Syntheses, characterizations, crystal structures, and biological activities of two new mixed ligand Ni(II) and Cu(II) Schiff base complexes

Article abstract of DOI:10.1080/00958972.2014.1000883

Two mixed-ligand complexes, [Cu(L)(2imi)] (1) and [Ni(L)(2imi)]?MeOH (2) [L = 2-(((5-chloro-2-oxyphenyl)imino)methyl)phenolato) and 2imi = 2-methyl imidazole], have been prepared by the reaction of appropriate metal salts with H₂L and 2-methyl

Full text of DOI:10.1080/00958972.2014.1000883

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Syntheses, characterizations, crystal structures and
biological activities of two new mixed ligand Ni(II) and
Cu(II) Schiff base complexes
S. Yousef Ebrahimipoura, Maryam Mohamadib, Jesús Castroc, Nasrin Mollaniad, Hadi Amiri
Rudbarie & Alessandro Saccáf
a Department of Chemistry, Faculty of Science, Shahid Bahonar University of Kerman,
Kerman, Iran
b Department of Chemistry, Faculty of Sciences, Islamic Azad University, Kerman Branch,
Kerman, Iran
c Departamento de Química Inorgánica, Universidade de Vigo, Facultade de Química,
Edificio de Ciencias Experimentais, 36310 Vigo, Galicia, Spain
d Department of Biology, Faculty of Basic Sciences, Hakim Sabzevari University, Sabzevar,
Click for updates
Iran
e Faculty of Chemistry, University of Isfahan, Isfahan 81746-73441, Iran
f Dipartimento di Scienze Chimiche, Universita′ di Messina, Viale F. Stagno d’Alcontres 31,
98166 Messina, Italy
Accepted author version posted online: 23 Dec 2014.
To cite this article: S. Yousef Ebrahimipour, Maryam Mohamadi, Jesús Castro, Nasrin Mollania, Hadi Amiri Rudbari &
Alessandro Saccá (2014): Syntheses, characterizations, crystal structures and biological activities of two new mixed ligand
Ni(II) and Cu(II) Schiff base complexes, Journal of Coordination Chemistry, DOI: 10.1080/00958972.2014.1000883
To link to this article: http://dx.doi.org/10.1080/00958972.2014.1000883
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Publisher: Taylor & Francis
Journal: Journal of Coordination Chemistry
DOI: http://dx.doi.org/10.1080/00958972.2014.1000883
Syntheses, characterizations, crystal structures and biological activities of two
new mixed ligand Ni(II) and Cu(II) Schiff base complexes
S. YOUSEF EBRAHIMIPOUR*a, MARYAM MOHAMADIb, JESÚS CASTROc, NASRIN MOLLANIAd,
HADI AMIRI RUDBARIe and ALESSANDRO SACCÁf
aDepartment of Chemistry, Faculty of Science, Shahid Bahonar University of Kerman, Kerman, Iran
bDepartment of Chemistry, Faculty of Sciences, Islamic Azad University, Kerman Branch, Kerman, Iran
cDepartamento de Química Inorgánica, Universidade de Vigo, Facultade de Química,
Edificio de Ciencias Experimentais, 36310 Vigo, Galicia, Spain
dDepartment of Biology, Faculty of Basic Sciences, Hakim Sabzevari University, Sabzevar, Iran
eFaculty of Chemistry, University of Isfahan, Isfahan 81746-73441, Iran
fDipartimento di Scienze Chimiche, Universita´ di Messina, Viale F. Stagno d’Alcontres 31, 98166 Messina, Italy
Two mixed-ligand complexes, [Cu(L)(2imi)] (1) and [Ni(L)(2imi)]·MeOH (2) [L = 2-(((5-
chloro-2-oxyphenyl)imino)methyl)phenolato) and 2imi = 2-methyl imidazole], have been
prepared by the reaction of appropriate metal salts with H2L and 2-methyl imidazole. Their
structures were characterized by microanalysis, FT-IR, UV-Vis, molar conductivity and 1H NMR
for [Ni(L)(2im)]·MeOH. The structures were determined using single crystal X-ray diffraction.
Each four-coordinate metal center, Cu(II) in 1 and Ni(II) in 2, is surrounded by donors of Schiff
base (L2-) and N of 2-methyl imidazole in square planar geometries. α-Amylase activities of
these compounds have also been investigated. The experimental data showed that α-amylase was
inhibited by Ni(II) complex while the Cu(II) complex causes a 1.3 fold decrease in Km value.
Antimicrobial results show that these compounds, especially the Cu(II) complex, have potential
for antibacterial activity against Gram negative and Gram positive bacteria and antifungal
activity against Aspergillus fumigatus.
Keywords: Schiff base; Ni(II) complex; Cu(II) complex; Anti-microbial activity; α-Amylase
activity; Enzyme inhibitory
*Corresponding author. Emails: Ebrahimipour@uk.ac.ir, Ebrahimipour@ymail.com
1

1. Introduction
Asymmetric Schiff bases, especially ONO tridentate ones, have attracted much attention because
of structural similarities with some biological units, their facile synthesis and applications in
different areas [1, 2].
These compounds form various complexes with metal ions in different oxidation states.
Among these, there has been increasing interest in research in Cu(II) and Ni(II) complexes due to
their applications in various fields [3, 4]. They are widely used in industrial processes [5, 6].
Investigation of their biological properties has been of more importance, since in many cases,
complexation can improve these properties of the resultant compounds [7, 8]. Furthermore,
copper exists in biological systems with asymmetric multidentate chelates [9, 10]. Many Cu(II)
Schiff base complexes are models for simulating and representing the function of copper ion in
copper-containing proteins and enzymes such as polyphenol oxidase [11]. Cu(II) complexes
containing ONO Schiff base ligands reveal other biological properties such as DNA binding,
anti-cancer, anti-oxidant and anti-microbial activities [12-15].
Nickel also plays a role in some redox enzymes such as urease [16]. Similar to Cu,
Ni(II)-ONO Schiff base complexes possess considerable biological activities [17-19].
On the other hand, α-amylase, an enzyme existing in plants, animals and microorganisms,
cleaves internal α-1,4-glycosidic linkages [20]. α-Amylase inhibitors reduce postprandial
hyperglycemia by partially inhibiting the enzymatic hydrolysis of complex carbohydrates, and
hence delay the absorption of glucose. Accordingly, α-amylase inhibitors are used as targets for
drug-designing in the treatment of diabetes, obesity and hyperlipidemia [21]. Herein, we describe
the synthesis, characterization, α-amylase inhibitory and antimicrobial activities of two new
square-planar mixed-ligand Ni(II) and Cu(II) complexes with tridentate ONO salicylidene
derivative ligand and 2-methyl imidazole.
2. Experimental
2.1. Materials and instrumentation
All chemicals were analytical grade and used as received. [H2L] was prepared according to our
previous report [22]. Elemental analyses of C, H and N were performed on a Heracuse CHN
rapid analyzer. The FT-IR spectrum was recorded on a Nicolet-Impact 400D spectrometer
(4000–400 cm-1) in KBr pellets. Conductance measurements were made with a Metrohm 712
2

Conductometer in DMSO. 1H-NMR spectrum was recorded at 25 °C with a Bruker BRX 100
AVANCE spectrometer. The electronic spectra were recorded in DMSO on a Cary 50
spectrophotometer. Melting point was determined on a Gallenkamp melting point apparatus.
2.2. Synthesis of 2-(((5-chloro-2-hydroxyphenyl)imino)methyl)phenol [H2L]
Ethanolic solution (5 mL) of 2-amino-4-chlorophenol (0.3 g, 2 mmol) was added to an ethanolic
solution (5 mL) of 2-hydroxy-benzaldehyde (0.2 g, 2 mmol) with vigorous stirring. After 15 min
heating, the solution was cooled and an orange precipitate was separated by filtration, washed
with cold ethanol and dried in a desiccator over anhydrous CaCl2. Yield: 0.41 g, 83%. m.p.:
156 °C. Anal. Calc. for C13H10ClNO2 (247.68 g mol-1): C, 63.04; H, 4.07; N, 5.66. Found: C,
63.18; H, 4.01; N, 5.73%. FT-IR (KBr) cm-1: ν(OH) 3047, ν(C=N) 1627, ν(C=Cring) 1496,
ν(C-O) 1307, ν(C-Cl) 655. 1H-NMR (100 MHz, DMSO-d6, 25 °C, ppm): δ = 13.4 (s, 1H; OH),
10 (s, 1H; OH), 9.0 (s, 1H; CH=N), 6.7-7.8 (m, 7H, rings). UV/Vis (DMSO) λmax, nm (log(ε), L
mol-1 cm-1): 271(4.41), 361(4.31), 460(4.24).
2.3. (2-Methylimidazole)(2-(((5-chloro-2-oxyphenyl)imino)methyl)phenolato))copper(II)
[Cu(L)(2imi)] (1)
Methanolic solution (4 mL) of Cu(NO3)2·4H2O (0.1 mmol, 0.02 g) was added to 3 mL solution
of H2L (0.1 mmol, 0.03 g) and NaOH (0.2 mmol, 0.01 g) with constant stirring and the mixture
was refluxed in a water bath for 5 min. Then, 4 mL methanolic solution of 2-methyl imidazole
(0.3 mmol, 0.02 g) was added to the reaction mixture and the reflux was continued for an
additional 2 h. With slow evaporation of the resulting solution, red crystals appeared. These were
separated, dried in air at room temperature and stored in a CaCl2 desiccator. Yield: 0.022 g, 56%.
m.p.: 206 °C. Molar conductance (10-3 M, DMSO) 23.0 Ohm-1 cm2 mol-1. Anal. Calc. for
C17H14ClCuN3O2 (391.31 g mol-1): C, 52.18; H, 3.61; N, 10.74. Found: C, 52.24; H, 3.48; N,
10.80%. FT-IR (KBr) cm-1: ν(NH) 3421, ν(C=N) 1608, ν(C=Cring) 1473, ν(C-O) 1281, ν(C-Cl)
651. UV/Vis (DMSO, λmax, nm (log(ε), L mol-1 cm-1): 306(3.92), 399(2.85), 450(3.06),
661(2.18).
2.4. (2-Methylimidazole)(2-(((5-chloro-2-oxyphenyl)imino)methyl)phenolato))nickel(II)
methanol solvate [Ni(L)(2imi)]·MeOH (2)
3

Ni(NO3)2·4H2O (0.1 mmol, 0.02 g) was added to 4 mL methanolic solution of H2L (0.1 mmol,
0.018 g) and the resulting brown mixture was refluxed in a water bath for 10 min. To the
mixture, 2-methyl imidazole (0.3 mmol, 0.02 g) was added giving a reddish solution that was
further refluxed for ca. 40 min. After cooling, the precipitate was separated and then
recrystallized from ethanol–methanol (30/70) to give red single crystals that were dried in a
vacuum desiccator over CaCl2. Yield: 0.027 g, 64%. m.p.: 211 °C. Molar conductance (10-3 M,
DMSO) 28.0 Ohm-1 cm2 mol-1. Anal. Calc. for C18H18ClN3NiO3 (418.50 g mol-1): C, 51.66; H,
4.34; N, 10.04. Found: C, 51.81; H, 4.25; N, 10.15%. FT-IR (KBr) cm-1: ν(NH) 3419, ν(OH)
3101, ν(C=N) 1608, ν(C=Cring) 1481, ν(C-O) 1284, ν(C-Cl) 675. 1H-NMR (100 MHz, DMSO-d6,
ppm): δ = 12.8 (s, 1H, NH), 9.7 (s, 1H; CH=N), 6.7-8.1 (m, 9H; rings), 3.9 (q, H; OH), 3.2 (d,
3H; CH3), 2.9 (s, 3H, CH3). UV/Vis (MeOH, λmax, nm (log(ε), L mol-1 cm-1): 305(3.81),
401(3.90), 454(4.16).
2.5. Crystal structure determination
X-ray data were collected at room temperature with a Bruker APEX II CCD area-detector
diffractometer using Mo Kα radiation (λ = 0.71073 Å). Data collection, cell refinement, data
reduction and absorption correction were performed using multi scan methods with Bruker
software [23]. The structures were solved by direct methods using SIR2004 [24]. The non-
hydrogen atoms were refined anisotropically by full matrix least squares on F2 using SHELXL
97 [25]. All hydrogens except N—H and O—H hydrogens were placed in calculated positions
and refined as isotropic with the “riding-model technique”. Hydrogens of N—H and O—H were
found in a difference Fourier map and refined isotropically with distance restraints of N—H =
0.86 Å and N—H = 0.82 Å. The complete conditions for data collection and structure solution
are given in table 1.
2.6. α-Amylase activity determination and biochemical characterization
α-Amylase activity was assessed at 37 °C using potato starch (1%) as a substrate in 20 mM
Tris−HCl, pH 7.4. The reaction was stopped by adding 3,5-dinitrosalicylic acid (DNS) and the
amount of reducing sugars released was determined by dinitrosalicylic acid method [26]. One
unit of α-amylase activity is defined as the amount of enzyme that produces reducing sugar
equivalent to 1 μmol maltose (as the standard) per minute under the assay conditions. A maltose
4

standard curve was constructed with different concentrations of maltose. Furthermore,
SDS-PAGE electrophoresis was performed using 10% acrylamide gel and then coomassie
brilliant blue staining was performed according to the Laemmli method [27]. The protein
concentration was determined by the Bradford assay using BSA as the standard [28]. Steady-
state kinetic parameters Km, Vmax, kcat for immobilized enzyme were determined and compared
with the free enzyme.
2.7. Effect of chemical compounds on the amylolytic activity
To investigate the effects of H2L and its complexes (1 and 2), these compounds were added to
the reaction tube. Tris-HCl (20 mM) was used as the buffer and the pH was finally adapted to
7.4. After addition of starch as the substrate to the reaction, the enzyme activity was determined
as described previously. The activity of the enzyme was determined as the initial activity (or
100% activity) before addition of the compounds.
2.8. Enzyme inhibitory effect of compounds
The effect of [Ni(L)(2imi)]·MeOH enzyme activity was determined using the standard assay in
the presence of 1 to 5 mM of these compounds as inhibitors and the type of inhibition for
enzyme was determined by Lineweaver-Burk plots. One of the ways in which inhibition of
enzyme-catalyzed reactions can be discussed is in terms of a general scheme shown below:
It is assumed that the enzyme-containing complexes are in equilibrium with each other, i.e., that
the breakdown of ES in order to generate product does not significantly disturb the equilibrium
[29, 30].
In the present work, all data obtained for a particular system were analyzed using a mixed
model of enzyme inhibition as described in the following equation:
5

ꢀ ꢁ
I
ͤ
ꢂ
Kͽ
(1 + (
))
⁄
KΛ(1 + ([I ] KΗ)) 1
1
ͤ
=
+
V
ͤ
VΛΏΦ
[S ]
ͤ
VΛΏΦ
where V0 is the initial reaction rate, Vmax the maximum reaction rate, Km the binding constant for
the starch, and Ki and KI are the inhibition constants for binding of inhibitor to enzyme and
enzyme-substrate complex, respectively. In this model, a mixed inhibitor displays finite but
unequal affinity for both the free enzyme and the ES complex; hence the dissociation constants
from each of these enzyme forms must be considered in the kinetic analysis of these inhibitors.
I0 and KI were determined using secondary plots as described by the following equation:
ꢀ ꢁ
I
Kͽ
ͤ
1 + (
)
1
=
V′ΛΏΦ
VΛΏΦ
Slope for inhibited reaction = slope for uninhibited reaction × (1+ [I0] / Ki). Hence a secondary
plot of 1/Vmax against [I0] will be linear and the intercept on the [I0] axis gives −KI. A graph of
slope of primary plot against [I0] will also be linear and the intercept on the [I0] axis would be
−Ki [29, 30].
2.9. Fluorescence measurements in the absence and presence of [Ni(L)(2imi)]·MeOH
The intrinsic fluorescences of the native enzyme in the absence and presence of compounds were
measured on a Perkin Elmer luminescence spectrometer LS 55. The excitation wavelength was
set at 280 nm and the emission spectra were recorded from 300 to 400 nm. All experiments were
carried out at room temperature with protein concentrations of 20 μM in 20 mM Tris buffer,
pH 7.4.
2.10. Antimicrobial activity
Antibacterial assays were done on Escherichia coli, Pseudomonas aeruginosa and
Agrobacterium tumifaciens by Agar and broth dilution methods. Luria Bertani (LB) broth/agar
medium was used to cultivate bacteria. A fresh overnight culture of inoculum of each culture was
prepared [31]. The standardization of the bacterial cell number used for susceptibility testing is
6

of critical importance for obtaining correct and reproducible results. For this reason, McFarland
0.5 BaSO4 turbidity standard was used. Sterile paper discs of 4 mm diameter with increasing
amount of [H2L] or [Cu(L)(2imi)] and [Ni(L)(2imi)]·MeOH compounds in each disc were used
for the antimicrobial assay in solid medium. Furthermore, bacteria are inoculated into a liquid
growth medium in the presence of different concentrations of compounds as an antimicrobial
agent.
3. Results and discussion
Schematic diagram for synthesis of [Cu(L)(2imi)] and [Ni(L)(2imi)]·MeOH complexes are
presented in figure 1. These ligands have two main tautomeric forms. These complexes were
obtained in yields of 56-64%. They are insoluble in water and n-hexane, partially soluble in
common organic solvents and have excellent solubility in DMF and DMSO. These complexes
showed non-electrolyte behavior in DMSO (10-3 M).
3.1. Spectral characterizations
Selected frequencies and vibrational assignments are listed in the Experimental section. In the
spectrum of H2L strong absorption at 1627 cm-1 is related to v(C=N) which shows a red shift
after coordination, indicating coordination takes place through the azomethineic nitrogen [32]. In
the spectrum of ligand, the band at 1307 cm-1 is due to the CO vibration that exhibits 26 cm-1 and
23 cm-1 shifts to lower frequencies upon complexation. These shifts indicate the ligand is
coordinated through phenolic oxygens in both complexes [33]. Bands due to NH stretch are
assigned at 3421 cm-1 for 1 and 3421 cm-1 for 2 [34]. Finally, the OH band vibrations of non-
coordinated methanol in [Ni(L)(2imi)]·MeOH are at 3101 cm-1 [35].
The electronic spectra of H2L and its complexes were recorded in DMSO. According to
our previous work [22], in the UV-Vis spectrum of H2L, a band at 268 nm corresponds to π→π*
transitions of aromatic rings. The azomethine π→π* and n→π* transitions are assigned at
350 nm and 438 nm, respectively [22]. In the electronic spectra of complexes all the bands shift
in comparison with free ligand, indicating coordination of Schiff base [33]. In the Ni(II)
complex, the band at 450 nm may be due to LMCT (O/N(p)→Ni(d)) transition and in case of
Cu(II) complex, the band at 450 nm with the epsilon value of loge = 3.06 is due to admixture of
LMCT and MLCT. Moreover, 1 with d9 and 2 with d8 electron configuration in square planar
7

geometry have three transitions. These transitions for 1 are 2A1g→2B1g, 2B2g→2B1g and 2Eg→2B1g
and for 2 are 1A1g→1A2g, 1A1g→1B1g and 1A1g→1Eg. The tailing of CT bands for 1 and 2 to the
visible region causes the disappearance of these bands and only the 2Eg→2B1g transition at
661 nm for [Cu(L)(2imi)] [36].
3.2. X-ray crystal structures
In 1 and 2 2-(((5-chloro-2-oxyphenyl)imino)methyl)phenolato) behaves as a dianionic tridentate
ligand, coordinating through two phenolate oxygens and the imino nitrogen (ONO donor).
Coordination of the metal (Cu or Ni) is completed with monodentate 2-methyl imidazole. The
coordination around both metals is square planar and the Schiff base is essentially planar.
ORTEP drawings of both compounds are given in figures 2 and 3, and selected geometrical
parameters are gathered in table 2.
The coordination behavior of the Schiff base is similar to that previously found in similar
nickel [37, 38] or copper complexes, with [CuO2N2] core [39-41] or even with [CuO2N3] core
(five coordinate) [35, 42]. In 1, the Cu-N distances are 1.9464(15) and 1.9936(16) Å and the
Cu-O distances are 1.8834(13) and 1.9296(13) Å. Cu-O distances are clearly shorter than those
found in [Cu(Br-SAA)(ImH)] [41]. The Cu-N(imi) bond length in 1 is longer than Cu-N(1),
contrasting to [Cu(Br-SAA)(ImH)] [41], where both bond distances are equal. In 2 the Ni-N
distances are 1.857(3) and 1.906(3) Å, in fact, same values that were found for imidazole(2-((5-
chloro-2-hydroxyphenylimino)methyl)phenolato)nickel(II) [38]. The Ni-O bond lengths are
1.822(2) and 1.865(2) Å, values also similar to those in the aforementioned complex.
In 1 and 2, coordination of the tridentate Schiff base generates a four cycle fused system,
two phenolates at both ends and, in the middle, two metallacycles, one five-membered and the
other one six-membered. The four cycle fused system is planar, although the copper complex is
much more distorted than the nickel one, as demonstrated by the dihedral angle between the
phenolate rings, 12.19(10)° for [Cu(L)(2imi)] and only 4.77(17)° for [Ni(L)(2imi)]·MeOH. The
dihedral angle between metallacycles is 9.51(7)° for 1 and only 3.13(13)° for 2.
The metal core [N2O2] is also much more distorted for 1 than in 2. The trans angles, for
example, are 168.36(7) and 167.61(6)° in [Cu(L)(2imi)], clearly deviating from 180°. In the case
of [Ni(L)(2imi)]·MeOH, those values are almost 180° [177.35(11) and 175.95(13)°]. This is in
contrast with the [Cu(Br-SAA)(ImH)] [41] with trans angles over 173.4(2)°.
8

The distortion in the core can be evaluated through the sum of the angles in the
coordination sphere. In a perfect square planar geometry the sum of the cis angles should be
360°, and also the sum of trans angles should be 360°. The sum of both values (720°) could be
used for comparison with a tetrahedral core, where the sum of the 6 angles (109.47°) should be
656.82 [43, 44]. For [Cu(L)(2imi)], the sum of those values is 698.38, whereas for
[Ni(L)(2imi)]·MeOH such sum resulted to be 713.29°. These values clearly indicate that the
copper complex is somewhat distorted towards a tetrahedral geometry, while the nickel complex
shows a square-planar geometry scarcely distorted.
The neutral 2-methyl imidazole behaves differently in the two compounds. In 1 neutral
imidazole is not in the plane, but forms a dihedral plane with the donor atom plane of 27.44(10)°.
However, in 2 this ligand is almost perpendicular, with a dihedral angle of 76.5(2). The angle
between the Cu-N(2) vector and the coordination plane is 5.86(7)°, as a consequence of the
mentioned distortion, while the corresponding angle between the Ni-N(2) vector and the [N2O2]
plane is only 0.37(12)°. This disposition is quite different from that found in the non-substituted
imidazole complex [38] as shown in figure 5, indicating that the presence of the methyl group in
the ligand determines the disposition of this ligand. The neutral 2-methyl imidazole ligand plays
an important role in the supramolecular structure of both compounds. In the case of 1 a classical
N-H…O hydrogen bond is formed between neighboring molecules, as shown in figure 4, with
the parameters found in table 3. This interaction causes a zig-zag growing in the z axis. Those 1D
zigzag structures are ordered in the unit cell as shown in figure 6; figure 7 shows the unit cell for
[Cu(L)(2imi)].
In the case of 2, the asymmetrical unit contains, in addition to the complex, also a
methanol, not coordinated, that plays an important role in the supramolecular network. Indeed, a
classical O-H…O hydrogen bond is formed between methanol and one coordinating oxygen of a
phenolate. Once more, the non-coordinating NH group of 2-methyl imidazole forms a classical
N-H…O hydrogen bond with methanol, similar to that found in imidazole(2-((5-chloro-2-
hydroxyphenylimino)methyl)phenolato)nickel(II) [38]. Thus, the methanol is implicated in two
hydrogen bonds as shown in figure 8, with the hydrogen bond parameters in table 4.
The disposition of the 2-methyl imidalole ring, almost perpendicular to the plane
containing the rest of the molecule (metal + Schiff base) (vide supra) allows a C-H-π interaction,
whose parameters are shown in table 5 (figures 9 and 10).
9

3.3. Kinetic parameters determination
The kinetic parameters of the enzyme were determined in the absence and presence of 1 as
described above. The Km of the enzyme was calculated from a Lineweaver-Burk plot and the
results compared to those obtained for the control without complex (figure 11). The Km values
were estimated as 3 and 4 mM for enzyme in the absence and presence of complex, respectively,
with a 1.3 fold decrease in Km for 1. Reduction of Km reflects the change made in molecular
recognition of the substrate by enzyme. This alteration was either due to slight conformational
changes of the enzyme in the presence of 1, resulting in a lower possibility of forming a
substrate-enzyme complex, or lower accessibility of the substrate to the active sites of the
enzyme. The presence of Ni has a slight effect on the enzyme catalysis accompanied with a
decrease of Km.
3.4. Inhibition of amylase by [Ni(L)(2imi)]·MeOH
The enzyme activity was measured in the presence of 1 and 2 at 37 °C as shown in table 6.
[Cu(L)(2imi)] (2 mM) had no effect on α-amylase activity but in the presence of
[Ni(L)(2imi)]·MeOH (2 and 5 mM) an inhibitory effect was detected. To investigate the
mechanism of action of [Ni(L)(2imi)]·MeOH complex, we used this compound in reaction
mixture. Reaction rates were determined for different substrate concentrations using enzyme and
measurements were carried out in the presence of 2 as the inhibitor. Enzyme deactivation or
enzyme inhibition was assumed to be the reason for the observed reduction in enzyme activity.
The activity was recovered by dilution and remained constant during the incubation time
indicating reversible inhibition and showed mixed inhibition patterns (figure 12). A mixed
inhibitor displays affinity for both the free enzyme and the enzyme-substrate complex. Mixed-
type inhibitors interfere with substrate binding and hamper catalysis in the ES complex. Since
there is no structural similarity between these compounds and substrate, it is likely that this
inhibitor interacts with the enzyme at a site separate from the substrate binding sites.
The structure changes using intrinsic fluorescence measurements were recorded to study
protein unfolding/denaturation at concentrations of compounds where inhibition occurs.
Denaturation was followed by both changes in the maximal intensity of fluorescence (Imax) and
the red shift of the maximal emission wavelength (λmax). As shown in figure 13, the intrinsic
10

fluorescence of enzyme in the presence of increased concentration of [Ni(L)(2imi)]·MeOH was
lower than the native enzyme. Fluorescence emission of a protein is the result of its intrinsic
fluorophores like Trp and Tyr residues. The intensity of emission is related to the protein
conformation which may expose, or bury, the internal fluorophores.
3.5. Antimicrobial activity
To test the antimicrobial properties for the compounds, we used cultured pathogenic bacteria.
The antimicrobial activity was assayed on LBA plates and liquid medium using the agar and
broth dilution methods. Different amounts of H2L, [Cu(L)(2imi)] and [Ni(L)(2imi)]·MeOH were
placed on the discs and the inhibition zones of the different pathogenic microbes were measured.
As seen in table 7, activity of the Cu(II) complex against all tested microorganisms was greater
than the other compounds. This enhancement in activity may be due to an efficient diffusion of
the complex into the cell and/or interaction with the cell [45]. For the Ni(II) complex, higher
antifungal activity was observed against A. fumigatus than the ligand, but the antibacterial
activity of the ligand was more than that of the Ni(II) complex.
4. Conclusion
In this study, two new Cu(II) and Ni(II) complexes were synthesized and characterized using
spectroscopic techniques. Both complexes have square planar geometry, from donors of Schiff
base ligand and nitrogen of 2-methyl imidazole. Biochemical activities of 1 and 2 were also
investigated. Furthermore, the effects of H2L, [Cu(L)(2imi)] and [Ni(L)(2imi)]·MeOH upon
α-amylase activity were carried out and biochemical characterization of enzyme in the presence
of the compounds was determined. Results showed a 1.3 fold decrease in Km value for
[Cu(L)(2imi)]. The experimental data showed that α-amylase was inhibited by the
[Ni(L)(2imi)]·MeOH. The reciprocal plot related to the reversible inhibitory effect of inhibitor
showed mixed inhibition patterns. According to the antimicrobial results, the [Cu(L)(2imi)]
shows more activity against all bacterial varieties in comparison to [Ni(L)(2imi)]·MeOH and
H2L.
Supplementary data
11

CCDC 961113 and 961114 contain the supplementary crystallographic data for 1 and 2,
respectively. A copy of this information may be obtained free of charge from The Director,
CCDC, 12 Union Road, Cambridge, CB2 1EZ, UK (Fax: +44 1223 336 033); web page:
http://www.ccdc.cam.ac.uk/cgi-bin/catreq.cgi.
Acknowledgements
Authors gratefully acknowledge the financial support provided for this work by the Shahid
Bahonar University of Kerman. We thank the University of Messina for provision of the
diffractometer.
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14

Table 1. Crystal data and structure refinement.
[Cu(L)(2imi)] (1)
[Ni(L)(2imi)]·MeOH (2)
Empirical formula
Formula weight
Temperature
C17H14ClCuN3O2
391.31
C18H18ClN3NiO3
418.51
296(2) K
296(2) K
Wavelength
0.71073 Å
0.71073 Å
Crystal system
Space group
Monoclinic
P21/c
Orthorhombic
P212121
Unit cell dimensions
a = 16.8758(9) Å
b = 10.8835(7) Å
c = 8.8026(5) Å
β = 100.030(3)°
1592.04(16) Å3
4
a = 4.54770(10) Å
b = 14.9634(3) Å
c = 26.8683(6) Å
β = 90°
Volume
1828.36(7) Å3
Z
4
Density (calculated)
Absorption coefficient
F(000)
1.633 Mg/m3
1.554 mm-1
796
1.520 Mg/m3
1.230 mm-1
864
Theta range for data collection
Index ranges
1.23 to 28.07°
-22≤h≤22
1.52 to 28.17°
-6≤h≤6
-14≤k≤14
-19≤k≤19
-11≤l≤11
-35≤l≤35
Reflections collected
52915
74371
Independent reflections
Reflections observed (>2σ)
Data completeness
3868 [R(int) = 0.0377]
3298
4453 [R(int) = 0.0636]
3603
0.996
0.997
Refinement method
Full-matrix least-squares on F2
3868 / 0 / 218
0.813
Full-matrix least-squares on F2
4453 / 0 / 237
1.109
Data / restraints / parameters
Goodness-of-fit on F2
Final R indices [I>2σ (I)]
R1 = 0.0249
wR2 = 0.0735
R1 = 0.0330
wR2 = 0.0847
R1 = 0.0369
wR2 = 0.0885
R1 = 0.0551
wR2 = 0.1077
-0.01(2)
R indices (all data)
Absolute structure parameter
Largest diff. peak and hole
0.331 and -0.287 e.Å-3
0.481 and -0.263 e.Å-3
15

Table 2. Selected bond lengths [Å] and angles [°].
[Cu(L)(2imi)]
[Ni(L)(2imi)]·MeOH
M*-O(1)
M-O(2)
M-N(1)
M-N(2)
1.8827(13)
1.9293(13)
1.9460(14)
1.9940(15)
1.822(2)
1.864(2)
1.857(3)
1.906(3)
O(1)-M-N(1)
O(2)-M-N(1)
O(1)-M-N(2)
O(2)-M-N(2)
∑ cis angles
O(1)-M-O(2)
N(1)-M-N(2)
∑ trans angles
Total sum
93.55(6)
84.50(6)
88.82(6)
95.54(6)
362.41
95.94(11)
86.45(11)
88.04(11)
89.56(11)
359.99
168.34(7)
167.60(6)
335.94
177.35(11)
175.95(13)
353.3
698.35
713.29
*M=Cu for [Cu(L)(2imi)] and Ni in case of [Ni(L)(2imi)]·MeOH
16

Table 3. Hydrogen bonds for [Cu(L)(2imi)] [Å and °].
D-H...A
d(D-H)
0.86
d(H...A)
1.92
d(D...A)
2.771(2)
<(DHA)
169.3
N(3)-H...O(2i)
Symmetry transformations used to generate equivalent atoms, i: x, 1/2-y,
1/2+z
17

Table 4. Hydrogen bonds for [Ni(L)(2imi)]·MeOH [Å and °].
D-H...A
N(3)-H(3A)...O(3i)
d(D-H)
0.86
d(H...A)
1.97
d(D...A)
2.807(4)
2.716(4)
<(DHA)
163
O(3)-H(3B)...O(2)
0.82
1.91
169
Symmetry transformations used to generate equivalent atoms, i: 2-x, 1/2+y, 3/2-z
18

Table 5. C-H-π interaction parameters for [Ni(L)(2imi)]·MeOH [Å and °].
Interaction
H...Ct
H-Perp
2.71
γ
<(X-H...Ct) X...Ct
138 3.508(5)
<(X-H, π)
C(17)-H(17C)···Ct1 2.74
8.02
55
γ is the angle between Ct-H vector and ring normal.
Symmetry transformations used to generate equivalent atoms, 1: x-1, y, z.
19

Table 6. The effect of metal ions and some reagents on enzyme activity.
Compounds
Relative activity (2 mM) Relative activity (5 mM)
Control (no addition)
H2L
1.00
0.88
1.00
0.8
1.00
0.85
0.86
0.6
[Cu(L)(2imi)]
[Ni(L)(2imi)]·MeOH
20

Table 7. Antimicrobial activity in the presence of H2L and its complexes.
Compounds A. tumefaciens P. aeruginosa E. coli S. pneumonia fumigatusA.
H2L
(30 ppm)
Intermediate
(14 mm)
Intermediate
(13.5 mm)
Sensitive
(24 mm)
Intermediate
(16 mm)
Resistance
(10 mm)
[Cu(L)(2imi)]
(30 ppm)
Sensitive
(26.5 mm)
Intermediate
(17 mm)
Sensitive
(22 mm)
Sensitive Intermediate
(20 mm) (19.5 mm)
[Ni(L)(2imi)]·MeOH
(30 ppm)
Intermediate
(18 mm)
Resistance
(13 mm)
Resistance
(11 mm)
Resistance Intermediate
(10 mm) (16 mm)
Chloramphenicol
Vancomycin
Sensitive
(18.5 mm)
Sensitive
(18 mm)
Sensitive
(22 mm)
Sensitive Intermediate
(21 mm)
(16 mm)
Sensitive
(16 mm)
Sensitive Intermediate
(18 mm) (11 mm)
Resistance
(6 mm)
Sensitive
(19 mm)
21

Figure 1. Schematic diagram for complexation processes.
22

Figure 2. ORTEP diagram of 1.
23

Figure 3. ORTEP diagram of 2.
24

Figure 4. Growing of [Cu(L)(2imi)] along the z axis.
25

Figure 5. Upper: comparative figure between [Ni(L)(2imi)] (blue) and [Ni(L)(imi)] [38] (pink).
Lower: comparative figure between [Cu(L)(2imi)] (red) and [Cu(Br-SAA)(ImH)] [41] (green).
26

Figure 6. The 1D zigzag arrangement.
27

Figure 7. Unit cell for [Cu(L)(2imi)].
28