Structure-guided design of A3 adenosine receptor-selective nucleosides: Combination of 2-arylethynyl and bicyclo[3.1.0]hexane substitutions

Article abstract of DOI:10.1021/jm300396n

(N)-Methanocarba adenosine 5′-methyluronamides containing known A₃ AR (adenosine receptor)-enhancing modifications, i.e., 2-(arylethynyl)adenine and N⁶-methyl or N⁶-(3-substituted- benzyl), were nanomolar full agonists of human (h) A₃AR and highly selective (K_i ～0.6 nM, N⁶-methyl 2-(halophenylethynyl) analogues 13 and 14). Combined 2-arylethynyl-N⁶-3-chlorobenzyl substitutions preserved A₃AR affinity/selectivity in the (N)-methanocarba series (e.g., 3,4-difluoro full agonist MRS5698 31, K _i 3 nM, human and mouse A₃) better than that for ribosides. Polyaromatic 2-ethynyl N⁶-3-chlorobenzyl analogues, such as potent linearly extended 2-p-biphenylethynyl MRS5679 34 (K_i hA₃ 3.1 nM; A₁, A_2A, inactive) and fluorescent 1-pyrene adduct MRS5704 35 (K_i hA₃ 68.3 nM), were conformationally rigid; receptor docking identified a large, mainly hydrophobic binding region. The vicinity of receptor-bound C2 groups was probed by homology modeling based on recent X-ray structure of an agonist-bound A_2AAR, with a predicted helical rearrangement requiring an agonist-specific outward displacement of TM2 resembling opsin. Thus, the X-ray structure of related A_2AAR is useful in guiding the design of new A₃AR agonists.

Full text of DOI:10.1021/jm300396n

Article
pubs.acs.org/jmc
Structure-Guided Design of A3 Adenosine Receptor-Selective
Nucleosides: Combination of 2-Arylethynyl and Bicyclo[3.1.0]hexane
Substitutions
Dilip K. Tosh,†, § Francesca Deflorian,†, § Khai Phan,† Zhan-Guo Gao,† Tina C. Wan,‡
Elizabeth Gizewski,‡ John A. Auchampach,‡ 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
‡Department of Pharmacology, Medical College of Wisconsin, 8701 Watertown Plank Road, Milwaukee, Wisconsin 53226, United
States
S
* Supporting Information
ABSTRACT: (N)-Methanocarba adenosine 5′-methylurona-
mides containing known A3 AR (adenosine receptor)-
enhancing modifications, i.e., 2-(arylethynyl)adenine and N6-
methyl or N6-(3-substituted-benzyl), were nanomolar full
agonists of human (h) A3AR and highly selective (Ki ∼0.6
nM, N6-methyl 2-(halophenylethynyl) analogues 13 and 14).
Combined 2-arylethynyl-N6-3-chlorobenzyl substitutions pre-
served A3AR affinity/selectivity in the (N)-methanocarba
series (e.g., 3,4-difluoro full agonist MRS5698 31, Ki 3 nM,
human and mouse A3) better than that for ribosides.
Polyaromatic 2-ethynyl N6-3-chlorobenzyl analogues, such as
potent linearly extended 2-p-biphenylethynyl MRS5679 34 (Ki
hA3 3.1 nM; A1, A2A, inactive) and fluorescent 1-pyrene adduct MRS5704 35 (Ki hA3 68.3 nM), were conformationally rigid;
receptor docking identified a large, mainly hydrophobic binding region. The vicinity of receptor-bound C2 groups was probed by
homology modeling based on recent X-ray structure of an agonist-bound A2AAR, with a predicted helical rearrangement requiring
an agonist-specific outward displacement of TM2 resembling opsin. Thus, the X-ray structure of related A2AAR is useful in
guiding the design of new A3AR agonists.
(often m-substituted benzyl groups) and on the ribose moiety
(often 5′-N-methyluronamide), such as occur on prototypical
agonists 1 and 2 (Chart 1).13 Replacement of the conforma-
tionally flexible ribose tetrahydrofuryl group with a rigid
bicyclo[3.1.0]hexane (methanocarba) ring system in an
isomeric form that enforces a North (N) envelope
conformation increases both the binding affinity and selectivity
of adenosine derivatives as A3AR agonists.14−16 For example,
analogues that combined N6-(3-halobenzyl) and (N)-meth-
anocarba modifications, such as 3 and 4, display anti-
inflammatory activity, protection in a model of lung injury,
and protection against chemotherapy-induced neuropathic
pain.17−19 Cristalli and co-workers have explored the SAR
leading to enhancement of A2AAR and A3AR affinity by alkyn-2-
yl groups, such as hexyn-2-yl, at the adenine C2 position of
adenine 9-ribosides.20 The enhancement of human (h) A3AR
affinity by such groups was also observed in the (N)-
methanocarba series.21 Because ligand affinity at the A3AR is
often dependent on species, it was particularly important to
INTRODUCTION
■
The structure−activity relationship (SAR) of nucleoside
agonists at the A3 adenosine receptor (AR), a G protein-
coupled receptor (GPCR) of the rhodopsin-like family A, has
been extensively explored.1 Several A3AR-selective agonists are
currently in advanced clinical trials for the treatment of
hepatocellular carcinoma, autoimmune inflammatory dis-
eases,2−4 such as rheumatoid arthritis, psoriasis, and dry eye
disease, and other conditions.5−8 Other potential applications
of such agonists could be osteoarthritis, Crohn’s disease,
ischemia, and other inflammatory disorders. As applied to
cancer models, there is evidence that multiple modes of benefit
from A3AR agonists are obtained: myeloprotection and
reduction of neuropathic pain, in addition to the antiprolifer-
ative effects on tumors in vivo. A3AR-selective antagonists are
also of interest for therapeutic applications in asthma,
glaucoma, septic shock, and other conditions.9−12 Thus, this
subtype of AR (others are A1AR, A2AAR, and A2BAR) has
provided numerous opportunities for translation to therapeu-
tics.
In order to achieve selectivity of adenosine derivatives for the
Received: March 21, 2012
Published: May 4, 2012
A3AR, modifications are typically introduced on the N6 position
© 2012 American Chemical Society
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study establishes that these two substitutions are fully additive
and conducive to high selectivity when applied to (N)-
methanocarba adenosine derivatives. We have also used
molecular modeling based on a recent X-ray structure of an
A2AAR complex with a 5′,2,N6 trisubstituted agonist25,26 and
with insights from other GPCRs26 to probe the vicinity of the
2-arylethynyl groups when docked to the A3AR. Thus, the
crystallographic structure of one AR subtype is used to guide
the design of new analogues at another AR subtype following
necessary customization for this ligand set.
Chart 1. Derivatives of Adenosine as A3AR-Selective
Agonists in the Ribose (1,2) and (N)-Methanocarba (3,4)
Series
RESULTS
■
Chemical Synthesis. We have explored 2-arylethynyl
substitution of the adenine ring of previously reported (N)-
methanocarba 5′-N-methyluronamide nucleoside derivatives
that act as selective A3AR agonists.14,15,21 Two series,
depending on the N6 substitution (N6-methyl, 8−26, and N6-
3-chlorobenzyl, 27−36), that are closely patterned on known
potent agonists are included (Scheme 1). N6-Methyladenosines
are typically significantly more potent at the hA3AR than at rat
(r) A3AR, while N6-(3-halobenzyl)adenosines tend to display
greater species-independent selectivity.
The synthetic route used to prepare (N)-methanocarba 5′-N-
methyluronamido derivatives containing a 2-arylethynyl group
involved a key Sonogashira reaction27 at a 2-iodoadenine
moiety of intermediate 43 or 44 (Scheme 1). L-Ribose 38 was
converted as previously reported into the 2′,3′-protected
establish the effect in species other than humans. The
combination of N6-(3-halobenzyl), 2-alkyn-2-yl, and 5′-N-
methyluronamide modifications was found to be particularly
well suited for application to agonists displaying A3AR
selectivity across species.
There is a precedent for the lack of compatibility in AR
recognition of multiple modifications of nucleoside derivatives
at the N6 (i.e., 3-halobenzyl) and C2 positions (i.e., aryl or large
hydrophobic groups), each of which may promote affinity at
A3AR and other subtypes.13,22 There is also indication that N6-
3-(substituted benzyl) and 2-arylethynyl (introduced by
Cristalli and colleagues23) modifications are not additive in
their effect on A3AR selectivity of 9-ribosides.24 The present
a
Scheme 1. Synthesis of (N)-Methanocarba Nucleoside Analogues
a
R1 = Me or 3-Cl-benzyl. Reagents: (i) MeNH2 or 3-Cl-benzylamine, Et3N, MeOH, rt; (ii) 40% MeNH2, MeOH, rt; (iii) HCCAr, Pd(PPh3)2Cl2,
CuI, Et3N, DMF, rt; (iv) 10% TFA, MeOH, 70 °C; (v) 1-ethynylpyrene or 4-ethynylpyrene 51, Pd(PPh3)2Cl2, CuI, Et3N, DMF, rt; (vi) 1 N, HCl,
dioxane, 60°C.
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intermediate 39 containing a 5-ethyl ester, which was then
subjected to a Mitsunobu condensation with 2-iodo-6-
chloropurine to give 40.21,28 A N6-methyl or N6-(3-
chlorobenzyl) group was added by nucleophilic substitution
of 6-chloro at room temperature to provide intermediates 41
and 42, respectively, followed by aminolysis of the ester at
room temperature leading to 5′-N-methyluronamides 43 and
44. A Sonogashira reaction was then carried out with a variety
of commercially available arylacetylenes to give protected
nucleosides 45−46 (N6-methyl) and 47a−f (N6-3-chloroben-
zyl). Finally, acid hydrolysis of the isopropylidene protecting
group provided N6-methyl 9−26 and N6-3-chlorobenzyl 27−36
nucleosides. The associated synthesis of a polyaromatic ethynyl
intermediate 51 is shown in Scheme 2.
hA3AR. The presence of a branched p-t-Bu group in 22 reduced
A3AR binding affinity by 12-fold compared to the H analogue
9. The most potent A3AR ligands in the N6-methyl series were
halo-substituted compounds 13 and 14 with Ki values of 0.5−
0.6 nM. A p-acetyl substitution in 23 was lower in hA3AR
affinity and higher in hA2AAR affinity than most other ring
substitutions, unlike in the riboside series of Cristalli and co-
workers,23a in which a 2(-p-acetylphenylethynyl) group favored
A3AR affinity and selectivity.
The variability of binding affinity upon substitution of the
arylethynyl moiety was greater at the hA2AAR than at the
hA1AR. For example, the hA2AAR affinity increased substan-
tially to a Ki value of 1.3 μM upon replacement of 3-F 12 with
3-Cl in 15. In contrast, the same change slightly decreased
hA3AR affinity. At the hA1AR, only 10 to 30% of binding
inhibition was typically seen at 10 μM for a variety of
substitutions.
Scheme 2. Synthesis of an Intermediate Arylalkynyl
a
Derivative
The SAR was extended by the analysis of 11 analogues
prepared in the N6-(3-chlorobenzyl) series (27−36). The effect
in 27 of adding a phenyl group to the ethynyl substituent of the
simpler acetylene derivative 6 was complete retention of the
affinity at hA3AR and a reduction of the hA1AR affinity from Ki
= 174 nM to >10 μM, thus providing high selectivity.
Furthermore, with only marginal binding of 27 at the
hA2AAR, the hA3AR selectivity was roughly 10,000-fold in
comparison to both hA1AR and hA2AAR. For comparison, by
adding a n-propyl chain in 7 rather than a phenyl ring, the
hA1AR affinity was reduced only 6-fold in comparison to 6, and
the hA2AAR affinity appeared to be increased. Thus, a flat aryl
ring rather than a flexible alkyl chain provides the appropriate
geometry for hA3AR selectivity. A potent A3AR ligand 34 in the
N6-(3-chlorobenzyl) series containing a rigid, linearly extended
2-p-biphenylethynyl group served as a model ligand for
receptor docking due to its steric and conformational
constraints. It displayed a Ki value at the hA3AR of 3.06 nM
and exceptionally high selectivity in comparison to the hA1 and
hA2AARs (no inhibition observed). The hA3AR affinities of
biphenyl derivative 34 and the corresponding N6-methyl
analogue 24 were equal. The affinity of the pyrene adducts
35 and 36 depended on the position of substitution. Thus, the
combination of 2-arylethynyl, 5′-N-methyluronamide, and N6-3-
chlorobenzyl substitution preserved the hA3AR selectivity of
(N)-methanocarba nucleosides, even for large 2-arylethynyl
moieties.
Species differences in the binding affinity of the nucleoside
derivatives were explored. Nanomolar A3AR affinity was
maintained in a murine species only for the N6-(substituted
benzyl) series (Figure 1). The N6-methyl-2-arylethynyl
derivatives 13 and 14 were determined to be ∼70-fold weaker
at the mA3AR than at the hA3AR, consistent with the previously
reported reduced affinity at mA3AR of N6-methyl-2-Cl
derivative 8.21 The mA3AR/mA1AR selectivity in the N6-
methyl series was enhanced with the elongation and rigid-
ification of the C2 substituent. However, with N6-3-
chlorobenzyl substitution, elongation at C2 did not significantly
reduce affinity at the mA3AR compared to hA3AR (except for a
3-fold reduction for the elongated biaryl derivative 34). In the
N6-3-chlorobenzyl series, particularly high selectivity compared
to mA1AR and mA2AAR was generally present. There was
greater variability in the degree of binding inhibition at mA1AR
than at mA2AAR. Elongation of a 2-ethynyl group in 6 with a
straight alkyl chain in 7 reduced mA3AR affinity by 7-fold, but
elongation with a phenyl ring in 27 only slightly reduced
a
(i) HCC-TMS, Pd(PPh3)2Cl2, CuI, Et3N, DMF, rt; (ii) TBAF,
THF.
Pharmacological Activity. Radioligand binding assays at
3
three hAR subtypes were carried out using standard H (52,
53) and 125I-labeled (54) nucleosides (Table 1).29−31 The
membrane preparations were obtained from Chinese hamster
ovary (CHO) cells (A1 and A3) or human embryonic kidney
(HEK293) cells (A2A) stably expressing a hAR sub-
type.28,29,32,33 A3AR binding curves generally showed a Hill
coefficient of ∼1. The nucleoside analogues were not all
screened for activity at the hA2BAR because other (N)-
methanocarba nucleosides were previously noted to be very
weak or inactive at that subtype.15 For example, in cyclic AMP
assays, agonist 3 displayed 30,000-fold selectivity for the hA3AR
in comparison to the hA2BAR.17 Some previously reported 2-
chloro and 2-alkynyl (N)-methanocarba agonists (3−8) were
used for comparison in the biological assays.20 For exploring
species differences in AR ligand recognition, binding assays
were also performed on selected nucleoside derivatives at three
mouse (m) ARs (Table 2) expressed in HEK293 cells.21,33b
The simplest N6-methyl 2-phenylethynyl analogue 9
displayed a subnanomolar Ki value at the hA3AR and was
nearly inactive at the hA1AR and hA2AAR, with <20% inhibition
of binding at 10 μM. Therefore, the degree of A3AR selectivity
of 9 was estimated to be >10,000-fold. The effects of phenyl
modification of the C2-arylethynyl group at the C2 position
were explored initially in the N6-methyl 5′-N-methyluronamide
series. These derivatives displayed great freedom of substitution
of the arylethynyl moiety, i.e., aza substitution of a CH of
phenyl (10), monohalo (11−17) and dihalo (19, 20) or other
(18, 21−23) substituent groups on the phenyl ring, and the
presence of additional aryl rings (24−26). The 3,4-difluor-
ophenyl analogue 19 was particularly A3AR-selective with
insignificant inhibition at A1 and A2AARs. Planar polyaromatic
groups, such as α-naphthyl 25 and the larger phenanthrene 26,
in the N6-methyl series did not interfere with the binding to the
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Table 1. Binding Affinity of a Series of (N)-Methanocarba Adenosine Derivatives at Three Subtypes of hARs and the Functional
Efficacy at the hA3AR
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Table 1. continued
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Table 1. continued
a
All experiments were done on CHO or HEK293 (A2A only) cells stably expressing one of three subtypes of the four hARs. The binding affinity for
A1, A2A, and A3ARs was expressed as Ki values (n = 3−5) and was determined by using agonist radioligands ([3H]N6-R-phenylisopropyladenosine 52
(R-PIA), [3H]2-[p-(2-carboxyethyl)phenyl-ethylamino]-5′-N-ethylcarboxamidoadenosine 53 (CGS21680), or [125I]N6-(4-amino-3-iodobenzyl)-
adenosine-5′-N-methyl-uronamide 54 (I-AB-MECA), respectively), unless noted.29−31 The percent value in parentheses refers to inhibition of
b
radioligand binding at 10 μM (n = 3). ND, not determined. Unless noted, the efficacy at the hA3AR was determined by inhibition of forskolin-
stimulated cAMP production in AR-transfected CHO cells.32−34 At a concentration of 10 μM, in comparison to the maximal effect of 5′-N-
c
ethylcarboxamidoadenosine 48 (=100%) at 10 μM. Data are expressed as the mean standard error (n = 3). Values from Lee et al. and Tchilibon
d
e
et al.15,16 Values from Melman et al.21 The relative efficacy (10 μM) at the hA2BAR (cyclic AMP accumulation) was 20−30% of full agonist 48.
mA3AR affinity. The most potent derivative at the mA3AR was
the p-aminophenyl analogue 32 with a Ki value of 0.82 nM.
Curiously, although this derivative remained selective, its A1AR
affinity was enhanced over other members of the series at both
in mouse (Ki 261 nM) and human (Ki 1520 nM).
Table 2. Binding Affinity of a Series of (N)-Methanocarba
Adenosine Derivatives at Three Subtypes of mARs
a
affinity (Ki, nM) or % inhibition
compd
mA1
mA2A
mA3
b
3
15.3 5.8
7.32 1.5
45.6 7.9
1390 430
55.3 6.0
(29 2%)
(55 5%)
(50 5%)
(65 3%)
(51 12%)
(35 3%)
(14 3%)
261 19
10,400 1,700
5,350 860
(41%)i
1.49 0.46
0.80 0.14
0.85 0.08
6.06 1.21
49.0 3.9
37.7 1.1
37.2 2.0
1.23 0.14
2.38 0.04
2.64 0.22
2.39 0.38
3.08 0.23
0.82 0.06
3.66 0.25
10.8 0.93
47.6 4.6
Functional data were determined in an assay consisting of
hA3AR-induced inhibition of the production of adenosine 3′,5′-
cyclic phosphate (cAMP) in membranes of CHO cells
expressing the hA3AR (Table 1).34 Inhibition by 10 μM
NECA (48, 5′-N-ethylcarboxamidoadenosine) was set at 100%
relative efficacy. The novel (N)-methanocarba 5′-N-methylur-
onamide derivatives at 10 μM were predominantly full agonists
at the A3AR, with a few analogues showing relative efficacy of
80% or less (p-fluoro 13, t-butyl-phenyl 22, and 1-pyrene 35
derivatives). A full concentration−response curve in hA3AR-
mediated inhibition of adenylate cyclase for 3,4-difluoro
analogue 31 provided an EC50 value of 1.2 0.7 nM (Figure
2), i.e., slightly more potent than its binding affinity. Although
weak in binding, the pyren-4-yl derivative 36 was fully
efficacious.
b
4
b
6
b
7
(42%)i
b
8
20,400 3,200
(0%)
13
14
27
28
29
30
31
32
33
34
35
(2 1%)
(2 1%)
(7 2%)
(19 3%)
(55%)
(27 2%)
(5 2%)
(22%)
(18 3%)
(41 6%)
(8 2%)
(6 1%)
(64%)
Molecular Modeling. A homology model of the hA3AR
based on an agonist-bound hA2AAR X-ray structure (PDB code
3QAK)25 was used to study the putative interactions between
the C2-substituted agonist 34 and the A3AR. The binding mode
of 34 obtained after Induced Fit Docking (IFD)35 revealed the
following binding interactions of the ligand (Figure 3). The key
a
Competition radioligand binding assays using [125I]54 (A1 and
A3ARs) and [3H]53 (A2AAR) were conducted with membranes
prepared from HEK293 cells expressing recombinant mA1, A2A, or
A3ARs. The data (n = 3−4) are expressed as Ki values. The percent
value in parentheses refers to the inhibition of radioligand binding at
10 μM. Values from Melman et al.21
b
Figure 2. Functional agonism tested in an assay of adenylate cyclase in
membranes of CHO cells expressing hA3AR. Activity of the 2-(3,4-
difluorophenylethynyl)-N6-3-chlorobenzyl-5′-N-methyluronamide-
(N)-methanocarba analogue 31 (EC50 value 1.2 0.7 nM). The full
agonist 5′-N-ethyluronamidoadenosine 48 was included for compar-
ison (representing 100% efficacy). The experiment was repeated three
times, and the average is shown.
Figure 1. Inhibition of binding of the radioligand [125I]54 (0.3 nM) at
the mA3AR by compounds 13, 31, 32, and 34. Ki values are found in
Table 2.
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Figure 3. Binding mode (two different views with (A) wire helices and (B) tube helices) of linearly extended N6-3-chlorobenzyl analogue 34
obtained after IFD to the A3AR homology model. The template was built based on the crystal structure of an agonist-bound A2AAR (purple
helices)25 and adjusted for movement of TM2 based on other templates (β2-adrenergic receptor, brown helices; opsin, green helices).40,41 The most
successful template for TM2 was the structure of opsin.
residues embedding the adenosine moiety of the agonists in the
receptor binding site are mostly conserved among AR subtypes.
The 3′- and 2′-hydroxyl groups were located in proximity to
Ser271 (7.42) and His272 (7.43), respectively, and could form
H-bonds with these residues. The NH group of the 5′-N-
methylcarboxamido moiety was involved in H-bonding with the
side chain hydroxyl group of Thr94 (3.36). Both the 6-amino
group and the N7 atom of the adenine ring H-bond with
Asn250 (6.55). A π−π interaction was observed between the
adenine ring of 34 and Phe168 (EL2), while the side chains of
Leu246 (6.51) and Ile268 (7.39) offered CH-π interactions to
the adenine moiety of 34. The ligand−receptor interactions
observed in the present model were in good agreement with
the data of site-directed mutagenesis and with our previously
published models of ARs, including the studies of AR agonists
docked to the A2AAR crystal structure.36−38 In the model
obtained, the 3-chlorobenzyl ring of docked 34 was located in
the hydrophobic pocket formed by Val169 (EL2), Met174
(5.35), Met172 (5.33), Ile253 (6.58), and Leu264 (7.35). More
precisely, the N6-3-chlorobenzyl substituent of 34 was locked in
the hydrophobic pocket of the A3AR among TM5, TM6, TM7,
and EL2 by CH-π interactions with the side chains of Val169
(EL2) and Ile253 (6.58). Ile253 (6.58) is not conserved among
the ARs: in A1, A2A, and A2B, the residue at position 6.58 is a
smaller threonine. Moreover, favorable interactions between
the backbone group of Met172 (5.33) and the chloro atom
stabilized the compound in the binding site.
The arylethynyl substituent at the C2 position of 34 was
oriented toward the extracellular part of the A3AR in close
proximity to TM2. A2AAR is characterized by four constraining
disulfide bridges in the extracellular domains, unlike the A3AR,
which has only one disulfide bond in that region. Thus, we
expected the A3AR to be more subject to reorganization of the
TMs. The disulfide bond between Cys77 (3.25) and Cys166
(EL2) is conserved not only among the AR subtypes but also
among the family A GPCRs, and it is crucial for the expression
and the function of the receptors. This disulfide bond involving
Cys166 holds in place the backbone of two neighboring EL2
residues that are important in ligand coordination, i.e., Gln167
and Phe168. Two other disulfide bridges of A2AAR are between
EL1 and EL2, namely, between Cys71 and Cys159 and
between Cys74 and Cys146. Another disulfide bond involves
the two cysteines in EL3, Cys259, and Cys262. It has been
speculated that the presence within the extracellular domains of
the three disulfide bridges unique to A2AAR is not crucial for
the tertiary structure and the stability of the receptor but is
indeed important for ligand recognition.39 From the structural
point of view, from a comparison between the A2AAR structure
and other GPCR structures, the presence of the two disulfide
bridges between EL1 and EL2 forced the extracellular terminal
of TM2 toward the TM bundle, thus reducing the size of the
pocket embedding the agonist C2-substituents.
The IFD procedure applied to these rigid, extended A3AR
agonists at the A2AAR-based model of A3AR was unable to
accommodate the long and straight arylethynyl substituent at
the C2 position due to a steric clash with the TM2 residues. In
particular, the phenyl ring of the N6-methyl 2-phenylethynyl
derivative 9, a high affinity agonist at A3AR, was clearly clashing
with Ser73 in TM2, suggesting that the orientations of the
extracellular terminal of TM2 and the EL1 in the A2AAR-based
model of A3AR were not optimal for the accessibility of these
methanocarba derivatives to the A3AR binding site.
New hybrid models of the A3AR (Figure 3) were built using
different templates for the homology modeling of the
extracellular part of TM2, namely, an agonist-bound human
β2 adrenergic receptor crystallographic structure (designated
A3AR-β2adr)40 and the structure of the opsin in the activated
state (designated A3AR-ops).41 In the hybrid A3AR model
based on both the A2AAR and the β2-adrenergic receptor
structures, the extracellular extremity of TM2 moved outward
by about 4 Å at the Cα atom of Ser73. This movement created
a larger pocket for the C2 arylethynyl substituents of the A3AR
agonists, thereby allowing derivatives such as agonist 9 to dock
in the binding cavity without steric clashes with TM2.
Nevertheless, the docking of the N6-methyl 2-biphenylethynyl
agonist 24 to the binding site of the A3AR-β2adr model did not
produce any reasonable pose due to the steric clash between
the distal phenyl ring of the compound and the residues in
TM2. The unfit docked pose of 24 to the A3AR-β2adr model of
A3AR suggested that a larger pocket for the longer C2-
substituted agonists was needed.
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The outward movement of the extracellular terminal of TM2
in the hybrid A3AR-ops model was by approximately 7 Å at the
Cα of Ser73, with the creation of a larger pocket for the
accessibility of C2 substituents of the present rigid chain-
extended A3AR agonists, such as compounds 24 and 34. The
docking poses of 24 and 34 in the A3AR-ops model showed the
biphenylethynyl moiety of the C2 chain pointing toward the
extracellular environment. The dihedral angle between the two
phenyl rings was approximately 30−40°, making the C2 chain
noncoplanar with the ethynyl moiety. The biphenylethynyl side
chain was stabilized by favorable hydrophobic interactions with
residues in TM2, EL2, and TM7, namely, Ile268 (7.39), Tyr265
(7.36), Val72 (2.64), Leu264 (7.35), Phe168 (EL2), and the
carbon chain of Gln167 (EL2). The side chain of Gln167, after
the IFD optimization, pointed away from the binding cavity,
opening the cavity to the agonists. The docking pose of 34
showed the distal phenyl ring of the C2 chain between the
carbon chain of Gln167 and the aromatic ring of Tyr265.
Tyr265 is in the wall of the binding pocket, but π interactions
with the ligand were not evident. Analogue 22, which deviated
from planarity because of the branched t-Bu group, was
significantly less potent in hA3AR binding. The presence of a
halogen atom at the o-, m-, or p-positions of the phenyl ring in
the C2 chain was studied in the binding sites of A3AR models
and the A2AAR crystal structure in order to understand the gain
of affinity of compounds 15 and 30 at the A2AAR. The m-Cl
atoms of the C2 side chain of 15 and 30 in the predicted
orientations in the A3AR-ops model were located between
Tyr271 and Gln167 (EL2), locked in the pocket by interactions
between the halogen atom of the compounds and the OH
group of Tyr271 and the side chain CO group of Gln167. The
corresponding residue of Gln167 in human A2AAR is the
hydrophobic and bulky Leu167. Nevertheless, in the docked
poses of compounds 15 and 30 in the binding site of A2AAR,
the m-Cl atom was able to generate strong interactions with the
hydroxyl groups and the aromatic moieties of Tyr271 (7.35)
and Tyr9 (1.35) of A2AAR. Instead, the orientation of the o-Cl
atom of the docked agonists 14 and 29 was not optimal to
create interactions with the side chain of Tyr271 (7.35) of
A2AAR. However, in the docked complexes of 14 and 29 with
the A3AR-ops model, the o-Cl atom was located between
Tyr265 (7.35) and Val169 (EL2), creating favorable
interactions with the OH group of Tyr265 and the backbone
NH group of Val169. The residue corresponding to Val169 in
the A2AAR is Glu169, whose side chain was oriented such that
the γ-carboxyl group interacted with the backbone NH group,
i.e., no longer available to interact with the o-Cl atom of 14 or
29.
agonists by Melman et al.,21 but we have introduced this
modification in the present study.
Previously, in the ribose-5′-uronamide series, combination of
2-phenylalkynyl groups with various bulky N6 substituents was
not additive in its effect on A3AR affinity. In a series of A3AR-
selective N6-arylurea derivatives, a 2-phenylalkynyl group
reduced the rA3AR affinity in comparison to a 2-chloro
analogue containing the same bulky N6-arylurea, although
selectivity was increased.42 The combination of N6-(substituted
benzyl) groups and a 2-phenylethynyl modification produced
much lower affinity in hA3AR binding than the same structure
with H at the C2 position.24 A study of 2-pyrazolyl-N6-
substituted adenosine derivatives concluded that bulky
substitutions at those two positions generally did not benefit
from additivity in hA3AR binding affinity.13c
We have now explored the effects of various 2-(arylethynyl)
groups at the adenine C2 position on AR affinity of (N)-
methanocarba 5′-N-methyluronamido nucleosides. The result-
ing analogues (both N6-methyl and N6-3-halobenzyl) were full
agonists of the hA3AR of nanomolar affinity that were
consistently highly selective (typically >1000-fold vs hA1AR
and hA2AAR). The most potent and selective N6-methyl
compounds were p-F 13 and o-Cl 14 analogues. At the mA3AR,
selectivity generally remained, but for N6-methyl derivatives, the
high affinities achievable were somewhat lower than those at
hA3AR. Thus, the combination of a 2-(arylethynyl) group and
large N6 substitutions was better tolerated at the A3AR in the
methanocarba series than in the 9-riboside series, as
characterized in earlier reports. There is also a broad flexibility
of substitution of the 2-(arylethynyl) moiety, with halo,
hydrophobic, and hydrophilic substitutions without losing
A3AR selectivity. This feature could also benefit specific
pharmacological characteristics, such as pharmacokinetic
properties. The 3,4-difluoro substitution of 31 might impede
possible in vivo metabolic transformation of this ring (cf. P2Y12
receptor antagonist Ticagrelor1b).
The pharmacokinetic properties of these analogues have not
been measured. However, they represent an increase in
hydrophobicity, as well as selectivity, in comparison to most
widely used A3AR agonists. For example, the cLog P values of
potent 2-chlorophenylethynyl 29, 4-fluorophenylethynyl 30,
and 3,4-fluorophenylethynyl 31 derivatives are 4.65, 4.08, and
4.15, respectively. The cLog P values of more polar ribosides 1
and 2 are 0.48 and 1.20, respectively, which is possibly less
desirable for full bioavailability. The total polar surface area of
29 is 121.9 Å2, which is a more favorable value for one of the
physical parameters that predicts drug-likeness compared to
131.1 Å2 for both 1 and 2.43
Even large, planar polyaromatic groups at C2 (separated by
an acetylene moiety) were tolerated and retained nanomolar
affinity. In binding to the A2AAR, a 2-(2-(naphth-1-yl)-
ethyloxy) analogue bound with high affinity, and the location
of the naphthyl group was predicted by docking.44 In the
present study, the size of the polycyclic C2 groups, i.e., in the
potent phenanthrene-ethynyl derivative 26, exceeded that of all
previous AR ligands. The 1-pyrene derivative 35 and its less
potent 4-pyrene isomer 36 represent different sites of
attachment of the 2-ethynyl moiety to the same large
polyaromatic group. In receptor binding experiments, differ-
ences were observed that could provide insight into the
geometry of the binding site considering the spatial and
conformational constraints of these agonists. The 4-pyrene
DISCUSSION
■
In previous studies,13 the SAR in AR binding was studied for
two series of ribonucleosides that are particularly suited for
selectivity at the hA3AR: N6-methyl and N6-3-halobenzyl. N6-3-
Halobenzyl substitution is greatly favored for maintaining the
selectivity of ribosides at the A3AR in mouse and rat. Upon
addition of the (N)-methanocarba modification,21 a rise in
affinity was observed at the mA1AR for certain 2-chloroadenine
analogues, leading to a reduction of AR selectivity in murine
species. However, straight chain alkynyl groups at the C2
position, such as pentynyl 7, alleviated this problem by reducing
affinity at the mA1AR. Substitution with 2-arylethynyl groups
was not examined in the (N)-methanocarba series of A3AR
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Article
derivative 36 (with one additional ring added) has a close
analogue in the N6-methyl series, i.e., 26.
could be differential effects on multiple signaling pathways and
might eventually provide a rational basis for the design of
biased GPCR agonists.
These observations were subjected to molecular modeling
analysis, which also predicted useful analogues to be
synthesized. The ribose moiety is tightly anchored through a
H-bond network involving TM3 and TM7, and the geometry of
the unnatural glycosidic bond to the nucleobase is restricted.
Thus, there is little angular flexibility of the extended, rigid C2
substituents. Thus, anchoring of the adenosine moiety to
conserved amino acid residues provides a framework for
exploring the region surrounding the linearly extended C2
substituent. The placement of a 2-arylethynyl substituent in the
hA3AR binding site was predicted by Dal Ben et al. using the
inactive structure of the hA2AAR as template, and the
orientation is similar to that found here.23b However, the
present study predicts the docking mode with greater detail and
confidence because the homology model is based on the
agonist-bound A2AAR structure.25 With the present set of
rigidified analogues, we have located a very large hydophobic
pocket on the receptor that depends on an outward
diplacement of TM2 in order to accommodate multiple fused
rings. Thus, the binding would be allowed by the plasticity of
the receptor structure to attain a ligand-specific reorganization.
The reduction of hA3AR affinity for the larger C2 substituents
in 35 and 36 suggests that although TM2 could shift position to
accommodate such groups, there is an energetic cost for these
more bulky planar polyaromatic groups. As the movement of
TM2 increases, some stabilizing interactions with other regions
of the receptor would be progressively lost.
The A3AR homology model built based exclusively on the
crystal structure of an agonist-bound A2AAR required an
adjustment of the position of TM2, which was based on the
orientation of TM2 in other agonist-bound or activated GPCR
templates, i.e., the β2-adrenergic receptor or opsin. The most
successful template for TM2 was the structure of opsin, which
displaced the upper part of TM2 relative to the agonist-bound
A2AAR by ∼7 Å. Thus, we predict an outward displacement of
TM2 similar to the opsin structure, which is specific for
agonists containing rigid C2 extensions. The inability of the
A2AAR to undergo this rearrangement might contribute to the
A3AR selectivity of these compounds.
The displacement of one or more TMs to accommodate a
sterically bulky ligand, assuming that there are no other
constraints on the helix such as a proximal disulfide bond,
might be a general phenomenon in GPCRs. The ability of the
helices to readjust position or “breathe” to enable a larger
ligand to bind has already been proposed, specifically with
respect to the “multi-conformational space of the antagonist-
like state of the human A3 receptor”.45 The example of altered
A2AAR conformation specific to the binding of a sterically
extended agonist 6-(2,2-diphenylethylamino)-9-
((2R,3R,4S,5S)-5-(ethylcarbamoyl)-3,4-dihydroxytetrahydrofur-
an-2-yl)-N-(2-(3-(1-(pyridin-2-yl)piperidin-4-yl)ureido)ethyl)-
9H-purine-2-carboxamide (59, UK-432097) was already
documented in the X-ray structure.25 In particular, outward
movements of EL3 and extracellular portion of TM7 were
associated exclusively with this bulky, multifunctionalized
agonist.
The enhancement of A2AAR affinity in the 2-(3-chlorophe-
nylethynyl) analogues 15 and 30 indicated a consistent
interaction with a specific site on the hA2AAR. Modeling
analysis suggested halogen−π interactions46 with two tyrosine
residues in the C2 binding cavity of A2AAR, namely, Tyr271
(7.36) and Tyr9 (1.35), to explain the activity of the 2-(3-
chlorophenylethynyl) methanocarba adenosine derivatives 15
and 30 at the A2AAR.
The freedom to insert polyaromatic ring systems on the 2-
ethynyl group suggests inclusion of reporter groups, such as
fluorescent dye moieties at this site. In fact, the pyren-1-yl
analogue 35 is highly fluorescent and could be explored as
spectroscopic probes of moderate affinity for receptor binding
experiments. The fluorescent properties of pyrene are sensitive
to the environment. The p-amino derivative 32 has a high
affinity at both the mA3AR and hA3AR, and could potentially be
derivatized for radioisotope incorporation or affinity cross-
linking. We are also interested in the design of radiofluorinated
nucleosides for positron emission tomographic (PET) imaging
of the A3AR in vivo.47 Several fluorinated species in this study
could provide the basis for incorporation of 18F into a high
affinity A3AR agonist.
In conclusion, we have found a new series of highly potent
and selective series of A3AR agonists containing combined
substitution of the (N)-methanocarba ring system and
arylethynyl groups at the adenine C2 position. The binding
affinities demonstrated high tolerance of steric bulk and ring
substitution by extension at the C2 position, with many aryl
groups providing Ki values in the nanomolar range. Molecular
modeling suggests that this reflects conformational plasticity of
the A3AR. Potent agonist ligands should be useful in future
pharmacological studies of the antiinflammatory, anticancer,
and antiischemic properties of A3AR agonists.
EXPERIMENTAL SECTION
■
Chemical Synthesis. Materials and Instrumentation. L-Ribose
and other reagents and solvents were purchased from Sigma-Aldrich
(St. Louis, MO). Alcohol derivative 39 was prepared as reported.28 1H
NMR spectra were obtained with a Bruker 400 spectrometer using
CDCl3 and CD3OD as solvents. Chemical shifts are expressed in δ
values (ppm) with tetramethylsilane (δ 0.00) for CDCl3 and water (δ
3.30) for CD3OD. TLC analysis was carried out on glass sheets
precoated with silica gel F254 (0.2 mm) from Aldrich. The purity of
final nucleoside derivatives was checked using a Hewlett−Packard
1100 HPLC equipped with a Zorbax SB-Aq 5 μm analytical column
(50 × 4.6 mm; Agilent Technologies Inc., Palo Alto, CA). Mobile
phase: linear gradient solvent system, 5 mM TBAP (tetrabutylammo-
nium dihydrogenphosphate)−CH3CN from 80:20 to 0:100 in 13 min;
the flow rate was 0.5 mL/min. Peaks were detected by UV absorption
with a diode array detector at 230, 254, and 280 nm. All derivatives
tested for biological activity showed >95% purity by HPLC analysis
(detection at 254 nm). Low-resolution mass spectrometry was
performed with a JEOL SX102 spectrometer with 6-kV Xe atoms
following desorption from a glycerol matrix or on an Agilent LC/MS
1100 MSD, with a Waters (Milford, MA) Atlantis C18 column. High
resolution mass spectroscopic (HRMS) measurements were per-
formed on a proteomics optimized Q-TOF-2 (Micromass-Waters)
using external calibration with polyalanine, unless noted. Observed
mass accuracies are those expected based on known performance of
the instrument as well as trends in masses of standard compounds
observed at intervals during the series of measurements. Reported
masses are observed masses uncorrected for this time-dependent drift
The implications for receptor coupling and signaling of this
type of displacement, such as the agonist-dependent movement
of TM2 that we predict here, are unknown. If this
conformational change associated with a family of ligands is
propagated to the intracellular regions, we speculate that there
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Journal of Medicinal Chemistry
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in mass accuracy. Synthetic procedures for 45−47 are in Supporting
Information. LogP and total polar surface area values were calculated
using ChemBioDraw Ultra (version 12.0.3, PerkinElmer, Boston,
MA).
(1S,2R,3S,4R,5S)-4-(2-((4-Chlorophenyl)ethynyl)-6-(methylami-
no)-9H-purin-9-yl)-2,3-dihydroxy-N-methylbicyclo[3.1.0]hexane-1-
carboxamide (16). Compound 16 (84%) was prepared from
compound 45g following the same method as that used for compound
1
9. H NMR (CD3OD) δ 8.10 (s, 1H), 7.64 (d, J = 8.4 Hz, 2H), 7.46
(1S,2R,3S,4R,5S)-2,3-Dihydroxy-N-methyl-4-(6-(methylamino)-2-
(phenylethynyl)-9H-purin-9-yl)bicyclo[3.1.0]hexane-1-carboxamide
(9). A solution of compound 45a (29 mg, 0.06 mmol) in methanol (2
mL) and 10% trifluoromethane sulfonic acid (2 mL) was heated at 70
°C for 5 h. The solvent was evaporated under vacuum, and the residue
was purified on flash silica gel column chromatography (CH2Cl2/
(d, J = 8.4 Hz, 2H), 5.06 (d, J = 5.6 Hz, 1H), 4.88 (s, 1H), 4.02 (d, J =
6.4 Hz, 1H), 3.15 (br s, 3H), 2.84 (s, 3H), 2.13−2.09 (m, 1H), 1.88 (t,
J = 4.8 Hz, 1H), 1.41−1.38 (m, 1H). HRMS calculated for
C22H22ClN6O3 (M + H)+: 453.1460; found, 453.1454.
(1S,2R,3S,4R,5S)-4-(2-((4-Bromophenyl)ethynyl)-6-(methylami-
no)-9H-purin-9-yl)-2,3-dihydroxy-N-methylbicyclo[3.1.0]hexane-1-
carboxamide (17). Compound 17 (76%) was prepared from
compound 45h following the same method as that used for compound
1
MeOH = 25:1) to give compound 9 (21 mg, 81%) as a syrup. H
NMR (CD3OD) δ 8.12 (s, 1H), 7.67−7.65 (m, 2H), 7.47−7.43 (m,
2H), 5.06 (d, J = 5.2 Hz, 1H), 4.08 (d, J = 6.4 Hz, 1H), 3.15 (br s,
3H), 2.84 (s, 3H), 2.13−2.10 (m, 1H), 1.88 (t, J = 5.2 Hz, 1H), 1.41−
1.39 (m, 1H). HRMS calculated for C22H23N6O3 (M + H)+: 419.1832;
found, 419.1818.
1
9. H NMR (CD3OD) δ 8.10 (s, 1H), 7.62 (d, J = 8.8 Hz, 2H), 7.56
(d, J = 8.4 Hz, 2H), 5.05 (d, J = 6.8 Hz, 1H), 4.88 (s, 1H), 4.02 (d, J =
6.4 Hz, 1H), 3.14 (br s, 3H), 2.84 (s, 3H), 2.13−2.09 (m, 1H), 1.88 (t,
J = 5.2 Hz, 1H), 1.41−1.38 (m, 1H). HRMS calculated for
C22H22BrN6O3 (M + H)+: 497.0937; found, 497.0948.
(1S,2R,3S,4R,5S)-4-(2-((3-Aminophenyl)ethynyl)-6-(methylami-
no)-9H-purin-9-yl)-2,3-dihydroxy-N-methylbicyclo[3.1.0]hexane-1-
carboxamide (18). Compound 18 (67%) was prepared from
compound 45i following the same method as that used for compound
9. 1H NMR (CD3OD) δ 8.09 (s, 1H), 7.14 (t, J = 8.0 Hz, 1H), 6.98−
6.94 (m, 2H), 6.80−6.78 (m, 1H), 5.06 (d, J = 5.6 Hz, 1H), 4.02 (d, J
= 6.4 Hz, 1H), 3.15 (br s, 3H), 2.85 (s, 3H), 2.12−2.09 (m, 1H), 1.87
(t, J = 4.8 Hz, 1H), 1.41−1.39 (m, 1H). HRMS calculated for
C22H22N7O3 (M + H)+: 432.1784; found, 432.1799.
(1S,2R,3S,4R,5S)-4-(2-((3,4-Difluorophenyl)ethynyl)-6-(methyla-
mino)-9H-purin-9-yl)-2,3-dihydroxy-N-methylbicyclo[3.1.0]hexane-
1-carboxamide (19). Compound 19 (83%) was prepared from
compound 45j following the same method as that used for compound
9. 1H NMR (CD3OD) δ 8.12 (s, 1H), 7.58−7.50 (m, 1H), 7.50−7.48
(m, 1H), 7.40−7.34 (m, 1H), 5.06 (d, J = 6.4 Hz, 1H), 4.02 (d, J = 6.4
Hz, 1H), 3.14 (br s, 3H), 2.84 (s, 3H), 2.13−2.09 (m, 1H), 1.88 (t, J =
4.8 Hz, 1H), 1.41−1.40 (m, 1H). HRMS calculated for C22H21F2N6O3
(M + H)+: 455.1643; found, 455.1639.
(1S,2R,3S,4R,5S)-2,3-Dihydroxy-N-methyl-4-(6-(methylamino)-2-
(pyridin-2-ylethynyl)-9H-purin-9-yl)bicyclo[3.1.0]hexane-1-carboxa-
mide (10). Compound 10 (80%) was prepared from compound 46
1
following the same method as that used for compound 9. H NMR
(CD3OD) δ 8.65 (s, 1H), 8.01 (s, 1H), 7.91−7.97 (m, 1H), 7.87 (d, J
= 8.8 Hz, 1H), 7.53−7.48 (m, 1H), 5.15 (d, J = 5.6 Hz, 1H), 4.09 (d, J
= 8.4 Hz, 1H), 3.14 (br s, 3H), 2.83 (s, 3H), 2.13−2.06 (m, 1H), 1.85
(t, J = 5.2 Hz, 1H), 1.42−1.40 (m, 1H). HRMS calculated for
C21H22N7O3 (M + H)+: 420.1784; found, 420.1797.
(1S,2R,3S,4R,5S)-4-(2-((2-Fluorophenyl)ethynyl)-6-(methylami-
no)-9H-purin-9-yl)-2,3-dihydroxy-N-methylbicyclo[3.1.0]hexane-1-
carboxamide (11). Compound 11 (82%) was prepared from
compound 45b following the same method as that used for compound
9.1H NMR (CD3OD) δ 8.11 (s, 1H), 7.49−7.39 (m, 3H), 7.25−7.20
(m, 1H), 5.06 (d, J = 5.2 Hz, 1H), 4.9 (s, 1H), 4.02 (d, J = 6.8 Hz,
1H), 3.15 (br s, 1H), 2.84 (s, 3H), 2.13−2.10 (m, 1H), 1.88 (t, J = 4.8
Hz, 1H), 1.41−1.38 (m, 1H). HRMS calculated for C22H22FN6O3 (M
+ H)+: 437.1737; found, 437.1753.
(1S,2R,3S,4R,5S)-4-(2-((3-Fluorophenyl)ethynyl)-6-(methylami-
no)-9H-purin-9-yl)-2,3-dihydroxy-N-methylbicyclo[3.1.0]hexane-1-
carboxamide (12). Compound 12 (78%) was prepared from
compound 45c following the same method as that used for compound
9. 1H NMR (CD3OD) δ 8.11 (s, 1H), 7.67 (t, J = 6.0 Hz, 1H), 7.52−
7.47 (m, 1H), 7.28−7.22 (m, 2H), 5.07 (d, J = 6.8 Hz, 1H), 4.03 (d, J
= 6.8 Hz, 1H), 3.13 (br s, 1H), 2.84 (s, 3H), 2.13−2.10 (m, 1H), 1.88
(t, J = 4.8 Hz, 1H), 1.41−1.40 (m, 1H). HRMS calculated for
C22H22FN6O3 (M + H)+: 437.1737; found, 437.1718.
(1S,2R,3S,4R,5S)-4-(2-((4-Fluorophenyl)ethynyl)-6-(methylami-
no)-9H-purin-9-yl)-2,3-dihydroxy-N-methylbicyclo[3.1.0]hexane-1-
carboxamide (13). Compound 13 (81%) was prepared from
compound 45d following the same method as that used for compound
9. 1H NMR (CD3OD) δ 8.11 (s, 1H), 7.75−7.68 (m, 2H), 7.23−7.18
(m, 2H), 5.05 (d, J = 6.0 Hz, 1H), 4.02 (d, J = 6.4 Hz), 1H), 3.14 (br
s, 3H), 2.84 (s, 3H), 2.13−2.10 (m, 1H), 1.89 (t, J = 4.8 Hz, 1H),
1.41−1.39 (m, 1H). HRMS calculated for C22H22FN6O3 (M + H)+:
437.1737; found, 437.1722.
(1S,2R,3S,4R,5S)-4-(2-((3,5-Difluorophenyl)ethynyl)-6-(methyla-
mino)-9H-purin-9-yl)-2,3-dihydroxy-N-methylbicyclo[3.1.0]hexane-
1-carboxamide (20). Compound 20 (84%) was prepared from
compound 45k following the same method as that used for compound
1
9. H NMR (CD3OD) δ 8.12 (s, 1H), 7.31−7.26 (s, 2H), 7.14−7.09
(m, 1H), 7.49−7.42 (m, 2H), 5.06 (d, J = 5.2 Hz, 1H), 4.89 (s, 1H),
4.03 (d, J = 6.4 Hz, 1H), 3.14 (br s, 3H), 2.84 (s, 3H), 2.12−2.09 (m,
1H), 1.88 (t, J = 4.8 Hz, 1H), 1.41−1.39 (m, 1H). HRMS calculated
for C22H21F2N6O3 (M + H)+: 455.1643; found, 455.1630.
(1S,2R,3S,4R,5S)-4-(2-((4-Ethylphenyl)ethynyl)-6-(methylamino)-
9H-purin-9-yl)-2,3-dihydroxy-N-methylbicyclo[3.1.0]hexane-1-car-
boxamide (21). Compound 21 (79%) was prepared from compound
45l following the same method as that used for compound 9. 1H NMR
(CD3OD) δ 8.09 (s, 1H), 7.57 (d, J = 8.0 Hz, 2H), 7.29 (d, J = 8.0 Hz,
2H), 5.06 (d, J = 5.2 Hz, 1H), 4.91 (s, 1H), 4.02 (d, J = 6.4 Hz, 1H),
3.15 (br s, 3H), 2.84 (s, 3H), 2.74−2.68 (m, 2H), 2.13−2.09 (m, 1H),
1.88 (t, J = 4.8 Hz, 1H), 1.41−1.38 (m, 1H), 1.30 (t, J=7.6 Hz, 3H).
HRMS calculated for C24H27N6O3 (M + H)+: 447.2145; found,
447.2130.
(1S,2R,3S,4R,5S)-4-(2-((2-Chlorophenyl)ethynyl)-6-(methylami-
no)-9H-purin-9-yl)-2,3-dihydroxy-N-methylbicyclo[3.1.0]hexane-1-
carboxamide (14). Compound 14 (85%) was prepared from
compound 45e following the same method as that used for compound
(1S,2R,3S,4R,5S)-4-(2-((4-tert-Butylphenyl)ethynyl)-6-(methylami-
no)-9H-purin-9-yl)-2,3-dihydroxy-N-methylbicyclo[3.1.0]hexane-1-
carboxamide (22). Compound 22 (83%) was prepared from
compound 45m following the same method as that used for
1
9. H NMR (CD3OD) δ 8.11 (s, 1H), 7.72 (d, J = 7.2 Hz, 1H), 7.54
(d, J = 7.2 Hz, 1H), 7.46−7.36 (m, 2H), 5.10 (d, J = 6.4 Hz, 1H), 4.04
(d, J = 6.8 Hz, 1H), 3.15 (br s, 3H), 2.83 (s, 3H), 2.12−2.08 (m, 1H),
1.86 (t, J = 4.8 Hz, 1H), 1.41−1.39 (m, 1H). HRMS calculated for
C22H22ClN6O3 (M + H)+: 453.1442; found, 453.1449.
1
compound 9. H NMR (CD3OD) δ 8.10 (s, 1H), 7.59 (d, J = 8.4
Hz, 2H), 7.50 (d, J = 8.4 Hz, 2H), 5.06 (d, J = 6.4 Hz, 1H), 4.02(d, J =
6.4 Hz, 1H), 3.15 (br s, 3H), 2.85 (s, 3H), 2.13−2.10 (m, 1H), 188(t, J
= 4.8 Hz, 1H), 1.41−1.39 (m, 10H). HRMS calculated for
C26H31N6O3 (M + H)+: 475.2458; found, 475.2450.
(1S,2R,3S,4R,5S)-4-(2-((3-Chlorophenyl)ethynyl)-6-(methylami-
no)-9H-purin-9-yl)-2,3-dihydroxy-N-methylbicyclo[3.1.0]hexane-1-
carboxamide (15). Compound 15 (82%) was prepared from
compound 45f following the same method as that used for compound
(1S,2R,3S,4R,5S)-4-(2-((4-Acetylphenyl)ethynyl)-6-(methylamino)-
9H-purin-9-yl)-2,3-dihydroxy-N-methylbicyclo[3.1.0]hexane-1-car-
boxamide (23). Compound 23 (80%) was prepared from compound
1
9. H NMR (CD3OD) δ 8.11 (s, 1H), 7.67 (s, 1H), 7.59 (d, J = 7.2
1
Hz, 1H), 7.49−7.42 (m, 2H), 5.06 (d, J = 6.4 Hz, 1H), 4.02 (d, J = 6.8
Hz, 1H), 3.14 (br s, 3H), 2.84 (s, 3H), 2.13−2.10 (m, 1H), 1.88 (t, J =
4.8 Hz, 1H), 1.41−1.39 (m, 1H). HRMS calculated for C22H22ClN6O3
(M + H)+: 453.1442; found, 453.1442.
45n following the same method as that used for compound 9. H
NMR (CD3OD) δ 8.11 (s, 1H), 8.06 (d, J = 8.4 Hz, 2H), 7.77 (d, J =
8.0 Hz, 2H), 5.06 (d, J = 6.4 Hz, 1H), 4.03 (d, J = 6.4 Hz, 1H), 3.15
(br s, 3H), 2.84 (s, 3H), 2.64 (s, 3H), 2.14−2.10 (m, 1H), 1.88 (t, J =
4856
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Journal of Medicinal Chemistry
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4.8 Hz, 1H), 1.41−1.39 (m, 1H). HRMS calculated for C24H25N6O4
(M + H)+: 461.1937; found, 461.1937.
methylbicyclo[3.1.0]hexane-1-carboxamide (30). Compound 30
(66%) was prepared from compound 44 following the same method
as that used for compound 27. H NMR (CD3OD) δ 8.15 (s, 1H),
7.66−7.57 (m, 2H), 7.48−7.26 (m, 6H), 5.07 (d, J = 6.4 Hz, 1H), 4.85
(s, 1H), 4.04 (d, J = 6.8 Hz, 1H), 2.84 (s, 3H), 2.14−2.10 (m, 1H),
1.88 (t, J = 4.8 Hz, 1H), 1.41−1.39 (m, 1H). HRMS calculated for
C28H25Cl2N6O3 (M + H)+: 563.1365; found, 563.1359.
1
(1S,2R,3S,4R,5S)-4-(2-(Biphenyl-4-ylethynyl)-6-(methylamino)-
9H-purin-9-yl)-2,3-dihydroxy-N-methylbicyclo[3.1.0]hexane-1-car-
boxamide (24). Compound 24 (74%) was prepared from compound
1
45o following the same method as that used for compound 9. H
NMR (CD3OD) δ 8.11 (s, 1H), 7.75−7.67 (m, 6H), 7.47 (t, J = 7.6
Hz, 2H), 7.38 (t, J = 7.2 Hz, 1H), 5.06 (d, J = 6.0 Hz, 1H), 4.89 (s,
1H), 4.03 (d, J = 6.4 Hz, 1H), 3.16 (br s, 3H), 2.84 (s, 3H), 2.13−2.10
(m, 1H), 189 (t, J = 4.8 Hz, 1H), 1.42−1.39 (m, 1H). HRMS
calculated for C28H27N6O3 (M + H)+: 495.2145; found, 495.2141.
(1S,2R,3S,4R,5S)-2,3-Dihydroxy-N-methyl-4-(6-(methylamino)-2-
(naphthalen-1-ylethynyl)-9H-purin-9-yl)bicyclo[3.1.0]hexane-1-car-
boxamide (25). Compound 25 (73%) was prepared from compound
(1S,2R,3S,4R,5S)-4-(6-(3-Chlorobenzylamino)-2-((3,4-
difluorophenyl)ethynyl)-9H-purin-9-yl)-2,3-dihydroxy-N-
methylbicyclo[3.1.0]hexane-1-carboxamide (31). Compound 31
(63%) was prepared from compound 44 following the same method
1
as that used for compound 27. H NMR (CD3OD) δ 8.14 (s, 1H),
7.58 (t, J = 8.8 Hz, 1H), 7.47−7.44 (m, 2H), 7.39−7.25 (m, 4H), 5.06
(d, J = 6.4 Hz, 1H), 4.89 (s, 1H), 4.04 (d, J = 6.4 Hz, 1H), 2.84 (s,
3H), 2.13−2.10 (m, 1H), 1.88 (t, J = 4.8 Hz, 1H), 1.41−1.38 (m, 1H).
HRMS calculated for C28H24F2ClN6O3 (M + H)+: 565.1566; found,
565.1559.
(1S,2R,3S,4R,5S)-4-(2-((4-Aminophenyl)ethynyl)-6-(3-chloroben-
zylamino)-9H-purin-9-yl)-2,3-dihydroxy-N-methylbicyclo[3.1.0]-
hexane-1-carboxamide (32). Compound 32 (59%) was prepared
from compound 44 following the same method as that used for
compound 27. 1H NMR (CD3OD) δ 8.02 (s, 1H), 7.84 (d, J = 8.8 Hz,
2H), 7.33−7.23 (m, 4H), 6.60 (d, J = 8.4 Hz, 2H), 5.17 (d, J = 6.4 Hz,
1H), 4.85 (s, 1H), 4.73 (br s, 2H), 4.02 (d, J = 6.8 Hz, 1H), 2.83 (s,
3H), 2.08−2.05 (m, 1H), 1.81 (t, J = 4.8 Hz, 1H), 1.41−1.37 (m, 1H).
HRMS calculated for C28H27ClN7O3 (M + H)+: 545.1041; found,
545.1045.
3-((6-(3-Chlorobenzylamino)-9-((1S,2R,3S,4R,5S)-3,4-dihydroxy-
5-(methyl-carbamoyl)bicyclo[3.1.0]hexan-2-yl)-9H-purin-2-yl)-
ethynyl)benzoic acid (33). PdCl2(PPh3)2 (3.0 mg, 0.004 mmol), CuI
(1.0 mg, 0.004 mmol), phenylacetylene (18.7 mg, 0.12 mmol), and
triethylamine (20 μL, 0.2 mmol) were added to a solution of
compound 44 (12.68 mg, 0.02 mmol) in anhydrous DMF (1 mL) and
stirred at room temperature overnight. The solvent was evaporated
under vacuum, and the residue was roughly purified on flash silica gel
column chromatography. The resulting compound was dissolved in
dioxane (2 mL) and 1 N HCl (1.5 mL), and heated at 60 °C for 2 h.
After completion of the starting material, the solvent was evaporated
under vacuum, and the residue was purified on flash silica gel column
chromatography (CH2Cl2/MeOH/TFA = 25:1:0.1) to give com-
pound 33 (7 mg, 61%) as a syrup. 1H NMR (CD3OD) δ 8.28 (s, 1H),
8.18−8.16 (m, 1H), 8.11−8.07 (m, 1H), 7.87 (d, J = 7.6 Hz, 1H),
7.59−7.54 (m, 2H), 7.47 (s, 1H), 7.41−7.26 (m, 2H), 5.09 (d, J = 6.4
Hz, 1H), 4.91 (s, 1H), 4.05 (d, J = 6.4 Hz, 1H), 2.85 (s, 3H), 2.14−
2.11 (m, 1H), 1.88 (t, J = 4.8 Hz, 1H), 1.42−1.38 (m, 1H). HRMS
calculated for C29H26ClN6O5 (M + H)+: 573.1653; found, 573.1646.
(1S,2R,3S,4R,5S)-4-(2-(Biphenyl-4-ylethynyl)-6-(3-chlorobenzyla-
mino)-9H-purin-9-yl)-2,3-dihydroxy-N-methylbicyclo[3.1.0]hexane-
1-carboxamide (34). Compound 34 (68%) was prepared from
compound 44 following the same method as that used for compound
27. 1H NMR (CD3OD) δ 8.13 (s, 1H), 7.74−7.66 (m, 7H), 7.49−7.45
(m, 2H), 7.40−7.26 (m, 4H), 5.07 (d, J = 5.6 Hz, 1H), 4.9 (s, 1H),
4.60 (br s, 2H), 4.05 (d, J = 6.4 Hz, 1H), 2.85 (s, 3H), 2.14−2.11 (m,
1H), 1.89 (t, J = 4.8 Hz, 1H), 1.42−1.40 (m, 1H). HRMS calculated
for C34H30ClN6O3 (M + H)+: 605.2068; found, 605.2083.
1
45p following the same method as that used for compound 9. H
NMR (CD3OD) δ 8.56 (d, J = 7.6 Hz, 1H), 8.11 (s, 1H), 8.00−7.90
(m, 3H), 7.68 (t, J = 5.6 Hz, 1H), 7.62−7.53 (m, 2H), 5.10 (d, J = 5.2
Hz, 1H), 4.93 (s, 1H), 4.06 (d, J = 6.4 Hz, 1H), 3.19 (br s, 3H), 2.79
(s, 3H), 2.15−2.12 (m, 1H), 189 (t, J = 4.8 Hz, 1H), 1.41−1.38 (m,
1H). HRMS calculated for C26H25N6O3 (M + H)+: 469.1988; found,
469.2005.
(1S,2R,3S,4R,5S)-2,3-Dihydroxy-N-methyl-4-(6-(methylamino)-2-
(phenanthren-9-ylethynyl)-9H-purin-9-yl)bicyclo[3.1.0]hexane-1-
carboxamide (26). Compound 26 (65%) was prepared from
compound 45q following the same method as that used for compound
9. 1H NMR (CD3OD) δ 8.84−8.77 (m, 2H), 8.66−8.63 (m, 1H), 8.26
(s, 1H), 8.12 (s, 1H), 7.97 (d, J = 7.6 Hz, 1H), 7.80−7.73 (m, 3H),
7.66 (t, J = 7.2 Hz, 1H), 5.10 (d, J = 6.0 Hz, 1H), 4.92 (s, 1H), 4.07 (d,
J = 6.4 Hz, 1H), 3.20 (br s, 3H), 2.81 (s, 3H), 2.15−2.12 (m, 1H),
1.90 (t, J = 4.8 Hz, 1H), 1.42−1.38 (m, 1H). HRMS calculated for
C30H27N6O3 (M + H)+: 519.2145; found, 519.2137.
(1S,2R,3S,4R,5S)-4-(6-(3-Chlorobenzylamino)-2-(phenylethynyl)-
9H-purin-9-yl)-2,3-dihydroxy-N-methylbicyclo[3.1.0]hexane-1-car-
boxamide (27). PdCl2(PPh3)2 (6.13 mg, 0.008 mmol), CuI (1.2 mg,
0.004 mmol), phenylacetylene (30 μL, 0.26 mmol), and triethylamine
(60 μL, 0.4 mmol) were added to a solution of compound 44 (26 mg,
0.04 mmol) in anhydrous DMF (1 mL) and stirred at room
temperature overnight. The solvent was evaporated under vacuum,
and the residue was roughly purified on flash silica gel column
chromatography. The resulting compound was dissolved in methanol
(2 mL) and 10% trifluoromethane sulfonic acid (2 mL), and heated at
70 °C for 5 h. The solvent was evaporated under vacuum, and the
residue was purified on flash silica gel column chromatography
(CH2Cl2/MeOH = 25:1) to give compound 27 (17 mg, 76%) as a
syrup. 1H NMR (CD3OD) δ 8.13 (s, 1H), 7.66−7.63 (m, 2H), 7.46−
7.42 (m, 4H), 7.37−7.26 (m, 3H), 5.06 (d, J = 5.6 Hz, 1H), 4.9 (br s,
2H) 4.04 (d, J = 6.4 Hz, 1H), 2.84 (s, 3H), 2.14−2.11 (m, 1H), 1.88
(t, J = 4.8 Hz, 1H), 1.41−1.37 (m, 1H). HRMS calculated for
C28H26ClN6O3 (M + H)+: 529.1755; found, 529.1740.
(1S,2R,3S,4R,5S)-4-(6-(3-Chlorobenzylamino)-2-((4-fluorophenyl)-
ethynyl)-9H-purin-9-yl)-2,3-dihydroxy-N-methylbicyclo[3.1.0]-
hexane-1-carboxamide (28). Compound 28 (68%) was prepared
from compound 44 following the same method as that used for
1
compound 27. H NMR (CD3OD) δ 8.13 (s, 1H), 7.70−7.63 (m,
2H), 7.59−7.55 (m, 1H), 7.45 (s, 1H), 7.37−7.21 (m, 3H), 7.19−7.16
(m, 1H), 5.06 (d, J = 5.6 Hz, 1H), 4.9 (s, 1H), 4.58 (br s, 2H), 4.04 (d,
J = 7.6 Hz, 1H), 2.84 (s, 3H), 2.17−2.10 (m, 1H), 1.88 (t, J = 4.8 Hz,
1H), 1.41−1.38 (m, 1H). HRMS calculated for C28H25ClFN6O3 (M +
H)+: 547.1661; found, 547.1652.
(1S,2R,3S,4R,5S)-4-(6-(3-Chlorobenzylamino)-2-(pyren-1-ylethyn-
yl)-9H-purin-9-yl)-2,3-dihydroxy-N-methylbicyclo[3.1.0]hexane-1-
carboxamide (35). Compound 35 (91%) was prepared from
compound 44 following the same method as that used for compound
1
33. H NMR (CD3OD) δ 8.71 (d, J = 9.2 Hz, 1H), 8.26−8.23 (m,
(1S,2R,3S,4R,5S)-4-(6-(3-Chlorobenzylamino)-2-((2-
chlorophenyl)ethynyl)-9H-purin-9-yl)-2,3-dihydroxy-N-
methylbicyclo[3.1.0]hexane-1-carboxamide (29). Compound 29
(65%) was prepared from compound 44 following the same method
4H), 8.16−8.13 (m, 2H), 8.08−8.03 (m, 3H), 7.54 (s, 1H), 7.43 (d, J
= 7.6 Hz, 1H), 7.34 (t, J = 8.0 Hz, 1H), 7.27 (d, J = 8.0 Hz, 1H), 5.06
(d, J = 6.4 Hz, 1H), 4.83 (s, 1H), 4.04 (d, J = 6.4 Hz, 1H), 2.81 (s,
3H), 2.10−2.07 (m, 1H), 1.89 (t, J = 4.8 Hz, 1H), 1.41−1.37 (m, 1H).
HRMS calculated for C38H30ClN6O3 (M + H)+: 653.2068; found,
653.2078.
1
as that used for compound 27. H NMR (CD3OD) δ 8.14 (s, 1H),
7.72−7.70 (m, 1H), 7.53 (d, J = 8.0 Hz, 1H), 7.47−7.25 (m, 6H), 5.11
(d, J = 6.8 Hz, 1H), 4.90 (s, 1H), 4.05 (d, J = 6.8 Hz, 1H), 2.82 (s,
3H), 2.12−2.09 (m, 1H), 1.86 (t, J = 4.8 Hz, 1H), 1.40−1.38 (m, 1H).
HRMS calculated for C28H24Cl2N6O3Na (M + Na)+: 585.1185; found,
585.1167.
(1S,2R,3S,4R,5S)-4-(6-(3-Chlorobenzylamino)-2-(pyren-4-ylethyn-
yl)-9H-purin-9-yl)-2,3-dihydroxy-N-methylbicyclo[3.1.0]hexane-1-
carboxamide (36). Compound 36 (69%) was prepared from
compound 44 following the same method as that used for compound
(1S,2R,3S,4R,5S)-4-(6-(3-Chlorobenzylamino)-2-((3-
chlorophenyl)ethynyl)-9H-purin-9-yl)-2,3-dihydroxy-N-
1
33. H NMR (CD3OD) δ 8.82 (d, J = 7.6 Hz, 1H), 8.51 (s, 1H),
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8.29−8.21 (m, 3H), 8.16−8.12 (m, 4H), 8.04 (t, J = 7.6 Hz, 1H), 7.54
(s, 1H), 7.43 (d, J = 7.6 Hz, 1H), 7.35 (t, J = 8.0 Hz, 1H), 7.28 (d, J =
8.4 Hz, 1H), 5.1 (d, J = 6.0 Hz, 1H), 4.06 (d, J = 6.0 Hz, 1H), 2.80 (s,
3H), 2.18−2.09 (m, 1H), 1.89 (t, J = 4.8 Hz, 1H), 1.42−1.39 (m, 1H).
HRMS calculated for C38H30ClN6O3 (M + H)+: 653.2068; found,
653.2056.
the centroid of the agonist molecules. The default values were used for
other parameters.
ASSOCIATED CONTENT
* Supporting Information
■
S
Docking figures for compounds 14, 15, and 34, procedures for
the synthesis of intermediates, detailed molecular modeling
procedures, and biological assays for novel nucleoside
derivatives, and coordinates of complexes with 13, 31, and
35. This material is available free of charge via the Internet at
http://pubs.acs.org.
(1S,2R,3S,4R,5S)-Ethyl-(2,3-O-isopropylidene)-4-(2-iodo-6-(meth-
ylamino)-9H-purin-9-yl)bicyclo[3.1.0]hexane-1-carboxylate (41).
Methylamine hydrochloride (0.353 g, 5.23 mmol) and triethylamine
(1.4 mL, 16.6 mmol) was added to a solution of compound 40 (0.528
g, 1.04 mmol) in anhydrous methanol (15 mL) and stirred at room
temperature overnight. The solvent was evaporated under vacuum,
and the residue was purified on flash silica gel column chromatography
(hexane/ethylacetate = 1:1) to give compound 41 (0.470 g, 94%) as a
AUTHOR INFORMATION
Corresponding Author
■
1
foamy solid. H NMR (CD3OD) δ 7.94 (s, 1H), 5.83 (d, J = 7.2 Hz,
1H), 4.94 (s, 1H), 4.80 (d, J = 6.0 Hz, 1H), 4.33−4.27 (m, 2H), 3.05
(br s, 3H), 2.25−2.21 (m, 1H), 1.65−1.61 (m, 1H), 1.53−1.49 (m,
4H), 1.34 (t, J = 7.2 Hz, 3H), 1.29 (s, 3H). HRMS calculated for
C18H23IN5O4 (M + H)+: 500.1072; found, 500.1075.
*Molecular Recognition Section, Bldg. 8A, Rm. B1A-19, NIH,
NIDDK, LBC, Bethesda, MD 20892-0810. Tel: 301-496-9024.
Fax: 301-480-8422. E-mail: kajacobs@helix.nih.gov.
(1S,2R,3S,4R,5S)-(2,3-O-Isopropylidene)-4-(2-iodo-6-(methylami-
no)-9H-purin-9-yl)-N-methylbicyclo[3.1.0]hexane-1-carboxamide
(43). 40% Methylamine solution (10 mL) was added to a solution of
compound 41 (0.470 g, 0.94 mmol) in methanol (15 mL) and stirred
at room temperature for 48 h. The solvent was evaporated under
vacuum, and the residue was purified on flash silica gel column
chromatography (CH2Cl2/MeOH = 40:1) to give compound 43
(0.360 g, 79%) as a syrup. 1H NMR (CD3OD) δ 7.95 (s, 1H), 5.72 (d,
J = 7.2 Hz, 1H), 4.93 (s, 1H), 4.84 (d, J = 7.2 Hz, 1H), 3.05 (br s, 3H),
2.90 (s, 3H), 2.17−2.11 (m, 1H), 1.54−1.49 (m, 4H), 1.39 (t, J = 5.2
Hz, 1H), 1.30 (s, 3H). HRMS calculated for C17H22IN6O3 (M + H)+:
485.0798; found, 485.0803.
Pharmacological Characterization. The nucleoside derivatives,
dissolved as stock solutions in DMSO (5 mM) and stored frozen, were
evaluated in binding29−31 and a functional assay34 at the A3AR, binding
assays at the A1AR and A2AAR, and a functional assay at the hA3AR
(details in Supporting Information). Use of heterologously expressed
mouse ARs was as reported.33b,49 Protein content was determined48
and IC50 values in binding inhibition transformed to Ki values50 as
reported.
Molecular Modeling. The Homology Model module of MOE was
utilized to build a new molecular model of the hA3AR (details in
Supporting Information). The recently reported model of the complex
of the nonselective AR agonist 59 docked to the crystal structure of
the A2AAR was used as a template for modeling of the A3AR.25,51 The
sequence alignment of the A2A and A3ARs was performed with MOE,
taking into account positions of highly conserved amino acid residues.
The position of full agonist 59 inside the A2AAR was taken into
account during the modeling. In the resulting initial homology model
of the A3AR (prior to movement of TM2), the complex 5′,2,N6
trisubstituted agonist 59 used to crystallize the A2AAR was removed
and the simpler 5′-uronamide 58 docked inside the receptor. The
Glide program of the Schrodinger package52 was used to dock the
other agonists to the A3AR model obtained. The receptor grid
generation was performed for the box with a center in the centroid of
58 in its initial position. The size of the box was determined
automatically. The extra precision mode (XP) of Glide was used for
the docking. The binding site was defined as 58 and all amino acid
residues located within 5 Å from 58. All A3AR residues located within
2 Å from the binding site were used as a shell. The following
parameters of energy minimization were used: OPLS2005 force field,
water was used as an implicit solvent, and a maximum of 5000
iterations of the Polak−Ribier conjugate gradient minimization
method was used with a convergence threshold of 0.01 kJ·mol−1·Å−1.
Another reference nucleoside 58 was also docked in this hA3AR
homology model.
Author Contributions
§These authors contributed equally to this work.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
We thank Dr. John Lloyd and Dr. Noel Whittaker (NIDDK)
for mass spectral determinations. This research was supported
by the Intramural Research Program of the NIH, National
Institute of Diabetes and Digestive and Kidney Diseases, and by
NIH R01 HL077707.
ABBREVIATIONS
■
AR, adenosine receptor; cAMP, adenosine 3′,5′-cyclic phos-
phate; CHO, Chinese hamster ovary; Cl-IB-MECA, 2-chloro-
N6-(3-iodobenzyl)-5′-N-methylcarboxamidoadenosine; DIPEA,
diisopropylethylamine; DCM, dichloromethane; DMF, N,N-
dimethylformamide; DMEM, Dulbecco’s modified Eagle’s
medium; EDTA, ethylenediaminetetraacetic acid; EL, extrac-
ellular loop; GPCR, G protein-coupled receptor; HEK, human
embryonic kidney; I-AB-MECA, N6-(4-amino-3-iodobenzyl)-
adenosine-5′-N-methyl-uronamide; IFD, induced fit docking;
NECA, 5′-N-ethylcarboxamidoadenosine; HEPES, 4-(2-hydrox-
yethyl)-1-piperazineethanesulfonic acid; HRMS, high resolu-
tion mass spectroscopy; NMR, nuclear magnetic resonance; R-
PIA, N6-R-phenylisopropyladenosine; TEA, triethylamine;
TLC, thin layer chromatography; TM, transmembrane domain
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