Derivatives of 4-amino-6-hydroxy-2-mercaptopyrimidine as novel, potent, and selective A3 adenosine receptor antagonists

Article abstract of DOI:10.1021/jm701159t

A number of derivatives of 4-amino-6-hydroxy-2-mercaptopyrimidine (5) were synthesized and biologically evaluated as A₃ adenosine receptor (A₃ AR) antagonists. The new compounds were designed as open chain analogues of a triazolopyrimidinone derivative displaying submicromolar affinity for the A₃ AR, which had been previously identified using a 3D database search. Substituents R, R′, and R″ attached to the parent compound 5 were chosen according to factorial design and stepwise lead optimization approaches, taking into account the essentially hydrophobic nature of the A₃ AR binding site. As a result, 5m (R = n-C₃H ₇, R′ = 4-ClC₆H₄CH₂, R″ = CH₃) was identified among the pyrimidine derivatives as the ligand featuring the best combination of potency and selectivity for the target receptor. This compound binds to the A₃ AR with a K_i of 3.5 nM and is devoid of appreciable affinity for the A₁, A _2A, and A_2B ARs.

Full text of DOI:10.1021/jm701159t

1764
J. Med. Chem. 2008, 51, 1764–1770
Derivatives of 4-Amino-6-hydroxy-2-mercaptopyrimidine as Novel, Potent, and Selective A3
Adenosine Receptor Antagonists
Barbara Cosimelli,*,† Giovanni Greco,† Marina Ehlardo,† Ettore Novellino,† Federico Da Settimo,§ Sabrina Taliani,§
Concettina La Motta,§ Marusca Bellandi,§ Tiziano Tuccinardi,§ Adriano Martinelli,§ Osele Ciampi,‡
Maria Letizia Trincavelli,‡ and Claudia Martini‡
Dipartimento di Chimica Farmaceutica e Tossicologica, UniVersità di Napoli “Federico II”, Via D. Montesano, 49, 80131 Napoli, Italy,
Dipartimento di Scienze Farmaceutiche, UniVersità di Pisa, Via Bonanno 6, 56126 Pisa, Italy, and Dipartimento di Psichiatria, Neurobiologia,
Farmacologia e Biotecnologie, UniVersità di Pisa, Via Bonanno 6, 56126 Pisa, Italy
ReceiVed September 17, 2007
A number of derivatives of 4-amino-6-hydroxy-2-mercaptopyrimidine (5) were synthesized and biologically
evaluated as A3 adenosine receptor (A3 AR) antagonists. The new compounds were designed as open chain
analogues of a triazolopyrimidinone derivative displaying submicromolar affinity for the A3 AR, which had
been previously identified using a 3D database search. Substituents R, R′, and R′′ attached to the parent
compound 5 were chosen according to factorial design and stepwise lead optimization approaches, taking
into account the essentially hydrophobic nature of the A3 AR binding site. As a result, 5m (R ) n-C3H7, R′
) 4-ClC6H4CH2, R′′ ) CH3) was identified among the pyrimidine derivatives as the ligand featuring the
best combination of potency and selectivity for the target receptor. This compound binds to the A3 AR with
a Ki of 3.5 nM and is devoid of appreciable affinity for the A1, A2A, and A2B ARs.
Introduction
The A3 AR is involved in a variety of important physiological
processes, including modulation of cerebral and cardiac ischemic
damage,11,12 inflammation,13 modulation of intraocular pres-
sure,14 regulation of normal and tumor cell growth,10,15,16 and
immunosuppression.2,17 Accordingly, A3 AR antagonists may
be employed in the acute treatment of stroke and glaucoma and
as antiasthmatic, antiallergenic, and cerebroprotective agents.17
Furthermore, the significant overexpression of A3 AR in several
types of tumor cells and the prosurvival and antiapoptotic effects
of A3 AR stimulation have recently led Baraldi et al. to propose
that antagonists for this receptor might sensitize tumor cells to
chemotherapeutic drugs.15,18
In the past 5 years, considerable progress has been made
toward the identification and development of potent and selective
ligands for A3 AR. The best known classes of A3 AR antagonists
(see Chart 1) are imidazopurinones I,19,20 triazoloquinazolines
II,21,22 pyrazolotriazolopyrimidines III,15,23,24 triazoloquinoxa-
lines IV and V,25–27 isoquinolines VI,28–30 and pyridines
VII.31–33
We have recently described the synthesis and the biological
evaluation of a number of A1 and A3 AR antagonists designed
using database search and lead optimization approaches (struc-
tures and binding data are reported in Table 1).34 Our work
proceeded through the following steps (Figure 1). Briefly, (i)
the Cambridge Structural Database (CSD)35 was searched using
two substructures as queries extracted by known A1 and A3 AR
antagonists; (ii) five potential leads (A-E) were selected to
purchase, synthesize, or translate synthetically into less bulky
or hydrophilic analogues; (iii) out of the five tested compounds,
two displayed micromolar to nanomolar affinity for the A1 AR
(B and C), four showed nanomolar affinity for the A3 R (B-E),
while one turned out to be inactive (A); (iv) lead optimization
efforts gave the best results in the series of 2-(benzimidazol-
2-yl)quinoxalines (C) yielding compounds C′ and C′′ as potent
and selective antagonists at the A1 and A3 ARs, respectively.
Although the triazolopyrimidinone E exhibited an interest-
ing selectivity profile at the A3 AR, simple modifications
about the thiazolidine ring yielded compounds equipotent or
Adenosine is a ubiquitous purine nucleoside that plays a
pivotal role in a large variety of physiological and pathophysi-
ological processes, which are modulated by its interaction with
specific receptors. Four different adenosine receptors (ARs) have
been identified, A1, A2A, A2B, and A3, all of which belong to
the superfamily of G-protein-coupled receptors.1–3 ARs from
different species show 82–92% amino acid sequence homology,
the only exception being the A3 AR, which exhibits 74%
homology between rats and humans.4–6
AR-mediated signaling occurs through inhibition (A1 and A3)
or stimulation (A2A and A2B) of adenylate cyclase, resulting in
a modification of the intracellular production of cAMP. Ad-
enosine receptor coupling to other second messenger systems
has also been described, such as stimulation of phospholipase
C (A1, A2B, and A3) or activation and inhibition of potassium
and calcium channels (A1).7
Each AR is considered an attractive target for pharmacologi-
cal intervention in many pathological conditions, including renal
failure, cardiac and cerebral ischemia, central nervous system
disorders, neurodegenerative diseases, and inflammatory pa-
thologies, such as asthma.8 The development of potent and
selective synthetic antagonists of ARs has been the subject of
medicinal chemistry research for more than 3 decades.9 In
particular, over the past few years, the interest in blocking the
A3 subtype increased after the discovery of its involvement in
cellular growth.10
The A3 AR is widely distributed in mammals, but pronounced
differences in expression levels exist between species. In
humans, the highest density of this receptor has been found in
lung and liver, with lower levels in aorta, brain, and testes.11
* To whom correspondence should be addressed. Phone: 081678614.
Fax: 081678630. E-mail: barbara.cosimelli@unina.it.
†
Università di Napoli “Federico II”.
Dipartimento di Scienze Farmaceutiche, Università di Pisa.
Dipartimento di Psichiatria, Neurobiologia, Farmacologia e Biotec-
§
‡
nologie, Università di Pisa.
10.1021/jm701159t CCC: $40.75
2008 American Chemical Society
Published on Web 02/13/2008

Mercaptopyrimidine DeriVatiVes as Antagonists
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 6 1765
Chart 1. Structures of Known A3 AR Antagonists
the R, R′, and R′′ substituents in the general formulas 5 and
6 were alkyl and phenylalkyl chains.
Preliminary binding assays of the first newly synthesized
compounds revealed that derivatives of series 6 were totally
devoid of any affinity toward ARs (data not shown) while
derivatives of series 5 displayed interesting binding profiles at
the A3 AR. A few precursors of series 5 (4d,e) and 6 (3d,e)
likewise turned out to be inactive (data not shown). Further
research focused on series 5 through the design, synthesis, and
testing of additional 4-amino-6-hydroxy-2-mercaptopyrimidines
to identify those characterized by the highest affinity and
selectivity as A3 AR antagonists.
Chemistry
The derivatives 5a-s were obtained in three steps starting
from the commercially available 4-amino-6-hydroxy-2-mercap-
topyrimidine monohydrate (1) (Scheme 1). The first was the
alkylation of the sulfur atom at the 2-position by reaction with
iodomethane or the opportune bromo(phenyl)alkane in 1 M
NaOH to give compounds 2a-e (Table 1 of Supporting
Information), which were collected by filtration and were
sufficiently pure to be used in the next step without further
purification. The second step, i.e., the alkylation of the 4-oxygen
atom of compounds 2a-e to prepare derivatives 4, always gave
a mixture of the isomeric products 3 and 4 because of the
keto–enol equilibrium of the pyrimidinone scaffold. The ex-
perimental conditions were extensively studied with regard to
solvent (anhydrous DMF, acetonitrile, ...), base quantities,
temperature, and time of reaction. The best results in terms of
4 yields were obtained when the reaction was carried out in
anhydrous DMF in the presence of an excess of K2CO3 as base,
which generally gave a 1:3 ratio of isomers 3 and 4, even though
the relative quantities of the two isomers obtained varied
depending on the stereoelectronic features of both reagents.
Conversely, for example, the use of acetonitrile as solvent
increased the formation of the 3 isomer. For only the case of
derivatives with R′ ) CH3, the use of methyl iodide always
gave an excess of the N-isomer 3 with respect to 4 (see Table
2 of Supporting Information for details). The 4 isomer was then
easily isolated by flash chromatography and characterized
(Tables 2 and 3 of Supporting Information). The final step was
the conversion of the 4-amino group of 4 into the amide group
of 5 by reaction with the opportune anhydride and concentrated
H2SO4 as catalyst (Table 4 of Supporting Information).
Table 1. Affinities at Human A1, A2A, and A3 ARs of Compounds
Designed as A1 AR and/or A3 AR Antagonists by Means of 3D
Database Searching34
Ki (nM)a
b
c
d
compd
hA1
hA2A
hA3
A
B
>10000
3336 ( 557
50 ( 15
>10000
>10000
561 ( 17
3440 ( 980
833 ( 67
>10000
>10000
337 ( 28
16 ( 3
>1000
125 ( 20
763 ( 13
955 ( 215
26 ( 9
C (R ) H)
C′ (R ) SC2H5)
C′′ (R ) CH3)
D
0.5 ( 0.01
8000 ( 567
>10000
130 ( 3
440 ( 33
>1000
73 ( 5
0.22 ( 0.02
Ee
>10000
DPCPXf
0.5 ( 0.03
14 ( 4
Biology
NECAg
The affinities of the new compounds 5a-s toward human
A1, A2A, and A3 ARs were evaluated by competition experiments
assessing their respective abilities to displace [3H]DPCPX,
[3H]NECA, or [125I]AB-MECA binding from transfected Chi-
nese hamster ovary (CHO) cells. Experiments were performed
as described elsewhere.36
Compounds 5m, 5o, 5p, and 5r, which all showed high A3
affinity, were also tested in functional assays at human A2B and
A3 ARs by measuring their effects on NECA-mediated cAMP
modulation in transfected CHO cells.37
Cl-IB-MECAh
890 ( 61
401 ( 25
a The Ki values are the mean ( SEM of at least three determinations
derived from an iterative curve-fitting procedure (Prism program, GraphPad,
San Diego, CA). b Displacement of specific [3H]DPCPX binding in
membranesobtainedfromCHOcellsstablyexpressinghA1 AR.c Displacement
of specific [3H]NECA binding in membranes obtained from CHO cells
stably expressing hA2A AR. d Displacement of specific [125I]AB-MECA
binding in membranes obtained from CHO cells stably expressing hA3 AR.
e Assayed with a purity degree of 96%. f DPCPX: 1,3-dipropyl-8-cyclopentyl-
xanthine. g NECA: 5′-N-ethylcarboxamidoadenosine. h Cl-IB-MECA: 2-chloro-
N6-(3-iodobenzyl)adenosine-5′-N-methylcarboxamide.
Results and Discussion
inactive in binding experiments. Because the triazolopyri-
midinone scaffold was not synthetically versatile, we turned
our attention to derivatives of 4-amino-6-hydroxy-2-mercap-
topyrimidine (5) and 6-amino-2-mercaptopyrimidin-4(3H)-
one (6) as open chain analogues of E, which were better
suited to lead optimization (Figure 2). Taking into account
that the binding site of A3 AR antagonists is mostly lipophilic,
Table 2 lists the binding affinities for the human A1, A2A
,
and A3 ARs of the new derivatives of 4-amino-6-hydroxy-2-
mercaptopyrimidine (5a-s). The first eight compounds (5a-h)
were prepared according to a factorial design in which the size/
lipophilicity of substituents R, R′, and R′′ was explored at two
levels. For R and R′ the low and high levels correspond to CH3

1766 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 6
Cosimelli et al.
Figure 1. Strategy adopted to identify lead compounds as antagonists at the A1 and/or A3 ARs by a 3D search of the CSD.
for A3 AR versus A2A AR is conferred by a large R′ and a
small R′′ (compare 5g vs 5h). Moreover, a pairwise comparison
of the couples 5c,d, 5e,f, and 5h,g indicates that switching of
R′′ from CH3 to C2H5 slightly lowers affinity for A3 AR. Thus,
further modifications of R′′ were not taken into consideration.
Taking 5g as the reference structure, further modifications
were made to increase potency and selectivity at the A3 AR.
First, we inserted a bulky benzyl group on the S-2 atom,
obtaining 5i, which showed a significant decrease in affinity
for the target receptor. Then, by keeping fixed R ) n-C3H7 and
R′′ ) CH3, we increased the weight of R′ through substituted
benzyl chains (compounds 5j-o). This modification produced
a general enhancement in A3 AR affinity, with 5o (R′ )
4-CH3OC6H4CH2) being the most active compound (Ki ) 1.8
nM). Within this subset, 5m (R′ ) 4-ClC6H4CH2) was the best-
performing derivative compared with 5g, showing a Ki of 3.5
nM at the A3 AR and no affinity for A1 and A2A ARs. Additional
modifications of R′ led to the synthesis of 5p and 5q, featuring
a PhCH2CH2, and a c-HexCH2 chain, respectively. Although
these two compounds showed excellent affinity (5p was the most
active compound of the whole series with a Ki of 1.4 nM) and
selectivity at the A3 AR, they were not significantly better than
5m.
Figure 2. Design of open-chain analogues of triazolopyrimidinone E
as potential A3 AR antagonists.
Scheme 1. Synthesis of 4-Amino-6-hydroxy-2-mercaptopyrimidine
Derivatives 5a-s (See Table 2 for the Substituents)a
Finally, to find the optimum size/lipophilicity of R, we took
5m as the reference structure and replaced the n-C3H7 with a
C2H5 or a n-C4H9 to obtain 5r and 5s, respectively. Both
compounds exhibited nanomolar potency at the A3 AR (Ki of
2.1 and 8.6 nM, respectively) and a high selectivity over the
A1 and A2A ARs, but they still do not possess the same
outstanding affinity/selectivity profile of their homologue, 5m.
Compounds 5m, 5o, 5p, and 5r were also tested at the A2B
AR by evaluating their inhibitory effects on NECA-mediated
cAMP accumulation in CHO cells stably expressing this receptor
subtype (Table 3). These derivatives were inactive in this assay,
thus indicating a complete lack of affinity for A2B AR.
Given the recent discovery of non-nucleoside AR agonists,38,39
the antagonistic activities of compounds 5m, 5o, 5p, and 5r
were evaluated in cAMP functional assays at the A3 AR (Table
3). As expected, all of these compounds displayed full antago-
nism with potencies comparable to their binding affinities.
Furthermore, when tested in the absence of the receptor agonist
NECA, they did not show any significant effect on cAMP levels,
even following stimulation by forskolin, indicating neutral
antagonism (Figure 3).
a Reagents and conditions: (i) methyl iodide or (phenyl)alkyl bromide,
1 M NaOH; (ii) methyl iodide or (substituted phenyl)alkyl bromide,
anhydrous DMF, excess K2CO3; (iii) acetic or propionic anhydride, catalytic
conc H2SO4.
and n-C3H7, respectively; for R′′ the low and high levels
correspond to CH3 and C2H5, respectively. The best combination
of substituents was found in 5g (R ) R′ ) n-C3H7, R′′ ) CH3),
which displayed a Ki value of 26 nM at the A3 AR, submicro-
molar affinity at the A1 AR, and no affinity at the A2A AR.
The binding data of 5a-h suggest that the effects of R, R′,
and R′′ on A3 AR affinity are fairly additive and that selectivity

Mercaptopyrimidine DeriVatiVes as Antagonists
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 6 1767
Table 2. Affinities at Human A1, A2A, and A3 ARs of Derivatives of 4-Amino-6-hydroxy-2-mercaptopyrimidines 5a-s
Ki (nM) or % inhibitiona
b
c
d
compd
R
R′
R′′
CH3
CH3CH2
CH3
CH3CH2
CH3
CH3CH2
CH3
CH3CH2
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
hA1
30%
2%
7%
19%
43%
hA2A
29%
21%
hA3
49%
51%
5a
5b
5c
5d
5e
5f
5g
5h
5i
5j
5k
5l
5m
5n
5o
5p
5q
5r
5s
CH3
CH3
CH3
CH3
CH3
CH3
CH3CH2CH2
CH3CH2CH2
CH3
1121 ( 88
398 ( 20
10%
31 ( 2.4
45 ( 0.9
166 ( 10
276 ( 24
26 ( 0.8
38 ( 0.7
525 ( 21
7.5 ( 0.22
6.3 ( 0.07
7.1 ( 0.48
3.5 ( 0.05
30 ( 2.7
1.8 ( 0.15
1.4 ( 0.14
22 ( 0.4
2.0 ( 0.2
8.6 ( 0.81
CH3CH2CH2
CH3CH2CH2
CH3CH2CH2
CH3CH2CH2
C6H5CH2
CH3CH2CH2
CH3CH2CH2
CH3CH2CH2
CH3CH2CH2
CH3CH2CH2
CH3CH2CH2
CH3CH2CH2
CH3CH2CH2
CH3CH2
CH3
486 ( 34
5474 ( 275
1462 ( 111
34%
2445 ( 110
1840 ( 18
582 ( 36.3
17%
4%
30%
CH3CH2CH2
CH3CH2CH2
CH3CH2CH2
C6H5CH2
2-ClC6H4CH2
3-ClC6H4CH2
4-ClC6H4CH2
2,4-Cl2-C6H3CH2
4-MeOC6H4CH2
C6H5CH2CH2
c-Hex-CH2
71 ( 3.4
23%
36%
4149 ( 400
41%
43%
29%
47%
27%
33%
56%
26%
46%
2443 ( 240
2080 ( 205
43%
4-ClC6H4CH2
4-ClC6H4CH2
4480 ( 440
CH3(CH2)3
0%
a The Ki values are the mean ( SEM of at least three determinations derived from an iterative curve-fitting procedure (Prism program, GraphPad, San
Diego, CA). The % inhibition of specific radioligand binding is determined at 10 µM (A1 and A2A ARs), and 1 µM (A3 ARs) ligand concentration. b Displacement
of specific [3H]DPCPX binding in membranes obtained from CHO cells stably expressing hA1 AR. See Ki values of reference ligands in Table 1. c Displace-
ment of specific [3H]NECA binding in membranes obtained from CHO cells stably expressing hA2A AR. See Ki values of reference ligands in Table 1.
d Displacement of specific [125I]AB-MECA binding in membranes obtained from CHO cells stably expressing hA3 AR. See Ki values of reference ligands
in Table 1.
Table 3. Potency of Compounds 5o, 5m, 5p, and 5r versus hA2B and
hA3 ARs
a
IC50 (nM) of cAMP assays
for hA2B
for hA3
compd
R
R′
R″
CHO cells
CHO cells
5m CH3CH2CH2 4-ClC6H4CH2
CH3 >10000 (0%)
9.2 ( 0.7
2.7 ( 0.2
5o
CH3CH2CH2 4-MeOC6H4CH2 CH3 >10000 (0%)
Figure 3. Effect of selected compounds (5m, 5o, 5p, and 5r) on
forskolin (FK) stimulated cAMP production in A3 CHO cells. Cells
were treated with 1 µM forskolin in the absence or in the presence of
100 nM NECA or 10 µM of each compound. Data are expressed as
percentage of cAMP production with respect to FK-stimulated cAMP
levels, set to 100%. Each data point represents the mean ( SEM of
three different experiments.
5p CH3CH2CH2 C6H5CH2CH2
CH3 >10000 (13%) 1.25 ( 0.09
CH3 >10000 (10%) 1.84 ( 0.1
5r
CH3CH2
4-ClC6H4CH2
a The data are expressed as the mean ( SEM of four independent
experiments performed in triplicate. In parentheses are indicated the
percentages of inhibition, at 10 µM compound, of the cAMP levels,
stimulated by 100 nM NECA for A2B adenosine receptors.
The most potent compound at the A3 AR, 5p, was docked
into our recently published model of this receptor40 according
to a procedure described in the Experimental Section. As shown
in Figure 4, 5p is engaged in several attractive interactions: the
ether oxygen receives an H-bond from the N250 (6.55) side
chain; the pyrimidine moiety π-stacks with the aromatic rings
of H95 (3.37) and W243 (6.48); the phenethyl and the propylthio
substituents fill two adjacent hydrophobic pockets delimited by
the side chains of F182 (5.43) and W185 (5.46) and of M99
(3.41), L102 (3.44), W185 (5.46), P189 (5.50), and F239 (6.44),
respectively. The putative binding mode of 5p is consistent with
the mutagenesis data supporting a key role of H95 (3.37), F182
(5.43), W243 (6.48), and N250 (6.55) in the recognition of
several A3 AR antagonists.41 This model will probably be
exploited to design new A3 AR antagonists similar to our
pyrimidine derivatives. A glance at the Y-like disposition of
the three substituents of 5p within the A3 AR binding cavity
may suggest why compounds of series 6 are devoid of any
affinity toward this receptor: their R′ and R substituents are too
close, with the result that the former is forced to clash with the
binding cleft boundaries.
In summary, we prepared derivatives of 4-amino-6-hydroxy-
2-mercaptopyrimidine (5) as open chain analogues of the weakly
potent A3 AR antagonist triazolopyrimidinone derivative (E).
The optimum size/lipophilicity of the R, R′, and R″ substituents
born by the new scaffold were sought by a preliminary factorial
design followed by a stepwise lead optimization. As a result,
we identified 5m as the ligand displaying the best combination

1768 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 6
Cosimelli et al.
Figure 4. Compound 5p docked into the A3 AR model.40
Biological Methods. Materials. [3H]DPCPX, [3H]NECA, and
of potency at the A3 AR (Ki ) 3.5 nM) and selectivity (Ki >
10 µM at the A1 and A2A ARs).
[
125I]AB-MECA were obtained from DuPont-NEN (Boston, MA).
Adenosine deaminase (ADA) was from Sigma Chemical Co. (St.
Louis, MO). All other reagents were from standard commercial
sources and of the highest commercially available grade. CHO cells
stably expressing human A1, A2A, A2B, and A3 receptors were kindly
supplied by Prof. K. N. Klotz, Wurzburg University, Germany.44
Experimental Section
Chemistry. Evaporation was performed in vacuo (rotary evapo-
rator). Analytical TLC was carried out on Merck 0.2 mm precoated
silica gel aluminum sheets (60 F-254). Silica gel 60 (230–400 mesh)
was used for column flash chromatography. Melting points were
determined using a Büchi apparatus B 540 and are uncorrected.
Routine nuclear magnetic resonance spectra were recorded on a
Varian Mercury 400 spectrometer operating at 400 MHz for the
proton and 100 MHz for the carbon in DMSO-d6 solution.
Elemental analyses were performed by our Analytical Laboratory
in Pisa, and the results agreed with theoretical values to within
(0.4%.
General Procedure for the Synthesis of 4-Amino-6-hydroxy-
2-(phenyl)alkylthiopyrimidines 2a-e. Haloalkane (12.4 mmol)
was added dropwise into a solution of 4-amino-6-hydroxy-2-
mercaptopyrimidine monohydrate (1g, 6.2 mmol) in 1 M NaOH
(12.4 mL). The reaction mixture was kept, under stirring, at 50–55
°C for 4 h and then was left overnight at room temperature. Next,
the mixture was neutralized with acetic acid, obtaining a solid that
was collected by filtration, washed with petroleum ether, and dried.
The solid, identified by 1H and 13C NMR spectra or by comparison
with published melting points42,43 (see Table 1 of Supporting
Information for details), was sufficiently pure to be used in the
next reaction without further purification.
General Procedure for the Synthesis of 6-Amino-3-(phenyl)
alkyl-2-(phenyl)alkylthiopyrimidin-4-ones 3a-p and 4-Amino-
6-(phenyl)alkyloxy-2-(phenyl)-alkylthiopyrimidines 4a-p. Ha-
loalkane (10.0 mmol) was added dropwise into a suspension of
the opportune compound 2a-e (10.0 mmol) in anhydrous DMF
(30 mL) and K2CO3 (30 mmol). The reaction mixture was kept,
under stirring, at 80–85 °C for 4 h. After the mixture was cooled,
the inorganic material was taken off by filtration and the solvent
was removed in vacuo. The solid residue, consisting of a mixture
of 3 and 4 isomers, was resolved by flash chromatography.
Purification conditions, yields, and physicochemical and spectral
data are reported in Tables 2 and 3 of the Supporting Information.
General Procedure for the Synthesis of Compounds 5a-s.
A mixture of amine 4a-p (1.2 mmol), acetic or propionic anhydride
(72 mmol), and a catalytic amount of concentrated H2SO4 was
warmed to 50 °C until disappearance of the substrate (40–120 min),
while the progress of the reaction was monitored by TLC. After
cooling at room temperature, the mixture was poured into ice/water
and extracted with chloroform. The organic phase was washed with
a saturated solution of NaHCO3, then with water, and dried on
Na2SO4. Removal of the solvent under reduced pressure gave the
crude products 5a-s, which were purified by recrystallization from
n-hexane (Table 4 of Supporting Information).
Adenosine Receptor Binding Assay. Human A1 Adenosine
Receptors. Aliquots of membranes (50 µg proteins) obtained from
A1 CHO cells were incubated at 25 °C for 180 min in 500 µL of
T1 buffer (50 mM Tris-HCl, 2 mM MgCl2, 2 units/mL ADA, pH
7.4) containing [3H]DPCPX (3 nM) and six different concentrations
of the newly synthesized compounds. Nonspecific binding was
determined in the presence of 50 µM RPIA.36 The dissociation
constant (Kd) of [3H]DPCPX A1 CHO cell membranes was 3 nM.
Human A2A Adenosine Receptors. Aliquots of cell membranes
(80 µg) were incubated at 25 °C for 90 min in 500 µL of T2 buffer
(50 mM Tris-HCl, 2 mM MgCl2, 2 units/mL ADA, pH 7.4) in the
presence of 30 nM of [3H]NECA and six different concentrations
of the newly synthesized compounds. Nonspecific binding was
determined in the presence of 100 µM NECA.36 The dissociation
constant (Kd) of [3H]NECA in A2A CHO cell membranes was 30
nM.
Human A3 Adenosine Receptors. Aliquots of cell membranes
(40 µg) were incubated at 25 °C for 90 min in 100 µL of T3 buffer
(50 mM Tris-HCl, 10 mM MgCl2, 1 mM EDTA, 2 units/mL ADA,
pH 7.4) in the presence of 1.4 nM [125I]ABMECA and six different
concentrations of the newly synthesized compounds. Nonspecific
binding was determined in the presence of 50 µM R-PIA.36 The
dissociation constant (Kd) of [125I]AB-MECA in A3 CHO cell
membranes was 1.4 nM.
Measurement of Cyclic AMP Levels on hA2B and hA3
CHO Cells. Intracellular cAMP levels were measured using a
competitive protein binding method.37 (Nordstedt) CHO cells
expressing recombinant hA2B or hA3 ARs were harvested by
trypsinization. After centrifugation and resuspension in medium,
cells (∼60 000) were plated in 24-well plates in 0.5 mL of medium.
After 48 h, the medium was removed, and the cells were incubated
at 37 °C for 15 min with 0.5 mL of DMEM in the presence of
Ro20-1724 (20 µM), as phosphodiesterase inhibitor, and ADA (1U/
mL). The antagonistic profile of the new compounds toward A2B
AR was evaluated assessing their ability to inhibit 100 nM NECA-
mediated accumulation of cAMP. The antagonistic profile of the
new compounds toward A3 AR was evaluated by assessing their
ability to counteract 100 nM NECA-mediated inhibition of cAMP
accumulation stimulated by 1 µM forskolin. Cells were incubated
in the reaction medium (15 min at 37 °C) with different compound
concentrations (1 nM to 10 µM) and then treated with NECA. In
parallel, aliquots of cells were treated with the compound alone
(10 µM) in the absence or in the presence of forskolin. The reaction

Mercaptopyrimidine DeriVatiVes as Antagonists
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 6 1769
was terminated by removal of the medium and addition of 0.4 N
HCl. After 30 min, lysates were neutralized with 4 N KOH, and
the suspension was centrifuged at 800g for 5 min. For determination
of cAMP production, cAMP binding protein isolated from bovine
adrenal glands was incubated with [3H]cAMP (2 nM), 50 µL of
cell lysate, or cAMP standard (0–16 pmol) at 4 °C for 150 min in
a total volume of 300 µL. Bound radioactivity was separated by
rapid filtration through GF/C glass fiber filters and washed twice
with 4 mL of 50 mM Tris-HCl, pH 7.4. The radioactivity was
measured by liquid scintillation spectrometry.
Klotz for his generous gift of transfected CHO cells expressing
human A1, A2A, A2B, and A3 receptors.
Supporting Information Available: Tables listing yields,
chemical-physical properties, and spectral data of compounds
2a-e, 3a-o, 4a-o, and 5a-s, as well as analytical data of 5a-s.
This material is available free of charge via the Internet at http://
pubs.acs.org.
References
All compounds were routinely dissolved in DMSO and diluted
with assay buffer to the final concentration, where the amount of
DMSO never exceeded 2%. Percentage inhibition values of specific
radiolabeled ligand binding at 1–10 µM concentration are the mean
( SEM of at least three determinations. For compound IC50
determination, at least six different ligand concentrations were used.
IC50 values, computer-generated using a nonlinear regression
formula on a computer program (Graph-Pad, San Diego, CA), were
converted to Ki values, based on the Kd values of radioligands in
the different tissues and using the Cheng and Prusoff equation.45
Ki values are the mean ( SEM of at least three determinations.
Computational Methods. The ligands were submitted to 1000
steps of Monte Carlo based conformational search employing the
MMFFs force field with a distance-dependent dielectric constant
of 1.0.46 The resulting molecules were then energy-minimized using
the conjugated gradient method until a convergence value of 0.05
kcal/(mol·Å) was reached. The obtained structures were docked
into a recently published40 model of the A3 receptor using the
AUTODOCK 3.0 program.47 The regions of interest used by
AUTODOCK were defined by considering N250 as the central
residue of a grid of 44, 54, and 50 points in the x, y, and z directions.
A grid spacing of 0.375 Å and a distance-dependent function of
the dielectric constant were used for the energetic map calculations.
By use of a Lamarckian genetic algorithm, the compounds were
subjected to 100 runs of the AUTODOCK search with 500 000
steps of energy evaluation; the other parameters were set to default
values. Cluster analysis was performed on the docked results,
applying an rms tolerance of 1.0 Å.
The best docking complexes were energy-minimized by applying
a constraint of 30 kcal/(mol ·Å) on the R carbons of the TMs. Each
minimization consisted of 5000 steps with a combined algorithm,
namely, the sequential steepest descent and conjugate gradient
methods for the first 1000 and the last 4000 steps, respectively.
The resulting geometry-optimized complexes were then subjected
to 1.2 ns of molecular dynamics (MD) simulation using AMBER
9.0.48 MD simulations were carried out at 300 K using the parm94
force field. Chlorine ions were added as counterions to neutralize
the system. The time step of the simulations was 2.0 fs, with a
cutoff of 12 Å for the nonbonded interaction, and the SHAKE
option was employed to keep all bonds involving hydrogen atoms
rigid. The MD simulation was carried out by gradually decreasing
the constraint on the R carbons of the TMs from 30 to 5 kcal/
(mol·Å).
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The final structure of each complex was obtained by minimizing
the average structure of the last 400 ps of MD using the conjugate
gradient method until a convergence of 0.05 kcal/(Å ·mol) was
reached. During the whole computational simulation, with the
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Acknowledgment. This work was financially supported by
MIUR (Cofin 2005, ex 40%). We thank Prof. Dr. Karl-Norbert

1770 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 6
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