Bitopic fluorescent antagonists of the A2A adenosine receptor based on pyrazolo[4,3-: E] [1,2,4]triazolo[1,5- c] pyrimidin-5-amine functionalized congeners

Article abstract of DOI:10.1039/c7md00247e

A pyrazolo[4,3-e][1,2,4]triazolo[1,5-c]pyrimidin-5-amine antagonist of the A_2A adenosine receptor (AR) was functionalized as amine congeners, fluorescent conjugates and a sulfonate, and the A_2AAR binding modes were predicted computationally. The optimal n-butyl spacer was incorporated into the following A_2AAR-selective (K_i, nM) conjugates: BODIPY630/650 derivative 11 (MRS7396, 24.6) and AlexaFluor488 derivative 12 (MRS7416, 30.3). Flow cytometry of 12 in hA_2AAR-expressing HEK-293 cells displayed saturable binding (low nonspecific) and inhibition by known A_2AAR antagonists. Water-soluble sulfonate 13 was a highly potent (K_i = 6.2 nM) and selective A_2AAR antagonist based on binding and functional assays. Docking and molecular dynamics simulations predicted the regions of interaction of the distal portions of these chain-extended ligands with the A_2AAR. The BODIPY630/650 fluorophore of 11 was buried in a hydrophobic interhelical (TM1/TM7) region, while AlexaFluor488 of 12 associated with the hydrophilic extracellular loops. In conclusion, we have identified novel high affinity antagonist probes for A_2AAR drug discovery and characterization.

Full text of DOI:10.1039/c7md00247e

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Ciancetta, P. Mannes, J. Yu, S. Boyapati, E. Gizewski, S. Yous, F. Ciruela, J. Auchampach, Z. Gao and K.
Jacobson, Med. Chem. Commun., 2017, DOI: 10.1039/C7MD00247E.
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Page 1 of 26
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Med. Chem. Comm.
Bitopic Fluorescent Antagonists of the A2A Adenosine Receptor Based on Pyrazolo[4,3-
e][1,2,4]triazolo[1,5-c]pyrimidin-5-amine Functionalized Congeners
Romain Duroux,a,b,f Antonella Ciancetta,a,f Philip Mannes,a Jinha Yu,a Shireesha Boyapati,a,c Elizabeth
Gizewski,d Said Yous,b Francisco Ciruela,e John A. Auchampach,d ZhanꢀGuo Gao,a
and Kenneth A. Jacobsona*
a Molecular Recognition Section, Laboratory of Bioorganic Chemistry, National Institute of Diabetes and
Digestive and Kidney Diseases, National Institutes of Health, Bethesda, MD 20892.
b Univ. Lille, Inserm, CHU Lille, UMRꢀS 1172 ꢀ JPArc ꢀ Centre de Recherche JeanꢀPierre AUBERT
Neurosciences et Cancer, Fꢀ59000 Lille, France.
c Department of Pharmaceutical Chemistry, Telangana University, Nizamabad, Telangana,
India 503322.
d Department of Pharmacology, Medical College of Wisconsin, 8701 Watertown Plank Road,
Milwaukee, Wisconsin 53226 USA.
e Unitat de Farmacologia, Departament Patologia i Terapeutica Experimental, Facultat de Medicina,
IDIBELL, Universitat de Barcelona, 08907 L'Hospitalet de Llobregat, Spain.
f Contributed equally.
Corresponding author: Dr. K.A. Jacobson, Chief, Molecular Recognition Section, Bldg. 8A, Rm.
B1Aꢀ19, NIH, NIDDK, LBC, Bethesda, MD 20892ꢀ0810. Tel: 301-496-9024. Fax: 301-480-8422;
Email: kennethj@niddk.nih.gov
ABBREVIATIONS: AN, acetonitrile; AR, adenosine receptor; BODIPY, 4,4ꢀdifluoroꢀ4ꢀboraꢀ3a,4aꢀ
diazaꢀsꢀindacene; CNS, central nervous system; DMF, N,Nꢀdimethylformamide; EDC, Nꢀ(3ꢀ
dimethylaminopropyl)ꢀN’ꢀethylcarbodiimide; HEK, human embryonic kidney; IE, interaction energy;
NECA, 5′ꢀNꢀethylcarboxamidoadenosine; PD, Parkinson’s disease; TBAP, tetrabutylammonium
dihydrogen phosphate; THF, tetrahydrofuran; TLC, thin layer chromatography; TM, transmembrane
domain.

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Abstract
A pyrazolo[4,3ꢀe][1,2,4]triazolo[1,5ꢀc]pyrimidinꢀ5ꢀamine antagonist of the A2A adenosine receptor (AR)
was functionalized as amine congeners, fluorescent conjugates and a sulfonate, and A2AAR binding
modes were predicted computationally. The optimal nꢀbutyl spacer was incorporated into the following
A2AARꢀselective (Ki, nM) conjugates: BODIPY630/650 derivative 11 (MRS7396, 24.6) and
Alexafluor488 derivative 12 (MRS7416, 30.3). Flow cytometry of 12 in hA2AARꢀexpressing HEKꢀ293
cells displayed saturable binding (low nonspecific) and inhibition by known A2AAR antagonists. Waterꢀ
soluble sulfonate 13 was a highly potent (Ki = 6.2 nM) and selective A2AAR antagonist based on binding
and functional assays. Docking and molecular dynamics simulations predicted the regions of interaction
of the distal portions of these chainꢀextended ligands with the A2AAR. The BODIPY630/650
fluorophore of 11 was buried in a hydrophobic interhelical (TM1/TM7) region, while AlexFluor488 of
12 associated with the hydrophilic extracellular loops. In conclusion, we have identified novel high
affinity antagonist probes for A2AAR drug discovery and characterization.
Keywords: adenosine antagonist; SCH442416; G proteinꢀcoupled receptor; fluorescence; radioligand
binding.

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Introduction
The development of selective agonists and antagonists of the four subtypes of adenosine receptors (ARs)
has been extensively explored.1ꢀ3 Antagonists of the Gs proteinꢀcoupled A2AAR are sought as agents for
treating neurodegenerative conditions such as Parkinson’s disease (PD) and Alzheimer’s disease (AD),
and for coadministration with cancer immunotherapy.4ꢀ7 Caffeine, the most consumed psychostimulant
in the world, acts as a nonselective AR antagonist and readily enters the brain to antagonize the A2AAR
at doses generally consumed. Epidemiological evidence showing a lower occurrence of AD and PD with
modest caffeine intake points to the possibility that caffeine consumption is neuroprotective. Indeed,
A2AAR antagonists can exert a neuroprotective effect against excitotoxicity in animal models; one
mechanism appears to be enhancing the activity of an A1AR, which forms a heterodimer with A2AAR, to
inhibit glutamate release.8 A2AR antagonists also control microgliaꢀmediated neuroinflammation.9
Therefore, it is possible that A2AAR antagonists may also delay neurodegenerative disease progression.
In the striatum, A2AAR antagonists act to boost dopaminergic signaling, and thus provide symptomatic
relief to reduce the motor deficits in PD without inducing dyskinesia. Certain selective A2AAR
antagonists, e.g. 1ꢀ4 (Chart 1), have been shown to enter the brain in sufficient levels for imaging and
efficacy in PD models.10ꢀ12 A caffeineꢀlike 1,3,7ꢀtrialkylxanthine, istradefylline 1 is approved in Japan
for treating PD, and its safety and efficacy in reducing in offꢀtime in levodopaꢀtreated patients were
established in a 52ꢀweek trial.10 A tricyclic pyrazoloꢀtriazolopyrimidine derivative 4, which binds
potently and selectively to the A2AAR, was in clinical trials for PD.12
In the periphery, elevated adenosine levels present in the microenvironment of tumors lead to a
suppressed immune response, to shift the T cell response away from an aggressive state capable of
attacking tumors.6 Increasing evidence supports the use of AR antagonists in cancer treatment, either

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with selectivity for A2AAR alone or with dual selectivity for A2AAR and A2BAR. Cytokine production in
CD8+ chimeric antigen receptor (CAR) T cells was increased, and both CD8+ and CD4+ CAR T cells
were activated upon blocking the A2AAR.7 Thus, there is great interest in the discovery of novel A2AAR
antagonists to act either centrally or peripherally, and new tools for ligand discovery are needed.
Various fluorescent probes have been developed over the past few years for characterizing ARs,13ꢀ15 and
their use in fluorescence polarization (FP), fluorescence resonance energy transfer (FRET) and flow
cytometry (FCM) has proven feasible. Moreover, these ligands can provide a better understanding of
receptor location, function and regulation. Most of the reported fluorescent A2AAR antagonists used 5ꢀ
aminoꢀ7ꢀ(3ꢀ(4ꢀmethoxy)phenylpropyl)ꢀ2ꢀ(2ꢀfuryl)pyrazolo[4,3ꢀe]ꢀ1,2,4ꢀtriazolo[1,5ꢀc]pyrimidine
(SCH442416, 3) as the pharmacophore due to a pꢀmethoxyphenylpropyl side chain moiety of the
antagonist, which served as the site for attachment of functionalized chains through an ether linkage.
Much of the ligand development for ARs now benefits from a detailed structural knowledge of the
human (h)A2AAR.16ꢀ20 More than two dozen highꢀresolution Xꢀray crystallographic A2AAR structures
with bound agonist or antagonist have been determined and used for the in silico screening of chemical
libraries. A2AAR
complexes with antagonist 4ꢀ[2ꢀ[7ꢀaminoꢀ2ꢀ(2ꢀfuryl)ꢀ1,2,4ꢀtriazolo[1,5ꢀ
a][1,3,5]triazinꢀ5ꢀylꢀamino]ethylphenol (ZM241385, 2) show that the heterocyclic pharmacophore binds
to residues in the orthosteric binding pocket that also coordinate the adenine moiety of AR agonists.16,17
Although there is no Xꢀray structure of a 3ꢀA2AAR complex, we used the 2ꢀA2AAR structure as
structural template for molecular modeling. The hypothetical docking pose of 3 and its AlexaFluor488
derivative 5 established most of the conserved interactions in the orthosteric binding site.13,14 The

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modeling also predicted stabilizing interactions of the tethered fluorophore with specific charged and Hꢀ
bonding residues of the second extracellular loop (EL2).
Conjugate 5 was not optimal for fluorescent binding due to its moderate hA2AAR affinity (Ki = 111
nM).13 Also, its green light emission presents difficulties in fluorescence microscopy due to cell
autofluorescence. Thus, there remains a need for A2AAR antagonist fluorescent probes of higher affinity
and compatibility with microscopy. A BODIPY650/655 conjugate containing a secondary amine in the
linking chain was reported to have a Ki value of 15 nM at the A2AAR, but its utility was not
established.13 With this aim, we explored further the SAR of the distal region of this chemical series by
varying the chain length of the spacer group and the terminal fluorophore in order to enhance affinity,
selectivity and photophysical properties.

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Chart 1. Structures of selective A2AAR antagonists and the target series: (A) Widely used
pharmacological probes and clinical candidates (1ꢀ4); (B) A reported fluorescent probe 5 derived from 3
and functionalized amine congeners of varied chain length 6 explored in this study.
Results and Discussion
We initially prepared a series of primary amine congeners of an A2AAR antagonist 3 by extending the pꢀ
methoxyphenylpropyl chain, which is predicted to lie outside the orthosteric binding site of the
pharmacophore and be able to reach accessory sites.13 The congener 6a (n = 2) was reported earlier,13

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and we hypothesized that extension of the alkyl spacer could enhance affinity or selectivity at this
receptor, perhaps by establishing Hꢀbond or electrostatic interactions with polar residues in the EL
region of the A2AAR. The synthesis of the pyrazolo[4,3ꢀe][1,2,4]triazolo[1,5ꢀc]pyrimidinꢀ5ꢀamine
derivatives is shown in Scheme 1.
The synthesis of fluorescent ligands 9-12 was accomplished in 4 steps from commercially available 3.
The latter was demethylated to 7 by the action of BBr3 and then treated with an excess of methylꢀ2ꢀ
bromoacetate (12 eq.) and Cs2CO3 as base to provide ester 8 in 90% yield. Other methods, such as using
NaH as base with 1 eq. of methyl 2ꢀbromoacetate in DMF, were attempted but led to the formation of di
and triꢀalkylated compounds. Amide synthesis was then performed by treatment of ester 8 with a series
of alkyl diamines to give the amine congeners 6a-e. Two of these amines, 6a (n = 2) and 6c (n = 4),
were reacted with activated fluorophore moieties in DMF in the presence of Et3N to afford, after semiꢀ
preparative HPLC, compounds 9-12 in good yield (40ꢀ60%): cyanine 5 red fluorescent AlexaFluor647
(n=2, 9; n=4, 10), and red fluorescent BODIPY630/650 (n = 4, 11) and green fluorescent AlexaFluor488
(n = 4, 12). Both fluorophores are commonly incorporated into ligand tools for chemical biology.21

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Scheme 1. Synthesis of A2AAR antagonist functionalized congeners 6 and their fluorescent derivatives
o
9ꢀ12. (a) BBr3, CH2Cl2, rt, 4h; (b) methyl 2ꢀbromoacetate, Cs2CO3, MeOH, 40 C, overnight; (c)
diaminoalkane, MeOH (9:1 v/v), rt, overnight; (d) activated fluorophore (AlexaFluor647 Nꢀ
hydroxysuccinimidyl ester for 9 and 10, BODIPY 630/650 Nꢀhydroxysuccinimidyl ester for 11,
AlexaFluor488 carboxylic acid, 2,3,5,6ꢀtetrafluorophenyl ester for 12), Et3N, DMF, rt, overnight.

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The affinity of the amine congeners and other antagonists was measured using standard radioligand
binding assays at hA1, A2A and A3ARs (Table 1).1,13,14,22 Membranes of human embryonic kidney
(HEK)293 cells expressing the AR of interest were used in the assay. Primary amine congeners 6a ꢀ 6d
containing spacers of 2 – 5 methylenes were similar in hA2AAR affinity (Ki 6 ꢀ 9 nM), but the
butylamino congener 6c displayed the highest A2AAR selectivity in the series. The affinity of this
compound at hA3AR was determined to be 6.0 µM, with only slightly higher hA1AR affinity.
Homologation to 6 methylenes in compound 6e lowered A2AAR affinity. Among the fluorescent
conjugates of butylamino congener 6c, i.e. 10, 11, and 12, the highest A2AAR affinity and selectivity
were observed with the BODIPY630/650 fluorophore 11 and AlexaFluor488 12 (Figure 1). Thus,
fluorescent 11 and 12 were only 4ꢀfold weaker in A2AAR affinity with respect to parent amino derivative
6c. Interestingly, AlexaFluor488 conjugate 12 was at least as potent in A2AAR affinity as its shorter
homologue 5. No dependence of affinity on chain length was evident when comparing AlexaFluor647
conjugates 9 and 10. Affinity at the mouse (m) ARs was also measured for selected compounds by
methods described,22 and 11 was particularly potent and selective at the mA2AAR (Ki = 2.1 nM), in
contrast to 12 (Ki 585 nM).
We performed a molecular modeling analysis to identify possible binding modes of fluorescent
conjugates 11 and 12 using the high resolution 2ꢀA2AAR Xꢀray structure (PDB ID: 4EIY).17 We first
docked reference compound 3 at the hA2AAR by retaining several water molecules observed in the Xꢀ
ray structure as described (Supporting Information). In the corresponding docking pose (Figure 2A), the
pyrazolotriazolopyrimidine core of 3 docked in the orthosteric binding site similarly to the
triazolopyrimidine core of 2, with a πꢀπ stacking interaction of the aromatic core with F168 (EL2), and a
Hꢀbonding network with N253 (6.55, using standard GPCR notation23) and E169 (EL2). The methoxyꢀ

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phenyl substituent of 2 pointed toward the extracellular (EC) side of the receptor and interacted with the
side chain of Glu169 (EL2) through a water molecule. The binding mode of 3 described above was
validated using 30 ns of molecular dynamics (MD) simulation. During the simulation (Video S1, replica
analysis reported in Table S1), the methoxyꢀphenyl moiety folded toward transmembrane domain 2
(TM2) and established a πꢀπ stacking interaction with Y271 (7.36), while the aromatic core maintained
the Hꢀbond network observed in the initial docking pose. Notably, the most energetically favored ligandꢀ
protein complex featured the same interaction pattern described above (Figure S1).
The potent fluorescent conjugate 11 was then docked in the A2AAR structure (Figure 2B) with the same
water molecules retained as in the 3ꢀA2AAR complex.17 The pyrazolotriazolopyrimidine core established
the same interactions observed for 3 in the orthosteric binding site. The fluorophore linker pointed
toward the EC side and folded back toward the TM bundles by directing the fluorophore group to an
aromatic pocket at the interface between TM1 and TM7 (transparent surface in Figure 2B). To explore
other orientations of the linker at the receptor’s EC side, we docked compound 6c, the amino precursor
of 11, at the hA2AAR. To sample all the possible Hꢀbond acceptor/donor partners on the EC side of the
receptor, we removed water molecules interacting with E169 during the docking. We then clustered the
binding modes obtained according to the linker orientation. This selection resulted in two alternative
binding modes (Figure S2) that were subsequently subjected to MD validation (30 ns of simulation run
in triplicate for each binding mode, see Table S1). In the most energetically favored docking mode
(hereby referred as “BM1”, orange carbon sticks in Figure S2, docking score = ꢀ12.077 kcal/mol), the
tail was oriented toward TM4 and TM5 with the amide moiety establishing a Hꢀbond with the side chain
of E169, while the terminal amine group Hꢀbonded with the backbone of E169 (EL2) and the side chain
of K150 (EL2). This latter Hꢀbond was expected to be unstable in a dynamic environment due to

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competition in the formation of a saltꢀbridge between K150 and D170 (EL2). In the alternative binding
mode (BM2, green carbon sticks in Figure S2, docking score = ꢀ10.994 kcal/mol), the tail was oriented
toward TM1 and TM2 and did not establish additional interactions. MD trajectory analysis revealed that
BM1 achieved ligandꢀprotein complexes that were more energetically favored (data not shown). Figure
S3 depicts the two 11ꢀA2AAR complexes with the most favorable ligandꢀprotein interaction energy (IE,
values differed by less than 2 kcal/mol and were considered equivalent) obtained for BM1 that feature
different orientations of the tail. We therefore searched for hydrophobic and aromatic regions in
proximity of the terminal amine moiety to investigate the compatibility of these orientations with the
fluorophore insertion. Specifically, we searched for receptor regions rich in hydrophobic and aromatic
residues at 5, 13, and 14 Å from the nitrogen atom of the terminal alkylamino group of 6c (Figure S3B).
The choice of distances reflected the analysis of the Nꢀgroup distance to aromatic rings in both the
docked and the energy minimized threeꢀdimensional structures of 11 (Figure S3A). Of the two possible
orientations of 11, only one displayed hydrophobic and aromatic residues at distances compatible with
the insertion of the aromatic fluorophore. Notably, the aromatic/hydrophobic region is located at the
interface between TM1 and TM7 (Figure S3C), thus suggesting the same orientation of the ligand as
resulted by the docking analysis. Nonetheless, we cannot exclude that 11 might explore different regions
on the receptor’s EC side.
The fluorescent conjugate 12 (considered as the species carrying a ꢀ2 net charge) was docked in the
A2AAR structure by following the same procedure described for compound 11 (Supporting Information).
The docking output suggested two equally plausible orientations of the fluorophore (Figure 2C and D).
In one docking pose (BM1, cyan carbon sticks in Figure 2C, docking score = ꢀ11.490 kcal/mol), the
fluorophore group was projected toward EL3. In the alternative binding mode (BM2, purple carbon

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sticks, docking score = ꢀ11.279 kcal/mol), the fluorophore group interacted with residues in EL2 and
EL3. The interaction pattern of the core was the same as for other members of this chemical series: Hꢀ
bonds with N253 and E169 (EL2); πꢀπ stacking with H252 (6.52), F168 (EL2), and Y271 (7.36). In the
MD simulation starting from BM1 (30 ns run in triplicate, see Table S1) the fluorophore group and the
linker fluctuated on the EC surface of the receptor without engaging in specific interactions except for a
Hꢀbond involving the fluorophore carboxylate moiety (data not shown). On the other hand, the
simulations starting from BM2 (30 ns, replica analysis in Table S1) achieved more energetically favored
ligandꢀprotein complexes. The simulations returned the ligandꢀprotein complex characterized by the
lowest IE value for all trajectories and converged in a unique binding mode (Figure S4) featuring the
fluorophore group stacked between EL2 and EL3. In such a conformation, the ligand established a tight
network of Hꢀbonds and saltꢀbridges between charged residues in EL2 and EL3 and polar/charged
counterparts in the fluorophore moiety. In particular, during the MD simulation (visualization of run3
trajectory selected as example, Video S2) the sulfonic groups established salt bridges with K153
(persistent) and K150 (intermittent) in EL2, one of the amine moieties interacted with the sidechain of
E169 (EL2), and the carboxylate moiety of the ligand replaced E169 (EL2) in the salt bridge with H264
(EL3). Moreover, for most of the total simulated time, the pyrazolotriazolopyrimidine core maintained
its interaction pattern with N253 (6.55) and F168 (EL2), while the linker between the core and the
fluorophore group was anchored to TM7, EL2 and TM2 through Hꢀbond interactions with the backbone
of S67 (2.65) and the sidechains of Q157 (EL2) and Y271 (7.36), respectively (Video S2).
Thus, both 11 and 12 are bitopic in the sense that each bridged two separate domains of the A2AAR, i.e.
the orthosteric binding site that is well defined in Xꢀray structures16,17 and an additional domain. The
BODIPY630/650 fluorophore of 11 is predicted to be buried in a hydrophobic region, while

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AlexFluor488 of 12 associates with the hydrophilic ELs. The bitopic nature of these conjugates does not
necessarily imply allosteric modulation of the orthosteric action,28 which is unexplored in this series.
In order to further explore ligand interaction with the EC surface, we prepared an aryl sulfonate 13,
which contained a terminal group capable of multiple polar interactions (Scheme S1, Supporting
Information). This terminal phenylsulfonic acid moiety also would allow for πꢀπ interactions with
aromatic residues. A further benefit of incorporating a sulfonate group is that it carries a permanent
negative charge at physiological pH and would prevent penetration of the blood brain barrier,22 a useful
characteristic in a pharmacological probe for in vivo studies.24 13 was potent and selective in binding to
the h and mA2AARs, with selectivity compared to the hA1AR and hA3AR of 671ꢀ and 426ꢀfold,
respectively. 13 binding to the mA1AR and mA3AR was insignificant.
The binding of 13 was modeled using the same docking procedure and MD validation described for 6c.
Figure 3 depicts the three alternative binding modes of 13. In the most energetically favorable pose
(BM1, blue carbon sticks) the sulfophenyl ring established a πꢀπ stacking interaction with H264 (EL3)
and the sulfonic group engaged in an Hꢀbond interaction with the residue backbone. In the other two
docking poses (BM2 and BM3, magenta and orange carbon sticks, respectively) the sulfophenyl ring
established a πꢀcation interaction with K153 (EL2) and the sulfonic group interacted with the side chain
of either K153 (EL2; BM2, magenta) or S156 (EL2; BM3, orange). During the MD simulation, the
ligand’s sulfophenyl tail fluctuated considerably (high averaged Root Mean Square Deviation values,
Table S1), thus demonstrating the instability of the interaction pattern predicted in the initial docking
poses. On the other hand, the three different initial poses converged in a unique binding mode, as the
ligandꢀprotein complexes with the lowest IE value returned by each different binding mode featured the

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same conformation (Figure S5). In particular, the 7ꢀphenylpropyl ring moved toward TM7 to establish a
πꢀπ stacking interaction with Y271 (7.36).
Two representative derivatives were shown to be A2AAR antagonists in a cyclic AMP assay (Figure S6).
The activation curve for known agonist 15 was rightꢀshifted in a parallel manner by fixed concentrations
of 11 and 13. The EC50 of 15 was shifted from 0.89±0.17 nM to 128±35 nM in the presence of 11 (1000
nM) and to 10.2±2.3 nM in the presence of 13 (100 nM). Offꢀtarget binding activities at 45 diverse
receptors were determined by the PDSP25 for selected compounds: 6c and 13 (Supporting information).
The only Ki values below 10 µM were: 6c, 3.36 µM, 5HT2A serotonin receptor; 1.92 µM, 5HT2B
receptor. Thus, the primary amine precursor of the fluorescent conjugates 10 ꢀ 12 was not promiscuous
in its interaction with other proteins, i.e. this chemical series is not associated with panꢀassay
interference compounds.26
Compounds 11 and 12 were tested as fluorescent tracers for flow cytometry of HEKꢀ293 cells
expressing the hA2AAR (Figure 4A). 11 proved to have high nonspecific binding, with a low level of
specific binding amounting to <25% of total and therefore was not optimal for flow cytometric analysis.
Thus, 11 tended to bind nonꢀspecifically to hydrophobic membranes by undetermined mechanisms.
However, the more hydrophilic 12 displayed low nonspecific binding by this method, and its binding
was saturable with a Kd value of 45.4 nM (Figure 4B). For inhibition studies, the cells were coꢀincubated
for one hour prior to flow cytometry with fluorescent probe 12 (10 nM) and a test antagonist. Binding of
12 was inhibited by two known A2AAR antagonists, nonxanthine 3 and xanthine 17, with the expected
range of potency. The Ki values were 3.8 and 17.2 nM (Figure 5), respectively, which corresponded

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closely to the reported values of 4.1 and 18 nM at hA2AAR.1,13 Agonist inhibition of competitive binding
of 12 was complex (Supporting information).
Conclusions
We succeeded in identifying useful A2AAR probes by systematically varying the chain length of amineꢀ
functionalized congeners of a potent antagonist and coupling the primary amine having an optimal
length to various fluorophores. We successfully enhanced the A2AAR affinity compared to known
fluorescent A2AAR ligands.15 Conjugates 11 and 12 were potent and selective antagonist probes for the
A2AAR, but 12 was more promising for characterization of the hA2AAR in whole cells by flow
cytometry. Molecular modeling suggested that the fluorophores of 11 and 12 interacted with the A2AAR
at distinct locations separate from the orthosteric binding site. The BODIPY630/650 moiety of 11 was
predicted to associate with hydrophobic regions between TMs. However, the hydrophilic fluorophore of
12 was coordinated to the ELs, consistent with its lower overall hydrophobicity and more favorable
whole cell binding characteristics compared to 11 under present conditions. An evaluation of 11 and 12
in confocal microscopy studies remains to be performed. Thus, we have introduced antagonist ligands
displaying high A2AAR affinity and selectivity that may serve as versatile tools to better study this
receptor.
Acknowledgements
This research was supported in part by the Intramural Research Program of the NIH, National Institute
of Diabetes and Digestive and Kidney Diseases. RD was supported by Inserm, Univ. Lille, France. We

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thank Dr. John Lloyd (NIDDK) for mass spectra. SB was supported by UGC, India. JA acknowledges
support from National Heart, Lung, and Blood Institute (R01HL133589).
Supporting information
Supplementary data (5 files) can be found, in the online version, at doi:xxxxxxx.
1) chemical synthesis, characterization data, HPLC analysis, pharmacological studies, off-target screening and
additional molecular modeling procedures/results.
2) 3D coordinates of the hA2AAR in complex with 11 (docking pose).
3) 3D coordinates of hA2AAR in complex with 12 (docking pose, BM2).
4) Video of 30 ns of MD simulations of hA2AAR in complex with 3. Video S1. Trajectory visualization (left panel)
and ligand-protein interaction energy profile (right panel) of 30 ns of membrane MD simulation of the 3-
hA2AAR complex.
5) Video of 30 ns of MD simulations of hA2AAR in complex with 12. Video S2. Trajectory visualization of 30 ns of
membrane MD simulation of the 12-hA2AAR complex starting from docking binding mode denoted as "BM2":
side view facing TM5, TM6, and TM7 (left panel), and side view facing TM4, TM5 and TM6 (right panel).
Conflict of interest
The authors declare no competing interests.
References and Notes
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Table 1. AR binding affinity determined for a series of pyrazolo[4,3ꢀ e][1,2,4]triazolo[1,5ꢀ c]pyrimidinꢀ
5ꢀamine derivatives (R1, as in Scheme 1). Human ARs, unless noted (m indicates mouse).
H
H
O
H
O
R2
N
N
N
H2N
R
C
n
n
NH2
N
O
O
O
N
N
O
N
N
R1
R1
N
3,7,13
6
5,9-12
Affinity, Ki, nM (or %inhib)
a
a
a
Compd
3b
Structure
A1
A2A
A3
R = OCH3
(35±5%)c
(20±3%)c
1270±140c
1300±350
2390±100
1910±120
6500±2830
(66±2%)c
(13±6%)c,
(3±1%)c (m)
(20±3%)c
(40±3%)c,
(0%)c (m)
1680±470,
(0%)c (m)
4190±750,
4.1e
111±16e
6.8±1.1e
(67±1%)c
(4±2%)c
5b
R2 = AlexaFluor488d, n = 2
6ab
6b
6c
n = 2
3970±120
2170±660
5990±2900
656±132
n = 3
9.29±7.92
6.46±1.63
6.36±3.58
22.8±8.45
48±28e
n = 4
n = 5
6d
n = 6
2070±850
(34±3%)c
(21±3%)c,
(4±1%)c (m)
(21±2%)c
(33±5%)c,
(2±2%)c (m)
(32±3%)c,
(5±2%)c (m)
2660±1250,
(4±1%)c (m)
6e
7b
R = ꢀOH
R2 = AlexaFluor647d, n = 2
332±165,
458±24 (m)
295±176
9
R2 = AlexaFluor647d, n = 4
10
11f
R2 = BODIPY630/650d, n = 4
R = AlexaFluor488d, n = 4
R = ꢀOCH2ꢀPhꢀpꢀSO3H
24.6±17.6,
2.09±0.16 (m)
30.3±4.9,
585±73 (m)
6.24±2.42,
64.1±5.3 (m)
12f
13f
(21±4%%)c (m)
a. Competition radioligand binding assays were conducted with membranes prepared from HEKꢀ293 cells
expressing recombinant A1, A2A, or A3AR, human, unless noted. Incubations were performed for 1 h at 25
oC. The radioligands used were: A1AR, [3H]8ꢀcyclopentylꢀ1,3ꢀdipropylxanthine ([3H]DPCPX, 0.5 nM)

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14; A2AAR, [3H]ZM241385 2 (1.0 nM) or if from published data13 (as noted) [3H]2ꢀ[pꢀ(2ꢀ
carboxyethyl)phenylꢀethylamino]ꢀ5′ꢀNꢀethylcarboxamidoadenosine ([3H]CGS21680, 10 nM) 15; A3AR,
[125I]N6ꢀ(4ꢀaminoꢀ3ꢀiodobenzyl)adenosineꢀ5′ꢀNꢀmethyluronamide ([125I]IꢀABꢀMECA, 0.2 nM) 16.
Nonspecific
binding
was
determined
using
10
µM
8ꢀ[4ꢀ[[[[(2ꢀ
aminoethyl)amino]carbonyl]methyl]oxy]phenyl]ꢀ1,3ꢀdipropylxanthine (XAC) 17 (A1AR and A2AAR) or
10 µM adenosineꢀ5′ꢀNꢀethyluronamide (NECA) 18 (A3AR). HEKꢀ293 cells expressing recombinant mA1,
mA2A, or mA3AR were used. Values are expressed as the mean ± SEM from 3 independent experiments.
The cell lines were from American Type Culture Collection (ATCC, Manassas, VA), and the cDNA for
the ARs was obtained from cdna.org.
b. Data from Kumar et al. and Kecskés et al.13,14
c. Percent inhibition at 10 ꢀM.
d. Fluorophore moiety, as shown in Scheme 1 (R2).
e. Using [3H]16 as radioligand.
f. 11, MRS7396; 12, MRS7416; 13, MRS7352.

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Figures and Legends
Figure 1. Antagonist radioligand ([3H]2, 1.0 nM) binding inhibition curves at the hA2AAR for four
antagonist fluorescent conjugates. Compound numbers: MRS7322 9, MRS7395 10, MRS7396 11,
MRS7416 12. Membranes from HEKꢀ293 cells expressing the hA2AAR were used, and incubations were
o
performed for 1 h at 25 C. Results are expressed as the mean ± SEM. The Ki values from three
independent experiments are listed in Table 1.

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Figure 2. Molecular modeling of antagonist binding to the hA2AAR. Details of the binding site of the Xꢀ
ray structure of the receptor modeled with various ligands docked: (A) known antagonist 3, with the
retention of a subset of water molecules found in the high resolution A2AAR structure17; (B) the
BODIPY630/650ꢀlabeled antagonist 11, showing the most energetically favorable orientation of the
terminal fluorophore chain; side (C) and top (D) view of the two possible orientations of the fluorophore
group of derivative 12. Residues establishing polar (dashed orange lines) and πꢀπ interactions with the
docked ligands are represented as sticks. Aromatic residues establishing hydrophobic contacts with the
terminal fluorophore of compound 11 are represented as transparent surfaces with colors matching the
corresponding TM domain. Nonꢀpolar hydrogen atoms are omitted.

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Figure 3. Modeling of antagonist 13 binding to the hA2AAR: side (C) and top (D) view of the three
possible orientations (BM1, blue; BM2, magenta; BM3, orange) of the pꢀsulfophenyl tail. Residues
establishing polar (dashed orange lines) and πꢀπ interactions with the docked ligands are represented as
sticks. Nonꢀpolar hydrogen atoms are omitted.

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Figure 4. Saturation of fluorescent antagonists 11 (A) and 12 (B) in hA2AARꢀexpressing HEKꢀ293 cells
measured using flow cytometry following a 1 h incubation at 37 °C. Nonꢀspecific binding was
determined with 10 µM 3. Results are expressed as the mean ± SEM. The Kd value calculated from from
Figure 4B is listed in the text.
A
B

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Figure 5. Inhibition of the specific binding of fluorescent antagonist 12 (10 nM, 1 h incubation at 37 °C)
in hA2AARꢀexpressing HEKꢀ293 cells measured using flow cytometry. Competing antagonists were:
nonxanthine 3 and xanthine 17. Nonꢀspecific binding was determined with 10 µM 3. Results are
expressed as the mean ± SEM. The Ki values are listed in the text.

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Table of contents entry
Bitopic Fluorescent Antagonists of the A2A Adenosine Receptor Based on Pyrazolo[4,3-
e][1,2,4]triazolo[1,5-c]pyrimidin-5-amine Functionalized Congeners
Romain Duroux, Antonella Ciancetta, Philip Mannes, Jinha Yu, Shireesha Boyapati, Elizabeth
Gizewski, Said Yous, Francisco Ciruela, John A. Auchampach, Zhan-Guo Gao, and Kenneth A.
Jacobson