Exploring the influence of the substituent at position 4 in a series of 3,4-dihydropyrimidin-2(1H)-one A2B adenosine receptor antagonists

Article abstract of DOI:10.1007/s10593-017-2054-4

(Figure Presented) In the context of a program to identify selective adenosine A_2B receptor antagonists, we have obtained a focused library of 4-substituted 3,4-dihydropyrimidin-2(1H)-ones and its affinity for the four human adenosine receptor

Full text of DOI:10.1007/s10593-017-2054-4

DOI 10.1007/s10593-017-2054-4
Chemistry of Heterocyclic Compounds 2017, 53(3), 316–322
Exploring the influence of the substituent at position 4
in a series of 3,4-dihydropyrimidin-2(1H)-one
A adenosine receptor antagonists
2B
1
,2
1,2
1,2
1,2
Abel Crespo , Abdelaziz El Maatougui , Jhonny Azuaje , Luz Escalante ,
1
,2
3
3
3
María Majellaro , María Isabel Loza , José Brea , María Isabel Cadavid ,
Hugo Gutiérrez-de-Terán , Eddy Sotelo *
4
1,2
1
Centro Singular de Investigación en Química Biolóxica e Materiais Moleculares (CIQUS)
Universidade de Santiago de Compostela,
1
578, Santiago de Compostela, Spain; e-mail: e.sotelo@usc.es
2
3
Departamento de Química Orgánica, Facultade de Farmacia, Universidade de Santiago de Compostela,
578, Santiago de Compostela, Spain; e-mail: e.sotelo@usc.es
1
Centro Singular de Investigación en Medicina Molecular y Enfermedades Crónicas (CIMUS)
Universidade de Santiago de Compostela,
1
578, Santiago de Compostela, Spain; e-mail: Mabel.loza@usc.es
4
Department of Cell and Molecular Biology, Uppsala Biomedicinska Centrum (BMC),
Uppsala University,
Husarg. 3, Uppsala SE-7512, Sweden; e-mail: Hugo.Gutierrez@icm.uu.se
Published in Khimiya Geterotsiklicheskikh Soedinenii,
017, 53(3), 316–322
Submitted January 9, 2017
Accepted February 17, 2017
2
In the context of a program to identify selective adenosine A2B receptor antagonists, we have obtained a focused library of 4-substituted
3
,4-dihydropyrimidin-2(1H)-ones and its affinity for the four human adenosine receptor subtypes was determined. The synthesis was
accomplished by using an experimentally simple and efficient Biginelli approach. The biological evaluation of the library revealed that
all the documented derivatives exhibit low or negligible affinity for the A2B receptor, thus highlighting the critical importance of the
substituent at position 4 of the 3,4-dihydropyrimidin-2(1H)-one chemotype.
Keywords: 3,4-dihydropyrimidin-2(1H)-ones, adenosine antagonists, A2B adenosine receptor, A2B antagonists, structure–activity relationship.
Adenosine, an endogenous nucleoside released from
rized AR. Recent evidences confirm that the A2BAR is
implicated in major biological processes (e.g., vascular
tone, cardiac myocyte contractility, glucose homeostasis,
pulmonary inflammation, inflammatory response, and
pain).11 Whereas the interest in A2BAR signalling is
1
almost all mammalian cells, is structurally and metabo-
2
lically related to the bioactive nucleotides, RNA and the
2
coenzymes A, NAD and FAD. Adenosine is also a key
signalling molecule, that mediates a plethora of physio-
logical effects through the stimulation of a family of
11
currently increasing significantly, the compendium of
ligands that selectively target the A2BAR remains scarce.12
The pursuit of A2BAR antagonists has traditionally focused
on the pharmacomodulation of naturally occurring xanthi-
G-protein-coupled receptors (GPCRs) named adenosine
3
receptors (ARs: A
1
, A2A, A2B, and A
3
). Human ARs,
4
exhibit high sequence homology and differ in their affinity
for adenosine, the localization, and the G-protein-mediated
5
7–9
nes (Fig. 1, compounds 1–5). In clear contrast, very few
downstream signalling pathways. Adenosine receptors have
proved to participate in diverse physiological processes,1
being considered attractive drug targets in several
examples of potent, selective, and structurally simple non-
xanthine A2BAR antagonists have been described (Fig. 1,
,2,6
6
–9,11,12
compounds 6–11).
Accordingly, there is a demand
6–9
pathophysiological events.
The A2B adenosine receptor (A2BAR) is ubiquitously
for novel scaffolds representative of diverse topologies,
physicochemical features, and binding modes. In this
context, we recently reported the discovery of a novel
1
0–11
expressed in the body
and remains the poorer characte-
0
009-3122/17/53(03)-0316©2017 Springer Science+Business Media New York
316

Chemistry of Heterocyclic Compounds 2017, 53(3), 316–322
O
O
O
O
O
O
O
O
Me
N
H
N
H
N
H
N
MeN
O
MeN
O
PrN
O
O
HN
CN
PrN
N
H
N
N
N
N
Me
N
N
O
N
N
N
O
N
Me
Me
Pr
Pr
1
2
3
4
Caffeine
K 33800 nM
Theofylline
K 9070 nM
MRS-1754
K 1.97 nM
MRE-2029-F20
K 5.50 nM
i
i
i
i
H
N
O
H
N
O
Me
O
NH
N
Cl
Me
O
N
N
H
N
O
N
N
O
N
O
N
N
N
Pr
N
N
N
6
Ph
Ph
5
Ph
OSIP339391
7
K 5.52 nM
i
K 0.50 nM
i
K 1.40 nM
i
S
O
F
N
O
O
H
O
O
X
N
N
O
HN
OEt
Me
HN
Oi-Pr
HN
OEt
Me
R1
O
N
H
O
N
O
N
H
Me
O
N
H
R2
Et
i-Pr
H
N
HN
O
8
9
10
11
O
N
H
Me
LAS-101057
K 24.00 nM
SYAF08635
SYAF01435
SYAF10135
K 23.60 nM
K 40.80 nM
K 39.60 nM
12a–n
i
i
i
i
Figure 1. Representative A2BAR antagonists (compounds 1–8), model A2BAR antagonists (compounds 9–11) and herein targeted
compounds 12a–n.
family of A2BAR antagonists based on the 3,4-dihydro-
Scheme 1. Briefly, the reaction of urea (15), the corres-
ponding carbaldehyde 13a–g, the selected alkyl aceto-
1
3
pyrimidin-2(1H)-one scaffold. As part of this study we
have demonstrated the excellent pharmacological profile of
this novel chemotype, preliminarily exploring the SAR at
positions 1, 2, 5, and 6. In the frame of this program, we
herein document the synthesis of a collection of 3,4-di-
hydropyrimidin-2(1H)-ones 12 designed to evaluate the
effect of substituting the heterocyclic ring at position 4 of
the scaffold by different groups.
acetate 14a,b, and ZnCl were heated at 70°C in THF for
2
12 h to afford the targeted 3,4-dihydropyrimidin-2(1H)-
ones 12a–n in satisfactory to excellent yields (Table 1).
The pharmacological profiles of compounds 12 obtained
were studied in vitro at the four human adenosine receptor
subtypes using radioligand-binding assays.1 The affinities
3,17
for the four human adenosine receptor subtypes (A , A2A, A2B,
1
With the aim to investigate the effect produced by the
replacement of the pentagonal heterocyclic moieties (e.g.,
and A ) of the 4-substituted 3,4-dihydropyrimidin-2(1H)-ones
3
12a–n were determined in vitro with radioligand binding
2
- or 3-furan and 2- or 3-thiophene) at position 4 of
assays according to experimental protocols described by our
group1 and reported in Table 1. In the previous and current
study, all compounds were evaluated as racemic mixtures.
Human adenosine receptors expressed in transfected CHO
3,17
13
previously identified hit compounds (Fig. 1, compounds 9–11)
we designed a small library of 3,4-dihydropyrimidin-
2
2
(1H)-ones (compounds 12). The 3,4-dihydropyrimidin-
1
(1H)-ones 12a–b (R = H) would allow to evaluate the
(A
1
AR), HeLa (A2AAR and A AR), and HEK-293 (A2BAR)
3
3
real contribution of the substituent at position 4 during the
interaction with the A2B receptor. In a similar fashion, the
introduction of cyclopentyl (Cyp) and cyclohexyl (Cy)
rings, phenyl and 2-substituted phenyl groups was
undertaken to explore new substituents providing different
topologies, physicochemical properties, and binding
modes. In this study, we retained the most advantageous
cells were employed. [ H]-1,3-Dipropyl-8-cyclopentyl-
3
3
xanthine ([ H]DPCPX) for A
1
AR and A2BAR, [ H]ZM241385
AR were employed as
3
for A2AAR, and [ H]NECA for A
3
radioligands in binding assays. The biological data are
expressed as K ± SEM in nM (n = 3) or percentage of
i
inhibition of specific binding at 1 mM (n = 2, average) for
those compounds that did not fully displace radioligand.
Examination of the binding data obtained for herein
documented compounds (Table 1) revealed that all synthe-
sized 4-substituted 3,4-dihydropyrimidin-2(1H)-ones were
deprived of any significant affinity for the A2BAR. The
obtained data unequivocally confirms the relevant role
exercised by pentagonal heterocyclic cores (i.e., 2- or 3-furan
or 3-thiophene)1 at position 4 during the interaction with
the A2BAR. Such a role was early detected when comparing
substituents at positions 2 (O), 3 (H), 6 (Me), and 5 (CO Et
2
or CO i-Pr).
2
The required 4-substituted 3,4-dihydropyrimidin-2(1H)-
ones 12a–n were assembled by following environmentally
14
friendly protocol using the highly reliable Biginelli
reaction,15 thus demonstrating the potential of the
multicomponent reactions as preparative tools in drug
discovery.16 The synthetic transformations are depicted in
3,17
3
17

Chemistry of Heterocyclic Compounds 2017, 53(3), 316–322
Scheme 1
Table 1. Structure and binding data for 3,4-dihydropyridin-2(1H)-ones 12a–n at the human adenosine receptors
i
K at 1 mM
Compound
2a18
2b*
2c*
R1
R2
Yield, %
hA
1
**
hA2A***
hA2B*4
3%
hA
3
*5
1
H
H
Et
i-Pr
Et
74
76
74
76
81
84
96
93
91
93
87
92
90
91
–
1%
2%
3%
5%
1%
2%
1
1%
1
Cyp
Cyp
Cy
Cy
Ph
3%
1%
2%
18%
7%
1
1
2d*
2e19
i-Pr
Et
1%
1%
3%
4%
10%
8%
2%
11%
9%
1
2f*
2g20
2h21
2i19
i-Pr
Et
2%
5%
1
1%
10%
1%
2%
2%
1
Ph
i-Pr
Et
2%
2%
2%
1
2-FC
2-FC
6
6
H
4
26%
12%
37%
11%
31%
1%
10%
3%
54%
38%
42%
18%
50%
19%
40%
12%
1722 ± 11
863 ± 4
1
2j*
H
4
i-Pr
Et
1
2k22
2-ClC
6
H
4
6%
1321 nM
32%
1
2l23
2m24
2n*
2-ClC
6
H
4
i-Pr
Et
13%
18%
4%
1
2-MeOC
6
H
4
4117 nM
42%
1
2-MeOC
6
H
4
i-Pr
–
DPCPX*6
ZM241385*7
–
–
2.2 ± 0.2
683 ± 4.1
157 ± 3
1.9 ± 0.1
73.2 ± 2
65.7 ± 1
–
–
*
*
New compound.
3
* Displacement of specific [ H]DPCPX binding in human CHO cells expressed as K
i
± SEM in nM (n = 3) or percentage displacement of specific
binding at a concentration of 1 mM (n = 2).
** Displacement of specific [ H]-4-{2-[7-amino-2-(2-furyl)[1,2,4]triazolo[2,3-a][1,3,5]triazin-5-ylamino]ethyl}phenol binding in human HeLa cells
3
*
expressed as K ± SEM in nM (n = 3) or percentage displacement of specific binding at a concentration of 1 mM (n = 2).
i
4
3
*
Displacement of specific [ H]DPCPX binding in human HEK-293 cells expressed as K
i
± SEM in nM (n = 3) or percentage displacement of specific
binding at a concentration of 1 mM (n = 2).
5
3
*
Displacement of specific [ H]NECA binding in human HeLa cells expressed as K
i
± SEM in nM (n = 3) or percentage displacement of specific binding
at a concentration of 1 mM (n = 2).
6
*
*
DPCPX: 1,3-dipropyl-8-cyclopentylxanthine, potent and selective A
ZM241385: 4-{2-[7-amino-2-(2-furyl)[1,2,4]triazolo[2,3-a][1,3,5]triazin-5-ylamino]ethyl}phenol, potent and selective A2A antagonist standard.
1
antagonist standard.
7
the excellent data experienced (K
i
23–40 nM) for the
ligands identified during this work. In any case, these ligands
probed to be 57- and 179-fold less potent that model ligand
9, respectively (Fig. 1). An additional observation emerging
from the obtained data highlights that the synthesized
ligands show a similar low affinity profile irrespectively of
the alkoxy residue (EtO, i-PrO) present at the ester moiety,
a fact that highlights the prominent influence of the residue
at position 4 during the interaction of the 3,4-dihydro-
pyrimidin-2(1H)-one scaffold with the A2BAR. In addition,
to reinforce the role of the pentagonal heterocyclic ring, the
obtained data corroborate the binding modes proposed for
model compounds 9–11 in previous papers.1
1
3,17
reference compounds (Fig. 1, compounds 9–11)
and the
1
negligible affinity observed for compounds 12a–b (R = H).
Examination of the binding data of the 3,4-dihydro-
pyrimidin-2(1H)-ones bearing cyclopentyl, cyclohexyl, and
phenyl groups at position 4 (Table 1, compounds 12c–f and
1
2g–h) reinforced the idea that the pentagonal heterocyclic
cores are the optimal substituents for this position. As
observed, the only 3,4-dihydropyrimidin-2(1H)-ones exhi-
biting a modest affinity (micromolar) are those bearing a
2
-substituted phenyl group at position 4 (i.e., compounds
2i–n), with ethyl 4-(2-chlorophenyl)-6-methyl-2-oxo-1,2,3,4-
tetrahydropyrimidine-5-carboxylate (12k) and ethyl
-(2-methoxyphenyl)-6-methyl-2-oxo-1,2,3,4-tetrahydro-
pyrimidine-5-carboxylate (12m) being the most attractive
3,17
1
In summary, a focused library of 4-substituted 3,4-dihydro-
pyrimidin-2(1H)-ones was synthesized and their affinity for
4
the four human adenosine receptor subtypes (A , A2A, A2B,
1
3
18

Chemistry of Heterocyclic Compounds 2017, 53(3), 316–322
and A
3
) determined in vitro with radioligand binding assays.
(CDCl
165.5.
3
), δ, ppm: 14.2; 17.4; 43.2; 59.0; 94.5; 148.5; 152.8;
The synthesis of the new derivatives was accomplished in
satisfactory to excellent yields by using an experimentally
simple and efficient Biginelli approach. The biological
evaluation of the library revealed that all the documented
derivatives exhibit low or negligible affinity at A2BAR. The
reported data highlight the critical importance of the
substituent at position 4 of the 3,4-dihydropyrimidin-2(1H)-
one scaffold during its interaction with the A2BAR. Novel
studies are currently in progress in our laboratories to
explore the biological activity of novel 3,4-dihydro-
pyrimidin-2(1H)-ones bearing heterocyclic scaffolds at
position 4.
(±)-Isopropyl 6-methyl-2-oxo-1,2,3,4-tetrahydropyri-
midine-5-carboxylate (12b). Yield 301 mg (76%), white
solid, mp 168–169°C. H NMR spectrum (CDCl
1
3
), δ, ppm
(J, Hz): 1.26 (3H, d, J = 1.8) and 1.29 (3H, d, J = 1.9,
OCH(CH ); 2.27 (3H, s, 6-CH ); 3.84 (2H, s, CH ); 4.97
(1H, dq, J = 12.7, J = 6.4, OCH(CH ); 6.74 (1H, br. s,
NH); 8.84 (1H, br. s, NH). C NMR spectrum (CDCl ),
δ, ppm: 14.2; 17.3; 44.2; 60.2; 97.1; 147.7; 151.9; 166.1.
3
)
2
3
2
)
3 2
13
3
+
Found, m/z: 198.1007 [M] . C
9
H
14
N
2
O . Calculated, m/z: 198.1004.
3
(±)-Ethyl 4-cyclopentyl-6-methyl-2-oxo-1,2,3,4-tetra-
hydropyrimidine-5-carboxylate (12c). Yield 373 mg
1
(
(
74%), white solid, mp 142–143°C. H NMR spectrum
Experimental
CDCl
3
), δ, ppm (J, Hz): 1.28 (3H, t, J = 7.1, CH ); 1.40–
3
1H and 13C NMR spectra were recorded on Bruker
AM300 (300 MHz) spectrometer using TMS as internal
standard. Due to long relaxation times of the quaternary
carbon atoms of some compounds, particular signals of the
quaternary carbon atoms were hardly observed. High-
resolution mass spectra were obtained on an Autospec
Micromass spectrometer (EI, 70 eV). Melting points were
determined on a Gallenkamp melting point apparatus and
are uncorrected. The reactions were monitored by thin-
layer chromatography (TLC) on 2.5 mm Merck silica gel
GF 254 strips, and the purified compounds each showed a
single spot unless stated otherwise, UV light and/or iodine
vapor were used to detect compounds. The Biginelli
reactions were performed in coated Kimble vials on a PLS
Organic Synthesizer with orbital stirring. Filtration and
washing protocols for supported reagents were performed in a
1.81 (8H, m, H Cyp); 2.00–2.20 (1H, m, H Cyp); 2.27 (3H,
s, CH
J = 5.5, J = 3.8, 6-CH); 5.86 (1H, br. s, NH); 7.92 (1H, br. s,
NH). 13C NMR spectrum (CDCl
), δ, ppm: 13.9; 16.4;
3
); 4.17 (2H, q, J = 7.0, OCH
2
CH
3
); 4.31 (1H, dd,
3
25.8; 25.9; 31.6; 31.7; 42.8; 54.7; 61.6; 96.9; 150.7; 154.9;
+
168.4. Found, m/z: 252.1476 [M] . C13
lated, m/z: 252.1474.
H
20
N
2
3
O . Calcu-
(±)-Isopropyl 4-cyclopentyl-6-methyl-2-oxo-1,2,3,4-
tetrahydropyrimidine-5-carboxylate (12d). Yield 405 mg
1
(76%), white solid, mp 138–139°C. H NMR spectrum
(CDCl ), δ, ppm (J, Hz): 1.25 (3H, d, J = 1.8) and 1.27
3
(3H, d, J = 1.8, OCH(CH
2.01–2.18 (1H, m, H Cyp); 2.27 (3H, s, CH
J = 5.8, J = 3.7, 6-CH); 4.99–5.12 (1H, m, OCH(CH
3
)
2
); 1.43–1.78 (8H, m, H Cyp);
); 4.29 (1H, dd,
);
5.87 (1H, br. s, NH); 7.86 (1H, br. s, NH). C NMR
spectrum (CDCl ), δ, ppm: 16.5; 21.6; 25.8; 31.6; 31.7;
43.5; 54.6; 71.2; 98.4; 153.3; 154.7; 167.2. Found, m/z:
3
)
3 2
13
3
1
2-channel vacuum manifold. Purification of isolated
products was carried out by column chromatography
Kieselgel 0.040–0.063 mm, E. Merck) or medium pressure
liquid chromatography on a CombiFlash Companion
+
266.1633 [M] . C14
H
22
N
2
O . Calculated, m/z: 266.1630.
3
(
(±)-Ethyl 4-cyclohexyl-6-methyl-2-oxo-1,2,3,4-tetra-
19
hydropyrimidine-5-carboxylate (12e). Yield 431 mg
1
(
Teledyne ISCO) with RediSep prepacked normal-phase
(81%), white solid, mp 138–139°C. H NMR spectrum
silica gel (35–60 µm) columns followed by recrystalli-
zation.
(CDCl
(3H, t, J = 7.1, OCH
1.81 (3H, m, H Cy); 2.28 (3H, s, CH
OCH CH , 6-CH); 5.67 (1H, br. s, NH); 7.83 (1H, br. s,
NH). C NMR spectrum (CDCl ), δ, ppm: 14.1; 16.6;
3
), δ, ppm (J, Hz): 0.96–1.21 (5H, m, H Cy); 1.28
2
CH
3
); 1.42–1.56 (3H, m, H Cy); 1.58–
); 4.05–4.28 (3H, m,
Unless otherwise indicated, all starting materials,
reagents, and solvents were purchased and used without
further purification.
3
2
3
1
3
3
The Biginelli synthesis of 4-substituted 3,4-dihydro-
25.7; 25.8; 26.3; 29.6; 29.7; 40.8; 55.6; 60.7; 96.9; 151.6;
+
pyrimidin-2(1H)-ones 12a–n (General method).
A
153.9; 169.5. Found, m/z: 266.1632 [M] . C14
Calculated, m/z: 266.1630.
H
22
N
2
3
O .
mixture of urea 15 (0.180 g, 3.0 mmol), the corresponding
carbaldehyde 13 (2.0 mmol), alkyl acetoacetate 14
(±)-Isopropyl 4-cyclohexyl-6-methyl-2-oxo-1,2,3,4-tetra-
(
2.0 mmol), and ZnCl
2
(0.027 g, 0.2 mmol) in THF (3 ml)
hydropyrimidine-5-carboxylate (12f). Yield 471 mg
1
was stirred at 70°C for 12 h. After completion of the
reaction, as indicated by TLC, the reaction mixture was
poured onto crushed ice and stirred for 5–10 min. The solid
separated was filtered under vacuum, washed with ice-cold
water (20 ml), dried in vacuum, purified by column
chromatography (n-hexane–EtOAc, 4:1) on silica gel, and
then recrystallized from EtOH.
(84%), white solid, mp 134–135°C. H NMR spectrum
(CDCl ), δ, ppm (J, Hz): 0.95–1.21 (5H, m, H Cy); 1.24
3
(3H, d, J = 1.8) and 1.27 (3H, d, J = 1.8, OCH(CH
1.56 (3H, m, H Cy); 1.58–1.81 (3H, m, H Cy); 2.28 (3H, s,
CH ); 4.18 (1H, t, J = 3.5, 6-CH); 5.05 (1H, dq, J = 12.7,
J = 6.4, OCH(CH ); 5.53 (1H, br. s, NH); 7.45 (1H, br. s,
NH). 13C NMR spectrum (CDCl
), δ, ppm: 16.5; 22.5; 22.6;
3
)
2
); 1.42–
3
)
3 2
3
(±)-Ethyl 6-methyl-2-oxo-1,2,3,4-tetrahydropyrimidine-
25.1; 25.2; 25.7; 29.6; 29.7; 41.8; 54.3; 71.1; 98.4; 152.4;
1
8
+
5
-carboxylate (12a). Yield 273 mg (74%), white solid,
153.8; 166.3. Found, m/z: 280.1791 [M] . C15
Calculated, m/z: 280.1787.
H
24
N
2
3
O .
1
mp 241–242°C. H NMR spectrum (CDCl
J, Hz): 1.19 (3H, t, J = 7.0, OCH CH ); 2.15 (3H, s, CH
.86 (2H, s, CH ); 4.03 (2H, q, J = 7.0, OCH CH ); 6.95
1H, br. s, NH); 8.87 (1H, br. s, NH). C NMR spectrum
3
), δ, ppm
(
2
3
3
);
(±)-Ethyl 6-methyl-2-oxo-4-phenyl-1,2,3,4-tetrahydro-
20
3
2
2
3
pyrimidine-5-carboxylate (12g). Yield 499 mg (96%),
1
1
3
(
white solid, mp 209–211°C. H NMR spectrum (DMSO-d ),
6
3
19

Chemistry of Heterocyclic Compounds 2017, 53(3), 316–322
+
δ, ppm (J, Hz): 1.05 (3H, t, J = 6.9, OCH
s, CH ); 3.96 (2H, q, J = 6.9, OCH CH ); 5.46 (1H, s, 6-CH);
.17–7.30 (5H, m, H Ar); 7.74 (1H, s, NH); 9.23 (1H, s, NH).
±)-Isopropyl 6-methyl-2-oxo-4-phenyl-1,2,3,4-tetra-
2
CH
3
); 2.24 (3H,
157.6; 163.8. Found, m/z: 304.1427 [M] . C16
Calculated, m/z: 304.1423.
H
20
N
2
O .
4
3
2
3
7
Pharmacology. Radioligand binding competition assays
were performed in vitro using A2A and A2B human recep-
tors expressed in transfected HeLa (hA2A) and HEK-293
(
2
1
hydropyrimidine-5-carboxylate (12h). Yield 510 mg
1
17
(
(
(
(
93%), white solid, mp 198–200°C. H NMR spectrum
CDCl ), δ, ppm (J, Hz): 1.24 (3H, d, J = 1.9) and 1.26
3H, d, J = 1.8, OCH(CH ); 2.34 (3H, s, CH ); 4.90–5.01
1H, m, OCH(CH ); 5.38 (1H, d, J = 2.7, CH); 5.65 (1H,
(hA2B) cells as described previously. A brief description
3
is given below. Adenosine A receptor competition binding
1
3
)
2
3
experiments were carried out in membranes from CHO–A
1
3
3
)
2
cells labelled with 2 nM [ H]DPCPX. Nonspecific binding
was determined in the presence of 10 µM R-PIA. The
reaction mixture was incubated at 25°C for 60 min. Adenosine
s, NH); 7.19–7.37 (5H, m, H Ar); 7.88 (1H, s, NH).
±)-Ethyl 4-(2-fluorophenyl)-6-methyl-2-oxo-1,2,3,4-
(
19
tetrahydropyrimidine-5-carboxylate (12i). Yield 506 mg
A2A receptor competition binding experiments were carried
1
(
(
91%), white solid, mp 234–236°C. H NMR spectrum
out in membranes from HeLa–A2A cells labelled with 3 nM
3
DMSO-d
6
), δ, ppm (J, Hz): 1.20 (3H, t, J = 7.1,
); 2.31 (3H, s, CH ); 4.07 (2H, q, J = 7.1,
); 5.62 (1H, s, 6-CH); 7.28–7.48 (4H, m, H Ar);
[ H]ZM241385. Nonspecific binding was determined in the
OCH
OCH
2
CH
CH
3
3
presence of 50 µM NECA. The reaction mixture was
incubated at 25°C for 30 min. Adenosine A2B receptor
competition binding experiments were carried out in
membranes from HEK-293–A2B cells (Euroscreen, Gosselies,
2
3
7
.87 (1H, s, NH); 9.43 (1H, s, NH).
(
±)-Isopropyl
4-(2-fluorophenyl)-6-methyl-2-oxo-
22
3
1
5
,2,3,4-tetrahydropyrimidine-5-carboxylate (12j). Yield
Belgium) labelled with 35 nM [ H]DPCPX. Nonspecific
1
43 mg (93%), white solid, mp 168–169°C. H NMR
binding was determined in the presence of 400 µM NECA.
The reaction mixture was incubated at 25°C for 30 min.
spectrum (DMSO-d
6
), δ, ppm (J, Hz): 1.27 (3H, d, J = 1.9)
and 1.29 (3H, d, J = 1.9, OCH(CH
3
)
2
); 2.32 (3H, s, CH
); 5.39 (1H, s, 6-CH); 7.21–
.36 (4H, m, H Ar); 7.80 (1H, s, NH); 9.38 (1H, s, NH).
±)-Ethyl 4-(2-chlorophenyl)-6-methyl-2-oxo-1,2,3,4-
3
);
Adenosine A
3
receptor competition binding experiments
cells
5
7
.08–5.12 (1H, m, OCH(CH
3
)
2
were carried out in membranes from HeLa–A
3
3
labelled with 30 nM [ H]NECA. Nonspecific binding was
determined in the presence of 100 µM R-PIA. The reaction
mixture was incubated at 25°C for 180 min. cAMP assays
were performed at adenosine receptors transfected using a
cAMP enzyme immunoassay kit (Amersham Biosciences).
HEK-293 cells were seeded (10000 cells/well) in 96-well
culture plates and incubated at 37°C in an atmosphere with
(
23
tetrahydropyrimidine-5-carboxylate (12k). Yield 513 mg
1
(
(
87%), white solid, mp 213–215°C. H NMR spectrum
DMSO-d
6
), δ, ppm (J, Hz): 1.08 (3H, t, J = 7.5,
); 2.32 (3H, s, CH ); 3.91 (2H, q, J = 7.5,
); 5.04 (1H, s, 6-CH); 7.16–7.23 (4H, m, H Ar);
OCH
OCH
2
CH
CH
3
3
2
3
7
.72 (1H, s, NH); 9.30 (1H, s, NH).
(
5% CO in Eagle's Medium Nutrient Mixture F-12 (EMEM
2
±)-Isopropyl 4-(2-chlorophenyl)-6-methyl-2-oxo-1,2,3,4-
tetrahydropyrimidine-5-carboxylate (12l). Yield 568 mg
F-12), containing 10% foetal calf serum (FCS) and 1%
L-glutamine. Cells were washed with 3 × 200 µl assay
medium (EMEM-F12 and 25 mM HEPES pH 7.4) and
preincubated with assay medium containing 30 µM
rolipram and test compounds at 37°C for 15 min. 10 µM
NECA was incubated for 15 min at 37°C (total incubation
time 30 min). Reaction was stopped with lysis buffer
supplied in the kit and the enzyme immunoassay was
carried out for detection of intracellular cAMP at 450 nm in
an Ultra Evolution detector (Tecan). Data analysis: IC50
values were obtained by fitting the data with non-linear
regression using Prism 2.1 software (GraphPad, San Diego,
CA). For those compounds that showed either little affinity
or poor solubility a percentage inhibition of specific
binding is reported. Results are the mean of 3 experiments
(n = 3) each performed in duplicate. The experimental
details and dose-response curves obtained during the
functional assays are described in the supporting
information.
1
(
92%), mp 168–169°C. H NMR spectrum (DMSO-d
δ, ppm (J, Hz): 1.21 (3H, d, J = 1.9) and 1.24 (3H, d,
J = 1.9, OCH(CH ); 2.31 (3H, s, CH ); 5.11–5.15 (1H, m,
OCH(CH ); 5.60 (1H, d, J = 2.6, 6-CH); 7.18–7.29 (4H,
m, H Ar); 7.70 (1H, s, NH); 9.34 (1H, s, NH). C NMR
6
),
3
)
2
3
)
3 2
1
3
spectrum (CDCl
3
), δ, ppm: 16.4; 21.7; 21.8; 50.8; 71.2;
9
1
7.8; 125.7; 129.7; 130.4; 131.7; 133.5; 138.7; 155.8;
+
57.6; 163.8. Found, m/z: 308.0931 [M] . C15
H
17ClN
2 3
O .
Calculated, m/z: 308.0928.
(±)-Ethyl
4-(2-methoxyphenyl)-6-methyl-2-oxo-1,2,3,4-
24
tetrahydropyrimidine-5-carboxylate (12m). Yield 522 mg
1
(
90%), white solid, mp 260–262°C. H NMR spectrum
DMSO-d ), δ, ppm (J, Hz): 1.08 (3H, t, J = 7.5, OCH CH );
.30 (3H, s, CH ); 3.80 (3H, s, OCH ); 3.93 (2H, q, J = 7.5,
CH ); 5.50 (1H, d, J = 3.0, 6-CH); 6.87–7.05 (3H, m,
H Ar); 7.14–7.21 (2H, m, NH, H Ar); 9.10 (1H, br. s, NH).
(
6
2
3
2
3
3
OCH
2
3
(
±)-Isopropyl 4-(2-methoxyphenyl)-6-methyl-2-oxo-
1
5
,2,3,4-tetrahydropyrimidine-5-carboxylate (12n). Yield
1
54 mg (91%), white solid, mp 239–241°C. H NMR
This work was financially supported by the Concellería
de Cultura, Educación e Ordenación Universitaria of the
Galician Government (grant GPC2014/03), Centro
Singular de Investigación de Galicia Accreditation 2016–2019
(ED431G/09), and the European Regional Development
Fund (ERDF). Our laboratories are part of the European
COST Actions CM1207 (GLISTEN) and CM15135
(MuTaLig).
spectrum (DMSO-d
6
), δ, ppm (J, Hz): 1.21 (3H, d, J = 2.0)
and 1.24 (3H, d, J = 2.1, OCH(CH
3
)
2
); 2.31 (3H, s, CH
); 5.04–5.09 (1H, m, OCH(CH ); 5.46
1H, d, J = 3.1, 6-CH); 6.85–7.08 (3H, m, H Ar); 7.11–7.23
3
);
3
(
(
.86 (3H, s, OCH
3
)
3 2
1
3
2H, m, NH, H Ar); 9.12 (1H, br. s, NH). C NMR
), δ, ppm: 16.5; 22.1; 22.2; 47.2; 55.4;
0.4; 97.6; 110.8; 121.1; 127.2; 129.9; 130.9; 155.2; 156.4;
spectrum (CDCl
3
7
3
20

Chemistry of Heterocyclic Compounds 2017, 53(3), 316–322
References
13. Crespo, A.; El Maatougui, A.; Biagini, P.; Azuaje, J.; Coelho, A.;
Brea, J.; Loza, M. I.; Cadavid, M. I.; García-Mera, X.
Gutiérrez-de-Terán, H.; Sotelo, E. ACS Med. Chem. Lett.
2013, 4, 1031.
1
. Fredholm, B. B.; Arslan, G.; Halldner, L.; Kull, B.; Shulte, G.;
Wasserman, W. Naunyn-Schmiedeberg's Arch. Pharmacol.
2
000, 362, 299.
14. Sun, Q.; Wang, Y.; Ge, Z.; Cheng, T.; Li, R. Synthesis 2004,
1047.
2
3
. Lennarz, W. J., Lane, M. D. Biol. Chem. 2004, 1, 34.
. Fredholm, B. B.; Ijzerman, A. P.; Jacobson, K. A.; Linden, J.;
Muller, C. E. Pharmacol. Rev. 2011, 63, 1.
15. Kappe, C. O. Eur. J. Med. Chem. 2000, 35, 1043.
16. Dömling, A. Chem. Rev. 2006, 106, 17.
17. Al Maatougui, A.; Azuaje, J.; González-Gómez, M.; Miguez, G.;
Crespo, A.; Carbajales, C.; Escalante, L.; García-Mera, X.;
Gutiérrez-de-Terán, H.; Sotelo, E. J. Med. Chem. 2016, 59,
1967.
4
5
6
. Moro, S.; Deflorian, F.; Bacilieri, M.; Spalluto, G. Curr.
Pharm. Des. 2006, 12, 2175.
. Fredholm, B. B.; Irenius, E.; Kull, B.; Schulte, G. Biochem.
Pharmacol. 2001, 61, 443.
. Adenosine Receptors in Health and Disease. Handbook of
Experimental Pharmacology; Wilson, C. N.; Mustafa, S. J.,
Eds.; Springer-Verlag: Berlin, Heidelberg, 2009, Vol. 193.
. Baraldi, P. G.; Tabrizi, M. A.; Gessi, S.; Borea, P. A. Chem.
Rev. 2008, 108, 238.
18. Xu, F.; Wang, J. J.; Tian, Y. P. Synth. Commun. 2008, 38,
1299.
19. Gholap, A. R.; Venkatesan, K.; Daniel, T.; Lahoti, R. J.;
Srinivasan, K. V. Green Chem. 2004, 6, 147.
20. Song, Q.; An, X.; Che, F.; Shen, T. J. Heterocycl. Chem.
2015, 52, 1496.
7
8
9
. Jacobson, K. A.; Gao, Z. G. Nat. Rev. Drug Discovery 2006, 5, 247.
. Pierce, K. D.; Furlong, T. J.; Selbie, L. A.; Shine, J. Biochem.
Biophys. Res. Commun. 1992, 187, 86.
21. Cepanec, I.; Litvic, M.; Bartolincic, A.; Lovric, M.
Tetrahedron 2005, 61, 4275.
1
1
1
0. Fredholm, B. B.; Ijzerman, A. P.; Jacobson, K. A.; Klotz, K. N.;
22. Hajipour, A. R.; Khazdooz, L.; Zarei, A. Synth. Commun.
2011, 41, 2200.
Linden, J. Pharmacol. Rev. 2001, 53, 527.
1. Aherne, C. M.; Kewley, E. M.; Eltzschig, H. K. Biochim.
Biophys. Acta 2011, 1808, 1329.
23. Chitra, S.; Devanathan, D.; Pandiarajan, K. Eur. J. Med.
Chem. 2010, 45, 367.
2. Cacciari, B.; Pastorin, G.; Bolcato, C.; Spalluto, G.; Bacilieri, M.;
Moro, S. Mini Rev. Med. Chem. 2005, 5, 1053.
24. Niknam, K.; Zolfigol, M. A.; Hossieninejad, Z.; Daneshvar, N.
Chin. J. Catal. 2007, 28, 591.
3
21