Design and synthesis of some thiazolyl and thiadiazolyl derivatives of antipyrine as potential non-acidic anti-inflammatory, analgesic and antimicrobial agents

Article abstract of DOI:10.1016/j.bmc.2008.11.035

The synthesis of two groups of structure hybrids comprising basically the antipyrine moiety attached to either polysubstituted thiazole or 2,5-disubstituted-1,3,4-thiadiazole counterparts through various linkages is described. Twelve out of the newly synt

Full text of DOI:10.1016/j.bmc.2008.11.035

Bioorganic & Medicinal Chemistry 17 (2009) 882–895
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Design and synthesis of some thiazolyl and thiadiazolyl derivatives
of antipyrine as potential non-acidic anti-inflammatory, analgesic
and antimicrobial agents q
b
c
Sherif A. F. Rostom a, , Ibrahim M. El-Ashmawy , Heba A. Abd El Razik ,
*
Mona H. Badr c, Hayam M. A. Ashour c
a Department of Medicinal Chemistry, Faculty of Medicine, King Abdulaziz University, PO Box 80205, Jeddah 21589, Saudi Arabia
b Department of Pharmacology, Faculty of Veterinary Medicine, University of Alexandria, Alexandria, Egypt
c Department of Pharmaceutical Chemistry, Faculty of Pharmacy, University of Alexandria, Alexandria 21521, Egypt
a r t i c l e i n f o
a b s t r a c t
Article history:
The synthesis of two groups of structure hybrids comprising basically the antipyrine moiety attached to
either polysubstituted thiazole or 2,5-disubstituted-1,3,4-thiadiazole counterparts through various link-
ages is described. Twelve out of the newly synthesized compounds were evaluated for their anti-inflam-
matory activity using two different screening protocols; namely, the formalin-induced paw edema and
the turpentine oil-induced granuloma pouch bioassays, using diclofenac Na as a reference standard.
The ulcerogenic effects and acute toxicity (ALD50) values of these compounds were also determined.
Meanwhile, the analgesic activity of the same compounds was evaluated using the rat tail withdrawal
technique. Additionally, the synthesized compounds were evaluated for their in vitro antimicrobial activ-
ity. In general, compounds belonging to the thiazolylantipyrine series exhibited better biological activi-
ties than their thiadiazolyl structure variants. Collectively, compounds6, 10, 26, and 27 proved to display
distinctive anti-inflammatory and analgesic profiles with a fast onset of action. All of the tested com-
pounds revealed super GI safety profile and are well tolerated by the experimental animals with high
safety margin (ALD50 > 3.0 g/kg). Meanwhile, compounds 7, 10, 11, and 23 are considered to be the most
active broad spectrum antimicrobial members in this study. Compound 10 could be identified as the most
biologically active member within this study with an interesting dual anti-inflammatory analgesic and
antibacterial profile.
Received 22 September 2008
Revised 4 November 2008
Accepted 12 November 2008
Available online 20 November 2008
Keywords:
Antipyrine
Thiazoles
1,3,4-Thiadiazoles
Anti-inflammatory activity
Ulcerogenic effect
Acute toxicity
Analgesic activity
Antimicrobial activity
Ó 2008 Elsevier Ltd. All rights reserved.
1. Introduction
kidneys, whereas, the inducible COX-2 is selectively activated by
pro-inflammatory stimuli and facilitates the release of prostaglan-
Non-steroidal anti-inflammatory drugs (NSAIDs) represent a
heterogeneous family of pharmacologically active compounds
used to alleviate acute and chronic inflammation, pain, and fever.
Their clinical efficacy is closely related to their ability to inhibit
both COX-1 and COX-2 isoforms of the enzyme cyclooxygenase
(COX) also referred to as prostaglandin H2 synthase since it cata-
lyzes the conversion of arachidonic acid to prostaglandin H2
(PGH2).1 The constitutive COX-1 isoform is mainly responsible for
the synthesis of prostaglandins which exert cytoprotective effect
in the gastrointestinal (GI) tract and control renal function in the
dins involved in the inflammatory process.2 Consequently, their
long-term clinical employment is associated with significant
side effects such as gastrointestinal lesions, bleeding, and
nephrotoxicity.3
Since the introduction of antipyrine; the first pyrazolone deriv-
ative used in the management of pain, inflammation and fever into
clinical use in 1884, great attention has been focused on pyrazole
derivatives as potent anti-inflammatory, analgesic and antipyretic
agents.4–11 As a result, a large number of pyrazoles have been
obtained and some have gained application on the clinical level.
Among the already marketed COX-2 inhibitors that comprise the
pyrazole nucleus, celecoxib; 4-[5-(4-methylphenyl)-3-(trifluor-
ophenyl)-1H-pyrazol-1-yl]benzenesulfonamide; proved to be a
potent and GI safe anti-inflammatory and analgesic agent.12
Furthermore, diverse chemotherapeutic activities have been
ascribed to pyrazoles as antimicrobial,13–16 antiparasitic,17 antivi-
ral,18 and antineoplastic agents.19–21 Interest in this field has been
q
This work was presented in the World Congress of Pharmacy and Pharmaceutical
Sciences, 68th International Congress of FIP (Reengineering Pharmacy Practice in a
Changing World), Basel, Switzerland, August 29th–September 4th, 2008, Poster No.
MC-P-009.
* Corresponding author. Tel.: +966 507654566.
E-mail address: sherifrostom@yahoo.com (S.A.F. Rostom).
0968-0896/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2008.11.035

S. A. F. Rostom et al. / Bioorg. Med. Chem. 17 (2009) 882–895
883
intensified after the discovery of the natural pyrazole C-glycoside
pyrazofurin; 4-hydroxy-3-b- -ribofuranosyl-1H-pyrazole-5-car-
structure leads A35 and B37 (Fig. 1) which comprise the thiazole
ring and C36 (Fig. 1) which encounter the antipyrine moiety.
In view of the above-mentioned facts, we report herein the
synthesis, anti-inflammatory, analgesic and antimicrobial evalu-
ation of some novel structure hybrids incorporating both the
antipyrine moiety with either the thiazole or thiadiazole ring
systems through different linkages having the general formulae
D–F (Fig. 1). This combination was suggested in an attempt to
investigate the influence of such hybridization and structure
variation on the anticipated biological activities, hoping to add
some synergistic biological significance to the target molecules.
The target compounds were rationalized so as to comprise
some pharmacophores that are believed to be responsible for
the biological activity of some relevant chemotherapeutic agents
such as the carboxamido, ureido and thioureido functionalities.
The substitution pattern of the thiazole and the thiadiazole
rings was carefully selected so as to confer different electronic
environment to the molecules. It should be pointed out that,
in addition to the targeted anti-inflammatory, analgesic and
antimicrobial activities, the ulcerogenic and acute toxicity pro-
files of some of the newly synthesized compounds will be also
evaluated.
D
boxamide. This antibiotic was reported to possess a broad spec-
trum of antimicrobial and antiviral activities in addition to being
active against several tumor cell lines.22 On the other hand, careful
literature survey revealed that thiazole and thiadiazole ring sys-
tems have occupied a unique position in the design and synthesis
of novel biologically active agents with remarkable analgesic and
anti-inflammatory activities,23–27 in addition to their well docu-
mented potential antimicrobial activities.28–32
Concomitant use of several drugs to treat inflammatory condi-
tions that might be associated with some microbial infections
may cause serious health problems, especially in patients with
impaired liver or kidney functions. In addition, from pharmaco-
economic viewpoint, and seeking better patient compliance, the
discovery of a dual anti-inflammatory–antimicrobial agent with
potential activity and fewer adverse effects is a priority of the cur-
rent global research, and consequently has occupied our prime
interest in recent years.
Being involved in a research program aiming at finding out new
structure leads that would act as potent dual anti-inflammatory–
antimicrobial agents, we have reported the synthesis, anti-inflam-
matory and antimicrobial activities of some lead compounds
comprising mainly the pyrazole counterpart substituted with
various functionalities and attached to different heterocyclic ring
systems through various linkages.33–37 In particular, potential dual
anti-inflammatory and antimicrobial activities were linked to the
2. Results and discussion
2.1. Chemistry
The synthetic strategies adopted for the synthesis of the interme-
diate and target compounds are depicted in Schemes 1–4. In Scheme
1, the starting compound 2-cyano-N-(2,5-dihydro-2,3-dimethyl-5-
R1
S
oxo-1-phenyl-1H-pyrazol-4-yl)acetamide38
2
was prepared by
R
N
H2N
O
N
N
N
R
heating
4-amino-2,3-dimethyl-1-phenyl-1,2-dihydropyrazol-3-
H2N
N
one (4-aminoantipyrine) 1 with ethyl cyanoacetate according to a
literature procedure.39 The synthesis of the key intermediate thiaz-
olyl derivatives 3 and 4 could be achieved according to the method
described by Gewald,40 by reacting amide 2 with sulfur and the
appropriate aryl isothiocyanate in the presence of triethyl amine
as a basic catalyst. Refluxing 3 with dimethylsulfate in acetonitrile
afforded the methyl thiothiazolium salt 5, which in its turn was con-
densed with either malononitrile or ethyl cyanoacetate in presence
of triethylamine to produce the methylidene derivatives 6 or 7,
respectively, according to Gewald’s method.41,42 Moreover, heating
4 with acetic anhydride resulted in the formation of the thiazolopyr-
imidine derivative 8 in a reasonable yield. Furthermore, the synthe-
sis of the ureido derivative 9 and thioureido analogs 10–13 could be
successfully achieved by heating a mixture of 3 or 4 with either the
appropriate isocyanate or isothiocyanate derivatives, respectively,
in dry dioxane containing anhydrous K2CO3. Shifting to Scheme 2,
the intermediates 14 and 15 were prepared by adopting the same
reaction conditions reported by Bukowski et al.43 for the synthesis
of similar derivatives that involves the reaction of the appropriate
arylisothiocyanate with theamide2 in drydimethylformamide con-
taining two equivalents of potassium hydroxide. Upon acidification
of compounds 14 and 15, the acrylamides 16 and 17, respectively,
could be obtained. The latter acrylamides were further reacted with
the selectedphenacyl bromide utilizing a previously reported proce-
dure44 to produce the corresponding thiazoline derivatives 18–21.
Moreover, the thiazolidinone derivatives 22 and 23 were prepared
by reacting the intermediates 14 and 15 with chloroacetyl chloride.
On the other hand, compound 2-(5-amino-1,3,4-thiadiazol-2-
ylthio)-N-(2,5-dihydro-2,3-dimethyl-5-oxo-1-phenyl-1H-pyrazol-
4-yl)acetamide 26 was served as a key intermediate in Scheme 3. It
was prepared by reacting 2-chloro-N-(2,5-dihydro-2,3-dimethyl-
5-oxo-1-phenyl-1H-pyrazol-4-yl)-acetamide 2445 with aminomer-
captothiadiazole 25. Formation of the N-acetyl derivative 27 was
H
S
N
N
N
N
N
N
N
S
H
O
O
B
A
N
N
N
O
R1
HN
O
N
N
N
N
R
N
O
H
S
N
S
N
NH2
O
S
C
O
D
N
N
N
O
N
O
R
S
N
NH
S
H
S
N
N
O
H
R1
O
N
N
R
NC
F
E
Figure 1. Structures of lead compounds A–C and general formulae of the novel
series of compounds D–F.

884
S. A. F. Rostom et al. / Bioorg. Med. Chem. 17 (2009) 882–895
N
CN
N
N
N
H
H
N
N
N
CN
X
N
X
O
S
NH2
S
N
O
O
O
O
SCH3
N
X = CN or COOEt
H2N
H2N
1
2
SO4
CN
2
6: X = CN
7: X = COOEt
5
COOEt
3: R = C6H5
(CH3)2SO4
N
N
CH3
N
N
N
H
N
N
N
N
S / RNCS
O
O
H
(CH3CO)2O
N
O
O
O
N
Cl
S
N
O
NC
S
4: R = 4-Cl-C6H4
S
2
S
H2N
R
8
3: R= C6H5
4: R = 4-Cl-C6H4
R1
NH
N
NH
O
O
HN
N
N
O
S
R
HN
N
N
N
N
H
C6H5-NCO
Cl
R1NCS
O
H
S
N
O
S
S
S
4: R = 4-Cl-C6H4
10: R= C6H5 , R1= C6H5
11: R= C6H5 , R1 = 4-Cl-C6H4
12: R = 4-Cl-C6H4 , R1 = C6H5
9
13: R= 4-Cl-C6H4 , R1 = 4-Cl-C6H4
Scheme 1.
N
N
N
RNCS / KOH
N
HCl
N
N
NH
NC
SH
NHR
O
NH
NC
NH
S
K
O
O
O
O
O
NHR
NC
16: R= C6H5
17: R = 4-Cl-C6H4
2
14: R= C6H5
15: R = 4-Cl-C6H4
R1COCH2Br
ClCH2COCl
N
N
N
N
NH
NC
NH
S
S
O
R1
O
O
O
N
O
N
R
R
CN
18: R= C6H5 , R1= 4-Cl-C6H4
19: R= C6H5 , R1 = 4-Br-C6H4
20: R = 4-Cl-C6H4, R1 = 4-Cl-C6H4
21: R= 4-Cl-C6H4 , R1 = 4-Br-C6H4
22: R= C6H5
23: R = 4-Cl-C6H4
Scheme 2.
achieved in an excellent yield by heating 26 in acetic anhydride,
whereas, the N-benzoyl derivative 28 was obtained in an excellent
yield by refluxing 26 with benzoyl chloride in dry pyridine. In addi-
tion, stirring a suspension of 26 with ethyl chloroformate in dry
pyridine afforded the ethoxycarbonylamino derivative 29. Further-
more, heating 26 with the appropriate aryl isothiocyanate in dry

S. A. F. Rostom et al. / Bioorg. Med. Chem. 17 (2009) 882–895
885
O
Cl
O
O
NH2
ClCH2COCl
N
H
N
N
N
N
24
1
N
N
HS
NH2
S
25
O
O
N
N
N
O
N
N N
O
O
O
O
S
NH2
N
O
S
S
N
S
S
N
S
N
N
H
N
N
H
H
H
N
H
N
N
N
(CH3CO)2O
C6H5COCl
27
26
28
S
N
N
O
O
R
N
N
O
O
N
O
S
N
O
S
S
N
N
H
S
N
H
ClCOOC2H5
N
RNCS
H
N
H
H
N
N
30: R = C6H5
31: R = 4-Cl-C6H4
29
32: R = 4-CH3-C6H4
Scheme 3.
R
N
N
O
O
N
S
S
N
N
H
N
33: R = 4-Br-C6H4
34: R = 4-Cl-C6H4
N
N
O
O
RCOCH2Br
S
NH2
S
N
N
H
N
R
O
26
N
N
S
O
N
S
35: R = 4-Br-C6H4
36: R = 4-Cl-C6H4
Scheme 4.
dioxane containing anhydrous potassium carbonate yielded the
targeted thioureido derivatives 30–32.
Whereas, their 1H NMR spectra were characterized by the com-
plete disappearance of the signals attributed to the aminoantipyri-
nyl moiety and the presence of a triplet and a quartet assigned for
the ester moiety.
At this stage, it was attempted to prepare the proposed imi-
dazothiadiazolyl derivatives of antipyrine 33 and 34, by refluxing
compound 26 with the appropriate phenacyl bromide in ethanol.
Surprisingly, the (5-arylimidazo[2,1-b][1,3,4]thiadiazol-2-ylsulfa-
nyl)acetic acid ethyl esters 35 and 36 were obtained instead. The
structures of such unexpected compounds 35 and 36 were sub-
stantiated on basis of their IR and 1H NMR spectral data. The IR
spectra were characterized by the presence of C@O absorption
band at 1730–1729 cmꢀ1 corresponding to the ester group.
2.2. Biological evaluation
2.2.1. Anti-inflammatory (AI) activity
To assess the AI activity of the designed compounds, selected
analogs (12 compounds) from both the thiazolyl- and thiadiazolyl-
antipyrine series namely; 3, 6, 7, 9, 10, 18, 20, 22, 26, 27, 29, and

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S. A. F. Rostom et al. / Bioorg. Med. Chem. 17 (2009) 882–895
30; were evaluated by two screening protocols; namely, the forma-
lin-induced paw edema46 and turpentine oil-induced granuloma
pouch47 bioassays, using diclofenac Na (10 mg/kg) as a reference
standard anti-inflammatory agent. The paw edema was employed
as a model for acute and sub-acute inflammation, while the tur-
pentine oil-induced granuloma pouch assay was utilized as
another model for sub-acute inflammatory condition. The data
obtained were presented in Tables 1–3 and expressed as
means SE. Statistical differences of control and test groups was
carried out using the Analysis of Variance (ANOVA) followed by
‘Student–Newman–Keuls Multiple Comparison Test’. They were
performed using computer package of the Statistical Analysis
System (SAS, 1987), SAS Incorporation Institute. The difference in
results was considered significant when P < 0.05.
antipyrine series, the highest anti-inflammatory activity was con-
fined to compound 27 (43%), whereas, the rest of this series
showed unreliable activities. After 2 h interval, the data indicated
that compounds 3, 6, 9, 10, 20, 26, and 27 were nearly effective
in inhibiting the paw edema with percentage activity of 37–43%,
when compared with that of diclofenac Na (40%). Taking the
anti-inflammatory activity after 3 h time interval as a criterion
for comparison, it can be concluded that compound 9 from the
thiazolylantipyrine series showed anti-inflammatory activity
(49%) higher than diclofenac Na (44%), whereas, compounds 3, 6,
7, 10, and 20 displayed a good anti-inflammatory activity (38–
41%), however, none of them was found to be superior over the ref-
erence drug. On the contrary, the thiadiazolylantipyrines 26 and 27
proved to be more effective (49% and 46%, respectively) than dic-
lofenac Na (44%) at the same time interval. After 4h, compounds
3, 6, 9, 10, 18, 26, and 27 were nearly equipotent (38–43%) to
the reference drug (45%).
2.2.1.1. Formalin-induced paw edema bioassay (acute inflam-
matory model). In this acute inflammatory model,46 each test
compound was dosed orally (20 mg/kg body weight) 1 h prior to
induction of inflammation by formalin injection. Diclofenac Na
was utilized as a reference anti-inflammatory drug at a dose of
10 mg/kg, po. the anti-inflammatory activity was then calculated
1–4 h after induction and presented in Table 1 as the mean paw
volume (mL) in addition to the percentage anti-inflammatory
activity (AI%).
A comparative study of the anti-inflammatory activity of test
compounds relative to the reference drug at different time inter-
vals indicated the following: after 1 h, two compounds showed dis-
tinctive pharmacokinetic profiles as revealed from their potent and
rapid onset of action which was higher than diclofenac Na at a dose
of 10 mg/kg, po. Concerning the thiazolylantipyrine series, out-
standing AI% was recorded for compound 20 (35%), when com-
pared with diclofenac Na (17%), whereas, other compounds were
insignificantly different from the control. Among the thiadiazolyl-
2.2.1.2. Formalin-induced paw edema bioassay (sub-acute
inflammatory model). For this sub-acute inflammatory mod-
el,46 inflammation was induced by formalin injection in the first
and third days, and test compounds were administered orally (at
20 mg/kg daily) for 7 days. Again, diclofenac Na was used as a ref-
erence anti-inflammatory agent in this assay. The anti-inflamma-
tory activity was calculated at 1st and 8th day after induction
and presented in Table 2 as the mean paw volume and the percent-
age anti-inflammatory activity (AI%).
The obtained data revealed that, at the 1st day, compounds 6,
10, and 18 from the thiazolylantipyrine series displayed anti-
inflammatory activity (37–40%) nearly similar to diclofenac Na
(40%). Other compounds from both series showed weak to moder-
ate anti-inflammatory activities noticeably less than the reference
drug. At the 8th day, compounds 9 and 18 from thiazolylantipyrine
Table 1
Anti-inflammatory activity (AI) of some selected compounds in formalin-induced rat paw edema bioassay (acute inflammatory model)
Compounda
Volume of edema (mL)b
2 h
0
1 h
3 h
4 h
Control
3
0.30 0.01
0.31 0.01
0.53 0.01
0.46 0.02
(35)c
0.48 0.03
(26)
0.47 0.02
(30)
0.48 0.01
(26)
0.65 0.01
0.53 0.02*
(37)
0.69 0.01
0.54 0.03*
(41)
0.72 0.02
0.55 0.01*
(43)
6
0.31 0.01
0.31 0.01
0.31 0.02
0.32 0.04
0.32 0.01
0.30 0.01
0.29 0.01
0.31 0.01
0.32 0.01
0.33 0.01
0.30 0.03
0.30 0.01
0.52 0.02*
(40)
0.55 0.02*
(38)
0.56 0.04*
(40)
7
0.57 0.01
(26)
0.55 0.03*
(38)
0.61 0.02*
(29)
9
0.53 0.02*
(37)
0.51 0.02*
(49)
0.56 0.02*
(40)
10
0.48 0.02
(30)
0.54 0.02*
(37)
0.55 0.03*
(41)
0.57 0.05*
(40)
18
0.46 0.01
(39)
0.57 0.01
(29)
0.58 0.04*
(33)
0.56 0.02*
(43)
20
0.45 0.01*
(35)
0.51 0.02*
(40)
0.53 0.01*
(41)
0.60 0.01*
(29)
22
0.49 0.02
(13)
0.53 0.03*
(31)
0.59 0.02
(23)
0.63 0.01*
(19)
26
0.48 0.02
(26)
0.51 0.01*
(43)
0.51 0.02*
(49)
0.57 0.02*
(38)
27
0.45 0.01*
(43)
0.54 0.02*
(37)
0.53 0.01*
(46)
0.56 0.03*
(43)
29
0.48 0.04
(35)
0.62 0.03
(17)
0.63 0.02
(23)
0.67 0.02
(19)
30
0.47 0.02
(26)
0.54 0.03*
(31)
0.61 0.02
(21)
0.61 0.02*
(26)
Diclofenac Na
0.49 0.01*
(17)
0.51 0.02*
(40)
0.52 0.02*
(44)
0.55 0.02*
(45)
*
Significantly different compared to respective control values, P < 0.05.
Dose levels, po: test compounds (20 mg/kg b.wt.), diclofenac Na (10 mg/kg b.wt.).
Values are expressed as mean SE (number of animals N = 5 rats).
Values between parentheses (percentage anti-inflammatory activity, AI%).
a
b
c

S. A. F. Rostom et al. / Bioorg. Med. Chem. 17 (2009) 882–895
887
Table 2
inhibition was calculated. Diclofenac Na (10 mg/kg) was used as a
reference drug. The results depicted in Table 3 revealed that, while
most of the test compounds from both series showed anti-inflam-
matory activity less than the reference drug, the thiazolylantipy-
rine 9 was nearly equipotent (72%) with diclofenac Na (73%).
Additionally, compounds 6, 10, and 26 showed moderate anti-
inflammatory activity in this bioassay with percentage of granu-
loma inhibition of 62%, 65%, and 62%, respectively.
A collective interpretation of the anti-inflammatory activity of
the test compounds in pre-mentioned screens (Tables 1–3) re-
vealed that the thiazolylantipyrines 6, 9, 10, and 18 and the thi-
adiazolylantipyrine analogs 26 and 27 showed pronounced
activity in the formalin paw edema screen (acute inflammatory
model), nevertheless they proved to be less active in formalin
paw edema and turpentine oil-induced granuloma pouch screens
(sub-acute inflammatory models). These facts would suggest that
such type of compounds might be effective in managing acute
inflammation, while they would be less effective in controlling
chronic inflammatory conditions.
Anti-inflammatory activity (AI) of some selected compounds in formalin-induced rat
paw edema bioassay (sub-acute inflammatory model)
Compounda
Volume of edema (mL)b
1st day
0
8th day
Control
3
0.30 0.01
0.31 0.01
0.65 0.02
0.59 0.01
(20)c
0.89 0.02
0.70 0.01*
(34)
6
0.31 0.01
0.31 0.01
0.31 0.02
0.32 0.02
0.32 0.01
0.30 0.01
0.29 0.01
0.31 0.01
0.32 0.01
0.33 0.01
0.30 0.03
0.30 0.01
0.52 0.03*
(40)
0.71 0.01*
(32)
7
0.56 0.01*
(29)
0.72 0.02*
(31)
9
0.55 0.02*
(31)
0.65 0.01*
(42)
10
0.53 0.02*
(40)
0.68 0.02*
(39)
18
0.54 0.02*
(37)
0.65 0.01*
(44)
20
0.55 0.02*
(29)
0.66 0.03*
(39)
22
0.54 0.02*
(29)
0.71 0.02*
(17)
26
0.56 0.02*
(29)
0.67 0.02*
(39)
2.2.1.4. Ulcerogenic activity. The twelve tested compounds
that exhibited variable anti-inflammatory profiles in the pre-men-
tioned animal models were further evaluated for their ulcerogenic
potential in rats.48 Gross observation of the isolated rat stomachs
showed a normal stomach texture for all of the tested compounds
with no observable hyperemia indicating a superior GI safety pro-
file (no ulceration) in the population of the test animals at an oral
dose of 300 mg/kg, when administered twice at 2 h interval in
fasted rats. It is worth-mentioning that, diclofenac Na; the refer-
ence standard anti-inflammatory drug; was found to cause 100%
ulceration under the same experimental conditions.
27
0.57 0.02*
(29)
0.68 0.02*
(39)
29
0.58 0.03
(29)
0.76 0.03*
(27)
30
0.57 0.02*
(23)
0.77 0.02*
(20)
Diclofenac Na
0.51 0.01*
(40)
0.62 0.01*
(46)
*
Significantly different compared to respective control values, P < 0.05.
Dose levels, po: test compounds (20 mg/kg b.wt.), diclofenac Na (10 mg/kg
a
b.wt.).
b
Values are expressed as mean SE (number of animals N = 5 rats).
Values between parentheses (Percentage anti-inflammatory activity, AI%).
c
2.2.1.5. Acute toxicity. All of the selected compounds were fur-
ther evaluated for their approximate acute lethal dose ALD50 in
male rats using a literature method.49 The results indicated that
all of the tested compounds proved to be non-toxic and are well
tolerated by the experimental animals. The compounds showed a
high safety margin when screened at graded doses (0.1–3.0 g/kg,
po), where their ALD50 values were found to be >3.0 g/kg.
series were nearly effective (42% and 44%, respectively) as diclofe-
nac Na (46%). Other compounds from both series displayed vari-
able activities, however, none of them was found to be superior
over the reference drug.
2.2.1.3. Turpentine oil-induced granuloma pouch bioassay
(sub-acute inflammatory model). In this bioassay,47 each test
compound was administered orally (20 mg/kg) 1h prior to turpen-
tine oil injection and continued for 7 days. At the 8th day, the exu-
dates volume (mL) was measured and the percentage of granuloma
2.2.2. Analgesic activity
The analgesic activity of the same selected compounds was
evaluated using the rat tail withdrawal technique in response to
immersion in water at 55 °C,50 using diclofenac Na as a reference
drug (10 mg/kg, po). The analgesic activity was measured at 1–
3 h time intervals after pain induction. The results were recorded
as the average values of five administrations and the percentage
increase of the reaction time in comparison with the basal values.
The results were presented in Table 4 and expressed as means SE.
Statistical differences of control and test groups was carried out as
described under Section 2.2.1.
A comparative study of the analgesic activity of the test com-
pounds relative to the reference drug at different time intervals re-
vealed the following: after 1h, six compounds namely;6, 7, 10, 20,
26, and 27, were found to exhibit fast analgesic activity similar to
or even higher than (percentage increase of reaction time 27–48%)
that of diclofenac Na (26%). Special high analgesic activity was dis-
played by the thiazolylantipyrine 10, which exhibited potent anal-
gesic activity almost twice (48%) as that of diclofenac Na (26%).
Whereas, among the thiadiazolylantipyrine analogs, compound
26 proved to be the most potent member with percentage increase
of reaction time 42%. Furthermore, the results showed that the
same pattern of analgesic activity of the investigated compounds
was maintained after 2 h time interval (Table 4). On the other
hand, after 3 h the thiazolylantipyrines 6 and 10, and the thiadiaz-
olylantipyrine 26 were found to be nearly equipotent (72%, 82%,
Table 3
Anti-inflammatory activity of some selected compounds in turpentine oil-induced
granuloma pouch in rats
Compounda
Volume of exudates (mL)b
Percentage of inhibition
Control
3
6
7
9
10
18
20
22
26
27
29
2.15 0.06
1.07 0.08*
0.82 0.02*
1.02 0.08*
0.60 0.08*
0.75 0.02*
0.90 0.09*
0.85 0.06*
1.00 0.16*
0.82 0.01*
0.87 0.10*
1.45 0.09*
1.75 0.13*
0.57 0.06*
—
50*
62*
53*
72*
65*
58*
60*
53*
62*
60*
33*
19*
73*
30
Diclofenac Na
*
Significantly different compared to respective control values, P < 0.05.
Dose levels, po: test compounds (20 mg/kg b.wt.), diclofenac Na (10 mg/kg
a
b.wt.).
b
Values are expressed as means SE (number of animals N = 5 rats).

888
S. A. F. Rostom et al. / Bioorg. Med. Chem. 17 (2009) 882–895
Table 4
stere 10 (R = R1 = C6H5), although it displayed significant activity
at the acute inflammatory model, yet the activity at the sub-acute
inflammatory model was remarkably decreased. Meanwhile, the
analgesic activity of this analog was greatly enhanced to exceed
that of diclofenac Na, the reference drug in this study. Shifting to
the 2,3-dihydro-3,4-diarylthiazoles and 3-arylthiazolidin-4-ones
members, it was found that, remarkable anti-inflammatory activity
was linked with the thiazoline derivative 18 (R = C6H5, R1 = 4-Cl–
C6H4), where it showed recognizable activity in both the acute
and sub-acute inflammatory models, however, with diminished
analgesic effect. Introduction of another chlorine atom to the same
structure as in 20 (R = R1 = 4-Cl–C6H4), resulted in a marked reduc-
tion in the anti-inflammatory activity at both models, meanwhile,
the analgesic activity was significantly enhanced. On the contrary,
the thiazolidinone derivative 22 (R = C6H5) proved to be the least
active anti-inflammatory and/or analgesic member within this ser-
ies of compounds.
On the other hand, regarding the thiadiazolylantipyrine struc-
ture variants, their anti-inflammatory and analgesic activities ap-
pear to be closely related to the nature of the substituent at
position-5 of the 1,3,4-thiadiazole counterpart. As can be noticed
from Tables 1–4, the aminothiadiazolyl key intermediate 26
proved to be the most active member within this series with an ob-
servable anti-inflammatory potency at both the acute and sub-
acute models, in addition to a potential analgesic profile. Acetyla-
tion of the amino group as in 27, led to a significant improvement
in the anti-inflammatory activity at the acute model comparable
with that of diclofenac Na, nevertheless, its analgesic activity was
noticeably reduced. Finally, conversion of the amino group to the
carbamate or phenylthioureido functionalities as in 29 and 30,
respectively, resulted in an obvious loss of both the anti-inflamma-
tory and analgesic activities.
Analgesic activity of some selected compounds using the rat tail withdrawal
technique
Compounda
Reaction time (s)b
2 h
0
1 h
3 h
Control
3
2.80 0.12
2.95 0.06
2.90 0.09
3.50 0.32
(19)c
3.00 0.40
4.43 0.40*
(50)
2.97 0.04
4.93 0.31*
(67)
6
2.83 0.18
2.87 0.07
2.77 0.16
2.57 0.14
2.95 0.13
2.92 0.04
2.88 0.80
2.60 0.29
2.82 0.21
3.00 0.40
2.50 0.20
2.82 0.44
3.80 0.29*
(27)
4.12 0.42*
(46)
4.87 0.42*
(72)
7
3.80 0.12*
(32)
4.42 0.19*
(50)
4.92 0.12*
(67)
9
3.27 0.18
(18)
4.13 0.42*
(49)
4.00 0.40*
(44)
10
3.80 0.14*
(48)
4.37 0.23*
(70)
4.67 0.26*
(82)
18
3.60 0.22
(22)
4.23 0.25*
(43)
4.22 0.25*
(43)
20
3.90 0.19*
(34)
4.80 0.27*
(64)
4.60 0.53*
(58)
22
3.50 0.16
(22)
3.47 0.27
(20)
3.85 0.15
(34)
26
3.70 0.18*
(42)
4.45 0.21*
(71)
4.57 0.25*
(76)
27
3.67 0.34*
(30)
4.50 0.28*
(60)
4.37 0.25*
(55)
29
3.50 0.17
(17)
3.45 0.25
(15)
3.85 0.25
(28)
30
3.17 0.20
(27)
3.57 0.35
(43)
3.65 0.36
(46)
Diclofenac Na
3.55 0.35*
(26)
4.37 0.23*
(55)
5.07 0.32*
(79)
*
Significantly different compared to respective control values, P < 0.05.
Dose levels, po: test compounds (20 mg/kg b.wt.), diclophenac Na (10 mg/kg
a
b.wt.).
b
Values are expressed as means SE (number of animals N = 5 rats).
Values between parentheses (percentage analgesic activity).
c
Collectively, these results highlight the synergistic role of the
thiazole rather than the 1,3,4-thiadiazole substituents, together
with their substitution pattern, in manipulating the enhanced bio-
logical activity of such model of compounds.
and 76%) with the reference drug (79%). However, the rest of the
investigated compounds showed variable degree of analgesic
activities ranging between weak to moderate (28–67%).
2.2.3. In vitro antimicrobial activity
All of the newly synthesized target compounds were evaluated
for their in vitro antibacterial activity against Staphylococcus aureus
(ATCC 6538), Bacillus subtilis (NRRL B-14819) and Micrococcus lu-
teus (ATCC 21881) as examples of Gram positive bacteria and Esch-
erichia coli (ATCC 25922), Pseudomonas aeruginosa (ATCC 27853)
and Klebsiella pneumonia (clinical isolate) as examples of Gram
negative bacteria. They were also evaluated for their in vitro anti-
fungal potential against Candida albicans (ATCC 10231) and Asper-
gillus niger (recultured) fungal strains. Agar-diffusion method was
used for the determination of the preliminary antibacterial and
antifungal activity. Ampicillin trihydrate and clotrimazole were
used as reference drugs. The results were recorded for each tested
compound as the average diameter of inhibition zones (IZ) of bac-
terial or fungal growth around the disks in mm. The minimum
inhibitory concentration (MIC) measurement was determined for
compounds that showed significant growth inhibition zones
(P14 mm) using the twofold serial dilution method.51 The MIC
A deep insight in the structures of the tested compounds re-
vealed that they represent two different main hybrids, namely
the thiazolylantipyrine series (Schemes 1 and 2), and the thiadiaz-
olylantipyrines (Scheme 3). Within the first series, the type of the
thiazole ring substituent seems to modulate the anti-inflammatory
and analgesic activities. Compounds comprising the 3-substituted-
4-aminothiazole-2-thiones and their derivatives (Scheme 1)
showed anti-inflammatory and analgesic activities better than
those belonging to the 2,3-dihydro-3,4-diarylthiazoles and 3-aryl-
thiazolidin-4-ones series presented in Scheme 2 (Tables 1–4). In
this respect, it could be recognized that the key intermediate ami-
nothiazole 3 revealed appreciable anti-inflammatory potency at
both the acute and sub-acute inflammatory models together with
good analgesic profile. Conversion of the 2-thioxo function at the
thiazole moiety to 2-dicyanomethylene moiety (6; X = CN) resulted
in an obvious improvement in the anti-inflammatory activity to-
wards both models, in addition to a better analgesic activity. How-
ever, replacing the cyano moiety with a carbethoxy one (7;
X = COOEt), did not offer any advantage in both activities over
the parent compound 3. On the other hand, derivatization of the
4-amino group at the thiazole ring into the ureido and thioureido
functionalities as in 9 and 10, resulted in a great enhancement in
both the anti-inflammatory and analgesic profiles. In this view,
the ureido derivative 9 exhibited potential anti-inflammatory
activity at both the acute and sub-acute inflammatory models
and was found to be almost equipotent with diclofenac Na, how-
ever, with weak analgesic effect. Regarding the thioureido bioiso-
(lg/mL) values are recorded in Table 5.
The results depicted in Table 5 revealed that, 19 out of the
tested 23 compounds displayed variable inhibitory effects on the
growth of the tested Gram positive and Gram negative bacterial
strains, while they were totally inactive against the two tested
strains of fungi. In general, most of the tested compounds revealed
better activity against the Gram positive rather than the Gram neg-
ative bacteria. Among the Gram positive bacteria tested, two
strains namely; S. aureus and B. subtilis showed relative high sensi-
tivity towards the tested compounds. It could also be noticed that
compounds belonging to the thiazolylantipyrine series (Schemes 1

S. A. F. Rostom et al. / Bioorg. Med. Chem. 17 (2009) 882–895
889
Table 5
Minimal inhibitory concentrations (MIC,
lg/mL) of the active newly synthesized compounds
Compound
S. aureus ATCC 6538
B. subtilis NRRL B-14819
M. luteus ATCC 21881
E. coli ATCC 25922
P. aeruginosa ATCC 27853
K. pneumonia (clinical isolate)
a
3
4
6
7
8
—
100
100
100
12.5
100
100
12.5
12.5
—
100
—
100
100
100
25
—
100
—
25
100
—
50
12.5
—
—
—
—
—
—
—
—
50
—
—
—
—
50
—
—
—
—
50
—
—
—
12.5
—
—
50
25
100
—
100
—
9
—
—
—
10
11
12
13
18
19
20
22
23
27
29
30
31
Ab
50
25
—
—
—
—
—
50
25
—
50
50
—
—
—
—
—
50
50
—
50
50
—
—
—
—
—
50
100
—
—
50
50
100
—
100
100
6.25
100
12.5
—
—
—
—
100
—
—
12.5
—
—
—
6.25
—
—
—
12.5
—
—
—
12.5
—
12.5
a
(—): totally inactive (MIC P 200
A: ampicillin trihydrate (standard broad spectrum antibiotic).
l
g/mL).
b
and 2) exhibited better antibacterial potentials than members of
the thiadiazolylantipyrine one (Scheme 3).
with a broad spectrum of antibacterial activity against both Gram
positive and Gram negative bacteria.
Regarding the activity of the thiazolylantipyrine series
(Schemes 1 and 2) against Gram positive bacteria, the results
revealed that compounds 7, 10, 11, 22, and 23 exhibited broad
spectrum antibacterial profile against the tested three organ-
isms. In this view, compound 7 was equipotent to ampicillin
in inhibiting the growth of B. subtilis (MIC 12.5 lg/mL), while
its activity was 50% lower than that of ampicillin against
S. aureus and M. luteus species. Compound 10 showed equal
3. Conclusion
The objective of the present study was to synthesize and
investigate the anti-inflammatory, analgesic and antimicrobial
activities of new pyrazole-containing compounds with the hope
of discovering new structure leads serving as dual anti-inflam-
matory–antimicrobial agents. Our aim has been verified by
the synthesis of two different groups of structure hybrids com-
prising basically the antipyrine moiety attached to either
polysubstituted thiazole or 2,5-disubstituted-1,3,4-thiadiazole
counterparts through various linkages for synergistic purpose.
The obtained results clearly revealed that compounds derived
from the thiazolylantipyrine series exhibited better anti-inflam-
matory and analgesic activities than their thiadiazolyl structure
variants. Among the tested analogs, compounds 6, 9, 10, 18,
26, and 27 showed pronounced anti-inflammatory activity
comparable with diclofenac Na in the acute inflammatory mod-
el rather than the sub-acute inflammatory models, suggesting
that they might be effective in managing acute inflammation
rather than controlling chronic inflammatory conditions. Mean-
while, compounds 6, 7, 10, 18, 20, 22, 26, and 27 were found
to exhibit fast analgesic activity (within 2 h time interval) sim-
ilar to or even higher than that of diclofenac Na. Collectively,
compounds 6, 10, 26, and 27 proved to display distinctive
anti-inflammatory and analgesic profiles with a fast onset of
action. Additionally, all of the tested compounds revealed super
GI safety profile and are well tolerated by the experimental
animals with high safety margin (ALD50 > 3.0 g/kg). On the
other hand, some of the newly synthesized compounds were
able to display variable inhibitory effects on the growth of
the Gram positive and Gram negative bacteria while they were
totally deprived of any antifungal activity. Compounds belong-
ing to the thiazolylantipyrine series exhibited better antibacte-
rial potencies than members of the thiadiazolylantipyrine one.
Among these, compounds 7, 10, 11, and 23 are considered to
be the most active antimicrobial members identified in this
study with a broad spectrum of antibacterial activity against
activity as ampicillin against B. subtilis (MIC 12.5
gether with a moderate activity against S. aureus and M. luteus
(MIC 50 g/mL). Moreover, distinctive anti-Gram positive pro-
lg/mL) to-
l
file was displayed by compound 11 where it proved to be
equipotent as ampicillin against B. subtilis and M. luteus (MIC
12.5
eus (MIC 25
ited moderate anti-Gram positive against the tested strains
(MIC 50–100 g/mL), yet its chloro derivative 23 (R = 4-Cl–
C6H4) showed potential inhibitory activity against M. luteus
equivalent to that of ampicillin (MIC 12.5 g/mL), whereas it
showed 50% of the activity of ampicillin (MIC 25 g/mL)
lg/mL), together with a significant activity against S. aur-
lg/mL). Although the analog 22 (R = C6H5) exhib-
l
l
l
against B. subtilis. On the other hand, compounds of the same
series namely; 7, 10, 11, 22, and 23 exhibited weak to moder-
ate growth inhibitory activity against Gram negative bacteria
as revealed from their MIC values (25–100 lg/mL). Among
these, compounds 11 and 23 showed relatively good growth
inhibitory profiles against E. coli and P. aeruginosa (MIC 25
and 50
activity of ampicillin against the same organisms (MIC 6.25
and 12.5 g/mL, respectively).
lg/mL, respectively) which were about 25% of the
l
Concerning the antibacterial activity of the compounds
belonging to the thiadiazolylantipyrine series (Scheme 3), com-
pounds 27, 29, 30, and 31, they revealed weak growth inhibi-
tory activity against the tested Gram positive bacteria (MIC
100 lg/mL), whereas they were totally deprived of any activity
against the Gram negative bacteria strains employed in this
study.
Collectively, compounds 7, 10, 11, and 23 are considered to be
the most active antimicrobial members identified in this study

890
S. A. F. Rostom et al. / Bioorg. Med. Chem. 17 (2009) 882–895
both Gram positive and Gram negative bacteria. Finally, com-
pound 10 could be identified as the most biologically active
member within this study with an interesting dual anti-inflam-
matory analgesic and antibacterial profile. Consequently, such
type of compounds would represent a fruitful matrix for the
future development of a new class of dual non-acidic anti-
inflammatory–antimicrobial agents that deserves further inves-
tigation and derivatization.
4.1.2. General procedure for the synthesis of 4-amino-2-
(dicyanomethylene)-2,3-dihydro-N-(2,5-dihydro-2,3-dimethyl-
5-oxo-1-phenyl-1H-pyrazol-4-yl)-3-phenylthiazole-5-
carboxamide (6) and ethyl 2-(5-(2,5-dihydro-2,3-dimethyl-5-
oxo-1-phenyl-1H-pyrazol-4-ylcarbamoyl)-4-amino-3-
phenylthiazol-2(3H)-ylidene)-2-cyanoacetate (7)
To a suspension of 3 (0.44 g, 0.001 mol) in CH3CN (10 mL), di-
methyl sulfate (0.19 g, 0.14 mL, 0.0015 mol) was added. The reac-
tion mixture was heated under reflux for 3 h then allowed to
cool. Malononitrile or ethyl cyanoacetate (0.0015 mol) and trieth-
ylamine (0.2 mL) were then added while stirring. Stirring was con-
tinued on a boiling water bath for 1 h then the reaction mixture
was allowed to attain room temperature. The separated solid prod-
uct was filtered, washed with cold EtOH, dried, and crystallized
from the proper solvent.
4. Experimental
4.1. Chemistry
Melting points were determined in open glass capillaries on a
Gallenkamp melting point apparatus and were uncorrected. The
infrared (IR) spectra were recorded on Perkin-Elmer 1430 infra-
red spectrophotometer using the KBr plate technique. 1H NMR
spectra were determined on Jeol spectrometer (500 MHz) at
the Microanalytical unit, Faculty of Science, Alexandria Univer-
sity and on a Varian spectrometer (300 MHz), Faculty of Science,
Cairo University using tetramethylsilane (TMS) as the internal
standard and DMSO-d6 as the solvent (Chemical shifts in d,
ppm). Splitting patterns were designated as follows: s: singlet;
d: doublet; m: multiplet. Microanalyses were performed at the
Microanalytical Unit, Faculty of Science, Cairo University and at
the Central lab, Faculty of Pharmacy, Alexandria University,
Egypt and the found values were within 0.4% of the theoretical
values. Follow up of the reactions and checking the homogeneity
of the compounds were made by TLC on silica gel-protected
glass plates and the spots were detected by exposure to UV-
lamp at k 254.
4.1.2.1. 4-Amino-2-(dicyanomethylene)-2,3-dihydro-N-(2,5-
dihydro-2,3-dimethyl-5-oxo-1-phenyl-1H-pyrazol-4-yl)-3-phe-
nylthiazole-5-carboxamide (6). Yield: 70%, mp 238–240 °C
(DMF/EtOH; 3:1). IR (cmꢀ1): 3364, 3259, 3204 (NH); 2205
(C„N); 1650 (C@O); 1631 (C@N); 1250, 1061 (C–S–C). 1H NMR
(d ppm): 2.18 (s, 3H, CH3–C); 3.11 (s, 3H, CH3–N); 7.00 (s, 2H,
NH2, D2O exchangeable); 7.37 (d, J = 7.5 Hz, 2H, phenyl-C2,6–H);
7.40 (t, J = 7.65 Hz, 2H, phenyl-C3,5–H); 7.6–7.75 (m, 6H, Ar-H);
8.85 (s, 1H, NH, D2O exchangeable). Anal. Calcd for C24H19N7O2S
(469.52): C, 61.39; H, 4.08; N, 20.88. Found: C, 61.81; H, 4.29; N,
20.76.
4.1.2.2. Ethyl 2-(5-(2,5-dihydro-2,3-dimethyl-5-oxo-1-phenyl-
1H-pyrazol-4-ylcarbamoyl)-4-amino-3-phenylthiazol-2(3H)-yli-
dene)-2-cyanoacetate (7). Yield: 72%, mp 243–245 °C (EtOH). IR
(cmꢀ1): 3374, 3269, 3208 (NH); 2199 (C„N); 1750, 1655 (C@O);
1642 (C@N); 1285, 1061 (C–S–C); 1213, 1024 (C–O–C). 1H NMR
(d ppm): 1.97 (t, J = 6.9 Hz, 3H, CH2CH3); 2.97 (s, 3H, CH3); 3.90
(s, 3H, CH3–N); 4.93 (q, J = 6.9 Hz, 2H, CH2CH3); 7.03 (br s, 2H,
NH2, D2D exchangeable); 7.15 (t, J = 7.65 Hz, 1H, phenyl-C4–H);
7.31 (d, J = 7.65 Hz, 2H, phenyl-C2,6–H); 7.40 (t, J = 7.65 Hz, 2H,
phenyl-C3,5–H); 7.56–7.66 (m, 5H, phenyl-H); 9.62 (br s, 1H, NH
D2O exchangeable). Anal. Calcd for C26H24N6O4S (516.57): C,
60.45; H, 4.68; N, 16.27. Found: C, 59.95; H, 4.43; N, 15.91.
4.1.1. 4-Amino-2,3-dihydro-N-(2,5-dihydro-2,3-dimethyl-5-
oxo-1-phenyl-1H-pyrazol-4-yl)-3-substituted-2-thioxothiazole-
5-carboxamides (3 and 4)
A mixture of compound 2 (0.23 g, 0.001 mol), finely divided sul-
fur (0.03 g, 0.001 mol) and triethylamine (0.1 g, 0.14 mL,
0.001 mol) in dry DMF/abs. EtOH mixture (1:1) (2 mL) was stirred
at 60 °C for 30 min. The appropriate isothiocyanate (0.001 mol)
was gradually added and stirring at 60 °C was continued for 6–
8 h during which a yellow precipitate was partially separated
out. The reaction mixture was diluted with EtOH and the obtained
precipitate was filtered, washed with EtOH, dried, and crystallized
from dioxane/EtOH (1:1).
4.1.3. 3-(4-Chlorophenyl)-6-(2,5-dihydro-2,3-dimethyl-5-oxo-
1-phenyl-1H-pyrazol-4-yl)-5-methyl-2-thioxo-2,3-dihydro-6H-
thiazolo[4,5-d]pyrimidin-7-one (8)
A suspension of 4 (0.47 g, 0.001 mol) in acetic anhydride (5 mL)
was heated under reflux for 2 h, cooled, poured into ice-cold H2O
then set aside for an over-night. The obtained precipitate was fil-
tered, washed with H2O, dried, and crystallized from benzene.
Yield: 70%, chars before melting. IR (cmꢀ1): 1698 (C@O), 1630
(C@N); 1248, 1064 (C–S–C). 1H NMR (d ppm): 1.90 (s, 3H, thiazol-
opyrimidine-C5-CH3); 2.13 (s, 3H, CH3–C); 2.97 (s, 3H, CH3–N);
7.15 (d, J = 8.4 Hz, 2H, C6H4Cl–C2,6–H); 7.25 (t, J = 7.65 Hz, 1H, phe-
nyl-C4–H); 7.31 (d, J = 7.65 Hz, 2H, phenyl-C2,6–H); 7.40 (t,
J = 7.65 Hz, 2H, phenyl-C3,5–H); 7.78 (d, J = 8.4 Hz, 2H, C6H4Cl-
C3,5–H). Anal. Calcd for C23H18ClN5O2S2 (496.01): C, 55.69; H,
3.66; N, 14.12. Found: C, 55.27; H, 3.41; N, 13.91.
4.1.1.1. 4-Amino-2,3-dihydro-N-(2,5-dihydro-2,3-dimethyl-5-
oxo-1-phenyl-1H-pyrazol-4-yl)-3-phenyl-2-thioxothiazole-5-
carboxamide (3). Yield: 62%, mp: 201–203 °C [reported 236–
238 °C].38 IR (cmꢀ1): 3326, 3243 (NH); 1652 (C@O); 1639, 1630
(C@N); 1248, 1045 (C–S–C). 1H NMR (d ppm): 2.19 (s, 3H, CH3–
C); 3.12 (s, 3H, CH3–N); 6.93 (s, 2H, NH2, D2O exchangeable);
7.2–7.8 (m, 10H, Ar-H); 8.66 (s, 1H, NH, D2O exchangeable). Anal.
Calcd for C21H19N5O2S2 (437.54): C, 57.65; H, 4.38; N, 16.01. Found:
C, 58.13; H, 4.65; N, 16.38.
4.1.1.2. 4-Amino-2,3-dihydro-N-(2,5-dihydro-2,3-dimethyl-5-
oxo-1-phenyl-1H-pyrazol-4-yl)-3-(4-chlorophenyl)-2-thioxo-
thiazole-5-carboxamide (4). Yield: 53%, mp 186–188 °C. IR
(cmꢀ1): 3374, 3300 (NH); 1650 (C@O); 1630, 1608 (C@N); 1245,
1073 (C–S–C). 1H NMR (d ppm): 2.12 (s, 3H, CH3–C); 3.06 (s, 3H,
CH3–N); 6.97 (s, 2H, NH2, D2O exchangeable); 7.32, 7.63 (two d,
J = 8.4 Hz, each 2H, C6H4Cl–H) 7.39 (d, J = 7.65 Hz, 2H, phenyl-
C2,6–H); 7.48 (m, 3H, phenyl-C3,4,5–H); 8.66 (s, 1H, NH, D2O
exchangeable). Anal. Calcd for C21H18ClN5O2S2 (471.98): C, 53.44;
H, 3.84; N, 14.84. Found: C, 53.78; H, 4.15; N, 15.13.
4.1.4. N-(2,5-Dihydro-2,3-dimethyl-5-oxo-1-phenyl-1H-
pyrazol-4-yl)-3-(4-chlorophenyl)-4-(3-phenylureido)-2-thioxo-
2,3-dihydrothiazole-5-carboxamide (9)
To a suspension of 4 (0.47g, 0.001 mol) in dry dioxane (5 mL)
containing anhydrous potassium carbonate (0.14 g, 0.001 mol),
phenyl isocyanate (0.12 g, 0.11 mL, 0.001 mol) was added. The
reaction mixture was heated under reflux for 12 h, cooled then
poured into ice-cold H2O. The obtained precipitate was filtered,
washed with H2O, dried, and crystallized from dioxane/EtOH

S. A. F. Rostom et al. / Bioorg. Med. Chem. 17 (2009) 882–895
891
(1:1); yield 71:%, mp above 300. IR (cmꢀ1): 3373 (NH); 1696, 1668
(C@O), 1630 (C@N); 1255, 1085 (C–S–C). 1H NMR (d ppm): 2.17
(s, 3H, CH3–C); 3.25 (s, 3H, CH3–N); 7.19, 7.29 (two d, each 2H,
J = 8.1 Hz, C6H4Cl); 7.35–7.40 (m, 4H, Ar-H); 7.48–7.55 (m, 4H,
Ar-H); 7.63 (d, J = 7.65 Hz, 2H, phenyl-C2,6–H). Anal. Calcd for
C28H23ClN6O3S2 (591.11): C, 56.89; H, 3.92; N, 14.22. Found: C,
56.85; H, 3.86; N, 13.87.
D2O exchangeable). Anal. Calcd for C28H22Cl2N6O2S3 (641.62): C,
52.41; H, 3.46; N, 13.10. Found: C, 52.20; H, 3.12; N, 13.33.
4.1.6. 2-Cyano-N-(2,5-dihydro-2,3-dimethyl-5-oxo-1-phenyl-
1H-pyrazol-4-yl)-3-mercapto-3-(arylamino)acrylamides (16
and 17)
To an ice-cooled suspension of finely powdered potassium
hydroxide (0.11 g, 0.002 mol) in dry DMF (5 mL), compound 2
(0.38 g, 0.001 mol) and then the appropriate isothiocyanate
(0.001 mol) were added in portions with stirring. After complete
addition, stirring was continued at room temperature for an
over-night. The reaction mixture was then poured onto ice/cold
H2O and acidified with 0.1 N HCl to pH 3–4. The obtained precipi-
tate was filtered, washed with H2O, dried, and crystallized from the
proper solvent.
4.1.5. N-(2,5-Dihydro-2,3-dimethyl-5-oxo-1-phenyl-1H-
pyrazol-4-yl)-3-aryl-4-(3-arylthioureido)-2-thioxo-2,3-
dihydrothiazole-5-carboxamides (10–13)
To a suspension of the appropriate aminothiazole derivative 3
or 4 (0.001 mol) in dry dioxane (5 mL) containing anhydrous potas-
sium carbonate (0.14 g, 0.001 mol), the appropriate isothiocyanate
(0.001 mol) was added. The reaction mixture was heated under re-
flux for 12 h, cooled then poured into ice-cold H2O. The separated
precipitate was filtered, washed with H2O, dried, and crystallized
from DMF/EtOH (3:1).
4.1.6.1. 2-Cyano-N-(2,5-dihydro-2,3-dimethyl-5-oxo-1-phenyl-
1H-pyrazol-4-yl)-3-mercapto-3-(phenylamino)acrylamide
(16). Yield: 70%, mp 188–190 °C (EtOH). IR (cmꢀ1): 3340, 3114
(NH); 2630 (SH); 2197 (C„N); 1665 (C@O, C@N); 1563, 1230,
1042, 999 (N–C@S). 1H NMR (d ppm): 2.93 (s, 3H, CH3–C); 3.84 (s,
3H, CH3–N); 7.38–7.47 (m, 2H, phenyl-C2,6–H); 7.8–8.6 (m, 8H, Ar-
H); 8.51, 10.25 (2s, each 1H, 2NH, D2O exchangeable); 11.82 (s,
1H, SH, D2O exchangeable). Anal. Calcd for C21H19N5O2S (405.47):
C, 62.20; H, 4.72; N, 17.27. Found: C, 61.91; H, 4.29; N, 17.02.
4.1.5.1. N-(2,5-Dihydro-2,3-dimethyl-5-oxo-1-phenyl-1H-pyra-
zol-4-yl)-3-phenyl-4-(3-phenylthioureido)-2-thioxo-2,3-dihy-
drothiazole-5-carboxamide (10). Yield: 53%, mp above 300 °C.
IR (cmꢀ1): 3317–3128 (NH); 2215 (C„N); 1665 (C@O); 1634
(C@N); 1267, 1059 (C–S–C). 1H NMR (d ppm): 2.21 (s, 3H, CH3–
C); 3.31 (s, 3H, CH3–N); 7.01 (t, J = 8.1 Hz, 1H, phenyl-C4–H); 7.13
(t, J = 8.1 Hz, 2H, phenyl-C3,5–H); 7.30 (d, J = 8.1 Hz, 2H, phenyl-
C2,6–H); 7.40–7.60 (m, 11H, 10Ar-H and NH); 9.08 (s, 1H, NH,
D2O exchangeable). Anal. Calcd for C28H24N6O2S3 (572.73): C,
58.72; H, 4.22; N, 14.67. Found: C, 59.20; H, 4.44; N, 14.38.
4.1.6.2. 2-Cyano-N-(2,5-dihydro-2,3-dimethyl-5-oxo-1-phenyl-
1H-pyrazol-4-yl)-3-mercapto-3-(4-chlorophenylamino)acryl-
amide (17). Yield: 74%, mp 193–195 °C (dioxane/EtOH; 1:1). IR
(cmꢀ1): 3334 (NH); 2693 (SH); 2184 (C„N); 1660 (C@O); 1642,
1625 (C@N); 1578, 1207, 1087, 972 (N–C@S). 1H NMR (d ppm):
2.16 (s, 3H, CH3–C); 3.0 (s, 3H, CH3–N); 7.2–7.3 (m, 2H, C6H4Cl–
C2,6–H); 7.33–7.37 (m, 3H, phenyl-C2,4,6–H); 7.49 (t, J = 7.8 Hz,
2H, phenyl-C3,5–H); 7.7–7.8 (m, 2H, chlorophenyl-C3,5–H). Anal.
Calcd for C21H18ClN5O2S (439.92): C, 57.33; H, 4.12; N, 15.92.
Found: C, 57.63; H, 4.43; N, 15.51.
4.1.5.2. N-(2,5-Dihydro-2,3-dimethyl-5-oxo-1-phenyl-1H-pyra-
zol-4-yl)-3-phenyl-4-(3-(4-chlorophenyl)thioureido)-2-thioxo-
2,3-dihydrothiazole-5-carboxamide (11). Yield: 58%, mp above
300 °C. IR (cmꢀ1): 3370 (NH); 2215 (C„N); 1671 (C@O); 1615
(C@N); 1290, 1088 (C–S–C) 830 (C–Cl). 1H NMR (d ppm): 2.16 (s,
3H, CH3–C); 3.27 (s, 3H, CH3–N); 7.14 (d, J = 8.4 Hz, 2H, C6H4Cl–
C2,6–H); 7.31 (d, J = 8.4 Hz, 2H, phenyl-C2,6–H); 7.34–7.58 (m,
11H, 10Ar-H and NH); 9.17 (s, 1H, NH, D2O exchangeable). Anal.
Calcd for C28H23ClN6O2S3 (607.17): C, 55.39; H, 3.82; N, 13.84.
Found: C, 55.01; H, 4.19; N, 13.39.
4.1.7. 2-Cyano-N-(2,5-dihydro-2,3-dimethyl-5-oxo-1-phenyl-
1H-pyrazol-4-yl)-2-(3,4-diarylthiazol-2(3H)-ylidene)acetamides
(18–21)
To a suspension of the appropriate acrylamide 16 or 17
(0.001 mol) in abs. EtOH (10 mL), the appropriate phenacyl bro-
mide (0.001 mol) was added. The reaction mixture was heated un-
der reflux for 2–3 h then allowed to cool. The obtained precipitate
was filtered, washed with EtOH, dried, and crystallized from DMF/
EtOH (3:1).
4.1.5.3. N-(2,5-Dihydro-2,3-dimethyl-5-oxo-1-phenyl-1H-pyra-
zol-4-yl)-3-(4-chlorophenyl)-4-(3-phenylthioureido)-2-thioxo-
2,3-dihydrothiazole-5-carboxamide (12). Yield: 61%, mp above
300 °C. IR (cmꢀ1): 3250 (NH); 2250 (C„N); 1670 (C@O); 1618
(C@N); 1292, 1055 (C–S–C) 799 (C–Cl). 1H NMR (d ppm): 2.16 (s,
3H, CH3–C); 3.27 (s, 3H, CH3–N); 7.04 (t, J = 8.1 Hz, 1H, phenyl-
C4–H); 7.14 (t, J = 7.65 Hz, 2H, phenyl-C3,5–H); 7.27 (d, J = 8.1 Hz,
2H, phenyl-C2,6–H); 7.36 (t, J = 8.1 Hz, 1H, phenyl-C4–H); 7.39 (t,
J = 8.1 Hz, 2H, phenyl-C3,5–H); 7.48–7.54 (m, 4H, C6H4Cl–C2,6–H &
phenyl C2,6–H); 7.60 (d, J = 8.4 Hz, 2H, C6H4Cl–C3,5–H); 9.15 (s,
1H, NH, D2O exchangeable). Anal. Calcd for C28H23ClN6O2S3
(607.17): C, 55.39; H, 3.82; N, 13.84. Found: C, 55.10; H, 4.13; N,
14.10.
4.1.7.1. 2-[4-(4-Chlorophenyl)-3-phenyl-3H-thiazol-2-ylidene]-
2-cyano-N-(2,5-dihydro-2,3-dimethyl-5-oxo-1-phenyl-1H-pyra-
zol-4-yl)acetamide (18). Yield: 70%, mp 278–280 °C. IR (cmꢀ1):
3125 (NH); 2215 (C„N); 1657 (C@O); 1625 (C@N); 1226, 1082 (C–
S–C) 857 (C–Cl). 1H NMR (d ppm): 2.13 (s, 3H, CH3–C); 3.04 (s, 3H,
CH3–N); 7.19–7.50 (m, 15H, Ar-H and thiazoline-C5–H); 7.66 (s, 1H,
NH, D2O exchangeable). Anal. Calcd for C29H22ClN5O2S (540.04): C,
64.50; H, 4.11; N, 12.97. Found: C, 64.17; H, 3.96; N, 12.88.
4.1.5.4. N-(2,5-Dihydro-2,3-dimethyl-5-oxo-1-phenyl-1H-pyra-
zol-4-yl)-3-(4-chlorophenyl)-4-(3-(4-chlorophenyl)thioureido)-
2-thioxo-2,3-dihydrothiazole-5-carboxamide (13). Yield: 63%,
mp above 300 °C. IR (cmꢀ1): 3300 (NH); 2247 (C„N); 1690
(C@O); 1609 (C@N); 1298, 1026 (C–S–C) 883 (C–Cl). 1H NMR
(d ppm): 2.16 (s, 3H, CH3–C); 3.27 (s, 3H, CH3–N); 7.19 (d,
J = 8.4 Hz, 2H, C6H4Cl–C2,6–H); 7.29 (d, J = 9.2 Hz, 2H, C6H4Cl–
C2,6–H); 7.36 (t, J = 7.65 Hz, 1H, phenyl-C4–H); 7.38 (t, J = 7.65 Hz,
2H, phenyl-C2,6–H); 7.48–7.54 (m, 4H, phenyl-C3,5–H and C6H4Cl–
C3,5–H); 7.63 (d, J = 8.4 Hz, 2H, C6H4Cl–C3,5–H); 9.22 (s, 1H, NH,
4.1.7.2. 2-[4-(4-Bromophenyl)-3-phenyl-3H-thiazol-2-ylidene]-
2-cyano-N-(2,5-dihydro-2,3-dimethyl-5-oxo-1H-pyrazol-4-yl)
(19). Yield: 72%, mp 278–280 °C. IR (cmꢀ1): 3357 (NH); 2192
(C„N); 1674 (C@O); 1637 (C@N); 1272, 1068 (C–S–C); 694
(C–Br). 1H NMR (d ppm): 2.13 (s, 3H, CH3–C); 3.04 (s, 3H, CH3–N);
7.10 (d, J = 8.4 Hz, 2H, phenyl-C2,6–H); 7.17 (s, 1H, thiazoline-
C5–H); 7.25–7.49 (m, 12H, Ar-H) 7.70 (s, 1H, NH, D2O exchange-
able). Anal. Calcd for C29H22BrN5O2S (584.49): C, 59.59; H, 3.79;
N, 11.98. Found: C, 59.59; H, 3.95; N, 12.06.

892
S. A. F. Rostom et al. / Bioorg. Med. Chem. 17 (2009) 882–895
4.1.7.3. 2-[3,4-Bis-(4-chlorophenyl)-3H-thiazol-2-ylidene]-2-
cyano-N-(2,5-dihydro-2,3-dimethyl-5-oxo-1-phenyl-1H-pyra-
zol-4-yl)acetamide (20). Yield: 75%, mp 272–74 °C. IR (cmꢀ1):
3307 (NH); 2200 (C„N); 1655 (C@O); 1612 (C@N); 1204, 1089
(C–S–C) 857 (C–Cl). 1H NMR (d ppm): 2.13 (s, 3H, CH3–C); 3.04
(s, 3H, CH3–N); 7.10 (d, J = 8.4 Hz, 2H, C6H4Cl–C2,6–H); 7.20 (s,
1H, thiazoline-C5–H); 7.27 (t, J = 7.65 Hz, phenyl-C4–H); 7.30–
7.49 (m, 10H, Ar-H) 7.70 (s, 1H, NH, D2O exchangeable). Anal. Calcd
for C29H21Cl2N5O2S (574.48): C, 60.63; H, 3.68; N, 12.19. Found: C,
60.44; H, 3.71; N, 12.11.
25 (0.54 g, 0.004 mol) in dry acetone (20 mL) was heated under re-
flux for 6 h then allowed to cool. The reaction mixture was filtered
and the obtained precipitate was washed with H2O, dried and puri-
fied by dissolving in glacial acetic acid and re-precipitation by dil.
NH4OH. Yield: 66%, mp 229–31 °C. IR (cmꢀ1): 3407, 3267, 3167
(NH); 1655 (C@O); 1645, 1608 (C@N); 1217, 1062 (C–S–C). 1H
NMR (d ppm): 2.90 (s, 3H, CH3–C); 3.84 (s, 3H, CH3–N); 4.75 (s,
2H, CH2); 8.08 (br s, 2H, NH2, D2O exchangeable); 8.12–8.16 (m,
3H, phenyl-C2,4,6–H); 7.95 (t, J = 8.1 Hz, 2H, phenyl-C3,5–H); 10.17
(s, 1H, NH, D2O exchangeable). Anal. Calcd for C15H16N6O2S2
(376.46): C, 47.86; H, 4.28; N, 22.32. Found: C, 47.81; H, 4.12; N,
22.32.
4.1.7.4. 2-[4-(4-Bromophenyl)-3-(4-chlorophenyl)-3H-thiazol-
2-ylidene]-2-cyano-N-(2,5-dihydro-2,3-dimethyl-5-oxo-1-phe-
nyl-1H-pyrazol-4-yl)-acetamide (21). Yield: 80%, mp 290–2 °C.
IR (cmꢀ1). 3373 (NH); 2176 (C„N); 1668 (C@O); 1624 (C@N);
1269, 1092 (C–S–C); 877 (C–Cl); 681 (C–Br). 1H NMR (d ppm):
2.08 (s, 3H, CH3–C); 3.01 (s, 3H, CH3–N); 7.12 (d, J = 7.65 Hz,
2H, C6H4Cl–C2,6–H); 7.17 (s, 1H, thiazoline-C5–H); 7.28 (t,
4.1.10. 2-(5-Acetamido-1,3,4-thiadiazol-2-ylthio)-N-(2,5-
dihydro-2,3-dimethyl-5-oxo-1-phen-yl-1H-pyrazol-4-yl)-
acetamide (27)
A suspension of 26 (0.38 g, 0.001 mol) in acetic anhydride
(5 mL) was heated under reflux for 10 min then allowed to cool.
The obtained solid was filtered, washed with EtOH, dried, and
crystallized from DMF. Yield: 72%, mp 244–6 °C. IR (cmꢀ1):
3245, 3203, 3147 (NH); 1674, 1652 (C@O), 1627 (C@N); 1257,
1050 (C–S–C). 1H NMR (d ppm): 2.10 (s, 3H, CH3–C); 2.18 (s,
3H, CH3CO); 3.04 (s, 3H, CH3–N); 4.14 (s, 2H, CH2); 7.29–7.36
(m, 3H, phenyl-C2,4,6–H); 7.85 (t, J = 7.8 Hz, 2H, phenyl-C3,5–H);
9.44, 12.57 (two s, each 1H, 2NH, D2O exchangeable). Anal. Calcd
for C17H18N6O3S2 (418.49): C, 48.79; H, 4.34; N, 20.08. Found: C,
49.17; H, 4.26; N, 20.24.
J = 7.65 Hz, phenyl-C4–H); 7.31 (d, J = 7.65 Hz, 2H, phenyl-C2,6
–
H); 7.42–7.49 (m, 6H, C6H4Cl–C3,5–1H–H, C6H4Br–C2,6–H and
phenyl-C3,5–H); 7.52 (d, J = 7.65 Hz, 2H, C6H4Br–C3,5–H); 7.77 (s,
1H, NH, D2O exchangeable). Anal. Calcd for C29H21BrClN5O2S
(618.93): C, 56.28; H, 3.42; N, 11.32. Found: C, 56.47; H, 3.50;
N, 11.32.
4.1.8. 2-Cyano-N-(2,5-dihydro-2,3-dimethyl-5-oxo-1-phenyl-
1H-pyrazol-4-yl)-2-(3-aryl-4-oxothiazolidin-2-ylidene)-
acetamides (22 and 23)
To an ice-cooled suspension of finely powdered potassium
hydroxide (0.11 g, 0.002 mol) in dry DMF (5 mL), compound 2
(0.38 g, 0.001 mol) and then the appropriate isothiocyanate
(0.001 mol) were added in portions with stirring. After complete
addition, stirring was continued at room temperature for an
over-night. Chloroacetyl chloride (0.11 g, 0.08 mL, 0.001 mol) was
then added to the ice-cooled reaction mixture and stirring was
continued at room temperature for an over-night. The reaction
mixture was then poured onto ice cold H2O and the separated solid
product was filtered, washed with H2O, dried, and crystallized
from the proper solvent.
4.1.11. N-(5-((2,5-Dihydro-2,3-dimethyl-5-oxo-1-phenyl-1H-
pyrazol-4-ylcarbamoyl)methylthio)-1,3,4-thiadiazol-2-yl)-
benzamide (28)
To a stirred ice-cooled suspension of 26 (0.38 g, 0.001 mol) in
dry pyridine (5 mL), benzoyl chloride (0.14 g, 0.12 mL, 0.001 mol)
was added drop-wise. The reaction mixture was heated under re-
flux for 6h then allowed to cool. The obtained precipitate was fil-
tered, washed with EtOH, dried, and crystallized from dioxane/
EtOH (3:1). Yield: 52%, mp 258–260 °C. IR (cmꢀ1): 3240, 3204
(NH); 1651 (C@O); 1626 (C@N); 1250, 1061 (C–S–C). 1H NMR
(d ppm): 2.08 (s, 3H, CH3–C); 3.01 (s, 3H, CH3–N); 4.16 (s, 2H,
CH2); 7.25–7.33 (m, 3H, phenyl-C2,4,6–H); 7.46 (t, J = 7.65 Hz, 2H,
phenyl-C3,5–H); 7.53 (t, J = 7.65 Hz, 2H, benzoyl-C3,5–H); 7.64 (t,
J = 7.65 Hz, 1H, benzoyl-C4–H) 8.07 (d, J = 7.65 Hz, 2H, benzoyl-
C2,6–H) 9.50, 13.12 (two s, each 1H, 2NH, D2O exchangeable). Anal.
Calcd for C22H20N6O3S2 (480.56): C, 54.98; H, 4.19; N, 17.49. Found:
C, 55.21; H, 4.29; N, 17.17.
4.1.8.1. 2-Cyano-N-(2,5-dihydro-2,3-dimethyl-5-oxo-1-phenyl-
1H-pyrazol-4-yl)-2-(4-oxo-3-phenylthiazolidin-2-ylidene)acet-
amide (22). Yield: 82%, chars before melting (EtOH). IR (cmꢀ1):
3310, 3226 (NH); 1660 (C@O); 1642, 1625 (C@N); 1236, 1046
(C–S–C). 1H NMR (d ppm): 2.11 (s, 3H, CH3–C); 3.05 (s, 3H, CH3–
N); 3.97 (s, 2H, thiazolidinone-C5-H2); 7.32–7.54 (m, 10H, Ar-H);
8.39 (s, 1H, NH, D2O exchangeable). Anal. Calcd for C23H19N5O3S
(445.49): C, 62.01; H, 4.30; N, 15.72. Found: C, 62.43; H, 3.97; N,
15.84.
4.1.12. Ethyl 5-((2,5-dihydro-2,3-dimethyl-5-oxo-1-phenyl-1H-
pyrazol-4-yl-carbamoyl)methylthio)-1,3,4-thiadiazol-2-
ylcarbamate (29)
4.1.8.2. 2-Cyano-N-(2,5-dihydro-2,3-dimethyl-5-oxo-1-phenyl-
1H-pyrazol-4-yl)-2-(3-(4-chlorophenyl)-4-oxothiazolidin-2-yli-
dene)acetamide (23). Yield: 85%, chars before melting (ethyl
acetate/pet. ether; 3:1). IR (cmꢀ1): 3310, 3226 (NH); 1660 (C@O);
1642, 1625 (C@N); 1236, 1046 (C–S–C). 1H NMR (d ppm): 2.04 (s,
3H, CH3–C); 3.00 (s, 3H, CH3–N); 3.91 (s, 2H, thiazolidinone-
C5–H2); 7.26–7.33 (m, 4H, phenyl-C2,6–H and C6H4Cl–C2,6–H);
7.43–7.50 (m, 3H, phenyl-C3,4,5–H); 7.55 (d, J = 8.4 Hz, 2H,
C6H4Cl-C3,5–H), 8.47 (s, 1H, NH, D2O exchangeable). Anal. Calcd
for C23H18ClN5O3S (479.94): C, 57.56; H, 3.78; N, 14.59. Found: C,
57.63; H, 4.03; N, 14.29.
To a stirred ice-cooled suspension of 26 (0.38 g, 0.001 mol) in
dry pyridine (5 mL), ethyl chloroformate (0.16 g, 0.14 mL,
0.0015 mol) was added dropwise. Stirring was maintained at
room temperature for an over-night then poured into ice-cold
H2O. The obtained precipitate was filtered, washed with H2O,
dried and crystallized from dioxane/EtOH (3:1). Yield: 66%, mp
234–6 °C. IR (cmꢀ1): 3254, 3209 (NH); 1718, 1678 (C@O);
1639, 1618 (C@N); 1247, 1061 (C–S–C); 1207, 1038 (C–O–C).
1H NMR (d ppm): 2.05 (t, J = 6.9 Hz, 3H, CH2CH3); 2.90 (s, 3H,
CH3–C); 3.84 (s, 3H, CH3–N); 4.15 (s, 2H, SCH2); 4.91 (q,
J = 6.9 Hz, 2H, CH2CH3); 8.12–8.16 (m, 3H, phenyl-C2,4,6–H);
8.29 (t, J = 8.1 Hz, 2H, phenyl-C3,5–H); 10.23, 13.01 (two s, each
1H, 2NH, D2O exchangeable). Anal. Calcd for C18H20N6O4S2
(448.52): C, 48.20; H, 4.49; N, 18.74. Found: C, 48.41; H, 4.16;
N, 18.68.
4.1.9. 2-(5-Amino-1,3,4-thiadiazol-2-ylthio)-N-(2,5-dihydro-
2,3-dimethyl-5-oxo-1-phenyl-1H-pyrazol-4-yl)acetamide (26)
A mixture of 2445 (1.08 g, 0.004 mol), anhydrous potassium car-
bonate (0.56 g, 0.004 mol) and 5-amino-1,3,4-thiadiazole-4-thiol

S. A. F. Rostom et al. / Bioorg. Med. Chem. 17 (2009) 882–895
893
4.1.13. 1-(5-((2,5-Dihydro-2,3-dimethyl-5-oxo-1-phenyl-1H-
4.2. Biological evaluation
pyrazol-4-yl-carbamoyl)methylthio)-1,3,4-thiadiazol-2-yl)-3-
arylthioureas (30–32)
4.2.1. Anti-inflammatory (AI) activity
To a suspension of 26 (0.38 g, 0.001 mol) in dry dioxane
(5 mL) containing anhydrous potassium carbonate (0.14 g,
0.001 mol), the appropriate isothiocyanate (0.001 mol) was
added. The reaction mixture was heated under reflux for 12 h,
cooled then poured into ice-cold H2O. The solid product formed
was filtered, washed with H2O, dried, and crystallized from
DMF/EtOH (3:1).
4.2.1.1. Formalin-induced paw edema bioassay (acute inflam-
matory model). Male albino rats weighing 120–150 g were
used throughout the assay. They were kept in the animal house un-
der standard condition of light and temperature with free access to
food and water. The animals were randomly divided into groups
each of five rats. One group of five rats was kept as a control and
another group received the standard drug diclofenac Na (at a dose
of 10 mg/kg body weight po). A solution of formalin (2%, 0.1 mL)
was injected into the subplanter region of the left hind paw under
light ether anesthesia 1h after oral administration (po) of the test
compound (at a dose level of 20 mg/kg body weight). The paw vol-
ume (ml) was measured by means of water plethysmometer and
re-measured again 1, 2, 3, and 4 h after administration of formalin.
The edema was expressed as an increase in the volume of paw, and
the percentage of edema inhibition for each rat and each group was
obtained as follows:
4.1.13.1. 1-(5-((2,5-Dihydro-2,3-dimethyl-5-oxo-1-phenyl-1H-
pyrazol-4-yl-carbamoyl)methyl-thio)-1,3,4-thiadiazol-2-yl)-3-
phenylthiourea (30). Yield: 40%, mp 230–2 °C. IR (cmꢀ1): 3325,
3225 (NH); 1661 (C@O); 1625 (C@N); 1258, 1091 (C–S–C). Anal.
Calcd for C22H21N7O2S3 (511.64): C, 51.64; H, 4.14; N, 19.16. Found:
C, 51.35; H, 4.19; N, 18.68.
4.1.13.2. 1-(5-((2,5-Dihydro-2,3-dimethyl-5-oxo-1-phenyl-1H-
pyrazol-4-yl-carbamoyl)methyl-thio)-1,3,4-thiadiazol-2-yl)-3-
(4-chlorophenyl)thiourea (31). Yield: 43%, mp 235–6 °C. IR
(cmꢀ1): 3320, 3237, 3193 (NH); 1671 (C@O); 1630 (C@N); 1234,
1095 (C–S–C) 819 (C–Cl). 1H NMR (d ppm): 2.90 (s, 3H, CH3–C);
3.84 (s, 3H, CH3–N); 4.92 (s, 2H, CH2); 8.08–8.19 (m, 4H, Ar-H);
8.27–8.32 (m, 3H, Ar-H); 8.59 (d, J = 9 Hz, 3H, p-C6H4Cl–C3,5–H);
10.16, 10.26, 11.34 (3s, each 1H, 3NH, D2O exchangable). Anal.
Calcd for C22H20ClN7O2S3 (546.09): C, 48.39; H, 3.69; N, 17.95.
Found: C, 48.43; H, 4.01; N, 17.90.
% Inhibition ¼ ðVt ꢀ VoÞ control ꢀ ðVt
ꢀ VoÞtested compound=ðVt ꢀ VoÞcontrol ꢁ 100
where Vt = volume of edema at specific time interval and
Vo = volume of edema at zero time interval.
4.2.1.2. Formalin-induced paw edema bioassay (sub-acute
inflammatory model). Rats in the first experiment were given
the same test compounds at a dose level of 20 mg/kg body
weight daily for 7 consecutive days. A solution of formalin (2%,
0.1 mL) was injected into the subplanter region of the left hind
paw under light ether anesthesia 1 h after oral administration
(po) of the test compound. A second injection of formalin (2%,
0.1 mL) was given on the third day. The changes in the volume
of paw were measured plethymographically at the first and
eighth days.
4.1.13.3. 1-(5-((2,5-Dihydro-2,3-dimethyl-5-oxo-1-phenyl-1H-
pyrazol-4-yl-carbamoyl)methyl-thio)-1,3,4-thiadiazol-2-yl)-3-
(4-tolyl)thiourea (32). Yield: 39%, mp 233–4 °C. IR (cmꢀ1):
3320–3109 (NH); 1690 (C@O); 1620 (C@N); 1260, 1090 (C–S–C).
1H NMR (d ppm): 2.07 (s, 3H, CH3–C); 2.20 (s, 3H, tolyl-CH3);
3.01 (s, 3H, CH3-N); 4.08 (s, 2H, CH2); 7.10 (d, J = 7.65 Hz, 2H, p-
C6H4CH3–C2,6–H); 7.27–7.33 (m, 3H, phenyl-C2,4,6–H); 7.43–7.50
(m, 4H, phenyl-C3,5–H and p-C6H4CH3–C3,5–H); 9.46, 10.35, 12.80
(3s, each 1H, 3NH, D2O exchangable). Anal. Calcd for C23H23N7O2S3
(525.67): C, 52.55; H, 4.41; N, 18.65. Found: C, 52.26; H, 4.23; N,
18.35.
4.2.1.3. Turpentine oil-induced granuloma pouch bioassay
(sub-acute inflammatory model). Male albino rats weighing
120–150 g were used throughout this assay. They were kept in
the animal house under standard condition of light and tempera-
ture with free access to food and water. One group of five rats
was kept as a control and another group received the standard
drug diclofenac Na (at a dose of 10 mg/kg body weight po). Subcu-
taneous dorsal granuloma pouch was made in ether-anesthetized
rats by injecting 2 mL of air, followed by injection of 0.5 ml of tur-
pentine oil into it. All of the test compounds were administered or-
ally (at a dose level of 20 mg/kg body weight) one hour prior to
turpentine oil injection and continued for seven consecutive days.
On the eighth day, the paw was opened under anesthesia and the
exudates were taken out with a syringe. The volume (mL) of the
exudates was measured and the percentage inhibition of inflam-
mation relative to the reference drug (diclofenac Na) was deter-
mined as follows:
4.1.14. Ethyl 2-(5-arylimidazo[2,1-b][1,3,4]thiadiazol-2-
ylsulfanyl)acetates (35 and 36)
A mixture of 26 (0.38 g, 0.001 mol) and the appropriate phena-
cyl bromide (0.0011 mol) in absolute EtOH (10 mL) was heated un-
der reflux for 24 h then allowed to cool. The separated precipitate
was filtered, washed with EtOH, dried, and crystallized from EtOH.
4.1.14.1. Ethyl 2-(5-(4-bromophenyl)imidazo [2,1-b][1,3,4]thia-
diazol-2-ylsulfanyl)acetate (35). Yield: 45%, mp 154–6 °C. IR
(cmꢀ1): 1730 (C@O), 1650 (C@N); 1250, 1069 (C–S–C); 1032 (C-
O-C); 732 (C-Br). 1H NMR (d ppm): 1.17 (t, J = 6.85 Hz, 3H, CH2CH3);
4.13 (q, J = 6.85 Hz, 2H, CH2CH3); 4.24 (s, 2H, CH2); 7.55, 7.76 (two
d, J = 8.4 Hz, each 2H, C6H4–Br); 8.67 (s, 1H, imidazothiadiazole-
C6–H). Anal. Calcd for C14H12BrN3O2S2 (398.30): C, 42.22; H, 3.04;
N, 10.55. Found: C, 42.53; H, 3.25; N, 10.56.
% Inhibition ¼ V control ꢀ V treated=V control ꢁ 100
4.2.1.4. Ulcerogenic activity. Male albino rats (180–200 g)
were divided into groups each of five animals and were fasted
for 12 h prior to the administration of the test compounds. Water
was given ad libitum. Control group received 1% gum acacia orally.
Other groups received diclofenac Na or the test compounds orally
in two equal doses at 0 and 12 h for three successive days at a dose
of 300 mg/kg per day. Animals were sacrificed by diethyl ether 6 h
after the last dose and their stomachs were removed. An opening at
the greater curvature was made and the stomach was cleaned by
4.1.14.2. E(5-(4-chlorophenyl)imidazo [2,1-b][1,3,4]thiadiazol-
2-ylsulfanyl)acetate (36). Yield: 40%, mp 155–157 °C. IR
(cmꢀ1): 1729 (C@O), 1650 (C@N); 1299, 1089 (C–S–C); 1032 (C–
O–C); 835 (C–Cl). 1H NMR (d ppm): 1.16 (t, J = 6.85 Hz, 3H,
CH2CH3); 4.13 (q, J = 6.85 Hz, 2H, CH2CH3); 4.24 (s, 2H, CH2);
7.42, 7.82 (two d, J = 8.4 Hz, each 2H, C6H4–Cl); 8.66 (s, 1H, imi-
dazothiadiazole-C6–H). Anal. Calcd for C14H12ClN3O2S2 (353.85):
C, 47.52; H, 3.42; N, 11.88. Found: C, 47.98; H, 3.73; N, 11.38.

894
S. A. F. Rostom et al. / Bioorg. Med. Chem. 17 (2009) 882–895
washing with cold saline and inspected with a 3ꢁ magnifying lens
for any evidence of hyperemia, haemorrhage, definite hemorrhagic
erosion or ulcer.
Acknowledgments
The authors are very grateful to Dr. Yasser R. Abdel-Fattah, Ge-
netic Engineering and Biotechnology Research Institute (GEBRI),
Mubarak City for Scientific Research and Technology Applications,
Bourg El-Arab, Alexandria, Egypt, for his great effort in carrying out
the microbiological screening.
4.2.1.5. Acute toxicity. Twelve groups of rats (180–200 g) each
consists of five animals, were used in this test. The animals were
fasted for 24 h prior to administration of the test compounds.
The compounds were given orally in graded doses of 0.1–3.0 g/kg
body weight, po. The compounds were screened at graded doses
for their acute lethal doses (ALD50) and the mortalities were re-
corded at each dose level after 24 h.
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