Structure-based design, synthesis by click chemistry and in vivo activity of highly selective A3 adenosine receptor agonists

Article abstract of DOI:10.1039/c4md00571f

2-Arylethynyl derivatives of (N)-methanocarba adenosine 5′-uronamides are selective A₃AR (adenosine receptor) agonists. Here we substitute a 1,2,3-triazol-1-yl linker in place of the rigid, linear ethynyl group to eliminate its potential metabo

Full text of DOI:10.1039/c4md00571f

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Medicinal Chemistry Communications
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Med. Chem. Comm.
Structure-Based Design, Synthesis by Click Chemistry and in Vivo
Activity of Highly Selective A3 Adenosine Receptor Agonists
Dilip K. Tosh,† Silvia Paoletta,† Zhoumou Chen,‡ Steven Crane,† John Lloyd,†
Zhan-Guo Gao,† Elizabeth T. Gizewski,§ John A. Auchampach,§
Daniela Salvemini,‡ and Kenneth A. Jacobson†*
†Laboratory of Bioorganic Chemistry, National Institute of Diabetes and Digestive and
Kidney Diseases, National Institutes of Health, Bethesda, MD 20892 USA.
‡Department of Pharmacological and Physiological Science, Saint Louis University
School of Medicine, St. Louis, Missouri 63104 USA.
§ Department of Pharmacology, Medical College of Wisconsin, 8701 Watertown Plank
Road, Milwaukee, Wisconsin 53226 USA.
Corresponding author: Dr. K. A. Jacobson, Chief, Molecular Recognition Section,
Bldg. 8A, Rm. B1A-19, NIH, NIDDK, LBC, Bethesda, MD 20892-0810 USA. Tel:
301-496-9024. Fax: 301-480-8422; Email: kennethj@niddk.nih.gov
ABBREVIATIONS: AR, adenosine receptor; cAMP, adenosine 3′,5′-cyclic
monophosphate; CCI, chronic constriction injury; CHO, Chinese hamster ovary; CNS,
central nervous system; DIC, N,Nʹ-diisopropylcarbodiimide; DMEM, Dulbecco’s
modified Eagle medium; DMF, N,N-dimethylformamide; EL, extracellular loop; GPCR,
G protein-coupled receptor; HEPES, 2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic
acid; HEK, human embryonic kidney; HPLC, high performance liquid chromatography;
HRMS, high resolution mass spectroscopy; MW, molecular weight; NECA, 5′-N-
ethylcarboxamidoadenosine; NMR, nuclear magnetic resonance; PBS, phosphate
buffered saline; TSPO, translocator protein; PWT, Paw Withdrawal Threshold; RMS,
root-mean-square; SAR, structure-activity relationship; TBAP, tetrabutylammonium
dihydrogenphosphate; TEA, triethylamine; THF, tetrahydrofuran; TLC, thin layer
chromatography; TM, transmembrane helix; tPSA, total polar surface area.

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Abstract
2-Arylethynyl derivatives of (N)-methanocarba adenosine 5′-uronamides are selective
A3AR (adenosine receptor) agonists. Here we substitute a 1,2,3-triazol-1-yl linker in
place of the rigid, linear ethynyl group to eliminate its potential metabolic liability.
Docking of nucleosides containing possible short linker moieties at the adenine C2
position using a hybrid molecular model of the A3AR (based on the A2AAR agonist-
bound structure) correctly predicted that a triazole would maintain the A3AR selectivity,
due to its ability to fit a narrow cleft in the receptor. The analogues with various N6 and
C2-aryltriazolyl substitution were synthesized and characterized in binding (Ki at hA3AR
0.3 – 12 nM) and in vivo to demonstrate efficacy in controlling chronic neuropathic pain
(chronic constriction injury). Among N6-methyl derivatives, a terminal pyrimidin-2-yl
group in 9 (MRS7116) increased duration of action (36% pain protection at 3 h) in vivo.
N6-Ethyl 5-chlorothien-2-yl analogue 15 (MRS7126) preserved in vivo efficacy (85%
protection at 1 h) with short duration. Larger N6 groups, e.g. 17 (MRS7138, >90%
protection at 1 and 3 h), greatly enhanced in vivo activity. Thus, we have combined
structure-based methods and phenotypic screening to identify nucleoside derivatives of
having translational potential.
Keywords: G protein-coupled receptor; nucleoside; click chemistry; neuropathic pain;
molecular modeling.

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Introduction
Two nucleoside agonists of the A3 adenosine receptor (A3AR) are currently in clinical
trials for the treatment of autoimmune inflammatory diseases (N6-(3-iodobenzyl)-5ʹ-N-
methylcarboxamidoadenosine, IB-MECA), including rheumatoid arthritis and plaque
psoriasis, and hepatocellular carcinoma (2-chloro analogue, Cl-IB-MECA).1,2 An even
broader range of conditions have been shown to be ameliorated by administration of
A3AR agonists in animal models, including osteoarthritis, neutropenia, and other
inflammatory and infective conditions and chronic neuropathic pain.3,4 Chronic
neuropathic pain can result from injury or diseases such as cancer and diabetes or from
pharmaceutical administration, and its treatment represents an unmet medical need. Thus,
the design, synthesis and pharmacological characterization of novel drug-like A3AR
agonists of high affinity, selectivity and in vivo efficacy are well justified.
The selective A3AR agonists that have advanced to clinical trials are adenine-9-riboside
derivatives.1,2 As an extension of the structure activity relationship (SAR) of AR agonists,
we have introduced nucleoside derivatives containing in place of ribose a
bicyclo[3.1.0]hexane (methanocarba) ring system, which display increased affinity and/or
selectivity for the A3AR compared to other AR subtypes.5 The rigid ribose ring substitute
maintains a receptor-preferred conformation and thus decreases an entropic energy
barrier for receptor binding. Prototypical nucleosides containing a methanocarba ring
system in a North (N) envelope conformation are typically >100-fold more potent at the

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A3AR than the corresponding isomers in the South (S) conformation.6 A combination of
A3AR-favoring modifications provided MRS5698 1 and MRS5980 2 (Chart 1A) as
highly potent agonists with favorable protective properties in the sciatic nerve chronic
constriction injury (CCI) model of neuropathic pain in mice.7-9 These rigidified
nucleosides contain a C2-arylethynyl group,10 which is thought to be responsible for the
high A3AR selectivity, based on the greater structural plasticity of the A3AR compared to
the A2AAR, which is more constrained by disulfide bridges in the extracellular loops
(ELs). In particular, we have proposed an outward movement of transmembrane helix 2
(TM2) in the A3AR in order to accommodate the C2-arylethynyl group and still to
maintain conserved H-bonding interactions of the hydroxyls of the ribose-like moiety
with the N6 and N7 of the adenine moiety.7,11
However, the presence of an arylethynyl group might be an indication of potential liver
toxicity due to its electrophilicity and possible reactivity with glutathione.12 Therefore,
we explored alternative structures that would still maintain the proper geometry of the
distal aryl group with respect to the adenosine core structure. A number of alternative C2
substituents were considered and compared by docking to an A3AR model: benzyl 3b,
phenylethyl 3c, phenylcyclopropyl 3d, phenyl-trans-ethenyl 3e, and phenyl-triazolyl 3f
(Chart 1A, Ar = C6H5). A 1,2,3-triazol-1-yl group was predicted to best maintain a
similar geometry compared to the potent and selective C2-arylethynyl derivatives.
This work has multiple goals: 1) to identify a general class of compounds that are highly
selective agonists of the A3AR but do not contain an arylethynyl group, based initially on

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a comparison of hypothetical structures using molecular modeling; 2) to determine the in
vivo efficacy and duration of action in a mouse model of neuropathic pain in order to
select preferred candidates for further development.
Results and discussion
We used molecular modeling to predict the effect of logical C2 modifications 3b-f (R =
CH3, Ar = C6H5) on human (h) A3AR recognition. Because the rigid, elongated C2
substituent of the series of general formula 3a is key to the >3000-fold A3AR selectivity
vs. the A2AAR, a substitution for the ethynyl group would need to maintain a similar
extended conformation when bound to the receptor. The receptor model used for docking
was our recently published hybrid homology model of the A3AR.7,11 The agonist-bound
hA2AAR X-ray structure13 was used as a template for all of the TMs, except for the upper
part of TM2.11 Figure 1 compares the effects of these substitutions when the nucleosides
are docked in the hybrid model, and Table 1 summarizes the features of the different
proposed linkers (substitutes for C≡C of 3a). Docking simulations showed that all the
derivatives bearing the proposed alternative C2 substituents were able to fit the binding
site of the A3AR hybrid model in an orientation analogous to the one observed for the
original arylethynyl derivatives. Such a binding pose has been well validated in previous
studies7,11 and shows all the interactions key for agonist binding at ARs.13 In particular,
the pseudo-sugar moiety forms H-bonds with Thr94 (3.36), Ser271 (7.42) and His272
(7.43) (numbers in parenthesis follow the Ballesteros-Weinstein numbering system),14 the
adenine core forms two H-bonds with Asn250 (6.55) and a π-π stacking with Phe168

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(EL2), the N6 substituent interacts with hydrophobic residues on EL2 such as Val169 and
the C2 group is directed towards TM2 (Figure 1A).
Thus, to select the best linker to be used in the new series of derivatives, we analyzed the
various alternatives in a detailed manner. The rigid, elongated C2 substituent is a key
feature for high A3AR selectivity, because of the greater structural plasticity of the A3AR
compared to the A2AAR as previously proposed,7 therefore we predicted higher
selectivity at the A3AR for those derivatives bearing rigid and elongated linkers, such as
3d-f. These substitutions might approximate the docked position of the terminal aryl
group of known selective agonist series 3a (Figure 1A).
The terminal phenyl ring need not be coplanar with the adenine ring, but a substantial
bending within the C2 substituent would not be favored. Compounds 3b and 3c, even
though predicted to bind the A3AR, would probably interact also with the A2AAR because
of flexibility in the position of the terminal phenyl ring, possibly resulting in reduced
selectivity. Moreover, the C2 linker is accommodated in a narrow region between Phe168
(EL2) and Ile268 (7.39) (~4 Å, Figure 1B). Therefore, we predicted a higher affinity at
the A3AR for those derivatives bearing a planar linker, such as 3a, 3e and 3f, compared to
those with wider and out-of-plane linkers, such as 3b-d. In light of these considerations,
compounds 3e and 3f would be the best alternatives to preserve the high affinity and
selectivity of the arylethynyl group. However, because of the potential chemical and
photochemical reactivity of a styryl group, we decided not to synthesize derivatives of
compound 3e but to focus on triazolyl derivatives. In fact, as highlighted in Table 1,

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triazole series 3f seemed to be the best alternative to alkynyl series 3a, because of its
stability, its rigid, planar and elongated C2 group (Figure S1, Supporting information)
and its synthetic feasibility. The terminal phenyl rings of 3a and 3f are largely
overlapping when docked to the A3AR. However, 3f is slightly more elongated than 3a
(by 0.8 Å), and it was shown previously that rigid C2 substituents more extended than
phenylethynyl are tolerated at the A3AR.7
Nucleoside derivatives 5 - 20 (Table 2) were synthesized by the route shown in Scheme
1. The previous series of arylethynyl derivatives was prepared by a Sonogashira
reaction15 of a substituted arylacetylene with a C2-iodo derivative of a (N)-methanocarba
adenosine. Here we access the triazole derivatives via a click reaction, [3+2]
cycloaddition of a congeneric set of 2-azidoadenosines (30 – 34) and arylacetylenes,16
which link adenine and C2-terminal aryl moieties (Scheme 1). The azide group is
introduced on adenine via reaction of sodium azide with the corresponding 2-iodo
intermediates (25 – 29), which were prepared as described from L-ribose.17 The product
2-(1,2,3-triazol-1-yl) nucleosides that were subsequently tested in binding assays were
demonstrated using HPLC to be of high purity (>95%). Click chemistry of (N)-
methanocarba adenosines was used previously to prepare various high affinity A3AR
ligands.18
Radioligand binding assays were performed at three subtypes of hARs that are relevant
for this chemical series and at the mouse (m) A3AR, which were stably expressed in
mammalian cells, as previously reported (Table 2).7,8 An unusual ferrocene-ethynyl

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derivative 4 was included as a reference compound due to its prolonged in vivo activity
and for comparison to its triazole analogue 20.8 Representative binding curves are shown
in Supporting Information. As predicted using the 3D model of the hA3AR, the affinity
and selectivity of the N6-methyl and N6-ethyl [1,2,3]triazolyl derivatives (5 – 15, 19, 20)
were extremely high. All of the derivatives (5 – 34) displayed Ki values in the range of
0.3 – 12 nM at the A3AR, but larger N6 groups, i.e. 16 – 18, reduced selectivity by
increasing A1AR affinity. The 2-iodo and 2-azido intermediates (25 – 34) were even less
selective in comparison to the A1AR and A2AAR. Thus, the triazolyl spacer constitutes a
suitable replacement for the ethynyl group with respect to the receptor binding profile.
Also, a triazole group like an alkyne is uncharged at physiological pH. A set of A3AR-
selective triazolyl derivatives in the 9-riboside series of adenosine agonists was reported
previously,19 and this (N)-methanocarba series extends the finding of high A3AR affinity
in the present set of analogues, and with comparable or greater selectivity. There is a
correlation (r2 = 0.66) between the A3AR binding affinity of twelve N6-methyl C2-
triazolyl derivatives reported here and the corresponding ethynyl derivatives (Figure 2).
This is consistent with the hypothesis that the terminal aryl ring occupies a similar
position in the two series when bound to the A3AR, as shown in Figure 2. Binding of
selected compounds at the mA3AR indicated that a C2-triazolyl-5-chlorothien-2-yl group
in compounds 15 – 17 maintained a relatively high affinity in the range of 7 – 14 nM with
various N6 groups. Curiously, the C2-triazolyl-pyrimidin-2-yl derivative 9 was much less
potent than other analogues at the mA3AR. The ligand efficiency20 with respect to A3AR
affinity was favorable: 1, 0.23; 2, 0.33; 9, 0.27; 15, 0.31; 17, 0.24.

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After confirming the A3AR-selectivity, the A3AR agonists were screened in vivo in the
mouse CCI model.21 The protection by A3AR agonists against chronic neuropathic pain
occurs at both central and peripheral sites of action.11 The methods used were as
reported,7,8 and the agonist was given by oral gavage (3 µmol/kg) on day 7 after sciatic
nerve ligation, i.e. the time when peak pain develops. The paw withdrawal threshold
(PWT) was measured up to 5 h after drug administration. The maximal efficacy in vivo
and the duration of action were indicated (Table 3). Each compound reached its peak
protection against chronic neuropathic pain by 1 h after administration. In the earlier
alkynyl series8 peak protection was achieved between 0.5 and 2 h after administration and
maintained up to 3 h (as with 5-chlorothienyl derivative 2, which suppressed neuropathic
pain to the degree of 83% protection at 3 h). By comparison, compound 1 at the same
dose displayed only 24% protection after 3 h.8 Here, at 1 h after drug treatment the
mechanoallodynia was a nearly completely reversed by some triazole derivatives, i.e.
pyrimidin-2-yl 9, N-methylpyrazolyl 11, 5-chlorothien-2-yl 15 and 5-bromothien-2-yl 19
analogues and compounds 16 – 18. For N6-methyl analogues, the majority of the pain
protection by the triazolyl derivatives waned by the 3 h time point (Figures 3A,B). In
analogues containing the same terminal 5-chlorothiophene group found in very
efficacious and long-acting agonist 2, an N6-ethyl group in 15 compared to the N6-methyl
homologue 14 produced a higher maximal protection at 1 h, i.e. 85% vs. 67%,
respectively. A 2-pyrimidyl group in 9 favored duration of action in vivo, with 36% of the
protection remaining at 3 h, which would not have been predicted by its relatively weak
mA3AR affinity. Larger N6 groups, e.g. 3-chlorobenzyl in 17 (>90% protection at 1 and 3
h), combined with a terminal chlorothienyl group greatly enhanced in vivo activity

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(Figure 3C). At a lower dose (1 µmol/kg), 17 produced the same duration of protection
but the maximal effect reached a plateau at ~70% (Figure S2, Supporting Information).
N6-Cyclobutyl 16 and N6-(2-phenylethyl) 18 derivatives were also prolonged in activity.
There is some consistency of the in vivo results between triazole and ethynyl series. For
example, a ferrocene group present in ethynyl derivative 4 and triazole 20 was associated
with high efficacy in vivo, although in vitro stability tests indicated that 4 was subject to
rapid degradation (t1/2 in human liver microsomes and pH 1.6 simulated stomach acid of
3.66 and 46.5 min, respectively).8 Therefore, we used mass spectrometry (Figures S3-S6,
Supporting information) to identify the immediate, transient product of acidic
decomposition of 4 upon exposure to aqueous HCl at pH 1.6. The result indicated a rapid
loss of Fe-cyclopentadiene as detected within 5 min by mass, leaving a cyclopentadiene
moiety remaining on the nucleoside (39, Chart 1B); however, 39 was highly unstable and
unable to be isolated. The corresponding cyclopentadiene 40 in the triazole series was not
detected upon incubation at pH 1.6 of 20, which was stable. Therefore, the substitution of
the 2-alkyne spacer with triazole increased the chemical stability of the ferrocene
derivative. This might result from greater delocalization of the ferrocene electron density
in the direction of adenine moiety in the extended triazole-linked aromatic system,
rendering the ferrocene moiety less readily protonated.
Off-target activities of several triazole derivatives (12, 14) were also determined in a
broad receptor binding screen (Supporting Information).22 The only interactions at IC50
<10 µM were with H2 histamine (12, 50% inhibition at 10 µM) and sigma2 receptors (12,

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50%; 14, 62%) and the peripheral benzodiazepine receptor (TSPO, 14, 54%). These few
interactions reinforced the A3AR selectivity of these representative compounds.
Conclusions
Derivatives of selective A3AR agonists containing a triazole linker in place of an ethynyl
group, a preferred substitution as predicted using molecular modeling, were prepared and
shown to parallel the ethynyl series in receptor affinity and selectivity. Docking of
possible linker moieties at the C2 position, using a molecular model of the A3AR,
correctly predicted that the receptor affinity and selectivity would be maintained due to
common structural features that favored selective binding at the A3AR. A planar C2-
triazole linker fit well in a narrow cleft when docked in the receptor binding site. The
effects of N6 substitution were also probed. All of these analogues contain a (N)-
methanocarba ring system to optimize A3AR affinity, which improves the selectivity in
comparison to the ribose series.5
The analogues were characterized in binding and in vivo assays to demonstrate efficacy
in controlling chronic neuropathic pain in agreement with A3AR selectivity. An N6-ethyl
group in 15 preserved in vivo activity compared to N6-methyl and binding selectivity,
while larger groups reduced A3AR selectivity. In comparison to the activities of the
corresponding 2-arylethynyl derivatives, the duration of action of many analogues tended
to be shorter.8 Nevertheless, among N6-methyl derivatives a terminal pyrimidin-2-yl
group in 9 extended the duration of action compared to other aryl groups. A highly
prolonged and full efficacy in reversing mechanoallodynia was achieved in a few

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derivatives, i.e. 16 – 18 that contained larger N6 substitution. The most enhanced in vivo
efficacy and duration was seen with N6-(3-chlorobenzyl derivative 17 (nearly full
protection up to 4 h).
The use of X-ray structures of closely related GPCRs in homology modeling, docking
and screening has facilitated understanding ligand recognition and drug discovery for the
A3AR and for other receptors.23 Here, the effects of potential structural changes were
probed computationally to select preferred structures to synthesize. The use of a
phenotypic assay, i.e. the CCI model, provides much information on efficacy, duration of
action and indirectly oral bioavailability. Thus, this study has progressed from the stage
of structure-based design to a comparison of molecules having the desired
pharmacological properties in in vitro and in vivo preclinical experiments. We have
combined efficient methods to narrow the group of nucleoside derivatives of potential
translational interest.
Acknowledgements
This research was supported in part by the Intramural Research Program of the NIH,
National Institute of Diabetes and Digestive and Kidney Diseases, and NIH R01
HL077707). We thank Noel Whittaker (NIDDK) for mass spectral determinations. We
thank Dr. Bryan L. Roth (Univ. North Carolina at Chapel Hill) and National Institute of
Mental Health's Psychoactive Drug Screening Program (Contract # HHSN-271-2008-
00025-C) for screening data.

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Supporting information
Supplementary data (chemical synthesis, characterization data, calculations and results of
in vivo experiments, modeling procedures and off-target screening) associated with this
article can be found in the online version, at doi:.

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Notes and references
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Rheumatol. 2008, 35, 41.
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25 Melman, A., Gao, Z. G., Kumar, D., Wan, T. C., Gizewski, E., Auchampach, J. A.,
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Figure and Scheme Legends
Chart 1. A. Structures of recently reported highly selective A3AR agonists (1, 2) and the
structural options considered in the present study (3). R is a small alkyl, cycloalkyl or
substituted arylalkyl group; Ar refers to substituted phenyl or heterocyclic groups or
ferrocene (See compound 4, Table 2). B. Hypothetical transient breakdown products
39 and 40 from acid treatment of the ferrocene derivatives 4 and 20, respectively.
Only 39 was detected by mass upon acidification of 4; compound 20 was stable at pH
1.6.
Scheme 1. Synthesis of A3AR agonists containing triazolyl spacers between the C2
position of adenine and a terminal aryl group. For the structures of 5 - 20 see Table 2.
Figure 1. Superposition of the docking poses of trial compounds 3a – f (R = CH3, Ar =
Ph) in the hA3AR hybrid homology model. A. Side view of the receptor (shown in
cyan ribbon). Ligands are shown in sticks with different carbon colors (3a, dark
green; 3b, orange; 3c, pink; 3d, yellow; 3e, magenta; 3f, light green). Side chains of
residues important for ligand recognition are shown in sticks (grey carbons), and H-
bonding interactions are indicated by red dashed lines. Non-polar hydrogen atoms are
not displayed. The view of TM7 is partially omitted. B. Top view of the receptor
showing the surface of the binding site residues in cyan. Ligands are shown in sticks
with different carbon colors (color scheme as in panel A). The distance between
Phe168 (EL2) and Ile268 (7.39) is highlighted.

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Figure 2. Correlation of the binding affinities at the hA3AR of parallel series of triazolyl
(Y-axis) and ethynyl (X-axis) derivatives.8 The values shown include those
determined for triazolyl compounds 5 – 14, 19, and 20.
Figure 3. Protection against hind paw mechanoallodynia in mice resulting from CCI of
the sciatic nerve. Triazolyl derivatives were administered at a single dose (p.o.) of 3
µmol/kg at the point of peak pain (7 days post surgery; representative curves of 2-4
determinations). A. Derivatives 5, 6 and 8 – 10, in which the terminal aryl group is a
phenyl, substituted phenyl or six-membered heterocycle. B. Derivatives 7, 11, 13, 19
and 20, in which the terminal aryl group is a 3,4-difluorophenyl or five-membered
heterocycle. C. Derivatives 16 – 18, with large N6 substitution and in which the
terminal aryl group is 5-chlorothien-2-yl. Data are mean ± SEM of n=2 or 4 and
analyzed by two-tailed, two-way ANOVA with Bonferroni comparisons to D0 or
D7/BL. *P<0.05 vs. D0 and †P<0.05 vs. D7/BL.

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Chart 1.
A
Cl
R
CH3
N
HN
N
HN
HN
N
N
N
N
N
N
N
N
N
a
N
H
H
H
N
O
N
O
F
F
N
O
S
H3C
H3C
H3C
Ar
Cl
-CH2-
b
c
1
3
2
OH
OH
OH
OH
OH
OH
-(CH2)2-
d
e
f
-CH=CH-
N
N
N
B
CH3
N
CH3
N
HN
HN
N
N
N
N
N
N
H
N
H
N
N
O
O
H3C
H3C
N
N
39
OH
OH
OH
OH
40

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Scheme 1.
R
R
Cl
HN
HN
N
N
N
N
N
N
N
N
N
O
O
O
N
N
I
N
I
EtOC
I
MeHNC
EtOC
i
ii
L-Ribose
21
O
O
O
O
O
O
22
23a - e
24a - e
R =
d
e
a CH3
Cl
b CH2CH3
c c-Bu
R
R
R
HN
N
HN
N
HN
N
N
N
N
N
N
N
N
iii
iv
O
O
v
O
N
Ar
I
N3
MeHNC
MeHNC
N
N
MeHNC
N
N
25-29
30-34
OH OH
OH OH
5-20
OH OH
Cl
R = CH3
25, 30
28, 33
29, 34
CH2CH3 26, 31
c-Bu 27, 32
Reagents and condition: (i) RNH2, Et3N, MeOH, rt; (ii) 40% MeNH2, MeOH, rt; (iii) 10% TFA, MeOH, 70 oC; (iv) NaN3,
t
Sod.ascorbate, CuSO4.5H2O, Na2CO3, L-proline, BuOH-H2O, 65 oC; (v) Ar-alkynes, Sod. ascorbate, CuSO4. 5H2O, TBTA,
tBuOH-H2O

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Figure 1.

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Figure 2.

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Figure 3.

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Table 1. Comparison of the features of C2 modifications 3a-f (Chart 1A, Ar = C6H5). A
check mark indicates favorable (planarity and rigidity, with respect to the binding cleft in
the C2-extended adenine region of the A3AR as in Figure 1) or unfavorable criteria
(reactivity) as applied to each linker. All of the maximum lengths shown are within the
range of the proposed outward displacement of TM2 in the hybrid A3AR model.
Linker Features
Linker
Planaritya Rigidityb Max Lengthc Potential Reactivityd
a
C≡C
8.00
✔
✔
✔
b
c
CH2
6.37
5.84
7.61
(CH2)2
d
cyclopropyl
✔
✔
✔
e
f
CH=CH
triazole
7.70
8.80
✔
✔
✔
a Ability to fit in a narrow space. This refers to the planarity of the linker only not of the
overall C2 substituent.
b Inability to bend.
c Distance measured between the C2 of the adenine core and the most distal hydrogen of
the phenyl ring.
d Potential toxicity and/or reactivity with nucleophiles in vivo.

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Table 2. Structures and binding affinitiesa of AR agonists, including reference
compounds 1-4, triazole derivatives 5-20 and their synthetic intermediates 25-34.
R1
N
R1
N
R1
N
HN
N
HN
N
HN
N
N
N
N
N
N
N
H
N
H
N
R3
N
N
R2
H
N
O
O
O
R2 H3C
H3C
H3C
N
OH OH
OH OH
OH OH
1 – 4
5 – 20
25 – 34
A1AR
A3AR
Compd.
A2AAR
R1
R2 or R3
% inhibition
% inhibition
% inhibitionc
or Ki (nM)a
or Ki (nM)a
F
Cl
1b,d
2b,d
3.49±1.84,
3.08±0.23 (m)
6 ± 4%
6 ± 1%,
41 ± 10%
F
S
Cl
0.70 ± 0.11,
36.1±4.7 (m)
CH3
CH3
24 ± 13%,
47 ± 2%
4b,d
25 ± 2%
2.68 ± 0.44
5d
6
CH3
CH3
18 ± 4%
20 ± 5%
35 ± 8%
31 ± 6%
0.96 ± 0.07
0.95 ± 0.50
Cl
F
7
CH3
9 ± 4%
39 ± 4%
1.06 ± 0.10
1.84 ± 0.38
F
N
8
9
CH3
CH3
29 ± 7%
14 ± 7%
25 ± 10%
26 ± 2%
27 ± 3%
8 ± 3%
N
3.48 ± 0.97,
334 ± 49 (m)
N
N
10
CH3
CH3
CH3
2.21 ± 0.34
2.16 ± 0.32
0.96 ± 0.09
N
CH3
11
12
N
2 ± 2%
1 ± 1%
N
O
43 ± 2%
51 ± 1%

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O
13
14
CH3
CH3
12 ± 7%
40 ± 6%
34 ± 5%
1.25 ± 0.27
0.73 ± 0.10
S
Cl
27 ± 8%
1.22 ± 0.26,
9.64 ± 0.84
(m)
7.05 ± 5.81,
14.2 ± 0.2 (m)
15d
16
S
Cl
C2H5
15 ± 11%
32 ± 5%
ND
S
Cl
77%
9.02 ± 5.78,
6.53 ± 0.58
(m)
Cl
S
Cl
17
65%
ND
S
Cl
18
19
58%
ND
6.66 ± 2.07
0.58 ± 0.17
S
Br
CH3
CH3
33 ± 7%
23 ± 3%
7 ± 6%
20
54 ± 2%
3.09 ± 0.21
25b
26
CH3
I
I
I
ND
1910 ± 300
93%
ND
1.91 ± 0.85
1.22 ± 0.21
1.53 ± 0.45
CH2CH3
11 ± 7%
>10,000
27
Cl
28b
29
2200e
I
I
>10,000
3.6
68%
42%
0.91 ± 0.14
30
31
32
CH3
N3
N3
N3
77 ± 1%
707 ± 152
100%
1 ± 1%
11 ± 6%
46%
0.54 ± 0.10
0.69 ± 0.08
0.85 ± 0.10
CH2CH3
Cl
33
2770e
N3
ND
1.08 ± 0.75

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34
1110e
N3
93%
0.85 ± 0.10
a
Binding in membranes prepared from CHO or HEK293 (A2A only) cells stably
expressing one of three hAR subtypes, unless noted. The binding affinity for hA1, A2A
and A3ARs was expressed as Ki values (n = 3−4, unless noted), measured using agonist
radioligands [3H]N6-R-phenylisopropyladenosine 35, [3H]2-[p-(2-carboxyethyl)phenyl-
ethylamino]-5ʹ-N-ethylcarboxamido-adenosine
36,
or
[125I]N6-(4-amino-3-
iodobenzyl)adenosine-5ʹ-N-methyl-uronamide 37, respectively. Additional values
designated (m) are for mouse ARs. A percent in italics refers to inhibition of binding at
10 µM. Nonspecific binding was determined using 38 (10 µM). Values are expressed as
the mean ± SEM. Ki values were calculated as reported.24
b Data from Tosh et al.7,8 and Melman et al.25
c Percent of inhibition at 10 µM.
d 1, MRS5698; 2, MRS5980; 4, MRS5979; 5, MRS7110; 15, MRS7126; 17, MRS7138.
e n = 1.
ND – not determined.

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Table 3. Activity of orally administered A3AR agonists (3 µmol/kg) in CCI model of
neuropathic pain (mechanoallodynia) in mice and physicochemical parameters.
Compound
Max. effect
Effect at 3h
n
MW cLogPb tPSA
Emax (%±SEM)a (%±SEM)
(D)
(Å2)
1
100 ± 0.0
93.3 ± 6.7
94.3 ± 5.7
77.4±4.5
69.1±8.6
75.2±12.8
60.9±11.9
92.7±9.8
76.6±0.9
82.5±5.9
65.3±8.2
74.0±10.7
66.8±19.4
84.9±14.0
90.7±6.0
94.7±3.1
94.1±5.9
69.1±8.6
23.7 ± 10.8
82.8 ± 11.2
78.1 ± 3.5
27.5±4.1
7.9±7.9
4
4
4
4
2
4
4
4
4
4
2
4
2
4
4
4
2
2
565
459
526
461
496
497
462
463
463
465
451
502
502
515
542
612
592
546
4.15 122
2.17 122
1.42 122
1.58 150
2.04 150
1.79 150
0.29 162
-0.67 175
-0.67 175
-0.31 165
0.96 159
2.34 159
2.19 150
2.72 150
3.10 150
4.35 150
4.29 150
2.34 150
2
4
5
6
7
27.0±7.4
21.1±3.3
36.2±6.8
26.6±6.3
19.1±5.9
21.1±7.0
22.3±8.3
8.4±7.2
8
9
10
11
12
13
14
15
16
17
18
19
17.9±12.6
85.2±9.0 4
91.0±4.5 4
82.9±5.3 2
13.5±2.3

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20
94.1±5.9
22.4±6.6
2
569
1.22 165
a Percent represents reversal of reduction in PWT of the ipsilateral hind paw. The time of
peak for compounds 5 – 20 is 1 h. Data for 1, 2 and 4 are from Tosh et al.8 No
mechano-allodynia or A3AR agonist effect on PWT was observed on the contralateral
side.
b calculated using ChemBioDraw, v. 14.0.