1,2,4-Triazolo[1,5-a]pyrimidines: Efficient one-step synthesis and functionalization as influenza polymerase PA-PB1 interaction disruptors

Article abstract of DOI:10.1016/j.ejmech.2021.113494

In the search for new anti-influenza virus (IV) compounds, we have identified the 1,2,4-triazolo[1,5-a]pyrimidine (TZP) as a very suitable scaffold to obtain compounds able to disrupt IV RNA-dependent RNA polymerase (RdRP) PA-PB1 subunits heterodimerization. In this work, in order to acquire further SAR insights for this class of compounds and identify more potent derivatives, we designed and synthesized additional series of analogues to investigate the role of the substituents around the TZP core. To this aim, we developed four facile and efficient one-step procedures for the synthesis of 5-phenyl-, 6-phenyl- and 7-phenyl-2-amino-[1,2,4]triazolo[1,5-a]pyrimidines, and 2-amino-5-phenyl-[1,2,4]triazolo[1,5-a]pyrimidin-7-ol. Two analogues having the ethyl carboxylate moiety at the C-2 position of the TZP were also prepared in good yields. Then, the scaffolds herein synthesized and two previous scaffolds were functionalized and evaluated for their anti-IAV activity, leading to the identification of compound 22 that showed both anti-PA-PB1 (IC₅₀ = 19.5 μM) and anti-IAV activity (EC₅₀ = 16 μM) at non-toxic concentrations, thus resulting among the most active TZP derivatives reported to date by us. A selection of the synthesized compounds, along with a set of in-house available analogues, was also tested against SARS-CoV-2. The most promising compound 49 from this series displayed an EC₅₀ value of 34.47 μM, highlighting the potential of the TPZ scaffold in the search for anti-CoV agents.

Full text of DOI:10.1016/j.ejmech.2021.113494

European Journal of Medicinal Chemistry 221 (2021) 113494
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
1,2,4-Triazolo[1,5-a]pyrimidines: Efficient one-step synthesis and
functionalization as influenza polymerase PA-PB1 interaction
disruptors
,
,
Maria Chiara Pismataro a 1, Tommaso Felicetti a 1, Chiara Bertagnin b, Maria Giulia Nizi a,
Anna Bonomini b, Maria Letizia Barreca a, Violetta Cecchetti a, Dirk Jochmans c,
Steven De Jonghe c, Johan Neyts c, Arianna Loregian b, Oriana Tabarrini a,
,
*
Serena Massari a
a Department of Pharmaceutical Sciences, University of Perugia, Via Del Liceo 1, 06123, Perugia, Italy
b Department of Molecular Medicine, University of Padua, Via Gabelli 63, 35121, Padua, Italy
c KU Leuven, Department of Microbiology, Immunology and Transplantation, Rega Institute for Medical Research, Laboratory of Virology and Chemotherapy,
Herestraat 49, Box 1043, 3000, Leuven, Belgium
a r t i c l e i n f o
a b s t r a c t
Article history:
In the search for new anti-influenza virus (IV) compounds, we have identified the 1,2,4-triazolo[1,5-a]
pyrimidine (TZP) as a very suitable scaffold to obtain compounds able to disrupt IV RNA-dependent RNA
polymerase (RdRP) PA-PB1 subunits heterodimerization. In this work, in order to acquire further SAR
insights for this class of compounds and identify more potent derivatives, we designed and synthesized
additional series of analogues to investigate the role of the substituents around the TZP core. To this aim,
we developed four facile and efficient one-step procedures for the synthesis of 5-phenyl-, 6-phenyl- and
7-phenyl-2-amino-[1,2,4]triazolo[1,5-a]pyrimidines, and 2-amino-5-phenyl-[1,2,4]triazolo[1,5-a]pyr-
imidin-7-ol. Two analogues having the ethyl carboxylate moiety at the C-2 position of the TZP were also
prepared in good yields. Then, the scaffolds herein synthesized and two previous scaffolds were func-
tionalized and evaluated for their anti-IAV activity, leading to the identification of compound 22 that
Received 9 February 2021
Received in revised form
30 March 2021
Accepted 18 April 2021
Available online 26 April 2021
Keywords:
1,2,4-triazolo[1,5-a]pyrimidines
Antiviral agents
Influenza virus
showed both anti-PA-PB1 (IC50 ¼ 19.5
m
M) and anti-IAV activity (EC50 ¼ 16
mM) at non-toxic concen-
SARS-CoV-2
Protein-protein interaction
Influenza polymerase
PA-PB1 interaction
trations, thus resulting among the most active TZP derivatives reported to date by us. A selection of the
synthesized compounds, along with a set of in-house available analogues, was also tested against SARS-
CoV-2. The most promising compound 49 from this series displayed an EC50 value of 34.47 mM, high-
lighting the potential of the TPZ scaffold in the search for anti-CoV agents.
© 2021 Elsevier Masson SAS. All rights reserved.
1. Introduction
During pandemic events, since long time is needed to produce a
new vaccine, antivirals are an important weapon. To date, the
antiviral armamentarium to treat IV infections is limited to a few
licensed drugs belonging to M2 ion channel inhibitors, which are
no longer recommended [1,2], and NA inhibitors [3], towards
which, however, many IAV strains developed resistance and IBV
showed reduced sensitivity.
Searching for new anti-IV drugs, the viral RNA-dependent RNA
polymerase (RdRP) is a really attractive drug target since i) it plays a
key role in the biological processes of virus transcription, replica-
tion, and evolution [4,5], ii) its structure is highly conserved among
IAV, IBV, and ICV strains, and iii) no homologue has been found in
mammalian cells. Regarding its structure, RdRP is a heterotrimeric
Among the four influenza virus (IV) types A, B, C and D, IAV and
IBV pose a severe threat for human health, circulating among
humans and causing seasonal epidemics. Moreover, pandemic
outbreaks can occur if highly virulent and pathogenic IAV strains,
deriving from antigenic shift, cross the species barrier and spread
easily among human beings that are naïve to the novel strain.
* Corresponding author. Department of Pharmaceutical Sciences, University of
Perugia, 06123, Perugia, Italy.
E-mail address: serena.massari@unipg.it (S. Massari).
Co-first authors.
1
https://doi.org/10.1016/j.ejmech.2021.113494
0223-5234/© 2021 Elsevier Masson SAS. All rights reserved.

M.C. Pismataro, T. Felicetti, C. Bertagnin et al.
European Journal of Medicinal Chemistry 221 (2021) 113494
complex formed by the viral polymerase basic protein 1 (PB1),
polymerase basic protein 2 (PB2), and polymerase acidic protein
(PA) [6e9]. To date, three compounds targeting each of the three
polymerase subunits have been recently approved or in clinical
trials: the nucleoside analog favipiravir (T-705, Avigan®) [10,11]
approved in 2014 in Japan, the PA endonuclease inhibitor baloxavir
marboxil (Xofluxa™) [12,13] approved in 2018 in both Japan and
the USA, and the PB2 cap-binding inhibitor pimodivir (VX-787, JNJ-
63623872) [14,15] that was recently halted after phase 3 evaluation
[16].
In the search for new RdRP inhibitors, we pursued the approach
to interfere with the correct assembly of the RdRP subunits by
developing protein-protein interaction (PPI) inhibitors. In partic-
ular, we focused our attention on the identification of small mol-
ecules as disruptors of RdRP PA-PB1 complex formation [17e19].
Compounds that interfere with PA-PB1 interaction have two major
advantages over other classes of anti-IV compounds [20,21]. First,
residues of both PB1 and PA that are crucial for subunits interaction
are highly conserved among IAV strains [22], suggesting that PA-
PB1 interaction inhibitors are expected to show broad-spectrum
antiviral activity against several human and animal IAV strains,
including new pandemic strains. Moreover, IVs showed a limited
ability to compensate for mutations deliberately introduced into
the PAC or PB1N termini [23], thus PA-PB1 inhibitors should be less
prone to drug resistance, overcoming the main limitation that
characterizes the majority of antiviral drugs.
carbamoyl derivative 3 [24,25] as well as hybrid molecules 4 and 5
(Fig. 1) [19] born by merging the TPZ core with the cyclo-
hepthathiophene-3-carboxamide moiety, which showed the best
anti-PA-PB1 and anti-IV activity.
The synthesis of the above anti-IV TZPs required the preparation
of 2-amino-[1,2,4]triazolo[1,5-a]pyrimidine and ethyl [1,2,4]tri-
azolo[1,5-a]pyrimidine-2-carboxylate intermediates unsymmetri-
cally substituted at the C-5 and C-7 positions. Since their synthesis
was scarcely explored, we were also interested in developing
suitable procedures for their preparation. In particular, we recently
reported two facile and efficient one-step procedures for the
regioselective synthesis of 2-amino-5-methyl-7-phenyl-[1,2,4]tri-
azolo[1,5-a]pyrimidine (6) and its isomer 2-amino-7-methyl-5-
phenyl-[1,2,4]triazolo[1,5-a]pyrimidine (7) [25] (Fig. 2).
TZP is a privileged structure in chemistry and an important
scaffold in medicinal chemistry, mimicking a wide range of natural
building blocks and exhibiting different interesting biological activ-
ities. A number of biologically active compounds based on the TZP
structure has been reported [26,27], such as CNS agents, antihista-
minic, anticancer, anti-inflammatory, cardiovascular agents, adeno-
sine receptor antagonists, diuretics, anxiolytic agents, platelet
aggregation inhibitors, anxiolytic agents, bronchodilators, antimi-
crobials, antimalarial, and antivirals. Focusing on antiviral TZP-based
compounds, they have been developed against different viruses [28],
such as HIV, HCV, HBV, HSV, Chikungunya virus, DENV and IV.
Continuing our search for anti-IV compounds based on inno-
vative TZP cores, in this work, we determined the best reaction
conditions for the synthesis of 2-amino-7-phenyl-[1,2,4]triazolo
[1,5-a]pyrimidine (8), 2-amino-5-phenyl-[1,2,4]triazolo[1,5-a]py-
rimidine (9), 2-amino-6-phenyl-[1,2,4]triazolo[1,5-a]pyrimidine
(10) and 2-amino-5-phenyl-[1,2,4]triazolo[1,5-a]pyrimidin-7-ol
(11) (Fig. 2). Analogously to the previously reported C-2 ethyl
carboxylate analogues 12 and 13 [25], attempts to synthesize
compounds 14e17 (Fig. 2) were also done, successfully providing
compounds 14 and 16.
Our previous studies on the discovery of PA-PB1 inhibitors
focused on the optimization of hit compound 1 (Fig.1), identified by
a virtual screening on the PAc crystal structure [17]. Aromatization
of the [1,2,4]triazolo[1,5-a]pyrimidine (TZP) core as well as inver-
sion of the C-5/C-7 substituents led to the identification of deriv-
ative 2 (Fig.1) [19], which showed an improved ability to inhibit PA-
PB1 interaction and, above all, acquired anti-IV activity at non-toxic
concentrations. The inversion of amide bond allowed the func-
tionalization of the C-2 position of the TZP scaffold affording
a
Fig. 1. Examples of [1,2,4]triazolo[1,5-a]pyrimidine-based compounds showing anti-PA-PB1 and anti-IV activity. The IC50 value represents the compound concentration that
b
c
reduces the PA-PB1 complex formation by 50% (ELISA assay); the EC50 value represents the compound concentration that inhibits 50% of IAV replication (PRA assay); the CC50
value represents the compound concentration that inhibits 50% of cell viability (MTT assay).
2

M.C. Pismataro, T. Felicetti, C. Bertagnin et al.
European Journal of Medicinal Chemistry 221 (2021) 113494
Fig. 2. Structures of 1,2,4-triazolo[1,5-a]pyrimidines variously functionalized at the C-5, C-6 and C-7 positions useful for the synthesis of IV RdRP PA-PB1 inhibitors.
Then, scaffolds 8e14 and 16 were used for the preparation of PA-
PB1 inhibitors. In particular, three set of compounds (Fig. 3) were
synthesized and evaluated for their ability to inhibit the PA-PB1
interaction and IAV replication, with the aim to study the role of
the substituent at the C-2, C-5, C-6 and C-7 positions of the TZP
core. Additionally, some compounds herein reported, along with a
small set of in-house TZP compounds, were evaluated for the anti-
SARS-CoV-2 activity.
regioselective synthesis was reported in literature via reaction of
3,5-diamonotriazole (32) with 3-(dimethylamino)-1-phenyl-2-
propen-1-one (33) both in glacial acetic acid at reflux (72% yield)
[29] and in EtOH at reflux (100% yield) [30] (Scheme 1a).
Therefore, we initially repeated the reaction in both the solvents
and, for this purpose, compound 33 [31] was synthesized as re-
ported in literature, via reaction of acetophenone with dime-
thylformamide dimethyl acetal at 160 ꢀC for 15 min in a microwave
oven. In agreement with Ribeiro et al. [29], the reaction in glacial
acetic acid at reflux (entry 1, Table 1) furnished 8 in 72% yield. On
the other hand, differently from Menet et al. [30], the reaction in
EtOH at reflux (entry 2) did not finish after 24 h (also waiting until
48h) furnishing 8 only in 42% yield.
2. Results and discussion
2.1. Synthesis of 2-amino-[1,2,4]triazolo[1,5-a]pyrimidines
Based on these results, we repeated the reaction in glacial acetic
acid using 2 equiv. of 32 (entry 3) and we found that the reaction
Focusing the attention on the synthesis of isomer 2-amino-7-
phenyl-[1,2,4]triazolo[1,5-a]pyrimidine (8), we found that its
Fig. 3. Structure of the compounds 18e31 designed, synthesized and evaluated as inhibitors of PA-PB1 binding and IAV replication.
3

M.C. Pismataro, T. Felicetti, C. Bertagnin et al.
European Journal of Medicinal Chemistry 221 (2021) 113494
Scheme 1. Known procedures for the synthesis of 8 and 9.
Table 1
Optimization of reaction conditions for 8a.
Entry
Solvent
32 (equiv.)
Et3N (equiv.)
Temperature
Time (h)
Yield (%)b
1
2
3
4
5
6
acetic acid
EtOH
acetic acid
EtOH (0.7 mL of acetic acid)
DMF
DMF
1.5
1
2
1
2
no base
no base
no base
no base
1
reflux
reflux
reflux
reflux
110 ꢀC
110 ꢀC
1
24
40 min
16
24
72c
42d
85
47
18
2
3
24
45
a
The reaction was performed on 1.0 mmol scale of 32 in 2.5 mL of solvent at reflux.
Isolated yields.
b
c
Yield reported in literature ¼ 72% [29].
d
Yield reported in literature ¼ 100% [30].
was slightly more rapid (40 min) and, above all, more efficient (85%
yield). Then, to understand whether the acid environment is
essential for the outcome of the reaction, the experiment was
repeated in EtOH but adding a catalytic amount of glacial acetic acid
(entry 4). Results showed that the reaction led to the formation of 8
but less efficiently (47% yield) and much more slowly (16h) than
that in glacial acetic acid. Then, we also repeated the reaction under
basic conditions in the presence of different equiv. of Et3N in DMF at
110 ꢀC (entries 5 and 6). Both reactions did not finish after 24h (also
waiting until 48h), furnishing 8 in 18% and 45% yield by using 1
equiv. and 3 equiv. of Et3N, respectively. Thus, the best reaction
conditions to synthesize compound 8 are treating 32 (2 equiv.)
and 33 (1 equiv.) in glacial acetic acid at reflux for 40 min (entry 3).
Through this one-step procedure, compound 8 was regioselectively
obtained in 85% yield.
The same authors reporting the synthesis of 8 in glacial acetic
acid, also described the preparation of 2-amino-5-phenyl-[1,2,4]
triazolo[1,5-a]pyrimidine (9) via cyclocondensation of 32 with 3-
(dimethylamino)-1-phenyl-1-propanone hydrochloride (34) in
glacial acetic acid at reflux, followed by heteroaromatization using
NBS with a 29% overall yield (Scheme 1b) [29].
Thus, after preparation of compound 34 [32] via reaction of
acetophenone with paraformaldehyde, dimethylamine hydrochlo-
ride and a catalytic amount of HCl in EtOH at reflux, we attempted
4

M.C. Pismataro, T. Felicetti, C. Bertagnin et al.
European Journal of Medicinal Chemistry 221 (2021) 113494
the synthesis of 9 by following the two-step procedure reported by
Ribeiro et al. Interestingly, after the first reaction, we observed the
formation of a mixture of 5-phenyl-4,7-dihydro-[1,2,4]triazolo[1,5-
a]pyrimidin-2-amine and oxidized compound 9. Prompted by this
observation, we repeated the reaction in glacial acetic acid but in an
open flask in order to promote the oxidation reaction, analogously
to the synthesis of isomers 6 and 7 [25]. We observed an increase in
the amount of 9, although not reaching the complete oxidation of
the 4,7-dihydro intermediate. Thus, we repeated the reaction under
basic conditions by treating 32 (1 or 2 equiv.) and 34 (1 equiv.) in
the presence of Et3N (1 equiv.) in DMF at 110 ꢀC in an open flask
(entries 1 and 2, Table 2). We were pleased to find that these
conditions regioselectively furnished only the already oxidized
compound 9, with the reaction performed by using 2 equiv. of 32
that was much more efficient (78% yield) and rapid (3h) than that
performed by using 1 equiv. of 32 (56% yield after 5h). With these
encouraging results, then, we studied the effect of the temperature
(entry 3) or of the equiv. of Et3N (entry 4), maintaining 2 equiv. of 32
and performing the reaction in DMF in an open flask. Results
showed that the reaction performed at 120 ꢀC is less efficient (55%
yield) and rapid (6h) than that performed at 110 ꢀC. On the other
hand, the reaction performed with 2 equiv. of Et3N is equally rapid
(3h) and slightly less efficient (72% yield) than that performed with
1 equiv. of Et3N. Finally, maintaining the best reaction conditions (2
equiv. of 32 and 1 equiv. of base at 110 ꢀC in DMF in an open flask),
we studied the effects of two different bases such as K2CO3 and
Cs2CO3 (entries 5 and 6). Both reactions were equally rapid (5h), but
the reaction performed with K2CO3 as base was more efficient (72%
yield) than that performed with Cs2CO3 (50% yield). Thus, the op-
timum reaction conditions to synthesize compound 9 are treat-
ing 32 (2 equiv.) and 34 (1 equiv.) in DMF at 110 ꢀC in an open flask
for 3h in presence of Et3N (1 equiv.) (entry 2). Through this one-step
procedure, compound 9 was regioselectively obtained in 78% yield.
Then, we turned the attention to the synthesis of compounds 2-
amino-6-phenyl-[1,2,4]triazolo[1,5-a]pyrimidine (10) and 2-
amino-5-phenyl-[1,2,4]triazolo[1,5-a]pyrimidin-7-ol (11), whose
syntheses are not known in literature.
observed. Thus, we repeated the reaction under basic conditions
treating 32 (2 equiv.) and 35 (1 equiv.) in the presence of Et3N (1
equiv.) in i-PrOH at room temperature (rt) (entry 2). No starting
material conversion was observed because of the insolubility of 32
in this solvent (also heating at 50 ꢀC). Thus, we repeated the re-
action in DMF, also studying the effect of different bases such as
Et3N (entry 3), NaH (entry 4) and K2CO3 (entry 5). We were pleased
to find that all the reactions took place at rt rapidly (1h) and effi-
ciently (92%, 54%, and 80% yield, respectively). Finally, the reaction
was repeated in DMF in absence of a base (entry 6) furnishing 10 in
39% yield after 24h, thus showing that the basic environment
significantly improves the reaction yield. Thus, the optimum reac-
tion conditions to synthesize compound 10 are treating 32 (2
equiv.) and 35 (1 equiv.) in the presence of Et3N (1 equiv.) in DMF at
rt for 1h (entry 3). Through this one-step procedure,
compound 10 was obtained in 92% yield.
Finally, we searched for a synthetic procedure for the prepara-
tion of 2-amino-5-phenyl-[1,2,4]triazolo[1,5-a]pyrimidin-7-ol (11)
and carried out the synthesis via reaction of 32 with ethyl 3-oxo-3-
phenylpropanoate (36) (Table 4). Also in this case, we initially
performed the reaction in glacial acetic acid at reflux (entry 1), but
still no significant reaction was observed after 24h. Thus, we
repeated the reaction in DMF at 110 ꢀC studying the effect of
different bases such as NaH (entry 2), Et3N (entry 3) and K2CO3
(entry 4). No significant reaction was observed in presence of Et3N
or NaH after 24h, while, after 12h, the reaction in presence of K2CO3
led to the regioselective formation of 11 although in 40% yield. To
further explore the reaction conditions, the experiment was
repeated in EtOH using EtONa as base (entry 5) and in tBuOH using
tBuOK as base (entry 6) at reflux. Results showed that the reaction
in EtOH was more rapid (12h) and efficient (66%) than that in
tBuOH (24h, 54% yield). Based on this result, we then studied the
effect of the equiv. of 32 (entry 7), of EtONa (entry 8), or both (entry
9) in EtOH at reflux. Both reactions performed with 2 equiv. of
EtONa were less efficient furnishing 11 after 12h in 38 and 39%
yield, while the reaction performed with 1 equiv. of 32 resulted the
most efficient, yielding 11 after 12h in 79% yield. Thus, the best
reaction conditions to synthesize compound 11 are treating 32 (1
equiv.) and 36 (1 equiv.) in EtOH in the presence of EtONa (1 equiv.)
at reflux for 12h (entry 7). Through this one-step procedure,
compound 11 was regioselectively obtained in 79% yield.
Regarding the preparation of compound 10, we carried out the
synthesis via reaction of 32 with N-[3-(dimethylamino)-2-
phenylprop-2-en-1-ylidene]-N-methylmethanaminium perchlo-
rate (35) (Table 3). Compound 35 [33] was synthesized as reported
in literature via reaction of 2-phenylacetic acid with DMF and POCl3
at 90 ꢀC. We initially performed the reaction between 32 and 35 in
glacial acetic acid at reflux (entry 1), but no significant reaction was
It is worthwhile to underline that all the compounds 8e11 were
obtained pure from their relative procedures and did not require
purification.
Table 2
Optimization of reaction conditions for 9a.
Entry
Solvent
32 (equiv.)
Base
Temperature
Time (h)
Yield (%)b
1
2
3
4
5
6
DMF
DMF
DMF
DMF
DMF
DMF
1
2
2
2
2
2
Et3N, 1 equiv.
Et3N, 1 equiv.
Et3N, 1 equiv.
Et3N, 2 equiv.
K2CO3, 1 equiv.
Cs2CO3, 1 equiv.
110 ꢀC
110 ꢀC
120 ꢀC
110 ꢀC
110 ꢀC
110 ꢀC
5
3
6
3
5
5
56
78
55
72
72
50
a
The reaction was performed on 1.0 mmol scale of 32 in 2.5 mL of solvent in an open flask.
Isolated yields.
b
5

M.C. Pismataro, T. Felicetti, C. Bertagnin et al.
European Journal of Medicinal Chemistry 221 (2021) 113494
Table 3
Optimization of reaction conditions for 10a.
Entry
Solvent
32 (equiv.)
Base (equiv.)
Temperature
Time (h)
Yield (%)b
1
2
3
4
5
6
acetic acid
i-PrOH
DMF
DMF
DMF
1
2
2
2
2
2
no base
reflux
50 ꢀC
r.t.
r.t.
r.t.
24
e
1
1
1
e
Et3N, 1 equiv.
Et3N, 1 equiv.
NaH, 1 equiv.
K2CO3, 1 equiv.
no base
e
92
54
80
39
DMF
r.t.
24
a
The reaction was performed on 1.0 mmol scale of 32 in 2.5 mL of solvent at reflux.
Isolated yields.
b
Table 4
Optimization of reaction conditions for 11a.
Entry
Solvent
32 (equiv.)
Base
Temperature
Time (h)
Yield (%)b
1
2
3
4
5
6
7
8
9
acetic acid
DMF
DMF
DMF
EtOH
tBuOH
EtOH
EtOH
1
2
2
2
2
2
1
2
1
no base
reflux
110 ꢀC
110 ꢀC
110 ꢀC
reflux
reflux
reflux
reflux
reflux
24
24
24
12
12
24
12
12
12
e
NaH, 1 equiv.
Et3N, 1 equiv.
K2CO3, 1 equiv.
EtONa, 1 equiv.
tBuOK, 1 equiv.
EtONa, 1 equiv.
EtONa, 2 equiv.
EtONa, 2 equiv.
e
e
40
66
54
79
39
38
EtOH
a
The reaction was performed on 1.0 mmol scale of 32 in 2.5 mL of solvent at reflux.
Isolated yields.
b
Regarding structural information, isomers 8 and 9 were previ-
2.2. Reactivity of 3,5-diamino-1,2,4-triazole toward ambident
electrophiles such as -diketones, -enones, -enaminones,
amino ketones and -ketoesters
ously described in literature [29], with the structure of compound 8
that was confirmed by crystallographic studies and was distin-
guished by that of compound 9 by NMR spectroscopy on the basis
of the chemical shifts of the pyrimidine hydrogens (7.25 (H-6) and
8.53 (H-5) ppm for 8 and 7.72 (H-6) and 9.07 (H-7) ppm for 9) and
the coupling constant of pyrimidine hydrogens (J ¼ 4.8 Hz for 8 and
J ¼ 6.9 Hz for 9). On the other hand, compounds 10 and 11 herein
reported were not described previously and their structures were
fully characterized by spectral data of 1H NMR, 13C NMR, and HRMS.
In order to demonstrate the correct regiosimoeric structure of
compound 11 (C-5 -Ph, C-7 eOH), by comparing its 13C NMR
spectrum to that of the analogue devoid of the amino group at the
C(2) position [34], it is essential to consider the chemical shift of
C(6) that is very similar for both 11 (98.4 ppm) and its close
analogue (97.3 ppm); on the other hand, the chemical shift of C(6)
of the 7-phenyl analogue devoid of the amino group at the C(2)
position is 106.8 [35].
b
a,
b
b
b-
b
Based on the regioselectivity of the above reported reactions
and those previously reported by us, we analyzed the reactivity of
3,5-diamino-1,2,4-triazole (32) towards different ambident elec-
trophiles (Fig. 4). About the reactivity of 32, we can assume that the
presence of a second electron-donating amino group on the 1,2,4-
triazole might be responsible for a higher nucleophilicity of the
C(3) amino group than the N(2). Considering this hypothesized
reactivity and based on the regioselectivity of previously reported
reactions [25], it can be assumed that, when reacting with a b-
diketone such 37, the triazole C(3) amino group undergoes a direct
addition at the C(3) carbonyl carbon and not at the C(1) carbonyl
carbon of 37, probably due to the steric hindrance of the phenyl
group at the C(1). Analogously, when 32 reacts with
a,b-enones
such as 38 and 39, independently from the hindrance of the
6

M.C. Pismataro, T. Felicetti, C. Bertagnin et al.
European Journal of Medicinal Chemistry 221 (2021) 113494
Fig. 4. Plausible reactivity of 3,5-diamino-1,2,4-triazole (32) toward ambident electrophiles such as b-diketone (37), a,b-enones (38 and 39), b-enaminone (33), b-amino ketone (34)
and b-ketoester (36).
substituent on the carbonyl carbon, the triazole C(3) amino group
seems to undergo the direct addition at the carbonyl carbon (C(2) of
the reaction between 40 and 35 afforded the target compound 16,
although in 34% yield. However, the use of microwave irradiation
improved the yield up to 70%, also reducing the time (40 min).
Prompted from this observation, we also repeated the reaction
between 40 and 33 in glacial acetic acid under microwave irradi-
ation, but compound 14 was obtained in a lower yield (58%) than
when using traditional heating. To complete the study, we also
reacted 40 and 33 in DMF and Et3N, observing that a basic envi-
ronment strongly impaired the reaction yield (20%).
Of note, the scarce reactivity of 40 toward ambident electrophiles,
due to the presence of an electron-withdrawing group at the C-2
position, was already observed by us during the preparation of ethyl
5-methyl-7-phenyl-[1,2,4]triazolo[1,5-a]pyrimidine-2-carboxylate
(12) and 7-methyl-5-phenyl-[1,2,4]triazolo[1,5-a]pyrimidine-2-
carboxylate (13) (Scheme 2b) [25]. In particular, the reaction of 40
38 and C(1) of 39) [25], instead of a conjugated addition at the
carbon (C(4) of 38 and C(3) of 39).
b-
Nevertheless, when 32 reacts with a
the triazole C(3) amino group seems to prefer to undergo nucleo-
philic substitution at the -carbon instead of the direct addition at
the C(1) carbonyl carbon. Obviously, in the reaction of 32 with a
b-enaminone such as 33,
b
b
-
amino ketone such as 34, the triazole C(3) amino group conceivably
undergoes a nucleophilic addition at the C(1) carbonyl carbon of 34.
Finally, it is worth to note that when reacting with a b-ketoester
such as 36, triazole C(3) amino group seems to prefer attacking the
C(3) carbonyl carbon instead of the C(1) carbonyl carbon under-
going a direct addition rather than a nucleophilic substitution.
2.3. Synthesis of ethyl [1,2,4]triazolo[1,5-a]pyrimidine-2-
carboxylates
with b-diketone 37 in glacial acetic acid at reflux was rapid (4h) and
efficient (80% yield) but showed a dramatically decreased regiose-
lectivity, furnishing 12 and 13 in the ratio of 3.7:1. On the other hand,
To explore if the best reaction conditions determined for the
synthesis of 8e11 could be applied for the preparation of ethyl
[1,2,4]triazolo[1,5-a]pyrimidine-2-carboxylate analogues 14e17,
we repeated the reactions starting from ethyl 5-amino-1,2,4-
triazole-3-carboxylate (40) (Scheme 2a). Results showed that the
reaction of 40 with 33 in glacial acetic acid at reflux was equally
rapid (40 min) as that starting from 3,5-diaminotriazole (32),
furnishing compound 14 only slightly less efficiently (75% yield). On
the contrary, the reactions of 40 with 34 and 35 in dry DMF at 110 ꢀC
and in the presence of Et3N did not finish after 24h, furnishing, the
first, a mixture of several products containing 15 only in traces and,
the second, compound 16 in 23% yield. Finally, no starting material
conversion was observed by reacting 40 with 36 in EtOH at reflux
and using EtONa as a base.
the reaction of 40 with a,b
-enones 38 and 39 (in DMF at 110 ꢀC in the
presence of Et3N and in an open vessel) furnished after 24h isomers
12 and 13 in traces and 19% yield, respectively.
2.4. Design of antiviral compounds based on the [1,2,4]triazolo[1,5-
a]pyrimidine structure
As a continuation of our previous efforts in identifying anti-IAV
compounds able to inhibit RdRP PA-PB1 subunits interaction, in
this work, we have synthesized additional analogues to better
investigate the role of the substituents on the TZP core. Previously,
starting from hit compound 1 [17], we synthesized a series of TZP
analogues, mainly as hybrid compounds in which the TZP nucleus
was joined with a thiophene-based core [19,24], while to date few
biological data are available about the sole TZP nucleus [19,25].
Therefore, herein, about the TZP C-2 position, we studied the
role of the 2-carbamoyl moiety on the (hetero)aromatic ring, which
To verify if acidic conditions could differently impact on reaction
performance to obtain derivatives 15e17, compound 40 was reac-
ted with 34e36 in glacial acetic acid at reflux. Unfortunately, only
7

M.C. Pismataro, T. Felicetti, C. Bertagnin et al.
European Journal of Medicinal Chemistry 221 (2021) 113494
Scheme 2. Synthesis of 14e17 via reaction of 40 with 33e36, respectively, and known procedures for the synthesis of 12 and 13. *Isolated yields.
previously emerged as important to achieve anti-PA-PB1 activity
[19,24] in the 5-methyl-7-phenyl- and 7-methyl-5-phenyl-[1,2,4]
triazolo[1,5-a]pyrimidine-2-carboxyamide series. In particular, the
carbamoyl moiety was replaced by a bioisosteric carboxylic acid in
compounds 18 and 19, also evaluating methyl ester intermediates
20 and 21. Moreover, the amide linkage and the C-2 carboxylic
group of compounds 18 and 19 were constrained into a benzo[d]
[1,3]oxazin-4-one ring, giving derivatives 22 and 23.
Focusing the attention on the C-5, C-6, and C-7 positions of the
TZP core, the presence of a sole phenyl ring in each of these posi-
tions was explored by using scaffolds 8e11, which were function-
alized with an unsubstituted benzoyl moiety at the C-2 position,
analogously to hit compound 2. Compounds 24e26 were thus
prepared along with compound 27, where the 5-phenyl ring was
coupled with a hydroxyl group at the C-7 position.
Unfortunately, synthetic issues impaired the functionalization of
scaffolds 8e11 with a 2-carbamoylphenyl moiety, thus, we decided
to use an inverse amide to introduce this moiety.
As reported above, we were able to synthesize only ethyl [1,2,4]
triazolo[1,5-a]pyrimidine-2-carboxylate 14 and 16, which were
functionalized to give compounds 28 and 29, respectively. Addi-
tionally, analogues 30 and 31 were prepared, selecting as C-2
substituent the cycloheptathiophene-3-carboxamide moiety char-
acterizing compounds 4 and 5.
3, by coupling reaction of 5-methyl-7-phenyl-[1,2,4]triazolo[1,5-a]
pyrimidine-2-carbonyl chloride 41 [19] and 7-methyl-5-phenyl-
[1,2,4]triazolo[1,5-a]pyrimidine-2-carbonyl chloride 42 [19] with
methyl 2-aminobenzoate in CH2Cl2 in presence of DIPEA at rt, to
give methyl esters 20 and 21. The successive hydrolysis of com-
pounds 20 and 21 in the presence of LiOH in H2O/THF mixture at
50 ꢀC furnished acid derivatives 18 and 19, of which 19 was in turn
cyclized in acetic anhydride at 100 ꢀC furnishing benzooxazinone
derivative 23. Actually, the synthesis of compounds 22 and 23 was
initially attempted via reaction of 41 [19] and 42 [19] with 2-
aminobenzoic acid in CH2Cl2 in presence of DIPEA at rt, but only
compound 22 was successfully obtained, while the reaction start-
ing from 42 furnished only acid intermediate 19.
Compounds 24e27 were synthesized via reaction of 8e11 with
benzoyl chloride in pyridine at 80 ꢀC, as reported in Scheme 4.
Finally, target compounds 28e31 were synthesized starting from
compounds 14 and 16, which were hydrolysed under basic condi-
tions, to give intermediates 43 and 44, chlorinated, and reacted
with 2-aminobenzamide or 2-amino-5,6,7,8-tetrahydro-4H-cyclo-
hepta[b]thiophene-3-carboxamide in CH2Cl2 in the presence of
DIPEA (Scheme 4).
2.6. Evaluation of antiviral activity
Compounds 18e31 were evaluated for the ability to inhibit the
physical interaction between IAV PA and PB1 subunits by ELISA
including the Tat-PB11-15 peptide as a positive control of inhibition.
In parallel, for all the synthesized compounds, the antiviral activity
2.5. Synthesis of the target compounds 18-31
Compounds 18 and 19 were synthesized, as reported in Scheme
8

M.C. Pismataro, T. Felicetti, C. Bertagnin et al.
European Journal of Medicinal Chemistry 221 (2021) 113494
Scheme 3. Synthesis of target compounds 18e23. Reagents: i) methyl 2-aminobenzoate, DIPEA, CH2Cl2, rt; ii) LiOH, H2O/THF (1:1), 50 ꢀC; iii) Ac2O, 100 ꢀC; iv) 2-aminobenzoic acid,
DIPEA, CH2Cl2, rt.
Scheme 4. Synthesis of target compounds 24e31. Reagents: i) pyridine 80 ꢀC; ii) NaOH, MeOH, reflux; iii) oxalyl chloride, CH2Cl2, DMF, rt; iv) 2-aminobenzamide or 2-amino-
5,6,7,8-tetrahydro-4H-cyclohepta[b]thiophene-3-carboxamide, DIPEA, CH2Cl2, rt.
was tested by plaque reduction assays (PRA) in Madin-Darby canine
kidney (MDCK) cells infected with a reference IAV, the A/PR/8/34
strain. Ribavirin (RBV), a known broad-spectrum inhibitor of RNA
viruse polymerases, 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, compounds 24e27 were devoid of both the
abilities to dissociate PA-PB1 interaction and inhibit viral
replication.
Finally, efficient ability to disrupt PA-PB1 heterodimerization
was also shown by four of the six compounds based on scaffolds 6
and 7, with IC50 values ranging from 19.5 to 32 mM. Also in this case,
the compounds did not show antiviral activity, with the exception
of compound 22 that besides displaying the best anti-PA-PB1 ac-
tivity (IC50 ¼ 19.5
tivity (EC50 ¼ 16
most active TZP derivative reported to date by us. Of note, all
compounds 18e31 were non-toxic up to 250 M concentration
(Figure S4, for compound 22), with the exception of compound 31
that showed mild cytotoxicity (CC50 ¼ 100 M). Based on these
m
M, Figure S1), also showed a good anti-IAV ac-
mM, Figure S2 and S3), thus resulting among the
m
On the other hand, when the TZP scaffolds having a C-6 or C-7
phenyl ring and the inverse amide were functionalized with a 2-
carbamoylphenyl moiety at the C-2 position (compounds 28 and
29), efficient inhibition of PA-PB1 interaction was achieved
m
data, some SAR insights can be drawn: i) the 7-methyl-5-phenyl-
and 5-methyl-7-phenyl-TZP series were better than compounds
having only a phenyl ring at the C-5, C-6 or C-7 positions of the TZP
core; ii) among the 7-methyl-5-phenyl-TZP and 5-methyl-7-
phenyl-TZP series, the activity of the compounds is strictly
dependent on the substituent at the C-2 position of the TZP nu-
cleus; iii) focusing on the C-2 substituent, the 2-carbamoylphenyl
ring was the best within the 7-methyl-5-phenyl-TZP series, while
(IC50
(IC50 ¼ 22
¼
21
mM). Of note, the same anti-PA-PB1 activity
mM) was shown by 6-phenyl TZP compound 31 func-
tionalized at the C-2 position with a cycloheptathiophene-3-
carboxamide moiety. Nevertheless, all compounds 28e31 were
devoid of antiviral activity.
9

Table 5
Structure and Anti-IAV Activity of [1,2,4]triazolo[1,5-a]pyrimidine derivatives.
Compd
R2
R5
R7
ELISA
PRA in MDCK cells EC50,
m
Mb
Cytotoxicity in MDCK cells CC50, m
Mc
PA-PB1
Interaction Assay IC50, m
Ma
18
CH3
C6H5
116 43
>100
>250
19
20
“
C6H5
CH3
CH3
C6H5
32 14
26.5 2.1
>100
>80
>250
>250
21
22
“
“
C6H5
CH3
CH3
C6H5
22.3 6.4
19.5 4.9
>100
16 5.7
>250
>250
23
C6H5
CH3
123 45
>100
>200
Compd
R5
R6
R7
ELISA
PA-PB1
PRA in MDCK cells
Cytotoxicity (MTT Assay) in MDCK cells
EC50,
m
Mb
CC50, m
Mc
Interaction Assay
IC50, m
Ma
24
25
26
H
C6H5
H
H
H
C6H5
C6H5
H
H
>200
165
>100
>100
>250
>250
>200
113 46
>100
>100
>250
>250
27
C6H5
H
OH
Compd
28
R2
R6
R7
ELISA
PA-PB1
PRA in MDCK cells
Cytotoxicity (MTT Assay) in MDCK cells
EC50,
m
Mb
CC50, m
Mc
Interaction Assay
IC50
, m
Ma
H
C6H5
21 4.2
>100
>250
29
30
“
“
C6H5
H
H
C6H5
21
>200
8
>100
>60
>250
>200
31
C6H5
H
22
35
3
4
>100
100
9
Tat-PB11-15
peptide
RBV
41
10
5
2
>100
>250
a
Activity of the compounds in ELISA PA-PB1 interaction assays. The IC50 value represents the compound concentration that reduces by 50% the interaction between PA and
PB1.
b
Activity of the compounds in plaque reduction assays with the IAV A/PR/8/34 strain. The EC50 value represents the compound concentration that inhibits 50% of plaque
formation.
c
Cytotoxicity of the compounds in MTT assays. The CC50 value represents the compound concentration that causes a decrease of cell viability by 50%. All the reported values
represent the means SD of data derived from at least three independent experiments in duplicate.
10

M.C. Pismataro, T. Felicetti, C. Bertagnin et al.
European Journal of Medicinal Chemistry 221 (2021) 113494
within the 5-methyl-7-phenyl-TZP series, the benzooxazinone
imparted the best activity thus emerging as a new moiety to be
further investigated; iv) regarding the amide linkage, an inverse
amide permitted a wider functionalization of the C-2 TZP, which
instead was not permitted staring from a 2-amino-TZP, thus it was
not possible to determine the best linkage by comparing strict
analogues.
Then, we investigated the ability of compound 22 to interfere
with the catalytic activity of IAV RdRP in a cellular context by a
minireplicon assay in HEK 293T cells. Compound 22 exhibited an
a]pyrimidines, and 2-amino-5-phenyl-[1,2,4]triazolo[1,5-a]pyr-
imidin-7-ol have been developed, under mild conditions, with high
yields (ranging from 85% to 92%), and regioselectivity for com-
pounds 8, 9 and 11. Of note, the one-step procedure used for the
synthesis of 9 resulted much more efficient (78%) than the two-
steps procedure reported in literature (29%), but, above all, avoi-
ded the oxidation reaction by NBS or Br2, which being highly
reactive limit the choice of substituents on the TZP. The synthesis of
the four analogues having the ethyl carboxylate moiety at the C-2
position of the TZP structure was also attempted, leading to syn-
thesize 7-phenyl derivative 14 and 6-phenyl derivative 16 in good
yields (75% and 70%, respectively).
EC50 of 21
3
mM
(Figure S5) at non-toxic concentration
(CC50 > 150
mM), thus confirming its ability to inhibit RdRP func-
tions by interfering with PA-PB1 heterodimerization; RBV was used
as reference control (EC50 ¼ 16 M, CC50 > 250 M).
Subsequently, the synthesized scaffolds have been used for the
synthesis of a set of TZP derivatives as anti-IAV compounds acting
by disrupting viral RdRP PA-PB1 subunits interaction. From the
biological evaluation, compound 22 inhibited PA-PB1 interaction
2
m
m
In the course of this work, the global population has been
affected by the COVID-19 pandemic, caused by the severe acute
respiratory syndrome coronavirus 2 (SARS-CoV-2). Developing new
treatments is typically a lengthy process, while repurposing exist-
ing compounds is a potential accelerated way to find an urgently
needed treatment. Having in house a library of drug-like com-
pounds, of which many were developed as antiviral agents, a set of
31 TZP-based compounds, including seven of the derivatives herein
reported (compounds 19, 21e23, 25, 26 and 31), was also evaluated
for their activity against SARS-CoV-2. In a first round of screening,
all compounds were evaluated in duplicate at 4 concentrations
with an IC50 of 19.5
viral replication with an EC50 of 16
icity up to 250 M.
m
M, RdRP functions with an EC50 of 21
mM and
mM, and not showing cytotox-
m
Although good results have been achieved in this work, further
efforts are required to enhance the antiviral potency and better
characterize PA-PB1 inhibitory properties through: i) co-
crystallization studies and mutagenesis experiments in order to
guide the hit-optimization; ii) co-administration with approved
agents; and iii) the evaluation of the anti-IAV activity in an animal
model.
(100, 20, 4, and 0.8
mM) as potential inhibitors of SARS-CoV-2
replication in a cell context. Initially, only antiviral activity, but no
cytotoxicity was measured. From this initial screening, five TZPs
(compounds 45e49) emerged as weak inhibitors of SARS-CoV-2,
Interestingly, the TZP derivative 49 also showed anti-SARS-CoV-
2 activity with an EC50 value of 34.47 mM and lacking cytotoxicity on
Vero E6 cells. This activity could be due to a mechanism of action
involving a PPI inhibition, as suggested from its ability to interfere
with extrapolated EC50 values in the range of 30e65
mM
(Table 6). One analogue from this series (compound 49) was sub-
jected to a full dose-response analysis (Figure S6) with simulta-
neous determination of the cytotoxicity in Vero E6 cells. This
compound showed anti-SARS-CoV-2 activity with an EC50 value of
with PA-PB1 heterodimerization (IC50 ¼ 17.5
mM). Further studies
will be devoted to investigating this hypothesis.
4. Experimental section
34.47
mM and lacked cytotoxicity at the highest tested concentra-
tion (i.e. 100
mM). GS-441524, the parent nucleoside analogue of
4.1. Chemistry
remdesivir [36], was included as positive control and reference
compound. This compound was endowed with an EC50 and CC50
values of 0.89 mM and 72.38 mM, respectively.
Commercially available starting materials, reagents, and sol-
vents were used as supplied. Compounds 3-(dimethylamino)-1-
phenyl-2-propen-1-one (33) [31], 3-(dimethylamino)-1-phenyl-1-
propanone hydrochloride (34) [32], and N-[3-(dimethylamino)-2-
phenylprop-2-en-1-ylidene]-N-methylmethanaminium (35) [33]
were synthesized as reported in literature. Compound 3-oxo-3-
phenylpropanoate (36) was purchased from Alfa Aesar. Com-
pound ethyl 5-amino-1,2,4-triazole-3-carboxylate (40) [38] was
synthesized as reported in literature. Compounds 5-methyl-7-
phenyl-[1,2,4]triazolo[1,5-a]pyrimidine-2-carbonyl chloride (41)
[19] and 7-methyl-5-phenyl-[1,2,4]triazolo[1,5-a]pyrimidine-2-
carbonyl chloride (42) [19] were synthesized as previously re-
ported by us. Synthesis of compound 49, which entailed the reac-
tion of 42 [19] with o-fluoroaniline in pyridine at 80 ꢀC, was not
reported previously and herein included.
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
230e400). 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 18e31 were determined by LC/MS
using an Agilent 1290 Infinity System machine equipped with DAD
detector from 190 to 640 nm. The purity was revealed at 254 nm
using a Phenomenex AERIS Widepore C4, 4.6 mm, 100 mm (6.6 lm)
with flow rate: 0.85 ml/min; acquisition time: 10 min; gradient:
Based on the emerged SAR insights, the compounds showing
anti-SARS-CoV-2 activity belong to the series of 5-phenyl-7-
methyl-TZPs, while compounds having a phenyl ring at the C-6 or
C-7 position resulted inactive. Regarding the C-2 position, an
unsubstituted phenyl ring (compound 45) imparted the same ac-
tivity of a p-NH2 phenyl ring (compound 48), which instead slightly
decreased with a p-OPr phenyl moiety (compound 46) and
completely fell with p-OMe, p-NO2 and 3,4-di-OH phenyl moieties
(compounds 50e52, respectively). On the other hand, the activity
slightly increased with a o-F phenyl moiety at the C-2 position
(compound 47) and this activity was maintained also inverting the
amide linkage, as shown by compound 49.
Finally, trying to highlight common SAR insights for IAV and
SARS-CoV-2, we herein evaluated the anti-IAV activity of com-
pounds 47 [37], 48 [37], 49, 51 [37] and 52 [37], and their activity
was reported in Table 6, along with those of compounds 45 [19], 46
[19] and 50 [19]. No common SAR insights have been found,
although derivatives 45 and 49, belonging to 5-phenyl-7-methyl-
TZP class, showed both antiviral activities. Of note, compound 49
also exhibited good anti-PA-PB1 activity with an IC50 value of
17.5 mM.
3. Conclusions
In this work, four efficient one-step procedures for the synthesis
of 5-phenyl-, 6-phenyl- and 7-phenyl-2-amino-[1,2,4]triazolo[1,5-
11

M.C. Pismataro, T. Felicetti, C. Bertagnin et al.
European Journal of Medicinal Chemistry 221 (2021) 113494
Table 6
Structure and anti-SARS-CoV-2 activity of [1,2,4]triazolo[1,5-a]pyrimidine derivatives.
Compd
R
Anti-SARS-CoV-2 activity EC50
M)a
VeroE6 cells CC50
M)b
PRA in MDCK cells EC50,
Mc
Cytotoxicity (MTT Assay) in MDCK cells CC50,
m
Mc
(m
(
m
m
45
44.36 10.35
NDd
25
>250
46
47
65.35 3.52
ND
>100
247
30.61 10.69
ND
>100
>250
48
43.96 7.80
34.47 2.99
ND
>100
>250
>250
49e
>100
83.8 19.8
50
51
52
>100
>100
>100
ND
ND
ND
>100
>100
>100
>250
>250
112
GS-
0.89 0.62
72.38 11.57
441524
a
EC50 ¼ concentration of compound that gives 50% rescue of the virus-reduced eGFP signals as compared to the untreated virus-infected control cells.
b
CC50 ¼ 50% cytotoxic concentration, as determined by measuring the cell viability with the colorimetric formazan-based MTS assay. The values represent the means SD
of data derived from duplicate experiments.
c
For the definition of EC50 and CC50, see Table 5.
d
ND ¼ not determined.
e
IC50 ¼ 17.5 3.5
mM (ELISA PA-PB1 Interaction Assay).
acetonitrile in water containing 0.1% of formic acid (0100% in
10 min); oven temperature, 30C. Peak retention time is given in
minutes. 1H NMR and 13C NMR spectra were recorded on Bruker
Avance DRX-400MHz using residual solvents such as dime-
4.1.2. 2-Amino-5-phenyl-[1,2,4]triazolo[1,5-a]pyrimidine (9) [29]
To a mixture of 32 (0.1 g, 1 mmol) and 34 [32] (0.1 g, 0.5 mmol)
in DMF (2.5 mL), Et3N (0.07 mL, 0.5 mmol) was added and the re-
action mixture was heated at 110 ꢀC for 3 h maintaining open the
flask. After cooling, the reaction mixture was poured into ice/water
obtaining a precipitate that was filtered. The filtrate was extracted
with EtOAc and the organic layers were evaporated to dryness,
giving a solid that was combined with the precipitate previously
filtered, to give 9 (0.083 g, 78%) a yellow solid. 1H NMR (400 MHz,
thylsulfoxide (
standard. Chemical shifts were recorded in ppm (
d
¼ 2.48) or chloroform (
d
¼ 7.26) as an internal
d
) and the spectral
data are consistent with the assigned structures. The spin multi-
plicities are indicated by the: symbols s (singolet), d (doublet), t
(triplet), q (quartet), m (multiplet), and bs (broad singolet).
DMSO‑d6)
J ¼ 6.9 Hz, 1H, H6), 8.13e8.15 (m, 2H, AreH), 8.95 (d, J ¼ 6.9 Hz, 1H,
H7); 13C NMR (101 MHz, DMSO‑d6)
: 105.0, 127.4, 129.3, 130.9,
d: 6.36 (s, 2H, NH2), 7.49e7.53 (m, 3H, AreH), 7.57 (d,
4.1.1. 2-Amino-7-phenyl-[1,2,4]triazolo[1,5-a]pyrimidine (8) [29]
A mixture of 32 (0.1 g, 1 mmol) and 33 [31] (0.09 g, 0.5 mmol) in
glacial acetic acid (2.5 mL) was refluxed for 40 min. After cooling,
the reaction mixture was evaporated to dryness, obtaining a solid
that was treated with ice/water and filtered to give 8 (0.18 g, 85%) as
d
135.1, 136.8, 155.3, 158.0, 168.3. HRMS: m/z calcd for C11H9N5
212.0937 (M þ Hþ), found 212.0935 (M þ Hþ).
a white solid. 1H NMR (400 MHz, DMSO‑d6)
d
: 6.42 (s, 2H, NH2), 7.21
4.1.3. 2-Amino-6-phenyl-[1,2,4]triazolo[1,5-a]pyrimidine (10)
To a mixture of 32 (0.1 g, 1 mmol) and 35 [33] (0.15 g, 0.5 mmol)
in dry DMF (2.5 mL), dry Et3N (0.07 mL, 0.5 mmol) was added under
nitrogen atmosphere. The reaction mixture was maintained at rt for
1h, and then evaporated to dryness obtaining a solid that was
(d, J ¼ 4.9 Hz, 1H, H-6), 7.52e7.58 (m, 3H, AreH), 8.10e8.14 (m, 2H,
AreH), 8.50 (d, J ¼ 4.9 Hz,1H, H-7); 13C NMR (101 MHz, DMSO‑d6)
d:
107.5, 128.8, 129.6, 130.6, 131.5, 144.9, 151.9, 156.4, 167.7. HRMS: m/z
calcd for C11H9N5 212.0937 (M þ Hþ), found 212.0936 (M þ Hþ).
12

M.C. Pismataro, T. Felicetti, C. Bertagnin et al.
European Journal of Medicinal Chemistry 221 (2021) 113494
treated with ice/water and filtered to give 10 (0.098 g, 92%) as a
water to give a solid that was filtered and purified as described
below.
white solid. 1H NMR (400 MHz, DMSO‑d6)
d: 6.45 (s, 2H, NH2), 7.38
(t, J ¼ 7.4 Hz, 1H, AreH), 7.46 (t, J ¼ 7.4 Hz, 2H, AreH), 7.75 (d,
J ¼ 7.4 Hz, 2H, AreH), 8.80 (d, J ¼ 2.0 Hz, 1H, H7), 9.28 (d, J ¼ 2.0 Hz,
4.1.8. Methyl 2-(5-methyl-7-phenyl-[1,2,4]triazolo[1,5-a]
pyrimidine-2-carboxamido)benzoate (20)
1H, H5); 13C NMR (101 MHz, DMSO‑d6)
d: 121.6, 127.0, 128.4, 129.5,
132.1, 133.9, 151.1, 154.6, 168.2. HRMS: m/z calcd for C11H9N5
The title compound was prepared starting from 41 [19] and
methyl 2-aminobenzoate by Method A and purified by flash chro-
matography eluting with CHCl3/MeOH (99:1) in 43% yield. 1H NMR
212.0937 (M þ Hþ), found 212.0935 (M þ Hþ).
4.1.4. 2-Amino-5-phenyl-[1,2,4]triazolo[1,5-a]pyrimidin-7-ol (11)
To a mixture of 32 (0.1 g, 1 mmol) and 36 (0.17 mL, 1 mmol) in
dry EtOH (2.5 mL), freshly prepared NaOEt (0.068 g, 1 mmol) was
added under a nitrogen atmosphere and the reaction mixture was
refluxed for 12h. After cooling, it was poured into ice/water and
acidified until pH 6 with a 2 N HCl, obtaining a precipitate that was
filtered to give 11 (0.18 g, 79%) as white solid. 1H NMR (400 MHz,
(400 MHz, CDCl3): d 2.86 (s, 3H, CH3), 4.06 (s, 3H, OCH3), 7.22 (t,
J ¼ 7.5 Hz, 1H, AreH), 7.31 (s, 1H, H6), 7.65e7.69 (m, 4H, AreH), 8.16
(d, J ¼ 8.0 Hz, 1H, AreH), 8.25e8.27 (m, 2H, AreH), 9.04 (d,
J ¼ 8.5 Hz, 1H, AreH), 12.94 (s, 1H, NH); 13C NMR (101 MHz, CDCl3):
d
25.5, 52.6, 111.1, 116.2, 120.9, 123.4, 129.1, 129.3, 129.7, 131.1, 132.1,
134.7, 140.8, 147.8, 156.5, 157.9, 159.9, 166.5, 168.2. HRMS: m/z calcd
for C21H17N5O3 388.1429 (M þ Hþ), found 388.1412(M þ Hþ). HPLC,
ret. time: 7.26 min, peak area: 98.89%.
DMSO‑d6)
d
: 6.16 (s, 1H, H-6), 7.45e7.48 (m, 3H, AreH), 7.81e7.84
(m, 2H, AreH); 13C NMR (101 MHz, DMSO‑d6)
d
: 98.4, 127.4, 129.1,
130.7, 134.7, 150.6, 153.0, 155.8, 159.9. HRMS: m/z calcd for
C
4.1.9. Methyl 2-(7-methyl-5-phenyl-[1,2,4]triazolo[1,5-a]
pyrimidine-2-carboxamido)benzoate (21)
The title compound was prepared starting from 42 [19] and
methyl 2-aminobenzoate by Method A and purified by flash chro-
matography eluting with CHCl3/MeOH (99:1) in 40% yield. 1H NMR
11H9N5O 228.0886 (M þ Hþ), found 228.0882 (M þ Hþ).
4.1.5. Ethyl 7-phenyl-[1,2,4]triazolo[1,5-a]pyrimidine-2-carboxylate
(14)
Method 1: the title compound was prepared by using the same
procedure as used for the synthesis of 8 starting from 40 [38]
(40 min), in 75% yield as white solid.
(400 MHz, CDCl3): d 3.02 (s, 3H, CH3), 4.04 (s, 3H, OCH3), 7.19 (t,
J ¼ 7.6 Hz, 1H, AreH), 7.52 (s, 1H, H6), 7.54e7.57 (m, 3H, AreH), 7.64
(t, J ¼ 7.2 Hz, 1H, AreH), 8.13 (dd, J ¼ 1.6 and 8.0 Hz, 1H, AreH),
8.23e8.25 (m, 2H, AreH), 9.00 (d, J ¼ 8.4 Hz,1H, AreH),12.88 (s, 1H,
Method 2: the title compound was prepared by using the same
conditions as used for the synthesis of 8 starting from 40 [38], but
performing the reaction under microwave irradiation; after 40 min
at 180 ꢀC, the reaction was poured into ice/water and a precipitate
was obtained and filtered to give 14 in 58% yield as white solid.
NH); 13C NMR (101 MHz, CDCl3):
d 17.8, 52.7, 108.4, 116.2, 121.1,
123.5, 128.0, 129.2, 131.2, 131.7, 134.7, 136.1, 140.7, 148.4, 155.7, 158.0,
160.2, 162.5, 168.2. HRMS: m/z calcd for C21H17N5O3 388.1427
(M þ Hþ), found 388.1411 (M þ Hþ). HPLC, ret. time: 9.60 min, peak
area: 99.68%.
1H NMR (400 MHz, DMSO‑d6)
d
: 1.35 (t, J ¼ 7.0 Hz, CH2CH3), 4.43
(q, J ¼ 7.0 Hz, CH2CH3), 7.64e7.67 (m, 3H, AreH), 7.71 (d, J ¼ 4.6 Hz,
1H, H-6), 8.13e8.15 (m, 2H, AreH), 9.05 (d, J ¼ 4.3 Hz, 1H, H-5); 13
C
4.1.10. 2-(5-Methyl-7-phenyl-[1,2,4]triazolo[1,5-a]pyrimidine-2-
carboxamido)benzoic acid (18)
NMR (101 MHz, DMSO‑d6) d: 14.0, 61.8, 111.3, 128.7, 129.3, 129.5,
131.8, 147.9, 155.9, 156.1, 156.6, 159.9. HRMS: m/z calcd for
C
A suspension of 20 (0.05 g, 0.13 mmol) and LiOH (0.02 g,
0.51 mmol) in a mixture H2O/THF (1:1, 5 mL) was maintained at
50 ꢀC for 3h. After cooling the reaction mixture was acidified (pH
5e6) with 2 N HCl obtaining a precipitate that was filtered and
crystallized by EtOH/DMF to give 18 (0.035 g, 73% yield). 1H NMR
14H12N4O2 269.1039 (M þ Hþ), found 269.1039 (M þ Hþ).
4.1.6. Ethyl 6-phenyl-[1,2,4]triazolo[1,5-a]pyrimidine-2-carboxylate
(16)
Method 1: The title compound was prepared by using the same
procedure as used for the synthesis of 8 starting from 40 [38] and
replacing 33 with 35 (24 h), in 34% yield as white solid.
Method 2: the title compound was prepared by using the same
conditions as used for the synthesis of 8 starting from 40 [38] and
replacing 33 with 35, but performing the reaction under microwave
irradiation; after 40 min at 180 ꢀC, the reaction was poured into ice/
water and a precipitate was obtained and filtered to give 16 in 70%
yield as white solid.
(400 MHz, DMSO‑d6):
d
2.74 (s, 3H, CH3), 7.26 (t, J ¼ 7.6 Hz, 1H,
AreH), 7.68e7.72 (m, 5H, AreH and H6), 8.09 (d, J ¼ 7.9 Hz, 1H,
AreH), 8.24e8.26 (m, 2H, AreH), 8.81 (d, J ¼ 8.4 Hz, 1H, AreH),
12.85 (s, 1H, NH), 13.75 (bs, 1H, COOH); 13C NMR (101 MHz,
DMSO‑d6): d 24.8, 111.8, 116.8, 119.9, 123.5, 128.8, 129.3, 129.6, 131.4,
131.8, 134.3, 140.1, 146.6, 155.6, 157.4, 158.3, 166.8, 169.1. HRMS: m/z
calcd for C20H15N5O3 374.1260 (M þ Hþ), found 374.1250 (M þ Hþ).
HPLC, ret. time: 3.97 min, peak area: 99.16%.
1H NMR (400 MHz, CDCl3)
d
: 1.51 (t, J ¼ 7.0 Hz, 3H, CH2CH3), 4.59
4.1.11. 2-(7-Methyl-5-phenyl-[1,2,4]triazolo[1,5-a]pyrimidine-2-
carboxamido)benzoic acid (19)
(q, J ¼ 7.0 Hz, 2H, CH2CH3), 7.54e7.62 (m, 5H, AreH), 9.02 (d, J ¼ 2.4,
1H, H7), 9.20 (d, J ¼ 2.4, 1H, H5); 13C NMR (101 MHz, DMSO‑d6)
d
:
The title compound was obtained starting from 21 by using the
same procedure as used for the synthesis of 18 in 43% yield after
14.0, 61.8, 125.4, 127.4, 129.1, 129.3, 132.4, 134.5, 154.0, 156.6, 156.7,
159.7. HRMS: m/z calcd for C14H12N4O2 269.1039 (M þ Hþ), found
269.1040 (M þ Hþ).
crystallization by EtOH/DMF. 1H NMR (400 MHz, DMSO‑d6):
d 2.91
(s, 3H, CH3), 7.27 (t, J ¼ 7.6 Hz, 1H, AreH), 7.62e7.63 (m, 3H, AreH),
7.71 (t, J ¼ 7.9 Hz, 1H, AreH), 8.09 (d, J ¼ 7.8 Hz, 1H, AreH), 8.14 (s,
1H, H6), 8.31e8.33 (m, 2H, AreH), 8.84 (d, J ¼ 8.3 Hz, 1H, AreH),
12.90 (s, 1H, NH), 13.79 (bs, 1H, COOH); 13C NMR (101 MHz,
4.1.7. General procedure for the synthesis of compounds 20e22 and
28e31 by amidation
(Method A). To a solution of the appropriate [1,2,4]triazolo[1,5-
a]pyrimidine-2-carboxylic acid (2 mmol) in dry CH2Cl2 (20 mL),
oxalyl chloride (12 mmol) was added and after 30 min dry DMF (2
drops) was added. After 2 h stirring at rt, the reaction mixture was
evaporated to dryness to give a residue that was dissolved in dry
CH2Cl2 and added of the appropriate aniline (2 mmol) and DIPEA
(2 mmol). The reaction was maintained at rt for 2 h and then
evaporated to dryness to give a residue that was treated with ice/
DMSO‑d6): d 16.9, 108.8, 116.9, 119.9, 123.5, 127.7, 129.1, 131.4, 131.6,
134.2, 135.8, 140.0, 149.1, 154.9, 157.3, 158.7, 161.3, 169.1. HRMS: m/z
calcd for C20H15N5O3 374.1275 (M þ Hþ), found 374.1258 (M þ Hþ).
HPLC, ret. time: 4.48 min, peak area: 98.21%.
4.1.12. 2-(5-Methyl-7-phenyl-[1,2,4]triazolo[1,5-a]pyrimidin-2-yl)-
4H-benzo[d][1,3]oxazin-4-one (22)
The title compound was prepared starting from 41 [19] and 2-
13

M.C. Pismataro, T. Felicetti, C. Bertagnin et al.
European Journal of Medicinal Chemistry 221 (2021) 113494
aminobenzoic acid by Method A and purified by crystallization by
Method B and purified by flash chromatography eluting with
CHCl3/MeOH (99:1) in 44% yield. 1H NMR (400 MHz, DMSO‑d6):
MeOH/DMF in 31% yield. 1H NMR (400 MHz, CDCl3):
d 2.82 (s, 3H,
CH3), 7.20 (s, 1H, H6), 7.60e7.65 (m, 4H, AreH), 7.87e7.94 (m, 2H,
d
7.44 (t, J ¼ 7.4 Hz, 1H, AreH), 7.48e7.53 (m, 4H, AreH), 7.59 (t,
AreH), 8.13e8.16 (m, 2H, AreH), 8.30 (d, J ¼ 7.7 Hz, 1H, AreH); 13
C
J ¼ 7.2 Hz, 1H, AreH), 7.84 (d, J ¼ 7.7 Hz, 2H, AreH), 8.01 (d,
J ¼ 8.1 Hz, 2H, AreH), 9.16 (d, J ¼ 2.3 Hz, 1H, H7), 9.72 (d, J ¼ 2.3 Hz,
1H, H5), 11.45 (s, 1H, NH); 13C NMR (101 MHz, DMSO‑d6): 123.7,
127.4, 128.5, 128.8, 128.9, 129.6, 132.6, 133.3, 133.9, 134.0, 153.4,
154.3, 161.1, 165.1. HRMS: m/z calcd for C18H13N5O 316.1199
(M þ Hþ), found 316.1194 (M þ Hþ). HPLC, ret. time: 3.82 min, peak
area: 99.84%.
NMR (101 MHz, DMSO‑d6):
d 25.7, 111.5, 118.1, 128.4, 128.9, 129.2,
129.3, 129.5, 129.7, 132.2, 136.8, 146.2, 147.7, 150.9, 156.8, 157.7,
158.5, 166.9. HRMS: m/z calcd for C20H13N5O2 356.1154 (M þ Hþ),
found 356.1145 (M þ Hþ). HPLC, ret. time: 3.78 min, peak area:
99.95%.
4.1.13. 2-(7-Methyl-5-phenyl-[1,2,4]triazolo[1,5-a]pyrimidin-2-yl)-
4H-benzo[d][1,3]oxazin-4-one (23)
4.1.18. N-(7-Hydroxy-5-phenyl-[1,2,4]triazolo[1,5-a]pyrimidin-2-
yl)benzamide (27)
The title compound was prepared starting from 11 (3 h) by
Method B and purified by flash chromatography eluting with
CHCl3/MeOH (99:1) in 45% yield. 1H NMR (400 MHz, DMSO‑d6):
A mixture of 19 (0.06 g, 0.16 mmol) and acetic anhydride
(0.016 g, 1.68 mmol) was heated at 100 ꢀC overnight. After cooling,
the reaction mixture was poured into ice/water obtaining a pre-
cipitate that was filtered and purified by flash chromatography
eluting with 100% CHCl3 to give 23 (0.026 g, 45% yield). 1H NMR
d
6.31 (s, 1H, H6), 7.47e7.49 (m, 5H, AreH), 7.57e7.60 (m, 1H,
AreH), 7.92e7.98 (m, 4H, AreH), 11.44 (s, 1H, NH); 13C NMR
(101 MHz, DMSO‑d6): 92.3, 127.6, 128.4, 128.8, 129.1, 130.6, 132.7,
(400 MHz, CDCl3):
d 3.02 (s, 3H, CH3), 7.51e7.55 (m, 4H, AreH and
H6), 7.61e7.65 (m, 1H, AreH), 7.87e7.95 (m, 2H, AreH), 8.21e8.24
d
(m, 2H, AreH), 8.31 (d, J ¼ 7.7 Hz, 1H, AreH); 13C NMR (101 MHz,
133.6, 134.4, 137.3, 156.6, 157.0, 161.1, 165.7. HRMS: m/z calcd for
CDCl3):
d
17.8, 108.5, 118.1, 128.0, 128.4, 128.9, 129.2, 129.7, 131.8,
C
18H13N5O2 332.1148 (M þ Hþ), found 332.1179 (M þ Hþ). HPLC, ret.
136.0,136.9,146.2,148.1,150.9,156.2,158.0,158.4,162.5. HRMS: m/z
calcd for C20H13N5O2 356.1158 (M þ Hþ), found 356.1148 (M þ Hþ).
HPLC, ret. time: 2.95 min, peak area: 99.83%.
time: 3.33 min, peak area: 95.21%.
4.1.19. 7-Phenyl-[1,2,4]triazolo[1,5-a]pyrimidine-2-carboxylic acid
(43)
4.1.14. General procedure for the synthesis of compounds 24e27 by
amidation (method B)
A mixture of 14 (1.2 g, 4.47 mmol) and NaOH (0.2 g, 5.82 mmol)
in MeOH (25 mL) was refluxed for 4 h. After cooling, the reaction
mixture was poured into ice/water and acidified with 2 N HCl
obtaining a precipitate that was filtered and treated with Et2O, to
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 (8e11) (1.0 mmol) in dry pyridine
(15 mL), and then the reaction mixture was maintained at 80 ꢀC
until no starting material was detected by TLC. After cooling, it was
poured into ice/water and acidified with CH3COOH (pH ¼ 5),
obtaining a precipitate that was filtered and purified as described
below.
give 43 (0.85g, 85%). 1H NMR (400 MHz, DMSO‑d6):
d 7.61e7.67 (m,
4H, AreH and H6), 8.10e8.12 (m, 2H, AreH), 8.99 (d, J ¼ 4.5 Hz, 1H,
H5).
4.1.20. 6-Phenyl-[1,2,4]triazolo[1,5-a]pyrimidine-2-carboxylic acid
(44)
The title compound was prepared starting from 16 following the
same procedure as used for the synthesis of 43 (1 h) in 100% yield.
4.1.15. N-(7-Phenyl-[1,2,4]triazolo[1,5-a]pyrimidin-2-yl)benzamide
(24)
1H NMR (400 MHz, DMSO‑d6):
d 7.44e7.48 (m, 1H, AreH), 7.53 (t,
The title compound was prepared starting from 8 (4 h) by
Method B and purified by flash chromatography eluting with
CH2Cl2/MeOH (97:3) in 64% yield. 1H NMR (400 MHz, DMSO‑d6):
J ¼ 7.4 Hz, 2H, AreH), 7.85 (d, J ¼ 7.5 Hz, 2H, AreH), 9.34 (s, 1H, H7),
9.80 (s, 1H, H5), 13.86 (s, 1H, COOH).
d
7.49 (t, J ¼ 7.8 Hz, 2H, AreH), 7.55e7.63 (m, 5H, H6 and AreH),
4.1.21. N-(2-Carbamoylphenyl)-7-phenyl-[1,2,4]triazolo[1,5-a]
pyrimidine-2-carboxamide (28)
7.99e8.00 (m, 2H, AreH), 8.21e8.23 (m, 2H, AreH), 8.83 (d,
J ¼ 4.8 Hz,1H, H-5),11.41 (s,1H, NH); 13C NMR (101 MHz, DMSO‑d6):
The title compound was prepared starting from 43 and 2-
aminobenzamide by Method A and purified by recrystallization
d
109.7, 128.5, 128.8, 129.0, 129.9, 130.0, 132.0, 132.6, 133.9, 146.9,
154.7, 155.3, 160.5, 165.3. HRMS: m/z calcd for C18H13N5O 316.1199
(M þ Hþ), found 316.11978 (M þ Hþ). HPLC, ret. time: 3.95 min,
peak area: 100%.
from MeOH/DMF in 42% yield. 1H NMR (400 MHz, DMSO‑d6):
d 7.19
(t, J ¼ 7.2 Hz, 1H, AreH), 7.56 (t, J ¼ 7.3 Hz, 1H AreH), 7.65e7.67 (m,
3H, AreH), 7.72e7.74 (m, 2H, CONH2 and AreH), 7.85 (d, J ¼ 7.9 Hz,
1H, AreH), 8.25e8.28 (m, 2H, AreH), 8.31 (bs, 1H, CONH2), 8.68 (d,
4.1.16. N-(5-Phenyl-[1,2,4]triazolo[1,5-a]pyrimidin-2-yl)benzamide
(25)
J ¼ 8.1 Hz, 1H, AreH), 9.02e9.03 (m, 1H, H5), 13.22 (s, 1H, NH); 13
C
NMR (101 MHz, DMSO‑d6): d 110.9, 120.3, 120.5, 123.3, 128.7, 128.8,
The title compound was prepared starting from 9 (24 h) by
Method B and purified by flash chromatography eluting with
CHCl3/MeOH (99:1) in 35% yield. 1HNMR (400 MHz, DMSO‑d6):
129.1, 129.7, 131.9, 132.3, 136.6, 147.8, 155.9, 156.4, 157.1, 158.7, 170.4.
HRMS: m/z calcd for C19H14N6O2 359.1270 (M þ Hþ), found
359.1257 (M þ Hþ). HPLC, ret. time: 2.23 min, peak area: 99.14%.
d
7.50 (t, J ¼ 7.3 Hz, 2H, AreH), 7.55e7.61 (m, 4H, AreH), 7.90 (d,
J ¼ 7.1 Hz, 1H, H6), 7.99 (d, J ¼ 7.3 Hz, 2H, AreH), 8.24e8.27 (m, 2H,
4.1.22. N-(2-Carbamoylphenyl)-6-phenyl-[1,2,4]triazolo[1,5-a]
pyrimidine-2-carboxamide (29)
The title compound was prepared starting from 44 and 2-
aminobenzamide by Method A and purified by flash chromatog-
raphy eluting with CHCl3/MeOH (98:2), in 38% yield. 1H NMR
AreH), 9.38 (d, J ¼ 7.1 Hz, 1H, H7), 11.41 (s, 1H, NH); 13C NMR
(101 MHz, DMSO‑d6):
d
107.6, 127.9, 128.5, 128.8, 129.5, 131.7, 132.5,
134.0, 136.3, 137.3, 154.2, 160.6, 161.2, 165.2. HRMS: m/z calcd for
C
18H13N5O 316.1199 (M þ Hþ), found 316.1195 (M þ Hþ). HPLC, ret.
time: 3.84 min, peak area: 99.57%.
(400 MHz, DMSO‑d6):
d
7.20 (t, J ¼ 7.6 Hz, 1H, AreH), 7.47 (t,
J ¼ 7.3 Hz, 1H, AreH), 7.52e7.58 (m, 3H, AreH), 7.75 (s, 1H, CONH2),
7.84 (d, J ¼ 7.8 Hz, 1H, AreH), 7.89 (d, J ¼ 7.6 Hz, 2H, AreH), 8.33 (s,
1H, CONH2), 8.70 (d, J ¼ 8.3 Hz, 1H, AreH), 9.16 (d, J ¼ 2.2 Hz, 1H,
H7), 9.86 (d, J ¼ 2.1 Hz, 1H, H5), 13.12 (s, 1H, NH); 13C NMR
4.1.17. N-(6-Phenyl-[1,2,4]triazolo[1,5-a]pyrimidin-2-yl)benzamide
(26)
The title compound was prepared starting from 10 (5 h) by
14

M.C. Pismataro, T. Felicetti, C. Bertagnin et al.
European Journal of Medicinal Chemistry 221 (2021) 113494
(101 MHz, DMSO‑d6):
d
120.8, 121.0, 123.7, 125.5, 127.7, 129.1, 129.3,
4.2.2. Cells and virus
129.7, 132.7, 132.8, 134.9, 138.9, 154.3, 156.7, 157.3, 159.7, 170.7.
HRMS: m/z calcd for C19H14N6O2 359.1257 (M þ Hþ), found
359.1236 (M þ Hþ). HPLC, ret. time: 3.89 min, peak area: 100%.
Madin-Darby canine kidney (MDCK) cells were grown in Dul-
becco’s modified Eagle’s medium (DMEM, Life Biotechnologies)
supplemented with 10% (v/v) fetal bovine serum (FBS, Life Tech-
nologies) and antibiotics (100 U/mL penicillin and 100
mg/mL
streptomycin, Life Technologies). Cells were maintained at 37 ꢀC in
a humidified atmosphere with 5% CO2. IAV A/PR/8/34 (H1N1,
Cambridge lineage) was kindly provided by P. Digard (Roslin
Institute, University of Edinburgh, United Kingdom).
4.1.23. N-(3-Carbamoyl-5,6,7,8-tetrahydro-4H-cyclohepta[b]
thiophen-2-yl)-7-phenyl-[1,2,4]triazolo[1,5-a]pyrimidine-2-
carboxamide (30)
The title compound was prepared starting from 43 and 2-
amino-5,6,7,8-tetrahydro-4H-cyclohepta[b]thiophene-3-
carboxamide [39] by Method A and purified by flash chromatog-
raphy eluting with CHCl3/MeOH (99:1) in 16% yield. 1H NMR
4.2.3. PA-PB1 interaction enzyme-linked immunosorbent assay
(ELISA)
The PAꢁPB1 interaction was detected by a procedure previously
described [17]. Briefly, 96-well microtiter plates (Nuova Aptaca)
were coated with 400 ng of 6His-PA(239ꢁ716) for 3h at 37 ꢀC and then
blocked with 2% BSA (Sigma) in PBS for 1h at 37 ꢀC. The 6His-
PA(239ꢁ716) protein was expressed in E. coli strain BL21(DE3)pLysS
and purified as already described [17]. After washing, 200 ng of
GST-PB1(1ꢁ25), or of GST alone as a control, in the absence or the
presence of test compounds at various concentrations, were added
and incubated O/N at room temperature. Escherichia coli-expressed,
purified GST and GST-PB1(1ꢁ25) proteins were obtained as previ-
ously described [17,41]. After washing, the interaction between
6His-PA(239ꢁ716) and GST-PB1(1ꢁ25) was detected with a horseradish
peroxidase-coupled anti-GST monoclonal antibody (GenScript)
diluted 1:4000 in PBS supplemented with 2% FBS. Following
washes, the substrate 3,30,5,50tetramethylbenzidine (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 set as 100% of PAꢁPB1 interaction.
(400 MHz, CDCl3):
d 1.66e1.73 (m, 4H, cycloheptane CH2 x 2),
1.86e1.90 (m, 2H, cycloheptane CH2), 2.76e2.80 (m, 4H, cyclo-
heptane CH2 x 2), 7.39 (d, J ¼ 4.5 Hz, 1H, H6), 7.60e7.66 (m, 3H,
AreH), 8.15e8.18 (m, 2H, AreH), 9.00 (d, J ¼ 4.5 Hz,1H, H5),10.14 (s,
1H, NH); 13C NMR (101 MHz, CDCl3):
d 26.4, 27.1, 28.2, 28.4, 31.1,
97.1, 109.6, 113.3, 127.9, 128.3, 128.7, 131.6, 132.4, 135.6, 141.8, 148.4,
154.4,155.2,155.3,156.7. HRMS: m/z calcd for C22H20N6O2S 437.1118
(M þ Naþ)þ[-H2O], found 437.1162 (M þ Naþ)þ[-H2O]. HPLC, ret.
time: 4.78 min, peak area: 97.82%
4.1.24. N-(3-Carbamoyl-5,6,7,8-tetrahydro-4H-cyclohepta[b]
thiophen-2-yl)-6-phenyl-[1,2,4]triazolo[1,5-a]pyrimidine-2-
carboxamide (31)
The title compound was prepared starting from 44 and 2-
amino-5,6,7,8-tetrahydro-4H-cyclohepta[b]thiophene-3-
carboxamide [39] by Method A and purified by flash chromatog-
raphy eluting with CHCl3/MeOH (95:5), in 42% yield. 1H NMR
(400 MHz, DMSO‑d6) d: 1.58e1.59 (m, 4H, cycloheptane CH2 x 2).
1.77e1.79 (m, 2H, cycloheptane CH2), 2.71e2.73 (m, 2H, cyclo-
heptane CH2), 2.79e2.85 (m, 2H, cycloheptane CH2), 7.45e7.49 (m,
2H, AreH and CONH), 7.52e7.56 (m, 2H, AreH), 7.72 (bs,1H, CONH),
7.88 (d, J ¼ 7.6 Hz, 2H, AreH), 9.40 (d, J ¼ 1.9 Hz, 1H, H7), 9.88 (d,
J ¼ 1.8 Hz, 1H, H5), 12.07 (s, 1H, NH); 13C NMR (101 MHz DMSO‑d6)
4.2.4. 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 [17,42]. Briefly, MDCK cells
(seeded at density of 2 ꢂ 104 per well) were grown in 96-well plates
for 24h 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 48h, 5 mg/mL of MTT (Sigma) in PBS was
added into each well and incubated at 37 ꢀC for further 4h. Suc-
cessively, a solubilization solution (10% SDS, 0.01 N HCl) 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
(Tecan Sunrise™).
d
: 27.5, 27.9, 28.5, 28.7, 31.9, 122.1, 125.6, 127.7, 129.4, 129.7, 131.7,
132.7, 135.0, 135.5, 136.6, 154.2, 155.2, 156.9, 158.2, 167.7. HRMS: m/z
calcd for C22H20N6O2S 455.1266 (M þ Naþ), found 455.12613
(M þ Naþ). HPLC, ret. time: 5.071 min, peak area: 100%.
4.1.25. N-(2-Fluorophenyl)-7-methyl-5-phenyl-[1,2,4]triazolo[1,5-
a]pyrimidine-2-carboxamide (49)
The title compound was prepared starting from 42 [19] and o-
fluoroaniline by Method A and purified by flash chromatography
eluting with CH2Cl2, in 45% yield. 1H NMR (400 MHz, CDCl3):
d 2.81
4.2.5. Plaque reduction assay (PRA)
(s, 3H, CH3), 7.17e7.28 and 7.53e7.61 (m, each 3H, AreH), 7.77 (t,
J ¼ 7.3 Hz, 1H, AreH), 8.11 (s, 1H, H-6), 8.23e8.27 (m, 2H, AreH),
The antiviral activity of test compounds against IAV was tested
by PRA as previously described [17,43]. MDCK cells were seeded at
5 ꢂ 105 cells/well into 12-well plates, and incubated at 37 ꢀC for
24h. The following day, the culture medium was removed and the
monolayers were first washed with serum-free DMEM and then
infected with the IAV A/PR/8/34 strain at 40 PFU/well in DMEM
10.48 (s, 1H, NH); 13C NMR (101 MHz, CDCl3):
d: 17.47,109.33, 116.26
(J ¼ 19.4 Hz), 125.06, 127.77 (J ¼ 19.4 Hz), 125.79, 127.26, 128.26,
129.70, 132.14, 136.47, 149.77, 155.31 (J ¼ 247.5 Hz), 155.45, 158.02,
159.21, 162.04. HRMS: m/z calcd for C19H14FN5O 348.1261 (M þ Hþ),
found 348.1263 (M þ Hþ). HPLC, Xterra C18; CH3CN 80%:H2O 20%;
ret. time: 2.18 min, peak area: 96.59%.
supplemented with 1 mg/mL of TPCK-treated trypsin (Worthington
Biochemical Corporation) and 0.14% BSA and incubated for 1 h at
37 ꢀC. The IAV infection was performed in the presence of different
concentrations of test compounds or solvent (DMSO) as a control.
4.2. Biology (IAV)
After virus adsorption, DMEM containing 1 mg/mL of TPCK-treated
4.2.1. Compounds and peptide
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%.
RBV (1-D-ribofuranosyl-1,2,4-triazole-3-carboxamide) was pur-
chased from Roche. Each test compound was dissolved in 100%
DMSO. The PB1(1e15)eTat 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 47e59), which allows the delivery into the cell [40].
4.2.6. Minireplicon assays
The minireplicon assay was performed as described [17], with
15

M.C. Pismataro, T. Felicetti, C. Bertagnin et al.
European Journal of Medicinal Chemistry 221 (2021) 113494
some modifications. Briefly, HEK 293T cells (2 ꢂ 105 cells per well)
were plated into 24-well plates and incubated overnight at 37 ꢀC.
The next day, cells were transfected using calcium phosphate co-
precipitation method with pcDNA-PB1, pcDNA-PB2, pcDNA-PA,
pcDNA-NP plasmids (100 ng/well of each) along with 50 ng/well
of the pPolI-Flu-ffLuc reporter plasmid and 50 ng/well of pRL-SV40
plasmid as a transfection control. Transfections were performed in
the presence of different concentrations of test compounds or
DMSO. RBV was used as a positive control for inhibition. Cell me-
dium was removed 5 h post-transfection and replaced with DMEM
financial interests or personal relationships that could have
appeared to influence the work reported in this paper.
Acknowledgements
This work was supported by grants “Excellent Departments” by
ꢀ
Ministero dell’Istruzione, dell’Universita e della Ricerca-MIUR (to S.
Massari), PRIN 2017 - cod. 2017BMK8JR (to S. Massari, T. Felicetti
and V. Cecchetti), Fondazione Cassa Risparmio Perugia - Ricerca
Scientifica e Tecnologica 2019: “Giovani ricercatori: risorsa per il
territorio” e cod. 3FCRPG19_SM (to M.C. Pismataro), Associazione
Italiana per la Ricerca sul Cancro, AIRC, grant n. IG18855 (to A.
Loregian); British Society for Antimicrobial Chemotherapy, UK,
BSAC-2018-0064 (to A. Loregian), Ministero dell’Istruzione,
containing the compounds, RBV, or DMSO. At 24
h post-
transfection, cells were harvested, lysed and both firefly and
Renilla luciferase activity were measured using the Dual Luciferase
Assay Kit (Promega). In each experiment, firefly luciferase activity
was normalized with that of the Renilla luciferase and relative
luciferase units (RLU) were obtained. The activity measured in
control transfection reactions containing DMSO was set at 100% of
polymerase activity.
ꢀ
dell’Universita e della Ricerca, PRIN 2017 - cod. 2017KM79NN (to A.
Loregian), Fondazione Cassa di Risparmio di Padova e Rovigo -
Bando Ricerca Covid-2019 Nr. 55777 2020.0162 (to A. Loregian). The
SARS-CoV-2 research was performed using the ‘Caps-It’ research
infrastructure (project ZW13-02) that was financially supported by
the Hercules Foundation and Rega Foundation, KU Leuven.
4.3. Biology (SARS-CoV-2)
4.3.1. Cells and viruses
Appendix A. Supplementary data
The SARS-CoV-2 isolate used in this study was the BetaCov/
Belgium/GHB-03021/2020 (EPI ISL407976|2020-02-03), which
was isolated from a Belgian patient returning from Wuhan in
February 2020. The isolate was passaged 7 times on Vero E6 cells
which introduced two series of amino acid deletions in the spike
protein [44]. The infectious content of the virus stock was deter-
mined by titration on Vero E6 cells. Vero E6 cells were maintained
in Dulbecco’s modified Eagle’s medium (DMEM; Gibco) supple-
mented with heat-inactivated 10% v/v fetal calf serum (FCS; Bio-
Supplementary data to this article can be found online at
https://doi.org/10.1016/j.ejmech.2021.113494.
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