Design and synthesis of 3-pyrazolyl-thiophene, thieno[2,3-d]pyrimidines as new bioactive and pharmacological activities

Article abstract of DOI:10.1016/j.bmcl.2008.08.071

Two series of 5-ethyl-2-amino-3-pyrazolyl-4-methylthiophenecarboxylate and 2-thioxo-N³-aminothieno[2,3-d]pyrimidines were prepared from 3,5-diethyl-2-amino-4-methylthio-phenecaboxylate and evaluated as anti-inflammatory, analgesic and ulcerogen

Full text of DOI:10.1016/j.bmcl.2008.08.071

Bioorganic & Medicinal Chemistry Letters 18 (2008) 5222–5227
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Design and synthesis of 3-pyrazolyl-thiophene, thieno[2,3-d]pyrimidines
as new bioactive and pharmacological activities
*
H. N. Hafez , A. B. A. El-Gazzar
Photochemistry Department, Heterocyclic and Nucleoside Unit, National Research Centre, Dokki, Giza 12622, Egypt
a r t i c l e i n f o
a b s t r a c t
Two series of 5-ethyl-2-amino-3-pyrazolyl-4-methylthiophenecarboxylate and 2-thioxo-N3-aminothie-
no[2,3-d]pyrimidines were prepared from 3,5-diethyl-2-amino-4-methylthio-phenecaboxylate and eval-
uated as anti-inflammatory, analgesic and ulcerogenic activities. Among the compounds studied,
compounds which containing the substituted hydrazide at C-3 position 7, 16, and 17a showed more
potent anti-inflammatory and analgesic activities than the standard drug (Indomethacin and Aspirin),
along without ulcerogenity. While compounds 2, 5, 9, 10, and 11c showed moderate activities. Some
of the newly synthesized compounds have good to excellent antimicrobial activity.
Article history:
Received 14 July 2008
Revised 17 August 2008
Accepted 20 August 2008
Available online 26 August 2008
Keywords:
3-Pyrazolyl-thiophene
Thieno[2,3-d]pyrimidines
Antimicrobial
Ó 2008 Elsevier Ltd. All rights reserved.
Anti-inflammatory
Analgesic
Ulcerogenic activities
Thiophene-3-ethylcarboxylate derivative represent important
building blocks in organic and medicinal chemistry. In addition,
they are interest in their own right, due to their pharmacological
properties. For example, 5-substituted 2-aminothiophenes and
2-amino-5-bromo-4-(3-nitrophenyl)thiophene are A1 adenosine
receptor agonists.1,2 Also, many thieno[2,3-d]pyrimidine deriva-
tives were covered by patents as phospho-diesterase inhibitors3
and various receptor antagonists.4,5 Various thieno[2,3-d]pyrimi-
dine derivatives show pronounced anti-tumor6 and radioprotec-
tive activities.7 Based on thieno[2,3-d]pyrimidine derivatives,
immunomodulators8 and compounds used for prophylaxis and
therapy of cerebral ischemia,9 malaria,10–12 tuberculosis,13 Alzhei-
mer’s disease,14 Parkinson’s disease,15 and other diseases were
designed.16,17
As a part of our continuing program on the synthesis of anti-
inflammatory and analgesic compounds as therapeutics agents,
we have earlier reported a series of heterocyclic moieties that have
biological and pharmacological activities.18,19 In the library screen-
ing of our new compounds as anti-inflammatory and analgesic as-
say system, several thiophenes and thieno[2,3-d]pyrimidines
provided inhibitory activity of inflammation even through some
others five or focused heterocycle rings showed low anti-inflam-
matory and analgesic activity.
oxylate substituted at the C-3 position and thieno-[2,3-d]pyrim-
idines substituted at C-2 with various groups. The interaction of
ethylacetoacetate with ethyl cyanoacetate and sulfur metal in
ethanol medium in the presence of diethylamine led to ethyl
thiophene-3-carboxylate 1.20 The hydrazide
2 obtained by
refluxing of ethyl carboxylate 1 with hydrazine hydrate in eth-
anol medium, was used as precursor for the synthesis of 3-(pyr-
azolyl)-4-methylthiophenes and 3-(1,3,4-oxa/thiadiazol-2-yl)-4-
methyltiophene as well as for the preparation of thieno[2,3-
d]pyrimidines.
Hydrazide 2 undergo different cyclization reactions to give five
member heterocycle compounds. The product of cyclization de-
pends on the reagent used. This cyclization leads to the formation
of 3-[(pyrazolyl derivatives)-carbonyl]-4-methylthiophene deriva-
tives 3–5, 3-(1,3,4-oxa/thiadiazol-2-yl)-4-methyltiophene deriva-
tives 8a, b and thieno[2,3-d]pyrimidines derivatives 10, 14, and
15. The reaction of hydrazide 2 with
a,b-diketones resulted in
2-amino-5-carboxyethyl-3-[(3,5-dimethylpyrazolyl derivatives)-
carbonyl]-4-methylthiophene 3a, b. The structure of the products
was assigned on the basis of their spectral analyses.21 The 1H
NMR spectrum of 3a, as an example showed the signals at d 1.30
(t, 3H, J = 7.0 Hz, CH3), 2.19 (s 3H CH3), 2.51(s, 3H, CH3), 2.67 (s,
3H, CH3), 4.25 (q, 2H, J = 7.10 Hz, CH2), 6.21 (s, 1H, pyrazole pro-
ton), and 7.98 (br, 2H, NH2, D2O exchangeable), while the pyrazole
proton disappeared in the 1H NMR of 3b. Also, the hydrazide 2
reacted with diethylmalonate afforded 5-ethyl-2-amino-3-[(3,5-
dioxopyrazo-lidine-1-yl)-carbonyl]-4-methylthiophenecarboxy-
late (5).
This report deals with the synthesis and the pharmacological
evaluation of a series of 5-ethyl-2-amino-4-methylthiophenecab-
* Corresponding author. Tel.: +20 2 373 18830; fax: +20 2 333 70931.
E-mail address: hendnagah2000@yahoo.com (H.N. Hafez).
0960-894X/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2008.08.071

H. N. Hafez, A. B. A. El-Gazzar / Bioorg. Med. Chem. Lett. 18 (2008) 5222–5227
5223
Treatment of compound
2
with formic acid yielded (N-
Also, compound 2 was treated with carbon disulfide to afford the
promising compound 3-amino-6-carboxyethyl-5-methyl-2-thi-
oxothieno[2,3-d]pyrimidin-4-one (15).
hydroxymethylene-carbohydrazide) derivative (7) which under-
goes cyclization to give 2-amino-5-carboxyethyl-3-(1,3,4-oxa/
thiadiazol-2-yl)-4-methyltyhiophene (8a, b) when treated with
P2O5 or P2S5. Not only that but give the same compound (8a)
when compound 2 was treated with triethylorthoformate to
afford 5-ethyl-2-amino-3-(N-ethoxymethylene-carbohydrazide)-
4-methyl-thiophene-carboxylate (6) which undergoes cyclization
by fusion to give the same compound (Scheme 1).
Scheme 2 reports cyclization procedure for the formation of 3-
amino-2,5-dimethyl-6-carboxyethyl-thieno[2,3-d]pyrimidin-4-one
(10) from compound 9. Compound 1 was treated with acetic anhy-
dride to afford 3,5-diethyl-2-acetylamino-4-methylthiophene-
dicarboxylate (9). This cyclization realized on treatment with
hydrazine hydrate.
Finally, compounds 16 was formed by the reaction of compound
2 with potassium thiocyanate, the reaction was carried out by heat-
ing the mixture in 10% HCl, then the product undergoes cyclization
when treated with chloroaceton or phenacyl bromide to afford
5-ethyl-2-amino-3-(4-methyl/or 4-phenyl-1,3-thiazol-2-yl)carbo-
hydrazide-4-methyl-thiophene carboxylate (17a, b) (Scheme 4).
Amongst the compounds 2–17a screened for antimicrobial
activity, compound 7 and 17a showed the highest activity against
all bacteria. It exhibited stronger activity than ampicillin also
towards Bacillus cereus and Salmonella typhi it was followed by
compound 16 which showed the highest activity against B. cereus
and S. typhi. As far as antifungal activity is concerned, all com-
pounds showed good to excellent activity against all the fungi.
Compounds 7 and 10 exhibited stronger activity than nystatin
against Alternaria alternata and Coillectotrium corchori, compounds
7 and 17a against Macrophomina phaseolina, compounds 7, 15, and
17a against Fusarium equiseti. Compounds 2–15 were either inac-
tive or moderately to fairly active against the tested bacteria
whereas compounds 2, 5, and 9 exhibited good to excellent results
against all the fungi. Introduction of thiophene or thiazole moiety
to the pyrimidine derivatives might be responsible for antimicro-
bial activity enhancement of these compounds (Tables 1 and 2).
Also, treatment of compound
2 with aromatic aldehydes
yielded arylmethylene hydrazide derivatives (11a–c). The cycliza-
tion of 11a–c with acetic anhydride to afford 12a–c was unsuccess-
ful, while compound 11a–c reacted with acetic anhydride to afford
5-ethyl-2-acetylamino-3-(arylmethylene
hydrazide)-4-methyl-
thiophenecarboxylate (13a–c) which undergoes cyclization upon
refluxing in sodiumethoxide solution to afford 6-carboxyethyl-
2,5-dimethylthieno[2,3-d]pyrimidin-(3H)-4-one (14) (Scheme 3),
which characterized by 1H NMR which revealed the disappearance
of azomethine proton and the protons of aromatic substitutions.
CH3
O
N
HC
COCH3
COCH3
N
X
X
H3C
NH2
EtOOC
S
EtOOCCH2COCH3 EtCOOCH2CN
3a,b
a, X = H
b, X = Cl
+
S
DEA
CH3
O
N
COOEt
COCH3
H3C
COOEt
H3C
N
O
NH2
EtOOC
EtOOC
S
NH2
S
4
H
1
O
O
N
H3C
COOEt
COOEt
N
NH2NH2
O
H3C
CONHNH2
NH2
NH2
EtOOC
H3C
S
5
O
OH
EtOOC
S
N
N
HCOOH
2
H
H
EtOOC
S
NH2
CH(OEt)3
7
P2O5
or P2S5
O
OEt
N
N
N
N
H3C
H3C
H
fusion
X= O
H
X
EtOOC
S
NH2
NH2
EtOOC
S
6
8a,b
a, X = O
b, X = S
Scheme 1.

5224
H. N. Hafez, A. B. A. El-Gazzar / Bioorg. Med. Chem. Lett. 18 (2008) 5222–5227
O
N
H3C
COOEt
O
H3C
H3C
COOEt
NH2
NH2
CH3
NH2NH2
Ac2O
N
EtOOC
N
H
CH3
S
EtOOC
S
EtOOC
S
10
9
1
Scheme 2.
O
Ar
O
N
H3C
H3C
N
N
Ar
Ac2O
ArCHO
H
N
2
H
H
NH2
EtOOC
S
EtOOC
S
N
CH3
a, Ar = phenyl
b, Ar = 4-chlorophenyl
c, Ar = 4-methoxyphenyl
11a-c
12a-c
Ac2O
O
Ar
O
N
H3C
H3C
N
NaOC H5
2
H
NH
H
CH3
N
EtOOC
S
EtOOC
S
N
CH3
O
H
14
13a-c
Scheme 3.
O
H3C
NH2
N
CS2
2
EtOOC
S
N
H
S
KSCN
15
H
H
O
NH2
S
O
N
N
H3C
H3C
N
N
S
RCOCH2X
N
H
H
R
EtOOC
EtOOC
S
NH2
S
NH2
17a,b
a, R = methyl
16
b, R = phenyl
Scheme 4.
Table 1
Antimicrobial activity of the some selected synthesized compounds
Table 2
Antifungal activity of the some selected synthesized compounds
Compound
MIC in l
g/mL, and zone of inhibition (mm)a
Compound
MIC in
M. phaseolina
l
g/mL, and zone of inhibition (mm)a
B. cereus
S. dysenteriae
S. typhi
F. equseti
A. alternata
C. corchori
2
3a
10
8
6
—
—
8
2
3a
5
7
9
10
11c
15
16
47.5
34.5
46.0
95.0
49.3
48.3
44.2
58.6
62.0
70.0
71.8
29.3
14.8
40.0
65.6
36.3
37.5
28.0
47.6
28.0
43.8
44.7
38.8
27.8
39.5
65.4
35.7
54.0
27.0
42.8
34.6
27.0
51.6
30.3
29.0
33.6
54.5
34.6
50.0
33.2
29.0
36.4
41.0
40.5
5
7
9
10
11c
15
16
17a
9
12
30
14
7
15
34
11
15
12
17
28
29
24
33
11
15
10
19
20
31
21
—
18
13
28
30
Ampicillin
17a
Nystatin
—, no inhibition; DMF, negative control.
a
a
1 mg mLÀ1 per disc.
mg mLÀ1 per disc.

H. N. Hafez, A. B. A. El-Gazzar / Bioorg. Med. Chem. Lett. 18 (2008) 5222–5227
5225
Table 3
Acute inflammation in rat using Planimeter for the synthesized compounds
Group
1 h
Edema rate (%)
2 h
Edema rate (%)
3 h
Edema rate (%)
4 h
Edema rate (%)
Pot. (%)
Pot. (%)
Pot. (%)
Pot. (%)
Control
2
89.07 7.46
77.11 6.48
(À13.43)
0
102.78 2.98
93.69 6.11
(À8.84)
0
106.14 3.29
95.74 5.97
(À9.80)
0
29.4
122.37 5.83
106.08 5.41
(À13.32)
0
37.85
22.22
32.39
3a
91.37 9.39
(2.58)
À7.27
74.8
110.88 0.88
(7.88)
—
19.80
78.01
115.11 4.31
(8.46)
—
25.38
65.5
122.07 4.46
(À0.25)
0.61
65.27
76.17
12.65
37.06
32.32
59.99
84.19
64.52
100
5
65.43 4.01*
(À26.54)
70.88 4.69*
(À31.04)
89.95 4.90
(À20.90)
89.53 8.32*
(À26.84)
7
43.48 3.78*
(À51.18)
144.25
42.36
84.05
40.59
85.09
103.04
127.23
100
63.52 1.87*
(À38.20)
96
74.74 4.88*
(À29.58)
88.75
0.12
84.05 4.90*
(À31.32)
9
75.68 5.30
(À15.03)
90.33 6.68
(À12.11)
30.44
45.06
3.82
101.90 7.85
(À4.00)
116.01 10.67
(À5.20)
10
62.51 4.84*
(À29.82)
84.35 3.37
(À17.93)
90.59 3.75
(À14.65)
43.95
11.73
31.74
88.21
75.99
100
103.73 4.65
(À15.24)
11c
76.25 4.11
(À14.40)
101.88 5.16
(À1.52)
101.99 2.97
(À3.91)
106.11 3.61
(À13.29)
15
62.18 4.37*
(À30.19)
87.83 5.03
(À14.54)
36.54
93.52
93.42
100
94.91 7.86
(À10.58)
97.19 9.93*
À24.67
16
56.51 5.40*
(36.56)
64.54 6.36*
(À37.21)
74.93 6.89*
(À29.40)
80.01 7.13*
(À34.62)
17a
48.87 2.49*
(À45.14)
64.58 6.41*
(À37.17)
79.25 3.69*
(À25.33)
89.91 6.01*
(À26.53)
Indomethacin
57.47 3.78*
(À35.48)
61.88 3.37*
(À39.79)
70.76 3.25*
(À33.33)
72.05 3.38*
(À41.12)
Values represent the mean SE of five animals for each group.
Each value in parenthesis indicates the percentage inhibition rate.
The potency (pot.) was calculated compared to the reference drug indomethacin.
*
P < 0.05: Statistically significant from control using one way ANOVA (Dunnett’s as post hoc test).
National Research Centre, Dokki, Cairo, Egypt, for performing phar-
macological activities evaluation.
Table 4
Central analgesic activity (hot plate test)
Group
Basal
30 min
60 min
90 min
References and notes
Control
Aspirin
2
3a
5
7
8.5 0.30
5.2 0.39
7.6 0.35
7.1 0.41
6.24 0.39
7.78 0.65
7.86 0.68
9.78 0.24*
10.68 0.22*
4.84 0.24*
6.08 0.42*
6.70 0.39
9.06 0.30
7.16 0.31
6.14 0.20*
4.70 0.42*
6.66 0.31
6.12 0.24
7.72 0.32
10.22 0.35*
6.08 0.58
7.34 0.17
6.46 0.22
6.42 0.16
6.60 0.41
5.44 0.42
6.68 0.33
6.56 0.52
6.20 0.18
7.62 0.35
12.42 0.70*
6.90 0.38
5.96 0.33
5.04 0.32
7.72 0.68
7.24 0.44
5.74 0.43
6.46 0.39
1. Aurelio, L.; Figler, H.; Flynn, B. L.; Linden, J.; Scammells, P. J. Bioorg. Med. Chem.
2008, 16, 1319.
2. Lütjens, H.; Zickgraf, A.; Figler, H.; Linden, J.; Olsson, R.; Scammells, P. J. J. Med.
Chem. 2003, 46, 1870.
3. Ghakraborti, A. K.; Gopalakrishnan, B.; Elizabeth-Sobhia, M.; Malde, A. Bioorg.
Med. Chem. Lett. 2003, 13, 1403.
4. Jules-Marie, H. R. G.; Marius, T. C.; Jan. K. PCT Int. Appl. WO 2003020727.; .
Chem. Abstr. 2003, 138, 238195.
5. Jules-Marie, H. R. G.; Marius, T. C. PCT Int. Appl. WO 03 20, 726 (Cl.C07D495/
04).; . Chem. Abstr. 2003, 138, 238194.
6. Sasaki, S.; Cho, N.; Nara, Y.; Harada, M.; Endo, S.; Suzuki, N.; Furuya, Sh.; Fujino,
M. J. Med. Chem. 2003, 46, 113.
5.18 0.50
5.10 0.47
5.85 0.59
5.60 0.54
5.54 0.16
5.02 0.27
4.90 0.36
4.38 0.40
4.80 0.28
4.80 0.28
9
10
11c
15
16
17a
7. Nassan, O. M.; Hassan, A. Y.; Heiba, H. I.; Ghorb, M. M. Al-Azhar Bull. Sci. 1997, 8,
435; . Chem. Abstr. 1999, 130, 110174.
8. Heiba, H. I.; Ghorab, M. M.; Amin, N. E.; Ramadan, L. Egypt. J. Biotechnol. 1998, 4,
16; . Chem. Abstr. 2000, 132, 49937.
Values represent the mean SE of five animals for each groups.
P < 0.05: Statistically significant from control using one way ANOVA (Dunnett’s
as post hoc test).
*
9. Gerd, S.; Kurt, S.; Wilfred, L.; Uta, H.; Dorothea, S.; Monika, K.; Laszlo, S.; Franz,
E.; Javi, G.-L.F.; Hans-Peter, H.; Liliane, U. Ger. Offen. DE 19, 900, 545(Cl.A61K31/
505).; . Chem. Abstr. 2000, 133, 89541.
10. Rosowsky, A.; Chaykovsky, M.; Chen, K. K. N.; Lin, M.; Modest, E. J. J. Med. Chem.
1973, 16, 185.
11. Chaykovsky, M.; Lin, M.; Rosowsky, A.; Modest, E. J. J. Med. Chem. 1973, 16, 188.
12. Rosowsky, A.; Chen, K. N. N.; Lin, M. J. Med. Chem. 1973, 16, 191.
13. Kapustina, M. V.; Omelkin, Yu. O.; Kharizomenova, I. A.; Shvedov, V. I.; Felitis, L.
N. Khim.-farm. Zh. 1991, 25, 38; . Pharm. Chem. J. 1991, 25, 7.
14. Shuichi, F.; Nobuhiro, S. PCT Int. Appl. WO 01 78,780 (Cl. A1K45/00).; . Chem.
Abstr. 2001, 135, 313627.
15. Hirotsune, I.; Takashi, K.; Eisuke, N.; Hidetaka, K.; Masamichi, O.; Takashi, T.;
Atsushi, N. PCT Int. Appl. WO 02 62,803 (Cl. C07D495/04).; . Chem. Abstr. 2002,
137, 169542.
16. Lichen, W.; Jinzhi, R.; Baolin, L.; Jiaxuan, L.; Jing, Y. J. Zhongguo Yaoke Daxue
Xuebao 1998, 29, 331; . Chem. Abstr. 1999, 130, 139309.
17. Henry, B. T.; John, I. R.; Andrew, L. C. Eur. Pat. 404,356 (Cl.C07D495/04).; . Chem.
Abstr. 1991, 115, 8821.
18. El-Gazzar, A. B. A.; Hussein, H. A. R.; Hafez, H. N. Acta. Pharm. 2007, 57, 395.
19. El-Gazzar, A. B. A.; El-Enany, M. M.; Mahmoud, M. N. Bioorg. Med. Chem. 2008,
16, 3261.
The anti-inflammatory activity data (Table 3) indicated that
compounds 15, 10, 5, 9, 11c, and 2 show significant anti-inflamma-
tory activity in descending order in comparison to the control
group, with 85%, 84%, 74%, 42%, 40%, and 37% potency with respect
to indomethacin. Compound 3a have no anti-inflammatory activ-
ity. Where compounds 17a, 16, and 7 show anti-inflammatory
activity in ascending order in comparison to the indomethacin
which give higher activity more than the indomethacin with
103%, 127%, and 144% potency.
The results of Analgesic activity (Table 4) indicated that 15, 10,
16, 9, 7, and 17a in descending order, where compounds 5, 3a, 11c,
and 2 show higher activity more than Acetyl salicylic acid. Also, the
group of rats of compounds 5, 2, and 3a were killed and their stom-
ach were checked for the ulcerogenic effect and show there is no
ulcers were found.
20. Gewald, A. K.; Schinke, E.; Bottcher, H. Chem. Ber. 1966, 99, 94.
21. All melting points were taken on Electrothermal IA 9100 series digital melting
point apparatus. Microanalytical data (in accord with the calculated values)
were performed by Vario, Elementar apparatus (Shimadzu). The IR spectra
(KBr) were recorded on a Perkin–Elmer 1650 spectrometer (USA). 1H NMR
spectra were determined on a JEOL EX-270 run for HNMR at 270 MHz and run
Acknowledgments
Author thanks Prof. Dr. El-Eraky W. Professor of Pharmacology
and Head of Pharmacological Unit, Pharmacology Department,

5226
H. N. Hafez, A. B. A. El-Gazzar / Bioorg. Med. Chem. Lett. 18 (2008) 5222–5227
for CNMR at 67.5 MHz; or on JEOL ECA-500 run for HNMR at 500 MHz and run
5-Ethyl-2-amino-3-(N-ethoxymethylene-carbohydrazide)-4-methylthiophene-
carboxylate (6). A solution of compound 2 (0.01 mol) and triethylorthoformate
(20 mL) was heated under reflux with stirring for 2 h. The reaction mixture
was filtered while hot and then cooled to rt. The solid formed was filtered off,
dried and crystallized from ethanol, as yellow powder in 78% yield, mp 182–
for CNMR at 125 MHz and Chemical shifts were expressed in ppm relative to
SiMe4 as internal standards. Mass spectra were recorded on 70 eV EI Ms-QP
1000 EX (Shimadzu, Japan). The Pharmacological evaluations of the products
were carried out in Pharmacological Unit, Pharmacology Department, National
Research Centre, Dokki, Cairo, Egypt. Biological activity screening was carried
out in The Biotechnology and Fermentation Centre, Al-Azhar University, Cairo,
Egypt.
184 °C: IR (cmÀ1
, m); 3390 (br s, NH), 2929 (CH alkyl), 1700, 1685, (2CO),
1572 (C@N), 1509 (C@C): 1H NMR (DMSO-d6, d, ppm): 1.21 (t, 3H, J = 14.3 Hz,
CH3), 1.35 (t, 3H, J = 7.03 Hz, CH3) 2.24 (s, 3H, CH2), 4.18 (q, 2H, J = 7.13 Hz,
CH2), 4.28 (q, 2H, J = 7.08 Hz, CH2), 6.88 (s, 1H, azomethine proton), 8.50 (br,
2H, NH2, D2O exchangeable), 10.02 (br, NH, D2O exchangeable); Its MS (m/z),
299 (M+); C12H17N3O4S (299.3); Requires (Found): C, 48.14 (48.11); H, 5.72
(5.69); N, 14.03 (13.98).
3,5-Diethyl-2-amino-4-methylthiophenedicarboxylate (1). According to Gewald
et al.20 and the developed method, a mixture of ethylacetoacetate (0.01 mol),
ethylcyano-acetate (0.01 mol), sulfur (0.01 mol) and diethylamine (0.01 mol)
was heated (70 °C) under stirring in absolute ethanol for 4 h, then leave the
mixture for 24 h at 0 °C. The formed solid was collected by filtration, washed
with ethanol (20 mL), dried and crystallized from absolute ethanol, as yellow
5-Ethyl-2-amino-3-(N-hydroxymethylene-carbohydrazide)-4-methylthiophene-
carboxylate (7). A mixture of 6c (0.01 mol) and formic acid (10 mL), was
heated under reflux for 6 h. The reaction mixture was allowed to cool to
room temperature and poured onto water (100 mL). The solid formed was
collected by filtration, dried, and crystallized from benzene, orange powder,
crystals in a 72% yield, mp 107–109 °C; IR (cmÀ1
, m); 3420 (br, NH), 3042, 2917
(CH alkyl), 1715, 1718 (2CO); 1H NMR (DMSO-d6, d, ppm): 1.58 (m, 6H, two
triplet overlapped, 2CH3), 2.49 (s, 3H, CH3), 4.08 (m, 4H, two quartet
overlapped, 2CH2), 7.73 (br, 2H, NH2, D2O exchangeable); Its MS (m/z), 257
(M+); C11H15NO4S (257.3); Requires (Found): C, 51.34 (51.31); H, 5.87 (5.85); N,
5.44 (5.39).
in 80% yield, mp 198–2001 °C; IR (cmÀ1
, m): 3415 (br, NH), 2920 (CH
alkyl),1724, 1715, 1680 (3CO), 1560 (C@N); 1H NMR (DMSO-d6, d, ppm): 1.33
(t, 3H, J = 7.03 Hz, CH3), 2.52 (s, 3H, CH2), 4.35 (q, 2H, J = 7.09 Hz, CH2), 8.05
(br, 2H, NH2, D2O exchangeable), 8.65 (br, NH, D2O exchangeable), 9.02 (s, 1H,
aldehydic proton), 11.30 (br, OH, D2O exchangeable); Its MS (m/z), 271.3
(M+); C10H13N3O4S (507.5); Requires (Found): C, 44.27 (44.24); H, 4.83 (4.81);
N, 15.49 (15.51).
5-Ethyl-2-amino-3-carbohydrazide-4-methylthiophenecarboxylate
(2).
A
suspension of dry compound 2 (2.57 g, 0.01 mol) in hydrazine hydrate (80%)
(5 mL) was stirred under gentle reflux. The insoluble solid dissolved within
10 min with copious evolution of hydrogen sulfide to form a clear solution.
After 30 min. when the solid product started separating out, heating was
continued for 8 h. The reaction mixture was then allowed to cool to room
temperature. The solid was filtered, washed with ethanol, dried and
crystallized from dioxane; as yellow crystals in a 80% yield, mp 278–279 °C;
5-Ethyl-2-amino-3-(1,3,4-oxa/thiadiazol-2-yl)-4-methylthiophenecarboxylate (8a,
b). General Procedure.
A mixture of compound 7 (0.005 mol) and
phosphorus pentaoxide or phosphorus pentasulfide (4.00 g) was heated
under reflux for 12 h, in dry xylene (30 mL). The solid that separated upon
cooling was filtered off, crystallized from appropriate solvent to produce (8a,
b) in good yield.
IR (cmÀ1 ); 3400 (br, NH), 2917 (CH alkyl), 1715, 1690 (2CO); 1H NMR
, m
(DMSO-d6, d, ppm): 1.25 (t, 3H, J = 7.1 Hz, CH3), 2.42 (s, 3H, CH3), 4.18 (q, 2H,
J = 7.09 Hz, CH2), 4.35 (br, 2H, NH2), 7.63 (br, 2H, NH2), 8.93 (br, 1H, NH) (NH,
2NH2, D2O exchangeable); Its MS (m/z), 243 (M+); C9H13N3O3S (243.3);
Requires (Found): C, 44.43 (44.39); H, 5.38 (5.37); N, 17.27 (17.29).
5-Ethyl-2-amino-3-(1,3,4-oxadiazol-2-yl)-4-methyltiophenecarboxylate
Method A. It was obtained from the reaction of with phosphorus
pentoxide, as yellow powder and crystallized from ethanol/dioxane (1:1) in
76% yield, mp 211–213 °C; IR (cmÀ1
, m): 3385 (br, NH), 3026 (CH aryl), 2920 (CH
(8a).
7
5-Ethyl-2-amino-3-[(3,5-dimethylpyrazolyl
derivatives)-carbonyl]-4-
methylthiophene-carboxylate (3a, b). General procedure.
A
mixture of
alkyl), 1712 (CO), 1560 (C@N); 1H NMR (DMSO-d6, d, ppm): 1.30 (t, 3H,
J = 7.01 Hz, CH3), 2.54 (s, 3H, CH2), 4.28 (q, 2H, J = 7.11 Hz, CH2),7.98 (s, CH,
oxadiazole proton), 8.25 (br, 2H, NH2, D2O exchangeable); Its MS (m/z), 253
(M+); C10H11N3O3S (253.2); Requires (Found): C, 47.42 (47.39); H, 4.37 (4.39);
N, 16.59 (16.62).Method B. Compound 6 (0.005 mol) was heated at 210 °C for
30 min. The reaction product was purified preparative TLC on silica gel using
chloroform/ethylacetate (80:20) as an eluent to give 8a.
compound 2 (0.01 mol) and the b-diketone (0.01 mol) in absolute ethanol
(30 mL) was stirred under reflux for 12 h. The reaction mixture was allowed to
cool to 0 °C for 24 h, the solid precipitate was filtered off, dried and crystallized
from an appropriate solvent to produce 3a, b in high yields.
5-Ethyl-2-amino-3-[(3,5-dimethylpyrazol-1-yl)-carbonyl]-4-methylthiophene-
carboxylate (3a). It was obtained from the reaction of 2 (0.01 mol) with pentan-
2,4-dione (0.01 mol), as yellow crystals, crystallized from ethanol in a 80%
5-Ethyl-2-amino-3-(1,3,4-thiadiazol-2-yl)-4-methyltiophenecarboxylate (8b). It was
yield, mp 152–153 °C; IR (cmÀ1
,
m
); 3380 (br, NH), 2917 (CH alkyl), 1710, 1690
obtained from the reaction of
powder and crystallized from ethanol/dioxane (1:1) in a 78% yield, mp 230–
231 °C; IR (cmÀ1
): 3392 (br, NH), 3029 (CH aryl), 2918 (CH alkyl), 1718 (CO),
7 with phosphorus pentasulfide, as yellow
(2CO), 1625 (C@N), 1540 (C@C); 1H NMR (DMSO-d6, d, ppm): 1.30 (t, 3H,
J = 7.0 Hz, CH3), 2.19 (s, 3H CH3), 2.51 (s, 3H, CH3), 2.67 (s, 3H, CH3), 4.25 (q, 2H,
J = 7.10 Hz, CH2), 6.21 (s, 1H, pyrazole proton), 7.98 (br, 2H, NH2, D2O
exchangeable); Its MS (m/z), 308 (M+, 12%), 307 (M+À1, 57%), 212 (M+-
C5H8N2, 100%); C14H18N3O3S (308.4); Requires (Found): C, 54.52 (54.49); H,
5.88 (5.86); N, 13.62 (13.59).
,
m
1568 (C@N); 1H NMR (DMSO-d6, d, ppm): 1.32 (t, 3H, J = 7.02 Hz, CH3), 2.51 (s,
3H, CH2), 4.30 (q, 2H, J = 7.09 Hz, CH2), 8.05 (s, CH, thiadiazole proton), 8.40 (br,
2H, NH2, D2O exchangeable); Its MS (m/z), 269 (M+, 100%); C10H11N3O2S2
(269.3); Requires (Found): C, 44.59 (44.57); H, 4.11 (4.09); N, 15.60 (15.57).
3,5-Diethyl-2-acetylamino-4-methylthiophenedicarboxylate (9). It was obtained
from the reaction of 1 (0.01 mol) with acetic anhydride (30 mL) was heated
under reflux for 3 h. The reaction mixture was allowed to cool to room
temperature and the solid formed was collected by filtration, dried, and
crystallized from ethanol, as yellow powder and crystallized from ethanol in a
5-Ethyl-2-amino-3-[(4-chloro-3,5-dimethylpyrazol-1-yl)-carbonyl]-4-methyl-
thiophene-carboxylate (3b). It was obtained from the reaction of 2 with 3-
chloropentan-2,4-dione (0.01 mol), as a light yellow powder and crystallized
from ethanol in a 76% yield, mp 103–105 °C; IR (cmÀ1
, m); 3400 (br, NH), 2929
(CH alkyl), 1712, 1688 (2CO), 1620 (C@N), 1550 (C@C); 1H NMR (DMSO-d6, d,
ppm): 1.32 (t, 3H, J = 7.1 Hz, CH3), 2.21 (s 3H CH3), 2.52 (s, 3H, CH3), 2.63 (s, 3H,
CH3), 4.22 (q, 2H, J = 7.08 Hz, CH2), 8.02 (br, 2H, NH2, D2O exchangeable); Its
MS (m/z), 342 (M+ + 1, 28%), 341 (M+, 56%), 212 (M+-C5H7ClN2, 100%);
90% yield, mp 129–130 °C; IR (cmÀ1
, m): 3400 (br, NH), 2920 (CH alkyl), 1718,
1685 (2CO), 1530 (C@C); 1H NMR (DMSO-d6, d, ppm): 1.29 (t, 3H, J = 7.02 Hz,
CH3), 1.35 (t, 3H, J = 14.00 Hz, CH3), 2.24 (s, 3H, CH3), 2.52 (s, 3H, CH3), 4.26 (q,
2H, J = 7.08 Hz, CH2), 4.36 (q, 2H, J = 7.10 Hz, CH2), 11.10 (br, NH, D2O
exchangeable); Its MS (m/z), 299 (M+, 100%); C13H17NO5S (299.3); Requires
(Found): C, 52.15 (52.17); H, 5.72 (5.74); N, 4.67 (4.59).
C14H16ClN3O3S (341.8); Requires (Found): C, 49.19 (49.17); H, 4.72 (4.71); N,
12.29 (12.26).
5-Ethyl-2-amino-3-[(3-methyl-5-oxo-4,5-dihydropyrazol-1-yl)carbonyl]-4-methyl-
thiophenecarboxylate (4).
A
solution of compound
2
(0.01 mol) and
6-Carboxyethyl-2,5-dimethyl-3-(N-amino)-thieno[2,3-d]pyrimidin-4-one (10).
It was obtained from the reaction of 9 (2.99 g, 0.01 mol) with hydrazine hydrate
(10 mL, 80%) in ethanol (30 mL) was heated under reflux for 2 h. The reaction
mixture was allowed to cool to room temperature and poured onto water
(100 mL). The solid formed was collected by filtration, dried and crystallized
from ethanol, as yellow powder and crystallized from ethanol in a 83% yield, mp
ethylacetoacetate (0.01 mol) in sodium ethoxide solution (prepared by
dissolving (0.01 mol) of sodium metal in (30 mL) absolute ethanol was
heated under reflux with stirring for 6 h. The reaction mixture was allowed
to cool and poured onto cold water (100 mL) and neutralized by acetic acid,
whereby a solid was precipitated, which was filtered off and crystallized from
ethanol, as a yellow powder in a 74% yield, mp 150–152 °C; IR (cmÀ1
,
m
); 3410
205–207 °C; IR (cmÀ1
, m): 3405 (br, NH), 2920 (CH alkyl), 1715, 1683 (2CO),
(br, NH), 2936 (CH alkyl), 1705, 1697, 1684 (3CO), 1550 (C@N), 1500 (C@C): 1
H
1570 (C@N); 1H NMR (DMSO-d6, d, ppm): 1.34 (t, 3H, J = 7.01 Hz, CH3), 2.29 (s,
3H, CH3), 2.53 (s, 3H, CH3), 4.35 (q, 2H, J = 7.12 Hz, CH2), 8.56 (br, 2H, NH2, D2O
exchangeable); Its MS (m/z), 267, (M+, 100%); C11H13N3O3S (267.3); Requires
(Found): C, 49.42 (49.39); H, 4.90 (4.93); N, 15.72 (15.69).
5-Ethyl-2-amino-3-(arylmethylenehydrazide)-4-methylthiophene-carboxylate (11a–
c). General procedure. A mixture from compound 2 (2.43 g, 0.01 mol) and the
appropriate aromatic aldehyde (0.01 mol) was stirred under reflux in glacial
acetic acid (10 mL) for 10 min. The reaction mixture was allowed to cool to
room temperature; the solid separated was filtered off and crystallized from
appropriate solvent to produces 11a–c in high yields.
NMR (DMSO-d6, d, ppm): 1.31 (t, 3H, J = 7.2 Hz, CH3), 2.23 (s, 3H, CH3), 2.52 (s,
2H, CH3), 2.86 (s, 2H, CH2), 4.23 (q, 2H, J = 7.09 Hz, CH2), 8.00 (br, 2H, NH2, D2O
exchangeable); Its MS (m/z), 309 (M+); C13H15N3O4S (309.3); Requires (Found):
C, 50.47 (50.49); H, 4.88 (4.86); N, 13.58 (13.55).
5-Ethyl-2-amino-3-[(3,5-dioxopyrazolidin-1-yl)carbonyl]-4-methylthiophene-
carboxylate (5). A solution of compound 2 (0.01 mol) and freshly distilled
diethylmalonate (0.01 mol) in sodium ethoxide solution (prepared by
dissolving (0.01 mol) of sodium metal in (30 mL) absolute ethanol was
heated under reflux with stirring for 5 h. The solvent was evaporated under
reduced pressure, and the crude was acidified with 10% hydrochloric acid.
The solid formed was filtered off, washed with cold water, and crystallized
5-Ethyl-2-amino-3-(phenylmethylenehydrazide)-4-methylthiophenecarboxylate
(11a). It was obtained from 2 and benzaldehyde (1.06 g), as yellow crystals and
from ethanol, as yellow powder in 70% yield, mp 212–214 °C; IR (cmÀ1
,
m
);
crystallized from ethanol in a 83% yield, mp 120–123 °C; IR (cmÀ1
, m); 3388 (br,
3400 (br, NH), 2928 (CH alkyl), 1700, 1690, 1684, 1678 (4CO), 1580 (C@N),
1520 (C@C); 1H NMR (DMSO-d6, d, ppm): 1.32 (t, 3H, J = 7.0 Hz, CH3), 2.23 (s,
3H, CH2), 2.80 (s, 2H, CH2), 4.25 (q, 2H, J = 7.11 Hz, CH2), 8.06 (br, 2H, NH2,
D2O exchangeable), 8.86 (br, NH, D2O exchangeable); Its MS (m/z), 311 (M+);
NH), 3038 (CH aryl), 2925 (CH alkyl), 1695, 1688 (2CO), 1625 (C@N); 1H NMR
(DMSO-d6, d, ppm): 1.27 (t, 3H, J = 7.0 Hz, CH3), 2.43 (s, 3H, CH3), 4.16 (q, 2H,
J = 7.06 Hz, CH2), 7.14–7.29 (m, 5H, Ar-H), 7.90 (br, 2H, NH2), 8.12 (s, 1H,
azomethine proton), 8.93 (br, 1H, NH) (NH, NH2, D2O exchangeable); Its MS (m/
z), 331 (M+, 100%); C16H17N3O3S (331.4); Requires (Found): C, 57.98 (57.96); H,
5.17 (5.68); N, 12.68 (12.66).
C12H13N3O5S (311.3); Requires (Found): C, 46.29 (46.27); H, 4.20 (4.18); N,
13.49 (13.46).

H. N. Hafez, A. B. A. El-Gazzar / Bioorg. Med. Chem. Lett. 18 (2008) 5222–5227
5227
5-Ethyl-2-amino-3-(4-chlorophenylmethylenehydrazide)-4-methylthiophene-
carboxylate (11b). It was obtained from 2 and 4-chlorobenzaldehyde (1.40 g), as
yellow crystals and crystallized from dioxane in a 86% yield, mp 195–197 °C; IR
andchloroacetoneorphenacylbromide(0.01 mol)wasrefluxedinethanol(50 mL)
for 8–10 h. The reaction mixture was then kept overnight,
the solid that separated was filtered off and crystallized from n-hexane to
produce (17a, b) in high yield .
(cmÀ1
, m); 3400 (br, NH), 3040 (CH aryl), 2929 (CH alkyl), 1700, 1686 (2CO),
1630 (C@N); 1H NMR (DMSO-d6, d, ppm): 1.24 (t, 3H, J = 7.0 Hz, CH3), 2.41 (s,
3H, CH3), 4.19 (q, 2H, J = 7.05 Hz, CH2), 7.14 (d, 2H, J = 8.02 Hz, Ar-H),7.69 (d, 2H,
J = 8.02 Hz, Ar-H), 8.02 (br, 2H, NH2), 8.10 (s, 1 H, azomethine proton), 9.10 (br,
1H, NH) (NH, NH2, D2O exchangeable); Its MS (m/z), 366 (M+ + 1, 28%), 365 (M+,
100%); C16H16ClN3O3S (365.8); Requires (Found): C, 52.52 (52.49); H, 4.41
(4.39); N, 11.48 (11.50)..
5-Ethyl-2-amino-3(4-methyl-1,3-thiazol-2-yl)carbohydrazide-4-methylthiophene-
carboxylate (17a). It was obtained from compound 16 and chloroacetone; as white
crystals, crystallized from n-hexane in 66% yield, mp 134–135 °C (dec): IR (cmÀ1
m
,
);3425 (br, NH), 2927 (CH alkyl), 1692, 1675 (2CO), 1635 (C@N);1H NMR(DMSO-
d6, d, ppm): 1.23 (t, 3H, J = 7.01 Hz, CH3),2.22 (s, 3H, CH3), 2.45 (s, 3H, CH3), 4.21 (q,
2H, J = 7.03 Hz, CH2), 7.90 (br, 2H, NH2), 8.16 (s, CH, thiazole proton), 9.05, 10.20
(2br, 2H, 2NH) (2NH, NH2, D2O exchangeable); Its MS (m/z), 340 (M+, 70%);
5-Ethyl-2-amino-3-(4-methoxyphenylmethylenehydrazide)-4-methylthiophene-
carboxylate (11c). It was obtained from 2 and 4-methoxybenzaldehyde (1.36 g),
as yellow powder, crystallized from dioxane in a 81% yield, mp 166–167 °C; IR
C13H16N4O3S2 (340.4); Requires (Found): C, 45.86 (45.83); H, 4.73 (4.69); N, 16.46
(16.42).
(cmÀ1
,
m
); 3396 (br, NH), 3025 (CH aryl), 2934 (CH alkyl), 1705, 1687 (2CO),
5-Ethyl-2-amino-3(4-phenyl-1,3-thiazol-2-yl)carbohydrazide-4-methylthiophene-
carboxylate (17b). It was obtained from compound 16 and phenacylbromide, as a
yellow powder, crystallized from benzene in 53% yield, mp 107–109 °C (melted);
1635 (C@N); 1H NMR (DMSO-d6, d, ppm): 1.25 (t, 3H, J = 7.02 Hz, CH3), 2.43 (s,
3H, CH3), 4.17 (q, 2H, J = 7.08 Hz, CH2), 4.34 (s, 3H, OCH3), 7.25 (d, 2H,
J = 8.00 Hz, Ar-H), 7.70 (d, 2H, J = 8.01 Hz, Ar-H), 7.98 (br, 2H, NH2), 8.18 (s, 1H,
azomethine proton), 9.40 (br, 1H, NH) (NH, NH2, D2O exchangeable); Its MS (m/
z), 361 (M+, 100%); C17H19N3O4S (361.4); Requires (Found): C, 56.49 (56.47); H,
5.29 (5.26); N, 11.62 (11.56).
3430 (br, NH),3035 (CH, aryl), 2915 (CH alkyl), 1689, 1670 (2CO), 1620 (C@N); 1
H
NMR (DMSO-d6, d, ppm): 1.28 (t, 3H, J = 7.02 Hz, CH3), 2.47 (s, 3H, CH3), 4.19 (q, 2H,
J = 7.05 Hz, CH2), 7.09–7.23 (m, 5H, Ar-H), 8.05 (br, 2H, NH2), 8.20 (s, CH, thiazole
proton), 9.00, 10.15 (2br, 2H, 2NH) (2NH, NH2, D2O exchangeable); Its MS (m/z),
402 (M+, 43%); C18H18N4O3S2 (402.4); Requires (Found): C, 53.71 (53.69); H, 4.50
(4.47); N, 13.92 (13.89).
5-Ethyl-2-acetamido-3-(arylmethylenehydrazide)-4-methylthiophenecarboxylate
(13a–c). General procedure. A solution of each of compound 11a–c (0.01 mol) in
30 mL acetic anhydride was stirred under reflux for 4 h (TLC). The excess of the
solvent was evaporated under vacuum to dryness. The solid product that formed
was crystalized from appropriate solvent to produces 13a–c in high yields.
5-Ethyl-2-acetamido-3-(phenylmethylenehydrazide)-4-methylthiophenecarboxylate
(13a). Itwas obtainedfrom 11a, asyellow crystals andcrystallizedfrom ethanolin
Antibacterial activity. The newly synthesized compounds were screened for their
antibacterial activity against pathogenic organisms; Bacillus cereus (BTCC19),
Shigella dysenteriae (AE 14396), and Salmonella typhi (AE 14612) (Table 1) and for
antifungalactivityagainst Macrophominaphaseolina (Tassi) Goid, Fusariumequiseti
(Corda) Sacc, Alternaria alternata (Fr.) Kedisslar and Coillectotrium corchori Ikata
(Yoshida) (Table 2). The disc diffusion method22 and poisoned-food techniques23
were used for antibacterial and antifungal activities, respectively. The tested
compounds were dissolved in N,N-dimethylformamide (DMF) to get a solution of
1 mg mLÀ1. The inhibition zones were measured in millimeters at the end of an
incubation period of 48 h at 28 °C. DMF alone showed no inhibition zone. Nutrient
agar (NA) and potato dextrose agar (PDA) were used as basal media to test the
bacteria and fungi, respectively. Commercial antibacterial ampicillin and
antifungal nystatin were also tested under similar conditions for comparison.
Anti-inflammatory activity (Carrageenin induced rat hind paw edema model). The
method adopted resembles essentially that described by Winter et al.24 (0.5 mL of
tween-80 in distilled water) was selected as vehicle to suspend the standard drugs
and the test compounds. The albino rats weighing between 150 and 180 g was
starved for 18 h prior to the experiment. The animals were weighed, marked for
identification and divided into 12 groups each group containing five animals.
Edema was induced in the left hind paw of all rats by subcutaneous injection of
0.1 mL of 1% (W/V) carrageenin in distilled water into their fotpads. The 1st group
was kept as control and was given the respective volume of the solvent (0.5 mL of
tween-80 in distilled water). The 2nd to 11th groups were orally administered
aqueous suspension of the synthesized compounds in dose of (15 mg/kg body
weight) 1 h before carrageenin injection. The last group (standard) was
78% yield, mp 190–193 °C; IR (cmÀ1
, m); 3386 (br, NH), 3032 (CH aryl), 2920 (CH
alkyl), 1696, 1685, 1680 (3CO), 1615 (C@N); 1H NMR (DMSO-d6, d, ppm): 1.23 (t,
3H, J = 7.01 Hz, CH3), 2.45 (s, 3H, CH3), 3.02 (s, 3H, CH3), 4.13 (q, 2H, J = 7.06 Hz,
CH2), 7.18–7.34 (m, 5H, Ar-H), 8.08 (s, 1H, azomethineproton), 8.95, 9.80 (2br, 2H,
2NH, D2O exchangeable); Its MS (m/z), 373 (M+, 100%); C18H19N3O4S (373.4);
Requires (Found): C, 57.89 (57.90); H, 5.12 (5.09); N, 11.25 (11.26).
5-Ethyl-2-acetamido-3-(4-chlorophenylmethylenehydrazide)-4-methylthiophene-
carboxylate (13b). It was obtained from 11b, as yellow crystals and crystallized
from dioxane in 70% yield, mp 213–215 °C; IR (cmÀ1
, m); 3410 (br, NH), 3025 (CH
aryl), 2932 (CH alkyl), 1705, 1688, 1680 (3CO), 1615 (C@N); 1H NMR (DMSO-d6, d,
ppm): 1.22 (t, 3H, J = 7.02 Hz, CH3), 2.40 (s, 3H, CH3), 3.05 (s, 3H, CH3), 4.16 (q, 2H,
J = 7.06 Hz, CH2), 7.22 (d, 2H, J = 8.01 Hz, Ar-H),7.75 (d, 2H, J = 8.01 Hz, Ar-H), 8.12
(s, 1H, azomethine proton), 9.20, 10.00 (2br, 2H, 2NH, D2O exchangeable); Its MS
(m/z), 408 (M+ + 1, 31%), 407 (M+, 100%); C18H18ClN3O4S (407.8); Requires
(Found): C, 53.00 (53.02); H, 4.44 (4.39); N, 10.30 (10.34).
5-Ethyl-2-acetamido-3-(4-methoxyphenylmethylenehydrazide)-4-methylthiophene-
carboxylate (13c). It was obtained from 11c, as yellow powder, crystallized from
ethanol in 73% yield, mp 231–233 °C; IR (cmÀ1
, m); 3400 (br, NH), 3034 (CH aryl),
2918 (CH alkyl), 1695, 1688, 1675 (3CO), 1620 (C@N); 1H NMR (DMSO-d6, d,
ppm): 1.26 (t, 3H, J = 7.0 Hz, CH3), 2.44 (s, 3H, CH3), 3.01 (s, 3H, CH3), 4.20 (q, 2H,
J = 7.06 Hz, CH2), 4.30 (s, 3H, OCH3), 7.28 (d, 2H, J = 8.04 Hz, Ar-H), 7.78 (d, 2H,
J = 8.04 Hz, Ar-H), 8.11 (s, 1H, azomethine proton), 9.00, 9.40 (2br, 2H, 2NH, D2O
exchangeable); Its MS (m/z), 403 (M+, 100%); C19H21N3O5S (403.4); Requires
(Found): C, 56.56 (56.53); H, 5.24 (5.22); N, 10.41 (10.39).
administered indomethacin in
a dose of 10 mg/kg body weight, orally as
aqueous suspension.25 The paw volume of each rat was measured immediately
by mercury plethysmometer, before carrageenin injection and then hourly for 4 h
post administration of aqueous suspension of the synthesized compounds. The
edema rate and inhibition rate of each group were calculated as follows, (Edema
rate (E) % = Vt À Vo/Vo, Inhibition rate (I) % = Ec À Et/Ec) where Vt is the volume
before carrageenin injection (mL), Vt is the volume at t hours after carrageenin
injection (mL) Ec, Et the edema rat of control group and treated group, respectively.
Analgesic activityusinghotplatetest. Theexperimentwascarried outasdescribed
by Turner26 using hot plate apparatus, maintained at 53 0.5 °C. The mice were
divided into 12 groups of five animals each. The reaction time of the albino mice to
the thermal stimulus was the time interval between placing the animal in the hot
plateandwhen itlickeditshind paworjumped. Reaction timewasmeasured prior
to aqueous suspension of synthesized compounds and drug treatment (0 min).
Group 1 was kept as normal control. The aqueous suspension of synthesized
compounds wasorallyadministeredtomice ofgroups 2to11 atdosesof20 mg/kg.
Mice of group 12 (reference) were orally treated with aspirin in a dose of 20 mg/kg
b.w. Thereaction time was againmeasured at 15 minandrepeated at 0, 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 s. The reaction time was
calculated for each synthesized compounds and drug-treated group.
Animals. Both sex of albino rats (150–200 g) were used in the study of
anti-inflammatory activity and both sex of Swiss albino mice weighing (25–
30 g) used in analgesic activity and, taking into account international
principle and local regulations concerning the care and used of laboratory
animals.27 The animals had free access to standard commercial diet and
water ad libitum and were kept in rooms maintained at 22 1 °C with 12 h
light dark cycle.
2,5-Dimethyl-5-carboxyethyl-thieno[2,3-d]pyrimidin-4(H)-one
(14).
General
procedure. A solution of each of compound 13a–c (0.01 mol) in an ethanolic
sodium ethoxide solution (prepared by dissolving 0.23 g of sodium metal in 30 mL
ethanol), was stirred under reflux for 6 h (TLC). The excess of the solvent was
evaporated under vacuum to dryness. The solid product that formed was
crystalized from dimethylformamide, as yellow crystals and crystallized from
dimethylformamidein72%yield, mp267–270 °C;IR(cmÀ1
,m);3385(br, NH),2918
(CH alkyl), 1700, 1687 (2CO), 1640 (C@N); 1H NMR (DMSO-d6, d, ppm): 1.30 (t, 3H,
J = 7.03 Hz, CH3), 2.40 (s, 3H, CH3), 2.79 (s, 3H, CH3), 4.21 (q, 2H, J = 7.06 Hz, CH2),
9.30 (br, 1H, NH, D2O exchangeable); Its MS (m/z), 252 (M+, 100%); C11H12N2O3S
(252.2); Requires (Found): C, 52.36 (52.29); H, 4.79 (4.76); N, 11.09 (11.12).
3-Amino-6-carboxyethyl-5-methyl-2-thioxo-thieno[2,3-d]pyrimidin-4-one (15). To a
warmed ethanolic sodium hydroxide solution (0.40 g in 50 mL ethanol),
compound 2 (2.43 g, 0.01 mol), and carbon disulfide (excess 5 mL) were added.
Themixture washeatedunderrefluxfor15 h. Thereaction mixturewasallowed to
cool to 0 °C, the deposited precipitate was filtered off, washed by water (20 mL),
dried, and crystallized from ethanol as brown crystals; in 68% yield, mp 192–
194 °C; IR (cmÀ1 ); 3430 (br, NH), 2923 (CH alkyl), 1690, 1675 (2CO); 1H NMR
, m
(DMSO-d6, d, ppm): 1.28 (t, 3H, J = 7.00 Hz, CH3), 2.44 (s, 3H, CH3), 4.19 (q, 2H,
J = 7.04 Hz, CH2), 7.54 (br, 2H, NH2), 9.30 (br, 1H, NH) (NH, NH2, D2O
exchangeable); Its MS (m/z), 285 (M+, 100%); C10H11N3O3S2 (285.3); Requires
(Found): C, 42.09 (42.07); H, 3.88 (3.85); N, 14.72 (14.76).
5-Ethyl-2-amino-3-carbonylthiosemicarbazide-4-methylthiophenecarboxylate (16). A
mixture of 2 (0.01 mol) was refluxed with 10 mL of 10% HCl and potassium
thiocyanate (0.015 mol) for 4 h. The reaction mixture was allowed to cool to room
temperature. The solid formed was collected by filtration, washed with water,
dried and then crystallized from dioxane/DMF (2:1), as a yellow powder in a 78%
22. Bauer, A. W.; Kirby, W. M. M.; Sherris, J. C.; Turck, M. Am. J. Clin. Pathol. 1966,
45, 493.
23. Grover, R. K.; Moore, J. D. Phytopathology 1952, 52, 876.
24. Winter, C. A.; Risley, E. A.; Nuss, G. W. Proc. Soc. Exp. Biol. Med. 1962, 111, 544.
25. Mino, J.; Moscatelli, V.; Hnatyszyn, O.; Gorzalczany, S.; Acevedo, C.; Ferraro, G.
G. J. Pharmacol. Res. 2004, 50, 59.
26. Turner, R. A. Analgesics. In Screening Methods in Pharmacology; Turner, R. A.,
Ed.; Academic Press: London, 1965; p 100.
yield, mp 226–227 °C; IR (cmÀ1
,
m
); 3400 (br, NH), 2915 (CH alkyl), 1690, 1678
(2CO); 1H NMR (DMSO-d6, d, ppm): 1.27 (t, 3H, J = 7.00 Hz, CH3), 2.45 (s, 3H, CH3),
4.16 (q, 2H, J = 7.05 Hz, CH2), 5.30 (br, 2 H, NH2), 7.60 (br, 2H, NH2), 8.95, 9.30 (2br,
2H, 2NH) (2NH, 2NH2, D2O exchangeable); Its MS (m/z), 302 (M+, 100%);
C10H14N4O3S2 (302.3); Requires (Found): C, 39.72 (39.67); H, 4.66 (4.64); N,
18.53 (18.46).
27. Olfert, E. D.; Cross, B. M.; McWilliam, A. A., Eds.; Canadian Council of
Animal Care Guide to the Care and Use of Experimental Animals; Vol. 1,
2nd ed., 1993.
5-Ethyl-2-amino-3(4-methyl/or phenyl-1,3-thiazol-2-yl)carbohydrazide-4-methylthio-
phenecarboxylate (17a, b), General procedure; A mixture of compound16 (0.01 mol)