Discovery of 2-aminoimidazopyridine adenosine A2A receptor antagonists

Article abstract of DOI:10.1016/j.bmcl.2010.08.064

A novel series of adenosine A_2A receptor antagonists was identified by high-throughput screening of an encoded combinatorial compound collection. The initial hits were optimized for A_2A binding affinity, A₁ selectivity, an

Full text of DOI:10.1016/j.bmcl.2010.08.064

Bioorganic & Medicinal Chemistry Letters 20 (2010) 6845–6849
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Discovery of 2-aminoimidazopyridine adenosine A2A receptor antagonists
Brian F. McGuinness , Andrew G. Cole, Guizhen Dong, Marc-Raleigh Brescia, Yuefei Shao, Ian Henderson,
Laura L. Rokosz, Tara M. Stauffer, Neelima Mannava, Earl F. Kimble, Catherine Hicks, Nicole White,
Pamela G. Wines, Elizabeth Quadros
Ligand Pharmaceuticals, Inc., 3000 Eastpark Boulevard, Cranbury, NJ 08512, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
A novel series of adenosine A2A receptor antagonists was identified by high-throughput screening of an
encoded combinatorial compound collection. The initial hits were optimized for A2A binding affinity, A1
selectivity, and in vitro microsomal stability generating orally available 2-aminoimidazo[4,5-b]pyridine-
based A2A antagonist leads.
Received 23 July 2010
Revised 12 August 2010
Accepted 12 August 2010
Available online 19 August 2010
Ó 2010 Published by Elsevier Ltd.
Keywords:
Adenosine
A2A receptor
Imidazopyridine
Combinatorial chemistry
Parkinson’s disease
Adenosine mediates various biological functions through its
interaction with a family of G-protein-coupled receptors.1 To date,
four subtypes of adenosine receptors (A1, A2A, A2B, and A3) have been
identified and each exhibits a unique expression pattern and physi-
ological role.1 Adenosine receptor subtype A2A, located in the stria-
tum of the brain, contributes to the regulation of motor activity by
modulating dopamine D2 receptor activation.2 Hence, inhibition of
the interaction of adenosine with the A2A receptor may provide a
potential treatment for Parkinson’s disease.3 Notably, A2A antago-
nists such as xanthine KW-6002,4 pyrazolotriazolopyrimidine
SCH420814,5 and triazolopyrimidine V2006/BIIB-0146 have ad-
vanced to human clinical trials for motor-related disorders. In
addition, chemotypes such as thiazolopyrimidines,7 benzofurans,8
and 6-furanyl-4-carboxamidylpyrimidines9 have been recently dis-
closed as A2A antagonists. In our efforts, high-throughput screening
of an extensive encoded combinatorial compound collection10 (ꢀ4.5
million compounds screened) also identified multiple active
chemotypes. We have recently reported the discovery of substituted
aminothiazoles11 and trisubstituted purinones12 as A2A receptor
antagonists identified through this screening campaign. Herein,
the optimization of a third chemotype, based on a 2-aminoimidazo-
pyridine scaffold, identified via this high-throughput screen is
discussed.
The initial hit, benzimidazole 1a, was an active A2A antagonist
(46 nM) but lacked microsomal stability (11% remaining after
0.5 h treatment with human liver microsomes).13 Intriguingly,
the hit lacked a furan moiety,14 a common feature of non-xanthine
A2A antagonists. Hence, a hit to lead effort was initiated with the
goal of converting this active in vitro hit into an orally available,
brain penetrant lead molecule. In addition, selectivity against the
A1 receptor subtype was considered desirable in light of the poten-
tial for cardiovascular or renal effects associated with antagonism
of the A1 subtype.15
Benzimidazoles 1a–1h were synthesized via the solid-phase
route outlined in Scheme 1. Acid-cleavable linker 4-(40-formyl-30-
methoxy)phenoxy butyric acid16 was coupled to amino-TentaGel
generating resin 6. Reductive amination with 3-methoxybenzyl-
amine followed by HATU-mediated coupling of 4-fluoro-3-nitro-
benzoic acid produced intermediate 8. Subsequent fluorine
displacement, nitro reduction, and cyclization with cyanogen
bromide yielded resin-bound aminobenzimidazole 11,
a key
intermediate which enabled access to various R3 substitutions on
the 2-amino group of the benzimidazole core.
The unsubstituted aminobenzimidazole 1b (generated via di-
rect cleavage of resin-bound intermediate 11) exhibited a dramatic
drop in binding affinity with the A2A receptor (Table 1). Addition-
ally, analogs containing smaller alkyl secondary amides or tertiary
amides in this R3 position were inactive (data not shown). Hence, a
focus was placed on optimizing the substitution of the benzoyl
amide.
⇑
Corresponding author. Tel.: +1 609 570 1000x1527; fax: +1 609 570 1050.
E-mail address: bmcguinness@venenumbiodesign.com (B.F. McGuinness).
In 2008, Pharmacopeia, Inc. was acquired by Ligand Pharmaceuticals, Inc.
0960-894X/$ - see front matter Ó 2010 Published by Elsevier Ltd.
doi:10.1016/j.bmcl.2010.08.064

6846
B. F. McGuinness et al. / Bioorg. Med. Chem. Lett. 20 (2010) 6845–6849
O
O
O
NO2
F
NO2
NH
a
c
b
N
N
NH
H
MeO
MeO
MeO
f,g
MeO
6
7
8
9
O
O
N
O
O
O
R3
NH2
NH
MeO
N
N
N
N
e
d
N
N
H
NH2
NH
MeO
O
O
10
11
O
1a-h
Scheme 1. Solid-phase synthesis of analogs 1a–1h. Initial resin 6 = TentaGel resin harboring an acid-cleavable 4-(40-formyl-30-methoxy)phenoxybutyric acid linker.17
Reagents and conditions: (a) 3-methoxybenzylamine, Na(OAc)3BH, DCE, rt; (b) 3-nitro-4-fluorobenzoic acid, HATU, DIEA, DMF, rt; (c) 3-methoxypropylamine, DMF, rt; (d)
SnCl2, DMF, rt; (e) BrCN, DMF, rt; (f) substituted benzoic acid, HATU, DIEA, DMF, rt; (g) 50% TFA/DCM (v/v), rt.
Table 1
As is demonstrated in Table 1, alternative electron-withdrawing
substituents such as 3-fluoro (1c) and 3-chloro (1d) suffered five or
SAR for R3 2-aminobenzimidazole analogs
O
10-fold reductions in binding affinity, respectively, when compared
to the 3-cyano parent (1a). The more lipophilic electron-withdraw-
ing 3-trifluoromethyl group (1e) was inactive in the assay. Substitu-
tion with an electron-donating methoxy group on the 3-position of
the benzamide also produced a weakly active analog (1f). Relocation
of the electron-withdrawing group to either the 2-position (1g) or
4-position (1h) on the ring also led to a drop in affinity.
Similarity between the 2-aminobenzamidazole core of these
compounds and the 2-aminobenzothiazole core of previously
disclosed A2A antagonists18 was noted. However, various benzoyl
substitution patterns were allowed on the previously disclosed
2-aminobenzothiazole scaffold.18 In contrast, as indicated in Table
1, the 3-cyano substitution on this 2-aminobenzamidazole (1a)
R3
NH
MeO
N
N
N
H
O
Compds
R3
Human A2A binding Ki (nM)16
1a
1b
1c
1d
1e
1f
3-Cyanobenzoyl
H
3-Fluorobenzoyl
3-Chlorobenzoyl
46
6770
210
439
3-Trifluoromethylbenzoyl
3-Methoxybenzoyl
2-Chlorobenzoyl
>10,000
2380
4710
1670
1g
1h
4-Cyanobenzoyl
Table 2
Effect of central ring system on in vitro microsome stability
CN
O
O
N
N
R1
NH
X
O
Compds
R1
X
Human A2A binding Ki (nM)
Liver microsome stability (% remaining-0.5 h)13
Human
Rat
2a
2b
2c
2d
n-Butylamino
n-Butylamino
Piperidinyl
CH
N
CH
N
48
40
48
9.6
38%
73%
16%
36%
27%
95%
67%
90%
Piperidinyl
O
NO2
O
O
O
O
O
NO2
NH2
N
F
a
c
b
O
O
O
O
or
N
NH2
NO2
N
X
X
NH
R2
X
NH
R2
R2
12
13
14
Cl
CN
CN
CN
O
O
O
O
O
O
d
e
N
f
R1RN
N
N
O
N
N
HO
NH
NH
NH
N
X
X
X
R2
R2
R2
15
16
2-4
Scheme 2. Solution-phase synthesis route for analogs 2–4. Reagents and conditions: (a) R2NH2, THF, rt; (b) Na2S2O4, NaHCO3, 1,4-dioxane/H2O; rt; (c) BrCN, MeOH, rt; (d) 3-
cyanobenzoic acid, EDC, HOBt, DCM, rt; (e) LiOH, 1,4-dioxane, H2O, 80 °C; (f) R1RNH, EDC, HOBt, DCM, rt.

B. F. McGuinness et al. / Bioorg. Med. Chem. Lett. 20 (2010) 6845–6849
6847
CN
O
O
O
O
NO2
N
N
NO2
Cl
R1RN
R1RN
a,b
c,d
Cl
NH
N
N
NH
R2
N
R2
17
18
2-4
Scheme 3. Alternative route for analogs 2–4. Reagents and conditions: (a) R1RNH, DIEA, DCM; rt; (b) R2NH2, THF, rt; (c) Na2S2O4, NaHCO3, 1,4-dioxane/H2O, rt; (d) 3-
cyanobenzoyl isothiocyanate18, DCM, EDC, Et3N, rt.
uniquely provided high binding affinity. This 3-cyanobenzoyl
group was held constant through further optimization efforts.
A limited set of examples indicated increased microsomal
stability when the benzimidazole core was replaced with an
imidazo[4,5-b]pyridine core (Table 2; compare 2a to 2b and 2c to
2d). Since this change did not negatively affect binding affinity
and the potential for added microsomal stability was desirable,
the imidazopyridine series was the main focus moving forward.
A solution-phase synthetic route (Scheme 2) was employed for
the generation of imidazopyridine analogs aimed at understanding
the SAR of the R1 and R2 moieties. The appropriate ortho-nitro, hal-
ogen-substituted benzoic ester was reacted with an amine to gen-
erate intermediate 12. Nitro reduction to diamine 13, cyclization
with cyanogen bromide to 14, and coupling with 3-cyanobenzoic
acid produced ester 15. Hydrolysis yielded acid 16 which was con-
verted to the analogs 2–4 via carbodiimide-mediated amide forma-
tion. Alternatively, as outlined in Scheme 3, acid chloride 17 could
be reacted with two sequential amines to generate intermediate
18. Nitro reduction and cyclization with 3-cyanobenzoyl isothiocy-
anate19 yielded the desired analogs 2–4.
amides were indeed tolerated. While the highly simplified dimeth-
ylamide analog 3b had less affinity than 1a, slightly larger alkyl
amides were more tightly bound (3c and 3d).
An in vitro cAMP functional assay was employed to further
characterize the activity of these analogs in rat cells (Table 3).20
Sterically crowded R1 alkylamides (as in analogs 3e–3g) imparted
enhanced activity in this cAMP functional assay. In addition, these
sterically crowded R1 analogs demonstrated improved selectivity
against the A1 receptor subtype.
The compounds discussed thus far have harbored a fixed
3-methoxypropyl moiety at the N3-position of the imidazole ring.
Changes to this R2 position were explored while maintaining a
simple N-piperidinylamide in the R1 position (Table 4; 4a–4j).
Table 4
SAR for R2 2-aminoimidazopyridine analogs
O
O
N
N
NH
Y
N
CN
N
R2
As suggested by the human A2A binding affinity of compounds
in Table 2, elimination of the original R1 3-methoxybenzyl moiety
was tolerated allowing a reduction of the molecular weight. While
simplification to the primary amide was not allowed (Table 3; 3a),
analog 2d suggested that tertiary amides might be preferred,
thereby removing one undesired hydrogen bond donor from this
potential CNS molecule. As demonstrated in Table 3, other tertiary
Compds
Y
R2
Human A2A binding Ki
(nM)
A1/A2A
ratio
O
2d
4a
CH2
CH2
9.6
93
22
147
CH3
4b
4c
CH2
CH2
904
51
3
Table 3
OH
SAR for R1 2-aminoimidazopyridine analogs
10
CN
O
O
NH
4d
CH2
179
10
R1
N
N
O
N
HN
O
4e
4f
CH2
CH2
37
>46
2
O
O
Compds R1
Human A2A binding
Ki (nM)
Rat A2A cAMP Ki
A1/A2A
ratio
(nM)19
325
H2N
3a
3b
2430
123
ND
ND
ND
30
N
4g
4h
4i
CH2
CH2
CH2
CH2
1220
2590
233
35
7
3
O
3c
N
N
25
16
24
59
48
97
F
3d
N
4
3e
2d
3f
13
12
52
6.4
237
93
N
4j
39
N
N
9.6
7.7
O
4k
4l
O
O
O
92
61
1.8
9
O
N
N
3300
1100
234
O
O
4m
3g
11
14
120
4n
O
>10,000
—

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B. F. McGuinness et al. / Bioorg. Med. Chem. Lett. 20 (2010) 6845–6849
the imidazopyridine core (4l) was detrimental to the binding
affinity. Extension of the methoxy group to an ethoxy (4m) or
isopropoxy group (4n) was also not tolerated.
NO2
Cl
NO2
NH
a
b
Cl
N
Cl
N
Clearly, the 3-methoxypropyl chain (2d) was providing an opti-
mal electronic and steric display to the A2A receptor. In addition,
none of the changes outlined in Table 4 enhanced selectivity
against the A1 receptor subtype.
19
20
O
O
NO2
NH
c
N
N
EtO
NH
R1RN
Since the R1 amide proved to be the most tolerant toward
change, the possibility of relocating this functional group from
the 6-position to the 5-position of the imidazopyridine was ex-
plored. The synthesis of these analogs (5a–5d) is outlined in
Scheme 4. Treatment of 2,6-dichloro-3-nitropyridine (19) with
3-methoxypropylamine led to intermediate 20. Palladium-cata-
lyzed carbonylation21 generated ester 21 which was converted to
the desired analogs via the steps outlined in Scheme 3.
N
CN
N
O
O
O
O
21
5
Scheme 4. Synthesis of analogs 5a–5d. Reagents and conditions: (a) 3-methoxy-
propylamine, DIEA, THF, rt, 83%; (b) CO, EtOH, Et3N, PdCl2(PPh3)2, 70 °C, 37%; (c)
procedures outlined in Scheme 3.
Truncation of the longer 3-methoxypropyl chain (2d) to a simpli-
fied methyl group (4a) resulted in a 15-fold drop in binding affinity
for the A2A receptor. Replacement of the oxygen in the methoxy-
propyl group with a methylene (4b) caused an even sharper drop
in affinity. However, the hydroxypropyl analog 4c exhibited only
a minor loss of affinity for the receptor. Hence, it was hypothesized
that the oxygen of this chain served as a hydrogen bond acceptor
with the A2A receptor. Replacement with a different functional
group capable of accepting a hydrogen bond, for instance an acet-
amide (4e), was tolerated lending support to the hypothesis. The
aromatic analogs in this series (4f–4j) also support the require-
ment for a hydrogen bond acceptor. Para-fluorobenzyl analog 4h
is a comparatively weak binder of the A2A receptor in relation to
the pyridinylmethyl analogs 4i and 4j. The location of the hydrogen
bond accepting moiety is important since the 3-pyridylmethyl ana-
log 4i is sixfold less tightly bound then the 4-pyridylmethyl analog
4j. Additionally, the 3-methoxyphenyl analog 4f is fourfold more
tightly bound than the 4-methoxybenzyl analog 4g. Surprisingly,
the 3-methoxybenzyl analog (not shown) was inactive in the assay.
In general the SAR for the 5-substituted amides was similar to
the 6-position amide analogs (Table 5). Again, small alkyl tertiary
amides were preferred at R1 with the N-methyl-N-isopropylamide
derivative 5d exhibiting high activity in both the A2A binding and
functional assays. The 5-carboxamido-substituted analogs were
generally less stable to microsomes than their 6-substituted coun-
terparts. However, analog 5c was particularly notable since it
maintained moderate stability in the liver microsome assay.
Selected compounds which exhibited activity in the cAMP func-
tional assay, A1 selectivity, and in vitro microsomal stability were
tested for brain levels in rat after oral dosing.22 A comparison of
two example analogs is provided in Table 6. Analog 5c exhibited
the best overall profile of desired in vitro activity, good plasma
exposure, and modest brain levels after oral dosing in the rat.
In summary, a novel class of A2A antagonists has been discov-
ered via a high-throughput screen. These analogs represent the
third chemotype of A2A antagonists identified from an encoded
combinatorial compound collection.11,12 Initial optimization of this
series led to the synthesis of 2-aminoimidazopyridine 5c, an orally
available A2A antagonist (Ki = 12 nM) which exhibits 80-fold
selectivity against the A1 receptor and activity in an in vitro cAMP
accumulation assay.
A
series of relatively conservative modifications to the
3-methoxypropyl R3 group was tested while maintaining a N-mor-
pholino amide in the R1 position (4k–4n). When compared to the
3-methoxypropyl analog 4k, relocation of the oxygen closer to
Table 5
SAR for 5-carboxamidoimidazopyridine analogs
CN
O
N
N
NH
O
R1
X
O
Compds
R1
X
hA2A binding Ki (nM)
rA2A cAMP Ki (nM)
A1/A2A ratio
Liver microsome stability (% remaining-0.5 h)
Rat
Human
5a
5b
5c
N
CH
CH
N
3.1
17
16
85
23
183
131
80
19%
12%
52%
36%
N
19%
N
N
12
52%
5d
N
2.5
4.3
88
14%
25%
Table 6
Brain levels in rat at 1 and 4 h (po; 30 mpk).22
Compds
Plasma level (ng/mL) 1 h
Plasma level (ng/mL) 4 h
Brain level (ng/g) 1 h
Brain level (ng/g) 4 h
Brain/plasma ratio 1 h/4 h
3c
5c
4360
4630
2290
2380
480
1050
210
450
0.11/0.09
0.23/0.19

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6849
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A rat functional assay was chosen to support in vivo rat model studies.
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in 1ꢁ Ham’s F12 medium (Invitrogen) supplemented with 10% horse
serum (Invitrogen), 2% FBS (Invitrogen), and 1ꢁ Penn/Strep (Invitrogen)
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final volume of 18
L. Assay was conducted in 1ꢁ HBSS (Invitrogen),
5 mM HEPES (Invitrogen), 0.05% BSA, (Sigma), 50 rolipram (Sigma),
a
l
l
M
pH 7.4 buffer at 37 °C/5% CO2. Final DMSO concentration was 0.5%.
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a
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22. Procedure for the rat brain level determination: male Sprague–Dawley rats
were used for all studies. Rats were acclimated to the vivarium for 5–7 days
before each study. On the day before the study, the rats were fasted overnight
(18–20 h) but allowed free access to water. The next day, each rat was weighed
and dosed (based on the individual animal’s body weight) via oral intubation
with test compounds suspended in 0.5%MC. One, 2 or 4 h after compound
administration rats (two/group) were euthanized by CO2 asphyxiation. Blood
was collected by cardiac puncture and plasma prepared. Brain tissue was
collected following organ perfusion to remove any excess blood. The brain
tissue was homogenized on ice in cold PBS. The plasma and homogenized brain
tissue were stored frozen until the time of analysis using LC/MS/MS. Standards
curves, quality control and appropriate blank samples were included and all
samples were analyzed. Results were expressed as the brain/plasma ratio (ng/
g) at the specified time.
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16. For details on the A2A and A1 binding assays see the footnotes of Ref. 11. The
values are reported as the mean of at least two determinations.
17. (a) Williams, G. M.; Carr, R. A. E.; Congreve, M. S.; Kay, C.; McKeown, S. C.;
Murray, P. J.; Scicinski, J. J.; Watson, S. P. Angew. Chem., Int. Ed. 2000, 39, 3293;