Synthesis, characterization and antibacterial activity of a Schiff base derived from cephalexin and sulphathiazole and its transition metal complexes

Article abstract of DOI:10.1016/j.saa.2014.03.019

Metal(II) coordination compounds of a cephalexin Schiff base (HL) derived from the condensation of cephalexin antibiotic with sulphathiazole were synthesized. The Schiff base ligand, mononuclear [ML(OAc)(H₂O)₂] (M(II) = Mn, Co, Ni, Zn) complexes and magnetically diluted trinuclear copper(II) complex [Cu₃L(OH)₅] 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 analytical and molar conductance values indicated that the acetate ions coordinate to the metal ions. The Schiff base ligand HL behaves as a monoanionic tridentate NNO and tetradentate NNOO chelating agent in the mono and trinuclear complexes respectively.

Full text of DOI:10.1016/j.saa.2014.03.019

Accepted Manuscript
Synthesis, characterization and antibacterial activity of a Schiff base derived
from cephalexin and sulphathiazole and its transition metal complexes
J.R. Anacona, Juan Luis Rodriguez, Juan Camus
PII:
S1386-1425(14)00430-2
http://dx.doi.org/10.1016/j.saa.2014.03.019
SAA 11852
DOI:
Reference:
Spectrochimica Acta Part A: Molecular and Biomo-
lecular Spectroscopy
To appear in:
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Accepted Date:
1 December 2013
15 March 2014
Please cite this article as: J.R. Anacona, J.L. Rodriguez, J. Camus, Synthesis, characterization and antibacterial
activity of a Schiff base derived from cephalexin and sulphathiazole and its transition metal complexes,
Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy (2014), doi: http://dx.doi.org/10.1016/j.saa.
2014.03.019
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1
Dear Editor
We would apréciate if you consider the attached manuscript to be published in
Spectrochimica Acta. The present study is of interest considering that the
emergence of resistant human pathogens is a major problem in current
antimicrobial therapy, encouraging efforts to develop novel drugs. In recent
years, intensive research has been carried out in order to obtain modified
cephalosporins with improved antimicrobial properties. Metal complexes were
found to be particularly useful in this matter, extending the landscape of drug
design and enabling novel mechanisms of action. The transport properties and
other biological and physiological properties of the complexes may be of
greater importance.
Sincerely,
J.R. Anacona

2
Graphical abstract
Synthesis, characterization and antibacterial activity of a Schiff base derived from
cephalexin and sulphathiazole and its transition metal complexes
J.R. Anaconaa,*, Juan Luis Rodrigueza and Juan Camusb
aDepartment of Chemistry, Universidad de Oriente, Cumana 6101. Venezuela
bFacultad de Ciencias, Universidad de Playa Ancha, Valparaíso, Chile
Transition coordination compounds with a Schiff base (HL) derived from the condensation
of cephalexin antibiotic with sulphathiazole were synthesized, characterized and screened
for antibacterial activity..

3
Spectrochimica Acta Part A
Highlights
We prepare a new Schiff base using cephalexin antibiotic
We prepare cephalexin Schiff base transition metal complexes.
We study magnetic, spectroscopic properties and antibacterial activity

4
Synthesis, characterization and antibacterial activity of a Schiff
base derived from cephalexin and sulphathiazole and its
transition metal complexes
J.R. Anaconaa,*, Juan Luis Rodrigueza and Juan Camusb
aDepartment of Chemistry, Universidad de Oriente, Cumana 6101. Venezuela
bFacultad de Ciencias, Universidad de Playa Ancha, Valparaíso, Chile
*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 cephalexin Schiff base (HL)
derived from the condensation of cephalexin antibiotic with sulphathiazole
were synthesized. The Schiff base ligand, mononuclear [ML(OAc)(H2O)2]
(M(II) = Mn, Co, Ni, Zn) complexes and magnetically diluted trinuclear
copper(II) complex [Cu3L(OH)5] were characterized by several techniques,
including elemental and thermal analysis, molar conductance and magnetic
1
susceptibility measurements, electronic, FT−IR, EPR and H NMR spectral
studies. The analytical and molar conductance values indicated that the acetate
ions coordinate to the metal ions. The Schiff base ligand HL behaves as a
monoanionic tridentate NNO and tetradentate NNOO chelating agent in the
mono and trinuclear complexes respectively.
Keywords: Schiff base metal complexes, Schiff base containing cephalexin,
synthesis cephalexin derivative, magnetic studies, spectroscopic studies,
synthesis
Introduction

5
The Schiff bases are widely employed as ligands in coordination chemistry [1,
2]. Schiff base ligands containing an amine group (–RC=N–) are usually
formed by the condensation of a primary amine with an active carbonyl. They
are readily available, versatile and depending on the nature of the starting
materials they exhibit various denticities and functionalities. Moreover, the
nature, number, and the relative position of the donor atoms of a Schiff base
ligand allow a good control over the stereochemistry of the metallic centers, as
well as over the number of the metal ions within homo and heteropolynuclear
complexes [3]. All these advantages make Schiff bases very good candidates
in the effort to synthesize metal complexes of interest in bioinorganic
chemistry, catalysis, encapsulation, transport and separation processes [3].
Schiff bases form an important class of organic compounds with a wide
variety of biological properties [4]. Development of a new chemotherapeutic
Schiff base is now attracting the attention of medicinal chemist [5]. Many
studies have been reported regarding the biological activities of Schiff bases,
including their anticancer [6], antibacterial [7], antifungal, and herbicidal
activities. Schiff bases derived from various amine and carbonyl derivatives
were reported to possess genotoxicity [8, 9], antimicrobial [10], and antifungal
activities [11]. 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 Schiff base ligand derived from the condensation of
cephalexin antibiotic, first generation cephalosporin, with sulphathiazole. The
chemical structure of cephalexin is shown in Figure 1.
Experimental
Materials and methods

6
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 near infrared region were taken with a Cary Recording
Spectrophotometer Model 17D, while a Perkin-Elmer spectrophotometer was
used for recording the visible and u.v. regions. The contents of C, H, N and S
were analysed on a LECO CHNS 932 model microanalytical instrument. The
compounds were decomposed by wet digestion at 340°C with sulphuric acid
and hydrogen peroxide. The metal ion contents in this solution were
determined by normal complexometric titration procedures with standard 0.01
mol L-1 EDTA solution using xilenol orange as an indicator [17]. The metal
contents as well as the coordinated water were also obtained from the TG
curves. Magnetic susceptibilities were measured on a Johnson Matthey
Magnetic Susceptibility balance at room temperature using HgCo(NCS)4 as
1
calibrant. H 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 10ºC 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.

7
Measurements were taken over the range of 2θ=2–70° using Ni filtered CuKα
radiation.
Synthesis of Schiff base ligand HL
To 1 mmol of cephalexin in 250 mL of hot methanol were added 1 mmol of
sulphathiazole 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 72%),
m.p. = 165°C.
Synthesis of Schiff base complexes.
Manganese(II), cobalt(II), nickel(II) and zinc(II) complexes were prepared by
the same general method. To a solution of 1 mmol of the appropriate M(OAc)2
metal salts 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 colored precipitates formed. The acetate 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. Copper(II) complex was
prepared by mixing HL (1 mmol) and copper(II) acetate (3 mmol) in methanol
(20 mL), then pH of the solution was adjusted to 8.0 with KOH solution and
the reaction mixture was stirred at room temperature for about 4 h and then
left to stand overnight. A green polymeric complex was formed but on adding
ethanol and scratching, the polymeric substance changed to a powder.
Complexes were purified by recrystallization from dimethylsulfoxide/water
mixture. Yield 55–75%, m.p. > 250°C.

8
Antibacterial activity
The antibacterial activity of Schiff base ligand and metal complexes was
tested against Staphylococcus aureus as a Gram-positive bacterium, and
Escherichia coli as a Gram-negative bacterium according to a modified
Kirby−Bauer disc diffusion method under standard conditions using
Mueller−Hinton agar medium, as previously reported [12−15].
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 in colour. All the complexes did not
melt/decompose when heated up to 250°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 1:1:1:2 metal: ligand: acetate:
coordinated water stoichiometry for the mononuclear complexes. Thus, the
general formulae [ML(OAc)(H2O)2] (M(II) = Mn, Co, Ni, Zn) have been
assigned to the mononuclear complexes and they are very air stable solids at
room temperature without decomposition for a long time. The trinuclear
copper(II) compound of formula [Cu3L(OH)5] was obtained by reaction of HL
with either copper(II) acetate or chloride in a 1:3 molar ratio. 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), copper(I) and mercury(II) ions were unsuccessful. The molar

9
conductance values measured in DMSO at room temperature vary from 1.20
to 20.2 S cm2 mol-1, revealing the non-electrolytic nature of the complexes
[18] and suggesting that the acetate ion in [ML(OAc)(H2O)2] complexes is
coordinated to the metal(II) ions.
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 three 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 second step at 210–395°C corresponds to removal of
acetate as acetic acid, which is well known in the literature [20]. 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. Thermal degradation of hydrated complexes are
given as follows:
[ML(OAc)(H2O)2] → [ML(OAc)]
[ML(OAc)]
[ML]
→
[ML]
→
[MO]

10
Thus, the overall thermogravimetric results are consistent with the formulation
of these complexes. The degradation process of the trinuclear copper(II)
complex is intricate and is not possible to distinguish intermediate solid
products. The solid residues obtained during thermal decomposition of
complexes are suitable metal oxides: MnO, Co3O4, 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 base
ligands reported in the literature [21]. In the 1H−NMR spectrum of Schiff base
ligand single peaks attributed to methyl, COOH and SO2NH groups appeared
at 1.95, 10.1 and 12.4 ppm respectively. Three groups of double peaks given
by N−CH and N=C–CH on the β-lactam ring and 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 3.15–3.45 ppm region with coupling constant 16.9 Hz for JAB.
Furthermore, coupling between NH2 and the adjacent CH could not be
distinguished and a broad single signal due to NH2 protons was observed at
4.97 ppm. A multiplet in the range 6.58–8.02 ppm due to aromatic ring
protons was also present. All the complexes are paramagnetic with the
1
exception of zinc(II) complex, therefore the H−NMR spectra of the
1
complexes could not be obtained Comparison of the H−NMR spectrum of
Schiff base with that of the diamagnetic zinc(II) complex, shows the absence
of the signal assigned to the COOH proton of Schiff base ligand indicating
deprotonation and suggests the formation of a metal–COO bond. Owing to

11
13
their low solubility it was not possible to record satisfactory C−NMR
spectrum for the diamagnetic complex.
Infrared spectra
The main infrared spectral bands of the ligand and its metal complexes are
presented in Table 2. The lactam ν(C=O) band appear at 1750 cm−1 in the
spectra of cephalexin. The IR spectrum of the Schiff base 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 1630 cm−1 attributed to ν(C=N) vibrations, is
consistent with the product being the expected Schiff base ligand [22, 23]. The
infrared spectra of the metal complexes display IR absorption bands in the
1640-1645 cm−1 range which can be assigned to the C=N stretching
frequencies of the coordinated ligand (HL). The shift of this band on
complexation towards higher wave numbers indicate coordination of the
azomethine nitrogen to the metal centre [23]. Three bands of the free Schiff
base ligand at 3340−3200 cm−1 due to ν(NH) and ν(NH2) are still present in
the complexes, strongly suggesting noninvolvement of these groups in
coordination. Also, disappearance of the stretching frequency at 1690 cm−1
assigned to ν(COOH) in the ligand and appearance in the complexes of new
νas and νs modes of the (COO−) group indicates that the Schiff base 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 tricopper(II) and mononuclear complexes exhibit strong bands
corresponding to νas(COO) at 1595 and 1580 cm−1, and νs(COO) at 1425 and

12
1370 cm−1 respectively (Table 1). The ∆ν value of 170 and 210 cm−1 in these
complexes are very similar to that reported for the copper(II) complexes with
both bidentate µ2-O,O’ and monodentate carboxylate groups [24], thus
indicating the carboxylate group behaves both as a bridging bidentate ligand
between copper(II) ions [25, 26], and as monodentate ligand in the
mononuclear complexes. A broad band centered at 3426 cm−1 for the complex
can be assigned to the ν(OH) stretch of coordinated water molecules.
The remaining carboxylate bands, namely νsym(COO), γ(COO), ω(COO) and
ρ(COO), formerly at 1400, 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 C=N moiety could be bonded to the metal ion through the nitrogen atom.
The bands in the 350–400 cm−1 region observed in the complexes, and absent
in the free Schiff base ligand, are tentatively assigned to ν(M–O) vibrations.
The metal(II) complexes also show bands in the 1420–1460, 1070–1100 and
720–740 cm−1 ranges which can be assigned to phenyl ring 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. The usual modes of sulphathiazol moiety are also present, as
expected, the bands at 1310, 1150, and 560 cm−1 attributed to an O=S=O
group remain unchanged with respect to those of the ligand, excluding the
coordination of the SO2 group. These overall data suggest that the
azomethine−N, thiazole−N and carboxylate−O group with monodentate mode
are involved in coordination in the mononuclear complexes and that the Schiff
base behaves as a tridentate monoanionic NNO chelating agent. In the

13
trinuclear copper(II) complex the Schiff base behaves as a tetradentate
monoanionic NNOO ligand having a carboxylate group with bidentate mode.
Magnetic properties
From the molar magnetic susceptibility values, corrected magnetic moments
were calculated using Pascal’s constants [27]. 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 5.83 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 5.13 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) [28].
The nickel(II) complex has a magnetic moment of 3.30 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
dx2-y2 orbital to the dxy orbital. The complex 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 [28]. A
magnetic moment of 2.84 µB. is observed for the copper(II) compound in the
solid state, indicative of the presence of a polynuclear complex with some
ferro/antiferromagnetic interactions, operating through Cu–Cu interactions
[29]. This observed value is slightly lower than the d9 spin-only magnetic
moment calculated value µeff = 3,00 µB for a tricopper complex with three

14
spin-system in the absence of an exchange interaction. This result suggests the
presence of a weak antiferromagnetic spin-exchange interaction in the
complex. Thus, the suggested formula for the copper(II) complex is
[Cu3L(OH)5].
According to Kambe’s approach [30], in trinuclear copper(II) system, first
couple the spins of the two copper(II) ions (SCu1 = SCu2 = 1/2) to give two spin
states of S’ = 1 and 0. Then couple S’ to the third copper(II) ion (SCu3 = 1/2) to
yield three total spin states of ST = 3/2, 1/2, 1/2. Thus for the case of
copper(II) trinuclear complex three different spin configurations can be found:
the high spin quartet, with a total spin of 3/2, and two degenerate doublets,
with total spin 1/2 separated by exchange coupling constants J. The exchange
coupling constant between adjacent metal ions comes from the contributions
of the sum of both ferromagnetic (JF) and antiferromagnetic (JAF) interactions.
The spin−exchange interaction will be antiferromagnetic (J < 0) if S=1/2 it is
the ground state; on the contrary if S=3/2 is the ground state, the
spin−exchange interaction will be ferromagnetic (J > 0) [31]. The observed
magnetic moment value of the tricopper(II) complex at room temperature is a
consequence of population of the doublet (S = 1/2) molecular state.
For the tricopper(II) complex the observed χMT product is 1.007 cm3 mol−1 K
at room temperature which corresponds to an effective magnetic moment of
2.84 µB. The observed χMT value is very close to the theoretical value
expected for three magnetically independent copper(II) ions (χMT = 3
(Nβ2g2/3kT) S(S+ 1)) = 1.24 cm3 mol−1 K, with g =2.1 and S =1/2) [32]. It is
thus possible that the copper(II) centres, are weakly coupled by the magnetic
exchange antiferromagnetic interactions. However, this could not be probed

15
further due to lack of facilities for variable temperature magnetic
measurements.
The X-band EPR spectrum of a powder sample of the Cu3L(OH)5 complex at
room temperature showed a single broad signal with poor resolution of the
hyperfine structure (Figure 2). The analysis of spectrum gives g|| value of 2.21
and g⊥ value of 2.06. The trend g|| > g⊥ > 2.0023 observed for the complex
indicate that the unpaired electron is localized in the dx2−y2 orbital of the Cu(II)
ion and is characteristic of the axial symmetry [33, 34]. The gav value was
calculated to be 2.11. The deviation of gav from that of the free electron
(2.0023) is due to covalence character as per Kivelson and Neiman [35]. The
parameter ‘G’ is calculated by using the expression, i.e., G = (g|| − 2) / (g⊥−2).
The G value of 3.5 indicates moderate exchange interaction between metal
centres in solid complex consistent with Hathaway approach [36]. The powder
EPR spectrum recorded at liquid nitrogen temperature does not show any
change compared to that of room temperature spectrum. The linewidth does
not change very much with decreasing temperature and should be the result of
the dipolar and exchange interactions between the copper ions [37]. In general,
polynuclear Cu(II) complexes give broad EPR peaks and the broadening is
assigned to a dipolar interaction [38]. The greater value of g║ compared to g⊥
proposes a distorted tetrahedral-square pyramidal structure and rules out the
possibility of a trigonal bipyramidal structure which is expected to have g⊥ >
g║. Also, the observed g║ value of less than 2.3 provide evidence for the
covalent character of bonding between Cu(II) ion and the ligand [37, 38].
Electronic spectra
A long-term UV–Vis study was carried out to verify the stability of new
complexes in DMSO solution. Compared with ligand, it is significant to note

16
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 spectra of the Schiff base ligand in 10-3 M DMSO solution showed
three broad bands at 250, 310 and 360 nm. The former two bands are due to
the π−π* transitions within the aromatic ring and remain almost unchanged in
the spectra of metal complexes while the third band is due to the π−π*
transitions within the >C=N– chromophore and shows a bathochromic shift in
the spectra of metal complexes due to the coordination of azomethine nitrogen
to the metal atom [39]. This band shifts slightly to the higher energy region in
the spectra of metal complexes due to the polarization within the >C=N
chromophore caused by the metal-ligand electron interaction.
The spectra of the metal complexes shows that the absorptions around 400–
800 nm is due to ligand to metal charge transfer and d–d transition bands of
the metal in the complexes [40]. The manganese(II) complex shows a very
weak absorption at 380 nm probably due to the coincidence of charge transfer,
d → π*, L → M and intraligand n → π* transitions [41, 42]. The visible
region spectrum of the cobalt(II) complex indicates additional two bands at
4
4
420 and 460 nm attributed to metal-ligand charge and T1g(F) → T2g(F)
transition respectively, suggesting octahedral stereochemistry around the
metal ion [43]. The UV−Vis spectrum of the nickel(II) complex presents two
major absorptions maxima, at 420 and 480 nm due to d−d bands which may be
assigned, considering that the immediate coordination sphere of the metal is
3
3
3
3
Oh symmetry, to the transitions A2g → T1g(P) and A2g → T1g(F)
respectively [44]. The electronic spectrum of trinuclear copper(II) complex
shows a broad band centered at 620 nm (ε = 560.1 cm−1 mol−1) assigned to the
2
2T2g → E2g transition in a distorted tetrahedral/planar geometry around the

17
copper(II) ion. According to the crystal field theory, the d-d transition
absorption band envelope shifts to lower energy as the planar copper(II)
complexes are twisted toward the distorted tetrahedral structure [40]. The
bands at 390 and 470 nm are assigned to charge-transfer, mainly of the L →
Cu type [40].
Coordination sites
The coordination chemistry of transition metal ions with ceftriaxone [45],
cefotaxime [46], cefepime [47] and ceftazidime [48] antibiotics have been
reported. In the present case, the Schiff base 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 Schiff
base would act as an efficient pseudo-encapsulating ligand, with N−thiazole
ring, C=N and carboxylate group with monodentate mode, presumably bound
to the octahedral ions. Thus, the metal ions in the [ML(OAc)(H2O)2]
complexes containing one coordinated acetate anion and two water molecules
at the vertices of an octahedron are hexacoordinate. For the trinuclear
copper(II), the adjacent coppers would be connected by hydroxo bridges and a
bidentate carboxylato group. The assumption that each Schiff base is bound to
three tetrahedral copper ions (with N−thiazole ring, C=N and the carboxylate
group with bidentate mode) seems likely from molecular models.
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 containing cephalosporin Schiff base
complexes have been reported. These studies represent a contribution to future

18
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). Reports on trinuclear and higher nuclearity copper compounds are rather
scarce. In the present case, the magnetic and spectroscopic data are sufficient
to deduce the ocurrence of a magnetically coupled trinuclear unit containing
three copper(II) ions.
Antibacterial activity
A long-term UV–Vis study was carried out to verify the stability of new
complexes in DMSO solution. Compared with ligands, 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.
Preliminary screening for antimicrobial activities of the stock solutions at 20
mg/mL 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 Schiff base ligand HL as well as its metal(II)
complexes were tested on against S. aureus as a Gram-positive and E. coli as a
Gram-negative microorganism and compared to cephalexin and sulphathiazol

19
used as standards. As expected, HL is significantly more toxic against Gram-
positive than Gram-negative bacteria, which may be due to the different cell
wall structure of the tested microorganisms, while the reference compounds
cephalexin and sulphathiazol show almost equal activity against both strains
tested.
The average results are shown in Table 3 where can be appreciated that the
Schiff base ligand and its metal complexes have different behaviour compared
with standard antibiotics against the same bacteria. Thus: (1) The Schiff base
ligand, zinc(II) and copper(II) complexes were found to have higher activity
than the two established drugs against the bacteria strains studied under the
test conditions, showing that they have a good activity as bactericides. (2)
While both cobalt(II) and nickel(II) complexes are inactive, the antibacterial
activity of manganese(II) complex shows to be less toxic than the two
referenced drugs and the Schiff base ligand.
According to Tweedy’s theory [49], chelation could enhance the lipophilic
character of the central metal atom, which subsequently favors its permeation
through the lipid layers of the cell membrane and blocking the metal binding
sites on enzymes of microorganism. In the present case, the in vitro
antibacterial activities demonstrated that copper(II) and zinc(II) complexes
have higher antimicrobial activity in comparison with that of the ligand HL,
but, ligand HL also showed highly biological activity against the tested strains
compared to the manganese(II), cobalt(II) and nickel(II) complexes. Therefore,
antimicrobial activity can also 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

20
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) [50].
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 Schiff base metal complexes may
change the stereochemistry required in solvolytic reactions on an enzyme
surface. The obtained results may highlight that the activity of the compounds
is most probably related to their conformational adaptability, depending on the
size and nature of the metal complexes and the geometrical constraints
induced by intramolecular H bonds. Thus, the bactericidal activity of
cephalexin Schiff base and cephalexin Schiff base metal complexes compared
to cephalexin antibacterial activity 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 complexes
is determined by the amount, nature and kinetic properties of the PBPs.
Conclusion
A Schiff base derived from cephalexin and sulphathiazole and its transition
metal complexes have been prepared. The coordination to metal occurs
through the carboxylate, imine and N-thiazole moieties. The solubility of the
Schiff base antibiotic and its metal complexes in water and common organic
solvents is reduced on complexation. The cephalexin Schiff base, zinc(II) and
copper(II) complexes were found to have higher bactericidal activity than the
uncomplexed cephalexin and the sulphathiazole against the bacteria strains,

21
showing that they have a good activity as bactericides. The cobalt(II) and
nickel(II) complexes show no activity at all against the bacteria while the
manganese(II) complex showed to be less toxic than the two referenced drugs
and the Schiff base ligand. Apart of membrane permeability, antibacterial
activity of cephalexin Schiff base and its metal complexes depends mainly on
the metal ion and the type of microorganism.
Supplementary data
Scheme S1 is available as PDF file from the authors and the journal.
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 and thermoanalytical (TG) results
Found (Calcd.) %

25
H2O M1
M2
Compound
C
N
H
S
[(HL)]
51.6
14.1
4.4
16.2
[(C25H24N6O5S3)]
[MnL(OAc)(H2O)2]
[Mn(C27H30N6O9S3)]
[CoL(OAc)(H2O)2]
[Co(C27H30N6O9S3)]
[NiL(OAc)(H2O)2]
[Ni(C27H30N6O9S3)]
[Cu3L(OH)5]
(51.4) (14.4) (4.1) (16.5)
44.3 11.9 3.9 13.4
(44.2) (11.5) (4.1) (13.1) (4.9) (7.5)
44.3 11.7 3.8 13.2 4.5 8.2
(44.0) (11.4) (4.1) (13.0) (4.9) (8.0)
44.2 11.6 3.9 13.5 5.1 8.4
(44.0) (11.4) (4.1) (13.0) (4.9) (8.0)
34.7 9.9 3.6 11.6
(3.4) (11.2) (4.2) (22.2) (22.2)
4.8 7.5
(7.7)
(7.5)
(8.2)
(8.0)
(8.1)
(8.0)
4.5 22.6 (22.4)
[Cu3(C25H29N6O10S3)] (34.9) (9.8)
[ZnL(OAc)(H2O)2]
[Zn(C27H30N6O9S3)]
43.8
11.7
3.9
13.4
4.4 9.2
(9.0)
(8.8)
(43.6) (11.3) (4.1) (13.0) (4.8) (8.8)
M1 = complexometric analysis, M2 = thermal analysis
Table 2. Main vibrational wavenumbers of the metal complexes (cm-1)
νC=O νC=O νC=N
lactam amide imino
νCOO νCOO
∆ν
Compound
asymm symm

26
Cephalexin
1750
1690
1690
1690
1690
1690
1690
1690
[HL]
1635
1620
1620
1620
1615
1620
[MnL(OAc)(H2O)2]
[CoL(OAc)(H2O)2]
[NiL(OAc)(H2O)2]
[Cu3L(OH)5]
1580
1580
1580
1595
1580
1370
1370
1370
1425
1370
210
210
210
170
210
[ZnL(OAc)(H2O)2]
Table 3 Antibacterial activity
Compound
Zone of inhibition (mm)
S.A
E.C.
Sulphathiazole
Cephalexin
7.0 1.0
8.0 1.0
12.0 1.5
15.0 1.0
7.0 1.0
0.0
10.0 1.0
12.0 1.0
6.0 1.0
0.0
[HL]
[MnL(OAc)(H2O)2]
[CoL(OAc)(H2O)2]
[NiL(OAc)(H2O)2]
[CuL(OH)5]
0.0
0.0
13.0 1.5
15.0 1.0
18.0 1.0
17.0 1.0
[ZnL(OAc)(H2O)2]
a S.A. Staphylococcus aureus ATCC 25923, E.C. Escherichia coli 35939, All
doses were 400 µg /disc. Values are the mean Standard deviation of the
mean.
FIGURE CAPTIONS
Figure 1. Chemical structure of cephalexin

27
Figure 2. EPR spectrum of [Cu3L(OH)5] complex at room temperature
Figure 3. Proposed structure of [ML(OAc)(H2O)2] complexes

28

29
Supplementary data
Scheme S1
SUGGESTED REFEREES
Jim R. Durig
Spectrochimica Acta
durigj@umkc.edu
1
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