Truncated nucleosides as A3 adenosine receptor ligands: Combined 2-arylethynyl and bicyclohexane substitutions

Article abstract of DOI:10.1021/ml300107e

C2-Arylethynyladenosine-5′-N-methyluronamides containing a bicyclo[3.1.0]hexane [(N)-methanocarba] ring are selective A₃ adenosine receptor (AR) agonists. Similar 4′-truncated C2-arylethynyl-(N)-methanocarba nucleosides containing alkyl or alkylaryl groups at the N⁶ position were low-efficacy agonists or antagonists of the human A₃AR with high selectivity. Higher hA₃AR affinity was associated with N⁶-methyl and ethyl (K_i = 3-6 nM) than with N⁶-arylalkyl groups. However, combined C2-phenylethynyl and N⁶-2-phenylethyl substitutions in selective antagonist 15 provided a K_i of 20 nM. Differences between 4′-truncated and nontruncated analogues of extended C2-p-biphenylethynyl substitution suggested a ligand reorientation in AR binding, dominated by bulky N⁶ groups in analogues lacking a stabilizing 5′-uronamide moiety. Thus, 4′-truncation of C2-arylethynyl-(N)-methanocarba adenosine derivatives is compatible with general preservation of A₃AR selectivity, especially with small N⁶ groups, but reduced efficacy in A₃AR-induced inhibition of adenylate cyclase. This article not subject to U.S. Copyright. Published 2012 by the American Chemical Society.

Full text of DOI:10.1021/ml300107e

Letter
pubs.acs.org/acsmedchemlett
Truncated Nucleosides as A3 Adenosine Receptor Ligands: Combined
2-Arylethynyl and Bicyclohexane Substitutions
Dilip K. Tosh, Silvia Paoletta, Khai Phan, Zhan-Guo Gao, and Kenneth A. Jacobson*
Molecular Recognition Section, Laboratory of Bioorganic Chemistry, National Institute of Diabetes and Digestive and Kidney
Diseases, National Institutes of Health, Bethesda, Maryland 20892-0810, United States
S
* Supporting Information
ABSTRACT: C2-Arylethynyladenosine-5′-N-methyluronamides containing a
bicyclo[3.1.0]hexane [(N)-methanocarba] ring are selective A3 adenosine receptor (AR)
agonists. Similar 4′-truncated C2-arylethynyl-(N)-methanocarba nucleosides containing
alkyl or alkylaryl groups at the N6 position were low-efficacy agonists or antagonists of the
human A3AR with high selectivity. Higher hA3AR affinity was associated with N6-methyl
and ethyl (Ki = 3−6 nM) than with N6-arylalkyl groups. However, combined C2-
phenylethynyl and N6-2-phenylethyl substitutions in selective antagonist 15 provided a Ki
of 20 nM. Differences between 4′-truncated and nontruncated analogues of extended C2-
p-biphenylethynyl substitution suggested a ligand reorientation in AR binding, dominated
by bulky N6 groups in analogues lacking a stabilizing 5′-uronamide moiety. Thus, 4′-
truncation of C2-arylethynyl-(N)-methanocarba adenosine derivatives is compatible with
general preservation of A3AR selectivity, especially with small N6 groups, but reduced
efficacy in A3AR-induced inhibition of adenylate cyclase.
KEYWORDS: G protein-coupled receptor, purines, molecular modeling, structure−activity relationship, radioligand binding,
adenosine receptor
xtracellular adenosine acts in cell signaling through four
subtypes of rhodopsin-like G protein-coupled receptors
Chart 1. (N)-Methanocarba-adenosine Derivatives as
Selective A3AR Ligands (hAR Affinity in nM): Full Agonists
in the 5′-N-Methyluronamide Series (1 and 3) and
Truncated Nucleosides (2) That Act as A3AR Antagonists
and Partial Agonists, Including the Present Target Structures
(4−21)
E
(GPCRs), that is, A1 adenosine receptor (AR), A2AAR, A2BAR,
and A3AR.1 The A3AR is the least widely distributed of the
subtypes in the body and has become a target in drug discovery
for various disease conditions.2−5 A3AR-selective agonists have
successfully progressed to phase 2 and 3 clinical trials for the
treatment of hepatocellular carcinoma (including subsequent to
hepatitis C viral infection), autoimmune inflammatory diseases,
such as rheumatoid arthritis, psoriasis, and osteoarthritis,
glaucoma, and dry eye disease. A3AR agonists are also under
consideration for treating uveitis, Crohn's disease, neuropathic
pain, loss of skin pigmentation, lung injury, and ischemia of
brain, heart, and skeletal muscle.2,6−8 A3AR-selective antago-
nists are being explored for treatment of asthma, septic shock,
glaucoma, and other conditions.9−15
The structure−activity relationship (SAR) of nucleosides at
the A3AR has been extensively explored.5 Selective agonists of
the A3AR are typically adenine-9-ribosides containing both 5′-
N-methyluronamide (facilitates complete activation of A3AR)
and N6-(3-halobenzyl) groups. A general means of enhancing
A3AR affinity and selectivity is the replacement of the flexible
ribose ring with a conformationally constrained bicyclo[3.1.0]-
hexane (methanocarba) ring system that enforces a receptor-
preferred North (N) conformation,16,17 as in the selective
agonist 1a (MRS3558) and its congeners (Chart 1).
The effects of truncation of adenosine derivatives at the 4′
carbon, that is, loss of the CH2OH of adenosine, have been
extensively explored in the 4′-thio, 4′-oxo, and (N)-
Received: May 3, 2012
Accepted: June 11, 2012
Published: June 11, 2012
This article not subject to U.S. Copyright.
Published 2012 by the American Chemical
Society
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Table 1. Affinity of a Series of (N)-Methanocarba adenosine Derivatives at hARs and Functional Efficacy at the hA3AR
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ACS Medicinal Chemistry Letters
Letter
Table 1. continued
a
Binding in membranes of CHO or HEK293 (A2A only) cells stably expressing one of three hAR subtypes. The binding affinity for hA1, A2A, and
A3ARs was expressed as Ki values using agonists [3H]N6-R-phenylisopropyladenosine, [3H]2-[p-(2-carboxyethyl)phenyl-ethylamino]-5′-N-
ethylcarboxamidoadenosine, or [125I]N6-(4-amino-3-iodobenzyl)adenosine-5′-N-methyluronamide, respectively. A percent in parentheses refers to
b
inhibition of binding at 10 μM. Inhibition of forskolin-stimulated cyclic AMP production in hA3AR-transfected CHO cells. At 10 μM, in
comparison to the maximal effect of 10 μM 5′-N-ethylcarboxamidoadenosine (=100%). Selected compounds (10 μM) were evaluated for stimulation
of cyclic AMP production (% of full agonist 5′-N-ethylcarboxamidoadenosine) in hA2BAR-transfected CHO cells: 3c, 28 3; 3e, 28 4; 5, 49 7;
c
6, 38 3; 9, 44 10; 10, 33 11; 13, 40 9; and 15, 46 14. Data are expressed as means standard errors (n = 3, unless noted). Data from
d
e
f
g
Melman et al.20 Data from Tosh et al.30 Data from Tosh et al.23,24 Inhibition of radioligand binding at 1 μM. n = 2.
methanocarba series of nucleosides. An effect of this truncation
is typically retention of affinity at the A3AR and reduction of
affinity at other AR subtypes.18−20 Specifically, in the (N)-
methanocarba series of N6-(3-halobenzyl) derivatives, that is,
full A3AR agonists 1, truncation resulted in series 2 that retains
hA3AR affinity and selectivity but is typically reduced in efficacy
at A3 and A1ARs but not A2AAR.21 The functional activity of 2a
(MRS5127) and its congeners range from full competitive
antagonism (KB = 8.9 nM,20 by Schild analysis in blocking
agonist-induced guanine nucleotide binding) to partial agonism
of ∼50% efficacy (in inhibition of adenylate cyclase).
Compound 2a was radioiodinated and studied in binding to
ARs in different species,22 and its high affinity and selectivity for
the A3AR were independent of species.
The synthetic route used to prepare truncated (N)-
methanocarba derivatives containing a C2-arylethynyl group
is shown in Scheme 1. Initially, D-ribose was converted as
Scheme 1. Synthesis of Truncated (N)-Methanocarba-
a
adenosine Analogues
We recently demonstrated that (N)-methanocarba adenosine
5′-N-methyluronamide derivatives tolerate the introduction of
rigid, extended C2-arylalkynyl groups.23,24 Previously, similar
groups were included in potent A3AR agonists in the relatively
flexible riboside series.25 In the rigid (N)-methanocarba series,
the much larger planar groups, such as large as 2-
pyrenylethynyl, than anticipated were tolerated at the human
(h) A3AR.
The present study establishes that truncation of the 4′ group
of 2-arylethynyl-(N)-methanocarba adenosine agonists in
general preserves selectivity for the A3AR, while the effects of
such truncation on the relative agonist efficacy at this receptor
are complex. These truncated partial agonists/antagonists of
the A3AR have favorable physicochemical properties, such as
low polarity, which will likely alter pharmacokinetic behavior in
vivo. The SAR in AR binding of 4′-truncated (N)-
methanocarba-adenosine derivatives was characterized by
Melman et al.20 and Tosh et al.24 The derivatives contained
mainly N6-3-halobenzyl groups and relatively flexible, straight
chain alkynyl groups at the C2 position, such as 2d (Table 1),
but substitution with rigid, linear C2-arylethynyl groups was
not included in the previous studies. We have explored such
rigid C2 substituents in the present study and also varied the N6
group.
a
Reagents: (i) 2-Iodo-6-chloropurine, Ph3P, DIAD, THF, rt. (ii)
R1NH2, Et3N, MeOH, rt. (iii) Substituted HCC-Ph-R2, Pd-
(PPh3)2Cl2, CuI, Et3N, DMF, rt. (iv) 50% TFA (aq.), MeOH:CH2Cl2
(3:1), rt.
previously reported into the 2′,3′-protected intermediate
cyclopentenone 22.27 Compound 22 was then converted
stereoselectively in two steps to the alcohol 23, which was
subjected to a Mitsunobu reaction with 2-iodo-6-chloropurine
The 4′-truncated analogues contain varied N6 substitution
(N6-methyl, methoxy, or ethyl: 4−9; N6-3-chlorobenzyl: 10−
14; and N6-2-phenylethyl or 2,2-diphenylethyl: 15−21) (Table
1). With small N6 substitution, only two phenylethynyl
variations were placed at the C2 position: unsubstituted and
2-chloro, which was previously shown to promote A3AR
affinity.23,24 However, in the N6-3-chlorobenzyl series, several
other C2-phenylethynyl substitutions were used, that is, 3,4-
difluoro (11), 3-chloro (13), and 4-phenyl (14). N6-Methyl and
adenosines are typically more potent at the hA3AR than rat (r)
A3AR, while N6-(3-halobenzyl)adenosines tend to display
greater species-independent selectivity.22 Thus, it is useful to
have a range of N6 groups present.
to give intermediate 24.28 Nucleophilic substitution with the
,30
appropriate amine at room temperature provided the N6-
substituted intermediates 25−30. A Sonogashira reaction26 was
then carried out with a variety of commercially available
phenylalkynes, followed by acid hydrolysis of the 2′,3′-
isopropylidene group to provide truncated nucleosides 4−21.
Radioligand binding assays were performed at three hAR
subtypes using standard 3H- and 125I-labeled nucleoside ligands
of high affinity (Table 1).20 A3AR binding curves of the
truncated nucleosides generally showed a Hill coefficient of ∼1.
Selected compounds in the (N)-methanocarba nucleoside
series were assayed at the hA2BAR and found to be weakly
active, as this ring system was previously found to reduce
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hA2BAR affinity.16 Some previously reported 2-chloro (2a−c)
and 2-alkynyl (2d and 3a−f) (N)-methanocarba-adenosine
derivatives were used for comparison in the biological
assays.17,2324,29
biological activities at the A3AR. For example, the cLog P values
of potent C2-arylethynyl derivatives having N6-methyl 5, N6-3-
chlorobenzyl 10, N6-2-phenylethyl 15, and N6-2,2-diphenyleth-
yl 17 substituents were 2.32, 3.76, 3.70, and 5.14, respectively.
The total polar surface area of all four derivatives was 92.8 Å2,
which is within the desired range.30 These physicochemical
parameters are predicitive of bioavailability, although in vivo
pharmacological and pharmacokinetic properties of these
analogues have not been measured.
Flexibility of a 5′-N-alkyluronamido moiety of adenosine
agonists, which is entirely lacking in the present analogues, is
closely associated with full A3AR activation. Consistently, these
analogues have low or minimal levels of efficacy in the A3AR-
induced inhibition of adenylate cyclase, but the functional
efficacy for other effector systems coupled to the same receptor
has not been measured. For comparison, other truncated (N)-
methanocarba-adenosine derivatives were shown to be full
antagonists in a guanine nucleotide binding assay, which is
relatively poorly coupled, and partial agonists in an assay of
cyclic AMP.22 The unusual shape of these nucleoside ligands
might translate to ligand-dependent differences in the
conformation of the nucleoside-bound A3AR, which could
have functional implications on effects induced by this receptor.
There is already an indication that nucleoside ligands of the
A3AR can display a functional bias.31
The observation of high affinity at the A3AR in the 5′-N-
methyluronamido series was explained in terms of a proposed
outward shift of transmembrane helix 2 (TM2) from its
position in a homology model based on the agonist-bound
A2AAR structure.23,24 The present 4′-truncated derivatives were
subjected to similar molecular modeling analysis (Supporting
Information) to predict a similar 7 Å shift of TM2, similar to its
position in opsin, to accommodate the bulky, extended C2
substituent. As for the 5′-N-methyluronamides,23,24 the 3′- and
2′-hydroxyl groups of docked 10 formed H-bonds with Ser271
(7.42) and His272 (7.43) side chains, respectively. The side
chain of Asn250 (6.55) strongly interacted with the 6-amino
group and the adenine N7 atom. Moreover, the adenine ring
was anchored inside the binding site by a π−π stacking
interaction with Phe168 (EL2) and strong hydrophobic
contacts with Leu246 (6.51) and Ile268 (7.39). Substituents
at the N6 (TM5, TM6, and EL2) and C2 positions (TM2 and
EL1) were located in hydrophobic extracellular portions of the
hA3AR binding pocket. While the 5′-N-methyluronamides23,24
were able to form a H-bond with the side chain hydroxyl group
of Thr94 (3.36), truncation prevented an interaction with this
key residue, a putative explanation of the low efficacy profile of
these 4′-truncated nucleosides.
In the 5′-N-methyluronamido series, an enhancement of
A2AAR affinity was observed for 2-(3-chlorophenylethynyl)
analogues, which suggested halogen−π interactions with Tyr9
(1.35) and Tyr272 (7.36) of the A2AAR.23,24 In the present
truncated series, compound 13 contained the same C2-(3-
chlorophenylethynyl) group, and the A2AAR affinity was
enhanced, likely reflecting a common interaction with the
same site on the hA2AAR. However, 2-(3-chlorophenylethynyl)
derivative 20 did not display this characteristic enhancement,
suggesting a reorientation of the bound ligand, likely stemming
from the bulky N6-2,2-diphenylethyl group and the lack of
stabilizing 5′-uronamido interactions.
The N6-methyl C2-phenylethynyl analogue 4 displayed a Ki
value of 5.5 nM in binding to the hA3AR and was nearly
inactive at the hA1AR and hA2AAR with only 18% inhibition of
binding at 10 μM. Therefore, the degree of A3AR selectivity of
4 was estimated to be >2000-fold. A variety of other 4′-
truncated C2-arylethynyl-(N)-methanocarba nucleosides con-
taining N6-methoxy, N6-ethyl, N6-3-chlorobenzyl, N6-2-phenyl-
ethyl, or N6-2,2-diphenylethyl groups displayed a range of high-
to-moderate affinities at the hA3AR and generally with A3AR
selectivity. The hA3AR affinity of these derivatives demon-
strated a freedom of substitution at C2. The smaller groups at
N6 in 4−9 were associated with higher affinity at the A3AR.
Thus, N6-methyl (5, Ki = 3.2 nM) and ethyl (9, Ki = 5.8 nM) in
the set of 2-(2-chlorophenylethynyl) derivatives provided the
highest hA3AR affinity with only weak binding to A1 and
A2AARs, implying a selectivity of ≥1000-fold, and a N6-methoxy
derivative 7 was slightly less potent. In the N6-3-chlorobenzyl
series, the unsubstituted phenylethynyl derivative 10 was the
most potent in binding at the A3AR (Ki = 39 nM). However,
unlike the 5′-N-methyluronamide series, halo substitution in
3,4-difluorophenylethynyl 11 and 2-chlorophenylethynyl 12
analogues significantly reduced A3AR affinity. N6-2-Phenylethyl
substitution in C2-phenylethynyl analogue 15 provided a Ki
value of 20 nM at the hA3AR. A 2-p-biphenylethynyl
substitution was well tolerated at the A3AR in the N6-3-
chlorobenzyl 14 but not N6-(2,2-diphenylethyl) 21 series.
At the hA1AR, only 10−40% of binding inhibition was
typically seen at 10 μM for a variety of substitutions, except for
the 2-chlorophenylethynyl analogue 12 (Ki = 2.7 μM).
However, the variability of affinity upon substitution of the
arylethynyl moiety was greater at hA2AAR than at hA1AR. For
example, three N6-3-chlorobenzyl compounds (10, 11, and 13)
had Ki values in the range of 0.7−1.8 μM at the hA2AAR. As in
the 5′-N-methyluronamide series,23,24 hA2AAR affinity increased
substantially upon replacement with a C2-(3-chlorophenyle-
thynyl) group in 13, leading to only 8.6-fold selectivity in A3AR
binding.
Functional data were determined at a single, saturating
concentration (10 μM) in an assay of hA3AR-induced
inhibition of the forskolin-stimulated production of adenosine
3′,5′-cyclic monophosphate (cyclic AMP) in membranes of
Chinese hamster ovary (CHO) cells expressing the hA3AR
(Table 1).28 Inhibition by 10 μM 5′-N-ethylcarboxamidoade-
nosine (NECA) was set at 100% relative efficacy. The novel
truncated derivatives were generally low-efficacy partial agonists
(e.g., 6, 12, and 16) or antagonists of the hA3AR. Although N6-
ethyl and 2,2-diphenylethyl derivatives tended to be antagonists
of the hA3AR, N6-methyl derivative 4 and N6-methoxy
derivative 6 had significant residual efficacy. The efficacy of
N6-Cl-benzyl and 2-phenylethyl derivatives 10−17 varied
depending on the substitution of the phenylethynyl group.
The highly selective ligands 10 and 15 displayed 14% and 4%
relative efficacy, respectively.
Because of the sterically bulky hydrocarbon group at the C2
position, these compounds display increased hydrophobicity in
comparison to previously described truncated adenosine
analogues. The large differences in steric bulk and hydro-
phobicity with respect to the conventional ribonucleoside series
could influence the pharmacokinetics and the spectrum of
In conclusion, we have found a new series of highly potent
and selective, low-efficacy partial agonists (e.g., 6 and 16) or
antagonists (e.g., 5, 8, 9, and 15) at the A3AR, containing
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varied N6 substitution. The binding affinities demonstrated
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substituents, allowed by a proposed plasticity of the
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ethyl, provided higher A3AR affinity than N6-arylalkyl
substitutuents. This difference contrasts with the analogues
having an additional receptor anchor, that is, the 5′-N-
methyluronamido group in the region of TMs 3 and 7, for
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arylethynyl-(N)-methanocarba adenosine derivatives was
shown to be compatible with retention of A3AR selectivity.
Substitution of the phenylethynyl group modulated the agonist
efficacy (0−30% of full agonism in adenylate cyclase inhibition)
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ASSOCIATED CONTENT
* Supporting Information
■
S
Procedures for chemical synthesis (with selected spectra),
biological assays, and molecular modeling. This material is
available free of charge via the Internet at http://pubs.acs.org.
AUTHOR INFORMATION
Corresponding Author
*Tel: 301-496-9024. Fax: 301-480-8422. E-mail: kajacobs@
helix.nih.gov.
■
Author Contributions
All authors contributed to this manuscript and have given
approval to its final version.
Funding
(14) Zhu, C. B.; Lindler, K. M.; Campbell, N. G.; Sutcliffe, J. S.;
Hewlett, W. A.; Blakely, R. D. Colocalization and regulated physical
association of presynaptic serotonin transporters with A3 adenosine
receptors. Mol. Pharmacol. 2011, 80, 458−465.
This research was supported by the Intramural Research
Program of the NIH, NIDDK.
Notes
(15) Ren, T.; Grants, I.; Alhaj, M.; McKiernan, M.; Jacobson, M.;
Hassanian, H. H.; Frankel, W.; Wunderlich, J.; Christofi, F. L. Impact
of disrupting adenosine A3 receptors (A3−/−AR) on colonic motility or
progression of colitis in the mouse. Inflamm. Bowel Dis. 2011, 17,
1698−1713.
(16) Jacobson, K. A.; Ji, X.-D.; Li, A. H.; Melman, N.; Siddiqui, M. A.;
Shin, K. J.; Marquez, V. E.; Ravi, R. G. Methanocarba analogues of
purine nucleosides as potent and selective adenosine receptor agonists.
J. Med. Chem. 2000, 43, 2196−2203.
(17) Tchilibon, S.; Joshi, B. V.; Kim, S. K.; Duong, H. T.; Gao, Z. G.;
Jacobson, K. A. Methanocarba 2,N6-disubstituted adenine nucleosides
as highly potent and selective A3 adenosine receptor agonists. J. Med.
Chem. 2005, 48, 1745−1758.
(18) Jacobson, K. A.; Siddiqi, S. M.; Olah, M. E.; Ji, X. D.; Melman,
N.; Bellamkonda, K.; Meshulam, Y.; Stiles, G. L.; Kim, H. O. Structure-
activity relationships of 9-alkyladenine and ribose-modified adenosine
derivatives at rat A3 adenosine receptors. J. Med. Chem. 1995, 38,
1720−1735.
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
We thank Dr. John Lloyd and Dr. Noel Whittaker (NIDDK)
for mass spectral determinations.
■
ABBREVIATIONS
■
AR, adenosine receptor; cyclic AMP, adenosine 3′,5′-cyclic
monophosphate; CHO, Chinese hamster ovary; DIAD,
diisopropyl azodicarboxylate; GPCR, G protein-coupled
receptor; HEK, human embryonic kidney; NECA, 5′-N-
ethylcarboxamidoadenosine; TFA, trifluoroacetic acid; TM,
transmembrane helix
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