Discovery of 1,2,4-triazine derivatives as adenosine A2A antagonists using structure based drug design

Article abstract of DOI:10.1021/jm201376w

Potent, ligand efficient, selective, and orally efficacious 1,2,4-triazine derivatives have been identified using structure based drug design approaches as antagonists of the adenosine A_2A receptor. The X-ray crystal structures of compounds 4e and 4g bound to the GPCR illustrate that the molecules bind deeply inside the orthosteric binding cavity. In vivo pharmacokinetic and efficacy data for compound 4k are presented, demonstrating the potential of this series of compounds for the treatment of Parkinson's disease.

Full text of DOI:10.1021/jm201376w

Article
pubs.acs.org/jmc
Discovery of 1,2,4-Triazine Derivatives as Adenosine A2A Antagonists
using Structure Based Drug Design
Miles Congreve,* Stephen P. Andrews, Andrew S. Dore, Kaspar Hollenstein, Edward Hurrell,
́
Christopher J. Langmead, Jonathan S. Mason, Irene W. Ng, Benjamin Tehan, Andrei Zhukov,
Malcolm Weir, and Fiona H. Marshall
Heptares Therapeutics Limited, BioPark, Broadwater Road, Welwyn Garden City, Hertfordshire AL7 3AX, U.K.
S
* Supporting Information
ABSTRACT: Potent, ligand efficient, selective, and orally efficacious 1,2,4-
triazine derivatives have been identified using structure based drug design
approaches as antagonists of the adenosine A2A receptor. The X-ray crystal
structures of compounds 4e and 4g bound to the GPCR illustrate that the
molecules bind deeply inside the orthosteric binding cavity. In vivo
pharmacokinetic and efficacy data for compound 4k are presented, demonstrating
the potential of this series of compounds for the treatment of Parkinson’s disease.
INTRODUCTION
RESULTS AND DISCUSSION
Design and Synthesis. The binding mode of 1,2,4-triazine
■
■
The adenosine A2A receptor is expressed in the basal ganglia
where it functionally opposes the actions of the dopamine D2
receptor, i.e., inhibition of the A2A receptor leads to
enhancement of D2 receptor function. Given that the primary
pathology in Parkinson’s disease is a loss of nigrostriatal
dopamine and hence reduced dopamine D2 receptor activation,
adenosine A2A receptor antagonism has emerged as a potential
nondopaminergic therapy for this disorder. Preclinically,
adenosine A2A receptor antagonists are effective in animal
models of Parkinson’s disease, ranging from the reversal of
haloperidol-induced catalepsy through to efficacy in more
disease-relevant models such as 6-hydroxydopamine lesioned
rats and MPTP-lesioned primates.1 Furthermore, a number of
these compounds have progressed into clinical development,
the most advanced of which is currently preladenant. This
compound was shown to be effective in a phase IIa trial of
patients with moderate-to-severe Parkinson’s disease when
administered in conjunction with levodopa, increasing on-time
with no concomitant increase in dyskinesias.2
In the preceding publication, hit molecules derived from a
virtual screening strategy were described.3 One series of 1,3,5-
triazine derivatives was identified and optimized to give potent
and selective adenosine A2A receptor antagonists. As part of the
optimization of this chemotype and by considering the
proposed binding mode to the receptor, we hypothesized
that the alternative 1,2,4-triazine isomers might also bind to the
receptor but could sit more deeply in the receptor pocket
accessing the region normally occupied by the ribose group of
the natural ligand adenosine, in addition to mimicking the
adenine ring itself. Upon testing of the commercially available
parent 5,6-diphenyl-1,2,4-triazine-3-amine 4a, we discovered
that the molecule was indeed an antagonist of the receptor
(Table 1, compound 1; pKi = 6.93). In this paper, we outline
our studies in this isomeric chemical series.
derivatives was initially derived from modeling of representative
compounds in an “experimentally enhanced” homology model
of the adenosine A2A receptor (described in the preceding
paper), refined using site directed mutagenesis data both from
the literature and our own Biophysical Mapping (BPM)
approach.3−6 Parts A and B of Figure 1 illustrate the proposed
binding mode of two analogues 4g and 4e (Table 1) in the
orthosteric binding site of the receptor and also show the BPM
binding fingerprint around these example ligands, used to refine
and improve the model. The residues that, when mutated to
alanine, reduce binding of each ligand are colored in red for
nonbinding, dark-orange for the largest effect (tier 1), orange
for the next largest effect (tier 2), yellow for the smallest effect
(tier 3), and in green if the mutation caused an increase in
binding of the ligand. The BPM studies have been reported
elsewhere and analogues 4e and 4g here equate to examples 3b
and 3d in the earlier publication.4 As well as rationalizing the
role of the aminoheterocyclic scaffold binding to Asn2536.55
(superscripts refer to Ballesteros−Weinstein numbering),7 in
particular the BPM fingerprints and modeling suggested that
presence of a hydrogen bond acceptor at the para position of
ring A of the ligands to His2787.43 with addition of one or more
flanking lipophilic substituents on the same ring to interact with
Ile662.64 and Ser2777.42 should be one focus of the SAR
program (R1, R2, R3 positions in Scheme 1 and Table 1). In
addition, careful 3D analysis of the GRID maps, calculated for
both A2A and an A1 homology model, enabled the
pharmacophoric preferences (hydrophobic, hydrogen-bond
donor and acceptor) and shape constrictions/differences to
be identified to allow an enhanced evaluation of each ligand
Received: October 12, 2011
Published: January 5, 2012
© 2012 American Chemical Society
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a
Table 1. SAR and in Vitro ADME Data for Compounds 4a to 4l
SPR data
kinetic
solubility
(μM)
A2A
pKi
A1
pKi
RLM
(min)
PPB
(%)
ID
formula
LE17
ka
kd
>1 × 100
1.68 × 10
2.43 × 10
ND
KD
pKD
−6
4a
4b
4c
4d
4e
4f
XC; R1−6H
6.93
7.29
8.40
7.67
8.85
8.39
8.11
7.81
7.56
7.98
8.46
8.34
6.56
7.25
7.36
6.71
9.79
7.78
7.07
6.40
6.77
6.96
7.50
6.93
0.50
0.50
0.55
0.50
0.57
0.52
0.53
0.48
0.45
0.49
0.48
0.45
23
29
ND
97.9
99.0
98.0
98.0
93.3
82.1
69.0
ND
87.0
92.0
93.0
>100
13
38
20
45
43
40
43
45
48
35
34
>5 × 107
3.79 × 105
5.32 × 105
ND
4.07 × 106
8.57 × 106
9.92 × 106
1.13 × 107
9.44 × 106
1.41 × 107
1.08 × 106
1.55 × 106
9.03 × 10
4.42 × 10
4.57 × 10
5.0
6.4
7.3
ND
9.6
9.8
8.9
8.0
8.0
8.5
8.5
7.6
XC; R1Cl; R2−6H
−1
−7
XC; R1R3Cl; R2,4,5,6 H
108
9
−2
−8
XC; R1R3Me; R2,4,5,6H
ND
XC; R1Cl; R2OH; R3,4,5,6 H
XC; R1R3Me; R2OH; R4,5,6H
XN; R1R3Me; R4,5,6H
69
1.01 × 10
1.36 × 10
1.15 × 10
1.15 × 10
8.84 × 10
4.27 × 10
3.73 × 10
4.09 × 10
2.48 × 10
1.59 × 10
1.16 × 10
1.02 × 10
9.37 × 10
3.03 × 10
3.45 × 10
2.63 × 10
−3
−10
−3
−10
75
−2
−9
4g
4h
4i
100
100
100
78
XN; R1R3Me; R5F; R4,6H
XN; R1R3Me; R4,6F; R5H
XN; R1R3Me; R4F; R5,6H
XN; R1Me; R3CF3; R4,5,6H
XN; R1Me; R3CF3; R5F; R4,6H
−1
−8
−2
−9
−2
−9
4j
−3
−9
4k
4l
86
−2
−8
97
a
RLM rat liver microsome half-life in mins; PPB rat plasma protein binding; SPR kinetics using A2A−StaR (see main text).
docked into the binding site.8,9 Very subtle binding site
differences, such as A2A−Ser7.42 to A1−Thr7.42 and A2A−Ala2.57
to A1−Val2.57 and also ligand preferences between the two
receptor models were exploited to overall enable the design of
high yield, under iridium catalysis, with bis(pinacolato)-
diboron.11−13
X-Ray Crystallography. The overall structure of the A2A−
StaR2 in complex with compounds 4e and 4g is in close
agreement to the previously solved structures in our laboratory
and methods for crystallization and a description of the general
receptor architecture are described elsewhere.10 Statistics for
data collection and refinement are given in Supporting
Information Table S1. The cocrystal structures of the A2A−
StaR2 in complex with compounds 4g and 4e (Figure 1C,D)
show clear positive omit density at 3.0σ (data not shown) for
the presence and position of the ligands in the receptor binding
pocket. The structure of A2A−StaR2−4g shows the amino-
triazine core makes two critical donor and acceptor H-bonding
interactions with the side chain of Asn2536.55 with bonding
distances of 2.85 and 2.76 Å, respectively. In addition, the
helical portion of extracellular loop (ECL) 2 is positioned for
Phe168 to form a perpendicular π−π stack on one side of the
core, while the side chain of Met2707.35 makes a hydrophobic
interaction on the opposite side, completing the receptor
interactions around this region of the ligand. The phenyl
substituent from the C5 position of the triazine core occupies a
hydrophobic pocket deeper inside the receptor flanked by
small, polar, selective, and ligand-efficient compounds.
During the SAR optimization process, a number of members
of the series were successfully cocrystallized in the receptor
using the published thermally stabilized adenosine A2A
construct A2A−StaR2 (stabilized receptor or StaR) developed
in our laboratories.10 This allowed, for the first time to our
knowledge, X-ray structure-directed optimization of a hit series
to derive potent and selective leads for a G protein-coupled
receptor. This reinforced our efforts focused on the
optimization of the substitution patterns around the pendent
aryl rings A and B in an atom efficient manner (Scheme 1 and
Table 1) to engineer high potency and reduce affinity for the
adenosine A1 receptor without significantly increasing molec-
ular weight or lipophilicity. The X-ray structures also validated
the BPM approach, which had successfully predicted the
binding mode of the compounds but add a further level of
understanding particularly with respect to teasing out selectivity
for A2A over the A1 receptor.
Synthesis of the target compounds began with a set of
purchased 3-amino-5-aryl-1,2,4-triazine derivatives 1, Scheme 1.
These building blocks were further elaborated following
treatment with NBS at room temperature to afford the
corresponding 6-bromotriazines 2. A set of conditions was
optimized to enable Suzuki cross-coupling of a diverse range of
bromotriazines with commercially available boronic acid
derivatives. Thus 2 and 3 were coupled at 150 °C in a sealed
vessel in the presence of catalytic quantities of Pd(PPh3)4 to
afford the biaryl triazines of interest (4). Access to bespoke 4-
pyridylboronic acid derivatives was required to synthesize
molecules suggested by the proposed binding mode in the
receptor. Using a method described by Hartwig et al.,
commercial 2,6-disubstituted pyridines 5 were borylated in
Leu843.32, Leu853.33, Met1775.38, Asn1815.42, Trp2466.48
,
Leu2496.51, and His2506.52. The second substituent, dimethyl-
pyridine, from the C6 carbon of the triazine core occupies the
ribose binding pocket (of the natural agonist adenosine)
defined by His2787.43 and Ser2777.42, with one methyl
substituent pointing toward a hydrophobic region defined by
Ala632.61 and Ile662.64 and the other pointing toward the
stabilizing mutation Ser2777.42Ala.14 Additionally, the side chain
of His2787.43 is positioned 4.03 Å away from N4 of the
dimethyl-pyridine substituent, precluding a direct H-bond
between the ligand and this receptor residue. Compound 4e
occupies a similar position overall to that of 4g in the A2A
receptor ligand binding site but with specific differences. The
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amino-triazine core makes a similar set of interactions to the
receptor as 4g, however, the bond lengths to Asn2536.55
increase to 3.12 and 3.1 Å in comparison. The driving force
for this is an additional hydrogen bond, 2.87 Å in length,
formed between the phenolic hydroxyl on the chloro-phenol
substituent to Nε of His2787.43. Additionally, Glu131.37 has
switched rotamer and is now poised to H-bond to Nδ1 of
His2787.43. The additional conjugation of the ligand through
the receptor pulls compound 4e ∼1.2 Å deeper into the ribose
binding pocket in comparison to 4g (pivoting on the common
phenyl group) and perhaps provides a basis for the slow off-rate
receptor kinetics of these phenolic compounds (see Table 1). It
is noted that two potential conformations for the phenyl and
dimethyl-pyridine substituents (compound 4g), and the phenyl
and chloro-phenol substituents (compound 4e), involving
concomitant ∼50° rotations of each around the bond to the
amino-triazine core, can exist in nature. In the structures
presented here the conformations with the lower b-factors were
submitted to the Research Collaboratory for Structural
Bioinformatics Protein Data Bank (RCSB).
An analysis of the A2A binding site (with ligand 4e removed)
using the WaterMap software (Schrodinger)15 shows that these
̈
highly ligand efficient ligands (LE = 0.57 for 4e) occupy exactly
the region where there is a cluster of “unhappy” waters (shown
as red and yellow balls in Figure 1E) and not other less
favorable regions (as for example less ligand efficient
compounds such as ZM241385 bind, reported elsewhere).16
The WaterMap software uses a molecular dynamics simulation
on a full explicit water network to calculate the enthalpic and
entropic energies of waters compared to bulk solvent. Finally, in
Figure 1, as the initial SAR work was based on our BPM-
optimized homology model, we include a comparison of a
docked structure of 4e into the homology model and the
protein−ligand X-ray structure, showing that the overall
orientation and key hydrogen bond interactions were correctly
predicted (Figure 1f). The biophysical mapping fingerprints of
compounds 4e and 4g (published previously by Zhukov et al, as
compounds 3b and 3d in Table 1), respectively, are in excellent
agreement with the crystal structures, highlighting the
Figure 1. (A,B) BPM fingerprint of 1,2,4-triazine adenosine A2A
antagonists. Compounds 4g (A) and 4e (B) are illustrated bound to
the orthosteric pocket of the receptor and the residues lining the
pocket that interact with the ligands are labeled. The tier 1, 2, and 3
designation is described in the main text. The key hydrogen bonding
to Asn2536.55 of the scaffold is highlighted by green dotted lines. (C,D)
Illustration of the A2A−StaR2 ligand binding site in complex with
compound 4g (C) and 4e (D). TM helices and visible extracellular
regions are depicted in the rainbow format. Ligands are represented as
stick models, carbon and chlorine atoms are green, oxygen atoms red,
and nitrogen atoms blue. Residues involved in ligand binding are
labeled and represented as gray sticks, oxygen atoms are red, and
nitrogen atoms are blue. Extracellular loop 2, the key binding site
residues and TM’s 1, 2, 5, and 6 are labeled for reference. Potential H-
bonds between the ligand and receptor are represented as dashed blue
lines. TM3 and TM4 have been omitted for clarity. (E) WaterMap
calculation on the binding site of compound 4e (ligand removed for
the calculation). Waters calculated are color coded to show the most
“unhappy” vs bulk solvent as red (>3.5 kcal/mol), then yellow (2.2−
3.5 kcal/mol), with gray intermediate (−1 to 2.2 kcal) and blue
“happy” (<−1 kcal/mol). The CPK surface of the ligand 4e is shown
as a red dot surface, clearly illustrating that the cluster of red and
yellow “unhappy” waters deep in the binding site have been displaced.
GRID maps are also shown that highlight the shape (Csp3 (C3) at 1
kcal/mol in light-gray), the lipophilic hotspots (aromatic CH probe
(C1) in yellow at −2.5 kcal/mol), and the water probe hotspots (in
green wire mesh at −6.6 kcal/mol). (F) Alignment of the A2A
homology model with 4e docked (cyan carbons) onto the crystal
structure of A2A−4e complex (green carbons). The alignment was
generated by the align algorithm in Pymol utilizing only helices where
hydrogen bonds are formed with the ligand, helices 6 and 7. Helices 2,
3, and 4 are removed for clarity.
significant interactions with Ile662.64, Leu853.33, Asn1815.42
,
and Asn2536.55 and confirming the initial deep placement of the
ligand in the ribose sugar pocket of the A2A receptor.4
Structure−Activity Relationship. The parent compound
4a was identified as a ligand efficient adenosine A2A receptor
antagonist (Table 1).17 Having examined the putative binding
mode in silico, SAR focused on simple substitution of rings A
and B (Table 1). During the optimization process, the binding
mode predictions were first improved using our BPM approach
and then by determination of the crystal structures of
compounds 4e and 4g (discussed above). The models
suggested only small groups would be tolerated, particularly
in ring B, and this was quickly evident because addition of
chlorine or methyl in ring A and fluorine in ring B was tolerated
or increased affinity (compounds 4a−d and 4h−j), but larger
groups tended to be detrimental to potency (data not shown).
The models also suggested the potential to form hydrogen
bonding interactions from ring A at the para position R2, and
introduction of either a phenolic hydroxyl (compounds 4e and
4f) or a 4-pyridyl nitrogen (compound 4g) were found to
increase potency (compare 4f and 4g with 4d). A challenging
aspect of the optimization was that affinity for the adenosine A1
receptor was generally also observed, and it was thought
desirable to reduce affinity against this target to avoid any
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a
Scheme 1. Synthesis of 5,6-Biaryl-1,2,4-triazine-3-amine Derivatives (4) and 4-Pyridylboronic Acid Derivatives (6)
a
Reagents and conditions: (a) NBS, DMF, RT; (b) 3, Pd(PPh3)4, K2CO3, 1,4-dioxane/H2O, 150 °C; (c) [Ir(COD)OMe]2, DTBPY, [B(pin)]2,
hexane, 50 °C.
potential side effects caused by cross reactivity with this
receptor. It was quickly noted, consistent with small differences
in the binding sites predicted from a GRID analysis of models
of the two receptors (discussed above), that 3,5-disubstitution
in ring A introduced a modest selectivity for A2A over A1
(compare 4b with 4c and 4e with 4f). Also, although a very
subtle effect, introduction of fluorine at R5 on ring B reduced
activity against both receptors but slightly improved the
selectivity window for A2A over A1 (compare 4g with 4h).
Introduction of fluorine in other substitution patterns tended to
reduce affinity further or had no additional selectivity benefit
(compounds 4i and 4j). Overall, the derivatives were found to
be highly selective over the adenosine A3 receptor, e.g.,
compounds 4c, 4g, and 4h had antagonist affinity of >10 μM.
The series was generally 10-fold selective over the A2B subtype,
and inhibition of this target was not thought to be detrimental
therapeutically; 4c, 4g, and 4h had pKi values of 7.4, 7.3, and
6.8, respectively (A3 and A2B data were generated by Ricerca
Biosciences, Taipei). Fine tuning of affinity by various
combinations of small lipophilic substituents quickly led us to
pyridyl analogues 4k and 4l, incorporating a CF3 group at the
3-position of ring A with methyl at the 5-position and with or
without fluorine at the para position of ring B. These two
compounds have the best balance of potency and selectivity,
derived by introduction of simple substituents on the scaffold,
of the examples shown in the Table.
Table 1 also details the binding kinetics of compounds from
the lead series as measured using the adenosine A2A−StaR by
surface plasmon resonance on a Biacore instrument (see
Supporting Information for full details). The pKD values were
generally in good agreement with the radioligand binding data,
and the method also allowed comparison of on rates (ka) and
off rates (kd) to the receptor between related compounds. Of
most note in the data presented here is that the phenolic
analogues 4e and 4f had very slow off rates, consistent with the
change in binding mode observed in the crystal structure of 4e
discussed above. Consideration of relative receptor kinetics was
part of the decision making process used for selection of
compounds for in vivo efficacy experiments.
nitrogen atom), relatively low plasma protein binding (PPB).
Another general trend was a lack of inhibition of cytochrome
P450 enzymes and the hERG channel, with only the occasional
outlier (data not shown). A number of compounds also
demonstrated good pharmacokinetic properties in rat dosed
either orally or intravenously. Data is given here for example 4k
in a rat pharmacokinetic (PK) experiment; PK parameters are
shown in Table 2. Compound 4k displayed moderate clearance
Table 2. PK Parameters of Compound 4k in Rat
4k, 1 mg/kg (IV)
4k, 2 mg/kg (PO)
plasma clearance
42 mL/min/kg
4.6 L/kg
1.2 h
Tmax
0.4 h
Vd (ss)
Cmax
244 ng/mL
terminal t1/2
AUCinf
terminal t1/2
AUCinf
Fpo
1.1 h
397 ng·h/mL
3.2
846 ng·h/mL
100%
brain:plasma (0.5 h)
CSF:brain (0.5 h)
0.036
(42 mL/min/kg), although due to a relatively high steady-state
volume of 4.6 L/kg, the compound had an acceptable half-life
of 1.2 h. Compound 4k was rapidly absorbed after oral dosing
(Tmax = 0.4 h) and displayed good exposure with AUC = 846
ng·h/mL, resulting in an estimated Fpo of 100%. The derivative
also displayed excellent brain penetration, as measured by
samples at 0.5 h post-IV dose (brain/plasma = 3.2).
Furthermore, measured levels in the CSF at the same time
point suggested that the unbound fraction of 4k in the brain
was 3.6%, reasonably consistent with the measured plasma
protein binding in vitro of 92% (unbound plasma fraction =
8%).
Given the good overall in vitro ADME and in vivo PK profile
of 4k, especially with respect to brain penetration and oral
bioavailability, the compound was tested for its ability to
reverse haloperidol-induced catalepsy in rats, a simple and well
validated in vivo pharmacodynamic model mimicking the loss
of striatal dopamine receptor function observed in Parkinson’s
disease.1 Compound 4k was found to very potently reverse
catalepsy induced by haloperidol, with ED50 values of 0.2 mg/
kg at both 1 and 2 h post dose time points (Figure 2).
Pharmacokinetics and in Vivo Efficacy. The examples in
Table 1 generally exhibited good physicochemical and in vitro
ADME properties, having moderate to high aqueous solubility,
good stability in rat liver microsomes (RLM) and, where a
polar substituent had been introduced (such as a pyridyl
CONCLUSIONS
■
The studies presented here have shown for the first time that
biophysical mapping and cocrystal X-ray structures of ligands to
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methylpyridine (5.9 g, 83%). HPLC: 96%, 7.57 min (210 nm). Mass
spectroscopy: m/z 287.8 [M + H]+. 1H NMR: (400 MHz, DMSO) δ:
1.31 (s, 12 H), 2.51 (s, 3H), 7.70 (s, 1H), 7.76 (s, 1H).
6-(2,6-Dimethylpyridin-4-yl)-5-phenyl-1,2,4-triazin-3-amine 4g: A
Typical Procedure for the Synthesis of 5,6-Biaryl-1,2,4-triazine-3-
amine derivatives. A solution of 6-bromo-5-phenyl-1,2,4-triazin-3-
amine, (90 mg, 0.358 mmol) in dioxane (2.0 mL) was treated with 2,6-
dimethyl-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)pyridine (96
mg, 0.412 mmol) and K2CO3 (148 mg, 1.07 mmol). The resulting
mixture was diluted with water (1.0 mL), degassed, treated with
tetrakis triphenylphosphinepalladium(0) (21 mg, 0.018 mmol), and
stirred for 2 h at 150 °C in a sealed vessel. Upon completion of the
reaction, the mixture was diluted with water (20 mL) and extracted
with ethyl acetate (3 × 20 mL); the combined organic extracts were
then dried over Na2SO4 and concentrated under reduced pressure.
The crude compound was purified by gradient flash chromatography,
eluting with mixtures of ethyl acetate and hexanes to afford 6-(2,6-
dimethylpyridin-4-yl)-5-phenyl-1,2,4-triazin-3-amine 4g (44 mg, 43%).
HPLC: 98%, 6.09 min (281 nm). Mass spectroscopy: m/z 278.1 [M +
Figure 2. In vivo efficacy of 4k. Dose-dependent effect of 4k (0.1−1
mg/kg, po; 1 and 2 h pretreatment time) to reverse haloperidol-
induced catalepsy in rats in comparison with the positive control,
istradefylline (1 mg/kg, po).
1
H]+. H NMR: (400 MHz, DMSO) δ: 2.33 (s, 6H), 6.97 (s, 2H),
7.37−7.43 (m, 4H), 7.48 (m, 1H), 7.58 (bs, 2H).
6-[2-Methyl-6-(trifluoromethyl)pyridin-4-yl]-5-phenyl-1,2,4-tria-
zin-3-amine 4k. 6-[2-Methyl-6-(trifluoromethyl)pyridin-4-yl]-5-phe-
nyl-1,2,4-triazin-3-amine 4k (0.32 g, 35%) was prepared from 6-
bromo-5-phenyl-1,2,4-triazin-3-amine (0.70 g, 2.78 mmol) and 2-
methyl-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-6-trifluoro-
methyl-pyridine (1.2 g, 4.1 mmol) according to the typical procedure
described above. HPLC purity: 99%, 10.29 min (269 nm). Mass
spectroscopy: m/z 332.0 [M + H]+. 1H NMR: (400 MHz, DMSO) δ:
2.48 (s, 3H), 7.38 (m, 5H), 7.47 (s, 1H), 7.58 (s, 1H), 7.73 (bs, 2H).
Biology Methods. Methods for determination of antagonist
potency against human adenosine A2A and A1 receptors, binding
constants, and receptor kinetics of compounds binding to the A2A−
StaR by surface plasmon resonance and the procedure for
determination of in vivo efficacy in rodents by reversal of haloperidol
induced catalepsy are detailed in the Supporting Information.
Diffraction Data Collection of A2A−StaR2 in Complex with
4e and 4g. Diffraction data from crystals of A2A−StaR2 in complex
with compounds 4e and 4g were collected at I24, Diamond Light
Source, Oxford, UK. Statistics for data collection and refinement are
given in Supporting Information Table S1. Atomic coordinates and
structure factors have been deposited in the RCSB under accession
codes 3UZC and 3UZA, respectively.
a G protein-coupled receptor can be used to direct optimization
of novel, low molecular weight hit molecules into highly potent
and selective lead compounds. Compound 4k, described above,
has desirable physicochemical and drug-like properties,
including high oral bioavailability and very potent in vivo
efficacy. Further optimization of this 1,2,4-triazine series of
antagonists of the adenosine A2A receptor has subsequently
allowed identification of a preclinical candidate for the potential
treatment of Parkinson’s disease, and details of the develop-
ment of this molecule will be the topic of future publications.
EXPERIMENTAL SECTION
■
Synthetic Methods. The purity of the final compounds was
determined by HPLC or LC/MS analysis to be >95%. Full
experimental details of all compounds in Table 1 are described in
the Supporting Information. Synthesis of compounds 4g and 4k are
described below.
6-Bromo-5-phenyl-1,2,4-triazin-3-amine. A solution of 5-phenyl-
1,2,4-triazin-3-amine (1.50 g, 8.70 mmol) in DMF (15 mL) was cooled
to −25 °C and treated with a solution of N-bromosuccinimide (4.50 g,
26.6 mmol) in DMF (10 mL) by dropwise addition. The reaction was
warmed gradually to room temperature and stirred overnight with
TLC monitoring. After completion of the reaction, the mixture was
poured into saturated bicarbonate solution (50 mL) and extracted with
diethyl ether (25 × 3 mL). The organic phases were combined, dried
over Na2SO4, and concentrated in vacuo. The crude compound was
purified by gradient flash chromatography, eluting with mixtures of
ethyl acetate in hexane to afford 6-bromo-5-phenyl-1,2,4-triazin-3-
amine (1.40 g, 64%). HPLC: 99%, 8.31 min (244 nm). Mass
spectroscopy: m/z 250.9 [M + H]+. 1H NMR: (400 MHz, DMSO) δ:
7.49−7.57 (m, 5H), 7.72 (m, 2H).
Computational Chemistry. Homology models were constructed
from the avian β1 adrenergic GPCR crystal structure bound to
cyanopindolol (PDB: 2VT4) using several computational approaches
as detailed in the preceding paper and refined/validated using site-
directed mutagenesis and BPM data and known ligands (see
Supporting Information for full details).3,4 Docking was done using
Glide SP and XP (Schrodinger), and GRID analyses of the binding
̈
sites was used to evaluate potential docking poses (using the Csp3
(C3) for shape, aromatic CH probe (C1) for lipophilic hotspots,
carbonyl group (O) for hydrogen-bond acceptor hotspots, and amide
NH (N1) for hydrogen-bond donor hotspots) and driving the
designs.8,9
2-(Trifluoromethyl)-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-
yl)-6-methylpyridine. Methoxy(cyclooctadiene)iridium(I) dimer (30
mg, 0.062 mmol), 4,4′-di-tert-butyl-2,2′-bipyridine (33 mg, 0.124
mmol), and bis(pinacolato)diboron (4.09 g, 16.1 mmol) were added
to a flask which had been thoroughly purged with nitrogen. The flask
was once more purged before adding hexane via syringe (30 mL). The
resulting mixture was heated at 50 °C for 10 min until the appearance
of a dark-red solution was observed. 2-Trifluoromethyl-6-methyl
pyridine (4.0 g, 24.8 mmol) was then added by syringe, and heating
continued for a further 6 h. After cooling to room temperature, the
crude reaction mixture was concentrated under reduced pressure. The
resulting residue was purified by column chromatography, eluting with
ethyl acetate/hexane mixtures to afford the target compound 2-
(trifluoromethyl)-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-6-
ASSOCIATED CONTENT
■
S
* Supporting Information
Crystallographic table of statistics. Synthesis protocols, 1H
NMR, purification details, yields, purities by HPLC and MS or
LCMS. Biological protocols for in vitro and in vivo experi-
ments. Computational chemistry methods. SPR binding and
kinetic data. This material is available free of charge via the
Internet at http://pubs.acs.org.
Accession Codes
The PDB codes for 4g and 4e are 3UZA and 3UZC,
respectively.
1902
dx.doi.org/10.1021/jm201376w | J. Med. Chem. 2012, 55, 1898−1903

Journal of Medicinal Chemistry
Article
pounds with bis(pinacolato)diboron: regioselective synthesis of
heteroarylboronates. Tetrahedron Lett. 2002, 43, 5649−5651.
(12) Murphy, J. M.; Liao, X.; Hartwig., J. F. Meta Halogenation of
1,3-Disubstituted Arenes via Iridium-Catalyzed Arene Borylation. J.
Am. Chem. Soc. 2007, 129, 15434−15435.
AUTHOR INFORMATION
■
Corresponding Author
*Phone +44 (0)1707 358638. E-mail: miles.congreve@
heptares.com.
(13) Ishiyama, T.; Takagi, J.; Nobuta, Y.; Miyaura., N. Iridium-
catalyzed C−H borylation of arenes and heteroarenes: 1-chloro-3-
iodo-5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzene and 2-
(4,4,5,5,-tetramethyl-1,3,2-dioxaborolan-2-yl)indole. Org. Synth. 2005,
82, 126−132.
(14) Lebon, G.; Warne, T.; Edwards, P. C.; Bennett, K.; Langmead,
C. J.; Leslie, A, G. W.; Tate, C. G. Agonist-bound adenosine A2A
receptor structures reveal common features of GPCR activation.
Nature 2011, 474, 521−523.
(15) Higgs, C.; Beuming, T.; Sherman, W. Hydration site
thermodynamics explain SARs for triazolylpurines analogues binding
to the A2A receptor. ACS Med. Chem. Lett. 2010, 1, 160−164.
(16) Congreve, M.; Langmead, C. J.; Mason, J. S.; Marshall, F. H.
Progress in Structure Based Drug Design for G Protein-Coupled
Receptors. J. Med. Chem. 2011, 54, 4283−4311.
(17) Hopkins, A. L.; Groom, C. R.; Alex, A. Ligand efficiency: a
useful metric for lead selection. Drug Discovery Today 2004, 9, 430−
431.
ACKNOWLEDGMENTS
■
We thank Chris Richardson and Bissan Al-Lazikani for help
with constructing the first generation of homology models and
ligand dockings, David Myszka for helping to establish
conditions for use of A2A StaRs on the Biacore format as a
precursor to the work in this manuscript, and Nathan
Robertson, Asma Baig, Jason Brown, Alistair O’Brien, and
Giles Brown at Heptares.
ABBREVIATIONS USED
■
StaR, stabilized receptor; BPM, biophysical mapping; GPCR, G
protein-coupled receptor; SPR, surface plasmon resonance;
ECL, extracellular loop; TM, transmembrane helix; SAR,
structure−activity relationship; PDB, Protein Data Bank; LE,
ligand efficiency; RLM, rat liver microsomal turnover; PPB, rat
plasma protein binding; RCSB, Research Collaboratory for
Structural Bioinformatics Protein Data Bank
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