P2X7 receptor inhibition by 2-amino-3-aryl-1,4-naphthoquinones

Article abstract of DOI:10.1016/j.bioorg.2020.104278

Extracellular ATP activates purinergic receptors such as P2X7, cationic channels for Ca²⁺, K⁺, and Na⁺. There is robust evidence of the involvement of these receptors in the immune response, so P2X7 receptors (P2X7R) are considered a potential therapeutic target for the development of anti-inflammatory drugs. Although there are many studies of the anti-inflammatory properties of naphthoquinones, these molecules have not yet been explored as P2X7 antagonists. In previous work, our group prepared 3-substituted (halogen or aryl) 2-hydroxy-1,4-naphthoquinones and studied their action on P2X7R. In this paper, eight 2-amino-3-aryl-1,4-naphthoquinones were evaluated to identify the inhibitory activity on P2X7R and the toxicological profile. Three analogues (AD-4CN, AD-4Me, and AD-4F) exhibited reduced toxicity for mammalian cells with CC₅₀ values higher than 500 μM. These three 3-substituted 2-amino-1,4-naphthoquinones inhibited murine P2X7R (mP2X7R) in vitro. However, the analogues AD-4CN and AD-4Me showed low selectivity index values. AD-4F inhibited both mP2X7R and human P2X7R (hP2X7R) with IC₅₀ values of 0.123 and 0.93 μM, respectively. Additionally, this analogue exhibited higher potency than BBG at inhibiting the ATP-induced release of IL-1β in vitro. Carrageenan-induced paw edema in vivo was reversed for AD-4F with an ID₅₀ value of 11.51 ng/kg. Although AD-4F was less potent than previous 3-substituted (halogen or aryl) 2-hydroxy-1,4-naphthoquinones such as AN-04 in vitro, this 3-substituted 2-amino-1,4-naphthoquinone revealed higher potency in vivo to reduce the edematogenic response. In silico analysis suggests that the binding site of the novel 2-amino-3-aryl-1,4-naphthoquinone derivatives, including all the tautomeric forms, is located in the pore area of the hP2X7R model. Based on these results, we considered AD-4F to be a satisfactory P2X7R inhibitor. AD-4F might be used as a scaffold structure to design a novel series of inhibitors with potential inhibitory activity on murine (mP2X7R) and human (hP2X7R) P2X7 receptors.

Full text of DOI:10.1016/j.bioorg.2020.104278

BioorganicChemistry104(2020)104278
Contents lists available at ScienceDirect
Bioorganic Chemistry
journal homepage: www.elsevier.com/locate/bioorg
P2X7 receptor inhibition by 2-amino-3-aryl-1,4-naphthoquinones
⁎
Daniela de Luna Martinsa, , Adriel Alves Borgesa, Nayane A. do A. e Silvaa, Juliana Vieira Fariab,c,
Lucas Villas Bôas Hoelzd, Hellen Valério Chaves Moura de Souzad, Murilo Lamim Belloe,
Nubia Boechatd, Vitor Francisco Ferreiraf, Robson Xavier Fariab,c
a Research Group on Catalysis and Synthesis (CSI), Universidade Federal Fluminense, Laboratório 413, Instituto de Química, Campus do Valonguinho, Centro, Niterói, RJ
24020-141, Brazil
b Postgraduate Program in Sciences and Biotechnology, Instituto de Biologia, Universidade Federal Fluminense, Niterói, RJ, Brazil
c Fundação Oswaldo Cruz, Instituto Oswaldo Cruz, Laboratório de Toxoplasmose e outras protozooses, Avenida Brasil 4365, Manguinhos CEP 21045-900, Rio de Janeiro,
RJ, Brazil
d Laboratorio de Sintese de Farmacos - LASFAR, Farmanguinhos - Fiocruz, Fundacao Oswaldo Cruz, Rua Sizenando Nabuco, 100 - Manguinhos, Rio de Janeiro, RJ 21041-
250, Brazil
e Laboratório de Planejamento Farmacêutico e Simulação Computacional, Faculdade de Farmácia, Universidade Federal do Rio de Janeiro, Rio de Janeiro, RJ 21941-590,
Brazil
f Departamento de Tecnologia Farmacêutica, Universidade Federal Fluminense, Faculdade de Farmácia, R. Dr Mario Vianna, 523 - Santa Rosa, Niterói, RJ 24241-002,
Brazil
A R T I C L E I N F O
A B S T R A C T
Keywords:
Extracellular ATP activates purinergic receptors such as P2X7, cationic channels for Ca2+, K+, and Na+. There is
robust evidence of the involvement of these receptors in the immune response, so P2X7 receptors (P2X7R) are
considered a potential therapeutic target for the development of anti-inflammatory drugs. Although there are
many studies of the anti-inflammatory properties of naphthoquinones, these molecules have not yet been ex-
plored as P2X7 antagonists. In previous work, our group prepared 3-substituted (halogen or aryl) 2-hydroxy-1,4-
naphthoquinones and studied their action on P2X7R. In this paper, eight 2-amino-3-aryl-1,4-naphthoquinones
were evaluated to identify the inhibitory activity on P2X7R and the toxicological profile. Three analogues (AD-
4CN, AD-4Me, and AD-4F) exhibited reduced toxicity for mammalian cells with CC50 values higher than
500 µM. These three 3-substituted 2-amino-1,4-naphthoquinones inhibited murine P2X7R (mP2X7R) in vitro.
However, the analogues AD-4CN and AD-4Me showed low selectivity index values. AD-4F inhibited both
mP2X7R and human P2X7R (hP2X7R) with IC50 values of 0.123 and 0.93 µM, respectively. Additionally, this
analogue exhibited higher potency than BBG at inhibiting the ATP-induced release of IL-1β in vitro. Carrageenan-
induced paw edema in vivo was reversed for AD-4F with an ID50 value of 11.51 ng/kg. Although AD-4F was less
potent than previous 3-substituted (halogen or aryl) 2-hydroxy-1,4-naphthoquinones such as AN-04 in vitro, this
3-substituted 2-amino-1,4-naphthoquinone revealed higher potency in vivo to reduce the edematogenic response.
In silico analysis suggests that the binding site of the novel 2-amino-3-aryl-1,4-naphthoquinone derivatives,
including all the tautomeric forms, is located in the pore area of the hP2X7R model. Based on these results, we
considered AD-4F to be a satisfactory P2X7R inhibitor. AD-4F might be used as a scaffold structure to design a
novel series of inhibitors with potential inhibitory activity on murine (mP2X7R) and human (hP2X7R) P2X7
receptors.
Quinone
Purinergic receptor
Anti-inflammatory
Cation channel
Suzuki coupling
1. Introduction
P2X7 receptor (P2X7R), after ATP binding, results in the passage of
small cations [1,2]. Extracellular ATP can act as a danger signal, in-
Members of the P2X family (P2X1-7) are ATP-gated receptors that
act as cation channels permeable to Na+, K+, and Ca2+. Overture of the
itiating inflammation by activating P2X purinergic receptors. Con-
cerning the immune response, P2X7 is the most investigated among the
⁎ Corresponding author.
E-mail address: dlmartins@id.uff.br (D. de Luna Martins).
URL: https://www.danielamartinsgroup.com.br (D. de Luna Martins).
@ (D. de Luna Martins)
https://doi.org/10.1016/j.bioorg.2020.104278
Received 10 March 2020; Received in revised form 10 September 2020; Accepted 11 September 2020
Availableonline17September2020
0045-2068/©2020ElsevierInc.Allrightsreserved.

D. de Luna Martins, et al.
BioorganicChemistry104(2020)104278
P2 family because it is involved with innate and adaptive immune re-
sponses. P2X7R is expressed in several cells of the immune system, such
as monocytes, macrophages, lymphocytes, and other cells [3]. Several
signaling pathways are connected to P2X7 activation, such as the cas-
pase-1-containing inflammasome NLRP3, which is associated with both
maturation and release of proinflammatory cytokines such as IL-1β [4].
Therefore, P2X7 receptors are an essential target for a plethora of in-
flammatory pathological conditions [3,5–8].
and using microwave irradiation proved to be an effective approach for
arylation of the 3-position of 2-hydroxy-1,4-naphthoquinone [31].
Thus, in this work, we adopted these conditions as a starting point for
the arylation of 2-amino-3-iodo-1,4-naphthoquinone to prepare the AD
series of derivatives. However, some modifications were necessary to be
made so that the reactions could succeed. In contrast to 3-aryl-3-hy-
droxy-1,4-naphthoquinones that were obtained by a fully aqueous
protocol, due to solubility issues, a mixture of ethanol and water (1:1)
was necessary to perform couplings with 2-amino-3-iodo-1,4-naphtho-
quinones to obtain 2-amino-3-aryl-1,4-naphthoquinones. Furthermore,
it was necessary to employ a 5-fold higher amount of the palladium
precatalyst and 2-fold higher amount of boronic acid. This procedure
seems to indicate that 2-amino-3-iodo-1,4-naphthoquinone is a weaker
electrophile for the Suzuki reaction than 2-hydroxy-3-iodo-1,4-naph-
thoquinone. A possible reason is that nitrogen is a better electron
density donor than oxygen, which makes the 3-position of the 2-amino-
3-iodo-1,4-naphthoquinone electronically richer than the same 3-posi-
tion of the 2-hydroxy-3-iodo-1,4-naphthoquinone. The higher density at
the 3-position of the 2-amino-3-iodo-1,4-naphthoquinone compared to
the 3-position density of 2-hydroxy-3-iodo-1,4-naphthoquinone can be
observed by the chemical shift values in the 13C spectrum of the com-
pounds. 2-Amino-3-iodo-1,4-naphthoquinone exhibits a chemical shift
of 82.2 ppm for carbon 3 (quinone ring), whereas 2-hydroxy-3-iodo-
1,4-naphthoquinone exhibits a chemical shift of 92.7 ppm, which
means that the latter is more deshielded, that is, poorer electronically.
Additionally, poor yields were achieved when an acid protocol was
employed for the work-up of the reaction. Better yields were found by
filtering the product and washing the solid with a basic aqueous solu-
tion, which contributed to removing some of the excesses of the boronic
acid, facilitating purification of the product by flash column chroma-
tography.
Actually, an expressive number of commercial and synthetic sub-
stances can target P2X7R and inhibit its function [9]. Although some
selective hP2X7R inhibitors exhibit promising results in clinical assays
[10,11], P2X7R inhibitors have not been approved for therapeutic
purposes. Consequently, there is a need to search for other inhibitors of
P2X7 receptors envisaging anti-inflammatory drugs [12–14].
Naphthoquinones are privileged structures present in several com-
pounds of biological interest [15–17]. Atovaquone, which contains the
1,4-naphthoquinone skeleton as a constituent part of its structure, is
present in the atovaquone-proguanil composition employed in the
treatment of malaria [18,19]. Another example of a member of the
naphthoquinone group of great importance in medicinal chemistry is
doxorubicin, an antineoplastic agent employed in the therapy of several
types of cancer (leukemia, sarcoma, lymphoma) [20–22]. Although
there are several reports of naphthoquinones with anti-inflammatory
activity [23–26], this class of compounds has not been explored con-
cerning its potential as a possible P2X7 receptor antagonist.
In our previous investigation, we prepared a series of 3-substituted
2-hydroxy-1,4-naphthoquinones through Suzuki cross-coupling reac-
tions and studied their anti-inflammatory activity and action on human
and murine P2X7 receptors. Some of these molecules have shown better
results than classic P2X7 antagonists such as BBG, inhibiting IL-1β re-
lease and the induced carrageenan paw edema [27].
In the present work, we synthesized 2-amino-3-aryl-1,4-napthoqui-
nones by Suzuki couplings. This new series of naphthoquinones was
evaluated concerning their capacity to inhibit P2X7R receptor function
in vitro and the acute inflammatory response in vivo. Additionally, we
used a molecular docking approach to study the binding mode of novel
naphthoquinone derivative ligands with the P2X7 receptor.
A series of 2-amino-3-aryl-1,4-naphthoquinones AD was obtained in
moderate yields by Suzuki couplings under microwave irradiation using
different arylboronic acids (Fig. 1). The decrease in yields occurred in
the purification step because of the difficulty of separating the re-
maining boronic acids from the product by column chromatography.
2.2. Biological assays
2. Results
2.2.1. Cytotoxicity studies
2.1. Chemistry
2-Amino-3-aryl-1,4-naphthoquinone derivatives affected the cyto-
plasmic and mitochondrial reducing enzymes from mouse peritoneal
macrophages when measured for the resazurin reduction assay [32].
After continuous treatment for 24 h at a concentration of 10 μM, all
naphthoquinone analogues reduced the metabolic activity (Fig. 2).
Only AD-3F and AD-3T caused a reduction higher than 25% when
compared with the negative control (CN). Therefore, most of the
naphthoquinones demonstrated low toxicity potential.
Using the concept of bioisosterism, through which a prototype can
be optimized in medicinal chemistry, exchanging an atom or group for
another with similar chemical and biological characteristics, we used as
a strategy in this work to exchange the hydroxy group for the amino
group, taking as prototypes the 3-aryl-2-hydroxy-1,4-naphthoquinones
from the previous work [27]. This change was also made because the
amino group can be easily introduced in position 2 of the 1,4-naph-
thoquinone nucleus and, once present, this group increases the re-
activity of the resulting naphthoquinone for iodination in position 3.
This halogenation is important because organic halides are the ideal
substrate for Suzuki coupling, which is our methodological strategy
adopted in the synthetic sequence to obtain aryls in position 3 of the
quinone nucleus.
The amino-1,4-naphthoquinones were evaluated for their ability to
inhibit P2X7R functionality in peritoneal macrophage cells. The ana-
logues AD-Ph, AD-3F, AD-3OMe, and AD-3T did not inhibit ATP-in-
duced dye uptake. However, AD-4CN, AD-4F, and AD-4Me inhibited
this effect (Fig. 3).
Thus, we evaluated the cellular toxicity (for LDH release assay) of
the three analogues that inhibited P2X7 receptor function (AD-4CN,
AD-4F, and AD-4Me). The treatment at crescent concentrations for 24 h
for determining the CC50 value indicated low toxicity for these analo-
gues (Fig. 4A1–A3). AD-4CN exhibited a CC50 value of 513 µM
(Fig. 4A1). The analogues AD-4Me and AD-4F exhibited values twice as
high as those of AD-4CN, 1000, and 1330 µM, respectively (Fig. 4A2
and A3). These three amino-1,4-naphthoquinone analogues showed a
low toxicological profile for mammalian cells.
Initially, 2-amino-1,4-naphthoquinone 2 (yield = 71%) was pre-
pared by the reaction of 1,4-naphthoquinone 1 with hydrazoic acid,
produced in situ by the reaction between sodium azide and acetic acid
[28]. 2-Amino-1,4-naphthoquinone 2 was iodinated using a morpho-
line-iodine complex as the electrophile (yield = 89%). This complex
was prepared from morpholine and iodine (yield = 96%), according to
the procedure reported in the literature [29,30]. 2-Amino-3-iodo-1,4-
naphthoquinone 3 was employed in a Suzuki cross-coupling reaction
using palladium acetate as the precatalyst under microwave irradiation
(Scheme 1).
2.2.2. 2-Amino-3-aryl-1,4-naphthoquinones inhibited P2X7 receptor
function dose-dependently
As previously reported, Suzuki coupling under aqueous conditions
AD-4CN dose-dependently reduced the ATP-induced dye uptake
2

D. de Luna Martins, et al.
BioorganicChemistry104(2020)104278
I
H
O
O
O
O
O
O
O
N
O
I
NH2
I
NH2
NH2
5% Pd(OAc)2
HOAc
NaN3
K2CO3
H2O
K2CO3
H2O/EtOH
(1:1)
Ar
O
1
2
3
AD
Ar-B(OH)2
Scheme 1. Synthetic sequence to 2-amino-3-aryl-1,4-naphthoquinone.
(Fig. 6). The IC50 value measured was 0.093 µM. Therefore, this
naphthoquinone was more potent at inhibiting hP2X7R.
ATP-induced IL-1β release in the human cell lineage was inhibited
by treatment with AD-4F added 30 min before ATP addition (Fig. 7).
The IC50 value calculated for this inhibition was 460.9 nM.
The AD-4F analogue was evaluated in relation to solubility, mi-
crosomal stability, and permeability in vitro. This compound was stable
in mouse and human microsomes (Table 1). Additionally, AD-4F ex-
hibited higher permeability in Caco-2 than propranolol. This control
exhibited 56% permeability, and the compound AD-4F exhibited 70%
permeability (Table 1).
Paw edema formation after 30 min of ATP administration suffered a
blunt reduction in the presence of the compound AD-4F. Pretreatment
with compound AD-4F for 60 min potently inhibited edema formation
(Fig. 8). The ID50 value measured for this naphthoquinone derivative
was 11.91 ng/kg. We observed similar AD-4F inhibitory activity when
paw edema was induced by carrageenan (Supplemental Fig. S1).
2.2.3. Molecular docking
Amino-1,4-naphthoquinone derivatives may exist in different tau-
tomeric forms, depending on the solvent type [33,34]. Considering the
fisiological aqueous medium, we have taken into account the tauto-
merization possibility. Thus, to study the interaction of 2-amino-3-aryl-
1,4-naphthoquinones to the P2X7 receptor, studies were performed for
two tautomeric structures of these compounds: the amino (TAU-1) and
the imino forms (TAU-2) (Scheme 2).
Fig. 1. 2-Amino-3-aryl-1,4-naphthoquinones obtained from Suzuki couplings.
The molecular complexes between the three inhibitors AD-4F, AD-
4CN, and AD-4Me, including all tautomeric forms (aminoquinone-
TAU1 and iminoquinone-TAU2 forms), and the hP2X7R model (Fig. 9)
were predicted using the molecular docking approach. Consequently,
the ligands with the lowest energy were selected for analysis.
The molecular docking results indicated that the predicted binding
site of the amino-naphthoquinone derivatives, including all the tauto-
meric forms, in the hP2X7R model was the same as that reported in our
previous study [27]. Thus, all the compounds interact with negative
values of energy within the cavity formed by Phe95(C), Phe103(C),
Tyr93(C), Tyr291(C), Ser101(C), Thr94(C), Gln98(C), Leu97(A),
Pro96(C), Gln98(A), and Phe102(C) (Fig. 10).
Among the inhibitors, AD-4CN-TAU1 and AD-4CN-TAU2 presented
the highest MolDock scores, −110.42 a.u., and −108.31 a.u., respec-
tively. AD-4F-TAU1 and AD-4F-TAU2 presented the lowest values of
the MolDock score, −121.07 a.u., and −121.27 a.u., respectively,
showing the highest affinity to the hP2X7R model. Similarly, AD-4Me-
TAU1 and AD-4Me-TAU2 presented MolDock score values comparable
to AD-4F-TAU1 and AD-4F-TAU2, −121.15 a.u., and −120.17 a.u.,
respectively. In general, the MolDock score values of the 2-amino-3-
aryl-1,4-naphthoquinone derivatives seem to be more favorable than
those related to the 3-aryl-2-hydroxy-1,4-naphthoquinone derivatives,
which were reported in our previous study [27] (Table 2).
Fig. 2. 2-Amino-3-aryl-1,4-naphthoquinones toxicity on peritoneal macro-
phages (5.0 x106 cells) treated for 24 h, CN- Negative control, CP- Positive
control. These data are representatives of 3–4 experiments in 3 distinct days.*
for p < 0.05, ** for a p < 0.01, *** for a p < 0.001.
assay. However, the IC50 value observed at 0.347 µM was near the CC50
value (Fig. 5B1). Thus, the selectivity index (CC50/IC50 = 1.47) was
low for this analogue. AD-4Me reduced the ATP effect with higher
potency than AD-4CN (Fig. 5B2); in contrast, the selectivity index was
also low: CC50/IC50 = 1.72. The analogue AD-4F potently inhibited
ATP-induced dye uptake when compared with the other naphthoqui-
none analogues (IC50 = 0.123 µM) (Fig. 5B3). The selectivity index
calculated for AD-4F was 10.83. Consequently, we focused the addi-
tional assays on AD-4F.
In addition, the hydrogen bonds (H-bonds) and steric interactions
were mapped using the ligand-map algorithm, generated by the MVD
program. The H-bonds are represented in Fig. 11A–E. AD-4CN-TAU1
and AD-4CN-TAU2 interact with the hP2X7R model via H-bonds with
Phe95(C) (Fig. 11A and B), presenting steric interactions with
Phe95(C), Phe103(C), Ser101(C) and Gln98(A) (Table 2). AD-4F-TAU1
forms H-bonds with Phe95 (C) and Tyr291 (C), while AD-4F-TAU2
BzATP-induced dye uptake in HEK 293 cells expressing the P2X7
receptor was impaired in the presence of crescent AD-4F concentrations
3

D. de Luna Martins, et al.
BioorganicChemistry104(2020)104278
Fig. 3. 2-Amino-3-aryl-1,4-naphthoquinones re-
duced ATP induced dye uptake via P2X7R acti-
vation on mice peritoneal macrophages (5.0
x106 cells). All 2-amino-3-aryl-1,4-naphthoqui-
nones were incubated for 5 min before 5 mM
ATP application for 20 min. PI dye was added in
the last 5 min of ATP treatment. These data are
representatives of 4–5 experiments in 3 distinct
days. * for p < 0.05, ** for a p < 0.01, *** for
a p < 0.001.
100
80
60
***
***
40
***
20
0
l
e
e
F
F
4
T
X
P
N
C
h
o
3
3
-
T
M
M
-
-
r
t
P
-
0
4
A
4
on
D
D
D
-
-
t
3
D
i
-
A
A
A
D
D
on
M
r
A
D
A
T
A
C
m
M
M
M
A
M
u
u
u
5
M
M
%
u
ve
M
u
i
1
u
t
u
0
10
10
10
0
a
10
10
1
g
10
Ne
forms hydrogen bonds with Phe95 (C), Tyr291 (C), Phe103 (C), and
Thr94 (C). These compounds also make steric interactions with
Phe95(C), Phe103(C), Tyr93(C), and Ser101(C) (Fig. 11C and D).
Nevertheless, AD-4Me-TAU1 makes H-bonds with Phe95(C) and
Tyr291(C), while AD-4Me-TAU2 interacts with Phe95(C).
A1
150
100
50
3. Discussion
Previously, we published 3-substituted 2-hydroxy-1,4-naphthoqui-
nones with inhibitory activity against P2X7 receptor function. Among
the tested compounds of this series, the analogues AN-03 and AN-04
showed better inhibitory activity and efficacy results in vitro and in vivo
to reduce the inflammatory response [27]. As explained previously
(Section 2), using the bioisosterism approach and employing 3-aryl-2-
hydroxy-1,4-napthoquinones as prototypes, the hydroxy group was
replaced by the amino group, giving rise to the 2-amino-3-aryl-1,4-
napthoquinone AD series proposed in the present work.
0
-7
-6
-5
-4
-3
-2
[AD-4f] M
150
100
50
A2
The novel 2-amino-1,4-naphthoquinone series obtained showed
higher toxicity compared to the series of 2-hydroxy-1,4-naphthoqui-
nones [27]. All amino-1,4-naphthoquinone derivatives inhibited me-
tabolic activity, a characteristic sometimes associated with a possible
reduction in cellular viability. The toxicity of quinones may be related
to their ability to induce oxidative stress in cells, which might lead to
consequences such as damage to nucleic acids, destruction of proteins,
lipid peroxidation and plasma membrane disruption [35].
0
The 2-hydroxy-1,4-naphthoquinone compounds evaluated are less
toxic than the 2-amino-1,4-naphthoquinone derivatives since the CC50
values obtained for the hydroxy-naphthoquinones tested were all
higher than 800 µM (844–2022 µM). These results corroborate those
obtained from the resazurin tests: 2-hydroxy-1,4-naphthoquinones are
less toxic than 2-amino-1,4-naphthoquinones. The presence of an amino
group at the 2-position of the quinone ring instead of a hydroxy group
leads to increased toxicity in the cells tested. Munday and coworkers
investigated the toxicity of 2-hydroxy-1,4-naphthoquinone, 2-amino-
1,4-naphthoquinone, and its derivatives against blood cells and renal
cells. In this study, in vivo, both compounds showed toxicity to the cells
and tissues surveyed [36]. Nevertheless, it was also observed that
substitution at the 3-position of the naphthoquinone nucleus sig-
nificantly decreased the toxicity, which was also observed in our pre-
vious work [27]. This reduction in toxicity due to substitution at the 3-
position has not yet been fully elucidated. However, steric implications
generated by the substituents might be among the causes, since studies
have shown that, generally, small substituents do not decrease the
toxicity [37]. Ring substitution of 3-aryl-2-hydroxy-1,4-naphthoqui-
nones did not affect the toxicity of these compounds in the resazurin
-8
-6
-4
-2
0
[AD-4CN] M
A3
150
100
50
0
-6
-4
-2
0
[AD-4Me] M
-50
Fig. 4. 2-Amino-3-aryl-1,4-naphthoquinones induced LDH release on perito-
neal macrophages (5.0 x106 cells) treated for 24 h, CN- Negative control, CP-
Positive control. These data are representatives of 3–4 experiments in 3 distinct
days.
4

D. de Luna Martins, et al.
BioorganicChemistry104(2020)104278
150
100
50
B1
150
100
50
0
-10
0
-12
-8
-6
-4
-2
-10
-8
-6
-4
-2
[AD-4F] M
[AD-4F] M
Fig. 6. AD-4F dose-dependently inhibit P2X7R function HEK 293 cells trans-
fected with P2X7 receptor (2.5 x106 cells). Crescent doses of AD-4F incubated
for 5 min before 5 mM ATP application for 20 min. PI dye was added in the last
5 min of ATP treatment. These data are representatives of 3 experiments in 3
distinct days.
150
100
50
B2
150
100
50
0
-10
-8
-6
-4
-2
[Ad-4CN] M
0
-10
-8
-6
-4
-2
B3
150
100
50
[AD-4F] M
Fig. 7. ATP-induced IL-1β release inhibition by 2-amino-3-aryl-1,4-naphtho-
quinones in THP-1 cells. (A) Differentiated THP-1 cells (5 x106 cells) treated
with 1 mM ATP (30 min) and LPS (4 h) in the presence of BBG, A740003, or
AD-4F. The curves are representative of 3–5 independent experiments.
Table 1
Liver microsomal (LM) stability and Caco-2 data for AD-4F.
Compound
LM stabilitya mouse
LM stabilitya human
Caco-2b
0
-10
AD-4F
46.7
59.3
69.7
2.6
-8
-6
-4
-2
a
Stability in mice and human liver microsomes. Data reported as CLint (µL
/minutes/mg protein).
[AD-4Me] M
b
Fig. 5. AD-4F, AD-4CN, and AD-4Me dose-dependently inhibit P2X7R func-
tion in peritoneal macrophages (2.5 x106 cells). Crescent doses of AD-4F, AD-
4CN, and AD-4Me were incubated for 5 min before 5 mM ATP application for
20 min. PI dye was added in the last 5 min of ATP treatment. These data are
representatives of 4–5 experiments in 3 distinct days.
Apparent permeability values (Papp) measured using low permeability and
high permeability absorption compounds, vinblastine, and propranolol, re-
spectively, as reference. Data are reported in 106 cm/s. These values indicate an
apical to basolateral (A - B) direction. They were tested at the same time as AD-
4F. Values are means
standard error of 3 experiments.
residues have already been indicated in other studies as important for
the binding of ligands at the allosteric site of P2X7R [38,39].
test. In contrast, the presence of a ring substituent in 2-amino-1,4-
naphthoquinones, regardless of the nature of the substituent, decreased
the toxicity of 2-amino-1,4-naphthoquinones compared to compound
AD-Ph (unsubstituted ring, Supplemental Fig. S1). Thus, for 2-amino-
1,4-naphthoquinone derivatives, the steric effect may be more critical
in reducing toxicity than the electronic effect.
As observed for compound AN-04, the inhibitory activity of com-
pound AD-4F is higher on hP2X7R than on mP2X7R. However, the
inhibitory activity of compound AD-4F is less than the inhibitory ac-
tivity of compound AN-04 on hP2X7R. The binding site indicated by
docking studies is located at the pore upper area between two different
subunits of the ion channel. The binding of ligands in that molecular
area of P2X7R might prevent the movement of the protein, thus
blocking the pore of the ion channel [38]. This binding pocket is in the
same allosteric binding site area of well-known ligands, such as
A740003, A804598, AZ10606120, GW791343 and JNJ47965567,
which is a binding pocket distal to the ATP-binding site [39].
Comparing 2-hydroxy-3-phenyl-1,4-naphthoquinone AN-04, which
was the most potent inhibitor from the hydroxy-1,4-naphthoquinone
series, to the most potent inhibitor AD-4F of the 2-amino-3-aryl-1,4-
naphthoquinone series, it is possible to observe that an amino sub-
stituent does not improve the inhibitory effect against the P2X7 re-
ceptor. The pharmacophore 1,4-naphthoquinone core seems to have
affinity to residues Phe95 and Phe103 by means of intermolecular in-
teractions such as hydrophobic and π-stacking interactions. These
Although the hit compound of the series of 3-aryl-2-hydroxy-1,4-
5

D. de Luna Martins, et al.
BioorganicChemistry104(2020)104278
150
paw edema in a previous publication [27]. AD-4F (1 mg/kg) inhibited
carrageenan-induced paw edema 2.42 times higher than sodium di-
clofenac (10 mg/kg) (Supplemental Fig. S1). In the previous publica-
tion, AN-04 (1 mg/kg) inhibited 0.4 times more than sodium diclofenac
(10 mg/kg). This reduced AD-4F concentration to inhibit the in-
flammatory response compared with AN-04 may be associated with the
higher permeability measured in the Caco-2 cell assay. Although AD-4F
was less potent in vitro to inhibit P2X7 receptor function than AN-04, its
higher permeability seems to compensate for this feature and cause a
more potent inhibitory effect in vivo.
100
50
Since pH 7.4 was applied in biological assays in vitro, it is possible
that the ionized form of compound AN-04 is predominant with de-
protonated hydroxyl groups. The ionized form of the compound is less
fat-soluble and better solvated in aqueous media, decreasing the mo-
lecular membrane permeability. On the other hand, the compound AD-
4F probably has a higher concentration of non-ionized state molecules
at pH 7.4, which might permeate more easily through the lipid bilayer
membrane. As the non-ionized form of compound AD-4F might be
passively transported through the lipid membranes, the acid-base
equilibrium shifts to facilitate the permeation of this 2-amino-1,4-
naphthoquinone derivative. The higher solvation of compound AN-04
may be responsible for hindering its passive transport across the lipid
membranes compared to compound AD-4F. This possible physical–-
chemical property difference between the compounds seems to be in
agreement with the better permeation in the Caco-2 assay presented by
derivative AD-4F.
0
-10
-9
-8
-7
-6
-5
-4
[AD-4F] mg/kg
Fig.8. AD-4F inhibits the ATP-induced paw edema. Groups containing five
Swiss Webster mice that were administered with intraplantar ATP. AD-4F
(0.001–1 mg/kg) (A) was administered 60 min before ATP administration, all
these as control. Paw edema was measured 30 min after the ATP application.
These results represent three distinct days and are expressed as mean
s.d.
O
O
NH2
NH
Ar
Ar
O
O
H
Molecular docking results showed a better score for the compound
AD-4F pose than the analogue AN-04 binding pose, while the in vitro
biological assay indicated a higher inhibitory activity of compound AN-
04 than the compound AD-4F on P2X7R. Most likely, after inter-
molecular interactions of ligands with P2X7R, compound AN-04 sta-
bilized the molecular complex more efficiently than compound AD-4F.
It is possible to observe in the ligand binding poses that the hydro-
phobic interactions are important for affinity to P2X7R, and the oxygen
atoms of the quinone moiety are involved in hydrogen bonds.
Therefore, the substitution of hydroxyl groups by amine groups, both
bioisoster substituents, in the novel 2-amino-1,4-naphthoquinone series
inserted a hydrogen bond donor group that reduced the affinity of
compound AD-4F to the binding site compared to compound AN-04.
Nevertheless, the hit compound AD-4F showed the best performance in
the in vivo biological evaluations.
TAU-1
TAU-2
Scheme 2. Tautomeric forms of 2-amino-3-aryl-1,4-naphthoquinones.
4. Conclusions
Suzuki cross-couplings proved to be an efficient methodology for
obtaining 2-amino-3-aryl-1,4-naphthoquinones, which were prepared
under microwave irradiation (30 min) in moderate yields. AD-4F, AD-
4Me and AD-4CN exhibited low toxicity with respect to mammalian
cells (CC50 > 500 μM), and AD-4F presented the best selectivity index
of the series (selectivity index CC50/IC50 = 10.83).
AD-4F potently inhibited mP2X7R and hP2X7R, being more active
in hP2X7R inhibition (BzATP-induced dye uptake in HEK 293 cells
expressing P2X7: IC50 = 0.093 μM). AD-4F was shown to be more
potent than the known inhibitor BBG in ATP-induced IL-1β release in-
hibition with activity on both mP2X7R (IC50 = 0.544 μM) and hP2X7R
(IC50 = 0.460 μM).
Fig. 9. Cartoon structural representation of the human P2X7 receptor
(hP2X7R), showing the A (in blue), B (in red), and C (in green) chains. (For
interpretation of the references to color in this figure legend, the reader is re-
ferred to the web version of this article.)
naphthoquinones showed better inhibitory activity than the hit com-
pound of the series 2-amino-3-aryl-1,4-naphthoquinone, compound
AD-4F exhibited microsomal stability 1.6 times higher (mouse and
human) than compound AN-04. The compound AD-4F also presented
better permeability (1.6 times higher) than the compound AN-04,
which was the best P2X7R inhibitor from the hydroxy-1,4-naphtho-
quinone series.
The replacement of the hydroxyl group of the 2-hydroxy-1,4-
naphthoquinone series used as a prototype by the amino group did not
result in an improvement in the inhibitory activity on P2X7 function in
vitro; however, the hit compound AD-4F of the novel 2-amino-1,4-
naphthoquinone series showed better performance in vivo. That is, the
presence of the amino group at position 2 of the quinone moiety re-
sulted in improved reversion of the paw edema caused by ATP
(11.51 ng/kg) and carrageenan (113 ng/kg). Derivative AD-4F also
showed better performance compared to sodium diclofenac in pro-
moting the reduction of edema. Improvement in the in vivo performance
The ID50 value calculated for carrageenan-induced paw edema in-
hibition was 113 ng/kg. The hydroxy-1,4-naphthoquinone AN-04 ex-
hibited an ID50 value of 294 ng/kg for inhibiting carrageenan-induced
6

D. de Luna Martins, et al.
BioorganicChemistry104(2020)104278
Fig. 10. Binding site of 2-amino-3-aryl-1,4-naphthoquinones derivatives in the human P2X7 receptor (hP2X7R) according to molecular docking results.
compared to the prototype compound may be related to its better mi-
crosomal stability and better permeability in the assay using Caco-2
cells.
5.1.1. Experimental procedure for the synthesis of 2-Amino-1,4-
naphthoquinone 2 from 1,4-naphthoquinone 1
A round-bottomflask (250 mL) equipped with a magnetic stir bar
was charged with 1,4-naphthoquinone (5.0 g, 32 mmol) and glacial
acetic acid (50 mL). This system was heated to 40 °C, and this tem-
perature was maintained until the end of the reaction. A second solu-
tion was prepared in a 50 mL beaker containing sodium azide (3,4 g,
52 mmol) and water (10 mL). This later solution was dropped over the
1,4-naphthoquinone solution. The resulting mixture was stirred at 40 °C
for an additional 1.5 h. After the reaction mixture reached room tem-
perature, cold water was added for precipitation of 2-amino-1,4-naph-
thoquinone, which was obtained after vacuum filtration and washing
with acetic acid and cold water. The product was recrystallized with
ethanol and obtained as a brown solid in 71% yield [28].
Molecular docking studies with tautomer molecules of the most
active compounds of the novel 2-amino-1,4-naphthoquinone series in-
dicated that the binding site is located in an allosteric pocket and that
hydrophobic interactions with Phe95 and Phe103 might be important
for binding affinity. Therefore, the hit compound AD-4F seems to be
promising for additional investigations.
5. Experimental section
5.1. Chemistry
2-Amino-1,4-naphthoquinone (2) – The compound was obtained
as a brown solid in 71% yield. IR (KBr) ʋmax/cm−1 3323, 3288 (ν NeH),
1615 (ν C]O), 1564 (νNH). δH (500 MHz; DMSO‑d6; J in Hz) 7.93 (1H,
dd, J = 1.0; 7.5), 7.89 (1H, dd, J = 1.0; 7.5), 7.70–7.68 (1H, m), 7.12
(2H, s, NH2), 5.82 (1H, s) ppm.
Common solvents were obtained from VETEC Química. Deuterated
solvents and the other reagents were purchased from Sigma Aldrich
Brazil LTDA. A Fisatom 413D apparatus was employed to determine the
noncorrected melting points of the products. KBr pellets were used in
the infrared analysis, and spectra were recorded on a Varian FT-IR 660
spectrophotometer. A Varian VNMRS (300 or 500 MHz) instrument was
utilized for recording the NMR spectra, using CDCl3 or DMSO‑d6 as
5.1.2. Experimental procedure for preparing 2-Amino-3-iodo-1,4-
naphthoquinone (3)
solvents, with chemical shifts represented in units of
δ ppm.
a) Morpholine-iodine Complex: a 125 mL separatory funnel was
charged with distilled water (50 mL), potassium iodide (KI, 47 g,
280 mmol) and iodine (I2, 25.4 g, 100 mmol). A second solution was
placed in an Erlenmeyer (250 mL) equipped with a magnetic stir bar
and containing distilled water (80 mL) and morpholine (10 mL,
Tetramethylsilane (TMS) was used as the internal standard. Coupling
constants (J, Hertz) refer to apparent multiplicities of the peak. Thin-
layer chromatoplates from Sillicycle Ultrapure Silica Gels (F254) were
employed to monitor the progress of the reaction.
Table 2
Summary of the interactions between all tautomeric forms of each inhibitor (AD-4CN, AD-4Me, and AD-4F) and the human P2X7 receptor (hP2X7R) model,
including the respective Moldock score values.
Inhibitors
hP2X7R
Residues (H-bond interaction)
Residues (steric interactions)
MolDock score (a.u.)a
AD-4CN-TAU1
AD-4CN-TAU2
AD-4F-TAU1
AD-4F-TAU2
AD-4Me-TAU1
AD-4Me-TAU2
AN02-TAU1
AN02-TAU2
AN03-TAU1
AN03-TAU2
AN04-TAU1
AN04-TAU2
Phe95(C)
Phe95(C), Phe103(C), Asp92(C), Ser101(C), Gln98(A)
Phe95(C), Phe103(C), Tyr93(C), Ser101(C), Gln98(A)
Phe95(C), Phe103(C), Tyr93(C), Ser101(C)
Phe95(C), Phe103(C), Tyr93(C), Ser101(C)
Phe95(C), Phe103(C), Tyr93(C), Ser101(C)
Phe95(C), Phe103(C), Tyr93(C), Ser101(C), Asp92(C)
Ser101(C), Leu97(A),Pro96(C)
−110.42
−108.31
−121.07
−121.27
−121.15
−120.17
−97.01
Phe95(C)
Phe95(C), Tyr291(C)
Phe95(C), Phe103(C), Tyr291(C), Thr94(C)
Phe95(C), Tyr291(C)
Phe95(C), Phe103(C), Tyr291(C), Thr94(C)
Tyr291(C), Ser101(C), Gln98(C)
Phe95(C), Thr94(C), Gln98(A)
Gln98(C), Ser101(C), Tyr291(C)
Gln98(A), Thr94(C), Phe95(C), Phe103(C)
Tyr291(C)
Gln98(A), Ser101(C), Phe102(C), Pro96(C)
Pro96(C), Ser101(C),Leu97(A)
−91.29
−97.29
Thr94(C), Phe103(C),Tyr93(C)
−92.44
Phe95(C), Ser101(C), Pro98(A), Phe293(C)
Phe95(C), Phe103(C), Ser101(C)
−104.96
−111.45
Phe95(C), Phe103(C), Thr94(C)
a
Arbitrary unities; bThe letter within the parenthesis indicates the chain of the respective residue.
7

D. de Luna Martins, et al.
BioorganicChemistry104(2020)104278
Fig. 11. Representation of the hydrogen bond interactions between the human P2X7 receptor model and the inhibitors: (A) AD-4CN-TAU1, (B) AD-4CN-TAU2, (C)
AD-4F-TAU1, (D) AD-4F-TAU2, (E) AD-4Me-TAU1, and (F) AD-4Me-TAU2. The respective chain are shown within the parenthesis. Black interrupted lines represent
the hydrogen bonds. The inhibitors and hP2X7R residue structures are the in stick model and colored by atom: the nitrogen atoms are shown in blue, the oxygen in
red, the fluor atoms in purple, and the carbon chain in white or cyan. The hydrogen atoms were omitted for clarity, except for those present in the heteroatoms and
the structure of the inhibitor. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
100 mmol). The iodine solution was dripped slowly onto the morpho-
line solution under vigorous agitation for 2 h. An orange solid was
obtained after vacuum filtration and after washing the crude product
with cold water in 96% yield. b) Iodination of 2-amino-1,4-naphtho-
quinone: An Erlenmeyer flask equipped with a magnetic stir bar was
charged with 2-amino-1,4-naphthoquinone (30 mmol, 5.2 g), potassium
carbonate (90 mmol, 12.44 g) and distilled water (300 mL). To this
mixture, the morpholine-iodine complex was added in small portions
every 15 min. After completion of the complex addition, the reaction
mixture remained for another 30 min under stirring. Then, the reaction
mixture was cooled with an ice bath and acidified with 85% H3PO4. The
product was obtained after vacuum filtration, after washing with dis-
tilled water and drying, as an orange solid in 89% yield [27,29].
2-Amino-3-iodo-1,4-naphthoquinone (3) – The compound was
obtained as an orange solid in 89% yield. mp 188–190 °C (lit.
201–202 °C) [26]. IR (KBr) ʋmax/cm−1 3447, 3346 (ν NH2), 1671 (νC]
O), 1579 (νNH). δH (500 MHz; DMSO‑d6; J in Hz) 8.13–8.10 (1H, m),
7.93–7.89 (1H, m), 7.88–7.85 (1H, s) ppm. δC (125 MHz; DMSO‑d6)
177.3, 176.5, 152.5, 134.5, 132.5, 131.5, 129.4, 126.4, 126.3,
82.2 ppm. HRMS (ESI+) m/z calc. for C10H6INNaO2 [M−Na]+ 322.04,
found 321.9328.
8

D. de Luna Martins, et al.
BioorganicChemistry104(2020)104278
5.1.3. Typical procedure for Suzuki couplings for preparing 2-amino-3-aryl-
1,4-naphthoquinones
158.4, 146.2, 134.7, 132.9, 132.3, 131.6, 130.2, 125.7, 125.4, 125.1, 114.5,
113.9, 55.9 ppm. HRMS (ESI+) m/z calc. for C17H13NNaO3
[M−Na]+302.0793, found 302.0774.
A Pyrex® 10 mL tube was charged with 2-amino-3-iodo-1,4-napht-
thoquinone (0,5 mmol, 150 mg), potassium carbonate (2.5 mmol,
346 mg), arylboronic acid (1.0 mmol), Pd(OAc)2 (5% mol, 0.025 mmol)
and water/ethanol 1:1 (5.0 mL). The reaction mixture was subjected to
microwave irradiation (100 °C, 30 min) using a monomode microwave
CEM Discover reactor. The solid product was filtered and washed with
aqueous sodium hydroxide 1.0 M solution and water. The crude pro-
ducts were purified by flash chromatography using 75% di-
chloromethane/hexane.
2-Amino-3-(4-methylphenyl)-1,4-naphthoquinone (AD-4Me) –
Red solid (31%), m.p. = 210–211 °C. IR (KBr) ʋmax/cm−1 3467, 3352
(νNH2), 1670, 1598 (νC]O), 2907 (νCeH). δH (500 MHz; DMSO‑d6; J
in Hz) 8.00 (1H, dd, J = 5.0 Hz), 7,97 (1H, dd, J = 5.0 Hz), 7.84 (1H,
td, J = 5.0 Hz), 7.73 (1H, td, J = 5.0 Hz), 7.25 (2H, d, J = 5.0 Hz),
7.18 (2H, d, J = 5.0 Hz), 2.36 (3H, s) ppm. δC (100 MHz; DMSO‑d6)
181.8, 180.3, 146.2, 136.4, 134.8, 132.0, 132.2, 130.3, 130.2, 129.0,
125.8, 114.7, 21.0 ppm. HRMS (ESI+) m/z calc. for C17H13NNaO2
[M−Na]+286.0844, found 286.0835.
2-Amino-3-phenyl-1,4-naphthoquinone (AD-Ph)
– The com-
pound was obtained as a red solid in 54% yield, m.p. = 177–178 °C. IR
2-Amino-3-(thiophen-3-yl)-1,4-naphthoquinone
(AD-3T)
–
(KBr)
ʋ
max/cm−1 3279 (νN-H), 1603 (νC]O), 1557 (νNH). δH
Purple solid (40%), m.p. = 210–211 °C. IR (KBr) ʋmax/cm−1 3351
(νNH2), 1670 (νC]O). δH (500 MHz; DMSO‑d6; J in Hz) 8.05 (2H, m),
7,94–7,937 (1H, m), 7.87 (1H, td, J = 15.0 Hz, 7.5 Hz), 7.82 (1H, td,
J = 15.0 Hz), 7.56–7.53 (2H, m) ppm. δC (125 MHz; CDCl3) 181.94,
180.71, 146.67, 135.15, 133.14, 133.10, 132.76, 130.15, 129.88,
127.01, 126.85, 125.20, 110.80 ppm. HRMS (ESI+) m/z calc. for
C14H9NNaO2S [M−Na]+278.0252, found 278.0238.
(500 MHz; DMSO‑d6; J in Hz) 8.13 (1H, d, J = 7.5 Hz); 8.10 (1H, d,
J = 7.0 Hz); 7.95 (1H, td, J = 1.0, 7.5 Hz); 7.86 (1H, td, J = 1.0,
7.5 Hz); 7.57 (2H, t, J = 7.5 Hz); 7.47 (1H, t, J = 7.5 Hz); 7.41–7.39
(2H, m) ppm. δC (125 MHz; DMSO‑d6) 181.7, 180.2, 146.1, 134.7,
130.4, 128.3, 127.3, 125.7, 125.5, 114.8 ppm. HRMS (ESI+) m/z calc.
for C16H11NNaO2 [M−Na]+272.0684, found 272.0669.
2-Amino-3-(4-fluorophenyl)-1,4-naphthoquinone (AD-4F) – The
compound was obtained as
a
red solid in 44% yield.,
5.2. Biological assays
m.p. = 193–194 °C. IR (KBr) ʋmax/cm−1 3470, 3359 (νNH2), 1674,
1674, 1601 (νC]O), 1348 (νCN). δH (500 MHz; DMSO‑d6; J in Hz) 8.00
(1H, dd, J = 5.0 Hz), 7,90 (1H, dd, J = 5.0 Hz), 7.83 (1H, td,
J = 5.0 Hz), 7.77 (1H, td, J = 5.0 Hz), 7.32 (2H, m, J = 5.0 Hz), 7.2
(1H, m, J = 10.0 Hz) ppm. δC (100 MHz; DMSO‑d6) 181.7, 180.2,
162.6, 160.20, 146.6, 134.8, 132.7, 132.6,132.3, 130.2, 129.6, 129.5,
125.7, 125.5, 115.4, 115.1, 113.7 ppm. HRMS (ESI+) m/z calc. for
5.2.1. Mouse peritoneal macrophages
We used male Swiss Webster mice for the collection of macrophages
from the peritoneal cavity. The protocols used were in agreement with
the Ethical Principles in Animal Experimentation adopted by the
Brazilian College of Animal Experimentation as well as with those ap-
proved by the FIOCRUZ Research Ethics Committee (number L039-16).
C
16H10FNNaO2 [M−Na]+290.0593, found 290.0578.
2-Amino-3-(3-fluorophenyl)-1,4-naphthoquinone (AD-3F) – The
5.2.2. HEK293 cells transfected with P2X7R
compound was obtained as
a
red solid in 45% yield.,
Cultivation of HEK293 cells expressing P2X7R was performed sup-
plemented with fetal bovine serum (FBS 10%), L-glutamine (2 mM) and
antibiotics (streptomycin 50 µg/mL, penicillin 50 U/mL) and main-
tained in DMEM (Dulbecco’s modified Eagle’s medium) using a humi-
dified CO2 atmosphere (5%) at 37 °C.
m.p. = 188–189 °C. IR (KBr) ʋmax/cm−1 3394, 3282 (νNH2), 1674,
1603 (νC]O), 1353 (νCN). δH (500 MHz; CDCl3; J in Hz) 8.14 (1H, dd,
J = 5.0 Hz), 8.07 (1H, dd, J = 5.0 Hz), 7.74 (1H, td, J = 5.0 Hz), 7.65
(1H, td, J = 5.0 Hz), 7.43 (1H, m, J = 10 Hz), 7.2 (1H, m, J = 15.0 Hz)
ppm. δC (100 MHz; DMSO‑d6) 181.7, 179.9, 163.4, 161.0, 146.5, 135.8,
135.7, 134.9, 132.8, 132.4, 130.2, 130.2, 130.1, 126.7, 126.7, 125.8,
125.6, 117.5, 117.3, 114.2, 114.0, 113.3., 113.2 ppm. HRMS (ESI+) m/
z calc. for C16H10FNNaO2 [M−Na]+290.0593, found 290.0577.
5.2.3. Dye uptake assay
Before treatment with ATP, naphthoquinones and antagonists of P2X7R
were preincubated for 10 min. Doses of the antagonists ranged from 1 ng to
500 µg/mL. ATP solution (5 mM) was added to mouse peritoneal macro-
phages (5.0 × 105 cells) or HEK293 cells (5.0 × 105 cells) for 25 min at
37 °C. Propidium iodide (PI) (5.0 × 10−2 mg/mL in PBS) was added in the
last five minutes of ATP treatment. After ATP treatment, we discarded the
medium and added a PBS solution with 4% paraformaldehyde until mea-
suring in an M5 Spectramax reader with excitation and emission wave-
lengths of 535 nm and 617 nm, respectively [27].
2-Amino-3-(4-cyanophenyl)-1,4-naphthoquinone (AD-4CN)
–
The compound was obtained as an orange solid in 23% yield,
m.p. = 229–230 °C. IR (KBr) ʋmax/cm−1 3579, 3414, 3282 (νNH2),
2233 (νC^N), 1682, 1637, 1603 (νC = O). δH (500 MHz; DMSO‑d6; J
in Hz) 8.15 (1H, dd, J = 5.0 Hz),8.12 (1H, dd, J = 5.0 Hz), 7.77 (3H,
M, J = 10.0 Hz), 7.65 (1H, td, J = 5.0 Hz), 7.70 (1H, td, J = 5.0 Hz),
7.52 (2H, d, J = 5.0 Hz) ppm. δC (100 MHz; DMSO‑d6) 181.8, 180.2,
147.0, 139.1, 135.4, 133.0, 132.9, 132.6, 132.1, 130.4, 126.2, 126.1,
119.5, 113.1, 110.1 ppm. HRMS (ESI+) m/z calc. for C17H10N2NaO2
[M−Na]+297.0640, found 297.0625.
5.2.4. IL-1β enzyme-linked immunosorbent assay (ELISA)
THP-1 cells were primed for 4 h with LPS (25 ng/mL) prior to the
experiments. At the last hour of incubation at 37 °C with LPS, AD-4F
was added. At the last 30 min, ATP (5 mM) was also added. The col-
lected treated samples were centrifuged (251.55 × g, 5 min, 4 °C), and
the supernatants were stored at −70 °C. IL-1β release was measured
using an ELISA Kit (ab46052-ABCAM, Cambridge).
2-Amino-3-(4-methoxylphenyl)-1,4-naphthoquinone
(AD-
4OMe)– Purple solid (53%), m.p. = 210–211 °C. IR (KBr) ʋmax/cm−1
3470, 3360 (νNH2), 1670, 1598 (νC]O), 2921 (νCeH). δH (500 MHz;
DMSO‑d6; J in Hz) 8.00 (1H, dd, J = 5.0 Hz), 7,96 (1H, dd, J = 5.0 Hz),
7.84 (1H, td, J = 5.0 Hz), 7.72 (1H, td, J = 5.0 Hz), 7.21 (2H, d,
J = 5.0 Hz), 7.19 (1H, d, J = 10.0 Hz), 3.84 (3H, s) ppm. δC (100 MHz;
DMSO‑d6) 181.8, 180.1, 159.2, 146.2, 134.8, 134.6, 132.9, 132.3,
130.2, 129.4, 125.8, 125.5, 122.7, 115.8, 114.6, 113.1, 55.0 ppm.
HRMS (ESI+) m/z calc. for C17H13NNaO3 [M−Na]+302.0793, found
302.0774.
5.2.5. pH dependent solubility of AD-4F analog
We measured the AD-4F kinetic solubility using DMSO solutions
(5 µL, in triplicate) containing concentrations ranging from 1 to
250 µM. These solutions were added to 995 µL buffer (pH 2.0 - hy-
drochloride, 4.0–100 mM citrate buffer and 7.4–100 mM phosphate
buffer) in a 96-well plate for 2 h at room temperature. Calibration
standards solutions were prepared with DMSO stock solutions (5 µL)
into 995 µL acetonitrile/buffer (1:1) mixture. The reaction samples
were centrifuged (1118 × g, 10 min, 25 °C) and diluted 1:1 with
acetonitrile [27].
2-Amino-3-(3-methoxylphenyl)-1,4-naphthoquinone (AD-3OMe) –
Red solid (25%), m.p. = 212–213 °C. IR (KBr) ʋmax/cm−1 3402, 3304 (ν
NH2), 1672, 1598 (νC]O), 2938 (νCeH). δH (500 MHz; CDCl3; J in Hz)
8.00 (1H, dd, J = 5.0 Hz), 7,97 (1H, dd, J = 5.0 Hz), 7.84 (1H, td,
J = 5.0 Hz), 7.73 (1H, td, J = 5.0 Hz), 7.21 (1H, t, J = 5.0 Hz), 7.19 (3H,
m, J = 9.0 Hz), 3.85 (3H, s) ppm. δC (100 MHz; DMSO‑d6) 181.8, 180.4,
9

D. de Luna Martins, et al.
BioorganicChemistry104(2020)104278
5.2.6. Caco-2 cell culture and treatments
5.3. In silico evaluation
Caco-2 cells (3 × 105 cells/well) bathed with DMEM supplemented
with 10% FSB were seeded on Corning®Costar®Transwell plates (Sigma-
Aldrich, St. Louis, MO, USA). This culture was conserved for up to
21 days in a humidified incubator at 37 °C and 5% CO2. AD-4F, vin-
blastine (poor permeability control), and propranolol (high perme-
ability control) stock solutions, all at a concentration of 100 mM, were
prepared in Hank's Balanced Salt Solution (HBSS) containing 25 mM
HEPES at pH 7.4 with 0.5% (v/v) DMSO. Transport buffer (0.3 mL) was
used to normalize the cells with the transport buffer in each well. A 24-
well enhanced repossession plate with 1 mL of transport buffer (pH 7.4)
substituted the feeder tray. The transport buffer in the superior wells
was replaced, and 0.3 mL of the AD-4F, vinblastine, or propranolol
solution was added. Then, the cells were substituted for incubation for
60 min. Lucifer yellow concentrations in the donor and acceptor wells
were evaluated at the last incubation time. The fluorescence measure-
ment was realized using a plate reader M5 Spectramax at excitation
485 nm and emission 530 nm.
5.3.1. Comparative modelling
The hP2X7R model was constructed using the approach previously
described [27].
5.3.2. Ligand preparation
Chemicalize® software (ChemAxon) was employed to support the
evaluation of tautomer forms and ionization states [40]. Chemical
structures of the tautomers of the inhibitors were built with the aid of
the program Spartan’10 v.1.0.1. (Wavefunction, Inc., Irvine, CA, USA),
where the semi-empirical RM1 (Recife Model 1) method was applied for
geometry optimization [41].
5.3.3. Molecular docking
Molecular docking was performed using the Molegro Virtual Docker
(MVD) 6.0 program (CLC Bio, 8200, Aarhus, Denmark) [42]. The
MolDock score algorithm was selected as the score function, and the
partial charges were assigned according to the MVD charges scheme.
The search algorithm, MolDock Optimizer, was used within a search
space of 40 Å around each detected cavity in the hP2X7R model. Fi-
nally, molecular docking was performed for each tautomer using the
following parameters set: runs = 100, population size = 50, max in-
teractions = 2000, scaling factor = 0.50, and crossover rate = 0.90),
followed by energy minimization. The poses with the lowest energy
values were selected and analyzed using the MVD program.
5.2.7. In vitro stability assays in liver microsomes
AD-4F stability was assessed in male mouse and human liver mi-
crosomes [27]. Both microsomes accommodated a final protein con-
centration of 0.5 mg/mL (0.1 M phosphate buffer) at pH 7.4. Micro-
somes were preincubated with AD-4F (1 μM) and DMSO (0.5 μM) at
37 °C prior to NADPH (1 mM) addition. A buffer containing 0.1 M
phosphate (50 μL) at pH 7.4 was used as a control for the reaction.
Positive controls for mouse and human microsomes were diazepam and
verapamil, respectively. Human and mouse microsomes were treated
with Ad-4F for 0, 5, 15, 30, and 45 min and the negative control (minus
NADPH) for 45 min. The reactions were stopped using methanol
(50 μL) at the appropriate time points. Subsequently, the samples were
centrifuged (1640 × g) for 20 min at 4 °C to prevent protein pre-
cipitation.
Declaration of Competing Interest
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influ-
ence the work reported in this paper.
Acknowledgements
We used the equations below for calculating in vitro intrinsic
clearance (CLint mic) for the metabolism of AD-4F in mouse and human
liver microsomes:
The authors are indebted to CNPq (National Council of Research of
Brazil, Fellowship Process Numbers: 308755/2018-9, 455739/2014-5),
CAPES and FAPERJ for funding this work and for Research fellowships
(Fellowship Process Number E-26/203.246/2017, E-26/010.001861/
2019; E-26/010.002125/2019). This study was financed in part by the
Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - Brasil
(CAPES) - Finance Code 001.
Half life(t1/2)(minutes) = 0.693/k
(1)
V(µL/mg) = volume of the incubation (µL)/protein in the incubation (mg)
(2)
Intrinsic clearance (CLint) (µL/minutes/ mg protein) = V × 0.693/t1/2
Appendix A. Supplementary material
according to the manufacturer’s instructions.
Supplementary data to this article can be found online at https://
doi.org/10.1016/j.bioorg.2020.104278.
5.2.8. Paw edema
References
AD-4F or sodium diclofenac were administered via i.p. at 60 min
before intrathecal ATP (2.5 mM/paw) or carrageenan (300 µM/paw)
administration. ATP and carrageenan-induced mouse paw edema for-
mation were measured using a plethysmometer (Insight, Brazil). This
protocol obeyed the Ethical Principles in Animal Experimentation and
was approved by the FIOCRUZ Research Ethics Committee (number
LW-58/14).
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