Synthesis of some urea and thiourea derivatives of 3-phenyl/ethyl-2-thioxo-2,3-dihydrothiazolo[4,5-d]pyrimidine and their antagonistic effects on haloperidol-induced catalepsy and oxidative stress in mice

Article abstract of DOI:10.1016/j.ejmech.2009.04.007

A series of 3-phenyl/ethyl-2-thioxo-2,3-dihydrothiazolo[4,5-d]pyrimidin-7-yl urea and thiourea derivatives were designed and synthesized. All the compounds have been evaluated for their antiparkinsonian activity in catalepsy induced by haloperidol in mice

Full text of DOI:10.1016/j.ejmech.2009.04.007

European Journal of Medicinal Chemistry 44 (2009) 3889–3897
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Synthesis of some urea and thiourea derivatives
of 3-phenyl/ethyl-2-thioxo-2,3-dihydrothiazolo[4,5-d]pyrimidine
and their antagonistic effects on haloperidol-induced catalepsy
and oxidative stress in mice
,
a
b
Faizul Azam a , Ismail A. Alkskas , Musa A. Ahmed
*
a Department of Pharmaceutical Chemistry, Faculty of Pharmacy, University of Seventh October, P.O. Box 2873, Misurata, Libya
b Department of Medicinal Chemistry, Faculty of Pharmacy, Al-Arab Medical University, Benghazi, Libya
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of 3-phenyl/ethyl-2-thioxo-2,3-dihydrothiazolo[4,5-d]pyrimidin-7-yl urea and thiourea deriva-
tives were designed and synthesized. All the compounds have been evaluated for their antiparkinsonian
activity in catalepsy induced by haloperidol in mice. A majority of the compounds exhibited significant
antiparkinsonian activity after intraperitoneal administration. The most active compound carries
methoxy group at 2-position of the phenyl ring. Some of the potent compounds were selected for
biochemical estimations of malondialdehyde, glutathione, superoxide dismutase and glutathione
peroxidase from brain homogenate to highlight the neuroprotective properties associated with them.
The results obtained in the present study may lead to the development of a suitable approach to the
treatment of Parkinson’s disease and may be the starting point for the future drug design.
Ó 2009 Elsevier Masson SAS. All rights reserved.
Received 30 August 2008
Received in revised form
24 March 2009
Accepted 2 April 2009
Available online 14 April 2009
Keywords:
Parkinson’s disease
Thiazolopyrimidine
Urea
Thiourea
Neuroprotection
1. Introduction
metabolism [5]. The predominant cellular free radicals are the
superoxide (Oꢀ2ꢁ) and hydroxyl (OHꢀ) species [4,6]. Other molecules,
Parkinson’s disease (PD) is considered as one of the major
progressive neurologic disorders of the elderly population and
about three percent of this age group over 65 years have developed
the overt illness [1]. Clinically, PD is characterized by the tetrad of
tremor at rest, slowness of voluntary movements, rigidity, and
postural instability [2]. The cardinal biochemical abnormality in PD
is the profound deficit in brain dopamine level, primarily attributed
to the loss of neurons of the nigrostriatal dopaminergic pathway
[3]. Over the last two decades, tremendous strides toward acquiring
a better knowledge of both the etiology and the pathogenesis of PD
revealed the free radical theory as one of the mechanisms involved
in the pathogenesis of this disease [4].
such as hydrogen peroxide (H2O2) and peroxynitrite (ONOOꢁ),
although not themselves free radicals, can lead to the generation of
free radicals through various chemical reactions. Thus H2O2, in the
presence of reduced metal, forms the highly reactive OHꢀ via the
Fenton reaction [6]. ONOOꢁ, formed by the reaction of nitric oxide
(NOꢀ) with Oꢀ2ꢁ, is a highly reactive molecule that also breaks down
to form OHꢀ. Together, these molecules are referred to as reactive
oxygen species (ROS) to signify their ability to lead to oxidative
changes within the cell [6]. Problems occur when the production of
ROS exceeds the ability of cells to defend themselves against these
substances. This imbalance between cellular production of ROS and
the ability of cells to defend themselves against them is referred to
as oxidative stress [6]. Oxidative stress can cause cellular damage
and ROS oxidize critical cellular components such as membrane
lipids, proteins, and DNA, thereby inducing apoptosis or necrosis
[7,8]. There is a large scientific literature regarding the relation
between ROS production, the induction of apoptosis or necrosis and
the pathogenesis of PD [9,10].
There is substantial evidence that the brain, which consumes
large amounts of oxygen, is particularly vulnerable to
oxidative damage. Free radicals are normal products of cellular
* Corresponding author. Tel.: þ218 92 7677641/þ218 91 3300265; fax: þ218 51
628149.
Superoxide dismutase (SOD) and glutathione peroxidase (GSH-
Px) are among the major antioxidant enzymes present in the
E-mail addresses: azamfaizul@yahoo.co.in, faizulazam@gmail.com (F. Azam).
0223-5234/$ – see front matter Ó 2009 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2009.04.007

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F. Azam et al. / European Journal of Medicinal Chemistry 44 (2009) 3889–3897
human body that protect against the oxygen toxicity [11]. Nonen-
zymatic antioxidant system includes glutathione (GSH), an oxy-
radical scavenger, thereby useful in protecting against oxidative
damage by free radicals and inhibiting lipid peroxidation and DNA
damage [12]. There are reports suggesting a decrease in SOD, GSH-
Px and other antioxidant enzyme activities in substantia nigra of PD
patients [13,14]. Consistent with this, a reduction of glutathione
[15] and polyunsaturated fatty acid contents, as well as an increase
in lipid peroxidation [16] has also been reported.
neuronal pathway [28]. In addition, cabergoline, a dopamine D2
receptor agonist which was launched in 1993 as an anti-Parkinson’s
agent containing urea functionality in its structure [29] while other
derivatives such as KHG21834 (Fig. 1) with urea and thiazole moiety
were capable of protecting PC12 cells and cortical and mesence-
phalic neurons from amyloid
some thiazol-2-yl urea derivatives (e.g. AR-A014418, Fig.1) are being
developed as an inhibitor of glycogen synthase kinase-3 as its
b-induced degeneration [30]. Likewise,
b
dysregulation is implicated in certain psychiatric and neurodegen-
erative diseases [31,32] and these agents are being considered as
a novel strategy to treat PD [33]. Also, a series of aryl/heteroaryl ureas
bearing thiazole moiety have emerged as a potent and selective
inhibitors of cyclin dependent kinases for the treatment of Alz-
heimer’s disease and other neurodegenerative disorders [34].
Thiazolo[4,5-d]pyrimidine derivatives, which can be considered
as thia-analogues of the natural purine bases such as adenine and
guanine, have acquired a growing importance in the field of
medicinal chemistry because of their biological potential while
some thiazolo[3,2-a]pyrimidines have been demonstrated to be
associated with potent immunomodulating properties [35].
Furthermore, the recently demonstrated adenosine A2A receptor
antagonistic activities of certain thiazoles with a urea moiety [36]
and thiazolopyrimidines (Fig. 1) [37] for the development of
a suitable approach to the treatment of PD may be the starting point
for the future drug design.
Led by above facts and coupled with our ongoing project aimed
at investigating new bioactive heterocycles derived from thiazole
nucleus [19–23], we have designed a series of compounds incor-
porating above moieties together in order to prepare the molecules
having enhanced biological activity. The compounds were evalu-
ated for haloperidol-induced catalepsy model of PD. The extent of
oxidative stress has been evaluated by measuring MDA level, GSH
content, SOD and GSH-Px activities from brain homogenate in
Dopamine-replacement therapy has dominated the treatment of
motor symptoms of PD since the early 1960s. The effects are
predictable (as are the side effects) and none of the more recently
introduced synthetic analogues has surpassed the clinical benefit
derived from levodopa [17]. Although levodopa is the most effective
drug for the control of motor symptoms, it also causes a high level of
motor complications, particularly dyskinesias [18]. However,
despite the many dopaminergic agents currently available, the
search for novel approaches based on dopamine-replacement
therapy continues. The multiplicity of dopamine receptors in the
brain offers a range of potential targets, but so far exploitation of
drugs acting on specific receptor subtypes has been disappointing.
Thiazole derivatives have evoked considerable attention in recent
years as these are endowed with wide range of biological activities
[19–21] as well as drugs of PD [22,23]. For example, pramipexole
(Fig. 1), an aminobenzothiazole analogue is a dopamine D2/D3
receptor agonist currently in clinical use for the treatment of PD [24].
It has been reported that pramipexole acts as a scavenger of ROS,
based on the findings of an in vivo microdialysis study of the rat
striatum [25]. Protective effect of pramipexole against H2O2-induced
PC12 cell death is also reported in literature [26]. Another example of
an analogue with an aminothiazole moiety is riluzole (Fig. 1), a Naþ
channel blocker with neuroprotective activity [27] effective against
MPTP-induced neurodegeneration of the nigrostriatal dopaminergic
H
N
H
N
O
N
N
H2N
OEt
C3H7
S
N
S
O
H
KHG21834 [30]
Pramipexole [24-26]
A
N
X
N
OCH3
H
N
H
N
N
O2N
S
R1
S
R2
N
O
AR-A014418 [33]
Thiazolopyrimidine derivatives [37]
H
H
N
N
N
F3CO
S
R
NH2
S
X
N
Thiazole derivatives with urea/thiourea moiety [23]
Riluzole [27]
Fig. 1. Structure of some cited molecules.

F. Azam et al. / European Journal of Medicinal Chemistry 44 (2009) 3889–3897
3891
selected compounds. Part of this work has been presented in an
abstract form [38].
hand, cataleptic mice, when placed in this awkward position, were
unable to come down from the bar over a period of 180 s or more.
Haloperidol (5 mg/kg) produced a profound increase in cata-
lepsy as shown by a progressive increase in the latency to step
down the rod over time as compared with controls (P < 0.001).
Efficacious compounds were defined as those that allowed the mice
to come down from the bar within 180 s. The results obtained from
acute administration of haloperidol are in agreement with previous
studies of our research group [22,23] as well as report from other
laboratory [41]. Acute subcutaneous administration of compounds
(3a–20a and 3b–14b) at a dose of 100 mg/kg significantly antago-
nized haloperidol-induced catalepsy as evident from the decrease
in mean descent time in treated animals when compared to mice
injected with haloperidol alone. The effect of title compounds and
standard drug in terms of mean descent time observed at different
time intervals, mean CDT [3 h] and % catalepsy are presented in
Table 2.
Compounds 5a and 14b showed better activity than standard
drug levodopa. The least active compound among the series was 10b
while 5a was most potent. Rest of the compounds exhibited
moderate activity and the results obtained are presented in Table 2.
When talking about the structure–activity relationship, electron
attractive groups decreased the antiparkinsonian activity, while
pronounced activity was observed in derivatives having electron
donors like –OCH3 groups especially at position 2 of the phenyl ring.
These results buttress the theoretical dopamine D2 receptor model
of Sukalovic et al. [42] where it is reported that methoxy group
increases the affinities of ligands if attached to position 2, since one
additional hydrogen bond could be formed with Trp 182. Similarly, it
is possible that compounds having –OCH3 group at position 2 may
interact with Trp 182 of dopamine D2 receptor binding site, making
the compound potent in terms of observed anticataleptic as well as
2. Synthesis
In the present study urea and thiourea derivatives of 3-phenyl/
ethyl-2-thioxo-2,3-dihydrothiazolo[4,5-d]pyrimidine were synthe-
sized as presented in Scheme 1. 4-Amino-3-phenyl/ethyl-2-thioxo-
2,3-dihydrothiazole-5-carbonitrile (1a, 1b) was prepared when to
a mixture of malononitrile, phenyl/ethyl isothiocyanate and finely
divided sulfur in DMF was added triethylamine very slowly with
constant stirring at room temperature. 1a or 1b was treated with
formamide and formic acid with heating to yield 7-amino-3-
phenyl/ethyl thiazolo[4,5-d]pyrimidine-2(3H)-thione (2a, 2b). The
urea and thiourea derivatives (3a–20a, 3b–14b) were obtained
when 2a or 2b and appropriate aryl isocyanate/isothiocyanate were
refluxed with stirring in dry acetonitrile. All compounds had IR, 1H
NMR and mass spectra in accord with their anticipated structure.
The physical characterization data of the compounds are given in
Table 1.
3. Results and discussion
Catalepsy is a behavioral condition characterized by the rigid
state of a part or all of the muscles which shares some similarity to
human PD [39,40]. The phenomenon of cataleptic immobility
induced in rodents by the use of a dopamine antagonist such as
haloperidol is a robust behavioral method for studying nigrostriatal
function. This is a behavioral condition in which the animal is
unable to correct an externally imposed posture. Catalepsy-free
mice, i.e., those without haloperidol, should be able to come down
from the horizontal bar within certain time period. On the other
X
NH2
N
N
C
X
S
Sulfur Powder
N
C
C
S
+
Triethylamine
N
N
S
C
1a, 1b
X
O
OH
NH2
O
N
N
NCY
S
N
S
R
X
N
Y
N
H
C
N
N
Acetonitrile
S
N
H
S
R
3a-20a
3b-14b
NH2
2a, 2b
a (X = Ph)
3a,b; 12a,b
4a,b; 13a,b
5a,b; 14a,b
b (X = Et)
(R = H)
(R = 4-OCH3)
(R = 2-OCH3)
Y = O or S
(R = 4-Cl)
6a,b; 15a
7a,b; 16a
8a,b; 17a
(R = 2-F)
9a,b; 18a
(R = 2-NO2)
(R = 4-NO2)
(R = 4-F)
10a,b; 19a
11a,b; 20a
(R = 2-Cl)
Scheme 1. Synthetic protocol of the title compounds.

3892
F. Azam et al. / European Journal of Medicinal Chemistry 44 (2009) 3889–3897
Table 1
Characterization data of the urea and thiourea derivatives 3a–20a and 3b–14b.
N
N
Y
C
R
N
H
N
H
X
N
S
S
Compound
X
Y
R
Yield (%)
m.p. (ꢂC)
Mol. formulaa
Mol. weight
log Pb
3a
4a
5a
6a
7a
8a
9a
10a
11a
12a
13a
14a
15a
16a
17a
18a
19a
20a
3b
4b
5b
6b
7b
8b
9b
10b
11b
12b
13b
14b
Ph
Ph
Ph
Ph
Ph
Ph
Ph
Ph
Ph
Ph
Ph
Ph
Ph
Ph
Ph
Ph
Ph
Ph
Et
Et
Et
Et
Et
Et
Et
Et
Et
O
O
O
O
O
O
O
O
O
S
S
S
S
S
S
S
S
S
O
O
O
O
O
O
O
O
O
S
H
79
86
63
55
90
85
91
83
76
67
82
85
83
59
88
65
80
76
66
78
72
65
73
66
58
63
77
71
78
82
260–262
310–312
306–308
199–200
255–256
242–243
234–236
284–286
265–267
250–252
304–305
287–288
271–273
261–263
270–272
278–280
284–286
256–257
265–267
249–250
258–260
271–273
283–284
225–227
286–288
220–222
264–265
268–270
298–300
282–283
C18H13N5OS2
C19H15N5O2S2
C19H15N5O2S2
C18H12FN5OS2
C18H12FN5OS2
C18H12ClN5OS2
C18H12ClN5OS2
C18H12N6O3S2
C18H12N6O3S2
C18H13N5S3
C19H15N5OS3
C19H15N5OS3
C18H12FN5S3
C18H12FN5S3
C18H12ClN5S3
C18H12ClN5S3
C18H12N6O2S3
C18H12N6O2S3
C14H13N5OS2
C15H15N5O2S2
C15H15N5O2S2
C14H12FN5OS2
C14H12FN5OS2
C14H12ClN5OS2
379.46
409.48
409.48
397.45
397.45
413.9
3.48
3.43
3.54
3.45
3.93
3.99
4.47
3.73
3.94
2.96
2.91
2.86
2.92
3.41
3.47
3.95
3.21
3.42
2.26
2.21
2.32
2.22
2.71
2.77
3.25
2.51
2.72
1.74
1.69
1.64
4-OCH3
2-OCH3
2-F
4-F
2-Cl
4-Cl
413.9
2-NO2
4-NO2
H
4-OCH3
2-OCH3
2-F
4-F
2-Cl
4-Cl
2-NO2
4-NO2
H
4-OCH3
2-OCH3
2-F
4-F
2-Cl
4-Cl
424.46
424.46
395.52
425.55
425.55
413.51
413.51
429.97
429.97
440.52
440.52
331.42
361.44
361.44
349.41
349.41
365.86
365.86
376.41
376.41
347.48
377.51
377.51
C14H12ClN5OS2
2-NO2
4-NO2
H
4-OCH3
2-OCH3
C14H12N6O3S2
C14H12N6O3S2
C14H13N5S3
C15H15N5OS3
C15H15N5OS3
Et
Et
Et
S
S
a
Elemental analyses for C, H and N were within ꢃ0.4% of the theoretical values.
b
log P was generated using ACD/log P 11.0 software (Advanced Chemistry Development Inc., Toronto, Canada).
antioxidant activities. In addition, the electron attractive groups
decrease the binding affinity, while electron donors like –OCH3
increase the affinity for the binding at the dopamine D2 receptor in
comparison with the unsubstituted analogues [43,44]. Also,
methoxy group is said to be linked with binding affinity of the drug
molecules at dopamine D2 and D3 receptor sites [43]. In a report of
Teeter and DuRand [45] and Simpson et al. [46] it was revealed that
benzimidazole structural motif interacts with the receptor through
hydrogen bonds that involve amino acids Ser 122 and Ser 141 inTM II
and TM III, that are a part of catechol binding site of the dopamine D2
receptor. Likewise it can be assumed that one of the nitrogen atoms
of pyrimidine ring may interact with the receptor through hydrogen
bond in a similar fashion. Based on these observations, the proposed
binding interactions of the title compounds at the receptor site are
presented in Fig. 2.
Some potent compounds (5a, 5b, 14a and 14b), were selected for
the measurement of antioxidant parameters from the brain
homogenate of mice to investigate their role in the pathophysi-
ology of PD. Oxidative stress to dopaminergic neurons of substantia
nigra pars compacta is believed to be one of the leading causes of
neurodegeneration in PD. Thus ROS scavenging antioxidants may
play an important role in the prevention of PD and combat against
oxidative stress-induced progressive neurodegeneration [47–49].
Our results revealed that haloperidol increased the lipid
peroxidation and reduced the GSH level, along with reduced
activities of antioxidant enzyme (SOD and GSH-Px) in the brain
homogenate. Many reports indicate that an excessive production of
free radicals is associated with haloperidol treatment [50,51]. This
abnormal free radical production often goes hand in hand with GSH
and ATP depletion [50,52], and a loss of detoxifying enzyme activity
such as SOD, catalase and GSH-Px [53,54]. Also, many preclinical
and clinical studies have proposed the production of ROS as causes
of haloperidol-induced toxicity [51] and a loss of detoxifying
enzymes activities [52,55].
Compounds 5a, 5b, 14a and 14b administered 30 min before
haloperidol significantly suppressed oxidative stress, restoring
enzyme activities as well as GSH and MDA contents to almost similar
to the controls (Table 3). The present data demonstrate that urea and
thiourea derivatives possess antioxidant effect on catalepsy model of
PD, offering protection by enhancing GSH content and antioxidant
enzyme(SODandGSH-Px)activitiesaswellasdecreasingLPOinmice
treated with haloperidol. Such regulation of oxidative stress markers
and antioxidant enzymes by the title compounds in the present study
may be well correlated with our previous reports, where thiazole
derivatives as well as urea/thiourea derivatives were effective in
restoring the levels of oxidative stress markers and antioxidative
enzymes up to the basal level against oxidative stress induced by
haloperidol [22,23] and pentylenetetrazole in mice brain [21].

F. Azam et al. / European Journal of Medicinal Chemistry 44 (2009) 3889–3897
3893
Table 2
Mean descent time observed at different time intervals.
Compound
Mean descent time (s) ꢃ S.E.M. at time (min)
Mean CDT [3 h] ꢃ SEM
Catalepsy (%)
30
60
90
120
150
180
3a
4a
5a
6a
7a
8a
9a
10a
11a
12a
13a
14a
15a
16a
17a
18a
19a
20a
3b
45 ꢃ 0.56a
128 ꢃ 2.86a
92 ꢃ 1.34a
49 ꢃ 0.75a
126 ꢃ 2.17b
114 ꢃ 2.45a
118 ꢃ 2.16a
177 ꢃ 2.12a
63 ꢃ 1.53a
79 ꢃ 1.27a
58 ꢃ 1.32a
66 ꢃ 1.53a
44 ꢃ 0.54a
99 ꢃ 1.43a
129 ꢃ 2.14b
116 ꢃ 3.19a
147 ꢃ 4.17a
69 ꢃ 1.36a
113 ꢃ 1.61b
122 ꢃ 1.75a
111 ꢃ 1.32a
31 ꢃ 0.73a
119 ꢃ 1.89a
99 ꢃ 1.71a
138 ꢃ 2.91b
173 ꢃ 4.12b
168 ꢃ 1.93a
140 ꢃ 2.55a
115 ꢃ 1.12a
43 ꢃ 0.33a
15 ꢃ 0.12a
38 ꢃ 1.54a
224 ꢃ 4.45c
5 ꢃ 0.21
98 ꢃ 1.61b
134 ꢃ 1.99a
135 ꢃ 2.32a
89 ꢃ 1.26a
141 ꢃ 2.13a
158 ꢃ 3.72a
167 ꢃ 2.76a
205 ꢃ 1.81a
135 ꢃ 2.65a
125 ꢃ 2.57a
192 ꢃ 2.76a
158 ꢃ 2.11a
108 ꢃ 1.17a
160 ꢃ 2.11a
195 ꢃ 3.22a
158 ꢃ 1.16a
168 ꢃ 2.57a
93 ꢃ 1.14a
200 ꢃ 1.69a
125 ꢃ 1.67a
83 ꢃ 2.54a
152 ꢃ 3.14a
153 ꢃ 2.89a
175 ꢃ 2.34a
206 ꢃ 1.58b
176 ꢃ 2.19a
191 ꢃ 1.29b
216 ꢃ 4.15a
172 ꢃ 2.67a
112 ꢃ 1.93a
208 ꢃ 3.61a
218 ꢃ 3.28a
241 ꢃ 2.62a
209 ꢃ 3.5b
234 ꢃ 2.82a
161 ꢃ 1.87a
133 ꢃ 3.31a
138 ꢃ 2.37a
135 ꢃ 3.12a
186 ꢃ 2.66a
187 ꢃ 4.14a
196 ꢃ 2.38b
226 ꢃ 4.77a
225 ꢃ 3.58a
210 ꢃ 4.88a
172 ꢃ 1.18a
126 ꢃ 2.16a
97 ꢃ 1.1a
195 ꢃ 2.60b
129 ꢃ 3.19b
94 ꢃ 1.11a
800 ꢃ 5.55a
638 ꢃ 3.68a
398 ꢃ 4.49a
826 ꢃ 5.12a
853 ꢃ 3.74a
854 ꢃ 4.29a
1045 ꢃ 5.65a
727 ꢃ 3.93a
803 ꢃ 4.81a
824 ꢃ 3.19a
702 ꢃ 4.47a
476 ꢃ 3.64a
944 ꢃ 3.44a
982 ꢃ 4.99a
1002 ꢃ 5.29a
1003 ꢃ 3.16a
737 ꢃ 4.15a
769 ꢃ 3.09a
774 ꢃ 5.52a
704 ꢃ 4.61a
488 ꢃ 4.01a
860 ꢃ 5.11a
886 ꢃ 4.36a
938 ꢃ 4.67a
1025 ꢃ 4.38a
1130 ꢃ 5.45a
1087 ꢃ 3.73a
810 ꢃ 3.71a
535 ꢃ 4.56a
402 ꢃ 4.24a
424 ꢃ 3.22a
1580 ꢃ 5.19c
36 ꢃ 1.04
49.5
39
23.4
51.2
52.9
53
65.3
44.8
49.7
51
43.1
28.5
58.8
61.3
62.6
62.6
45.4
47.5
47.8
43.3
29.3
53.4
55.1
58.4
64.1
70.9
68.1
50.1
32.3
23.7
25.1
100
–
57 ꢃ 1.1a
26 ꢃ 2.18a
83 ꢃ 1.25a
85 ꢃ 1.18a
76 ꢃ 1.25a
76 ꢃ 1.12b
55 ꢃ 1.12a
53 ꢃ 1.17a
66 ꢃ 0.88a
70 ꢃ 1.12a
25 ꢃ 0.15a
78 ꢃ 0.81b
75 ꢃ 1.17a
64 ꢃ 1.56a
58 ꢃ 1.06b
42 ꢃ 1.01a
58 ꢃ 0.81a
49 ꢃ 1.12a
77 ꢃ 0.17a
35 ꢃ 0.31a
67 ꢃ 1.14a
66 ꢃ 1.02b
127 ꢃ 1.98a
64 ꢃ 1.31a
102 ꢃ 1.82a
100 ꢃ 2.37b
73 ꢃ 0.69a
26 ꢃ 0.19a
18 ꢃ 0.18a
19 ꢃ 0.51b
183 ꢃ 3.68c
8 ꢃ 0.06
100 ꢃ 2.01a
57 ꢃ 1.18a
166 ꢃ 2.19b
115 ꢃ 2.51a
144 ꢃ 2.53a
139 ꢃ 2.53b
121 ꢃ 1.57a
119 ꢃ 1.7a
158 ꢃ 2.56a
228 ꢃ 4.88a
174 ꢃ 2.33a
242 ꢃ 2.72a
177 ꢃ 3.61a
236 ꢃ 3.35b
217 ꢃ 3.79a
133 ꢃ 2.34a
109 ꢃ 1.87a
257 ꢃ 3.45b
200 ꢃ 2.74a
238 ꢃ 2.61a
268 ꢃ 4.12a
232 ꢃ 3.23b
159 ꢃ 2.58a
199 ꢃ 2.36a
143 ꢃ 2.0a
75 ꢃ 1.67a
103 ꢃ 1.74a
78 ꢃ 0.93a
142 ꢃ 2.81a
165 ꢃ 2.27b
185 ꢃ 2.21a
153 ꢃ 2.66a
69 ꢃ 2.34a
131 ꢃ 1.98a
127 ꢃ 1.33b
114 ꢃ 3.15a
40 ꢃ 0.56a
138 ꢃ 1.66a
113 ꢃ 1.58a
131 ꢃ 2.48a
163 ꢃ 2.74a
209 ꢃ 2.91a
194 ꢃ 3.12a
139 ꢃ 2.31a
92 ꢃ 2.11a
53 ꢃ 0.19a
86 ꢃ 1.54a
273 ꢃ 3.82c
5 ꢃ 0.36
147 ꢃ 2.32a
144 ꢃ 2.95a
121 ꢃ 1.44b
90 ꢃ 2.65a
173 ꢃ 2.37a
184 ꢃ 2.31a
157 ꢃ 2.79a
152 ꢃ 2.16a
198 ꢃ 3.53a
212 ꢃ 4.11a
140 ꢃ 2.49a
114 ꢃ 1.86a
75 ꢃ 0.81a
88 ꢃ 0.68b
300 ꢃ 4.23c
5 ꢃ 0.08
4b
5b
6b
7b
8b
9b
157 ꢃ 3.19a
177 ꢃ 2.37a
237 ꢃ 3.9a
189 ꢃ 2.17a
247 ꢃ 2.38b
228 ꢃ 2.62a
231 ꢃ 3.34b
171 ꢃ 2.73a
134 ꢃ 2.24a
144 ꢃ 4.22a
108 ꢃ 1.51a
300 ꢃ 4.96c
7 ꢃ 0.64
10b
11b
12b
13b
14b
Levodopa
Haloperidol
Control
85 ꢃ 1.62a
300 ꢃ 4.31c
6 ꢃ 0.57
Data are presented as mean descent time (s) ꢃ S.E.M. Number of animals per group (n) ¼ 5, Haloperidol (5 mg/kg), compounds 3a–20a, 3b–14b and levodopa at the dose of
100 mg/kg 30 min prior to haloperidol injection. The sum of the descent time values measured every 30 min during the 3 h after haloperidol or vehicle is defined as the
cumulative descent time (CDT [3 h]). Data were analyzed by one-way ANOVA followed by Bonferroni post hoc analysis.
a
P < 0.001 as compared with haloperidol group.
P < 0.01 as compared with haloperidol group.
P < 0.001 as compared with control group.
b
c
Poor solubility and poor permeability are among the main
causes for failure during drug development [56,57]. It is therefore
important to determine these physicochemical properties associ-
ated with a drug, before synthetic work is undertaken. The esti-
mated log P values (P is the partition coefficient of the molecule in
the water/octanol system), which can be used as an indicator of
passive diffusion across cell membranes and cellular uptake
[58–60], were determined for all urea and thiourea derivatives and
are presented in Table 1. The lipophilicity study showed that most
of compounds possess optimum lipophilicities (log P 1.64–4.47)
required for oral absorption and biomembrane penetration, even
for BBB penetration according to Lipinski’s drug-likeness ‘rules of
five’ [61]. This bioavailability feature makes it possible to use these
compounds in treatment of neurodegenerative diseases. A precise
correlation between lipophilicity and biological activity was not
observed which reflects that the lipophilicity has an influence on
the activity, but it does not solely determine the antiparkinsonian
activity of these compounds.
Ser 141
Trp 182
OH
H3C
H2N
O
OH
H
N
O
O
Table 3
Y
OH
Biochemical estimation from brain homogenate.
C
N
H2N
Compound
LPO (nmol MDA/mg GSH (
m
g/gm
GSH-Px (U/g SOD (U/mg
N
protein)
tissue)
protein)
protein)
NH
H
O
OH
5a
5b
14a
14b
3.201 ꢃ 0.47a
2.806 ꢃ 0.39a
2.746 ꢃ 0.14a
3.123 ꢃ 0.38c
14.362 ꢃ 2.34c 1.134 ꢃ 0.1a
1.992 ꢃ 0.11a
13.981 ꢃ 1.71a 1.544 ꢃ 0.08a 1.815 ꢃ 0.12a
14.156 ꢃ 1.18a 1.597 ꢃ 0.14a 2.345 ꢃ 0.09a
11.672 ꢃ 0.97a 1.331 ꢃ 0.24a 1.866 ꢃ 0.14a
6.753 ꢃ 0.15b 0.765 ꢃ 0.13b 0.941 ꢃ 0.15b
N
OH
S
H2N
Haloperidol 5.521 ꢃ 0.23b
X = Ph or Et
Y = O or S
N
Ser 122
Control
0.958 ꢃ 0.08
24.74 ꢃ 0.29
1.893 ꢃ 0.12
2.327 ꢃ 0.06
X
S
The data are expressed as mean ꢃ S.E.M (n ¼ 5).
a
P < 0.001 compared with corresponding value for haloperidol-treated mice.
P < 0.001 compared with corresponding value for control mice.
P < 0.01 compared with corresponding value for haloperidol-treated mice.
b
c
Fig. 2. Proposed binding interactions of the title compounds at the receptor site.
Dashed lines indicate H-bond.

3894
F. Azam et al. / European Journal of Medicinal Chemistry 44 (2009) 3889–3897
4. Conclusions
716 cmꢁ1; 1H NMR (CDCl3)
d 5.12 (br s, 2H, NH2, D2O exchangeable),
6.7–7.56 (m, 5H, ArH), 8.52 (s, 1H, CH pyrimidine); MS (FAB) m/z
Finally, we have recognized a novel series of 3-phenyl/ethyl-2-
259 (M þ 1)þ.
thioxo-2,3-dihydrothiazolo[4,5-d]pyrimidin-7-yl
urea/thiourea
derivatives that reduced haloperidol-induced catalepsy and
oxidative stress in an animal model of PD which could be important
for future development of new antiparkinsonian drugs or it could as
well be an important treatment armamentarium for PD.
5.1.4. Synthesis of 7-amino-3-ethylthiazolo[4,5-d]pyrimidin-2(3H)-
thione (2b)
A mixture of 2a (0.01 mol), formamide (40 ml) and formic acid
(10 ml) was heated at 110 ꢂC for 6 h, cooled, poured onto ice-water
to give precipitates, which were filtered off, dried and recrystallized
from ethanol to afford 2b. Yield 64%; m.p. 181–183 ꢂC; IR (KBr) ymax
3403, 3151, 2952, 2834, 1665, 1586, 1540, 1472, 1450, 1256, 1121,
5. Experimental protocols
5.1. Chemistry
1068, 892, 767, 699 cmꢁ1; 1H NMR (CDCl3)
d 1.34 (t, 3H, CH3), 3.61
(q, 2H, CH2), 4.74 (s, 2H, NH2, D2O exchangeable), 9.44 (s, 1H, CH
Synthetic starting material, reagents and solvents were of
analytical reagent grade or of the highest quality commercially
available and were purchased from Aldrich Chemical Co., Merck
Chemical Co. and were dried when necessary. The progress of the
reactions was monitored by thin layer chromatography with F254
silica-gel precoated sheets (Merck) using chloroform/methanol 95/
5 as eluent; UV light and iodine vapours were used for detection. IR
spectra were recorded, as KBr pellets, on a Shimadzu 8201 PC FT-IR
spectrophotometer and wave numbers are given in cmꢁ1. The mass
spectra were recorded on Jeol SX-102 (FAB). 1H NMR spectra, in
DMSO-d6 and CDCl3 solutions, were recorded on a Bruker DRX-300
instrument at 298 K. Chemical shifts are reported as ppm relative to
TMS as internal standard. Melting points (ꢂC) were determined
with an open glass capillary tube and are uncorrected. Elemental
analyses were performed on Elementar Vario EL III instrument.
pyrimidine); MS (FAB) m/z 213 (M þ 1)þ.
5.1.5. General method for the synthesis of urea and thiourea
derivatives of 3-phenyl/ethyl-2-thioxo-2,3-dihydrothiazolo[4,5-
d]pyrimidine (3a,b–20a,b)
To a solution of 2a or 2b (0.002 mol) in acetonitrile (20 ml), an
appropriate isocyanate or isothiocyanate (0.0024 mol) in acetoni-
trile (5 ml) was added dropwise with stirring at room temperature.
The mixture was refluxed with stirring for 1–4 h until the
completion of the reaction and the resultant precipitate was
filtered, washed with acetonitrile, dried and recrystallized from
95% ethanol. The physical data of the compounds are presented in
Table 1. The spectral data of the compounds are as follows.
5.1.5.1. 1-Phenyl-3-(3-phenyl-2-thioxo-2,3-dihydrothiazolo[4,5-d]pyr-
imidin-7-yl)urea (3a). IR (KBr) ymax 3360, 3170, 3029, 2928, 1700,
5.1.1. Synthesis of 4-amino-3-phenyl-2-thioxo-2,3-dihydrothiazol-
5-carbonitrile (1a)
1608, 1547, 1188 cmꢁ1 1H NMR (DMSO-d6)
; d 6.69–7.13 (m, 10H,
ArH), 8.48 (s, 1H, CH pyrimidine), 9.63 (s, 1H, NH), 10.51 (s, 1H, NH);
To phenyl isothiocyanate (0.05 mol) in 50 ml DMF was added
finely divided sulfur (0.05 mol) and malononitrile (0.05 mol). Trie-
thylamine (0.1 mol) was added dropwise with constant stirring to
the above mixture at room temperature and the stirring was
continued for 3 h. The deep red thick solution so obtained was
poured into a mixture of 90 ml ice-cold water and 10 ml methanol.
The colorless solid so obtained was filtered, washed with water,
dried and recrystallized from methanol. Yield 46%; m.p. 263 ꢂC; IR
(KBr) ymax 3370, 3300, 3229, 3184, 2206,1637,1580,1437,1244,1037,
MS (FAB) m/z 380 (M þ 1)þ.
5.1.5.2. 1-(4-Methoxyphenyl)-3-(3-phenyl-2-thioxo-2,3-dihydrothia-
zolo[4,5-d]pyrimidin-7-yl)urea (4a). IR (KBr) ymax 3378, 3206, 3092,
2957,1715,1602,1532,1507,1389,1223, 790 cmꢁ1; 1H NMR (DMSO-
d6)
d 3.66 (s, 3H, OCH3), 6.46–7.64 (m, 9H, ArH), 8.71 (s, 1H, CH
pyrimidine), 8.95 (s, 1H, NH), 9.98 (s, 1H, NH); MS (FAB) m/z 410
(M þ 1)þ.
722 cmꢁ1; 1H NMR (DMSO-d6)
d
3.78(s, 2H, NH2, D2Oexchangeable),
5.1.5.3. 1-(2-Methoxyphenyl)-3-(3-phenyl-2-thioxo-2,3-dihydrothia-
zolo[4,5-d]pyrimidin-7-yl)urea (5a). IR (KBr) ymax 3392, 3235, 3082,
3001, 2876, 1701, 1577, 1542, 1376, 1134, 796 cmꢁ1; 1H NMR (DMSO-
6.54–7.28 (m, 5H, ArH); MS (FAB) m/z 234 (M þ 1)þ.
5.1.2. Synthesis of 4-amino-3-ethyl-2-thioxo-2,3-dihydrothiazol-5-
carbonitrile (1b)
d6) d 3.89 (s, 3H, OCH3), 6.74–7.54 (m, 8H, ArH), 8.65 (d, 1H,
J ¼ 7.4 Hz), 8.96 (s, 1H, CH pyrimidine), 9.92 (br s, 1H, NH), 11.13 (s,
To ethyl isothiocyanate (0.05 mol) in 50 ml DMF was added
finely divided sulfur (0.05 mol) and malononitrile (0.05 mol).
Triethylamine (0.1 mol) was added dropwise with constant stirring
to the above mixture at room temperature and the stirring was
continued for 3 h. The deep red thick solution so obtained was
poured into a mixture of 90 ml ice-cold water and 10 ml methanol.
The colorless solid so obtained was filtered, washed with water,
dried and recrystallized from methanol. Yield 39%; m.p. 168 ꢂC; IR
(KBr) ymax 3308, 3225, 3184, 2972, 2207, 1644, 1576, 1466, 1435,
1H, NH); MS (FAB) m/z 410 (M þ 1)þ.
5.1.5.4. 1-(2-Fluorophenyl)-3-(3-phenyl-2-thioxo-2,3-dihydrothiazolo-
[4,5-d]pyrimidin-7-yl) urea (6a). IR (KBr) ymax 3355, 3253, 3164,
2985, 1689, 1600, 1532, 1247, 804 cmꢁ1; 1H NMR (DMSO-d6)
d 6.72–
7.24 (m, 7H, ArH), 8.36 (d, 1H, J ¼ 7.42 Hz), 8.74 (d, 1H, J ¼ 7.66 Hz),
8.96 (s, 1H, CH pyrimidine), 9.38 (s, 1H, NH),10.46 (br s, 1H, NH); MS
(FAB) m/z 398 (M þ 1)þ.
1341, 1238, 1118, 1015, 804 cmꢁ1; 1H NMR (DMSO-d6)
d
1.76 (t, 3H,
5.1.5.5. 1-(4-Fluorophenyl)-3-(3-phenyl-2-thioxo-2,3-dihydrothiazolo-
CH3), 2.74 (q, 2H, CH2), 3.92 (s, 2H, NH2, D2O exchangeable); MS
[4,5-d]pyrimidin-7-yl) urea (7a). IR (KBr) ymax 3408, 3251, 3166,
(FAB) m/z 186 (M þ 1)þ.
2990, 1699, 1613, 1533, 1248, 804 cmꢁ1; 1H NMR (DMSO-d6)
d 6.46–
7.44 (m, 9H, ArH), 8.32 (s, 1H, CH pyrimidine), 8.96 (s, 1H, NH), 11.32
5.1.3. Synthesis of 7-amino-3-phenylthiazolo[4,5-d]pyrimidin-
2(3H)-thione (2a)
(s, 1H, NH); MS (FAB) m/z 398 (M þ 1)þ.
A mixture of 1a (0.01 mol), formamide (40 ml) and formic acid
(10 ml) was heated at 110 ꢂC for 6 h, cooled, poured onto ice-water
to give precipitates, which were filtered off, dried and recrystallized
from ethanol to afford 2a. Yield 72%; m.p. 204–206 ꢂC; IR (KBr) ymax
3435, 3049, 2927, 2842, 1666, 1587, 1539, 1420, 1256, 1090, 882,
5.1.5.6. 1-(2-Chlorophenyl)-3-(3-phenyl-2-thioxo-2,3-dihydrothiazolo-
[4,5-d]pyrimidin-7-yl) urea (8a). IR (KBr) ymax 3474, 3259, 3130,
2854,1676,1396, 790 cmꢁ1; 1H NMR (DMSO-d6)
d 6.61–7.58 (m, 9H,
ArH), 8.10 (s, 1H, CH pyrimidine), 8.82 (s, 1H, NH), 10.50 (s, 1H, NH);
MS (FAB) m/z 414 (M þ 1)þ.

F. Azam et al. / European Journal of Medicinal Chemistry 44 (2009) 3889–3897
3895
5.1.5.7. 1-(4-Chlorophenyl)-3-(3-phenyl-2-thioxo-2,3-dihydrothiazolo-
(m, 9H, ArH), 8.57 (s, 1H, CH pyrimidine), 8.97 (s, 1H, NH), 11.01
[4,5-d]pyrimidin-7-yl) urea (9a). IR (KBr) ymax 3372, 3091, 2983,
(s, 1H, NH); MS (FAB) m/z 441 (M þ 1)þ.
1703, 1584, 1541, 1377, 1256, 1142, 1085, 818, 790 cmꢁ1 1H NMR
;
(DMSO-d6)
d
7.13–8.12 (m, 9H, ArH), 8.65 (s, 1H, CH pyrimidine),
5.1.5.18. 1-(4-Nitrophenyl)-3-(3-phenyl-2-thioxo-2,3-dihydrothiazolo-
9.36 (s, 1H, NH), 10.84 (s, 1H, NH); MS (FAB) m/z 414 (M þ 1)þ.
[4,5-d]pyrimidin-7-yl) thiourea (20a). IR (KBr) ymax 3316, 3203,
2972, 1701, 1588, 1575, 1334, 1160, 805 cmꢁ1 1H NMR (DMSO-d6)
;
5.1.5.8. 1-(2-Nitrophenyl)-3-(3-phenyl-2-thioxo-2,3-dihydrothiazolo-
d 7.38–7.91 (m, 9H, ArH), 8.80 (s, 1H, CH pyrimidine), 10.11 (br s, 1H,
[4,5-d]pyrimidin-7-yl) urea (10a). IR (KBr) ymax 3352, 3231, 1690,
NH), 11.43 (s, 1H, NH); MS (FAB) m/z 441 (M þ 1)þ.
1583, 1344, 1152 cmꢁ1 1H NMR (DMSO-d6)
; d 7.28–8.36 (m, 9H,
ArH), 8.98 (s, 1H, CH pyrimidine), 9.59 (s, 1H, NH), 11.26 (s, 1H, NH);
5.1.5.19. 1-(3-Ethyl-2-thioxo-2,3-dihydrothiazolo[4,5-d]pyrimidin-7-
yl)-3-phenylurea (3b). IR (KBr) ymax 3346, 3252, 2991, 1608, 1521,
MS (FAB) m/z 425 (M þ 1)þ.
692 cmꢁ1; 1H NMR (DMSO-d6)
d 1.01 (t, 3H, CH3), 3.12 (q, 2H, CH2),
5.1.5.9. 1-(4-Nitrophenyl)-3-(3-phenyl-2-thioxo-2,3-dihydrothiazolo-
6.98–7.66 (m, 5H, ArH), 8.82 (s, 1H, CH pyrimidine), 9.43 (s, 1H, NH),
[4,5-d]pyrimidin-7-yl) urea (11a). IR (KBr) ymax 3340, 3215, 3156,
10.67 (br s, 1H, NH); MS (FAB) m/z 332 (M þ 1)þ.
1587, 1363, 1180, 768 cmꢁ1 1H NMR (DMSO-d6)
; d 6.4–7.2 (m, 7H,
ArH), 8.3 (d, 1H, J ¼ 7.0 Hz), 8.76 (d, 1H, J ¼ 7.31 Hz), 9.25 (s, 1H, CH
pyrimidine), 9.98 (s, 1H, NH), 10.86 (s, 1H, NH); MS (FAB) m/z 425
(M þ 1)þ.
5.1.5.20. 1-(3-Ethyl-2-thioxo-2,3-dihydrothiazolo[4,5-d]pyrimidin-7-
yl)-3-(4-methoxyphenyl) urea (4b). IR (KBr) ymax 3279, 3102, 3078,
2998, 2937, 1701, 1547, 1364, 1243, 732 cmꢁ1 1H NMR (DMSO-d6)
;
d
1.39 (t, 3H, CH3), 2.78 (q, 2H, CH2), 3.74 (s, 3H, OCH3), 7.00–7.59
5.1.5.10. 1-Phenyl-3-(3-phenyl-2-thioxo-2,3-dihydrothiazolo[4,5-d]-
pyrimidin-7-yl)thiourea (12a). IR (KBr) ymax 3406, 3259, 3130, 1674,
(m, 4H, ArH), 8.48 (s, 1H, CH pyrimidine), 9.39 (br s, 1H, NH), 10.47
(s, 1H, NH); MS (FAB) m/z 362 (M þ 1)þ.
1365, 1199 cmꢁ1 1H NMR (DMSO-d6)
; d 6.77–7.63 (m, 10H, ArH),
7.81 (s, 1H, CH pyrimidine), 8.24 (s, 1H, NH), 9.33 (s, 1H, NH); MS
5.1.5.21. 1-(3-Ethyl-2-thioxo-2,3-dihydrothiazolo[4,5-d]pyrimidin-7-
yl)-3-(2-methoxyphenyl) urea (5b). IR (KBr) ymax 3319, 3207, 3088,
(FAB) m/z 396 (M þ 1)þ.
2945, 1699, 1584, 1541, 1377, 1012, 734 cmꢁ1 1H NMR (DMSO-d6)
;
5.1.5.11. 1-(4-Methoxyphenyl)-3-(3-phenyl-2-thioxo-2,3-dihydrothia-
d 1.17 (t, 3H, CH3), 2.68 (q, 2H, CH2), 3.89 (s, 3H, OCH3), 7.34–7.79 (m,
zolo[4,5-d]pyrimidin-7-yl)thiourea (13a). IR (KBr) ymax 3360, 3308,
4H, ArH), 8.91 (s, 1H, CH pyrimidine), 9.01 (br s, 1H, NH), 10.33 (s,
3200, 1605, 1360, 1170 cmꢁ1
;
1H NMR (DMSO-d6)
d
3.26 (s, 3H,
1H, NH); MS (FAB) m/z 362 (M þ 1)þ.
OCH3), 6.35–7.01 (m, 9H, ArH), 7.66 (s, 1H, CH pyrimidine), 8.22 (s,
1H, NH), 8.95 (s, 1H, NH); MS (FAB) m/z 426 (M þ 1)þ.
5.1.5.22. 1-(3-Ethyl-2-thioxo-2,3-dihydrothiazolo[4,5-d]pyrimidin-7-
yl)-3-(2-fluorophenyl) urea (6b). IR (KBr) ymax 3220, 3110, 2983,
5.1.5.12. 1-(2-Methoxyphenyl)-3-(3-phenyl-2-thioxo-2,3-dihydrothia-
2894,1680, 1350,1050 cmꢁ1; 1H NMR (DMSO-d6)
d 1.22 (t, 3H, CH3),
zolo[4,5-d]pyrimidin-7-yl)thiourea (14a). IR (KBr) ymax 3440, 3325,
1.46 (q, 2H, CH2), 6.72 (1H, d, J ¼ 7.9 Hz), 7.08 (2H, d, J ¼ 8.2 Hz), 7.51
(1H, dd, J ¼ 1.6, 8.3 Hz), 7.76 (s, 1H, CH pyrimidine), 7.95 (s, 1H, NH),
9.81 (s, 1H, NH); MS (FAB) m/z 350 (M þ 1)þ.
3200, 1585, 1360, 1170 cmꢁ1 1H NMR (DMSO-d6)
; d 3.94 (s, 3H,
OCH3), 6.85–7.81 (m, 9H, ArH), 8.79 (s, 1H, CH pyrimidine), 9.5 (s,
1H, NH), 10.91 (s, 1H, NH); MS (FAB) m/z 426 (M þ 1)þ.
5.1.5.23. 1-(3-Ethyl-2-thioxo-2,3-dihydrothiazolo[4,5-d]pyrimidin-7-
yl)-3-(4-fluorophenyl) urea (7b). IR (KBr) ymax 3250, 3120, 3100,
5.1.5.13. 1-(2-Fluorophenyl)-3-(3-phenyl-2-thioxo-2,3-dihydrothiazolo-
[4,5-d]pyrimidin-7-yl) thiourea (15a). IR (KBr) ymax 3313, 3206,
3080,1245,1350, 1070 cmꢁ1; 1H NMR (DMSO-d6)
d 1.39 (t, 3H, CH3),
3081, 1691, 1587, 1480, 1364, 1276, 1218, 953, 820 cmꢁ1
;
1H NMR
1.74 (q, 2H, CH2), 6.97 (2H, d, J ¼ 7.7 Hz), 7.88 (1H, d, J ¼ 7.6 Hz), 8.32
(s, 1H, CH pyrimidine), 9.95 (br s, 1H, NH), 11.25 (s, 1H, NH); MS
(FAB) m/z 350 (M þ 1)þ.
(DMSO-d6)
d
6.74 (d, 1H, J ¼ 7.14 Hz), 6.96 (d, 1H, J ¼ 7.22 Hz), 7.48–
7.85 (m, 7H, ArH), 8.84 (s, 1H, CH pyrimidine), 10.52 (s, 1H, NH),
11.23 (s, 1H, NH); MS (FAB) m/z 414 (M þ 1)þ.
5.1.5.24. 1-(2-Chlorophenyl)-3-(3-ethyl-2-thioxo-2,3-dihydrothiazolo-
5.1.5.14. 1-(4-Fluorophenyl)-3-(3-phenyl-2-thioxo-2,3-dihydrothiazolo-
[4,5-d]pyrimidin-7-yl) urea (8b). IR (KBr) ymax 3367, 3288, 3195,
[4,5-d]pyrimidin-7-yl) thiourea (16a). IR (KBr) ymax 3300, 3190,
3071, 2962, 1689, 1585–1277, 1375, 1018, 800 cmꢁ1 1H NMR
;
3011, 1590, 1360, 1220, 687 cmꢁ1
;
1H NMR (DMSO-d6)
d
7.42–7.91
(DMSO-d6) d 1.01 (t, 3H, CH3), 1.8 (q, 2H, CH2), 6.64–7.62 (m, 4H,
(m, 9H, ArH), 9.15 (s, 1H, CH pyrimidine), 10.32 (br s, 1H, NH), 10.88
ArH), 8.93 (s,1H, CH pyrimidine),10.68 (s,1H, NH),11.56 (s,1H, NH);
(s, 1H, NH); MS (FAB) m/z 414 (M þ 1)þ.
MS (FAB) m/z 366 (M þ 1)þ.
5.1.5.15. 1-(2-Chlorophenyl)-3-(3-phenyl-2-thioxo-2,3-dihydrothiazolo-
5.1.5.25. 1-(4-Chlorophenyl)-3-(3-ethyl-2-thioxo-2,3-dihydrothiazolo-
[4,5-d]pyrimidin-7-yl) thiourea (17a). IR (KBr) ymax 3235, 3161,
[4,5-d]pyrimidin-7-yl) urea (9b). IR (KBr) ymax 3356, 3329, 3189,
2992, 1621, 1509, 682 cmꢁ1; 1H NMR (DMSO-d6)
d
7.27–7.88 (m, 9H,
3045, 1702, 1541, 1266, 1154, 834, cmꢁ1; 1H NMR (DMSO-d6)
d 1.62
ArH), 8.93 (s, 1H, CH pyrimidine), 9.31 (s, 1H, NH), 9.86 (br s, 1H,
(t, 3H, CH3), 1.95 (q, 2H, CH2), 6.44–7.12 (m, 4H, ArH), 7.96 (s, 1H, CH
pyrimidine), 8.30 (s, 1H, NH), 10.93 (s, 1H, NH); MS (FAB) m/z 366
(M þ 1)þ.
NH); MS (FAB) m/z 431 (M þ 1)þ.
5.1.5.16. 1-(4-Chlorophenyl)-3-(3-phenyl-2-thioxo-2,3-dihydrothiazolo-
[4,5-d]pyrimidin-7-yl) thiourea (18a). IR (KBr) ymax 3221, 3023,
5.1.5.26. 1-(3-Ethyl-2-thioxo-2,3-dihydrothiazolo[4,5-d]pyrimidin-7-
yl)-3-(2-nitrophenyl) urea (10b). IR(KBr)ymax 3317, 3238,3136,1699,
2998, 1614, 1522, 691 cmꢁ1; 1H NMR (DMSO-d6)
d 7.71–7.85 (m, 9H,
ArH), 9.01 (s, 1H, CH pyrimidine), 9.55 (s, 1H, NH), 10.22 (s, 1H, NH);
1580, 1540, 1287, 1150, 756 cmꢁ1; 1H NMR (DMSO-d6)
d 1.27 (t, 3H,
MS (FAB) m/z 431 (M þ 1)þ.
CH3), 2.3 (q, 2H, CH2), 6.86–7.72 (m, 4H, ArH), 7.89 (1H, d, J ¼ 7.5 Hz),
9.79 (s, 1H, NH), 11.85 (s, 1H, NH); MS (FAB) m/z 377 (M þ 1)þ.
5.1.5.17. 1-(2-Nitrophenyl)-3-(3-phenyl-2-thioxo-2,3-dihydrothiazolo-
[4,5-d]pyrimidin-7-yl) thiourea (19a). IR (KBr) ymax 3288, 3219,
5.1.5.27. 1-(3-Ethyl-2-thioxo-2,3-dihydrothiazolo[4,5-d]pyrimidin-7-
yl)-3-(4-nitrophenyl) urea (11b). IR (KBr) ymax 3330, 3260, 3150,
3137, 3009, 1617, 1511, 682 cmꢁ1 1H NMR (DMSO-d6)
; d 7.22–8.09

3896
F. Azam et al. / European Journal of Medicinal Chemistry 44 (2009) 3889–3897
3150, 1251, 1350, 1071 cmꢁ1
;
1H NMR (DMSO-d6)
d
1.67 (t,
a maximum of 300 s, was defined as the descent time. The sum of
the descent time values measured every 30 min during the 3 h after
haloperidol or vehicle was defined as the cumulative descent time
(CDT [3 h]). The mean CDTs measured in animals treated by the
vehicle in which haloperidol was dissolved were subtracted from
the mean CDTs recorded in mice treated with haloperidol. This
difference was taken as 100% of catalepsy, and served as a reference
value to calculate the percent inhibition of drugs on catalepsy
intensity.
3H, CH3), 2.94 (q, 2H, CH2), 7.22–8.03 (m, 5H, ArH þ CH
pyrimidine), 10.37 (br s, 1H, NH), 11.34 (s, 1H, NH); MS (FAB)
m/z 377 (M þ 1)þ.
5.1.5.28. 1-(3-Ethyl-2-thioxo-2,3-dihydrothiazolo[4,5-d]pyrimidin-7-
yl)-3-phenylthiourea (12b). IR (KBr) ymax 3298, 3182, 3076, 2960,
1692, 1580, 1363, 1336, 792, 752 cmꢁ1 1H NMR (DMSO-d6)
; d 1.85
(t, 3H, CH3), 3.22 (q, 2H, CH2), 6.98–8.19 (m, 6H, ArH þ CH
pyrimidine), 9.78 (s, 1H, NH), 10.59 (s, 1H, NH); MS (FAB) m/z 348
(M þ 1)þ.
5.3. Biochemical evaluation
5.1.5.29. 1-(3-Ethyl-2-thioxo-2,3-dihydrothiazolo[4,5-d]pyrimidin-7-
yl)-3-(4-methoxyphenyl) thiourea (13b). IR (KBr) ymax 3372, 3205,
Mice were sacrificed by decapitation 3 h after the last injection.
The brains were quickly removed and were washed twice with ice-
cold saline solution, placed into glass bottles, labeled, and stored in
a deep freezer (ꢁ30 ꢂC) until processing (maximum 10 h). Tissues
were homogenized in four volumes of ice-cold Tris–HCl buffer
(50 mM, pH 7.4) using a glass Teflon homogenizer (Ultra Turrax IKA
T18 Basic, USA) after cutting up the brain into small pieces with
scissors (for 2 min at 5000 rpm). Malondialdehyde (MDA) and
protein levels were carried out at this stage. The homogenate was
then centrifuged at 10,500 ꢄ g for 20 min to remove nuclear debris.
Clear supernatant fluid was taken and GSH-Px activity was carried
out in this stage. The supernatant solution was then extracted with
an equal volume of an ethanol/chloroform mixture (5:3, v/v). After
centrifugation at 5000 ꢄ g for 30 min, the clear upper layer (the
ethanol phase) was taken and used in the SOD activity. All prepa-
ration procedures were performed at þ4 ꢂC.
3093, 2928, 1703, 1607, 1542, 1236, 806 cmꢁ1 1H NMR (DMSO-d6)
;
d
1.13 (t, 3H, CH3), 2.28 (q, 2H, CH2), 3.37 (s, 3H, OCH3), 6.83–7.61 (m,
4H, ArH), 7.93 (s, 1H, CH pyrimidine), 8.42 (s, 1H, NH), 10.61 (s, 1H,
NH); MS (FAB) m/z 378 (M þ 1)þ.
5.1.5.30. 1-(3-Ethyl-2-thioxo-2,3-dihydrothiazolo[4,5-d]pyrimidin-7-
yl)-3-(2-methoxyphenyl) thiourea (14b). IR (KBr) ymax 3378, 3200,
3082, 2974, 1698, 1578, 1362, 1270, 1018, 804, 748 cmꢁ1 1H NMR
;
(DMSO-d6)
d 1.27 (t, 3H, CH3), 2.74 (q, 2H, CH2), 3.96 (s, 3H, OCH3),
6.89–7.76 (m, 5H, ArH þ CH pyrimidine), 9.36 (s, 1H, NH), 10.65
(s, 1H, NH); MS (FAB) m/z 378 (M þ 1)þ.
5.2. Pharmacological evaluation
All the compounds were screened for antiparkinsonian activity
in haloperidol-induced catalepsy test in mice. The results obtained
are summarized in Table 2.
5.3.1. LPO assay
The extent of LPO in brain homogenate was determined by
measuring the release of thiobarbituric acid reactive substance
(TBARS) in terms of MDA equivalent using a molar extinction
coefficient of 1.56 ꢄ 105/min/cm as described by Ohkawa et al. [62].
Briefly, the homogenate was centrifuged at 3000 ꢄ g for 15 min and
supernatant was used for assay. Samples of 0.1 ml homogenate
were mixed with 0.2 ml of 8.1% SDS, 1.5 ml 20% glacial acetic acid
and 1.5 ml of 0.8% thiobarbituric acid (TBA). Following these addi-
tions, tubes were mixed and heated at 95 ꢂC for 1 h on a water bath
and cooled under tap water before mixing 1 ml of distilled water
and 5 ml mixture of n-butanol and pyridine (15:1). The mixture was
centrifuged at 2200 g for 10 min. The amount of MDA formed was
measured by the absorbance of upper organic layer at a wavelength
of 532 nm in Perkin Elmer spectrophotometer using appropriate
controls. The results are expressed as nmol MDA/mg protein.
5.2.1. Animals and drugs
Adult male pathogen-free Swiss albino mice weighing 18–25 g
were used. All animal experimentation was conducted in accor-
dance with the Animal Ethics Committee of the Institute. The
procedures adhered to the NIH Guidelines for the Care and Use of
Laboratory Animals. Levodopa (Sigma–Aldrich) was injected i.p. in
dose of 100 mg/kg. Haloperidol (Sigma–Aldrich) was administered
in a dose of 5 mg/kg i.p. Synthesized compounds 3a–20a and 3b–
14b were administered at 100 mg/kg i.p. All the drugs and
synthesized compounds were suspended in 0.5% gum acacia in
redistilled water and administered at a volume of 0.1 ml/100 g.
5.2.2. Haloperidol-induced catalepsy test
Haloperidol-induced catalepsy was measured with the standard
bar test [40], in a wooden chamber (length, 23 cm; width, 10.5 cm;
height, 9 cm) with a horizontal metal bar (diameter, 0.4 cm; length,
10.5 cm) fixed at 9 cm above the floor, and at 4 cm from the back of
the box. All experiments were carried out between 8:00 and
15:30 h in a room with controlled temperature (23 ꢃ1 ꢂC), and
light intensity of 20 lux. Five mice were taken in each group.
Control animals received 0.5% gum acacia in redistilled water.
Synthesized compounds and levodopa were injected intraperito-
neally at 100 mg/kg, 30 min prior to haloperidol injection. Animals
were used only once. Catalepsy was measured every 30 min during
the whole session that lasted 3 h after haloperidol injection. To
assess whether the repeated handling of animals could have any
influence on catalepsy intensity over time [40], the bar test was
performed in groups of mice that were injected only with the
vehicle in which haloperidol was dissolved.
5.3.2. GSH determination
The amount of GSH in brain was measured according to the
method of Sedlak and Lindsay [63]. Briefly, brain tissue was
deproteinized with an equal volume of 10% TCA and was allowed to
stand at 4 ꢂC for 2 h. The contents were centrifuged at 2000 g for
15 min. The supernatant was added to 2 ml of 0.4 M Tris buffer (pH
8.9) containing 0.02 M EDTA (pH 8.9) followed by the addition of
0.01 M DTNB {5,50-dithiobis(2-nitrobenzoic acid)}. Finally, the
mixture was diluted with 0.5 ml of distilled water, to make the total
mixture to 3 ml and absorbance was read in a spectrophotometer at
412 nm and results are expressed as mg GSH/gm tissue.
5.3.3. SOD activity determination
Cu,Zn-SOD activity was determined according to the method of
Sun et al. [64]. In this method, a xanthine–xanthine oxidase
complex produces superoxide radicals, which react with nitro-
bluetetrazolium (NBT) to form the farmazan compound. In brief,
a reactive was prepared with 0.1 mM of xanthine, 0.1 mM of EDTA,
To measure catalepsy, the mouse was gently lifted until its
forepaws firmly grasped the metal bar. Then, the mouse body was
released and simultaneously a stopwatch was started. The time
elapsed until the animal released both forepaws from the bar, up to
50 mg of bovine serum albumin, 25 mM of NBT and 40 mM of

F. Azam et al. / European Journal of Medicinal Chemistry 44 (2009) 3889–3897
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