Molecular probes for the A2A adenosine receptor based on a pyrazolo[4,3-e][1,2,4]triazolo[1,5-c]pyrimidin-5-amine scaffold

Article abstract of DOI:10.1016/j.bmcl.2010.11.082

Pyrazolo[4,3-e][1,2,4]triazolo[1,5-c]pyrimidin-5-amine derivatives such as SCH 442416 display high affinity and selectivity as antagonists for the human A_2A adenosine receptor (AR). We extended ether-linked chain substituents at the p-position of the phenyl group using optimized O-alkylation. The conjugates included an ester, carboxylic acid and amines (for amide condensation), an alkyne (for click chemistry), a fluoropropyl group (for ¹⁸F incorporation), and fluorophore reporter groups (e.g., BODIPY conjugate 14, K_i 15 nM). The potent and A_2AAR-selective N-aminoethylacetamide 7 and N-[2-(2-aminoethyl)-aminoethyl]acetamide 8 congeners were coupled to polyamidoamine (PAMAM) G3.5 dendrimers, and the multivalent conjugates displayed high A_2AAR affinity. Theoretical docking of an AlexaFluor conjugate to the receptor X-ray structure highlighted the key interactions between the heterocyclic core and the binding pocket of the A _2AAR as well as the distal anchoring of the fluorophore. In conclusion, we have synthesized a family of high affinity functionalized congeners as pharmacological probes for studying the A_2AAR.

Full text of DOI:10.1016/j.bmcl.2010.11.082

Bioorganic & Medicinal Chemistry Letters 21 (2011) 2740–2745
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Molecular probes for the A2A adenosine receptor based
on a pyrazolo[4,3-e][1,2,4]triazolo[1,5-c]pyrimidin-5-amine scaffold
T. Santhosh Kumar a, Shilpi Mishra a, Francesca Deflorian a, Lena S. Yoo a, Khai Phan a, Miklos Kecskés a,
Angela Szabo a, Bidhan Shinkre b, Zhan-Guo Gao a, William Trenkle b, Kenneth A. Jacobson a,
⇑
a Molecular Recognition Section, Laboratory of Bioorganic Chemistry, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bldg. 8A, Rm.
B1A-19, Bethesda, MD 20892, USA
b Chemical Biology Unit, Laboratory of Cell Biochemistry and Biology, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda,
MD 20892, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
Pyrazolo[4,3-e][1,2,4]triazolo[1,5-c]pyrimidin-5-amine derivatives such as SCH 442416 display high
affinity and selectivity as antagonists for the human A2A adenosine receptor (AR). We extended ether-
linked chain substituents at the p-position of the phenyl group using optimized O-alkylation. The conju-
gates included an ester, carboxylic acid and amines (for amide condensation), an alkyne (for click chem-
istry), a fluoropropyl group (for 18F incorporation), and fluorophore reporter groups (e.g., BODIPY
conjugate 14, Ki 15 nM). The potent and A2AAR-selective N-aminoethylacetamide 7 and N-[2-(2-amino-
ethyl)-aminoethyl]acetamide 8 congeners were coupled to polyamidoamine (PAMAM) G3.5 dendrimers,
and the multivalent conjugates displayed high A2AAR affinity. Theoretical docking of an AlexaFluor con-
jugate to the receptor X-ray structure highlighted the key interactions between the heterocyclic core and
the binding pocket of the A2AAR as well as the distal anchoring of the fluorophore. In conclusion, we have
synthesized a family of high affinity functionalized congeners as pharmacological probes for studying the
A2AAR.
Received 22 October 2010
Accepted 16 November 2010
Available online 21 November 2010
Keywords:
SCH 442416
G protein-coupled receptor
Fluorescence
Dendrimer
Radioligand binding
Published by Elsevier Ltd.
Adenosine acts as a neuromodulator and has been termed as an
endogenous cerebroprotective agent with respect to epilepsy and
ischemia. Selective ligands for the adenosine receptors (ARs) are
considered to have potential in the treatment of conditions of the
central nervous system (CNS).1,2 Adenosine activates four receptor
subtypes—A1, A2A, A2B, and A3—the first two of which have been as-
signed specific roles in the central nervous system.3 The A2AAR is
coupled to stimulation of adenylate cyclase and in the brain is
highly expressed in the striatum and olfactory tubercle. A2AAR
antagonism is suggested to mediate many of the CNS stimulatory
effects of caffeine in man.4 Its blockade by potent and selective
antagonists is under development as an approach to the symptom-
atic treatment of Parkinson’s disease (PD), and such antagonists
might be useful against progressive neurodegeneration.2,3,5 The
selective A2AAR antagonists 1a, a 1,3,7-trialkylxanthine derivative,
and 1b, a triazolopyrimidine derivative (Chart 1), have been in clin-
ical trials.6,7 Triazolo[1,5-c]pyrimidin-5-amine antagonists 2 (SCH
442416) and Preladenant (SCH 420814), have also shown promise
for the treatment of PD.8,9 Another envisioned disease application
of A2AAR antagonists is in cancer treatment.10
The recently reported X-ray structure of the A2AAR11 has been
utilized for the in silico screening of chemical libraries for the
Abbreviations: AR, adenosine receptor; BODIPY, 4,4-difluoro-4-bora-3a,4a-
diaza-s-indacene; CHO, chinese hamster ovary; CNS, central nervous system;
DMF, N,N-dimethylformamide; EDC, N-(3-dimethylaminopropyl)-N0-ethylcarbodi-
imide; FP, fluorescence polarization; HEK, human embryonic kidney; KW6002, (E)-
1,3-diethyl-8-(3,4-dimethoxystyryl)-7-methyl-3,7-dhydro-1H-purine-2,6-dione;
MCMM, Monte Carlo Multiple Minima; MES, 2-(N-morpholino)-ethanesulfonic
acid; NECA, 50-N-ethylcarboxamidoadenosine; PAMAM, polyamidoamine dendri-
mer; PD, Parkinson’s disease; PET, positron emission tomography; SCH 442416,
5-amino-7-(3-(4-methoxy)phenylpropyl)-2-(2-furyl)pyrazolo[4,3-e]-1,2,4-triaz-
olo[1,5-c]pyrimidine; TAMRA, tetramethylrhodamine; THF, tetrahydrofuran; TLC,
thin layer chromatography; TM, transmembrane helical domain; ZM241385, 4-
[2-[7-amino-2-(2-furyl)-1,2,4-triazolo[1,5-a][1,3,5]triazin-5-yl-amino]ethylphenol.
O
N
O
CH3
N
CH3CH2
O
N
N
N
N
N
N
OCH3
OCH3
H2N
N
CH2CH3
NH2
CH3
1a, KW6002
1b, V2006
⇑
Corresponding author. Tel.: +1 301 496 9024; fax: +1 301 480 8422.
E-mail address: kajacobs@helix.nih.gov (K.A. Jacobson).
Chart 1. Representative selective A2AAR antagonists that have been in clinical trials
for PD: Xanthine derivative 1 and nonxanthine heterocycle 2.
0960-894X/$ - see front matter Published by Elsevier Ltd.
doi:10.1016/j.bmcl.2010.11.082

T. S. Kumar et al. / Bioorg. Med. Chem. Lett. 21 (2011) 2740–2745
2741
identification of new leads for selective antagonists of this recep-
tor, based on novel chemotypes.12,13 The screening of in silico hits
is routinely carried out using radioligand binding assays. It would
be desirable to have new pharmacological probes for this receptor
that would not require the use of radioactivity and instead would
be based on fluorescence and other spectroscopic methods. We
have explored chain functionalization of the A2AAR antagonist 2
for this purpose to arrive at a series of functionalized congeners
that provide a means of covalent attachment of sterically bulky
groups without losing the ability to bind to the A2AAR with high
affinity. Also, a biotin handle was included in this approach for
complexation to streptavidin. We continued our efforts to develop
receptor imaging agents for positron emission tomography (PET)
that selectively target the most relevant subtype of ARs that is
highly expressed in the striatum, that is, the A2AAR. We developed
a O-(3-fluoropropyl) analogue, intended for incorporation of 18F. It
was essential to identify an insensitive position on 2 that would
accommodate a chain. The methoxy group of the phenyl ring of 2
was selected for this purpose because it has been shown to display
flexibility of substitution and conformation with respect to binding
to the A2AAR, as deduced from structure–activity relationship
(SAR) studies in conjunction with molecular modeling and ligand
docking.14
Table 1 lists the structures of the pyrazolo[4,3-e][1,2,4]triazol-
o[1,5-c]pyrimidin-5-amine derivatives that were assayed for bind-
ing affinity at human ARs. The various 4-ether derivatives were
chemically functionalized with amino, carboxylic acid, fluoropropyl
or extended alkynyl chains, making them functionalized congeners
for conjugation to other biologically active moieties or to carriers.15
Synthetic routes to various pyrazolo[4,3-e][1,2,4]triazolo[1,5-
c]pyrimidin-5-amine derivatives (3–17) in which extended chain
substituents have replaced the methoxy group of SCH 442416
are described in Schemes 1–3. We have reported the removal of
the methyl ether of 2 as a means of further derivatizing this series
of A2AAR antagonists.14 Commercially available SCH 442416 was
demethylated using BBr3 in CH2Cl2 to provide phenol 3, which
was used in the next reaction without further purification. Various
conditions to chemoselectively alkylate phenol 3 in the presence of
the exocyclic amine functionality were compared. Reaction with
methyl 2-bromoacetate and Cs2CO3 as a base in MeOH at room
temperature and addition of 5 equiv of electrophile at 1 h intervals
formed 5 as the major product and considerably decreased the for-
mation of di- and tri-alkylated products from ꢀ25% to ꢀ10% and
the formation of polar byproducts. However, our attempts to first
protect the amine of 3 as a tert-butyl carbamate (Boc) using Boc2O
and triethylamine in DMF resulted in the exclusive formation of
the Boc-protected phenol (data not shown).
Fluoropropyl derivative 4 was synthesized in satisfactory yields
with the optimized chemo-selective alkylation conditions using 1-
bromo-3-fluoropropane and Cs2CO3 in MeOH (Scheme 1). Hydrolysis
of the methyl ester of the O-alkylated product 5 with aqueous 1 N
NaOH in MeOH and THF provided the corresponding carboxylic acid
derivative 6 (Scheme 1). Alternatively, methyl phenoxyacetate 5
could be reacted with either neat ethylenediamine or diethylenetria-
mine in DMF at room temperature (Scheme 1) to form N-aminoethy-
lacetamide 7 (55%) and N-[2-(2-aminoethyl)-aminoethyl]acetamide
8 (56%), respectively.
N-Aminoethylacetamide 7 was used as a common key interme-
diate to anchor two fluorophore moieties viz. AlexaFluorÒ 488 in 9
or 6-carboxytetramethylrhodamine in 10 by active ester coupling
in a mixture of DMF and aqueous Na2B4O7 at pH 8.5 with purifica-
tion by semi-preparative HPLC (Scheme 2). Similarly, diamine 8
was labeled with BODIPYÒ 650/66523 to yield compound 14.
As an alternative coupling approach, the primary amine of
N-aminoethylacetamide 7 was regio-selectively coupled to 6-
heptynoic acid using EDCꢁHCl in DMF to afford compound 11 with
Table 1
Affinity of a series of pyrazolo[4,3-e][1,2,4]triazolo[1,5-c]pyrimidin-5-amine derivatives at three subtypes of human ARs
Compound
Structure
Affinity, Ki, nM (or %inhib)
a
a
a
A1
A2A
A3
2
3
4
5
6
R = –OCH3
R = –OH
R = –O(CH2)3F
R = –OCH2CO2CH3
(35 5%)c
4440 810
1000 40
4840 430
1700 250
1270 140
(20 4%)c
(20 3%)c
(7 5%)c
1100 130
1300 330
1700 290
(58 4%)c
4180 920
(7 5%)d
4.1b
(67 1%)c
(34 3%)c
1320 200
(50 6%)c
(29 2%)c
3970 120
3290 1500
(4 2%)c
48 28b
53.6 25.5
96 48
R = –OCH2CO2H
10.2 2.2
6.8 1.1
10.7 4.6
111 16
357 130
75.4 23.3
330 15
220 14
15.1 1.8
31.7 1.0
38.1 4.3
7
8
R = –OCH2CONH(CH2)2NH2
R = –OCH2CONH(CH2)2NH(CH2)2NH2
AlexaFluor conjugate of 7
TAMRA conjugate of 7
R = –OCH2CONH(CH2)2NHCO(CH2)4C„CH
Biotin-PEG4 conjugate of 11
N-(Ethylene)acetamide conjugate of 11
BODIPY conjugate of 8
9b,e
10
11
12
13
14e
16e
17e
(24 1%)c
1280 80
>1000
302 50
(28 3%)c
1250 160
(26 4%)d
PAMAM conjugate of 7 (13% substitution)
PAMAM conjugate of 8 (16% substitution)
a
Competition radioligand binding assays were conducted with membranes prepared from mammalian cells expressing recombinant human A1, A2A, or A3AR. The
radioligands used were: A1, [3H]8-cyclopentyl-1,3-dipropylxanthine; A2AAR, [3H]2-[p-(2-carboxyethyl)phenyl-ethylamino]-50-N-ethylcarboxamidoadenosine; A3AR, [125I]N6-
(4-amino-3-iodobenzyl)adenosine-50-N-methyl-uronamide. Values for the multivalent conjugates 16 and 17 are calculated on the basis of dendrimer concentration. Values
are expressed as the mean SEM. ND, not determined.
b
Data from Shinkre et al.14 and Kecskés et al.19
c
Percent inhibition at 10
l
M.
M.
9, MRS5346; 14, MRS5418; 16, MRS5475; 17, MRS5383.
d
e
Percent inhibition at 1
l

2742
T. S. Kumar et al. / Bioorg. Med. Chem. Lett. 21 (2011) 2740–2745
Me
H
O
O
O
F
NH2
N
NH2
NH2
N
a
b
N
N
N
N
N
N
O
O
O
N
N
N
N
N
N
N
N
N
N
3
4
7
2, SCH 442416
c
H
O
N
OH
N
O
Me
O
NH2
R =
O
O
6
d
NH2
N
NH2
N
H
3
N
NH2
N
N
N
O
O
N
N
H
8
N
N
N
N
5
N
Scheme 1. Synthesis of fluoropropyl, amine and carboxylic acid functionalized congeners derived from SCH 442416. Reagents and conditions: (a) BBr3, CH2Cl2, rt, 2 h; (b) 1-
bromo-3-fluoropropane, Cs2CO3, MeOH, rt, 18 h; (c) methyl 2-bromoacetate, Cs2CO3, MeOH, rt, 16 h; (d) aq 1 N NaOH, MeOH/THF (1 mL, 1:1, v/v), rt, 2 h (for 6);
ethylenediamine/MeOH (9:1, v/v), rt, 36 h (for 7); diethylenetriamine, DMF, rt, 32 h (for 8).
Me
O
R =
H
N
NH2
SO3
N
Me
O
N
R
H
a
O
O
7
O
O
O
2 (Et3NH)+
O
NH2
N
O
N
SO3
O
N
NH2
N
Me
Me
N
N
9
10
11
N
9 - 11
O
H
N
N
R =
O
N
N
H
O
O
O
H
H
N
S
N
N
Me
b
R
O
4
6
4
H
NH2
N
11
H
H
NH
O
N
O
HN
N
12
13
N
O
N
N
12, 13
O
H
N
H
HN
N
O
N
N
H
H
F
O
O
O
c
F
+
N
B
-
8
N
NH2
N
N
O
N
N
N
14
N
Scheme 2. Synthesis of conjugates of pyrazolo[4,3-e][1,2,4]triazolo[1,5-c]pyrimidin-5-amines with fluorescent moieties (9, 10, and 14) and other acyl derivatives using
standard amide coupling protocols and click chemistry. Reagents and conditions: (a) activated fluorophore (AlexaFluor 488 carboxylic acid, 2,3,5,6-tetrafluorophenyl ester for
9; 6-carboxytetramethylrhodamine succinimidyl ester for 10), DMF, 0.1 M aq solution of Na2B4O7 (pH 8.5), rt, 20 h; 6-heptynoic acid, EDCꢁHCl, DMF/ CH2Cl2 (1:1, v/v), rt, 16 h
for 11; (b) corresponding azide (PEG4 carboxamide-6-azidohexanyl biotin for 12, N-(2-azidoethyl)acetamide for 13, CuSO4ꢁ5H2O, sodium ascorbate, DMSO/H2O (1:1, v/v), rt
16 h; (c) 6-(((4,4-difluoro-5-(2-pyrrolyl)-4-bora-3a,4a-diaza-s-indacene-3-yl)styryloxy)acetyl)aminohexanoic acid, succinimidyl ester (BODIPY 650/665-X), DMF, 0.1 M aq
Na2B4O7 (pH 8.5), rt, 18 h.
a terminal alkyne, which is suitable for the derivatization using a
click cycloaddition reaction.20 The alkyne 11 reacted with PEG4
carboxamide-6-azidohexanyl biotin and N-(2-azidoethyl)acetam-
ide16 using the Cu(I)-catalyzed 2+3 cyclization to form correspond-
ing triazole derivatives 12 and 13, respectively.
Finally, the amine congeners 7 and 8 were conjugated to the
peripheral carboxylic acids of polyamidoamine (PAMAM) G3.5 den-
drimer 15 (containing a butane core) using the EDC-mediated cou-
pling protocol in MES buffer to obtain 16 (35%) and 17 (69%),
respectively (Scheme 3). Mass analysis of the conjugates 16 and 17

T. S. Kumar et al. / Bioorg. Med. Chem. Lett. 21 (2011) 2740–2745
2743
O
H
N
N
O
H
O
3.5
HO2C
a, 7
55.9
NH2
N
CO2H
64
N
N
O
N
N
3.5
8.1
N
16
PAMAM dendrimer
carrier
H
N
H
N
O
N
O
a, 8
H
15
O
NH2
N
3.5
HO2C
N
O
N
54
N
N
17
N
10
Scheme 3. Synthesis of conjugates of pyrazolo[4,3-e][1,2,4]triazolo[1,5-c]pyrimidin-5-amines with a PAMAM dendrimer. Reagents and conditions: (a) EDCꢁHCl, DMSO,
freshly prepared 0.1 M MES aq buffer (pH 5, adjusted with 1 N NaOH or 10% HCl).
(MW of 16–17 K) indicated an average loading of 8.1 and 10 mono-
mer moieties, respectively, out of a possible 64 sites. Consistently,
UV spectroscopic analysis indicated the loading of approximately
6.8 and 7.8 ligands on 16 and 17, respectively (Supplementary data).
Binding assays were carried out using standard radioligands in
Chinese hamster ovary (CHO) cells expressing the human A1 or
A3ARs and in HEK293 cells expressing the human A2AAR.15,17,18 High
selectivity for the A2AAR was generally achieved (e.g., amine congen-
ers 7 and 8 at the A3AR), with binding at the A1 or A3ARs in the micro-
molar range at best. This selectivity was evidently a function of the
robust pharmacophore present in SCH 442416 and the insensitivity
in receptor binding at the phenolic position. However, some modu-
lation of the A2AAR affinity, depending on the terminal groups, was
observed. For example, the fluoropropyl derivative bound to the
A2AAR in transfected or control cells with the advantage of a long
wavelength of emission. In fluorescent microscopy, one of the main
problems is the autofluorescence of the cells in the green wave-
length range obscuring the signal from the noise, which can be
eliminated with a fluorophore emitting at a longer wavelength.
These two fluorophores are in this range, and compound 14 with
a Ki value of 15 nM is likely suitable for cell labeling experiments.
The multivalent dendrimer conjugates 16 and 17 were highly
potent and selective in binding to the A2AAR. The two spacer chains
were indistinguishable by A2AAR affinity. Previously, various AR
agonists were tethered from PAMAM to form GPCR ligand-dendri-
mer (GLiDe) conjugates that retained high affinity, selectivity, and
efficacy as agonists.24 This is the first example of PAMAM dendri-
mer conjugates of a strategically designed, functionalized AR
antagonist.
A
2AAR with a Ki value of 53.6 nM. An ester derivative 5 was less po-
tent at the A2AAR than either carboxylic 6 or amino derivatives 7 and
8. There was no preference at the A2AAR for cationic versus anionic
terminal groups in 6–8, and the addition of a second amine in the
chain in 8 did not increase the affinity. The introduction of sterically
bulky groups in conjugates 9, 10, 12, and 14 had variable effects on
the affinity at the A2AAR. Compound 14 retained nearly full affinity
with respect to the precursor amine 8, supporting the expectation
that this chain extends into an external region of the receptor. Inter-
estingly, 14 contained a secondary amine in the chain, suggesting
this positively charged group as a source of the relatively high affin-
ity of the conjugate. Amide conjugates 11–13, including 12 and 13
with a triazole linker from click coupling, showed reduced A2AAR
affinity relative to precursor amine 7.
The three fluorescent conjugates 9, 10, and 14 were considered
for use as fluorescent probes in A2AAR assays. Compound 9 proved
to be a useful probe in fluorescence polarization (FP) assays.19 The
fluorescent dye, AlexaFluor 488 is a suitable fluorophore for FP be-
cause of its prolonged lifetime of 4.1 ns, which proved to be appro-
priate in our experiments and in other studies.19,21,22 If the
fluorescent lifetime is either too short or too long, a significant
FP signal is unattainable. Compounds 10 and 14 contained as fluo-
rophores tetramethylrhodamine (TAMRA) and BODIPY 650/665-X,
respectively, which are not often applied in FP experiments be-
cause of their short lifetime. In preliminary experiments, it was
not possible to get a significant FP signal with compound 14 in
our assay system. Thus, compounds 10 and 14 were mainly de-
signed for fluorescence microscopy experiments to detect the
A molecular modeling study was carried out to explore the rec-
ognition of the AlexaFluor-labeled antagonist 9 in binding to the X-
ray crystal structure of the A2AAR. The impeded free rotation of the
molecule, which leads to a fluorescence polarization signal,19 could
be explained by the anchoring of the AlexaFluor moiety to the
receptor through interactions with specific residues in the extra-
cellular region of the A2AAR. A flexible molecular docking was per-
formed to find a likely binding pose of the antagonist in the A2AAR
binding cavity and to predict its putative interactions. The recently
released 2.4 Å resolution structure of the A2AAR in complex with
the triazolotriazine antagonist ZM241385 (PDB code: 1EML) was
used as the starting structure.11
The AlexaFluor-labeled antagonist 9 was built and energy min-
imized in MacroModel and then automatically docked in the bind-
ing pocket of the unoccupied receptor by means of Glide. The
docking box was centered on the bicyclic core of ZM241385, and
the box dimensions were sufficiently large to accommodate 9,
which is of greater volume than ZM241385. The most favorable
pose from the Glide docking results was chosen as a representative
binding mode for the antagonist, and the ligand–receptor complex
was further subjected to conformational search via Monte Carlo
Multiple Minima (MCMM) in MacroModel.26 Both the ligand and
the amino acid side chains in the binding pocket were considered
fully flexible during the MCMM conformational search calculations
in order to extensively explore the conformational space. The low-
est energy conformation of the ligand–receptor complex was cho-
sen as the preferred binding mode of the antagonist.

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T. S. Kumar et al. / Bioorg. Med. Chem. Lett. 21 (2011) 2740–2745
Figure 1. Molecular modeling of ligand binding to the human A2AAR. Details of the binding site of the X-ray structure of the receptor complexed with the AlexaFluor-labeled
antagonist 9 as obtained after a fully flexible MCMM conformational search. (A) View from the plane of the phospholipid bilayer. (B) View from the extracellular side (putative
H-bonds between the ligand and receptor are shown). The ligand is represented as a ball and stick structure, colored by element (carbon atoms colored in cyan). For clarity,
the residues located in proximity to the ligand are labeled and represented as sticks, colored by element (carbon atoms colored in green).
As expected, the pyrazolotriazolopyrimidine core of our antag-
onist was oriented within the A2AAR binding cavity, with the triaz-
F257(6.59) was in proximity to the uncharged amino group of
the AlexaFluor moiety, while the positively charged ketimine
group formed an ionic interaction with the carboxyl group of
D170 in EL2.
In conclusion, we have identified a path to design novel phar-
macological probes for the A2AAR. The introduction of a chemically
functionalized chain at an insensitive site on a pyrazolotriazolo-
pyrimidine antagonist allows the incorporation of sterically large
reporter groups, such as fluorophores that avoid the use of radioac-
tivity in drug screening. Some of these analogues have retained the
selectivity and high affinity of the parent compound, for example,
fluorescent BODIPY conjugate 14. An analogue of moderate affinity
contained a fluoroalkyl chain for PET imaging. The potent and
olopyrimidine moiety binding in
a manner similar to the
triazolotriazine ring of ZM241385. The ligand–receptor binding
mode corresponding to 9 is shown in Figure 1. In detail, the N3 of
the pyrazolotriazolopyrimidine accepted an H-bond from the
CONH2 of N253 in transmembrane helical domain (TM) 6, desig-
nated residue 6.55.25 Moreover, the exocyclic amino group of the
antagonist was anchored by strong H-bonding to the side chain
carbonyl group of N253 and to the carboxyl group of E169 in the
second extracellular loop (EL2). The pyrazolotriazolopyrimidine
moiety was further stabilized by a
p-stacking interaction with
the aromatic side chain of F168 located in EL2. Hydrophobic resi-
dues such as M174(5.35), M177(5.38), I252(6.54), L267(7.32),
and M270(7.35) created a favorable environment for the heterocy-
clic moiety of the ligand. The furan substituent common to both
antagonist classes interacted with the NH2 group of N253 and
was also coordinated by favorable aromatic or hydrophobic resi-
dues such as H250(6.52), L249(6.51), W246(6.48), L85(3.31), and
V84(3.32). The docking pose of the antagonist showed the phenoxy
extension protruding toward the extracellular region of the recep-
tor, allowing the linker chain and the AlexaFluor moiety to be sit-
uated at the top of the receptor with the xanthene ring system
lying almost parallel to the plane of the cell membrane. The phen-
oxy ring was close to hydrophobic side chains, including L167 in
EL2 and L267 at the extracellular portion of TM7. The two carbox-
amido groups in the linker chain between the phenoxy group and
the xanthene were solvent exposed and located between EL2 and
EL3. The negatively charged carboxyl group of the phenyl ring
pointed up, presumably interacting with the solvent molecules.
Figure 1B shows, from a top view, 9 docked in the binding site of
A
2AAR-selective N-aminoethylacetamide 7 and N-[2-(2-amino-
ethyl)-aminoethyl]acetamide 8 congeners were coupled to pol-
yamidoamine (PAMAM) G3.5 dendrimers, and the multivalent
conjugates displayed high A2AAR affinity. The modeling results
highlighted the key interactions between the heterocyclic core
and the binding pocket of the receptor. The results also predicted
the binding mode of a fluorescent AlexaFluor moiety attached to
the pharmacophore in conjugate 9 to find putative stabilizing
interactions with residues of the ELs, consistent with the experi-
mental data indicating reduced rotation of the fluorophore when
bound to the receptor. Moreover, with high molecular weight den-
drimer conjugates that bound potently and selectively at the
A2AAR, we have confirmed experimentally that the functionalized
chain is accessing the extracellular regions of the receptor.
Acknowledgments
This research was supported in part by the Intramural Research
Program of the NIH, National Institute of Diabetes and Digestive
and Kidney Diseases. We thank Dr. Dale Kiesewetter (NIBIB, NIH)
for helpful discussions.
A2AAR, depicting the strong interactions that anchor the AlexaFluor
xanthene core to the extracellular region of the receptor. The
charged sulfonate groups of the AlexaFluor engaged in interactions
with N145 in EL2 on one side and with N175(5.36), at the top of
TM5, on the other side of the xanthene ring. In addition, the back-
bone NH of M174(5.35) acted as a H-bond donor to one of the sul-
fonate groups, while the hydrophobic side chain of the same
residue was oriented in a favorable position underneath the xan-
thene ring. The hydrophilic backbone carbonyl group of
Supplementary data
Supplementary data (chemical synthesis, characterization data,
HPLC purification and purity analysis procedures, Cell culture and
membrane preparation, Radioligand membrane binding studies,

T. S. Kumar et al. / Bioorg. Med. Chem. Lett. 21 (2011) 2740–2745
2745
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UV determination of the number of amine monomers on G3.5
dendrimers 16 and 17 and additional modeling information) asso-
ciated with this article can be found, in the online version, at
doi:10.1016/j.bmcl.2010.11.082.
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