Synthesis, reactions, and anti-inflammatory activity of heterocyclic systems fused to a thiophene moiety using citrazinic acid as synthon

Article abstract of DOI:10.1007/s00706-007-0651-0

A series of pyridines, pyrimidinones, and oxazinones were synthesized as anti-inflammatory agents using citrazinic acid (2,6-dihydroxyisonicotinic acid) as a starting material. Acryloyl pyridine was treated with cyanothioacetamide to give cyano pyridine-thione, which was reacted with ethyl chloroacetate to yield the corresponding amino ester. The ester was hydrolysed to the sodium salt, which was treated with acetic anhydride to afford 2-methyloxazinone, which was treated with ammonium acetate to afford 2-methylpyrimidinone followed by methylation with methyl iodide to yield 2,3-dimethylpyrimidinone. In addition, the oxazinone derivative was reacted with aniline or hydrazine hydrate to give 3-phenyl- or 3-aminopyrimidinones. The latter reacted with thiophene-2- carboxaldehyde or phenylisothiocyanate to afford Schiff's bases or thiosemicarbazides. 3-Aminopyrimidinone was treated with phthalic anhydride or 1,2,4,5-benzenetetracarboxylic acid dianhydride or toluene-3,5-diisocyanate to afford the corresponding imide, bis-imide, and bis-semicarbazide derivatives. The pharmacological screening showed that many of these compounds have good anti-inflammatory activity comparable to Prednisolone as reference drug. Springer-Verlag 2007.

Full text of DOI:10.1007/s00706-007-0651-0

Monatshefte fu¨r Chemie 138, 699–707 (2007)
DOI 10.1007/s00706-007-0651-0
Printed in The Netherlands
Synthesis, Reactions, and Anti-inflammatory Activity of Heterocyclic Systems
Fused to a Thiophene Moiety Using Citrazinic Acid As Synthon
Abd El-Galil E. Amr1;ꢀ, Nermien M. Sabry1, and Mohamed M. Abdulla2
1
National Research Center, Applied Organic Chemistry Department, Cairo, Dokki, Egypt
2
Research Units, Hi-Care Pharmaceutical Co., Cairo, Egypt
Received January 3, 2007; accepted January 10, 2007; published online May 11, 2007
# Springer-Verlag 2007
Summary. A series of pyridines, pyrimidinones, and oxazi-
nones were synthesized as anti-inflammatory agents using
citrazinic acid (2,6-dihydroxyisonicotinic acid) as a starting
cyclic compounds containing a sulfur atom have
material. Acryloyl pyridine was treated with cyanothioacet-
and antitumor activities [6, 7]. In addition, the bio-
logical and analgesic activities of many hetero-
been reviewed [8–11]. On the other hand, thieno-
amide to give cyano pyridine-thione, which was reacted with
pyrimidine and thioxopyrimidine derivatives have
promising biological [12, 13] and anticancer activ-
ities [14, 15]. Recently, some new pyridine, pyrim-
idine and their derivatives have been synthesized
and used as analgesic, anticonvulsant, and anti-
parkinsonian agents [16–22]. In view of these ob-
servations and in continuation of our previous work
in pyridine chemistry, we synthesized some new
heterocyclic compounds containing the thiophene
ring fused with a pyridine, oxazinone, or pyrimidi-
none nucleus and tested their anti-inflammatory
activity.
ethyl chloroacetate to yield the corresponding amino ester.
The ester was hydrolysed to the sodium salt, which was
treated with acetic anhydride to afford 2-methyloxazinone,
which was treated with ammonium acetate to afford 2-meth-
ylpyrimidinone followed by methylation with methyl iodide
to yield 2,3-dimethylpyrimidinone. In addition, the oxazinone
derivative was reacted with aniline or hydrazine hydrate to
give 3-phenyl- or 3-aminopyrimidinones. The latter reacted
with thiophene-2-carboxaldehyde or phenylisothiocyanate
to afford Schiff’s bases or thiosemicarbazides. 3-Amino-
pyrimidinone was treated with phthalic anhydride or
1,2,4,5-benzenetetracarboxylic acid dianhydride or toluene-
3,5-diisocyanate to afford the corresponding imide, bis-imide,
and bis-semicarbazide derivatives. The pharmacological
screening showed that many of these compounds have good
anti-inflammatory activity comparable to Prednisolone+ as
reference drug.
Results and Discussion
Keywords. Citrazinic acid; Oxazinone; Pyrimidinone; Anti-
inflammatory; Prednisolone+.
Synthesis
The starting materials 3 and 4 were prepared from
citrazinic acid (2,6-dihydroxyisonicotinic acid, 1) via
the corresponding 2-chloro-6-ethoxy-4-acetylpyri-
dine (2) according to literature methods [1, 23].
Thionation of 4 to the corresponding thione deriva-
tive 5 was achieved by the action of P2S5 in dry
pyridine (method A), which was prepared directly
from 3 with cyanothioacetamide in the presence of
Introduction
In our previous work we have found that certain
substituted pyridines and their derivatives show an-
timicrobial and pharmacological properties [1–5]
ꢀ
Corresponding author. E-mail: aamr1963@yahoo.com

700
A. E.-G. E. Amr et al.
ammonium acetate in refluxing ethanol (method B).
Condensation of 5 with ethyl chloroacetate in the
presence of anhydrous K2CO3 gave the ethyl ester
derivative 6, which was cyclized by sodium methox-
ide in methanol to give the amino ester derivative 7
(Scheme 1). The IR spectra of 7 showed the absence
Scheme 1

Heterocyclic Systems Fused to a Thiophene Moiety
701
Scheme 2
of ꢀꢀ (CꢁN) for 6 and the presence of broad band
corresponding to ꢀꢀ (NH2).
under reflux afforded the 3-phenyl- and 3-amino-
pyrimidines 11 and 12. Condensation of 12 with
thiophene-2-carboxaldehyde in refluxing ethanol
containing a few drops of pipridine yielded the cor-
responding Schiff’s base 13. Also, 12 was treated with
phenylisothiocyanate in refluxing dioxane to give the
thiosemicarbazide 14 (Scheme 3).
On the other hand, reaction of 12 with phthal-
ic anhydride or 1,2,4,5-benzenetetracarboxylic acid
dianhydride in refluxing acetic acid afforded the
corresponding imide 15 and bis-imide 16. Upon re-
fluxing with toluene-3,5-diisocyanate in dioxane it
afforded the bis-semicarbazide 17 (Scheme 4).
Compound 7 was hydrolyzed by refluxing with etha-
nolic NaOH solution to the corresponding sodium salt
A, which was treated with refluxing acetic anhydride
to give the oxazinone derivative 8. Reaction of 8 with
ammonium acetate in refluxing acetic acid afforded
the corresponding pyrimidinone derivative 9, which
was treated with methyl iodide in N,N-dimethylform-
amide in the presence of anhydrous K2CO3 to yield
the 3-methyl-pyrimidinone derivative 10 (Scheme 2).
Similarly, reaction of oxazinone 8 with aniline
in acetic acid or with hydrazine hydrate in ethanol

702
A. E.-G. E. Amr et al.
Scheme 3
Pharmacological Screening
Anti-inflammatory Potency
Then the newly synthesized compounds were phar-
macologically screened for their anti-inflammatory
potency using male albino rats (Tables 2 and 3).
Initially the acute toxicity of the compounds was
assayed determining their LD50. Interestingly, all
the synthesized compounds and starting materials
were less toxic than the reference drug (Table 1).
Purpose and Rational
For the determination of the antiphlogistic poten-
cy of the synthesized compounds, two standard

Heterocyclic Systems Fused to a Thiophene Moiety
703
Scheme 4
tests were realized at 25 and 50 mg=kg body
weight of the rats, namely the protection against
carrageenan-induced edema according to Winter
et al. [24] and the inhibition of plasma PGE2. The
latter is known as a good confirming indicator for
the carragenan-induced rat paw edema [25]. Re-
garding the protection against carrageenan-induced
edema, four compounds, namely 9, 10, 12, and
14, were found to be more potent (120%) than
Prednisolone+. For these compounds, a similar ac-
tivity profile was realized for the inhibition of
plasma PGE2.

704
A. E.-G. E. Amr et al.
Table 1. Acute toxicity LD50 of the synthesized and starting
compounds
Table 3. Anti-inflammatory potency of the synthesized com-
pounds (inhibition of plasma PGE2)
Compound no.
LD50=mg kgꢂ1
Compound no.
Dose=mg kgꢂ1
% Inhibition of
plasma PGE2
Prednisolone+
1.618
1.780
1.965
1.875
2.350
3.715
2.500
1.800
1.882
2.970
2.680
2.615
2.751
1.980
1.920
4.186
2.872
2.264
Prednisolone+
25
50
25
50
25
50
25
50
25
50
25
50
25
50
25
50
25
50
25
50
25
50
25
50
25
50
25
50
77.00
91.00
–
40.98
–
54.46
–
38.50
–
66.80
94.65
96.58
82.65
93.63
55.76
65.00
93.30
98.10
68.12
59.73
80.36
93.45
70.08
82.75
–
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
5
6
7
8
9
10
11
12
13
14
15
16
17
Table 2. Anti-inflammatory potency of the synthesized com-
pounds (protection against carragenan-induced edema)
Compound
no.
Dose=
mg kgꢂ1
% Protection against
carrageenan-induced edema
Prednisolone+
25
50
25
50
25
50
25
50
25
50
25
50
25
50
25
50
25
50
25
50
25
50
25
50
25
50
25
50
81.00
93.00
–
38.28
–
58.26
–
36.49
–
42.38
88.54
97.15
86.25
97.05
47.50
51.60
86.84
99.41
65.80
88.16
85.52
96.10
75.38
85.24
–
50.68
54.88
66.98
5
6
7
Experimental
8
All melting points were taken on Electrothermal IA 9000
series digital melting point apparatus. Elemental analytical
data (in accord with the calculated values) were obtained from
the microanalytical unit, Cairo University, Cairo, Egypt. The
IR spectra (KBr) were recorded on a Pye Unicam SP-1000
spectrophotometer. The 1H NMR spectra were recorded at
270 MHz on Varian EM-360 Spectrometer using TMS as an
internal standard. The Central Services Laboratory, Cairo
University, Egypt. The mass spectra were performed using
VG 2AB-3F spectrometer (70 eV). All reactions were follow-
ed by TLC (silica gel, aluminum sheets 60 F254, Merck).
Starting materials 2–4 were prepared from citrazinic acid 1
according to published procedures [1, 23].
9
10
11
12
13
14
15
16
17
2-Chloro-6-ethoxy-4-[3-cyano-4-(2-thienyl)-2-thioxopyridin-
6-yl]pyridine (5, C17H12ClN3OS2)
Method A: A mixture of 0.357 g 4 (1mmol) and 2.23 g P2S5
(10 mmol) in 50 cm3 dry pyridine was heated under reflux for
6 h with stirring. The reaction mixture was cooled, and then
poured into ice, the separated solid was collected by filtration,
64.10
62.90
64.80

Heterocyclic Systems Fused to a Thiophene Moiety
705
washed with H2O, dried under vacuum, and crystallized to
afford 0.24 g (65%) 5.
(m, 3 thiophene-H), 8.20–8.26 (m, 2 pyr-H), 8.55 (s, pyr-50-H)
ppm; MS (EI, 70eV): m=z (%) ¼ 456 [Mþ, 22] and at 353
[100, base peak].
Method B: A mixture of 0.3 g 3 (1mmol), 0.10g ethyl
cyanothioacetamide (1mmol) and 0.6 g ammonium acetate
(8mmol) in 30 cm3 absolute ethanol was refluxed for 5 h.
After cooling, the formed product was collected by filtration,
washed with ethanol, dried and crystallized to give 0.28g
(76%) 5. Mp 214–217ꢃC (AcOH); IR (film): ꢀꢀ¼ 3365 (NH),
2224 (CꢁN), 1235 (C¼S) cmꢂ1; 1H NMR (270 MHz, DMSO-
d6): ꢁ ¼ 1.35 (t, CH3), 3.85 (q, CH2), 7.15–7.25 (m, 3 thio-
phene-H), 8.22–8.27 (m, 2 pyr-H), 8.58 (s, pyr-50-H), 9.24 (s,
NH, exchangeable with D2O) ppm; MS (EI, 70 eV): m=z
(%) ¼ 374 [Mþ, 12] and at 267 [100, base peak].
2-Chloro-6-ethoxy-4-[2-methyl-4-oxo-9-(2-thienyl)-3,4-
dihydropyrido[30,20:4,5]thieno-[3,2-d]pyrimidin-7-yl]
pyridine (9, C21H15ClN4O2S2)
A mixture of 0.455 g 8 (1 mmol) and 0.6 g ammonium acetate
(8mmol) in 100 cm3 glacial acetic acid was refluxed for 6 h.
The reaction mixture was concentrated under reduced pres-
sure, then poured into H2O, and the solid formed was col-
lected by filtration and crystallized to afford 0.30g (70%) 9.
Mp 165–167ꢃC (EtOH=H2O); IR (film): ꢀꢀ¼ 3420 (NH), 1669
1
(C¼O) cmꢂ1; H NMR (270MHz, DMSO-d6): ꢁ ¼ 1.10 (s,
2-Chloro-6-ethoxy-4-[3-cyano-2-ethylthioglycolate-4-(2-
thienyl)pyridin-6-yl]pyridine (6, C21H18ClN3O3S2)
CH3), 1.35 (t, CH3), 3.84 (q, CH2), 7.05–7.24 (m, 3 thio-
phene-H), 8.12–8.21 (m, 2 pyr-H), 8.62 (s, pyr-50-H), 8.35
(s, NH exchangeable with D2O) ppm; MS (EI, 70eV): m=z
(%) ¼ 455 [Mþ, 42] and at 298 [100, base peak].
To a mixture of 0.373 g 5 (1mmol) and 0.18g anhydrous
K2CO3 (1mmol) in 25 cm3 N-dimethylformamide was stirred
at room temperature for 2 h, 0.18 g ethyl chloroacetate
(1.5 mmol) was added with stirring. The reaction mixture
was heated at 60ꢃC for 2 h and after cooling poured into ice.
The solid formed was collected by filtration and crystallized
to afford 0.3 g (68%) 6. Mp 191–193ꢃC (dioxane); IR (film):
2-Chloro-6-ethoxy-4-[2,3-dimethyl-4-oxo-9-(2-thienyl)-3,4-
dihydropyrido[30,2:4,5]thieno-[3,2-d]pyrimidin-7-yl]
pyridine (10, C22H17ClN4O2S2)
A solution of 0.454g 9 (1mmol) in 20cm3 DMF was stirred
with 0.19 g anhydrous K2CO3 (1mmol) for 10min at room
temperature, then 0.28g methyl iodide (2mmol) in 5 cm3
DMF were added. The reaction mixture was heated at 60ꢃC
for 4 h, after cooling poured into H2O, and the precipitate
was filtered off and crystallized to afford 0.3 g (62%) 10.
Mp 197–199ꢃC (DMF=H2O); IR (film): ꢀꢀ¼ 1672 (C¼O)
ꢀꢀ¼ 2219 (CꢁN), 1736 (C¼O, ester) cmꢂ1
;
1H NMR
(270 MHz, DMSO-d6): ꢁ ¼ 1.32, 1.38 (2t, 2CH3), 3.85, 3.98
(2q, 2CH2), 4.70 (s, S–CH2), 6.95–7.24 (m, 3 thiophene-H),
8.15–8.18 (m, 2 pyr-H), 8.60 (s, pyr-50-H) ppm; MS (EI,
70eV): m=z (%) ¼ 460 [Mþ, 8] and at 414 [100, base peak].
2-Chloro-6-ethoxy-4-[3-amino-2-carbethoxy-4-(2-thienyl)
thieno[2,3-b]pyridin-6-yl]pyridine (7, C21H18ClN3O3S2)
A mixture of 0.459 g 6 (1mmol) in 20cm3 sodium methoxide
solution (2%) was refluxed for 1 h on a water bath at 70ꢃC
with stirring. The reaction mixture was evaporated under re-
duced pressure, the obtained residue was dissolved in CH2Cl2,
washed with H2O, 10 cm3 1 N HCl and then H2O. The solvent
was dried over anhydrous CaCl2, evaporated under reduced
pressure, and the product was crystallized to afford 0.38g
(82%) 7. Mp 206–208ꢃC (EtOH); IR (film): ꢀꢀ¼ 3442–3310
(NH2), 1742 (C¼O, ester) cmꢂ1; 1H NMR (270 MHz, DMSO-
d6): ꢁ ¼ 1.32, 1.38 (2t, 2CH3), 3.84, 3.96 (2q, 2CH2), 4.32
(brs, NH2, exchangeable with D2O), 7.10–7.25 (m, 3 thio-
phene-H), 8.18–8.25 (m, 2 pyr-H), 8.62 (s, pyr-50-H) ppm;
MS (EI, 70 eV): m=z (%) ¼ 460 [Mþ, 4] and at 334 [100,
base peak].
cmꢂ1
;
1H NMR (270MHz, DMSO-d6): ꢁ ¼ 1.05 (s, CH3),
1.37 (t, CH3), 2.48 (s, N–CH3), 3.90 (q, CH2), 6.96–7.15
(m, 3 thiophene-H), 8.10–8.17 (m, 2 pyr-H), 8.42 (s, pyr-50-
H) ppm; MS (EI, 70eV): m=z (%) ¼ 469 [Mþ, 32] and at 242
[100, base peak].
2-Chloro-6-ethoxy-4-[2-methyl-4-oxo-3-phenyl-9-(2-
thienyl)-3,4-dihydropyrido[30,20:4,5]-thieno[3,2-d]
pyrimidin-7-yl]pyridine (11, C27H19ClN4O2S2)
A mixture of 0.456 g 8 (1mmol) and ꢄ0.1g aniline (1mmol)
in 50cm3 glacial acetic acid was heated under reflux for 6 h.
The reaction mixture was concentrated, poured onto ice, and
the formed solid was filtered off and crystallized to afford
0.4 g (75%) 11. Mp 156–158ꢃC (MeOH=H2O); IR (film):
ꢀꢀ¼ 1679 (C¼O) cmꢂ1
;
1H NMR (270 MHz, DMSO-d6):
ꢁ ¼ 1.15 (s, CH3), 1.33 (t, CH3), 3.88 (q, CH2), 6.90–7.60
(m, 8 phenyl-H and thiophene-H), 8.22–8.25 (m, 2 pyr-H),
8.52 (s, pyr-50-H) ppm; MS (EI, 70eV): m=z (%) ¼ 531 [Mþ,
16] and at 201 [100, base peak].
2-Chloro-6-ethoxy-4-[(2-methyl-4-oxo-9-(2-thienyl)
pyrido[30,20:4,5]thieno[3,2-d]oxazin-7-yl]pyridine
(8, C21H14ClN3O3S2)
A mixture of 0.459 g 7 (1mmol) in 100 cm3 ethanolic NaOH
(5%) was heated under reflux for 4 h. The solvent was evapo-
rated under reduced pressure, the obtained sodium salt [A]
was dissolved in 100 cm3 acetic anhydride and refluxed for
6 h. The reaction mixture was concentrated and allowed to
cool. The obtained solid was collected and crystallized to
afford 0.34 g (74%) 8. Mp 149–151ꢃC (AcOH=H2O); IR
2-Chloro-6-ethoxy-4-[3-amino-2-methyl-4-oxo-9-(2-thienyl)-
3,4-dihydropyrido[30,20:4,5]-thieno[3,2-d]pyrimidin-7-yl]
pyridine (12, C21H16ClN5O2S2)
A mixture of 0.454g 8 (1mmol) and 0.4 cm3 hydrazine
hydrate (8mmol) in 100cm3 absolute ethanol was refluxed
for 4 h. After cooling the solid formed was collected and
crystallized to afford 0.3g (64%) 12. Mp>250ꢃC (AcOH=
1
(film): ꢀꢀ¼ 1735 (C¼O) cmꢂ1; H NMR (270MHz, DMSO-
d6): ꢁ ¼ 1.15 (s, CH3), 1.32 (t, CH3), 3.85 (q, CH2), 6.98–7.25
H2O); IR (film): ꢀꢀ¼ 3365–3300 (NH2), 1670 (C¼O) cmꢂ1
;

706
A. E.-G. E. Amr et al.
1,2,4,5-Bis-{2-chloro-6-ethoxy-4-[2-methyl-4-oxo-3-
1H NMR (270 MHz, DMSO-d6): ꢁ ¼ 1.12 (s, CH3), 1.34 (t,
CH3), 3.85 (q, CH2), 4.35 (brs, NH2, exchangeable with D2O),
7.00–7.25 (m, 3 thiophene-H), 8.16–8.26 (m, 2 pyr-H), 8.56
(s, pyr-50-H) ppm; MS (EI, 70eV): m=z (%) ¼ 470 [Mþ, 100,
base peak].
(phthalimido)-9-(2-thienyl)-3,4-di-hydropyrido[30,20:4,5]-
thieno[3,2-d]-pyrimidin-7-yl]pyridine}phthalimide
(16, C52H30Cl2N10O8S4)
Mp >300ꢃC (DMF); IR (film): ꢀꢀ¼ 1668, 1676 (C¼O) cmꢂ1
;
1H NMR (270MHz, DMSO-d6): ꢁ ¼ 1.10 (s, 2CH3), 1.35 (t,
2CH3), 3.88 (q, 2CH2), 7.05–7.55 (m, 8 thiophene-H and
phenyl-H), 8.25–8.34 (m, 4 py-H), 8.58 (s, 2 pyr-50-H) ppm;
MS (EI, 70 eV): m=z (%) ¼ 1122 [Mþ, 6] and at 380 [100,
base peak].
2-Chloro-6-ethoxy-4-[2-methyl-4-oxo-3-(2-
thienylmethylidene)amino)-9-(2-thienyl)-3,4-dihydro-
pyrido[30,20:4,5]thieno[3,2-d]pyrimidin-7-yl]pyridine
(13, C26H18ClN5O2S3)
A mixture of 0.469 g 12 (1mmol) and 0.12 g thiophene-2-
carbaldehyde (1mmol) in 25 cm3 absolute ethanol was re-
fluxed for 6 h. The obtained solid was filtered off, washed
with ethanol, and crystallized to afford 0.48 g (86%) 13.
Mp>250ꢃC (EtOH=H2O); IR (film): ꢀꢀ¼ 1688 (C¼O), 1660
3,5-Bis-{2-chloro-6-ethoxy-4-[2-methyl-4-oxo-3-
(phenysemicarbazido)-9-(2-thienyl)-3,4-di-
hydropyrido[30,20:4,5]thieno[3,2-d]pyrimidin-7-yl]
pyridine}toluene (17, C51H38Cl2N12O6S4)
1
(C¼N) cmꢂ1; H NMR (270MHz, DMSO-d6): ꢁ ¼ 1.16 (s,
A mixture of 1.10g 12 (2mmol) and 0.174g toluene-3,5-di-
isocyanate (1mmol) in 50cm3 dry dioxane containing 2 cm3
TEA was refluxed for 12h. The solvent was evaporated under
reduced pressure and the oily product was triturated with n-
hexane and petroleum ether (40–60ꢃC). The obtained solid
was filtered off, dried under vacuum and crystallized to afford
0.62g (56%) 17. Mp>300ꢃC (AcOH); IR (film): ꢀꢀ¼ 3346–
CH3), 1.38 (t, CH3), 3.92 (q, CH2), 7.10–7.45 (m, 7 thio-
phene-H þ CH¼N), 8.14–8.20 (m, 2 pyr-H), 8.48 (s, pyr-50-
H) ppm; MS (EI, 70eV): m=z (%) ¼ 564 [Mþ, 10] and at 164
[100, base peak].
2-Chloro-6-ethoxy-4-[2-methyl-4-oxo-3-(2-
1
3298 (NH), 1680 (C¼O) cmꢂ1; H NMR (270MHz, DMSO-
phenylthiosemicarbazido)amino)-9-(2-thienyl)-3,4-
dihydropyrido[30,20:4,5]thieno[3,2-d]pyrimidin-7-yl]
pyridine (14, C28H21ClN6O2S3)
d6): ꢁ ¼ 1.08 (s, 2CH3), 1.35 (t, 2CH3), 2.25 (s, CH3), 3.85 (q,
2CH2), 7.05–7.56 (m, 9 thiophene-H and phenyl-H), 8.15–
8.26 (m, 4 pyr-H), 8.54 (s, 2 pyr-50-H), 8.65 (brs, 4 NHCO,
exchangeable with D2O) ppm; MS (EI, 70eV): m=z (%) ¼
1114 [Mþ, 14] and at 176 [100, base peak].
A mixture of 0.469 g 12 (1mmol) and 014 g phenyl isothio-
cyanate (1mmol) in 50cm3 dry dioxane containing 2 cm3
triethylamine was heated under reflux for 10 h. The solvent
was evaporated under reduced pressure, the obtained residue
was solidified with n-hexane. The obtained solid was filtered
off, washed with diethyl ether, dried and crystallized to afford
0.4 g (65%) 14. Mp 142–144ꢃC (AcOH); IR (film): ꢀꢀ¼ 3336–
Pharmacological Screening
Determination of Acute Toxicity (LD50
)
The LD50 was determined by using rats. They were injected
with different increasing doses of the synthesized compounds.
The dose that killed 50% of the animal was calculated accord-
ing to Austen et al. [26].
3250 (NH), 1678 (C¼O), 1232 (C¼S) cmꢂ1
;
1H NMR
(270 MHz, DMSO-d6): ꢁ ¼ 1.14 (s, CH3), 1.34 (t, CH3), 3.88
(q, CH2), 4.15–4.25 (bs, 2 NH–CS, exchangeable with D2O),
6.94–7.55 (m, 8 thiophene-H and phenyl-H), 8.06–8.16 (m,
2 pyr-H), 8.48 (s, pyr-50-H) ppm; MS (EI, 70 eV): m=z
(%) ¼ 605 [Mþ, 6] and at 467 [100, base peak].
Anti-inflammatory Activity
Carrageenan-Induced Edema (Rats Paw Test)
Groups of adult male albino rats (150–180 g), each of eight
animals were orally dosed with tested compounds at a dose lev-
el of 25–50mg=kg one hour before carrageenan challenge.
All animals were obtained from the Animal House Colony,
Research Institute of Ophthalmology, Giza, Egypt. Foot paw
edema was induced by subplantar injection of 0.05 cm3 of a 1%
suspension of carrageenan in saline into the plantar tissue of one
hind paw. An equal volume of saline was injected to the other
hand paw and served as control. Four hours after drug admin-
istration the animals were decapitated, blood was collected, and
the paws were rapidly excised. The average weight of edema
was examinedfor the treated as well as the control groupand the
percentage inhibition of weight of edema was also evaluated.
Prednisolone+ (5mg=kg) was employed as standard reference
against which the tested compounds were compared.
Synthesis of 15 and 16
A mixture of 1.10 g 12 (2 mmol) and 0.29 g phthalic anhy-
dride (2 mmol) or 0.22 g 1,2,4,5-benzenetetracarboxylic
acid dianhydride (1 mmol) was refluxed in 50 cm3 glacial
acetic acid for 6 h. The reaction mixture was cooled, the ob-
tained product was collected by filtration, dried under vac-
uum, and crystallized to afford 0.5 g (84%) 15 and 0.98 g
(88%) 16.
2-Chloro-6-ethoxy-4-[2-methyl-4-oxo-3-(phthalimido)-9-(2-
thienyl)-3,4-dihydropyrido-[30,20:4,5]thieno[3,2-d]-
pyrimidin-7-yl]pyridine (15, C29H18ClN5O4S2)
Mp>300ꢃC (DMF); IR (film): ꢀꢀ¼ 1678, 1682 (C¼O) cmꢂ1
;
1H NMR (270 MHz, DMSO-d6): ꢁ ¼ 1.12 (s, CH3), 1.36 (t,
CH3), 3.82 (q, CH2), 7.15–3.68 (m, 7 thiophene-H and phe-
nyl-H), 8.14–8.16 (m, 2 pyr-H), 8.52 (s, pyr-50-H) ppm; MS
(EI, 70eV): m=z (%) ¼ 600 [Mþ, 16] and at 315 [100, base
peak].
Estimation of Plasma Prostaglandin E2 (PGE2)
Heparinized blood samples were collected from rats (n ¼ 8),
plasma was separated by centrifugation at 12000g for 2 min at

Heterocyclic Systems Fused to a Thiophene Moiety
707
40ꢃC, immediately frozen, and stored at 20ꢃC until use. The
design correlate EIA prostaglandin E2 (PGE2) kit (Aldrich,
Steinheim, Germany) is a competitive immuno assay for the
quantitative determination of PGE2 in biological fluids. The kit
uses a monoclonal antibody to PGE2 to bind, in a competitive
manner, the PGE2 in the sample after a simultaneous incuba-
tion at room temperature. The excess reagents were washed
away and the substrate was added, after a short incubation time
the enzyme reaction was stopped, and the yellow color gener-
ated was read on a microplate reader DYNATech, MR 5000 at
405 nm (Dynatech Industries Inc., McLean, VA, USA). The
intensity of the bound yellow color is inversely proportional
to the concentration of PGE2 in either standard or samples.
[9] Chakrabarti JK, Horsman L, Hotten TM, Pullar IA,
Tupper DE, Wright FC (1980) J Med Chem 23: 878
[10] Ram VJ, Pandey HK, Vlietinck AJ (1981) J Heterocycl
Chem 18: 1277
[11] Fahmy HH, El-Eraqy W (2001) Arch Pharm Res 24:
171
[12] DeClercq E (1986) Anticancer Res 6: 549
[13] DeClercq E (1986) J Med Chem 29: 156
[14] Hammam AG, Sharaf M, Abdel-Hafez NA (2001) Ind J
Chem 40B: 213
[15] Hammam AG, Fahmy AFM, Amr AE, Mohamed AM
(2003) Ind J Chem 42B: 1985
[16] Amr AE, Hegab MI, Ibrahiem AA, Abdulla MM (2003)
Monatsh Chem 134: 1395
[17] Nehad AA, Amr AE, Alhusain AI (2006) Monatsh
Chem (Accepted 23-11-2006)
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