Synthesis of some heterofunctionalized penicillanic acid derivatives and investigation of their Biological Activities

Article abstract of DOI:10.1002/ardp.201300280

6-Substituted amino-penicillanic acid esters were synthesized starting with 6-apa. The compounds containing a 1,3-thiazole- or 1,3-thiazolidinone nucleus linked to the penicillanic acid skeleton via a hydrazino linkage were obtained from 6-apa. The treatment of carbonylamino and carbonothioylamino compounds with 4-chlorophenacyl bromide or ethyl bromoacetate gave 6-bis{4-[1,3- thiazol(idinone)amino]benzoyl}amino derivatives of 6-apa. Benzyl derivatives were synthesized in several steps, starting with 4-aminobenzoyl chloride. The treatment of 4-{[3-benzyl-4-oxo-1,3-thia(oxa)zolidin-2-ylidene]amino}benzoyl chlorides with 6-apa in ethanolic solution produced the 6-[bis(4-{[3-benzyl-4- oxo-1,3-thiazolidin-2-ylidene]amino}benzoyl)amino] derivative of penicillanic acid, while the reaction of the same intermediates in DMF gave the mono-substituted amino derivative of 6-apa. The synthesized compounds were screened for their biological activities, and some of them were found to possess good to moderate antimicrobial activity. Moreover, some of the compounds displayed antiurease, anti-β-lactamase, and/or antilipase activities. Synthesis of some penicillanic acid derivatives including several heterocyclic rings was performed and the biological activities of the synthesized compounds were investigated. Some of them were found to possess antimicrobial, anti-β-lactamase, antiurease, and/or antilipase activity.

Full text of DOI:10.1002/ardp.201300280

200
Arch. Pharm. Chem. Life Sci. 2014, 347, 200–220
Full Paper
Synthesis of Some Heterofunctionalized Penicillanic Acid
Derivatives and Investigation of Their Biological Activities
Serpil Demirci1, Ahmet Demirbas1, Serdar Ulker2, Sengul Alpay-Karaoglu2, and Neslihan Demirbas1
1
Department of Chemistry, Karadeniz Technical University, Trabzon, Turkey
Department of Biology, Recep Tayyip Erdoğan University, Rize, Turkey
2
6-Substituted amino-penicillanic acid esters were synthesized starting with 6-apa. The compounds
containing a 1,3-thiazole- or 1,3-thiazolidinone nucleus linked to the penicillanic acid skeleton via a
hydrazino linkage were obtained from 6-apa. The treatment of carbonylamino and carbono-
thioylamino compounds with 4-chlorophenacyl bromide or ethyl bromoacetate gave 6-bis{4-[1,3-
thiazol(idinone)amino]benzoyl}amino derivatives of 6-apa. Benzyl derivatives were synthesized in
several steps, starting with 4-aminobenzoyl chloride. The treatment of 4-{[3-benzyl-4-oxo-1,3-thia(oxa)-
zolidin-2-ylidene]amino}benzoyl chlorides with 6-apa in ethanolic solution produced the 6-[bis(4-{[3-
benzyl-4-oxo-1,3-thiazolidin-2-ylidene]amino}benzoyl)amino] derivative of penicillanic acid, while the
reaction of the same intermediates in DMF gave the mono-substituted amino derivative of 6-apa. The
synthesized compounds were screened for their biological activities, and some of them were found to
possess good to moderate antimicrobial activity. Moreover, some of the compounds displayed
antiurease, anti-b-lactamase, and/or antilipase activities.
Keywords: 6-Amino penicillanic acid / Anti b-lactamase activity / Antilipase activity / Antimicrobial activity / Anti-urease
activity / 1,3-Oxa(thia)zole
Received: July 25, 2013; Revised: September 12, 2013; Accepted: September 17, 2013
DOI 10.1002/ardp.201300280
Introduction
because the use of antimicrobial drugs to treat with infections
causes several problems in some cases, especially patients
with impaired liver or kidney functions [6, 7]. Recent
studies have shown that administration of highly potent
antimicrobial agents at appropriate dosage will not only
eradicate bacterial growth, but also minimize the probability
of resistance formation [1].
Design and synthesis of new combinational chemother-
apeutics constitute a major challenge of medicinal chemistry,
which aims to overcome the antimicrobial resistance [8].
Besides the development of completely new agents possessing
different chemical properties than those of the existing
ones, another approach is to combine two or more
pharmacophores into a single molecule. Therefore, a single
molecule containing more than one pharmacophore, each
As a result of enhanced antimicrobial resistance, infectious
diseases account for a substantial part of deaths worldwide.
Although World Health Organization has defined the
antimicrobial agents as miracle drugs and leading weapons
for the treatment of infectious diseases, a number of
clinically efficacious antimicrobial agents, which are actively
used, have become less effective due to antimicrobial
resistance [1–5]. Furthermore, treatment of infectious diseases
is more difficult in immunodeficient patients such as those
infected with human immunodeficiency virus (HIV) [1]. The
rising prevalence of multi-drug-resistant bacteria has served
the medicinal chemists to design and synthesize novel
antimicrobial agents, which are active against these resistant
strains in current treatment. Besides, the newly synthesized
agents should have fewer side effects on the human body
with
a different mode of action, could be beneficial
for the treatment of microbial infectious [9–13]. 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 [14].
Correspondence: Prof. Neslihan Demirbas, Department of Chemistry,
Karadeniz Technical University, 61080 Trabzon, Turkey.
E-mail: neslihan@ktu.edu.tr
Fax: þ90 4623253196
ß 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

Arch. Pharm. Chem. Life Sci. 2014, 347, 200–220
Heterofunctionalized Penicillanic Acid Derivatives
201
b-Lactam derivatives constitute
a
class of the most
such as pyelonephritis, hepatic coma, peptic ulceration,
injection-induced urinary stones, and stomach cancer [27, 28].
Moreover, the mixing of NH3 to the atmosphere from urea is
subsequently deposited on land or water. The result of this is
eutrophication and acidification of natural ecosystems on a
regional scale [29].
Obesity can lead to a series of serious diseases including but
not limited to hypertension, hyperlipidemia, arteriosclerosis,
and type II diabetes [30]. Pancreatic lipase plays an important
role in digestion of fat. Pancreatic lipase inhibitors, such as
orlistat, have been used as therapeutic agents for curing
obesity [31]. However, some side effects including fecal
incontinence, flatulence, and steatorrhea have been reported
for orlistat [32, 33]. The compounds possessing anti-lipase
activity can be the alternatives of orlistat.
important antibacterial agents in the current clinical
regimen, although the threat of resistance is getting stronger.
Besides their applications as antibacterials, b-lactams are
increasingly used as inhibitor of other medicinally important
targets [15]. Nowadays, the industrial production of semi-
synthetic b-lactam antibiotics is mostly carried out by
chemical syntheses containing complex routes in which
extreme temperatures (i.e., ꢀ30°C) and organochloride
solvents are involved. But, these methods are being criticized
since they are potential sources of environmental dam-
ages [16]. Accordingly, enzymatic synthesis of b-lactam
antibiotics received great attention from industry and
academy as a green chemistry alternative. However, low
yield and selectivity restricted the industrial application of
the enzymatic methods [17].
In the present study, as a part of our ongoing study on the
synthesis of bioactive hybrid molecules, we aimed to obtain
new penicillanic acid derivatives containing azole moieties in
a more environment-friendly way.
Among several privileged structures, thiazolidinone ring is
one of the most attractive targets for combinatorial synthesis,
since its structure–activity relationship belongs to an
important class of N- and S-containing heterocycles, which
are widely used in drug design and synthesis studies [18, 19]. Results and discussion
Oxazolidinones are reported as a unique class of anti-
bacterials interacting with the bacterial ribosome to inhibit
bacterial protein synthesis by preventing formation of 70S
initiation complex [20–22]. This mechanism of action allows
organisms, which are resistant to other antimicrobials, to
remain susceptible to oxazolidinone inhibition [23].
Substituted piperazines were reported as important
pharmacophores, which are involved in a number of
therapeutically important drugs such as crixivan, an HIV
protease inhibitor, norfloxacine, ciprofloxacine, levofloxa-
cine, second-generation fluoroquinolones, eperezolid, ranbe-
zolid, which are oxazolidinone class antibacterials, and an
antifungal drug, itraconazole [24].
It is known that the production of enzymes such as the
serine-b-lactamase (SBLs) and metallo-b-lactamase (MBLs) in
bacteria increasingly causes the resistance against a broader
range of common b-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, b-lactamase inhibitors have gained importance to
overcome the antibacterial resistance [25, 26]. In this context,
a number of natural and synthetic compounds have been
reported to possess anti b-lactamase activity, which catalyzes
the hydrolysis of the CO–N bond in the molecules of
penicillins and cephalosporins. However, only a few of them
have found a field of use at clinical settings.
Chemistry
The main aim of the present study is to design and synthesize
new penicillanic acid derivatives incorporating piperazine,
1,3-thiazol(idinone), or 1,3-oxazol(idinone) ring within a
single structure, and to investigate their biological activities.
The core structure of the present study, penicillanic acid,
belongs to the b-lactam class compounds, which constitute
one of the most important antibacterial agents, besides their
other applications including b-lactamase inhibitory effect,
etc. [15–17]. Moreover, the pharmacological importance of
other groups, namely 1,3-oxazole, 1,3-thiazole, and pipera-
zine, which were combined with penicillanic acid skeleton in
the present study, was well documented [18–24]. In addition,
in one of our previous studies [34] the discovery of some azole
compounds as antiurease agents served to merge the azole
groups, namely 1,3-oxazol(idin)e and 1,3-thiazol(idin)e, with
the penicillanic acid skeleton. Another reason for choosing
these azole groups for hybridization is that some hybrid
compounds carrying 1,3-oxazol(idin)e or 1,3-thiazol(idin)e
nucleus were obtained as antilipase agents in our laborato-
ry [35, 36].
The intermediate and target compounds were synthesized
according to the reactions outlined in Schemes 1–5. The
starting compound (þ)-6-aminopenicillanic acid (6-apa, 1) was
provided commercially.
Compound 3 was synthesized via the reaction of (þ)-6-
amino penicillanic acid (6-apa, 1) with 2-fluoro-4-amino-
benzoic acid in the presence of ethyl chloroformate and
triethylamine at low temperature. This idea originated from
the need to incorporate an amide function to molecule,
Bacterial urease enzymes, which accelerate hydrolysis of
urea to ammonia gas with the reaction rate at least 1014 over
the spontaneous reaction, have been reported as important
virulent factors including several important pathogeneses
ß 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.archpharm.com

202
S. Demirci et al.
Arch. Pharm. Chem. Life Sci. 2014, 347, 200–220
NO2
Si HN
O
S
F
N
O
O2N
N
O
S
C
O
N
O
F
C
O
4
O
iii
NH
H2N
S
H2N
S
S
H2N
O2N
F
N
N
ii
N
O
i
O
O
O
C
C
C
O
O
OH
O
OH
2
3
1
H
N
H
S
HN
N
N
S
N
O
iv
N
O
O
O
C
C
O
O
O
O
O
O
6
7
_
+
TsO H3N
v
S
vi
N
O
O
C
O
O
O
vii
5
viii
F
O2N
H
O2N
N
N
N
H
N
S
S
S
O2
N
O
N
O
O
O
C
C
O
O
O
O
O
O
9
8
Scheme 1. i: Ethylchloroformate, 2-fluoro-4-aminobenzoic acid, Et3N, 6-apa, stirring in THF at room temperature; ii: catalytic amount
of H2SO4, reflux in ethanol; iii: Et3N, 6-apa, H2NCONH2 in dry DCM, trimethyl chlorosilane, n-BuLi at N2 atm, 4-bromo-2-fluoronitrobenzene;
iv: Et3N, 6-apa, chloromethylpivalate and then p-TSA; v: Et3N, 4-bromonitrobenzene in DCM; vi: piperazine in DCM at room temperature;
vii: Et3N and 1-(2-fluoro-4-nitrophenyl)piperazine, stirring in DCM at room temperature; viii: Et3N, 4-nitrobenzene sulfonyl chloride, stirring in
DCM at room temperature.
because amide bond is essential for antibacterial activity,
especially in b-lactam antibiotics. For instance, ampicillin,
penicillin G, penicillin V, nafcillin, meticillin, oxacillin,
pivampicillin, and talampicillin incorporate an amide
function instead of amine [24].
The treatment of 6-aminopenicillanic acid ethyl ester (2)
with 3,4-difluoro nitrobenzene resulted in disubstitution of
NH2 protons and generated compound 4, when 6-apa itself
was used in this reaction; no change could be observed due
to the low reactivity of compound 1 toward nucleophilic
ß 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.archpharm.com

Arch. Pharm. Chem. Life Sci. 2014, 347, 200–220
Heterofunctionalized Penicillanic Acid Derivatives
203
H2N
O
S
i
NHR
HN
N
X
COOH
S
1
N
O
_
+
COOHN
_
10a e
10a: X= S, R=
10b: X= O, R=
10c: X= S, R=
10d: X= O, R=
CH2Ph
CH2Ph
C6H4OCH3(p
Ph
)
10e: X= S, R= C(CH3)3
iii
ii
N
N
N HN
O
S
N HN
O
S
X
O
X
N
N
_
_
+
+
COOHN
COOHN
12a: X= S
12b: X= O
11a: X= S
11b: X= O
Cl
Scheme 2. i: RNCX, Et3N, DMF, room temperature; ii: (4-)ClC6H4COCH2Br, CH3COONa, reflux in ethanol; iii: BrCH2CO2Et, CH3COONa,
reflux in ethanol.
aromatic substitution. It was reported that the conversion of
6-apa to compound 5 increases the reactivity of the amine-
nitrogen of 6-apa toward nucleophilic substitution [37]. The
substitution of one of two amine protons by 4-nitrophenyl
nucleus was carried out starting from compound 5, and thus,
compound 6 was obtained.
The signals derived from phenyl moiety were present at the
aromatic region in the 1H and 13C NMR spectra of compounds
3, 4, and 6. The mass spectra and elemental analysis were
consistent with mono-substitution of amine protons of 6-apa,
leading to the formation of compounds 3 and 6. On the other
hand, the mass spectrum and the elemental analysis
supported the disubstitution for compound 4.
amines in dichloromethane, contain a piperazine nucleus as
an additional pharmacophore linked to the penicillanic acid
skeleton. In the 1H and 13C NMR spectra of compounds 7 and
8, the signals originating from the piperazine moiety were
observed at the related chemical shift value. In addition,
1H and 13C NMR spectra of compound 8 displayed additional
signals appearing in the aromatic region due to 4-nitrophenyl
moiety. The stretching bands due to nitro group were present
in the FT-IR spectra of compounds 6 and 8.
Compounds 10a–e were obtained from the reaction of
compound 1 with several isocyanates and isothiocyanates as
important intermediates leading to the formation of 1,3-
oxazol(idinone) and 1,3-thiazol(idinone) derivatives.
With the aim to bring
a
sulfonamide function to
As different from compound 1, 1H and 13C NMR spectra of
compounds 10a–e exhibited additional signals due to (thio)-
urea and triethylammonium moieties at the related chemical
shift values. In addition, these compounds gave relatively
stable [M]þ, [Mþ1]þ, [Mꢀ1]þ, or [Mþ2]þ ion peaks and
elemental analysis data. The intermolecular cyclization of
compounds 10a,b with ethyl bromoacetate in boiling absolute
penicillanic acid core structure, compound 9 was obtained
by the reaction of compound 5 with 4-nitrobenzene sulfonyl-
chloride in anhydrous DMF; this idea originated from the fact
that the antimicrobial activities of sulfonamides were well
documented [24]. Compounds 7 and 8, which were obtained
by the reaction of compound 5 with the corresponding
ß 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.archpharm.com

204
S. Demirci et al.
Arch. Pharm. Chem. Life Sci. 2014, 347, 200–220
O
O
H2N
H
N
Cl
S
O2N
N
iii
O
COOH
NH2
NO2
H2N
O
O
O
S
i
ii
O
O
N
N
O
N
S
S
N
COOH
N
O
COOH
COOH
H2N
O2N
1
14
13
iv
Cl
S
N
S
HN
NH
N
O
v
O
O
O
N
O
S
N
S
N
N
O
COOH
S
HN
NH
COOH
N
O
N
16
N
S
15
S
N
vi
O
Cl
O
N
S
N
O
COOH
N
N
S
O
17
Scheme 3. i: K2CO3, (4-)NO2C6H4COCl, acetone–water, rt; ii: Pd-C, H2NNH2, reflux in n-butanol; iii: 6-apa, K2CO3, acetone–water, rt;
iv: PhCNS, reflux in ethanol; v: (4-)ClC6H4COCH2Br, CH3COONa, reflux in ethanol; vi: BrCH2CO2Et, CH3COONa, MW 200 W.
ß 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.archpharm.com

Arch. Pharm. Chem. Life Sci. 2014, 347, 200–220
Heterofunctionalized Penicillanic Acid Derivatives
205
NH2
O
O
N
O
S
N
COOH
H2N
14
X
i
N
H
NH
O
O
X
O
O
N
O
N
H
HN
S
X
Cl
H2N
Cl
ii
i
N
N
H
HN
19a: X= S
19b: X= O
18a: X= S
18b: X= O
COOH
iv
iii
O
O
N
O
N
v
N
N
N
Cl
S
N
N
O
X
O
S
O
O
20a: X= S
20b: X= O
S
N
21
HOOC
vi
O
H
N
N
N
S
X
O
N
O
22a: X= S
22b: X= O
COOH
Scheme 4. i: C6H5CH2CNS(O) MW at 150 W or reflux in ethanol; ii: 6-apa, K2CO3, acetone–water, stirring at rt; iii and iv: BrCH2CO2Et,
CH3COONa, reflux in ethanol; v: 6-apa, K2CO3, in DMF, rt; vi: 6-apa, K2CO3, acetone–water, rt or MW (for 22b) 150 W.
ethanol afforded the corresponding 5-oxo-1,3-thiazole (12a) or
5-oxo-1,3-oxazole (12b) derivatives, while the cyclocondensa-
tion of the same intermediates (10a,b) with 4-chloropenacyl-
bromide produced 1,3-thiazole (11a) or 1,3-oxazole (11b)
compounds. With the conversion of compounds 10a,b into
11a,b, one of two NH signals disappeared in the 1H NMR
spectra, while additional signals originating from chloro-
phenyl nucleus were recorded in the aromatic region in the
1H and 13C NMR spectra of compounds 11a,b. Moreover,
compounds 11 and 12 displayed reasonable elemental
analysis data consistent with the assigned structures.
With the aim to obtain
a useful intermediate for
further cyclizations and also to bring an amide function,
the NH2 position of 6-apa (1) was functionalized to two
4-nitrophenylbenzoyl groups in acetone–water at cold-
room temperature; thus, compound 13 was prepared. In
this reaction, monoacyl derivative of 6-apa could not be
obtained.
ß 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.archpharm.com

206
S. Demirci et al.
Arch. Pharm. Chem. Life Sci. 2014, 347, 200–220
X
O
N
H
N
H
O
X
N
S
N
HN
N
H
O
18a: X= S
18b: X= O
COOH
i
N
N
O
O
X
N
N
S
N
Cl
N
X
O
COOH
24a: X= S
Cl
24b: X= O
iii
X
O
O
N
N
HN
ii
Cl
N
H
Cl
X
19a: X= S
19b: X= O
23a: X= S
23b: X= O
Cl
Scheme 5. i: (4-)ClC6H4COCH2Br, CH3COONa, reflux in ethanol; ii: (4-)ClC6H4COCH2Br, CH3COONa, MW (150 W for 23a) or reflux
(for 23b); iii: 6-apa, K2CO3 in DMF, rt.
Our efforts on the reduction of nitro groups to amino
groups in different reduction conditions resulted in failure
due to decomposition of b-lactam ring. The synthesis of the
amine 14 was achieved by the reaction of 4-nitrobezoyl
chloride with 6-apa at cold-room temperature. The FT-IR
spectrum of 13 demonstrated two sharp signals pointing the
presence of nitro groups, whereas the FT-IR spectrum of 14
displayed absorption bands derived from amine functions.
The signals belonging to two phenyl nuclei were recorded at
aromatic region in the 1H and 13C NMR spectra of compounds
13 and 14. In addition, these compounds displayed reasonable
mass fragmentation; and their elemental analyses are
consistent with the assigned structures.
The treatment of compound 14 with phenylisothiocyanate
yielded the corresponding bithiourea derivative, 15. However
our attempt to transform the amino groups of 14 into
ß 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.archpharm.com

Arch. Pharm. Chem. Life Sci. 2014, 347, 200–220
Heterofunctionalized Penicillanic Acid Derivatives
207
benzylthiourea and benzylurea function was unsuccessful
because obtained viscous reaction mixture was very complex
and the components could not be separated and identified.
However, the synthesis of (thio)urea derivatives, 18a and 18b,
could be achieved by the treatment of 6-apa with 19a,b, which
were obtained starting from 4-aminobenzoyl chloride.
In the FT-IR spectra of compounds 19a,b, the absence of any
–
corresponding m/z values, which match with their molecular
formulas.
The cyclocondensation of 15 with 4-chlorophenacyl bro-
mide produced 1,3-thiazol-2(3H)-ylidene-penicillanic acid
derivative (16). On the other hand, the treatment of the
same intermediate (15) with ethyl bromoacetate afforded the
corresponding 4-oxo-3-phenyl-1,3-thiazolidine derivative (17).
–
signal due to –NH group and the presence of additional C O
The disappearance of C S stretching band in the FT-IR spectra
–
–
2
–
(or C S) stretching bands constitute an evidence for
of compounds 16 and 17 supported the cyclization. Moreover,
in the NMR spectrum of 16, the additional signals resonating
for 8 protons in aromatic region were attributed to two
chlorophenyl moieties. Elemental analysis of these com-
pounds was in accordance with their structures, while these
compound displayed reasonable fragmentation in the
mass spectra including [M–COOH]þ, [Mþ2–C6H5]þ, [M–PhCl]þ,
and/or [M–PhCl–COOH]þ molecular ion peaks, which match
with their molecular formulas.
Compounds 24a,b were obtained by microwave irradiation
of compounds 19a,b with 4-chlorophenacylbromide, followed
by the treatment of the obtained 1,3-thiazole derivatives
(23a,b) with 6-apa.
As different from 23a and 23b, the NMR spectra of
compounds 24a and 24b exhibited additional signals derived
from penicillanic acid nucleus at the related chemical shift
values. Moreover, the presence of [Mꢀ2Cl]þ or [Mþ1]þ ion
peak in the mass spectra at the related m/z values and
elemental analysis supported their molecular masses.
–
condensation between 4-aminobenzoyl chloride and benzyl-
isocyanate or benzylisothiocyanate. In the 1H NMR spectra of
compounds 19a,b, two D2O exchangeable peaks at 6.77–8.44
and 8.98–9.93 ppm attributed to two NH protons. Moreover,
in the 1H and 13C NMR spectra of these compounds, the
crowding in the aromatic region that originated from
additional phenyl ring supported the condensation. When
compounds 19a,b were converted to 18a,b, additional signals
were observed at the related chemical shift values in the
1H NMR spectra, pointing to the presence of penicillanic acid
core. In the 13C NMR spectra, these carbons resonated at the
expected chemical shift values in accordance with other 6-apa
derivatives synthesized in this study. When compounds 18a,b
were treated with ethyl bromoacetate with the aim to obtain
the corresponding 4-oxo-1,3-thiazolidine derivatives (21a,b),
an unidentified viscous mixture was obtained. But still
the synthesis of compound 21 was achieved by the reaction
of 6-apa with the intermediate 20a that was obtained
from the condensation between 19a and ethyl bromoacetate.
It is interesting to note that the condensation between 6-apa
and 20a generated the diacylated product (21) in DMF,
while similar condensation between 6-apa and 20a,b in
ethanolic solution resulted in the monoacylation; thus,
compounds 22a,b were obtained. The structures of com-
pounds 21 and 22a,b were elucidated by FT-IR, 1H NMR,
13C NMR, EI-MS spectroscopic methods, and elemental
analysis.
To introduce the 1,3-thiazole nucleus into penicillanic acid
molecular framework, compound 19a was treated with 4-
chlorophenacyl bromide, followed by the reaction with 6-apa;
thus, compounds 22a,b were obtained. 1H NMR spectra of
compounds 19a,b revealed the presence of two NH groups due
to the signals observed at 8.44 ppm (for 19a) and 7.87 ppm (for
19b) as D2O exchangeable peaks, while 1H NMR spectra of
compounds 22a,b displayed only one D2O exchangeable peak
at 9.23 (for 22a) and 8.78 ppm (for 22b) corresponding to one
NH proton. Furthermore, in the NMR spectra of these
compounds (22a,b), additional signals derived from 4-oxo-
1,3-thiazolidine- and penicillanic acid moieties appeared at
the related chemical shift values. In addition, elemental
analyses were consistent with the assigned structures for
these compounds, and the mass spectra of the cyclization
products (22a,b) showed [M]þ or [MþH2O]þ ion peaks at the
Biological activity
Antimicrobial activity
Compound 3 that contains a 4-amino-2-fluorophenylbenzo-
ylamino moiety at the position 6 of penicillanic acid
framework exhibited activity toward the test microorganisms
except Mycobacterium smegmatis (Ms), Candida albicans (Ca), and
Saccharomyces cerevisiae (Sc). With the conversion of amid
function to amine group in the structures of compounds 4
and 6, the antimicrobial activity diminished; only slight
activity was observed on Ca and Sc, which are yeast-like fungi.
Similarly, the introduction of piperazine nucleus into the
structure of 6-apa resulted in no activity on the test
microorganisms. On the other hand, compound 8 that
contains a 4-(4-nitrophenyl)piperazine nucleus in the pen-
icillanic acid structure displayed activity against Pseudomonas
aeruginosa (Pa), Staphylococcus aureus (Sa), Enterococcus faecalis (Ef),
which are Gram positive cocci, Bacillus cereus (Bc) that is Gram
positive spore bacillus, Mycobacterium smegmatis (Ms) that is an
atypical tuberculosis factor, and Ca. This compound (8) can be
considered as the derivative of compound 7 including an
additional 4-nitrophenyl group or a derivative of 6 with
piperazine nucleus. Similarly, compound 9 that is sulfon-
amide derivative of 6 demonstrated moderate activity on the
same test microorganisms with compound 8. The carbo(thio)-
ß 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.archpharm.com

208
S. Demirci et al.
Arch. Pharm. Chem. Life Sci. 2014, 347, 200–220
nylamino derivatives (10a–e) showed better antimicrobial
activity than compounds 3–9. Among these compounds, the
activity of compound 10b towards Sa was the same as that of
standard drug ampicillin. 10d, which contains a phenylamino
nucleus in the carbonylamino side chain, displayed moderate
activity against Ef with the inhibition zone of 8 mm. Other
carbono(thio)ylamino compounds (10a, 10b, 10c, and 10d)
exhibited better activities than standard drug ampicillin, with
the inhibition zones varying between 12 and 30 mm.
Compounds 10a and 10e were found to possess the same
activity on Bc as ampicillin. Among compounds 10a–e, anti-Ca
and -Sc activity was observed for compounds 10a and 10c,
which are the compounds containing benzylamino (10a) or
4-methoxyphenylamino (10c) moiety in the carbonothioyl-
amino side chain linked to penicillanic acid core. The activity
of 10c was better than 10a. Other carbo(thio)nylamino
compounds 18a,b and 19a,b displayed good-to-moderate
inhibition activity on some of the test microorganisms. The
penicillanic acid derivatives functionalized with 3,5-disubsti-
tuted 1,3-thiazole nucleus at the position 6 (11a) were
found to be active on the test microorganisms except Ec,
Yp, and Pa. On the other hand, compound 11b, which
contains a 1,3-oxazole ring instead of 1,3-thiazole moiety,
displayed only slight activity on Sa with the inhibition
zone of 8 mm. Among other 1,3-thiazole derivatives (16, 23a,
and 24a) the best antibacterial activity was observed
for compound 16 with the inhibition zones between 10
and 20 mm. Compound 24 carrying a 1,3-oxazole ring
instead of 1,3-thiazole exhibited good-to-moderate activity
on Ec, Yp, Pa, Bc, and Sc, while no inhibitory effect was
observed for other 1,3-oxazole compound 23b that did not
contain any penicillanic acid framework. Among the com-
pounds 12a,b, 17, 20a,b, 21, and 22a,b, which are the 6-apa
derivatives incorporating 5-oxo-1,3-thiazolidine (12a, 17, 20a,
21, and 22a) or 5-oxo-1,3-oxazolidine (12b, 20b, and 22b)
moiety, compounds 17, 21, 22a,b were found to be active
against some of the test microorganisms. Compound 13 that
b-lactamase inhibition. HgCl2 with IC50 value 0.093 mM was
used as standard inhibitor.
Anti-urease activity
The synthesized compounds were assayed for their in vitro
inhibitory activity against Jack bean urease. Three of those
compounds, 10c, 10d, and 10e, which are alkylamino-
carbono(thio)yl derivatives of 6-apa, showed potent urease
inhibition. Thiourea with IC50 value 51.62 ꢁ 7.28 mg mLꢀ1 was
used as standard inhibitor. Among tested compounds 10e
was found to have the maximum inhibitory effect against
urease with an IC50 value of 54.14 ꢁ 1.45 mg mLꢀ1. Com-
pounds 10c and 10d have moderate inhibitory activity. Dose-
dependent inhibitory effects of compounds are depicted in
Fig. 1. These compounds might be considered as potential
antibiotics to treat infections. Urease inhibitor activity
of the synthesized compounds may be associated with
inhibition of H. pylori, a key cause of stomach infections.
Anti-lipase activity results
All compounds were evaluated with regard to pancreatic
lipase activity, and 11a, 18b, and 20a showed anti-lipase
activities at various concentrations. No significant inhibitory
effect was detected for other compounds. Dose-dependent
pancreatic lipase activity is shown in Fig. 2. Among the tested
compounds, 20a showed the best anti-lipase activity. The
compound inhibited pancreatic lipase activity by 62, 91, and
94% at concentrations of 1.25, 3.75, and 6.25 mg mLꢀ1
,
respectively. Orlistat, a known pancreatic lipase inhibitor
used as anti-obesity drug, showed inhibitory effect by 80, 87,
and 98% at the same concentrations. Orlistat and compound
20a IC50 values were calculated as 0.61 and 0.84 mg mLꢀ1
,
respectively. Orlistat is the only approved anti-obesity
medication [31] but it has some side effects, such as fecal
incontinence, flatulence, and steatorrhea [32]. Compound 20a
can be considered as a good alternative to orlistat.
can be considered as disubstituted derivative of 6 displayed Conclusions
moderate activity against the test microorganisms except Ef,
Ca, and Sc. With the conversion of compound 13 to 14 by
the reduction of two nitro groups, no activity was observed
on Ec, Yp, and Ms; instead, additional activities were
noted on Bc, Ca, and Sc. Compound 14 and its carbono-
thioylamino derivative 15 displayed the same activity on Sc
with the inhibition zone 25 mm that is the same as with
the standard drug fluconazole.
This study reports the successful synthesis of some new
penicillanic acid derivatives containing piperazine, 1,3-
oxazole, or 1,3-thiazole moiety. The antimicrobial, anti-
b-lactamase, anti-urease, and anti-lipase activity screening
studies were also performed in the study. Penicillanic acid
nucleus is one of the active components present in many
standard drugs and it is known to increase the pharmacologi-
cal activity of the molecules. The presence of piperazine, 1,3-
oxazol(idin)e, or 1,3-thiazol(idin)e moiety is also instrumental
in contributing to the net biological activity of a system.
Moreover, the therapeutic efficiency of hybrid molecules
containing different pharmacophore groups was well
Anti-b-lactamase activity
The synthesized compounds were assayed for their in vitro
inhibitory activity against B. cereus b-lactamase. Compounds
10a, 10b, and 10d, which are 6-apa derivatives carrying
an alkylaminocarbo(thio)nylamino side change, showed low
documented for
a
number of pathological conditions.
ß 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.archpharm.com

Arch. Pharm. Chem. Life Sci. 2014, 347, 200–220
Heterofunctionalized Penicillanic Acid Derivatives
209
120
100
80
60
40
20
0
10c
10d
10e
Thiourea
0
50
100
150
200
250
Conc. (µg/mL)
Figure 1. Dose-dependent inhibitory effect of some of the synthesized compounds. Thiourea was used as standard inhibitor. Inhibitory
effect of all compounds and thiourea were measured in the range of 250–0.114 mg mLꢀ1 concentrations. Residual activities of compounds
are expressed as the mean ꢁ S.D. in triplicate.
11a
18b
20a
100
80
60
40
20
0
Orlistat
0
1
2
3
4
5
6
7
Conc. (µg/mL)
Figure 2. Dose-dependent inhibitory effects of the compounds. Orlistat was used positive control. All compounds and orlistat were
measured at final concentrations of 1.25, 3.75, and 6.25 mg mLꢀ1. Residual activities of compounds are expressed as the mean ꢁ S.D. in
triplicate.
ß 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.archpharm.com

210
S. Demirci et al.
Arch. Pharm. Chem. Life Sci. 2014, 347, 200–220
Hence, herein we combined all these potential units, namely
penicillanic acid, piperazine, 1,3-oxazol(idin)e, and 1,3-
thiazol(idin)e rings or sulfonamide function. Compounds
16 and 17, which incorporate a phenyl substituent at the
position 3 of 1,3-thiazole or 1,3-thiazolidine ring, could
be obtained from condensation of compound 15 with 4-
chlorophenacyl bromide (for compound 16) or ethyl bromo-
acetate (for compound 17). On the other hand, compounds
18a and 18b, which are the intermediates incorporating a
benzyl substituent instead of phenyl, gave unidentified
products at the same condensation conditions. It might be
speculated that this result was obtained probably due to the
–
unreacted 6-apa was removed by filtration. After evaporating the
solvent under reduced pressure, a solid was obtained. This crude
product was recrystallized from ethyl acetate to give the pure
compound. Yield 62%, m.p.: 140–141°C. FT-IR (ymax, cmꢀ1): 3060
–
–
(aromatic C–H), 2902 (aliphatic C–H), 1722 (C O), 1625 (C O),
–
–
1235 (C–O). 1H NMR (DMSO-d6): d (ppm) ¼ 1.17 (brs, 3H, CH3), 1.37
(brs, 3H, CH3), 1.54 (s, 3H, CH3), 3.42 (d, 2H, CH2), 3.73 (s, 1H, CH),
4.41 (s, 2H, CH), 8.50 (brs, 2H, NH2, D2O exch.). 13C NMR (DMSO-d6):
d (ppm) ¼ 12.11 (CH3), 27.90 (2CH3), 36.59 (CH2), 61.38 (CH), 62.54
–
–
–
(CH), 65.63 (CH), 72.76 (C), 168.19 (C O), 172.59 (C O). EI MS m/z
–
(%): 468.49 ([Mþ2]þ, 30), 467.55 ([Mþ1]þ, 100), 404.33 (23), 388.32
(25), 366.35 (35), 350.35 (32). Elemental analysis: for C10H16N2O3S
calcd. % C, 49.16; H, 6.60; N, 11.47. Found: % C, 48.86; H, 6.68; N,
11.46.
steric hindrance of the benzyl group on C S function of
–
compounds 18a and 18b. In this way, penicillanic acid core,
which already contains a stretched and unstable b-lactam
ring, underwent decomposition in the reaction conditions.
Thus, compounds 24a,b were obtained by another way
presented in Scheme 5.
The antimicrobial screening suggests that most of the
synthesized compounds exhibited good-to-moderate activities
against the test microorganisms. In addition, compounds
10a, 10b, and 10d, which are 6-apa derivatives carrying an
alkylaminocarbonylamino or alkylaminocarbonothioyl-
amino side chain, showed low b-lactamase inhibition.
Furthermore, three compounds, 10c, 10d, and 10e, displayed
perfect urease inhibition activity, while one compound, 20a,
demonstrated good anti-lipase activity.
(2S,5R,6R)-6-[(4-Amino-2-fluorobenzoyl)amino]-
penicillanic acid (3)
Ethylchloroformate (10 mmol) was added into the solution of
2-fluoro-4-aminobenzoic acid in dry tetrahydrofuran in the
presence of triethylamine (10 mmol) at ꢀ15°C, drop by drop,
and the mixture was stirred for 10 min. Then, the suspension of
6-apa (10 mmol) and triethylamine (10 mmol) in 10 mL of water
was added into it, and the reaction was maintained at room
temperature for 22 h. After the reaction was completed, water
was added into the reaction mixture and acidified to pH 6 with
diluted HCl. The acidified mixture was extracted with 15 mL of
dichloromethane (three times). The combined organic layer was
dried over sodium sulfate, and the solvent was evaporated under
reduced pressure. The obtained solid was recrystallized from n-
butyl acetate/diethyl ether (1:1) to give the pure product. Yield
21%, m.p.: 169°C. FT-IR (ymax, cmꢀ1): 3200 (NH2), 3104 (NH), 3063
–
–
–
(aromatic C–H), 2932 (aliphatic C–H), 1727 (C O), 1655 (2C O),
–
1233 (C–O). 1H NMR (DMSO-d6): d (ppm) ¼ 1.15 (brs, 6H, 2CH3), 1.97
(s, 1H, CH), 4.00 (brs, 2H, CH), 6.21–6.37 (m, 2H, NH2, D2O exch.),
7.53–7.58 (m, 3H Ar–H), 10.67 (brs, 1H, NH, D2O exch.). 13C NMR
(DMSO-d6): d (ppm) ¼ 27.19 (CH3), 31.30 (CH3), 58.79 (CH), 59.36(C),
65.57 (CH), 69.43 (CH), 112.45 (CH), 112.87 (CH), 125.19(CH),
Experimental
General
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 chromatog-
raphy (TLC) on silica-gel 60 F254 aluminum sheets. The mobile
phase was ethanol/ethyl ether 1:1, and detection was made using
UV light. FT-IR spectra were recorded using a Perkin Elmer 1600
series FTIR spectrometer. 1H NMR and 13C NMR spectra were
registered in DMSO-d6 on a BRUKER AVENE II 400 MHz NMR
spectrometer (400.13 MHz for 1H and 100.62 MHz for 13C) or
Varian-Mercury 200 MHz NMR spectrometer (200 MHz for 1H and
50 MHz for 13C). The chemical shifts are given in ppm relative to
Me4Si as an internal reference; J values are given in Hz. The
elemental analysis was performed on a Costech Elemental
Combustion System CHNS-O elemental analyzer. All the com-
pounds 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. Compound 5 and 1-(2-fluoro-4-nitrophenyl)pipera-
zine were obtained by the way reported earlier [34, 37].
–
–
–
–
137.03 (C), 142.3 (2C), 150.68 (C O), 153.42 (C O), 161.96 (C O). EI
–
–
MS m/z (%): 355.78 (42), 354.16 (37), 353.89 ([M]þ, 52), 265.19 (100).
Elemental analysis: for C15H16FN3O4S calcd. % C, 50.98; H, 4.56; N,
11.86. Found: % C, 51.01; H, 4.83; N, 11.56.
Ethyl (2S,5R,6R)-6-[bis(2-fluoro-4-nitrophenyl)amino]-
penicillanate (4)
Triethylamine (30 mmol) was added to the suspension of
6-aminopenicillanic acid (6-apa) (10 mmol) and urea (20 mmol)
in dry dichloromethane and the mixture was stirred at 10°C for
10 min, and at room temperature for 30 min. Then, trimethyl
chlorosilane (30 mmol) was added dropwise at 5°C, the reaction
content was stirred at room temperature for 30 min and an
additional 1 h at 40°C. n-BuLi (10.3 mmol) was added dropwise to
the solution of the obtained silyl ester of 6-apa [38] at ꢀ78°C in the
presence of nitrogen atmosphere, and the mixture was stirred at
the same temperature for 1 h. Then, the solution of 4-bromo-
2-fluoronitrobenzene in dichloromethane was added into the
reaction mixture and the stirring was continued at room
temperature for additional 7 h. The reaction mixture was cooled
to 0°C and 80 mL of water was added dropwise. The two layers
were separated and the water layer was extracted with 40 mL of
ethyl acetate two times. The combined organic layer was dried on
Ethyl (2S,5R,6R)-6-amino-penicillanate (2)
The suspension of 6-apa (10 mmol) in ethanol was refluxed in
the presence of catalytic amount of H2SO4 for 12 h. Then, the
ß 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.archpharm.com

Arch. Pharm. Chem. Life Sci. 2014, 347, 200–220
Heterofunctionalized Penicillanic Acid Derivatives
211
Na2SO4 and Na2SO4 was removed by filtration. Upon cooling the
mixture overnight, a solid formed. This crude product was
recrystallized from acetone to give the pure compound. Yield
429.31 (37), 413.31 (37). Elemental analysis: for C20H25N3O7S
calcd. % C, 53.20; H, 5.58; N, 9.31. Found: % C, 53.25; H, 5.59; N,
9.42.
57%, m.p.: 77–78°C. FT-IR (ymax, cmꢀ1): 1725 (C O), 1651 (C O),
–
–
–
–
1517 and 1341 (NO2), 1248 (C–O). 1H NMR (DMSO-d6):
d (ppm) ¼ 1.33 (t, 3H, CH3, J ¼ 7.0 Hz), 1.71 (s, 3H, CH3), 1.81
(s, 3H, CH3), 4.26 (m, 5H, 3CHþ CH2), 7.32 (d, 2H, Ar–H, J ¼ 8.6 Hz),
7.60 (s, 2H, Ar–H), 7.83 (d, 2H, Ar–H, J ¼ 8.6 Hz). 13C NMR
(DMSO-d6): d (ppm) ¼ 14.19 (3CH3), 65.70 (CH2), 58.98 (CH), 59.58
(C), 65.51 (CH), 68.58 (CH), arC: [118.16 (2CH), 123.33 (2CH), 126.48
(2CH), 126.98 (2C), 127.42 (C), 132.30 (C), 138.58 (2C)], 151.90
[(2,2-Dimethylpropanoyl)oxy]methyl (2S,5R,6R)-6-
(piperazin-1-yl)aminopenicillanate (7)
Piperazine (2.2 mmol) was added into the solution of compound 5
(2.2 mmol) in dichloromethane at 0°C and the mixture was
stirred at room temperature for 5 h. Water was added, and two
layers were separated. Water layer was extracted with 5 mL of
dichloromethane two times and the combined organic layer was
dried over NaSO4. On evaporating the solvent under reduced
pressure, a solid formed. This was recrystallized from ethyl
acetate/hexane (1:1) to obtain the target compound. Yield
25%, m.p.: 94–95°C. FT-IR (ymax, cmꢀ1): 3353 and 3300 (2NH),
–
þ
–
(2C O). EI MS m/z (%): 517.31 (21), 522.54 ([M] , 100), 523.54
–
([M þ 1]þ, 26). Elemental analysis: for C22H20F2N4O7S calcd. % C,
50.57; H, 3.86; N, 10.72. Found: % C, 50.59; H, 3.83; N, 10.42.
(2S,5R,6R)-2-[(2,2-Dimethylpropanoyl)oxy]-
methylpenicillanat-6-aminium-4-methylbenzenesulfonate
(5)
2973 (aliphatic C–H), 1760, 1738 and 1642 (3C O), 1279 (C–O).
–
1H NMR (DMSO-d6): d (ppm) ¼ 1.15 (s, 9H, 3CH3), 1.48 (s, 6H, 2CH3),
3.65–3.88 (m, 8H, 4CH2), 5.31–5.65 (m, 2H, 2CH), 5.90 (brs, 1H, CH).
13C NMR (DMSO-d6): d (ppm) ¼ 27.21 (2CH3), 27.68 (3CH3), 37.98
(C), 47.21 (2CH2), 52.63 (2CH2), 59.71 (C), 61.52 (CH), 71.90 (2CH),
Triethylamine (10 mmol) was added to the suspension of 6-apa
(10 mmol) in 9 mL of anhydrous DMF and the mixture was stirred
at room temperature for 30 min, then, chloromethylpivalate
(20 mmol) was added into it. After stirring at 25°C for 4 h, the
mixture was diluted with 50 mL of EtOAc. The precipitate was
filtered off and the filtrate was washed with H2O and petroleum
ether to remove DMF and unreacted 6-apa. The organic layer was
dried over Na2SO4 and freshly recrystallized p-toluenesulfonic
acid (10 mmol) was added into it. The solid formed [39] was
filtered and washed with cold EtOAc. Yield 43%, m.p.: 150–151°C.
FT-IR (ymax, cmꢀ1): 2972 (aliphatic C–H), 1750, 1763, and 1694
–
–
–
80.11 (CH ), 168.55 (C O), 176.86 (C O), 180.09 (C O). EI MS
–
–
–
2
m/z (%): 437.21 ([MþNa]þ, 32), 431.08 (30), 408.88 (29), 407.14 (100).
Elemental analysis: for C18H30N4O5S calcd. % C, 52.15; H, 7.29; N,
13.52. Found: % C, 52.25; H, 7.23; N, 13.41.
[(2,2-Dimethylpropanoyl)oxy]methyl (2S,5R,6R)-6-{[4-(2-
fluoro-4-nitrophenyl)piperazin-1-yl]aminopenicillanate (8)
The solution of compound 5 (2.2 mmol) in 9 mL of anhydrous
dichloromethane was stirred at 0°C in the presence of triethyl-
amine (2.2 mmol) for 30 min. 1-(2-Fluoro-4-nitrophenyl)piperazine
(2.2 mmol) was added and the mixture was stirred at room
temperature for 10 h. Water was added into it and extracted with
dichloromethane three times. The combined organic layers was
dried on Na2SO4 and evaporated under reduced pressure. The
obtained crude product was recrystallized from dichlorome-
thane. Yield 25%, m.p.: 83°C. FT-IR (ymax, cmꢀ1): 3287 (NH), 2970
(3C O). 1H NMR (DMSO-d6): d (ppm) ¼ 1.12 (brs, 9H, 3CH3), 1.41
–
–
(brs, 3H, CH3), 1.59 (brs, 3H, CH3), 2.26 (brs, 3H, CH3), 4.57 (s, 1H,
CH), 5.13 (s, 1H, CH), 5.52 (s, 1H, CH), 5.74–5.83 (m, 2H, CH2), 7.12
(d, 2H, Ar–H, J ¼ 7.4 Hz), 7.46 (d, 2H, Ar–H, J ¼ 7.4 Hz), 8.85 (brs,
NH3þ, D2O exch.). 13C NMR (DMSO-d6): d (ppm) ¼ 21.47 (2CH3),
26.55 (CH3), 27.21 (CH3), 27.23 (CH3), 34.12 (CH3), 57.84 (CH), 60.17
(CH2), 64.49 (CH), 71.71 (CH), 80.33 (2C), arC: [126.17 (CH), 126.75
–
–
–
(CH), 128.77 (2CH)], 146.21 (C O), 168.31 (C O), 169.61 (C O).
–
–
–
–
–
(aliphatic C–H), 1751 (C O), 1638 (2C O), 1514 and 1330 (NO ),
–
–
2
1234 (C–O). 1H NMR (DMSO-d6): d (ppm) ¼ 1.04 (brs, 9H, 3CH3), 1.46
(brs, 6H, 2CH3), 3.37 (brs, 6H, 3CH2 þ H2O), 3.61–3.87 (m, 4H,
2CH2), 4.80 (brs, 1H, CH), 5.71 (brs, 1H, CH), 5.87 (brs, 1H, CH), 7.20
(d, 1H, Ar–H, J ¼ 8.8 Hz), 7.99–8.06 (m, 2H, Ar–H). 13C NMR (DMSO-
d6): d (ppm) ¼ 26.81 (CH3), 27.21 (3CH3), 28.44 (CH3), 38.91–41.37
(DMSO-d6 þ C-(CH3)3), 46.43 (2CH2), 58.32 (CH2), 59.52 (CH2), 64.48
(C(CH3)2), 69.57 (CH), 70.35 (CH), 73.13 (CH), 78.92 (CH2), arC:
[114.51 and 114.94 (d, CH, J ¼ 21.5 Hz), 119.02 and 119.40 (d, CH,
J ¼ 19.0 Hz), 122.13 and 122.23 (d, CH, J ¼ 5.0 Hz), 126.17 and
128.72 (d, C, J ¼ 127.5 Hz), 138.14 and 138.46 (d, C, J ¼ 16.0 Hz),
[(2,2-Dimethylpropanoyl)oxy]methyl (2S,5R,6R)-6-[(4-
nitrophenyl)amino]-penicillanate (6)
To a solution of compound 5 (2.2 mmol) in 1.7 mL of dimethyl
formamide, triethylamine (2.2 mmol) was added at ꢀ5°C and the
mixture was stirred at the same temperature for 30 min. The
solution of 4-bromonitrobenzene in dichloromethane was added
dropwise and the stirring was continued at room temperature for
24 h. Water was added into it and extracted with dichloro-
methane (3 ꢂ 5 mL). The combined organic layers were dried on
Na2SO4 and evaporated under reduced pressure. The obtained
oily product was recrystallized from ethyl acetate–hexane (1:1) to
afford the desired compound. Yield 30%, m.p.: 114–115°C. FT-IR
(ymax, cmꢀ1): 3373 (NH), 3097 (aromatic C–H), 2976 (aliphatic
–
–
–
147.64 and 152.01 (d, C, J ¼ 218 Hz)], 165.61 (C O), 166.97 (C O),
–
þ
–
176.77 (C O). EI MS m/z (%): 554.27 ([Mþ1] , 63), 428.16 (25),
–
242.27 (27), 196.16 (42), 138.12 (100), 102.12 (50). Elemental
analysis: for C24H32FN5O7S calcd. % C, 52.07; H, 5.83; N, 12.65.
Found: % C, 52.38; H, 6.09; N, 12.22.
C–H), 1749 and 1669 (2C O), 1354 (C–O). 1H NMR (DMSO-d6): d
–
–
(ppm) ¼ 1.12–1.25 (m, 15H, 5CH3), 1.46 (s, 2H, CH2), 5.31 (s, 1H,
CH), 5.60 (d, 1H, CH, J ¼ 5.8 Hz), 5.87 (d, 1H, CH, J ¼ 6.6 Hz), 7.84–
7.90 (m, 2H, Ar–H), 8.10–8.17 (m, 2H, Ar–H). 13C NMR (DMSO-d6): d
(ppm) ¼ 7.87 (2CH3), 27.18 (2CH3), 28.43 (CH3), 46.12 (C), 52.74
(CH2), 59.39 (C), 70.02 (CH), 73.05 (2CH), arC: [125.98 (2CH), 130.04
[(2,2-Dimethylpropanoyl)oxy]methyl (2S,5R,6R)-6-{[(4-
nitrophenyl)sulfonyl]amino}penicillanate (9)
Triethylamine (10 mmol) and 4-nitrobenzene sulfonyl chloride
were added to a solution of compound 5 (10 mmol) in anhydrous
dichloromethane at 0°C and the mixture was stirred at room
temperature for 13 h. Water was added into it, two layers
were separated, and the water layer was extracted with
–
–
(C), 133.49 (2CH), 147.42 (C)], 165.61 (2C O), 168.81 (C O). EI MS
–
–
m/z (%): 531.38 (65), 526.42 (38), 517.36 (37), 501.37 (38), 487.35
([Mꢀ2þK]þ, 74), 473.34 ([MþNa]þ, 37), 457.37 (37), 443.33 (100),
ß 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.archpharm.com

212
S. Demirci et al.
Arch. Pharm. Chem. Life Sci. 2014, 347, 200–220
dichloromethane two times and the combined organic layers
were dried on Na2SO4. After evaporating the solvent under
reduced pressure, a solid formed. This was recrystallized from
ethanol/water (1:1) to afford the desired compound. Yield
50%, m.p.: 62°C. FT-IR (ymax, cmꢀ1): 3108 (NH), 2976 (aliphatic
calcd. % C, 58.64; H, 7.61; N, 12.43. Found: % C, 58.61; H, 7.69;
N, 12.40.
Triethylammonium (2S,5R,6R)-6-({[(4-methoxyphenyl)-
amino]carbonothioyl}amino)penicillanate (10c)
–
–
CH), 1752 (2C O), 1675 (C O), 1530 and 1348 (NO ), 1281 (C–O).
–
–
2
Yield 82%, m.p.: 139–140°C. FT-IR (ymax, cmꢀ1): 3150 (3NH), 3077
1H NMR (DMSO-d6): d (ppm) ¼ 1.12 (brs, 15H, 5CH3), 3.59 (brs, 2H,
CH2), 5.38 (brs, 1H, NH), 5.70 (brs, 3H, CH), 8.07 (brs, 2H, Ar–H),
8.36 (brs, 2H, Ar–H). 13C NMR (DMSO-d6): d (ppm) ¼ 27.21 (CH3),
31.23 (CH3), 35.03 (3CH3), 38.64 (C-(CH3)3), 41.86 (C-(CH3)2), 46.21
(CH2), 59.90 (2CH), arC: [125.71 (2CH), 128.83 (2CH), 140.64 (2C)],
–
–
(aromatic C–H), 2921 (aliphatic C–H), 1754 (C O), 1609 (C O),
–
–
1517 (C S). 1H NMR (DMSO-d6): d (ppm) ¼ 1.357 (brs, 9H, 3CH3),
–
–
1.47–1.55 (m, 6H, 2CH3), 2.507 (s, 6H, 3CH2 þ DMSO-d6), 3.01 (brs,
1H, CH), 3.51 (brs, 2H, 2CH þ H2O), 3.79 (s, 3H, OCH3), 7.01 (d, 2H,
Ar–H, J ¼ 8.8 Hz), 7.19 (d, 2H, Ar–H, J ¼ 7.6 Hz). 13C NMR (DMSO-d6):
d (ppm) ¼ 8.63 (3CH3), 23.13 (2CH3), 22.07 (CH2), 25.62 (CH2), 31.26
(CH2), 55.30 (OCH3), 65.42 (CH), 68.96 (C), 69.22 (CH), 69.73 (CH),
arC: [131.87 (2CH), 129.75 (2CH), 158.59 (C), 159.10 (C)], 162.40
–
–
–
148.26 (C O), 168.27 (C O), 171.06 (C O). EI MS m/z (%): 547.62
–
–
–
(25), 538.61 ([MþNa]þ, 21), 386.56 (68), 359.65 (31), 358.59 (100),
338.38 (34), 322.36 (37), 320.29 (71). Elemental analysis: for
C20H25N3O9S2 calcd. % C, 46.59; H, 4.89; N, 8.15. Found: % C, 46.51;
–
–
–
–
–
–
(C O), 172.32 (C O), 183.66 (C S). EI MS m/z (%): 500.98
H, 4.85; N, 8.42.
([MþH2O]þ, 23), 483.71 ([Mþ1]þ, 40), 420.55 (100), 215.09 (23).
Elemental analysis: for C22H34N4O4S2 calcd. % C, 54.75; H, 7.10; N,
11.61. Found: % C, 54.39; H, 6.98; N, 11.52.
General method for the synthesis of compounds 10a–e
Triethylamine (35 mmol) was added to a solution of 6-apa
(10 mmol) in dimethyl formamide at ꢀ5°C and stirred for 30 min.
The solution of the corresponding alkyliso(thio)cyanate
(10 mmol) in dimethyl formamide was added into it dropwise
and stirred for additional 15 min. Then, temperature was allowed
to reach to room temperature and the stirring was continued
additional 3 h. The unreacted 6-apa was removed by filtration.
Diethyl ether was added into the filtrate. The solution was stirred
rapidly and left in cold overnight. The solid formed was filtered
off and recrystallized from petroleum ether.
Triethylammonium (2S,5R,6R)-6-[(anilinocarbonyl)amino]-
penicillanate (10d)
Yield 92%, m.p.: 170–171°C. FT-IR (ymax, cmꢀ1): 3328 (2NH), 3290
(NH), 3137, 3091 (aromatic C–H), 2981 (aliphatic C–H), 1767
(2C O), 1704 (C O), 1212 (C–O). 1H NMR (DMSO-d6): d (ppm)
–
–
–
–
¼ 1.122 (t, 9H, 3CH3, J ¼ 7.2 Hz), 1.45 (s, 3H, CH3), 1.59 (s, 3H, CH3),
2.92 (t, 6H, 3CH2, J ¼ 7.2 Hz), 4.02 (s, 1H, CH), 5.47 (s, 2H, 2CH), 6.93
(t, 2H, Ar–H, J ¼ 7.6 Hz), 7.24 (t, 2H, Ar–H, J ¼ 8.0 Hz), 7.39 (t, 2H,
Ar–H, J ¼ 7.6 Hz), 9.04 (s, 2H, NH, D2O exch.). 13C NMR (DMSO-d6):
d (ppm) ¼ 8.90 (CH3), 27.13 (CH3), 31.95 (CH3), 38.83 (3CH2), 58.49
(CH), 64.23(C), 67.72 (CH), 72.67 (CH), arC: [121.56 (2CH), 126.99
Triethylammonium (2S,5R,6R)-6-{[(benzylamino)-
–
–
carbonothioyl]amino}penicillanate (10a)
(CH), 128.59 (2CH), 139.30 (C)], 153.40 (C O), 170.56 (C O), 174.41
–
–
–
Yield 55%, m.p.: 132°C. FT-IR (ymax, cmꢀ1): 3125 (2NH), 3104 (NH),
(C O). EI MS m/z (%): 438.49 ([Mþ2] , 32), 436.55 ([M] , 11), 404.59
þ
þ
–
–
3068 (aromatic C–H), 2932 (aliphatic C–H), 1727 (2C O), 1108
(10), 369.35 (43), 350.35 (100). Elemental analysis: for C21H32N4O4S
calcd. % C, 57.77; H, 7.39; N, 12.83. Found: % C, 57.47; H, 7.21; N,
12.80.
–
1
–
(C S), 1233 (C–O). H NMR (DMSO-d ): d (ppm) ¼ 1.11 (t, 9H, 3CH
–
6
3
J ¼ 7.0 Hz), 1.46 (s, 3H, CH3), 1.53 (s, 3H, CH3), 2.92 (q, 6H, 3CH2,
J ¼ 8.2 Hz), 4.67 (s, 2H, CH2), 5.45 (s, 1H, CH), 5.83 (brs, 2H, CH),
7.29 (brs, 5H, Ar–H), 8.19 (brs, 1H, NH, D2O exch.), 8.47 (s, 1H, NH,
D2O exch.). 13C NMR (DMSO-d6): d (ppm) ¼ 9.31 (3CH3), 27.93 (CH3),
31.57 (CH3), 42.60 (CH2), 45.78 (3CH2), 59.44 (CH), 64.93 (C), 69.82
(CH), 73.59 (CH), arC: [126.74 (CH), 126.92 (CH), 127.01 (CH), 128.88
Triethylammonium (2S,5R,6R)-6-{[(tert-butylamino)-
carbonothioyl]amino}penicillanate (10e)
Yield 91%, m.p.: 129–130°C. FT-IR (ymax, cmꢀ1): 3302 (2NH), 3173
–
–
–
(NH), 2927 (aliphatic C–H), 1792 (C O), 1766 (C O), 1506 (C S).
–
–
–
–
–
–
–
(CH), 128.95 (CH), 141.98 (C)], 157.52 (C S), 170.13 (C O), 172.59
1H NMR (DMSO-d6): d (ppm) ¼ 1.165 (t, 9H, 3CH3, J ¼ 7.2 Hz), 3.02
(t, 6H, 3CH2, J ¼ 7.2 Hz), 4.53 (d, 1H, CH, J ¼ 4.2 Hz), 5.39 (d, 2H,
2CH, J ¼ 4.2 Hz). 13C NMR (DMSO-d6): d (ppm) ¼ 8.46 (3CH3), 26.49
(CH3), 26.86 (CH3), 27.63 (CH3), 28.65 (CH3), 33.45 (CH3), 45.07
–
þ
þ
–
–
(C O). EI MS m/z (%): 468.49 ([Mþ2] , 30), 467.55 ([Mþ1] , 100),
404.33 (23), 388.32 (25), 366.35 (35), 350.35 (32). Elemental
analysis: for C22H34N4O3S2 calcd. % C, 56.62; H, 7.34; N, 12.01.
Found: % C, 56.89; H, 7.73; N, 12.42.
(3CH ), 57.49 (C), 57.87 (CH), 62.29 (2CH), 63.08 (C), 170.61 (C O),
–
2
þ
–
–
–
–
172.42 (C O), 178.45 (C S). EI MS m/z (%): 431.70 ([Mꢀ1] , 28),
420.55 (10), 402.33 (54), 388.32 (25), 266.75 (35). Elemental
analysis: for C19H36N4O3S2 calcd. % C, 52.75; H, 8.39; N, 12.95.
Found: % C, 52.68; H, 8.51; N, 12.78.
Triethylammonium (2S,5R,6R)-6-{[(benzylamino)carbonyl]-
amino}penicillanate (10b)
Yield 40%, m.p.: 188°C. FT-IR (ymax, cmꢀ1): 3325 (3NH), 2980
(aliphatic C–H), 1764 (2C O), 1679 (C O), 1276 (C–O). 1H NMR
–
–
–
–
General method for the synthesis of compounds 11a,b
4-Chlorophenacyl bromide (10 mmol) and dried sodium acetate
(200 mmol) were added to the solution of the corresponding
compound 10 in absolute ethanol, and the reaction mixture was
refluxed for 9 h. Then, the mixture was cooled to room
temperature, poured into ice-cold water while stirring, and left
overnight in cold. The formed solid was filtered, washed with
water three times, and recrystallized from dimethyl formamide/
diethylether (1:2) (for 11a) or ethyl acetate (for 11b) to afford the
desired product.
(DMSO-d6): d (ppm) ¼ 1.12 (t, 9H, 3CH3, J ¼ 7.4 Hz), 1.46 (s, 3H, CH3),
1.54 (s, 3H, CH3), 2.93 (q, 6H, 3CH2, J ¼ 7.2 Hz), 3.97 (s, 1H, CH), 4.21
(s, 2H, CH), 5.39 (s, 2H, CH2), 6.65 (d, 1H, NH, D2O exch.), 6.86
(s, 1H, NH, D2O exch.), 7.21–7.39 (m, 5H, Ar–H). 13C NMR
(DMSO-d6): d (ppm) ¼ 9.32 (3CH3), 27.92 (CH3), 32.37 (CH3), 43.61
(CH2), 45.58 (3CH2), 59.45 (CH), 64.83 (C), 68.62 (CH), 73.54 (CH),
arC:[127.44 (CH), 127.62 (CH), 127.77 (CH), 128.83 (CH), 129.00
–
–
–
(CH), 140.91 (C)], 157.12 (C O), 171.17 (C O), 176.29 (C O). EI MS
–
–
–
m/z (%): 452.39 ([Mþ2]þ, 13), 449.23 (26), 447.22 (100), 432.27 (12),
405.26 (27), 354.23 (10). Elemental analysis: for C22H34N4O4S
ß 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.archpharm.com

Arch. Pharm. Chem. Life Sci. 2014, 347, 200–220
Heterofunctionalized Penicillanic Acid Derivatives
213
Triethylammonium (2S,5R,6R)-6-{2-[3-benzyl-5-(4-
chlorobenzyl)-1,3-thiazol-2(3H)-ylidene]hydrazino}-
Triethylammonium (2S,5R,6R)-6-[2-(3-benzyl)-5-oxo-1,3-
oxazolidin-2-ylidene)hydrazino]penicillanate (12b)
Yield 91%, m.p.: 225°C. FT-IR (ymax, cmꢀ1): 3405 (2NH), 3170
penicillanate (11a)
Yield 78%, m.p.: 95–96°C. FT-IR (ymax, cmꢀ1): 3412 (2NH), 3170
–
–
(aromatic C–H), 2979 (aliphatic C–H), 1719 (C O), 1634 (2C O),
–
–
1
–
1548 (C N), 1279 (C–O). H NMR (DMSO-d ): d (ppm) ¼ 2.17–2.37
–
–
–
–
–
6
(Ar–H), 2974 (aliphatic C–H), 1728 (C O), 1659 (C O), 1598 (C N),
–
–
1342 (C–S). 1H NMR (DMSO-d6): d (ppm) ¼ 0.88–0.95 (m, 7H,
2CH2 þ CH3), 1.13 (brs, 3H, CH3), 1.27–1.35 (m, 5H, CH2 þ CH3),
1.76 (brs, 6H, 2CH3), 2.40–2.56 (m, 4H, 4CH), 4.88 (s, 2H, CH2),
7.22–7.34 (m, 5H, Ar–H). 13C NMR (DMSO-d6): d (ppm) ¼ 15.08
(2CH3), 44.71 (3CH3), 53.03 (4CH2), 55.15 (C), 67.71 (CH), 72.04
(2CH), 116.21 (C), arC: [126.99 (3CH), 128.59 (CH), 130.19 (2CH),
130.57 (2CH), 131.01(CH), 131.97 (C), 135.67 (C), 137.03 (CH),
(m, 21H, 3CH2 þ 5CH3), 3.90–4.01 (m, 7H, 2CH2 þ 3CH), 7.65 (brs,
5H, Ar–H). 13C NMR (DMSO-d6): d (ppm) ¼ 13.15 (CH3), 13.29 (CH3),
44.32 (3CH3), 51.69 (2CH2), 54.71 (2CH2), 55.67 (CH2), 56.35 (CH),
65.41 (CH), 72.61 (CH), 117.15 (C), arC: [125.79 (3CH), 129.54 (CH),
–
–
–
–
130.89 (CH), 133.97 (C)], 161.21 (C N), 174.64 (C O), 176.45
þ
þ
–
–
(2C O)]. EI MS m/z (%): 505.63 ([M] , 19), 487.58 ([MꢀH O] , 54),
2
389.65 (89), 234.27 (100). Elemental analysis: for C24H35N5O5S
calcd. % C, 57.01; H, 6.98; N, 13.85. Found: % C, 57.25; H, 6.99; N,
13.84.
–
–
–
138.30 (C)], 159.40 (N C), 176.56 (C O), 178.41 (C O). EI MS
–
–
–
m/z (%): 634.70 ([MþH2O]þ, 25), 616.55 ([M]þ, 18), 489.33 (51),
258.32 (100), 241.75 (35). Elemental analysis: for C30H38ClN5O3S2
calcd. % C, 58.47; H, 6.22; N, 11.36. Found: % C, 58.40; H, 6.35; N,
11.36.
(2S,5R,6R)-6-[Bis(4-nitrobenzoyl)amino]penicillanic
acid (13)
The solution of K2CO3 (10 mmol) in 12 mL of acetone and 14 mL of
water was added to the suspension of 6-apa (10 mmol) in 10 mL of
acetone at ꢀ5°C and the mixture was stirred for 10 min. The
solution of 4-nitrobenzoyl chloride (10 mmol) in 10 mL of acetone
was added into it dropwise in 3 h. Then, temperature was allowed
to reach to room temperature and the stirring was continued for
an additional 1 h. Ethyl acetate was added and the reaction
mixture was acidified to pH 3 with 37% HCl. The mixture was
extracted with petroleum ether; the organic layer was separated
and dried over Na2SO4. After evaporating the solvent under
reduced pressure, a solid was obtained. The crude product was
recrystallized from chloroform. Yield 33%, m.p.: 165–166°C. FT-IR
(ymax, cmꢀ1): 3163 (aromatic C–H), 2952 (aliphatic C–H), 1727
Triethylammonium (2S,5R,6R)-6-{2-[3-benzyl-5-(4-
chlorobenzyl)-1,3-oxazol-2(3H)-ylidene]hydrazino}-
penicillanate (11b)
Yield 85%, m.p.: 110–111°C. FT-IR (ymax, cmꢀ1): 3381 (2NH),
–
3170 (aromatic C–H), 2979 (aliphatic C–H), 1763 (C O), 1634
–
(C O), 1279 (C–O). 1H NMR (DMSO-d6): d (ppm) ¼ 1.12–1.68
–
–
(m, 21H, 5CH3 þ 3CH2), 3.82–4.18 (m, 6H, CH2 þ 4CH), 7.24
(brs, 9H, Ar–H). 13C NMR (DMSO-d6): d (ppm) ¼ 14.79 (2CH3), 25.11
(3CH3), 43.49 (CH2), 51.18 (C), 63.85 (CH), 73.52 (2CH), 118.14 (C),
127.03 (CH), 127.20 (CH), 127.61 (CH), 127.93 (CH), 128.75 (CH),
128.85 (CH), 128.96 (CH), 129.08 (2CH), 130.92 (C), 134.87 (C),
–
þ
–
141.86 (CH), 156.47 (C), 158.82 (N C), 173.36 (C O), 175.75
–
–
–
–
–
–
(C O), 1655 (C O), 1623 (C O), 1475 and 1335 (NO ), 1233(C–O).
–
–
–
2
–
(C O). EI MS m/z (%): 600.15 ([M] , 78), 586.33 (31), 326.52
1H NMR (DMSO-d6): d (ppm) ¼ 1.43 (brs, 6H, 2CH3), 4.26 (s, 1H, CH),
4.35 (s, 1H, CH), 5.47 (s, 1H, CH), 8.13 (d, 4H, H-2 and H-6,
J ¼ 8.6 Hz), 8.29 (d, 4H, H-3 and H-5, J ¼ 8.6 Hz). 13C NMR (DMSO-d6):
d (ppm) ¼ 27.04 (CH3), 33.90 (CH3), 57.69 (CH), 58.38 (C), 69.37 (CH),
71.41 (CH), arC: [124.40 (2CH), 131.37 (2CH), 137.03 (2C)], 150.68
(41), 245.32 (100). Elemental analysis: for
C30H38ClN5O4S
calcd. % C, 60.04; H, 6.38; N, 11.67. Found: % C, 59.96; H, 6.36;
N, 11.59.
–
–
–
–
(C O), 161.96 (C O), 166.50 (2C), 171.00 (C O), 172.70 (C O).
General method for the synthesis of compounds 12a,b
Ethyl bromoacetate was added to the solution of the correspond-
ing compound 10 in absolute ethanol (10 mmol) and the
mixture was refluxed in the presence of dried sodium acetate
(16.4 g, 200 mmol) for 20 h. Then, the mixture was cooled
to room temperature, poured into ice-cold water while stirring
and left overnight in cold. The solid formed was filtered,
washed with water three times, and recrystallized from
acetone.
–
–
–
–
EI MS m/z (%): 543.36 (29), 537.61 ([MþNa]þ, 10), 415.47 (30),
283.19 (100), 140.08 (69). Elemental analysis: for C22H18N4O9S
calcd. % C, 51.36; H, 3.53; N, 10.80. Found: % C, 51.01; H, 3.28; N,
10.77.
(2S,5R,6R)-6-[Bis(4-aminobenzoyl)amino]penicillanic
acid (14)
The solution of K2CO3 (10 mmol) in 12 mL of acetone and 14 mL of
water was added to the suspension of 6-apa (10 mmol) in 10 mL of
acetone at ꢀ5°C and the mixture was stirred for 10 min. The
solution of 4-aminobenzoyl chloride (10 mmol) in 10 mL of
acetone was added into it dropwise in 3 h. Then, temperature was
allowed to reach to room temperature and stirring was continued
for an additional 1 h. Ethyl acetate was added and acidified to pH
3 with 37% HCl. The mixture was extracted with petroleum ether;
the organic layer was separated and dried over Na2SO4. After
evaporating the solvent under reduced pressure, a solid was
obtained. The crude product was recrystallized from chloroform.
Yield 55%, m.p.: 213–214°C. FT-IR (ymax, cmꢀ1): 3459 (OH), 3381
and 3361 (NH2), 3230 (aromatic C–H), 2967 (aliphatic C–H), 1659,
Triethylammonium (2S,5R,6R)-6-[(2-(3-benzyl)-5-oxo-1,3-
thiazolidin-2-ylidene)hydrazino]penicillanate (12a)
Yield 95%, m.p.: 100–101°C. FT-IR (ymax, cmꢀ1): 3379 (NH), 3342
(NH), 3088, 3065, 3032 (aromatic C–H), 2970 (aliphatic C–H), 1713
(3C O), 1535 (C N), 1327 (C–S). 1H NMR (DMSO-d6): d (ppm)
–
–
–
–
¼ 0.93–1.28 (m, 15H, 5CH3), 1.56–1.86 (m, 6H, 3CH2), 2.56 (s, 4H,
2CH2), 4.34 (brs, 3H, 3CH), 7.22 (brs, 5H, Ar–H). 13C NMR (DMSO-
d6): d (ppm) ¼ 12.21 (2CH3), 30.63 (CH2), 46.91 (CH2), 47.21 (3CH3),
52.21 (CH), 65.71 (CH), 72.05 (CH), arC: [127.21 (2CH), 128.61 (CH),
–
þ
–
129.00 (2CH), 133.21 (C), 150.33 (C)], 167.03 (C O), 170.51 (C O),
–
–
–
176.21 (C O). EI MS m/z (%): 523.63 ([Mþ2] , 41), 407.56 (74),
–
1635, and 1622 (3C O). 1H NMR (DMSO-d6): d (ppm) ¼ 1.45 (brs,
–
395.26 (32), 383.56 (82), 235.87 (100). Elemental analysis: for
C24H35N5O4S2 calcd. % C, 52.25; H, 6.76; N, 13.42. Found: % C,
52.24; H, 6.71; N, 13.49.
–
6H, 2CH3), 4.28 (s, 1H, CH), 4,37 (s, 1H, CH), 5.55 (s, 1H, CH), 6.53
(d, 2H, NH2, J ¼ 8.2 Hz, exch. D2O), 7.60 (d, 2H, NH2, J ¼ 7.0 Hz,
ß 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.archpharm.com

214
S. Demirci et al.
Arch. Pharm. Chem. Life Sci. 2014, 347, 200–220
exch. D2O), 8.10 (d, 4H, Ar–H, J ¼ 7.0 Hz), 8.31 (d, 4H, Ar–H,
J ¼ 6.0 Hz). 13C NMR (DMSO-d6): d (ppm) ¼ 26.30 (CH3), 34.50 (CH3),
57.70 (CH), 58.38 (C), 71.41 (CH), 113.21 (CH), arC: [124.43 (4CH),
131.40 (4CH), 137.11 (C), 150.70 (C), 153.84 (C), 161.96 (C)], 166.52
(2S,5R,6R)-6-[Bis(4-{[4-oxo-3-phenyl-1,3-thiazolidin-2-
ylidene]amino}benzoyl)amino]penicillanic acid (17)
A mixture of compound 15 (10 mmol), dried sodium acetate
(50 mmol), and ethylbromoacetate (20 mmol) in absolute ethanol
was irradiated with CEM brand, microwave device at 200°C,
200 W, for 50 min. Then, the mixture was cooled to room
temperature, poured into ice-cold water while stirring, and left
overnight in cold. The formed solid was filtered and recrystallized
from ethyl acetate/hexane (1:2). Yield 53%, m.p.: 181–182°C. FT-IR
(ymax, cmꢀ1): 3459 (OH), 3042 (aromatic C–H), 2986 (aliphatic
–
–
–
–
(C O), 168.19 (C O), 171.01 (C O), 172.71 (C O). EI MS m/z (%):
–
–
–
–
575.39 (15), 519.05 ([Mþ2þKþNa]þ, 34), 443.32 (19), 399.29 (11),
305.14 (10), 242.27 (34), 212.11 (100), 108.07 (27). Elemental
analysis: for C22H22N4O5S calcd. % C, 58.14; H, 4.88; N, 12.33.
Found: % C, 58.14; H, 4.81; N, 12.36.
C–H), 1727, 1675, 1637 (6C O), 1420 (C N). 1H NMR (DMSO-d6):
(2S,5R,6R)-6-(Bis{4-[(anilinocarbonothioyl)amino]-
–
–
–
–
d (ppm) ¼ 2.50 (s, 6H, 2CH3 þ DMSO-d6), 3.51 (s, 2H, CH2 þ H2O),
4.78 (s, 3H, CH þ CH2), 6.58 (s, 2H, 2CH), 7.61–7.74 (m, 9H, Ar–H),
7.86–7.97 (m, 9H, Ar–H), 10.11 (s, 1H, OH, D2O exch.). 13C NMR
(DMSO-d6): d (ppm) ¼ 14.79 (CH3), 15.09 (CH3), 44.82 (2CH2), 61.13
(C), 61.29 (CH), 62.44 (CH), 63.52 (CH), arC: [111.83 (2CH), 113.35
(CH), 117.93 (CH), 118.12 (2CH), 118.83 (CH), 119.03 (CH), 122.84
(2C), 131.12 (2CH), 131.31 (2CH), 131.48 (2CH), 131.69 (2CH),
benzoyl}amino)penicillanic acid (15)
The solution of compound 14 (10 mmol) in absolute ethanol was
refluxed in the presence of phenylisothiocyanate (20 mmol) for
22 h. After evaporating the solvent under reduced pressure, a
solid was obtained. This was recrystallized from ethyl acetate to
afford the desired product. Yield 93%, m.p.: 149–150°C. FT-IR
(ymax, cmꢀ1): 3460, 3362, and 3207 (4NH), 3112 (aromatic C–H),
–
131.87 (CH), 132.09 (CH), 144.99 (2C), 152.68 (2C)], 154.01 (2C N),
–
–
–
–
2985 (aliphatic C–H), 1686 and 1623 (C O), 1311 and 1289 (C S).
–
1H NMR (DMSO-d6): d (ppm) ¼ 1.22–1.32 (m, 6H, 2CH3), 4.48 (d, 2H,
CH, J ¼ 6.6 Hz), 6.55 (d, 1H, CH, J ¼ 8.2 Hz), 7.12 (d, 2H, Ar–H,
J ¼ 3.2 Hz), 7.31 (d, 7H, Ar–H, J ¼ 6.6 Hz), 7.43 (d, 3H, Ar–H,
J ¼ 7.4 Hz), 7.59–7.65 (m, 4H, Ar–H), 7.88 (d, 2H, Ar–H, J ¼ 8.2 Hz),
9.70 (s, 1H, NH, D2O exch.), 10.95 (s, 1H, NH, exch. D2O), 11.24 (s,
1H, NH, D2O exch.). 13C NMR (DMSO-d6): d (ppm) ¼ 13.91(2CH3),
112.50 (3CH), 116.80 (C), arC: [121.77 (CH), 121.97 (CH), 123.60
(2CH), 123.64 (2CH), 124.37 (CH), 124.65 (CH), 125.62 (C), 128.39
(CH), 128.48 (CH), 128.75 (CH), 128.96 (CH), 129.84 (2CH), 130.08
(CH), 131.17 (3CH), 139.18 (C), 139.41 (2C), 143.77 (C), 153.11 (C)],
–
–
–
165.61 (2C O), 168.10 (2C O), 168.56 (2C O). EI MS m/z (%): 760.99
–
–
–
([M]ꢀCOOH]þ, 11), 686.83 ([Mþ2]–C6H5]þ, 15), 410.96 (25), 331.86
(81), 317.86 (89), 260.81 (100). Elemental analysis: for
C54H42Cl2N6O5S3 calcd. % C, 63.46; H, 4.14; N, 8.22. Found:
% C, 63.44; H, 4.21; N, 8.12.
General method for the synthesis of compounds 18a,b
6-Apa (10 mmol) was added to the solution of K2CO3 (10 mmol) in
water (8 mL) and acetone (6 mL) at ꢀ5°C and the mixture was
stirred for 10 min. Then, the solution of the corresponding
compound 19 in acetone (10 mL) was added in a period for 3 h.
Temperature was allowed to reach to room temperature and the
mixture was stirred for 13 h. 12 mL of water was added and stirred
for an additional 1 h. The reaction content was acidified to pH 3
with diluted HCl and extracted with ethyl acetate (3 ꢂ 10 mL).
Organic phase was dried on Na2SO4 and evaporated under
reduced pressure. The obtained crude product was recrystallized
from petroleum ether to afford the desired compound.
–
–
–
–
–
166.74 (C O), 166.91 (C O), 167.45 (C O), 179.32 (C O), 179.56
–
–
–
þ
–
–
(C S), 187.49 (C S). EI MS m/z (%): 531.38 ([(Mþ1)–PhN CH Ph] ,
–
–
2
2
15), 461.31 (33), 399.29 (15), 242.26 (78), 212.10 (100). Elemental
analysis: for C37H33N5O5S3 calcd. % C, 61.39; H, 4.59; N, 9.67.
Found: % C, 61.03; H, 4.51; N, 9.32.
(2S,5R,6R)-6-[Bis(4-{[5-(4-chlorophenyl)-3-phenyl-1,3-
thiazol-2(3H)-ylidene]amino}benzoyl)amino]penicillanic
acid (16)
A mixture of compound 15 (10 mmol) and 4-chlorophenacyl-
bromide (20 mmol) in absolute ethanol was refluxed in the
presence of dried sodium acetate (50 mmol) for 8 h. The reaction
content was then cooled to room temperature and the salt was
separated by filtration. After the solvent was evaporated under
reduced pressure, the solid formed was recrystallized from ethyl
acetate. Yield 45%, m.p.: 119–120°C. FT-IR (ymax, cmꢀ1): 3065
(aromatic C–H), 2951 (aliphatic C–H), 1727, 1673, 1632, and 1606
(2S,5R,6R)-6-[Bis(4-{[(benzylamino)carbonothioyl]-
amino}benzoyl)amino]penicillanic acid (18a)
Yield 43%, m.p.: 184–185°C. FT-IR (ymax, cmꢀ1): 3317, 3250, 3180
–
(4NH), 3064 (aromatic C–H), 2921 (aliphatic C–H), 1668 (4C O),
–
1
–
1297, 1263 (2C S). H NMR (DMSO-d ): d (ppm) ¼ 1.47 (s, 3H, CH ),
–
6
3
1.94 (s, 3H, CH3), 4.39 (s, 1H, CH), 4.73 (d, 4H, 2CH2 þ 2CH,
J ¼ 8.0 Hz), 7.20–7.36 (m, 18H, Ar–H), 8.62 (brs, 2H, NH exch. D2O),
9.49 (brs, 2H, NH exch. D2O). 13C NMR (DMSO-d6): d (ppm) ¼ 26.35
(CH3), 33.22 (CH3), 46.90 (CH2), 47.46 (CH2), 51.42 (C), 57.08 (CH),
68.72 (CH), 70.73 (CH), arC: [126.52 (3CH), 126.83 (CH), 127.22
(5CH), 127.45 (CH), 128.10 (5CH), 128.39 (3CH), 139.07 (5C), 139.36
(C O), 1510 and 1467 (C N). 1H NMR (DMSO-d6): d (ppm) ¼ 1.06
–
–
–
–
(s, 3H, CH3), 1.32 (s, 3H, CH3), 1.84 (s, 1H, CH), 2.09 (s, 1H, CH), 2.50
(s, 1H, CH þ DMSO-d6), 6.52 (s, 2H, Ar–H), 6.92 (s, 3H, Ar–H,), 7.02
(brs, 2H, Ar–H), 7.18 (brs, 4H, Ar–H), 7.30–7.40 (m, 15H, Ar–H), 9.63
(s, 2H, Ar–H). 13C NMR (DMSO-d6): d (ppm) ¼ 22.53 (2CH3), 55.98
(C), 98.44 (CH), 98.58 (2CH), arC: [111.41 (2CH), 117.59 (2CH),
119.75 (2CH), 120.90 (2CH), 122.96 (2CH), 127.70 (2CH), 128.27
(2CH), 128.87 (2CH), 128.99 (2CH), 129.50 (2CH), 129.87 (2CH),
129.93 (2C), 130.00 (2CH), 130.62 (2CH), 130.98 (2CH), 133.04 (2C),
–
–
–
–
–
–
–
(C), 153.11 (C)], 159.62 (C O), 161.23 (C O), 170.28 (C O), 170.62
(C O), 171.99 (C O), 182.62 (C O). EI MS m/z (%): 752.95 ([M] , 21),
þ
–
–
–
–
–
–
427.06 (13), 395.12 (41), 321.11 (73), 297.05 (100), 148.07 (66),
102.09 (89). Elemental analysis: for C39H37N5O5S3 calcd. % C,
62.29; H, 4.96; N, 9.31. Found: % C, 62.57; H, 5.13; N, 9.69.
137.61 (2C), 137.86 (2C), 151.27 (2C), 152.44 (2C)], 159.00 (2C N),
–
þ
(2S,5R,6R)-6-[Bis(4-{[(benzylamino)carbonyl]-
–
–
–
–
159.17 (2C O), 169.42 (C O). EI MS m/z (%): 883.45 ([M]ꢀPhCl] ,
13), 728.32 ([M]ꢀPhClꢀCOOH]þ, 11), 707.38 (25), 663.36 (41),
567.04 (34), 501.08 (100). Elemental analysis: for C52H38Cl2N6O5S3
calcd. % C, 62.83; H, 3.85; N, 8.45. Found: % C, 62.74; H, 3.81; N,
8.49.
amino}benzoyl)amino]penicillanic acid (18b)
Yield 22%, m.p.: 218–219. FT-IR (ymax, cmꢀ1): 3287 (4NH), 3064
–
–
–
(aromatic C–H), 2931 (aliphatic C–H), 1661 (3C O), 1637 (3C O).
–
1H NMR (DMSO-d6): d (ppm) ¼ 2.08 (s, 3H, CH3), 2.50 (s, 3H, CH3),
ß 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.archpharm.com

Arch. Pharm. Chem. Life Sci. 2014, 347, 200–220
Heterofunctionalized Penicillanic Acid Derivatives
215
4.25 (s,1H, CH), 4.15 (d, 4H, 2CH2, J ¼ 5.9 Hz), 6.85 (s, 1H, CH), 6.98
(s, 1H, CH), 7.21–7.36 (m, 10H, Ar–H), 7.51 (t, 4H, Ar–H, J ¼ 8.6 Hz),
7.83 (t, 4H, Ar–H, J ¼ 8.6 Hz), 8.94 (s, H, NH exch. D2O), 9.03 (s, 2H,
2NH, exch. D2O), 10.00 (s, 1H, NH, exch. D2O), 12.57 (brs, H, OH,
exch. D2O). 13C NMR (DMSO-d6): d (ppm) ¼ 29.00 (2CH3), 42.70
(2CH2), 50.03 (C), 52.08 (CH), 65.72 (CH), 68.73 (CH), arC: [116.63
(3CH), 122.88 (C), 124.90 (C), 126.41 (2CH), 126.73 (4CH), 126.89
(2CH), 127.11 (3CH), 128.02 (2CH), 130.44 (2CH), 140.05 (C), 140.10
was refluxed in the presence of dried sodium acetate (200 mmol)
for 14 h. Then, the mixture was cooled to room temperature,
poured into ice-cold water while stirring, and left overnight in
cold. The solid formed was filtered, washed with water three
times, and recrystallized from ethyl acetate.
4-{[3-Benzyl-4-oxo-1,3-thiazolidin-2-ylidene]amino}-
benzoyl chloride (20a)
–
–
–
(2C), 144.72 (C)], 158.63 (2C O), 158.74 (2C O), 166.02 (C S),
–
–
–
Yield 65%, m.p.: 189–200°C. FT-IR (ymax, cmꢀ1): 3065 (aromatic
þ
–
–
167.09 (C S). EI MS m/z (%): 737.39 ([Mꢀ1þH O] , 13), 721.39
2
–
–
–
C–H), 2964 (aliphatic C–H), 1712 (C O), 1674 (C O), 1510 (C N).
([Mþ1]þ, 22), 707.38 (25), 663.36 (32), 619.34 (39), 567.04 (100),
531.29 (48). Elemental analysis: for C38H36N6O7S calcd. % C, 63.32;
H, 5.03; N, 11.66. Found: % C, 63.30; H, 5.01; N, 11.59.
–
–
–
1H NMR (DMSO-d6): d (ppm) ¼ 4.10 (s, 2H, CH2), 4.91 (s, 2H, CH2),
6.76 (d, 2H, Ar–H, J ¼ 7.8 Hz), 7.34 (s, 5H, Ar–H), 7.81 (d, 2H, Ar–H,
J ¼ 8.2 Hz. 13C NMR (DMSO-d6): d (ppm) ¼ 32.24 (CH2), 33.03 (CH2),
127.05 (CH), 127.43 (CH), 127.61 (CH), 127.89 (CH), 128.14 (CH),
128.26 (CH), 128.30 (CH), 128.32 (2CH), 135.02 (C), 136.44 (C),
General method for the synthesis of compounds 19a,b
–
–
–
–
–
Method 1: The solution of 4-aminobenzoyl chloride (10 mmol)
in ethanol was refluxed in the presence of benzyl iso(thio)cyanate
(10 mmol) for 8 h. The solid obtained upon cooling the reaction
mixture in cold was filtered off and recrystallized from ethanol to
give the pure compound.
139.05 (C), 171.74 (C N), 171.98 (C O), 178.49 (C O). EI MS m/z (%):
–
347.13 (19), 344.52 ([M]þ, 19), 311.19 (13), 251.63 (100). Elemental
analysis: for C17H13ClN2O2S calcd. % C, 59.21; H, 3.80; N, 8.12.
Found: % C, 59.24; H, 3.80; N, 8.10.
4-{[3-Benzyl-4-oxo-1,3-oxazolidin-2-ylidene]amino}-
Method 2: The mixture of 4-aminobenzoyl chloride (10 mmol)
and benzyl iso(thio)cyanate (10 mmol) was irradiated with CEM
brand, monopod microwave device at 125°C, 150 W, for 10 min.
The obtained solid was recrystallized from ethanol.
benzoyl chloride (20b)
Yield 67%, m.p.: 240–241°C. FT-IR (ymax, cmꢀ1): 3086 (aromatic
–
–
–
–
C–H), 2929 (aliphatic C–H), 1756 (C O), 1656 (C O), 1547 (C N).
–
–
1H NMR (DMSO-d6): d (ppm) ¼ 4.22 (d, 4H, 2CH2, J ¼ 6.1 Hz), 7.20
(brs, 2H, Ar–H), 7.28 (s, 5H, Ar–H), 7.78 (s, 2H, Ar–H). 13C NMR
(DMSO-d6): d (ppm) ¼ 40.09 (2CH2), arC: [126.41 (3CH), 126.94
4-{[(Benzylamino)carbonothioyl]amino}benzoyl
–
–
–
(3CH), 128.02 (3CH), 140.57 (C N), 158.74 (2C O). EI MS m/z (%):
–
chloride (19a)
328.71 ([M]þ, 15), 321.19 (19), 259.47 (55), 209.21 (100). Elemental
analysis: for C17H13ClN2O3 calcd. % C, 62.11; H, 3.99; N, 8.52.
Found: % C, 62.01; H, 3.86; N, 8.51.
Yield 70% (for method 1), 82% (for method 2), m.p.: 206–207°C.
FT-IR (ymax, cmꢀ1): 3247 and 3135 (NH), 3080 (aromatic C–H), 2977
(aliphatic C–H), 1668 (C O), 1286 (C S). 1H NMR (DMSO-d6):
–
–
–
–
d (ppm) ¼ 4.72 (s, 2H, CH2), 7.32 (brs, 5H, Ar–H), 7.64 (d, 2H, Ar–H,
J ¼ 8.6 Hz), 7.86 (d, 2H, Ar–H, J ¼ 8.6 Hz), 8.44 (s, 1H, NH, exch.
D2O), 9.93(s, 1H, NH exch. D2O). 13C NMR (DMSO-d6):
d (ppm) ¼ 47.65 (CH2), arC: [121.78 (CH), 127.72 (2CH), 128.18
(2S,5R,6R)-6-[Bis(4-{[3-benzyl-4-oxo-1,3-thiazolidin-2-
ylidene]amino}benzoyl)amino]penicillanic acid (21)
The solution of K2CO3 (10 mmol) in 3 mL of DMF and 14 mL of
water was added to the suspension of 6-apa (10 mmol) in 3 mL of
DMF at ꢀ5°C and the mixture was stirred for 10 min. The solution
of 20a (10 mmol) in 3 mL of DMF was added into it dropwise in 3 h
and the mixture was stirred for 12 h. Then, temperature was
allowed to reach to room temperature and stirring was continued
for an additional 1 h. Ethyl acetate was added into it and acidified
to pH 3 with 37% HCl. The mixture was extracted with
ethylacetate; the organic layer was separated and dried over
Na2SO4. The solvent was removed under reduce pressure and a
solid was obtained. The crude product was recrystallized from
ethyl acetate/n-hexane (1:2). Yield 53%, m.p.: 181–182°C. FT-IR
(ymax, cmꢀ1): 3459 (OH), 3068 (aromatic C–H), 2986 (aliphatic
(2CH), 129.05 (2CH), 130,72 (2CH), 139.21 (C), 144.33 (C), 144.44
þ
–
–
(C)], 167.57 (C O), 181.03 (C S). EI MS m/z (%): 305.15 ([Mþ1] , 15),
–
–
301.14 (20), 293.09 (15), 289.16 ([Mþ2ꢀH2O]þ, 20), 271.10 (100).
Elemental analysis: for C15H13ClN2OS calcd. % C, 59.11; H, 4.30; N,
9.19. Found: % C, 59.50, H, 4.54; N, 9.44.
4-{[(Benzylamino)carbonyl]amino}benzoyl chloride (19b)
Yield 44% (method 1), 47% (method 2), m.p.: 235–236°C. FT-IR
(ymax, cmꢀ1): 3285 and 3221 (NH), 3088 (aromatic C–H), 2929
(aliphatic C–H), 1786 and 1655 (C O). 1H NMR (DMSO-d6):
–
–
d (ppm) ¼ 4.29 (brs, 2H, CH2), 7.29 (brs, 5H, Ar–H), 7.49 (d, 2H,
Ar–H, J ¼ 8.2 Hz), 7.80 (d, 2H, Ar–H, J ¼ 8.4 Hz), 7.87 (s, 1H, NH,
exch. D2O), 8.98 (s, 1H, NH, exch. D2O). 13C NMR (DMSO-d6):
d (ppm) ¼ 43.25 (CH2), arC: [117.43 (2CH), 123.53 (C), 127.55 (2CH),
127.79 (2CH), 129.06(CH), 131.21 (2CH), 140.57 (C), 145.20 (C)],
C–H), 1727, 1675, 1637 (6C O), 1420 (C N). 1H NMR (DMSO-d6):
–
–
–
–
d (ppm) ¼ 1.19 (s, 3H, CH3), 1.23 (s, 3H, CH3), 3.51 (s, 2H, CH2), 4.18
(s, 5H, 2CH2 þ CH), 4.93 (s, 4H, 2CH þ CH2), 6.97 (d, 4H, Ar–H,
J ¼ 8.4 Hz), 7.91 (d, 10H, Ar–H, J ¼ 8.4 Hz), 7.99 (d, 1H, Ar–H,
J ¼ 8.4 Hz), 12.95 (s, 1H, OH, D2O exch.). 13C NMR (DMSO-d6):
d (ppm) ¼ 26.13 (CH3), 30.51 (CH3), 31.22 (2CH2), 39.15 (2CH2),
59.29 (C), 60.23 (CH), 61.88 (CH), 67.13 (CH), 125.75 (4CH),
128.06 (4CH), 129.45 (4CH), 130.53 (4CH), 132.81 (2C), 133.14 (2C),
–
–
155.50 (C O), 167.84 (C O). EI MS m/z (%): 349.11 (19), 329.16
–
–
([Mþ2þK]þ, 19), 321.11 (90), 315.01 (71), 305.09 ([Mꢀ1þH2O]þ, 14),
301.08 (33), 293.03 (43), 271.05 ([Mþ1ꢀH2O]þ, 100). Elemental
analysis: for C15H13ClN2O2 calcd. % C, 62.40; H, 4.54; N, 9.70.
Found: % C, 62.78; H, 4.58; N, 9.94.
–
–
–
158.01 (2C), 160.65 (2C N), 166.54 (C O), 171.27 (C O), 171.53
–
–
–
(C O), 171.77 (C O). EI MS m/z (%): 833.70 ([M]þ, 22),
–
–
–
–
818.71 ([M–CH3]þ, 15). Elemental analysis: for C40H32N6O7S3
calcd. % C, 59.69; H, 4.01; N, 10.44. Found: % C, 59.51; H, 3.89; N,
10.61.
General method for the synthesis of compounds 20a,b
Ethyl bromoacetate was added to the solution of the correspond-
ing compound 19 in absolute ethanol (10 mmol) and the mixture
ß 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.archpharm.com

216
S. Demirci et al.
Arch. Pharm. Chem. Life Sci. 2014, 347, 200–220
(2S,5R,6R)-6-[(4-{[3-Benzyl-4-oxo-1,3-thiazolidin-2-
ylidene]amino}benzoyl)amino]penicillanic acid (22a)
left overnight in cold. The formed solid was filtered and
recrystallized from ethyl acetate. Yield 85%, m.p.: 240–241°C.
FT-IR (ymax, cmꢀ1): 3102 (aromatic C–H), 2668 (aliphatic C–H), 1674
The solution of K2CO3 (10 mmol) in 12 mL of acetone and 14 mL of
water was added to the suspension of 6-apa (10 mmol) in 10 mL of
acetone at ꢀ5°C and the mixture was stirred for 10 min. The
solution of 20a (10 mmol) in 10 mL of ethanol was added into it
dropwise in 3 h and stirred at room temperature for 30 h. Ethyl
acetate was added into it and acidified to pH 3 with 37% HCl. The
mixture was extracted with petroleum ether; the organic layer
was separated and dried over NaSO4. The solvent was removed
under reduce pressure and a solid was obtained. The crude
product was recrystallized from ethyl acetate. Yield 57%, m.p.:
179–180°C. FT-IR (ymax, cmꢀ1): 3456 (OH), 3168 (aromatic C–H),
1
–
–
(C O), 1490 (C N). H NMR (DMSO-d ): d (ppm) ¼ 5.08 (s, 2H, CH ),
–
–
6
2
6.42 (s, 1H, CH), 6.98–7.23 (m, 4H, Ar–H), 7.26–7.48 (m, 5H, Ar–H),
7.46 (d, 2H, Ar–H, J ¼ 8.2 Hz), 7.89 (d, 2H, Ar–H, J ¼ 9.8 Hz). 13C NMR
(DMSO-d6): d (ppm) ¼ 50.74 (CH2), 100.93 (CH), 123.83 (CH), 127.60
(C), 129.26 (CH), 130.04 (CH), 130.40 (CH), 130.74 (CH), 130.90 (CH),
131.37 (CH), 131.98 (CH), 132.40 (CH), 132.63 (CH), 133.45 (CH),
134.00 (CH), 134.15 (CH), 137.01 (C), 139.72 (2C), 141.46 (C), 157.50
þ
–
–
(C), 162.54 (C N), 169.97 (C O). EI MS m/z (%): 440.35 ([Mþ1] , 41),
–
–
351.48 (34), 421.35 ([MꢀH2O]þ, 71), 245.49 (100). Elemental
analysis: for C23H16Cl2N2OS calcd. % C, 62.88; H, 3.67; N, 6.38.
Found: % C, 62.82; H, 3.68; N, 8.39.
–
–
–
2667 (aliphatic C–H), 1727, 1676, 1638 (4C O), 1427 (C N).
–
1H NMR (DMSO-d6): d (ppm) ¼ 1.20 (s, 3H, CH3), 1.42 (s, 3H, CH3),
3.52 (brs, 4H, 2CH2 þ H2O) 4.01 (s, 1H, CH), 4.96 (s, 2H, 2CH),
7.21–7.50 (m, 9H, Ar–H), 8,78 (s, 1H, NH). 13C NMR (DMSO-d6):
d (ppm) ¼ 24.13 (CH3), 29.51 (CH3), 32.22 (CH2), 39.65 (C), 45.15
(CH2), 55.29 (CH), 58.88 (CH), 67.13 (CH), 126.75 (2CH), 127.06 (CH),
127.45 (CH), 127.53 (CH), 127.82 (CH), 127.95 (CH), 128.05 (CH),
–
4-{[3-Benzyl-5-(4-chlorophenyl)-1,3-oxazol-2(3H)-ylidene]amino}-
benzoyl chloride (23b). 4-Chlorophenacyl bromide (10 mmol) and
dried sodium acetate (200 mmol) were added to the solution of
compound 19b in absolute ethanol, and the reaction mixture was
refluxed for 14 h. Then, the mixture was cooled to room
temperature, after the solvent was evaporated under reduced
pressure, poured into ice-cold water while stirring, and left
overnight in cold. The formed solid was filtered and recrystallized
from ethyl acetate. Yield 65%, m.p.: 247–248°C. FT-IR (ymax, cmꢀ1):
–
128.16 (CH), 135.81 (C), 139.14 (C), 158.01 (C), 169.54 (C O), 169.89
–
–
–
–
–
(C N), 170.27 (C O), 171.53 (C O), 171.77 (C O). EI MS m/z (%):
–
–
–
–
524.13 ([M]þ, 11), 321.08 (10), 245.47 (35), 206.51 (100). Elemental
analysis: for C25H24N4O6S calcd. % C, 57.24; H, 4.61; N, 10.68.
Found: % C, 57.13; H, 4.61; N, 11.01.
3065 (aromatic C–H), 2930 (aliphatic C–H), 1656 (C O), 1454
–
1
–
(C N). H NMR (DMSO-d ): d (ppm) ¼ 4.24 (d, 3H, CH þ CH), 6.98
–
6
2
(brs, 2H, Ar–H), 7.28 (s, 9H, Ar–H), 7.78 (s, 2H, Ar–H). 13C NMR
(DMSO-d6): d (ppm) ¼ 32.24 (CH2), 33.03 (CH2), 126.41 (2CH), 126.97
(3CH), 128.02 (3CH), 127.99 (3CH), 128.87 (3CH), 137.02 (2C), 138.44
(2S,5R,6R)-6-[(4-{[3-Benzyl-4-oxo-1,3-oxazolidin-2-
ylidene]amino}benzoyl)amino]penicillanic acid (22b)
The solution of K2CO3 (10 mmol) in 3 mL of acetone and 14 mL of
water was added to the suspension of 6-apa (10 mmol) in 3 mL of
acetone at ꢀ5°C and the mixture was stirred for 10 min. 20b
(10 mmol) in 3 mL of acetone was added and the reaction mixture
was irradiated under reflux with CEM brand, monopod micro-
wave device at 150°C, 150 W, for 90 min. Then, temperature was
allowed to reach room temperature and stirring was continued
for an additional 1 h. Ethyl acetate was added and acidified to pH
3 with 37% HCl. The mixture was extracted with ethyl acetate; the
organic layer was separated and dried over NaSO4. The solvent
was removed under reduce pressure and a solid was obtained. The
crude product was recrystallized from methanol. Yield 57%, m.p.:
184–185°C. FT-IR (ymax, cmꢀ1): 3286 (NH), 3030 (aromatic C-H),
–
–
–
(2C), 140.05 (2C), 170.84 (C N), 178.23 (C O). EI MS m/z (%): 423.13
–
([M]þ, 51), 402.08 (11), 344.79 (89), 308.51 (100). Elemental
analysis: for C23H16Cl2N2O2 calcd. % C, 65.26; H, 3.81; N, 6.62.
Found: % C, 65.37; H, 3.84; N, 6.63.
General method for the synthesis of compounds 24a,b
The solution of K2CO3 (10 mmol) in 3 mL of DMF and 14 mL of
water was added to the suspension of 6-apa (10 mmol) in 3 mL of
DMF at ꢀ5°C and the mixture was stirred for 10 min. The
corresponding compound 23 (10 mmol) was added into the
reaction mixture and stirred at room temperature for 12 h. Then,
temperature was allowed to reach room temperature and stirring
was continued for an additional 1 h. Ethyl acetate was added and
acidified to pH 3 with 37% HCl. The mixture was extracted with
ethyl acetate; the organic layer was separated and dried over
NaSO4. The solvent was removed under reduce pressure and a
solid was obtained. The crude product was recrystallized from
ethyl acetate/hexane (1:2).
–
–
–
2958 (aliphatic C-H), 1714 (2C O), 1664 (2C O), 1413 (C N).
–
–
–
1H NMR (DMSO-d6): d (ppm) ¼ 1.18–1.54 (m, 6H, 2CH3), 1.82 (s, 4H,
2CH2), 3.99 (s, 1H, CH), 4.21 (s, 1H, CH), 4.38 (s, 1H, CH), 7.26 (brs,
9H, Ar–H), 9.23 (s, 1H, NH). 13C NMR (DMSO-d6): d (ppm) ¼ 25.73
(CH3), 29.91 (CH3), 33.26 (CH2), 39.54 (C), 41.15 (CH2), 59.26 (CH),
62.18 (CH), 67.23 (CH), 125.75 (2CH), 126.54 (CH), 128.45 (2CH),
129.53 (2CH), 129.98 (2CH), 135.11 (C), 139.14 (C), 158.01 (C),
(2S,5R,6R)-6-[Bis(4-{3-benzyl-5-(4-chlorophenyl)-1,3-
thiazol-2(3H)-ylidene]amino}benzoyl)amino]penicillanic
–
–
–
–
–
–
–
–
162.23 (C O), 169.54 (C N), 170.29 (C O), 171.58 (C O), 172.89
þ
–
–
(C O). EI MS m/z (%): 526.35 ([MþH O] , 61), 451.67 (94),
2
acid (24a)
421.30 (49), 243.49 (100). Elemental analysis: for C25H24N4O6S
calcd. % C, 59.04; H, 4.76; N, 11.02. Found: % C, 59.13; H, 4.78; N,
11.02.
Yield 57%, m.p.: 165–166°C. FT-IR (ymax, cmꢀ1): 3061 (aromatic
–
–
–
C–H), 2957 (aliphatic C–H), 1795 (C O), 1730 (C O), 1674, (2C O),
–
–
–
1
–
1492 (2C N). H NMR (DMSO-d ): d (ppm) ¼ 2.73 (s, 6H, 2CH ), 3.34
4-{[3-Benzyl-5-(4-chlorophenyl)-1,3-thiazol-2(3H)-ylidene]amino}-
benzoylchloride (23a). 4-Chlorophenacyl bromide (10 mmol) and
dried sodium acetate (200 mmol) were added to the solution of the
corresponding compound 19a in absolute ethanol, and the
reaction mixture was irradiated with CEM brand, microwave
device at 125°C, 150 W, for 60 min. Then, the mixture was cooled
to room temperature, after the solvent was evaporated under
reduced pressure, poured into ice-cold water while stirring, and
–
6
3
(s, 4H, 2CH2 þ D2O), 4.76 (s, 2H, 2CH), 5.12 (s, 1H, CH), 5.71 (s, 1H,
CH), 6.49 (s, 1H, CH), 7.01–7.22 (m, 3H, Ar–H), 7.24–7.38 (m, 13H,
Ar–H), 7.47 (brs, 1H, Ar–H), 7.67 (d, 4H, Ar–H, J ¼ 8.2 Hz), 7.89
(d, 4H, Ar–H, J ¼ 7.6 Hz), 7.99 (s, 1H, Ar–H). 13C NMR (DMSO-d6):
d (ppm) ¼ 39.56 (2CH3), 47.84 (CH2), 48.76 (CH2), 67.84 (C), 121.57
(2CH), 121.88 (2CH), 126.08 (2C), 127.75 (2CH), 127.68 (3CH),
127.82 (CH), 128.22 (2CH), 129.70 (3CH), 129.27 (2CH), 129.87
ß 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.archpharm.com

Arch. Pharm. Chem. Life Sci. 2014, 347, 200–220
Heterofunctionalized Penicillanic Acid Derivatives
217
(2CH), 129.99 (CH), 130.29 (2C), 130.87 (2CH), 130.67 (2CH), 131.00
(2CH), 131.62 (CH), 131.98 (2CH), 131.04 (2CH), 134.61 (2C), 136.86
7.21–7.25 (m, 10H, Ar–H,), 7.35 (d, 2H, Ar–H, J ¼ 7.2 Hz), 7.46
(d, 2H, Ar–H, J ¼ 7.8 Hz), 7.63 (d, 4H, Ar–H, J ¼ 8.1 Hz), 7.72 (d, 2H,
Ar–H, J ¼ 7.2 Hz), 7.81 (d, 2H, Ar–H, J ¼ 7.8 Hz), 7.87 (d, 4H, Ar–H,
J ¼ 7.2 Hz). 13C NMR (DMSO-d6): d (ppm) ¼ 21.08 (2CH3), 43.29
(CH2), 43.42 (CH2), 62.79 (CH), 67.06 (C), 68.22 (CH), 88.75 (CH),
117.33 (CH), 117.53 (CH), 121.75 (C), 125.63 (C), 127.13 (2CH),
127.45 (2CH), 127.61 (2CH), 127.69 (2CH), 127.83 (2CH), 128.73
(2CH), 129.00 (2CH), 129.26 (CH), 129.73 (2CH), 129.76 (2CH),
130.39 (2CH), 130.44 (2CH), 130.81 (CH), 131.12 (CH), 131,34 (CH),
133.23 (C), 139.52 (C), 140.72 (C), 140.78 (C), 141.27 (C), 144.37 (C),
–
–
–
(C), 139.26 (2C), 144.44 (3C), 158.00 (N C), 161.20 (N C), 167.72
–
–
þ
–
–
–
–
(2C O), 181.16 (2C O). EI MS m/z (%): 953.16 ([Mꢀ2Cl] , 13), 527.81
(12), 707.38 (25), 420.70 (100), 359.83 (48). Elemental analysis:
for C54H42Cl2N6O5S3 calcd. % C, 63.46; H, 4.14; N, 8.22. Found: % C,
63.50; H, 4.13; N, 8.18.
(2S,5R,6R)-6-[Bis(4-{[3-benzyl-5-(4-chlorophenyl)-1,3-
oxazol-2(3H)-ylidene]amino}benzoyl)amino]penicillanic
145.31 (C), 146.22 (C), 155.48 (C), 155.55 (C), 159.46 (C N), 165.64
–
–
–
–
–
–
acid (24b)
(C N), 167.95 (C O), 170.55 (C O), 192.67 (C O), 192.91 (C O).
–
–
–
–
–
Yield 70%, m.p.: 170–171°C. FT-IR (ymax, cmꢀ1): 3032 (aromatic
EI MS m/z (%): 990.16 ([Mþ1]þ, 23), 890.45 (15), 767.38 (30),
401.75 (100), 270.89 (23). Elemental analysis: for C54H42Cl2N6O7S
calcd. % C, 65.52; H, 4.28; N, 8.49. Found: % C, 65.50; H, 4.29;
N, 8.41.
–
–
–
C–H), 2919 (aliphatic C–H), 1744 (C O), 1692 (C O), 1494 (2C N).
–
–
–
1H NMR (DMSO-d6): d (ppm) ¼ 2.53(s, 6H, 2CH3), 3.34 (s, 4H,
2CH2 þ D2O), 4.86 (s, 1H, CH), 5.89 (s, 3H, 3CH), 5.98 (s, 1H, CH),
Table 1. Screening for antimicrobial activity of the compounds (50 mL).
Microorganisms and inhibition zone (mm)
Comp. no
Ec
Yp
Pa
Sa
Ef
Bc
Ms
Ca
Sc
3
4
6
7
8
9
10a
10b
10c
10d
10e
11a
8
–
10
–
10
–
25
–
10
–
6
–
-
–
-
–
-
–
9
–
–
6
–
–
–
–
–
–
–
8
–
–
–
–
–
–
–
–
–
–
6
6
8
8
10
6
12
30
10
8
8
–
20
10
13
–
24
10
6
–
–
–
–
12
12
12
12
12
–
22
35
13
30
18
20
8
14
8
10
10
15
11
–
8
–
8
–
–
20
–
24
–
10
–
8
20
–
8
19
–
–
20
8
–
–
–
10
–
8
–
6
–
11b
–
–
–
–
12a
–
–
–
–
–
–
–
–
–
12b
–
–
–
–
–
–
–
–
–
13
14
15
16
17
18a
18b
19a
8
–
8
–
10
10
10
10
8
8
6
10
–
25
11
–
–
10
8
8
10
12
10
–
15
–
–
–
8
8
10
8
8
10
10
–
25
25
8
12
12
10
10
–
–
10
10
10
–
12
12
13
–
20
25
10
–
–
12
15
–
25
25
–
–
–
–
10
–
10
–
12
–
20
–
19b
–
–
–
–
20a
20b
21
22a
22b
23a
23b
–
–
6
–
–
–
–
10
–
10
–
–
–
10
12
8
10
12
–
–
–
–
–
10
10
8
12
10
8
15
20
25
–
8
8
10
10
12
–
20
15
18
6
10
10
–
10
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
24a
24b
Amp. (10 mg)
Strep. (10 mg)
Flu. (5 mg)
10
8
10
12
10
18
8
10
18
–
–
6
–
–
–
10
–
–
–
–
–
35
10
15
35
25
25
Ec: Escherichia coli ATCC 25922, Yp: Yersinia pseudotuberculosis ATCC 911, Pa: Pseudomonas aeruginosa ATCC 43288, Sa: Staphylococcus aureus
ATCC 25923, Ef: Enterococcus faecalis ATCC 29212, Bc: Bacillus cereus 702 Roma, Ms: M. smegmatis ATCC607, Ca: Candida albicans ATCC 60193,
Sc: Saccharomyces cerevisiae RSKK 251, Amp.: ampicillin, Strep.: streptomycin, Flu.: fluconazole, (–): no activity.
ß 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.archpharm.com

218
S. Demirci et al.
Arch. Pharm. Chem. Life Sci. 2014, 347, 200–220
Biological activity assessment
Table 2. Inhibitory activities of the synthesized compounds
against B. cereus b-lactamase. All compounds and HgCl2 were
assayed at final concentrations of 1 mM.
Antimicrobial activity
All test microorganisms were obtained from the Hifzissihha
Institute of Refik Saydam (Ankara, Turkey) and were as
follows: Escherichia coli (E. coli) ATCC25922, Enterobacter aerogenes
(E. aerogenes) ATCC13048, Yersinia pseudotuberculosis (Y. pseudotu-
berculosis) ATCC911, Pseudomonas aeruginosa (P. aeruginosa)
ATCC43288, Staphylococcus aureus (S. aureus) ATCC25923,
Enterococcus faecalis (E. faecalis) ATCC29212, Bacillus cereus
(B. cereus) 702 Roma, Mycobacterium smegmatis (M. smegmatis)
ATCC607, Candida albicans (C. albicans) ATCC60193, Candida
tropicalis (C. tropicalis) ATCC 13803, Aspergillus niger (A. niger)
RSKK 4017, and Saccharomyces cerevisiae (S. cerevisiae) RSKK 251.
All the newly synthesized compounds were weighed and
dissolved in dimethylsulphoxide to prepare extract stock
solution of 5.000 microgram/milliliter (mg mLꢀ1).
Compound
Inhibition (%)
IC50 (mM)
10a
10b
10d
HgCl2
10
18
22
76
nd
nd
nd
0.093
triplicate. The IC50 value was determined as the concentration
of compound that give 50% inhibition of maximal activity [41]
and results are presented in Table 2.
Urease inhibition assay
Reaction mixtures comprising 25 mL of Jack bean urease,
55 mL of buffer (100 mM urea, 0.01 M K2HPO4, 1 mM EDTA, and
0.01 M LiCl, pH 8.2), and 100 mM urea were incubated with
5 mL 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 determine the urease
inhibitory activity. The phenol reagent (45 mL, 1% w/v phenol
and 0.005% w/v sodium nitroprusside) and alkali reagent
(70 mL, 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
Agar-well diffusion method
Screening test using agar-well diffusion method [40] as
adapted earlier was used for all newly synthesized com-
pounds. Each microorganism was suspended in Mueller
Hinton (MH) (Difco, Detroit, MI) broth and diluted approxi-
mately to 106 colony forming units (cfu) mLꢀ1. They were
“flood-inoculated” onto the surface of MH agar and Sabouraud
Dextrose Agar (SDA) (Difco, Detriot, MI) and then dried. For
C. albicans and S. cerevisiae, SDA were used. Five-millimeter
diameter wells were cut from the agar using a sterile cork-
borer, and 50 mL of the extract substances was delivered into
the wells. The plates were incubated for 18 h at 35°C.
Antimicrobial activity was evaluated by measuring the zone
of inhibition against the test organism. Ampicillin (10 mg),
streptomycin (10 mg), and fluconazole (5 mg) were standard
drugs. Dimethyl sulfoxide was used as solvent control. The
antimicrobial activity results are summarized in Table 1.
calculated from the equation 100 ꢀ (ODtestwell/ODcontrol
)
ꢂ 100. Thiourea was used as the standard inhibitor. In order
to calculate IC50 values, different concentrations of synthe-
sized compounds and standard were assayed at the same
reaction conditions [42] and the obtained results are
presented in Table 3.
Table 3. Inhibitory activities of the synthesized compounds
against Jack bean urease. All compounds and thiourea were
assayed at final concentrations of 250 and 100 mg mLꢀ1
.
b-Lactamase assay
In vitro b-lactamase (Bacillus cereus MBLs, Sigma) activity was
determined by monitoring the hydrolysis of reporter
substrate Nitrocefin (Calbiochem, Darmstadt, Germany) by
b-lactamase, at 486 nm (Louie et al. 2012). Enzyme assays were
performed in 25 mM piperazine-N,N0-bis(2-ethane sulfonic
acid) (PIPES) buffer, pH 7.0, with 100 mM ZnSO4. 50 mL of
1.5 mM enzyme solution, 50 mL of 150 mM Nitrocefin, and
50 mL of 1 mM synthesized compound were recorded
continuously for 20 min at 37°C against the buffer alone by
using microplate reader (Multiskan GO microplate spectro-
photometer, Thermo Scientific) at 486 nm. The inhibitory
activities of those compounds and HgCl2, a positive control
against b-lactamase, were measured at various concentra-
tions. Residual activities were calculated by comparing to
control without inhibitor (Tþ). The assays were done in
Inhibition (%)
IC50 ꢁ S.D.
Compound
250 mg mLꢀ1
100 mg mLꢀ1
(mg mLꢀ1
)
a)
5
7
44
56
100
98
100
20
25
15
31
6
100
–
ndb)
nd
–
70.0
56.0
81.8
–
10c
10d
10e
18a
18b
20a
21
70.95 ꢁ 7.14
74.31 ꢁ 5.83
54.14 ꢁ 1.45
nd
–
nd
nd
nd
–
–
24a
Thiourea
–
nd
92.2
51.62 ꢁ 7.28
a)
–: No inhibition.
nd: Not determined.
b)
ß 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.archpharm.com

Arch. Pharm. Chem. Life Sci. 2014, 347, 200–220
Heterofunctionalized Penicillanic Acid Derivatives
219
Table 4. Residual lipase activity and IC50 values of synthesized
[2] O. A. Phillips, E. E. Udo, A. A. M. Alic, N. Al-Hassawi, Bioorg.
Med. Chem. 2003, 11, 35–41.
chemical compounds. All compounds and orlistat were assayed
at a final concentration of 6.25 mg mLꢀ1
.
[3] G. L. Almajan, S.-Felicia Barbuceanu, E. Ruxandra Almajan,
C. Draghici, G. Saramet, Eur. J. Med. Chem. 2009, 44, 3083–
3089.
Compound
no.
Inhibition (%)
IC50
(6.25 mg mLꢀ1
)
(mg mLꢀ1
)
[4] P. P. Dixit, V. J. Patil, P. S. Nair, S. Jain, N. Sinha, S. K. Arora,
nda)
nd
nd
4.85
nd
nd
Eur. J. Med. Chem. 2006, 41, 423–428.
6
66
4
62
89
8
67
6
100
98
4
10a
10b
11a
11b
16
18a
18b
20a
20b
23b
24a
Orlistat
[5] X. Jin, C. J. Zheng, M. X. Song, Y. Wu, L. P. Sun, Y. J. Li, L. J. Yu,
H. R. Piao, Eur. J. Med. Chem. 2012, 56, 203–209.
[6] A. A. Bekhit, O. A. El-Sayed, E. Aboulmagd, J. Y. Park, Eur. J.
Med. Chem. 2004, 39, 249–255.
[7] E. Hassan, M. I. Al-Ashmawi, B. Abdel-Fattah, Pharmazie 1983,
38, 833–835.
nd
4.18
0.84
nd
nd
nd
[8] H. Bayrak, A. Demirbas, N. Demirbas, N. S. Alpay-Karaoglu,
Eur. J. Med. Chem. 2010, 45, 4726–4732.
9
68
100
[9] V. R. Solomon, C. Hu, H. Lee, Bioorg. Med. Chem. 2010, 18,
1563–1572.
0.61
[10] C. Hu, V. R. Solomon, G. Ulibarri, H. Lee, Bioorg. Med. Chem.
2008, 16, 7888–7893.
a)
: Not determined.
[11] C. Hu, V. R. Solomon, P. Cano, H. Lee, Eur. J. Med. Chem. 2010,
45, 705–709.
[12] V. V. Kouznetsov, A. Gomez-Barrio, Eur. J. Med. Chem. 2009, 44,
3091–3113.
Anti-lipase activity assay
[13] J. Adamec, R. Beckert, D. Weiss, V. Klimesova, K. Waisser,
U. Mollmann, J. Kaustova, V. Buchta, Bioorg. Med. Chem. 2007,
15, 2898–2906.
The inhibitory effects of those compounds were evaluated
against porcine pancreatic lipase (PPL) (15 ng mLꢀ1). Lipase
activity assays were done according to Pencreac’h et al. [43].
Microtiter plates were coated with purified tung oil TAGs.
Compounds were mixed with PPL 1:2 v/v and incubated for
30 min. The microtiter plates containing purified tung oil,
lipase solution, and assay buffer (10 mM Tris-HCl buffer, pH
8.0, containing 150 mM NaCl, 6 mM CaCl2, 1 mM EDTA, and
3 mg mLꢀ1 b-cyclodextrin) were recorded continuously for
40 min against the buffer alone by using a microplate reader
(SpectraMax M5, Molecular Devices) at 272 nm. The inhibitory
activity of those compounds and orlistat, a positive control
against pancreatic lipase, were measured at concentrations of
[14] A. Polak, Mycoses 1999, 42, 355–370.
[15] T. Fekner, J. E. Baldwin, R. M. Adlington, T. W. Jones,
C. K. Prout, C. J. Schofield, Tetrahedron 2000, 56, 6053–6074.
[16] M. P. A. Ribeiro, T. F. Pádua, O. D. Leite, R. L. C. Giordano,
R. C. Giordano, Chemom. Int. Lab. Sys. 2008, 90, 169–177.
[17] A. Bruggink, P. D. Roy, Synthesis of b-Lactam Antibiotics, Kluwer
Academic Publishers, The Netherlands 2001.
[18] A. Verma, S. K. Saraf, Eur. J. Med. Chem. 2008, 43, 897–905.
[19] K. Arya, D. S. Rawat, A. Dandia, H. Sasai, J. Fluorine Chem. 2012,
137, 117–122.
[20] T. R. Anderegg, R. N. Jones, Int. J. Antimic. Agents 2004, 23, 6–10.
[21] S. J. Brickner, D. K. Hutchinson, M. R. Barbachyn,
P. R. Manninen, D. A. Ulanowicz, S. A. Garmon, J. Med.
Chem. 1996, 39, 673–679.
6.25, 2.08, and 1.04 mg mLꢀ1
. Residual activities were
calculated by comparing to control without inhibitor (Tþ).
The assays were done in triplicate. The IC50 value was
determined as the concentration of compound that gives 50%
inhibition of maximal activity. The results are presented in
Table 4.
[22] C. G. Gemmell, J. Antimicrob. Chemother. 2001, 48, 47–52.
[23] D. J. Diekema, R. N. Jones, Lancet, 358, 2001, 1975–1982.
[24] E. Palaska, Farmasötik Kimya, 2nd Edn, Hacettepe University
Press, Ankara 2004.
[25] M. S. Mohamed, W. M. Hussein, R. P. Mc Geary, P. Vella,
G. S. Rania, H. Abd El-hameed, Eur. J. Med. Chem. 2011, 46,
6075–6082.
The support provided by Scientific and Technological Research Council
of Turkey (TUBITAK, Project no: 111T427) is gratefully acknowledged.
[26] B. Di Giacomo, G. Tarzia, A. Bedini, G. Gatti, F. Bartoccini,
C. Balsamini, A. Tontini, W. Baffone, E. Di Modugno, A. Felici,
Il Farmaco 2002, 57, 273–283.
The author have declared no conflict of interest.
[27] M. Kot, W. Karcz, W. Zaborska, Bioorg. Chem. 2010, 38, 132–
137.
References
[28] S. Demirci, S. Basoglu, A. Bozdereci, N. Demirbas, Med. Chem.
Res. 2013, 22, 4030–4945.
[1] V. Raparti, T. Chitre, K. Bothara, V. Kumar, S. Dangre,
C. Khachane, S. Gore, B. Deshmane, Eur. J. Med. Chem. 2009, 44,
3954–3960.
[29] A. S. Cobena, T. H. Misselbrook, A. Arce, J. I. Mingot, J. A. Diez,
A. Vallejo, Agric. Ecosyst. Environ. 2008, 126, 243–249.
ß 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.archpharm.com

220
S. Demirci et al.
Arch. Pharm. Chem. Life Sci. 2014, 347, 200–220
[30] L. F. Van Gaal, I. L. Mertens, C. E. De Block, Nature 2006, 444,
875–880.
N. Adibpour, A. Shafiee, A. Foroumadi, Eur. J. Med. Chem. 2011,
46, 65–70.
[31] R. J. Jandacek, S. C. Woods, Drug Discov. Today 2004, 15, 874–
[38] L. S. Changov, E. D. Naydenova, G. I. Ivanova, R. T. Gergova,
E. E. Keuleyan, B. V. Aleksiev, Bioorg. Med. Chem. 1999, 7, 2899–
2904.
880.
[32] R. B. Birari, K. K. Bhutani, Drug Discov. Today 2007, 12, 879–
889.
[39] A. Favre, J. Grugier, A. Brans, J. Marchand-Brynaert,
Tetrahedron 2012, 68, 10818–10826.
[33] D. S. Weigle, J. Clin. Endocrinol. Metab. 2003, 88, 2462–2469.
[40] I. Ahmad, Z. Mehmood, F. Mohammed, J. Ethnopharmacol.
1998, 62, 183–193.
[34] H. Bektas, S. Ceylan, N. Demirbas, S. Alpay-Karaoglu,
B. Bilgin-Sokmen, Med. Chem. Res. 2013, 22, 3629–3639.
[41] A. Louie, B. D. VanScoy, D. L. Brown, R. W. Kulawy,
H. S. Heine, G. L. Drusano, Antimic. Agents Chemother. 2012,
56, 258–270.
[35] S. Ceylan, H. Bektas, H. Bayrak, N. Demirbas, S. Alpay-Karaoglu,
Serdar. Ulker, Arch. Pharmazie 2013, 346, 1–14.
[36] S. Basoglu, S. Ulker, S. Alpay-Karaoglu, N. Demirbas, Med.
Chem. Res. 2013, (in press, Ref no: MCRE-4100).
[42] M. W. Weatherburn, Anal. Chem. 1967, 39, 971–974.
[43] G. Pencreac’h, J. Graille, M. Pina, R. Verger. Anal. Biochem. 2002,
303, 17–24.
[37] A. Khalaj, M. Nakhjiri, A. S. Negahbani, M. Samadizadeh,
L. Firoozpour, S. Rajabaliand, N. Samadi, M. A. Faramarzi,
ß 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.archpharm.com