New metal complexes with diclofenac containing 2-pyridineethanol or 2-pyridinepropanol: synthesis, structural, spectroscopic, thermal properties, catechol oxidase and carbonic anhydrase activities

Article abstract of DOI:10.1080/00958972.2016.1227802

Four new neutral diclofenac-based complexes, [Co(dicl)2(2-pyet)2] 1, [Ni(dicl)2(2-pyet)2] 2, [Cu2(dicl)2(2-pyet)2] 3, and [Cu2(dicl)2(2-pypr)2] 4 have been synthesized and characterized by elemental analysis, FT-IR, thermal analysis. Complexes 1, 3, and 4 have also been characterized by X-ray single-crystal structural analysis. The compounds of Co(II) and Ni(II) have octahedral geometry with two diclofenac and two 2-pyridineethanol ligands in the coordination sphere. The compounds of Cu(II) have square-pyramidal geometry and Cu(II) ions are linked via oxygens to the bridging 2-pyridineethanol or 2-pyridinepropanol ligands. The Δν values acquired by FT-IR are in agreement with the single XRD data. Studies on the thermal properties are reported and the complexes are stable to 196, 216, 215, and 201?°C in air, respectively. Two dinuclear Cu(II) complexes have demonstrated catalytic activity on oxidation of 3,5-di-tert-butylcatechol to 3,5-di-tert-butylquinone showing saturation kinetics at high substrate concentrations. The diclofenac complexes are investigated as inhibitors of the human cytosolic isoforms hCA I and II. The complexes are good as hCA I inhibitors (Kis of 1.52–55.06?μM) but only moderately efficient as hCA II inhibitors (Kis of 0.23–5.61?μM).

Full text of DOI:10.1080/00958972.2016.1227802

Journal of Coordination Chemistry
ISSN: 0095-8972 (Print) 1029-0389 (Online) Journal homepage: http://www.tandfonline.com/loi/gcoo20
New metal complexes with diclofenac containing
2-pyridineethanol or 2-pyridinepropanol:
Synthesis, structural, spectroscopic, thermal
properties, catechol oxidase and carbonic
anhydrase activities
Sema Caglar, Esra Dilek, Bulent Caglar, Ekrem Adiguzel, Ersin Temel, Orhan
Buyukgungor & Ahmet Tabak
To cite this article: Sema Caglar, Esra Dilek, Bulent Caglar, Ekrem Adiguzel, Ersin Temel,
Orhan Buyukgungor & Ahmet Tabak (2016): New metal complexes with diclofenac containing
2-pyridineethanol or 2-pyridinepropanol: Synthesis, structural, spectroscopic, thermal
properties, catechol oxidase and carbonic anhydrase activities, Journal of Coordination
Chemistry, DOI: 10.1080/00958972.2016.1227802
To link to this article: http://dx.doi.org/10.1080/00958972.2016.1227802
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Date: 29 August 2016, At: 07:28

Publisher: Taylor & Francis
Journal: Journal of Coordination Chemistry
DOI: http://dx.doi.org/10.1080/00958972.2016.1227802
New metal complexes with diclofenac containing 2-pyridineethanol or 2-
pyridinepropanol: Synthesis, structural, spectroscopic, thermal properties,
catechol oxidase and carbonic anhydrase activities
Sema Caglar†*, Esra Dilek‡, Bulent Caglar†, Ekrem Adiguzel†, Ersin Temel§,
Orhan Buyukgungor¶, Ahmet Tabak
†Department of Chemistry, Faculty of Arts and Sciences, Erzincan University, 24030, Erzincan,
Turkey
‡Department of Biochemistry, Division of Pharmaceutical Basic Sciences, Faculty of Pharmacy,
Erzincan University, 24030, Erzincan, Turkey
§Department of Electric and Energy, Technical Science Vocational High School,Giresun University,
28100,Giresun, Turkey
¶Department of Physics, Faculty of Arts and Sciences, Ondokuz Mayis University, 55200, Atakum,
Samsun, Turkey
Department of Chemistry, Faculty of Arts and Sciences, Sinop University, 57000 Sinop, Turkey
Abstract
Four new neutral diclofenac-based complexes, [Co(dicl)2(2-pyet)2] 1, [Ni(dicl)2(2-pyet)2]
2, [Cu2(dicl)2(2-pyet)2] 3 and [Cu2(dicl)2(2-pypr)2] 4 have been synthesized and characterized
by elemental analysis, FT-IR, thermal analysis. Complexes 1, 3 and 4 have also been
characterized by X-ray single crystal structural analysis. The compounds of Co(II) and Ni(II)
have octahedral geometry with two diclofenac and two 2-pyridineethanol ligands in the
coordination sphere. The compounds of Cu(II) have square-pyramidal geometry and Cu(II)
ions are linked via oxygens to the bridging 2-pyridineethanol or 2-pyridinepropanol ligands.
The Δν values acquired by FT-IR are in agreement with the single XRD data. Studies on the
thermal properties are reported and the complexes are stable to 196, 216, 215 and 201oC in

air, respectively. Two dinuclear Cu(II) complexes have demonstrated catalytic activity on
oxidation of 3,5-di-tert-butylcatechol to 3,5-di-tert-butylquinone showing saturation kinetics
at high substrate concentrations. The diclofenac complexes are investigated as inhibitors of
the human cytosolic isoforms hCA I and II. The complexes are good as hCA I inhibitors (Kis
of 1.52-55.06 µM) but only moderately efficient as hCA II inhibitors (Kis of 0.23-5.61 µM).
Keywords: Diclofenac; Crystal structure; FTIR; Catechol oxidation; Carbonic anhydrases
* Corresponding author. Tel.: +90 446 224 30 97 Fax: +90 446 224 30 16
E-mail address: semacaglar2002@hotmail.com (S. Caglar)
1. Introduction
Non-steroidal anti-inflammatory drugs (NSAIDs) form various chemical groupings:
oxicams family such as meloxicam, piroxicam, tenoxicam and lornoxicam, and carboxylic
acid family such as tolfenamic, diclofenac, mefenamic, flufenamic, ibuprofen and naproxen
[1]. They are widely used as analgesics, anti-inflammatories, antipyretics and antitumor drugs
[2-5]. The action of the NSAID is inhibition of cyclooxygenase (COX) production of
prostaglandins [6]. Inhibition of COX also decreases prostaglandins in the epithelium of the
stomach, making it more sensitive to corrosion by gastric acid.
Most anti-inflammatory drugs contain the carboxylate group. Carboxylates create an
important class of ligands in inorganic and bioinorganic chemistry for metal-ligand interaction
[7] with the ability to bind to metal ions in different ways (monodentate, bidentate or
bridging) [8,9].
Diclofenac (dicl), 2-(2,6-dicholoroanilino)phenylacetic acid, belongs to the derivatives of
phenylalkanoic acids. Dicl is widely used to treat pain, inflammatory disorders and acute
migraines [8, 10-15]. Study of metal complexes with non-steroidal anti-inflammatory drugs as
ligands is increasing. The crystal structures of metal(II) complexes with dicl have been

reported and biological evaluation of metal complexes were investigated [10, 15, 16-21]. It
has been also reported that the metal complexes of some NSAIDs are better or have different
pharmacological profile than free ligand [15-17, 22, 23]. Furthermore, these studies indicated
that copper, manganese and nickel complexes showed enhanced biological properties and
antioxidant activities compared to free Nadicl. [4, 10, 16].
Catechol oxidase enzyme exists widely in nature such as in plants, insects and
crustaceans [24]. The crystal structures of catechol oxidase obtained from sweet potato shows
that catechol oxidases present a dinuclear copper center in their active site and the copper
center is surrounded by three histidines via three nitrogens [25-27]. The copper centers are
bridged via hydroxyl groups in a trigonal pyramidal geometry. Therefore, mononuclear and
dinuclear copper(II) complexes formed by oxygen bridge receive much attention [28-34].
This enzyme catalyzes the substrate specific oxidation of o-diphenols to o-diquinones in
plants [35, 36].
Many of the enzymes found in living organisms are inhibited or activated with drugs.
Inhibitions or activations of enzymes are important for the continuity of functions in living
organisms. Carbonic anhydrases (CA; Carbonate hydrolyses, EC 4.2.1.1) are a family of
-
metalloenzyme in mammals. These enzymes catalyze the conversion of CO2 to HCO3 in
cells. This conversion normally occurs very slowy. But they increase to reaction rate
approximately 104-106 per second. By this reaction, they play important roles in several
physiological/pathological processes such as electrolyte secretion, acid-base balance, bone
resorption, respiration, carbon dioxide and ion transport, gluconeogenesis, ureagenesis and
lipogenesis [37, 38]. Sixteen CA isozymes have been identified in mammals that differ in
subcellular localization and catalytic activity [39]. Many such CA isozymes which assist these
processes are important therapeutic targets with the potential to be inhibited/activated for the
treatment of diseases such as glaucoma, edema, obesity, osteoporosis, epilepsy and cancer

[40-42]. Carbonic anhydrase inhibitors (CAI) are used in eye diseases, and have intraocular
pressure lowering effects among the most potent drugs. Therefore, many scientists have
focused on inhibitors of CA isozymes [43-47].
Inhibitory effects of different complexes, sulfonamide derivatives, anions, phenols,
drugs,various chemicals and pesticides have been investigated against CA [21, 39, 43-52].
However, it is still important to discover further classes of potential CAIs in order to develop
compounds with distinct inhibition profiles as compared to the known molecules [39, 43-46].
In the current study, we report the synthesis, structural and thermal characterization of
metal(II) complexes with dicl in the presence of 2-pyridineethanol (2-pyet) or 2-
pyridinepropanol (2-pypr). We also studied kinetics of the catalytic oxidation of 3,5-di-tert-
butylcatechol (DTBCH2) to 3,5-di-tert-butyl-1,2-benzoquinone (DTBQ) with dioxygen via
spectrophotometric studies and CA inhibitory activities of the compounds were evaluated.
2. Experimental
2.1. Methods of sample characterization
CoCl2·6H2O (Sigma), NiCl2·6H2O (Sigma), Cu(CH3CO2)2·H2O (Sigma), sodium diclofenac
(Sigma, 99.00%), 2-pyridineethanol (Aldrich, 98.00%), 2-pyridinepropanol (Aldrich, 96.00%)
were used for the preparation of the complexes. FT-IR spectra of the complexes were
recorded on a Thermo Nicolet 6700 spectrophotometer from 4000-450 cm-1 at 4 cm-1
resolution using KBr pellets. UV-Vis spectra were recorded on a PG 80+ from 200-1100 nm
in DMF. Elemental analyses (C, H and N) of complexes were determined on a LECO CHNS-
932 elemental analyzer. Thermal analysis runs were carried out on a PRIS Diamond
TG/DTG/DTA apparatus from 30 to 1000 oC at a heating rate of 10 oC min-1 under dynamic
air atmosphere (platinum crucibles and mass ∼10 mg).
2.2. Synthesis of Complexes
2.2.1. [Co(dicl)2(2-pyet)2], 1

Nadicl (sodium diclofenac) (0.18 g, 0.6 mmol) and CoCl2·6H2O (0.06 g, 0.3 mmol)
were dissolved in methanol (40 cm3) with continuous stirring at 50 oC. Then 2-pyet (0.07 g,
0.6 mmol) was added. The solution was left for slow evaporation at room temperature and X-
ray quality purple crystals appeared after five days. The purple crystals were filtered, washed
with water and dried at room temperature.
Yield for 1: 78 %; Analytical data for [C42H38Cl4N4O6Co]: Found: C, 56.32; H, 4.23;
N, 6.31 %; calcd: C, 56.28; H, 4.24; N, 6.25 %).
2.2.2. [Ni(dicl)2(2-pyet)2], 2
Complex 2 was prepared in a similar way to 1 with the use of NiCl2·6H2O (0.06 g, 0.3
mmol) instead of CoCl2·6H2O. The resulting solution was left for slow evaporation at room
temperature and green micro crystals appeared after one week. The green micro crystalline
product was filtered, washed with water and dried at room temperature.
Yield for 2: 89 %; Analytical data for [C42H38Cl4N4O6Ni]: Found: C, 56.32; H, 4.23;
N, 6.31 %; calcd: C, 56.29; H, 4.24; N, 6.25 %).
2.2.3. [Cu2(dicl)2(2-pyet)2], 3
Cu(CH3CO2)2·H2O (0.06 g, 0.3 mmol) and Nadicl ligand (0.18 g, 0.6 mmol) were
dissolved in methanol (40 cm3) with continuous stirring at 50 oC. Then 2-pyet (0.07 g, 0.6
mmol) was added. The resulting solution was left for slow evaporation at room temperature,
giving blue crystals after four days. The formed crystals were filtered, washed with water and
dried at room temperature.
Yield for 3: 91 %; Analytical data for [C42H36Cl4N4O6Cu2]: Found: C, 52.40; H, 3.78;
N, 5.80 %; calcd: C, 52.15; H, 3.74; N, 5.82 %).
2.2.4. [Cu2(dicl)2(2-pypr)2], 4
Complex 4 was synthesized in a similar way to 3 with the use of 2-pypr (0.08 g, 0.6
mmol) instead of 2-pyet. The resulting solution was left for slow evaporation at room

temperature and X-ray quality blue crystals appeared after two weeks. The formed crystals
were filtered, washed with water and dried at room temperature.
Yield for 4: 84 %; Analytical data for [C44H40Cl4N4O6Cu2]: Found: C, 53.28; H, 4.20;
N, 5.61 %; calcd: C, 53.34; H, 4.04; N, 5.65 %).
2.3. X-ray crystallography
The diffraction data were collected on a STOE IPDSII image plate detector using Mo
Kα radiation (α= 0.71073Å, T = 293 K). A summary of the key crystallographic information
is given in Table 1. Data collection: Stoe X-AREA [53]. Cell refinement: Stoe X-AREA [53].
Data reduction: Stoe X-RED [54]. The structure was solved by direct-methods using
SHELXS-97 [54] and anisotropic displacement parameters were applied to non-hydrogen
atoms in a full-matrix least-squares refinement based on F2 using SHELXL-97 [54]. All
hydrogens attached to carbon were positioned geometrically and refined by a riding model
with Uiso 1.2 times than that of attached atoms and remaining hydrogens were located from
the Fourier difference map.
2.4. Catechol oxidase activity
The catecholase activities of 3 and 4 were evaluated by reaction with 3,5-di-tert-
butylcatechol at 25 oC in DMF. The absorption at 395 nm, characteristic of the formed 3,5-di-
tert-butylquinone (DTBQ), was measured as a function of time. The initial reaction rate was
determined from the slope of the trace at 395 nm in the first minute of the reaction. For this
purpose seven different ratios of complex and substrate were studied [20]. The minimum
complex to substrate ratio was 1:5 and the maximum complex to substrate ratio was 1:50.
Copper(II) complexes exhibited saturation kinetics. The data satisfied the Michaelis-Menten
equation; Km, Vmax, and kcat values were determined from Lineweaver-Burk plots.
2.5. Purification and enzymatic activity of hCA I and hCA II

The CA isozymes have been purified many times from different organisms [43, 45, 51,
52, 55, 56]. In this study, the purification of the two CA isozymes was performed with a
simple one step method by a Sepharose-4B-tyrosine-sulfanilamide affinity column from
human erythrocytes [45, 47]. Then the activity of hCA I and hCA II were determined by using
the esterase activity method with 4-nitrophenyl acetate (NPA) as substrate [45]. The
inhibitory effects of the diclofenac complexes were tested in vitro. The inhibitor
concentrations that caused 50% inhibition (IC50) were determined from % activity versus
[Inhibitor] plots and the Ki values were calculated from Lineweaver-Burk plots [20].
2.6. Protein determination
Bradford method was used to carry out quantitative protein assay. Sodium dodecyl sulfate-
polyacrylamide gel electrophoresis (SDS-PAGE) was performed after purification of the
enzymes according to the Laemmli’s procedure as in previous studies [45, 57-58]. Samples
were applied as 20 µg to the electrophoresis. Gels were smudged for 1.5 h in 0.1% Coomassie
Brilliant Blue R-250 in 50% methanol and 10% acetic acid, then stored in the same solvent
without dye.
3. Results and discussion
3.1. FT-IR Spectra
In all complexes, the stretching vibrations of N-H are at 3266, 3270, 3247 and 3232
cm-1, respectively. This band is at 3250 cm-1 in Nadicl. The aromatic and aliphatic C-H
stretching bands of Nadicl, 2-pyridineethanol and 2-pyridinepropanol ligands are 3071-2857
cm-1.
In [Co(dicl)2(2-pyet)2], [Ni(dicl)2(2-pyet)2], [Cu2(dicl)2(2-pyet)2] and [Cu2(dicl)2(2-
pypr)2], the νasym(COO) band is at 1572, 1572, 1577 and 1574 cm-1 and the νsym(COO) at
1368, 1369, 1452 and 1449 cm-1, respectively. The ligands which are included in carboxylate
groups have two donor sites and coordinate to metal ions with carboxyl oxygens. The

coordination mode of the carboxylate groups can be determined from IR spectra by
comparing the Δν [νasym(COO) - νsym(COO)] value. The Δν [νasym(COO) - νsym(COO)] values
are 204, 203, 120 and 125 cm-1, respectively. The position of these bands suggest
monodentate for 1 and 2 and bidentate for 3 and 4 [59-60].
When 2-pyet or 2-pypr was bound to metal via nitrogen of pyridine ring, the pyridine
ring vibration of free 2-pyet or 2-pypr at 1592 and 1423 cm-1 shift to higher frequencies. In
the IR spectra of 1-4, these peaks exist at 1603 and 1453 cm-1. The medium intensity bands at
1307-1303 cm−1 and 1232-1249 cm−1 belong to the anti-symmetric and symmetric stretching
ν(C-N-C) of dicl ligand, respectively. The band attributed to in plane deformation of the C-H
groups occurs at 940 cm-1. The bands at 780 cm-1 correspond to ν(C-Cl) vibration of dicl. The
bands at 600-450 cm-1 are attributed to metal-oxygen and metal-nitrogen stretching vibrations.
3.2. Crystal Structures
3.2.1. [Co(dicl)2(2-pyet)2] 1
Fig.1 shows an ORTEP with the labelling scheme. Selected bond distances and angles
are found in Table 2. The single crystal X-ray analysis shows that [Co(dicl)2(2-pyet)2], 1, is
neutral and crystallizes in monoclinic space group P21/c.
The six coordinate Co(II) has slightly distorted octahedral geometry with two
carboxylate oxygens, O(2) and O(2i) coming from the two dicl ligands and two nitrogens and
two ethanoic oxygens (N(1) and N(1i); O(1) and O(1i)) coming from the two 2-pyet ligands.
2-Pyet is bidentate forming a six-membered chelating ring with cobalt. Intramolecular O-
H…O, N-H…O and C-H…O type hydrogen bonds generate S(6), S(7) and S(6) rings,
respectively. The hydrogen bonding and intermolecular interactions are given in Table 3.
Intermolecular π…π interactions form a 2-D supramolecular structure parallel to the bc-plane
(Fig. 2).

Co-Odicl bond distance is 2.0934(13) Å, which is similar with corresponding values in
[Co(dicl)2(2-aepyr)2] (2.077(2)-2.095 Å) [21]. The Co-Odicl bond distance is slightly shorter
than the 2.080(2)-2.228(2) Å found in [Co(dicl)2(3-pic)2] [21], where 2-aepyr, 3-pic are 1-(2-
aminoethyl)pyrrolidine and 3-picoline.
3.2.2. [Cu2(dicl)2(2-pyet)2] 3 and [Cu2(dicl)2(2-pypr)2] 4
The ORTEP viewes of the asymmetric units of 3 and 4, along with the atomic
numbering scheme, are shown in Fig. 3 and 5, respectively. Selected bond lengths and angles
together with the hydrogen bonding geometries are given in Tables 2 and 3. Complexes
crystallize in monoclinic space group P21/c. The Cu(II) is formed by CuNO4 group with
approximate square pyramidal geometry. The structural distortion index tau (τ) was calculated
as 0.12 for 3; 0.30 for 4 and the τ-value is appropriate for square-pyramidal geometry.
Cu(II) ions are linked via oxygens to the bridging ligands (2-pyet for 3; 2-pypr for 4)
and 2-pyet or 2-pypr is also connected to each metal ion through the nitrogen. The five
coordinate geometry of each Cu(II) is brought about by ligation of the metal by a dicl as the
second ligand. In 3, intramolecular N-H…O type hydrogen bonds generate S(7) rings.
2
Intermolecular C-H…π interactions form a riband of edge-fused R2 (22) rings running through
the b-axis. In 4, intramolecular N-H…O type hydorgen bonds generate S(7) rings.
Intermolecular C-Cl…π interactions form a 2-D supramolecular structure parallel to bc-plane.
C-H…π, C-Cl…π and hydrogen bonding interactions of 3 and 4 are shown in Fig. 4 and 6,
respectively.
Cu-Odicl bond distance is 1.921(2) Å for 3 and 1.924(2) Å for 4, which is shorter than
the 1.966(5)-2.609(6) Å found in [Cu(dicl)2(3-pic)2] [20] and in [Cu(dicl)2(4-pic)2] (1.941(2)-
2.1934(2) Å) [20]. Cu-Odicl bond distance is similar with a corresponding values in
[Cu(dicl)2(py)2] (1.9446(15) Å) [61].
3.3. Thermal Analyses

Thermal gravimetric analyses are used to analyze the thermal stabilities of 1-4. The
results demonstrate that the complexes are stable to ~ 200 oC in air. Complex 1 decomposes in
two steps. The first step between 30-203 oC corresponds to the loss of one coordinated 2-pyet
with the endothermic DTA signal at 192 oC (DTGmax. = 196 oC, found 14.20%, calc. 13.75%).
The second stage contains a major mass loss extending from 203 °C to 687 °C, suggesting
decomposition of one 2-pyet and two dicl ligands with a mass loss of 77.1% (DTGmax
=
279°C, calc. 79.66%). The final weight of residue observed as 8.7% (calc. 8.36%) can be
attributed to the CoO.
For 2, the endothermic peak of DTA (223 °C) curve from 30-261 °C is related to loss
of two 2-pyet ligands (DTGmax = 216, 226 °C; found 26.20%; calc. 27.51%). The second
stage between 261-436 °C corresponds to the release of one dicl ligand by giving an
exothermic peak (DTGmax = 292 °C; found 32.80%; calc. 32.96%). The last stage involves
decomposition of one dicl (DTGmax = 516 °C; found 32.49%; calc. 32.96%). The final
decomposition product is NiO (found = 8.51%, calc. = 8.34%).
Complex 3 shows three steps of decomposition. The first stage between 30 and 227 oC
is related to removal of two 2-pyet ligands (DTGmax = 215 °C; DTA = 212 °C; found 25.20%;
calc. 25.56%). The second stage from 227-557 °C is assigned to decomposition of one dicl
ligand, observed mass loss 31.30% (DTGmax = 329 °C, DTA = 360 °C (exo) calc. 30.63%).
The third stage weight loss of 28.9% between 557 and 780 °C corresponds to loss of one dicl
ligand (calcd. 30.63%). The end products can be assigned to CuO by considering calculated
mass (14.6%) and found weights (16.49%).
For 4, the first peak between 30 and 228 °C is related to removal of two 2-pypr ligands
(DTGmax = 201, 209 °C; DTA = 199 (endo) °C; found 26.66%; calc. 27.66%). In the second
stage, exothermic degradation of one dicl occurs at 228-477 oC with exothermic DTA peaks at
321 and 343 oC (found 30.24%; calc. 29.76%). The next stage, from 477-650 oC, corresponds

to loss of one dicl ligand (found 27.65%; calc. 29.76%). The final decomposition product is
CuO (found 15.45%; calc. 16.03%).
3.4. Catechol oxidase activity
The oxidase biomimetic catalytic reactivity of the reported dinuclear copper
complexes (3 and 4) towards the aerobic oxidation of DTBCH2 under catalytic conditions was
studied using a UV-Vis spectrophotometer by following the appearance of the absorption
maximum of quinone at 395 nm.
In order to determine the catechol oxidase activity of the dinuclear copper complexes,
10−4 M solutions of 3 and 4 in DMF were treated with 50 equiv. of DTBCH2 under aerobic
conditions at room temperature. Time-dependent increases in quinone absorbance were
examined at 300-600 nm. In this study, 10-4 M complex and 5×10−3 M substrate were stirred at
equal volumes and quinone formation was determined by UV-Vis spectrophotometer at 395
nm. A blank experiment without catalyst did not show formation of the quinone up to 3 h in
DMF. Kinetic measurements were made spectrophotometrically on a UV-Vis
spectrophotometer, following the appearance of DTBQ (395 nm absorbance maximum,
ε = 1621 Lmol−1cm−1 in DMF). The kinetic experiments were conducted at 25 °C. The rate
determining step was found to change with the substrate to complex ratio. While the complex
concentrations were kept constant (1x10-4 M), substrate concentration was changed (50-40-
30-20-15-10-5x10-4 M). At least three runs were taken for each concentration and the initial
rates were determined from the slope of the tangent to the absorbance vs time curve at t = 0
(Fig. 7).
Turnover number (kcat), Michaelis-Menten constant (KM) and maximum velocity
(Vmax) can be calculated by means of Lineweaver-Burk plot. The Vmax values are 2.06x10-6
and 1.87x10-6 Ms-1; KM values 7x10-4 and 2.32x10-4 M; kcat values are 148.10 and 135.24
h−1 for 3 and 4, respectively. The kcat values for 3 and 4 have been compared with

reported dinuclear copper(II) complexes [62-66]. Our result is quite comparable with
literature, but much lower than that of the native enzyme catechol oxidase isolated from sweet
potatoes (Ipomoea batatas) (Kcat = 8.25x106 h−1) [67].
3.5. Carbonic Anhydrase Activity Assay
We synthesized four diclofenac complexes and evaluated their ability to inhibit hCA I
and hCA II over a wide range of concentrations (0.1–100 µM). IC50 values were determined
for the in vitro inhibition of hCA I and hCA II with the complexes, by the esterase method
with 4-NPA as substrate. These results are given in Table 4. Complexes 1-4 showed in vitro
inhibitory effects on hCA I and hCA II. 3 had the strongest inhibitory effects on hCA I and
hCA II. Inhibition types and Ki values were determined for the complexes using Lineweaver-
Burk curves for hCA I and hCA II (Fig. 8 and 9). 1 and 3 acted as uncompetitive inhibitors
with 4-NPA as substrate, 2 and 4 acted as noncompetitive inhibitors for hCA I. 1 and 2 acted
as uncompetitive inhibitors with 4-NPA as substrate, 3 and 4 acted as noncompetitive
inhibitors for hCA II. These complexes showed inhibition constants in the range of 1.52-55.06
µM for hCA I and 0.23-5.61 µM for hCA II ( Table 5). 1-4 showed more inhibitory effect
compared to the our previously reported Co(II) / Ni(II)-diclofenac complexes [21]. This
situtation indicated that the type of N-donor heterocyclic ligands affected the carbonic
anhydrase activity. In addition these complexes exhibited significant inhibition at low
concentration. Interaction of most CA isozymes with several types of phenols, such as simple
phenol and its substituted derivatives, clioquinol, salicyclates, some of their derivatives and
complexes derived from drugs has been investigated [21, 45, 50, 68].
4. Conclusions
The synthesis and characterization of the mononuclear cobalt(II), nickel(II) and
dinuclear copper(II) complexes with the non-steroidal anti-inflammatory drug diclofenac in
the presence of 2-pyridineethanol or 2-pyridinepropanol have been reported. Diclofenac is

bound to metal(II) ions via carboxylate oxygens. This coordination mode is supported with X-
ray diffraction data and FT-IR spectral results. The purity of the complexes was confirmed by
elemental analysis data. Thermal analysis data and characteristic IR vibrational bands of 1
were similar to those obtained for 2. These results demonstrated that 1 and 2 have similar
coordination mode and crystal structure. Structurally characterized [Co(dicl)2(2-pyet)2] and
[Ni(dicl)2(2-pyet)2] have distorted octahedral geometry; [Cu2(dicl)2(2-pyet)2] and
[Cu2(dicl)2(2-pypr)2] have square pyramidal geometry. Complexes are stable to approximately
200oC. Catechol oxidase activities of copper complexes have been investigated by using
DTBCH2 as substrate in DMF. 3 and 4 showed good catechol oxidase activity. The diclofenac
complexes represent a different class of hCA I and hCA II, and the results discussed in this
study may help design new analogs with enhanced activity. Furthermore, similar metal
complexes by preparing the NSAID and/or by adding mixed-atom donors as co-ligands could
be synthesized and characterized.
5. Supplementary Material
Supplementary data CCDC-1440607, 1440608 and 1440609 contains the
supplementary crystallographic data for complex 1, 3 and 4 respectively. 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.
Acknowledgement
The Research Foundation of Erzincan University is acknowledged for the financial
support to this work under the Project Number 110205.
Disclosure statement
No potential conflict of interest was reported by the authors.
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Figure Legends
Fig. 1. The molecular structure of the 1 showing the atom numbering scheme [symmetry
code: (i) 1-x, 1-y, 1-z]
Fig. 2. 2D layer formed by π…π interactions of 1.
Fig. 3. The molecular structure of the 3 showing the atom numbering scheme [symmetry
code: (i) 1-x, 1-y, 1-z]
Fig. 4. The packing formed by C-H…π interactions of 3.
Fig. 5. The molecular structure of the 4 showing the atom numbering scheme [symmetry
code: (i) 1-x, 1-y, 1-z]
Fig. 6. 2D layer formed by C-Cl…π interactions of 4.
Fig. 7. (a) Dependence of the reaction rates on DTBCH2 concentrations for the oxidation
reaction catalyzed by 3 and 4. (b) Lineweaver-Burk plot for the catalysis by 3 and 4.
Fig. 8. Determination of inhibition types and Ki values for the complexes using Lineweaver-
Burk curves (for hCA I).
Fig. 9. Determination of inhibition types and Ki values for the complexes using Lineweaver-
Burk curves (for hCA II).

Fig. 1

Fig. 2

Fig. 3

Fig. 4

Fig. 5

Fig. 6

Fig. 7

Fig. 8

Fig. 9

Table 1. Crystal data and structure refinement parameters for 1, 3 and 4
(1)
(3)
(4)
Empirical formula
Formula weight
Temperature (K)
Wavelength (Å)
Crytal system
Space group
Unit cell dimensions
a (Å)
C42H38Cl4N4O6Co
895.49
C42H36Cl4N4O6Cu2
961.63
C44H40Cl4N4O6Cu2
989.73
293
293
293
0.71073
monoclinic
P21/c
0.71073
monoclinic
P 21/c
0.71073
monoclinic
P 21/c
8.0798(3)
20.4071(7)
13.1154(5)
90
11.7479(5)
8.2759(2)
21.1225(9)
90
10.7735(4)
8.61585(23)
22.9349(8)
90
b (Å)
c (Å)
α (o)
β (o)
92.495(3)
90
114.011(3)
90
99.815(3)
90
γ (o)
V (Å)3
1975.43(13)
4
2023.57(13)
2
2126.86(12)
2
Z
A. coefficient (mm-1) 0.76
1.369
1.305
Dcalc (mg m-3)
1.506
1.578
1.5454
Crystal size (mm)
0.18; 0.42; 0.80
0.25;0.36;0.48
0.14; 0.42; 0.80
Theta range for data
collection (o)
1.70;28.03
1.76;27.31
1.78;27.31
Measured reflections 22334
17369
27147
Indepen. reflections
Absorpt. correction
11671
9880
12920
Integrationa
Integrationa
Integrationa
Full-matrix least-
squares on F2
Full-matrix least-squares
on F2
Full-matrix least-
squares on F2
Refinement method
Final R indices
0.0368
0.0454
1.156
0.0371
1.029
[F2 > 2σ(F2)]
Goodness-of-fit on F2 1.055
aStoe and Cie (2002) [54]