New, non-adenosine, high-potency agonists for the human adenosine A 2B receptor with an improved selectivity profile compared to the reference agonist N-ethylcarboxamidoadenosine

Article abstract of DOI:10.1021/jm049947s

The adenosine A_2B receptor is the least well characterized of the four known adenosine receptor subtypes because of the absence of potent, selective agonists. Here, we present five non-adenosine agonists. Among them, 2-amino-4-(4-hydroxyphenyl)

Full text of DOI:10.1021/jm049947s

©
Copyright 2004 by the American Chemical Society
Volume 47, Number 15
J uly 15, 2004
Letters
Sch em e 1. Synthetic Route to 16-20a
New , Non -Ad en osin e, High -P oten cy
Agon ists for th e Hu m a n Ad en osin e A2B
Recep tor w ith a n Im p r oved Selectivity
P r ofile Com p a r ed to th e Refer en ce
Agon ist N-Eth ylca r boxa m id oa d en osin e
Margot W. Beukers,* Lisa C. W. Chang,
J acobien K. von Frijtag Drabbe K u¨ nzel,
Thea Mulder-Krieger, Ronald F. Spanjersberg,
J ohannes Brussee, and Ad P. IJ zerman
Division of Medicinal Chemistry, LACDR,
Gorlaeus Laboratories, P.O. Box 9502, 2300 RA,
Leiden, The Netherlands
Received J anuary 19, 2004
a Reagents: (a) piperidine, EtOH; (b) malononitrile, thiophenol,
triethylamine, EtOH; (c) (i) Na2S, DMF, (ii) 1 M HCl; (d) 2-bro-
momethylimidazole, NaHCO3, DMF.
Abst r a ct : The adenosine A2B receptor is the least well
characterized of the four known adenosine receptor subtypes
because of the absence of potent, selective agonists. Here, we
present five non-adenosine agonists. Among them, 2-amino-
7
A receptors. A selective high-potency agonist for the
3
adenosine A2B receptor would be very useful to delineate
the precise role of the adenosine A2B versus the A3
receptor in this mast-cell-mediated stimulation of an-
giogenesis.
4
-(4-hydroxyphenyl)-6-(1H-imidazol-2-ylmethylsulfanyl)pyri-
dine-3,5-dicarbonitrile, 17, LUF5834, is a high-efficacy partial
agonist with EC50 ) 12 nM and 45-fold selectivity over the
adenosine A
2A subtypes. Compound 18, LUF5835, the 3-hydroxyphenyl
analogue, is a full agonist with EC50 ) 10 nM.
3 1
receptor but lacking selectivity versus the A and
Recently, Stasch et al. and Kerstin et al. reported on
the synthesis of a series of substituted 2-amino-4-
phenyl-6-phenylsulfanylpyridine-3,5-dicarbonitriles as
A
Adenosine receptors belong to the family of G-protein-
coupled receptors and can be subdivided into A1, A2A,
A2B, and A3 receptors. In contrast to the other three
subtypes, no high-affinity adenosine analogues have so
8,9
agonists for adenosine receptors. From their patent
data, we deduced that certain members of this class of
compounds might be interesting as agonists for the
adenosine A2B receptor. In this study we have synthe-
sized five 4-phenyl-substituted 2-amino-4-phenyl-6-phen-
ylsulfanylpyridine-3,5-dicarbonitriles. We determined
their ability to activate the human adenosine A2B
receptor through stimulation of cAMP production in
CHO cells stably expressing this receptor. In addition,
we determined the affinity and efficacy of these com-
pounds for the other three human adenosine receptors.
Compounds 16-20 were synthesized according to
1
,2
far been identified for the adenosine A2B receptor.
Hence, this receptor is also known as the low-affinity
3
receptor. To date, the most potent, albeit nonselective,
agonist for this receptor is N-ethylcarboxamidoadeno-
4
sine, NECA, with affinity in the micromolar range.
The adenosine A2B receptor has been implied in cell
proliferation and/or differentiation and in mast-cell-
mediated activation of angiogenesis.5 This last effect
is the result of a cooperative action with the adenosine
-7
8
Scheme 1. The aldehyde was reacted with malononitrile
in the presence of a few drops of piperidine to give the
intermediates (1-5) in moderate to good yields (40-
83%). The pyridine ring was formed by refluxing the
*
To whom correspondence should be addressed. Phone:
31-(0)715274607. Fax: +31-(0)715274537. E-mail: beukers@
chem.leidenuniv.nl.
+
1
0.1021/jm049947s CCC: $27.50 © 2004 American Chemical Society
Published on Web 06/15/2004

3
708 J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 15
Letters
Ta ble 1. Interaction of NECA and the Five Newly Synthesized
a
Compounds with Human Adenosine Receptors
K
i
, nM
EC50, nM
hA2B
compd
R
hA
1
hA2A
hA
3
1
1
6
7
phenyl
p-OH-
phenyl
2.4 ( 1.0
2.6 ( 0.3
28 ( 4
28 ( 4
171 ( 109
538 ( 210
19 ( 7
12 ( 2
1
1
2
8
9
0
m-OH-
phenyl
m-OCH
phenyl
p-OCH
phenyl
4.4 ( 2.0
2.0 ( 1.0
7.0 ( 0.8
21 ( 2
104 ( 49
74 ( 21
24 ( 7.6
10 ( 3
34 ( 24
9 ( 3
F igu r e 1. Stimulation of cAMP production via the human
adenosine A2B receptor stably expressed on CHO cells by
NECA (9), 16 (4), 17 (2), 18 (b), 19 (3), and 20 (1).
3
-
105 ( 22
214 ( 37
3
-
NECA
12 (9.6-15)12 60 ( 1012 11 ( 0.812 104 ( 15
1 3 2A
Ta ble 2. Inhibition (A and A Receptors) and Stimulation (A
and A2B Receptors) of cAMP Production by the Five Newly
a
Radioligand binding experiments were carried out on mem-
branes made from CHO cells stably expressing the human A1 and
from HEK293 cells stably expressing the A2A and A3 receptors with
a
Synthesized Compounds Compared to Reference Agonists
b
efficacy, %
3
3
125
[
H]DPCPX, [ H]ZM241385, and [ I]I-ABMECA as radioligands,
compd
hA1
hA2A
hA3
hA2B
respectively. To determine the ability of these compounds to
activate the human A2B receptor, cAMP experiments were carried
out on CHO cells stably transfected with this receptor (n ) 3-12).
The expression level of the A2B receptor on these cells amounted
16
17
18
19
109 ( 11
103 ( 6
112 ( 6
80 ( 13
46 ( 28
55 ( 20
55 ( 12
80 ( 6
49 ( 18
32 ( 5
84 ( 0.5
23 ( 4
95 ( 4
39 ( 2
73 ( 3
81 ( 3
74 ( 2
92 ( 3
68 ( 3
33 ( 1
6
to approximately 300 fmol/10 cells.
2
0
a
functionalized malononitrile with another equivalent of
malononitrile and an equivalent of thiophenol in ethanol
and triethylamine, resulting in 6-10 (20-43% yield).
To obtain the free thiol in the 6-position of the pyridine
ring, we added 3.3 equiv of sodium sulfide in DMF at
The production of cAMP was studied in CHO cells stably
expressing the adenosine receptors (n ) 3). b Efficacy is expressed
with respect to the following reference agonists: N -cyclopentyl-
adenosine (CPA), CGS21680, NECA, and 2Cl-IBMECA for the
human adenosine A1, A2A, A2B, and A3 receptors, respectively.
Compounds were tested at 100 times their Ki values for the A1,
A2A, and A3 receptors. To study the inhibitory effect of the agonists
on the A1 and A3 receptors, cAMP production was stimulated with
6
8
0 °C for 2 h, resulting in quantitative yields of 11-15.
-Bromomethylimidazole was synthesized by reducing
2
1
0 µM forskolin.
the commercially available 2-imidazole carboxaldehyde
with LiAlH4 in THF. The resulting alcohol function was
then substituted for bromine through the action of a
solution of hydrobromic acid in glacial acetic acid
according to literature procedures.1 The final step was
the reaction of the free thiol with 2-bromomethylimi-
dazole in the presence of NaHCO3 in DMF at room
temperature to give 16-20 in modest yields.
CHO cells expressing the human adenosine A2B
receptor were used to generate and measure cAMP
production as described before.11 For comparison, the
affinity of these compounds for the human adenosine
A1, A2A, and A3 receptors stably expressed on CHO cells
all four human adenosine receptors is summarized. As
expected, NECA was nonselective and bound not only
to the A2B receptor but also (with higher affinity) to the
A1, A2A, and A3 receptors. All five compounds interacted
with the human adenosine A2B receptor with EC50
ranging from 9 to 34 nM.
Interestingly, the Emax value of these compounds
varied greatly, indicating that substituents at the
phenyl ring are important for the efficacy (Figure 1 and
Table 2). The unsubstituted 16 had an efficacy of 81%
compared with NECA. The para-substituted compounds
had a relatively low efficacy, 74% for 17 (p-OH), and
0
(
A1) or HEK293 cells (A2A, A3) was determined in
33% for 20 (p-OCH ), compared with NECA. Substitu-
3
3
radioligand binding studies with [ H]DPCPX (KD ) 1.6
tion on the meta position yielded compounds with
efficacies of 92% for 18 (m-OH) and 68% for 19 (m-
3
125
nM), [ H]ZM241385 (KD ) 1.0 nM), and [ I]I-ABMECA
1
2
(KD ) 5.0 nM) as radioligands, respectively. To deter-
OCH ). Overall, 18 displayed the highest efficacy of the
3
mine whether the compounds possessed agonistic activ-
ity on the adenosine A1, A2A, and A3 receptors, cAMP
series, 92% compared with the reference agonist NECA,
combined with a low EC50 of 10 nM. The increased
cAMP production at low concentrations of 19 was
reproducible, although we have no explanation for this
phenomenon. Compound 20 had a similar EC50 of 9 nM
and was a partial agonist with an efficacy of 33%
compared to NECA. In the past we have successfully
synthesized potent partial agonists for the adenosine
experiments were performed on CHO cells essentially
as previously described.1
1,12
The CHO cells expressing
the human A2A, A2B, and A3 receptor were provided by
Dr. Steve Rees, GlaxoSmithKline, U.K. HEK293 cells
expressing the human adenosine A2A or the A3 receptor
were provided by Dr. J . Wang, Biogen, and Dr. K.-N.
Klotz, University of W u¨ rzburg, Germany, respectively.
CHO cells expressing the human adenosine A1 receptor
were provided by Dr. Andrea Townsend-Nicholson,
University College of London, U.K. To analyze the data,
PRISM software (GraphPad, San Diego, CA) was used.
1
3-15
A , A , and A receptors.
1
2A
3
Here, we demonstrate for
the first time the synthesis of potent partial agonists
for the adenosine A2B receptor.
To confirm that the cAMP production in the CHO cells
was due to activation of the adenosine A2B receptor, we
investigated whether the cAMP production could be
antagonized by the potent adenosine receptor antagonist
In Table 1, the interaction of the reference compound
NECA and our five newly synthesized compounds with

Letters
J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 15 3709
4
CGS15943. CGS15943 dose-dependently (0.1-10 µM)
antagonized the cAMP production induced by NECA
and by 16-20 with a pKB of 8.3 ( 0.6 (data not shown).
This value corresponds to literature data in which a pA2
angiogenesis. Moreover, this series of atypical non-
ribose compounds provides a new structural class of
agonists for the adenosine receptors, challenging the
rule that adenosine receptor agonists require a more or
less intact ribose function.
In conclusion, we have discovered a series of agonists
for the human adenosine A2B receptor. Moreover, we
have identified the first partial agonist for this receptor
subtype. Next to improved EC50 values for cAMP
production compared with NECA, these new compounds
also show improved selectivity over the other adenosine
receptors.
1
6
value for CGS15943 of 8.0 ( 0.3 was reported. Hence,
the new compounds are indeed agonists for the adeno-
sine A2B receptor.
Besides the activity of this series of compounds on the
adenosine A2B receptor, we also looked at the selectivity
with respect to the other subtypes of adenosine recep-
tors. In the absence of radioligand binding data on the
adenosine A2B receptor, this selectivity is defined as the
ratio of Ki (A1, A2A, A3) to EC50 (A2B). The nonselective
reference compound NECA prefers the Gi-coupled A1
and A3 receptors over the Gs-coupled A2A and A2B
receptors. Interestingly, the new compounds provided
more promising data concerning selectivity. Compound
Su p p or tin g In for m a tion Ava ila ble: Synthetic proce-
1
13
dures for 16-20 as well as H and C NMR, MS, and
combustion analysis data. This material is available free of
charge via the Internet at http://pubs.acs.org.
2
0 for example had a 24-fold lower EC50 value for the
Refer en ces
adenosine A2B receptor compared to its Ki value for the
adenosine A2A receptor. In addition, this compound was
equipotent on the adenosine A1 receptor, whereas the
other compounds preferred the adenosine A1 receptor
over the adenosine A2B receptor. Substantial selectivity
was obtained with respect to the adenosine A3 receptor.
Compound 17 had a 45-fold lower EC50 value on the
adenosine A2B receptor compared to its affinity for the
adenosine A3 receptor.
To verify whether the new compounds also acted as
agonists on the adenosine A1, A2A, and A3 receptors, we
performed cAMP studies with CHO cells expressing
these receptors. The compounds were tested at a
concentration of 100 times their Ki values to determine
their maximal effect (Table 2). At this concentration the
receptor is fully occupied, and as a result, the maximal
efficacy of the compounds can be determined.
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least to some extent, on all four adenosine receptors.
However, strong differences between receptor subtypes
were observed. Whereas the unsubstituted 16 had a
high efficacy for all receptors except the A2A receptor,
introduction of a m-OH group (18) resulted in almost
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position increased the efficacy on the A3 receptor but
decreased the efficacy on the A1 and A2B receptors.
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group increased the efficacy on all adenosine receptors
except for the A3 receptor. Apparently, the substitution
pattern on the phenyl ring of the newly synthesized
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J M049947S