Preparation, physicochemical characterization and biological
Received August 26, 2008Accepted March 18, 2009
evaluation of cefodizime metal ion complexes
Sayed H. Audaa,b, Y. Mrestanic, Dietrich H. Niesd, Cornelia Großed
Institutes of aPharmacy, cApplied Dermatopharmacy and dBiology, Martin-Luther-University Halle-Wittenberg,Halle (Saale), Germany, and bDepartment of Pharmaceutics and Industrial Pharmacy, Faculty of Pharmacy,Al-Azhar University, Assiut Branch, Assiut, Egypt
Objectives Cefodizime is a broad spectrum cephalosporin belonging to the thirdgeneration agents. In this study, attention has been paid to the preparation, physicochemicalcharacterization and biological evaluation of new Cu2+, Zn2+, Fe3+, Co2+ and Al3+complexes of cefodizime. Methods The stoichiometrics and the mode of bonding of the complexes were deducedfrom their elemental and metal analysis, electrical conductivity measurements, UV-vis, IRand Raman spectroscopic investigations. Study of the stoichiometry of these complexesreferred to the formation of 1 : 1 ratios of metal to ligand. Antimicrobial activity of thecomplexes was determined using two strains of Gram-positive (Bacillus subtilis andProteus vulgaris) and two strains of Gram-negative (Escherichia coli W3110 andPseudomonas putida) bacteria. The minimal inhibitory concentration was determined asthe lowest concentration inhibiting bacterial growth on solid Luria Bertani medium. Key findings The spectra gave evidence as to the position of binding. In addition, theaqueous solubility of cefodizime was strongly reduced by complexation. Conclusions The antibacterial activity of cefodizime was not affected by complexationwith Al3+ but it was reduced by complexation with the other tested metal ions against thebacteria under study. Keywords antibacterial activity; cefodizime; metal complexes; physicochemicalcharacterization
The cephalosporin antibiotics are semisynthetic antibacterials derived from cephalosporin C,a natural antibiotic produced by the mould Cephalosporium acremonium. The most widelyused system of classification of cephalosporins is by generations. According to theirantimicrobial spectrum of activity, they are classified into four generations. The firstgeneration cephalosporins are very active against Gram-positive cocci. They have limitedactivity against Gram-negative bacteria. The second generation cephalosporins havesomewhat increased activity against Gam-negative microorganisms but are much less activethan the third and fourth generation agents.[3,4]
Cefodizime is a broad-spectrum, third-generation, parenteral cephalosporin which
possesses a prolonged elimination half-life, >3.5 h.[5,6] The chemical structure ofcefodizime disodium salt is shown in Figure 1.
Cephalosporin antibiotics have long been known to behave as relatively efficient
chelating agents. A list of clinically used chelating agents may be found in mostpharmacopoeia, while new chelating agents continue to be sought.[8,9]
The medicinal uses of metal complexes are of increasing clinical and commercial
Correspondence: Reinhard H. H. Neubert, Institute of Pharmacy,
importance. Fluorouracil-oxaliplatin complex is used in Europe and the USA for treatment
of colorectal cancer.[10,11] Ranitidine-bismuth citrate complex is marketed in the USA as
ranitidine bismutrex for the management of peptic ulcer and ulcers associated with
Helicobacter pylori. Gold and ruthenium complexes of chloroquine and clotrimazole
have been investigated for their antiparasitic activity.[13,14] Furthermore, it was found that
some chloroquine complexes are useful even in chloroquine-resistant cases. Iqbal et al.
reported that copper–cephalexin complex exhibited a good anti-inflammatory activity and
their colours the other complexes were not suitable for the
An HP 8452 A (Hewlett-Packard, Waldbronn, Germany) was
used to determine the UV–vis spectra of cefodizime and its
metal complexes in phosphate buffer (pH 7.4).
The chemical structure of cefodizime disodium.
C, H, N and S contents were analysed using an elemen-tal analyser, CHN -932, Leco Corporation (St Joseph,
had more antibacterial effect than the free cephalexin. The
effect of metal ions on drug activity was confirmed byseveral studies.[16,17]
The aim of this work was the preparation and physico-
Metal contents were determined by direct titration against
chemical characterization of cefodizime metal complexes as
standard EDTA (for the Zn2+ complex) or by a back titration
well as the investigation of their antibacterial activity.
technique using standard Zn solution (for the remainingcomplexes) after complete decomposition of the complexes
achieved by boiling with concentrated nitric acid for 10 min.
Water content determinationWater content in the prepared complexes was determined
All chemicals were of reagent grade and were used without
using the Karl-Fischer method using the Karl-Fischer-
any further purification. Cefodizime disodium was obtai-
Titrator AQUA 40.00 instrument, Elektrochemie Halle
ned from Hoechst (Frankfurt, Germany). CuSO4 and
ZnSO4.7H2O were purchased from Sigma-Aldrich GmbH(Seelza, Germany). FeCl3.6H2O was supplied by Roanal
(Budapest, Hungary). Co(NO3)2.6H2O was obtained from
Quantitative solubility of the complexes was determined
Gruessing (Filsum, Germany). NiCl3.7H2O and AlCl3 were
spectrophotometrically in phosphate buffer (pH 7.4) by the
purchased from Germed (Dresden, Germany). Dimethyl
equilibrium solubility method, which employs a saturated
sulfoxide (DMSO) was obtained from Carl Roth GmbH &
solution of the material, obtained by stirring an excess of the
Co. (Karlsruhe, Germany). Ethylene diamine tetraacetate
material in the solvent for a prolonged period until equilibrium
(EDTA), nitric acid, dipotassium hydrogen phosphate and
is achieved. At 270 nm and room temperature the extinction
potassium dihydrogen phosphate were supplied by E. Merck
coefficient was between 6.51 and 6.70 l/mmol/cm.
(Darmstadt, Germany). Methanol, ethanol, diethyl ether,acetonitrile, acetone and dimethylformamide were obtained
from Riedel-de Haen AG (Seelze, Germany).
The buffer solution was prepared by dissolving 1.237 gdipotassium hydrogen phosphate and 0.394 g potassium
dihydrogen phosphate in 600 ml distilled water, and then
Cefodizime disodium (2 mmol) was dissolved in 20 ml
adding distilled water to reach a volume of 1000 ml. The pH
methanol. Metal salts, CuSO4, ZnSO4.7H2O, FeCl3.6H2O,
of the buffer was measured at 25oC using a microprocessor
Co(NO3)2.6H2O and AlCl3 (1 mmol) were separately
pH meter obtained from Testo GmbH and Co. (Lenzkirch,
dissolved in 10 ml methanol. The two solutions were
mixed while stirring for 30 min. Coloured products pre-cipitated and were isolated by filtration. The products were
washed with water, acetone and dimethyl ether, and dried in
Saturated solutions of cefodizime and its complexes were
prepared by adding an excess mass of powder to a constantvolume (2 ml) of phosphate buffer, pH 7.4. Saturated
solutions of cefodizime and its complexes were kept on a
magnetic stirrer in closed glass tubes for 24 h at 25oC. Before
The FTIR spectra of cefodizime and its metal complexes
the analysis all samples were filtered through 0.45-mm
were recorded using a FTIR spectrometer Vertex 70 by
Millipore PTFE filters (Millipore Corp., Bedford, MA, USA).
Bruker Optics (Ettlingen, Germany). The samples were
diluted with an adequate amount of KBr and compressed topellets. The pellets were measured in the range from 370 to
Antimicrobial activity of the complexes was determined
using two strains of Gram-positive (Bacillus subtilis andProteus vulgaris) and two strains of Gram-negative (Escher-
ichia coli W3110 and Pseudomonas putida) bacteria. The
The FT-Raman measurements of cefodizime and its Zn2+ and
minimal inhibitory concentration (MIC) was determined as
Al3+ complexes were acquired by using the RFS 100/S
the lowest concentration inhibiting bacterial growth on solid
spectrometer (Bruker Optics, Karlsruhe, Germany). Due to
Luria Bertani medium (DifcoTM Lennox; Becton Dickinson,
Germany). A preculture for each bacterium was incubated at
30∞C, 250 rev/min, for 17 h. This was then diluted 1 : 400 in
Evidence for complex formation was obtained by comparing
fresh medium and incubated for 2 h at 30∞C, 250 rev/min.
the most characteristic infrared (IR) spectral bands of the free
This 2 h-culture was used for streaking onto plates contain-
cefodizime and its complexes. In general, cephalosporins
ing the complexes (dissolved in DMSO) in different
have three characteristic C=O absorptions for the stretching
concentrations. The plates were incubated at 30∞C for 17 h.
vibrations of the b-lactam ring, the carboxylate and amide I.
Using the paper disc diffusion method on solid Luria
Cephalosporins have a zwitterionic character. Thus, their
Bertani medium another technique was performed. The
spectra of free ligand show bands of antisymmetric (nas) and
complexes were tested at a concentration of 3 mg/ml in
symmetric (ns) vibrations of the carboxylate group. Disap-
DMSO. As a control, DMSO alone was applied to the paper
pearance of one or more of such bands may indicate the
discs. A preculture and a 2 h-culture (as described above)
participation of it or them in metal coordination. The
were done. A 500 ml sample of the 2 h-culture were plated
important IR frequencies of cefodizime and its metal
onto nutrient agar (Carl Roth, Germany), dried and paper
complexes along with their assignments are given in Table 2.
discs with 10 ml of the complex solution were applied. After17 h at 30∞C the inhibition zone was measured.
The main Raman spectra of cefodizime and its Zn2+ and Al3+
The one-way analysis of variance post-hoc test was used for
complexes are listed in Table 3. Due to their colours the rest
the determination of significant differences in the study
of the complexes were not suitable for Raman investigation.
(OriginPro 7.5). The analysis of variance post-hoc Tukey’stest was used to compare all the samples with one other
(antibacterial activity of cefodizime and its complexes
Five-point standard calibration curves of cefodizime and
its complexes in phosphate buffer, pH 7.4, gave linearitycorrelation coefficients ranging from 0.996 to 0.999. The
compounds were determined spectrophotometrically at
lmax = 270 nm using the same solvent medium as the
The Fe3+ and Co2+ complexes were brown and rose,respectively. The Zn2+ and Al3+ complexes were white,
while the Cu2+ complex was green. Microanalytical and
The results of the MIC test are shown in Table 5. Cefodizime
complexometric titration data (Table 1) confirmed the
interferes with cell-wall synthesis of bacteria, leading to lysis
formation of 1 : 1 metal to ligand ratio. Water content
of the infectious microorganisms. In-vitro antibacterial
determined by the Karl-Fischer method showed that the Cu2+
activity of cefodizime and its complexes were tested using
complex was tetrahydrated, the Al3+ complex was penta-
the MIC and the paper disc diffusion method using two
hydrated, whilst the Zn2+, Fe3+, and Co2+ complexes were
strains of Gram-positive (B. subtilis and P. vulgaris) and two
hexahydrated. UV-vis spectra showed no significant differ-
strains of Gram-negative (E. coli W3110 and P. putida)
ence between cefodizime and its metal complexes.
bacteria. Antibacterial activity of cephalosporin metal ion
Elemental analysis data of cefodizime metal complexes
n = 4 (contents in % ± SE). Found (calcd.).
The main FTIR spectra of cefodizime and its metal complexes
The main FT-Raman spectra of cefodizime and its Zn2+ and Al3+ complexes
The aqueous solubility of cefodizime and its complexes in
either as a monodentate or a bidentate ligand, giving changes
in the relative positions of the antisymmetric and symmetricstretching vibrations. The FT-IR spectra of the complexes
gave a separation value of >200 cm-1 suggesting mono-dentate bonding for the carboxylate group.
of cefodizime at 1041 cm-1 which showed no significant
changes in any of the complexes, suggesting that there was
no coordination through this group to the metal ion. In
addition, IR spectra of cefodizime exhibited a band at
Phosphate buffer was pH 7.4. SE, standard error.
1356 cm-1 due to n(C–N) of the b-lactam and thiazole ringnitrogen atom. This band appeared in all studiedcomplexes without further change, indicating that the
complexes depends mainly on the type of cephalosporin
b-lactam and thiazole ring nitrogen atom were not participat-
used, the type of metal ion and the type of microorganism
ing in the bonding. Bands at 3191 and 3310 cm-1 in the
spectra of cefodizime were due to antisymmetric andsymmetric NH stretching of the carbamate NH2 group. These
bands appeared relatively at the same position in the spectraof all the complexes with the exception of the Co2+ complex.
In the FT-IR spectra (Table 2) of cefodizime, a characteristic
This may provide evidence to the participation of this group
band arising from stretching vibrations of the carbonyl group
in the coordination only in the case of the Co2+ complex and
of the b-lactam ring appeared at 1777 cm-1. This band
its inertness towards coordination in the other complexes.
appeared in all studied complexes almost at the same wave
Similar to the IR spectra, Raman spectra of cefodizime
number. This may suggest that the carbonyl oxygen atom
showed a characteristic band arising from stretching vibra-
from the b-lactam ring was not engaged in metal binding.
tions of the carbonyl group of the b-lactam ring at 1764 cm-1
Furthermore, the FT-IR spectra of cefodizime revealed a
(Table 3). This band appeared in all the studied complexes
band at 1659 cm-1 due to stretching vibrations of the amide
almost at the same wave number. It may indicate that the
carbonyl group. This band either vanished or appeared as a
carbonyl oxygen atom from the b-lactam ring was not
shoulder in metal complexes suggesting the coordination of
engaged in metal binding. Raman spectra of cefodizime
revealed a band at 1623 cm-1 due to stretching vibrations of
The bands of antisymmetric (as) and symmetric (s)
the amide carbonyl group. This band was significantly
vibrations of the carboxylate group arose at 1588 and
shifted in metal complexes suggesting the coordination of
1375 cm-1, respectively. In spectra of metal complexes,
metals through this group. The bands of antisymmetric (as)
these two bands were shifted towards the higher wave
and symmetric (s) vibrations of carboxylate groups of
number. This suggested interaction between the metal ions
cefodizime arose at 1585 and 1397 cm-1, respectively.
and the carboxylate group of cefodizime. On the other
Although the shift of these bands in the Raman spectra of
hand, a carboxylate ligand could bind to the metal atom
metal complexes was small, they gave a significant shift in
Antibacterial activity of cefodizime and its complexes against different bacteria*
*Bacteria were incubated (30∞C) in solid Luria Bertani medium containing increasing concentrations of cefodizime or its complexes. Minimalinhibitory concentration, the lowest concentration that inhibited the formation of single colonies. The experiment was performed in triplicate.
the FT-IR spectra. This may suggest interaction between
exhibited significant differences at the 0.05 level only for
metal ions and the carboxylate group of cefodizime.
B. subtilis, B. vulgaris and E. coli with P. putida. The
The solubility values of complexes that were calculated
population means were significantly different (one-way
from these determinations showed significant decrease when
compared with the solubility of cefodizime. These valuesare listed in Table 4. The solubility of cefodizime in the
phosphate buffer (pH 7.4) was 164.41 mg/ml while thesolubility of complexes ranged from 1.963 to 2.58 mg/ml.
Cefodizime formed complexes with different metal ions. The
The decreased solubility of metal complexes may have been
stoichiometric ratio of these complexes was 1 : 1 metal to
attributed to the decrease in their hydrophilicity compared
ligand. Furthermore, the coordination of ligand with metal
with cefodizime. According to this study we could arrange
ions occurred through carboxylate and amide carbonyl groups.
the solubility of complexes in phosphate buffer (pH 7.4) in the
In addition, the aqueous solubility of the cefodizime was
following order, with the highest solubility for the cefodizime-
strongly affected by complexation. With the exception of the
Co2+ complex and the least solubility for the cefodizime–Fe3+
cefodizime-Al complex, all tested metal ion complexes were
complex: cefodizime-Co2+ > cefodizime-Zn2+ > cefodizime-
less active than cefodizime against the bacteria under study.
Al3+ > cefodizime-Cu2+> cefodizime-Fe3+.
Except for the cefodizime-Al3+ complex, all tested metal
ion complexes were shown to be less active than cefodizime
against the bacteria investigated. Cefodizime-Al3+ complex
exhibited an antibacterial activity similar to the parent drugagainst B. subtilis, P. vulgaris and E. coli and 1.16-times less
The Author(s) declare(s) that they have no conflicts of
than cefodizime against P. putida. The antimicrobial activity
of the other complexes ranged from 1.3 to 5.8-times less
active than the pure antibiotic. Cefodizime-Zn2+ andcefodizime-Fe3+ complexes had a middling activity, while
This research received no specific grant from any funding
the cefodizime-Cu2+ and cefodizime-Co2+ complexes had the
agency in the public, commercial or not-for-profit sectors.
On the other hand, the inhibition zone of cefodizime and its
metal ion complexes against the same bacteria was measuredusing the paper disc diffusion method on solid Luria Bertani
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Q1: Please check whether it is (upsilon) or n (nu) symbol to be used for symmetric vibrations throughout the article.
Blake Lapthorn Tarlo Lyons' Professional Regulatory Law update – July 2008 Welcome to this month’s edition of the Blake Lapthorn Tarlo Lyons’ Professional Regulatory Law update – our at-a-glance guide to the important case law and news in the Professional Regulatory field. This month's ebulletin sees the introduction of two new areas of commentary: 'in the news' and 'hot topics'
Gabriel Maislos, DPM, FACFAS BIOGRAPHICAL DATA: Date of Birth: June 24, 1974 Place of Birth: Detroit, MI HIGH SCHOOL EDUCATION Kinkaid High School Houston, TX 1988-1992 UNDERGRADUATE EDUCATION: Tufts University Medford, MA Bachelor of Science, Biology 1992-1996 MEDICAL EDUCATION: Dr. William M. Scholl College of Podiatric Chicago, IL Medicine