
around gold show slight deviations from the ideal 90. The gold(III)
atoms are practically in the same plane generated by the three chloride
atoms and the pyridine nitrogen (average metal center deviation from
the planes formed by the four donor atoms is 0.010 ). As expected,
the asymmetric carbon atoms C7 in 7 and 8 retained the S conguration
of the commercial amino acid methyl ester used in the synthesis of L1
and L2.
2.3. Absorption and emission spectra
UVvis spectra for the ligands and the complexes were recorded at
ca. 105 M concentration in dichloromethane solution. The absorption
spectra of the ligands display one strong absorption at 230 nm and a
weaker one at 263 nm. The spectra for all the complexes resemble
those of the ligands with a slight variation in the energy, the strongest
absorption has a maximum at ca. 235 nm and a weaker absorption at
ca. 265 nm. Those complexes bearing phosphine ligands, especially
PPh2py display more intense and dened absorptions. As these absorp-
tions are present in the ligands and in all the complexes, including those
with no phosphine ligands, we attributed them to intraligand * or
n * transition characteristic of aromatic systems.
The gold and silver complexes bearing 2-diphenylphosphino pyri-
dine ligands are emissive in solution and in the solid state, the date is
summarized in Table 1. In dichloromethane solutions the gold com-
plexes, 2, 4 and 6, and the silver derivatives, 12, 15 and 18, show an
emission around 370 and 400 nm, respectively. The same behavior is
observed in the solid state, where the complexes display intense
136 L. Ortego et al. / Journal of Inorganic Biochemistry 156 (2016) 133144
Fig. 1. 31P{1H} NMR (CD3)2CO spectrum of [Ag(L1)(PPh3)](OTf) (10) at 203 K.
emissions at about 440 nm. In both, solution and solid state, these emis-
sions are assigned to intraligand transitions modied by coordination to
the metal center. In these sense a redshift is observed on going from
gold to silver species in solutions, whereas in the solid state this redshift
is only noticeable in complex 18.
2.4. Biological evaluation
2.4.1. Cytotoxic studies
The in vitro cytotoxic activity of metal complexes (except complexes
11, 14, 17) was tested against four tumor human cell lines, A-549
(human lung), Hep-G2 (human liver cancer), HeLa (human cervix epi-
thelial carcinoma), MCF-7 (breast cancer), and one tumor mouse cell
line, NIH-3T3 (mouse broblast), and IC50 values compared with those
of cisplatin [52,53]. As control, metal-free ligands L1, L2 and L3 were
tested for cytotoxicity against all the cell lines except the MCF-7 cells.
Compounds are not soluble in water, but they are soluble in DMSO
and in the DMSO/water mixtures used in the cytotoxicity tests, which
contain a small amount of DMSO (5%). We did not observe any precip-
itation of the complexes while performing the tests. The colorless
(Au(I), Ag(I) Cu(I)) or yellow Au(III)-DMSO solutions are very stable
at room temperature for weeks.
Cells were exposed to each compound for 48 h. By using the colori-
metric mitochondrial function-based MTT viability assay the IC50 values
(nal concentration 5% DMSO) were calculated from doseresponse
curves obtained by nonlinear regression analysis. IC50 values are con-
centrations of a drug required to inhibit tumor cell proliferation by
50%, compared to the control viability. Controls were performed using
cells grown in medium with 5% DMSO. IC50 values at 48 h are listed in
Table 2.
As can be seen in Table 2, the tested ligands and the gold(III)
complexes showed no cytotoxic activity. By contrast, some gold(I),
Fig. 2. A) Proposed structures for complexes 12, 15 and 18 based on the structure of the compound [Ag2(OTf)2(-PPh2py)2]; B) 31P{1H}NMR spectrum (300 MHz, (CD3)2CO, 203 K) of
complex 15.
silver(I) and copper(I) complexes appeared to be quite effective as cyto-
toxic agents in vitro against the growth of the cancer cell lines tested.
The gold(I) complexes showed in general to be effective as cytotoxic
agents against the in vitro growth of all the tested cancer cell lines. They
are particularly effective against HeLa (IC50 values range between 4.33
and 11.71 M) and MCF-7 (IC50 values range between 0.45 and
20.05 M) cell lines being 2, 4 and 5 the most potent agents against
MCF-7 with IC50 values of 0.65, 1.78 and 0.45 M, respectively. The var-
iation of the tertiary phosphine from PPh3 to PPh2py provided higher
cytotoxicity. Also if we consider the amino acid ester derivative in the
pyridine moiety, the lower IC50 values have been achieved with the va-
line methyl ester.
Silver(I) derivatives presented globally lower activities in all the
cancer cell lines compared with those of gold(I). Again an increase in cy-
totoxicity is achieved when changing the phosphine from PPh3 to
PPh2py. Specially, complex 18 with PPh2py is a powerful agent against
the HeLa cell line (IC50 value of 3.40 M), and the species 16,
[Ag(L3)(PPh3)](OTf), and 18, [Ag(L3)(PPh2py)](OTf), showed strong
cytotoxic activity with IC50 values of 5.63 and 2.81 M, respectively,
against MCF-7 cell line. Considering the amino acid ester derivative in
the pyridine moiety, the lower IC50 values have been achieved with
the phenyl alanine methyl ester.
Copper(I) complexes also showed remarkable activities against NIH-
3T3, HeLa and MCF-7 cell lines with IC50 values ranging between 5.27
and 24.83 M. In this case and considering the amino acid ester deriva-
tive in the pyridine moiety, the lower IC50 values have been achieved
with the valine and phenyl alanine methyl esters.
In summary, almost all the gold(I) complexes are cytotoxic against
all the cell lines, being the stronger agents the PPh2py derivatives. In
general, a lower activity was observed for the silver(I) complexes
although some of them showed high antiproliferative activity, particu-
larly complexes with the L3 (phenyl alanine methyl esther) ligand.
Also, a signicant antiproliferative activity can be observed for
 L. Ortego et al. / Journal of Inorganic Biochemistry 156 (2016) 133144 137

Fig. 3. Diagram of complex 7 (the second molecule within the asymmetric unit is omitted for clarity). Hydrogen atoms (except NH and C7-H) are omitted for clarity. Selected bond lengths
[] and angles []: Au(1)Cl(1) 2.257 (8); Au(1) Cl(2) 2.273(8); Au(1) Cl(3) 2.281(8); Au(1)N(1) 2.04(3); Cl(1)Au(1)N(1) 90.2(9); Cl(1)Au(1)Cl(2) 90.7(3); Cl(2)Au(1)
Cl(3) 91.2(3); N(1)Au(1)Cl(3) 88.0(9).

copper(I) complexes especially against the NIH-3T3 cell line. Although
it is not possible to establish a reliable relation between composition
and activity, the measured data showed that the MCF-7 and HeLa cells
are very sensitive to the metallic complexes tested, with most of the
IC50 values being lower than those found for cisplatin, the antitumoral
agent taken as reference. We hypothesize that these complexes may
enter the cells, as could be observed for the rhenium complexes with
these para-amino acid ester ligands that showed a signicant uptake
in MCF-7 cells [43]. Moreover these para rhenium derivatives induced
damage in the cell structure and cell death was observed. In contrast,
cells incubated with the meta ligands remained healthy. Unfortunately,
the biological activity of the meta gold(I) complexes could not be inves-
tigated due to their poor stability in solution at room temperature.
2.4.2. Detection of cell death using FITC-conjugated annexin V (ow
cytometry)
Apoptosis is the nal effect of the treatment with multiple
chemotherapeutical agents in cancer. Many anticancer drugs (as cis-
platin) are effective against cancerous cells by causing apoptosis by
the generation of reactive oxygen species (ROS) and DNA damage [15,
28,54]. Apoptosis is a programmed mode of cell death that is accompa-
nied by numerous morphological as well as biochemical changes in the
cellular architecture that include cell surface changes. In apoptotic cells,
phosphatidylserine (PS) is translocated from the inner to the outer leaf-
let of the plasma membrane, where it can be detected by its binding to
the intracellular protein Annexin V, labeled with a uorophore. The
Annexin V assay is able to distinguish apoptotic cells from viable or ne-
crotic cells on the basis of the resulting uorescence of the former cells.
The simultaneous use of a dead cell stain employs the uorochrome
propidium iodide (PI), which can only penetrate dead or dying cells
that have lost or are losing membrane integrity. PI intercalates into
DNA to produce a highly uorescent adduct, whose observation is
indicative of late apoptosis or necrosis [5558]. To determine whether
antiproliferation and cell death are associated with apoptosis, NIH-3T3
cells were exposed for 24 h to IC50 concentrations of complexes 2
(Au), 16 (Ag), 19 (Cu), 20 (Cu) or 21 (Cu). Annexin V labeled with the
uorophore FITC, and the uorochrome propidium iodide PI were
added and cells were analyzed by ow cytometry. Flow cytometry stud-
ies allow us to know whether cells are dying via apoptosis or necrosis. In
Fig. 5, the lower-right panel corresponds to early apoptotic cells
(Annexin V+ FITC), the upper-left panel to necrotic cells (PI) and the
upper right panel to cells in a necrotic process but whose death started
as apoptotic (late apoptosis, Annexin V + PI). NIH-3T3 cell studies
showed that 80% of untreated cells were alive after incubation of 24 h
in 5% DMSO (Fig. 5a). Incubation with the complexes at the concentra-
tions corresponding to the IC50 values showed that for complexes
[Au(L1)(PPh2py)](OTf) (2) (Fig. 5b) and [Ag(L3)(PPh3)](OTf) (16)
(Fig. 5c) 100% of cells were in 100% late apoptosis. Cells incubated
with [Cu(L)(PPh3)](NO3) (L = L1, 19; L2, 20; L3, 21) (Fig. 5df) com-
plexes were alive (2519%), in late apoptosis (5257%) and in early ap-
optosis (1623%). All these observations suggest that the mechanism by
which the complexes inhibit cell proliferation in NIH-3T3 cells inducing
cell death, is mainly apoptotic. Moreover, it can be observed that com-
plexes 1921 induced a slower cytotoxic mechanism, although they
are more effective cytotoxic agents (smaller IC50 values) than 2 and 16.
3. Conclusions
A series of gold, silver and copper complexes, [M(L)(PR3)]+ or
[AuCl3(L)] (L = L-valine-N-(4-pyridylcarbonyl) methyl ester (L1), L-ala-
nine-N-(4-pyridylcarbonyl) methyl ester (L2), L-phenylalanine-N-
(4-pyridylcarbonyl) methyl ester (L3); M = Au(I), Ag(I), Cu(I),
PR3 = PPh3, PPh2py), with modied amino acid esters and phosphine
ligands have been prepared. The in vitro cytotoxic activity of metal

Fig. 4. Diagram of complex 8. Hydrogen atoms (except NH and C7-H) are omitted for clarity. Selected bond lengths [] and angles [] for complex 8: Au(1)Cl(1) 2.2838(11); Au(1) Cl
(2) 2.2599(10); Au(1) Cl(3) 2.2766(11); Au(1)N(1) 2.028(3); Cl(1)Au(1)N(1) 90.14(9); Cl(1)Au(1)Cl(2) 92.33(4); Cl(2)Au(1)Cl(3) 90.72(4); N(1)Au(1)Cl(3) 86.82(9).
138 L. Ortego et al. / Journal of Inorganic Biochemistry 156 (2016) 133144

Table 1 prepared according to published procedures. [Au(OTf)(PPh3)] or

Excitation and emission values for complexes 2, 4, 6, 12, 15 and 18. [Au(OTf)(PPh2py)] was obtained by reaction of [AuCl(PPh3)] [59] or
Solution (CH2Cl2 7 104 M) Solid [AuCl(PPh2py)] [60] with Ag(OTf) in dichloromethane and used in
Excitation (nm) Emission (nm) Excitation (nm) Emission (nm)
situ. All other reagents were commercially available and used without
further purication. C, H, and N analyses were carried out with a
2 330 370 395 440
PerkinElmer 2400 microanalyzer. 1H, 13C{1H} and 31P{1H}, including
4 320 376 390 440
6 320 365 395 440 2D experiments, were recorded at room temperature on a Bruker
12 337 390 392 440 Avance 400 and a Bruker ARX 300 spectrometers. Chemical shifts
15 337 390 390 440 (, ppm) were reported relative to the solvent peaks in the 1H, 13C
18 362 440 357 480 spectra or external 85% H3PO4 in 31P spectra. UVvis spectra were re-
corded with a 1 cm quartz cells on an Evolution 600 spectrophotometer.
Room temperature steady-state emission and excitation spectra were
complexes was tested against four tumor human cell lines and one recorded with a Jobin-Yvon-Horiba uorolog FL3-11 spectrometer
tumor mouse cell line. They were A-549 (human lung), Hep-G2 tted with a JY TBX picosecond detection module.
(human liver cancer), HeLa (human cervix epithelial carcinoma),
MCF-7 (breast cancer) and NIH-3T3 (mouse broblast). A metabolic ac-
tivity test (3-(4,5-dimethyl-thiazol-2-yl)-2,5-diphenyl tetrazolium bro- 4.2. General synthesis of L1 L3
mide, MTT) was used and IC50 values were compared to cisplatin.
Almost all the gold(I) complexes are cytotoxic against all the cell lines, Isonicotinic acid chloride (4.08 g, 20 mmol), triethylamine (3.42 mL,
being the stronger agents the PPh2py derivatives. In general, a lower ac- 22 mmol) and the correspondent L-amino acid methyl ester (20 mmol)
tivity was observed for the silver(I) complexes although some of them were stirred in dry DCM for 24 h at room temperature under an argon
showed high antiproliferative activity, specially complexes with the L3 atmosphere. Then an aqueous solution of NaHCO3 was added to the
ligand. Also, a signicant cytotoxic activity can be observed for mixture and an extraction was performed (3 20 mL DCM). The com-
copper(I) complexes especially against the NIH-3T3 cell line. Although bined organic layers were dried over MgSO4 and ltered over celite.
it is not possible to establish a reliable relation between composition The volume was reduced to 10 mL, and addition of hexane and/or
and activity, the measured data showed that the MCF-7 and HeLa cells diethyl ether afforded L1 as orange solid, L2 as beige solid and L3 as a
are very sensitive to the metallic complexes tested, with most of the brown oil.
IC50 values being lower than those found for cisplatin, the reference
used. Flow cytometry studies of NIH-3T3 cells exposed to IC50 concen-
trations of ve selected complexes, showed that the mechanism by 4.3. L-Valine-N-(4-pyridylcarbonyl) methyl ester (L1)
which the complexes inhibit cell proliferation inducing cell death is
mainly apoptotic. Copper complexes tested induced a slower cytotoxic Yield: 4.30 g, 91%. Anal. Calcd. (%) for C12H16N2O3 (236.26): C, 61.00;
mechanism than gold and silver derivatives, in spite of being more H, 6.83; N, 11.86. Found: C, 61.12; H, 6.84; N, 11.95. IR (cm 1):
cytotoxic. (NH) = 3245, (COcarboxylate) = 1729, (COamide) = 1660. 1H NMR
(400 MHz, (CD3)2CO) : 8.71 (m, 2H, H2), 8.05 (s br, 1H, NH), 7.78 (m,
4. Experimental section 2H, H3), 4.57 (dd, 1H, NHCH, 3JHH 8.4 Hz, 3JHH 6.4 Hz), 3.72 (s, 3H,
COOCH3), 2.26 (m, 1H, CH(CH3)2), 1.02 (d, 3H, CH(CH3)2, 3JHH 6.8 Hz),
4.1. General procedures 1.00 (d, 3H, CH(CH3)2, 3JHH 6.8 Hz). 13C{1H}NMR (400 MHz, (CD3)2CO)
: 173.62 (s, 1C, COOCH3), 167.44 (s, 1C, CONH), 152.15 (s, 2C, C2),
The synthesis of the copper complexes 1921 were carried out 143.19 (s, 1C, C4), 123.17 (s, 2C, C3), 60.20 (s, 1C, NHCH), 53.19 (s, 1C,
under Ar atmosphere, in solvents dried and degassed prior to use. The COOCH3), 32.37 (s, 1C, CH(CH3)2), 20.45 (s, 1C, CH(CH3)2), 19.77 (s,
starting material [Ag(OTf)(PPh3)] [45] or [Ag(OTf)(PPh2py)] [51] was 1C, CH(CH3)2). UVvis, max nm: 230, 263.

Table 2
IC50 (48 h) values of complexes and ligands against tumor cells.

IC50 A-549 Hep-G2 NIH-3 T3 HeLa MCF-7

L Cisplatin 10.22 0.44 [52] 25 3.1 [53] nt 10 2.2 [52] 21.65 0.22 [51]
L1 N50 N50 N50 N50 nt
L2 N50 N50 N50 N50 nt
L3 N50 N50 N50 N50 nt
AuPPh3 1 24.56 0.34 nt 4.76 0.13 4.43 1.43 6.04 0.33
3 25.35 0.40 10.50 0.46 10.75 0.43 11.71 1.06 20.05 0.34
5 N50 25.95 0.16 3.71 0.64 10.66 0.33 0.45 0.20
AuPPh2py 2 10.21 0.60 10.18 0.41 13.41 1.37 4.33 0.46 0.65 0.34
4 25.22 0.59 10.81 0.78 10.31 0.37 5.58 0.86 1.78 0.05
6 25.35 0.44 10.85 0.54 6.43 1.55 6.93 0.50 10.14 0.69
AuCl3 7 N50 N50 N50 N50 nt
8 N50 N50 N50 N50 nt
9 N50 N50 N50 N50 nt
AgPPh3 10 25.22 0.37 10.50 0.45 24.92 0.07 48.91 0.13 nt
13 50 N50 10.08 0.14 25.78 0.82 nt
16 N50 25.18 0.36 10.59 0.22 13.98 1.69 5.63 0.93
AgPPh2py 12 25.16 0.34 5.47 0.27 25.07 0.25 N50 nt
15 N50 N50 25.31 0.18 N50 nt
18 N50 25.42 0.42 4.78 0.13 3.40 0.09 2.81 0.66
CuPPh3 19 24.50 0.35 35.12 0.34 1.82 0.30 8.13 1.05 12.94 0.75
20 24.56 0.32 25.02 0.38 1.98 0.32 24.83 2.70 5.63 0.93
21 10.47 0.23 25.17 0.23 2.12 0.38 5.27 1.07 10.55 0.45

nt: not tested.

 L. Ortego et al. / Journal of Inorganic Biochemistry 156 (2016) 133144
Fig. 5. NIH-3T3 control cells grown for 24 h in 5% DMSO (a); NIH-3 T3 cells treated for 24 h with compounds 2 (b); 16 (c); 19 (d); 20 (e); or 21 (f) (concentrations corresponded to IC50
values). Apoptosis/necrosis hallmarks were analyzed as described in Experimental section. The percentage of cells in each region is indicated.
4.4. L-Alanine-N-(4-pyridylcarbonyl) methyl ester (L2)
Yield: 3.70 g, 89%. Anal. Calcd. (%) for C10H12N2O3 (208.21): C, 57.68,
H, 5.81, N, 13.45. Found: C, 57.42; H, 5.92; N, 13.46. IR (cm1): (NH) =
3296, (COcarboxylate) = 1731, (COamide) = 1645, sym(C = N) = 1537.
1
H NMR (400 MHz, (CD3)2CO) : 8.72 (m, 2H, H2), 8.22 (s br, 1H, NH),
7.78 (m, 2H, H3), 4.66 (qd, 1H, NHCH, 3JHH 7.3 Hz, 3JHH 7.3 Hz), 3.70 (s,
3H, COOCH3), 1.48 (d, 3H, CHCH3, 3JHH 7.3 Hz). 13C{1H}NMR (400 MHz,
(CD3)2CO) : 174.39 (s, 1C, COOCH3), 166.61 (s, 1C, CONH), 152.08 (s,
2C, C2), 142.83 (s, 1C, C4), 122.87 (s, 2C, C3), 53.25 (s, 1C, NHCH), 50.31
(s, 1C, COOCH3), 18.27 (s, 1C, CHCH3). UVvis, max nm: 230, 263.
4.5. L-Phenylalanine-N-(4-pyridylcarbonyl) methyl ester (L3)
Yield: 5.42 g, 95%. Anal. Calcd. (%) for C16H16N2O3 (284.31): C, 67.59;
H, 5.67; N, 9.85. Found: C, 67.17; H, 5.50; N, 9.81. IR (cm1): (NH) =
3282, (COcarboxylate) = 1740, (COamide) = 1647, sym (C_N) =
1532. 1H NMR (400 MHz, (CD3)2CO) : 8.67 (m, 2H, H2), 8.39 (d, 1H,
NH, 3JHH 7.9 Hz), 7.69 (m, 2H, H3), 7.27 (m, 5H, CH2Ph), 4.96 (m, 1H,
NHCH), 3.69 (s, 3H, COOCH3), A = 3.31 B = 3.17 (2H, system ABX,
CH2Ph, JAX 5.4 Hz, JBX 9.4 Hz, JAB 13.9 Hz). 13C{1H}NMR (400 MHz,
(CD3)2CO) : 173.46 (s, 1C, COOCH3), 167.03 (s br, 1C, CONH), 152.05
(s, 2C, C2), 142.88 (s, 1C, C4), 139.02 (s, 1C, CipsoCH2Ph), 130.95 (s, 2C,
CorthoCH2Ph), 130.16 (s, 1C, CparaCH2Ph), 128.49 (s, 2C, CmetaCH2Ph), 122.93
(s, 2C, C3), 56.17 (s, 1C, NHCH), 53.47 (s, 1C, COOCH3), 38.82 (s, 1C,
CH2Ph). UVvis, max nm: 230, 263.
4.6. Synthesis of [Au(L)(PR3)](OTf) (L1, R3_Ph3 (1), Ph2py (2); L2, R3_Ph3
(3), Ph2py (4); L3, R3_Ph3 (5), Ph2py (6))
A solution of [Au(OTf)(PPh3)] (127.68 mg, 0.2 mmol) or
[Au(OTf)(PPh2py)] (128.67 mg, 0.21 mmol) prepared in situ and L-
139
alanine-N-(4-pyridylcarbonyl) methyl ester (L1; 47 mg, 0.2 mmol) or
L-valine-N-(4-pyridylcarbonyl) methyl ester (L2; 42 mg, 0.2 mmol) or
L-phenylalanine-N-(4-pyridylcarbonyl) methyl ester (L3; 64 mg,
0.2 mmol) were stirred in DCM (20 mL) for 2 h at room temperature.
The volume was reduced to 5 mL, and addition of hexane afforded com-
pounds 16. 1 and 3 as white solids, 2 and 4 as beige solids and 5 and 6
as brown oils.
4.6.1. [Au(L1)(PPh3)](OTf) (1)
Yield: 132 mg, 78%. Anal. Calcd. (%) for C31H31AuF3N2O6PS (844.59):
C, 44.09; H, 3.70; N, 3.32; S, 3.80. Found: C, 44.18; H, 3.55; N, 3.29; S,
3.63. IR (cm1): (NH) = 3290, (COcarboxylate) = 1737, (COamide) =
1666, sym(C_N) = 1537, asym(SO3) = 1252, sym(CF3) = 1221,
asym(CF3) = 1151, sym(SO3) = 1028. 1H NMR (300 MHz, (CD3)2CO)
: 9.15 (m, 2H, H2), 8.40 (d, 1H, NH, 3JHH 8.2 Hz), 8.33 (m, 2H, H3),
7.68 (m, 15H, HPh), 4.57 (m, 1H, NHCH), 3.72 (s, 3H, COOCH3), 2.29
(m, 1H, CH(CH3)2), 1.03 (m, 6H, CH(CH3)2). 19F NMR (400 MHz,
(CD3)2CO) : -78.82 (s, 1F, OTf). 31P{1H}NMR (400 MHz, (CD3)2CO) :
29.8 (s, 1P, AuPPh3). 13C{1H}NMR (300 MHz, (CD3)2CO) : 173.22 (s,
1C, COOCH3), 165.59 (s, 1C, CONH), 154.14 (s, 2C, C2), 147.60 (s, 1C,
C4), 136.22 (d, 6C, CorthoPh, 2JCP 13.6 Hz), 136.56 (d, 3C, CparaPh, 4JCP
2.6 Hz), 131.58 (d, 6C, CmetaPh, 3JCP 12.1 Hz), 129.51 (d, 3C, CipsoPh, 1JCP
66.4 Hz), 126.48 (s, 2C, C3), 60.72 (s, 1C, NHCH), 53.29 (s, 1C,
COOCH3), 32.33 (s, 1C, CH(CH3)2), 20.43 (s, 1C, CH(CH3)2), 19.88 (s,
1C, CH(CH3)2). UVvis, max nm: 233, 265 (br).
4.6.2. [Au(L1)(PPh2py)](OTf) (2)
Yield: 137 mg, 81%. Anal. Calcd. (%) for C30H30AuF3N3O6PS (845.58):
C, 42.61; H, 3.58; N, 4.97; S, 3.79. Found: C, 42.83; H, 3.42; N, 5.15; S,
3.93. IR (cm1): (NH) = 3287, (COcarboxylate) = 1731, (COamide) =
1661, sym(C_N) = 1536, asym(SO3) = 1253, sym(CF3) = 1221,
asym(CF3) = 1153, sym(SO3) = 1028. 1H NMR (400 MHz, (CD3)2CO)
140 L. Ortego et al. / Journal of Inorganic Biochemistry 156 (2016) 133144
: 9.04 (m, 1H, H6), 8.84 (m, 2H, H2), 8.20 (m, 1H, NH), 8.11 (m, 1H, H4),
7.93 (m, 2H, H3), 7.66 (m, 12H, HPh, 3, 5), 4.56 (m, 1H, NHCH), 3.72 (s,
3H, COOCH3), 2.27 (m, 1H, CH(CH3)2), 1.02 (m, 6H, CH(CH3)2). 19F
NMR (400 MHz, (CD3)2CO) : 80.06 (s, 1F, OTf). 31P{1H}NMR
(400 MHz, (CD3)2CO) : 31.3 (s, 1P, AuPPh2py). 13C{1H}NMR
(400 MHz, (CD3)2CO) : 172.54 (s, 1C, COOCH3), 165.74 (s, 1C, CONH),
153.59 (d, 1C, C6, 3JCP 16.8 Hz), 151.80 (s, 2C, C2), 143.84 (s, 1C, C4),
139.56 (d, 1C, C4, 3JCP 9.0 Hz), 135.53 (d, 4C, CorthoPh, 2JCP 13.6 Hz),
133.68 (d, 2C, CparaPh, 4JCP 2.8 Hz), 132.16 (d, 1C, C3, 2JCP 25.2 Hz),
130.53 (d, 4C, CmetaPPh, 3JCP 12.0 Hz), 127.63 (s, 1C, C5), 123.44 (s, 2C,
C3), 59.50 (s, 1C, NHCH), 52.30 (s, 1C, COOCH3), 31.36 (s, 1C,
CH(CH3)2), 19.51 (s, 1C, CH(CH3)2), 18.91 (s, 1C, CH(CH3)2). UVvis,
max nm: 232, 265.
4.6.3. [Au(L2)(PPh3)](OTf) (3)
Yield: 160 mg, 98%. Anal. Calcd. (%) for C29H27AuF3N2O6PS (816.53):
C, 42.66; H, 3.33; N, 3.43; S, 3.93. Found: C, 42.94; H, 3.54; N, 3.29; S,
3.72. IR (cm1): (NH) = 3314, (COcarboxylate) = 1740, (COamide) =
1666, sym(C_N) = 1539, asym(SO3) = 1268 and 1250, sym(CF3) =
1221, asym(CF3) = 1154, sym(SO3) = 1028. 1H NMR (400 MHz,
(CD3)2CO) : 9.10 (m, 2H, H2), 8.61 (s br, 1H, NH), 8.27 (m, 2H, H3),
7.67 (m, 15H, HPh), 4.66 (qd, 1H, NHCH, 3JHH 7.3 Hz, 3JHH 7.3 Hz), 3.71
(s, 3H, COOCH3), 1.51 (d, 3 H, CHCH3, 3JHH 7.3 Hz). 19F NMR (400 MHz,
(CD3)2CO) : 80.09 (s, 1F, OTf). 31P{1H}NMR (400 MHz, (CD3)2CO)
: 29.7 (s br, 1P, AuPPh3). 13C{1H}NMR (300 MHz, (CD3)2CO) : 174.18
(s, 1C, COOCH3), 165.63 (s, 1C, CONH), 153.82 (s, 2C, C2), 146.71 (s, 1C,
C4), 136.15 (d, 6C, CorthoPh, 2JCP 13.8 Hz), 134.45 (s, 3C, CparaPh), 131.53
(d, 6C, CmetaPh, 3JCP 12.1 Hz), 125.84 (s, 2C, C3), 53.46 (s, 1C, NHCH),
50.81 (s, 1C, COOCH3), 18.21 (s, 1C, CHCH3). UVvis, max nm: 235,
265 (br).
4.6.4. [Au(L2)(PPh2py)](OTf) (4)
Yield: 157 mg, 96%. Anal. Calcd. (%) for C28H26AuF3N3O6PS (817.53):
C, 41.14; H, 3.21; N, 5.14; S, 3.92. Found: C, 41.55; H, 3.17; N, 4.84; S,
3.63. IR (cm1): (NH) = 3313, (COcarboxylate) = 1740, (COamide) =
1664, sym(C_N) = 1540, asym(SO3) = 1268 and 1250, sym
(CF3) = 1221, asym(CF3) = 1155, sym(SO3) = 1028. 1H NMR
(400 MHz, (CD3)2CO) : 9.13 (s br, 1H, H6), 8.87 (m, 2H, H2), 8.42 (m,
1H, NH), 8.15 (m, 1H, H4), 7.97 (m, 2H, H3), 7.76 (m, 12H, HPh, 3, 5),
4.66 (qd, 1H, NHCH, 3JHH 7.3 Hz, 3JHH 7.3 Hz), 3.71 (s, 3H, COOCH3),
1.49 (d, 3H, CHCH3, 3JHH 7.3 Hz). 19F NMR (400 MHz, (CD3)2CO) :
80.07 (s, 1F, OTf). 31P{1H} NMR (400 MHz, (CD3)2CO) : 30.0 (s br,
AuPPh2py). 13C{1H}NMR (400 MHz, (CD3)2CO) : 174.34 (s, 1C,
COOCH3), 165.67 (s, 1C, CONH), 154.92 (d, 1C, C6, 3JCP 16.9 Hz), 154.08
(s, 1C, C2), 153.02 (s, 2C, C2), 144.97 (s, 1C, C4), 140.90 (d, 1C, C4, 3JHH
8.8 Hz), 136.51 (d, 4C, CorthoPh, 3JCP 13.7 Hz), 134.81 (s, 2C, CparaPh),
133.16 (d, 1C, C3, 2JCP 23.7 Hz), 131.57 (d, 4C, CmetaPh, 4JCP 12.2 Hz),
128.83 (s, 1C, C5), 128.23 (s, 2C, CipsoPh, 3JCP 66.2 Hz), 124.59 (s, 2C,
C3), 53.44 (s, 1C, NHCH), 50.67 (s, 1C, COOCH3, 1C), 18.26 (s, 1C,
CHCH3). UVvis, max nm: 232, 265.
4.6.5. [Au(L3)(PPh3)](OTf) (5)
Yield: 130 mg, 73%. Anal. Calcd. (%) for C35H31AuF3N2O6PS (892.63):
C, 47.09; H, 3.50; N, 3.14; S, 3.59. Found: C, 46.99; H, 3.42; N, 2.93; S,
3.23. IR (cm1): (NH) = 3313, (COcarboxylate) = 1738, (COamide) =
1667, sym(C_N) = 1539, asym(SO3) = 1268 and 1251, sym(CF3) =
1222, asym(CF3) = 1151, sym(SO3) = 1028. 1H NMR (300 MHz,
(CD3)2CO) : 8.97 (m, 2H, H2), 8.51 (d, 1H, NH, 3JHH 8.0 Hz), 8.04 (m,
2H, H3), 7.66 (m, 15H, HPh), 7.28 (m, 5H, CH2Ph), 4.93 (m, 1H, NHCH),
3.70 (s, 3H, COOCH3), A = 3.30 B = 3.16 (2H, system ABX, CH2Ph,
JAX 5.4Hz, JBX 9.4 Hz, JAB 13.9 Hz). 31P{1H}NMR (400 MHz, (CD3)2CO)
: 29.5 (s br, 1P, AuPPh3). 13C{1H}NMR (300 MHz, (CD3)2CO) : 173.09
(s, 1C, COOCH3), 165.11 (s br, 1C, CONH), 154.06 (s, 2C, C2), 146.97 (s,
1C, C4), 139.13 (s, 1C, CipsoCH2Ph), 136.17 (d, 6C, CorthoPh, 2JCP 13.6 Hz),
134.50 (d, 3C, CparaPPh3 4JCP 2.8 Hz), 131.54 (d, 6C, CmetaPh, 3JCP 12.1 Hz),
131.08 (s, 2C, CorthoCH2Ph), 130.24 (s, 1C, CparaCH2Ph), 128.58 (s, 2C,
CmetaCH2Ph), 129.58 (d, 3C, CipsoPh, 1JCP 61.0 Hz), 125.97 (s, 2C, C3), 56.60
(s, 1C, NHCH), 53.54 (s, 1C, COOCH3), 38.76 (s, 1C, CH2Ph). UVvis,
max nm: 235, 265 (br).
4.6.6. [Au(L3)(PPh2py)](OTf) (6)
Yield: 127 mg, 71%. Anal. Calcd. (%) for C34H30AuF3N3O6PS (893.62):
C, 45.70; H, 3.38; N, 4.70; S, 3.59. 1028. Found: C, 45.72; H, 3.44; N, 4.34;
S, 3.77. IR (cm1): (NH) = 3313, (COcarboxylate) = 1741, (COamide) =
1668, sym(C_N) = 1541, asym(SO3) = 1270 and 1251, sym(CF3) =
1222, asym(CF3) = 1151, sym(SO3) = 1028. 1H NMR (400 MHz,
(CD3)2CO) : 9.08 (s br, 1H, H6), 8.83 (s br, 2H, H2), 8.40 (s br, 1H,
NH), 8.13 (m, 1H, H4), 7.86 (m, 2H, H3), 7.65 (m, 12H, HPh, 3, 5), 7.28
(m, 5H, CH2Ph), 4.92 (m, 1H, NHCH), 3.71 (s, 3H, COOCH3), A = 3.30
B = 3.16 (2H, system ABX, CH2Ph, JAX 5.4 Hz, JBX 9.4 Hz, JAB 13.9 Hz).
31 1
P{ H}NMR 400 MHz, (CD3)2CO) : 30.1 (s, 1P, AuPPh2py).
13 1
C{ H}NMR (400 MHz, (CD3)2CO) : 173.29 (s, 1C, COOCH3), 165.94
(s, 1C, CONH), 154.77 (d, 1C, H6, 1JCP 53.7 Hz), 152.77 (s, 2C, C2),
144.87 (s br, 1C, C4), 140.77 (s, 1C, C4), 139.18 (s, 1C, CipsoCH2Ph),
136.49 (d, 4C, CorthoPh, 2JCP 13.7 Hz), 134.72 (s br, 2C, CparaPh), 133.14
(d, 1C, C3, 2JCP 24.2 Hz), 131.53 (d, 4C, CmetaPh, 3JCP 12.2 Hz), 131.04 (s,
2C, CorthoCH2Ph), 130.23 (s, 1C, CparaCH2Ph), 128.73 (s br, 1C, C5), 128.56
(s, 2C, CmetaCH2Ph), 124.39 (s, 2C, C3), 56.38 (s, 1C, NHCH), 53.51 (s, 1C,
COOCH3), 38.73 (s, 1C, CH2Ph). UVvis, max nm: 230, 265.
4.7. Synthesis of [AuCl3(L)] (L1 (7), L2 (8), L3 (9))
To a methanol solution (25 mL) of L1 (47 mg, 0.2 mmol), L2 (42 mg,
0.2 mmol), or L3 (57 mg, 0.2 mmol) was added AuCl3 (47 mg,
0.2 mmol), at room temperature. After the addition, the reaction mix-
ture was stirred for 2 h. The volume was concentrated under vacuum
to 5 mL and addition of distilled water gave yellow precipitates. The
resulting yellow precipitates were ltered off and dried under vacuum.
4.7.1. [AuCl3(L1)] (7)
Yield: 74 mg, 68%. Anal. Calcd. (%) for C12H16AuCl3N2O3 (539.59): C,
26.71; H, 2.99; N, 5.19. Found: C, 27.17; H, 3.11; N, 5.27. IR (cm 1):
(NH) = 3276, (COcarboxylate) = 1743, (COamide) = 1645,
(AuCl) = 363. 1H NMR (400 MHz, (CD3)2CO) : 9.30 (m, 2H, H2),
8.43 (m, 1H, NH), 8.32 (m, 2H, H3), 4.62 (dd, 1H, NHCH, 3JHH 8.4 Hz,
3
JHH 6.4 Hz), 3.76 (s, 3H, COOCH3), 2.31 (m, 1H, CH(CH3)2), 1.05 (m,
6H, CH(CH3)2). 13C{1H}NMR (400 MHz, (CD3)2CO) : 173.08 (s, 1C,
COOCH), 165.34 (s, 1C, CONH), 153.01 (s, 2C, C2), 149.05 (s, 1C, C4),
128.03 (s, 2C, C3), 60.54 (s, 1C, NHCH), 53.36 (s, 1C, COOCH3), 32.50 (s,
1C, CH(CH3)2), 20.40 (s, 1C, CH(CH3)2), 19.60 (s, 1C, CH(CH3)2). UV
vis, max nm: 232, 265 (br).
4.7.2. [AuCl3(L2)] (8)
Yield: 96 mg, 94%. Anal. Calcd. (%) for C10H12AuCl3N2O3 (511.54): C,
23.48; H, 2.36; N, 5.48. Found: C, 23.75; H, 2.38; N, 5.53. IR (cm 1):
(NH) = 3342, (COcarboxylate) = 1729, (COamide) = 1654,
(AuCl) = 363. 1H NMR (400 MHz, (CD3)2CO) : 9.29 (m, 2H, H2),
8.63 (m, 1H, NH), 8.31 (m, 2H, H3), 4.69 (qd, 1H, NHCH, 3JHH 7.3 Hz,
3
JHH 7.3 Hz), 3.72 (s, 3H, COOCH3), 1.51 (d, 3H, CHCH3, 3JHH 7.3 Hz).
13 1
C{ H}NMR (300 MHz, (CD3)2CO) : 174.02 (s, 1C, COOCH3), 164.48
(s, 1C, CONH), 153.16 (s, 2C, C2), 148.66 (s, 1C, C4), 127.80 (s, 2C, C3),
53.58 (s, 1C, NHCH), 50.85 (s, 1C, COOCH3), 18.36 (s, 1C, CHCH3). UV
vis, max nm: 232, 265 (br).
 L. Ortego et al. / Journal of Inorganic Biochemistry 156 (2016) 133144
4.7.3. [AuCl3(L3)] (9)
Yield: 97 mg, 83%. Anal. Calcd. (%) for C16H16AuN2O3Cl3 (587.63): C,
32.70; H, 2.74; N, 4.77. %. Found: C, 33.13; H, 2.50; N, 4.75. IR (cm1):
(NH) = 3342, (COcarboxylate) = 1737, (COamide) = 1653,
(AuCl) = 363. 1H NMR (400 MHz, (CD3)2CO) : 9.26 (m, 2H, H2),
8.64 (d, 1H, NH, 3JHH 7.9 Hz), 8.20 (m, 2H, H3), 7.27 (m, 5H, CH2Ph),
4.95 (m, 1H, NHCH), 3.72 (s, 3H, COOCH3), A = 3.32 B = 3.15 (2H, sys-
tem ABX, CH2Ph, JAX 5.4 Hz, JBX 9.4 Hz, JAB 13.9 Hz). 13C{1H}NMR
(400 MHz, (CD3)2CO) : 172.91 (s, 1C, COOCH3), 164.67 (s, 1C, CONH),
153.14 (s, 2C, C2), 148.62 (s, 1C, C4), 138.78 (s, 1C, CipsoCH2Ph), 131.06
(s, 2C, CorthoCH2Ph), 130.30 (s, 1C, CparaCH2Ph), 128.68 (s, 2C, C3), 127.73
(s, 2C, CmetaCH2Ph), 56.52 (s, 1C, NHCH), 53.62 (s, 1C, COOCH3), 38.88 (s,
1C, CH2Ph). UVvis, max nm: 232, 265 (br).
4.8. Synthesis of [Ag(L)(PPh3)n](OTf) (L1, n = 1 (10), n = 2 (11); L2, n = 1
(13), n = 2 (14); L3, n = 1 (16), n = 2 (17))
To a DMC solution (25 mL) of L1 (47 mg, 0.2 mmol), or L2 (42 mg,
0.2 mmol), or L3 (57 mg, 0.2 mmol) was added 0.104 g (0.2 mmol) of
[Ag(OTf)PPh3], or 0.156 g (0.2 mmol) of [Ag(OTf)(PPh3)2] at room tem-
perature. After the addition, the reaction mixture was stirred for 1 h. The
volume was reduced to 5 mL, and addition of hexane afforded white
solids of 10, 11, 13, 14, 16 or 17.
4.8.1. [Ag(L1)(PPh3)](OTf) (10)
Yield: 127 mg, 84%. Anal. Calcd. (%) for C31H31AgF3N2O6PS (755.49):
C, 49.28, H, 4.14; N, 3.71; S, 4.24. Found: C, 49.71; H, 3.88; N, 3.76; S,
4.63. IR (cm1): (NH) = 3314, (COcarboxylate) = 1738, (COamide) =
1650, sym(C_N) = 1532, asym(SO3) = 1273 and 1240, sym(CF3) =
1220, asym(CF3) = 1154, sym(SO3) = 1024. 1H NMR (400 MHz,
(CD3)2CO) : 8.94 (m, 2H, H2), 8.18 (d, 1H, NH, 3JHH 8.2 Hz), 8.01 (m,
2H, H3), 7.59 (m, 15H, HPh), 4.58 (dd, 1H, NHCH, 3JHH 6.4 Hz, 3JHH
8.3 Hz), 3.74 (s, 3H, COOCH3), 2.28 (m, 1H, CH(CH3)2), 1.03 (m, 6H,
CH(CH3)2). 19F NMR (400 MHz, (CD3)2CO) : 79.93 (s, 1F, OTf).
31 1
P{ H}NMR (400 MHz, (CD3)2CO) : 17.4 and 12.9 (2 s br, 1P,
AgPPh3). 31P{1H}NMR (300 MHz, (CD3)2CO, 203 K), 12.4 (2 d, 1P,
AgPPh3, J109AgP 735.7 Hz, J107AgP 637.4 Hz), 9.6 (2 d, 2P, Ag(PPh3)2,
J109AgP 512.1 Hz, J107AgP 443.3 Hz). 13C{1H}NMR (400 MHz,
(CD3)2CO) : 172.48 (s, 1C, COOCH3), 165.69 (s, 1C, CONH), 152.67 (s,
2C, C2), 144.22 (s, 1C, C4), 134.83 (d, 6C, CorthoPh, 2JCP 16.1 Hz), 132.13
(s, 3C, CparaPh), 131.64 (d, 3C, CipsoPh, 1JCP 30.1 Hz), 130.24 (d, 6C, CmetaPh,
3
JCP 9.9 Hz), 123.66 (s, 2C, C3), 59.49 (s, 1C, NHCH), 52.29 (s, 1C,
COOCH3), 31.41 (s, 1C, CH(CH3)2), 19.50 (s, 1C, CH(CH3)2), 18.86 (s,
1C, CH(CH3)2). UVvis, max nm: 247, 275 (br).
4.8.2. [Ag(L1)(PPh3)2](OTf) (11)
Yield: 173 mg, 85%. Anal. Calcd. (%) for C49H46AgF3N2O6P2S
(1017.78): C, 57.82; H, 4.56; N, 2.75; S, 3.15. Found: C, 57.43; H, 4.30;
N, 2.85; S, 3.52. IR (cm1): (NH) = 3301, (COcarboxylate) = 1739,
(COamide) = 1664, sym(C_N) = 1532, asym(SO3) = 1282 and
1242, sym(CF3) = 1221, asym(CF3) = 1152, sym(SO3) = 1027. 1H
NMR (300 MHz, (CD3)2CO) : 8.76 (m, 2H, H2), 8.03 (m, 1H, NH), 7.85
(m, 2H, H3), 7.54 (m, 30H, HPh), 4.58 (dd, 1H, NHCH, 3JHH 8.4 Hz, 3JHH
6.4 Hz), 3.73 (s, 3H, COOCH3), 2.27 (m, 1H, CH(CH3)2), 1.02 (m, 6H,
CH(CH3)2). 19F NMR (400 MHz, (CD3)2CO) : 79.93 (s, 1F, OTf).
31 1
P{ H}NMR (300 MHz, (CD3)2CO) : 11.5 (s br, 2P, AgPPh3).
31 1
P{ H}NMR (300 MHz, (CD3)2CO, 203 K), 12.3 (2d, 1P, AgPPh3, J109Ag
P 716.9 Hz, J107AgP 622.1 Hz), 9.5 (2d, 1P, Ag(PPh3)2, J109AgP 492.7 Hz,
J107AgP 426.9 Hz, 8.8 (2d, 3P, Ag(PPh3)3, J107 AgP 367.1 Hz).
13
C{1H}NMR (300 MHz, (CD3)2CO) : 152.61 (s, 2C, C2), 135.70 (d,
12C, CorthoPh, 2JCP 15.4 Hz,), 132.13 (s, 6C, CparaPh), 132.97 (d, 12C, CmetaPh,
3
JCP 8.8 Hz), 123.65 (s, 2C, C3), 60.30 (s, 1C, NHCH), 53.20 (s, 1C,
COOCH3), 32.41 (s, 1C, CH(CH3)2), 20.47 (s, 1C, CH(CH3)2), 19.80 (s,
1C, CH(CH3)2).
141
4.8.3. [Ag(L2)(PPh3)](OTf) (13)
Yield: 125 mg, 86%. Anal. Calcd. (%) for C29H27AgF3N2O6PS (727.44):
C, 47.88; H, 3.74; N, 3.85; S, 4.41. Found: C, 47.52; H, 3.75; N, 3.90; S,
4.72. IR (cm1): (NH) = 3312, (COcarboxylate) = 1740, (COamide) =
1649, sym(C_N) = 1537, asym(SO3) = 1281 and 1241, sym(CF3) =
1220, asym(CF3) = 1157, sym(SO3) = 1025. 1H NMR (400 MHz,
(CD3)2CO) : 8.93 (m, 2H, H2), 8.42 (d, NH, 3JHH 7.3 Hz), 8.01 (m, 2H,
H3), 7.57 (m, 15H, HPh), 4.66 (qd, 1H, NHCH, 3JHH 7.3 Hz, 3JHH 7.3 Hz),
3.71 (s, 3H, COOCH3), 1.50 (d, 3H, CHCH3, 3JHH 7.4 Hz). 19F NMR
(400 MHz, (CD3)2CO) : 79.91 (s, 1F, OTf). 31P{1H}NMR (400 MHz,
(CD3)2CO) : 17.0 and 12.74 (2 s br, 1P, AgPPh3). 31P{1H}NMR
(300 MHz, (CD3)2CO, 203 K) : 12.5 (2d, 1P, AgPPh3, J109AgP 732.8 Hz,
J107AgP 635.0 Hz), 9.7 (2d, 2P, Ag(PPh3)2, J109AgP 507.3 Hz, J107AgP
439.6 Hz). 13C{1H}NMR (300 MHz, (CD3)2CO) : 174.36 (s, 1C,
COOCH3), 166.01 (s, 1C, CONH), 153.39 (s, 2C, C2), 144.59 (s, 1C, C4),
135.75 (d, 6C, CorthoPh, 2JCP 15.5 Hz), 133.02 (s, 3C, CparaPh), 132.40
(s br, 3C, CipsoPh), 131.17 (d, 6C, CmetaPh, 3JCP 8.8 Hz), 124.23 (s, 2C, C3),
53.42 (s, 1C, NHCH), 50.61 (s, 1C, COOCH3), 18.31 (s, 1C, CHCH3). UV
vis, max nm: 248, 275.
4.8.4. [Ag(L2)(PPh3)2](OTf) (14)
Yield: 180 mg, 91%. Anal. Calcd. (%) for C47H42AgF3N2O6P2S
(989.72): C, 57.04; H, 4.28; N, 2.83; S, 3.24. Found: C, 57.17; H, 4.22;
N, 2.85; S, 3.24. IR (cm 1): (NH) = 3312, (COcarboxylate) = 1740,
(COamide) = 1665, sym(C_N) = 1538, asym(SO3) = 1284 and
1242, sym(CF3) = 1220, asym(CF3) = 1152, sym(SO3) = 1026. 1H
NMR (400 MHz, (CD3)2CO) : 8.77 (m, 2H, H2), 8.29 (s br, 1H, NH),
7.84 (m, 2H, H3), 7.53 (m, 30H, HPh), 4.66 (qd, 1H, NHCH, 3JHH 7.3 Hz,
3
JHH 7.3 Hz), 3.71 (s, 3H, COOCH3), 1.49 (d, 3H, CHCH3, 3JHH 7.3 Hz).
19
F NMR (400 MHz, (CD3)2CO) : 80.03 (s, 1F, OTf). 31P{1H}NMR
(400 MHz, (CD3)2CO) : 12.6 (s br, 1P, AgPPh3). 31P{1H}NMR
(400 MHz, (CD3)2CO), 203 K) : 12.5 (2d, 1P, AgPPh3, J109AgP
726.6 Hz, J107AgP 629.2 Hz), 9.7 (2d, 2P, Ag(PPh3)2, J109AgP 500.6 Hz,
J107AgP 317.7 Hz), 8.8 (2d, 3P, Ag(PPh3)3, J109AgP 433.7 Hz, J107AgP
369.9 Hz). 13C{1H}NMR (400 MHz, (CD3)2CO) : 174.43 (s, 1C,
COOCH3), 166.30 (s, 1C, CONH), 152.76 (s, 2C, C2), 143.84 (s, 1C, C4),
135.67 (d, 12C, CorthoPh, 2JCP 15.7 Hz), 132.02 (s, 6C, CparaPh), 132.61
(s br, 6C, CipsoPh), 131.15 (d, 12C, CmetaPh, 3JCP 9.2 Hz), 123.70 (s, 2C,
C3), 53.40 (s, 1C, NHCH), 50.55 (s, 1C, COOCH3), 18.36 (s, 1C, CHCH3).
4.8.5. [Ag(L3)(PPh3)](OTf) (16)
Yield: 159 mg, 99%. Anal. Calcd. (%) for C35H31AgF3N2O6PS (803.53):
C, 52.32; H, 3.89; N, 3.49; S, 3.99. Found: C, 52.43; H, 3.99; N, 3.80; S,
4.25. IR (cm1): (NH) = 3299, (COcarboxylate) = 1739, (COamide) =
1654, sym(C = N) = 1536, asym(SO3) = 1283 and 1241, sym(CF3) =
1220, asym(CF3) = 1155, sym(SO3) = 1025. 1H NMR (400 MHz,
(CD3)2CO) : 8.87 (s br, 2H, H2), 8.39 (d, 1H, NH, 3JHH 7.9 Hz), 7.88
(s br, 2 H, H3), 7.65 (m, 15H, HPh), 7.56 (m, 5H, CH2Ph), 4.93 (m, 1H,
NHCH), 3.70 (s, 3H, COOCH3), A = 3.30 B = 3.15 (2H, system ABX,
CH2Ph, JAX 5.4 Hz, JBX 9.4 Hz, JAB 13.9 Hz). 31P{1H}NMR (400 MHz,
(CD3)2CO) : 12.4 (s br, 1P, AgPPh3). 31P{1H}NMR (300 MHz,
(CD3)2CO), 203 K) : 12.7 (2d, 1P, AgPPh3, J109AgP 754.0 Hz, J107AgP
653.0 Hz), 9.8 (2d, 2P, Ag(PPh3)2, J109AgP 539.9 Hz, J107AgP 468.12 Hz).
13 1
C{ H}NMR (400 MHz, (CD3)2CO) : 173.31 (s, 1C, COOCH3), 166.21
(s, 1C, CONH), 153.40 (s, 2C, C2), 144.54 (s, 1C, C4), 139.17 (s, 1C,
CipsoCH2Ph), 135.74 (d, 6C, CorthoPh, 2JCP 14.9 Hz), 133.02 (s, 3C, CparaPh),
132.61 (d, 3C, CipsoPh, 1JCP 33.9 Hz), 131.17 (d, 6C, CmetaPh, 3JCP 7.5 Hz),
131.04 (s, 2C, CorthoCH2Ph), 130.22 (s, 1C, CparaCH2Ph), 128.55 (s, 2C,
CmetaCH2Ph), 124.16 (s br, 2C, C3), 56.38 (s, 1C, NHCH), 53.49 (s, 1C,
COOCH3), 38.77 (s, 1C, CH2Ph). UVvis, max nm: 248, 275.
4.8.6. [Ag(L3)(PPh3)2](OTf) (17)
Yield: 204 mg, 96%. Anal. Calcd. (%) for C53H46AgF3N2O6P2S
(1065.82): C, 59.73; H, 4.35; N, 2.63; S, 3.01. Found: C, 59.45; H, 4.36;
N, 2.93; S, 3.29. IR (cm 1): (NH) = 3313, (COcarboxylate) = 1741,
(COamide) = 1667, sym(C_N) = 1539, asym(SO3) = 1282 and
142 L. Ortego et al. / Journal of Inorganic Biochemistry 156 (2016) 133144
1243, sym(CF3) = 1221, asym(CF3) = 1152, sym(SO3) = 1027. 1H
NMR (400 MHz, (CD3)2CO) : 8.74 (m, 2H, H2), 8.29 (s br, 1H, NH),
8.03 (m, 2H, H3), 7.39 (m, 30H, HPh), 7.28 (m, 5H, CH2Ph), 4.90 (m, 1H,
NHCH), 3.70 (s, 3H, COOCH3), A = 3.30 B = 3.15 (2H, system ABX,
CH2Ph, JAX 5.4 Hz, JBX 9.4 Hz, JAB 13.9 Hz). 31P{1H}NMR (400 MHz,
(CD3)2CO) : 12.3 (s br, 2P, Ag(PPh3)2). 31P{1H}NMR (300 MHz,
(CD3)2CO), 203 K) : 12.6 (2d, 1P, AgPPh3, J109AgP 737.3 Hz, J107AgP
638.4 Hz), 9.9 (2d, 2P, Ag(PPh3)2, J109AgP 513.7 Hz, J107AgP 445.0 Hz).
13
C{1H}NMR (400 MHz, (CD3)2CO) : 173.37 (s, 1C, COOCH3), 166.47
(s, 1C, CONH), 152.85 (s, 2C, C2), 143.81 (s, 1C, C4), 139.20 (s, 1C,
CipsoCH2Ph), 135.67 (d, 12C, CorthoPh, 2JCP 15.7 Hz), 132.90 (s, 6C, CparaPh),
132.62 (s br, 6C, CipsoPh), 131.14 (d, 12C, CmetaPh, 3JCP 9.0 Hz), 131.04 (s,
2C, CorthoCH2Ph), 130.22 (s, 1C, CparaCH2Ph), 128.54 (s, 2C, CmetaCH2Ph),
123.67 (s br, 2C, C3), 56.31 (s, 1C, NHCH), 53.46 (s, 1C, COOCH3),
38.80(s, 2C, CH2Ph).
4.9. Synthesis of [Ag(L)(PPh2py)](OTf) (L1 (12), L2 (15), L3 (18))
To a DMC solution (25 mL) of L1 (47 mg, 0.2 mmol), L2 (42 mg,
0.2 mmol), or L3 (57 mg, 0.2 mmol) was added [Ag(OTf)(PPh2py)]
(105 mg, 0.2 mmol) at room temperature. After the addition, the reac-
tion mixture was stirred for 1 h. The volume was reduced to 5 mL, and
addition of hexane afforded compounds 12, 15 and 18.
4.9.1. [Ag(L1)(PPh2py)](OTf) (12)
Yield: 124 mg, 82%. Anal. Calcd. (%) for C30H30AgF3N3O2PS (756.48):
C, 47.63; H, 4.00; N, 5.55; S, 4.24. Found: C, 47.55; H, 3.90; N, 5.43; S,
3.94. IR (cm1): (NH) = 3287, (COcarboxylate) = 1736, (COamide) =
1662, sym(C_N) = 1534, asym(SO3) = 1277 and 1242, sym(CF3) =
1221, asym(CF3) = 1153, sym(SO3) = 1027. 1H NMR (400 MHz,
(CD3)2CO) : 9.27 (s br, 1H, H6), 8.69 (m, 2H, H2), 8.22 (d, 1H, NH,
3
JHH 7.9 Hz), 8.14 (m, 1H, H4), 7.89 (m, 2H, H3), 7.80 (m, 1H, H5), 7.40
(m, 11H, HPh,3), 4.54 (m, 1H, NHCH), 3.72 (s, 3H, COOCH3), 2.27 (m,
1H, CH(CH3)2), 1.02 (m, 6H, CH(CH3)2). 19F NMR (400 MHz,
(CD3)2CO) : 80.07 (s, 1F, OTf). 31P{1H}NMR (400 MHz, (CD3)2CO)
: 18.7 (s br, 1P, AgPPh2py). 31P{1H}NMR (300 MHz, (CD3)2CO, 203 K)
: 20.0 and 15.0 (2 m, 1P, AgPPh2py, Javerage 666.5Hz). 13C{1H}NMR
(400 MHz, (CD3)2CO) : 173.42 (s, 1C, COOCH3), 166.78 (s, 1C, CONH),
157.93 (d, 1C, C2, 1JCP 55.8 Hz), 155.20 (d, 1C, C6, 3JCP 16.0 Hz), 151.59
(s, 2C, C2), 144.94 (s, 1C, C4), 141.43 (s, 1C, C4), 135.48 (d, 4C, CorthoPh,
2
JCP 14.2 Hz), 133.40 (s, 2C, CparaPh), 132.35 (m, 1C, H3), 131.46 (d, 4C,
CmetaPh, 3JCP 8.2 Hz), 130.48 (m, 2C, CipsoPh), 128.38 (s, 1C, C5), 124.32
(s, 2C, C3), 60.45 (s, 1C, NHCH), 53.21 (s, 1C, COOCH3), 32.21 (s, 1C,
CH(CH3)2), 20.43 (s, 1C, CH(CH3)2), 19.84 (s, 1C, CH(CH3)2). UVvis,
max nm: 230, 264.
4.9.2. [Ag(L2)(PPh2py)](OTf) (15)
Yield: 128 mg, 88%. Anal. Calcd. (%) for C28H26AgF3N3O6PS (728.43):
C, 46.17; H, 3.60; N, 5.77; S, 4.40. Found: C, 46.42; H, 3.74; N, 5.61; S,
4.04. IR (cm1): (NH) = 3316, (COcarboxylate) = 1740, (COamide) =
1663, sym(C_N) = 1539, asym(SO3) = 1275 and 1243, sym(CF3) =
1221, asym(CF3) = 1154, sym(SO3) = 1027. 1H NMR (400 MHz,
(CD3)2CO) : 9.25 (s br, 1H, H6), 8.73 (m, 2H, H2), 8.34 (m, 1H, NH),
8.16 (m, 1H, H4), 7.89 (m, 2H, H3), 7.81 (m, 1H, H5), 7.46 (m, 11H,
HPh,3), 4.64 (qd, 1H, NHCH, 3JHH 7.3 Hz, 3JHH 7.3 Hz), 3.70 (s, 3H,
COOCH3), 1.49 (d, 3H, CHCH3, 3JHH 7.3 Hz). 19F NMR (400 MHz,
(CD3)2CO) : 80.06 (s, 1F, OTf). 31P{1H}NMR (400 MHz, (CD3)2CO)
: 20.4 and 14.4 (s br, 1P, AgPPh2py). 31P{1H}NMR (400 MHz,
(CD3)2CO), 203 K) : 17.4 (2 m, AgPPh2py, Javerage 695.8 Hz).
13
C{1H}NMR (400 MHz, (CD3)2CO) : 174.36 (s, 1C, COOCH3), 166.01
(s, 1C, CONH), 157.91 (d, 1C, C2, 1JCP 53.71 Hz), 155.20 (s br, 1C, C6),
151.59 (s, 2C, C2), 144.77 (s, 1C, C4), 141.53 (s, 1C, C4), 135.56 (s br,
4C, CorthoPh), 133.49 (s, 2C, CparaPh), 132.47 (m, 2C, C3), 131.49 (s br, 4C,
CmetaPh), 128.45 (s, 1C, C5), 124.15 (s, 2C, C3), 53.42 (s, 1C, NHCH),
50.61 (s, 1C, COOCH3), 18.28 (s, 1C, CHCH3). UVvis, max nm: 234, 264.
4.9.3. [Ag(L3)(PPh2py)](OTf) (18)
Yield: 157 mg, 97%. Anal. Calcd. (%) for C34H30AgF3N3O6PS (804.52):
C, 50.76; H, 3.76; N, 5.22; S, 3.99. Found: C, 50.83; H, 3.84; N, 5.65; S,
4.04. IR (cm1): (NH) = 3322, (COcarboxylate) = 1740, (COamide) =
1666, sym(C_N) = 1540, asym(SO3) = 1265 and 1250, sym(CF3) =
1221, asym(CF3) = 1151, sym(SO3) = 1028. 1H NMR (400 MHz,
(CD3)2CO) : 9.14 (s br, 1H, H6), 8.84 (m, 2H, H2), 8.35 (m, 1H, NH),
8.17 (m, 1H, H4), 7.89 (m, 3H, H3, 5), 7.63 (m, 11H, HPh,3), 7.28 (m,
5H, CH2Ph), 4.93 (m, 1H, NHCH), 3.72 (s, 3H, COOCH3), A = 3.31
B = 3.16 (2H, system ABX, CH2Ph, JAX 5.4 Hz, JBX 9.4 Hz, JAB 13.9 Hz).
31 1
P{ H}NMR (400 MHz, (CD3)2CO) : 30.1 (s br, 1P, AgPPh2py).
31 1
P{ H}NMR (300 MHz, (CD3)2CO), 203 K) : 17.9 (2 m, 1P, AgPPh2py,
Javerage 623.4 Hz). 13C{1H}NMR (400 MHz, (CD3)2CO) : 173.36 (s, 1C,
COOCH3), 166.31 (s, 1C, CONH), 155.37 (s br, 1C, C6), 151.56 (s, 2C,
C2), 144.56 (s, 1C, C4), 141.65 (s, 1C, C4), 139.17 (s, 1C, CipsoCH2Ph),
135.57 (s br, 4C, CorthoPh), 133.54 (s br, 2C, CparaPh), 132.55 (s br, 1C,
C3), 131.54 (s br, 4C, CmetaPh), 131.05 (s, 2C, CorthoCH2Ph), 130.25 (s, 1C,
CparaCH2Ph), 128.57 (s, 2C, CmetaCH2Ph), 123.91 (s, 2C, C3), 56.32 (s, 1C,
NHCH), 53.49 (s, 1C, COOCH3), 38.80 (s, 1C, CH2Ph). UVvis, max nm:
230, 264.
4.10. Synthesis of [Cu(L)(PPh3)]NO3 (L1 (19), L2 (20), L3 (21))
These reactions were carried out under Ar, in dried solvents and
degassed prior to use. To a DMC solution (25 mL) was added L1
(47 mg, 0.2 mmol), L2 (42 mg, 0.2 mmol), or L3 (57 mg, 0.2 mmol)
and [Cu(NO3)(PPh3)2] (130 mg, 0.2 mmol), at room temperature.
After the addition, the reaction mixture was stirred for 1 h and then
was concentrated under vacuum. The addition of hexane gave yellow
precipitates that were ltered off.
4.10.1. [Cu(L1)(PPh3)](NO3) (19)
Yield: 97 mg, 78%. Anal. Calcd. (%) for C30H31CuN3O6P (624.10): C,
57.73; H, 5.01; N, 6.73. Found: C, 57.69; H, 5.46; N, 6.83. IR (cm 1):
(NH) = 3203, (COcarboxylate) = 1732, (COamide) = 1662,
sym(C_N) = 1537, (NO3) = 1095, 1022, 1009, 828, 692. 1H NMR
(400 MHz, (CD3)2CO) : 8.71 (m, 1H, H2), 7.99 (s br, 1H, NH), 7.79 (m,
2H, H3), 7.42 (m, 15H, HPh), 4.57 (dd, 1H, NHCH, 3JHH 4.9, 3JHH 8.5 Hz),
3.72 (s, 3H, COOCH3), 2.26 (m, 1H, CH(CH3)2), 1.02 (m, 6H, CH(CH3)2).
31 1
P{ H}NMR (400 MHz, (CD3)2CO) : 0.0 (s, CuPPh3). 13C{1H}NMR
(300 MHz, CDCl3) : 172.19 (s, 1C, COOCH3), 165.31 (s, 1C, CONH),
150.63 (s, 2C, C2), 133.60 (d, 6C, CorthoPh, 2JCP 13.0 Hz), 131.73 (d, 3C,
CipsoPh, 1JCP 32.9 Hz), 130.18 (s, 3C, CparaPh), 128.82 (d, 6C, CmetaPh, 3JCP
5.6 Hz), 57.59 (s, 1C, NHCH), 52.42 (s, 1C, COOCH3), 31.56 (s, 1C,
CH(CH3)2), 18.94 (s, 1C, CH(CH3)2), 17.93 (s, 1C, CH(CH3)2).
4.10.2. [Cu(L2)(PPh3)](NO3) (20)
Yield: 82 mg, 69%. Anal. Calcd. (%) for C28H27CuN3O6P (596.05): C,
56.42; H, 4.57; N, 7.05. Found: C, 56.65; H, 4.61; N, 4.79. IR (cm 1):
(NH) = 3296, (COcarboxylate) = 1741, (COamide) = 1665,
sym(C_N) = 1540, (NO3) = 1095, 1024, 1024, 849, 692. 1H NMR
(400 MHz, CDCl3) : 8.55 (m, 2H, H2), 7.66 (m, 2H, H3), 7.43 (d, 1H,
NH, 3JHH 7.0 Hz), 7.27 (m, 15H, HPh), 4.73 (qd, 1H, NHCH, 3JHH 7.2 Hz,
3
JHH 7.2 Hz), 3.77 (s, 3H, COOCH3), 1.51 (d, 3H, CHCH3, 3JHH 7.2 Hz).
31 1
P{ H}NMR (400 MHz, CDCl3) : 0.0 (s, 1P, CuPPh3). 13C{1H}NMR
(400 MHz, CDCl3) : 174.15 (s, 1C, COOCH3), 164.83 (s, 1C, CONH),
135.58 (d, 6C, CorthoPh, 2JCP 13.9 Hz), 132.61 (d, 3C, CipsoPh, 1JCP 33.8 Hz),
130.17 (s, 3C, CparaPh), 128.82 (d, 6C, CmetaPh, 3JCP 7.5 Hz), 52.70 (s, 1C,
NHCH), 48.70 (s, 1C, COOCH3), 18.25 (s, 1C, CHCH3).
 L. Ortego et al. / Journal of Inorganic Biochemistry 156 (2016) 133144 143

4.10.3. [Cu(L3)(PPh3)](NO3) (21) Munchen, Germany). The IC50 was calculated by nonlinear regression
Yield: 100 mg, 76%. Anal. Calcd. (%) for C34H31CuN3O6P (672.16): C, analysis.
60.76; H, 4.65; N, 6.25. Found: C, 60.43; H, 4.16; N, 6.71. IR (cm 1):
(NH) = 3246, (COcarboxylate) = 1742, (COamide) = 1666, 4.14. Flow cytometry analysis of cell death
sym(C_N) = 1539, (NO3) = 1094, 1023, 1023, 844, 692. 1H NMR
(400 MHz, DMSO) : 9.25 (m, 2H, H2), 8.82 (s br, 1H, NH), 7.77 (s br, NIH-3 T3 cells were seeded in 6-well plate format at 100,000 cells/
2H, H3), 7.40 (m, 20H, CH2Ph, HPh), 4.75 (m, 1H, NHCH), 3.71 (s, 3H, well using 1 mL DMEM media supplemented (see Table 3). Cells were
COOCH3), A = 3.25 B = 3.14 (2H, system ABX, CH2Ph, JAB 5.2 Hz, JBX exposed for 24 h to IC50 concentrations of complexes and ligands.
10.2 Hz, JAX 13.7 Hz). 31P{1H}NMR (400 MHz, DMSO) : 3.7 (s, 1P, Cells from each individual well were centrifuged 400 x g for 5 min,
CuPPh3). 13C{1H}NMR (300 MHz, (CD3)2CO) : 173.46 (s, 1C, COOCH3), washed two times with cold PBS and then resuspended in 200 L of
166.86 (s br, 1C, CONH), 152.32 (s br, 2C, C2), 139.17 (s, 1C, CipsoCH2Ph), binding buffer. 100 L of each cell solution was transferred to a culture
135.41 (d, 6C, CorthoPh, 2JCP 14.9 Hz), 134.17 (d, 3C, CipsoPh, 1JCP 32.0 Hz), tube and added 5 L of Annexin V-FITC and 5 L of PI (stock solution
132.22 (s, 3C, CparaPh), 131.04 (s, 2C, CorthoCH2Ph), 130.80 (d, 6C, CmetaPh, 50 g/mL). After incubation for 20 min in the dark, cell suspensions
3
JCP 9.3 Hz), 130.24 (s, 1C, CparaCH2Ph), 128.57 (s, 2C, CmetaCH2Ph), 56.23 were added to 400 L of binding buffer, gently homogenized, and ana-
(s, 1C, NHCH), 53.45 (s, 1C, COOCH3), 38.88 (s, 1C, CH2Ph). lyzed by ow cytometry (Coulter Epics XL-MCL, Coulter, Krefeld,
Germany).
4.11. Crystal structure determination
Abbreviations
Data were registered on a Bruker Smart APEX CCD (7) or Xcalibur
Oxford Diffraction (8) diffractometers. The crystals were mounted in asym asymmetry
inert oil on glass bers and transferred to the cold gas stream of the CDCl3 chloroform-d
diffractometers. Data were collected using monochromated Mo K ra- (CD3)2CO acetone-d6
diation ( = 0.710 73) in scans. Absorption corrections based on mul- COSY correlation spectroscopy
tiple scans were applied with the program SADABS [61]. The structures DMC dichlorometane
were solved by direct methods and rened on F2 using the program DMEM dulbecco's modied eagle's medium
SHELXL-97 [62]. All non-hydrogen atoms were rened anisotropically. HSQC heteronuclear single quantum coherence spectroscopy
Hydrogen atoms were included by using a riding model. CCDC- Hz Hertz
1,423,474 (7) and CCDC-1,423,475 (8) contain the supplementary crys- FCS fetal calf serum
tallographic data for this paper. These data can be obtained free of FITC uorescein isothiocyanate
charge from The Cambridge Crystallographic Data Centre via www. IC50 half maximal inhibitory concentration
ccdc.cam.ac.uk/data_request/cif. IR infrared
MTT 3-(4,5-dimethyl-thiazol-2-yl)-2,5-diphenyl tetrazolium
bromide
4.12. Cell cultures
NCS newborn calf serum
NMR nuclear magnetic resonance
Cell lines were cultivated in Dulbecco's Modied Eagle's medium
OTf triuoromethanesulfonate
(DMEM, D7777 Sigma-Aldrich, Steinheim, Germany) supplemented
P penicillin
with the according serum (FCS, fetal calf serum or NCS, newborn calf
PBS phosphate buffered saline
serum) (Table 3) and 1% antibiotics (penicillin/100 U/mL and strepto-
PI propidium iodide
mycin/100 g/mL) in a humidied atmosphere of 95% air/37 C/5%
PS phosphatidylserine
CO2. After 34 days the cells had grown to conuence and were then de-
Py pyridine
tached with trypsin and cultured in a new cell culture ask.
ROS reactive oxygen species
S streptomycin
4.13. Cytotoxicity assays by MTT sym symmetry

The MTT assay was used to determine cell viability as an indicator for Acknowledgments
cell sensitivity to the complexes (MTT, Sigma-Aldrich, Steinheim,
Germany). The authors thank the Ministerio de Economa y Competitividad-
Exponentially growing cells were seeded with a dened number of FEDER CTQ2013-48635-C2-1-P and DGA-FSE (E77) for nancial
cells (see Table 3), in a 96-well at-bottomed microplate and 48 h support.
later they were incubated with the compounds. The complexes were
dissolved in DMSO and tested in concentrations ranging from 1 to
Appendix A. Supplementary data
50 M. Cytotoxicity of tested compounds was evaluated by the MTT
method [63]. Controls were made by incubating cells with supplement-
Supplementary data to this article can be found online at http://dx.
ed media and supplemented media with 5% DMSO. Optical density was
doi.org/10.1016/j.jinorgbio.2015.12.018.
measured at 570 nm using a 96-well multiscanner autoreader (Bio-Rad,

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