A new generation of adenosine receptor antagonists: From di- to trisubstituted aminopyrimidines

Article abstract of DOI:10.1016/j.bmc.2008.01.013

New adenosine receptor ligands were designed as hybrid structures between previously synthesized substituted dicyanopyridines and aminopyrimidines, yielding two series of cyano-substituted diphenylaminopyrimidines. We were interested in assessing the effect of this substitution pattern on both affinity and intrinsic activity, as the dicyanopyridines comprised both agonists and inverse agonists, whereas the original aminopyrimidines were exclusively inverse agonists. It was found that the new compounds were generally selective for adenosine A₁ receptors, although affinity for the adenosine A_2A receptor was also noticed for some of the compounds. In a cAMP second messenger assay the compounds behaved as inverse agonists rather than agonists. Among the more A₁ receptor-selective compounds were 5 (LUF6048), 27 (LUF6040) and 53 (LUF6056) with K_i values of 8.1, 1.2 and 5.7 nM, respectively.

Full text of DOI:10.1016/j.bmc.2008.01.013

Available online at www.sciencedirect.com
Bioorganic & Medicinal Chemistry 16 (2008) 2741–2752
A new generation of adenosine receptor antagonists:
From di- to trisubstituted aminopyrimidines
Jacobus P. D. van Veldhoven, Lisa C. W. Chang, Jacobien K. von Frijtag Drabbe Kunzel,
¨
Thea Mulder-Krieger, Regina Struensee-Link, Margot W. Beukers,
Johannes Brussee and Adriaan P. IJzerman*
Leiden/Amsterdam Center for Drug Research, Division of Medicinal Chemistry, PO Box 9502, 2300 RA Leiden, The Netherlands
Received 28 September 2007; revised 3 January 2008; accepted 8 January 2008
Available online 12 January 2008
Abstract—New adenosine receptor ligands were designed as hybrid structures between previously synthesized substituted dicyano-
pyridines and aminopyrimidines, yielding two series of cyano-substituted diphenylaminopyrimidines. We were interested in assess-
ing the effect of this substitution pattern on both affinity and intrinsic activity, as the dicyanopyridines comprised both agonists and
inverse agonists, whereas the original aminopyrimidines were exclusively inverse agonists. It was found that the new compounds
were generally selective for adenosine A1 receptors, although affinity for the adenosine A2A receptor was also noticed for some
of the compounds. In a cAMP second messenger assay the compounds behaved as inverse agonists rather than agonists. Among
the more A1 receptor-selective compounds were 5 (LUF6048), 27 (LUF6040) and 53 (LUF6056) with Ki values of 8.1, 1.2 and
5.7 nM, respectively.
ꢀ 2008 Elsevier Ltd. All rights reserved.
R1
R2
1. Introduction
X = C, Y = N
X = N, Y = C
Adenosine receptors belong to the large super family of
G protein-coupled receptors. Four subtypes have been
cloned and characterized, A1, A2A, A2B and A3. Their
individual physiological roles and pharmacology have
been extensively reviewed.1 Traditionally, agonists for
adenosine receptors were derivatives of adenosine, and
antagonists were often xanthines, such as caffeine, theo-
phylline and structural analogues. This simple scenario
has changed dramatically, most particularly for the
adenosine A1 receptor. For instance, many different
classes of adenosine A1 receptor antagonists have now
been designed, synthesized and tested. These include
all kinds of different mono-, bi-, tri- and even quadri-
cyclic mostly nitrogen containing aromatic compounds.2
Among our own efforts were two classes of substituted
pyrimidines, exemplified as A in Figure 1.3 Very interest-
ingly, also agonists have greater structural diversity than
originally anticipated. The ribose group in adenosine,
long thought to be mandatory for agonistic activity,
was shown to be absent in a series of 2-aminopyridine-
NC
S
CN
X
Y
O
HO
N
N
R
N
NH2
H
A
B
R1
R1
R2
R2
C
and
CN
NC
N
O
N
O
N
N
H
R
N
N
R
H
Figure 1. Rationale for the design of the di- and trisubstituted
aminopyrimidines (compound C). A: diphenyl substituted aminopyr-
imidines as adenosine receptor antagonists;3 B: general structure of
non-ribose agonists for the adenosine receptors.4,5
Keywords: Adenosine; Adenosine A1 receptor; Inverse agonist;
Cyanopyrimidines.
*
Corresponding author. Tel.: +31 0 71 527 4651; fax: +31 0 71 527
4565; e-mail: ijzerman@lacdr.leidenuniv.nl
0968-0896/$ - see front matter ꢀ 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2008.01.013

2742
J. P. D. van Veldhoven et al. / Bioorg. Med. Chem. 16 (2008) 2741–2752
3,5-dicarbonitriles. Nevertheless, Rosentreter et al. in a
series of patents4 disclosed these compounds to be ago-
nists for adenosine receptors. On these findings we elab-
orated and synthesized analogous compounds,5 indeed
confirming the agonistic activity on adenosine receptors
of some of these derivatives (B in Fig. 1). In particular,
LUF5853, in which R1 and R2 combined into a dioxy-
methylene bridge, was a potent and selective full agonist
for the adenosine A1 receptor (Ki hA1 = 11 nM).
Bromination was done at the 5-position of the 2-amino-
pyrimidine with Br2 in the presence of CaCO3 in CHCl3
(8 and 9),9 followed by reaction with acid chlorides in
pyridine at room temperature to obtain the correspond-
ing amides (10–22) in modest to good yields (20–92%
yield).8 The carbonitrile was introduced by substitution
of the bromine with CuCN in pyridine in a sealed tube
at 250 ꢁC under microwave conditions.10 This final step
gave the 2-amido-5-cyanopyrimidines (23–37) in 17–
66% yield. Compounds 43–47 were synthesized accord-
ing to Scheme 2. The amine 42, the unsubstituted
analogue of compound 43, was previously described
by Chang3 starting from ethyl benzoylacetate and ben-
zamidine. To synthesize its methylenedioxy analogue
43, we chose a new pathway starting from piperonal.
Nucleophilic addition of hydroxylamine to piperonal
(38) gave the arylnitrile (39) in high yield via dehydra-
tion of the oxime intermediate.11 The enaminonitrile
(40) was obtained in high yield by reaction of acetoni-
trile with 39 in the presence of potassium tert-butox-
ide.12 The pyrimidine ring was created by refluxing 40
and N,N-dimethylbenzamide in phosphorous oxychlo-
ride.13 Subsequent substitution with ammonia in a
sealed vessel gave the aminopyrimidine 43 in a good
yield.3 Pyrimidine 43 was reacted at room temperature
with several acid chlorides and gave good yields of the
screening compounds 44–47. Unexpectedly the subse-
quent bromination of 44–47 failed with various meth-
ods. Analogous to Scheme 1, bromination of 43
followed by microwave-assisted amide formation at
100 ꢁC failed. Hence the procedure of Scheme 2 was
not suitable for the synthesis of the cyano products
51–58; these compounds were synthesized according to
Scheme 3. Functionalised malononitrile5 was refluxed
in methanol in the presence of benzamidine hydrochlo-
ride and K2CO3 to yield only 6% of 51. The yield was
improved by an additional oxidation with KMnO4 to
oxidize the dihydropyrimidine-intermediate resulting in
In the present study, hybrid structures of A and B were
designed, predominantly to explore the role of (i) cyano
substitution at the pyrimidine core structure and (ii) the
introduction of a dioxymethylene bridge on one of the
two phenyl rings (compound C). We were interested in
assessing the effect of this substitution pattern on both
affinity and intrinsic activity. It was learned that such
compounds were generally selective for adenosine A1
receptors, although affinity for the adenosine A2A recep-
tor was also noticed for some of the compounds. In a
cAMP second messenger assay the compounds behaved
as inverse agonists rather than agonists.
2. Results and discussion
2.1. Chemistry
Compounds 3–7 and 23–37 were synthesized according
to Scheme 1. Chalcone 2 was prepared via an aldol con-
densation in the presence of NaOH6 while 1 was com-
mercially available. Guanidine was freed from its
hydrochloride salt form with aqueous NaOH and re-
acted with the appropriate chalcone according to a
method described by Al-Hajjar and Sabri.3,7 The pri-
mary amines 3 and 4 were obtained in 29–32% yield.
Acid chlorides were reacted with the 2-aminopyrimi-
dines (3 and 4) in pyridine to give the desired 2-amido-
pyrimidines 5–7 in 33–73% yield.8
a
51% yield of the aminopyrimidine–carbonitrile
R1
1
R
R1
R1
O
R1
b
a
N
R1
N
O
R2
N
NH2
N
N
H
1. R1 = H
2. R1 = OCH2O
3. R1 = H
4. R1 = OCH2O
c
d
5-7
R1
R1
R1
R1
R1
R1
N
Br
e
Br
N
O
N
O
N
N
N
R2
R2
N
N
N
NH2
H
H
23-37 See Table 1
8,9
10-22
Scheme 1. Synthetic route to 2-amido-4,6-disubstituted-pyrimidine-5-carbonitriles. Reagents and conditions: (a) guanidineÆHCl, NaOH, H2O,
ethanol, 2 h, 130 ꢁC, MW; (b) RCOCl, pyridine; (c) Br2, CaCO3, CHCl3; (d) RCOCl, pyridine; (e) CuCN, pyridine, 20 min, 250 ꢁC, MW.

J. P. D. van Veldhoven et al. / Bioorg. Med. Chem. 16 (2008) 2741–2752
2743
O
O
O
O
O
N
O
b
O
a
O
c
N
O
N
Cl
N
NH2
38
40
41
39
d
O
O
O
O
O
O
f
e
Br
N
O
N
O
N
N
N
H
R2
N
N
H
R2
N
NH2
48
43
44-47
Scheme 2. Synthetic route to 4-amido-2,6-disubstituted-pyrimidines. Reagents and conditions: (a) hydroxylamineÆHCl, N-methylpyrrolidone,
115 ꢁC, 4 h; (b) acetonitrile, t-BuOK, benzene, 20 h; (c) N,N-dimethylbenzamide, POCl3, 120 ꢁC, 1 h; (d) NH3, ethanol, sealed vessel, 20 h; (e)
RCOCl, pyridine, 100 ꢁC, 1 h; (f) Br2, CaCO3, CHCl3, reflux or Br2, AcOH, 100 ꢁC.
R1
R1
R1
R1
N
R1
R1
N
N
O
a, b
c
+
N
N
R2
N
H2N
NH
N
NH2
N
N
H
49. R1 = H
50. R1 = OCH2O
51. R1 = H
53-58 See Table 2
52. R1 = OCH2O
Scheme 3. Synthetic route to 4-amido-2,6-disubstituted-pyrimidine-5-carbonitriles. Reagents and conditions: (a) K2CO3, MeOH, reflux, 2 h; (b)
KMnO4, acetone, 15 min; (c) RCOCl, pyridine, MW.
51.14,15 The reaction of 51 and 52 with aliphatic acid
chlorides gave only low yields of 53–58 and required
heating in the microwave synthesizer,8 whereas aromatic
acid chlorides and tert-butyryl chloride failed.
affinity of 8.1 nM for the adenosine A1 receptor. As
the major focus of this study the introduction of a cyano
group on the 5-position in both series (23–37 and 51–58)
often led to an increase in both A1 and A2A receptor
affinity, while maintaining the low or negligible affinity
for the adenosine A3 receptor. Fine examples are 23
and 51 when compared to 3 and 42, respectively. A
slight increase in A1 receptor affinity was paralleled by
a more substantial gain in A2A receptor affinity, render-
ing 23 and 51 less selective overall. Further substitution
of 23 and 51 with again the dioxymethylene moiety on
one of the phenyl groups, yielding 24 (Ki = 1.8 nM)
and 52 (Ki = 4.9 nM), respectively, improved A1 recep-
tor affinity somewhat without affecting A2A receptor
affinity substantially. Substitution of the free amino
group of 24 and 52 yielded carboxamido compounds
25, 26 and 53–55. The latter three compounds showed
a remarkable decrease in A2A receptor affinity, while
having also little affinity for the adenosine A3 receptor.
As a result cyclopentyl derivative 53 in particular was
very selective (>175-fold) for the adenosine A1 receptor
with a Ki value of 5.7 nM. We then returned to the di-
phenyl substituted pyrimidines, that is, without the
dioxymethylene moiety but with the cyano substituent,
2.2. Structure–activity relationships
The two series of pyrimidine derivatives were tested in
radioligand binding and second messenger assays, and
the results of these experiments are presented in Tables
1 and 2. The unsubstituted aminodiphenylpyrimidine 3
showed appreciable affinity for the adenosine A1 recep-
tor already, only 2- to 3-fold lower than the standard
reference ligands CPA (agonist) and DPCPX (antago-
nist/inverse agonist). The same held true for position
isomer 423 in Table 2. Both compounds also shared a
modest selectivity for the A1 receptor when compared
to the A2A receptor. Substitution of one of the phenyl
groups with a dioxymethylene moiety (4 and 43) did
not affect the pharmacological profile very much. Addi-
tional substitution of the free amine group to yield ali-
phatic carboxamides (5–7 and 44–47) generally
decreased A2A receptor affinity, except for 47. The most
selective compound in this comparison was 5 with an

2744
J. P. D. van Veldhoven et al. / Bioorg. Med. Chem. 16 (2008) 2741–2752
Table 1. Affinities of the substituted-pyrimidines 3–7 and 23–37 in radioligand binding assays of human adenosine receptors
R1
R1
R3
N
R2
N
N
H
Compound
R1
R2
R3
Ki (nM) or % displacementa
% Change in cAMPe
b
c
d
hA1
hA2A
hA3
CPA
DPCPX
—
—
—
—
—
—
10 1.3
6.1 1.6
17.7 4.6
34.8 14.1
8.1 1.8
43.0 15.0
17.6 8.7
3.3 1.0
1.8 0.1
2.8 1.3
8.3 0.2
1.2 0.3
2.9 0.3
6.4 0.3
2.9 1.0
9.4 3.1
92.3 38.2
26%
1652 344
129
281 56
1700 170
34%
48%
52%
25%
46%
8%
51
1
133
123
1
8
3
H
OCH2O
H
H
405 183
381 62
26%
4
5 LUF6048
H
H
118 11
OCH2O
OCH2O
OCH2O
CO-c-Pent
CO-Pr
H
121
125
125
3
3
5
6
H
8%
33%
7
23
CO-1-EtPr
H
H
H
OCH2O
CN
CN
CN
CN
CN
CN
CN
CN
CN
CN
CN
CN
CN
CN
CN
22.7 2.3
5.2 0.2
27.2 5.8
21.3 5.2
159 30
48.2 6.8
54.5 9.4
35.3 12.7
45.5 8.2
32.9 19.2
42%
123 11
24
25
H
46%
26%
21%
26%
31%
31%
43%
45%
21%
0%
120
138
5
6
OCH2O
OCH2O
CO-c-Pent
CO-Pr
26
27 LUF6040
146 14
H
H
H
H
H
H
H
H
H
H
H
CO-c-Pent
CO-Pr
121
132
128
134
5
3
6
4
28
29
30
31
32
33
34
35
36
37
CO-1-EtPr
CO-i-Pr
CO-c-Pr
CO-4-Cl-Ph
CO-2-furan
CO-4-MeO-Ph
CO-4-Me-Ph
CO-Ph
121 11
116 10
—
102 3.4
115 85
38%
300 67
158 25
48%
13%
4%
114
110
—
6
3
6%
0%
CO-3,4-diCl-Ph
58%
35%
—
a Ki SEM (n = 3), % displacement (n = 2).
b Displacement of specific [3H]DPCPX binding in CHO cell membranes expressing human adenosine A1 receptors or % displacement of specific
binding at 1 lM concentrations.
c Displacement of specific [3H]ZM 241385 binding in HEK 293 cell membranes expressing human adenosine A2A receptors or % displacement of
specific binding at 1 lM concentrations.
d Displacement of specific [125I]AB-MECA binding in HEK 293 cell membranes expressing human adenosine A3 receptors or % displacement of
specific binding at 1 lM concentrations.
e % Change of cAMP production SEM (n = 3) in CHO cell membranes expressing human adenosine A1 receptors, with 0% as basal cAMP
production in the absence of any ligand, 100% representing forskolin-stimulated (10 lM) cAMP production. The forskolin-induced cAMP
production was inhibited by CPA (full agonist, reduction from 100% to 51%) and further stimulated by DPCPX (full inverse agonist, increase from
100% to 133%). All compounds were tested at 100· their Ki value in the presence of forskolin, as was the case for CPA and DPCPX (see text).
yielding compounds 27–37 (Table 1) and 56–58 (Table
2). Various substitution patterns on the amido group
were explored. Either introduction of 2-furan (33) or
aromatic substitution according to Topliss16 (32, 34–
37) was not favored by any of the three receptor sub-
types. Alkyl, branched alkyl or cycloalkyl substitution
was better accommodated by the 5-cyanoamidopyrimi-
dines, as is evident from the radioligand binding data
for 27–31 and 56–58. Again cyclopentyl substitution ap-
peared favorable, compound 27 displaying nanomolar
affinity for the adenosine A1 receptor (Ki = 1.2 nM) with
132-fold (A2A) and >833-fold (A3) selectivity. Similarly,
although less prominent, 56 was a potent and selective
A1 receptor ligand.
In fact the current two series of compounds were de-
signed as hybrids between these non-ribose agonists
and pyrimidine antagonists as detailed in Figure 1. Pro-
duction of cAMP was determined in the same cells as
used for the preparation of membranes for our radioli-
gand binding studies on the A1 receptor. In these cells
the reference agonist N6-cyclopentyladenosine (CPA)
inhibited the (forskolin-stimulated) cAMP production
by almost 50% (Tables 1 and 2). In contrast, the refer-
ence inverse agonist 1,3-dipropyl-8-cyclopentylxanthine
(DPCPX) further increased the forskolin-induced
cAMP production by approximately 30%. When we
tested all compounds in this setup at a single concentra-
tion of 100· their Ki value (to ensure full receptor occu-
pancy), we learned that all compounds resembled
DPCPX much more than CPA. In all cases a further
enhancement of cAMP production was observed com-
pared to the cAMP concentrations in the presence of
The recent discoveries of non-ribose agonists4,5 required
our attention to also focus on performing functional as-
says at the adenosine A1 receptor with these compounds.

J. P. D. van Veldhoven et al. / Bioorg. Med. Chem. 16 (2008) 2741–2752
2745
Table 2. Affinities of the substituted-pyrimidines 42–58 in radioligand binding assays of human adenosine receptors
R1
R1
R3
N
R2
N
N
H
Compound
R1
R2
R3
Ki (nM) or % displacementa
% Change in cAMPe
b
c
d
hA1
hA2A
hA3
CPA
DPCPX
—
—
—
—
—
—
10 1.3
6.1 1.6
1652 344
129
281 56
1700 170
42%
51
133
123
131
136
135
1
1
4
9
7
6
42
43
H
H
H
25.2 12.3
15.6 5.7
17.3 5.6
6.8 2.6
169 15
557 92
339 31
222 58
574 49
69.2 12.7
19.0 3.6
44.3 2.5
14%
OCH2O
OCH2O
OCH2O
OCH2O
OCH2O
H
H
H
15%
44
45
CO-c-Pent
CO-Pr
CO-1-EtPr
CO-c-Pr
H
H
186 25
53.9 2.4
634 34
30.7 2.7
58%
H
46
47
H
27.9 11.6
13.0 6.8
10.8 6.0
4.9 1.6
139 14
142 16
H
51
52
CN
CN
CN
CN
CN
CN
CN
CN
122
128
126
131
140
131
8
5
4
4
7
9
OCH2O
OCH2O
OCH2O
OCH2O
H
H
50%
27%
53 LUF 6056
CO-c-Pent
CO-Pr
CO-1-EtPr
CO-c-Pent
CO-Pr
CO-c-Pr
5.7 1.6
54
55
56
57
58
13.4 6.1
18.4 0.6
5.3 2.5
23%
0%
50%
6%
280 35
69.5 7.2
49.8 2.6
22%
38%
H
12.6 1.3
27.9 9.3
125 12
125 11
H
27%
a Ki SEM (n = 3), % displacement (n = 2).
b Displacement of specific [3H]DPCPX binding in CHO cell membranes expressing human adenosine A1 receptors or % displacement of specific
binding at 1 lM concentrations.
c Displacement of specific [3H]ZM 241385 binding in HEK 293 cell membranes expressing human adenosine A2A receptors or % displacement of
specific binding at 1 lM concentrations.
d Displacement of specific [125I]AB-MECA binding in HEK 293 cell membranes expressing human adenosine A3 receptors or % displacement of
specific binding at 1 lM concentrations.
e % Change of cAMP production SEM (n = 3) in CHO cell membranes expressing human adenosine A1 receptors, with 0% as basal cAMP
production in the absence of any ligand, 100% representing forskolin-stimulated (10 lM) cAMP production. The forskolin-induced cAMP
production was inhibited by CPA (full agonist, reduction from 100% to 51%) and further stimulated by DPCPX (full inverse agonist, increase from
100% to 133%). All compounds were tested at 100· their Ki value in the presence of forskolin, as was the case for CPA and DPCPX (see text).
110
forskolin only. These results suggest that the compounds
are all antagonists/inverse agonists for the adenosine A1
90
receptor. This claim was substantiated with a more de-
tailed analysis of the behavior of two of the more potent
70
compounds, 27 and 53. Concentration–effect curves
were recorded for both compounds in the presence of
50
10 lM forskolin and 100 nM CPA (Fig. 2), yielding
IC50 values (n = 3) of 0.15 0.02 lM for 27 and
1.4 0.2 lM for 53. This corresponds to apparent affin-
ities (KB-values) of approximately 3 and 28 nM, respec-
tively, quite comparable to the Ki values of the two
compounds obtained in radioligand binding studies.
30
10
-10
-11
-10
-9
-8
-7
-6
-5
-4
log [ligand (M)]
Figure 2. Concentration–effect curves for compounds 27 ( ) and 53 (m)
inhibiting the CPA (100 nM)-induced decrease in forskolin (10 lM)-
stimulated cAMP production in CHO cells expressing the human
adenosine A1 receptor (n = 3).
3. Conclusion
This series of pyrimidine derivatives with affinity for
adenosine receptors are hybrid structures based on cya-
nopyridine agonists and pyrimidine antagonists/inverse
agonists. All compounds synthesized were inverse ago-
nists rather than agonists. Among the more A1 receptor
selective compounds were 5, 27 and 53 with Ki values of
8.1, 1.2 and 5.7 nM, respectively.
4. Experimental
4.1. Chemistry
All reagents used were obtained from commercial
sources and all solvents were of analytical grade. 1H

2746
J. P. D. van Veldhoven et al. / Bioorg. Med. Chem. 16 (2008) 2741–2752
and 13C NMR spectra were recorded on a Bruker AC
200 (1H NMR, 200 MHz; 13C NMR, 50.29 MHz) spec-
trometer with tetramethylsilane as an internal standard.
Chemical shifts are reported in d (ppm) and the follow-
ing abbreviations are used: s, singlet; d, doublet; dd,
double doublet; t, triplet; m, multiplet; br, broad; ar,
aromatic protons. Melting points were determined on
tion from methanol gave the corresponding amide in
crystalline form.
4.1.3.1.
Cyclopentane
carboxylic
acid
(4-
benzo[1,3]dioxol-5yl-6-phenyl-pyrimidin-2-yl)-amide (5)
LUF 6048. Yield 51%; white solid. 1H NMR d (DMSO):
8.13–8.08 (m, 3H, phenyl-H + NH), 7.70 (dd, 1H,
J1 = 4.9 Hz, J2 = 1.8 Hz, Ar-H), 7.70 (s, 1H, pyrimidi-
nyl-H), 7.64 (d, 1H, J = 1.5 Hz, Ar-H), 7.55–7.50 (m,
3H, phenyl-H), 6.93 (d, 1H, J = 8.0 Hz, Ar-H), 6.06 (s,
2H, –OCH2O–), 3.54–3.47 (m, 1H, CH-cyclopentyl),
2.04–1.58 (m, 8H, CH2-cyclopentyl). 13C NMR d
(CDCl3): 176.1, 165.5, 165.0, 157.5, 149.9, 148.2,
136.5, 130.7, 128.6, 126.9, 121.7, 108.2, 107.1, 106.4,
101.4, 45.6, 29.9, 25.8. Anal. Calcd for C23H21N3O3:
C, 71.30; H, 5.46; N, 10.85. Found: C, 71.24; H, 5.61;
N, 11.21.
a Buchi melting point apparatus and are uncorrected.
¨
Elemental analyses were performed by the Leiden Insti-
tute of Chemistry and are within 0.4% of the theoretical
values unless otherwise stated. Reactions were routinely
monitored on TLC using Merck silica gel F254 plates.
Microwave reactions were performed in an Emrys Opti-
mizer (Biotage AB, formerly Personal Chemistry). Watt-
age was automatically adjusted so as to maintain the
desired temperature. The yields of all products were
not optimized. All the final products (Tables 1 and 2)
were purified by column chromatography followed by
recrystallization.
4.1.3.2. N-(4-Benzo[1,3]dioxol-5yl-6-phenyl-pyrimidin-
2-yl)-butyramide (6). Yield 33%; off-white solid. 1H
NMR d (CDCl3): 8.14–8.08 (m, 2H, phenyl-H), 8.04
(br s, 1H, NH), 7.70 (dd, 1H, J1 = 6.4 Hz,
J2 = 1.8 Hz, Ar-H), 7.70 (s, 1H, pyrimidinyl-H), 7.64
(d, 1H, J = 1.8 Hz, Ar-H), 7.56–7.50 (m, 3H, phe-
nyl-H), 6.94 (d, 1H, J = 8.0 Hz, Ar-H), 6.07 (s, 2H,
–OCH2O–), 3.00 (t, 2H, J = 8.9 Hz, –CH2CH2CH3),
1.94–1.75 (m, 2H, –CH2CH2CH3), 1.07 (t, 3H,
J = 7.3 Hz, –CH2CH2CH3). 13C NMR d (CDCl3):
173.7, 165.6, 165.1, 157.5, 150.1, 148.2, 136.5, 130.8,
128.6, 126.9, 121.7, 108.3, 107.1, 106.4, 101.5, 39.1,
18.1, 13.7. Anal. Calcd for C21H19N3O3: C, 69.79;
H, 5.30; N, 11.63. Found: C, 70.16; H, 5.43; N,
12.01.
4.1.1. Synthesis of 3,4-methylenedioxychalcone (2). Pre-
pared as described in the literature.6
4.1.2. General procedure for the synthesis of 2-amino-4,6-
disubstituted-pyrimidines.3 To a mixture of the chalcone
(4 mmol, 1 equiv) and guanidine hydrochloride
(4.1 mmol, 1.05 equiv) in EtOH (3 mL) was added an
aqueous solution (1 mL) of NaOH (12 mmol,
3.05 equiv). This mixture was heated at 130 ꢁC in a
sealed tube in the microwave for 2 h. The solvent was re-
moved in vacuo and the organics were extracted with
hot acetone. The crude product was purified by recrys-
tallization from methanol or column chromatography
on SiO2, eluting with a methanol–dichloromethane sol-
vent system. The same system was used for TLC evalu-
ation, usually yielding Rf values in between 0.3 and 0.5.
4.1.3.3. N-(4-Benzo[1,3]dioxol-5yl-6-phenyl-pyrimidin-
2-yl)-2-ethyl-butyramide (7). Yield 73%; white solid. H
1
NMR d (CDCl3): 8.15–8.10 (m, 2H, phenyl-H), 8.08
(br s, 1H, NH), 7.78–7.70 (m, 2H, Ar-H + pyrimidi-
nyl-H), 7.67 (d, 1H, J = 1.8 Hz, Ar-H), 7.55–7.50 (m,
3H, phenyl-H), 6.94 (d, 1H, J = 8.4 Hz, Ar-H), 6.06
4.1.2.1. 2-Amino-4,6-diphenyl-pyrimidine (3). Pre-
pared as described in the literature.3
4.1.2.2. 2-Amino-4-benzo[1,3]dioxol-5yl-6-phenyl-pyrim-
(s,
2H,
–OCH2O–),
3.07–2.84
(m,
1H,
1
idine (4).7 Yield 32%; white solid. H NMR d (CDCl3):
8.08–7.99 (m, 2H, phenyl-H), 7.65–7.59 (m, 2H, Ar-H),
7.52–7.45 (m, 3H, phenyl-H), 7.36 (s, 1H, pyrimidinyl-
–CH(CH2CH3)2), 1.96–1.55 (m, 4H, –CH(CH2CH3)2),
1.00 (t, 6H, J = 7.3 Hz, –CH(CH2CH3)2). 13C NMR
d (CDCl3): 175.3, 165.6, 165.1, 157.5, 150.0, 148.2,
136.6, 130.7, 128.7, 127.0, 121.8, 108.3, 107.2, 106.7,
101.4, 49.9, 24.8, 11.7. Anal. Calcd for C23H23N3O3:
C, 70.93; H, 5.95; N, 10.79. Found: C, 70.72; H,
6.06; N, 10.97.
H),
6.91
(d,
1H,
Ar-H),
6.04
(s,
2H,
–OCH2O–), 5.13 (br s, 2H, NH2). 13C NMR d (DMSO):
164.8, 164.2, 163.9, 149.3, 147.9, 137.5, 131.6, 130.4,
128.6, 127.0, 121.7, 108.3, 107.0, 101.6. Anal. Calcd for
C17H13N3O2Æ0.2 H2O: C, 69.26; H, 4.57; N, 14.25. Found:
C, 69.20; H, 4.46; N, 14.49.
4.1.4. General procedure for the synthesis of 2-amino-5-
bromo-4,6-disubstituted-pyrimidines.9 To a mixture of
the 2-amino-4,6-disubstituted-pyrimidine (14.96 mmol,
1.0 equiv) and CaCO3 (8.98 mmol, 0.6 equiv) in CHCl3
(70 mL) was added drop wise to a solution of bromine
(16.47 mmol, 1.1 equiv) in CHCl3 (10 mL). The reaction
mixture was stirred at room temperature overnight.
Upon completion as judged by TLC (ethylacetate/petro-
leum ether, 1:3) the reaction mixture was separated be-
tween water (pH 9 with 2 M NaOH solution) and
dichloromethane (3· 50 mL). The combined organic
layers were dried over MgSO4, concentrated in vacuo
and recrystallized from ethanol resulting in the corre-
sponding crystalline form.
4.1.3. General procedure for the synthesis of 2-amido-4-
benzo[1,3]dioxol-5yl-6-phenyl-pyrimidines.8 To a solu-
tion of the 2-amino-4-benzo[1,3]dioxol-5yl-6-phenylpyr-
imidine (1.4 mmol, 1.0 equiv) in anhydrous pyridine
(3.5 mL) was added the appropriate acid chloride
(2.1 mmol, 1.5 equiv) and stirred at room temperature
for 2 h, after which according to TLC no starting mate-
rial was visible. The solvent was evaporated under re-
duced pressure and the crude product purified by
column chromatography, eluting with dichloromethane.
The same solvent was used for TLC evaluation, usually
yielding Rf values in between 0.3 and 0.5. Recrystalliza-

J. P. D. van Veldhoven et al. / Bioorg. Med. Chem. 16 (2008) 2741–2752
2747
4.1.4.1. 2-Amino-5-bromo-4,6-phenyl-pyrimidine (8).
–CH(CH2CH3)2), 0.95 (t, 6H, J = 7.2 Hz, –CH(CH2
CH3)2).
1
Yield 70%; white solid. H NMR d (CDCl3): 7.70–7.61
(m, 4H, phenyl-H), 7.52–7.42 (m, 6H, phenyl-H), 5.49
(br s, 2H, NH2). MS (ESI): 327.0.
4.1.5.6. N-(5-Bromo-4,6-diphenyl-pyrimidin-2-yl)-iso-
butyramide (15). Yield 36%; white solid. 1H NMR d
(CDCl3): 8.05 (br s, 1H, NH), 7.83–7.76 (m, 4H, phe-
nyl-H), 7.54–7.48 (m, 6H, phenyl-H), 3.18–3.04 (m,
1H, –CH(CH3)2), 1.27 (s, 3H, –CH(CH3)2), 1.24 (s,
3H, –CH(CH3)2). 13C NMR d (CDCl3): 176.5, 167.6,
155.3, 137.8, 129.8, 129.3, 127.8, 110.6, 35.2, 18.9.
4.1.4.2.
2-Amino-4-benzo[1,3]dioxol-5yl-5-bromo-6-
phenyl-pyrimidine (9). Yield 70%; white solid. 1H
NMR d (DMSO): 7.62–7.59 (m, 2H, phenyl-H), 7.51–
7.47 (m, 3H, phenyl-H), 7.22–7.16 (m, 2H, Ar-H), 7.03
(d, 2H, Ar-H), 7.01 (br s, 2H, NH2), 6.12 (s, 2H,
–OCH2O–). 13C NMR d (DMSO): 167.0, 166.1, 148.1,
146.7, 139.1, 132.6, 129.1, 128.9, 127.8, 123.4, 109.5,
107.8, 103.0, 101.5.
4.1.5.7. Cyclopropane carboxylic acid (5-bromo-4,6-
diphenyl-pyrimidin-2-yl)-amide (16). Yield 71%; white so-
lid. 1H NMR d (CDCl3): 8.25 (br s, 1H, NH), 7.78–7.73
(m, 4H, phenyl-H), 7.56–7.45 (m, 6H, phenyl-H), 2.58–
2.40 (m, 1H, –CHCH2CH2–), 1.25–1.14 (m, 2H,
–CHCH2CH2–), 0.96–0.87 (m, 2H, –CHCH2CH2).
4.1.5. General procedure for the synthesis of 2-amido-5-
bromo-4,6-disubstituted-pyrimidines.8
Starting from
compound 8. The experimental procedure was identical
to the general procedure of compounds 5–7.
4.1.5.8. N-(5-Bromo-4,6-diphenyl-pyrimidin-2-yl)-4-
chloro-benzamide (17). Yield 92%; white solid. 1H
NMR d (CDCl3): 8.92 (br s, 1H, NH), 7.81–7.69 (m,
6H, phenyl-H + Ar-H), 7.47–7.23 (m, 8H, phenyl-
H + Ar-H).
4.1.5.1. Cyclopentane carboxylic acid (4-benzo-
[1,3]dioxol-5-yl)-5-bromo-6-phenyl-pyrimidin-2-yl-amide
(10). Yield 65%; white solid. H NMR d (CDCl3): 8.00
1
(br s, 1H, NH), 7.75–7.71 (m, 2H, phenyl-H), 7.51–
7.47 (m, 3H, phenyl-H), 7.39 (dd, 1H, J1 = 8.0 Hz
J2 = 1.8 Hz, Ar-H), 7.32 (d, 1H, J = 1.8 Hz, Ar-H),
6.92 (d, 1H, J = 8.0 Hz, Ar-H), 6.05 (s, 1H, –OCH2O–),
3.38–3.20 (m, 1H, CH-cyclopentyl), 2.05–1.51 (m, 8H,
CH2-cyclopentyl). 13C NMR d (CDCl3): 175.4, 167.8,
166.7, 155.1, 148.9, 147.2, 137.9, 131.5, 129.7,
129.1, 127.8, 124.3, 110.1, 109.9, 107.7, 101.3, 45.6,
29.9, 25.8.
4.1.5.9. Furan-2-carboxylic acid (5-bromo-4,6-diphe-
nyl-pyrimidin-2-yl)-amide (18). Yield 54%; white solid.
1H NMR d (CDCl3): 8.92 (br s, 1H, NH), 7.85–7.81
(m, 4H, phenyl-H), 7.52–7.47 (m, 7H, phenyl-H + fur-
an-H), 7.32 (d, 1H, J = 3.7 Hz, furan-H), 6.57 (dd, 1H,
J1 = 1.8 Hz J2 = 1.8 Hz, furan-H). 13C NMR d (CDCl3):
167.7, 155.0, 154.6, 147.1, 144.7, 137.7, 129.8, 129.4,
127.9, 116.4, 112.6, 111.2.
4.1.5.2. N-(4-Benzo[1,3]dioxol-5yl)-5-bromo-6-phenyl-
pyrimidin-2-yl-butyramide (11). Yield 79%; white solid.
1H NMR d (CDCl3): 8.02 (br s, 1H, NH), 7.75–7.70
(m, 2H, phenyl-H), 7.53–7.48 (m, 3H, phenyl-H), 7.39–
7.30 (m, 2H, Ar-H), 6.92 (d, 1H, J = 8.0 Hz, Ar-H),
6.06 (s, 1H, –OCH2O–), 2.79 (t, 2H, J = 7.7 Hz,
–CH2CH2CH3), 1.85–1.60 (m, 2H, –CH2CH2CH3),
0.97 (t, 3H, J = 7.3 Hz, –CH2CH2CH3).
4.1.5.10. N-(5-Bromo-4,6-diphenyl-pyrimidin-2-yl)-4-
methoxy-benzamide (19). Yield 29%; white solid. 1H
NMR d (CDCl3): 8.61 (br s, 1H, NH), 7.92–7.80 (m,
6H, phenyl-H + Ar-H), 7.52–7.46 (m, 6H, phenyl-H),
6.97 (d, 2H, J = 5.1 Hz, Ar-H), 3.87 (s, 3H, –OCH3).
13C NMR d (CDCl3): 167.6, 164.2, 162.8, 155.7, 137.8,
129.8, 129.4, 129.3, 127.9, 126.0, 113.8, 111.0, 55.2.
4.1.5.3. Cyclopentane carboxylic acid (5-bromo-4,6-
diphenyl-pyrimidin-2-yl)-amide (12). Yield 20%; white so-
lid. 1H NMR d (CDCl3): 8.02 (br s, 1H, NH), 7.80–7.74
(m, 4H, phenyl-H), 7.51–7.48 (m, 6H, phenyl-H), 3.32–
3.24 (m, 1H, CH-cyclopentyl), 1.99–1.50 (m, 8H, CH2-
cyclopentyl). 13C NMR d (DMSO): 175.5, 167.6,
155.2, 137.8, 129.7, 129.2, 127.8, 45.6, 29.9, 25.8. MS
(ESI): 423.0.
4.1.5.11. N-(5-Bromo-4,6-diphenyl-pyrimidin-2-yl)-4-
methyl-benzamide (20). Yield 63%; white solid. 1H
NMR d (CDCl3): 8.63 (br s, 1H, NH), 7.86–7.81 (m,
6H, phenyl-H + Ar-H), 7.52–7.48 (m, 6H, phenyl-H),
7.28 (d, 2H, J = 7.6 Hz, Ar-H), 2.42 (s, 3H, CH3). 13C
NMR d (CDCl3): 167.7, 164.7, 162.8, 155.7, 142.8,
137.8, 131.0, 129.8, 129.3, 129.2, 127.9, 127.4, 111.0,
21.3.
4.1.5.4.
N-(5-Bromo-4,6-diphenyl-pyrimidin-2-yl)-
4.1.5.12. N-(5-Bromo-4,6-diphenyl-pyrimidin-2-yl)-benza-
mide (21). Yield 26%; white solid. H NMR d (CDCl3):
8.66 (br s, 1H, NH), 7.94–7.90 (m, 2H, phenyl-H), 7.88–
7.79 (m, 4H, phenyl-H), 7.58–7.44 (m, 9H, phenyl-H).
13C NMR d (CDCl3): 167.7, 164.8, 155.6, 137.7, 133.9,
132.2, 129.8, 129.3, 128.6, 127.9, 127.3, 111.2.
1
butyramide (13). Yield 63%; white solid. 1H NMR d
(CDCl3): 8.03 (br s, 1H, NH), 7.82–7.73 (m, 4H,
phenyl-H), 7.55–7.47 (m, 6H, phenyl-H), 2.79 (t, 1H,
J = 7.3 Hz, –CH2CH2CH3), 1.87–1.70 (m, 2H,
–CH2CH2CH3),
–CH2CH2CH3).
0.97
(t,
3H,
J = 7.7 Hz,
4.1.5.13. N-(5-Bromo-4,6-diphenyl-pyrimidin-2-yl)-3,4-
dichloro-benzamide (22). Yield 37%; white solid. 1H
NMR d (CDCl3): 8.65 (br s, 1H, NH), 7.99 (d, 1H,
J = 1.8 Hz, Ar-H), 7.81–7.77 (m, 4H, phenyl-H), 7.71
(dd, 1H, J1 = 6.2 Hz J2 = 2.2 Hz, Ar-H), 7.56 (s, 1H,
Ar-H), 7.51–7.48 (m, 6H, phenyl-H). 13C NMR d
4.1.5.5. N-(5-Bromo-4,6-diphenyl-pyrimidin-2-yl)-2-
ethyl-butyramide (14). Yield 46%; white solid. 1H
NMR d (CDCl3): 8.01 (br s, 1H, NH), 7.83–7.75
(m, 4H, phenyl-H), 7.53–7.48 (m, 6H, phenyl-H), 2.87–
2.60 (m, 1H, –CH(CH2CH3)2), 1.85–1.47 (m, 4H,

2748
J. P. D. van Veldhoven et al. / Bioorg. Med. Chem. 16 (2008) 2741–2752
(CDCl3): 167.7, 162.9, 155.3, 137.5, 136.5, 133.5, 132.8,
130.4, 129.8, 129.7, 129.3, 128.0, 126.7, 111.6.
H), 6.97 (d, 1H, J = 8.4 Hz, Ar-H), 6.09 (s, 2H,
–OCH2O–), 2.91 (t, 2H, J = 7.3 Hz, –CH2CH2CH3),
1.89–1.71 (m, 2H, –CH2CH2CH3), 1.03 (t, 3H,
J = 7.7 Hz, –CH2CH2CH3). 13C NMR d (DMSO-d6):
172.1, 170.7, 169.2, 157.5, 150.4, 147.6, 135.9, 131.5,
129.5, 129.3, 128.6, 124.7, 117.7, 109.3, 108.4, 102.1,
96.8, 40.9, 17.9, 13.6. Anal. Calcd for C22H18N4O3: C,
68.38; H, 4.70; N, 14.50. Found: C, 68.28; H, 4.70; N,
14.45.
4.1.6. General procedure for the synthesis of 2-amido-4,6-
disubstituted-pyrimidin-5-carbonitriles. To a solution of
2-amido-5-bromo-disubstituted-pyrimidine (0.9 mmol,
1.0 equiv) in pyridine (3 mL) was added CuCN
(1.0 mmol, 1.1 equiv). This was heated at 250 ꢁC under
microwave conditions10 for 20 min, after which no start-
ing material was visible according to TLC (a mixture
of petroleum ether/tetrahydrofuran/dichloromethane,
4:1:1). The solvent was evaporated under reduced pres-
sure, dissolved in dichloromethane (75 mL) and back-
washed with a 5% NH4OH solution in water (3·
20 mL) and water (1· 20 mL). The organic layer was
dried over MgSO4 and the mixture was concentrated
in vacuo. This crude product was purified by column
chromatography, eluting with a mixture of petroleum
ether/tetrahydrofuran/dichloromethane (4:1:1) and
recrystallized from methanol or n-heptane.
4.1.6.5. Cyclopentane carboxylic acid (5-carbonitrile-
4,6-diphenyl-pyrimidin-2-yl)-amide (27) LUF6040. Yield
1
51%; white solid. H NMR d (CDCl3): 8.22 (br s, 1H,
NH), 8.11–8.00 (m, 4H, phenyl-H), 7.65–7.51 (m, 6H,
phenyl-H), 3.50–3.36 (m, 1H, –CH-cyclopentyl), 2.03–
1.54 (m, 8H, CH2-cyclopentyl). 13C NMR d (CDCl3):
177.7, 172.9, 158.8, 137.0, 133.4, 130.8, 130.3, 118.8,
98.9, 47.6, 31.7, 27.6. Anal. Calcd for C23H20N4O: C,
74.98; H, 5.47; N, 15.21. Found: C, 74.61; H, 5.49; N,
15.19.
4.1.6.1. 2-Amino-4,6-diphenyl-pyrimidin-5-carbonitrile
(23). Yield 32%; off-white solid. H NMR d (CDCl3):
4.1.6.6. N-(5-Carbonitrile-4,6-diphenyl-pyrimidin-2-
yl)-butyramide (28). Yield 25%; white solid. H NMR
1
1
8.00–7.90 (m, 4H, phenyl-H), 7.55–7.48 (m, 6H, phe-
nyl-H), 5.86 (br s, 2H, NH2). 13C NMR d (acetone-d6):
206.1, 172.0, 164.1, 137.8, 131.6, 129.8, 129.1, 118.9,
93.1.
d (CDCl3): 8.24 (br s, 1H, NH), 8.06–8.02 (m, 4H, phe-
nyl-H), 7.64–7.51 (m, 6H, phenyl-H), 2.92 (t, 1H,
J = 7.3 Hz, –CH2CH2CH3), 1.86–1.75 (m, 2H,
–CH2CH2CH3), 1.02 (t, 3H, J = 7.3 Hz, –CH2CH2CH3).
13C NMR d (CDCl3): 173.2, 171.3, 156.9, 135.2, 131.6,
129.0, 128.5, 116.9, 39.4, 17.9, 13.5. MS (ESI): 342.0.
Anal. Calcd for C21H18N4O: C, 73.67; H, 5.30; N,
16.36. Found: C, 73.37; H, 5.16; N, 16.67.
MS (ESI): 271.7. Anal. Calcd for C17H12N4Æ1.2 H2O: C,
69.33; H, 4.95; N, 19.02. Found: C, 69.32; H, 5.33; N,
19.18.
4.1.6.2. 2-Amino-4-benzo[1,3]dioxol-5yl-6-phenyl-pyr-
imidin-5-carbonitrile (24). Yield 64%; off-white solid. H
4.1.6.7. N-(5-Carbonitrile-4,6-diphenyl-pyrimidin-2-
yl)-2-ethyl-butyramide (29). Yield 42%; white solid. H
1
1
NMR
d
(DMSO-d6): 7.91–7.86 (m, 4H, phenyl-
NMR d (CDCl3): 8.23 (br s, 1H, NH), 8.09–8.04
(m, 4H, phenyl-H), 7.64–7.51 (m, 6H, phenyl-H),
2.93–2.75 (m, 1H, –CH(CH2CH3)2), 1.93–1.52 (m,
4H, –CH(CH2CH3)2), 0.98 (t, 6H, J = 7.3 Hz,
–CH(CH2CH3)2). 13C NMR d (CDCl3): 175.2, 171.2,
156.9, 135.2, 131.6, 129.1, 128.5, 116.9, 97.4, 50.2,
24.7, 11,6. MS (ESI): 370.0. Anal. Calcd for
C23H22N4O: C, 74.57; H, 5.99; N, 15.12. Found: C,
74.47; H, 5.94; N, 15.46.
H + NH2), 7.62–7.52 (m, 4H, phenyl-H + Ar-H), 7.47
(dd, 1H, J1 = 2.6 Hz, J2 = 1.8 Hz, Ar-H), 7.12 (d, 1H,
J = 8.0 Hz, Ar-H), 6.17 (s, 2H, –OCH2O–). 13C NMR
d (DMSO-d6): 170.9, 169.6, 162.7, 149.7, 147.4, 136.6,
130.8, 130.4, 128.8, 128.3, 123.9, 118.8, 108.9, 108.2,
101.9, 90.7. Anal. Calcd for C18H12N4O2Æ0.2 H2O: C,
67.61; H, 3.90; N, 17.52. Found: C, 67.58; H, 3.67; N,
17.54.
4.1.6.3. Cyclopentane carboxylic acid (4-benzo-
[1,3]dioxol-5yl-5-carbonitrile-6-phenyl-pyrimidin-2-yl)-amide
(25). Yield 66%; white solid. H NMR d (CDCl3): 8.15
4.1.6.8. N-(5-Carbonitrile-4,6-diphenyl-pyrimidin-2-
yl)-isobutyramide (30). Yield 26%; white solid. 1H
NMR d (CDCl3): 8.22 (br s, 1H, NH), 8.09–8.00 (m,
4H, phenyl-H), 7.65–7.51 (m, 6H, phenyl-H), 3.33–3.19
(m, 1H, –CH(CH3)2), 1.32 (s, 3H, –CH(CH3)2), 1.29
(s, 3H, –CH(CH3)2). 13C NMR d (CDCl3): 176.5,
171.2, 157.0, 135.2, 131.6, 129.1, 128.5, 117.0, 97.3,
35.5, 24.7, 18.9. MS (ESI): 342.0 Anal. Calcd for
C21H18N4O: C, 73.67; H, 5.30; N, 16.36. Found: C,
73.34; H, 5.16; N, 16.69.
1
(br s, 1H, NH), 8.04–8.00 (m, 2H, phenyl-H), 7.72 (dd,
1H, J1 = 6.6 Hz, J2 = 1.8 Hz, Ar-H), 7.60–7.50 (m, 4H,
phenyl-H + Ar-H), 6.98 (d, 1H, J = 8.0 Hz, Ar-H),
6.05 (s, 2H, –OCH2O–), 3.50–3.35 (m, 1H, CH-cyclo-
pentyl), 2.03–1.60 (m, 8H, CH2-cyclopentyl). 13C
NMR d (CDCl3): 175.8, 171.3, 169.9, 156.8, 150.7,
147.9, 135.3, 131.5, 129.0, 128.4, 124.6, 117.3, 109.1,
108.1, 101.7, 96.4, 45.8, 29.9, 25.8. Anal. Calcd for
C24H20N4O3: C, 69.89; H, 4.89; N, 13.58. Found: C,
70.20; H, 4.95; N, 13.78.
4.1.6.9. Cyclopropane carboxalic acid (5-carbonitrile-
4,6-diphenyl-pyrimidin-2-yl)-amide (31). Yield 29%;
1
white solid. H NMR d (CDCl3): 8.33 (br s, 1H, NH),
4.1.6.4. N-(4-Benzo[1,3]dioxol-5yl-5-carbonitrile-6-
phenyl-pyrimidin-2-yl)-butyramide (26). Yield 59%; white
solid. H NMR d (CDCl3): 8.16 (br s, 1H, NH), 8.03–
7.99 (m, 2H, phenyl-H), 7.71 (dd, 1H, J1 = 6.2 Hz,
J2 = 1.8 Hz, Ar-H), 7.61–7.50 (m, 4H, phenyl-H + Ar-
8.07–8.02 (m, 4H, phenyl-H), 7.78–7.51 (m, 6H, phe-
nyl-H), 2.71–2.58 (m, 1H, –CHCH2CH2–), 1.29–1.21
(m, 2H, –CHCH2CH2–), 1.06–0.96 (m, 2H,
–CHCH2CH2–). 13C NMR d (CDCl3): 173.8, 171.2,
157.1, 135.2, 131.6, 129.0, 128.5, 116.9, 97.3, 15.0, 9.9.
1

J. P. D. van Veldhoven et al. / Bioorg. Med. Chem. 16 (2008) 2741–2752
2749
MS (ESI): 340.1. Anal. Calcd for C21H16N4OÆ0.7 H2O:
C, 71.47; H, 4.97; N, 15.88. Found: C, 71.46; H, 4.92;
N, 15.94.
J2 = 2.2 Hz, Ar-H), 7.65–7.51 (m, 7H, phenyl-H + Ar-
H). 13C NMR d (CDCl3): 171.3, 162.8, 157.1, 137.2,
134.9, 133.2, 131.8, 130.7, 129.6, 129.1, 128.6, 126.6,
116.7, 98.2. Anal. Calcd for C24H14Cl2N4OÆ0.6 H2O:
C, 63.20; H, 3.36; N, 12.28. Found: C, 63.21; H, 3.32;
N, 12.23.
4.1.6.10. N-(5-Carbonitrile-4,6-diphenyl-pyrimidin-2-
yl)-4-chloro-benzamide (32). Yield 27%; white solid. H
1
NMR d (CDCl3): 8.75 (br s, 1H, NH), 8.12–8.07 (m,
4H, phenyl-H), 7.90 (d, 2H, J = 8.4 Hz, Ar-H), 7.65–
7.54 (m, 6H, phenyl-H), 7.51 (d, 2H, J = 12.4 Hz, Ar-
H). 13C NMR d (CDCl3): 171.2, 163.8, 157.3, 139.0,
135.0, 131.9, 131.7, 129.1, 128.9, 128.5, 116.8, 97.9.
MS (ESI): 410.1. Anal. Calcd for C24H15ClN4OÆ1.1
H2O: C, 66.95; H, 4.02; N, 13.01. Found: C, 66.92; H,
4.04; N, 13.15.
4.1.7. Benzo[1,3]dioxole-5-carbonitrile (39). Prepared as
described in the literature.11
4.1.8. 3-Amino-3-benzo[1,3]dioxol-5-yl-acrylonitrile (40).
Prepared as described in the literature.12
4.1.9. 4-Benzo[1,3]dioxol-5-yl-6-chloro-2-phenyl-pyrimi-
dine (41). Prepared as described in the literature.13
4.1.6.11. Furan-2-carboxylic acid (5-carbonitrile-4,6-
diphenyl-pyrimidin-2-yl)-amide (33). Yield 35%; white so-
lid. 1H NMR d (CDCl3): 9.09 (br s, 1H, NH), 8.17–8.12
(m, 4H, phenyl-H), 7.62–7.54 (m, 7H, phenyl-H + furan-
H), 7.39 (d, 1H, J = 3.3 Hz, furan-H), 6.62 (dd, 1H,
J1 = 1.8 Hz J2 = 1.5 Hz, furan-H). 13C NMR d (CDCl3):
171.3, 156.9, 154.3, 146.7, 145.0, 135.2, 131.7, 129.2,
128.6, 117.1, 117.0, 112.9, 97.9. Anal. Calcd for
C22H14N4O2Æ1.0 H2O: C, 68.59; H, 3.76; N, 14.36.
Found: C, 68.56; H, 3.88; N, 14.48.
4.1.10. 2,6-Diphenyl-pyrimidin-4-ylamine (42).3 Yield
68%, white solid. H NMR d (CDCl3): 8.52–8.47 (m,
1
2H, phenyl-H), 8.10–8,05 (m, 2H, phenyl-H), 7.52–7.42
(m, 6H, phenyl-H), 6.63 (s, 1H, pyrimidine-H), 5.08
(br s, 2H, NH2). 13C NMR d (CDCl3): 163.9, 162.9,
138.0, 137.2, 130.1, 129.9, 128.3, 128.1, 127.9, 126.6,
98.2. MS (ESI): 248.4. Anal. Calcd for C16H13N3: C,
77.71; H, 5.29; N, 16.99. Found: C, 77.39; H, 5.09; N,
17.33.
4.1.6.12. N-(5-Carbonitrile-4,6-diphenyl-pyrimidin-2-
yl)-4-methoxy-benzamide (34). Yield 21%; white solid.
1H NMR d (CDCl3): 8.76 (br s, 1H, NH), 8.11–8.08
(m, 4H, phenyl-H), 7.94 (dd, 2H, J1 = 4.7 Hz
J2 = 1.8 Hz, Ar-H), 7.62–7.52 (m, 6H, phenyl-H), 7.00
(dd, 2H, J1 = 4.8 Hz J2 = 1.8 Hz, Ar-H), 3.90 (s, 3H,
–OCH3). 13C NMR d (CDCl3): 171.1, 164.0, 163.1,
157.6, 135.2, 131.6, 129.6, 129.2, 128.5, 125.6, 117.0,
113.9, 97.5, 55.3. Anal. Calcd for C25H18N4O2 Æ0.5
H2O: C, 72.29; H, 4.61; N, 13.49. Found: C, 72.27; H,
4.27; N, 13.45.
4.1.11. 6-Benzo[1,3]dioxol-5-yl-2-phenyl-pyrimidin-4-yla-
mine (43).3 Yield 64%; white solid. 1H NMR d (CDCl3):
8.50–8.44 (m, 2H, phenyl-H), 7.74 (s, 1H, Ar-H), 7.66
(d, 1H, J = 5.4 Hz, Ar-H), 7.53–7.43 (m, 3H, phenyl-
H), 6.95 (dd, 1H, J1 = 8.8 Hz J2 = 1.5 Hz, Ar-H), 6.64
(s, 1H, pyrimidine-H), 6.04 (s, 2H, –OCH2O–). 13C
NMR d (DMSO): 164.9, 162.9, 160.8, 149.0, 148.0,
138.4, 131.9, 130.1, 128.3, 127.7, 120.9, 108.5, 106.6,
101.5, 97.4. Anal. Calcd for C17H13N3O2: C, 70.09; H,
4.50; N, 14.42. Found: C, 69.86; H, 4.64; N, 14.39.
4.1.12. General procedure for the synthesis of 2,6-disub-
stituted-pyrimidin-4-yl-amides. Procedure identical to
compounds 5–7, starting from 43.
4.1.6.13. N-(5-Carbonitrile-4,6-diphenyl-pyrimidin-2-
yl)-4-methyl-benzamide (35). Yield 17%; white solid. H
1
NMR d (CDCl3): 8.81 (br s, 1H, NH), 8.13–8.09 (m,
4H, phenyl-H), 7.86 (d, 2H, J = 8.0 Hz, Ar-H), 7.62–
7.54 (m, 6H, phenyl-H), 7.32 (d, 2H, J = 8.0 Hz, Ar-
H), 2.45 (s, 3H, CH3). 13C NMR d (CDCl3): 171.2,
164.5, 157.5, 143.5, 135.2, 131.6, 130.6, 129.4, 129.2,
128.5, 127.5, 116.9, 97.7, 21.4. Anal. Calcd for
C25H18N4OÆ0.7 H2O: C, 74.60; H, 4.84; N, 13.92.
Found: C, 74.67; H, 5.15; N, 13.67.
4.1.12.1. Cyclopentane carboxylic acid (6-benzo-
(44).
[1,3]dioxol-5-yl-2-phenyl-pyrimidin-4-yl)-amide
1
Yield 48%; white solid. H NMR d (CDCl3): 8.51–8.46
(m, 3H, phenyl-H + pyrimidinyl-H), 8.06 (br s, 1H,
NH), 7.83–7.78 (m, 2H, Ar-H), 7.51–7.46 (m, 3H, phe-
nyl-H), 6.92 (d, 1H, J = 8.8 Hz, Ar-H), 6.05 (s, 2H,
–OCH2O–), 2.83–2.71 (m, 1H, CH-cyclopentane), 1.98–
1.60 (m, 8H, CH2-cyclopentane). 13C NMR d (CDCl3):
176.9, 163.8, 163.0, 159.3, 149.8, 148.2, 137.4, 131.0,
130.8, 128.6, 127.9, 121.7, 108.7, 106.8, 102.2, 101.8,
89.7, 88.7. Anal. Calcd for C23H21N3O3: C, 71.30; H,
5.46; N, 10.85. Found: C, 70.99; H, 5.70; N, 10.83.
4.1.6.14. N-(5-Carbonitrile-4,6-diphenyl-pyrimidin-2-
1
yl)-benzamide (36). Yield 60%; white solid. H NMR d
(CDCl3): 8.85 (br s, 1H, NH), 8.13–8.08 (m, 4H, phe-
nyl-H), 7.95 (d, 2H, J = 6.6 Hz, phenyl-H), 7.66–7.48
(m, 9H, phenyl-H). 13C NMR d (CDCl3): 171.2, 164.7,
157.4, 135.2, 133.5, 132.6, 131.7, 129.2, 128.7, 128.6,
127.5, 116.9, 97.7. Anal. Calcd for C24H16N4OÆ0.4
H2O: C, 74.98; H, 4.43; N, 14.57. Found: C, 75.00; H,
4.47; N, 14.54.
4.1.12.2. N-(6-Benzo[1,3]dioxol-5-yl-2-phenyl-pyrimi-
din-4-yl)-butyramide (45). Yield 46%; white solid. Anal.
1H NMR d (CDCl3): 8.50–8.45 (m, 3H, phenyl-
H + pyrimidinyl-H), 8.05 (br s, 1H, NH), 7.84–7.79
(m, 2H, Ar-H), 7.51–7.48 (m, 3H, phenyl-H), 6.93 (d,
1H, J = 8.4 Hz, Ar-H), 6.06 (s, 2H, –OCH2O–), 2.44
(t, 2H, J = 7.3 Hz, –CH2CH2CH3), 1.85–1.74 (m, 2H,
–CH2CH2CH3), 1.04 (t, 3H, J = 7.7 Hz, –CH2CH2CH3).
13C NMR d (CDCl3): 172.4, 164.9, 163.5, 158.0, 149.8,
4.1.6.15. N-(5-Carbonitrile-4,6-diphenyl-pyrimidin-2-
yl)-3,4-dichloro-benzamide (37). Yield 25%; white solid.
1H NMR d (CDCl3): 8.77 (br s, 1H, NH), 8.10–8.03
(m, 5H, phenyl-H + Ar-H), 7.75 (dd, 1H, J1 = 6.2 Hz

2750
J. P. D. van Veldhoven et al. / Bioorg. Med. Chem. 16 (2008) 2741–2752
148.1, 137.3, 131.3, 130.5, 128.3, 127.9, 121.9, 108.2,
107.4, 102.2, 101.3, 39.3, 18.2, 13.3. Anal. Calcd for
C21H19N3O3: C, 69.79; H, 5.30; N, 11.63. Found: C,
69.72; H, 5.50; N, 11.65.
164.1, 136.7, 136.7, 131.6, 130.9, 128.6, 116.5, 84.5.
MS (ESI): 272.0. Anal. Calcd for C17H12N4Æ0.5 H2O:
C, 72.31; H, 4.68; N, 19.84. Found: C, 72.33; H, 4.49;
N, 19.69.
4.1.12.3. N-(6-Benzo[1,3]dioxol-5-yl-2-phenyl-pyrimi-
din-4-yl)-2-ethyl-butyramide (46). Yield 52%; white solid.
1H NMR d (CDCl3): 8.55 (s, 1H, pyrimidinyl-H), 8.50–
8.45 (m, 2H, phenyl-H), 8.16 (br s, 1H, NH), 7.85–7.80
(m, 2H, Ar-H), 7.51–7.46 (m, 3H, phenyl-H), 6.92 (d,
1H, J = 8.8 Hz, Ar-H), 6.05 (s, 2H, –OCH2O–),
2.18–2.07 (m, 1H, –CH(CH2CH3)2), 1.82–1.53 (m, 4H,
4.1.15.2. 4-Amino-6-benzo[1,3]dioxol-5-yl-2-phenyl-
pyrimidine-5-carbonitrile (52). Yield 39%, white solid.
1H NMR d (CDCl3): 8.50–8.46 (m, 2H, phenyl-H),
7.76 (dd, 1H, J1 = 6.6 Hz, J2 = 1.8 Hz, Ar-H), 7.60 (d,
1H, J = 1.8 Hz, Ar-H), 7.54–7.45 (m, 3H, phenyl-H),
6.96 (d, 1H, J = 8.4 Hz, Ar-H), 6.08 (s, 1H, –OCH2O–
), 5.68 (br s, 2H, NH2). 13C NMR d (DMSO): 167.1,
164.9, 163.9, 149.8, 147.6, 136.7, 131.5, 130.5, 128.5,
123.7, 116.7, 108.8, 108.2, 101.9, 83.7. Anal. Calcd for
C18H12N4O2: C, 68.35; H, 3.82; N, 17.71. Found: C,
68.33; H, 3.88; N, 17.91.
–CH(CH2CH3)2),
0.95
(t,
6H,
J = 7.3 Hz,
–CH(CH2CH3)2). 13C NMR d (CDCl3): 175.8, 165.1,
163.6, 158.1, 149.9, 148.1, 137.3, 131.3, 130.6, 128.2,
127.9, 122.0, 108.2, 107.4, 102.3, 101.3, 51.9, 25.2,
11.6. Anal. Calcd for C23H23N3O3: C, 70.93; H, 5.95;
N, 10.79. Found: C, 70.60; H, 5.83; N, 10.75.
4.1.16. General procedure for the synthesis of 4-amido-
2,6-disubstituted-pyrimidine-5-carbonitriles.8 To a solu-
tion of the 4-amino-2,6-disubstituted-pyrimidine
(1 mmol, 1 equiv) in anhydrous pyridine (4 mL) was
added the appropriate acid chloride (5 mmol, 5 equiv)
and heated at 100 ꢁC in a sealed tube in the microwave
for 20 min. After completion (TLC, dichloromethane),
the solvent was evaporated under reduced pressure,
the crude product was purified by column chromatogra-
phy, eluting with dichloromethane and crystallized from
methanol or acetone.
4.1.12.4.
benzo[1,3]dioxol-5-yl-2-phenyl-pyrimidin-4-yl)-amide (47).
Cyclopropane
carboxylic
acid
(6-
1
Yield 51%; white solid. H NMR d (CDCl3): 8.51–8.47
(m, 2H, phenyl-H), 8.42 (s, 1H, pyrimidinyl-H), 8.32
(br s, 1H, NH), 7.82–7.77 (m, 2H, Ar-H), 7.51–7.47
(m, 3H, phenyl-H), 6.91 (d, 1H, J = 7.7 Hz, Ar-H),
6.05 (s, 2H, –OCH2O–), 1.67–1.55 (m, 1H, CH-cyclo-
propane), 1.19–1.12 (m, 2H, CH2-cyclopropane), 0.98–
0.90 (m, 2H, CH2-cyclopropane). 13C NMR d (CDCl3):
173.1, 164.9, 163.6, 158.0, 149.8, 148.1, 137.3, 131.3,
130.5, 128.2, 127.9, 121.9, 108.2, 107.4, 102.2, 101.3,
15.8, 8.6. Anal. Calcd for C21H17N3O3: C, 70.18; H,
4.77; N, 11.69. Found: C, 69.81; H, 4.50; N, 11.67.
4.1.16.1.
Cyclopentane
carboxylic
acid
(6-
benzo[1,3]dioxol-5yl-5-carbonitrile-2-phenyl-pyrimidin-4-
yl)-amide (53) LUF6056. Yield 33%; white solid. 1H
NMR d (CDCl3): 8.53–8.48 (m, 2H, phenyl-H), 8.07
(br s, 1H, NH), 7.80 (dd, 1H, J1 = 5.8 Hz, J2 = 1.8 Hz,
Ar-H), 7.68 (d, 1H, J = 1.8 Hz, Ar-H), 7.60–7.46 (m,
3H, phenyl-H), 6.98 (d, 1H, J = 8.0 Hz, Ar-H), 6.09 (s,
2H, –OCH2O–), 3.45–3.29 (m, 1H, CH-cyclopentyl),
2.06–1.62 (m, 8H, CH2-cyclopentyl). 13C NMR d
(CDCl3): 174.8, 167.7, 164.6, 159.7, 150.7, 148.0,
135.7, 132.0, 129.3, 128.8, 128.4, 124.5, 115.4, 109.0,
108.2, 101.7, 91.4, 46.1, 29.9, 25.7. Anal. Calcd for
C24H20N4O3: C, 69.89; H, 4.89; N, 13.58. Found: C,
69.68; H, 4.87; N, 13.88.
4.1.13. 2-Benzylidene-malononitrile (49). Prepared as de-
scribed in the literature.5
4.1.14. 2-Benzo[1,3]dioxol-5-ylmethylene-malononitrile
(50). Yield 97%, yellow solid. 1H NMR d (CDCl3):
7.61 (s, 1H, Ar-H), 7.60 (s, 1H, CHC(CN)2), 7.32 (dd,
1H, J1 = 6.6 Hz J2 = 1.5 Hz, Ar-H), 6.93 (d, 1H,
J = 8.4 Hz, Ar-H), 6.13 (s, 2H, OCH2O).
4.1.15. General procedure for the synthesis of 4-amino-
2,6-disubstituted-pyrimidine-5-carbonitriles.14 A mixture
of the functionalized malononitrile (45 mmol, 1 equiv),
benzamidin hydrochloride (45 mmol, 1 equiv) and
K2CO3 (95 mmol, 2.05 equiv) was refluxed in methanol
(35 mL) for 2 h. The methanol was removed in vacuo
and the residue was slurried in hot acetone. KMnO4
(2.5 g, 16 mmol) was portion wise added and monitored
by TLC (dichloromethane). The reaction mixture was
filtered over Celite, the excess of KMnO4 in the filtrate
was reduced with NaHSO3 and followed by a second fil-
tration over Celite. The filtrate was concentrated in va-
cuo. Recrystallization from ethyl acetate–petroleum
ether or a methanol–acetone mixture gave white
crystals.
4.1.16.2. N-(6-Benzo[1,3]dioxol-5-yl-5-cyano-2-phe-
nyl-pyrimidin-4-yl)-butyramide (54). Yield 49%; white
1
solid. H NMR d (CDCl3): 8.50–8.47 (m, 2H, phenyl-
H), 8.07 (br s, 1H, NH), 7.78 (dd, 1H, J1 = 6.2 Hz,
J2 = 2.2 Hz, Ar-H), 7.66 (d, 1H, J = 1.8 Hz, Ar-H),
7.60–7.46 (m, 3H, phenyl-H), 6.97 (d, 1H, J = 8.0 Hz,
Ar-H), 6.09 (s, 2H, –OCH2O–), 2.95 (t, 2H,
J = 6.9 Hz, CH2CH2CH3), 1.95–1.77 (m, 2H,
CH2CH2CH3), 1.09 (t, 3H, J = 7.3 Hz, CH2CH2CH3).
13C NMR d (CDCl3): 172.2, 167.7, 164.5, 159.6, 150.8,
148.0, 135.7, 132.1, 129.3, 128.9, 128.4, 124.4, 115.3,
109.0, 108.2, 101.7, 90.1, 39.6, 17.9, 13.5. Anal. Calcd
for C22H18N4O3: C, 68.38; H, 4.70; N, 14.50. Found:
C, 68.78; H, 4.74; N, 14.83.
4.1.15.1. 4-Amino-2,6-diphenyl-pyrimidine-5-carboni-
trile (51). Yield 51%, off-white solid. 1H NMR d
(CDCl3): 8.51–8.48 (m, 2H, phenyl-H), 8.15–8.10 (m,
2H, phenyl-H), 7.60–7.44 (m, 6H, phenyl-H), 5.70 (br
s, 2H, NH2). 13C NMR d (DMSO): 168.2, 164.7,
4.1.16.3. N-(6-Benzo[1,3]dioxol-5-yl-5-cyano-2-phe-
nyl-pyrimidin-4-yl)-2-ethyl-butyramide (55). Yield 65%;
white solid. 1H NMR d (CDCl3): 8.53–8.50 (m, 2H, phe-
nyl-H), 8.05 (br s, 1H, NH), 7.81 (dd, 1H, J1 = 6.2 Hz,

J. P. D. van Veldhoven et al. / Bioorg. Med. Chem. 16 (2008) 2741–2752
2751
J2 = 1.8 Hz, Ar-H), 7.68 (d, 1H, J = 1.8 Hz, Ar-H),
7.60–7.46 (m, 3H, phenyl-H), 6.98 (d, 1H, J = 8.4 Hz,
Ar-H), 6.09 (s, 2H, –OCH2O–), 2.71 (m, 1H,
CH(CH2CH3)2), 1.83–1.59 (m, 4H, CH(CH2CH3)2),
1.05 (t, 3H, J = 7.3 Hz, CH(CH2CH3)2). 13C NMR d
(CDCl3): 174.2, 164.8, 159.6, 150.8, 148.0, 135.7,
132.0, 129.4, 128.9, 128.4, 124.7, 115.4, 109.2, 108.2,
101.6, 92.0, 50.8, 24.9, 11.5. Anal. Calcd for
C24H22N4O3: C, 69.55; H, 5.35; N, 13.52. Found: C,
69.46; H, 5.43; N, 13.31.
was determined in the presence of 10 lM CGS21680 in-
stead of 100 lM CPA. The human A1 receptors were ex-
pressed in CHO cells, and [3H]DPCPX was used as the
radioligand. The A2A and A3 receptors were expressed
in HEK 293 cells, and [3H]ZM 241385 and [125I]AB-
MECA were used as the respective radioligands.
4.2.1.2. cAMP determinations. A number of com-
pounds specified in the text were tested in functional as-
says for their ability to influence the levels of cAMP in
the test system under various experimental conditions,
(i) in the presence of forskolin (10 lM, Tables 1 and
2), and (ii) in the presence of both forskolin (10 lM)
and CPA (100 nM, Fig. 2).
4.1.16.4. Cyclopentane carboxylic acid (5-carbonitrile-
2,6-diphenyl-pyrimidin-4-yl)-amide (56). Yield 25%;
white solid. 1H NMR d (CDCl3): 8.60–8.50 (m, 2H, phe-
nyl-H), 8.17–8.13 (m, 2H, phenyl-H), 8.08 (s, 1H, NH),
7.62–7.47 (m, 6H, phenyl-H), 3.47–3.32 (m, 1H, CH-
cyclopentyl), 2.20–1.95 (m, 4H, CH2-cyclopentyl),
1.95–1.60 (m, 4H, CH2-cyclopentyl).
A suspension in PBS buffer (supplemented with 5 mM
Hepes and 0.1% BSA) of CHO cells expressing the hu-
man adenosine A1 receptor was seeded into a 384-well
plate (white Optiplates, Perkin Elmer, NL; approxi-
mately 2500 cells/well). To each well were added the
compounds of interest, adenosine deaminase (0.8 IU/
mL), rolipram (50 lM), cilostamide (50 lM) in a total
volume of 7.5 ll, after which the plate was centrifuged
at 600g for 1 min. This mixture was then incubated for
15 min at room temperature followed by the addition
(2.5 ll) of forskolin (10 lM final concentration). After
a subsequent 45 min incubation at room temperature,
cAMP antibody solution (5 ll) and detection mix
(5 ll) were added, and the plate was centrifuged once
more. After a further incubation of at least 2 h the
amount of cAMP was determined in a TR-FRET assay
on a Victor spectrometer (Perkin-Elmer, NL) according
to the instructions of the supplier (Lance cAMP assay,
Perkin-Elmer, NL). All data reflect at least three inde-
pendent experiments performed in duplicate.
MS (ESI): 369.2. Anal. Calcd for C23H20N4O: C, 74.98;
H, 5.47; N, 15.21. Found: C, 74.67; H, 5.42; N, 15.15.
4.1.16.5. Cyclopropane carboxylic acid (5-carbonitrile-
2,6-diphenyl-pyrimidin-4-yl)-amide (57). Yield 18%;
white solid. 1H NMR d (CDCl3): 8.56–8.51 (m, 4H, phe-
nyl-H), 8.24 (br s, 1H, NH), 7.17–8.13 (m, 6H, phenyl-
H), 2.53–2.41 (m, 1H, CH-cyclopropyl), 1.37–1.29 (m,
2H, CH2-cyclopropyl), 1.14–1.06 (m, 2H, CH2-cyclo-
propyl). Anal. Calcd for C21H16N4OÆ0.2 H2O: C,
73.29; H, 4.81; N, 16.28. Found: C, 73.28; H, 4.74; N,
16.24.
4.1.16.6. N-(5-Carbonitrile-2,6-diphenyl-pyrimidin-4-
yl)-butyramide (58). Yield 37%; white solid. H NMR
1
d (CDCl3): 8.60–8.45 (m, 2H, phenyl-H), 8.16–8.12 (m,
2H, phenyl-H), 8.10 (br s, 1H, NH), 7.64–7.48 (m, 6H,
phenyl-H), 3.29 (t, 2H, J = 7.3 Hz, CH2CH2CH3),
1.97–1.79 (m, 2H, CH2CH2CH3), 1.10 (t, 3H,
J = 7.3 Hz, CH2CH2CH3). 13C NMR d (DMSO):
172.5, 169.1, 165.1, 159.7, 135.9, 135.6, 132.4, 131.9,
129.2, 129.1, 128.8, 128.7, 115.2, 91.1, 39.9, 18.1, 13.7.
MS (ESI): 343.0. Anal. Calcd for C21H18N4O: C,
73.66; H, 5.29; N, 16.36. Found: C, 73.34; H, 5.16; N,
16.74.
4.2.1.3. Data analysis. Ki values were calculated using
a non-linear regression curve-fitting program (Graph-
Pad Prism 4, GraphPad Software Inc., San Diego,
CA, USA). KD values of the radioligands were 1.6, 1.0
and 5.0 nM for [3H]DPCPX (A1 receptor), [3H]ZM
241385 (A2A receptor) and [125I]AB-MECA (A3 recep-
tor), respectively. The data from the functional assays
with compounds 27 and 53 were also analyzed with
GraphPad Prism and Figure 2 was generated by evalu-
ating the data to relate to the reference agonist CPA
(set at 0%) and the compounds’ maximum effects (set
at 100%).
4.2. Biology
4.2.1. Materials and methods. [3H]DPCPX and [125I]AB-
MECA were purchased from Amersham Biosciences
(NL). [3H]ZM 241385 was obtained from Tocris Cook-
son, Ltd, UK. CHO cells expressing the human adeno-
sine A1 receptor were provided by Dr. Andrea
Townsend-Nicholson, University College London,
UK. HEK 293 cells stably expressing the human adeno-
sine A2A and A3 receptor were gifts from Dr. J. Wang
(Biogen, USA) and Dr. K.-N. Klotz (University of
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