Synthesis and evaluation of 1,2,4-triazolo[1,5-c]pyrimidine derivatives as A2A receptor-selective antagonists

Article abstract of DOI:10.1016/j.bmcl.2010.08.021

Movement disorders such as Parkinson's disease and Huntington's disease are serious life-limiting and debilitating movement disorders. Their onset typically occurs from mid-life to late in life, and effective diagnostic techniques for detecting and following the disease course are lacking. Our goal is to develop receptor imaging agents for positron emission tomography (PET) that selectively target the most relevant subtype of adenosine receptors (AR) that are highly expressed in the striatum, that is, the A_2A AR. To further this goal, we have synthesized and characterized pharmacologically a family of high affinity A_2A AR ligands, based on the known antagonist, SCH 442416 (R = -Me), which have structural variability on the terminus (R = -Et, -i-Pr, -allyl, and others). A O-fluoroethyl analogue suitable for use as a PET tracer had a K_i value of 12.4 nM and was highly selective for the A_2A AR in comparison to the A₁ and A₃ ARs.

Full text of DOI:10.1016/j.bmcl.2010.08.021

Bioorganic & Medicinal Chemistry Letters 20 (2010) 5690–5694
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis and evaluation of 1,2,4-triazolo[1,5-c]pyrimidine derivatives as A2A
receptor-selective antagonists
Bidhan A. Shinkre a, T. Santhosh Kumar b, Zhan-Guo Gao b, Francesca Deflorian b, Kenneth A. Jacobson b,
,
⇑
William C. Trenkle a,
⇑
a Chemical Biology Unit, Laboratory of Cell Biology and Biochemistry, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bldg. 8, Rm. 121,
NIH, NIDDK, LCBB, Bethesda, MD 20892-0810, United States
b Molecular Recognition Section, Laboratory of Bioorganic Chemistry, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda,
MD 20892, United States
a r t i c l e i n f o
a b s t r a c t
Article history:
Movement disorders such as Parkinson’s disease and Huntington’s disease are serious life-limiting and
debilitating movement disorders. Their onset typically occurs from mid-life to late in life, and effective
diagnostic techniques for detecting and following the disease course are lacking. Our goal is to develop
receptor imaging agents for positron emission tomography (PET) that selectively target the most relevant
subtype of adenosine receptors (AR) that are highly expressed in the striatum, that is, the A2A AR. To fur-
ther this goal, we have synthesized and characterized pharmacologically a family of high affinity A2A AR
ligands, based on the known antagonist, SCH 442416 (R = –Me), which have structural variability on the
terminus (R = –Et, –i-Pr, –allyl, and others). A O-fluoroethyl analogue suitable for use as a PET tracer had a
Ki value of 12.4 nM and was highly selective for the A2A AR in comparison to the A1 and A3 ARs.
Published by Elsevier Ltd.
Received 9 July 2010
Revised 3 August 2010
Accepted 4 August 2010
Available online 10 August 2010
Keywords:
Nucleoside
G Protein-coupled receptor
Molecular modelling
Adenosine receptor
Radioligand binding
Movement disorders such as Parkinson’s disease (PD) and Hun-
tington’s disease (HD) are serious life-limiting and debilitating
movement disorders affecting roughly 1% of the US population
over age 50.1,2 The onset of these neurodegenerative diseases typ-
ically occurs from mid-life to late in life. By the time a diagnosis is
made, damage to the neuronal pathways in the brain governing
movement is well-advanced. One problem is a lack of adequate
diagnostic techniques for detecting and following the course of
these diseases. This clearly holds true for Parkinson’s disease for
which there currently is no predictive genetic test. Even with re-
spect to Huntington’s disease, which is entirely of a genetic origin,
there remains a need for new tools to follow the progress of the
neurodegeneration and to assess the effectiveness of experimental
drugs by objective criteria.
ported selective A2A AR antagonists could act as starting points for
development of a PET ligand including the pyrazolotriazolopyrim-
idine SCH 442416 (1a, Chart 1) and its analogue SCH 420814 (1b,
Chart 1), which is currently in advanced clinical trials.4–9 The A2A
AR in the brain is distributed mainly in the striatum and the olfac-
tory tubercle. In the striatum, adenosine acting at the postsynaptic
A2A AR has antagonistic molecular and behavioral interactions with
dopamine acting at the postsynaptic D2 receptor. Thus, an antago-
nist of this subtype administered to a PD patient acts in a manner
similar to dopamine agonists. To further the goal of developing a
PET tracer, we have synthesized a family of A2A AR ligands based
on the known antagonist, SCH 442416 (general structure 3, R = –
Me, Scheme 1), which has structural variability on the terminus
(R = –Et, –i-Pr, –allyl, and others). The preparation and binding
selectivity of these unreported ligands is reported herein.
The SCH 442416 pharmacophore has been previously utilized
for radiolabeling experiments.7 Specifically, the 11C-labeled SCH
442416 has been successfully utilized as a PET imaging agent for
the visualization of A2A AR in macaques (M. nemestrina)7 and PD
patients.8 However, this agent suffers due to the short half-life
(20.4 min) of the 11C isotope. Our studies focused on developing
a route to an agent that would incorporate 18F, which has a longer
half-life (110 min). We chose to examine the core of SCH 442416 as
a starting point since it had already been demonstrated to cleanly
cross the blood/brain barrier as well as possessing high A2A AR
Our goal is to develop receptor imaging agents for positron
emission tomography (PET) that selectively target the most rele-
vant subtype of adenosine receptors that are highly expressed in
the striatum, that is, the A2A adenosine receptor (AR).3 Several re-
Abbreviations: AR, adenosine receptor; CGS21680, 2-[p-(2-carboxyethyl)phen-
ylethylamino]-50-N-ethylcarboxamido-adenosine; CHO, Chinese hamster ovary;
HEK, human embryonic kidney; SCH 442416, 5-amino-7-(3-(4-methoxy)phenyl-
propyl)-2-(2-furyl)pyrazolo[4,3-e]-1,2,4-triazolo[1,5-c]pyrimidine; ZM241385, 4-
[2-[7-amino-2-(2-furyl)-1,2,4-triazolo[1,5-a][1,3,5]triazin-5-yl-amino]ethylphenol.
⇑
Corresponding author. Tel.: +1 301 402 2699; fax: +1 301 402 2639.
E-mail address: wtrenkle@niddk.nih.gov (W.C. Trenkle).
0960-894X/$ - see front matter Published by Elsevier Ltd.
doi:10.1016/j.bmcl.2010.08.021

B. A. Shinkre et al. / Bioorg. Med. Chem. Lett. 20 (2010) 5690–5694
5691
Chart 1. Antagonist ligands of the pyrazolo[4,3-e]-1,2,4-triazolo[1,5-c]pyrimidine class that have been in development for the treatment of PD.
Scheme 1. Retrosynthetic strategy for facile preparation of analogues.
selectivity over the A1 and A3 ARs. In this study, we shall describe
our modeling, preparation, and SAR studies of a new set of A2A AR
ligands.
A2A ARs are G protein-coupled receptors (GPCR) with a defined
binding site for an antagonist. The X-ray structure of the A2A AR in
complex with the triazolotriazine ZM241385, a high affinity A2A
selective antagonist,10 was recently released (PDB ID: 3EML) and
used here as a basis for ligand docking.11 The coordinates of the
protrude towards a more polar, solvent exposed area at the top
of the receptor. A conformational flexibility was detected for the
terminal phenolic side chains, and substituents larger than the
methoxy group, in our fluorinated compounds series, were found
to be well tolerated in the docking models (Fig. 1 shows fluoroethyl
analogue 5). The modeling results, combined with the know ability
of SCH 442416 to cross the blood brain barrier,7 encouraged us to
explore the structural variability that is tolerated on the phenolic
position.
A
2A AR crystal structure were used to investigate the binding prop-
erties of fluoroethyl ligand 5 (Table 1). The active pocket of A2A AR
is located in the transmembrane (TM) bundle, involving residues
from TM3, TM5, TM6, and TM7. Residues from the second and third
extracellular loops (EL2 and EL3) also delineate the upper part of
the binding cavity (Fig. 1a). A flexible docking of ligands in the
binding site for adenosine of the A2A AR was performed using the
Induce Fit Docking (IFD) protocol in the Schrödinger software
package.12 This protocol combines the flexible docking, by means
of the GLIDE docking program,13 of the ligands in the active site
and the refinement, by means of the Prime module,14 of the bind-
ing site residues in order to accommodate the correct binding con-
formation of the ligands. Our IFD model of the fluorinated analog 5
shows a binding mode similar to the co-crystallized ZM241385
conformation reproducing the key interactions that were observed
in the X-ray structure of the A2A AR with residues like N253 in TM6
and E169 and F168 in EL2 (see Experimental section for amino
acids numeration). The orientation adopted by the pyrazolotriazol-
opyrimidine analogues is almost perpendicular to the cell mem-
brane plane (Fig. 1a). The exocyclic amino group of the
heterocyclic unit makes strong H-bond interactions with the side
chains of N253 and E169. The central pyrazolotriazolopyrimidine
It was envisioned that elaboration of the phenoxymethyl moi-
ety into larger ether substituents would be well tolerated by the
A2A AR. To prepare our new analogues of SCH 442416, we tested
the approach that a family of compounds could arise from a com-
mon phenolic intermediate (2) via selective alkylation of the phe-
nol in the presence of the nucleophilic aromatic amine core under
basic conditions. The initial phenol (2) would be prepared from
commercially available SCH 442416 through
a nucleophilic
demethylation. This concise route would allow the facile prepara-
tion and screening of new analogues.
We were gratified to find that demethylation of SCH 442416 (1)
proceeded in near quantitative yield with boron tribromide to pro-
vide known phenol 2 (Scheme 2).15,16 With phenol 2 in hand, we
examined a variety of conditions for the selective alkylation of
the phenol. Alkylation of the phenol was achieved in high regio-
and chemoselectivity using methanol as the solvent. Alternate sol-
vents were substantially less efficacious. Polar aprotic solvents,
such as DMF, lead to contamination with polyalkylation and/or
decomposition, while solubility was poor in other alcoholic/ke-
tonic solvents. A variety of bases furnished the desired products
(such as Bu4NOH, K2CO3, and others) but Cs2CO3 provided the
highest yields and cleanest reaction mixtures. It was found that
highly selective alkylation of the phenol could be obtained with
Cs2CO3 as the base in a solution of methanol for a variety of elec-
trophiles including primary, secondary and allylic bromides. Using
the optimized reaction conditions, we were able to produce a fam-
ily of new analogues (Scheme 2).
core forms an aromatic
p-stacking with the phenyl side chain of
F168 in EL2 and other hydrophobic interactions with the side
chains of L249 in TM6 and I274 in TM7 (Fig. 1b). The furan ring
is engaged in polar and hydrophobic interactions with N253,
H250 and W246 in TM6, and M177 in TM5. The substituted pheno-
lic side chain of the pyrazolotriazolopyrimidine derivatives is lo-
cated in the extracellular region of the binding pocket. The
phenyl–propyl linker is embedded by hydrophobic residues such
as L167 in EL2, H264 in EL3, L267 and M270 in the extracellular
part of TM7. The terminal phenolic side chains (–OR, Scheme 1)
During our alkylation studies, we wished to access the homolo-
gated glycolated product 13 and corresponding mesylate 14
(Scheme 3). It is reasonably well precedented that this can be ob-
tained directly through reaction with ethylene oxide,17 ethylene

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B. A. Shinkre et al. / Bioorg. Med. Chem. Lett. 20 (2010) 5690–5694
Table 1
Affinity of a series of pyrazolotriazolopyrimidine derivatives at three subtypes of ARs in various species
Compound
R
Affinity (Ki, nM, or % inhibition at 10 lM)
a
a
a
A1 (%)
A2A (nM)
A3 (%)
1a
2
4
5
6
7
8
9
10
11
12
13
14
–Me
–H
–Et
–CH2CH2F
–n-Pr
–i-Pr
–sec-Bu
–Allyl
–Homoallyl
–Cinnamyl
–Hydrocinnamyl
–CH2CH2OH
–CH2CH2–OMs
38.4 1.9
44.3 0.9
4.8 3.3
42.7 0.6
17.9 3.1
35 4.6
16.8 2.8
32.8 1.2
25.5 1.3
7.3 2.7
4.1 0.4
48.3 28
43.9 9.3
12.4 0.8
11.7 2.2
13.3 2.6
22.1 0.4
9.0 2.3
14.4 5.2
308 141
78.9 15.5
23.9 3.3
45.7 9.4
67.4 0.5
34.2 2.6
4940 1700b
59.6 4.7
36.5 5.6
72.8 3.8
41.9 3.0
62.5 2.3
44.8 0.8
17.8 3.4
18.2 2.6
57.8 6.4
62.4 1.7
6.9 2.0
47.9 8.2
27.8 6.2
a
All experiments were done on CHO or HEK293 (A2A only) cells stably expressing one of three subtypes of human ARs. The
binding affinity for A1, A2A and A3ARs was expressed as Ki values (n = 3–5) and was determined by using agonist radioligands
([3H]CCPA; [3H]CGS21680; or [125I]I-AB-MECA, respectively), unless noted.
b
Ki expressed as nM.
Figure 1. Docking pose of fluorinated analogue 5 in the binding pocket of the A2A AR. Panel a: view from the plane of the cellular membrane of the crystallographic structure
of A2A AR bound to the ligand. The binding pocket is located in the TM bundle region of the receptor defined by residues from TM3, TM5, TM6, and TM7 and residues from EL2
and EL3. Panel b: Close-up view showing the best scoring pose for compound 5 in the active pocket of A2A AR. The ligand is anchored to the residues in the binding pocket by
strong H-bond interactions and stabilized by hydrophobic interactions. In the image the carbon atoms are colored gray for the protein and yellow for the ligand. The non-
polar hydrogen atoms are not shown for clarity. Key interactions of side chains residues within the binding site are highlighted as dotted lines.
oxide precursors such bromoethanol,18 or through a sequence of
reactions with ethyl iodoacetate follow by reduction/deprotec-
tion.19 We found that the homologation reaction was more compli-
cated in the presence of the reactive heterocyclic core. However,
fluoroethanol with tetrabutylammonium hydroxide provided a
good isolated yield of glycol 13 with the fewest side products. This
was an unexpected discovery, because fluoroethanol has not been
previously reported as a precursor to ethylene oxide. Although gly-
col 13 can be isolated via preparative TLC, it was found that direct
submission of the highly polar unpurified glycol 13 to mesylation
conditions furnished 14 in 42% isolated overall yield for the two
steps.
Binding assays at three hAR subtypes were carried out on
the 1,2,4-triazolo[1,5-c]pyrimidine derivatives using standard
radioligands and membrane preparations from Chinese hamster
ovary (CHO) cells (A1 and A3) or human embryonic kidney
(HEK293) cells (A2A
)
stably expressing
a
hAR subtype
(Table 1).20–23

B. A. Shinkre et al. / Bioorg. Med. Chem. Lett. 20 (2010) 5690–5694
5693
Scheme 2. Rapid and facile synthetic preparation of SCH 442416 analogues.
Scheme 3. Glycolation of phenol 2 with fluoroethanol.
In general, our substrates show that there is a wide range of
structural tolerance of substitution on the phenolic position. The
newly synthesized analogues gave binding inhibition constants
(Ki) in the range of 9.0–300 nM (Table 1), which is consistent with
the observed binding affinity of SCH 442416 (1a), which was mea-
sured to be 4.1 nM.24 These results agree with the ligand docking,
which indicated that the phenolic substituent protrudes toward
the extracellular matrix and is close to the flexible extracelluar
loops of the A2A AR.
We have synthesized a family of A2A AR ligands, based on the
known antagonist, SCH 442416 (1a), which has structural variabil-
ity on the side chain phenolic terminus (including –Et, –i-Pr, –allyl,
and others). Our facile preparation of this family of A2A antagonists
was achieved through a highly phenol-selective alkylation in the
presence of nucleophilic heterocyclic core. The binding properties
of the newly prepared analogues were consistent with our predic-
tions based on molecular modeling of these antagonists with the
A2A AR. The goal of preparing a fluorine-substituted A2A selective
ligand was achieved with the fluoroethyl analogue 5. The prepara-
tion of the 18F-labeled ligand and its utility as a PET ligand are the
subject of ongoing studies.25
Small structural variation of the phenoxy substituent gave ana-
logues with binding affinity similar to the parent ligand. It was
found that removal of the –methyl substituent from the phenoxy
position resulted in reduced binding affinity, while simple n-alkyl
(saturated and unsaturated analogues) retained activity as the
chain length increased. Addition of a fluorine to an ethyl substitu-
ent (ligand 5) displayed comparable binding relative to the unsub-
stituted ethyl (4) and the n-propyl (6) analogues. The O-fluoroethyl
analogue 5 appeared to be suitable for use as a PET tracer based on
its favorable binding affinity. It displayed a Ki value of 12.4 nM at
the A2A AR and was highly selective in comparison to the A1 and
A3 ARs, at which the affinities were estimated to be approximately
Acknowledgements
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 Dale Kiesewetter, Abesh Bhatta-
charjee, and Lixin Lang (NIH, NIBIB) for helpful discussion.
10 l
M. The preparation of 18F-5 and studies of its use as a PET tra-
Supplementary data
cer have been completed and will be reported elsewhere.25
As the size of the substituent increased (–R, Table 1), variation
was observed in the binding affinity. Alpha branching did not dra-
matically affect the affinity (i-Pr analogue 7 and s-Bu analogue 8)
nor did the introduction of polarity to a short chain (hydroxylethyl
analogue 13 and mesylate analogue 14). Although, cinnamyl deriv-
ative 11 and hydrocinnamyl derivate 12 are tolerated, they show a
reduced binding affinity at the A2A AR. The observed reduction in
affinity may be a function of the non-polar character of these
groups, which necessarily project into the polar extracellular loop
region (Fig. 1). Due to the polar character of this loop region, it
may be expected to better accept charged and/or H-bonding sub-
stituents as compared to aromatic rings. Future experiments will
examine more polar side chains to evaluate what functionality is
most complementary to the polarity of this region.
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmcl.2010.08.021.
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