Efficient and regioselective one-step synthesis of 7-aryl-5-methyl- and 5-aryl-7-methyl-2-amino-[1,2,4]triazolo[1,5-a] pyrimidine derivatives

Article abstract of DOI:10.1039/c7ob02085f

Two facile and efficient one-step procedures for the regioselective synthesis of 7-aryl-5-methyl- and 5-aryl-7-methyl-2-amino-[1,2,4]triazolo[1,5-a]pyrimidines have been developed, via reactions of 3,5-diamino-1,2,4-triazole with variously substituted 1-a

Full text of DOI:10.1039/c7ob02085f

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Biomol. Chem., 2017, DOI: 10.1039/C7OB02085F.
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Efficient and regioselective one-step synthesis of 7-aryl-5-methyl- and 5-aryl-7-
methyl-2-amino-[1,2,4]triazolo[1,5-a]pyrimidine derivatives.
Serena Massari,*a Jenny Desantis,a Giulio Nannetti,b Stefano Sabatini,a Sara Tortorella,c Laura Goracci,c
Violetta Cecchetti,a Arianna Loregianb and Oriana Tabarrinia
a Department of Pharmaceutical Sciences, University of Perugia, 06123 Perugia, Italy.
E-mail: serena.massari@unipg.it; Tel: +39 0755855146
b Department of Molecular Medicine, University of Padua, 35121 Padua, Italy.
c Department of Chemistry, Biology and Biotechnology, University of Perugia, 06123 Perugia, Italy.
Abstract
Two facile and efficient one-step procedures for the regioselective synthesis of 7-aryl-5-methyl- and 5-aryl-
7-methyl-2-amino-[1,2,4]triazolo[1,5-a]pyrimidines have been developed, via reactions of 3,5-diamino-
1,2,4-triazole with variously substituted 1-aryl-1,3-butanediones and 1-aryl-2-buten-1-ones, respectively.
The excellent yield and/or regioselectivity shown by the reactions decreased when ethyl 5-amino-1,2,4-
triazole-3-carboxylate was used. Being the [1,2,4]triazolo[1,5-a]pyrimidine a privileged scaffold, the
procedure herein reported may be useful for the preparation of biologically active compounds. In this
study, the preparation of a set of compounds based on the [1,2,4]triazolo[1,5-a]pyrimidine scaffold let to
the identification of compound 20 endowed with a very promising ability to inhibit influenza virus RNA
polymerase PA-PB1 subunits heterodimerization.
1

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Introduction
[1,2,4]triazolo[1,5-a]pyrimidine is a privileged structure with numerous chemical and biological
applications. Beside to their great versatility in the interactions with metal ions, [1,2,4]triazolo[1,5-
a]pyrimidines showed a wide range of biological activities both in agriculture and in medicine.1a,b Examples
of biologically active compounds include trapidil (Rocornal®, Fig. 1), a platelet-derived growth factor
antagonist that has been used to treat patients with ischemic coronary heart, liver, and kidney disease,1c
and filibuvir (Fig. 1), a non-nucleoside inhibitor of HCV NS5B polymerase that passed stage II clinical trials,
although its clinical development program was then suspended.1d Focusing on the most recent literature,
compounds based on the [1,2,4]triazolo[1,5-a]pyrimidine core have been reported as phosphodiesterase 2
(PDE2a) inhibitors for the treatment of memory disorders,2a anti-Alzheimer's disease,2b-d anticancer,2e,f
antimalarial,2g,h antitubercular,2i antileishmanial,2j antibacterial,2k antiviral,2l hypnotic,2m and CB2
cannabinoid receptor inverse agonists.2n
Figure 1. Examples of biologically active compounds with a [1,2,4]triazolo[1,5-a]pyrimidine scaffold.
We have also been involved in the synthesis of a series of [1,2,4]triazolo[1,5-a]pyrimidines (compound I
and structures II and III, Fig. 1)3a,b within our research program on the development of influenza virus (flu)
RNA-dependent RNA polymerase (RdRP) PA-PB1 subunits interaction inhibitors.3 In particular, the synthesis
2

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of the anti-flu compounds entailed the preparation of the key intermediate 2-amino-5-methyl-7-phenyl-
[1,2,4]triazolo[1,5-a]pyrimidine (1a) and its isomer 2-amino-7-methyl-5-phenyl-[1,2,4]triazolo[1,5-
a]pyrimidine (2a) (Table 1).
A major contribution to the chemistry of 2-amino-[1,2,4]triazolo[1,5-a]pyrimidines has been provided by
Desenko and co-workers, who were the first to report on their synthesis,4 and Chernyshev’s research
group, who investigated their synthesis further,5a-c their reactivity,5d and their use for the preparation of
polycondensed heterocycles.5e-h Nevertheless, the synthesis of 7-aryl-5-methyl- and 5-aryl-7-methyl-2-
amino-[1,2,4]triazolo[1,5-a]pyrimidine derivatives has been scarcely explored. In this work, we reported
two approaches for their preparation via reaction between 3,5-diamino-1,2,4-triazole and variously
functionalized 1-aryl-1,3-butanediones and 1-aryl-2-buten-1-ones, respectively. Both strategies allowed the
synthesis of the desired isomer under mild conditions, with high yields, and regioselectively.
Results and discussion
The synthetic method known for the regioselective preparation of compound 1a, as well as of some 5,7-
diaryl-2-amino-[1,2,4]triazolo[1,5-a]pyrimidines, involves a two-step procedure (Scheme 1) entailing: i)
cyclocondensation of 3,5-diamino-1,2,4-triazole (3a) with chalcone 4-phenylbut-3-en-2-one (4a) giving2-
amino-5-methyl-7-phenyl-4,7-dihydro-[1,2,4]triazolo[1,5-a]pyrimidine,4a-c and ii) heteroaromatization using
either N-bromosuccinimide (NBS) or Br2.4a However, the overall yields do not exceed 25-30%. Moreover,
NBS and Br2 are highly reactive and, thus, this method can only be used to prepare [1,2,4]triazolo[1,5-
a]pyrimidines with limited substitutions at the C-5 and C-7 positions. Increased overall yields up to 40-77%
were achieved by acetyl protection of the C-2 amino group, by adding Ac2O to the reaction mixture after
the completion of cyclocondensation, preventing oxidation of the amino group in the successive step.5a
Through this three-step procedure, compound 1a was regioselectively obtained in 77% yield (Scheme 1).
3

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Scheme 1. Known procedures for the synthesis of 1a.
The reaction of 3-amino-1,2,4-triazole bearing different substituents at the C-5 position with
unsymmetrically 1,3-diketones where one of the substituents is a methyl group and the other is a group
different from methyl, was known from literature to form a mixture of 5-methyl and 7-methyl isomers. The
two isomers can be distinguished by NMR on the basis of the chemical shifts of the pyrimidine methyl
carbon appearing at 24-25 ppm and 16-17 ppm for the 5-methyl and 7-methyl isomers, respectively.6
Although 5-methyl isomer is always the main product of the reaction, the ratio of isomers is influenced: i)
by the steric hindrance of the substituent on the 1,3-diketone, with a more bulky substituent that gives a
higher ratio of 5-methyl isomer, and ii) by the inductive effect of the substituent at the C-5 position of the
3-amino-1,2,4-triazole, which influences the ratio of isomers much more than the inductive effect of the
substituent on the 1,3-diketone different from the methyl.6b
Based on these facts, we hypothesized that, the presumable high nucleophilicity of 3a owing to the
presence of a second electron-donating amino groups in the molecule,7 might led to an efficient synthesis
of 2-amino-7-aryl-5-methyl-[1,2,4]triazolo[1,5-a]pyrimidines by cyclocondensation with 1-aryl-1,3-
butanediones. To test this idea, we first tried the reaction of 3a with 1-phenyl-1,3-butanedione (5a) in
glacial acetic acid at reflux (Table 1, entry 1). Actually, Kreutzberger and Risse reported in 1979 that this
reaction condition provided a mixture of isomers 1a and 2a in 2% and 26% yield, respectively.8 In contrast
to what reported by Kreutzberger and Risse who mistakenly inverted the assignment of the structures, we
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were pleased to find that the reaction took place rapidly (4 h), highly efficiently, and, more interestingly,
highly regioselectively. Indeed, 2-amino-5-methyl-7-phenyl-[1,2,4]triazolo[1,5-a]pyrimidine (1a) was
obtained in 88% yield while its regioisomer 2-amino-7-methyl-5-phenyl-[1,2,4]triazolo[1,5-a]pyrimidine (2a)
was formed only in traces.
To further explore the reaction conditions, the reaction was then carried out in different solvents. In
particular, the reaction was repeated in protic and aprotic solvents, i.e. EtOH, DMF, CHCl3, THF, and
acetone (Table 1), in order to understand whether the acid environment of glacial acetic acid could be
essential or not for regioselectivity. We also aimed at evaluating the possible influence on tautomerism of
5a, that is well known to be extremely sensitive to solvent effect.9 In particular, the enolic form of 5a is
much greater in nonpolar solvents than in polar or hydrogen-bond donor solvents, since the first ones do
not compete with hydrogen-bond formation.9a For example, in water compound 5a is extimated to be a
mixture of both tautomers, with the keto-form being more favoured than the enol one (about 60% and
40%, respectively).9b Although data on tautomerism of 5a in glacial acetic acid are not available, it is
reasonable to hypothesize that in this solvent the keto-form is predominant over the enol one, while, for
example, in CHCl3 it is known that this compound exists as two kinds of cis-enol forms.9c No significant
reaction was observed in CHCl3 (entry 2), acetone (entry 3), and THF (entry 4) at reflux after 24 h, because
of the insolubility of 3a in these solvents at the used concentrations. On the other hand, the reaction in
EtOH (entry 5) and DMF (entry 6) led to the formation of isomer 1a as the main product but in lower yield
(57% and 66%, respectively) and much more slowly than in acetic acid. Since the reactions did not go to
completion under both conditions after 24 h, we studied the effect of the equiv of 3a (entry 7) as well as
the influence of the addition of a base such as triethylamine (entries 8) in DMF. Analogously, the reactions
were much more slow and less efficient (69% and 61%, respectively), but, most importantly, they showed a
dramatically decreased regioselectivity. Finally, the reaction was carried out in EtOH with the addition of a
catalytic amount of acetic acid (entry 9), to verify whether a protic solvent in acid conditions could lead to a
similar regioselectivity compared to that obtained with glacial acetic acid. The presence of acetic acid did
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not influence the outcome of the reaction, meaning that the simple catalytic effect by acid conditions in the
condensation reaction is not sufficient to obtain a high regioselectivity.
Thus, the best reaction conditions are treating 3a (1 equiv) and 5a (1 equiv) in acetic acid at reflux for 4 h.
Through this one-step procedure, compound 1a was regioselectively obtained in 88% yield.
Table 1. Optimization of reaction conditions for 1aa
Yield (%)b
3a
(equiv)
Entry
Solvent
Time (h)
1a
88
-
2a
1
2
3
4
5
6
7
1
1
1
1
1
1
2
Acetic Acid
CHCl3
4
traces
24
24
24
24
24
24
-
-
Acetone
THF
-
-
-
EtOH
57
66
39
4
3
30
DMF
DMF
DMF
(1 equiv of Et3N)
8
9
1
1
24
48
13
5
EtOH
24
62
(0.5 mL of Acetic Acid)
a The reaction was performed on 1.0 mmol scale of 5a in 2.5 mL of solvent at reflux.
b Determined by 1H NMR.
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Utilizing the optimized conditions, we then studied the scope of the reaction (Table 2). Thus, 1-aryl-1,3-
butanediones (5b-k) bearing different electron-donating and electron-withdrawing substituents on the
phenyl ring were prepared and reacted with 3a. 1-Aryl-1,3-butanediones were in turn synthesized through
a Claisen condensation by reacting aryl-methyl ketones with ethyl acetate in the presence of sodium10 (for
the synthesis and characterization of compounds 5b-k, see SI).
The results listed in Table 2 show that 1-aryl-1,3-butanediones bearing both electron-donating (entries 2
and 3) and electron-withdrawing (entry 4) substituents on the phenyl ring are suitable substrates for this
reaction, reacting smoothly with 3a to give products 1b-d. The effect of the position of electron-
withdrawing substituents on the phenyl ring was also studied (entries 5 and 6), and the reaction gave
consistently good yields for compounds 1e and 1f. Multiple electron-donating (entry 7) and electron-
withdrawing (entry 8) substituents on the 1-aryl-1,3-butanedione phenyl ring gave goods yields. Finally,
using 1-(naphthalen-1-yl)butane-1,3-dione (entry 9) and 1-(pyridin-4-yl)butane-1,3-dione (entry 10), the
reaction gave compounds 1i and 1k in modest yields.
Table 2. Preparation of 2-amino-7-aryl-5-methyl-[1,2,4]triazolo[1,5-a]pyrimidinesa
Entry
R
Time (h)
Product
1a
Yield (%)b
1
2
3
4
5
6
C6H5
4
3
4
3
2
3
88
93
98
98
90
86
p-CH3C6H5
p-CH3SC6H5
p-BrC6H5
o-ClC6H5
m-CF3C6H5
1b
1c
1d
1e
1f
7

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7
8
m,p-Di-CH3OC6H5
m,p-Di-ClC6H5
1-Naphthyl
3
2
2
5
1g
1h
1i
80
83
62
65
9
10
4-Pyridinyl
1k
a The reaction was performed on 1.0 mmol scale of 3a and 1 equiv of 5 in 2.5 mL of glacial acetic
acid at reflux.
b Isolated yields.
To further explore the scope of the reaction, 5a was reacted with 5-amino-1,2,4-triazole-3-carboxylic acid,
characterized by the presence of an electron-withdrawing group at the C-2 position, under the same
reaction conditions. Unfortunately, the reaction was accompanied by decarboxylation, thus, it was
repeated starting from ethyl 5-amino-1,2,4-triazole-3-carboxylate (6a). The reaction was equally rapid (4 h)
and efficient (80% yield) but showed a dramatically decreased regioselectivity, in that ethyl 5-methyl-7-
phenyl-[1,2,4]triazolo[1,5-a]pyrimidine-2-carboxylate (7a) and ethyl 7-methyl-5-phenyl-[1,2,4]triazolo[1,5-
a]pyrimidine-2-carboxylate (8a) were obtained in the ratio of 3.7:1 (Scheme 2). Of note, although not
regioselective, this reaction permitted to obtain derivatives 7a in acceptable yield (63%) with respect to the
procedure entailing the reaction of 6a with 4-phenylbut-3-en-2-one (4a) followed by heteroaromatization
(Scheme 2), which provided compound 7a in a very lower yield (10%).3a
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Scheme 2. Synthesis of 7a via reaction of 6a with 5a and 4a. *Isolated yields.
Plausible pathways accounting for the formation of compounds 7a and 8a through the reaction of 6a with
5a are speculatively reported in Scheme 3. In particular, an initial direct addition of the amino group at the
C(5) position of 6a on the carbonyl carbon C(3) of 5a to give a β-aminovinyl ketone, followed by
intramolecular cyclization of the latter at the nucleophilic N(1) center of 6a on the carbonyl C(1) of 5a
would give 7a (Scheme 3a). The same pathway may be responsible for the formation of 1a starting from 3a.
On the other hand, compound 8a could be obtained by: i) an initial direct addition of N(1) of 6a on the
carbonyl carbon C(3) of 5a followed by intramolecular cyclization of the latter by direct addition of the C(5)
amino group of 6a on the carbonyl C(1) of 5a (Scheme 3b); or ii) an initial direct addition of the C(5) amino
group of 6a on the carbonyl carbon C(1) of 5a followed by intramolecular cyclization of the latter by direct
addition of N(1) of 6a on the carbonyl C(3) of 5a (Scheme 3c).
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a)
NH2
H
O
CH3
OH
CH3
N
N
N
+
R
- H2O
N
O
R
N
HN
N
H
O
3a R = NH2
6a R = CO2Et
5a
H
N
CH3
N
N
N
CH3
N
N
N
N
CH3
R
- H2O
R
N
R
N
N
HN
O
OH
1a R = NH2
7a
R = CO2Et
b)
O
NH2
O
NH2
NH2
N
N
- H2O
N
+
O
EtO2C
EtO2C
N
N
N
N
N
EtO2C
N
O
CH3
OH
H
H3C
CH3
5a
6a
H
N
N
N
N
- H2O
N
N
EtO2C
OH
EtO2C
N
N
CH3
8a
CH3
c)
H
N
N
N
NH2
N
- H2O
EtO2C
EtO2C
OH
N
+
N
HN
N
O
HN
N
EtO2C
O
N
O
H
O
CH3
CH3
CH3
6a
5a
H
N
N
N
N
N
N
- H2O
EtO2C
EtO2C
N
N
OH
H3C
CH3
8a
Scheme 3. Plausible reaction mechanisms toward 1a, 7a, and 8a.
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Although a deep investigation of the mechanisms involved in this reaction is beyond the scope of this study,
the mechanism reported in Scheme 3b was hypothesized as more likely to occur compared to that in
Scheme 3c. Indeed, if the steric hindrance of the phenyl group in 5a was negligible in driving the
nucleophilic attack of the amino group, the high regioselectivity observed in the reaction of 3a could not be
explained. Thus, the difference in the regioselectivity shown by the reactions of 3a and 6a with 5a might
depend on the different nucleophilicity of the two aminotriazoles due to the effect of the substituent. In
particular, while the presence of a second electron-donating amino group in 3a might be responsible for a
higher nucleophilicity of the C(3) amino group than N(2), the electron-withdrawing ethyl carboxylate
moiety in 6a might led to a smaller difference of nucleophilicity between the C(5) amino group and N(1),
resulting in a decreased selectivity in forming 7a and 8a. Moreover, in the acidic conditions used (pH in
glacial acetic acid is reported to be 2.4) compound 3a should be almost fully protonated, while about 34%
of 6a is in its neutral form according to MoKa predictions,11 and this might explain why the nucleophilic
attack of N(1) can occur for compound 6a (although this remains the minor pathway). Finally, the presence
of two amino groups in 3a with a presumable comparable nucleophilicity would make their initial attack
more probable in 3a than in 6a, as also confirmed by lost of regioselectivity observed when one of the two
amino groups in 3a was dimethylated.6b
As shown above, the reaction of 3a and 5a under a few different conditions (entries 7 and 8 Table 1) gave a
mixture of 1a and 2a, of which, however, the latter is always obtained in very low yield (no more than 30%
yield). Thus, we searched for an alternative synthetic procedure to prepare 2-amino-5-aryl-7-methyl-
[1,2,4]triazolo[1,5-a]pyrimidine derivatives, taking into account the regioselective cyclocondensation of 3a
with chalcones.
The study started by reacting 3a with phenyl-1-propenyl-ketone (9a) in DMF at reflux and by adding Ac2O,
in order to obtain N-(7-methyl-5-phenyl-4,7-dihydro-[1,2,4]triazolo[1,5-a]pyrimidin-2-yl)acetamide to be
then heteroaromatized and hydrolysed, to give 2a. After 30 min, the reaction gave a mixture compounds,
of which one was the already oxidized N-(7-methyl-5-phenyl-[1,2,4]triazolo[1,5-a]pyrimidin-2-yl)acetamide
derivative. Thus, the reaction was repeated without adding Ac2O, in order to directly obtain 2a (Table 3).
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After 30 min, compound 2a was obtained in 44% yield (entry 1),3a which increased up to 50% yield after 4 h
(entry 2).
With these encouraging results, we further explored the reaction conditions. Based on the fact that
atmospheric O2 can enhance oxidation of 4,7-dihydro-[1,2,4]triazolo[1,5-a]pyrimidines,13 we hypothesized
that an higher yield might be achieved by carrying out the reaction in an open flask. Thus, the reaction was
initially carried out in different solvents (Table 3), i.e. toluene, dioxane, N-methylpyrrolidone (NMP), and
DMF (CHCl3, acetone, and THF have not been used because of the insolubility of 3a), at 110 °C in an open
flask. No significant reaction was observed in toluene (entry 3) and dioxane (entry 4) after 24 h, while the
reaction in NMP (entry 5) provided compound 2a in 25% yield after 4 h. On the other hand, the reaction in
DMF (entry 6) led to the formation of 2a in 57% yield after 2 h. Then, we studied the effect of the equiv of
9a (entry 7), the presence of a base such as Et3N (entry 8), and both of them (entry 9) in DMF, and found
that the optimum reaction conditions are treating 3a (2 equiv) and 9a (1 equiv) in the presence of Et3N (1
equiv) in DMF at 110 °C for 2 h (entry 9) in an open flask. Through this one-step procedure, compound 2a
was regioselectively obtained in 80% yield.
Table 3. Optimization of reaction conditions for 2a.a
3a
(equiv)
Et3N
(equiv)
Entry
Solvent
Time (h)
Yield (%)b
1
2
3
1
1
1
no base
no base
no base
DM c
DMFc
0.5
4
44
50
-
Toluene
24
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4
5
6
7
8
9
1
1
1
2
1
2
no base
no base
no base
no base
1
Dioxane
NMP
DMF
24
4
-
25
2
57
67
71
80
DMF
2
DMF
2
1
DMF
2
a
Unless otherwise indicated, the reaction was performed on 1.0 mmol scale of 9a in 2.5 mL of
solvent in an open flask at 110 °C.
b Isolated yields.
c Using the condenser.
With these optimized reaction conditions, we next studied the scope of the reaction (Table 4). Thus, 1-aryl-
2-buten-1-ones (9b-i) containing different electron withdrawing as well as electron-donating groups on the
phenyl ring were synthesized and reacted with 3a. 1-Aryl-2-buten-1-ones were synthesized through a
Witting reaction. In particular, 2-bromoacetophenones were treated with triphenylphosphine in toluene
giving triphenylphosphonium bromides,13 which were then reacted with aqueous NaOH in dichloromethane
to afford 1-aryl-2-(triphenylphosphoranylidene) ethanones, and then with acetaldehyde in a Witting
reaction to give the corresponding 1-aryl-2-buten-1-ones14 (for the synthesis and characterization of
compounds 9b-i, see SI).
The results shown in Table 4 highlight that 1-aryl-2-buten-1-ones bearing both electron-donating and
electron-withdrawing substituents on the phenyl ring are suitable substrates for this reaction. Indeed, with
the exception of 4-nitrophenyl- (entry 4) and 4-pyridinyl- (entry 9) 2-buten-1-ones, which gave derivatives
2d and 2i in modest yields, all the other studied 1-aryl-2-buten-1-ones (entries 2, 3, and 5-7) reacted
efficiently and regioselectively with 3a to give 2-amino-5-aryl-7-methyl-[1,2,4]triazolo[1,5-a]pyrimidines in
consistently good yields.
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Table 4. Preparation of 2-amino-5-aryl-7-methyl-[1,2,4]triazolo[1,5-a]pyrimidines.a
Entry
R
Time (h)
Product
2a
Yield (%)b
1
2
3
4
5
6
7
8
9
C6H5
2
3
2
2
2
2
3
2
2
80
83
92
73
80
87
80
88
60
p-CH3C6H5
p-CH3OC6H5
p-NO2C6H5
p-ClC6H5
m-BrC6H5
o-FC6H5
2b
2c
2d
2e
2f
2g
o,p-Di-FC6H5
4-Pyridinyl
2h
2i
a The reaction was carried out on 1.0 mmol scale of 3a, 0.5 mmol of 9, and 0.5 mmol of Et3N in 2.5
mL of DMF in an open flask at 110 °C.
b Isolated yields.
To further explore the scope of the reaction, by using the same reaction conditions, 3a was reacted with 4-
phenylbut-3-en-2-one (4a) giving isomer 1a regioselectively in 72% yield after 6 h (Scheme 4a). Of note,
although in lower yield (72%) than the reaction of 3a with 5a (88% yield), the reaction of 3a with 4a under
these conditions is an alternative one-step procedure for the regioselective synthesis of 1a, which was
obtained with comparable yield to that of the known three-step procedure (77% yield).5a Finally, by using
the same reaction conditions, ethyl 5-amino-1,2,4-triazole-3-carboxylate (6a) was also reacted with both 4a
and 9a. Surprisingly, isomers 7a and 8a were obtained in traces and 19% yield, respectively, after 24 h
(Scheme 4b).
14

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Scheme 4 a) Synthesis of 1a via reaction of 3a with 4a; b) Synthesis of 7a and 8a via reaction of 3a with 4a
and 8a respectively. *Isolated yields.
All the compounds herein reported, with the exception of 1a, were not described previously and their
1
structures were fully characterized by spectra data of H NMR, 13C NMR, and HRMS. It is worthwhile to
underline that all the products obtained from both the procedures were purified by simple crystallization
without the involvement of chromatography.
[1,2,4]triazolo[1,5-a]pyrimidines as anti-flu compounds
As mentioned above, we have recently identified a series of potent anti-flu compounds based on the
[1,2,4]triazolo[1,5-a]pyrimidine scaffold that act by inhibiting flu RdRP PA-PB1 subunits interaction.3a In
particular, within the optimization of compound I (Fig. 1) we prepared a large series of analogues, along
with a few derivatives characterized by the oxidized [1,2,4]triazolo[1,5-a]pyrimidine scaffold (structure II
and III, Fig. 1).3a Among them, derivatives 10 and 11 (Table 5) showed a good ability to inhibit flu replication
(EC50 = 42 and 25 μM, respectively) at non toxic concentrations (CC50 > 250 μM). Compound 11 also
inhibited PA-PB1 interaction with a comparable IC50 (26 μM).
In order to add structure-activity relationship insights, we decided to exploit the scaffolds herein
synthesized by preparing an additional set of compounds to study the effect of modifications on the C-5, C-
15

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7, and C-2 positions of the [1,2,4]triazolo[1,5-a]pyrimidine core. Thus, 2-amino derivatives 1e, 1f, 2c, 2d,
and 2e variously functionalized at the C-5 or C-7 position were reacted with benzoyl chloride in pyridine at
80 °C providing target derivatives 12-16 (Table 5). To study the C-2 position, target compounds 19 and 20
(Table 5) were prepared starting from 2-carboxylate scaffolds 7a and 8a, which were hydrolyzed to give
intermediates 17 and 18, chlorinated, and then reacted with 2-aminobenzamide in CH2Cl2 in the presence
of DIPEA.
The synthesized compounds were first evaluated for the ability to inhibit the physical interaction between
fluA PA and PB1 subunits by ELISA including the Tat-PB11-15 peptide15 as a positive control of inhibition. In
parallel, for all the synthesized compounds the antiviral activity was tested by plaque reduction assays
(PRA) in Mardin-Darby canine kidney (MDCK) cells infected with a reference fluA virus, the A/PR/8/34
strain. Ribavirin (RBV), a known broad-spectrum inhibitor of RNA viruses polymerase,16 was also included.
To exclude that the observed antiviral activities could be due to toxic effects on the target cells, the
compounds were also tested by MTT assays in MDCK cells.
As shown in Table 5, derivatives 12-16, which were functionalized on the phenyl ring at the C-5 or C-7
position, although nontoxic, resulted unable to inhibit the viral growth at low micromolar concentrations.
Nevertheless, p-nitrophenyl and p-chlorophenyl derivatives 15 and 16 showed a good ability to interfere
with PA-PB1 heterodimerization (IC50 = 25 and 40 μM, respectively). The lack of antiviral activity was shown
also by compound 19, in which however the presence of the benzamide moiety at the C-2 position led to
increase of about 15 folds the anti-PA-PB1 activity (IC50 = 11 μM) with respect to the strict analogue
compound 10 (IC50 = 160 μM). The best and most balanced results was achieved with compound 20, which
showed both the ability to inhibit viral replication and PA-PB1 heterodimerization at non toxic
concentrations. In particular, derivative 20 showed a slightly decreased anti-flu activity (EC50 = 31 μM) but
an enhanced ability to inhibit PA-PB1 complex formation (IC50 = 11 μM) with respect to its analogue 11 (IC50
= 25 μM), and even better than the reference PB11–15–Tat peptide (IC50 = 41 μM).
Table 5. Synthesis and biological activity of [1,2,4]triazolo[1,5-a]pyrimidine derivatives.
16

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ELISA
Cytotoxicity
PRA
in MDCK cells
EC50, µMb
PA-PB1
Interaction Assay
IC50, µMa
(MTT Assay) in
MDCK cells
CC50, µMc
Compd
R
R’
10
11
12
13
14
15
16
C6H5
CH3
CH3
C6H5
160 ± 11
26 ± 5
42 ± 5
25 ± 1
>100
>250
>250
>250
>250
>250
>250
>250
o-ClC6H5
m-CF3C6H5
CH3
CH3
>200
CH3
163 ± 20
160 ± 1
25 ± 2
>100
p-CH3OC6H5
p-NO2C6H5
p-ClC6H5
92 ± 5
92 ± 5
99 ± 2
CH3
CH3
40 ± 4
ELISA
PA-PB1
Interaction Assay
IC50, µMa
Cytotoxicity
(MTT Assay) in
MDCK cells CC50,
µMc
PRA
in MDCK cells
EC50, µMb
Compd
R
R’
19
20
C6H5
CH3
CH3
11 ± 3
7 ± 1
>100
>250
>250
C6H5
31 ± 10
Tat-PB11-15
peptide
35 ± 4
41 ± 5
10 ± 2
>100
>250
RBV
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a Compounds activity in ELISA PA−PB1 interaction assays. The IC50 value is defined as the compound
b
concentration that reduces the PA-PB1 interaction by 50%. Antiviral activity of the compounds
against the fluA A/PR/8/34 strain in plaque reduction assays. The EC50 value represents the effective
c
compound concentration required to reduce virus plaque formation by 50%. Citotoxicity of the
compounds in MTT assays. The CC50 value represents the compound concentration resulting in 50%
inhibition of MDCK cell viability. All the reported values represent the means ± SD of data obtained
from at least three independent experiments in duplicate.
Conclusions
In summary, two facile and efficient one-step procedures for the regioselective synthesis of 7-aryl-5-
methyl- and 5-aryl-7-methyl-2-amino-[1,2,4]triazolo[1,5-a]pyrimidines have been developed. These
procedures have proven to be suitable for 1-aryl-1,3-butanediones and 1-aryl-2-buten-1-ones with different
substitution patterns on the phenyl ring, permitting to obtain 2-amino-[1,2,4]triazolo[1,5-a]pyrimidines
variously functionalized at the C-5 and C-7 positions, respectively. The synthesized derivatives may be
useful for the preparation of biologically active compounds. In this study, they have been used for the
synthesis of a set of [1,2,4]triazolo[1,5-a]pyrimidine derivatives as anti-flu compounds. From this study,
derivative 20 emerged as a new potential antiviral compound endowed with a very good ability to inhibit
flu RNA polymerase complex formation.
Experimental
Material and methods
Commercially available starting materials, reagents, and solvents were used as supplied. Compounds 4-
phenylbut-3-en-2-one (4a), 1-phenyl-1,3-butanedione (5a), and phenyl-1-propenyl-ketone (9a) were
purchased from Alfa Aesar and Apollo Scientific. Synthesis of 1-aryl-1,3-butanediones (5b-k) and 1-aryl-2-
buten-1-ones (9b-i) was reported in the SI. Compound 5-amino-1,2,4-triazole-3-carboxylate (6a) was
synthesized as reported in literature.17 Hydrolysis of compounds 8a and 9a to [1,2,4]triazolo[1,5-
a]pyrimidine-2-carboxylic acid 16 and 17, respectively, was carried out as previously reported by us.3a
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All reactions were routinely monitored by TLC on silica gel 60F254 (Merck) and visualized by using UV or
iodine. Flash column chromatography was performed on Merck silica gel 60 (mesh 230-400). After
extraction, organic solutions were dried over anhydrous Na2SO4, filtered, and concentrated with a Büchi
rotary evaporator at reduced pressure. Yields are of purified product and were not optimized. HRMS
spectra were registered on Agilent Technologies 6540 UHD Accurate Mass Q-TOF LC/MS, HPLC 1290
Infinity. Purities of compounds 12-16, 19, and 20 were determined by UHPLC on Agilent Technologies 6540
UHD Accurate Mass Q-TOF LC/MS, HPLC 1290 Infinity with DAD detector and evaluated to be 100% pure.
HPLC conditions to assess the purity of final compounds were as follows: column, Phenomenex AERIS
Widepore C4, 4.6mm × 100 mm (6.6 μm); flow rate, 0.85 mL/min; acquisition time, 10 min; DAD 254 nm;
oven temperature, 30 °C; gradient of acetonitrile in water containing 0.1% of formic acid (0−100% in 10
1
min). H NMR and 13C NMR spectra were recorded on Bruker Avance DRX-400MHz using residual solvents
such as dimethylsulfoxide (δ = 2.48) or chloroform (δ = 7.26) as an internal standard. Chemical shifts were
recorded in ppm (δ) and the spectral data are consistent with the assigned structures. The spin
multiplicities are indicated by the: symbols s (singolet), d (doublet), t (triplet), q (quartet), m (multiplet),
and bs (broad singolet).
General procedure for the synthesis of 2-amino-7-aryl-5-methyl-[1,2,4]triazolo[1,5-a]pyrimidines (1a-k).
A mixture of the appropriate 1-aryl-1,3-butanedione (5a-k) (1 mmol) and 3a (1 mmol) in glacial acetic acid
(2.5 mL) was refluxed until no starting material was detected by TLC (2-5h). After cooling, the reaction
mixture was poured into ice/water and neutralized with 10% NaOH, obtaining a precipitate that was
filtered and crystallized by EtOH/DMF.
5-Methyl-7-phenyl-[1,2,4]triazolo[1,5-a]pyrimidin-2-amine (1a). White crystals. 1H NMR (400 MHz, DMSO-
d6)
δ: 2.52 (s, 3H, CH3), 6.32 (s, 2H, NH2), 7.14 (s, 1H, H-6), 7.56-7.57 (m, 3H, aromatic CH), 8.10-8.12 (m, 2H,
aromatic CH); 13C NMR (101 MHz, DMSO-d6)
δ
: 24.1, 107.3, 128.4, 129.1, 130.3, 131.0, 143.8, 155.7, 161.6,
167.1; HRMS: m/z calcd for C12H11N5 226.1093 (M + H+), found 226.1021 (M + H+).
5-Methyl-7-p-tolyl-[1,2,4]triazolo[1,5-a]pyrimidin-2-amine (1b). White crystals. 1H NMR (400 MHz, DMSO-
d6)
δ: 2.38 and 2.51 (s, each 3H, CH3), 6.32 (s, 2H, NH2), 7.15 (s, 1H, H-6), 7.37 and 8.06 (d, J = 8.0 Hz, each
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2H, aromatic CH); 13C NMR (101 MHz, DMSO-d6)
δ: 21.1, 24.1, 106.9, 127.4, 129.0, 129.1, 141.2, 143.8,
155.8, 161.5, 167.1; HRMS: m/z calcd for C13H13N5 240.1250 (M + H+), found 240.1249 (M + H+).
1
5-Methyl-7-(4-(methylthio)phenyl)-[1,2,4]triazolo[1,5-a]pyrimidin-2-amine (1c). Light yellow crystals. H
NMR (400 MHz, DMSO-d6) : 2.51 and 2.54 (s, each 3H, CH3), 6.32 (s, 2H, NH2), 7.17 (s, 1H, H-6), 7.41 and
8.13 (d, J = 8.5 Hz, each 2H, aromatic CH); 13C NMR (101 MHz, DMSO-d6)
: 14.1, 24.1, 106.7, 124.9, 126.1,
δ
δ
129.5, 142.7, 143.3, 155.8, 161.5, 167.0; HRMS: m/z calcd for C13H13N5S 272.0971 (M + H+), found 272.0968
(M + H+).
1
7-(4-Bromophenyl)-5-methyl-[1,2,4]triazolo[1,5-a]pyrimidin-2-amine (1d). Light yellow crystals. H NMR
(400 MHz, DMSO-d6) δ: 2.52 (s, 3H, CH3), 6.35 (s, 2H, NH2), 7.19 (s, 1H, H-6), 7.78 and 8.09 (d, J = 8.5 Hz,
each 2H, aromatic CH); 13C NMR (101 MHz, DMSO-d6)
δ: 24.1, 107.3, 124.7, 129.5, 131.2, 131.5, 142.6,
155.7, 161.6, 167.1; HRMS: m/z calcd for C12H10BrN5 304.0199 (M + H+), found 304.0199 (M + H+).
1
7-(2-Chlorophenyl)-5-methyl-[1,2,4]triazolo[1,5-a]pyrimidin-2-amine (1e). Light yellow powder. H NMR
(400 MHz, DMSO-d6) : 2.53 (s, 3H, CH3), 6.31 (s, 2H, NH2), 6.99 (s, 1H, H-6), 7.51-7.65 (m, 4H, aromatic CH);
13C NMR (101 MHz, DMSO-d6)
: 24.1, 109.0, 127.4, 129.5, 130.3, 131.4, 131.9, 132.1, 142.2, 154.9, 161.5,
δ
δ
167.2; HRMS: m/z calcd for C12H10ClN5 260.0704 (M + H+), found 260.0702 (M + H+).
5-Methyl-7-(3-(trifluoromethyl)phenyl)-[1,2,4]triazolo[1,5-a]pyrimidin-2-amine (1f). Light yellow crystals.
1H NMR (400 MHz, DMSO-d6)
δ: 2.54 (s, 3H, CH3), 6.38 (bs, 2H, NH2), 7.29 (s, 1H, H-6), 7.82 (t, J = 7.8 Hz, 1H,
aromatic CH), 7.95 and 8.40 (d, J = 7.8 Hz, each 1H, aromatic CH), 8.48 (s, 1H, aromatic CH); 13C NMR (101
MHz, DMSO-d6) δ: 24.1, 107.9, 125.8 (q, JC-F = 3.03 Hz), 126.6 (q, JC-F = 2.72.7 Hz), 127.5 (q, JC-F = 3.03 Hz),
129.2 (d, JC-F = 32.3 Hz), 129.7, 131.4, 133.2, 142.2, 155.6, 161.8, 167.0; HRMS: m/z calcd for C13H10F3N5
294.0961 (M + H+), found 294.0967 (M + H+).
1
7-(3,4-Dimethoxyphenyl)-5-methyl-[1,2,4]triazolo[1,5-a]pyrimidin-2-amine (1g). Light yellow crystals. H
NMR (400 MHz, DMSO-d6)
aromatic CH), 7.20 (s, 1H, H-6), 7.77 (d, J = 1.8 Hz, 1H, aromatic CH), 7.90 (dd, J = 1.8 and 8.5 Hz, 1H,
aromatic CH); 13C NMR (101 MHz, DMSO-d6)
: 24.1, 55.7, 55.7, 106.5, 111.2, 112.6, 122.3, 122.9, 143.6,
δ: 2.50 (s, 3H, CH3), 3.84 (s, 6H, OCH3), 6.28 (s, 2H, NH2), 7.11 (d, J = 8.5 Hz, 1H,
δ
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148.3, 151.1, 155.9, 161.3, 166.9; HRMS: m/z calcd for C14H15N5O2 286.1305 (M + H+), found 286.1303 (M +
H+).
7-(3,4-Dichlorophenyl)-5-methyl-[1,2,4]triazolo[1,5-a]pyrimidin-2-amine (1h). Light yellow crystals. 1H
NMR (400 MHz, DMSO-d6)
δ: 2.52 (s, 3H, CH3), 6.41 (s, 2H, NH2), 7.27 (s, 1H, H-6), 7.85 (d, J = 8.5 Hz, 1H,
aromatic CH), 8.12 (dd, J = 2.0 and 8.5 Hz, 1H, aromatic CH) 8.47 (d, J = 2.0 Hz, 1H, aromatic CH); 13C NMR
(101 MHz, DMSO-d6)
δ: 24.1, 107.6, 129.3, 130.7, 131.7, 131.0, 131.3, 133.7, 141.1, 155.7, 161.7, 167.1;
HRMS: m/z calcd for C12H9Cl2N5 294.0314 (M + H+), found 294.0315 (M + H+).
1
5-Methyl-7-(naphthalen-1-yl)-[1,2,4]triazolo[1,5-a]pyrimidin-2-amine (1i). White powder. H NMR (400
MHz, DMSO-d6) : 2.56 (s, 3H, CH3), 6.17 (s, 2H, NH2), 7.03 (s, 1H, H-6), 7.36 (d, J = 8.3 Hz, 1H, naphthalene
CH), 7.45-7.49 and 7.54-7.58 (m, each 1H, naphthalene CH), 7.62-7.69 (m, 2H, naphthalene CH), 8.03 and
8.12 (d, J = 8.0 Hz, each 1H, naphthalene CH); 13C NMR (101 MHz, DMSO-d6)
: 24.3, 110.0, 125.2, 125.5,
δ
δ
126.6, 127.3, 127.9, 128.5, 129.0, 129.8, 130.6, 133.0, 144.2, 155.1, 161.9, 167.1; HRMS: m/z calcd for
C16H13N5 276.125 (M + H+), found 276.1247 (M + H+).
1
5-Methyl-7-(pyridin-4-yl)-[1,2,4]triazolo[1,5-a]pyrimidin-2-amine (1k). White crystals. H NMR (400 MHz,
DMSO-d6)
δ: 2.54 (s, 3H, CH3), 6.42 (s, 2H, NH2), 7.30 (s, 1H, H-6), 8.10 and 8.80 (d, J = 4.6 Hz, each 2H,
pyridine CH); 13C NMR (101 MHz, DMSO-d6)
δ
: 24.2, 107.7, 123.0, 137.7, 141.1, 150.1, 155.6, 161.8, 167.2;
HRMS: m/z calcd for C11H10N6 227.1046 (M + H+), found 227.1044 (M + H+).
Ethyl 5-methyl-7-phenyl-[1,2,4]triazolo[1,5-a]pyrimidine-2-carboxylate (7a) and ethyl 7-methyl-5-phenyl-
[1,2,4]triazolo[1,5-a]pyrimidine-2-carboxylate (8a). The title compounds were prepared through the
general procedure for the synthesis of 2-amino-5-methyl[1,2,4]triazolo[1,5-a]pyrimidines by replacing 3a
with ethyl 5-amino-1,2,4-triazole-3-carboxylate (6a),15 and were separated by flash chromatography eluting
1
with CHCl3/acetone (9:1). 7a: white solid (63% yield); H NMR (400 MHz, DMSO-d6)
δ: 1.31 (t, J = 7.0 Hz,
CH2CH3), 2.69 (s, 3H, CH3), 4.38 (q, J = 7.0 Hz, CH2CH3), 7.63-7.65 (m, 4H, H-6 and aromatic CH), 8.07-8.09
(m, 2H, aromatic CH); 13C NMR (101 MHz, DMSO-d6)
: 14.1, 24.8, 61.7, 112.2, 128.7, 129.3, 129.5, 131.7,
δ
146.7, 155.6, 155.9, 160.0, 166.9; HRMS: m/z calcd for C15H14N4O2 283.1196 (M + H+), found 283.1196 (M +
1
H+); 8a: white solid (17% yield); H NMR (400 MHz, DMSO-d6)
δ: 1.35 (t, J = 7.0 Hz, CH2CH3), 2.84 (s, 3H,
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CH3), 4.41 (q, J = 7.0 Hz, CH2CH3), 7.59-7.60 (m, 3H, aromatic CH), 8.10 (s, 1H, H-6), 8.25-8.27 (m, 2H,
aromatic CH); 13C NMR (101 MHz, DMSO-d6)
: 14.1, 17.0, 61.7, 108.9, 127.7, 129.2, 131.7, 135.7, 149.1,
δ
155.0, 156.3, 159.9, 161.3; HRMS: m/z calcd for C15H14N4O2 283.1196 (M + H+), found 283.1194 (M + H+).
General procedure for the synthesis of 2-amino-5-aryl-7-methyl-[1,2,4]triazolo[1,5-a]pyrimidines (2a-i).
To a mixture of the appropriate 1-aryl-2-buten-1-ones (9a-i) (0.5 mmol) and 3a (1 mmol) in dry DMF (2.5
mL), dry Et3N (0.5 mmol) was added and the reaction mixture was heated at 110 °C until no starting
material was detected by TLC (2-3h). After cooling, the reaction mixture was poured into ice/water,
obtaining a precipitate that was filtered and crystallized by EtOH/DMF.
1
7-Methyl-5-phenyl-[1,2,4]triazolo[1,5-a]pyrimidin-2-amine (2a). Light yellow crystals. H NMR (400 MHz,
DMSO-d6) δ: 2.64 (s, 3H, CH3), 6.41 (s, 2H, NH2), 7.49-7.54 (m, 3H, aromatic CH), 7.59 (s, 1H, H-6), 8.13-8.15
(m, 2H, aromatic CH); 13C NMR (101 MHz, DMSO-d6)
δ: 17.1, 104.7, 127.0, 128.9, 130.4, 136.7, 145.4, 155.1,
156.8, 167.5; HRMS: m/z calcd for C12H11N5 226.1093 (M + H+), found 266.1088 (M + H+).
1
7-Methyl-5-p-tolyl-[1,2,4]triazolo[1,5-a]pyrimidin-2-amine (2b). Light yellow crystals. H NMR (400 MHz,
DMSO-d6) : 2.36 and 2.63 (s, each 3H, CH3), 6.38 (s, 2H, NH2), 7.32 (d, J = 6.0 Hz, each 1H, aromatic CH),
7.55 (s, 1H, H-6), 8.05 (d, J = 6.0 Hz, each 1H, aromatic CH); 13C NMR (101 MHz, DMSO-d6)
: 17.4, 21.3,
δ
δ
104.7, 127.2, 129.8, 134.2, 140.6, 145.7, 155.4, 157.2, 167.7; HRMS: m/z calcd for C13H13N5 240.1250 (M +
H+), found 240.1251 (M + H+).
1
5-(4-Methoxyphenyl)-7-methyl-[1,2,4]triazolo[1,5-a]pyrimidin-2-amine (2c). Yellow crystals. H NMR (400
MHz, DMSO-d6)
δ: 2.62 (s, 3H, CH3), 3.82 (s, 3H, OCH3), 6.34 (s, 2H, NH2), 7.06 (d, J = 8.8 Hz, 2H, aromatic
CH), 7.51 (s, 1H, H-6), 8.11 (d, J = 8.8 Hz, 2H, aromatic CH); 13C NMR (101 MHz, DMSO-d6)
δ: 17.0, 55.4,
104.0, 114.0, 128.6, 129.0, 145.2, 155.1, 156.7, 161.2, 167.3; HRMS: m/z calcd for C13H13N5O 256.1199 (M +
H+), found 256.1193 (M + H+).
1
7-Methyl-5-(4-nitrophenyl)-[1,2,4]triazolo[1,5-a]pyrimidin-2-amine (2d). Yellow crystals. H NMR (400
MHz, DMSO-d6) δ: 2.66 (s, 3H, CH3), 6.54 (s, 2H, NH2), 7.71 (s, 1H, H-6), 8.33 and 8.38 (d, J = 8.9 Hz, each 2H,
aromatic CH); 13C NMR (101 MHz, DMSO-d6)
δ: 17.3, 105.7, 124.2, 128.3, 142.7, 146.0, 148.4, 154.2, 155.1,
167.9; m/z calcd for C12H10N6O2 271.0944 (M + H+), found 271.0944 (M + H+).
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1
5-(4-Chlorophenyl)-7-methyl-[1,2,4]triazolo[1,5-a]pyrimidin-2-amine (2e). Yellow crystals. H NMR (400
MHz, DMSO-d6) δ: 2.64 (s, 3H, CH3), 6.45 (s, 2H, NH2), 7.57-7.61 (m, 3H, aromatic CH and H-6), 8.17 (d, J =
8.4 Hz, 2H, aromatic CH); 13C NMR (101 MHz, DMSO-d6)
δ: 17.1, 104.6, 128.8, 129.0, 135.2, 135.5, 145.6,
155.0, 155.4, 167.6; HRMS: m/z calcd for C12H10ClN5 260.0704 (M + H+), found 260.0681 (M + H+).
1
5-(3-Bromophenyl)-7-methyl-[1,2,4]triazolo[1,5-a]pyrimidin-2-amine (2f). Light brown crystals. H NMR
(400 MHz, DMSO-d6)
δ: 2.63 (s, 3H, CH3), 6.42 (s, 2H, NH2), 7.47 (t, J = 7.8 Hz, 1H, aromatic CH), 7.61 (s, 1H,
H-6), 7.67 and 8.12 (d, J = 7.8 Hz, each 1H, aromatic CH), 8.29 (s, 1H, aromatic CH); 13C NMR (101 MHz,
DMSO-d6)
δ: 17.3, 105.2, 122.6, 126.2, 129.7, 131.4, 133.3, 139.1, 146.1, 155.2, 155.4, 167.7; HRMS: m/z
calcd for C12H10BrN5 304.0199 (M + H+), found 304.0194 (M + H+).
1
5-(2-Fluorophenyl)-7-methyl-[1,2,4]triazolo[1,5-a]pyrimidin-2-amine (2g). Yellow crystals. H NMR (400
MHz, DMSO-d6) : 2.66 (s, 3H, CH3), 6.49 (s, 2H, NH2), 7.33-7.51 (m, 3H, aromatic CH and H-6), 7.52-7.57 (m,
1H, aromatic CH), 7.97 (dt, J = 1.5 and 7.8 Hz, 1H, aromatic CH); 13C NMR (101 MHz, DMSO-d6)
: 17.1, 108.2
δ
δ
(d, J = 10.1 Hz), 116.5 (d, J = 22.2 Hz), 125.0, 125.1 (d, J = 22.2 Hz), 130.9 (d, J = 3.0 Hz), 132.1 (d, J = 8.0 Hz),
145.2, 153.4, 154.9, 160.0 (d, J = 251.4 Hz), 167.6; HRMS: m/z calcd for C12H10FN5 244.0999 (M + H+), found
244.0997 (M + H+).
5-(2,4-Difluorophenyl)-7-methyl-[1,2,4]triazolo[1,5-a]pyrimidin-2-amine (2h). Light yellow crystals. 1H
NMR (400 MHz, DMSO-d6)
δ
: 2.65 (s, 3H, CH3), 6.49 (s, 2H, NH2), 7.24-7.29 (m, 2H, aromatic CH), 7.31 (s, 1H,
: 17.1, 104.9 (t, JC-F
H-6), 7.41-7.47 and 8.02-8.08 (m, each 1H, aromatic CH); 13C NMR (101 MHz, DMSO-d6)
δ
= 26.7 Hz), 107.9 (d, JC-F = 10.1 Hz), 112.4 (dd, JC-F = 3.0 and 21.2 Hz), 122.0 (dd, JC-F = 4.0 and 13.1 Hz), 132.4
(dd, JC-F = 4.0 and 11.1 Hz), 145.3, 152.5, 154.9, 160.3 (dd, JC-F = 13.1 and 251.4 Hz), 163.2 (dd, JC-F = 13.1 and
252.5 Hz), 167.3; HRMS: m/z calcd for C12H9F2N5 262.0905 (M + H+), found 262.0899 (M + H+).
1
7-Methyl-5-(pyridin-4-yl)-[1,2,4]triazolo[1,5-a]pyrimidin-2-amine (2i). Yellow crystals. H NMR (400 MHz,
DMSO-d6) δ: 2.68 (s, 3H, CH3), 6.56 (s, 2H, NH2), 7.73 (s, 1H, H-6), 8.07 and 8.73 (dd, J = 1.6 and 4.6 Hz, each
2H, pyridine CH); 13C NMR (101 MHz, DMSO-d6)
δ: 17.2, 105.2, 120.9, 143.7, 145.9, 150.5, 154.0, 155.0,
167.8; HRMS: m/z calcd for C11H10N6 227.1046 (M + H+), found 277.1041 (M + H+).
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General procedure for the synthesis of compounds 12-16 by amidation. A solution of benzoyl chloride (2.0
mmol) in dry pyridine (5 mL) was added dropwise to a solution of the appropriate [1,2,4]triazolo[1,5-
a]pyrimidine-2-amine (1e, 1f, 2c, 2d, or 2e) (1.0 mmol) in dry pyridine (15 mL), and then the reaction
mixture was maintained at 80 °C overnight. After cooling, it was poured into ice/water, obtaining a
precipitate that was filtered and purified as described below.
N-(7-(2-chlorophenyl)-5-methyl-[1,2,4]triazolo[1,5-a]pyrimidin-2-yl)benzamide (12). The title compound
was prepared starting from 1e and purified by flash chromatography eluting with CH2Cl2/MeOH (98:2) in
1
71% yield as white solid. H NMR (400 MHz, DMSO-d6)
δ: 2.67 (s, 3H, CH3), 7.34 (s, 1H, H-6), 7.46-7.50 and
7.57-7.58 (m, each 2H, aromatic CH), 7.64 (t, J = 7.2 Hz, 1H, aromatic CH), 7.70-7.72 (m, 2H, aromatic CH),
7.96 (d, J = 7.5 Hz, 2H, aromatic CH), 11.30 (s, 1H, NH); 13C NMR (101 MHz, DMSO-d6)
: 24.6, 112.0, 127.5,
δ
128.0, 128.3, 129.6, 129.7, 131.5, 132.1, 132.3, 132.3, 133.4, 143.9, 154.0, 160.1, 164.6, 164.8.; HRMS: m/z
calcd for C19H14ClN5O 364.0966 (M + H+), found 364.0965 (M + H+).
N-(5-methyl-7-(3-(trifluoromethyl)phenyl)-[1,2,4]triazolo[1,5-a]pyrimidin-2-yl)benzamide (13). The title
compound was prepared starting from 1f and purified by flash chromatography eluting with CH2Cl2/MeOH
1
(98:2) in 45% yield as white solid. H NMR (400 MHz, DMSO-d6)
δ
: 2.67 (s, 3H, CH3), 7.48-7.52 (m, 2H,
aromatic CH), 7.57-7.61 (m, 1H, aromatic CH), 7.63 (s, 1H, H-6), 7.87 (t, J = 7.8 Hz, 1H, aromatic CH), 7.99-
8.01 (m, 3H, aromatic CH), 8.51 (d, J = 7.9 Hz, 1H, aromatic CH), 8.59 (s, 1H, aromatic CH), 11.34 (s, 1H, NH);
13C NMR (101 MHz, DMSO-d6)
δ: 24.5, 110.3, 123.8 (q, J = 273.7 Hz), 126.2 (q, J = 3.0 Hz), 127.9 (q, J = 3.0
Hz), 128.0, 128.3, 129.4 (q, J = 32.3 Hz), 129.8, 130.7, 132.1, 133.4, 133.5, 154.6, 159.9, 164.6, 164.8; HRMS:
m/z calcd for C20H14F3N5O 398.1229 (M + H+), found 398.1227 (M + H+).
N-(5-(4-methoxyphenyl)-7-methyl-[1,2,4]triazolo[1,5-a]pyrimidin-2-yl)benzamide
compound was prepared starting from 2c and purified by flash chromatography eluting with
CH2Cl2/acetone (8:2) in 62% yield as white solid. 1H NMR (400 MHz, DMSO-d6)
: 2.78 and 3.84 (s, each 3H,
(14).
The
title
δ
CH3), 7.11 (d, J = 8.8 Hz, 2H), 7.50-7.54 (m, 2H, aromatic CH), 7.60 (d, J = 7.2 Hz, 1H, aromatic CH), 7.84 (s,
1H, H-6), 8.02 (d, J = 7.4 Hz, 2H, aromatic CH), 8.22 (d, J = 8.8 Hz, 2H, aromatic CH), 11.34 (s, 1H, NH); 13C
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NMR (101 MHz, DMSO-d6) δ: 17.4, 55.8, 106.6, 114.8, 128.4, 128.8, 128.8, 129.4, 132.5, 134.0, 147.7, 154.5,
159.4, 160.5, 162.1, 165.2; HRMS: m/z calcd for C20H17N5O2 360.1461 (M + H+), found 360.1460 (M + H+).
N-(7-methyl-5-(4-nitrophenyl)-[1,2,4]triazolo[1,5-a]pyrimidin-2-yl)benzamide (15). The title compound
was prepared starting from 2d and purified by flash chromatography eluting with CH2Cl2/acetone (8:2) in
64% yield as white solid. 1H NMR (400 MHz, DMSO-d6)
7.60 (t, J = 7.2 Hz, 1H, aromatic CH), 8.01-8.03 (m, 3H, H-6 and aromatic CH), 8.39 and 8.46 (d, J = 8.8 Hz,
each 2H aromatic CH), 11.47 (s, 1H, NH); 13C NMR (101 MHz, DMSO-d6)
: 17.2, 107.6, 124.1, 128.1, 128.4,
δ: 2.82 (s, 3H, CH3), 7.50-7.54 (m, 2H, aromatic CH),
δ
128.6, 132.2, 133.6, 142.0, 148.4, 148.7, 153.9, 156.7, 160.9, 164.8; HRMS: m/z calcd for C19H14N6O3
375.1206 (M + H+), found 375.1201 (M + H+).
N-(5-(4-chlorophenyl)-7-methyl-[1,2,4]triazolo[1,5-a]pyrimidin-2-yl)benzamide (16). The title compound
was prepared starting from 2e and purified by flash chromatography eluting with CH2Cl2/acetone (8:2) in
52% yield as white solid. 1H NMR (400 MHz, DMSO-d6)
7.57-7.63 (m, 3H, aromatic CH), 7.90 (s, 1H, H-6), 8.00 and 8.23 (d, J = 8.2 Hz, each 2H, aromatic CH), 11.38
(s, 1H, NH); 13C NMR (101 MHz, DMSO-d6)
: 17.1, 106.8, 128.1, 128.4, 129.1, 129.1, 132.1, 133.6, 135.0,
δ: 2.78 (s, 3H, CH3), 7.48-7.52 (m, 2H, aromatic CH),
δ
136.0, 147.9, 153.9, 158.0, 160.5, 164.8; HRMS: m/z calcd for C19H13F2N5O 366.1167 (M + H+), found
C19H14ClN5O 364.0966 (M + H+), found 364.0963 (M + H+).
General procedure for the synthesis of compounds 19 and 20 by amidation. To a solution of the
appropriate [1,2,4]triazolo[1,5-a]pyrimidine-2-carboxylic acid (173a or 183a) (2 mmol) in well dry CH2Cl2 (20
mL), oxalyl chloride (12 mmol) was added and after 30 min dry DMF (2 drops) was added. After 2 h, the
reaction mixture was evaporated to dryness to give a residue that was dissolved in well dry CH2Cl2 and
added of the appropriate aniline (2 mmol) and DIPEA (2 mmol). The reaction was maintained at rt until no
starting material was detected by TLC (4h for 19 and 1h for 20). The work up of the reaction and compound
purification are reported below.
N-(2-carbamoylphenyl)-5-methyl-7-phenyl-[1,2,4]triazolo[1,5-a]pyrimidine-2-carboxamide (19). The
reaction mixture was evaporated to dryness and treated with ice/water obtaining a precipitate that was
filtered and purified by flash chromatography eluting with CH2Cl2/MeOH (98:2), to give 19 in 52% yield; 1H-
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NMR (DMSO-d6) δ: 2.70 (s, 3H, CH3), 7.20 and 7.57 (t, J = 7.5 Hz, each 1H, aromatic CH), 7.69-7.62 (m, 4H,
H-6 and aromatic CH), 7.75 (bs, 1H, CONH2), 7.84 (d, J = 7.7 Hz, 1H, aromatic CH), 8.25-8.19 (m, 2H,
aromatic CH), 8.33 (bs, 1H, CONH2), 8.66 (d, J = 8.2 Hz, 1H, aromatic CH), 13.14 (s, 1H, NH); 13C NMR (101
MHz, DMSO-d6) δ: 25.0, 111.9, 120.5, 120.8, 123.6, 128.9, 129.0, 129.4, 129.8, 132.0, 132.6, 138.7, 146.8,
155.8, 157.4, 158.6, 167.0, 170.6; HRMS: m/z calcd for C20H16N6O2 373.1414 (M + H+), found 373.1411 (M +
H+).
N-(2-carbamoylphenyl)-7-methyl-5-phenyl-[1,2,4]triazolo[1,5-a]pyrimidine-2-carboxamide (20). The
reaction mixture was filtered and the precipitate was washed with Et2O, and then purified by flash
chromatography eluting with CH2Cl2/MeOH (98:2), to give 20 in 59% yield as white solid. 1H NMR (400 MHz,
DMSO-d6)
1H, CONH2), 7.86 (d, J = 7.8 Hz, 1H, aromatic CH), 8.10 (s, 1H, H-6), 8.38 – 8.25 (m, 3H, aromatic CH and
CONH2), 8.73 (d, J = 8.3 Hz, 1H, aromatic CH, 13.16 (s, 1H, NH); 13C NMR (101 MHz, DMSO-d6)
: 17.0, 108.7,
δ: 2.88 (s, 3H, CH3), 7.22 (t, J = 7.5 Hz, 1H, aromatic CH), 7.57-7.60 (m, 4H, aromatic CH), 7.78 (bs,
δ
120.4, 120.6, 123.3, 127.7, 128.7, 129.2, 131.6, 132.3, 135.8, 138.6, 149.1, 154.9, 157.2, 158.9, 161.2,
170.4; HRMS: m/z calcd for C20H16N6O2 373.1414 (M + H+), found 373.1413 (M + H+).
Biological assays
Compounds and peptide. RBV (1-D-ribofuranosyl-1,2,4-triazole-3-carboxamide) was purchased from
Roche. Each test compound was dissolved in 100% DMSO. The PB11–15–Tat peptide was synthesized and
purified by the Peptide Facility of CRIBI Biotechnology Center (University of Padua, Padua, Italy). This
peptide corresponds to the first 15 amino acids of PB1 protein fused to a short sequence of HIV Tat protein
(amino acids 47−59), which allows the delivery into the cell.18
Cells and virus. Mardin-Darby canine kidney (MDCK) cells were grown in Dulbecco’s modified Eagle’s
medium (DMEM, Life Biotechnologies) supplemented with 10% (v/v) fetal bovine serum (FBS, Life
Technologies) and antibiotics (100 U/mL penicillin and 100 µg/mL streptomycin, Life Technologies). The
cells were maintained at 37 °C in a humidified atmosphere with 5% CO2. Influenza virus strain A/PR/8/34
(H1N1, Cambridge lineage) was kindly provided by P. Digard (Roslin Institute, University of Edinburgh,
United Kingdom).
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PA-PB1 interaction enzyme-linked immunosorbent assay (ELISA). The PA−PB1 interacꢀon was detected as
described,3c with some modifications.19 Briefly, 96-well microtiter plates (Nuova Aptca) were coated with
400 ng of 6His--PA(239−716) for 3 h at 37 °C and then blocked with 2% BSA (Sigma) in PBS for 1 h at 37 °C. The
6His--PA(239−716) protein was expressed in E. coli strain BL21(DE3)pLysS and purified as already
described.3cAfter washing, 200 ng of GST-PB1(1−25), or of GST alone as a control, in the absence or the
presence of test compounds at various concentrations, were incubated in serum-free DMEM O/N at room
temperature as described.19 Escherichia coli-expressed, purified GST and GST-PB1(1−25) proteins were
obtained as previously described.3c,20 After washing, the interaction between 6His--PA(239−716) and GST-
PB1(1−25) was detected with a horseradish peroxidase-coupled anti-GST monoclonal antibody (GenScript)
diluted 1:4,000 in PBS supplemented with 2% FBS. Following washes, the substrate
3,3′,5,5′tetramethylbenzidine (TMB, KPL) was added and absorbance was measured at 450 nm by an ELISA
plate reader (Tecan Sunrise™). Values obtained from the samples treated with only DMSO were used to set
as 100% of PA−PB1 interacꢀon.
Cytotoxicity assay. Cytotoxicity of compounds was tested in MDCK cells by the 3-(4,5-dimethylthiazol-2-yl)-
2,5-diphenyl tetrazolium bromide (MTT) method, as previously reported.3c,21 Briefly, MDCK cells (seeded at
density of 2 x 104 per well) were grown in 96-well plates for 24 h and then treated with serial dilutions of
test compounds, or DMSO as a control, in DMEM supplemented with 10% FBS. After incubation at 37 °C for
48 h, 5 mg/mL of MTT (Sigma) in PBS was added into each well and incubated at 37 °C for further 4 h.
Successively, a solubilization solution was added to lyse the cells and incubated O/N at 37 °C. Finally, optical
density was read at the wavelength of 620 nm on a microtiter plate reader.
Plaque reduction assay (PRA). The antiviral activity of test compounds against influenza A virus was tested
by PRA as previously described.3c MDCK cells were seeded at 5 x 105 cells/well into 12-well plates, and
incubated at 37°C for 24 h. The following day, the culture medium was removed and the monolayers were
first washed with serum-free DMEM and then infected with the flu A/PR/8/34 strain at 40 PFU/well in
DMEM supplemented with 1 μg/mL of TPCK-treated trypsin (Worthington Biochemical Corporation) and
0.14% BSA and incubated for 1 h at 37 °C. The influenza virus infection was performed in the presence of
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different concentrations of test compounds or solvent (DMSO) as a control. After virus adsorption, DMEM
containing 1 μg/mL of TPCK-treated trypsin, 0.14% BSA, 1.2% Avicel, and DMSO or test compounds was
added to the cells. At 48 h post-infection, cells were fixed with 4% formaldehyde and stained with 0.1%
toluidine blue. Viral plaques were counted, and the mean plaque number in the DMSO-treated control was
set at 100%.
Conflicts of interest
There are no conflicts of interest to declare.
Acknowledgements
This work was supported by University of Perugia, Ricerca di base 2015 – cod. 3RICBASEOT (to OT and SM),
by the University of Padova, Progetto di Ricerca di Ateneo 2014 grant n. CPDA141311 and Ex-60% (to AL),
and by Associazione Italiana per la Ricerca sul Cancro (AIRC), Investigator Grant n. 18855 (to AL).
Notes and references
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