Multi-target heteroleptic palladium bisphosphonate complexes

Article abstract of DOI:10.1007/s00775-020-01779-y

Abstract: Bisphosphonates are the most commonly prescribed drugs for the treatment of osteoporosis and other bone illnesses. Some of them have also shown antiparasitic activity. In search of improving the pharmacological profile of commercial bisphosphona

Full text of DOI:10.1007/s00775-020-01779-y

JBIC Journal of Biological Inorganic Chemistry
https://doi.org/10.1007/s00775-020-01779-y
ORIGINAL PAPER
Multi‑target heteroleptic palladium bisphosphonate complexes
Micaella Cipriani1 · Santiago Rostán1 · Ignacio León2 · Zhu‑Hong Li3 · Jorge S. Ganchef1 · Ulrike Kemmerling4 ·
Claudio Olea Azar5 · Susana Etcheverry2 · Roberto Docampo3 · Dinorah Gambino1 · Lucía Otero1
Received: 20 December 2019 / Accepted: 16 March 2020
© Society for Biological Inorganic Chemistry (SBIC) 2020
Abstract
Bisphosphonates are the most commonly prescribed drugs for the treatment of osteoporosis and other bone illnesses. Some
of them have also shown antiparasitic activity. In search of improving the pharmacological profle of commercial bis-
phosphonates, our group had previously developed frst row transition metal complexes with N-containing bisphospho-
nates (NBPs). In this work, we extended our studies to heteroleptic palladium–NBP complexes including DNA interca-
lating polypyridyl co-ligands (NN) with the aim of obtaining potential multi-target species. Complexes of the formula
[Pd(NBP)2(NN)]·2NaCl·xH2O with NBP=alendronate (ale) or pamidronate (pam) and NN=1,10 phenanthroline (phen) or
2,2′-bipyridine (bpy) were synthesized and fully characterized. All the obtained compounds were much more active in vitro
against T. cruzi (amastigote form) than the corresponding NBP ligands. In addition, complexes were nontoxic to mammalian
cells up to 50–100 µM. Compounds with phen as ligand were 15 times more active than their bpy analogous. Related to the
potential mechanism of action, all complexes were potent inhibitors of two parasitic enzymes of the isoprenoid biosynthetic
pathway. No correlation between the anti-T. cruzi activity and the enzymatic inhibition results was observed. On the con-
trary, the high antiparasitic activity of phen-containing complexes could be related to their ability to interact with DNA in
an intercalative-like mode. These rationally designed compounds are good candidates for further studies and good leaders
for future drug developments.
Graphic abstract
Four new palladium heteroleptic complexes with N-containing commercial bisphosphonates and DNA intercalating polypyri-
dyl co-ligands were synthesized and fully characterized. All complexes displayed high anti-T. cruzi activity which could be
related to the inhibition of the parasitic farnesyl diphosphate synthase enzyme but mainly to their ability to interact DNA.
NN = 1,10 phenathroline
NBP = pamidronate (n = 0)
alendronate (n = 1)
NN = 2,2´bipyridine
Keywords Bisphosphonate · Palladium · DNA · Chagas · Toxoplasmosis
Introduction
Electronic supplementary material The online version of this
article (https://doi.org/10.1007/s00775-020-01779-y) contains
Bisphosphonates are structural analogs of inorganic pyroph-
supplementary material, which is available to authorized users.
osphate that are resistant to enzymatic and chemical break-
down. These compounds are well-established drugs for the
prevention and treatment of diseases associated with exces-
sive bone resorption, including Paget’s disease of bone,
* Lucía Otero
luotero@fq.edu.uy
Extended author information available on the last page of the article
Vol.:(0123456789)
1 3

JBIC Journal of Biological Inorganic Chemistry
multi-target therapy by designing a single chemical entity
that acts simultaneously on multiple targets through, not
only the metal, but the diferent coordinated ligands [15, 16]
(Fig. 2). Palladium was selected as metal center and, besides
NBPs, polypyridyl compounds (NN, Fig. 1) were chosen as
ligands. On the one hand, as stated, parasitic FPPS enzyme
could be a potential target for the proposed complexes as it
is for the free NBPs and these ligands would contribute to
the selectivity of the compounds through accumulation in
acidocalcisomes [8]. On the other hand, the selection of both
the metal ion and the NN co-ligands is intended for pointing
out DNA as target. The reason why this biomolecule could
be a potential parasitic target is based on that DNA has clas-
sically been involved in the mode of action of metal-based
anticancer compounds [17]. Due to the observed metabolic
similarities between tumor cells and parasites, a parallel-
ism between the antitumor and antiparasitic efects has
been proposed [18–21]. In particular, as an alternative to
the classic covalent DNA interaction, metal complexes with
extended planar aromatic ligands can also bind reversibly
to DNA through intercalation of the planar aromatic moiety
between nucleobases [22, 23]. The most widely investigated
intercalating ligands are derivatives of 2,2′-bipyridine (bpy)
or 1,10-phenanthroline (phen). In addition, some of us had
demonstrated that metal complexes with this kind of ligands
also showed antiparasitic activity [24–26].
(a)
(d)
(b)
(c)
(e)
Fig. 1 Top: a pyrophosphate; b general structure of bisphosphonates;
c pamidronate (pam, n=1, acid form), alendronate (ale, n=2, acid
form); Bottom: polypyridyl compounds (NN=1,10-phenanthroline
(phen, (d)), 2,2-bipyridine (bpy, (e))
myeloma, bone metastases, and osteoporosis [1]. The chemi-
cal structure of bisphosphonates (Fig. 1) allows changes in
the two lateral chains (R1 and R2) leading to variations in
physicochemical and biological properties as well as to dif-
ferent mechanisms of action. The simplest, non-nitrogen-
containing bisphosphonates can be metabolically incorpo-
rated as non-hydrolysable analogs of adenosine triphosphate
(ATP). In contrast, the more potent nitrogen-containing
bisphosphonates (NBPs) inhibit a key enzyme in the meva-
lonate pathway, farnesyl pyrophosphate synthase (FPPS),
leading to osteoclast apoptosis [2–4].
The selection of palladium as the metal center for the
proposed complexes is based on that Pd(II) complexes have
been widely studied as an alternative to Pt(II) ones in cancer
therapy [27]. In addition, both palladium and platinum com-
plexes have shown activity against trypanosomatid parasites
[28–30]. Besides the chemical resemblance between both
metal ions, Pd(II) complexes are kinetically less stable than
their Pt(II) analogs tending to dissociate readily in solution
leading to toxicity. To stabilize Pd(II) complexes, chelating
ligands are often envisaged and in particular, NN heterocy-
clic derivatives [31]. Moreover, as Pd(II) ions usually adopt
square planar geometries, planarity of the ligands could be
extended to the square planar complex making the complex
more susceptible to intercalate in DNA [32].
NBPs are also potent inhibitors of the proliferation of
trypanosomatid (Trypanosoma cruzi, Trypanosoma brucei
rhodesiense and Leishmania donovani) and Apicomplexan
(Toxoplasma gondii and Plasmodium falciparum) parasites
[5–7]. These parasites have some organelles called acido-
calcisomes that are equal in composition to bone mineral
and that could accumulate bisphosphonates aiding to their
antiparasitic activity [8]. In addition, the isoprenoid biosyn-
thesis is also the antiparasitic target of NBPs through the
inhibition of the enzymatic activity of FPPS [9–11].
Our group had previously developed frst row transition
metal complexes with some commercial NBPs, i.e., rise-
dronate, alendronate, pamidronate and ibandronate in the
search of more efective anti-Trypanosoma cruzi (T. cruzi)
agents. Obtained compounds showed IC50 values in vitro
against the amastigote form of the parasite in the low micro
molar levels with low toxicity on mammalian Vero cells. As
a consequence of metal complexation, the activity of the
free NBPs increased and the obtained complexes were able
to selectively inhibit T. cruzi FPPS [12–14].
In this work, four new complexes of the formula [Pd(NN)
(NBP)2] with NN=2,2-bipyridine (bpy) or 1,10-phenanth-
roline (phen) (Fig. 1) and NBP=alendronate or pamidronate
Based on these results, we have decided to rationally
design multi-target metal complexes including NBPs as
ligands with the purpose of developing new potential anti-
T. cruzi agents. Metal complexes are ideal candidates for a
Fig. 2 Rational design of proposed multi-target palladium complexes
1 3

JBIC Journal of Biological Inorganic Chemistry
(Fig. 1) were synthesized and fully characterized in the solid
state and in solution. Computational studies at the density
functional level of theory (DFT) were performed to calcu-
late the vibrational spectra of the complexes at the opti-
mized geometries. The in vitro antiproliferative efect of the
obtained compounds on T. cruzi amastigotes, their ability to
inhibit two parasitic enzymes of the mevalonate pathway and
to interact with DNA were evaluated. Additionally, activity
on Toxoplasma gondii (T. gondii) parasite and preliminary
anticancer activity were also studied.
[Pd(pam)2(bpy)]·7H2O·2NaCl, Pd–pam–bpy (yield:
92 mg, 63%). Anal. (%) calc. for C16H42Cl2N4Na2O21P4Pd:
C, 19.74; H, 4.35; N, 5.75; H2O 13.4. Found: C, 20.01; H,
1
4.25; N, 5.69; H2O 12.9; H-NMR: δ (ppm): 8.56 (d, 1H,
H-C6, bpy moiety), 8.34 (t, 1H, H-C5, bpy moiety); 8.23(d,
1H, H-C6′, bpy moiety); 8.23 (d, 1H, H-C3, bpy moiety);
8.00 (t, 1H, H-C5′, bpy moiety); 7.82 (d, 1H, H-C3′, bpy
moiety); 7.69 (t, 1H, H-C4, bpy moiety); 7.43 (t, 1H, H-C4′,
bpy moiety); 3.38 (m, 4H, N-CH2, pam moiety); 2.31 (m,
4H, N-C-CH2-C, pam moiety); 31P-NMR: δ: 34.8 ppm,
18.6 ppm.
[Pd(ale)2(phen)]·9H2O·2NaCl, Pd–ale–phen (yield:
65 mg, 42%). Anal. (%) calc. for C20H50Cl2N4Na2O23P4Pd:
C, 22.62; H, 4.75; N, 5.28; 15.2 H2O. Found: C, 22.94; H,
Materials and methods
1
Materials
4.65; N, 5.38; 15.4 H2O. H-NMR: δ (ppm): 8.42 (d, 2H,
H-C2,H-C9, phen mioety); 8.19 (d, 2H, H-C4, H-C7, phen
moiety); 7.51 (s, 2H, H-C5, H-C6, phen moiety); 7.35 (dd,
2H, H-C3, H-C8, phen moiety); 3.13 (m, 8H, N-C-CH2-
CH2, ale moiety); 2.14 (m, 4H, N-CH2, ale moiety). 31P-
NMR: δ: 35.3 ppm, 18.1 ppm.
All common laboratory chemicals were purchased from
commercial sources and were used without any further
purifcation. [PdCl2(dmso)2] was prepared according to a
published procedure [33]. Sodium alendronate trihydrate
(4-amino-1-hydroxybutane-1,1-diylphosphonic acid sodium
salt, Naale) and sodium pamidronate pentahydrate (3-amino-
1-hydroxypropane-1,1-diylphosphonic acid disodium salt,
Na2pam), 2,2-bipyridine (bpy) and 1,10-phenanthroline
were purchased from commercial sources and were used as
received.
[Pd(pam)2(phen)]·8H2O·2NaCl, Pd–pam–phen (yield:
67 mg, 44%). Anal. (%) calc. for C18H44Cl2N4Na2O22P4Pd:
C, 21.28; H, 4.37; N, 5.52; 14.1 H2O. Found: C, 21.56; H,
1
4.25; N, 5.32; 14.1 H2O. H-NMR: δ (ppm): 8.36 (d, 1H,
H-C2,phen moiety); 8.17 (t, 2H, H-C3 and H-C8, phen moi-
ety) 8.15 (d, 1H, H-C9, phen moiety) 7.51 (bs, 1H, H-C5,
phen moiety); 7.45 (dd, 1H, H-C7, phen moiety); 7.43 (bs,
1H, H-C6, phen moiety); 7.38 (dd, 1H, H-C3, phen moiety);
3.34 (t, 4H, N-CH2, pam moeity); 2.29 (m, 4H, N-C-CH2,
pam).31P-NMR: δ: 35.3 ppm, 18.0 ppm.
Synthesis of the Pd–NBP–NN complexes
All compounds were prepared by dropwise addition of 5 mL
of an aqueous solution containing 0.15 mmol of the NN
ligand (bpy or phen) over a solution containing 0.15 mmol
of [PdCl2(dmso)2] or Na2[PdCl4] dissolved in 5 ml of H2O.
The mixture containing a white solid was stirred at room
temperature for 1 h and then, 10 mL of an aqueous solution
containing 0.30 mmol of the corresponding NBP (Naale o
Na2pam) and 0.30 mmol of triethylamine (TEA) was added
dropwise. The mixture was stirred at 60 °C until a clear dark-
brown solution was observed and then it was concentrated
to 10 mL. The compounds were isolated by adding 10 mL
of acetone and washed of with ethyl alcohol and diethyl
ether. All the obtained products were microcrystalline yel-
low solids.
Physicochemical characterization
C, H and N analyses were carried out with a Thermo Sci-
entifc Flash 2000 elemental analyzer. Analytical determi-
nation of Na was performed by FAAS on a Perkin Elmer
AAnalyst 200 with a 10-cm burner and operated at 589 nm.
The hollow cathode lamp (Photron) was operated under the
manufacturer’s recommendations. The composition of the
fame was acetylene (2.5 L/min)–air (10.0 L/min). UV–Vis
spectra were recorded in a Shimadzu 1603 spectropho-
tometer. The FTIR absorption spectra (4000–400 cm−1
)
of the complexes and free ligands were measured as KBr
pellets with a Shimadzu IR Prestige-21 instrument. Ther-
mogravimetric measurements (TGA) were done on a Shi-
madzu TGA 50 thermobalance, with a platinum cell, work-
ing under fowing nitrogen (50 mL min−1) and at a heating
rate of 0.5 °C min−1 (room temperature to 80 °C range) and
[Pd(ale)2(bpy)]·7H2O·2NaCl, Pd–ale–bpy (yield: 77 mg,
51%). Anal. (%) calc. for C18H46Cl2N4Na2O21P4Pd: C,
21.58; H, 4.63; N, 5.59; H2O 12.6. Found: C, 21.78; H, 4.63;
N, 5.54; H2O 13.0. 1H-NMR: δ (ppm): 8.39 (d, 2H, H-C6,
H-C6′, bpy moiety); 8.00 (t, 2H, H-C4, H-C4′, bpy moiety);
7.85 (d, 2H, H-C3, H- C3′, bpy moiety); 7.44 (t, 2H, H-C5,
H-C5′, bpy moiety); 3.10 (m, 4H, N-CH2, ale moiety); 2.12
(m, 8H, N–C–CH2 and N–C–C–CH2, ale moiety); 31P-
NMR: δ: 34.8 ppm, 18.5 ppm.
1
1.0 °C min−1 (80–350 °C range). H-NMR spectra were
recorded with a Bruker Avance 400 MHz spectrometer in
D2O at 27 °C. The peak for residual solvent (H2O, 4.79 ppm)
was used as reference [34]. 31P-NMR spectra were obtained
1 3

JBIC Journal of Biological Inorganic Chemistry
with the same spectrometer at 161,976 MHz and referred to
H3PO4 85% (0.00 ppm).
Biological studies
Drug screening assays in Vero cells and T. cruzi intracellular
Computational details
amastigotes
Gamma-irradiated (2000 Rads) Vero cells (3.4×104 cells/
well) were seeded in 96-well plates (black, clear bottom
plates from Greiner Bio-One) in 100 μL RPMI (Roswell
Park Memorial Institute from Sigma) with 10% FBS (Fetal
Bovine Serum). Gamma irradiation was performed to pre-
vent host cell division and detachment of the culture. Plates
were incubated overnight at 35 °C and 7% CO2. After over-
night incubation, Vero cells were challenged with 3.4×105
trypomastigotes/well (CL strain overexpressing a tdTomato
red fuorescent protein) [48] in 50 μL volume and incubated
for 5 h at 35 °C and 7% CO2. After infection, wells were
washed once with Hanks solution (150 μL/well) to elimi-
nate any extracellular parasites and tested compounds were
immediately added in serial dilutions in RPMI media in
150 μL volumes and left for the whole period of the experi-
ment. Each dilution was tested in quadruplicate. Each plate
also contained controls with host cells and no parasites (for
background check), controls with two representative drug
dilutions and no parasites (for cytotoxicity assays), and con-
trols with parasites and no drugs (positive control). After
drug addition, plates were incubated at 35 °C and 7% CO2.
At day 3 post-infection, plates were assayed for fuorescence.
IC50 values were determined by non-linear regression analy-
sis using SigmaPlot. Vero cell cytotoxicity was measured by
the Alamar blue assay as described previously [49].
All computational studies were performed at the density
functional level of theory (DFT). First, pamidronate (as an
example) was theoretically studied to shed light on its pre-
dominant chemical form in the solid state. The molecular
geometry of pam (two isomers) interacting with two sodium
atoms and fve water molecules (Fig. S1) was optimized
employing B3LYP functional [35–37] in combination with
6–31 + G*base [38–41]. Calculations on Pd–pam–phen
(taken as example) were conducted aiming at confrming the
coordination behavior of pam in these systems. The absence
of crystallographic data prompted us to study complexes
derived from two diferent coordination possibilities, i.e.,
employing N-donor atoms (N-pam), or O-donor atoms (O-
pam). For the last option, the commonly observed bidentate
monoanionic (zwitterionic) form of coordination was sup-
posed [42]. Thus, the geometries of Pd–(N-pam)–phen and
Pd–(O-pam)–phen complexes were optimized in a closed-
shell singlet (S = 1) employing LANL2DZ base [40, 41,
43–45] on palladium atom, and 6–31+G*base [38–41] on
non-metal atoms. LANL2DZ takes scalar relativistic efects
into account, especially important when systems with heavy
atoms are studied [46]. Water molecules were included in
all cases and for Pd–(N-pam)–phen system in particular, two
sodium atoms were also added (further details in Fig. 3). The
nature of the stationary point was verifed through a vibra-
tional analysis (no imaginary frequencies). All theoretical
studies reported in this work were conducted by employing
the program package Gaussian 09, Rev. A.01 [47].
Drug screening in Toxoplasma gondii
Cell cultures Human foreskin fbroblasts (HFF) (ATCC®
SCRC-1041) were grown in Dulbecco’s modifed Eagle’s
medium (DMEM) supplemented with 10% fetal bovine
serum (FBS) and antibiotics. Cell cultures were maintained
at 37 °C in a humidifed atmosphere with 5% CO2 and with
replacement of the culture medium every 24–48 h [51].
Fig. 3 Model systems
{[Pd(phen)(O-pam)](H2O)4}+
(left), and {[Pd(phen)(N-
pam)2](Na)2(H2O)8}2+ (right)
as calculated at the B3LYP/
LANL2DZ/6–31+G* level
(T=298 K)
1 3

JBIC Journal of Biological Inorganic Chemistry
Parasite culture and harvesting Semi-confuent HFF were
infected with T. gondii tachyzoites (RH strain) at a multi-
plicity of infection of 3–5 parasites per cell. After 40 h of
infection, infected cells were washed and monolayers were
scraped from the fasks and passed through 20-, 23-, and
25-gauge needles. Tachyzoites were purifed from host cell
debris with a 3.0-μm Isopore flter (Merck Millipore) [51].
Effect of the complexes on tachyzoite viability was
evaluated through the tetrazolium salt (MTT) reduction
assay (see above). Briefy, 10 mL of 5 mg/mL MTT dye
(3[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bro-
mide) plus 0.22 mg/mL phenazine methosulfate were added
to each well containing 106 parasites in 100 mL of RPMI
1640 medium, without phenol red. After incubating the cells
for 4 h at 37ºC, the generated formazan crystals were dis-
solved with 100 mL of 10% (w/v) SDS in 0.01 M HCl. The
plates were kept overnight at 37 °C, and the optical density
(OD) was determined using a microplate reader (Labsystems
Multiskan MS, Finland) at 570 nm. Under these conditions,
the OD is directly proportional to the viable cell number in
each well. All experiments were performed at least three
times.
complexes were used for the inhibition assay. Positive (no
inhibitor, only enzyme) and negative controls (no inhibitor
and no enzyme) for each experiment were performed.
TcSPPS assay The activity of the enzyme was determined
by a radiometric assay. Briefy, 100 µL of assay bufer
(100 mM Tris–HCl bufer, pH 7.4, 1 mM MgCl2, 1% (v/v)
Triton X-100, 7.07 µM [4-14C]IPP (10 µCi/lmol)) and 50 µM
GGPP (geranylgeranyl diphosphate) were prewarmed to
37 °C. The assay was initiated by the addition of 10–20 ng
of recombinant protein. The assay was allowed to proceed
for 30 min at 37 °C and was quenched by chilling quickly in
an ice bath. The reaction products were extracted with 1 mL
of 1-butanol saturated with water. The organic layer was
washed with water saturated with NaCl, and transferred to
a scintillation vial with 4 mL of scintillation solution Ecol-
ume for counting. One unit of enzyme activity was defned
as the activity required to incorporate 1 nmol of [4-14C]IPP
into [4-14C]FPP in 1 min [54].
DNA interaction studies (fuorescence quenching assay)
Experiments for competitive binding to calf thymus DNA
(ctDNA, SIGMA, Type I, No. D-1501) with ethidium bro-
mide (EB, SIGMA) were carried out in 10 mM Tris–HCl
bufer at pH 7.4. Millipore® water was used for the prepara-
tion of all aqueous solutions. Fluorescence measurements
were carried out on individually prepared samples to ensure
the same pre-incubation time for all samples in each assay.
The complexes’ solutions were prepared in purifed water
followed by appropriate dilution to obtain the targeted con-
centration. DNA stock solutions were prepared by hydrating
ctDNA in Tris–HCl bufer (1 mg/mL, ca. 2 mMnuc–1) for
3–4 days at 4 °C, swirling the solution about 4–5 times a
day until full dissolution was attained, and a clear solution
was obtained. This solution was kept at 4 °C (in the fridge)
in-between measurements and discarded after 4 days. The
concentration of each stock solution was determined by UV
spectrophotometry using the molar extinction coefcient ε
(260 nm)=6600 M–1 cm–1 nuc–1 [55]. An EB 5 mM solution
was prepared in Tris–HCl bufer. ctDNA was pre-incubated
with EB at 4 °C for 24 h. Samples were prepared with a
total concentration of DNA and of EB of 20 μM nuc–1 and
10 μM, respectively, by varying the total complex concen-
tration from 2 to 100 μM. They were incubated at 37 °C
for 30 min. Samples with complex alone and samples with
complex and EB but no DNA were used as blanks. Fluores-
cence spectra were recorded from 520 to 650 nm at an exci-
tation wavelength of 510 nm with a Shimadzu RF-5301PC
spectrofuorometer. Fluorescence emission intensity was
corrected for the absorption and emission inner flter efect
at the maximum emission wavelength (594 nm) using the
Preliminary anti‑tumor screening
Experimental details on drug screening in MG-63 and A549
tumor cell lines are included in the Supplementary material.
Enzymatic inhibition assays
FPPSassay(bothT.cruziandhuman) HumanFPPS(HsFPPS)
was expressed and purifed as previously described [52, 53].
T. cruzi FPPS (TcFPPS) was obtained as described before
[9]. The FPPS inhibition radiometric assay was performed
essentially as previously reported [53]. Briefy, 100 µL of
assay bufer (10 mM Hepes (4-(2-hydroxyethyl)-1-pip-
erazineethanesulfonic acid), pH 7.4, 5 mM MgCl2, 2 mM
dithiothreitol, 4.7 µM [4-14C]IPP (isopentenyl diphosphate),
10 µCi/µmol, and 55 µM DMAPP (dimethylallyl pyrophos-
phate) were prewarmed to 37 °C. The assay was initiated
by the addition of recombinant protein (10–20 ng). The
assay was allowed to proceed for 30 min at 37 °C and was
quenched by the addition of 6 M HCl (10 µL). The reaction
media were made alkaline with 6.0 M NaOH (15 µL), diluted
in water (0.7 mL), and extracted with hexane (1 mL). The
hexane solution was washed with water and transferred to a
scintillation vial for counting. One unit of enzyme activity
was defned as the activity required to incorporate 1 nmol
of [4-14C]IPP into [4-14C]FPP (farnesyl diphosphate) in
1 min. IC50 values were determined by non-linear regression
analysis using SigmaPlot. 10 µL of several dilutions of the
1 3

JBIC Journal of Biological Inorganic Chemistry
UV–Vis absorption data recorded for each sample according
to the following equation [56]:
addition, O-coordination in which the terminal basic amine
group is protonated (zwitterion form) was obtained when
neutral or acidic synthetic conditions were used. However,
N-coordination is strongly favored by basic conditions as
those used in this work. At high pH values, the amine group
deprotonates favoring coordination to the metal center [57,
59].
IFcorr = IF × 10(Abs(ꢀexc)+Abs(ꢀem)∕2
.
Results and discussion
FTIR spectra showed characteristic band patterns con-
frming the presence of the corresponding NN and NBP
ligands in the obtained palladium complexes. According
to the similarities between the spectra of ale and pam, for
each NN ligand, the spectral pattern of Pd–pam–NN and
Pd–ale–NN were almost identical (Figure S2). This fact evi-
dences that, in the solid state, the analogous compounds pre-
sent the same chemical structure. The characteristic bands
of the NN ligand corresponding to ν(C = C) and ν(C = N)
between 1200 and 1600 cm−1 [61] and some of the bispho-
sphonate ligand bands appear overlapped in the complexes
which makes it difcult to do a complete accurate assign-
ment in this spectral region. In fact, DFT calculations (see
below) confrmed that most of these bands correspond to
coupled vibrational modes. In the region of 900–1300 cm−1,
the obtained compounds show the typical band pattern of
phosphonates [62]. Modifcations in the frequency values of
these bands in the complexes could be attributed to sodium
and hydrogen bond interactions [12, 13, 63]. The bands
near 3350 and 3260 cm−1 assigned to νas(NH2) and νs(NH2),
respectively, in the NBP ligands collapse in a broad band as
a consequence of coordination through the ammine nitro-
gen [60]. In addition, the band near 550 cm−1 assigned to
ν(Pd–O) is not present in the obtained complexes confrming
the N-coordination of the NBP ligands [60].
Synthesis and solid‑state characterization
Four new palladium complexes of the formula
[Pd(NBP)2(NN)]·2NaCl·xH2O with NBP=ale or pam and
NN=phen or bpy have been synthesized and fully character-
ized in the solid state and in solution. The new complexes
were obtained as yellow microcrystalline solids and all
of them were soluble in water. Analytical data, including
thermogravimetric analysis results confrmed the proposed
formula. The presence of Na+ in the molecular formula of
the compounds neutralizes the charge of some of the non-
coordinated phosphonate groups and it was confrmed by
atomic absorption measurements (Fig. 4). In fact, the pres-
ence of NaCl to stabilize this kind of structure in the solid
state had been previously reported for some NBPs [57].
The common synthetic procedure for all the compounds
is also shown in Fig. 4. The frst step leads to the corre-
sponding [PdCl2(NN)2] intermediate insoluble compound.
In the second step, a basic medium was used both to dissolve
these intermediates by forming μ-hydroxo bridged species
[58] and to favor coordination of the bisphosphonate ligands
through the terminal ammine nitrogen (see below) [59]. Pre-
viously reported palladium–amine–NBP complexes showed
dimeric structures with O-coordinated bridging NBP ligands
[57, 60]. Unlike the compounds included in this work,
O-coordination of NBP to palladium or other metal cent-
ers, leads to water insoluble complexes [12–14, 57, 60]. In
The mode of coordination of the NBP ligands in the
obtained complexes was also confrmed using a theoretical
approach through the analysis of the vibrational spectrum of
compounds with N- and O-coordinated ligands.
Theoretical calculations
As mentioned in ‘Computational Details’, the absence of
crystallographic data for the title complexes motivated theo-
retical studies on geometry and vibrational IR spectrum. The
system involving Pd–pam–phen was selected as an exam-
ple. Hypothetical compounds with pam coordinated through
either nitrogen (Pd–N-pam) or oxygen (Pd–O-pam) were
included in the study.
Vibrational IR spectra of Pd–O-pam, and Pd–N-pam as
calculated at the B3LYP/LANL2DZ/6–31+G* level in the
range 1800–500 cm−1 are displayed in Fig. S3, which also
exhibits the experimental spectrum. When the theoretical
spectra are compared to the experimental evidence, a closer
match of the one obtained for Pd–N-pam seems to emerge.
This observation becomes clearer by inspection of the region
Fig. 4 Synthetic procedure (NN=2,2-bipyridine (bpy) or 1,10-phen-
anthroline, NBP=pamidronate (n=1) or alendronate (n=2), RT
room temperature, TEA triethylamine)
1 3

JBIC Journal of Biological Inorganic Chemistry
1000–1300 cm−1, in which the absorption-band profle of
Pd–N-pam system reasonably reproduces the features of
the experimental fndings. In this region, two main bands
with similar intensities were observed both in the calculated
Pd–N-pam and in the experimental spectra. The origin of
these bands can be traced to highly coupled modes involv-
ing P–O–H bending, O–P–O stretching and H–N–H wag-
ging modes. In addition, non-negligible contributions from
several C=C and C=N stretching bands of the phen ligand
were also observed.
deshielding of the phosphorus nucleus should be expected
due to the loss of the zwitterionic character of the NBP
ligands due to coordination [66]. In addition, the diferent
levels of protonation of the phosphonate groups as well as
possible sodium interaction in solution could account for the
observed results [57].
The stability of complexes in water was assessed by
1
UV–Vis spectrophotometry and H and 31P NMR spec-
troscopy. The results obtained demonstrated that both com-
pounds were stable in aqueous solutions during 24 h.
The theoretical results already discussed allow us to infer
the N-coordination mode of pam to Pd2+ in the obtained
complexes with a 1:2 metal to pam molar ratio. The equi-
librium geometry for this system exhibits a slight distorted
square planar coordination geometry, being the phen ligand
quasi-planar. The interaction of each pam ligand with the
metal via N-atoms implies that the ligand must change its
zwitterionic character to promote coordination leading to
a monoanion whose total charge is compensated with a
sodium ion (experimentally, this sodium ions are provided
by disodium pamidronate). In our model, eight water mol-
ecules—that were added to simulate coordination geometry
of sodium atoms—were calculated interacting with cations
and phosphonate groups through several H-bonds (Fig. 3).
It is to be mentioned that no remarkable diferences in equi-
librium geometry were obtained in going from the gas phase
to aqueous solution (taken into account through the C-PCM
[64, 65] methodology).
Biological results
Anti‑T. cruzi activity
The activity against intracellular amastigote form of T.
cruzi of the palladium complexes was studied. Results are
shown in Table 1. All obtained complexes were inhibitors
of T. cruzi’s growth and they resulted more active than the
corresponding free NBP ligands. On the other hand, the
antiparasitic activity seems to be dependent of the nature of
the NN ligand. In fact, complexes with phen as ligand were
15 times more active than those including bpy. Pd–ale–phen
and Pd–pam–phen were even more active than the refer-
ence drug benznidazole and almost 30 times more active
than ale- and pam-free ligands. It also should be noted that
these Pd–NBP–phen complexes only wed signs of unspecifc
toxicity on mammalian Vero cells at concentrations 40–70
times higher than the corresponding IC50 values (Table 1).
An increase in the anti-T. cruzi activity and in selectivity as
a consequence of complexation had already been reported
by us for other metal–NBP compounds [12–14].
In‑solution characterization and stability
1H-NMR spectra in D2O of all the complexes showed narrow
signals typical for diamagnetic square planar palladium(II)
complexes (Fig. S4). A complete assignation of the 1H-NMR
spectral bands was performed and it is shown in the experi-
mental section. The numbered scheme for NN ligands is
shown in Fig. 1. The integration of the signals corresponding
to the NN ligands with respect to the NBP ones confrmed
the stoichiometry of the new compounds in solution. The
chemical shift of the signals corresponding to the aliphatic
protons of the NBP ligands (3.03 ppm (t, 2H), 2.00 (m, 4H)
and 3.32 ppm (t, 2H), 2.26 (m, 2H) for ale and pam, respec-
tively) was barely modifed upon coordination as previously
described for other NBP complexes [57, 60]. However, in all
cases, the shape of the signals sufered from some modif-
cations (broadening, changes in multiplicity) that could be
attributed to the efect of palladium coordination.
Anti‑T. gondii activity
NBPs are also inhibitors of the growth of the protozoan
parasite Toxoplasma gondii, causative agent of toxoplas-
mosis, a widespread disease that afects primarily immu-
nocompromised and pregnant individuals [5]. In particular,
ale and pam resulted efective in inhibiting intracellular
proliferation of T. gondii showing IC50 values of 37 µM
and 42 µM, respectively [67]. Based on that, obtained pal-
ladium complexes were tested against T. gondii tachyzoites.
Pd–ale–phen and Pd–pam–phen complexes showed IC50 val-
ues of 15.2 1.1 and 30.6 1.2 µM, respectively. A slight
increase in the activity was observed as a consequence of
complexes’ formation (IC50 = 35 µM for both free ale and
free pam) [6]. Interestingly, no activity was observed for
Pd–ale–bpy and Pd–pam–bpy complexes. A similar behavior
was observed for the anti-T. cruzi activity, being complexes
with phen as ligand more active than those with bpy.
31P-{1H}-NMR spectra of all the obtained complexes
showed two signals at around 18 and 35 ppm (Fig.S5).
This result is in accordance with two phosphorous popula-
tions for each compound that could be attributed to two not
equivalent phosphorous atoms of each coordinated NBP but
magnetically equivalent to the second coordinated NBP. A
1 3

JBIC Journal of Biological Inorganic Chemistry
Table 1 Results of biological
Compound
IC50 (μM)a
Toxicity (μM)b
IC50 (μM)c
IC50 (μM)d
IC50 (μM)e
and enzymatic assays
T. cruzi
Vero cells
TcFPPS
hFPPS
TcSPPS
ale
38.3 2.3f
1.44 0.86
17.36 0.48
38.8 5.0f
1.30 0.77
21.4 0.80
2.02 0.32
>100
100
100
>100
50
50
–
0.20 0.05
0.12 0.01
0.33 0.08
0.05 0.01
0.22 0.02
nd
1.52 0.29
0.40 0.03
0.52 0.04
0.46 0.07
0.41 0.09
nd
5.88 1.53
1.35 0.12
0.96 0.06
0.99 0.10
0.57 0.06
nd
Pd–ale–phen
Pd–ale–bpy
pam
Pd–pam–phen
Pd–pam–bpy
Benznidazol
–
–
–
Where indicated values are means s.d. of three independent experiments (n=3)
nd not determined because of unspecifc toxicity
aConcentration of compounds inhibiting 50% growth of T. cruzi amastigotes
bConcentration of compounds showing signs of toxicity on Vero cells (visual assay)
cConcentration of compounds inhibiting 50% activity of TcFPPS
dConcentration of compounds inhibiting 50% activity of HsFPPS
eConcentration of compounds inhibiting 50% activity of TcSPPS
f[12]
Correlation between anti‑tumor and antiparasitic activities
12–14]. Therefore, the ability of the obtained palladium
complexes to inhibit TcFPPS was assayed. Results are
depicted in Table 1. Studied complexes resulted in inhibi-
tors of TcFPPS but obtained IC50 values were comparable
to those observed for the free NBP ligands. For other pre-
viously obtained metal compounds with NBPs as ligands,
a fairly good correlation between the ability of the com-
plexes to inhibit both intracellular T. cruzi amastigotes
growth and TcFPPS activity had been found [12, 13]. How-
ever, for the complexes described herein, TcFPPS inhibi-
tion values could not explain the increase of the anti-T.
cruzi activity as a consequence of complexes’ formation.
On the other hand, solanesyl-diphosphate synthase
(TcSPPS) is an enzyme belonging to the prenyltransferase
family involved in the biosynthesis of solanesyl diphos-
phate, a precursor of the side chains of ubiquinones.
Because ubiquinones play a central role in energy pro-
duction in parasites, TcSPPS could be a promising chemo-
therapeutic target, in particular for bisphosphonates [54].
Both the free NBP ligands and palladium complexes
showed inhibition of TcSPPS (Table 1). The inhibition by
Pd–ale–phen and Pd–ale–bpy were larger than the inhi-
bition by ale ligand alone but did not correlate with the
growth inhibition results. So, the inhibition of this enzyme
could not either explain the diferences between the anti-T.
cruzi activity of the diferent palladium compounds.
Finally, complexes were also assayed on their ability to
inhibit human farnesyl pyrophosphate synthase enzyme
(HsFPPS) (Table 1). Results showed that FPPS inhibition
is not selective. In spite of that, most obtained palladium
compounds were non-toxic for mammalian Vero cells at
low concentrations.
Based on the proposed parallelism between the antitumor
and antiparasitic efects [18–21], the cytotoxicity of the
complexes on MG-63 osteosarcoma and the A549 lung
adenocarcinoma cells was preliminary investigated. Results
are shown in Fig. S6.
In A549 cells, Pd–ale–phen and Pd–pam–phen showed
the highest cytotoxic activities. For this cell line, the ten-
dency in cytotoxicity was similar to that observed for
the antiparasitic activity. On the other hand, in MG-63
cells, only Pd–pam–phen showed cytotoxic efect while
Pd–pam–bpy did not show any efects and Pd–ale–phen and
Pd–ale–bpy only provoked a slight efect in the cell viability.
As expected, on this bone-related cell line, the cytotoxic
activity was dependent not only on the NN ligand but also
on the NBP.
Although Pd–NBP–NN compounds were designed as
potential antiparasitic agents, some of the obtained cyto-
toxic results were similar to those previously reported
for some palladium and platinum complexes with NBPs
as ligands [60, 68] and to the reference drug cispl-
atin (IC50 for A549 = 114.0 9.1 μM [69] and IC50 for
MG-63=39.0 1.2 μM [70]). In addition, a quite good cor-
relation between both activities was observed.
Insight into the potential targets
TcFPPS, HsFPPS and TcSPPS inhibition assays
The main mode of anti-T. cruzi action of NBPs and their
metal complexes has been related to the inhibition of
farnesyl pyrophosphate synthase enzyme (TcFPPS) [5,
1 3

JBIC Journal of Biological Inorganic Chemistry
DNA interaction
plot curves downward towards the x-axis at the highest
concentrations which is a characteristic feature of two fuo-
rophore populations, one of which is not accessible to the
quencher [56].
Fluorescence experiments
Obtained results showed that, as expected, the nature of
the NN ligand seems to determine the complexes’ interaction
with DNA. In fact, both Pd–NBP–phen complexes show a
much higher afnity for DNA in the EB displacement reac-
tion experiments than their Pd–NBP–bpy counterparts.
As stated, DNA could be a potential target for the title
metal compounds not only through the palladium ion by
covalent binding but also through the NN ligand in an inter-
calative-like mode. Therefore, the fuorescent DNA probe,
ethidium bromide (EB), was used to access the interaction
of the obtained complexes with DNA. EB is a conjugate
planar molecule with weak intrinsic fuorescence emission.
At pH 7.4 in Tris–HCl bufer, its spectrum shows an emis-
sion maximum at 601 nm (λexc. = 510 nm). Intercalation
of EB into double-stranded DNA induces an increase of
the fuorescence quantum yield [71]. In the spectrum of the
{DNA–EB} adduct, the emission maximum is shifted to
594 nm under these experimental conditions. Neither DNA
nor the palladium complexes are fuorescent and no fuores-
cence emission results from compounds’ direct interaction
with DNA.
Conclusions
Four new palladium complexes with the bioactive bisphos-
phonates alendronate and pamidronate as ligands and phen-
anthroline and bipyridine as co-ligands were synthesized
and fully characterized. All the obtained compounds showed
an increased anti-T. cruzi activity when compared to the
free NBP ligands. In particular, Pd–NBP–phen complexes
resulted 15 times more active than the corresponding bpy
analogs. This tendency was also observed for the anti-T.
gondii and antitumor activities. However, all the complexes
were able to similarly inhibit TcFPPS and TcSPPS enzymes
suggesting that enzymatic inhibition could not be responsi-
ble for the observed diferences in the biological activity of
the obtained compounds. On the contrary, such diferences
could be related to their ability to interact with DNA in an
intercalative-like manner. In the search of better compounds
for the treatment of parasitic illnesses, the title palladium
compounds are good candidates for further developments,
in particular, for in vivo studies.
Obtained results for the titration of the {DNA–EB}
adduct with Pd–ale–phen and Pd–ale–bpy are shown as an
example in Fig. S7. For Pd–ale–phen, a quenching of the
emission of {DNA–EB} adduct up to 50% was observed
upon increasing the complex’s concentration. The decrease
in emission intensity was accompanied by a small red shift
of around 2 nm at the highest concentrations in accord-
ance to the change from the characteristic emission of the
{DNA–EB} adduct (594 nm) to the one of the free EB
(601 nm) (Fig. S8). For Pd–ale–bpy, only a very slight efect
on the emission intensity was observed (Fig. S7). A quite
similar behavior is observed for Pd–pam–phen when com-
pared to Pd–pam–bpy (Fig. S9). These results are consist-
ent with an EB displacement from the {DNA–EB} adduct
brought upon by the binding of Pd–NBP–phen complexes
in an intercalative-like mode or by inducing DNA confor-
mational changes, both mechanisms promoting the partial
release of EB.
Acknowledgements We thank Melina Galizzi for technical help.
Authors thank CSIC, PEDECIBA and ANII-SNI, Uruguay, and FON-
DECYT 1190340, Chile. Z-H Li and R.D. work was funded by the U.S.
National Institute of Health (Grant AI082542 to R.D).
Compliance with ethical standards
The extent of the interaction involved in the fuorescence
quenching process for Pd–NBP–phen complexes can be
assessed by a Stern–Volmer analysis according to the below
equation:
Conflict of interest All authors declare that they have no confict of
interest.
IF0∕IF = 1 + KSV[Q].
(1)
References
In Eq. (1), IF0 and IF are the emission fuorescence inten-
sity of the {DNA–EB} adduct in the absence and in the pres-
ence of the complex, respectively, KSV is the Stern–Volmer
constant, and [Q] is the concentration of the quencher (in
this case, the complexes) [56]. For both studied complexes,
the plot IF0/IF vs. [complex] showed a linear ft with logKSV
values of 4.6 and 3.5 for Pd–ale–phen and Pd–pam–phen,
respectively. In the case of Pd–ale–phen the Stern–Volmer
1. Graham Russell R (2011) Bone 49:2–19
2. Gałęzowska J (2018) ChemMedChem 13(4):289–302
3. Ebetino FH, Hogan AML, Sun S, Tsoumpra MK, Duan X, Triftt
JT, Kwaasi AA, Dunford JE, Barnett BL, Oppermann U, Lundy
MW, Boyde A, Kashemirov BA, McKenna ChE, Russell RG
(2011) Bone 49:20–33
4. Cremers S, Drake MT, Ebetino FH, Bilezikian JP, Russell RGG
(2019) Br J Clin Pharmacol 85(6):1052–1062
5. Docampo R, Moreno SN (2011) Curr Drug Target-Infect Disord
1:51–61
1 3

JBIC Journal of Biological Inorganic Chemistry
32. Mansouri-Torshizi H, Saeidifar M, Divsalar A, Saboury AA
(2010) Spectrochim Acta A 77(1):312–318
6. Martin MB, Grimley JS, Lewis JC, Heath HT, Bailey BN,
Kendrick H, Yardley V, Caldera A, Lira R, Urbina JA, Moreno
SNJ, Docampo R, Croft SL, Oldfeld E (2001) J Med Chem
44(6):909–916
33. Kukushkin YN, Vlasova RA, Pazukhina YL (1968) Zh Prikl Khim
41(11):2381–2385
34. Fulmer GR, Miller AJ, Sherden NH, Gottlieb HE, Nudelman
A, Stoltz BM, Bercaw JE, Goldberg KI (2010) Organometallics
29(9):2176–2179
7. Urbina JA, Docampo R (2003) Trends Parasitol 19:495–501
8. Docampo R, Moreno SNJ (2008) Curr Pharm Des 14:882–888
9. Montalvetti A, Bailey BN, Martin MB, Severin GW, Oldfeld E,
Docampo R (2001) J Biol Chem 276:33930–33937
10. Rodriguez JB, Falcone BN, Szajnman SH (2016) Expert Opin
Drug Discov 11:307–320
35. Becke AD (1993) J Chem Phys 98:1372–1377
36. Becke AD (1988) Phys Rev A 38:3098–3100
37. Lee C, Yang W, Parr RG (1988) Phys Rev B 37:785–789
38. Hariharan PC, Pople JA (1973) Theoret Chim Acta 28:213–222
39. Francl MM, Petro WJ, Hehre WJ, Binkley JS, Gordon MS,
DeFrees DJ, Pople JA (1982) J Chem Phys 77:3654–3665
40. Feller D (1996) J Comp Chem 17:1571–1586
11. Mukherjee S, Basu S, Zhang K (2019) Mol Biochem Parasit
230:8–15
12. Demoro B, Caruso F, Rossi M, Benítez D, Gonzalez M, Cerecetto
H, Parajón-Costa B, Castiglioni J, Gallizi M, Docampo R, Otero
L, Gambino D (2010) J Inorg Biochem 104:1252–1258
13. Demoro B, Caruso F, Rossi M, Benítez D, González M, Cerecetto
H, Galizzi M, Malayil L, Docampo R, Faccio R, Mombrú AW,
Gambino D, Otero L (2012) Dalton Trans 41(21):6468–6476
14. Demoro B, Rostán S, Moncada M, Li ZH, Docampo R, Olea Azar
C, Maya JD, Torres J, Gambino D, Otero L (2018) J Biol Inorg
Chem 23(2):303–312
41. Schuchardt KL, Didier BT, Elsethagen T, Sun L, Gurumoorthi V,
Chase J, Li J, Windus TL (2007) J Chem Inf Model 47:1045–1052
42. Matczak-Jon E, Videnova-Adrabinska V (2005) Coord Chem Rev
249:2458–2488
43. Hay PJ, Wadt WR (1985) J Chem Phys 82:270–283
44. Hay PJ, Wadt WR (1985) J Chem Phys 82:284–298
45. Hay PJ, Wadt WR (1985) J Chem Phys 82:299–310
46. Pyykkö P (1990) The efect of relativity in atoms, molecules and
the solid state. Plenum, New York
15. Cavalli A, Bolognesi ML (2009) J Med Chem 52:7339–7359
16. Zheng W, Zhao Y, Luo Q, Zhang Y, Wu K, Wang F (2016) Sci
China Chem 59:1240–1249
47. Frisch MJ, Trucks GW, Schlegel HB, Scuseria GE, Robb MA,
Cheeseman JR, Scalmani G, Barone V, Petersson GA, Nakat-
suji H, Li X, Caricato M, Marenich A, Bloino J, Janesko BG,
Gomperts R, Mennucci B, Hratchian HP, Ortiz JV, Izmaylov AF,
Sonnenberg JL, Williams-Young D, Ding F, Lipparini F, Egidi
F, Goings J, Peng B, Petrone A, Henderson T, Ranasinghe D,
Zakrzewski VG, Gao J, Rega N, Zheng G, Liang W, Hada M,
Ehara M, Toyota K, Fukuda R, Hasegawa J, Ishida M, Nakajima
T, Honda Y, Kitao O, Nakai H, Vreven T, Throssell K, Montgom-
ery JA, Peralta JE Jr, Ogliaro F, Bearpark M, Heyd JJ, Brothers
E, Kudin KN, Staroverov VN, Keith T, Kobayashi R, Normand
J, Raghavachari K, Rendell A, Burant JC, Iyengar SS, Tomasi J,
Cossi M, Millam JM, Klene M, Adamo C, Cammi R, Ochterski
JW, Martin RL, Morokuma K, Farkas O, Foresman JB, Fox DJ
(2013) Gaussian 09, Rev. A.01. Gaussian Inc, Wallingford
48. Canavaci AM, Bustamante JM, Padilla AM, Pereza Brandan CM,
Simpson LJ, Xu D, Boehlke CL, Tarleton RL (2010) PLOS Negl
Trop Dis 4:e740–e745
17. Huang R, Wallqvist A, Covell DG (2005) Biochem Pharmacol
69:1009–1039
18. Kinnamon K, Steck EA, Rane ES (1979) Antimicrob Agents
Chemother 15:157–160
19. Dorosti Z, Yousef M, Maryam Sharaf S, Darani HY (2014)
Future Oncol 10:2529–2539
20. Sánchez-Delgado RA, Anzellotti A, Suárez L (2004) Metal ions
in biological systems. In: Sigel H, Sigel A (eds) 41: metal ions
and their complexes in medication. Marcel Dekker, New York, pp
379–419
21. Otero L, Gambino D (2019) Metal compounds in the develop-
ment of antiparasitic agents: rational design from basic chemistry
to the clinic. In: Carver P (ed) 19: essential metals in medicine:
therapeutic use and toxicity of metal ions in the clinic metal ions
in life sciences. de Gruyter, Berlin, pp 331–358
22. Pages BJ, Garbutcheon-Singh KB, Aldrich-Wright JR (2017) Eur
J Inorg Chem 2017(12):1613–1624
49. Recher M, Barboza AP, Li Z-H, Galizzi M, Ferrer-Casal M, Szajn-
man SH, Docampo R, Moreno SNJ, Rodriguez JB (2013) Eur J
Med Chem 60:431–440
23. Fanelli M, Formica M, Fusi V, Giorgi L, Micheloni M, Paoli P
(2016) Coord Chem Rev 310:41–79
24. Scalese G, Benítez J, Rostán S, Correia I, Bradford L, Vieites M,
Minini L, Merlino A, Coitiño EL, Birriel E, Varela J, Cerecetto H,
González M, Costa Pessoa J, Gambino D (2015) J Inorg Biochem
147:116–125
50. Mosmann TJ (1983) Immunol Methods 65(1–2):55–63
51. Nardelli SC, Che FY, Silmon de Monerri NC, Xiao H, Nieves E,
Madrid-Aliste C, Angel SO, Sullivan WJ Jr, Angeletti RH, Kim
K, Weiss LM (2013) MBio 4(6):e00922–1013
25. Gambino D (2011) Coord Chem Rev 255(19–20):2193–2203
26. Scalese G, Mosquillo MF, Rostán S, Castiglioni J, Alho I, Pérez L,
Correia I, Marques F, Costa Pessoa J, Gambino D (2017) J Inorg
Biochem 175:154–166
52. Kavanagh KL, Guo K, Dunford JE, Wu X, Knapp S, Ebetino FH,
Rogers MJ, Russell RG, Oppermann U (2006) Proc Natl Acad Sci
USA 103:7829–7834
53. Rilling HC (1985) Methods Enzymol 110:145–152
54. Ferella M, Montalvetti A, Rohlof P, Miranda K, Fang J, Reina
S, Kawamukai M, Búa J, Nilsson D, Pravia C, Katzin A, Cassera
MB, Åslund L, Andersson B, Docampo R, Bontempi EJ (2006) J
Biol Chem 281(51):39339–39348
27. Alam MN, Huq F (2016) Coord Chem Rev 316:36–67
28. Otero L, Vieites M, Boiani L, Denicola A, Rigol Olsen C, Opazo
L, Olea Azar C, Maya Arango J, Morello Casté A, Krauth Siegel
R, Piro O, Castellano E, González M, Gambino D, Cerecetto H
(2006) J Med Chem 49(11):3322–3331
55. Barton JK, Goldberg JM, Kumar ChV, Turro NJ (1986) J Am
Chem Soc 108:2081–2088
29. Rodríguez Arce E, Mosquillo MF, Pérez-Díaz L, Echeverría
GA, Piro OE, Merlino A, Coitiño EL, Maríngolo Ribeiro C,
Leite CQF, Pavan FR, Otero L, Gambino D (2015) Dalton Trans
44:14453–14464
56. Lakowicz JR (2006) Principles of fuorescence spectroscopy, 3rd
edn. Springer, New York (ch. 8)
57. Margiotta N, Capitelli F, Ostuni R, Natile G (2008) J Inorg Bio-
chem 102(12):2078–2086
30. Rodríguez Arce E, Putzu E, Lapier M, Maya JD, Olea Azar C,
Echeverría GA, Piro OE, Medeiros A, Sardi F, Comini M, Risi
G, Correia I, Costa Pessoa J, Otero L, Gambino D (2019) Dalton
Trans 48:7644–7658
58. Wimmer S, Castan P, Wimmer FL, Johnson NP (1989) J Chem
Soc Dalton Trans 403–412
59. Galezowska J, Gumienna-Kontecka E (2012) Coord Chem Rev
256:105–124
31. Garoufs A, Hadjikakou SK, Hadjiliadis N (2009) Coord Chem
Rev 253(9–10):1384–1397
1 3

JBIC Journal of Biological Inorganic Chemistry
60. Fathy AA, Butler IS, Abd Elrahman M, Jean-Claude BJ, Mostafa
SI (2018) Inorg Chim Acta 473:44–50
69. Frik M, Martínez A, Elie BT, Gonzalo O, Ramírez de Mingo D,
Sanaú M, Sánchez Delgado R, Sadhukha T, Prabha S, Ramos JB,
Marzo I, Contel M (2014) J Med Chem 57(23):9995–10012
70. Burgos Lopez Y, Del Plá J, Balsa LM, León IE, Echeverría GA,
Piro OE, García Tojal J, Pis Diez R, González Baró AC, Parajón-
Costa BS (2019) Inorg Chim Acta 487:31–40
61. Campos-Vallette MM, Clavijo RE, Mendizabal F, Baraona R,
Zamudio W, Diaz G (1996) Vib Spectrosc 12(1):37–44
62. Gong Y, Tang W, Hou W, Zha Z, Hu C (2006) Inorg Chem
45:4987–4995
63. Juribašić M, Tušek-Božić L (2009) J Mol Struct 924–926:66–72
64. Barone V, Cossi M (1998) J Phys Chem A 102:1995–2001
65. Cossi M, Rega N, Scalmani G, Barone V (2003) J Comp Chem
24:669–681
71. Tan C, Liu J, Chen LM, Shi S, Ji LN (2008) J Inorg Biochem
102:1644–1653
Publisher’s Note Springer Nature remains neutral with regard to
jurisdictional claims in published maps and institutional afliations.
66. Appleton G, Hall JR, McMahon I (1986) Inorg Chem 25:726–734
67. Rodrigues CD, Scott DA, Bailey BN, De Souza W, Benchimol M,
Moreno B, Urbina JA, Oldfeld E, Moreno SNJ (2000) Biochem J
349(3):737–745
68. Alvarez-Valdes A, Matesanz AI, Perles J, Fernandes C, Cor-
reia JDG, Mendes F, Quiroga AG (2019) J Inorg Biochem
191:112–119
Afliations
Micaella Cipriani1 · Santiago Rostán1 · Ignacio León2 · Zhu‑Hong Li3 · Jorge S. Ganchef1 · Ulrike Kemmerling4 ·
Claudio Olea Azar5 · Susana Etcheverry2 · Roberto Docampo3 · Dinorah Gambino1 · Lucía Otero1
1
4
Química Inorgánica, Facultad de Química, UdelaR,
Montevideo, Uruguay
Programa de Anatomía Y Biología del Desarrollo, Facultad
de Medicina, ICBM, Universidad de Chile, Santiago, Chile
2
5
Facultad de Ciencias Exactas, Centro de Química Inorgánica
(CONICET-UNLP), Universidad Nacional de La Plata,
La Plata, Argentina
Departamento de Química Inorgánica Y Analítica, Facultad
de Ciencias Químicas Y Farmacéuticas, Universidad de
Chile, Santiago, Chile
3
Center for Tropical and Emerging Global Diseases
and Department of Cellular Biology, University of Georgia,
Athens, USA
1 3