Chalcones and their B-aryl analogues as myeloperoxidase inhibitors: In silico, in vitro and ex vivo investigations

Article abstract of DOI:10.1016/j.bioorg.2021.104773

In the present study, a series of chalcones and their B-aryl analogues were prepared and evaluate as inhibitors of myeloperoxidase (MPO) chlorinating activity, using in vitro and ex vivo assays. Among these, B-thiophenyl chalcone (analogue 9) demonstrated inhibition of in vitro and ex vivo MPO chlorinating activity, exhibiting IC₅₀ value of 0.53 and 19.2 μM, respectively. Potent ex vivo MPO inhibitors 5, 8 and 9 were not toxic to human neutrophils at 50 μM, as well as displayed weak 2,2-diphenyl-1-pycrylhydrazyl radical (DPPH?) and hypochlorous acid (HOCl) scavenger abilities. Docking simulations indicated binding mode of MPO inhibitors, evidencing hydrogen bonds between the amino group at 4′position (ring A) of chalcones with Gln91, Asp94, and Hys95 MPO residues. In this regard, the efficacy and low toxicity promoted aminochalcones and arylic analogues to the rank of hit compounds in the search for new non-steroidal anti-inflammatory compounds.

Full text of DOI:10.1016/j.bioorg.2021.104773

Bioorganic Chemistry 110 (2021) 104773
Contents lists available at ScienceDirect
Bioorganic Chemistry
journal homepage: www.elsevier.com/locate/bioorg
Chalcones and their B-aryl analogues as myeloperoxidase inhibitors: In
silico, in vitro and ex vivo investigations
Mariana Bastos dos Santosa, Beatriz Carvalho Marquesa, Gabriela Miranda Ayussoa,
Mayara Aparecida Rocha Garciaa, Luana Chiquetto Paracatub, Ivani Paulic,
Vanderlan Silva Bolzanid, Adriano Defini Andricopuloc, Valdecir Farias Ximenesb,
,
,*
Maria Luiza Zeraike *, Luis Octavio Regasinia
a
˜
˜
´
Institute of Biosciences, Humanities and Exact Sciences, Sao Paulo State University (UNESP), 15054-000 Sao Jose do Rio Preto, SP, Brazil
b
c
˜
Department of Chemistry, Faculty of Sciences, Sao Paulo State University (UNESP), 17033-360 Bauru, SP, Brazil
˜
˜
˜
Physics Institute of Sao Carlos, University of Sao Paulo, 13563-120 Sao Carlos, SP, Brazil
d
˜
Department of Organic Chemistry, Institute of Chemistry, Sao Paulo State University, 14800-900 Araraquara, SP, Brazil
e Department of Chemistry, State University of Londrina (UEL), 86051-990 Londrina, PR, Brazil
A R T I C L E I N F O
A B S T R A C T
Keywords:
In the present study, a series of chalcones and their B-aryl analogues were prepared and evaluate as inhibitors of
myeloperoxidase (MPO) chlorinating activity, using in vitro and ex vivo assays. Among these, B-thiophenyl
chalcone (analogue 9) demonstrated inhibition of in vitro and ex vivo MPO chlorinating activity, exhibiting IC50
value of 0.53 and 19.2 µM, respectively. Potent ex vivo MPO inhibitors 5, 8 and 9 were not toxic to human
neutrophils at 50 µM, as well as displayed weak 2,2-diphenyl-1-pycrylhydrazyl radical (DPPH•) and hypo-
chlorous acid (HOCl) scavenger abilities. Docking simulations indicated binding mode of MPO inhibitors,
evidencing hydrogen bonds between the amino group at 4′position (ring A) of chalcones with Gln91, Asp94, and
Hys95 MPO residues. In this regard, the efficacy and low toxicity promoted aminochalcones and arylic analogues
to the rank of hit compounds in the search for new non-steroidal anti-inflammatory compounds.
Chalcone
Myeloperoxidase (MPO)
In vitro
In silico
Structure-activity relationship (SAR)
1. Introduction
combines both desirable function in immune response and undesirable
effects due to the high reactivity of its products [7].
Myeloperoxidase (MPO) is a heme-peroxidase, belonging to cyclo-
oxygenases superfamily. MPO is the most abundant protein stored in
azurophilic granules of monocytes, neutrophils and tissue macrophages,
and plays a main role in human antimicrobial and antiviral defense
[1,2]. When a pathogen is phagocyted by polymorphonuclear cells
(PMNs), MPO and its substrates, hydrogen peroxide (H2O2) and halides
(mainly Cl–), are released into phagolysosomes [3,4]. Catalytic activity
of MPO produces hypochlorous acid (HOCl) mainly, which display
similar reactivity to reactive oxygen species (ROS), as well as the
capability to damage microbial and viral components [3,5]. On the other
hand, MPO overexpression and high production of HOCl, caused by
pathological conditions, conduces to damage on human tissues, which is
related to MPO discharged outside the PMNs [6]. HOCl induces irre-
versible nonspecific transformations on biomolecule structures,
including halogenation, nitration and oxidative reactions. Thus, MPO
Destructive MPO effects have been correlated to several chronic
dysfunctions, such as atherosclerosis [8,9], rheumatoid arthritis [10],
Alzheimer’s disease [11], Parkinson’s disease [11,12]. In this way, the
search for MPO inhibitors can provide prototypes for the innovative
drugs useful to multifactorial and chronic diseases [13,14]. Soubhye and
co-authors exhaustively overviewed MPO inhibitors as potential drugs,
including natural and synthetic compounds [14]. Among these, aro-
matic hydrazides [15,16] and 2-thiopyrimidinones [17] are irreversible
inhibitors of MPO chlorinating activity. Salicylhydroxamic acid is a
reversible MPO inhibitor, which acts modifying the heme group [18].
Chalcones are compounds bearing the 1,3-diphenyl-2-propren-1-one
skeleton, which has a huge importance in medicinal chemistry as a
privileged scaffold [19]. Two phenyl rings (A and B) and
α
,β-unsaturated ketone are versatile subunits for the design of bioactive
congener library [20]. Few studies have investigated the potential of
* Corresponding authors.
E-mail addresses: zeraiklm@uel.br (M.L. Zeraik), luis.regasini@unesp.br (L.O. Regasini).
https://doi.org/10.1016/j.bioorg.2021.104773
Received 2 April 2020; Received in revised form 29 January 2021; Accepted 22 February 2021
Available online 2 March 2021
0045-2068/© 2021 Elsevier Inc. All rights reserved.

M.B. Santos et al.
Bioorganic Chemistry 110 (2021) 104773
chalcones as MPO inhibitors [21]. In our previous study, we identified
the potent inhibition of MPO chlorinating activity by fluorinated and
methylated chalcones bearing an amino group on ring A [22].
As part of our ongoing search for new MPO inhibitors, we evaluated a
series of chalcones bearing amino group on ring A and their B-aryl. MPO
inhibitory activity of these compounds was evaluated by in vitro assays
to derive preliminary structure–activity relationships (SAR). In order to
rationalize SAR and interaction of inhibitors with the catalytic site of
MPO, in silico studies were carried out. Selected MPO inhibitors 5, 8 and
9 were subjected to ex vivo MPO inhibitory activity and cytotoxicity
evaluations using human neutrophils. Additionally, their scavenger
abilities on 2,2-diphenyl-1-pycrylhydrazyl radical (DPPH•) and hypo-
chlorous acid (HOCl) were determined.
intestinal absorption (HIA), was measured by PreAdmet tools available
online [30,31]. All chalcones and B-aryl analogues respected the phys-
icochemical boundaries delineated by Veber and Lipinski indicating
they may not have restrictions on oral bioavailability based on these
boundaries.
Human intestinal absorption (HIA) is a relevant process in compound
pharmacokinetic determination. Yee and coauthors [32] classified
compounds according to their percentage of HIA in poorly absorbed
(0–20%), moderately absorbed (20–70%) and well absorbed
(70–100%), as demonstrated in Table 1, all synthesized compounds will
be well absorbed.
2.3. In-vitro MPO inhibitory activity
2. Results and discussion
In order to determine the inhibitory activities of chalcones (1–7) and
their B-aryl analogues (8–11), in vitro assays were performed with MPO
from human polymorphonuclear leukocytes (EC 1.11.1.7), purchase
from Planta Natural Products (Vienna, Austria). The effects on the
chlorinating activity of MPO was determined based on the reaction
between HOCl and taurine (Tau) to generate taurine chloramine
(TauNHCl), which oxidized 3,3′,5,5′-tetramethylbenzidine (TMB) to a
strongly absorbing blue product served as enzyme substrate. By carrying
out these experiments in the presence of various concentrations of
compounds (0.125–1.00 µM), plots of the percentage of inhibition
versus inhibitor concentrations were constructed. From these plots IC50
values were calculated, using linear regression. The resulting equation
was used to calculate the concentration of inhibitor that decreased HOCl
production by 50% (IC50). For comparison, 5-fluorotryptamine (FT), a
known MPO inhibitor was used as a reference compound [22].
The IC50 values for the inhibition of the human MPO are given in
Table 2. Out of eleven screened chalcones and their B-aryl analogues
(1–11), seven compounds presented MPO chlorinating inhibitory ac-
tivity. The most active compound 5 exhibited IC50 value of 0.52 µM, 3-
fold less potent than FT (IC50 = 0.19 µM). Chalcones 1 and 2, which
possess amino group at 2′ and 3′positions, inhibit MPO with IC50 values
of 1.05 and 0.97 µM, respectively. Chalcone 3, bearing amino group at 4′
position, displayed inhibition of MPO chlorinating activity, demon-
strating an IC50 value of 0.56 µM. These data suggested the position of
amino group influences MPO inhibitory activity, and position 4′ is more
active, corroborating our previous report. Our preliminary structur-
e–activity relationship report indicated an amino group on ring A was
crucial for MPO chlorinating inhibitory activity. Its absence or
replacement by the hydroxyl group had completely blocked MPO inhi-
bition [22]. (See Table 3)
2.1. Chemistry
Chalcones and their B-aryl analogues (1–11) were synthesized by
Claisen-Schmidt condensation reaction, and the synthesis of compounds
3–11 was reported in our previous studies [23,24]. Reactions to obtain
compounds 1 and 2, between aminoacetophenones and aromatic alde-
hydes solubilized in ethanol were performed under basic catalysis, at
room temperature, culminating in yields of 82 and 55%, respectively.
The structures of compounds were confirmed by 1H NMR and 13C
NMR spectra analyses. The most characteristic signals observed in the
1H NMR spectrum were those of the double bond hydrogens, which were
present as a pair of doublets with coupling constants ranging from 15.0
to 15.6 Hz, indicating E configuration of olefin. In the 13C NMR spec-
trum, the most downfield signals with chemical shifts ranging from
190.9 to 190.2 ppm were attributed to the carbonyl group of the
α
,β-unsaturated ketone subunit. For all compounds, NMR data (chemical
shifts, integrations, multiplicities and coupling constants) corresponded
with the proposed structures.
2.2. In silico prediction of drug likeness properties of chalcones
Chalcones and B-aryl analogues physicochemical properties are
important to achieve the desirable compound’s pharmacokinetics [25].
Lipinski developed rules about the physicochemical properties of com-
pounds (Lipinski’s rule of five), to predict appropriated pharmacoki-
netics [26]. Lipinsky’s rules suggest compounds structure should have:
(i) less than 5 hydrogen bonds donors (HBD); (ii) less than 10 hydrogen
bond acceptors (HBA); (iii) molecular weight (MW) less than 500 g
molꢀ 1; (iv) maximum lipophilicity (milog P) of 5; v) not violate more
than one of the previous rules [27]. Veber and coauthors proposed
criteria for appropriated bioavailability of a compound structure: (a)
number of rotatable bond (NROBT) ≤ 10; (b) topological polar surface
area (TPSA) ≤ 140 Å2 [28]. Compounds physicochemical features were
predicted by Molinspiration Tools for Chemoinformatics available on-
line (Table 1) [29]. Additionally, compounds percentage of human
The major requirement for a molecule to act as an MPO inhibitor is
the presence of oxidizable substituents in the aromatic rings. Indeed, it is
not any substituents, but those that provoke high reactive with the in-
termediate compound I of MPO (MPO-I) and low reactivity with the
compound II (MPO-II) [33]. Aromatic amines, such as anilines, bearing
electron-withdrawing substituents, has been reported able to fulfill this
requirement [34].
Table 1
Drug-likeness properties.
Cpd
HIA
TPSA
milog P
MW
HBD
HBA
Lipinki’s Violations
NROBT
Veber’s Violation
LogS
1
2
96.08
96.08
96.08
96.77
96.00
95.84
96.13
96.25
97.02
96.71
96.71
43.09
43.09
43.09
66.89
52.33
88.92
43.09
56.23
43.09
43.09
43.09
3.25
2.86
2.89
2.64
2.97
2.85
3.78
1.97
2.61
3.87
4.07
223.28
223.28
223.28
248.28
253.30
268.27
291.27
213.24
229.30
273.33
273.33
2
2
2
2
2
2
2
2
2
2
2
2
2
2
3
3
5
2
3
2
2
2
0
0
0
0
0
0
0
0
0
0
0
3
3
3
3
4
4
4
3
3
3
3
0
0
0
0
0
0
0
0
0
0
0
ꢀ 3.60
ꢀ 4.28
ꢀ 4.30
ꢀ 4.86
ꢀ 4.51
ꢀ 4.99
ꢀ 5.13
ꢀ 3.75
ꢀ 4.04
ꢀ 6.04
ꢀ 6.16
3
5
4
6
7
8
9
10
11
2

M.B. Santos et al.
Bioorganic Chemistry 110 (2021) 104773
hydrophobic region of MPO binding site (Glu115, Pro220, Phe366,
Phe407, Val410, and Met411 residues). In this way, variations of elec-
tronic density caused by electron-withdrawing and electron-donating
groups on ring B did not significantly affect MPO inhibition (Fig. 1).
Table 2
In vitro MPO inhibitory activity of chalcones.
R
Ar
Inactive chalcones 10 and 11 display
α- and β-naphthalene rings,
O
respectively, which are hydrophobic rings. These rings interact with
hydrophobic MPO binding site, hindering formation hydrogen bonds
between the amino group (ring A) and MPO residues (Fig. 2).
Compound
R
Ar
IC50(µM)
1
2′-NH2
3′-NH2
4′-NH2
4′-NH2
4′-NH2
4′-NH2
4′-NH2
4′-NH2
4′-NH2
4′-NH2
4′-NH2
–
phenyl
phenyl
phenyl
1.05 ± 0.08
0.97 ± 0.11
0.56 ± 0.05
0.56 ± 0.06
0.52 ± 0.04
0.63 ± 0.01
0.63 ± 0.07
0.54 ± 0.04
0.53 ± 0.08
> 10
2.5. Ex vivo assays
2
3
4
4-methoxyphenyl
4-cyanophenyl
4-nitrophenyl
4-trifluoromethylphenyl
2-furyl
Compounds 3–9 were selected to ex vivo assays using human neu-
trophils, which exhibiting in vitro MPO inhibition. Poor solubility of
compounds in cell culture medium restricted their ex vivo evaluations.
Thus, three soluble compounds (5, 8 and 9) were submitted to MPO
inhibitory activity in a cellular system. Neutrophils were stimulated by
phorbol myristate acetate (PMA), a protein kinase C (PKC) agonist, and
incubated for an additional 30 min to release MPO and HOCl. The
chlorinating activity of MPO in neutrophils was also based on the re-
action between HOCl with Tau to furnish TauNHCl, which oxidizes
TMB. This experiment differs from in vitro MPO assays in that H2O2 is
produced by stimulated neutrophils instead of being added to the re-
action. Compounds were solubilized in DMSO on 96-well plates at 50,
20, 10 and 5 µmol Lꢀ 1. Production of TauNHCl by tested compounds was
used to calculate the percentage of inhibition. Plots of percentage of
inhibition versus inhibitor concentrations were constructed. Resulting
equation and linear regression analysis were used to calculated IC50
values. In agreement with our expectations, compounds 5, 8 and 9 were
identified as inhibitors of HOCl production by activated neutrophils.
Compounds 8 and 9 exhibited potent ex vivo MPO chlorination inhibi-
tory activity, demonstrating IC50 values ranging from 19.2 to 21.2 µM.
These compounds were approximately two times less potent than 5-flu-
ortryptamine (FT) (IC50 = 10.1 µM). However, even though 5-fluortrypt-
amine is a potent MPO inhibitor, it cannot be administered in vivo due to
severe side effects, such as eye lacrimation, convulsions or effect on
seizure threshold [37]. Analysis data confirmed bioisosteric correlations
among 8 and 9 as observed by in vitro evaluations.
5
6
7
8
9
2-tiophenyl
10
11
FT*
α-naphthyl
β-naphthyl
> 10
–
0.16 ± 0.07
*
FT: 5-fluorotryptamine.
Table 3
IC50 values for inhibition of MPO assay in cell system.
Compound
IC50
(
μ
mol Lꢀ 1) ex vivo
5
>50
8
21.2 ± 1.1
19.2 ± 0.5
10.1 ± 0.4
9
5-fluorotryptamine
In the second step, we studied the influence of the electronic density
on ring B on MPO inhibition. Interestingly, 4′-aminochalcones 4–7
substituted by electron-withdrawing or electron-donating groups on
ring B presented very closely IC50 values, ranging from 0.52 to 0.63 µM.
Aminochalcones 6 and 7, substituted by strong electron-withdrawing
groups (–NO2 and –CF3), were slightly less active than aminochalcone
5 and 4, which possess moderate electron-withdrawing (–CN) and
electron-donating (–OCH3) substituents on ring B, respectively. Then, it
is concluded that electron donating or withdrawing groups replaced on
phenyl ring B does not influence chalcone MPO inhibition activity. Also,
aryl analogues chalcone 8 and 9 demonstrated IC50 values of 0.54 and
0.53 µM, respectively, which were very similar to 3 (IC50 = 0.52 µM).
These results suggest a ring bioisosteric correlation among three com-
Interestingly, in vitro active compound 5 was not able to inhibit MPO
chlorinating activity in cell-system, displaying IC50 > 50 µM. These
findings can be related to the hydrophilic effect of the nitrile group on
ring B, which reduces neutrophil membrane transport by passive diffu-
sion [38]. Thus, the octanol–water partition coefficient (log Po/w) of
compounds 3, 5, 8 and 9 were determined by RP-HPLC using OECD
protocol. Log Po/w values were, respectively, 2.30, 1.70, 1.22 and 1.64.
The toxic effect of selected compounds 5, 8 and 9 against human
neutrophils was evaluated by using trypan blue exclusion assay based on
counting of cells. Compounds were dissolved in DMSO at a final con-
pounds (3, 8 and 9), which exhibited six
π electrons on ring B. Benzene-
heterocyclic bioisosteric replacements have been employed to design
several drug candidates, including isoxazoles and isothiazoles [35].
Naphthylchalcones 10 and 11 were not able to inhibit chlorinating MPO
activity (IC50 > 50 µM).
centration of 50
μ
mol Lꢀ 1. All compounds were not cytotoxic at 50
μmol
L
ꢀ 1 even after 90 min. This data indicated ex vivo chlorinating inhibitory
Other compounds, such as, nitroxides have been described as potent
inhibitors of MPO activity [36]. However, we presented in this paper the
chalcones derivatives 3–9 with higher inhibitory activity than nitroxides
described (4-aminoTEMPO, with IC50 = 1.2 µmol Lꢀ 1, and 3-amino-
PROXYL, IC50 = 2.7 µmol Lꢀ 1).
activity was not mediated by death cell.
It is important to note that the chlorinating activity of MPO was
determined by measuring the taurine chloramine accumulated, gener-
ated by the reaction between HOCl and taurine, with the TMB. There-
fore, the direct effect of these compounds as scavengers of taurine
chloramine was also studied, once this reaction could influence on the
results of the measurement of chlorinating MPO activity using taurine
chloramine. However, these compounds did not react with taurine-
chloramine in meaningful rates; the scavenged capacity was lower
than 15% at 25.0 µmol Lꢀ 1 for all compounds studied (Fig. 3a), rein-
forcing the inhibitory action of chalcones derivative on enzymatic ac-
tivity and not as direct scavenger of taurine-chloramine.
2.4. Molecular docking investigations
In order to understand preliminary structure–activity relationships
(SAR) data, we investigated inhibitors using in silico evaluations with
human MPO (PDB 1DNW), using GOLD software. Docking simulation
studies supported our biochemical experiments, indicating two pivotal
binding sites between MPO and aromatic rings of chalcones. The amino
group at C-4′position interacts by hydrogen bonds with MPO residues,
such as Gln91, Asp94, and His95, which are crucial to its catalysis. These
hydrogen bonds were observed for MPO inhibitors 3–9. Simulations for
HClO is the main product of MPO activity and presents a deleterious
effect on tissues. Lazarevic-Pasti and collaborators reported compounds
able directly to reduce HClO scavenger ability can be a strategy to
enhance the therapeutic application of MPO inhibitors. Chalcones 1–11
were subjected to HClO scavenger ability evaluations in the
inhibitors 3–9 indicated their ring
B interacts predominantly
3

M.B. Santos et al.
Bioorganic Chemistry 110 (2021) 104773
Fig. 1. Conformation of the most active compounds
into the MPO binding cavity. Highlighted are the
hydrogen bonds (yellow dashes) of the amino group
at C-4′position. Important interacting MPO amino
acids are represented as sticks. Compound 3: cyan;
compound 4: yellow; compound 5: pink; compound
6: light blue; compound 7: light green; compound 8:
dark green; compound 9: orange. (For interpretation
of the references to colour in this figure legend, the
reader is referred to the web version of this article.)
Fig. 2. Conformation of compound 11 into the MPO binding cavity, highlighting the absence of the hydrogen bonds between the amino group at C-4′position and
MPO cavity amino acids.
concentration of 10.0
μ
mol Lꢀ 1, compounds were not able to inhibit
scavenger ability, which were evaluated against 2,2-diphenyl-1-pycryl-
hydrazyl radical (DPPH•). This assay evaluates the antiradical capac-
ity by direct reduction via electron and hydrogen atom transfer.
Compounds 1–11 were assayed in concentrations used for MPO assays
(micromolar quantities) and millimolar levels. At maximum concen-
tration (1.0 mmol Lꢀ 1), selected compounds demonstrated the per-
centage of DPPH scavenged ranging from 1 to 30%, indicating their very
weak radical scavenger ability (Fig. 4). Quercetin was used as a positive
control (IC50 = 5.37 µmol L–1).
HClO, displaying the percentage of scavenged HClO bellow to 15%
(Fig. 3b).
2.6. Antiradical assay
Production of HOCl from oxidation of halides involves successive
radical reactions, which are mediated by highly reactive species. We
hypothesized effect of MPO inhibitors can be related to their radical
4

M.B. Santos et al.
Bioorganic Chemistry 110 (2021) 104773
Fig. 3. (a) Scavenging effect of chalcone derivatives (25.0 µmol Lꢀ 1) on taurine chloramine, used as control of MPO assay, reaction contained taurine (5 mmol Lꢀ 1
)
and HOCl at 25 µmol Lꢀ 1. The assays were performed in triplicate. (b) Effect of chalcone derivatives as HOCl scavengers. HOCl (10
μ
mol Lꢀ 1) was incubated 30 min
at 25 ◦C in the presence of chalcones derivatives (1
μ
mol Lꢀ 1).
fluorotryptamine (reference’s inhibitor) at final concentrations of 2.5,
0.25 and 0.025 µmol Lꢀ 1 were added, followed by purified MPO (20
nmol Lꢀ 1), and H2O2 (50 µmol Lꢀ 1). The reactions were initiated by
H2O2 and incubated for 30 min at 37 ◦C (final volume of 200
μL). After
incubation, the reactions were stopped by adding 5 μL of catalase (1 mg
mLꢀ 1), and the accumulated taurine chloramine was measured by
adding 50 μ
L of 3,3′,5,5′-tetramethylbenzidine (TMB) solution (14 mmol
Lꢀ 1) dissolved in 50% N,N-dimethylformamide (DMF), 100 µmol Lꢀ 1 KI
and 400 mmol Lꢀ 1 acetic acid. The resulting blue product was detected
by a microplate reader (Synergy 2 Multi-Mode, BioTek, USA) at 655 nm.
The percentage of inhibition was calculated by accounting for the
amount of taurine chloramine produced in the control, in which the
tested compounds were absent. A line was fitted to the concen-
tration–response curve for each inhibitor using linear regression. The
resulting equation was used to calculate the concentration of inhibitor
that decreased HOCl production by 50% (IC50) [22].
Fig. 4. Effect of chalcone derivatives as DPPH scavengers. The compounds
were incubated 30 min with DPPH (10
μ
mol Lꢀ 1) at 25 ◦C in the presence of
chalcones derivatives (1 mmol Lꢀ 1).
3.3. DPPH• scavenging capacity assay
The antioxidant capacity of chalcone derivatives was evaluated using
the DPPH• method [39,40], with some modification [22]. In 96-well
microtiter plates, an aliquot of 35 µL of test compounds (at a final
concentration of 1 mmol Lꢀ 1) in ethanol was added to 210 µL of DPPH•
3. Experimental
3.1. Chemistry
ethanol solution (100
μ
mol Lꢀ 1). The mixture was shaken and incubated
Chalcones bearing the amino group on ring A (1 and 2) were syn-
thesized by Claisen-Schmidt reactions under basic catalyzes according to
previously described protocols. Compounds 3–11 synthesis were re-
ported previously [23,24].
in the dark at 25 ◦C for 30 min. The absorbance of DPPH• was deter-
mined at 517 nm using a microplate reader (Synergy 2 Multi-Mode,
BioTek). Quercetin was used as a positive control. Ethanol was used as
a blank solution and DPPH• solution (10 µmol Lꢀ 1) in ethanol as the
control. The radical scavenging activity of the chalcone derivatives was
calculated as: [(absorbance of control ꢀ absorbance of sample)/
(absorbance of control)] × 100 and expressed in terms of EC50 (amount
of antioxidant necessary to decrease the initial concentration of DPPH•
by 50%).
Compounds were prepared using 5.0 mmol of aminoacetophenones
and benzaldehydes on the ethanolic solution of sodium hydroxide (1.0
mol L–1). Mixtures were stirred at room temperature and monitored by
TLC, using hexanes/ethyl acetate as the mobile phase. Crude products
were filtered and purified by chromatography on a silica gel column
using hexanes and ethyl acetate. All compounds were identified using 1H
and 13C NMR spectral data obtained from a Bruker Avance III spec-
trometer (14.0 T), using DMSO‑d6 as solvent.
3.4. HOCl reduction assay
3.2. Measurement of chlorination activity of purified MPO
The reduction ability of potent oxidant HOCl (generated in neutro-
phils by MPO) by chalcone derivatives was assayed. For the assay, HOCl
(50 µmol Lꢀ 1) was incubated with PBS for 5 min at 25 ◦C in the presence
The chlorination activity of MPO (cell-free system) was determined
using the taurine chloramine (TauNHCl) assay to measure the HOCl
produced. The fundamental of this test is the oxidation of TMB by
TauNHCl to a strongly absorbing blue product [1]. The procedure was
performed according to Zeraik et al. (2012) [22], in a 96-well microtiter
plate containing PBS, taurine (14 mmol Lꢀ 1), chalcone derivatives at
final concentrations of 1.0, 0.5, 0.25 and 0.125 µmol Lꢀ 1, and 5-
or absence of tested compounds (1
μ
mol Lꢀ 1). After incubation, 20 µL of
taurine (5 mmol Lꢀ 1) was added, and the accumulated taurine chlora-
mine was measured by adding 50
μ
L of a TMB solution (14 mmol Lꢀ 1
)
dissolved in 50% DMF, 100 µmol Lꢀ 1 potassium iodide and 400 mmol
Lꢀ 1 acetic acid [1]; the final volume was 200
μL. The ability of the
chalcones to scavenge HOCl was determined by measuring the oxidation
5

M.B. Santos et al.
Bioorganic Chemistry 110 (2021) 104773
of TMB by taurine chloramine at 655 nm using a microplate reader
Synergy 2 Multi-Mode, BioTek
[44], available at the Protein Data Bank (PDB). The protein was pre-
pared for docking studies by adding hydrogen atoms, removing water
and co-crystallized inhibitors. Enzyme-inhibitor interactions within a
radius of 12 Å centered on HIS95 NE atoms were evaluated. Docking
poses were ranked by the ChemScore fitness function. Ligplot [45] was
used to identify contacts among inhibitors and myeloperoxidase.
3.5. Ex vivo assays
3.5.1. Isolation of human neutrophils
Blood samples were obtained from healthy volunteers using heparin
as an anticoagulant, following regulations of the Research Ethics Com-
mittee (CEP/FCF-UNESP no. 29/2011), Faculty of Pharmaceutical Sci-
4. Conclusion
˜
˜
ences, University of the State of Sao Paulo (UNESP), Sao Paulo, Brazil.
Neutrophils were isolated by density gradient centrifugation on a His-
topaque®-1077/1119 at 700g for 30 min at room temperature [41]. The
supernatant was discarded and the pellet washed twice with PBS buffer
(700g, 10 min, 20 ◦C). The resulting pellet, which mainly contained
neutrophils, was suspended in supplemented PBS with 1 mmol Lꢀ 1
calcium chloride, 0.5 mmol Lꢀ 1 magnesium chloride, and 1 mg mLꢀ 1
glucose. The neutrophil concentration was determined using a diluted
cell suspension distributed into a Neubauer chamber. Each batch of
neutrophils was prepared from 20 mL of blood. The cells were used
within 4 h after isolation. Each experiment was repeated at least three
times with different cell batches.
In summary, a series of chalcones bearing the amino group on ring A
and several substituents on ring B were prepared as part of our ongoing
search for MPO inhibitors designed. Two B-aryl analogues (8 and 9)
display in vitro and ex vivo MPO inhibitory activity. Preliminary SAR and
docking simulations indicated the relevance of steric and hydropho-
bicity parameters to MPO inhibition. Besides, low toxicity of chalcones
against human neutrophils corroborates their potential as hits useful for
new non-steroidal MPO inhibitors.
Declaration of Competing Interest
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influence
the work reported in this paper.
3.5.2. Measurement of the chlorination activity of MPO produced by
stimulated neutrophils
The chlorination activity of MPO by neutrophils was also based on
the reaction of the produced HOCl with taurine to give taurine chlora-
mines, which oxidize TMB [1]. This procedure differs from that
described on item 3.1, in this assay MPO and H2O2 will be released by
stimulated neutrophils, instead of being added to the reaction. Suspen-
sions of neutrophil (4.106 cells mLꢀ 1) were incubated in supplemented
PBS, 5 mmol Lꢀ 1 taurine, chalcone derivatives and 5-fluorotryptamine
at final concentrations of 1, 10, 20 and 50 µmol Lꢀ 1 for 15 min, at
37 ◦C. Then, the cells were stimulated by PMA (100 nmol Lꢀ 1) and
incubated for an additional 30 min at 37 ◦C to release MPO and H2O2.
The reactions were stopped by adding catalase (30 µg mLꢀ 1) and
Acknowledgments
The authors gratefully acknowledge financial support from the Co-
ordination for the Improvement of Higher Education Personnel
(CAPES), the National Research Council (CNPq) (Grants 471129/2013-
˜
5; 306251/2016-7, 429322/2018-6 and 309957/2019-2), and the Sao
Paulo Research Foundation (FAPESP) (Grants 2014/18330-0 and 2018/
15083-2). Also, we would like to thank the National Institute for Sci-
ence and Technology of Biodiversity and Natural Products (INCT-Bio-
Nat) (Grants Fapesp 2014/50926-0 and CNPq 465337/2014-0), the
Multiuser Centre for Biomolecular Innovation (CMIB/Fapesp Grant
2009/53989-4) for NMR experiments. MBS thanks Fapesp for her
scholarship (2016/00580-5).
centrifuged (200g, 5 min, 20 ◦C). Supernatants aliquot of 200
μ
L were
transferred to a 96-well microtiter plate, and 50
μL of TMB (14 mmol
Lꢀ 1) was added. The production of taurine chloramine was quantified at
655 nm by a microplate reader Synergy 2 Multi-Mode, BioTek. The
production of taurine chloramine with the tested compounds omitted
(control) was used to calculate the percentage of inhibition. The IC50
values were calculated by linear regression analysis using GraphPad
Prism software.
Appendix A. Supplementary material
Supplementary data to this article can be found online at https://doi.
org/10.1016/j.bioorg.2021.104773.
References
3.5.3. Cellular viability assay
In the presence of chalcone derivatives, the viability of neutrophils
was evaluated using the trypan blue exclusion method based on the
counting of 100 cells [42]. Neutrophils (1x106 cell mLꢀ 1) were incu-
bated for 30, 60 and 90 min at 37 ◦C in the presence of chalcone de-
rivatives at final concentrations of 50 µmol Lꢀ 1. Then, this mixture was
incubated with trypan blue (0.5%) in a ratio of 1:1 for 5 min. Cells were
observed under a microscope, and stained and unstained cells were
counted. The viable cell ratios were then calculated as percentages of
viable cells.
[1] E. Malle, P.G. Furtmüller, W. Sattler, C. Obinger, Myeloperoxidase: a target for new
drug development? Br. J. Pharmacol. 152 (2007) 838–854.
[2] C. Nussbaum, A. Klinke, M. Adam, S. Baldus, M. Sperandio, Myeloperoxidase: A
Leukocyte-Derived Protagonist of Inflammation and Cardiovascular Disease,
Antioxid. Redox Signal. 18 (2013) 692–713.
[3] M.J. Davies, Myeloperoxidase-derived oxidation: mechanisms of biological damage
and its prevention, J. Clin. Biochem. Nutr. 48 (2010) 8–19.
[4] Y. Aratani, Myeloperoxidase: Its role for host defense, inflammation, and
neutrophil function, Arch. Biochem. Biophys. 640 (2018) 47–52.
[5] I.V. Gorudko, D.V. Grigorieva, A.V. Sokolov, E.V. Shamova, V.A. Kostevich, I.
V. Kudryavtsev, E.D. Syromiatnikova, V.B. Vasilyev, S.N. Cherenkevich, O.
M. Panasenko, Neutrophil activation in response to monomeric myeloperoxidase,
Biochem. Cell Biol. 96 (2018) 592–601.
3.6. Docking simulations
[6] F.J. Olorunniji, M.O. Iniaghe, J.O. Adebayo, S.O. Malomo, S.A. Adediran,
Mechanism-Based Inhibition of Myeloperoxidase by Hydrogen Peroxide:
Enhancement of Inactivation Rate by Organic Donor Substrates, TOEIJ. 2 (2009)
28–35.
The 3D chemical structure of each compound was generated using
standard tools available in the molecular modeling software package
Sybyl 8.0 (Tripos, St. Louis, MO, USA). Tripos force field and Powell’s
method were used for conformational energy minimization. Partial
atomic charges were calculated considering the molecules to be in an
implicit aqueous environment (dielectric constant of 80.0), using the
Gasteiger-Hückel method. Simulations were carried out using GOLD
[43], a software based on a genetic algorithm to explore the ligand
conformational space. Protein coordinates were obtained from 1DNW
[7] D. Lau, S. Baldus, Myeloperoxidase and its contributory role in inflammatory
vascular disease, Pharmacol. Ther. 111 (2006) 16–26.
¨
[8] D. Cheng, J. Talib, C.P. Stanley, I. Rashid, E. Michaelsson, E.-L. Lindstedt, K.
D. Croft, A.J. Kettle, G.J. Maghzal, R. Stocker, Inhibition of MPO
(Myeloperoxidase) Attenuates Endothelial Dysfunction in Mouse Models of
Vascular Inflammation and Atherosclerosis, ATVB. 39 (2019) 1448–1457.
[9] N. Anatoliotakis, S. Deftereos, G. Bouras, G. Giannopoulos, D. Tsounis,
C. Angelidis, A. Kaoukis, C. Stefanadis, Myeloperoxidase: expressing inflammation
and oxidative stress in cardiovascular disease, Curr. Top Med. Chem. 13 (2013)
115–138.
6

M.B. Santos et al.
Bioorganic Chemistry 110 (2021) 104773
[10] W. Wang, Z. Jian, J. Guo, X. Ning, Increased levels of serum myeloperoxidase in
patients with active rheumatoid arthritis, Life Sci. 117 (2014) 19–23.
[11] S. Gellhaar, D. Sunnemark, H. Eriksson, L. Olson, D. Galter, Myeloperoxidase-
immunoreactive cells are significantly increased in brain areas affected by
neurodegeneration in Parkinson’s and Alzheimer’s disease, Cell Tissue Res. 369
(2017) 445–454.
[25] T. Tuntland, B. Ethell, T. Kosaka, F. Blasco, R.X. Zang, M. Jain, T. Gould,
K. Hoffmaster, Implementation of pharmacokinetic and pharmacodynamic
strategies in early research phases of drug discovery and development at Novartis
Institute of Biomedical Research, Front. Pharmacol. 5 (2014) 1–16.
[26] C.A. Lipinski, Lead- and drug-like compounds: the rule-of-five revolution, Drug
Discov. Today: Technol. 1 (2004) 337–341.
[12] R.A. Maki, M. Holzer, K. Motamedchaboki, E. Malle, E. Masliah, G. Marsche, W.
F. Reynolds, Human myeloperoxidase (hMPO) is expressed in neurons in the
[27] C.A. Lipinski, F. Lombardo, B.W. Dominy, P.J. Feeney, Experimental and
computational approaches to estimate solubility and permeability in drug
discovery and development settings, Adv. Drug Deliv. Rev. 23 (1997) 3–25.
[28] D.F. Veber, S.R. Johnson, H.-Y. Cheng, B.R. Smith, K.W. Ward, K.D. Kopple,
Molecular Properties That Influence the Oral Bioavailability of Drug Candidates,
J. Med. Chem. 45 (2002) 2615–2623.
substantia nigra in Parkinson’s disease and in the hMPO-
α
-synuclein-A53T mouse
model, correlating with increased nitration and aggregation of
α
-synuclein and
exacerbation of motor impairment, Free Radical Biol. Med. 141 (2019) 115–140.
[13] A. Strzepa, K.A. Pritchard, B.N. Dittel, Myeloperoxidase: A new player in
autoimmunity, Cell. Immunol. 317 (2017) 1–8.
[29] Molinspiration Cheminformatics, (n.d.). https://www.molinspiration.com/
(accessed March 26, 2020).
´
[14] J. Soubhye, M. Prevost, P. Van Antwerpen, K. Zouaoui Boudjeltia, A. Rousseau, P.
`
G. Furtmüller, C. Obinger, M. Vanhaeverbeek, J. Ducobu, J. Neve, M. Gelbcke,
[30] P. Ertl, B. Rohde, P. Selzer, Fast Calculation of Molecular Polar Surface Area as a
Sum of Fragment-Based Contributions and Its Application to the Prediction of Drug
Transport Properties, J. Med. Chem. 43 (2000) 3714–3717.
F. Dufrasne, Structure-Based Design, Synthesis, and Pharmacological Evaluation of
3-(Aminoalkyl)-5-fluoroindoles as Myeloperoxidase Inhibitors, J. Med. Chem. 53
(2010) 8747–8759.
[31] ADME, PreADMET | Prediction of ADME/Tox. (2014). https://preadmet.bmdrc.kr/
adme/ (accessed March 26, 2020).
[15] A.J. Kettle, C.A. Gedye, C.C. Winterbourn, Mechanism of inactivation of
myeloperoxidase by 4-aminobenzoic acid hydrazide, Biochem. J 321 (1997)
503–508.
[32] S. Yee, No title found, Pharm. Res. 14 (1997) 763–766.
[33] J. Soubhye, P. Van Antwerpen, F. Dufrasne, A patent review of myeloperoxidase
inhibitors for treating chronic inflammatory syndromes (focus on cardiovascular
diseases, 2013–2019), Expert Opin. Ther. Pat. 30 (2020) 595–608.
[34] A.J. Kettle, C.C. Winterbourn, Mechanism of inhibition of myeloperoxidase by anti-
inflammatory drugs, Biochem. Pharmacol. 41 (1991) 1485–1492.
[35] D.S. Garvey, J.T. Wasicak, R.L. Elliott, S. Lebold, A.-M. Hettinger, G.M. Carrera, N.-
H. Lin, Y. He, M.W. Holladay, Ligands for Brain Cholinergic Channel Receptors:
Synthesis and in Vitro Characterization of Novel Isoxazoles and Isothiazoles as
Bioisosteric Replacements for the Pyridine Ring in Nicotine, J. Med. Chem. 37
(1994) 4455–4463.
[16] L.V. Forbes, P.G. Furtmüller, I. Khalilova, R. Turner, C. Obinger, A.J. Kettle,
Isoniazid as a substrate and inhibitor of myeloperoxidase: Identification of amine
adducts and the influence of superoxide dismutase on their formation, Biochem.
Pharmacol. 84 (2012) 949–960.
´
¨
`
[17] A.-K. Tiden, T. Sjogren, M. Svensson, A. Bernlind, R. Senthilmohan, F. Auchere,
H. Norman, P.-O. Markgren, S. Gustavsson, S. Schmidt, S. Lundquist, L.V. Forbes,
N.J. Magon, L.N. Paton, G.N.L. Jameson, H. Eriksson, A.J. Kettle, 2-Thioxanthines
Are Mechanism-based Inactivators of Myeloperoxidase That Block Oxidative Stress
during Inflammation, J. Biol. Chem. 286 (2011) 37578–37589.
¨
`
[18] L.V. Forbes, T. Sjogren, F. Auchere, D.W. Jenkins, B. Thong, D. Laughton,
P. Hemsley, G. Pairaudeau, R. Turner, H. Eriksson, J.F. Unitt, A.J. Kettle, Potent
Reversible Inhibition of Myeloperoxidase by Aromatic Hydroxamates, J. Biol.
Chem. 288 (2013) 36636–36647.
[36] M.D. Rees, S.E. Bottle, K.E. Fairfull-Smith, E. Malle, J.M. Whitelock, M.J. Davies,
Inhibition of myeloperoxidase-mediated hypochlorous acid production by
nitroxides, Biochem. J 421 (2009) 79–86.
[37] H. Edery, G. Schatzberg-Porath, Some biological properties of two fluoro-
derivatives of tryptamine, Experientia 16 (1960), 200 200.
[19] C. Zhuang, W. Zhang, C. Sheng, W. Zhang, C. Xing, Z. Miao, Chalcone: A Privileged
Structure in Medicinal Chemistry, Chem. Rev. 117 (2017) 7762–7810.
[20] M. Gomes, E. Muratov, M. Pereira, J. Peixoto, L. Rosseto, P. Cravo, C. Andrade,
B. Neves, Chalcone Derivatives: Promising Starting Points for Drug Design,
Molecules 22 (2017) 1210.
[38] F.F. Fleming, L. Yao, P.C. Ravikumar, L. Funk, B.C. Shook, Nitrile-Containing
Pharmaceuticals: Efficacious Roles of the Nitrile Pharmacophore, J. Med. Chem. 53
(2010) 7902–7917.
[39] A.J. Kettle, Detection of 3-chlorotyrosine in proteins exposed to neutrophil
oxidants, in: Methods in Enzymology, Elsevier, 1999: pp. 111–120.
[40] W. Brand-Williams, M.E. Cuvelier, C. Berset, Use of a free radical method to
evaluate antioxidant activity, LWT - Food Sci. Technol. 28 (1995) 25–30.
[41] D. English, B.R. Andersen, Single-step separation of red blood cells, granulocytes
and mononuclear leukocytes on discontinuous density gradients of Ficoll-Hypaque,
J. Immunol. Methods 5 (1974) 249–252.
[21] R.J. de M. Albuquerque, L.K.A.M. Leal, M.A. Bandeira, G.S. de B. Viana, L.V.
Rodrigues, Chalcones from Myracrodruon urundeuva are efficacious in guinea pig
ovalbumin-induced allergic conjunctivitis, Rev. Bras. Farmacogn. 21 (2011)
953–962.
[22] M.L. Zeraik, V.F. Ximenes, L.O. Regasini, L.A. Dutra, D.H.S. Silva, L.M. Fonseca, D.
Coelho, S.a.S. Machado, V.S. Bolzani, 4’-Aminochalcones as novel inhibitors of the
chlorinating activity of myeloperoxidase, Curr. Med. Chem. 19 (2012) 5405–5413.
[23] M.B. Santos, V.C. Pinhanelli, M.A.R. Garcia, G. Silva, S.J. Baek, S.C. França, A.
L. Fachin, M. Marins, L.O. Regasini, Antiproliferative and pro-apoptotic activities
of 2′- and 4′-aminochalcones against tumor canine cells, Eur. J. Med. Chem. 138
(2017) 884–889.
[42] R.R. Kitagawa, M.S.G. Raddi, N.M. Khalil, W. Vilegas, L.M. da Fonseca, Effect of the
Isocoumarin Paepalantine on the Luminol and Lucigenin Amplified
Chemiluminescence of Rat Neutrophils, Biol. Pharm. Bull. 26 (2003) 905–908.
[43] M.L. Verdonk, J.C. Cole, M.J. Hartshorn, C.W. Murray, R.D. Taylor, Improved
protein-ligand docking using GOLD, Proteins. 52 (2003) 609–623.
[44] M. Blair-Johnson, T. Fiedler, R. Fenna, Human Myeloperoxidase: Structure of a
Cyanide Complex and Its Interaction with Bromide and Thiocyanate Substrates at
1.9 Å Resolution †, Biochemistry 40 (2001) 13990–13997.
[24] M.B. dos Santos, D. Bertholin Anselmo, J.G. de Oliveira, B.V. Jardim-Perassi, D.
Alves Monteiro, G. Silva, E. Gomes, A. Lucia Fachin, M. Marins, D.A.P. de Campos
Zuccari, L. Octavio Regasini, Antiproliferative activity and p53 upregulation effects
of chalcones on human breast cancer cells, J. Enzyme Inhibition Med. Chem. 34
(2019) 1093–1099.
[45] A.C. Wallace, R.A. Laskowski, J.M. Thornton, LIGPLOT: a program to generate
schematic diagrams of protein-ligand interactions, Prot. Eng. Des. Sel. 8 (1995)
127–134.
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