Chemistry and anti-herpes simplex virus type 1 evaluation of 4-substituted-1H-1,2,3-triazole-nitroxyl-linked hybrids

Article abstract of DOI:10.1007/s11030-020-10094-2

Abstract: HSV disease is distributed worldwide. Anti-herpesvirus drugs are a problem in clinical settings, particularly in immunocompromised individuals undergoing herpes simplex virus type 1 infection. In this work, 4-substituted-1,2,3-1H-1,2,3-triazole linked nitroxyl radical derived from TEMPOL were synthesized, and their ability to inhibit the in vitro replication of HSV-1 was evaluated. The nitroxide derivatives were characterized by infrared spectroscopy and elemental analysis, and three of them had their crystal structures determined by single-crystal X-ray diffraction. Four hybrid molecules showed important anti-HSV-1 activity with IC₅₀ values ranged from 0.80 to 1.32?μM. In particular, one of the nitroxide derivatives was more active than Acyclovir (IC₅₀ = 0.99?μM). All compounds tested were more selective inhibitors than the reference antiviral drug. Among them, two compounds were 4.5 (IC₅₀ 0.80?μM; selectivity index CC₅₀/IC₅₀ 3886) and 7.7 times (IC₅₀ 1.10?μM; selectivity index CC₅₀/IC₅₀ 6698) more selective than acyclovir (IC₅₀ 0.99?μM; selectivity index CC₅₀/IC₅₀: 869). These nitroxide derivatives may be elected as leading compounds due to their antiherpetic activities and good selectivity. Graphic abstract: [Figure not available: see fulltext.]

Full text of DOI:10.1007/s11030-020-10094-2

Molecular Diversity
https://doi.org/10.1007/s11030-020-10094-2
ORIGINAL ARTICLE
Chemistry and anti‑herpes simplex virus type 1 evaluation
of 4‑substituted‑1H‑1,2,3‑triazole‑nitroxyl‑linked hybrids
1
2
3
4
5,6
Anna C. Cunha · Vitor F. Ferreira · Maria G. F. Vaz · Rafael A. Allão Cassaro · Jackson A. L. C. Resende
·
7
7
7
7,8
9
Carolina Q. Sacramento · Jéssica Costa · Juliana L. Abrantes · Thiago Moreno L. Souza · Alessandro K. Jordão
Received: 17 December 2019 / Accepted: 23 April 2020
©
Springer Nature Switzerland AG 2020
Abstract
HSV disease is distributed worldwide. Anti-herpesvirus drugs are a problem in clinical settings, particularly in immunocom-
promised individuals undergoing herpes simplex virus type 1 infection. In this work, 4-substituted-1,2,3-1H-1,2,3-triazole
linked nitroxyl radical derived from TEMPOL were synthesized, and their ability to inhibit the in vitro replication of HSV-1
was evaluated. The nitroxide derivatives were characterized by infrared spectroscopy and elemental analysis, and three of
them had their crystal structures determined by single-crystal X-ray diꢀraction. Four hybrid molecules showed important
anti-HSV-1 activity with IC values ranged from 0.80 to 1.32 µM. In particular, one of the nitroxide derivatives was more
5
0
active than Acyclovir (IC =0.99 µM). All compounds tested were more selective inhibitors than the reference antiviral
5
0
drug. Among them, two compounds were 4.5 (IC 0.80 µM; selectivity index CC /IC 3886) and 7.7 times (IC 1.10 µM;
5
0
50
50
50
selectivity index CC /IC 6698) more selective than acyclovir (IC 0.99 µM; selectivity index CC /IC : 869). These
5
0
50
50
50
50
nitroxide derivatives may be elected as leading compounds due to their antiherpetic activities and good selectivity.
Graphic abstract
Keywords 1H-1,2,3-triazole derivatives · TEMPOL-based radicals · Anti-HSV-1 activity · Crystal structure
Introduction
Herpes simplex virus (HSV) is a highly prevalent infec-
Electronic supplementary material The online version of this
tion globally. DNA virus replication may establish latency
in sensory neuron innervation, periodically reactivating to
produce localized recurrent lesions [1]. In addition, HSV
virus can cause serious illnesses such as recurrent keratitis
which is the most common source of infectious blindness in
article (https://doi.org/10.1007/s11030-020-10094-2) contains
supplementary material, which is available to authorized users.
*
Alessandro K. Jordão
akjordao@ccs.ufrn.br
Extended author information available on the last page of the article
Vol.:(0
1
123456789)
3

Molecular Diversity
the western world. Other diseases associated with HSV virus
include encephalitis and systemic infection in neonates and
immunocompromised patients [2].
cases [22]. Since the treatment of viral diseases is the sup-
pression of viral replication followed by cell survival, the
development of antiviral molecules with antioxidant prop-
erties may be a promising adjunct in the treatment of viral
diseases [22, 23].
Ribavirin (1-β-D-ribofuranosyl-1,2,4-1H-1,2,3-triazole-
3
-carboxamide) is a nucleoside analogue with a broad anti-
viral spectrum. It is active against many viral infections such
as herpes simplex virus (HSV), human immunodeꢁciency
virus (HIV-1), inꢂuenza, respiratory syncytial, and hepatitis
C among other viruses [3, 4]. The clinical use of ribavirin
has been limited by its marked toxicology proꢁle. Others
nucleoside drugs as acyclovir and its prodrug valacyclovir
and famciclovir must be phosphorylated in order to be active
Nitroxide radicals (NR) are largely studied because of
their diverse properties such as antioxidants [27]. Many
of them exhibited antitumoral [28, 29], antihypertension
[30], anti-inꢂammatory [31] and antiviral activities [32].
Among nitroxide radicals, TEMPOL (6, 4-hydroxy-
2,2,6,6-tetramethylpiperidinyl-1-oxy) (Eq. 1) is one of
the most explored radical [33] mainly due to its relative
simple synthesis, stability and versatility. In fact, in vivo
tests using TEMPOL showed eꢀective reduction of tissue
injury in animal models of inꢂammation.
[
5]. The exigency of antiviral agents is still inevitable and
the emergence of virus strains that are resistant to commonly
used anti-herpesvirus drugs is a problem [6–8]. Several
classes of non-nucleoside inhibitors [9–12] have been devel-
oped as an alternative strategy for the identiꢁcation of poten-
tial new antiviral drugs with diꢀerent mechanisms of action
from those of the currently used compounds. Among non-
nucleoside compounds, the 1H-1,2,3-triazole derivatives
TEMPOL (Eq. 1) has been studied in animal models of
oxidative stress. This compound protected many organs,
including the heart and brain, from ischemia/reperfusion
damage. This nitroxide (Eq. 1) and related compounds
decrease the oxidative damage via reacting with free radi-
cals, such as reactive oxygen species (Eq. 1), directly or by
oxidizing the reduced metals (Eq. 3) thereby inhibiting the
Fenton and the metal-catalyzed Haber–Weiss reactions.
Nitroxides can prevent generation of strong oxidant radical
1
and 3 (Fig. 1) represent a promising class of lead com-
pounds for future antiviral drug design [13–15]. The suc-
cess of 1H-1,2,3-triazole heterocyclics can be assessed by
the number of drugs that are on the pharmaceutical market
for various diseases: tazobactam, cefatrizine and ruꢁnamide
•
OH generates by the Fenton reaction (Eq. 4) by accepting
[
16–18].
H-1,2,3-Triazole derivatives are also known to exhibit
diꢀerentiated biological activities, including antibacterial
the electron from the reduced metal complex as shown in
Eq. 3 (Scheme 1) [34].
1
Molecular hybridization of pharmacophoric moieties of
diꢀerent bioactive substances is a powerful tool that has
been utilized by several research groups to develop hybrid
molecules with improved aꢃnity and eꢃcacy, when com-
pared to the parent compounds [35, 36].
[
19], antiviral [20], antiplatelet [21], anti-cancer [22], anti-
inꢂammatory [23] and antitubercular [24].
Reports in the literature showed that viral infections and
other human diseases are under chronic oxidative stress [25,
2
6]. Reactive oxygen and nitrogen species (ROS and RNS,
Against this background, we describe the synthesis
and antiviral profile against HSV-1 virus of 4-substi-
tuted-1H-1,2,3-triazole-nitroxyl-linked hybrids 9a–j
formed by copper(I)-catalyzed 1,3-dipolar cycloaddition
reaction between TEMPOL azide 8 and terminal alkynes
(Scheme 2).
respectively) metabolites play a complex role in many dis-
eases and in metabolic regulation. Uncontrolled production
of reactive species results in oxidative stress and causes
damage in proteins, lipids, DNA, and cellular structures.
Oxidative processes contribute to several aspects of
viral disease pathogenesis as loss of immune function,
viral replication in infected cells, decrease cell prolifera-
tion, and induce cell apoptosis. Antioxidant compounds
may prevent or treat various diseases that are associated
with oxidative stress, having a therapeutic eꢀect in many
Fig. 1 Examples of inhibitors
of herpes virus with anti-HSV
activity
1
3

Molecular Diversity
Scheme 1 Redox-cycling reactions of TEMPOL involving the nitroxide radical
Scheme 2 Structural planning
of TEMPOL 1H-1,2,3-triazoles
1
3

Molecular Diversity
Results and discussion
in yields ranging from 44 to 80%, by the 1,3-dipolar
cycloaddition reaction of azide compound 8 with various
alkynes using CuBr as catalyst.
Synthesis
For the preparation of the 1H-1,2,3-triazole derivatives
Structure of 4‑substituted‑1H‑1,2,3‑tria‑
9
a–j, 4-hydroxy-2,2,6,6-tetramethylpiperidinyl-1-oxy
zole‑nitroxyl‑linked hybrids
(
TEMPOL) (6) has been used as the stable nitroxide
radical, according to Scheme 3. The triacetonamine (4)
was converted to 2,2,6,6-tetramethyl-4-piperidinol (5)
by treatment with sodium borohydride [37]. 4-Hydroxy-
Nitroxide compounds 9b, 9c and 9d (Fig. 2 and Table S1)
were obtained as single crystals and had their structures
determined by single-crystal X-ray diffraction analysis.
Other characterization methods, such as infrared spectros-
copy and elemental analysis also were realized.
2
,2,6,6-tetramethylpiperidine-1-oxyl (6) was obtained by
catalytic oxidation reaction of 4-hydroxy-2,2,6,6-tetra-
methylpiperidine (5) with sodium tungstate and hydrogen
peroxide in 70% yield [38]. The reaction of TEMPOL
Figure 2 shows the ORTEP diagrams of these com-
pounds, and crystal data and reꢁnements are provided in
Table S1 in Supplementary Information.
(
6) and benzenesulfonyl chloride in pyridine produced
4
-(4′-toluenlesulphonate)-2,2,6,6-tetramethylpiperi-
The piperidinoxyl ring adopts a chair conformation in all
crystal structures. In the compound 9b the N–O bond length
value is 1.291(4) Å. The crystalline packing is formed by
C–H⋯N and C–H⋯O interactions. The short contacts C(1
dine-N-oxyl (7), which was subsequently converted into
4
-azido-2,2,6,6-tetramethylpiperidine-1-oxyl (8) using
sodium azide in DMF [39].
(
x,−1+y,z)
(1+x,−2−y,−
A series of novel 1H-1,2,3-triazoles derivatives 9a–j
containing a stable nitroxide group were synthesized,
0)–H(10)⋯N(3)
and C(16)–H(16)⋯O(1)
describe intermolecular interaction chains in direc-
tions [010] and [− 201], respectively. The compound 9c
1
/2+z)
Scheme 3 Synthesis of the compounds 9a–j
1
3

Molecular Diversity
Table 1 Antiviral activity for
H-1,2,3-triazole derivatives
Compounds
% of inhibi-
tion of HSV-1
yields
1
9
9
9
9
9
9
9
9
9
9
a
b
c
d
e
f
38
18
NP
84
83
NP
45
86
NP
NP
95
g
h
i
j
ACV
NP Not performed
compounds inhibited virus replication by more than 80% at
5
0 µM after 72 h post-infection.
The bioactive compounds 9d, 9e and 9h inhibited HSV-1
replication by 84%, 83% and 86%, in this order. In this anti-
viral evaluation, the drug acyclovir (ACV) was used as a
positive control. This drug, in the same experimental condi-
tions as nitroxides, inhibits 95% of HSV-1 replication.
The hybrid compounds 9a, 9b, 9d, 9e, 9g and 9h were
submitted to cytotoxicity and anti-HSV-1 potency evalua-
tion (Table 2).
Fig. 2 Molecular structure of compound 9b, 9c and 9d (Ellipsoids are
drawn at 50% probability). Occupational disorder in compounds 9c
and 9d has been omitted to clarify
All compounds were less cytoxic (CC = 1612, 6103,
5
0
6
765, 2177, 3109, and 1811 µM) than was acyclovir, which
crystallizes in a special position on the mirror symmetry
element. The N–O bond length is 1.288(3) Å. Occupational
disorder was observed in the ethyl group. The carboxylate
group and 1H-1,2,3-triazole ring are coplanar and all atoms
from these groups lie in m mirror plane. Short C–H⋯O
intermolecular contacts between C(10)–H(10A) and
presented a CC of 860 ± 54 µM, under our assay condi-
50
tions (Table 2). These derivatives were 2–7.8-times less
cytotoxic than acyclovir. A change in the substitution pat-
tern of 1H-1,2,3-triazole ring of these compounds caused
(
−1+x,y,−1+z)
O(1)
from nitroxide group along direction [101].
Table 2 Cytotoxicity, anti-HSV-1 activity, and selectivity index in
vero cells for hybrid compounds 9a, 9b, 9d, 9e, 9g and 9h
(
1+x,y,z)
It was also observed as well as C(6)–H(6)⋯N(3)
between 1H-1,2,3-triazole moities along crystallographic
axis a. These intermolecular interactions contribute to
stabilize the crystal packing. In the crystal of compound
CC50a (µM)
IC50b (µM)
SIc
Compounds
9
9
9
9
9
9
a
b
d
e
g
h
1612± 101
6103± 28
6765± 152
2177± 240
3109± 85
1811± 78
860± 25
1.32± 0.20
3.80± 0.80
1.01± 0.50
1.80± 0.09
0.80± 0.08
1.17± 0.10
0.99± 0.02
1221
1606
6698
1209
3886
1547
869
9
d was observed twining and occupational disorder in
-hydroxypropan-2-yl group. The N–O bond length is
.285(3) Å. The main intermolecular interactions in the
2
1
(
1+x,y,z)
crystal are formed by the contacts C(10)–H(10)⋯N3
(
2.5−x,0.5+y,0.5−z)
and O(2)–H(2)⋯O1
along crystallographic
ACV
axis a.and b, respectively.
Acyclovir (ACV) drug was used for the comparative study
a
CC : cytotoxic concentration or concentration required to reduce
5
0
Anti‑HSV‑1 activity
the viability of host cells by 50%
b
IC : 50% Eꢀective concentration or concentration required to
5
0
The synthetic nitroxides compounds were evaluated for
their antiviral inhibitory eꢀect on HSV-1 (Table 1). Three
inhibit HSV-1
c
SI: selectivity index (CC /IC )
5
0
50
1
3

Molecular Diversity
distinguishable diꢀerence in their cytotoxic eꢀects on cell
vero.
The vero cells were purchase from the cell bank of the
Federal University of Rio de Janeiro: https://bcrj.org.br/
pagina/bcrj. The Fetal bovine serum was purchased from
GE Healthcare HyClone: https://www.ꢁshersci.com/shop/
products/hyclone-fetal-bovine-serum-u-s-standard-500ml/
sh3008803.
The compounds 9a, 9d, 9g and 9h, with IC values of
5
0
1
.32, 1.01, 0.80 and 1.17 µM, respectively, displayed potent
activity against HSV-1. The 1H-1,2,3-triazole derivative 9g
was more eꢀective than acyclovir.
The results show that the structural diꢀerence between
compound 9a and 9b inꢂuenced in the antiviral activity.
In the compound 9b, the presence of one electronegative
heteroatom attached between two aromatic rings promoted
decrease in anti-HSV activity. Selective Index (SI; the ratio
of cellular toxicity to antiviral potency in vitro) values,
which represent the in vitro antiviral potency and safety of
a candidate antiviral drug; all compounds were more selec-
tive inhibitors than the reference antiviral drug against HSV.
The best candidates, 9d and 9g, were 7.7 and 4.5 times more
selective than acyclovir (Table 2).
Synthesis
General procedure for the preparation of NRs (9) using click
chemistry with diꢀerent alkynes
To a solution of 0.150 g (0.7 mmol) 8 in CH Cl (5 mL)
2
2
were added 0.110 g (0.9 mmol) of CuBr and (0.9 mmol) of
desired alkyne and stirred for 12 h. Then the reaction mix-
ture was puriꢁed into column chromatography using ethyl
acetate/hexane (7:3) as eluent. Compound 2,2,6,6-tetrame-
thyl-4-(4-phenyl-1H-1,2,3-triazol-1-yl)piperidin-1-oxyl (9a)
was synthesized and its properties were previously described
[31]. The other compounds data are shown below:
The choice of substituent at the C-4 position of 1H-1,2,3-
triazole ring can signiꢁcantly change the toxicity proꢁle as
well as antiviral activity of the compounds synthesized.
2
,2,6,6‑tetramethyl‑4‑(4‑(phenoxymethyl)‑1H‑1,2,3‑tria‑
zol‑1‑yl)piperidin‑1‑oxi (9b)
Conclusion
As a yellow solid in 61% yield (0.152 g), m. p.=177–180 °C.
In summary, a new series of hybrid compounds nitrox-
ide-1H-1,2,3-triazoles 9a–j have been synthesized, charac-
terized by spectroscopic methods and crystal structure of
some of them were determined by single-crystal X-Ray dif-
fraction data and evaluated for their antiviral HSV-1 activi-
ties. The compounds 9a, 9d, 9g and 9h exhibited the highest
anti-HSV-1 activity, being the compound 9g the best result
−
1
IR ν (KBr, cm ): 2921 (=C–H); 1236, 1171 (C–O–C);
max
Anal. Calc. for C H N O : C, 64.74; H, 7.35; N, 17.76.
1
7
23
4
2
Found: C, 65.73; H, 8.45; N, 19.18. Crystals were obtained
from the slow evaporation of its dichloromethane. The struc-
ture of compound 9b was described by X-ray single-crystal
analyses (CCDC: 1954301).
(
IC 0.80 µM). All the compounds exhibited low cytotox-
50
Ethyl 1‑(1‑oxi‑2,2,6,6‑tetramethylpi‑
peridin‑4‑yl)‑1H‑1,2,3‑triazole‑4‑carboxylate (9c)
icity and high selectivity index (SI). The next step is the
evaluation in vivo assay.
As a yellow solid in 80% yield (0.179 g), m. p.=198–199 °C.
−
1
IR ν (KBr, cm ): 1737 (C=O); 1236, 1221 (C–O); Anal.
max
Experimental section
Calc. for C H N O : C, 56.93; H, 7.85; N, 18.97. Found:
1
4
23
4
3
C, 57.02; H, 8.01; N, 19.31. Crystals were obtained from the
slow evaporation of its dichloromethane. The structure of
compound 9c was described by X-ray single-crystal analyses
(CCDC: 1954302).
General methods
All chemicals were reagent grade. Melting points were
determined on a Fisher-Johns instrument and are uncor-
rected. Infrared (IR) spectra were recorded on Perkin-Elmer
4‑(4‑(2‑Hydroxypropan‑2‑yl)‑1H‑1,2,3‑tria‑
zol‑1‑yl)‑2,2,6,6‑tetramethylpiperidin‑1‑ol (9d)
1
420 spectrophotometer in KBr pellets. Microanalyses were
performed on a Perkin-Elmer Model 2400 instrument and
all values were within ± 0.4% of the calculated composi-
tions. X-ray diꢀraction data collections were performed on
a Bruker Kappa-CCD diꢀractometer using graphite-mono-
chromatized Mo Kα radiation (λ=0.71069 Å) at room tem-
perature. All the structures were solved by direct methods
As a yellow solid in 61% yield (0.131 g), m. p.=207–208 °C.
−
1
IR ν (KBr, cm ): 3428 (O–H); 2977 (C–H); Anal. Calc.
max
for C H N O : C, 59.76; H, 8.96; N, 19.91. Found: C,
1
4
25
4
2
60.01; H, 8.50; N, 19.24. Crystals were obtained from the
slow evaporation of its dichloromethane. The structure of
compound 9d was described by X-ray single-crystal analyses
(CCDC: 1954303).
2
and reꢁned by full-matrix least-squares on F with SHELX
software package [40].
1
3

Molecular Diversity
2
,2,6,6‑Tetramethyl‑4‑(4‑((tetrahydro‑2H‑pyran‑2‑yl)
Anti‑HSV‑1 activity
oxy)‑1H‑1,2,3‑triazol‑1‑yl)piperidin‑1‑oxi (9e)
Cells and virus
As a yellow solid in 61% yield (0.151 g), m. p.=167–169 °C.
−1
IR ν (KBr, cm ): 2929 (C–H); 1124, 1033 (C–O–C); Anal.
Compounds were tested as inhibitors of HSV-1 in vero
(African green monkey kidney cells) purchased from the
cell bank of the Federal University of Rio de Janeiro:
https://bcrj.org.br/pagina/bcrj. These cells were grown in
Dulbecco’s modiꢁed Eagle’s medium (DMEM) with 5%
fetal bovine serum (FBS), purchased from GE Healthcare
HyClone: https://www.ꢁshersci.com/shop/products/hyclo
ne-fetal-bovine-serum-u-s-standard-500ml/sh3008803. The
culture medium was supplemented with antibiotics (penicil-
lin, 100 U/mL, and streptomycin, 100 g/mL). Cells were
max
Calc. for C H N O : C, 59.42; H, 8.41; N, 17.32. Found: C,
1
6
27
4
3
6
1.34; H, 8.40; N, 16.14.
4‑(4‑(3‑Aminophenyl)‑1H‑1,2,3‑triazol‑1‑yl)‑2,2,6,6‑tetra‑
methylpiperidin‑1‑oxi (9f)
As a yellow solid in 42% yield (0.100 g), m. p.=224–229 °C.
−
1
IR ν
(KBr, cm ): 2922 (=C–H); Anal. Calc. for
max
C H N O: C, 68.43; H, 7.43; N, 18.78. Found: C, 65.04; H,
17
22
4
7
.56; N, 17.00.
kept at 37 °C in 5% CO . HSV-1 infection was performed
2
in vero cells under the multiplicity of infection (MOI) of
0.1 for 1 h, at temperature described above. After 2 days
post-infection (p.i.), cellular supernatants were harvested
and stored at −70 °C.
4
‑(4‑(3‑Chloropropyl)‑1H‑1,2,3‑triazol‑1‑yl)‑2,2,6,6‑tetra‑
methylpiperidin‑1‑ol (9g)
As a yellow solid in 64% yield (0.146 g), m. p.=186–189 °C.
−
1
IR ν
(KBr, cm ): 2926 (C–H); Anal. Calc. for
max
C H ClN O: C, 56.08; H, 8.07; N, 18.69. Found: C, 56.65;
Cytotoxicity assay
14
24
4
H, 7.77; N, 17.73.
4
Conꢂuent vero cells, at density of 10 cells/well in 96-mul-
4‑(4‑(1‑Hydroxycyclohexyl)‑1H‑1,2,3‑tria‑
tiwell plates, were exposed to various concentrations of the
compounds under investigation or Acyclovir, the positive
control. After 3 days, a marker of viable cells, 2,3-bis(2-
methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxan-
ilide (XTT), at concentration of 2.5 mg/mL, as added in
association with phenazine methosulfate (PMS) at 32 µg/
mL. Optical density was quantiꢁed at 492 and 620 nm wave-
lengths, 4 h after the incubation period. The classical phar-
macological parameter to determine the cellular cytotoxicity
by 50% (CC ) was used to indicate the compound safety
zol‑1‑yl)‑2,2,6,6‑tetramethylpiperidin‑1‑oxi (9h)
As a yellow solid in 66% yield (0.161 g), m. p.=187–189 °C.
−1
IR ν (KBr, cm ): 3398 (O–H); 2934 (C–H); Anal. Calc.
max
for C H N O : C, 63.52; H, 9.09; N, 17.43. Found: C, 62.37;
17 29 4 2
H, 8.48; N, 16.26.
4‑(4‑(2‑Hydroxy‑4‑methylpentan‑2‑yl)‑1H‑1,2,3‑tria‑
zol‑1‑yl)‑2,2,6,6‑tetramethylpiperidin‑1‑ol (9i)
5
0
for cell culture. CC was determined based on nonlinear
5
0
As a yellow solid in 58% yield (0.129 g), m. p.=163–166 °C.
regression curves of the dose–response data.
−1
IR ν (KBr, cm ): 3489 (O–H); 2974 (C–H); Anal. Calc.
max
for C H N O : C, 63.13; H, 9.66; N, 17.32. Found: C, 61.73;
17 31 4 2
H, 9.85; N, 19.95.
Anti‑HSV‑1 assays
4
4
‑(4‑(Cyclohex‑1‑en‑1‑yl)‑1H‑1,2,3‑tria‑
Monolayers of vero cells, at density of 10 cells in each/well
zol‑1‑yl)‑2,2,6,6‑tetramethylpiperidin‑1‑oxi (9j)
(96-well plates), were infected with HSV-1 for 1 h, with
inoculum at dose of 100 CCID /well. Inoculum was then
5
0
As a yellow solid in 44% yield (0.102 g), m. p.=205–208 °C.
removed, cells were washed and fresh medium with 50 µM
of the investigated compounds was added. After 3 days, the
XTT/PMS mixture described above was injected into the
well to monitor cells viability, which indirectly measures
HSV-1-induced cytopathic eꢀect [41].
−1
IR ν (KBr, cm ): 2926 (C–H); Anal. Calc. for C H N O:
max
17 27 4
C, 67.29; H, 8.97; N, 18.46. Found: C, 67.82; H, 8.60; N,
17.69.
The best compounds were further tested at diꢀerent con-
centrations, to determine the antiviral potency, also known
as inhibitory activity by 50% (IC ). Conꢂuent vero cells in
5
0
6
-well plates were exposed to HSV-1 inoculum at MOI of 5
for 1 h at 37 °C. Next, cells were washed, treated for 24 h,
1
3

Molecular Diversity
lysed and centrifuged. The supernatant was harvested and
tittered [36]. All assays were also performed with acyclovir,
synthesized substituted pyrimidine, thiopyrimidine and thiazo-
lopyrimidine derivatives. Eur J Med Chem 45:1494–1501. https
:
//doi.org/10.1016/j.ejmech.2009.12.057
the positive control. The IC was established by nonlinear
5
0
13. Wen Y, Zhang Z, Liu N-N, Andrei G, Snoeck R, Xiang Y-H,
Schols D, Chen X, Zhang Z-Y, Zhang Q-S, Wu Q-P (2017)
Synthesis and antiviral activity of 5-(benzylthio)-4-carba-
myl-1,2,3-triazoles against human cytomegalovirus (CMV) and
varicella-zoster virus (VZV). Med Chem 13:453–464. https://
doi.org/10.2174/1573406413666170307165236
regression from dose–response curves.
Acknowledgements Fellowships granted by CAPES and by CNPq
(
Brazil) are gratefully acknowledged. This work was partially sup-
ported by CNPq, FINEP, FAPERJ and UFF. The authors acknowledge
LDRX-UFF for using its laboratory facilities.
14. Mohamed SF, Abbas EMH, Khalaf HS, Farghaly TA, Abd
El-Shafy D (2017) Triazolopyrimidines and thiazolopyrimi-
dines: synthesis, anti-HSV-1, cytotoxicity and mechanism
of action. Mini-Rev Med Chem 18:794–802. https://doi.
org/10.2174/1389557518666171207161542
Compliance with ethical standards
Conflict of interest The authors declare no competing or ꢁnancial con-
15. Jordão AK, Ferreira VF, Souza TML, Faria GGS, Machado
V, Abrantes JL, de Souza MCBV, Cunha AC (2011) Synthe-
sis and anti-HSV-1 activity of new 1,2,3-triazole derivatives.
Bioorg Med Chem 19:1860–1865. https://doi.org/10.1016/j.
bmc.2011.02.007
ꢂict of interest.
References
16. Khan FY, Elhiday A, Khudair IF, Youssef H, Omran AH, Alsam-
man SH, Elhamid M (2012) Evaluation of the use of piperacillin/
tazobactam (Tazocin®) at Hamad General Hospital, Qatar: are
there unjustiꢁed prescriptions? Infect Drug Resist 5:17–21. https
1
2
3
.
.
.
Nicoll MP, Proença JT, Efstathiou S (2012) The molecular basis of
herpes simplex virus latency. FEMS Microbiol Rev 36:684–705.
https://doi.org/10.1111/j.1574-6976.2011.00320.x
:
//doi.org/10.2147/idr.s27965
1
7. Long TE, Williams JT (2014) Cephalosporins currently in early
clinical trials for the treatment of bacterial infections. Expert Opin
Investig Drugs 23:1375–1387. https://doi.org/10.1517/13543
Rajasagi NK, Rouse BT (2019) The role of T cells in herpes stro-
mal keratitis. Front Immunol 10:1–7. https://doi.org/10.3389/
ꢁ
mmu.2019.00512
7
84.2014.930127
Zhurilo NI, Chudinov MV, Matveev AV, Smirnova OS, Konstan-
tinova ID, Miroshnikov AI, Prutkov AN, Grebenkina LE, Pulkova
NV, Shvets VI (2018) Isosteric ribavirin analogues: synthesis and
antiviral activities. Bioorg Med Chem Lett 28:11–14. https://doi.
org/10.1016/j.bmcl.2017.11.029
1
1
8. Wheless JW, Vazquez B (2010) Ruꢁnamide: a novel broad-spec-
trum antiepileptic drug. Epilepsy Curr 10:1–6. https://doi.org/10
.
1111/j.1535-7511.2009.01336.x
9. Wang Y, Cong C, Chai WC, Dong R, Jia L, Song D, Zhou Z,
Ma S (2017) Synthesis and antibacterial activity of novel
4
5
6
.
.
.
Dheer D, Singh V, Shankar R (2017) Medicinal attributes of
4
″-O-(1-aralkyl-1,2,3-triazol-4-methyl-carbamoyl) azithromy-
1
,2,3-triazoles: Current developments. Bioorg Chem 71:30–54.
cin analogs. Bioorg Med Chem Lett 27:3872–3877. https://doi.
org/10.1016/j.bmcl.2017.06.044
https://doi.org/10.1016/j.bioorg.2017.01.010
Poole CL, James SH (2018) Antiviral therapies for herpesviruses:
current agents and new directions. Clin Ther 40:1282–1298. https
2
2
0. Gonzaga DTG, Souza TML, Andrade VMM, Ferreira VF, da Silva
FC (2018) Identiꢁcation of 1-Aryl-1H-1,2,3-triazoles as Potential
New Antiretroviral Agents. Med Chem 14:242–248. https://doi.
org/10.2174/1573406413666170906121318
:
//doi.org/10.1016/j.clinthera.2018.07.006
Czartoski T, Liu C, Koelle DM, Schmechel S, Kalus A, Wald A
(
2006) Fulminant, acyclovir-resistant, herpes simplex virus type
1. Jordão AK, Ferreira VF, Lima ES, de Souza MCBV, Carlos ECL,
Castro HC, Geraldo RB, Rodrigues CR, Almeida MCB, Cunha
AC (2009) Synthesis, antiplatelet and in silico evaluations of
novel N-substituted-phenylamino-5-methyl-1H-1,2,3-triazole-
2
hepatitis in an immunocompetent woman. J Clin Microbiol
4:1584–1586. https://doi.org/10.1128/JCM.44.4.1584-1586.2006
4
7
.
Chen Y, Scieux C, Garrait V, Socié G, Rocha V, Molina JM, Thou-
venot D, Morꢁn F, Hocqueloux L, Garderet L, Espérou H, Sélimi
F, Devergie A, Leleu G, Aymard M, Morinet F, Gluckman E,
Ribaud P (2000) Resistant herpes simplex virus type 1 infection:
an emerging concern after allogeneic stem cell transplantation.
Clin Infect Dis 31:927–935. https://doi.org/10.1086/314052
Reusser P (2002) Management of viral infections in immunocom-
promised cancer patients. Swiss Med Wkly 132:374–378. https://
doi.org/10.4414/smw.2002.09875
4
-carbohydrazides. Bioorg Med Chem 17:3713–3719. https://
doi.org/10.1016/j.bmc.2009.03.053
2
2
2
2. An R, Hou Z, Li J-T, Yu H-N, Mou Y-H, Guo C (2018) Design,
synthesis and biological evaluation of novel 4-substituted cou-
marin derivatives as antitumor agents. Molecules 23:2281–2292.
https://doi.org/10.3390/molecules23092281
8
9
.
.
3. Kaur P, Chawla A (2018) Green synthesis, structural characteri-
zation and pharmacological evaluation for analgesic and anti-
inꢂammatory activities of salicylic acid based triazolothiadiazole
derivatives. Eur J Biomed Pharm Sci 5:472–479
Wathen MW (2002) Non-nucleoside inhibitors of herpesviruses.
Rev Med Virol 12:167–178. https://doi.org/10.1002/rmv.354
1
0. De Clerq E (2003) New inhibitors of human cytomegalovirus
4. Jordão AK, Sathler PC, Ferreira VF, Campos VR, de Souza
MCBV, Castro HC, Lannes A, Lourenço A, Rodrigues CR, Bello
ML, Lourenço MCS, Carvalho GSL, Almeida MCB, Cunha
AC (2011) Synthesis, antitubercular activity, and SAR study
of N-substituted-phenylamino-5-methyl-1H-1,2,3-triazole-
(
HCMV) on the horizon. J Antimicrob Chemother 51:1079–1083.
https://doi.org/10.1093/jac/dkg205
1
1. Hammond JL, Koontz DL, Bazmi HZ, Beadle JR, Hostetler
SE, Kini GD, Aldern KA, Richman DD, Hostetler KY, Mel-
lors JW (2001) Alkylglycerol prodrugs of phosphonoformate
are potent in vitro inhibitors of nucleoside-resistant human
immunodeficiency virus type 1 and select for resistance
mutations that suppress zidovudine resistance. Antimicrob
Agents Chemother 45:1621–1628. https://doi.org/10.1128/
AAC.45.6.1621-1628.2001
4
-carbohydrazides. Bioorg Med Chem 19:5605–5611. https://
doi.org/10.1016/j.bmc.2011.07.035
2
5. Fedoreyev SA, Krylova NV, Mishchenko NP, Vasileva EA, Pisly-
agin EA, Iunikhina NP, Lavrov VF, Svitich AO, Ebralidze LK,
Loenova GN (2018) Antiviral and antioxidant properties of echi-
nochrome A. Mar Drugs 16:509–518. https://doi.org/10.3390/
md16120509
1
2. Mohamed SF, Flefel EM, Amr AEGE, Abd El-Shafy DN (2010)
Anti-HSV-1 activity and mechanism of action of some new
1
3

Molecular Diversity
2
2
6. Rehman ZU, Meng C, Sun Y, Safdar A, Pasha RH, Munir M, Ding
C (2018) Oxidative stress in poultry: lessons from the viral infec-
tions. Oxid Med Cell Longev. https://doi.org/10.1155/2018/51231
34. Wilcox CS (2010) Eꢀects of tempol and redox-cycling nitroxides
in models of oxidative stress. Pharmacol Ther 126:119–145. https
://doi.org/10.1016/j.pharmthera.2010.01.003
4
7
35. Howieson VM, Tran E, Hoegl A, Fam HL, Fu J, Sivonen K, Li
XX, Auclair K, Saliba KJ (2016) Triazole substitution of a labile
amide bond stabilizes pantothenamides and improves their anti-
plasmodial potency. Antimicrob Agents Chemother 60:7146–
7152. https://doi.org/10.1128/AAC.01436-16
7. Linares E, Giorgio S, Augusto O (2008) Inhibition of in vivo leish-
manicidal mechanisms by tempol: Nitric oxide down-regulation
and oxidant scavenging. Free Radic Biol Med 44:1668–1676.
https://doi.org/10.1016/j.freeradbiomed.2008.01.027
2
2
8. Lewandowski M, Gwozdzinski K (2017) Nitroxides as antioxi-
dants and anticancer drugs. Int J Mol Sci 18:2490–2515. https://
doi.org/10.3390/ijms18112490
36. Bahia SBBB, Reis WJ, Jardim GAM, Souto FT, de Simone CA,
Gatto CC, Menna-Barreto RFS, de Castro SL, Cavalcanti BC,
Pessoa C, Araújo MH, da Silva Júnior EN (2016) Molecular
hybridization as a powerful tool towards multitarget quinoidal
systems: synthesis, trypanocidal and antitumor activities of naph-
thoquinone-based 5-iodo-1,4-disubstituted-, 1,4- and 1,5-disubsti-
tuted-1,2,3-triazoles. MedChemComm 7:1555–1563. https://doi.
org/10.1039/c6md00216a
9. Thomas K, Moody TW, Jensen RT, Tong J, Rayner CL, Barnett
NL, Fairfull-Smith KE, Ridnour LA, Wink DA, Bottle SE (2018)
Design, synthesis and biological evaluation of hybrid nitroxide-
based non-steroidal anti-inꢂammatory drugs. Eur J Med Chem
1
47:34–47. https://doi.org/10.1016/j.ejmech.2018.01.077
3
3
0. Nunes DVQ, Costa CA, De Bem GF, Cordeiro VSC, Santos IB,
Carvalho LCRM, Jordão AK, Cunha AC, Ferreira VF, Moura
RS, Resende AC, Ognibene DT (2018) Tempol, a superoxide
dismutase-mimetic drug, prevents chronic ischemic renal injury
in two-kidney, one-clip hypertensive rats. Clin Exp Hypertens
37. Lutz WB, Lazarus S, Meltzer RI (1962) New derivatives of
2,2,6,6-tetramethylpiperidine. J Org Chem 27:1695–1703. https
://doi.org/10.1021/jo01052a050
38. Rauckman EJ, Rosen GM, Abou-Donia MB (1975) Improved
methods for the oxidation of secondary amines to nitroxides.
Synth Commun 5:409–413. https://doi.org/10.1080/0039791750
8065573
4
0:721–729. https://doi.org/10.1080/10641963.2018.1425423
1. Queiroz RF, Jordão AK, Cunha AC, Ferreira VF, Brigagão MRPL,
Malvezzi A, de Amaral AT, Augusto O (2012) Nitroxides attenu-
ate carrageenan-induced inꢂammation in rat paws by reducing
neutrophil inꢁltration and the resulting myeloperoxidase-medi-
ated damage. Free Radic Biol Med 53:1942–1953. https://doi.
org/10.1016/j.freeradbiomed.2012.09.001
39. Zhou B-H, Chen Y-F, Yin G-D, Wu A-X (2006) Synthesis and
crystal structure of 4-azido-2,2,6,6-tetramethylpiperidine-1-oxyl
free radical. Chinese J Struct Chem 25:127–130
40. Sheldrick GM (2015) Crystal structure reꢁnement with SHELXL.
Acta Cryst Section C Struct Chem C71:3–8. https://doi.
org/10.1107/S2053229614024218
3
3
2. Wang H, Gao P, Jing L, Qin X, Sun X (2012) The heart-protective
mechanism of nitronyl nitroxide radicals on murine viral myo-
carditis induced by CVB3. Biochimie 94:1951–1959. https://doi.
org/10.1016/j.biochi.2012.05.015
41. Pauwels R, Balzarini J, Baba M, Snoeck R, Schols D, Herd-
ewijn P, Desmyter J, De Clercq E (1988) Rapid and automated
tetrazolium-based colorimetric assay for the detection of anti-
HIV compounds. J Virol Methods 20:309–321. https://doi.
org/10.1016/0166-0934(88)90134-6
3. Soule BP, Hyodo F, Matsumoto K-I, Simone NL, Cook JA,
Krishna MC, Mitchell JB (2007) The chemistry and biology of
nitroxide compounds. Free Radic Biol Med 42:1632–1650. https
:
//doi.org/10.1016/j.freeradbiomed.2007.02.030
Aꢀliations
1
2
3
4
5,6
Anna C. Cunha · Vitor F. Ferreira · Maria G. F. Vaz · Rafael A. Allão Cassaro · Jackson A. L. C. Resende
·
7
7
7
7,8
9
Carolina Q. Sacramento · Jéssica Costa · Juliana L. Abrantes · Thiago Moreno L. Souza · Alessandro K. Jordão
1
2
3
4
5
6
7
8
Departamento de Química Orgânica, Instituto de Química,
Outeiro de São João Batista, Universidade Federal
Fluminense, Niterói, RJ 24020-141, Brazil
Instituto de Ciências Exatas E da Terra, Universidade Federal
do Mato Grosso, Barra do Garças, MT 78698-000, Brazil
Laboratório de Imunofarmacologia, Instituto Oswaldo Cruz,
FIOCRUZ, Rio de Janeiro, Brazil
Departamento de Tecnologia Farmacêutica, Faculdade de
Farmácia, Universidade Federal Fluminense, Rua Dr. Mário
Vianna 523, Niterói, RJ 24241-002, Brazil
National Institute for Science and Technology On Innovation
On Diseases of Neglected Populations (INCT/IDPN), Center
for Technological Development in Health (CDTS), Fiocruz,
Rio de Janeiro, RJ, Brazil
Departamento de Química Inorgânica, Instituto de Química,
Outeiro de São João Batista, Universidade Federal
Fluminense, Niterói, RJ 24020-141, Brazil
9
Departamento de Farmácia, Universidade Federal do Rio
Grande do Norte, Natal, RN 59012-570, Brazil
Departamento de Química Inorgânica, Instituto de Química,
Universidade Federal Do Rio de Janeiro, Rio de Janeiro,
RJ 21941-972, Brazil
Laboratório de difração de Raios X, Programa de
Pós-Graduação Em Química, Universidade Federal
Fluminense, Niterói, RJ 24020-141, Brazil
1
3