Synthesis and adenosine receptors binding studies of new fluorinated analogues of pyrido[2,3-d]pyrimidines and quinazolines

Article abstract of DOI:10.1007/s00044-017-2099-z

A series of new fluorine containing pyrido[2,3-d]pyrimidines and imidazo[1,2-c]pyrido[3,2-e]pyrimidines along with a series of bioisosteric fluorinated quinazolines were synthesised following appropriate synthetic schemes and characterised by spectral analytical means. X-ray crystal structure of the key precursor 1 (2-amino-3-cyano-4-trifluoro-methyl-6-phenyl-pyridine) was also determined to gain insight into its reactivity. Binding affinity data of all the compounds for adenosine receptors (ARs) showed that pyrido[2,3-d]pyrimidine scaffold with free amino (NH₂) group at 2- and 4-position (2a) exhibited the maximum binding affinity for hA₃ AR with similar affinity for the hA₁ and somewhat lower affinity for hA_2A ARs resulting in a compound with no A₃ selectivity vs. A₁ and moderate selectivity vs. A_2A AR (K_i hA₁ = 0.62 μM, hA_2A = 3.59 μM and hA₃ = 0.42 μM). Interestingly, the replacement of both the amino groups with carbonyl (C=O) groups (compound 4) resulted in significantly improved affinity for hA₁ AR but with moderate selectivity against hA_2A and hA₃ ARs (K_i hA₁ = 0.17 μM, hA_2A = 0.67 μM and hA_{3 =}0.68 μM). In case of fluorinated quinazolines, only compound 18a showed remarkable affinity for hA₁ AR with significant selectivity against hA_2A and hA₃ ARs (K_i hA₁ = 0.73 μM, hA_2A CloseSPigtSPi 30 μM and hA₃ = 9.27 μM). The preliminary results of these compounds demonstrate that the fluorinated pyrido[2,3-d]pyrimidine and imidazo[1,2-c]pyrido[3,2-e]pyrimidine can be considered as promising scaffolds for further optimisation in search of potential antagonists with better affinity and selectivity towards hA₁ and hA₃ ARs.

Full text of DOI:10.1007/s00044-017-2099-z

MEDICINAL
CHEMISTRY
RESEARCH
Med Chem Res
DOI 10.1007/s00044-017-2099-z
ORIGINAL RESEARCH
Synthesis and adenosine receptors binding studies of new
fluorinated analogues of pyrido[2,3-d]pyrimidines and
quinazolines
Balakumar Chandrasekaran1 Pran Kishore Deb2 Sonja Kachler3
●
●
●
Raghuram Rao Akkinepalli1,5 Raghuprasad Mailavaram4 Karl-Norbert Klotz3
●
●
Received: 31 July 2017 / Accepted: 12 October 2017
© Springer Science+Business Media, LLC 2017
Abstract A series of new fluorine containing pyrido[2,3-d]
pyrimidines and imidazo[1,2-c]pyrido[3,2-e]pyrimidines
along with a series of bioisosteric fluorinated quinazolines
were synthesised following appropriate synthetic schemes
and characterised by spectral analytical means. X-ray
crystal structure of the key precursor 1 (2-amino-3-cyano-
4-trifluoro-methyl-6-phenyl-pyridine) was also determined
to gain insight into its reactivity. Binding affinity data of all
the compounds for adenosine receptors (ARs) showed that
pyrido[2,3-d]pyrimidine scaffold with free amino (NH2)
group at 2- and 4-position (2a) exhibited the maximum
binding affinity for hA3 AR with similar affinity for the hA1
and somewhat lower affinity for hA2A ARs resulting in a
compound with no A3 selectivity vs. A1 and moderate
selectivity vs. A2A AR (Ki hA1 = 0.62 µM, hA2A = 3.59 µM
and hA3 = 0.42 µM). Interestingly, the replacement of both
the amino groups with carbonyl (C=O) groups (compound
4) resulted in significantly improved affinity for hA1 AR but
with moderate selectivity against hA2A and hA3 ARs (Ki
hA1 = 0.17 µM, hA2A = 0.67 µM and hA3 = 0.68 µM). In
case of fluorinated quinazolines, only compound 18a
showed remarkable affinity for hA1 AR with significant
selectivity against hA2A and hA3 ARs (Ki hA1 = 0.73 µM,
hA2A > 30 µM and hA3 = 9.27 µM). The preliminary results
of these compounds demonstrate that the fluorinated pyrido
[2,3-d]pyrimidine and imidazo[1,2-c]pyrido[3,2-e]pyr-
imidine can be considered as promising scaffolds for further
optimisation in search of potential antagonists with better
affinity and selectivity towards hA1 and hA3 ARs.
Electronic supplementary material The online version of this article
(https://doi.org/10.1007/s00044-017-2099-z) contains supplementary
material, which is available to authorized users.
* Pran Kishore Deb
●
Keywords Fluorinated pyrido[2,3-d]pyrimidines
pdeb@philadelphia.edu.jo
prankishore1@gmail.com
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●
Fluorinated quinazolines Adenosine receptors binding
* Raghuram Rao Akkinepalli
X-ray crystallography
raghumed@gmail.com
1
Pharmaceutical Chemistry Division, University Institute of
Pharmaceutical Sciences and UGC Center of Advanced Study in
Pharmaceutical Sciences (UGC-CAS), Panjab University,
Chandigarh 160 014, India
Introduction
2
Faculty of Pharmacy, Philadelphia University-Jordan, P.
Adenosine receptors (ARs) belong to the super family of G-
protein-coupled receptors (GPCRs) and are classified into
four subtypes A1, A2A, A2B, and A3 ARs, respectively, all
of which exhibit distinct physiological functions (Fredholm
et al. 2011). A1 ARs are mainly present in the brain with
minimum levels in the heart, lungs, kidney, adipose tissue,
stomach, spleen, and liver (Ribeiro et al. 2002). A2A ARs
are highly distributed in blood platelets, striatum, nucleus
O. BOX (1), Philadelphia University 19392, Jordan
3
Institut für Pharmakologie und Toxikologie, Universität
Würzburg, Versbacher Strasse 9, Würzburg 97078, Germany
4
Pharmaceutical Chemistry Division, Sri Vishnu College of
Pharmacy, Vishnupur, Bhimavaram, Andhra Pradesh, India
5
Present address: National Institute of Pharmaceutical Education
and Research (NIPER), Sector 67, S.A.S. Nagar, Mohali, Punjab
160 062, India

Med Chem Res
accumbens, and olfactory tubercle (Preti et al. 2015), while
A2B ARs regulate a varied number of physiological and
pathological events that involve lungs, blood vessels, and
bladder (Kalla and Zablocki 2009). A3 ARs are highly
expressed in immune cells, lung and liver and at lower
densities in heart, aorta and brain (Borea et al. 2009).
Agonists selective for A1 AR showed antinociceptive
activity and are thought to be useful in the treatment of
stroke, epilepsy, migraine, pain, cardiac ischemia, and
arrhythmias (Cosimelli et al. 2016) while antagonists might
be useful in conditions such as cognitive dysfunction
associated with ageing, stroke-induced brain damage or
neurodegenerative disorders like Alzheimer’s disease (AD)
(Ribeiro et al. 2002; Baraldi et al. 2008). A2A AR antago-
nists are being considered as potential therapeutic alter-
native for the treatment of Parkinson’s disease (PD) (Preti
et al. 2015). Recent studies reported the use of highly potent
and selective antagonists such as ZM241385, SCH58261
and some xanthine derivatives (istradefylline) as pharma-
cological tools for this receptor subtype (Bahreyni et al.
2017; Dong et al. 2017; Dungo and Deeks 2013; Kazemi
et al. 2017; Shook et al. 2011; van Rhee et al. 1996; Wardas
et al. 2001). Recently, istradefylline has been approved for
marketing in Japan as an anti-PD agent (Venkatesan et al.
2014). Selective A2B antagonists were shown to decrease
pain (inflammatory), and are promising candidates for the
treatment of asthma (Brown et al. 2008) and diabetes (Kalla
and Zablocki 2009; Baraldi et al. 2008). Agonists of the A3
AR are thought to be useful for the treatment of stroke, lung
injury (asthma and COPD), cardiac ischemia, rheumatoid
arthritis, and cancer (Borea et al. 2009; Jacobson et al.
2009). The blockade of A3 ARs could be beneficial for the
treatment of glaucoma, stroke, asthma, and renal failure
(Borea et al. 2009; Jacobson et al. 2009). During the past
years a number of potent and selective AR antagonists have
been developed, including xanthines and non-xanthine
derivatives (Baraldi et al. 2008). Recently reported AR
antagonists under different stages of clinical and pre-clinical
trial are presented in Figs. 1 and 2 (Muller and Jacobson
2011).
The derivatives of pyrido[2,3-d]pyrimidine have shown
remarkable biological and pharmacological activities, in
particular for the treatment of various inflammatory condi-
tions such as allergy, arthritis, and asthma (Suhagia et al.
Fig. 1 Recently reported potent
hA1 and hA2A ARs antagonists
under different stages of
development
A1 AR Antagonists
N
N
O
OH
HN
N
N
N
H
N
HO
SLV-320 (Ki = 1 nM)
FK-453 (Ki = 18 nM)
(Preclinical-Heart failure)
(Preclinical-Acute renal failure)
A
2A AR Antagonists
OCH3
N
O
N
CH3
N
H3C
N
NH
OH
S
N
N
OCH3
OCH3
O
CH3
O
N
O
H3C
Istradefylline (Ki = 12 nM)
Tozadenant (Ki = 11.5 nM)
(Antiparkinsonian drug-Marketed)
(Antiparkinsonian agent-Phase-II)
N
O
N
N
OCH3
N
N
N
N
N
NH2
O
N
N
N
O
N
N
NH2
CH3
H2N
Preladenant (Ki = 0.9 nM)
(Antiparkinsonian agent-Phase-III; Discontinued)
Vipadenant (Ki = 1.3 nM)
(Antiparkinsonian agent- Phase-III; Discontinued)

Med Chem Res
2006; Bulicz et al. 2006; Hafez et al. 2008; Nam et al.
2001). Recently, a series of pyrido[2,3-d]pyrimidine deri-
vatives have also been reported as selective bicyclic A2B
AR antagonists (Eastwood et al. 2011). Thus, it was thought
of interest to further explore this pyrido[2,3-d]pyrimidine
scaffold and evaluate some novel analogues as potent AR
antagonists.
Further, quinazolines are considered as bioisosters of
pyrido[2,3-d]pyrimidines. Quinazoline derivatives exhib-
ited various interesting pharmacological properties includ-
ing adenosine receptor antagonism (Bertelli et al. 2000;
Khan et al. 2014; Smutny et al. 2016). It has been observed
that the presence of a fluoro or trifluoromethyl group at a
strategic position on the heterocyclic nucleus enhances the
activity of a molecule by increasing the lipophilicity
(Balakumar et al. 2010). It is interesting to note that the
pyrimidine core being part of the endogenous ligand of ARs
(adenosine), is a recurrent substructural motif of various
heterofused bicyclic and tricyclic AR antagonists. Based on
these observations, we earlier designed (Pran Kishore et al.
2011; Balakumar et al. 2017), synthesized and reported a
series of triazolothienopyrimidines (Raghu Prasad et al.
2008),
4H-pyrimido[2,1-b]benzothiazole-2-arylamino-3-
cyano-4-ones (Balakumar et al. 2012), pyrido[3,2-e][1,2,4]
triazolo[1,5-c]pyrimidines (Veeraswamy et al. 2013), 2-
amino[1,2,4]triazolo[1,5-c]quinazolines (Burbiel et al.
2016) as possible ARs antagonists. In continuation to our
ongoing efforts, we are reporting a convenient method for
the synthesis of trifluoromethyl substituted pyrido[2,3-d]
pyrimidine analogues from a versatile precursor 1 (2-amino-
3-cyano-4-trifluoro-methyl-6-phenyl-pyridine). In order to
understand and illustrate the reactivity of this key precursor
(1), we examined the X-ray crystal structure to gain insight
into its structural features. All the newly synthesized
Fig. 2 Recently reported potent
hA2B and hA3 ARs antagonists
under different stages of
A2B AR Antagonists
CN
development
N
N
N
N
N
O
NH
N
S
N
N
H
N
N
N
CH3
LAS-38096 (Ki = 3.5 nM)
QAF-805 (Ki = 3.4 nM)
(Antiinflammatory-Preclinical)
(Asthma- Phase-I)
A3 AR Antagonists
O
O
N
H
NH
N
N
H3CO
NH
N
N
N
N
Br
N
O
N
N
CH3
CH3
KF-26777 (Ki = 0.2 nM)
MRE-3008-F-20 (Ki = 0.82 nM)
(Asthma- Preclinical)
(Asthma- Preclinical)
F3C
OCH3
O
HN
N
N
H
N
N
N
N
N
H3C
N
H
N
N
OT-7999 (Ki = 0.95 nM)
VUF-5574 (Ki = 4.03 nM)
(Glaucoma-Preclinical)
(Asthma- Preclinical)

Med Chem Res
compounds (2a–c, 4, 6a–b) along with the previously
reported bioisosteric fluorinated fused quinazolines (17a–c
and 18a–c) have been evaluated for their binding affinity
towards all the subtype of ARs.
ppm; MS (ESI): m/z (%) = 306.1 (15) [M + H]+, 265.0
42]. Elemental analysis: calcd. For
C14H10F3N5: C, 55.08; H, 3.30; N, 22.94%. Found: C,
55.14; H, 3.36; N, 22.87%.
_
(100) [M+
4-Amino-7-phenyl-5-(trifluoromethyl)-pyrido[2,3-d]
pyrimidin-2(1H)-thione (2b)
Experimental
Reactions were routinely monitored by thin layer chroma-
tography (TLC) on silica gel (precoated F254 Merck plates)
and the spots were visualized under UV light (254 nm).
Melting points were recorded in open capillaries on
LABINDIA melting point apparatus and were uncorrected.
IR spectra were recorded on Perkin Elmer FT-IR Spectro-
Yellow solid, yield: 64%, M.P.: 236–237 °C, IR: 3468,
3395, 3301, 3180, 1637, 1581, 1371, 1261, 1198, 1131
cm−1; 1H NMR (DMSO-d6, 400 MHz) δ 6.30 (s, 2H, NH2),
7.46–7.51 (m, 3H, Ar–H), 7.77 (s, 1H, NH), 8.06–8.10 (m,
3H, Ar–H) ppm; 13C NMR (DMSO-d6, 100 MHz) δ 104.8,
113.3, 121.4, 126.6, 128.8, 129.9, 135.4, 137.9, 155.6,
156.8, 166.6 ppm; MS (ESI): m/z (%) = 323.1 (15) [M +
H]+, 280.1 (100) [M+ _ 42]. Elemental analysis: calcd. For
C14H9F3N4S: C, 52.17; H, 2.81; N, 17.38%. Found: C,
52.12; H, 2.87; N, 17.44%.
1
meter (Spectrum RX I) using KBr pellet technique. H and
13C NMR spectra were determined in CDCl3 or DMSO-d6
with a Bruker Avance II [400 MHz (1H) and 100 MHz
(13C)] spectrometer and signals were recorded in parts per
million (δ) downfield from tetramethylsilane as an internal
standard. Mass spectra (ESI) were recorded on Waters
Micromass Q-TOF Micro. Silica gel (60–120 mesh) was
used for column chromatography. The X-ray crystal-
lographic study was performed for precursor 1. All mea-
surements were performed on an Oxford X Calibur Mova
diffractometer using graphite-monochromated MoKα
radiation (λ = 0.71069 Ǻ) and equipped with a CCD
detector.
4-Amino-7-phenyl-5-(trifluoromethyl)-pyrido[2,3-d]
pyrimidin-2(1H)-one (2c)
White solid, yield: 68%, M.P.: 267–269 °C, IR: 3467, 3395,
3302, 3178, 1637, 1370, 1198, 1133 cm−1 1H NMR
;
(DMSO-d6, 400 MHz) δ 6.29 (s, 2H, NH2), 7.45–7.51 (m,
3H, Ar–H), 7.77 (s, 1H, NH), 8.06–8.09 (m, 3H, Ar–H)
ppm; 13C NMR (DMSO-d6, 100 MHz) δ 103.6, 113.3,
121.6, 126.6, 128.6, 129.5, 135.1, 137.5, 155.6, 156.4,
General procedure for the synthesis of compounds
(2a–c)
166.6 ppm; MS (ESI): m/z (%) = 307.1 (15) [M + H]+,
_
264.1 (100) [M+
42]. Elemental analysis: calcd. For
C14H9F3N4O: C, 54.91; H, 2.96; N, 18.29%. Found: C,
54.85; H, 2.99; N, 18.22%.
Dried and pulverized guanidine (0.354 g)/thiourea (0.456
g)/urea (0.36 g) [6 mmol] was added to a solution prepared
by dissolving sodium metal (0.138 g, 6 mmol) in absolute
ethanol (20 mL) and the mixture was stirred vigorously at
25 °C for 30 min before adding the precursor 1 (0.789 g, 3
mmol). The resulting mixture was heated under reflux for
48–50 h. After completion of the reaction (monitored by
TLC), the mixture was poured on to crushed ice, the solid
separated was filtered, washed with water. The crude pro-
duct was dried and purified through column of silica gel
(60–120 mesh) using n-hexane/ethyl acetate (4:1) as eluent.
General procedure for the synthesis of 2-Amino-3-
carboxamido-4-trifluoromethyl-6-phenyl pyridine (3)
It was prepared by following the reported method (Bhalerao
and Krishnaiah 1995).
General procedure for the synthesis of 7-Phenyl-5-
(trifluoromethyl)-pyrido[2,3-d]pyrimidin-2,4(1H, 3H)-
dione (4)
7-Phenyl-5-(trifluoromethyl)-pyrido[2,3-d]pyrimidin-2,4-
diamine (2a)
A solution of triethylamine (0.218 g, 2.16 mmol) in anhy-
drous tetrahydrofuran (3 mL) was added dropwise to a
mixture of 3 (0.258 g, 0.92 mmol) and triphosgene (0.106 g,
0.36 mmol) in anhydrous tetrahydrofuran (20 mL). The
mixture was heated under reflux for 4 h, then cooled to
room temperature and poured on to crushed ice. The solid
separated was collected by filtration and washed with water.
The crude product was dried and purified through column
of silica gel (60–120 mesh) using n-hexane/ethyl acetate
(4:1).
White solid, yield: 68%, M.P.: 226–228 °C, IR: 3399, 3220,
2926, 1582, 1458, 1371, 1260, 1177, 1132 cm−1; 1H NMR
(DMSO-d6, 400 MHz) δ 6.27 (s, 2H, NH2), 7.32 (s, 1H, Ar–
H), 7.43–7.53 (m, 3H, Ar–H), 7.75 (s, 1H, NH), 8.04–8.12
(m, 2H, Ar–H and 1H, NH) ppm; 13C NMR (DMSO-d6,
100 MHz,) δ 103.78, 113.35, 124.36, 126.60, 128.83,
129.51, 130.46, 135.07, 137.48, 155.60, 156.41, 166.67

Med Chem Res
Pale yellow, yield: 68%, M.P.: > 300 °C, IR: 3267, 3132,
3053, 2847, 1739, 1694, 1595, 1570, 1402, 1368, 1130
cm−1; 1H NMR (DMSO-d6, 400 MHz) δ 7.53–7.55 (m, 3H,
Ar–H), 7.89 (s, 1H, Ar–H), 8.13–8.16 (m, 2H, Ar–H), 11.55
(s, 1H, NH), 11.92 (s, 1H, NH) ppm; 13C NMR (DMSO-d6,
100 MHz) δ 104.62, 112.65, 123.49, 127.59, 128.98,
131.36, 135.76, 137.74, 149.87, 154.35, 159.4, 160.98
ppm; MS (ESI): m/z (%) = 308.1 (100) [M + H]+, 264.1
8.16–8.19 (m, 2H, Ar–H) ppm; MS (ESI): m/z (%) = 339.4
(100) [M + Na+]+, 317.4 (65) [M + H]+. Elemental ana-
lysis: calcd. For C16H11F3N4: C, 60.76; H, 3.51; N, 17.71%.
Found: C, 60.81; H, 3.58; N, 17.66%.
5-Methyl-8-phenyl-10-(trifluoromethyl)-2,3-dihydroimidazo
[1,2-c]pyrido[3,2-e]pyrimidine (6b)
_
(78) [M 43]+. Elemental analysis: calcd. For C14H8F3N3
White solid, yield: 52%, M.P.: 282–283 °C, IR: 1633, 1561,
1467, 1304, 1265, 1129 cm−1; 1H NMR (CDCl3, 400 MHz)
δ 2.64 (s, 3H, CH3), 4.14 (t, 2H, CH2), 4.31 (t, 2H, CH2),
7.47–7.51 (m, 3H, Ar–H), 7.83 (s, 1H, Ar–H), 8.11 (m, 2H,
Ar–H) ppm; MS (ESI): m/z (%) = 353.1 (100) [M +
Na+]+, 331.1 (65) [M + H]+. Elemental analysis: calcd. For
C17H13F3N4: C, 61.82; H, 3.97; N, 16.96%. Found: C,
61.89; H, 3.91; N, 16.88%.
O2: C, 54.73; H, 2.62; N, 13.68%. Found: C, 54.79; H,
2.68; N, 13.61%.
General procedure for the synthesis of 3-(4,5-Dihydro-
1H-imidazol-2-yl)-6-phenyl-4-(trifluoromethyl)pyridin-
2-amine (5)
A mixture of compound 1 (1.052 g, 4 mmol), ethylenediamine
(0.96 g, 16 mmol) and sulfur (0.032 g, 1 mmol) was heated
under reflux on an oil bath (120 °C) for 4 h. The progress of
the reaction was monitored by TLC (EtOAc/MeOH, 4:1).
After completion of the reaction, the mixture was cooled to
room temperature and cold water was added and extracted
with chloroform. The organic layer was dried over anhydrous
Na2SO4 and evaporated under reduced pressure. The residue
obtained was recrystallized using cyclohexane.
X-ray crystallographic study of the precursor compound
(1)
The intensities were corrected for Lorentz and polarization
effects, and a multi-scan absorption correction was also
performed. The structure was solved by direct methods
using SHELXS97 (Sheldrick 1997), which revealed the
positions of all non-hydrogen atoms. The non-hydrogen
atoms were refined anisotropically. All of the hydrogen
atoms were fixed at geometrically calculated positions and
each was assigned a fixed isotropic displacement parameter
with a value equal to 1.2 Ueq of its parent atom. They were
riding with their bonded atoms. A weighting scheme of the
Yellow solids, yield: 74%, M.P.: 232–234 °C, IR: 3473,
3346, 3180, 2931, 2864, 1625, 1582, 1497, 1373, 1267, 1133
1
cm−1; H NMR(CDCl3, 400 MHz) δ 3.75 (m, 4H, 2CH2),
5.77 (s, 2H, NH2), 7.19 (s, 1H, NH), 7.24 (s, 1H, Ar–H), 7.41
(m, 3H, Ar–H), 7.90 (m, 2H, Ar–H) ppm; 13C NMR (CDCl3,
100 MHz) δ 25.05, 28.12, 104.48, 112.61, 120.63, 126.20,
127.43, 128.79, 136.81, 137.13, 156.92, 157.02, 160.07 ppm;
MS (ESI): m/z (%) = 307.1 (100) [M + H]+. Elemental ana-
lysis: calcd. For C15H13F3N4: C, 58.82; H, 4.28; N, 18.29%.
Found: C, 58.77; H, 4.35; N, 18.23%.
form w = 1/[σ2(F0 ) + (ap)2 + bp] with a = 0.0291 and b =
2
0.0 was used. The refinement converged to a final R value of
0.0438 (wR2 = 0.0810 for 2276 reflections, [I > 2σ(I)]. The
final difference map was featureless. The data collection and
refinement parameters are given in supporting information.
General procedure for the synthesis of compounds
Biological activity
(6a–b)
Binding at human A1, A2A and A3 ARs
A mixture of compound 5 (1.53 g, 5 mmol) and triethy-
lorthoformate/triethylorthoacetate (15 mL) was heated
under reflux for 3 h. After cooling, the reaction mixture was
poured on to cold water. The solid product was isolated by
filtration, washed with water, dried, and crystallized using
cyclohexane.
Binding studies at hA1, hA2A, and hA3 ARs were carried
out by employing our previously reported procedures (Klotz
et al. 1998). Chinese hamster ovary (CHO) cells stably
transfected with human (h) A1, A2A, and A3 ARs subtypes
were used for the preparation of membranes for radioligand
binding studies. Further, 1 nM [3H]-2-chloro-6-cyclopentyl
adenosine ([3H]CCPA), 10 nM [3H]-5’-N-ethylcarboxamido
adenosine ([3H]NECA) and 1 nM [3H]-2-(1-hexynyl)-N6-
methyl adenosine ([3H]HEMADO) were used as radi-
oligands at the hA1, hA2A and hA3 Ars, respectively.
Nonspecific binding of [3H]CCPA was determined in pre-
sence of 1 mM theophylline, while 100 µM (R)-N6-pheny-
liso-propyladenosine (R-PIA) was used for [3H]NECA and
8-Phenyl-10-(trifluoromethyl)-2,3-dihydroimidazo[1,2-c]
pyrido[3,2-e]pyrimidine (6a)
White solid, yield: 61%, M.P.: 262–263 °C, IR: 1632, 1560,
1463, 1361, 1198, 1148, 1115 cm−1; 1H NMR (CDCl3, 400
MHz) δ 4.08–4.13 (m, 2H, CH2), 4.24–4.29 (m, 2H, CH2),
7.49–7.52 (m, 3H, Ar–H), 8.01–8.03 (m, 2H, Ar–H),

Med Chem Res
[3H]HEMADO, respectively (Klotz et al. 2007). Calculation
of Ki values from competition experiments was carried out
by using the program SCIFIT (Delean et al. 1982).
shown in Scheme 1, the precursor 1 (2-amino-3-cyano-4-
trifluoromethyl-6-phenyl-pyridine) on a cyclocondensation
reaction with guanidine/thiourea/urea under basic condi-
tions (sodium ethoxide) resulted in the formation of sub-
stituted pyrido[2,3-d]pyrimidines (2a–c). When these
reactions were carried out in the absence of a base, the
product formation was not observed. This could be due to
the low nucleophilicity of 2-amino group of the precursor
which may be attributed to two possibilities: the presence of
a highly electron-withdrawing nitrile group in ortho posi-
tion to the amino group and the distinct possibility of imine
formation between the amino group and pyridine ring
nitrogen due to tautomerism. Thus, nucleophilicity of the
amino group in precursor is less when compared with aro-
matic amines. The complete disappearance of the nitrile
absorption band at 2216 cm−1 in the IR spectra of 2a–c
indicated the utilization of the α-aminonitrile (1). The pre-
sence of amino groups at δ 6.27 ppm (C-4 NH2) and
7.75–8.04 ppm (NH) in the 1H NMR spectra of 2a–c further
confirmed the cyclization. The presence of a pyrimidine
ring system was further confirmed by D2O exchange of
amino protons along with the presence of carbon signals at
156.41–166.67 ppm in 13C NMR spectra. The formation of
2a–c indicated that 2-amino group was actively participat-
ing in the nucleophilic attack with the selected reagents
(guanidine/thiourea/urea). Further, it also provided an
Adenylyl cyclase activity
Due to the lack of a high affinity radioligand for A2B AR
adenylyl cyclase experiments were carried out as a measure of
affinity for hA2B AR by employing previously reported pro-
cedures with minor modifications (Klotz et al. 1985, 1998).
Membranes were prepared from CHO cells stably transfected
with hA2B ARs followed by incubation with 100 nM NECA
as well as 150,000 cpm of [α-32P] ATP. All the target com-
pounds were tested at different concentration for 20 min in the
incubation mixture without using EGTA (ethylene glycolte-
traacetic acid) and NaCl (Klotz et al. 1985). None of the
compounds showed measureable interaction with the hA2B
AR (IC50 values > 90 µM; data not shown).
Results and discussion
Chemistry
The required precursor (1) was synthesized following an
earlier reported protocol (Narsaiah et al. 1993, 1994). As
Scheme 1 Synthesis of
substituted and heterofused
pyrido[2,3-d]pyrimidines.
Reagent and conditions: (i)
guanidine/thiourea/urea,
C2H5ONa/EtOH, reflux 48–50
h; (ii) KOH, reflux, 4 h; (iii)
triphosgene, triethylamine/
tetrahydrofuran, reflux; (iv)
ethylenediamine, sulfur, 120 °C,
4 h; (v) triethylorthoformate/
triethylorthoacetate, reflux 3 h
CF3
CF3 NH2
Comp. No.
R
N
2a
2b
2c
NH
S
Ph
N
N
H
R
O
2a-c
X
(i)
H2N
NH2
CF3
CF3
N
CONH2
CN
N
H
NH2
(iv)
(ii)
Ph
N
3
NH2
Ph
N
Ph
N
1
NH2
5
(v)
(iii)
CF3
N
N
CF3
O
Comp. No.
R
N
NH
6a
6b
H
CH3
Ph
N
R
Ph
N
4
N
H
O
6a-b

Med Chem Res
interesting clue that the nucleophilicity of the 2-amino
group could be the rate limiting factor in these reactions.
Pyrido[2,3-d]pyrimidine analogue 4 was obtained by the
cyclization of an intermediate 2-amino-3-carboxamido-4-
trifluoromethyl-6-phenylpyridine 3 (Bhalerao and Krish-
naiah 1995) using triphosgene in the presence of basic
medium (triethylamine). The carbonyl group showed a
1
strong signal at 1694 cm−1 in its IR spectrum and the H
NMR spectrum exhibited two signals at δ 11.55 ppm and δ
11.92 ppm corresponding to two NH protons. Further, fused
pyrido[2,3-d]pyrimidines (6a and 6b) were obtained by the
cyclization of 3-(4,5-dihydro-1H-imidazol-2-yl)-6-phenyl-
4-(trifluoromethyl)pyridin-2-amine (5) a new intermediate
with one carbon donor reagents like triethylorthoformate/
triethylorthoacetate. The intermediate 5 was synthesized by
the functional conversion of cyano group in 1 to an imi-
dazoline group by using 1,2-ethylene- diamine in the pre-
sence of sulfur as a catalyst. The IR spectrum of compound
5 showed disappearance of C≡N stretch and an absorption
Fig. 3 An ORTEP view of compound 1 with atom numbering scheme
employed
1
band at 3180 cm−1 related to the N–H stretch. Its H NMR
spectrum showed a multiplet signal at δ 3.75 ppm for
methylene protons and a singlet at δ 7.19 ppm corre-
sponding to the imidazoline –NH. The IR and H NMR
One of the noteworthy features in the molecular structure
of 1 is the near planar orientation of the phenyl group with
the pyridyl ring, the torsional angle N1-C5-C8-C13 is only
18.6°, even though there can be complete flexibility in
rotation of C5-C8 bond. The cause for such an orientation is
two-fold, the better resonance stabilization as the two aro-
matic rings become co-planar and the structure stabilization
through an intramolecular C–H…N hydrogen bonding
between N1 and one of the ortho hydrogen atoms (H13A)
of the phenyl ring (N1…H13A = 2.509 Å, C13-H13A…
N1 = 99.6°). In the crystal lattice, molecules are packed in a
herringbone manner as shown in Fig. 4. In the wedge shape
region of the herringbone two molecules form a cen-
trosymmetric dimer through a pair of N–H…N hydrogen
bonds (N3…H2B = 2.212 Å, N2-H2B…N3 = 160.01°)
between the adjacent –NH2 and –CN functionality (Fig. 5a).
Further, these dimers stack one over the other through a
sheared manner (Fig. 5b) in which the pyridyl and phenyl
rings are 3.84 Å apart that shows π…π stacking interactions
between the aromatic moieties. There are no other short
contacts observed in the crystal lattice.
The observed coplanarity of the two rings (phenyl and
pyridyl) of the precursor might have contributed towards its
improved reactivity of both amino and nitrile groups located
on the pyridine ring. This could probably be the reason why
several reactions on nitrile and amino groups individually as
well as together in one step have been successfully con-
ducted. Our observation of the quantitative yields with such
condensations using appropriate reagents is noteworthy.
Earlier, we reported reactions involving pyridopyrimidine
system with low to moderate yields (Suma et al. 2000).
Now, it can be understood that the strategically located
1
spectra of 6a–b showed the absence of characteristic
absorption bands for primary and secondary amino groups
which authenticated the formation of cyclized compounds.
The conversion of cyano to other groups (carboxamido or
imidazolino) improved the nucleophilicity of the amino
group of the compound 5 as compared to the precursor (1),
facilitating subsequent convenient cyclization reaction
under mild conditions to result in the formation of fused-
pyrimidine analogues.
X-ray diffraction analysis of compound 1
To explore the versatility of this precursor compound (1), a
crystallization process with various solvents was attempted.
Interestingly, distinct monoclinic crystals were obtained
when absolute ethanol was used as a crystallizing solvent. It
prompted us to investigate further its X-ray crystallographic
data to understand the reactivity of this key intermediate
towards a variety of reagents. Compound 1 crystallizes in a
monoclinic crystal system (space group = P21/c) with four
molecules in the unit cell. A perspective view of the
molecule with numbering scheme is shown in Fig. 3.
All the bond lengths and angles observed in the structure
are normal. The cyano group, as expected, is almost linear
with a C2-C7-N3 angle of 177.8 (2)°. The trifluoromethyl
group orients with the pyridyl ring in such a fashion that
there is a short contact of 2.391 Å between one of the
fluorine atoms and the adjacent hydrogen (F2…H4A =
2.391 Å, C4-H4A… F2 = 100.08°) which can be con-
sidered as in intramolecular C–H…F interaction.

Med Chem Res
phenyl substituent at 6-position of pyridine nucleus could
perhaps be responsible for these unexpectedly high yields of
these type of reactions. The fluorinated fused quinazolines
(17a–c and 18a–c) were synthesized and characterized as
per our earlier report (Balakumar et al. 2010) by following
the synthetic route as shown in Scheme 2.
compounds towards hA2B AR was investigated by mea-
suring adenylyl cyclyse activity, while the amount of inhi-
bition of NECA-stimulated adenylyl cyclase activity would
provide a measurement of an antagonistic nature. Interest-
ingly, none of these compounds showed detectable inter-
action with the hA2B AR (data not shown in Table 1) in an
agonistic or antagonistic fashion (EC50 or IC50 values > 90
µM).
Biological activity
In the series of fluorinated pyrido[2,3-d]pyrimidines
(2a–c and 4), compound 2a exhibited the maximum binding
affinity for hA3 AR with similar affinity for the hA1 and
somewhat lower affinity for hA2A ARs resulting in a
compound with no A3 selectivity vs. A1 and moderate
selectivity vs. A2A AR (Ki hA1 = 0.62 µM, hA2A = 3.59 µM
and hA3 = 0.42 µM). This could be attributed to the presence
of free amino group at 2-position of pyrido[2,3-d]pyr-
imidine nucleus in addition to amino group at 4-position of
the scaffold. Replacement of the free amino (NH2) group
with sulphur or oxygen atom in 2-position of the pyrimidine
moiety of compounds 2b and 2c drastically lowered the
affinity for all the AR subtypes. Interestingly, in case of
compound 4, the presence of a carbonyl (C=O) group at 2-
position and 4-position of the pyrimidine moiety improved
the affinity for hA1 AR but showed poor selectivity against
hA2A and hA3 ARs (Ki hA1 = 0.17 µM, hA2A = 0.67 µM
and hA3 = 0.68 µM). The fusion of an imidazole ring with
the pyrimido moiety of the scaffold in case of compounds
6a and 6b further showed detrimental effect on the affinity
for all the ARs.
All the newly synthesised fluorinated pyrido[2,3-d]pyr-
imidines (2a–c, 4 and 6a–b) and previously reported
fluorinated quinazolines (17a–c and 18a–c) were evaluated
for their binding affinity and selectivity towards hA1, A2A
2B, and A3 ARs which were expressed in CHO cells.
Three different radioligands such as [3H]CCPA,
[3H]NECA, and [3H]HEMADO were used for hA1, hA2A
,
A
,
and hA3 ARs, respectively. The AR binding affinity and
selectivity data of compounds 2a–c, 4 and 6a–b are pro-
vided in Table 1. A potential agonistic activity of these
There are numerous examples of more potent antagonists
for A1, A2A, and A3AR (Fredholm et al. 2001, 2011),
however, many structurally novel compounds show affi-
nities in the low micromolar or high nanomolar range
(Cagide et al. 2015). Although further development of such
ligands is usually aiming at improved affinity this is not
essential as many successfully used drugs show affinities in
this range such as Ranitidine (0.20 µM) and Loratadine
(0.16 µM) (Hill et al. 1997).
Fig. 4 Packing of 1 viewed down a axis showing the herring-bone
arrangement of molecules in the crystal lattice. The dimer formation
through N–H…N hydrogen bonds are shown as dashed lines
A
B
Fig. 5 a The dimer formation through N–H…N hydrogen bonding (dashed line) in 1. The dashed solid line shows the π…π interaction between
the pyridyl andphenyl moieties. b The stacking pattern of 1. The dashed solid line shows π…π interaction between the pyridyl and phenyl moieties

Med Chem Res
Scheme 2 Synthesis of
CF3
CF3
fluorinated quinazolines.
Reagents and conditions: (i)
methanol, reflux, 6 h; (ii) 20%
KOH, reflux, 7 h; (iii) glacial
acetic acid, r.t., 4 h; (iv) MnO2,
dichloromethane, r.t., 2 h; (v)
POCl3, reflux, 5 h (Balakumar
et al. 2010)
O
O
CN
CN
O
CN
CN
(i)
+
+
C6H5
CF3
C6H5
NH2
C6H5
N
H
10
7
8
CN
9
(ii)
CF3
C6H5 O
N
CONH2
13a-c
CF3
O
CONH2
NH2
NH
NH
R
(iii)
+
C6H5
RCHO
+
6
F3C
R
C6H5
N
H
H
CONH2
11
CONH2
12a-c
(iv)
C6H5 O
(iv)
Comp. No.
R
CF3
O
N
O
a
NH
NH
F3C
N
CONH2
15a-c
R
C6H5
R
b
c
CONH2
14a-c
OH
Cl
Cl
. HCl
. HCl
(v)
(v)
S
NH2
NH2
16
16
C6H5
N
N
CF3
N
N
F3C
N
R
C6H5
N
R
CONH2
CONH2
17a-c
18a-c
In the quinazoline series, compound 18a was found to be
the most active with substantial binding affinity for A1 AR
as well as significant selectivity against A2A and A3 ARs (Ki
hA1 = 0.73 µM, hA2A > 30 µM and hA3 = 9.27 µM). Sur-
prisingly, other quinazoline derivatives (17a–c, 18b and
18c) did not show remarkable affinity for any of the AR
subtypes.
in quantitative yields by using 2-amino-4-trifluoromethyl-6-
phenyl nicotinonitrile (1) as a precursor following an
appropriate synthetic Scheme 1. In order to gain insight into
the structural features as well as to illustrate the reactivity of
the key precursor (1), we also examined the X-ray crystal
structure. Further, a series of fluorinated heterofused qui-
nazolines (17a–c and 18a–c) were also synthesized and
characterized as they are bioisosteres to pyrido[2,3-d]pyr-
imidines based on our earlier report. Radioligand binding
affinity studies of all these compounds for ARs showed that
pyrido[2,3-d]pyrimidine scaffold with free amino (NH2)
group at 2-position and 4-position (compound 2a) exhibited
good binding affinity for hA3 AR with no selectivity against
hA1, but moderate hA2A selectivity. Interestingly, the
Conclusion
In conclusion, we have synthesized and characterised some
new fluorinated pyrido[2,3-d]pyrimidines (2a–c and 4) and
fluorinated imidazo[1,2-c]pyrido[3,2-e]pyrimidines (6a–b)

Med Chem Res
Table 1 Binding affinity (Ki) of compounds (2a–c, 4, 6a–b, 17a–c and 18a–c) at hA1, hA2A, and hA3 ARs and selectivity against hA1 and hA2A
ARs
CF3 NH2
N
CF3
C6H5
N
N
CF3
O
CF3
N
N
N
N
NH
N
N
Ph
N
N
H
R
C6H5
R
N
R
F3C
R
Ph
N
N
H
O
Ph
N
CONH2
CONH2
6a-b
2a-c
4
17a-c
18a-c
CN
R
Ki, µM (95 % Cl)
Selectivity
hA1/hA2A
a
b
c
hA1
hA2A
hA3
hA1/hA3
hA2A/hA3
2a
2b
2c
4
NH2
S
0.623 (0.561–0.692)
12.9 (7.52–22.1)
11.1 (6.16–19.9)
0.176 (0.161–0.192)
3.08 (1.65–5.73)
>30
3.59 (1.76–7.33)
0.429 (0.310–0.595)
5.85 (3.45–9.91)
19.5 (10.3–37.2)
0.685 (0.444–1.06)
3.19 (2.43–4.18)
13.40 (8.43–21.4)
2.34 (1.42–3.87)
0.17
>0.43
>0.37
0.26
0.44
>1
1.5
8.4
>30
>30
2.2
>5.1
>1.5
0.99
2.2
O
0.57
0.26
0.96
2.2
–
0.678 (0.469–0.980)
7.03 (6.36–7.77)
27.3 (22.1–33.7)
22.6 (14.4–35.2)
6a
6b
17a
H
CH3
2.0
5.84 (4.69–7.27)
0.25
2.5
9.7
O
O
18a
17b
0.73 (0.65–0.82)
7.80 (6.81–8.94)
>30
9.27 (4.71–18.3)
2.52 (1.25–5.09)
0.02
0.31
0.07
3.09
3.2
9.7
24.5 (17.0–35.3)
OH
18b
2.57 (1.35–4.91)
8.62 (4.86–15.3)
3.96 (3.16–4.97)
0.29
0.64
2.2
OH
17c
18c
10.6 (7.72–14.4)
1.97 (1.77–2.19)
26.0 (22.5–30.1)
6.62 (4.49–9.76)
3.85 (3.09–4.78)
1.87 (1.23–2.82)
0.40
0.29
2.8
6.8
3.5
S
S
1.05
Data are expressed as geometric means, with 95% confidence limits in parentheses
a
Displacement of specific [3H]CCPA binding at human A1 AR expressed in CHO cells (n = 3–6)
b
Displacement of specific [3H]NECA binding at human A2A AR expressed in CHO cells (n = 3–6)
c
Displacement of specific [3H]HEMADO binding at human A3 AR expressed in CHO cells (n = 3–6)

Med Chem Res
related compounds by using active MnO2. Indian J Chem
34B:587–590
replacement of both the amino groups with carbonyl (C=O)
groups (compound 4) resulted in significantly improved
affinity for hA1 AR but with moderate selectivity against
hA2A and hA3 ARs. Further, fusion of an imidazole ring
with the pyrimidine moiety of the scaffold (compounds 6a
and 6b) showed detrimental effect in affinity for all the
ARs. In case of fluorinated quinazolines, only compound
18a showed good affinity for hA1 AR with significant
selectivity against hA2A and hA3 ARs. The preliminary
result of these compounds demonstrate that the fluorinated
pyrido[2,3-d]pyrimidine and imidazo[1,2-c]pyrido[3,2-e]
pyrimidine can be considered as promising scaffolds for
further optimisation in search of potential antagonists with
better affinity and selectivity towards hA1 and hA3 ARs.
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Conflict of interest The authors declare that they have no competing
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