Alkyl sulfonic acide hydrazides: Synthesis, characterization, computational studies and anticancer, antibacterial, anticarbonic anhydrase II (hCA II) activities

Article abstract of DOI:10.1016/j.molstruc.2015.07.054

Abstract Methane sulfonic acide hydrazide, CH₃SO₂NHNH₂ (1), ethane sulfonic acide hydrazide, CH₃CH₂SO₂NHNH₂ (2), propane sulfonic acide hydrazide, CH₃CH₂CH₂SO₂NHNH₂ (3) and butane sulfonic acide hydrazide, CH₃CH₂CH₂CH₂SO₂NHNH₂ (4) have been synthesized as homologous series and characterized by using elemental analysis, spectrophotometric methods (¹H-¹³C NMR, FT-IR, LC-MS). In order to gain insight into the structure of the compounds, we have performed computational studies by using 6-311G(d, p) functional in which B3LYP functional were implemented. The geometry of the sulfonic acide hydrazides were optimized at the DFT method with Gaussian 09 program package. A conformational analysis of compounds were performed by using NMR theoretical calculations with DFT/B3LYP/6-311++G(2d, 2p) level of theory by applying the (GIAO) approach. The anticancer activities of these compounds on MCF-7 human breast cancer cell line investigated by comparing IC₅₀ values. The antibacterial activities of synthesized compounds were studied against Gram positive bacteria; Staphylococcus aureus ATCC 6538, Bacillus subtilis ATCC 6633, Bacillus cereus NRRL-B-3711, Enterococcus faecalis ATCC 29212 and Gram negative bacteria; Escherichia coli ATCC 11230, Pseudomonas aeruginosa ATCC 15442, Klebsiella pneumonia ATCC 70063 by using the disc diffusion method. The inhibition activities of these compounds on carbonic anhydrase II enzyme (hCA II) have been investigated by comparing IC₅₀ and Ki values. The biological activity screening shows that butane sulfonic acide hydrazide (4) has more activity than the others against tested breast cancer cell lines MCF-7, Gram negative/Gram positive bacteria and carbonic anhydrase II (hCA II) isoenzyme.

Full text of DOI:10.1016/j.molstruc.2015.07.054

Accepted Manuscript
Alkyl Sulfonic Acide Hydrazides: Synthesis, Characterization, Computational Studies
and Anticancer, Antibacterial, anticarbonic Anhydrase II (hCA II) Activities
Ummuhan O. Ozdemir, Dr., Firdevs İlbiz, Ayla Balaban Gunduzalp, Neslihan Ozbek,
Zuhal Karagoz Genç, Fatma Hamurcu, Suat Tekin
PII:
S0022-2860(15)30165-4
10.1016/j.molstruc.2015.07.054
MOLSTR 21709
DOI:
Reference:
To appear in: Journal of Molecular Structure
Received Date: 1 May 2015
Revised Date: 26 July 2015
Accepted Date: 27 July 2015
Please cite this article as: U.O. Ozdemir, F. İlbiz A.B. Gunduzalp, N. Ozbek, Z.K. Genç, F. Hamurcu,
S. Tekin, Alkyl Sulfonic Acide Hydrazides: Synthesis, Characterization, Computational Studies and
Anticancer, Antibacterial, anticarbonic Anhydrase II (hCA II) Activities, Journal of Molecular Structure
(2015), doi: 10.1016/j.molstruc.2015.07.054.
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Graphical abstract
The alkyl sulfonic acid hydrazides (1–4) homologous series were synthesized and characterized
by theorical and spectroscopic method. Their anticancer, antibacterial, anticarbonic anhydrase
activities were studied. Moleculer structures and HOMO - LUMO energy levels are given below

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Alkyl Sulfonic Acide Hydrazides: Synthesis, Characterization,
Computational Studies and Anticancer, Antibacterial, anticarbonic
Anhydrase II (hCA II) Activities
a
Ummuhan O. Ozdemira
*
,
Firdevs İlbiz , Ayla Balaban Gunduzalpa, Neslihan
Ozbekb, Zuhal Karagoz Gençc, Fatma Hamurcud Suat Tekine
,
aDepartment of Chemistry, Faculty of Science, Gazi University, Ankara, Turkey.
bDepartment of Chemistry Faculty of Education, Ahi Evran University, Kırşehir, Turkey.
cDepartment of Materials Engineering, Faculty of Engineering, Adıyaman University,
Adıyaman, Turkey.
dDepartment of Molecular Biology and Genetics, Faculty of Science, Bartın University,
Bartın, Turkey
eDepartment of Physiology, Faculty of Medicine, Inonu University, Malatya, Turkey
*Reprint requests to Dr. Ümmühan Ö. Özdemir
Fax: 90 312 2122279
E-mail: ummuhan@gazi.edu.tr(Ümmühan Ö. Özdemir)
Abstract:
Methane sulfonic acide hydrazide, CH3SO2NHNH2 (1), ethane sulfonic acide hydrazide,
CH3CH2SO2NHNH2 (2), propane sulfonic acide hydrazide, CH3CH2CH2SO2NHNH2 (3) and
butane sulfonic acide hydrazide, CH3CH2CH2CH2SO2NHNH2 (4) have been synthesized as
homologous series and characterized by using elemental analysis, spectrophotometric
methods (1H-13C NMR, FT-IR, LC-MS). In order to gain insight into the structure of the
compounds, we have performed computational studies by using 6–311G(d,p) functional in
which B3LYP functional were implemented. The geometry of the sulfonic acide hydrazides
were optimized at the DFT method with Gaussian 09 program package. A conformational
analysis of compounds were performed by using NMR theoretical calculations with
DFT/B3LYP/6–311++G(2d,2p) level of theory by applying the (GIAO) approach. The

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anticancer activities of these compounds on MCF-7 human breast cancer cell line
investigated by comparing IC50 values. The antibacterial activities of synthesized compounds
were studied against Gram positive bacteria; Staphylococcus aureus ATCC 6538, Bacillus
subtilis ATCC 6633, Bacillus cereus NRRL-B-3711, Enterococcus faecalis ATCC 29212and
Gram negative bacteria; Escherichia coli ATCC 11230, Pseudomonas aeruginosa ATCC
15442, Klebsiella pneumonia ATCC 70063 by using the disc diffusion method. The inhibition
activities of these compounds on carbonic anhydrase II enzyme (hCA II) have been
investigated by comparing IC50 and Ki values. The biological activity screening shows that
butane sulfonic acide hydrazide (4) has more activity than the others against tested breast
cancer cell lines MCF-7, Gram negative/Gram positive bacteria and carbonic anhydrase II
(hCA II) isoenzyme.
Keywords: Alkyl sulfonic acide hydrazide, sulfonamide, anticancer, antibacterial, anti
carbonic anhydrase II isoenzyme, computational studies
1. Introduction
The molecules of sulfonic acide hydrazide involve two pharmacophoric fragments:
sulfonamide group and hydrazine residue. Some of them exhibit strong cytostatic activity
[1,2], antibacterial [3], anti-inflammatory and analgesic [4,5] activity, as well as carbonic
anhydrase activating properties [6]. Special attention has been paid to the relationship
between the cytotoxic effect of sulfonamides and their enzyme (carbonic anhydrases,
cyclooxygenase-2 and dihydrofolate reductase) inhibitory activity [7-9]. Sulfonamides are of
considerable interest and have been used as pharmaceutical agents for the treatment of
different diseases such as infections [10], Alzheimer’s disease [11] and HIV [12]. 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 and saluretic action, among others [13]. Due to Sulfonamides/sulfonylhydrazines/
sulfonylhydrazonessignificant pharmacology applications and widespread use in medicine,
these compounds have gained importance in bioinorganic and metalbased drug chemistry.

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In our previous studies, we reported the antibacterial and cytotoxic effect of methane
sulfonic acid hydrazide (CH3SO2NHNH2) and its sulfonylhydrazone derivatives [14,15], as
well as its metal complexes [16,17]. Methane, ethane and propane sulfonylhydrazone
derivatives and their transition metal complexes were synthesized and screened for their
antimicrobial activities [18-22]. Furthermore, ethanesulfonylhydrazone derivatives and their
transition metal complexes, and different aromatic/heteroaromatic sulfonylhydrazone
derivatives were investigated for inhibitory effects on carbonic anhydrase II (hCA II) enzyme
[21,23].
As part of the ongoing studies, different alkyl sulfonyl hydrazides (compounds 1-4)
(Alkyl=methane, ethane, propane, butane) were synthesized. The structure of compound 1was
reported in previous work [14] and also its conformational analysis and vibrational
spectroscopic investigation was reported before [24]. Compounds 2-4 were synthesized for
1
the first time and characterized by using elemental analysis, H/13C-NMR, FT-IR, LC–MS,
spectroscopic methods. Gaussian 09 soft ware was used to obtain the most stable
conformation of the compounds 1-4 based on DFT/B3LYP/6-311G (d,p) method. The
geometrical parameters (bond lenghts, bond angles and torsion angles) and electronic
parameters (HOMO-LOMO energies)were calculated by this quantum set. NMR calculations
were performed by 6-311++G (2d,2p).
The anticancer activities of these compounds on MCF-7 human breast cancer cell line
were investigated by comparing IC50 values. The antibacterial activities of synthesized
compounds were studied against Gram positive bacteria; Staphylococcus aureus ATCC 6538,
Bacillus subtilis ATCC 6633, Bacillus cereus NRRL-B-3711, Enterococcus faecalis ATCC
29212and Gram negative bacteria; Escherichia coli ATCC 11230, Pseudomonas aeruginosa
ATCC 15442, Klebsiella pneumonia ATCC 70063 by using the disc diffusion method. In
addition, the inhibition activities of these compounds on carbonic anhydrase II enzyme (hCA
II) have been investigated by comparing IC50 and Ki values.
2. Experimental
2.1. Physical measurements
The solvents used were purified and distilled according to routine procedures.
Methane/ethane/propane and butane sulfonyl chloride, hydrazine hydrate were commercial
products (purum). Elemental analyses were performed according to standard micro analytical

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1
13
procedures by Leco CHNS-932, H and C-NMR spectra of dimethylsulfoxide-d6 (DMSO-
d6)solutions of the compounds were registered on a Bruker WM-400 spectrometer (400 MHz)
using tetra methyl silane as internal standard. The infrared spectra of the compounds as KBr-
disks were recorded in the range of 4000-400 cm-1 with a Mattson 1000 FT-IR spectrometer.
UV-Vis spectra were recorded on UNICAM-UV 2-100 spectrophotometer. Melting points
ofalkyl sulfonic acide hydrazides were determined with a Gallenkamp melting point
apparatus. The anticancer activities of these compounds on MCF-7 human breast cancer cell
lineinvestigated by comparing IC50 values. The disc diffusion method were used to determine
the antibacterial activity of compounds against Gram positive bacteria; Staphylococcus aureus
ATCC 6538, Bacillus subtilis ATCC 6633, Bacillus cereus NRRL-B-3711, Enterococcus
faecalis ATCC 29212 and Gram negative bacteria; Escherichia coli ATCC 11230,
Pseudomonas aeruginosa ATCC 15442, Klebsiella pneumonia ATCC 70063. The inhibition
activities of synthesized compounds on carbonic anhydrase II (hCAII) were investigated by
comparing Ki and IC50 values.
2.2. General procedure for the synthesis
The nucleophilic substitution reactions of the hydrazine hydrate with various alkyl
sulfonyl chloride (alkyl=methane, ethane, propane, butane ) were carried out as follows
[14,22]: An ethanol solution of the alkyl sulfonyl chloride, R-SO2Cl (0.12 mol) was added
dropwise to the ethanol solution of hydrazine hydrate (0.62 mol), maintaining the temperature
between 10-12oC. Then, the reaction mixture was stirred for 1 h at room temperature. After
the completion of the reaction, the solvent was removed under vacuum and the viscose
residue was takento ether phaseusinga continuousextraction methodin 5 days. Then the
etherwas removed withrotary evaporator. The resulting product recrystallised from
ethylacetateandthen allowed to standin the freze and crystals were obtainedafter a few weeks.
2.3. Computational section
Molecular geometry optimizations, geometrical parameters such as bond lenghts, bond
angles and torsion angles and electronic parameters consist of HOMO-LUMO energies were
examined with Becke’sthree-parameterexchange functional [25] in combination with the Lee–
Yang–Parrcorrelation functional (B3LYP) [26] density functional theory (DFT)

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method with 6-311G (d,p) basis set by using the Gaussian 09 program package [27,28]. NMR
chemical shifts were calculated by DFT/B3LYP/6–311++G(2d,2p) method
.
2.4. Biological activity
2.4.1. Cell culture and cytotoxicity determination
The cells were incubated under 5% CO2/air at 37°C conditions at Nuaire humidified
carbon dioxide incubator (Playmouth, MN, USA). Cells’ state was controlled by inverted
microscope (Soif Optical Inc. China) and results are expressed as Mean±STD. Statistical
analysis and comparison between mean values for cytotoxicity were performed by Tukey
variance analysis (SPSS 10.0 for Windows; Chicago, IL, USA).
A colorimetric cell viability assay under usage of the tetrazole 3-(4,5-dimethylthiazol-
2yl)-2,5-diphenyltetrazolium bromide (MTT) was used to evaluate the cytotoxic effects of the
test compounds [29]. MCF-7 cancer cell line were grown as monolayer culture in a high
glucose concentration (4.5 g/l) DMEM medium supplemented with 10% fetal calf serum
(FCS), 1% L-glutamine (200 mM), 1% of mixture penicillin (100 IU/ml) and streptomycin
(100 lg/ml) incubated at 37 oC in an atmosphere of 5% CO2–95% air mixture. Briefly, 5x104
MCF-7 tumor cells were plated in triplicate in 96-well flat bottom tissue culture plates, and
treated with different concentrations of drugs for the time indicated. MTT (0.005 g=mL in
phosphate buffer saline) was added to the cell culture and incubated for 4 h at 37 °C in 5%
CO2 humidified incubator. The formazan crystals formed during the reaction of active
mitochondria with MTT, were dissolved in 0.04 N (100 mL) in isopropanol and readings were
taken in a microtiter plate reader using a 570 nm filter. Cytotoxic activity results was
expressed as the IC50 (ꢀM) and Docetaxel was used as positive control.

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2.4.2. Procedure for antibacterial activity
The in vitro antibacterial activity of the alkyl sulfonic acide hydrazide were tested
against the Gram positive bacteria; Staphylococcus aureus ATCC 25923, Bacillus subtilis
RSKK 244, Bacillus magaterium RSKK 5117 and Gram negative bacteria, Salmonella
enteritidis ATCC 13076, Escherichia coli ATCC 11230 by using the disc diffusion method.
The Bacteria cultures were obtained from Gazi University, Biology Department. Bacterial
strains were cultured overnight at 310 K in Nutrient Broth. During the survey, these stock
cultures were stored in the dark at 277 K. The inocula of microorganisms were prepared from
12 h broth cultures and suspensions were adjusted to 0.5 McFarland standard turbidity.
The alkyl sulfonic acide hydrazides were dissolved in dimethylsulfoxide (20% DMSO)
to a final concentration of 5.0 mg mL−1 and sterilized by filtration by 0.45 ꢀm millipore
filters. Antimicrobial tests were then carried out by the disc diffusion method using 100 ꢀL of
suspension containing 108 CFU mL−1 bacteria spred on a nutrient agar (NA) medium. The
discs (6 mm in diameter) were impregnated with 20 ꢀL of each compound (100 ꢀg/disc) at the
concentration of 5.0 mg mL−1 and placed on the inoculated agar. DMSO impregnated discs
were used as negative control. Sulfamethoxazole (300 µg/disk) and sulfisoxazole (300
µg/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 37oC
for 24 h for bacterial strains isolates. Antimicrobial activity in the disc diffusion assay was
evaluated by measuring the zone of inhibition against the test organisms. Each assay in this
experiment was repeated twice. Percentage of inhibition by comparing distance of the
compounds to the positive control using (Sulfamethoxazole) the equation below [30].
ꢇꢂꢈꢉꢊꢄꢊꢋ ꢅꢌ ꢄℎꢊ ꢍꢈꢉꢎꢏꢊ
% ꢀꢁℎꢂꢃꢂꢄꢂꢅꢁ = ꢆ
ꢒ ∙ 100
ꢇꢂꢈꢉꢊꢄꢊꢋ ꢅꢌ ꢄℎꢊ ꢎꢅꢍꢂꢄꢂꢐꢊ ꢑꢅꢁꢄꢋꢅꢏ
2.4.3. Procedure for hCA II enzyme inhibitor activity
Carbonic anhydrase activity was assayed by the hydrolysis of p-nitrophenylacetate [31].
IC50 and Ki values of compounds were determined on hCA II enzyme. Acetazolamide (5-
acetamido-1,3,4-thiadiazole-2-sulfonamide) AAZ, a clinically used in hCA II inhibition has
also been investigated as standard inhibitor.
In order to determine IC50 values, 100 µL of 3.0 mM p-nitrophenylacetate as substrate
and four different concentrations (3×10-2; 3×10-3; 5×10-4; 3×10-4 M) of inhibitors were used.

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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 total volume of 300 µL. The absorbance was determined at 348 nm after
6 min [32]. This study was repeated three times for each inhibitor. In order to determine IC50
values, Graphs were drawn by using inhibition % values by a statistical packing program on a
computer. The IC50 concentrations of the compounds were determined from graphs [33]. This
method was applied to determine Ki values. In the media with or without inhibitor, the
substrate concentrations were 0.3, 0.6, 1.0, 3.0 mM. For this aim, inhibitor solutions were
used for the reaction medium in four different concentrations (3×10-2; 3×10-3; 5×10-4; 3×10-4
M). The Linewear-Burk graphs were obtained and Ki values were calculated according to
Cheng Prusoff equation.
3. Results and Discussion
Analytical data and some physical properties of the alkyl sulfonic acide hydrazides are
summarized in Table 1. The general synthetic route used to prepare the compounds are
illustrated in Fig. 1-2. The exothermic nucleophilic substitution reaction of corresponding
alkyl sulfonyl chloride with hydrazine hydrate were employed to form alkyl sulfonic acide
hydrazides (methane sulfonic acide hydrazide (1), ethane sulfonic acide hydrazide (2),
propane sulfonic acide hydrazide (3) and butane sulfonic acide hydrazide (4)).
The geometry optimization was performed on DFT/B3LYP/6-311G(d,p) methods with
Gaussian 09 program. Optimized structure of the sulfonic acide hydrazides are given in Fig.3.
The calculated geometrical parameters of these possible stable conformers for compounds 1-4
are presented in Table 2-5 with respect to the bond lenghts, bond angles and torsion angles.
3.1. Characterization of compounds
3.1.1 NMR spectra
1H-13C NMR spectra of compounds 1-4 were obtained in DMSO-d6 at room temperature
using TMS as an internal standard (Fig. 4). 1H-13C NMR Chemical shifts of the compounds 1-
4 were calculated by Gauge-Independent Atomic Orbital (GIAO) method [34] at
1
DFT/B3LYP/6-311++G(2d,2p) level in DMSO. The experimental and calculated H-13C
NMR assignments in DMSO-d6 are listed in Tables 6 and 7.
The terminal CH3 protons of alkyl chain for compounds 1–4 as -CH3 (three H
intensities) are observed at 2.88 (3H, singlet), 1.18 ppm (3H, triplet), 0.98 (3H, triplet), 0.87

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ppm (3H, triplet) and corresponding calculation values are 3.05 ppm, 1.15 ppm, 0.98 ppm,
1.00 ppm, respectively. CH2 protons directly linked to terminal CH3 for compounds 3 and 4 as
–CH2-CH3 (two H intensities) are observed at 1.65 ppm (2H, multiplet) and 1.37 ppm (2H,
multiplet) and corresponding calculation values are 1.58 ppm and 1.29 ppm, respectively.
CH2 protons between two CH2 groups for compounds 4 as CH2–CH2-CH2 (two H intensities)
are observed at 1.57 ppm (2H, multiplet) and corresponding calculation values are 1.29 ppm,
respectively. And also CH2 protons directly linked to SO2 for compounds 2-4 as –CH2-SO2-
(two H intensities) are observed at 3.11 ppm (2H, multiplet), 3.02 ppm (2H, triplet), 3.06 ppm
(2H, triplet) and corresponding calculation values are 3.02 ppm, 3.14 ppm, 3.12 ppm,
respectively. In addition, the experimental primer NH2 protons and seconder NH protons shift
of compounds 1–4 are observed in the range of 4.32-4.82 ppm and 7.67-7.81 ppm [35,36].
This results showed poor correlation with the calculated proton shifts (3.34-3.47 ppm and
4.48-5.29 ppm) in DMSO-d6. This suggests that NH2 and seconder NH protons have both
intramolecular and intermolecular hydrogen bonding [37].
CH3 carbons of alkyl moiety, (C1) of compounds 1–4 are observed in the range of 8.24-
49.50 ppm corresponding calculation values are 10.49-50.56 ppm, respectively. CH2 carbons
of alkyl moiety directly linked to terminal CH3 for compounds 3 and 4 are observed at 24.10
ppm and 21.38 ppm and corresponding calculation values are 23.13 ppm and 27.09 ppm. In
addition, CH2 carbonsdirectly linked to SO2 for compounds 2-4 are observed between 44.52-
53.80 ppm and corresponding calculation values are between 49.35-56.87 ppm, respectively.
3.1.2 FT-IR spectra
The selected vibration frequencies of alkyl sulfonic acide hydrazides are listed in
Table 8. The assignment of the bands was made by taking into consideration the literature
data for compounds containing appropriate structural fragments; sulfonylhydrazines,
methane/ethane/propane sulfonylhydrazones derivatives [7-10]. NH2 vibrations in the
prepared compounds 1-4 are observed between 3360- 3249 cm-1 as double bonds. And also
the presence of a single bands observed at 3210 cm-1 and 3211 cm-1 confirm the presence of
secondary amine groups (NHs) for compounds 2 and 4.The other NH vibrations in compound
1 and 3 could not be observed because of overlapping.υas (SO2) and υs(SO2) stretching
vibrations are observed between 1321–1317 cm-1 and 1156–1133 cm-1 for compounds 1–4.

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3.1.3 LC/MS spectra
LC/MS shows that molecular ion peaks for compounds 1 ([M]+), compound
2([M+H]+), compound 3 ([M]+), ([M-H]+) andcompound 4 ([2M+H]+) are exhibited at m/z;
110.1, 124.2, 138.3 and 304.80, respectively.
3.1.3. Frontier molecular orbital analysis
The highest occupied molecular orbital (HOMO) and the lowest unoccupied molecular
orbital (LUMO) are very important for quantum chemistry [37,38]. HOMO and LUMO are
the main orbitals in chemical stability. The HOMO represents the ability to donate an
electron, LUMO as an electron acceptor representing the ability to obtain an electron. These
orbitals play an important role in the electric and optical properties. The HOMO and LUMO
are the main orbital taking part in chemical reaction. The HOMO energy is directly related to
the ionization potential and LUMO energy is directly related to the electron affinity. The
frontier molecular orbitals (HOMO and LUMO) are mostly the π-antibonding type molecular
orbitals in the structure. The frontier molecular orbital distributions and energy levels of the
HOMO-1, HOMO, LUMO and LUMO+1 orbitals, which computed at B3LYP/6-311G (d,p)
level of the title molecule are shown in Fig.5.
3.2.Biological studies
3.2.1. In vitro antitumor activity
To study the growth inhibitory effects of compounds1-4 on breast cancer, MCF-7 cells
were treated with compounds and the growth of the cells were examined with MTT assay.
Table 9 illustrates the cytotoxic activities as 50% inhibitory concentration (IC50) values. The
compounds 1 and 2 have no significant inhibition of proliferation being observed at
concentrations up to 100 µM. The compounds 3 and 4 elicited a rather similar cytotoxicity
(average IC50 values of 16.65 µM and 15.41µM). Compound 4, having the highest activity in
alkyl series, shows the closest activity against standard docetaxel (average IC50 value of 7.24
µM). Viewing their structures, it can be assumed that the increasing methyl units provides
enhanced activity.

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3.2.2. Antibacterial activity results
The test compounds were screened in vitro for their antibacterial activities against four
Gram-positive species (S. aureus, B. subtilis B. cereus and E. faecalis) and three Gram-
negative species (E. Coli, P. Aeruginosa and K. pneumonia) by the disc diffusion method.
The antibacterial results were given in Table 10 by disc diffusion and Table 11. The results
were compared with those of the standard drugs as sulfamethoxazole and sulfisoxazole (Fig.6
and 7).The size of the inhibition zone depends upon the culture medium, incubation
conditions, rate of diffusion and the concentration of the antibacterial agent (the activity
increases as the concentration increases). In the present study, the alkylsulfonicacide
hydrazides are active against both types of the Gram positive and Gram negative bacteria.
As the disc diffusion assay results evidently show that compound 4 exhibits generally
strong inhibition effect against tested bacteria whereas the other compound 1-3 have moderate
activity (Table 10, Fig. 6 and 7). The alkyl sulfonic acide hydrazides show the highest
activities against Gram positive bacteria; E. Faecalis in the diameter zone of 8-10 mm
whereas sulfisoxazole and sulfamethaxazole, the drug used as standard, have been found
inactive. This result is interesting that the compounds 1-4 have selectivity against E. Faecalis.
Percentage of inhibition for the compounds exhibited in Fig.7 that is expressed as
excellent activity (120–200% inhibition), good activity (90–100% inhibition), moderate
activity (75–85% inhibition), significant activity (50–60% inhibition), negligible activity (20–
30% inhibition) and no activity [39]. As seen in Fig.7, the compound 4 (200%) Show
excellent activity against bacteria especially P.aeruginosa (Sulfisoxazole is accepted 100%
inhibition).
The results obtained by the disc diffusion method indicates that the number of carbon atoms
in alkyl sulfonic acide hydrazide (Fig. 7) may play an important role in the antibacterial
activity. The highest antibacterial activity was observed with an aliphatic carbon chain of
compound 4.
3.2.3. hCA II inhibition result
In this study, the aim was to determine the inhibitory effects of the alkyl sulfonic acide
hydrazides. The inhibitory effects of new compounds were evaluated by using IC50 (IC50
represents the molarity of inhibition as 50% decrease of enzyme activity) and Ki (inhibitor–
enzyme dissociation constant) values which are two of the most appropriate parameters of the
inhibitors (Table 8) [40]. Acetazolamide (5-acetamido-1,3,4-thiadiazole-2-sulfonamide)
(AAZ) has also been investigated as standard inhibitor, clinically used against CA II. As seen

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in Table 8 (Fig. 6a and 6b), the compounds behave as inhibitors against hCA II enzyme.
Compound 4 shows enhanged inhibition effect (Ki: 7.96×10-6M, IC50: 3.10×10-4) on hCA II
than the other compound. This is probably due to the further effect of the length of an alkyl
chain that alters the lipophilicity of a lead and its ability to cross the cell membrane on the
histidine residue in the active site of CA II enzyme [41] .
Alkyl groups are very often investigated as different R-groups during drug discovery.
The various alkyl groups tend to be easy to prepare and provide useful information. Analog
series are made by repeatedly adding an extra carbon to a alkyl chain and are called
homologues. If the homologues have a longer and longer chain with no branching, they are
called a homologous series. Within a homologous series, activity often increases to a
maximum as a side chain is lengthened. This behavior is often seen in both biochemical and
cell-based analysis.
Increasing the length of an alkyl chain changes the lipophilicity of a lead and its ability
to cross the cell membrane. In many of the homologous series, the lipophilicity has an
optimal value. If lipophilicity is too low, the compound hardly crosses the membranes and if
the lipophilicity is too high, the compound enters the membrane easily. Testing a homologous
series can help discover the suitable lipophilic properties of a lead. This activity trend has less
to do with target binding, which can be determined through a biochemical assay. Instead, the
trend is related to the ability of the lead to reach the target [40].
4. Conclusions
In this study, the researchers have reported the synthesis of alkyl sulfonic acide
hydrazides. The structural characterizations of the synthesized compounds were made by
using the elemental analyses and spectroscopic methods. Based on physicochemical
evidence, the proposed structure of compounds 1-4 are exhibited in Fig. 2. The biological
activity screening shows that compound 4 has high inhibition effect against breast cancer cell
lines MCF-7, tested bacteria and hCA II enzyme. The remarkable activity of compound 4 may
be arising from increasing methyl units, which may play an important role in biological
activies. Changing the alkyl chain increases the lipophilicity and easily passes the cell
membrane.
Acknowledgements
This research was supported by Gazi University Research Found under Project No
05/2010-92. We thank Ankara University, Faculty of Pharmacy. Central Labs. for allocation
of time at the Mass Spectra and Elemental Analyses.

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ACCEPTED MANUSCRIPT
Table 1
Analytical and physical data for alkyl sulfonic acide hidrazides
Found
(calculated)
%H
5,25
(5,45)
Empirical Formula
(Formula Weight)
Yield
(%)
m.p. (oC)
colour
Compound
%C
10,80
(10,91)
%N
25,35
(25,45) (29,09)
%S
28,50
CH6N2SO2
110,05 g/mol
white
white
50-51
40-42
50
1
2
C2H8 N2SO2
124,14 g/mol
60
65
70
19,03
(19,36)
6,15
(6,45)
22,04
(22,58) (25,81)
24,65
C3H10 N2SO2
138,18 g/mol
white
white
40-41
45-48
25,92
(26,07)
7,10
(7,29)
19,85
(20,27) (23,20)
22,80
3
4
C4H12 N2SO2
152,31 g/mol
30,90
(31,58)
7,09
(7,89)
17,38
(18,42) (21,05)
20,25
Table2
Optimized geometric parameters of compound 1 by using B3LYP-6-311G(d,p) basic set
Bond lengths (Å)
Bond angles (o)
10H-9N-11H
10H-9N-4N
11H-9N-4N
9N-4N-5H
9N-4N-6S
4N-6S-7O
4N-6S-8O
7O-6S-8O
7O-6S-1C
Torsion angles (o)
10H-9N-4N-15H
11H-9N-4N-15H
10H-9N-4N-6S
11H-9N-4N-6S
9N-4N-6S-7O
9N-4N-6S-8O
9N-4N-6S-1C
5H-4N-6S-7O
5H-4N-6S-8O
5H-4N-6S-1C
4N-6S-1C-2H
4N-6S-1C-3H
4N-6S-1C-12H
9N-10H
9N-11H
9N-4N
4N-5H
4N-6S
6S-7O
6S-8O
6S-1C
1.0140
110.3739
112.0571
109.4309
118.5500
116.0504
108.0207
103.4225
103.4225
106.0981
109.4772
104.9729
103. 7590
113.8230
-25.3404
1.0126
1.4045
1.0147
1.6987
1.4538
1.4617
1.8177
1.0918
1.0700
1.0923
97.4290
111.5934
-125.6372
62.2019
-167.2273
-52.0349
-157.9417
153.1209
-98.7782
56.9062
1C-2H
1C-12H
1C-3H
8O-6S-1C
6S-1C-2H
6S-1C-3H
6S-1C-12H
170.6086
-67.3870

ACCEPTED MANUSCRIPT
Table 3
Optimized geometric parameters of compound 2 by using B3LYP-6-311G(d,p) basic set
Bond lengths (Å)
Bond angles (o)
13H-12N-14H
13H-12N-7N
14H-12N-7N
12N-7N-8H
12N-7N-9S
7N-9S-10O
7N-9S-11O
8H-7N-9S
10O-9S-1C
11O-9S-1C
9S-1C-2H
9S-1C-3H
9S-1C-4C
Torsion angles (o)
13H-12N-7N-8H
14H-12N-7N-8H
12N-7N-9S-10O
12N-7N-9S-11O
12N-7N-9S-1C
7N-9S-1C-2H
7N-9S-1C-3H
9S-1C-4C-15H
9S-1C-4C-5H
12N-13H
12N-14H
12N-7N
7N-8H
7N-9S
0.9985
108.8825
106.1289
106.1143
107.8193
119.4341
107.4512
104.9825
114.7419
108.4132
109.7618
110.3219
108.4143
116.1186
108.9658
108.4169
111.2333
110.1261
110.4466
47.7103
163.4455
57.1721
-173.9170
-58.1511
66.6720
179.8091
-177.0512
-55.2836
-126.7633
57.6746
179.4422
-63.9219
-54.7707
66.9969
0.9993
1.4499
1.0027
1.7726
1.4586
1.4589
1.8163
1.1097
1.1105
1.5183
1.0700
1.1091
1.1095
1.0700
9S-10O
9S-11O
9S-1C
1C-2H
1C-3H
9S-1C-4C-6H
1C-4C
2H-1C-4C-15H
2H-1C-4C-5H
2H-1C-4C-6H
3H-1C-4C-15H
3H-1C-4C-5H
3H-1C-4C-6H
4C-15H
4C-6H
4C-5H
4C-15H
2H-1C-14C
3H-1C-14C
1C-4C-15H
1C-4C-5H
-176.3672
1C-4C-6H
Table 4
Optimized geometric parameters of compound 3 by using B3LYP-6-311G(d,p) basic set
Bond lengths (Å)
Bond angles (o)
16H-15N-17H
10N-15N-16H
10N-15N-17H
15N-10N-11H
15N-10N-12S
11H-10N-12S
16H-15N-10N
10N-12S-13O
10N-12S-14O
130-12S-14O
10N-12S-1C
12S-1C-4C
12S-1C-2H
12S-1C-3H
2H-1C-4C
14O-12S-1C
13O-12S-14O
3H-1C-4C
1C-4C-7C
1C-4C-5H
1C-4C-6H
6H-4C-5H
6H-4C-7C
5H-4C-7C
Torsion angles (o)
17H-15N-10N-11H
16H-15N-10N-12S
17H-15N-10N-12S
15N-10N-12S-13O
15N-10N-12S-14O
11H-10N-12S-14O
11H-10N-12S-13O
10N-12S-1C-2H
10N-12S-1C-13H
10N-12S-1C-4C
13O-12S-1C-2H
13O-12S-1C-3H
13O-12S-1C-4C
14O-12S-1C-4C
12S-1C-4C-7C
12S-1C-4C-6H
12S-1C-4C-5H
2H-1C-4C-7C
2H-1C-4C-6H
2H-1C-4C-5H
3H-1C-4C-7C
3H-1C-4C-5H
3H-1C-4C-6H
1C-4C-7C-18H
1C-4C-7C-8H
1C-4C-7C-9H
6H-4C-7C-18H
6H-4C-7C-8H
15N-16H
15N-17H
15N-10N
10N-11H
10N-12S
12S-13O
12S-14O
12S-1C
1C-2H
0.9983
108.8946
106.2943
105.9997
108.0264
119.2989
114.5713
106.2943
107.4813
104.8960
119.9841
104.6616
115.7496
110.6630
108.6220
108.9587
110.2971
108.3884
108.3935
111.3058
110.0246
110.0904
105.8470
109.6100
109.8175
111.7956
111.7314
111.0259
107.4038
107.3560
158.8473
176.2359
-67.9912
-121.5073
-168.1788
-37.8816
-166.6337
54.0249
167.3003
-70.5446
-60.4432
52.8322
174.9874
-19.7980
-177.3669
60.8825
-129.3138
57.1870
-64.5636
179.1553
-55.0904
66.8779
-176.8410
-179.7049
-59.7845
60.4768
0.9993
1.4490
1.0026
1.7735
1.4582
1.4595
1.8151
1.1097
1.1106
1.5189
1.1091
1.1087
1.5130
1.0983
1.0983
1.0976
1C-3H
1C-4C
4C-5H
4C-6H
4C-7C
7C-8H
7C-9H
7C-18H
4C-7C-8H
4C-7C-9H
4C-7C-18H
8H-7C-18H
9H-7C-18H
127.7377
62.2449
6H-4C-7C-9H
-177.4939
58.2069
-61.6115
178.1273
5H-4C-7C-18H
5H-4C-7C-9H
5H-4C-7C-8H

ACCEPTED MANUSCRIPT
Table 5
Optimized geometric parameters of compound 4 by using B3LYP-6-311G(d,p) basic set
Bond lengths (Å)
Bond angles (o)
20H-19N-21H
20H-19N-10N
21H-19N-10N
19N-10N-11H
19N-10N-16S
11H-10N-16S
10N-16S-17O
10N-16S-18O
10N-16S-1C
17O-16S-1C
18O-16S-1C
16S-1C-2H
16S-1C-3H
16S-1C-4C
2H-1C-3H
1C-4C-5H
1C-4C-6H
1C-4C-7C
5H-4C-7C
6H-4C-7C
Torsion angles (o)
20H-19N-10N-11H
20H-19N-10N-16S
21H-19N-10N-11H
21H-19N-10N-16S
19N-10N-16S-17O
19N-10N-16S-18O
19N-10N-16S-1C
11H-10N-16S-17O
11H-10N-16S-18O
10N-16S-1C-2H
10N-16S-1C-3H
10N-16S-1C-4C
17O-16S-1C-2H
17O-16S-1C-3H
17O-16S-1C-4C
18O-16S-1C-2H
18O-16S-1C-3H
16S-1C-4C-5C
16S-1C-4C-6C
16S-1C-4C-7C
2H-1C-4C-6H
20H-19N
21H-19N
19N-10N
10N-11H
10N-16S
16S-17O
16S-18O
16S-1C
1C-2H
0.9985
108.8792
106.1350
44.8232
47.6042
-179.2285
-22.8373
-63.4982
56.3370
-174.7531
-58.7766
-173.4959
-44.5859
55.2414
168.4877
-69.3232
-59.1693
54.0770
176.2660
167.6423
-79.1114
-56.2041
60.2030
0.9993
1.4498
1.0026
1.7729
1.4587
1.4588
1.8158
1.1098
1.1106
1.5183
1.1096
1.1093
1.5218
1.1084
1.1084
1.5121
1.0980
1.0974
1.0983
109.8331
119.3379
114.6885
107.5211
105.1382
104.3330
108.5176
39.2610
110.5685
108.6187
115.6789
103.7696
110.1663
110.2458
111.2749
109.7269
109.4732
111.2523
105.8065
105.5463
109.9950
109.9527
111.1841
111.6905
111.6715
36.3585
1C-3H
1C-4C
4C-5H
4C-6H
4C-7C
7C-8H
7C-9H
7C-12C
12C-13H
12C-14H
12C-15H
-178.1340
-65.1418
178.4511
56.5212
4C-7C-12C
6H-4C-5H
8H-7C-9H
2H-1C-4C-5H
2H-1C-4C-7C
3H-1C-4C-5H
3H-1C-4C-6H
3H-1C-4C-7C
1C-4C-7C-12C
1C-4C-7C-8H
1C-4C-7C-9H
6H-4C-7C-8H
8H-7C-12C
9H-7C-12C
7C-12C-14H
7C-12C-13H
7C-12C-15H
13H-12C-15H
13H-12C-14H
15H-12C-14H
66.0699
-177.5230
-55.8600
-179.7868
-57.6377
58.1765
107.3761
107.3976
64.4738
6H-4C-7C-9H
-178.7120
-57.6752
-179.8223
121.8672
58.0287
60.3041
-179.7723
-59.8341
6H-4C-7C-12C
5H-4C-7C-8H
5H-4C-7C-9H
5H-4C-7C-12C
4C-7C-12C-13H
4C-7C-12C-14H
4C-7C-12C-15H

ACCEPTED MANUSCRIPT
Table 6
The experimental and theoretical 1H-NMR data (ppm) of the alkyl sulfonic acide hidrazides
1
2
3
4
Assign.
δ(exp.)
δ(calc.)a
δ(exp.)
δ(calc.)a
1.15
-
δ(exp.)
δ(calc.)a
0.98
δ(exp.)
δ(calc.)a
0.87 (t,3H)
1.37(m, 2H)
1,57(m,2H)
1.00
CH3
2,88 (s,3H)
3.05
1.18 (t,3H)
-
0.98 (t,3H)
1.65(m,2H)
-
1.29
1.46
CH2-CH3
-CH2-
-
-
1.58
-
-
-
-
SO2-CH2
-
-
3.11 (m,2H)
4,32(s,2H)
7,67(s,1H)
3.22
3.35
5.29
3.02 (t,2H)
4,52(s,2H)
7,81(s,1H)
3.14
3.34
5.24
3.06 (t, 2H)
4,82(s,2H)
7,75(s,1H)
3.12
4,32(s,2H)
3.47
NH2
NH
3.42
5.23
7,67(s,1H)
4.48
a Calculated using GIAO/B3LYP/6311++G(2d,2p) method.
Table 7
The experimental and theoretical 13C-NMR data (ppm) of the alkyl sulfonic acide hidrazides
4
1
2
3
Assign.
δ(calc.)a
δ(exp.)
δ(calc.)a
δ(exp.)
13.50
24.10
-
δ(calc.)a
14.13
23.13
-
δ(exp.)
δ(calc.)a
δ(exp.)
13,94
16.17
27.09
32.14
55.76
CH3
49.50
50.56
8.24
10.49
21,38
25,29
47,17
-
-
-
-
-
-
-
-
CH2-CH3
CH2
-
-
44.52
49.35
53.80
56.87
SO2-CH2
a Calculated using GIAO/B3LYP/6311++G(2d,2p) method.

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Table 8
Wave number (cm-1) of selected vibration of the alkyl sulfonic acide hidrazides.
Assign.
υas(NH2)
υs(NH2)
υ(NH)
1
2
3
4
3360m
3300m
-
3334s
3276s
3210s
1317s
1143s
1637m
542m
577m
3335m
3300m
-
3383m
3249m
3211w
1317s
1133s
1639m
525
υas(SO2)
υs(SO2)
δ(NH2)
δ(SO2)
δ(NH)
1320s
1156s
1640m
526s
1321s
1145s
1625m
526w
635w
650m
630
Table 9
Cytotoxicity compounds (1-4), Docetaxel against MCF-7 cell lines
IC50 (ꢀmolL-1) for MCF-7
cell lines
>100
Compound
1
2
3
>100
16.65
15.41
7.24
4
Docetaxel
Refererence : Docetaxel

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Table 10
Inhibition zone values (mm,100 µg/disc)of the alkyl sulfonic acide hydrazides by disc diffusion
method
Gram
Gram
positive
negative
Compound
B.subtilis B. cereus E. faecalis S. aureus P.aeruginosa K.pneuonia
E. coli
ATCC
8
ATCC
10
NRRL-B-
ATCC
8
ATCC
9
ATCC
ATCC
8
8
9
9
1
8
9
10
9
9
9
9
2
8
9
10
10
17
18
10
14
8
10
10
20
17
9
3
13
25
15
12
18
15
10
-
12
28
24
4
Sulfisoxazole
Sulfamethaxazole
-
17
Reference : Sulfisoxazole, Sulfamethaxazole
Table 11
The result of inhibition studies for alkyl sulfonic acide hidrazides on carbonic anhydrase II.
hCA II
Esterase activity
Compound
IC50 (M)
3,78×10-4
Ki (M)
6,94.10-5
1
2
3
4
6,44.10-5
5,96.10-5
5,80.10-5
2,11.10-5
3.44×10-4
3.17×10-4
3.10×10-4
1.13×10-4
Acetazolamide
Reference : Acetazolamide

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Fig.1. Preparation of different alkyl sulfonic acid hydrazide (1-4).
Fig.2. Structures of alkyl sulfonic acid hydrazides (1–4) as homologous series.
Fig.3. Optimized geometries of the the alkyl sulfonic acid hydrazides (1–4).

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.
1
Fig.4. H NMR spectra of the alkyl sulfonic acid hydrazides (1–4).

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Fig.5. HOMO and LUMO shapes of the alkyl sulfonic acid hydrazides (1–4).

ACCEPTED MANUSCRIPT
Fig. 6. Comparison of antibacterial activite of the alkyl sulfonic acid hydrazides (1–4) and antibiotics.
Fig. 7. Percentage of inhibition of the alkyl sulfonic acid hydrazides (1–4) against sulfisoxazol

ACCEPTED MANUSCRIPT
1
2
3
4
Fig 8. Activity% vs [I] regression analysis graphs for hCA II in the presence of different concentrations of 1-4

ACCEPTED MANUSCRIPT
Highlights
● Homolog series were synthesized
● Computational studies were evaluated
● Biological activities were studied