Synthesis, characterization, antibacterial and antitumoral activities of mononuclear zinc complexes containing tridentate amine based ligands with N3 or N2O donor groups

Article abstract of DOI:10.1016/j.ica.2014.02.040

The synthesis and characterization of the four zinc(II) complexes [Zn(HL₁)Cl₂] (1), [Zn(H₂L₂)Cl ₂](2), [Zn(H₂L₃)Cl₂] (3) and[Zn(H₂L₄)Cl₂

Full text of DOI:10.1016/j.ica.2014.02.040

Accepted Manuscript
Synthesis, characterization, antibacterial and antitumoral activities of mononu-
clear zinc complexes containing tridentade amine based ligands with N3 or N2O
donor groups
Christiane Fernandes, Adolfo Horn Jr, Olney Vieira-da-Motta, Milton M.
Kanashiro, Michelle R. Rocha, Rafaela O. Moreira, Samila R. Morcelli, Bruna
F. Lopes, Luciana da S. Mathias, Franz V. Borges, Layla J.H. Borges, William
R. Freitas, Lorenzo C. Visentin, João C.A. de Almeida, Gerhard Schenk
PII:
S0020-1693(14)00143-1
http://dx.doi.org/10.1016/j.ica.2014.02.040
ICA 15898
DOI:
Reference:
To appear in:
Inorganica Chimica Acta
Received Date:
Revised Date:
Accepted Date:
15 August 2013
14 February 2014
26 February 2014
Please cite this article as: C. Fernandes, A. Horn Jr, O. Vieira-da-Motta, M.M. Kanashiro, M.R. Rocha, R.O. Moreira,
S.R. Morcelli, B.F. Lopes, L.d.S. Mathias, F.V. Borges, L.J.H. Borges, W.R. Freitas, L.C. Visentin, J.C.A. de
Almeida, G. Schenk, Synthesis, characterization, antibacterial and antitumoral activities of mononuclear zinc
complexes containing tridentade amine based ligands with N3 or N2O donor groups, Inorganica Chimica Acta
(
2014), doi: http://dx.doi.org/10.1016/j.ica.2014.02.040
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1
Synthesis, characterization, antibacterial and antitumoral activities of mononuclear zinc complexes
containing tridentade amine based ligands with N3 or N2O donor groups
a,*
a
b
c
Christiane Fernandes , Adolfo Horn Jr , Olney Vieira-da-Motta , Milton M. Kanashiro , Michelle
a
a
a
a
b
R. Rocha , Rafaela O. Moreira , Samila R. Morcelli , Bruna F. Lopes , Luciana da S. Mathias , Franz
c
c
c
d
e
V. Borges , Layla J. H. Borges , William R. Freitas , Lorenzo C. Visentin , João C. A. de Almeida
f
and Gerhard Schenk
a
Laboratório de Ciências Químicas, Universidade Estadual do Norte Fluminense Darcy Ribeiro,
2
8013-602, Campos dos Goytacazes/RJ, Brazil.
b
Laboratório de Sanidade Animal, Universidade Estadual do Norte Fluminense Darcy Ribeiro, 28013-
6
02, Campos dos Goytacazes/RJ, Brazil.
c
Laboratório de Biologia do Reconhecer, Universidade Estadual do Norte Fluminense Darcy Ribeiro,
8013-602, Campos dos Goytacazes/RJ, Brazil.
2
d
P&D NanoBusiness e-Diffraction Pharma, CEP: 22451-900, Rio de Janeiro/RJ, Brazil.
e
Laboratório de Fisiologia e Bioquímica de Microrganismos, Universidade Estadual do Norte
Fluminense Darcy Ribeiro, 28013-602, Campos dos Goytacazes/RJ, Brazil.
f
School of Chemistry and Molecular Biosciences, The University of Queensland, Brisbane, QLD 4072,
Australia.
To whom correspondence should be addressed: Phone: +55 22 2739 7213. Fax:+55 22 2739 7046. E-
mail: chrisf@uenf.br

2
Abstract
The synthesis and characterization of the four zinc(II) complexes [Zn(HL
)Cl ](2), [Zn(H )Cl ] (3) and[Zn(H )Cl ] (4), where HL
= (2-hydroxybenzyl-2-pyridylmethyl)amine, H = N-2[(pyridine-2-ylmethyl)amino)ethanol,
= 1-[(pyridine-2-ylmethyl)-amino]-propan-2-ol are reported; (3) and (4) are new while (2) was
1
)Cl
2
] (1),
[
Zn(H
2
L
2
2
2
L
3
2
2
L
4
2
1
= (bis-2-pyridylmethyl)amine,
H
2
2
L
2
4
L
2 3
H
L
reported previously but its structure had not been determined. The complexes were characterized by
elemental analysis, IR, UV-Vis and NMR spectroscopies, electrospray ionization mass spectrometry
(
ESI(+)-MS) and tandem mass spectrometry ESI(+)-MS/MS). X-ray diffraction studies were performed
for complexes (1)-(3) revealing the presence of mononuclear structures in the solid state. The x-ray
analyses of (1) and (3) demonstrate that HL and HL act as tridentate ligands, while the ligand H in
2) is bidentate. The cytotoxic properties of the ligands and of all the complexes were examined using
human leukemia THP-1, U937 and Molt-4 cells. Complex (4) exhibited the highest cytotoxicity in this
1
2
L
2 2
(
-1
series with an IC50 value of 75 ± 1 µmol L against U937 cells. Transmission electron microscopy
TEM) reveals ultrastructural changes typical of apoptotic cells. The induction of apoptosis was
(
confirmed by the annexin V assay. The antimicrobial activity of complexes (1)-(4) was also
investigated in vitro against four Gram-positive bacteria (ATCC10832, ATCC25923, COL) and the
clinical Staphylococcus aureus isolate LSA88 (SEC/SEF/TSST-1+). Complex (2) showed the most
potent inhibitory activity, reaching almost 100% of inhibition against all strains tested. Morphological
investigations using TEM indicate that the antibacterial activity of complex (2) may be associated with
the inhibition of cell wall and therefore cell division.
Keywords: Mononuclear Zn complexes; ESI(+)-MS/MS; Crystal structure; Antibacterial activity;
Cytotoxic activity; Transmission electron microscopy (TEM).

3
E-mail: chrisf@uenf.br Fax: +55 22 2739 7046. Phone: +55 22 2739 7213.

4
1
. Introduction
Zinc has a vital importance in metabolism, being required for proper functioning of the immune
system, the formation of red blood cells, organ, muscle and bone function, cell membrane stability, as
well as cell growth, division, differentiation and genetics [1]. Zinc compounds are employed as
radioprotective agents [2], tumor photosensitizers [3], antidiabetic insulin mimetics [4] and
antimicrobial agents [5,6,7]. In 2012, Muthmary and co-workers reported the cytotoxicity of di-
zinc(II) complexes for human hepatoma HepG2 cancer cells, where these compounds were able to
induce caspase-dependent apoptosis [8]. In 2013, Kessissoglou and co-workers reported the
antimicrobial activity of zinc(II) complexes on five different microorganisms with IC50 values in the
-1
range of 1-4 µg.ml [9]. Another established application for a zinc compound is as an anti-dandruff
agent; zinc pyrithione (ZPT) is an antimicrobial compound used since 1960 in anti-dandruff shampoos
and also anti-fouling paints [10]. In 2011, Saunders and co-workers reported that the ZPT efficacy
against fungi is related to both the increasing concentrations of intracellular copper and the inactivation
of Fe-S cluster-containing proteins [11].
Recently, we reported the antibacterial activity of iron(III), cobalt(II), copper(II) and zinc
complexes, obtained with the ligand (N-(2-hydroxybenzyl)-N-(2-pyridylmethyl)[(3-chloro)(2-2-
hydroxy)]propylamine), against nine strains of S. aureus. Complexes containing cobalt and zinc
showed the most potent inhibitory activity, reaching almost 100% of inhibition against the nine strains
under investigation. Based on TEM studies, it was proposed that the zinc complex interacts directly
with the genetic material of the S. aureus cells, promoting cell wall and cytoplasmic membrane
damages, which result in the leakage of cellular content and inhibiting further division processes [12].
In that study, the metal ions were coordinated by tetradentate ligands which provide a pyridine, a
phenol, an alcohol and a tertiary amine as coordinating groups. Due to the encouraging results obtained

5
with the zinc complex, we decided here to investigate simpler ligands, i.e. the tridentate bipodal ligands
HL1= (bis-2-pyridylmethyl)amine, H2L2= (2-hydroxybenzyl-2-pyridylmethyl)amine, H2L3= N-
2
[(pyridine-2-ylmethyl)amino)ethanol and H2L4= 1-[(pyridine-2-ylmethyl)-amino]-propan-2-ol). We
also aimed to identify structural features related to the antibacterial activity of the corresponding zinc
complexes. Furthermore, we also investigated the antitumor activity of these zinc complexes to probe a
possible connection between these two biological activities. It is worth noting that complex (1), the
crystal structure of which is reported in this manuscript, was recently identified as an inhibitor of the
activated RAS protein [13]. In cancer research RAS represents a target of high interest owing to its
critical involvement in about 30% of all human malignancies [14].
2
. Experimental
2
.1 Materials and methods
All the reagents and solvents were commercially available of high purity and used as such.
The CHN elemental analysis for the complexes was performed on a Thermo Scientific FLASH 2000
CHNS/O analyzer. Infrared spectra were recorded with KBr disks on a Shimadzu FT-IR 8300. Nuclear
magnetic resonance (NMR) data were obtained using a JEOL eclipse 400+ spectrometer and deuterated
-3
-3
dimethylsulfoxide (d6-DMSO) as solvent. The electrical conductivity of a 1x10 mol.dm solution of
each complex was measured with a Biocristal conductometer, in MeCN. Melting points (MP) were
measured on a Microquimica MQAPF-301 apparatus. Full scan mass spectra (MS mode) were obtained
on a MicroTOF LC Bruker Daltonics spectrometer equipped with an electrospray source operating in
positive ion mode. Samples were dissolved in a MeOH/H O (1:1) solution and injected in the apparatus
2
by direct infusion. To prepare the samples for transmission electron microscopy (TEM) the cells were
cultured until reaching their final exponential and initial stationary phases. Pellets of cells were then
harvested and processed for transmission electron microscopy (TEM) according to routine procedures

6
as follows: the control and cells treated with the coordination compounds were fixed in 2.5%
-1
glutaraldehyde, 4% paraformaldehyde diluited in 0.1 mol L cacodilic buffer (pH = 7.2) and post-fixed
in 1% osmium tetroxide and 0.8% potassium ferrocyanide, at room temperature, in the dark for 20
minutes. After the fixing step, the dehydration process was conducted with 30, 50, 70, 80, 95 and 100%
®
of acetone and samples were embedded in a crescent series of epoxy resin (Polybed 812 , EUA). Small
®
blocks of the obtained polymer were cut with an ultramicrotome (Reichert Ultracut’s, Leica
Switzerland). Ultrathin sections were then negatively stained with uranyl acetate (5%) and lead citrate
for observation in the microscopy (TEM 900 Zeiss, Germany), at 80 kV.
2
2
.2 Syntheses
.2.1 Synthesis of the ligands
The ligands employed in this work are presented in Scheme 1 and were synthesized as
described previously [15, 16, 17].
2
.2.2. Syntheses and characterizations of the complexes
The syntheses of the zinc complexes followed a similar synthetic route as exemplified below for
the synthesis of the compound [Zn(HL1)Cl2] (1) (the syntheses and partial characterizations of
complexes (1) and (2) were previously described in the literature [18,19]). Table 1 shows
crystallographic data and Table 2 shows some data concerning the synthesis and characterization of the
compounds (1)-(4).
3
[
Zn(HL1)Cl
2
] (1): An isopropanol solution (15 cm ) of ZnCl
2
.2H O (136 mg, 1 mmol) was added to a
2
3
methanol solution (15 cm ) of the ligand HL1 (200 mg, 1 mmol), resulting in a white solid. The
solution was heated (50-60ºC) until complete dissolution of the precipitate. The solution was

7
refrigerated for some days and white single crystals were formed, filtered off and washed with
methanol.
2
.3 Crystal data collection and refinement
The molecular structures of the complexes (1), (2) and (3) were solved by single crystal x-ray
diffraction. Crystal data were collected at 295 K from an Enraf-Nonius Kappa-CCD diffractometer
with graphite monochromatized Mo K radiation [20]. The cell parameters were obtained using the
α
COLLECTA [21] and PHICHI [22] programs, and were refined using the DIRAX program [23].
Intensities were corrected by Lorentz polarization using the EvalCCD program [24], and absorption
correction with the SADABS program [25]. The structures were solved as described in reference [26].
The positional parameters of the H atoms bonded to C atoms in the methylene moiety were obtained
3
geometrically, with the C-H distances fixed at 0.96Å for Csp atoms, and their respective C atoms were
3
refined with Uiso(H) = -1.2Ueq(Csp ). The positional parameters of the H atoms bonded to C atoms in
2
the aromatic rings were obtained geometrically, with the C-H distances fixed at 0.93Å for Csp atoms,
2
and set to ride on their respective C atoms, with Uiso(H) = 1.2Ueq(Csp ). The hydrogen atoms of the
aminic and hydroxyl groups were located in Fourier maps and free refined to position.
Crystallographic and structure refinement data for complexes (1)-(3) are shown in Table 1.
Table 1. Crystallographic data and structure refinement for complexes (1), (2) and (3).
Complexes
(1)
(2)
(3)
Empirical formula
Formula weight
C
12
H
13
N
3
ZnCl
2
C
13
H
14
N
2
OZnCl
2
C
8
H
12
N
2
OZnCl
2
335.52
295(2) K
0.71073 Å
Monoclinic, P2
350.56
295(2) K
288.47
T /K
295(2) K
0.71073 Å
Monoclinic, P2
Radiation, λ/Å
Crystal System, space group
0.71073 Å
1
/n Triclinic, P-1
1
/c

8
Unit cell dimensions in (Å) a = 8.4981(17)
a = 8.3502(17)
b = 9.4156(19)
c = 11.005(2)
α = 99.79(3)
β = 111.48(3)
γ = 108.24(3)
723.9(3)
a = 10.131(2)
b = 8.0967(16)
c = 14.367(3)
β = 108.99(3)
and (°)
b = 11.914(2)
c = 14.405(3)
β = 105.74(3)
3
Volume /Å
Z
1403.8(5)
4
1114.3(4
4
2
-
3
Calculated density/g.cm
1.588
2.114
680
1.608
2.057
356
1.720
2.652
584
-1
Absorption coefficient /mm
F(000)
3
Crystal size / mm
Theta range /°
0.17 x 0.12 x 0.10 0.76 x 0.47 x 0.27
0.22 x 0.21 x 0.15
3.29 to 25.00
19363
3.05 to 25.49
2.10 to 25.50
Reflections collected
Independent reflections
Completeness to θ max.
29432
20491
2614[R(int)=0.0726] 2667[R(int)=0.0520] 1954 [R(int) = 0.0406]
99.8 %
99.1 %
99.6 %
Max. and min. transmission
0.8148 and 0.7204 0.6066 and 0.3039 0.6981 and 0.5931
Data / restraints / parameters
2614 / 0 / 167
1.043
2667 / 0 / 181
1.123
1954 / 0 / 136
1.041
2
Goodness-of-fit on F
Final Rindices [I>2sigma(I)]
R indices (all data)
R
1
= 0.0272
wR = 0.0490
= 0.0478
wR = 0.0531
None
R
1
= 0.032
2
R
1
= 0.0199
wR = 0.0425
= 0.0258
wR = 0.0440
wR = 0.0681
2
2
R
1
R
1
= 0.0550
2
R
1
wR = 0.0809
2
2
Extinction coefficient
0.0174(18)
0.0009(3)
-
Largest diff. peak and hole (e 0.232 and -0.220
0.487 and -0.628
0.271 and -0.238
-3
.
Å )
2
2
.4.Antileukemic activity studies
.4.1. Culture media
Human leukemia cell lines THP-1 (acute monocytic), U937 (histiocytic lymphoma) and Molt-4
acute lymphoblastic) were cultured routinely in DMEM-F12 medium (Gibco, BRL), supplemented
with 10% fetal calf serum and gentamicin (20 µg/ml; Gibco, BRL) at 37 °C in a humidified atmosphere
containing 5% CO . Culture media were changed every 2–3 days. Blood samples were collected from
healthy donors in Sodium Heparin glass tubes "VacutainerTM" (Becton Dikinson). Peripherical blood
(
2

9
mononuclear cells (PBMC) were isolated over Ficoll-Paque™ Plus (1.08 g/mL) in a 50 mL conical
tube (2:1 - blood:ficoll). Twenty milliliters of a fresh, heparinized blood sample were diluted in
phosphate-buffered saline (PBS), gently laid over 10 ml of Ficoll and centrifuged at 500xg for 20 min
at 25 °C. PBMC rings were collected from the interface of blood samples and washed three times with
PBS (phosphate buffered saline) by centrifugation at 500xg for 10 min at 4 °C. The supernatant was
discarded and the cells were suspended in DMEM-F12 medium (Gibco, BRL). Trypan blue solution
0
.4% (Sigma, Germany) was used to count the cells into an appropriate concentration and the viability
of cells was checked; the required range of cell viability was 95-99%.
2
.4.1. Screening for antileukemic activity
In order to evaluate the in vitro antitumoral activity of complexes (1-4), their respective ligands
and metallic salt were dissolved in DMSO and tested using THP-1, U937 and Molt-4 cells lines.
Cisplatin and DMSO were used, respectively, as a positive and negative control of the assay. To
determine cell viability the colorimetric MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium
bromide) metabolic activity assay was used and IC50 values were calculated in order to evaluate the
relationship between the structure and the activity of these synthetic compounds (IC50 is the
concentration required for 50% of inhibition). The MTT assay assesses the capacity of the
mitochondrial enzyme succinate dehydrogenase to transform the MTT salt into a purple colored
6
product (formazan), which is proportional to the number of viable cells [27]. Leukemia cells (1 x 10
-1
-1
cells mL ) in DMEM-F12 medium supplemented with 10% fetal calf serum and 20 µg mL
gentamicin were plated (100 µL/ well) in 96 well plates and treated with different concentrations of
o
complexes (1-4) for 36 h and incubated at 37 C with 5% of CO
2
and humidified atmosphere. Twenty
-1
microliters of MTT working solution (5 mg mL ) were added into each well, and the cells were
incubated at 37 °C for 4 h. Then, the MTT-formazan crystals produced by viable cells were dissolved in

1
0
an isopropanol-HCl solution. The optical density (OD) values were measured by spectrophotometry at
5
70 nm using a Microplate Reader (Multiskan-EX). The OD values are presented as relative viable cell
®
numbers. For apoptosis analysis by Annexin V-FITC Apoptosis Detection kit (Sigma ) U-937 cells
6
-1
were seeded in 24-well plates at a density of 1 x 10 cells.mL and treated with compound (4) at
-1
concentrations of 200 and 400 µmol L and incubated at 37 ºC for 24 h in a humidified atmosphere
with 5% CO . After 24 h the cells were collected and washed in DPBS and suspended in binding
2
buffer containing 5 µL of annexin V – FITC and 10 µL of propidium iodide. The cells were incubated
for exactly 10 minutes (protected from light) at room temperature, according to the manufacturer's
recommendations. Data acquisition was performed by BD FACSCalibur with CELLQuest software and
for data analysis, WinMDI software version 2.9 was used. For analysis of cell viability and apoptosis
the mean and standard error were calculated from the raw data and then subjected to the One-way
ANOVA- Turkey´s Multiple Comparison Test (for ZnCl
Bonferroni posttests (for all the complexes, ligands and metallic salt), software Graphpad version 5.0
analysis of variance). Significant difference was taken as *p < 0.05, ** p <0.01 and *** p <0.001.
2
and cisplatin) and two-way ANOVA-
(
2
.5. Antibacterial activity studies
2
.5.1. Microorganisms
Bacterial strains: S. aureus (ATCC10832, ATCC25923, COL) and the clinical S. aureus LSA88 isolate
(
(
SEC/SEF/TSST-1+) were obtained from the bacterial collection of the Laboratory of Animal Health
Laboratorio de Sanidade Animal- LSA), Universidade Estadual do Norte Fluminense Darcy Ribeiro,
RJ/ Brazil.
2
.5.2 Growth inhibition
The inhibition assay was conducted by adding bacterial cells to grow in BHI (Brain Heart Infusion

11
8
Broth) medium during 24 h at 37 ºC. An aliquot of 0.1 mL of innoculum containing 10 cells/mL (0.5
McFarland, Densimat (bioMérieux, France) was added to 1.85 mL of medium and 0.5 mL of each
-2
-1
compound (ligands, coordination compounds and ZnCl at concentration of 10 mol L ). As positive
2
-2
-1
and negative controls, gentamicin at concentration of 10 mol L and DMSO were used, respectively.
The growth curves were measured with a densitometer D.O.550nm (Densimat - bioMérieux, France) in
intervals of 60 min. The assays were stopped when cell density reached 7.5 McFarland. Each
experiment was performed in triplicates and repeated twice. Inhibition percentages were thus reported
as averaged values.
2
.5.3 Minimal Inhibitory Concentration (MIC)
In vitro antibacterial activity of the complexes was investigated by the broth microdilution
method according to the Clinical and Laboratory Standards Institute (CLSI 2009) document M7-A8
28]. Mueller Hinton broth (Acumedia, USA), supplemented with 2% glucose, was employed as the
bacterial growth medium. Stock solutions of the compounds were prepared in DMSO and diluted in the
[
-4
-3
-1
same culture medium in serial, twofold concentrations in the range of 1.25x10 –10 mol L . Through
8
-1
photometric readings (Densimat - bioMérrieux, France) the inoculum containing 1x10 CFU mL
corresponding to 0.5 McFarland, were obtained from the broth cultures in the log phase of growth and
6
-1
diluted to 1x10 CFU mL . Subsequently, a 96 wells cell culture plate (TPP, USA) was used for MIC
assays, and serial diluted chemical compound solutions (0.1 mL), inoculated in each sample of each
bacterium strain (0.1mL), were incubated in a humid chamber at 37 ºC for 24 h. The MIC of each
tested compound was defined as the lowest concentration exhibiting no visible growth compared to the
drug-free control wells seen after the addition of 30 µL (1 mg/mL) of the dye resazurin (Sigma, USA).
Gentamicin (Sigma, USA) was used as a reference antibacterial agent. A column of plate wells
containing only inoculated medium was kept as the negative control. Metallic salt (ZnCl
2
) and ligand
controls were also conducted simultaneously. All assays were performed in triplicates [28].

1
2
3
. Results and discussion
In our attempt to generate compounds with effective biological activity (i.e. antitumoral and
antibacterial), we focused on Zn(II) complexes with distinct N,O-donor ligands, containing one or two
aromatic chelating groups (Scheme 1). We have selected those ligands since they allow us (i) to probe
if different bipodal ligands have similar coordination behavior and (ii) to investigate the influence of
the ligands' structure on the antibacterial and antitumor activities of these complexes.
While the bipodal ligand HL1 induces a N3 coordination environment on the metal center, the
bipodal ligands H2L2, H2L3 and H2L4 may generate a N2O coordination, where the O-donor center is
provided by a phenol, a primary alcohol and a secondary alcohol, respectively. Ligands H2L3 and
H2L4 are similar, but the ligand H2L4 presents a methyl group, which is absent in ligand H2L3.
The ligands and their respective zinc complexes are very stable in air. They are remarkably
soluble in polar aprotic solvents such as DMF, DMSO and MeCN; slightly soluble in ethanol, methanol
and chloroform; insoluble in water.
The reactions between the ligands described and ZnCl
2
.2H O allowed the isolation of crystals
2
for all compounds. It was possible to determine the molecular structure through x-ray diffraction for
compounds (1)-(3). Table 2 shows relevant data from the synthesis and characterization of the
compounds (1)-(4).The conductivity behavior of complexes in MeCN was examined. The low
conductivity values observed for complexes (1)-(4) suggest that they are non-electrolyte and indicate
that the arrangement observed in solid state is the same in solution.
The data presented in Table 2 indicate that the compounds are pure and that compounds (1)-(4)
are neutral species. Scheme 1 presents the proposed structures of complexes (1)-(4), based on
spectroscopic and structural data.
Scheme 1 here

1
3
Table 2. Yield, melting point (M.P.), elemental analysis (C, H, N) and conductivity data for complexes
1)-(4), in MeCN.
(
Complex Composition
M.W
g/mol) (%)
Yield M. P.
%C
%H
%N
Λ
M
o
2
−1
-1
(
( C) (Found/Calcd.) (Found/Calcd.) (Found/Calcd.)
(
cm Ω mol )
(
(
(
(
1)
2)
3)
4)
Zn(HL1)Cl
Zn(H L2)Cl
Zn(H
Zn(H
2
335.54
78
79
63
68
286
193
169
175
42.86/42.96
44.57/44.54
33.47/33.31
35.40/35.73
3.95/3.91
4.03/4.03
4.21/4.19
4.89/4.66
12.56/12.52
8.24/7.99
9.98/9.71
9.16/9.26
5.6
17.3
3.8
2
2
2
2
350.55
288.48
302.51
2
L3)Cl
L4)Cl
2
16.8
3
.1. IR and NMR spectra
The IR spectra of complexes (1)-(4) were analyzed in comparison with those of their common
-1
free ligands HL
1
-H
2
L in the region 4000-400 cm . The IR spectra of free ligands are similar and show
4
characteristic absorption bands: νN-H ,νC=C, νC=N and δ(pyridine ring). For ligand HL1, characteristic bands
-1
of the pyridine group are observed at 1593, 1435 and 758 cm , assigned to νC=N, νC=C and δ(pyridine ring)
,
-1
respectively. The νN-H band is observed at 3344 cm for ligand HL1. For ligand H2L2, characteristic
-1
bands of the pyridine group are observed at 1593, 1430 and 756 cm , assigned to νC=N, νC=C and
-1
-1
δ
(pyridine ring), respectively. The νN-H band is observed at 3263 cm . There is a band at 1280 cm ,
attributed to the phenol group (νC-O). For ligand HL3, characteristic bands of the pyridine group are
-1
observed at 1595, 1435 and 762 cm , assigned to νC=N, νC=C and δ(pyridine ring), respectively. The νN-H
-1
band is observed at 3300 cm . For ligand H2L4, characteristic bands of the pyridine group are
-1
observed at 1595, 1437 and 764 cm , assigned to νC=N, νC=C and δ(pyridine ring), respectively. The νN-H
-1
band is observed at 3300 cm . For all the complexes, the bands due to νN-H and δ(pyridine ring) exhibited
downward and upward shifts, respectively, after coordination to ZnCl . For complexes (1)-(4), the νN-H
are observed at 3244, 3226, 3228 and 3178 cm , and δ(pyridine ring) are observed at 760, 760,779 and 767
2
-1
-1
cm , respectively. The bands attributed to νC=N exhibited a downward shift, resulting in bands at 1570,
-1
1
570 and 1573 cm , respectively for complexes (1), (3) and (4). For complex (2), no shift was
-1
observed for the band νC=N; furthermore, the presence of a band at 1273 cm indicates the presence of

1
4
the phenol group in the structure of the complex, in good agreement with the crystal data. Table 3
presents the characteristic bands of L1-L4 and their respective complexes.
-1
Table 3. The characteristic IR bands of the ligands L1-L4 and their respective complexes, in cm .
HL1
3344
1593
1435
758
(1)
H2L2
3263
1593
1430
756
(2)
H2L3
3300
1595
1435
762
(3)
H2L4
3300
1595
1437
764
(4)
ν
N-H
3244
1570
1442
760
3226
1593
1435
760
3228
1570
1446
779
3178
1573
1442
767
ν
C=N
,
ν
C=C
δ
(pyridine
ring)
phenol
νC-O)
The H NMR spectra for the complexes are presented in Figure 1. To facilitate the
---
1280
----
---
1273
(
1
interpretation, the carbon atoms in the ligands are numbered, and the numbers on the signals indicate
the hydrogen atom attached to these carbon atoms. The spectra for complexes (1), (3) and (4) show a
similar profile related to the aromatic region (7.0-9.0 ppm), which is attributed to the hydrogen atoms
of the pyridine group. Interestingly, while these complexes show only three signals related to these
aromatic hydrogen atoms, their free ligands display four signals (see Table 4). The integral ratio and the
coupling constant indicate that the signals of the hydrogen atoms bound to the carbon atoms 2 and 4
were merged in a multiplet around 7.56 ppm.
For complex (1) the methylenic hydrogen (CH ) from the ligand HL1 is observed at 4.13 ppm
2
with a very broad shape, while in the free ligand it appears as a narrow singlet at 3.90 ppm. For the
same complex, the amine hydrogen atom was observed at 2.90 ppm in the free ligand and appears at
4
.93 ppm in the complex. This shift is in agreement with the fact that the electron density on the
nitrogen atom is lower in the complex than in the free ligand.

1
5
The spectrum of complex (3) shows the methylenic group attached to the aromatic ring as a
singlet at 4.06 ppm (3.92 ppm in the free ligand). There are also two triplets (J = 5.5 Hz) associated
with the other two methylenic groups, at 3.51 and 2.78 ppm. The former is the one bound to the alcohol
group while the latter is connected to the amine nitrogen atom.
For complex (4), the hydrogen atoms from the py-CH -N unit were split in two doublets with J
2
values of 15.6 Hz, typical of geminal hydrogen atoms. The hydrogen atom bound to C8 appears at 3.74
ppm as a broad singlet, while the hydrogen atoms from the methylenic and methyl groups are observed
at 2.77 ppm (dd, J
1
= 11.8 Hz; J = 3.13 Hz) and 1.07 ppm (d, J = 6.3 Hz), respectively.
2
The spectrum of complex (2) differs significantly from the others, since it possesses two
1
different aromatic rings (pyridine and phenol), resulting in a larger number of signals in the H NMR
spectrum between 6 and 9 ppm. The assignment of the hydrogen atoms from the pyridinic group may
be established by comparison to the compounds described above. However, it should be pointed out
that some of the aromatic hydrogen atoms are presenting two different signals, accounting to a ratio of
2
0-30/70-80 %. For example, the hydrogen atom attached to C1 presents just one signal in compounds
(
1), (3) and (4). However, in (2) there are two signals associated with this hydrogen atom (8.75 ppm, d,
J = 4.0 Hz and 8.59 ppm, d, J = 3.1 Hz). Similarly, there are other signals in the aromatic range which
present the same ratio and are associated with the same hydrogen atoms (C3-H, C4-H, C11-H). On the
other hand, there are some signals that are not split (C2-H, C9-H, C10-H, C12-H). We have already
observed the same behavior with another zinc compound synthesized with a ligand which also contains
pyridine and phenol groups; those studies seem to indicate the presence of different isomers in solution
[
9]. The aliphatic region is dominated by two intense broad signals at 3.95 ppm and 4.08 ppm, which
are associated with the methylenic hydrogen atoms. There are also some side peaks that again suggest
the presence of different isomers in solution. In the free ligand the methylenic hydrogen atoms appear
1
at 4.00 and 3.83 ppm. Table 4 shows the relevant H NMR spectral data with assignments.

1
6
1
Table 4. H NMR spectral data with assignments for ligands HL1-H2L4 and their respective
complexes.

1
7
H
1
2
3
4
6
L1
/M/J (Hz)
8.48/dd/ 4.0; 1.8
ZnL1 (1)
L2
/M/J (Hz)
8.58/dd/5.3; 1.7
ZnL2 (2)
L3
/M/J (Hz)
8.47/d/4.2
ZnL3 (3)
L4
/M/J (Hz)
ZnL4 (4)
δ
δ/M/J (Hz)
δ
δ/M/J (Hz)
δ
δ/M/J (Hz)
δ
δ/M/J (Hz)
8.78/d/4.7
8.76/d/4.0
8.56/d/5.08
8.54/d/4.2
7.15/dd/7.7; 4.8
7.63/dt/7.7; 1.8
7,26/d/7.7
8.55/d/4.7
8
.59/d/3.1
7.07/m
7.55/m
7.19/m
7.50/m
7.11/dd/7.7;
7.55/m
7.53/m
8.01/dt/7.8; 1.2
7.53/m
4
.4
7.56/dt/7.7; 1.8 8.02/dt/7.6; 1.6 7.65/dt/7.7; 1.7
7.96/t/7.6
.70/dt/7.8; 1.6
7.50/m
7.59/dt/7.7; 1.5 8.03/dt/8.0; 1.5
7
7.28/d/7.7
3.90/s
7.55/m
4.13/s
7.19/m
3.83/s
7.23/d/7.7
3.87/s
7.55/m
4.06/s
7
.08/d/7.4
3.95/s
3.91/s
4.06/d/15.6
3.98/d/15.6
7
9
-
-
-
-
-
-
-
-
4.0/s
6.97/d/7.3
6.77/dt/7.3; 0.9
7.19/m
4.08/s
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
7.25/d/6.6
6.78/t/7.4
6.36/t/7.0
10
11
7
.17/t/7.6
12
13
14
15
-
-
-
-
-
-
-
-
6.86/m
6.86/d//6,9
-
-
-
-
6
,89/d/6,9
-
-
-
-
2.76/t/5.3
3.61/t/5.3
-
2.78/t/5.5
3.51/t/5.5
-
2.74/dd/12.9; 2.2 2.76/dd/12.0; 3.1
.41/dd/12.9; 9.5
2
-
-
3.81/m
3.73/ m
1.13/d/6.2
1.06/d/6.2
δ= chemical shift; M = signal multiplicity, J = coupling constant.

1
8
3.2. Structure of the complexes
3.2.1. Description of the structure of complexes (1)-(3)
The single crystal x-ray studies are in agreement with the above spectroscopic
analysis and show three new structures of mononuclear zinc complexes with σ-
(HL1), σ-N (H2L2) andσ-N (H2L3) donor sets. The ORTEP-3 projection of (1),
2) and (3) are shown in Figures 2-4, respectively. Bond lengths and bond angles are
N
3
2
2 1
O
(
listed in Table 5. These parameters are in agreement with literature values [29] and
within the expected range for compounds of this class, as presented by the International
Tables for X-ray Crystallography [30].
Table 5. Selected bond lengths [Å] and angles [°] for complexes (1), (2) and (3).
Bond lengths
Zn1-N1
Zn1-N2
Zn1-N3
Zn1-O1
Zn-Cl1
(1)
(2)
(3)
2.1548(19) 2.031(2)
2.1244(16)
2.0971(17)
-----
2.146(2)
2.158(2)
-----
2.057(3)
-----
-----
2.3150(17)
2.2650(7) 2.1988(10) 2.2180(7)
2.2598(7) 2.2214(13) 2.2679(7)
Zn-Cl2
N2-C6
1.453(3)
1.461(3)
1.332(3)
1.335(3)
-----
1.475(4)
1.475(4)
1.343(4)
1.315(4)
-----
1.463(2)
1.463(3)
1.334(2)
1.327(3)
1.410(3)
-----
N2-C7
N1-C5
N1-C1
O1-C8
O1-C9
-----
1.337(4)
(2)
Bond angles
C6-N2-C7
N2-Zn1-N1
N2-Zn1-N3
(1)
(3)
115.7(2)
76.00(7)
76.44(8)
113.7(2)
81.66(10)
-----
114.22(16)
77.95(6)
-----

1
9
N1-Zn1-N3
N2-Zn1-Cl2
N1-Zn1-Cl2
N3-Zn1-Cl2
N2-Zn1-Cl1
N1-Zn1-Cl1
N3-Zn1-Cl1
Cl2-Zn1-Cl1
N2-Zn1-O1
N1-Zn1-O1
Cl1-Zn1-O1
Cl2-Zn1-O1
150.22(8)
-----
-----
139.63(6) 113.74(9)
124.64(5)
98.30(5)
-----
96.53(5)
97.16(6)
109.13(8)
-----
103.56(6) 112.38(8)
115.11(5)
103.69(5)
-----
97.76(6)
99.43(6)
116.80(4)
-----
116.17(8)
-----
118.25(4)
-----
119.26(2)
74.00(6)
151.08(6)
94.51(5)
91.94(5)
-----
-----
-----
-----
-----
-----
Incomplete structural data for complex (1) were previously reported in 1992
[
[
31]. In 2007, Kang and co-workers reported the structural data for the complex
2 3
ZnCl (HL1)].CHCl , revealing the presence of a chloroform as solvate in the molecular
structure [32]. Here, we are reporting the structural data for complex (1) without solvate
molecule (Figure 2). In (1), the zinc center is coordinated by a σ-N donor set provided
by the ligand HL1, via one aminic nitrogen atom (N2H), two pyridinic nitrogen atoms
N1 and N3) and two chloride anions (Cl1 and Cl2), resulting in a pentacoordinated
3
(
zinc(II) complex with a N3Cl2 coordination environment around the zinc(II) center. The
tridentate ligand HL1 is linked to the zinc(II) ion by two five-membered rings, with the
following bite angles: 76.00(7°) from N1-Zn-N3 and 76.44(8°) from N2-Zn-N3. In this
complex the τ parameter (τ = (β−α)/60 = 0.18) indicates a slightly distorted square
pyramidal geometry [33]. The basal positions are occupied by N1, N2, N3 and Cl2,
while the axial position is occupied by Cl1 atom. The base of the pyramid shows the
following angles: β = 150.22(8)° from N1-Zn1-N3 and α = 139.63(6)° from N2-Zn1-
Cl2. The zinc center shows a dihedral angle with N1/C5/C6/N2 plane of 30.4(1)° and a

2
0
torsion angle for this fragment (N1-C5-C6-N2) of -24.2(3)°. The zinc atom shows a
deviation of 0.594 Å out of the N1/N2/N3/Cl2 plane.
Although it has been shown that the ligand H2L2 usually provides a N2O1
2 2
donor set, when reacted with to ZnCl .2H O [34, 16, 35], it acts as a bidentate ligand
through the aminic (N2) and the pyridinic nitrogen (N1) atoms (Figure 3). The distorted
tetrahedral geometry presented by the zinc center is completed by two chloro ligands.
The angles around the zinc center are 81.7(1)° for N1-Zn1-N2 and 118.26(4)° for Cl1-
Zn1-Cl2. The phenol group remains protonated after coordination and away from the
metal center, indicating that the hydrogen bonds of the O1H from the phenolate group
with H6B (from C6) and H7B (from C7) are strong. This behavior is different from
II
what we observed previously in the compound [Zn (HBPClNOL)Cl], in which the
phenol group is coordinated to the zinc center as an anion [12]. This suggests that the
II
Zn(II) ion in (2) has a lower acidity than in the complex [Zn (HBPClNOL)Cl]. The
structure for complex (2) is in excellent agreement with that proposed by Tandon and
co-workers in 1994 [19].The molecular structure of the complex (3) reveals a
pentacoordinated zinc center (Figure 4), which adopts a strongly distorted square
pyramidal geometry (τ = 0.44). The basal positions are occupied by N1, N2, O1 and Cl2
atoms, and the axial position is occupied by the Cl1 anion. The basal angles are β =
151.08° for N1-Zn1-O1 and α = 124.64° for N2-Zn1-Cl2. Although the ligand H2L3 is
very similar to H2L2, also providing a N2O donor set, it is able to act as a tridentate
ligand, while the latter showed a bidentate coordination behavior. Thus, the Zn(II) ion
coordinates to the H2L3 ligand via the pyridinic nitrogen atom (N2), aminic nitrogen
atom (N1) and by the alcohol group (O1H). Two chloride anions (Cl1 and Cl2) are also
bound to the Zn(II) center, resulting in a N
2 2
OCl coordination environment. The two
five-membered rings observed in the complex have bite angles of 77.95(6)° in N2-Zn1-

2
1
N1 and 74.00(6)° for N2-Zn1-O1, which are very similar to those observed in complex
1). The zinc atom shows a deviation of 0.715 Å out of the N1/N2/O1/Cl2 equatorial
(
plane. The metal atom forms a dihedral angle with the N1/C5/C6/N2 plane of 27.2(1)°
and an N1-C5-C6-N2 torsion angle of -31.6(2)°. The dihedral angle formed by
N2/C7/C8/O1 is 17.1(1)° and the torsion angle in this N2-C7-C8-O1 moiety is
52.43(2)°.
3.3. ESI(+)-MS and ESI(+)-MS/MS studies
For complex (1), in a water/methanol solution (1:1), signals associated with
+
mononuclear and dinuclear species are observed ([Zn(NaL1)Cl] (m/z 322) and
+
[
(HL1)(OH
2
)(OH)Zn-(µ-Cl)-Zn(HL1)(OH)(H
2
O)] (m/z 635)), as well as signals related
+
to the free ligand [H2L1] (m/z 200). The observation of the m/z 635 species in the
MS/MS spectrum suggests the formation of the mononuclear cation
+
[
[
Zn(HL1)(OH)(OH
2
)]
(m/z 300), by the loss of the neutral molecule
)] (m/z 335).
Zn(HL1)(Cl)(OH)(OH
2
As complex (2) is neutral, i.e. [Zn(HL2)Cl ], no signal associated with this
2
species is observed. However, in a water/methanol (1:1) solution, twelve additional
signals and their characteristic set of iso-topolog ions, due mainly to the presence of Zn
and Cl atoms, are clearly detectable (data not shown). These signals are related to
+
+
mono- and dinuclear species containing Zn, e.g: [Zn(HL2)] (m/z 277), [Zn(H2L2)Cl]
+
+
(
[
m/z 313), [Zn(NaHL2)Cl] (m/z 335), [Zn(NaH2L2)(OH)(Cl)(CH
3
OH)] (m/z 385),
+
+
Zn(H2L2)(HL2)] (m/z 493), [Zn(H2L2)(HL2)(CH
3
OH) ] (m/z 555), [(HL2)Zn-(µ-
2
+
+
OH)-Zn(HL2)] (m/z 571), [(HL2)Zn-(µ-Cl)-Zn(HL2)] (m/z 589) and [(NaHL2)Zn-(µ-
+
Cl)-Zn(HL2)Cl] (m/z 647). In addition, a signal attributable to the ligand H2L2 (i.e.
+
[
H3L2] (m/z 215)) is observed. MS/MS studies indicate that the ion of m/z 533 may

2
2
+
yield the cation [Zn(H2L2)(HL2)] (m/z 493) by the loss of solvent, suggesting that the
+
m/z 533 species is probably a solvent cluster of the [Zn(H2L2)(HL2)] (m/z 493) ion.
+
MS/MS analysis of the species with m/z 493 yields the cation [Zn(HL2)] (m/z 279) and
the neutral molecule of the ligand H2L2 (m/z 214). The ion corresponding to m/z 482
may be associated with dimerization of the H2L2 ligand and solvent molecules. MS/MS
7 6
data of the ion with m/z 482 indicates the formation of C H O (m/z 106), as reported
previously [24]. In water/methanol solutions interesting association processes are
+
observed rendering the species [(HL2)Zn-(µ-OH)-Zn(HL2)] (m/z 571), [(HL2)Zn-(µ-
+
+
Cl)-Zn(HL2)] (m/z 589) and [(NaHL2)Zn-(µ-Cl)-Zn(HL2)] (m/z 647) as a result of the
+
interaction between the two [Zn(HL2)] m/z 277 ions. The ion with m/z 571 is formed
by the interaction between two species of m/z 277 via a hydroxo bridge; the ion with
m/z 589 is formed by an interaction between two species with m/z 277 involving a
chloride bridge and for the ion with m/z 647it is suggested that it may contain the
species with m/z 589 plus a chloride and a sodium ion. ESI(+)-MS/MS of these ions
were unsuccessful due the low intensity of the majority of the signals. ESI(+)-MS/MS
for the species with m/z 277, 482 and 491 are also structurally characteristic, indicating
only the presence of the ligand H2L2 in these fragments.
ESI(+)-MS data for complex (3) indicate the presence of five cations in
+
+
water/methanol (1:1) solution: [H3L3] (m/z 153), [Zn(HL3)] (m/z 215),
+
+
[
[
Zn(H2L3)2(Cl)] (m/z 403), [(HL3)Zn-(µ-Cl)-Zn(H2L3)Cl] (m/z 505) and
+
(H2L3)(Cl)Zn- (µ-Cl)-Zn(H2L3)(Cl)] (m/z 541). The ESI(+)-MS/MS of the ion with
m/z 541 is also structurally characteristic; the ion loses an HCl molecule to form a
+
fragment ion with m/z 505 [Zn
2
(HL3)(H2L3)Cl
2
] . The loss of two HCl molecules leads
+
to the formation of the fragment ion with m/z 469, [Zn
2
(HL3)
2
Cl] . The loss of the
neutral complex [Zn(H2L3)Cl
2
] (m/z 288) results in a fragment ion with m/z 253

2
3
+
[
Zn(H2L3)Cl] .
For complex (4) five species were detected in the ESI(+)-MS spectrum:
+
+
+
[
[
NaH2L4] (m/z 189), [NaZn(HL4)Cl)] (m/z 287), [Zn(H2L4)(HL4)] (m/z 395),
+
+
2 2 2 2
NaZn (L4) ] (m/z 480) and [Zn (HL4) Cl] (m/z 495). The ESI(+)-MS/MS data for
these species indicate the presence of the ligand H2L4. Figure 5 illustrates some
species detected in the ESI(+)-MS and ESI(+)-MS/MS spectra for compounds (2) and
(
2
4) in H O/MeOH. Based on the ESI(+)-MS and ESI(+)-MS/MS data, it is suggested
that complex (4) is mononuclear in the solid state, with the zinc center coordinating to
one molecule of the H2L4 ligand and to chloride ions, thus resulting in a neutral
complex, in good agreement with other results (i.e. conductivity measurements, NMR
and elemental analysis, see above). In solution it can however dimerize, as observed for
the other compounds.
The spectra and the proposed structures based on the isotopic pattern for the
main ion of each complex are supplied in the Supplementary material.
3.4. Antileukemic activity of the complexes
In order to study the antitumoral activity of the zinc complexes (1)-(4), their
cytotoxicity against THP-1, U937 and Molt-4 human leukemia cell lines was
-1
investigated (Table 6). Complex (4) has a lower IC50 value (75 ± 1 µmol L ) for the
U937 cell line, and is the complex with the highest activity of this series. Ligand HL1 is
the only ligand with measurable antitumoral activity in the three cell lines under
-1
investigation, with an IC50 value of 52 ± 1 µmol L in the Molt-4 cell line. No
potentiating effect is observed upon coordination of HL1 to ZnCl since complex (1)
exhibits very low activity against U937 and Molt-4 and moderate activity against THP-1
2
-1
cell lines (116 ± 1 µmol L ). Based on their IC50 values, the zinc complexes are only

2
4
weakly active, at best over an order of magnitude less potent than cisplatin.
Table 6. The 50% inhibitory concentration (IC50) of the zinc complexes described in this
study and their respective ligands, as well as DMSO and cisplatin, for leukemia cancer
cell lines (THP-1, U937 and Molt-4).
-1
Compound
IC50 (µmol L )
U937
THP-1
118.5 ± 10.5
103.5 ± 12.5
> 400
Molt-4
55 ± 3
> 400
HL1
141 ± 1
> 400
(
1)
H2L2
2)
H2L3
3)
H2L4
4)
Cisplatin
ZnCl
> 400
> 400
(
107 ± 19
> 400
> 400
> 400
> 400
> 400
(
108.5 ± 6.5
> 400
122 ±1
> 400
210 ± 8
> 400
(
98.5 ± 8.5
9.5 ± 1.5
302 ± 8
75 ± 1
235.5 ± 5.5
6 ± 1
7.5 ± 0.5
123± 13
2
137 ± 1
The cytotoxicity of the complexes was determined after 36 h of incubation, using the
MTT method [27]. * Data represent the mean of a triplicate experiment which was
repeated twice.
Based on the moderate activity of complex (4) in the U937 cell line, we decided
to investigate the mechanism of cell death promoted by this complex. For this purpose,
the Annexin V-FITC/PI staining assay was employed. In this assay, U937 cells stained
single-positive for Annexin V-FITC are considered mostly early apoptotic cells and cells
stained single-positive for PI are predominantly necrotic cells. Cells that are stained

2
5
double-positive are considered as late apoptotic or secondary necrotic cells [36]. The
Annexin V-FITC/PI staining data indicate that the percentage of apoptosis was 90.8%
-1
and 99.1%, respectively, after treatment for 24h with 200 and 400 µmol L (4),
respectively (see Figure 6c and 6d). For comparison, Figure 6a presents the results for a
control group and Figure 6b those collected in presence of cisplatin. For cisplatin, the
apoptosis and necrosis rates are 41 % and 16 %, respectively, while the normal cells
-1
reaches 44%, at a concentration of 50 µmol L after 24h of incubation. These data
strongly suggest that complex (4) could effectively induce apoptosis in U937 cells in a
concentration-dependent manner. It is worth noting that apoptosis is a desired pathway
for cell death to occur when developing anticancer agents since apoptosis or
programmed cell death is central to the development of homeostasis of metazoans [37].
3.5. Transmission Electron Microscopy
3.5. 1.Morphological changes of the leukemia cell line U937 after treatment with
complex (4)
In order to directly observe the morphological changes of the internal structure
of human leukemia cells (THP-1 and U937) after the treatment with complex (4),
transmission electron microscopy (TEM) was employed.
After incubation times, which varied from 6 h to 12 h, samples were collected to
measure cell density and morphological changes. The concentration of complex (4) was
-1
also varied (100 and 200 µmol L , respectively) to assess its effect on the cells.
Figure 7 shows the morphological changes promoted by complex (4), at a
-1
concentration of 100 µmol.L after 6 h of incubation. A clear association between
mitochondria (M) and endoplasmic reticulum (ER) was observed, indicating the
-1
initiation of apoptosis. At a concentration of 200 µmol. L and after 12 h of incubation

2
6
(
(
Figure 8), an intense vacuolization of the cells, condensation of the nuclear chromatin
NC) and the formation of vacuoles (V) were clearly observed. Figure 8 also
demonstrates the intense association between mitochondria (M) and the endoplasmic
reticulum (ER), as well as the formation of membrane blebs (B). Blebs are typical of
apoptotic cells, indicating that complex (4) is promoting cell death via an apoptotic
mechanism.
NC
.5.2. Morphological changes of the leukemia cell line THP-1 after treatment
3
with complex (4)
THP-1 cells were treated similarly (6-12 h incubations at complex
-1
concentrations of 100 and 200 µmol L ). An increased association between
mitochondria (M) and the endoplasmic reticulum (ER) was also observed, as well as a
large number of structurally altered mitochondria and large vacuoles with membranous
structures, shown in Figures 9 and 10, respectively. In Figure 9, ER-like elements in
close proximity to highly altered mitochondria (M) appear with an empty matrix (star),
displaying only cristae structures, or possibly extruding their content, resulting in the
formation of a vacuole (arrow). Different stages of cell death are observed, leading
ultimately to the appearance of abnormal mitochondria (M) and their subsequent
destruction, as well as the formation of vacuoles with membranous content (V). Figure
10 shows a multitude of long vacuoles (V) with membranous structures inside (star).
3.6. Antibacterial activity of the complexes
Since we have previously described that the zinc complex [Zn(HBPClNOL)Cl]
was very effective on different S. aureus strains, we were interested in evaluating if
simpler zinc complexes would show similar activity. Table presents the
7

2
7
antibacterial activities of the complexes (1)-(4), of the metallic salt, and of their
respective ligands and gentamicin (standard drug), which were evaluated in terms of
growth inhibition curves (i.e. percentage of inhibition) of S. aureus strains
(
ATCC10832, ATCC25923, COL and LSA88).
-2
-1
Complex (2), at a concentration of 10 mol L , showed the best inhibitory
activity reaching 100% of inhibition on each of the four strains under investigation. In
contrast, complex (4) was the least effective, showing very low inhibitory activity
against ATCC25923 (0%), COL (17%) and LSA88 (16%). However, this complex
showed high antibacterial activity against ATCC10832 (96%), demonstrating the effect
of the coordination of zinc to the ligand, since no activity was observed for the ligand
H2L4 alone (see Table 7). The values for the percentage of inhibition of complex (4) are
very similar to those of its respective ligand H2L4: ATCC25923 (0%), COL (12%) and
LSA88 (5%). Ligands H2L3, H2L3 and H2L4 showed low inhibitory activities against
this group of tested bacteria (ATCC10832 (5, 0 and 0%, respectively), ATCC25923 (2.4,
0
and 0 %, respectively), COL (11, 12 and 12%, respectively) and LSA88 (6.2, 4 and
%, respectively)), thus demonstrating that the antibacterial activity of zinc complexes
5
against ATCC10832 is the result of a potentiating effect between the ligands H2L2,
H2L3 and H2L4 and zinc(II) ion. For complex (1), high activity was observed only
against ATCC10832 (96%). Since complex (2) was the most active, experiments to
determine its minimum inhibitory concentration (MIC) were carried out against the
same strains of S. aureus, resulting in values of 512 µg/mL, 512 µg/mL, 512 µg/mL and
>
512 µg/mL to ATCC 10832, ATCC 25923, COL and LSA88, respectively (512 µg/mL
-1
≈
1.46 mmol L ).
The values obtained for ATCC10832 and ATCC25923 are 32 times higher than
those determined with gentamicin, a clinical drug. However, for COL, the MIC was

2
8
only twice the value determined for gentamicin. Previously reported MIC values for the
complex [Zn(HBPClNOL)Cl], the most active against nine strains of S. aureus, are
cited, which were all shown to be ~100 times higher than those obtained with
chloroamphenicol [12]. As presented in Table 7, complex (2) was the most active and its
antibacterial activity could be related to the phenol group and to the potentiating effect
2
between the ligand H2L2 and ZnCl . The same effects were observed for complex
[
Zn(HBPClNOL)Cl], reported previously [12]. This complex presents a phenol group
coordinated to the zinc(II) center and showed the best inhibitory activity, reaching
almost 100% of inhibition against nine strains of S. aureus under investigation.
Table 7. Antibacterial activities of the complexes (1)-(4), their respective ligands,
metallic salt and gentamicin against pathogenic bacteria (S. aureus) after 10 h of
incubation.
Compound
% inhibition promoted by the compounds
S. aureus
ATCC10832
ATCC25923
COL
15.3
67
LSA88
14.6
52
HL1
1)
H2L2
2)
H2L3
3)
H2L4
4)
ZnCl
Gentamicin
8
96
5
7
51
2.4
100
0
(
11
6.2
100
4
(
100
0
100
12
(
92
0
15
0
14
23
12
5
(
96
0
0
17
16
1
5.6
99
0
2
100
100
100

2
9
3.6.1. Morphological changes of S. aureus cells after treatment with complexes (1) and
(
2)
To directly observe the morphological changes of the internal structure of
bacterial cells after the treatment with complexes (1) and (2), TEM was employed.
Complexes (1) and (2) were chosen since complex (1) exerts strong inhibition and
complex (2) approximately half that of complex (1). As shown in Figure 11, when S.
aureus (LSA88) was treated with complex (1), the majority of the cells presented debris
with small structures resembling cell wall parts; some cells suggested the condensation
of DNA (indicated by a star), in agreement with the moderate inhibition promoted by
this complex on S.aureus cells (see Figure 11B). Figure 11C reveals the formation of
membrane-derived vesicles (MVs) (indicated by an arrow), which can be a kind of
defense mechanism against the drug, as suggested by Lee and co-workers [38].
According to their study, membrane-derived vesicles (MVs) are produced by Gram-
positive bacteria and the subsequent delivery to host cells could be related to the
virulence since many virulence-associated proteins were identified in the S. aureus
MVs. This was the first report describing the importance of MVs as vehicle for delivery
of bacterial effector molecules to host cells. Figure 11D reveals the presence of DNA
condensation (indicated by a star) and inner walls with aberrant division planes
(
indicated by arrows) which could be related to the inhibition of wall protein synthesis
which, in turn, is directly related to the cellular division process [39]. Figure 12A shows
control cells of S. aureus with a well defined cell wall and DNA during cell division
after 24 h of incubation with complex (2). As shown in Figure 12B, the treatment with
complex (2) promotes disruption of the cell, with the presence of holes and the
extrusion of the cytosolic material. Thus, it is possible to suggest that the high inhibitory
activity of complex (2) may be associated with the inhibition of the cell division process