Truncated (N)-methanocarba nucleosides as A1 adenosine receptor agonists and partial agonists: Overcoming lack of a recognition element

Article abstract of DOI:10.1021/ml200114q

A₁ adenosine receptor (AR) agonists are neuroprotective, cardioprotective, and anxiolytic. (N)-Methanocarba adenine nucleosides designed to bind to human A₁AR were truncated to eliminate 5′-CH ₂OH. This modification previously converted A₃AR agonists into antagonists, but the comparable effect at A₁AR is unknown. In comparison to ribosides, affinity at the A₁AR was less well preserved than that at the A₃AR, although a few derivatives were moderately A₁AR selective, notably full agonist 21 (N⁶- dicyclopropylmethyl, K_i 47.9 nM). Thus, at the A₁AR, recognition elements for nucleoside binding depend more on 5′ region interactions, and in their absence, A₃AR selectivity predominates. Based on the recently reported agonist-bound AR structure, this difference between subtypes likely correlates with an essential His residue in transmembrane domain 6 of A₁ but not A₃AR. The derivatives ranged from partial to full agonists in A₁AR-mediated adenylate cyclase inhibition. Truncated derivatives have more druglike physical properties than other A₁AR agonists; this approach is appealing for preclinical development. This article not subject to U.S. Copyright. Published 2011 by the American Chemical Society.

Full text of DOI:10.1021/ml200114q

LETTER
pubs.acs.org/acsmedchemlett
Truncated (N)-Methanocarba Nucleosides as A1 Adenosine Receptor
Agonists and Partial Agonists: Overcoming Lack of a Recognition Element
Dilip K. Tosh, Khai Phan, Francesca Deflorian, Qiang Wei, 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, United States
S Supporting Information
b
ABSTRACT: A1 adenosine receptor (AR) agonists are neuroprotective, cardioprotective,
and anxiolytic. (N)-Methanocarba adenine nucleosides designed to bind to human A1AR
were truncated to eliminate 50-CH2OH. This modification previously converted A3AR
agonists into antagonists, but the comparable effect at A1AR is unknown. In comparison to
ribosides, affinity at the A1AR was less well preserved than that at the A3AR, although a few
derivatives were moderately A1AR selective, notably full agonist 21 (N6-dicyclopropyl-
methyl, Ki 47.9 nM). Thus, at the A1AR, recognition elements for nucleoside binding
depend more on 50 region interactions, and in their absence, A3AR selectivity predominates. Based on the recently reported agonist-
bound AR structure, this difference between subtypes likely correlates with an essential His residue in transmembrane domain 6 of
A1 but not A3AR. The derivatives ranged from partial to full agonists in A1AR-mediated adenylate cyclase inhibition. Truncated
derivatives have more druglike physical properties than other A1AR agonists; this approach is appealing for preclinical development.
KEYWORDS: G protein-coupled receptor, purines, molecular modeling, radioligand binding, adenylate cyclase
denosine modulates many physiological processes by acti-
vating one or more of four subtypes of G protein-coupled
convert A3AR agonists into selective antagonists in a guanine
nucleotide binding assay.10 Subsequently, partial agonism in a
functional assay of adenosine 30,50-cyclic phosphate (cyclic
AMP) at the Gi-coupled A3AR was shown for MRS5127 (5)
and congeners.11
A
receptors (GPCRs).1 The medicinal chemistry of adenosine
receptors (ARs) is now well advanced in comparison to the
cases of many other GPCRs, with the existence of numerous
selective agonists and antagonists, allosteric modulators, pro-
drugs, radioligands for imaging, fluorescent probes, and macro-
molecular ligand conjugates.2 A1AR ligands have been con-
sidered clinically for a variety of conditions: agonists (diabetes,
pain), partial agonists (arrhythmias), and antagonists (heart failure,
renal protection).3
In contrast to the reduced A3AR efficacy of 50-truncated
nucleosides, at the Gs-coupled A2AAR, full agonism is retained,
as shown recently for the 40-thio series.12 The effects of
truncation on A1AR efficacy are unknown. Our major objective
was to probe the effects of truncated (N)-methanocarba
nucleosides at the human (h) A1AR, both pharmacologically
and with insight into the structural basis for receptor recogni-
tion. Therefore, we have incorporated N6 substituents that are
expected to promote A1AR affinity. For example, nucleoside 6,
previously characterized at the A3AR,12 contains N6-cyclopen-
tyl, which generally produces A1AR selectivity in the riboside
series. The Gi-coupled A1AR is more homologous in primary
sequence and effector coupling to the Gi-coupled A3AR than to
the Gs-coupled A2AAR. If it more closely resembles A3AR in
ligand binding and activation mechanism, the truncated ana-
logues such as 6 will be A1AR antagonists or partial agonists.
However, if its activation more closely resembles the A2AAR,
then these truncated derivatives will be full A1AR agonists.
With the recent structural elucidation of an A2AAR active
state,15 it is feasible to relate these findings to specific binding
site interactions.
The adenosine structure has been extensively modified, on
both the nucleobase and ribose, for pharmacological optimiza-
tion to selectively activate ARs. One means of achieving AR
subtype selectivity has been the replacement of ribose with a
sterically constrained methanocarba ([3.1.0]-bicyclohexane)
ring system. This bicyclic system adopts a North (N)-envelope
conformation in MRS3558 (1) (Chart 1) to maintain a receptor-
preferred conformation and enhanced affinity at the A3AR.6 The
(N)-methanocarba modification is also tolerated at A1AR, but
without affinity enhancement. Consequently, the cardioprotec-
tive MRS3630 (2) containing the A1AR-favoring N6-cyclopentyl
substituent is a mixed A1/A3AR agonist.7
Another useful modification is truncation of the ribose at the
40 position, that is, removing the 50-CH2OH moiety, while
retaining all other features of the ribose-like moiety and its
stereochemistry. Thus, 40-thionucleoside antagonist LJ-1251
(3) and 40-oxo antagonist 4 preserve affinity and selectivity at
the A3AR, while removing the ability to induce the required
conformational change for receptor activation.8,9 Truncation of
(N)-methanocarba nucleosides originally was reported to
Received: May 10, 2011
Accepted: June 1, 2011
Published: June 01, 2011
This article not subject to U.S. Copyright.
Published 2011 by the American Chemical Society
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Chart 1. Structures of Representative Ribosides and Ring-Constrained Methanocarba Nucleoside Derivatives That Have Been
Characterized as Agonist, Partial Agonists, and Antagonists at the A3AR (Ki, nM, in Binding to the hA3AR in Italics)6ꢀ10
’ RESULTS
methanocarba analogues (Ki, in nM) at the hA1AR (with Ki
of the corresponding N6 derivatized 9-ribosides at rat A1AR in
parentheses13) are the following: cyclobutyl 9, 51.6 (0.7);
isopropyl 17, 72.2 (1.9); cyclopropylmethyl 19, 68.4 (0.8);
and di(cyclopropyl)methyl 21, 47.9 (0.8). The A1AR affinity
of the N6-ethyl analogue 15 was more substantially reduced,
by 141-fold, with Ki values of 930 and 6.6 nM for the truncated
and 9-riboside analogues, respectively. Other N6 analogues
that were more significantly reduced in their hA1AR affinity
in comparison to the corresponding riboside at rat A1AR
(Ki, nM) are as follows: cyclohexyl 10, 131-fold (0.9); endo-
norbornyl 13, 113-fold (0.34); exo-norbornyl 14, 231-fold
(0.7); and 2-fluorobenzyl 22, 800-fold (6). Because of this
reduced affinity, the selectivity for the A1AR was diminished
overall. Only a few derivatives tended toward A1AR selectivity
in comparison to the A3AR: di(cyclopropyl)-methyl 21,
10-fold; endo-norbornyl 13, 4-fold; and large cycloalkyl deriva-
tives 10ꢀ12, 2ꢀ3-fold. The degree of selectivity vs A2AAR was
higher: 21, 74-fold; 13, 30-fold. Many of the other derivatives
were equipotent in binding to A1 and A3ARs, and the substituted
N6-benzyladenosine derivatives (22ꢀ28) were generally selec-
tive for the A3AR, similar to previous observations with truncated
N6-benzyl derivatives.10,12
Functional data determined at a single concentration (10
μM) in an assay of adenylate cyclase (A1AR-induced inhibi-
tion of cyclic AMP) are reported in Table 1. The potent and
selective agonist CPA was used as the standard full agonist,
and the nonselective AR agonist 50-N-ethylcarboxamidoade-
nosine (NECA) was also a full agonist in this assay. Most of
the analogues were partial agonists of the A1AR. However,
concentrationꢀresponse curve for dicyclopropylmethyl ana-
logue 21 in A1AR-mediated inhibition of cyclic AMP indi-
cated full agonism compared to NECA (Figure S1 of the
Supporting Information). The EC50 values for 21 and NECA
were 40.7 ( 19.7 and 10.2 ( 3.3 nM, respectively, in close
agreement with the A1AR binding affinities.
With the objective of increasing A1AR affinity, we explored N6
substitution of 40-truncated (N)-methanocarba nucleoside deri-
vatives, which were shown previously to be selective A3AR
antagonists/partial agonists.12 Therefore, the series included
N6-cycloalkyl (8ꢀ12) and N6-bicycloalkyl (13,14), and N6-
acyclic alkyl and N6-cyclopropylalkyl (15ꢀ21) substitutions
associated previously with A1AR selectivity of ribosides
(Table 1).13 Finally, certain substituted N6-benzyladenosine
derivatives, such as 2-fluorobenzyl, were reported to have
enhanced affinity at the A1AR.23 Therefore, a variety of fluori-
nated and nonfluorinated N6-benzyl derivatives (22ꢀ28) were
prepared.
The synthetic route to the truncated derivatives involves
nucleophilic displacement by the appropriate amine of a 6-chlor-
oadenine group in a 20,30-isopropylidene-protected precursor 29
(Scheme S1 of the Supporting Information). All of the analogues
contain a 2-chloro substitution of the adenine ring, which has
been shown to increase affinity at either or both A1AR and A3AR.
2-Chloro substitution of the A1AR agonist N6-cyclopentylade-
nosine (CPA, 30) also was shown to reduce agonist efficacy at
the A3 but not A1AR.14
Several previously reported 2-chloro (N)-methanocarba deri-
vatives (1, 2, 5, and 6) were used for comparison in the biological
assays (Table 1). Binding assays at three hAR subtypes were
carried out using standard radioligands and membrane prep-
arations from Chinese hamster ovary (CHO) cells (A1 and A3)
or human embryonic kidney (HEK) 293 cells (A2A) stably
expressing a hAR subtype (Table 1).12 Since activity within the
class of (N)-methanocarba nucleosides was previously noted to
be very weak or absent at the hA2BAR,16 we did not include this
receptor in the screening protocol.
Generally, the 50-truncated (N)-methanocarba-adenosine
derivatives, in comparison to the corresponding 9-ribosides,
maintained affinity at the A3AR more effectively than at the
A1AR. Nevertheless, affinities of <100 nM at the A1AR were
achieved for certain N6-alkyl and cycloalkyl members of this
series. Among the most potently binding 2-chloro (N)-
A correlation plot of the hA3AR affinity of truncated
2-chloro-(N)-methanocarba analogues in the present study
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LETTER
(x-axis) vs the hA3AR affinity of 2-unsubstituted adenine-7-
riboside analogues (y-axis)13
Compound 22 was 115-fold selective for the A3AR in com-
parison to the A1AR. A 2-fluoroethyl ether 23 was a potent and
selective ligand at the A3AR (Ki 10.3 nM), which might be
sufficient affinity for use in radiofluorination for positron
emission tomography.
There is a general phenomenon of reduced AR efficacy by
removing H-bonding groups from the ribose moiety, espe-
cially at the A3AR but also at the A1AR.5,16 A precedent for
reduced A1AR efficacy in nucleoside derivatives is the series of
xanthine 7-ribosides. The 50-uronamide analogue 50-N-methyl
1,3-dibutylxanthine 7-β-D-ribofuronamide (DBXRM) was a
full agonist at both A1 and A3ARs. However, removal of the 30-
hydroxyl group produced antagonism at the rat adipocytes
A1AR and partial agonism at the rat A3AR. In examining the
question of whether 50-truncation in the present series of rigid
(N)-methanocarba analogues provides agonists or antagonists
at the A1AR, we found that the answer lies in the middle; that
is, the agonist efficacy is partially maintained for many of the
derivatives, as shown in Table 1, but the pattern is not
uniform.
To examine docking of truncated (N)-methanocarba nu-
cleosides, homology models of the hA1AR and hA3AR were
produced from the agonist-bound A2AAR X-ray structure
(3QAK).15 It was noted that key interactions of the nucleoside
ligand with His251 in transmembrane domain (TM) 6, con-
served between A1AR and A2AAR, and with Thr91 in TM3 are
necessarily missing in the truncated derivatives. In the A2AAR
structure, this essential His residue, which is absent in the
A3AR, and Thr91 both H-bonded to a 50-CO-NH-alkyl
moiety of a cocrystallized NECA-like agonist.15 We pre-
dict that the difference in this key His residue is related to
the dramatically altered binding of the truncated ring-
constrained nucleosides at A1 and A3ARs. Thus, in general,
A3AR recognition would depend more on interactions of
groups other than the nucleoside 50 substituent; A1AR recog-
nition depends more on 50 region interactions, and in their
absence, A3AR selectivity predominates. This is consistent
with the truncated derivatives maintaining A3AR, but not
A1AR, affinity.
demonstrated a parallel in these parameters (Figure 1B). The
affinity at the A3AR subtype was relatively well maintained
(correlation coefficient of 0.55). The enhanced A3AR affinity
of a 50-truncated ribonucleoside derivative was first noted for
the 2-chloro-N6-(3-iodobenzyl) derivative16 and has been
validated consistently in SAR studies that also showed low-
ered efficacy in this series.8ꢀ10 However, a similar plot of
A1AR affinity comparing ribonucleosides and truncated ring-
constrained nucleosides illustrated the trend of consistently
lower affinity with truncation (Figure 1A), but without corre-
lation of Ki values (correlation coefficient of 0.33). Therefore,
in comparison to the case of ribosides, the affinity of the
truncated ring-constrained analogues at the A1AR was less
well preserved than that at the A3AR.
One derivative that tended toward A1AR-selectivity, the full
agonist 21, contained a N6-dicyclopropylmethyl group. Cur-
iously, truncated analogues of N6-cyclopentyl and N6-benzyl
derivatives were notably reduced in A1AR affinity, even in the
case of a 2-fluoro analogue 22, a modification previously found
to enhance A1AR selectivity in the riboside series.13
Furthermore, the hydrophilic region of the receptor asso-
ciated with ribose binding is critical for activation. This step
likely involves essential residues of TM3, TM6, and TM7
throughout the AR family, as in the A2AAR.15 Multiple H-
bonding groups in this region promote agonism at the A3AR,14
such as the interactions of the 50-CH2OH of the ribose moiety or
the corresponding 50-CO-NH-alkyl in NECA-like analogues.
Thus, loss of the 50 substituent is expected to reduce AR efficacy,
which it clearly does at the A3AR. However, the effect at the
A1AR is highly variable, with relative maximal efficacies of the
truncated (N)-methanocarba analogues ranging from low
(20ꢀ30% in compounds 14, 16, and 18) to high (80ꢀ100%
in compounds 8, 19, and 21). The structural basis for the full
agonism of 21, in contrast to closely related compounds such as
the N6-(3-pentyl) derivative 18, which has greatly reduced
efficacy at this subtype, must arise from conformational effects
of the N6 group.
The docking poses of nonselective agonist NECA, full
agonist 21, and low efficacy partial agonist 16 in the putative
binding site of the A1AR were compared (Figure 2 and Figure
S2 of the Supporting Information). The H-bond interactions
between the 50-CO-NH-ethyl group of NECA and the two
residues, His251(6.52) and Thr91(3.36), could lock the ribose
Figure 1. (A) Correlation of the affinity of truncated 2-chloro-(N)-
methanocarba analogues (x-axis, at the hA1AR) and 2-unsubstituted
adenine-7-riboside analogues (y-axis, at the rat A1AR).13 (B) Correlation
of the hA3AR affinity of truncated 2-chloro-(N)-methanocarba analo-
gues (x-axis) and 2-unsubstituted adenine-7-riboside analogues
(y-axis).13
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Table 1. Potency of a Series of Truncated (N)-Methanocarba Adenosine Derivatives at Three Subtypes of hARs and Relative
Efficacy at hA1AR
a Using CHO or HEK293 (A2A only) cells stably expressing a hAR (Supporting Information); affinity was expressed as Ki value (n = 3ꢀ5) or percent
inhibition of radioligand binding at 10 μM. b Values from refs 6, 7, and 12. 5 and 6 were prepared previously.12 c 20 is a diastereomeric mixture. d Maximal
efficacy (at 10 μM) in an A1AR functional assay, determined by inhibition of forskolin-stimulated cyclic AMP production in AR-transfected CHO cells,
expressed as percent inhibition (mean ( standard error, n = 3ꢀ5) in comparison to effect (100%) of full agonist CPA 30 at 10 μM. The value for NECA
was 100 ( 15.
moiety in an active conformation, with the 20,30-hydroxyl
groups of the ribose ring correctly directed toward Thr277-
(7.42) and His278(7.43) in order to pull TM7 toward TM3 to
efficiently activate the receptor.15 In the absence of an inter-
action with His251(6.52) and/or Thr91(3.36) due to the lack
of the 50 substituent, the orientation of the rigid methanocarba
moiety could be less effective in forming the H-bond interac-
tions with Thr277(7.42) and His278(7.43) needed to attract
TM7. In the case of A3AR, the activation process could be
slightly different from that for the A1AR, due to some differe-
nces in the key residues of the binding pocket. Position 3.32 in
A3AR consists of a nonconserved bulky and hydrophobic
leucine residue, while a smaller valine is present in the other
AR subtypes. Residue 3.32 was close to the ribose ring of the
docked NECA in both A1AR and A3AR binding sites. The
longer side chain of Leu90(3.32) in A3AR, in comparison to
Val87(3.32) of A1AR, could contribute to a stronger hydro-
phobic interaction with the methanocarba moiety of the
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50-truncated agonists. At the same time, the different nature of
the substituents at the N6 position of the adenine ring could
influence the selectivity and the efficacy of agonists at both the
A1AR and the A3AR. The N6 groups in the docking poses of
the studied agonists lay in a region of the pocket formed by
residues in the upper part of TM6 and TM7. The A1AR and
A3AR differ in the nature of the residues lining this pocket.
The variation of the affinity and potency of this series of 50
truncated methanocarba analogues at the A1AR indicates that
the N6 substituent can greatly affect these parameters, in some
cases compensating for the lack of H-bonding interactions in
the ribose 40 region. From the docking pose of full agonist 21
in the A1AR model (Figure 2B), the favorable interactions of
the N6-dicyclopropylmethyl substituent and the residues in
the upper part of TM6 and TM7 (such as Thr257, Leu253,
and Thr270) could maintain the adenine and methanocarba
moieties in an efficacious active conformation with strong
H-bond and hydrophobic interactions with residues in TM6,
TM3, TM7, and EL2. A smaller and more flexible N6 group,
such as the 3-fluoropropyl substituent of 16, in addition to
the lack of interactions with His251(6.52) and Thr91(3.36),
could negatively affect the ability to fully activate the recep-
tor through conformational affects originating at the upper pocket
of TM6 and TM7 (Figure S3 of the Supporting Informa-
tion).
would favor bioavailability. The molecular weight of 21 of 376
is comfortably within the preferred range. Therefore, by
several criteria, the full agonist of the A1AR 21 is more
druglike than CPA.
The bioavailability of peripherally administered 21 in the
brain, i.e. whether its altered physicochemical properties may
facilitate its passage across the blood brain barrier, is unde-
termined. Many of the efforts to develop A1 agonists have
attempted to limit central nervous system (CNS) penetration
to avoid central mediated side effects, but other envisioned
applications of A1 agonists depend on brain entry. In previous
studies of the activity of A1AR agonists in the CNS, only a
small fraction of a peripherally administered agent crossed the
blood brain barrier.4 However, a similar attempt to alter the
biodistribution by removing the 20-hydroxyl group of CPA did
not enhance brain uptake.4
In conclusion, truncated (N)-methanocarba adenine nu-
cleosides display highly variable degrees of binding affinity and
activation at the hA1AR. Based on the recently reported
agonist-bound AR X-ray structure, this difference between
subtypes likely correlates with an essential His residue in TM6
of A1 but not A3AR. By overcoming the lack of an important
recognition element for receptor binding, i.e., the 50 substi-
tuent, a N6-dicyclopropylmethyl derivative 21 was empirically
identified as a moderately A1AR selective, full agonist. This
is counterintuitive given that many 50-modified analogues
are partial agonists at the A1AR.18 A1AR agonists hold
interest therapeutically for their cardio- and neuroprotective,
antiarrhythmic, antiseizure, antilipolytic, antiglaucoma, and
anxiolytic actions. It is conceivable that the expanded
range of physical properties in the present series of truncated
derivatives would offer pharmacokinetic advantages. There-
fore, this approach is appealing for preclinical development.
This hypothesis will have to be evaluated in the future in vivo
studies.
The physicochemical properties of nucleosides that act as
AR agonists often lead to limited in vivo bioavailability. The
C-log p of compound 21 is 1.41, with the optimal for small
molecular pharmaceutical substances being typically 2ꢀ3.17
The comparable parameter for the related A1AR-selective
riboside and prototypical agonist CPA is 0.14, which is less
desirable. Also, the polar surface area (PSA) values for 21 and
CPA are calculated to be 92.8 and 122 Å2, respectively. Most
druglike small molecules have a PSA smaller than 120 Å2.
Compound 21 has fewer hydroxyl groups than CPA, which
Figure 2. Docking poses of NECA (A) and truncated nucleoside 21 (B) in the binding site of the hA1AR homology model based on the X-ray structure
of an agonist-bound hA2AAR, indicating the main H-bond interactions.
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LETTER
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’ AUTHOR INFORMATION
Corresponding Author
*Molecular Recognition Section, Bldg. 8A, Rm. B1A-19, NIH,
NIDDK, LBC, Bethesda, MD 20892-0810. E-mail: kajacobs@-
helix.nih.gov. Telephone: 301-496-9024. Fax: 301-480-8422.
Funding Sources
The authors thank the Intramural Research Program of the NIH,
NIDDK, for support.
’ ACKNOWLEDGMENT
We thank Prof. Ray Stevens (Scripps Research Institute,
La Jolla, CA) and Dr. Vsevolod Katrich (University of California,
San Diego) for helpful discussions and Dr. Noel Whittaker
(NIDDK) for mass spectral determinations.
’ ABBREVIATIONS
AR, adenosine receptor; cyclic AMP, adenosine 30,50-cyclic phos-
phate; CPA, N6-cyclopentyladenosine; CHO, Chinese hamster
ovary; GPCR, G protein-coupled receptor; HEK, human embry-
onic kidney; MRS3558, (10S,20R,30S,40R,50S)-40-{2-chloro-6-[(3-
chlorophenylmethyl)amino]purin-9-yl}-1-(methylaminocarbonyl)-
bicyclo[3.1.0]hexane-2,3-diol; MRS3630, (10S,20R,30S,40R,50S)-
4-(2-chloro-6-(cyclopentylamino)-9H-purin-9-yl)-2,3-dihydroxy-
N-methylbicyclo[3.1.0]hexane-1-carboxamide; MRS5127, (10S,20R,
30S,40R,50S)-40-[2-chloro-6-(3-iodobenzylamino)-purine]-20,30-
O-dihydroxybicyclo[3.1.0]hexane; NECA, 50-N-ethylcarboxami-
doadenosine; TM, transmembrane domain
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A
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The X-ray structure of NECA bound to a thermostabilized
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