Design, synthesis, anti-inflammatory, analgesic screening, and molecular docking of some novel 2-pyridyl (3H)-quinazolin-4-one derivatives

Article abstract of DOI:10.1007/s00044-012-0097-8

A novel series of 6-bromo-2-(4-pyridyl)-quinazolin-4(3H)-ones were synthesized by reacting 5-bromo anthranilic acid with isonictinoly chloride in the presence of acetic anhydride, which were further reacted with p-amino acetophenone to obtain 3-(4-acetylphenyl)-6-bromo-2-(pyridin-4-yl)quinazolin- 4(3H)-one (3). Compound 3 underwent further reactions with different aldehydes to afford chalcone derivatives 4-10, which in turn underwent various cyclization reactions to afford cyclized products 15-18. 2-aminothiazole derivatives 12 obtained by reaction of 3 with bromine then with thiourea. Compound 14 obtained by treatment of 11 with KSCN followed by cyclization. Some of the synthesized compounds 4, 5, 12, 14, 15 and 18 were screened for both analgesic and anti-inflammatory activities. All tested compounds showed good analgesic and anti-inflammatory activity in comparison to the reference standard indomethacin. Compounds 4 and 5 showed the highest anti-inflammatory activity, while compounds 14 and 15 showed the highest analgesic activity among all the tested compounds.

Full text of DOI:10.1007/s00044-012-0097-8

MEDICINAL
CHEMISTRY
RESEARCH
Med Chem Res (2013) 22:1011–1020
DOI 10.1007/s00044-012-0097-8
ORIGINAL RESEARCH
Design, synthesis, anti-inflammatory, analgesic screening, and
molecular docking of some novel 2-pyridyl (3H)-quinazolin-4-one
derivatives
•
Ahmad F. Eweas Ahmed O. H. El-Nezhawy
•
•
Ayman R. Baiuomy Mohamed M. Awad
Received: 21 December 2011 / Accepted: 14 May 2012 / Published online: 27 May 2012
Ó Springer Science+Business Media, LLC 2012
Abstract A novel series of 6-bromo-2-(4-pyridyl)-quinaz-
olin-4(3H)-ones were synthesized by reacting 5-bromo
anthranilic acid with isonictinoly chloride in the presence of
acetic anhydride, which were further reacted with p-amino
acetophenone to obtain 3-(4-acetylphenyl)-6-bromo-2-(pyri-
din-4-yl)quinazolin-4(3H)-one (3). Compound 3 underwent
further reactions with different aldehydes to afford chalcone
derivatives 4–10, which in turn underwent various cyclization
reactions to afford cyclized products 15–18. 2-aminothiazole
derivatives 12 obtained by reaction of 3 with bromine then
with thiourea. Compound 14 obtained by treatment of 11 with
KSCN followed by cyclization. Some of the synthesized
compounds 4, 5, 12, 14, 15 and 18 were screened for both
analgesic and anti-inflammatory activities. All tested com-
poundsshowedgoodanalgesic and anti-inflammatoryactivity
in comparison to the reference standard indomethacin. Com-
pounds4 and 5 showedthe highest anti-inflammatoryactivity,
while compounds 14 and 15 showed the highest analgesic
activity among all the tested compounds.
Keywords (3H)-Quinazolin-4-ones ꢀ Molecular docking ꢀ
Anti-inflammatory
A. F. Eweas (&)
Department of Medicinal Chemistry, National Research Center,
Dokki, Cairo, Egypt
e-mail: eweas1@mailcity.com
Introduction
A. F. Eweas ꢀ A. O. H. El-Nezhawy
Nonsteroidal anti-inflammatory drugs (NSAIDs) are con-
sidered among the most commonly used drugs for the
treatment of both acute and chronic inflammation, pain,
and fever. Some of the major side effects NSAIDs are
gastrointestinal lesions, bleeding, and nephrotoxicity which
are commonly associated with long-term medications of
NSAIDs. Therefore the development of new analgesic and
anti-inflammatory active drugs with less ulcerogenic side
effects is still a challenging target for the drug industry
(Van Ryn et al., 2000), (Carter, 2000). In recent years
3H-quinazolin-4-one and their derivatives have drawn
great attention in the field of synthetic medicinal chemistry
as they were reported to possess significant activity as
antihypertensive (Ram et al., 1990), anti-fibrotic, chole-
retic, antiphlogistic (Bekhit et al., 2001), antimitotic anti-
cancer (Chandrika et al., 2008) antifungal, (Lopez et al.,
2000) (Farghaly and Moharram, 1999) and anticonvulsant
agents (Usifoh and Scriba, 2000).
Department of Pharmaceutical Chemistry, College of Pharmacy,
Taif University, Taif, KSA
A. O. H. El-Nezhawy
Department of Chemistry of Natural and Microbial Products,
National Research Center, Dokki, Cairo, Egypt
A. R. Baiuomy
Department of Pharmacology, National Research Center,
Dokki, Cairo, Egypt
A. R. Baiuomy
Department of Toxicology, College of Medicine,
Taif University, Taif, KSA
M. M. Awad
Department of Pharmacology & Toxicology,
College of Pharmacy, Helwan University, Helwan, Egypt
M. M. Awad
Department of Pharmacology & Toxicology,
College of Pharmacy, Taif University, Taif, KSA
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Med Chem Res (2013) 22:1011–1020
Quinazolinones in general were reported to possess
Results and discussion
diverse pharmacological activities such as a CNS depres-
sant (Tani et al., 1979), hypnotic, anti-inflammatory
(Plescia et al., 1984), antitumor (Singh, 1978), muscle
relaxants (Ochiai and Ishida, 1981), and antineoplastic
activity (Raffa et al., 1999). Quinazolines and condensed
quinazoline derivatives such as hoquizil, prazosin, and
buqineran, possess PDE inhibitory (Rotella et al., 2000)
antimicrobial (Bekhit et al., 2001), anti-inflammatory
(Maggio et al., 2001), anticonvulsant (Lasztoczi et al.,
2002), (Lopez-Farre et al., 2002), and antihypertensive
activity. Afloqualone and diproqualone which are deriva-
tives of quinazoline-4-one are being used as successful
anti-inflammatory agents in the management of lower back
pain as well as a centrally acting muscle relaxants (Ochiai
and Ishida, 1982) (Fig. 1).
Chemistry
Synthesisofthestartingcompound6-bromo-2-(pyridin-4-yl)-
4H-benzo[d][1,3]oxazin-4-one2 wasachievedbythereaction
of5-bromoanthranilicacidwithisonictionylchloridetoafford
5-bromo-2-(isonicotinamido)benzoic acid 1 which was then
refluxed with excess acetic anhydride to afford 2 in good yield
(Table 1). Compound 2 underwent fusion reaction with
P-aminoacetophenone to afford 3-(4-acetylphenyl)-6-bromo-
2-(pyridin-4-yl)quinazolin-4(3H)-one 3. The same compound
3 was prepared via alternative method by reacting 2 with
P-aminoacetophenone in dry pyridine which was considered
as a chemical prove of structure of 3. The structure of 3 was
further proved via instrumental analysis including elemental
analysis and NMR.
Based on these findings it is rationalized to design and
synthesize new substituted 3H-quinazolin-4-ones and
screen their anti-inflammatory and analgesic activities.
a,b-unsaturated ketones (chalcones), represent active
intermediates for several heterocyclic ring systems of
Fig. 1 Quinazoline drugs
Proquazone
Fluproquazone
Afloqualone
Diproqualone
Table 1 The docking energy
Cpd. no.
Docking
score
(Kcal/mol)
No. of
hydrogen
bonds
Amino acid residues forming
˚
hydrogen bonds in A
scores of compounds 4, 5, 12,
14, 15, and 18 with the amino
acid residues forming hydrogen
bonds in comparison with
Ligand
(indomethacin)
-67.53
2
R374 hh21–m M o1
S143 o–m M h13
reference ligand indomethacin
4
5
-86.37
-88.55
1
3
R374 hh21–m M o1
Arg 374 he–m Res 1 o2
Arg 374 hh22–m Res 1 o2
Asn 537 hd21–m Res 1 o3
R374 hh22–m M o1
R374 he–m M o1
12
14
-79.62
-80.06
3
3
R374 hh22–m M o1
R374 hh22–m M n4
N375 hn–m M o1
R374 hh21–m M o1
R374 hh21–m M o1
R374 hh22–m M o1
G225 hn–m M o2
15
18
-92.88
2
4
104.56
R374 hh21–m M o1
N375 hn–m M o3
N375 hn–m M o1
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Med Chem Res (2013) 22:1011–1020
1013
biological importance, such as pyrazolines, pyridones, and
pyrimidone or pyrimidine-thiones (Ram et al., 1990). So,
Clause-Schmidt condensation of ketene 3 with the various
aromatic aldehydes namely p-chloro, p-nitro, p-fluoro, p-N,
N-dimethyl amino, p-methoxy benzaldehydes, 2-thiophene
aldehyde, and 2-furane aldehyde were carried out to produce
the corresponding chlacone derivatives 4–10 (Scheme 1).
Compound 3 underwent a substitution reaction with
bromine water to afford the a-bromoketone 11 which in
turn reacted with thiourea in acetone under reflux condi-
tions to afford the 2-aminothiazole derivatives 12
(Scheme 1). Structure of 12 was elucidated via its correct
elemental analysis in addition to NMR spectrum which
shows the appearance of peak at d 6.23 (br, 2H NH2) and d
6.99 (s, 1H; methylene H of the thiazole ring).
Chalcone derivatives 4, 5, and 6 underwent various
cyclization reactions with hydroxyl amine hydrochloride,
urea, methyl hydrazine, and hydrazine hydrate hydrochlo-
ride to afford the corresponding cyclized products 15, 16,
17, and 18. The structures of the aforementioned com-
pounds were elucidated via their correct elemental analysis
in addition to their NMR spectra (Scheme 2).
Pharmacology
Anti-inflammatory activity
The activity of the newly synthesized compounds 4, 5, 12,
14, 15, and 18 compared to indomethacin as a reference
was measured before and 1, 2, 3, and 4 h after carrageenan
injection (Fig. 2a, b).
Compound 11 reacted with potassium thiocynate in
ethanol in water bath to afford the methylene thioccynate
derivative 13 which was used directly to react with glacial
acetic acid under reflux to afford the 2-hydroxythiazole
derivative 14. Structure of 14 was elucidated via its correct
elemental analysis in addition to NMR spectrum which
shows the appearance of peak at d 5.53 (s, 1H; methylene
H of the thiazole ring) and d 7.28 (br, 1H OH).
After i.p. administration, compounds 4, 5, 12, 14, 15,
and 18 (50 mg/kg for each tested compound) significantly
inhibited the paw edema response compared with the car-
rageenan control group at all time points and showed sig-
nificant anti-inflammatory activities (Fig. 2a, b).
The maximal effects of compounds 4, 15 and 18, and
indomethacin were observed at 1 h time period after
Scheme 1 Reagents and
conditions: a TEA, CH2Cl2;
b (CH3CO)O, reflux 6 h;
c p-aminoaceophenone, 150 °C
or p-aminoaceophenone,
pyridine, reflux 8 h;
d appropriate aromatic/
heterocyclic aldehyde, 10 %
ethanolic sodium hydroxide
reflux 1 h; e CH3COOH, Br2;
f thiourea, NaOH; g KSCN,
C2H5OH; h glacial acetic acid,
sulfuric acid
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Med Chem Res (2013) 22:1011–1020
Scheme 2 Reagents and
conditions: a Urea, C2H5OH,
KOH, reflux; b CH3NH2NH2,
CH3COOH, reflux;
c NH2OH.HCl, NaOH,
C2H5OH, reflux; d NH2NH2,
CH3COOH, reflux
carrageenan by 73.8, 62.3, 64.5, and 62.9 % inhibition,
respectively. While the maximal effect of compound 5, 12,
and 14 was observed at 2 h time period after carrageenan
by 70.5, 62.8, and 67.3 % inhibition, respectively.
Compound 5 showed the highest anti-inflammatory
activity 4 h after carrageenan injection with ED50 of
22.3 mg/kg.
calculations is prediction of correct binding geometry for
each binder. The scoring functions and hydrogen bonds
formed with the surrounding amino acids of the receptor
COX-1 are used to predict tested compounds binding
modes. Indomethacin was used as reference drug for the
anti-inflammatory activity of the new (3H)-quinazolin-4-
one derivatives. It is well known that indomethacin is a
nonselective COX inhibitor with preferential binding
selectivity for COX-1 over COX-2 receptor (Perrone et al.,
2010). We evaluated the highest anti-inflammatory and
analgesic active new compounds 4, 5, 12, 14, 15, and 18
through molecular modeling and docking techniques
against COX-I crystal structure which was downloaded
from PDB website PDB id (2OYE) (Fig. 4) shows binding
mode of the original ligand (indomethacin) into its binding
site while (Figs. 5, 6) show binding modes of compounds
12 and 18, respectively. As shown in (Table 1). The
following results can be drawn.
Analgesic activity
The anti-nociceptive activity of the newly synthesized
compounds 4, 5, 12, 14, 15, and 18 compared to indo-
methacin as a reference was also investigated. It was
assessed by the chemical model using acetic acid-induced
writhing in mice.
After s.c. administration, compounds 4, 5, 12, 14, 15,
and 18 (50 mg/kg for each tested compound) significantly
inhibited the acetic acid-induced writhes compared with
the control untreated group, and showed significant anti-
nociceptive activities (Fig. 3).
Indomethacin ‘‘the reference drug’’ docking results to
COX-1 reveals docking score of DG = -67.53 kcal/mol
and 2 H-bonds between O-1 of the ligand and H21 of Arg
374 and H13 of the ligand and O of the carbonyl group of
Ser 143 (Fig. 4). The new quinazolinone compounds were
docked against COX-1, all docked compounds showed
higher binding affinity toward COX-1 compared to indo-
methacin (Table 1), where compound no. 18 showed the
highest binding score of DG = -104.56 kcal/mol (Fig. 6)
while compound 12 showed the lowest binding score of
DG = -79.62 kcal/mol (Fig. 5). All docked compounds
showed H-bonding with COX-1 Arg 374 residue similar to
the reference drug. From that we can assume that COX-1
Arg 374 is essential in fitting the ligand to COX-1 binding
site.
The percentages of maximal protection were noted with
the tested compounds 14 and 15 by 75.67 and 75.40 %,
respectively. 14 showed the highest anti-nociceptive
activity with PD50 of 15 mg/kg3.
Molecular docking study
To understand both the anti-inflammatory and analgesic
data on a structural basis, molecular docking studies were
carried out using Mol soft ICM 3.5-0a software. ICM
docking is probably the most accurate predictive tool of
binding geometry today (Kroemer, 2003), (Cavasotto and
Abagyan, 2004). The aim of the flexible docking
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Med Chem Res (2013) 22:1011–1020
1015
Fig. 3 Effect of administration of compounds 4, 5, 12, 14, 15, and
18 (50 mg/kg, s.c.) and indomethacin (18 mg/kg, s.c.) on writhing
induced in mice by intraperitoneal injection of acetic acid. Each bar
a
represents mean SE of six mice per group. p \ 0.05, statistically
b
significant from the control group. p \ 0.05, statistically significant
from the indomethacin-treated group (Tukey test)
Fig. 4 Binding mode of the reference ligand indomethacin into its
binding site of COX-1
Fig. 2 Anti-inflammatory effect of compounds 4, 5, 12 (a), 14, 15, 18
(b) (50 mg/kg, i.p.) and indomethacin (18 mg/kg, i.p.) on the rat paw
edema, induced by sub-plantar injection of 100 ll of 1 % sterile
carrageenan. Results are expressed as a percentage increase in paw
volume from control (pre-drug) values, each point represents
a
mean SE of six rats per group. p \ 0.05, statistically significant
from the carrageenan control group. bp \ 0.05, statistically significant
from the indomethacin-treated group (Tukey test)
Experimental
Chemistry
The purity of the newly synthesized compounds was evi-
denced by TLC. All solvents which were used for crys-
tallization were of analytical grade. All melting points were
determined on a Gallenkamp apparatus and uncorrected.
Elemental analysis were carried out at the Micro analytical
Laboratory of the National Research Centre, Cairo, Egypt
(satisfactory microanalyses were obtained C; 0.40, H;
0.02; and N; 0.30, The 1H NMR were recorded
(DMSO-d6), on JOEL-JNM-EX 270 FTNMR system
(NRC) and chemical shifts (d) are expressed in ppm using
TMS as an internal standard. Splitting patterns are indi-
cated as s, singlet; d, doublet; m, multiple, b, broad signal.
Fig. 5 Binding mode of the Compound 12 into its binding site of
COX-1
5-Bromo-2-(isonicotinamido)benzoic acid 1
Isonicotinyl chloride (0.2 mol) was added drop wise to a
stirred solution of 5-bromoanthranilic acid (0.2 mol) with
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Med Chem Res (2013) 22:1011–1020
crystallized from ethanol/H2O to give 3 in 72 % yield mp:
218–220 °C.
Method 2
A mixture of equimolar amounts 6-bromo-2-(pyridin-4-yl)-
4H-benzo[d][1,3]oxazin-4-one 2 and P-aminoaceophenone
(0.01 mol) in dry pyridine (15 ml) was heated under reflux
in a sand bath for 8 h. The mixture was then cooled to
ambient temperature, acidified with diluted HCl. The solid
formed was filtered off, dried and crystallized from etha-
1
nol/H2O to give 3 in 75 % yield; H NMR (300 MHz,
DMSO-d6) d [ppm] = 2.50 (s, 3H); 7.28–8.66 (m, 11H);
Anal. Calcd. For C21H14BrN3O2: C, 60.02; H, 3.36; N,
10.00. Found: C, 60.13; H, 3.32; N, 10.12.
Fig. 6 Binding mode of the Compound 18 into its binding site of
COX-1
General procedure for 6-bromo-2-(pyridin-4-yl)-3-(4-
(3-(Aryl/Heteryl)acryloyl) phenyl) quinazolin-4(3H)-
ones 4–10
(0.2 mol) triethyl amine in 200 ml dichloromethane. The
reaction mixture was stirred at room temperature for 10–12 h.
The solid formed was filtered off, washed with water several
times and crystallized from DMF to give 1 as pale yellow
crystals yield 72 %; m.p. 297 °C. 1H NMR (300 MHz,
DMSO-d6) d [ppm] = 7.80–8.89 (m, 7H), 9.15 (s, br., NH);
11.01(s, br., OH) Anal. Calcd. For C13H9BrN2O3. C, 48.62;H,
2.82; N, 8.72. Found: C, 48.65; H, 2.87; N, 8.73.
A mixture of 3-(4-acetylphenyl)-6-bromo-2-(pyridin-4-
yl)quinazolin-4(3H)-one
3
(2.1 g, 0.005 mol) and
(0.005 mol) of the appropriate aromatic/heterocyclic alde-
hyde in 10 % ethanolic sodium hydroxide (20 ml) was
stirred at room temperature for 10 h then refluxed for 1 h.
The reaction mixture was then poured over crushed ice
(100 g), neutralized with diluted HCl. The resulted solid
was filtered off, washed with cold water, dried, and crys-
tallized from the suitable solvent (Table 1) to give com-
pounds 4–10.
6-Bromo-2-(pyridin-4-yl)-4H-benzo[d][1,3]oxazin-4-
one 2
A mixture of 5-bromo-2-(isonicotinamido)benzoic acid 1
(3.2 g, 0.01 mol) and acetic anhydride (10 ml) was heated
under reflux for 5 h. The excess acetic anhydride was
distilled off under reduced pressure the residue was tritu-
rated with petroleum ether 40–60 and the solid formed was
filtered off, dried, and crystallized from ethanol to afford 2
as colorless crystals yield 75 %; m.p. 169–171 °C; 1H
NMR (300 MHz, DMSO-d6) d [ppm] = 7.6 (d, 1H); 8–8.7
(m, 6H); Anal. Calcd. For C13H7BrN2O2, 51.51; H, 2.33;
N, 9.24. Found: C, C, 51.53; H, 2.39; N, 9.27.
(E)-6-Bromo-3-(4-(3-(4-
chlorophenyl)acryloyl)phenyl)-2-(pyridin-4-
yl)quinazolin-4(3H)-one 4
Crystallized from EtOH/H2O; colorless solid; yield: 76%
m.p. 174–76 °C; 1H NMR (DMSO-d6) d (ppm): 7.36–8.22
(m, 15H); 8.66 (d, 2H, 2,20-pyridyl protons); C13 NMR
(DMSO-d6) d (ppm): 55.3, 114.4, 120.9, 122.2, 122.9,
124.3, 124.9, 127.8, 130.4, 131.6,132.3, 136.0, 136.7,
138.5, 143.5, 145.1, 149.7, 156.2, 159.8, 160.6, 189.7;
Anal. Calcd. For C28H17BrClN3O2; C, 61.96; H, 3.16; N,
7.74. Found: C, 61.93; H, 3.19; N, 7.70.
3-(4-Acetylphenyl)-6-bromo-2-(pyridin-4-
yl)quinazolin-4(3H)-one 3
(E)-6-Bromo-3-(4-(3-(4-nitrophenyl)acryloyl)phenyl)-
Method 1
2-(pyridin-4-yl)quinazolin-4(3H)-one 5
A
mixture of p-aminoaceophenone (0.01 mol) and
Crystalized from EtOH/H2O; yellowish solid; yield 59 %
1
6-bromo-2-(pyridin-4-yl)-4H-benzo[d][1,3]oxazin-4-one 2
(0.01 mol) was fused at 150–160 °C in an oil path for 1 h.
The mixture was then allowed to cool to ambient temper-
ature, washed with diluted HCl then several times with
water. The solid formed was filtered off dried and
m.p. 150–151 °C; H NMR (DMSO-d6) d (ppm): 7.36–
8.22(m, 15H); 8.66 (d, 2H, 2,20-pyridyl protons); Anal.
Calcd. For C28H17BrN4O4; C, 60.77; H, 3.10; N, 10.12.
Found: 60.75; H, 3.08; N, 10.09.
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Med Chem Res (2013) 22:1011–1020
1017
(E)-6-Bromo-3-(4-(3-(4-fluorophenyl)acryloyl)phenyl)-
6-Bromo-3-(4-(2-bromoacetyl)phenyl)-2-(pyridin-4-
2-(pyridin-4-yl)quinazolin-4(3H)-one 6
yl)quinazolin-4(3H)-one 11
Crystalized from EtOH; colorless solid, yield 62 % m.p.
155 °C; 1H NMR (DMSO-d6) d (ppm): 7.19–8.22(m,
15H); 8.67 (d, 2H, 2,20-pyridyl protons); Anal. Calcd. For
C28H17BrFN3O2; C, 63.89; H, 3.26; N, 7.98. Found: C,
63.29; H, 3.23; N, 7.93.
To a solution of 3 (4.3 g, 0.01 mol) in acetic acid 25 ml
was added bromine in water 8 ml. The reaction mixture
was allowed to stir for 5 h at 40 °C. The resultant solid was
filtered off, washed with cold ethanol several times, air
dried, and crystallized from Ethanol/water to give com-
pound 11 as a colorless solid, yield 75 % m.p. 167–169 °C;
1H NMR (DMSO-d6) d (ppm): 4.58 (s, 2H); 7.28–8.23 (m,
9H); 8.66 (d, 2H, 2,20-pyridyl protons); Anal. Calcd. For
C21H13Br2N3O2; C, 50.53; H, 2.63; N, 8.42. Found: C,
50.56; H, 2.67; N, 8.38.
(E)-6-Bromo-3-(4-(3-(4-
(dimethylamino)phenyl)acryloyl)phenyl)-2-(pyridin-4-
yl)quinazolin-4(3H)-one 7
Crystalized from MeOH; colorless solid, yield 73 % m.p.
1
137–139 °C; H NMR (DMSO-d6) d (ppm): 3.04 (s, 6H
3-(4-(2-Aminothiazol-4-yl)phenyl)-6-bromo-2-
N,N-dimethyl protons); 6.78 (d, 2H); 7.36–8.21(m, 13H);
8.67(d, 2H, 2,20-pyridyl protons); C13 NMR (DMSO-d6) d
(ppm): 41.5, 110.8, 121.3, 122.5, 123.6, 124.3, 124.9,
125.4, 125.9, 129.7, 131.7, 132.8, 133.1, 133.9, 136.0,
136.7, 143.5, 145.1, 150.1, 151.3, 156.2, 160.5, 189.9;
Anal. Calcd. For C30H23BrN4O2; C, 65.34; H, 4.20; N,
10.16. Found: C, 65.30; H, 4.23; N, 10.13.
(pyridin-4-yl)quinazolin-4(3H)-one 12
A mixture of 11 (0.005 mol) and thiourea (0.5 g) in ace-
tone 30 ml was refluxed for 10 h. The excess solvent was
removed under reduced pressure. The residue was poured
into crushed ice (100 g). Sodium hydroxide solution
(10 %) was added and the mixture was heated to boiling
for few minutes. The mixture was then neutralized with
dilute hydrochloric acid. After cooling to ambient tem-
perature the mixture was rendered just alkaline with
ammonia solution. The formed precipitate was filtered off,
dried, and crystallized from ethanol to give compound 12
as a colorless solid, Yield 79 % m.p. 228–230 °C; 1H NMR
(DMSO-d6) d (ppm): 6.23 (br, 2H NH2); d 6.99 (s, 1H;
methylene H of the thiazole ring); 7.12–8.66 (m, 11H); C13
NMR (DMSO-d6) d (ppm): 100.3, 121.1, 122.5, 123.4,
124.6, 127.7, 129.2, 130.4, 132.3, 136.4, 143.5, 149.7,
156.2, 160.6, 189.7; Anal. Calcd. For C22H14BrN5OS; C,
55.47; H, 2.96; N, 14.70. Found: C, 55.42; H, 3.02; N,
14.73.
(E)-6-bromo-3-(4-(3-(4-
methoxyphenyl)acryloyl)phenyl)-2-(pyridin-4-
yl)quinazolin-4(3H)-one 8
Crystalized from AcOH/H2O; pale yellow solid; yield
1
68 %; m.p. 216–218 °C; H NMR (DMSO-d6) d (ppm):
3.84 (s, 3H o-methyl protons); 6.96–8.22 (m, 15H); 8.62(d,
2H, 2,20-pyridyl protons); Anal. Calcd. For C29H20BrN3O3;
C, 64.69; H, 3.74; N, 7.80. Found: C, 64.64; H, 3.72; N,
7.48.
(E)-6-Bromo-2-(pyridin-4-yl)-3-(4-(3-(thiophen-2-
yl)acryloyl)phenyl)quinazolin-4(3H)-one 9
Crystalized from AcOH/H2O; white solid; yield 53 %; m.p.
212–214 °C; 1H NMR (DMSO-d6) d (ppm): 7.53–8.25 (m,
14H); 8.68 (d, 2H, 2,20-pyridyl protons); Anal. Calcd. For
C26H16BrN3O2S; C, 60.71; H, 3.14; N, 8.17. Found: C,
60.86; H, 3.17; N, 8.18.
6-Bromo-2-(pyridin-4-yl)-3-(4-(2-
thiocyanatoacetyl)phenyl)quinazolin-4(3H)-one 13
A solution of (0.001 mol, 0.5 g) of 12 in 10 ml absolute
ethanol was added to a solution of (0.007 mol, 0.87 g) of
potassium thiocyante in 5 ml absolute ethanol. The reac-
tion mixture was heated over steam bath for 4 h. the
mixture was then allowed to cool to ambient temperature.
The formed solid was filtered off, air dried and recrystal-
lized from isopropanol to afford compound 13 in 83 %
(E)-6-Bromo-3-(4-(3-(furan-2-yl)acryloyl)phenyl)-2-
(pyridin-4-yl)quinazolin-4(3H)-one 10
Crystalized from EtOH/H2O; colorless solid; yield 65 %;
1
1
m.p. 120–122 °C; H NMR (DMSO-d6) d (ppm): 6.85–
yield; m.p.: 205–206 °C; H NMR (DMSO-d6) d (ppm):
8.27 (m, 14H); 8.25 (d, 2H, 2,20-pyridyl protons); Anal.
Calcd. For C26H16BrN3O3; C, 62.67; H, 3.24; N, 8.43.
Found: C, 62.62; H, 3.21; N, 8.39.
5.02 (s, 2H); 7.28–8.66 (m, 11H); Anal Calcd. For
C22H13BrN4O2S; C, 55.36; H, 2.75; N, 11.74. Found: C,
55.41; H, 2.72; N, 11.81.
123

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Med Chem Res (2013) 22:1011–1020
6-Bromo-3-(4-(2-hydroxythiazol-4-yl)phenyl)-2-
NMR (300 MHz, DMSO-d6) d [ppm] = 2.2 (s, 3H, N–
CH3); 3.1, 3.7 (d, d, 2H CH2 of the dihydro-1H-pyrazol
ring); 4.6 (t, 1H, CH of the dihydro-1H-pyrazol ring);
7.1–8.5 (m, 15H, aromatic protons). C13 NMR (DMSO-d6)
d (ppm): 39.2, 41.1, 60.2, 121.7, 122.6, 123.2, 123.9, 125.7,
129.8, 132.0, 135.4, 136.4, 141.3, 143.5, 146.4, 149.8,
151.6, 157.2, 160.4; Anal. Calcd. For C, 59.91; H, 3.64; N,
14.45. Found C, 59.95; H, 3.60; N, 14.48.
(pyridin-4-yl)quinazolin-4(3H)-one 14
A mixture of (0.001 mol, 0.477 g) of compound 13 in
20 ml of glacial acetic acid and 0.15 ml of conc. Sulfuric
acid was heated under reflux for 3 h. during stirring. The
reaction mixture was then allowed to cool to ambient
temperature, the solid formed solid was filtered off and
recrystallized from EtOH/H2O (1:1) to afford colorless
crystals of compound 14 in 68 % yield; m.p.: 261–263°C;
1H NMR (DMSO-d6) d (ppm): d 5.53 (s, 1H; methylene H
of the thiazole ring); d 7.18 (br, 1H OH); d 7.32–8.66 (m,
11H); C13 NMR (DMSO-d6) d (ppm): 108.9, 121.7, 122.8,
123.4, 124.6, 127.5, 128.6, 129.7, 132.7, 136.4, 143.5,
149.5, 152.3, 155.0, 156.4, 160.4; Anal Calcd. For
C22H13BrN4O2S; C, 55.36; H, 2.75; N, 11.74. Found: C,
55.42; H, 2.68; N, 11.79.
6-Bromo-3-(4-(5-(4-(dimethylamino)phenyl)-4,5-
dihydroisoxazol-3-yl)phenyl)-2-(pyridin-4-
yl)quinazolin-4(3H)-one 17
A mixture of 7 (0.003 mol), hydroxylamine hydrochloride
(0.35 g, 0.005 mol) and ethanolic sodium hydroxide (0.5 g
in 60 ml absolute ethanol) was refluxed for 8 h. The
reaction mixture was then cooled to ambient temperature,
poured into ice cold water. The formed solid was filtered
off, dried, and crystallized from ethanol/water 1:1 to give
6-Bromo-3-(4-(6-(4-chlorophenyl)-2-oxo-1,2,5,6-
tetrahydropyrimidin-4-yl)phenyl)-2-(pyridin-4-
yl)quinazolin-4(3H)-one 15
1
compound 17; 58 % yield; m.p.: 167–168 °C. H NMR
(300 MHz, DMSO-d6) d [ppm] = 3.1 (s, 6H); 3.6, 3.8 (d,
d, 2H, CH2 of dihydroisoxazol ring); 5.9 (t, 1H, CH of the
dihydroisoxazol ring); 6.8–8.7 (m, 15H, aromatic protons)
Anal. Calcd. For C30H24BrN5O2 : C, 63.61; H, 4.27; N,
12.36. Found: C, 63.63; H, 4.29; N, 12.33.
Alcoholic solution of urea (0.01 mol) in 15 ml absolute
ethanol was added to a solution of 4 (0.01 mol) and
potassium hydroxide (0.01 mol) in 15 ml absolute ethanol.
The reaction mixture was heated under reflux for 15 h
during stirring. The mixture was then allowed to cool down
to ambient temperature and the excess solvent was evap-
orated under reduced pressure. The residue was dissolved
in 50 ml distilled water and the formed solid was filtered
off, dried and crystallized from ethyl alcohol/water mixture
1:1 to produce 13 as yellow crystals; 67 % yield m.p.:
3-(4-(1-Acetyl-5-(4-methoxyphenyl)-4,5-dihydro-1H-
pyrazol-3-yl)phenyl)-6-bromo-2-(pyridin-4-
yl)quinazolin-4(3H)-one 18
A mixture of (1.6 g, 0.003 mol) of compound 8 and hydra-
zine hydrate (0.006 mol) in 10 ml glacial acetic acid was
heated under reflux for 8 h. after cooling to ambient tem-
perature the reaction mixture was poured into ice cold water
(50 ml). The formed precipitate was filtered off, dried, and
crystallized from methanol/water 1:1 to give compound 18
as yellow crystals, yield 59 %, m.p.: 212–214 °C. 1H NMR
(300 MHz, DMSO-d6) d [ppm] = 2.3 (s, 3H, CO–CH3);
2.5, 3.0(d, d, 2H, CH2 of the dihydro-1H-pyrazole ring); 3.9
(s, 3H, O–CH3); 5.5 (dd, 1H, CH of the dihydro-1H-pyrazole
ring); 7.0–8.5 (m, 15H, aromatic protons); C13 NMR
(DMSO-d6) d (ppm): 23.5, 40.1, 55.8, 66.2, 114.1, 121.6,
122.2, 123.0, 124.5, 126.6, 129.6, 132.2, 134.0, 136.6, 143.5,
149.8, 151.6, 156.4, 160.5, 168.3; Anal. Calcd. For
C31H24BrN5O3 C, 62.63; H, 4.07; N, 11.78. Found: C, 62.69;
H, 4.10; N, 11.73.
1
212–213 °C. H NMR (300 MHz, DMSO-d6) d [ppm] =
2.8–2.9 (dd, dd 2H pyrimidinone ring, J = 12.8, 7.0 Hz);
7.0-8.8 (m, 17H, aromatic protons, overlapped with CH
proton of pyrimidinone ring); 10.95 (1H, s, NH); Anal.
Calcd. For C29H19BrClN5O2 C, 59.56; H, 3.27; N, 11.97.
Found: C, 59.49; H, 3.22; N, 11.96.
6-Bromo-3-(4-(1-methyl-5-(4-nitrophenyl)-4,5-
dihydro-1H-pyrazol-3-yl)phenyl)-2-(pyridin-4-
yl)quinazolin-4(3H)-one 16
A mixture of (0,82 g, 0.003 mol) of compound 5 and
(6.15 ml, 0.006 mol) of methyl hydrazine in 10 ml acetic
acid was heated under reflux for 10 h after cooling to
ambient temperature the reaction mixture was poured into
ice cold water (50 ml). The formed solid was filtered off,
extracted with chloroform (75 ml), dried over magnesium
sulfate anhydrous. The excess solvent was removed under
reduced pressure. The solid residue was collected, crys-
tallized from Methyl alcohol/water mixture 1:1 to produce
Pharmacological assay
Animals
Adult albino Sprague-Dawely rats (120–150 g) and Swiss
mice (20–25 g) of either sex, supplied by the Animal
1
16 as yellowish crystals; 62 yield m.p.: 162–163 °C. H
123

Med Chem Res (2013) 22:1011–1020
1019
House Colony of the National Research Centre, Cairo,
Egypt were used throughout the experiments.
kg, s.c.), indomethacin (18 mg/kg, s.c.) and control normal
saline (10 ml/kg, s.c.). After 1 h pretreatment interval, an
i.p. injection of 0.6 % acetic acid was carried out (1 ml/
100 g, i.p.). Each mouse was then placed in an individual
clear plastic observational chamber, and 5 min after the
administration of acetic acid the total number of writhes
(abdominal constrictions) made by each mouse was coun-
ted for 30 min. The results of the treatment groups were
compared with those of normal saline pre-treated control.
Several doses of the tested compounds were used to
construct the dose–response curve for each compound. The
protective dose fifty (PD50) for each one was determined.
Animals were kept in raised mesh bottom colony cages
to prevent coprophagy; and maintained at 25 2 °C, rel-
ative humidity 50–55 %, and under 12:12 h light and dark
cycle. They were fed with standard animal feed and water
ad libitum. Animals were divided into groups, six animals
each in all experiments.
All animal procedures were performed after approval
from the Ethics Committee of the National Research
Centre and in accordance with the recommendations for the
proper care and use of laboratory animals.
Anti-inflammatory activity
Statistical analysis
Carrageenan-induced paw edema assay
Results expressed as mean SEM significance of the
difference of the responses to treatment group in compar-
ison to controls was determined by one-way analysis of
variance (ANOVA) followed by Tukey test p \ 0.05 was
considered significant.
Paw edema was induced by sub-plantar injection of 100 ll
of 1 % sterile carrageenan lambda in saline into the right
hind paw of rats (Winter et al., 1962). Contralateral paw
received an equal volume of saline. Paw volume was
determined immediately before carrageenan injection and
at selected times thereafter using a plethysmometer (Ugo
Basile, Milan, Italy). The edema component of inflamma-
tion was quantified by measuring the paw volume (ml) at
zero time (before carrageenan injection) and at 1, 2, 3, and
4 h after carrageenan injection and comparing it with the
pre-injection value for each animal. The effect of systemic
administration of tested compounds 4, 5, 12, 14, 15, and 18
(50 mg/kg, i.p., 0.2 ml, n = 6/group) given 30 min before
induction of inflammation by sub-plantar carrageenan was
studied. The control group of carrageenan-treated rats
received an equal volume of saline 30 min before sub-
plantar carrageenan injection (n = 6 each). A further group
administered indomethacin (18 mg/kg, i.p.) served as a
positive control. Edema was expressed as a percentage
increase in paw volume from control (pre-drug, zero time)
values. The % inhibition was calculated for each point in
order to compare the potencies of the tested compounds.
Several doses of the tested compounds were used to
construct the dose–response curve for each compound by
plotting the dose against the % inhibition. The effective
dose fifty (ED50) for each one was determined after 4 h
from carrageenan injection.
Molecular modeling studies
All docking studies were performed using ‘Internal Coor-
dinate Mechanics (Molsoft ICM 3.4–8C).
Preparation of small molecule
A set of (3H)-quinazolin-4-one derivatives synthesized to
inhibit cyclooxygenase I was compiled by us using
ChemDraw 3D structures were constructed using Chem 3D
ultra 12.0 software [Molecular Modeling and Analysis;
Cambridge Soft Corporation, USA (2010)], and then they
were energetically minimized by using MOPAC (semi-
empirical quantum mechanics), Job Type with 100 itera-
tions and minimum RMS gradient of 0.01, and saved as
MDL MolFile (*.mol).
Generation of ligand and enzyme structures
The crystal structure of target protein cyclooxygenase
(2OYE) is a COX-I complexed with indomethacin was
retrieved from the Protein Data Bank (http://www.rcsb.org/
pdb/welcome.do). All bound waters ligands and cofactors
were removed from the protein.
Analgesic activity
Acetic acid-induce writhing in mice
Docking using Molsoft ICM 3.4-8C program
The analgesic investigation was carried out according to
the method (Koster et al., 1959). The mice were divided
into different groups (six mice each). They were pretreated
with the tested compounds 4, 5, 12, 14, 15, and 18 (50 mg/
1- Convert our PDB file into an ICM object: This con-
version involves addition of hydrogen bonds, assign-
ment of atoms types, and charges from the residue
templates.
123

1020
Med Chem Res (2013) 22:1011–1020
(2000) Synthesis and preliminary cytotoxic and antifungal
evaluation of some 6-N,N-dialkyl 2-aryl-4(3H)-quinazolinone
derivatives. Heterocycl Commun 7:473–480
2- To perform ICM small molecule docking:
a) Setup docking project:
1) Set project name,
Lopez-Farre A, Rodriguez-Feo JA, Garcia-Colis E (2002) Reduction
of the soluble cyclic GMP vasorelaxing system in the vascular
wall of stroke-prone spontaneously hypertensive rats: effect of
the a1-receptor blocker doxazosin. J Hypertens 20:463
Maggio B, Daidone G, Raffa D (2001) Synthesis and pharmacological
study of ethyl 1-methyl-5-(substituted 3,4-dihydro-4-oxoquina-
zolin-3-yl)-1H-pyrazole-4-acetates. Eur J Med Chem 36:737–742
Ochiai T, Ishida R (1981) Effects of afloqualone, a new centrally
acting muscle relaxant, on DRL response and CER in rats. Jpn J
Pharmacol 78:381–392
Ochiai T, Ishida R (1982) Pharmacological studies on 6-amino-2-
fluoromethyl-3-(O-tolyl)-4(3H)-quinazolinone (afloqualone), a
new centrally acting muscle relaxant. (II) Effects on the spinal
reflex potential and the rigidity. Jpn J Pharmacol 32(3):427–438
Perrone MG, Scilimati A, Simone L, Vitale P (2010) Selective COX-
1 inhibition: a therapeutic target to be reconsidered. Curr Med
Chem 17:3769
2) Setup the receptor,
3) Review and adjust binding site,
4) Make receptor maps,
b) Start docking simulation:
3- Display the result: ICM stochastic global optimization
algorithm attempts to find the global minimum of the
energy function that include five grid potentials
describing interaction of the flexible ligand with the
receptor and internal conformational energy of the
ligand, during this process a stack of alternative low
energy conformations is saved (Table 1).
The mode of interaction of the Indomethacin within COX-
1 was used as a standard docked model. All inhibitors were
compared according to the best binding-free energy (min-
imum) obtained among all the run.
Plescia S, Daiolone G, Ceraulo L, Bajardr ML, Reina R (1984)
Studies in organic mass spectrometry. IV. Electron impact
induced fragmentation of 2-substituted 3-(5-isoxazolyl)-4(3H)-
quinazolinones of pharmaceutical interest. Farmaco ED Sci
39:120
Raffa D, Daidone G, Schillaci D, Maggio B, Plescia F (1999)
Synthesis of new 3-(3-phenyl-isoxazol-5-yl) or 3-[(3-phenyl-
isoxazol-5-yl)-amino] substituted 4(3H)-quinazolinone deriva-
tives with antineoplastic activity. Pharmazie 54:251–254
Ram VJ, Srimal RC, Kushwaha DS, Mishra L (1990) Chemothera-
peutic agents. XIX. Synthesis of [1,2,4]-triazoloquinazolinones
and related compounds as antihypertensive agents. J Prakt Chem
323:629–639
Rotella DP, Sun Z, Zhu Y, Krupinski J, Pongrac R, Seliger L,
Normandin D, Macor JE (2000) N-3-substituted imidazoquinaz-
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agents for treatment of erectile dysfunction. J Med Chem
43:1257–1263
Singh P (1978) Study in nitrogen mustards. Part II: synthesis of some
2-Alkyl-3-aryl-4(3H)-quinazolinone derivatives as possible anti-
tumour agents. J Ind Chem Soc 55:801–805
Tani J, Yamada Y, Oine T, Ochiai T, Ishida R, Inoue I (1979) Studies
on biologically active halogenated compounds. 1. Synthesis and
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