Design, synthesis, characterization, enzymatic inhibition evaluations, and docking study of novel quinazolinone derivatives

Article abstract of DOI:10.1016/j.ijbiomac.2020.12.121

In this study, novel quinazolinone derivatives 7a-n were synthesized and evaluated against metabolic enzymes including α-glycosidase, acetylcholinesterase, butyrylcholinesterase, human carbonic anhydrase I, and II. These compounds exhibited high inhibitory activities in comparison to used standard inhibitors with K_i values in the range of 19.28–135.88 nM for α-glycosidase (K_i value for standard inhibitor = 187.71 nM), 0.68–23.01 nM for acetylcholinesterase (K_i value for standard inhibitor = 53.31 nM), 1.01–29.56 nM for butyrylcholinesterase (K_i value for standard inhibitor = 58.16 nM), 10.25–126.05 nM for human carbonic anhydrase I (K_i value for standard inhibitor = 248.18 nM), and 13.46–178.35 nM for human carbonic anhydrase II (K_i value for standard inhibitor = 323.72). Furthermore, the most potent compounds against each enzyme were selected in order to evaluate interaction modes of these compounds in the active site of the target enzyme. Cytotoxicity assay of the title compounds 7a-n against cancer cell lines MCF-7 and LNCaP demonstrated that these compounds do not show significant cytotoxic effects.

Full text of DOI:10.1016/j.ijbiomac.2020.12.121

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Design, synthesis, characterization, enzymatic inhibition
evaluations, and docking study of novel quinazolinone derivatives
Keyvan Pedrood, Maedeh Sherafati, Maryam Mohammadi-
Khanaposhtani, Mohammad Sadegh Asgari, Samanesadat
Hosseini, Hossein Rastegar, Bagher Larijani, Mohammad
Mahdavi, Parham Taslimi, Yavuz Erden, Sevilay Günay, İlhami
Gulçin
PII:
S0141-8130(20)35292-2
DOI:
https://doi.org/10.1016/j.ijbiomac.2020.12.121
BIOMAC 17501
Reference:
To appear in:
International Journal of Biological Macromolecules
Received date:
Revised date:
Accepted date:
21 September 2020
12 December 2020
15 December 2020
Please cite this article as: K. Pedrood, M. Sherafati, M. Mohammadi-Khanaposhtani, et al.,
Design, synthesis, characterization, enzymatic inhibition evaluations, and docking study
of novel quinazolinone derivatives, International Journal of Biological Macromolecules
(2020), https://doi.org/10.1016/j.ijbiomac.2020.12.121
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Design, Synthesis, Characterization, Enzymatic Inhibition Evaluations, and Docking Study of
Novel Quinazolinone Derivatives
1
1
2
Keyvan Pedrood , Maedeh Sherafati , Maryam Mohammadi‐Khanaposhtani , Mohammad Sadegh
3
4
5
1
1,
Asgari , Samanesadat Hosseini , Hossein Rastegar , Bagher Larijani , Mohammad Mahdavi *,
6
7
7
8
Parham Taslimi *, Yavuz Erden , Sevilay Günay , İlhami Gulçin
1
Endocrinology and Metabolism Research Center, Endocrinology and Metabolism Clinical Sciences
Institute, Tehran University of Medical Sciences, Tehran, Iran
Cellular and Molecular Biology Research Center, Health Research Institute, Babol University of
2
Medical Sciences, Babol, Iran
3
School of Chemistry, College of Science, University of Tehran, Tehran, Iran
4
Department of Pharmaceutical Chemistry, School of Pharmacy, Shahid Beheshti University of
Medical Sciences, Tehran, Iran
5
Cosmetic products research center, Iranian food and drug administration, MOHE, Tehran, Iran
Department of Biotechnology, Faculty of Science, Bartin University, 74100 Bartin, Turkey
Department of Molecular Biology and Genetics, Faculty of Science, Bartin University, 74100 Bartin,
6
7
Turkey
8
Department of Chemistry, Faculty of Sciences, Ataturk University, 25240, Erzurum, Turkey
*
Corresponding authors.
E-mail addresses: momahdavi@tums.ac.ir (M. Mahdavi) and ptaslimi@bartin.edu.tr (P. Taslimi)
ABSTRACT
In this study, novel quinazolinone derivatives 7a-n were synthesized and evaluated against
metabolic enzymes α-glycosidase, acetylcholinesterase, butyrylcholinesterase, human
carbonic anhydrase I, and II. These compounds exhibited high inhibitory activities in
comparison to used standard inhibitors with K values in the range of 19.28–135.88 nM for α-
i
glycosidase (K value for standard inhibitor = 187.71 nM), 0.68–23.01 nM for
i
acetylcholinesterase (K value for standard inhibitor = 53.31 nM), 1.01-29.56 nM for
i
butyrylcholinesterase (K value for standard inhibitor = 58.16 nM), 10.25-126.05 nM for
i
human carbonic anhydrase I (K value for standard inhibitor = 248.18 nM), and 13.46-178.35
i
nM for human carbonic anhydrase II (K value for standard inhibitor = 323.72). Furthermore,
i
the most potent compounds againt each enzyme were selected in order to evaluate interaction
modes of these compounds in the active site of the target enzyme. Cytotoxicity assay of the

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title compounds 7a-n against cancer cell lines MCF-7 and LNCaP demonstrated that these
compounds do not show significant cytotoxic effects.
Keywords: Quinazolinone; Metronidazole; Enzyme inhibition; Cytotoxicity; Molecular
docking
1
. Introduction
Quinazolinone and its derivatives have received a great deal of attention, due to their
therapeutic and pharmaceutical properties, such as anti-inflammatory, antibacterial, antitumor,
antifungal, and antitubercular activities [1-17]. Moreover, quinazolinone scaffold also found
in the potent α-glycosidase inhibitors such as compounds A-C (Fig. 1) [18-20]. α-Glycosidase
involved in the hydrolyzation of oligo- and disaccharides into monosaccharides and control of
glucose level in blood [21]. Therefore, inhibition of this enzyme tends to slow breakdown and
release of sugars into the bloodstream and can be used as a therapeutic method in the
treatment of diabetes and obesity [22]. In particular, some α-glycosidase inhibitors such as
acarbose, voglibose, and miglitol are used in the treatment of diabetes [23, 24].
One of the important biological effects of quinazolinone derivatives is acetylcholinesterase
(AChE) and butyrylcholinesterase (BChE) inhibition as two target enzymes used in treatment
of Alzheimer's disease (AD). For example, compounds D-F exhibited high inhibitory
activities against the latter enzymes (Fig. 1) [25-27]. One of the plenty of extensively
recorded statements of AD development is the cholinergic hypothesis, which offers clear
aspects of therapy strategies [28, 29]. Cholinesterase (ChE) enzymes are a family group that
mainly catalyzes the hydrolysis of the neurotransmitter acetylcholine (ACh) at the end of the
nerve transmission in the centric neural system [30, 31]. There are two main forms of ChEs:
AChE and BChE. There are documents that show while AChE activity prevails to BChE
activity in a healthy brain, but in patients with AD, BChE activity increased and AChE
activity decreased [32, 33].

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Furthermore, several compounds containing quinazolinone core with human carbonic
anhydrase I, and II (CA I and CA II) inhibitory properties were also reported (Fig. 1,
compounds G-I) [34, 35]. CA enzymes are a family of zinc-containing metalloenzymes that
composed of sixteen isoenzymes, which differ in function, kinetic properties, inhibition
profiles, and tissue expression patterns [36]. The overexpression and sluggishness of several
CA isoenzymes are responsible for plenty of diseases in human beings [37]. The CA I and CA
II isoforms had plenty of significant normal physiological mechanisms like regulation of the
acid-base homeostasis made them act as worthy drug targets in cerebral edema, glaucoma,
and epilepsy [38, 39].
Fig. 1. α-Glycosidase inhibitors A-C, cholinesterase inhibitors D-F, and carbonic anhydrase
inhibitors G-I bearing quinazolinone core
In light of this information, the aim of this study was to synthesis new derivatives of
quinazolinone and investigates these derivatives on α-glycosidase, AChE, BChE, hCA I, and
hCA II inhibitory activities. Docking studies of the most potent compounds against each
enzyme were also performed. Moreover, considering the several reports of cytotoxic effects
of quinazolinone derivatives, cytotoxicity of all the synthesized compounds was evaluated by
the MTT assay against cancer cell lines MCF-7 and LNCaP [40, 41].

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2
. Result and discussion
2
.1. Chemistry
The synthetic route for the synthesis of new quinazolinone derivatives 7a-n has been depicted
in Scheme 1 [42]. It was started from the reaction between 2-aminobenzamide 1 and aromatic
aldehydes 2 in the presence of Na S O in DMF at 100 °C to give quinazolinone derivatives
2
2
5
3
. On the other hand, by a nucleophilic substitution reaction between tosyl chloride 4 and
metronidazole 5, tosyl-metronidazole 6 was obtained. In the final step, the latter compound
reacted with quinazolinone derivatives 3 in presence of K CO in DMF at 80 °C for provided
2
3
the title compounds 7a-n.
o
Scheme 1. Synthesis of the new quinazolinone derivatives 7a-n: (a) DMF, Na S O , 100 C, 5
2
2
5
h; (b) Pyridine, 50 °C, 4h; (c) DMF, K CO , H O, 80 °C, 24 h
2
3
2
1
13
The structures of the synthesized compounds 7a-n were determined by H and C NMR, IR,
1
and elemental analysis. For example, H NMR spectrum of compound 7a showed a signal at
2
.42 ppm corresponding to protons related to methyl group attached to imidazole ring, besides
two triplets in the region of 5.14 and 4.93 ppm attributed to ethyl group between

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quinazolinone and imidazole rings. In addition, aromatic hydrogen related to the imidazole
ring appeared at 8.00 ppm. The number of other aromatic hydrogens of compound 7a is fully
1
compatible with the aromatic region picks in H NMR spectrum.
2
.2. Enzyme inhibition results
It was established that different CA isoforms are involved in numerous physiological and
pathological processes associated with CO hydration reaction such as bone resorption,
2
calcification, electrolyte secretion, respiration, lipogenesis, gluconeogenesis, tumorigenicity,
and many others [43]. CA Inhibitors (CAIs) for the diverse human isoforms (i.e. 16 to date)
have recorded clinical applications for the handling of diseases like epilepsy, obesity, ocular
hypertension in glaucoma, hypoxic cancers, and neuropathic pain [44]. The major hurdle in
CAI development is relevant to the isoenzyme selectivity issue, which thrived in novel
chemotypes. In this context, our research group recently reported various aromatic
compounds (i.e. 1,3,5-trisubstituted-pyrazolines, 4-phenylbutenone bromophenols, novel
NHC Precursors, and cyclic thioureas) as potent and novel CAIs [45-48]. Therefore, the novel
quinazolinone derivatives 7a-n were evaluated against CAs and obtained results were listed in
Table 1. As can be seen in the latter table, our results demonstrated that novel quinazolinone
derivatives 7a-n had high inhibitory activity against hCA I isoform in comparison to standard
CA inhibitor acetazolamide. The obtained K values for the newly synthesized compounds
i
were in range of 10.25±1.26 to 126.05±25.40 nM while acetazolamide had K value of
i
2
48.18±23.13 nM against hCA I. As can be seen in Scheme 1, in order to better evaluation of
enzymatic activities of title compounds, various aromatic groups were applied for synthesis of
new derivatives of the novel quinazolinone scaffold 7. Among the synthesized compounds,
the most potent compound was compound 7j with 2-chloro-5-nitrophenyl group with K value
i
of 10.25±1.26 that was around 25-folds more potent than acetazolamide. The second potent
compound among the synthesized compounds was compound 7g with 4-chlorophenyl group.

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Replacement of chloro substituent in compound 7g with methyl or methoxy group, led to a
moderate decrease in the 4-methyl derivative 7b and a significant decrease in 4-methoxy
derivative 7f while replacement of chloro with phenoxy group, as in compound 7l, created a
negligible decrease in inhibitory activity. It is worthy to note that in addition to 4-methoxy
derivative 7f, other methoxy derivatives 7d, 7e, and 7k were moderate inhibitors against CA I
in comparison to chloro derivatives 7g and 7j. The third most potent compound was 3-
hydroxyphenyl derivative 7c. Replacement of Bromo substituent instead of hydroxyl group
slightly decreased inhibitory activity (compound 7h vs. compound 7c). As can be seen in
Table 1 and Scheme 1, 2-nitro-3-methoxy derivative 7i had inhibitory activity similar to 3-
Bromo derivative 7h. Order of inhibitory activities of un-substituted aromatic groups
demonstrated that 1-naphthalene group exhibited the best activity in comparison to phenyl
and thiophene groups (compound 7n vs. compound 7a and 7m, respectively).
The novel quinazolinone derivatives 7a-n exhibited K values varying from 13.46±4.13 to
i
1
78.35±17.94 nM against hCA II isoform (Table 1). These results revealed that title
compounds 7a-n inhibited hCA II better than standard inhibitor acetazolamide with Ki value
of 323.72±51.31. Like the effects on hCA I, chlorine derivatives of 7j and 7g acted better than
others against hCA II. The third most potent hCA II inhibitor among the title compounds was
4
-phenoxy derivative 7l while methoxy derivatives 7d-f and 7k demonstrated moderate
inhibitory activities in comparison to 4-phenoxy derivative 7l. As can be seen in Table 1 and
Scheme 1, replacement of phenoxy group of compound 7l with methyl group, as in compound
7
b, led to a slight decrease in the inhibitory activity. Observed data also demonstrated that
compounds with substituents 4-methyl, 3-hydroxy, 3-bromo, and or 2-nitro-3-methoxy
showed approximately same inhibitory activity against hCA II. The order of inhibitory
activity of unsubstituted aromatic derivatives against hCA II is similar to it against hCA I: 1-

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naphthalene derivative 7n was more potent than phenyl derivative 7a and thiophene
derivative 7m.
BChE and AChE inhibition have been documented as critical goals for the effective
management of AD. Cholinesterase inhibitors (ChEIs) have various benefits in improvement
of brain ACh amounts resulting in a raised cholinergic transmission [49]. Nowadays, ChEIs
represent the major therapy for behavioral dysfunctions associated with AD and ameliorate
the cognitive [50]. AChE and BChE inhibition properties of novel quinazolinone derivatives
7
a-n were determined according to Ellman’s procedure as previously described [51]. Novel
quinazolinone derivatives 7a-n exhibited high inhibitory activities against cholinesterase
enzymes AChE (K values = 0.68±0.04-23.01±4.81 nM) and BChE (K values = 1.01±0.21-
i
i
2
9.56±2.95 nM) when compared with standard cholinesterase inhibitor tacrine (K value
i
against AChE = 53.31±11.32 nM and K value against BChE = 58.16±7.24 nM). Obtained
i
results revealed that 1-naphthalene derivative 7n, 4-phenoxyphenyl derivative 7l, and 4-
chloro derivative 7g were the most potent compounds among the newly synthesized
compounds against both AChE and BChE. On the other hand, the less potent compound
against both AChE and BChE was thiophene derivative 7m. The remaining compounds
showed different activities against AChE and BChE depending on the type of substitutions on
phenyl group. In this regard, in the term of AChE inhibitory activity, 3-bromo derivative 7h,
4
-methyl derivative 7b, and 2-nitro-3-methoxy derivative 7i with approximately same IC50
values showed good inhibitory activity, but remaining compounds in comparison to the
compounds 7h, 7b, and 7i exhibited moderate anti-AChE activity. On the other hand, in the
term of BChE inhibitory activity, 3-hydroxy derivative 7c and 3-bromo derivative 7h with
inhibitory activities approximately same, after the compounds 7n, 7l, and 7g were the most
active compounds. Like observed anti-AChE activity, 4-methyl derivative 7b and 2-nitro-3-
methoxy derivative 7i also exhibited high inhibitory activities against BChE and remaining

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compounds were moderate inhibitors against BChE when compared with the mentioned most
potent compounds.
The α-glycosidase enzyme is located at the brush border of intestine, where it is involved in
the breakdown of dietary sugars and starches to glucose [52]. Inhibition of this enzyme is an
attractive target for managing blood glucose levels in type 2-diabetes [53-58]. In the final step
of our in vitro enzymatic inhibition assays, α-glycosidase inhibitory activity of the newly
synthesized compounds 7a-n was evaluated against yeast form of this enzyme and obtained
results were compared with acarbose as a standard α-glycosidase inhibitor. Our results
demonstrated that all the novel quinazolinone derivatives 7a-n (K values = 19.28±1.88-
i
1
35.88±14.92 nM) had more inhibitory activities than acarbose (K value = 187.71±28.40
i
nM). Among the synthesized compounds, 3-bromo derivative 7h, 2-chloro-5-nitro derivative
j, and 3-hydroxy derivative 7c were the most potent compounds. Replacement of hydroxyl
7
substituent in compound 7c with methoxy group, as in compound 7e, led to a significant
decrease in the inhibitory activity. Moreover, changing the position of methoxy group of 3-
position in compound 7e to 2-position, as in compound 7d, and or introduction of 2-nitro
group and or 4-ethoxy group on 3-methoxy derivative 7e, as in compounds 7i and 7k,
respectively, led to increasing in the inhibitory activity. The fourth potent compound among
the synthesized compounds was 4-chloro derivative 7g. Replacement of chloro substituent in
the latter compound with methoxy and or methyl, as in compound 7f and 7b, respectively, led
to a dramatic decrease in the inhibitory activity while replacement of chloro of compound 7g
with phenoxy group, as in compound 7l, led to a negligible decrease in the inhibitory activity.
Inhibitory activities of un-substituted aromatic groups revealed that un-substituted phenyl
derivative 7a and 1-naphthalene derivative 7n had good anti-α-glycosidase activity while 2-
thiophene derivative 7m was one of the weakest compounds against α-glycosidase.

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Table 1. Inhibition results of novel quinazolinone derivatives (7a-n) on some metabolic
enzymes including α-glycosidase (α-Gly), acetylcholinesterase (AChE), butyrylcholinesterase
(BChE), human carbonic anhydrase I, and II isoenzymes (hCA I, and hCA II)
IC50 (nM)
i
K (nM)
Compoun
d
hC
A I
2
hC
A II
2
AC
hE
2
BC
hE
2
α-
Gly
2
r
r
r
r
r
hCA I
hCA II
AChE
BChE
α-Gly
6
2
7.
1
0.9
624
56.
04
0.9
725
15.
47
0.9
093
10.
51
0.9
325
101
.47
0.9
841
82.54±1 62.45±6 12.66±
0.41 .22 0.96
7.43±
1.13
78.46±1
5.70
7
7
7
7
7
7
7
7
7
7
7
7
7
7
a
b
c
d
e
f
2
4
6.
5
0.9
482
20.
55
0.9
511
4.0
6
0.9
037
6.0
3
0.9
490
105
.46
0.9
506
34.12±6 26.93±3 3.13±0. 4.96±
.07 .94 93 0.94
135.88±
14.92
1
8
5.
2
0.9
638
19.
41
0.9
876
5.8
3
0.9
236
4.3
7
0.9
533
22.
50
0.9
277
18.04±3 26.57±2 7.23±1. 3.78±
27.01±4
.73
.77
.36
34
0.65
9
0
1.
5
0.9
041
76.
16
0.9
889
17.
32
0.9
587
14.
03
0.9
805
67.
18
0.9
440
98.01±1 84.01±1 14.70±
3.56 3.73 2.52
13.01
±0.97
70.47±1
0.03
9
2
0.
4
0.9
581
69.
08
0.9
682
8.2
7
0.9
882
11.
21
0.9
410
91.
83
0.9
583
93.06±1 78.36±8 5.98±0. 9.45±
3.87 .43 94 1.47
99.16±1
5.94
8
2
3.
5
0.9
915
63.
08
0.9
699
10.
24
0.9
581
13.
95
0.9
094
78.
61
0.9
624
98.04±1 79.33±9 7.94±0. 10.42
3.76 .42 56 ±1.93
78.04±1
2.57
1
7
2.
8
0.9
635
10.
27
0.9
827
1.8
7
0.9
637
2.3
6
0.9
834
26.
96
0.9
736
16.83±2 14.37±1 1.45±0. 2.01±
.25 .87 24 0.24
30.76±4
.85
g
h
i
3
8
3.
1
0.9
948
28.
36
0.9
633
3.7
8
0.9
735
4.7
5
0.9
683
13.
78
0.9
925
39.62±9 30.23±2 3.05±0. 3.94±
.45 .84 67 0.67
19.28±1
.88
3
4
0.
7
0.9
611
25.
73
0.9
947
3.8
7
0.9
310
8.1
4
0.9
126
37.
38
0.9
327
35.87±7 28.94±3 3.26±0. 5.98±
.32 .12 53 1.03
44.72±4
.78
7
3
.9
0.9
518
10.
25
0.9
224
8.3
1
0.9
528
12.
67
0.9
790
18.
36
0.9
943
10.25±1 13.46±4 6.25±1. 9.14±
.26 .13 01 0.94
23.25±2
.76
j
4
7
6.
1
0.9
882
40.
26
0.9
424
12.
47
0.9
603
16.
34
0.9
023
46.
04
0.9
167
58.02±5 52.94±5 9.27±1. 13.66
.47 .47 21 ±2.78
50.01±1
3.88
k
l
1
0
9.
5
0.9
506
12.
05
0.9
932
1.0
5
0.9
325
1.9
8
0.9
912
29.
36
0.9
532
24.35±0 15.33±1 0.96±0. 1.15±
35.20±3
.65
.98
.24
12
0.20
1
11
0.9
830
146
.98
0.9
483
26.
01
0.9
598
34.
61
0.9
501
94.
60
0.9
701
126.05± 178.35± 23.01±
25.40 17.94 4.81
29.56
±2.95
103.04±
14.90
m
n
.
58
2
4
2.
8
0.9
889
16.
58
0.9
548
0.9
5
0.9
721
1.2
4
0.9
598
68.
52
0.9
290
28.55±5 21.76±2 0.68±0. 1.01±
77.95±9
.90
.62
.56
04
0.21
Acetazol
amide*
218
.65
0.9
892
281
.87
0.9
233
-
-
-
-
248.18± 323.72±
-
-
-
-
-
-
-
-
23.13
-
51.31
-
6
2
7.
1
0.9
643
83.
24
0.9
719
53.31±
11.32
58.16
±7.24
Tacrine*
-
-
-
-
-
-
-
-
Acarbose
*
154
.36
0.9
983
187.71±
28.40
-
-
-
-
-
-
-
-
(*They were used as control compounds for some metabolic enzymes)
2
.3. Molecular modeling study

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After in vitro enzymatic assays, the most potent compounds against studied enzymes hCA I,
hCA II, AChE, BChE, and α-glucosidase were docked in the active site of these enzymes by
Autodock Tools 1.5.6 software. Compounds 7j and 7g as the most potent compounds against
hCA I were screened in the active site of this enzyme. As was mentioned in our previous
works, acetazolamide as a standard inhibitor against hCA I interacted with Zn301 and His200
in hCA I active site [59]. As can be seen in Table 1, compound 7j inhibited hCA I about 25-
fold stronger than acetazolamide. Docking study of this compound in hCA I active site
showed that compound 7j interacted with Zn301 and His200 via π-cation interactions and
Gln92 and His67 via hydrogen bonds (Fig. 2). Compound 7j also formed two non-classic
hydrogen bonds with Thr199 and Pro202. Furthermore, hydrophobic interactions between this
compound and active site residues Val143, Ala121, His44, Leu198, His200, Ala135, Phe91,
and Leu131 were observed in Fig. 2.
The second potent hCA I inhibitor 7g formed only a π-cation interaction with His67. On the
other hand, this compound established three hydrogen bonds with His64, His67, and His200
and a non-classic hydrogen bond with Gln92. Furthermore; compound 7g also interacted with
His119, His94, Leu198, Leu131, Ala135, and Val62 via hydrophobic interactions. Further
studies showed that compound 7j has a lower free binding energy (-7.92 kcal/mol) than
compound 7g (-7.56 kcal/mol) and therefore binds easily to hCA I than does compound 7g.
7
j
7g

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Fig. 2. Docking poses of compounds 7j and 7g in the active site of hCA I isoenzyme.
Compounds of 7j and 7g also acted as the most potent inhibitors against hCA II isoenzyme.
Therefore, these compounds were also docked in the active site of this enzyme (Fig. 3).
Standard hCA II inhibitor of acetazolamide interacted with Zn265, Asn67, and Thr199 in the
active site hCA II. Compound 7j formed three hydrogen bonds with Thr199 and Thr200 and
several hydrophobic interactions with His94, Ala65, Val121, Leu198, Pro202, Val135, and
Phe131. The compound of 7g, with hCA II inhibitory activity approximately same with
compound of 7j, formed hydrogen bonds with Gln92 and Asn62 and hydrophobic interactions
with residues His96, His94, His119, Trp209, His64, Val121, Leu198, Pro202, Val135, and
Phe131. Binding energies of these compounds, like inhibitory activities against hCA II, are
not significantly different (binding energy Compound of 7j = -7.45 kcal/mol and binding
energy Compound of 7j = - 7.35 kcal/mol).
7
j
7g

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Fig. 3. Docking poses of compounds of 7j and 7g in the active site of hCA II isoenzyme.
The most potent compounds against cholinesterase enzymes of AChE and BChE were
compounds of 7n and 7l that were docked into active site of these enzymes. The active site of
AChE and BChE includes two important components: 1) catalytic anionic site (CAS) that
itself consists of two parts, anionic site (AS) and catalytic triad (CT), and 2) peripheral
anionic site (PAS) [60]. Interaction of cholinesterase inhibitors with both components CAS
and PAS has an important role in therapeutic effects of these compounds [61].
Molecular modeling of the compound 7n as most potent AChE inhibitor among the
synthesized compounds showed that this compound interacted with CAS residues Trp84 (AS)
and Phe330 (AS) via hydrophobic interactions and PAS residues Asp72 via π-anion
interaction (Fig. 4). This compound also formed a π-cation interaction with CAS residues
His440 (CT) and hydrogen bonds with other active site residues Gly119, Gly118, and Ala201.

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The binding energy of this compound in the AChE active site was -10.82 kcal/mol. The
second potent AChE inhibitor 7l interacted with CAS residues Phe330 (AS), Phe331 (AS),
Trp84 (AS), His440 (CT), and Ser200 (CT) and PAS residues Tyr121 and Tyr334.
Furthermore, compound 7l also formed an interaction with active site residue Glu199. The
binding energy of this compound was -10.64 kcal/mol.
7
n
7l
Fig. 4. Interaction modes of compounds of 7n and 7l in the active site of AChE
Compound 7n established interactions with CAS residues Trp82 (AS), Phe329 (AS), and
His438 (CT) in the BChE active site (Fig. 6). This compound also interacted with Gly117,
Gly116, Ala199, Pro84, and Ala328 in the active site of this enzyme. Other strong BChE
inhibitor 7l established interactions with BChE active site residues Trp82 (AS), Tyr128 (AS),
His438 (CT), Gly117, Gly116, Ala199, His438, Thr120, Ala328, Trp430, and Trp231 (Fig.
5
). Binding energies of compounds 7n and 7l were -10.17 and -8.64 kcal/mol.
n 7l
7

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Fig. 5. Interaction modes of compounds 7n and 7l in the active site of BChE
The α-glycosidase inhibitory activities of synthesized compounds of 7a-n exhibited that
compounds of 7h and 7j acted better than others (Table 1). Docking study of compound of 7h
demonstrated that this compound established a hydrogen bond with Gln322 and two non-
classical hydrogen bonds with Thr301 and Gly306 in the α-glucosidase active site.
Furthermore, several hydrophobic interactions between compound of 7h and active site
residues Val305, Pro309, and His279 were also observed (Fig. 6). The second potent α-
glucosidase inhibitor of 7j formed following interactions with α-glucosidase active site: two
hydrogen bonds with residues Gly306 and Thr307Gln322, two non-classical hydrogen bond
with Thr301 and Glu304, a π-anion interaction with Glu304, and several hydrophobic
interactions Pro309 and His279. Binding energies of compounds of 7h and 7j were -7.45 and
-7.37 kcal/mol.
7
h
7j

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Fig. 6. Interaction modes of compounds of 7h and 7j in the active site of α-glucosidase
enzyme
2
.4. In vitro cytotoxicity assay
Given that several series of quinazolinone derivatives with high cytotoxic effects were
reported, all the newly synthesized compounds 7a-n were evaluated against human cancer cell
lines LNCaP and MCF-7 by MTT assay [62-64]. Obtained results revealed that title
compounds do not show significant cytotoxic effects against studied cell lines (Table 2).
Table 2. In vitro cytotoxicity effects of newly synthesized compounds 7a-n
Compound
Cytotoxicity (LogIC μM)
Compound
Cytotoxicity (LogIC μM)
50
5
0
LNCaP
2.81±0.67
2.55±0.2
3.15±1.21
MCF-7
LNCaP
MCF-7
7
a
1.63±0.07
3.71±0.33
3.93±0.51
7h
7i
3.46±0.56
2.58±0.19
3.58±1.09
1.82±0.32
2.49±0.15
2.86±0.41
7
b
7
c
7j

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7
d
2.38±0.1
2.59±0.14
2.71±0.37
2.85±0.32
2.69±0.15
2.05±0.04
2.00±0.03
1.95±0.32
7k
7l
2.33±0.09
2.38±0.1
3.56±0.32
1.76±0.08
2.99±0.26
3.4±0.23
7
e
7
f
7m
7n
5.75±2.21
3.57±0.73
7
g
3
. Materials and methods
.1. General Procedure for the Synthesis of quinazolinone derivatives 3
3
A mixture of 2-aminobenzamide 1 (1 mmol), aromatic aldehydes 2 (1 mmol), and Na S O
5
2
2
o
(
1.1 mmol) in DMF (20 mL) was stirred at 100 C for 5 h at the closed condition. Then, the
mixture was poured in the cold water and a pure quinazolinone derivative 3 was filtered off.
3
.2. Synthesis of tosyl-metronidazole 6
A solution of tosyl chloride 4 (1 mmol) and metronidazole 5 (1 mmol) in pyridine (15 mL)
was stirred at 50 °C for 4 h at the closed condition. Then, the mixture was poured in water and
was filtered off. The obtained residue was washed with water to obtain pure tosyl-
metronidazole (6).
3
.3. General Procedure for the Synthesis of quinazolinone derivatives linked to metronidazole
7
a-n
Finally, quinazolinone derivatives 3 (1 mmol), tosyl-metronidazole 6 (1 mmol), and K CO in
2
3
DMF (10 mL) were stirred at 80 °C for 24 h at the closed condition. After that, reaction
mixture was poured into water and formed products were filtered off, washed with water, and
purified by recrystallization (in ethylacetate) to give target compounds of 7a-n.
3
-(2-(2-methyl-5-nitro-1H-imidazol-1-yl)ethyl)-2-phenylquinazolin-4(3H)-one 7a

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-1
1
White solid (87%); mp > 250 °C; IR (KBr) ṽ [cm ] = 3073, 2925, 1641, 1299. H NMR (301
2
3
MHz, DMSO-d 25 °C, TMS): δ = 8.36 (d, J = 8.2 Hz, 2H), 8.00 (s, 1H, H-C ), 7.97 – 7.93
6
,
(m, 4H), 7.65 (dd, J = 8.5, 3.9 Hz, 1H), 7.35 (d, J = 8.0 Hz, 2H), 5.14 (t, J = 4.9 Hz, 2H,
12
13
13
H C ), 4.93 (t, J = 4.9 Hz, 2H, H C ), 2.42 (s, 3H, CH ) ppm. C NMR (76 MHz, DMSO-
2
2
3
d6): δ = 166.22 (C-carbonyl), 159.10, 151.95, 151.74, 139.16, 137.50, 134.94, 133.57, 131.36,
12
13
26
1
29.01, 128.52, 128.44, 128.22, 127.86, 123.24, 114.64, 50.52 (C ), 45.49 (C ), 14.49 (C )
ppm. Anal. Calcd for C H N O (375): C, 63.99; H, 4.56; N, 18.66. found: C, 64.02; H,
2
0
17
5
3
4
.51; N, 18.64.
-(2-(2-methyl-5-nitro-1H-imidazol-1-yl)ethyl)-2-(p-tolyl)quinazolin-4(3H)-one 7b
3
-1
1
White solid (94%); mp 214-216 °C; IR (KBr) ṽ [cm ] = 3074, 2925, 1640, 1298. H NMR
(
301 MHz, DMSO-d 25 °C, TMS): δ = 8.50 – 8.43 (m, 2H), 8.03 – 7.93 (m, 3H), 7.67 (dd, J
6,
1
2
=
8.0, 4.7 Hz, 1H), 7.58 – 7.52 (m, 3H), 5.16 (t, J = 4.8 Hz, 2H, H C ), 4.94 (t, J = 4.8 Hz,
2
1
3
13
2
1
H, H C ), 2.41 (s, 3H, CH ), 2.31 (s, 3H, CH ) ppm. C NMR (76 MHz, DMSO-d ): δ =
2 3 3 6
66.11 (C-carbonyl), 159.12, 151.94, 151.76, 141.19, 139.16, 134.84, 133.58, 133.47, 129.60,

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12
13
25
1
28.42, 128.42, 128.12, 127.59, 123.22, 114.51, 50.37 (C ), 45.48 (C ), 21.53 (C ), 14.48
2
6
(
C ) ppm. Anal. Calcd for C H N O (389): C, 64.77; H, 4.92; N, 17.98. found: C, 64.72;
21 19 5 3
H, 4.93; N, 17.97.
2
-(3-hydroxyphenyl)-3-(2-(2-methyl-5-nitro-1H-imidazol-1-yl)ethyl)quinazolin-4(3H)-one 7c
-1
1
White solid (87%); mp 208-210 °C; IR (KBr) ṽ [cm ] = 3073, 2937, 1643, 1239. H NMR
(
301 MHz, DMSO-d 25 °C, TMS): δ = 9.66 (s, 1H), 7.98 (s, 1H), 7.95 – 7.93 (m, 3H), 7.69
6,
–
1
7.61 (m, 1H), 7.53 (d, J = 7.9 Hz, 2H), 7.34 (t, J = 7.9 Hz, 1H), 6.96 (dd, J = 8.0, 1.7 Hz,
1
2
13
H), 5.10 (m, J = 4.8 Hz, 2H, H C ), 4.92 (t, J = 4.7 Hz, 2H, H C ), 2.29 (s, 3H, CH ) ppm.
2
2
3
1
3
C NMR (76 MHz, DMSO-d ): δ = 166.06 (C-carbonyl), 159.18, 158.05, 151.96, 151.69,
6
1
38.92, 138.56, 134.83, 133.56, 129.93, 128.68, 128.16, 127.73, 125.94, 123.17, 119.43,
12
13
25
1
14.62, 57.18 (C ), 45.41 (C ), 14.51 (C ) ppm. Anal. Calcd for C H N O (391):
20 17 5 4
C H N O , C, 61.38; H, 4.38; N, 17.89. found: C, 61.41; H, 4.34; N, 17.88.
20
17
5
4
2
-(2-methoxyphenyl)-3-(2-(2-methyl-5-nitro-1H-imidazol-1-yl)ethyl)quinazolin-4(3H)-one 7d

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-1
1
White solid (94%); mp 205-207 °C; IR (KBr) ṽ [cm ] = 3086, 2963, 1659, 1293. H NMR
(
301 MHz, DMSO-d 25 °C, TMS): δ = 8.33 (d, J = 8.1 Hz, 2H), 7.99 – 7.89 (m, 4H), 7.67 –
6,
1
2
7
.58 (m, 1H), 7.32 (d, J = 8.1 Hz, 2H), 5.11 (t, J = 4.7 Hz, 2H, H C ), 4.91 (t, J = 4.7 Hz, 2H,
2
13
13
H C ), 4.03 (s, 3H, OCH ), 2.40 (s, 3H, CH ) ppm. C NMR (76 MHz, DMSO-d ): δ =
2
3
3
6
1
66.05 (C-carbonyl), 159.11, 151.92, 151.74, 141.14, 139.14, 134.81, 134.78, 133.57, 129.56,
2
7
12
13
1
29.56, 128.50, 128.40, 128.08, 127.53, 123.18, 114.51, 65.23 (C ), 51.01 (C ), 45.48 (C ),
2
0
1
4.48 (C ) ppm. Anal. Calcd for C H N O (405): C H N O , C, 66.80; H, 4.53; N, 14.98.
26 21 5 4 26 21 5 4
found: C, 66.83; H, 4.54; N, 14.91.
2
-(3-methoxyphenyl)-3-(2-(2-methyl-5-nitro-1H-imidazol-1-yl)ethyl)quinazolin-4(3H)-one 7e
-1
1
White solid (91%); mp 234-236 °C; IR (KBr) ṽ [cm ] = 2952, 1646, 1258. H NMR (301
MHz, DMSO-d 25 °C, TMS): δ = 8.05 (d, J = 7.8 Hz, 1H), 8.02 – 7.89 (m, 5H), 7.70 – 7.62
6,
(m, 1H), 7.46 (t, J = 7.9 Hz, 1H), 7.13 (dd, J = 8.1, 2.0 Hz, 1H), 5.15 (t, J = 4.8 Hz, 2H,
12
13
13
H C ), 4.92 (t, J = 4.8 Hz, 2H, H C ), 3.89 (s, 3H, OCH ), 2.41 (s, 3H, CH ) ppm. C NMR
2
2
3
3
(
76 MHz, DMSO-d ): δ = 166.13 (C-carbonyl), 159.96, 158.84, 151.93, 151.65, 139.15,
6
1
38.98, 134.90, 133.54, 130.06, 128.23, 127.87, 123.21, 120.90, 117.10, 114.66, 113.44,
2
7
12
13
25
6
5.27 (C ), 55.63 (C ), 45.47 (C ), 14.49 (C ) ppm. Anal. Calcd for C H N O (405):
26 21 5 4
C H N O , C, 66.80; H, 4.53; N, 14.98. found: C, 66.82; H, 4.58; N, 14.96.
26
21
5
4
2
-(4-methoxyphenyl)-3-(2-(2-methyl-5-nitro-1H-imidazol-1-yl)ethyl)quinazolin-4(3H)-one 7f

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-1
1
White solid (89%); mp 209-211 °C; IR (KBr) ṽ [cm ] = 2956, 1642, 1298. H NMR (301
MHz, DMSO-d 25 °C, TMS): δ = 8.41 (d, J = 8.9 Hz, 2H), 7.97 – 7.91 (m, 3H), 7.65 – 7.58
6,
1
2
(
m, 1H), 7.50 (d, J = 8.0 Hz, 1H), 7.08 (d, J = 8.9 Hz, 2H), 5.13 (t, J = 4.8 Hz, 2H, H C ),
2
13
13
4
.93 (t, J = 4.8 Hz, 2H, H C ), 3.87 (s, 3H, OCH ), 2.41 (s, 3H, CH ) ppm. C NMR (76
2 3 3
MHz, DMSO-d ): δ = 166.02 (C-carbonyl), 162.07, 158.98, 151.86, 139.16, 134.81, 133.59,
6
2
7
12
1
4
4
30.16, 130.01, 128.60, 127.96, 127.30, 125.93, 123.21, 114.34, 65.20 (C ), 55.82 (C ),
1
3
26
5.50 (C ), 14.50 (C ) ppm. Anal. Calcd for C H N O (405): C H N O , C, 66.80; H,
2
6
21
5
4
26 21
5
4
.53; N, 14.98. found: C, 66.83; H, 4.53; N, 14.97.
2
-(4-chlorophenyl)-3-(2-(2-methyl-5-nitro-1H-imidazol-1-yl)ethyl)quinazolin-4(3H)-one 7g
-1
1
White solid (95%); mp 239-241 °C; IR (KBr) ṽ [cm ] = 2983, 1647, 1291. H NMR (301
MHz, DMSO-d 25 °C, TMS): δ = 8.42 (d, J = 8.5 Hz, 2H), 7.99 – 7.91 (m, 4H), 7.69 – 7.63
6,
12
(
m, 1H), 7.57 (d, J = 8.6 Hz, 2H), 5.11 (t, J = 4.6 Hz, 2H, H C ), 4.92 (t, J = 4.5 Hz, 2H,
2
13
13
H C ), 2.40 (s, 3H, CH ) ppm. C NMR (76 MHz, DMSO-d ): δ = 166.24 (C-carbonyl),
2
3
6
1
58.03, 151.96, 151.57, 139.14, 136.32, 136.19, 134.99, 133.59, 130.10, 129.04, 128.17,

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1
2
13
25
1
28.02, 123.23, 114.61, 55.24 (C ), 45.44 (C ), 14.50 (C ) ppm. Anal. Calcd for
C H ClN O (409): C, 58.61; H, 3.94; N, 17.09. found: C, 58.57; H, 3.91; N, 17.06.
20
16
5
3
2
-(3-bromophenyl)-3-(2-(2-methyl-5-nitro-1H-imidazol-1-yl)ethyl)quinazolin-4(3H)-one 7h
-1
1
White solid (88%); mp 248-250 °C; IR (KBr) ṽ [cm ] = 2905, 1742, 1156. H NMR (301
MHz, DMSO-d 25 °C, TMS): δ = 8.34 (d, J = 8.1 Hz, 2H), 7.97 (m, 1H), 7.94 (d, J = 3.4 Hz,
6
,
1
2
2
4
H), 7.68 – 7.59 (m, 1H), 7.33 (d, J = 8.1 Hz, 2H), 5.12 (t, J = 4.7 Hz, 2H, H C ), 4.92 (t, J =
2
1
3
13
.7 Hz, 2H, H C ), 2.41 (s, 3H, CH ) ppm. C NMR (76 MHz, DMSO-d ): δ = 166.38 (C-
2
3
6
carbonyl), 163.07, 157.58, 151.94, 149.04, 139.83, 134.92, 133.53, 130.80, 128.31, 127.93,
12
13
26
1
27.24, 126.33, 123.29, 122.53, 121.64, 114.80, 52.39 (C ), 45.43 (C ), 14.52 (C ) ppm.
Anal. Calcd for C H BrN O (454): C, 52.88; H, 3.55; N, 15.42. found: C, 52.83; H, 3.52;
2
0
16
5
3
N, 15.47.
2
4
-(3-methoxy-2-nitrophenyl)-3-(2-(2-methyl-5-nitro-1H-imidazol-1-yl)ethyl)quinazolin-
(3H)-one 7i

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-1
1
White solid (81%); mp 174-176 °C; IR (KBr) ṽ [cm ] = 3082, 2967, 2924, 1657, 1224. H
NMR (301 MHz, DMSO-d 25 °C, TMS): δ = 8.04 – 7.99 (m, 2H), 7.97 (d, J = 4.8 Hz, 2H),
6
,
7
.92 (d, J = 8.1 Hz, 1H), 7.76 – 7.68 (m, 2H), 7.55 (d, J = 7.8 Hz, 1H), 4.99 – 4.86 (m, 4H,
1
3
CH -CH ), 3.98 (s, 3H, OCH ), 2.41 (s, 3H, CH ) ppm. C NMR (76 MHz, DMSO-d ): δ =
2
2
3
3
6
1
1
1
5
66.13 (C-carbonyl), 156.10, 151.90, 151.25, 140.47, 139.05, 135.35, 133.56, 131.67, 131.60,
2
7
12
13
30.48, 128.92, 128.29, 123.25, 122.38, 116.13, 114.56, 66.08 (C ), 57.43 (C ), 45.25 (C ),
2
0
4.46 (C ) ppm. Anal. Calcd for C H N O (450): C, 56.00; H, 4.03; N, 18.66. found: C,
2
1
18
6
6
5.98; H, 4.01; N, 18.64.
-(2-(2-methyl-5-nitro-1H-imidazol-1-yl)ethyl)-2-(3-nitrophenyl)quinazolin-4(3H)-one 7j
3
-1
1
White solid (92%); mp 185-187 °C; IR (KBr) ṽ [cm ] = 3082, 2965, 1651, 1247. H NMR
(
301 MHz, DMSO-d 25 °C, TMS): δ = 8.62 (d, J = 2.8 Hz, 1H), 8.37 (dd, J = 8.8, 2.8 Hz,
6,
1
H), 8.11 – 8.03 (m, 3H), 7.94 (d, J = 8.8 Hz, 1H), 7.88 (s, 1H), 7.84 – 7.77 (m, 1H), 5.08 (t, J

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1
2
13
13
=
4.8 Hz, 2H, H C ), 4.90 (t, J = 4.8 Hz, 2H, H C ), 2.47 (s, 3H, CH ) ppm. C NMR (76
2
2
3
MHz, DMSO-d ): δ = 166.19 (C-carbonyl), 158.17, 151.86, 151.27, 146.73, 139.21, 139.17,
6
1
2
1
4
1
38.55, 135.36, 133.42, 132.59, 129.12, 128.35, 126.76, 125.74, 123.27, 114.45, 54.27 (C ),
13
20
5.42 (C ), 14.43 (C ) ppm. Anal. Calcd for C H ClN O (454): C, 52.81; H, 3.32; N,
20
15
6
5
7.48. found: C, 52.85; H, 3.31; N, 17.53.
2
4
-(4-ethoxy-3-methoxyphenyl)-3-(2-(2-methyl-5-nitro-1H-imidazol-1-yl)ethyl)quinazolin-
(3H)-one 7k
-1
1
White solid (86%); mp 199-201 °C; IR (KBr) ṽ [cm ] = 2972, 2938,1634, 1252. H NMR
(
301 MHz, DMSO-d 25 °C, TMS): δ = 8.04 – 7.86 (m, 6H), 7.60 (dd, J = 8.0, 4.0 Hz, 1H),
6,
1
2
13
7
6
.05 (d, J = 8.5 Hz, 1H), 5.18 – 5.09 (m, 2H, H C ), 4.96 – 4.85 (m, 2H, H C ), 4.11 (q, J =
2 2
29
30
.8 Hz, 2H, H C ), 3.91 (s, 3H, OCH ), 2.49 (s, 3H, H C), 1.39 (t, J = 6.8 Hz, 3H, H C )
2
3
3
3
1
3
ppm. C NMR (76 MHz, DMSO-d ): δ = 165.87 (C-carbonyl), 158.94, 151.91, 151.80,
6
1
1
51.10, 149.10, 139.13, 134.73, 133.54, 129.93, 127.93, 127.21, 123.17, 121.98, 114.28,
27
29
12
13
30
25
12.47, 111.30, 64.94 (C ), 64.21 (C ), 55.82 (C ), 45.55 (C ), 15.16 (C ), 14.47 (C )
ppm. Anal. Calcd for C H N O (449): C, 61.46; H, 5.16; N, 15.58. found: C, 61.43; H,
2
3
23
5
5
5
.13; N, 15.62.
-(2-(2-methyl-5-nitro-1H-imidazol-1-yl)ethyl)-2-(4-phenoxyphenyl)quinazolin-4(3H)-one 7l
3

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-1
1
White solid (84%); mp 189-191°C; IR (KBr) ṽ [cm ] =3056, 2938, 1632, 1309. H NMR
2
3
(
301 MHz, DMSO-d 25 °C, TMS): δ = 8.25 (d, J = 7.6 Hz, 1H), 8.10 (s, 1H, H-C ), 8.01 –
6,
7
7
.93 (m, 4H), 7.68 (s, 1H), 7.57 (d, J = 7.9 Hz, 1H), 7.49 – 7.43 (m, 2H), 7.24 – 7.18 (m, 2H),
1
2
13
.13 (d, J = 7.8 Hz, 2H), 5.14 – 5.03 (m, 2H, H C ), 4.98 – 4.86 (m, 2H, H C ), 2.42 (s, 3H,
2
2
13
CH ) ppm. C NMR (76 MHz, DMSO-d ): δ = 166.25 (C-carbonyl), 158.40, 157.52, 157.08,
3
6
1
1
51.93, 151.60, 143.98, 139.66, 134.99, 133.55, 130.61, 128.26, 124.12, 123.60, 123.24,
1
2
13
26
21.54, 119.25, 118.35, 114.73, 50.04 (C ), 45.38 (C ), 14.49 (C ) ppm. Anal. Calcd for
C H N O (467): C, 66.80; H, 4.53; N, 14.98. found: C, 66.84; H, 4.52; N, 14.9.
26
21
5
4
3
-(2-(2-methyl-5-nitro-1H-imidazol-1-yl)ethyl)-2-(thiophen-2-yl)quinazolin-4(3H)-one 7m
-1
1
White solid (84%); mp >250°C; IR (KBr) ṽ [cm ] = 3075, 2938, 1644, 1299. H NMR (301
MHz, DMSO-d 25 °C, TMS): δ = 8.00 – 7.95 (m, 3H), 7.92 (dd, J = 6.8, 1.4 Hz, 1H), 7.87
6
,
(d, J = 7.7 Hz, 1H), 7.80 (dd, J = 5.0, 1.2 Hz, 1H), 7.66 – 7.59 (m, 1H), 7.24 (dd, J = 5.0, 3.7
12
13
Hz, 1H), 5.12 (t, J = 4.9 Hz, 2H, H C ), 4.91 (t, J = 4.9 Hz, 2H, H C ), 2.41 (s, 3H, CH )
2
2
3
1
3
ppm. C NMR (76 MHz, DMSO-d ): δ = 166.01 (C-carbonyl), 156.08, 151.90, 151.59,
6
1
43.31, 139.15, 135.05, 133.58, 131.42, 129.75, 128.84, 127.65, 127.51, 123.36, 114.47,

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1
2
13
20
5
3
4.74 (C ), 45.47 (C ), 14.49 (C ) ppm. Anal. Calcd for C H N O S (381): C, 56.68; H,
18 15 5 3
.96; N, 18.36. found: C, 56.74; H, 3.92; N, 18.31.
-(2-(2-methyl-5-nitro-1H-imidazol-1-yl)ethyl)-2-(naphthalen-2-yl)quinazolin-4(3H)-one 7n
3
-1
1
White solid (92%); mp 177-179 °C; IR (KBr) ṽ [cm ] = 3071, 2936, 1632, 1297. H NMR
2
3
(
301 MHz, DMSO-d 25 °C, TMS): δ = 8.06 – 8.01 (m, 3H), 7.95 (s, 1H, H-C ), 7.89 (s,
6,
1
H), 7.75 – 7.72 (m, 1H), 7.71 – 7.65 (m, 2H), 7.62 – 7.59 (m, 2H), 7.58 – 7.53 (m, 2H), 5.07
12
13
13
(
t, J = 4.8 Hz, 2H, H C ), 4.92 (t, J = 4.8 Hz, 2H, H C ), 2.38 (s, 1H, CH ) ppm. C NMR
2 2 3
(
76 MHz, DMSO-d ): δ = 166.00 (C-carbonyl), 161.59, 155.01, 151.95, 151.58, 135.91,
6
1
1
35.36, 134.96, 134.06, 133.51, 131.93, 131.08, 130.74, 129.68, 128.87, 128.22, 127.19,
12
13
26
26.73, 126.39, 125.63, 123.17, 121.02, 114.26, 50.63 (C ), 45.33 (C ), 14.49 (C ) ppm.
Anal. Calcd for C H N O (425): C, 67.76; H, 4.50; N, 16.46. found: C, 67.72; H, 4.43; N,
2
4
19
5
3
1
6.47.
.4. Enzymatic assays
3
In the present work, hCA I, and II isoenzymes were purified by Sepharose-4B-L-Tyrosine-
sulfanilamide affinity column chromatography [65]. On the other hand, inhibition effects of
novel quinazolinone derivatives 7a-n on CA isoenzymes activity was determined according to
the spectrophotometric method of Verpoorte et al. as described in our previous studies [66-
7
0]. The inhibitory effect of novel quinazolinone derivatives 7a-n on AChE and BChE

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activities was performed according to spectrophotometric method of Ellman as described
previously [71-74]. Butyrylcholinesterase from equine serum and acetylcholinesterase from
Electrophorus electricus (electric eel) have been purchased from Sigma-Aldrich. α-
Glycosidase inhibition effect of novel quinazolinone derivatives (7a-n) was evaluated
according to the method of Tao et al [75-79]. The absorbance of samples was recorded at 405
nm.
3
.5. Docking study
Docking studies of the compounds 7j and 7g as most potent compounds against hCA I (pdb
code: 4WR7) and hCA II (pdb code: 5AML), compounds 7n and 7l as most potent
compounds against AChE (pdb code: 1EVE), and BChE (pdb code: 1P0I), and compounds 7h
and 7j as most potent compound against α-glucosidase (modeled enzyme) were performed by
Autodock Tools 1.5.6 [80, 81]. The 3D structures of the selected inhibitors were constructed
by MarvineSketch 5.10.4, and then the pdbqt formats of these entries were prepare by
Autodock Tools 1.5.6. By using the latter software, the pdbqt structures of the target enzymes
were also constructed and Autodock Tools parameters for them were set as follows: 1) hCA I:
box size: 40×40×40 Å, the center of box: x = 1.8085, y = 72.66, z = 55.3515, 2) hCA II: box
size: 60×60×60 Å, the center of box: x = -3.8315, y = 3.9065, z = 15.043, 3) AChE: box size:
4
0×40×40 Å, the center of box: x = 2.023, y = 63.295, z = 67.062, 4) BChE: box size:
5
6×56×56 Å, the center of box: x = 137.985, y = 122.725, z = 38.78, 5) α-glucosidase: box
size: 40×40×40 Å, the center of box: x = 12.5825, y = -7.8955, z = 12.519 [82]. Each docked
system was carried out by 25 runs of the AUTODOCK search (by the Lamarckian genetic
algorithm). Finally, the best-docked pose of each selected compound was analyzed by
Discovery Studio 2019 Client (Accelrys, Inc., San Diego, CA).
3
3
.6. Cytotoxicity effect
.6.1. Proliferation of cells

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Human prostate and breast cancer cell lines (LNCaP and MCF-7, respectively) were used to
determine the cytotoxic effects of the tested compounds. LNCaP and MCF-7 cells were
cultured in RPMI-1640 medium (Sigma-Aldrich, USA) and DMEM medium (Gibco, UK),
respectively. This medium supplemented with 10% fetal bovine serum (Biowest, USA) and
2
1
% penicillin/streptomycin solution (Gibco, UK). Cell plates were maintained in 75-cm
culture flasks (TPP) and were placed in 5% CO humidified atmosphere at 37 °C (Thermo
2
Forma II CO incubator, USA).
2
3
.6.2. Cell Viability
The cytotoxicity of compounds was tested using 3-(4,5-dimethyl thiazol-2-il)-2,5-
diphenyltetrazolium bromide (MTT) assays [82]. LNCaP and MCF-7 cells were seeded in 96-
3
well microplate (15×10 ) and cultured for 24 h. Cells were treated with 1, 5, 25, 50 and 100
µM concentrations of test compounds (DMSO for Vehicle control) at 24 h. After incubated,
5
0 μL of MTT (0.5 mg/mL) solution was added to the wells and incubated further for 3 h.
Then medium was removed and 100 μL of DMSO was added to each wells. A microplate
reader was used for measurement of absorbance of each plate at 570 nm (Thermo Multiskan
Go, USA). Absorbances from the control wells (only cells and culture medium) were
measured and the mean absorbance values obtained were considered as 100% viable cells.
Viability values in treated groups were calculated according to the control group. These
experiments were repeated 5 times independently on different days [83].
3
.7. Statistical Analysis
Statistical analysis was performed using SigmaPlot 12.0 software package. Homogeneity of
variance was evaluated by Shapiro-Wilk test. In case of equal variances, ANOVA was used to
determine differences between the two groups and Bonferroni test was used for multiple
comparisons. In case of unequal variances (or non-normal distributions), Kruskal-Wallis H

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test followed by post-hoc Tukey test was used. Quantitative data were expressed as the mean
of standard deviation (Mean±SD) and were considered significant at p<0.05 [84].
4
. Conclusion
New quinazolinone derivatives 7a-n were synthesized and screened for their inhibitory
activities on metabolic enzymes hCA I, II, AChE, BChE, and α-glycosidase. All the
synthesized compounds exhibited better enzymatic inhibitory activities than acetazolamide as
standard CA inhibitor, tacrine as standard ChEI, and acarbose as standard α-glycosidase
inhibitor. Molecular docking analysis of the most potent derivatives against each enzyme was
performed and obtained data revealed that our new potent synthesized compounds interacted
with key residues in the active site of studied target enzymes. Furthermore, in vitro
cytotoxicity study of the title compounds 7a-n demonstrated that these compound were
almost inactive against cancer cell line MCF-7 and LNCaP.
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