The 1,2,4-Triazolo[4,3-a]pyrazin-3-one as a Versatile Scaffold for the Design of Potent Adenosine Human Receptor Antagonists. Structural Investigations to Target the A2A Receptor Subtype

Article abstract of DOI:10.1021/acs.jmedchem.7b00457

In this work, we describe the identification of the 1,2,4-triazolo[4,3-a]pyrazin-3-one as a new versatile scaffold for the development of adenosine human (h) receptor antagonists. The new chemotype ensued from a molecular simplification approach applied to our previously reported 1,2,4-triazolo[4,3-a]quinoxalin-1-one series. Hence, a set of novel 8-amino-2-aryl-1,2,4-triazolopyrazin-3-one derivatives, featured by different substituents on the 2-phenyl ring (R) and at position 6 (R₆), was synthesized with the main purpose of targeting the hA_2A adenosine receptor (AR). Several compounds possessed nanomolar affinity for the hA_2A AR (K_i = 2.9-10 nM) and some, very interestingly, also showed high selectivity for the target. One selected potent hA_2A AR antagonist (12, R = H, R₆ = 4-methoxyphenyl) demonstrated some ability to counteract MPP⁺-induced neurotoxicity in cultured human neuroblastoma SH-SY5Y cells, a widely used in vitro Parkinson's disease model. Docking studies at hAR structures were performed to rationalize the observed affinity data.

Full text of DOI:10.1021/acs.jmedchem.7b00457

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Article
The 1,2,4-Triazolo[4,3-a]pyrazin-3-one as a Versatile Scaffold for
the Design of Potent Adenosine Human Receptor Antagonists.
2A
Structural Investigations to Target the A Receptor Subtype
Matteo Falsini, Lucia Squarcialupi, Daniela Catarzi, Flavia Varano, Marco Betti, Diego Dal Ben, Gabriella
Marucci, Michela Buccioni, Rosaria Volpini, Teresa De Vita, Andrea Cavalli, and Vittoria Colotta
J. Med. Chem., Just Accepted Manuscript • Publication Date (Web): 07 Jun 2017
Downloaded from http://pubs.acs.org on June 8, 2017
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The 1,2,4-Triazolo[4,3-a]pyrazin-3-one as a Versatile Scaffold
for the Design of Potent Adenosine Human Receptor
Antagonists. Structural Investigations to Target the A2A
Receptor Subtype
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Matteo Falsini, Lucia Squarcialupi, Daniela Catarzi, Flavia Varano, Marco Betti, Diego Dal
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b
b
b
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c,d
Ben, Gabriella Marucci, Michela Buccioni, Rosaria Volpini, Teresa De Vita, Andrea Cavalli,
a*
Vittoria Colotta
a
Dipartimento di Neuroscienze, Psicologia, Area del Farmaco e Salute del Bambino, Sezione di Farmaceutica e
Nutraceutica, Università degli Studi di Firenze, Via Ugo Schiff, 6, 50019 Sesto Fiorentino, Italy.
b
Scuola di Scienze del Farmaco e dei Prodotti della Salute, Università degli Studi di Camerino, via S. Agostino 1,
6
2032 Camerino (MC), Italy.
c
CompuNet, Istituto Italiano di Tecnologia (IIT), Via Morego 30, 16163 Genova, Italy.
d
Dipartimento di Farmacia e Biotecnologia, Università degli Studi di Bologna, Via Belmeloro 6, 40126 Bologna, Italy.
Key words: G proteinꢀcoupled receptors, A2A adenosine receptor antagonists, 1,2,4ꢀtriazolo[4,3ꢀ
a]pyrazinꢀ3ꢀone, ligandꢀadenosine receptor modeling studies.
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ABSTRACT
In this work we describe the identification of the 1,2,4ꢀtriazolo[4,3ꢀa]pyrazinꢀ3ꢀone as a new
versatile scaffold for the development of adenosine human (h) receptor antagonists. The new
chemotype ensued from a molecular simplification approach applied to our previously reported
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,2,4ꢀtriazolo[4,3ꢀa]quinoxalinꢀ1ꢀone series. Hence, a set of novel 8ꢀaminoꢀ2ꢀarylꢀ1,2,4ꢀ
triazolopyrazinꢀ3ꢀone derivatives, featured by different substituents on the 2ꢀphenyl ring (R) and at
position 6 (R ), was synthesized with the main purpose of targeting the hA adenosine receptor
6
2A
i
(AR). Several compounds possessed nanomolar affinity for the hA2A AR (K = 2.9ꢀ10 nM) and
some, very interestingly, also showed high selectivity for the target. One selected potent hA2A AR
+
antagonist (12, R= H, R
6
= 4ꢀmethoxyphenyl) demonstrated some ability to counteract MPP ꢀ
induced neurotoxicity in cultured human neuroblastoma SHꢀSY5Y cells, a widely used in vitro
Parkinson’s disease model. Docking studies at hAR structures were performed to rationalize the
observed affinity data.
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INTRODUCTION
The neuromodulatorꢀꢀadenosineꢀꢀexerts important physiological functions through activation of
1,2
specific G proteinꢀcoupled receptors, subdivided into the four subtypes A
1
, A2A, A2B and A
3
.
A
2A
adenosine receptor (AR) is ubiquitous in the human body and are expressed in a variety of
peripheral organs such as the heart, liver and lung. In the central nervous system, a higher density of
the A2A AR has been found in the striatum, nucleus accumbens and olfactory tuberculum and lower
density in the cortex and hippocampus. A2A AR is highly expressed in platelets, leukocytes,
immune and endothelial cells, implying a role of this receptor in the regulation of inflammatory
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1,2
processes.
The A2A AR is coupled to Gs proteins that activate adenylate cyclase, thus enhancing intracellular
cAMP levels. The subsequent stimulation of protein kinase A causes phosphorylation and activation
of different proteins, such as receptors, enzymes and ion channels depending on the cell type and
tissue. A2A AR has attracted much interest as druggable target for therapeutic intervention in
3
4,5
3,6,7
neurodegenerative diseases, such as Huntington’s disease and cerebral ischemia.
Related to
them, the ability of A2A AR antagonists in inducing protection is well known and is largely
attributed to the control of excessive glutamatergic transmission in striatal and nigral neurons,
4,7
which prevents neuronal death. A large body of evidence has clearly shown that A2A AR
8
,9
antagonists are beneficial in animal models of Parkinson's disease (PD) and, as a result,
pharmaceutical industries have progressed A2A antagonists to clinical trials, such as Istradefylline
10
which has been approved for marketing in Japan.
A2A AR antagonists have demonstrated
therapeutic value in the treatment of PD because they potentiate dopamine D receptorꢀmediated
2
neurotransmission. At a striatal level, in fact, the A2A AR is coꢀexpressed with the D dopamine
2
receptor on GABAergic neurons of the indirect pathways of motor control where adenosine and
dopamine elicit opposite effects in the modulation of adenylate cyclase activity, thus behaving as
1
1,12
functional antagonists in the regulation of motor function.
Moreover, in the striatum, due to
allosteric receptorꢀreceptor interactions in the A ꢀD heteroreceptor complexes, adenosine reduces
2
A
2
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the affinity of agonists for the D receptor, thus behaving as a negative modulator of D receptorꢀ
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mediated neurotransmission.
However, interest in the use of A2A AR antagonists in PD has increased because recent studies have
highlighted that, besides being effective in relieving motor symptoms, they might help control
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neuropsychiatric impairments of the disease, such as anxiety and cognitive deficiency
but, more
16,17
importantly, help counteract neurodegeneration.
Although therapies are available to treat the
signs and symptoms of PD, the progression of neuronal death remains relentless. Hence, there is a
need to develop neuroprotective or diseaseꢀmodifying treatments that stabilize this degeneration. In
animal models, A2A AR antagonists protected nigral dopaminergic neurons from death induced by
17
both 1ꢀmethylꢀ4ꢀphenylꢀ1,2,3,6ꢀtetrahydropyridine (MPTP) and 6ꢀhydroxydopamine, slowing the
deterioration of dopamineꢀproducing cells and modifying PD progression. However, the precise
molecular mechanisms underlying the A2A AR blockadeꢀinduced neuroprotection is far from being
clearly understood. Extraꢀstriatal A2A ARs, e.g. those on the forebrain neurons, probably modulate
1
8
17
neurodegeneration and possibly exert protective effects mediated by extraꢀneuronal A2A ARs. In
particular, MPTP toxicity is probably attenuated through a mechanism counteracting
1
7,19
neuroinflammation and involving A2A AR on glial cells.
MPTP is transformed into the toxic
+
metabolite 1ꢀmethylꢀ4ꢀphenylꢀpyridinium (MPP ) which produces cell death due to inhibition of
mitochondrial respiratory chain.
2
0ꢀ25
Based on these data, most of our recent research in the field of adenosine
has been focused on
identifying selective antagonists of the hA2A AR subtype. To this aim, different sets of 7ꢀ
22,23,25
aminopyrazolo[4,3ꢀa]pyrimidines have been synthesized recently
as bicyclic analogues of our
26,27
tricyclic pyrazolo[3,4ꢀc]quinoline derivatives
(Chart 1). The synthetic versatility of the bicyclic
scaffold permitted us to replace the fused benzo ring of the leads with many different substituents
(alkyl, aryl, arylalkyl) and to obtain several pyrazolo[4,3ꢀa]pyrimidines endowed with nanomolar
2
2,23
affinity for the hA2A receptor.
Hence, searching for a new bicyclic chemotype to develop
selective hA2A AR antagonists, we applied the same simplification approach to another tricyclic
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scaffold, i.e. the 1,2,4ꢀtriazolo[4,3ꢀa]quinoxalinꢀ1ꢀone (Chart 1, TQX series) which we have
successfully employed in the past to obtain potent and selective antagonists of A , A2A and A3
1
28ꢀ32
ARs.
Thus, we designed the 2ꢀarylꢀ8ꢀaminoꢀ1,2,4ꢀtriazolo[4,3ꢀa]pyrazinꢀ3ꢀone series (Chart 1)
considering that its synthetic accessibility would have permitted us to introduce diverse substituents
) with different lipophilic and steric properties at the 6ꢀposition. Hence, a set of 8ꢀaminoꢀ1,2,4ꢀ
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(R
6
triazolopyrazinꢀ3ꢀone derivatives (Chart 1, 1-26) was synthesized and biologically assayed at hARs
to investigate the SARs of this new series and to shed light on the structural requirements of
targeting the hA2A AR subtype. Binding studies at hARs were also carried out on the intermediates
1
,2,4ꢀtriazolo[4,3ꢀa]pyrazineꢀ3,8ꢀdione derivatives 48-62 (see Scheme 1) because they were
envisaged to possess affinity for the hA AR subtype which was still of our interest, although being
3
offꢀtarget for the work (see Results and Discussion for details).
RESULTS AND DISCUSSION
Chemistry
The new 8ꢀaminoꢀ1,2,4ꢀtriazolo[4,3ꢀa]pyrazinꢀ3ꢀone derivatives 1-15 and 16-26 were prepared as
outlined in Schemes 1 and 2, respectively. The first set was synthesized starting from the ethyl 2ꢀ
23,33,34
arylhydrazonoꢀ2ꢀchloroacetates 27-30,
prepared by reacting the suitable aryldiazonium
chloride with ethyl 2ꢀchloroacetoacetate, in the presence of sodium acetate in MeOH. Treatment of
27-30 with 33% aqueous ammonia in dioxane yielded the corresponding aminoꢀderivatives 31-
3
5,36
3
4,
which were cyclized with triphosgene to yield the key intermediates ethyl 5ꢀoxoꢀ1ꢀarylꢀ1Hꢀ
37
4
1
,2,4ꢀtriazoleꢀ3ꢀcarboxylates 35-38. Their N ꢀalkylation with the suitable αꢀhaloketones in
DMF/CH CN, and in the presence of potassium carbonate, afforded the ethyl 1ꢀarylꢀ5ꢀoxoꢀ1,2,4ꢀ
3
triazoleꢀ3ꢀcarboxylate derivatives 39-47 whose cyclization with ammonium acetate, performed by
conventional heating or under microwave irradiation, gave the 1,2,4ꢀtriazolo[4,3ꢀa]pyrazineꢀ3,8ꢀ
3
dione derivatives 48-53, 58-60. The methoxy derivatives were demethylated with BBr (50-51, 53
and 58) or with 48% HBr in glacial acetic acid (59), to yield the corresponding hydroxyꢀsubstituted
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compounds 54-56, 61 and 62. Catalytic (Pd/C) hydrogenation in a Parr apparatus of the 2ꢀ(4ꢀ
nitrophenyl) derivative 52 furnished the 2ꢀ(4ꢀaminophenyl) derivative 57. The 3,8ꢀdiones 48-53, 58-
6
0 were chlorinated with phosphorus oxychloride, under microwave irradiation, to obtain the
corresponding 8ꢀchloro derivatives 63-71 which gave the desired 8ꢀaminoꢀ1,2,4ꢀtriazolo[4,3ꢀ
a]pyrazineꢀ3ꢀone derivatives 1-6, 11-13 upon treatment with a saturated solution of ammonia in
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absolute ethanol. Demethylation of the methoxyphenyl derivatives 3, 4, 6, 11 and 12 with BBr
gave the corresponding hydroxyphenylꢀsubstituted compounds 7-9, 14 and 15. The 2ꢀ(4ꢀ
nitrophenyl) derivative 5 was reduced (H , Pd/C) in a Parr apparatus to yield the corresponding 2ꢀ
4ꢀaminophenyl) derivative 10. The synthesis of compounds 16-27 is outlined in Scheme 2. Briefly,
3
2
(
the derivatives were prepared by alkylation of the 6ꢀ(hydroxyphenyl) derivatives 14 or 15 with the
suitable alkyl bromides, in refluxing 2ꢀbutanone and in the presence of potassium carbonate.
Pharmacological Assays
The 8ꢀaminoꢀ2ꢀarylꢀ1,2,4ꢀtriazolo[4,3ꢀa]pyrazinꢀ3ꢀones 1-26 and the 3,8ꢀdione derivatives 48-62
1 3
were evaluated for their affinity to hA , hA2A and hA ARs, stably transfected in Chinese hamster
ovary (CHO) cells, and were also tested at the hA2B AR subtype by measuring their inhibitory
effects on 5’ꢀ(Nꢀethylꢀcarboxamido)adenosine (NECA)ꢀstimulated cAMP levels in hA CHO cells.
2B
Moreover, derivatives 12 and 20, showing high hA2A AR affinity and selectivity, were studied by
evaluating their effect on cAMP production in CHO cells, stably expressing hA2A AR, in order to
assess the ability of these compounds to inhibit or stimulate the A2A AR. All pharmacological data
are presented in Tables 1ꢀ2. The selected derivative 12 was also profiled for its protective effect
+
against MPP ꢀneurotoxicity in cultured human neuroblastoma SHꢀSY5Y cell lines, a widely used
38,39
cellular PD model.
The results of these experiments are reported in Figures 1ꢀ3.
Structure-affinity relationship studies
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The biological data reported in Tables 1ꢀ2 show that several new derivatives exhibited nanomolar
affinity for hA , hA and hA ARs thus indicating that the 1,2,4ꢀtriazolo[4,3ꢀa]pyrazineꢀ3ꢀone is a
1
2A
3
valuable scaffold for developing new potent antagonists for these AR subtypes. All the tested
compounds proved to be inactive (IC50> 30000 nM) in inhibiting the NECAꢀstimulated cAMP
levels in hA2B CHO cells. These results enabled us to deduce that compounds 1-29 and 48-62
lacked affinity for the hA2B AR. In fact, the IC50 values obtained by our cAMP assays on reference
10
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6
ligands, such as 8ꢀcyclopentylꢀ1,3ꢀdipropylꢀxanthine (DPCPX), NECA and 2ꢀchloroꢀN –
i
cyclopentyladenosine (CCPA) (Tables 1 and 2) well correlated with the K values obtained from
40,41
radioligand binding studies.
8-Amino-2-aryl-1,2,4-triazolo[4,3-a]pyrazin-3-one derivatives 1-26
As anticipated above, the main purpose of the work was to perform a preliminary investigation of
the SARs of this new series and to identify new hA2A AR antagonists. This aim has been completely
satisfied because four triazolopyrazines (12, 17, 20 and 22) possess high affinity (K
and a complete selectivity for the hA2A AR subtype. Several compounds (2, 11, 14, 16) show high
affinity for the hA2A receptor (K < 10 nM) and also bind to the hA subtype with similar or just 5ꢀ
i
= 2.9ꢀ10.6 nM)
i
1
to 6ꢀfold reduced affinity. This behavior makes these derivatives interesting as well, because dual
targeted antagonists of hA and hA2A ARs have emerged as promising agents for the treatment of
1
4
2ꢀ44
PD.
dopamine release.
release (A ) and potentiate the postꢀsynaptic response to dopamine (A2A). Moreover, hA
1
The hA AR is presynaptically expressed on striatal dopaminergic neurons where it inhibits
4
5ꢀ46
Hence, dual hA
1
and hA2A AR antagonism would both facilitate dopamine
ARs are
1
1
also expressed in the hippocampus, neocortex and limbic system that are brain areas implicated in
1
the control of cognitive and emotive functions. Thus, hA AR antagonists could ameliorate
1
cognitive impairments associated with PD since they improve performance in an animal model of
43,44
learning and memory.
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Some triazolopyrazines, possessing high affinity for both hA
1
and hA2A ARs, were also able to bind
efficiently to the hA AR subtype (2, 11, 15, 16), the most active compound, being the 2,6ꢀdiphenyl
3
i
derivative 2 (K = 11 nM).
Retracing the diverse phases of our work, we first synthesized a set of compounds (1-4, 7-8) to
evaluate which group, between methyl and phenyl, was the better for the 6ꢀposition. The 2,6ꢀ
diphenylꢀsubstituted derivative 2 turned out to be notable, showing high and comparable affinities
for hA , hA and hA ARs (K = 10ꢀ13 nM). The 6ꢀmethylꢀ2ꢀphenyl derivative 1 was significantly
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i
less active, in particular for the targeted hA2A receptor. The para hydroxy substituent inserted on the
2
8
2
ꢀphenyl ring of 1 and 2, was chosen because in the TQX series it was profitable for A2A AR
affinity. The 2ꢀ(4ꢀhydroxyphenyl)ꢀsubstituted 7 and 8 were less active at the hA2A AR than the
unsubstituted derivatives 1 and 2 and also than the respective methoxy derivatives 3 and 4, their
synthetic precursors. The last two derivatives showed, on the whole, lower affinities for both hA1
and hA2A ARs than their parent compounds 1 and 2.
The comparison of AR affinities of the 6ꢀmethyl derivatives 1, 3 and 7 with those of the
corresponding 6ꢀphenyl derivatives 2, 4 and 8 highlighted that the 6ꢀphenyl residue was more
advantageous than the 6ꢀmethyl one, probably due to its higher lipophilicity and/or capability to
enhance the structural complementarity of the whole molecule with the receptor binding site.
Hence, subsequent investigations were carried out on the 6ꢀphenylꢀsubstituted compound 2 which
was modified by introduction of a para amino group on the 2ꢀphenyl residue (compound 10), as
2
8
suggested by affinity data of the TQX derivatives. Compound 10 did not show enhanced affinity
for the hA2A AR, with respect to the lead 2, and the same applies to its synthetic precursor 2ꢀ(4ꢀ
nitrophenyl) derivative 5. On the contrary, hA AR affinities of 5 and 10 were very high (K = 8.1
1
i
and 8.9 nM) and similar to that of 2. The presence of a methoxy or hydroxy group at the ortho
position of the 2ꢀphenyl ring proved to be disadvantageous for the recognition of both hA2A and hA
ARs. In fact, compounds 6 and 9 are significantly less active than 2, even if the hA2A affinity of the
1
2
ꢀ(2ꢀhydroxyphenyl) derivative 9 still remained good (K = 47 nM).
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The binding data obtained within these first sets of compounds indicated that the best group for the
2
ꢀposition was the unsubstituted phenyl ring. Thus, in the subsequent derivatives the 2ꢀphenyl ring
was maintained unmodified while structural changes were made at the 6ꢀphenyl level. Introduction
of a methoxy or hydroxy group at the meta (11 and 14) and para (12 and 15) positions on this
moiety achieved interesting results, the most relevant being the identification of the 6ꢀ(4ꢀ
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
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45
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48
49
50
51
52
53
54
55
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57
58
59
60
methoxyphenyl) derivative 12 possessing nanomolar affinity (K
selectivity for the hA2A AR. Demethylation of compound 12 completely changed the affinity
profile. The hydroxy derivative 15 shows, in fact, a 6ꢀfold reduced hA2A AR affinity (K = 45 nM)
and, above all, a nulled selectivity, being able to bind also hA and hA ARs with similar K values.
Moving the methoxy substituent from the para to the meta position (derivative 11) maintained a
high hA2A AR affinity (K = 6.8 nM) but lost selectivity, showing 11 to have considerable affinity
also for the hA AR and hA ARs (K = 44 and 42 nM). Demethylation of compound 11 did not
i
= 7.2 nM) and a complete
i
1
3
i
i
1
3
i
modify the affinity profile much, although compound 14 had a high affinity ligand for hA
1
and
hA2A ARs (K = 14 and 3.5 nM, respectively) and quite a good one for hA AR. Replacement of the
i
3
4ꢀmethoxy group with a methyl elicited a detrimental effect, which was difficult to explain,
compound 13 being totally inactive.
In trying to enhance hA2A AR affinity or selectivity, we replaced the 3ꢀ and 4ꢀmethoxy group of 11
and 12 with propargyloxy (compounds 16 and 17) and benzyloxy residues (derivatives 18 and 19).
These modifications were suggested by the SARs of different series of hA2A AR antagonists of
similar size and shape, indicating that the presence of hindering substituents in suitable positions
3
was often profitable for an effective and selective recognition of the hA2A AR. Interestingly,
affinities of the propargyloxyꢀderivatives 16 and 17 resemble those of the respective methoxyꢀ
i
substituted compounds 11 and 12: both 16 and 17 showed nanomolar affinity for the hA2A AR (K =
5.1 and 10.6 nM) and 17 was also totally selective. On the contrary, the benzyloxyꢀderivatives 18
and 19 turned out to be significantly less active than 11 and 12, in particular, the 3ꢀbenzyloxy
derivative 18 was completely devoid of affinity for all the ARs. Given that the preferred position for
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2
2
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2
2
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5
5
5
5
5
6
a substituent on the 6ꢀphenyl ring seemed to be the para and that the steric bulk of the substituent
seemed to play a key role in the binding, in the last step of the work, we modified derivative 12 by
replacing the 4ꢀmethoxy group with other small alkoxy residues, containing either linear,
unsaturated, branched or cyclic alkyl chains (compounds 20-26). Within this new set of ligands, two
other potent and completely selective hA2A AR antagonists were identified, i.e. the 4ꢀethoxyphenyl
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
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2
3
4
5
6
7
8
9
0
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5
6
7
8
9
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1
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4
5
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7
8
9
0
i i
derivative 20 (K = 2.9 nM) and the 4ꢀisopropoxyphenyl derivative 22 (K = 7.4 nM), bearing the
smallest alkyl groups, among those evaluated. Instead, compounds 24 and 25, bearing the
cyclopropylmethoxy and cyclobutylmethoxy moieties, turned out to be inactive at the hA2A AR, as
well as at the other ARs. For derivatives 23 and 26 no biological data are available since we met
some difficulties in testing them, probably due to their scarce solubility in the assay medium. The
same applies to 21 for the assays at the hA2A AR.
Finally, the selected derivatives 12 and 20, showing high hA2A AR affinity and selectivity, were
tested to evaluate their effect on cAMP production in CHO cells, stably expressing the hA2A AR.
The obtained IC50 values (Table 1) showed that 12 and 20 inhibited the NECAꢀinduced increase of
cAMP accumulation, thus behaving as antagonists.
2
-Aryl-1,2,4-triazolo[4,3-a]pyrazine-3,8-dione derivatives 48-62
The triazolopyrazineꢀ3,8ꢀdione derivatives 48-62 were evaluated for their affinity for ARs since
they were thought capable of binding the hA AR subtype on the basis of overall nanomolar hA
3
3
26
AR affinities of the 1,4ꢀdione derivatives of the TQX series. Hence, since we were interested in
identifying new hA AR antagonists, although this was not the main purpose of this work, we
3
decided to test the 3,8ꢀdione derivatives 48-53, 58ꢀ60 prepared as synthetic intermediates.
Moreover, to further explore the SARs in this small set of derivatives, we synthesized compounds
54-56, 57, 61-62. The obtained binding data (Table 2) partly confirmed our hypothesis. Although
3
some derivatives showed null (58-60) or scarce affinity (53, 56) for the hA ARs, as for the other
ARs, the remaining compounds displayed some affinities for the hA AR subtype which are, on the
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whole, higher. The most interesting was the 2ꢀ(4ꢀhydroxyphenyl) derivative 55, being a highly
potent and selective ligand of the hA AR (K = 0.37 nM). Also the 2ꢀphenyl derivatives 48 (R =
3
i
6
Me) and 49 (R
showing good affinity (K
Instead, the 2ꢀ(4ꢀmethoxyphenyl)ꢀ derivative 51 (R
AR ligand (K = 214 nM). Demethylation of the 2ꢀ(4ꢀmethoxyphenyl) derivative 50, as well as
introduction of orthoꢀsubstituents (OMe, OH) on the 2ꢀphenyl ring of 49, significantly reduced the
hA AR affinity (see compounds 54, and 53, 56, respectively). Good hA AR binding activity (K
3 nM) was displayed by the 2ꢀ(4ꢀaminophenyl)ꢀ derivative 57 (R = Ph) which, however, is
scarcely selective versus hA and hA2A ARs. Insertion of OMe, Me, and OH substituents on the 6ꢀ
phenyl moiety (compounds 58-62) was detrimental to hA AR affinity, with the exception of the 4ꢀ
6
= Ph) and the 2ꢀ(4ꢀmethoxyphenyl)ꢀderivative 50 (R
6
= Me) are worth noting
i
= 60, 96 and 50 nM, respectively) and high selectivity for the hA
3
AR.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
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48
49
50
51
52
53
54
55
56
57
58
59
60
6
= Ph), even if selective, is less active as a hA
3
i
3
3
i
=
6
6
1
3
hydroxy group (compound 62) which afforded a moderate affinity for this AR subtype (K = 207
i
nM).
It was not possible to test the 4ꢀnitro derivative 52 at any of the ARs due to its scarce solubility in
the assay medium. The same applies to the 4ꢀhydroxy derivative 54 for the assays at the hA2A AR,
since its low solubility did not allow it to reach high enough concentrations to obtain the doseꢀ
response curve.
Neuroprotection studies in SH-SY5Y cell lines
Parkinson's disease is the second most common neurodegenerative disorder of aging, characterized
47
by motor bradykinesia, rigidity, resting tremor, and postural instability. The progressive loss of
dopaminergic neurons in the substantia nigra is the main cause of these deficits. Several
mechanisms have been implicated as crucial to PD pathogenesis: oxidative stress, mitochondrial
dysfunction, protein aggregation and misfolding, inflammation, excitotoxicity, apoptosis and other
cell death pathways, and loss of trophic support. The aim of the current study was to examine the
protective effects of the selected triazolopyrazine 12 on SHꢀSY5Y cells in an experimental in
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+
vitro PD model. Several neurotoxins are employed to study neurodegeneration. In particular, MPP
is widely accepted as inducing neurotoxicity. The neurotoxin is able to induce dopaminergic
toxicity through oxidation, hydrogen peroxide formation, and direct inhibition of the mitochondrial
4
8
respiratory chain. Much evidence indicates that SHꢀSY5Y cells possess many features of
dopaminergic neurons and have been widely employed for the study of neuroprotection against PDꢀ
related neurotoxins. A pilot study was conducted to evaluate the neurotoxic effect produced by
0
1
2
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4
5
6
7
8
9
0
1
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1
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0
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2
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4
5
6
7
8
9
0
+
+
MPP on SHꢀSY5Y cells. Cells were treated for 24 h with increasing doses of MPP (50 μM to 3
+
mM). The results in Figure 1 show that MPP produced a significant and concentrationꢀdependent
neurotoxic effect in this cell line. The dose of 1.5 mM, which caused 50% of cell death was chosen
for the subsequent neuroprotection studies. Compound 12, when administered alone, did not modify
cell viability (Figure 2A) while at the concentration of 15 nM it was able to partially counteract
MPP+ꢀinduced neurotoxicity (Figure 2B). In order to verify that the protective effect of 12 was due
to the selective blockade of the A2A AR, we compared the effects of the compound with those of the
wellꢀknown selective hA2A AR antagonist 4ꢀ(2ꢀ[7ꢀaminoꢀ2ꢀ(2ꢀfuryl[1,2,4]ꢀtriazolo[2,3ꢀ
4
9
a][1,3,5]triazinꢀ5ylamino]ethyl)phenol 72 (ZM241385) and we evaluated the effects of 12 in the
presence of the selective hA2A AR agonist 2ꢀ[pꢀ(2ꢀcarboxyethyl)phenethylamino]ꢀ5’ꢀNꢀ
50
ethylcarboxamido adenosine 73 (CGS21680). As shown in Figure 3A, the hA2A AR antagonist 72,
38
used at the concentration of 0.5 nM, presented a neuroprotective effect on SHꢀSY5Y cells, thus
+
counteracting MPP toxicity. To validate the involvement of the A2A AR in the neuroprotective
+
activity of 12 against MPP toxicity, we evaluated the ability of the hA2A AR agonist 73 to reverse
the effects of compound 12. SHꢀSY5Y cells were treated with 12 (15 nM) in the presence of
different 73 concentrations ranging from 10 to 100 nM. As shown in Figure 3B, the hA2A agonist 73
was able to suppress the protective effects of 12, thus confirming that the effects of 12 may be
attributed to the selective blockade of the A2A AR.
Molecular modeling studies
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The binding mode of the synthesized compounds at the hA2A AR cavity was simulated with docking
analysis by using the Molecular Operating Environment (MOE, 2014.09) docking tool and Gold
51ꢀ54
and Autodock software.
The MOE software analysis was made by selecting the induced fit
docking and optimization protocol (schematically, a preliminary docking analysis provides a set of
ligand conformations that are energy minimized, including in this step the side chains of the
receptor residues in proximity). For the docking tasks, two crystal structures of the hA2A AR in
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
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48
49
50
51
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53
54
55
56
57
58
59
60
complex with the antagonist/inverse agonist 72 were employed (http://www.rcsb.org; pdb code:
55ꢀ57
3
EML; 2.6ꢀÅ resolution and pdb code: 4EIY; 1.8ꢀÅ resolution
). The docking analysis was
performed with different docking tools and two different crystal structures of the target to get an
average prediction of the binding mode of the synthesized compounds at the binding cavity. For a
1
subset of compounds, the binding modes at the hA AR crystal structure (pdb code: 5UEN; 3.2ꢀÅ
58
resolution) and at a homology model of the hA AR were also simulated with the same tools and
3
protocols.
The docking results at the hA2A AR show that the molecules could bind to the pocket of this
receptor with a preferred orientation (“typeꢀone” conformations), presenting the substituent at the 2ꢀ
position (R
2
) located in the depth of the cavity and the R
6
group at the entrance of the binding site
6.55
(Figure 4A). The scaffold adopts a position that makes it able to interact with Asn253
and
Glu169 (EL2) through Hꢀbond contacts, while a πꢀπ interaction is present between the phenyl ring
of Phe168 (EL2) and the bicyclic core of the compounds (Figure 4B). This interaction is very
55,57
similar to the one given by the coꢀcrystallized compound 72
and by other structural classes of
22,23
hA AR ligands previously described.
2
A
A second binding mode (“typeꢀtwo” conformations) simulated by docking experiments presents the
scaffold oriented in the opposite way with respect to the conformations described above, the 6ꢀ
substituent being located in the depth of the cavity and the 2ꢀsubstituent pointing toward the
extracellular environment. This second binding mode is generally not preferred at the hA2A AR,
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being associated with lower docking scores than those of the above described docking
conformations, except for derivatives presenting orthoꢀsubstituent on the 2ꢀphenyl ring (see below).
Compound 2 can be considered the reference ligand of the series, as it bears two unsubstituted
phenyl rings at the 2ꢀ and 6ꢀ positions. This derivative showed good affinity at the hA
1
AR, hA2A
0
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0
AR and hA AR and this makes it a sort of passeꢀpartout for the three ARs. Docking results of
3
compound 2 at the three AR structures showed that it may be inserted in the receptor cavities with
the two binding modes, both associated with good docking scores (the “typeꢀone” generally
preferred). The possibility of making more than one complex with the same receptor could result in
good affinity and this feature could be applied at the three ARs.
The substituents inserted on the 2ꢀphenyl group modulate the interaction with the binding pocket. In
detail, the presence of small groups at the paraꢀposition generally affords decreased hA2A AR
affinity, with respect to the corresponding analogues with an unsubstituted 2ꢀphenyl ring (compare
derivatives 3 and 7 with 1 and compounds 4, 5, 8, and 10 with 2). This result was interpreted
considering various parameters. The first is the topological complementarity of the ligand with the
binding pocket (Figure 4C). The presence of a paraꢀsubstituent on the 2ꢀphenyl ring seems to cause
a slight displacement of the ligand that decreases its ability in establishing some crucial interactions
6.55
with the receptor (i.e. Hꢀbonds with Asn253 and Glu169) with respect to the compounds with an
unsubstituted 2ꢀphenyl ring.
Secondly, hA2A AR affinities may be rationalized by docking results also considering that the depth
of the binding cavity is mainly hydrophobic. Hence, a nonꢀpolar group at the paraꢀposition of the 2ꢀ
phenyl ring, such as the methoxy group of 4 (K = 78 nM), would afford a slightly better interaction
i
with the target, when compared to a more polar group at the same position, such as the OH group of
8
(K
unsubstituted 2ꢀphenyl ring (1 and 2) with those of the corresponding derivatives presenting paraꢀ
substituents on this ring. We observed a wellꢀconserved trend of logP and pK values for the two
i i
= 138 nM). In this sense, we compared logP and pK values of compounds bearing an
i
groups of ligands (compare derivatives 3 and 7 to 1 and derivatives 4, 5, 8, 10 to 2, Figure 5AꢀB).
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On this basis, we conclude that a paraꢀsubstituent on the 2ꢀphenyl ring is fairly allowed for these
compounds and, if present, should be a small hydrophobic function.
To evaluate the interaction of compounds bearing a 2ꢀphenyl (derivative 2) and a 2ꢀparaꢀ
substitutedꢀphenyl moiety (compounds 4, 5, 8, and 10) with the receptor residues, in proximity of
the 2ꢀsubstituent, we also employed the IF-E 6.0 tool (see Experimental section for details) that
calculates the atomic and residue interaction forces and the perꢀresidue interaction energies
10
11
12
13
14
15
16
17
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19
20
21
22
23
24
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48
49
50
51
52
53
54
55
56
57
58
59
60
ꢀ1
59ꢀ61
(
expressed as kcal x mol ). This tool has been previously used for analogue analyses at hARs.
The results are graphically displayed in Figure 5CꢀD and indicate that a higher ligandꢀtarget
interaction is observed when the 2ꢀphenyl ring is unsubstituted (2) or presents a nonꢀpolar paraꢀ
substituent (4). Polar or electronꢀrich paraꢀsubstituents, such as nitro (5), hydroxyl (8), or amino
6
.52
(
10), provide some interaction only with polar receptor residues, such as His250 . These results
are in good agreement with the affinity data at the hA AR for the analyzed compounds.
2
A
Interestingly, several derivatives featuring polar paraꢀsubstituents on the 2ꢀphenyl ring (5, 8 and 10)
are endowed with high affinity for the hA AR, compounds 5 and 10 being the most active. Docking
results showed that these ligands present both the binding modes at the hA AR with corresponding
good scores. The “type two” binding mode at the hA AR is favoured by polar interactions with
1
1
1
2.65
7.36
Asn70 , Glu170 (EL2), Ser267 (EL3) and Tyr271 . While the last residue is conserved among
2
.65
ARs, the others, i.e. Asn70 , Glu170 and Ser267, are specific of the hA
1
AR, being substituted by
2
.65
a serine and two leucines in the hA2A AR (Ser67 , Leu167 and Leu267) and by a serine and two
2
.65
glutamines in the hA
3
AR (Ser73 , Gln167 and Gln261). The possibility of giving two good score
binding modes at the hA AR, not observed at hA and hA ARs, could help to explain the hA AR
1
2A
3
1
affinity and selectivity of compounds 5, 8 and 10. The “typeꢀtwo” docking conformation of
compound 10 at the hA AR is reported in the Supporting information (Figure S1).
1
Introduction of a substituent (OMe, OH) at the orthoꢀposition of the 2ꢀphenyl ring of compound 2
afforded derivatives 6 and 9, endowed with 20ꢀ to 30ꢀfold reduced hA2A AR affinity. When 6 and 9
are inserted into the binding site with the 2ꢀaryl located in the depth of the cavity, the orthoꢀ
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6
substituent may be oriented toward the 3ꢀcarbonyl group or the Nꢀ1 scaffold atom. In the first case,
the orthoꢀhydroxy substituent (derivative 9) makes an internal Hꢀbond interaction with the carbonyl
group but this leads to a reorientation of the 2ꢀphenyl ring with respect to the bicyclic scaffold
plane. The presence of the orthoꢀmethoxy substituent (derivative 6) makes the reorientation of the
0
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0
2
ꢀphenyl ring even more evident due to the repelling effect between the orthoꢀmethoxy and the 3ꢀ
carbonyl function. Consequently, these docking conformations are associated with a low docking
score for all the employed software. When the orthoꢀsubstituent is oriented toward the Nꢀ1 atom,
the reorientation of the 2ꢀphenyl ring, with respect to the bicyclic core, is greatly reduced, but the
6
.55
ligands get displaced with respect to compound 2, due to the steric clash with Asn253 (even if a
6
.55
polar interaction might occur between the orthoꢀsubstituent and the Asn253
side chain). Even
these docking conformations appear associated with a low score. Docking results suggest that
compounds 6 and 9 preferentially adopt the “typeꢀtwo” orientation with the 2ꢀaryl pendant located
at the entrance of the cavity and the R
this way, the orthoꢀhydroxy group of derivative 9 could give some polar interaction with Glu169
(EL2). An analogue arrangement was observed at the hA AR, where the orthoꢀhydroxy group of 9
could interact with Glu172 (corresponding to Glu169 of the hA2A AR) and also with Glu170
corresponding to Leu167 of the hA2A AR). This binding mode could give some interpretation of
the slight hA AR selectivity of compound 9.
Docking results of compounds bearing an unsubstituted 2ꢀphenyl ring and various aryl substituents
at the R position (11-26) again show a preferential binding mode with the 2ꢀgroup located in the
6
group positioned in the depth of the pocket (Figure 6A). In
1
(
1
6
depths of the cavity and the 6ꢀsubstituent pointing toward the external environment. Hence, the
substituents on the 6ꢀphenyl ring are generally located at the entrance of the cavity, providing
different degrees of interaction with the receptor residues in proximity. Considering the effects of
substituents at the paraꢀposition of the 6ꢀphenyl ring, the introduction of a polar hydroxyl group led
i
to a decrease in hA2A AR affinity (15, K = 45 nM) with respect to the unsubstituted analogue 2.
Docking results suggest that this hydroxy group is inserted within a set of hydrophobic amino acid
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residues, such as Leu167 (EL2), Leu267 (EL3) and Tyr271 , thus helping to explain the nonꢀ
optimal interaction of the 6ꢀ(4ꢀphenol) group (15) with the receptor site, with respect to the phenyl
ring (2). On the same basis, we may interpret why the introduction of nonꢀpolar groups at the paraꢀ
position of the 6ꢀphenyl ring is generally well tolerated, leading to compounds (12, 17, 20, and 22)
with similar affinity with respect to the corresponding analogue 2, lacking a substituent on the 6ꢀ
phenyl ring. Figure 6B shows the binding mode of compound 20 with a focus on the residues
10
11
12
13
14
15
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located in proximity of the R substituent. The paraꢀethoxy substituent of 20 appears inserted
6
7.36
among the above cited hydrophobic residues Leu167 (EL2), Leu267 (EL3), and Tyr271
.
Insertion of a phenyl ring on the methoxy group of 12, to give compound 19, lowered the hA2A AR
affinity even if it was still in the high nanomolar range (K = 708 nM). Interestingly, compounds 24
i
and 25 bearing a smaller moiety at the same position, such as cyclopropylmethoxy and a
cyclobutyloxy group, respectively, completely lack binding activity, not only at the hA2A AR but
also at the other hARs. Docking results show that the benzyloxy substituent of 19 is partially
exposed to the external environment, in a position that makes it able to give πꢀπ interaction with
7
.36
7.36
Tyr271 and also to interact with the polar hydrogen atoms of Lys153 (EL2) and Tyr271 side
chains. Hence, this docking pose could justify both the decreased affinity, considering the exposure
of the phenyl group to the solvent, and the maintenance of at least high nanomolar binding.
In the case of compounds 24 and 25, the cycloalkyl groups are in a comparable position to that of
the external phenyl group of 19. They are exposed to the solvent but they are not able, as the benzyl,
to provide the above cited interactions with the receptor residues. This could help to interpret the
lower affinity of these two compounds. Nevertheless, modeling results do not clearly depict the loss
of affinity of these compounds with respect to other analogues presenting slightly smaller alkyl
groups and very high affinity (such as compound 22, K
i
= 7.45 nM).
Considering the docking results at the hA AR, it has to be noted that in this AR subtype the above
1
7
.36
cited Leu167 (EL2), Leu267 (EL3), and Tyr271
of the hA2A AR are replaced by Glu170 (EL2),
7.36
Ser267 (EL3), and Tyr271 . As a consequence, the presence of hydrophobic substituents at the
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paraꢀposition of the 6ꢀphenyl ring was less tolerated by the hA
1
AR, with respect to the hA2A AR,
thus justifying the low to null hA AR affinity of compounds 16-26.
1
In the case of derivatives with substituents at the metaꢀposition of the 6ꢀphenyl ring, the affinity
data show that the nature of the substituent does not significantly influence the receptorꢀligand
interaction, since the presence of a polar hydroxyl group (14) or nonꢀpolar functions, such as
methoxy or propargyloxy groups (11, and 16, respectively) leads to analogue affinities at the hA2A
AR. Figure 6B shows the presence on the receptor of nonꢀpolar groups (i.e. the alkyl chain of
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
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2
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7
8
9
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2
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8
9
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2
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7
8
9
0
2.64
2.64
2.65
Ile66 ) as well as polar functions (i.e. the carbonyl groups of Ile66 and Ser67 ) in proximity
with the metaꢀposition of the 6ꢀphenyl ring. On the other hand, while the introduction of a
benzyloxy group at the paraꢀposition of the 6ꢀphenyl ring of 2, to obtain 19, was fairly tolerated,
although causing a significant decrease of hA2A AR affinity, the introduction of the same group at
the metaꢀposition led to an inactive compound (18). This chain may be oriented toward either the
transꢀmembrane (TM) domains or the extracellular environment. In the first case, there would be a
clash with the TM atoms, while in the second case the hydrophobic benzyloxy chain would be
completely exposed to the solvent without interacting significantly with receptor residues. Similar
considerations can be made for the hA
1
AR, where the presence of polar residues at the entrance of
the receptor cavity (see above) makes hydrophobic substituents at the metaꢀposition of the 6ꢀphenyl
1
ring less beneficial for the hA AR binding than for the hA2A receptorꢀligand interaction.
CONCLUSION
This work has led to the identification of the 1,2,4ꢀtriazolo[4,3ꢀa]pyrazinꢀ3ꢀone as a new versatile
scaffold for the development of hAR antagonists. In fact, several synthesized compounds are
endowed with nanomolar affinities and have different degrees of selectivity for hA
AR subtypes. Very interestingly, some 8ꢀaminoꢀ2ꢀphenylꢀ1,2,4ꢀtriazolopyrazinꢀ3ꢀone derivatives,
featuring small paraꢀalkoxy substituents on the 6ꢀphenyl ring, show high affinity (K = 2.9ꢀ10.6 nM)
1 3
, hA2A and hA
i
and a complete selectivity for the targeted hA2A AR subtype. The potent and selective hA2A AR
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antagonist 12 is able to partially counteract MPP ꢀinduced neurotoxicity in cultured human
neuroblastoma SHꢀSY5Y cells, a widely used cellular PD model. The results of the docking studies
at hAR structures permit us to explain some of the observed affinity data in terms of ligandꢀreceptor
molecular interactions, and provide useful indications for the design of new 8ꢀaminoꢀtriazolo[4,3ꢀ
a]pyrazinꢀ3ꢀone derivatives as hA2A AR antagonists.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
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60
EXPERIMENTAL PROCEDURES
Chemistry. The microwaveꢀassisted syntheses were performed using an Initiator EXP Microwave
Biotage instrument (frequency of irradiation: 2.45 GHz). Analytical silica gel plates (Merck F254)
and silica gel 60 (Merck, 70ꢀ230 mesh) were used for analytical TLC and for column
chromatography, respectively. All melting points were determined on a Gallenkamp melting point
apparatus and are uncorrected. Elemental analyses were performed with a Flash E1112
Thermofinnigan elemental analyzer for C, H, N and the results were within ± 0.4% of the
theoretical values. All final compounds revealed purity not less than 95%. The IR spectra were
ꢀ
recorded with a PerkinꢀElmer Spectrum RX I spectrometer in Nujol mulls and are expressed in cm
1
.
NMR spectra were recorded on a Bruker Avance 400 spectrometer (400 MHz). The chemical
shifts are reported in δ (ppm) and are relative to the central peak of the residual nondeuterated
solvent, which was CDCl or DMSOd . The following abbreviations are used: s= singlet, d=
3
6
doublet, t= triplet, q= quartet, m= multiplet, br= broad and ar= aromatic protons.
General Procedure for the Synthesis of 8-Amino-2-aryl-1,2,4-triazolo[4,3-a]pyrazin-3(2H)-one
derivatives (1-6).
A suspension of the 8ꢀchloroꢀtriazolopyrazine derivatives 63-71 (1 mmol) in a saturated solution of
NH
3
in absolute ethanol (30 mL) was heated at 120 °C in a sealed tube for 16 h. The mixture was
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2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
cooled at room temperature and the solid was collected by filtration, washed with water (about 5ꢀ10
mL), dried and recrystallized.
8
-Amino-6-methyl-2-phenyl-1,2,4-triazolo[4,3-a]pyrazin-3(2H)-one (1). Yield 92%; mp 258ꢀ259
1
°
C (Toluene). H NMR (DMSOꢀd
6
3
) 2.11 (s, 3H, CH ), 7.09 (s, 1H, Hꢀ5), 7.35 (t, 1H, ar, J = 6.6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Hz), 7.40 (br s, 2H, NH
2
), 7.54 (t, 2H, ar, J = 7.7 Hz), 8.05 (d, 2H, ar, J = 7.7 Hz). Anal. Calc. for
12 11 5
C H N O.
8
-Amino-2,6-diphenyl-1,2,4-triazolo[4,3-a]pyrazin-3(2H)-one (2). Yield 50%; mp 276ꢀ277 °C
1
(
CH
3
NO
2
). H NMR (DMSOꢀd
6
) 7.33ꢀ7.38 (m, 2H, ar), 7.43 (t, 2H, ar, J = 7.4 Hz), 7.55ꢀ7.59 (m,
), 7.77 (s, 1H, Hꢀ5), 7.98 (d, 2H, ar, J = 7.5 Hz), 8.08 (d, 2H, ar, J = 7.9 Hz). Anal.
O.
-Amino-6-methyl-2-(4-methoxyphenyl)-1,2,4-triazolo[4,3-a]pyrazin-3(2H)-one (3). Yield 83%; mp
4
H, 2 ar + NH
2
13 5
Calc. for C17H N
8
1
2
43ꢀ244 °C (EtOH/2ꢀMethoxyethanol). H NMR (DMSOꢀd ) 2.11 (s, 3H, CH ), 3.81 (s, 3H,
6
3
OCH
3
), 7.07 (s, 1H, Hꢀ5), 7.09 (d, 2H, ar, J = 9.1 Hz), 7.36 (br s, 2H, NH
2
), 7.90 (d, 2H, ar, J = 9.1
13 5 2
Hz). Anal. Calc. for C13H N O .
8-Amino-2-(4-methoxyphenyl)-6-phenyl-1,2,4-triazolo[4,3-a]pyrazin-3(2H)-one (4). Yield 84 %;
1
mp 254ꢀ255 °C (CH
3
NO
2
). H NMR (DMSOꢀd
6
) 3.82 (s, 3H, OCH
3
), 7.13 (d, 2H, ar, J = 8.8 Hz),
7
1
.33ꢀ7.45 (m, 3H, ar), 7.56 (br s, 2H, NH ), 7.75 (s, 1H, Hꢀ5), 7.92ꢀ7.99 (m, 4H, ar). IR 3366, 3311,
2
15 5 2
644. Anal. Calc. for C18H N O .
8-Amino-2-(4-nitrophenyl)-6-phenyl-1,2,4-triazolo[4,3-a]pyrazin-3(2H)-one (5). Yield 62%; mp
1
2
90ꢀ291 °C (Cyclohexane/EtOAc). H NMR (DMSOꢀd
6
) 7.13 (t, 1H, ar, J = 8.8 Hz), 7.35 (t, 2H, ar,
J = 7.5 Hz), 7.69 (br s, 2H, NH ), 7.79 (s, 1H, Hꢀ5), 7.99 (d, 2H, ar, J = 7.5 Hz), 8.38 (d, 2H, ar, J =
2
9
.3 Hz), 8.48 (d, 2H, ar, J = 9.3 Hz). IR 3366, 3311, 1644. Anal. Calc. for C H N O .
17 12 6 3
8
-Amino-2-(2-methoxyphenyl)-6-phenyl-1,2,4-triazolo[4,3-a]pyrazin-3(2H)-one (6). Yield 74 %;
1
mp 257ꢀ258 °C (CH
3
NO
2
). H NMR (DMSOꢀd
6
) 3.80 (s, 3H, OCH
3
), 7.11 (t, 1H, ar, J= 7.6 Hz),
7
4
.26 (d, 1H, ar, J = 8.3 Hz), 7.35 (t, 1H, ar, J = 7.3 Hz), 7.43 (t, 2H, ar, J = 7.3 Hz), 7.49ꢀ7.56 (m,
H, 2ar + NH ), 7.73 (s, 1H, Hꢀ5), 7.97 (d, 2H, ar, J = 7.5 Hz). Anal. Calc. for C H N O .
2
18 15
5
2
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General Procedure for the Synthesis of 2-(Hydroxyphenyl)-substituted 8-amino-2-phenyl-
1
,2,4-triazolo[4,3-a]pyrazin-3(2H)-one (7-9).
M solution of BBr in dichloromethane (5.1 mL) was slowly added at 0 °C, under nitrogen
1
3
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
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30
31
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36
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48
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atmosphere, to a suspension of the methoxyꢀsubstituted triazolopyrazines 3, 4, 6 (1.02 mmol) in
anhydrous dichloromethane (20 mL). The mixture was stirred at rt until the disappearance of the
starting material (TLC monitoring, 5ꢀ16 h), then was diluted with water (10 mL) and neutralized
3
with a NaHCO saturated solution. The organic solvent was removed by evaporation at reduced
pressure and the solid was collected by filtration. The crude derivatives were dried and purified by
recrystallization.
8
-Amino-2-(4-hydroxyphenyl)-6-methyl-1,2,4-triazolo[4,3-a]pyrazin-3(2H)-one (7). Yield 65%; mp
1
>
300 °C (EtOH/2ꢀMethoxyethanol). H NMR (DMSOꢀd ) 2.11 (s, 3H, CH ), 6.88 (d, 2H, ar, J =
6
3
6
2
.8 Hz), 7.06 (s, 1H, Hꢀ5), 7.33 (br s, 2H, NH ), 7.75 (d, 2H, ar, J = 6.8 Hz). 9.69 (br s, 1H, OH).
11 5 2
Anal. Calc. for C12H N O .
8-Amino-2-(4-hydroxyphenyl)-6-phenyl-1,2,4-triazolo[4,3-a]pyrazin-3(2H)-one (8). Yield 72%; mp
1
>
300 °C (EtOH). H NMR (DMSOꢀd
6
) 6.91 (d, 2H, ar, J = 8.5 Hz), 7.34 (t, 1H, ar, J = 7.5 Hz),
7
.43 (t, 2H, ar, J = 7.5 Hz), 7.53 (br s, 2H, NH ), 7.74 (s, 1H, Hꢀ5), 7.78 (d, 2H, ar, J = 8.5 Hz), 7.97
2
13 5 2.
(d, 2H, ar, J = 7.8 Hz), 9.72 (s, 1H, OH). IR 3393ꢀ3122, 1642, 1667. Anal. Calc. for C17H N O
8-Amino-2-(2-hydroxyphenyl)-6-phenyl-1,2,4-triazolo[4,3-a]pyrazin-3(2H)-one (9). Yield 67%; mp
1
2
71ꢀ273 °C (2ꢀMethoxyethanol). H NMR (DMSOꢀd
6
) 6.95 (t, 1H, ar, J = 7.5 Hz), 7.03 (d, 1H, ar, J
=
8.1 Hz), 7.33ꢀ7.44 (m, 5H, ar), 7.55 (br s, 2H, NH ), 7.75 (s, 1H, Hꢀ5), 7.98 (d, 2H, ar, J = 7.5
2
Hz), 9.88 (s, 1H, OH). Anal. Calc. for C H N O .
17
13
5
2
8-Amino-2-(4-aminophenyl)-6-phenyl-1,2,4-triazolo[4,3-a]pyrazin-3-(2H)one (10).
0 % Pd/C (20% w/w with respect to the nitro derivative) was added to a solution of the 2ꢀ(4ꢀ
1
nitrophenyl) derivative 5 (0.8 mmol) in DMF (4 mL). The mixture was hydrogenated in a Parr
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apparatus at 45 psi for 24 h. Then the catalyst was filtered off and the clear solution was diluted
with water (about 50 mL) to obtain a solid that was collected by filtration, washed with water and
1
Et
.33 (br s, 2H, NH
Hz), 7.49 (br s, 2H, NH
14 6
Anal. Calc. for. C17H N
2
O, dried and recrystallized. Yield 39 %; mp >300 °C (2ꢀMethoxyethanol). H NMR (DMSOꢀd
6
)
5
2
), 6.68 (d, 2H, ar, J = 8.7 Hz), 7.34 (t, 1H, ar, J = 7.2 Hz), 7.42 (t, 2H, J = 7.4
), 7.57 (d, 2H, J = 8.7 Hz), 7.72 (s, 1H, Hꢀ5), 7.96 (d, 2H, ar, J = 7.4 Hz).
O.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
2
General Procedure for the Synthesis of 8-Amino-2-aryl-1,2,4-triazolo[4,3-a]pyrazin-3(2H)-one
derivatives (11-13).
The tile compounds were obtained by reacting the 8ꢀchloroꢀtriazolopyrazine derivatives 69-71 (1
mmol) in the same experimental conditions as described above to prepare 1-6. The crude
derivatives were purified by recrystallization.
8
-Amino-6-(3-methoxyphenyl)-2-phenyl-1,2,4-triazolo[4,3-a]pyrazin-3(2H)-one (11). Yield 66%;
1
mp 249ꢀ251 °C (EtOH). H NMR (DMSOꢀd
6
) 3.83 (s, 3H, OCH
3
), 6.90ꢀ6.93 (m, 1H, ar), 7.31ꢀ7.37
(m, 2H, ar), 7.53ꢀ7.58 (m, 6H, 4 ar + NH
2
), 7.81 (s, 1H, Hꢀ5), 8.07 (d, 2H, ar, J = 7.5 Hz). IR 3358,
3
15 5 2
312, 1703, 1645. Anal. Calc. for C18H N O .
8
-Amino-6-(4-methoxyphenyl)-2-phenyl-1,2,4-triazolo[4,3-a]pyrazin-3(2H)-one (12). Yield 94%;
1
mp 251ꢀ252 °C (EtOH/2ꢀMethoxyethanol). H NMR (DMSOꢀd
6
) 3.80 (s, 3H, OCH
), 7.67 (s, 1H, H ), 7.92 (d,
H, ar, J = 8.7 Hz), 8.08 (d, 2H, ar, J = 8.5 Hz). IR 3381, 3308, 1697, 1649. Anal. Calc. for
C H N O .
3
), 6.99 (d, 2H,
ar, J = 8.7 Hz), 7.36 (t, 1H, ar, J = 7.5 Hz), 7.54ꢀ7.58 (m, 4H, 2ar + NH
2
2
18
15
5
2
8
-Amino-6-(4-methylphenyl)-2-phenyl-1,2,4-triazolo[4,3-a]pyrazin-3(2H)-one (13). Yield 75%; mp
1
2
87ꢀ288 °C (2ꢀMethoxyethanol). H NMR (DMSOꢀd
6
) 2.34 (s, 3H, CH
), 7.70 (s, 1H, Hꢀ5), 7.87 (d, 2H, ar, J
7.9 Hz), 8.07 (d, 2H, ar, J = 7.6 Hz). IR 3366, 3310, 1701, 1651. Anal. Calc. for C18
3
), 7.24 (d, 2H, ar, J = 7.9
Hz), 7.36 (t, 1H, ar, J = 7.4 Hz), 7.54ꢀ7.58 (m, 4H, 2 ar + NH
2
=
15 5 .
H N O
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General Procedure for the Synthesis of 6-(Hydroxyphenyl)-substituted 8-amino-2-phenyl-
1
,2,4-triazolo[4,3-a]pyrazin-3(2H)-one (14, 15).
The tile compounds were obtained by reacting the corresponding methoxy derivatives 11, 12 (1.02
mmol) with BBr in the same experimental conditions as described above to prepare compounds 7-
. The crude derivatives were dried and purified by recrystallization.
3
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
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58
59
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9
8-Amino-6-(3-hydroxyphenyl)-2-phenyl-1,2,4-triazolo[4,3-a]pyrazin-3(2H)-one (14). Yield 93%;
1
mp >300 °C (EtOH/2ꢀMethoxyethanol). H NMR (DMSOꢀd ) 6.75 (d, 1H, ar J = 7.3 Hz), 7.21 (t,
6
1
2
H, ar, J = 7.4 Hz), 7.36ꢀ7.38 (m, 3H, ar), 7.55ꢀ7.61 (m, 4H, 2 ar + NH ), 7.61 (s, 1H, Hꢀ5), 8.08 (d,
2
H, ar, J = 8.1 Hz), 9.45 (s, 1H, OH). Anal. Calc. for C17
13 5 2
H N O .
8-Amino-6-(4-hydroxyphenyl)-2-phenyl-1,2,4-triazolo[4,3-a]pyrazin-3(2H)-one (15). Yield 85%;
1
mp 285ꢀ286 °C (EtOH/2ꢀMethoxyethanol). H NMR (DMSOꢀd
6
) 6.81 (d, 2H, ar, J = 6.8 Hz), 7.35
(
t, 1H, ar, J = 7.4 Hz), 7.40 (t, 2H, ar, J = 7.6 Hz), 7.53 (br s, 2H, NH ), 7.73 (s, 1H, Hꢀ5), 7.79 (d,
2
ꢀ1
2
H, ar, J = 6.8 Hz), 8.08 (d, 2H, ar, J = 7.6 Hz), 9.58 (s, 1H, OH). IR 3379ꢀ3294, 1694, 1643 cm .
13 5 2
Anal. Calc. for C17H N O .
General Procedure for the Synthesis of 8-Amino-6-(alkyloxyphenyl)-2-phenyl-1,2,4-
triazolo[4,3-a]pyrazin-3(2H)-ones (16-26).
A solution of the suitable alkyl bromide (1.2 mmol) in butanꢀ2ꢀone (3 mL) was added dropwise to a
mixture of the hydroxyphenylꢀ derivative 14 or 15 (1 mmol) and K
mL). The mixture was heated at reflux until the disappearance of the starting hydroxyꢀderivative
TLC monitoring, 7ꢀ58 h). After cooling at room temperature, the solid was collected by filtration,
washed with water, dried and recrystallized.
2 3
CO (2 mmol) in butanꢀ2ꢀone (5
(
8
-Amino-2-phenyl-6-(3-propargyloxyphenyl)-1,2,4-triazolo[4,3-a]pyrazin-3(2H)-one (16). Yield
1
58%; mp 217ꢀ219 °C (EtOH/2ꢀMethoxyethanol). H NMR (DMSOꢀd
6
) 3.58 (t, 1H, CH, J = 2.3
Hz), 4.89 (d, 2H, CH
2
, J = 2.3 Hz), 6.97 (d, 1H, ar, J = 6.5 Hz), 7.36 (t, 2H, ar, J = 7.6 Hz), 7.55ꢀ
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1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
7.63 (m, 6H, 4ar + NH
2
), 7.84 (s, 1H, Hꢀ5), 8.08 (d, 2H, ar, J = 7.6 Hz). IR 3443, 3298, 1721, 1643
ꢀ
1
cm . Anal. Calc. for C H N O .
2
0
15
5
2
8
-Amino-2-phenyl-6-(4-propargyloxy)phenyl-1,2,4-triazolo[4,3-a]pyrazin-3(2H)-one (17). Yield
1
5
6%; mp 244ꢀ245 °C (AcOH). H NMR (DMSOꢀd
6
) 3.59 (t, 1H, CH, J = 2.3 Hz), 4.85 (d, 2H, CH
2
,
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
J = 2.4 Hz), 7.04 (d, 2H, ar, J = 6.9 Hz), 7.36 (t, 1H, ar, J = 7.4 Hz), 7.54ꢀ7.59 (m, 4H, 2ar + NH
2
),
7.69 (s, 1H, Hꢀ5), 7.93 (d, 2H, ar, J = 6.9 Hz), 8.08 (d, 2H, ar, J = 7.6 Hz). IR 3458, 3331, 3219,
1
695, 1620. Anal. Calc. for C H N O
20 15 5 2.
8
-Amino-6-(3-benzyloxyphenyl)-2-phenyl-1,2,4-triazolo[4,3-a]pyrazin-3(2H)-one (18). Yield 78%;
1
mp 264ꢀ266 °C (2ꢀMethoxyethanol). H NMR (DMSOꢀd
6
) 5.19 (s, 2H, CH
2
), 6.99 (d, 1H, ar, J =
8.3 Hz), 7.32ꢀ7.65 (m, 13H, 11ar + NH
2
), 7.84 (s, 1H, Hꢀ5), 8.07 (d, 2H, ar, J = 8.0 Hz). Anal. Calc.
19 5 2
for C24H N O .
8
-Amino-6-(4-benzyloxyphenyl)-2-phenyl-1,2,4-triazolo[4,3-a]pyrazin-3(2H)-one (19). Yield 78%;
1
mp 284ꢀ285 °C (2ꢀMethoxyethanol). H NMR (DMSOꢀd
6
) 5.16 (s, 2H, CH
), 7.67 (s, 1H, Hꢀ5), 7.92 (d, 2H, ar, J =
8.9 Hz), 8.08 (d, 2H, ar, J = 7.6 Hz). IR 3362, 3310, 1697, 1647. Anal. Calc. for C24
2
), 7.07 (d, 2H, ar, J =
8
.9 Hz), 7.34ꢀ7.48 (m, 6H, ar), 7.51ꢀ7.58 (m, 4H, 2ar + NH
2
19 5 2
H N O .
8
2
2
-Amino-6-(4-ethoxyphenyl)-2-phenyl-1,2,4-triazolo[4,3-a]pyrazin-3(2H)-one (20). Yield 72%; mp
1
67ꢀ268 °C (EtOH/2ꢀMethoxyethanol). H NMR (DMSOꢀd ) 1.35 (t, 3H, CH , J = 7.0 Hz), 4.07 (q,
6
3
H, CH
), 7.65 (s, 1H, Hꢀ5), 7.90 (d, 2H, ar, J = 6.8 Hz), 8.08 (d, 2H, ar, J = 8.8 Hz). IR 3119, 3102,
697, 1647. Anal. Calc. for C19
2
, J = 7.0 Hz), 6.96 (d, 2H, ar, J = 6.8 Hz), 7.36 (t, 1H, ar, J = 8.8 Hz), 7.53ꢀ7.58 (m, 4H, 2ar
+ NH
2
1
8
6
1
17 5 2
H N O .
-Amino-6-(4-n-propyloxyphenyl)-2-phenyl-1,2,4-triazolo[4,3-a]pyrazin-3(2H)-one (21). Yield
1
7%; mp 266ꢀ267 °C (2ꢀMethoxyethanol). H NMR (DMSOꢀd ) 0.99 (t, 3H, CH , J = 7.4 Hz),
6
3
.72ꢀ1.77 (m, 2H, CH
2
), 3.97 (t, 2H, CH
2
, J = 6.5 Hz), 6.97 (d, 2H, ar, J = 8.8 Hz), 7.35 (t, 1H, ar, J
= 7.3 Hz), 7.54ꢀ7.58 (m, 4H, 2ar + NH
2
), 7.65 (s, 1H, Hꢀ5), 7.89 (d, 2H, ar, J = 8.8 Hz), 8.07 (d, 2H,
ar, J = 7.9 Hz). Anal. Calc. for C20H N O .
19 5 2
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8-Amino-6-(4-isopropyloxyphenyl)-2-phenyl-1,2,4-triazolo[4,3-a]pyrazin-3(2H)-one (22). Yield
1
7
0%; mp 240ꢀ241 °C (EtOH/2ꢀMethoxyethanol). H NMR (DMSOꢀd ) 1.30 (d, 6H, 2CH , J = 6.0
6
3
Hz), 4.63ꢀ4.69 (m, 1H, CH), 6.96 (d, 2H, ar, J = 8.7 Hz), 7.36 (t, 1H, ar, J = 7.4 Hz), 7.52 (br s, 2H,
NH ), 7.58 (m, 2H, ar, J = 7.7 Hz), 7.64 (s, 1H, Hꢀ5), 7.88 (d, 2H, ar, J = 8.7 Hz), 8.08 (d, 2H, ar, J
7.9 Hz). IR 3385, 3310, 1701, 1638. Anal. Calc. for C20
2
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
=
19 5 2
H N O .
8-Amino-6-(4-isobutyloxyphenyl)-2-phenyl-1,2,4-triazolo[4,3-a]pyrazin-3(2H)-one (23). Yield
1
6
1
7
4%; mp 249ꢀ251 °C (2ꢀMethoxyethanol). H NMR (DMSOꢀd ) 1.00 (d, 6H, 2CH , J= 6.5 Hz),
6
3
.99ꢀ2.06 (m, 1H, CH), 3.78 (d, 2H, CH
2
, J = 6.5 Hz), 6.97 (d, 2H, J = 8.7 Hz), 7.35 (t, 1H, ar, J =
.3 Hz), 7.54ꢀ7.57 (m, 4H, 2ar + NH ), 7.65 (s, 1H, Hꢀ5), 7.98 (d, 2H, ar, J = 8.7 Hz), 8.07 (d, 2H,
2
21 5 2
ar, J = 7.9 Hz). Anal. Calc. for C21H N O .
8
-Amino-6-[(4-cyclopropylmethoxy)phenyl]-2-phenyl-1,2,4-triazolo[4,3-a]pyrazin-3(2H)-one (24).
1
Yield 86%; mp 276ꢀ278 °C (EtOH/2ꢀMethoxyethanol). H NMR (DMSOꢀd ) 0.32ꢀ0.36 (m, 2H,
6
2
CH), 0.57ꢀ0.60 (m, 2H, 2CH), 1.21ꢀ1.24 (m, 1H, CH), 3.85 (d, 2H, CH
ar, J = 8.9 Hz), 7.36 (t, 1H, ar, J = 7.4 Hz), 7.54 (br s, 2H, NH ), 7.56 (t, 2H, ar, J = 7.4 Hz), 7.65 (s,
1H, Hꢀ5), 7.91 (d, 2H, ar, J = 8.9 Hz), 8.08 (d, 2H, ar, J = 7.4 Hz). IR 3362, 3316, 1669, 1649. Anal.
Calc. for C21
-Amino-6-[(4-cyclobutylmethoxy)phenyl]-2-phenyl-1,2,4-triazolo[4,3-a]pyrazin-3(2H)-one (25).
2
, J = 7.0 Hz), 6.97 (d, 2H,
2
19 5 2
H N O .
8
1
Yield 45%; mp 266ꢀ268 °C (EtOH/2ꢀMethoxyethanol). H NMR (DMSOꢀd
4CH), 2.06ꢀ2.13 (m, 2H, 2CH), 2.72ꢀ2.75 (m, 1H, CH), 3.99 (d, 2H, CH , J = 6.7 Hz), 6.98 (d, 2H,
ar, J = 8.8 Hz), 7.36 (t, 1H, ar, J = 7.4 Hz), 7.56 (br s, 2H, NH ), 7.60 (t, 2H, ar, J = 7.8 Hz), 7.65 (s,
H, Hꢀ5), 7.90 (d, 2H, ar, J = 8.8 Hz), 8.08 (d, 2H, ar, J = 7.8 Hz). IR 3354, 3312, 1699, 1647. Anal.
Calc. for C H N O .
6
) 1.88ꢀ1.96 (m, 4H,
2
2
1
22
21
5
2
8
-Amino-2-phenyl-6-(4-allyloxy)phenyl-1,2,4-triazolo[4,3-a]pyrazin-3(2H)-one (26). Yield 75%;
1
mp 260ꢀ261 °C (2ꢀMethoxyethanol). H NMR (DMSOꢀd
6
) 4.60 (d, 2H, CH
2
), 5.27 (d, 1H, geminal
CH=, J = 10.4 Hz), 5.42 (d, 1H, geminal CH=, J = 17.4 Hz), 6.02ꢀ6.11 (m, 1H, CH=), 7.00 (d, 2H,
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2
2
2
2
2
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5
5
5
5
6
2
ar, J = 8.8 Hz), 7.35 (t, 1H, ar, J = 7.9 Hz), 7.54ꢀ7.58 (m, 4H, 2ar + NH ), 7.66 (s, 1H, Hꢀ5), 7.90 (d,
2
H, ar, J = 8.8 Hz), 8.07 (d, 2H, ar, J = 7.9 Hz). Anal. Calc. for C H N O .
20 17 5 2
General Procedure for the Synthesis of Ethyl 2-amino-2-arylhydrazonoacetates (31-34).
2
Ethyl 2ꢀaminoꢀ2ꢀarylhydrazonoacetates 32 (R = 4ꢀOMe), 33 (R = 2ꢀOMe) and 34 (R = 4ꢀNO )
0
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2
3
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7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
36
2
2
2
3
5
were prepared as previously described for 31 (R= H) i.e. from the corresponding 2ꢀchloro
derivatives 27-30. Briefly, 33% aqueous ammonia (3 mL) in dioxane (5 mL) was added dropwise to
23,33,34
a solution of derivatives 27-30
(13.3 mmol) in dioxane (15 mL) and the reaction mixture was
stirred for 4 h at rt. The white solid was filtered off and the mother liquor was concentrated at
reduced pressure. The obtained precipitate was collected by filtration, washed with water (30 mL),
dried and recrystallized. The crude compounds 32 and 33 were obtained as oily residues which were
purified on silica gel column chromatography (eluent cyclohexane/EtOAc, 7:3 and 1:1,
respectively). After evaporation of the eluent, derivative 32 was an oily product which was used as
such for the next step while 33 solidified.
3
5
Ethyl 2-amino-2-(phenylhydrazono)acetate (31). Yield 73%; mp 129ꢀ130 °C (lit 128 °C)
1
(
Cyclohexane/EtOAc). H NMR (DMSOꢀd
6
) 1.28 (t, 3H, ar, J = 7.1 Hz), 4.23 (q, 2H, CH
2
, J = 7.1
Hz), 5.88 (br s, 2H, NH ), 6.72 (t, 1H, ar, J = 7.3 Hz), 7.01 (d, 2H, ar, J = 7.6 Hz), 7.18 (t, 1H, ar, J
2
=
8.2 Hz), 8.66 (br s, 1H, NH).
Ethyl 2-amino-2-(4-methoxyphenylhydrazono)acetate (32). Yield 55%; brownish oil; H NMR
CDCl ) 1.42 (t, 3H, CH , J = 7.1 Hz), 3.87 (s, 3H, OCH ), 4.38 (q, 2H, CH , J = 7.1 Hz), 4.69 (br s,
H, NH ), 6.45 (br s, 1H, NH), 6.88 (d, 2H, ar, J= 9.0 Hz), 7.11 (d, 2H, ar, J= 9.0 Hz), 8.27 (s, 1H,
1
(
3
3
3
2
2
2
NH).
Ethyl 2-amino-2-(2-methoxyphenylhydrazono)acetate (33). Yield 95%; mp 99ꢀ101 °C
1
(Cyclohexane/EtOAc). Hꢀ NMR (DMSOꢀd
6
) 1.28 (t, 3H, ar, J = 7.1 Hz), 3.82 (s, 3H, OCH
), 6.73 (t, 1H, ar, J = 7.6 Hz), 6.84ꢀ6.92 (m, 2H, ar),
.28 (d, 1H, ar, J = 7.9 Hz), 7.86 (s, 1H, NH). Anal. Calcd. for C H N O .
3
), 4.23
(
2 2
q, 2H, CH , J = 7.1 Hz), 6.15 (br s, 2H, NH
7
11
15
3
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Ethyl 2-amino-2-(4-nitrophenylhydrazono)acetate (34). Yield 65 %; mp 192ꢀ193 °C (EtOH) (lit
1
1
90ꢀ191 °C). H NMR (DMSOꢀd ) 1.29 (t, 3H, CH , J = 7.2 Hz), 4.27 (q, 2H, CH , J = 7.2 Hz),
6
3
2
6
2
.39 (br s, 2H, NH ), 7.07 (d, 2H, ar, J = 9.2 Hz), 8.20 (d, 2H, ar, J = 9.2 Hz), 9.66 (br s, 1H, NH).
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
General Procedure for the Synthesis of Ethyl 5-oxo-1-aryl-4,5-dihydro-1H-1,2,4-triazole-3-
carboxylates (35-38).
A solution of triphosgene (4.2 mmol) in anhydrous THF (10 mL) was added dropwise to a stirred
solution of ethyl 2ꢀaminoꢀ2ꢀ(arylhydrazono)acetate derivatives 31-34 (4.6 mmol) in anhydrous THF
(15 mL) at 0 °C. After the addition was completed, the mixture was stirred 2ꢀ3 h at rt. Then, most of
the solvent was removed at reduced pressure and water (20 mL) was added to the residue to give a
solid which was collected by filtration, washed with water (20 mL), dried and recrystallized.
Ethyl 5-oxo-1-phenyl-4,5-dihydro-1H-1,2,4-triazole-3-carboxylate (35). Yield 62%; mp 200ꢀ202 °C
37
1
(
lit. 193ꢀ194 °C) (Cyclohexane/EtOAc). H NMR (DMSOꢀd
6 3
) 1.33 (t, 3H, CH , J = 7.1 Hz), 4.38
(
q, 2H, CH , J = 7.1 Hz), 7.30 (t, 1H, ar, J = 7.4 Hz), 7.49 (t, 2H, ar, J = 7.4 Hz), 7.89 (d, 2H, ar, J =
2
7.6 Hz), 7.90 (s, 1H, Hꢀ9), 12.99 (br s, 1H, NH).
Ethyl 1-(4-methoxyphenyl)-5-oxo-4,5-dihydro-1H-1,2,4-triazole-3-carboxylate (36). Yield 42%; mp
37
1
1
86ꢀ187 °C (lit 179 °C) (EtOH). H NMR (CDCl ) 1.48 (t, 3H, CH , J = 7.2 Hz), 3.80 (s, 3H,
3 3
3 2
OCH ),4.50 (q, 2H, CH , J = 7.2 Hz), 6.98 (d, 2H, ar, J = 9.1 Hz), 7.87 (d, 2H, ar, J = 9.1 Hz), 10.65
(br s, 1H, NH).
Ethyl 1-(2-methoxyphenyl)-5-oxo-4,5-dihydro-1H-1,2,4-triazole-3-carboxylate (37). Yield 79%; mp
1
1
31ꢀ133 °C (EtOAc). H NMR (DMSOꢀd ) 1.30 (t, 3H, CH , J = 7.1 Hz), 3.78 (s, 3H, OCH ), 4.34
6
3
3
(
q, 2H, CH , J = 7.1 Hz), 7.06 (t, 1H, ar, J = 7.6 Hz), 7.21 (d, 1H, ar, J= 8.3 Hz), 7.36 (d, 1H, J = 7.6
2
13 3 4
Hz), 7.49 (t, 1H, J = 7.6 Hz), 12.69 (br s, 1H, NH). Anal. Calcd. for C12H N O .
Ethyl 1-(4-nitrophenyl)-5-oxo-4,5-dihydro-1H-1,2,4-triazole-3-carboxylate (38). Yield 65%; mp
1
2
41ꢀ242 °C (Cyclohexane/EtOAc). H NMR (CDCl
3
)
1
3
.50 (t, 3H, CH , J = 7.1 Hz,), 4.55 (q, 2H,
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2
2
2
2
2
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4
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5
5
5
5
5
5
5
6
CH
2
, J = 7.1 Hz), 8.29ꢀ8.37 (m, 4H, ar), 10.31 (br s, 1H, NH). IR 3369, 1755, 1698, 1513, 1375.
Anal. Calcd. for C H N O
5.
1
1
10
4
General Procedure for the Synthesis of Ethyl 1-aryl-5-oxo-4-(2-oxopropyl)-4,5-dihydro-1H-
,2,4-triazole-3-carboxylates (39, 41) and Ethyl 1-aryl-5-oxo-4-(2-oxoarylethyl)-4,5-dihydro-
0
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2
3
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7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
1H-1,2,4-triazole-3-carboxylates (40, 42-47).
Chloroacetone (1.2 mmol) or the suitable αꢀbromoketone (1.2 mmol) was added to a mixture of
ethyl 1ꢀarylꢀ5ꢀoxoꢀ1,2,4ꢀtriazoleꢀ3ꢀcarboxylate derivatives 35-38 (1 mmol) and potassium carbonate
3
(2 mmol) in DMF/CH CN (1:9 ratio, 10 mL). The suspension was stirred at rt until the
disappearance of the starting material (TLC monitoring, 2ꢀ24 h). The solvent was removed at
reduced pressure and the residue was treated with water (50ꢀ70 mL). The solid was collected by
filtration, washed with water (20 mL), then with Et O (10 mL) and recrystallized. To isolate
2
compound 44, the reaction mixture was treated with water (50ꢀ70 mL) and extracted with CH
2 2
Cl
(30 mL x 3). The organic phase was anhydrified (Na SO ) and reduced to dryness under vacuum to
2
4
give a solid made up of a mixture of derivative 44 and its Oꢀalkylated isomer 44a (about 2:0.8 ratio,
1
from H NMR spectrum) that were separated on silica gel column (eluent nꢀhexane/EtOAc/Et
2
O
5
:4:7). Compounds 44 and 44a were obtained, respectively from the second and first eluates.
Ethyl 5-oxo-4-(2-oxopropyl)-1-phenyl-4,5-dihydro-1H-1,2,4-triazole-3-carboxylate (39). Yield
1
58%; mp 104ꢀ105 °C (EtOH). H NMR (DMSOꢀd
6
) 1.31 (t, 3H, CH
), 7.35 (t, 1H, ar, J = 7.4 Hz), 7.54 (t, 2H, ar, J
7.4 Hz), 7.91 (d, 2H, ar, J = 7.7 Hz). Anal. Calc. for C H N O
3
, J = 7.1 Hz), 2.28 (s, 3H,
3 2 2
CH ), 4.35 (q, 2H, CH , J = 7.1 Hz), 4.92 (s, 2H, CH
=
14
15
3
4.
Ethyl 5-oxo-4-(2-oxo-2-phenylethyl)-1-phenyl-4,5-dihydro-1H-1,2,4-triazole-3-carboxylate (40).
1
Yield 75%; mp 157ꢀ159 °C (EtOAc/EtOH). H NMR (DMSOꢀd
6
) 1.21 (t, 3H, ar, J = 7.1 Hz), 4.29
(q, 2H, CH
2 2
, J = 7.1 Hz), 5.59 (s, 2H, CH ), 7.37 (t, 1H, ar, J = 7.4 Hz), 7.55 (t, 2H, ar, J = 7.6 Hz),
7
.63 (t, 2H, ar, J = 7.7 Hz), 7.76 (t, 1H, ar, J = 7.4 Hz), 7.95 (d, 2H, ar, J = 7.7 Hz), 8.11 (d, 2H, ar, J
=
7.8 Hz). Anal. Calc. for C H N O
19 17 3 4.
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Ethyl
1-(4-methoxyphenyl)-5-oxo-4-(2-oxopropyl)-4,5-dihydro-1H-1,2,4-triazole-3-carboxylate
1
(41). Yield 92%; mp 98ꢀ100 °C (EtOH). H NMR (DMSOꢀd ) 1.30 (t, 3H, CH , J = 7.1 Hz), 2.27
6
3
3 3 2 2
(s, 3H, CH ), 3.80 (s, 3H, OCH ), 4.34 (q, 2H, CH , J = 7.1 Hz), 4.91 (s, 2H, CH ), 7.08 (d, 2H, ar,
17 3 5.
J = 9.1 Hz), 7.76 (d, 2H, ar, J = 9.1 Hz). Anal. Calc. for C15H N O
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
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56
57
58
59
60
Ethyl
1-(4-methoxyphenyl)-5-oxo-4-(2-oxo-2-phenylethyl)-4,5-dihydro-1H-1,2,4-triazole-3-
1
carboxylate (42). Yield 58%; mp 127ꢀ128 °C (Cyclohexane/EtOAc). H NMR (CDCl
3
) 1.38 (t, 3H,
CH , J = 7.1 Hz), 3.86 (s, 3H, OCH ), 4.40 (q, 2H, CH , J = 7.1 Hz), 5.57 (s, 2H, CH ), 6.99 (d, 2H,
3
3
2
2
ar, J = 9.1 Hz), 7.55 (t, 2H, ar, J = 7.5 Hz), 7.68 (t, 1H, ar, J = 7.4 Hz), 7.88 (d, 2H, ar, J = 9.1 Hz),
.03 (d, 2H, ar, J = 7.4 Hz). IR 1732, 1711, 1694. Anal. Calc. for C20
8
19 3 5.
H N O
Ethyl 1-(4-nitrophenyl)-5-oxo-4-(2-oxo-2-phenylethyl)-4,5-dihydro-1H-1,2,4-triazole-3-carboxylate
1
(43). Yield 92 %; mp 147ꢀ148 °C (MeOH). H NMR (CDCl
3
) 1.40 (t, 3H, CH
3
, J = 7.2 Hz), 4.44 (q,
2
H, CH , J = 7.2 Hz), 5.59 (s, 2H, CH ), 7.58 (t, 2H, ar, J = 7.2 Hz), 7.70 (t, 1H, ar, J = 8.4 Hz),
2 2
8
.04 (d, 2H, ar, J = 7.2 Hz), 8.35ꢀ8.38 (m, 4H, ar). IR 1736, 1725, 1700, 1463, 1375. Anal. Calc. for
19 16 4 6.
C H N O
Ethyl
1-(2-methoxyphenyl)-5-oxo-4-(2-oxo-2-phenylethyl)-4,5-dihydro-1H-1,2,4-triazole-3-
1
carboxylate (44). Yield 65 %; mp 88ꢀ90 °C (Cyclohexane/EtOAc). H NMR (DMSOꢀd
6
) 1.18 (t,
3
7
H, CH , J = 7.1 Hz), 3.81 (s, 3H, OCH ), 4.24 (q, 2H, CH , J = 7.1 Hz), 5.55 (s, 2H, CH ), 7.04ꢀ
3 3 2 2
.09 (m, 2H, ar), 7.42ꢀ7.48 (m, 2H, ar), 7.57 (t, 2H, ar, J = 7.5 Hz), 7.67 (t, 1H, ar, J = 7.4 Hz), 8.04
19 3 5.
(d, 2H, ar, J = 7.1 Hz). Anal. Calc. for C20H N O
Ethyl 1-(2-methoxyphenyl)-5-(2-oxo-2-phenylethoxy)-1H-1,2,4-triazole-3-carboxylate (44a). Yield
1
3
3
7
0 %; mp 138ꢀ140 °C (Cyclohexane/EtOAc). H NMR (DMSOꢀd ) 1.27 (t, 3H, CH , J= 7.1 Hz),
6
3
.84 (s, 3H, OCH ), 4.29 (q, 2H, CH , J = 7.1 Hz), 5.94 (s, 2H, CH ), 7.15 (t, 1H, ar, J = 7.6 Hz),
3
2
2
.30 (d, 1H, ar, J = 7.8 Hz), 7.52 (d, 1H, ar, J = 7.8 Hz), 7.58 (t, 2H, ar, J = 7.8 Hz), 7.73 (t, 1H, ar, J
= 7.4 Hz), 7.98 (d, 2H, ar, J = 7.8 Hz). Anal. Calc. for C20
19 3 5.
H N O
Ethyl 4-[2-(3-methoxyphenyl)-2-oxoethyl]-5-oxo-1-phenyl-4,5-dihydro-1H-1,2,4-triazole-3-
1
carboxylate (45). Yield 80%; mp 123ꢀ125 °C (EtOH). H NMR (DMSOꢀd ) 1.20 (t, 3H, ar, J = 7.1
6
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