Structure-activity relationships and molecular modeling of 1,2,4-triazoles as adenosine receptor antagonists

Article abstract of DOI:10.1021/ml300097g

The structure-activity relationship (SAR) for a novel class of 1,2,4-triazole antagonists of the human A_2A adenosine receptor (hA_2AAR) was explored. Thirty-three analogs of a ligand that was discovered in a structure-based virtual sc

Full text of DOI:10.1021/ml300097g

Letter
pubs.acs.org/acsmedchemlett
Structure−Activity Relationships and Molecular Modeling of 1,2,4-
Triazoles as Adenosine Receptor Antagonists
Jens Carlsson,*,† Dilip K. Tosh,‡ Khai Phan,‡ Zhan-Guo Gao,‡ and Kenneth A. Jacobson*,‡
†Center for Biomembrane Research, Department of Biochemistry and Biophysics, Stockholm University, SE-10691 Stockholm,
Sweden
‡Molecular Recognition Section, Laboratory of Bioorganic Chemistry, National Institute of Diabetes and Digestive and Kidney
Diseases, National Institutes of Health, Bethesda, Maryland 20892, United States
S
* Supporting Information
ABSTRACT: The structure−activity relationship (SAR) for a novel class of 1,2,4-
triazole antagonists of the human A2A adenosine receptor (hA2AAR) was explored.
Thirty-three analogs of a ligand that was discovered in a structure-based virtual screen
against the hA2AAR were tested in hA1, A2A, and A3 radioligand binding assays and in
functional assays for the A2BAR subtype. As a series of closely related analogs of the initial
lead, 1, did not display improved binding affinity or selectivity, molecular docking was
used to guide the selection of more distantly related molecules. This resulted in the
discovery of 32, a hA2AAR antagonist (Ki 200 nM) with high ligand efficiency. In light of
the SAR for the 1,2,4-triazole scaffold, we also investigated the binding mode of these
compounds based on docking to several A2AAR crystal structures.
KEYWORDS: 1,2,4-Triazole, A2A adenosine receptor, antagonist, molecular docking, structure−activity relationship
screen large chemical libraries against the binding site of a
protein.10 Two independent docking screens that were carried
out against the first crystal structure of the hA2AAR were
remarkably successful, with hit-rates of 35 and 41%,
respectively.11,12
xtracellular adenosine regulates numerous physiological
Eprocesses via activation of four G protein-coupled
receptors (GPCRs).1 The A1, A2A, A2B, and A3 adenosine
receptor (AR) subtypes display varying affinities for adenosine
and act via different signaling pathways. The A2A and A2BAR
subtypes are primarily coupled to Gs and thereby increase
intracellular cAMP levels, whereas the A1 and A3ARs inhibit
cAMP production via activation of Gi. The human (h) A2AAR is
expressed in both the periphery and the central nervous system
(CNS). The extracellular adenosine concentration increases in
response to cell stress or damage, and activation of the hA2AAR
protects tissues by reducing inflammation.2 In the CNS, a
postsynaptic striatal hA2AAR regulates the effects of other
neurotransmitters via interactions with D2 dopamine receptors
and metabotropic glutamate receptor-5.3 There is a growing
interest in the hA2AAR as a drug target. Agonists are explored as
anti-inflammatory drugs, and antagonists are developed for the
treatment of neurodegenerative disorders such as Parkinson’s
disease.4,5
Until recently, drug discovery efforts targeting the hA2AAR
have been limited to ligand-based medicinal chemistry
approaches.6,7 Many compound series that display high affinity
for the A2A subtype have been developed based on adenosine or
naturally occurring antagonists, e.g. caffeine.8 In late 2008, the
determination of the first atomic-resolution structures of the
hA2AAR9 led to an increasing interest in the use of structure-
based approaches in ligand discovery. One of these, the
molecular docking method, can be used to computationally
The starting point of this study, compound 1, was discovered
based on a docking screen of 1.4 million compounds against the
first high-resolution crystal structure of the hA2AAR (Table 1,
Figure 1A). The molecule was ranked as number 88 based on
its score for complementarity to the orthosteric site and was
selected for experimental evaluation together with 19 other
compounds from the in silico screen. Seven of these molecules
were shown to bind to the A2AAR with inhibition constant (Ki)
values lower than 10 μM. Among these, compound 1 was one
of the most potent and represented a novel class of 1,2,4-
triazole antagonists.11 Herein we explore the structure−activity
relationship (SAR) for 1,2,4-triazole antagonists by testing a
series of 33 analogs of 1 in radioligand binding assays against
the A1, A2A, and A3AR subtypes and functional assays for the
A2B receptor. Molecular docking to hA2AAR receptor crystal
structures (PDB accession codes 3eml9 and 3pwh13) was used
to guide the selection of compounds for experimental testing
and to investigate the binding modes of the ligands.
Received: April 20, 2012
Accepted: July 29, 2012
Published: July 30, 2012
© 2012 American Chemical Society
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Table 1. Binding Affinities of a Series of 1,2,4-Triazole Derivatives at the Human A1, A2A, and A3ARs Measured in Radioligand
Binding Assays and Percent Inhibition of cAMP Accumulation in the Presence of 300 nM NECA in a Functional Assay for the
A2BAR
a
Ki (μM) or % inhibition at 10 μM
compd
R
X
Y
A1
19%
A2A
A3
A2B
1
CH3
3-CH3
3-Cl
H
H
H
H
H
H
H
H
H
H
H
H
H
H
1.2 0.1
1.0 0.2
1.0 0.1
6.4 0.9
4.0 0.9
1.0 0.2
8.8 0.6
1.4 0.2
2.0 0.4
1.8 0.5
1.7 0.3
1.7 0.5
3.9 0.2
3.2 0.8
2.2 1.0
3.4 0.7
5.0 0.1
3.5 0.6
2%
3.0 0.2
2.6 0.5
3.3 0.6
1.4 0.2
2.5 0.1
2.6 0.7
0.5 0.1
1.9 0.3
1.5 0.3
2.3 0.1
1.0 0.1
5.5 0.3
2.2 0.8
2.7 0.9
1.9 0.2
8.3 0.6
4.6 0.9
1.5 0.3
50%
18%
28%
23%
−6%
51%
31%
6%
2
CH3
CH3
CH3
CH3
CH3
(CH3)2
CH2CH3
H
1.1 0.1
5.2 0.8
38%
3
H
4
4-CH3
4-Cl
5
3.9 0.4
3.7 0.4
41%
6
2-F
7
4-Cl
8
H
1.5 0.5
2.3 0.7
20%
−9%
8%
9
3-CH3
H
10
11
12
13
14
15
16
17
18
19
20
21
22
23a
23b
23c
24
H
16%
13%
43%
9%
H
2-CH3
2,6-CH3
3-(CH2)3-4
3,5-CH3
16%
H
49%
H
46%
H
1.6 0.7
3.3 0.9
40%
−5%
21%
40%
7%
8.1 0.7
2%
H
H
4-F
−10%
11%
−10%
23%
−3%
5%
H
2-F
4-CH3
4-CH3
4-CH3
2-CH3
3-CH3
3-CH3
3-CH3
2%
H
2-OCH3
8%
49%
1.0 0.1
3.2 0.4
48%
H
H
H
H
H
H
6.5 1.5
6%
3.6 0.4
26%
CH3
CH3
CH3 (R)
CH3 (S)
8%
2.0 0.1
1.8 0.3
23%
5.9 1.2
6.3 1.4
32%
12%
13%
37%
38%
5.3 1.5
−15%
a
Measured in three independent experiments.
A series of closely related derivatives of compound 1 was first
ortho and meta substitutions on the phenoxy ring did not affect
binding, while para-substitutions led to a 2-fold loss of affinity.
Replacing the phenoxy group with a phenylsulfanyl, benzyl, or
benzyloxy group (15−17) led to 2−4-fold reductions of affinity
compared to compound 1. The predicted binding mode of
compound 1 (Figure 1A) suggested that the phenoxy group
interacted with residues in the extracellular loops (ELs).
Although several residues in EL2 have been shown to be critical
for ligand binding to the hA2AAR,14,15 the inherent flexibility of
the GPCR loop regions makes it difficult to relate the SAR to
the predicted binding modes for these analogs, a point to be
discussed below. In the next step, substitutions on the 3-phenyl
of the triazole ring were tested. Methyl substitutions in the para
(19−21) and ortho (22) positions reduced hA2AAR affinity,
while the meta-methylated analog (23a) displayed a Ki of 2 μM.
The reduction of affinity observed for all compounds with
substitutions on the 3-phenyl ring suggested that this part of
tested to obtain an SAR that could serve as a starting point for
further structural optimization (Table 1, compounds 1−24).
Compound 1 displayed a Ki of 1.2 μM in binding to the
hA2AAR. This differed from that reported previously by a factor
of 6,11 which was likely due to differences in the experimental
conditions. Replacing the 3-methyl group on the phenoxy ring
of compound 1 with either a chloride or hydrogen (2 and 3,
respectively) did not change hA2AAR affinity, while para-
substituted compounds displayed 3−5-fold reductions of the
binding affinity (4−5). Modifications at the chiral center did
not improve affinity in the series (7−8). The 1,2,4-triazole
scaffold was further explored starting from compound 9, which
lacked the chiral center of compound 1 and displayed a Ki of 2
μM at the hA2AAR. Mono- and dimethyl substitutions on the
phenoxy ring (10−14) did not significantly improve the
affinity. In agreement with observations for compounds 2−5,
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Figure 1. (A−D) Predicted binding modes for the A2AAR crystal structure with PDB accession code 3eml: (A) 1, (B) 27, (C) 32, (D) 33. (E)
Alignment of two A2AAR crystal structures, PDB accession codes 3eml and 3pwh. Key residues are shown in sticks (orange, 3eml; white, 3pwh). (F−
H) Predicted binding modes for the A2AAR crystal structure with PDB accession code 3pwh: (F) 1, (G) 27, (H) 32. The binding site is shown in
white ribbons with selected side chains shown in sticks. Ligands are depicted with orange carbon atoms. Black dotted lines indicate hydrogen bonds.
Table 2. Binding Affinities of a Series of 1,2,4-Triazole Derivatives at the Human A1, A2A, and A3ARs Measured in Radioligand
Binding Assays and Percent Inhibition of cAMP Accumulation in the Presence of 300 nM NECA in a Functional Assay for the
A2BAR
a
Ki (μM) or % inhibition at 10 μM
compd
R
A1
18%
A2A
43%
A3
A2B
25
26
27
28
CH3
0.3 0.01
0.3 0.2
0.2 0.04
3.1 0.7
10%
8%
CH2CH3
39%
2.5 0.6
1.1 0.2
5.9 0.5
(CH2)2CH3
0.8 0.2
3.2 0.7
5%
−7%
X
29
30
31
32
33
34
H
1.9 0.3
1.3 0.4
31%
0.4 0.02
0.3 0.1
2.8 0.8
0.2 0.06
0.2 0.04
1.0 0.2
0.3 0.2
0.1 0.04
0.2 0.1
0.6 0.2
0.3 0.2
1.0 0.3
72%
29%
−2%
16%
10%
−6%
4-CH3
4-Cl
2-OCH3
0.5 0.1
22%
29%
a
Measured in three independent experiments.
the ligand was buried in a sterically limited pocket, consistent
with the predicted binding mode of compound 1. Docking of
compounds 1 and 19 suggested that a para substituent clashed
with Val84 at the bottom of the orthosteric site (Figure 1A).
This would push compound 19 upward toward the extracellular
side and reduce favorable hydrogen bond interactions with
Asn253. To test the possible benefit of a smaller substituent,
the 3-phenyl was replaced with a furyl ring (24), but this led to
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reduced binding affinity. A possible reason for the loss of
activity for compound 24 is that the smaller size of the furyl
ring leads to a loss of van der Waals interactions in the
orthosteric site. As the initial hit and several analogs were chiral
(1−6, 8, 15, 22, 23a−c, 24), the pure enantiomers (23b and
23c) were synthesized for compound 23a. This revealed that
the active form of these ligands is the R-form, which is
consistent with the docking predictions for both compound 23
and the initial hit in ref 11 (1, Figure 1A).
would expect similar affinities for the A1 and A2A subtypes; the
binding sites of the A1 and A2A subtypes differ by only a few
residues in the orthosteric site, while 10 out of 20 binding
cavity residues are unique to the A3 receptor.21 Based on a
comparison of the A2AAR crystal structure to models of the A1
and A3 subtypes, we identified that Leu167 in EL2 (Glu and
Gln in A1 and A3, respectively) and Met270 in helix 7 (Thr and
Val in A1 and A3, respectively) were likely responsible for the
observed A2A and A1 selectivity. The 1,2,4-triazole series was
predicted to mainly have nonpolar interactions with Leu167
and Met270 in the A2AAR, and in both cases, the most polar
residue in this position is found in the A1 subtype, which may
reduce binding to this receptor (Figure S1, Supporting
Information). As the size of the ligands was reduced, affinities
typically increased at all subtypes for several of the most potent
compounds (e.g., compounds 27, 30, and 32). To further
improve A2AAR selectivity, it is likely necessary to increase the
compound size and extend substituents further toward
nonconserved residues in the outer regions of the orthosteric
site.
Subsequent to the testing of the 34 compounds described
here, several new high-resolution structures of the A2AAR have
been reported.13 Interestingly, two of these are cocrystallized
with the same antagonist, but the orientations of the ligand and
residues in the ELs differ. Since the docking screen was carried
out against a rigid receptor structure, the structural
reorganization at the opening of the orthosteric site could
significantly affect the predicted binding modes of the ligands.
For this reason, representative ligands in the series were docked
to an alternative crystal structure of the A2AAR (PDB accession
code 3pwh13) to further investigate the binding mode of the
1,2,4-triazole antagonists. Docking to the first hA2AAR crystal
structure (PDB accession code 3eml14) favored a conformation
where the 1,2,4-triazole and amide nitrogens hydrogen bonded
to Asn253 and Glu169, respectively (Figure 1A). In the
alternative antagonist-bound crystal structure (PDB accession
code 3pwh13), a hydrogen bond between the side chains of
Glu169 and His264 was broken, which opened a hydrophobic
pocket (Figure 1E). The predicted binding modes for three
representative compounds to the alternative crystal structure
are shown in Figure 1F−H. Compounds 1 and 32 were
predicted to bind in the same overall binding mode in both
structures, but compound 27 docked in an alternative
conformation. In the case of compound 27, the amide nitrogen
interacts directly with Asn253 instead of Glu169, and an
internal hydrogen bond was formed between the triazole ring
and the amide carbonyl (Figure 1G). This binding mode was
not accessible in the crystal structure used in the docking
screen, because the ligands would clash with Glu169 in EL2. To
test if this second binding mode was also energetically favored
for 1 and 32, docking calculations with restricted conforma-
tional sampling parameters were carried out for these two
compounds. Both compounds 1 and 32 did fit in the alternative
conformation (Figure 2). However, the docking energy of the
alternative conformation was less favorable by more than 7
kcal/mol, in support of the first binding mode. The docking
energies also tend to favor the first crystal structure (PDB
accession code 3eml9) because of the strong electrostatic
interaction energy between the ligands and Glu169. However,
because the internal energy contribution of a receptor is
notoriously difficult to estimate and is not taken into account in
the docking energy calculations, the relative free energy of
binding for the two conformations could not be calculated
As the 23 close analogs of compound 1 did not display
improved affinity, focus was placed on exploring more distantly
related molecules. In this step, molecular docking calculations
with DOCK3.616−18 were used to guide selection of molecules
from commercially available libraries. Docking of compounds
1−24 to the hA2AAR crystal structure revealed that the phenoxy
group was solvent exposed in several cases, and this conclusion
was also supported by the affinity of several analogs being
unaffected by substitutions of the phenyl ring. Based on these
observations, it appeared likely that the phenoxy group did not
contribute significantly to binding. To test this hypothesis, we
explored compounds where the phenoxy group was replaced by
a series of small aliphatic substituents (25−28). The ethyl- and
propyl-substituted compounds displayed the same levels of
affinity as the close analogs of compound 1, confirming our
prediction that the phenoxy group was not essential for binding
(Table 2). The propyl substituted compound (27) displayed a
Ki of 1.3 μM at the A2AAR, which was equipotent to compound
1 (Figure 1B). However, it should be noted that 27 was
significantly smaller than compound 1, making it a more
promising lead structure. The ligand efficiency (LE)19 of
compound 27, calculated as its free energy of binding divided
by the number of heavy atoms of the molecule, was 0.48, which
put this fragment-sized compound in a promising range for
further optimization.20 In comparison, compound 1 displayed
essentially the same affinity, but had a ligand efficiency of only
0.34 per atom.
To explore the possibility to further improve affinity while
retaining relatively high ligand efficiency, another series of
commercially available analogues was docked to the hA2AAR
orthosteric binding site. A set of compounds with substituted
phenyl rings that docked in the same overall binding mode as
the other ligands was identified (Table 2, 29−34). Compared
to compound 1, the molecules had a more compact structure
and did not extend as far toward the ELs. Compound 29
displayed a Ki of 0.4 μM, a 3-fold improvement compared to
27. This compound also retained a good LE of 0.44. A series of
substitutions at the benzamide ring was explored, and the 2-
methoxy substituted analog (32) led to another 2-fold
improvement of affinity to a Ki of 200 nM (Figure 1C).
Interestingly, replacing the amide of 32 with a urea group
resulted in compound 33, which also displayed a Ki of 200 nM
at the A2AAR (Figure 1D).
In parallel to our efforts to identify a potent 1,2,4-triazole
antagonist of the hA2AAR, the 34 compounds were also
screened at the hA1, A3, and A2BAR subtypes. Compound 1 was
quite selective for the hA2A and A3AR with only 19% inhibition
of A1AR radioligand binding at 10 μM. The functional assays
carried out for the A2BAR subtype showed no significant activity
for compound 1 or any of the close analogs (Table 1). An
unusual property of compounds 1−24 was that most of them
bound with affinities in the 1−5 μM range at the A2A and A3AR
subtypes, but 14 of them had ≤50% inhibition of the A1AR at
10 μM. Based on sequence identity in the binding pocket, one
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Letter
ABBREVIATIONS
■
AR, adenosine receptor; CNS, Central Nervous System; EL,
extracellular loop; GPCR, G protein-coupled Receptor; LE,
ligand efficiency; SAR, Structure−Activity Relationship
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ASSOCIATED CONTENT
* Supporting Information
Descriptions of the molecular modeling and experimental
procedures. This material is available free of charge via the
Internet at http://pubs.acs.org.
■
S
AUTHOR INFORMATION
Corresponding Author
*J.C.: phone, +46-8-16-4672; e-mail, jens.carlsson@dbb.su.se.
K.A.J.: phone, +1-301-496-9024; fax, +1-301-480-8422; e-mail,
kajacobs@helix.nih.gov.
■
Author Contributions
J.C. performed the molecular modeling studies. D.K.T., K.P.,
and Z.-G.G. carried out the experiments. The manuscript was
written by J.C. and K.A.J.
Funding
(18) Irwin, J. J.; Shoichet, B. K.; Mysinger, M. M.; Huang, N.; Colizzi,
F.; Wassam, P.; Cao, Y. Q. Automated Docking Screens: A Feasibility
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Supported by the NIDDK Intramural Res. Program (to K.A.J.)
and the Knut and Alice Wallenberg Foundation (to J.C.).
Notes
The authors declare no competing financial interest.
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