Synthesis of novel 7-imino-2-thioxo-3,7-dihydro-2H-thiazolo [4,5-d] pyrimidine derivatives as adenosine A2A receptor antagonists

Article abstract of DOI:10.1016/j.bmcl.2009.11.133

Novel bicyclic thiazolopyrimidine compounds (15-26) were synthesized to develop adenosine A_2A receptor (A_2AR) antagonist for the treatment of Parkinson's disease (PD). The binding affinity of the compounds (15-26) with A_2A

Full text of DOI:10.1016/j.bmcl.2009.11.133

Bioorganic & Medicinal Chemistry Letters 20 (2010) 1214–1218
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis of novel 7-imino-2-thioxo-3,7-dihydro-2H-thiazolo [4,5-d]
pyrimidine derivatives as adenosine A2A receptor antagonists
*
Pratibha Mehta Luthra , Chandra Bhushan Mishra, Pawan Kumar Jha, Sandeep Kumar Barodia
Medicinal Chemistry Division, Dr. B.R. Ambedkar Center for Biomedical Research, University of Delhi, 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:
Novel bicyclic thiazolopyrimidine compounds (15–26) were synthesized to develop adenosine A2A recep-
tor (A2AR) antagonist for the treatment of Parkinson’s disease (PD). The binding affinity of the compounds
(15–26) with A2AR was evaluated using radioligand binding assay on isolated membranes from stably
transfected HEK293 cells. Selectivity of the compounds towards A2AR was assessed by comparing their
binding affinities with A1 receptors (A1R). cAMP concentrations were measured from HEK293 cells
treated with compounds (15–26) as compared to NECA (A2AR agonist). The compound (16) possessed
strongest A2AR binding affinity (Ki value = 0.0038 nM) and selectivity (737-fold) versus A1R. Decrease
in A2AR-coupled release of endogenous cAMP from HEK293 cells treated with compounds (15–26) is
evocative of their potential as A2AR antagonist.
Received 28 August 2009
Revised 10 November 2009
Accepted 27 November 2009
Available online 4 December 2009
Keywords:
Adenosine
A2AR
Combinatorial chemistry
GPCR
Ó 2009 Elsevier Ltd. All rights reserved.
Radioligand binding
Parkinson’s disease
Thiazolopyrimidines
Parkinson’s disease (PD) is a neurodegenerative disorder caused
by the degeneration of dopaminergic neurons in nigrostriatal re-
gion of the brain and characterized by loss of motor coordination
manifested as tremor and rigidity of the limbs and trunk.1 Dopa-
mine depletion due to degeneration of dopaminergic neurons can
output neurons.8 A2AR antagonist activated nigrostriatal pathway
via D2 receptor leading to antiparkinsonian effect. A2AR and D2R
interact antagonistically in striatopallidal neurons. The effect of
A2AR activated adenylyl cyclase by persistent activation of D2
receptor was antagonized at the level of the striatopallidal GABA
neurons suggesting D2 receptor mediated anti-parkinsonian role
of A2AR antagonists.9 Besides providing symptomatic relief in PD,
A2AR antagonists have also been shown to be neuroprotective in
animal models of PD.10
be supplemented by administration of the dopamine precursor L-
DOPA, one of the major treatments for PD.2 However, undesirable
side effects such as dyskinesia (abnormal involuntary movements)
induced by L-dopa therapy are one of the major challenges for the
treatment of PD.3 Other available treatments (dopaminergic ther-
apy) of PD include dopamine agonists and inhibitors of endoge-
nous dopamine degradation by monoamine oxidase [MAO]B and
catechol-O-methyltransferase[COMT]), and anticholinergics. As an
alternative therapeutic approach, PD therapeutics involving A2ARs
antagonists are gaining importance due to their capability to give
partial symptomatic relief.4–6 Blockade of the A2ARs in striatopalli-
dal neurons diminished postsynaptic effects of dopamine deple-
Several xanthine and non-xanthine derivatives were synthe-
sized in last decade in search of potent A2AR antagonist as novel
antiparkinsonian agent (Fig. 1), however, not a single drug could
be accomplished for the treatment of PD. Xanthine-based A2A
R
antagonists such as KW6002 showed efficacy in models of PD
without inducing hyperactivity or inducing dyskinesia but failed
in clinical trials.11–15 Non-xanthine compounds such as
ZM241385 and SCH58261 possessed strong A2AR antagonist activ-
ity.16–19 However, suffered from several drawbacks including low-
er selectivity, poor solubility and poor pharmacokinetic profile.20
The discovery of selective and potent A2AR antagonist still remains
a challenge.
tion, and in turn reduced the motor deficits of PD.7 A2A
R
antagonists might partially improve not only the symptoms of
PD but also its course, by lessening the prime neurodegeneration
and reducing the maladaptive neuroplasticity3 A2AR belongs to
the family of seven trans-membrane G-protein coupled receptors
(GPCRs) and is highly expressed in the nigrostriatum (basal gan-
glia) co-localized with dopamine D2 receptors on striatopallidal
Thiazoles have emerged as an important class of compounds
due to their anti-oxidant,21 anti-inflammatory,22 and neuroprotec-
tive effect.23 A series of aryl/heteroaryl urea bearing thiazole com-
pounds possessed selective cycline dependent kinase inhibiting
activity.24 Recently, thiazole derivatives with urea moiety have
* Corresponding author. Tel.: +91 11 27666272; fax: +91 11 27666248.
E-mail addresses: pmluthra@acbr.du.ac.in, pmlsci@yahoo.com (P.M. Luthra).
demonstrated A2A
R
antagonist activity.25 ECLiPS™ (Encoded
0960-894X/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2009.11.133

P. M. Luthra et al. / Bioorg. Med. Chem. Lett. 20 (2010) 1214–1218
1215
O
N
N
N
O
N
O
O
ZM 241385
KW 6002
NH2
N
N
N
N
N
N
O
SCH 58261
Figure 1. Structures of A2AR antagonists ZM241385, KW6002, and SCH58261. Binding affinities are as follows: (a) ZM241385 (Ki value; A2AR = 0.8 nM, A1R = 260 nM, and hA1/
hA2 = 325)33 (b) KW6002 (Ki value; A2AR = 2.2 nM, A1R = 150 nM, and hA1/hA2 = 68),34 and (c) SCH58261 (Ki value; A2AR = 1.23 nM, A1R = 594.1 nM, and hA1/hA2 = 483).
combinatorial libraries on Polymeric Support) libraries showed
aminothiazole core structure as active A2AR antagonists26 Several
pyrazolo-triazolo-pyrimidine derivatives were synthesized as po-
tent A2AR antagonist such as SCH58261.16 In our efforts to develop
potent A2AR antagonist, pyrazolo-triazolo-pyrimidine pharmaco-
phore of known A2AR antagonists (SCH58261) was replaced by
thiazolopyrimidine pharmacophore, considered as thiazolo-analog
of natural purine base along with open triazole ring to obtain 7-
imino-2-thioxo-3,7-dihydro-2H-thiazolo [4,5-d] pyrimidine phar-
macophore with urea and furonamide moiety. Novel thiazolopyr-
imidine pharmacophore with urea moiety possessing aliphatic
flexible groups, and aromatic planar structure was synthesized to
give the compounds (15–21). Furan ring has been found essential
for the activity of several non-xanthine derivatives to hold the mol-
ecule in active site cavity of A2AR,27 therefore, amino group of urea
was replaced with furan ring to give furonamide series of the com-
pounds (22–26). Since, non-xanthine compounds showed consid-
erable selectivity for A2AR subtype, but suffered from low water
solubility. We thought that thiazolopyrimidine ring possessing
urea and furonamide derivatives would increase water solubility
due to flexible polar interactions.
The binding affinity of the compounds (15–26) with human
A2AR was evaluated in vitro using competitive binding with
[3H]ZM241385. The A2AR selectivity of the compounds was deter-
mined using the A1R selective antagonist [3H]DPCPX. A2AR-coupled
cAMP concentrations were measured in HEK293 cells treated with
compounds (15–26).28
Synthesis of the compounds (15–26) was carried out according
to scheme 1.29 An equimolar mixture of isothiocyanate, malononit-
rile and sulfur powder in DMF was stirred in ice bath. After 15 min,
triethylamine was added drop-wise to the mixture, and the reac-
tion was continued for 4 h to give 4-amino-3-substituted-2-thi-
oxo-2,3-dihydro-thiazole-5-carbonitrile derivatives (1–7). The
carbonitrile compounds (1–7) were refluxed in toluene with tri-
ethylorthoformate (equimolar ratio) and p-toluene sulfonic acid
(catalytic amount) for 6 h to yield imino-ether derivatives (8–14).
The disappearance from IR spectra of an intense absorption band
at 3230–3369 cmꢀ1 (free amino group), and the appearance of a
singlet at 8.1–8.4 ppm (N@CH of methylene amine) in the 1H spec-
tra validated the formation of Schiff’s bases.
The disappearance from IR spectra of an intense absorption band at
2205 cmꢀ1, and the appearance of a singlet at 8.1–8.8 ppm (N@CH
of pyrimidine ring) in the 1H spectra confirmed the formation of
bicyclic compounds. All the compounds have been fully character-
ized and their purity was checked by HPLC (Ref. 29 and Supple-
mentary data).
The result of A2AR binding assay are expressed as inhibition
constants (Ki in nM). The A1R/A2AR describes their selectivity over
A1R.30 In the set of thiazolopyrimidine urea derivatives (15–21,
R2 = NH2), ethyl substitution (15, R1 = –C2H5) exhibited signifi-
cantly higher binding affinity with A1R (0.00016 0.007 nM) as
compared to A2AR (0.09 0.01 nM). Homologation of one carbon
in compound 15 gave the propyl derivative of thiazolo pyrimidine
urea (16, R1 = –C3H7). In competitive radioligand binding assay, the
displacement of [3H]ZM241385 with 16 was significantly in-
creased (ꢁ25 times) as compared to 15, and with very high selec-
tivity for A2AR (737-fold selectivity over A1R), and was better than
the known antagonist SCH58261 (Ki = 1.23 0.016, hA1/hA2 =
483).31 However 3-carbon chain with
p-overlap in allyl derivative
(18, R1 = –C3H5) displayed good binding affinity (Ki = 0.092 0.01)
but reduced selectivity (hA1/hA2 = 5). Further extending the alkyl
chain to give butyl derivative of thiazolopyrimidine urea (17,
R1 = –C4H9) resulted in decreased selectivity over A1R. Incorpora-
tion of aromatic ring (phenyl) substituent in thiazolopyrimidine
urea (19, R1 = –C6H5) gave enhanced binding affinity and selectiv-
ity. Moreover, p-iodophenyl substitution (20, R1 = –C6H4I) on the
pharmacophore improved both binding affinity and selectivity
(144-fold). Insertion of one carbon homologation in planar aro-
matic ring of thiazolopyrimidine urea (21, R1 = –CH2C6H5) led de-
creased selectivity. We can conclude that both 19 and 20 possess
promising activity, yet the compound (16) is the most active
among all thiazolopyrimidine urea derivatives. The amino (NH2)
group of urea moiety of thiazolopyrimidine pharmacophore was
replaced by furan ring to give another set of compounds (22–26,
R2 = –furyl). Overall substituent effects to binding affinity (pro-
pyl > butyl > allyl > aryl > ethyl) and selectivity (propyl > allyl >
butyl > aryl > ethyl) profile of thiazolopyrimidine furonamide
(22–26) decreased, however in the set of compound (22–26) pro-
pyl derivative (23, R1 = –C3H7) showed maximum binding and
selectivity to A2AR.
Mixture of imino-ether derivatives (8–14), semicarbazide
hydrochloride/furoic acid hydrazide (equal mol) and TEA (catalyst)
in ethanol was stirred at rt for 12 h to give the compounds (15–26).
The finding clearly demonstrated that bicyclic thiazolopyrimi-
dine urea derivatives (15–21) were more potent and selective
than the corresponding bicyclic thiazolopyrimidine furonamide

1216
P. M. Luthra et al. / Bioorg. Med. Chem. Lett. 20 (2010) 1214–1218
R1
NH2
CN
CN
CN
N
A
R1 NCS
+
S
+
S
S
1-7
B
R1
N=CH O
CN
N
S
S
C
D
8-14
R1
N
O
R1
N
N
O
N
NH
N
N
NH
S
R2
S
S
R2
NH
S
NH
15-21
22-26
O
O
O
22, R1 = -C2H5;
23, R1 = -C3H7;
24, R1 = -C4H9;
25, R1 = -C3H5;
26, R1 = -C6H5;
R2
R2
R2
R2
R2
=
=
=
=
=
15, R1 = -C2H5;
16, R1 = -C3H7;
17, R1 = -C4H9;
18, R1 = -C3H5;
19, R1 = -C6H5;
20,R1 = -C6H4I;
R2 = NH2
R2 = NH2
R2 = NH2
R2 = NH2
R2 = NH2
R2 = NH2
O
O
21, R1 = -CH2C6H5; R2 = NH2
Scheme 1. Reagents and conditions: (A) triethyl amine (TEA), rt; (B) triethyl orthoformate, p-toluene sulfonic acid (PTSA), reflux; (C) furoic acid hydrazide, TEA, rt; and
(D) semi-carbazide HCl, TEA, rt.
derivatives (22–26) suggesting that one H-bond acceptor and three
H-bond donors are required for high affinity of A2AR. The low affin-
ity and selectivity of furonamide derivatives suggested that basic
amino group of urea at the active site is essentially required
(Fig. 2).
A2AR mediate cellular response through multiple signal trans-
duction pathways and a generic functional assay for Gi- and Gs-
protein-coupled receptor directly measures adenosine 30,50-cyclic
monophosphate (cAMP) production and inhibition in live cells.
We extended our investigation to study the effect of compounds
(15–26) on A2AR mediated inhibition of cAMP accumulation.28
Functional antagonism study points that all the compounds (15–
26) significantly decreased the cAMP concentration compared to
agonist NECA (0.75 pmol/ml) and showed better inhibition of
cAMP than SCH58261 (Table 1).
In summary, all the urea derivatives (15–21) showed binding
affinity with A2AR in nanomolar range. However, the compound
16 displayed Ki value of 0.0038 nM with (737-fold) binding selec-
tivity versus A1R and compound 20 exhibited reasonably good
A2AR binding affinity (Ki value 0.017) and (>100-fold) selectivity
over A1R. The inhibition of cAMP accumulation with the compound
16 and 20 was 0.14 pmol and 0.076 pmol, respectively, better than
Hydrophobic
Binding
H-bond donor
H-bond donor
H-bond acceptor
H-bond acceptor
Hydrophobic
Binding
N
N
O
N
O
N
N
NH
N
NH
S
Hydrophobic binding
S
S
NH2
S
NH
O
NH
H-bond donor
H-bond donor
H-bond acceptor
H-bond donor
Figure 2. Potential binding site of thiazolopyrimidine urea derivative 16 and thiazolopyrimidine furonamide derivative 23.

P. M. Luthra et al. / Bioorg. Med. Chem. Lett. 20 (2010) 1214–1218
1217
Table 1
Radioligand binding and cAMP functional assay of thiazolopyrimidine compounds (15–26)
R1
N
O
N
N
NH
R2
S
S
NH
Compounds
R1
R2
hA2A binding
hA1 binding
hA1/hA2A
Ratio
cAMP
(pmol/ml)
Ki SDa (nM)
Ki SDb (nM)
15
16
17
18
–CH2CH3
–NH2
–NH2
–NH2
–NH2
0.09 0.01
0.00016 0.007
2.8 0.8
1.04 0.84
0.47 0.1
0.002
737
11
0.085
0.14
0.083
0.08
–CH2CH2CH3
–CH2CH2CH2CH3
–CHCHCH3
0.0038 0.001
0.089 0.01
0.092 0.01
5
19
20
21
–NH2
–NH2
–NH2
0.063 0.008
0.017 0.01
0.023 0.014
1.5 1.10
2.5 1.1
24
147
6
0.078
0.076
0.067
I
0.14 0.086
22
23
24
25
26
–CH2CH3
1.2 1.10
0.016 0.01
0.087 0.05
0.0053 0.001
0.59 0.11
0.01
3
0.12
O
–CH2CH2CH3
–CH2CH2CH2CH3
CHCHCH3
0.024 0.01
0.029 0.01
0.17 0.1
0.092
0.06
O
O
O
O
0.2
3
0.084
0.048
0.33 0.75
0.0085 0.001
0.02
SCH58261
NECA
1.23
14
594.1
20
483
0.25
0.75
0.4
0.732
Untreated HEK293 cells (stably expressing A2AR)
a
Standard deviation (SD) of binding affinity (Ki in nM) of compound 15-26 for human A2AR.
Standard deviation (SD) of binding affinity (Ki in nM) of compound 15-26 for human A1R.
b
9. Fuxe, K.; Marcellino, D.; Genedani, S.; Agnati, L. Mov. Disord. 2007, 22, 1990.
10. Chen, J. F.; Sonsalla, P. K.; Pedata, F.; Melani, A.; Domenici, M. R.; Popoli, P.;
Geiger, J.; Lopes, L. V.; Mendonça, A. Prog. Neurobiol. 2007, 83, 310.
11. Shiozaki, S.; Ichikawa, S.; Nakamura, J.; Kitamura, S.; Yamada, K.; Kuwana, Y.
Actions Psychopharmacol. 1999, 147, 90.
SCH58261 (0.25 pmol). The results demonstrated that the com-
pound 16 was most potent A2AR antagonist among all compounds
(15–26) and thiazolopyrimidine with urea extension represent a
novel pharmacophore template for further development of selec-
tive and potent A2AR antagonist.
12. Kanda, T.; Jackson, M. J.; Smith, L. A.; Pearce, R. K. B.; Nakamura, J.; Kase, H.;
Kuwana, Y.; Jenner, P. Exp. Neurol. 2000, 162, 321.
13. Hauser, R. A.; Schwarzschild, M. A. Drugs Aging 2005, 22, 471.
14. LeWitt, P. A.; Guttman, M.; Tetrud, J. W.; Tuire, P. J.; Mori, A.; Chaikin, P.;
Sussman, N. M. Ann. Neurol. 2008, 63, 295.
Acknowledgment
15. http://www.kyowa-kirin.co.jp/english/news/2009/e20090115_01.html.
16. Caulkett, P. W. R.; Jones, G.; McPartlin, M.; Renshaw, N. D.; Stewart, S. K.;
Wright, B. J. J. Chem. Soc., Perkin Trans.1 1995, 801.
17. Baraldi, P. G.; Cacciari, B.; Spalluto, G.; Pineda de Villatoro, M. J.; Zocchi, C.;
Dionisotti, S.; Ongini, E. J. Med. Chem. 1996, 39, 1164.
Authors are thankful to Council of Scientific and Industrial Re-
search, Delhi, India, for providing the funds to carry this work.
Supplementary data
18. Zocchi, C.; Ongini, E.; Conti, A.; Monopoli, A.; Negretti, A.; Baraldi, P. G.;
Dionisotti, S. J. Pharmacol. Exp. Ther. 1996, 276, 398.
19. Baraldi, P. G.; Cacciari, B.; Romagnoli, R.; Spalluto, G.; Monopoli, A.; Ongini, E.;
Varani, K.; Borea, P. A. J. Med. Chem. 2002, 45, 115.
20. Kisselgof, E.; Tulshian, D. B.; Arik, L.; Zhang, H.; Fawzi, A. Bioorg. Med. Chem.
Lett. 2005, 15, 2119.
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmcl.2009.11.133.
21. Hirota, T.; Sasaki, K.; Tashima, Y.; Nakayaama, T. J. Hetrocyclic Chem. 1991, 28, 263.
22. Nagamatsu, T.; kinoshita, K.; Sasaki, K.; Nakayama, T.; Hirota, T. J. Hetrocyclic
Chem. 1991, 28, 513.
23. Hirota, T.; Leno, K.; Sasaki, K. J. Heterocycl. Chem. 1986, 23, 1685.
24. Helal, C. J.; Sanner, M. A.; Cooper, C. B.; Gant, T.; Adam, M.; Lucas, J. C.; Kang, Z.;
Kupchinsky, S.; Ahlijanian, M. K.; Tate, B.; Menniti, F. S.; Kelly, K.; Peterson, M.
Bioorg. Med. Chem. Lett. 2004, 14, 5521.
25. Slee, D.; Lanier M.; Vong, B.G.; Chen, Y.; Zhang, X.; Lin, E.; Moorjani, M.; Castro,
P.; Laria, J.C. US Patent 20, 080, 275, 064, 06 November 2008.
26. Ohlmeyer, M. H. J.; Swanson, R. N.; Dillard, L.; Reader, J. C.; Asouline, G.;
Kobayishi, R.; Wigler, M.; Still, W. C. Proc. Natl. Acad. Sci. U.S.A. 1993, 90, 10922.
27. Jaakola, V. P.; Griffith, M. T.; Hanson, M. A.; Cherezov, V.; Chien, E. Y. T.; Lane, J.
R.; Ijzerman, A. P.; Stevens, R. C. Science 2008, 322, 1211.
References and notes
1. Jenner, P. Neurology 2003, 61, S32.
2. Bossy-Wetzel, E.; Schwarzenbacher, R.; Lipton, S. Nat. Med. 2004, 10, S2.
3. Olanow, C. W.; Obeso, J. A. Ann. Neurol. 2000, 47, S167.
4. Kanda, T.; Shiozaki, S.; Shimada, J.; Suzuki, F.; Nakamura, J. Eur. J. Pharmacol.
1994, 256, 263.
5. Fenu, S.; Pinna, A.; Ongini, E.; Morelli, M. Eur. J. Pharmacol. 1997, 321, 143.
6. Kanda, T.; Jackson, M. J.; Smith, L. A.; Pearce, R. K. B.; Nakamura, J.; Kase, H.;
Kuwana, Y.; Jenner, P. Ann. Neurol. 1998, 43, 507.
7. Aoyama, S.; Kase, H.; Borrelli, E. J. Neurosci. 2000, 20, 5848.
8. Svenningsson, P.; LeMoine, C.; Fisone, G.; Fredholm, B. B. Prog. Neurobiol. 1999,
59, 355.

1218
P. M. Luthra et al. / Bioorg. Med. Chem. Lett. 20 (2010) 1214–1218
28. In the functional assay, cAMP concentrations were determined using direct
cAMP EIA kit (Assay designs, USA) in treated HEK293 cells (stably transfected
with cDNA encoding human A2AR). Cells (1 ꢂ 106) were treated with 100 nM of
compounds (15–26), 100 nM SCH58261 and 100 nM of NECA for 24 h, 0.1 N
HCl was added to release endogenous cAMP, centrifuged at 1000g for 5 min at
4 °C. cAMP concentrations were measured (P. Tijssen, ‘Practice & theory of
enzyme immunoassays’, (1985) Amsterdam, Elsevier) in the treated cells, and
the results were compared with untreated cells (representing basal level of
cAMP in the cells).
(s, 1H, NH), 13C NMR (DMSO-d6): d 10.9, 20.2, 46.3, 98.1, 154.1, 155.8, 158.5,
159.0, 190.3, LC–MS: m/z 284 (M+), 285 (M+1).
30. Adenosine A2AR and A1R binding assays: [3H]ZM241385 and [3H]DPCPX binding
assays for adenosine A2AR and A1Rs, respectively, were performed in HEK293
cells (procured from National Center for Cell Sciences, Pune, India). Briefly,
10
with human A2AR and A1R cDNA were incubated with different concentrations
(1 pM to 1 l incubation
M) of compounds and 1 nM [3H]ZM241385 in 200
buffer containing 50 mM Tris–Cl, 1 mM EDTA, pH 7.4 and 2.5 U/ml adenosine
deaminase. Adenosine A1R assays were performed on 10 g of HEK293 cell
membranes expressing human adenosine A1Rs and 1 nM [3H]DPCPX in 200
lg HEK293 cell membranes isolated from stably transfected HEK293 cells
l
l
29.
A
mixture of isothiocyanate (1 equiv), malononitrile (1 equiv), and sulfur
l
powder (1 equiv), in DMF (50 ml) was stirred in ice bath. After 15 min,
triethylamine (5 ml) was added, and the reaction was continued for 4 h. The
reaction mixture was poured in water and the resulting precipitate
was dissolved in absolute ethanol to give pure crystalline 4-amino-3-
substituted -2-thioxo-2,3-dihydro-thiazole-5-carbonitrile derivatives (1–7).
The carbonitrile compounds 1–7 (1 equiv) were refluxed with
triethylorthoformate (1 equiv), and p-toluene sulfonic acid (catalytic amount)
in toluene (20 ml) for 6 h. Toluene was evaporated under reduced pressure and
crude product was dissolved in ethyl acetate, washed with water and dried
with Na2SO4. Solution was concentrated under reduced pressure to give pure
ll
incubation 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
(Millipore, USA). Filters were washed three times with 300 ll of cold washing
buffer containing 50 mM Tris–Cl, 10 mM 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,
respectively, obtained by saturation binding assays. Nonspecific binding for
adenosine A2AR and A1R were determined in the presence of 50
lM NECA and
crystalline imino-ether derivatives (8–14).
A
Mixture of imino-ether
50 lM CPA, respectively. Assays were performed in duplicates and compounds
derivatives 8–14 (1 equiv), semicarbazide hydrochloride(1 equiv), and TEA
(1–2 ml) in ethanol (10 ml) was stirred at rt for 12 h. The precipitate was
filtered, washed with absolute ethanol and water to give pure compounds (15–
21). imino-ether derivatives 8–11(1 equiv), were stirred with furoic acid
hydrazide (1 equiv), and TEA (1–2 ml) in ethanol (10 ml) at rt for 12 h. The
precipitate was filtered, washed with ethanol and water to give the compounds
(22–26). Spectroscopic data for 16. 7-Imino-3-propyl-2-thioxo-3,7-dihydro-2H-
thiazolo[4,5-d]pyrimidin-6-yl)-urea Yield: 86% (100% purity by HPLC), mp:
were tested twice. 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) and Ki values were calculated using Cheng and Prusoff
formula.
31. Ongini, E. Drug Dev. Res. 1998, 42, 63.
32. Ongini, E.; Dionisotti, S.; Gessi, S.; Irenius, E.; Fredholm, B. B. Naunyn
Schmiedebergs Arch. Pharmacol. 1999, 359, 7.
33. Linden, J.; Thai, T.; Figler, H.; Jin, X.; Robeva, A. S. Mol. Pharmacol. 1999, 56, 705.
34. Shimada, J.; Koike, N.; Nonaka, H.; Shiozaki, S.; Yanagawa, K.; Kanda, T.;
Kobayashi, S.; Ichimura, M.; Nakamura, J.; Kase, H.; Suzuki, F. Bioorg. Med.
Chem. Lett. 1997, 7, 2349.
220 °C, IR (KBr): 3249, 3163 (NH2), 2957, 2782 (alkyl), 1673 (C@O) cmꢀ1 1H
,
NMR (DMSO-d6): d 0.89 (t, 3H, J = 7.2 Hz, CH3), 9.70 (s, 1H, NH), 1.67–1.79 (m,
2H, CH2), 4.28 (t, 2H, J = 7.2 Hz, CH2), 6.35 (s, 2H, NH2), 8.40 (s, 1H, N@CH), 9.70