3'-aminoadenosine-5'-uronamides: Discovery of the first highly selective agonist at the human adenosine A3 receptor

Article abstract of DOI:10.1021/jm0255724

Selective adenosine A₃ agonists have potential utility for the prevention of perioperative myocardial ischemic injury. Herein, we report on the discovery and synthesis of compound 7. This amino nucleoside agonist possesses unprecedented levels

Full text of DOI:10.1021/jm0255724

J . Med. Chem. 2003, 46, 353-355
353
3′-Am in oa d en osin e-5′-u r on a m id es:
Discover y of th e F ir st High ly Selective
Agon ist a t th e Hu m a n Ad en osin e A3
Recep tor
Michael P. DeNinno,* Hiroko Masamune,
Lois K. Chenard, Kenneth J . DiRico, Cynthia Eller,
J ohn B. Etienne, J eanene E. Tickner,
Scott P. Kennedy, Delvin R. Knight, J immy Kong,
J oseph J . Oleynek, W. Ross Tracey, and Roger J . Hill
PGRD Groton Laboratories, Pfizer Inc.,
Groton, Connecticut 06340
Received August 13, 2002
Abstr a ct: Selective adenosine A3 agonists have potential
utility for the prevention of perioperative myocardial ischemic
injury. Herein, we report on the discovery and synthesis of
compound 7. This amino nucleoside agonist possesses unprec-
edented levels of selectivity for the human adenosine A3
receptor.
Patients undergoing cardiovascular surgery have an
increased risk for a myocardial damaging ischemic event
during or within a few days after the procedure.1 In
addition, noncardiovascular surgeries involving patients
with risk factors for cardiovascular disease also carry
an increased incidence of mortality and morbidity
associated with ischemic injury.2 Presently, there is no
approved therapy for the prevention of myocardial
ischemic injury despite the large number of exploratory
approaches being investigated. One of these approaches
involves a concept known as ischemic preconditioning
(IP), which originated from the observation that re-
peated short episodes of ischemia will protect the heart
from a subsequent longer, infarct-producing ischemic
event.3 Research around this phenomenon ultimately
led to pharmacological mimics of IP, which includes the
activation of cardiac adenosine receptors. Initial efforts
focused on the preparation of selective A1 receptor
agonists.4 Despite being cardioprotective, activation of
the A1 receptor leads to decreases in blood pressure and
heart rate, effects deemed unacceptable in a surgical
setting. More recently it has been shown that agonists
at the A3 receptor have cardioprotective properties
without inducing hemodynamic effects in a rabbit model
of ischemic injury.5 Our goal was to discover a potent
(<10 nM) agonist at the human A3 receptor with >100-
fold selectivity against the other human adenosine
receptors (A1, A2A, A2B) while possessing pharmaceutical
properties suitable for iv administration. Herein, we
report on the discovery of the amino nucleoside 7 (CP-
608039), the first highly selective full agonist at the
human adenosine A3 receptor that in addition has water
solubility suitable for developing an iv formulation.
Resu lts. Since the first report on the isolation of the
A3 receptor in 1992, numerous agonists and antagonists
have been reported for this target.6 In the agonist arena,
IB-MECA6a (1) and Cl-IB-MECA6b (2) have been used
F igu r e 1. Compounds 1-7.
Ta ble 1. Binding to Human A1 and A3 Receptorsa
b
Ki (nM)
ratio
A1/A3
compd
hA1
20
99
4600c
65
hA3
4.4
14
72
4.8
120
1.6
5.8
1
2
3
4
5
6
7
4.5
6.9
64
13
194
42
23000
68c
7300
1260
a
b
125I-ABA was used as the radioligand in these assays. Values
represent the average of at least three determinations unless
otherwise noted. Average SEM is (15%. c n ) 2.
as prototypical selective A3 agonists (Figure 1). How-
ever, although these analogues show selectivity against
the rat receptors, there is poor correlation between
species, an observation supported by the low receptor
sequence homology.7 Thus, as shown in Table 1, these
compounds have only modest selectivity at the human
receptors. Our discovery efforts began with the simpli-
fied MECA derivative 4, which displayed potency and
selectivity similar to those of 1. Modifications to all
positions of the ribose and adenine portions of compound
4 were achieved. These included atom substitutions in
the base and sugar, as well as amide isosteres at C5′.
Many of these changes gave disappointing results,
leading to analogues with reduced potency, selectivity,
or functional activity. One notable exception to this
trend was the 3′-R-amino derivative 5, which provided
the desired level of selectivity at the expense of lower
potency.
Compound 5, as well as other 3′-amino analogues, was
prepared as outlined in Scheme 1. Earlier strategies
employing commercially available nucleosides (e.g.,
inosine) as starting materials proved to be unsatisfac-
tory. The preparation of the required amino sugar in
the “ribose” stereochemical configuration was readily
achieved through an azide displacement of the triflate
* To whom correspondence should be addressed. Phone: (860)
441-5858. Fax: (860) 686-0620. E-mail: michael_p_deninno@
groton.pfizer.com.
10.1021/jm0255724 CCC: $25.00 © 2003 American Chemical Society
Published on Web 12/20/2002

354 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 3
Letters
Sch em e 1a
F igu r e 2. Concentration-response curve for the inhibition
of isoproterenol-stimulated cAMP accumulation by 7 (9, EC50
) 3.4 nM) or IB-MECA (2, EC50 ) 1.0 nM) in HEK 293 cells
expressing recombinant human A3 receptors. Data are pre-
sented as the mean and SEM of three experiments, each run
in duplicate. Maximal inhibition in this system is ∼70%.
possessed only moderate levels of selectivity and po-
tency. Clearly the 3′ amino group and N-6 substituent
work in concert to provide the high levels of both A3
binding affinity and selectivity seen in 7.
a
(a) Tf2O, pyridine, CH2Cl2, -20 °C; (b) NaN3, DMF, room temp;
(c) HIO4, THF-H2O; (d) RuO2, NaIO4, CHCl3, CH3CN, H2O; (e)
(COCl)2, CH2Cl2; (f) MeNH2, CH2Cl2; (g) HOAc, Ac2O, H2SO4; (h)
TMS-6-chloropurine, TMSOTf, DCE, 60 °C; (i) Et3N, MeOH; (j)
MeNH2, EtOH, 60 °C; (k) Ph3P, NH4OH, THF-H2O.
In summary, the discovery of the first highly potent
and selective human adenosine A3 agonist has been
achieved. Key to this discovery was the introduction of
an amino group at the 3′ position of the nucleoside,
which improved not only the selectivity but also the
aqueous solubility. High water solubility is a prerequi-
site for the development of an agent for perioperative
ischemic injury given the preferred parenteral route of
administration. Further details of the pharmacology of
compound 7 will be published elsewhere.
derived from glucose diacetonide (8). Selective manipu-
lation of the 5,6-acetonide provided the amide 10.
Hydrolysis of the remaining acetonide followed by
Vorbru¨ggen8 glycosidation using 6-chloro-9-trimethyl-
silylpurine afforded the intermediate 11. Reaction of 11
with methylamine and reduction of the azide group with
triphenylphosphine furnished the amino nucleoside 5.
Attempts to recapture the high level of potency seen
in 4 were made by elaborating the N-6 substituent. This
region of the A3 receptor is known to be highly tolerant
to substitution,6 and analogues were readily prepared
from the late-stage intermediate 11 using the appropri-
ate amine. Numerous incremental improvements ulti-
mately led to the optimized structure 7, which was
chosen for clinical development. This compound binds
to the human A3 receptor with a Ki of 5.8 nM and
possesses over 1000-fold selectivity versus the human
A1 receptor (Table 1). Even greater selectivity was
observed over the human A2A and A2B receptors (Ki >
50 µM). Functional activity was measured in separate
cell-based cAMP assays in which the human adenosine
receptors were expressed. Compound 7 displayed full
agonist activity at the hA3 receptor, inhibiting the
isoproterenol-stimulated increase in cAMP with an EC50
of 3.4 nM (Figure 2). Remarkably, greater than 1000-
fold functional selectivity was also observed over the A1,
Su p p or tin g In for m a tion Ava ila ble: Detailed chemistry
and pharmacology experimental procedures and spectral data
for all new compounds. This material is available free of charge
via the Internet at http://pubs.acs.org.
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A
2A, and A2B receptors. Further, the compound showed
no antagonist activity at any of the receptors. In
addition to enhancing A3 selectivity, the 3′-amino group
also endowed the molecule with favorable pharmaceuti-
cal properties. In particular, compound 7 has a pKa of
6.25, resulting in acceptable aqueous solubility when
formulated in acidic buffers (e.g., 2.5-5 mg/mL in 20
mM lactic acid).
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of 7 (compound 6) and the 3′-amino analogue of IB-
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