Synthesis, anti-inflammatory, and structure antioxidant activity relationship of novel 4-quinazoline

Article abstract of DOI:10.1007/s00044-013-0468-9

The practice of medicinal chemistry is devoted to the discovery and development of new agents for treating disease. A new derivative of methyl 2-((E)-3-(3,4-dihydroxyphenyl)acrylamido)benzoate 2 was synthesized by reacting the amino group of methyl anthranilate 1 with caffeic acid in the presence of PCl₃. Cyclcondensation of 2 with hydrazine hydrate afforded the corresponding 2,3-dihydro-2-(3,4-dihydroxyphenyl) pyrazolo[5,1-b]quinazolin- 9(1H)-one 3. The median lethal doses (LD_50s) of compounds 2 and 3 in mice were 1,135 and 495 mg/kg b.w., respectively. The anti-inflammatory, reducing power, chelating activity on Fe²⁺, free radical-scavenging, and total antioxidant activities were more pronounced in compound 2 compared to compound 3. On the other hand, antipyretic activity was more pronounced in compound 3 compared to compound 2. Antioxidant activity of compounds 2 and 3 increased with increased concentrations. Total antioxidant activity of compounds 2, 3 and both standards decreased in the order of α-tocopherol > compound 2 > trolox > BHA > BHT > compound 3. Administration of compounds 2 and 3 orally to the rats at dose of 50, 100, and 150 mg/kg b.w., for 10 days showed non-significant changes in serum level of GOT, GPT, ALP, γ-GT, and LDH as compared with the control group. In addition, oral administration of the compound 2 at a concentration of 100 and 150 mg/kg b.w. and compound 3 at a concentration of 150 mg/kg b.w. daily to normal rats for 10 days showed a significant increase in liver GSH, GPx, GR, and GST activities and significant decrease in TBARS level. But, administration of diclofenac sodium (30 mg/kg b.w.) orally to the rats daily for 10 days to rats showed significant increase in serum SGOT, SGPT, ALP, γ-GT, and LDH and significant decrease in liver GSH, GPx, GR, and GST activities. These findings suggest that compounds 2 and 3 exhibited good antioxidant and anti-inflammatory activity and also showed effects on liver enzymes.

Full text of DOI:10.1007/s00044-013-0468-9

MEDICINAL
CHEMISTRY
RESEARCH
Med Chem Res (2013) 22:4641–4653
DOI 10.1007/s00044-013-0468-9
ORIGINAL RESEARCH
Synthesis, anti-inflammatory, and structure antioxidant activity
relationship of novel 4-quinazoline
Mohammed Abdalla Hussein
Received: 10 August 2012 / Accepted: 3 January 2013 / Published online: 17 January 2013
ꢀ Springer Science+Business Media New York 2013
Abstract The practice of medicinal chemistry is devoted
to the discovery and development of new agents for
treating disease. A new derivative of methyl 2-((E)-3-(3,4-
dihydroxyphenyl)acrylamido)benzoate 2 was synthesized
by reacting the amino group of methyl anthranilate 1 with
caffeic acid in the presence of PCl3. Cyclcondensation of 2
with hydrazine hydrate afforded the corresponding
2,3-dihydro-2-(3,4-dihydroxyphenyl) pyrazolo[5,1-b]qui-
nazolin-9(1H)-one 3. The median lethal doses (LD50s) of
compounds 2 and 3 in mice were 1,135 and 495 mg/kg
b.w., respectively. The anti-inflammatory, reducing power,
chelating activity on Fe2?, free radical-scavenging, and
total antioxidant activities were more pronounced in
compound 2 compared to compound 3. On the other hand,
antipyretic activity was more pronounced in compound 3
compared to compound 2. Antioxidant activity of com-
pounds 2 and 3 increased with increased concentrations.
Total antioxidant activity of compounds 2, 3 and both
standards decreased in the order of a-tocopherol [
compound 2 [ trolox [ BHA [ BHT [ compound 3.
Administration of compounds 2 and 3 orally to the rats at
dose of 50, 100, and 150 mg/kg b.w., for 10 days showed
non-significant changes in serum level of GOT, GPT, ALP,
c-GT, and LDH as compared with the control group. In
addition, oral administration of the compound 2 at a con-
centration of 100 and 150 mg/kg b.w. and compound 3 at a
concentration of 150 mg/kg b.w. daily to normal rats for
10 days showed a significant increase in liver GSH, GPx,
GR, and GST activities and significant decrease in TBARS
level. But, administration of diclofenac sodium (30 mg/kg
b.w.) orally to the rats daily for 10 days to rats showed
significant increase in serum SGOT, SGPT, ALP, c-GT,
and LDH and significant decrease in liver GSH, GPx, GR,
and GST activities. These findings suggest that compounds
2 and 3 exhibited good antioxidant and anti-inflammatory
activity and also showed effects on liver enzymes.
Keywords Quinazolines ꢀ Caffeic acid ꢀ Antioxidant ꢀ
Anti-inflammatory ꢀ Oxidative stress biomarkers
Introduction
Nonsteroidal anti-inflammatory drugs (NSAIDs) are com-
monly prescribed for the treatment of acute and chronic
inflammation, pain, and fever. In medicinal chemistry, quina-
zolines have been very well known for their therapeutic
applications. Quinazolin-ones with 2,3-substitution are reported
to possess significant analgesic, anti-inflammatory (Abdel-
Rahman et al., 2003; Chambhare et al., 2003), and anticon-
vulsant activities (Santagati et al., 1995). Recently, we have
documented some lead 2-phenyl-3-substituted quinazolines
(Hussein, 2012). Caffeic acid, 3,4-dihydroxycinnamic acid
exert beneficial effects on human health through prevention
of degenerative pathologies such as cardiovascular diseases
and cancer (Bendini et al., 2007; Jiang et al., 2005). Caffeic
acid is well known to show antioxidant activity (Toda, 2002).
As an extension of our studies on the synthesis of some
new biologically active heterocyclic compounds (Hussein,
2011, 2012), now we wish to report the synthesis of
combining form of these two structural features into one
molecule might produce compounds with promising anti-
oxidant, anti-inflammatory and antipyretic effects.
M. A. Hussein (&)
Department of Biochemistry, Faculty of Pharmacy,
October 6th University, 6th of October City, Egypt
e-mail: abdallamohammed_304@yahoo.com;
abdallamohammed_405@hotmail.com
URL: www.Scitopics.com
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Med Chem Res (2013) 22:4641–4653
Experimental
Chemistry
114.663(C-6), 119.102(C-60), 165.021(C-7), 144.63(C-70),
55.520(C-8), 109.21(C-80), 199.47(C-90).
2-Synthesis of 2,3-dihydro-2-(30,40-dihydroxyphenyl)
Melting points were determined on Gallenkamp melting
point apparatus and are uncorrected. The infrared (IR)
spectra were recorded on Shimadzu MR 470 infrared
spectrophotometer using the KBr pellets. 1H- and 13C
NMR spectra were recorded in DMSO-d6 and CD3OD,
respectively, on a Varian EM 360 (1H NMR at 240 MHz)
and (13C NMR at 75 MHz). The chemical shifts are
reported in part per million (d ppm) downfield from
internal tetramethylsilane (TMS). Mass spectra were run
using HP Model MS-5988. Microanalytical data (C, H, N)
were determined at the Microanalytical Center, Cairo
University, Egypt.
pyrazolo[5,1-b]quinazolin-9(1H)-one (3)
A mixture of methyl 2-((E)-3-(3,4-dihydroxyphenyl)acry-
lamido)benzoate 2 (0.01 mol) and hydrazine hydrate
(95 %) (0.05 mol) were dissolved in n-butanol (30 ml) and
refluxed for 3–5 h. The solvent was concentrated and the
residue was recrystallized from ethanol to give 3. Physi-
cochemical and analytical data are listed in Table 1. IR
(KBr, cm-1) 3: 3500 cm-1 (OH), 3146 cm-1 (NH), 3041
and 2927 cm-1 (CH-arom), 2862 cm-1 (CH-aliph.), 1683
(C=O), 1591 cm-1 (C=N). MS (m/z) 3: 294 (M?,
25.11 %), 154 (50.18 %), 103 (46.52 %), 65 (100 %), 55
(46.42 %).1H NMR (DMSO-d6) 3: 4.6 [s, 2H, CH2], 6.6 [s,
1H, CH], 7.3–8.0 [m, 7H, Ar–H], 11.6 [s, 1H, NH]. 13C
Caffeic acid, a-tocopherol, butylated hydroxytolu-
ene(BHT), butylated hydroxyanisole (BHA), and trolox
were from Sigma, USA.
NMR(75 MHz,
CD3OD)
3:121.98(C-10),
146.722
(C-2),115.06(C-20), 135.72(C-3), 145.10(C-30), 78.04(C-300),
147.602(C-40), 156.225(C-400), 131.215(C-5), 115.6(C-50),
127.221(C-6), 119.98(C-60), 118.214(C-7), 125.568(C-8),
161.515(C-800), 175.78(C-9).
1-Synthesis of methyl 2-((E)-3-(3,4-
dihydroxyphenyl)acrylamido)benzoate (2)
To a solution of caffeic acid (0.01 mol) and methyl
anthranilate 1 (2.31 g, 0.01 mol) in xylene (50 ml), phos-
phorus trichloride (3 ml) was added. The reaction mixture
was heated under reflux for 3–4 h. The crude product was
recrystallized from ethanol to give (2). Physicochemical
and analytical data are listed in Table 1. IR (KBr, cm-1) 2:
3332 cm-1 (OH), 3133 cm-1 (NH), 2962 cm-1 (CH-arom.),
2854 cm-1 (CH-aliph.), 1715, 1685 (2C=O), 1520 cm-1
(C=N). MS (m/z) 2: 313 (M?, 10.57 %), 276 (10.25 %),
224 (14.29 %), 183 (21.42 %), 137 (25.05 %), 97 (100 %),
55 (46.42 %).1H NMR (DMSO-d6) 2: 2.4 [s, 3H, COCH3],
6.1–6.2 and 7.3–7.4 [d, 2H, CH=CH, two trans-olefinic
protons], 6.7–8.0 [m, 7H, Ar–H], 10.2 [s, 1H, NH]. 13C
NMR(75 MHz, CD3OD) 2:140.322(C-1), 121.392(C-10),
123.303(C-2),115.529(C-20), 130.500(C-3), 145.452(C-30),
125.521(C-4), 145.702(C-40), 129.859(C-5), 115.929(C-50),
Biological testing
Animals
Male albino mice weighing around 18–20 g and male
Wistar rats weight around 180–200 g were purchased from
Faculty of Veterinary Medicine, Cairo University. They
were acclimatized to animal house conditions. Animals
were provided with standard diet and water adlibrium.
Animals were kept under constant environmental condition
and observed daily throughout the experimental work.
Anti-inflammatory activity
The anti-inflammatory activity was carried out following
the method of Winter et al. (1962). Rats (180–200 g) were
divided into five different groups each of eight animals. At
the beginning, the thickness of the left paw was measured.
They were treated orally with the tested compounds, at 50,
100, and 150 mg/kg body weight or diclofenac sodium
(30 mg/kg b.w.) as a reference standard. After 30 min of
administration, the inflammation was induced by S.C.
injection of 0.1 ml of 1 % carrageenan in normal saline.
The right hind paw was injected with an equal volume of
saline.
Table 1 Physico-chemical properties and molecular formulae of the
synthesized compounds
Compd. mp (ꢁC) Yield
Mol. formula
(Mol. Wt.)
Elemental
analyses
no.
(%)
Calcd./found (%)
C
H
N
2
3
178–180 77
C18H17NO5
(313)
65.17 4.79 4.47
66.20 4.25 4.15
65.30 4.08 14.28
65.75 4.15 14.10
The difference in thickness between the two paws gave
the swelling induced by formalin. The anti-inflammatory
efficacy was estimated by comparing the swelling of the
245–247 85
C
16H12N3O3
(294)
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Med Chem Res (2013) 22:4641–4653
4643
treated with the control. The difference in thickness was
recorded after 30, 60, 90, 120, and 180 min.
2 and 3 in dimethyl sulfoxide (DMSO) was prepared for
intragastric intubation of rats. Groups of animals each con-
sisting of six rats in each were treated daily for 10 days as
follows:
Antipyretic activity
Group A Control (8 rats, was given 1 ml of saline p.o.)
Group B Normal (8 rats, was given 1 ml of DMSO, 1 %
p.o.)
Hyperthermia was induced in rats (150–160 g) by s.c.
injection of 15 % Brewer’s yeast solution at a dose of
20 ml/kg by the method of Loux et al. (1972). The body
temperature was monitored rectally using a digital ther-
mometer inserted into the rectum for 2 cm distance. The
initial temperature was taken just before yeast injection.
After elapsing 15 h, the 5 groups of animals, each com-
prising 8 rats, received oral administration of either acet-
aminophen (20 mg/100 g) as a reference standard or the
tested compounds 2 and 3 of (50, 100 and 150 mg/kg b.w.).
The body temperature was recorded at 1, 2, and 3 h after
administration.
Group C ‘Test’ (consisted of 3 sub groups, 8 rats in each
subgroup) received compound 2 (50, 100, and 150 mg/kg
orally suspended in DMSO orally in a single daily dose
(Lavergne et al., 2005).
Group D ‘Test’ (consisted of 3 sub groups, 8 rats in each
subgroup) received compound 3 (50, 100, and 150 mg/kg
orally suspended in DMSO orally in a single daily dose
(Lavergne et al., 2005).
Group E Was treated with diclofenac (30 mg/kg b.w.)
dissolved in DMSO orally in a single daily dose
(Lavergne et al., 2005)
Determination of LD50 of compounds 2 and 3
After 10 days of treatment, animals were killed by
cervical dislocation, blood samples were withdrawn from
the retro-orbital vein of each animal. The separated blood
was used for the estimation of SGOT, SGPT, c-GT, ALP,
LDH, and TBARS. Their livers were removed, perfused
immediately with ice-cold saline and homogenized in
chilled phosphate buffer (0.1 M, pH 7.4) using a Potter–
Elvehjem homogenizer. The 10 % of homogenate was used
to estimate the activities of the antioxidant enzymes, GSH,
GPx, GR, and GST.
Preliminary experiments were carried out on six main
groups (10 mice/each dose/each group). Compounds 2 and
3 were injected in different doses to find out the range of
doses which cause 0 and 100 % mortality of animals. A
range of doses was determined for each compound.
In group of ten animals each, compound 2 was
given i.p. in doses of 650, 800, 1150, 1350, 1500, and
1800 mg/kg b.w. Also, LD50 was determined by
i.p. injection of compound 3 in different doses 150, 300,
450, 600, 750, and 900 mg/kg b.w. The LD50 was evalu-
ated by Spearman and Karber method (Finney 1964) on
groups of mice, each of ten animals. The test compounds
were administrated i.p. at different doses. The number of
animals which died within 24 h was recorded.
Biochemical assays
Serum levels of glutamic-oxaloacetictransaminase (GOT)and
glutamic-pyruvate transaminase (GPT) were carried out by
Reitman and Frankel (1975); alkaline phosphatase (ALP) was
carried out by King and Armstrong (1988); Gamma glutamyl
transferase c-GT was carried out by Fiala et al. (1972); lactate
dehydrogenase was carried out by Buhl and Jackson (1978);
andTBARS in serumwas carried out byUchiyama andMihara
(1978). Liver Reduced glutathione, glutathione peroxidase
(GPx), glutathione reductase, and glutathione-S-transferase
activities were carried out by Moron et al. (1979), Marklund
and Marklund (1974), Staal et al. (1969), and Habig et al.
(1974), respectively. Liver total protein was determined
according to the method of Lowry et al. (1951).
The LD50 was then calculated by the application of the
following formula:
P
ðZ dÞ
LD50 ¼ Dm ꢁ
;
n
where Dm is the dose which killed all the mice in the group,
Z is half the sum of the dead mice from two successive
groups, d is the difference between two successive doses,
and n is the number of animals in each group.
Biochemical studies of anti-inflammatory compounds
2 and 3 on liver enzymes in rats
Antioxidant activity
Experimental design
Determination of reducing power
This experiment was carried out to examine the effect of anti-
inflammatory compounds, 2 and 3 on liver enzymes at doses
of (10, 20 and 30 mg/kg b.w.). A solution of 50, 100, and
150 mg/kg b.w. for each anti-inflammatory compounds,
The reducing power of compounds 2 and 3 were measured
according to the method of Oyaizu (1986). Various con-
centrations of compounds 2 and 3 (20–140 lg) in 1 ml of
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Med Chem Res (2013) 22:4641–4653
distilled water were mixed with 2.5 ml of phosphate buffer
(0.2 M, pH 7.6) and 2.5-ml potassium ferricyanide [K3
Fe(CN)6] (1 %, w/v), and then the mixture was incubated
at 50 ꢁC for 30 min. Afterward, 2.5 ml of trichloroacetic
acid (10 %, w/v) was added to the mixture, which was then
centrifuged at 3,000 rpm for 10 min. Finally, 2.5 ml of
upper-layer solution was mixed with 2.5-ml distilled water
and 0.5-ml FeCl3 (0.1 %, w/v), and the absorbance was
measured at 700 nm. a- tocopherol, BHA and BHT were
used as standard antioxidants. Higher absorbance of the
reaction mixture indicated greater reducing power.
where A0 is the absorbance of the control reaction and A1 is
the absorbance in the presence of compounds 2 and 3
samples.
Determination of total antioxidant activity
The antioxidant activity was determined according to the
thiocyanate method of Osawa and Namiki (1981) with
slight modifications. For the stock solution, 10 mg of
compounds 2 and 3 was dissolved in 10 ml water. Then,
the solution of compounds 2 and 3 or standards samples
(tocopherol, trolox, BHA and BHT) [100 mg/l] in 2.5 ml
of potassium phosphate buffer (0.04 M, pH 7.6) was added
to 2.5 ml of linoleic acid emulsion. Fifty-ml linoleic acid
emulsion contained Tween-20 (175 lg), linoleic acid
(155 ll), and potassium phosphate buffer (0.04 M, pH 7.6).
On the other hand, 5.0 ml of control contained 2.5 ml of
linoleic acid emulsion and 2.5 ml of potassium phosphate
buffer (0.04 M, pH 7.6). Each solution was then incubated
at 37 ꢁC in a glass flask in the dark. At 24 h intervals
during incubation, 0.1 ml of this incubation solution was
added to 4.7 ml of 75 % (v/v) ethanol and 0.1 ml of 30 %
(w/v) ammonium thiocyanate. Precisely, 3 min after 0.1 ml
of 0.02 M FeCl2 in 3.5 % (w/v) HCl was added to the
reaction mixture, the absorbance of the red color was
measured at 500 nm in a spectrophotometer. The solutions
without added compounds 2 and 3 or standards were used
as the control. The inhibition of lipid peroxidation in per-
centage was calculated by the following equation:
Determination of chelating activity on Fe2?
The chelating activity of compounds 2 and 3 on ferrous
ions (Fe2?) were measured according to the method of
Decker and Welch (1990). Aliquots of 1 ml of different
concentrations (0.25, 0.50, 1.0, 1.25, and 1.5 mg/ml) of the
samples were mixed with 3.7 ml of deionized water. The
mixture was incubated with FeCl2 (2 mM, 0.1 ml) for 5,
10, 30, and 60 min. After incubation, the reaction was
initiated by addition of ferrozine (5 mM and 0.2 ml) for
10 min at room temperature, and then the absorbance was
measured at 562 nm in a spectrophotometer. A lower
absorbance indicates a higher chelating power. The che-
lating activity of compounds 2 and 3 on Fe2? were com-
pared with that of EDTA at a level of 0.037 mg/ml.
Chelating activity was calculated using the following
formula:
Inhibition % ¼ ½ðA0 ꢁ A1Þ=A0 ꢃ 100ꢂ;
where A0 is the absorbance of the control reaction and A1 is
the absorbance in the presence of compounds 2 and 3 or
standards.
Chelating activity (% ) = [1
ꢁ ðAbsorbance of sample/Absorbance of controlÞꢂ
ꢃ 100ꢂ
Control test was performed without addition of
compounds 2 and 3.
Statistical analysis
Determination of free radical-scavenging activity
All the grouped data were statistically evaluated with
SPSS/13 software. Hypothesis testing methods included
one way analysis of variance (ANOVA). P \ 0.05 was
considered to indicate statistical significance. All the
results were expressed as mean SD for eight animals in
each group.
The free radical-scavenging activity of compounds 2 and 3
were measured with 1,1-diphenyl-2-picrylhydrazil (DPPHꢀ)
using the slightly modified methods of Brand-Williams
et al. (1995) and Takashira and Ohtake (1998). Briefly,
6 9 10-5 mol/l DPPH• solution in ethanol was prepared
and 3.9 ml of this solution was added to 0.1 ml of the
compounds 2 and 3 (2–6 mg/ml) and trolox solution (0.02–
0.06 mg/ml). The mixture was shaken vigorously and the
decrease in absorbance at 517 nm was measured at 5, 10,
30, and 60 min. Water (0.1 ml) in place of compounds 2
and 3 were used as control. The percent inhibition activity
was calculated using the following equation:
Results and discussion
Chemistry
The synthesis of the target compounds methyl 2-((E)-
3-(3,4-dihydroxyphenyl)acrylamido)benzoate
2 and
Inhibition activity ð% Þ ¼ ½ðA0 ꢁ A1Þ=A0 ꢃ 100ꢂ;
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Med Chem Res (2013) 22:4641–4653
4645
O
O
8
CH3
5
2
7
O
O
C
4
3
OCH3
6
1
Caffeic Acid
H
9'
N
NH2
8'
PCl3/ xylen e
2'
5'
H
OH
OH
3'
4'
7'
1'
H
1
2
6'
NH2NH2.H2O
O
O
8
NH2
8'
1
NH
9
7
6
N
N
H
2'
2
3
3'
OH
OH
4''
3''
N
4
N
1'
6'
5
H
H
OH
OH
4'
5'
3
4
Scheme 1 Rationalized formation of compounds 2 and 3
Scheme 2 Mechanism of
compound 3 formation
O
H3CO
H H
-EtOH
2 + NH2NH2
HO
N
HO
H
O
O
H
H
N
N
-H2O
OH
OH
N
3
2,3-dihydro-2-(3,4-dihydroxyphenyl)pyrazolo[5,1-b]quinazolin-
9(1H)-one 3 were achieved by the route depicted in
Scheme 1.
The structures of compounds 2 and 3 were proved on the
basis of elemental analyses, IR, mass and 1H NMR spectral
data. The IR spectrum of compound 2 showed bands at
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Med Chem Res (2013) 22:4641–4653
Table 2 Anti-inflammatory activity of the different doses of compounds 2 and 3
Experimental group dose (orally)
Time (in min) and paw diameter (in mm)
30
60
90
120
180
Control Group A (untreated)
20.27 5.78
22.67 5.23
24.61 6.28
25.66 4.46
27.11 4.22
30.62 3.75
33.70 4.75
34.11 4.36
29.16 2.56
28.52 4.22
Control Group B (distilled water-
treated) 3 ml/kg
Group C compound 2 (mg/kg)
50
15.26 1.84*
(24.72 %)
14.38 1.96*
(41.56 %)
12.71 3.85**
(53.12 %)
12.21 2.61**
(63.77 %)
10.11 3.15**
(65.33 %)
100
150
14.84 2.51*
(26.78 %)
13.44 1.82*
(45.39 %)
13.75 1.08**
(49.28 %)
12.04 2.08**
(64.27 %)
8.53 2.28**
(70.74 %)
12.58 1.08*
(37.94 %)
11.63 1.68*
(52.74 %)
10.14 2.01**
(62.59 %)
8.07 1.27**
(76.05 %)
6.14 1.16**
(78.94 %)
Group D compound 3 (mg/kg)
50
20.53 2.45
(1.28 %)
17.7 3.43*
(28.08 %)
16.4 0.67*
(39.51 %)
15.6 2.84**
(53.71 %)
15.46 2.32**
(46.98 %)
100
150
19.34 2.37
(4.58 %)
15.26 1.38*
(37.99 %)
14.66 2.60*
(45.92 %)
15.26 2.26**
(54.72 %)
15.08 2.08**
(48.28 %)
18.55 1.24
(8.48 %)
16.20 1.33*
(34.17 %)
15.12 1.45*
(44.22 %)
14.10 2.27**
(58.16 %)
13.25 0.2**
(54.56 %)
Group E (diclofenac) (mg/kg)
30
11.89 0.03*
(41.34 %)
4.43 0.05***
(81.99 %)
3.72 0.05***
(86.28 %)
2.44 0.04***
(92.76 %)
0.00 0.00***
(100 %)
Diclofenac sodium is used as a reference
Significant at * P \ 0.05; ** P \ 0.01; *** P \ 0.005
Table 3 Antipyretic activity of
the different doses of
compounds 2 and 3
Experimental group dose (orally)
Initial body
temp.
Recorded temperature after (h)
2
1
3
Control Group A (untreated)
37.00 2.30
38.30 2.5
37.00 3.55
38.50 1.25
37.00 1.70
38.70 3.40
36.80 1.90
38.80 3.50
Control Group B (distilled
water-treated) 3 ml/kg
Group C compound 2 (mg/kg)
50
38.10 2.80
37.70 2.50
38.50 3.4
37.70 3.50
37.20 1.90
36.90 3.60
37.00 2.30
36.70 3.55
36.50 3.10*
36.80 3.10
36.80 2.60
36.50 1.95*
100
150
Group D compound 3 (mg/kg)
50
37.60 2.90
38.35 2.50
38.45 2.25
37.60 3.25
37.80 2.10
37.50 2.30
36.50 2.70
36.00 3.00*
37.00 2.60
36.10 2.40
36.80 1.35
36.60 1.90*
100
Acetaminophen is used as a
reference
150
Group E (acetaminophen) (mg/kg)
20
Significant at * P \ 0.05;
** P \ 0.01
37.75 1.90
37.00 2.30
36.80 2.30*
36.40 2.45*
3332 cm-1 (OH), 3133 cm-1 (NH), 2962 cm-1 (CH-arom.),
2854 cm-1 (CH-aliph.), 1715, 1685 (2C=O), 1520 cm-1
(C=N). Mass spectrum of compound 2 exhibited molecu-
lar ion peak 313 (M?, 10.57 %) with base peak at 97
(100 %) and other significant peaks at 276 (10.25 %), 224
(14.29 %), 183 (21.42 %), 137 (25.05 %) and 55
CH=CH, two trans-olefinic protons], 6.7–8.0 [m, 7H,
Ar–H], 10.2 [s, 1H, NH]. Also, 13C NMR spectrum of
compound 2 in (CD3OD) exhibited signals at 144.63(C-70),
109.21(C-80) and 199.47(C-90) indicated that the presence
of double bond between C-70 and C-80 as well as the
presence of (C=O) group at (C-90).
1
(46.42 %). H NMR spectrum of compound 2 in (DMSO-
d6) exhibited signals at 2.4 [s, 3H, COCH3], 6.2 [d, 2H,
The formation of compound 3 is assumed to proceed via
formation of intermediate (acid hydrazide) followed by
123

Med Chem Res (2013) 22:4641–4653
4647
nucleophilic addition with elimination of mole of water, to
give the intermediate which was cyclized via nucleophilic
addition to give compound 3 Scheme 2.
Table 4 Determination of LD50 of compound 2 given orally in adult
mice
Group
number
Dose
(mg/kg) animals/group animals
No. of
No. of dead (Z) (d) (Z.d)
The IR spectrum of compound 3 showed bands at
3500 cm-1 (OH), 3146 cm-1 (NH), 3041 and 2927 cm-1
(CH-arom.), 2862 cm-1 (CH-aliph.), 1683 (C=O),
1591 cm-1 (C=N). Mass spectrum of compound 3 exhibited
molecular ion peak 294 (M?, 25.11 %) at with base peak at 65
(100 %) and other significant peaks at 154 (50.18 %), 103
(46.52 %),, 55 (46.42 %). 1H NMR spectrum of compound 3 in
(DMSO-d6) exhibited signals at 4.6 [s, 2H, CH2], 6.6 [s, 1H,
CH], 7.3–8.0 [m, 7H, Ar–H], 11.6 [s, 1H, NH]. 13C NMR
spectrum of compound 3 in (CD3OD) exhibited signals at
121.98(C-10), 146.722(C-2), 115.06(C-20), 135.72(C-3),
145.10(C-30) and 78.04(C-300), and disappear of 144.63(C-70),
109.21(C-80) and 199.47(C-90) signals due to formation of
pyrazol ring.
1
2
3
4
5
6
650
800
10
10
10
10
10
10
0
3
1.5 150 225
4.0 350 1,400
6.0 200 1,200
7.5 150 1,125
9.0 300 2,700
1,150
1,350
1,500
1,800
5
7
8
10
0
00
00
P
ðZdÞ
LD50 ¼ Dm
LD50 ¼ 1800 ꢁ
ꢁ
n
ꢀ
ꢁ
6650
10
¼ 1135 mg/kg b. w.
Table 5 Determination of LD50 of compound 3 given orally in adult
mice
Biological activity
Group
number
Dose
No. of
(mg/kg) animals/group animals
No. of dead (Z) (d) (Z.d)
It is well known from the literature that the tested quina-
zolines exhibit a wide range of biological activities. So, it
was of interest to design new compounds containing both
these biologically active phenolic moieties and to study
their anti-inflammatory activities.
1
2
3
4
5
6
150
300
450
600
750
900
10
10
10
10
10
10
0
2
1.0 150 150
3.0 150 450
5.5 150 825
8.0 150 1,200
9.5 150 1,425
4
7
9
10
0
00
00
Anti-inflammatory activity
P
ðZꢀdÞ
LD50 ¼ Dm
ꢁ
n
4050
10
Table 2 listed that compounds 2 and 3 have promising anti-
inflammatory activity compared with the reference
ꢀ
ꢁ
LD50 ¼ 900 ꢁ
¼ 495 mg/kg b. w.
Table 6 Levels of glutamic-oxaloacetic transaminase (GOT), glutamic-pyruvate transaminase (GPT), alkaline phosphatase (ALP), Gamma
glutamyl transferase (c-GT), lactate dehydrogenase (LDH), and lipid peroxides (TBARS) in serum of normal and experimental groups of rats
Group
SGOT (U/l)
SGPT (U/l)
ALP (U/l)
c-GT (U/l)
LDH (U/l)
TBARs
(nmol/ml)
Group A normal (distilled water-
treated) 1 ml/kg
145.50 13.86 38.77 4.25
39.50 3.86
42.26 6.24
0.57 0.04
249.89 13.26 0.82 0.073
Group B control (1 ml DMSO, 1 %) 146.76 9.11 39.18 5.81
0.56 0.026 245.37 10.27 0.81 0.050
Group C compound 2 (mg/kg)
50
135.62 10.94 42.47 6.38
133.79 15.26 43.21 4.57
129.32 14.65 40.84 3.92
38.30 5.84
35.64 4.99
31.17 6.24
0.53 0.032 240.38 15.07 0.77 0.084
0.51 0.046 239.19 13.58 0.75 0.062
0.49 0.052 232.48 17.28 0.70 0.054
100
150
Group D compound 3 (mg/kg)
50
139.53 12.36 41.97 5.08
136.34 13.55 36.15 4.36
134.55 10.06 33.26 3.84
40.77 3.15
35.64 5.62
37.58 4.78
0.55 0.0081 246.29 9.16
0.51 0.063 243.46 7.08
0.82 0.09250
0.83 0.07450
100
150
0.50 0.057 238.21 11.65 0.80 0.0650
Group E (diclofenac) (mg/kg)
30
201.27 9.74* 84.43 4.35** 75.25 6.46** 1.37 0.064* 320.69 15.47* 1.41 0.130*
Compounds 2, 3 and diclofenac sodium were given orally as a single daily dose for 10 days. Control group was compared to normal group.
Experimental groups were compared to control group. Values are given as mean SD for groups of eight animals each
* Significantly different from control group at P \ 0.05
** Significantly different from control group at P \ 0.01
123

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Med Chem Res (2013) 22:4641–4653
Table 7 Level of reduced glutathione (GSH), glutathione peroxidase (GPx), glutathione reductase (GR), and glutathione-S- transferase (GST)
activities in liver of normal and experimental groups of rats
Group
GSH
GPx
GR
GST
Group A normal (distilled water-treated) 1 ml/kg
1.84 0.092
1.76 0.085
47.47 3.86
46.83 5.24
4.19 0.42
4.07 0.35
0.86 0.08
0.87 0.06
Group B control (1 ml DMSO, 1 %)
Group C compound 2 (mg/kg)
50
1.94 0.069
2.39 0.17*
2.95 0.15*
49.11 4.9
4.85 0.28
5.59 0.19*
6.36 0.26*
1.30 0.13
1.54 0.19*
1.97 0.14*
100
53.74 5.17*
57.89 4.85*
150
Group D compound 3 (mg/kg)
50
1.80 0.18
1.85 0.094
2.39 0.19*
49.25 4.6
4.33 0.19
4.25 0.21
5.14 0.34*
0.97 0.09
1.15 0.16
1.34 0.14*
100
51.38 5.22
53.45 4.74*
150
Group E (diclofenac) (mg/kg)
30
0.79 0.15*
32.09 4.55*
3.28 0.12*
0.63 0.16*
Compounds 2, 3 and diclofenac sodium were given orally as a single daily dose for 10 days. Control group was compared to normal group.
Experimental groups were compared to control group. Values are given as mean SD for groups of eight animals each. GSH is expressed as
mg/mg protein, GPx as mmoles of GSH oxidized/min per mg protein, GR as nmoles of NADPH oxidized/min per mg protein, and GST as
mmoles of CDNB conjugated/min per mg protein
* Significantly different from control group at P \ 0.05
Fig. 1 Reducing power of
compounds 2 and 3, BHA, BHT
and a-tocopherol
0.5
0.4
0.3
0.2
0.1
0
BHA
Compound 2
Compound 3
BHT
α- tocopherol
20
40
60
80
100
120
140
Concentration (ug/ml)
anti-inflammatory drug, diclofenac sodium. Also, com-
pound 2 more pronounced anti-inflammatory activity than
compound 3.
1800 mg/kg b.w. resulted in mortalities of 0, 3, 5, 7, 8, and 10
respectively. The dose of compound 2 that killed half of the
mice (LD50) was 1130 mg/kg b.w. The results are given in
Table 5shows thati.p. injection of compound 3 in doses of150,
300, 450, 600, 750, and 900 mg/kg b.w. resulted in mortalities
of 0, 2, 4, 7, 9, and 10, respectively. The dose of compound 3
that killed half of the mice (LD50) was 495 mg/kg b.w.
Antipyretic activity
Table 3 showed that compounds 2 and 3 have good anti-
pyretic activities compared with the reference antipyretic
acetaminophen. Also, compound 3 more pronounced anti-
inflammatory activity than compound 2.
Toxic symptoms Compounds 2 and 3 injected mice
exhibited an increase in heart rate, rapid respiration with in
1–2 h. There is a general depression in activity with
tremors in hind limbs. The mucous of the eye become
brownish in color and the skin and toes bluish. The tem-
perature of the animals extremities dropped with the toes
and tail being cool.
Determination of LD50 of compounds 2 and 3 in adult mice
The results are given in Table 4 shows that i.p. injection of
compound 2 in doses of 650, 800, 1150, 1350, 1500, and
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Med Chem Res (2013) 22:4641–4653
4649
Fig. 2 a Chelating effects of
different concentrations of
compound 2 on Fe2? ions at
different incubation times with
FeCl2. b Chelating effects of
different concentrations of
compound 3 on Fe2? ions at
different incubation times with
FeCl2
50
45
40
35
30
25
20
15
10
5
(a)
0.25 mg/ml
0.5 mg/ml
1.0 mg/ml
1.25 mg/ml
1.5 mg/ml
0
10
30
60
90
120
Time (min.)
50
45
40
35
30
25
20
15
10
5
(b)
0.25 mg/ml
0.5 mg/ml
1.0 mg/ml
1.25 mg/ml
1.5 mg/ml
0
10
30
60
90
120
Time (min.)
Fig. 3 Scavenging activities of
different concentrations of
Compound 2 (6mg)
Trolox(0.06mg)
80
compounds 2 and 3 and trolox
against the 1,1-diphenyl-2-
picryl-hydrazil (DPPHꢀ) radical
70
60
50
40
30
20
10
0
Trolox(0.04mg)
Trolox(0.02mg)
Compound 2 (4mg)
Compound 3 (6mg)
Compound 2 (2mg)
Compound 3 (4mg)
Compound 3 (2mg)
Biochemical studies of anti-inflammatory compounds
group (Table 6). In addition, oral administration of the
compound 2 at a concentration of 100 and 150 mg/kg b.w.
and compound 3 at a concentration of 150 mg/kg b.w.
daily to normal rats for 10 days showed a significant
increase in liver GSH, GPx, GR, and GST activities and
significant decrease in TBARS level (Table 7). But,
administration of diclofenac sodium (30 mg/kg b.w.)
2 and 3
Administration of compounds 2 and 3 orally to the rats at
dose of 50, 100, and 150 mg/kg b.w., for 10 days showed
non-significant changes in serum level of GOT, GPT, ALP,
c-GT, LDH, and TBARS as compared with the control
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4650
Med Chem Res (2013) 22:4641–4653
α-tocopherol
Fig. 4 Total antioxidant
activities of compounds 2 and 3,
a-tocopherol, trolox and BHA,
BHT (100 mg/l concentration)
on peroxidation of linoleic acid
emulsion
Compound 2
90
80
70
60
50
40
30
20
10
0
Trolox
BHA
Compound 3
BHT
orally to the rats daily for 10 days to rats showed signifi-
cant increase in serum SGOT, SGPT, ALP, c-GT, and
LDH and significant decrease in liver GSH, GPx, GR, and
GST activities (Tables 6 and 7).
transition metal ion (Fe2?/Fe3?) that play an essential role
in the formation of reactive oxygen species in Fenton’s
reactions.
The DPPHꢀ radical-scavenging effects of compounds 2
and 3 are presented in Fig. 3 and showed appreciable free
radical-scavenging activities. The free radical-scavenging
activity of compounds 2 and 3 were compared to trolox, as
a synthetic antioxidant. Compounds 2 and 3 of 6 mg/ml
had the highest radical-scavenging activity when compared
with 0.06 mg/ml trolox. The effects of 100 mg/l of com-
pounds 2 and 3 on peroxidation of linoleic acid emulsion
are shown in Fig. 4. Compound 2 showed higher antioxi-
dant activity when compared to trolox, BHA, and BHT.
Total antioxidant activity of compounds 2 and 3 and both
standards decreased in the order of a-tocopherol [ com-
pound 2 > trolox [ BHA [ compound 3 [ BHT.
The antioxidant activity of compounds 2 (four resonating
structures)more pronounced than compound3 (two resonating
structures) due to the presence of conjugated double bond,
which makes the electrons more delocalized from C ring to the
carbonyl group (Scheme 3). Free radicals are known to be a
major factor in biological damages, and DPPH• has been used
to evaluate the free radical-scavenging activity of natural
antioxidants (Yokozawa et al., 1998). DPPH•, which is a
molecule containing a stable free radical with a purple color,
changesintoastablecompoundwithayellowcolorbyreacting
with an antioxidant which can donate an electron to DPPH•. In
such case, the purple color typical of the free DPPH• radical
decays, a change which can be followed either spectrophoto-
metrically (517 nm). The proton radical-scavenging action is
known as an important mechanism of antioxidation. 1,
1-Diphenyl-2-picrylhydrazil (DPPH•) is used as a free radical
to evaluate the antioxidative activity of some natural sources
(Chung et al., 2005). The DPPH• radical-scavenging effects of
compounds 2 and 3 are presented in Fig. 3. From these results,
it can be stated that compounds 2 have the ability to scavenge
free radicalsandcould serveas astrong freeradicalinhibitoror
scavenger according to trolox. Many attempts at explaining the
structure–activity relationships of some phenolic compounds
Antioxidant activity of compounds 2 and 3
Figure 1 shows the reducing power of compounds 2 and 3.
The reducing power of compounds 2 and 3 increased with
increasing concentration. Based on a comparison of the
absorbance at 700 nm, the reducing power of compounds 2
and 3 at a concentration of 20 lg/ml were nearly similar to
that of BHA and BHT, respectively. This indicates that
compounds 2 and 3 were electron donors and could also
react with free radicals, converting them to more stable
products and terminate the radical chain reaction. Also,
120 lg/ml compounds 2 and 3 are the best concentration
which exhibits the most reducing power. In the reducing
power assay, the presence of reductants (antioxidants)
resulted in the reduction of the Fe3?/ferricyanide complex
to the ferrous form (Fe2?) Fig. 1. The amount of Fe2?
complex can therefore be monitored by measuring the
formation of Perl’s Prussian Blue at 700 nm2. The reducing
power of compounds 2 and 3 and both standards decreased
in the order of BHA [ compound 2 [ compound
3 [ BHT [ a-tocopherol.
Figure 2a, b shows the chelating effect of compounds 2
and 3. All samples at 1.50 mg/ml concentration showed the
best chelating effect 30 and 22 % on ferrous ions at an
incubation time of 60 min, respectively. The chelating
activity of samples increased with increasing incubation
times with FeCl2. However, the chelating activity of
compounds 2 of 1.25 mg/ml (30 %) was lower than EDTA
at 0.037 mg/ml (43.67 %) for an incubation time of
90 min. This indicates that the chelation property of the
compounds 2 on Fe2? ions may afford protection against
oxidative damage. The results in Fig. 2a, b indicated that a
significant property of compounds 2 and 3 is its capability
for blocking the oxidative activity of systems with
123

Med Chem Res (2013) 22:4641–4653
4651
O
O
OC2 H5
N
NH
D
A
H
C
O
C
A
3
OH
OH
N
N
H
B
3
3
OH
4
2
B
R, RO, ROO
4
OH
O
R, RO, ROO
N
NH
A
D
H
C
O
3
OH
N
OC2 H5
B
O
O
C
A
4
(I)
C
N
O
H
3
4
OH
B
N
NH
A
D
H
3
O
OH
O
N
(I)
B
O
4
(II)
OC2 H5
O
C
A
N
O
H
OH
B
3
N
NH
C
A
H
D
4
3
O
OH
N
(II)
B
O
O
4
OC2 H5
A
O
C
N
H
OH
B
3
4
O
(III)
O
OC2 H5
O
A
O
C
OC2H5
O
A
N
(IV)
H
OH
N
B
3
H
OH
B
4
3
O
4
O
Scheme 3 Proposal mechanism of compounds 2 and 3 antioxidant activity
have beenreported in theliterature. It has been reported that the
antioxidant activity of phenolic compounds may result from
the neutralization of free radicals initiating oxidation
processes, or from the termination of radical chain reactions,
due to their hydrogen donating ability (Baumann et al., 1979).
It is also known that the antioxidant activity of phenolic
123

4652
Med Chem Res (2013) 22:4641–4653
Cao G, Sofic E, Prior RL (1997) Antioxidant and prooxidant
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Chambhare RV, Khadse BG, Bobde AS, Bahekar RH (2003)
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compounds is closely associated with their structures, such as
substitutions on the aromatic ring and side chain structure.
Their accessibility to the radical center of DPPH• could also
influence the order of the antioxidant power. Free radical-
scavenging activity of phenolic compounds is believed to be
influenced by the number and position of phenolic hydrogen in
their molecules (Rice-Evans et al., 1996). It is also proposed
that the higher antioxidant activity of compound 2 and 3 is
related to the presence of hydroxyl groups (Cao et al., 1997).
The structural requirement considered essential for effective
radical scavenging by compounds 2 and 3 is the presence of
P-dihydroxyl groups in B ring and conjugated double bond.
The presence of double bond between B ring and carbonyl
group in compound 2 makes the electrons more delocalized to
form quinone structure which possesses electron donating
properties and is a radical target (Hussein and Samir 2010)
(Scheme 3).
imidin-4-ones as antimicrobial agents. Eur
38:89–100
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Finney DJ (1964) Statistical methods in biological assay. Charles
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novel anti-inflammatory quinazolines. Int J Org Bioorg Chem
1:12–20
Hussein MA (2012) Synthesis of some novel triazoloquinazolines and
triazinoquinazolines and their evaluation for anti-inflammatory
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011-9707-0
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Jiang RW, Lau KM, Hon PM, Mak T, Woo K, Fung K (2005)
Chemistry and biological activities of caffeic acid derivatives
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King EJ, Armstrong AR (1988) Calcium, phosphorus and phosphate.
Synthesis and structure antioxidant activity relationship
effect of newly anti-inflammatory quinazolines bearing
caffeic acid moiety has not been reported earlier to our
knowledge, and this study is perhaps the first observation
of its kind.
In conclusion, quinazoline derivatives when combining
with caffeic acid exhibited promising antioxidant, anti-
inflammatory activity, high LD50 value, and more safe on
liver enzymes. Also, the present study showed that the
effects of antioxidative activity of compound 2 more pro-
nounced than 3 depend on their resonating structures. More
studies are needed to prove their medicinal and biological
importance which may pave the way for possible thera-
peutic applications.
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