Design, synthesis and biological activities of some 7-aminocephalosporanic acid derivatives

Article abstract of DOI:10.1016/j.ejmech.2013.07.040

The treatment of 7-ACA with 4-substituted benzensulfonyl chlorides afforded the compounds containing 4-nitro/aminophenyl sulfonylamino moiety in the cephalosporanic acid skeleton (2, 4). The synthesis of the cephalosporanic acid derivatives containing 1,3

Full text of DOI:10.1016/j.ejmech.2013.07.040

Accepted Manuscript
Design, Synthesis and Biological Activities of Some 7-Amino Cephalosporanic Acid
Derivatives
Serap Basoglu, Ahmet Demirbas, Serdar Ulker, Sengul Alpay-Karaoglu, Neslihan
Demirbas
PII:
S0223-5234(13)00489-3
10.1016/j.ejmech.2013.07.040
EJMECH 6328
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 12 April 2013
Revised Date: 23 July 2013
Accepted Date: 25 July 2013
Please cite this article as: S. Basoglu, A. Demirbas, S. Ulker, S. Alpay-Karaoglu, N. Demirbas, Design,
Synthesis and Biological Activities of Some 7-Amino Cephalosporanic Acid Derivatives, European
Journal of Medicinal Chemistry (2013), doi: 10.1016/j.ejmech.2013.07.040.
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ACCEPTED MANUSCRIPT
Design, Synthesis and Biological Activity Evaluation of Some Cephalosporanic Acid Derivatives
Cl
O
NH
O
S
S
N
S
N
SO
2
NH
S
N
SO
2
NH
O
N
N
O
N
S
OAc
N
R
N
OAc
COOH
N
OAc
O
2
O N
F
COOH
COOH
Serap Basoglua, Serdar Ülker, Sengul Alpay-Karaoglu, Ahmet Demirbas, Neslihan Demirbas
Synthesis and biological activities of some cephalosporanic acid derivatives including 1,3-thiazol(idinon)e or piperazine ring were
described.

ACCEPTED MANUSCRIPT
Design, Synthesis and Biological Activities of Some 7-Amino
Cephalosporanic Acid Derivatives
a
a
b
b
Serap Basoglu , Ahmet Demirbas , Serdar Ulker , Sengul Alpay-Karaoglu ,
a*
Neslihan Demirbas
a)
Karadeniz Technical University, Department of Chemistry, 61080, Trabzon, TURKEY
b)
Recep Tayyip Erdoğan University, Department of Biology, 53100, Rize, TURKEY
*
Address correspondence to: Neslihan Demirbas,
Karadeniz Technical University,
Department of Chemistry 61080 Trabzon-Turkey
Phone: +90 462 3772600
Fax: +90 4623253196
E-Mail: neslihan@ktu.edu.tr
Abstract- The treatment of 7-ACA with 4-substitutedbenzensulfonyl chlorides afforded the
compounds containing 4-nitro/aminophenyl sulfonylamino moiety in the cephalosporanic acid
skeleton (2, 4). The synthesis of the cephalosporanic acid derivatives containing 1,3-thiazole
or 5-oxo-1,3-thiazolidine nucleus and sulfonamide function (8a,8b, 10) was performed
starting from 7-ACA by several steps. The reaction of 7-ACA with [4-(2-fluoro-4-
nitrophenyl)piperazin-1-yl]acetyl chloride afforded the corresponding 7-{[4-(2-fluoro-4-
nitrophenyl)piperazin-1-yl]acetyl}amino derivative (13).
The synthesized compounds were screened for their antimicrobial and antiurease
activities. Some of them were found to possess good-moderate antimicrobial activity against
the test microorganisms. Compound 5d was observed to have moderate anti-urease activity.
Keywords: 7-amino cephalosporanic acid, 1,3-oxazole, 1,3-thiazole, antimicrobial activity,
anti-urease activity, anti β-lactamase activity.
1

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1
. Introduction
In recent decades, the growing incidence of bacterial resistance towards the present
antibacterials has become the most serious clinical and socio-economical problem worldwide.
Multidrug-resistant Gram-positive pathogens, such as methicillin-resistant Staphylococcus
aureus (MRSA) and Staphylococcus epidermis (MRSE), vancomycin-resistant Enterococci
(VRE), cephalosporin-resistant Streptococcus pneumoniae have been leading significant
morbidity and mortality of infected patients [1-3]. Other pathogenic microorganism,
Streptococcus pneumoniae have been reported responsible for approximately 3 million deaths
each year worldwide due to pneumonia, meningitis and sepsis, and cause serious upper airway
infections such as sinusitis and otitis media. Penicillin resistance on S. pneumoniae is another
significant clinical problem with the resistance rate of 39 % [4-7]. Therefore, design and
synthesis of new and potent antibacterial agents without cross-resistance with the present
antibacterials is a crucial task for the effective treatment of bacterial infections.
Beside the development of completely new agents possessing different chemical
properties than those of the existing ones, there is another approach containing to combine
two or more pharmacophores into a single molecule. Therefore, a single molecule including
more than one pharmacophore, each with different mode of action, could be beneficial for the
treatment of microbial infectious. These synergistic antimicrobial combinations have several
major advantages, including the potential to slow down the development of drug resistance, a
broader antimicrobial spectrum, and a potential reduction in the dose and toxicity of each drug
[
5-11].
Cephalosporins and carbapenems belonging to a growing class of β-lactam antibiotics,
continue to play an important role in antibacterial therapy due to their high efficacy and safety
profile. They are the most versatile class of antibiotics used in whole world. Among
2

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cephalosporins, cefotaxime, cephalothin, and cefazolin are well known antibiotics used
currently. Since their discovery, many advances have been made in the synthesis, chemical
modification and biology of these fascinating molecules [12-19], some of which contains
different heterocyclic moieties in their structures [20, 21].
Other important pharmacophores, substituted piperazines constitute a molecular part
of some important drugs such as Crixivan, a HIV protease inhibitor, piperazinyl linked
Ciprofloxacin dimers which are potent antibacterial agents. Eperezolid that belongs
oxazolidinone class antibacterials, can be given as another important example containing a
piperazine nucleus [22, 23]. The drugs used as cardiovascular agents, Prazosin, Lidoflazine
and Urapidil contain a piperazine nucleus in their structures [24, 25].
1
,3-Thiazole and oxo-1,3-thiazolidine derivatives which are other important classes of
pharmaceuticals have been reported to possess a broad spectrum of biological activities such
as anti-mycobacterial [26], anti-fungal [26-28], anti-cancer [26, 28], anti-inflammatory [28,
2
9], anti-tuberculosis [26, 28, 30], anti-HIV [31], analgesic properties [28] and antibacterial
32, 33].
Bacterial urease enzymes, which accelerate hydrolysis of urea to ammonia gas with
[
1
4
the reaction rate at least 10 over the spontaneous reaction, have been reported as important
virulent factors including several important pathogenesis such as pyelonephritis, hepatic
coma, peptic ulceration, injection-induced urinary stones and stomach cancer [34-36]. The
detrimental impact of ureases is not only on human health. As a result of urease activity, the
NH lost from fertilizers is an economic impact for farmers. Moreover, the interference of
3
NH to the atmosphere from urea will subsequently be deposited to land or water. The result
3
of this is eutrophication and acidification of natural ecosystems on a regional scale [37].
Hydroxamic acids, phosphoramidates [38], polyphenols [34], 1,2,4-triazoles [39],
1
,3,4-oxadiazoles and 1,3,4-thiadiazoles [40] have been reported as the compounds
3

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possessing antiurease activity. Although some Schiff base-metal complexes have been found
to display urease inhibitory effects along with other metal complexes, as well, the presence of
heavy-metal atoms has restricted of their applications as drugs in the human body [41, 42].
It is known that the production of enzymes such as the serine- β-lactamases (SBLs)
and metallo-β-lactamases (MBLs) in bacteria increasingly causes the resistance against a
broader range of common β-lactam antibiotics such as penams, carbapenems and
cephalosporins. Thus, the development of an inhibitor for SBLs and MBLs is an attractive
approach to maintain the usefulness of existing antibiotics. Due to this reason, β-lactamase
inhibitors have gained importance to overcome the antibacterial resistance [43, 44]. In this
context, a number of natural and synthetic compounds have been reported to possess anti β-
lactamase activity, which catalyzes the hydrolysis of the CO-N bond in the molecules of
penicillins and cephalosporins. However, only a few of them has found field of use at clinical
settings.
Motivated by these findings and in continuation of our ongoing efforts on the
synthesis of new hybride molecules with potential chemotherapeutic activities, we would like
to report here the synthesis, antiurease and antimicrobial activities of some new
cephalosporanic acid derivatives incorporating also 1,3-oxazole, 1,3-thiazole, 5-oxo-1,3-
oxazolidine and 5-oxo-1,3-thiazolidine moieties.
2
2
. Results and Discussion
.1. Chemistry
The main aim of the present study is to synthesize and investigate the antimicrobial
and antiurease activities of some new cephalosporanic acid derivatives also containing 1,3-
thiazole, 1,3-oxazole or piperazine nucleus and/or a sulfonamide function in the one
4

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molecular structure. Synthesis of the intermediate and target compounds was performed
according to the reactions outlined in Scheme 1 and Scheme 2. The starting compound 4-
nitrobenzensulfonyl chloride (1) was provided commercially. 4-Aminobenzensulfonyl
chloride (3) obtained by the reduction of the nitro group of compound 1 is available
commercially.
In the present study, compound 2 was obtained from the reaction of compound 1 with
7
-ACA in the mild reaction conditions with the aim to introduce a sulfonamide function to the
cephalosporanic acid skeleton. In addition, the presence of the phenyl ring in the structure of
compound 2 is important to increase the lipophilicity of the molecule, because it is well
known that the lipophilic character of a bioactive molecule facilitates the penetration of it into
the cell [45, 46]. Moreover, the presence of nitro group on phenyl ring was necessary to
obtain compounds 6a-d via the formation of compound 4. However, our efforts on the
synthesis of compound 4 via the reduction of the nitro group of compound 2 did not succeed
due to the decomposition of 2 at every turn, although a number of different reduction
conditions and reagents were applied. The possible underlying reason of this is to contain the
cephalosporanic acid moiety a β-lactam nucleus that is a strained and easy-decomposable ring
in acidic and basic conditions [47].
We obtained compound 4 from the reaction of compound 3 with 7-ACA in the mild
reaction conditions. Similarly, the treatment of compound 4 with phenyl/benzylisocyanates or
phenyl/benzylisothiocyanates resulted in decomposition. The synthesis of compounds 6a-d
were achieved by the condensation of 7-ACA with compounds 5a-d, which were obtained
from the reaction of compound
3
with
phenyl/benzylisocyanates or
phenyl/benzylisothiocyanate. The synthesis of compounds 7a,b was performed from the
reaction of compounds 5a,b with ethyl bromoacetate in ethanolic solution. With the aim to
5

ACCEPTED MANUSCRIPT
merge the 1,3-thiazole nucleus and cephalosporanic acid skeleton via a sulfonamide linkage,
compounds 8a,b were synthesized by the treatment of 7a,b with 7-ACA at room temperature.
The reaction of compound 5a with ethyl bromoacetate generated the corresponding
1
,3-thiazolidinone derivative (9); then, this compound was converted to (6R,7R)-3-
(acetyloxy)methyl]-7-{[(4-{[3-benzyl-5-oxo-1,3-thiazolidin-2-ylidene]amino}phenyl)
sulfonyl]amino}-8-oxo-5-thia-1-azabicyclo[4.2.0]oct-2-ene-2-carboxylic acid (10).
-(2-Fluoro-4-nitrophenyl)piperazine (11), that was obtained from the reaction of
[
1
piperazine with 3,4-difluoronitrobenzene is a commercially known compound. As a linker
group between piperazine and cephalosporanic acid moieties, acetyl function was introduced
to the structure of compound 12 by the reaction of compound 11 with chloroethanoyl
chloride. Compound 13, that is a hybrid molecule incorporating a cephalosporanic acid core
linked to the (2-fluoro-4-nitrophenyl)piperazin-1-yl nucleus via an amide function, was
obtained from the reaction of 12 with 7-ACA. It is well known that the presence of an amide
group in the structure of β-lactam antibiotics is necessary for antimicrobial activity [47].
Moreover, it was reported that fluorophenylenpiperazinyl nucleus constitutes one of the active
parts of Eperezolid that belongs to oxazolidinone class antibacterial agents [48].
1
The FT-IR and H NMR spectra of compound 3 displayed signals due to amine
function. When compound 3 was converted to compound 4, additional signals derived from
cephalosporanic acid moiety appeared at the related regions in the FT-IR and NMR spectra of
compound 4.
1
13
As different from compound 4, H and C NMR spectra of compounds 5a-d exhibited
additional signals due to carbonothioyl- or carbonylamino moiety at the related chemical shift
values, while the signal originated from any amino group was not recorded. Instead, new
signals due to two NH groups appeared. In the FT-IR spectra of compounds 5a-d, the
6

ACCEPTED MANUSCRIPT
absorption bands belonging to the NH groups were observed. In addition, compounds 5a-c
gave Mass spectral data and elemental analysis consistent with the assigned structures.
1
13
The H and C NMR spectra of compounds 6a-d exhibited additional signals representing
the presence of a cephalosporanic acid moiety as a result of the condensation between
1
13
compounds 5a-d and 7-ACA. Compounds 6a-d displayed H and C NMR spectra consistent
with the assigned structures. The additional support for the formation of the targeted
+
compounds was obtained by the appearance of [M-1] ion peaks at corresponding m/z values
confirming their molecular masses, besides elemental analysis data.
With the conversion of compound 5a-c into 4-{[5-(4-chlorophenyl)-3-phenyl(benzyl)-
1
,3-thiazol-2(3H)-ylidene]amino}benzene sulfonyl chlorides (7a-c), the signals due to two
NH function were disappeared, instead, new signal derived from chlorophenyl nucleus
1
13
appeared at the aromatic region in the H NMR and C NMR spectra. Furthermore, in the FT-
IR spectra, the absence of any signal due to NH absorption supported the ring closure. In the
+
+
+
+
EI-MS spectra of these compounds, [M] , [M+1] , [M+Na] and/or [M+K] ion peaks are
present at the related m/z values.
1
3
1
Similarly, compounds 8a-c displayed FT-IR, C NMR and H NMR spectra representing
the presence a cephalosporanic acid moiety in their structures. Moreover, elemental analysis
results and mass spectral data supported the proposed structures.
The structure of compound 13 was elucidated on the basis of spectroscopic techniques.
2
In the FT-IR spectrum, the –NO stretching bands and the -OH vibration derived from
carboxyl group were present. The absorption bands due to carbonyl groups were recorded as
1
13
separate signals. The H and C NMR spectra and elemental analysis results of compound 13
+
supported the proposed structure. In addition, compound 13 gave relatively stable [M+Na]
ion peak in the EI-MS spectrum.
7

ACCEPTED MANUSCRIPT
2
2
.2. Biological activity
.2.1. Antimicrobial activity
All the compounds were tested for their antimicrobial activities and the results were
presented in Table1. Compound 2 that is a cephalosporanic acid derivative containing a 4-
nitrophenylsulfonylamino moiety exhibited moderate activity selectively towards
Staphylococcus aureus (Sa), Enterococcus faecalis (Ef) which are Gram positive cocci and
Bacillus cereus (Bc) that is Gram positive spore bacillus. Whereas, compound 4 that is the
amino derivative of nitro compound 3, displayed good antimicrobial activities against the test
microorganisms with the inhibition zones varying between 10-20 mm. For this compound (4),
the highest activities were observed on enteric bacteria, Escherichia coli (Ec); Gram negative
bacillus, Pseudomonas aeruginosa (Pa); Gram positive coccus, Enterococcus faecalis (Ef)
and Mycobacterium smegmatis (Ms) that is an atipic tuberculosis factor.
When the activities were compared with each other, it can be seen that
carbonothioylamino (5a and 5b) derivatives demonstrated better activity than carbonylamino
(5c and 5d) compounds. Moreover, as can be seen in Table 1, compounds 5a,b have better
activity than the standard drug Ampicillin. When compounds 5a,b were converted to the
corresponding cephalosporanic acid derivatives (6a,b), the antibacterial activities decreased,
surprisingly, while no important change were observed for antifungal activities of them.
However nonetheless, when the inhibition zones were compared with Ampicillin, it can be
concluded that these compounds (6a,b) possess better antibacterial activity against Ec and Ef.
On the other hand, the conversion of 5c,d to 6c,d resulted in the increase in antimicrobial
activities of compounds 6c,d.
4
-{[3-Alkyl-5-(4-chlorophenyl)-1,3-thia(oxa)zol-2(3H)-ylidene]amino}benzene sulfonyl
chlorides (7a-c) displayed good-moderate activities against the test microorganisms excepted,
Ec, Yp and Pa. On the other hand, as different from 7a and 7b, compounds 8a and 8b were
8

ACCEPTED MANUSCRIPT
found to be active towards all the test microorganisms. The 5-oxo-1,3-thiazolidine
compounds, 9 and 10 demonstrated similar antimicrobial activities with the corresponding
1
,3-thiazole derivatives (7a and 8a).
Compound 13, that is a cephalosporanic acid derivative containing the 7-({[4-(2-fluoro-4-
nitrophenyl)piperazin-1-yl]acetyl}amino) moiety was found to have activity towards the test
microorganisms except Pa. For compounds 12 and 13, the highest activity was observed on
Saccharomyces cerevisiae (Sc) that is yeast like fungus.
2
.2.2. Anti-urease activity
The synthesized compounds were assayed for their in vitro inhibitory activity against Jack Bean
urease. Thiourea with IC50 value 51.62±7.28 µg/mL was used as standard inhibitor. Initially, all
synthesized compounds were screened 250 µg/mL final concentration. The compounds that showed
more than 80 % inhibition were assayed at different concentration for calculation IC values. Among
5
0
the synthesized compounds, 4-[(anilinocarbonyl)amino]benzenesulfonyl chloride (5d) displayed the
best inhibitory effect against urease with an IC50 value of 120.69±0.98 µg/mL. The other compounds
show no significant inhibition (Table 2).
2
.2.3. Anti-β-Lactamase activity
The synthesized compounds were assayed for their in vitro inhibitory activity against B.
cereus β-lactamase. Three of those compounds showed β-lactamase inhibition. HgCl with
2
IC50 value 0.093 mM was used as standard inhibitor. Among tested compounds B7 was
found to be the best inhibitory effect against β-lactamase with an IC50 value of 0.543 mM.
Compounds B5 and B9 have moderate inhibitory activity (Table 3). These compounds might
2
be considered as potential β-lactamase inhibitors. All compounds and HgCl were assayed at
final concentrations of 1 mM.
9

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3
. Experimental
.1. General
3
All the chemicals were purchased from Fluka Chemie AG Buchs (Switzerland) and used
without further purification. Melting points of the synthesized compounds were determined in
open capillaries on a Büchi B-540 melting point apparatus and are uncorrected. Reactions
were monitored by thin-layer chromatography (TLC) on silica gel 60 F254 aluminium sheets.
The mobile phase was ethyl acetate:diethyl ether (1:1), and detection was made using UV
1
light. FT-IR spectra were recorded using a Perkin Elmer 1600 series FTIR spectrometer. H
13
NMR and C NMR spectra were registered in DMSO-d
6
on a BRUKER AVENE II 400 MHz
1
13
NMR Spectrometer (400.13 MHz for H and 100.62 MHz for C). The chemical shifts are
given in ppm relative to Me Si as an internal reference, J values are given in Hz. The
4
elemental analysis was performed on a Costech Elemental Combustion System CHNS-O
elemental analyzer. All the compounds gave C, H and N analysis within ±0.4% of the
theoretical values. The Mass spectra were obtained on a Quattro LC-MS (70 eV) Instrument.
3
.1.1. (6R,7R)-3-[(Acetyloxy)methyl]-7-{[(4-nitrophenyl)sulfonyl]amino}-8-oxo-5-
thia-1-azabicyclo[4.2.0]oct-2-ene-2-carboxylic acid (2)
7
-Aminocephalosporanic acid (7-ACA) (10 mmol) was added to the solution of
2 3
K CO (11 mmol) in 4 mL of water and 3 mL of acetone cooled to -5 °C, and the resulting
solution was stirred for 10 min. The solution of 4-nitrobenzenesulfonyl chloride (11 mmol) in
acetone was added drop wise in a period of 2.5-3 h. Then, the temperature was allowed to
reach to room temperature, and the reaction was continued for 7 hours by stirring. 2-3 mL of
water was added, and the reaction mixture was stirred for additional 1 h. 2-3 Drop of ethyl
acetate was added into it, and the reaction mixture was acidified to pH 3.0-3.5 with 10 % HCl.
The solution was extracted with 5 mL of ethyl acetate there times, the combined organic
10

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layers were dried over anhydrous Na SO , and the solvent was removed under reduced
2
4
pressure. The oily product obtained was recrystallized from petroleum ether. M.p.: 181 °C,
-1
yield: 34 %. FT-IR (υmax, cm ): 3267 (NH + OH), 2972 (aliphatic CH), 1736 (C=O), 1528
and 1350 (NO
2
). Elemental analysis for C16
H
15
N
3
O
9
S
2
, Calculated (%), C: 42.01; H: 3.31; N:
) δ ppm: 1.97 (s, 3H, CH ),
1
9
2
2
.19. Found (%), C: 42.28; H: 3.43; N: 9.55. H NMR (DMSO-d
6
3
.48 (s, 2H, CH ), 4.66 (brs, 1H, CH), 5.00-5.04 (m, 2H, CH ), 5.68 (brs, 1H, CH), 8.10 (d,
2
2
1
3
H, ar-H, J= 8.2 Hz), 8.44 (d, 2H, ar-H, J= 9.0 Hz), 9.54 (brs, 1H, NH). C NMR (DMSO-
d ) δ ppm: 16.88 (CH ), 25.35 (CH ), 60.57 (CH ), 82.63 (2CH), arC:[125.25 (2CH), 128.89
6
3
2
2
(2CH), 140.57 (C), 151.36 (C)], 132.78 (2C), 158.71 (2C=O), 165.57 (C=O). EI-MS: 477.85
+
+
(
[M+Na] , 58), 455.65 ([M+1] , 32), 312.02 (100).
3
.1.2. 4-Aminobenzenesulfonyl chloride (3)
Pd–C (5 mmol) catalyst was added to solution of the 4-nitrobenzenesulfonyl chloride 1
(10 mmol) in ethanol, and the mixture was run under microwave irradiation at 100°C, 150 W
for 20 min. in the presence of hydrazine hydrate (50 mmol). The progress of the reaction was
monitored by TLC. After completion of the reaction, the catalyst was separated by filtration.
Upon evaporating the reaction solvent under reduced pressure, a liquid product was obtained.
-1
2
This was used without further purification. FT-IR (υmax, cm ): 3331 and 3286 (NH +OH),
1
1
8
346 (S=O). H NMR (DMSO-d
6
) δ ppm: 7.26 (brs, 2H, NH
2
), 7.68- 7.74 (m, 2H, ar-H),
13
.16-8.22 (m, 2H, ar-H). C NMR (DMSO-d
6
) δ ppm: 120.25 (2CH), 134.26 (2CH), 137.98
(C), 145.10 (C).
11

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3
.1.3. (6R,7R)-3-[(Acetyloxy)methyl]-7-{[(4-aminophenyl)sulfonyl]amino}-8-oxo-5-thia-1-
azabicyclo[4.2.0]oct-2-ene-2-carboxylic acid (4)
-Aminocephalosporanic acid (7-ACA) (10 mmol) was added to the solution of K
11 mmol) in 4 mL of water and 3 mL of acetone cooled to -5 °C, and the resulting solution
7
2 3
CO
(
was stirred at this temperature for 10 min. The solution of compound 3 (11 mmol) in acetone
was added into it drop wise in a period of 2.5-3 h. After the addition was completed, 2 mL of
water was added, and the reaction mixture was stirred at room temperature for another 1 h.
The reaction mixture was acidified to pH 3.0-3.5 with 10% HCl and the acidified solution was
extracted with 5 mL of ethyl acetate three times. The combined organic layers were dried over
anhydrous Na
2 4
SO , and filtered. Upon evaporating the solvent under reduced pressure, an oily
product was obtained. This was recrystallized from dimethyl sulfoxide:water (1:2) to afford
-1
the desired compound. M.p.: 68 °C, yield: 12 %. FT-IR (υmax, cm ): 3260 and 3102
(
OH+NH+NH
%), C: 44.96; H: 4.01; N: 9.83. Found (%), C: 44.88; H: 3.93; N: 9.55. H NMR (DMSO-d
), 2.92 (s, 2H, CH ), 4.22 (brs, 3H, CH +CH), 4.92 (s, 1H, CH), 7.81-
.85 (m, 2H, ar-H), 7.96-8.07 (m, 3H, NH +NH), 8.27-8.38 (m, 2H, ar-H). C NMR (DMSO-
2 17 3 7 2
), 1723 (C=O), 1345 (S=O). Elemental analysis for C16H N O S , Calculated
1
(
6
)
δ ppm: 2.02 (s, 3H, CH
3
2
2
1
3
7
2
d
6 3 2 2
) δ ppm: 20.52 (CH ), 25.12 (CH ), 51.90 (CH ), 63.46 (CH), 64.13 (CH), arC: [113.23
(
(
(
2CH), 124.08 (2CH), 140.57 (C), 144.27 (C)], 128.11 (C), 129.94 (C), 151.39 (C=O), 156.34
+
+
C=O), 179.67 (C=O). EI-MS: 427.45 ([M] , 28), 338.16 ([M+2-(NH
2
CH
2
OAc] , 15), 102.12
100).
3
.1.4. General Method for The Synthesis of Compounds 5a and 5b
The corresponding alkylisothiocyanate (10 mmol) was added to the solution of
compound 3 (10 mmol) in ethanol, and the reaction was performed under microwave
irradiation at 120°C, 150 W for 20 min. On cooling the reaction mixture to room temperature,
12

ACCEPTED MANUSCRIPT
a solid appeared. This crude product was recrystallized from ethanol to give the target
compound.
3
.1.4.1. 4-{[(Benzylamino)carbonothioyl]amino}benzenesulfonyl chloride (5a)
-1
M.p.: 103-105 °C, yield 75 %. FT-IR (υmax, cm ): 3383 (NH+OH), 3304 (NH), 3059
aromatic CH), 1278 (S=O), 1210 (C=S). Elemental analysis for C H ClN O S , Calculated
(
(
14
13
2
2 2
1
%), C: 49.33; H: 3.84; N: 8.22. Found (%), C: 49.68; H: 3.93; N: 8.55. H NMR (DMSO-d )
6
δ ppm: 4.69 (d, 2H, CH
2
, J= 6.2 Hz), 7.19-7.29 (m, 9H, ar-H), 8.30 (s, 1H, NH), 8.75 (s, 1H,
NH). C NMR (DMSO-d ) δ ppm: 46.43 (CH ), arC: [125.49 (2CH), 126.60 (2CH), 127.27
2CH), 128.05 (2CH), 128.09 (CH), 139.39 (C), 139.76 (C), 151.73 (C)], 181.44 (C=S). EI-
1
3
6
2
(
+
+
MS: 340.87 ([M] , 48), 389.16 (56), 242.24 ([M+1-SO Cl] , 85), 212.07 (100).
2
3
.1.4.2. 4-[(Anilinocarbonothioyl)amino]benzenesulfonyl chloride (5b)
-1
M.p.: 158-160 °C, yield 75 %. FT-IR (υmax, cm ): 3302 (NH+OH), 3241 (NH), 1310
S=O), 1252 (C=S). Elemental analysis for C H ClN O S , Calculated (%), C: 47.77; H:
(
13
11
2
2 2
1
3
6
.39; N: 8.57. Found (%), C: 47.68; H: 3.63; N: 8.55. H NMR (DMSO-d ) δ ppm: 6.91-6.96
(m, 1H, ar-H), 7.24- 7.33 (m, 2H, ar-H), 7.52-7.57 (m, 2H, ar-H), 7.77-7.81 (m, 1H, ar-H),
1
3
7
6
.92- 7.97 (m, 2H, ar-H), 8.20-8.26 (m, 1H, ar-H), 9.87 (s, 2H, 2NH). C NMR (DMSO-d ) δ
ppm: 117.42 (2CH), 121.92 (2CH), 125.21 (CH), 127.45 (2CH), 129.74 (2CH), 141.61 (C),
+
+
1
44.30 (C), 156.37 (C). EI-MS: 349.87 ([M+Na] , 67), 292.30 ([M-Cl] , 23), 269.08 (100).
3
.1.5. General Method For The Synthesis of Compounds 5c and 5d
The corresponding alkylisocyanate (10 mmol) was added to the solution of compound
(10 mmol) in ethanol, and the reaction mixture was refluxed for 13 h (for 5c) or 10 h (for
d). On cooling it to room temperature, a solid appeared. The crude product was
3
5
13

ACCEPTED MANUSCRIPT
recrystallized from ethyl acetate (for 5c) or acetone:diethyl ether (1:2) (for 5d) to give the
desired product.
3
.1.5.1. 4-{[(Benzylamino)carbonyl]amino}benzenesulfonyl chloride (5c)
-1
M.P.: 203-205 °C, yield 75 %. FT-IR (υmax, cm ): 3285, 3195 (2NH+OH), 1657
C=O), 1294 (S=O). Elemental analysis for C H ClN O S, Calculated (%), C: 51.77; H:
(
14
13
2
3
1
4
2
2
.03; N: 8.63. Found (%), C: 51.68; H: 3.83; N: 8.55. H NMR (DMSO-d
6
) δ ppm: 4.21 (d,
H, CH
2
3
, J= 5.6 Hz), 6.67 (brs, 2H, arH), 6.99 (s, 2H, arH), 7.25 (brs, 5H, arH), 7.79 (s, 2H,
1
NH). C NMR (DMSO-d
6
, δ ppm): 42.56 (CH
2
), arC:[112.50 (2CH), 125.50 (CH), 126.42
(CH), 126.63 (CH), 128.03 (2CH), 128.75 (2CH), 149.08 (C), 150.01 (C), 153.91 (C)],
+
+
1
58.84 (C=O). EI-MS: 346.85 ([M-1+Na] , 65), 324.80 ([M] , 84), 271.00 (100).
3
.1.5.2.
4-[(Anilinocarbonyl)amino]benzenesulfonyl chloride (5d)
-1
M.p.: 226-227 °C, yield 47 %. FT-IR (υmax, cm ): 3294, 3211 (2NH+OH), 1667 (C=O),
1
9
309 (S=O). Elemental analysis for C13
H
2 3
11ClN O S, Calculated (%), C: 50.24; H: 3.57; N:
1
.01. Found (%), C: 50.68; H: 3.63; N: 8.85. H NMR (DMSO-d , δ ppm): 6.94 (t, 3H, arH,
6
J= 7.2 Hz), 7.25 (t, 3H, arH, J= 7.6 Hz), 7.50 (d, 3H, arH, J= 7.9 Hz), 8.18 (s, 1H, NH), 8.98
13
(
s, 1H, NH). C NMR (DMSO-d
6
, δ ppm): arC:[119.02 (2CH), 122.34 (2CH), 126.04
+
(
2CH), 129.26 (3CH), 136.87 (C), 140.56 (2C)], 156.82 (C=O). EI-MS: 359.41 ([M+K] , 68),
+
+
3
34.98 8[M+1+Na] , 78), 310.79 ([M] , 15), 165.48 (100).
3
.1.6. General Method For The Synthesis of Compounds 6a and 6b
7
2 3
-ACA (10 mmol) was added to the solution of K CO (11 mmol) in 4 mL of water
and 3 mL of acetone cooled to -5 °C, and the resulting solution was stirred at this temperature
for 10 min. Then, the solution of the corresponding compound 5 (11 mmol) in acetone was
14

ACCEPTED MANUSCRIPT
added into it drop wise in a period of 2,5-3 h. After the addition was completed, the
temperature was allowed to reach to room temperature, and the reaction mixture was stirred
for 14 h (for 6a) or 5 h (for 6b). Then, water was added, and the reaction mixture was stirred
for an additional 1 h. 2-3 Drop of ethyl acetate was added, and the reaction mixture was
acidified to pH 3.0-3.5 with 10% HCl. The acidified solution was extracted with 5 mL of
ethyl acetate there times. The combined organic layers were dried over anhydrous Na SO ,
2
4
and filtered. Upon evaporating the solvent under reduced pressure, a solid obtained. The oily
product was recrystallized from acetone (for 6a) or petroleum ether (for 6b).
3
.1.6.1. (6R,7R)-3-[(Acetyloxy)methyl]-7-{[(4-{[(benzylamino)carbonothioyl]amino} phenyl)
sulfonyl]amino}-8-oxo-5-thia-1-azabicyclo[4.2.0]oct-2-ene-2-carboxylic acid (6a)
-1
M.p.: 128-129 °C, yield: 22 %. FT-IR (υmax, cm ): 3675 (OH), 3294 and 3280 (3NH),
2
971 (aliphatic CH), 1655 (3C=O), 1270 (S=O), 1248 (C=S). Elemental analysis for
C
24
H
24
N
4
O
7
S
3
Calculated (%) C: 49.99; H. 4.19; N: 9.72. Found (%) C: 50.37; H: 4.57; N:
) δ ppm: 1.92 (s, 3H, CH ), 2.48 (s, 2H CH + DMSO), 3.34 (s,
1
1
2
1
6
1
1
2
0.06. H NMR (DMSO-d
6
3
2
H, CH +H O), 4.76 (s, 4H, 2CH+CH ), 7.23-7.30 (m, 9H, ar-H), 8.65 (s, 1H, NH), 10.03 (s,
2
2
2
1
3
H, NH). C NMR (DMSO-d
1.45 (CH
28.10 (2CH), 139.42 (C), 148.40 (C), 151.67 (C)], 132.34 (C), 134.68 (C), 164.78 (C=O),
6
) δ ppm: 25.00 (CH
3 2 2
), 28.50 (CH ), 56.95 (CH ), 60.00 (CH),
2
), 64.20 (CH), arC: [117.50 (2CH), 120.83 (2CH), 126.68 (CH), 127.31 (2CH),
+
+
67.68 (C=O), 178.11 (C=S + C=O). EI-MS: 575.37 ([M-1] , 34), 531.34 ([(M- CO H] , 40),
2
12.08 (100).
15

ACCEPTED MANUSCRIPT
3
.1.6.2.
(6R,7R)-3-[(Acetyloxy)methyl]-7-[({4-[(anilinocarbonothioyl)amino]phenyl}
sulfonyl)amino]-8-oxo-5-thia-1-azabicyclo[4.2.0]oct-2-ene-2-carboxylic acid (6b)
-1
M.p.: 202-203 °C, yield 22 %. FT-IR (υmax, cm ): 3524 (OH), 3184 and 3130 (3NH),
2
972 (aliphatic CH), 1794, 1735 (3C=O), 1229 (C-O). Elemental analysis for C23
H
22
N
4
O
7
S
3
1
Calculated (%) C: 49.10; H: 3.94; N: 9.96. Found (%) C: 49.17; H: 4.33; N: 10.06. H NMR
(
DMSO-d
2.6 Hz), 4.88 (d, 1H, CH, J= 5.2 Hz), 4.99 ( t, 2H, CH
Hz), 7.30 (t, 3H, ar-H, J= 8.0 Hz ), 7.56 (d, 4H, ar-H, J= 7.7 Hz), 9.88 (brs, 2H, 2NH). C
NMR (DMSO-d ) δ ppm: 20.55 (CH ), 25.11 (CH ), 58.81 (CH), 62.83 (CH ), 63.48 (CH),
6
) δ ppm: 2.03 (s, 3H, CH
3
), 3.51 (q, 2H, CH2, J= 20.0 Hz ), 4.73 (d, 1H, CH, J=
1
2
, J= 7.7 Hz) 6.93 (t, 2H, ar-H, J= 8.0
1
3
6
3
2
2
arC: [116.73 (3CH), 120.96 (3CH), 128.93 (3CH), 141.15 (2C), 155.61 (C)], 125.56 (C),
+
1
5
26.54 (C), 163.16 (C=O), 169.82 (C=O), 170.21 (C=S+C=O). EI-MS: 561.32 ([M-1] , 13),
+
31.32 (33), 487.29 ([M-Ph] , 43), 212.07 (100).
3
.1.7. General Method For The Synthesis of Compounds 6c and 6d
7
-ACA (10 mmol) was added to the solution of K CO (11 mmol) in 4 mL of water
2 3
and 3 mL of acetone cooled to -5 °C, and the resulting solution was stirred at this temperature
for 10 min. The solution of corresponding compound 5 (11 mmol) in acetone was added drop
wise in a period of 2 hours. Then, temperature was allowed to reach to room temperature, and
the mixture was stirred for 48 h (for 6c) or 24 h (for 6d). 2-3 mL of water was added into it
and the mixture was stirred for an additional 1 h. The reaction content was acidified to pH 3
with 10 % HCl, and the acidified solution was extracted with 5 mL of ethyl acetate three
4
times. The combined organic layers were dried on anhydrous NaSO and filtered. On
evaporating the solvent under reduced pressure, a solid obtained. This was recrystallized from
acetone (for 6c) or acetone:water (1:2) (for 6d) to afford the desired compound.
16

ACCEPTED MANUSCRIPT
3
.1.7.1.
(6R,7R)-3-[(Acetyloxy)methyl]-7-{[(4-{[(benzylamino)carbonyl]amino}
phenyl)sulfonyl]amino}-8-oxo-5-thia-1-azabicyclo[4.2.0]oct-2-ene-2-carboxylic acid (6c)
-1
M.p.: 229-230 °C, yield: 20 %. FT-IR (υmax, cm ): 3643 (OH), 3286, 3177 (3NH), 1798,
1
5
737, 1658 (4C=O), 1335 (S=O). Elemental analysis for C24
24 4 8 2
H N O S Calculated (%) C:
1
1.42; H: 4.32; N: 9.99. Found (%) C: 51.17; H: 4.33; N: 10.26. H NMR (DMSO-d
6
δ
ppm): 2.01 (s, 3H, CH ), 3.50 (q, 2H, CH , J= 16.0 Hz), 4.22 (brs, 2H, CH ), 4.67 (brs, 1H,
3
2
2
CH), 4.81 (brs, 1H, CH), 4.93-4.99 (m, 2H, CH
2
), 7.25 (brs, 9H, arH), 7.77 (s, 1H, NH), 7.93
1
3
(
s, 1H, NH). C NMR (DMSO-d
CH), 62.85 (CH ), 63.31 (CH), 122.47 (C), 126.69 (C), arC: [126.34 (CH), 126.41 (2CH),
26.92 (2CH), 127.97 (2CH), 128.02 (2CH), 140.56 (C), 158.75 (C), 158.79 (C)], 163.19
6 3 2 2
, δ ppm): 21.05 (CH ), 25.12 (CH ), 62.56 (CH ), 58.84
(
2
1
+
(
C=O), 169.54 (C=O), 170.22 (C=O), 172.00 (C=O). EI-MS: 560.65 ([M] , 74), 583.79
+
(
[M+Na] , 39), 325.70 (100).
3
.1.7.2.
(6R,7R)-3-[(Acetyloxy)methyl]-7-[({4-[(anilinocarbonyl)amino]phenyl}
sulfonyl)amino]-8-oxo-5-thia-1-azabicyclo[4.2.0]oct-2-ene-2-carboxylic acid (6d)
-1
M.p.: 235 °C, yield 20 %. FT-IR (υmax, cm ): 3676 (OH), 3296, 3218 (2NH), 3096
(aromatic CH), 2988, 2902 (aliphatic CH), 1799, 1737, 1668 (4C=O), 1333 (S=O). Elemental
analysis for C23
H
22
N
4
O
8
S
2
Calculated (%) C: 50.54; H: 4.06; N: 10.25. Found (%) C: 50.17;
, δ ppm): 2.02 (s, 3H, CH ) 3.54 (q, 2H, CH , J= 16.0
1
H: 4.33; N: 10.06. H NMR (DMSO-d
6
3
2
Hz), 4.67 (d, 1H, CH, J= 12.6 Hz), 4.79 (d, 1H, CH, J= 4.7 Hz), 4.92-5.10 (m, 2H, CH
2
),
6
.93 (t, 3H, arH, J= 7.2 Hz), 7.24 (t, 3H, arH, J= 7.4 Hz), 7.49 (d, 3H, arH, J= 7.6 Hz), 8.18
1
3
(
6 3
s, 1H, NH), 9.10 (s, 1H, NH). C NMR (DMSO-d , δ ppm): 21.06 (CH ), 25.10 (CH
2
),
6
1
2.90 (CH
2
), 63.30 (CH), 64.40 (CH), 122.10 (2C), arC: [118.19 (2CH), 121.58 (2CH),
28.54 (3CH), 128.75 (2CH), 139.85 (C), 156.07 (2C)], 163.23 (C=O), 169.51 (C=O), 170.22
+
+
(
C=O), 171.98 (C=O). EI-MS: 570.14 ([M+1+Na] ,45), 546.78 ([M] , 68), 236.15 (100).
17

ACCEPTED MANUSCRIPT
3
.1.8. General Method For The Synthesis of Compounds 7a-c
4
- Chlorophenacylbromide (10 mmol) and dried sodium acetate (50 mmol) was added
to the solution of the corresponding compound 5 in absolute ethanol, and the reaction mixture
was refluxed for 15 h (for 7a), 18 h (for 7b) or 20 h (for 7c). Then, the solvent was removed
under reduced pressure and the solid formed was washed with water. The obtained oily
product was extracted with 5 mL of ethyl acetate there times. The combined organic layers
were dried over anhydrous Na SO , and filtered. Upon evaporating the solvent under reduced
2
4
pressure, an oily product obtained. This was recrystallized from acetone:diethyl ether (1:2)
for 7a) or ethanol:water (1:2) (for 7b and 7c) to afford the desired product.
(
3
.1.8.1. 4-{[3-Benzyl-5-(4-chlorophenyl)-1,3-thiazol-2(3H)-ylidene]amino}benzene sulfonyl
chloride (7a)
M.p.: 106-108 °C, yield: 28 %. FT-IR (υmax, cm ): 3068, 3031 (aromatic CH), 1496
-1
(
C=N), 1396 (S=O). Elemental analysis for C22
H
16Cl
2
N
2
O
2
S
2
Calculated (%) C: 55.58; H:
, δ ppm): 4.78 (d,
1
3
2
.39; N: 5.89. Found (%) C: 55.17; H: 3.03; N: 6.06. H NMR (DMSO-d
6
1
3
H, CH , J= 6.2 Hz), 6.75 (s, 1H, CH), 7.30-7.46 (m, 13H, ar-H). C NMR (DMSO-d , δ
2
6
ppm): 56.00 (CH ), 126.35 (CH), 131.63 (C), arC: [127.21 (CH), 127.26 (CH), 127.31 (2CH),
2
1
27.47 (2CH), 127.55 (CH), 127.61 (CH), 128.69 (CH), 128.77 (2CH), 128.83 (2CH), 141.25
+
+
(
2C), 149.00 (C), 153.45 (2C)], 156.72 (C=N). EI-MS: 476.48 ([M+1] , 87), 475.46 ([M] ,
7
5), 125.54 (100).
3
.1.8.2. 4-{[5-(4-Chlorophenyl)-3-phenyl-1,3-thiazol-2(3H)-ylidene]amino}benzene sulfonyl
chloride (7b)
M.p.: 98-100 °C, yield 55 %. FT-IR (υmax, cm ): 3093, 3028 (aromatic CH), 1543
C=N), 1336 (S=O). Elemental analysis for C21 Calculated (%) C: 54.67; H:
-1
(
2 2 2 2
H14Cl N O S
18

ACCEPTED MANUSCRIPT
1
3
.06; N: 6.07. Found (%) C: 54.87; H: 3.13; N: 6.36. H NMR (DMSO-d , δ ppm): 6.86-6.95
6
1
3
(
m, 2H, CH+ar-H), 7.21 (t, 6H, ar-H, J= 8.0 Hz), 7.78-7.58 (m, 6H, ar-H). C NMR (DMSO-
, δ ppm): 104. 99 (C), 124.67 (CH), arC: [117.46 (2CH), 122.74 (2CH), 125.16 (C), 125.60
d
6
(CH), 128.55 (2CH), 129.76 (2CH), 130.91 (2CH), 132.00 (2CH), 136.00 (2C), 141.52 (C),
+
1
2
43.95 (C)], 156.47 (C=N). EI-MS: 484.31 ([M+Na] , 16), 242.24 ([M+2-(C12
6 2
H Cl )], 94),
12.08 (100).
3
.1.8.3. 4-{[3-Benzyl-5-(4-chlorophenyl)-1,3-oxazol-2(3H)-ylidene]amino}benzene sulfonyl
chloride (7c)
M.p.: 223-225 °C, yield: 44 %. FT-IR (υmax, cm ): 3064 (Aromatic CH), 1489 (C=N), 1296
S=O). Elemental analysis for C H Cl N O S Calculated (%) C: 57.52; H: 3.51; N: 6.10.
-1
(
22
16
2
2
3
1
Found (%) C: 57.87; H: 3.13; N: 6.36. H NMR (DMSO-d , δ ppm): 3.69 (brs, 2H, CH +
6
2
2
H O), 6.40-6.45 (m, 1H, CH), 7.23-7.29 (m, 2H, ar-H), 7.72 (s, 1H, ar-H), 7.80-7.86 (m, 4H,
1
3
ar-H), 8.13-8.25 (m, 4H, ar-H), 8.26 (s, 2H, ar-H). C NMR (DMSO-d
6
, δ ppm): 53.53
), 112.90 (CH), arC:[123.85 (3CH), 123.93 (2CH), 126.17 (CH), 127.35 (2CH), 127.61
3CH), 128.90 (2CH), 148.00 (2C), 149.72 (2C), 154.44 (C)], 135.83 (C), 166.49 (C=N). EI-
(CH
2
(
+
+
MS: 498. 68 ([M+K] , 89), 459.35 ([M] , 65), 234.81 (100).
3
.1.9. General Method For The Synthesis of Compounds 8a and 8b
-ACA (10 mmol) was added to the solution of K CO (11 mmol) in 4 mL of water
7
2
3
and 3 mL of acetone cooled to -5 °C and the resulting solution was stirred at this temperature
for 10 min. The solution of the corresponding compound 7a, 7b (11 mmol) in acetone was
added drop wise in a period of 2,5-3 h. After the addition was completed, the reaction mixture
was stirred at room temperature for 24 h (for 8a) or 6 h (for 8b). Then, water was added into
it, and the mixture was stirred for another 1 h. The reaction mixture was acidified to pH 3
19

ACCEPTED MANUSCRIPT
with 10% HCl and the acidified solution was extracted with 5 mL of ethyl acetate three times.
The combined organic layers were dried over anhydrous Na SO , and filtered. After
2
4
evaporating the solvent under reduced pressure, an oily product was obtained. This was
recrystallized from acetone:diethyl ether (1:2) (for 8a) or acetone: water (1:2) (for 8b) to give
the target compound.
3
.1.9.1.
thiazol-2(3H)-ylidene]amino}phenyl)sulfonyl]amino}-8-oxo-5-thia-1-azabicyclo[4.2.0]oct-
-ene-2-carboxylic acid (8a)
M.p.: 158-160 °C, yield 12 %. FT-IR (υmax, cm ): 3275 (NH+OH), 2987, 2923
aliphatic CH), 1801, 1735 (3C=O), 1535 (C=N), 1335 (S=O). Elemental analysis for
C H ClN O S Calculated (%) C: 54.04; H: 3.83; N: 7.88. Found (%) C: 54.27; H: 3.73;
(6R,7R)-3-[(Acetyloxy)methyl]-7-{[(4-{[3-benzyl-5-(4-chlorophenyl)-1,3-
2
-1
(
32
27
4
7 3
1
N: 7.46. H NMR (DMSO-d
d, 1H, CH, J= 12.7 Hz), 4.77 - 4.80 (m, 1H, CH), 4.96-5.01 (m, 4H, 2CH
CH), 7.27-7.41 (m, 5H, ar-H), 7.59-7.71 (m, 5H, ar-H), 7.87- 7.96 (m, 3H, ar-H). C NMR
6
, δ ppm): 2.03 (s, 3H, CH
3
), 3.51 (q, 2H, CH
2
, J= 8.0 Hz), 4.67
(
2
), 6.77 (brs, 1H,
1
3
(
(
(
DMSO-d , δ ppm): 20.54 (CH ), 25.61 (CH ), 46.46 (CH ), 58.64 (CH), 62.84 (CH ), 63.29
6 3 2 2 2
CH), 122.57 (2C), 126.61 (C), 126.68 (CH), arC: [126.89 (2CH), 127.25 (2CH), 128.16
2CH), 128.30 (2CH), 128.58 (2CH), 128.77 (CH), 128.84 (CH), 131.10 (CH) 139.41 (2C),
1
7
51.66 (2C), 163.18 (C)] 169.47 (C=N), 170.20 (C=O), 172.00 (C=O), 178.10 (C=O). EI-MS:
+
+
34.45 ([M+Na] , 85), 713.21 ([M+2] , 35), 220.68 (100).
20

ACCEPTED MANUSCRIPT
3
.1.9.2.
(6R,7R)-3-[(Acetyloxy)methyl]-7-{[(4-{[5-(4-chlorophenyl)-3-phenyl-1,3-
thiazol-2(3H)-ylidene]amino}phenyl)sulfonyl]amino}-8-oxo-5-thia-1-azabicyclo[4.2.0]oct-
-ene-2-carboxylic acid (8b)
M.p.: 202-203 °C, yield 22 %. FT-IR (υmax, cm ): 3183 (NH+OH), 3006 (aromatic
2
-1
CH), 2922, 2849 (aliphatic CH), 1801, 1736 (3C=O), 1540 (C=N), 1335 (S=O). Elemental
analysis for C H ClN O S Calculated (%) C: 53.41; H: 3.61; N: 8.04. Found (%) C:
31
25
4
7 3
1
5
3.77; H: 3.43; N: 8.36. H NMR (DMSO-d
J= 20.0 Hz), 4.67 (d, 1H, CH, J= 12.7 Hz), 4.84 (d, 1H, CH, J= 4.7 Hz), 4.97-5.01 (m, 2H,
CH ), 6.83 (brs, 1H, CH), 6.91 (t, 1H, ar-H, J= 7.2 Hz), 7.22-7.38 (m, 4H, ar-H), 7.40-7.81
6 3
) δ ppm: 2.03 (s, 3H, CH ), 3.51 (q, 2H, CH2,
2
1
3
(
(
(
6 3 2
m, 8H, ar-H). C NMR (DMSO-d , δ ppm): 20.54 (CH ), 25.17 (CH ), 58.35 (CH), 62.78
CH ), 62.96 (CH), 114.73 (CH), 122.93 (C), arC: [116.26 (2CH), 116.72 (2CH), 120.90
2
2CH), 123.75 (CH), 124.69 (2CH), 126.47 (2C), 128.89 (2CH), 131.29 (2CH), 133.14 (C),
1
41.19 (C), 142.32 (C)], 133.84 (2C), 155.63 (C=N), 163.09 (C=O), 168.93 (C=O), 170.20
+
+
(
C=O). EI-MS: 720.01 ([M+Na] , 75 ), 697.28 ([M] , 95), 156.45 (100).
3
.1.10. 4-{[3-Benzyl-5-oxo-1,3-thiazolidin-2-ylidene]amino}benzenesulfonyl chloride (9)
The solution of compound 5a (10 mmol) in acetic acid was refluxed with etilbromo
acetate (10 mmol) in the presence of dried sodium acetate (30 mmol) for 24 h. Then, the
reaction mixture was poured into water, and a white solid appeared. This crude product was
filtered and recrystallized from ethyl acetate:diethyl ether (1:2). M.p.: 213-215. Yield: 25 %.
-1
FT-IR (υmax, cm ): 3033 (aromatic CH), 1705 (C=O), 1525 (C=N), 1341 (S=O). Elemental
analysis for C16
H
13ClN
2
O
3
S
2
Calculated (%) C: 50.46; H: 3.44; N: 7.36. Found (%) C:
, δ ppm): 4.03 (s, 2H, CH ), 4.79 (s, 2H, CH ),
, δ ppm): 32.91 (CH ), 46.52 (CH ), arC: [128.26
1
5
7
0.77; H: 3.73; N: 8.00. H NMR (DMSO-d
6
2
2
13
.35 (brs, 9H, ar-H). C-NMR (DMSO-d
6
2
2
21

ACCEPTED MANUSCRIPT
(3CH), 128.97 (3CH), 129.27 (3CH), 136.57 (C), 157.35 (C), 160.40 (C)], 168.33 (C=N),
+
+
1
72.48 (C=O). EI-MS: 402.15 ([M-1+Na] , 56), 381.98 ([M+1] , 67), 178.02 (100).
3
.1.11. (6R,7R)-3-[(Acetyloxy)methyl]-7-{[(4-{[3-benzyl-5-oxo-1,3-thiazolidin-2-
ylidene]amino}phenyl)sulfonyl]amino}-8-oxo-5-thia-1-azabicyclo[4.2.0]oct-2-ene-2-
carboxylic acid (10)
7
-ACA (10 mmol) was added to the solution of K CO (11 mmol) in 4 mL of water and
2 3
3
1
mL of acetone cooled to -5 °C and the resulting solution was stirred at this temperature for
0 min. The solution of the corresponding compound 9 (11 mmol) in acetone was added drop
wise in a period of 2,5-3 h. After the addition was completed, the reaction mixture was stirred
at room temperature for 9 h. Then, water was added into it, and the mixture was stirred for
another 1 h. The reaction mixture was acidified to pH 3 with 10% HCl and the acidified
solution was extracted with 5 mL of ethyl acetate three times. The combined organic layers
were dried over anhydrous Na
2 4
SO , and filtered. After evaporating the solvent under reduced
pressure, an oily product was obtained. This crude product was crystallized from butyl
-1
acetate: diethyl ether (1:3). M.p.: 207-208 °C, yield: 35 %. FT-IR (υmax, cm ): 3450 (OH),
065 (aromatic CH), 2985, 2875 (aliphatic CH), 1705, 1685, 1617 (4C=O), 1341 (S=O).
Elementel analysis for C26 Calculated (%) C: 50.64; H: 3.92; N: 9.09. Found: C:
0.25; H: 4.12; N: 9.28. H-NMR (DMSO-d , δ ppm): 1.91 (s, 3H, CH ), 3.38 (brs, 4H,
CH ), 3.97 (s, 2H, CH ), 4.39 (s, 2H, CH ), 4.83 (s, 1H, CH), 5.49 (s, 1H, CH), 7.31 (brs,
H, arH). ). C-NMR (DMSO-d , δ ppm): 14.68 (CH ), 32.57 (CH ), 46.45 (CH ), 52.26
3
24 4 8 3
H N O S
1
5
2
9
6
3
2
2
2
1
3
6
3
2
2
(
2CH), 56.50 (2CH ) 117.20 (2C), arC:[127.50 (CH), 127.95 (2CH), 128.18 (2CH), 128.59
2
(CH), 128.81 (CH), 129.05 (2CH), 134.65 (2C), 138.26 (C)], 155.65 (C=N), 166.72 (C=O),
+
+
1
68.25 (C=O), 174.67 (2C=O). EI-MS: 639.40 ([M+Na] 25), 616.70 ([M] ,72).
22

ACCEPTED MANUSCRIPT
3
.1.12. 1-(2-Fluoro-4-nitrophenyl)piperazine (11)
The solution of 3,4-difluoronitrobenzene (10 mmol) in acetonitrile was refluxed in the
presence of piperazine (50 mmol) for 5 h. After evaporating the solvent under reduced
pressure, an oily mass was obtained. The crude product was treated with water and extracted
with 5 mL of ethyl acetate three times. The combined organic layers were dried on anhydrous
NaSO and filtered. On evaporating the solvent under reduced pressure, a crude product was
4
obtained. This was crystallized from butyl acetate: diethyl ether (1:3). M.p.: 66-68°C [47].
3
.1.13. [4-(2-Fluoro-4-nitrophenyl)piperazin-1-yl]acetyl chloride (12)
Chloroethanoylchloride (15 mmol) was added to the mixture of compound 11 (10
o
mmol) and triethylamine (30 mmol) in THF cooled to -5 C drop wise over a 2-hour period.
Then, the temperature was allowed to reach to room temperature and the mixture was stirred
for 4 h. The precipitated salt was removed by filtration, the solvent was evaporated under
reduced pressure and water was added into it. The resulting yellow oily product was
recrystallized form ethyl acetate: hexane (1:3) to afford the desired compound. M.p.: 118-119
-1
°
C. 60 % yield. FT-IR (υmax, cm ): 1661 (C=O), 1494 and 1330 (NO ). Elemental analysis for
2
C H ClFN O Calculated (%) C: 47.77; H: 4.34; N: 13.93. Found (%) C: 47.57; H: 3.99;
12
13
3
3
1
N: 13.66. H NMR (DMSO-d
H, CH ), 7.18 (t, 1H, ar-H, J= 4.0 Hz), 7.98- 8.07 (m, 2H, ar-H). C-NMR (DMSO-d
ppm: 45.59 (CH ), 46.05 (CH ), 49.49 (CH ), 49.66 (CH ), 60.83 (CH ), arC: [112.71 and
6
) δ ppm: 3.31 (brs, H
2
O+2CH
2
), 3.61 (s, 4H, 2CH
2
), 4.43 (s,
1
3
2
2
6
) δ
2
2
2
2
2
1
1
2
13.23 (d, CH, J= 26.0 Hz), 118.83 (CH), 121.93 and 121.97 (d, CH, J= 2.2 Hz), 140.24 and
40.42 (d, C, J= 9.0 Hz), 145.59 and 145.74 (d, C, J= 7.5 Hz), 150.26 and 155.18 (d, C, J=
+
46 Hz)], 170.93 (C=O). EI-MS: 301.08 ([M] , 82), 217.05 (100).
23

ACCEPTED MANUSCRIPT
3
.1.14. (6R,7R)-3-[(Acetyloxy)methyl]-7-({[4-(2-fluoro-4-nitrophenyl)piperazin-1-
yl]acetyl}amino)-8-oxo-5-thia-1-azabicyclo[4.2.0]oct-2-ene-2-carboxylic acid (13)
7
2 3
-ACA (10 mmol) was added to the solution of K CO (11 mmol) in 4 mL of water and
3
1
mL of acetone cooled to -5 °C and the resulting solution was stirred at this temperature for
0 min. Then, the solution of the corresponding compound 12 (11 mmol) in acetone was
added drop wise in a period of 2,5-3 h. After the addition was completed, the reaction mixture
was stirred at room temperature for 1h. Water was added into it, and the mixture was stirred
for another 1 h. The reaction mixture was acidified to pH 3 with 10% HCl, the resulting
precipitate was filtered off and recrystallized from acetone. M.p.: 178-180 °C, yield: 86 %.
-1
FT- IR (υmax, cm ): 3145 (NH+ OH), 1794 (C=O), 1735 (C=O), 1661 (2C=O), 1511 and 1334
NO ), 1228 (C-O). Elementel analysis for C H FN O S Calculated (%) C: 49.16; H: 4.50;
(
2
22 24
5
8
1
N: 13.03. Found (%) C: 49.18; H: 4.40; N: 12.63. H-NMR (DMSO-d
CH ), 3.30 (brs, 4H, 2CH ), 3.54 (brs, 6H, 3CH +H O), 4.43 (s, 2H, CH
.82 (s, 1H, CH), 4.99 (s, 2H, CH ), 7.17 (t, 1H, ar-H, J= 9.0 Hz), 8.0-8.05 (m, 2H, ar-H).
) δ ppm: 21.03 (CH ), 25.18 (CH ), 41.30 (CH ), 41.88 (CH ), 44.87
), 49.00 (CH ), 58.21 (CH), 62.76 (CH ), 62.81 (CH), 112.13 and 112.39
6
) δ ppm: 2.01 (s, 3H,
3
2
2
2
2
), 4.67 (s, 1H, CH),
4
2
1
3
C-NMR (DMSO-d
6
3
2
2
2
(
(
(
CH ), 48.96 (CH
2
2
2
2
d, CH, J= 26 Hz), 118.12 and 118.16 (d, CH, J= 4.0 Hz), 121.23 (CH), 123.04 (C), 126.43
C), 139.58 and 139.66 (d, C, J= 8.0 Hz), 144.92 and 144.97 (d, C, J=5.0 Hz), 150.77 and
1
53.22 (d, C, J= 245.0 Hz), 163.05 (C=O), 164.78 (C=O), 168.68 (C=O), 170.20 (C=O). LC-
+
+
MS: 560.10 ([M+Na] , 22), 491.17 ([(M-1)-CO H] , 22), 313.07 (100).
2
3
3
.2.
Biological activity
.2.1. Antimicrobial activity
The test microorganisms were obtained from the Hifzissihha Institute of Refik Saydam
(Ankara, Turkey) and were as follows: Escherichia coli (E. coli) ATCC35218, Yersinia
24

ACCEPTED MANUSCRIPT
pseudotuberculosis (Y. pseudotuberculosis) ATCC911, Pseudomonas aeruginosa (P.
aeruginosa) ATCC43288, Enterococcus faecalis (E. faecalis) ATCC29212, Staphylococcus
aureus (S. aureus) ATCC25923, Bacillus cereus (B. cereus) 709 Roma, Mycobacterium
smegmatis (M. smegmatis) ATCC607, Candida albicans (C. albicans) ATCC60193 and
Saccharomyces cerevisiae (S. cerevisia) RSKK 251, Ar: Arthrobacter oxydans (laboratory
strain), Ct: Candida tropicalis, ATCC 13803, Pv: Proteus vulgaris ATCC 13315, Ac:
Acinetobacter sp (laboratory strain), except Serretia marcescens (Sm), Acinetobacter sp (Ac)
and Klebsiella oxitoka (Ko) which are laboratry strains. All the newly synthesized compounds
were weighed and dissolved in hexane to prepare extract stock solution of 20.000
microgram/milliliter (ꢀg/mL).
The antimicrobial effects of the substances were tested quantitatively in respective broth
media by using double microdilution and the minimal inhibition concentration (MIC) values
(µg/mL) were determined. The antibacterial and antifungal assays were performed in Mueller-
Hinton broth (MH) (Difco, Detroit, MI) at pH.7.3 and buffered Yeast Nitrogen Base (Difco,
Detroit, MI) at pH 7.0, respectively. The micro dilution test plates were incubated for 18-24 h
at 35 °C. Brain Heart Infusion broth (BHI) (Difco, Detriot, MI) was used for M. smegmatis,
and incubated for 48-72 h at 35 °C [50]. Ampicillin (10 ꢀg) and fluconazole (5ꢀg) were used
as standard antibacterial and antifungal drugs, respectively. Dimethylsulphoxide with dilution
of 1:10 was used as solvent control.
3
.2.2. Urease inhibition assay
Reaction mixtures comprising 25 µL of Jack Bean Urease, 55 µL of buffer (100 mM
2 4
urea, 0.01 M K HPO , 1 mM EDTA and 0.01 M LiCl, pH 8.2) and 100 mM urea were
incubated with 5 µL of the test compounds at room temperature for 15 min in microtiter
plates. The production of ammonia was measured by indophenol method and used to
25

ACCEPTED MANUSCRIPT
determine the urease inhibitory activity. The phenol reagent (45 µL, 1% w/v phenol and 0.005
%
%
w/v sodium nitroprusside) and alkali reagent (70 µL, 0.5 % w/v sodium hydroxide and 0.1
v/v NaOCl) were added to each well and the increasing absorbance at 625 nm was
measured after 20 min, using a microplate reader (Molecular Device, USA). The percentage
inhibition was calculated from the formula 100 – (ODtestwell/ODcontrol) × 100. Thiourea
was used as the standard inhibitor. In order to calculate IC values, different concentrations of
50
synthesized compounds and standard were assayed at the same reaction conditions [51].
3
.2.3. β-Lactamase assay
In vitro β-lactamase (Bacillus cereus metallo β-lactamase, Sigma) activity was
determined by monitoring the hydrolysis of reporter substrate Nitrocefin (Calbiochem,
Darmstadt, Germany) by β-lactamase, at 486 nm (Louie et al. 2012). Enzyme assays were
performed in 25 mM piperazine-N,N’-bis (2-ethane sulfonic acid) (PIPES) buffer pH 7.0 with
1
4
00 µM ZnSO . 50 µL 1.5 µM enzyme solution, 50 µL 150 µM Nitrocefin and 50 µL 1 mM
synthesized compound were recorded continuously for 20 minutes at 37 °C against the buffer
alone by using microplate reader (Multiskan GO Microplate Spectrophotometer, Thermo
Scientific) at 486 nm. The inhibitory activity of those compounds and HgCl , a positive
2
control against β-lactamase were measured at various concentrations. Residual activities were
calculated by comparing to control without inhibitor (T+). The assays were done in triplicate.
The IC50 value was determined as the concentration of compound that give 50% inhibition of
maximal activity [52].
Acknowledgement
The support provided by Scientific and Technological Research Council of Turkey
(TUBITAK, Project no: 111T427).
26

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