Synthesis, spectral analysis, stability constants, antioxidant and biological activities of Co (II), Ni (II) and Cu (II) mixed ligand complexes of nicotinamide, theophylline and thiocyanate

Article abstract of DOI:10.1016/j.molstruc.2017.07.069

In the present study, transition metal complexes or coordination entities with metal precursors such as Co (II), Ni (II) and Cu (II) of mixed ligands namely, nicotinamide (NA), theophylline (TP) and KSCN in water were synthesized under refluxing condition

Full text of DOI:10.1016/j.molstruc.2017.07.069

Accepted Manuscript
Synthesis, spectral analysis, stability constants, antioxidant and biological activities
of Co (II), Ni (II) and Cu (II) mixed ligand complexes of nicotinamide, theophylline and
thiocyanate
Özlen Altun, Mehtap Şuözer
PII:
S0022-2860(17)31013-X
10.1016/j.molstruc.2017.07.069
MOLSTR 24096
DOI:
Reference:
To appear in: Journal of Molecular Structure
Received Date: 21 April 2017
Revised Date: 14 July 2017
Accepted Date: 21 July 2017
Please cite this article as: Ö. Altun, M. Şuözer, Synthesis, spectral analysis, stability constants,
antioxidant and biological activities of Co (II), Ni (II) and Cu (II) mixed ligand complexes of
nicotinamide, theophylline and thiocyanate, Journal of Molecular Structure (2017), doi: 10.1016/
j.molstruc.2017.07.069.
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Graphical Abstract
Title Page: Synthesis, spectral analysis, stability constants, antioxidant and biological
activities of Co (II), Ni (II) and Cu (II) mixed ligand complexes of nicotinamide, theophylline
and thiocyanate
Authors: Özlen ALTUN*, Mehtap Şuözer
Affiliations: Ph.D. and Research student
Trakya University, Department of Chemistry 22030 Edirne/TURKEY
0,40
[Co2+] /[Co2+] + [Mixed Ligand]
[Ni2+] /[Ni2+] + [Mixed Ligand]
0,35
0,30
[Cu2+] /[Cu2+] + [Mixed Ligand]
0,25
0,20
0,15
0,10
0,05
0,00
0,0
0,2
0,4
0,6
0,8
1,0
Molar Ratio
Fig. 2. The determination of molar ratio. Job’s method of continuous variations produced one
maximum for all complexes and is [M2+]/{[M2+] + [Mixed Ligand] ~ 0.352. Two molecules
of ligand react with one molecule of metal(II) ion.

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Synthesis, spectral analysis, stability constants, antioxidant and biological
activities of Co (II), Ni (II) and Cu (II) mixed ligand complexes of
nicotinamide, theophylline and thiocyanate
Özlen ALTUN*, Mehtap Şuözer
Trakya University, Department of Chemistry, 22030 Edirne/TURKEY
*Corresponding author: Trakya University, Department of Chemistry, 22030
Edirne/TURKEY
e-mail: ozlenaltun@yahoo.com
Abstract
In the present study, transition metal complexes or coordination entities with metal
precursors such as Co (II), Ni (II) and Cu (II) of mixed ligands namely, nicotinamide (NA),
theophylline (TP) and KSCN in water were synthesized under refluxing conditions. The
optimization of the reactions to obtain the composition of complexes was performed. The
structural elucidation of the complexes was undertaken by using physico-chemical and
spectroscopic methods (UV/Vis, FT-IR, Mass and XRD) and thermal analysis. The spectral
measurements of mixed ligands with the Co (II), Ni (II) and Cu (II) complexes are compared
with each other in determining which atoms of the ligand are coordinated to the metal ion.
Based on spectral and magnetic moment measurements, all the coordination entities were
identified as in distorted octahedral structure and have the form [M(NA)2(TP)2(SCN)2].xH2O.
In addition, K (stability constant) and ꢀG (Gibbs free energy) values were calculated by using
the Babko and Stanley & Turner’s methods. Antioxidant, antibacterial and antifungal
activities of the complexes were studied.
Keywords: Spectrophotometry, Nicotinamide, Theophylline, Stability Constant, Antioxidant
Activity, Biological Activity

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1.Introduction
Nicotinamide (NA) is a component of both vitamin B complex and co-enzyme,
nicotinamide adenine dinucleotide (NAD) [1]. These are very important to transfer hydrogen
in cell respiration. The structure of nicotinamide has been the subject of many studies. As
pyridine derivatives are associated with important biological activity such as fungicidal,
antitumor and antibacterial activities, nicotinamide is a pharmacologically and physiologically
active reagent [2-6].
The purines includes theophylline (TP), theobromine and caffeine constitute an
important class of anti-inflammatory agents [7]. Theophyline is well known as a hydrotropic
agent and has the ability to solubilize a wide variety of therapeutic drugs. Thus, it can be used
as a drug in therapy for respiratory such as COPD or asthma under a variety of brand names
and anticancer drugs [8, 9]. A few metal-theophylline complexes have shown significant
antitumor activity [10]. The anion derived from theophylline has often been used as a model
ligand in studying the interaction with metal ions [11].
The chemical structures of theophylline and nicotinamide are shown below.
Structurally, theophylline contains two aromatic rings that affect the solubility of the aromatic
anti-malarial agent [2, 4, 6]. In Scheme 1, theophylline has a methyl group at the N1 and N3
sites, which block the interaction of metal cations with N1 and N3 atoms. Thus, the major
binding site in theophylline is the N9 atom, while in nicotinamide only the N1 atom is the
target of metal cation interaction [11, 12].
Theophylline
Nicotinamide
Scheme 1
Mixed-ligand complexes play a key role in biological inorganic chemistry [13]. Also,
mixed-ligand complexes have been extensively studied for their thermodynamic [14], as well
as kinetic stabilities [15]. Metal-based antioxidants have received attention recently for their
capacity to protect organisms and cells from damage induced by oxidative stress. An effective

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antioxidant should be able to terminate the attack of reactive species like free radicals and
prevent them from attacking body cells. The antioxidant activity of a synthetic compound can
be measured by the scavenging ability of that compound to trap free radicals [16].
In the present study, the preparation and structural elucidation of the transition metal
complexes of a mixed ligand of nicotinamide, theophylline and thiocyanate ions are
undertaken by using different methods such as physico-chemical, spectroscopic methods
(UV/Vis, FT-IR, Mass and XRD) and thermal analysis. In addition, optimum conditions and
the molar ratio for the reactions are determined by spectral measurements. K (stability
constant) and ꢀG (Gibbs free energy) values are calculated by using the Babko and Stanley &
Turner’s methods. Moreover, in this paper, the antibacterial, antifungal and antioxidant
activities of the complexes are studied.
2. Experimental
2.1. Material and measurements
All reagents were purchased from commercial sources and used as supplied. Elemental
analyses for C, H, N, O and S were obtained on dried samples using a Perkin Elmer 2400
elemental analyzer. Conductivity measurements were determined in DMSO using an Inolab
Thermal 740P. The magnetic susceptibilities were measured with a MK-1 Sherwood scientific
magnetic susceptibility balance. All pH measurements were made using a calibrated Metrohm
654 digital pH meter with a Sensorex combination pH glass electrode assembly. The pH
meter was calibrated daily before use with pH 4 and 7 Metrohm AG CH 9100 Hersau buffers.
UV/Vis spectra were recorded at 25 °C with a Shimadzu UV-1700 Pharma spectrophotometer
in the 200-800 nm range. IR spectra were determined on a Perkin Elmer BXII spectrometer as
KBr pellets in the frequency range 400-4000 cm−1. A Shimadzu XRD-6000 was employed for
the XRD analyses. Mass spectra were investigated on an AB-SCIEX Triple TOF 4600
System. Thermal analysis curves (TG and DTA) were obtained from a Seiko Exstar TG/DTA
6200 thermal analyzer in a flowing air atmosphere with a heating rate of 10 °C min−1 using a
sample size of 5-10 mg and platinum crucibles.
2.2. General procedure
In the spectroscopic studies, in order to determine the wavelength for the reactions,
various solutions of mixed ligand (NA + TP + KSCN) in the presence of Co (II), Ni (II) or Cu
(II) between pH 1-10 were prepared in the proportion of 1 : 2 (M2+: Mixed Ligand) and the

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spectra were measured after waiting for 10min. In various pH ranges, metal complexes
showed absorption peaks at λ = 647, 649 and 510 nm, respectively. We observed that the
wavelength changed very little in the pH range 6~7; a pH value of 7 was chosen for all
experiments at 80-85 °C [17-20].
2.3. Synthesis of complexes
TP of 0.97 g (5 mmol) was dissolved in water. An aqueous solution of the
metal salt was added and placed in a magnetically-stirred 25 ml round bottom flask. This was
followed by the addition of an ethanolic solution (0.61 g, 5 mmol) of NA and an aqueous
solution (0.48 g, 5 mmnol) of KSCN at the molar ratio of 1:2:2:2 (M:NA:TP:KSCN). The pH
of the solution was adjusted to ~ 7 by adding 0.1 M NaOH in water and the system was
refluxed for two hours at 80-85 °C. The solid material obtained were washed in water,
recrystallized with ethanol:water mixture and dried. The yields were obtained between 70-85
%. All complexes were air stable and soluble only in organic solvents.
[Co(NA)2(TP)2(SCN)2].6H2O (1): Yield (%): 85. Color: Orange. M.P. (°C): 162. Elemental
Analysis (%): Calcd.: C 37.86, H 4.51, N 22.08, O 21.63, S 7.21, Co 6.71; Found: C 37.80, H
4.41, N 22.01, O 21.53, S 7.17, Co 6.68, FT-IR (cm-1): 3302.95 ν(NH), 2117.83 ν(SCN),
1666.84 ν(C=O), 1626.78 ν(C=N),1610.00 ν(C=C), 1251.62 ν(C-N), 714.06 ν(CS), 555.84
ν(M-N), 466.82, ν(MSCN). UV-Vis. 647, 388, 239. Magnetic Moment (BM): 4.55.
Conductivity (ꢁ-1cm2mol-1): 20.81. MS (m/z, EI): 888.0; Found: 887.54.
[Ni(NA)2(TP)2(SCN)2].6H2O (2): Yield (%): 82. Color: Blue. M.P. (°C): 210. Elemental
Analysis (%): Calcd.: C 37.85, H 4.51, N 22.08, O 21.63, S 7.21, Ni 6.74; Found: C 37.77, H
4.36, N 22.05, O 21.58, S 7.17, Ni 6.70. FT-IR (cm-1): 3304.50 ν(NH), 2117.82 ν(SCN),
1666.74 ν(C=O), 1627.66 ν(C=N), 1610.02 ν(C=C), 1250.48 ν(C-N), 714.16 ν(CS), 556.30
ν(M-N), 466.03 ν(MSCN). UV-Vis. (nm): 649, 389, 298. Magnetic Moment (BM): 3.40.
Conductivity (ꢁ-1cm2mol-1): 19.72. MS (m/z, EI): 888.10; Found: 887.77.
[Cu(NA)2(TP)2(SCN)2].2H2O (3): Yield (%): 75. Color: Yellow-Brown M.P. (°C): 285,
Elemental Analysis (%): Calcd.: C 40.96, H 3.90, N 23.89, O 15.60, S 7.80, Cu 7.84; Found:
C 40.91, H 3.78, N 23.85, O 15.54, S 7.75, Cu 7.79. FT-IR (cm-1): 3304.28 ν(NH), 2117.88
ν(SCN), 1667.78 ν(C=O), 1627.51 ν(C=N), 1609.98 ν(C=C), 1251.13 ν(C-N), 714.23 ν(CS),
555.54 ν(M-N), 466.89 ν(MSCN). UV-Vis. (nm): 510, 251. Magnetic Moment (BM): 2.30.
Conductivity (ꢁ-1cm2mol-1): 19.22. MS (m/z, EI): 821.00; Found: 820.33.

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2.4. Antimicrobial Activity Procedure
The biological and medicinal potency of complexes is established by their
antitumor, antiviral and antimalarial activities. This characteristic property is related to the
stability of the metal ion to form complexes with the ligand containing nitrogen donor atoms.
In this study, to determine antimicrobial activities [21-24], gram negative Escherichia coli
ATCC 25922, Escherichia coli 0157:H7, Salmonella thyphimirium ATCC 14028, Listeria
monocytogenes ATCC19115, gram positive Staphylococcus aureus ATCC 25923 and fungal
Candida albicans ATCC 1023 were used. As antibiotic, Ampicillin and Amphoreicin B were
used in bacteria and fungal cultures, respectively. These were obtained from American Type
Culture Collection (ATCC). Minimal inhibition concentration (MIC) values were determined
by CLSI (Clinical and Laboratory Standards Institute) mikro broth delusion method. Briefly,
the test micro-organisms were prepared at different concentration ( 100-3.125 ppm) and were
transferred onto 96-well sterile microplate. The microplate was incubated at 37 °C for 24 h.
for bacteria and fungi respectively and the absorbance was measured at 600 nm with a
Thermo Multiscan GO Microplate Reader Spectrophotometer (Thermo Scientific, USA).
For cell culture and treatment conditions, cells were cultured in a 1:1 mixture of
Dulbecco’s Modified Eagle Medium (DMEM) and HAM’s Nutrient Mixture F12 with 5 %
heat-inactivated foetal bovine serum (FBS), 1 % L glutamine and 100 IU/ml penicillin-
streptomycin. Cells were maintained at 37 °C in a CO2 incubator with a humidified
atmosphere of 5 % CO2 in 75 ml sterile cell culture flasks. The experiments works began at
the 5 th passage of all cells.
2.5. Antioxidant activity procedure
The antioxidant activity [25-28] of the complexes was determined in vitro with slight
modifications as follows: The compounds were serially diluted in DMSO to obtain a
concentration range of 1.25–20 mM. On a 96 well plate the reaction mixture consisted of 100
ꢂl of 0.5 mM 2,2-diphenyl-1-picrylhydrazyl radical (DPPH), and 100 ꢂl of each concentration
of the test compounds using DMSO as solvent. For positive control, 2,6-di-tert-butyl-4-
methylphenol (BHT) was used at a concentration range of 0.0625–2 mM in methanol. The
solvents, DMSO and methanol, were used as blanks. Duplicate experiments were performed.
The plates were covered with aluminum foil and kept in the dark for 20 minutes after which
the absorbance was read on a Tecan-PC infinite M200 Pro Plate reader at the absorbance
wavelength of 517 nm. The percent antioxidant activity was calculated as follows:

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% ꢀꢁꢂꢃꢄꢅꢃꢆꢇꢁꢂ = ꢈꢉꢊꢈꢋ
(1)
ꢈꢉ
where A0 is the absorbance of the blank and A1 is the absorbance in the presence of the
sample or positive control.
3. Results and Discussion
Elemental analysis C, H, N, O, S and metal determination are in good agreement with
the general formula given for the complexes. The molar conductance values obtained for
these coordination entities at the concentration of 10-3 M are in the range of 10-25 ohm-1 mol-1
cm2. These values are too low to account for any dissociation of the complexes in DMSO.
Hence, these complexes can be regarded as non-electrolytes [29, 30]. All the obtained
complexes are solids, insoluble in water but soluble in some organic solvents such as DMSO,
DMF and THF.
Mass (LC-MS) spectrum of complexes are given Fig 1. In Fig 1, there is one peak in
all coordination entities and this is in agreement with the molecular weights of the complexes.
These results indicate that is a complexation.
3.1. Magnetic Susceptibility
The magnetic moment of Co (II) complex was 4.55 BM indicating that the Co (II)
complex is typically a high spin complex with octahedral structure. The Ni (II) complex has
magnetic moment value of 3.40 BM, indicating octahedral coordination of the ligands around
a Ni (II) ion. The Cu (II) complex exhibits magnetic moment of 2.30 BM, suggesting a
distorted octahedral nature for this complex. The values of µeff for Co2+, Ni2+ and Cu2+
complexes are closer to spin-only models [31]. The absorption bands observed in the
electronic spectra of Co (II), Ni (II) and Cu (II) complexes also support an octahedral
geometry [5].
3.2. Conductance
The observed molar conductance values of the complexes in DMSO are in the range
10-25 ohm-1 mol-1 cm2 at room temperature. The value for 1:1 electrolyte in DMSO is
between 50-70 ohm-1 mol-1 cm2. Hence, it is concluded that the nicotinamide, theophylline and
thiocyanate are covalently bonded to the metal ions, which indicates that they act as ligands.
Based on the metal-ligand ratio calculated from analytical data and the nature of the

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electrolytes given by the conductance measurements, compositions are assigned for the
complexes. From the magnetic and conductometric analysis, it is predicted that the complexes
are in the form of [M(NA)2(TP)2(SCN)2].x H2O (M:Co(II), Ni(II) or Cu(II)).
3.3. UV-Visible spectra
The UV-Visible spectra of the ligands and their metal complexes in DMSO showed
absorption bands between 290-850 nm (Table 1). In the electronic spectra of the ligands and
their metal complexes, the presence of a wide range of bands are due to both π-π* and n-π*
and also due to a charge transfer transition arising from π electron interactions between the
metal and ligand that involves either a metal-to-ligand or ligand-to-metal electron transfer.
The absorption spectra of the Co (II) complex shows an absorption band at 239 nm,
4
4
which is attributed to the electronic transition of T1g (F) → A2g (F), while the band at 388
4
4
nm is caused by the electronic transition of T1g (F) → T1g (P). The electronic transition of
4
4T1g (F) → T2g (F) appeared at 647 nm. These transitions and assignments indicate that the
Co (II) complex has a distorted octahedral geometry.
The spectrum of the Ni (II) complex exhibits three bands at 298, 389 and 649 nm,
3
3
3
3
which are attributed to the electronic transitions of A2g (F) → T1g (P), A2g (F) → T1g (F)
and 3A2g (F) → 3T2g (F), respectively, suggesting a distorted octahedral geometry around a Ni
(II) ion.
The electronic spectrum of the Cu (II) complex has a band in the visible region at 510
2
2
nm, which is attributed to the electronic transition between Eg (D) → T2g (D), suggesting
tetragonal geometry [32, 33].
3.5. FT-IR spectra
The FT-IR specra of the free ligands and complexes are presented in Table 2.
In the FT-IR spectrum of NA [34, 35], the band at 1680 cm-1 assigned to nicotinamide due to
the ν(C=O) stretching vibration in free nicotinamide is also observed at the frequency range
1666.8-1667.8 cm-1 in the metal complexes. The vibrations at 1612 cm-1 and 1255 cm-1 due to
ν(C=N) and ν(C-N) stretching move to higher wave numbers (1626.7-1627.5 cm-1) and (1250.4-
1251.6 cm-1) compared to free nicotinamide. The coordination through the nitrogen atom in
ν(C=N) and ν(C-N) groups is further supported by the occurrence of new bands at 555.5-556.3
cm-1 in the spectra of the complexes which may be assigned to ν(M-N) [36]. The free

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nicotinamide shows a medium intensity band at 3210 cm-1 assigned to ν(NH) vibrations, which
is observed in the 3303.9-3304.5 cm-1 region for the complexes. There is no considerable shift
in the ν(NH) vibrations in the complexes, compared to nicotinamide, indicating non-
involvement of the amide NH function in the coordination.
The FT-IR spectra of TP shows strong bands at 1720 and 1650 cm-1 that may be
attributed to the ν(C=O) and ν(C=N) stretching frequencies, respectively [34, 35]. These bands are
shifted to lower frequencies in all the complexes by (1666.8-1667.8 cm-1) and (1626.7-1627.5
cm-1), respectively. These results indicate that the coordination of TP to the metal ions is
through the nitrogen atom N9 [37].
The KSCN shows a strong band at 2048 cm-1 which is caused by ν(CN). Moreover, on
complex formation, this band is shifted to higher frequencies by (2117.8-2117.9 cm-1),
respectively [34, 35]. The S-bonded SCN is the weak band appearing at 400-412 cm-1, which
may be due to the bending of the absorbed thiocynate ν(M-SCN). The bending of N-bonded NCS
is expected at higher frequencies [38].
3.6. XRD spectra
The XRD (powder pattern) of the complexes were indexed in an X-Ray diffractometer
and the unit cell parameters were calculated from 2θ values with the help of a computer.
Powder XRD pattern of all complexes consist of four important peaks in the range 5-50 °C
(2θ). The inter planar spacing (d) was calculated from the positions of intense peaks using
Bragg’s relationship. The 2θ values with maximum intensity of the peaks for Co (II) complex
are found to be 17.2793, 22.4790, 23.8355 and 27.8986 (2θ) which corresponds to d: 5.
12194, 3.95207, 3.73013 and 3.19543, respectively. For the Ni (II) complex, the 2θ values are
determined to be 12.4200, 14.0800, 22.5388 and 25.5200 which corresponds to d: 7.12101,
6.28497, 3.94172 and 3.48761, respectively. Finally, for the Cu (II) complex, 2θ and d values
are 16.5246, 27.6015, 33.0423, 47.4688 and 5.36028, 3.22914, 2.70880 and 1.91380,
respectively. All the important peaks indexed on the observed values of inter planar distance
are compared with the calculated ones. The presence of forbidden number confirms the
tetragonal systems. This implies that Co (II), Ni (II) and Cu (II) complexes have distorted
octahedral geometry.

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3.7. Thermal Analysis
TG-DTA analysis was completed in temperature range of 25 and 1200 °C in static
atmosphere of dry air to examine thermal stabilities of complexes. The TG-DTA curves of the
Co (II), Ni (II) and Cu (II) complexes are very similar to the same metals. Thermal analysis
data of the complexes are summarized in Table 3.
The thermal behavior of the Co (II) complex is discussed as an example; the first stage
decomposition is endothermic in the range of 25-200 °C with a mass loss of 12.16 ℅, which
corresponds to the loss of 6 moles of water. The second stage of decomposition is also
endothermic in the range 200-500 °C, which is the decomposed organic component (81.03
℅). The remaining compound over 500 °C is determined as CoO (6.80 ℅). These results are
in good agreement with the suggested structures of the complexes. It is confirmed that most of
the complexes contain water in the structure. As a result of the thermal study, the qualitative
conclusions can be made for the stability of the complexes.
3.8. Composition and stability constants of complexes
Job’s method of continuous variations [39-41], and the methods of Babko [42] and
Turner & Anderson [43], was used in the investigation of the composition and stability
constants of the complexes during the reaction. Various solutions were prepared at total
concentrations of 1.33 x 10−3 M, under the general procedure, including mixed ligand group
(NA + TP + KSCN) and Co (II), Ni (II) or Cu (II). Firstly, the perpetual change graph was
plotted after the necessary absorbance adjustment was made and is illustrated in Fig. 2.
According to Fig. 2, Job’s method of continuous variations produced one maximum for all
complexes and is [M2+]/{[M2+] + [Mixed Ligand] ~ 0.352. Two molecules of ligand react
with one molecule of metal (II) ion.
Secondary, dilution curves were drawn to determine the stability constants with the
help of solutions that give equal absorption [39-43]. The measured absorbance values of
solutions of mixed ligand with Ni (II), Co (II) or Cu (II) at different concentrations are shown
in Table 4 and Fig. 3. The plots of absorbance (nm) against [M2+] = [Mixed Ligand] are
linear.
In the 1:2 stoichiometric reactions, the stability constant (K) formula [41, 42] are

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(ꢌꢐꢊꢍ)(ꢎꢐꢊꢍ)
K = (ꢌꢋꢊꢍ)(ꢎꢋꢊꢍ)
=
(2)
(3)
ꢏ
ꢏ
2
2
2
2
4(a1-a2+b1-b2)x2+(b2 -b1 +4a2b2-4a1b1)x+a1b1 –a2b2 =0
where a’s and b’s denote initial concentration of M2+ (M = Co, Ni or Cu) and ligands,
respectively. Here, a1 is the concentration of metal and is calculated from [M2+] /[M2+] +
[Mixed Ligand], b1 is the concentration of ligands and is computed from [M2+] + [Mixed
Ligand] = 1.33 x10-3 M. Further, a2 and b2 are [M2+] and [Mixed Ligand], respectively. They
are calculated from dilution curves in Table 4 and x denote the concentration of the complex
formed. K values calculated by this method have the same order of magnitude as those
obtained by the first method. The values of K for the reactions of ligands with metal (II) are
calculated from the above equations.
The Gibbs free energy of formation of the complex may be calculated readily from the
relationship;
ꢀG = -RTlnK
(4)
Where R is the gas constant (8.314 j / mol K), T is temperature (273.16 + 20 = 293.16 K) and
K is the stability constant. The values of K (stability constant) and ꢀG (Gibbs free energy) for
the reactions of mixed ligand with Co (II), Ni (II) or Cu (II) are calculated by means of the
above equations [44]. K and ꢀG values of the complexes are presented in Table 5.
According to Job’s method, K values are calculated from perpetual change and
dilution curves as 14.780, 22.499 and 28.367, respectively. Finally, ꢀG values of the
complexes are -25.692,40 j / mol for Co (II) + Mixed Ligand, -25.214,93 j / mol for Ni (II) +
Mixed Ligand and -24.740,14 j / mol for Cu (II) + Mixed Ligand, respectively. The negative
values of ꢀG mean that the kinetic process is a spontaneous one.
3.9. Antimicrobial Activity
The results for CLSI (Clinical and Laboratory Standards Institute) mikro broth
delusion tests show that only Co (II) complex significant antimicrobial activity and that gram
negative Escherichia coli 0157:H7, Salmonella thyphimirium ATCC 14028, Listeria
monocytogenes ATCC19115 and fungal Candida albicans ATCC 1023. The results are given
Fig. 4.

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3.10. Antioxidant Activity
The antioxidant results of the free ligands and their metal complexes are given in Fig.
5 and Fig. 6. According to Fig. 5, the antioxidant activity of the free ligands was between 10
% and 15 % but, upon complexation (Fig. 6), it increased significantly. As reported [16], this
phenomenon is a result of the electron withdrawing effect of the M (II) ion which facilitates
the release of hydrogen to reduce the DPPH radical. All the complexes also show significant
free radical scavenging ability when tested against DPPH.
4. Conclusion
As a result of experiments, small changes in the spectral analyses were
observed in all complexes. According to physico-chemical, spectrophotometric and thermal
analysis results, the reactions of a mixed ligand (NA + TP + SCN) with Co (II), Ni (II) and Cu
(II) are complexation reactions. Two molecules of mixed ligand react with one molecule of
metal(II) ion. The negative values of ꢀG (Gibbs free energy) mean that the kinetic process is
a spontaneous one. The mixed ligand complexes with Co (II) is active against some of the
representative bacterial and fungal strains. According to test results, the antimicrobial
activities of the complex is risen in comparison with the antimicrobial activity of ligands
.
Also, the complexes also have significant antioxidant activity. Accordingly, the following is
the proposed structure of mixed ligand complexes of nicotinamide, theophylline and
thiocyanate in the presence of Co (II), Ni (II) and Cu (II),
Scheme 2

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Acknowledgement
We thank Trakya University TUBAP-2010/72 for financial support.We (Assoc Prof
Özlen ALTUN and Research Student Mehtap ŞUÖZER) confirm that “Trakya University”
just provided financial support within the scope of the project for chemicals in the manuscript
and we declare that the support received did not lead to any conflict of interests regarding the
publication of this manuscript. There is no need for Trakya University’s confirmation to
publish this manuscript.
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Table Legends
Table 1. UV-Visible spectra of the free ligands and their metal complexes
Table 2. Characteristic IR bands (cm-1) of the free ligands and their metal complexes
Table 3. Thermal analysis data of the metal complexes
Table 4. The values of dilution curves
Table 5. The values of K (stabilityconstant) and ꢀG (Gibbs free energy)

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Table 1. UV-Visible spectra of the free ligands and their metal complexes
Compound
NA
λ max(nm)
260
ABS
1.052
0.553
1.774
0.006
0.014
0.529
0.329
3.957
Assignment
π-π*
340
n-π*
TP
275
π-π*
316
n-π*
365
n-π*
KSCN
1
276
π-π*
360
n-π*
239
π→π* ve n→π*
4T1g (F) → 4A2g (F)
π→π* ve n→π*
4T1g (F) → 4T1g (P)
4T1g (F) → 4T2g (F)
π→π* ve n→π*
3A2g (F) → 3T1g (P)
π→π* ve n→π*
3A2g (F) → 3T1g (F)
3A2g (F) → 3T2g (F)
2Eg (D) → 2T2g (D)
388
0.469
647
298
0.239
3.968
2
3
389
0.573
649
510
0.185
0.958
Table 2. Characteristic FT-IR bands (cm-1) of the free ligands and their metal complexes
Comp. ν(NH) ν(SCN) ν(C=O) ν(C=N) ν(C=C) ν(C-N) ν(CS) ν(M-N) ν(MSCN)
NA
3210.0
3350.0
-
-
-
1680.0 1612.0 1542.0 1255.0
-
-
-
-
-
TP
1720.0 1658.0 1599.0
-
-
-
-
KSCN
2048.0
-
-
-
-
-
1
2
3
3303.9 2117.8 1666.8 1626.7 1610.0 1251.6 714.1
3304.5 2117.8 1667.7 1627.6 1610.0 1250.4 714.2
3304.3 2117.9 1667.8 1627.5 1609.9 1251.1 714.2
555.8
556.3
555.5
466.8
466.0
466.9

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Table 3. Thermal analysis data of the metal complexes
Compound
First Step
Decomp.
Second
Step
Decomp.
Weight loss
(℅)
Final
Produc
t
Tb-Tc
(°C)
Weight loss
(℅)
Tb-Tc
(°C)
200-
500
Tb-Tc
(°C)
500-
750
(℅)
1
2
3
25-200
12.16
81.03
6.80
CoO
11.80
NiO
25-200
25-200
12.16
4.39
200-
600
76.04
81.71
600-
800
200-
850
850-
950
13.90
CuO
Table 4. The values of dilution curves
[M2+]x10-3
[Mixed
Absorbance
Ligand]x 10-3
(1) λ647
(2) λ649
(3) λ510
0.069
0.078
0.118
0.168
0.218
0.062
0.069
0.112
0.173
0.213
0.053
0.058
0.106
0.156
0.196
1
2
3
4
5
2
4
6
8
10
Table 5. The values of K (stability constant) and ꢀG (Gibbs free energy)
Compound
K (stability constant)
ꢀG (Gibbs free
energy)
1
2
3
14.780
22.499
28.367
-25.692,40
-25.214,93
-24.740,14

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Figure Legends
Fig 1. Mass (LC-MS) spectrum for Co (II) complex (a), Ni (II) complex and Cu (II) complex
(c), respectively.
Fig. 2. The determination of molar ratio
Fig. 3. The determination of dilution curves
Fig. 4. The absorbance values of Co (II) complex at different concentration (100-3.125 ppm)
for E. coli 0157:H7 (a), Salmonella thyphimirium (b), Listeria monocytogenes (c) and
Candida albicans (d), respectively.
Fig. 5. Percent antioxidant activity of the free ligands
Fig. 6. Percent antioxidant activity of the complexes

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(a)
(b)
(c)
Fig 1. Mass (LC-MS) spectrum for Co (II) complex (a), Ni (II) complex and Cu (II) complex
(c), respectively.

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0,35
0,30
0,25
0,20
0,15
0,10
0,05
0,00
Co (II) (
λ
)
)
647 nm
Ni (II) (
λ
)
649 nm
Cu (II) (
λ
510 nm
0,0
0,2
0,4
0,6
0,8
1,0
CM / CM + CL (M)
Fig. 2. The determination of molar ratio
0,20
0,15
0,10
0,05
Co (II) (
λ
)
647 nm
)
Ni (II) (
λ
649 nm
510 nm
Cu (II) (
λ
)
2
4
6
8
10
[M2+] = 2[Mixed Ligand] x 10-3 (M)
Fig. 3. The determination of dilution curves

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E. coli o157:h7
1.5000
1.0000
0.5000
0.0000
complex 1
antibiotic
controldmso
(a)
Salmonella thyphimirium
1.5000
1.0000
0.5000
0.0000
complex 1
antibiotic
controldmso
(b)
Listeria monocytogenes
1.0000
0.8000
0.6000
0.4000
0.2000
0.0000
complex 1
antibiotic
controldmso
(c)
Candida albicans
0.3000
0.2000
0.1000
0.0000
complex 1
antibiotic
controldmso
(d)
Fig. 4. The absorbance values of Co (II) complex at different concentration (100-3.125 ppm)
for E. coli 0157:H7 (a), Salmonella thyphimirium (b), Listeria monocytogenes (c) and
Candida albicans (d), respectively.

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KSCN
NA
CA
16
12
8
4
0
0,0
0,5
1,0
1,5
2,0
2,5
Concentration (µM)
Fig. 5. Percent antioxidant activity of the free ligands
100
80
60
40
20
0
BHT
Co (II) complex
Ni (II) complex
Cu (II) complex
0,0
0,5
1,0
1,5
2,0
2,5
Concentration (µΜ)
Fig. 6. Percent antioxidant activity of the complexes

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HIGHLIGHTS
1. Synthesis, spectral and thermal analysis of new complexes of nicotinamide,
theophylline and thiocyanate in the presence Co (II), Ni (II) and Cu (II).
2. Optimization of Reactions
3. Composition, Stability Constants and Gibbs free energies of complexes
4. Antibacterial, antifungal and antioxidant activities of the free ligands and their
complexes