Synthesis, characterization, antimicrobial activity and carbonic anhydrase enzyme inhibitor effects of salicilaldehyde-N-methyl p-toluenesulfonylhydrazone and its Palladium(II), Cobalt(II) complexes

Article abstract of DOI:10.1016/j.saa.2014.04.121

We report the synthesis of the ligand, salicilaldehyde-N-methyl p-toluenesulfonylhydrazone (salptsmh) derived from p-toluenesulfonicacid-1- methylhydrazide (ptsmh) and its Pd(II) and Co(II) metal complexes were synthesized for the first time. The structure of the ligand and their complexes were investigated using elemental analysis, magnetic susceptibility, molar conductance and spectral (IR, NMR and LC-MS) measurements. Salptsmh has also been characterized by single crystal X-ray diffraction. ¹H and ¹³C shielding tensors for crystal structure were calculated with GIAO/DFT/B3LYP/6-311++G(d,p) methods in CDCl₃. The complexes were found to have general composition [ML₂]. The results of elemental analysis showed 1:2 (metal/ligand) stoichiometry for all the complex. Magnetic and spectral data indicate a square planar geometry for Pd(II) complex and a distorted tetrahedral geometry for Co(II) complexes. The ligand and its metal chelates have been screened for their antimicrobial activities using the disk diffusion method against the selected Gram positive bacteria: Bacillus subtilis, Bacillus cereus, Staphylococcus aureus, Enterococcus faecalis, Gram negative bacteria: Eschericha coli, Pseudomonas aeruginosa, Klebsiella pneumonia. The inhibition activities of these compounds on carbonic anhydrase II (CA II) and carbonic anhydrase I (CA I) have been investigated by comparing IC₅₀ and Ki values and it has been found that Pd(II) complex have more enzyme inhibition efficiency than salptsmh and Co(II) complex.

Full text of DOI:10.1016/j.saa.2014.04.121

Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 131 (2014) 294–302
Contents lists available at ScienceDirect
Spectrochimica Acta Part A: Molecular and
Biomolecular Spectroscopy
journal homepage: www.elsevier.com/locate/saa
Synthesis, characterization, antimicrobial activity and carbonic
anhydrase enzyme inhibitor effects of salicilaldehyde-N-methyl
p-toluenesulfonylhydrazone and its Palladium(II), Cobalt(II) complexes
⇑
Saliha Alyar , Sßevki Adem
Department of Chemistry, Science Faculty, Karatekin University, 18100 Çankırı, Turkey
h i g h l i g h t s
g r a p h i c a l a b s t r a c t
Salicilaldehyde-N-methyl p-toluenesulfonylhydrazone (salptsmh) derived from p-toluenesulfonicacid-
1-methylhydrazide (ptsmh) and its Pd(II) and Co(II) metal complexes were synthesized for the first time
and investigated their antibacterial activities and carbonic anhydrase enzyme inhibitor effects.
ꢀ
ꢀ
Synthesis of p-toluenesulfonicacid-1-
methylhydrazide (ptsmh).
Synthesis of salicilaldehyde-N-methyl
p-toluenesulfonylhydrazone
(salptsmh).
ꢀ
ꢀ
ꢀ
Synthesis of Co(salptsmh)2 and
Pd(salptsmh)2 complexes.
Characterization and antimicrobial
activities of compounds.
Carbonic anhydrase enzyme inhibitor
effects of compounds.
a r t i c l e i n f o
a b s t r a c t
Article history:
We report the synthesis of the ligand, salicilaldehyde-N-methyl p-toluenesulfonylhydrazone (salptsmh)
derived from p-toluenesulfonicacid-1-methylhydrazide (ptsmh) and its Pd(II) and Co(II) metal complexes
were synthesized for the first time. The structure of the ligand and their complexes were investigated
using elemental analysis, magnetic susceptibility, molar conductance and spectral (IR, NMR and LC-MS)
measurements. Salptsmh has also been characterized by single crystal X-ray diffraction. 1H and 13C
shielding tensors for crystal structure were calculated with GIAO/DFT/B3LYP/6-311++G(d,p) methods
in CDCl3. The complexes were found to have general composition [ML2]. The results of elemental analysis
showed 1:2 (metal/ligand) stoichiometry for all the complex. Magnetic and spectral data indicate a
square planar geometry for Pd(II) complex and a distorted tetrahedral geometry for Co(II) complexes.
The ligand and its metal chelates have been screened for their antimicrobial activities using the disk dif-
fusion method against the selected Gram positive bacteria: Bacillus subtilis, Bacillus cereus, Staphylococcus
aureus, Enterococcus faecalis, Gram negative bacteria: Eschericha coli, Pseudomonas aeruginosa, Klebsiella
pneumonia. The inhibition activities of these compounds on carbonic anhydrase II (CA II) and carbonic
anhydrase I (CA I) have been investigated by comparing IC50 and Ki values and it has been found that
Pd(II) complex have more enzyme inhibition efficiency than salptsmh and Co(II) complex.
Received 27 March 2014
Received in revised form 12 April 2014
Accepted 22 April 2014
Available online 29 April 2014
Keywords:
Sulfonamides
Pd(II)
Co(II) complexes
Disk diffusion method
CA I
CA II
Ó 2014 Elsevier B.V. All rights reserved.
⇑
Corresponding author. Tel.: +90 5396070947.
E-mail address: saliha@gazi.edu.tr (S. Alyar).
http://dx.doi.org/10.1016/j.saa.2014.04.121
1386-1425/Ó 2014 Elsevier B.V. All rights reserved.

S. Alyar, Sß. Adem / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 131 (2014) 294–302
295
(4000–400 cmÀ1) were recorded on a Mattson 1000 FT-IR Spectro-
photometer with samples prepared as KBr pellets. LC/MS-APCl was
recorded on an AGILENT 1100 Spectrometer. The melting points
were measured using an Opti Melt apparatus. TLC was conducted
on 0.25 mm silica gel plates (60F 254, Merck). The molar magnetic
susceptibilities were measured on powdered samples using Gouy
method. The molar conductance measurements were carried out
using a Siemens WPA CM 35 conductometer. All solvents were pur-
chased from Merck and reagents were obtained from Aldrich
Chem. Co. (ACS grade) and used as received. The experiments were
carried out in dynamic nitrogen atmosphere (20 mL minÀ1) with a
heating rate of 10 °C minÀ1 in the temperature range 30–400 °C
using platinum crucibles. The microdilution broth method was
used to determine the antibacterial activity of compounds
against the Gram positive bacteria B. subtilis ATCC 6633,
B. cereus NRRL-B-3711, S. aureus ATCC 6538, E. faecalis ATCC
29212, Gram negative bacteria: E. coli ATCC 11230, P. aeruginosa
ATCC 15442, K. pneumonia ATCC 70063.
Introduction
The importance of sulfonamide was realized [1] when sulfonyla-
mide, a key analog of sulfonamide, was reported [2] to be the first
antibacterial drug. Sulfonamides were the first effective chemo-
therapeutic agents employed systematically for the prevention
and the cure of bacterial infections in humans and other animal sys-
tems [3,4]. Later on, many thousands of molecules containing the
sulfanilamide structure have been created since its discovery, yield-
ing improved formulations with greater effectiveness and less tox-
icity. Sulfa drugs are still widely used for conditions such as acne
and urinary tract infections, and are receiving renewed interest
for the treatment of infections caused by bacteria resistant to other
antibiotics. Also, a number of other activities, some of which
have been recently observed, include endotelin antagonism, anti-
inflammatory activity, tubular transport inhibition, insulin release,
carbonic anhydrase and saluretic action, among others [5].
In order to find better compounds, some metal sulfonamides
have attracted much attention due to the fact that complexes
showed more activity than both free ligands and the corresponding
metallic salts. In particular Ag-sulfadiazine has proved to be an
effective topical antimicrobial agent, of significance in burn
therapy, better than the free ligand or than AgNO3 [6]. Moreover
several Cu(II), Ce(III), Bi(III), Cd(II) and Hg(II) sulfonamide com-
plexes have shown antibacterial activity [7–9]. Specially, a series
of copper complexes with heterocyclic sulfonamides was studied
and a plausible explanation of their activities was presented [10].
Carbonic anhydrases (CAs, EC 4.2.1.1) form a family of metalloen-
zymes that play an important function in various physiological
and pathological processes [11]. These enzymes are very efficient
catalysts for the reversible hydration of carbon dioxide to bicar-
bonate. Because of the wide distribution of the CAs in many cells,
tissues and organs, CAIs are widely used as target enzymes to treat
or prevent a multitude of diseases. For this reason, the discovery of
new CA inhibitors is very significant [12].
Synthesis
General synthesis method of the compounds was depicted
schematically in Fig. 1S. (Supporting Information).
Salicilaldehyde-N-methyl p-toluenesulfonylhydrazone (salptsmh)
Ethanol/ethyl acetate (1:1) solution of p-toluenesulfonic acid
1-methylhydrazide (1.5 g, 4.72 mmol) was added drop wise to an
ethanol/ethyl acetate (1:1) solution of salicilaldehyde (0.52 g,
5.0 mmol), maintaining the temperature at about 323 K. Then,
the mixture was stirred for 24 h at room temperature. The precip-
itated product was crystallized from ethanol/n-hexane (2:1) mix-
ture. The yellow crystalline solid was dried in vacuo and stored
at ethanol/n-hexane vapor (2:1). Yield 65%; mp: 155–157 °C.
Elemental analysis: Anal. Calcd. For C15H16SO3N2: C, 60.36; H,
5.70; N, 8.80; S, 10.07. Found: C, 59.80; H, 5.74; N, 9.20; S, 10.22.
In our previous studies, aliphatic/aromatic bis sulfonamides
were synthesized and tested for antimicrobial activity [13–16].
Also, we have reported conformational analysis and c spectroscopic
investigation of the methanesulfonic acid hydrazide [17] methane-
sulfonic acid 1-methylhydrazide [18] some methanesulfonylhyd-
razone derivatives [19–21]. In this work, Pd(II) and Co(II)
complexes of salicilaldehyde-N-methyl-p-toluenesulfonylhydraz-
one(salptsmh) derived from p-toluenesulfonicacid-1-methylhyd-
razide (ptsmh) were synthesized and characterized by using
elemental analyses, FT-IR, LC-MS spectrometric methods, magnetic
susceptibility and conductivity measurements method for the
compounds. Salptsmh has also been characterized by single crystal
X-ray diffraction. 1H and 13C shielding tensors for crystal structure
were calculated with GIAO/DFT/B3LYP/6-311++G(d,p) methods in
CDCl3. The antibacterial activities of synthesized compounds were
studied against Gram positive bacteria: B. subtilis ATCC 6633,
B. cereus NRRL-B-3711, S. aureus ATCC 6538, E. faecalis ATCC
29212, Gram negative bacteria: E. coli ATCC 11230, P. aeruginosa
ATCC 15442, K. pneumonia ATCC 70063 by using microdilution
method (as MICs) and disk diffusion method. The inhibition
activities of these compounds on carbonic anhydrase II (CA II) and
carbonic anhydrase I (CA I) have been investigated by comparing
IC50 and Ki values.
Synthesis of Pd(II), Co(II) complexes
All complexes are prepared by the following general method: a
sample of anhydrous 0.80 mmol MCl2, where M: Pd(II) and Co(II),
were dissolved in a mixture of methanol and acetonitrile (2/1,
30 mL) and a solution of salptsmh (2.0 mmol) in a mixture of
acetonitrile (2.0 mL) and NaOH solution in methanol (2.0 mL)
was added. The reaction mixture was heated at 60 °C for 1 h. The
complexes precipitated quickly after stirring the mixture at room
temperature and filtered off, dried in a desiccator over CaCl2.
Pd(salptsmh)2 (C30H30S2O6N4Pd): Yield 70%; mp: 287–289 °C.
Elemental analysis: Calcd. for C, 51.86; H, 4.62; N, 7.56; S, 8.65.
Found: C, 50.16; H, 4.45; N, 7.14; S, 8.85.
Co(salptsmh)2 (C30H30S2O6N4Co): Yield 80%; mp: 284–286 °C.
Elemental analysis: Calcd. for C, 55.40; H, 4.94; N, 8.08; S, 9.24.
Found: C, 55.31; H, 4.55; N, 8.22; S, 8.95.
Crystallography
For the crystal structure determination, single-crystal of
salptsmh was used for data collection on a four-circle Rigaku
R-AXIS RAPID-S diffractometer (equipped with a two-dimensional
area IP detector). Graphite-monochromated Mo
(k = 0.71073 Å) and oscillation scans technique with
K
a
D
radiation
w = 5° for
one image were used for data collection. The lattice parameters
Experimental
were determined by the least-squares methods on the basis of all
reflections with F2 > 2 (F2). Integration of the intensities, correc-
r
Physical measurements
tion for Lorentz and polarization effects and cell refinement was
performed using CrystalClear software [22]. The structures
were solved by direct methods using SHELXS-97 [23], and refined
The elemental analyses (C, H, N and S) were performed on a
LECO CHNS 9320 type elemental analyzer. The IR spectra
by
a full-matrix least-squares procedure using the program

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S. Alyar, Sß. Adem / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 131 (2014) 294–302
SHELXL-97 [23]. H atoms were positioned geometrically and
refined using a riding model. The final difference Fourier maps
showed no peaks of chemical significance.
enzyme activity under comparable assay and laboratory conditions
were calculated from inhibitor dose response curves. On esterase
activity, the Ki constants were calculated using the Cheng–Prusoff
equation.
Antimicrobial activity
Theoretical calculations
B. subtilis ATCC 6633, B. cereus NRRL-B-3711, S. aureus ATCC
6538, E. faecalis ATCC 29212, E. coli ATCC 11230, P. aeruginosa ATCC
15442, K. pneumonia ATCC 70063 cultures were obtained from Gazi
University, Biology Department. Bacterial strains were cultured
overnight at 37 °C in Nutrient Broth. During the survey, these stock
cultures were stored in the dark at 4 °C.
The molecular geometry optimizations and vibration frequency
calculations were performed with the Gaussian 03W software
package by using DFT approaches in addition to the determination
of crystal structure [24]. The split valence 6-311++G(d,p) basis set
was used for the expansion of the molecular orbital [25]. The
geometries were fully optimized without any constraint with the
help of an analytical gradient procedure implemented within the
Gaussian 03W program. All the parameters were allowed to relax
and all the calculations converged to an optimized geometry which
corresponds to a true energy minimum as revealed by the lack of
imaginary values in the wave number calculations. The 1H and
13C NMR chemical shifts of the compounds were calculated in
CDCl3 using the GIAO method. The energy gap of HOMO–LUMO
explains the prospective charge transfer interaction within the
molecule, and in this study, the frontier orbital energy gap has
obtained at B3LYP method using LanL2DZ basis set.
Disk diffusion method
The synthesize compounds and complexes were dissolved in
dimethylsulfoxide (20% DMSO) to
10 mg mLÀ1 and sterilized by filtration by 0.45
Antimicrobial tests were then carried out by the disk diffusion
method using 100
L of suspension containing 108 CFU mLÀ1 bac-
teria spread on a nutrient agar (NA) medium. The disks (6 mm in
diameter) were impregnated with 20 of each compound
(200
g/disk) at the concentration of 10 mg mLÀ1 and placed on
the inoculated agar. DMSO impregnated disks were used as nega-
tive control. Sulfamethoxazole (300 g/disk) sulfioxazole (300 g/
a
final concentration of
lm millipore filters.
l
l
L
l
l
l
disk) were used as positive reference standards to determine the
sensitivity of one strain/isolate in each microbial species tested.
The inoculated plates were incubated at 37 °C for 24 h for bacterial
strains isolates. Antimicrobial activity in the disk diffusion assay
was evaluated by measuring the zone of inhibition against the test
organisms. Each assay in this experiment was repeated twice [26].
Results and discussion
Crystal structure analysis
Molecular structure with the atom-numbering scheme of
salptsmsh was given in Fig. 1(a). Crystal data and structure refine-
ment parameters of salptsmsh were given in Table 1. Experimental
geometric parameters are given in Table 2. The crystal structure
contains intramolecular hydrogen bonds which play an important
role in stabilization, O4AH4Á Á ÁN2 = 2.588(5) Å, <(N3AH3Á Á ÁO3i) =
145.7°. The presence of an O4AH4Á Á ÁN2 intramolecular hydrogen
bond has been found to be a general feature in the molecular struc-
tures of sulfonylhydrazones, thiocarbazides and thiocarbazones
[30–33]. Thus, the length of the CAO bond is 1.364 Å which is con-
sistent with a single bond; while the C@N bond distance is 1.279 Å.
The geometry around S1 atom is significantly deviated from that of
regular tetrahedral, The maximum and minimum angles around S1
are 120.7(3)° and 105.6(2)°, respectively. In all essential details, the
geometry of the molecule regarding bond lengths and angles of
the compound are in good agreement with the values observed in
similar structures [20,34]. Unit cell content indicating the crystal
structure of the molecule is given in Fig. 1(b).
Micro-dilution assays
The minimal inhibition concentration (MIC) values, except one,
were also studied for the microorganisms sensitive to at least one
of the five compounds determined in the disk diffusion assay. The
inocula of microorganisms were prepared from 12 h broth cultures
and suspensions were adjusted to 0.5 McFarland standard turbid-
ity. The test compounds dissolved in dimethylsulfoxide (DMSO)
were first diluted to the highest concentration (2000
be tested, and then serial, twofold dilutions were made in a con-
centration range from 15.625 to 2000
g mLÀ1 in 10 mL sterile test
l
g mLÀ1) to
l
tubes containing nutrient broth. The MIC values of each compound
against bacterial strains were determined based on a micro-well
dilution method [27]. The 96-well plates were prepared by dis-
pensing 95
each well. One hundred
tially prepared at the concentration of 2400
into the first wells. Then, 100 L from each of their serial dilutions
was transferred into eight consecutive wells. The last well contain-
ing 195 L of nutrient broth without compound, and 5 L of the
inoculums on each strip, was used as negative control. The final
volume in each well was 200 L. The contents of the wells were
l
L of nutrient broth and 5
L from each of the test compounds ini-
g mLÀ1 was added
lL of the inoculums into
l
l
l
FT-IR spectra
l
l
IR spectra support the structure of the complexes by the
determination of the coordination modes. The changes in the char-
acteristic vibrations of the ligands were compared with complexes.
The important IR spectral bands of the compounds along with their
tentative assignments are given in Table 3. In the IR spectrum of the
ptsmh, strong bands observed at 3345, 3261 and 1640 cmÀ1 are
l
mixed and the micro-plates were incubated at 37 °C for 24 h. All
compounds tested in this study were screened twice against each
microorganism. The MIC was defined as the lowest concentration
of the compounds to inhibit the growth of microorganisms.
assigned to the
tas(NH2),
ts(NH2) and d(NH2) modes, respectively.
Enzyme assay and inhibition studies
Shifting of the
m
(C@N) frequency at 1620 cmÀ1 of the salptsmh to
a lower frequency by ꢁ18–24 cmÀ1 (1596–1602 cmÀ1) in all of its
metal complexes (Table 3) is the evidence of the nitrogen bonding
of the (C@N) group to the central metal atom. Shifting of the
(CAO) stretching frequency at 1244 cmÀ1 to a higher frequency
by ꢁ36–51 cmÀ1 (1280–1295 cmÀ1) in the complexes is also evi-
dence of the oxygen bonding of the (CAO) group to the metal atom.
This is further confirmed by the appearance of the new band at
555–570 cmÀ1 due to (MAO) stretching modes in the metal
Carbonic anhydrase activity was measured using esterase and
hydratase methods as described earlier [28,29]. To determine the
inhibitory potency of effects of compounds on hCA I and hCA II, five
different concentrations of compounds were added to the reaction
mixture and the enzyme activity was measured. All compounds
were examined in triplicate at each concentration used. The IC50
values as the inhibitor concentration that causes a 50% decline in

S. Alyar, Sß. Adem / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 131 (2014) 294–302
297
Fig. 1. The molecular structure of salptsmh with the atom-numbering scheme; displacement ellipsoids are drawn at the 50% probability level (a) and the crystal packing (b) of
salptsmh.
complexes. In the IR spectra of salptsmh, vibrational band observed
at 1144 cmÀ1 and 1333 cmÀ1 are assigned to asymmetric and sym-
metric SO2 stretching modes, respectively. Not shifting of symmet-
ric and asymmetric SO2 modes in the complexes is attributed to not
participating in coordination.
CH3AAr and NACH3 protons appeared at 2.425 ppm and
3.214 ppm were calculated at 2.354 ppm and 3.160 ppm. A signal
was also observed at d = 10.816 ppm of the phenolic HAOA group
and calculated at 5.101 ppm. The singlet peak belonging to the
azomethine CH@N proton at 8.749 ppm (calculated 7.26 ppm)
indicates the predominance of the phenolic-imine tautomer in
the salptsmh [20,36]. The 13C NMR spectrum of ptsmh, CH3AAr
and NACH3 carbon signals gave the following results: 20.390 ppm
and 33.476 ppm respectively. The 13C NMR spectra of salptsmh were
assigned at d 21.616 ppm, 33.590 ppm (calculated 21.025 ppm,
29.477 ppm respectively) for CH3AAr and NACH3 groups.
Azomethine CH@N carbon peak at 144.782 ppm (calculated
137.245 ppm) indicates the predominance for salptsmh. Signals
in the d 117.208–158.136 ppm region belong to aromatic ArAC-
atoms.
NMR spectra
The NMR spectra (1H, 13C) of ptsmh and salptsmh were measured
and interpreted in CDCI3. In order to facilitate the interpretation of
the NMR spectra, quantum-chemical calculations were performed
using B3LYP/6-311G++(d,p) basis set salptsmh in CDCI3 phase. Iso-
tropic shielding tensors of 13C and 1H were changed into chemical
shifts by using a linear relationship suggested by Ünal et al. [35].
The experimental and calculated chemical shift values are shown
in Table 4. The 13C NMR and 1H NMR spectrum of the salptsmh in
chloroform are given in Fig. 2. 1H NMR spectrum of ptsmh, CH3AAr
and NACH3 proton signals gave the following results: 2.263 ppm
and 3.419 ppm respectively. The NH2 proton shifts displayed as
singlet at 4.6 ppm. In Table 4, the 1H NMR spectrum of salptsmh,
Mass spectra
Fragmentation steps of all compounds in LC-MS spectra are
exhibitited in Figs. S2 and S3 (Supporting Information). The

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S. Alyar, Sß. Adem / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 131 (2014) 294–302
Table 1
Table 4
Crystal data and structure refinement for salptsmh.
The experimental and theoretical 1H and 13C NMR chemical shifts
d (ppm) for
salptsmh.
Empirical formula
Formula weight
Temperature
C15H16N2O3S
304.37
293(2) K
Assignment
B3LYP/6-311++G(d,p)a
d (exp.)
d (calc.)
Wavelength
Crystal system
Space group
0.71073 Å
Tetragonal
I 41/a
a = 28.9612(2) Å a = 90°
b = 28.9612(2) Å b = 90°
C-1
C-2
C-3
C-4
C-5
C-6
C-7(CH3)
C-8(CH3AN)
C-9(C@N)
C-10
C-11
C-12
C-13
C-14
C-15
H-2
H-3
H-5
132.267
131.206
131.842
149.211
131.842
131.206
21.616
33.590
144.782
119.439
158.136
117.548
128.035
117.208
129.958
7.762
7.219
7.215
7.742
2.425
3.214
8.749
6.926
7.285
138.925
128.789
129.079
146.652
128.800
128.789
21.025
29.477
137.245
122.321
155.32
115.569
129.823
119.789
133.967
8.387
7.218
7.460
7.747
2.356
3.160
7.26
6.751
7.201
Unit cell dimensions
c = 7.2364(1) Å
6069.54(12) Å3
16
c = 90°
Volume
Z
Density (calculated)
Absorption coefficient
F(000)
Crystal
Crystal size
h Range for data collection
Index ranges
1.332 g/cm3
0.224 mmÀ1
2560
Needle; yellow
0.20 Â 0.02 Â 0.02 mm3
2.8–26.4°
À36 6 h 6 36, À36 6 k 6 36, À9 6 l 6 7
20,324
1321[Rint = 0.1166]
99.4%
0.948 and 0.978
Full-matrix least-squares on F2
1321/0/193
Reflections collected
Independent reflections
Completeness to h = 26.4°
Max. and min. transmission
Refinement method
Data/restraints/parameters
Goodness-of-fit on F2
H-6
H-7(CH3)
H-8(CH3AN)
H-9(CH@N)
H-12
H-13
H-14
0.989
Final R indices [F2 > 2
R indices (all data)
Extinction coefficient
r
(F2)]
R1 = 0.076, wR2 = 0.167
R1 = 0.180, wR2 = 0.220
0.00
6.908
7.291
6.895
7.212
Largest diff. peak and hole
0.243 and À0.234 e ÅÀ3
H-15
OH
10.816
5.101
a
r
Transform into d using equations given in Ref. [35]; d13C = 175.7–0.963
r
13C
and d1H = 31.0–0.970 1H.
r
Table 2
Selected bond distances (Å) and angles (°) for salptsmh.
The bond lengths (Å)
S(1)AO(2) 1.421(4)
S(1)AC(1) 1.748(5)
N(2)AN(1) 1.374(6)
C(2)AH(2) 0.930(5)
C(2)AC(3) 1.385(8)
C(12)AH(12) 0.930(6)
C(12)AC(13) 1.390(9)
C(11)AO(4) 1.364(6)
S(1)AO(1) 1.427(4)
S(1)AN(1) 1.680(4)
N(2)AC(9) 1.278(7)
C(2)AC(1) 1.372(7)
C(1)AC(6) 1.384(7)
C(12)AC(11) 1.372(7)
N(1)AC(8) 1.467(7)
atom and fragment III is observed 303.5 (56%) from the removal
of the two (phen+ ÀM) groups.
Electronic spectra and magnetic behavior
The reaction of Schiff base with transition metal ions in 1:2 M
ratio lead to the formation of Schiff base sulfonamides complexes
of the types, [ML2] (M = Co(II), Pd(II)) (Fig. S1) (Supporting
Information). The molar conductivities (Km) of 10À3 M solutions
of the complexes were measured in DMSO at room temperature.
Conductivity results show that complexes are non-electrolyte.
The significant electronic spectra of the ligand and complexes were
recorded in DMSO. The important bands of the ligands and the com-
plexes were observed in the region of 293–260 and 430–332 nm.
The magnetic moments of the complexes (as B.M.) were measured
at room temperature. The electronic spectrum of the Pd(II) complex
353–354 nm, attributed to the 1A2g ? 1B1g and 3B1g ? 1A1g transi-
tions respectively, indicated a square planar environment around
the metal ion. The observed magnetic moments of the Palladium(II)
complex were 0 B.M. The magnetic moment value (3.89 B.M.) for
Co(II) complex, the broad band in its electronic spectrum centered
at 425 nm, suggested a tetrahedral geometry around the Co(II) ion.
Also, the 4T1(F) ? 4A2 transition observed in the tetrahedral
geometry [37].
The bond angles (°)
O(2)AS(1)AO(1) 120.7(3)
O(2)AS(1)AN(1) 105.6(2)
O(1)AS(1)AN(1) 105.9(2)
N(1)AN(2)AC(9) 122.6(4)
C(1)AC(2)AC(3) 119.7(5)
S(1)AC(1)AC(6) 120.1(4)
O(2)AS(1)AC(1) 109.4(3)
O(1)AS(1)AC(1) 108.5(3)
C(1)AS(1)AN(1) 105.7(2)
N(2)AN(1)AC(8) 120.2(4)
S(1)AC(1)AC(2) 119.9(4)
Table 3
Major IR absorption bands (cmÀ1) of the compounds.
Assignment
Ptsmh
Salptsmh
Co(salptsmh)2
Pd(salptsmh)2
t
t
t
t
t
t
t
(CAH)ar
(C@N)
(C@C)
as(SO2)
(CAO)
s(SO2)
3040
–
–
1330
–
1140
–
3045
1620
1575
1333
1244
1144
–
2940
1602
1572
1331
1295
1148
555
2935
1596
1570
1329
1280
1142
570
(MAO)
Frontier molecular orbital analysis
salptsmh gives the following fragmentation peaks: 304.8(15.5%) as
molecular ion peak by losing of one proton, 289.9(18.9%) which
occurs by losing of methyl group, 198.6 (100%) belongs to ptsmh
(C8H12SO2N2). Pd(salptsmh)2 gives the following peaks: 711.8
(8.9%) and 605.8 (20.5%) which occurs by losing of Pd atom and
fragment III is observed 303.1 (100%) from the removal of the
two (phen+ ÀM) groups. Co(salptsmh)2 gives the following peaks:
664.90 (12.6%) and 604.9 (27.1%) which occurs by losing of Co
The calculations indicate that the ligand and complex com-
pounds have 80 and 158 occupied molecular orbital, respectively.
The energy band gaps between the highest occupied molecular
orbital (HOMO) and the lowest unoccupied molecular orbital
(LUMO) energies for mentioned compounds, have been calculated
and is given in Fig. S4. The frontier molecular orbitals play an
important role in the electronic and optical properties, as well as

S. Alyar, Sß. Adem / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 131 (2014) 294–302
299
Fig. 2. (a) 1H NMR spectrum of the salptsmh, (b) 13C NMR spectrum of the salptsmh.
in UV–vis spectra and chemical reactions [38]. Also, the energy
band gap between HOMO and LUMO is a critical parameter in
determining molecular electrical transport properties and elec-
tronic systems with a larger HOMO–LUMO gap should be less reac-
tive than those with a smaller gap [39]. The energy band gap of
HOMO–LUMO explains the prospective charge transfer interaction
within the molecule, and in this study, the frontier orbital energy
band gap in case of ligand compound is found to be 4.18 eV
obtained at B3LYP method using LanL2DZ basis set. Also, the fron-
tier orbital energy gap in case of Co(II) and Pd(II) complex was
found to be 2.54 and 2.14 eV obtained at B3LYP method using
LanL2DZ basis set, respectively. The case of complex energy band
gap is reduced by about 40–50%.
LUMO energy and DE(HOMO–LUMO) band gap are important factors
for activities of sulfonamide compounds.
The biological activity is reverse relationship to the
DE(HOMO–
band gap. The biological activity and Carbonic anhydrase
LUMO)
activity of the studied compounds increase with the lower
E(HOMO–LUMO) band gap. Respective values of E(HOMO–LUMO)
band gap and biological activity are in following orders. For
E(HOMO–LUMO) band gap: salptsmh > Co(salptsmh)2 > Pd(salptsmh)2
D
D
D
and for biological activity and Carbonic anhydrase activity:
salptsmh < Co(salptsmh)2 < Pd(salptsmh)2.
Biological activity
The LUMO energy is very important descriptors which describe
electrophilicity of the compound and its level has the importance
because of the donor–acceptor interactions. Molecules with lower
LUMO energy values accept the electrons more easily than high-
The test compounds were screened in vitro for their antibacte-
rial activity against three Gram-positive species (B. subtilis,
S. aureus, E. faecalis) and three Gram-negative species (E. coli, P.
aeruginosa, K. pneumonia) of bacterial strains by the microdilution
and disk diffusion methods (Tables 5 and 6). The results were
compared with those of the standard drugs sulfamethoxazole
er’s. The lower LUMO energy and larger
DE(HOMO–LUMO) band gap
affect the binding affinities to the biologic molecules, therefore

300
S. Alyar, Sß. Adem / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 131 (2014) 294–302
Table 5
The MICs of antibacterial activity of the compounds.
Bacteria strains
MIC lg/mL (mM)
Salptsmh
Co(salptsmh)2
Pd(salptsmh)2
Sulfamethoxazole
Sulfisoxazole
Gram-negative
Eschericha coli ATCC 11230
Pseudomonas aeruginosa ATCC 15442
Klebsiella pneumonia ATCC 70063
Gram-positive
500 (1.64)
500 (1.64)
250 (0.82)
125 (0.187)
125 (0.187)
125 (0.187)
62.5 (0.087)
125 (0.175)
62.5 (0.087)
64 (0.25)
64 (0.25)
16 (0.063)
23.4 (0.088)
375 (1.40)
23.4 (0.088)
Bacillus subtilis ATCC 6633
Bacillus cereus NRRL-B-3711
Staphylococcus aureus ATCC 6538
Enterococcus faecalis ATCC 29212
250 (0.82)
500 (1.64)
500 (1.64)
250 (0.82)
62.5 (0.094)
62.5 (0.094)
125 (0.187)
250 (0.0374)
62.5 (0.087)
250 (0.350)
62.5 (0.087)
250 (0.350)
1500 (5.92)
16 (0.063)
32 (0.126)
32 (0.126)
–
375 (0.088)
93.75 (0.35)
93.75 (0.35)
Table 6
Measured inhibition zone diameter (mm) of the compounds and antibiotics by disk diffusion method.
Bacteria strains
Diameter inhibition zone a(mm.200
lg/disk)
Salptsmh
Co(salptsmh)2
Pd(salptsmh)2
Sulfamethoxazole
Sulfisoxazole
Gram-negative
Eschericha coli ATCC 11230
Pseudomonas aeruginosa ATCC 15442
Klebsiella pneumonia ATCC 70063
10
13
11
16
15
16
19
16
21
17
17
24
20
8
28
Gram-positive
Bacillus subtilis ATCC 6633
Bacillus cereus NRRL-B-3711
Staphylococcus aureus ATCC 6538
Enterococcus faecalis ATCC 29212
14
11
10
12
17
16
15
12
20
15
19
16
–
–
28
15
15
17
25
18
<10: weak; >10: moderate; >16: significant.
a
Average values.
Fig. 3. (a) Comparison of antibacterial activite (MIC) and (b) Comparison of antibacterial activite (disk).

S. Alyar, Sß. Adem / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 131 (2014) 294–302
301
Table 7
Inhibitor effects of compounds for the esterase and hydratase activities of human CA-I and II.
Compounds
hCA I
hCA II
Esterase activity
Hydratase activity
IC50 M)
Esterase activity
Hydratase activity
IC50
(lM)
Ki (
l
M)
(l
IC50
(lM)
Ki (
l
M)
IC50
74
(lM)
Salptsmh
431 22 (32–164)
69 1.2 (15–90)
402 26 (179–539)
0.360 0.05 (0.072–0.72)
280
2.32
13.49
0.240
Uneffect
9.6 0.03
82.6 0.03
0.108 0.02 (0.042–0.21)
113 3.8 (32–164)
25 1.4 (7–38)
130 14 (85–426)
0.200
0.044
3
Pd(salptsmh)2
Co(salptsmh)2
Acetazolamide
1.5 0.6
13 0.1
0.0053 0.0009 (0.0021–0.01)
0.230
0.015 0.003 (0.0036–0.036)
2.8 Â 10À5
and sulfioxazole (Fig. 3). As the disk diffusion assay results evi-
dently show that Pd(salptsmh)2 has exhibited the strong inhibition
effect against most of test bacteria whereas salptsmh has weaker
activity. Similar results were also reported by Özmen et al. [40].
The ligand and its complexes show the highest activities against
B. subtilis which is the mostly effected by Pd(salptsmh)2 having the
diameter zone of 20 mm. All compounds have moderate activity
against P. aeruginosa at in the diameter zone of 13–16 mm whereas
sulfisoxazole, the drug used as standard, has been found less active
(8 mm) against the bacteria mentioned above. According to the
MIC’s results shown in Table 5 the compounds possess a broad
spectrum of activity against the tested bacteria at the concentra-
moderately inhibited by Co(salptsmh)2. Synthesized sulfonamide
showed weak inhibitory effect on most abundant and rapid iso-
form hCA II, whereas this derivative did not show any inhibitor
effect on isoform hCA I. The new compounds showed weak hCA I
and II inhibitory efficiency as compared to the clinically used acet-
azolamide. These data clearly inform that palladium may be
enhanced the activity of the sulfonamides complexes.
Conclusions
In this study we have reported the synthesis of salptsmh and its
Co(II) and Pd (II) complexes. The structural characterizations of the
synthesized compounds were made by using the elemental analy-
ses, spectroscopic methods, magnetic and conductance studies.
The biological activity screening showed that complexes have
more activity than ligands against the tested bacteria. All com-
pounds have excellent activity against P. aeruginosa. Pd(salptsmh)2
shows good activity while Co(salptsmh)2 has moderate activity
against E. coli. Co(salptsmh)2 and salptsmh showed poor inhibitory
activity when compared to Pd(salptsmh)2 against CA II. Although
tions of 62.5–500
have shown activity against P. aeruginosa and B. cereus at a concen-
tration of 62.5 g/mL; 125 g/mL whereas sulfisoxazole, the drug
lg/mL [41]. The Pd(salptsmh)2 and Co(salptsmh)2
l
l
used as standard, has been found less active against the bacteria.
Also, the antimicrobial activity is highly influenced by the nature
of the salptsmh and its complexes the order of the activity in mM
for all test bacteria is as follows (Table 5).
Percentage of inhibition for the compounds exhibited in
Fig. 3(a) that was expressed as excellent activity (120–200% inhibi-
tion), good activity (90–100% inhibition), moderate activity (75–
85% inhibition), significant activity (50–60% inhibition), negligible
activity (20–30% inhibition) and no activity [42]. As seen in
Fig. 3, all compounds have excellent activity against P. aeruginosa.
Pd(salptsmh)2 shows good activity while Co(salptsmh)2 has moder-
ate activity against E. coli.
the case of complex the
DE(HOMO–LUMO energy band gap is reduced
by about 40–50%, the biological activity and Carbonic anhydrase
activity of the studied compounds increased in a manner inversely
proportional.
Acknowledgments
The authors would like to thank Karatekin University BAP
(Grant No: 05/2011-05) for the financial support of this project.
We would like to thank also to Dr. Ertan Sßahin for XRD
measurement.
Carbonic anhydrase activity results
Sulfonamides compose an important class of carbonic anhy-
drase inhibitors. Some compounds such as sulfapyridine, sulfadia-
zine and acetazolamide are used as medicaments. Therefore,
different sulfonamide derivatives have been synthesized and
investigated for their inhibition activity on the carbonic anhydrase
isoenzymes. These new compounds were tested to determine their
inhibitory effects on the hydratase and esterase activities of cyto-
solic CA isozymes. Table 7 depicted the in vitro effects of com-
pounds on esterase and hydratase activities of hCAI and hCAII.
Compounds showed different inhibitory effect on the esterase
and hydratase activity of enzymes.
Appendix A. Supplementary material
Crystallographic data that were deposited in CSD under CCDC
registration number 894662 contain the supplementary crystallo-
graphic data for this Letter. These data can be obtained free of
charge from the Cambridge Crystallographic Data Centre (CCDC)
via www.ccdc.cam.ac.uk/data_request/cif and are available free of
charge upon request to CCDC, 12 Union Road, Cambridge, UK
(fax: +44 1223 336033, e-mail: deposit@ccdc.cam.ac.uk). Supple-
mentary data associated with this article can be found, in the
online version, at http://dx.doi.org/10.1016/j.saa.2014.04.121.
For esterase activity, the compound Pd(salptsmh)2 behave as
potent inhibitors against hCA I and hCA II with IC50 value 69 and
25 lM, respectively, whereas compound Co(salptsmh)2 and sal-
ptsmh showed poor inhibitory activity when compared to Pd(sal-
ptsmh)2. Effect of the Pd(salptsmh)2 at different concentrations on
esterase and hydratase activity of hCA-I and hCA-II given in
Fig. S5. Compounds Co(salptsmh)2 and salptsmh exhibit the same
potency as hCA II inhibitors, with IC50 values in the range of
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