Synthesis, characterization and antimicrobial activity of copper(II) complexes with hydrazone derived from 3-hydroxy-5-(hydroxymethyl)-2-methylpyridine-4-carbaldehyde

Article abstract of DOI:10.1016/j.poly.2015.10.035

Pyridoxal isonicotinoyl hydrazone (HL) and its copper(II) complexes: [CuLCl(H₂O)₂] (1), [CuL(OAc)](H₂O) (2), [CuL(OAcac)] (3), [CuL(NO₃)](H₂O) (4), [CuL₂](H₂O) (5) were synthesized and characterized. The ligand has been obtained by condensation of 3-hydroxy-5-(hydroxymethyl)-2-methylpyridine-4-carbaldehyde (pyridoxal) hydrochloride with isonicotinoyl hydrazide. The characterization of the formed compounds was done by ¹H NMR, ¹³C NMR, UV-Vis, IR and EPR spectroscopy as well by thermal studies and elemental analysis. The crystal structure of HL has been determined by single crystal X-ray diffraction studies. The microbiological effect of the ligand and all five complexes against both Gram-positive and Gram-negative strains, Staphylococcus aureus ATTC 6538, Bacillus cereus ATCC 14579, Escherichia coli W3110 and Pseudomonas aeruginosa ATCC 9027 was tested.

Full text of DOI:10.1016/j.poly.2015.10.035

Accepted Manuscript
Synthesis, characterisation and antimicrobial activity of copper(II) complexes
with hydrazone derived from 3-hydroxy-5-(hydroxymethyl)-2-methylpyri-
dine-4-carbaldehyde
Reka-Ștefana Mezey, István Máthé, Sergiu Shova, Maria-Nicoleta Grecu, Tudor
Roșu
PII:
S0277-5387(15)00616-6
DOI:
http://dx.doi.org/10.1016/j.poly.2015.10.035
POLY 11620
Reference:
Polyhedron
To appear in:
Received Date:
Accepted Date:
12 July 2015
22 October 2015
Please cite this article as: R. Mezey, I. Máthé, S. Shova, M-N. Grecu, T. Roșu, Synthesis, characterisation and
antimicrobial activity of copper(II) complexes with hydrazone derived from 3-hydroxy-5-(hydroxymethyl)-2-
methylpyridine-4-carbaldehyde, Polyhedron (2015), doi: http://dx.doi.org/10.1016/j.poly.2015.10.035
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers
we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and
review of the resulting proof before it is published in its final form. Please note that during the production process
errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Synthesis, characterisation and antimicrobial activity of copper(II) complexes with hydrazone derived from 3-
hydroxy-5-(hydroxymethyl)-2-methylpyridine-4-carbaldehyde
Reka-Ștefana Mezeya*, István Máthéb, Sergiu Shovac, Maria-Nicoleta Grecud, Tudor Roșua
aDepartment of Inorganic Chemistry, Faculty of Chemistry, University of Bucharest, 23 Dumbrava Rosie, 050107
Bucharest, Romania
b Department of Bioengineering, Faculty of Technical and Social Sciences, Sapientia Hungarian University of
Transylvania, Libertatii Square 1, 530104, Miercurea Ciuc, Romania
cInstitute of Macromolecular Chemistry ”Petru Poni”, 41A Grigore Ghica Voda, 400786 Iasi, Romania
dNational Institute of Materials Physics, 077125 Măgurele-Ilfov, Romania
Keywords: copper(II) complexes, pyridoxal isonicotinoyl hydrazone, crystal structure, antimicrobial activity
ABSTRACT
Pyridoxal isonicotinoyl hydrazone (HL) and its copper(II) complexes: [CuLCl(H2O)2] (1), [CuL(OAc)](H2O) (2),
[CuL(OAcac)] (3), [CuL(NO3)](H2O) (4), [CuL2](H2O) (5) were synthesized and characterized. The ligand has been
obtained by condensation of 3-hydroxy-5-(hydroxymethyl)-2-methylpyridine-4-carbaldehyde (pyridoxal)
hydrochloride with isonicotinoyl hydrazide.
The characterization of the formed compounds was done by 1H NMR, 13C NMR, UV–Vis, IR and EPR spectroscopy
as well by thermal studies and elemental analysis. The crystal structure of HL has been determined by single crystal
X-ray diffraction studies. The microbiological effect of the ligand and all five complexes against both Gram-positive
and Gram-negative strains, Staphylococcus aureus ATTC 6538, Bacillus cereus ATCC 14579, Escherichia coli
W3110 and Pseudomonas aeruginosa ATCC 9027 was tested.
1. Introduction
During the past decay significant researches concerning the metal complexes of hydrazone-type ligands have been
undertaken. Especially the hydrazones containing a pyridine ring moiety are of great interest due to their extended
aplicability in biology and pharmacology as anticonvulsant, antimicrobial, antitubercular, antitumor and analgesic
agents [1-5]. Due to their coordination potential to different metal ions and their remarkable versatility, such
compounds atracted the interest of the inorganic and bioinorganic chemists as well. Different metal complexes of
hydrazone-type ligands were synthesized and proven to possess a wide range of application in catalysis, optic and
luminecense field and of course in medicine [6-11].
One of the most interesting class of complexes with hydrazone ligands is the one derived from isonicotinic acid
hydrazide. Isonicotinoyl hydrazide, also known as isoniazid, have been largely used as an antimocrobial agent,
especially in the treatment of tuberculosys caused by the bacteria Mycobacterium tuberculosis [12a, 12b, 12c]. Also,
the hydrazones obtained by condensation of isoniazid with various carbonilic compounds have been proven to
exhibit antimicrobial effect which in some case was superior to the one of the original hydrazide [13-15]. Further,

the resulting hydrazone can be used as ligand in various complexes structures, the structure of the final coordination
compound depending mainly on the number and position of the donor atoms in the molecule. Moreover, some
Ni(II), Co(II), Mn (II) complexes have been shown to exhibit enhaced microbiological activity compared to the
original ligands [9,11a].
3-hydroxy-5-(hydroxymethyl)-2-methylpyridine-4-carbaldehyde (pyridoxal) is one of the five naturally inter-
convertible forms of vitamin B6 [16]. The reason for choosing pyridoxal as derivative agent of isoniazid is linked to
its recognized physiological effect as agent influencing the aminoacids metabolism, antioxidant compound and
coenzyme involved in numerous metabolic processes [17-20]. Moreover, it was reported that the introduction of the
pyridoxal moiety in an azomethine compounds represents a major factor in the synthesis of therapeutic agents [21-
23]. On the other hand, pyridoxal isonicotinoyl hydrazone is an interesting reagent for the design of metal
complexes, acting as a tridentat ligand. Pyridoxal isonicotinoyl hydrazone was synthesized and reported in previous
literature materials [24] but in its unsolvated form.
This paper is a continuation of our previous research and it describes the synthesis and characterization of new
complexes of Cu(II) with the hydrazone (Scheme. 1) obtained by the condensation of 3-hydroxy-5-
(hydroxymethyl)-2-methylpyridine-4-carbaldehyde with isonicotinoyl hydrazide. Additionally, the crystal structure
of the ligand has been studied by X-ray diffraction. The ligand and complexes were tested in vitro for their
antibacterial activity against both Gram-positive and Gram-negative strains like Staphylococcus aureus ATTC
6538, Bacillus cereus ATCC 14579, Escherichia coli W3110 and Pseudomonas aeruginosa ATCC 9027.
2. Experimental
2.1. Materials
Pyridoxal hydrochloride (Sigma) and isonicotinoyl hydrazide (Sigma) were used as purchased without any pre-
synthesis purification step. The copper(II) salts CuCl2·2H2O, Cu(OAc)2·H2O, Cu(acac)2, Cu(NO3)2·3H2O,
CuSO4·5H2O were supplied by Merck and used as such. The solvents were purified before synthesis by standard
procedure. The pyridine and dimethylsulfoxide (DMSO) used during the antimicrobian screening tests were
sterilized before use.
2.2. Synthesis of ligand pyridoxal isonicotinoyl hydrazone (HL)
The ligand HL was prepared by refluxing an equimolar mixture of pyridoxal hydrochloride and isonicotinoyl
hydrazide. The amount of pyridoxal hydrochloride (0.204 g, 1 mmol) was dissolved at room temperature in
methanol (10 mL). Paralelly, the corresponding amount of isonicotinoyl hydrazide (0.137 g, 1 mmol) was dissolved
in the same solvent (10 mL). The pale yellow mixture formed by the two methanolic solutions was refluxed for one
hour. Afterwards, it was left to cool at room temperature. The resulting orange precipitate was filtered, washed with
alcohol and dried in vacuum. In order to obtain the orange single crystals suitable for X-ray diffraction, a certain
amount of solid ligand was re-dissolved in a mixture methanol:DMSO (3:1, v/v). After the slow evaporation of the
obtained solution the single crystals were isolated and filtered.

Yield: 63%; M. wt: 322.5 g/mol; Anal. Calc. for C14H15N4O3Cl: C, 52.09; H, 4.65; N, 17.36; Cl, 11.00%. Found: C,
52.85, H, 4.30, N, 17.08, Cl, 10.57%. Main IR peaks (KBr, cm-1): The condensation of the two reagents with the
formation of the hydrazone HL is confirmed by the appearance in the IR spectrum of the signal at 1605 cm-1. This
signal is typical for the vibration of the azomethine bond [25,26]; ν(OH) 3595, 3247; ν(C=O)1685; ν(C=N) 1605;
1
ν(Ar-OH) 1267; ν(N-NH) 1093; ν(CH2-OH) 1029. H NMR (DMSO-d6, δ (ppm), J (Hz)): The formation of the
azomethine group is emphasized by the presence of the signal at δ 9.18 ppm in the 1H NMR spectrum. 2.63 (s, 3H,
CH3); 4.77 (s, 2H, CH2-OH); 7.85 (d, 2H, H-2, H-6); 8.03 (d, 2H, H-3, H-5); 8.17 (s, 1H, OH); 8.86 (s, 1H, NH);
9.18 (s, 1H, CH=N). 13C NMR (DMSO-d6, δ (ppm), J (Hz)): The presence of the imine fragment in the molecule of
hydrazone HL is confirmed also by the signal registered at δ 149.94 ppm in the 13C NMR spectrum. 38.66 (CH3);
58.19 (C5); 122.00 (C6); 126.25 (C17, C21); 130.19 (C18, C20); 136.69 (C16); 149.94 (HC=N); 152.82 (C3);
161.68 (C=O).
Please insert Scheme 1 here
2.3. Synthesis of the complexes
All the complexes have been synthesized by the direction reaction between ligand HL and each of the copper salts.
2.3.1. Synthesis of complex [CuLCl(H2O)2] (1)
To a solution of ligand pyridoxal isonycotinoyl hydrazone (0.286 g, 1 mmol) in methanol (25 mL) was added a
solution of CuCl2·2H2O (0.17 g, 1 mmol) in methanol (15 mL). The pH of the solution was adjusted by adding
dropwise an alcoholic solution of NaOH. The mixture was refluxed for 3 hours. The dark green solid obtained was
filtered, washed with alcohol and dried in vacuum. The complex is soluble in pyridine, slightly soluble in DMSO
and insoluble in methanol and ethanol. Yield: 71%; M.wt.: 420.5 g/mol. Anal. calc. for CuC14H17N4O5Cl: C, 39.95;
H, 4.04; N, 13.31; Cu, 15.21%. Found: C, 39.36; H, 4.32; N, 13.08; Cu, 15.36%. Main IR peaks (KBr, cm-1): 3418,
ν(OH); 1611, ν(C=O); 1583, ν(C=N); 1217, ν(Ar-OH); 1095, ν(N-NH); 1029, ν(CH2-OH). µeff= 1.7.
2.3.2. Synthesis of complex [CuL(OAc)](H2O) (2)
A solution of Cu(OAc)2·H2O (0.199 g, 1 mmol) in methanol (10 mL) was added to a warm solution of HL (0.286 g,
1 mmol) in methanol (10 mL). The mixture was refluxed for 3-4 hours. A solid brown precipitate was isolated and
dried. Complex 2 is soluble in pyridine, slightly soluble in DMSO and DMF and insoluble in alcohols. Yield: 78%;
M.wt.: 427 g/mol. Anal. calc. for CuC16H18N4O6: C, 44.96; H, 4.44; N, 13.11; Cu, 14.98%. Found: C, 45.31; H,
4.01; N, 12.25; Cu, 14.10%. Main IR peaks (KBr, cm-1): 3349, ν(OH); 1606, ν(C=O); 1575, ν(C=N); 1207, ν(Ar-
OH); 1097, ν(N-NH); 1030, ν(CH2-OH). µeff=1.5.
2.3.3. Synthesis of complex [CuL(OAcac)] (3)
This complex was synthesized by refluxing a 1:1 mixture of ligand and copper(II) acetylacetonate. The Cu(acac)2
(0.261 g, 1 mmol) was dissolved in a small volume of chloroform (5 mL) and resulting solution was added to a

methanolic solution of corresponding amount of ligand HL (0.286 g, 1 mmol). The mixture was heated at 50⁰C for 5
hours. Once the solution was left to cool at room temperature a brown solid compound was isolated, washed with a
chloroform:methanol mixture and dried. The obtained complex is soluble in pyridine and slightly soluble in DMSO.
Yield: 60%; M.wt.: 448 g/mol. Anal. calc. for CuC19H20N4O5: C, 50.89; H, 4.46; N, 12.50; Cu, 14.28%. Found: C,
51.25; H, 4.18; N, 11.37; Cu, 13.77%. Main IR peaks (KBr, cm-1): 3324, ν(OH); 1606, ν(C=O); 1560, ν(C=N);
1204, ν(Ar-OH); 1092, ν(N-NH); 1025, ν(CH2-OH). µeff=1.9.
2.3.4. Synthesis of complex [CuL(NO3)](H2O) (4)
Complex 4 was prepared in a similar fashion as complex 2. To a warm solution of ligand (0.286 g, 1 mmol)
dissolved in methanol was slowly added a solution of Cu(NO3)2·3H2O (0.241 g, 1 mmol). The resulting solution was
heated under continous stirring for 3 hours. A brown solid was obtained. Finally, it was filtered, washed with
alcohol and dried. Complex 4 is soluble in pyridine and insoluble in alcohols, acetonitril or acetone. Yield: 85%; M.
wt.: 430 g/mol. Anal. calc. for CuC14H15N5O7: C, 39.06; H, 3.48; N, 16.27; Cu, 14.88%. Found: C, 39.55; H, 3.02;
N, 16.20; Cu, 14.49%. Main IR peaks (KBr, cm-1): 3349, ν(OH); 1606, ν(C=O); 1575, ν(C=N); 1207, ν(Ar-OH);
1089, ν(N-NH); 1030, ν(CH2-OH); 1090, ν(O-NO2); 1280, 1430, ν(NO3). µeff=1.6.
2.3.5. Synthesis of complex [CuL2](H2O) (5)
Complex 5 was prepared by stirring at reflux temperature a 2:1 mixture of ligand HL (0.286 g, 1 mmol) and
CuSO4·5H2O (0.249 g, 1 mmol). After one hour a solid green compound was obtained. The mixture was stirred for
another 2 hours in order to increase the yield of complex formation. Afterwards, the obtained compound was filtered
and dried. The novel complex is soluble in pyridine. Complex 5 is soluble in pyridine and insoluble in alcohols.
Yield: 65%; M.wt.: 654. Anal. calc. for CuC28H28N8O7: C, 51.37; H, 4.28; N, 17.12; Cu, 9.78%. Found: C, 51.74; H,
3.96; N, 16.94; Cu, 9.63%. Main IR peaks (KBr, cm-1): 3349, ν(OH); 1606, ν(C=O); 1575, ν(C=N); 1207, ν(Ar-
OH); 1100, ν(N-NH); 1030, ν(CH2-OH). µeff=1.8.
Please insert Scheme 2 here
2.4. Physical measurements
The percentages of C, H and N were determined by elemental analysis using a Carlo-Erba microdosimeter. The
amount of copper(II) was determined by atomic absorbtion spectroscopy. The IR spectra were measured by using
KBr pelletizing technique and registered with a BioRad FTS 135 spectrophotometer. The NMR results were
obtained using a solution of ligand HL dissolved in DMSO; the spectra was recorded using a Bruker DRX 400
spectrometer. The electronic spectra were registered with a Jasco V-670 spectrophotometer using samples optically
diluted with MgO. The X-band (9.2-9.6 GHz) EPR spectra were performed at room temperature on weighted
microcrystalline powder samples, in the same conditions of modulation, amplitude and microwave power, using a
CMS 4800 Adani EPR spectrometer (details in [27]). Spectra simulations were done with Bruker Simfonia and SIM

software. The magnetic susceptibility measurements were performed at 20⁰C on polycrystalline powders of all five
complexes using a home-made Faraday magnetic balance. The thermal analysis studies were made by using certain
amounts of samples in polycrystalline state. The analysis were conducted in nitrogen atmosphere by gradually
increasing the temperature between 30⁰C-900⁰C with 10⁰C/minute. The analysis were conducted with a Perkin
Elmer STA-600 thermal analyzer.
2.5. X-ray diffraction
Crystallographic measurements for the ligand HL were carried out with an Oxford-Diffraction XCALIBUR E CCD
diffractometer equipped with graphite-monochromated Mo-Kα radiation. Single crystals were positioned at 40 mm
from the detector and 374 frames were measured each for 10 s over 1° scan width. The unit cell determination and
data integration were carried out using the CrysAlis package of Oxford Diffraction [28a]. The structures were solved
by direct methods using Olex2 [28b] software with the SHELXS structure solution program and refined by full-
matrix least-squares method on F² with SHELXL-97 [28c]. Atomic displacements for non-hydrogen atoms were
refined using an anisotropic model. Hydrogen atoms were placed in fixed, idealized positions and refined as rigidly
bonded to the corresponding carbon atoms. Hydrogen atoms for NH and OH groups have been placed by Fourier
Difference, accounting for the hybridization and the hydrogen bonds parameters. The molecular plots were obtained
using the Olex2 program. The main crystallographic data data together with refinement details are summarized in
Table 1, while the bond distances and angles in table 1S. The crystallographic measurements for complexes 1-5
were carried out on polycrystalline powders with a X-ray diffractometer Empyrean which uses a characteristic X-
rays bean Cu Kα1 (λ = 1.540598). The diffractometer was equipped with a hybrid monochromator 2xGe(220) for
Copper and a detector PIXcel3D. The analysis was made by applying the Bragg-Brentano geometry (“theta-2-
theta”) for angles 2θ between 5⁰-60⁰, with an increasing step of 0,04⁰ and acquisition time per step of 500 seconds.
A small amount of complex sample was positioned on a silica monocrystal support. The complex polycrystalline
sample was pressed on the support in order to obtain a thin, flat layer. The crystal system was identified by using the
McMaille method, while the space group was determined the LeBail method, followed by structure refinement. The
selection of the parameters for structures refinement was based on the “goodness of fit” (GOF) index. After the
identification of crystal system and space group one additional step was to realize the fit profile by using the Pawley
method. For the identification of all parameters it was considered that the each sample contains only one
mineralogical phase.
Please insert Table 1 here
2.6. Antimicrobial activity
The ligand HL and the corresponding Cu(II) complexes were tested in respect to their antimicrobial potential. We
assessed the inhibition capacity of the ligand, solvents (pyridine and dimethylsulfoxide) and the five newly
synthesized Cu(II) hydrazone complex compounds against Escherichia coli W3110, Pseudomonas aeruginosa

ATCC 9027 (Gram-positive) and Bacillus cereus ATCC 14579, Staphylococcus aureus ATTC 6538 (Gram-negative)
bacterial strains. As control drug two aminoglycoside antibiotics (streptomycin sulfate salt, kanamycin sulfate) and
one beta-lactam antibiotic (ampicillin sodium salt) were used (all from Sigma- Aldrich). The minimal inhibitory
concentrations (MIC) were determined by Mueller-Hinton Broth (MHB) microdilution method proposed by
Wiegand et al. 2008 [29]. The MIC of ligand HL and complexes 1-5 were compared against the ones of the
antibiotics used as standards. In order to determine the MIC (µg/mL) for ligand and complexes a stock solution with
concentration 2048 µg/mL was prepared by dissolving the appropriate amount of compound in a pyridine:DMSO
(1:5, v/v) mixture. Further, by diluting the stock solution with the same solvents mixture was obtained a series of
subsequent solutions with gradually decreasing concentrations: 1024, 512, 256, 128, 64, 32, 16, 8, 4, 2, 1, 0.5, 0.25,
0.125, 0.063, 0.03, 0.015 µg/mL. Other materials used for the quantitative screening were 96-well plates,
suspension of microorganism (0.5 McFarland) and Muller-Hinton broth. After incubation at 37⁰C for 24-48 hours of
the inoculated plates, the MIC of all complexes against each microbial strain was determined by macroscopic
observation.
3. Results and discussion
The hydrazone ligand HL was prepared by a condensation method which was used in our working group for the
synthesis of other classes of compounds containing the azomethine group as well [30, 30a].
The new compound was obtained with a good yield and its molecular formula and structure were proven based on
1
the results of the IR, H and 13C RMN spectroscopy and elemental analysis. Moreover, based on the X-ray
diffraction results the firstly reported structure of this hydrazone was obtained.
All the complexes were prepared by the direct reaction between the ligand HL and each of the copper(II) salts. Dark
green and brown powders were obtained in significant yield (higher than 60%). The structures of the complexes
were influenced by the ligand coordination capacity to the metal ion, the nature of the anionic groups and the
working conditions. The results of the elemental analysis, spectral data and decomposition pattern confirm the
predicted molecular formula. For complex 1 the formula [CuLCl(H2O)2] is proposed. Complexes 2 and 4 share a
-
common general formula [CuLX](H2O), where X=OAc- (2) and X=NO3 (4). In case of complex 3 the IR spectra
indicates the coordination of an acetylacetonate group to the Cu(II) ion confirming the formula [CuL(acac)].
Complex 5 is different than all the previous compounds of this class by having two molecules of ligand coordinated
to a single copper ion – [CuCl2](H2O).
3.1. Crystal structure of ligand HL
As can be seen from Fig. 1, the crystal structure of the ligand comprises chlorhydrite units H4LCl and DMSO as
solvate molecules in 1:2 ratio. All the components of the structure are strogly associated via numerous O-H···Cl, O-
H···N, O-H···Cl, N-H···Cl, N-H···O and C-H···O hydrogen bonding. The parameters of the hydrogen bonds are
summarized in Table 2. Beside the above mentioned hydrogen bonding π-π stacking interactions are also observed
in the packing of H4LCl. It is realized through the Cg1 and Cg1΄ centroids of two centro-symmetrically related

C1/C2/C3/C4/N1/C5 rings, separated at 4.039 Å. The crystal structure motif of H4LCl is characterized as the
parallel packing of the colummn-like architecture, as shown in Fig. 2.
Please insert Fig. 1 here
Please insert Fig. 2 here
Please insert Table 2 here
3.2. X-ray powder diffraction
The elemental cell parameters, crystallin system and spatial group for complexes 1-5 are presented in Table 2S. The
crystallographic data indicate the same triclinic crystallin system for complexes 1 and 5 while in case of complexes
2 and 4 the monoclinic system is identified. Complex 4 is different than all the other compounds presenting a
hexagonal crystallin system. Among the serie of complexes it can be observed similarities between the crystallin
system and complexes geometries proposed based on the results of other techniques.
3.3. Infrared spectra and coordination mode
The assignment of the bands identified in the infrared spectra of the ligand and all complexes have been made based
on the relevant literature materials [31]. In case of the complexes, the comparision of their infrared spectra with the
spectra of ligand provided valuable information about the coordination mode to the copper ion. The main bands
identified in the infrared spectra of the ligand and complexes are summarized in the Experimental section.
The condensation of the two reagents with the formation of the hydrazone ligand is proven by the presence of the
band at 1605 cm-1 in the spectra of ligand. This signal is specific to the imine streching frequency ν(C=N) [5, 11a,
25]. Moreover, the absence of the bands characteristic to the carbonyl and amino groups indicates once again the
formation of the proposed hydrazone. The presence of the hydroxyl group is emphasized by the sharp signals at
1267 cm-1 (ν(Ar-OH)) and 1024 cm-1 (ν(CH2-OH)). Additionally, the infrared spectra exhibits a broad signal
between 3250-3450 cm-1 which is assigned to a hydrogen intramolecular bond (N···OH) [10]. Due to the presence
of –C=N-NH-C=O group the ligand HL is potentially subject to keto-enol tautomerism (Scheme 3). The region
between 1270-1690 cm-1 in the IR spectra of the free ligand provides important information about the tautomeric
form stabilized by the ligand molecule. The presence of the strong signal characteristic to the vibration of the C=O
group and the lack of band assigned to ν(=C-OH) in the spectra of the free ligand indicates that the hydrazone HL
stabilizes the keto tautomeric form. The signal recorded at 1548 cm-1 is the result of the coupled effect of the
streching vibration mode of the N-C=O group and the NH bond deformation. The deformation mode δ(NH) is also
connected to the weak signal at 1315 cm-1 [32]. These observations are confirmed also by the presence of the signal
characteristic to the C=O signal in the 13C NMR spectra. Moreover, the X-ray diffraction analysis support the keto
form proposed based on the spectral analysis.
A similar group of signals is emphasized in the infrared spectra of the copper complexes derived from the hydrazone
HL. The signal specific to the vibration of the C=O group is clearly visible in the spectra of all complexes between

1606-1611 cm-1 indicating that the structure of the ligand does not undergo any structural change after coordinating
to the metal ion. On the other hand, the band assigned to the carbonilic group is shifted to lower wavenumbers in the
infrared spectra of all complexes compared to the spectra of free ligand. Such behaviour indicates that the oxygen
atom of the C=O group is involved in the coordination. The band associated to the streching vibration of the C=N
group appears in the region 1546-1583 cm-1 in the spectra of complexes 1-5. The negative shift by 22-59 cm-1 in the
spectra of the complexes compared to the spectra of ligand suggest a decreasement of the streching force constant
due to the coordination via the nitrogen atom of the azomethine bond [25]. In a similar manner, the band specific to
the deformation vibration of Ar-OH group is moved from 1267 cm-1 in the spectra of ligand to 1230-1204 cm-1 in
the spectra of complexes indicating that a third position in the coordination sphere is occupied by the oxygen atom
of the deprotonated hydroxy group. The band associated to the second hydroxyl group (-CH2-OH) does not
undergoes any change in respect to its position in the spectra of complexes compared to the spectra of HL thus
suggesting that this fragment of the molecule is not involved into coordination. Therefore, it can be concluded that
the hydrazone HL coordinated to the copper(II) ion in the keto form as a monobasic tridentate ligand, generating a
five-atoms chelate ring and a six-atoms one. This finding is in accord with the behaviour of previously reported
similar mononuclear hydrazone derived complexes [10, 11a, 33]. In case of complex 2 the vibrational spectra shows
two bands at 1601 cm-1 and 1411 cm-1. The position and the difference between these characteristic frequecies of
these two signals suggest that the acetate anion participates to coordination as monodentat agent [31].The IR spectra
-
of complex 4 presents two additional signals at 1280 cm-1 and 1430 cm-1 associated to NO3 group. The difference
between the frequencies of these signals ∆ν=150 cm-1 indicates a monodentate coordination manner of the nitro
group [31,34]. A different pattern can be observed for complexes 1 and 5 in case of which the IR spectra do not
show the specific signals associated to the coordinated chloride ion and sulphate ion, respectively. This is an
indication that none of the mentioned anions participate into the coordination sphere of complexes 1 and 5.
Please insert Scheme 3 here
3.4. Electronic spectra and magnetic susceptibility
The electronic spectroscopy coupled with the results of magnetic susceptibility analysis was an effective method for
the determination of the geometry of complexes. The electronic absorbtion spectra of ligand and complexes were
recorded on policrystalline samples diluted with an optically inactive matrix (MgO). The electronic spectra of the
hydrazone HL exhibits one intense absorbtion band at 26.300 cm-1 with an additional shoulder at 22.200 cm-1. These
signals are assigned to the n→π* and π→π* transitions of the azomethine cromophore [35]. The broad and lower
intensity band recorded in the region of the spectra below 38.000 cm-1 is specific to the transition associated to the
pyridine moiety. In the spectra of the complexes the bands generated by the intraligand charge transfer are shifted to
lower frequencies indicating the coordination of the hydrazone via the nitrogen atom of the azomethine bond. In
addition, the complexes spectra display supplementary absorbtion bands specific to the d-d transitions. Complete
data are provided in Table 3. The magnetic moments recorded at 20⁰C are characteristic to the system containing
one unpaired spin and suggest that all complexes present paramagnetic properties (Table 3). This is also an evidence

of the mononuclear structure of all five complexes. The spectra of complexes 2 and 4 have a very similar layout,
displaying a very wide band with absorbtion maxima between 15.100-15.800 cm-1. Due to the strong dynamic Jahn-
Teller effect the geometry of these complexes is significantly distorded [36a]. Consequently, the bands specific to
2
2
the d-d transitions from the ground state B1g (dx -y2) can be widened and overlapped resulting in a single broad
signal. For these complexes the magnetic moments were determined between 1.5 – 1.6 MB. This is in accord with
the previously reported and expected values for Cu(II) complexes in a distorted square-planar environment [36b].
The electronic spectra of complex 3 presents two well-defined bands with absorbtion maxima at 9.850 cm-1 and
2
2
15.130 cm-1. These signals can be assigned to the d-d transitions 2B2→2E (dxy→dxz, yz) and 2B2→B1,2A1 (dxy→dx -y ,
2) which are tipical for tetrahedral complexes [37]. In addition, the magnetic moment value (1.9 MB) confirms the
z
proposed geometry an is also linked with the lower simmetry of the complex molecule. Complexes 1 and 5 are quite
different than the others in respect to the layout of electronic spectra and magnetic susceptibility. The band with a
2
2
maxima around 11.000 cm-1 is assigned to the transition B1g→2A1g (dx -y2→dz2). The next theoretically possible
2
2 2
transition B1g→B2g (dx -y →dxy) is not visible, most probably being embedded by the wider band at lower
frequency. The cause for this overlapping is connected with the very close energies of these two transitions and also
with the Jahn-Teller distorsion. The second observed band, higher in intensity, recorded around 15.000 cm-1 is
2
tipically associated to the transition 2B1g→2Eg (dx -y2→dxz,yz). These data suggest a distorted octahedral geometry of
these complexes and is in accord with previously reported observation for similar compounds [38]. Moreover, the
magnetic moment values measured for these complexes (1.7 MB and 1.8 MB) are very similar to the one of 1.73
MB expected for a single unpaired spin confirming the octahedral geometry [39]. The proposed structures are
supported by the EPR spectroscopy and thermal analysis as well.
Please insert Table 3 here
3.5. EPR spectra
To correlate the above data with the local symmetry of copper in the complex lattice, EPR spectroscopy was used as
a method-of-choice for its remarkable sensitivity and selectivity [40]. The room temperature EPR spectra recorded
on the complexes 1 – 5 are presented in Fig. 3a. The main EPR feature of these spectra is their anisotropic line shape
(of orthorhombic symmetry). The complexes 1 and 5 show an axial-like shape spectrum while the complexes 2, 3
and 4 show only a single asymmetric broad line with different line width of 15.5 mT, 13.0 mT, and 14.1 mT,
respectively. The main EPR parameters, g-tensor, were obtained by simulation of the experimental spectra. Figure
3b shows the EPR simulation spectra and their characterstic parameters are given in Table 4. It can be observed that
the room temperature spectra do not reflect the static local configuration around Cu ion. It is well known the
fluxionality of Cu complexes [41]. Such molecular motions can mediate the anisotropic parts of EPR spectra. Only
low temperature measurements, at which molecular motions become frozen, can provide correct interaction tensors
reflecting the static local configurations. A special comment can be although made for complexes 1 and 5. Their
EPR spectra (with g2 ≈ g3) correspond approximately to an orthorhombic local symmetry. That suggests a more rigid

structure of the complexes, a conclusion in accordance with the proposed distorted octahedral coordination. Besides,
the g-values of complexes 2 and 4 could imply a distorted square-planar geometry.
Please insert Fig. 3 here
Please insert Table 4 here
3.6. Thermal studies
In order to obtain further information about the thermal stability as well as the decomposition model we investigated
the thermal behaviour of all complexes. For this purpose the TG, DTG and DTA curves were recorded by applying
creadually increasing temperature to the policrystalline samples of complexes. This technique have been proven
very helpful especially in the determination of the small molecules presence. The results of the thermal analysis are
presented in Table 5.
The general decomposition pattern describes few steps associated to the loss of smaller fragments (H2O, Cl2, OAc-,
-
NO3 ) as well as two mass loss recorded at high temperature generated by the decomposition of the ligand
(overlapped in case of complexes 2, 3 and 5). TG curve for complex 1 describes two well defined steps associated to
the loss of two water molecules (251-270⁰C) and one chloride (278-300⁰C). The sequence of these molecules loss as
well as the loss temperature indicate that both the anion chloride and the H2O molecules are involved in the
coordination sphere. The thermal curves for complexes 2 and 4 are similar, indicating the exothermic elimination of
one water molecule and one coordinated acetate anion (complex 2) and nitrate anion (complex 4) respectively. The
low temperature at which the first decomposition process is observed demonstrates the crystallization nature of the
water molecule [42]. Still, in case of complex 4 this process occurs at slightly higher temperature which suggest the
involvement of crystallization water in intermolecular interactions [43]. For complex 3 the DTA and TG curves
present three exothermic processes. The one at lowest temperature (278-300⁰C) corresponds to the elimination of
the coordinated acetylacetonate fragment, while the overlapped ones occuring at higher temperatures (>530⁰C )
describe the degradation of the ligand. The TG curve of complex 5 describes a mass loss at low temperature (50-
97⁰C ) associated to one molecule of crystallization water. Between 265-420⁰C a significant mass loss occurs due to
the loss of one of the two molecules of ligand. In case of all complexes the degradation processes lead to CuO
formation.
Please insert Table 5 here
3.7. Antimicrobial activity
The antimicrobial activity of the ligand HL and complexes 1-5 was tested against a spectrum of Gram-positive and
Gram-negative bacteria. The minimal inhibitory concentrations (MICs) are indicated in Table 6. The five newly
synthesized Cu(II) complexes showed significantly higher antimicrobial activity compared to the uncoordinated
ligand HL. The value of the minimal inhibitory concentration (MIC) in case of complexes 1-5 varies between 64-
128 µg/mL, the observed values being 8-16 times smaller than those observed in the case of the ligand (MIC: 1024
µg/mL). The observed values of the MICs for utilized solvents (pyridine and DMSO) is 1024 µg/mL, showing a

higher value than the amount of the used solvents in experiments. Thus the solvents had no influence on the MIC
values obtained for ligand and Cu(II) complexes.
The increased antimicrobial potential of the complexes compared to the ligand can be explained in view of two
major concepts. When entering in direct contact with the pathogen bacteria the Cu(II) compounds cause cell damage
by membrane ruptures leading to loss of membrane potential and cytoplasmic content. So they inhibit the growth of
bacteria ultimately causing their death [44]. On the other hand, the chelation theory by Tweedy [45] as well as the
theory of Searl [46] can be considered relevant to explain the enhanced microbiological effect of the complexes.
The obtained results are in accord with similar microbiology studies for other hydrazones. Previously, Angelusiu et
al. evaluated the in vitro antibacterial effect of some aroyl-hydrazones and their Cu(II), Co(II) and Ni(II) complexes
against Gram-positive bacteria (Staphylococcus aureus, Bacillus subtilis) and Gram-negative bacteria (Pseudomonas
aeruginosa, Escherichia coli), using MICs method. The obtained results showed that the ligand had a weak action
on all the tested microorganisms while the activity become more pronounced when coordinated to the metal ions
[47]. The values of the MIC varied between 32-512 µg/mL (Angelusiu et al.), being higher than those observed by
us in case of complexes 1-5. Also, Özmen and Olgun demonstrated that sulfonyl hydrazone derivatives and their
nickel complexes were exhibited antibacterial activity against Gram-positive bacteria (Staphylococcus aureus,
Bacillus megaterium, Bacillus subtilis) and Gram-negative bacteria (Salmonella enteritidis, Escherichia coli) and
possess broad spectrum of activity [48]. The MIC values varied between 145 and 683µg/mL (Özmen and Olgun),
those compounds having an inferior antimicrobial capacity than the complexes described in the present study.
The inhibitory potential of ligand HL and complexes 1-5 was evaluated comparatively with the effect of three
antibiotics currently used in therapy. In the study of the antimicrobial effect of the control drugs the most sensible
bacterial strain was E. coli W3110 showing MIC values of 1 µg/mL for kanamycin, 2 µg/mL for ampicillin and 1
µg/mL for streptomycin. The next bacterial strain regarding the susceptibility was the S. aureus ATTC 6538 strain
with the observed MIC values of 0.5-32 µg/mL. The smallest antimicrobial susceptibility were observed in case of
B. cereus ATCC 14579 (MIC values 8-64 µg/mL) and P. aeruginosa ATCC 9027 (MIC values 256-1024 µg/mL)
bacterial strains. In our study both B. cereus ATCC 14579 and P. aeruginosa ATCC 9027 bacterial strains showed
ampicillin resistance. P. aeruginosa is often resistant to multiple antibiotics and consequently has joined the ranks of
‘superbugs’ due to its enormous capacity to engender resistance. Compared with other pathogens, P. aeruginosa is
very difficult to eradicate as it displays high intrinsic resistance to a wide variety of antibiotics, including
aminoglycosides, fluoroquinolones and β-lactams [49]. In case of the four studied bacterial species strains showing a
broad spectum of resistance against antibiotics was observed [50-53]. On the other hand, it was observed that
complexes 1, 2 and 4 present a good inhibitory effect against B. cereus ATCC 14579 and P. aeruginosa ATCC 9027
strains their MIC values (64 µg/mL) being 8 times lower than the MIC of ampicillin and streptomycin. In view of
these results, the presented Cu(II) complexes can be considered an important alternative class of compounds for new
drug development.
Please insert Table 6 here

4. Conclusion
In order to develop new effective antimicrobial agents we sinthesized and structurally characterized five Cu(II)
complexes with the ligand obtained by condensation of pyridoxal hydrochloride and isonicotinoyl hydrazide (HL).
The physico-chemical data confirm the molecular structure of compound HL while the stereochemistry of the
complex 1-5 was proposed based on IR, UV-VIS, EPR spectroscopy and magnetic moments values. The nature and
number of additionally coordinated small fragments as well as the thermal stability were determined by analizing the
TG, DTG and DTA curves. In case of all chelate compounds, HL acts as tridentate monoanionic ligand keeping the
same keto tautomeric form as prior coordination. The effect of the contraion from the Cu(II) salts is clearly
observed. For complexes 1 and 5 the spectral data indicate an octahedral geometry meanwhile compounds 2 and 4
adopts a square-planar surrounding of the Cu(II) ion. Complex 3 presents a lower-simetry tetrahedral
stereochemistry.
The single crystal X-ray diffraction data for HL show several intermolecular interactions via hydrogen bonds
between ligand and neighboring HCl and DMSO molecules. In addition, the π-π stacking interactions between
aromatic rings strengthen the resulting supramolecular structure.
The antimicrobial effect against E. coli W3110, P. aeruginosa ATCC 9027, B. cereus ATCC 14579 and S. aureus
ATTC 6538 strains was tested from qualitative and quantitative perspective. Although the ligand had a low effect on
the tested pathogen microorganisms the MIC values for complexes indicate up to 16 time increased antibacterial
activity after coordination. This behaviour underlines the influence of the Cu(II) ion and the structure of complex
molecules on the microbiological potential. The complexes are characterized by a wide antimicrobian spectrum
exerting inhibitory effect against both Gram-positive and Gram-negative bacteria.
The activity of complexes 1-5 was compared with the one of other reference antibiotics. While B. cereus ATCC
14579 and P. aeruginosa ATCC 9027 can be considered resistant to ampicillin and streptomycin the complexes 1, 2
and 4 were able to inhibit their growth at concentration of 64 µg/mL. In view of these results, the presented Cu(II)
complexes can be considered an important potential alternative class of compounds for new antimicrobial agents
development.
Appendix A. Supplementary data
CCDC 934828 contains the supplementary crystallographic data for C18H27ClN4O5S2 (HL). These 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.
Acknowledgements
The authors thank the Organic Chemistry Department, ICECHIM Bucharest (Romania) for microanalysis and
Faculty of Technical and Social Sciences, Sapientia Hungarian University of Transylvania (Romania) for

antimicrobial activity. This research was financially supported by European Regional Development Fund, Sectorial
Operational Program „Increase of Economic Competitiveness”, Priority Axis 2 (SOP IEC-A2-O2.1.2-2009-2, ID
570, COD SMIS-CSNR:12473, Contract 129/2010-POLISILMET.

References
[1] (a) C.M. Armstrong, P.V. Bernhardt, P. Chin, D.R. Richardson, Eur. J. Inorg. Chem. 6 (2003) 1145;
(b) J.R. Anacona, M. Rincones, Spectrochimica Acta A 141 (2015) 169;
(c) E.N. Nfor, A. Husian, F. Majoumo-Mbe, I.N. Njah, O.E. Offiong, S.A. Bourne, Polyhedron 63 (2013) 207;
[2] P. Vicini, M. Incerti, I.A. Doytchinova, P. La Colla, B. Busonera, R. Loddo, Eur. J. Med. Chem. 41 (2006) 624;
[3] S. Rollas, S.G. Kucukguzel, Molecules 12 (2007) 1910;
[4] A.A. Recio Despaigne, J.G. da Silva, A. C.M. do Carmo, F. Sives, O.E. Piro, E.E. Castellano, H. Beraldo,
Polyhedron 28 (2009) 3797;
[5] N.A. Mangalam, S. Sivakumar, M.R. Prathapachandra Kurup, E. Suresh, Spectrochimica Acta A 75 (2010) 686;
[6] O. Pouralimardan, A.C. Chamayou, C. Janiak, H. Hosseini-Monfared, Inorg. Chim. Acta 360 (2007) 1599;
[7] C. Basu, S. Chowdhury, R. Banerjee, H.S. Evans, S. Mukherjee, Polyhedron 26 (2007) 3617;
[8] M. Bakir, O. Green, W.H. Mulder, J. Mol. Struct, 873 (2008) 17;
[9] O.A. El-Gammal, G.A. El-Reash, S.F. Ahmed, J. Mol. Struct. 1007 (2012) 1;
[10] H. Hosseini-Monfared, H. Falakian, R. Bikas, P. Mayer, Inor. Chim. Acta 394 (2013) 526;
[11] (a) K.S. Abou-Melha, Spectrochimica Acta A 70 (2008) 162;
(b) H.N. Fox, Science 116 (1952) 129;
(c) J.R. Dilworth, Coord. Chem. Rev. 21 (1976) 29;
[12] (a). S. Kakimoto, K. Yashamoto, Pharm. Bull. 4 (1956) 4;
(b). V.J. Negi, A.Q. Sharma, J.S. Negi, V. Ram. Int. J. Pharm. Chem. 2 (2012) 100;
(c). M. Asif. Int. J. Adv. Chem. 2 (2014) 85;
[13] (a) G. Durgaprasad, C.C. Patel, Indian J. Chem. 11A (1973) 1300;
(b) F. Martins, S. Santos, C. Ventura, R. Elvas-Leitao, L. Santos, S. Vitorino, M. Reis, V. Miranda, H.F.
Correia, J. Aires-de-Sousa, V. Kovalishyn, D. A.R.S.Latino, J. Ramos, M. Viveiros, Eur. J. Med. Chem. 81 (2014)
119;

(c) E. Mackova, K. Hruskova, P. Bendova, A. Vavrova, H. Jansova, P. Haskova, P. Kovarikova, K. Vavrova,
T. Simunek, Chem-Biol. Interct. 197 (2012) 69;
[14] R.K. Agarwal, D. Sharma, L. Singh, H. Argawal, Bioorg. Chem. Appl. (2006) 1;
[15] F.R. Pavan, P.I. da S. Maia, S.R.A. Leite, V.M. Deflon, A.A. Batista, D.N. Sato, S.G. Franzblau, C.Q.F. Leite,
Eur. J. Med. Chem. 45 (2010) 1898;
[16] J.S. Casas, M.D. Couce, J. Sordo, Coord. Chem. Rev. 256 (2012) 3036;
[17] R. Chumnantana, N. Yokochi, T. Yagi, Biochim. Biophys. Acta 1722 (2005) 84;
[18] M. Adrover, B. Vilanova, F. Munoz, J. Donoso, Bioorg. Chem. 37 (2009) 26;
[19] S. Mann, O. Ploux, Biochim. Biophys. Acta 1814 (2011) 1459;
[20] H. Ota, M. Mushiga, T. Yoshimura, K. Yoshimune, J. Biosci. Bioeng. 120 (2015) 117;
[21] V.M.Leovac, M.D. Joksovic, V. Divjaskovic, L.S. Jovanonic, Z. Saranovic, A. Pevec, J. Inorg. Biochem. 101
(2007) 1094;
[22] M.S. Iqbal, A.H. Khan, B.A. Loothar, I.H. Bukhari, Med. Chem. Res. 18 (2009) 31;
[23] J.L. Buss, J. Neuzil, P. Ponka, Biochem. Soc. T. 30 (2002) 755;
[24] (a) J.P. Souron, M. Quarton, F. Robert, A. Lyubchova, A. Cosse-Barbi, J.P. Doucet, Acta Crystallog. C51
(1995), 2179;
(b) M.R. Maurya, S. Agarwal, C. Bader, D. Rehder, Eur. J. Inorg. Chem. 147 (2005);
[25] K. Nakamoto, Infrared Spectra of Inorganic and Coordination Compounds, 5th edition, Wiley-Interscience,
New York, 1997;
[26] S. Naskar, S. Naskar, R.J. Butcher, S.K. Chattopadhyay, Inorg. Chim. Acta 363 (2010) 404;
[27] F. Patrascu, M. Badea, M.N. Grecu, N. Stanica, L. Marutescu, D. Marinescu, C. Spinu, C. Tigae, R. Olar, J.
Therm. Anal. Calorim. 113 (2013) 1421;
[28] (a) CrysAlis RED, Oxford Diffraction Ltd., Version 1.171.36.32, 2003 ;
(b) O.V. Dolomanov, L.J. Bourhis; R.J. Gildea, J. Howard, A. K. H. Puschmann, OLEX2: a complete structure
solution, refinement and analysis program. J. Appl. Cryst. 42 (2009) 339;
(c) G.M. Sheldrick, SHELXS, Acta Cryst. A64 (2008) 112;

[29] I. Wiegand, K. Hilpert, E.W.R. Hancock, Nat. Protoc. 3 (2008) 163;
[30] (a) T, Rosu, S. Pasculescu, V. Lazar, C. Chifiriuc, C. Cernat, Molecules 11 (2006) 904;
(b) T. Rosu, E. Pahontu, R.S. Mezey, D.C. Ilies, R. Georgescu, S. Shova, A. Gulea, Polyhedron 31 (2012) 252;
[31] K. Nakamoto, Infrared and Raman Spectra of Inorganic and Coordination Compounds, Wiley and Sons, New
York 1986;
[32] N. Galic, I. Brodanac, D. Kontrec, S. Miljanic, Spectrochim. Acta A 107 (2013) 263;
[33] N. Galic, M. Rubcic, K. Magdic, M. Cindric, V. Tomisic, Inorg. Chim. Acta 366 (2011) 98;
[34] N.F.Curtis, Y.M.Curtis, Inorg. Chemistry 4 (1965) 804;
[35] A. Sreekanth, S. Sivakumar, M.R.P. Kurup, J. Mol. Struct. 655 (2003) 47;
[36] (a) R. Selwin Joseyphus, M. Sivasankaran Nair, Arabian J. Chem. 3 (2010) 195;
(b) D. Banerjea, Coordination Chemistry, Tata McGrwo-Hill Publisher, 1993;
[37] A.B.P. Lever, Inorganic Electronic Spectroscopy, 2nd ed. Elsevier Science, New York, 1984;
[38] R.B. Sumathi, M.B. Halli, Bioinorg. Chem. Appl. 2014 (2014);
[39] D.W. Smith, Inorg. Chem. 5 (1966) 2236;
[40] A. Lund, M. Shiotani, S. Shimada, Principles and Applications of ESR Spectroscopy, Springer Ed. 2011;
[41] C.C. Chou, H.J. Liu, L.H.C. Chao, H.B. Syu, T.S. Kuo, Polyhedron 37 (2012) 60;
[42] F. Patrascu, M. Badea, M.N. Grecu, N. Stanica, L. Marutescu, D. Marinescu, C. Spinu, C. Tigae, R. Olar, J.
Therm. Anal. Calorim. 113 (2013) 1421;
[43] D.B. Ninkovic, G.V. Janjic, S.D. Zaric. Cryst. Growth Des. 12 (2012) 1060;
[44] G. Grass, C. Rensing, M. Solioz. Appl. Environ. Microb. 77 (2011) 1541;
[45] B.G. Tweedy, Phytopathology 55 (1964) 910;
[46] J.W. Searl, R.C. Smith, S.J. Wyard, Proc. Phys. Soc. 78 (1961) 1174;
[47] M.V. Angelusiu, S.F. Barbuceanu, C. Draghici, G.L. Almajan. Eur. J. Med. Chem. 45 (2010) 2055;
[48] U.Ol. Ozmen, G. Olgun. Spectrochim. Acta A 70 (2008) 641;
[49] E.B. Breidenstein, C. de la Fuente-Nunez, R.E. Hancock. Trends Microbiol. 19 (2011) 419;

[50] L.B. Jensen, S. Baloda, M. Boye, F.M. Aarestrup. Environ. Int. 26 (2001) 581;
[51] G.G. Zhanel, T.L. Hisanaga, N.M. Laing, M.R. DeCorby, K.A. Nichol, B. Weshnoweski, J. Johnson, A.
Noreddin, D.E. Low, J.A. Karlowsky. Int. J. Antimicrob. Ag. 27 (2006) 468;
[52] H.F. Chambers, F. DeLeo. Nat. Rev. Microbiol. 7 (2009) 629;
[53] R.J. Anderson, P.W. Groundwater, A. Todd, A.J. Worsley. Wiley & Sons, Ltd. United Kingdom (2012);

Figures and Tables
Scheme 1. Ligand pyridoxal isonicotinoyl hydrazone (HL)
Scheme 2. Proposed structures for complexes 1-5
Scheme 3. Tautomeric equilibrium for ligand HL

Fig. 1. View of the asymmetric part in the crystal structure H4LCl, with thermal ellipsoids at 50% probability level.
Fig. 2. The columm-like supramolecular architecture in the crystal structure of H4LCl.
Fig. 3. EPR experimental spectra (a) and simulation spectra (b) for complexes 1-5

Table 1. Crystallographic data, details of data collection and structure refinement parameters for ligand HL.
Empirical formula
Formula weight
Temperature/K
Crystal system
Space group
a/Å
C18H27ClN4O5S2
479.01
200
triclinic
P-1
5.9740(5)
b/Å
13.1953(19)
c/Å
α/°
β/°
15.3609(18)
71.755(12)
86.141(8)
84.047(9)
1143.0(2)
2
γ/°
V/Å3
Z
Dcalc/mg/mm3
µ/mm-1
1.392
0.386
Crystal size/mm3
θmin, θ max(o)
Reflections collected
Independent reflections
Data/restraints/parameters
0.30 × 0.15 × 0.15
4.9 to 52.74
8318
4677 [Rint = 0.0261]
4677/0/278
0.0447
a
R1 (I > 2σ(I)
b
wR2 (all data)
0.1019
1.043
0.25/-0.31
GOFc
Largest diff. peak/hole/e Å‒3
a R1 = Σ||Fo| - |Fc||/Σ|Fo|.
b wR2 = {Σ[w (Fo2 - Fc2)2] /Σ[w(Fo2)2 ]}1/2.
c GOF = {Σ[w(Fo2 - Fc2)2] /(n – p)}1/2, where n is the number of reflections and p is the total number of
parameters refined.
Table 2. Characteristics of hydrogen bonds
Distance, Å
H···A
DHA angle, Symmetry code
D–H···A
deg
for A
D–H
0.82
D···A
-1 - x, -2 - y, 2 –
z
O2-H···Cl1
2.24
3.053(2)
172.7
N3-H···O4
N1-H···Cl1
O1-H···N2
C8-H···O4
C14-H···O4
C13-H···O5
0.86
0.86
0.82
0.93
0.93
0.93
1.95
2.20
1.86
2.37
2.49
2.56
2.770(2)
3.048(2)
2.574(2)
3.139(2)
3.377(3)
3.452(3)
158.8
168.6
145.5
139.7
158.4
160.2
x, y, z
x, y, z
x, y, z
x, y, z
x, y, z
x, y, z

Table 3. Electronic spectra and magnetic moments for complexes 1-5
Complex
[CuLCl(H2O)2] (1)
Transitions d-d (cm-1)
µeff B.M.
1.7
Geometry
Octahedral
2
2
2
dx -y2 → dz2
dx -y2 → dxy
dx -y2 → dxz, yz
11.840
-
15.400
2
2
[CuL(OAc)](H2O) (2) dx -y2 → dxy
dx -y2 → dyz, xz
1.5
1.9
Square-planar
Tetrahedral
-
15.100
2
2
[CuL(OAcac)] (3)
d
xy → dxz,yz
dxy→ d x -y
15.130
dxy→ dz2
-
9.850
2
2
[CuL(NO3)](H2O) (4) dx -y2 → dxy
dx -y2 → dyz, xz
1.6
1.8
Square-planar
Octahedral
-
15.750
2
2
2
[CuL2](H2O) (5)
dx -y2 → dz2
11.560
dx -y2 → dxy
dx -y2 → dxz, yz
-
14.920
Table 4. Principal values of g-tensor for complexes 1- 5
Complex
g1
g2
g3
2.211
2.170
2.152
2.143
2.235
0.003
2.051
2.105
2.093
2.113
2.068
2.045
2.043
2.044
2.048
2.052
0.003
1
2
3
4
5
Errors:
0.003
Table 5. Thermal analysis data for complexes 1-5
Complex
[CuLCl(H2O)2] (1)
Temperature
244-262°C
279-294°C
433-593°C
∆m
8.27%
9.21%
Fragment lost
2·H2O
Cl2
Ligand decomposition
[CuL(OAc)](H2O) (2) 58-100°C
270-330°C
4.90%
13.36%
H2O
OAc-
554-607°C
231-353°C
534-615°C
Ligand decomposition
22.32%
Acac-
Ligand decomposition
[CuL(OAcac)] (3)
[CuL(NO3)](H2O) (4) 70-105°C
258-343°C
4.97%
12.96%
H2O
NO3
-
524-601°C
Ligand decomposition
[CuL2](H2O) (5)
50-97°C
2.80%
H2O
265-420°C
43.73%
Ligand
433-582°C
Ligand decomposition

Table 6. MIC (µg/mL) values determined for ligand HL, complexes 1-5 and standard antibiotics
Compound
Staphylococcus aureus
var. Oxford ATCC
6538
Bacillus cereus
ATCC 14579
Pseudomonas
aeruginosa
ATCC 9027
Escherichia coli
W3110
1024
64
64
64
64
128
0.5
4
1024
64
64
64
1024
64
64
128
64
128
1024
HL
[CuLCl] (1)
128
128
128
128
128
2
[CuL(OAc)](H2O) (2)
[CuL(OAcac)] (3)
[CuL(NO3)](H2O) (4)
[CuL2](H2O) (5)
Ampicillin
64
128
resistant
8
resistant
256
Kanamycin
1
Streptomycin
Pyridine
DMSO
32
1024
1024
64
1024
1024
256
1024
1024
1
1024
1024

The characterization of pyridoxal isonicotinoyl hydrazone (HL) and its Cu(II) complexes was
done by 1H NMR, 13C NMR, UV–Vis, IR and EPR spectroscopy, thermal studies and elemental
analysis. The structure of HL was determined by single crystal X-ray diffraction. The
microbiological effect of ligand and complexes against Gram-positive and Gram-negative
strains was tested.