Synthesis, 3D pharmacophore, QSAR and docking studies of novel quinazoline derivatives with nitric oxide release moiety as preferential COX-2 inhibitors

Article abstract of DOI:10.1039/c4md00392f

Four novel series of 1-substituted-3-(2-methyl-4-oxo-4H-quinazolin-3-yl) urea and/or thiourea IIIa-c, 4-substituted-N-(2-methyl-4-oxo-4H-quinazolin-3-yl) benzene sulfonamide VIa-c and their NO-hybrid molecules as nitrate esters Va-c and VIIIa-c have been

Full text of DOI:10.1039/c4md00392f

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Medicinal Chemistry Communications
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DOI: 10.1039/C4MD00392F
Synthesis, 3D pharmacophore, QSAR and docking studies of novel quinazoline
derivatives with nitric oxide release moiety as preferential COX-2 inhibitors.
Doaa Boshra Faraga, Nahla A. Faraga, Ahmed Esmatb, Sally A. Abuelezzc, Eman Abdel-Salam
Ibrahimd and Dalal A. Abou El Ellae
aPharmaceutical Chemistry Department, Faculty of Pharmacy, Misr International University, Cairo, Egypt 11431
bPharmacology &Toxicology Department, Faculty of Pharmacy, Ain Shams University, Cairo, Egypt 11566
cPharmacology &Therapeutics Department, Faculty of Medicine, Ain Shams University, Cairo, Egypt 11566
dPathology Department, Faculty of Medicine, Ain Shams University, Cairo, Egypt 11566
ePharmaceutical Chemistry Department, Faculty of Pharmacy, Ain Shams University, Cairo, Egypt 11566
Keywords:
Anti-inflammatory activity
Preferential COX-2 inhibitors
Nitric oxide release moiety
3D pharmacophore modeling
QSAR study
Docking
Abstract
Four novel series of 1-substituted-3-(2-methyl-4-oxo-4H-quinazolin-3-yl) urea and/or thiourea IIIa-c, 4-substituted-
N-(2-methyl-4-oxo-4H-quinazolin-3-yl) benzene sulfonamide VIa-c and their NO-hybrid molecules as nitrate esters
Va-c, VIIIa-c have been synthesized and evaluated for their anti-inflammatory activity in-vivo and in-vitro. Also, the
nitrate ester derivatives Va-c, VIIIa-c have been assayed for releasing nitric oxide in serum using Griess
diazotization method by nitric oxide detection kit, and the results were correlated with their gastric protective activity
through determination of ulcer index, and the histopathological investigations of the gastric mucosa under light
microscope.
All the tested compounds showed potent to moderate anti-inflammatory when compared to the reference drug
Meloxicam. Whereby comparing IIIb and its nitrate ester Vb; for COX-2 inhibition assay, IIIb showed IC50= 5 µM,
and Vb showed IC50= 6.86 µM with selectivity index of 4.74 and 5.03, respectively. As for the IC50 of COX-1 assay,
it was found that the structures carrying nitric oxide moieties had less affinity to inhibit COX-1 than their
corresponding starting intermediates, as in the case of compound IIIb that showed IC50= 23.76 µM, and its nitrate
ester Vb that showed IC50= 34.6 µM which conclude that the alcoholic metabolites of NO esters had less tendency to
inhibit COX-1 leading to less gastric ulcerative side effects. This was confirmed by measuring the nitric oxide
amount release from Vb which was found to be 28.53 µmol/L with the least ulcer index of 0.4, showing the least
tendency to induce stomach ulcerations in rats.
According to these results, we can conclude that quinazoline derivatives with nitric oxide release moiety seem
potentially attractive as preferential COX-2 inhibitors.
1. Introduction
The most notable achievement in the development of NSAIDs has been in the area of selective COX-2
inhibitors (coxibs) without interfering with COX-1 enzymatic activity, where COX-1 is beneficial in
maintaining normal processes in the GI tract by producing cytoprotective prostaglandins, stimulating
———
 Corresponding author: Tel.: +20-100-566-7779; fax: +20-247-720-38; e-mail: doaa.boshra@miuegypt.edu.eg
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DOI: 10.1039/C4MD00392F
bicarbonate secretion and mucus, thereby producing an overall reduction in acid secretion, thus these
selective COX-2 inhibitors showed reduced gastrointestinal side effects as compared to traditional NSAIDs
[1]. Despite the promise of therapeutic effectiveness of the selective COX-2 inhibitors, the potential for
severe cardiovascular effects prompted a critical review of these drugs. This concern was initiated through
the revelation of long-term clinical trials of Rofecoxib that indicated an increased risk of heart attack.
These effects may be related to the different consequences of inhibition of COX-1 and COX-2 [2-8]. This
alteration resulted in increased incidences of an adverse cardiovascular thrombotic event such as
myocardial infarction [9-11]. That ultimately prompted the withdrawal of Rofecoxib and Valdecoxib [12,
13]. Other class was reported to have COX-2 selectivity but meanwhile it is accepted as so-called
preferential COX-2 inhibitor with certain COX-2 specificity, and a lower incidence of gastric and renal
side-effects than associated with classic NSAIDs. This group contained compounds such as Etodolac,
Meloxicam, and Nimesulide, all of which show a preferential selectivity toward COX-2 [14,15].
Moreover, it was reported that different series of NO/aspirin and NO/NSAIDs acquired anti-
inflammatory effects devoid of acute gastrotoxicity principally as a consequence of their NO donor ability
[16], where NO plays an important cytoprotective role in GI homeostasis by helping to maintain mucosal
blood flow, by optimizing mucus secretion [17-21]. Therefore, NO release provides an attractive method to
suppress vascular side effects associated with NSAID use [22, 23]. Recent strategies are now devoted to
minimize the ulcerogenic side effects of non-steroidal anti-inflammatory drugs (NSAIDs) by binding them
to NO donating moieties [24]. These actions of NO could enhance the gastro-sparing features of selective
COX-2 inhibitors and potentially induce peripheral vasodilation to circumvent the elevated blood pressure
exhibited by selective COX-2 inhibitors that decrease the physiological level of PGI2. In this regard, hybrid
selective COX-2 inhibitors possessing a NO-donor moiety have been investigated as a method to increase
the clinical safety of COX-2 inhibitors [25,26].
Throughout this research, both strategies of preferential selectivity towards COX-2 as well as addition of
NO release moiety were combined and novel analogues of 2,3-disubstituted-4-oxo-4H-quinazoline and
their NO-hybrid analogues have been synthesized, evaluated for their anti-inflammatory activity, their
ability to inhibit cyclooxygenase I, II, quantitative structure activity relationship (QSAR) and docking
studies for validation of the observed pharmacological properties of the investigated anti-inflammatory
compounds and for determination of the most important parameters controlling these properties.
2. Rational and design
After the withdrawal of some highly selective COX-2 inhibitors due to their severe cardiovascular
effects, and our knowledge about the class “preferential COX-2 inhibitors” with certain COX-2 specificity,
and a lower incidence of gastric and renal side-effects than associated with classic NSAIDs. This class
contained compounds such as Etodolac, Meloxicam, and Nimesulide, all of which show a preferential
selectivity toward COX-2. Meloxicam showed a lower incidence of gastric and renal side-effects than
associated with classic NSAIDs, and without the cardiovascular side effects of the highly selective COX-2
inhibitors.
Moreover, as previously discussed it was reported that different series of Aspirin-NO and NSAIDs-NO
acquired anti-inflammatory effects devoid of acute gastrotoxicity principally as a consequence of their NO
donor ability.
Also after the work presented by Alagarsamy et al.[27], Giri et al.[28], Alafeefy et al.[29], and Abbas et
al.[30],who tested different series of quinazoline derivatives for their anti-inflammatory derivatives and
showed promising activities.
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These findings led to our thought of combining both strategies of preferential selectiDvOitI:y10t.1o0w39a/Cr4dMsDC00O39X2F-2
as well as addition of NO release moiety to quinazoline derivatives to produce potentially active anti-
inflammatory agents with fewer side effects.
Structure-based design strategies for new scaffolds were based on:
Exploration of the previous revealed SAR studies and bioisosteric modifications of the lead
compound “Meloxicam”
OH
O
N
S
N
H
Meloxicam
N
S
O
O
Target Compounds
O
N
O
N
R
NH
H
H
N
O
N
N
S
N
X= O, S
O
X
R
1-substituted quinazolin-3-yl urea
or thiourea derivatives (IIIa-c)
4-substituted quinazolin-3-yl benzene
sulfonamide derivatives (VIa-c)
Bioisosteric replacement of 4-hydroxybenzthiazine-1,1-dioxide ring with 4-oxoquinazoline
as a planar area to bind to the flat area in the COX receptor.
Maintaining the amide group or its bioisosteric replacement with, thioamide or sulfonamide
groups
The second area of lipophilicity that is noncoplanar with the 4-hydroxybenzthiazine-1,1-
dioxide ring with 4-oxoquinazoline generally enhances activity
2-methyl substituent which is essential for the steric clash that causes the non-coplanarity
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Addition of NO moieties to the target compounds as the reference compounds AsDpOiIr: i1n0.10N39O/C,4MSDu0l0i3n9d2Fac
NO, and Naproxcinod (Naproxen NO)
F
O
O
O
O
O
O
N
O
O
O
O
N+
O
O
S
O
O
O
O-
H
N+
O-
O
O
Aspirin NO
Sulindac NO
Target Compounds
Naproxcinod (Naproxen NO)
O
O
R
H
O
H
N
N
NH
N
S
N
O
X
N
-O
N
O
R
O
N+
O
O
X= O, S
N+
O-
4-substituted-N-(2-nitooxy-4-oxo-4H-
quinazolin-3-yl benzenesulfonamide
derivatives VIIIa-c
1-substituted-3-[(2-nitooxy)-4-oxo-4H-
quinazolin-3-yl]urea and thiourea
derivatives Va-c
3. Results and discussion
3.1. Chemistry
The synthetic routes used to synthesize the four series of 1-substituted-3-(2-methyl-4-oxo-4H-quinazolin-
3-yl) urea and/or thiourea IIIa-c, 4-substituted-N-(2-methyl-4-oxo-4H-quinazolin-3-yl) benzene
sulfonamide VIa-c and their NO-hybrid molecules as nitrate esters Va-c, VIIIa-c are outlined in Scheme 1.
Synthesis of 4-quinazolinone derivatives were carried out through acetylation of anthranilic acid to yield
2-methyl-4-benzoxazinone I [31], followed by reaction with hydrazine hydrate to form 3-amino-2-methyl-
3H-quinazolin-4-one II [32]. Ureido and thioureido quinazolin-4-one derivatives IIIa-c were obtained by
reaction of II with the appropriate isocyanates and/or isothiocyanates in absolute ethanol in the presence of
triethylamine [33]. In addition, aryl sulfonamide derivatives VIa-c were synthesized by treatment of II
with sulphonic acid chloride derivatives in dry pyridine [34-36].
Bromination of III a-c and VIa-c with N-bromosuccinimide in chloroform, in the presence of catalytic
amount of benzoyl peroxide [37] yielded the bromomethyl derivatives IVa-c and VIIa-c. Addition of a
nitric oxide releasing moiety attached to the previously synthesized structures, through reaction of IVa-c
and VIIa-c with silver nitrate in dry acetonitrile to produce nitrate esters [38] Va-c,and VIIIa-c as NO-
hybrid molecules.
The structures of the newly synthesized compounds were confirmed by the spectroscopic techniques such
as IR, 1H-NMR, mass spectra and elemental analysis.
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DOI: 10.1039/C4MD00392F
O
O
O
NH2
b
a
N
OH
O
N
NH2
N
II
d
I
c
Anthranilic acid
O
N
O
R
H
O
H
N
N
NH
N
S
N
O
X
N
R'
III
VI
a R' = H
b R' = CH3
c R' = NHCOCH3
a
b
c
X=O, R = t-butyl
X=O, R= cyclohexyl
X=S, R= phenyl
e
e
O
O
N
R
H
O
H
N
N
NH
N
S
N
O
X
N
R'
Br
Br
IV
VII
a
b
c
X=O, R = t-butyl
X=O, R= cyclohexyl
X=S, R= phenyl
a R' = H
b R'= CH3
c R' = NHCOCH3
f
f
O
R
O
H
N
H
O
NH
N
N
N
S
X
O
N
O
N
O
R'
O
N+
O-
O
N+
O-
V
VIII
a
b
c
X=O, R = t-butyl
X=O, R= cyclohexyl
X=S, R= phenyl
a R' = H
b R' = CH3
c R' = NHCOCH3
Scheme 1. Reagents and conditions: (a) acetic anhydride, reflux, 3hrs; (b) hydrazine hydrate, reflux, 2 hrs; (c) The
appropriate isocyanate or isothiocyanate, absolute ethanol, TEA, reflux, 24 hrs; (d) The appropriate substituted
benzene sulfonyl acid chloride, dry pyridine, 25oC, 72 hrs ; (e) NBS, benzoyl peroxide, CHCl3, reflux, 4 hrs; (f)
AgNO3, dry acetonitrile, 80oC, 24 hrs.
3.2. In-vivo anti-inflammatory activity
The inhibitory activity of the synthesized compounds was evaluated on carrageenan induced rat paw
edema. Inflammation induced by carrageenan is acute, non-immune, well-researched, and highly
reproducible. Cardinal signs of inflammation—edema, hyperalgesia, and erythema—develop immediately
following subcutaneous injection, resulting from action of pro-inflammatory agents. The inflammatory
response is usually quantified by increase in paw size (edema) and is modulated by inhibitors of specific
molecules within the inflammatory cascade [39]. The anti-inflammatory properties of the synthesized
compounds were compared to that of Meloxicam (in a dose 80 mg/kg) as a reference standard of 100%
potency, and the results are displayed in Table 1.
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DOI: 10.1039/C4MD00392F
Table 1. Anti-inflammatory activity of the test compounds assessed in comparison to Meloxicam as reference. Data are
presented as Mean ± SD, and n=6.
3Hr
potency
1 Hr
2 Hr
% Edema
3 Hr
Group
% Edema
Inhibition
Paw Vol.
(ml)
% Edema
Inhibition
Paw Vol. (ml)
Paw Vol. (ml)
Inhibition
0.635 ± 0.050 b
0.821 ± 0.153
0.866 ± 0.085 a
0.826 ± 0.064
0.823 ± 0.075
0.918 ± 0.041 a
0.903 ± 0.083 a
0.880 ± 0.104 a
0.891 ± 0.074 a
0.885 ± 0.124 a
0.841 ± 0.142 a
0.926 ± 0.055 a
0.913 ± 0.122 a
0.831 ± 0.057 a
0.633 ± 0.106 b
0.850 ± 0.118 a,b
0.911 ± 0.131 a,b
0.890 ± 0.051 a,b
0.850 ± 0.044 a,b
0.953 ± 0.048 a,b
0.918 ± 0.095 a,b
0.910 ± 0.084 a,b
0.930 ± 0.094 a,b
0.906 ± 0.067 a,b
0.865 ± 0.042 a,b
1.008 ± 0.058 a
0.950 ± 0.072 a,b
0.833 ± 0.108 a,b
Control
Meloxicam
IIIa
0.660 ± 0.072 b ----
----
----
----
0.825 ± 0.092
0.846 ± 0.108
0.843 ± 0.092
0.838 ± 0.087
0.855 ± 0.112
0.851 ± 0.074
0.855 ± 0.150
0.856 ± 0.086
0.861 ± 0.133
0.825 ± 0.105
0.863 ± 0.090
0.855 ± 0.120
0.836 ± 0.038
20.80
43.43
29.80
41.92
42.93
14.14
18.69
25.76
22.22
24.24
37.37
11.62
15.66
40.40
61.88
51.03
54.84
61.88
43.70
49.85
51.32
47.80
51.91
59.24
34.02
44.28
64.81
100%
10.40
12.00
14.40
6.40
82.47%
88.62%
100%
IIIb
IIIc
Va
70.62%
80.56%
82.93%
77.25%
83.89%
95.73%
54.98%
71.56%
>100%
Vb
8.00
Vc
6.40
VIa
5.60
VIb
3.20
VIc
20.80
2.40
VIIIa
VIIIb
VIIIc
6.40
15.20
a Statistically significant difference from the control group at p < 0.05
b Statistically significant difference from the carrageenan-induced group at p < 0.05
It was found in this time-effect study that after 3 hours, all the tested compounds showed significant
difference from carrageenan group except compound VIIIa. Compound VIIIc showed higher potency than
Meloxicam, while compound IIIc showed 100% potency, and compound VIc showed potency of 95.73%.
In addition, the rest of the compounds showed moderate potency ranging from 88.62 to 70.62%. Only
VIIIa showed about 55% potency.
Results are reported as mean ± S.D. Statistical analysis was performed using one-way analysis of
variance (ANOVA). If the overall F-Value was found statistically significant (P < 0.05), further
comparisons among groups were made according to post hoc Tukey’s test. All statistical analyses were
performed using GraphPad Instat software version 3. (ISI® software, CA, USA).
3.3. In-vitro cyclooxygenase inhibition assay
The ability of the test compounds to inhibit ovine COX-1 and COX-2 was determined using an enzyme
immunoassay (EIA) (kit catalog number 560101, Cayman Chemical, Ann Arbor, MI, USA) according to
the manufacturer’s instructions [40].
Cyclooxygenase catalyzes the first step in the biosynthesis of arachidonic acid (AA) to PGH2. PGF2α,
produced from PGH2 by reduction with stannous chloride, is measured by enzyme immunoassay (ACE™
competitive EIA). Percent inhibition was calculated by the comparison of compound treated to various
control incubations.
The concentrations of the test compounds causing 50% inhibition (IC50, µM) was calculated from the
concentration–inhibition response curve (triplet determinations) and are represented in Table 2 as mean ±
S.D.
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Table 2. Data of the in-vitro COX-1/COX-2 inhibition assay
IC50a, µM (COX-1)
IC50 a, µM (COX-2)
SIb
Test Compound
IIIa
34.53 ± 5.20
23.76 ± 4.37
18.86 ± 3.35
44.6 ± 1.35
6.53 ± 0.65
5.00 ± 0.18
3.67 ± 0.77
12.46 ± 0.60
6.86 ± 0.30
5.93 ± 1.15
8.58 ± 0.85
5.75 ± 1.63
4.41 ± 0.50
42.10 ± 1.96
11.66 ± 1.55
3.59 ± 0.24
1.37 ± 0.30
5.28
4.74
5.13
3.57
5.03
4.11
3.50
5.67
5.16
2.41
3.32
6.72
14.16
IIIb
IIIc
Va
Vb
34.60 ± 3.95
24.40 ± 4.20
30.13 ± 1.49
32.66 ± 8.46
22.76 ± 4.60
98.63 ± 5.48
38.83 ±10.87
24.13 ± 3.70
19.40 ± 0.40
Vc
VIa
VIb
VIc
VIIIa
VIIIb
VIIIc
Meloxicam
a IC50 value is the compound concentration required to produce 50% inhibition of COX-1 or COX-2 for means of three determinations with the
standard deviation.
b Selectivity index (SI) is defined as IC50 (COX-1) / IC50 (COX-2)
From table 2, according to the IC50 of COX-2 data, it was found that thiourea derivatives IIIc and Vc are
more active as anti- inflammatory than cyclohexyl urea derivatives IIIb and Vb, which are more active
than t-butyl urea derivatives IIIa and Va. In addition, p-acetamido benzene sulfonamide derivatives VIc
and VIIIc are more active than p-tolyl derivatives VIb and VIIIb, which are also more active than the
unsubstituted benzene sulfonamide derivatives VIa and VIIIa.
In general, addition of the nitrate ester moieties showed less anti-inflammatory potency than their
corresponding starting intermediates except in case of VIIIc, which was found to be the most active one as
anti-inflammatory.
As for the IC50 of COX-1 data, it was found that the structures carrying nitric oxide moieties Va-c and
VIIIa-c had less affinity to inhibit COX-1 than their corresponding starting intermediates IIIa-c,and VIa-c,
which conclude that the alcoholic metabolites of NO esters had less tendency to inhibit COX-1 leading to
less gastric ulcerative side effects.
3.4. Evaluation of nitric oxide release in serum
Nitric oxide released in serum of rats was determined using Nitric Oxide detection kit (Enzo Life
Sciences, USA, Catalog No. ADI-917-010), for the synthesized structures carrying NO moiety Va-c, and
VIIIa-c, using the same procedure as indicated in the kit. The kit involves the enzymatic conversion of
nitrate to nitrite, by the enzyme Nitrate Reductase, followed by the colorimetric detection of nitrite as a
colored azo dye product of the Griess reaction that absorbs visible light at 540 nm. All the test compounds
carrying a nitrate moiety were assayed through this procedure and the results are shown in Table 3 as mean
± S.D.
Table 3. Nitric oxide amount released from synthesized structures Va-c, VIIIa-c. Data is represented as mean± S.D.
Test Compound
Nitric oxide (µmol/L)
Va
Vb
Vc
VIIIa
VIIIb
VIIIc
10.76 ± 0.51
28.53 ± 1.40
22.80 ± 2.12
16.73 ± 1.56
10.40 ± 0.81
7.90 ± 0.72
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From table 3, it was found that the nitric oxide ester of 3-cyclohexylureido quinazoline derivative Vb
released the highest concentration of nitric oxide in serum 28.53 µmol/L, and the phenylthioureido
derivative Vc released 22.8 µmol/L, while compound VIIIc (p-acetamido benzene sulfonamide derivative)
release the least amount of NO in serum equals to 7.9 µmol/L.
These results were found to correlate with the ability of nitric oxide to decrease the ulcerations produced
by these test compounds, this ability was discussed later through the calculated ulcer index.
3.5. Gastric protective investigation
2.5.1. Assessment of gastric mucosal ulcerogenicity
The ulcerogenic effect of the tested compounds Va-c, and VIIIa-c is presented together with the
reference compounds Indomethacin (the known ulcerative drug) and Meloxicam after 3 successive daily
doses as discussed later in the experimental section. Severity of ulcers is determined by giving scores 0.0-
4.0, where 0.0, normal (no injury, bleeding and latent injury); 0.5, latent injury or widespread bleeding; 1.0,
slight injury (2 to 3 dotted lines); 2.0, severe injury (5-6 dotted injuries); 3.0, very severe injury (several
continuous lined injuries) and 4.0, widespread lined injury or widened injury [16]. The partial scores were
then summed to obtain the ulcer index for the examined animal. The ulcer index (UI) for each group was
taken as the mean lesion score for all rats in those groups and represented in Figure 1 and Table 4 as mean
± S.D.
Table 4. Ulcerogenic effects (Ulcer Index) of Indomethacin,
Meloxicam and tested compounds (Va-c, VIIIa-c) in
comparison to the control group. Data is represented as mean
± S.D.
Compound
Ulcer Index (UI)
Control
Indomethacin
Meloxicam
Va
Vb
Vc
VIIIa
VIIIb
VIIIc
0.2 ± 0.01
1.8 ± 0.2
0.6 ± 0.1
0.9 ± 0.2
0.4 ± 0.02
0.6 ± 0.1
0.5 ± 0.05
1.0 ± 0.2
1.3 ± 0.1
Figure 1. Ulcerogenic effects (ulcer index) of
Indomethacin, Meloxicam and tested compounds
(Va-c, VIIIa-c) in comparison to the control group
From table 4, and figure 1, it was revealed that compounds Vb,Vc had the least ulcer index of 0.4, and
0.6, respectively, while compound VIIIc had the highest ulcer index of 1.3.
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2.5.2. Histopathological investigation
The histological slides were prepared as discussed in the experimental section. The slides obVtiaewinAertidcle fOrnolinme
DOI: 10.1039/C4MD00392F
the control group showed no lesions and characterized by continuous mucosal layer as shown in Figure 2-
A. The slide of Indomethacin in Figure 2-B showed greater irregular mucosa with ulcers of approximately
higher number and severity with UI value of 1.8, while Meloxicam showed ulcers with lower severity with
UI value of 0.6 as shown in Figure 2-C. The slide of the test compound Va showed mild erosion with UI
value of 0.9 in Figure 3-A, and compound Vb showed intact mucosa with the least UI of 0.4 as shown in
Figure 3-B, and compound Vc showed minimal ulceration of UI 0.6 as shown in Figure 3-C. Figure 4
showed mild erosion after using the test compounds VIIIb, and VIIIc with UI 1.0 and 1.3, respectively,
while showed minimal ulceration with VIIIa with UI value 0.5.
Figure 2. A] Fundic gastric mucosa of a control rat showing normally looking fundic glands. B] And C] showing
gastric mucosal ulcerations after using Indomethacin and Meloxicam drugs consecutively, H & E x10
Figure 3. Fundic gastric mucosa of rats groups after using the newly synthesized derivatives Va, Vb, and Vc
consecutively; A] mild erosion, B] intact mucosa without ulceration, C] minimal ulceration. H & E x10.
Figure 4. Fundic gastric mucosa of rats groups after using the newly synthesized derivatives VIIIa, VIIIb, and
VIIIc consecutively; A] minimal ulceration, B] and C] mild erosion. H & E x10.
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3.6. Molecular modeling studies
2.6.1. Pharmacophore modeling
A pharmacophore is the ensemble of steric and electronic features necessary to ensure the optimal
supramolecular interactions with a specific biological target, and to trigger or to block its biological
response. Typical pharmacophore features indicate regions in a molecule that are hydrophobic, aromatic,
can participate in hydrogen bonding (whether hydrogen bond donor or hydrogen bond acceptor), positive or
negative ionizable, which permits pharmacophore generation; structural alignment; activity prediction and
3D database creation. It uses the Catalyst HypoGen algorithm [41] to derive SAR hypothesis models
(pharmacophores) from a set of ligands with known activity values on a given biological target.
In this study, the 3D QSAR Pharmacophore Generation protocol (HypoGen protocol of CATALYST) was
used using Discovery Studio 2.5 software to generate ten predictive pharmacophore models via aligning
different conformations in which the molecules are likely to bind with the receptor pharmacophore models.
The given hypothesis was combined with a known activity data to create a 3D-QSAR model that identifies
overall aspects of molecular structure governing activity. Pharmacophores explain the variability of
bioactivity with respect to the geometric localization of the chemical features present in the molecules.
During hypothesis generation, the structure and activity correlations in the training set were rigorously
examined. HypoGen identifies features common to the active compounds and excludes features common to
the inactive compounds within conformationally allowable regions of space. It further estimates the activity
of our newly synthesized and tested compounds using regression parameters.
The parameters were computed by regression analysis using the relationship of geometric fit value versus
the activity. The better the geometric fit the greater the activity prediction of the compound. The fit function
checks if the feature is mapped. It also contains a distance term, which measures the distance separating the
feature on the molecule from the centroid of the feature in the pharmacophore hypothesis. Both terms are
used to calculate the geometric fit value.
Pharmacophore validation
In general, pharmacophore models are used as 3D queries to search chemical databases to identify new
and highly potent drug leads. These pharmacophore models should be statistically significant, able to predict
the activities of new chemical compounds and retrieve active compounds from the database. The selected
pharmacophore model was validated using four methods; cost analysis, activity prediction, Fischer
validation test, and mapping of Meloxicam (the conformation used was the co-crystallized Meloxicam with
COX-2, pdb code 4M11) as a reported external reference to the generated pharmacophore.
HypoGen selects the best hypotheses by applying a cost analysis. The overall cost of each hypothesis is
calculated by summing three cost factors - a weight cost, an error cost, and a configuration cost. HypoGen
also calculates two theoretical costs, the null and fixed costs that can be used to determine the significance
of the selected hypotheses. The cost values of the optimized hypotheses should lie somewhere between
these two costs. A larger difference between the fixed and null costs than that between the fixed and total
costs signifies the quality of a pharmacophore model.
The closer the cost value to the fixed cost and the further away it is from the null cost, the more
statistically significant the hypothesis is believed to be.
Fischer validation is another approach for pharmacophore model validation. This validation method
checks the correlation between the chemical structures and biological activity. This method generates
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pharmacophore hypotheses using the same parameters as those used to develop the original pharmacophore
hypothesis by randomizing the activity data of the training set compounds.
Further validation for the pharmacophore model generated was performed through mapping of Meloxicam
(the conformation used was the co-crystallized Meloxicam with COX-2, pdb code 4M11), using ligand
pharmacophore mapping protocol, and comparing its fit value with the tested compounds.
Pharmacophore study results
In this study, pharmacophore models were generated using hydrogen bond acceptor (HBA), hydrogen
bond donor (HBD), hydrophobic (HYP), positive ionizable (PosIon) and ring aromatic (RA) features, and
nine pharmacophore models were exported for further studies. All of the generated pharmacophore models
contained at least four chemical features. The best generated pharmacophore contained 2 HBA features, 1
HBD, and 1 HYP. The constraint distances and angles between the different features of the generated top
pharmacophore are presented in Table 5 and Figure 5.
Table 5. Constraint distances and angles between features of the generated top pharmacophore model.
Constraint distances (Ao)
(HBA1-HBA2): 5.23; (HBA1-
HBD): 5.08; (HBA1-HYP):
Constraint angles ( o)
(HBA1-HBA2-HBD): 68.62;
(HBA2-HBD-HYP): 131.14;
(HYP-HBD-HBA1): 126.29
7.48;
(HBA2-HBD):
3.33;
(HBA2-HYP): 5.99;
HYP): 3.25
(HBD-
Figure 5. Constraint distances and angles between features of the generated top pharmacophore model with the
features considered hydrogen bond donor (HBD) colored in magenta, hydrogen bond acceptors (HBA1 & HBA2)
colored in green, and hydrophobic (HYP) colored in cyan.
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The Fischer validation confidence level chosen is 95%, and the best generated pharmacophore
significance was 90%. The top pharmacophore hypothesis generated was developed with a total cost value
of 53.695, null cost 98.471, and fixed cost 52.535. Further evaluation of the generated pharmacophore
models was based on the correlation coefficient, which was found to be 0.82 that indicate the capability of
the pharmacophore model to predict the activity of the training set compounds.
In addition to cost analysis and Fischer validation, the pharmacophore model was validated through
activity prediction of the synthesized structures as training set. The predicted activities through the
pharmacophore model are represented in Table 6 as well as their fit values.
Moreover Meloxicam (the co-crysallized conformation of Meloxicam with COX-2 in pdb code 4M11)
was mapped on the generated pharmacophore for further validation and its fit value was detected as 5.77.
The best generated pharmacophore fitted with Meloxicam as well as the synthesized compound Vb as
shown in Figure 6. The pharmacophore features (hydrogen bond donor HBD, hydrogen bond acceptors
HBA1 & HBA2, and hydrophobic feature HYP) mapped with the synthesized compounds and Meloxicam,
as well as their fit values are represented in Table 7.
Figure 6. The best generated pharmacophore hypothesis with the features considered hydrogen bond
donor (HBD) colored in magenta, hydrogen bond acceptors (HBA1 and HBA2) colored in green, and
hydrophobic (HYP) colored in cyan, with Meloxicam (A) fitted in the pharmacophore with fit value 5.77,
and the synthesized structure Vb (B) fitted in the pharmacophore with fit value 5.73.
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Table 6. Fit values and predicted activities for the synthesized compounds (IIIa-c,Va-c,VIa-c,VIIIa-c) mapped
with the generated 3D-pharmacophore model.
Compd.
Predicted Experimenta
Fit
activity
(IC50 µM)
5.95
l activity
(IC50 µM)
6.63
5.00
3.67
12.46
6.86
5.93
8.58
5.75
4.41
42.10
11.66
3.59
value
IIIa
IIIb
IIIc
Va
Vb
Vc
VIa
VIb
VIc
5.68
5.69
5.78
5.67
5.73
5.75
5.11
5.53
5.57
5.12
5.62
5.72
5.77
5.76
5.38
6.06
5.23
4.97
21.86
8.26
7.65
21.47
6.75
VIIIa
VIIIb
VIIIc
Meloxicama
3.54
1.19
1.37
aMeloxicam mapping on the generated pharmacophore was used for further validation of the model, its fit value was found to be
5.77.
Table 7. The pharmacophore features (hydrogen bond donor HBD, hydrogen bond acceptors HBA1, HBA2, and
hydrophobic feature HYP mapped with the synthesized compounds and Meloxicam, as well as their fit values.
Fit
value
Cpd
IIIa
IIIb
IIIc
HBD
HBA1
HBA2
HYP
4-oxo group of
quinazoline ring
4-oxo group of
5.68
NH of urea side chain
NH of urea side chain
NH of thiourea side chain
-
-
-
2-methyl group
2-methyl group
5.69
5.78
quinazoline ring
O of the thio group in the
side chain
Phenyl group of side
chain
O of CH2OH
(alcoholic group after
NO release)
O of CH2OH
(alcoholic group after
NO release)
O of CH2OH
(alcoholic group after
NO release)
4-oxo group of
quinazoline ring
5.67
5.73
5.75
NH of urea side chain
NH of urea side chain
NH of thiourea side chain
t-butyl side chain
Cyclohexyl side chain
phenyl side chain
Va
Vb
Vc
4-oxo group of
quinazoline ring
4-oxo group of
quinazoline ring
NH of sulfonamide side
chain
NH of sulfonamide side
chain
NH of sulfonamide side
chain
O of SO2 of sulfonamide
side chain
O of SO2 of sulfonamide
side chain
O of SO2 of sulfonamide
side chain
5.11
5.53
5.57
-
-
2-methyl group
2-methyl group
2-methyl group
VIa
VIb
VIc
O of acetamide side
chain
O of CH2OH
(alcoholic group after
NO release)
NH of sulfonamide side
chain
O of SO2 of sulfonamide
side chain
5.12
5.62
phenyl side chain
VIIIa
VIIIb
O of CH2OH
(alcoholic group after
NO release)
NH of sulfonamide side
chain
O of SO2 of sulfonamide
side chain
Methyl group of tolyl
side chain
O of CH2OH
(alcoholic group after
NO release)
O of acetamide side
chain
Phenyl ring of
quinazoline
5.72
5.77
N1 of quinazoline ring
NH of amide side chain
VIIIc
4-hydroxy group of
benzthiazine ring
O of benzthiazine-
1,1-dioxide
Thiazole ring (side
chain)
Meloxicam
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2.6.2. QSAR study
The QSAR study was performed using Discovery Studio 2.5 Software. The training set was composed of
the twelve synthesized compounds for the present QSAR modeling, while the validation for the QSAR
model employed was leave one-out cross-validation (internal validation), external validation using
Meloxicam, as well as residuals between the predicted and experimental activity of Meloxicam and the
training set. “Calculate Molecular Properties” module was used for calculating the 2D molecular properties
like AlogP, fingerprints, molecular properties, surface area and volume, as well as topological descriptors,
also energies of highest occupied and lowest unoccupied molecular orbitals (HOMO and LUMO) [42] of
the training set compounds were calculated.
Furthermore, the training set compounds were fitted against representative pharmacophores and their fit
values were added as additional descriptors. The fit values of the training set compounds were calculated
automatically using the previously described “3D QSAR Pharmacophore Generation” module.
Partial Least Squares (PLS) model was employed to search for optimal QSAR models that combine high
quality binding pharmacophores with other molecular descriptors and being capable of correlating
bioactivity variation across the used training set collection. The trials were held while changing the
independent properties till the best model with the least variables was obtained.
QSAR model was validated employing leave one-out cross-validation, r2 (squared correlation coefficient
value), and external validation using Meloxicam, as well as residuals between the predicted and
experimental activity of Meloxicam and the training set and the results are displayed in Table 8 [43].
QSAR study results
Equation 1 represents the best performing QSAR model
-logIC50 = 21.492 - 0.030 ALogP - 58.220 Molecular_FractionalPolarSurfaceArea
-logIC50: (the negative logarithmic value of the concentration required to produce 50% inhibition of
cyclooxygenase-2 enzyme).
The scatter plots of the experimental versus the predicted activity values (-log IC50) according to Equation 1
are presented in Figure 7.
Figure 7 shows the corresponding scatter plots of the experimental versus the predicted bioactivity values
-logIC50 for the training set compounds according to Equation 1. (r2 = 0.983).
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Abbreviations used: ALogP is a measure of the hydrophobicity of the molecule; it is calculated in
Discovery Studio as the Log of the octanol-water partition coefficient using Ghose and Crippen's method
[43]; Molecular_FractionalPolarSurfaceArea is the ratio of the polar surface area divided by the total surface
area of the molecule.
QSAR validation
QSAR models were validated employing leave one-out cross-validation (internal validation), calculating
the residuals between experimental activities and those predicted by the QSAR model, as well as calculating
the predicted activity for Meloxicam through running as an external test compound on the constructed
QSAR model using “Calculate Molecular Properties” protocol and selecting the model from the “Other” set.
The regression values were as follows: r=0.991, r2 = 0.983, r2 (adjusted) = 1.023, and Least-squared error =
0.001366. The experimental activities and those predicted by QSAR studies are presented in Table 8.
Table 8. Experimental activity of the synthesized compounds against the predicted activity according to Equation 1a.
Experimental
activity
(-log IC50)
-0.822
Predicted
activity
(-log IC50)
-0.836
-0.631
-0.619
-1.082
-0.904
-0.718
-0.946
-0.760
-0.630
-1.610
-1.067
-0.568
-0.165
Compd.
Residual
0.014
-0.068
0.054
-0.014
0.068
-0.055
0.013
0.000
-0.014
-0.014
0.000
0.013
0.028
IIIa
IIIb
IIIc
Va
Vb
Vc
VIa
VIb
VIc
-0.699
-0.565
-1.096
-0.836
-0.773
-0.933
-0.760
-0.644
-1.624
-1.067
-0.555
-0.137
VIIIa
VIIIb
VIIIc
Meloxicama
aMeloxicam was used for external validation through calculating its predicted activity from the QSAR model constructed
using the 12 synthesized compounds.
It should be noted that the predicated anti-inflammatory activities by our QSAR models were very close
to those experimentally observed, indicating that these models can be safely applied for prediction of more
effective hits having the same skeletal framework.
2.6.3. Docking Study
The docking study was performed using Dock Ligands (CDOCKER) protocol which is one of the
Receptor-Ligand Interactions protocols in Acclerys Discovery Studio 2.5. CDOCKER is a grid-based
molecular docking method that employs CHARMm. CDOCKER score was reported as the negative value
(i.e., -CDOCKER_ENERGY), where a higher value indicates a more favorable binding. This score
includes internal ligand strain energy and receptor-ligand interaction energy, and is used to sort the poses
of each input ligand.
The docking protocol was chosen and validated after comparing different results from different docking
protocols with the co-crystallized Meloxicam in COX-2 (pdb code 4M11), and COX-1 (pdb code 4O1Z),
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through alignment of the docked Meloxicam with the co-crystallized one and calculating the RMSD value
which was 0.97, as well as comparing the binding interactions formed with the receptor’s amino acids.
Docking methodology
The synthesized compounds as well as Meloxicam, were exported to Discovery Studio 2.5, prepared to
be docked, using Prepare Ligands protocol. The Prepare Ligands protocol helps to prepare ligands for input
into other protocols, performing tasks such as removing duplicates, enumerating isomers and tautomers,
and generating 3D conformations.
The pdb codes used for COX-2 is (4M11), and for COX-1 is (4O1Z) loaded from the Protein Data Bank.
These proteins were optimized and prepared through a Prepare Protein protocol, which is one of the
general purpose protocols. The Prepare Protein protocol prepares proteins for input into other protocols,
performing tasks such as inserting missing atoms in incomplete residues, modeling missing loop regions,
deleting alternate conformations, removing un-needed waters, standardize atom names, and protonating
titratable residues using predicted pKs.
Docking results
The docking results are presented in Table 9 and Figures 8 –11. It was found that the results of docking
and 3D pharmacophore studies were coincide, by comparing the results shown in Table 7 and Table 9, we
found that most of the synthesized compounds and Meloxicam interact with the same binding orientations
inside COX-1 and COX-2 active sites, as well as fitting with the pharmacophore features of the best
generated pharmacophore.
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Table 9. Docking results for the synthesized compounds as well as Meloxicam in the binding sites of COX-2 (4M11) and COX-
1 (4O1Z), showing the H-bonds formed with the binding site residues.
COX-2 (pdb code 4M11)
COX-1 (pdb code 4O1Z)
Test
Compound
-CDOCKER
-CDOCKER
H-bonds
H-bonds
energy
energy
2 H-bonds
1 H-bond
Ser530: 4-hydroxy group of
benzthiazine ring
Ser530: 4-hydroxy group of
benzthiazine ring, Arg120: O of
benzthiazine-1,1-dioxide
1 H-bond
25.08
18.35
Meloxicam
1 H-bond
29.76
33.93
38.65
26.79
29.29
Ser530: 4-oxo group of quinazoline
ring
1 H-bond
Ser530: 4-oxo group of quinazoline
ring
1 H-bond
Ser530: 4-oxo group of quinazoline
ring
1 H-bond
Ser530: O of CH2OH (alcoholic
group after NO release)
1 H-bond
Ser530: 4-oxo group of quinazoline
ring
23.81
24.59
29.98
18.24
20.99
Ser530: 4-oxo group of quinazoline
ring
IIIa
IIIb
IIIc
Va
No H-bonds formed
1 H-bond
Ser530: thiourea side chain
1 H-bond
Arg120: O of CH2OH (alcoholic
group after NO release)
1 H-bond
Ser530: 4-oxo group of quinazoline
ring
Vb
2 H-bonds
Ser530: 4-oxo group of quinazoline
ring, Arg120: O of CH2OH
(alcoholic group after the release of
NO)
1 H-bond
Ser530: 4-oxo group of quinazoline
ring
30.02
25.87
Vc
1 H-bond
1 H-bond
29.24
33.34
38.33
26.54
Ser530: 4-oxo group of quinazoline
ring
1 H-bond
Arg120 and O of sulfonamide side
chain
2 H-bonds
Arg120: N & O of sulfonamide side
chain
2 H-bonds
Arg120: O of CH2OH (alcoholic
group after NO release)
2 H-bonds
Arg120: O of CH2OH (alcoholic
group after NO release); and O of
sulfonamide side chain
2 H-bonds
19.18
24.07
24.99
18.24
Arg120 and O of sulfonamide side
chain
VIa
VIb
-
1 H-bond
Arg120 and O of sulfonamide side
chain
1 H-bond
Arg120: O of CH2OH (alcoholic
group after NO release)
VIc
VIIIa
1 H-bond
Arg120: O of CH2OH (alcoholic
group after NO release)
27.72
38.97
19.16
30.70
VIIIb
VIIIc
1 H-bond
Arg120: O of CH2OH (alcoholic
group after NO release)
Arg120: O of CH2OH (alcoholic
group after NO release), Tyr385: O
of acetamido side chain
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Figure 8. Docking view of Meloxicam in the binding site of COX-2, forming 2 H-bonds between 4-
hydroxy group of the benzthiazine ring and Ser530 with distance 2.26 Ao as well as between O of
benzthiazine-1,1-dioxide and Arg120 with distance 2.49 Ao
Figure 9. Docking view of Vb in the binding site of COX-2, forming 1 H-bond between 4-oxo group of
the quinazoline ring and Ser530 with distance 2.45 Ao.
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Figure 10. Docking view of Meloxicam in the binding site of COX-1, forming 1 H-bond between 4-
hydroxy group of the benzthiazine ring and Ser530 with distance 2.26 Ao
Figure 11. Docking view of Vb in the binding site of COX-1, forming 1 H-bond between 4-oxo group of
the quinazoline ring and Ser530 with distance 2.46 Ao.
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4. Conclusion
Throughout this research, both strategies of preferential selectivity towards COX-2 as well as addition of
NO release moiety were combined to quinazoline derivatives to produce potentially active anti-
inflammatory agents with fewer side effects.
All the tested compounds showed potent to moderate anti-inflammatory activity where all the tested
compounds showed significant difference from carrageenan group except compound VIIIa. Compound
IIIb showed 86.62% potency, and its nitrate ester Vb showed 80.56% potency
According to the IC50 of COX-2 data, IIIb showed IC50= 5 µM, and its nitrate ester Vb showed IC50= 6.86
µM with selectivity index of 4.74 and 5.03, respectively.
As for the IC50 of COX-1 data, it was found that the structures carrying nitric oxide moieties had less
affinity to inhibit COX-1 than their corresponding starting intermediates, as in the case of compound IIIb
that showed IC50= 23.76 µM, and its nitrate ester Vb that showed IC50= 34.6 µM which conclude that the
alcoholic metabolites of NO esters had less tendency to inhibit COX-1 leading to less gastric ulcerative side
effects.
These findings were further empathized through measuring the amount of nitric oxide released and their
ulcer index, the organic nitrate analogue Vb exhibits the highest ability to release nitric oxide of 28.53
µmol/L, and the least ulcer index of 0.4, showing the least tendency to induce stomach ulcerations in rats.
These results were found to correlate with the ability of nitric oxide to decrease the ulcerations produced by
these test compounds.
Molecular modeling studies, including generation of 3D-pharmacophore model, Partial Least Squares
(PLS) model to search for optimal 2D-QSAR model, and docking into cyclooxygenase (COX-2, and COX-
1) active sites were carried out. These studies suggested the same binding orientation inside the COX
binding pockets for these analogues compared to Meloxicam, and these studies showed high correlation
with the experimental results. The predicated anti-inflammatory activities by our QSAR models were very
close to those experimentally observed, indicating that these models can be safely applied for prediction of
more effective hits having the same skeletal framework.
According to these results, we can conclude that quinazoline derivatives with nitric oxide release moiety
seem potentially attractive as preferential COX-2 inhibitors with fewer gastric side effects.
5. Experimental
5.1. Materials and instrumentation
Starting materials and reagents were purchased from Sigma-Aldrich and Merck and used without further
purification. Melting points were recorded on Stuart Scientific apparatus and were uncorrected. Reactions
were monitored using thin layer chromatography (TLC), performed on 0.255 mm silica gel plates, with
visualization under U.V. light (254 nm). FT-IR spectra were recorded on a 4100 Jasco spectrophotometer
using KBr disc in the Microanalytical center, Cairo University. 1H NMR spectra were recorded in δ scale
given in ppm on Varian Mercury VX-300 NMR spectrometer on (DMSO-d6) in NMR Lab, Chemistry
Department, Faculty of Science, Cairo University. Coupling patterns are described as follows: s, singlet; d,
doublet; m, multiplet; and 1H, 2H, 3H, etc. MS spectra mass were recorded on Shimadzu GCMS-QP 1000
EX gas chromatograph mass spectrometer in the Microanalytical center, Cairo University. Elemental
analyses were performed at the Microanalytical Center, Cairo University and Azhar University, Cairo,
Egypt.
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5.2. Chemistry
2-methyl-4H-benzo[1,3]oxazin-4-one (I)
A mixture of anthranilic acid (2.74 g, 20 mmol) and acetic anhydride (40 mmol) was refluxed for 3 hours.
Excess acetic anhydride was evaporated under vacuum. The collected solid was washed with petroleum
ether. The produced solid was fluffy buff powder having yield (85%), m.p.= 78-80 °C as reported [31]
3-amino-2-methyl-3H-quinazolin-4-one (II)
Compound I (2.3 g, 8 mmol) was rapidly refluxed with hydrazine hydrate 100% (16 mmol) for 2 hours.
Excess hydrazine hydrate was evaporated under vacuum then the collected solid was washed with dilute
HCl. The collected white solid was filtered and recrystallized from ethanol. Yield (93%) and m.p.= 140-
142 °C as reported [32].
1-substituted-3-(2-methyl)-4-oxo-4H-quinazolin-3-yl) urea and thiourea derivatives (III a-c)
General procedure:
A mixture of II (1.55 g, 5 mmol) and the appropriate substituted isocyanate or isothiocyanate (viz: tert-
butyl isocyanate, cyclohexyl isocyanate and phenyl isothiocyanate) (6 mmol) in absolute ethanol and in
presence of triethylamine (0.5 ml) was refluxed for 24 hours. The reaction mixture was concentrated under
vacuum and then poured on ice-cold water acidified with HCl and stirred for 30 minutes. The precipitate
was filtered and the resultant solid was crystallized from ethanol.
1-tert-butyl-3-(2-methyl-4-oxo-4H-quinazolin-3-yl) urea (IIIa)
Yield 70 %, m.p.=133-135 oC. FT-IR: (KBr, cm-1): 3305, 3208 (NH), 3100 (CH arom.), 2969, 2928 (CH
aliph.), 1667 (C=O), 1602 (NH bending). 1H-NMR (300MHz) (DMSO-d6) δ: 1.198 (s, 9H, t-butyl), 2.580
(s, 3H, CH3), 5.794 (s, 1H, NH, exchangeable by D2O), 7.750-7.807 (m, 1H, H8 quinazoline), 7.453-7.507
(m, 1H, H7 quinazoline)7.581-7.608 (d, 1H, H6 quinazoline, J =8.1 Hz), 8.088-8.114 (d, 1H, H5
quinazoline, J =7.8 Hz), 11.021 (s, 1H, NH, exchangeable by D2O). MS: m/z (%): 275.10 (M+, 30.13%) &
base peak at 175 (100%). Anal. Calcd. for C14H18N4O2: C 61.30, H 6.61, N 20.42; Found: C 61.36, H 6.64,
N 20.57.
1-cyclohexyl-3-(2-methyl-4-oxo-4H-quinazolin-3-yl) urea (IIIb)
Yield 75 %, m.p.=183-185 oC. FT-IR: (KBr, cm-1): 3422 (NH), 3160 (CH arom.), 3031, 2974 (CH aliph.),
1
1683 (C=O) 1611 (NH bending). H-NMR (300MHz) (DMSO-d6) δ: 0.90 (s, 3H, CH3), 1.142-1.329 (m,
10H, cyclohexyl), 4.404 (s, 1H, cyclohexyl), 6.928 (s, 1H, NH, exchangeable by D2O), 7.500-7.623 (m,
2H, H7,H8 quinazoline), 7.930-7.935 (d, 1H, H6 quinazoline), 8.103-8.108 (d, 1H, H5 quinazoline), 11.032
(s, 1H, NH, exchangeable by D2O). MS: m/z (%): 300.00 (M+, 24.93%), 301.00 (M++1, 5.19%) & base
peak at 174.95 (100%). Anal. Calcd. for C16H20N4O2: C 63.98, H 6.71, N 18.65; Found: C 63.81, H 6.89, N
20.02.
1-(2-methyl-4-oxo-4H-quinazolin-3-yl-3-phenylthiourea (IIIc)
Yield (80%) and m.p.= 193-195 °C as reported [45]. MS: m/z (%): 309.85 (M+-1, 14.22%), 310.85 (M+,
43.91%) & base peak at 76.95 (100%).
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1-(2-bromomethyl-4-oxo-4H-quinazolin-3-yl-3- urea and thiourea derivatives (IV a-c):
General procedure:
A mixture of the IIIa-c (6.27mmol), N-bromo succinimide (1.15g, 6.27mmol), and benzoyl peroxide
(0.11g, 0.44mmol) in chloroform (40mL) was refluxed for 4hr, cooled to 40°C, and then stirred for 48
hours at 40°C. Excess N-bromo succinimide (1.15g, 6.27mmol), and benzoyl peroxide (0.11g, 0.44mmol)
were added after 24 hours. The reaction was filtered. The filtrate was evaporated in vacuo to afford the
crude product which was washed with petroleum ether then diethyl ether then used without further
purification. However, the product crystallized from ethanol to give the brominated derivative.
1-(2-bromomethyl-4-oxo-4H-quinazolin-3-yl)-3-tert-butylurea (IVa)
Yield (50%) and m.p.= 163-165 °C. FT-IR: (KBr, cm-1): 3432, 3298 (NH), 3199 (CH arom.), 3052, 2982,
1
2924 (CH aliph.), 1668 (C=O). H-NMR (300MHz) (DMSO-d6) δ: 1.198 (s, 9H, t-butyl), 3.424 (s, 2H,
CH2-Br), 6.929 (s, 1H, NH, exchangeable by D2O), 7.662-8.231 (m, 4H, quinazoline), 11.021 (s, 1H, NH,
exchangeable by D2O). MS: m/z (%): 353.10 (M+, 17.21%), 355.10 (M++2, 17.19%) & base peak at 63.95
(100%). Anal. Calcd. for C14H17BrN4O2: C 47.61, H 4.85, N 15.86; Found: C 47.66, H 5.15, N 15.95.
1-(2-bromomethyl-4-oxo-4H-quinazolin-3-yl) -3- cyclohexylurea (IVb)
Yield (50%) and m.p.= 228-230 °C. FT-IR: (KBr, cm-1): 3432 (NH), 3170 (CH arom.), 3039, 2978 (CH
aliph.), 1693 (C=O) 1613 (NH bending). 1H-NMR (300MHz) (DMSO-d6) δ: 1.142-1.329 (m, 10H,
cyclohexyl), 3.451 (s, 2H, CH2-Br), 4.552 (s, 1H, cyclohexyl), 6.828 (s, 1H, NH, exchangeable by D2O),
7.518-8.103 (m, 4H, quinazoline), 11.032 (s, 1H, NH, exchangeable by D2O). MS: m/z (%):377.70 (M+-1,
0.05%), 378.70 (M+, 15.23%), 380.70 (M++2, 15.22%) & base peak at 132.00 (100%). Anal. Calcd. for
C16H19BrN4O2: C 50.67, H 5.05, N 14.77; Found: C 50.74, H 5.12, N 14.89.
1-(2-bromomethyl-4-oxo-4H-quinazolin-3-yl)-3-phenyl thiourea (IVc)
Yield (50%) and m.p.= 213-215 °C. FT-IR: (KBr, cm-1): 3432 (NH), 3162 (CH arom.), 3073 (CH aliph.),
1701 (C=O), 1293 (C=S). 1H-NMR (300MHz) (DMSO-d6) δ: 3.576 (s, 2H, CH2-Br), 7.126-7.641 (m, 5H,
H2´,3´,4´,5’,6´ phenyl), 7.587-7.592 (d, 1H, H7 quinazoline), 7.922-7.927 (d, 1H, H8 quinazoline), 7.946-7.950
(d, 1H, H6 quinazoline), 7.952-7.955 (d, 1H, H5 quinazoline, J =9 Hz), 11.020 (s, 1H, NH, exchangeable by
D2O). MS: m/z (%): 389.1 (M+, 18.46%), 391.1 (M++2, 18.44%) & base peak at 239.1 (100%). Anal.
Calcd. for C16H13BrN4OS: C 49.37, H 3.37, N 14.39; Found: C 49.60, H 3.28, N 9.49.
1-substituted-3-(2-nitrooxy)-4-oxo-4H-quinazolin-3-yl) urea and thiourea derivatives (Va-c)
General procedure:
A mixture of the brominated derivative IVa-c (3 mmol), and AgNO3 (2.03 g, 12 mmol) was heated at
o
reflux in 15 mL dry acetonitrile overnight at 80 C. The inorganic solid was filtered and discarded. The
filtrate was evaporated till dryness, and the residue was washed with diethyl ether and then crystallized
from absolute ethanol.
1-(2-nitrooxy)-4-oxo-4H-quinazolin-3-yl)-3-tert-butylurea (Va)
Yield (60%) and m.p.= 148-150 °C. FT-IR: (KBr, cm-1): 3440 (NH), 3162 (CH arom.), 2938 (CH aliph.),
1
1700 (C=O), 1375 (N-O). H-NMR (300MHz) (DMSO-d6) δ: 1.068 (s, 9H, t-butyl), 3.921 (s, 2H, CH2-
ONO2), 5.697 (s, 1H, NH, exchangeable by D2O), 7.461-8.148 (m, 4H, quinazoline), 11.021 (s, 1H, NH,
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exchangeable by D2O). MS: m/z (%): 337.90 (M++2, 21.70%) & base peak at 54.80 (100%). Anal. Calcd.
for C14H17N5O5: C 50.15, H 5.11, N 20.89; Found: C 50.22, H 5.13, N 21.04.
1-cyclohexyl-3-(2-nitrooxy)-4-oxo-4H-quinazolin-3-yl) urea (Vb)
Yield (60%) and m.p.= 203-205 °C. FT-IR: (KBr, cm-1): 3414 (NH), 3157 (CH arom.), 3074, 2938 (CH
1
aliph.), 1700 (C=O), 1362 (N-O). H-NMR (300MHz) (DMSO-d6) δ: 1.142-1.329 (m, 10H, cyclohexyl),
4.039 (s, 2H, CH2-ONO2), 4.362 (s, 1H, cyclohexyl), 6.929 (s, 1H, NH, exchangeable by D2O), 7.518-
7.680 (m, 2H, H7,H8 quinazoline), 7.829-7.834 (d, 1H, H6 quinazoline), 8.103-8.108 (d, 1H, H5
quinazoline), 11.032 (s, 1H, NH, exchangeable by D2O). MS: m/z (%): 358.80 (M+-2, 18.90%) & base
peak at 56.01 (100%). Anal. Calcd. for C16H19N5O5: C 53.18, H 5.30, N 19.38; Found: C 53.23, H 5.28, N
19.51.
1-(2-nitrooxy)-4-oxo-4H-quinazolin-3-yl)-3-phenyl thiourea (Vc)
Yield (60%) and m.p.= 203-205 °C. FT-IR: (KBr, cm-1): 3419 (NH), 3157 (CH arom.), 3072 (CH aliph.),
1
1700 (C=O), 1374 (N-O), 1293 (C=S). H-NMR (300MHz) (DMSO-d6) δ: 3.777 (s, 2H, CH2-ONO2),
7.137-7.510 (m, 5H, H2´,3´,4’,5´,6´ phenyl), 7.484 (s, 1H, H7 quinazoline), 7.578-7.582 (d, 1H, H8
quinazoline), 7.899-7.903 (d, 1H, H6 quinazoline), 7.927-7.931 (d, 1H, H5 quinazoline), 11.020 (s, 1H, NH,
exchangeable by D2O). MS: m/z (%): 370.75 (M+, 22.21%) & base peak at 63.90 (100%). Anal. Calcd. for
C16H13N5O4S: C 51.75, H 3.53, N 18.86; Found: C 51.83, H 3.55, N 20.02.
4-substituted-N-(2-methyl-4-oxo-4H-quinazolin-3-yl) benzenesulfonamide (VIa-c)
General procedure:
A mixture of II (1.55 g, 5 mmol) and the appropriate substituted benzene sulfonyl acid chloride (6 mmol)
(viz: benzene sulfonyl chloride, p-methylbenzene sulfonyl chloride and p-acetamidobenzene sulfonyl
chloride) in dry pyridine, was stirred for 72 hours. The reaction mixture was concentrated under vacuum
and then poured on ice-cold water acidified with HCl and stirred for 30 minutes. The precipitate was
filtered and the resultant solid was crystallized from ethanol.
N-(2-methyl-4-oxo-4H-quinazolin-3-yl) benzenesulfonamide (VIa)
Yield (70%) and m.p.= 160-162 °C as reported [46]. MS: m/z (%): 314.90 (M+-1, 2.42%), 315.85 (M+,
30.50%), 316.80 (M++1, 15.18%) & base peak at 76.95 (100%).
4-methyl-N-(2-methyl-4-oxo-4H-quinazolin-3-yl) benzenesulfonamide (VIb)
Yield (70%) and m.p.= 202-204 °C as reported [47] . MS: m/z (%): 329.90 (M+, 40.05%), 330.90 (M++1,
10.02%) & base peak at 174.95 (100%).
N-[4-(2-methyl-4-oxo-4H-quinazolin-3-ylsulfamoyl)-phenyl] acetamide (VIc)
Yield (70%) and m.p.= 238-240 °C as reported [47]. MS: m/z (%): 371.20 (M+, 29.01%) & base peak at
78.95 (100%).
4-substituted-N-(2-bromomethyl-4-oxo-4H-quinazolin-3-yl) benzenesulfonamide (VII a-c)
General procedure:
As discussed under the synthesis of compounds IVa-c.
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N-(2-bromomethyl-4-oxo-4H-quinazolin-3-yl) benzenesulfonamide (VIIa)
Yield (50%) and m.p.= 188-190 °C. FT-IR: (KBr, cm-1): 3424, 3298 (NH), 3168 (CH arom.), 3062, (CH
1
aliph.), 1757 (C=O), 1225 (S=O). H-NMR (300MHz) (DMSO-d6) δ: 3.140 (s, 2H, CH2-Br), 7.471-8.167
(m, 9H, aromatic), 11.021 (s, 1H, NH exchangeable by D2O). MS: m/z (%): 392.90 (M+, 13.99%), 394.90
(M++2, 13.97%) & base peak at 63.90 (100%). Anal. Calcd. for C15H12BrN3O3S: C 45.70, H 3.07, N 10.66;
Found: C 45.77, H 3.11, N 10.78.
N-(2-bromomethyl-4-oxo-4H-quinazolin-3-yl)methylbenzenesulfonamide (VIIb)
Yield (50%) and m.p.= 233-235 °C. FT-IR: (KBr, cm-1): 3384 (NH), 3156 (CH arom.), 3077 (CH aliph.),
1704 (C=O), 1293 (S=O). 1H-NMR (300MHz) (DMSO-d6) δ: 2.284 (s, 3H, tolyl CH3), 3.469 (s, 2H, CH2-
Br), 6.940-8.126 (m, 8H, quinazoline), 11.021 (s, 1H, NH, exchangeable by D2O). MS: m/z (%):407.80
(M+-1, 7.96%), 408.80 (M+, 8.20%), 410.80 (M++2, 8.19%) & base peak at 79.00 (100%). Anal. Calcd. for
C16H14BrN3O3S: C 47.07, H 3.46, N 10.29; Found: C 46.91, H 4.15, N 7.11.
N-[4-(2-bromomethyl-4-oxo-4H-quinazolin-3-ylsulfamoyl) –phenyl]acetamide (VIIc)
Yield (50%) and m.p.= 263-265 °C. FT-IR: (KBr, cm-1): 3452 (NH), 3161 (CH arom.), 3080, 2955 (CH
1
aliph.), 1773, 1694 (C=O), 1294 (O=S=O). H-NMR (300MHz) (DMSO-d6) δ: 1.904 (s, 3H, acetamido
CH3), 3.447 (s, 2H, CH2-Br), 7.471-7.951 (m, 8H, aromatic), 11.031 (s, 1H, NH, exchangeable by D2O).
MS: m/z (%): 451.80 (M+, 40.10%), 453.80 (M++2, 40.00%) & base peak at 54.90 (100%). Anal. Calcd.
for C17H15BrN4O4S: C 45.24, H 3.35, N 12.41; Found: C 45.64, H 3.19, N 12.45.
4-substituted-N-(2-nitrooxy-4-oxo-4H-quinazolin-3-yl) benzenesulfonamide (VIII a-c)
General procedure:
As discussed under the synthesis of compounds Va-c.
N-(2-nitrooxy-4-oxo-4H-quinazolin-3-yl) benzenesulfonamide (VIIIa)
Yield (60%) and m.p.= 178-180 °C. FT-IR: (KBr, cm-1): 3437 (NH), 3160 (CH arom.), 3076, 2958 (CH
1
aliph.), 1698 (C=O), 1364 (N-O), 1294 (S=O). H-NMR (300MHz) (DMSO-d6) δ: 3.763 (s, 2H, CH2-
ONO2), 7.313-7.951 (m, 9H, aromatic), 11.021 (s, 1H, NH exchangeable by D2O). MS: m/z (%): 375.50
(M+-1, 2.30%), 376.60 (M+, 14.70%) & base peak at 60.00 (100%). Anal. Calcd. for C15H12N4O6S: C
47.87, H 3.21, N 14.89; Found: C 47.93, H 3.19, N 14.97.
N-(2-nitrooxy-4-oxo-4H-quinazolin-3-yl)-4-methylbenzenesulfonamide (VIIIb)
Yield (60%) and m.p.= 213-215 °C. FT-IR: (KBr, cm-1): 3442 (NH), 3164 (CH arom.), 3076 (CH aliph.),
1702 (C=O), 1372 (N-O), 1293 (S=O). 1H-NMR (300MHz) (DMSO-d6) δ: 2.284 (s, 3H, tolyl CH3), 3.917
(s, 2H, CH2-ONO2), 7.134-8.105 (m, 8H, quinazoline), 11.021 (s, 1H, NH, exchangeable by D2O). MS:
m/z (%): 388.40 (M+-2, 12.20%) & base peak at 160.10 (100%). Anal. Calcd. for C16H14N4O6S: C 49.23, H
3.61, N 14.35; Found: C 49.93, H 3.19, N 14.97.
N-[4-(2-nitrooxy-4-oxo-4H-quinazolin-3-ylsulfamoyl) –phenyl] acetamide (VIIIc)
Yield (60%) and m.p.= 253-255 °C. FT-IR: (KBr, cm-1): 3439 (NH), 3195 (CH arom.), 3074 (CH aliph.),
1
1777, 1698 (C=O), 1370 (N-O), 1293 (O=S=O). H-NMR (300MHz) (DMSO-d6) δ: 2.087 (s, 3H,
acetamido CH3), 3.927 (s, 2H, CH2-ONO2), 7.449-7.925 (m, 8H, aromatic), 11.031 (s, 1H, NH,
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exchangeable by D2O). MS: m/z (%): 432.40 (M+-1, 17.90%) & base peak at 55.00 (100%). Anal. Calcd.
for C17H15N5O7S: C 47.11, H 3.49, N 16.16; Found: C 47.18, H 3.52, N 16.32.
5.3. In-vivo biological evaluation
The experimental tests on animals have been performed in accordance to the guidelines of the Ethical
Committee of the Faculty of Pharmacy, Ain Shams University, for Animal Use.
4.3.1. Anti-inflammatory activity
The inhibitory activities of the synthesized compounds were evaluated using carrageenan induced rat paw
edema model.
Materials and methods
Chemicals
Inflammatory-grade carrageenan was purchased from FMC (Rockland, ME, USA). All other chemicals
were of the highest available commercial grade. All test compounds were dissolved in DMSO before being
injected into the animals.
Animals
The experimental tests on animals have been performed in accordance to the guidelines of the Ethical
Committee of the Faculty of Pharmacy, Ain Shams University, for Animal Use.
Throughout the experiments, adult male Sprague–Dawley rats weighing 180–200 g were used. Animals
were housed at a temperature of 23 ± 2 ºC with free access to water and standard food pellets. Rats were
acclimatized in our animal facility for at least 1 week prior to the experiment.
Measurement of paw volume in carrageenan-induced rat edema model
Rats were equally divided into fifteen groups (6 animals/group) and assigned Latin numbers I–XV.
Animals were fasted, with free access to water, 16 h before the experiment. Groups I and II were given
CMC-Na by intragastric tube. Animals in group III were orally treated with Meloxicam as standard anti-
inflammatory drug (80 mg/kg). Groups (IV to XV) received the test compounds (IIIa-c, Va-c, VIa-c, VIIIa-
c) at oral doses of 80 mg/kg, respectively. Dosing volume was kept constant (10 ml/kg). Thirty minutes
after oral treatment, animals in group I were injected s.c. with 0.05 ml saline, while animals in groups II–
XV were injected s.c. with 0.05 ml carrageenan (1% solution in saline) on the plantar surface of the right
hind paw. The right hind paw volume was measured at 1, 2, and 3 hours after carrageenan injection by
saline displacement using UGO-BASILE 7140 plethysmometer (Comerio, Italy). The measured volumes
were used to calculate % edema inhibition for each compound [39].
4.3.2. Ulcerogenic investigation (Ulcer Index)
Ulcerogenic activity of Meloxicam, Indomethacin and tested compounds were studied in Albino rats of
wistar strain weighing 100–120 g of either sex and divided into nine groups each of five-six animals. The
first group served as control group treated with 0.2 mL DMSO. All experiments were performed at the
same time of the day to avoid variations in the gastric functions.
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Indomethacin was given by a dose of 5 mg/kg/day while the dose of Meloxicam and other tested
compounds was 10 mg/kg/day. All groups were treated for three consecutive days by oral tube. During
three days of dosage treatment, the animals were starved for 18 h but water was provided ad libitum. Food
was allowed 2 hours post administration of the drugs. Two hours following the last doses, rats were
sacrificed.
Each group was divided into two subgroups. For the first subgroup, the stomach of each rat was removed,
opened along the greater curvature, rinsed with 0.9% sodium chloride (isotonic solution) and stretched by
pins on a cork board. The lesions in gastric mucosa were determined by using a magnifying lens(x10).
Hemorrhagic lesions were evaluated by scores: 0.0, Normal (no injury, bleeding and latent injury); 0.5,
Latent injury or widespread bleeding; 1.0, Slight injury (2 to 3 dotted lines); 2.0, Severe injury (5-6 dotted
injuries); 3.0, Very severe injury (several continuous lined injuries) and 4.0, Widespread lined injury or
widened injury [16].
4.3.3. Histopathological investigation
The second subgroup of rats was used in performing this study. The stomachs of rats were opened, fixed
in 10% formaline, longitudinally sectioned, washed with ice-cold saline and scored for macroscopic gross
mucosal lesions [48].
They were routinely processed and embedded in paraffin blocks. 5 µm thick sections were cut on glass
slides and stained with haematoxylin and eosin.
The histological slides were prepared according to the reported procedures [49]. A site of the slide was
identified on which the section was applied by scratching wax around section with a needle. Dewax
hydrated sections by using graded alcohols to water. Slides were stained with haematoxylin for 5–7 min,
washed with tap water until sectioning for 5 min. and immersed for 5–10 seconds in solution of (1% HCl in
70% alcohol), then washed well with tap water for 10–15 min. Followed by staining with 1% Eosin for 10
min, washed with running tap water for 1–5 min. The slide was then dehydrated using alcohols, cleaned by
using xylene, covered by glass cover using Canda balsam then examined under microscope. The slides
were examined by a pathologist who was unaware of the nature of the drug the rat treated with.
5.4. In vitro biological evaluation
4.4.1. Cyclooxygenase inhibition assay
The ability of the test compounds to inhibit ovine COX-1 and COX-2 was determined using an enzyme
immunoassay (EIA) (kit catalog number 560101, Cayman Chemical, Ann Arbor, MI, USA) according to
the manufacturer’s instructions [39].
4.4.2. Evaluation of nitric oxide release in serum
Nitric oxide released in serum of rats is determined using Nitric Oxide detection kit (Enzo Life Sciences,
USA, Catalog No. ADI-917-010) using the same procedure as indicated in the kit. The kit involves the
enzymatic conversion of nitrate to nitrite, by the enzyme Nitrate Reductase, followed by the colorimetric
detection of nitrite as a colored azo dye product of the Griess reaction that absorbs visible light at 540 nm.
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