2-Amino-6-furan-2-yl-4-substituted nicotinonitriles as A2a adenosine receptor antagonists

Article abstract of DOI:10.1021/jm701594y

A_2A adenosine receptor antagonists usually have bi- or tricyclic N aromatic systems with varying substitution patterns to achieve desired receptor affinity and selectivity. Using a pharmacophore model designed by overlap of nonxanthine type of previously known A_2A antagonists, we synthesized a new class of compounds having a 2-amino nicotinonitrile core moiety. From our data, we conclude that the presence of at least one furan group rather than phenyl is beneficial for high affinity on the A_2A adenosine receptor. Compounds 39 (LUF6050) and 44 (LUF6080) of the series had K_i values of 1.4 and 1.0 nM, respectively, with reasonable selectivity toward the other adenosine receptor subtypes, A₁, A _2B, and A₃. The high affinity of 44 was corroborated in a cAMP second messenger assay, yielding subnanomolar potency for this compound.

Full text of DOI:10.1021/jm701594y

J. Med. Chem. 2008, 51, 4449–4455
4449
2-Amino-6-furan-2-yl-4-substituted Nicotinonitriles as A2A Adenosine Receptor Antagonists
Monica Mantri, Olivier de Graaf, Jacobus van Veldhoven, Anikó Go¨blyo¨s, Jacobien K. von Frijtag Drabbe Ku¨nzel,
Thea Mulder-Krieger, Regina Link, Henk de Vries, Margot W. Beukers, Johannes Brussee, and Adriaan P. IJzerman*
Leiden/Amsterdam Center for Drug Research, DiVision of Medicinal Chemistry, P.O. Box 9502, 2300 RA Leiden, The Netherlands
ReceiVed December 19, 2007
A2A adenosine receptor antagonists usually have bi- or tricyclic N aromatic systems with varying substitution
patterns to achieve desired receptor affinity and selectivity. Using a pharmacophore model designed by
overlap of nonxanthine type of previously known A2A antagonists, we synthesized a new class of compounds
having a 2-amino nicotinonitrile core moiety. From our data, we conclude that the presence of at least one
furan group rather than phenyl is beneficial for high affinity on the A2A adenosine receptor. Compounds 39
(LUF6050) and 44 (LUF6080) of the series had Ki values of 1.4 and 1.0 nM, respectively, with reasonable
selectivity toward the other adenosine receptor subtypes, A1, A2B, and A3. The high affinity of 44 was
corroborated in a cAMP second messenger assay, yielding subnanomolar potency for this compound.
Introduction
Adenosine receptors have adenosine as their endogenous
ligand and they belong to the class of G protein-coupled
receptors (GPCRs). They occur as four different subtypes, A1,
A2A, A2B, and A3, all of which have been cloned successfully
into artificial cell lines. All adenosine receptors are associated
with the cAMPa second messenger system. Activation of A1
Figure 1. Istradefylline (KW-6002).
and A3 receptors mediates inhibition of adenylate cyclase, while
A2A and A2B receptors, when activated, increase the intracellular
of A2A antagonists from a pharmacophore model, which we
constructedusingpreviouslyreportedA2A nonxanthineantagonists.
cyclic AMP level. In contrast to the widespread distribution of
A1 and A2B receptors in the brain, the A2A receptor is highly
expressed only in striatum, nucleus accumbens, olfactory
tubercles, and globus pallidus pars externa in the rat and human
brain.1 The A2A receptor is coexpressed with D2 receptors in
striatum. It is involved in the regulation of functional activity
of D2 dopamine receptors2 as heterodimerization of A2A and
D2 receptor subtypes inhibits D2 receptor functions. It has been
found that A2A adenosine receptor antagonists improve motor
function in animal models of basal ganglia disorders. Thus, such
compounds are thought to serve as a therapeutic modality in
Parkinson’s disease.
Results and Discussion
Molecular Modeling. A molecular superimposition model
was constructed using previously published antagonist ligands
having high affinity and selectivity for the A2A adenosine
receptor. All these molecules were selected with the criteria of
having diverse scaffolds based on the heterocyclic aromatic core
of each molecule. The actual ligand taken from a specific
scaffold class was the one that had a combination of high affinity
and selectivity for the A2A adenosine receptor. We also included
in the overlap four prototypic nonxanthine A2A adenosine
receptor antagonists, viz. CGS15943 (in fact a nonselective
compound), 8FB-PTP, SCH58261, and ZM241385.
The molecules shown in Figure 2 were drawn in the
SPARTAN ’0421 molecular modeling package, and each
molecule was energy-minimized to yield the lowest energy
conformer. Their electrostatic potentials were sampled over the
entire Van der Waal’s surface of each molecule and then electron
density was mapped over it. As an example, SCH58261 is
shown in Figure 3: shades of red and blue indicate relative
electronegative and electropositive regions, respectively. After
plotting the electrostatic potential over each individual molecule,
we noticed that all of them share common features, i.e., an
electron deficient region (blue) on the “top” of the molecule,
accompanied by an electron rich region (red) on the “right-
hand” side of the molecule. The former represents the H-bond
donating free amino group in most of the cases, while the latter
usually constitutes the electron rich region of the oxygen atom
in a furan ring.
Previously known A2A antagonists can be generally classified
as xanthine-like and nonxanthine like structures. KW-6002
(istradefylline) is a xanthine derivative having a styryl moiety
on the 8 position and is already in phase III clinical trials for
the treatment of Parkinson’s disease3 (Figure 1). The 8-styryl
xanthine moiety is associated with photochemical instability;4
however, also, xanthine-based structures have already been
optimized and explored to a high extent. This led us to find
new plausible A2A antagonists from nonxanthine like structures.
In this paper, we report the synthesis and SAR of a new class
* To whom correspondence should be addressed. Phone: +31 (0)71 527
4651. Fax: +31 (0)71 527 4565. E-mail: ijzerman@lacdr.leidenuniv.nl.
a Abbreviations: GPCR, G-protein-coupled receptor; cAMP, cyclic
adenosine monophosphate; SAR, structure-activity relationship; CHO,
chinese hamster ovary; HEK, human embryonic kidney; ADA, adenosine
deaminase; AB-MECA, N6-(4-aminobenzyl)adenosine-5′-methyluronamide;
CPA, N6-cyclopentyladenosine; DPCPX, 1,3-dipropyl-8-cyclopentylxan-
thine;NECA,5′-N-ethylcarboxamidoadenosine;TLC,thinlayerchromatography.
All molecules were superimposed on each other by consider-
ing these two areas of electron density together with the aromatic
10.1021/jm701594y CCC: $40.75
2008 American Chemical Society
Published on Web 07/19/2008

4450 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 15
Mantri et al.
Figure 2. Set of molecules selected for their different scaffold structure. a CGS15943 Ki (hA2A) ) 0.4 nM,5 b 8FB-PTP, c SCH58261 Ki (hA2A
)
) 0.6 nM,5 d ZM241385 Ki (hA2A) ) 0.8 nM,5 e Ki (hA2A) ) 0.8 nM,6 f Ki (rA2A) ) 1 nM,7 g Ki (hA2A) ) 4.3 nM,8 h Ki (hA2A) ) 0.5 nM,9 i
Ki (hA2A) ) 1.1 nM,10 j Ki (hA2A) ) 0.2 nM,11 k Ki (hA2A) ) 6.5 nM,12 l Ki (hA2A) ) 20 nM,13 m Ki (hA2A) ) 1.2 nM,14 n Ki (hA2A) ) 0.12 nM,15
o Ki (hA2A) ) 4 nM,16 p Ki (hA2A) ) 1.1 nM,17 q Ki (hA2A) ) 6.6 nM,18 r Ki (hA2A) ) 1.4 nM,19 s Ki (hA2A) ) 0.1 nM.20
nitrogen-containing ring system as a further basis of overlap
(Figure 4a). Because molecules c, d, f, h, i, k, n, s, and o (Figure
2) had bulky and flexible side chains, the orientation of these
side chains, at the “left side” of the molecules, was very diverse
(Figure 4a). To enhance the model for better overlap in this
region, the single conformation of each of the molecules
mentioned above compatible with the best overlap was chosen
from the set of all possible conformers within a limit of 5 kcal/
mol above the lowest energy conformation (see Experimental
Section for details). These selected conformations were over-
lapped with the other molecules used in the superimposition
from Figure 4a to yield Figure 4b. A simplified version with
only four molecules (e, j, m, and r) is shown in Figure 5 in
which the electrostatic potential energy surface of m was chosen
to show the general pattern of superimposition.
Superimposition of these molecules led to a pharmacophore
model shown in Figure 6. From the superimposition, it is clear
that for a molecule to be an A2A adenosine receptor antagonist,
it requires a hydrogen bond donor (A) and electron rich regions
(B and C), present on an N-containing aromatic ring (A and B)
and a largely lipophilic L1 group (C). These areas comply with
the recently published 3D pharmacophore model for A2A
receptor antagonism by Wei et al.22 In addition to these regions,
the N-containing ring also possesses an H-bond accepting region
D. Furthermore, the lipophilic region L2 seems to be desirable
for good binding to the receptor. To this area an appropriately
oriented heteroatom may be added (see also Figure 4b).
We decided to explore a monocyclic core ring structure in
particular because very few A2A adenosine receptor antagonists
with such a scaffold have been reported (compound l in Figure

2-Amino-6-furan-2-yl-4-Substituted Nicotinonitriles
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 15 4451
Figure 5. A simplified picture showing only four of the antagonists
(e, j, m, r) superimposed on each other, also showing the electrostatic
potential energy surface of m.
Figure 3. (I) SCH58261 in its lowest energy conformer as calculated
with SPARTAN. (II) SCH58261 with electrostatic potential energy
surface mapped as transparent cloud around stick model. (III) Molecular
electrostatic potential energy surface of SCH58261.
Figure 6. Simplified pharmacophore derived from superimposition of
the investigated molecules. A represents a H-bond donating region, B,
C and D represent electron-rich and H-bond accepting regions,
respectively. L1 and L2 represent lipophilic regions. A molecule with
a simple, monocyclic core and obeying the pharmacophore is shown
below it.
carry a safety liability as it is prone to oxidative metabolism,
hence we also searched for other rings at the L1 lipophilic region.
Chemistry. The compounds were synthesized according to
Scheme 1.24 The aldehyde was reacted with malononitrile by
adding a few drops of piperidine to give reaction intermediates
1-16 according to a Knoevenagel condensation.25 In the case
of heteroaromatic aldehydes, reactions were carried out at room
temperature so as to avoid decomposition of the intermediate.
In the next step, the malononitrile derivative and an aromatic
acetyl ketone were fused with ammonium acetate in a micro-
wave reactor at 120 °C for 1 h to form the nicotinonitriles
17-45.26 The reaction was optimized for microwave conditions
for this temperature and time to give maximum yield. The final
product was purified by column chromatography and recrys-
tallization.
Structure-Activity Relationships. The results of radioligand
binding assays performed on the substituted nicotinonitriles
17-45 are shown in Table 1.
The unsubstituted phenyl derivative (17) had 11 nM affinity
for the A2A receptor but was not much selective against the A1
receptor (Ki ) 28 nM). It also showed 170 nM affinity for the
A2B receptor. The biphenyl derivative 18 was inactive at both
Figure 4. (a) Superimposition of all the molecules in their lowest
energy conformation. (b) Optimized superimposition of molecules after
selecting a conformer with better overlap in L2 lipophilic region for
highly flexible molecules.
2 is one of the few examples). From the pharmacophore model
in Figure 6, we learned that a monocyclic ring should have an
aromatic character and a nitrogen atom at position B. At position
D, we chose the nitrile function instead of an extra nitrogen
atom in the ring to obtain a better distance separation between
D and C. For exploring lipophilic region L1, we tried various
aromatic rings with substitutions that are discussed later in Table
1 and Table 2 Thus we came up with the nicotinonitrile template,
having an amino group at position 2 as an H-bond donor. For
the exploration of lipophilic region L1, we included various
aromatic rings that are discussed later in Table 2. It has been
recently reported by Richardson et al.23 that a furan group may

4452 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 15
Mantri et al.
Table 1. Affinities of 2-Amino-4,6-substituted Nicotinonitriles in Radioligand Binding Assays of Human Adenosine Receptors
Ki (nM) or % displacementa
b
c
d
e
compound
R1
R2
hA1
hA2A
hA2B
hA3
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
NECA
CPA
DPCPX
H
furan-2-yl
furan-2-yl
furan-2-yl
furan-2-yl
furan-2-yl
furan-2-yl
furan-2-yl
furan-2-yl
furan-2-yl
furan-2-yl
furan-2-yl
furan-2-yl
furan-2-yl
phenyl
phenyl
phenyl
phenyl
phenyl
phenyl
phenyl
28 ( 7
0%
48 ( 7
68 ( 23
47%
34 ( 11
250 ( 99
11 ( 2
54 ( 18
11%
11 ( 5
170 ( 39
17%
33%
12%
15%
33%
31%
24%
370 ( 52
2%
8%
29%
620 ( 220
26%
13%
0%
28%
4%
21%
14%
21%
21%
20%
390 ( 110
34%
17%
7%
13%
18%
53%
32%
4-phenyl
4-Cl
3,4-diCl
4-CH3
4-OCH3
3,4-diOCH3
3,4-OCH2O-
4-OH
4-N(CH3)2
4-OCH(CH3)2
3-OCF3
3-CH3
0%
140 ( 35
190 ( 35
45%
41 ( 6
92 ( 4
26 ( 2
41 ( 15
3.8%
19%
6%
270 ( 24
64 ( 18
14 ( 1
66 ( 39
7.0 ( 0.4
12%
34 ( 2
15 ( 1.5
61 ( 19
7.8 ( 0.7
10 ( 1.3
6.1 ( 1.6
260 ( 13
140 ( 60
130 ( 10
160 ( 10
34%
H
3,4-diOCH3
3,4-OCH2O-
4-N(CH3)2
3-OCF3
3-CH3
48%
2%
28%
33%
1%
4%
7%
37%
18%
58 ( 6
130 ( 10
17%
4-OH
18%
1270 ( 160
11 ( 1
280 ( 60
1700 ( 170
1700 ( 300
130 (n ) 2)
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 µM concentrations. c Displacement of specific [3H]ZM241385 binding in HEK293 cell membranes
expressing human adenosine A2A receptors or % displacement of specific binding at 1 µM concentrations. d Displacement of specific [3H]MRS1754 binding
in CHO cell membranes expressing human adenosine A2B receptors or % displacement of specific binding at 1 µM concentrations. e Displacement of specific
[
125I]AB-MECA binding in HEK293 cell membranes expressing human adenosine A3 receptors or % displacement of specific binding at 1 µM concentrations.
Table 2. Affinities of 2-Amino-4,6-heteroaromatic-substituted Nicotinonitriles in Radioligand Binding Assays of Human Adenosine Receptors
Ki (nM) or % displacementa
b
c
d
e
compound
R1
R2
phenyl
thiophen-2-yl
furan-2-yl
thiophen-2-yl
pyrrol-2-yl
thiophen-2-yl
phenyl
hA1
hA2A
hA2B
hA3
37
38
39
40
41
42
43
44
45
furan-2-yl
5.8 ( 1.5
43 ( 6
28 ( 6
12 ( 2
28%
29 ( 5
22 ( 10
12 ( 2
100 ( 30
19 ( 1
220 ( 40
19%
75 ( 15
29%
31%
51%
32%
0%
39%
11%
33%
24%
thiophen-2-yl
thiophen-2-yl
phenyl
32 ( 15
1.4 ( 0.2
88 ( 25
640 ( 370
12 ( 1
41%
35%
phenyl
furan-2-yl
thiophen-2-yl
furan-2yl
380 ( 90
54 ( 3
44%
furan-2-yl
furan-2-yl
1.0 ( 0.1
15 ( 1
34 ( 2
440 ( 110
5-Me-furan-2-yl
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 µM concentrations. c Displacement of specific [3H]ZM241385 binding in HEK293 cell membranes
expressing human adenosine A2A receptors or % displacement of specific binding at 1 µM concentrations. d Displacement of specific [3H]MRS1754 binding
in CHO cell membranes expressing human adenosine A2B receptors or % displacement of specific binding at 1 µM concentrations. e Displacement of specific
[
125I]AB-MECA binding in HEK293 cell membranes expressing human adenosine A3 receptors or % displacement of specific binding at 1 µM concentrations.
A2A and A1 receptors, indicating that the L2 pocket cannot
accommodate extended bulky lipophilic groups. Further sys-
tematic evaluation of substitutions as suggested from the Topliss
scheme27 provided detailed insight in the requirements for A2A
adenosine receptor affinity. Both chloro substitution at the para
position (19) and 3,4-dichloro substitution (20) reduced affinity
for the A2A and A1 receptors as compared to unsubstituted 17.
Further changing substitution to methyl at the para position on
the phenyl ring (21) appeared even more unfavorable with
respect to affinity for A2A and A1 receptors with a displacement
of radioligand of only 45% and 47%, respectively, at a
concentration of 1 µM. Apparently, increased hydrophobicity
and electron withdrawing character are detrimental for the
affinity for the A2A receptor. The introduction of a 4-methoxy

2-Amino-6-furan-2-yl-4-Substituted Nicotinonitriles
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 15 4453
Scheme 1. Synthetic Route to 2-Amino-4,6-substituted
and 58 nM for A2A adenosine receptors. This represented
improved affinity over its furan analogue (28), but no significant
difference in selectivity was observed. The meta-methyl-
substituted derivative (35) actually showed more selectivity
toward A1 as compared to A2A adenosine receptors with a Ki
value 15 nM for A1 and 130 nM for A2A receptors. Compound
36 with a para-substituted hydroxy group on the phenyl ring
showed almost complete loss of affinity (17% displacement only
at 1 µM) for A2A adenosine receptor. Comparing 36 with 25 is
another confirmation that in case of nicotinonitriles the presence
of a furan ring in the L1 pocket is preferred over a phenyl ring
with respect to A2A adenosine receptor affinity. Concluding, the
L2 region can be substituted with various phenyl substituents.
However, in most cases, the selectivity for the A2A receptor is
decreased in favor of the A1 receptor.
Nicotinonitrilesa
Table 2 shows the results of radioligand binding assays
performed on heteroaromatic substituted nicotinonitriles
(37-45).
We optimized the L1 and L2 regions of the pharmacophore
by substituting the core molecule with various combinations of
heteroaromatic ring systems. All compounds had affinities for
A2A receptors in the low nanomolar range. Compounds 37 and
43, however, displayed even higher affinity toward A1 than A2A
receptors with Ki values of 5.8 and 19 nM for 37 and 22 and
54 nM for 43. Compound 38 with two thiophene rings had 32
nM affinity for the A2A adenosine receptor but was not selective
(43 nM on the A1 receptor). Replacing one of the rings by furan
as in 39 and 42 rendered these molecules more selective against
the A1 receptor. The Ki values of 39 and 42 for the A2A
adenosine receptor were 1.4 and 12 nM with 20- and 2.4-fold
selectivity, respectively. Also, compound 39 showed 75 nM
affinity for the A2B adenosine receptor. The only pyrrole
derivative made in this series (41) displaced only 28% of the
A1 receptor radioligand, making it selective for A2A, but its Ki
value for this receptor subtype was modest (640 nM). These
findings suggest that a five-membered heterocyclic ring is
favored over a phenyl ring in the L1 region, preferably with an
H-bond accepting rather than donating heteroatom. Compound
44 with two furan rings yielded the highest affinity in this series
for the A2A receptor with a Ki value of 1.0 nM and 12-fold
selectivity against the A1 receptor. It also displayed 34-fold
selectivity against the A2B receptor (34 nM affinity). Recently,
compound 44 has also been described as an inhibitor of mitogen-
activated protein kinase-activated protein kinase-2.28 Compound
45 with a 5-methyl substituent on the furan ring in the L2 pocket
showed somewhat reduced affinity for the A2A (15 nM) and A1
adenosine receptor (100 nM). However, this substituted furan
group is more metabolically stable and might thus be preferred
over the simple furan moiety. Other less “reactive” heterocyclic
moieties may also be explored, although the furan ring per se
is a very common feature in A2A-selective adenosine receptor
antagonists as found in many different research programs.
a Reagents and conditions: (i) Piperidine, EtOH, 1 h reflux; (ii) NH4OAc,
toluene, 120 °C in microwave.
group on the phenyl ring (22) showed restored affinity for the
2A receptor but without selectivity against the A1 receptor (Ki
A
values of 41 nM and 34 nM, respectively). The 3,4-dimethoxy
derivative 23 did show improvement in selectivity with respect
to the A1 receptor (Ki ) 250 nM), however, its affinity for the
A2A receptor was decreased to 92 nM. On the other hand,
compound 24 with a 3,4-dioxymethylene substituent showed
11 nM affinity for A1 and 26 nM affinity for the A2A receptor,
indicating the subtle receptor interactions at this part of the
molecule.
Compound 25 also displayed nanomolar affinity but less
selectivity (41 nM for the A2A and 54 nM for the A1 receptor).
Substitution at the para position by dimethyl amine (26) or an
isopropoxy group (27) showed a complete loss of affinity for
A
2A and A1 receptors, again indicating that bulky groups in the
L2 pocket are not favorable. Compounds 28 and 29, which have
trifluoromethoxy and methyl groups at the meta position,
respectively, showed 260 and 140 nM affinity for the A2A
adenosine receptor. However, these compounds were not
selective as their affinity values for A1 receptor were 270 nM
(28) and 64 nM (29). Compounds 30-36 were synthesized to
compare 2-amino-2-yl-6-furyl nicotinonitriles with 2-amino-2-
yl-6-phenyl nicotinonitriles. Compound 30 with two phenyl
substituents showed 14 nM affinity for the A1 and 130 nM
affinity for the A2A receptor. These data compare unfavorably
with furan-substituted 17, suggesting that the presence of a furan
moiety in the L1 pocket is preferred for A2A receptor affinity.
This observation also holds true in case of the 3,4-dimethoxy
substituent; the affinity of 23 changed from 92 to 160 nM after
replacing the furan by a phenyl ring (31). In the case of the
dioxymethylene ring, the change was even more prominent as
compound 32 showed only 34% displacement of the A2A
receptor radioligand. Substitution at the para position by
dimethyl amine (33) in the case of the phenyl series, also caused
a complete loss of affinity for A2A and A1 receptors due to the
unfavorable interaction with the bulky groups in the L2 pocket.
Compound 34 with a trifluoromethoxy substituent at the meta
position on the phenyl ring showed an affinity of 34 nM for A1
All compounds listed in both tables displayed little affinity
for the human A3 receptor. In most cases, there was less than
50% displacement of radioligand when compounds were tested
at 1 µM concentration.
Functional Assay at A2A Adenosine Receptors. For a
functional evaluation, intact CHO cells expressing the human
adenosine A2A receptor were used. The cells were stimulated
with increasing concentrations of the prototypic A2A receptor
agonist CGS21680 (2-[4-(2-carboxyethyl)phenylethylamino]-
5′-N-ethylcarboxamidoadenosine). Further CGS21680 concent-
ration-effect curves were recorded (n ) 3) in the presence of
two concentrations (10 and 100 nM) of 44 (Figure 7), causing

4454 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 15
Mantri et al.
NMR, 200 MHz; 13C NMR, 50.29 MHz) spectrometer with
tetramethylsilane as an internal standard. Chemical shifts are
reported in δ (ppm). Melting points were determined by Bu¨chi
melting point apparatus and are uncorrected. Elemental analyses
were performed by Leiden Institute of Chemistry and are within
0.4% of theoretical values unless otherwise stated. Reactions were
routinely monitored by TLC using Merck silica gel F254 plates.
Microwave reactions were performed on an Emrys Optimizer
(Biotage AB). Wattage was automatically adjusted to maintain the
desired temperature.
General Procedure for the Preparation of Malononitriles
(1-16). To malononitrile (20 mmol) dissolved in EtOH (40 mL)
was added an aromatic aldehyde (20 mmol) followed by 2 drops
of piperidine. In the case of substituted benzaldehydes, the reaction
mixture was refluxed for 1 h. In the case of heterocyclic aromatic
aldehydes, the reaction was carried out for 1 h at room temperature.
The precipitate was formed on cooling the reaction mixture to room
temperature. The crude product was filtered, and it was fairly pure
to carry out the further reactions.
General Procedure for the Preparation of Nicotinonitriles
(17-45). To a solution of previously synthesized benzylidene
malononitrile (3 mmol, 1 equiv) in toluene was added 2-acetyl furan
(3 mmol, 1 equiv) and ammonium acetate (4.5 mmol, 1.5 equiv).
The mixture was heated in a microwave at 120 °C for 1 h. The
reaction mixture was purified by column chromatography using
dichloromethane-methanol solvent system. Recrystallization from
methanol or ethanol gave the corresponding nicotinonitrile in pure
form. The yield of reaction varied from 40-70%.
Figure 7. The effect of increasing concentrations of compound 44
(10 and 100 nM) on the cAMP production induced by increasing
concentrations of CGS21680 in HEK 293 cells expressing the human
A2A adenosine receptor.
a rightward shift. These data were analyzed according to
Gaddum and Schild, yielding a pA2 value of 9.87 ( 0.36,
corresponding to an apparent affinity (KB value) of 0.14 nM
for 44. This value is in fair agreement with the binding data (Ki
value of 1 nM).
Conclusion
In this paper, we presented a new series of 2-amino-6-furan-
2-yl-4-substituted nicotinonitriles, designed and synthesized on
the basis of a pharmacophore model derived through molecular
modeling of previously known ligands. Comparing this series
of compounds with a number of 2-amino-6-phenyl-2-yl-4-
substituted nicotinonitriles demonstrated the importance of the
furan moiety to render these nicotinonitriles potent for the A2A
adenosine receptor. Compound 44 is highly potent on this
adenosine receptor subtype in both a radioligand binding and a
second messenger assay. One possible advantage of these small
molecules over some of the polycyclic antagonists resides in
their more favorable physicochemical properties. With the
Molecular Evoluator software,29,30 the following parameters for
compound 44 were calculated: LogP 3.06; MW 251; hydrogen
bond acceptors 5; hydrogen bond donors 2. These values comply
with the often-used Lipinski Rule of Five.31
2-Amino-4-thiophen-2-yl-6-furan-2-yl-nicotinonitrile (39,
LUF6050). mp: 180 °C. 1H NMR δ(CDCl3): 5.36 (s, 2H), 6.55-6.58
(m, 1H), 7.21 (s, 1H), 7.12-7.22 (m, 2H), 7.51-7.59 (m, 2H),
7.85-7.86 (d, 1H). Anal. (C14H9N3OS) C, H, N.
2-Amino-4,6-difuran-2-yl-nicotinonitrile (44, LUF6080). mp:
1
199 °C. H NMR δ(CDCl3): 5.30 (s, 2H), 6.55 (m, 2H), 7.46 (d,
1H), 7.51 (s, 1H), 7.58 (d, 1H). Anal. (C14H9N3O2) C, H, N.
Biology
Binding Studies. [3H]DPCPX and [125I]AB-MECA were
purchased from Amersham Biosciences (NL). [3H]ZM241385
(4-(2-[7-amino-2-(2-furyl)[1,2,4]triazolo[2,3-R][1,3,5]triazin-5-
ylamino]ethyl)phenol) and [3H]MRS1754 (N-(4-cyanophenyl)-
2-[4-(2,6-dioxo-1,3-dipropyl-2,3,4,5,6,7-hexahydro-1H-purin-8-
yl)-phenoxy]acetamide) were obtained from Tocris Cookson,
Ltd. (UK). CHO cells expressing the human adenosine A1
receptor were provided by Dr. Andrea Townsend-Nicholson,
University College London, UK. Dr. S. Rees (GSK, Stevenage,
UK) kindly provided CHO cells expressing the human A2B
receptor. HEK293 cells stably expressing the human adenosine
Experimental Section
Molecular Modeling. Molecular modeling was performed with
the SPARTAN ’0421 software package (Wave Function Inc.).
Default values in the Merck Force field were used in molecular
mechanics minimizations. Conjugate gradient energy minimization
was continued until the rms energy derivative was less than 0.001
kcal·mol-1 Å-1. Conformers were generated using the systematic
search method, and the lowest energy conformer was used for
further calculations. The molecular electrostatic potential of these
conformers was calculated with the semiempirical molecular orbital
program AM1. The energy and molecular electrostatic potentials
were sampled over the entire accessible surface of the molecules
(equal to the van der Waals contact surface). In the figures, the
most negative electrostatic potential is colored red and the most
positive one is blue.
For molecules c, d, f, h, i, k, n, s, and o, all possible
conformations were generated by calculating their conformation
distribution at ground-state by molecular mechanics. All the
conformations for each molecule were sorted on the basis of the
energy parameter to which a cutoff value of 5 kcal/mol from the
lowest energy was applied. From the remaining conformers, one
was chosen that had a side chain orientation that was most
compatible with rigid compound j in Figure 2.
A2A and A3 receptor were kind gifts from Dr. J. Wang (Biogen/
IDEC, Cambridge, MA) and Dr. K. N. Klotz (University of
Wu¨rzburg, Germany), respectively.
All compounds were tested in radioligand binding assays to
determine their affinities at human adenosine A1 ([3H]DPCPX),
A2A ([3H]ZM241385), and A3 ([125I]AB-MECA) receptors as
described previously in literature with the exception that
nonspecific binding to the A2A receptor was determined in the
presence of 10 µM CGS21680 instead of 100 µM CPA. For
the adenosine A2B receptor assay membranes containing 11 µg
of protein were incubated in a total volume of 100 µL of 50
mM Tris/HCl, 0.1% CHAPS, ADA 0.8 IU/mL (pH 7.4) and
[3H[MRS1754 (final concentration, 1.2 nM) for 1 h at 25 °C in
a shaking water bath. Nonspecific binding was determined in
the presence of 1 µM NECA. The incubation was terminated
by filtration over Whatman GF/C filters under reduced pressure
with a Brandel harvester (Gaithersburg, MD). Filters were
washed three times with ice cold 50 mM Tris/HCl pH 7.4,
placed in vials and counted.
Chemistry, Materials, and Methods. All reagents were obtained
from commercial sources and all solvents were of analytical grade.
1H and 13C NMR spectra were recorded on a Bruker AC 200 (1H

2-Amino-6-furan-2-yl-4-Substituted Nicotinonitriles
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 15 4455
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M. D. Synthesis and SAR evaluation of 1,2,4-triazoles as A2A receptor
antagonists. Bioorg. Med. Chem. Lett. 2004, 14, 817–821.
(14) Baraldi, P. G.; Fruttarolo, F.; Tabirizi, M. A.; Preti, D.; Romagnoli,
R.; El-Kashef, H.; Moorman, A.; Varani, K.; Gessi, S.; Merighi, S.;
Borea, P. A. Design, Synthesis, and Biological Evaluation of C9- and
C2-Substituted Pyrazolo[4,3-e]-1,2,4-triazolo[1,5-c]pyrimidines as New
A2A and A3 Adenosine Receptors Antagonists. J. Med. Chem. 2003,
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(17) Weiss, S. M.; Benwell, K.; Cliffe, I. A.; Gillespie, R. J.; Knight, A. R.;
Lerpiniere, J.; Misra, A.; Pratt, R. M.; Revell, D.; Upton, R.; Dourish,
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(18) Minetti, P.; Tinti, M. O.; Carminati, P.; Castorina, M.; Di Cesare,
M. A.; Di Serio, S.; Gallo, G.; Ghirardi, O.; Giorgi, F.; Giorgi, L.;
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cAMP assay. HEK293 cells expressing the human A2A
adenosine receptor were grown as a monolayer on 6 cm culture
plates. The cells were harvested and centrifuged two times for
5 min/1000 rpm. For cAMP production and determination, 7500
cells/well were used on 384-well plates. The cells were
incubated for 45 min at room temperature with either CGS21680
alone or together with compound 44 in different concentrations.
The assay medium also contained cilostamide (50 µM), rolipram
(50 µM), and adenosine deaminase (0.8 IU/mL). Incubation was
stopped with detection mix and antibody solution was added,
these two steps according to the instructions of the supplier.
The assay was performed with the Lance cAMP 384 kit from
Perkin-Elmer based on the competition of the sample’s cAMP
with a europium-labeled cAMP tracer complex for binding sites
on cAMP-specific antibodies labeled with Alexa Fluor dye.
Data Analysis. Ki values were calculated using a nonlinear
regression curve fitting program (GraphPad Prism 4.0, GraphPad
Software Inc., San Diego, CA). Ki values of radioligands were
1.6, 1.0, 1.3, and 5.0 nM for [3H]DPCPX, [3H]ZM 241385,
[3H]MRS1754, and [125I]AB-MECA, respectively.
Supporting Information Available: 1H NMR data of com-
pounds 1-16. Physical data and elemental analysis of the target
compounds 17-45. This material is available free of charge via
the Internet at http://pubs.acs.org.
(19) Yang, M.; Soohoo, D.; Soelaiman, S.; Kalla, R.; Zablocki, J.; Chu,
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