Analgesic and anti-inflammatory activities of salicylaldehyde 2-chlorobenzoyl hydrazone (H2LASSBio-466), salicylaldehyde 4-chlorobenzoyl hydrazone (H2LASSBio-1064) and their zinc(II) complexes

Article abstract of DOI:10.3390/molecules16086902

Salicylaldehyde 2-chlorobenzoyl hydrazone (H₂LASSBio-466), salicylaldehyde 4-chlorobenzoyl hydrazone (H₂LASSBio-1064) and their complexes [Zn(LASSBio- 466)H₂O]₂ (1) and [Zn(HLASSBio-1064)Cl]₂ (2) were

Full text of DOI:10.3390/molecules16086902

Molecules 2011, 16, 6902-6915; doi:10.3390/molecules16086902
OPEN ACCESS
molecules
ISSN 1420-3049
www.mdpi.com/journal/molecules
Article
Analgesic and Anti-Inflammatory Activities of Salicylaldehyde
2-Chlorobenzoyl Hydrazone (H2LASSBio-466), Salicylaldehyde
4-Chlorobenzoyl Hydrazone (H2LASSBio-1064) and Their
Zinc(II) Complexes
Walfrido Bispo Júnior 1, Magna S. Alexandre-Moreira 1, Marina A. Alves 2, Anayive
Perez-Rebolledo 3, Gabrieli L. Parrilha 3, Eduardo E. Castellano 4, Oscar E. Piro 5, Eliezer J.
Barreiro 2, Lídia Moreira Lima 2 and Heloisa Beraldo 3,*
1
LaFI Laboratório de Farmacologia e Imunidade, Instituto de Ciências Biológicas e da Saúde,
Universidade Federal de Alagoas, Maceió, AL, Brazil
2
LASSBio Laboratório de Avaliação e Síntese de Substâncias Bioativas (LASSBio,
http://www.farmacia.ufrj.br/lassbio/), Faculdade de Farmácia, Universidade Federal do Rio de
Janeiro, P. O. Box 68024, 21944-971, Rio de Janeiro, RJ, Brazil
3
Departamento de Química, Universidade Federal de Minas Gerais, 31270-901, Belo Horizonte,
Brazil
4
Instituto de Física de São Carlos, Universidade de São Paulo, 13560-970, São Carlos, SP, Brazil
5
Departamento de Física, Facultad de Ciencias Exactas, Universidad Nacional de La Plata and
Instituto IFLP (CONICET – CCT La Plata), C.C. 67, 1900 La Plata, Argentina
* Author to whom correspondence should be addressed; E-Mail: hberaldo@ufmg.br;
Tel.: 55-31-3409-5740; Fax: 55-31-3409-5700.
Received: 27 June 2011; in revised form: 2 August 2011 / Accepted: 11 August 2011 /
Published: 15 August 2011
Abstract: Salicylaldehyde 2-chlorobenzoyl hydrazone (H2LASSBio-466), salicylaldehyde
4-chlorobenzoyl hydrazone (H2LASSBio-1064) and their complexes [Zn(LASSBio-
466)H2O]2 (1) and [Zn(HLASSBio-1064)Cl]2 (2) were evaluated in animal models of
peripheral and central nociception, and acute inflammation. All studied compounds
significantly inhibited acetic acid-induced writhing response. Upon coordination the anti-
nociceptive activity was favored in the complex 1. H2LASSBio-466 inhibited only the first
phase of the formalin test, while 1 was active in the second phase, like indomethacin,
indicating its ability to inhibit nociception associated with the inflammatory response.
Hence coordination to zinc(II) altered the pharmacological profile of H2LASSBio-466.

6903
Molecules 2011, 16
H2LASSBio-1064 inhibited both phases but this effect was not improved by coordination.
The studied compounds did not increase the latency of response in the hot plate model,
indicating their lack of central anti-nociceptive activity. All compounds showed levels of
inhibition of zymosan-induced peritonitis comparable or superior to indomethacin,
indicating an expressive anti-inflammatory profile.
Keywords: acylhydrazones; zinc(II) complexes; analgesic activity; anti-inflammatory
activity
1. Introduction
Inflammation is a response of the immune system to physical and/or chemical and/or biological
injury, understanding by injury any process able to cause tissue or cellular damages. The types of
inflammation (i.e. acute or chronic) differ by cause, mechanism, outcome, and intensity. It is well
known that while acute inflammation has a physiological role in normal circumstances, chronic
inflammation exerts detrimental effects on the functional status of cells and tissues. As a consequence
inflammatory processes take part in a huge number of diseases such as atherosclerosis, Alzheimer
disease, Parkinson disease, cancer, asthma, arthritis and so on [1-3].
Zinc is one of the most prevalent trace elements in the human body. It has been shown to be
essential to the structure and function of a large number of macromolecules and for a variety of
enzymatic reactions, which mediate a wide range of physiological processes [4,5]. These include
production of collagen and other extracellular matrix proteins, modulation of immunoregulatory
(e.g., T and B lymphocytes, macrophages, and antigen-presenting dendritic cells) and inflammatory
(e.g., eosinophils, neutrophils, and mast cells) cells function. As a consequence, zinc may be considered
an important immunoregulatory agent with anti-apoptotic and anti-inflammatory activities [6].
Figure 1. Generic structure of salicylaldehyde 2-chlorobenzoyl hydrazone (H2LASSBio-
466) and its regioisomer salicylaldehyde 4-chlorobenzoyl hydrazone (H2LASSBio-1064).
In the context of a research program that aims to contribute to the discovery of new anti-
inflammatory and analgesic drug candidates, we describe the synthesis of zinc(II) complexes with
salicylaldehyde 2-chlorobenzoyl hydrazone (H2LASSBio-466) and its regioisomer salicylaldehyde 4-

6904
Molecules 2011, 16
chlorobenzoyl hydrazone (H2LASSBio-1064) (Figure 1), together with a pharmacological evaluation
of all acylhydrazones and zinc(II) complexes in animal models of peripheral and central nociception
and acute inflammation.
2. Results and Discussion
2.1. Formation of the Zinc(II) Complexes 1 and 2
Microanalyses and molar conductivity data are compatible with the formation of [Zn(LASSBio-
466)H2O] and [Zn(HLASSBio-1064)Cl]. In the first compound a di-anionic acylhydrazone is attached
to the metal center upon deprotonation at N(2)-H and O(1)-H. The remaining coordination site is
occupied by a water molecule, as indicated by the infrared spectrum of the compound (Section 2.3). In
the second, a mono-anionic acylhydrazone is attached to the metal upon deprotonation at O(1)-H,
together with a chloride ion.
Upon recrystallization of [Zn(HLASSBio-1064)Cl] in 1:9 DMSO-acetone, crystals of
[Zn2(LASSBio-1064)2(H2O)2]·[Zn2(LASSBio-1064)2(DMSO)4] were obtained. The compound
consists of two center symmetric binuclear zinc(II) complexes hosted in the same lattice, as previously
reported by some of us [7]. We had also obtained [Zn(LASSBio-1064)]2, which is a phenoxo-bridged
dimer since the monomer would contain zinc(II) with coordination number three, which is very
unlikely [7]. Hence [Zn(HLASSBio-1064)Cl] most probably also exists as a dimer, [Zn(HLASSBio-
1064)Cl]2.
We now obtained [Zn(LASSBio-466)(H2O)], in which a water molecule occupies the fourth
coordination position, like in the first center symmetric unit of [Zn2(LASSBio-
1064)2(H2O)2]·[Zn2(LASSBio-1064)2(DMSO)4]. Therefore, [Zn(LASSBio-466)(H2O)] is also
probably a phenoxo-bridged dimer, [Zn(LASSBio-466)(H2O)]2. Considering that dimerization is
favored in this class of compounds [7], the zinc(II) complexes are hereafter formulated as
[Zn(LASSBio-466)(H2O)]2 (1) and [Zn(HLASSBio-1064)Cl]2 (2).
Figure 2. Molecular plot of LASSBio-466 showing the labeling scheme of the non-H
atoms and their displacement ellipsoids at the 50% probability level.

6905
Molecules 2011, 16
2.2. Crystal Structure of H2LASSBio-466
Crystal data and refinement results are summarized in Table 1. Figure 2 is an ORTEP [8] drawing
of the molecule and Table 2 shows the corresponding intra-molecular bond distances and angles.
Table 1. Crystal data and structure solution methods and refinement results for
salicylaldehyde 2-chlorobenzoyl hydrazone (H2LASSBio-466).
Compound
H2LASSBio-466
C14H11ClN2O2
274.70
Empirical Formula
Formula Weight
Temperature, K
Crystal System
Space Group
294(2)
Monoclinic
P21/c
a, Å
b, Å
c, Å
α, °
β, °
γ, °
9.889(1)
13.360(2)
10.079(1)
90
Unit cell dimensions
93.10(1)
90
1329.7(3)
4, 1.372
0.286
568
Volume, Å3
Z, Density calc., Mg/m3
Absorption coefficient, mm−1
F(000)
Crystal size, mm
Crystal color / shape
θ range for data coll.
0.20 × 0.12 × 0.08
Colorless / prism
3.05 to 24.08º
−10 ≤ h ≤11
−15 ≤ k ≤ 15
−11 ≤ l ≤ 11
99.6 %
Index range
Completeness, θ = 26.37°
Max. / min. transmission
Goodness–of–fit on F2
0.977 / 0.945
1.016
Reflec. collect./unique (Rint)
Observed reflections [I > 2σ(I)]
5646/2095 (0.032)
1612
2
[σ2(Fo ) + (0.088P)2 + 0.22P]-1
Weights, w
2
P = [Max((Fo ,0) + 2Fc2]/3
Data / restraints / parameters
Final R indexesa [I > 2σ(I)]
R indices (all data)
2095 / 0 / 184
R1 = 0.0474, wR2 = 0.1296
R1 = 0.0637, wR2 = 0.1479
0.07(1)
Extinction coefficient
Larg. peak & hole, e Å−3
0.180 / -0.246
a R indices defined as: R1=Σ||Fo|-|Fc||/Σ|Fo|, wR2=[Σw(Fo -Fc2)2/Σw(Fo ) ] .
2
2 2 1/2
The phenyl rings show a delocalized bonding structure, with C-C distances ranging from 1.370(3)
to 1.388(3) Å for the chlorine-containing phenyl, where d(Cph-Cl) = 1.724(3) Å, and from 1.359(4) to
1.400(3) Å for the phenol ring, where d(Cph-OH) = 1.353(3) Å. We found for the carbonyl C(8)=O(2)
and C(7)=N(1) groups distances of 1.226(2) and 1.272(3) Å, respectively, as expected for formal
double bonds. The N(1)-N(2) length of 1.376(3) Å agrees with the single bond character of this link.
The molecule conformation is stabilized by a strong intra-molecular O-H…N bond [d(O(1)···N(1)) =

6906
Molecules 2011, 16
2.632 Å, ∠(O(1)-H(1)···N(1)) = 145.7°]. As shown in Figure 2 the hydrazone adopts the E
configuration in the crystal state.
Table 2. Bond lengths [Å] and angles [°] for salicylaldehyde 2-chlorobenzoyl hydrazone
(H2LASSBio-466).
Atoms
Cl-C(10)
Bond lengths (Å)
1.724(3)
1.353(3)
1.226(3)
1.376(3)
1.272(3)
1.346(3)
1.400(3)
1.384(4)
1.390(3)
1.445(3)
1.374(4)
1.371(5)
1.359(4)
1.494(3)
1.393(3)
1.388(3)
1.381(4)
1.376(4)
1.371(4)
1.370(3)
Atoms
Angles (°)
123.2(2)
121.1(2)
114.7(2)
118.6(2)
117.2(2)
118.3(2)
122.2(2)
120.5(3)
119.7(2)
121.1(3)
119.5(3)
119.5(2)
120.8(3)
122.4(2)
119.9(2)
120.8(2)
121.1(3)
121.5(3)
117.6(2)
119.5(3)
118.1(2)
120.3(3)
119.9(3)
122.4(2)
118.0(2)
122.1(2)
O(2)-C(8)-C(9)
N(1)-C(7)-C(2)
N(2)-C(8)-C(9)
N(1)-N(2)-C(8)
N(2)-N(1)-C(7)
C(1)-C(2)-C(3)
C(1)-C(2)-C(7)
C(1)-C(6)-C(5)
C(2)-C(1)-C(6)
C(2)-C(3)-C(4)
C(3)-C(4)-C(5)
C(3)-C(2)-C(7)
C(4)-C(5)-C(6)
C(8)-C(9)-C(10)
C(8)-C(9)-C(14)
C(9)-C(10)-Cl
C(9)-C(10)-C(11)
C(9)-C(14)-C(13)
C(10)-C(9)-C(14)
C(10)-C(11)-C(12)
C(11)-C(10)-Cl
C(11)-C(12)-C(13)
C(12)-C(13)-C(14)
O(1)-C(1)-C(2)
O(1)-C(1)-C(6)
O(2)-C(8)-N(2)
O(1)-C(1)
O(2)-C(8)
N(1)-N(2)
N(1)-C(7)
N(2)-C(8)
C(1)-C(2)
C(1)-C(6)
C(2)-C(3)
C(2)-C(7)
C(3)-C(4)
C(4)-C(5)
C(5)-C(6)
C(8)-C(9)
C(9)-C(10)
C(9)-C(14)
C(10)-C(11)
C(11)-C(12)
C(12)-C(13)
C(13)-C(14)
The solid is further stabilized by a medium-strength inter-molecular N-H···Ocarbonyl bond
[d(N(2)···O(2)) = 2.885 Å, ∠(N(2)-H(2)···O(2’)) = 166.2°] between neighboring molecules, symmetry-
related to each other through a glide plane, involving the imine group of a molecule as a donor and the
carbonyl oxygen of the other as an acceptor. This gives rise to a polymeric structure that extends along
the crystal c-axis (see Figure 2).
2.3. Spectroscopic Characterization
The vibrations at 3450–3436 cm−1 in the infrared spectra of the free acylhydrazones attributed to
ν(O-H) disappear in those of the complexes, in accordance with deprotonation of the phenol group [9].
The ν(C=O) absorption at 1657 cm−1 in the spectrum of the H2LASSBio-466 was not found in the
spectrum of the complex 1, indicating coordination of an enolate oxygen [10,11]. The ν(C=O)
absorption at 1676 cm−1 in the spectrum of the H2LASSBio-1064 shifts to 1618 cm−1 in the spectrum
of 2, indicating coordination through the carbonyl oxygen [10-13]. The vibrations attributed to ν(C=N)
at 1625 and 1624 cm−1 in the infrared spectra of the hydrazones shift to 1614 and 1611 cm−1,
respectively, in the spectra of the complexes, in agreement with coordination of the azomethine

6907
Molecules 2011, 16
nitrogen [10-13]. A new absorption at 1604 cm−1 in the spectrum of complex 1 was attributed to the
ν(OH2) vibration.
The NMR spectra of the acylhydrazones and their zinc(II) complexes were recorded in DMSO-d6.
1
The H resonances were assigned on the basis of chemical shifts and multiplicities. The carbon type
(C, CH) was determined by using distortionless enhancement by polarization transfer (DEPT 135)
experiments. The assignments of the protonated carbons were made by 2D heteronuclear multiple
quantum coherence experiments (HMQC).
1
The signals of all hydrogens and carbons are duplicated in the H- and 13C-NMR spectra of
H2LASSBio-466, indicating the presence of the E and Z configurational isomers in the DMSO-d6
solution. In fact, two N(2)-H signals were observed at δ 11.05 and 9.84 ppm, which were attributed to
the Z and E isomers, respectively. In the first N(2)-H is hydrogen bonded to the phenol oxygen, while
in the latter it is hydrogen bonded to the solvent [10-13]. Only one signal was found for each hydrogen
1
13
and each carbon in the H- and C-NMR spectra of H2LASSBio-1064. These signals are compatible
with the presence of the E configurational isomer.
Only one signal was observed for each hydrogen and each carbon in the spectra of complexes 1-2.
The O(1)-H signals were absent in the spectra of all complexes, in agreement with deprotonation and
formation of a phenolate group. In the spectrum of 1 the N(2)-H signal disappears, suggesting
deprotonation of the hydrazone upon coordination. Hence in 1 a di-anionic hydrazone ligand is
attached to the zinc(II) center. In the spectrum of 2 the N(2)-H signal was observed, according with the
presence of a mono-anionic hydrazone ligand. In the 1H-NMR spectra of the complexes the signals of
all hydrogens undergo significant shifts in relation to their positions in the free hydrazones. Similarly,
the signals of C=N, C=O and the phenol carbons undergo significant shifts in complexes 1 and 2,
indicating coordination through the Ophenol-N-O chelating system. Hence in 1-2 the hydrazones adopt
the E configuration.
2.4. Anti-Nociceptive Activity of H2LASSBio-466, H2LASSBio-1064, [Zn(LASSBio-466)H2O]2 (1) and
[Zn(HLASSBio-1064)Cl]2 (2)
The anti-nociceptive profiles of the free ligands H2LASSBio-466, H2LASSBio-1064 and their
zinc(II) complexes 1 and 2 were evaluated using three well-accepted pain models, namely acetic acid-
induced writhing, formalin-induced nociception and hot plate test. The acetic acid-induced abdominal
writhing and hot-plate test have been reported to be useful to investigate peripheral and central
activity, respectively, while the formalin-induced nociception is valuable to detect both effects,
including inflammatory pain.
All compounds were evaluated at a dose of 100 μmol/kg (p.o). Indomethacin, a COX-1 selective
inhibitor (100 μmol·kg−1, p.o.) and dipyrone, a COX-3 selective inhibitor (100 μmol·kg−1, p.o.) were
used as standard drugs in the peripheral nociception models, while morphine (15 μmol·kg−1, i.p.) was
used as standard in the hot-plate test. The analgesic activity of H2LASSBio-466, its regioisomer
H2LASSBio-1064, and their complexes [Zn(LASSBio-466)H2O]2 (1) and [Zn(HLASSBio-1064)Cl]2
(2) was initially evaluated employing the acetic acid-induced abdominal writhing model in mice and
compared with those of the standards [14].

6908
Molecules 2011, 16
As shown in Table 3, all compounds produced marked inhibition of acetic acid-induced writhing
response. However, the anti-nociceptive activity appears to have been favored by complex formation
in the case of complex 1, given its increased activity compared to the H2LASSBio-466 free ligand.
Table 3. Effect of H2LASSBio-466, its regioisomer H2LASSBio-1064, their zinc(II)
complexes, indomethacin and dipyrone (100 µmol·kg−1, p.o.) on the 0.6% acetic acid-
induced abdominal constrictions in mice, for a period of 25 min.
Writhing number
Mean ± S.E.M.
37.5 ± 1.4
% of inhibition
Mean ± S.E.M.
___
84.0 ± 8.3 **
77.8 ± 7.2 **
60.4 ± 6.0 **
81.3 ± 3.0 **
82.3 ± 3.7 **
70.9 ± 6.2 **
Substance
n
Control
Indomethacin
Dipyrone
H2LASSBio-466
H2LASSBio-1064
[Zn(LASSBio-466)H2O]2 (1)
[Zn(HLASSBio-1064)Cl]2 (2)
6
6
6
6
6
6
6
6.0 ± 3.1 **
8.3 2.7 **
14.8 ± 2.2 **
7.0 ± 1.1 **
6.6 ± 1.4 **
10.8 ± 2.3 **
Data are expressed as mean ± S.E.M. Statistical differences between the treated and the control
groups were evaluated by ANOVA and Dunnett tests and the asterisks denote the levels of
significance in comparison with control groups, ** P < 0.01.
The neurogenic and inflammatory pain was evaluated using the formalin test and analyzing the first
and the second phases of the nociceptive response, respectively [15]. In this model, H2LASSBio-466
was effective in inhibiting only the first phase, while [Zn(LASSBio-466)H2O]2 (1), like indomethacin,
was active in the second phase, indicating its ability to inhibit nociception associated with
inflammatory response (Table 4). H2LASSBio-1064 was able to inhibit both neurogenic and
inflammatory phases anticipating a distinct pharmacological profile. However this effect decreases on
coordination to zinc(II) in complex 2. These results suggest that coordination seems to be a good
strategy to improve the antinociception profile of the prototype H2LASSBio-466 associated with an
inflammatory pain.
Table 4. Effect of prototypes H2LASSBio-466, H2LASSBio-1064, their zinc(II) complexes
and indomethacin (100 µmol·kg−1, p.o.) on formalin (2.5%) test in mice.
% of inhibition (Mean ± S.E.M.)
Phase 1
Phase 2
Substance
n
Mean ± S.E.M. Mean ± S.E.M.
Phase 1
—
Phase 2
—
Control
Indomethacin
H2LASSBio-466
H2LASSBio-1064
[Zn(LASSBio-466)H2O]2 (1)
[Zn(HLASSBio-1064)Cl]2 (2)
5
5
5
5
5
5
54.8 ± 2,3
57.1 ± 8.5
29.3 ± 8.3 **
25.7 ± 4.9 *
40.6 ± 12.7
39.6 ± 7.8
227.6 ± 22.7
115.9 ± 3.3 *
182.4 ± 17.4
117.0 ± 19.1 *
142.8 ± 23.1 *
161.3 ± 36.0
0
49.1 ± 1.4 *
19.8 ± 9.7
48.5 ± 8.4 *
37.3 ± 10.1 *
29.2 ± 13.9
46.5 ± 8.3 **
53.1 ± 8.9 *
25.9 ± 15.7
27.7 ± 13.1
Data are expressed as mean ± S.E.M. Statistical differences between the treated and the control
groups were evaluated by test t and Mann-Whitney tests and the asterisks denote the levels of
significance in comparison with control groups, * P < 0.05 and ** P < 0.01.
In order to investigate an occasional central anti-nociceptive activity for prototypes H2LASSBio-
466, H2LASSBio-1064 and their zinc(II) complexes, the studied compounds were evaluated in the hot

6909
Molecules 2011, 16
plate test using morphine (15 µmol·kg−1, i.p.) as standard [16]. As shown in Table 5, these compounds
did not increase the latency of response significantly, showing that they do not present activity in the
supra-spinal analgesia, while morphine induced a marked increase in the latency of the animals at
60 min (9.0 ± 1.6 s), 90 min (7.4 ± 0.8 s), 120 min (5.3 ± 0.8 s) and 150 min (2.5 ± 0.2 s). These
results indicate that the studied compounds do not have any central anti-nociceptive activity.
Table 5. Time course effect of prototypes H2LASSBio-466, H2LASSBio-1064, their
zinc(II) complexes (100 µmol·kg−1, p.o.) and morphine (15 µmol·kg−1, i.p.) on hot-plate
test in mice.
Mean ± S.E.M.
Substance
n
0 min
60 min
90 min
120 min
150 min
Control
Morphine
H2LASSBio-466
H2LASSBio-1064
[Zn(LASSBio-466)H2O]2 (1)
[Zn(HLASSBio-1064)Cl]2 (2)
6
6
6
6
6
6
2.8 ± 0.2
1.8 ± 0.5
2.1 ± 0.21
1.7 ± 0.18
2.1 ± 0.2
1.5 ± 0.1
2.9 ± 0.3
9.0 ± 1.6 *
2.3 ± 0.4
2.6 ± 0.2
4.2 ± 0.8
2.0 ± 0.5
1.6 ± 0.2
7.4 ± 0.8 *
2.1 ± 0.2
2.1 ± 0.2
2.4 ± 0.6
2.3 ± 0.5
2.3 ± 0.40
5.3 ± 0.8 *
2.2 ± 0.2
2.5 ± 0.3
3.6 ± 0.6
2.9 ± 1.0
1.7 ± 0.1
2.5 ± 0.2
2.6 ± 0.1
3.5 ± 0.3
5.4 ± 1.2
1.9 ± 0.2
Data are expressed as mean ± S.E.M. Statistical differences between the treated and the control
groups were evaluated by ANOVA and Dunnett tests and the asterisks denote the levels of
significance in comparison with control groups, * P < 0.05.
To better assess the potential anti-inflammatory activity of the free acylhydrazones and their
zinc(II) complexes, the zymosan-induced peritonitis assay was performed [17]. As seen in Table 6, all
compounds showed some level of inhibition in this cell migration model comparable or superior to
indomethacin.
Table 6. Effect of prototypes H2LASSBio-466, H2LASSBio-1064, their zinc(II) complexes
and indomethacin (100 µmol·kg−1, p.o.) on the zymosan-induced peritonitis in mice.
Cell Number × 106/mL
Mean ± S.E.M.
38.0 ± 1.0
% of inhibition
Mean ± S.E.M.
___
Substance
n
Control
Saline
Indomethacin
H2LASSBio-466
H2LASSBio-1064
[Zn(LASSBio-466)H2O]2 (1)
[Zn(HLASSBio-1064)Cl]2 (2)
7
7
7
7
7
7
7
5.0 ± 0.8
___
17.7± 1.0 **
11.4 ± 1.4 **
8.4 ± 0.9 **
10.7 ± 1.8 **
13.4 ± 1.5 **
53.4 ± 2.7 **
70.0 ± 3.8 **
77.8 ± 2.4 **
71.8 ± 4.9 **
64.7 ± 4.0 **
Data are expressed as mean ± S.E.M. Statistical differences between the treated and the control
groups were evaluated by ANOVA and Dunnett tests and the asterisks denote the levels of
significance in comparison with control groups, ** P < 0.01.
In fact, H2LASSBio-466, H2LASSBio-1064, [Zn(LASSBio-466)H2O]2 and [Zn(HLASSBio-
1064)Cl]2, presented 70.0%, 77.8%, 71.8% and 64.7% of inhibition, respectively, while indomethacin
inhibited cell-migration by 53.4%.

6910
Molecules 2011, 16
3. Experimental
3.1. General
All common chemicals were purchased from Aldrich and used without further purification. Partial
elemental analyses were performed on a Perkin Elmer CHN 2400 analyzer. A YSI model 31
conductivity bridge was employed for molar conductivity measurements (1 × 10−3 mol L−1, DMF).
Infrared spectra (4000-400 cm-1) were recorded on a Perkin Elmer FT-IR Spectrum GX spectrometer
using KBr plates. NMR spectra were obtained with a Bruker DPX-200 Avance (200 MHz)
spectrometer using DMSO-d6 as the solvent and TMS as internal reference.
3.2. Synthesis of H2LASSBio-466 and H2LASSBio-1064
The synthesis of H2LASSBio-466 and H2LASSBio-1064 were performed using a previously
described methodology [18]. Briefly, salicylaldehyde (2-hydroxybenzaldehyde) was added to a
solution of 2-chlorobenzohydrazide or 4-chlorobenzohydrazide (an equimolar amount) in absolute
ethanol containing one drop of 37% hydrochloric acid. The mixture was stirred at room temperature
for 2 hours until extensive precipitation was observed. Next, the solvent was partially concentrated at
reduced pressure and the resulting mixture was poured into cold water. The precipitate formed was
filtered out and dried under vacuum producing the desired (E,Z)-salicylaldehyde 2-chlorobenzoyl
hydrazone (H2LASSBio-466) and (E)-salicylaldehyde 4-chlorobenzoyl hydrazone (H2LASSBio-1064)
in 87% and 91% yield, respectively. The melting point and 1H-NMR data for both compounds were in
agreement with previous reports [19,20].
3.3. Synthesis of Zinc(II) Complexes
3.3.1. Synthesis of [Zn(LASSBio-466)H2O]2 (1) and [Zn(HLASSBio-1064)Cl]2 (2)
Complexes 1 and 2 were obtained by mixing an ethanol solution of the desired hydrazone with zinc
chloride and triethylamine in 1:1:1 ligand-to-metal-to-triethylamine molar ratio. The resulting solids
were washed with ethanol followed by diethylether and then dried in vacuo.
[Zn(LASSBio-466)H2O]2 (1). Yield 68%.Yellow solid. Anal. Calc. for C28H22N4O6Cl2Zn2 (712.18): C,
47.26%; H, 2.85%; N, 7.40%. Found: C, 47.22%; H, 3.11%; N, 7.87%. M.P. > 300 °C. Molar
1
conductivity: 8.23 Ω−1cm2 mol−1. IR: ν(C=N) 1614, ν(Zn-OH2) 1604, ν(phenolic, CO) 1329. H-
NMR: δ (ppm) = 7.54 (1H, H7), 7.47–7.59 (1H, H14), 7.52 (1H, H11), 7.51 (1H, H5), 7.44–7.47 (1H,
H3), 7.32 (1H, H13), 7.28 (1H, H13), 6.79 (1H, H6), 6.70 (1H, H4). 13C-NMR: δ (ppm) = 169.73 (C8),
158.02 (C1), 150.59(C7), 135.87 (C10), 135.09 (C12), 134.37 (C13), 131.38 (C5), 130.35 (C11),
129.81 (C14), 127.48 (C3), 119.69 (C9, C2), 119.35 (C4), 118.37 (C6).
[Zn(HLASSBio-1064)Cl]2 (2). Yield 71%. Yellow solid. Anal. Calc. for C28H20N4O4Cl4Zn2 (749.11):
C, 44.72%; H, 2.24%; N, 7.10%. Found: C, 44.89%; H,2.69%; N, 7.48%. M.P. > 300 °C. Molar
conductivity: 11.35 Ω−1cm2 mol−1. IR: ν(N-H) 3179, ν(C=O) 1618, ν(C=N) 1611, ν(phenolic, CO)
1
1281. H-NMR: δ (ppm) = 8.99 (1H, H7), 8.01 (2H, H11,H13), 7.59 (2H, H10,H14), 7.38 (1H, H3),

6911
Molecules 2011, 16
7.24 (1H, H5), 6.73 (1H, H6), 6.89 (1H, H4). 13C-NMR: δ (ppm) = 168.88 (C8), 166.81 (C1), 155.61
(C7), 136.83 (C12), 131.62 (C5), 129.68 (C10,C14), 129.58 (C3), 128.79 (C11, C13), 119.53 (C4),
118.75 (C9, C2), 116.54 (C6).
3.4. X-ray Crystallography
The X-ray diffraction measurements were performed on an Enraf-Nonius Kappa-CCD
diffractometer with graphite-monochromated MoKα (λ = 0.71073 Å) radiation. Diffraction data were
collected (φ and ω scans with κ-offsets) with COLLECT [21]. Integration and scaling of the reflections
were performed with HKL DENZO-SCALEPACK [22] suite of programs. The unit cell parameters
were obtained by least-squares refinement based on the angular settings for all collected reflections
using HKL SCALEPACK. The structure was solved by direct methods with SHELXS-97 [23] and the
molecular model refined by full-matrix least-squares procedure on F2 with SHELXL-97 [24]. The
hydrogen atoms were included in the molecular model at stereo-chemical positions and refined with
the riding model.
3.5. Anti-nociceptive and anti-inflammatory activities of H2LASSBio-466, H2LASSBio-1064,
[Zn(LassBio-466)H2O]2 (1) and [Zn(HLassBio-1064)Cl]2 (2)
3.5.1. Animals
Experiments were conducted using Swiss mice obtained from the BIOCEN - UFAL breeding unit,
weighing 20–30 g each, males or females, adult, with 6 to 8 weeks of age, distributed in groups of up
to 6–8 animals for treatment. The animals were maintained with free access to food and water and kept
at 25–28 °C under a controlled 12 h light·dark−1 cycle. All animals were manipulated according to the
norms established by the Ethics Commission - UFAL for handling animals (protocol number:
026681/2009-23).
3.5.2. Writhing Test
This test was performed as described by Collier and coworkers [14]. Acetic acid (0.6%, v/v) was
administered i.p. in a volume of 0.1 mL·10 g−1. The number of writhes, a response consisting of
contraction of an abdominal wall, pelvic rotation followed by hind limb extension, was counted during
continuous observation for 20 min. beginning 5 min. after the acetic acid injection. The prototypes
H2LASSBio-466, H2LASSBio-1064 and their zinc(II) complexes, indomethacin and dipyrone were
administered at the dose of 100 µmol·kg−1 (p.o), 40 min. before the acetic acid injection. Control group
received 10 mL·kg−1 of vehicle (arabic gum, p.o.). Anti-nociceptive activity was expressed as
inhibition percentage of the usual number of writhing observed in control animals.
3.5.3. Formalin-Induced Pain in Mice
The formalin test was performed as described by Hunskaar and Hole [15]. Animals received 20 µL
of a 2.5% formalin solution (0.92% formaldehyde in saline) in the ventral surface of the right hind
paw. They were observed from 0 to 5 min. (neurogenic phase) and from 15 to 30 min. (inflammatory
phase) after injection and the time they spent licking the injected paw was recorded and considered as

6912
Molecules 2011, 16
indicative of nociception. The prototypes H2LASSBio-466, H2LASSBio-1064, their zinc(II)
complexes and indomethacin were administered at the dose of 100 µmol·kg−1 (p.o), 40 min. before
formalin injection. Control group received 10 mL·kg−1 of vehicle (arabic gum, p.o.).
3.5.4. Hot-Plate Test
Mice were treated according to the method described by Eddy and Leimbach [16]. Each mouse was
placed on the hot plate set at 54 ± 1.0 °C and the time of paw licking was recorded before and 30 min.
after oral administration of the tested compounds. The prototypes H2LASSBio-466, H2LASSBio-1064
and their zinc(II) complexes were administered at the dose of 100 µmol·kg−1 (p.o). Control group
received 10 mL·kg−1 of vehicle (arabic gum, p.o.). Morphine was also used as a drug standard at the
dose of 15 μmol·kg−1 (i.p.). Analgesia was defined as an increase in the latency of paw licking, and the
latency times were compared with the values obtained for control. Sixty seconds were taken as the cut-
off time to avoid mouse tissue damage.
3.5.5. Zymosan-Induced Peritonitis
Peritoneal inflammation was induced according to the method described by Doherty [17]. A
solution of zymosan A (Sigma-Aldrich, 2 mg·mL−1) was prepared in saline (0.9% NaCl) and injected
into the peritoneal cavity of mice (0.5 mL). Six hours after injection of zymosan A, the animals were
sacrificed by cervical dislocation and the peritoneal cavity was washed with cold Hank’s solution
(3 mL). The prototypes H2LASSBio-466, H2LASSBio-1064, their zinc(II) complexes and
indomethacin were administered at the dose of 100 µmol·kg−1 (p.o), 40 min. before zymosan A
injection. The control group received 10 mL·kg−1 of vehicle (arabic gum, p.o.). The number of cells
was quantified using an optical microscope, and a 100 x lens.
3.5.6. Statistical Analysis
Data obtained from animal experiments are represented by mean ± standard error of the mean
(Mean ± S.E.M.). Statistical differences between the treated and the control groups were evaluated by
test t of Student or ANOVA in the tutorial Prisma®. Values were considered significant if * P < 0.05,
** P < 0.01 and *** P < 0.001.
4. Conclusions
The studied compounds, evaluated at a dose of 100 μmol/kg (p.o), showed marked inhibition of
acetic acid-induced writhing response, with the anti-nociceptive activity being favored by zinc(II)
complex formation in the case of complex 1. The compounds also have antinociception profile
associated with inflammatory pain, with no activity in murine analgesic model of central pain. In the
formalin model, H2LASSBio-466 was effective in inhibiting only the first phase, while its zinc(II)
complex 1, like indomethacin, was active in the second phase, indicating its ability to inhibit
nociception associated with inflammatory response. Hence coordination to zinc(II) altered the
pharmacological profile of H2LASSBio-466. Moreover, the salicyladehyde N-acylhydrazone

6913
Molecules 2011, 16
derivatives and their zinc(II) complexes showed comparable or superior inhibition of cell-migration
process to indomethacin, indicating an expressive anti-inflammatory profile.
The different activities of the complexes could be explained by their releasing hydrazone at
different rates during the assays. In addition, complex 1 contains a di-anionic hydrazone while 2
contains a mono-anionic hydrazone. Hence different electronic effects could also influence the distinct
pharmacological profiles of complexes 1 and 2. In addition, 1 contains a water molecule in the metal
coordination sphere while 2 contains a chloride ion. Finally, coordination could in principle alter the
bioavailability of the two hydrazones in different manners. The above-mentioned results suggest that
further studies on the anti-inflammatory properties of this class of compounds and their zinc(II)
complexes should be carried out in order to investigate their mechanism of action
Acknowledgements
The authors are grateful to CNPq, INCT-INOFAR (CNPq proc.573.364/2008-6), FAPESP (Brazil)
and to CONICET (Argentina) for financial support. O. E. P. is a Research Fellow of CONICET,
Argentina.
Supplementary Material
CCDC 805145 contains the supplementary crystallographic data for salicylaldehyde 2-
chlorobenzoyl hydrazone (H2Lassbio-466). This data can be obtained free of charge via
http://www.ccdc.cam.ac.uk/conts/retrieving.html, or from the Cambridge Crystallographic Data
Centre, 12 Union Road, Cambridge CB2 1EZ, UK; fax: (+44) 1223-336-033; or e-mail:
deposit@ccdc.cam.ac.uk.
References
1. Yong, V.W. Inflammation in neurological disorders: A help or a hindrance? Neuroscientist 2010,
16, 408-420.
2. Kominsky, D.J.; Campbell, E.L.; Colgan, S.P. Metabolic shifts in immunity and inflammation. J.
Immunol. 2010, 184, 4062-4068.
3. Grivennikov, S.I.; Greten, F.R.; Karin, M. Immunity, inflammation, and cancer. Cell 2010, 140,
883-899.
4. Tapiero, H.; Tew, K.D. Trace elements in human physiology and pathology: Zinc and
metallothioneins. Biomed. Pharmacother. 2003, 57, 399-411.
5. Szewczyk, B.; Kubera, M.; Nowak, G. The role of zinc in neurodegenerative inflammatory
pathways in depression. Prog. Neuro-Psycopharmacol. Biol. Psychiatry 2011, 35, 693-701.
6. Zalewski, P.D.; Truong-Tran, A.Q.; Grosser, D.; Jayaram, L.; Murgia, C.; Ruffin, R.E. Zinc
metabolism in airway epithelium and airway inflammtion: basic mechanism and clinical targets.
A review. Pharmacol. Ther. 2005, 105, 127-149.
7. Parrilha, G.L.; Vieira, R.P.; Rebolledo, A.P.; Mendes, I.C.; Lima, L.M.; Barreiro, E.J.; Piro, O.E.;
Castellano, E.E.; Beraldo, H. Binuclear zinc(II) complexes with the anti-inflammatory

6914
Molecules 2011, 16
compounds salicylaldehyde semicarbazone and salicylaldehyde-4-chlorobenzoyl hydrazone.
Polyhedron 2011, 30, 1891-1898 and references therein.
8. Johnson, C.K. ORTEP; Report ORNL-3794; Oak Ridge National Laboratory: Tennessee, 1965.
9. Salavati-Niasari, M.; Amiri, A. Synthesis and characterization of alumina-supported Mn(II),
Co(II), Ni(II) and Cu(II) complexes of bis(salicylaldiminato)hydrazone as catalysts for oxidation
of cyclohexene with tert-buthylhydroperoxide. Appl. Catal. A Gen. 2005, 290, 46-53.
10. Despaigne, A.A.R.; Da Silva, J.G.; Do Carmo, A.C.M.; Sives, F.; Piro, O.E.; Castellano, E.E.;
Beraldo, H. Copper(II) and zinc(II) complexes with 2-formylpyridine-derived hydrazones.
Polyhedron 2009, 28, 3797-3803.
11. Despaigne, A.A.R.; Vieira, L.F.; Mendes, I.C.; Da Costa, F.B.; Speziali, N.L.; Beraldo, H.
Organotin(IV) complexes with 2-acetylpyridine benzoyl hydrazones: Antimicrobial activity. J.
Braz. Chem. Soc. 2010, 21, 1247-1257.
12. Despaigne, A.A.R.; Da Silva, J.G.; Do Carmo, A.C.M.; Piro, O.E.; Castellano, E.E.; Beraldo, H.
Structural studies on zinc(II) complexes with 2-benzoylpyridine-derived hydrazones. Inorg. Chim.
Acta 2009, 362, 2117-2122.
13. Despaigne, A.A.R.; Da Silva, J.G.; Do Carmo, A.C.M.; Piro, O.E.; Castellano, E.E.; Beraldo, H.
Copper(II) and zinc(II) complexes with 2-benzoylpyridine-methyl hydrazone. J. Mol. Struct.
2009, 920, 97-102.
14. Coolier, H.O.J.; Dinneen, L.C.; Johnson, C.A.; Schneider, C. Abdominal constriction response
and its suppression by analgesic drugs in the mouse. Brit. J. Pharmacol. 1968, 32, 295-310.
15. Hunskaar, S.; Hole, K. The formalin test in mice: Dissociation between inflammatory and
noninflammatory pain. Pain 1987, 30, 103-114.
16. Eddy, N.B.; Leimbach, D. Synthetic analgesics. II. Dithienylbutenylamines and
dithienylbutylamines. J. Pharmacol. Exp. Ther. 1953, 107, 385-393.
17. Doherty, N.S.; Poubelle, P.; Borgeat, P.; Beaver, T.H.; Westrich, G.L.; Schrader, N.L.
Intraperitoneal injection of zymosan in mice induces pain, inflammation and the synthesis of
peptidoleukotrienes and prostaglandin E2. Prostaglandins 1985, 30, 769-789.
18. Da Silva, Y.K.C.; Augusto, C.V.; Barbosa, M.L.C.; Melo, G.M.A.; De Queiroz, A.C.; Dias,
T.L.M.F.; Bispo, W., Jr.; Barreiro, E.J.; Lima, L.M.; Alexandre-Moreira, M.S. Synthesis and
pharmacological evaluation of pyrazine N-acylhydrazone derivatives designed as novel analgesic
and anti-inflammatory drug candidates. Bioorg. Med. Chem. 2010, 18, 5007-5015.
19. Dutta, M.M.; Goswami, B.N.; Kataky, J.C.S. Studies on biologically-active heterocycles. III.
Synthesis and antibacterial activity of some 2-aryl/arakyl-3-substituted-4-thiazoldinones. J. Indian
Chem. Soc. 1990, 67, 332-334.
20. Ainscough, E.W.; Brodie, A.M.; Denny, W.A.; Finlay, G.J.; Gothe, S.A.; Ranford, J.D.
Cytotoxicity of salicylaldehyde benzoylhydrazone analogs and their transition metal complexes:
quantitative structure-activity relationships. J. Inorg. Biochem. 1999, 77, 125-133.
21. Enraf-Nonius. COLLECT; Nonius BV: Delft, The Netherlands, 1997-2000.
22. Otwinowski, Z.; Minor, W. Methods in Enzymology; Carter, C.W., Jr., Sweet, R.M., Eds.;
Academic Press: New York, NY, USA, 1997; pp. 307-326.
23. Sheldrick, G.M. SHELXS-97. Program for Crystal Structure Resolution; University of Göttingen:
Göttingen, Germany 1997.

6915
Molecules 2011, 16
24. Sheldrick, G.M. SHELXL-97. Program for Crystal Structures Analysis; University of Göttingen:
Göttingen, Germany 1997.
Sample Availability: Samples of the compounds H2LASSBio-466, H2LASSBio-1064 and complexes
[Zn(LASSBio-466)H2O]2 (1) and [Zn(HLASSBio-1064)Cl]2 (2) are available from the authors.
© 2011 by the authors; licensee MDPI, Basel, Switzerland. This article is an open access article
distributed under the terms and conditions of the Creative Commons Attribution license
(http://creativecommons.org/licenses/by/3.0/).