Synthesis and SAR evaluation of 1,2,4-triazoles as A2A receptor antagonists

Article abstract of DOI:10.1016/j.bmcl.2003.09.095

The synthesis and in vitro structure-activity relationships (SAR) of a series of triazoles as A_2A receptor antagonists is reported. This resulted in the identification of potent, selective and permeable 1,2,4-triazoles such as 42 for further op

Full text of DOI:10.1016/j.bmcl.2003.09.095

Bioorganic & Medicinal Chemistry Letters 14 (2004) 817–821
Synthesis and SAR evaluation of 1,2,4-triazoles
as A2A receptor antagonists
Alexander Alanine,a Lilli Anselm,a Lucinda Steward,b Stefan Thomi,a Walter Vifiana
and Michael D. Groaninga,*
aF. Hoffmann-La Roche AG, Pharmaceuticals Division, Discovery Chemistry, Lead Generation, Basel, CH 4070, Switzerland
bF. Hoffmann-La Roche AG, Pharmaceuticals Division, Discovery Biology, Central Nervous System Disorders,
Basel, CH 4070, Switzerland
Received 1 June 2003; revised 25 August 2003; accepted 9 September 2003
Abstract—The synthesis and in vitro structure–activity relationships (SAR) of a series of triazoles as A2A receptor antagonists is
reported. This resulted in the identification of potent, selective and permeable 1,2,4-triazoles such as 42 for further optimization and
evaluation in vivo.
# 2003 Elsevier Ltd. All rights reserved.
The neurotransmitter adenosine interacts with four G-
protein coupled receptors the A1, A2A, A2B and A3
receptors.1 The A2A receptor, which is Gs coupled, is of
particular interest in the area of psychiatry research
with high receptor densities found to be located in the
dopamine-rich areas of the striatum and nucleus
accumbens.2 More recently A2A receptors have been
shown to be co-localized and interact with both dopamine
D2 and metabotropic glutamate 5 (mGlu5) receptors.3
Thus, antagonism of the A2A receptor may provide a
subtle means of modulating the dopaminergic system,
without the associated motor side effects of direct D2
receptor interaction. Furthermore, animal behavioral
studies have revealed the A2A receptor as a potential
new target for the development of antidepressants4 and
anti-Parkinsonian treatments.5 Several drug canditates,
which all contain a distinct purine motif, are currently
being evaluated in the hope to exploit this receptor target.
These compounds suffer from a variety of drawbacks
that call for the development of new classes of ligands
for the A2A receptor, due to the following: (a) the pre-
sence of the 2-mono-substituted furan moiety in 1 and 2
which carries metabolic liability, but is required for both
high affinity and selectivity; (b) the styryl moiety in 3 is
highly susceptible to photoisomerization, wherein the
thermodynamically more stable cis photoisomer is almost
1000 times weaker in the rat A2a [rA2a] binding assay;
finally (c) poly-annulated xanthinic and tricyclic structures
invariably suffer from poor physiochemical character-
istics (i.e., solubility, lipophilicity) that provide the oppor-
tunity to develop compounds with improved properties.
Keywords: 1,2,4-Triazoles; A2A receptor antagonists; SAR.
*Corresponding author at current address: Roche Colorado Corporation, 2075 North 55th Street, Boulder, CO 80301, USA. E-mail: michael.
groaning@roche.com
0960-894X/$ - see front matter # 2003 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2003.09.095

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A. Alanine et al. / Bioorg. Med. Chem. Lett. 14 (2004) 817–821
Scheme 1. (a) Hydrazine, EtOH, Á, 73%; (b) ArCHO, EtOH, 55–89%.
In order to identify and develop novel, biologically
active compounds in the A2 receptor class, we chose to
approach this task in two ways; (i) through an HTS
screening campaign and (ii) de novo design based on a
ligand derived pharmacophore model. The later
approach will be described in more detail elsewhere.
In order to address the problem with isomerization
about the hydrazone double-bond, N-acyl dihydropyr-
azoles 19 was synthesized but was found to be com-
pletely inactive (Ki>5.2 mM). Synthesis of the oxygen
variant of thioacylhydrazone 20 eliminated the metabo-
lically unstable thiocarbonyl and showed no isomeriza-
tion, but unfortunately it was also inactive. One
explanation for this finding might be the requirement
for a certain minimal NH acidity, since there is con-
siderable difference in the pKa (6.6) 8 compared to the
oxo analogue pKa (2.9) 20. In order to investigate this
possibility, the sulfone derivative 18 (pKa 6.5) was pre-
pared but found to be inactive.
Screening the Roche compound depository using a
scintilation proximity assay with [3H]SCH-58261 resul-
ted among other compounds in the identification of a
series of thioacylhydrazones (7, 12) as validated hits.6
We were aware of the metabolic liabilities of such com-
pounds but viewed this as a tractable entry point for
further investigation of the binding and selectivity
requirements (SAR), thus adding to our knowledge of
the A2A pharmacophore. In the initial synthetic approach
to thioacylhydrazones 6–13, we varied the appended
aryl group, by construction of the thioacylhydrazide 5
from 4, followed by condensation with a variety of
aldehydes to afford the respective hydrazones (Scheme
1). By analysis of the first small array we uncovered
some important findings: (a) geometry of the hydrazone
double bond was labile, isomerizing at room tempera-
ture; and (b) there was a surprisingly large range of
radioligand binding affinity (1000-fold) for these com-
pounds despite rather conservative variations (i.e., 8 vs
12), which could not be easily rationalized (Table 1).
Steric bulk was not well tolerated as demonstrated by
the loss in binding affinity with compounds 15 versus 8
and 9; and 6 versus 13.
Due to these findings we decided to terminate this series
of compounds. Since there exists a number of surrogates
of the amide and hydrazide moiety, amongst them the
triazole is described, our attention was drawn to a small
series of modestly active biaryl 1,2,4-triazole hits 21–23
bearing a basic meta side-chain or an ortho phenolic
moiety reminiscent of the thiohydrazides. Their profile
was considered promising with regard to affinity, selec-
tivity, stability and chemical tractibility. Interestingly,
compound 21 had the best affinity despite the lack of an
acidic NH. This suggested that the triazoles may offer
an alternative binding mode or an additional exit vector
that could be exploited.
Despite the attraction of a terminal polar function for
solubility purposes, we chose to remove this in order to
Table 1. Human recombinant A2A [hA2A] receptor affinities of compounds 6–13 [variation of R], determined by displacement of [3H]SCH-58261
radioligand binding7
Compd
R
Ki [mM]a Compd
R
Ki [mM]a Compd
R
Ki [mM]a Compd
R
Ki [mM]a
6
0.005
0.01
0.02
9
0.1
0.1
1.2
12
13
14
1.4
2.0
4.2
15
16
17
4.7
7
8
10
11
>5.2
>5.2
a Binding affinities are quoted as Ki values and are the mean of at least two experiments.

A. Alanine et al. / Bioorg. Med. Chem. Lett. 14 (2004) 817–821
819
Scheme 2. Route to 30–33 via acyl hydrazide; Reagents and conditions: (a) CDI, THF, reflux, 82–93%; (b) EtOH, reflux, 17–97%.
avoid introducing an amphiphilic vector resulting in
potential phospholipidosis. It was rapidly discovered
that significant binding affinity could be gained by
simply introducing a spacer atom between the tri-
azole core and the aryl side chain in 24, however
this effect was sensitive to further elongation 26 or
substitution and the optimum was found with a
methylene linker 25.
With these preliminary findings, a second focused array
was built around the benzyl, aryl substituted triazoles
30–33 and was constructed via condensation of the
acylhydrazide 28 with an ethyl benzylimidate 29 in
refluxing ethanol, providing the triazole, Scheme 2. The
starting acylhydrazide was obtained from the acid 27
with hydrazine using standard coupling conditions with
CDI which consistently provided high yields.

820
A. Alanine et al. / Bioorg. Med. Chem. Lett. 14 (2004) 817–821
Scheme 3. Route to 38–47 via thioimidate; Reagents and conditions: (a) CDI, THF, reflux; (b) Hydrazine, 80–96% over two steps; (c) NaSH,
NH4Cl, MeOH, 98%; (d) MeI, acetone, 89%; (e) EtOH, reflux, 60–75%.
The results revealed that the meta-methoxy group on
the aryl ring is essential for good A2A receptor binding.
This finding prompted us to concentrate on the benzyl
portion of the triazoles and that required a change in
the synthetic approach to allow expedient synthesis of
the second-generation compounds for evaluation. The
thioimidate 37 could be accessed by addition of sodium
sulfide to 3-methoxy benzonitrile 36 followed by
S-methylation of the resulting thioamide using methyl-
iodide in acetone. Coupling of the thioimidate 37 with
the respective acylhydrazide 35, provided our second
generation focused library of 3-m-methoxyphenyl-5-
benzyl triazoles 38–47 (Scheme 3).
The intial SAR showed that the m-methoxyphenyl moi-
ety was optimal with very little variation permitted. The
benzyl moiety provided more scope for variation pro-
vided substituents are hydrophobic in nature but intro-
duction of a polar group 47 dramatically abolishes
binding activity (Tables 2 and 3).
A set of the active compounds 38, 40, 42, 45 were fur-
ther profiled for their selectivity against the human A1
[hA1] receptor as well as their activity in the rat A2A
receptor binding. We observed moderate selectivities as
well as sufficient activity at the rat receptor to be con-
sidered for testing in the animal models. Additionally,
these compounds showed good to medium permeability
in the PAMPA assay and satisfactory solubility (2–20
mg/mL)9 (Table 4).
Table 2. hA2A receptor affinities of compounds 38–47 [variation of R],
determined by displacement of [3H]SCH-58261 radioligand binding7
In summary, we have identified a series of triazoles that
show significant activity at the A2A receptor and display
attractive physiochemical properties compared to 1–3.
We are currently working towards refining these com-
pounds and analyzing their in vivo activity and will
report these results in due course.
Compd
R2
Ki [mM]a Compd
R2
Ki [mM]a
Table 3. hA2A receptor affinities of compounds 30–33 [variation of R],
determined by displacement of [3H]SCH-58261 radioligand binding7
38
0.01
0.04
43
44
0.07
39
0.08
Compd
R
Ki [mM]a
Compd
R
Ki [mM]a
40
41
42
0.02
0.02
0.02
45
46
47
0.1
0.7
30
0.06
32
0.1
>5.2
31
0.09
33
>5.2
a Binding affinities are quoted as Ki values and are the mean of at least
two experiments.
a Binding affinities are quoted as Ki values and are the mean of at least
two experiments.

A. Alanine et al. / Bioorg. Med. Chem. Lett. 14 (2004) 817–821
821
Table 4. Recombinant hA2A, rA2A and hA1 receptor affinities of
compounds 38, 40, 42, 45 [variation of R], determined by displacement
of [3H]SCH-58261 [A2A] or [3H] DPCPX [A1] radioligand binding7
Ferre, S.; Karcz-Kubicha, M.; Hope, B. T.; Popoli, P.;
Burgueno, J.; Gutierrez, M. A.; Casado, V.; Fuxe, K.;
Goldberg, S. R.; Lluis, C.; Franco, R.; Ciruela, F. Proc.
Natl. Acad. Sci. 2002, 99, 11940.
4. Yacoubi, M. E.; Ledent, C.; Parmentier, M.; Bertorelli,
R.; Ongini, E.; Costentin, J.; Vaugeois, J.-M. Brit. J.
Pharmacol. 2001, 134, 68.
5. Richardson, P. J.; Kase, H.; Jenner, P. G. Trends Phar-
macol. Sci. 1997, 18, 338.
6. The original hit was identified as the cyclic version and
was later confirmed to be the open chain form.
Compd
R
hA2A Ki hA1 Ki sel. rA2A Ki PAMPA8
[mM]a
[mM]a
[mM]a
10À6 s/cm
38
40
42
45
0.01
0.1
7
51
69
20
0.04
1.9 (high)
7. hA2A, hA1 and rA2A receptor affinities were determined in
a 96 well plate assay, using [3H]SCH-58261 (hA2A and
rA2A; final concentration 1 and 0.4 nM, respectively)
and [3H] DPCPX (final concentration 0.6 nM) to radi-
olabel the receptors in the presence of 10 concentrations
of competing compound or buffer. Non-specific binding
was determined using 2 mM Xanthine amine congener
(XAC). Assay buffer consisted of Tris–HCl (50 mM, pH
0.02
0.02
0.1
0.9
1.7
2.5
0.04
0.09
0.4
3.8 (high)
0.9 (high)
0.3 (med.)
.
7.4), NaCl 120 mM, KCl 5 mM, CaCl2 2H2O 2 mM,
.
MgCl2 6H2O 10 mM. Membrane preparations of recep-
tors (approximately 2 mg/well in a 96-well plate) were
mixed with adenosine deaminase (10 mg/mL) and scintil-
lation proximity beads (SPA; 0.5 mg/well; Amersham)
and added to the wells to initiate the incubation for 60
min (hA2A and hA1) or 90 min (rA2A) at room temper-
ature. The assay was terminated by centrifugation (3000
rpm, 3 min) and counted in a scintillation counter (Top-
count, Packard). All assays were performed in duplicate
in at least two separate experiments. Graphs were plotted
with the % specific binding using the iterative curve fitting
program XL-fit.
References and notes
1. Fredholm, B. B.; Ijzerman, A. P.; Jacobson, K. A.; Klotz,
K.-N.; Linden, J. Pharmcol. Rev. 2001, 53, 527.
2. (a) Svenningsson, P.; Le Moine, C.; Fisone, G.; Fred-
holm, B. B. Progress in Neurobiology 1999, 59, 355. (b)
Hettinger, B. D.; Lee, A.; Linden, J.; Rosin, D. L. J.
Comp. Neurology 2001, 431, 331.
3. (a) Hillion, J.; Canalx, M.; Torvinen, M.; Casado, V.;
Scott, R.; Terasmaa, A.; Hansson, A.; Watson, S.; Olah,
M. E.; Mallol, J.; Canela, E. I.; Zoli, M.; Agnati, L. F.;
Ibanez, C. F.; Lluis, C.; Franco, R.; Ferre, S.; Fuxe, K. J.
Biol. Chem. 2002, 277, 18091. (b) Johansson, B.; Geor-
giev, V.; Fredholm, B. B. Neuroscience 1997, 80, 1187. (c)
8. PAMPA=Parallel Artificial Membrane Permeation
Assay: Kansy, M.; Senner, F.; Gubernator, K. J. Med.
Chem. 1998, 41, 1007.
9. Solubility determined in 0.05 M phosphate buffer at pH
6.5.