The synthesis of highly potent, selective, and water-soluble agonists at the human adenosine A3 receptor

Article abstract of DOI:10.1016/j.bmcl.2006.01.088

Using a combination of parallel and directed synthesis, the discovery of a highly potent and selective series of adenosine A₃ agonists was achieved. High aqueous solubility, required for the intended parenteral route of administration, was achi

Full text of DOI:10.1016/j.bmcl.2006.01.088

Bioorganic & Medicinal Chemistry Letters 16 (2006) 2525–2527
The synthesis of highly potent, selective, and water-soluble
agonists at the human adenosine A3 receptor
Michael P. DeNinno,* Hiroko Masamune, Lois K. Chenard, Kenneth J. DiRico,
Cynthia Eller, John B. Etienne, Jeanene E. Tickner, Scott P. Kennedy, Delvin R. Knight,
Jimmy Kong, Joseph J. Oleynek, W. Ross Tracey and Roger J. Hill
Pfizer Global Research and Development, Groton/NewLondon Laboratories, Groton, CT 06340, USA
Received 12 December 2005; revised 19 January 2006; accepted 19 January 2006
Available online 7 February 2006
Abstract—Using a combination of parallel and directed synthesis, the discovery of a highly potent and selective series of adenosine
A3 agonists was achieved. High aqueous solubility, required for the intended parenteral route of administration, was achieved by the
presence of one or two basic amine functional groups.
Ó 2006 Elsevier Ltd. All rights reserved.
Adenosine is a ubiquitous neurotransmitter present in all
cell types which exerts its actions through binding to four
distinct G-protein-coupled receptors (A1, A2A, A2B, and
A3). The elucidation of the biological functions of these
receptors has been achieved in part by the availability of
selective agonists and antagonists at the target proteins.1
Not surprisingly, the state of understanding at each of the
receptors accurately reflects the success of the medicinal
chemistry efforts. Our interest in A3 receptor agonists
was initially driven not only by their cardioprotective
properties,2 but other potential indications as well,
including cancer.3 The pioneering work of Jacobson4
has advanced our understanding of the functions of the
A3 receptor through the discovery of selective ligands,
such as the highly studied agonist, IB-MECA (1). Build-
ing upon this work we have earlier reported on the identi-
fication of the first highly human selective A3 agonist 2.5
Since that time, additional series of selective A3 agonists
have been published.6
Herein we describe the discovery of a related series of
highly potent, selective, and water-soluble adenosine
A3 receptor agonists.
Our initial lead was the N-6 methyl uronamide analog
4a. It possessed high potency for the A3 receptor but
only moderate selectivity over A1. Our primary assay
was for human A1 and A3 receptor binding. At the time
of this work, there were no human A2A or A2B binding
assays available, so functional assays were used to access
activity at these receptors. In general, it was found that
A2A and A2B functional activity tracked well with A1
potency for these series of compounds.
Early on in the program it was found that the chemistry
used to install the N-6 substituent on the adenosine tem-
plate 3 was amenable to high-speed techniques. Conse-
quently, a library of approximately 500 compounds
was produced in a 96-well format. Several challenges
emerged from this approach. At the time, parallel chem-
istry was in its infancy, and final products were not puri-
fied. Moreover, it was not potency but selectivity we
were seeking, but we lacked the screening capacity to
do full dose-downs on every analog. As a compromise,
each analog was tested at two concentrations at both
the A3 (10, 100 nM) and A1 (100, 1000 nM) receptors.
Compounds that appeared particularly potent, and/or
selective, were resynthesized for full characterization.
N
O
O
I
HN
HN
N
N
N
N
N
N
N
O
O
Me
Cl
Me
N
O
O
N
H
N
H
OH
H2N
HO
OH
2
1
From these 500 analogs, only two emerged as particular-
ly interesting (Table 1). The 3,5-dichloro benzyl analog
4b was shown to be highly potent, but without improved
Keywords: Adenosine; Agonist; A3; Cardioprotection.
*
Corresponding author. Tel.: +1 860 441 5858; fax: +1 860 686
0620; e-mail: michael.p.deninno@pfizer.com
0960-894X/$ - see front matter Ó 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2006.01.088

2526
M. P. DeNinno et al. / Bioorg. Med. Chem. Lett. 16 (2006) 2525–2527
Table 1. Selected compounds identified from library of N-6 analogs
Table 3. N-6 Modifications in the 30-amino series
NHR
NHR
Cl
N
N
N
N
N
N
6
O
O
O
1) RNH2
2) TFA
Me
N
Me
N
N
Me
N
N
O
N
O
O
N
H
N
N
H
H
HO
OH
O
O
H2N
OH
6
4
3
Compound R
hA3 Ki (nM) hA1/hA3
b
Compound
R
hA3 Kia (nM) hA1/hA3
6a
6b
6c
6d
3,5-Dichloro benzyl
2,5-Dimethoxy benzyl
2-Methoxy-5-chloro benzyl
25 ( 2.4) 148
160 ( 5.8) 294
15 ( 2.9) 317
1
3-Iodo benzyl
Methyl
3,5-Dichloro benzyl
4.4 ( 0.32)
4.8 ( 0.61)
2.5 ( 0.21)
4.5
13
9.4
45
4a
4b
4c
2-Benzyloxy-5-chloro benzyl 16 ( 4.2) 691
2,5-Dimethoxy benzyl 3.9 ( 0.3)
a Values represent means ( SEM) of at least three determinations
unless noted.
b Selectivity versus the human A1 receptor.
Cl
O
N
N
O
N
O
O
Table 2. 20-30 Analogs
Me
H2N
N
O
a,b
N
H
NHMe
+
N
N
Cl
N3
OH
O
Me
N
N
O
7
N
H
OH
3' 2'
O
O
R1
R2
HN
N
5
d
N
N
N
9a
O
Cl
Compound
R1
H
R2
hA3 Ki (nM)
hA1/hA3
Me
59a
87a
6
12.6
—
O
5a
5b
5c
5d
5e
5f
OH
H
N
H
c,d
OH
NHAc
9b-f
OH
OH
OH
OH
>3000
>3000
>3000
120 ( 10.8)
N3
OH
NHSO2Me
NHCONHEt
NH2
—
—
8
194
Scheme 1. Reagents and conditions: (a) Et3N, EtOH, 80°C; (b) TFA,
CH2Cl2; (c) R1R2NH, EDCI, HOBt, DMF; (d) PPh3, NH4Cl, THF,
H2O.
a n = 2.
selectivity, whereas, the 2,5-dimethoxy benzyl derivative
4c had an A1/A3 ratio of 45. The benzyl substitution pat-
tern in 4c proved to be crucial later in the program (vide
infra).
to the N-6 region in an attempt to recapture the single
digit nanomolar potency of the diol lead.
The N-6 methyl group was first replaced with the amines
identified in Table 1 (Table 3). The SAR appeared to be
additive, with compounds 6a and 6b displaying improve-
ments in potency and selectivity, respectively. Further
modification of the 2,5 substitution pattern on the benzyl
group revealed that large substituents were tolerated at
the 2 position, whereas smaller groups, particularly halo-
gen, were optimal at C5. This SAR is exemplified in com-
pound 6d, which served as a springboard to multiple sub-
series, including one that led to compound 2.
Concurrent with the library work, virtually all other re-
gions of the lead were explored by directed synthesis.
One area producing interesting SAR was the 20,30-diol
(Table 2). Conventional wisdom suggested that these
alcohols were critical for binding and agonist activity.
Therefore, an emphasis was placed on analogs that
maintained at least one hydrogen bond donor. Removal
of either of the hydroxyl groups (5a, 5b) did lead to a
substantial loss of potency, although partial agonist
activity was maintained. An amino series was envisioned
to provide the necessary hydrogen bond, while allowing
for additional substitution. In practice, no substitution
on the amino group was allowed. Compounds 5c–e, as
well as N-alkyl derivatives (not shown) were all essen-
tially inactive at the human A3 receptor. Fortunately,
the simple 30-amine analog 5f did show a dramatic
improvement in selectivity, despite losing ꢀ20-fold in
potency.5 Importantly, this analog maintained full ago-
nist functional activity. Efforts were then directed back
One direction of research sought to replace the benzyl
group in 6d with more solubilizing functional groups.
To achieve this goal, the carboxylic acid 9a was pre-
pared as described in Scheme 1. The synthesis of com-
pound 7 was described previously.5 It was encouraging
to see that this compound was active, which prompted
further derivatization with bifunctional amines. These
amides displayed excellent levels of potency and selectiv-
ity (Table 4).

M. P. DeNinno et al. / Bioorg. Med. Chem. Lett. 16 (2006) 2525–2527
Table 4. Amide analogs in the 30-amino series
2527
References and notes
R
O
1. (a) Hess, S. Expert Opin. Ther. Pat. 2001, 11, 1533; (b)
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HN
N
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¨
N
N
N
Curr. Top. Med. Chem. 2003, 3, 445; (d) Jacobson, M. A.
Expert Opin. Ther. Pat. 2002, 12, 489; (e) Hutchinson, S.
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Compound
R
hA3 Ki (nM)
hA1/hA3
9a
9b
OH
NH2
130 ( 12.4)
6.8 ( 0.65)
222
526
2. (a) Liang, B. T.; Jacobson, K. A. Proc. Natl. Acad. Sci.
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9c
9d
9e
18 ( 2.6)
20 ( 2.2)
9.4 ( 0.98)
720
700
445
N
N
N
O
NH
9f
9.1 ( 0.03)
456
N
NMe2
Table 5. Functional activitya of compound 9e
Compound
EC50 or % control at highest dose
hA2A
hA2B
hA3
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d.; Jacobson, K. A. J. Med. Chem. 1996, 39, 4142.
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9e
16% at 3 lM
1% at 3 lM
8.1 nM
a Functional assays measured the increase of cAMP (A2A and A2B) or
the inhibition of isoproterenol-induced increase in cAMP (A3) in
HEK293 cells expressing the appropriate human receptor.
Analog 9e was profiled further and was shown to be a
potent, full agonist at the A3 receptor, but functionally
inactive at the A2A and A2B receptors (Table 5). It was
negative in the in vitro micronucleus and Ames gene
tox assays. As expected, 9e has high aqueous solubility,
particularly in buffered media (50–100 mg/mL in pH 4
citrate buffer). This compound was selected as a poten-
tial back-up to the earlier A3 agonist candidate CP-
608039, 2.
In summary, a combination of both high speed and tra-
ditional medicinal chemistry techniques was used to
identify a series of highly potent, selective, and water-
soluble agonists at the human adenosine A3 receptor.
Supplementary data
Supplementary data associated with this article can be
found, in the online version, at doi:10.1016/j.bmcl.2006.
01.088.