Synthesis of novel Schiff bases using green chemistry techniques; antimicrobial, antioxidant, antiurease activity screening and molecular docking studies

Article abstract of DOI:10.1016/j.molstruc.2018.12.114

Schiff base derivatives were synthesized in this study via conventional, microwave irradiation and ultrasound sonication methods. Optimization conditions were examined for several parameter such as solvent, reaction time and yield. After determining the o

Full text of DOI:10.1016/j.molstruc.2018.12.114

Accepted Manuscript
Synthesis of novel Schiff bases using green chemistry techniques; antimicrobial,
antioxidant, antiurease activity screening and molecular docking studies
Arif Mermer, Neslihan Demirbas, Harun Uslu, Ahmet Demirbas, Sule Ceylan, Yakup
Sirin
PII:
S0022-2860(18)31547-3
DOI:
https://doi.org/10.1016/j.molstruc.2018.12.114
MOLSTR 26051
Reference:
To appear in: Journal of Molecular Structure
Received Date: 17 November 2018
Revised Date: 29 December 2018
Accepted Date: 31 December 2018
Please cite this article as: A. Mermer, N. Demirbas, H. Uslu, A. Demirbas, S. Ceylan, Y. Sirin, Synthesis
of novel Schiff bases using green chemistry techniques; antimicrobial, antioxidant, antiurease
activity screening and molecular docking studies, Journal of Molecular Structure (2019), doi: https://
doi.org/10.1016/j.molstruc.2018.12.114.
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ACCEPTED MANUSCRIPT
Synthesis of novel Schiff bases using green chemistry techniques; antimicrobial,
antioxidant, antiurease activity screening and molecular docking studies
1
1*
2
1
3
Arif Mermer , Neslihan Demirbas , Harun Uslu , Ahmet Demirbas , Sule Ceylan and Yakup
Sirin
1
1
Karadeniz Technical University, Department of Chemistry, 61000 Trabzon, Turkey
2
Inonu University, Faculty of Pharmacy, Department of Pharmaceutical Chemistry, 44280
Malatya, Turkey
3
Department of Occupational Health and Safety, Artvin Çoruh University, Faculty of Medical
Sciences, 08000 Artvin, Turkey
*
Address correspondence to: Prof. Neslihan Demirbas, Phone: +90 4623774252, Fax: +90
4
623253196, E-Mail: neslihan@ktu.edu.tr
Abstract
Schiff base derivatives were synthesized in this study via conventional, microwave irradiation
and ultrasound sonication methods. Optimization conditions were examined for several
parameter such as solvent, reaction time and yield. After determining the optimization
conditions, the compounds were synthesized by using ultrasound sonication. The structures of
the synthesized compounds were examined by spectral data, and the antiurease, antioxidant
and antimicrobial activities of the Schiff bases derivatives were investigated due to the imine
group (-C=N-) and promising results were obtained. The enzyme inhibitory potentials of these
compounds were further validated through molecular docking studies. Also, In Silico ADME
prediction studies were calculated for compounds.
Keywords: Ultrasound sonication, microwave irradiation, biological activity, ADME
prediction.
1
. Introduction
Schiff bases present a crucial group of organic compounds in many aspects and they have a
wide variety of biological activities such as antibacterial, anti-inflammatory, antifungal,
antimalarial, antitubercular and anti-urease [1-10]. Schiff bases containing the imine (-C=N-)
group are formed by the reaction of an aldehyde or ketone with a primary amine and imine
group offer for this kind of compounds is important for their biological activities. Therefore,
this moiety has been extensively explored for the development of new bioactive compounds
[
11, 12].

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Green chemistry techniques have attracted in many areas, especially in the field of
synthetic organic chemistry. Microwave and ultrasound mediated organic synthesis have
become an increasingly used techniques for the production of new molecules [13]. They
usually cause shorter reaction time, high reaction yield and purity. The main advantage of
thesemethods is the almost instantaneous ‘in-core’ heating of compounds in a homogenous
and selective conduct [14].
Urease, which posses nickel ions is the first crystallized and an important enzyme used in
agriculture and medicine industry. The urease enzyme allows rapid formation of ammonia and
carbamic acid by hydrolyzing the urea [15]. However, at the same time, the by-products
resulting from the reaction lead to an increase in pH which is responsible for the adverse
effects of urease activity in human health, leading to diseases such as gastric ulcers, stomach
cancer. The urease causes in pathologies by Helicobacter pylori (HP), by helping the bacteria
to stand at low pH of the stomach during colonization. Thus, it plays a vital role in the
pathogenesis of the gastric as well as peptic ulcers which may cause cancer [16,17].
Additionally, urease not only causes kidney stones formations [18], but also engages in the
growth of urolithiasis, pyelonephritis and hepatic encephalopathy [19]. In agriculture, during
urea fertilization, high urease activity results in significant environmental and economic
losses by discharge of abnormally huge amounts of ammonia in atmosphere. This also leads
to plant damage by depriving them from essential nutrients, secondary ammonia toxicity and
increase in pH of the soil [20]. Urease inhibition, therefore, has been identified as first line of
treatment of diseases caused by ureolytic bacteria [21]. To remove these adverse effects, it is
interesting to control the urease activity by the use of inhibitors [22]. The -C=N- imine bond
in Schiff bases plays a unique role in conferring broad-spectrum biological activities to these
compounds. The electrophilic carbon and nucleophilic nitrogen in -C=N- imine bond provides
excellent binding opportunities with different nucleophiles and electrophiles, thereby
inhibiting targeted diseases, enzymes or DNA replication.
Antioxidants are generally hydrogen donors or electron donors to the reactive site in
neutralizing free radicals. Antioxidants are extensively studied for their capacity for protect
organism and cell from damage that is induced by oxidative stress. Scientists in many
different disciplines become more interested in new compounds, either synthesized or
obtained from natural sources that could provide active components to prevent or reduce the
impact of oxidative stress on cell [23-25]. The scavenging activity of different organic
compounds can be assessed using DPPH, hydrogen peroxide, superoxide anion radical. Many
organic compounds already reported before have showed very good antioxidant capacity, thus

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it is important to understand the mode of action and efficiency of these antioxidants. There are
large numbers of natural and synthetic antioxidants which have been explored, and their
antioxidant capacity has been assessed by different methods.
In this paper, we reported the synthesis of basic Schiff base derivatives using green
chemistry techniques which are microwave irradiation and ultrasound sonication.
Antimicrobial, antioxidant and urease inhibition studies were investigated and carried out
molecular docking studies of newly synthesized compounds. At the same time, In Silico
ADME prediction studies were calculated for all compounds.
2
2
. Results and Discussion
.1. Chemistry
In the present study, the ecofriendly synthesis, urease enzyme inhibition, antioxidant
activity screening and molecular docking studies of new simple Schiff base derivatives were
contemplated. The structures of newly synthesized compounds were established on spectral
1
13
data H NMR, C NMR, FT IR and MS spectra. Eighteen aromatic Schiff bases were
synthesized (Table 1) and characterized.
Scheme 1. Preparation of Schiff bases
Table 1. Molecular structure, melting point and yield of synthesized compounds
Compound
Melting
Point ( C)
Yield (%)
o
a
9
2
b
c
9
4-95
97
9
8
1
a

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a
9
6-97
87
b
c
92
6
9
1
b
a
b
c
7
3-75
89
93
6
9
1
c
a
b
c
1
9
24-126
3-94
81
8
8
6
9
1
d
a
b
c
86
9
9
0
3
1
e
a
b
c
7
4-76
84
89
0
9
1
f
a
b
c
9
6
3-94
0-61
85
9
9
0
2
1
g
a
b
c
90
9
9
3
6
1
h

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a
1
07-108
89
b
c
92
4
9
1
i
a
b
c
1
37-139
87
91
4
9
2
a
a
b
c
9
0-91
89
92
3
9
2
b
a
b
c
7
1
9-81
87
8
9
9
2
2
c
a
b
c
48-150
86
8
9
8
1
2
d
a
b
c
1
39-141
85
87
1
9
2
e

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a
1
59-161
86
b
c
89
0
9
2
f
a
b
c
9
5-96
84
87
1
9
2
g
a
b
c
9
8
3-95
0-82
90
9
9
3
5
N
N
F
Cl
2
h
a
b
c
89
9
9
1
4
2
i
a
b
c
Conventioanl Method, Microwave Irradiation Method, Ultrasound sonication method
Initially, to optimize the solvent effect for this reaction, 4-methyl aniline (1 mmol), 4-
nitrobenzaldehyde (1 mmol), H SO (3-4 drops) and for solvents four solvents were tried;
2
4
ethanol, tetrahydrofurane, MeOH:H O (3:1) and acetonitrile in conventional method. The
2
reaction completed in 3 hours (the progress of reaction was determined by TLC) and it was
determined that the solvent was effective on the reaction (Table 2). Accordingly,
methanol:water (3:1) mixture is the most effective solvent.
Table 2. Solvent effect on reaction yield
Compound
No.
Solvent
Time (h) Yield
(%)

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1d
1d
1d
1d
Ethanol
THF
Methanol:H O
3
3
3
3
67
72
81
56
2
Acetonitrile
After determination of the solvent, the time and yield of this reaction were examined by
applying the green chemistry techniques to this reaction. For this purpose, we used two green
chemistry techniques; Microwave irradiation and ultrasound sonication. To optimize reaction
conditions in microwave method, the same reagents were chosen as model reaction and
microwave (MW) irradiation was implemented at same power (100 W), different time and
temperature (the progress of reaction was monitored by TLC) (Table 3). The highest reaction
o
yield (86%) was obtain in this method 100 W, 100 C and 3 min. In this optimization method,
o
after 100 C the reaction yield decreased from 86 to 82%.
Table 3. Optimization conditions of the reaction in Microwave Irradiation
Entry
Solvent
Power (W)
Temp.
Time
(min)
Yield
(%)
76
81
86
82
78
73
o
(
C)
50
75
1
2
3
4
5
6
MeOH:H
MeOH:H
2
O
O
100
100
100
100
100
100
5
3
3
3
5
3
2
MeOH:H O
100
100
150
200
2
MeOH:H
MeOH:H
MeOH:H
O
O
O
2
2
2
The second ecofriendly method were applied ultrasound soication. Again we also
optimized the conditions in US. By changing the parameters of temperature and time, we
determined in which conditions the reaction takes place with the highest yield. As a result of
o
the optimization study, it was determined that the maximum yield (89%) was 50 C and 1.5
min (Table 4).
When all the results are examined, it was realized that the green chemistry techniques are
superior to the conventional method. This reaction, which took place in 180 minutes in the
conventional method, took place in 3 min in the microwave irradiated method and 1.5 min in
the ultrasound sonication method. At the same time, when the reaction yield is evaluated in
these three methods, the highest reaction yield was obtained by the ultrasonic method.
Table 4. Optimization condtions of the reaction in Ultrasound Sonication
Entry
1
Solvent
MeOH:H
Temp
Time
(min)
3
Yield
(%)
75
o
(
C)
25
O
2

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2
3
4
5
6
MeOH:H
MeOH:H
MeOH:H O
2
O
O
25
50
50
75
75
1.5
3
1.5
3
77
82
89
83
80
2
2
MeOH:H
MeOH:H
O
O
2
2
3
In the FT IR spectra of the azomethine groups in the Schiff bases, HC=N bands are
-1
1
observed at 1600-1650 cm [26-31]. The H NMR spectra of all synthesized compounds were
recorded. The appearance of singlet proton of the azomethine group at 8.53-9.06 ppm in the
1
H NMR spectrum supported the structures of Schiff bases obtained. The protons of the
hydroxyl group in the compounds 1a and 2a were observed at 13.23 and 12.60 ppm,
1
3
respectively. Aromatic protons were resonated in the region 6.95-8.77 ppm. In C NMR
spectra of new Schiff bases, the carbon atom (HC=N) were observed between 151.56 and
1
65.04 ppm. Another spectroscopic evidence supporting the constructions of Schiff bases
obtained is the [M], [M+1] and [M+2] ion signals observed in the mass spectra of the
compounds. The compounds having imine group may exist as E/Z geometrical isomers about
–
C=N double bond and cis–trans amide conformers (Scheme 2). According to the literature,
the compounds containing imine bond are present in higher percentage in dimethyl-D6
sulfoxide solution in the form of geometrical E isomer about –C=N double bond. The Z
isomer can be stabilized in less polar solvents by an intramolecular hydrogen bond [32, 33]. In
the present study, the spectral data were obtained in dimethyl-d6 sulfoxide solution and no
signal belonging to Z isomer was observed. On the other hand, the cis–trans conformers of E
isomer were present in the dimethyl-d sulfoxide solution of compounds 1a-i and 2a-i.
6
2
.2. Antimicrobial activity screening
The synthesized compounds were screened for their antimicrobial activity using the disk
diffusion method and the results obtained were showed in Table 5. Amphicilin and
Gentamicin were used as standart antibiotic. All synthesized compounds showed
antimicrobial activity against test microorganisms. Among these, 1d, 1e and 2a exhibited
good-modarate activity against S. Aureus, all compounds except 1b, 1h and 2g showed good-
modarate activity against E. faecalis.1a, 1d, 2a and 2d exhibited good-modarate activity
against E. coli, P. aeruginosa, K. pneumoniae, A. haemolyticus. Among the compounds, 2e
exhibited excellent activity against all test microorganisms with the MIC values varying 0.5–4
µg/mL.
Table 5. Screening forantimicrobial activity of newly synthesized compounds

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Comp.
No
Microorganisms and Minimal Inhibition Concentration (µg/mL)
Sa
4
8
16
2
2
Ef
16
32
16
8
Ec
2
4
8
2
Pa
8
16
16
2
Kp
1
8
8
0.5
1
Ah
2
8
4
4
1
a
1
b
1
c
1
d
1
e
4
4
4
1
1
f
8
8
8
32
16
4
16
8
16
32
2
8
4
8
32
4
16
8
16
8
8
8
16
8
2
1
g
16
32
32
2
1
h
1
i
2
a
2
2
2
2
b
c
d
4
8
4
8
16
8
32
16
1
16
32
2
16
16
2
16
32
2
2
2
e
f
1
4
8
16
32
1.56
-
4
0.5
8
16
32
32
-
1
0.5
8
16
16
16
-
1
8
8
32
16
-
16
32
16
16
12.25
-
16
16
16
8
2
g
2
h
2
i
Amp.
Gen.
-
0.78
1.56
0.39
0.78
Sa: Staphylococcus aureus ATCC 25923, Ef: Enterococcus faecalis ATCC 29212, Ec: Escherichiacoli ATCC
2
5922, Pa: Pseudomonas aeruginosa ATCC 27853, Kp: Klebsiella pneumoniae ATCC 13883, Ah: Acinetobacter
haemolyticus ATCC 19002. Amp.: Amphicilin, Gen.: Gentamicin
35
30
25
20
15
10
5
0
Sa
Ef
Ec
Pa
Kp
Ah
1
a
1b 1c 1d 1e 1f 1g 1h 1i 2a 2b 2c 2d 2e 2f 2g 2h 2i Amp. Gen.
Figure 1. Antimicrobial activity of the synthesized compounds.

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2
.3. Antioxidant capacity
The newly synthesized Schiff base analogues of 4-methyl aniline and 3-chloro,4-fluoro
aniline were evaluated for their antioxidant capacity by DPPH, CUPRAC and FRAP (Table
). When the antioxidant results of the synthesized compounds were examined, it was
6
determined that the Schiff base derivatives obtained from 3-chloro-4-fluoro aniline showed
better activity. The compounds 2c, 2f and 2b exhibited good-moderate activities with IC50
values 0,15±0,01, 0,19±0,00 and 0,22±0,00 µM/mL, respectively in DPPH assay much better
than standart Trolox (IC =0,04±0,00). For FRAP and CUPRAC assays, the same compounds
5
0
2
c, 2f and 2bshowed best activities with the values 4486,91±18,22, 4225,13±9,67 and
4
155,32±14,78 (for FRAP), 8887,63±12,12, 8880,98±23,55 and 8156,13±15,65 (for
CUPRAC). On the basis of the above observation, compounds having –F (fluorine), –2,6-Cl
chlororine) and –OCH (methoxy) groups in the phenyl ring (2c, 2f and 2b) were found to be
(
3
the most potent antioxidants. Schiff bases containing fluorine, methoxy and chlorine groups
showed higher activity in this study, while it was expected that antioxidant activities of
hydroxyl group-containing structures would be expected to be higher, and electron-
withdrawing groups would have lower activity [34]. 2b has a fluorine atom in para position
on phenyl ring and 2c has methoxy group in para position on phenyl ring. 2f has chlorine
atom in ortho and para position on phenyl ring. Other compounds also displayed remarkable
antioxidant activity for FRAP, CUPRAC and DPPH.
2
.4. Urease inhibition assay
Obtained compounds which are Schiff base derivatives were evaluated against Jack bean
urease in vitro. At first, the compounds were screened at a concentration of 1 mM.
Compounds with greater than 50% inhibition for full characterization were also selected. All
of compounds were potent inhibitors of Jack bean urease. Thiourea was selected and used as
the standart compound for the assay, its affinity is also comprising in Table 6. The
synthesized compounds carry various functional groups in the phenyl ring came from
aldehyde. All results were represented in Table 6. The compounds displayed excellent
inhibitory activity in milimolar range. Second series (2a-i) of synthesized compunds showed
better inhibition than first series (1a-i). Among the tested compounds 2c was the most
vigoroues having 0,19±0,02 IC value. Other potent compounds were 2f and 2b having IC
5
0
50
values 0,21±0,02 and 0,25±0,02, respectively. The other compounds also exhibted better
activity than thiourea used as standart against Jack bean urease with IC values ranging from
5
0
0
,42±0,02 to 8,46±0,02. The anti-urease activities of the synthesized compounds vary due to

ACCEPTED MANUSCRIPT
the functional groups present in both the amine and the aldehyde group [35]. The activity of
schiff bases obtained using toluidine as amine was found to be less active than those elicited
using 3-chloro-4-fluoroaniline. Furthermore, the functional groups in the para position of the
aldehydes used are more active than the ortho and meta positions.
Table 6. Antioxidant capacity (AC) values and anti-urease activity of 18 synthesized novel
compounds
Compound
FRAP
DPPH
CUPRAC
Urease Inh.
(µmol TE/g)
IC50
(µmol TE/g)
(IC )
50
1
a
322.25±7.18
407.33±1.12
369.37±6.21
433.51±4.68
447.91±7.27
359.55±5.18
488.48±6.14
376.57±3.17
470.16±8.11
2728.62±10.14
4155.32±14.78
4486.91±18.22
3640.49±16.45
3858.64±12.25
4225.13±9.67
3448.52±13.43
3243.46±19.11
4015.71±11.18
6.12±0.04
4.91±0.03
5.29±0.06
4.83±0.01
4.61±0.02
5.86±0.03
4.45±0.06
5.18±0.00
4.48±0.06
1.01±0.04
0.22±0.00
0.15±0.01
0.42±0.01
0.36±0.02
0.19±0.00
0.51±0.03
0.67±0.02
0.29±0.00
0.04±0.00
613.13±34.34
1866.62±15.12
1186.74±12.66
2001.32±6.76
2156.47±21.54
986.85±8.19
2574.63±5.10
1425.74±9.98
2345.45±5.43
6875.13±14.10
8156.13±15.65
8887.63±12.12
7645.13±7.44
7854.46±16.81
8880.98±23.55
7312.42±27.76
7125.69±5.34
8012.56±15.21
8.46±0.02
5.41±0.02
6.14±0.02
5.36±0.02
4.18±0.02
7.12±0.02
1.24±0.02
5.53±0.02
3.13±0.02
1.13±0.02
0.25±0.02
0.19±0.02
0.52±0.02
0.48±0.02
0.21±0.02
0.64±0.02
0.89±0.02
0.42±0.02
1b
1
c
1d
1
1
1
e
f
g
1h
1
2
i
a
2b
2
c
2d
2
2
2
e
f
g
2h
2
i
Trolox
Thiourea
12.02±0.06
2
.5. In Silico ADME prediction study
Table 7. Pharmacokinetic parameters important for good oral bioavailability of synthesized
compound 1a-i and 2a-i.
Entry
%
ABS
TPSA
(A ) ROTB
n-
MV
MW
miLog
n-ON
acceptors OHNH
donors
n-
Nviolations
Drug
Likeness
Model
Score
2
P
-
-
-
-
≤500
≤5
<10
2
2
1
4
1
1
1
2
<5
1
0
0
0
0
0
0
0
≤1
0
0
0
0
1
1
0
0
1
a
97.75 32.59
101.54 21.60
104.74 12.36
88.92 58.19
104.74 12.36
104.74 12.36
104.74 12.36
100.28 25.26
100.28 25.26
97.75 32.59
2
3
2
3
2
2
2
2
2
2
203.29 211.26
220.82 225.29
200.21 213.25
218.61 240.26
222.34 264.15
222.34 264.15
213.74 247.70
191.12 196.25
191.12 196.25
205.20 249.67
3.86
3.98
4.08
3.88
5.20
5.18
4.67
2.68
2.63
4.18
-0.47
-0.51
-0.54
-0.53
-0.52
-0.47
-0.61
-0.43
-0.48
-0.46
1
b
1
c
1
d
1
1
1
e
f
g
1
h
1
2
i
a
2
2
0
1
0
0

ACCEPTED MANUSCRIPT
2
2
2
2
2
2
2
2
b
c
d
e
f
g
h
i
101.54 21.60
104.74 12.36
88.92 58.19
104.74 12.36
104.74 12.36
104.74 12.36
100.28 25.26
100.28 25.26
52.05
3
2
3
2
2
2
2
2
0
222.72 263.70
202.11 251.66
220.51 278.67
224.25 302.56
224.25 302.56
215.65 286.11
193.02 234.66
193.02 234.66
4.30
4.40
4.20
5.52
5.50
4.99
3.00
2.95
-0.46
2
1
4
1
1
1
2
2
2
0
0
0
0
0
0
0
0
4
0
0
0
1
1
0
0
0
0
-0.51
-0.49
-0.52
-0.47
-0.43
-0.53
-0.41
-0.46
-3.78
Thiourea
63.07
76.12
%
ABS: percentage absorption, TPSA: topological polar surface area, n-ROTB: number of
rotatable bonds, MV: molecular volume, MW: molecular weight, miLog P: logarithm of
partition coefficient of compound between n-octanol and water, n-ON acceptors: number of
hydrogen bond acceptors, n-OHNH donors: number of hydrogen bonds donors.
Many biologically active compounds fail to achieve the clinic because of their inadequate
absorption, distribution, metabolism, and elimination (ADME) parameters. For this reason, a
computational study of synthesized compounds 1a-i and 2a-i werestudied for evaluation of
ADME properties and value obtained is represent in Table 7. Polar surface area (TPSA),
number of rotatable bonds (n-ROTB), molecular volume (MV), and Lipinski’s rule of five
were calculated using Molinspiration online property calculation toolkit [36]. When the
results are examined, all the synthesized compounds showed excellent % absorption.
Moreover, none of the compounds trespass on Lipinski’s rule of five and thus showing
potential utility of series for improving the compound with good drug like properties. A
compound probably likely to be developed as an orally active drug candidate should
demonstrate 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 [37]. All the synthesized compounds complied the
standart for orally active drug and therefore, these compounds can be further advanced as oral
drug candidates. The results of this in silico ADME prediction analysis propose that the
obtained compounds follow the computational assessment and thus indicate a
pharmacologically active framework that should be considered on progressing further
potential hits. Drug-likeness model score (a combined effect of physico-chemical properties,
pharmacokinetics and pharmacodynamics of a compound and is displayed by a numerical
value) was calculated by Molinspiration software (http://www.molinspiration.com) for the 18
synthesized compounds. The best drug-likeness score was found to be -0.41 and -0.43 for
compounds 2h and 2f, respectively.

ACCEPTED MANUSCRIPT
Figure 2. Drug likeness score of compound 2h
.6. Molecular docking studies
According to the X-ray crystallographic structure of Urease (PDB ID:1E9Y),main binding
2
site has been determined around Ni atoms including Ni3001, Ni3002, His221, Asp362,
Ala365 [www.rcsb.org]. It has been reported that acetohydroxamic acid interacts with active
site in the gorge concordantly binding site [38]. Analyses of binding modes of the
acetohydroxamic acid indicate that the carbonyl, amino and hydroxyl groups are in H-bonds
with His221, Asp362, Ala365 in binding cavity [39]. The formation of hydrogen bond
between the nitrogen atom in the imine group of compound 2c and Cys321 showed binding to
the active site. The pyridine of compound 2i has a position in the gorge to interact with
His221 by doing a hydrogen bond. 2c, 2f and 2i interacted with nickel, especially Ni3001.
Docking studies were performed for the most active compounds 2c and 2f interaction modes
with enzyme active sites were determined. Docking studies revealed that there is a strong
interaction between the active sites of Helicobacter Pylori Urease enzyme and these
compounds. Ideally, a Helicobacter Pylori Urease inhibitor is expected to effectively interact
with these sites (Figure 3-4) (Table 8). The binding mode was produced by AutoDock and
showed by Maestro (Figure 5).

ACCEPTED MANUSCRIPT
Figure 3. Compounds 2c (pink) and 2f (blue) are presented into the Helicobacter pylori
+
2
urease (PDB ID: 1E9Y) binding cavity. Acetohydroxamic acid (orange), Ni atoms
NI3001, NI3002) are shown as sphere (red). For clarity, receptor residues are shown as
(
cartoon.
Figure 4. Compound 2c (pink sticks) in the binding site (molecular surface rendered) of
urease

ACCEPTED MANUSCRIPT
Figure 5. 2D interaction diagram from Glide for some compounds
Table 8. Molecular docking binding scores of some compounds, within the Helicobacter
pylori urease (PDB ID: 1E9Y) active site. Residues participating in hydrogen bonds and Pi-
cation contacts with the compounds are shown.
Estimated Inhibition
Constant, Ki,
Temp. = 298.15 K
Residue
Comp. Estimated Free Energy
of Binding (kcal/mol)
H-bond
Pi-cation
2
c
-5.93
-6.02
-5.77
45.33 uM
38.49 uM
58.96 uM
Cys321
Cys321
His221
-
2
f
Arg338
-
2
i
uM: micromolar
3
. Experimental

ACCEPTED MANUSCRIPT
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 (1:1), and detection was made using UV
1
light. FT-IR spectra were recorded using a Perkin Elmer 1600 series FTIR spectrometer. H
1
3
NMR and C NMR spectra were registered in DMSO-d on a BRUKER AVENE II 400 MHz
6
1
13
NMR Spectrometer (400.13 MHz for H and 100.62 MHz for C). The chemical shifts are
given in ppm relative to Me Si as an internal reference, J values are given in Hz. Microwave
4
and ultrasound mediated syntheses were carried out using monomod CEM-Discover
microwave apparatus and BandelinSonorex Super RK102H ultrasonic bath, respectively. The
Mass spectra were obtained on a Quattro LC-MS (70 eV) Instrument. Compound 1a and 2a
are known [40, 41]. In literature, 1a and 2a was synthesized by microwave irradiation and
obtained 97%, 79% yield in 1.5 minutes and 6 minutes, respectively (melting points of
o
compounds 1a and 2a are 93-94 and 138 C, respectively).
3
.1. General method for the synthesis of compounds 1a-i and 2a-i
Method 1. The amine compounds (10 mmol) (4-methyl aniline or 3-chloro-4-fluoro
aniline) was added in a solution of suitably substituted benzaldehyde (10 mmol) in
methanol:H O (3:1) (10 ml). And conc. sulphuric acid was dropped in catalytic amount to the
2
solution. The reaction mixture was refluxed for 3-5 hours (The reaction progress was
followed by TLC). After evaporating the solvent under reduced pressure, a solid was
obtained. The obtained compound was recrystallized from an appropriate solvent to afford the
desired product.
Method 2. The solution of suitably substituted benzaldehyde (10 mmol) and amine
compound (10 mmol) in methanol:H O (3:1) (10 mL) and conc. H SO (3-4 drops) was
2
2
4
o
irradiated in closed vessels at 100 C, 100 W, for 20 min. (The reaction progress was followed
by TLC). After evaporating the solvent under reduced pressure, a solid was obtained. The
obtained compound was recrystallized from an appropriate solvent to afford the desired
product.
Method 3. The solution of suitably substituted benzaldehyde (10 mmol) and amine
compound (10 mmol) in methanol:H O (3:1) (10 mL) and conc. H SO (3-4 drops) was
2
2
4
o
sonicated at 50 C for 20 min. (The reaction progress was followed by TLC). After

ACCEPTED MANUSCRIPT
evaporating the solvent under reduced pressure, a solid was obtained. The obtained compound
was recrystallized from an appropriate solvent to afford the desired product.
(E)-2-((p-tolylimino)methyl)phenol (1a)
-1
1
FT-IR (υmax,cm ): 3052 (ar-CH), 1613 (C=N). H NMR (DMSO-d , δppm): 2.35 (s, 3H,CH ),
6
3
6
.95-7.00 (m, 2H, arH), 7.28 (d, 2H, J=8.0 Hz, arH), 7.34 (d, 2H, J=8.0 Hz, arH), 7.39-7.43
1
3
(
m, 1H, arH), 7.64 (d, 1H, J=8.0 Hz, arH), 8.96 (s, 1H, CH), 13.23 (s, 1H, OH). C NMR
DMSO-d , δppm): 21.08 (CH ), arC: [117.02 (CH), 119.55 (CH), 119.78, 121.69 (2CH),
(
6
3
1
30.42 (2CH), 132.94 (CH), 133.54 (CH), 136.99, 145.86, 163.03], 160.74 (CH). EI MS m/z
+
(
%): 212.25 ([M+1] , 100).
3
.1.1. (E)-N-(4-methoxybenzylidene)-4-methylaniline (1b)
-1
1
FT-IR (υmax,cm ): 3007 (ar-CH), 1621 (C=N), 1217 (C-O). H NMR (DMSO-d , δppm): 2.34
6
(
s, 3H,CH ), 3.84 (s, 3H, OCH ), 7.07 (d, 2H, J=8.0 Hz, arH), 7.18 (q, 4H, J=16.0 Hz, arH),
3 3
1
3
7
5
1
.88 (d, 2H, J=8.0 Hz, arH), 8.53 (s, 1H, CH). C NMR (DMSO-d , δppm): 21.02 (CH ),
6 3
5.83 (OCH ),arC: [114.70 (2CH), 121.31 (2CH), 129.53, 130.11 (2CH), 130.78 (2CH),
3
+
35.30, 149.58, 162.24], 159.39 (CH). EI MS m/z (%):226.18 ([M+1] , 100).
3
.1.2. (E)-N-(4-fluorobenzylidene)-4-methylaniline (1c)
-1
1
FT-IR (υmax,cm ): 3027 (ar-CH), 1625 (C=N). H NMR (DMSO-d , δppm): 2.33 (s, 3H,CH ),
6
3
7
.21 (q, 4H, J=24.0 Hz, arH), 7.35 (t, 2H, J=16.0 Hz, arH), 8.00 (q, 2H, J=16.0 Hz, arH), 8.62
1
3
(
s, 1H, CH). C NMR (DMSO-d , δppm): 21.03 (CH ), arC: [116.22 (CH), 116.44 (CH),
6 3
1
21.42 (2CH), 130.15 (2CH), 131.25and 131.34 (d, J=9.0 Hz, 2CH), 133.32, 135.87, 149.09,
+
1
63.12 and 165.59 (d , J=247.0 Hz)], 158.86 (CH). EI MS m/z (%): 214.24 ([M+1] , 100).
C-F
3
.1.3. (E)-4-methyl-N-(4-nitrobenzylidene)aniline (1d)
-1
1
FT-IR (υmax,cm ): 3084 (ar-CH), 1623 (C=N), 1505 and 1337 (-NO ). H NMR (DMSO-d ,
2
6
δppm): 2.34 (s, 3H,CH ), 7.27 (t, 4H, J=16.0 Hz, arH), 8.17 (d, 2H, J=8.0 Hz, arH), 8.35 (d,
3
13
2
H, J=8.0 Hz, arH), 8.81 (s, 1H, CH). C NMR (DMSO-d , δppm): 21.10 (CH ), arC:
6 3
[
121.78 (2CH), 124.46 (2CH), 129.96 (2CH), 130.28 (2CH), 137.05, 142.17, 148.36, 149.18],
+
1
58.18 (CH). EI MS m/z (%): 241.28 ([M+1] , 100).
3
.1.4. (E)-N-(2,4-dichlorobenzylidene)-4-methylaniline (1e)
-1
1
FT-IR (υmax,cm ): 3064 (ar-CH), 1617 (C=N). H NMR (DMSO-d , δppm): 2.34 (s, 3H,CH ),
6
3
7
.25 (s, 4H, arH), 7.57 (d, 1H, J=8.0 Hz, arH), 7.78 (s, 1H, arH), 8.16 (d, 1H, J=8.0 Hz, arH),
1
3
8
.83 (s, 1H, CH). C NMR (DMSO-d , δppm): 21.08 (CH ), arC: [121.60 (2CH), 128.50
6
3

ACCEPTED MANUSCRIPT
(CH), 129.68 (CH), 130.00 (CH), 130.02 (CH), 130.52 (2CH), 132.25, 136.10, 136.85,
+
+
1
36.98, 148.67], 154.70 (CH). EI MS m/z (%): 264.14 ([M] ,100), 266.03 ([M+2] , 76).
3
.1.5. (E)-N-(2,6-dichlorobenzylidene)-4-methylaniline (1f)
-1
1
FT-IR (υmax,cm ): 3083 (ar-CH), 1632 (C=N). H NMR (DMSO-d , δppm): 2.33 (s, 3H,CH ),
6
3
7
.18 (d, 2H, J=8.0 Hz, arH), 7.24 (d, 2H, J=8.0 Hz, arH), 7.48 (d, 1H, J=4.0 Hz, arH), 7.57 (d,
1
3
2
H, J=8.0 Hz, arH),8.69 (s, 1H, CH). C NMR (DMSO-d , δppm): 21.06 (CH ), arC: [121.20
6
3
(2CH), 129.55 (2CH), 130.26 (2CH), 132.11 (CH), 132.83, 134.45, 136.78, 148.73], 156.07
+
(
CH). EI MS m/z (%):265.12 ([M+1] , 100).
3
.1.6. (E)-N-(2-chloro-6-fluorobenzylidene)-4-methylaniline (1g)
-1
1
FT-IR (υmax,cm ): 3084 (ar-CH), 1620 (C=N)). H NMR (DMSO-d , δppm): 2.33 (s,
6
3
H,CH ), 7.18 (d, 2H, J=8.0 Hz, arH), 7.25 (d, 2H, J=8.0 Hz, arH), 7.36 (t, 1H, J=16.0 Hz,
3
1
3
arH), 7.45 (d, 1H, J=8.0 Hz, arH), 7.55 (d, 1H, J=4.0 Hz, arH),8.71 (s, 1H, CH). C NMR
(
DMSO-d , δppm): 21.04 (CH ), arC: [116.04and 116.26 (d, J=22.0 Hz, CH), 121.25 (2CH),
6 3
1
1
1
22.70 and 122.83 (d, J=13.0 Hz,), 126.62 and 126.66 (d, J=4.0 Hz, CH), 130.27 (2CH),
33.06 and 133.16 (d, J=10.0 Hz, CH), 135.16 and 135.20 (d, J=4.0 Hz), 136.71, 149.27,
59.95 and 162.51 (d , J=256.0 Hz), 145.86, 163.03], 151.56 (CH). EI MS m/z (%):247.04
C-F
+
+
(
[M] , 100), 249.10 ([M+2] , 69).
3
.1.7. (E)-4-methyl-N-(pyridin-3-ylmethylene)aniline (1h)
-1
1
FT-IR (υmax,cm ): 3017 (ar-CH), 1625 (C=N). H NMR (DMSO-d , δppm): 2.33 (s, 3H,CH ),
6
3
7
2
1
.24 (s, 4H, arH), 7.54 (q, 1H, J=12.0 Hz, arH), 8.31 (d, 1H, J=8.0 Hz, arH), 8.69-8.72 (m,
1
3
H, arH), 9.05 (s, 1H, CH). C NMR (DMSO-d , δppm): 21.06 (CH ), arC: [121.53 (2CH),
6
3
24.50 (CH), 130.21 (2CH), 132.13, 135.32 (CH), 136.38, 148.86, 150.78 (CH), 152.24
+
(
CH)], 157.93 (CH).EI MS m/z (%): 219.23 ([M+Na] , 100).
3
.1.8. (E)-4-methyl-N-(pyridin-4-ylmethylene)aniline (1i)
-1
1
FT-IR (υmax,cm ): 3028 (ar-CH), 1622 (C=N). H NMR (DMSO-d , δppm): 2.34 (s, 3H,CH ),
6
3
7
.27 (s, 4H, arH), 7.84 (d, 2H, J=4.0 Hz, arH), 8.70 (s, 1H,CH), 8.74 (d, 2H, J=4.0 Hz, arH).
C NMR (DMSO-d , δppm): 21.09 (CH ), arC: [121.72 (2CH), 122.58 (2CH), 130.27 (2CH),
1
3
6
3
+
1
1
37.02, 143.10, 148.33, 150.90 (2CH)], 158.59 (CH).EI MS m/z (%): 220.19 ([M+1+Na] ,
00).
3
.1.9. (E)-2-(((3-chloro-4-fluorophenyl)imino)methyl)phenol (2a)

ACCEPTED MANUSCRIPT
-1
1
FT-IR (υmax,cm ): 3089 (ar-CH), 1614 (C=N). H NMR (DMSO-d , δppm): 6.97-7.02 (m,
6
2
H, arH), 7.42-7.52 (m, 3H, arH), 7.66 (d, 1H, J=4.0 Hz, arH), 7.73 (d, 1H, J=8.0 Hz, arH),
1
3
8
.96 (s, 1H, CH), 12.60 (s, 1H, OH). C NMR (DMSO-d , δppm): arC: [117.12 (CH), 118.03
6
(CH), 119.68, 120.60 and 120.70 (d, J=10.0 Hz), 122.97 and 123.04 (d, J=7.0 Hz, CH),
1
23.45 (CH), 133.07 (CH), 134.11 (CH), 146.07, 155.17 and 157.62 (d , J=245.0 Hz),
C-F
+
1
60.59], 165.04 (CH). EI MS m/z (%): 128.47 (100), 250.55 ([M+1] , 94).
3
.1.10. (E)-3-chloro-4-fluoro-N-(4-methoxybenzylidene)aniline (2b)
-1
1
FT-IR (υmax,cm ): 3066 (ar-CH), 1624 (C=N). H NMR (DMSO-d , δppm): 3.84 (s, 3H,
6
OCH ), 7.08 (d, 2H, J=8.0 Hz, arH), 7.25-7.28 (m, 1H, arH), 7.43 (t, 1H, J=16.0 Hz, arH),
3
1
3
7
.49 (d, 1H, J=8.0 Hz, arH), 7.88 (d, 2H, J=8.0 Hz, arH), 8.57 (s, 1H, CH). C NMR (DMSO-
d , δppm): 55.90 (OCH ), arC: [114.78 (2CH), 117.76 (CH), 122.80 (CH), 129.03, 131.18
6
3
(2CH), 132.27 (CH), 146.77, 149.36, 154.50 and 156.93 (d , J=243.0 Hz), 162.66], 161.86
C-F
+
+
(
CH). EI MS m/z (%): 156.29 (52), 252.66(45), ([M+1] , 58), 254.19 ([M+Na] , 100).
3
.1.11. (E)-3-chloro-4-fluoro-N-(4-fluorobenzylidene)aniline (2c)
-1
1
FT-IR (υmax,cm ): 3082 (ar-CH), 1629 (C=N). H NMR (DMSO-d , δppm): 7.29-7.32 (m,
6
1
H, arH),7.37 (t, 2H, J=16.0 Hz, arH), 7.46 (t, 1H, J=16.0 Hz, arH), 7.53 (d, 1H, J=4.0 Hz,
1
3
arH), 7.99 (t, 2H, J=12.0 Hz, arH), 8.66 (s, 1H, CH). C NMR (DMSO-d , δppm):arC:
6
[
116.33 and 116.55 (d, J=22.0 Hz, 2CH), 117.83 (CH), 122.62 and 122.70 (d, J=8.0 Hz, CH),
22.94, 131.64 and 131.73 (d, J=9.0 Hz, 2CH), 132.83, 146.75, 148.84 and 144.88 (d, J=4.0
Hz), 154.78 and 157.21 (d , J=243.0 Hz), 163.41 and 165.90 (d , J=249.0 Hz)], 161.43
1
C-F
C-F
+
(
CH). EI MS m/z (%): 252.64 ([M+1] , 100), 254.58 (36).
3
.1.12. (E)-3-chloro-4-fluoro-N-(4-nitrobenzylidene)aniline (2d)
-1
1
FT-IR (υmax,cm ): 3116 (ar-CH), 1627 (C=N), 1514 and 1347 (-NO ). H NMR (DMSO-d ,
2
6
δppm): 7.38-7.42 (m, 1H, arH),7.51 (t, 1H, J=16.0 Hz, arH), 7.65 (d, 1H, J=4.0 Hz, arH), 8.18
1
3
(
d, 2H, J=8.0 Hz, arH), 8.38 (d, 2H, J=8.0 Hz, arH), 8.85 (s, 1H, CH). C NMR (DMSO-d ,
6
δppm):arC: [117.77 and 117.99 (d, J=22.0 Hz, CH), 120.51 and 120.70 (d, J=19.0 Hz),
23.24 (CH), 124.51 (2CH), 130.26 (2CH), 131.10 (CH), 141.58, 148.13 and 148.16 (d, J=3.0
1
Hz), 149.51, 152.28 and 157.73 (d , J=245.0 Hz)], 160.96 (CH).EI MS m/z (%): 111.45
C-F
+
(
41), 128.15 (53), 233.60 (100), 250 (50), 279.62 ([M+1] , 82).
3
.1.13. (E)-3-chloro-N-(2,4-dichlorobenzylidene)-4-fluoroaniline (2e)
-1
1
FT-IR (υmax,cm ): 3090 (ar-CH), 1615 (C=N). H NMR (DMSO-d , δppm): 7.33-7.37 (m,
6
1
H, arH),7.48 (t, 1H, J=16.0 Hz, arH), 7.58 (d, 2H, J=8.0 Hz, arH), 7.78 (s, 1H, arH), 8.13 (d,

ACCEPTED MANUSCRIPT
13
1
H, J=8.0 Hz, arH), 8.83 (s, 1H, CH). C NMR (DMSO-d , δppm):arC: [117.77 and 117.98
6
(d, J=21.0 Hz, CH), 122.74 and 122.81 (d, J=7.0 Hz, CH), 123.31 (CH), 128.53 (CH), 130.09
(
CH), 130.24 (CH), 131.88, 136.49, 137.54, 148.40, 155.19 and 157.63 (d , J=244.0 Hz)],
C-F
+
+
1
57.38 (CH). EI MS m/z (%): 302.54([M] , 99), 304.35 ([M+2] , 100).
3
.1.14. (E)-3-chloro-N-(2,6-dichlorobenzylidene)-4-fluoroaniline (2f)
-1
1
FT-IR (υmax,cm ): 3081 (ar-CH), 1635 (C=N). H NMR (DMSO-d , δppm): 7.33 (s, 1H,
6
1
3
arH),7.54 (s, 3H, arH), 7.60 (s, 2H, arH), 8.79 (s, 1H, CH). C NMR (DMSO-d , δppm):arC:
6
[
117.88 and 118.10 (d, J=22.0 Hz, CH), 122.46 (CH), 123.00 (CH), 129.66 (2CH), 132.34,
32.59 (CH), 148.17, 154.54 (2C), 155.32 and 157.77 (d , J=245.0 Hz)], 158.82 (CH). EI
1
C-F
+
+
MS m/z (%): 264.38 (100), 302.55([M] , 80), 304.24 ([M+2] , 76).
3
.1.15. (E)-3-chloro-N-(2-chloro-6-fluorobenzylidene)-4-fluoroaniline (2g)
-1
1
FT-IR (υmax,cm ): 3091 (ar-CH), 1629 (C=N). H NMR (DMSO-d , δppm): 7.30-7.34 (m,
6
1
8
1
H, arH), 7.39 (t, 1H, J=16.0 Hz, arH), 7.48 (d, 2H, J=8.0 Hz, arH), 7.55-7.60 (m, 2H, arH),
13
.75 (s, 1H, CH). C NMR (DMSO-d , δppm):arC: [116.12 and 116.34 (d, J=22.0 Hz, CH),
6
17.81 and 118.03 (d, J=22.0 Hz, CH),120.71, 122.43 and 122.51 (d, J=8.0 Hz, CH),123.00
(CH), 126.77 and 126.81 (d, J=4.0 Hz, CH), 133.66 and 133.77 (d, J=11.0 Hz, CH), 135.34,
1
48.84, 155.21 and 157.66 (d , J=245.0 Hz), 160.06 and 162.63 (d , J=257.0 Hz)], 156.22
C-F
C-F
+
+
(
CH). EI MS m/z (%):286.09 ([M] , 100), 288.02 ([M+2] , 62).
3
.1.16. (E)-3-chloro-4-fluoro-N-(pyridin-3-ylmethylene)aniline (2h)
-1
1
FT-IR (υmax,cm ): 3034 (ar-CH), 1629 (C=N). H NMR (DMSO-d , δppm): 7.34-7.37 (m,
6
1
8
H, arH),7.49 (t, 1H, J=16.0 Hz, arH), 7.55-7.60 (m, 2H, arH), 8.31 (d, 1H, J=8.0 Hz, arH),
1
3
.72 (d, 1H, J=4.0 Hz, arH), 8.76 (s, 1H, arH), 9.06 (s, 1H, CH). C NMR (DMSO-d6,
δppm):arC: [117.70 and 117.91 (d, J=21.0 Hz, CH), 120.41 and 120.59 (d, J=18.0 Hz),122.80
and 122.88 (d, J=8.0 Hz, CH), 123.07 (CH), 124.56 (CH), 131.68, 135.59 (CH), 148.62 and
1
48.65 (d, J=3.0 Hz), 151.04 (CH), 152.71 (CH), 155.01 and 157.45 (d , J=244.0 Hz)],
C-F
+
1
60.69 (CH). EI MS m/z (%): 235.62 ([M+1] , 100).
3
.1.17. (E)-3-chloro-4-fluoro-N-(pyridin-4-ylmethylene)aniline (2i)
-1
1
FT-IR (υmax,cm ): 3100 (ar-CH), 1626 (C=N). H NMR (DMSO-d , δppm): 7.37-7.41 (m,
6
1
8
1
H, arH), 7.49-7.53 (m, 1H, arH), 7.64 (d, 1H, J=8.0 Hz, arH), 7.84 (d, 2H, J=4.0 Hz, arH),
1
3
.74 (s, 1H, CH), 8.77 (d, 2H, J=8.0 Hz, arH), C NMR (DMSO-d , δppm): arC: [117.78 and
6
17.99 (d, J=21.0 Hz, CH), 122.61 (CH), 122.74 (2CH), 123.24 (CH),135.09, 142.59, 148.11

ACCEPTED MANUSCRIPT
and 148.14 (d, J=3.0 Hz), 151.00 (2CH), 155.29 and 157.73 (d , J=244.0 Hz)], 161.24 (CH).
C-F
+
EI MS m/z (%): 235.58 ([M+1] , 100).
3
.2. Antimicrobial activity
The test microorganisms which are gram-positive and gram-negative as follows: Sa:
Staphylococcus aureus ATCC 25923, Ef: Enterococcus faecalis ATCC 29212, Ec:
Escherichiacoli ATCC 25922, Pa: Pseudomonas aeruginosa ATCC 27853, Kp: Klebsiella
pneumoniae ATCC 13883, Ah: Acinetobacterhaemolyticus ATCC 19002 were supplied from
the Hifzissihha Institute of Refik Saydam (Ankara, Turkey) and were. Amphicilin and
Gentamicin was used as standart compounds. For antimicrobial activity test, the obtained
compounds were dissolved in ethanol to prepare extract stock solution of 10.000
microgram/milliliter (µg/mL). The antimicrobial activities of the compounds were
quantitatively tested in the corresponding broth media by using double micro-dilution and the
minimal inhibitory concentration (MIC) values (ꢀg/mL) were detected. Mueller-Hinton broth
(MH) (Difco, Detroit, MI) at pH.7.3 and buffered Yeast Nitrogen Base (Difco, Detroit, MI) at
pH 7.0 were used for the assay. The micro dilution test plates were incubated for 18–24 h at
o
3
5 C [42]. All results were presented in Table 5.
3
.3. Antioxidant capacity
DPPH (2,2-diphenyl-1-picrylhydrazyl) radical scavenging activity: A 100 mL: chemical
solution was mixed with 1 mL of freshly prepared methanolic DPPH solution and then the
reaction mixture was incubated for 30 min at room temperature in the dark and was measured
at 520 nm as described by Blois [43]. The activity was given IC values.
5
0
FRAP (the ferric reducing ability of plasma): To 100 µL of each sample was added 2.9 mL
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.6H O in proportions of 10:1:1
2
o
(
v/v/v). The mixture was incubated for 30 min at 37 C and measured at 593 nm [44]. The
values were expressed as µmol of Trolox/g.
CUPRAC (cupric ion reducing antioxidant capacity): 100 µL of each chemical solution
was mixed with 900 µL bi-distilled water, 1 mL acetate buffer solution (1 mmol/L, pH: 7.0), 1
mL CuCl (10 mmol/L) and 1 mL 7.5 mmol/L neocuproine to a final volume of 4 mL. The
2
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 [45].

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Trolox was used as the standard calibration curves, and the results were expressed as µmol
Trolox equivalent per g.
3
.4. Urease inhibition assay [20]
Reaction mixture including 25 µL of Jack Bean urease, 55 µL of buffer (0.01 mol/L
K HPO , 1 mmol/L EDTA and 0.01 mol/L LiCl, pH 8.2) and 10 mmol/L urea were incubated
2
4
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
.1% v/v NaOCl) were added to each well. This mixture was incubated for 15 minutes more
0
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.
3
.5. Molecular Docking
Ligands were energy-minimized using GAMESS [46] module for ChemOffice version
Ultra 8.0.3 on an Intel®(Core™ i7-3632QM CPU @ 2.20GHz 2.20GHz) using Widows 8.1
operating system. Appropriate grid box points were determined by centering on Ni atoms for
3
each compound. Grid box for all ligands, defined with a size of 70*70*70 Å and a regular
space of 0.375 Å, was considered for docking.
"Crystal structure of Helicobacter pylori urease in complex with acetohydroxamic acid" pdb
file (PDB ID: 1E9Y) was get (www.rcsb.org) and was modified using the Maestro [47].
Subsequently, Gasteiger charges were calculated and the generated pdbqt files were saved by
ADT package version 1.5.6rc3. The Lamarckian Genetic Algorithm was used in medium type
so docking score and calculated Ki values were obtained using function of AutoDock 4.2
release 4.2.5.1 [48] software.
4
. Conclusions
In this study, we designed ecofriendly synthesis of novel Schiff bases. In synthesis process,
we applied there different method, conventional, microwave irradiation and ultasound
sonication, and among these, we obtained the best conditions in ultrasound sonication method.
By this method, the reaction time took 3h in conventional method decreased to 1.5 min and a

ACCEPTED MANUSCRIPT
significant increase in yield. The main purpose of this study is to have a lack of studies on the
synthesis of Schiff bases by ultrasonic sonication in the literature. Also, antimicrobail activity,
antioxidant capacity and antiurease activity of the synthesized compounds were investigated.
2
c was the most potent compound having IC =0,19±0,02 value. In antioxidant capacity assay
50
which are DPPH, CUPRAC and FRAP, 2c, 2f and 2b exhibited the best results. In particular,
almost all of the compounds exhibited excellent antiurease activity. In silico ADME
prediction were performed and drug likeness model score showed that 2a-i were found to
have higher drug likeness model score than the compounds 1a-i. And also, the theoretical
results obtained by ADME prediction and experimental inhibition studies were overlapped.
Interactions with key residues such as His221, Asp362, Ala365, NI3001, NI3002 of urease
were observed. The binding mode was produced and some compounds in the series showed
similar binding.
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Highlights
•
•
Urease inhibition of newly synthesized Schiff base derivatives
Screening antimicrobial and antioxidant activityi and in silico ADME prediction study
of novel drug-like compounds.
•
•
Docking studies were performed for the most active compounds and interaction modes
with enzyme active sites were determined.
Conventional, microwave and ultrasound prompted synthesis of new Schiff base
derivatives.