Design and synthesis of (4E)-4-(4-substitutedbenzylideneamino)-3- substituted-2,3-dihydro-2-thioxothiazole-5-carbonitrile as novel A2A receptor antagonists

Article abstract of DOI:10.1016/j.bmc.2013.07.005

Novel 2-thioxothiazole derivatives (6-19) as potential adenosine A _2A receptor (A_2AR) antagonists were synthesized. The strong interaction of the compounds (6-19) with A_2AR in docking study was confirmed by high binding af

Full text of DOI:10.1016/j.bmc.2013.07.005

Bioorganic & Medicinal Chemistry 21 (2013) 6077–6083
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Bioorganic & Medicinal Chemistry
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Design and synthesis of (4E)-4-(4-substitutedbenzylideneamino)-3-
substituted-2,3-dihydro-2-thioxothiazole-5-carbonitrile as novel A2A
receptor antagonists
Chandra Bhushan Mishra, Dimpy Sharma, Amresh Prakash, Namrata Kumari, Nitin Kumar,
⇑
Pratibha Mehta Luthra
Neuropharmaceutical Laboratory, Dr. B.R. Ambedkar Center for Biomedical Research, University of Delhi, North Campus, Mall Road, Delhi 110 007, India
a r t i c l e i n f o
a b s t r a c t
Article history:
Available online 13 July 2013
Novel 2-thioxothiazole derivatives (6–19) as potential adenosine A2A receptor (A2AR) antagonists were
synthesized. The strong interaction of the compounds (6–19) with A2AR in docking study was confirmed
by high binding affinity with human A2AR expressed in HEK293T cells using radioligand-binding assay.
The compound 19 demonstrated very high selectivity for A2AR as compared to standard A2AR antagonist
SCH58261. Decrease in A2AR-coupled release of endogenous cAMP in treated HEK293T cells demon-
strated in vitro A2AR antagonist potential of the compound 19. Attenuation in haloperidol-induced
impairment (catalepsy) in Swiss albino male mice pre-treated with compound 19 is evocative to explore
its prospective in therapy of PD.
Keywords:
A
2AR antagonist
Radioligand-binding assay
cAMP assay
Haloperidol
2-Thioxothiazole
Ó 2013 Elsevier Ltd. All rights reserved.
1. Introduction
carried the synthesis of the substituted thioxo-thiazoles as novel
A2AR antagonists.
Adenosine, an endogenous ligand interacts with adenosine
receptor to perform variety of physiological and pathological con-
ditions.1 Four mammalian adenosine receptor subtypes (A1R, A2AR,
Thiazole ring imparts vital role in metabolic pathways and
emerged as important synthon to generate variety of NCEs (new
chemical entities) due to highly reactive acidic C-2. Diverse modi-
fications of thiazole ring at various positions led to variety of novel
compounds, with wide spectrum of biological activities such as
antioxidant,11 anti inflammatory,11 and neuro protective.12 PI3
kinase modulatory thiazolo[4,5-g]dihydroindazoles, HIV-integrase
inhibitory thiazolo[5,4-b]pyrimidine-5(4H)-ones and GSK-3 inhib-
itory thiazole[5,4-f]quinazolin-9-ones13 revealed therapeutic
potential in various diseases. Riluzole, a novel neuroprotective
drug, approved for the treatment of amyotrophic lateral scleroses
possesses aminothiazole moiety.14 Hofman Le Roche has devel-
oped benzothiazole derivative ASN115 (tozadent) as potent A2AR
antagonist, which is in phase II clinical trials now.15 Previously,
we have reported bicyclic thiazolo-pyrimidines and tricyclic
thiazolo-triazolo-pyrimides as potent A2AR antagonists.16–19 In
the course of development, it was found that thioxothiazole moiety
of bicyclic thiazolo-pyrimidine and tricyclic triazolo-thiazolo-
pyrimidine was actively involved in interaction with receptor. Ring
sulfur of 2-thioxo thaizole moiety formed key interaction with
amino acid N253 at active site of A2AR. Therefore, novel monocyclic
compounds possessing 2-thioxo thiazole moiety were developed
as novel A2AR antagonists. The criterion of terminal hydrophoric
cores with polar central features from pharmacophore modeling
studies was incorporated in the design of novel monocyclic
thiazole derivatives. (4E)-4-(4-Substitutedbenzylideneamino)-3-
A
2BR, and A3R), belonging to GPCR superfamily, have been charac-
terized.2,3 Adenosine A2A receptors (A2AR) have emerged as prom-
ising nondopaminergic target to control the motor impairment
effect of PD.4 A2AR antagonists reversed Parkinsonian motor defi-
cits in preclinical models without inducing or exacerbating dyski-
nesias in nonhuman primate models5 due to blockade of A2A
R
co-expressed with D2R in striatopallidal neurons, which inhibited
the release of GABA in the globus pallidus, ultimately leading to
enhanced motor function through indirect motor pathway of the
basal ganglia.6
Several potent and selective A2AR antagonists of structural var-
iability have been synthesized7,8 (Fig. 1). However, xanthine com-
pounds such as KW6002 were unsuccessful in clinical trials,9 and
nonxanthine compounds such as SCH58261 suffered from lower
selectivity, solubility and pharmacokinetic profile.10 To overcome
the existing drawbacks, the development of novel A2A receptor
antagonists is essentially desired. Our research group has been
intensely engaged to the development of potent and selective
A
2AR antagonist. In the present paper, we have designed and
⇑
Corresponding author. Tel.: +91 11 27662778; fax: +91 11 27666248.
E-mail address: pmlsci@yahoo.com (P.M. Luthra).
0968-0896/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmc.2013.07.005

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C. B. Mishra et al. / Bioorg. Med. Chem. 21 (2013) 6077–6083
NH2
NH2
N
N
N
N
N
N
HO
N
N
O
N
O
N
N
N
H
ZM241385
SCH58261
Br
N
N
S
CN
S
19
Figure 1. Chemical structure of standard A2AR antagonist and synthesized compound 19.
substituted-2,3-dihydro-2-thioxothiazole-5-carbonitrile
(6–19)
2.2. Docking analysis
The interaction of thiazole derivatives 6–19 and known A2A
were synthesized. N3-hydrophobic and electron rich polar feature
of thiazole ring were used as starting material to incorporate N-4
hydrophobic terminal for optimal interaction at active site of A2AR.
In vitro radioligand-binding assay was carried using mem-
branes isolated from transfected HEK293T cells with human A2AR
to evaluate the binding affinity and selectivity of the compounds
(6–19), the results were comparable to docking analysis. Decrease
in endogenous cAMP concentrations in treated HEK293T cells and
restoration of locomotor activity in haloperidol-induced mice
model was determined for the compound 19, which possessed
maximum binding affinity and selectivity in vitro study.
R
antagonist ZM241385 was studied with recently solved X-ray crys-
tal structure of human A2AR (PDB ID: 3EML). The A2AR (3EML)
binding cavity is comprised of TM2 (A63, I66), TM3 (A81, V84,
L85), ECL2 (F168, E169), ECL3 (H 264, W268), TM5 (M174, M177)
and TM6 (W246, L249, N253). Docking simulation results showed
that the synthesized (4E)-4-(4-substitutedbenzylideneamino)-
3-substituted-2,3-dihydro-2-thioxothiazole-5-carbonitrile deriva-
tives (6–19) and ZM241385 shared a similar binding motif inside
the transmembrane (TM) region and extracellular loops of the
human A2AR similar to the co-crystallized ZM241385. The binding
interaction of (4E)-4-(4-bromobenzylideneamino)-N3-phenyl-2,3-
dihydro-2-thioxothiazole-5-carbonitrile (19 as one of the repre-
sentatives) showed that the sulfur atom of thiazole ring was
oriented towards the N253, M177 and V178 (Fig. 2). The involve-
ment of exocyclic sulfur to form H-bond interaction with amide
moiety of N253 was analogous to N-1 nitrogen at 7-position of
ZM241385. Furthermore, the key receptor residues F168, E169
from ECL2 formed aromatic stacking and hydrophobic polar inter-
action with N3 position substituent of 2-thioxo-thiazole deriva-
tives 6–19. Overall, thiazole scaffold possessed orientation and
contacts almost similar to ZM241385. The bromobenzylidene
(hydrophobic group) of 19 was found to be tilted and deep fixed
in lower hydrophobic domain of A2AR. Nitrogen of carbonitrile
and bromobenzylideneamine was involved in polar interaction
2. Results and discussion
2.1. Synthesis
The synthesis of compounds 1–5 was carried according to pre-
viously reported method.16 The compounds (4E)-4-(4-substitut-
edbenzylideneamino)-3-substituted-2,3-dihydro-2-thioxothiaz-
ole-5-carbonitrile (6–19) were prepared using equimolar mixture
of 4-amino-N3-substituted-2, 3-dihydro-2-thioxothiazole-5-car-
bonitrile (1–5) and p-substituted benzaldehyde with small amount
of AlCl3 in chloroform. Crude product was purified by column
chromatography using petroleum ether and ethyl acetate (2:1) as
solvent. The compounds (6–19) were characterized by IR, NMR
and mass spectroscopy (Scheme 1).
R1
R
NH2
CN
CN
CN
A
R
B
N
N
N
R-NCS+ S+
S
S
S
CN
S
1-5
6-19
6.R= Ethyl, R1= F
7. R= Propyl, R1=F
8. R=Butyl, R1= F
11.R= Ethyl , R1= Cl
12. R=Propyl, R1= Cl
13.R=Butyl, R1= Cl
16. R=Propyl, R1= Br
17. R=Butyl, R1= Br
18. Phenyl ethyl, R1= Br
19. R=Phenyl, R1= Br
9.R= Phenyl ethyl, R1= F 14.R= Phenyl , R1= Cl
10. R= Phenyl, R1= F 15. R=Ethyl, R1= Br
Scheme 1. Reagents and conditions: (A) TEA, DMF, RT. (B) Substituted benzaldehyde, chloroform, AlCl3, RT.

C. B. Mishra et al. / Bioorg. Med. Chem. 21 (2013) 6077–6083
6079
Figure 2. Docking analysis of ZM241385 and compound 19 with crystal structure of A2AR (cartoon view at PyMOL). (a) Standard antagonist ZM241385. (b) The compound 19
(polar interactions are represented with black and green dotted lines). Yellow dotes represented the water molecules present in A2AR active site.
Table 1
with N181 and H250, respectively. The N3-phenyl ring exhibited
In-vitro radioligand binding assay for the compounds (6–19)
p–p hydrophobic interaction with F168.
R1
2.3. Structure–activity relationship
Radioligand binding study of (4E)-4-(4-fluorobenzylideneamino)-
N3-substituted-2,3-dihydro-2-thioxothiazole-5-carbonitrile series
(6–10) revealed that N3-propyl, N3-butyl and N3-ethylphenyl deriv-
ativespossessed selectivity for A2AR, however, N3-ethyl derivative(6)
was highly selective for A1 receptor (A1/A2A = 0.000053) and N3-phe-
nyl derivative (10) showed strong affinity with both A2AR (Ki = 0.0047
nm)andA1R(Ki = 0.00063 nm) with someselectivity for A2AR receptor
(A1/A2A = 0.13). Overall order of selectivity profile of the compounds
6–10 for A2AR was C3H7 (A1/A2A = 3393) > C4H9 (A1/A2A = 618) > CH2
R
N
N
S
S
CN
No R group
R1 group Ki A2A (nM) Ki A1 (nM)
A1/A2A
0.000053
3393.3
618.51
336.81
0.13
6
7
8
9
Ethyl
Propyl
Butyl
Fluro
Fluro
Fluro
10.98
0.03
0.027
2.64
0.0047
1.44
0.00059
101.8
16.7
889.2
0.00063
19.82
2.32
24630.0
412.7
0.00074
606.7
0.10
0.25
186.0
Phenyl ethyl Fluro
CH2Ph (A1/A2A = 336) > Ph (A1/A2A = 0.13) > C2H5 (A1/A2A
=
10 Phenyl
11 Ethyl
12 Propyl
13 Butyl
14 Phenyl
15 Ethyl
16 Propyl
17 Butyl
Fluro
0.000053). In (4E)-4-(4-chlorobenzylideneamino)-N3-substituted-
2,3-dihydro-2-thioxothiazole-5-carbonitrile series (11–14), the
N3-butyl (A1/A2AR = 2565) and N3-phenyl (A1/A2AR = 2947) deriva-
tives were highly selective for A2AR. Moreover, the selectivity order
of N3-ethyl (11) and N3-propyl (12) derivatives was found to be
reversed. N-3-propyl derivative (12) exhibited more binding affinity
for A1R (Ki = 2 nM) as compared to A2AR (Ki = 55 nM; A1/A2A
R = 0.042), however, ethyl derivative (11) showed more affinity
(Ki = 1.44 nm; A1/A2A = 13.76) for A2AR (Table 1). N-butyl (13, Ki =
Chloro
Chloro
Chloro
Chloro
Bromo
Bromo
Bromo
13.76
0.042
54.8
9.60
0.14
0.01
2565.62
2947.85
0.074
337.05
0.013
1.8
7.61
0.067
0.0042
18 Phenyl ethyl Bromo
19 Phenyl Bromo
3.73
44285.71
9.6 nM, A1/A2A = 2565) and N-phenyl (14, Ki
= 0.14 nM,
A1/A2A = 2947) showed high selectivity and affinity for A2AR over
A1R. (4E)-4-(4-Bromobenzylideneamino)-N3-substituted-2,3-dihy-
dro-2-thioxothiazole-5-carbonitrile series (15–19), the order of bind-
ing affinity is Ph (19, Ki = 0.0042 nM) > C2H5 (15, Ki = 0.01 nM)
> CH2CH2Ph (18, Ki = 0.067 nM) > C3H7 (16, Ki = 1.8 nM) > C4H9 (17,
Ki = 7.61), however selectivity profile for A2AR is 19 (A1/A2A = 44285)
> 16 (A1/A2A = 337) > 18 (A1/A2A = 4) > 15 (A1/A2A = 0.074) > 17
(A1/A2A = 0.13).
The results showed that amongst the (4E)-4-(4-fluorobenzy-
lideneamino)-N3-substituted-2,3-dihydro-2-thioxothiazole-5-car-
bonitrile (6–10), (4E)-4-(4-chlorobenzylideneamino)-3-substituted-2,
3-dihydro-2-thioxothiazole-5-carbonitrile (11–14), and (4E)-4-(4-
bromobenzylideneamino)-3-substituted-2,3-dihydro-2-thioxo-
thiazole-5-carbonitrile (15–19), the fluoro substitution contributes
only -I effect, chloro substitution imparts -I and mesomeric effect
and bromo substitution exhibit mostly mesomeric effect. The opti-
mum carbon length at N terminal is propyl if -I effect prevails
(compound 7), butyl or phenyl if -I and mesomeric effects operate
(compounds 13 and 14), however, only mesomeric effect in N3-
phenyl (19) makes most selective compound in the series. The
comparison of N3-phenyl substituent in fluoro (10), chloro (14)
and bromo (19) series showed that the change from -I effect of
the fluoro (compound 10, Ki = 0.0047 nm, A1/A2A = 0.13) followed
by -I effect and mesomeric effect of chloro (14, Ki = 0.14 nM, A1/
A2A = 2947) to mesomeric effect of bromo (19, Ki = 0.0042 nM,
A1/A2A = 44285) essentially induced the modulation in the confor-
mation of adenosine receptors. Moreover, prevalent mesomeric ef-
fect did not affect the affinity with A2AR, but decreased the affinity
with A1 receptor (106 times) from compounds 6–19 (Table 1, such
as 10, Ki = 0.00063 nm; 14, Ki = 412 nm; 19, Ki = 186). In the total 14
compounds, four compounds (7, 13, 14, and 19) possessed both

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high binding affinity and selectivity, though 19 was most selective
compound for A2AR in this series. It has been reported that the
communication between ligand and receptor is based on electro-
static interaction, although a stacking interaction operating in the
molecular recognition event has been hypothesized. The fluoro-
substituent has been reported to alter the electronic distribution
in the substrate and changes the conformation without affecting
the main values. The size of the substituent modulates the molec-
ular volume determined on the C(aromatic)-substituent bond
length of the substituent, that is, chloro (1.747 Å) versus bromo
(1.899 Å), resulting in increased steric interaction and therefore
an increased energy barrier.20 In agreement of reported studies,
our work has demonstrated that the changes in the electron distri-
bution significantly influenced the electrostatic interaction to in-
duce conformational changes in the adenosine receptors.
(0.75 0.144) was significantly higher as compared to
SCH58261 (0.65 0.125).
2.5. Conclusion
Novel 2-thioxo thiazole derivatives (6–19) demonstrated their
potential as potent and selective A2AR antagonists. The compounds
(6–19) shared active site binding pocket identical to ZM241385 in
docking study. In vitro radioligand binding results showed that the
electrostatic interactions largely influenced the binding affinity.
However, (4E)-4-(4-bromobenzylideneamino)-3-phenyl-2,3-dihy-
dro-2-thioxothiazole-5-carbonitrile 19 displayed both higher bind-
ing affinity and selectivity for A2AR in comparison to standard A2A
R
antagonist SCH58261, antagonized the A2AR-coupled release of
endogenous cAMP from HEK293T cells and restored haloperidol-
induced hypo-locomotors behavior (catalepsy) in vivo to produce
motor stimulant effects, further accentuate the potential of 2-thi-
oxo-thiazoles (6–19) in the treatment of PD.
Most active and selective compound 19 was selected for cAMP
functional assay. In functional assay, binding of ligands to A2ARs
promotes GPCR mediated conformational change via Gas or Gai/o
subfamily to modulate the activity of adenylate cyclase and alter
the concentration of cAMP. An antagonist generally interacts with
3. Experimental methodology
A
2AR via Gai/o subfamily to inhibit the activity of adenylate cyclase
leading to decrease in the cAMP concentration. In this study, NECA
stimulated HEK293T cells treated with compound 19 displayed
reduction in cAMP concentration (0.56 pmol/ml) as compared to
NECA (cAMP concentrations 0.65 pmol/ml). The results were com-
parable to NECA stimulated HEK293T cells treated with SCH58261
(0.55 pmol/ml).
The compounds were characterized by TLC, IR, 1H NMR, 13C
NMR, and mass spectroscopy. TLC was carried out on commercially
available TLC plates (Silica Gel 60 F254, Merck, Germany). IR spec-
tra were performed on Spectrum BX FTIR, PerkinElmer. NMR spec-
tra were recorded on AC Bruker 400 MHz spectrometer in CDCl3 or
DMSO. Mass spectra were recorded on a QSTAR XL LC–MS–MS, Ap-
plied Biosystem. Melting points were determined using model
KSPII, KRUSS, Germany. Elemental analysis was performed on
Ementar analysen systeme. Column chromatography purifications
were performed using silica gel (100–200 mesh, Merck).
2.4. Haloperidol induced catalepsy
Four different doses of compound 19 (5, 10 15 and 20 mg/kg)
were considered to study haloperidol (2.5 mg/kg) induced cata-
lepsy in Swiss albino male mice. The haloperidol (2.5 mg/kg) trea-
ted mice showed high catalepsy score of 2.875 0.125 after 120
min. SCH 58261 pre-treated mice exhibited significant recovery in
3.1. General procedure for synthesis of 4-amino-N3-
substituted-2-thioxo-1,3-thiazole-5-carbonitrile (1–5)
haloperidol-induced catalepsy (0.625
0.125) after 120 min.
The compounds 1–5 were synthesized according to reported
method of Luthra et al.16 Briefly, an equimolar mixture of N-substi-
tuted isothiocyanates, malononitrile and sulfur powder in dimeth-
ylformamide was stirred in ice bath, added triethylamine drop-
wise after 15 min, and the stirring was continued for 4 h. The reac-
tion mixture was poured in water, the obtained precipitate was
crystallized from absolute ethanol to give 4-amino-3-substituted-
2-thioxo-2,3-dihydro-thiazole-5-carbonitrile derivatives (1–5).
Compound 19 pre-treated mice at the dose of 5, 10, 15, 20 mg/
kg exhibited catalepsy score of 0.75 0.144, respectively, after
120 min (Fig. 3). The saline treated and 1% acacia in saline treated
(control) mice as well as mice treated with compound 19 alone
exhibited zero catalepsy score up to 120 min. The results demon-
strated that the attenuation of catalepsy score in compound 19
pre-treated haloperidol-induced mice at the dose of 10 mg/kg
4
3
2
1
Saline
1%Acacia
Haloperidol(2.5mg/kg)
SCH58261(10mg/Kg)
ACPT(5mg/kg)
**
ACPT(10mg/kg)
ACPT(15mg/kg)
ACPT(20mg/kg)
**
0
0
5
0
5
0
0
1
3
4
6
9
20
1
Time(minutes)
Figure 3. Effect of (4E)-4-(4-bromobenzylideneamino)-3-phenyl-2,3-dihydro-2-thioxothiazole-5-carbonitrile at 5, 10, 15 and 20 mg/kg on haloperidol (2.5 mg/kg) induced
catalepsy in mice. Results are given as mean SEM, n = 4 (one-way ANOVA: p 6 0.0001); Kruskal–Wallis test: ⁄⁄p 6 0.0026 vs control).

C. B. Mishra et al. / Bioorg. Med. Chem. 21 (2013) 6077–6083
6081
3.1.1. 4-Amino-N3-ethyl-2-thioxo-1,3-thiazole-5-carbonitrile (1)
Yield: 74%. White solid; mp: 205 °C. IR (KBr) 3308.95, 3227.11,
2973, 2206.84, 1193.14 cmÀ1; 1H NMR (CDCl3): d ppm 1.27–1.32 (t,
J = 7.2 Hz, 3H, CH3), 4.22–4.29 (q, J = 7.2 Hz, 2H, CH2), 6.38 (s, 2H,
NH2). LC–MS (m/z): 185 (M+), 186 (M+1).
3.2.3. (4E)-4-(4-Flurobenzylideneamino)-N3-butyl-2,3-dihydro-
2-thioxothiazole-5-carbonitrile (8)
Yield: 75%; yellow solid; mp: 125–127 °C; IR (KBr): 2965, 2932
(alkyl), 2206 (CN) cmÀ1 1H NMR (400 MHz, CDCl3): d ppm 0.94–
;
0.96 (t, 3H, CH3), 1.38–1.44 (m, 2H, CH2), 1.74–1.78 (m, 2H, CH2),
4.25–4.28 (m, 2H, CH2), 7.25–7.29 (m, 2H, Ar), 8.01–8.04 (d, 2H,
Ar), 8.94 (s, 1H, Ar); 13C NMR (CDCl3): 13.6, 19.9, 29.3, 47.3,
111.9, 116.7, 117.0, 130.1, 132.8, 132.9, 156.4, 165.4, 166.8,
186.0; LC–MS (m/z): 320 (M++1); Analysis calculated for C15H14
FN3S2: C, 56.40; H, 4.42; N, 13.16; S, 20.08; Found: C, 56.51; H,
4.49; N, 13.27; S, 20.17.
3.1.2. 4-Amino-N3-propyl-2-thioxo-1,3-thiazole-5-carbonitrile (2)
Yield: 85%. White solid; mp: 160 °C. IR (KBr): 3319.65, 3234.59,
2205.95, 1198.14 cmÀ1; 1H NMR (CDCl3): d ppm 1.03 (t, J = 7.5, 3H,
CH3), 1.72–1.854 (m, 2H, CH2), 4.10 (t, J = 7.8 Hz, 2H,CH2), 4.83 (s,
2H, NH2); LC–MS (m/z): 199 (M+), 200 (M+1).
3.2.4. (4E)-4-(4-Flurobenzylideneamino)-N3-Phenylethyl-2,3-
dihydro-2-thioxothiazole-5-carbonitrile (9)
3.1.3. 4-Amino-N3-butyl-2-thioxo-1,3-thiazole-5-carbonitrile
(3)
Yield: 78%; yellow solid. mp: 174–176 °C; IR (KBr): 3078 (aro-
Yield: 84%. Brown solid; mp: 155 °C. IR (KBr): 3320.05, 3234.19,
matic), 2206 (CN) cmÀ1 1H NMR (400 MHz, CDCl3): d ppm 3.08–
;
2208.95, 1199.14 cmÀ1 1H NMR (300 MHz, CDCl3): d ppm 0.97 (t,
;
3.12 (t, 2H, CH2), 4.49–4.52 (t, 2H, CH2), 6.99–7.00 (d, 2H, Ar),
7.14–7.28 (m, 5H, Ar), 7.79–7.82 (m, 2H, Ar) 8.04 (s, 1H, Ar); 13C
NMR (CDCl3): 32.4, 48.8, 111.8, 116.5, 116.7, 127.0, 128.7, 129.0,
130.0, 132.6, 132.7, 137.6, 140.6, 156.9, 165.8, 166.6, 185.0. LC–MS
(m/z): 367 (M+), 368 (M++1); Analysis calculated for C19H14FN3S2:
C, 62.10; H, 3.84; N, 11.44; S, 17.45. Found C, 62.22; H, 3.72; N,
11.32; S, 17.52.
J = 7.2 Hz, 3H, CH3), 1.353–1.476 (m, 2H, CH2), 1.66–1.74 (m, 2H,
CH2), 4.16 (t, J = 7.8 Hz, 2H, CH2), 6.120 (s, 2H, NH2); LC–MS
(m/z): 213 (M+), 214 (M+1).
3.1.4. 4-Amino-N3-(phenylethyl)-2-thioxo-1,3-thiazole-5-
carbonitrile (4)
Yield: 75%, yellow solid; mp: 245–246 °C; IR (KBr): 3312, 3188,
2207, 684 cm-1; 1H NMR (300 MHz, CDCl3): d ppm 2.94(t, 2H, CH2),
4.32(t, 2H, CH2), 6.9(s, 2H, Ar), 7.24–7.33(m, 3H, Ar), 7.79(s, 2H,
NH2); LC–MS (m/z): 263 (M+), 264 (M+1).
3.2.5. (4E)-4-(4-Flurobenzylideneamino)-N3-phenyl-2,3-
dihydro-2-thioxothiazole-5-carbonitrile (10)
Yield: 72%; yellow solid; mp: 184–186 °C; IR (KBr): 3075 (aro-
matic), 2208 (CN) cmÀ1 1H NMR (400 MHz, CDCl3): d ppm 7.26–
;
3.1.5. 4-Amino-N3-phenyl-2-thioxo-1,3-thiazole-5-carbonitrile (5)
7.27 (m, 2H, Ar), 7.51–7.56 (m, 7H, Ar), 8.83 (s, 1H, Ar); 13C NMR
(CDCl3): 111.7, 116.5, 116.7, 128.3, 129.5, 130.0, 130.1, 132.7,
132.8, 135.9, 156.9, 165.2, 166.3, 187.4; LC–MS (m/z): 339 (M+),
340 (M++1); Analysis calculated for C17H10FN3S2: C, 60.16; H,
2.97; N, 12.38; S, 18.89. Found C, 60.27; H, 3.07; N, 12.29; S, 18.99.
Yield: 75%. Yellow solid; mp: 286 °C. IR (KBr): 3369, 3230, 2204,
684 cmÀ1 1H NMR (300 MHz, CDCl3): d ppm 5.90 (s, 2H, NH2)
;
7.12–7.47 (m, 5H, Ar); LC–MS (m/z): 233 (M+).
3.2. General procedure for synthesis of (4E)-4-(4-
substitutedbenzylideneamino)-N3-substituted-2,3-dihydro-2-
thioxothiazole-5-carbonitrile (6–19)
3.2.6. (4E)-4-(4-Chlorobenzylideneamino)-N3-ethyl-2,3-
dihydro-2-thioxothiazole-5-carbonitrile (11)
Yield: 68%; yellow solid; mp: 178–180 °C; IR (KBr): 2848, 2922
(alkyl) 2204 (CN) cmÀ1 1H NMR (400 MHz, CDCl3): d ppm 1.32–
;
An equimolar mixture of 4-amino-3-substituted-2,3-dihydro-2-
thioxothiazole-5-carbonitrile (1–5) and p-substituted benzaldehyde
(3.78 g, 27 mmol) with pinch of AlCl3 was stirred in chloroform for
8 h. After completion of the reaction, the solvent was evaporated.
The reaction mixture was dissolved in ethylacetate, washed with
water and dried with anhydrous sodium sulfate. Crude product
was purified by column chromatography using petroleum ether
and ethyl acetate (98:2) as solvent.
1.35 (t, 3H, CH3), 4.31–4.37 (q, 4H, CH2), 7.52–7.57 (d, 2H, Ar),
7.94–7.96 (d, 2H, Ar), 8.96 (s, 1H, Ar); 13C NMR (CDCl3): 12.6,
42.9, 111.8, 116.6, 129.4, 129.8, 131.5, 132.1, 141.1, 156.0, 167.2,
185.9; LC–MS (m/z): 308 (M++1); Analysis calculated for C13H10-
ClN3S2: C, 50.72; H, 3.27; N, 13.65; S, 20.83. Found: C, 50.66; H,
3.32; N, 13.72; S, 20.90.
3.2.7. (4E)-4-(4-Chlorobenzylideneamino)-3-propyl-2,3-
dihydro-2-thioxothiazole-5-carbonitrile (12)
3.2.1. (4E)-4-(4-Flurobenzylideneamino)-N3-ethyl-2,3-dihydro-
2-thioxothiazole-5-carbonitrile (6)
Yield: 72%; yellow solid; mp: 142–144 °C. IR (KBr): 2954,
2974(alkyl), 2211 (CN) cmÀ1 1H NMR (400 MHz, CDCl3) d ppm
;
Yield: 65%; yellow solid; mp: 175–177 °C; IR (KBr): 2978, 2934,
2874 (alkyl) 2211 (CN) cm-1; 1H NMR (400 MHz, CDCl3): d ppm
1.27 (t, 3H, CH3), 4.31–4.37 (q, 4H, CH2), 7.27–7.29 (m, 2H, Ar),
8.02–8.05 (m, 2H, Ar), 8.95 (s, 1H, Ar); 13C NMR (CDCl3): 12.7,
43.0, 112.1, 116.7, 116.9, 130.3, 133.1, 156.6, 165.5, 167.2, 185.9;
LC–MS (m/z): 291 (M+), 292 (M++1). Analysis calculated for C13H10-
FN3S2: C, 53.59; H, 3.46; N, 14.42; S, 22.01; Found: C, 53.48; H,
3.33; N, 14.53; S, 22.17.
0.91–0.99 (t, 3H, CH3), 2.07–2.18 (m, 2H, CH2), 4.21–4.25 (t, 2H,
CH2), 7.25–7.29 (m, 2H, Ar), 8.01–8.03 (m, 2H, Ar), 8.94 (s, 1H,
Ar); 13C NMR (CDCl3): 11.2, 20.7, 49.0, 111.9, 116.7, 130.1, 132.8,
132.9, 141.4, 156.4, 166.8, 186.1; LC–MS (m/z): 321 (M+), 322
(M++1); Analysis calculated for C14H12ClN3S2: C, 52.25; H, 3.76;
N, 13.06; S, 19.93. Found C, 52.36; H, 3.66; N, 13.12; S, 20.09.
3.2.8. (4E)-4-(4-Chlorobenzylideneamino)-N3-butyl-2,3-
dihydro-2-thioxothiazole-5-carbonitrile (13)
3.2.2. (4E)-4-(4-Flurobenzylideneamino)-N3-propyl-2,3-
dihydro-2-thioxothiazole-5-carbonitrile (7)
Yield: 76%; yellow solid; mp: 135–137 °C; IR (KBr): 2856, 2922
(alkyl), 2206 (CN) cmÀ1 1H NMR (400 MHz, CDCl3): d ppm 0.094–
;
Yield: 69%; yellow solid; mp: 135–137 °C; IR (KBr): 2859, 2952
(alkyl) 2206 (CN) cmÀ1 1H NMR (400 MHz, CDCl3): d ppm 0.92–
;
0.095 (t, 3H, CH3), 1.40–1.43 (m, 2H, CH2), 1.72–1.78 (m, 2H, CH2),
4.24–4.28 (t, 2H, CH2), 7.55–7.579 (d, 2H, Ar), 7.93–7.95 (d, 2H, Ar),
8.95(s, 1H, Ar); 13C NMR (CDCl3): 13.6, 19.9, 29.3, 47.4, 111.8,
116.8, 129.8, 131.5, 132.1, 141.2, 156.2, 166.9, 186.0; LC–MS (m/
z): 335 (M+), 336 (M++1); Analysis calculated for C15H14ClN3S2: C,
53.64; H, 4.20; N, 12.51; S, 19.09. Found C, 53.76; H, 4.33; N,
12.40; S, 19.18.
0.95 (t, 3H, CH3), 1.74–1.79 (m, 2H, CH2), 4.18–4.21 (t, 2H, CH2),
7.21–7.24 (m, 2H, Ar), 7.97–7.99 (m, 2H, Ar), 8.90 (s, 1H, Ar); 13C
NMR (CDCl3): 11.2, 20.7, 49.0, 111.9, 116.9, 130.1, 132.8, 132.9,
156.4, 165.8, 166.8, 168.4, 186.1; LC–MS: m/z 306 (M++1); Analysis
calculated for C14H12FN3S2: C, 55.06; H, 3.96; N, 13.76; S, 21.00;
Found: C, 55. 18; H, 3.87; N, 13.86; S, 21.14.

6082
C. B. Mishra et al. / Bioorg. Med. Chem. 21 (2013) 6077–6083
3.2.9. (4E)-4-(4-Chlorobenzylideneamino)-N3-phenyl-2,3-
dihydro-2-thioxothiazole-5-carbonitrile (14)
3.3. Molecular docking
Yield: 72%; yellow solid; mp: 187–189 °C; IR (KBr): 3059 (aro-
Docking simulations were performed with AutoDock3.0.5
according to our previous report.18 The recently solved X-ray crys-
tal structure of the human A2AR (pdb code: 3EML; 2.6 Å resolu-
tion) in complex with ZM241385 was retrieved from the RCSB
Protein Data Bank and all heteroat-oms were removed. The A2AR
was set up for docking with standard protocol. Polar hydrogens
were added using PROTONATE utility distributed with Auto-
Dock3.0.5 then protein co-ordinates were subjected to minimize
with CHARMm force field potential.21,22 Steps (1000) of steepest
descent followed by conjugate gradient minimization until the
RMS gradient of the potential energy was less than 0.05 kJ molÀ1
ÅÀ1 performed the minimizations. Kollman united atom charges
and solvation parameters were added to the final protein file.
The known A2AR antagonists and thiazole derivatives were drawn
on BUILDER module (insight II). DISCOVER molecular dynamics
(CHARMm force field) was run to carry the optimization of the
ligands, which were saved in mol2 format with the aid of BABEL
programs (Pat Walters and Matt Stahl, NCI). Full hydrogens were
added to the ligands and Gasteiger–Marsili partial atomic charges
were computed using the BABEL program and saved in the PDBQ
format. All possible flexible torsions of the resultant ligand mole-
cules were defined by using AUTOTORS. The prepared ligands in
PDBQ format were used as input files for AutoDock3.0.5 in the next
step. Docking simulation was carried out using the Lamarckian
Genetic Algorithm.21 For binding energy, three terms were taken
into account in the docking step: the van der Waals interaction
represented as a Lennard–Jones 12–6 dispersion/repulsion term,
the hydrogen bonding represented as a directional 12–10 term,
and the Coulombic electro-static potential. The resulting docking
orientations lying within 2.0 Å in the root-mean square deviation
(rmsd) tolerance of each other were clustered together represented
by the result with the most favorable free energy of binding. Final-
ly, the obtained top-posed docking conformations were subjected
to post-docking energy minimization on insight-II/accelrys.22
matic), 2201 (CN) cmÀ1 1H NMR (400 MHz, CDCl3): d ppm 7.28–
;
7.30 (m, 2H, Ar), 7.54–7.68 (m, 7H, Ar), 8.86 (s, 1H, Ar); 13C NMR
(CDCl3): 111.7, 116.5, 128.3, 128.8, 129.5, 130.0, 130.1, 132.7,
132.8, 135.9, 156.9, 166.3, 187.4; LC–MS (m/z): 356 (M++1);
Analysis calculated for C17H10ClN3S2: C, 57.38; H, 2.83; N, 11.81;
S, 18.02. Found: C, 57.50; H, 2.72; N, 11.90; S, 18.11.
3.2.10. (4E)-4-(4-Bromobenzylideneamino)-N3-ethyl-2,3-
dihydro-2-thioxothiazole-5-carbonitrile (15)
Yield: 75%; yellow solid; mp: 182–184 °C; IR (KBr): 2954 (alkyl),
2211 (CN) cmÀ1 1H NMR (400 MHz, CDCl3): d ppm 1.32–1.35
;
(t, 3H, CH3), 4.31–4.36 (q, 4H, CH2), 7.71 (d, 2H, Ar), 7.86–7.88
(d, 2H, Ar), 8.95 (s, 1H, Ar); 13C NMR (CDCl3): 12.6, 42.9, 111.8,
116.8, 130.0, 131.5, 132.5, 132.8, 156.0, 167.2; 185.8. LC–MS
(m/z): 352 (M++1); Analysis calculated for C13H10BrN3S2: C,
44.32; H, 2.86; N, 11.93; S, 18.20. Found: C, 44.41; H, 2.72; N,
12.04; S, 18.32.
3.2.11. (4E)-4-(4-Bromobenzylideneamino)-N3-propyl-2,3-
dihydro-2-thioxothiazole-5-carbonitrile (16)
Yield: 72%; yellow solid; mp: 155–157 °C; IR (KBr): 2923, 2852
(alkyl), 2210 (CN) cmÀ1 1H NMR (400 MHz, CDCl3): d ppm 0.90–
;
0.94 (t, 3H, CH3), 1.72–1.76 (m, 2H, CH2), 4.16–4.20 (t, 2H, CH2),
7.67–7.69 (d, 2H, Ar), 7.81–7.82 (d, 2H, Ar), 8.89 (s, 1H, Ar);
LC–MS (m/z): 366 (M++1); Analysis calculated for C14H12BrN3S2:
C, 45.91, H, 3.30; N, 11.47; S, 17.51. Found: C, 46.02; H, 3.41; N,
11.36; S, 17.59.
3.2.12. (4E)-4-(4-Bromobenzylideneamino)-N3-butyl-2,3-
dihydro-2-thioxothiazole-5-carbonitrile (17)
Yield: 66%; yellow solid; mp: 138–140 °C; IR (KBr): 2923, 2852
(alkyl), 2211 (CN) cmÀ1 1H NMR (400 MHz, CDCl3): d ppm 0.94–
;
0.97 (t, 3H, CH3), 1.36–1.43 (m, 2H, CH2), 1.73–1.77 (m, 2H, CH2),
4.24–4.28 (t, 2H, CH2), 7.72 (d, 2H, Ar), 7.87 (d, 2H, Ar), 8.94
(d, 2H, Ar), 8.94 (s, 1H, Ar); 13C NMR (CDCl3): 13.7, 19.9, 29.2,
47.4, 111.8, 116.7, 129.9, 131.5, 132.6, 132.7, 156.2, 167.2, 185.9;
LC–MS (m/z): 379 (M+), 380 (M++1); Analysis calculated for
C15H14BrN3S2: C, 47.37; H, 3.71; N, 11.05; S, 16.86. Found: C,
47.50; H, 3.63; N, 11.14; S, 16.97.
3.4. Radioligand binding assay
Adenosine A2AR and A1R binding assays: [3H]ZM241385 and
[3H]DPCPX binding assays for adenosine A2AR and A1Rs, respec-
tively, were performed in transfected HEK293T cells with human
A
2AR and A1R according Luthra et al.16 Briefly, 10
l
g HEK293T cell
membranes isolated from were incubated with different concen-
trations (1 pM–1
M) of compounds and 1 nM [3H]ZM241385 in
200 l incubation buffer containing 50 mM Tris–Cl, 1 mM EDTA,
pH 7.4 and 2.5 U/ml adenosine deaminase. Adenosine A1R assay
were performed on 10 g of HEK293T cell membranes expressing
human adenosine A1Rs and 1 nM [3H]DPCPX in 200
l incubation
3.2.13. (4E)-4-(4-Bromobenzylideneamino)-N3-phenylethyl-
2,3-dihydro-2-thioxothiazole-5-carbonitrile (18)
l
Yield: 79%; yellow solid; mp: 180–182 °C; IR (KBr): 3072 (aro-
l
matic), 2202 (CN) cmÀ1 1H NMR (400 MHz, CDCl3): d ppm 3.08–
;
3.11 (t, 2H, CH2), 4.48–4.52 (t, 2H, CH2), 6.97–7.08 (m, 2H, Ar),
7.11–7.28 (m, 3H, Ar), 7.50–7.52 (d, 2H, Ar), 7.71–7.73 (d, 2H,
Ar), 8.02 (s, 1H, Ar); 13C NMR (CDCl3): 32.4, 48.8, 111.7, 116.7,
127.0, 128.7, 129.1, 129.5, 131.3, 132.1, 137.6, 140.8, 156.7,
165.9, 185.9; LC–MS (m/z): 429 (M++1); Analysis calculated for
l
l
buffer. Reactions were carried out for 60 min at 26 °C and were
terminated by rapid filtration over 96-well plates equipped with
GF/B filters (Milipore, USA). Filters were washed three times with
C19H14BrN3S2: C, 53.27; H, 3.29; N, 9.81; S, 14.97. Found: C,
300 ll of cold washing buffer containing 50 mM Tris–Cl, 10 mM
53.39; H, 3.38; N, 9.92; S, 14.86.
MgCl2, pH 7.4, air dried, and radioactivity retained on filters were
counted in 1450 LSC & Luminescence counter (Wallac Microbeta
Trilux, Perkin–Elmer, USA). The Kd values for radioligands
[3H]ZM241385 and [3H]DPCPX were 0.99 and 1.79 nM, respec-
tively, obtained by saturation binding assays. Nonspecific binding
for adenosine A2AR and A1R was determined in the presence of
3.2.14. (4E)-4-(4-Bromobenzylideneamino)-N3-phenyl-2,3-
dihydro-2-thioxothiazole-5-carbonitrile (19)
Yield: 72%; yellow solid; mp: 185–187 °C; IR (KBr): 3065
(aromatic), 2208 (CN) cmÀ1 1H NMR (400 MHz, CDCl3): d ppm
;
7.27–7.28 (m, 2H, Ar), 7.51–7.60 (m, 7H, Ar), 8.85 (s, 1H, Ar);
13C NMR (CDCl3): 117.7, 116.8, 128.3, 129.5, 129.5, 129.8, 130.0,
131.4, 132.5, 132.6, 135.8, 156.7, 166.6, 187.3; LC–MS (m/z):
400 (M++1); Analysis calculated for C17H10BrN3S2: C, 51.01; H,
2.52; N, 10.50; S, 16.02. Found C, 51.19; H, 2.60; N, 10.38; S,
16.14.
50 lM NECA and 50 lM CPA, respectively. Assays were performed
in duplicates and compounds were tested thrice. Data were fitted
in one site competition-binding model for IC50 determination using
the program GraphPad Prism 4.0 (GraphPad Software, Inc., San
Diego, CA). Ki Values were calculated using Cheng and Prusoff
formula.23

C. B. Mishra et al. / Bioorg. Med. Chem. 21 (2013) 6077–6083
6083
3.5. Functional assay
4.3. Statistical analysis
HEK293T cells (1 Â 106/ml) washed with ice-cold PBS (pH 7.4)
were treated with adenosine deaminase (2 U/ml) for 30 min at
room temperature (to remove endogenously present adenosine
in the cells), followed by treatment with 1 lM standard A2AR ago-
nist (NECA) separately in three eppendorf tubes. The tubes were
The data for behavioral studies were statistically evaluated for
significance employing nonparametric analysis of variance
(Kruskal–Wallis test) for catalepsy and akinesia employing statisti-
cal package, Graph Pad Prism 4.0. Values of p 60.05 were consid-
ered significant. Results are given as mean SEM values.
incubated for 15 min in CO2 incubator to stimulate the cAMP re-
lease, 1
l
M of each PTTP and SCH58261 was added to the two
Acknowledgments
tubes and incubated for 15 min. The concentration of the cAMP
in the samples was determined according to Luthra et al.18
P.M.L. is thankful to Council of Scientific and Industrial Research
(CSIR), Delhi, India, for Grant-in-aid to carry this work. The
students C.B.M., A.P., N.K. and N.K. are thankful to CSIR, Delhi,
India, for providing the financial support.
4. Animals
Adult Swiss Albino male mice (4–6 weeks, 20–30 g) were
procured from National Institute of Communicable Diseases,
Delhi, India and were kept under controlled conditions of
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmc.2013.07.005.
temperature (22
1 °C), humidity (60
5%), and illumination
(12 h light; 12 h darkness) at the animal house, Dr. B.R. Ambed-
kar Centre for Bio-medical Research, University of Delhi, Delhi,
India. The experimental protocol met the National Guidelines
on the ‘Proper Care and Use of Animals in Laboratory Research’
(Indian National Science Academy, New Delhi) and was approved
by the Animal Ethics Committee of the department. The proce-
dures adhered to the NIH Guidelines for the Care and Use of
Laboratory Animals.
References and notes
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4.1. Drugs
Haloperidol and SCH58261 were purchased from Sigma
Chemicals Co. (St. Louis, Mo) and Tocris Bioscience, respectively.
Double distilled filtered and deionized water (Milli-Q-system
Waters, Milford, MA) was used throughout the study. Haloperidol,
SCH58261 and compounds 6–19 were dissolved in 1% acacia in
saline. Mice were divided into seven groups of four mice each.
SCH58261 (10 mg/kg) and compound 19 (5, 10 and 20 mg/kg)
were administered ip to each mice of the assigned group. Saline
and 1% acacia in saline were injected to two control groups. After
30 min of pre-treatment, haloperidol (2.5 mg/kg) was injected to
one group and all pre-treated groups.
16. Luthra, P. M.; Mishra, C. B.; Jha, P. K.; Barodia, S. K. Bioorg. Med. Chem. Lett. 2010,
3, 1214.
17. Luthra, P. M.; Mishra, C. B. U.S. Patent Appl. US2012/264937, 2012.
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Med. Chem. 2010, 18, 2491.
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http://www.accelrys.com/doc/life/insight2000.1/ffbs/FFSimulTOC.html.
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4.2. Catalepsy
The inability of an animal to correct an externally imposed
posture (catalepsy score) was measured at different time inter-
vals with both limbs on a square wooden block (3 cm high)
by placing the animals on a flat horizontal surface.18 The length
of time that animals held the bar without any voluntary move-
ment was recorded, with a cutoff time of 3 min. Catalepsy score
of each mice in a group was taken to compute the mean value of
the group.