Conventional and microwave prompted synthesis, antioxidant, anticholinesterase activity screening and molecular docking studies of new quinolone-triazole hybrids

Article abstract of DOI:10.1016/j.bioorg.2018.03.017

The synthesis of ethyl 4-oxo-1,4-dihydroquinoline-3-carboxylates (4, 5) was performed via the reaction of corresponding anilines with diethyl ethoxymethylenemalonate under conventional and also microwave promoted conditions. The treatment of 4 and 5 affor

Full text of DOI:10.1016/j.bioorg.2018.03.017

Accepted Manuscript
Conventional and Microwave Prompted Synthesis, Antioxidant, Anticholines-
terase Activity Screening and Molecular Docking Studies of New Quinolone-
Triazole Hybrids
Arif Mermer, Neslihan Demirbaş, Yakup Şirin, Harun Uslu, Zeynep Özdemir,
Ahmet Demirbas
PII:
S0045-2068(17)30903-3
DOI:
https://doi.org/10.1016/j.bioorg.2018.03.017
YBIOO 2307
Reference:
Bioorganic Chemistry
To appear in:
Received Date:
Revised Date:
Accepted Date:
27 November 2017
9 March 2018
18 March 2018
Please cite this article as: A. Mermer, N. Demirbaş, Y. Şirin, H. Uslu, Z. Özdemir, A. Demirbas, Conventional and
Microwave Prompted Synthesis, Antioxidant, Anticholinesterase Activity Screening and Molecular Docking
Studies of New Quinolone-Triazole Hybrids, Bioorganic Chemistry (2018), doi: https://doi.org/10.1016/j.bioorg.
2018.03.017
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Conventional and Microwave Prompted Synthesis, Antioxidant,
Anticholinesterase Activity Screening and Molecular Docking Studies of
New Quinolone-Triazole Hybrids
Arif Mermer1, Neslihan Demirbaş1*, Yakup Şirin1, Harun Uslu2, Zeynep Özdemir2,
Ahmet Demirbas1
1Karadeniz Technical University, Department of Chemistry, 61000, Trabzon, Turkey
2Inonu University, Faculty of Pharmacy, Department of Pharmaceutical Chemistry, 44280
Malatya, Turkey
Abstract. The synthesis of ethyl 4-oxo-1,4-dihydroquinoline-3-carboxylates (4, 5) was
performed via the reaction of corresponding anilines with diethyl ethoxymethylenemalonate
under conventional and also microwave promoted conditions. The treatment of 4 and 5
afforded the corresponding hydrazides (6 and 7). These hydrazides were converted to the
corresponding carbo(thio)amides (9a-f and 10a-e) which were then subjected to an
intramolecular cyclisation leading to the formation of quinolone-triazole hybrids (11a-f and
12a-e). The newly synthesized compounds were screened for their biological activities such
as antioxidant capacity (AC) and acetylcholinesterase Activity. Inhibition of cholinesterases is
an effective method to curb Alzheimer’s disease, a progressive and fatal neurological
disorder. A series of some novel quinolone derivatives were designed, synthesized, and their
inhibitory effects on AChE were evaluated. We obtained our compounds and determined their
anticholinesterase activities according to the Ellman’s method. 9b and 10c showed the best
AChE inhibition with 0.48±0.02 and 0.52±0.07, respectively. Docking studies were
performed for the most active compounds (9b, 10c) and interaction modes with enzyme active
sites were determined. As a result of these studies, a strong interaction between these
compounds and the active sites of AChE enzyme was revealed.
Keywords. Quinolone, 1,2,4-triazole, molecular docking, antioxidant capacity,
anticholinesterase, microwave irradiation.
1. INTRODUCTION
Quinolones are one of most important kind of synthetic antibacterial agents due to their
well tolerability with excellent safety profile, favorable pharmacokinetic characteristics, broad
antibacterial spectrum and good treatment effectiveness [1-3]. The quinolone structures
include motifs exhibiting a wide variety of biological activities and they have been shown to
possess various pharmacological activities such as anticonvulsant [4], antitumor [5] and

antiviral [6]. These compounds have also been extensively used to treat genitourinary
infections, prostatitis, respiratory diseases, gastroenteritis, sexually transmitted diseases, as
well as skin and soft tissue infections [7, 8]. Another biological activity of quinolones is ROS
scavenging ability [9] and they have already been efficiently used for design of dual AChE
inhibitor [10]. Acetylcholinesterase (AChE) is known as serine hydrolase enzyme managing
on the hydrolysis of acetylcholine (ACh), which is a significant neurotransmitter for
arrangement of cognition in animals [11-14].Inhibition of AChE leads to the rise of ACh
levels in cholinergic synapses [15]. Thus, cholinesterase inhibitors are used in the treatment of
various neuromuscular disorders which occur as a result of reduced cortical and hippocampal
levels of ACh such as Alzheimer’s disease (AD) which is a complex neurodegenerative
disorder characterized by synapse dysfunction, neuronal death, and loss of memory and
learning ability [16-18]. Current treatment approaches for AD continue to be principally
symptomatic, with the major therapeutic AD, known as the cholinergic hypothesis, and
specifically on cholinesterase inhibition [19, 20]. Various AChE inhibitors such as tacrine,
donepezil, rivastigmine, and galantamine (Figure 1) have been used contemporarily for the
symptomatic treatment of AD [21-24]. In recent years, novel cholinesterase inhibitors from
natural resources or synthetic ways, which have coumarin, benzofuran, berberine, β-carboline,
benzophenone, ferulic acid, naphthyridine, triazine, and quinolone scaffolds as the main
pharmacophoric groups, have been reported [25-29].
Figure 1. Molecular structure of tacrine, donepezil, rivastigmine, and galantamine

Figure 2. Structural hypothesis for AChE inhibitors.
According to the X-ray crystallographic structure of AChE (PDB ID:4EY7), two main
binding sites has been determined: the catalytic anionic site (CAS) including Trp86, Tyr130,
Tyr133, Ser203, Glu334, Tyr337, Phe338, His447 and the peripheral anionic site (PAS)
consisting of Tyr72, Asp74, Tyr124, Trp286, Tyr341 [30, 31]. It has been reported that DNP
interacts with both PAS and CAS and thus it is situated in the active site concordantly owing
to the feature of dual binding site (DBS) [32, 33]. Analyses of binding modes of the DNP
indicate that the benzyl moiety is in a pi-pi interaction with the indole of Trp86 in the CAS.
The formation of hydrogen bond between the oxygen atom of the carbonyl group in the 1-
indanone and the amino group of Phe338 is a very significant interaction in terms of binding
to the active site. The 1-indanone constitutes a pi-pi interaction with the indole of Trp286 in
the PAS region. The piperidine has a position in the gorge to interact with Tyr337 and Tyr341
by doing a hydrogen bond. It also set up a van der Waals interaction with amino acids in both
CAS and PAS [34, 35]. Docking studies were performed for the most active compounds 9b
and 10c interaction modes with enzyme active sites were determined. Docking studies
revealed that there is a strong interaction between the active sites of AChE enzyme and these
compounds. Ideally, a cholinesterase inhibitor is expected to effectively interact with these
sites (Figure 2). Using molecular docking approach, we tried to get insights into binding
interactions of our derivatives in comparison with 9b, 10c in the active site of enzyme and
understand the facts that underlie the relationships between structural modifications on these
ligands and their efficacy.

Due to its multifactorial pathogenesis, the current strategy for the development of new
drugs for AD is focusing on multipotent molecules acting in a complementary manner, in
different neural and biochemical targets, which could be more efficacious for AD patients
[36]. Many quinolone derivatives have been studied for their biological activity in AD. They
are used as radical scavengers, such as vitamin E (a tocopherol), as copper or iron chelators
such as clioquinol, or as inhibitors of AChE such as tacrine. Following our studies on the
synthesis of quinolone carried out in our laboratory [37], we now report the synthesis of 4-
oxo-1,4-dihydroquinoline-3-carbonyl)hydrazine carbothioamide, 4-oxo-1,4-dihydroquinoline-
3-carbonyl)hydrazine carboxamide 3-(5-mercapto-4H-1,2,4-triazol-3-yl)quinolin-4(1H)-one
and 3-(5-oxo-4,5-dihydro-1H-1,2,4-triazol-3-yl)quinolin-4(1H)-one. These new compounds
were tested as AChE inhibitors and investigated antioxidant capacity. Finally, and thanks to
molecular docking, we have identified the interactions with AChE.
2. Results and discussion
2.1. Chemistry
In the present study, the ecofriendly synthesis, acetylcholinenzyme inhibition and
antioxidant activity screening, and molecular docking studies of new hybrid molecules was
contemplated. The structures of newly synthesized compounds were established on the basis
1
of physicochemical, elemental analysis and spectral data (FT IR, H NMR, 13C NMR and EI-
MS).
Compounds 3, 5 and 7 were synthesized in the following procedure [38]. The complete
conversion of hydrazides 8 was observed after microwave irradiation at 100 W for 25 min.
Synthesis of corresponding carbo(thio)amides (9a-f) and (10a-e) was accomplished by
nucleophilic attack of the 7 and 8 hydrazide compounds to the alkyliso(thio) cyanates
(Scheme 1). The reaction conditions were investigated in ethanol under reflux and also
microwave irradiation conditions to maximize the yield of the product. To optimize reaction
conditions, the synthesis of compound 9a was chosen as model reaction and microwave
(MW) irradiation was implemented at different power values of 50, 100, 150, 200 and 250 W
(the progress of reaction was monitored by TLC) (Table 1). When compared to conventional
method, microwave irradiation reduced the reaction time from 5 h to 10 min. It is notable to
underline that shorter reaction time, lower microwave energy or very high microwave energy
power give rise to lower conversion rate, while increasing reaction time or MW power
resulted in fragmentation of the target product as revealed by TLC analysis. In the FT IR

spectra, the signal originated from –C=S function of compounds 9a-d and 10a-dappeared
between 1223-1263 cm-1. In the 13C NMR spectra of these compounds (9a–f, 10a-e), –C=S or
–C=O function resonated between 153.05–175.37 ppm. Further evidence for the formation of
carbo(thio)amides was the presence of three –NH- signals (exchangeable with D2O) at 7.04–
1
12.75 ppm in the H NMR spectra. The elemental analysis data of carbon, hydrogen and
nitrogen were formed to be satisfactory and within the permissible limit error.
The synthesis of triazoles (11a–f, 12a-e) was achieved by the treatment of NaOH in
ethanol-water under reflux condition as well as under microwave conditions in order to
introduce the 1,2,4-triazole nucleus to piperazine skeleton, because, according to molecular
hybridization concept, more efficacious bioactive compounds can be designed by combining
two or more biologically active heterocyclic systems together in a single molecular
framework [39, 40]. In order to maximize product yield in the microwave irradiation method,
the optimization operation at power values of 50, 100, 150, 200, 250 was carried out by
o
selecting compound 11a while keeping the temperature at 150 C (Table 1). The best product
yield was obtained at 200 Watt. The conversion of carbo(thio)amides to triazoles did not take
place completely at low temperature and low power values, while high temperature and power
values were applied, compounds started to decompose and product yields decreased.
Furthermore, compared with conventional thermal heating, microwave irradiation decreased

the reaction time from 6–12 h to 20 min and increased the yields from 71%–82% to 79%–
90% (Table 2).
Table 1. Optimization conditions for 9a and 11a in microwave irradiation
Power
(W)
9a
Time
(min)
9a
5
10
20
30
40
Yield
(%)
9a
70
84
78
75
60
11a
50
100
150
200
250
11a
5
10
20
30
40
11a
50
57
68
81
74
50
100
150
200
250
Table 2. Time, power and yield for the synthesized compounds.
Comp
d.
Microwave irradiation
method
Conventional
method
Melting
Crystallization
Solvent
point oC
Yield Time (h)
Time
(min)
Power
(W)
Yield
(%)
(%)
86
84
79
84
86
80
87
84
79
82
79
86
88
5
225
50
1
10
10
3
76
79
71
76
80
72
79
81
69
74
69
78
80
143-144
>300
DMF:H2O (1:3)
Ethyl acetate
Ethanol
4
6
20
25
20
20
20
20
25
25
20
20
20
20
100
100
100
100
100
100
100
100
100
100
100
260
8
223-224
231-232
244
Ethyl acetate
Ethyl acetate
Ethyl acetate
Ethyl acetate
Methanol
9a
9b
9c
4
6
3
216-217
245-246
241-242
286
9d
9e
5
6
Methanol
9f
4
Acetone
10a
10b
10c
10d
5
236-237
236
Ethanol
5
Ethanol
4
209-210
Methanol

25
30
30
30
30
35
35
30
30
30
35
35
100
200
200
200
200
200
200
200
200
200
200
200
80
81
80
89
86
82
78
83
81
90
82
79
6
8
66
70
75
82
80
76
71
76
72
78
81
73
211-212
>300
Methanol
Ethanol
10e
11a
11b
11c
11d
11e
11f
9
280-281
254
Methanol
Methanol
Ethanol
7
6
>300
7
>300
Acetone
10
7
>300
Methanol
Ethyl acetate
Ethanol
>300
12a
12b
12c
12d
12e
9
228
9
253-254
223-224
227
Ethyl acetate
Ethyl acetate
Methanol
11
12
It is well known that 1,2,4-triazol-3-(thi)ol derivatives can exist as mercapto-thioxo (or
enol-keto) tautomeric forms. The –SH proton due to mercapto form resonates at about 13–14
ppm, while NH signal originated from thioxo tautomeric form appears between 9–12 ppm as
D2O exchangeable signals [41-45]. In the FT-IR spectra of compounds 11a-d and 12a-d, the
presence of stretching bands ranging from 2830 cm-1 to 2853 cm-1 originated from -SH
function supported the mercapto form for these compounds. The -SH proton of these
1
compounds resonated between 13.85 and 14.13 ppm in the H NMR spectra. On the other
hand, our previous studies showed that the compounds having 5-oxo-4-alkyl-4,5-dihydro-1H-
1,2,4-triazoles exist almost in keto form [37,41-45]. Another evidence supporting the
formation of the targeted compounds, 11a-f and 12a-e were obtained by the appearance of
[M+1], [M+2], [M+Na] and/or [M+K] ion peaks at corresponding m/z values confirming their
molecular masses; and these compounds have given elemental analysis results consistent with
the proposed structures.
According to the X-ray crystallographic structure of AChE (PDB ID:4EY7), two main
binding sites has been determined: the catalytic anionic site (CAS) including Trp86, Tyr130,
Tyr133, Ser203, Glu334, Tyr337, Phe338, His447 and the peripheral anionic site (PAS)
consisting of Tyr72, Asp74, Tyr124, Trp286, Tyr341, [45, 46]. It has been reported that DNP
interacts with both PAS and CAS and thus it is situated in the active site concordantly owing

to the feature of dual binding site (DBS) [32, 33, 46]. Analyses of binding modes of the DNP
indicate that the benzyl moiety is in a pi-pi interaction with the indole of Trp86 in the CAS.
The formation of hydrogen bond between the oxygen atom of the carbonyl group in the 1-
indanone and the amino group of Phe338 is a very significant interaction in terms of binding
to the active site. The 1-indanone constitutes a pi-pi interaction with the indole of Trp286 in
the PAS region. The piperidine has a position in the gorge to interact with Tyr337 and Tyr341
by doing a hydrogen bond. It also set up a van der Waals interaction with amino acids in both
CAS and PAS [34, 35, 47]. Docking studies were performed for the most active compounds
9b and 10c interaction modes with enzyme active sites were determined. Docking studies
revealed that there is a strong interaction between the active sites of AChE enzyme and these
compounds. Ideally, a cholinesterase inhibitor is expected to effectively interact with these
sites (Figure 2). Using molecular docking approach, we tried to get insights into binding
interactions of our derivatives in comparison with 9b, 10c in the active site of enzyme and
understand the facts that underlie the relationships between structural modifications on these
ligands and their efficacy.
Due to its multifactorial pathogenesis, the current strategy for the development of new
drugs for AD is focusing on multipotent molecules acting in a complementary manner, in
different neural and biochemical targets, which could be more efficacious for AD patients
[36]. Many quinolone derivatives have been studied for their biological activity in AD. They
are used as radical scavengers, such as vitamin E (a tocopherol), as copper or iron chelators
such as clioquinol, or as inhibitors of AChE such as tacrine. Following our studies on the
synthesis of quinolone carried out in our laboratory [37], we now report the synthesis of 4-
oxo-1,4-dihydroquinoline-3-carbonyl)hydrazine carbothioamide, 4-oxo-1,4-dihydroquinoline-
3-carbonyl)hydrazine carboxamide 3-(5-mercapto-4H-1,2,4-triazol-3-yl)quinolin-4(1H)-one
and 3-(5-oxo-4,5-dihydro-1H-1,2,4-triazol-3-yl)quinolin-4(1H)-one. These new compounds
were tested as AChE inhibitors and investigated antioxidant capacity. Finally, and thanks to
molecular docking, we have identified the interactions with AChE.
2.2. Biological Activity
2.2.1. Antioxidant Activity
To determine antioxidant capacity (AC, µmol TE/g) value of the newly synthesized
compounds, DPPH, FRAP and CUPRAC assays were assayed. Compounds 10c
(3912.33±4.71 µmol TE/g), followed 9 and 10e, 12e (3460.55±8.35 µmol TE/g), followed 9e
(3379.89±13.00 µmol TE/g), 11e (3336.03±16.00 µmol TE/g) and 9b (3219.07±3.00 µmol

TE/g) for FRAP, 10e (7241.13±28.53µmol TE/g), followed 10c and 11a, 9c (6266.43±50.12
µmol TE/g), followed 8 (6185.61±21.01 µmol TE/g), 11e (5194.36±15.66 µmol TE/g) and 9b
(4392.85±11.01 µmol TE/g) for CUPRAC. Trolox was used as standard for DPPH radical
scavenging method and the results are given SC50 value. According to this method, 9b, 11e,
10c, 9e, 10e, 8 and 9c had the SC50 values, respectively. On the other hand, 12b
(123.17±1.18 µmol TE/g) and 10b (949.83±3.54 µmol TE/g) for FRAP, 9d (491.86±1.01
µmol TE/g) and 9d (3.86±0.02) had the lowest AC values among the synthesized compounds
(Table 5). In the table, for 12c no AC value and acetylcholinesterase activity was determined.
Also, while 10b had no activity for AChE and CUPRAC, 12b had no activity for CUPRAC,
DPPH and AChE.
Table 5. Antioxidant capacity (AC) values and acetylcholinesterase activity of 23 synthesized
novel compound.
EeAChE
Activity
Compound
FRAP
DPPH
SC50
CUPRAC
IC50 (mg/mL)
(µmol TE/g)
(µmol TE/g)
8
3711.50±1.18
1606.16±7.00
3219.07±3.00
1396.42±2.00
1378.99±5.00
0.12±0.01 6185.61±21.01
3.12±0.07 1860.79±3.54
0.75±0.05
5.26±0.01
0.48±0.02
0.81±0.01
-
9a
9b
0.07±0.00 4392.85±11.01
0.24±0.00 6266.43±50.12
9c
3.86±0.02
491.86±1.01
9d
9e
3379.89±13.00 0.10±0.00 3272.21±18.69
0.98±0.02
0.76±0.01
5.51±0.08
-
1436.38±4.00
1535.67±2.36
949.83±3.54
3912.33±4.71
1370.67±7.07
3770.67±9.43
1042.74±6.00
9f
0.36±0.00 4247.32±29.12
1.24±0.03 3842.64±10.85
10a
10b
10c
10d
10e
11a
2.21±0.07
-
0.09±0.01 6987.45±11.14
1.39±0.04 2896.12±22.14
0.11±0.00 7241.13±28.53
0.52±0.07
6.85±0.07
0.68±0.02
1.86±0.03
0.39±0.00
6723.57±5.05

1448.16±4.00
987.07±4.00
1219.30±3.00
1.13±0.01 1444.57±10.30
1.54±0.03
3.43±0.01
2.47±0.01
0.57±0.02
2.25±0.01
3.29±0.05
-
11b
11c
2.18±0.03
2709.29±5.09
11d
0.98±0.00
3165.43±8.08
11e
3336.03±16.00 0.09±0.00 5194.36±15.66
1187.12±4.00
1233.46±3.67
123.17±1.18
-
3.15±0.02
2234.17±5.34
11f
2.01±0.02
2348.24±9.65
12a
-
-
-
-
12b
-
12c
1454.00±9.43
3460.55±8.35
1.19±0.03 2965.13±32.12
0.54±0.00 4278.15±22.75
0.04±0.00
4.18±0.01
0.64±0.02
12d
12e
Trolox
Donepezil
0.03±0.00
2.2.2. Anticholinesterase activity
In vitro AChE inhibition of synthesized compounds was determined by the method of
Ellman et al. [48] using donepezil as reference and the activity data of the compounds were
summarized as IC50 value ± standard deviation (SD) values at 100 µg/ml concentration in
Table 3. None of the test compounds showed higher AChE inhibition than donepezil. All
compounds showed inhibitory activity against AChE. 9b and 10c showed the best AChE
inhibition with 0.48±0.02 and 0.52±0.07, respectively. In the structure of the 4-member
group compound we have synthesized, there are groups that can interact with the choline
binding site (CAS) and peripheral anionic site (PAS) and nitrogen atoms that can be
protonated. This allows the compounds to interact with the AChE enzyme and thus to show
activity. It is also evidenced by molecular modeling studies in which the compounds are
linked to the CAS and PAS regions of the enzyme. The compounds with the highest activity
are in group 9. This indicates that the hydrazide structure, including the protonatable
nitrogen atom between the quinolone ring attached to the PAS and the group bound to the
CAS, contributes to the activity more than the triazole ring. The electron attracting halogen
substituents added to the quinolone ring did not change significant activity in the molecule.

Table 3. AChE inhibition values (IC50) of the title compounds at 100 µg/mL
AChE Inhibition
Compound
IC50 (mg/mL) ± SD*
0.75±0.05
5.26±0.01
0.48±0.02
0.81±0.01
**
8
9a
9b
9c
9d
0.98±0.02
0.76±0.01
5.51±0.08
**
9e
9f
10a
10b
10c
10d
10e
11a
11b
11c
11d
11e
11f
12a
0.52±0.07
6.85±0.07
0.68±0.02
1.86±0.03
1.54±0.03
3.43±0.01
2.47±0.01
0.57±0.02
2.25±0.01
3.29±0.05

**
12b
12c
**
4.18±0.01
0.64±0.02
0.03±0.00
12d
12e
Donepezil
*Standart deviation, **IC50 value not detected
The fluorine atom in the 4th position of the phenyl ring, which is thought to be located in
the CAS, decreases activity markedly (9d, 11d, 10d, 12d). Compounds which are attached to
the phenyl ring of the nitrogen atom which may be protonated are generally more active than
ethyl chain-bearing derivatives. This suggests that the phenyl ring enhances the interaction
of van der Waals interactively with this region. It has been observed that activity is generally
higher in the nitrogen atom bearing benzyl structures bound by a methyl bridge instead of
the compounds directly attached by the phenyl ring. This suggests that the unpaired electrons
of the nitrogen atom bound to the phenyl ring are introduced into the ring to resonance and
that the nitrogen is less bound to the enzyme by reducing the probability of protonation.
Molecular modelling studies also demonstrate this decrease in the phenyl ring relative to the
benzyl.
2.2.3. Molecular docking studies
Glide predicted good fit for 9b, 10c in the active site of human AChE (PDB ID: 4EY7)
along the gorge with some characteristic interactions previously defined for some known
inhibitors (Figure 3).

9b
10c
Figure 3. Binding mode of compound 9b and 10c into the AChE (PDB ID: 4EY7) binding
cavity. For clarity, only interacting residues are displayed. Ligand (green), interacting key
residues (RasMol amino color) are represented as stick models. H-bonds are shown as spheres
(turquoise) lines and π- π interactions are shown as cylinders (yellow).
The 1-ethylquinolin-4(1H)-one-3-yl moiety of 9b and 10c occupied the choline-binding
pocket and engaged in strong pi-pi interactions against Trp286 aromatic side chain
furthermore weak close van der Waals interactions with Tyr72, Asp74, Tyr124, Tyr341,
Trp286 in the PAS region and Trp86, Ser203, Tyr337, Phe338in the CAS region. These
residues are reportedly involved in ligand-receptor complexes of tacrine, galantamaine,
huperzine A, and donepezil [47, 49]. Donepezil was reported to form water-mediated H-bonds
with Tyr337 and Tyr341 via its quaternary N of the piperidine ring. Instead, via its carbonyl
oxygen of the quinolin-4(1H)-one ring, 10c formed a direct H-bond to Phe295 backbone NH.
The latter interaction was reportedly observed with the carbonyl oxygen of the indanone
moiety of donepezil [47]. The quinolin-4(1H)-one ring of 10c occupied the lower PAS and π-
π interactions with Tyr341 aromatic side chain (Table 4).

Table 4. Molecular docking binding scores and binding interactions of compounds 9b and
10c, and within the AChE (PDB ID: 4EY7) active site. Residues participating in hydrogen
bonds and close van der Waals contacts (<3,75 Å) with the inhibitors are shown and distances
detected by selecting Closer Than VDW algorithms.
H-bonds
Close van der Waals contacts
Estimated
Inhibition
Estimated
Free
Comp.
Distance
Number of
Constant, Ki,
Residue
Residue
Distance (Å)
Energy of
Binding
(kcal/mol)
(Å)
Interaction
Temp. = 298.15
K
Tyr72
Asp74
Trp86
Tyr124
Ser203
3,318-3,638
2
3
2
5
1
7
2
4
3
6
9
2
1
6
1
in 3,094-3,598 range
3,677-3,7
in 1,776-3,234 range
2,827
Tyr124
1,766
2,12
(C=O--)
-10.25
30.74 nM
Trp286+ in 3,18-3,602 range
9b
Phe295
(N-H--)
Ser293
Phe297
Tyr337
Phe338
Tyr341
Asp74
Trp86
2,863-3,657
in 2,214-3,387 range
in 3,232-3,434 range
in 2,847-3,452 range
in 2,914-3,592 range
2,929-3,021
3,671
-8.38
716.31 nM
-
10c
Tyr124
in 2,622-3,094 range
Trp286+ 3,027

Ser293
Val294
Phe295
3,664
1
3
1
2
1
6
7
9
3
in 3,17-3,568 range
2,767
Arg296 2,509-3,249
Phe297 2,766
Tyr337+ in 3,086-3,678 range
Phe338 in 2,941-3,676 range
Tyr341+ in 2,784-3,603 range
His447 in 2,85-3,169 range
+ : π- π interactions; nM: nanomolar
Binding modes of 9b, 10c and co-crystal ligand Donepezil superimposed with the rest in
AChE active side that shown 9b’s and 10c’s binding mode and interactions in human AChE
active side very similar to E20 (Figure 4).

Figure 4. Binding modes of 9b, 10c, and co-crystal ligand Donepezil (Pdb ID: E20)
superimposed with the rest in AChE active site. View of the active site show ligand E20 and
binding-site interacting residues as green, 9b as red, 10c as blue by PyMOL (The PyMOL
Molecular Graphics System, Version 1.8 Schrödinger, LLC).
3. Experimental
3.1. General
All the chemicals were purchased from Fluka Chemie AG Buchs (Switzerland) and used
without further purification. Melting points of the synthesized compounds were determined in
open capillaries on a Büchi B-540 melting point apparatus and are uncorrected. Reactions
were monitored by thin-layer 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
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 relative to Me4Si as an internal reference, J values are given in Hz. The
elemental analysis was performed on a Costech Elemental Combustion System CHNS-O
elemental analyzer. All the compounds gave C, H and N analysis within -0.4 % of the
theoretical values. The mass spectra were obtained on a Quattro EI-MS (70 eV) Instrument.
Microwave irradiated reactions were performed in a CEM Discovery mono-mode synthesis
reactor.
3.1.1. General Method for the Preparation of Compounds 3 and 4
Method 1. The mixture of the corresponding aniline (10 mmol) and diethyl ethoxymethylene
malonate (10 mmol) in diphenyl ether (10 mL) was heated at 250 oC in an oil bath for 1 h. On
cooling the mixture to room temperature, a white solid formed. This crude product was
filtered off, washed with hexane and purified through recrystallization from DMF to give
corresponding compound.
Method 2. The mixture of the corresponding aniline (10 mmol) and diethyl ethoxymethylene
malonate (10 mmol) in diphenyl ether (4 mL) was irradiated in monomod microwave reactor
in closed vessel at 225 oC, 200 W, for 5 min. The white solid formed was filtered off, washed
with hexane and purified through recrystallization from DMF to give corresponding
compound.

Ethyl 7-chloro-6-fluoro-4-oxo-1,4-dihydroquinoline-3-carboxylate (3) [38]: FT-IR(υmax
,
cm-1): 3389 (NH), 3065 (ar-CH), 1687 (C=O). Mp. 138 oC.
3.1.1.1. Ethyl 6-methyl-4-oxo-1,4-dihydroquinoline-3-carboxylate (4)
Elemental analysis for C13H13NO3, calculated (%), C, 67.52; H, 5.67; N, 6.06, found (%), C,
67,12; H, 5,28; N, 5,99. FT-IR (υmax, cm-1): 3152 (NH), 3094 (ar-CH), 1690 (C=O), 1581
1
(C=N). H NMR (DMSO-d6, δppm): 1.28 (t, 3H, J= 4 Hz, CH3), 2.43 (s, 3H, CH3), 4.22 (s,
2H, CH2), 7.00 (s, 1H, ArH), 7.39 (s, 1H, ArH), 7.96 (s, 1H, CH), 8.49 (s, 1H, CH), 12.19 (s,
1H, NH). 13C NMR (DMSO-d6, δppm): 14.80 (CH3), 21.26 (CH3), 59.94 (CH2), arC: [109.98
(C), 119.07 (CH), 123.89 (CH), 127.67 (C), 130.50 (CH), 134.59 (C), 137.42 (C)], 144.84
(CH), 165.39 (C=O), 173.76 (C=O). EI MS m/z (%): 186.11 (25), 232.23 ([M+1]+, 58),
254.19 ([M+Na]+, 100).
3.1.2. General Method for The Synthesis of Compounds 5 and 6
Method 1. A mixture of compound 3 or 4 (10 mmol), K2CO3 (50 mmol) and ethyl bromide
o
(50 mmol) in 20 mL DMF was heated at 90 C for 10 h. After evaporating the solvent under
reduced pressure to dryness an oily mass appeared. This was treated with water and extracted
with 10 mL of chloroform three times. The combined organic layer was dried over Na2SO4
and evaporated to dryness. The crude product was recrystallized from ethyl acetate to afford
the corresponding compound.
Method 2. A mixture of compound 3 or 4 (10 mmol), K2CO3 (50 mmol) and ethyl bromide
(50 mmol) in 5 mL of DMF was irradiated in monomod microwave reactor in closed vessel at
o
80 C, 50 W for 20 min. After evaporating the solvent under reduced pressure to dryness an
oily mass formed. This was dissolved in water and extracted with 10 mL of chloroform three
times. The combined organic layer was dried over Na2SO4 and evaporated to dryness. The
crude product was recrystallized from ethyl acetate to afford the corresponding compound.
Ethyl 7-chloro-1-ethyl-6-fluoro-4-oxo-1,4-dihydroquinoline-3-carboxylate (5) [38]: FT-IR
(υmax, cm-1): 3048 (ar-CH), 1678 (C=O). Mp. >300oC.
3.1.2.1. Ethyl 1-ethyl-6-methyl-4-oxo-1,4-dihydroquinoline-3-carboxylate (6)
Elemental analysis for C15H17NO3, calculated (%), C, 69.48; H, 6.61; N, 5.40, found (%), C,
1
69,18; H, 6,42; N, 5,11. FT-IR (υmax, cm-1): 3050 (ar-CH), 1675 (C=O), 1595 (C=N). H
NMR (DMSO-d6, δppm): 1.22 (t, 3H, J= 12 Hz, CH3), 1.38 (t, 3H, J= 8 Hz, CH3), 2.38 (s,

3H, CH3), 4.10 (q, 2H, J=16 Hz, CH2), 4.23 (q, 2H, J=12 Hz, CH2), 6.70 (d, 1H, J=8 Hz,
ArH), 7.27 (dd, 1H, J=8 Hz, ArH), 7.71 (d, 1H, J=5 Hz, ArH), 8.33 (s, 1H, CH). 13C NMR
(DMSO-d6, δppm): 12.40 (CH3), 14.69 (CH3), 21.20 (CH3), 48.85 (CH2), 61.50 (CH2), arC:
[109.25 (C), 118.80 (CH), 127.78 (C), 129.04 (CH), 132.59 (CH), 133.87 (C), 140.82 (C)],
147.60 (CH), 164.77 (C=O) , 174.70 (C=O). EI MS m/z (%): 282.33 ([M+Na]+, 31), 298.30
([M+K]+, 100).
3.1.3. General method for the synthesis of compounds 7 and 8
Method 1. NH2NH2 (25 mmol) was added the solution of the corresponding compound 5 or 6
(10 mmol) in ethanol and the mixture was refluxed for 10 h. The product obtained upon
evaporating the solvent under reduced pressure was washed with water and recrystallized
from ethanol to afford the desired product.
Method 2. NH2NH2 (25 mmol) was added the solution of the corresponding compound 5or 6
(10 mmol) in ethanol and the mixture was irradiated in monomod microwave reactor in closed
o
vessel at 100 C, 80 W for 25 min. The product obtained upon evaporating the solvent under
reduced pressure was washed with water and recrystallized from ethanol to afford the desired
product.
7-chloro-1-ethyl-6-fluoro-4-oxo-1,4-dihydroquinoline-3-carbohydrazide (7) [38]: FT-IR
(υmax, cm-1): 3323 (NH), 3248 and 3212 (NH2), 3013 (ar-CH). Mp. 265oC.
3.1.3.1. 1-Ethyl-6-methyl-4-oxo-1,4-dihydroquinoline-3-carbohydrazide (8)
Elemental analysis for C15H15N3O2, calculated (%), C, 63.66; H, 6.16; N, 17.13; found (%), C,
63,06; H, 6,09; N, 6,07. FT-IR (υmax, cm-1): 3319 (NH), 3198-3170 NH2), 3047 (ar-CH), 1650
1
(C=O), 1590 (C=N). H NMR (DMSO-d6, δppm): 1.31 (t, 3H, J= 16 Hz, CH3), 2.0 (bs, 2H,
NH2), 2.34 (s, 3H, CH3), 4.62 (q, 2H, J=16 Hz, CH2), 6.68 (d, 1H, J=8 Hz, ArH), 7.29 (d, 1H,
J=8 Hz, ArH), 7.38 (s, 1H, ArH), 8.0 (s, 1H, NH), 8.96 (s, 1H, CH). 13C NMR (DMSO-d6,
δppm): 14.37 (CH3), 20.70 (CH3), 48.21 (CH2), arC: [113.50 (CH), 117.51 (C), 125.73 (C),
126.63 (CH), 131.47 (C), 134.255 (CH), 138.59 (C), 181.80 (C=O)], 141.95 (CH), 162.14
(C=O). EI MS m/z (%): 214.15 (90), 246.16 ([M+1]+, 88), 286.29 (100).
3.1.4. General method for the synthesis of compounds 9a-f and 10a-e
Method 1. A mixture of compound 7 or 8 (10 mmol) and the corresponding iso(thio)cyanate
(15 mmol) in ethanol was refluxed for 3-6 h (Table 2). After evaporating the solvent under

reduced pressure, a solid obtained. The crude product was purified by recrystallisation from
an appropriate solvent.
Method 2. A mixture of compound 7 or 8 (10 mmol) and the corresponding iso(thio)cyanate
o
(15 mmol) was irradiated in monomod microwave reactor in closed vessel at 100 C, 100
Watt for 20-25 min. The solid obtained upon the evaporation of solvent under reduced
pressure was purified by crystallization from an appropriate solvent.
3.1.4.1. N-Phenyl-2-(7-chloro-1-ethyl-6-fluoro-4-oxo-1,4-dihydroquinoline-3-carbonyl)
hydrazinecarbothioamide (9a)
Elemental analysis for C19H16ClFN4O2S, calculated (%), C, 54.48; H, 3.85; N, 13.38; found
(%), C, 54,16; H, 3,55; N, 13,15. FT-IR (υmax, cm-1): 3277 (NH), 3070 (ar-CH), 1645 (C=O),
1552 (C=N). 1H NMR (DMSO-d6, δppm): 1.22 (t, 3H, J=8 Hz, CH3), 4.74 (s, 2H, CH2), 7.23
(s, 1H, J=7 Hz, ArH), 7.31 (s, 3H, ArH), 7.61 (s, 2H, ArH), 8.06 (s, 1H, ArH), 8.58 (bs, 1H,
13
NH), 8.73 (s, 1H, CH), 9.51 (bs, 1H, NH), 11.58 (bs, 1H, NH). C NMR (DMSO-d6, δppm):
15.73 (CH3), 48.47 (CH2), arC: [117.78 (CH), 126.09 (CH), 127.54 (C), 128.86 (2CH),
131.55 (CH), 134.89 (2CH), 135.43 (C), 137.38 (C), 139.76 (C), 142.34 (C), 153.85-156.88
(dC-F, J=303 Hz, C)], 147.39 (CH), 157.31 (C=O), 171.73 (C=S), 175.38 (C=O). EI MS m/z
(%): 441.34 ([M+Na]+, 100).
3.1.4.2.
N-Benzyl-2-(7-chloro-1-ethyl-6-fluoro-4-oxo-1,4-dihydroquinoline-3-carbonyl)
hydrazinecarbothioamide (9b)
Elemental analysis for C20H18ClFN4O2S, calculated (%), C, 55.49; H, 4.19; N, 12.94; found
(%), C, 55,21; H, 4,02; N, 12,76. FT-IR (υmax, cm-1): 3255 and 3146 (NH), 3030 (ar-CH),
1
1668 (C=O), 1544 (C=N). H NMR (DMSO-d6, δppm): 1.37 (t, 3H, J=16 Hz, CH3), 4.57 (d,
2H, J=8 Hz, CH2), 4.73 (d, 2H, J=4 Hz, CH2), 7.23 (d, 1H, J=7 Hz, ArH), 7.30 (d, 4H, J=7
Hz, ArH), 8.16 (d, 1H, J=8 Hz, ArH), 8.31 (d, 1H, J=7 Hz, ArH), 8.60 (bs, 1H, NH), 8.94 (s,
13
1H, CH), 9.59 (bs, 1H, NH), 11.17 (bs, 1H, NH). C NMR (DMSO-d6, δppm):15.08 (CH3),
47.31 (CH2), 49.22 (CH2), arC: [110.94 (C), 112.83 (d, J=22 Hz, CH), 120.87 (CH), 121.81
(CH), 127.11 (CH), 127.58 (CH), 127.90 (d, J=6 Hz, C), 128.55 (CH), 136.19 (C), 139.70
(C), 145.34 (C), 153.75-156.21 (dC-F, J=246 Hz, C)], 148.92 (CH), 160.64 (C=O), 174.18
(C=S), 179.26 (C=O). EI MS m/z (%): 395.39 (32), 433.89 ([M+1]+, 100).

3.1.4.3.
2-(7-Chloro-1-ethyl-6-fluoro-4-oxo-1,4-dihydroquinoline-3-carbonyl)-N-ethyl
hydrazinecarbothioamide (9c)
Elemental analysis for C15H16ClFN4O2S, calculated (%), C, 48.58; H, 4.35; N, 15.11; found
(%), C, 48,20; H, 4,19; N, 14,98. FT-IR (υmax, cm-1): 3267 and 3202 (NH), 3029 (ar-CH),
1677 (C=O), 1545 (C=N). 1H NMR (DMSO-d6, δppm): 1.08 (s, 3H, CH3), 1.39 (s, 3H, CH3),
3.29 (s, 2H, CH2), 4.57 (s, 2H, CH2), 7.99 (s, 1H, ArH), 8.17 (d, 1H, J=8 Hz, ArH), 8.30 (s,
13
1H, CH), 8.91 (s, 1H, NH), 9.47 (bs, 1H, NH), 11.33 (bs, 1H, NH). C NMR (DMSO-d6,
δppm): 14.85 (CH3), 15.107 (CH3), 39.03 (CH2), 49.24 (CH2), arC: [110.92 (C), 112.82 (d,
J=22 Hz, CH), 120.86 (CH), 126.80 (d, J=20 Hz, C), 127.90 (d, J=6 Hz, C), 136.20 (C),
153.75-156.21 (dC-F, J=246 Hz, C)], 148.91 (CH), 162.31 (C=O), 174.17 (C=S), 181.10
(C=O).EI MS m/z (%): 403.46 ([M+Na]+, 100), 404.40 ([M+1+Na]+, 26).
3.1.4.4. 2-(7-Chloro-1-ethyl-6-fluoro-4-oxo-1,4-dihydroquinoline-3-carbonyl)-N-(4-fluoro
phenyl) hydrazinecarbothioamide (9d)
Elemental analysis for C19H15ClF2N4O2S, calculated (%), C, 52.24; H, 3.46; N, 12.82; found
(%), C, 52,08; H, 3,21; N, 12,69. FT-IR (υmax, cm-1): 3282 and 3158 (NH), 3039 (ar-CH),
1686 (C=O), 1542 (C=N). 1H NMR (DMSO-d6, δppm): 1.21 (t, 3H, J=8 Hz, CH3), 4.10-4.13
(m, 2H, CH2), 6.75 (d, 1H, J=4 Hz, ArH), 7.04 (s, 1H, ArH), 7.08 (d, 1H, J=7.1 Hz, ArH),
7.27 (t, 2H, J=7.2 Hz, ArH), 7.45 (d, 1H, J=8 Hz, ArH), 8.44 (s, 1H, CH), 8.65 (bs, 1H, NH),
9.14 (bs, 1H, NH), 9.84 (bs, 1H, NH). 13C NMR (DMSO-d6, δppm): 14.51 (CH3), 49.40
(CH2), arC: [114.17 (CH), 116.39 (C), 117.88 (2CH), 119.17 (2CH), 121.18 (d, J=21.0 Hz,
C), 127.26 (C), 128.09 (C), 137.16 (C), 152.22-154.55 (dC-F, J=233 Hz, C), 155.58-157.91
(dC-F, J=233 Hz, C)], 147.56 (CH), 162.06 (C=O), 175.29 (C=S), 179.94 (C=O). EI MS m/z
(%): 196.36 (42), 205.37 (100), 459.40 ([M+Na]+, 78).
3.1.4.5. 2-(7-Chloro-1-ethyl-6-fluoro-4-oxo-1,4-dihydroquinoline-3-carbonyl)-N-phenyl
hydrazinecarboxamide (9e)
Elemental analysis for C19H16ClFN4O3, calculated (%), C, 56.65; H, 4.00; N, 13.91; found
(%), C, 56,31; H, 3,89; N, 13,76. FT-IR (υmax, cm-1): 3301 (NH), 3048 (ar-CH), 1677 (C=O),
1537 (C=N). 1H NMR (DMSO-d6, δppm): 1.41 (s, 3H, CH3), 4.57 (d, 2H, J=8 Hz, CH2), 6.96
(s, 1H, ArH), 7.26 (s, 1H, ArH), 7.46 (d, 1H, J=8 Hz, ArH), 8.16 (d, 2H, J=8 Hz, ArH), 8.28
(s, 1H, CH), 8.84 (s, 1H, NH), 8.96 (s, 1H, NH), 12.53 (bs, 1H, NH). 13C NMR (DMSO-d6,

δppm): 15.04 (CH3), 49.36 (CH2), arC: [111.24 (C), 112.79 (d, J=22 Hz, CH), 118.82 (CH),
120.80 (2CH), 122.33 (CH), 126.75 (d, J=20 Hz, 2C), 129.12 (2CH), 136.19 (C), 140.08 (C),
153.69-156.15 (dC-F, J=246 Hz, C)], 149.14 (CH), 154.11 (C=O), 160.30 (C=O), 174.62
(C=O).EI MS m/z (%): 102 (41), 314 (100), 316 (46), 402 ([M]+, 26).
3.1.4.6.
N-Benzyl-2-(7-chloro-1-ethyl-6-fluoro-4-oxo-1,4-dihydroquinoline-3-carbonyl)
hydrazinecarboxamide (9f)
Elemental analysis for C19H16ClFN4O3, calculated (%), C, 56.65; H, 4.00; N, 13.91; found
(%), C, 56,31; H, 3,89; N, 13,76. FT-IR (υmax, cm-1): 3319 and 3203 (NH), 3033 (ar-CH),
1
1675 (C=O), 1547 (C=N). H NMR (DMSO-d6, δppm): 1.37 (s, 3H, CH3), 4.26 (d, 2H, J=6
Hz, CH2), 4.55 (d, 2H, J=8 Hz, CH2), 7.04 (s, 1H, NH), 7.23 (d, 1H, J=8 Hz, ArH), 7.30 (s,
4H, ArH), 8.13 (d, 1H, J=8 Hz, ArH), 8.26 (s, 1H, CH), 8.28 (d, 1H, J=7 Hz, ArH), 8.91 (s,
1H, NH), 11.06 (s, 1H, NH). 13C NMR (DMSO-d6, δppm): 15.04 (CH3), 43.19 (CH2), 49.21
(CH2), arC: [110.92 (C), 112.77 (d, J=22 Hz, CH), 120.80 (CH), 126.62 (C), 127.01 (CH),
127.44 (2CH), 127.90 (C), 128.61 (2CH), 136.20 (C), 140.92 (C), 153.70-156.16 (dC-F, J=246
Hz, C)], 148.86 (CH), 158.26 (C=O), 164.01 (C=O), 174.19 (C=O).EI MS m/z (%): 371 (48),
417 ([M+1]+, 100).
3.1.4.7.
2-[(1-Ethyl-6-methyl-4-oxo-1,4-dihydroquinolin-3-yl)carbonyl]-N-phenyl
hydrazinecarbothioamide (10a)
Elemental analysis for C20H20N4O2S, calculated (%), C, 63.14; H, 5.30; N, 14.73; found (%),
C, 63,02; H, 5,18; N, 14,49. FT-IR (υmax, cm-1): 3273-3198 (NH), 3060 (ar-CH), 1647
(C=O).1H NMR (DMSO-d6, δppm): 1.40 (t, 3H, J=16 Hz, CH3), 2.49 (s, 3H, CH3), 4.54 (d,
2H, J=4 Hz, CH2), 7.14 (t, 1H, J=16 Hz, ArH), 7.33 (t, 2H, J=16 Hz, ArH), 7.71 (d, 2H, J=8
Hz, ArH), 7.86 (d, 1H, J=8 Hz, ArH), 8.20 (s, 1H, ArH), 8.90 (s, 1H, CH), 10.58 (bs, 1H,
13
NH), 11.48 (bs, 1H, NH), 12.75 (bs, 1H, NH). C NMR (DMSO-d6, δppm): 15.09 (CH3),
21.05 (CH3), 48.81 (CH2), arC: [117.88 (CH), 126.17 (2CH), 127.54 (C), 128.65 (CH),
131.25 (CH), 135.03 (2CH), 135.50 (C), 137.16 (C), 139.76 (C), 143.99 (C)], 147.70 (CH),
161.74 (C=O), 170.74 (C=S), 175.26 (C=O). EI MS m/z (%): 106 (63), 135 (38), 149 (100),
213 (47), 409 (25).
3.1.4.8.
2-[(1-Ethyl-6-methyl-4-oxo-1,4-dihydroquinolin-3-yl)carbonyl]-N-benzyl-
hydrazinecarbothioamide (10b)

Elemental analysis for C21H22N4O2S, calculated (%), C, 63.94; H, 5.62; N, 14.20; found (%),
C, 63,77; H, 5,45; N, 14,04. FT-IR (υmax, cm-1): 3291-3246 (NH), 3036 (ar-CH), 1663 (C=O).
1H NMR (DMSO-d6, δppm): 1.38 (t, 3H, J=16 Hz, CH3), 2.48 (d, 3H, J=8 Hz, CH3), 4.55 (q,
2H, J=20 Hz, CH2), 4.73 (d, 2H, J=4 Hz, CH2), 7.23 (d, 1H, J=4 Hz, ArH), 7.31 (s, 3H, ArH),
7.65 (s, 1H, ArH), 7.72 (t, 1H, J=8 Hz, ArH), 7.86 (d, 1H, J=8 Hz, ArH), 8.18 (s, 1H, ArH),
8.65 (s, 1H, NH), 8.91 (s, 1H, CH), 9.56 (bs, 1H, NH), 11.44 (bs, 1H, NH).13C NMR (DMSO-
d6, δppm): 15.11 (CH3), 21.04 (CH3), 47.27 (CH2), 48.75 (CH2), arC: [117.87 (CH), 119.46
(CH), 125.22 (CH), 126.16 (CH), 127.57 (CH), 128.54 (CH), 134.73 (CH), 133.05 (CH),
135.51 (C), 137.14 (C), 137.60 (C), 140.07 (C), 153.49 (C)], 147.82 (CH), 161.24 (C=O),
175.37 (C=S), 176.44 (C=O). EI MS m/z (%): 102 (92), 395 ([M+1]+, 100), 396 ([M+2]+, 90).
3.1.4.9.
2-[(1-Ethyl-6-methyl-4-oxo-1,4-dihydroquinolin-3-yl)carbonyl]-N-ethyl-
hydrazinecarbothioamide (10c)
Elemental analysis for C16H20N4O2S, calculated (%), C, 57.81; H, 6.06; N, 16.85; found (%),
C, 57,64; H, 5,93; N, 16,62. FT-IR (υmax, cm-1): 3302-3237 (NH), 3033 (ar-CH), 1664 (C=O).
1H NMR (DMSO-d6, δppm): 1.06 (t, 3H, J=12 Hz, CH3), 1.39 (t, 3H, J=16 Hz, CH3), 2.49 (s,
3H, CH3), 3.46 (t, 2H, J=12 Hz, CH2), 4.55 (q, 2H, J=20 Hz, CH2), 7.72 (d, 1H, J=8 Hz,
ArH), 7.86 (d, 1H, J=8 Hz, ArH), 8.06 (s, 1H, NH), 8.19 (s, 1H, ArH), 8.89 (s, 1H, CH), 9.45
13
(bs, 1H, NH), 11.49 (bs, 1H, NH). C NMR (DMSO-d6, δppm): 13.88 (CH3), 14.97 (CH3),
20.95 (CH3), 40.57 (CH2), 48.13 (CH2), arC: [107.56 (C), 117.61 (CH), 125.93 (CH), 126.92
(C), 134.78 (CH), 134.84 (C), 137.67 (C)], 146.19 (CH), 160.25 (C=O), 166.94 (C=S), 174.19
(C=O). EI MS m/z (%): 117 (38), 135 (100), 149 (69), 333 ([M+1]+, 10).
3.1.4.10.
2-[(1-Ethyl-6-methyl-4-oxo-1,4-dihydroquinolin-3-yl)carbonyl]-N-(4-fluoro
phenyl) hydrazinecarbothioamide (10d)
Elemental analysis for C20H19FN4O2S, calculated (%), C, 60.29; H, 4.81; N, 14.06, found (%),
C, 60,10; H, 4,56; N, 13,88. FT-IR (υmax, cm-1): 3280-3173 (NH), 3049 (ar-CH), 1646
(C=O).1H NMR (DMSO-d6, δppm): 1.39 (t, 3H, J=16 Hz, CH3), 2.48 (s, 3H, CH3), 4.54 (d,
2H, J=8 Hz, CH2), 7.16 (d, 1H, J=8 Hz, ArH), 7.19 (s, 1H, ArH), 7.50 (s, 2H, ArH), 7.71 (d,
2H, J=12 Hz, ArH), 7.84 (d, 1H, J=12 Hz, ArH), 8.18 (s, 1H, ArH), 8.89 (s, 1H, CH), 9.85
13
(bs, 1H, NH), 11.55 (bs, 1H, NH), 12.68 (bs, 1H, NH). C NMR (DMSO-d6, δppm): 15.09
(CH3), 21.05 (CH3), 48.80 (CH2), arC:[117.88 (2CH), 119.33 (CH), 125.09 (CH), 126.17

(2CH), 127.54 (C), 129.25 (C), 131.54 (C), 135.04 (CH), 135.78 (d, J=55 Hz, C), 137.16 (C),
158.64-160.23 (dC-F, J=159 Hz, C)], 147.74 (CH), 153.05 (C=O), 170.49 (C=S), 175.29
(C=O). EI MS m/z (%): 349 (28), 365 (63), 409 (100), 437 ([M+K]+, 35).
3.1.4.11.
2-[(1-Ethyl-6-methyl-4-oxo-1,4-dihydroquinolin-3-yl)carbonyl]-N-phenyl
hydrazinecarboxamide (10e)
Elemental analysis for C20H20N4O3, calculated (%), C, 65.92; H, 5.53; N, 15.38; found (%), C,
65,78; H, 5,38; N, 15,12. FT-IR (υmax, cm-1): 3320-3272 (NH), 3042 (ar-CH), 1675 (C=O).1H
NMR (DMSO-d6, δppm): 1.39 (t, 3H, J=12 Hz, CH3), 2.50 (d, 3H, J=8 Hz, CH3), 4.54 (q, 2H,
J=16 Hz, CH2), 6.96 (t, 1H, J=16 Hz, ArH), 7.26 (d, 1H, J=8 Hz, ArH), 7.28 (s, 1H, ArH),
7.47 (d, 2H, J=8 Hz, ArH), 7.72 (dd, 1H, J=12 Hz, ArH), 7.86 (d, 1H, J=8 Hz, ArH), 8.19 (s,
13
1H, ArH), 8.74 (bs, 2H, 2NH), 8.90 (d, 1H, J=8 Hz, CH), 11.44 (bs, 1H, NH). C NMR
(DMSO-d6, δppm): 15.07 (CH3), 21.06 (CH3), 48.79 (CH2), arC: [110.42 (C), 117.87 (CH),
118.78 (CH), 119.46 (CH), 122.30 (CH), 125.14 (CH), 126.14 (CH), 127.60 (C), 129.14
(CH), 135.10 (CH), 135.44 (C), 137.20 (C), 140.13 (C)], 147.80 (CH), 164.44 (C=O), 175.37
(C=O), 176.01 (C=O). EI MS m/z (%): 117 (100), 321 (36), 365 ([M+1]+, 85).
3.1.5. General method for the synthesis of compounds 11a-f and 12a-e
Method 1. A solution of corresponding carbo(thio)amide 9a-f or 10a-e (10 mmol) in
ethanol/water (1:1) was refluxed in the presence of 2N NaOH (20 mmol) 6-12 h (Table 2).
Then, the resulting solution was cooled to room temperature and acidified to pH 5 with 37 %
HCl. The precipitate formed was filtered off, washed with water, and recrystallized from an
appropriate solvent to give the target compound.
Method 2. The mixture of compound 9a-f or 10a-e (10mmol) and 2N NaOH (20mmol) in
o
ethanol (10 mL) was irradiated in monomod microwave reactor in closed vessel at 150 C,
200 Watt for 30-35 min (the progress of reaction was monitored by TLC). Then the resulting
solution was cooled to room temperature and acidified to pH 5 with 37 % HCl. The
precipitate formed was filtered off, wash with water, and recrystallized from an appropriate
solvent to give the target compounds.
3.1.5.1. 7-Chloro-1-ethyl-6-fluoro-3-(5-mercapto-4-phenyl-4H-1,2,4-triazol-3-yl)quinolin-
4(1H)-one (11a)

Elemental analysis for C19H14ClFN4OS, calculated (%), C, 56.93; H, 3.52; N, 13.98; found
(%), C, 56,74; H, 3,29; N, 13,69. FT-IR(υmax, cm-1): 3060 (ar-CH), 1690 (C=O), 1595 (C=N).
1H NMR (DMSO-d6, δppm): 1.22 (t, 3H, J=8 Hz, CH3), 4.09-4.13 (m, 2H, CH2), 6.75 (d, 2H,
J=7.2 Hz, ArH), 7.27 (t, 3H, J=8 Hz, ArH), 7.51 (d, 1H, J=8 Hz, ArH), 7.61 (dd, 2H, J=7.1
Hz, ArH), 8.11 (s, 1H, ArH), 8.11 (s, 1H, CH), 14.13 (s, 1H, SH). 13C NMR (DMSO-d6,
δppm):14.95 (CH3), 47.77 (CH2), arC: [113.51 (C), 116.64 (CH), 118.25 (CH), 123.67 (CH),
125.93 (2CH), 126.95 (C), 131.07 (2CH), 134.49 (C), 135.12 (C), 136.15 (C), 153.36-155.66
(dC-F, J=233 Hz, C)], 142.66 (triazole C-3), 145.53 (CH), 166.42 (triazole C-5), 174.03
(C=O). EI MS m/z (%): 112.95 (32), 417.36 (44), 439.99 ([M+K]+, 100).
3.1.5.2. 7-chloro-1-ethyl-6-fluoro-3-(4-Benzyl-5-mercapto-4H-1,2,4-triazol-3-yl)quinolin-
4(1H)-one (11b)
Elemental analysis for C20H16ClFN4OS, calculated (%), C, 57.90; H, 3.89; N, 13.50; found
(%), C, 57,58; H, 3,68; N, 13,33. FT-IR (υmax, cm-1): 3116 (NH), 3062 (ar-CH), 1632 (C=O),
1
1546 (C=N). H NMR (DMSO-d6, δppm): 1.42 (t, 3H, J=8 Hz, CH3), 4.29 (d, 2H, J=8 Hz,
CH2), 5.28 (d, 2H, J=8 Hz, CH2), 6.96 (d, 2H, J=8 Hz, ArH), 7.14 (d, 3H, J=8 Hz, ArH), 8.11
(s, 1H, ArH), 8.18-8.21(m, 1H, ArH), 8.27 (s, 1H, CH), 14.03 (s, 1H, SH). 13C NMR (DMSO-
d6, δppm):19.60 (CH3), 52.01 (CH2), 52.78 (CH2), arC: [112.23 (C), 112.50 (CH), 124.16
(CH), 131.73 (2C), 132.30 (d, J=3 Hz, 2CH), 132.66 (d, J=3 Hz, 2CH), 133.52 (CH), 138.41
(C), 141.30 (C), 154.00-156.46 (dC-F, J=246 Hz, C)], 133.67 (triazole C-3), 150.48 (CH),
172.80 (triazole C-5), 177.80 (C=O). EI MS m/z (%): 205.11 (82), 307.28 (39), 414.32 ([M]+,
100).
3.1.5.3. 7-Chloro-1-ethyl-3-(4-ethyl-5-mercapto-4H-1,2,4-triazol-3-yl)-6-fluoroquinolin-
4(1H)-one (11c)
Elemental analysis for C15H14ClFN4OS, calculated (%), C, 51.06; H, 4.00; N, 15.88; found
1
(%), C, 50,94; H, 3,87; N, 15,62. FT-IR (υmax, cm-1): 3079 (ar-CH), 1545 (C=N). H NMR
(DMSO-d6, δppm): 1.15 (s, 3H, CH3), 1.38 (s, 3H, CH3), 3.92 (s, 2H, CH2), 4.43 (s, 2H, CH2),
8.09 (d, 1H, J=7 Hz, ArH), 8.26 (d, 1H, J=7 Hz, ArH), 8.52 (s, 1H, CH), 13.87 (s, 1H, SH).
13C NMR (DMSO-d6, δppm):13.85 (CH3), 14.91 (CH3), 48.65 (CH2), 65.76 (CH2), arC:
[107.67 (C), 112.61 (d, J=22 Hz, CH), 120.58 (CH), 127.19 (d, J=6 Hz, 2C), 148.37 (C),
153.50-155.96 (dC-F, J=246 Hz, C)], 136.81 (triazole C-3), 147.31 (CH), 167.12 (triazole C-
5), 173.15 (C=O). EI MS m/z (%): 205.16 (80), 266.35 (41), 307.30 (40), 352.85 ([M]+, 100).

3.1.5.4. 7-Chloro-1-ethyl-6-fluoro-3-(4-(4-fluorophenyl)-5-mercapto-4H-1,2,4-triazol-3-
yl)quinolin-4(1H)-one (11d)
Elemental analysis for C19H13ClF2N4OS, calculated (%), C, 54.48; H, 3.13; N, 13.38; found
(%), C, 54,22; H, 3,02; N, 13,10. FT-IR (υmax, cm-1): 3007 (ar-CH), 1672 (C=O), 1511 (C=N).
1H NMR (DMSO-d6, δppm): 1.22 (t, 3H, J=8 Hz, CH3), 4.14 (q, 2H, J=12 Hz, CH2), 6.76 (s,
1H, ArH), 7.14 (t, 2H, J=12 Hz, ArH), 7.50 (d, 1H, J=8 Hz, ArH), 7.60 (dd, 2H, J=8 Hz,
ArH), 8.11 (s, 1H, ArH), 8.12 (s, 1H, CH), 13.87 (s, 1H, SH). 13C NMR (DMSO-d6,
δppm):14.85 (CH3), 49.22 (CH2), arC: [107.10 (C), 115.11 (CH), 117.47 (CH), 118.77 (2CH),
125.69 (2CH), 128.12 (C), 129.58 (C), 131.73 (C), 135.90 (C), 152.55-154.60 (dC-F, J=205
Hz, C), 156.34-158.85 (dC-F, J=251 Hz, C)], 141.66 (triazole C-3), 147.09 (CH), 168.51
(triazole C-5), 177.94 (C=O). EI MS m/z (%): 394.94 (70), 441.64 ([M+Na]+, 100).
3.1.5.5.
7-Chloro-1-ethyl-6-fluoro-3-(5-oxo-4-phenyl-4,5-dihydro-1H-1,2,4-triazol-3-
yl)quinolin-4(1H)-one (11e)
Elemental analysis for C19H14ClFN4O2, calculated (%), C, 59.31; H, 3.67; N, 14.56; found
(%), C, 59,08; H, 3,32; N, 14,28. FT-IR (υmax, cm-1): 3070 (ar-CH), 1673 (C=O), 1541 (C=N).
1H NMR (DMSO-d6, δppm): 1.39 (t, 3H, J=16 Hz, CH3), 4.43 (t, 2H, J=12 Hz, CH2), 6.96 (t,
1H, J=16 Hz, ArH), 7.26 (t, 1H, J=16 Hz, ArH), 7.46 (d, 1H, J=8 Hz, ArH), 7.99 (d, 2H, J=8
Hz, ArH), 8.13 (d, 1H, J=7.1 Hz, ArH), 8.68 (s, 1H, CH), 14.78 (s, 1H, NH). 13C NMR
(DMSO-d6, δppm):15.44 (CH3), 48.19 (CH2), arC: [108.17 (C), 117.22 (CH), 119.32 (CH),
125.90 (CH), 126.71 (C), 127.28 (2CH), 128.76 (2CH), 129.84 (2C), 135.02 (C), 152.10-
155.10 (dC-F, J=300 Hz, C)], 143.49 (triazole C-3), 148.18 (CH), 160.61 (triazole C-5), 174.91
(C=O). EI MS m/z (%): 134.98 (44), 208.18 (55), 384.41 ([M]+, 100).
3.1.5.6.
7-Chloro-1-ethyl-6-fluoro-3-(5-oxo-4-benzyl-4,5-dihydro-1H-1,2,4-triazol-3-
yl)quinolin-4(1H)-one (11f)
Elemental analysis for C20H16ClFN4O2, calculated (%), 60.23; H, 4.04; N, 14.05; found (%),
C, 60,04; H, 3,91; N, 14,01. FT-IR (υmax, cm-1): 3177 (OH), 3060 (ar-CH), 1691 (C=O), 1548
1
(C=N). H NMR (DMSO-d6, δppm): 1.23 (s, 3H, CH3), 4.31 (s, 2H,CH2), 4.82 (s, 2H,CH2),
6.99 (s, 2H, ArH), 7.15 (s, 2H, ArH), 7.30 (s, 1H, ArH), 8.09-8.15 (m, 2H, ArH), 8.22 (s, 1H,
CH), 11.89 (bs, 1H, OH). 13C NMR (DMSO-d6, δppm):14.87 (CH3), 45.54 (CH2), 48.80
(CH2), arC: [110.47 (C), 117.70 (CH), 118.51 (CH), 122.29 (CH), 126.01 (2CH), 127.52 (C),

129.13 (2CH), 135.56 (C), 137.20 (C), 139.89 (C), 150.06-152.44 (dC-F, J=238 Hz, C)],
147.92 (CH), 155.21 (triazole C-3), 159.33 (triazole C-5), 175.95 (C=O). EI MS m/z (%):
415.41 (84), 437.63 ([M+K]+, 100).
3.1.5.7. 1-Ethyl-3-(5-mercapto-4-phenyl-4H-1,2,4-triazol-3-yl)-6-methylquinolin-4(1H)-
one (12a)
Elemental analysis for C20H18N4OS, calculated (%), C, 66.28; H, 5.01; N, 15.46; found (%),
1
C, 66,11; H, 4,91; N, 15,20. FT-IR (υmax, cm-1): 3085 (ar-CH), 1710 (C=O), 1567 (C=N). H
NMR (DMSO-d6, δppm): 1.31 (s, 3H, CH3), 2.37 (s, 3H, CH3), 4.30 (s, 2H, CH2), 7.35 (s, 5H,
ArH), 7.58 (s, 1H, ArH), 7.67 (s, 1H, ArH), 7.83 (s, 1H, ArH), 8.42 (s, 1H, CH), 14.08 (s, 1H,
SH). 13C NMR (DMSO-d6, δppm): 14.84 (CH3), 20.83 (CH3), 47.98 (CH2), arC: [107.39 (C),
117.41 (CH), 125.71 (CH), 126.66 (C), 128.30 (2CH), 128.94 (2CH), 129.14 (CH), 134.52
(CH), 134.55 (C), 134.93 (C), 137.52 (C)], 146.07 (CH), 148.99 (triazole C-3), 168.36 (C=S),
173.58 (C=O). EI MS m/z (%): 135 (100), 246 (39), 320 (38), 363 ([M+1]+, 40).
3.1.5.8. 1-Ethyl-3-(5-mercapto-4-benzyl-4H-1,2,4-triazol-3-yl)-6-methylquinolin-4(1H)-
one (12b)
Elemental analysis for C21H20N4OS, calculated (%), C, 67.00; H, 5.35; N, 14.88; found (%),
1
C, 66,87; H, 5,17; N, 14,49. FT-IR (υmax, cm-1): 3084 (ar-CH), 1551 (C=N). H NMR
(DMSO-d6, δppm): 1.18 (t, 3H, J=12 Hz, CH3), 2.47 (s, 3H, CH3), 4.27 (d, 2H, J=8 Hz, CH2),
5.29 (s, 2H, CH2), 6.95 (d, 2H, J=4 Hz, ArH), 7.15 (d, 3H, J=4 Hz, ArH), 7.67 (d, 1H, J=8
Hz, ArH), 7.74 (d, 1H, J=8 Hz, ArH), 8.12 (s, 1H, ArH), 8.19 (s, 1H, CH), 14.03 (s, 1H, SH).
13C NMR (DMSO-d6, δppm): 14.87 (CH3), 20.98 (CH3), 47.23 (CH2), 47.82 (CH2), arC:
[107.77 (C), 117.57 (CH), 125.90 (CH), 126.89 (C), 127.48 (2CH), 127.88 (CH), 128.76
(2CH), 134.83 (CH), 134.93 (C), 136.60 (C), 137.42 (C)], 145.93 (CH), 149.46 (triazole C-3),
168.01 (C=S), 174.02 (C=O). EI MS m/z (%): 135 (100), 149 (39), 213 (37), 377 ([M+1]+,
32).
3.1.5.9. 1-Ethyl-3-(5-mercapto-4-ethyl-4H-1,2,4-triazol-3-yl)-6-methylquinolin-4(1H)-one
(12c)
Elemental analysis for C16H18N4OS, calculated (%), C, 61.12; H, 5.77; N, 17.82; found (%),
C, 60,95; H, 5,32; N, 17,48. FT-IR (υmax, cm-1): 3112 (NH), 3088 (ar-CH), 1602 (C=N).1H
NMR (DMSO-d6, δppm): 1.14 (t, 3H, J=16 Hz, CH3), 1.38 (t, 3H, J=12 Hz, CH3), 2.47 (s,

3H, CH3), 3.94 (q, 2H, J=20 Hz, CH2), 4.40 (q, 2H, J=16 Hz, CH2), 7.69 (d, 1H, J=8 Hz,
13
ArH), 7.80 (d, 1H, J=8 Hz, ArH) 8.10 (s, 1H, ArH), 8.45 (s, 1H, CH), 13.85 (s, 1H, SH). C
NMR (DMSO-d6, δppm): 13.86 (CH3), 14.95 (CH3), 20.95 (CH3), 40.62 (CH2), 48.12 (CH2),
arC: [107.39 (C), 117.60 (CH), 125.93 (CH), 126.95 (C), 134.77 (CH), 134.83 (C), 137.70
(C)], 146.16 (CH), 149.06 (triazole C-3), 166.99 (C=S), 174.20 (C=O). EI MS m/z (%): 135
(100), 246 (22), 320 (25).
3.1.5.10.
1-Ethyl-3-[4-(4-fluorophenyl)-5-mercapto-4H-1,2,4-triazol-3-yl]-6-methyl
quinolin-4(1H)-one (12d)
Elemental analysis for C20H17FN4OS, calculated (%), C, 63.14; H, 4.50; N, 14.73; found (%),
C, 63,01; H, 4,29; N, 14,56. FT-IR (υmax, cm-1): 3121 (NH), 3046 (ar-CH), 1541 (C=N).1H
NMR (DMSO-d6, δppm): 1.33 (t, 3H, J=16 Hz, CH3), 2.38 (s, 3H, CH3), 4.33 (t, 2H, J=16
Hz, CH2), 7.21 (d, 1H, J=8 Hz, ArH), 7.24 (s, 1H, ArH), 7.39 (dd, 2H, J=16 Hz, ArH), 7.59
(t, 1H, J=8 Hz, ArH), 7.71 (d, 1H, J=8 Hz, ArH), 7.83 (s, 1H, ArH), 8.43 (s, 1H, CH), 14.09
13
(s, 1H, SH). C NMR (DMSO-d6, δppm): 14.85 (CH3), 20.83 (CH3), 48.06 (CH2), arC:
[107.18 (C), 115.88 (d, J=13 Hz, 2CH), 117.47 (CH), 125.69 (2CH), 126.67 (C), 130.50
(CH), 130.59 (C), 134.59 (CH), 137.55 (2C), 162.06 (dC-F, J=244 Hz, C)], 146.17 (CH),
149.13 (triazole C-3), 168.46 (C=S), 173.47 (C=O). EI MS m/z (%): 135 (100), 381 ([M+1]+,
30), 408 (21).
3.1.5.11. 1-Ethyl-3-(5-hydroxy-4-phenyl-4H-1,2,4-triazol-3-yl)-6-methylquinolin-4(1H)-
one (12e)
Elemental analysis for C20H18N4O2, calculated (%), C, 69.35; H, 5.24; N, 16.17; found (%), C,
69,19; H, 5,15; N, 16,02. FT-IR (υmax, cm-1): 3273 (OH), 3042 (ar-CH), 1679 (C=O), 1553
1
(C=N). H NMR (DMSO-d6, δppm): 1.39 (t, 3H, J=16 Hz, CH3), 2.51 (s, 3H, CH3), 4.54 (q,
2H, J=20 Hz, CH2), 6.95 (t, 1H, J=16 Hz, ArH), 7.26 (t, 2H, J=16 Hz, ArH), 7.47 (d, 2H, J=8
Hz, ArH), 7.71 (d, 1H, J=8 Hz, ArH), 7.86 (d, 1H, J=8 Hz, ArH), 8.19 (s, 1H, ArH), 8.56
(s,1H, ArH), 8.88 (s, 1H, CH), 11.42 (s, 1H, OH).13C NMR (DMSO-d6, δppm): 15.07 (CH3),
21.06 (CH3), 48.79 (CH2), arC: [110.42 (C), 117.87 (CH), 118.78 (CH), 119.46 (CH), 122.30
(CH), 125.14 (CH), 126.14 (CH), 127.60 (C), 129.14 (CH), 135.00 (CH), 135.43 (C), 137.20
(C), 140.21 (C)], 147.81 (CH), 155.16 (triazole C-3), 164.37 (C=O), 175.37 (C=O). EI MS
m/z (%): 346 ([M+1]+, 30).

3.2. Antioxidant activity
3.2.1. Antioxidant activity studies
DPPH (2,2-diphenyl-1-picrylhydrazyl) radical scavenging activity: The scavenging activity
of different chemicals was determined using the free radical DPPH (2,2-diphenyl-1-
picrylhydrazyl), as described by Blois [50]. A 100 mL 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 graph showing
the concentrations against the absorbances is drawn, and the the concentration (mg/mL) of the
sample that removes 50% of the total amount of the DPPH radical is accepted as SC50.
FRAP (the ferric reducing ability of plasma): FRAP was measured using the method
described by Benzie & Strain [51] with some modification. To 100 mL 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.6H2O in proportions of
o
10:1:1 (v/v/v). The mixture was incubated for 30 min at 37 C and measured at 593 nm. The
values were expressed as mmol of Trolox/g.
CUPRAC (cupric ion reducing antioxidant capacity): CUPRAC was measured following
the procedure described by Apak et al. [52] with some modification. Briefly, 100 mL of each
chemical solution was mixed with 900 mL bi-distilled 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 mmol Trolox equivalent per g.
3.2.2. Measurement of AChE activity
The AChE inhibitory activity of the compounds was determined using Ellman’s method
[48], using AChE from E. electricus (Sigma) and acetylthiocholine iodide (0.35 mM) as a
substrate. The reaction took place in the final volume of 3 mL of a phosphate-buffered
solution at pH 8, containing 0.035 U/mL of EeAChE and 0.35 mM of 5,50-dithiobis-2-
nitrobenzoic acid (DTNB), which produced yellow anion 5-thio-2-nitrobenzoic acid.
Inhibition curves were made by incubating with the different compounds for 15 min; a sample
without any compound was always used to determine the 100% of enzymatic activity. After

the 15 min incubation period, the production of color, as an indicator of enzymatic activity,
was evaluated by measuring absorbance at 412 nm in a spectrophotometer plate reader.
3.2.4. Molecular docking
Ligands were energy-minimized using GAMESS (M.W. Schmidt) module for ChemOffice
version Ultra 8.0.3 (PerkinElmer Inc.) on an Intel® (Core™ i7-3632QM CPU @ 2.20GHz
2.20GHz) using Windows 8.1 operating system. These modified ligand pdb files saved as
pdbqt files. Appropriate grid box points were determined by centering on ligand separately for
each compound. Ligand-centred grid box for all ligands, defined with a size of 126*126*126
Å3 and a regular space of 0.375 Å, was considered for docking.
"Crystal Structure of Recombinant Human acetylcholinesterase in complex with
Donepezil" [47] pdb file (PDB ID: 4EY7) was get (www.rcsb.org) and was modified using
the ADT package version 1.5.6rc3. All water molecules were deleted and polar hydrogens
were added. Subsequently, Gasteiger charges were calculated and the generated pdbqt files
were saved. To validate the docking program, the co-crystallized ligand (PDB ID: E20) was
redocked on the target enzyme and RMSD value of 0.709 was found for donepzil-bound
acetylcholinesterase. RMSD value were obtained using Lamarckian Genetic Algorithm and
scoring function of AutoDock 4.2 release 4.2.5.1 [53] software.
4. Conclusions
We have designed the synthesis, antioxidant and anti-acetylcholinesterase screening
studies of new quinolone-triazole hybrids. Also, the intermediate products which are
quinolones-carbo(thio)amides have investigated of their antioxidant capacity and AChE
inhibition activity. All compounds except 9d, 10b, 12b and 12c show promising AChE
inhibition activity. Among the active compounds against AChE, 9b and 10c are the most
potent. Surprisingly, while the quinolone-triazole compounds were expected to be more
active, the inhibition results showed that the most active compounds were quinolone-
carbothioamide derivatives. In antioxidant capacity, 10c is for FRAP, 10e is for CUPRAC and
9b is for DPPH showed the best results. Our work established to synthesize new multipotent
hybrid molecules which are quinolone-triazole for the treatment of Alzheimer’s Disease.