Synthesis of novel pyrido[3,2-e][1,2,4]triazolo[1,5-c]pyrimidine derivatives: Potent and selective adenosine A3 receptor antagonists

Article abstract of DOI:10.1002/ardp.201300003

A series of novel pyrido[3,2-e][1,2,4]triazolo[1,5-c]pyrimidine derivatives 5 was prepared from 2-amino-3-cyano-4-trifluoromethyl-6-phenylpyridine 1 in two steps via formation of iminoether 3 followed by reaction with different aroylhydrazides 4. Represen

Full text of DOI:10.1002/ardp.201300003

Arch. Pharm. Chem. Life Sci. 2013, 346, 699–707
699
Full Paper
Synthesis of Novel Pyrido[3,2-e][1,2,4]triazolo[1,5-c]pyrimidine
Derivatives: Potent and Selective Adenosine A Receptor
3
Antagonists
1
2
3
3
Veeraswamy Banda , Balakumar Chandrasekaran , Meryem Köse , Christin Vielmuth ,
3
1
1
1
Christa E. Müller , Kurumurthy Chavva , Santhosh Kumar Gautham , Sambasivarao Pillalamarri ,
4
5
1
Raghuprasad Mylavaram , Raghuramarao Akkinepally , Shanthanrao Pamulaparthy ,
1
and Narsaiah Banda
1
Fluoroorganic Division, Indian Institute of Chemical Technology, Tarnaka, Hyderabad, Andhra Pradesh, India
University Institute of Pharmaceutical Sciences and UGC Centre of Advanced Study in Pharmaceutical
2
Sciences (UGC-CAS), Panjab University, Chandigarh, Punjab, India
PharmaCenter Bonn, University of Bonn, Pharmaceutical Institute, Pharmaceutical Chemistry I, Bonn,
3
Germany
4
Vishnu College of Pharmacy, Bhimavaram, West Godavari, Andhra Pradesh, India
University College of Pharmaceutical Sciences, Warangal, Andhra Pradesh, India
5
A series of novel pyrido[3,2-e][1,2,4]triazolo[1,5-c]pyrimidine derivatives 5 was prepared from 2-amino-3-
cyano-4-trifluoromethyl-6-phenylpyridine 1 in two steps via formation of iminoether 3 followed by
reaction with different aroylhydrazides 4. Representative products 5 were evaluated for their affinity
towards all four subtypes of human adenosine receptors. Compounds 2-(3-fluorophenyl)-8-phenyl-10-
(trifluoromethyl)pyrido[3,2-e][1,2,4]triazolo[1,5-c]pyrimidine (5b), 2-(furan-2-yl)-8-phenyl-10-(trifluoro-
methyl)pyrido[3,2-e][1,2,4]triazolo[1,5-c]pyrimidine (5d), and 2-(furan-2-yl)-5-methyl-8-phenyl-10-(tri-
fluoromethyl)pyrido[3,2-e][1,2,4]triazolo[1,5-c]pyrimidine (5j) showed high affinity for the A3
receptors, with K values of 8.1, 10.4, and 12.1 nM, respectively, and were >1000-fold selective versus
i
all other adenosine receptor subtypes.
Keywords: Adenosine receptors / 2-Aminonicotinonitrile / Aroyl hydrazide / Orthoacetate / Orthoformate
Received: January 11, 2013; Revised: July 18, 2013; Accepted: July 19, 2013
DOI 10.1002/ardp.201300003
Introduction
1
and mast cells. A and A2A ARs are generally 60% identical
within the transmembrane domains [8–10] and represent
potential pharmacological targets for the treatment of
asthma, psychosis, chronic inflammation, anxiety, and
neurodegenerative disorders [11–13]. Thus, discovery and
development of adenosine receptor antagonists have been an
attractive field of research from the perspective of identifying
new drugs for the treatment of widespread disorders such as
inflammation, asthma, and Parkinson’s disease. Efforts to find
selective ligands for A2A receptors led to the identification
of pyrazolotriazolopyrimidines such as SCH 58261 (5-amino-7-
(2-phenylethyl)-2-(2-furyl)pyrazolo[4,3-e]1,2,4-triazolo[1,5-c]py-
rimidine) and SCH 63390 (5-amino-7-(3-phenylpropyl)-2-(2-
furyl)pyrazolo[4,3-e]1,2,4-triazolo[1,5-c]pyrimidine) [14]. Com-
pounds which possess hydrophilic groups in the para- and
ortho-position of the aromatic ring have been found to be
potent and selective for A2A ARs [15, 16]. Like A2A ARs, A2B ARs
1 3
Adenosine receptors (ARs) designated as A , A2A, A2B, and A ,
belong to the superfamily of G protein-coupled receptors
GPCRs), which modulate wide range of biological
(
a
functions including CNS, cardiac, and immune suppressive
functions [1, 2]. A2A ARs, for example, are present in high
density in the basal ganglia of the brain where they are co-
expressed with dopamine receptors forming heteromeric
receptor complexes [3]. A2A receptors are also present on
tissues of the peripheral system, e.g., on platelets [4],
lymphocytes [5], neutrophils [6, 7], monocytes, macrophages,
Correspondence: Dr. Narsaiah Banda, Fluoroorganic Division, Indian
Institute of Chemical Technology, Tarnaka, Hyderabad, Andhra Pradesh,
India.
E-mail: narsaiah@iict.res.in
Fax: þ91 40 27160387
ß 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

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00
V. Banda et al.
Arch. Pharm. Chem. Life Sci. 2013, 346, 699–707
stimulate adenylate cyclase activity in the presence of Results and discussion
adenosine. In contrast, A and A ARs inhibit adenylate
1
3
cyclase, and A antagonists have been proposed as novel anti-
Chemistry
3
cancer drugs [17], e.g., for the treatment of human
glioblastomas. Efforts to find selective ligands acting as
antagonists at A3 ARs led to the discovery of various
structures, such as KF-26777, MRS-1191, MRS-1220, MRS-
The 2-amino-3-cyano-4-trifluoromethyl-6-phenylpyridine 1 [22]
was reacted with triethylorthoformate/orthoacetate 2 in the
presence of catalytic amounts of acetic acid at reflux
temperature for 8 h yielding the imine ether derivatives
3 [23]. The compounds 3a and 3b were independently reacted
with different aroylhydrazides in toluene using catalytic
amounts of isobutyric acid at reflux temperature forming
products 5a–d while at 60–70°C in toluene or at 120°C in DMF
products 5e–o were obtained. The sequence of the reaction is
mainly an amination on the imine carbon followed by
cyclization of the amine with the nitrile carbon to result in
products 5. The reaction of compound 3a with various
aroylhydrazides is found to be sluggish. The structures of the
1
1
334, MRS-1523, MRS-3777, MRE-3005-F20, MRE-3008-F20, PSB
0, PSB-11, OT-7999, and VUF-5574 with high affinity [20, 21].
However none have so far been successfully evaluated in
clinical trials, which may partially be attributed to their poor
pharmacokinetic profile. Recent studies report that the
pyrrolo[3,4-e][1,2,4]triazolo[1,5-c]pyrimidine and the pyrazolo-
[
4,3-e][1,2,4]triazolo[1,5-c]pyrimidine structures have emerged
as useful templates for targeting A ARs [22–24]. Thus, based
3
on our recent results in the synthesis of such compounds [18,
1
13
1
[
9, 25], we focussed our attention on the preparation of pyrido-
3,2-e][1,2,4]triazolo[1,5-c]pyrimidine derivatives as bioisosteric
modification of the pyrrolo[3,4-e]/pyrazolo[4,3-e][1,2,4]triazolo-
1,5-c]pyrimidine structure, and evaluated the newly designed
and synthesized compounds in radioligand binding studies for
their affinity towards A , A2A, A2B, and A ARs and we thereby
identified promising new compounds.
synthesized compounds were confirmed by H and C NMR
spectroscopy, in addition to HPLC analysis coupled to
electrospray ionization mass spectrometry (LC/ESI-MS), which
was also used to determine the purity that was in all cases
shown to be >95%.
[
1
3
The reactions outlined in Scheme 1 and products collected
are presented in Table 1.
Scheme 1. Preparation of pyrido[3,2-e][1,2,4]triazolo[1,5-c]pyrimidine derivatives 5.
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Arch. Pharm. Chem. Life Sci. 2013, 346, 699–707
Selective Adenosine A
3
Receptor Antagonists
701
Table 1. Physical properties of pyrido[3,2-e][1,2,4]triazolo[1,5-c]pyrimidine derivatives 5.
S. no
Compd. no.
R
R
1
mp (°C)
Yield (%)
1
2
3
4
5
6
7
8
9
5a
5b
5c
5d
5e
5f
5g
5h
5i
5j
5k
5l
5m
5n
5o
H
H
H
C
6
H
5
248
273
256
250
263
290
275
276
264
296
259
274
269
271
284
54
60
45
56
70
73
81
73
68
65
74
68
64
69
74
3-FC
3-ClC
2-furyl
C
3-FC
6
H
4
6
H
4
H
CH
CH
CH
CH
CH
CH
CH
CH
CH
CH
CH
3
3
3
3
3
3
3
3
3
3
3
H
6 5
6
H
4
3-ClC
4-ClC
6
H
H
4
4
6
4-FC
2-furyl
2-ClC
2-OHC
4-NO
6
H
4
10
11
12
13
14
15
6
H
4
6 4
H
2
C
6
H
4
2-thienyl
3-pyridyl
Radioligand binding studies
The selected pyrido[3,2-e][1,2,4]triazolo[1,5-c]pyrimidine deriv-
residue, it was replaced with fluoro/chlorophenyl substitu-
ents. The 3-fluorophenyl was found to yield very high affinity
as well, with compound 5b displaying a K value of 8.1 nM.
i
Thus, the furyl residue is not an essential substituent at the
triazole ring. Further optimization of the substituent is
currently in progress to identify even more promising
compounds.
3
atives (5b, 5d–j) were tested for their ability to displace [ H]2-
6
3
chloro-N -cyclopentyladenosine ([ H]CCPA) from A
1
AR in rat
3
cortical membranes and [ H]MSX-2 from A AR in rat striatal
2
A
3
membranes, [ H]PSB-603 from human recombinant A2B ARs
3
and [ H]PSB-11 from human recombinant A
radioligand binding affinities of the compounds (5b, 5d–j)
were expressed as K
values or in percent inhibition (at 10 mM Conclusion
and A2A), or at 1 mM (A2B and A ) concentration,
3
ARs. The
i
(
A
1
3
respectively). The binding affinity data of the compounds
are presented in Table 2.
A series of novel pyrido[3,2-e][1,2,4]triazolo[1,5-c]pyrimidine
derivatives 5 was prepared and evaluated for affinity in
radioligand binding studies towards all adenosine receptor
subtypes, A , A , A , and A . Compounds 5b, 5d, and 5j
All the investigated compounds did not show binding
affinity towards either A or A ARs at 10 mM or towards A2B
1
2A
1
2A
2B
3
ARs at 1.0 mM concentration. However, compounds 5b, 5d,
and 5j were found to show high binding affinity towards A
showed high selectivity for A versus all other AR subtypes
3
3
with K values of 8.1, 10.4, and 12.1 nM, respectively. This new
i
ARs with K values of 8.1, 10.4, and 12.1 nM, respectively.
Figure 1 shows the radioligand competition binding curves of
class of A3 antagonists shows promising properties and
further investigations appear warranted.
i
the most potent compounds 5b, 5d, and 5j. Primarily it
appeared that the presence of a furyl substituent on the Experimental
triazole ring of compound 5d (10.4 nM) and 5j (12.1 nM)
promotes binding affinity towards the A
3
receptor. This was
General
not surprising since it had been shown in recent X-ray
crystallographic studies of the adenosine A2A receptor in
complex with the furyl-substituted triazolotriazine derivative
ZM241385 (4-(2-(7-amino-2-(2-furyl)[1,2,4]triazolo[2,3-a][1,3,5]-
triazin-5-yl)amino)ethyl)phenol) that an asparagine residue
Melting points of all the compounds were recorded on a Casia-
Siamia (VMP-AM) melting point apparatus and are uncorrected. IR
spectra were recorded on a Perkin-Elmer FT-IR 240-C spectropho-
1
13
tometer using KBr discs. H NMR and C NMR spectra were
recorded on 400, 300, and 75 MHz spectrometer, respectively in
CDCl and CF COOD using TMS as an internal standard. Electron
3
3
6
.55
in transmembranal helix 6 (N253 corresponding to N
)
impact (EI) and chemical ionization mass spectra were recorded
on a VG 7070 H instrument at 70 eV. All high-resolution spectra
were recorded on QSTARXL hybrid MS/MS system (Applied Bio-
systems, USA) under electron spray ionization. All the reactions
were monitored by thin layer chromatography (TLC) on pre-
coated silica gel 60 F254 (mesh) plates; spots were visualized with
UV light. Merck silica gel (60–120 mesh) was used for column
formed a hydrogen bond with the oxygen atom of the furanyl
residue [26, 27].
This asparagine residue is conserved in all four AR subtypes.
Therefore it is likely that a similar interaction can be found in
3
A ARs. In order to investigate the necessity of the furanyl
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7
02
V. Banda et al.
Arch. Pharm. Chem. Life Sci. 2013, 346, 699–707
Table 2. Activity data of compound 5 on radioligand binding affinity towards adenosine receptors.
A
1
rat brain
A2A rat brain
A2B human
3
A human
cortical membranes striatal membranes
recombinant
[ H]PSB-603
recombinant
[ H]PSB-11
3
3
3
3
[
H]CCPA
[ H]MSX-2
K
i
ꢀ SEM
K
i
ꢀ SEM
(nM) (n ¼ 3)
(% inhibition ꢀ SEM (% inhibition ꢀ SEM (% inhibition ꢀ SEM
K
i
ꢀ SEM
K
i
ꢀ SEM
(
nM) (n ¼ 3)
(nM) (n ꢁ 3)
(nM) (n ꢁ 3)
(
% inhibition ꢀ SEM
a)
a)
a)
a)
Code
Compound
at 10 mM) (n ¼ 2)
at 10 mM) (n ¼ 2)
at 1 mM) (n ¼ 2)
at 1 mM) (n ¼ 2)
5b
>10000 (13 ꢀ 10)
>10000 (ꢂ74 ꢀ 27)
>1000 (ꢂ4 ꢀ 17)
8.10 ꢀ 0.96 (n ¼ 5)
5d
3600 ꢀ 2180
113 ꢀ 50
>1000 (17 ꢀ 13)
10.4 ꢀ 1.60 (n ¼ 4)
5e
>10000 (38 ꢀ 1)
>10000 (ꢂ77 ꢀ 2)
>1000 (8 ꢀ 3)
>1000 (44 ꢀ 3)
5f
>10000 (2 ꢀ 11)
>10000 (ꢂ162 ꢀ 9)
>1000 (ꢂ5 ꢀ 1)
>1000 (30 ꢀ 3)
5g
>10000 (6 ꢀ 6)
>10000 (ꢂ52 ꢀ 2)
>1000 (ꢂ26 ꢀ 9)
>1000 (21 ꢀ 6)
5h
>10000 (ꢂ6 ꢀ 4)
>10000 (ꢂ51 ꢀ 11)
>1000 (ꢂ1 ꢀ 3)
>1000 (46 ꢀ 6)
(Continued)
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Arch. Pharm. Chem. Life Sci. 2013, 346, 699–707
Table 2. (Continued)
Selective Adenosine A
3
Receptor Antagonists
703
A
1
rat brain
A2A rat brain
A2B human
3
A human
cortical membranes striatal membranes
recombinant
[ H]PSB-603
recombinant
[ H]PSB-11
3
3
3
3
[
H]CCPA
[ H]MSX-2
K
i
ꢀ SEM
K
i
ꢀ SEM
K
i
ꢀ SEM
K
i
ꢀ SEM
(
nM) (n ¼ 3)
(nM) (n ¼ 3)
(nM) (n ꢁ 3)
(nM) (n ꢁ 3)
(
% inhibition ꢀ SEM
(% inhibition ꢀ SEM (% inhibition ꢀ SEM (% inhibition ꢀ SEM
a)
a)
a)
a)
Code
Compound
at 10 mM) (n ¼ 2)
at 10 mM) (n ¼ 2)
at 1 mM) (n ¼ 2)
>1000 (ꢂ9 ꢀ 2)
at 1 mM) (n ¼ 2)
137 ꢀ 7 (n ¼ 4)
5i
3640 ꢀ 2560
>10000 (ꢂ22 ꢀ 9)
5j
>10000 (9 ꢀ 5)
>10000 (ꢂ6 ꢀ 6)
>1000 (8 ꢀ 3)
12.1 ꢀ 1.6 (n ¼ 4)
a)
Unless otherwise noted.
þ
chromatography. Radioligand binding affinity of representative
pyrido[3,2-e][1,2,4]triazolo[1,5-c]pyrimidine derivatives was evalu-
(m, 4H, Ar–H), 8.7 (s, 1H, Ar–H), 9.6 (s, 1H, Ar–H). EI-mass: 391 (M ),
þ
HRMS m/z calcd. for C21
H
13
N
5
F
3
([MþH] ): 392.1123. Found
ated towards A
1
, A2A, A2B, and A
3
ARs.
392.1131.
Preparation of ethyl N-[3-cyano-6-phenyl-4-(trifluoromethyl)-
-pyridyl]imido formate (3a) was done as described in [23].
Preparation of ethyl N-[3-cyano-6-phenyl-4-(trifluoromethyl)-2-
pyridyl]ethane imidate (3b) was performed as described in [23].
2
2
-(3-Fluorophenyl)-8-phenyl-10-(trifluoromethyl)pyrido-
[
3,2-e][1,2,4]triazolo[1,5-c]pyrimidine (5b)
ꢂ1:
Brown solid, yield: 116 mg (60.2%), mp: 256 C, IR (KBr) cm 1596
C–N), 1H NMR (CDCl
3
) d: 7.16–7.23 (m, 1H, Ar–H), 7.46–7.63
(
(
General procedure for the preparation of 2-aryl-8-phenyl-
m, 4H, Ar–H.), 8.03–8.10 (d, 1H, J ¼ 8.4, Ar–H), 8.15–8.20 (d, 1H,
10-(trifluoromethyl)pyrido[3,2-e][1,2,4]triazolo[1,5-c]-
J ¼ 7.74, Ar–H), 8.28–8.36 (m, 2H, Ar–H), 8.46 (s, 1H, Ar–H), 9.53
þ
(s, 1H, Ar–H). ESI-mass: 410 (M þH), HRMS m/z calcd. for
pyrimidine (5a–d)
Compound 3a (142.4 mg, 0.47 mmol) and aroylhydrazide
0.47 mmol) were dissolved in toluene (3 mL) along with catalytic
þ
C
21
H
12
N
5
F
4
([MþH] ): 410. 1029. Found 410.1037.
(
amount of isobutyric acid (0.15 mL) and stirred at 60–70°C for 2 h;
as a result, solid separated from the solution. Toluene was
removed by filtration and washing was done with CHCl . The
3
crude product was dissolved on heating in toluene (5 mL)
and refluxed for 10–12 h, cooled to room temperature and
toluene was removed under vacuum. The crude product residue
was purified by passing through a column packed with silica gel
2-(3-Chlorophenyl)-8-phenyl-10-(trifluoromethyl)pyrido-
[3,2-e][1,2,4]triazolo[1,5-c]pyrimidine (5c)
ꢂ1:
Yellow solid, yield: 90 mg (45%), mp: 273 C, IR (KBr) cm
1598
1
(
3
C–N), H NMR (CDCl ) d: 7.5 (m, 2H, Ar–H), 7.5–7.7 (m, 3H, Ar–H),
–
8
4
.2–8.4 (m, 4H, Ar–H), 8.25 (s, 1H, Ar–H), 9.5 (s, 1H, Ar–H). EI-mass:
þ
þ
25 (M ), HRMS m/z calcd. for C21
H
12
N
5
ClF
3
([MþH] ): 426.0733.
Found 426.0742.
6
0–120 using 10% ethyl acetate in n-hexane.
2
-(Furan-2-yl)-8-phenyl-10-(trifluoromethyl)pyrido[3,2-e]-
2
,8-Diphenyl-10-(trifluoromethyl)pyrido[3,2-e][1,2,4]-
[
1,2,4]triazolo[1,5-c]pyrimidine (5d)
ꢂ1:
triazolo[1,5-c]pyrimidine (5a)
White solid, yield: 100 mg (55.8%), mp: 250 C, IR (KBr) cm
ꢂ1:
1599 (C–N), 1H NMR (CDCl
3
White solid, yield: 100 mg (54.4%), mp: 248 C, IR (KBr) cm
) d: 6.59–6.62 (dd, 1H, J ¼ 3.77 and
598 (C–N), 1H NMR (CDCl
1.51 –C–H), 7.33–7.39 (d, 1H, J ¼ 3.77, –C–H), 7.56–7.62 (m, 3H,
1
3
) d: 7.5–7.7 (m, 6H, Ar–H). 8.2–8.4
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7
04
V. Banda et al.
Arch. Pharm. Chem. Life Sci. 2013, 346, 699–707
A
B
C
1
1
2 5
0 0
1 2 5
1
1
2 5
0 0
1 0 0
7 5
7
5
2
5
0
5
7
5
5
0
5 0
2
5
2 5
0
0
1 0
0
1
-
1 2
1 0 - 1 0 1 0 - 8 1 0 - 6 1 0 - 4 1 0 - 2
5 j], M
0 - 1 2 1 0 - 1 0 1 0 - 8 1 0 - 6 1 0 - 4 1 0 - 2
- 1 2
1 0 - 1 0 1 0 - 8 1 0 - 6 1 0 - 4 1 0 - 2
1 0
[
[
5 b ], M
[5 d ], M
3
Figure 1. Competition binding curves of compounds 5b, 5d, and 5j at the human A AR expressed in CHO cell membranes versus
3
[
H]PSB-11.
Ar–H), 7.67 (d, 1H, J ¼ 1.51, –C–H), 8.29–8.34 (m, 2H, Ar–H), 8.44
2-(3-Chlorophenyl)-5-methyl-8-phenyl-10-(trifluoromethyl)-
pyrido[3,2-e][1,2,4]triazolo[1,5-c]pyrimidine (5g)
þ
(
s, 1H, Ar–H), 9.51 (s, 1H, Ar–H). ESI-mass: 404 (M þNa), HRMS
þ
m/z calcd. for C19
H
N
10 5
OF
3
([MþNa] ): 404.0735. Found 404.0743.
ꢂ1:
White solid, yield: 160 mg (81%), mp: 275 C, IR (KBr) cm 1623
C–N), 1H NMR (CDCl
(
3
) d: 3.23 (s, 3H, CH
3
), 7.46–7.49 (m, 2H,
Ar–H), 7.54–7.59 (m, 3H, Ar–H), 8.27–8.33 (m, 3H, Ar–H), 8.36 (s,
General procedure for the preparation of 2-aryl-5-methyl-
-phenyl-10-(trifluoromethyl)pyrido[3,2-e][1,2,4]triazolo-
1,5-c]pyrimidine (5e–j)
1
3
1
1
1
1
1
3
H, Ar–H), 8.4 (s, 1H, Ar–H). C NMR (CF COOD, 75 MHz): d 20.92,
8
[
11.84, 114.02, 122.67 (q, J ¼ 276.19 Hz), 123.11, 128.27, 129.89,
30.83, 131.01 (2-carbons), 131.72, 132.26, 132.71 (2-carbons),
34.04, 137.26, 137.99, 145.35 (q, J ¼ 37.41 Hz), 149.39, 161.33,
Compound 3b (142.65 mg, 0.45 mmol) and aroylhydrazide
0.45 mmol) were dissolved in toluene (3 mL) and catalytic
amount of isobutyric acid (0.15 mL) was added. After stirring at
0–70°C for 3 h, a solid separated from the solution. The solid was
(
þ
61.58, 168.81. EI-mass: 439 (M ), HRMS m/z calcd. for
þ
C
22
H
14
N
5
F
3
Cl ([MþH] ): 440.0889. Found 440.0882.
6
filtered and washed with hot ethanol. The crude product was
dried and purified by passing through a column packed with
silica gel 60–120 mesh using 10% ethyl acetate in n-hexane.
2
-(4-Chlorophenyl)-5-methyl-8-phenyl-10-(trifluoromethyl)-
pyrido[3,2-e][1,2,4]triazolo[1,5-c]pyrimidine (5h)
ꢂ1:
White solid, yield: 145 mg (73.4%), mp: 276 C, IR (KBr) cm 1628
C–N), 1H NMR (CDCl
) d: 3.23 (s, 3H, CH ), 7.49–7.58 (m, 5H,
(
3
3
1
3
5-Methyl-2,8-diphenyl-10-(trifluoromethyl)pyrido[3,2-e]-
Ar–H), 8.35–8.40 (m, 5H, Ar–H). C NMR (CF COOD, 75 MHz): d
3
[
1,2,4]triazolo[1,5-c]pyrimidine (5e)
20.56, 122.87, 123.27 (q, J ¼ 277.84 Hz), 128.29, 130.92 (3-carbons),
ꢂ1:
White solid, yield: 127 mg (69.6%), mp: 263 C, IR (KBr) cm 1629
C–N), 1HNMR (CD Cl
) d: 3.23 (s, 3H, CH ), 7.47–7.57 (m, 6H,
Ar–H), 8.28–8.32 (m, 2H, Ar–H), 8.36–8.40 (m, 3H, Ar–H). C NMR
CF
131.28 (3-carbons), 131.50 (3-carbons), 132.59 (3-carbons), 137.90,
1
4
41.15, 145.09 (q, J ¼ 37.41 Hz), 149.33, 161.51, 169.50. ESI-mass:
(
3
3
1
3
þ
þ
40 (M þH), HRMS m/z calcd. for C22
H
14
N
5
F
3
Cl ([MþH] ):
(
3
COOD, 75 MHz): d 20.84, 111.54, 122.72 (q, J ¼ 276.74 Hz),
440.0889. Found 440.0875.
1
1
1
22.98, 129.57, 130.19 (2-carbons), 130.96 (6-carbons), 131.06,
32.65 (2-carbons), 134.36, 137.88, 145.05 (q, J ¼ 37.41 Hz), 149.38,
2-(4-Fluorophenyl)-5-methyl-8-phenyl-10-(trifluoromethyl)-
þ
61.45, 170.20. ESI-mass: 406 (M þH), HRMS m/z calcd. for
pyrido[3,2-e][1,2,4]triazolo[1,5-c]pyrimidine (5i)
þ
C
22
H
15
N
5
F
3
([MþH] ): 406.1279. Found 406.1271.
ꢂ1:
Orange yellow, yield: 130 mg (68.28%), mp: 264 C, IR (KBr) cm
1
1
596 (C–N), H NMR (CDCl
3
) d: 3.21 (s, 3H, CH
3
), 7.17–7.24 (t, 2H,
2
-(3-Fluorophenyl)-5-methyl-8-phenyl-10-(trifluoromethyl)-
J ¼ 8.4, Ar–H), 7.52–7.61 (m, 3H, Ar–H), 8.27–8.34 (m, 2H, Ar–H),
13
8
3
.35–8.44 (m, 3H, Ar–H). C NMR (CF COOD, 75 MHz): d 20.79,
pyrido[3,2-e][1,2,4]triazolo[1,5-c]pyrimidine (5f)
ꢂ1:
111.64, 117.86, 118.13, 122.72 (q, J ¼ 275.64 Hz), 122.97, 126.18,
White solid, yield: 140 mg (73.5%), mp: 290 C, IR (KBr) cm 1624
C–N), 1H NMR (CDCl
130.95 (4-carbons), 132.54, 132.63 (4-carbons), 137.84, 145.16
(
3
) d: 3.22 (s, 3H, CH ), 7.15–7.24 (m, 1H,
3
(
(
q, J ¼ 37.96 Hz), 149.44, 161.46, 166.21, 169.57. ESI-mass: 424
Ar–H), 7.49–7.62 (m, 4H, Ar–H), 8.02–8.13 (dd, 1H, J ¼ 1.51 and 8.6
þ
þ
M þH), HRMS m/z calcd. for C22
H
14
N
5
F
4
([MþH] ): 424.1185.
Ar–H), 8.16–8.23 (d, 1H, J ¼ 7.74, Ar–H), 8.27–8.36 (m, 2H, Ar–H),
1
3
Found 424.1267.
8
1
1
1
1
3
.4 (s, 1H, Ar–H). C NMR (CF COOD, 75 MHz): 20.86, 111.88,
16.77, 117.08, 120.93, 121.22, 122.69 (q, J ¼ 278.94 Hz), 123.05,
2-(Furan-2-yl)-5-methyl-8-phenyl-10-(trifluoromethyl)-
26.07, 130.99 (2-carbons), 132.06, 132.69 (2-carbons), 132.82,
37.95, 145.35 (q, J ¼ 36.86 Hz), 149.45, 161.36, 161.70, 167.07,
pyrido[3,2-e][1,2,4]triazolo[1,5-c]pyrimidine (5j)
þ
ꢂ1:
White solid, yield: 115 g (64.68%), mp: 296 C, IR (KBr) cm 1599
69.34. ESI-mass: 424 ([MþH] ), HRMS m/z calcd. for C22
H
14
N
5
F
4
þ
1
([MþH] ): 424.1185. Found 424.1177.
(C–N), H NMR (CDCl
3
) d: 3.2 (s, 3H, CH
3
), 6.58–6.62 (m, 1H, –C–H),
ß 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
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Arch. Pharm. Chem. Life Sci. 2013, 346, 699–707
Selective Adenosine A
3
Receptor Antagonists
705
7
7
.31–7.38 (d, 1H, J ¼ 3.2, –C–H), 7.52–7.58 (m, 3H, Ar–H),
5-Methyl-8-phenyl-2-(thiophen-2-yl)-10-(trifluoromethyl)-
pyrido[3,2-e][1,2,4]triazolo[1,5-c]pyrimidine (5n)
1
.66 (m, 1H, –C–H), 8.25–8.35 (m, 2H, Ar–H), 8.38 (s, 1H, Ar–H).
1
3
C NMR (CF
3
COOD, 75 MHz): d 21.13, 111.49, 114.57, 119.15,
ꢂ1
–
Yield: 0.685 g (69%), mp: 270–272 C, IR (KBr) cm : 1627 (C–N), H
NMR (400 MHz, CDCl ) d: 3.20 (s, 3H, CH ), 7.18–7.20 (m, 1H, HC-
thienyl), 7.51–7.52 (m, 1H, HC-thienyl), 7.55–7.59 (m, 3H, Ar–H),
.02–8.04 (m, 1H, HC-thienyl), 8.30–8.32 (m, 2H, Ar–H), 8.39 (s, 1H,
1
1
1
22.64 (q, J ¼ 276.19 Hz), 123.17, 130.75, 131.08 (3-carbons),
3
3
32.68 (3-carbons), 138.10, 145.25, 145.61 (q, J ¼ 37.96Hz),
þ
48.80, 149.74, 161.08, 161.84. ESI-mass: 396 (M þH), HRMS
8
þ
m/z calcd. for
96.1082.
C
20
H
13
N
5
OF
3
([MþH] ): 396.1072. Found
13
3
Ar–H). C NMR (CF COOD, 75 MHz): d 20.60, 110.75, 122.44 (q,
3
J ¼ 275.09 Hz), 122.68, 128.89, 130.24, 130.77 (2-carbons), 131.47,
1
32.12 (2-carbons), 132.49, 132.87, 133.11, 137.06, 143.72
þ
General procedure for the preparation of 2-aryl-5-methyl-
-phenyl-10-(trifluoromethyl)pyrido[3,2-e][1,2,4]triazolo-
1,5-c]pyrimidine (5k–o)
(q, J ¼ 37.41 Hz), 148.98, 149.06, 160.36, 165.47. MS [ES (m/z, %
þ
relative intensity)]: 412.1 (M þH, 100).
8
[
5-Methyl-8-phenyl-2-(pyridin-3-yl)-10-(trifluoromethyl)-
To a solution of ethyl-3-cyano-6-phenyl-4-(trifluoromethyl)pyri-
dine-2-ethanamidate 3b (2.40 mmoles) in N,N-dimethyl formam-
ide (5 mL) was added aroylhydrazide (2.7 mmol) and the resulting
mixture was heated at 120°C for 2–4 h, under a nitrogen
atmosphere. After completion of reaction, it was filtered, washed
with hot ethanol, and dried. The crude product was further
purified by passing through a column packed with silica gel
pyrido[3,2-e][1,2,4]triazolo[1,5-c]pyrimidine (5o)
ꢂ1
1
Yield: 0.722 g (74%), mp: 283–285 C, IR (KBr) cm : 1625 (C–N), H
NMR (400 MHz, CDCl
3 3
) d: 3.24 (s, 3H, CH ), 7.57–7.59 (m, 3H,
Ar–H), 8.26–8.28 (m, 2H, Ar–H), 8.30–8.33 (m, 2H, Ar–H), 8.42
1
3
(
3
s, 1H, Ar–H), 8.82–8.83 (m, 2H, Ar–H). C NMR (CF COOD,
75 MHz): 21.02, 109.45, 112.06, 121.79 (q, J ¼ 276.19 Hz), 123.83,
1
26.80, 129.72, 131.15, 131.60, 132.10, 135.56 (5-carbons), 144.03
6
0–120 mesh using 10% ethylacetate in n-hexane.
þ
(3-carbons), 145.05 (q, J ¼ 37.96 Hz), 158.18, 158.57. MS [ES (m/z, %
þ
relative intensity)]: 407.1 (M þH, 100).
2-(2-Chlorophenyl)-5-methyl-8-phenyl-10-(trifluoromethyl)-
pyrido[3,2-e][1,2,4]triazolo[1,5-c]pyrimidine (5k)
ꢂ1
Radioligand binding studies
Yield: 0.780 g (74%), mp: 258–260 C, IR (KBr) cm : 1594 (C–N),
1
Materials
3 3
H NMR (400 MHz, CDCl ) d: 3.16 (s, 3H, CH ), 7.36–7.37 (m, 2H,
3
3
6
[
H]CCPA ([ H]2-chloro-N -cyclopentyladenosine) was obtained
Ar–H), 7.49–7.52 (m, 4H, Ar–H), 8.15–8.16 (m, 1H, Ar–H), 8.23–8.26
3
3
1
3
(
7
1
m, 2H, Ar–H), 8.34 (s, 1H, Ar–H) ppm. C NMR (CF
3
COOD,
5 MHz): d 20.81, 111.16, 112.26, 117.40, 122.45 (q, J ¼ 277.01 Hz),
28.85 (2-carbons), 128.98, 129.83, 130.90, 132.12, 132.48 (2-
from NEN Life Sciences (58 Ci/mmol), [ H]MSX-2 ([ H]3-(3-hydroxy-
propyl)-7-methyl-8-(m-methoxystyryl)-1-propargylxanthine) from
3
Amersham (84 Ci/mmol), [ H]PSB-603 (8-(4-(4-(4-chlorophenyl)-
carbons), 132.95, 133.08, 134.37, 135.95, 136.78, 143.68
piperazine-1-sulfonyl)phenyl)-1-propylxanthine) from GE Health-
þ
3
3
care (73 Ci/mmol), and [ H]PSB-11 ([ H]-8-ethyl-4-methyl-2-phenyl-
(8R)-4,5,7,8-tetrahydro-1H-imidazo[2,1-i]-purin-5-one) from Quo-
tient Biosciences (53 Ci/mmol). The precursors of the radioligands
were synthesized at the University of Bonn [24, 28, 29].
(
q, J ¼ 37.23 Hz), 151.80, 156.41, 167.56. MS [ES (m/z, % relative
þ
þ
intensity)]: 480.1 (M þ1þK, 30), 482.2 (M þ2þK, 10), 462.1
þ
þ
þ
(
(
M þ1þNa, 100), 464.1 (M þ2þNa, 25), 440.1 (MþH , 30), 442.2
þ
M þ2, 6).
Membrane preparations for A1, A2A, A2B, and
2
-[5-Methyl-8-phenyl-10-(trifluoromethyl)pyrido[3,2-e]-
A receptor assays
3
[
1,2,4]triazolo[1,5-c]pyrimidin-2-yl]phenol (5l)
Frozen rat brains were obtained from PelFreez (Rogers, AR, USA).
Rat brains were dissected to obtain cortical membrane prepara-
ꢂ1
Yield: 0.680 g (68%), mp: 272–275 C, IR (KBr) cm : 3030 (–OH),
1
1
621 (C–N), H NMR (400 MHz, CDCl
6.99 (m, 2H, Ar–H), 7.33–7.42 (m, 2H, Ar–H), 7.53–7.54 (m, 2H,
3 3
) d: 3.18 (s, 3H, CH ), 6.93–
tions for A
previously described [28, 30–34]. Membranes of CHO cells
expressing the human A2B receptor or the human A receptor
1
and striatal membrane preparations for A2A assays as
Ar–H), 8.22 (m, 1H, Ar–H). 8.27–8.29 (m, 2H, Ar–H), 8.44 (s, 1H,
Ar–H), 11.10 (s, 1H, OH). C NMR (CF COOD, 75 MHz): d 20.78,
3
1
3
3
were prepared by scraping the cells off the previously frozen cell
culture dishes in ice-cold hypotonic buffer (5 mM Tris–HCl, 2 mM
EDTA, pH 7.4). The cell suspension was homogenized on ice for
1
1
1
1
4
13.38, 119.20, 122.44 (q, J ¼ 277.84 Hz), 129.02 (2-carbons),
30.97 (2-carbons), 132.12 (2-carbons), 132.47 (2-carbons),
36.10,137.18, 138.77, 145.73 (q, J ¼ 37.41 Hz), 152.18, 155.38,
2
1
0 s with an Ultra-Turrax and spun down for 10 min (4°C) at
000g. The supernatant was subsequently centrifuged for 60 min
þ
58.12, 158.64, 160.36, 166.60. MS [ES (m/z, % relative intensity)]:
þ
22.1 (M þH, 100).
at 48000g. The obtained membrane pellets were re-suspended in
0 mL of 50 mM Tris–HCl buffer, pH 7.4 and centrifuged once
1
5
-Methyl-2-(4-nitrophenyl)-8-phenyl-10-(trifluoromethyl)-
more under the same conditions. Then the membrane pellets
were re-suspended and homogenized in the required amount of
50 mM Tris–HCl buffer, pH 7.4 to obtain a protein concentration
of 1–3 mg/mL. The protein concentration was determined by the
method of Lowry et al. using bovine serum albumin as a standard
reference. Aliquots of the membrane preparation (1 mL each)
were stored at ꢂ80°C until they were used in the binding assays.
pyrido[3,2-e][1,2,4]triazolo[1,5-c]pyrimidine (5m)
Yield: 0.640 g (64%), mp: 268–270 C, IR (KBr) cm : 1629 (C–N), H
NMR (400 MHz, CDCl
Ar–H), 8.28–8.30 (m, 2H, Ar–H), 8.34–8.36 (m, 2H, Ar–H), 8.45
ꢂ1
1
3 3
): d 3.33 (s, 3H, CH ), 7.52–7.54 (m, 3H,
1
3
3
(s, 1H, Ar–H), 8.52–8.55 (m, 2H, Ar–H). C NMR (CF COOD,
7
5 MHz): d 20.81, 111.84, 122.47 (q, J ¼ 275.64 Hz), 123.12, 125.95
(
(
2-carbons), 130.72, 130.90 (2-carbons), 131.06 (2-carbons), 132.39
2-carbons), 136.80, 137.60, 145.01 (q, J ¼ 37.96 Hz), 149.43,
A and A radioligand binding assays
1
2A
þ
1
51.43, 151.81, 161.19, 161.54, 167.40. MS [ES (m/z, % relative
Binding assays for A1 and A were performed essentially as
2A
þ
intensity)]: 451.1 (M þH, 100).
described in the literature [29, 30]. Stock solutions of the
ß 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.archpharm.com

7
06
V. Banda et al.
Arch. Pharm. Chem. Life Sci. 2013, 346, 699–707
compounds were prepared in dimethyl sulfoxide (DMSO), the
final concentration of DMSO in the assay being 2.5%. Membrane
preparations (30 mg/vial) were incubated in 96-well plates with
Authors are thankful to the Director, IICT, Hyderabad, India for her
constant encouragement. Authors (B. Veeraswamy, C. Kurumurthy,
G. Santhosh Kumar, P. SambasivaRao) are also thankful to CSIR, New
Delhi for the financial support in the form of senior research fellowship
and contingency grant. Author M. Raghuprasad is thankful to the
management of Dr. B. V. Raju foundation and Sri Vishnu educational
society, Bhimavaram, East Godavari Dist, Andhra Pradesh, India for
their sponsorship of a research stay at the University of Bonn.
3
3
0
.5 nM [ H]CCPA (rat A
Tris–HCl, pH 7.4 in a total volume of 200 mL. Incubation was
carried out at room temperature for 90 (rat A ) or 30 min (rat A2A),
respectively. Nonspecific binding was determined in the presence
of 10 mM CADO (2–chloroadenosine) for rat A or 10 mM CGS15943
9-chloro-2-(2-furanyl)-[1,2,4]triazolo[1,5-c]quinazolin-5-amine) for
1
) or 1.0 nM [ H]MSX-2 (rat A2A) in 50 mM
1
1
(
rat A2A. The incubation was terminated by rapid filtration
through GF/B glass filters (Whatman, Dassel, Germany) using a
Brandel cell harvester (Brandel, Gaithersburg, MD). Scintillation
cocktail was added to the filter plates (40 mL Microscent-20 per References
well) and after an incubation of 9 h filter-bound radioactivity was
measured by liquid scintillation counting.
[1] B. B. Fredholm, G. Arslan, L. Halldner, B. Kull, G. Schulte,
W. Wasserman, Naunyn Schmeideberg’s Arch. Pharmacol. 2000,
362, 364–374.
A2B radioligand binding assays
3
Competition experiments with [ H]PSB-603 were performed in a
[2] B. B. Fredholm, A. P. IJzerman, K. A. Jacobson, J. Linden,
final volume of 1000 mL containing 10 mL of test compound,
C. E. Müller, Pharmacol. Rev. 2011, 63, 1–34.
7
90 mL of buffer (50 mM Tris–HCl, pH 7.4), 100 mL of radioligand
[
[
[
3] S. Ferre, B. B. Fredholm, M. Morelli, P. Popoli, K. Fuxe, Trends
Neurosci. 1997, 20, 482–487.
solution (final concentration 0.3 nM), and 100 mL of membrane
preparation (30 mg protein per vial, pre-incubated with 2 U ADA/
mg protein). The final DMSO concentrations did not exceed 1%.
Nonspecific binding was determined in the presence of 10 mM
4] K. Varani, S. Gessi, A. Dalpiaz, P. A. Borea, Br. J. Pharmacol.
1
996, 117, 1693–1701.
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Pharmacol. 1997, 122, 386–392.
8
-cyclopentyl-1,3-dipropylxanthine (DPCPX). After an incubation
time of 75 min at room temperature, the assay mixture was
filtered through GF/B glass fiber filters using a Brandel harvester
[6] K. Varani, S. Gessi, S. Dionisotti, E. Ongini, P. A. Borea, Br. J.
Pharmacol. 1998, 123, 1723–1731.
(Brandel). Filters were washed four times (3–4 mL each) with ice-
cold 50 mM Tris–HCl buffer, pH 7.4, containing 0.1% bovine
serum albumin (BSA) and transferred to mini vials. Two milliliters
scintillation cocktail (Ultima Gold, Canberra Packard) was added
and after an incubation of 9 h radioactivity was counted in a
liquid scintillation counter at an efficiency of 54%.
[
7] S. Gessi, K. Varani, S. Merighi, E. Ongini, P. A. Borea, Br. J.
Pharmacol. 2000, 129, 2–11.
[
8] F. Libert, J. Van Sande, A. Lefort, A. Czernilofsky,
J. E. Dumont, G. Vassart, H. A. Ensinger, K. D. Mendla,
Biochem. Biophys. Res. Commun. 1992, 187, 919–926.
[
9] A. Townsend-Nicholson, J. Shine, Brain Res. Mol. Brain Res.
A radioligand binding assays
3
3
1992, 16, 365–370.
Binding studies with the A
3
AR radioligand [ H]phenyl-8-ethyl-4-
3
methyl-(8R)-4,5,7,8-tetrahydro-1H-imidazo[2,1-i]purine-5-one ([ H]-
PSB-11, 1 nM) were performed in Tris–HCl buffer (50 mM Tris, pH
[10] J. S. Fink, D. R. Weaver, S. A. Rivkees, R. A. Peterfreund,
A. E. Pollack, E. M. Adler, S. M. Reppert, Brain Res. Mol. Brain
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7
2
.4) containing 100 mg of protein per vial (pre-incubated with
U ADA/mg protein) in a final volume of 400 mL. Nonspecific
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6
binding was determined in the presence 100 mM R-PIA (N -[(R)-
phenylisopropyl]adenosine). After 45 min of incubation at room
temperature to allow equilibrium to be reached, bound and free
radioactivity were separated by filtering the assay mixture
through Whatman GF/B glass fiber filters using a Brandelcell
harvester (Brandel). Filters were rinsed three times with 2 mL of
ice-cold Tris–HCl buffer (50 mM, pH 7.4) each and incubated for
[
[
[
12] C. E. Müller, B. Stein, Curr. Pharm. Des. 1996, 2, 501–530.
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9
h with scintillation cocktail (Ultima Gold, Canberra Packard)
before radioactivity was counted by liquid scintillation counting.
The compounds that inhibited the specific [ H]PSB-11 binding by
[
16] P. G. Baraldi, B. Cacciari, R. Romagnoli, G. Spalluto,
3
A. Monopoli, E. Ongini, K. Varani, P. A. Borea, J. Med. Chem.
>
50% at a concentration of 1 mM in the initial screening were
2
002, 45, 115–126.
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subsequently re-analyzed at a range of concentrations to
determine their IC50 values. Curves were determined using seven
to nine different concentrations of test compounds spanning 3
orders of magnitude.
[
[
2
18] B. Sirisha, B. Narsaiah, T. Yakaiah, G. Gayatri, G. Narahari
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010, 45, 1739–1745.
Data analysis
Data were analyzed using Graph Pad PRISM version 6.0 (San
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Diego, CA). For nonlinear regression analysis, the Cheng–Prusoff
3
equation and K
D
values of 0.2 nM (rat A
1
) for [ H]CCPA and 4.9 nM
3
3
(human A ) for [ H]PSB-11, respectively, were used to calculate
K
i
values from IC50 values.
[20] C. E. Müller, Curr. Top. Med. Chem. 2003, 3, 445–462.
ß 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.archpharm.com

Arch. Pharm. Chem. Life Sci. 2013, 346, 699–707
Selective Adenosine A
3
Receptor Antagonists
707
[
[
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