Synthesis of novel Azol-β-lactam derivatives starting from phenyl piperazine and investigation of their antiurease activity and antioxidant capacity comparing with their molecular docking studies

Article abstract of DOI:10.1016/j.molstruc.2019.04.039

This study reports the synthesis, biological investigation and molecular docking of novel β-lactam derivatives bearing 1,3,4-thiadiazole and 1,3,4-oxadiazole ring system. The synthesized compounds were evaluated for in vitro antiurease activity and antiox

Full text of DOI:10.1016/j.molstruc.2019.04.039

Accepted Manuscript
Synthesis of novel Azol-β-lactam derivatives starting from phenyl piperazine and
investigation of their antiurease activity and antioxidant capacity comparing with their
molecular docking studies
Arif Mermer, Hacer Bayrak, Yakup Şirin, Mustafa Emirik, Neslihan Demirbaş
PII:
S0022-2860(19)30434-X
DOI:
https://doi.org/10.1016/j.molstruc.2019.04.039
MOLSTR 26411
Reference:
To appear in: Journal of Molecular Structure
Received Date: 21 January 2019
Revised Date: 1 April 2019
Accepted Date: 9 April 2019
Please cite this article as: A. Mermer, H. Bayrak, Y. Şirin, M. Emirik, N. Demirbaş, Synthesis of novel
Azol-β-lactam derivatives starting from phenyl piperazine and investigation of their antiurease activity
and antioxidant capacity comparing with their molecular docking studies, Journal of Molecular Structure
(2019), doi: https://doi.org/10.1016/j.molstruc.2019.04.039.
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ACCEPTED MANUSCRIPT
Synthesis of Novel Azol-β-Lactam Derivatives starting from phenyl piperazine and Investigation
of their Antiurease activity and Antioxidant Capacity Comparing with their Molecular Docking
Studies
Arif Mermer1, Hacer Bayrak2*, Yakup Şirin1, Mustafa Emirik3, Neslihan Demirbaş1
1Department of Chemistry, Karadeniz Technical University, 61080, Trabzon, Turkey
2Department of Chemistry and Chemical Processing Technology, Karadeniz Technical
University, 61080, Trabzon, Turkey
3Department of Chemistry, Recep Tayyip Erdogan University, 53100, Rize, Turkey
*Address correspondence to: Hacer Bayrak, Karadeniz Technical University, Maçka
Vocational School, Department of Chemistry and Chemical Processing Technology, 61750
Trabzon, Turkey, Phone: +90 462 3777639, Fax: +90 4625123552, e-mail:
h.bayrak@ktu.edu.tr
Abstract: This study reports the synthesis, biological investigation and molecular docking of
novel β-lactam derivatives bearing 1,3,4-thiadiazole and 1,3,4-oxadiazole ring system. The
synthesized compounds were evaluated for in vitro antiurease activity and antioxidant
capacity. Almost all compounds showed excellent antiurease activity compared to thiourea,
standard drug. Also, in silico ADME (absorption, distribution, metabolism, elimination)
prediction and molecular docking studies were performed.
Keywords: Azole, phenyl piperazine, β-Lactam, Antioxidant Capacity, Antiurease activity,
Molecular Docking
1. Introduction
β-Lactams are a major class of antibiotics defined by the presence of an azetidine-2-one ring,
which is the nucleus of biological activity [1]. The azetidine-2-one ring system is a
widespread structural character of a number of broad spectrum β-lactam antibiotics, such as
penicillins, cephalosporins, carbapenems, nocardicins and monobactams that have been
widely used as chemotherapeutic agents for treating microbial diseases [2]. This ring system
also possess many biological activities such as cholesterol absorption inhibitors [3], human
cytomegalovirus protease inhibitors [4], thrombin inhibitors [5], antihyperglycemic [6], anti-
tumour [7], anti-HIV [8], antiinflammatory, analgesic activities [9] and serine-dependent
enzyme inhibitors [10]. However, microorganisms have developed resistance to compounds
containing beta-lactam ring as in any drug class. Therefore, the synthesis of new compounds
with more effective and broad spectrum than the existing ones has gained importance. In this
context, a kind of varied techniques have been reported for the synthesis of β-lactam

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derivatives, including cyclization reactions, carbene insertion reactions, rearrangement of
heterocyclic compounds, Reformatsky reaction, Ugi 4CR/cyclization and [2 + 2] ketene-imine
cycloaddition [11-17].
Azoles are one of the crucial classes of nitrogen containing heterocycles that displayed
diverse biological activities such as anti-bacterial, anti-malarial, anti-fungal, anti-HIV,
antiinflammatory and anti-TB properties [18-20]. Among these class, thiadiazole and
oxadiazole moieties have an important role in the fields of heterocyclic and medicinal
chemistry because of their nitrogen, sulfur and oxygen atoms. Thiadiazole has chamred a
great deal of concern as a favored scaffold due to its considerable therapeutic potential. The
sulfur atom of thiadiazole ring gives improved liposolubility and the mesoionic nature of
1,3,4-thiadiazoles also allows these compounds to cross cellular membranes and interact with
biological targets with distinct affinities [21-24]. Thiadiazoles show a wide spectrum of
biological activities such as the inhibition of CAs, cyclooxygenases (COXs), neutral
endopeptidase (NEP), aminopeptidase N (APN), matrix metalloproteinases (MMPs),
phosphodiesterases (PDEs) and c-Src/Abl tyrosine kinase [25]. 1,3,4-Oxadiazole structure is a
crucial group in heterocyclic chemistry due to their significant bioactivities and they have a
broad application in organic synthesis [26, 27]. These compounds are affectingly being used
as antibacterial agents, anticancer, anti-Parkinson, anti-HIV, and anti-proliferative agents [28-
33].
Urease play a vital role in pathogenesis of peptide ulcers which cause cancer as helping the
helicobacter pylori baceria to standat low pH of the stomach during colonizaition [34. 35].
Antioxidants are generally hydrogen donors or electron donorsto the reactive site in
neutralizing free radicals. Antioxidants areextensively studied for their capacity for protect
organism and cellfrom damage that is induced by oxidative stress. Scientists in manydifferent
disciplines become more interested in new compounds,either synthesized or obtained from
natural sources that couldprovide active components to prevent or reduce the impact
ofoxidative stress on cell [36-38].
Our group has already reported oxadiazole and thiadiazole scaffold as potent antiurease, α-
glucosidase inhibitor and antimicrobial activity [39, 40]. Therefore, we considered the
synthesis of new hybrid compounds carrying oxadiazole, thiadiazole and β-lactam rings in the
same structure, and these new compounds were thought to be more potent inhibitors. In the
present research, the designed hybrid molecules were screened for their antioxidant capacity
and potential of enzyme inhibition against urease enzyme. In silico studies were also

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performed to figure out different kinds of interactions with active pocket of urease enzyme. In
addition, one of the computational studies, ADME prediction were applied for all compounds.
2. Experimental
2.1. Chemistry
All the chemicals were purchased from FlukaChemie AG Buchs (Switzerland) and used
without further purification. Melting points of the synthesized compounds were determined in
open capillaries on a Büchi B-540 melting point apparatus and are uncorrected. Reactions
were monitored by thin-layer chromatography (TLC) on silica gel 60 F254 aluminium sheets.
The mobile phase was ethyl acetate:diethyl ether (1:2), and detection was made using UV
1
light. FT-IR spectra were recorded using a Perkin Elmer 1600 series FTIR spectrometer. H
NMR and 13C NMR spectra were registered in DMSO-d6 on a BRUKER AVENE II 400 MHz
1
13
NMR Spectrometer (400.13 MHz for H and 100.62 MHz for C). The chemical shifts are
given in ppm, J values are given in Hz. The Mass spectra were obtained on a Quattro LC-MS
(70 eV) Instrument. The yields, melting points and crystallization solvent of all compounds
were given in Table 1.

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O
i
N
NH
N
N
OCH2CH3
1
R=
OCH3
6a, 7a
HO
ii
O
iii
N
N
R=
R=
NHNH2
2
6b, 7b
OH
N NH
O
iv
N
N
S
OCH3
3
CN
6c, 7c
N
N
v
N
N
N
R=
R=
R=
S
O
4
6d, 7d
N
N
N
NH2
S
vi
N
N
6e, 7e
N
N
S
O
5
N
N
N
6f, 7f
N
R
S
N
N
vii
N
N
S
R=
R=
F
O
6a-6h
6h, 7h
R
N
N
Cl
Cl
N
O
S
N
N
Cl
N
N
S
7a-7h
O
6g, 7g
Scheme 1. Synthetic pathway for the compounds. i: BrCH2COOEt, NEt3, THF, ii:
NH2NH2.H2O, EtOH, iii: CS2, KOH, EtOH, iv: CH2CHCN, NEt3, EtOH, v: NH2CSNHNH2,
CF3COOH, vi: RHCO, vii: ClCH2COCl, 1,4-dioxane, NEt3.
2.2. 5-((4-phenylpiperazin-1-yl)methyl)-1,3,4-oxadiazole-2(3H)-thione (3)
Compound 2 (10 mmol) and CS2 (20 mmol) were added to a solution of KOH (10 mmol) in
20 mL H2O and 20 mL ethanol and the reaction mixture was refluxed for 13 h. Then, the
reaction content was acidified with conc. HCl to pH 6. The precipitate formed was filtered
off, washed with H2O and recrystallized from ethanol to afford the desired compound.
1
FT-IR (υmax, cm-1): 3011 (aromatic CH), 2841 (SH), 1442 (C=N). H NMR (DMSO-d6, δ
ppm): 2.51 (4H, d, J= 1.6 Hz, 2CH2), 3.15 (4H, s, 2CH2), 3.44 (2H, d, J= 2.0 Hz, CH2), 6.79
(1H, t, J= 12.0 Hz, arH), 6.93 (2H, d, J= 8.0 Hz, arH), 7.21 (2H, t, J= 12.0 Hz, arH), 14.42

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(1H, brs, SH). 13C NMR (DMSO-d6, δ ppm): 48.52 (2CH2), 51.56 (CH2), 52.41 (2CH2), arC:
[116.00 (2CH), 119.45 (CH), 129.36 (2CH), 151.29 (C)], 161.09 (oxadiazole C-5), 178.48
(oxadiazole C-2). EI MS m/z (%): 315.22 ([M+K]+, 40, 277.23 ([M + 1]+, 100), 175.16 (47),
106.04 (45).
2.3.
3-(5-((4-phenylpiperazin-1-yl)methyl)-2-thioxo-1,3,4-oxadiazol-3(2H)-yl)propane
nitrile (4)
To a solution of the compound 3 (10 mmol) in ethanol was added acrylonitrile (50 mmol) in
the presence of triethylamine (50 mmol) and reaction mixture was refluxed for 6h. After
evaporating the solvent under reduced pressure, a solid obtained. The crude product was
recrystallized from ethanol to afford the desired product.
1
FT-IR (υmax, cm-1): 3073 (aromatic CH), 2253 (CN), 1461 (C=N), 1220 (C=S). H NMR
(DMSO-d6, δ ppm): 2.48 (2H, s, CH2), 2.64 (4H, s, 2CH2), 3.12 (2H, s, CH2), 3.30 (2H, s,
CH2), 3.77 (2H, s, CH2), 4.28 (2H, t, J= 12.4 Hz, CH2), 6.75 (1H, t, J= 13.6 Hz, arH), 6.91
13
(2H, d, J= 8.0 Hz, arH), 7.18 (2H, t, J= 12.0 Hz, arH). C NMR (DMSO-d6, δ ppm): 16.13
(CH2), 44.62 (CH2), 48.57 (2CH2), 51.54 (CH2), 52.40 (2CH2), arC: [115.99 (2CH), 118.37
(CH), 129.36 (2CH), 151.36 (C)], 119.43 (CN), 159.91 (oxadiazole C-5), 176.77 (oxadiazole
C-2). EI MS m/z (%): 368.36 ([M + K] +, 100), 175.28 (25), 113.02 (60).
2.4. 3-(2-(5-amino-1,3,4-thiadiazol-2-yl)ethyl)-5-((4-phenylpiperazin-1-yl)methyl)-1,3,4-
oxadiazole-2(3H)-thione (5)
A mixture of the compound 4 (10 mmol) and thiosemicarbazide (10 mmol) were combined in
ethanol in the presence of trifluoroacetic acid (5 mL) in an oil bath and then was refluxed for
9 h. Then the reaction content was neutralized with diluted NH3 to pH 6. Water was decanted
from the mixture and the remaining fatty fraction was treated with ether to solidify the crude
product. The resulting solid was filtered and purified by crystallization from acetone.
1
FT-IR (υmax, cm-1): 3279 (NH2), 3084 (aromatic CH),1527 (C=N), 1299 (C=S). H NMR
(DMSO-d6, δ ppm): 2.48 (2H, s, CH2), 2.74 (4H, s, 2CH2), 3.17 (4H, s, 2CH2), 3.88 (2H, s,
CH2), 4.32 (2H, s, CH2), 6.78 (1H, s, arH), 6.93 (1H, d, J= 6.0 Hz, arH), 7.02 (1H, s, arH),
7.17 (2H, d, J= 19.2 Hz, arH), 7.27 (2H, s, NH2). 13C NMR (DMSO-d6, δ ppm): 27.59 (CH2),
47.95 (2CH2), 48.47 (CH2), 51.38 (CH2), 52.39 (2CH2), arC: [116.02 (2CH), 119.46 (CH),
129.37 (2CH), 151.27 (C)], 153.89 (oxadiazole C-5), 161.94 (thiadiazole C-2), 169.28
(thiadiazole C-5), 178.60 (oxadiazole C-2). EI MS m/z (%): 405.40 ([M + 2] +, 22), 404.40
([M + 1]+, 100), 305.54 (21), 304.47 (88), 175.21 (25).

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2.5. General method for the synthesis of compounds 6a-h
To a solution of compound 5 (10 mmol) and corresponding aromatic aldehyde (20 mmol) was
refluxed for 2 h at 125 ºC in an oil bath. After completion of the reaction, pure water was
added to the flask and exracted with dichloromethane (1:3). Organic phase was dried with
Na2SO4. After filtration off the solvent was evaporated under reduced pressure, a solid
obtained. The crude product was recrystallized from an appropriate solvent (Table 1) to afford
the desired product.
2.5.1.
3-(2-(5-((4-methoxybenzylidene)amino)-1,3,4-thiadiazol-2-yl)ethyl)-5-((4-phenyl
piperazin-1-yl)methyl)-1,3,4-oxadiazole-2(3H)-thione (6a)
1
FT-IR (υmax, cm-1): 3044 (aromatic CH), 1576 (C=N), 1256 (C=S). H NMR (DMSO-d6, δ
ppm): 2.67 (4H, s, 2CH2), 3.04 (2H, s, CH2), 3.65 (4H, s, 2CH2), 3.80 (3H, s, OCH3), 3.97
(2H, s, CH2), 5.12 (2H, s, CH2), 6.61 (3H, t, J= 4.0 Hz, arH), 6.92 (2H, d, J= 8.0 Hz, arH),
7.06 (2H, d, J= 4.0 Hz, arH), 7.17 (2H, d, J= 4.0 Hz, arH), 8.36 (1H, s, CH). 13C NMR
(DMSO-d6, δ ppm): 29.56 (CH2), 46.65 (CH2), 50.47 (2CH2), 52.63 (2CH2), 55.89 (CH2),
59.92 (OCH3), ar: [113.02 (2CH), 118.86 (CH), 122.40 (CH), 126.18 (2CH), 129.09 (CH),
129.15 (2CH), 137.27 (C), 151.14 (C), 164.34 (C)], 155.11 (thiadiazole C-2), 161.86
(oxadiazole C-2), 166.70 (thiadiazole C-5), 169.66 (CH), 178.11 (oxadiazole C-5). EI MS m/z
(%): 128 (50), 175 (100), 404 (69), 522 ([M+1]+, 59).
2.5.2.
3-(2-(5-((2-hydroxybenzylidene)amino)-1,3,4-thiadiazol-2-yl)ethyl)-5-((4-phenyl
piperazin-1-yl)methyl)-1,3,4-oxadiazole-2(3H)-thione (6b)
1
FT-IR (υmax, cm-1): 3044 (aromatic CH),1573 (C=N), 1271 (C=S). H NMR (DMSO-d6, δ
ppm): 2.57 (4H, s, 2CH2), 3.12 (2H, s, CH2), 3.59 (4H, s, 2CH2), 3.78 (2H, s, CH2), 4.52 (2H,
s, CH2), 6.71 (2H, d, J= 4.0 Hz, arH), 6.74 (1H, s, arH), 6.82 (1H, d, J= 4.0 Hz, arH), 6.92-
7.01 (2H, m, arH), 7.11-7.18 (2H, m, arH), 7.47-7.51 (1H, m, arH), 9.17 (1H, s, CH), 11.27
(1H, s, OH). 13C NMR (DMSO-d6, δ ppm): 29.81 (CH2), 43.14 (CH2), 46.68 (CH2), 52.64
(2CH2), 59.89 (2CH2), ar: [113.05 (CH), 116.71 (2CH), 120.83 (C), 126.18 (CH), 127.02
(CH), 127.41 (CH), 128.59 (2CH), 133.48 (CH), 151.04 (C), 158.65 (C)], 155.12 (thiadiazole
C-2), 162.13 (oxadiazole C-2), 166.25 (thiadiazole C-5), 169.53 (CH), 178.69 (oxadiazole C-
5). EI MS m/z (%):102.27 (53), 323.32 (76), 327.35 (35), 447.60 (100), 448.48 (29).

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2.5.3. 3-(2-(5-((3-hydroxy-4-methoxybenzylidene)amino)-1,3,4-thiadiazol-2-yl)ethyl)-5-
((4-phenylpiperazin-1-yl)methyl)-1,3,4-oxadiazole-2(3H)-thione (6c)
1
FT-IR (υmax, cm-1): 3125 (OH), 3024 (aromatic CH), 1587 (C=N), 1236 (C=S). H NMR
(DMSO-d6, δ ppm): 2.79 (4H, t, J= 8.0 Hz, 2CH2), 2.97 (2H, q, J= 12.0 Hz, CH2), 3.23 (4H, t,
J= 16.0 Hz, 2CH2), 3.49 (2H, s, CH2), 3.98 (3H, s, OCH3), 4.36 (2H, q, J= 16.0 Hz, CH2),
6.84 (1H, d, J= 8.0 Hz, arH), 6.89 (1H, d, J= 8.0 Hz, arH), 6.90-6.99 (3H, m, arH), 7.28 (1H,
13
t, J= 16.0 Hz, arH), 7.43 (2H, d, J= 8.0 Hz, arH), 8.74 (1H, s, CH), 9.84 (1H, s, OH). C
NMR (DMSO-d6, δ ppm): 27.29 (CH2), 46.51 (CH2), 47.70 (CH2), 51.51 (2CH2), 52.29
(2CH2), 56.98 (OCH3), ar: [109.95 (CH), 113.10 (CH), 126.17 (2CH), 127.87 (CH), 128.25
(CH), 129.13 (2CH), 132.53 (C), 150.72 (C), 152.44 (C), 155.10 (C)], 159.98 (thiadiazole C-
2), 162.83 (oxadiazole C-2), 163.77 (CH), 169.79 (thiadiazole C-5), 180.51 (oxadiazole C-5).
EI MS m/z (%): 560.39 (M+, 100).
2.5.4. 5-((4-phenylpiperazin-1-yl)methyl)-3-(2-(5-((pyridin-2-ylmethylene)amino)-1,3,4-
thiadiazol-2-yl)ethyl)-1,3,4-oxadiazole-2(3H)-thione (6d)
1
FT-IR (υmax, cm-1): 3042 (aromatic CH), 1598 (C=N), 1218 (C=S). H NMR (DMSO-d6, δ
ppm): 2.61 (4H, s, 2CH2), 3.12 (4H, d, J= 8.0 Hz, 2CH2), 3.32 (2H, s, CH2), 3.73 (2H, s,
CH2), 4.33 (2H, d, J= 8.0 Hz, CH2), 6.79 (1H, d, J= 8.0 Hz, arH), 6.92 (2H, t, J= 16.0 Hz,
arH), 7.13 (1H, s, arH), 7.21 (1H, s, arH), 7.70-7.84 (1H, m, arH), 7.93-8.05 (1H, m, arH),
8.32 (1H, s, CH). 13C NMR (DMSO-d6, δ ppm): 24.38 (CH2), 46.70 (CH2), 51.88 (2CH2),
52.62 (2CH2), 59.89 (CH2), ar: [118.73 (CH), 122.29 (CH), 126.21 (2CH), 127.63 (CH),
129.13 (2CH), 136.31 (CH), 148.81 (CH), 151.49 (C), 155.74 (C)], 157.69 (thiadiazole C-2),
161.95 (oxadiazole C-2), 167.38 (thiadiazole C-5), 169.59 (CH), 179.66 (oxadiazole C-5). EI
MS m/z (%): 106 (63), 175 (100), 285 (63), 301 (62), 315 (60), 369 (50), 493 ([M+1]+, 20).
2.5.5. 5-((4-phenylpiperazin-1-yl)methyl)-3-(2-(5-((pyridin-3-ylmethylene)amino)-1,3,4-
thiadiazol-2-yl)ethyl)-1,3,4-oxadiazole-2(3H)-thione (6e)
1
FT-IR (υmax, cm-1): 3065 (aromatic CH), 1599 (C=N), 1220 (C=S). H NMR (DMSO-d6, δ
ppm): 2.51 (4H, s, 2CH2), 3.18 (4H, s, 2CH2), 3.39 (2H, s, CH2), 3.66 (2H, s, CH2), 4.38 (2H,
t, J= 16.0 Hz, CH2), 6.85 (1H, t, J= 16.0 Hz, arH), 7.0 (2H, d, J= 8.0 Hz, arH), 7.25 (2H, t, J=
12.0 Hz, arH), 7.67 (1H, q, J= 12.0 Hz, arH), 7.77 (1H, q, J= 16.0 Hz, arH), 8.17 (1H, d, J=
8.0 Hz, arH), 8.28 (1H, d, J= 8.0 Hz, arH), 8.74 (1H, s, CH). 13C NMR (DMSO-d6, δ ppm):
26.08 (CH2), 46.49 (CH2), 51.82 (CH2), 52.45 (2CH2), 52.64 (2CH2), ar: [118.82 (2CH),
122.32 (CH), 126.13 (2CH), 129.59 (CH), 135.48 (CH), 137.45 (C), 150.76 (C), 151.37 (CH),

ACCEPTED MANUSCRIPT
154.45 (C)], 157.44 (thiadiazole C-2), 162.36 (oxadiazole C-2), 165.97 (CH), 169.52
(thiadiazole C-5), 180.30 (oxadiazole C-5). EI MS m/z (%): 478 (100), 482 (53), 493 ([M+2]+,
90), 494 (43), 512 (53).
2.5.6. 5-((4-phenylpiperazin-1-yl)methyl)-3-(2-(5-((pyridin-4-ylmethylene)amino)-1,3,4-
thiadiazol-2-yl)ethyl)-1,3,4-oxadiazole-2(3H)-thione (6f)
1
FT-IR (υmax, cm-1): 3097 (aromatic CH), 1599 (C=N), 1215 (C=S). H NMR (DMSO-d6, δ
ppm): 2.51 (4H, s, 2CH2), 3.23 (4H, s, 2CH2), 3.29 (2H, s, CH2), 4.0 (2H, s, CH2), 4.35 (2H, t,
J= 16.0 Hz, CH2), 6.82 (1H, t, J= 16.0 Hz, arH), 6.97 (2H, d, J= 8.0 Hz, arH), 7.24 (2H, d, J=
8.0 Hz, arH), 7.57 (1H, d, J= 8.0 Hz, arH), 7.83 (1H, d, J= 8.0 Hz, arH), 8.71-8.75 (2H, m,
arH), 8.90 (1H, s, CH). 13C NMR (DMSO-d6, δ ppm): 27.97 (CH2), 44.25 (CH2), 46.41
(CH2), 52.13 (2CH2), 53.28 (2CH2), ar: [113.00 (2CH), 126.13 (CH), 127.48 (CH), 128.87
(2CH), 136.85 (C), 144.65 (CH), 150.36 (C), 154.96 (CH), 156.06 (CH)], 154.96 (thiadiazole
C-2), 160.51 (oxadiazole C-2), 164.83 (CH), 168.10 (thiadiazole C-5), 174.88 (oxadiazole C-
5). EI MS m/z (%): 108 (100), 140 (43), 404 (32), 493 ([M+2]+, 25).
2.5.7. 3-(2-(5-((2,6-dichlorobenzylidene)amino)-1,3,4-thiadiazol-2-yl)ethyl)-5-((4-phenyl
piperazin-1-yl)methyl)-1,3,4-oxadiazole-2(3H)-thione (6g)
1
FT-IR (υmax, cm-1): 3054 (aromatic CH), 1586 (C=N), 1225 (C=S). H NMR (DMSO-d6, δ
ppm): 2.80 (4H, s, 2CH2), 2.96 (4H, s, 2CH2), 3.25 (2H, s, CH2), 3.73 (2H, d, J= 16.0 Hz,
CH2), 4.36 (2H, q, J= 20.0 Hz, CH2), 6.89-6.98 (3H, m, arH), 7.13 (1H, s, arH), 7.25-7.30
(4H, m, arH), 8.75 (1H, s, CH). 13C NMR (DMSO-d6, δ ppm): 26.45 (CH2), 46.67 (CH2),
51.49 (CH2), 51.98 (2CH2), 54.13 (2CH2), ar: [126.11 (2CH), 127.64 (CH), 128.31 (2CH),
128.48 (CH), 129.93 (2CH), 132.30 (C), 133.73 (C), 151.91 (C)], 152.88 (thiadiazole C-2),
155.00 (oxadiazole C-2), 164.15 (CH), 169.33 (thiadiazole C-5), 176.57 (oxadiazole C-5). EI
MS m/z (%): 153.17 (100), 188.91 (82), 205.28 (77), 307.22 (75), 582.39 ([M+Na]+, 37).
2.5.8.
3-(2-(5-((4-fluorobenzylidene)amino)-1,3,4-thiadiazol-2-yl)ethyl)-5-((4-phenyl
piperazin-1-yl)methyl)-1,3,4-oxadiazole-2(3H)-thione (6h)
1
FT-IR (υmax, cm-1): 3063 (aromatic CH), 1599 (C=N), 1222 (C=S). H NMR (DMSO-d6, δ
ppm): 2.51 (4H, s, 2CH2), 3.17 (4H, s, 2CH2), 3.39 (2H, s, CH2), 4.41 (2H, s, CH2), 4.62 (2H,
s, CH2), 6.90 (1H, t, J= 16.0 Hz, arH), 7.05 (3H, d, J= 8.0 Hz, arH), 7.28 (3H, t, J= 16.0 Hz,
arH), 7.45 (1H, t, J= 16.0 Hz, arH), 8.01 (1H, t, J= 16.0 Hz, arH), 9.99 (1H, s, CH). 13C NMR
(DMSO-d6, δ ppm): 28.33 (CH2), 42.30 (CH2), 46.39 (CH2), 52.10 (2CH2), 53.16 (2CH2), ar:

ACCEPTED MANUSCRIPT
[113.08 (2CH), 126.20 (2CH), 127.43 (2CH), 127.75 (CH), 128.91 (2CH), 137.64 (C), 144.81
(C), 161.75-164.18 (dC-F, J=243.0 Hz, C)], 155.02 (thiadiazole C-2), 160.63 (oxadiazole C-2),
162.80 (thiadiazole C-5), 169.19 (CH), 177.75 (oxadiazole C-5). EI MS m/z (%): 205.18
(100), 486.25 (18), 509.21 ([M]+, 22).
2.6. General method for the synthesis of compounds 7a-h
A mixture of compound 6 (10 mmol), triethylamine (5 mmol) and chloroacetyl chloride (5
mmol) were combined in dioxane and stirred in an ice bath (0-5 °C) for 10 h. The reaction
mixture was poured into ice-water, the resulting solid was filtered, dried and crystallized from
an appropriate solvent.
2.6.1.
3-Chloro-4-(4-methoxyphenyl)-1-(5-(2-(5-((4-phenylpiperazin-1-yl)methyl)-2-
thioxo-1,3,4-oxadiazol-3(2H)-yl)ethyl)-1,3,4-thiadiazol-2-yl)azetidin-2-one (7a)
1
FT-IR (υmax, cm-1): 3032 (aromatic CH), 1698 (C=O), 1600 (C=N), 1496 (C=S). H NMR
(DMSO-d6, δ ppm): 2.37 (4H, t, J=12.0 Hz, 2CH2), 2.93 (2H, s, CH2), 3.28 (4H, t, J=8.0,
2CH2), 3.42 (2H, s, CH2), 3.74 (3H, s, OCH3), 4.24 (2H, s, CH2), 4.74 (1H, s, CH), 5.04 (1H,
s, CH), 6.97 (2H, d, J=8.0 Hz, arH), 7.36 (1H, s, arH), 7.40 (2H, d, J= 8.0 Hz, arH), 7.53-7.57
(m, 2H, arH), 8.03 (2H, d, J= 8.0 Hz, arH). 13C NMR (DMSO-d6, δ ppm): 24.39 (CH2), 46.66
(CH2), 50.29 (CH2), 51.89 (2CH2), 53.83 (2CH2), 56.27 (OCH3), 60.13 (CH), 67.57 (CH), ar:
[113.13 (2CH), 116.39 (2CH), 121.35 (CH), 126.13 (2CH), 129.13 (2CH), 137.41 (C), 149.59
(C), 155.05 (C)], 145.07 (thiadiazole C-5), 161.29 (oxadiazole C-2), 166.48 (thiadiazole C-2),
168.90 (C=O), 180.51 (oxadiazole C-5). EI MS m/z (%): 313.25 (30), 462.27 (100), 637.23
([M+K]+, 57).
2.6.2.
3-Chloro-4-(2-hydroxyphenyl)-1-(5-(2-(5-((4-phenylpiperazin-1-yl)methyl)-2-
thioxo-1,3,4-oxadiazol-3(2H)-yl)ethyl)-1,3,4-thiadiazol-2-yl)azetidin-2-one (7b)
1
FT-IR (υmax, cm-1): 3026 (aromatic CH), 1703 (C=O), 1578 (C=N), 1458 (C=S). H NMR
(DMSO-d6, δ ppm): 2.41 (4H, s, 2CH2), 2.64 (2H, s, CH2), 3.29 (4H, s, 2CH2), 3.42 (2H, s,
CH2), 4.29 (2H, s, CH2), 4.98 (1H, s, CH), 5.45 (1H, s, CH), 6.98 (2H, d, J=8.0 Hz, arH), 7.04
(1H, d, J= 8.0 Hz, arH), 7.25 (1H, t, J= 12.0 Hz, arH), 7.44-7.48 (m, 2H, arH), 7.54 (2H, d, J=
13
7.0 Hz, arH), 7.67 (1H, d, J= 8.0 Hz, arH), 9.62 (1H, s, OH). C NMR (DMSO-d6, δ ppm):
21.26 (CH2), 50.82 (CH2), 53.24 (2CH2), 55.49 (2CH2), 59.894(CH), 62.11 (CH), ar: [119.17
(2CH), 120.58 (CH), 123.89 (2CH), 125.42 (CH), 127.67 (C), 130.50 (2CH), 131.67 (CH),
153.23 (C), 157.12 (C)], 144.84 (thiadiazole C-5), 163.21 (oxadiazole C-2), 167.43

ACCEPTED MANUSCRIPT
(thiadiazole C-2), 171.47 (C=O), 180.12 (oxadiazole C-5). EI MS m/z (%): 606.24 (100),
608.25 ([M+1]+, 83), 609.32 (24).
2.6.3.
3-Chloro-4-(3-hydroxy-4-methoxyphenyl)-1-(5-(2-(5-((4-phenylpiperazin-1-
yl)methyl)-2-thioxo-1,3,4-oxadiazol-3(2H)-yl)ethyl)-1,3,4-thiadiazol-2-yl)azetidin-2-one
(7c)
FT-IR (υmax, cm-1): 3319 (-OH), 3063 (aromatic CH), 1678 (C=O), 1595 (C=N), 1245 (C=S).
1H NMR (DMSO-d6, δ ppm): 2.41 (4H, s, 2CH2), 2.87 (2H, s, CH2), 3.16 (4H, s, 2CH2), 3.54
(2H, s, CH2), 3.81 (3H, s, OCH3), 4.31 (2H, s, CH2), 5.19 (1H, s, CH), 5.41 (1H, s, CH), 6.30
(1H, bs, OH), 6.78 (1H, t, J=12.0 Hz, arH), 6.92-6.95 (3H, m, arH), 7.20-7.25 (3H, m, arH),
7.27-7.35 (1H, m, arH). 13C NMR (DMSO-d6, δ ppm): 26.86 (CH2), 46.71 (CH2), 51.51
(CH2), 52.71 (2CH2), 55.21 (2CH2), 57.67 (OCH3), 59.95 (CH), 66.08 (CH), ar: [113.11
(CH), 117.61 (CH), 119.60 (2CH), 122.22 (2CH), 126.19 (2CH), 137.31 (C), 143.04 (C),
150.90 (C)], 145.19 (thiadiazole C-5), 160.05 (oxadiazole C-2), 166.18 (thiadiazole C-2),
169.63 (C=O), 178.40 (oxadiazole C-5). EI MS m/z (%): 556.21 (100), 556.47 (91), 600.33
(22), 614.42 (M+, 26).
2.6.4.
3-Chloro-1-(5-(2-(5-((4-phenylpiperazin-1-yl)methyl)-2-thioxo-1,3,4-oxadiazol-
3(2H)-yl)ethyl)-1,3,4-thiadiazol-2-yl)-4-(pyridin-2-yl)azetidin-2-one (7d)
1
FT-IR (υmax, cm-1): 3061 (aromatic CH), 1681 (C=O), 1587 (C=N), 1239 (C=S). H NMR
(DMSO-d6, δ ppm): 2.59 (4H, s, 2CH2), 2.82 (2H, s, CH2), 3.13 (4H, s, 2CH2), 3.46 (2H, s,
CH2), 4.12 (2H, s, CH2), 5.09 (1H, s, CH), 5.38 (1H, s, CH), 6.77 (1H, t, J=16.0 Hz, arH),
6.91 (2H, d, J=8.0 Hz, arH), 7.02 (2H, d, J= 12.0 Hz, arH), 7.20 (2H, t, J=12.0 Hz, arH), 8.05
(2H, d, J= 12.0 Hz, arH). 13C NMR (DMSO-d6, δ ppm): 26.71 (CH2), 46.38 (CH2), 49.78
(CH2), 52.12 (2CH2), 52.97 (2CH2), 58.92 (CH), 66.05 (CH), ar: [116.13 (2CH), 119.86 (CH),
121.35 (CH), 126.13 (CH), 128.92 (2CH), 138.45 (CH), 151.22 (C), 153.17 (CH), 165.14
(C)], 147.95 (thiadiazole C-5), 161.64 (oxadiazole C-2), 166.59 (thiadiazole C-2), 169.96
(C=O), 176.58 (oxadiazole C-5). EI MS m/z (%): 101.13 (36), 554.32 (100), 570.28 ([M+1]+,
48).
2.6.5.
3-Chloro-1-(5-(2-(5-((4-phenylpiperazin-1-yl)methyl)-2-thioxo-1,3,4-oxadiazol-
3(2H)-yl)ethyl)-1,3,4-thiadiazol-2-yl)-4-(pyridin-3-yl)azetidin-2-one (7e)
1
FT-IR (υmax, cm-1): 3019 (aromatic CH), 1702 (C=O), 1579 (C=N), 1460 (C=S). H NMR
(DMSO-d6, δ ppm): 2.44 (4H, s, 2CH2), 2.68 (2H, s, CH2), 3.22 (4H, s, 2CH2), 3.52 (2H, s,

ACCEPTED MANUSCRIPT
CH2), 4.23 (2H, s, CH2), 5.06 (1H, s, CH), 5.41 (1H, s, CH), 6.97 (2H, d, J=12.0 Hz, arH),
7.27 (3H, d, J=8.0 Hz, arH), 7.34 (2H, t, J= 12.0 Hz, arH), 8.04 (2H, d, J= 12.0 Hz, arH). 13C
NMR (DMSO-d6, δ ppm): 25.60 (CH2), 46.66 (CH2), 51.89 (CH2), 52.46 (2CH2), 53.35
(2CH2), 58.72 (CH), 67.64 (CH), ar: [116.73 (2CH), 119.09 (CH), 126.09 (CH), 127.65 (CH),
129.49 (CH), 134.03 (CH), 141.60 (C), 147.89 (CH), 148.94 (CH), 152.54 (C)], 145.29
(thiadiazole C-5), 161.93 (oxadiazole C-2), 166.57 (thiadiazole C-2), 169.18 (C=O), 176.61
(oxadiazole C-5). EI MS m/z (%): 168.84 (100), 205.15 (96), 360.51 (46), 570.31 ([M+1]+,
68).
2.6.6.
3-Chloro-1-(5-(2-(5-((4-phenylpiperazin-1-yl)methyl)-2-thioxo-1,3,4-oxadiazol-
3(2H)-yl)ethyl)-1,3,4-thiadiazol-2-yl)-4-(pyridin-4-yl)azetidin-2-one (7f)
1
FT-IR (υmax, cm-1): 3030 (aromatic CH), 1701 (C=O), 1579 (C=N), 1496 (C=S). H NMR
(DMSO-d6, δ ppm): 2.39 (4H, s, 2CH2), 2.84 (2H, s, CH2), 3.26 (4H, s, 2CH2), 3.54 (2H, s,
CH2), 4.20 (2H, s, CH2), 4.93 (1H, s, CH), 5.95 (1H, s, CH), 6.92 (1H, d, J=12.0 Hz, arH),
13
7.37-7.40 (7H, m, arH), 8.01 (1H, d, J= 8.0 Hz, arH). C NMR (DMSO-d6, δ ppm): 25.93
(CH2), 47.78 (CH2), 51.74 (CH2), 52.03 (2CH2), 54.05 (2CH2), 60.49 (CH), 70.14 (CH), ar:
[117.77 (2CH), 121.64 (CH), 124.58 (2CH), 128.89 (2CH), 148.22 (C), 151.31 (2CH), 154.94
(C)], 145.48 (thiadiazole C-5), 161.70 (oxadiazole C-2), 166.65 (thiadiazole C-2), 169.62
(C=O), 178.69 (oxadiazole C-5). EI MS m/z (%): 205.21 (42), 447.54 (38), 606.24 (100),
608.38 ([M+K]+, 69).
2.6.7.
3-Chloro-4-(2,6-dichlorophenyl)-1-(5-(2-(5-((4-phenylpiperazin-1-yl)methyl)-2-
thioxo-1,3,4-oxadiazol-3(2H)-yl)ethyl)-1,3,4-thiadiazol-2-yl)azetidin-2-one (7g)
1
FT-IR (υmax, cm-1): 3058 (aromatic CH), 1681 (C=O), 1588 (C=N), 1239 (C=S). H NMR
(DMSO-d6, δ ppm): 2.52 (4H, s, 2CH2), 2.87 (2H, s, CH2), 3.41 (4H, s, 2CH2), 3.69 (2H, s,
CH2), 4.93 (2H, s, CH2), 5.71 (1H, s, CH), 6.02 (1H, s, CH), 6.98 (2H, d, J=8.0 Hz, arH), 7.39
13
(4H, t, J=20.0 Hz, arH), 8.03 (2H, d, J= 8.0 Hz, arH). C NMR (DMSO-d6, δ ppm): 24.56
(CH2), 46.72 (CH2), 48.65 (CH2), 50.29 (2CH2), 52.35 (2CH2), 59.98 (CH), 62.18 (CH), ar:
[115.99 (2CH), 121.71 (CH), 126.17 (2CH), 129.38 (2CH), 132.59 (CH), 134.46 (2C), 135.90
(C), 151.36 (C)], 145.18 (thiadiazole C-5), 160.50 (oxadiazole C-2), 165.27 (thiadiazole C-2),
168.34 (C=O), 179.61 (oxadiazole C-5). EI MS m/z (%): 191.93 (40), 239.95 (55), 242.02
(60), 443.26 (73), 613.35 (52), 637.27 (M+, 100).

ACCEPTED MANUSCRIPT
2.6.8. 3-Chloro-4-(4fluorophenyl)-1-(5-(2-(5-((4-phenylpiperazin-1-yl)methyl)-2-thioxo-
1,3,4-oxadiazol-3(2H)-yl)ethyl)-1,3,4-thiadiazol-2-yl)azetidin-2-one (7h)
1
FT-IR (υmax, cm-1): 3065 (aromatic CH), 1696 (C=O), 1582 (C=N), 1238 (C=S). H NMR
(DMSO-d6, δ ppm): 2.65 (4H, s, 2CH2), 2.96 (2H, s, CH2), 3.16 (4H, s, 2CH2), 3.52 (2H, s,
CH2), 4.31 (2H, s, CH2), 4.92 (1H, s, CH), 5.26 (1H, s, CH), 7.05 (2H, d, J=8.0 Hz, arH), 7.16
(1H, s, arH), 7.19 (2H, d, J= 8.0 Hz, arH), 7.42 (2H, s, arH), 7.78 (2H, d, J= 8.0 Hz, arH). 13C
NMR (DMSO-d6, δ ppm): 25.81 (CH2), 46.29 (CH2), 48.65 (CH2), 50.28 (2CH2), 52.03
(2CH2), 60.61 (CH), 67.41 (CH), ar: [115.98 (2CH), 118.59 (CH), 121.60 (CH), 126.17 (CH),
127.13 (CH), 128.89 (CH), 129.39 (2CH), 137.36 (C), 151.31 (C), 159.47-161.72 (dC-F,
J=245.0 Hz, C)], 144.15 (thiadiazole C-5), 164.30 (oxadiazole C-2), 167.64 (thiadiazole C-2),
169.81 (C=O), 179.62 (oxadiazole C-5). EI MS m/z (%): 120 (67), 213 (55), 314 (40), 452
(31), 587.42 ([M+1]+, 100), 588 ([M+2]+, 52).
3. Results and Discussion
3.1. Chemistry
1,3,4-thiadiazole-β-lactam hybrid compounds 7a-h have been prepared according to the
synthetic procedure introduced in Scheme 1. Compounds 1 and 2 were synthesized with the
known substitution reactions, separately with ethyl bromoacetate and hydrazine hydrate.
Compound 3, containing 1,3,4-oxadiazole ring, was obtained by cyclization of compound 2
with CS2 in basic media. 3-(5-((4-phenylpiperazin-1-yl)methyl)-2-thioxo-1,3,4-oxadiazol-
3(2H)-yl)propan nitrile (4) was synthesized by the reaction of compound 3 and acrylonitrile.
The CH2 signals in the structure formed by the addition of the acrylonitrile group to 1,3,4-
1
oxadiazole were observed between 3.12 and 4.28 ppm in H NMR spectrum. The quaternary
carbon atom of –CN group was resonated in 119.43 ppm in 13C NMR spectrum. These
findings approved the formation of the compound 4. Compound 5, comprising a 1,3,4-
thiadiazol-5-amino ring, was achieved by reacting compound 4 with thiosemicarbazide in the
presence of trifluoroacetic acid, as a catalyst. The appearance of -NH2 group was shown in
1
7.27 ppm in H NMR spectrum and this supported the formation of the thiadiazole ring of
compound 4 with thiosemicarbazide. In addition, the absence of nitrile carbon in the 13C
NMR spectrum and the resonance of the 1,3,4-thiadiazole C-2 and C-2 carbons at the relevant
sites promoted the compound 5.
The free amino group of compound 5 in 1,3,4-thiadiazole ring at C-2 position, were reacted
with various aldehydes, and corresponding Schiff base derivatives (6a-h) were obtained. The

ACCEPTED MANUSCRIPT
disappearance of amino group in the FT-IR spectrum for compounds 6a-h and the resonance
of the CH protons in the imine group (-N=CH) between 8.32 and 9.17 ppm showed that the
structure of Schiff bases was formed. It is interesting to note that the compounds having imine
group may exist as E/Z geometrical isomers due to the C=N double bond and as cis/trans
amide conformers [41-43]. According to the previous studies [42], in dimethyl-d6 sulfoxide
solution, this kind of compounds are present in higher percentage in the form of geometrical E
isomer about C=N double bond. Another evidence for supporting the formation of compounds
13
6a-h, carbon atom signals of HC=N were monitored between 163.87 and 169.66 ppm in C
NMR spectrum. Schiff base derivatives of compounds 6a-h were reacted by dropwise
addition of chloroacetyl chloride in an ice bath in the presence of triethylamine in dioxane
solvent to afford the target compounds of β-lactam derivatives of compounds 7a-h (Scheme
1). The protons in the imine group disappeared and the protons on the carbon atom to which
the Cl atom bound in the beta-lactam were resonated between 5.04 and 6.02 ppm in 1H NMR
spectrum. Meanwhile, this carbon atom and carbonyl carbon atom appeared in 62.11-70.14
and 168.34-171.47 ppm in 13C NMR, respectively.
Table 1. Reaction yields, melting points and crystallization solvents of all compounds.
Compound Yield
(%)
Melting
Point (oC)
178-179
145-146
155-156
178-179
181-182
188-189
200-201
175-177
189-190
205-206
165-166
202-203
205-206
166-167
175-176
199-200
160-161
181-182
192-193
Crystallization
Solvent
EtOH
86
82
56
79
89
81
83
78
82
76
69
64
68
72
58
65
70
73
66
3
4
5
EtOH
Acetone
EtOH
EtOH
EtOH
6a
6b
6c
6d
6e
6f
6g
6h
7a
7b
7c
7d
7e
7f
MeOH
MeOH:H2O (1:3)
MeOH:H2O (1:3)
EtOH
MeOH
Acetone
EtOH
EtOH
MeOH
MeOH
Acetone
EtOH
7g
7h
EtOH
3.2. Biological Activity
3.2.1. Antioxidant Capacity

ACCEPTED MANUSCRIPT
DPPH (2.2-diphenyl-1-picrylhydrazyl) radical scavenging activity was performed the
synthesized compounds with different chemicals was determined using the free radical DPPH
(2.2-diphenyl-1-picrylhydrazyl), as described by Blois [50]. A 100-ꢀL: chemical solution was
mixed with 1 mL of freshly prepared methanolic DPPH solution. The reaction mixture was
incubated for 30 min at room temperature in the dark and was then measured at 520 nm. The
activity was expressed as ꢀmol Trolox equivalent. FRAP (the ferric reducing ability of
plasma) was measured using the method described by Benzie & Strain [51] with some
modification. To 100 ꢀL of each sample was added 2900 ꢀL freshly prepared FRAP reagent
containing 300 mmol/L acetate buffer (pH 3.6), 10 mmol/L TPTZ (2,4,6-tripyridyle-s-
triazine) and 20 mmol/L FeCl.6H2O in proportions of 10:1:1 (v/v/v). The mixture was
incubated for 30 min at 37 ºC and measured at 593 nm. The values were given as ꢀmol of
Trolox/g. CUPRAC (cupric ion reducing antioxidant capacity) was measured following the
procedure described by Apak et al. [52] with some modification. Briefly, 100 ꢀL of each
chemical solution was mixed with 900 ꢀL bidistilled water, 1 mL acetate buffer solution (1
mmol/L, pH: 7.0), 1 mL CuCl2 (10 mmol/L) and 1 mL 7.5 mmol/L neocuproine to a final
volume of 4 mL. The reaction mixture was then incubated in the dark for 30 min at room
temperature and the absorbance of the reaction mixture was measured at 450 nm against a
water blank. Trolox was used as the standard calibration curves and the results were expressed
as ꢀmol Trolox equivalent per g.
DPPH, FRAP and CUPRAC assays were performed to determine the antioxidant capacity
(AC, µmol TE/g) of the synthesized compounds. All compounds displayed good-moderate
antioxidant activity (Table 2). Trolox was used as standard for DPPH radical scavenging
method and the results are given SC50 value. According to DPPH method, 6a, 6e and 6h
exhibited good activity with 0,12 ± 0,01, 0,26 ± 0,01 and 0,38± 0,01 SC50 values,
respectively. On the other hand, 7b (7529.53 ± 2.57 µmol TE/g) and 6e (7175.01 ± 20.00
µmol TE/g) for CUPRAC, 7b (4,453 ± 0,006) for FRAP showed the highest AC values
among the synthesized compounds (Table 2).
Table 2. Antioxidant capacity of newly synthesized compounds
Compound
No
CUPRAC
(µmol TE/g)
DPPH
(mg/mL) SC50
FRAP
(µmol TE/g)
1986.28±18.56
29.54±2.57
2431.99±16.10
3950.34±16.25
365.14±2.36
3,24±0,01
7,85±0,02
3,18±0,04
0,12±0,01
3,28±0,02
1,269±0,002
0,434±0,006
0,459±0,004
4,557±0,021
0,136±0,003
3
4
5
6a
6b

ACCEPTED MANUSCRIPT
348.64±26.00
5,06±0,01
2,61±0,02
0,26±0,01
1,18±0,01
3,18±0,04
0,38±0,01
4,29±0,03
0,81±0,00
1,18±0,01
2,13±0,02
3,23±0,06
1,79±0,05
2,13±0,03
4,29±0,01
0,04±0,00
0,947±0,016
1,912±0,032
1,710±0,056
0,421±0,009
0,459±0,004
0,564±0,004
1,119±0,030
4,453±0,006
0,828±0,018
0,486±0,020
0,592±0,000
0,673±0,004
0,218±0,001
1,229±0,016
6c
6d
6e
6f
6g
6h
7a
7b
7c
7d
7e
7f
7g
875.41±10.00
7175.01±20.00
123.97±4.61
431.98± 16.10
1843.56±3.00
1986.11±23.42
7529.53±2.57
756.14±10.00
1745.12± 1.00
1954.34± 4.58
1265.29±2.00
1048.11±6.00
1278.35±14.00
7h
Trolox
3.2.2. Urease Inhibitory Activity [53]
Urease activity was defined by using Weatherburn process. Reaction mixtures including 25
µL of Jack Bean urease, 55 µL of buffer (0.01 mol/L K2HPO4, 1 mmol/L EDTA and 0.01
mol/L LiCl, pH 8.2) and 10 mmol/L urea were incubated with 5 µL of the test compounds at
room temperature for 15 min in microtiter plates. The production of ammonia was measured
following the indophenol method and was used to determine the urease inhibitory activity.
The phenol reagent (45 µL, 1% w/v phenol and 0.005% w/v sodium nitroprusside) and alkali
reagent (70 µL, 0.5% w/v sodium hydroxide and 0.1% v/v NaOCl) were added to each well.
This mixture was incubated for 15 minutes more at 35 °C and optical density was measured at
625 nm against a blank solution including distilled water instead of enzyme. For the
determination of the IC50 value of the extracts, activity assays were conducted at five different
extract concentration and dose response curve was generated. Thiourea was used as standard
inhibitor.
The obtained compounds were examined for their in vitro urease inhibitory activity against
Jack bean urease. All the compounds displayed range from good to excellent activity
compared to thiourea used as standard drug. Especially, among the compounds 6a, 7b and 7f
displayed excellent inhibition with 0.98 ± 0.03; 1.15 ± 0.00 and 1.54 ± 0.01 IC50 values,
respectively (Table 3).
Table 3. Inhibitory activities of the synthesized compounds against Jack bean urease
Compound
Antiurease
IC50 (mg/mL)
6.87 ± 0.01
Compound
Antiurease
IC50 (mg/mL)
3.18 ± 0.02
No
3
No
6h

ACCEPTED MANUSCRIPT
8.54 ± 0.02
3.45 ± 0.04
0.98 ± 0.03
2.54 ± 0.02
3.72 ± 0.01
6.43 ± 0.02
5.62 ± 0.04
3.27 ± 0.02
3.45 ± 0.04
2.55 ± 0.03
4
5
7a
7b
7c
7d
7e
7f
7g
1.15 ± 0.00
4.75 ± 0.01
5.36 ± 0.02
2.85 ± 0.02
1.54 ± 0.01
5.84 ± 0.03
6.54 ± 0.01
12,02 ± 0,06
6a
6b
6c
6d
6e
6f
6g
7h
Thiourea
3.3. ADME Prediction
In silico ADME study, which is one of the computational calculations, was applied for the
synthesized compounds. In this study, we calculated molecular volume (MV), molecular
weight (MW), logarithm of partition coefficient (miLogP), number of hydrogen bond
acceptors (n-ON), number of hydrogen bonds donors (n-OHNH), topological polar surface
area (TPSA), number of rotatable bonds (n-ROTB) and Lipinski’s rule of five [45] using
Molinspiration online property calculation toolkit [45]. Absorption (% ABS) was calculated
by: % ABS =109 x(0.345xTPSA) [46]. From all these parameters (Table 4), all synthesized
compounds exhibited a good % ABS (75.73–92.33%). It was monitored that none of the
compound violated Lipinski’s rule of five except for 7c (Table 4). It violated due the number
of oxygen and nitrogen atoms in the structure and molecular weight. It can be suggested that
other compounds may be developed as good drug candidates. A molecule probably to be
evolved as an orally active drug candidate should exhibit no more than one violation of the
following four criteria: logP (octanol–water partition coefficient) ≤5, molecular weight ≤500,
number of hydrogen bond acceptors ≤10 and number of hydrogen bond donors ≤5 [47]. Since
the obtained compounds (except for 7c) followed this rule for orally active drug and therefore
can be promoted as oral drug candidates.
Table 4. Pharmacokinetic parameters for good oral bioavailability of synthesized compound
3-7.
Entry
%
ABS
TPSA
n-
MV
MW miLog
n-ON
acceptors OHNH
donors
n-
Nviolations
(A2) ROTB
P
-
-
-
3
5
6
9
8
12
11
-
≤500
≤5
≤10
5
6
8
9
9
10
9
≤5
1
0
2
0
1
2
1
≤1
0
0
0
1
1
1
0
92.33 48.30
87.88 61.23
78.21 89.25
79.74 84.82
76.21 95.02
69.90 113.34
77.16 97.27
244.45 276.37 0.62
295.29 329.43 0.83
343.53 403.54 1.11
452.45 521.67 3.22
434.93 507.64 3.10
453.89 525.66 2.40
416.17 480.62 1.89
3
4
5
6a
6b
6c
6d

ACCEPTED MANUSCRIPT
77.16 97.27
77.16 97.27
79.89 84.38
79.89 84.38
76.99 92.77
73.20 103.76
70.02 113.00
75.73 96.43
75.73 96.43
75.73 96.43
80.18 83.53
80.18 83.53
11
11
11
11
9
8
9
8
8
416.17 480.62 1.82
416.17 480.62 1.77
447.40 548.52 4.32
425.26 497.62 3.22
497.31 598.15 3.15
479.78 584.13 3.03
505.33 614.15 2.43
467.61 569.12 1.92
467.61 569.12 1.85
467.61 569.12 1.80
498.83 637.02 4.35
476.69 586.12 3.25
9
9
8
1
1
1
1
0
1
1
0
0
0
0
0
0
0
1
0
1
1
2
1
1
1
1
1
6e
6f
6g
6h
7a
7b
7c
7d
7e
7f
8
10
10
11
10
10
10
9
8
8
8
7g
7h
9
3.4. Molecular Docking Studies
To investigate possible binding interactions as well as to support the in vitro activity of the
studied compounds, molecular docking studies were performed using Maestro Molecular
Modeling platform (version10.5) by Schrödinger [48] for all newly synthesized β-lactam
derivatives. The protocol of docking study was given in Supplementary File.
The most active compounds (6a, 7b and 7f) were further investigated to clarify the probable
binding modes and provide straightforward knowledge for binding interactions of the protein-
ligand complexes. The active site residues of the Jack bean urease (JBU) consist of His407,
His409, Arg439, Ala440, Lys490, His492, Asp494, His519, His545, Cys592, His593, His594,
Arg609, Asp633, Ala636, and Met637 [49]. All compounds showed better docking scores
ranging from -5.33 kcal/mol to -9.21 kcal/mol than the reference inhibitor thiourea in Table 5.
Table 5. Docking scores, Lipophilic contact plus phobic attractive term, Hydrogen-bonding
term and polar interactions term in the active site
Comp. Docking
Glide
Lipo
Glide
Hbond Esite
Glide
Key Interacting Residues
No
Score
H-Bond
Ala636
Pi-Pi Stacking
3
4
-5.329
-5.943
-5.984
-8.856
-7.192
-7.6
-0.667
-2.419
-2.355
-3.673
-2.441
-2.922
-1.534
-0.32 -0.1
Arg609
0
-0.103
-
Arg439
5
-0.14 -0.307
Asp633, Arg439
Cys592, His593
Gly638, Gln635
Thr571, Asp633
Arg609, Cys592
His593
-0.101
0
His594, His593
6a
6b
6c
6d
-0.325
-0.456
-
-0.303 -0.299
-0.32 -0.331
-
-5.862
His594, Arg609

ACCEPTED MANUSCRIPT
6e
6f
-7.81
-7.342
-7.332
-6.595
-9.199
-9.208
-8.569
-7.279
-7.85
-3.249
-2.531
-3.695
-1.222
-4.479
-0.467
-1.003
-2.705
-3.443
-4.136
-3.971
-3.884
0
0
-0.173
-
His585
Arg439
-0.379 -0.095
Cys592, Gly638
6g
-0.176
-0.372
0
0
His593
Arg639
6h
0
Arg639, Met637
Arg439
7a
-0.104
-0.237
-
His585
Ala440, Arg439
-
7b
-0.292
7c
-0.247 -0.103
-0.429 -0.138
Cys592, Ala636
Arg439
7d
Cys592, Arg439,
Arg609, His594
His593
7e
0
-0.179
-0.023
-0.021
-0.037
-
Arg609, Thr571
His593
7f
-8.111
-7.945
-7.541
-4.981
-0.589
7g
0
0
-
His545, His585
His593, His585
-
7h
-
Thiourea
-0.49 -0.377
Gly550, His409
According to the docking studies, the top scoring poses and ligand interaction diagrams of
most active compounds were shown in Figure 1. The compound 6a forms a strong hydrogen
bond between the His593 residue and the nitrogen of the thiadiazole ring (NH–N) at distance
of 2.31 Å. The Cys592 (marked as CME592), which located at the mobile flap covering the
active site is forming hydrogen bond with the oxygen of phenoxy moiety (OH–O) at distance
of 2.10 Å. The pi-pi stacking interactions with the amino acid residues of His592 and His593
through the phenoxy ring and thiadiazole of 6a was also observed in protein-ligand complex
in Figure 1. The phenoxy ring and thiadiazole moiety of 6a were embedded in an exposed
solvent area as well. The best orientation of compound 7b was located the active site through
two hydrogen bonds with amino acid residues Arg439 and Ala440. The phenol ring of
compound 7b was embedded towards the Ni+2 cations of JBU. It was clearly observed that
compound 7f formed two hydrogen bond interactions with Arg609 and Thr571 residues and
formed pi-pi stacking interaction with His593 residue of JBU. The 7f-enzyme complex
showed that Z-shape form of ligand penetrated toward the catalytic site of enzyme and located
within binding pocket of urease. The benzyl group of compound 7f was extended with a
displacement towards the exposed solvent region. Similar interactions could be seen for the
other newly synthesized β-lactam derivatives with docking scores, lipophilic contact plus

ACCEPTED MANUSCRIPT
phobic attractive term, Hydrogen-bonding term and polar interactions term of Glide Scores in
Table 5.
Figure 1. Schematic view and 3D top-docking poses of compound 6a, 7b and 7f in the active
sites of JBU

ACCEPTED MANUSCRIPT
According to in silico results, the compounds containing β-lactam ring had better docking
score than their analog compounds without β-lactam ring. It is mostly due to fact that β-
lactam ring was gained to molecules two more rotatable bonds and one more hydrogen bond
acceptor so that the compounds containing β-lactam ring had more strong binding interaction.
3. Conclusion
In summary we report here the synthesis of some new kind of β-lactam derivatives containing
azole rings such as oxadiazole and thiadiazole with phenyl piperazine ring in one molecule.
And investigate their antiurease activity and antioxidant capacity comparing with their
molecular docking studies. And beside this compound 6a, which is a Schiff base containing a
4-methoxyphenyl group, compound 7b which is a β-lactam derivative containing 2-
hydoxyphenyl group and compound 7f contains a 4-pyridine ring showed excellent antiureas
activity and have excellent antioxidant capacity.
Acknowledgement
The support was provided by Scientific and Technological Research Council of Turkey
(TUBITAK, No. 115Z770).
Conflicts of interest
There are no conflicts to declare
.
References
[1] F. Broccolo, G. Cainelli, G. Caltabiano, C.E.A. Cocuzza, C.G. Fortuna, P. Galletti, D.
Giacomini, G. Musumarra, R. Musumeci, A. Quintavalla, J. Med. Chem. 49 (2006),
2804.
[2] A.K. Halve, D. Bhadauria, R. Dubey, Bioorg. Med. Chem. Lett. 17 (2007) 341.
[3] S.B. Rosenblum, T. Huynh, A. Afonso, HR. Jr. Davis, N. Yumibe, J.W. Clader, D.A.
Burnett, J. Med. Chem. 41 (1998) 973.
[4] A.D. Borthwick, G. Weingarte, T.M. Haley, M. Tomaszewski, W. Wang, Z. Hu, J.
Bedard, H. Jin, L. Yuen, T.S. Mansour, Bioorg. Med. Chem. Lett. 8 (1998) 365.
[5] W.T. Han, A.K. Trehan, J.J.K. Wright, M.E. Federici, S.M. Seiler, N.A. Meanwell,
Bioorg. Med. Chem. 3 (1995) 1123.
[6] R.K. Goel, M.P. Mahajan, S.K.J. Kulkarni, Pharm. Pharm. Sci. 7 (2004) 80.

ACCEPTED MANUSCRIPT
[7] G. Veinberg, I. Shestakova, M. Verma, I. Kenepe, E. Lukevics, Bioorg. Med. Chem. Lett.
14 (2004) 147.
[8] T. Sperka, J. Pitlik, P. Bagossi, J. Tozser, Bioorg. Med. Chem. Lett. 15 (2005) 3086.
[9] C. Saturnino, B. Fusco, P. Saturnino, G. De Martino, F. Rocco, J.C. Lanceolat, Biol.
Pharm. Bull. 23 (2000) 654.
[10] (a) M.I. Konaklieva, Curr. Med. Chem.-Anti-Infect. Agents 1 (2002) 215; (b) A.
Clemente, A. Domingos, A.P. Grancho, J. Iley, R. Moreira, J. Neres, N. Palma, A.B.
Santana, E. Valente, Bioorg. Med. Chem. Lett. 11 (2001) 1065.
[11] G.S. Singh, M. D’hooghe, N. De Kimpe, Azetidines, azetines and azetes: monocyclic. In:
Stevens CV, ed. Comprehensive Heterocyclic Chemistry III. Oxford, UK: Elsevier, 1
(2008) vol. 2.
[12] J.C. Walton. Top Curr. Chem. 264 (2006) 163.
[13] P.A. Magriotis. Angew. Chem. Int. Ed. 40 (2001) 4377.
[14] M. Gomez-Gallego, M.J. Mancheno, A.M. Sierra. Tetrahedron. 56 (2000) 5743.
[15] R.G. Soengas, Y. Segade, C. Jimenez, J. Rodríguez. Tetrahedron. 67 (2011) 2617.
[16] E. Ghabraie, S. Balalaie, S. Mehrparvar, F. Rominger. J Org. Chem. 17 (2014) 7926.
[17] R. Ocampo, J.W.R. Dolbier. Tetrahedron. 60 (2004) 9325.
[18] G.B. Bagihalli, P.G. Avaji, S.A. Patil, P.S. Badami. Eur. J Med. Chem. 43 (2008) 2639–
2649.
[19] T. Akhtar, S. Hameed, N.A. Al-Masoudi, K.M. Khan Heteroat. Chem. 18 (2007) 316–
322.
[20] M. Wujec, A. Siwek, M. Swatko-Ossor, L. Mazur, Z. Rzaczynska. J Heterocycl. Chem.
45 (2008) 1893–1896.
[21] Y. Li, J. Geng, Y. Liu, S. Yu, G. Zhao. ChemMedChem. 8 (2013) 27.
[22] S. Haider, M.S. Alam, H. Hamid. Eur. J Med. Chem. 92 (2015) 156.
[23] A.K. Jain, S. Sharma, A. Vaidya, V. Ravichandran, R.K. Agrawal. Chem. Biol. Drug.
Des. 81 (2013) 557.
[24] Y. Hu, C-Y. Li, X-M. Wang, Y-H. Yang, H-L. Zhu. Chem. Rev. 114 (2014) 5572.
[25] J. Matysiak. Mini-Rev. Med. Chem. 15 (2015) 762.
[26] M.S. Chande, A.A. Godbole, E. Coutinho, P. Desai, Ind. J. Chem. 42B (2004) 397.
[27] M. Yar, M. Bajda, R.A. Mehmood, L.R. Sidra, N. Ullah, L. Shahzadi, Lett. Drug. Des.
Discov. 11 (2014) 331–338.
[28] M. Amir, S. Shahani, Ind. J. Heterocycl. Chem. 8 (1998) 107–110.
[29] J. Hazarika, J.C.S. Kataky, Ind. J. Heterocycl. Chem. 7 (1998) 83–92.

ACCEPTED MANUSCRIPT
[30] B.S. Holla, C.S. Prasanna, B. Poojary, K.S. Rao, K. Shridhara, U.G. Bhat, Ind. J.
Chem. 43B (2004) 864–868.
[31] M.A. Abbasi, B. Shahzad, Aziz-ur-Rehman, S. Rasool, K. Nafeesa, M.A. Ashraf,
World J. Pharm. Sci. 2 (2014) 32–40.
[32] K. Rubab, M.A. Abbasi, S.Z. Siddiqui Aziz-ur-Rehman, M.N. Akhtar, Trop. J. Pharm.
Res. 15 (2016) 1515–1524.
[33] K. Rubab, M.A. Abbasi, Aziz-ur-Rehman, M. Ashraf, S.Z. Siddiqui, A. Shaukat, Trop.
J. Pharm. Res. 15 (2016) 1525–1533.
[34] S.S. Devesa, W.J. Blot, J.F. Fraumeni, Changing patterns in the incidence ofesophageal
and gastriccarcinoma in the United States, J. Canc. 83 (1998)2049e2053.
[35] C.P. Howson, T. Hiyama, E.L. Wynder, The decline of gastric cancer: epide-miology of
an unplanned triumph, J. Epidemiol. Rev. 8 (1986) 1e27.
[36]A.C. Bustamante, J.V. Paredes, A.F. Parra, A.P. Peraza, J.M. Martínez,M.S. Martínez,
A.R. Organillo, Antioxidant activity of butyl- and phenyl-stannoxanes derived from 2-, 3-
and 4-pyridinecarboxylic acids, Molecules 15(2010) 5445e5459.
[37] N. Dharmaraj, P. Viswanathamurthi, K. Natarajan, Ru(II) complexes
containingbidendate Schiff bases and their antifungal activity, Transit. Met. Chem.
26(2001) 105e110.
[38] C. Basu, S. Chowdhury, R. Banerjee, H.S. Evans, S. Mukherjee, Polyhedron 26(2007)
3617e3624.[
[39] A. Demirbas, D. Sahin, N. Demirbas, S.A. Karaoglu. Eur. J Med. Chem. 44 (2009)
2896–2903.
[40] H. Bayrak, A. Demirbas, N. Demirbas, S.A. Karaoglu. Eur. J Med. Chem. 45 (2010)
4726-4732.
[41] N. Galic, B. Peric, B. Kojic-Prodic, Z. Cimerman, J. Mol. Struct. 559 (2001) 187-194.
[42] E. Wyrzykiewicz, D. Prukah. J. Heterocycl. Chem. 35 (1998) 381-387.
[43] H. Bayrak, A. Demirbas, S.A. Karaoglu, N. Demirbas, Eur. J Med. Chem. 44 (2009)
1057-1066.
[44] Lipinski, C. A.; Lombardo, L.; Dominy, B. W.; Feeney, P. J. Adv. Drug Delivery Rev.
46 (2001) 3.
[45] Molinspiration Chemoinformatics, Brastislava, Slovak Republic, Available from:
http://www.molinspiration.com/cgi-bin/properties, 2015.
[46] Y. Zhao, M.H. Abraham, J. Lee, A. Hersey, N.C. Luscombe, G. Beck, B. Sherborne, I.
Cooper. Pharm. Res. 19 (2002) 1446.

ACCEPTED MANUSCRIPT
[47] P. Ertl, B. Rohde, P.J. Selzer. Med. Chem. 43 (2000) 3714.
[48] Schrödinger Suite 2009 Induced Fit Docking protocol; Glide version 5.5, S., LLC, New
York, NY, 2009; Prime version 2.1, Schrödinger, LLC, New York, NY, 2009.
[49] G. Akyüz, E. Menteşe, M. Emirik, N. Baltaş. Bioorganic Chemistry. 80 (2018) 121-128.
[50] M.S. Blois. Nature 181 (1958) 1199-1200.
[51] I.F.F. Benzie, J.J. Strain, Methods Enzymol. 299 (1999) 15-27.
[52] R. Apak, K. Guclu, M. Ozyurek, S.E. Karademir, J. Agric. Food Chem. 52 (2004) 7970-
7981.
[53] M.W. Weatherburn, Anal. Chem. 39 (1967) 971-974.
[54] S.W.I. Siu, K. Pluhackova, R.A. Böckmann, J. Chem. Theory Comput., 8 (2012), 1459-
1470
[55] A. Balasubramanian, K. Ponnuraj. J. Mol. Biol. 400 (2010) 274-283.
[56] R.A. Friesner, R.B. Murphy, M.P. Repasky, L.L. Frye, J.R. Greenwood, T.A. Halgren,
P.C. Sanschagrin, D.T. Mainz. J. Med. Chem. 49 (2006) 6177-6196.