Novel click modifiable thioquinazolinones as anti-inflammatory agents: Design, synthesis, biological evaluation and docking study

Article abstract of DOI:10.1016/j.ejmech.2017.12.065

Click chemistry was used to synthesize a new series of thioquinazolinone molecules equipped with propargyl moiety,1,2,3-triazolyl and isoxazolyl rings. Our design was based on merging pharmacophores previously reported to exhibit COX-2 inhibitory activities to a thioquinazolinone-privileged scaffold. The synthesized compounds were subjected to in vitro cyclooxygenase COX-1/COX-2 and 15-LOX inhibition assays. Compounds 2c, 3b, 3h, 3j, and 3k showed COX-2 inhibition with IC₅₀ (μM) 0.18, 0.19, 0.11, 0.16 and 0.17 respectively. These values were compared to celecoxib (IC₅₀ 0.05 μM), diclofenac (IC₅₀ 0.8 μM) and indomethacin (IC₅₀ 0.49 μM) reference drugs. They also showed 15-LOX inhibition with IC₅₀ (μM) 6.21, 4.33, 7.62, 5.21 and 3.98 respectively. These values were compared with Zileuton (IC₅₀ 2.41 μM) and Meclofenamate sodium (IC₅₀ 5.64 μM) as positive controls. These compounds were further challenged by PMA-induced THP-1 differentiation assay where compounds 2c and 3j inhibited monocyte to macrophage differentiation efficiently with IC₅₀ values of 4.78 μM and 5.63 μM, respectively, compared to that of diclofenac sodium (4.86 μM). On the other hand, 3h demonstrated a significantly increased potency compared to diclofenac in this assay (IC₅₀ = 0.13 μM). The same compounds exhibited significant in vivo anti-inflammatory effect as indicated by the formalin-induced rat-paw edema test. Docking experiments of compounds 2c, 3b, 3h, 3j, and 3k into COX-2 binding pocket have been conducted, where strong binding interactions have been identified and effective overall docking scores have been recorded. Their drug-likeness has been assessed using Molinspiration, Molsoft and Pre-ADMET software products.

Full text of DOI:10.1016/j.ejmech.2017.12.065

Accepted Manuscript
Novel click modifiable thioquinazolinones as anti-inflammatory agents: Design,
synthesis, biological evaluation and docking study
Ghandoura Moussa, Rana Alaaeddine, Lynn M. Alaeddine, Rasha Nassra, Ahmed
S.F. Belal, Azza Ismail, Ahmed F. El-Yazbi, Yasser S. Abdel-Ghany, Aly Hazzaa
PII:
S0223-5234(17)31096-6
10.1016/j.ejmech.2017.12.065
EJMECH 10043
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 4 October 2017
Revised Date: 2 December 2017
Accepted Date: 17 December 2017
Please cite this article as: G. Moussa, R. Alaaeddine, L.M. Alaeddine, R. Nassra, A.S.F. Belal, A.
Ismail, A.F. El-Yazbi, Y.S. Abdel-Ghany, A. Hazzaa, Novel click modifiable thioquinazolinones as anti-
inflammatory agents: Design, synthesis, biological evaluation and docking study, European Journal of
Medicinal Chemistry (2018), doi: 10.1016/j.ejmech.2017.12.065.
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ACCEPTED MANUSCRIPT
Graphical abstract:
A new series of propargyl and 1,2,3-Triazolyl thioquinazolinone showed dual
COX-2 and LOX inhibitory activity.

ACCEPTED MANUSCRIPT
Novel Click Modifiable Thioquinazolinones as Anti-Inflammatory
agents: Design, Synthesis, Biological Evaluation and Docking
Study.
1
3
3
Ghandoura Moussa , Rana Alaaeddine , Lynn M. Alaeddine , Rasha
2
1
1
3,4
Nassra , Ahmed S. F. Belal *, Azza Ismail , Ahmed F. El-Yazbi *, Yasser S.
Abdel-Ghany , Aly Hazzaa
Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Alexandria
University, Alexandria 21521, Egypt.
1
1
1
2
Department of Medical Biochemistry, Faculty of Medicine, Alexandria University,
Alexandria, Egypt.
3
Department of Pharmacology and Toxicology, Faculty of Medicine and Medical
Centre, American University of Beirut, Beirut, Lebanon.
4
Department of Pharmacology and Toxicology, Faculty of Pharmacy, Alexandria
University, Alexandria 21521, Egypt.
ABSTRACT
Click chemistry was used to synthesize a new series of thioquinazolinone
molecules equipped with propargyl moiety,1,2,3-triazolyl and isoxazolyl rings.
Our design was based on merging pharmacophores previously reported to
exhibit COX-2 inhibitory activities to a thioquinazolinone-privileged scaffold. The
synthesized compounds were subjected to in vitro cyclooxygenase COX-1/COX-
2
and 15-LOX inhibition assays. Compounds 2c, 3b, 3h, 3j, and 3k showed
COX-2 inhibition with IC50 (µM) 0.18, 0.19, 0.11, 0.16 and 0.17 respectively.
These values were compared to celecoxib (IC50 0.05 µM), diclofenac (IC50 0.8
µM) and indomethacin (IC50 0.49 µM) reference drugs. They also showed 15-
LOX inhibition with IC (µM) 6.21, 4.33, 7.62, 5.21 and 3.98 respectively. These
50
values were compared with Zileuton (IC 2.41 µM) and Meclofenamate sodium
50
(IC50 5.64 µM) as positive controls. These compounds were further challenged by
PMA-induced THP-1 differentiation assay where compounds 2c and 3j inhibited
monocyte to macrophage differentiation efficiently with IC50 values of 4.78 µM
and 5.63 µM, respectively, compared to that of diclofenac sodium (4.86 µM). On
the other hand, 3h demonstrated a significantly increased potency compared to
diclofenac in this assay (IC50 = 0.13 µM) .The same compounds exhibited

ACCEPTED MANUSCRIPT
significant in vivo anti-inflammatory effect as indicated by the formalin-induced
rat-paw edema test. Docking experiments of compounds 2c, 3b, 3h, 3j, and 3k
into COX-2 binding pocket have been conducted, where strong binding
interactions have been identified and effective overall docking scores have been
recorded. Their drug-likeness has been assessed using Molinspiration, Molsoft
and Pre-ADMET software products.
Keywords: Click reaction, Thioquinazolinone, Cyclooxygenase 1/Cyclooxygenase
2, Lipooxygenase, Monocyte / Macrophage, Docking, Drug-likeness.
*
Corresponding authors:
Ahmed S. F. Belal, Department of Pharmaceutical Chemistry, Faculty of
Pharmacy, Alexandria University, Alexandria, Egypt.
Email: ahmed.belal@alexu.edu.eg
Ahmed F. El-Yazbi, Department of Pharmacology and Toxicology, Faculty of
Medicine and Medical Centre, American University of Beirut, Beirut, Lebanon.
Email: ae88@aub.edu.lb
1. Introduction:
Arachidonic acid (AA) is the biological precursor for a plethora of inflammatory
mediators that are produced by two metabolic pathway key enzymes, namely:
cyclooxygenase (COX) and lipooxygenase (LOX) [1–3]. COX-1 and COX-2 are
responsible for converting AA to the hydroxyendoperoxide PGH2, which is further
metabolized to prostaglandins (PGs), prostacyclin (PGI2) and thromboxane A2
(TXA2) [1–3]. COX-1 is constitutively active and synthesizes prostaglandins in
the context of gastrointestinal tract cytoprotection, regulation of platelet
aggregation and renal function maintenance [1–5]. COX-2 expression is induced
by pro-inflammatory stimuli and it generates inflammatory signaling PGs [1–
3,5,6]. On the other hand, LOX converts AA to hydroperoxyeicosatetraenoic
acids (HPETE), subsequently to hydroxyeicosatetraenoic acids (HETE) and then
to the leukotrienes (LTs) [1,7–9]. Pathogenesis of several inflammatory
conditions, like osteoarthritis, rheumatoid arthritis, psoriasis, COPD and multiple
sclerosis; is associated with induction of PGs and LTs [1,7–9]. Of note, selective
COX-2 inhibitors gained much popularity in the late 1990s and early 2000s as

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anti-inflammatories that exhibited minimal GIT side effects [10]. However clinical
practice proved them to be associated with myocardial infraction and
cardiovascular thrombotic events [11–13]. Moreover, blocking only COX-2 would
shunt the inflammatory pathway towards more production of LTs and hence,
leads to more untoward side effects such as asthma [7–9]. As such, having a
dual COX-2/LOX inhibitor would be a more judicious alternative to produce an
efficacious anti-inflammatory with a better safety profile.
Quinazolines are privileged scaffolds [14–16] that display a wide range of
biological activities such as analgesic [17–19], anti-inflammatory [17–19],
antimicrobial [20–22], anticonvulsant [23,24] and anticancer [25–27] activities.
Proquazone [28] (Structure I, Figure 1a) and Fluproquazone [29] (structure II,
Figure 1a) are anti-prostaglandin quinazolines that are used to alleviate
inflammatory symptoms in rheumatoid arthritis. Moreover, recently reported
quinazolinones and thioquinazolinones either modified with small heterocyclic
motif at C-2 [30] (structure III, Figure 1a) or substituted on Sulfur atom with
acetic acid hydrazide [31](structure IV, Figure 1a), showed strong anti-
inflammatory properties. Furthermore, Converso et al reported the synthesis of
thioquinazolinone-S-acetamide derivative (Structure V, Figure 1a) that exhibited
a strong inhibition of checkpoint kinase 1 (Chk1) (IC50 = 1.3 µM) which should
sensitize cancerous cells to DNA damaging agents without negatively affecting
non-tumor cells [32]. Based on the structural information of the latter 5 structures,
we reasoned that thioquinazolinone scaffold would be an appropriate skeleton to
build on to reach potential active anti-inflammatory agents.
The discovery of the ability of Cu(I) to catalyze azide alkyne Huisgen
cycloaddition (CuAAC) reaction, reported independently by the Sharpless-Fokin
[
33] and the Meldal groups [34] in 2002, represented a major breakthrough that
revolutionized the applications of 1,2,3-triazole and illustrated a distinct example
of click reactions [35]. Click reactions are broad in scope, high in yield,
stereospecific (giving only 1,4-disubstituted derivative with Cu(I) catalysis), and
carried out in aqueous or green solvents with minimal and/or inoffensive

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byproducts [35]. 1,4-disubstituted 1,2,3-triazoles are stable to extreme redox and
pH conditions, which highlights their aromatic stabilization [36–38]. Their dipole
moment is 5.06 D, which enables them to be involved in efficient hydrogen
bonding and/or π-π stacking with biological targets [36–38]. Over and above,
they are non-trivial bioisosteres of the amide group [37]. All of these features
prompted Nobel Laureate K. Barry Sharpless to describe 1,2,3-triazoles as
aggressive pharmacophores [37]. Several anti-inflammatory disubstituted 1,2,3-
triazoles have been reported such as compound IV in Figure 1a, which exhibited
better in vivo anti-inflammatory activity than indomethacin in rat carrageenan-
induced foot, paw edema model and displayed selective COX-2 inhibitory activity
[
39].
The toolbox for click reactions has been recently extended to include the
cycloaddition reaction of nitrile oxides with alkynes to give Isoxazoles [40]. Such
reaction shares many advantages of the CuAAC such as rapid kinetics,
regioselectivity (giving only 3,5-disubstituted products) and synthetic ease [40].
Besides, isoxazoles, similar to 1,2,3-triazoles, represent a non-classical
bioisostere of amide bond and have a capability of engaging in hydrogen bonding
and/or π-π stacking with biological targets [40,41]. Of particular interest is the
presence of isoxazole pharmacophore in the selective COX-2 inhibitor
Valdecoxib (Figure 1a).
Given the need for a dual selective COX-2/LOX inhibitor that is capable of
maintaining the anti-inflammatory benefit without any untoward GI and asthma
side effects, and in light of the above-mentioned facts; we decided to synthesize
new thioquinazolinone molecules equipped with 1,2,3-triazolyl and isoxazolyl
rings (Figure 1b), via click chemistry, for evaluation as anti-inflammatory agents.
2. Results and discussion
2.1. Chemistry:
The synthetic strategy to prepare the target compounds is illustrated in
scheme1. 2-Mercapto-3-substituted phenyl-quinazolin-4(3H)-ones 1 (a-d) were
prepared by refluxing different isothiocyanates with anthranilic acid in ethanol

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giving yields from 35-85 % (Scheme 1). Non-commercially available
isothiocyanates were prepared according to the method reported by Muccioli et
al that involved the reaction of substituted aniline with carbon disulfide in
presence of NaOH to give dithiocarbamate, which in turn gives the corresponding
isothiocyanate upon reacting with ethyl chloroformate [42]. We also tried the
method reported by Abdel-Megeed et al to prepare intermediates 1 (a-d) which
involved one pot reaction of anthranilic acid, substituted aniline and carbon
disulfide in presence of methanolic KOH [43], which was found to give
comparable yields in some derivatives to the former method. Reaction of 1 (a-d)
with propargyl bromide in the presence of anhydrous potassium carbonate in
1
DMF gave the S-propargyl derivatives 2 (a-d) in yields 70-83 %. The H NMR of
2
(a-d) showed a propargylic characteristic triplet in the range of δ 3.1-3.17 ppm
and doublet in the range of δ 3.9-4.01 ppm, corresponding to terminal CH and
CH respectively. Both protons underwent a long range coupling of 2.7 Hz. The
C NMR of 2 (a-d) showed propargylic characteristic peaks at δ 20.3-20.9,
4.02-74.4 and 79.06-79.7 ppm corresponding to CH , CH and quaternary
2
13
7
2
carbon respectively. Other characteristic peaks appeared at their expected
chemical shifts such as C=O that appeared at the range of δ 160.3-160.5 ppm.
The IR spectra of 2 (a-d) displayed characteristic sharp acetylenic-CH stretching
-
1
in the range of 3226.74-3254.42 cm and C≡C stretching in the range of 2288-
-
1
2
365 cm .
1,3-dipolar cycloaddition reactions between S-propargyl derivatives 2 (a-d) and
aromatic azides were carried out by refluxing in DMF in the presence of
CuSO /ascorbic acid as a catalyst; producing 16 novel 1,4-disubstituted 1,2,3-
4
1
triazoles 3 (a-p) in good to excellent yield. The H NMR of 3 (a-p) showed
triazole C -H aromatic singlet around 8.7-9 ppm, along with the disappearance of
5
13
propargylic terminal CH. Furthermore, C NMR of 3 (a-p) displayed the triazole
and C peaks at 136.19-144 and 144.35-147.7 ppm, respectively, along with
C
4
5
the disappearance of previously mentioned propargylic CH and quaternary
carbon peaks. Moreover the IR spectra of 3 (a-p) were associated with the

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disappearance of ethynyl CH and C≡C stretching bands, clearly confirming the
formation of the triazole products. We also tried the one pot multicomponent
reaction, via refluxing thioquinazolinones, propargyl bromide and aromatic azides
in aqueous ethanol as reported [44], which turned out to be unproductive.
Consequently we adopted the stepwise approach that also enabled us to screen
for the biological activities of the S-propargyl derivatives 2 (a-d).
On the other hand, subjecting 2 (a-d) to 3+2 cycloaddition reaction with various
aldoximes in the presence of chloramine-T, CuSO and Cu powder furnished 3,5-
4
1
disubstituted isoxazoles 4 (a-l) in good overall yields. Again, H NMR of 4 (a-l)
contain isoxazole C -H singlet in the range of 5.54-7.01 along with the
4
13
disappearance of propargylic terminal CH. Furthermore, C NMR of 4 (a-l)
displayed the isoxazole C , C and C peaks at 170.32-196.27, 101.87-102.15
3
4
5
and 161.16-161.37 ppm, respectively, along with the disappearance of previously
mentioned propargylic CH and quaternary carbon peaks. EI-MS of some
representative examples of the synthesized derivatives showed both molecular
ion and base peaks at their expected values.
2.2. Biological activity:
2.2.1. In vitro COX inhibition assay:
All synthesized compounds were subjected to in vitro COX-1/ COX-2 inhibition
assay using an ovine COX-1/ human recombinant COX-2 assay kit (catalog no.
560131; Cayman Chemicals Inc., Ann Arbor, MI, USA). The half-maximal
inhibitor concentrations (IC50 µM) were determined and the selectivity index (SI)
values were calculated as IC (COX-1)/IC (COX-2) (Table 1).
50
50
Table 1. In vitro COX-1/COX-2 inhibition results.
a
a
b
COX-1
COX-2
Selectivity
Index
Code
µM IC50
µM IC50
Celecoxib
14.8
0.05
296
Diclofenac
sodium
3
.9
0.8
4.87

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Indomethacin
0.039
3.78
4.26
3.87
6.54
7.33
4.36
5.89
5.89
8.24
6.32
3.67
2.97
4.78
5.27
3.89
8.67
10.52
4.22
0.49
0.27
0.39
0.18
0.44
0.89
0.19
0.54
0.67
0.96
0.53
0.24
0.11
0.25
0.16
0.17
0.89
1.04
0.52
0.07
14
2a
2b
2c
2d
3a
3b
3c
3d
3e
10.92
21.5
14.86
8.24
22.95
10.91
8.79
8.58
11.92
15.29
27.00
19.12
32.94
22.88
9.74
3f
3
3
g
h
3i
3j
3k
3l
3
m
10.12
8.12
3
n

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3
3
4
4
4
4
4
o
p
a
b
c
d
e
7.63
11.86
4.67
9.23
8.79
11.23
10.22
5.61
5.34
4.57
9.14
6.23
6.53
7.68
0.67
1.34
0.37
1.02
0.56
1.34
1.33
0.84
0.56
0.34
0.56
0.65
0.77
0.63
11.39
8.85
12.62
9.04
15.69
8.38
7.68
4
f
6.67
4
4
g
h
9.53
13.44
16.32
9.58
4
i
4
j
4k
8.48
4
l
12.19
a
IC is the concentration (µM) needed to cause 50 % inhibition of COX-1 and
50
COX-2 enzymatic activity. All values are expressed as a mean of three replicates
b
with standard deviation less than 10 % of the mean. Selectivity index = (COX-1
IC / COX-2 IC ).
50
50
The in vitro COX inhibitory testing for the newly synthesized compounds
revealed that the 2-S-propagyl-3-aryl-4-thioquinazolinones 2a-d have potent
inhibitory activities against COX-2 in comparison to the two reference drugs
indomethacin and diclofenac sodium. They showed about 2-4 folds the
inhibitory activity of diclofenac sodium with 3 to 4 times its selectivity index

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towards COX-2. They reached about twice the activity of indomethacin with
about 150 to 300 times its selectivity towards COX-2. On the other hand, the
activity and selectivity of the new compounds 2a-d were significantly lower than
that of celecoxib, which could be envisioned as an advantage potentially allowing
the avoidance of cardiovascular side effects of highly selective COX-2 inhibitors
[
11,45]. Within the series, it was noticed that substitution with lipophilic π-
deficient substituents as was seen in the 3-p-chlorophenyl and p-bromophenyl
derivatives (2b & 2c with IC values of 0.39 and 0.018 µM respectively against
50
COX-2) was favored over pi-rich substitution as was seen in the 3-p-
methoxyphenyl derivative (2d with IC value of 0.44 µM).
50
Generally, cycloaddition of the propargyl thioquinazolinones 2a-d with p-
substituted phenyl azides to give the triazolo counterparts enhanced or at least
retained the COX-2 inhibitory activities of the compounds. A general pattern was
noticed that 1-(4-chloro or 4-bromophenyl) triazolo derivatives showed the
highest activities within the series as was shown in the triazolo derivatives 3j, 3k,
3b, 3c, 3n and 3o with IC50 values of (0.16, 0.17, 0.19, 0.54, 0.52 and 0.67 µM
respectively). The only exception was the triazolo compound 3h, which was
obtained from the 4-carboxyphenylazide that showed the best COX 2 inhibitory
activity of the entire study (IC50 = 0.11 µM with SI of 27). The previously noticed
pattern of decreasing the activity as we move from the π-deficient to the π-rich
aryl substitution on position 3 of the quinazolinone ring system is still noticed.
In addition, all of the isoxazolyl derivatives 4a-l were of generally lower COX-2
inhibitory activities and possessed lower selectivity compared to the triazolo
counterparts and even their parent S-propagyl derivatives. The most active
isoxazole derivative was 4h (IC = 0.34 µM), which originated from cycloaddition
50
with nitrile oxide from veratraldehyde oxime. Yet, it was of lower activity
compared to its parent S-propagyl derivative 2c. Furthermore, cycloaddtion
reaction of 2b with the nitrile oxides coming from anisaldehyde or veratraldehyde
to give the isoxazolyl derivatives 4d and 4e respectively, led to the least active
derivatives in the entire study (IC = 1.34, and 1.33 µM respectively).
50

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2.2.2. In vitro 15-LOX inhibition assay:
Compounds (2c, 3b, 3h, 3j, and 3k), that showed the highest in vitro COX-2
inhibitory activity were also subjected to in vitro lipoxygenase inhibition assay
using LOX assay kit (catalog no. 760700; Cayman Chemicals Inc., Ann Arbor,
MI, USA). In vitro 15-LOX enzyme inhibitory activities and IC50 of the tested
compounds are shown in table 2.
Table 2. In vitro 15-LOX enzyme inhibitory activities of compounds 2c, 3b, 3h, 3j,
and 3k.
LOX
µM IC50
Code
a
Zileuton
2.41
Meclofenamate
Na
5
.64
2
c
6.21
4.33
7.62
5.21
3.98
3
b
h
3
3
j
3k

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a
IC50 is the concentration (µM) needed to cause 50 % inhibition of 15-LOX
enzymatic activity. All values are expressed as a mean of three replicates with
standard deviation less than 10 % of the mean.
All five compounds showed substantial LOX inhibitory activity with IC50 in the
range of 3.98-7.62 µM. All the five compounds operate in the same order of
magnitude as Zileuton despite being of lower activity. Compounds 3b, 3j and 3k
showed superior LOX inhibitory activity to that of meclofenamate sodium.
Consequently, the above results showed that all five compounds have a
promising LOX inhibitory activity.
2.2.3. Inhibition of monocyte to macrophage differentiation:
Monocyte to macrophage differentiation is widely recognized as an early step of
the inflammatory response [46]. Particular interest was given to the involvement
of this process in the development of atherosclerosis [47]. Several reports
indicated that COX-2 expression is induced during monocyte to macrophage
activation, which in turn contributes both to the inflammatory response and
oxidant/anti-oxidant cellular imbalance [48–51]. Of particular interest
prostaglandin E2, via its action on EP2 and EP4 receptors, was shown to
stimulate the production and release of Interleukin-10 (IL-10), which is an
inflammatory cytokine that causes functional reprogramming of monocytes and
macrophages [52]. Along the same track, the role of cysteinyl leukotrienes
(cysLTs) in monocyte chemotaxis and differentiation plus its direct link with IL-10
regulatory mechanisms in inflammation are well established [53]. As such, we
anticipated that the five compounds (2c, 3b, 3h, 3k, and 3j) that showed the
highest dual COX-2 and LOX inhibitory activities might inhibit THP-1 monocyte
differentiation into macrophages. Towards this end, we used the PMA-induced
THP-1 differentiation assay as an accepted in vitro model of this process [54].
The effects of these compounds in the assay were compared to diclofenac as a
reference COX1/COX2 inhibitor (Figure 2).
The five compounds demonstrated varying potencies in inhibiting this process
compared to diclofenac. Figure 2B depicts representative micrographs of the

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density of differentiated macrophages after incubation with 100 µM of each of the
five compounds. As shown in Figure 2C & 2D, compounds 2c and 3j showed an
inhibitory pattern that is almost identical to diclofenac (IC50 values of 4.78 µM,
5.63 µM, and 4.86 µM, respectively). On the other hand, 3h demonstrated a
significantly increased potency compared to diclofenac in this assay (IC = 0.13
50
µM), while 3k yielded less inhibition of monocyte to macrophage differentiation
(IC = 99.39 µM). A trend towards an increased inhibition was observed for 3b
50
(
IC = 1.24 µM) compared to diclofenac; however, this result was not statistically
50
significant.
2.2.4. In vivo anti-inflammatory activity: Formalin-induced rat
paw edema test:
Based on the previous in vitro test results; eight compounds were selected to test
for their in vivo anti-inflammatory activity using formalin-induced rat paw edema
protocol as an acute inflammation model. Compounds (2c, 3b, 3h, 3j, 3k, 4a, 4g
and 4h) were administered orally in a dose of 5 mg/kg body weight, and then
inflammation was induced by injecting formalin subcutaneously. Percentage
inhibition of edema was determined after 4 h and potencies of synthesized
compounds relative to the negative control were determined, along with standard
celecoxib and diclofenac sodium as positive control. Interestingly, all the tested
compounds showed higher % inhibition than celecoxib (Supplementary material
Table SM1 and Figure 3). Four compounds; namely 2c, 3b, 3h and 3j; exhibited
more than double the % inhibition exhibited by celecoxib. Compounds 2c, 3b, 3h
and 3j exhibited slightly higher % inhibition than that of Diclofenac sodium.
2.2.5. Gastic ulcerogenic activity:
Histopathological observation of rat intestinal mucosa for the presence of lesions
following oral administration of 50 mmol/Kg of Compounds (2c, 3b, 3h, 3j and
3k) as well as reference compound celecoxib was used to gauge the ulcerogenic
potential of the tested compounds. Gross observation of the isolated rat
stomachs showed a normal stomach texture for the tested compounds 2c and 3b

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(Figure 4). While for compounds 3h and 3j variable degrees of hyperemia
without gross ulceration were observed (Figure 4) and the same pattern was
observed with compound 3k (not shown).
Visualization of the degree of inflammatory reaction in the gastric layers of the
treated rats stomachs revealed that compounds 2c and 3b showed no ulceration
with normal gastro-esophageal junction (Figure 5, panels E and F); a behavior
that is similar to that observed with celecoxib (not shown). On other hand,
compound 3j (Figure 5, panel G) and 3h (not shown) caused markedly
congested blood vessels in the esophagus, nonetheless without any maceration
of gastric mucosa, necrosis or lymphocytic infiltration. However, mucosal surface
of the rat stomachs revealed gastro-esophageal inflammatory infiltrate in case of
compound 3k (Figure 5, Panel H)
2
.3. Molecular modeling and in silico study
.3.1. Molecular docking study:
2
In order to gain an insight of the possible binding interactions of the most active
compounds in the series with the active site of COX-2, a molecular modeling was
performed. Cyclooxygenase-2 enzyme (COX-2) co-crystallized with SC-558
(PDB entry 1CX2) was used to perform the docking study using Molecular
Operating Environment (MOE) version 2016.0802 (Chemical Computing Group,
Montreal, CA). The protein was docked with all compounds of propargyl, triazole
and isoxazole series and the best stable docking poses were selected according
to the best-scored conformation predicted by the MOE scoring function. The
scoring functions, hydrogen bonds formed with the surrounding amino acids, and
the relative orientation of the docked compounds with respect to the co-
crystallized ligand SC-558 were used to estimate binding affinities to the active
site of COX-2 (Supplementary material Table SM2). Re-docking of the co-
crystallized ligand SC-558 into COX-2 active site validated the docking protocol
(Figure 6) and the docking pose was compared with the initial pose using root

ACCEPTED MANUSCRIPT
mean square deviation (RMSD). SC-558 docked almost at the same position
(RMSD = 1.0246 Å) with docking score of – 8.4441 kcal/mol (Figure 6).
Generally speaking, docking scores of the propargyl (from -7.9271 to -10.7236
kcal/mol) and triazole series (from -7.0416 to -12.0528 kcal/mol) were higher
than that of the isoxazole series (from -6.0456 to -9.6319 kcal/mol) with
exception of compound 4g (-9.8875 kcal/mol). This is in agreement with in vitro
COX2 data. Hence, we focused our attention to the molecular interactions
exhibited by the compounds of both the highest docking scores and in vitro
biological activities.
Out of the propargyl series, compound 2c had the highest score (-10.7236
Kcal/mol) and displayed both π-π edge to face interaction and hydrogen bond
between nitrogen N-1 of quinazolinone with Phe518 (Figure 7). Other hydrogen
bonds were found with Leu352, Met522, Phe518, Ser530 and Val523 (Figure 7).
As expected, triazoles 3b, 3h, 3j and 3k showed highest affinity towards COX-2
active site in the whole study (-11.4569, -12.0529, -10.4121 and -10.6398
Kcal/mol). Triazole 3b formed 3 hydrogen bonds with Phe518, Val523 and
Ala527 (Figure 8). Triazole 3h showed 3 important hydrogen bonding
interactions that involved Phe518 (with quinazolinone N-1), Ser530 (with
quinazolinone carbonyl oxygen) and Asp515 with the carboxylic group (Figure
8), in addition to hydrophobic interactions with Ala516, Met522, Val523 and
Gly526 (Figure 8).
Compounds 3j and 3k shared an interesting common feature, which is the
involvement of the 1,4-disubstituted-1,2,3-triazole in 2 peculiar hydrogen bonding
interactions (Figure 9). The first involves position 3-Nitrogen (with Arg513 in
compound 3j and Val523 in compound 3k), and the other involves exceptionally
5
acidic C -H (with Leu352 in both compounds) (Figure 9). This highlights our
expectation that 1,2,3-triazole is not a passive linker but an effective
pharmacophore that is involved in target recognition.

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An overlay of the docking poses of triazoles 3b, 3h, 3j and 3k showed that they
are almost superimposable with respect to each other which indicates that the
COX-2 active site is capable of accommodating all of the quinazolinone nucleus,
triazolyl side chain and orthogonal N -phenyl ring with almost the same binding
3
pattern (Figure 10). COX-2 active site is of larger size than that of COX-1, which
is attributed to the replacement of the amino acid residue Ile523 in COX-1 with
the less bulky Val523 in COX-2 [55]. Hence, COX-2 active site accommodates
bulkier structures and might allow for additional binding interactions. An overlay
of triazoles 3b, 3h, 3j and 3k with the co-crystallized ligand SC-558 showed a
perfect superimposition of the quinazolinone nucleus with the pyrazole of SC-558
and the orthogonal N -phenyl ring with the orthogonal 4-bromophenyl group in
3
SC-558 (Figure 10). This correlates well with the ability of these compounds to fit
in COX-2 active site and hence to display remarkable COX-2 inhibitory activity
and selectivity.
2.3.2 In silico prediction of physicochemical properties and
pharmacokinetic profile:
We used Molinspiration [56], Molsoft [57] and Pre-ADMET [58] software to
assess whether our most active compounds possess the correct parameters to
exhibit drug-likeness or not. It is necessary to point out drug-likeness metrics do
not guarantee that a compound would be an optimum drug for an ailment.
Nonetheless, compounds that fail the drug-likeness criteria often do not qualify to
be an effective clinical candidate due to poor bioavailability, excessive toxicity or
other concerns. We used Molinspiration Chemo-informatics server to predict
molecular descriptors that were used by Lipinski in formulating his rule of five
which are lipophilicity (LogP), molecular weight (MW), number of hydrogen bond
3
donors (HBD) and acceptors (HBA), molecular volume (A) , number of non-
rotatable bonds (NROTB). According to Lipinski, a compound is more likely to be

ACCEPTED MANUSCRIPT
membrane permeable and easily absorbed by the body if it matches the following
criteria:
1
2
3
. The lipophilicity of compound is less than 5.
. Its molecular weight is less than 500.
. The number of groups in the molecule that can donate hydrogen atoms
to hydrogen bonds is less than 5.
4. The number of groups in the molecule that can accept hydrogen atoms
to form hydrogen bonds is less than 10.
̕
Molinspiration is also used for calculation of Lipiniski s violation. In addition, it
2
calculates Topological Polar Surface Area (TPSA)(Å ). The values of TPSA are
used to calculate the percentage of oral absorption (%ABS) using the following
equation:
%ABS = 109 – 0.345 TPSA
2
A molecule usually requires a TPSA below 140-150Å to show acceptable
bioavailability. While for CNS acting molecules this value should be below 70-
2
8
0Å . A combination of TPSA and the number of rotatable bonds in molecule
was found to affect the oral bioavailability in such a way that compounds with 10
2
or fewer rotatable bonds and a TPSA less than 140Å have a high probability of
being orally bioavailable in rats. The in silico physicochemical properties data of
the active compounds are recorded in Table 3.
Table 3: In silico physicochemical properties date of active compounds as
predicted by Molinspiration.
̕
Lipinsks
Volumes
a
b
c
d
e
f
g
Code
LogP
M.Wt
HBA
HBD
PSA
%ABS
NROTB
3
Violation
0
(A)
2
3
3
c
b
h
4.21
4.95
371.26
445.94
3
6
0
0
34.90
65.61
96.95
86.36
274.71
367.98
3
5
0
4.86
5.75
5.89
489.94
524.83
569.28
8
6
6
1
0
0
0
2
2
102.91
65.61
65.61
73.49
86.36
86.36
394.98
385.86
390.21
6
5
5
3
j
3
k

ACCEPTED MANUSCRIPT
a
LogP: logarithm of compound partition coefficient between n-octanol and water.
b
c
d
M.Wt: molecular weight. HBA: number of hydrogen bond acceptors. HBD:
e
e
number of hydrogen bond donors. PSA: polar surface area. %ABS: percentage
of absorption. NROTB: number of rotatable bonds.
g
Two compounds namely 3j and 3k are in a slight violation regarding the
molecular weight (less than 500) and log P (less than 5). The other 3 compounds
(2c, 3b, 3h) are in full accordance to Lipinski’s rule of five. TPSA values of all
2
tested compounds were < 140Å . In addition, all five compounds displayed %
ABS in the range 73.49-96.95%, which indicates a good predicted oral
bioavailability.
Molsoft software was used to predict drug solubility and drug likeness model
score. It is well established that more than 80% of the marketed drugs have an
estimated solubility value greater than 0.0001mg/L. Drug-likeness is expressed
by a numerical value which determines whether a particular molecule can
behave as a drug or not, the more positive the numerical value the more it is
likely for a compound to act as a drug. Molsoft predictions are illustrated in
Table 4.
Table 4: In silico drug-likeness data of active compounds (Molsoft).
a
Code
S (mg/L)
Drug-likeness model score
2
c
0.63
0.28
1.11
3
b
h
0.04
3
0.00
1.01
3
j
0.00
0.00
0.78
0.66
3k
Celecoxib
0.27
-1.03
0.96
Diclofenac
sodium
1.72
a
S aqueous solubility.

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Although 3 compounds did not fulfill the solubility requirement, yet all of the
five compounds showed positive values in the drug-likeness model score, which
indicates that, they have a predicted drug-likeness potential.
Pre-ADMET software was used to calculate the predicted values of the
following pharmacokinetic parameters: CaCo2 cell permeability coefficient,
MDCK cell permeability coefficient, Human intestinal absorption (HIA), Brain-
blood partition coefficient (BBB), Binding to human plasma protein (PPB) and
Inhibition to cytochrome P450 2D6 (CYP2D6). The details of the predicted ADME
parameters are presented in Table 5.
Table 5: In silico ADME data of active compounds (Pre-ADMET).
a
b
c
d
e
Code
CaCo2
52.89
39.19
20.47
34.00
35.10
MDCK
0.053
0.051
0.050
0.018
0.018
HIA
BBB
PPB
2c
97.67
97.70
99.73
98.07
98.22
1.11
0.16
0.61
0.12
0.12
100
3
b
h
95.18
95.49
100
3
3j
3k
100
a
CaCo2: permeability through cells derived from human colon adenocarcinoma;
CaCo2 values < 4nm/sec (low permeability), values ranged from 4 to 70nm/sec
b
(medium permeability) and values >70nm/sec (high permeability), MDCK:
permeability through Madin-Darby Canin kidney cells tool for rapid permeability;
MDCK values < 25nm/sec (low permeability), values ranged from 25 to
5
00nm/sec (medium permeability) and values > 500nm/sec (high permeability),
c
HIA: percentage human intestinal absorption; HIA values ranged from 0 to 20%
(
poorly absorbed), values ranged from 20 to 70% (moderately absorbed) and
d
ranged from 70 to 100% (well absorbed), BBB: blood-brain barrier penetration;
BBB values < 0.1 (low CNS absorption), values ranged from 0.1 to 2 (medium
CNS absorption) and values > 2 (high CNS absorption), PPB: plasma protein
e
binding; PPB values < 90% (poorly bound) and values > 90% (strongly bound).
The tested compounds showed medium cell permeability in the CaCo2 cell
model with values from (20.47-52.89 nm/sec). On the other hand, all tested
compounds showed low cell permeability in the MDCK cell model with values
from (0.018-0.053 nm/sec).

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All five compounds showed extremely high HIA values from (97.50-99.73%)
indicating a reasonable oral absorption pattern. On the other hand all of the
tested compounds exhibited medium CNS absorption from (0.12-1.74).
Furthermore, all the tested compounds were shown to be strongly bound to
plasma proteins with values ranging from (93.72-100%). Moreover, all five
compounds were found to be non-inhibitors of CYP2D6 enzyme and hence they
are expected to be not involved in drug-drug interactions with CYP2D6 inhibitors
and/or inducers. In brief, we conclude that the most active five compounds in
this study exhibited reasonable drug-likeness and physicochemical properties.
Moreover, they obeyed Lipinski’s rule of five with few slight violations and
showed good predicted pharmacokinetic values, which might raise them to be
drug candidates.
3. Conclusion:
A new series of thioquinazolinone molecules decorated with propargyl group,
1
,2,3-triazolyl and isoxazolyl rings were synthesized for evaluation as dual COX-
and LOX inhibiting anti-inflammatory drug candidates. The latter two series
2
were prepared via click chemistry. Compounds 2c, 3b, 3h, 3j, and 3k showed
COX-2 inhibition with IC50 (µM) 0.18, 0.19, 0.11, 0.16 and 0.17 respectively.
These values were compared to celecoxib (IC50 0.05 µM), diclofenac (IC50 0.8
µM) and indomethacin (IC50 0.49 µM) reference drugs. They also showed 15-
LOX inhibition with IC50 (µM) 6.21, 4.33, 7.62, 5.21 and 3.98 respectively. These
values were compared with Zileuton (IC 2.41 µM) and Meclofenamate sodium
50
(IC50 5.64 µM) as positive controls. Moreover, compounds 2c, 3b, 3h and 3j
inhibited monocyte to macrophage differentiation, which is as an early key step of
the inflammatory response, with IC50 (µM) 4.78, 1.24, 0.13, 5.63 compared to
Diclofenac sodium (IC50 (µM) equals 4.86). Formalin-induced rat paw edema
protocol as an acute inflammation model was employed, in which compounds 2c,
3b, 3h and 3j; exhibited more than double the % inhibition of edema formation
exhibited by celecoxib and slightly higher % inhibition than that of Diclofenac
sodium. Gross observation of the isolated rat stomachs and histopathological
observation of rat intestinal mucosa confirmed the gastro-intestinal safety of

ACCEPTED MANUSCRIPT
compounds 2c and 3b. On other hand, compound 3j and 3h showed some blood
vessels congestion in the esophagus, nonetheless without any maceration of
gastric mucosa, necrosis or lymphocytic infiltration. Compounds 2c, 3b, 3h, 3j,
and 3k were docked into the active site of COX-2 and showed perfect fitting
within the binding pocket and substantial interactions with key amino acid
residues. Their drug-likeness were assessed using Molinspiration, Molsoft and
Pre-ADMET software and elucidated reasonable physicochemical properties and
a satisfactory predicted pharmacokinetic parameters. Accordingly, these active
compounds represent promising hits/leads to pursue as potential simultaneous
COX-2 and LOX inhibitors.
4. Experimental
4.1. Chemistry:
Melting points were recorded on electrotherm capillary tube Stuart melting point
apparatus SMP10 and are all uncorrected. Follow up of the reactions rates were
performed by thin-layer chromatography (TLC) on silica gel (60 GF254) coated
glass plates and the spots were visualized by exposure to iodine vapors or UV-
lamp at λ 254nm for few seconds. Infrared spectra (IR) were recorded, using KBr
-
1
discs on a FT-IR Perkin-Elmer 1430 Infrared spectrophotometer v-(cm ) in
Central Laboratory, Faculty of Pharmacy, Alexandria University. Nuclear
1
13
magnetic resonance ( H NMR and C NMR) spectra were recorded on a
Mercury 300 MHz spectrophotometer, Faculty of Science, Cairo University or on
a Bruker 400 MHz spectrophotometer, Faculty of Pharmacy; Cairo University
using deuterated Dimethylsulfoxide (DMSO-d ) as solvent. The data were
6
recorded as chemical shifts expressed in δ (ppm) relative to Tetramethylsilane
(TMS) as internal standard. Signal splitting are expressed by the following
abbreviations: s = singlet, d = doublet, t = triplet, q = quartet and m = multiplet.
Electron impact mass spectra (EIMS) were run on a gas chromatograph/mass
spectrophotometer at Al-Azhar University; (The Regional Center for Mycobiology
and Biotechnology). Relative intensity % to the base peak of the most
characteristic fragments was recorded. Microanalytical data (C, H, N and S) were

ACCEPTED MANUSCRIPT
performed at the microanalytical unit, Faculty of Science, Al-Azhar University.
The preparation of compounds 1 (a-d) [32,43], appropriate azides [59] and
appropriate aldoximes [60] were performed according to reported procedures.
4
.1.1. 3-Aryl-2(2-propynyl)thioquinazolin-4(3H)-ones (2)
To a solution of 3-aryl-2-mercaptoquinazolin-4(3H)-ones 1 (a-d) (4 mmol) in 10
mL of DMF, were added propargyl bromide (0.47 g, 4 mmol) and K CO (0.55 g,
mmol). The reaction mixture was heated for 3 h at 50 to 60° C, poured into ice-
2
3
4
water and allowed to stand overnight. The precipitate was filtered, washed, and
recrystallized from ethanol to give 2 (a-d).
4.1.1.1. 3-Phenyl-2-(prop-2-yn-1-ylthio)quinazolin-4(3H)-one (2a)
-
1
Yield 83%, m.p. 176 °C, IR (KBr, cm ): 1541.28 (C=C, Ar), 1602.28 (C=N),
1
1
679.42 (C=O), 2288-2347 (C≡C), 3226 (CH-acetylenic). H NMR (DMSO-d ): δ
6
13
3
.14 (t, 1H, J=2.7 Hz, CH), 4.01 (d, 2H, J=2.7 Hz,CH2), 7.4-8.1 (m, 9H, Ar). C
): 20.09 (CH
), 74.49 (CH), 79.70 (C≡C), 120.08-147.56 (12C,
Ar), 156.18 (C-2 quinazolinone), 161.12 (C=O). Anal. Calcd. for C H N OS: C,
NMR (DMSO-d
6
2
17
12
2
69.84, H, 4.14, N, 9.58, S, 10.97. Found: C, 70.08, H, 4.19, N, 9.70, S, 11.13.
4.1.1.2 3-(4-Chlorophenyl)-2-(prop-2-yn-1-ylthio)quinazolin-4(3H)-one (2b)
-
1
Yield 76%, m.p. 186-187 °C, IR (KBr, cm ): 1545.56 (C=C, Ar), 1604.16 (C=N),
1
1
688.97 (C=O), 2338-2362 (C≡C), 3254 (CH, acetylenic). H NMR (DMSO-d
6
): δ
13
3
.16 (t, 1H, J=2.7 Hz, CH), 4.02 (d, 2H, J=2.7 Hz, CH ), 7.3-8.1 (m, 8H, Ar). C
2
NMR (DMSO-d
6 2
): δ 20.42 (CH ), 74.02(CH), 79.07 (C≡C), 115.6-146.9 (12C, Ar),
1
6
55.2 (C-2 quinazolinone), 160.5 (C=O). Anal. Calcd. for C H ClN OS: C,
17
11
2
2.48, H, 3.39, N, 8.57, S, 9.81, Found: C, 62.65, H, 3.45, N, 8.69, S, 9.93. EI-
MS: [ M+ 326.97 and base peak 277.00].
.1.1.3 3-(4-Bromophenyl)-2-(prop-2-yn-1-ylthio)quinazolin-4(3H)-one (2c)
4
-
1
Yield 70%, m.p. 189-190 °C, IR (KBr, cm ): 1546.45 (C=C, Ar), 1603.99 (C=N),
1
1
688.86 (C=O), 2364-2340 (C≡C), 3251.04 (CH, acetylenic). H NMR (DMSO-
d ): δ 3.17 (t, 1H, J=2.7 Hz, CH), 4.02 (d, 2H, J=2.7 Hz, CH ), 7.4-8.1 (m, 8H,
6
2
13
Ar). C NMR (DMSO-d
6
): δ 20.39 (CH
), 74.02 (CH), 79.06 (C≡C), 119.5-146.9
2
(12C, Ar), 155.1 (C-2 quinazolinone), 160.49 (C=O). Anal. Calcd. for

ACCEPTED MANUSCRIPT
C
17
H
11BrN
2
OS : C, 55.00, H, 2.99, N, 7.55, S, 8.64, Found: C, 55.17, H, 2.96, N,
7.68, S, 8.71.
4.1.1.4 3-(4-Methoxyphenyl)-2-(prop-2-yn-1-ylthio)quinazolin-4(3H)-one (2d)
-
1
Yield 80%, m.p. 183-184 °C, IR (KBr, cm ): 1550.22 (C=C, Ar), 1605.72 (C=N),
1
1
d
7
7
686,79 (C=O), 2363-2312 (C≡C), 3253.42 (CH, acetylenic). H NMR (DMSO-
6
): δ 3.17 (t, 1H, J=2.7 Hz, CH), 3.8 (s, 3H, CH
3
), 3.9 (d, 2H, J=2.7 Hz, CH
2
),
13
.0-8.1 (m, 8H, Ar). C NMR (DMSO-d ): δ 20.9 (CH ), 55.9 (OCH ), 74.4 (CH),
6
2
3
9.7 (C≡C), 114.5-147 (12C, Ar), 156.8 (C-2 quinazolinone), 160.3 (C=O). Anal.
S: C, 67.06, H, 4.38, N, 8.69, S, 9.94, Found: C, 67.35, H,
.46, N, 8.84, S, 10.01.
.1.2. 3(4-Substituted phenyl)-2((1(4-substituted phenyl)-1H-1,2,3-triazol-4-
14 2 2
Calcd. for C18H N O
4
4
yl)methylene)thioquinazolin-4(3H)-ones (3)
To a mixture of 3-aryl-(2-propynyl)thioquinzolin-4(3H)-ones 2 (a-d) ( 1.7 mmol)
and p-azido phenyl derivative (1.7 mmol) in 10 mL of DMF, was added an
aqueous solution (5 mL) of ascorbic acid (0.06 g, 0.34 mmol) and CuSO4.5H2O
reaction (0.02 g, 0.085 mmol). The mixture was refluxed for 10-12 h. After
cooling, the mixture was poured on ice, filtered, washed with cold water, dried
and recrystallized from DMF and washed with methanol to obtain 3 (a-p).
4.1.2.1. 3-Phenyl-2-(((1-phenyl-1H-1,2,3-triazol-4-yl)methylene) thio)
quinazolin-4(3H)-one (3a)
-
1
Yield 70%, m.p. 140-142 °C, IR (KBr, cm ): 1546.40 (C=C, Ar), 1608.49 (C=N),
1
1
674.40 (C=O). H NMR (DMSO-d ): δ 4.5 (s, 2H, CH ), 7.4-8.1 (m, 14C, Ar), 9.0
6
2
(
s, 1H, C -triazole). Anal. Calcd. for C H N OS: C, 67.14, H, 4.16, N, 17.02, S,
5 23 17 5
7.79. Found: C, 67.42, H, 4.22, N, 17.36, S, 7.87.
4.1.2.2. 2-(((1-(4-Chlorophenyl)-1H-1,2,3-triazol-4-yl)methylene)thio)-3-
phenylquinazolin-4(3H)-one (3b)
-
1
Yield 83%, m.p. 238 °C, IR (KBr, cm ): 1546.89 (C=C, Ar), 1602.46 (C=N),
1
1
688.66 (C=O). H NMR (DMSO-d ): δ 4.5 (s, 2H, CH ), 7.3-8.3 (m, 13H, Ar), 8.7
6
2
13
(
s, 1H, C
5
- triazole). C NMR (DMSO-d
6 2
): δ 26.55 (CH ), 119-135.2 (18C, Ar),
(135.6 and 147) (2C, triazole), 156.3 (C-2 quinazolinone), 160.6 (C=O). Anal.

ACCEPTED MANUSCRIPT
5
Calcd. for C23H16ClN OS: C, 61.85, H, 3.62, N, 15.71, S, 7.19. Found: C, 62.18,
+
H, 3.95, N, 15.89, S, 7.26. EI-MS: [ M 445.14 and base peak 55.12 ].
4.1.2.3. 2-(((1-(4-Bromophenyl)-1H-1,2,3-triazol-4-yl)methylene)thio)-3-
phenylquinazolin-4(3H)-one (3c)
-
1
Yield 70%, m.p. 242 °C IR (KBr, cm ): 1493.85 (C=C, Ar), 1602.22 (C=N),
1
1
689.59 (C=O). H NMR (DMSO-d
6
): δ 4.5 (s, 2H, CH
2
), (s, 1H, C
5
-triazole), 7.3-
13
8
.1 (m, 13H, Ar). C NMR (DMSO-d ): δ 27.08 (CH ), 120.14-135 (18C, Ar),
6
2
(
136.19 and 144.35)(2C, triazole), 156 (C-2 quinazolinone), 161.2 (C=O). Anal.
Calcd. for C23 OS: C, 56.33, H, 3.29, N, 14.28, S, 6.54 Found: C, 56.48,
H, 3.34, N,14.52, S, 6.62.
H16BrN
5
4.1.2.4. 4-(4-(((4-Oxo-3-phenyl-3,4-dihydroquinazoline-2-yl)thio)methylene)-
1H-1,2,3-triazol-1-yl)benzoic acid (3d)
-
1
Yield 96%, m.p. 248 °C, IR (KBr, cm ): 1547.44 (C=C, Ar), 1605.05 (C=N),
1
1
674.66 (C=O), 2500-3500 (-OH, broad band). H NMR (DMSO-d
6
): δ 4.5 (s, 2H,
13
CH2), 7.3-8.1 (m, 13H, Ar), 8.8 (s, 1H, C -triazole), 13.0 (s, 1H, COOH).
C
5
6 2
NMR (DMSO-d ): δ 26.57 (CH ), δ 119.6-135 (18C, Ar), (139.3 and 144) (2C,
triazole), 156.3 (C-2 quinazolinone), 160.6 (C=O), 166.3 (C=O, acidic). Anal.
Calcd. for C H N O S: C, 63.29, H, 3.76, N, 15.64, S, 7.04 Found: C, 63.43, H,
24
17
5
3
3.74, N, 15.64, S, 7.12.
4.1.2.5. 3-(4-Chlorophenyl)-2-(((1-phenyl-1H-1,2,3-triazol-4-
yl)methylene)thio)quinazolin-4(3H)-one (3e)
-
1
Yield 81%, m.p. 220 °C, IR (KBr, cm ): 1547.95 (C=C, Ar), 1605.09 (C=N),
1
1
667.92 (C=O). H NMR (DMSO-d ): δ 4.5 (s, 2H, CH ), 7.4-8.1 (m, 13H, Ar), 8.7
6
2
5
(s, 1H, C -triazole). Anal. Calc. for C23H15BrClN
5
OS: C, 52.64, H, 2.88, N, 13.34,
+
S, 6.11. Found: C, 62.19, H, 3.68, N, 15.89, S, 7.31. EI-MS: [M 445.32 and base
peak 283.21].
4.1.2.6. 3-(4-Chlorophenyl)-2-(((1-(4-chlorophenyl)-1H-1,2,3-triazol-4-
yl)methylene)thio) quinazolin-4(3H)-one (3f)
-
1
Yield 84%, m.p. 199 °C, IR (KBr, cm ): 1544.00 (C=C, Ar), 1600.53 (C=N),
1
1
672.93 (C=O). H NMR (DMSO-d ): δ 4.5 (s, 2H, CH ), 7.2-8.1 (m, 12H, Ar), 8.7
6
2

ACCEPTED MANUSCRIPT
(
s, 1H, C
S, 6.67. Found: C, 57.80, H, 3.12, N, 14.79, S,6.75.
.1.2.7. 2-(((1-(4-Bromophenyl)-1H-1,2,3-triazol-4-yl)methylene)thio)-3-(4-
chlorophenyl)quinazolin-4(3H)-one (3g)
5 2 5
-triazole). Anal. Calcd. for C23H15Cl N OS: C, 57.51, H, 3.15, N, 14.58,
4
-
1
Yield 87%, m.p. >300 °C, IR (KBr, cm ): 1544.13 (C=C, Ar), 1603.92 (C=N),
1
1
683.41 (C=O). H NMR (DMSO-d
6
): δ 4.5 (s, 2H, CH
2
), 7.3-8.3 (m, 12H, Ar), 8.7
(
s, 1H, C -triazole). Anal. Calcd. for C H BrClN OS: C, 52.64, H, 2.88, N,
5 23 15 5
13.34, S, 6.11. Found: C, 52.81, H, 2.89, N, 13.62, S, 6.24.
4.1.2.8. 4-(4-(((3-Chlorophenyl)-4-oxo-3,4-dihydroquinazolin-2-
yl)thio)methylene)-1H-1,2,3-triazol-1-yl)benzoic acid (3h)
-
1
Yield 81%, m.p. >300 °C, IR (KBr, cm ): 1564.81 (C=C, Ar), 1606.28 (C=N),
1
1
663.93 (C=O), 2500-3500 (-OH, broad band). H NMR (DMSO-d ): δ 4.5 (s, 2H,
6
CH ), 7.4-8.1 (m, 12H, Ar), 8.8 (s, 1H, C -triazole), 13.0 (s, 1H, COOH). Anal.
2
5
5 3
Calcd. for C24H16ClN O S: C, 58.84, H, 3.29, N, 14.29, S, 6.54. Found: C, 59.12,
H, 3.33, N, 14.54, S, 6.62.
.1.2.9. 3-(4-Bromophenyl)-2-(((1-phenyl-1H-1,2,3-triazol-4-
yl)methylene)thio)quinazolin-4(3H)-one (3i)
4
-
1
1
Yield 73%, m.p. 195 °C, IR (KBr, cm ): 1545 (C=C, Ar), 1666 (C=N). H NMR
DMSO-d ): δ 4.5 (s, 2H, CH ), 7.2-8.1 (m, 13H, Ar), 8.7 (s, 1H, C -triazole).
Anal. Calcd. for C H BrN OS: C, 56.33, H, 3.29, N, 14.28, S, 6.54. Found: C,
(
6
2
5
23
16
5
5
6.57, H, 3.31, N, 14.53, S, 6.61. EI-MS: [M+ 490.05, (M+2)+ 492.24 and base
peak 83.10].
.1.2.10. 3-(4-Bromophenyl)-2-(((1-(4-chlorophenyl)-1H-1,2,3-triazol-4-
yl)methylene)thio)quinazolin-4(3H)-one (3j)
4
-
1
Yield 91%, m.p. 216-217 °C, IR (KBr, cm ): 1547.10 (C=C, Ar), 1607.01 (C=N),
1
1
680.04 (C=O). H NMR (DMSO-d
6
): δ 4.5 (s, 2H, CH
2
), 7.4-8.0 (m, 12H, Ar), 8.7
13
(
s, 1H, C -triazole). C NMR (DMSO-d ): δ 27.1 (CH ), 120-135.7 (s, 18C, Ar),
5 6 2
(
144.2 and 147.6) (2C, triazole), 156.4 (C-2 quinazolinone), 161 (C=O). Anal.
OS: C, 52.64, H, 2.88, N, 13.34, S, 6.11. Found: C,
2.91, H, 2.87, N, 13.62, S, 6.20.
Calcd. for C23H15BrClN
5
5

ACCEPTED MANUSCRIPT
4.1.2.11. 3-(4-Bromophenyl)-2-(((1-(4-bromophenyl)-1H-1,2,3-triazol-4-
yl)methylene)thio)quinazolin-4(3H)-one (3k)
-
1
Yield 87%, m.p. 210-212 °C, IR (KBr, cm ): 1581.99 (C=C, Ar), 1604.88 (C=N),
1
1
682.51 (C=O). H NMR (DMSO-d
6
): δ 4.5 (s, 2H, CH
2
), 7.2-8.0 (m, 12H, Ar), 8.1
13
(
s, 1H, C -triazole). C NMR (DMSO-d ): δ 26.6 (CH ), 119-135.2 (18C, Ar),
5 6 2
(135.6 and 147.1) (2C, triazole), 155.87 (C-2 quinazolinone), 160.6 (C=O). Anal.
Calcd. for C H Br N OS: C, 48.53, H, 2.66, N, 12.30, S, 5.63. Found: C, 48.79,
23
15
2
5
H, 2.65, N, 12.48, S, 5.74.
.1.2.12. 4-(4-(((3-(4-Bromophenyl)-4-oxo-3,4-dihydroquinazolin-2-
yl)thio)methylene)-1H-1,2,3-triazol-1-yl)benzoic acid (3l)
4
-
1
Yield 70%, m.p. 258-260 °C, IR (KBr, cm ): 1528.00 (C=C, Ar), 1606.16 (C=N),
1
1
670.22 (C=O, amide), 1707.81 (C=O, acidic), 2500-3500 (-OH, broad band). H
NMR (DMSO-d ): δ 4.5 (s, 2H, CH ), 7.29-8.15 (m, 12H, Ar), 8.8 (s, 1H, C -
6
2
5
5 3
triazole), 13.01 (s, 1H, COOH). Anal. Calcd. for C24H16BrN O S: C, 53.94, H,
3.02, N, 13.11, S, 6.00. Found: C, 54.12, H, 3.09, N, 13.24, S, 6.08.
4.1.2.13. 3-(4-Methoxyphenyl)-2-(((1-phenyl-1H-1,2,3-triazol-4-
yl)methylene)thio)quinazolin-4(3H)-one (3m)
-
1
Yield 79%, m.p. 120 °C, IR (KBr, cm ): 1547.62 (C=C, Ar), 1606.33 (C=N),
1
1
8
6
4
677.26 (C=O). H NMR (DMSO-d
6
): δ 3.8 (s, 3H, CH
3
), 4.5 (s, 2H, CH
2
), 7.0-
.01 (m, 13H, Ar), 8.7 (s, 1H, C -triazole). Anal. Calcd. for C H N O S: C,
5
24 19
5
2
5.29, H, 4.34, N, 15.86, S, 7.26. Found: C, 65.47, H, 4.41, N, 16.09, S, 7.31.
.1.2.14. 3-(4-Methoxyphenyl)-2-(((1-(4-chlorophenyl)-1H-1,2,3-triazol-4-
yl)methylene)thio)quinazolin-4(3H)-one (3n)
-
1
Yield 75%, m.p. 215-217 °C, IR (KBr, cm ): 1547.01 (C=C, Ar), 1606.97 (C=N),
1
1
8
5
671.59 (C=O). H NMR (DMSO-d ): δ 3.8 (s, 3H, CH ), 4.5 (s, 2H, CH ), 7.07-
6
3
2
1
3
.09 (m, 12H, Ar), 8.7 (s, 1H, C
5
-triazole). C NMR (DMSO-d
6 2
): δ 27.1 (CH ),
5.9 (CH ), 115-135(18C,Ar), (144 and 147.7) (2C, triazole), 157.6 (C-2
3
quinazolinone), 160.5 (C=O). Anal. Calcd. for C H ClN O S: C, 60.57, H, 3.81,
24
18
5
2
N, 14.71, S, 6.74. Found: C, 60.79, H, 3.88, N, 14.97, S, 6.80.
.1.2.15. 3-(4-Methoxyphenyl)-2-(((1-(4-bromophenyl)-1H-1,2,3-triazol-4-
yl)methylene)thio) quinazolin-4(3H)-one (3o)
4

ACCEPTED MANUSCRIPT
-
1
Yield 88%, m.p. 207 °C IR (KBr, cm ): 1548.02 (C=C, Ar), 1606.99 (C=N),
1
1
682.26 (C=O). H NMR (DMSO-d ): δ 3.8 (s, 3H, CH ), 4.5 (s, 2H, CH ), 7.1-8.1
6
3
2
13
(
m, 12H, Ar), 8.8 (s, 1H, C -triazole). C NMR (DMSO-d ): δ 27.1 (CH ), 56.5
5 6 2
3
(CH ), 115-136 (18C, Ar), (144 and 147) (2C, triazole), 157.6 (C-2
quinazolinone), 160.5 (C=O). Anal. Calcd. for C H BrN O S: C, 55.39, H, 3.49,
24
18
5
2
N, 13.46, S, 6.16. Found: C, 55.62, H, 3.54, N, 13.70, S, 6.28. EI-MS: [M+
518.92, (M+2)+ 520.96 and base peak 283.05].
4.1.2.16 4-(4-(((3-(4-Methoxyphenyl)-4-oxo-3,4-dihydroquinazolin-2-
yl)thio)methylene)-1H-1,2,3-triazol-1-yl)benzoic acid (3p)
-
1
Yield 80%, m.p. >300 °C, IR (KBr, cm ): 1542.49 (C=C, Ar), 1605.35 (C=N),
1
1
675.15 (C=O, amide), 1698.68 (C=O, acidic), 2500-3500 (-OH, broad band). H
NMR (DMSO-d ): δ 3.8 (s, 3H, CH ), 4.5 (s, 2H, CH ), 7.1-8.1 (m, 12H, Ar), 8.9
6
3
2
(
s, 1H, C -triazole), 12.7 (s, 1H, COOH). Anal. Calcd. for C H N O S: C, 61.85,
5 25 19 5 4
H, 3.94, N, 14.42, S, 6.60. Found: C, 62.09, H, 3.96, N, 14.65, S, 6.69.
.1.3. 3-(4-Substituted phenyl)-2(((3-(3,4-disubstitutedphenyl)isoxazol-5-
4
yl)methylene)thio)quinazolin-4(3H)-ones (4)
To the appropriate oxime (2 mmol) dissolved in 4 mL of t-BuOH/H2O (1:1),
Chloramine-T trihydrate (0.45 g, 2 mmol) was added portionwise over 3 min
4 2
followed by CuSO .5H O (6 mol %) and copper powder (12 mol %). To the
aforementioned solution, the appropriate alkyne 2 (a-d) (2 mmol) in 5mL DMSO
was added, the mixture was refluxed for 9-12 h. The reaction mixture was
poured onto ice, and dilute NH OH was added. The formed precipitates were
4
filtered, crystallized from DMF and washed with methanol to give the required
isoxazoles 4 (a-l).
4.1.3.1. 2-(((3-(4-Methoxyphenyl)isoxazol-5-yl)methylene)thio)-3-
phenylquinazolin-4(3H)-one (4a)
-
1
Yield 98%, m.p. 200 °C, IR (KBr, cm ): 1548.12 (C=C, Ar), 1608.72 (C=N),
1
1
5
1
676.91 (C=O). H NMR (DMSO-d ): δ 3.80 (s, 3H, OCH ), 4.61 (s, 2H, CH ),
6
3
2
.83 (s, 1H, CH), 5.54 (s, 1H, isoxazole), 6.97 (s, 1H, isoxazole), 7.02-8.10 (m,
3H, Ar). Anal. Calcd. for C H N O S: C, 68.01, H, 4.34, N, 9.52, S, 7.26.
25
19
3
3
Found: C, 67.96, H, 4.29, N, 10.14, S, 7.15.

ACCEPTED MANUSCRIPT
4.1.3.2. 2-(((3-(3,4-Dimethoxyphenyl)isoxazol-5-yl)methylene)thio)-3-
phenylquinazolin-4(3H)-one (4b)
-
1
Yield 64%, m.p. 166-168 °C IR (KBr, cm ): 1589.34 (C=C, Ar), 1608.63 (C=N),
1
1
662.64 (C=O). H NMR (DMSO-d
6 3
): δ3.83 (s, 6H, 2CH ), 3.59 (s, 2H,
isoxazole), 5.54 (s, 1H, isoxazole), 5.84 (s, 1H, CH), 5.88 (s, 1H, CH), 7.29-8.05
m, 12H, Ar). Anal. Calcd. for C26 S: C, 66.23, H, 4.49, N, 8.91, S, 6.80.
Found: C, 66.34, H, 4.64, N, 9.07 S, 6.98.
.1.3.3. 2-(((3-(4-Chlorophenyl)isoxazol)-5-yl)methylene)thio)-3-
phenylquinazolin-4(3H)-one (4c)
Yield 98%, m.p. 186-188 °C, IR (KBr, cm ): 1608.63 (C=N), 1670.35 (C=O). H
NMR (DMSO-d ): δ 3.59 (s, 2H, isoxazole), 5.54 (s, 1H, isoxazole), 5.83 (s, 1H,
CH ), 5.87 (s, 1H, CH), 7.29-8.04 (m, 13H, Ar). Anal. Calcd. for C H ClN O S:
(
21 3 4
H N O
4
-
1
1
6
2
24 16
3
2
C, 64.64, H, 3.62, N, 9.42, S, 7.19. Found: C, 64.92, H, 3.57, N, 9.68, S, 7.11.
Ms: [M+ 445.09 and base peak 55.08].
4.1.3.4. 3-(4-Chlorophenyl)-2-(((3-(4-methoxyphenyl)isoxazol-5-
yl)methylene)thio)quinazolin-4(3H)-one (4d)
-
1
Yield 73%, m.p. >300 °C, IR (KBr, cm ): 1543.05 (C=C, Ar), 1608.63 (C=N),
1
1
685.79 (C=O). H NMR (DMSO-d ): δ 1.24 (s, 1H, NH-isoxazole), 3.80 (s, 3H,
6
3
CH ), 4.63 (s, 2H, CH
2
), 5.53 (s, 1H, isoxazole), 6.98 (s, 1H, isoxazole), 7.02-
8
6
4
.08 (m, 12H, Ar). Anal. Calcd for C H ClN O S: C, 63.09, H, 3.81, N, 8.83, S,
25
18
3
3
.74 Found: C, 63.50, H, 3.79, N, 8.89, S, 6.80.
.1.3.5. 3-(4-Chlorophenyl)-2-(((3,4-dimethoxyphenyl)isoxazol-5-
yl)methylene)thio)quinazolin-4(3H)-one (4e)
-
1
Yield 78%, m.p. 197-198 °C, IR (KBr, cm ): 1550.77 (C=C, Ar), 1604.77 (C=N),
1
1
662.64 (C=O). H NMR (DMSO-d ): δ3.85 (s, 6H, 2CH ), 4.64 (s, 2H, CH ), 5.89
6
3
2
(s, 1H, CH), 5.53 (s, 1H, isoxazole), 7.02 (s, 1H, isoxazole), 7.03-8.10 (m, 11H,
13
Ar). C NMR (DMSO-d ): δ 26.94 (CH ), 56.03 (CH ), 56.08 (CH ), 102.15 (C-4
6
2
3
3
isoxazole), 109.95-150.92 (Ar, 17C), 161.16 (C-5 isoxazole), 161.77 (C-2
quinazolinone), 162.28 (C=O), 169.27 (C-3 isoxazole). Anal. Calcd for
C H ClN O S: C, 61.72, H, 3.98, N, 8.30, S, 6.34. Found: C, 62.08, H, 4.01, N,
26
20
3
4
8.15, S, 6.48. EI-MS [M+ 504.90 and base peak 310.99].

ACCEPTED MANUSCRIPT
4.1.3.6. 3-(4-Chlorophenyl)-2-(((3-(4-chlorophenyl)isoxazol-5-
yl)methylene)thio)quinazolin-4(3H)-one (4f)
-
1
Yield 85%, m.p. 194-196 °C, IR (KBr, cm ): 1519.82 (C=C, Ar), 1607.37 (C=N),
1
1
665.20 (C=O). H NMR (DMSO-d
6 2
): δ 4.65 (s, 2H, CH ), 5.58 (s, 1H, CH), 5.88
(s, 1H, CH), 3.58 (s, 2H, isoxazole), 5.53 (s, 1H, isoxazole), 7.08 (s, 1H,
isoxazole), 7.31-8.20 (m, 12H,Ar). Anal. Calcd. for C24 S: C, 60.01, H,
3 2
H15ClN O
3.15, N, 8.75, S, 6.67. Found: C, 60.12, H, 3.17, N, 8.85, S, 6.72.
4.1.3.7. 3-(4-Bromophenyl)-2-(((3-(4-methoxyphenyl)isoxazol-5-
yl)methylene)thio)quinazolin-4(3H)-one (4g)
-
1
1
Yield 75%, m.p. 226-228 °C, IR (KBr, cm ): 1604.77 (C=N), 1670.35 (C=O). H
NMR (DMSO-d ): δ 3.89 (s, 3H, CH ), 3.57 (s, 2H, isoxazole), 5.53 (s, 1H,
6
3
isoxazole), 5.83 (s, 2H, CH), 5.88 (s, 1H, CH), 7.28-8.05 (m, 12H, Ar). Anal.
Calcd. for C H BrN O S: C, 57.70, H, 3.49, N, 8.07, S, 6.16. Found: C, 57.86,
25
18
3
3
H, 3.56, N, 8.32, S, 6.29. EI-MS: [M+ 520.89, (M+2)+ 523.23 and base peak
354.92].
4.1.3.8. 3-(4-Bromophenyl)-2-(((3-(3,4-dimethoxyphenyl)isoxazol-5-
yl)methylene)thio)quinazolin-4(3H)-one (4h)
-
1
1
Yield 64%, m.p. 228-229 °C, IR (KBr, cm ): 1604.77 (C=N), 1670.35 (C=O). H
NMR (DMSO-d ): δ 3.85 (s, 6H, 2CH ), 4.63 (s, 2H, CH ), 5.82 (s, 1H, CH), 5.88
s, 1H, CH), 3.57 (s, 2H, isoxazole), 5.53 (s, 1H, isoxazole), 7.02 (s, 1H,
isoxazole), 7.27-8.06 (m, 11H, Ar). Anal. Calcd. for C26 S: C, 56.74, H,
6
3
2
(
3 4
H20BrN O
3.66, N, 7.63, S, 5.82. Found: C, 57.02, H, 3.71, N, 6.62, S, 5.97.
4.1.3.9. 3-(4-Bromophenyl)-2-(((3-(4-chlorophenyl)isoxazol-5-
yl)methylene)thio)quinazolin-4(3H)-one (4i)
-
1
Yield 55%, m.p. 228-230 °C, IR (KBr, cm ): 1524.10 (C=C, Ar), 1605.05 (C=N),
1
1
667.35 (C=O). H NMR (DMSO-d
6
): δ 5.82 (s, 1H, CH), 5.88 (s, 1H, CH), 3.58
(s, 2H, isoxazole), 5.53 (s, 1H, isoxazole), 7.28-8.04 (m, 12H, Ar). Anal. Calcd.
for C H ClBrN O S: C, 54.93, H, 2.88, N, 8.01, S, 6.90. Found: C, 55.19, H,
24
15
3
2
2.93, N, 9.17, S, 6.90.
4.1.3.10. 3-(4-Methoxyphenyl)-2-(((3-(4-methoxyphenyl)isoxazol-5-
yl)methylene)thio)quinazolin-4(3H)-one (4j)