Synthesis, characterization, computational studies, antimicrobial activities and carbonic anhydrase inhibitor effects of 2-hydroxy acetophenone-N-methyl p-toluenesulfonylhydrazone and its Co(II), Pd(II), Pt(II) complexes

Article abstract of DOI:10.1016/j.molstruc.2016.07.122

2-Hydroxyacetophenone-N-methyl p-toluenesulfonylhydrazone (afptsmh) derived from p-toluenesulfonicacid-1-methylhydrazide (ptsmh) and its Co(II), Pd(II), Pt(II) complexes were synthesized for the first time. Synthesized compounds were characterized by spec

Full text of DOI:10.1016/j.molstruc.2016.07.122

Accepted Manuscript
Synthesis, characterization, computational studies, antimicrobial activities and
carbonic anhydrase inhibitor effects of 2-hydroxy acetophenone-N-methyl p-
toluenesulfonylhydrazone and its Co(II), Pd(II), Pt(II) complexes
Neslihan Özbek, Saliha Alyar, Burcu Koçak Memmi, Ayla Balaban Gündüzalp, Zafer
Bahçeci, Hamit Alyar
PII:
S0022-2860(16)30816-X
10.1016/j.molstruc.2016.07.122
MOLSTR 22825
DOI:
Reference:
To appear in: Journal of Molecular Structure
Received Date: 19 March 2016
Revised Date: 28 July 2016
Accepted Date: 31 July 2016
Please cite this article as: N. Özbek, S. Alyar, B.K. Memmi, A.B. Gündüzalp, Z. Bahçeci, H. Alyar,
Synthesis, characterization, computational studies, antimicrobial activities and carbonic anhydrase
inhibitor effects of 2-hydroxy acetophenone-N-methyl p-toluenesulfonylhydrazone and its Co(II), Pd(II),
Pt(II) complexes, Journal of Molecular Structure (2016), doi: 10.1016/j.molstruc.2016.07.122.
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ACCEPTED MANUSCRIPT
2-Hydroxyacetophenone-N-methyl p-toluenesulfonylhydrazone (afptsmh) and its Co(II),
Pd(II) and Pt(II) metal complexes were synthesized for the first time and investigated their
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antibacterial activities and carbonic anhydrase enzyme inhibitor effects. Also H and 13C
shielding tensors for crystal structure were calculated with GIAO/DFT/B3LYP/6-
311++G(d,p) methods in CDCl3.

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Synthesis, characterization, computational studies, antimicrobial activities
and carbonic anhydrase inhibitor effects of 2-hydroxy acetophenone-N-
methyl p-toluenesulfonylhydrazone and its Co(II), Pd(II), Pt(II) complexes
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Neslihan Özbek1✉, Saliha Alyar2, Burcu Koçak Memmi3, Ayla Balaban Gündüzalp4,
Zafer Bahçeci 1, Hamit Alyar5
✉ Neslihan Özbek: ege_nesliozbek@hotmail.com
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1Department of Primary Education, Faculty of Education, Ahi Evran University, 40100
Kırşehir, Turkey
2Department of Chemistry, Science Faculty, Karatekin University, 18100 Çankırı, Turkey
3 Insecticide Test Laboratory, Hacettepe University, 06800 Ankara, Turkey
4Department of Chemistry, Science Faculty, Gazi University, 06500, Ankara, Turkey
5Department of Physics, Science Faculty, Karatekin University, 18100 Çankırı, Turkey
ABSTRACT
2-Hydroxyacetophenone-N-methyl p-toluenesulfonylhydrazone (afptsmh) derived from p-
toluenesulfonicacid-1-methylhydrazide (ptsmh) and its Co(II), Pd(II), Pt(II) complexes were
synthesized for the first time. Synthesized compounds were characterized by spectroscopic
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methods (FT-IR, H-13C NMR, LC-MS, UV-vis), magnetic susceptibility and conductivity
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measurements. H and 13C shielding tensors for crystal structure of ligand were calculated
with GIAO/DFT/B3LYP/6-311++G(d,p) methods in CDCl3. The vibrational band
assignments were performed at B3LYP/6-311++G(d,p) theory level combined with scaled
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quantum mechanics force field (SQMFF) methodology. The antibacterial activities of
synthesized compounds were studied against some Gram positive and Gram negative bacteria
by using microdilution and disc diffusion methods. In vitro enzyme inhibitory effects of the
compounds were measured by UV–vis spectrophotometer. The enzyme activities against
human carbonic anhydrase II (hCA II) were evaluated as IC50 (the half maximal inhibitory
concentration) values. It was found that afptsmh and its metal complexes have inhibitory
effects on hCA II isoenzyme. General esterase activities were determined using alpha and
beta naphtyl acetate substrates (α- and β-NAs) of Drosophila melanogaster (D.
melanogaster). Activity results show that afptsmh does not strongly affect the bacteria strains
and also shows poor inhibitory activity against hCAII isoenzyme whereas all complexes
posses higher biological activities.
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Keywords: Sulfonamides, B3LYP, disc diffusion method, MICs, hCA II, D. melanogaster
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1. Introduction
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Sulfonamides have been intensively investigated as the first effective antibacterial [1,2] and
chemotherapeutic agents employed systematically for the prevention and the cure of bacterial
infections in humans and other animal systems [3,4]. Sulfamethoxazole with trimethoprim
(SMX/TMP) is a drug combination with broad-spectrum of antibacterial activities against
both Gram positive and Gram negative organisms. SMX/TMP is currently the most effective
therapeutic agent against P.J. pneumonia in patients with AIDS [5]. Later on, many
thousands of molecules containing sulfanilamide group have been created yielding
formulations with greater effectiveness and less toxicity. Sulfa drugs are still widely used for
conditions such as acne and urinary tract infections and they have great interest for the
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treatment of infections having bacterial resistant to other antibiotics. Also, the other activities
include endotelin antagonism, anti-inflammatory activity, tubular transport inhibition, insulin
release, carbonic anhydrase and saluretic actions [8]. Some metal sulfonamides have been
attracted much attention due to higher activities than free ligands and the corresponding
metallic salts. In particular, Ag-sulfadiazine has been proved to be an effective topical
antimicrobial agent having significance in burn therapy with better activities than free ligand
or AgNO3 [9]. Moreover, several Cu(II), Ce(III), Bi(III), Cd(II) and Hg(II) sulfonamide
complexes were evaluated for their antibacterial activities [10-12]. One of the most important
activity of the sulfonamides is the enzyme inhibition effect to zinc containing metalloenzyme
named as carbonic anhydrase (CA). Many sulfonamide CA inhibitors have been used as
antiglaucoma, diuretic, antiobesity and antitumor agents and various neurological disorders
[13-15].
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In our previous studies, aliphatic/aromatic bis sulfonamides were synthesized and tested for
antimicrobial activities [16-29]. Also, we have reported conformational analysis and
vibrational spectroscopic investigation of the methanesulfonic acid hydrazide, [20]
methanesulfonic acid 1-methylhydrazide [21] and some methanesulfonylhydrazone
derivatives
[22,23].
In
this
work,
2-hydroxyacetophenone-N-methyl
p-
toluenesulfonylhydrazone (afptsmh) derived from p-toluenesulfonicacid-1-methylhydrazide
(ptsmh) and its Co(II), Pd(II), Pt(II) complexes were synthesized and characterized by using
elemental analyses, spectrometric methods (FT-IR, 1H-13C NMR, LC–MS, UV-vis), magnetic
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susceptibility and conductivity measurements. H and 13C shielding tensors for crystal
structure of ligand were calculated with GIAO/DFT/B3LYP/6-311++G(d,p) methods in
CDCl3. The vibrational band assignments were performed at B3LYP/6-311++G(d,p) theory
level combined with scaled quantum mechanics force field (SQMFF) methodology. The
antibacterial activities of compounds were studied against Gram positive species; B.subtilis
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ATCC 6633, B. cereus NRRL-B-3711, S. aureus ATCC 6538, E. faecalis ATCC 29212, S.
agalactiae ATCC 13813 and Gram negative species; E. coli ATCC 11230, P. aeruginosa
ATCC 15442, K. pneumonia ATCC 70063 by using microdilution (as MICs) and disc
diffusion (as mm zone) methods. The inhibition degrees of the compounds on carbonic
anhydrase II (hCA II) have been evaluated as IC50 (the half maximal inhibitory concentration)
and we also report the α esterase activities and the β- esterase activities from the model of
insect D. melanogaster.
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2. Experimental
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2.1. Instrumentation
The elemental analyses (C, H, N and S) were performed on a LECO CHNS 9320 type
elemental analyzer. The IR spectra (4000-400 cm-1) were recorded on a Mattson 1000 FT-IR
Spectrophotometer with samples prepared as KBr pellets. LC/MS-APCI 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 purchased from Merck and reagents were obtained from
Aldrich Chem. Co. (ACS grade) and used as received. The microdilution broth method was
used to determine antibacterial activities of the compounds against Gram positive species; B.
subtilis ATCC 6633, B. cereus NRRL-B-3711, S. aureus ATCC 6538, E. faecalis ATCC
29212, S. agalactiae ATCC 13813 and Gram negative species; E. coli ATCC 11230, P.
aeruginosa ATCC 15442, K. pneumonia ATCC 70063. The enzyme activity measurements
were also performed by using microplate reader (Biotek spectrophotometer, Vermont, USA).
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2.2. Synthesis
p-Toluenesulfonic acid 1-methylhydrazide(ptsmh)
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p-Toluenesulfonyl chloride (0.04 mol, 7.626 g ) in tetrahydrofuran (30 mL) was added
dropwise to solution of methylhydrazine (0.05 mol, 1.60 mL) in ethanol/ethyl acetate (1:1)
while the temperature was maintained between 268-273 K. The mixture was stirred for 24 h
mean while the completion of the reaction was monitored by TLC and then, the solvent was
evaporated. The colorless crude compound was purified in THF/n-hexane (1:1) by column
chromatography and then the product was recrystallized from THF/n-hexane mixture (1:1)
[24]. Yield 72%; mp: 115–1170C. Elemental analysis results for C8H12N2O2S: (Calc.%) C,
47.98; H, 6.04; N, 13.99; S, 16.01. (Found%): C, 45.55; H, 6.20; N, 14.25; S, 17.10. LC-MS:
m/z (abudance %) [M+1]+ :200.15 (54%), [M-N(CH3)-NH2]+ :155.85 (100%).
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2-Hydroxyacetophenone-N-methyl p-toluenesulfonylhydrazone (afptsmh)
p-Toluenesulfonic acid 1-methylhydrazide (1.5 g, 4.72 mmol) in ethanol/ethyl acetate (1:1)
solution was added dropwise to solution of 2'-hydroxyacetophenone (0.52 g, 5.0 mmol) in
ethanol/ethyl acetate (1:1) maintaining the temperature at about 323 K. Then, the mixture was
stirred for 24 h at room temperature. The precipitated product was recrystallized from
ethanol/n-hexane (2:1) mixture. The yellow crystalline solid was dried in vacuo to remove
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ethanol/n-hexane vapour. Yield 65%. Mp. 155-157 C. Elemental analysis for C16H18N2O3S
(Calc.%) 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. LC-
MS: m/z (abudance %) 319.8 [M+2]+:319.8 (25.5%), [M-AfOH]+: 198.6 (100%).
Synthesis of the complexes
All metal complexes were prepared by the following general method: To solution of afptsmh
(2.0 mmol) in acetonitrile (2.0 mL), anhydrous 0.80 mmol MCl2 (where M: Pt(II), Pd(II) and
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Co(II)) dissolved in methanol/acetonitrile (2:1, 30 mL) was added and then, NaOH in
methanol (2 mL) was added to obtain alkaline media. The reaction mixture was heated at 60
0C for one hour by stirring on magnetic stirrer. The metal complexes were precipitated at
room temperature and filtered off, dried in a desiccator over CaCl2.
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Trans-bis(2-Hydroxyacetophenone-N-methyl
p-toluenesulfonylhydrazonato)platinum(II)
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(Pt(afptsmh)2) Yield 85%. Mp. 317-319 C. Elementel analysis for C32H34N4O6S2Pt (Calc.%)
C 46.32, H 4.13, N 6.75, S 7.73, Pt 23.51. (Found%) C 48.10, H 3.80, N 6.95, S 7.24, Pt
23.48. APCL-MS: m/z (abudance %) [M+1]+: 830.2 (14.8%).
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Trans-bis(2-Hydroxyacetophenone-N-methyl
p-toluenesulfonylhydrazonato)palladium(II)
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(Pd(afptsmh)2) Yield 70%. Mp. 287-289 0C. Elemental analysis for C32H34N4O6S2Pd (Calc.%)
C 51.86, H 4.62, N 7.56, S 8.65, Pd 14.36 (Found%) C 50.16, H 4.45, N 7.14, S 8.85, Pd
14.28. APCL-MS m/z (abudance%) [M+1]+: 741.8 (19.9%)
Trans-bis(2-Hydroxyacetophenone-N-methyl p-toluenesulfonylhydrazonato)cobalt(II)
(Co(afptsmh)2) Yield 80%. Mp. 284-286 0C. µeff:3.89 BM. Elemental analysis for
C32H34N4O6S2Co (Calc.%) C 55.40, H 4.94, N 8.08, S 9.24, Co 8.50. (Found%) C 55.31, H
4.55, N 8.22, S 8.95, Co 8.45. APCL-MS m/z (abudance%) [M+1]+: 694.1(12.6%).
2.3. Computational Details
The molecular geometry optimizations, frontier molecular orbital (HOMO and LUMO)
energies and vibration frequency calculations for afptsmh were performed with Gaussian 03W
software package by using DFT approaches [25]. The split valence 6-311++G (d, p) basis set
was used for the expansion of the molecular orbitals [26]. The geometries were fully
optimized (as seen in Fig.1a) without any constraint with the help of analytical gradient
procedure implemented within Gaussian 03W program. All the parameters were allowed to
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relax and all the calculations were converged for optimized geometry which corresponds to
true energy minimum as revealed by the lack of imaginary values in the wave number
calculations. The vibrational band assignments were performed at B3LYP/6-311++G (d,p)
theory level combined with scaled quantum mechanics force field (SQMFF) methodology.
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The H and C NMR chemical shifts of the compounds were calculated in CDCl3 using the
GIAO method. Also, HOMO and LUMO energies of the compounds were calculated using
DFT procedure with the B3LYP/LanL2DZ level. The structure sketches of the complexes
were presented at Fig. 1b.
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2.4. Procedure for Antibacterial Activity
Microdilution assay
The minimal inhibition concentration (MIC) is defined as the lowest concentration of the
compounds to inhibit the growth of the microorganisms. The inocula of microorganisms were
prepared from 12 h broth cultures and suspensions were adjusted to 0.5 McFarland standard
turbidity. The tested compounds dissolved in 20% DMSO were first diluted to the highest
concentration (2000 ꢀg mL−1) and then serial two-fold dilutions were applied at the
concentration range from 15.625 to 2000 ꢀg mL−1 in 10 mL sterile test tubes containing
nutrient broth. The MIC value of each compound against bacterial strain was determined
based on a microdilution method [27]. The 96-well plates were prepared by dispensing 95 ꢀL
of nutrient broth and 5 ꢀL of the inoculums into each well. 100 ꢀL from each of the test
compounds initially prepared at the concentration of 2400 ꢀg mL−1 was added into the first
well in the plate. Then, 100 ꢀL from each of serial dilutions was added to eight consecutive
wells. The last well containing 195 ꢀL of nutrient broth without compound and 5 ꢀL of
inoculum was used as negative control. The contents of the wells were mixed and the micro
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plates were incubated at 37 C for 24 h. All compounds tested in this study were screened
twice against each microorganism.
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Disc diffusion method
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The synthesized ligand and its complexes were dissolved in dimethylsulfoxide (20% DMSO)
to final concentration of 10 mg mL−1 and sterilized by filtration through 0.45 ꢀm millipore
filters. Antimicrobial activity tests were then carried out by disc diffusion method using 100
ꢀL of suspension containing 108 CFU mL−1 bacterial spread on a nutrient agar (NA) medium.
The discs (6 mm in diameter) were impregnated with 20 ꢀL of each compound (200 ꢀg/disc)
at the concentration of 10 mg mL−1 and placed on the inoculated agar. The discs impregnated
with 20% DMSO were used as negative control. Sulfamethoxazole (SD1, 300 ꢀg/disc),
sulfioxazole (SD2, 300 ꢀg/disc) were used as positive controls (standard drugs) to determine
the sensitivity of one strain/isolate in each microbial species. The inoculated plates were
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incubated at 37 C for 24 h for bacterial strain isolates. Antimicrobial activities in the disc
diffusion assay were evaluated by measuring mm zone of inhibition against tested
microorganisms. Each assay in this study was repeated twice [28, 29].
2.5. Procedure for hCA II inhibition
The organism and environmental conditions
Hikone-R marker stock collected from minimum 40 D. melanogaster culture was obtained
from Bloomington Stock Center. The flies were kept in D. melanogaster culture room
(Hacettepe University, Ankara/Turkey) at 25±10C and relative humidity of 50-60% and in 12h
light, 12 h dark periods on a standard Carolina D. melanogaster medium.
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General esterase activity assay for D. melanogaster
General esterase experiments were adapted to D. melanogaster from the World Health
Organization (WHO) manual for the determination of insecticide resistance mechanisms.
Activity measurements were done only on male individuals collected from egg and kept at -80
0C [30]. Briefly, D. melanogaster samples were taken from the freezer, daily and one
individual was placed into each eppendorf tube and homogenized with the addition of 200 mL
homogenization buffer (50 mM sodium phosphate buffer, pH 7.5 containing 1% triton X-
100). The homogenate was centrifuged for 10 minutes at 14000 rpm at 40C in a refrigerated
centrifuge. The supernatant was taken with micropipette and placed in a 96–well microplate
and used as the enzyme source. α- and β- naphtyl acetates (α- and β-NAs) were used as
substrates. Reaction was stopped by adding fast blue B-SDS solution and absorbance was
determined at 600 nm for α-NA and at 560 nm for β-NA.
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The antibacterial activity experiments were performed for 5 different compounds (ptsmh
MD1, afptsmh MD2, Pd(afptsmh)2 MD3, Pt(afptsmh)2 MD4, Co(afptsmh)2 MD5) and positive
controls.
Protein assay
The protocol from Bradford (1976) was followed. General esterase activities were determined
using the alpha and beta naphtyl acetate substrates (α- and β-NAs). The reaction was
conducted for 20 min and stopped with fast blue. Esterases were identified as bands
developed by the catalysis of α- and β-NA’s as substrates. Based on the results, they were
named as α-esterases and β-esterases, respectively [31,32].
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Esterase activity for Carbonic anhydrase II (hCA II)
The carbonic anhydrase activity was assayed at 348 nm by following the change of 4-
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nitrophenylacetate (NPA) to 4-nitrophenylate ion over a period of 80 sec at 25 C using
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spectrophotometer according to the method described in the literature. The substrate was
added to 120 µL volume of six different concentrations of inhibitors. Reaction was started by
adding of 170 µL of 0.05 M tris-SO4 buffer (pH: 7.6) and 0.1 µL enzyme solution for a final
volume of 300 µL [33, 34].
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Determination of IC50 values
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Esterase activity of carbonic anhydrase II (hCA II) was assayed by the hydrolysis of p-nitro
phenyl acetate (PNPA) to p-nitro phenolate (PNP). IC50 (the half maximal inhibitory
concentration) values of the inhibitors [35] were determined on hCA II isoenzyme. Four
different concentrations of compounds (16.6 µM, 8.3 µM, 3.33 µM, 0.033 µM,) as inhibitors
and 100 µL of 3 mM p-nitro phenyl acetate as substrate were used for enzyme inhibition
studies. Reaction was started by addition of 170 µL of 0.05 M tris-SO4 buffer (pH: 7.6) and 10
µL enzyme solutions. The absorbance was determined at 348 nm after 80 sec. and it was
repeated three times for each inhibitor. In order to determine IC50 values, percent inhibition
graphs were drawn by using statistical packing program on a computer [36, 37].
3. Results and Discussion
3.1. Characterization of compounds
In IR spectra of ptsmh, the strong bands observed at 3345 cm−1, 3261 cm−1 and 1640 cm−1 are
assigned to the υas(NH2), υs(NH ) and δ(NH ) modes, respectively. Shifting of ν(C=N) frequency at
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1651 cm-1 of afptsmh to lower frequency by ~45-51 cm−1 (1600–1606 cm−1) and also, ν(C-O)
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frequency at 1240 cm−1 to higher frequency by ~47-63 cm-1 (1287-1303 cm-1) are the
evidence of the complexation through imine-N and carbonyl-O with metal ions. This is
further confirmed by the appearance of the new band at 555-570 cm−1 due to (M-O) stretching
modes in the metal complexes. In IR spectra of afptsmh, vibrational bands observed at 1268
cm−1 and 1089 cm−1 are assigned to υas(SO ) and υs(SO ) stretching modes, respectively. No
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shifting of symmetric and asymmetric SO2 modes in the complexes is attributed to
nonparticipating in coordination.
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3.1.1. Vibrational spectral analysis
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Vibrational frequencies and corresponding vibrational assignments of afptsmh have been
investigated, experimentally and theoretically. Vibrational frequencies of afptsmh were
calculated at the DFT levels with B3LYP (Becke-Lee-Yang-Parr three parameters) hybrid
functional [38]. DFT/B3LYP is the most used method in providing reasonable acceptable
vibrational wave numbers for organic molecules. Theoretical calculations were performed
using the Gaussian 03W software package. The vibrational band assignments were performed
at B3LYP/6-311++G(d,p) theory level combined with scaled quantum mechanics force field
(SQMFF) methodology to compare the experimental and calculated vibrational frequencies of
molecule. The vibrational modes were assigned on the basis of PED analysis using SQM
program [39]. The visual check for the vibrational band assignments were also performed by
using Gauss-View program. In order to enable assignment of observed peaks, we evaluated
some important characteristic vibrational frequencies of afptsmh and compared with
experimental values. The calculated vibrational data show good agreement with the
experimental results as seen in Table 1.
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O-H vibrations
In this study, the experimental O–H stretching vibration was observed at 3376 cm-1 and
calculated at 3743 cm-1 [40]. The largest differences between experimental and theoretical
values are evaluated due to the intermolecular hydrogen bonding interactions. We note that
our experimental results are belong to solid phase having intermolecular interactions and also,
theoretical calculations were performed for isolated molecule in the gaseous phase.
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C–H and CH3 vibrations
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The characteristic C–H stretching vibrations of aromatic compounds occur above 3000 cm-1
[42-44]. In present study, symmetric C–H stretching vibrations were observed at 3306 cm-1
and calculated at 3066 cm-1. Asymmetric C–H stretching vibrations were observed at 3067
cm-1 and calculated between 3064-3028 cm-1. The C-H in plane bending vibrations were
observed at 1485, 1240, 1157, 1102, 1089 cm-1 and calculated at 1432, 1430, 1232, 1154,
1098, 1065 cm-1, respectively. [42,43]. Our molecule has two benzene rings with different
chemical environment, C-H vibrations were also observed in the form of doublet pairs as
reported in our previous study [50].
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The vibration bands at 3004 cm-1 and 2917 cm-1 correspond to the asymmetric and
symmetric stretching of CH3 groups were calculated in the region 3028-2945 cm-1, as well as
at 2897, 2898, 2896 cm-1, respectively(Table 1).
C–C vibrations
Aromatic C–C stretching vibrations, ν(C-C) occur in 1625-1400 cm-1 region with variable
intensities. ν(C-C) bands are observed at 1468-1583, 1625-1590, 1590-1575, 1540-1470, 1460-
1430,1380-1280 cm-1 as reported by Varsanyi [45]. In this study, ν(C-C) bands were observed
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at 1645, 1586, 1485, 1338 cm-1 and calculated at 1620, 1611, 1598, 1592, 1489, 1477, 1432,
1430, 1331 cm-1, respectively.
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S=O vibrations
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The SO2 symmetric and asymmetric stretching vibrations occur in the range 1160±30 cm-1
and 1330±30 cm-1 [46,47]. While the asymmetric SO2 vibration was observed at 1268 cm-1,
the symmetric SO2 vibration was observed at 1089 cm-1. The asymmetric and symmetric SO2
vibrations were calculated at 1272 and 1097 cm-1, respectively.
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C–S and C–O vibrations
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The assignment of C–S stretching vibrations is very difficult for compounds. The C–S
stretching vibrations of 3-(4-fluorophenyl)tiophene,3-(4-nitrophenyl)tiophene,3-(4-cyano
phenyl)tiophene were observed at 867, 869, 873 cm-1 as reported by Mei-Rong [48]. The C–S
stretching vibrations of some methanesulfonamides [49] and n-(substituted phenyl)-
methanesulfonamide derivatives [50] are also assigned in the region of 789-740 cm-1. In our
previous study, ν(C-S) stretching vibration was observed at 664 cm-1 [44]. For our synthesized
molecule, the C–S stretching vibration was observed at 671 cm-1 and calculated at 650 cm-1,
respectively. Similarly, C–O stretching vibration was observed at 841 cm-1 and calculated at
823 cm-1, respectiverly.
S–N and C–N vibrations
The S–N stretching vibrations are observed in different frequencies, generally occur between
970-800 cm-1 region [47, 50, 51]. In this study, S–N stretching vibrations were observed at
702 cm-1 and calculated at 689 cm-1, respectively.
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The assignments of C–N stretching vibrations were studied for 1,2-diaminoethane [52-54] and
observed between 878-1100 cm-1. In our study, C–N stretching vibration was observed at
1014 cm-1.
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3.1.2. NMR spectra
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NMR spectra (1H-13C) of ptsmh and afptsmh were measured and interpreted in CDCI3. In
order to facilitate the interpretation of NMR spectra, quantum-chemical calculations were
performed using B3LYP/6-311G++(d,p) basis set in CDCI3 phase. Isotropic shielding tensors
of 13C NMR were changed into chemical shifts by using linear relationship suggested by
Blanco et al. [55]. A similar relationship proposed by Silva et al. [56] was used to obtain
chemical shifts for 1H NMR. 1H and 13C NMR spectra of afptsmh in CDCI3 were given in Fig.
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1
2. The experimental and calculated chemical shift values were presented in Table 2. In H
NMR spectra of ptsmh, Ar-CH3 and N-CH3 proton signals were observed at 2.263 ppm and
3.419 ppm, respectively. The chemical shift belongs to NH2 was observed at 4.6 ppm as
1
singlet. In H NMR spectra of afptsmh, Ar-CH3, N-CH3 and N=C-CH3 protons appeared at
1.292 ppm, 2.198 ppm and 3.780 ppm were calculated at 1.477 ppm, 2.380 ppm and 3.101
ppm, respectively. A signal belong to phenolic O-H proton was observed at δ =10.459 ppm
and calculated at 9.465 ppm, respectively. This difference is attributed to existence of
intramolecular hydrogen bonding between OH and CN groups. Signals in the range of 6.908-
7.351 ppm are belong to aromatic protons. In 13C NMR spectra of ptsmh, Ar-CH3 and N-CH3
carbon signals were observed at 20.390 ppm and 33.476 ppm, respectively. 13C-NMR spectra
of afptsmh were assigned at 18.817 ppm, 27.222 ppm and 39.988 ppm, calculated at 15.2072
ppm, 21.631 ppm and 35.586 ppm for Ar-CH3, N-CH3 and N=C-CH3 groups, respectively.
Imine (C=N) carbon was observed at 155.210 ppm and also calculated at 174.299 ppm in
weak field. Signals in 124.199-155.210 ppm region are belong to aromatic carbons.
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3.1.3. Electronic spectra, conductivity and magnetic behavior
The significant electronic spectra of ligand and its complexes were recorded in DMSO
between 200-800 nm. The important bands for ligand and its complexes were observed in the
region of 290-272 nm and 438-330 nm. Pt2+ and Pd2+ complexes show two weaker bands at
353-354 nm and 319-335 nm assigned as spin-forbidden d–d transitions. These two bands are
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attributed to A1g →1A2g (ν1) and A1g →1B1g (ν2) transitions [24,57] that indicates a square
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planar environment around the metal ions. In the electronic spectra of tetrahedral Co(II)
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complex, three spin-allowed bands are expected for A2(F)→4T2(F), A2(F)→4T1(F) and
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4A2(F)→4T1(P) transitions. In general, such complexes show two bands which can be
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assigned to A2(F)→4T2(F) and A2(F)→4T1(F) electronic transitions, A2(F)→4T1(P) is also
observed as shoulder. In this study, we couldn’t detect A2(F)→4T2(F) transition usually
4
observed out of visible field (over 800 nm), whereas 4A2(F)→4T1(F) transition was observed
at 425 nm [58].
The molar conductivities (Λm) of metal complexes in DMSO were measured at room
temperature. Conductivity results show that metal chelates are non-electrolyte.
The magnetic moments of metal complexes were measured at room temperature. Pd(II) and
Pt(II) complexes have diamagnetism as expected, but Co(II) complex has paramagnetism
(µeff=3.89 B.M.) which is correspond to 3 unpaired electrons of high-spin Co(II) complex in
Td geometry.
3.1.4. Frontier molecular orbital (FMO)analysis
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The FMO calculations indicate that ligand and its complexes have 79 and 167
occupied molecular orbitals, respectively. The energy band gaps between the highest occupied
molecular orbital (HOMO) and the lowest unoccupied molecular orbital (LUMO) for
mentioned compounds are given in Fig. 3. The HOMOs are as electron donors representing
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the ability to donate an electron and LUMOs are as electron acceptors representing the ability
to obtain an electron. The frontier molecular orbitals (FMOs) play an important role in
electronic and optical properties, as well as in UV-vis spectra and chemical reactions [59].
Also, the energy band gap between HOMO and LUMO is a critical parameter in determining
molecular electrical transport properties. The electronic system with a larger HOMO-LUMO
gap, ꢁE(HOMO-LUMO) should be less reactive than smaller energy gap [60]. The energy values
of other HOMO and LUMO levels are listed in Table 2. In this study, FMO energy band gap
of afptsmh was found to be 4.55 eV obtained by B3LYP method using LanL2DZ basis set.
FMO energy band gaps for Co(II), Pd(II) and Pt (II) complexes were also found 1.57 eV, 0.87
eV and 0.41 eV with the same basis set. The energy band gaps of metal complexes are
reduced by about 65%, 81% and 90%, respectively.
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The LUMO energy describes electrophilicity of the compound and its level has the
importance because of the donor-acceptor interaction. Molecule with lower LUMO energy
value accepts the electron more easily than higher’s and by the way the reactivity of the
molecule increases. The lower band gap affects the binding affinities to the biologic
molecules, therefore energy gap is one of the most important descriptor for structure-activity
relationship. Metal complexes having lower energy gaps show stronger activites than ligand
with higher energy gap. Pd(II) complex was found to have the highest activity against CA II
enzyme, although Pt(II) complex has the lowest energy gap calculated as 0.41 eV. There is no
correlation between biological activitiy and ꢁE(HOMO-LUMO) band gap for metal complexes.
3.2. Antibacterial activities
The compounds were screened in vitro for their antibacterial activities against Gram positive
species; B.subtilis ATCC 6633, B. cereus NRRL-B-3711, S. aureus ATCC 6538, E. faecalis
ATCC 29212, S. agalactiae ATCC 13813 and Gram negative species; E. coli ATCC 11230,
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P. aeruginosa ATCC 15442, K. pneumonia ATCC 70063 of bacterial strains by the
microdilution (Table 3) and disc diffusion (Table 4) methods. The activity results were
compared with those of the standard drugs; sulfamethoxazole and sulfioxazole (Fig. 4).
As seen in antibacterial activity results, the compounds have broad spectrum of activities
against tested bacteria at the concentrations of 15.62–2000 µg/mL. ptsmh exhibits enhanged
inhibition against most of tested bacteria, but afptsmh shows the weakest activity against all
bacterial strains.
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Pd(afptsmh)2 and Pt(afptsmh)2 complexes show more activities against P. aeruginosa and
Bacillus cereus at the concentration of 125 µg/mL and 250 µg/mL whereas sulfisoxazole is
found less active against bacteria mentioned above. The disc diffusion assay results evidently
show that ligand and its complexes have better inhibition activities against B. subtilis than
sulfamethoxazole and sulfisoxazole. In general, synthesized compounds have higher
inhibition effects against Gram positive bacteria than Gram negative bacteria, the
antimicrobial activities of the complexes increase in the order of Pd(II)>Pt(II)>Co(II),
respectively.
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Obtained bacterial results are in accord with similar microbiologic studies for other
hydrazones. Previously, Arslan et al. evaluated in vitro antibacterial effect of aroylhydrazones
and their Co(II), Ni(II), Cu(II), Pt(II) and Pd(II) complexes against Gram positive bacteria (S.
aureus, B.subtillis, B.cereus, E. feacalis,S. epdermidis) and Gram negative bacteria (P.
aeruginosa, K. pneumonia, E. coli, P. fluorescens) using disc diffusion method [57]. Activity
results show that afptsmh and its Pd(II), Pt(II) complexes have efficiencies up to 18 mm
diameter zone against all the test microorganisms. Also, Alyar and Erdem demonstrated that
sulfonylhydrazone and its Co(II), Pd(II) complexes possess broad spectrum of activities
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against Gram positive bacteria (S. aureus, B.subtillis, B.cereus, E. feacalis) and Gram
negative bacteria (P. aeruginosa,K. pneumonia, E. coli) [24]. Activity results were found
between 500-62.5 µg/mL by microdillution method and 11-18 mm by disc diffusion method.
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3.3. Carbonic anhydrase II (hCA II) activities
6
It is well-known that metal complexing anions and sulfonamides bind to human carbonic
anhydrase II (hCA II) isoenzyme and represent well investigated classes of inhibitors against
this isoenzyme [61]. In our study, we examined the inhibitory actions of sulfonamides on CA
II from human by assaying the inhibition of the esterase activity mentioned above. Esterase
activities of afptsmh and Co(II), Pd(II), Pt(II) complexes at different concentrations are given
in Fig. 5. Metal complexes especially Pd(afptsmh)2 with IC50 value of 2.15x10-2 mM has
higher inhibition effects than afptsmh as seen in Table 5. Ozdemir et al. evaluated
anticarbonic anhydrase II activities of alkyl sulfonic acide hydrazides. The inhibition results
indicate that those compounds have lower activities than our synthesized compounds [22].
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General esterase activities were determined using alpha naphtyl acetate (α-NA) and beta
naphtyl acetate (β-NA) substrates against D. Melanogaster Hikone R6. It was found that
Pd(afptsmh)2 (1,713nmol α naphthol/min/mg) has the strongest activity against D.
melanogaster populations for α-NA activity, ptsmh (0,891 nmol β-NA/min/mg) and
Pd(afptsmh)2 (0,731nmol β-NA/min/mg) have higher activities than afptsmh (0,595 nmol β-
NA/min/mg) towards D. melanogaster for β-NA activities as seen in Fig. 6.
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Conclusions
Theoretical studies were performed to afptsmh using B3LYP/6-311G++(d,p) basis set
combined with SQMFF for the vibrational band assignments. Excellent agreement was found
between the computed and experimental results in electronic spectra, which was recorded
between 290-272 nm, was converted 4.28-4.56 eV and calculated at 4.55 eV. The reaction of
Schiff base with metal ions leads to the formation of Schiff base complexes, [ML2] (where,
M:Co(II), Pd(II) and Pt(II)). Diamagnetic Pt(II) and Pd(II) complexes indicate a square planar
environment around the metal ions, while Co(II) complex has high spin Td geometry with
paramagnetic behavior. FMO (HOMO and LUMO) energies of ligand and metal complexes
were calculated using LanL2DZ basis set in B3LYP method. The FMO energy levels are very
important parameters in chemical and pharmacological processes giving information on the
electron donating and accepting characters of the compounds. The energy band gap between
HOMO and LUMO is a critical parameter in determining molecular electrical transport
properties. The lower band gap affects the binding affinities to the biologic molecules. FMO
energy gaps were found 4.55 eV for afptsmh and 0.41-1.57 eV for metal complexes. Metal
complexes with lower energy band gaps have stronger antibacterial activites than parent
ligand up to MIC’s of 62.5 µg/mL in microdillution method and 20 mm zone diameter in disc
diffusion method. And also, carbonic anhydrase II (hCA II) inhibition results show that all
complexes posses higher biological activities than ligand with increasing order of
Pd(II)>Pt(II)>Co(II) and also, it can be mentioned that Pd(II) complex is the strongest hCAII
enzyme inhibitor with IC50 of 2.15x10-2 mM.
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Acknowledgments:
The authors would like to thank Ahi Evran University BAP (Grant No: FBA-11-30) for the
financial support of this project. Authors also thank Prof. Figen Erkoç for their valuable
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suggestions. I'm thankful to Zafer Bahceci (Zafer Bahceci, died in February 17, 2016) for
scientific support.
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Table 1.The vibrational assignments of the afptsmh by normal mode analysis based on SQM
force field calculations
Exp.
IR
3376
3306
B3LYP/6-31G(d,p)
Unscaledfreq.
3835
3202
3202
3201
3189
3175
3169
3164
3157
3148
3126
3108
3107
3104
3078
PotentialEnergyDistributions (P.E.D.)
Description (%)
υ (OH) (100)
υs (CH)ring2 (99)
υs (CH)ring1 (97)
υas (CH)ring1 (95)
υas (CH)ring2 (98)
υas (CH)ring2 (100)
υas (CH)ring1 (99)
υas (CH)ring1 (99)
υas (C24-H3) (100)
υas (CH)ring2 (99)
υas (C20-H3) (99)
υas (C11-H3) (100)
υas (C25-H3) (100)
Scaledfreq.
3743
3066
3066
3064
3053
3040
3033
3028
3022
3013
2992
2975
2974
2970
2945
2898
2897
2896
1650
1620
3067
3004
2917
υas (C20-H3) (100)
υas (C11-H3) (100)
3028
3027
3026
1662
υs (C25-H3) (84) + υs (C20-H3) (11)
υas (C11-H3) (100)
υs (C20-H3) (83) + υs (C25-H3) (14)
υ (C24=N) (78) + υ (C24-C25) (5)
υ (CC)ring2 (73)
1651
1645
1644
1636/1613/1523
1620/1528
1495/1488
1492/1487
1478/1475
1395
1611/1592/1477 υ (CC)ring1 (70) + δ(CCH)ring1 (10)
1586
1563
1556
1485
1343
1338
1598/1489
1446/1440
1444/1439
1432/1430
1336
υ (CC)ring2 (73) + δ(CCH)ring2 (8)
δ (C11H3) (91)
δ (C20H3) (30) + δ (C25H3) (52)
υ (CC)ring2 (27) + υ (C-O39) (5) + δ(CCH)ring2 (36) + δ (C25H3) (5)
υ (C24-C25) (7) + γ (C25H3) (80)
1355
1331
υ (CC)ring2 (75) + δ(COH39) (10)
1289/1273
1272
1326/1308
1293
υ (C24-C29) (35) + υ (C24-C25) (8) + δ(CCH)ring2 (21) + γ (C24C28H28) (11)
υas (SO2) (89)
1268
1240
1157
1265
1195
1137
1232
1154
1107
υ (C30-O39) (43) + υ (CC)ring2 (23) + δ(CCH)ring2 (21)
υ (CC)ring2 (12) + δ(CO39-H) (43) + δ(CCH)ring2 (21)
υ (CC)ring1 (23) + δ(CCH)ring1 (61)
1102
1089
1098
1097/1065
1052
1132
1124/1078
1068
υ (CC)ring2 (24) + δ(CCH)ring2 (22) + δ(CO39-H) (8) + γ (C20H3) (8)
υ (CC)ring1 (35) + υs (SO2) (22) + υ (CS) (9) + δ(CCH)ring1 (8)
υ (CC)ring2 (24) + υ (CN18) (21) + υ (NN) (5) + δ(CCH)ring2 (14) + δ (C25H3) (6)
γ (C11H3) (89)
1039
1062
1033
1025
1060
1053
υ (CC)ring2 (36) + υ (CN18) (6) + δ(CCH)ring2 (12) + γ (C25H3) (10)
γ (C25H3) (80)
1014
984
1002
υ (C24-C25) (10) + γ (C25H3) (56)
981/970/815
976/948/759
860
823
738
993/973/826
984/950/760
873
836
744
γ (CH)ring1 (85)
γ (CH)ring2 (87)
951/900
865
841
υ (NN) (33) + υ (CN18) (8) + δ(CCH)ring2 (10)
υ (CO39) (21) + υ (CC)ring2 (12) + υ (C24C25) (5) + υ (NN) (6) + δ(CCC)ring2 (37)
τ(CCCH)ring2 (67)
751
721
715
τ(CCCH)ring1 (70)
702
671
654
699
664
647
689
650
619
υ (SN) (25) + υ (C24-C29) (8) + υ (C29-C30) (8) + δ(CCC)ring2 (7)
υ (CS) (17) + υ (C3-C11) (9) + δ(CCC)ring1 (7) + δ(CCC)ring2 (6)
δ(CCC)ring1 (73)
637
597
υ (SN) (12) + υ (SC) (5) + δ(CCC)ring2 (16) + δ(NC24C25) (5)
563
δ(NC24C29) (5) + δ(NC24C25) (5) +
δ(SO2) (10) + δ(OSN) (7) + δ(SNC) (6) +
(CCOH) (67)
τ
(CCCC)ring2 (23) +
τ
(CCCH)ring2 (12)
573
485
316
476
314
τ(CCSO)(15) +
τ(CCCH)ring1 (14) + τ(CCCC)ring1 (9)
τ
ν: bond stretching, δ: in-plane angle bending, γ: out-of-plane angle bending, τ: torsion, as: antisymmetric and s:
symmetric.

ACCEPTED MANUSCRIPT
Table 2.The experimental and theoretical 1H and 13C NMR chemical shifts δ(ppm) for
afptsmh
B3LYP/6-311++G(d,p)a
Assignment
δ(exp.)
138.888
135.204
δ(calc.)
139.404
129.358
128.373
C-1
C-2
136.842
151.002
136.842
135.204
18.817
27.222
39.988
163.101
126.438
155.210
124.554
133.029
124.199
134.950
7.742
C-3
C-4
147.006
130.162
128.874
15.2072
21.631
35.586
174.299
127.231
155.271
114.461
131.182
120.425
132.101
7.851
C-5
C-6
C-7
C-8
C-10
C-9
C-11
C-12
C-13
C-14
C-15
C-16
H-2
7.312
H-3
7.423
7.219
H-5
7.521
7.762
H-6
7.809
1.292
H-7(CH3)Ar
H-8(CH3-N)
H-9
1.477
2.198
2.380
3.780
3.101
6.929
H-13
H-14
H-15
H-16
OH
6.707
7.351
7.270
6.908
7.122
7.330
7.498
10.459
9.465
σ Transform into using equations given in Ref. [55,56]; δ13C = 175.7–0.963 σ13C and δ1H = 31.0–0.970 σ1H.

ACCEPTED MANUSCRIPT
Table3. The MICs of antibacterial activity of the compounds
MIC µg/mL
Bacteria strains
(mM)
Gram- negative
ptsmh afptsmh Pt(afptsmh) Pd(afptsmh) Co(afptsmh)2 SD1
SD2
250
(1.24) (1.57)
250 500
(1.24) (1.57)
125 250
(0.62) (0.78)
500
250
250
500
64
23.4
Eschericha coli ATCC 11230
(0.301)
250
(0.337)
125
(0.720)
125
(0.25)
64
(0.088)
375
Pseudomonas aeruginosaATCC 154
Klebsiella pneumonia ATCC 70063
(0.301)
125
(0.168)
500
(0.180)
250
(0.25)
16
(1.403)
23.4
0.150)
(0.674)
(0.360)
(0.063) (0.088)
Gram -positive
Bacillus subtilis ATCC 6633
250
(1.24) (0.78)
250 500
(1.24) (1.57)
500 500
(2.48) (1.57)
500 250
250
62.5
250
62.5
1500
-
(0.075)
250
(0.337)
125
(0.090)
62.5
(5.92)
Bacillus cereus NRRL-B-3711
16
(0.063) (1,403)
32 93.75
(0.126) (0.35)
32 93.75
(0.126) (0.35)
16 11.7
(0.063) (0.044)
375
(0.301)
250
(0.168)
250
(0.090)
62.5
Staphylococcus aureusATCC 6538
Enterococcus faecalisATCC 29212
StrepococcusagalactiaeATCC 1381
(0.301)
250
(0.337)
250
(0.090)
250
(2.48) (0.78)
15.62 125
(0.301)
31.25
(0.0376)
(0.337)
125
(0.360)
125
(0.078) (0.39)
(0.168)
(0.180)

ACCEPTED MANUSCRIPT
Table 4. Measured inhibition zone diameter (mm) of the compounds and antibiotics by disc
diffusion method
Bacteria strains
Diameter inhibition zone (mm,200 µg/disk)
Gram- negative
ptsmh afptsmh Pt(afptsmh)2 Pd(afptsmh)2 Co(afptsmh)2 SD1 SD2
Eschericha coli ATCC 11230
P.aeruginosaATCC 15442
Klebsiella pneumonia ATCC 70063
13
13
18
12
11
15
14
13
16
14
17
13
14
16
11
17
17
24
20
8
28
Gram -positive
Bacillus subtilis ATCC 6633
13
15
12
13
16
14
14
16
16
17
-
-
Bacillus cereus NRRL-B-3711
28
17
Staphylococcus aureusATCC 6538
Enterococcus faecalisATCC 29212
StrepococcusagalactiaeATCC 13813
13
14
23
13
13
15
15
14
17
16
16
14
18
12
14
15
15
23
25
18
21