Tridentate hydrazone metal complexes derived from cephalexin and 2-hydrazinopyridine: Synthesis, characterization and antibacterial activity

Article abstract of DOI:10.1016/j.saa.2015.01.009

Metal(II) coordination compounds of a tridentate hydrazone ligand (HL) derived from the condensation of cephalexin antibiotic with 2-hydrazinopyridine were synthesized. The hydrazone ligand and mononuclear [ML(OAc)(H₂O)] (M(II) = Mn, Co, Ni, Cu, Zn, Ag) complexes were characterized by several techniques, including elemental and thermal analysis, molar conductance and magnetic susceptibility measurements, electronic, FT-IR, EPR and ¹H NMR spectral studies. The cephalexin 2-pyridinylhydrazone ligand HL behaves as a monoanionic tridentate NNO chelating agent. The biological applications of complexes have been studied on three bacteria strains (Escherichia coli, Acinetobacter baumannii and Enterococcus faecalis) by agar diffusion disc method.

Full text of DOI:10.1016/j.saa.2015.01.009

Accepted Manuscript
Tridentate hydrazone metal complexes derived from cephalexin and 2-hydra-
zinopyridine: Synthesis, characterization and antibacterial activity
J.R. Anacona, Maria Rincones
PII:
S1386-1425(15)00019-0
http://dx.doi.org/10.1016/j.saa.2015.01.009
SAA 13177
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Spectrochimica Acta Part A: Molecular and Biomo-
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17 November 2014
5 January 2015
Please cite this article as: J.R. Anacona, M. Rincones, Tridentate hydrazone metal complexes derived from
cephalexin and 2-hydrazinopyridine: Synthesis, characterization and antibacterial activity, Spectrochimica Acta
Part A: Molecular and Biomolecular Spectroscopy (2015), doi: http://dx.doi.org/10.1016/j.saa.2015.01.009
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Tridentate hydrazone metal complexes derived from
cephalexin and 2−hydrazinopyridine: Synthesis,
characterization and antibacterial activity
J.R. Anacona*, Maria Rincones
Department of Chemistry, Universidad de Oriente, Cumana 6101.
Venezuela
*Corresponding author at: Department of Chemistry, Universidad de Oriente, Cumana 6101.
Venezuela
Tel.: +(58)(293)4002405
Fax: +(58)(293)4002405
E-mail address: juananacona@hotmail.com (J. Anacona)
Abstract: Metal(II) coordination compounds of a tridentate hydrazone
ligand (HL) derived from the condensation of cephalexin antibiotic with
2−hydrazinopyridine were synthesized. The hydrazone ligand and
mononuclear [ML(OAc)(H2O)] (M(II) = Mn, Co, Ni, Cu, Zn, Ag)
complexes were characterized by several techniques, including elemental
and thermal analysis, molar conductance and magnetic susceptibility
1
measurements, electronic, FT−IR, EPR and H NMR spectral studies. The
cephalexin 2−pyridinylhydrazone ligand HL behaves as a monoanionic
tridentate NNO chelating agent. The biological applications of complexes
have been studied on three bacteria strains (Escherichia coli, Acinetobacter
baumannii and Enterococcus faecalis) by agar diffusion disc method.
Keywords: hydrazone metal complexes, cephalexin 2−pyridinylhydrazone
ligand, synthesis hydrazone containing cephalexin, magnetic and spectral
studies, antibacterial activity
Introduction
In view of their applicability in various fields, hydrazones, a member of the
Schiff base family with triatomic >C=N–NH− linkage, takes the forefront

position in the development of coordination chemistry. Reports on the
synthesis, characterization and structural studies on hydrazone ligands
show the importance of hydrazone complexes in various fields, including
analytical and biological fields [1, 2]. Many studies have been reported
regarding the biological activities of hydrazones, including their anticancer
[3−5], antibacterial [6, 7] and antidepressant activities [8].
The β−lactam ring, has largely been recognized as a useful building block
in the synthesis of biologically important compounds. The activity of
famous antibiotic classes such as the penicillins and cephalosporins is
attributed to the presence of a β−lactam ring [9]. Unfortunately, the wide
use of the antibiotics resulted in the serious medical problem of drugs
resistance and public health concern [10, 11]. Preparation of new synthetic
derivatives of antibiotics with novel mechanism of action has become an
important task to cope with drug resistance problems.
Although hydrazones have extensively been studied and widely employed
in coordination chemistry, rather less is known about hydrazone ligands
containing β−lactam antibiotics. Continuing with metal−based antibiotics
studies in order to establish whether complexation affects the
pharmacological properties of the ligand and to derive additional
fundamental knowledge about antibiotic action [12−16], we report here the
isolation and characterization of metal(II) complexes containing a
hydrazone ligand derived from the condensation of cephalexin antibiotic,
first generation cephalosporin, with 2−hydrazinopyridine. The chemical
structure of cephalexin is shown in Figure 1.
Experimental
Materials and methods
All necessary precautions were taken to exclude oxygen and moisture
during the synthesis and handling of the compounds. Analytical grade

chemicals were used as received for all experiments. Fourier transform
infrared (FTIR) spectra of the ligand and its metal complexes as KBr
pellets were recorded in the spectral range 4,000–400 cm−1 with a Perkin-
Elmer Series 2000 apparatus. FT−IR spectra as polyethylene pellets were
recorded between 450 and 120 cm−1 using a Bruker IFS 66V
spectrophotometer. EPR spectra were recorded on a Bruker ECS 106
spectrometer operating in the X−band (9.76 GHz). DPPH free radical was
used as the g marker. Measurements of d–d transitions in the visible and
and u.v. regions were taken with a Perkin−Elmer spectrophotometer. The
contents of C, H, N and S were analysed on a LECO CHNS 932 model
microanalytical instrument. To determine the metal contents, the
compounds were decomposed by wet digestion at 350 °C with sulphuric
acid and hydrogen peroxide and determined by normal complexometric
titration procedures with standard EDTA solution [17]. The metal contents
as well as the coordinated water were also obtained from the TGA curves.
Molecular weight determinations were carried out by the Rast camphor
method. Magnetic susceptibilities were measured on a Johnson Matthey
Magnetic Susceptibility balance at room temperature using HgCo(NCS)4 as
calibrant. 1H NMR spectra were run at 80 MHz on a Varian spectrometer in
DMSO against tetramethylsilane (TMS) as internal reference.
Thermograms were recorded on a simultaneous thermal analyzer,
STA−6000 (Perkin Elmer) instrument at a heating rate of 10 ºC min−1 up to
800 °C. X−ray powder diffraction patterns for the studied complexes and
final solid product of thermal decomposition were recorded on a HZG 4
diffractometer. Measurements were taken over the range of 2θ = 2–70°
using Ni filtered CuKα radiation.
Synthesis of 2−hydrazinopyridine

One mmol liquid 2−chloropyridine was dissolved in 2.0 mL 85% hydrazine
hydrate and methanol (5 mL) and the mixture was refluxed at 90°C for 6 h
upon cooling, a yellow solid was separated out and recrystallized with
ethanol−DMF to get the pure 2−hydrazinopyridine. Yield 60%.
Synthesis of cephalexin 2−pyridinyl hydrazone HL
To 1 mmol of cephalexin in 25 mL of hot methanol were added 1 mmol of
2−hydrazinopyridine. The solution was refluxed under nitrogen atmosphere
at 70 °C for 3 h to give a dark yellow precipitate. This material was filtered
off and washed with methanol and ether, and dried under reduced pressure.
The product was purified by recrystallization from the same solvent. Yield
54%.
Synthesis of hydrazone metal complexes
Metal(II) complexes were prepared by the same general method. To a
solution of 1 mmol of the metallic acetate in 20 mL of water was slowly
added with stirring a solution of 1 mmol of HL in 10 mL of ethanol. To this
solution KOH (0.1% in methanol) was added to adjust the pH of the
solution at 7–8 and the mixture was then refluxed for 4 h and coloured
precipitates formed. The metal(II) complexes of HL were separated from
the reaction mixture as amorphous solids and washed several times with
water, methanol and ether and dried under reduced pressure at room
temperature. Complexes were purified by recrystallization from
dimethylsulfoxide/water mixture. Yield 65–78%,
Antibacterial activity
The antibacterial activity of hydrazone ligand and hydrazone metal
complexes was tested against Enterococcus faecalis as a Gram−positive
bacterium and Escherichia coli and Acinetobacter baumannii as

Gram−negative bacteria, according to a modified Kirby−Bauer disc
diffusion method under standard conditions using Mueller−Hinton agar
medium, as previously reported [12−15]. The test compounds in DMSO
solutions (5 x 10−2 M) were added dropwise (10 µL) to a 5 mm diameter
filter paper disc placed at the centre of each agar plate.
Results and discussion
The ligand and the metal(II) complexes were isolated pure in very good
yields and they are of various colours. The ligand, manganese(II) and
zinc(II) complexes are yellow, cobalt(II) complex is red wine, nickel(II)
and copper(II) complexes are green, and silver(II) is orange in colour. All
the complexes did not melt/decompose when heated up to 158 °C. The
synthetic route of HL ligand is given in Scheme S1. The elemental analyses
of the ligand and complexes are contained in Table 1 and they agree well
with a 1:1:1:1 metal: ligand: acetate: coordinated water stoichiometry. Thus,
the general formulae [ML(OAc)(H2O)] (M(II) = Mn, Co, Ni, Cu, Zn, Ag)
have been assigned to the complexes and they are very air stable solids at
room temperature without decomposition for a long time. The complexes
are insoluble in water and other common organic solvents such as ethanol,
chloroform, benzene, acetone, dichloromethane, DMF, acetonitrile and
ether but soluble in DMSO. Attempts to form complexes of a well−defined
stoichiometry, under the above−mentioned conditions, with chromium(III),
iron(II) and mercury(II) ions were unsuccessful. The molar conductance
values measured in DMSO at room temperature vary from 10.4 to 12.8 S
cm2 mol−1, revealing the non−electrolytic nature of the complexes [18].
Thermal analysis

By thermal analysis, information on their properties, nature of intermediate
and final products of their thermal decomposition can be obtained [19].
From TGA curves, the mass loss was calculated for the different steps and
compared with those theoretically calculated for the suggested formulae
based on the results of elemental analyses as well as molar conductance
measurements. TGA indicated the formation of metal oxide as the end
product from which the metal content could be calculated and compared
with that obtained from analytical determination. Thermograms of the
hydrated metal complexes indicate endothermic decompositions in two
steps and also reveal that the complexes are stable with no hydration water
and solvent molecules. The first step in the 158 to 175 °C range is assigned
to loss of coordinated water molecules (Table 1). The final decomposition
step includes complete evaporation of the ligand as well as formation of
metal oxide as final product from which the metal content was found to be
in very good agreement with the data obtained from complexometric
analyses. Thus, the overall thermogravimetric results are consistent with
the formulation of these complexes. The solid residues obtained during
thermal decomposition of complexes are suitable metal oxides: MnO, CoO,
NiO, CuO and ZnO. Their compositions have been confirmed by X−ray
diffraction measurement. The diffraction patterns of obtained residues have
been compared with reference patterns.
1H−NMR
The values of the chemical shifts obtained were similar to those of Schiff
1
base ligands reported in the literature [20]. In the H−NMR spectrum of
hydrazone ligand single peaks attributed to methyl, COOH and =N−NH
groups appeared at 1.95, 10.1 and 11.2 ppm respectively. Three groups of
double peaks given by N=C−CH and N–CH on the β−lactam ring and
O=C−NH− appeared at 4.90, 5.45 and 9.01 ppm, respectively. One group

of four resonance signals consistent with an AB system attributed to S−CH2
on the dihydrothiazine ring, was observed in the 3.18−3.49 ppm region
with coupling constant 16.9 Hz for JAB. Furthermore, coupling between
NH2 and the adjacent CH moieties could not be distinguished and a broad
single signal due to NH2 protons was observed at 5.05 ppm. A multiplet in
the range 6.70–7.90 ppm due to aromatic ring protons was also present. All
the complexes are paramagnetic with the exception of zinc(II) complex,
1
therefore the H−NMR spectra of the complexes could not be obtained
Comparison of the 1H−NMR spectrum of hydrazone ligand with that of the
diamagnetic zinc(II) complex, shows the absence of the proton signals
assigned to the COOH moiety of hydrazone ligand indicating deprotonation
and suggests the formation of COO−metal bond. No significant changes in
1H chemical shifts were observed for other atoms upon complexation
suggesting that, in solution, the aromatic rings are not involved in stacking
interactions. Owing to their low solubility it was not possible to record
satisfactory 13C−NMR spectrum for the diamagnetic complex.
Infrared spectra
The IR spectrum of the complexes were recorded down to the far−IR
region of 200 cm−1 and tentative band assignments of some characteristic
bands of hydrazone ligand were made by analogy with other related
systems [21, 22]. The main infrared spectral bands of the hydrazone ligand
and its metal complexes are presented in Table 2. The lactam ν(C=O) band
appear at 1735 cm−1 in the spectra of cephalexin. The IR spectrum of the
hydrazone ligand shows no absorption bands which can be assigned to
lactam ν(C=O) vibrational mode coming from cephalexin. The absence of
such absorption together with the appearance of a new band at 1635 cm−1
attributed to ν(C=N−NH) vibrations, is consistent with the product being

the expected hydrazone ligand [23, 24]. The infrared spectra of the metal
complexes display IR absorption bands in the 1620−1625 cm−1 range which
can be assigned to the C=N stretching frequencies of the coordinated ligand
(HL), strongly suggesting involvement of this group in coordination. The
ν(N–N) of the hydrazone complexes are found at 1110 cm−1. The increase
in the frequency of this band in the spectra of the complexes, due to the
increase in the bond strength, again confirms the coordination via the
azomethine nitrogen [25].
Disappearance of the stretching frequency at 1690 cm−1 assigned to
ν(COOH) in the ligand and appearance in the complexes of new νasym and
νsym modes of the (COO−) group indicates that the hydrazone has reacted.
Since the carboxylate group can coordinate to the metal ion in either
bidentate or monodentate fashion, the ‘‘∆ν criterion’’ [∆ν = νasym(COO) −
νsym(COO)] was employed to determine the coordination mode of the
carboxylate group. The hydrazone metal complexes exhibit strong bands
corresponding to νasym(COO) at 1600−1605 and 1580−1585 cm−1, and
νsym(COO) at 1420−1425 and 1370−1375 cm−1. The ∆ν value of 175−180
and 205−215 cm−1 in our complexes are very similar to that reported for the
metal complexes with both bidentate µ2−O,O’ and monodentate
carboxylate groups respectively [26], thus indicating the carboxylate
groups behave both as bidentate [27, 28] and monodentate ligands.
The remaining carboxylate bands, namely γ(COO), ω(COO) and ρ(COO),
formerly at 785, 610 and 530 cm−1, respectively, also change as a result of
coordination. Furthermore, the appearance of new bands in the 450–490
cm−1 ranges attributed to ν(M–N) stretching vibrations, observed in the
spectra of the complexes (absent in the free ligand) provide evidence that
the pyridine moiety can be bonded to the metal ion through the nitrogen
atom. The metal(II) complexes also display bands in the 1590–1595 and

1015–1025 cm−1 ranges due to ν(C=N) of coordinated pyridine ring and
pyridine ring breathing mode of vibration [29].
The bands in the 350–400 cm−1 region observed in the complexes, and
absent in the free hydrazone ligand, are tentatively assigned to ν(M–O)
vibrations. Medium intensity band appearing in the 2830–2950 cm−1 region
corresponds to aliphatic ν(C–H), while aromatic ν(C–H) stretches appear in
the 3000–3100 cm−1 region. A broad band centered at 3426 cm−1 for the
complex can be assigned to the ν(OH) stretch of coordinated water
molecules. These overall data suggest that the bonding sites are the
pyridine nitrogen, azomethine nitrogen and carboxylate oxygen atoms are
involved in coordination in the complexes and that the hydrazone behaves
as a tridentate monoanionic NNO chelating agent.
Magnetic properties
Magnetic susceptibility is the degree of the magnetization of a material in
response to a magnet. The method measures the Boltzmann occupation of
all energy levels. From the molar magnetic susceptibility values, corrected
magnetic moments were calculated using Pascal’s constants [30]. The
magnitudes of the magnetic moments for the paramagnetic complexes fall
within the ranges associated with spin−free high spin ions in octahedral
fields.The manganese(II) complex has a magnetic moment value of 5.90 µB
which is typical of high spin d5 systems with five unpaired electrons and S
= 5/2 ground state. The cobalt(II) complex has a magnetic moment of 4.60
µB which is a typical value of a d7 system with three unpaired electrons
indicating a quartet state in an octahedral arrangement around the metal, as
compared with the reported values for octahedral complexes of cobalt(II)
(4.7−5.2 µB) [31]. The nickel(II) complex has a magnetic moment of 3.24
µB characteristic of two unpaired electrons and greater than the spin−only

value, presumably due to the orbital contribution resulting from the transfer
of an electron from the dx −y orbital to the dxy orbital. The complex
2
2
therefore probably has distorted octahedral geometry. At room temperature
a magnetic moment of 1.9–2.2 µB is usually observed for mononuclear
copper(II) complexes, regardless of stereochemistry [31]. A magnetic
moment of 2.10 µB is observed for the copper(II) compound in the solid
state.
When silver(I) acetate or nitrate was added to an aqueous suspension or to
a 50% MeOH solution of the hydrazone ligand, performed under aerobic
conditions, we observed an obvious color change from light yellow to dark
orange. This observation suggests the occurrence of oxidation process,
(Ag(I) d10 → Ag(II) d9 complex), induced by the oxygen in air at an
observable and rather rapid rate, depending on conditions of concentration
and temperature. The starting Ag(I)−material does not yield an EPR
spectrum, however, its oxidation was followed by EPR spectroscopy which
showed the gradual gain of the signal assigned to the oxidised product; on
completion of the oxidation a EPR signal could be appreciated. The
silver(II) complex, isolated as [AgL(OAc)(H2O)], is an orange solid which
can be conveniently recrystallized from water, alcohol or acetonitrile. The
compound is paramagnetic, having an effective magnetic moment of 2.10
µB characteristic of one unpaired electron. Thus the magnetic data supports
a mononuclear structure. In a control experiment it was shown that no EPR
signal occurred when hydrazone ligand was employed in the absence of
silver(I) ions. The propensity of divalent silver to act as an oxidazing agent
is well documented [32] and silver(II) complexes have been rare and stable
only when formed with ligands which could survive the strong oxidizing
capabilities of silver(II). For the most part these ligands have been nitrogen
containing unsaturated heterocycles, such as pyridines and related

compounds. Ligand oxidation did not occur in the above reaction mixtures
as shown by IR and UV−Vis spectra, and the compound appears to be
stable in the solid state. The formation of this novel complex illustrates
further the effect of the exceptionally strong ligand field exerted by the
hydrazone ligand. This serves to raise antibonding level (dx −y
)* of the
2
2
Ag(I) ion of hydrazone complex to such a high energy that electron is
easily removed.
The powder EPR spectrum of the orange product at room temperature
shows an axial spectrum with principal g−factor values at 2.17 and 2.04 for
parallel and perpendicular tensors respectively, and they coincide with
other reported values for silver(II) complexes [33−35]. The experimental
hyperfine structure observed is presented in Figure 2. The spectrum is a
superposition of the hyperfine doublet structure characteristic of the silver
nuclear spin (I = 1/2) and the superhyperfine couplings due to 14N nuclei (I
= 1). The number of the superhyperfine lines due to azomethine and
pyridine nitrogen nuclei coordinated to the silver center is expected to be
two sets of five (according to the known rule for the number of the
superhyperfine lines to be equal to 2nI + 1, where I is the nuclear magnetic
moment and n is the number of the corresponding nuclei) with relative
intensity of the superhyperfine lines, 1:2:3:2:1. It could be even more
complicated considering the presence of superhyperfine structure due to
magnetic interaction in both, parallel and in perpendicular directions, with
the nuclei of silver, 107Ag, I = 1/2, 51.82% and 109Ag, I = 1/2, 48.18%,
which have the magnetic dipole moment of −0.1135 and −0.1305,
respectively. The EPR line has a width of ∼ 550 G. The line width does not
change very much with decreasing temperature (down to 120 K) and
should be the result of the dipolar and exchange interactions between the
silver ions. Superhyperfine structure is also observed at both temperatures.

From the g values of a d9 metal complex, we can obtain very important
information about the structure of the complex. The geometric parameter G,
which is a measure of the exchange interaction between the silver(II)
centers in the polycrystalline compound, is calculated using the equation: G
= (g║ − 2.0023) / (g⊥ − 2.0023) for axial spectrum. If G < 4.0 considerable
exchange interaction is indicated in the solid complex [36−39]. The G
parameter has the value of 4.25 suggesting that there is no exchange
interaction between silver(II) centres.
Electronic spectra
A long−term UV–Vis study was carried out to verify the stability of new
complexes in DMSO solution. Compared with hydrazone ligand, it is
significant to note that the absorption wavelengths of new complexes
hardly varied for up to 1 month, meaning that new complexes were stable
in DMSO solution. The electronic spectrum of the hydrazone ligand in
DMSO solution showed three broad bands at 32,250, 30,300 and 27,780
cm−1. The former two bands are due to the π−π* transitions within the
aromatic rings and remains almost unchanged in the spectra of metal
complexes, while the third band, due to the π−π* transition within the
>C=N–NH− chromophore, is shifted to a longer wavelength as a
consequence of coordination when binding with the metal atom,
confirming the formation of metal complexes and reflecting that
azomethine nitrogen is involved in coordination [40].
The UV–Vis spectra of the metal complexes in DMSO solutions present
absorption maxima attributable to the hydrazone ligand together with the
absorptions, around 25,000–12,500 cm−1, due to ligand to metal charge
transfer and d–d transitions of the metal in the complexes [41]. The
manganese(II) complex shows a very broad absorption at 22,220−20,830

cm−1 probably due to the coincidence of charge transfer, d → π*, L → M
and intraligand n → π* transitions [42, 43]. The visible region spectrum of
the cobalt(II) complex indicates additional two bands at 25,640 and 20,000
4
4
cm−1, attributed to metal−ligand charge and T1g(F) → A2g(F) (ν2)
transitions respectively, suggesting octahedral stereochemistry around the
metal ion [44]. The UV−Vis spectrum of the nickel(II) complex presents
two major absorptions maxima, at 25,000 and 18,182 cm−1 due to d−d
bands which may be assigned, considering that the immediate coordination
sphere of the metal is Oh symmetry, to the spin allowed transitions 3A2g →
3
3
3T1g(P) (ν3) and A2g → T1g(F) (ν2) respectively [45]. Ligand field
parameters (10Dq, B, β,) and ν1 have been calculated for Ni(II) complex
by using ν2 , ν3 and the procedure of Lever [41]. The results are satisfactory
owing to the ratio ν2/ν1 is found to be 1.57 as required for the octahedral
Ni(II) complexes [46]. The values of ν1 = 11,570 cm−1, the magnetic
moment (µspin only < µobs ~ µS+L) and the calculated ligand field parameters of
Dq = 1,157 cm−1, B = 565 cm−1 and β = 0.54 are also consistent with the
octahedral geometry. The low value of Racah parameter (B) for the
complexes compared to the free ion value indicates significant covalent
character of the metal–ligand bonds. The nephelauxetic ratio β also support
covalent character in this complex. The electronic spectrum of copper(II)
complex exhibits a broad band centered at 14,280 cm−1 (ε = 280.1 M−1
2
2
cm−1) assigned to the E2g → T2g transition in a distorted octahedral
geometry around the copper(II) ion. A band at 24,400 cm−1 is assigned to
charge−transfer, mainly of the L → Cu type [41]. The electronic spectrum
of silver(II) complex presents a broad band at 25,000 cm−1 assigned to
charge transfer of the L → Ag type. The spectrum of the Zn(II) complex
exhibits a strong intense charge transfer transition (LMCT) band at 27,000
cm−1.

Coordination sites
The coordination chemistry of transition metal ions with ceftriaxone [47],
cefotaxime [48], cefepime [49] and ceftazidime [50] antibiotics have been
reported. In the present case, the hydrazone ligand containing cephalexin
has a number of potential donor atoms in various positions which can bind
to the metal ions forming multinuclear chelates. From the data it appears
that each metal ion lies in a distorted octahedron coordination sphere and
the hydrazone would act as an efficient pseudo−encapsulating ligand, with
pyridine nitrogen, azomethine nitrogen and carboxylate oxygen atoms,
presumably bound to the octahedral ions. Thus, the metal ions in the
[ML(OAc)(H2O)] complexes containing one water molecule and acetate
with bidentate mode at the vertices of an octahedron are hexacoordinate.
We have attempted to grow single crystals of the metal chelates but in no
case have we had any success, due to their insolubility in common organic
solvents. The complexes only form amorphous materials as revealed by
their XRD patterns. Up to now no crystal structures of hydrazone
complexes containing cephalexin have been reported. These studies
represent a contribution to future crystallographic analyses, which are
complicated by the difficulties in obtaining X−ray quality crystals of
cephalosporin derived complexes. Although crystal structure of the
complexes are not known, the coordination environment of mononuclear
complexes, may tentatively be proposed (Figure 3).
Antibacterial activity
Preliminary screening for antimicrobial activities of the stock solutions
were performed qualitatively using the disc diffusion assay. In vitro
antimicrobial activities were measured from the diameter of clear inhibition
zones caused by samples against the same bacteria and under the identical

experimental conditions. As assessed by colour, the complexes remain
intact during biological testing. In order to clarify role of DMSO any
participating and metal(II) acetate salts in the biological screening, separate
studies were carried out with the solutions alone of DMSO and the free
metal salt and they have been found that they have no effect on the growth
of any microorganisms taken. The antibacterial activity of hydrazone
ligand HL as well as its metal(II) complexes were tested on against
Enterococcus faecalis, Escherichia coli and Acinetobacter baumannii
bacteria and compared to cephalexin used as standard.
The average results are shown in Table 3 where can be appreciated that the
hydrazone ligand and its metal complexes have different behaviour
compared with standard antibiotics against the same bacteria. Thus: (1)
Cephalexin and the hydrazone ligand have similar antibacterial activity. (2)
Manganese(II), cobalt(II) and silver(II) complexes were found to have
higher activity than the established drug and HL against the bacteria strains
studied under the test conditions, showing that they have good activity as
bactericides. (3) The antibacterial activity of nickel(II), copper(II) and
zinc(II) complexes show to be less toxic than the reference drug and
hydrazone ligand.
According to Tweedy’s theory [51], the enlarged activity of complexes
compared to ligands may be attributed to increased delocalisation of π
electrons of ligand over the whole chelate ring. Chelation reduces the
polarity of metal ions and increases the lipophilic character of the chelate,
which subsequently favors its permeation through the lipid layers of the
cell membrane and blocking the metal binding sites on enzymes of
microorganism. However, in the present case, the in vitro antibacterial
activities demonstrated that manganese(II), cobalt(II) and silver(II)
complexes have higher antimicrobial activity in comparison with that of the
ligand HL, whereas nickel(II), copper(II) and zinc(II) complexes showed

lower activity against the tested strains compared to hydrazone ligand.
Therefore, antimicrobial activity must be influenced by other factors
beyond membrane permeability.
The targets for β−lactam antibiotics are cell wall−synthesizing enzymes
(penicillin binding proteins, PBPs) which are found as both
membrane−bound and cytoplasmic enzymes that catalyze cross−linking
reactions. β−lactam antibiotics, interfere with cell wall synthesis by binding
covalently to the PBPs catalytic site. PBPs are present in almost all bacteria,
but they vary from species to species differing in amount, molecular weight,
affinity for β−lactam antibiotics and enzymatic function (e.g.,
transpeptidase, carboxypeptidase, or endopeptidase) [52].
The results in Table 3 can be understood considering that the enzyme
probably serves primarily to hold catalytic groups or the substrate in the
proper positions and is possible to expect that hydrazone metal complexes
may change the stereochemistry required in solvolytic reactions on an
enzyme surface. The obtained results may highlight that the bactericidal
activity of hydrazone metal complexes compared to hydrazone ligand and
cephalexin may reflect a different mechanistic pathway by which they react
with the PBP active sites to achieve formation of a stable PBP−inhibitor
adduct. The level of resistance to β−lactam metal complexes is determined
by the amount, nature and kinetic properties of the PBPs.
Conclusions
A hydrazone ligand derived from cephalexin and 2−hydrazinopyridine and
its transition metal complexes have been prepared. The hydrazone
coordination to metal occurs through the pyridine−N, azomethine−N and
carboxylate−O atoms. Manganese(II), cobalt(II) and silver(II) complexes
were found to have higher bactericidal activity than the uncomplexed

cephalexin and the hydrazone ligand against the bacteria strains, showing
that they have a good activity as bactericides. The nickel(II), copper(II) and
zinc(II) complexes showed to be less toxic than the reference drug and the
hydrazone ligand. Apart of membrane permeability, antibacterial activity of
metal complexes depends mainly on the metal ion and the type of
microorganism.
Acknowledgements. The authors express their sincere thanks to Comision
de Investigación from the Universidad de Oriente for financial support.
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Table 1. Analytical, thermoanalytical (TG) and physical data

Found (Calcd.) %
Mol
wt.
Compound
C
N
H
S
H2O
M1
M2
[(HL)]
57.6
19.2 4.4
7.2
427.0
[(C21H21N6O3S)]
(57.7) (19.2) (4.8) (7.3)
(437.5)
[MnL(OAc)(H2O)] 48.7
14.9 4.9
5.4 2.8
9.5
9.7 553.2
[Mn(C23H25N6O6S)] (48.6) (14.8) (4.4) (5.6) (3.2) (9.7) (9.7) (568.5)
[CoL(OAc)(H2O)] 48.5 14.9 4.8 5.3 3.5 10.2 10.5 560.3
[Co(C23H25N6O6S)] (48.3) (14.7) (4.4) (5.6) (3.1) (10.3)(10.3) (572.5)
[NiL(Oac)(H2O)] 48.7 14.8 4.2 5.5 2.9 10.4 10.1 561.4
[Ni(C23H25N6O6S)] (48.3) (14.7) (4.4) (5.6) (3.1) (10.3)(10.3) (572.3)
[CuL(OAc)(H2O)] 47.7 14.2 4.7 5.4 3.5 10.7 11.4 564.8
[Cu(C23H25N6O6S)] (47.9) (14.6) (4.4) (5.6) (3.1) (11.0)(11.0) (577.1)
[ZnL(OAc)(H2O)] 48.1 14.7 4.1 5.8 3.2 11.2 11.0 565.3
[Zn(C23H25N6O6S)] (47.7) (14.5) (4.4) (5.5) (3.1) (11.3)(11.3) (578.9)
[AgL(OAc)(H2O) 44.8 13.2 3.8 5.4 3.0 17.1 17.7 610.7
[Ag(C23H25N6O6S)] (44.5) (13.5) (4.1) (5.2) (2.9) (17.4)(17.4) (621.4)
M1 = complexometric analysis, M2 = thermal analysis

22
Table 2. Main vibrational wavenumbers of the metal complexes (cm−1)
νC=O νC=N νCOO νCOO ∆ν νCOO νCOO ∆ν
Compound
amide imino asym sym
asym sym
Cephalexin
HL
1657
1656 1635
[MnL(OAc)(H2O)] 1656 1625 1600
[CoL(OAc)(H2O)] 1653 1620 1600
[NiL(OAc)(H2O)] 1653 1620 1605
[CuL(OAc)(H2O)] 1658 1625 1605
[ZnL(OAc)(H2O)] 1654 1620 1600
[AgL(OAc)(H2O)] 1656 1620 1600
1420 180 1580
1370 210
1375 210
1370 215
1370 210
1375 205
1375 210
1420 180 1585
1425 180 1585
1425 180 1580
1425 175 1580
1425 175 1585

23
Table 3 Antibacterial activity
Zone of inhibition (mm)
A.B. E.F.
Compound
E.C.
Cephalexin
40.0 1.0
38.0 1.0
45.0 1.0
45.0 1.0
15.0 1.0
20.0 1.5
5.0 1.0
50.0 1.0
50.0 1.0
52.0 1.0
55.0 1.0
35.0 1.0
40.0 1.0
10.0 1.0
52.0 1.0
20.0 1.5
21.0 1.0
30.0 1.0
50.0 1.0
5.0 1.0
5.0 1.0
5.0 1.0
25.0 1.0
[HL]
[MnL(Oac)(H2O)]
[CoL(OAc)(H2O)]
[NiL(OAc)(H2O)]
[CuL(OAc)(H2O)]
[ZnL(OAc)(H2O)]
[AgL(OAc)(H2O)]
42.0 1.0
E.C. Escherichia coli, A.B. Acinetobacter baumannii, E.F. Enterococcus
faecalis. Values are the mean standard deviation of the mean.
FIGURE CAPTIONS
Figure 1. Chemical structure of cephalexin
Figure 2. EPR spectrum of [AgL(OAc)(H2O)] complex at room temperature
Figure 3. Proposed structure of [ML(OAc)(H2O)] complexes

24

25

26
Supplementary data
Scheme S1
SUGGESTED REFEREES

27
1
Name and Surname
Institution Address
E-mail
Juan Camus
Universidad Playa Ancha
jcamus@upla.cl
Phone
Country
Chile
2
3
4
5
Name and Surname
Institution Address
E-mail
Phone
Country
Name and Surname
Institution Address
E-mail
Phone
Country
Name and Surname
Institution Address
E-mail
Phone
Country
Name and Surname
Institution Address
E-mail
Dinorah Gambino
Universidad La Republica
dgambino@fq.edu.uy
Uruguay
Luca Fadini
Universidad Nacional
lfadini@unal.edu.co
Colombia
Ana Burgos
Universidad Nacional
anesbc@unal.edu.co
Colombia
V.E. Marquez
Instituto Universitario Tecnología
Victoriamarquez1308@hotmail.com
Phone
Country
Venezuela

28
Graphical abstract
Tridentate hydrazone metal complexes derived from cephalexin and
2−hydrazinopyridine: Synthesis, characterization and antibacterial activity
J.R. Anacona*, Maria Rincones
Department of Chemistry, Universidad de Oriente, Cumana 6101. Venezuela
Transition coordination compounds with a tridentate hydrazone ligand (HL) derived from
the condensation of cephalexin antibiotic with 2−hydrazinopyridine were synthesized,
characterized and screened for antibacterial activity.

29
Spectrochimica Acta Part A
Highlights
A new Schiff base using cephalexin antibiotic was prepared
Cephalexin Schiff base transition metal complexes were prepared.
Magnetic, spectroscopic properties and antibacterial activity were studied