Antioxidant and anti-sickling activity of glucal-based triazoles compounds – An in vitro and in silico study

Article abstract of DOI:10.1016/j.bioorg.2021.104709

The sickle cell disease (SCD) has a genetic cause, characterized by a replacement of glutamic acid to valine in the β-chain of hemoglobin. The disease has no effective treatment so far, and patients suffer a range from acute to chronic complications that include chronic hemolytic anemia, vaso-occlusive ischemia, pain, acute thoracic syndrome, cerebrovascular accident, nephropathy, osteonecrosis and reduced lifetime. The oxidation in certain regions of the hemoglobin favors the reactive oxygen species (ROS) formation, which is the cause of many clinical manifestations. Antioxidants have been studied to reduce the hemoglobin ROS levels, and in this sense, we have searched for new antioxidants glucal-based triazoles compounds with anti-sickling activity. Thirty analogues were synthetized and tested in in vitro antioxidant assays. Two of them were selected based in their effects and concentration–response activity and conducted to in cell assays. Both molecules did not cause any hemolysis and could reduce the red blood cell damage caused by hydrogen peroxide, in a model of oxidative stress induction that mimics the SCD. Moreover, one molecule (termed 11m), besides reducing the hemolysis, was able to prevent the cell damage caused by the hydrogen peroxide. Later on, by in silico pharmacokinetics analysis, we could see that 11m has appropriated proprieties for druggability and the probable mechanism of action is the binding to Peroxiredoxin-5, an antioxidant enzyme that reduces the hydrogen peroxide levels, verified after molecular docking assays. Thus, starting from 30 glucal-based triazoles molecules in a structure–activity relationship, we could select one with antioxidant proprieties that could act on RBC to reduce the oxidative stress, being useful for the treatment of SCD.

Full text of DOI:10.1016/j.bioorg.2021.104709

Bioorganic Chemistry 109 (2021) 104709
Contents lists available at ScienceDirect
Bioorganic Chemistry
journal homepage: www.elsevier.com/locate/bioorg
Antioxidant and anti-sickling activity of glucal-based triazoles compounds –
An in vitro and in silico study
a
b
c
b,*
´
Rodinei Vieira Veloso , Anwar Shamim , Yann Lamarrey , Helio A. Stefani
,
,
*
Juliana Mozer Sciania
a
´
˜
Laboratorio Multidisciplinar de Pesquisa, Universidade Sao Francisco, Bragança Paulista, SP, Brazil
b
c
´
ˆ
ˆ
˜
˜
Departamento de Farmacia, Faculdade de Ciencias Farmaceuticas, Universidade de Sao Paulo, Sao Paulo, SP, Brazil
´
˜
˜
˜
Laboratorio de Biologia Molecular, Hemocentro de Ribeirao Preto, Universidade de Sao Paulo, Ribeirao Preto, SP, Brazil
A R T I C L E I N F O
A B S T R A C T
Keywords:
The sickle cell disease (SCD) has a genetic cause, characterized by a replacement of glutamic acid to valine in the
β-chain of hemoglobin. The disease has no effective treatment so far, and patients suffer a range from acute to
chronic complications that include chronic hemolytic anemia, vaso-occlusive ischemia, pain, acute thoracic
syndrome, cerebrovascular accident, nephropathy, osteonecrosis and reduced lifetime. The oxidation in certain
regions of the hemoglobin favors the reactive oxygen species (ROS) formation, which is the cause of many
clinical manifestations. Antioxidants have been studied to reduce the hemoglobin ROS levels, and in this sense,
we have searched for new antioxidants glucal-based triazoles compounds with anti-sickling activity. Thirty
analogues were synthetized and tested in in vitro antioxidant assays. Two of them were selected based in their
effects and concentration–response activity and conducted to in cell assays. Both molecules did not cause any
hemolysis and could reduce the red blood cell damage caused by hydrogen peroxide, in a model of oxidative
stress induction that mimics the SCD. Moreover, one molecule (termed 11m), besides reducing the hemolysis,
was able to prevent the cell damage caused by the hydrogen peroxide. Later on, by in silico pharmacokinetics
analysis, we could see that 11m has appropriated proprieties for druggability and the probable mechanism of
action is the binding to Peroxiredoxin-5, an antioxidant enzyme that reduces the hydrogen peroxide levels,
verified after molecular docking assays. Thus, starting from 30 glucal-based triazoles molecules in a structur-
e–activity relationship, we could select one with antioxidant proprieties that could act on RBC to reduce the
oxidative stress, being useful for the treatment of SCD.
Sickle cell disease
Antioxidant
Glucal-based triazoles compounds
1. Introduction
cells (RBC), constituted by a tetramer (2 subunits α e 2 subunits β), with
function of oxygen transportation. Each subunit has a covalent-bound
heme group, constituted by a protoporphyrin and one iron atom in its
Free radicals or reactive oxygen species (ROS) are atoms with one or
more unpaired electrons in their orbit, turning instable as highly reac-
tive, by donating or receiving electrons [45].
center. The oxygen binds irreversible to Fe2+ and produce Fe3+
.
Nevertheless, the heme group protects the Fe2+ oxidation by make the
bind reversible and restoring the protein oxygen binding capacity [59].
The hemoglobin mutation in the SCD causes protein polymerization
of tetramers and consequently instability, due to the change of the
protein electric charge. Consequently, the red blood cells (RBC) become
deformed and rigid, in elongated and bending shape [41]. Moreover, the
ability of RBC in transporting oxygen is reduced, especially in the
microvasculature, which causes hemolysis, elimination of nitric oxide,
and alteration in the RBC permeability [56,43,13].
Several diseases are related to the ROS formation, which can cause
several cell damages, intra or extracellular protein alterations, and DNA
modification. Thus, the harmful role of ROS has been associated to
neurodegenerative and cardiovascular diseases, cancer, and sickle cell
disease (SCD) [31].
The SCD has a genetic cause, characterized by the replacement of
glutamic acid to a valine in the β-chain of hemoglobin, being this mu-
tation homozygotic (HbSS) or heterozygotic (HbSC) [3].
Hemoglobin is a 64.4 kDa vascular protein present in the red blood
The symptoms suffered by such patients range from acute to chronic
˜
˜
˜
* Corresponding authorsat: Universidade Sao Francisco, Avenida Sao Francisco de Assis, 218, Bragança Paulista, Sao Paulo CEP: 12916-900. Brazil.
E-mail address: juliana.sciani@usf.edu.br (J. Mozer Sciani).
https://doi.org/10.1016/j.bioorg.2021.104709
Received 8 October 2020; Received in revised form 4 January 2021; Accepted 28 January 2021
Available online 9 February 2021
0045-2068/© 2021 Elsevier Inc. All rights reserved.

R. Vieira Veloso et al.
Bioorganic Chemistry 109 (2021) 104709
R1
N
TMS
TMS
BF3K
O
PhN3 (1.2 eq.)
CuI ( 7 mol%)
(1.2 equiv.)
N
AcO
O
O
N
AcO
AcO
AcO
AcO
BF3 .OEt2
AcO
ACN, 0 oC, 30 min.
TBAF ( 1.2 eq.)
DMF, 80 oC, 2 h
up to 95 %
OAc
1
80 %
3a-c
2
Pd(PPh3) (5 mol%)
NaN3 (1.2 eq.)
THF:H2O (7:3)
R1
R1
N
N
R1
N
N
R1
N
N
N
N
O
O
Ph
(1.2 eq.)
N
N
N
AcO
AcO
OAc
OAc
CuI (7 %)
N
N
PMDETA (1.2 eq.)
O
O
N
N
N
N
N
DMF, 70 oC, 2 h
N3
N3
4a-c
up to 82%
(over two steps)
R2
R2
5a-q
Scheme 1. Synthesis of bis-triazolyl glycosal derivatives from 3,4,6-tri-O-acetyl-D-glucal.
complications, and include chronic hemolytic anemia, vaso-occlusive
ischemia, pain, acute thoracic syndrome, cerebrovascular accident, ne-
phropathy, osteonecrosis and reduced lifetime [23,1].
and neuroregenerative activities [28]. Antioxidant activity has also been
associated with this structure, with an unknown mechanism [2].
Its pharmacokinetics and pharmacodynamics properties are favor-
able, and generally binds easily with their therapeutic targets. For
example, 1,4 disubstituted 1,2,3-triazol has a CH-bond as a hydrogen
donor and N-3 pair as hydrogen acceptor [5].
Besides the alteration in RBC morphology, the HbS hemoglobin
isoform results in the ROS production inside the RCB, in the prosthetic
group heme containing iron atoms, a highly oxidative region in the
hemoglobin. This region, called “oxidative hotspots”, contains certain
amino acids that favor the ROS formation, such as βCys93, able to be
irreversibly oxidized, which contributes to the collapse, denaturation
and degradation of the hemoglobin β subunit, causing the release of the
heme group, essential for the oxygen binding. It is important to mention
that this region is important for the transition of R (the relaxed state) to
T (the tense state), representing a higher and lower affinity for oxygen,
respectively [20].
Moreover, the presence of glucal in their structure tends to increase
the molecule stability, increases the polarity, and makes them compat-
ible with the biologic fluids due to the stereogenic center of carbohy-
drates [10].
Here we have evaluated the antioxidant activity of 30 glucal-based
triazole compounds and studied their neutralization effect on the
oxidative stress induced in RBC, simulating the SCD. Moreover, the
pharmacokinetics and molecular target were studied, in order to check
their druggability and the possible mechanism of action.
Toxic radicals O2 and H2O2 are frequently formed, which increases
the antioxidant enzymes activity (catalase and dismutase superoxide) as
a response for this oxidative stress. The increase of NADPH oxidase
expression is another internal source for toxic ROS production, besides
active mitochondria retention by mature mutated hemoglobin-RBCs [1].
The RBC membrane is a target for oxidative stress, as it is abundant
in -SH groups, converted into disulfide binds (-R-SSG) from the oxidant
agents, which causes proteins’ denaturation and lipoperoxidation of
lipidic components of the membrane, inducing cell hemolysis [9].
Due to the importance of oxidative stress in the SCD context, anti-
oxidants have been considered for the treatment of such disease, which
today is based on drugs to treat the symptoms (e.g. analgesics) and blood
transfusion.
2. Material and methods
2.1. Synthesis
The synthesis of the libraries of compounds below are categorized
based on the difference in synthetic procedures.
2.1.1. Synthesis of glycosal based bis-triazole derivatives
The synthesis of glycosal based bis-triazole derivatives started with
3,4,6-tri-O-acetyl-D-glucal 1, using Ferrier rearrangement to obtain
glycosal alkyne 2, followed by coper catalyzed azide-alkyne cycloaddi-
tion (CuAAC) click reaction to obtain the glycosyl-triazoles 3a-c
[51,47]. These glycosyl triazoles 3a-c, were then used in palladium-
catalyzed Tsuji-Trost type allylic azidation to obtain regioisomeric glu-
cal based allylic azides 4a-c followed by a second CuAAC click reaction
to obtain 17 bis-triazole compounds 5a-q as shown in Scheme 1 [50].
Hydroxyurea and L-glutamine are drugs approved by FDA for SCD,
although the mechanism has not been completely elucidated yet [11]. It
is known that the hydroxyurea inhibits the hemoglobin polymerization
and protects the βCys93, while the L-glutamine increases the NAD syn-
thesis, reducing the ROS production [39].
Classic antioxidants available, such as ascorbic acid, have inadequate
physical–chemical properties, and consequently poor pharmacokinetics,
impairing the molecule to reach the molecular target in a therapeutic
concentration. Therefore, new druggable antioxidants need to be
studied.
2.1.2. Synthesis 2-alkenyl mono-triazole derivatives of D-glucal
The trimethylsilyl protected alkyne derivatives of glucal 10a-g were
synthesized from 3,4,6-tri-O-acetyl-D-glucal based on the reported
methods [49,46,58]. This includes the synthesis of 2-iodo-3,4,6-tri-O-
acetyl-D-glucal 6 starting from the acetylated D-glucal, followed by the
copper and ligand free Sonogashira coupling and regioselective
palladium-catalyzed hydrostannation to obtain the corresponding
alkynyl derivatives 7a-e and stannyl derivatives 8a-e of D-glucal,
respectively. The stannyl derivatives were subjected to iodination to
afford 9a-f and Sonogashira coupling to obtain 10a-g which were ulti-
mately applied in the CuAAC click reaction to obtain the desired triazole
Triazoles are heterocyclic compounds with five-membered unsatu-
rated ring containing 3 nitrogen atoms, with several pharmacological
proprieties [21]. Antifungal activity is the most known, and the
important functional groups for such structure are the 1,2,3-triazole and
1,2,4-triazole rings [29].
However, besides being antifungal, this scaffold has been used for the
synthesis of molecules applied for antimicrobial, antiviral, antitumor
2

R. Vieira Veloso et al.
Bioorganic Chemistry 109 (2021) 104709
NIS (1.2 eq.)
AgNO3 (0.2 eq.)
R, (1.2 eq.)
O
O
O
AcO
AcO
AcO
AcO
AcO
AcO
Pd(OAc)2 (5 mol%)
ACN, 80 oC,
4-5 h
I
Cs2CO3 (1.5 eq.)
DMF, rt, 0.3-6 h
OAc
6
OAc
7a-e
R1
OAc
65%
77-96
% yield
PdCl2(PPh3)2 (2 mol%)
HSnBu3 (1.2 eq.)
60-85%
THF, r.t,
3-6 h
TMS, (1.2 eq.)
O
O
I2 (1.2 eq.)
DCM, rt, 2-5 h
AcO
AcO
R1
AcO
AcO
R1
PdCl2(PPh3)2 (5 mol%)
I
SnBu3
CuI (5 mol%), DMF:NEt3 (1:1)
rt, 12h
up to 98 %
OAc
OAc
OAc
OH
9a-f
8a-e
K2CO3, MeOH
91-95%
R2N3 (1.2 eq.)
CuI (5%), PMDETA(1.2 eq.)
O
O
Ac/HO
Ac/HO
R1
Ac/HO
Ac/HO
R1
TBAF (1.1 eq.), DMF, rt, 12 h
N
N
N
OH/Ac
OH/Ac
11a-m
75-95%
10a-g
TMS
R2
Scheme 2. Synthesis of 2-alkenyl mono-triazole derivative of D-glucal.
based glucal compounds 11a-m as summarized in Scheme 2 [48].
transferred to 96-well plate for the hemoglobin measure by spectro-
photometer at 414 nm. Data were compared with negative control (PBS
buffer) and a positive control (Triton X-100 0.1%). Experiments were
performed in triplicate and the percent hemolysis was calculated as
follows: (%) = (Asample ꢀ Abuffer)/(Atriton ꢀ Abuffer) × 100.
2.2. Antioxidant activity
The antioxidant activity of molecules was determined by 1,1-
diphenyl-2-picrylhydrazyl radical (DPPH) assay and e ferric reducing
antioxidant power (FRAP) assays.
2.4. Oxidative stress in RBC
The DPPH test was conducted in 96-well plate with 30 min incuba-
tion of 0.5, 1 or 2 mM of samples with 0.1 mM DPPH reagent, diluted in
methanol, in the dark. After this period, the mixture had its absorbance
read by a spectrophotometer at λ = 515 nm. The % of radical scavenging
activity was calculated by: [(Ac - As)/ Ac)] × 100, where Ac = absor-
bance of the control and As = absorbance of the test sample.
For the PFRAP assay, phosphate-saline buffer (PBS 50 mM pH 7.3)
was incubated with 1% potassium ferrocyanide and samples (0.5, 1 or
2 mM) for 20 min at 50 ◦C. After that, 10% trichloroacetic acid was
added and the solution centrifuged at 3000 rpm for 10 min. An aliquot
was added to distilled water and 0.1% ferric chloride, and the solution
had its absorbance read by a spectrophotometer at λ = 700 nm. The
relative % of reducing power was calculated as (A- Amin) / Amax -
Amin) × 100, where A = absorbance of the sample, Amin = absorbance
of control and Amax = Highest absorbance of standard.
The molecules were tested in RBC induced with oxidative stress in
order to evaluate the prevention or treatment effect (impairment or
removal of free radicals).
For prevention test, RBC (20
with 50
μL of 4% suspension) were incubated
μ
L PBS and 1 mM molecules for 60 min at 37 ◦C. After that, 1%
H2O2 was added for 10 min and then the mixture was centrifuged for
4000 rpm for 5 min at room temperature. An aliquot of supernatant was
transferred to a 96-well plate and the absorbance was read at
λ = 414 nm.
For treatment test, RBC (20 μL of 4% suspension) were incubated
with 50 μL PBS and 1% H2O2 for 10 min. After that, 1 mM molecules
were incubated for 60 min at 37 ◦C. The mixture was centrifuged and
read at spectrophotometer at the same protocol described above.
Experiments were performed in triplicate and compared to a nega-
tive (PBS) and positive (H2O2) control.
For both assays, a control (methanol or buffer instead the sample)
was added and ascorbic acid was used as reference standard. The sam-
ples were analyzed in triplicate and the results are shown as featuring
potential antioxidant activity.
2.5. In silico ADME parameters, toxicity, physicochemical descriptors and
druglike nature of compounds
ADME parameters (gastrointestinal absorption, blood–brain barrier
permeation and liver enzymes inhibition) of molecules were predicted
by Swiss ADME. Moreover, the physicochemical properties and drug-
likeness was predicted, following the Lipinski rules [30].
2.3. Hemolytic activity
The hemolysis was performed in human erythrocytes, obtained from
a pool of 20 healthy volunteers (approved by the Ethics Committee from
˜
The toxicity was predicted by admetSAR 2.0 server [54]. Parameters
evaluated were acute oral toxicity and carcinogenicity [57].
Universidade Sao Francisco, CAAE 25441719.0.0000.5514). The blood
was collected in EDTA tubes and centrifuged at 1000g for 10 min at
room temperature. Plasma was removed and red blood cells (RBC) were
washed with phosphate-saline buffer (PBS, 50 mM, pH 7.3). One mL of
RBC was diluted with 9 mL of PBS to give a 4% suspension. Samples
2.6. Molecular docking
(0.5 µM, diluted in PBS) were incubated to 20
μ
L of 4% RBC and 50
μL
The 11m had its 2D structure drawn in OpenBabel Cheminformatics
and saved into mol2 format after being converted into 3D with pH 7 and
no changes in adding or deleting hydrogens.
PBS for 60 min at 37 ◦C. The mixture was centrifuged at 4000 rpm for
5 min at room temperature and an aliquot of supernatant were
3

R. Vieira Veloso et al.
Bioorganic Chemistry 109 (2021) 104709
O
O
S
N
S
N
OAc
N
OAc
N
N
N
N
N
N
OAc
N
OAc
N
O
N
O
N
N
N
N
N
OAc
N
N
O
N
OAc
O
N
N
N
N
O
O
N
N
F
N
N
N
N
N
N
N
N
N
N
N
N
5a
5c
5e
5g
5i
5k
F
O
O
S
S
N
N
N
OAc
N
N
N
N
N
OAc
OAc
N
N
N
N
OAc
N
N
O
OAc
O
O
N
N
O
N
N
OAc
O
N
N
N
N
N
N
O
N
N
N
N
N
F
N
N
N
N
N
N
N
N
N
5b
5d
5f
5h
5j
5l
F
N
N
N
N
N
N
OAc
N
N
N
OAc
N
OAc
OAc
N
N
N
N
N
O
O
O
O
O
N
N
AcO
N
N
N
N
N
N
N
N
N
N
N
N
N
5p
5m
5n
5q
5o
O
O
AcO
OAc
AcO
OAc
(A)
O
O
O
O
O
AcO
AcO
AcO
AcO
AcO
AcO
AcO
N
N
AcO
N
N
N
N
AcO
N
AcO
N
N
OAc
OAc
OAc
N
N
OAc
OAc
N
N
N
N
11c
11d
11a
11b
11e
OMe
O2N
NH2
NH2
F
N
O
O
O
O
AcO
AcO
AcO
AcO
AcO
AcO
AcO
F
N
N
N
N
AcO
N
N
N
N
OAc
OAc
OAc
N
OAc
11i
N
N
N
S
S
11f
11g
F
F
N
N
11h
NO2
F
O
O
O
O
HO
HO
AcO
AcO
F
AcO
AcO
AcO
AcO
F
N
N
N
N
N
N
N
N
OH
OAc
OAc
N
OAc
N
N
N
S
N
11m
11l
11k
11j
CF3
(B)
Fig. 1. Chemical structures of triazolyl glucal derivatives (A) bis-triazoles, (B) mono-triazoles.
4

R. Vieira Veloso et al.
Bioorganic Chemistry 109 (2021) 104709
Fig. 2. Antioxidant tests for the molecule’s evaluation. A - DPPH assay, shown as percentage of radical scavenging activity. B – PFRAP assay, shown as percentage of
relative reducing power activity.
The molecular docking was performed in the Swiss Dock webserver
(http://www.swissdock.ch/docking), with parameters set to automatic
search, considering the CHARMM method. Receptors and ligands were
protonated at pH 7.0 and proteins were chosen in the Protein Data Bank
(PDB) as follows: Superoxide Dismutase (1CB4 -chain A + B), Catalase
(1DGB - chain A), Glutathione peroxidase 3 (2R37 - chain A), NADPH
oxidase (2CDU - chain B), Lipoxygenase (1N8Q - chain A) and
Peroxiredoxin-5 (3MNG - chain A).
In this sense, some triazole-derived molecules have been described:
4-amino-5-aryl-3H-1,2,4-triazole-3-tiones with methoxybenzyl and
methoxyphenyl groups [16], 4,5-Dihydro-1H-1,2,4-triazole-5-one [15],
1,2,4-triazols [26,6] and 4-[3,4-di-(4-nitrobenzoxy)-benzylidenamino]-
4,5-dihydro-1H-1,2,4-triazol-5-one [15].
TD3 (4,4′-di(1,2,3-triazolyl) disulfide) is under pre-clinical trials for
its promising activity of saline-bind formations that is able to stabilize
the R state of hemoglobin and destabilize the T state, which favors the
oxygen bind to the protein [36].
TD1, a TD3 analogue(di(5-(2,3-di-hydro-1,4-benzodioxin-2-yl) ꢀ 4H-
1,2,4-triazol-3-yl) disulfide) was developed as an antioxidant focused on
anemia, with mechanism of βCys93 protection [22].
2.7. Statistical analysis
Cell experiments were submitted to statistical analysis for groups
Carbohydrates also have been used in several synthetic compounds,
as it facilitates the cell recognition and can activate intracellular path-
ways for cell proliferation or apoptosis. Moreover, the addition of this
group can add interesting proprieties, such as the increase of hydro-
philicity, reduction of the toxicity and increase of the bioavailability
[50].
comparison. For that, ANOVA test was used, with Tukeýs post-test.
Differences were considered statistically significant when p < 0.05,
indicated by asterisks in the figures.
3. Results and discussion
Thirty compounds were synthesized starting from 3,4,6-tri-O-acetyl-
D-glucal a commercially available reagent purchased from Sigma-Aldrich.
The main reactions performed included Ferrier rearrangement, Stille
reaction, copper(I) catalyzed azide alkyne cycloaddition (CuAAC), Tsuji-
Trost azidation, Sonogashira coupling and various functional groups
3.1. Chemistry
The triazole’s scaffold has been associated to the antioxidant activity
due to functional groups that favors the radical scavenging, such as –OH,
–
–
-SH, –COOH, -PO H , C O, -NR , -S-, -O- [14].
3
2
2
5

R. Vieira Veloso et al.
Bioorganic Chemistry 109 (2021) 104709
Fig. 3. Concentration-response effect of 11g and 11m: A – DPPH test and B – PFRAP test.
transformation to afford these multicyclic glucal based triazole com-
pounds. All the obtained compounds were stable, easy to handle and
were achieved with good to excellent yields. The structural character-
ization was performed using NMR, FTIR, and HRMS and the detailed
experimental procedures and characterization is already reported in
literature that is appropriately cited in the corresponding sections.
All the compounds are summarized in Fig. 1 and subdivided into
glucal-derived bis-triazoles (Fig. 1A) and glucal based mono-triazoles
(Fig. 1B). The analogues had variation in their chemical structures to
increase stability and biological effect and were submitted to antioxi-
dant activity in the first screening. This rationale in the medicinal
chemistry was applied for the search of new molecules with better ac-
tivity, selectivity, and low adverse effects, through the study of struc-
ture–activity relationship (SAR) [32].
have physico-chemical proprieties that impairs their cell membrane
penetration and consequently facilitates its elimination. On the other
hand, alpha tocopherol, another known antioxidant, is highly hydro-
phobic and accumulates in tissues, not reaching the pharmacological
target.
Here we have tested 30 molecules derived from triazole and glucal in
the DPPH oxidant test. It was possible to identify 8 molecules with
radical scavenging activity, in different intensity. The compound 11m,
for example, caused the most intense activity ꢀ 69.75% of antioxidant
effect with 0.5 mM (Fig. 2A). For comparison, it was used 1 mM ascorbic
acid, which caused 85.01% of radical scavenging activity.
The DPPH is a classic assay for determining antioxidant activity by
radical scavenging, and it is appropriated for a screening, as it is fast and
easy to conduct and analyze [12]. However, to determine an antioxidant
activity, one complementary test with a different mechanism should be
performed, and in this sense, we choose PFRAP, in order to evaluate the
reduction of Fe3+ to Fe2+ in the presence of the antioxidant, condition
that happens in the SCD [34].
3.2. Antioxidant activity
Antioxidants are being considered in the treatment of several dis-
eases, including cancer, neurodegenerative and sickle cell diseases [1].
Due to their success in the clinics, new antioxidants have been
searched for a better activity and appropriated pharmacokinetics fea-
tures. Ascorbic acid is the most known but is highly hydrophilic and
Thus, 5 molecules that presented higher activity in the DPPH test
were selected for PFRAP (also with 0.5 mM). It was observed that three
of them caused relative reducing power effect (Fig. 2B), being two (11g
and 11m) with important activity in both antioxidant tests, which were
6

R. Vieira Veloso et al.
Bioorganic Chemistry 109 (2021) 104709
3.4. Oxidative stress in RBC
The two molecules (11g and 11m) were tested in RBC induced with
oxidative stress in a prevention or treatment effect. This condition
induced by hydrogen peroxide mimics the oxidative stress in RBC when
the disease is installed and therefore it is being used as a SCD model, as it
is simple and rapid way to verify the antioxidants effects [38,1].
Both molecules were effective in significantly reducing the hemolysis
in the treatment effect, i.g. the incubation of samples after the oxidative
stress induction – 11g reduced 28.6% of hemolysis, and 11m reduced
61% (Fig. 5A).
The use of 11m molecule in a preventive effect, i.g. the incubation of
molecules before the oxidative stress induction, could reduce 40% of
hemolysis. The 11g did not cause any preventive effect (Fig. 5B).
The active compounds, 11g and 11m, do not contain functional
groups that favor the radical scavenging in the triazole group, thus this
substitution could not increase the activity in a SAR study. On the other
hand, the addition of glucal seemed to contribute for the activity, with
the presence of NH2 and -O- probably responsible for the activity besides
their contribution for the solubility proprieties.
Fig. 4. Hemolytic activity of 11g and 11m. For comparison, positive control
(0.1% Triton X-100) and negative control (PBS). *p < 0.05 in comparison to
Triton-X 100 group.
selected for further studies.
In order to verify a concentration–response effect, 11g and 11m
molecules were tested in DPPH and PFRAP assay with 0, 0.5, 1 and
2 mM. As shown in Fig. 3A and 3B, both molecules had their activity
increased according to the concentration. Using the same concentration,
(1 mM), 11m had the same activity as ascorbic acid (85%) in the DPPH
assay.
This method allowed the conclusion that the molecules, especially
11m, significantly reduced the hemolysis caused by the hydrogen
peroxide by its radical scavenging capacity, besides preventing the he-
molysis. This result confirms the antioxidant propriety of the molecule
and its application in a cellular model, being a positive effect in the RBC
damage caused by the ROS release.
Hydroxyurea is the main drug currently used for SCD treatment that
contributes for the reduction of ROS. However, other antioxidants are
currently being studied for this disease. Quercetin is one example, and it
is able to scavenge the free radicals through di-hydroxy orto structures,
protecting the RBC from oxidative damages [35,18,42]. Kassa et al [23]
showed that TD3 reduced 22% of ferryl heme caused by H2O2, which
represents a reduction in the βCys93 oxidation.
Thus, as the 2 molecules were selected by DPPH and PFRAP tests, it is
expected that the radical scavenging capacity of the molecules con-
tributes to the stabilization of RBC structure and to the preservation of
Fe ions oxidation, contributing to the hemoglobin maintenance and
reduction of hemolysis.
3.3. Hemolytic activity
The 11g and 11m were submitted to a hemolysis test in order to
check their cell toxicity. As shown in Fig. 4, the addition of Triton-X100
was able to disrupt the cell, causing hemolysis, as expected. On the other
hand, the negative control, PBS incubation, caused no hemolytic activ-
ity. Both molecules, 11g and 11m, caused no hemolytic effect as well, as
no hemoglobin release was observed, indicating low cytotoxicity in vitro.
In a study that used the SAR method to modify the resveratrol
structure, a known natural antioxidant, 9 analogues were obtained
aiming to reduce the cytotoxicity and induce the fetal hemoglobin (HbF)
expression [52] ꢀ 3 of them could induce the HbF with low cell toxicity.
In our study, two antioxidant molecules (11g and 11m) did not cause
cell toxicity. Thus, further experiments were performed to verify their
activity on oxidative stress induced in such cell type.
Table 1
Pharmacokinetics and toxicity parameters predicted for the molecules 11g and
́
11m and their fits on the Lipinskis rules.
Parameter
Criterion
11 g
11m
Molecular weight
< 500 g/mol
<10
550.53 g/mol
391.42 g/mol
Hydrogen-bond acceptors (HBA)
Hydrogen-bond donors (HBD)
LogP
11
6
<5
2
3
2 a 5
3.75
2.98
TPSA
40 a 100 A2
Yes
128.32A2
100.63A2
Gastrointestinal absorption
Blood-brain barrier permeant
Metabolism enzymes inhibitors
Acute oral toxicity
Yes
No
2
Yes
No
0
No
< 2
III or IV*
No
III
III
Carcinogenicity
No
No
*
These criteria indicate LD50 values in a range of 500 mg/kg – 5000 mg/kg
(category III) and greater than 5000 mg/kg (category IV).
Fig. 5. Oxidative stress induction in RBC and treatment with 11g and 11m molecules. A – preventive effect. B – treatment effect.
7

R. Vieira Veloso et al.
Bioorganic Chemistry 109 (2021) 104709
Fig. 6. Molecular docking interaction between the 11m ligand and the antioxidant proteins. A – SOD; B – Catalase; C - Glutathione peroxidase 3; D - NADPH oxidase;
E – Lipoxygenase; F - Peroxiredoxin-5.
3.5. ADME in silico properties
Moreover, the n-octanol amphipathic nature is considered an agent
that mimics the phospholipidic membrane and it is considered a
parameter to determine the druggability of a molecule [7]. The LogP
between 2 and 5 reflects the lipophilicity of the molecule and also in-
dicates its ability to cross membranes, in which the main component is a
The two molecules, selected by their antioxidant activity, in vitro and
by cellular effect, were analyzed by their pharmacokinetic properties.
The physico-chemical parameters evaluated followed the Lipinski’s
rules, that estimate values through computational analysis for finding
out the molecular mass, TPSA, LogP, hydrogen donor/acceptor, in order
to give the molecule a status of “druggable”. These parameters reflect
the cell permeability, gastrointestinal (GI) absorption, blood–brain
barrier (BBB) permeation and hepatic enzymes inhibition.
The values for each category were determined by the Lipinski’s
study, which are validated with several known compounds in high
throughput screening analysis [30].
́
lipid layer. The value of LogP 2.98 for 11m follows the Lipinskis rules
and explain its good activity on RBC – the molecule could permeate the
cell with short periods of time and exert its antioxidant activity in cells.
We also estimated the gastrointestinal absorption and blood–brain
barrier permeation by the BOILED-Egg method (Brain or intestinal
estimated permeation), as a predictive model of lipophilicity and po-
larity of small molecules, using LogP and TPSA parameters [8]. It was
confirmed for both compounds good cell permeation and high GI ab-
sorption, which indicates that the molecules could be administered by
oral route, a common, easy, and cheap way of drug administration,
convenient for the patient.
When both molecules were analyzed for ADME proprieties, the 11g
did not fit all requirements, being the 11m the most relevant one
(Table 1).
The 11m molecule has a molecular mass of 391.42 g/mol, 6
hydrogen-bond acceptors (HBA), 3 hydrogen-bond donors (HBD), n-
octanol/water partition coefficients (LogP) 2.98 and TPSA 100.63 A2.
The molecular mass below 500 g/mol indicates that the molecule can
cross passively plasmatic membranes, besides its ability to enter in the
target pocket, which makes it possible to develop its pharmacological
potential [33].
Besides, the TPSA value (100.63 A2) for 11m reflects high polarity
and low permeability to the BBB, although the LogP is in the adequate
range. In this case, the inability of 11m to penetrate the BBB indicates a
peripherical effect, without affecting the central nervous system, which
is positive for the systemic SCD.
HBA and HBD parameters reflect the interaction with ligands and
–
–
determine the bioavailability for the N H or O H bind prediction in
8

R. Vieira Veloso et al.
Bioorganic Chemistry 109 (2021) 104709
3.6. Molecular docking studies
Table 2
Protein targets and parameters of binding after molecular docking with the 11m
molecule.
Protein
After identifying the activity and pharmacokinetics proprieties, 11m
was chosen for a virtual screening by molecular docking for predicting
the interactions between a target protein and the molecule and elucidate
its possible mechanism of action. Six protein targets were selected based
on their participation on the antioxidant activity and simulated with
11m (Fig. 6). The parameters resulting from the protein target and
ligand interaction are shown in Table 2.
PDB
Binding
energy
(kcal/
mol)
Full
Binding
interaction
(ΔG)
Bond
length
(Å)
code
Fitness
(kcal/
mol)
Superoxide
Dismutase
(SOD)
1CB4
(chain
A + B)
1DGB
(chain
A)
ꢀ 9.14
ꢀ 1369.18
LigOH-
3.62
3.70
Asp11
LigN2-Lys9
LigNH-
Although the lowest binding energy has been reached in the inter-
action with superoxide dismutase (ꢀ 9.14 kcal/mol), the ligand was
positioned out of the catalytic site, indicating that the ligand does not
interfere with the enzyme antioxidant activity. The glutathione peroxi-
dase 3, NADPH oxidase, catalase and lipoxygenase active site were not
covered by 11m as well, indicating that these proteins are not the target,
even having low binding energy and/or full fitness.
Catalase
ꢀ 8.56
ꢀ 2144.19
3.15
2.79
3.36
Arg431
LigOH-
Asp157
LigOH-
Lys349
Glutathione
peroxidase 3
2R37
(chain
A)
ꢀ 8.90
ꢀ 8.60
ꢀ 1049.03
ꢀ 2349.03
LigNH-
2.42
3.93
3.50
Thr165
LigO2-
On the other hand, 11m interacted with the peroxiredoxin-5 cata-
lytic pocket, with a strong hydrogen bond (ꢀ 8.59 kcal/mol) identified in
the side chain of the key residues Arg127, and Gly46 with low bond
length (4.29 and 2.20 Å, respectively) [4]. Thus, we have an indication
that peroxiredoxin-5 could be the molecular target for 11m.
Peroxiredoxins are thiol-dependent peroxidases, able to reduce
hydrogen peroxide, alkyl hydroperoxides, and peroxynitrite [19]. They
are well conserved enzymes, mammalian, found in both cytosol and
mitochondria, already identified in the liver and lung. These enzymes
were related to several pathologies involving tendons, cartilages, nerves,
carcinomas (ovarian, breast, colon, adrenocortical), thyroid, astrocytes,
retina, and also blood cells [25].
Arg168(NH)
LigO2-
Leu162(NH)
LigO2-
NADPH
oxidase
2CDU
(chain
B)
3.67
5.44
3.13
Arg308(NH)
LigO-Arg308
(NH2)
LigN1-
Arg305
(NH2)
Lipoxygenase
1N8Q
(chain
A)
ꢀ 6.56
ꢀ 8.59
ꢀ 3881.73
ꢀ 783.09
LigO-Arg621
(NH)
3.19
3.38
4.46
LigO-
Arg621-N
LigN2-
The Peroxiredoxin-5 (PRDX5) expression was related to acute in-
flammatory effects, as it was upregulated in rats after the injection of
lipopolysaccharides and during stress of endothelial cells and macro-
phages [24]. The increase in activity was also found in neutrophils and
monocytes [27].
Glu625(O)
LigN-Gly46
(HN)
Peroxiredoxin-
5
3MNG
(chain
A)
2.20
4.29
Arg127
(NH2)
The molecular docking was performed using the PRDX5 because it is
the most available crystalized protein isoform, the opposite of PRDX2,
related to SCD. However, the catalytic site is conserved, which indicates
that 11m could interact with the PRDX2 as well.
the intra or extracellular environment [17]. According to Lipinski’s
rules, both values are in accordance with the desirable one for a mole-
cule being a drug. These binding predictions also help the verification of
the interactive capacity of a molecule with the enzymes for the drug
metabolism (CYP P450 superfamily). The 11m does not have potential
to inhibit any of the enzymes evaluated (CYP1A2, CYP2C19, CYP2C9,
CYP2D6, CYP3A4), which indicates low hepatotoxicity and drug inter-
action. On the other hand, 11g has potential to inhibit 2 CYPs, indicating
possible drug interaction and effects on xenobiotics metabolism.
The acute oral toxicity of both molecules (11g and 11m) was esti-
mated as category III (Table 1). This parameter is important because
indicates the possible adverse effects occurring within a short time after
an oral administration of a substance and the category III is referred as
“slightly toxic and slightly irritating” according to US EPA for chemicals
[53].
In the SCD, there is a huge quantity of ROS being produced by the
RBC, such as H2O2, and consequently the activities of antioxidant en-
zymes are increased, such as superoxide dismutase, catalase, glutathione
peroxidase, and three isoforms of peroxiredoxin (I, II, and VI). So, it is
expected that the PRDX5 activity would be increased, compatible to this
́
radicals production, trying to eliminate intracellular concentration of
H2O2 [44].
Indeed, we have seen that the 11m molecule reduced the hemolysis
caused by H2O2, and besides the scavenging mechanism, it can be acting
in the enzymatic activity of PRDX5 trying to remove the ROS.
Peroxiredoxins can be inactivated during catalysis because the
cysteine residue, important for the active site, is oxidated to sulfinic acid
(Cys-SO2H), being their reactivation achieved by reduction of the sul-
finic moiety [55]. Thus, by acting on the PRDX, the 11m molecule could
act coordinating the active site of the enzyme, increasing its catalytic
efficiency, consequently increasing the H2O2 removal, as demonstrated
here.
In the classification stablished by OECD (Organization for Economic
Co-operation and Development) [40], four categories are considered
regarding lethal dose 50% (LD50) values: Category I contains com-
pounds with LD50 values less than or equal to 50 mg/kg; Category II
compounds with LD50 values >50 through 500 mg/kg; Category III
compounds with LD50 values >500 through 5000 mg/kg; Category IV
compounds with LD50 values greater than 5000 mg/kg.
4. Conclusion
The LD50 values for 11g and 11m, 500–5000 mg/kg predicted, are
comparable to hydroxyurea, which was experimentally calculated as
5760 mg/kg after an oral administration, in rats. Considering that a
chosen drug for sickle cell disease, our molecules have potential for a
drug development program [37].
After evaluating 30 molecules, we could select one (11m) according
to its antioxidant activity, evaluated by two different methods, which
was able to reduce the hemolysis caused by reactive oxygen species in
red blood cell, a model that mimics the sickle cell disease. The radical
scavenging activity of 11m can be added to the possible activation of
peroxiredoxin-5, an antioxidant enzyme, which could remove H2O2 in
both preventive and treatment action. Moreover, this molecule has
adequate pharmacokinetics proprieties, hence making it a good candi-
date as a drug to treat sickle cell disease.
This feature, added to the one hydrogen-bond acceptors surplus and
the molecular weight above 500 g/mol, made us eliminate 11g for the
study, being the 11m the chosen one for the molecular docking
evaluation.
9

R. Vieira Veloso et al.
Bioorganic Chemistry 109 (2021) 104709
[24] B. Knoops, A. Clippe, C. Bogard, K. Arsalane, R. Wattiez, C. Hermans,
Funding
E. Duconseille, P. Falmagne, A. Bernard, Cloning and characterization of AOEB166,
a novel mammalian antioxidant enzyme of the peroxiredoxin family, J. Biol. Chem.
274 (1999) 30451–30458.
This research did not receive any specific grant from funding
agencies in the public, commercial, or not-for-profit sectors. Grant
received by Professor Stefani (FAPESP 2017/24821-4).
[25] B. Knoops, J. Goemaere, V. van der Eecken, J.P. Declercq, Peroxiredoxin 5:
structure, mechanism, and function of the mammalian atypical 2-Cys
peroxiredoxin, Antioxid Redox Signal. 15 (3) (2011) 817–829.
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Declaration of Competing Interest
[27] R.I. Krutilina, A.V. Kropotov, C. Leutenegger, V.B. Serikov, Migrating leukocytes
are the source of peroxiredoxin V during inflammation in the airways, J Inflamm.,
v. 3, n. 13, 2006.
A competing interest statement is provided, even if the authors have
no competing interests to declare.
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derivatives – A review, Mini-Rev. Org. Chem. v.10, 2013.
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