1,4-Naphthoquinones potently inhibiting P2X7 receptor activity

Article abstract of DOI:10.1016/j.ejmech.2017.10.033

P2X7 receptor (P2X7R) is an ATP-gated ion-channel with potential therapeutic applications. In this study, we prepared and searched a series of 1,4-naphthoquinones derivatives to evaluate their antagonistic effect on both human and murine P2X7 receptors. W

Full text of DOI:10.1016/j.ejmech.2017.10.033

Accepted Manuscript
1,4-Naphthoquinones potently inhibiting P2X7 receptor activity
R.X. Faria, F.H. Oliveira, J.P. Salles, A.S. Oliveira, N.L. von Ranke, M.L. Bello, C.R.
Rodrigues, H.C. Castro, A.R. Louvis, D.L. Martins, V.F. Ferreira
PII:
S0223-5234(17)30825-5
10.1016/j.ejmech.2017.10.033
EJMECH 9825
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 7 August 2017
Revised Date: 9 October 2017
Accepted Date: 11 October 2017
Please cite this article as: R.X. Faria, F.H. Oliveira, J.P. Salles, A.S. Oliveira, N.L. von Ranke, M.L.
Bello, C.R. Rodrigues, H.C. Castro, A.R. Louvis, D.L. Martins, V.F. Ferreira, 1,4-Naphthoquinones
potently inhibiting P2X7 receptor activity, European Journal of Medicinal Chemistry (2017), doi: 10.1016/
j.ejmech.2017.10.033.
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ACCEPTED MANUSCRIPT
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Log [ATP]M
Log [ATP]M + 500 nM AN-03
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Log [ATP]M + 500 nM A740003
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AN-03
AN-04
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Treatments
Residues interacting
with AN-03, and AN-04
analogs in the ATP-
binding site.
AN-03, and AN-04 interaction in the
human P2X7R.
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Log [ATP]M
Log [ATP]M + 500 nM AN-04
Log [ATP]M + 500 nM A740003
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-6
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-1
Treatments

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1,4-Naphthoquinones potently inhibiting
P2X7 receptor activity
a*
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Faria RX , Oliveira FH , Salles JP , Oliveira AS , von Ranke NL , Bello ML ,
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Rodrigues CR , Castro HC , Louvis AR Martins DL , Ferreira VF
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Laboratório de Toxoplasmose e outras protozooses, Instituto Oswaldo Cruz, Fiocruz,
Brazil.
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Programa de Pós-graduação em Ciências e Biotecnologia, Universidade Federal
Fluminense, Campus do Valonguinho, Centro, Niterói, RJ 24020-141, Brazil.
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Laboratório de Modelagem Molecular e QSAR, Departamento de Fármacos e
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Medicamentos, Faculdade de Farmácia, Universidade Federal do Rio de Janeiro, CEP
21941-902, RJ, Brazil.
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Grupo de Pesquisas em Catálise e Síntese, Laboratório 413, Universidade Federal
Fluminense, Instituto de Química, Campus do Valonguinho, Centro, Niterói, RJ 24020-
141, Brazil.
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Universidade Federal Fluminense, Departamento de Química Orgânica, Instituto de
Química, Campus do Valonguinho, CEP 24020-150, Niterói-RJ, Brazil.
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*Corresponding author: Robson Xavier Faria, robson.xavier@gmail.com.
Laboratório de Toxoplasmose e outras protozooses, Instituto Oswaldo Cruz,
Laboratório de Toxoplasmose e outras protozooses, Avenida Brasil, 4365, Pavilhão
108, sala 32, CEP 21045-900, Rio de Janeiro, Fiocruz, Brazil.
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Abbreviations
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ATP,
adenosine
5′-triphosphate;
HEPES,
4-(2-hydroxyethyl)-1-
piperazineethanesulfonic acid; ELISA, enzyme-linked immunosorbent assay; SDS,
sodium dodecyl sulfate; IC50, half-maximum inhibitory concentration; IL, interleukin;
LPS, lipopolysaccharide; ESI, electrospray ionization; NMR, nuclear magnetic

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resonance, LDH, lactate dehydrogenase; EGTA, ethylene glycol tetraacetic acid; PBS,
phosphate-buffered saline; NAD+, Nicotinamide adenine dinucleotide; IL-1β,
Interleukin 1β; PI, Propidium iodide; BBG, Brilliant Blue G; DMEM, Dulbecco's
Modified Eagle's Medium; IR, infrared spectroscopy; TMS, tetramethylsilane; TLC,
Thin Layer Chromatography; ROS, Reactive oxygen species; iNOS, inducible nitric
oxide synthase; A740003, N-(1-(((Cyanoamino)(5-quinolinylamino)methylene)amino)-
2,2-dimethylpropyl)-3,4-dimethoxybenzeneacetamide; A804598, AZ 10606120
dihydrochloride, N-Cyano-N"-((1S)-1-phenylethyl)-N'-5-quinolinyl-guanidine; N-(2-
((2-((2-Hydroxyethyl)amino)ethyl)amino)-5-quinolinyl)-2-tricyclo(3.3.1.13,7)dec-1-
ylacetamide dihydrochloride; A438079, 3-((5-(2,3-Dichlorophenyl)-1H-tetrazol-1-
yl)methyl)pyridine hydrochloride; AZ11645373, 3-(1-(((3'-Nitro(1,1'-biphenyl)-4-
yl)oxy)methyl)-3-(4-pyridinyl)propyl)-2,4-thiazolidinedione; THP-1 cell, human
monocytic cell line derived from an acute monocytic leukemia patient; and SAR,
Structure-activity relationship.
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Abstract
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P2X7 receptor (P2X7R) is an ATP-gated ion-channel with potential therapeutic
applications. In this study, we prepared and searched a series of 1,4-naphthoquinones
derivatives to evaluate their antagonistic effect on both human and murine P2X7
receptors. We explored the structure-activity relationship and binding mode of the most
active compounds using a molecular modeling approach. Biological analysis of this
series (eight analogues and two compounds) revealed significant in vitro inhibition
against both human and murine P2X7R. Further characterization revealed that AN-03
and AN-04 had greater potency than BBG and A740003 in inhibiting dye uptake, IL-1β
release, and carrageenan-induced paw edema in vivo. Moreover, we used
electrophysiology and molecular docking analysis for characterizing AN-03 and AN-04
action mechanism. These results suggest 1,4-napthoquinones, mainly AN-04, as
potential leads to design new P2X7R blockers and anti-inflammatory drugs.
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Keywords: P2X7 receptor, naphthoquinones, dye uptake, IL-1β release, paw edema,
molecular docking.
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1. Introduction
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The P2X7 receptor (P2X7R) is an ATP-gated ion-channel expressed in cells originating
from the hematopoietic lineage [1]. This receptor has been a target of prominent interest
in novel scientific literature and patents focusing on potential therapeutic application
[2–4]. During P2X7R activation, an extensive number of reactions occur, including
non-specific cation flux [5] and pore opening with flux of large molecules [5]. Activated
P2X7R participates in inflammasome formation, and processing and release of
interleukin-1β (IL-1β) [6]. Additionally, distinct cells in the central nervous system
express P2X7R [7] and this receptor mediates glutamate release in glial cells [8].
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The combination of both strategic localization of molecular target in the immune system
and the ability to stimulate release of inflammatory mediators [9] suggests a potential
role of P2X7R blockers in the treatment of pathological conditions including
inflammatory diseases, pain, and neurodegenerative disorders. Preclinical in vivo studies
have precisely suggested P2X7R participation in both inflammatory and neuropathic
pain [10] whereas new small P2X7R inhibitors have been demonstrated as efficacious
in animal models of neuropathic pain [11–14].
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According to literature, P2X7R inhibitors may function as novel therapeutic agents for
diseases [14-15]. However, clinical trials in patients with rheumatoid arthritis using
AZD-9056 and CE-224535 showed no improvement in the disease compared to the
current clinical treatment [15-17], which reinforces the need for exploring novel
selective P2X7R pore inhibitors. Accordingly, the quinones, which are widely present
in nature and show pivotal effects on the life cycles of cells, might be an important
scaffold to design new P2X7R inhibitors. Currently, many compounds, which possess
the quinone moiety, display different biological profiles including anti-inflammatory
activity (Fig. 1) [18, 19, 20, 21].
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Among the quinone core containing compounds, lapachol [18, 19] is a naturally
occurring quinone with anticancer activity. Taking lapachol as the structural base, there
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are reports describing naphthoquinones with anticancer, antileishmanial, antifungal, and
anti-inflammatory bioactivities (Fig. 1) [20-22]. In addition, Mishra and co-workers
reported the isolation of a naphthoquinone from Dendryphionnanum culture, an
endophytic fungus present in Ficus religiosa leaves collected from Mumbai, India [23].
The isolated naphthoquinone showed anti-inflammatory activity inhibiting LPS induced
cytokine production in the human mononuclear cell line (THP-1).
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Fig. 1. Lapachol (1) and naturally occurring 1,4-naphthoquinones (2-3) with anti-
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inflammatory activities.
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Synthetic bioactive quinone derivatives also were reported in literature. Chia and co-
workers reported thiazine-quinoline-quinones [24] capable of inhibiting neutrophil
superoxide production. The naphthoquinone series showed high anti-inflammatory
activity in vitro, with IC values around 60 µM. They inhibited neutrophil infiltration
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and superoxide production at doses of 2.5 µM/kg in a mouse model of acute
inflammation. Currently, naphthoquinones are considered a potential scaffold in
medicinal chemistry. However, few studies have investigated naphthoquinones as
P2X7R inhibitors.
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In this study, we synthesized and evaluated a new 3-halo and 3-aryl-2-hydroxy-1,4-
naphthoquinone series in the attempt to discover P2X7R inhibitors. Thus, we tested
these analogs against human and murine P2X7R, exploring their binding mode and
structure-activity relationship using a molecular modeling approach.
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2. Experimental Section
2.1 Chemistry
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Solvents, bases, and lawsone (2-Hydroxy-1,4-naphthoquinone) were purchased
from Sigma Aldrich Brazil LTDA; boronic acids were purchased from Combi-Blocks.
These reagents were used as received, without further purification. The 3-Aryl-1,4-
naphthoquinones were purified by column flash chromatography (silica gel 60: 35-70
µm). Yields refer to isolated yields after flash chromatography. The melting points of
the pure products were recorded on a Fisatom 413D apparatus. Reaction progress was
monitored using analytical thin-layer chromatography (Silicycle Ultrapure Silica Gels,
F254) and the spots were visualized by UV light or iodine vapors. Infrared spectra (KBr
pellets) were measured on a Perkin-Elmer 1420 FT-IR Spectrometer. NMR spectra were
recorded on a Varian VNMRS (300 MHz or 500 MHz) instrument in DMSO-d or
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CDCl . Chemical shift data were represented in units of δ (ppm) downfield from
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Tetramethyl silane (TMS), an internal standard. Coupling constants (J) refer to apparent
peak multiplicities reported in Hertz. Gas chromatography/mass spectrometry (GC/MS)
analysis was conducted on an Agilent® 6890 chromatograph (column: Agilent® 122-
5532 DB-5MS) coupled to the Agilent® 5973 mass spectrometer. Fragmentation values
and molecular ions were reported in m/z units.
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2.1.1. Lawsone bromination (AN-02)
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Bromine (0.5 mL) was added to a solution containing lawsone (1.16 g, 6.7 mmol) and
acetonitrile (100 mL) and the solution was refluxed for 2 h. Then, 1.2 mL of
cyclohexene was added to the resulting mixture. The solvent was removed under
vacuum using a rotary evaporator. The residue in the flask was triturated with hexane,
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filtered, re-solubilized in ether, and treated with charcoal for posterior filtration. The
solvent was removed under vacuum.
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2.1.2. 2-Hydroxy-3-iodonaphthalene-1,4-dione (AN-02)
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Yield: 77%, yellow solid, mp 203-205°C. IR (KBr) ʋmax/cm 3176 (O-H), 1672 (C=O),
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1580 and 1454 (C=C), 3161 (OH). δH (500 MHz; DMSO-d ; J in Hz) 8.03-8.00 (2H,
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m); 7.85-7.80 (2H, m). δC (125 MHz; DMSO-d ) 179.2, 178.6, 158.7, 135.1, 134.0,
131.9, 130.2, 126.9, 126.8, 111.1 ppm.
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2.1.3. Lawsone iodination (AN-03)
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The morpholine-iodine complex (iodinating agent) was prepared according to the
protocol described by Rice and Heights (38-40). The complex (12.8 g, 37.5 mmol) was
added in parts, over a period of 2 h, to an Erlenmeyer flask containing lawsone (30
mmol) and a basic aqueous solution of K CO (12.4 g, 90 mmol)/distilled water (300
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mL). After morpholine-iodine complex addition, the reaction mixture was vigorously
stirred for 30 min at 25- 30ºC. Next, with an ice bath, the reaction mixture was cooled
and acidified to pH = 2 using concentrated H PO , and stirred for 24 h. The solid was
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removed by filtration, washed with cold water, and dried at room temperature protected
from light.
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2.1.4. 2-Hydroxy-3-iodonaphthalene-1,4-dione (AN-03)
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Yield: 82%, orange solid, mp 172-173°C. IR (KBr) ʋmax/cm 1620 (C=O), 1670 (C=O),
3161 (OH). δH (500 MHz; DMSO-d ; TMS) 8.06-7.98 (2H, m), 7.85-7.78 (2H, m). δC
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(125 MHz; DMSO-d ) 179.5, 177.2, 162.2, 134.4, 133.3, 130.7, 129.6, 126.6, 126.3,
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92.7 ppm.
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2.1.5. 1,4-Napthoquinones arylation/general procedure
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2-Hydroxy-3-iodo-1,4-naphthoquinone (0.5 mmol) was added to a 10 mL Pyrex tube,
together with K CO (0.346 g, 2.5 mmol), arylboronic acid (0.75 mmol), water (5 mL),
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and Pd(OAc) (0.001 g, 1 mol%). The reaction mixture was magnetically stirred and
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irradiated for 10 min at 100°C. It was then acidified (pH ≅ 2, H PO ) and filtered. The
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solid obtained was purified by flash column chromatography (hexane/CH Cl ).
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2.1.6. 2-Hydroxy-3-phenylnaphthalene-1,4-dione (AN-04)
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Yield: 71%, yellow solid, mp 145-146°C (lit. 146°C). IR (KBr) ʋ /cm 1649 (C=O),
max
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1659 (C=O), 3346 (OH). δH (500 MHz; DMSO-d ; J in Hz) 8.19 (1H, dd, J = 1.0, 7.5
Hz); 8.15 (1H, dd, J = 1.0, 7.5 Hz); 8.02-7.99 (1H, m), 7.95 (1H, td, J = 1.5, 7.5 Hz);
7,55-7.45 (5H, m, Ph). δC (125 MHz; CDCl ) 183.7, 181.8, 152.2, 135.3, 133.1, 132.8,
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130.6, 130.0, 129.3, 128.6, 127.9, 127.3, 126.1, 122.2 ppm. MS (EI ) m/z (%): 250 (M ,
100), 221 (19) 194 (21), 165 (47).
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2.1.7. 2-Hydroxy-3-(4-tolyl)naphthalene-1,4-dione (AN-12)
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Yield: 77%, orange solid, mp 169-171°C. IR (KBr) ʋmax/cm 1645 (C=O), 3361 (OH).
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δH (500 MHz; DMSO-d ; J in Hz) 8.06-8.05 (1H, m), 8.03-8.02 (1H, m), 7.89-7.86
(1H, m), 7.83 (1H, td, J = 1.0, 7.5 Hz), 7.27 (2H,d, J = 8.5 Hz), 7.22 (2H,d, J = 8.0 Hz),
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2.35 (3H, s). δC (125 MHz; DMSO-d ) 183.5, 181.3, 154.5, 136.7, 134.5, 133.0, 131.9,
130.4, 129.8, 128.2, 127.8, 125.9, 125.4, 122.0, 20.8 ppm.
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2.1.8. 2-(3-Fluorophenyl)-3-phenylnaphthalene-1,4-dione(AN-13)
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Yield: 78%, orange solid, mp 163-165°C. IR (KBr) ʋmax/cm 1648 (C=O), 1672 (C=O),
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3246 (OH). δH (500 MHz; DMSO-d ; J in Hz) 8.07 (1H, dd, J = 1.5, 7.5 Hz); 8.04
(1H, dd, J = 1.0, 7.5 Hz); 7.89 (1H, td, J = 1.5, 7.5 Hz); 7.84 (1H, td, J = 1.5, 7.5
Hz); 7.48-7.43 (1H, m); 7.23 (1H, dd, J = 1.0, 7.5 Hz); 7.20-7.17 (2H, m).δC (125
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MHz; DMSO-d ) 183.4, 181.5, 162.6, 160.7, 155.3, 135.0, 133.9, 133.5, 132.1, 130.1,
129.5, 127.1, 126.3, 125.8, 121.0, 117.6, 114.6 ppm.
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2.1.9. 2-Hydroxy-3-(4-methoxyphenyl)naphthalene-1,4-dione(AN-14)
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Yield: 75%, red darkness solid, mp 180-182°C. IR (KBr) ʋmax/cm 1609 (C=O), 1635
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(C=O), 3366 (OH). δH (500 MHz; DMSO-d ; J in Hz) 8.07-8.01 (2H, m), 7.90-7.79
(2H, m), 7.37-7.32 (2H, m), 7.00-6.95 (2H, m), 3.80 (3H, s, CH ). δC (125 MHz;
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DMSO-d ) 184.4, 181.8, 159.2, 154.9, 135.2, 133.8, 132.5, 132.4, 130.3, 126.6, 126.0,
123.7, 122.5, 113.5, 55.6 ppm.
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2.1.10.2-Hydroxy-3-(3-methoxyphenyl)naphthalene-1,4-dione(AN-15)
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Yield: 66%, yellow solid, mp 126-127°C. IR (KBr) ʋmax/cm 1652 (C=O), 1661 (C=O),
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3359 (OH). δH (500 MHz; DMSO-d ; J in Hz) 8.06 (1H, dd, J = 0.5, 7.5 Hz), 8.03
(1H,dd, J = 1.0, 7.5 Hz); 7.88 (1H, td, J = 1.5, 7.5 Hz); 7.83 (1H, td, J = 1.0, 7.5 Hz);
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7.34-7.31 (1H, m); 6.94-6.92 (3H, m); 3.76 (3H, s, CH ). δC (125 MHz; DMSO-d )
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183.4, 181.4, 158.4, 154.8, 134.6, 133.1, 132.7, 132.0, 129.9, 128.3, 126.0, 125.5,
123.0, 122.1, 116.4, 113.0, 55.0 ppm.
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2.1.11. 4-(3-Hydroxy-14-dioxo-1,4-dioxo-1,4-dihydronaphthalen-2-
yl)benzonitrile(AN-16)
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Yield: 43%, yellow solid, mp 253-255°C. IR (KBr) ʋmax/cm 1653 (C=O), 1674 (C=O),
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2239 (C≡N), 3211 (OH). δH (500 MHz; DMSO-d ; J in Hz) 8.07 (1H, dd, J = 1.5,
7.5 Hz), 8.05-8.03 (1H, m), 7.91-7.83 (4H, m), 7.59 (2H, d, J = 8,0 Hz). δC (125 MHz;
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DMSO-d ) 182.9, 181.1, 155.5, 136.8, 134.8, 133.3, 131.9, 131.7, 131.2, 130.0, 126.0,
125.6, 120.4, 118.8, 110.0 ppm.
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2.2. Biological Assays
2.2.1. In vitro experiments
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2.2.1.1. Mice peritoneal macrophages. We harvested peritoneal macrophages from male
Swiss Webster mice through peritoneal cavity lavage with 10 mL of Dulbecco’s
modified Eagle's medium (DMEM). Our protocols adhered to the Ethical Principles in
Animal Experimentation adopted by the Brazilian College of Animal Experimentation
and approved by the FIOCRUZ Research Ethics Committee (number LW-033/12 and
LW-58/14). The cell suspension was centrifuged, suspended, added to microplate wells
(0.5 mL), and incubated in a humidified atmosphere (37°C, 5% CO ) for 30 min. Non-
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serum (FBS) and gentamycin (1 µL/mL). The firmly adhering cells were re-suspended
in phenol red-free DMEM for subsequent experimental procedures.
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2.2.1.2. HEK293 cells transfected with P2X7R. HEK293 cells expressing P2X7R were
maintained in DMEM supplemented with 10% fetal bovine serum (FBS), 2 mM L-
glutamine, and antibiotics (50 U/mL penicillin and 50 mg/ml streptomycin) in
humidified 5% CO atmosphere at 37°C. The cell suspension was prepared at a density
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of 2.5 × 10 cells/mL and added to each well (80 µL for a final volume of 200 µL) of
96-well culture plates. Until completion of the experiments, the cells were maintained in
a humidified 5% CO atmosphere at 37°C.
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2.2.1.3. Dye uptake assay. Mouse peritoneal macrophages (5.0 × 10 cells) were
incubated with 5 mM ATP for 25 min and propidium iodide (PI) (0.05 mg/mL in PBS)
was added in the last 5 min at 37°C. Before ATP treatment, the cells were pre-incubated
with P2X7R antagonists or naphthoquinone for 10 min. Low forward, side scatters, and
an extremely high fluorescence profile were used to rule out dead cells and cellular
debris using flow cytometry. Naphthoquinone derivative doses varied from 0.01 ng/mL
to 10 µg/mL and P2X7R antagonist doses ranged from 1 ng/mL to 500 µg/mL.
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HEK293 cells expressing P2X7R were suspended in a 10 mL cell suspension and added
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to each well of 96-well culture plates at a density of 2.5 × 10 cells/mL. After 24 h, the
cells were treated with the compounds and ATP as described for peritoneal
macrophages in addition to a fluorescent dye (ethidium bromide – 25 µM in PBS). After
incubation, we measured fluorescence with a Gemini fluorescence plate reader at an
excitation wavelength of 530 nm and an emission wavelength of 620 nm. Ethidium
uptake inhibition (percent) was expressed as the value relative to the maximum
accumulation when stimulated with ATP. To calculate IC values, we obtained dose-
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response data using nonlinear regression analysis (i.e., ethidium bromide accumulation
percentage vs. compound concentration).
2.1.1.4. IL-1b enzyme-linked immunosorbent assay (ELISA)
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THP-1 cells were grown in Roswell Park Memorial Institute 1640 medium (RPMI
medium), supplemented with 10% FBS, penicillin (100 U/mL), and streptomycin (100
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mg/mL) in a humidified 5% CO atmosphere at 37°C. Cells were plated at 2 × 10
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cells/well in 96-well culture plates, with 500 ng/mL of phorbol 12-myristate 13-acetate
(PMA) and 10 ng/mL of IFN-γ. This co-treatment for 24 h differentiated the human
monocytic THP-1 cells into macrophage-like cells. The THP-1 cells were primed with
LPS (25 ng/mL) for 4 h. The differentiated and primed THP-1 cells were incubated with
BBG, A740003, and naphthoquinones, followed by 5 mM ATP for 30 min at 37°C. The
treated samples were collected, centrifuged at 1000 rmp for 5 min at 4°C, and the
supernatants were stored at -70°C. To measure IL-1β release, the Human IL-1 beta
ELISA Kit (ab46052 -ABCAM, Cambridge) was used.
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Peritoneal macrophages were primed for 4 h with LPS 100 ng/mL. In the last 30 min of
LPS incubation, the cells were untreated or treated with 1 mM ATP. In some assays,
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cells were treated with BBG, A740003, and naphthoquinones at 15 min before LPS
addition, ATP treatment, or both. The mature IL released from the macrophages was
quantified by sandwich ELISA following the manufacturers protocol (eBioscience, San
Diego, CA, USA).
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2.2.1.5. Caco-2 cells culture and treatments. Caco-2 cells were seeded in 96-well
polyester Corning® Costar® transwell plates (Sigma-Aldrich, St. Louis, MO, USA) at a
5
density of 3 × 10 cells/well with medium (DMEM with 10% fetal calf serum). Caco-2
cells were cultured for up to 21 days in a humidified incubator maintained at 37°C in an
atmosphere of 5% CO . The medium and monolayer integrity were checked every 3
2
days. On the day of the experiment, working solutions of AN-03 and AN-04,
vinblastine (poor permeability control), and propranolol (high permeability control) at
100 µM were prepared in the transport buffer (HBSS and 25 mM HEPES) at pH 7.4 or
6.5 with 0.5% (v/v) DMSO. Cells were washed with prewarmed (37ºC) transport buffer
at the corresponding pH. To equilibrate the cells with the transport buffer, 0.3 mL of
transport buffer was added to the wells. The feeder tray was substituted for a 24-well
Enhanced Recovery Plate with 1 mL of transport buffer at pH 7.4. These plates are
treated with a hydrophilic covalent coating to resist nonspecific adsorption of
compounds. Cells were then incubated for 30 min at 37°C and 5% CO . After this, the
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transport buffer in the apical wells was removed and 0.3 mL of the corresponding AN-
03 and AN-04, vinblastine, or propranolol solution was added. The cells were again
placed in the incubator for 60 min. The plates were then separated, and the Lucifer
Yellow concentrations in the donor and acceptor wells and the initial solution were
measured. For Lucifer Yellow, fluorescence was measured on the plate reader M5
(molecular probes) at excitation 485 nm and emission 530 nm.
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2.2.1.6. pH Dependent Solubility of AN-03 and AN-04. The kinetic solubility of AN-03
and AN-04 was assessed from 1 to 250 µM by spiking DMSO stock solutions (5 µL, in
triplicate) into 995 µL buffer (pH 2.0-hydrochloride, 4.0-100 mM citrate buffer and 7.4-
100 mM phosphate buffer) in a 96-well plate and placing at room temperature for 2 h.
Calibration standards were prepared by spiking 5 µL of DMSO stock solutions into 995
µL acetonitrile/buffer (1:1) mixture. After centrifugation (10,000 rpm, 10 min, 25ºC)
the reaction samples were diluted 1:1 with acetonitrile.
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2.2.1.7. Distribution coefficient (Log D) in octanol/PBS pH 7.4. Octanol and PBS pH
7.4 at a ratio of 1:1 (v/v) were taken in a flask and shaken mechanically for 24 h to pre-
saturate the PBS with octanol and octanol with PBS. Naphthoquinones AN-03 and AN-
04 in a volume of 4 µL and at a concentration of 25 mM were added to 396 µL PBS for
partitioning with different volumes of octanol (100-400 µl). After 2 h with vigorous
shaking for mixing, centrifugation at 3000 rpm for 5 min for separation, followed by 1 h
of standing without disturbance, the PBS layer was taken out. Acetonitrile (100 µl) was
added to a 100 µl aliquot of the PBS layer and its absorbance was measured (396 µL
PBS containing 4 µL of 25 mM AN-03 or AN-04 + 400 µL acetonitrile). Two standard
compounds were studied similarly to validate the assay.
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2.2.1.8. In vitro stability assays in liver microsomes. Liver microsomes from male mice
and humans with a final protein concentration of 0.5 mg/ml in 0.1 M phosphate buffer
at pH 7.4. Naphthoquinones AN-03 and AN-04 at a final concentration of 1 µM and
DMSO at a concentration of 0.5 µM were pre-incubated with the microsomes at 37°C
before NADPH addition at a final concentration of 1 mM to initiate the reaction. The
final incubation volume was 50 µl. A buffer containing 0.1 M phosphate at pH 7.4 was
used as a control replacing NADPH (minus NADPH). Diazepam and verapamil were
used as positive controls for mice and humans, respectively. The naphthoquinones AN-
03 and AN-04, and controls were incubated with the microsomes for 0, 5, 15, 30, and 45
min; the negative control (minus NADPH) was incubated for 45 min. Methanol (50 µL)
was used to stop the reactions at the appropriate time points. Samples incubated in
plates were centrifuged at 1640 × g for 20 min at 4°C to aid protein precipitation.
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In vitro intrinsic clearance (CLint mic) for the metabolism of AN-03 and AN-04 in
mouse and human liver microsomes was calculated using the equations below:
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Half life (t1/2) (min) = 0.693/k (1)
V(µL/mg) = volume of the incubation (µL)/protein in the incubation (mg) (2)
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Intrinsic Clearance (CLint) (µL/min/ mg protein) = V x 0.693/ t1/2 (3)
according to Biosystem instructions.
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2.2.1.9. Electrophysiological measurements. A whole-cell configuration was set up as
described by Faria and coworkers [25]. The series resistance was set as 5-11 MΏ for all
the experiments in standard saline (bath and pipette solutions), and no compensation
was applied for currents less than 1500 pA. Above this level, the currents were
compensated by 90%. Measurements were discarded when the series resistance was
increased substantially. Macrophage (mean ± s.d., 18.2 ± 5.02 pF; n = 68) cell
capacitance was measured by applying a 20 mV hyperpolarizing pulse from a holding
potential of 20 mV; the capacitive transient was then integrated and divided by the
amplitude of the voltage step (20 mV). All recordings were obtained at a holding
potential of -60 mV at 37°C.
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2.2.1.10. Saline solutions for electrophysiology. Different saline solutions were used in
the pipette or bath, depending on the protocol. The bath solution (in mM) consisted of
the following: 150 NaCl, 5 KCl, 1 MgCl2, 1 CaCl2, and 10 HEPES (pH, 7.4). The
pipette solution (in mM) consisted of the following: 150 KCl, 5 NaCl, 1 MgCl2, 10
HEPES, and 0.1 EGTA (pH, 7.4).
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2.2.1.11. Drug application. Patch clamp experiments were performed under perfusion
(RC-24 chamber, Warner Instrument Corp.) at a rate of 1 mL/min to confirm the data
obtained by micropipette application. All the drugs were dissolved in saline solution
immediately before use. Ion currents were studied by applying 1 mM ATP (for 300 s) in
the presence or absence of AN-03, AN-04, or BBG.
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2.2.2. In vivo experiments
2.2.1. Paw edema
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Mouse paw edema, an acute inflammatory model, was used to investigate the
antagonistic action of AN-03 and AN-04. In these experiments, naphthoquinones were
administered intraperitoneally at 60 min prior to intrathecal ATP (1 mM/paw) or
carrageenan (µM/paw) administration in saline suspension. After 30 min, paw edema
was measured using a plethysmometer (Insight, Brazil). Animals were euthanized by
CO inhalation.
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2.2.2. Statistical analyses
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The measurements were obtained in triplicate and performed on at least three
independent days unless otherwise specified in the figure legends. All data are presented
as means ± standard deviations of the means (S.D.M.). To test whether the samples
followed a Gaussian distribution, D’Agostino and Pearson normality tests were used.
When the data followed a Gaussian distribution, we applied analysis of variance
(ANOVA), followed by Tukey’s test. Otherwise, we applied the non-parametric
Kruskal–Wallis test, followed by Dunn’s test. All tests were two-tailed and are specified
®
in the figure legends. PRISM software (GraphPad, Inc., San Diego, CA, USA) was
used for all statistical analyses. P values < 0.05 were considered statistically significant.
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2.3 In silico evaluation
2.3.1. ADMET properties
®
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The ADMET properties in silico investigation was performed using the Osiris
®
(Actelion Pharmaceuticals Ltda.) and ADMET Predictor (Simulation Plus) programs.
Osiris was used to investigate the toxicological parameters as well as the drugscore
values, which compute the drug likeness and physicochemical parameters of the
®
compound molecules. ADMET Predictor was used to investigate the pharmacokinetic
properties including naphthoquinone derivatives metabolism profiles.
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2.3.2. Homology modeling
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Homology modeling was performed using Modeller 9.16 program and applying
the satisfaction of spatial restrains [26,27] to develop the structure of the human model
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trimeric P2X7 protein. The human ATP-gated cation channel P2X7R amino acid
sequence (accession code: CAA73360.1) was retrieved from the National Center for
Biotechnology Information (NCBI) [28] and the deposited P2X7 crystal structure (PDB
ID: 5U1L) was used as a template [29]. For this purpose, 50 models were generated
with a repeat optimization. The models were then ranked based on the Modeller internal
scoring function in order to select the best structure generated. Finally, the Swiss PDB
viewer program [30] was used for energy minimization for the best human P2X7
protein model. In order to validate the model, a structural quality assessment was
performed using the Procheck program to generate the Ramachandran plot [31, 32] and
the ProsA program to calculate the z-score and knowledge-based energy plots.
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2.3.3. Ligand preparation
®
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The ionization states of each molecule were assessed using Chemicalize software
(ChemAxon), which indicated that the hydroxyl group attached to naphthoquinone is
ionized at physiological pH. Thus, the selected ionized species were used for
constructing the three most active derivatives
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The ligand theoretical chemical structures were constructed using Spartan´10 v.1.0.1
program. The MMFF (Merck Molecular Force Field) [33] was used to obtain the
conformers local energy minimum. The geometry optimization that specifies the nearest
energy minimum was then applied with the semi-empirical RM1 method [34]. Finally,
we used a single point quantum energy calculation by applying the density function
theory (DFT) with the hybrid B3LYP/6-31G* quantum basis set [35-37].
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2.3.4. Molecular docking
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Molecular docking was performed using the AutoDockVina program [38, 39] The
ligand and human model P2X7 were previously prepared using AutoDock tools 4.2.6
which included addition of hydrogen atoms as well as assigned Gasteiger partial
charges. The grid box poses search was performed around the P2X7R molecular
structure in a blind molecular docking search. When the principal cavities were found,
molecular docking was directed to these protein regions in order to refine the results.
The ATP binding cavity has known conserved residues involved in ATP binding such
as: Lys64, Lys66, Phe188, and Thr189 from one subunit, and Asn292, Phe293, Arg294,
and Lys311 from an adjacent subunit [40, 41]. In order to support this molecular
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docking approach, a blind docking of the ligand A740003 [29, 42] and the ATP
molecule was performed with the human model P2X7. Furthermore, another molecular
docking was performed centered on a more favorable cavity for each ligand from the
blind molecular docking.
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3.
Results
3.1. Synthesis
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3-Bromo-2-hydroxy-1,4-naphthoquinone (AN-02) was obtained at 77% yield from
lawsone bromination (AN-01) (2-hydroxy-1,4-naphthoquinone) using bromine in
acetonitrile (Scheme 1).
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Scheme 1. Bromination of lawsone
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2-Hydroxy-3-iodo-1,4-naphthoquinone (AN-03) was prepared through lawsone
iodination (AN-01) in an aqueous basic medium using the morpholine-iodine complex,
prepared according to the methodology described by Rice and Heights [43-45] (Scheme
2).
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Scheme 2. Iodination of lawsone to prepare 2-hydroxy-3-iodo-1,4-naphthoquinone
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Five 3-aryl-2-hydroxy-1,4-naphthoquinones were prepared via palladium-
catalyzed Suzuki-Miyaura C-C coupling between 2-hydroxy-3-iodo-1,4-napthoquinone
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(AN-03) and different arylboronic acids, using microwave irradiation (Scheme 3).
Palladium acetate (1 mol%) was used as a pre-catalyst, in an aqueous system with
K CO as the base. These 3-Aryl-2-hydroxy-1,4-napthoquinones exhibited moderate to
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good yields after flash column chromatography.
O
O
O
OH
OH
Ar
Pd(OAc)2
+
Ar-B(OH)2
I
K2CO H O
3,
2
O
MW, 10 min
AN-03
AN-12-16
O
O
O
O
O
OH
OH
OH
F
O
AN-04
1%
AN-12
AN-13
7
77%
78%
O
O
O
OH
OH
OH
OMe
O
O
OMe
O
CN
AN-14
5%
AN-15
AN-16
7
66%
43%
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Scheme 3. Arylation of 2-hydroxy-3-halogeno-1,4-naphthoquinone
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3.2 Biological assay
3.2.1. In vitro
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Based on pathological conditions related to P2X7R activation, new antagonists have
been searched to treat inflammation and chronic pain [46]. Among the P2X7R-related
diseases, rheumatoid arthritis is extremely well-studied. Although, rheumatoid arthritis
is an autoimmune disease, IL-1β release is a central factor to maintain the disease and
P2X7R activation may mature and release this cytokine [47]. In the last years, new
selective P2X7R antagonists have failed in clinical trials [48-49]. We then explored the
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potential of naphthoquinones as feasible P2X7R antagonists by testing eight 1,4-
napthoquinone derivatives against P2X7R functional activity to identify prototypes for a
new treatment option (Fig. 2).
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Fig. 2. 1,4-naphthoquinones (AN) tested as P2X7R inhibitors.
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We tested their toxicity against mammalian cells by exposing mouse peritoneal
macrophages to 100 µM 1,4-naphthoquinones derivatives continuously for 72 h (Fig. 3).
The results indicate that naphthoquinones showed low toxicity, except for a discrete
Resazurin reduction caused by the AN-01 analogue.
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Fig. 3. Cytotoxicity in mammalian cells. (A) Mice peritoneal macrophages were treated
with 100 µM of the AN derivatives (AN-01, AN-02, AN-03, AN-04, AN-12, AN-13,
AN-14, AN-15, and AN-16) for 72 h. The graphic results represent 3-5 independent
Alamar blue assays. These results are expressed as mean ± s.d. *significantly different
from the control value at P < 0.05. CP – positive control; CN – negative control.
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We used a dye uptake assay to evaluate the antagonistic action 1,4-naphthoquinones
against mice P2X7R (Table 1). Therefore, AN-01, AN-12, AN-13, AN-14, and AN-16
exhibited IC values superior to BBG and A740003, whereas AN-15 showed an IC
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value lower than BBG, but higher than that of A740003. Notably, AN-02, AN-03, and
AN-04 analogues reduced ATP-induced P2X7R dye uptake (IC = 0.712 uM, 0.0484
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uM and 0.0231 uM, respectively) dose dependently, with IC50 values inferior to
A740003.
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Therefore, we selected AN-02, AN-03, and AN-04 to measure LDH release in
increasing concentrations in comparison with P2X7R antagonists (Table 2). BBG was
the most toxic with the lowest CC value of 87.32 µM, whereas A740003 exhibited a
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AN-04 showed a better toxicity profile (844 µM, 2022 µM, and 1988 µM, respectively)
with CC values 10 to 20 times higher than BGG and 3 to 6 times higher than
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A740003. Overall, all three 1,4-naphthoquinones exhibited low toxicity in comparison
to P2X7R antagonists with superior selectivity indices (Table 2).
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Table 1. Antagonistic effects of P2X7R antagonists and naphthoquinones against
ATP-induced dye uptake in mice peritoneal macrophages.
a
IC = 50 % inhibitory concentrations were obtained from concentration– response curves.
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Values are means of 2–4 experiments. Compounds tested at the mice peritoneal macrophages.
Table 2. Index selectivity of naphtoquinones.
a
CC = 50 % inhibitory concentrations were obtained from concentration response curves.
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Thus, we examined the effect of AN-02, AN-03, and AN-04 on ATP-induced IL-1β
release. Human THP-1 monocytes differentiated with INF-γ and PMA were primed
with LPS for 4 h, with ATP added in the last 30 min of LPS incubation. P2X7R
antagonists and 1,4-naphthoquinones were added 30 min before ATP addition.
Naphthoquinone derivatives inhibited ATP-induced IL-1β release in a similar manner in
both, human and mouse P2X7R (Fig. 4 and Table 3). In THP-1 cells, AN-02 presented
higher potency than BBG, but lower than that of A740003, whereas AN-03 and AN-04
were more potent than both P2X7R antagonists (Fig. 4A). We observed a similar
antagonistic profile for ATP-induced IL-1β release with mouse P2X7R (Fig. 4B and
Table 3). Interestingly, AN-02, AN-03, and AN-04 did not inhibit LPS-induced IL-1β
release though they reduced ATP-induced IL-1β release (Fig. Suppl. 1). These results
indicate a selective inhibition mechanism inhibiting IL-1β release mediated by P2X7R
activation.
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Fig. 4. ATP induced IL-1β release inhibition by naphthoquinones in human and mouse
cells. (A) Differentiated THP-1 cells treated with 1 mM ATP (30 min) and LPS (4 h) in
the presence of increasing doses of BBG, A740003, AN-02, AN-03, or AN-04. (B)
Mouse peritoneal macrophages treated with 1 mM ATP (30 min) and LPS (4 h) in the
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presence of increasing doses of BBG, A740003, AN-02, AN-03, or AN-04. Curves are
representative of 3-5 independent experiments.
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Fig. Suppl. 1. ATP dependent pathway of IL-1β release inhibition for AN-02, AN-03,
and AN-04 molecules. LPS induced IL-1β release was not inhibited by
naphthoquinones, in contrast to ATP-induced IL-1β release. Peritoneal macrophages
were treated as previously described. The graphic results represent 3-5 independent
experiments and are expressed as mean ± s.d. *significantly different from the cells
value at P < 0.05.
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In the current literature, a large number of novel synthetic drugs exhibit P2X7R
antagonist activity at nanomolar concentrations [50-51]. AN-03 and AN-04 potently
inhibited dye uptake and IL-1β release assays with results better than BBG and
A740003. This prompted us to characterize these molecules using HEK 293 cells
transfected with the human P2X7 receptor. The 1,4-naphthoquinones derivatives
inhibited dye uptake with IC values of 0.414 µM (AN-02), 0.0712 µM (AN-03), and
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0.0224 µM (AN-04) (Table 3). The compounds AN-03 and AN-04 conserved a high
potency for inhibiting P2X7R in human receptors as observed in mouse P2X7R.
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Table 3. Antagonistic effects of P2X7R antagonists and
naphthoquinones against ATP-induced IL-1β release in
LPS/IFNγ-differentiated human THP-1 cells.
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Ionic currents were measured by varying the ATP and 1,4-naphthoquinone
derivative concentrations. The ATP concentrations ranged from 100 µM to 25 mM in
the medium containing AN-03 or AN-04 and in medium without these compounds. The
dose of both 1,4-naphthoquinone derivatives was fixed at 500 nM. When treated with
AN-03, the EC value for the ATP dose-response curve augmented in comparison with
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to that of AN-03 (Fig. 5B).
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Fig. 5. Evaluation of the inhibitory mechanism of AN-03 and AN-04 as P2X7R
antagonists. Competitive mechanisms were evaluated by recording the ionic currents of
mice peritoneal macrophages with (●) ATP alone or (■) with 500 ng/mL of AN-03 (A)
or AN-04 (B) at 30-37°C. Graphics are representative of 3-4 independent experiments.
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Both 1,4-naphthoquinone derivatives AN-03 and AN-04, were compared with
A740003 inhibitor, since this compound interferes with ATP binding to P2X7 receptor.
Honore and co-workers reported that A740003 is a P2X7 receptor competitive inhibitor
acting on the ATP binding site, whereas Karasawa and Kawate suggested that this
compound is a non-competitive inhibitor binding at an allosteric site [29, 42, 52, 53].
The 1,4-naphthoquinone derivatives AN-03 and AN-04, showed action profiles similar
to the inhibitor A740003 (Fig. 5A and 5B). This result indicates a P2X7R inhibition
mechanism for AN-03 and AN-04 similar to inhibitor A740003.
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3.2.2. In silico studies
3.2.2.1. ADMET studies
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Theoretical data obtained using Osiris software showed low mutagenic, tumorigenic,
irritability, and reproductive risks for the 1,4-naphthoquinone derivatives AN-02, AN-
03, and AN-04. Moreover, the 1,4-naphthoquinone derivatives presented comparable
drug score values, when compared with commercial anti-inflammatory drugs
(potassium diclofenac, ibuprofen, and naproxen) (Fig. Suppl. 2).
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Fig. Suppl. 2. Comparison of drug score values of three naphthoquinones (AN-02, AN-
03, and AN-04) and commercial anti-inflammatory drugs calculated using Osiris
software.
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The pharmacokinetics parameters were analyzed for 1,4-naphthoquinones derivatives
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using ADMET Predictor software. Though the derivatives AN-02, AN-03, and AN-04
are slightly less lipophilic than commercial anti-inflammatory drugs, all of them
presented a high profile crossing the blood-brain barrier. Furthermore, 1,4-
naphthoquinone derivatives showed favorable results compared to commercial anti-
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inflammatory drugs regarding their solubility in intestinal fluid and jejunal permeability
simulations (Fig. Suppl. 3).
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Although AN-02 and AN-03 presented a lower in vitro anti-inflammatory profile
compared to AN-04, in silico analyses indicate higher level of AN-04 binding with
blood plasma proteins compared to AN-02 and AN-03 (Fig. Suppl. 3). These theoretical
results indicate that the derivatives AN-02 and AN-03 can reach the molecular target
with less difficulties than compound AN-04. However, this pharmacokinetic profile
should be investigated in more detail in future studies.
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Fig. Suppl. 3. Pharmacokinetic parameters measured by ADMET Predictor®
(Simulation Plus) of AN-02, AN-03, and AN-04 compared to commercial anti-
inflammatory drugs (Diclofenac, Ibuprofen, and Naproxen). Indicating the capability of
naphthoquinones to cross the blood-brain barrier, to be absorbed by human intestinal
cells (High or Low), and the percent value estimation of unbound molecules to plasma
proteins.
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Regarding the in silico metabolism parameters, the compounds AN-02, AN-03,
and AN-04 presented similar profiles, though distinct from the commercial anti-
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inflammatory drugs used as controls. Despite this, the results indicated that these
compounds undergo phase I and phase II metabolism, since all these compounds are
theoretically metabolized by at least one CYP oxygenase and UDP-glucuronosyl
transferase isoform, (Fig. Suppl. 4). Overall, these data suggest the glucuronidation
reaction as an inactivation and elimination mechanism for the selected 1,4-
naphthoquinone derivatives.
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Fig. Suppl. 4. Comparison of the qualitative evaluation of metabolic liability of the
selected naphthoquinones and commercial anti-inflammatory drugs measured for human
isoforms of CYP oxygenase and UDP-glucuronosyltransferase.
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3.2. Solubility, microsomal stability, and permeability in vitro.
Naphthoquinones AN-03 and AN-04 were tested for microsomal stability in mouse and
human liver microsomes. Both molecules were stable in mouse and human microsomes
(Table 4). Additionally, AN-03 was permeable in Caco-2 at percentages up to 50%
(Table 14) and AN-04 was permeable in a range superior to 60%, both when compared
with propranolol.
Table 4. Liver microsomal stability and Caco-2 data for AN-03 and AN-04.
Liver Microsomes
LM
LM
Caco-2(b)
stability(a) stability(a)
mouse
24.6
27.7
human
31.1
36.8
AN-03
AN-04
54.2 ± 3.3
61.13 ± 2
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(a)
protein).
(b)
Stability in mice and human liver microsomes. Data reported as CLint (µL/min/ mg
Apparent permeability values (Papp) measured using low permeability and high
permeability absorption compounds, vinblastine and propranolol, respectively, as reference. Data are
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reported in 10 cm/s. These values indicate an apical to basolateral (A-B) direction. They were tested at
the same time as AN-03 and AN-04. Values are means ± standard error of 3 experiments.
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When solubilized in solutions with pH values ranging from 2 to 10, AN-03
demonstrated solubilities in turn of 300 µM (Table 5). LogD7.4 measured for AN-03
gave a value of -0.11 ± 0.03 (Table 6). In relation to AN-04, it demonstrated solubility
at concentrations above 500 µM (Table 5). The LogD7.4 value for AN-04 was -0.39 ±
0.02 indicating good solubility in aqueous solutions (Table 6).
This reduced lipophilicity favored microsomal stability and almost did not affect
the Caco-2 permeability results measured for both molecules.
Table 5. Solubility of AN-03 and AN-04 at various pH conditions.
Drug
pH 2(a) pH 4(b) pH 7.4(c) pH 10(d)
300 µM <300 µM 300 µM
AN-03 300 µM
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AN-04 <500 µM <500 µM <500 µM <500 µM
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(a) pH 2: hydrochloride buffer; (b) pH 4: citrate buffer; (c) pH 7.4: phosphate buffer; (d) pH 10:
sodium hydroxide buffer, n = 3 on distinct days.
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Table 6. Log D of AN-03 and AN-04 naphthoquinones.
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.4
Compound
LogD (a)
7.4
AN-03
AN-04
-0.11 ± 0.03
-0.39 ± 0.02
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(a) Results are average of three experiments and in all cases individual Log D values were within
± 0.3 log units of the average Log D Propranolol HC.
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3.3. In vivo assays
We investigated the in vivo anti-inflammatory properties of AN-03 and AN-04
using the carrageenan induced paw edema model in mice. Sodium diclofenac and
naphthoquinones were administered intraperitoneally at 1 h before carrageenan treatment
for 1 h. The oxidized ATP inhibited the carrageenan induced inflammatory response
(AN-03, Fig. 6A and AN-04, Fig. 6B). As expected, sodium diclofenac inhibited
carrageenan induced paw edema (Fig. 6). Surprisingly, AN-03 and AN-04 dose-
dependently inhibited the carrageenan effect. AN-03 and AN-04 produced inhibition
levels similar to diclofenac and oxidized ATP (Fig. 6). In accordance with in vitro assays,
AN-04 inhibited the carrageenan-induced paw edema with higher potency compared to
the AN-03 analog (Fig. 6B).
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