Synthesis and antioxidant, anti-inflammatory, and analgesic activity of novel polycyclic pyrimido[4,5-b]quinolines

Article abstract of DOI:10.1080/10426500802167027

The behavior of the 2-methylthio-pyrimido[4,5-b]quinolin-4-one towards differently substituted amines is reported. Also, the reactivity of 3-aminothiazolo[3′,2′ :1,2]-pyrimido[4,5-b]quinoline-2-carbonitrile towards formic acid, urea, thiourea, formamide,

Full text of DOI:10.1080/10426500802167027

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Synthesis and Antioxidant,
Anti-Inflammatory, and
Analgesic Activity of Novel
Polycyclic Pyrimido[4,5-
b]quinolines
A. B.A. El-Gazzar a , H. N. Hafez a , A. A. Abu-
Hashem a & A. S. Aly a
a Photochemistry Department (Heterocyclic and
Nucleosides Unit) , National Research Centre ,
Dokki, Giza, Egypt
Published online: 03 Feb 2009.
To cite this article: A. B.A. El-Gazzar , H. N. Hafez , A. A. Abu-Hashem & A. S. Aly
(2009) Synthesis and Antioxidant, Anti-Inflammatory, and Analgesic Activity of Novel
Polycyclic Pyrimido[4,5-b]quinolines, Phosphorus, Sulfur, and Silicon and the Related
Elements, 184:2, 379-405, DOI: 10.1080/10426500802167027
To link to this article: http://dx.doi.org/10.1080/10426500802167027
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Phosphorus, Sulfur, and Silicon, 184:379–405, 2009
Copyright © Taylor & Francis Group, LLC
ISSN: 1042-6507 print / 1563-5325 online
DOI: 10.1080/10426500802167027
Synthesis and Antioxidant, Anti-Inflammatory,
and Analgesic Activity of Novel Polycyclic
Pyrimido[4,5-b]quinolines
A. B. A. El-Gazzar, H. N. Hafez, A. A. Abu-Hashem,
and A. S. Aly
Photochemistry Department (Heterocyclic and Nucleosides Unit),
National Research Centre, Dokki, Giza, Egypt
The behavior of the 2-methylthio-pyrimido[4,5-b]quinolin-4-one towards differently
substituted amines is reported. Also, the reactivity of 3-aminothiazolo[3ꢀ,2ꢀ:1,2]-
pyrimido[4,5-b]quinoline-2-carbonitrile towards formic acid, urea, thiourea, for-
mamide, and carbon disulfide is discussed. Some of the synthesized derivatives pos-
sess biological activities as anti-inflammatory and analgesic agents. Some of these
selective biologically active compounds were screened for antioxidant properties.
Keywords 3-Aminothiazolo-[3ꢀ,2ꢀ:1,2]pyrimido[4,5-b]quinoline-2-carbonitrile; anal-
gesic activities; anti-inflammatory; antioxidant; 2-arylaminopyrimido[4,5-b]quinoline; 2-
methylthio
INTRODUCTION
Various polysubstituted 2-aminopyrimidines exhibit important phar-
macological properties. For example, derivative A was reported as a
5HT2 receptor antagonist,1 whereas the bicyclic compound B shows sor-
bitol dehydrogenase inhibition properties2 (Figure 1). In addition, many
bicyclic- and tricyclic compounds that are either natural (phytoestro-
gens) or obtained by synthesis and that exhibit a quinoline ring, have
been studied for their biological activities. They are especially used as
radical scavengers such as quercetol or coumestrol3 or the copper- or
iron-chelating molecules such as clioquinol.4 Also, pyrimidine amide
derivatives are novel anti-allergic agents.5 S-Alkylated derivatives
are potent antiviral agents.6 6-Alkylamino derivatives are inhibitors
of Bacillus subtilis DNA polymerase III.7 Aziridino derivatives are
Received 19 November 2007; accepted 28 April 2008.
Address correspondence to A. B. A. El-Gazzar, Photochemistry Department (Hete-
rocyclic and Nucleosides Unit), National Research Centre, 12622 Dokki, Giza, Egypt.
E-mail: profelgazzar@yahoo.com
379

380
A. B. A. El-Gazzar et al.
FIGURE 1 Some polysubstituted pyrimidines exhibited pharmacological
properties.
new cytotoxic agents with tumor-inhibitory activity.8 Also, arylamino
derivatives of pyrimidines are potential anti-cytomegalovirus agents.9
We recently reported the identification of pyrimido[4,5-b]quinoline
as a selective antioxidant,10 with anti-inflammatory and highly
analgesic properties. We report here
a one-pot synthesis of
new tetrahydropyrimido[4,5-b]quinoline, 2-arylamino-, 2-piperazino-,
morpholino-, and 3-amino-thiazolo[3ꢀ,2ꢀ:1,2]pyrimido[4,5-b]quinolines.
Their antioxidant properties were also evaluated by the 2,2ꢀ-azino-
bis(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS) method and the ery-
throcyte hemolysis method. Also, the anti-inflammatory activity was
evaluated by carrageenan-induced paw edema test in rats, and the
analgesic activity was performed by the Turner and Collier technique.
RESULTS AND DISCUSSION
A one-pot synthesis of 5-(4-chlorophenyl)-9-(4-chlorophenylmethylene)-
2-thioxo-6,7,8,9-tetrahydropyrimido[4,5-b]quinolin-4-one (4) is de-
scribed below. 4-Chlorobenz-aldehyde 1 was condensed with cyclohex-
anone 2 and 6-aminothiouracil 3 in dimethylformamide (DMF) solu-
tion. Stirring for a long time under thin layer chromatography (TLC)
control afforded 4 in good yield (Scheme 1).
First, a condensation reaction of 2 mol of 4-chlorobenzaldehyde
with cyclohexanone with loss of 2 mol of water under formation of
an α,β-unsaturated ketone occurs. This ketone reacts with 3 under
formation of a Schiff base, which undergoes an interamolecular [4+2]-
cycloaddition with subsequent spontaneous dehydrogenation to form
the aromatic pyrimido[4,5-b]qinoline 4.
It is well known that positions 2 and 4 in pyrimidine and fused
pyrimidines show distinct activities towards nucleophiles. Therefore,

Synthesis of Pyrimido[4,5-b]quinolines
381
SCHEME 1
2-methylthio-5-(4-chlorophenyl)-9-(4-chlorophenylmethylene)-6,7, 8, 9-
tetrahydropyrimido[4,5-b]quinolin-4-one (5) was prepared, and its
activity towards nucleophiles such as primary aromatic amines and
secondary aliphatic amines (piperazine, morpholine) was investigated.
Thus heating under reflux of 2-methylthio-pyrimido[4,5-b]quinolin-4-
one (5) with aniline, 4-chloroaniline, and p-anisidine in methanol pro-
duced 2-arylamino-5-(4-chloro-phenyl)-9-(4-chlorophenylmethylene)-
6,7,8,9-tetrahydropyrimido[4,5-b]quinolin-4-one derivatives (6a–c)
with evolution of methanethiol (Scheme 2). The IR spectra of 6a–c
displayed absorption bands around 3365–3420 cm−1 (NH) and 1680–
1
1685 cm−1 (C O). Moreover, the H-NMR spectra of 6a–c,7 revealed
the absence of a CH3S signal. For example, the 1H-NMR spectrum
of 6c showed signals at 1.63–1.67, 2.27–2.33, and 2.67–2.72 for the
three CH2 groups, a signal at 3.89 (OCH3), signals due to the aromatic
protons (6.92–7.69), and the signal for the vinyl proton at 8.40. The
broad band at 10.00 belongs to NH (exchangeable by D2O). Its mass
spectrum showed the molecular ion peak [M+], m/z 555, (100%).
Reaction of 5 with piperazine and morpholine in methanol produced
the 2-piperazino- and 2-morpholino-2-methylthio-5-(4-chlorophenyl)-
9 - (4 - chlorophenyl - methylene) - 6, 7, 8, 9 - tetrahydropyrimido[4, 5 - b]-
quinolin-4-one derivatives (7a,b).The 1H-NMR spectrum of 7a showed
a triplet at 3.29–3.33, a multiplet at 3.35–3.38, and a triplet at 3.45–
3.49 corresponding to the protons of four CH2 groups of morpholine,
in addition to the remaining protons, which supported the proposed
structure of compound 7a.
Moreover, the reaction of 5 with anthranilic acid afforded 2-(o-
carboxyphenylamino)-5-(4-chlorophenyl)-9-(4-chlorophenylmethylene)
-6,7,8,9-tetrahydropyrimido[4,5-b]-quinolin-4-one (8). The IR spectrum
of 8 displayed bands at 3510 cm−1 (OH), 3368 (NH), and 1716 (C O).
1
Its H-NMR spectrum showed signals at δ 1.66–1.70, 2.24–2.30, and
2.69–2.76 for the three methylene groups; a multiplet for the aromatic
protons around 6.92–7.72; and a singlet for CH at 8.25 (s, 1H, CH),

382
A. B. A. El-Gazzar et al.
SCHEME 2
in addition to two broad bands at 9.10, 12.30 corresponding to the
NH and hydroxyl groups. Compound 8 was cyclized by boiling with
glacial acetic acid in the presence of a few drops of sulfuric acid to give
12-(4-chlorophenyl)-8-(4-chlorophenylmethylene)-8,9,10,11-terahydro-
quino[2ꢀ,3ꢀ:4,5]pyrimido[2,1-b]quinazoline-13(H),15(H)-dione (9) (Sche-
me 2). Structure 9 is preferred on the basis of elemental analysis and
spectral data. Its IR spectrum revealed the absence of any absorp-
tion bands in the OH region, and a shift of the carbonyl frequency
from 1716 to 1683 indicates the formation of the quinazoline ring.
Also, its 1H-NMR spectrum supported the proposed structure (see
Experimental).
4-Chloro-2-methylthio-5-(4-chlorophenyl)-9-(4-chlorophenylmethyl-
ene)-6,7,8,9-tetrahydropyrimido[4,5-b]quinoline (10) was prepared by
gentle heating of 5 with phosphorus oxychloride in dry dioxane,12,13
and its reactivity towards hydrazine hydrate was investigated. The
IR spectrum of 10 revealed the absence of any absorption bands in
the NH and CO region. Its 1H-NMR spectrum supported the proposed
structure. Also, its mass spectrum showed the molecular ion peak
[M+], m/z 498, (78%).

Synthesis of Pyrimido[4,5-b]quinolines
383
Refluxing a mixture of 10 and hydrazine hydrate afforded 5-(4-
chloro-phenyl) - 9 -(4 - chlorophenylmethylene)-2,4-dihydrazino-6,7,8,9-
tetrahydropyrimido[4,5-b]quinoline (11). The IR spectrum of 11 dis-
played absorption bands around 3438–3350 cm−1 (NH) groups. Its 1H-
NMR spectrum showed broad singles at 2.45, 8.54 indicating the pres-
ence of two amino groups. Compound 11 reacted with formic and acetic
acid to yield the 6-(4-chlorophenylmethylene)-10-(4-chlorophenyl)-
6,7,8,9 - tetrahydro - s-tria-zolo[3ꢀ,4ꢀ:6, 1]-s-triazolo[3ꢀꢀ,4ꢀꢀ,:2, 3]pyrimido-
[4,5-b]quinolines (12a,b), with a new ring system. Besides the correct
values of the elemental analyses, the spectral data of 12 are in agree-
ment with the assigned structure. The IR spectrum of 12 revealed
1
the absence of NH groups. Also, the H-NMR spectrum revealed the
absence of NH2 and NH groups, while the two signals at 9.23 and 9.39
for 12a correspond to the triazole protons. They are replaced in 12b
by two singlets at 2.86, 2.89 due to the methyl groups. In agreement
with the structure, the 13C-NMR spectrum of 12a showed signals at δ
22.25, 26.67, 27.20 due to three sp3 carbon atoms and 23 sp2 carbon
atoms around 105.71–153.27 ppm, and its mass spectrum showed the
molecular ion peak [M+] at m/z 498 (100%).
The reaction of 4 with bromo-malononitrile in an ethanolic
potassium carbonate solution gave 3-amino-6-(4-chlorophenyl)-10- (4-
chlorophenylmethylene) - 5 - oxo -7,8,-9,10-tetrahydrothiazolo[3ꢀ,2ꢀ:1,2]
pyrimido[4,5-b]quinoline-2-carbonitrile (13). The IR spectrum of 13
displayed absorption bands corresponding to amino, nitrile, and car-
bonyl groups, and the 1H-NMR spectrum showed signals at 1.63–1.66,
2.30–2.32, 2.73–2.78 due to the three methylene groups, the aromatic
protons around 7.10–7.43, and a singlet due to CH at 8.19, in addition
to the broad peak at 8.90 corresponding to the amino group. Its
13C-NMR spectrum showed signals 24.30, 26.97, 28.06 for three sp3
carbon atoms, one signal at 68.5 (CN), 18 signals corresponding to sp2
carbon atoms around 108.5–158.7, and the signal of (C O) at 164.9.
Its mass spectrum showed the absorption molecular ion peak [M+],
m/z 530, (100%).
Compound 13, as a typical β-enaminonitrile derivative, reacted
with aliphatic acids to afford 8-(4-chlorophenyl methylene)-12-(4-
chlorophenyl)-8,9,10,11-tetrahydropyr-imido[4ꢀꢀ, 5ꢀꢀ,:4ꢀ, 5ꢀ]thiazolo[3ꢀ,2ꢀ:
1,2]pyrimido[4,5-b]quinoline-4,13-diones (14a,b). The IR spectrum of
14 revealed the absence of amino and nitrile groups. The 1H-NMR
spectrum of 14b showed a signal at 2.32 for the methyl group
and a broad signal at 10.00 for the NH group. Similarly, com-
pound 13 reacted with formamide, in the presence of DMF, to yield
4 - amino - 8 - (4 - chlorophenylmethylene) - 12 - (4-chloro-phenyl)-8,9,10,

384
A. B. A. El-Gazzar et al.
11 - tetrahydropyrimido[4ꢀꢀ, 5ꢀꢀ, :4ꢀ, 5ꢀ]thiazolo[3ꢀ, 2ꢀ:1, 2]pyrimido[4,5-b]-
quinolin-13-one (15). The 1H-NMR exhibited a singlet at 8.15 cor-
responding to the pyrimidine proton and a broad band at 8.35 for
the amino group. When 13 was heated with urea or thiourea, 4-
amino-8-(4-chlorophenylmethylene)- 12-(4-chlorophenyl)-8,9,10,11-tet-
rahydropyrimido[4ꢀꢀ, 5ꢀꢀ, : 4ꢀ, 5ꢀ]thiazolo[3ꢀ, 2ꢀ:1, 2]pyrimido[4,5-b]quinol-
ine-2,13-dione (16a) and 4-amino-8-(4-chlorophenylmethylene)-12
-(4-chlorophenyl)-8,9,10,11-tetrahydropyrimido[4ꢀꢀ,5ꢀꢀ,:4ꢀ,5ꢀ]thiazolo [3ꢀ,
2ꢀ:1,2]pyrimido[4,5-b]quinolin-2-thioxo-13-one (16b), respectively, were
formed (Scheme 3). The 1H-NMR spectrum of 16b showed signals
around δ 1.64–2.78 due to the three methylene groups, at 7.06–7.47 for
the arene protons, a singlet signal at 8.02 for CH, and broad bands at
8.75, 9.50 due to the NH2 and NH groups.
Alternatively, heating of 13 with carbon disulfide in pyridine
led to 8-(4-chlorophenylmethylene)-12-(4-chlorophenyl)-4-imino-8,9,
10, 11-tetrahydroquino[2ꢀꢀ, -3ꢀꢀ, : 4ꢀ, 5ꢀ]pyrimido[2ꢀ, 1ꢀ:2, 3]thiazolo[4, 5-d]
[1,3]thiazine-13-one (17). The 1H-NMR spectrum of 17 showed signals
around δ 1.63–2.81, three methylene groups around 7.05–7.51 (Ar-H),
and a singlet signal at 8.12 for CH, in addition to broad bands at
9.75, 10.50 due to two NH groups. The 13C-NMR showed signals at δ
22.23, 26.65, 27.33 due to three sp3 carbon atoms; 23 sp2 carbon atoms
around 108.6–155.3; and two bands at 163.5 and 176.8 corresponding
to the carbonyl and thioxo group, respectively.
Condensation of 13 with triethyl orthoformate yielded the cor-
responding 3-ethoxy-methyleneamino derivative 18, with expected
IR and 1H-NMR data. Compound 18 reacted with hydrazine hy-
drate in boiling dioxane to give 3-hydrazino-methylene-amino-
6-(4-chlorophenyl) - 10 - (4 - chlorophenylmethylene) - 5 - oxo - 7, 8, 9, 10 -
tetrahydro - thiazolo[3ꢀ, 2ꢀ : 1, 2]pyrimido[4, 5-b]quinoline-2-carbonitrile
(19), which on boiling in an ethanolic sodium ethoxide solution un-
derwent cyclization to give 3-amino-8-(4-chlorophenylmethylene)-12-
(4-chlorophenyl)-4 -imino - 8, 9, 10, 11-tetrahydropyrimido-[4ꢀꢀ, 5ꢀꢀ,:4ꢀ, 5ꢀ]
thiazolo[3ꢀ,2ꢀ:1,2]pyrimido[4,5-b]quinolin-13-one (20) (Scheme 3). The
IR spectrum of 18 displayed absorption bands at 2215 cm−1(CN),
1
while that of 20 revealed the absence of a nitrile group. The H-NMR
spectrum of 20 showed the standard signals for the methylene and
aromatic protons, the broad signals corresponding to the NH2 and NH
at 8.76 and 10.82, and a singlet at 8.29 for the pyrimidine proton.
Finally, compound 18 was heated with aqueous ammo-
nia
to
give
3-aminomethyleneamino-6-(4-chlorophenyl)-10-(4-
chlorophenylmethylene)-5-oxo-7, 8, -9, 10-tetrahydrothiazolo[3ꢀ, 2ꢀ :1, 2]
pyrimido[4,5-b]quinoline-2-carbonitrile (21). The IR spectrum of

Synthesis of Pyrimido[4,5-b]quinolines
385
SCHEME 3

386
A. B. A. El-Gazzar et al.
21 displayed absorption bands at 3400 cm−1 (NH2), 2216 cm−1
(CN), and 1694 cm−1 (CO), and the 1H-NMR revealed that the
absence of an ethyl group and showed the broad signal at 8.90,
characteristic of the NH2 group. Compound 21 was cyclized to
4-amino-8-(4-chlorophenylmethylene)-12-(4-chlorophenyl) - 8, 9, 10, 11-
tetrahydropyrimido[4ꢀꢀ, 5ꢀꢀ,:4ꢀ, 5ꢀ]thiazolo[3ꢀ,2ꢀ:1, 2]pyrimido[4,5-b]quin-
olin-13-one (15) upon heating in an ethanolic sodium ethoxide solution.
Antioxidant Activity Screening
The antioxidant activity of pyrimido[4,5-b]quinoline and their deriva-
tives was determined with three standard assays. The antioxidant ac-
tivity was measured as the ability of pyrimido[4,5-b]quinoline and its
derivatives to react with the preformed radical monocation of ABTS+.
This assay is also known as the total radical-trapping antioxidant pa-
rameter assay (TRAP assay). The antioxidant activity was also mea-
sured by erythrocyte hemolysis (Table I) and by bleomycin-dependent
DNA damage.
In all three assays, ascorbic acid was used as a control. The results of
the ABTS assay of the antioxidant activity are shown in Table II. This
indicates that compounds 7a–c, 8a, 8b, and 15 exhibit high activity due
to the presence of arylamino, piperazino, or morpholino groups. While
the other synthesized compounds showed moderate activity in compari-
son to the control, the compounds that exhibit high activity were chosen
to assay for bleomycin-dependent DNA damage. The results indicate
that they may have some protective activity to DNA by a certain mech-
anism. The series of compounds 7a–c, 8a, 8b, 13, and 15 exhibited a
high antioxidant activity. On the other hand, these compounds protect
the DNA from the induced damage by bleomycin (Table III).
Anti-Inflammatory Activity
The anti-inflammatory activity was evaluated by the carrageenan-
induced paw edema test in rats. The anti-inflammatory activity data
(Table IV) indicated that all the test compounds protected rats from
carrageenan-induced inflammation, and some of the tested compounds
are more potent than our earlier reported ones. Compounds 6a–c, 7a,b,
13, and 15 showed similar or higher anti-inflammatory activity than
diclofenac sodium.

Synthesis of Pyrimido[4,5-b]quinolines
387
TABLE I Antioxidant Assays by Erythrocyte Hemolysis
Erythrocyte hemolysis 1-A/B × 100
Absorbance of samples (A) % Hemolysis
Methods
Compounds
Complete hemolysis with Dist-H2O (B)
Ascorbic acid
4
5
10
6a
6b
6c
7a
7b
8
0.660
0.026
0.042
0.048
0.062
0.236
0.284
0.154
0.232
0.281
0.062
0.048
0.031
0.028
0.218
0.052
0.055
0.236
0.027
0.055
0.056
0.038
0.032
3.93
6.36
7.27
9.39
35.75
36.72
19.18
35.70
36.68
9.39
7.27
4.64
4.24
30.60
7.87
8.33
35.75
4.09
8.19
8.34
9
11
12
13
14a
14b
15
16a
16b
17
18
20
5.76
4.55
Analgesic Activity
The analgesic activity was determined by the hot plate test (central
analgesic activity) and acetic-acid–induced writhing assay. The results
(Tables V and VI) reveal that all test compounds exhibited significant
activity. Most of the tested compounds have nearly the same activity
as the reference drug, and the remaining tested compounds have good
activities in central analgesic activity. Also 2-arylaminopyrimido[4,5-b]-
quinoline (6a–c), 2-piperazino-, 2-morphlinopyrimidoquinoline (7a–c),
and 3-amino-thiazolo[3ꢀ,2ꢀ,1,2]pyrimido[4,5-b]quinoline-2-carbonitrile
(13) exhibit activities higher than the reference drug in peripheral
analgesic activity testing. The remaining compounds have the same
activity in peripheral analgesic activity testing.

388
A. B. A. El-Gazzar et al.
TABLE II Antioxidant Assays by ABTS Method
ABTS Abs (control)-Abs (Test)/Abs (control) × 100
Methods
Compounds
Absorbance of samples
% Inhibition
Control of ABTS
Ascorbic acid
4
5
10
6a
6b
6c
7a
7b
8
0.54
0.06
3.37
0.42
0.36
0.09
0.20
0.16
0.26
0.31
0.36
0.34
0.31
0.35
0.27
0.37
0.34
0.14
0.35
0.32
0.28
0.34
0.37
0
88.9
33.5
26.8
37.8
82.5
62.3
68.7
50.4
42.7
33.3
37.0
42.6
35.2
48.3
31.5
37.0
62.5
35.2
40.7
48.1
37.0
31.5
9
11
12
13
14a
14b
15
16a
16b
17
18
20
TABLE III Assay for Bleomycin–Dependent
DNA Damage (DNA)
Methods
Bleomycin-dependent DNA damage
Compounds
Absorbance of samples
Ascorbic acid
0.020
0.024
0.022
0.034
0.032
0.030
0.025
0.028
6a
6b
6c
7a
7b
13
15

Synthesis of Pyrimido[4,5-b]quinolines
389
TABLE IV Percent Inflammatory Activity of the Tested
Compounds (Carrageenan-Induced Paw Edema Test in Rats)
Percent protection
Compd. No.
1 hour
2 hours
3 hours
6a
6b
6c
7a
7b
9
11
12a
13
14a
14b
15
49.0 1.27∗∗∗
56.3 1.37
59.5 1.06∗∗
59.8 1.41∗∗
49.6 2.41∗
46.3 1.53∗
46.7 2.28∗
41.2 1.38∗
42.3 1.63∗
44.1 1.83∗
39.8 1.42∗
43.1 162∗
6.1 0.27
53.2 1.32∗∗
62.0 1.76∗∗
62.4 2.03∗∗
59.3 1.32∗
57.1 1.63∗
48.6 139∗
44.2 1.83∗
42.5 1.46∗
45.8 1.47∗
49.0 1.14
42.3 1.42∗
52.0 1.95∗∗
5.7 0.32
41.0 1.31∗
40.2 1.05∗
46.5 1.26∗
42.3 1.26∗
44.8 1.83∗
38.6 1.39∗
39.2 1.04∗
31.1 1.33∗
42.9 1.63∗
30.5 1.83∗
25.3 1.32∗
42.0 1.92∗
3.2 0.93
Control
Diclofenac Sodium
52.4 0.92∗
60.3 1.52∗∗
42.0 1.36∗
Each value represents the mean S.E (n = 6).
Significance levels ∗p < 0.5, ∗∗p < 0.001 as compared with respective
control.
Dose (20 mg/kg) for the selected tested compound.
TABLE V Central Analgesic Activity (Hot Plate Test)
Reaction time (min.)
Group
0 min
30 min
60 min
90 min
Control
6a
6b
6c
7a
7b
9
11
12a
13
8.24 0.33
6.08 0.90
7.08 0.13
8.09 0.34
7.27 0.40
9.38 0.30
8.43 0.61
8.26 0.40
6.64 0.20
8.26 0.40
8.91 0.65
6.24 0.57
7.42 0.36
8.11 0.36b
7.93 0.74
8.65 0.87
8.83 0.35a
8.50 0.48
10.14 0.26b
8.62 0.34
8.68 0.48
7.54 0.26b
9.68 0.48
9.19 0.57a
7.08 0.78a
8.43 0.45a
10.05 0.12a
8.62 0.41b
10.15 1.20
10.98 0.91a
9.87 0.58a
9.86 0.52
9.52 0.40b
11.51 0.40a
11.12 0.75a,b
10.46 0.48b
10.47 0.18b
7.63 0.60b
10.74 0.28b
9.06 0.56b
9.80 0.52
9.22 0.47b
10.40 0.18b
12.00 0.36a
10.43 0.18b
11.08 0.24b
12.68 0.61a
10.68 0.59a,b
13.20 0.38a
8.61 0.60a
9.82 0.52
14a
14b
15
10.78 0.45a
9.52 0.82a
10.43 0.29a
11.40 0.53a
Diclofanc sodium 6.51 0.40
Values represent the mean S.E. of 6 animals for each groups.
aP< 0.05: Statistically significant from control (Dunnett’s test).
bP< 0.05: Statistically significant from ASA (Dunnett’s test).
∗Significant at p < 0.05.

390
A. B. A. El-Gazzar et al.
TABLE VI Percent Analgesic Activity (Peripheral, Writhing Test)
Percent protection
Compd. No.
30 min
1 hour
2 hours
3 hours
6a
6b
6c
7a
7b
62.2 1.15∗∗∗
61.4 1.51∗∗
70.0 1.05
73.4 1.05∗∗
46.7 1.51∗
50.4 1.34∗
46.0 1.93∗
43.5 1.92∗
41.4 1.16∗
43.4 1.21∗
43.2 1.32∗
41.5 1.43∗
02.0 0.35
65.3 1.31∗∗
69 1.56∗∗
74 1.93∗∗
76.3 1.39∗∗
53 1.49∗∗
52 1.02∗
54 1.41∗
49 1.37∗
47 1.32∗
48 1.42∗
45 1.03∗
52 1.26∗
06.0 0.50
55.2 1.16∗
69.4 1.03∗∗
74.3 1.84∗∗
75.4 1.51∗∗
77.6 1.31
59.3 1.69
55.5 1.37
56.6 1.39
53.1 1.16
48.8 1.39
51.3 1.60
46.1 1.39
49.3 1.92
04.0 0.59
62 1.49∗
50.4 1.73∗
48.3 172∗∗
58.2 1.17
63.3 1.39∗∗
65.3 1.31∗
37.6 1.49∗
37.3 1.23∗
38.4 1.81∗
67.3 1.53∗
39.6 1.83∗
34.7 1.15∗
37.3 1.39∗
04.0 0.90
39 1.13∗
9
11
12a
13
14a
14b
15
Control
Diclfenac sodium 46.0 095∗
Each value represents the mean S.E (n = 6).
Significance levels ∗p < 0.5, ∗∗p < 0.001 as compared with respective control.
Dose (20 mg/kg) for the selected tested compound.
CONCLUSIONS
The new ring systems seem to be interesting for biological activity stud-
ies. Furthermore, the present investigation offers rapid and effective
new procedures for the synthesis of the polycondensed new hetero-
cyclic ring systems. Compounds (6a–c) showed the highest inhibitory
antioxidant activity either using erythrocytes hemolysis or ABTS meth-
ods. These quinolones are the phenylamino, 4-chlorophenylamino, or
4-methoxyphenylamino derivatives. This means that the presence of
the phenyl group will potentiate the activity, which may increase by
introducing an electron-donating group, e.g. OCH3, or an electron-
withdrawing group, e.g., Cl in the p-position. Compounds 6a–c, 7b,13,
and 15 manifested the best protective effect against DNA damage in-
duced by bleomycin. Compounds 6a–c, 7a,b, and 13 exhibited a po-
tent anti-inflammatory activity according to the carrageenan-induced
paw edema test in rats. The p-methoxy derivative of 6 exhibited the
best activity in all tested assays. This suggests the mechanism of anti-
inflammatory and analgesic activities of compounds 6a–c, especially
6c, to be due to the potent antioxidant activity to maintain the in-
tegrity of both the cell membrane and DNA of the cells.

Synthesis of Pyrimido[4,5-b]quinolines
391
EXPERIMENTAL
All melting points are uncorrected and measured using an Electrother-
mal IA 9100 apparatus (Shimadzu, Japan). The 1H-NMR and 13C-NMR
spectra were recorded on JEOL JNM-LA-400 FT NMR Spectrome-
ter (University of Konstanz, Germany) and chemical shifts were ex-
pressed as δ values against SiMe4 as internal standards. IR spectra
were recorded as KBr pellets on a Perkin-Elmer 1430 spectrometer
(National Research Center). Mass spectra were recorded on GCMS-Q
P1000 EX, Shimadzu, Japan (cas chromatography-mass spectrometer).
Microanalytical data were performed by the Microanalytical Center at
Cairo University (Egypt). The starting materials are prepared accord-
ing to El-Gazzar et al.14,15 The biological evaluation of the products was
carried out at the Fermentation Biotechnology & Applied Microbiology
(Ferm-BAM) Center at Al-Azhar University, Cairo, Egypt.
5-(4-Chlorophenyl)-9-(4-chlorophenylmethylene)-2-thioxo-6,7,
8,9-tetrahydro-pyrimido[4,5-b]quinolin-4-one (4)
A mixture of 4-chlorobenzaldehyde (1.40 g, 10 mmol), cyclohexanone
(1.12 g, 10 mmol), and 6-aminothiouracil 3 (1.43 g, 0.01 mol) was re-
fluxed in 50 mL DMF for 50 h (under TLC control). The reaction mix-
ture was cooled; the precipitate was filtered off, washed with ethanol,
dried, and crystallized from DMF, as a yellow powder, in 80 % yield,
m.p. 297–299◦C; IR, cm−1: 3361 (brs, NH), 3025 (CH aryl), 2911 (CH
1
alkyl), 1688 (CO), 1631 (C N). H-NMR (DMSO-d6) ppm: δ 1.64–1.67
(m, 2H, CH2), 2.29–2.32 (t, 2H, CH2) 2.75–2.97 (t, 2H, CH2), 7.10–
7.12 (d, 2H, Ar-H), 7.14–7.15 (d, 2H, Ar-H), 7.16–7.17 (dd, 4H, Ar-H),
8.19 (s, 1H, CH), 11.50 (brs, NH). 13C-NMR (DMSO-d6) ppm: δ 22.27,
26.73, 27.18 (3C, 3CH2), 108.35, 127.95, 128.29, 128.52, 128.58, 129.44,
130.14, 131.17, 132.34, 132.43, 135.53, 135.70, 136.22, 149.71, 158.47
(15C, SP2 carbon atoms), 162.25 (CO), 175.42 (CS); MS, [M+], m/z 465
(100%). C24H17Cl2N3OS (465.5); Requires (Found): C, 61.92 (62.01); H,
3.68 (3.64); N, 9.03 (9.11).
5-(4-Chlorophenyl)-9-(4-chlorophenylmethylene)-2-methylthio-
6,7,8,9-tetra-hydropyrimido[4,5-b]quinolin-4-one (5)
To a warmed ethanolic KOH solution (prepared by dissolving 0.01 mol
of KOH in 50 mL ethanol), 4 (4.65 g, 10 mmol) was added, the heating
was continued for 30 min, the mixture was allowed to cool to room
temperature, and methyl iodide (0.012 mol) was added. The mixture
was stirred under reflux for 5 h, then cooled to room temperature and

392
A. B. A. El-Gazzar et al.
poured into cold water (100 mL). The solid product precipitated was
filtered off and washed with 100 mL water, and the product was dried
and crystallized from dioxane in 80% yield, m.p. 327–330◦C; IR, cm−1
:
3403 (br, NH), 3036 (CH aryl), 2925 (CH alkyl), 1687 (CO), 1652 (C N);
1H-NMR (DMSO-d6, δ, ppm): 1.65–1.68 (t, 2H, CH2), 2.30–2.36 (t, 2H,
CH2), 2.73–2.77 (m, 2H, CH2), 2.84 (s, 3H, SCH3), 7.18–7.23 (d, 2H, Ar-
H), 7.31–7.35 (d, 2H, Ar-H), 7.43–7.48 (d, 2H, Ar-H), 7.56–7.63 (d, 2H,
Ar-H), 8.34 (s, 1H, CH), 9.20 (br, NH, D2O exchangeable); MS: [M+],
m/z 480, (100%), [M+-SCH3], m/z 433 (44%); C25H19Cl2N3OS (480.4);
Requires (Found): C, 62.50 (62.47); H, 3.98 (3.94); N, 8.75 (8.79).
2-Arylamino-5-(4-chlorophenyl)-9-(4-chlorophenylmethylene)-
6,7,8,9-tetra-hydropyrimido[4,5-b]quinoline (6a–c or 7a,b):
General Procedure
Into a warm solution of 5 (4.79 g, 10 mmol) in methanol (100 mL) was
added the freshly distilled amine (10 mmol). The reaction mixture was
stirred under reflux for 5 h, then allowed to cool to 0◦C for 12 h. The
solid obtained was filtered, washed with water (100 mL), dried, and
recrystallized from appropriate solvent to produce (6a–c) and (7a,b).
5-(4-Chlorophenyl)-9-(4-chlorophenylmethylene)-2-
phenylamino-6,7,8,9-tetra-hydropyrimido[4,5-b]quinolin-4-
one (6a)
It was obtained from aniline (0.93 g, 10 mmol) as yellow crystals,
crystallized from dioxane; in 76% yield, m. p. 215–217◦C; IR, cm−1: 3400
(brs NH), 3030 (CH aryl), 2915 (CH alkyl), 1683 (C O), 1600 (C N).
1H-NMR (DMSO-d6, δ, ppm): 1.63–1.67 (t, 2H, CH2) 2.28–2.35 (t, 2H,
CH2), 2.70–2.74 (m, 2H, CH2), 6.97–7.13 (m, 3H, Ar-H), 7.19–7.25 (d,
2H, Ar-H), 7.37–7.43 (m, 2H, Ar-H), 7.47–7.53 (dd, 4H, Ar-H), 7.60–7.64
(d, 2H, Ar-H), 8.30 (s, 1H, CH), 9.20 (br, NH, D2O exchangeable), 10.30
(br, NH, D2O exchangeable); MS: [M+], m/z 525, (73%), [M++1], m/z
526 (29%); C30H22Cl2N4O (525.4); Requires (Found): C, 68.57 (68.49);
H, 4.22 (4.19); N, 10.66 (10.57).
2-(4-Chlorophenylamino)-5-(4-chlorophenyl)-9-(4-chloropheny-
lmethylene)-6,7,-8,9-tetrahydropyrimido[4,5-b]quinolin-4-
one (6b)
It was obtained from 4-chloro-aniline (1.27 g, 10 mmol) as yellow
crystals, crystallized from ethanol; in 73% yield, m.p. 221–223◦C; IR,
cm−1: 3395 (brs, NH), 3039 (CH aryl), 2924 (CH alkyl), 1685 (C O),
1605 (C N); 1H-NMR (DMSO-d6, δ, ppm): 1.65–1.68 (t, 2H, CH2) 2.25–

Synthesis of Pyrimido[4,5-b]quinolines
393
2.34 (t, 2H, CH2), 2.66–2.72 (m, 2H, CH2), 7.00–7.18 (dd, 4H, Ar-H),
7.21–7.26 (d, 2H, Ar-H), 7.33–7.40 (2d, 4H, Ar-H), 7.63–7.69 (d, 2H,
Ar-H), 8.33 (s, 1H, CH), 9.50 (br, NH, D2O exchangeable), 10.00 (br,
NH, D2O exchangeable); MS: [M+], m/z 559, (100%), [M++1], m/z 560
(27%); C30H21Cl3N4O (559.9); Requires (Found): C, 64.35 (64.32); H,
3.78 (3.74); N, 10.01 (9.97).
5-(4-Chlorophenyl)-9-(4-chlorophenylmethylene)-2-(4-
methoxyphenylamino)-6,7,8,9-tetrahydropyrimido[4,5-b]
quinolin-4-one (6c)
The compound was obtained from 4-anisidine (1.23 g, 10 mmol) as
pale yellow crystals, crystallized from ethanol; in 77% yield, m.p. 217–
219◦C; IR, cm−1: 3420 (brs, NH), 3045 (CH aryl), 2928 (CH alkyl), 1683
(C O), 1600 (C N); 1H-NMR (DMSO-d6, δ, ppm): 1.63–1.68 (t, 2H, CH2)
2.28–2.33 (t, 2H, CH2), 2.68–2.73 (m, 2H, CH2), 3.86 (s, 3H, OCH3),
6.96–7.14 (dd, 4H, Ar-H), 7.20–7.26 (d, 2H, Ar-H), 7.30–7.34 (d, 2H, Ar-
H), 7.43–7.48 (d, 2H, Ar-H), 7.62–7.66 (d, 2H, Ar-H), 8.43 (s, 1H, CH),
9.60 (br, NH, D2O exchangeable), 10.30 (br, NH, D2O exchangeable);
MS: [M+], m/z 555, (100%); C31H24Cl2N4O2 (555.4); Requires (Found):
C, 67.03 (67.00); H, 4.35 (4.37); N, 10.08 (10.11).
5-(4-Chlorophenyl)-9-(4-chlorophenylmethylene)-2-piperazino-
6,7,8,9-tetra-hydropyrimido[4,5-b]quinolin-4-one (7a)
The compound was obtained from piperazine (0.86 g, 10 mmol) as
pale yellow crystals, crystallized from ethanol in 70% yield, m.p. 201–
203◦C; IR, cm−1: 3380 (br, NH), 3053 (CH aryl), 2930 (CH alkyl), 1684
(C O), 1590 (C N); 1H-NMR (DMSO-d6, δ, ppm): 1.65–1.69 (t, 2H, CH2)
2.24–2.32 (t, 2H, CH2), 2.67–2.73 (m, 2H, CH2), 3.26–3.31 (t, 2H, CH2),
3.38–3.41 (m, 4H, CH2), 3.46–3.50 (t, 2H, CH2), 6.98–7.16 (d, 2H, Ar-
H), 7.28–7.32 (d, 2H, Ar-H), 7.36–7.40 (d, 2H, Ar-H), 7.51–7.55 (d, 2H,
Ar-H), 8.35 (s, 1H, CH), 9.60 (br, NH, D2O exchangeable), 10.50 (br,
NH, D2O exchangeable); MS: [M+], m/z 518, (100%), [M++1], m/z 519
(26%); C28H25Cl2N5O (518.4); Requires (Found): C, 64.86 (64.79); H,
4.86 (4.84); N, 13.51 (13.48).
5-(4-Chlorophenyl)-9-(4-chlorophenylmethylene)-2-
morpholino-6,7,8,9-tetra-hydropyrimido[4,5-b]quinolin-4-one
(7b)
The compound was obtained from morpholine (0.87 g, 10 mmol) as
yellow crystals, crystallized from ethanol; in 62% yield, m.p. 187–190◦C;
IR, cm−1: 3415 (brs, NH), 3036 (CH aryl), 2929 (CH alkyl), 1688 (C O),
1620 (C N); 1H-NMR (DMSO-d6, δ, ppm): 1.68–1.75 (t, 2H, CH2) 2.26–

394
A. B. A. El-Gazzar et al.
2.32 (t, 2H, CH2), 2.67–2.73 (m, 2H, CH2), 3.27–3.32 (t, 2H, CH2), 3.40–
3.42 (t, 2H, CH2), 3.84–3.88 (m, 4H, 2CH2), 6.96–7.12 (d, 2H, Ar-H),
7.24–7.29 (d, 2H, Ar-H), 7.38–7.44 (d, 2H, Ar-H), 7.56–7.61 (d, 2H, Ar-
H), 8.36 (s, 1H, CH), 9.60 (br, NH, D2O exchangeable); MS: [M+], m/z
519, (68%), [M++1], m/z 520 (30%); C28H24Cl2N4O2(519.4); Requires
(Found): C, 64.74 (64.69); H, 4.66 (4.64); N, 10.78 (10.88).
2-(o-Carboxyphenylamino)-5-(4-chlorophenyl)-9-(4-
chlorophenylmethylene)-6,-7,8,9-tetrahydropyrimido[4,5-
b]quinolin-4-one (8)
The compound was obtained from anthranilic acid (1.37 g, 10 mmol)
as yellow powder, crystallized from ethanol; in 75% yield, m.p. 243–
245◦C; IR, cm−1; 3520 (br, OH), 3390 (br, NH), 3027 (CH aryl), 2919
1
(CH alkyl), 1718 (C O), 1685 (C O), 1600 (C N); H-NMR (DMSO-
d6, δ, ppm); 1.66–1.71 (t, 2H, CH2) 2.23–2.31 (t, 2H, CH2), 2.69–2.76
(m, 2H, CH2), 6.97–7.14 (dd, 4H, Ar-H), 7.20–7.25 (d, 2H, Ar-H), 7.36–
7.40 (m, 2H, Ar-H), 7.47–7.53 (d, 1H, Ar-H), 7.55–7.61 (d, 2H, Ar-H),
7.66–7.74 (d, 1H, Ar-H), 8.30 (s, 1H, CH), 9.20, 10.30 (2br, 2NH, D2O
exchangeable), 12.50 (brs, OH, D2O exchangeable); MS: [M+], m/z 569,
(100%); C31H22Cl2N4O3 (569.4); Requires (Found): C, 65.38 (65.29); H,
3.89 (3.92); N, 9.84 (9.87).
12-(4-Chlorophenyl)-8-(4-chlorophenylmethylene)-8,9,10,11-
terahydroquino[2ꢀ,-3ꢀ:4,5]pyrimido[2,1-b]quinazoline-13,14-
dione (9)
A solution of 9 (2.84, 5 mmol) in glacial acetic acid (40 mL) and a
catalytic amount of sulfuric acid (1 mL) was stirred under reflux for
8 h. The reaction mixture was allowed to cool, poured into cold water
(100 mL) and neutralized with ammonia solution. The solid precipitate
was filtered off, washed with water, dried, and crystallized from DMF
as yellow crystals; in 76% yield, m.p. 229–231◦C; IR, cm−1: 3028 (CH
aryl), 2927 (CH alkyl), 1705 (C O), 1692 (C O), 1620 (C N); 1H-NMR
(DMSO-d6, δ, ppm): 1.67–1.73 (t, 2H, CH2), 2.26–2.34 (t, 2H, CH2),
2.69–2.76 (m, 2H, CH2), 6.99–7.17 (dd, 4H, Ar-H), 7.21–7.25 (d, 2H,
Ar-H), 7.33–7.40 (m, 2H, Ar-H), 7.50–7.54 (d, 1H, Ar-H), 7.60–7.65 (d,
2H, Ar-H), 7.72–7.78 (d, 1H, Ar-H), 8.43 (s, 1H, CH), 10.25 (br, NH,
D2O exchangeable); MS: [M+], m/z 551, (100%); C31H20Cl2N4O2 (551.4);
Requires (Found): C, 67.52 (67.49); H, 3.65 (3.59); N, 10.16 (10.12).

Synthesis of Pyrimido[4,5-b]quinolines
395
4-Chloro-5-(4-chlorophenyl)-9-(4-chlorophenylmethylene)-2-
methylthio-6,7,8,9-tetrahydropyrimido[4,5-b]quinoline (10)
A solution of 5 (4.79 g, 10 mmol) in dry dioxane (30 mL) was treated
with 10 mL of phosphorus oxychloride, and the mixture was stirred
under reflux for 3 h. The reaction mixture was allowed to cool to room
temperature, and poured into cold water (100 mL), whereby a solid was
separated, filtered off, and crystallized from ethanol (yellow powder),
in 80% yield, m.p. 360–362◦C; IR, cm−1; 3049 (CH aryl), 2919 (CH
alkyl), 1600 (C N), 1160 (C-Cl); 1H-NMR (CDCl3, δ, ppm); 1.65–1.69 (t,
2H, CH2) 2.31–2.39 (t, 2H, CH2), 2.65–2.72 (m, 2H, CH2), 2.84 (s, 3H,
SCH3), 6.91–7.11 (d, 2H, Ar-H), 7.15–7.26 (d, 2H, Ar-H), 7.34–7.40 (d,
2H, Ar-H), 7.42–7.53 (d, 2H, Ar-H), 8.25 (s, 1H, CH); MS: [M+], m/z 498,
(78%), [M++1], m/z 499 (29%); C25H18Cl3N3S(498.8); Requires (Found):
C, 60.19 (60.17); H, 3.63 (3.65); N, 8.42 (8.43).
5-(4-Chlorophenyl)-9-(4-chlorophenylmethylene)-2,4-
dihydrazino-6,7,8,9-tetra-hydropyrimido[4,5-b]quinoline (11)
A mixture of 6 (4.98 g, 10 mmol) and hydrazine hydrate (99–100%,
20 mL) was stirred under reflux in dioxane (50 mL) and ethanol (20
mL) for 12 h. The reaction mixture was allowed to cool to 0◦C for 5 h,
and the solid was collected by filtration and crystallized from dioxane
as pale yellow powder; in 90% yield, m.p. 298–300◦C; IR, cm−1: 3438–
3350 (brs, NH), 3035 (CH aryl), 2923 (CH alkyl), 1625 (C N); 1H-NMR
(DMSO-d6, δ, ppm); 1.67–1.75 (t, 2H, CH2), 2.26–2.34 (m, 2H, CH2), 2.45
(br, NH2, D2O exchangeable), 2.75–2.78 (m, 2H, CH2), 7.05–7.09 (d, 2H,
Ar-H), 7.14–7.18 (d, 2H, Ar-H), 7.27–7.34 (d, 2H, Ar-H), 7.40–7.45 (d,
2H, Ar-H), 8.32 (s, 1H, CH), 8.54–8.80 (br, NH2, D2O exchangeable),
10.70, 11.60 (two, brs, 2NH, D2O exchangeable); C24H21Cl2N7 (478.4);
Requires (Found): C, 60.25(60.23); H, 4.43 (4.45); N, 20.50 (20.52).
6-(4-Chlorophenylmethylene)-10-(4-chlorophenyl)-6,7,8,9-
tetrahydro-s-triazolo-[3ꢀ,4ꢀ:6,1]-s-triazolo[3ꢀꢀ,4ꢀꢀ,:2,3]
pyrimido[4,5-b]quinoline (12a)
A mixture of compound 11 (2.39 g, 5 mmol) and formic acid (30 mL)
was heated under reflux for 18 h. The reaction mixture was allowed to
cool to room temperature and poured into water (100 mL). The solid
formed was collected by filtration, washed with ethanol (20 mL), dried,
and crystallized from ethanol; in 69 % yield, m.p. 289–291◦C; IR, cm−1
:
1
3025 (CH aryl), 2943 (CH alkyl), 1605 (C N), 1560 (C=C). H-NMR
(DMSO-d6, δ, ppm): 1.69–1.73 (m, 2H, CH2), 2.36–2.40 (t, 2H, CH2),

396
A. B. A. El-Gazzar et al.
2.77–2.83 (t, 2H, CH2), 7.14–7.18 (d, 2H, Ar-H), 7.40–7.47 (dd, 4H, Ar-
H), 7.52–7.56 (d, 2H, Ar-H), 8.15 (s, 1H, CH), 8.25 (s, 1H, triazole), and
8.67 (s, 1H, triazole). 13C-NMR (DMSO-d6) ppm: δ 22.83, 26.36, 27.09
(3C, 3CH2), 108.6, 128.1, 128.2, 128.4, 129.3, 130.9, 131.3, 131.5, 132.4,
132.6, 134.3, 135.8, 136.4, 143.1, 144.5, 152.6, 154.6, 155.7, 158.7 (23
sp2 carbon atoms); C26H17Cl2N7 (498.4); Requires (Found): C, 62.65
(62.57); H, 3.44 (3.41); N, 14.23 (14.19).
6-(4-Chlorophenylmethylene)-10-(4-chlorophenyl)-6,7,8,9-
tetrahydro-3,14-dimethyl-s-triazolo[3ꢀ,4ꢀ:6,1]-s-triazolo
[3ꢀꢀ,4ꢀꢀ,:2,3]pyrimido[4,5-b]quinoline (12b)
A mixture of 11 (2.39 g, 5 mmol) and glacial acetic acid (80 mL) was
stirred under reflux for 20 h (TLC control). The reaction mixture was al-
lowed to cool to room temperature and poured into water (100 mL). The
solid formed was collected by filtration, washed with ethanol (20 mL),
dried, and crystallized from DMF; in 72 % yield, m.p. 256–258◦C;
IR,cm−1: 3038 (CH aryl), 2972 (CH alkyl), 1698 (CO), 1636 (C N), 1565
(C=C). 1H-NMR (DMSO-d6, δ ppm): 1.62–1.66 (m, 2H, CH2), 2.27–2.32
(t, 2H, CH2), 2.41 (s, 3H, CH3), 2.44 (s, 3H CH3), 2.76-2.79 (t, 2H, CH2),
7.09–7.14 (d, 2H, Ar-H), 7.40–7.45 (d, 2H, Ar-H), 7.48–7.52 (dd, 4H, Ar-
H), 8.12 (s, 1H, CH); C28H21Cl2N7 (526.4); Requires (Found): C, 63.88
(63.86); H, 4.02 (3.99); N, 18.63 (18.66).
3-Amino-6-(4-chlorophenyl)-10-(4-chlorophenylmethylene)-5-
oxo-7,8,9,10-tetra-hydrothiazolo[3ꢀ,2ꢀ:1,2]pyrimido[4,5-b]
quinoline-2-carbonitrile (13)
A mixture of compound 4 (4.65 g, 10 mmol), bromomalononitrile (1.45 g,
10 m mol) and anhydrous potassium carbonate (2.76 g, 20 mmol) was
stirred under reflux in 30 mL ethanol for 12 h (TLC control). The reac-
tion mixture was allowed to cool to room temperature and then poured
into cold water (100 mL). The precipitate was filtered off, dried, and
crystallized from benzene as brown crystals, crystallized from ethanol;
in 68% yield, m.p. 262–264◦C; IR, cm−1: 3410 (brs, NH), 3053 (CH aryl),
1
2915 (CH, alkyl), 2215 (CN), 1686 (CO), 1610 (C N), 1520 (C=C); H-
NMR (DMSO-d6, δ, ppm): 1.63, 1.74 (m, 2H, CH2), 2.25–2,34 (m, 2H,
CH2), 2.72–2.78 (m, 2H, CH2), 7.10–7.19 (dd, 4H, Ar-H), 7.28–7.33 (d,
2H, Ar-H), 7.35–7.43 (d, 2H, Ar-H), 8.19 (s, 1H, CH), 8.90 (br, NH2,
D2O exchangeable); 13C-NMR; 24.30, 26.97, 28.06 (3C, 3CH2), 68.50
(1C, CN), 108.5, 125.1, 127.8, 128.1, 128.7, 128.8, 129.3, 129.5, 130.8,

Synthesis of Pyrimido[4,5-b]quinolines
397
131.0, 132.6, 135.3, 135.6, 135.9, 141.6, 147.9, 152.8, 158.7 (22C, sp2
carbon atoms) and 163.88 (CO); MS: [M+], m/z 530, (100%), [M+2-H2N-
C=C-CN], m/z 464 (17%); C27H17Cl2N5OS (530.5); Requires (Found):
C, 61.14 (61.18); H, 3,23 (3.19); N, 13.20 (13.16).
8-(4-Chlorophenylmethylene)-12-(4-chlorophenyl)-8,9,10,11-
tetrahydropyrim-ido[4ꢀꢀ,5ꢀꢀ,:4ꢀ,5ꢀ]thiazolo[3ꢀ,2ꢀ:1,2]pyrimido
[4,5-b]quinoline-4,13-dione (14a)
A mixture of compound 13 (2.65 g, 5 mmol), formic acid (10 mL), and
a catalytic amount of concentrated hydrochloric acid was heated under
reflux for 16 h. The reaction mixture was allowed to cool to room tem-
perature, and poured into cold water (100 mL). The formed solid was
collected by filtration, washed by ethanol (20 mL), dried, and crystal-
lized from DMF (yellow) in 79% yield, m.p 233–235◦C; IR, cm−1: 3380
(brs, NH), 3064 (CH aryl), 2940 (CH alkyl), 1690, 1679 (2CO), 1615
1
(C N), 1525 (C=C); H-NMR (DMSO-d6, δ, ppm): 1.65–1.73 (m, 2H,
CH2), 2.28–2.36 (m, 2H, CH2), 2.82–2.87 (m, 2H, CH2), 7.15–7.22 (d,
2H, Ar-H), 7.24–7.29 (dd, 4H, Ar-H), 7.51–7.55 (d, 2H, Ar-H), 8.22 (s, 1H,
CH), 8.88 (s, 1H, CH pyrimidine) and 11.20 (brs, NH, D2O exchange-
able); C28H17Cl2N5O2S (558.4); Requires (Found): C, 60.23 (60.26); H,
3.06 (2.99); N, 12.54 (12.49).
8-(4-Chlorophenylmethylene)-12-(4-chlorophenyl)-2-methyl-
8,9,10,11-tetra-hydropyrimido[4ꢀꢀ,5ꢀꢀ,:4ꢀ,5ꢀ]thiazolo[3ꢀ,2ꢀ:1,2]
pyrimido[4,5-b]quinoline-4,13-dione (14b)
A mixture of compound 13 (5.30 g, 0.01 mol) and acetic acid (80 mL)
was heated under reflux for 20 h. The reaction mixture was allowed
to cool to room temperature and poured into cold water (100 mL).
The formed solid was collected by filtration, washed with ethanol (20
mL), dried, and crystallized from ethanol as yellow powder; in 77%
yield, m.p. 223–225◦C; IR, cm−1: 3400 (brs, NH), 3054 (CH aryl),
2932 (CH alkyl), 1689, 1676 (2CO), 1625 (C N), 1510 (C=C); 1H-
NMR (DMSO-d6, δ, ppm): δ 1.65–1.76 (m, 2H, CH2), 2.25–2.28 (t,
2H, CH2), 2.33 (s, 3H, CH3), 2.75–2.79 (t, 2H, CH2), 7.10-7.14 (d,
2H, Ar-H), 7.29-7.34 (dd, 4H, Ar-H), 7.42–7.44 (d, 2H, Ar-H), 8.02
(s, 1H, CH), 10.00 (brs, NH, D2O exchangeable); C29H19Cl2N5O2S
(572.5); Requires (Found): C, 60.85 (60.78); H, 3.34 (3.29); N, 12.34
(12.29).

398
A. B. A. El-Gazzar et al.
4-Amino-8-(4-chlorophenylmethylene)-12-(4-chlorophenyl)-
8,9,10,11-tetrahydro-pyrimido[4ꢀꢀ,5ꢀꢀ,:4ꢀ,5ꢀ]thiazolo[3ꢀ,2ꢀ:1,2]
pyrimido[4,5-b]quinolin-13-one (15)
Method A
A mixture of compound 13 (5.30 g, 0.01 mol), formamide (10 mL), and
formic acid (2 mL) was stirred under reflux in DMF (50 mL) for 6 h.
The reaction mixture was allowed to cool to room temperature, poured
into water (100 mL), and neutralized with ammonia solution. The pre-
cipitate was collected by filtration, washed with water and ethanol,
dried, and crystallized from dioxane in 64% yield, m.p. 295◦C (dec.);
IR, cm−1: 3390 (brs, NH), 3073 (CH aryl), 2934 (CH alkyl), 1682 (CO),
1630 (C N), 1525 (C=C); 1H-NMR (DMSO-d6, δ, ppm): 1.65–1.69 (t, 2H,
CH2), 2.28–2.33 (m, 2H, CH2), 2.76–2.81 (t, 2H, CH2), 7.11–7.16 (d, 2H,
Ar-H), 7.28–7.34 (d, 2H, Ar-H), 7.41–7.44 (d, 2H, Ar-H), 7.50–7.54 (d,
2H, Ar-H), 8.05 (s, 1H, CH), 8.25 (s, 1H, CH pyrimidine), and 8.90 (br,
NH2, D2O exchangeable); C28H18Cl2N6OS (557.4); Requires (Found): C,
60.32 (60.29); H, 3.25 (3.22); N, 15.08 (15.11).
Method B
To a warmed ethanolic sodium ethoxide solution (prepared by dis-
solving (0.23 g, 10 mmol), sodium metal in 50 mL absolute ethanol)
compound 21 (2.78 g, 5 mmol) was added. The mixture was stirred un-
der reflux for 5 h. The reaction mixture was allowed to cool to room
temperature, poured into cold water (100 mL), and neutralized with
acetic acid. The precipitate was filtered off, dried, and crystallized from
dioxane in 63% yield, with identical data.
4-Amino-8-(4-chlorophenylmethylene)-12-(4-chlorophenyl)-
8,9,10,11-tetrahydro-pyrimido[4ꢀꢀ,5ꢀꢀ,:4ꢀ,5ꢀ]thiazolo[3ꢀ,2ꢀ:1,2]
pyrimido[4,5-b]quinolin-2-(oxo/or thioxo)-13-ones (16a,b):
General Procedure
A mixture of compound 13 (5.30 g, 10 mmol) and urea or thiourea
(0.01 mol) was heated at 180◦C in a test tube on a sand-bath for 4 h.
The mixture was allowed to cool to room temperature; the product
was solidified by cooling and adding methanol (50 mL). The precipitate
was collected by filtration and crystallized from the proper solvent to
produce (16a,b).

Synthesis of Pyrimido[4,5-b]quinolines
399
4-Amino-8-(4-chlorophenylmethylene)-12-(4-chlorophenyl)-
8,9,10,11-tetrahydro-pyrimido[4ꢀꢀ,5ꢀꢀ,:4ꢀ,5ꢀ]thiazolo[3ꢀ,2ꢀ:1,2]
pyrimido[4,5-b]quinolin-2,13-dione (16a)
The compound was obtained from urea (0.61 g, 0.01 mol) as a dark
brown substance and crystallized from ethanol, in 82% yield, m.p. 216–
218◦C; IR, cm−1: 3400 (brs, NH), 3079 (CH aryl), 2938 (CH alkyl), 1694,
1686 (2CO), 1654 (C N), 1530 (C=C); 1H-NMR (DMSO-d6, δ, ppm):
1.64–1.69 (m, 2H, CH2), 2.29–2.32 (t, 2H, CH2), 2.76–2.78 (t, 2H, CH2),
7.06–7.12 (d, 2H, Ar-H), 7.39–7.41 (d, 2H, Ar-H), 7.43–7.47 (dd, 4H,
Ar-H), 8.02 (s, 1H, CH), 8.75 (brs, NH2, D2O exchangeable), 9.50 (brs,
NH, D2O exchangeable); C28H18Cl2N6O2S (573.4); Requires (Found): C,
58.64 (58.65); H, 3.16 (3.12); N, 14.66 (14.62).
4-Amino-8-(4-chlorophenylmethylene)-12-(chlorophenyl)-
8,9,10,11-tetrahydro-pyrimido[4ꢀꢀ,5ꢀꢀ,:4ꢀ,5ꢀ]thiazolo[3ꢀ,2ꢀ:1,2]
pyrimido[4,5-b]quinolin-2-thioxo-13-one (16b)
The compound was obtained from thiourea (0.77 g, 0.01 mol) as
brown crystals and crystallized from ethanol; in 80% yield, m.p. 240–
242◦C (dec.); IR, cm−1: 3397 (brs, NH), 3084 (CH aryl), 2929 (CH alkyl),
1683 (CO), 1658 (C N), 1526 (C=C); 1H-NMR (DMSO-d6, δ, ppm): 1.65–
1.69 (m, 2H, CH2), 2.31–2.36 (t, 2H, CH2), 2.69–2.74 (t, 2H, CH2), 7.09–
7.18 (dd, 4H, Ar-H), 7.21–7.29 (d, 2H, Ar-H), 7.35–7.39 (d, 2H, Ar-H),
8.16 (s, 1H, CH), 8.82 (br, NH2) and 11.50 (br, NH). C28H18Cl2N6OS2
(589.5); Requires (Found): C, 57.04 (57.03); H, 3.08 (2.99); N, 14.26
(14.19).
8-(4-Chlorophenylmethylene)-12-(4-chlorophenyl)-4-imino-
8,9,10,11-tetrahydro-quino[2ꢀꢀ,3ꢀꢀ,:4ꢀ,5ꢀ]pyrimido[2ꢀ,1ꢀ:2,3]
thiazolo[4,5-d][1,3]thiazine-13-one (17)
A mixture of compound 13 (2.65 g,5 m mol) and carbon disulfide (excess
10 mL) was heated under reflux on a water-bath (80◦C) in 40 mL pyri-
dine for 10 h (TLC control). The reaction mixture was allowed to cool
to 0◦C for 12 h, the precipitate was filtered off, washed with ethanol
(40 mL), dried, and crystallized from ethanol; in 64% yield, m.p. 272–
275◦C (dec.); IR,cm−1: 3420 (brs, NH), 3085 (CH aryl), 2932 (CH alkyl),
1678 (CO), 1625 (C N), 1520 (C=C); 1H-NMR (DMSO-d6) ppm: δ 1.63–
1.74 (m, 2H, CH2), 2.33–2.38 (t, 2H, CH2), 2.71–2.81 (t, 2H, CH2),
7.05–7.11 (d, 2H, Ar-H), 7.19–7.22 (d, 2H, Ar-H), 7.36–7.41 (d, 2H, Ar-
H), 7.46–7.51 (d, 2H, Ar-H), 8.12 (s, 1H, CH), and 9.75, 10.50 (2brs,
2NH, D2O exchangeable); 13C-NMR; 22.23, 26.65, 27.33 (3C, 3CH2),
108.6, 125.2, 127.6, 128.4, 128.6, 128.9, 129.4, 129.8, 130.6, 131.3, 132.7,

400
A. B. A. El-Gazzar et al.
135.6, 135.8, 135.5, 141.4, 147.6, 152.8, 154.3, 155.3 (23C, sp2 carbon
atoms) and 163.5 (CO), 176.8 (C=S); C28H17Cl2N5OS3 (606.5); Requires
(Found): C, 55.44 (55.39); H, 2.82 (2.84); N, 11.55 (11.49).
3-Ethoxymethyleneamino-6-(4-chlorophenyl)-10-(4-
chlorophenylmethylene)-5-oxo-7,8,9,10-tetrahydrothiazolo
[3ꢀ,2ꢀ:1,2]pyrimido[4,5-b]quinoline-2-carbonitrile (18)
A mixture of compound 13 (2.65 g, 5 mmol) and triethyl orthoformate
(2.96 g, 20 mmol) was stirred under reflux in acetic anhydride (30 mL)
for 6 h. The reaction mixture was allowed to cool to room temperature,
poured into cold water (100 mL), and neutralized with ammonia so-
lution. The precipitate was collected by filtration, washed with water,
dried and crystallized from ethanol in 77% yield, m.p. 191–193◦C; IR,
cm−1: 3066 (CH aryl), 2922 (CH alkyl), 2215 (CN), 1690 (CO), 1620
(C N), 1510 (C=C). 1H-NMR (DMSO-d6, δ, ppm): 1.24–1.28 (t, 3H,
CH3), 1.65–1.71 (t, 2H CH2), 2.26–2.32 (t, 2H, CH2) 2.70–2.76 (t, 2H,
CH2), 4.39–4.45 (q, 2H, CH2), 7.14–7.18 (d, 2H, Ar-H), 7.28–7.33 (d,
2H, Ar-H), 7.41–7.45 (d, 2H, Ar-H), 7.50–7.56 (d, 2H, Ar-H), 8.20 (s, 1H,
CH), 8.50 (s, 1H, CH); C30H21Cl2N5O2S (586.5); Requires (Found): C,
61.43 (61.39); H, 3.61 (3.58); N, 11.94 (11.78).
6-(4-Chlorophenyl)-10-(4-chlorophenylmethylene)-3-
hydrazinomethyleneamino-5-oxo-7,8,9,10-tetrahydrothiazolo
[3ꢀ,2ꢀ:1,2]pyrimido[4,5-b]quinoline-2-carbonitrile (19)
A mixture of compound 18 (2.93 g, 5 mmol) and hydrazine hydrate (99–
100%) (20 mL) was stirred under reflux in a mixture of dioxane/ethanol
(80 mL/20 mL) for 12 h. The reaction mixture was allowed to cool to
room temperature, poured into cold water (100 mL), and neutralized
by acetic acid. The precipitate was collected by filtration, washed with
water, dried, and crystallized from ethanol; in 69% yield, m.p. 299–
301◦C; IR, cm−1: 3420 (brs, NH), 3054 (CH aryl), 2926 (CH alkyl),
2216 (CN), 1683 (CO), 1630 (C N), 1505 (C=C).1H-NMR (DMSO-d6,
δ, ppm): 1.68–1.76 (t, 2H, CH2), 2.27–2.37 (m, 2H, CH2), 2.69–2.76 (t,
2H, CH2), 7.18–7.21 (d, 2H, Ar-H), 7.31–7.35 (dd, 4H, Ar-H), 7.40–7.56
(d, 2H, Ar-H), 8.06 (s, 1H, CH), 8.45 (s, 1H, CH), 8.80 (br, NH2, D2O
exchangeable) and 10.00 (br, NH, D2O exchangeable); C28H19Cl2N7OS
(572.4); Requires (Found): C, 58.74 (58.75); H, 3.34 (3.32); N, 17.13
(17.11).

Synthesis of Pyrimido[4,5-b]quinolines
401
3-Amino-8-(4-chlorophenylmethylene)-12-(4-chlorophenyl)-4-
imino-8,9,10,11-tetrahydropyrimido[4ꢀꢀ,5ꢀꢀ,:4ꢀ,5ꢀ]thiazolo
[3ꢀ,2ꢀ:1,2]pyrimido[4,5-b]quinolin-13-one (20)
Compound 19 (2.86 g, 5 mmol) was added to a warmed ethanolic sodium
ethoxide solution (prepared by dissolving (0.23 g, 10 mmol) sodium
metal in 50 mL absolute ethanol). The mixture was stirred under reflux
for 8 h. The reaction mixture was allowed to cool to room temperature,
poured into cold water (100 mL), and neutralized with acetic acid. The
precipitate was filtered off, dried, and crystallized from benzene in 59 %
yield, m.p. 267–270◦C; IR, cm−1: 3400 (brs, NH), 3091 (CH, aryl), 2937
1
(CH, alkyl), 1692 (CO), 1635 (C N), 1546 (C=C); H-NMR (DMSO-d6,
δ, ppm): 1.69–1.75 (t, 2H, CH2), 2.28–2.33 (t, 2H, CH2), 2.68–2.76 (t,
2H, CH2), 7.10–7.15 (d, 2H, Ar-H), 7.23–7.28 (d, 2H, Ar-H), 7.40–7.43
(d, 2H, Ar-H), 7.44–7.46 (d, 2H, Ar-H), 8.28 (s, 1H, CH), 8.49 (s, 1H, CH
pyrimidine), 8.85 (br, 2H, NH2), and 11.30 (br, NH); C28H19Cl2N7OS
(572.4); Requires (Found): C, 58.74 (58.69); H, 3.34 (3.31); N, 17.13
(17.09).
3-Aminomethyleneamino-6-(4-chlorophenyl)-10-(4-
chlorophenylmethylene)-5-oxo-7,8,9,10-tetrahydrothiazolo
[3ꢀ,2ꢀ:1,2]pyrimido[4,5-b]quinoline-2-carbonitrile (21)
A mixture of compound 18 (2.93 g, 5 mmol) and ammonia solution (40–
50%, 10 mL) was stirred under reflux in 50 mL ethanol for 12 h. The
reaction mixture was allowed to cool to room temperature, poured into
cold water (100 mL), and neutralized with acetic acid. The precipitate
was filtered off, washed with water, dried, and crystallized from DMF
in 55 % yield, m.p. 287–290◦C (dec.); IR, cm−1: 3395 (br, NH2), 3061
(CH aryl), 2929 (CH alkyl), 2217 (CN), 1690 (CO), 1620 (C N), 1530
(C=C).1H-NMR (DMSO-d6, δ, ppm): 1.65–1.71 (t, 2H, CH2), 2.26–2.31
(t, 2H, CH2), 2.71–2.78 (t, 2H, CH2), 6.97–7.00 (d, 2H, Ar-H), 7.11–7.16
(d, 2H, Ar-H), 7.23–7.31 (d, 2H, Ar-H), 7.40–7.44 (d, 2H, Ar-H), 8.08
(s, 1H, CH), 8.54 (s, 1H, CH), and 9.05 (br, NH2, D2O exchangeable);
C28H18Cl2N6OS (557.4); Requires (Found): C, 60.32 (60.29); H, 3.25
(3.21); N, 15.08 (15.11).
Pharmacological Screening
Animals
Male Sprague-Dawley rats (150–200 g) were used in the study of
antioxidant activity, but adult females were used in anti-inflammatory
activity. Both sex of Swiss mice weighing 25–30 g were used in analgesic

402
A. B. A. El-Gazzar et al.
activity, taking into account international principles and local regula-
tions concerning the care and use of laboratory animals.16 The animals
had free access to standard commercial diet and water ad libitum, and
were kept in rooms maintained at 22 1◦C with a 12 h light/dark cycle.
Reagents
DNA (Type 1. Calf Thymus), bleomycin sulfate, butylated hydrox-
yanisole (BHA) and L-ascorbic acid were obtained from Sigma. 2,2ꢀ-
Azo-bis-(2-amidinopropane) dihydrochloride (AAPH), 2,2ꢀ-Azino-bis(3-
ethylbenzthiazoline-6-sulfonic acid) (ABTS) was purchased from Wak.
All other chemicals were of the highest quality available.
Antioxidant Sreening
Assay for Erythrocyte Hemolysis
Blood was obtained from rats by cardiac puncture and collected
in heparinized tubes. Erythrocytes were separated from plasma and
the Buffy coat and washed three times with 10 volumes of 0.15 M
NaCl. During the last washing, the erythrocytes were centrifuged at
2.500 rpm for 10 min to obtain a constantly packed cell preparation.
Erythrocyte hemolysis was mediated by peroxyl radicals in this as-
say system.17 A 10% suspension of erythrocytes in pH 7.4 phosphate
buffered saline (PBS) was added to the same volume of 200 mM 2.2ꢀ-
azobis (2-amidinopropane) dihydrochloride (AAPH) solution (in PBS)
containing samples to be tested at different concentrations. The reac-
tion mixture was shaken gently while being incubated at 37◦C for ∼h.
The reaction mixture was then removed, diluted with 8 volumes of PBS,
and centrifuged at 2,500 rpm for 10 min. The absorbance A of the super-
natant was read at 540 nm. Similarly, the reaction mixture was treated
with 8 volumes of distilled water to achieve complete hemolysis, and
the absorbance B of the supernatant obtained after centrifugation was
measured at 540 nm. The percentage hemolysis was calculated by the
equation (1-A/B) × 100%. The data were expressed as mean standard
deviation. L-Ascorbic acid was used as a positive control.
Antioxidant Activity Screening Assay ABTS Method
For each of the investigated compounds, 2 mL of ABTS solution
(60 µM) were added to 3 M MnO2 solution (25 mg/mL) all prepared in
phosphate buffer (PH.7, 0.1 M). The mixture was shaken, centrifuged,
filtered, and the absorbance (Acontrol) of the resulting green-blue solu-
tion (ABTS radical solution) was adjusted at ca. 0.5 at λ 734 nm. Then,
50 µL of (2 mM) solution of the test compound in spectroscopic grade

Synthesis of Pyrimido[4,5-b]quinolines
403
MeOH/phosphate buffer (1:1) were added. The absorbance (Atest) was
measured, and the reduction in color intensity was expressed as % in-
hibition. The % inhibition for each compound is calculated from the
following equation18:
Acontrol − Atest
The % inhibition =
× 100
AControl
Ascorbic acid (Vitamin C) was used as standard antioxidant (pos-
itive control). A blank sample was run without ABTS and using
MeOH/Phosphate buffer (1:1) instead of the sample. A negative con-
trol sample was run with MeOH/phosphate buffer (1:1) instead of the
tested compound.
Bleomycin-Dependent DNA Damage
The assay was done according to Aeschlach et al.19 with minor mod-
ifications. The reaction mixture (0.5 mL) contained DNA (0.5 mg/mL),
bleomycin sulfate (0.05 mg/mL), MgCl2 (5 mM), FeCl3 (50 µM), and
samples to be tested at different concentrations. L-Ascorbic acid was
used as a positive control. The mixture was incubated at 37◦C for 1 h.
The reaction was terminated by addition of 0.05 mL EDTA (0.1M). The
color was developed by adding 0.5 mL thiobarbituric acid (TBA)(1%,
w/v) and 0.5 mL HCI (25%, v/v) followed by heating at 80◦C for 10 min.
After centrifugation, the extent of DNA damage was measured by in-
crease in absorbance at 532 nm.
Anti-Inflammatory Activity (Carrageenan Induced Rat Hind
Paw Edema Model)
The method adopted resembles essentially that described by Winter
et al.20 (distilled water was selected as vehicle to suspend the stan-
dard drugs and the test compounds). The albino rats weighing between
150–180 g were starved for 18 h prior to the experiment. The animals
were weighed, marked for identification, and divided into 14 groups,
each group containing 6 animals. Edema was induced in the left hind
paw of all rats by subcutaneous injection of 0.1 mL of 1% (w/v) car-
rageenan in distilled water into their footpads. The first group was
kept as control and was given the respective volume of the solvent
(0.5 mL distilled water). Groups 2–13 were orally administered aque-
ous suspension of the synthesized compounds in doses of 20 mg/kg
1 h before carrageenan injection. The last group (standard) was ad-
ministered diclofenc sodium in a dose of 20 mg/kg, orally as aqueous
suspension.21 The paw volume of each rat was measured immediately

404
A. B. A. El-Gazzar et al.
by mercury plethysmometer, before carrageenan injection, and then
hourly for 4 h post-administration of aqueous suspension of the syn-
thesized compounds. The edema rate and inhibition rate of each group
were calculated as follows: Edema rate (E)% = Vt – Vo/Vo, Inhibition
rate (I)% = Ec – Et/Ec where Vo is the volume before carrageenan in-
jection (mL), Vt is the volume at t h after carrageenin injection (mL),
and Ec, Et are the edema rat of control group and treated group, re-
spectively.
Analgesic Activity Using Hot-Plate Test
The experiment was carried out as described by Turner22 using a hot-
plate apparatus, maintained at 53 0.5◦C. The mice were divided into
14 groups of 6 animals each. The reaction time of the mice to the ther-
mal stimulus was the time interval between placing the animal in the
hot plate and when it licked its hind paw or jumped. The reaction time
was measured prior to aqueous suspension of synthesized compounds
and drug treatment (0 min). The first group was kept as normal con-
trol. The aqueous suspension of the synthesized compounds was orally
administered to the mice of the groups (2–13) at doses of 20 mg/kg. The
mice of the last group were orally treated with diclofenac sodium in
a dose of 20 mg/kg. The reaction time was again measured at 15 min
and repeated at 30, 60, and 90 min after treatment. To avoid tissue
damage to the mice paws, cut-off time for the response to the thermal
stimulus was set at 60 seconds. The reaction time was calculated for
each synthesized compounds and drug-treated group.
Analgesic Activity (Acetic Acid Induced Writhing Response
Model)
The compounds were selected for investigating their analgesic ac-
tivity in acetic acid induced writhing response in Swiss albino mice,
following the method of Collier et al.23 The mice were divided into 14
groups (6 in each group), starved for 16 h, and pretreated as follows: The
first group, which served as control positive, orally received distilled
water in appropriate volumes. The last group orally received diclofenac
sodium in a dose of 20 mg/kg. The remaining groups received the aque-
ous suspension of synthesized compounds orally at doses (20 mg/kg).
After 30 min, each mouse was administrated 0.7% of an aqueous solu-
tion of acetic acid (10 mL/kg), and the mice were then placed in trans-
parent boxes for observation. The number of writhes was counted for
20 min after acetic acid injection. The number of writhes in each treated
group was compared to that of a control group. The number of writhes
was recorded, and the percentage protection was calculated using the

Synthesis of Pyrimido[4,5-b]quinolines
405
following ratio (%) protection = (control mean – treated mean/control
mean) × 100.
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