1,4-Dihydroquinazolin-3(2H)-yl benzamide derivatives as anti-inflammatory and analgesic agents with an improved gastric profile: Design, synthesis, COX-1/2 inhibitory activity and molecular docking study

Article abstract of DOI:10.1016/j.bioorg.2018.11.030

The design and synthesis of a new series of 1,4-dihydroquinazolin-3(2H)-yl benzamide derivatives (4a–o) as anti-inflammatory and analgesic agents and COX-1/2 inhibitors are reported. The target compounds (4a–o) were synthesized using a two-step scheme, an

Full text of DOI:10.1016/j.bioorg.2018.11.030

BioorganicChemistry84(2019)76–86
Contents lists available at ScienceDirect
Bioorganic Chemistry
journal homepage: www.elsevier.com/locate/bioorg
1,4-Dihydroquinazolin-3(2H)-yl benzamide derivatives as anti-
inflammatory and analgesic agents with an improved gastric profile: Design,
synthesis, COX-1/2 inhibitory activity and molecular docking study
⁎
Asmaa Sakra,1, Hend Kothayera, ,1, Samy M. Ibrahima, Mohamed M. Barakaa, Samar Rezqb
a Department of Medicinal Chemistry, Faculty of Pharmacy, Zagazig University, Egypt
b Department of Pharmacology, Faculty of Pharmacy, Zagazig University, Egypt
A R T I C L E I N F O
A B S T R A C T
Keywords:
The design and synthesis of a new series of 1,4-dihydroquinazolin-3(2H)-yl benzamide derivatives (4a–o) as
anti-inflammatory and analgesic agents and COX-1/2 inhibitors are reported. The target compounds (4a–o) were
synthesized using a two-step scheme, and their chemical structures were confirmed with 1H NMR, 13C NMR, and
mass spectra and elemental analysis. Compounds 4b, 4d, 4h, 4l, 4n and 4o showed the best in vitro COX-2
inhibitory activity (IC50 0.04–0.07 μM), which was nearly the same as that of the reference drug celecoxib (IC50
0.049 μM), but had a lower selectivity index, as dictated in our target design. In the in vivo anti-inflammatory
inhibition assay, compounds 4b, 4c, 4e, 4f, 4m and 4o showed better oedema inhibition percentages, ranging
from 38.1% to 54.1%, than did diclofenac sodium (37.8%). An in vivo analgesic assay revealed that compounds
4b and 4n had a potential analgesic effect 4- to 21-fold more potent than that of indomethacin and diclofenac
sodium. All the tested compounds showed an improved ulcerogenic index when compared to indomethacin. In
the synthesized series, compound 4b showed the best biological activity in all the experiments. The docking
study results agreed with the in vitro COX inhibition assay results. Moreover, the predicted in silico studies of all
the compounds support their potential as drug candidates.
COX-1/2 inhibition
Dihydroquinazolinyl benzamide
Inflammation
Molecular modeling
1. Introduction
has a protective mechanism in the stomach and kidney, and blocking this
pathway elicits the main side effects of NSAIDs, varying from bleeding to
Non-steroidal anti-inflammatory drugs (NSAIDs) have been used for
decades worldwide for symptomatic treatment of acute and chronic pain,
and these drugs are known for their effects on fever, inflammation and
rheumatic disorders [1–3]. The inflammation process is mainly con-
trolled by the cyclooxygenase (COX) enzyme, which is the key enzyme in
prostaglandin biosynthesis and is responsible for inflammation and pain
[4]. The discovery of two COX isoforms, COX-1 and COX-2, provided
another facet for the exploration of this enzyme [5,6]. COX-1 and COX-2
are the main targets of most traditional anti-inflammatory drugs [7,8].
Studies of the different effects of the two isoforms revealed that COX-1
peptic ulcers [9]. Accordingly, the new approach was to make selective
COX-2 inhibitors lacking these side effects. Rofecoxib and valdecoxib
(Fig. 1) are the most famous and highly selective coxibs, or COX-2 in-
hibitors; unfortunately, they were withdrawn from the market due to
their cardiovascular side effects [10,11]. Celecoxib (Fig. 2) is still on the
market, and current evidence supports it as a therapeutic option due to
its lower toxicity than other coxibs [12]. Recent studies have also focused
on the promising applications of selective COX-2 inhibitors in the
treatment of cancer and neurodegenerative disorders, such as Alzhei-
mer’s disease and Parkinson’s disease [13].
⁎ Corresponding author at: Department of Medicinal Chemistry, Faculty of Pharmacy, Zagazig University, 44519 Zagazig, Egypt.
E-mail addresses: HKElhamalawy@pharmacy.zu.edu.eg, hendo1311@hotmail.com (H. Kothayer).
1 Equal contribution.
https://doi.org/10.1016/j.bioorg.2018.11.030
Received 23 September 2018; Received in revised form 14 November 2018; Accepted 19 November 2018
Availableonline22November2018
0045-2068/©2018ElsevierInc.Allrightsreserved.

A. Sakr et al.
BioorganicChemistry84(2019)76–86
Fig. 1. The chemical structures of the withdrawn highly selective COX-2 inhibitors.
Fig. 2. The molecular design through rationale prospective to obtain the novel target compounds (4a–o).
77

A. Sakr et al.
BioorganicChemistry84(2019)76–86
Scheme 1. Synthetic route of target compounds, reagent, and conditions: (a) C2H5OH/2 ml glacial acetic acid, reflux, 12 h; (b) Appropriate aromatic aldehyde,
glacial acetic acid, reflux, 8 h.
The highly selective inhibition of COX-2 leads to potential cardiac
side effects, while non-selective (i.e., COX-1/COX-2) inhibition with
greater affinity for COX-1 is responsible for gastric and kidney problems
[9], albeit with reduced or even absent cardiovascular risk.
sulfonamide and methyl sulfonyl groups present in celecoxib and
etoricoxib, respectively.
Previous studies highlighted the potential essential role of the para-
substituted sulfonyl group in boosting selectivity for COX-2 by perfectly
fitting in the smaller pocket and interacting with the crucial amino acid
Arg513, which is found in the active site of only COX-2 [19,29].
The newly synthesized compounds 4a–o were evaluated for their
COX-1/COX-2 selectivity using in vitro and in vivo assays of anti-in-
flammatory and analgesic activity and for their ulcer index (UI) profile.
Molecular modelling and in silico studies were used to predict their
binding modes with COX-1/COX2, physicochemical properties and
pharmacokinetic profiles.
Large numbers of compounds have been designed, synthesized and
evaluated for their selectivity towards COX-2. The common structural
features are the presence of two neighbouring aryl rings attached to a
central heterocyclic moiety (V-shape) and a sulfonamide or sulfone
substituent at the para position of one of the rings, which provides the
high selectivity [14–16]. Recent trials introduced a linker, either an
ester as in compound I (Fig. 2) [17] or an amide as in compound II
(Fig. 2) [18], between one of the aryl rings and the central heterocycle.
This linker was supposed to improve hydrogen bond formation with the
active site of the enzyme, and inclusion of the linker generated potent
anti-inflammatory and selective anti-COX-2 compounds, as reported for
compounds I and II [17,18].
2. Results and discussion
2.1. Chemistry
To avoid the side effects of both highly selective COX-2 inhibitors
and non-selective COX inhibitors, we designed compounds with mod-
erately selective COX-2 inhibitory activity [7,9,15,19]. The main goal
was achieved by designing compounds that retained some structural
features of highly selective COX-2 inhibitors but lacked others. The
The pathways of the synthetic compounds are illustrated in Scheme
1.
N-Methyl-isatoic anhydride is a versatile starting material in the
preparation of N′-benzoyl-2-(methylamino)benzohydrazide, an im-
portant intermediate for the synthesis of several fused heterocyclic
compounds with robust biological activity. Our target intermediate N′-
design included i) the incorporation of
a large central anti-in-
flammatory ring (quinazolinone), ii) the maintenance of the vicinal
shape characteristic of selective COX-2 inhibitors, iii) the removal of the
carboxylic acid group common to non-selective COX inhibitors, and iv)
the removal of the sulfonyl group, which is responsible for the high
COX-2 selectivity. The quinazolin-4(3H)-one scaffold was chosen as a
central heterocyclic moiety for this study because of its remarkable
anti-inflammatory and analgesic activities [20]. Proquazone (structure
III, Fig. 2) and fluproquazone (structure IV, Fig. 2) both contain a
quinazolinone moiety and are well-known NSAIDs on the market,
especially used to treat gout and rheumatoid arthritis [21]. Rutae-
carpine (structure V, Fig. 2) is a natural quinazolinone derivative that
possesses anti-inflammatory and analgesic activity and high selectivity
towards COX-2 inhibition [22–24]. To maintain the common V-shaped
structural integrity, two neighbouring phenyl rings were attached at
positions 2 and 3 [17–19]. The latter was bounded through an amide
linker that may potentiate target interactions and make the compounds
slightly bulkier and thus more favourable for entry to the COX-2 active
site, which is 20% larger than the COX-1 active site [19,25,26]. On the
other hand, we avoided the incorporation of the carboxylic acid group,
which is characteristic of many traditional NSAIDs, such as in-
domethacin and reported compound VI (Fig. 2), and is responsible for
the gastric mucosal side effects [27,28]; we also removed the
(4-(substituted)-benzoyl)-2-(methylamino)benzohydrazides
(3a–c)
[30] were prepared by condensation of N-methyl-isatoic anhydride
with 4-(substituted)-benzohydrazide and were identified by their re-
ported melting points [30]. The final target compounds, 1,4-dihy-
droquinazolin-3(2H)-yl benzamide (4a–o), were prepared by cycliza-
tion of intermediates 3a–c with different aldehydes.
2.2. Biological activity
2.2.1. In vitro COX inhibition assay
All synthesized compounds were tested by in vitro COX-1/COX-2
inhibition assays using an ovine COX-1/human recombinant COX-2
assay kit as previously reported [31–33]. The half-maximal inhibitory
concentration (IC50, μM) values were determined, and the selectivity
index (SI) values were calculated as IC50 (COX-1)/IC50 (COX-2).
(Table 1).
Testing of the newly synthesized compounds revealed that they all
showed more potent COX-2 inhibitory activity than did the two re-
ference drugs diclofenac sodium and indomethacin; compounds 4b, 4d,
4h, 4l, 4n and 4o showed the greatest COX-2 inhibitory activity, with
IC50 values ranging from 0.04 to 0.07 μM, which is nearly the value as
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A. Sakr et al.
BioorganicChemistry84(2019)76–86
Table 1
26.7% to 54.1%, compared to diclofenac sodium and celecoxib, which
had average oedema inhibition percentages of 37.8% and 53.7%, re-
spectively (Table 2).
The in vitro COX-1/COX-2 inhibition assay results.
Compound Code
aCox-1 mM IC50
aCox-2 mM IC50
bSelectivity Index (SI)
Compound 4d showed the best oedema inhibition percentage of
54.1%, which was better than that of both reference drugs. Compounds
4b, 4c, 4e, 4f, 4m and 4o showed better oedema inhibition percen-
tages, ranging from 38.1% to 48%, than did diclofenac sodium (37.8%);
however, these values were lower than that of celecoxib (53.7%).
Celecoxib
15.1
4.23
0.04
9.67
4.21
8.11
5.11
8.74
7.54
6.21
5.98
7.34
9.23
6.52
3.89
5.16
4.21
5.66
0.23
0.25
0.00
0.19
0.18
0.36
0.30
0.01
0.23
0.13
0.25
0.18
0.12
0.13
0.07
0.01
0.06
0.06
0.05
0.79
0.51
0.21
0.05
0.15
0.07
0.19
0.11
0.10
0.06
0.19
0.16
0.08
0.04
0.11
0.05
0.07
0.00
0.03
0.08
0.01
0.01
0.01
0.01
0.01
0.01
0.01
0.01
0.01
0.01
0.01
0.01
0.01
0.01
0.01
302.0
5.35
Diclofenac sod.
Indomethacin
0.08
4a
4b
4c
4d
4e
4f
46.05
84.20
54.07
73.00
46.00
68.55
62.10
99.67
38.63
57.69
81.50
97.25
46.91
84.20
80.86
2.2.4. Gastric ulcerogenic activity
Ulceration and damage of the gastric mucosa are considered the
major serious side effects of NSAIDs [36,37]. Stomach lacerations were
detected and examined according to a previously reported method after
the oral administration of 100 mmol/kg test compound, 50 mmol/kg
celecoxib (first reference drug), or 20 mmol/kg indomethacin (second
reference drug) to determine if the test compounds induce ulcers
[38,39]. The results revealed that all the test compounds had a mag-
nificent safety profile when compared to indomethacin because the UI
range (zero - 10.42) was safer than that for the reference indomethacin
(UI = 19.3), as shown in Table 3.
4g
4h
4i
4j
4k
4l
4m
4n
4o
a
IC50
(mM) concentration as expressed as mean
SED, for three re-
in
Compounds 4a, 4b, 4g, and 4n had a UI of 2.3–2.7, similar to the
value of the reference drug celecoxib (2.4). Interestingly, compound 4 k
showed no ulceration (UI = zero) (Table 3, Fig. 4).
plications, (n = 3).
Selectivity index = (COX-1 IC50/COX-2 IC50).
b
the reference drug celecoxib (IC50 0.049 μM). All the synthesized
compounds had better COX-1 inhibitory activity than the reference
drug celecoxib; compounds 4b and 4l showed the most COX-1 in-
hibitory activity (IC50 4.21 μM and 3.89 μM, respectively), which was
greater than that of diclofenac sodium (IC50 4.23 μM). All the synthe-
sized compounds were 7–17 times more selective than diclofenac so-
dium (reference drug) towards COX-2.
2.3. Structure activity relationship (SAR)
To expand the SAR study, we added different substituents to both of
the neighbouring phenyl rings present in our compounds (4a–o) R and
R′ (as shown in scheme 1). We utilized chloride, fluoride, methoxy or
nitro groups as substituents.
In general, substitution of both phenyl rings was favourable as
compounds 4a, 4f and 4k with unsubstituted phenyl ring showed lower
activities in all the tests compared to their substituted analogues.
Compound 4b carrying two chloride atoms on both of the phenyl
rings showed the best activities in either the invitro COX inhibitory
activity or the in vivo anti-inflammatory activity.
The selectivity of the new compounds was significantly lower than
that of celecoxib, which could be envisioned as an advantage in
avoiding the cardiovascular side effects of highly selective COX-2 in-
hibitors [19,34].
2.2.2. In vivo analgesic assay: Acetic acid-induced writhing test
Substitution with either chloride or methoxy also showed good in
vitro and in vivo activities (compounds 4d and 4l).
The analgesic activity of the newly synthesized compounds was
evaluated by the acetic acid-induced writhing test using indomethacin,
diclofenac sodium and celecoxib as positive controls according to the
reported method [35]. The significant decrease in the number of acetic
acid-induced writhes indicated the activity of each compound. Two
compounds, 4b and 4n, had a potential analgesic effect, with 3.4 and
0.8 writhes, respectively; these compounds were 4- to 21-fold more
potent than indomethacin and diclofenac sodium (12.6 and 17.4
writhes, respectively), and 8- to 36-fold more effective than celecoxib
(28.8 writhes). Additionally, compounds 4m (12 writhes) and 4j (15.2
writhes) showed better analgesic activity than did diclofenac sodium or
celecoxib. The seven compounds, 4c, 4d, 4g, 4h, 4i, 4l, and 4o, de-
monstrated analgesic activity with the number of writhes ranging from
20.2 to 28.8, which was slightly higher than or equal to the number
after treatment with celecoxib (28.8). (Fig. 3.)
Compound 4h carrying two nitro groups showed good in vitro COX
inhibitory activity but with lower in vivo anti-inflammatory activity,
this is may be due the high polarity of the two nitro groups that de-
creases the lipophilicity of the compound and hence decreases its
bioavailability.
It is also notable that for a good in vivo anti-inflammatory activity, at
least one of the substituents should be a halogen. This is may be due to
the lipophilicity of the halogen that improves the bioavailability.
2.4. Molecular modelling and in silico study
2.4.1. Docking study
Docking studies of compounds 4a–o were performed using
Molecular Operating Environment (MOE) 2018 software. The 2D/3D
tools, scoring function and ligand interactions with the amino acid pool
for the selected proteins COX-1 (PDB code: 1EQG) and COX-2 (PDB
code: 1CX2) provided a good estimate of the behaviour of each com-
pound in the active sites. The best score was chosen for each compound.
The validation process was performed by re-docking SC-558 into the
COX-2 active site and ibuprofen into the COX-1 active site, and their
original conformations were reproduced (RMSD: 1.34 Å and 1.16 Å
Score −9.39 and −7.56 respectively).
2.2.3. In vivo anti-inflammatory inhibitory assay: Carrageenan-induced rat
paw oedema assay
The anti-inflammatory activity of the new compounds was mea-
sured by the carrageenan-induced rat paw oedema assay as previously
reported [35], and diclofenac sodium and celecoxib were used as po-
sitive reference drugs. The test compounds were administered to the
animals orally as a suspension in vehicle (1% tween 80 in 10 ml H2O
per kg), and the mean hind paw oedema thickness of rats pretreated
with the test compounds was measured at 0, 1, 2, 3, 4, 5 and 24 h after
the induction of inflammation. The percentage inhibition of oedema
thickness was calculated and compared to that with diclofenac sodium
and celecoxib. All tested compounds showed moderate anti-in-
flammatory activity, with average oedema inhibition percentages from
Ser353, Tyr355, His90, Arg513 and Arg120 are the key residues in
the binding mode of SC-558.
Comparison of the binding sites of COX-1 and COX-2 reveals an
additional side pocket in the COX-2 binding site, making it larger. This
side pocket is surrounded by His90, Gln192, Tyr355 and Arg513 (the
last residue is replaced by His513 in COX-1). Binding with Arg513 is
79

A. Sakr et al.
BioorganicChemistry84(2019)76–86
Fig. 3. Effect of the tested compounds (100 mg/kg, p.o.) indomethacin, diclofenac sodium (20 mg/kg, p.o) or celecoxib (50 mg/kg) on acetic acid-induced writhing
(0.7%, 1 ml/100 g) in mice. Statistical analysis was performed using one way ANOVA followed by Tukey’s post hoc test or student’s t test. Data is expressed as
mean
S.E.M (n = 5). *P < 0.05 vs. control values.(*) for Control, (#) for indomethacin, (@) for diclofenac sod., (&) for celecoxib.
responsible for the high selectivity of COX-2 inhibitors (usually through
the sulfone of sulfonamide groups) [18,40]. In contrast, non-selective
COX inhibition mainly results from salt bridge formation between
Arg120 and the carboxylate ion present in most non-selective inhibitors
[41].
(4a–o) revealed that the scores ranged from −3.6527 to 1.9031,
whereas the score of SC-558 was −1.3609, indicating a slight increase
in selectivity towards COX-1 compared with that of SC-558 (Supp. Data
Table 1). Compounds 4a, 4b, 4d, 4g, 4j and 4n had no interactions
with the surrounding amino acids. The central quinazolinone moiety
made the compounds too bulky to enter the site. Only compounds 4c
and 4i interacted with Arg120, Tyr355 and Val116; however, the
central ring expansion with the vicinal shape may be responsible for the
decreased selectivity and the scores being nearer those of SC-558 than
ibuprofen (1EQG).
The docking scores for the series 4a–o on COX-2 ranged from
−4.3668 to −5.9426. All the compounds 4a–o showed interactions
with Ser353 amino acid except compounds 4g and 4j. (Supp. Data
Table 1).
Beside Ser353 compounds 4c, 4e, 4h, 4m and 4o made hydrogen
bonds with His90 (Fig. 5), while compounds 4f formed hydrogen bonds
with Tyr355 and Gln192.
The results of the comparison of binding modes for each compound
with the COX-1/2 active sites met the target rationale of this study:
there was no binding to Arg513 in the COX-2 active site (responsible for
high selectivity) or Arg120 (responsible for non-selectivity). The limited
or absent hydrogen bond formation with COX-1 active site residues,
Compounds 4g and 4j showed only one hydrogen bond interaction
with Tyr355 or Gln192 respectively.
Analysis of the docking results and scores for COX-1 with the series
Table 2
Effect of the tested compounds on carrageenan-induced hind paw edema in rats.
Compound
Mean edema thickness (mm)
SEM
Average edema inhibition%
0 h
1 h
2 h
3 h
4 h
5 h
24 h
Control
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
4.18
2.18
1.67
1.09
1.12
1.09
1.19
2.36
2.14
2.35
1.56
1.34
2.09
2.58
3.00
1.05
1.49
1.15
1.99
0.64
4.88
3.82
2.71
1.68
2.17
1.68
1.32
3.33
3.14
2.98
1.67
1.57
3.03
3.24
4.00
1.02
2.13
1.26
2.2
0.58
0.21*
0.26*
0.12*
0.26*
0.12*
0.39*
0.39*
0.17*
0.54*
0.36*
0.35*
0.36*
0.39*
0.22
4.64
3.70
2.87
3.03
2.69
3.03
2.87
3.46
3.81
3.42
3.04
3.03
3.45
3.26
3.83
2.22
2.45
2.05
1.8
0.28
0.13
4.52
3.88
2.96
3.19
2.62
3.19
3.01
3.89
3.69
3.62
3.17
3.24
3.16
3.49
3.55
3.05
2.45
2.08
2.02
0.21
4.55
3.49
2.61
2.76
2.59
2.76
2.92
3.80
3.27
3.24
2.99
2.93
3.17
2.96
3.09
2.78
2.80
2.28
1.9
0.23
0.27*
0.08*
0.22*
0.26*
0.22*
0.41*
0.26
2.82
1.87
1.13
2.04
0.73
2.04
1.86
1.73
1.54
1.75
2.53
2.37
1.63
1.45
1.78
2.01
2.08
1.31
0.49
0.38
0.16
0.16*
0.27
0.11*
0.27*
0.37
0.21*
0.20*
0.21*
0.29
0.35
0.34*
0.19*
0.31*
0.29
0.36
0.41*
0.1*
–
4a
0.12*
0.19*
0.09*
0.09*
0.08*
0.32*
0.18*
0.16*
0.32*
0.14*
0.25*
0.25*
0.43*
0.23*
0.23*
0.18*
0.13*
0.14*
0.26
26.7
48
4b
0.19*
0.27*
0.24*
0.27*
0.56*
0.11*
0.24
0.28*
0.12*
0.31*
0.12*
0.36*
0.19
4c
38.1
54.1
38.1
38.8
25.4
33.5
32.2
29.9
32.5
34.8
38.2
31.2
43.4
37.8
53.7
65.4
4d
4e
4f
4g
4h
0.38
0.48*
0.27*
0.27*
0.35*
0.48*
0.21*
0.29*
0.15*
0.28*
0.44*
0.18*
4i
0.37*
0.27*
0.33*
0.33*
0.16*
0.07
0.28
4j
0.26*
0.29*
0.41*
0.24
4k
4l
4m
4n
0.26
4o
0.18*
0.35*
0.23*
0.11*
0.14*
0.43*
0.45*
0.13*
0.17*
0.43*
0.36*
0.16*
Diclofenac sod
Celecoxib
Indomethacin
All synthesized compounds were tested in comparison of reference drugs diclofenac sodium, celecoxib and indomethacin in dose of 100 mg/kg, 20 mg/kg, 50 mg/kg
and 20 mg/kg, respectively. Drugs were given orally 1 h before carrageenan injection. Paw edema thickness was measured at 0, 1, 2, 3, 4, 5 and 24 h from the
induction of inflammation. The percentage inhibition of edema thickness was calculated from the AUC data of the paw-thickness time course curves. Data are
mean
SEM (n = 5 each).
* P < 0.05 versus control values.
80

A. Sakr et al.
BioorganicChemistry84(2019)76–86
than or equal to 10 and 140 Å2, respectively. All data for our series are
provided in Supp. Data Table 2.
Table 3
Acute Ulcerogenecity study of the new compounds with two reference drugs
indomethacin and celecoxib.
The synthesized compounds 4a–4g and 4i–4o did not violate
Lipinski’s rule and had TPSA values of less than 140 Å2 (range,
52.65–107.70), which indicated high oral bioavailability. The only
exception was compound 4h, which slightly violated the parameters
with HBA = 11 and TPSA = 144.29 Å2.
Compounds
Number of rats
Lesion
Average Ulcer
number
Ulcer Index
(UI)a
with ulcer
incidence (%)
Control
0
1
1
2
3
2
2
1
3
1
2
0
3
1
1
3
5
1
–
–
Nil
4a
20
20
40
60
40
40
20
60
20
40
–
0.2
0.6
1.6
1.6
0.6
0.6
0.2
1.6
0.5
1.4
–
2.4
MolSoft software [45] was used to test the druglikeness model score
and drug water solubility. The more positive the number is, the more it
behaves as a drug. All the synthesized compounds gave scores from
1.87 to 1.06, indicating that they will act as drugs (Supp.Data Table 3).
All the tested compounds showed good water solubility values >
0.0001 (80% of drugs on the market have a solubility value greater
than 0.0001). They also had positive drug likeliness scores, and this
supports their potential as drug candidates.
4b
2.7
4c
10.42
8.1
4d
4e
5.15
5.6
4f
4g
2.3
4h
8.15
4.7
4i
4j
10.32
Nil
4k
The PreADMET calculator [46] was used in the prediction of five
more pharmacokinetic parameters. The first two models, Caco-2 and
MDCK, provide parameters of cell permeability, human intestinal ab-
sorption (HIA), brain-blood barrier (BBB) partition coefficient, plasma
protein binding (PPB) and inhibition of cytochrome P450 2D6
(CYP2D6) (Supp.Data Table 4). All compounds in series 4a–o showed
moderate cell permeability in the Caco-2 cell model (17.76–34.44 nm/
s) and low cell permeability in the MDCK cell model (0.04–0.26 nm/s)
All the synthesized compounds had high HIA values
(96.35–97.74%), which assured high oral bioavailability as observed
with the ABS%. Moreover, the compounds had a low to moderate
ability to penetrate the BBB (0.01–0.96). Furthermore, all compounds
in the series showed strong PPB, with values ranging from 90.30 to
98.77%. Finally, the inhibitory effect of compounds 4a–o on CYP2D6
was the same as that of the 3 reference drugs (celecoxib, diclofenac
sodium and indomethacin), which do not inhibit CYP2D6; thus, the test
compounds are expected to not be included in any drug-drug interac-
tions involving inhibitors and/or inducers of this enzyme. Taken to-
gether, the results demonstrated that the newly synthesized compounds
had acceptable druglikeness and physicochemical properties and ful-
filled Lipinski’s rule of five, except for one drug, 4h, which had one
violation. According to the pharmacokinetics predictions, these com-
pounds are future drug candidates.
4l
60
20
20
60
100
20
1.2
0.3
0.2
0.6
12.2
1
7.9
4m
7.1
4n
2.3
4o
9.7
Indomethacin
Celecoxib
23.6
2.4
where ulcer index is the sum of average severity (US), average number (UN)
and % lesion incidence (UP). For orally administrated dose of tested compound
100 mg/kg, celecoxib 50 mg/kg and indomethacin with 20 mg/kg. (N = 5).
a
The ulcer index (UI) was calculated according to the equation
(UI = UN + US + UPX10−1).
along with ligand exposure to every bulky group, e.g., the methoxy,
chloro, nitro and two aryl groups, especially the central aromatic group,
made it difficult for the compounds to enter the site.
2.4.2. In silico prediction of pharmacokinetic and physiochemical properties
Molinspiration software [42] was used to predict the oral bioa-
vailability of our compounds through Lipinski’s rule of five.
Molinspiration gives a clear result not only about violations of the
rule of five but also about the topological polar surface area (TPSA)
(Å2). The bioavailability is acceptable for a drug with a TPSA value
below 140–150 Å2, but drugs with a value below 70–80 Å2 will pene-
trate the BBB and act in the CNS. The TPSA was also used in the cal-
culation of oral bioavailability (%ABS) by the following previously re-
ported equation: (%ABS) = 109–0.345 TPSA [43,44]. The two
parameters, TPSA and number of rotatable bonds (NROTB), together
affect oral bioavailability in the tested animal; compounds are found to
have high oral bioavailability if the NROTB and TPSA values are less
3. Conclusion
A library of 15 compounds (4a–o) was designed to be moderately
selective COX-2 inhibitors and synthesized. Their anti-inflammatory,
analgesic, ulcerogenic and in vitro COX inhibition activities were
Fig. 4. Showed the presence or absence of ulcer comparing with two reference drugs indomethacin and celecoxib.
81

A. Sakr et al.
BioorganicChemistry84(2019)76–86
Fig. 5. 2D and 3D interaction image of compound 4o in the active site of COX-2.
evaluated. Compound 4b showed the best results in most aspects be-
cause it had an oedema inhibition percentage (48%) better than that of
the reference drug diclofenac sodium (37.8%) and an analgesic effect
(3.4 writhes) better than that of both indomethacin (12.6 writhes) and
diclofenac sodium (17.4 writhes). Moreover, its UI of 2.7 was 7-fold
safer than that of indomethacin (19.3) and nearly the same as that of
celecoxib (2.4). These results were in complete accordance with the in
vitro COX-1/COX-2 enzyme assay results: COX-1 IC50 = 4.21, COX-2
IC50 = 0.05, and SI = 84.20. Moreover, a docking study revealed that
these compounds recognized the essential amino acids in the COX-2
active site, except for Arg120 and Arg513, and their inability to enter
COX-1 with zero reactions with amino acids; moreover, the physico-
chemical and pharmacodynamic studies predicted these compounds to
have promising medicinal value.
formed precipitate was filtered, washed with cold petroleum ether,
dried and then crystallized from dioxane/H2O drops to generate the
desired products 4a–o at 60–90% yield.
4.1.2. 4-Chloro-N-(1-methyl-4-oxo-2-phenyl-1,4-dihydroquinazolin-
3(2H)-yl)benzamide (4a)
White powder, 90% yield. M.p., 243–248 °C. 1H NMR (DMSO‑d6) δ
2.81 (3H, s, NCH3), 5.93 (1H, s, NCHN), 6.76 (1H, d, J = 8.28 Hz, ArH),
6.88 (1H, t, J = 7.48 Hz, ArH), 7.32–7.36 (5H, m, ArH), 7.47 (1H, t,
J = 8.48 Hz, ArH), 7.57 (2H, d, J = 8.56 Hz, ArH), 7.79–7.84 (3H, m,
ArH), 10.91 (1H, s, CONH, exch). 13C NMR (DMSO‑d6) δ 35.62 (NCH3),
80.28 (NCHN), 112.96 (ArCH), 115.40 (ArC), 118.40 (ArCH), 127.35
(ArC), 128.50 (ArCH), 128.98 (ArCH), 129.05 (ArCH), 129.52 (ArCH),
130.09 (ArCH), 131.31 (ArCH), 135.07 (ArCH), 137.41 (ArC), 137.55
(ArC), 147.56 (ArC), 161.00 (ArC), 164.98 (ArC). MS, m/z: 392 (M+),
394 (M++2). Analysis calcd. for C18H18ClN3O2: C, 67.43; H, 4.63; N,
10.72. Found: C, 67.54; H, 4.61; N, 10.75.
4. Materials and methods
4.1. Chemistry
4.1.3. 4-Chloro-N-(2-(4-chlorophenyl)-1-methyl-4-oxo-1,4-
All reagents were obtained commercially with the high percent
purity, especially for synthesis, unless otherwise mentioned. Melting
dihydroquinazolin-3(2H)-yl)benzamide (4b)
Pale yellow powder, 89% yield. M.p., 257–261 °C. 1H NMR
(DMSO‑d6) δ 2.81 (3H, s, NCH3) 5.97 (1H, s, NCHN), 6.76 (1H, d,
J = 8.28 Hz, ArH), 6.88 (1H, t, J = 7.40 Hz, ArH), 7.35 (2H, d,
J = 8.52 Hz, ArH), 7.42 (2H, d, J = 8.48 Hz, ArH), 7.47 (1H, t,
J = 8.52 Hz, ArH), 7.58 (2H, d, J = 8.56 Hz, ArH), 7.79–7.86 (3H, m,
ArH), 10.90 (1H, s, CONH, exch). 13C NMR (DMSO‑d6) δ 35.55 (NCH3),
79.53 (NCHN), 113.04 (ArCH), 115.29 (ArC), 118.54 (ArCH), 128.55
(ArC), 129.00 (ArCH), 129.09 (ArCH), 129.24 (ArCH), 130.08 (ArC),
131.24 (ArC), 134.17 (ArCH), 135.14 (ArCH), 136.61 (ArCH), 137.48
(ArC), 147.30 (ArC), 160.82 (ArC), 164.96 (ArC). MS, m/z: 426.05
(M+), 428 (M++2). Analysis calcd. for C22H17Cl2N3O2: C, 61.99; H,
4.02; N, 9.86. Found: C, 61.81; H, 4.03; N, 10.00.
points (°C) were determined using open capillary tubes on
a
Gallenkamp melting point apparatus (London, UK), and the results are
uncorrected. Elemental analyses (C, H, N) and mass spectra were per-
formed in the Microanalytical Center, Faculty of Science, Cairo
University, Giza, Egypt. NMR spectra were performed in the
Microanalytical Center, Faculty of Pharmacy, Cairo University, Egypt.
All NMR spectra were generated on a Varian Mercury-300 (300 MHz)
(Palo Alto, CA, USA) using dimethyl sulfoxide (DMSO)-d6 as the sol-
vent. Chemical shifts are reported in d ppm units with respect to TMS as
an internal standard (chemical shift in δ, ppm). Mass spectra were de-
tected using a GC/MS Shimadzu Qp-2010 plus (Shimadzu Corporation,
Tokyo, Japan). Elemental analyses were determined using the Vario EL-
III (Elementar) CHNS analyzer (Hanau, Germany). All reactions were
observed continuously by thin layer chromatography (TLC) (Rf) on si-
lica gel 60 GF245 (E-Merck, Germany) and were detected by a UV-lamp
at a wavelength (λ) of 254 nm.
4.1.4. 4-Chloro-N-(1-methyl-2-(4-nitrophenyl)-4-oxo-1,4-
dihydroquinazolin-3(2H)-yl)benzamide (4c)
White powder, 71% yield. M.p., 268–271 °C. 1H NMR (DMSO‑d6) δ
2.88 (3H, s, NCH3), 6.17 (1H, s, NCHN), 6.78 (1H, d, J = 8.32 Hz, ArH),
6.91 (1H, t, J = 7.44 Hz, ArH), 7.49 (1H, t, J = 8.40 Hz, ArH), 7.62
(4H, q, J = 8.70 Hz, ArH), 7.82–7.87 (3H, m, ArH), 8.22 (2H, d,
J = 8.72 Hz, ArH), 10.98 (1H, s, CONH, exch). 13C NMR (DMSO‑d6) δ
35.76 (NCH3), 79.22 (NCHN), 113.29 (ArCH), 115.36 (ArC), 118.87
(ArCH), 124.19 (ArCH), 128.65 (ArCH), 128.73 (ArCH), 129.11
(ArCH), 130.08 (ArC), 131.12 (ArCH), 135.25 (ArCH), 137.57 (ArC),
144.75 (ArC), 147.06 (ArC), 148.38 (ArC), 160.71 (ArC), 165.05 (ArC).
MS, m/z: 437.25 (M+), 439.30 (M++2). Analysis calcd. for
Compounds 1, 2a–c and 3a–c were synthesized according to pre-
viously reported methods [47 48 30].
4.1.1. General method for the synthesis of 1,4-dihydroquinazolin-3(2H)-yl
benzamide (4a–o)
A mixture of N-(4-(substituted)-benzoyl)-2-(methylamino) benzo-
hydrazide (3a–c, 0.003 mol) and the appropriate aldehyde (0.003 mol)
in glacial acetic acid was heated under reflux for 8 h. After cooling, the
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A. Sakr et al.
BioorganicChemistry84(2019)76–86
C22H17ClN4O4: C, 60.49; H, 3.92; N, 12.83. Found: C, 60.51; H, 3.97; N,
12.92.
4.1.9. N-(1-Methyl-2-(4-nitrophenyl)-4-oxo-1,4-dihydroquinazolin-
3(2H)-yl)-4-nitrobenzamide (4h)
Pale yellow powder, 70% yield. M.p., 267–271 °C. 1H NMR
(DMSO‑d6) δ 2.89 (3H, s, NCH3), 6.21 (1H, s, NCHN), 6.80 (1H, d,
J = 8.28 Hz, ArH), 6.92 (1H, t, J = 7.48 Hz, ArH), 7.50 (1H, t,
J = 8.52 Hz, ArH), 7.64 (2H, d, J = 8.72 Hz, ArH), 7.83 (1H, d,
J = 9.08 Hz, ArH), 8.05 (2H, d, J = 8.8 Hz, ArH), 8.23 (2H, d,
J = 8.72 Hz, ArH), 8.35 (2H, d, J = 8.84 Hz, ArH), 11.23 (1H, s, CONH,
exch). 13C NMR (DMSO‑d6) δ 35.79 (NCH3), 79.13 (NCHN), 113.36
(ArCH), 115.24 (ArC), 118.92 (ArCH), 124.20 (ArCH), 124.24 (ArCH),
128.67 (ArCH), 128.73 (ArCH), 129.69 (ArC), 135.35 (ArCH), 137.99
(ArC), 144.66 (ArCH), 147.09 (ArC), 148.42 (ArC), 150.03 (ArC),
160.65 (ArC), 164.59 (ArC). MS, m/z: 447.10 (M+). Analysis calcd. for
4.1.5. 4-Chloro-N-(2-(4-methoxyphenyl)-1-methyl-4-oxo-1,4-
dihydroquinazolin-3(2H)-yl)benzamide (4d)
Pale yellow crystals, 74% yield. M.p., 215–219 °C. 1H NMR
(DMSO‑d6) δ 2.77 (3H, s, NCH3), 3.73 (3H, s, OCH3), 5.88 (1H, s,
NCHN), 6.74 (1H, d, J = 8.28 Hz, ArH), 6.85–6.90 (3H, m, ArH), 7.25
(2H, d, J = 8.72 Hz, ArH), 7.46 (1H, t, J = 8.48 Hz, ArH), 7.57 (2H, d,
J = 8.60 Hz, ArH), 7.79–7.85 (3H, m, ArH), 10.87 (1H, s, CONH, exch).
13C NMR (DMSO‑d6) δ 35.44 (NCH3), 79.97 (NCHN), 112.88 (ArCH),
114.27 (ArCH), 115.38 (ArC), 118.27 (ArCH), 128.48 (ArCH), 128.74
(ArCH), 129.03 (ArCH), 129.65 (ArCH), 130.10 (ArC), 131.38 (ArC),
134.99 (ArCH), 137.40 (ArC), 147.60 (ArC), 160.23 (ArC), 161.04
(ArC), 164.90 (ArC). MS, m/z: 422 (M+), 424 (M++2). Analysis calcd.
for C23H20ClN3O3: C, 65.48; H, 4.78; N, 9.96. Found: C, 65.70; H, 5.05;
N, 10.06.
C
22H17N5O6: C, 59.06; H, 3.83; N, 15.65. Found: C, 59.30; H, 3.91; N,
15.62.
4.1.10. N-(2-(4-Methoxyphenyl)-1-methyl-4-oxo-1,4-dihydroquinazolin-
3(2H)-yl)-4-nitrobenzamide (4i)
Yellow crystals, 60% yield. M.p., 245–250 °C. 1H NMR (DMSO‑d6) δ
2.79 (3H, s, NCH3), 3.73 (3H, s, OCH3), 5.92 (1H, s, NCHN), 6.76 (1H,
d, J = 8.32 Hz, ArH), 6.86–6.91 (3H, m, ArH), 7.27 (2H, d, J = 8.64 Hz,
ArH), 7.47 (1H, t, J = 7.74 Hz, ArH), 7.82 (1H, d, J = 7.64 Hz, ArH),
8.03 (2H, d, J = 8.76 Hz, ArH), 8.34 (2H, d, J = 8.76 Hz, ArH), 11.12
(1H, s, CONH, exch). 13C NMR (DMSO‑d6) δ 35.46 (NCH3), 55.57
(OCH3), 79.91 (NCHN), 112.94 (ArCH), 114.31 (ArCH), 115.25 (ArC),
118.32 (ArCH), 124.12 (ArCH), 128.51 (ArCH), 128.74 (ArCH), 129.57
(ArCH), 129.69 (ArC), 135.08 (ArCH), 138.26 (ArC), 147.63 (ArC),
149.95 (ArC), 160.27 (ArC), 161.00 (ArC), 164.43 (ArC). MS, m/z: 432
(M+). Analysis calcd. for C23H20N4O5: C, 63.88; H, 4.66; N, 12.96.
Found: C, 64.06; H, 4.91; N, 13.14.
4.1.6. 4-Chloro-N-(2-(4-fluorophenyl)-1-methyl-4-oxo-1,4-
dihydroquinazolin-3(2H)-yl)benzamide (4e)
White powder, 74% yield. M.p., 219–223 °C. 1H NMR (DMSO‑d6) δ
2.80 (3H, s, NCH3), 5.97 (1H, s, NCHN), 6.76 (1H, d, J = 8.32 Hz, ArH),
6.89 (1H, t, J = 6.44 Hz, ArH), 7.19 (2H, t, J = 8.88 Hz, ArH),
7.37–7.40 (2H, m, ArH), 7.48 (1H, t, J = 8.52 Hz, ArH), 7.58 (2H, d,
J = 8.56 Hz, ArH), 7.80–7.85 (3H, m, ArH), 10.89 (1H, s, CONH, exch).
13C NMR (DMSO‑d6) δ 35.51 (NCH3), 79.55 (NCHN), 113.02 (ArCH),
115.29 (ArC), 115.92 (d, J = 21 Hz, ArCH), 118.50 (ArCH), 128.53
(ArC), 129.07 (ArC), 129.59 (d, J = 8 Hz, ArCH), 130.06 (ArCH),
131.27 (ArCH), 133.98 (d, J = 3 Hz, ArC), 135.13 (ArCH), 137.45
(ArC), 147.40 (ArC), 160.90 (ArC), 164.14 (d, J = 245 Hz, ArC), 164.96
(ArC). Analysis calcd. for C22H17ClFN3O2: C, 64.47; H, 4.18; N, 10.25.
Found: C, 64.28; H, 4.24; N, 10.42.
4.1.11. N-(2-(4-Fluorophenyl)-1-methyl-4-oxo-1,4-dihydroquinazolin-
3(2H)-yl)-4-nitrobenzamide (4j)
Yellow powder, 75% yield. M.p., 230–236 °C. 1H NMR (DMSO‑d6) δ
2.81 (3H, s, NCH3), 6.00 (1H, s, NCHN), 6.78 (1H, d, J = 8.32 Hz, ArH),
6.89 (1H, t, J = 7.44 Hz, ArH), 7.19 (2H, t, J = 8.88 Hz, ArH),
7.38–7.41 (2H, m, ArH), 7.49 (1H, t, J = 8.48 Hz, ArH), 7.82 (1H, d,
J = 8.80 Hz, ArH), 8.03 (2H, d, J = 8.80 Hz, ArH), 8.35 (2H, d,
J = 8.80 Hz, ArH), 11.15 (1H, s, CONH, exch). 13C NMR (DMSO‑d6) δ
35.54 (NCH3), 79.48 (NCHN), 113.09 (ArCH), 115.16 (ArC), 115.98 (d,
J = 22 Hz, ArCH), 118.56 (ArCH), 124.16 (ArCH), 128.56 (ArCH),
129.59 (d, J = 8 Hz, ArCH), 133.90 (d, J = 3 Hz, ArC), 135.23 (ArCH),
138.15 (ArCH), 147.42 (ArC), 149.98 (ArC), 160.85 (ArC), 164.17 (d,
J = 245 Hz, ArC), 164.49 (ArC). MS, m/z: 420 (M+), 422 (M++2).
Analysis calcd. for: C22H17FN4O4: C, 62.85; H, 4.08; N, 13.33. Found: C,
62.70; H, 4.09; N, 13.32.
4.1.7. N-(1-Methyl-4-oxo-2-phenyl-1,4-dihydroquinazolin-3(2H)-yl)-4-
nitrobenzamide (4f)
Yellow powder, 62% yield. M.p., 264–269 °C. 1H NMR (DMSO‑d6) δ
2.81 (3H, s, NCH3), 5.97 (1H, s, NCHN), 6.77 (1H, d, J = 8.32 Hz, ArH),
6.89 (1H, t, J = 7.44 Hz, ArH), 7.36 (5H, bs, ArH), 7.48 (1H, t,
J = 8.44 Hz, ArH), 7.81 (1H, d, J = 7.64 Hz, ArH), 8.03 (2H, d,
J = 8.76 Hz, ArH), 8.34 (2H, d, J = 8.76 Hz, ArH), 11.17 (1H, s, CONH,
exch). 13C NMR (DMSO‑d6) δ 35.65 (NCH3), 80.22 (NCHN), 113.03
(ArCH), 115.27 (ArC), 118.46 (ArCH), 124.13 (ArC), 127.34 (ArCH),
128.53 (ArCH), 129.03 (ArCH), 129.60 (ArCH), 129.69 (ArCH), 135.17
(ArCH), 137.47 (ArCH), 138.21 (ArC), 147.59 (ArC), 149.96 (ArC),
160.97 (ArC), 164.53 (ArC). MS, m/z: 402 (M+). Analysis calcd. for
4.1.12. 4-Methoxy-N-(1-methyl-4-oxo-2-phenyl-1,4-dihydroquinazolin-
3(2H)-yl)benzamide (4k)
C
22H18N4O4: C, 65.66; H, 4.51; N, 13.92. Found: C, 65.50; H, 4.38; N,
13.64.
White powder, 77% yield. M.p., 279–284 °C. 1H NMR (DMSO‑d6) δ
2.80 (3H, s, NCH3), 3.82 (3H, s, OCH3), 5.91 (1H, s, NCHN), 6.75 (1H,
d, J = 8.28 Hz, ArH), 6.87 (1H, t, J = 7.44 Hz, ArH), 7.01 (2H, d,
J = 8.8 Hz, ArH), 7.34 (5H, bs, ArH), 7.46 (1H, t, J = 8.48 Hz, ArH),
7.79–7.83 (3H, m, ArH), 10.66 (1H, s, CONH, exch). 13C NMR
(DMSO‑d6) δ 35.61 (NCH3), 55.88 (OCH3), 80.38 (NCHN), 112.89
(ArCH), 114.11 (ArCH), 115.57 (ArC), 118.33 (ArCH), 124.67 (ArC),
127.36 (ArCH), 128.46 (ArCH), 128.93 (ArCH), 129.45 (ArCH), 130.13
(ArCH), 134.96 (ArCH), 137.66 (ArC), 147.52 (ArC), 161.05 (ArC),
162.65 (ArC), 165.36 (ArC). MS, m/z: 387 (M+). Analysis calcd. for:
4.1.8. N-(2-(4-Chlorophenyl)-1-methyl-4-oxo-1,4-dihydroquinazolin-
3(2H)-yl)-4-nitrobenzamide (4g)
Yellow powder, 75% yield. M.p., 272–277 °C. 1H NMR (DMSO‑d6) δ
2.83 (3H, s, NCH3), 6.02 (1H, s, NCHN), 6.78 (1H, d, J = 8.32 Hz, ArH),
6.89 (1H, t, J = 7.44 Hz, ArH), 7.37 (2H, d, J = 8.44 Hz, ArH), 7.43
(2H, d, J = 8.36 Hz, ArH), 7.49 (1H, t, J = 7.48 Hz, ArH), 7.81 (1H, d,
J = 7.32 Hz, ArH), 8.05 (2H, d, J = 8.56 Hz, ArH), 8.35 (2H, d,
J = 8.56 Hz, ArH), 11.16 (1H, s, CONH, exch). 13C NMR (DMSO‑d6) δ
35.58 (NCH3), 79.46 (NCHN), 113.10 (ArCH), 115.16 (ArC), 118.60
(ArCH), 124.17 (ArCH), 128.57 (ArCH), 129.05 (ArCH), 129.23
(ArCH), 129.69 (ArC), 134.23 (ArCH), 135.24 (ArCH), 136.53 (ArC),
138.12 (ArC), 147.32 (ArC), 150.00 (ArC), 160.77 (ArC), 164.50 (ArC).
MS, m/z: 437.05 (M+), 439.05 (M++2). Analysis calcd. for
C
23H21N3O3: C, 71.30; H, 5.46; N, 10.85. Found: C, 71.00; H, 5.35; N,
10.53.
4.1.13. N-(2-(4-Chlorophenyl)-1-methyl-4-oxo-1,4-dihydroquinazolin-
3(2H)-yl)-4-methoxybenzamide (4l)
C
22H17ClN4O4: C, 60.49; H, 3.92; N, 12.83. Found: C, 60.21; H, 4.13; N,
White powder, 85% yield. M.p., 255–259 °C. 1H NMR (DMSO‑d6) δ
2.81 (3H, s, NCH3), 3.82 (3H, s, OCH3), 5.95 (1H, s, NCHN), 6.75 (1H,
12.92.
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d, J = 8.28 Hz, ArH), 6.88 (1H, t, J = 7.40 Hz, ArH), 7.02 (2H, d,
J = 8.88 Hz, ArH), 7.34 (2H, d, J = 8.52 Hz, ArH), 7.42 (2H, d,
J = 8.52 Hz, ArH), 7.47 (1H, t, J = 8.16 Hz, ArH), 7.79–7.84 (3H, m,
ArH), 10.66 (1H, s, CONH, exch). 13C NMR (DMSO‑d6) δ 35.53 (NCH3),
55.89 (OCH3), 79.62 (NCHN), 112.98 (ArCH), 114.15 (ArCH), 115.47
(ArC), 118.49 (ArCH), 124.60 (ArCH), 128.51 (ArCH), 128.95 (ArCH),
129.26 (ArCH), 130.12 (ArC), 134.08 (ArCH), 135.03 (ArC), 136.71
(ArC), 147.28 (ArC), 160.88 (ArC), 162.70 (ArC), 165.33 (ArC). MS, m/
z: 422 (M+). Analysis calcd. for: C23H20ClN3O3: C, 65.48; H, 4.78; N,
9.96. Found: C, 65.21; H, 4.85; N, 9.95.
was repeated three times (n = 3), and the data are presented as the
average of three values
standard error of the mean (SEM). The half-
maximal inhibitory concentration (IC50) was measured, and the SI va-
lues were calculated as IC50 (COX-1)/IC50 (COX-2).
4.2.2. In vivo analgesic activity: acetic acid-induced writhing test
The analgesic activity was measured using an acetic acid-induced
abdominal writhing test in mice as described by Nakamura et al. [49].
The test was performed in groups of five mice each. The control group
(1) was given vehicle, which consisted of 1% Tween 80 (10 ml/kg,
p.o.). The newly synthesized drugs were administered p.o. at 100 mg/
kg 1 h before the injection of 0.7% acetic acid (1 ml/100 mg, i.p.). Then,
the number of writhes over 30 min was counted. Diclofenac and in-
domethacin were used as reference drugs (20 mg/kg).
4.1.14. 4-Methoxy-N-(1-methyl-2-(4-nitrophenyl)-4-oxo-1,4-
dihydroquinazolin-3(2H)-yl)benzamide (4m)
Pale yellow powder, 71% yield. M.p., 193–197 °C. 1H NMR
(DMSO‑d6) δ 2.87 (3H, s, NCH3), 3.82 (3H, s, OCH3), 6.14 (1H, s,
NCHN), 6.77 (1H, d, J = 8.3 Hz, ArH), 6.91 (1H, t, J = 7.32 Hz, ArH),
7.02 (2H, d, J = 8.92 Hz, ArH), 7.48 (1H, t, J = 7.72 Hz, ArH), 7.62
(2H, d, J = 8.76 Hz, ArH), 7.81–7.84 (3H, m, ArH), 8.22 (2H, d,
J = 8.76 Hz, ArH), 10.72 (1H, s, CONH, exch). 13C NMR (DMSO‑d6) δ
35.72 (NCH3), 55.89 (OCH3), 79.32 (NCHN), 113.24 (ArCH), 114.18
(ArCH), 115.52 (ArC), 118.81 (ArCH), 124.15 (ArCH), 124.47 (ArCH),
128.61 (ArCH), 128.75 (ArCH), 130.13 (ArC), 135.15 (ArC), 144.87
(ArCH), 147.04 (ArC), 148.35 (ArC), 160.77 (ArC), 162.76 (ArC),
165.42 (ArC). MS, m/z: 432 (M+). Analysis calcd. for: C23H20N4O5: C,
63.88; H, 4.66; N, 12.96. Found: C, 64.00; H, 4.66; N, 12.98.
4.2.3. In vivo anti-inflammatory activity: carrageenan-induced rat paw
oedema test
The anti-inflammatory activity of the test compounds was in-
vestigated using carrageenan-induced rat paw oedema as described by
Sobeh et al. [35]. Albino male rats weighing 200–250 g were housed at
23–25 °C (room temperature) with good ventilation, an appropriate
dark/light cycle and ad libitum access to food/water. The rats were
divided into 18 groups (five rats each). Group 1 was the control group
and was given vehicle (1% Tween 80, 10 ml/kg). The remaining groups
each received one of the synthesized drugs (100 mg/kg) or one of the
two reference drugs, diclofenac sodium (20 mg/kg) and celecoxib
(50 mg/kg). All drugs were administered orally one hour prior to the
injection of carrageenan solution (1% in 0.9% NaCl, 0.1 ml) (Sigma-
Aldrich, USA) into the sub-planter tissue of the right hind paw. The
increase in thickness (mm) was determined using callipers before and
after the carrageenan injection at 0, 1, 2, 3, 4, 5 and 24 h. The % in-
hibition of paw oedema thickness was calculated as (control − drug/
control) × 100. The cumulative anti-inflammatory effect during the
entire observation period was estimated by calculating the area under
the curve (AUC). Both the analgesic and anti-inflammatory screening
were carried out in accordance with the guidelines of the Faculty of
Pharmacy, Zagazig University, Egypt, and the whole study was ap-
proved by the local authorities, the Ethical Committee for Animal
Handling at Zagazig University (ECAHZU), Faculty of Pharmacy, Za-
gazig University, Egypt, with a registration number (P15-12-2017).
4.1.15. 4-Methoxy-N-(2-(4-methoxyphenyl)-1-methyl-4-oxo-1,4-
dihydroquinazolin-3(2H)-yl)benzamide (4n)
White powder, 65% yield. M.p., 265–269 °C. 1H NMR (DMSO‑d6) δ
2.77 (3H, s, NCH3), 3.73 (3H, s, OCH3), 3.82 (3H, s, OCH3), 5.85 (1H, s,
NCHN), 6.73 (1H, d, J = 8.32 Hz, ArH), 6.85–6.89 (3H, m, ArH), 7.01
(2H, d, J = 8.84 Hz, ArH), 7.24 (2H, d, J = 8.68 Hz, ArH), 7.46 (1H, t,
J = 8.44 Hz, ArH), 7.78–7.83 (3H, m, ArH), 10.62 (1H, s, CONH, exch).
13C NMR (DMSO‑d6) δ 35.44 (NCH3), 55.57 (OCH3), 55.89 (OCH3),
80.04 (NCHN), 112.83 (ArCH), 114.11 (ArCH), 114.22 (ArCH), 115.55
(ArC), 118.21 (ArCH), 124.74 (ArCH), 128.43 (ArCH), 128.73 (ArCH),
129.77 (ArCH), 130.13 (ArC), 134.88 (ArC), 147.56 (ArC), 160.17
(ArC), 161.06 (ArC), 162.64 (ArC), 165.27 (ArC). MS, m/z: 418 (M+).
Analysis calcd. for: C24H23N3O4: C, 69.05; H, 5.55; N, 10.07. Found: C,
69.33; H, 5.73; N, 10.10.
4.1.16. N-(2-(4-Fluorophenyl)-1-methyl-4-oxo-1,4-dihydroquinazolin-
3(2H)-yl)-4-methoxybenzamide (4o)
White powder, 66% yield. M.p., 255–260 °C. 1H NMR (DMSO‑d6) δ
2.79 (3H, s, NCH3), 3.82 (3H, s, OCH3), 5.94 (1H, s, NCHN), 6.75 (1H,
d, J = 8.28 Hz, ArH), 6.88 (1H, t, J = 7.4 Hz, ArH), 7.02 (2H, d,
J = 8.8 Hz, ArH), 7.18 (2H, t, J = 8.88 Hz, ArH), 7.35–7.39 (2H, m,
ArH), 7.47 (1H, t, J = 6.96 Hz, ArH), 7.79–7.82 (3H, m, ArH), 10.64
(1H, s, CONH, exch). 13C NMR (DMSO‑d6) δ 35.50 (NCH3), 55.88
(OCH3), 79.64 (NCHN), 112.97 (ArCH), 114.14 (ArCH), 115.46 (ArC),
115.86 (d, J = 21 Hz, ArCH), 118.45 (ArCH), 124.62 (ArC), 128.49
(ArCH), 129.59 (d, J = 8 Hz, ArCH), 130.10 (ArCH), 134.07 (d,
J = 3 Hz, ArC), 135.02 (ArCH), 147.37 (ArC), 160.96 (ArC), 162.68
(ArC),164.10 (d, J = 245 Hz, ArC), 165.33 (ArC). MS, m/z: 405 (M+),
407 (M++2). Analysis calcd. for: C23H20FN3O3: C, 68.14; H, 4.97; N,
10.36. Found: C, 67.94; H, 5.00; N, 10.23.
4.2.4. Acute ulcerogenic activity
All new test compounds (4a–o) were subjected to an investigation
of their ulcerogenic activity using indomethacin and celecoxib as re-
ference drugs [38]. The rats from the previous experiment were fasted
for 12 h, followed by the administration of additional doses of the test
compounds (4a–o) for two additional days. Six hours after the last dose,
the animals were sacrificed, and the stomachs were removed and ex-
amined for ulceration after being washed with saline solution (0.9%).
The ulcer scores were calculated according to the method prescribed by
Kulkarni and in a previous study [38] as follows:
– Zero for normal coloured stomach
– (0.5) for red colouration
– for a spot ulcer
4.2. Biological screening
– (1.5) for haemorrhagic streaks
– for an ulcer > 3 but < 5 mm
– for ulcers > 5 mm.
4.2.1. In vitro COX-1/COX-2 inhibition assay
The inhibition of both cyclooxygenase enzymes (COX-1/COX-2) was
evaluated using the colorimetric ovine COX-1/human recombinant
COX-2 assay (catalogue No. 560131; Cayman Chemicals Inc., Ann
Arbor, MI, USA) according to the manufacturer’s instructions and pre-
viously reported studies [31–33]. All the compounds were tested in
comparison with the reference drugs at the same time. The experiment
The UI was calculated according to the following equation:
[UI = UN + US + UP × 10−1], where UN is the average number of
ulcers, US is the average severity score, and UP is the percentage of
animals with an ulcer.
84

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BioorganicChemistry84(2019)76–86
4.3. Molecular modelling and in silico study
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