Next Article in Journal
212Pb: Production Approaches and Targeted Therapy Applications
Next Article in Special Issue
In Vitro Validation of Antiparasitic Activity of PLA-Nanoparticles of Sodium Diethyldithiocarbamate against Trypanosoma cruzi
Previous Article in Journal
Restoration of the Phenotype of Dedifferentiated Rabbit Chondrocytes by Sesquiterpene Farnesol
 
 
Search for Articles:
Title / Keyword
Author / Affiliation / Email
Journal
Article Type
 
 
Advanced Search
 
Section
Special Issue
Volume
Issue
Number
Page
 
Journals
Pharmaceutics
Volume 14
Issue 1
10.3390/pharmaceutics14010188
pharmaceutics-logo
Submit to this Journal Review for this Journal Propose a Special Issue
► Article Menu

Article Menu

share Share announcement Help format_quote Cite question_answer Discuss in SciProfiles thumb_up
...
Endorse textsms
...
Comment
first_page
settings
Order Article Reprints
Font Type:
Arial Georgia Verdana
Font Size:
Aa Aa Aa
Line Spacing:
Column Width:
Background:
Article

The Compound (E)-2-Cyano-N,3-diphenylacrylamide (JMPR-01): A Potential Drug for Treatment of Inflammatory Diseases

by
Pablo Rayff da Silva
1,2,3,
Renan Fernandes do Espírito Santo
4,5,
Camila de Oliveira Melo
1,3,
Fábio Emanuel Pachú Cavalcante
2,
Thássia Borges Costa
2,
Yasmim Vilarim Barbosa
2,
Yvnni M. S. de Medeiros e Silva
3,
Natália Ferreira de Sousa
6,
Cristiane Flora Villarreal
4,5,
Ricardo Olímpio de Moura
1,3 and
Vanda Lucia dos Santos
1,2,*
1
Programa de Pós Graduação em Ciências Farmacêuticas, Universidade Estadual da Paraíba, Campina Grande 58429-500, PB, Brazil
2
Laboratório de Ensaios Farmacológicos, Departamento de Farmácia, Universidade Estadual da Paraíba, Campina Grande 58429-500, PB, Brazil
3
Laboratório de Desenvolvimento e Síntese de Fármacos, Departamento de Farmácia, Universidade Estadual da Paraíba, Campina Grande 58429-500, PB, Brazil
4
Instituto Gonçalo Moniz, Fundação Osvaldo Cruz, Salvador 40296-710, BA, Brazil
5
Faculdade de Farmácia, Universidade Federal da Bahia, Salvador 40170-290, BA, Brazil
6
Programa de Pós Graduação em Produtos Naturais, Sintéticos e Bioativos, Universidade Federal da Paraiba, João Pessoa 58051-900, PB, Brazil
*
Author to whom correspondence should be addressed.
Pharmaceutics 2022, 14(1), 188; https://doi.org/10.3390/pharmaceutics14010188
Submission received: 16 December 2021 / Revised: 7 January 2022 / Accepted: 10 January 2022 / Published: 13 January 2022
(This article belongs to the Special Issue Drug Discovery and Drug Delivery System for Biological Application)
Browse Figures
Review Reports Versions Notes

Abstract

:
The compound (E)-2-cyano-N,3-diphenylacrylamide (JMPR-01) was structurally developed using bioisosteric modifications of a hybrid prototype as formed from fragments of indomethacin and paracetamol. Initially, in vitro assays were performed to determine cell viability (in macrophage cultures), and its ability to modulate the synthesis of nitrite and cytokines (IL-1β and TNFα) in non-cytotoxic concentrations. In vivo, anti-inflammatory activity was explored using the CFA-induced paw edema and zymosan-induced peritonitis models. To investigate possible molecular targets, molecular docking was performed with the following crystallographic structures: LT-A4-H, PDE4B, COX-2, 5-LOX, and iNOS. As results, we observed a significant reduction in the production of nitrite and IL-1β at all concentrations used, and also for TNFα with JMPR-01 at 50 and 25 μM. The anti-edematogenic activity of JMPR-01 (100 mg/kg) was significant, reducing edema at 2–6 h, similar to the dexamethasone control. In induced peritonitis, JMPR-01 reduced leukocyte migration by 61.8, 68.5, and 90.5% at respective doses of 5, 10, and 50 mg/kg. In silico, JMPR-01 presented satisfactory coupling; mainly with LT-A4-H, PDE4B, and iNOS. These preliminary results demonstrate the strong potential of JMPR-01 to become a drug for the treatment of inflammatory diseases.

Graphical Abstract

1. Introduction

Inflammation is the body’s reaction to aggressive chemical, physical, or biological agents, promoting coordinated activation of signaling pathways with the aim of instituting repair processes. The process is initiated by activation of standard cell surface receptors, which recognize stimuli, and activate intracellular signaling cascades, inducing the translocation of transcription factors and resulting in the expression of various inflammatory mediators [1,2]. At the tissue level, during the inflammatory response, typical acute symptoms such as edema, redness, heat, and pain, which may progress to loss of tissue function, are observed [3,4].
An acute process, if unresolved, can become both chronic and part of disease pathogenesis. According to the World Health Organization (WHO), inflammatory diseases are the third leading cause of death, accounting for about 3.46 million deaths, corresponding to 10.8% of the world total [5]. According to Global Business Intelligence Research, Non-steroidal anti-inflammatory drugs (NSAIDs) and steroids constitute one of the main classes of drugs used worldwide, mobilizing USD 85.9 billion in 2017 [6].
Many classes of drugs, such as non-steroidal anti-inflammatory drugs (NSAIDs) and corticosteroids have been shown to be effective in the treatment of inflammatory disorders [5,7]. However, when used for prolonged periods or in non-therapeutic doses, these agents can promote gastrointestinal, renal, and liver disorders, and in the case of steroids, immunosuppression and metabolic changes. Research for new clinically acceptable compounds and bioactive molecules against inflammation is therefore a continuing demand.
As for the development of promising drugs with anti-inflammatory potential, medicinal chemistry has become an important tool in planning new molecules with therapeutic potential, aimed at maintaining pharmacological response and with better toxicity profiles than previously described for conventional anti-inflammatory drugs. In this context, new structures are synthesized using privileged carbon skeletons as a basis, such as the phenylacrylamide function. Our research aimed to synthesize and investigate the anti-inflammatory potential of (E)-2-cyano-N,3-diphenylacrylamide, obtained by organic synthesis from bioisosteric modifications of a previously studied drug [8] and resulting from the hybridization of molecular fragments of the anti-inflammatory drugs indomethacin and paracetamol.

2. Materials and Methods

2.1. General Procedure for the Synthesis of Compound (E)-2-Cyano-N,3-diphenylacrylamide (JMPR-01)

To obtain the JMPR-01, the following products were used in equimolar (1 mmol): 2-cyano-N-phenylacetamide (JM-01), previously synthesized; and 2-carboxyaldehyde (Sigma-Aldrich, St. Louis, MO, USA). Toluene (10 mL) (Vetec, Rio de Janeiro, Brazil) was used as reaction medium, added with 5–10 drops of the catalyst, triethylamine (Sigma-Aldrich, St. Louis, MO, USA) (Scheme 1). The reaction was kept under magnetic stirring, for 24 h at a temperature between 105–110 °C, and its end was observed by Analytical Thin Chromatography (CCDA). The reaction was filtered and the obtained crystals were washed with ice-cold distilled water, and then recrystallized in ethanol–water 1:1. The product was then analyzed by 1H and 13C NMR (Agilent NMR spectrometer, model Mercury Plus 500 MHz, OXFORD 300 magneto-NMR), infrared spectroscopy (IRPrestige-21 Spectrophotometer) and mass spectrometry (Shimadzu® AXIMA series MALDI-TOF/MS).

2.2. Physical-Chemical Properties and Spectroscopic Data of (E)-2-Cyano-N,3-diphenylacrylamide (JMPR-01)

JMPR-01 was obtained as gray powder (72.37%). M.p. (°C): 200,26. Rf (7:3 AcOEt/n-hexane): 0.60. IR (ATR): 1596 (NH, amide folding), 1682 (C=O), 2227 (CN), 3317 (NH, secondary amide stretch), 3053 (=C-H), 1531–1440 (C=C, Ar) cm−1. NMR 1H (500 MHz, DMSOd6): 10.43 (1H, s, NH amide); 8.30 (1H, s, C=CH); 8.05–7.96 (2H, m, Ar phenylacetamide); 7.72–7.66 (2H, m, Ar phenylacetamide); 7.39 (2H, t, J = 7.9 Hz, Ar phenylacetamide) 7.67–7.57 (3H, m, Ar phenylacetamide); 7.19–7.12 (2H, m, Ar phenylacetamide). NMR de 13C (124 MHz, DMSOd6): δ 160.97 (C, C=O); 151.27 138,68 (C, N-Ar phenylacetamide), 132,94, (C, Ar phenylacetamide), 132,37 (C, Ar phenylacetamide), 130,52 (C, Ar phenylacetamide), 129,77 (C, Ar phenylacetamide) 129,24 (C, Ar phenylacetamide), 124,84 (C, Ar phenylacetamide), 121,02 (C, Ar phenylacetamide), 116,68 (C, CN), 107,81 (C, adjacent to CN). HRMS m/z [M + H] + calculated for C16 H12N2O; 248.09 found: 249.01.

2.3. Thermal Characterization

2.3.1. Differential Scanning Calorimetry (DSC)

The Differential Scanning Calorimetry curve was obtained in a differential exploratory module of the Q20 (TA®—Instruments, New Castle, DE, USA). Samples of 2.00 ± 0.05 mg were used, placed in hermetically sealed aluminum crucibles, analyzed at a heating ratio of 10 °C min−1 with a temperature of 25 °C to 400 °C, under a nitrogen atmosphere with flow 50 mL min−1.

2.3.2. Thermogravimetric (TG)

The thermogravimetric curve of JMPR-01 was obtained in a Pyris 1 TGA (Perkin Elmer®, Boston, MA, USA) thermal analyzer using alumina crucibles with 8 ± 0.1 mg samples, under nitrogen atmosphere at a 50 mL min−1 flow rate. The experiments were carried out in the temperature range of 25–900 °C, with a heating rate of 10 °C min−1.

2.4. Biological Activity

2.4.1. Macrophages Cytotoxicity

The cytotoxicity of JMPR-01 was determined using the cell line J774 according to the methodology proposed by Mosmann (1983) [9], adapted according to Espirito–Santo (2017) [10]. Macrophages were seeded in 96-well plates (2 × 105 cells/well) in Dulbecco’s Modified Eagle Medium (DMEM; Life Technologies®, Gibco BRL®, Gaithersburg, MD, USA), supplemented with 10% fetal bovine serum (Gibco BRL®, Gaithersburg, MD, USA) and 50 µg/mL of gentamicin (Novafarma, Anápolis, GO, Brazil). Afterwards, the plates were incubated for 2 h at 37° in an atmosphere of 5% CO2. The test molecule was added to the wells in triplicate at concentrations of 100, 50, 25, 12.5 and 6.25 µM, and incubated again for 72 h. As a positive control, 10 μM gentian violet (Synth, São Paulo, Brazil) was used. Finally, 20 µL/well of Alamarblue Cell Viability Reagent (Invitrogen®, Carlsbad, CA, USA) was added to the plate, holding for 12 h. Colorimetric scanning was performed at a length from 570 to 600 nm.

2.4.2. Assessment of Cytokine and Nitric Oxide Production by Macrophages

To determine the influence of JMPR-01 on cytokines and nitric oxide (NO), J774 macrophages were seeded in 96-well culture plates at a concentration of 2 × 105 cells per well in DMEM medium supplemented with 10% FBS and 50 μg/mL of gentamicin for 2 h at 37 °C under an atmosphere of 5% CO2. After seeding, the cells were treated with the molecule, at non-cytotoxic concentrations, with the vehicle (negative control) and with dexamethasone 40 µM (positive control), and then stimulated with LPS (500 ng/mL, Sigma, St. Louis, MO, USA) and IFN-γ (5 ng/mL; Sigma, St. Louis, MO, USA) and incubated at 37 °C. After an incubation period of 4 h (to measure TNF-α) and 24 h (to quantify IL-1β and nitrite), cell supernatants were collected, and kept at −80 °C. Then, cytokine concentrations were determined by enzyme-linked immunosorbent assay (ELISA), using the DuoSet kit from R & D Systems (Minneapolis, MN, USA). The quantification of nitric oxide was performed using the Griess method according to Green et al. (1982) [11].

2.4.3. Animals

Adult male Swiss Webster mice weighing between 25 and 35 g, obtained from the Prof. Eduardo Barbosa Beserra Animal Facilities at the Paraíba State University and the Gonçalo Moniz Institute (FIOCRUZ; Salvador, Brazil), were used. In the vivarium, the animals were kept in plastic cages, under room temperature and humidity (23 ± 2 °C), with a 12 h light-dark cycle, and fed with feed and water ad libitum. All studies were performed between 08:00 and 17:00 p.m. The animal care and handling procedures were followed in strict accordance with the recommendations of the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health and the Brazilian College of Animal Experimentation. Previously, the project was approved by the Ethics Committee on Animal Use under number 003/2021. All procedures from the beginning of the study until the time of euthanasia were performed to avoid suffering and reduce the discomfort and pain of the animals.

2.4.4. Inflammatory Model

Zymosan-Induced Acute Peritonitis in Mice

Mice were divided into groups: positive control, negative control and test groups, which were treated orally with saline (5% DMSO), indomethacin 10 mg/kg and JMPR-01 at doses of 5, 10 and 50 mg/kg, respectively. After 1 h of the treatments, 0.25 mL of 2% zymosan was injected intraperitoneally. Four hours after induction of inflammation, the animals were euthanized by administering 2 mL of heparinized phosphate-buffered saline into the intraperitoneal cavity [12,13]. At the end, an incision was made, collecting the exudate, whose cells were resuspended in 500 μL of PBS and 10 μL of Turk’s fluid (1:20). To count the leukocytes, a Neubauer chamber was used under light microscopy, examining the four external quadrants.

Plesthismometer Test

Mice were lightly anesthetized with halothane and received an intraplantar injection of complete Freund’s adjuvant (CFA) (Sigma) in the right paw in a final volume of 20 μL, according to previously reported method [14]. JMPR-01 (50 and 100 mg/kg) or vehicle (5% DMSO in saline; control group) was administered by p.o. route 40 min before phlogiston agent. Dexamethasone (2 mg/kg, i.p) was used as standard. The paw volume was assessed (mm3) by plesthismometer (Ugo Basile, Comerio, Italy) as described above. The amount of paw swelling was determined for each mouse and data were represented as paw volume variation (Δ, mm3).

2.5. Docking Studies

The structure of the JMPR-01 was built with Chemdraw professional 3D 15.0 software, optimized by a minimization of the molecular energy using molecular mechanics (MM2) and then saved as MOL2 files. With the use of AutoDockTools-1.5.6, non-polar hydrogens were merged with the corresponding carbons, and then partial charges of atoms were calculated using the Gasteiger procedure implemented in the AutoDockTools package. Finally, the rotatable bonds of the ligands were defined, the structures were saved as pdbqt and used for docking studies.
The crystal structures of Inducible nitric oxide synthase (PDB ID: 3E7G), Phosphodiesterase 4B (PDB ID: 1XMU), Leukotriene A4 hydrolase (PDB ID: 1HS6), 5-Lipoxygenase (PDB ID: 6NCF) and Cyclooxygenase-2 (PDB ID: 3LN1) were retrieved from RCSB Protein Data Bank as described [15,16]. With the use of Molecular Graphics System, PyMOL, water molecules and other heteroatoms were removed. Then, using AutoDockTools, non-polar hydrogens were merged, and polar hydrogens added to the structures of the proteins. Kollman charges were added and the structures were saved as pdbqt for the docking studies.
The Lamarckian genetic algorithm in AutoDock 4.2.6 was applied to search the best conformation and orientation of the ligands. The global optimization was started with a population of 150 randomly positioned individuals with a maximum of 2,500,000 energy evaluations and a maximum of 27,000 generations. During each docking experiment, 100 runs were carried out. The resulting docking poses were analyzed using AutoDockTools and Discovery Studio Visualizer 2021 Client. To validate the docking procedure, the co-crystalized ligand was previously docked to the protein, obtaining a RMSD value of ≤2 Ǻ in the redocking procedure.

2.6. Statistical Analysis

Data are presented as means ± SEM of measurements made on 6 animals in each group. Comparisons across three or more treatments were made using one-way ANOVA with Tukey’s post hoc test or repeated measures two-way ANOVA with Bonferroni’s post hoc test, when appropriate. All data were analyzed using the Prism 5.01 computer software (GraphPad, San Diego, CA, USA). Statistical differences were considered to be significant at p < 0.05.

3. Results

3.1. Synthesis

The intermediate 2-cyano-N-phenylacetamide (JM-01) (previously synthesized and characterized by Silva (2018) was used to synthesize the phenylacrylamide derivative 2-cyano-N-3-diphenylacrylamide (JMPR-01). The final molecule was obtained through the Knoevenagel condensation reaction presented in Scheme 1. The Knoevenagel condensation is an important organic reaction in the formation of a carbon–carbon double bond between the carbonyl function and activated methylene groups. Many α, β-unsaturated products obtained by this type of condensation have been widely used as intermediates in the synthesis of drugs, chemicals, cosmetics, foodstuffs, and agrochemicals [17,18,19].
Due to its aprotic nature which favors catalytic activity at higher temperatures, toluene was used as a reaction medium. The catalyst used was triethylamine (NEt3), whose function in the reaction medium was to abstract a carbon proton directly linked to nitrile and carbonyl, which, due to a negative mesomeric effect, is deficient in electrons acquiring a partially positive charge. The electronic effect exerted by the ligands makes the hydrogen more acidic, facilitating the loss of the proton to form the carbanion. Knovenagel condensation results from the nucleophilic attack of the carbanion on the carbonyl group of the aldehyde, due to its electron-deficient nature. Protonated triethylamine transfers its proton to the oxygen of the aldehyde, forms a hydroxyl, and restores the base.
The reaction’s completion is characterized by protonation of the hydroxyl and its full exit in the form of water. This process allows the carbon to oxidize and results in the formation of a double bond. After synthesis of JMPR-01, spectroscopic and spectrometric methods were performed for its structural elucidation and determination of physicochemical parameters: theoretical partition coefficient (LogP), molar mass (MM) (Chemdraw), retention factor (Rf), and yield, shown in Table 1.

3.2. Structural Elucidation

Through 1H NMR analysis, it was possible to identify a singlet referring to the NH group of the amide that were detected with displacement at δ 10.43 ppm. This signal is outside the characteristic range of 5.0 to 9.0 ppm due to the anisotropic effect, which leaves the H less shielded. It was observed peaks characteristic of singlet (s) at δ 8.30 ppm indicative of condensation between the aldehyde and methylene group, culminating in the formation of the vinyl group (C=CH). Other aromatic hydrogens could be found with displacements ranging between δ 7.12 to 8.05 in triplet (t) and multiplet (m) form. The 13C NMR spectrum also confirmed the structure by the presence of signals correspondent to the compound. Two signals were observed in the negative region at 160.97 and 138.68 ppm, corresponding respectively to the carbonyl carbon and the quaternary carbon, and indicating Knoevenagel condensation. Likewise, it was possible to detect signals at 151.27 ppm, indicative of vinyl carbons, and at 116.68 ppm, which is diagnostic of the nitrile function. Infrared results also assisted in the structural characterization of the JMPR-01. The following bands were observed: 1682, 2227, and 3017 cm−1, characteristic of elongation of the carbonyl, nitrile, and amide groups, respectively. Ultimately, mass spectrometry (MS) was useful to confirm the structure of the novel synthesized compound, exhibiting the result of m/z = 249.10 (Spectroscopic and spectrometric data can be found in the Supplementary Materials, Figures S1–S4).

3.3. Thermal Characterization (DSC and TGA)

The DSC curve for JMPR-01 presents a well-defined endothermic peak, characteristic of melting, occurring at a temperature of 200.26 °C (ΔH = 222.5 J g−1) and a purity of 99.82%. Regarding the thermogravimetric curve, we observed a single stage of degradation between 174.35–319.92 °C with a mass loss of 96.74%. At the end of the analysis, the presence of inorganic residues (0.3545%) was verified which did not entirely decompose until 900 °C (The DSC and TGA curves are found in the Supplementary Materials, Figure S5).

4. Biological Activity

4.1. In Vitro Tests

The anti-inflammatory and immunomodulatory potential of JMPR-01 was explored in a series of cell culture assays using J774 murine macrophages. The first test performed was cell viability using the Alamarblue method, which indicates impairment of cell metabolism through color change by mitochondrial enzymes [20]. For this test, JMPR-01 was used at concentrations of 6.25, 12.5, 25, 50, and 100 μM, as shown in Figure 1. Based on the results, induction of cytotoxic effects occurred at a concentration of 100 µM. Determining the cytotoxic concentration is important, since it allows targeting concentrations of JMPR-01 for use in other tests, such as dosing for nitrite and cytokines. For these tests, concentrations of up to 50 μM were tested, with no changes in cell viability after 72 h.
Macrophages are a principal cell of the immune system, which, when activated by microbial products (such as LPS and IFN-γ), produce mediators such as nitric oxide, proteolytic enzymes, and inflammatory cytokines, among others. These substances are part of the pathophysiological inflammation process promoted in tissue and vascular injury [21]. Nitrite is a decomposition product of nitric oxide, important in the cell oxidation process, and quantified in cultures of stimulated macrophages. In Figure 2, JMPR-01 at concentrations of 50–3.125 μM promoted a significant reduction (p < 0.05) in nitrite production compared to the un-stimulated control group. Dexamethasone (40 μM) was used as a standard.
Nitric oxide (NO) is a gas molecule which, when crossing biological membranes, produces reactive species through association with oxygen and superoxides. Its production involves nitric oxide synthase (NOS) enzymes, which are presented in three isoforms: neuronal (nNOS), endothelial (eNOS), and inducible (iNOS), using L-arginine, NADPH, and oxygen as substrates. The inducible form (iNOS) is expressed in the highest concentration in response to inflammatory stress, producing higher concentrations of nitric oxide. In macrophages, iNOs can be induced by LPS, a microbial product. NO promotes the release of leukocytes, macrophages, mast cells, endothelial cells, and platelets. NO also promotes blood flow modulation, leukocyte adhesion to vascular endothelium, and the activity of numerous enzymes which impact inflammatory responses [22,23].
The capacity of JMPR-01 to suppress production of cytokines expressed by macrophage stimulation (interleukin 1 beta (IL-1β) and tumor necrosis factor alpha (TNF-α)) was also evaluated. Based on the results, there was a significant reduction (p < 0.05) of TNF-α after the treatment of cultures with JMPR-01 at concentrations of 50 and 25 μM (Figure 3A). For IL-1β, JMPR-01 inhibited TNF-α at all concentrations (50 to 3.125 μM) (Figure 3B), which demonstrates its pharmacological potential even at its lowest tested concentration. Dexamethasone at a concentration of 40 μM was used as the standard drug for this assay as well.
From the dose-response curve of JMPR-01, standardized by TNF-α inhibition in J774 macrophage cultures, the CC50, EC50 and Selectivity Index (S.I.) values were obtained. The cytotoxic concentration that reduced cell viability by 50% compared to the control group was 977.25 ± 655.36 µM. The concentration of the compound for which 50% of the effect was observed was 7.02 ± 4.24 µM. From the ratio (CC50/EC50) was determined (S.I.), which showed a value of 139.20 (Table S1 in the Supplementary Materials. Given these results, JMPR-01 proves to be a strong candidate as an anti-inflammatory agent, encouraging subsequent in vivo assays.
Based on the observed reduced expression of TNF-α, IL-1β, and nitrite in vitro, it can be inferred that a regulatory mechanism is correlated with this transcriptional pathway effect, including a possible decrease in oxidative stress, and inhibition of key regulators of the NF-κB pathway. There are few studies evaluating the performance of phenylacrylamide derivatives in this effect, but they do suggest that their Michael acceptor regions bind to cysteine residues in Keap-1 cytosolic proteins, which act as redox sensors and negative regulators of Nrf2 (nuclear factor erythhroid 2 related factor 2), activating the expression of antioxidant and anti-inflammatory enzymes [24,25,26]. Our results suggest possible mechanisms of action of phenylacrylamide derivatives towards the inhibition of inflammatory mediators involved in inflammatory signaling, and allow for the future development of promising drugs to treat inflammatory diseases.

4.2. In Vivo Tests

4.2.1. Peritonitis Induced Zymosan

Leukocyte recruitment involves intercellular communication between endothelium and defense cells, forming the leukocyte adhesion cascade. Subsequent phases involve the release of inflammatory mediators into the vessel lumen, activation of resident cells, and leukocyte chemotaxis towards the inflammation site [27,28]. An in vivo peritonitis assay was used to investigate the anti-inflammatory pharmacology and potential of the test drug to decrease leukocyte migration.
Zymosan (obtained from the polysaccharide wall of the fungus Saccharomyces cerevisiae) was used as a phlogiston agent [29]. Its action occurs through activation of components of the inflammatory machinery, such as complement system proteins, prostaglandins, leukotrienes, ROS, and platelet aggregation factor [30]. Through direct macrophage stimulation mediated by Toll-like TLR-2 and TLR6 receptors, it was observed that Zymosan was able to induce phagocytosis and the production of inflammatory mediators such as TNF-α, IL-1β, by activating the NF-κB pathway [31].
As can be seen in Figure 4 of the peritonitis model, JMPR-01 significantly (p < 0.05) inhibited leukocyte migration by 61.8, 68.5, and 90.5%, at respective doses of 5, 10, and 50 mg/kg. For the group treated with the standard indomethacin (10 mg/kg), inhibition was 45%. All groups were compared to control (DMSO 5% + saline).
Studies involving Zymosan-induced peritonitis reveal that in the initial phase of administration, animals present vascular permeability and increased levels of myeloperoxidase, demonstrating massive activity involving recruitment and activation of leukocytes [31,32]. In the inflammatory process, leukocyte chemotaxis involves marginalization, capture, rollover, activation, adhesion, and transmigration to the inflammation center [33]. Depending on the microenvironment where inflammation occurs, these processes include different signaling pathways and chemo-attractants [34]. These data corroborate in vitro human umbilical vein endothelial cell studies, where it was observed that IL1-β and TNF-α mediate eosinophil adhesion and trans-endothelial migration, stimulating the release of ICAM-1 (intercellular adhesion molecule-1) E-selectin, and VCAM-1 (vascular cell adhesion molecule-1) [35].

4.2.2. Paw Edema

Vascular responses are the first changes seen in the inflammatory cascade. They involve vasodilation, hyperemia, and increased vascular permeability, to enable the flow of proteins, electrolytes, and exudate into the interstitium [36]. Vascular responses are mediated directly or indirectly by chemical factors caused by inflammatory stimulus in plasma cells and proteins, resulting in what can be seen clinically as edema [37].
Complete Freund’s Adjuvant (CFA) consists of inactivated Mycobacterium tuberculosis and mineral oil. It is an inducer of inflammatory processes, acting through mobilization and activation of antigen-presenting cells (APCs), and increasing the response of T cells [38]. In addition to immunological manifestations, CFA can trigger the release of inflammatory mediators such as PGE2, NO, leukotriene B4 (LTB4), TNF-α, IL-2, and IL-17 [39].
According to Ben et al. [40] and Gris et al. [41] in models of rheumatoid arthritis and nociception, CFA is commonly used [38,42] in experimental inflammation models as an inducing agent, having shown that it induces a chronic inflammatory response after intraplantar administration edema with a peak within 24 h occurs.
Figure 5 presents the results of evaluation of JMPR-01 anti-inflammatory activity in the CFA-induced paw edema model. Pre-treatment carried out with JMPR-01 at a dose of 50 mg/kg inhibited formation of edema from 6 h onwards, with activity ceasing by 24 h. Using the compound in a higher dose (100 mg/kg), a satisfactory inhibition was observed from 2 to 6 h, with similar activity to the Dexamethasone control, yet without being significantly maintained after 24 h.
In vitro, JMPR-01 was shown to suppress TNF-α and IL-1β production by macrophages stimulated with LPS and IFN-γ; making it plausible that this effect may contribute to the anti-edema activity obtained. This assumption is based on modulation (by TNF-α and IL-1β) of gene expression responsible for encoding the iNOS enzyme, through the p38 MAPK pathway and activation of the nuclear factor kappa-B (NF-κB) [42,43], as well as the significant reduction in nitrite production (a metabolite used to measure NO in vitro). Once expressed, iNOS catalyzes synthesis of NO, (a molecule with a stimulating action on the soluble guanylate cyclase enzyme (sGC) with consequent formation of cyclic guanosine monophosphate (cGMP)—especially in this context, due to its vasodilator activity) [44]. With the Zymosan-induced paw edema test being nonspecific, the action of JMPR-01 may result from inhibition of many mediators that contribute to edema promoting activity. These in silico results may contribute to predict its behavior in important targets for anti-inflammatory activity.

4.3. Docking Studies

In silico studies use molecular docking to predict the most favorable conformation, and orientation or “pose” assumed by a ligand, as well as the interactions established between molecules and biological targets. Consequently, molecular docking allows elucidation of fundamental biochemical processes through characterization of the behavior of small molecules when inserted in the active site [45,46].
Free binding energy (ΔG) estimates the binding affinity of the ligand–target complex and is obtained through the Gibbs–Helmholtz equation. Lower free binding energy values indicate greater stability for the complex, favoring interaction of the ligand in the active site when it assumes the particular pose [47]. The results obtained through in silico evaluation of relevant molecular targets and delineation of possible mechanisms related to the anti-inflammatory and immunomodulatory potential of JMPR-01 are described in Table 2 and Figure 6. Validation of the selected targets occurred during redocking of the co-crystallized ligands, presenting RMSD values ≤ 2.0 Å [48].
According to Table 2, JMPR-01 presents greater affinity than the co-crystallized inhibitors to the inducible nitric oxide synthase (iNOS) and phosphodiesterase 4B (PDE4B) targets. The iNOS isoform (with crucial importance to the inflammatory process) is regulated at the transcriptional level through stimulation of immune cells by pro-inflammatory cytokines or endotoxins. The regulation of iNOS isoform activity is important to maintain physiological responses and control the deleterious effects mediated by NO [49]; thus, inhibition is part of clinical management in many inflammatory diseases. This means that iNOS inhibition, as a result of the suppression of cytokines, chemokines, and adhesion molecules, in addition to the maintenance of vascular tone and permeability, (as suggested in the present paw edema model in this study, and as well as the affinity of the tested compound for iNOS) is related to the observed in vitro modulation of nitrite production [37,50].
Figure 7 presents JMPR-01 and the AR-C95791 ligand—iNOS target interactions; it is noteworthy that JMPR-01 established additional interactions with the amino acid residues Gln 263 and Glu 377, justifying greater affinity with the active site. According to Garcin et al. (2008), [51] these residues are essential for the inhibitory activity of this target, since substitutions resulted in binding affinity reductions (Kd), especially in relation to residue Glu 377, whose Kd ranged from 0.4 μM to above 100 µM. In addition to having interactions with residues in common (Val 352 and Pro 350) with the co-crystallized target inhibitor IC50 = 0.35 μM—iNOS isoenzyme [51], the pose established parallel to the Heme group (Figure 6A) also favored interaction with the cofactor (Hem 901).
Delineation of the affinity of JMPR-01 with PDE4 (an isoform that has specific hydrolyzing capacity on cAMP—a second messenger with modulatory action on effector cells in the pathogenesis of inflammation) was also tested [52]. PDE4 as an inhibited pharmacological target is correlated with a consequent intracellular accumulation of cAMP, which can directly activate protein kinase A (PKA) (responsible for phosphorylation of cAMP-responsive protein (CREB) and activating factor of transcription (ATF-1)), leading to increased synthesis of anti-inflammatory cytokines [53].
Roflumilast is a potent selective PDE4 inhibitor, presenting IC50 = 0.8 nM against PDE4 expressed in human neutrophils [54]. Figure 8 presents roflumilast and JMPR-01 with the PDE4B target. Certain shared interactions with amino acid residues (Ile 410, Asn 395, Asp 392, and His 234) are highlighted. JMPR-01 interacted in a complementary way with Ser 394 and Pro 396, as well as presented higher quality interactions at the active site, through hydrogen bonds and shorter distance charge transfer interactions, resulting in a higher interactive force and lower free binding energy values as compared to roflumilast.
The influence of COX-2 in anti-inflammatory therapy is suggested, which highlights its specific up-regulation at the site of inflammation, mainly in response to the local release of cytokines such as IL-1 and TNF-α, activators of the NF-kB transcription factor [55]. Through the metabolism of arachidonic acid, these generate pro-inflammatory and vasoactive eicosanoids such as prostaglandins (PGD2, PGE2, PGF2α), prostacyclins (PGI2), and thromboxanes (TXA2) [56]. Thus, its selective inhibition is an effective way to obtain anti-inflammatory, analgesic, and antipyretic activity without harming the physiological actions inherent to COX-1, a target inhibited by traditional NSAIDs [57].
The selective inhibitor Celecoxib, (IC50 = 0.132 ± 0.005 μM against COX-2), establishes interactions with amino acid residues related to selective inhibition of the enzyme, such as Ser 339, Arg 499, Phe 504 and Val 509, as observed in Figure 9 [58]. JMPR-01, being directed to the more selective region (Val 509), also shares interactions with residues Trp 373, Met 508, and Leu 370, as well as with Ala 502 and Tyr 371. Therefore, JMPR-01 demonstrated inhibition potential that corroborates the results obtained in the in vivo assays, where leukocyte migration and edema formation are significantly induced by PGE2, together with the chemotactic and vasoactive action of leukotrienes [56].
Arachidonic acid is also a 5-LOX substrate, and characterized as a crucial point in synthesis of leukotrienes and lipoxins, which are, respectively, (initiation and resolution) considered regulators of inflammation through association with the 5-LO activating protein (FLAP), the metabolite is 5-hydroperoxyeicosatetraenoic acid (5-HPETE) and, in a subsequent dehydration reaction, the leukotrienes A4 (LTA4). These, in turn, are substrates for the enzyme LTC4 synthase, responsible for the biosynthesis of cystenyl-leukotrienes (CisLT) and for LTA4H in catalysis of the final step in LTB4 biosynthesis, which is a recognized lipid chemotactic mediator and leukocyte activator [59,60].
Further, the action of JMPR-01 in 5-LOX and LTA4 hydrolase enhances its inhibitory effect on the synthesis of leukotrienes and consequent reduction in leukocyte migration, which was indicated in the zymosan induced peritonitis model. According to Sailer et al. (1996) [61], the AKBA ligand co-crystallized to the target 5-LOX presents an IC50 = 1.5 μM in relation to the enzyme expressed by peritoneal polymorphonuclear leukocytes. The active AKBA site is allosteric to the catalytic portion, where it binds to residues His 130, Arg 101, and Thr 137 through hydrogen bonds, shown in Figure 10 [62].
JMPR-01 interacted with two highlighted residues (His 130 and Arg 101) and also shared interactions with Val 110 and Arg 138. Further, the complementary portion composed of Glu 134, Ala 388, and Pro 98 was indicated. Yet the compound under study presented greater affinity for the enzyme LTA4 hydrolase, with a free binding energy close to the co-crystallized ligand, Bestatin, which is a reversible inhibitor of this enzyme with IC50 = 4.0 ± 0.8 μM [63]. In Figure 11, an interactive overlap suggests influence on the catalytic domain of the target (Zn 2+ coordinated by His 295, His 299, and Glu 318) from interactions with His 295, and Zn2+ that are essential for LTB4 biosynthesis of [64].
Through in silico studies, carried out in molecular docking, it was possible to predict the affinity and behavior of JMPR-01 in the catalytic sites of important inflammation targets, with satisfactory results in the enzymes: iNOs, PDE 4B and LTA4H. Inhibition of these targets suggests possible molecular mechanisms for further investigation in more specific in vitro and in vivo assays.

5. Discussion

The compound under study is a synthetic phenylacrylamide: 2-cyano-N-3-diphenylacrylamide derivative (JMPR-01), obtained by the Knoevenagel condensation reaction, structurally elucidated by 1H and 13C NMR techniques, in addition to infrared (IR) and mass spectrometry techniques, showing signs that characterized their main substituent groups, namely, the amide group, the benzene ring systems and the cyano group. It is noteworthy that phenylacrylamide derivatives have already been reported in the literature showing a promising result, affirming the molecule as a good drug candidate. Existing studies include the evaluation of the cytotoxic activity of 2-phenylacrylamide derivatives [65] and the evaluation of the anti-inflammatory activity of cyano-phenylacrylamide hybrid derived from indomethacin and paracetamol [8].
The inflammatory process is mediated by multiple agents. Experimental evaluation in inflammation models is necessary for a good understanding at the molecular level of the substance’s action. Cytotoxicity assays performed demonstrated that the compound JMPR-1 initiated the induction of cytotoxic effects at a concentration of 100 µM, without significant changes up to a period of 72 h. In addition to the assessment of cytotoxicity and cell viability alteration, the study compound at concentrations of 50–3.125 μM promoted a significant reduction (p < 0.05) in nitrite production compared to the unstimulated control group, and also inhibited the TNF-α at all concentrations (50 to 3.125 μM), thus demonstrating a possible modulation of nitric oxide synthases and reduced expression of TNF-α.
TNF-α is a pleiotropic cytokine, inducing the production of IL-1 and IL-6. Together, these factors promote cellular processes such as chemotaxis, increased vascular permeability and hyperalgesia. IL-1β is directly related to increased excitability and sensitization of nociceptive endings, in addition to inducing apoptosis in the responses of inflammatory and carcinogenic processes [66]. These cytokines, as well as endotoxins, such as lipopolysaccharides (LPS), promote the activation of the endothelium cells through the Toll-like receptor 4 (TLR4) and the RIG-I pathway, which promotes cytokine secretion and the expression of molecules of adhesion, such as E-selectin, VCAM-1 and ICAM-1 to facilitate leukocyte extravasation. Tissue damage arises with increased leukocyte migration due to the secretion of large amounts of inflammatory mediators and reactive molecules, which aggravate the inflammatory process [67].
Cytokines are essential mediators for the development of autoimmune and inflammatory pathologies, a result of the induction of the TNF-α signal, including the activation of NF-κB, ERK, p38 MAPK and JNK, which are gene activation pathways for IL-1β, IL-8, IL-6, chemokines, adhesion molecules and immunological enzymes, such as iNOs itself [68,69]. TNF-α, as well as IL-1, influence the activity of the signaling pathway for the NF-κB transcription factor, which regulates the gene expression of inflammatory mediators (these cytokines are overexpressed when this signaling pathway is activated by stress oxidative). This family of cytokines is composed of structurally related members, including NF-κB1 (p50/p105), NF-κB2 (dp52/p100), RelA (p65), RelB and c-Rel that are normally inactivated in the cytoplasm by inhibitory IκB proteins, mainly IκBα [70,71].
These cytokines, in turn, act on both canonical and non-canonical NF-κB activation pathways, activating the IκB kinase complex (IKK), which then mediates phosphorylation, ubiquitination and degradation of IκB and free NF-κB dimer translocation to the nucleus to initiate gene transcription (canonical pathway). The non-canonical pathway involves phosphorylation of the inhibitory domains of p100 and p105 and also results in the release of the NF-κB dimer. Activation of this signaling pathway is important, as it results in the expression of several inflammatory mediators, including many enzymes, such as inducible nitric oxide synthase (iNOS), cyclooxygenases and lipoxygenases that exert pro-inflammatory effects [72,73].
The action of the compound JMPR-1 as a modulator of nitric oxide (NO) synthases was also visualized through Molecular Docking simulations, since the score obtained by the test compound was more negative than that obtained by the co-crystallized ligand; besides that, it also established important interactions with the amino acid residues Gln 263 and Glu 377 that are essential for the inhibitory activity of this target.
The regulation of the activity of iNOS isoforms is important to maintain the physiological responses and control the deleterious effects mediated by NO [49]; thus, inhibition is part of the clinical management in many inflammatory diseases. Therefore, the inhibition of iNOS, as a result of the suppression of cytokines, chemokines and adhesion molecules, in addition to the maintenance of vascular tone and permeability, (as suggested in the present paw edema model in this study, and also the affinity of the compound tested for iNOS) is related to the observed in vitro modulation of nitrite production [37,74].
Based on the observed reduced expression of TNF-α, IL-1β, and nitrite in vitro, it can be inferred that a regulatory mechanism is correlated with this transcriptional pathway effect, including a possible decrease in oxidative stress, and inhibition of key regulators of the NF-κB pathway. There are few studies evaluating the performance of phenylacryla-mide derivatives in this effect, but they do suggest that their Michael acceptor regions bind to cysteine residues in Keap-1 cytosolic proteins, which act as redox sensors and negative regulators of Nrf2 (nuclear factor erythhroid 2 related factor 2), activating the expression of antioxidant and anti-inflammatory enzymes [23,24,25].
In evaluating the anti-inflammatory activity in in vivo models, JMPR-01 significantly inhibited (p < 0.05) the migration of leukocytes by 61.8, 68.5 and 90.5%, at the respective doses of 5, 10 and 50 mg/kg, assuming a better performance when compared to the control, which corresponded to indomethacin. Similarly, in the in situ inflammation model, the paw edema that JMPR-01 at a dose of 50 mg/kg inhibited the formation of edema from 6 h onwards, with cessation of activity within 24 h, could be visualized. Using the compound at a higher dose (100 mg/kg), satisfactory inhibition was observed from 2 to 6 h, with activity similar to that of Dexamethasone control, but not significantly maintained after 24 h.
Inflammatory mediators directly involved in the recruitment of leukocytes have been widely studied with the aim of elucidating their mechanisms of action. Despite a well elucidated chemotactic activity [75], Tager et al. [76] identified the involvement of Leukotriene B4 (LTB4) in the recruitment of CD-4 + T lymphocytes through the BLT-1 receptor and demonstrated that LTB4/BLT-1 T cell-mediated chemotactic activity compared to CXCL12 is one of the most effective T cell chemoattractants. An in vitro complementary study also revealed that CD-8+ T cells, after stimulation by IL-2, acquired chemotactic sensitivity to LTB4 (when released by previously activated mast cells) [77].
Cytokines such as TNF-α, IL-1, and IL-4 also play important roles in chemotaxis, acting through the up-regulation of vascular adhesion molecules, specifically E-selectin, P-selectin, and the cell adhesion molecule (ICAM -1), promoting fixation and rolling of leukocytes inside the vessel [78]. These data corroborate in vitro human umbilical vein endothelial cell studies, where it was observed that IL1-β and TNF-α mediate eosinophil adhesion and trans endothelial migration, thereby stimulating the release of ICAM-1 (intercellular adhesion molecule-1) E-selectin, and VCAM-1 (vascular cell adhesion molecule-1) [34].
The reduction in cytokine production disfavors COX-2 transcription through the NFκB pathway. In the edematogenic activity, COX-2 transcription stands out for the biosynthesis of eicosanoids, including prostaglandin E1 (PGE1), prostaglandin E2 (PGE2) and prostacyclin I2 [56]. Together, they participate in the vascular phase of inflammation, promoting vasodilation and increased permeability of the venule with consequent plasma exudation [36,56]. These, in particular PGE1 and PGE2, are capable of sensitizing afferent nociceptive nerve terminals to endogenous pain mediators, such as bradykinin, histamine and serotonin, which are intrinsically related to hyperalgesia [79].
Dexamethasone was chosen as the standard for in vitro tests of nitrite and cytokine dosages, as well as in paw edema, due to its good performance in anti-inflammatory and immunomodulatory therapy. Its mechanism involves the action on nuclear receptors involved in the transcription of anti-inflammatory mediators [80], among which we highlight those of protein origin, such as Annexin A1, known as a potent multifunctional anti-inflammatory mediator. It is known that the activity of Annexin A1 comes mainly from its active grouping called N-terminal peptide Ac2-26, which binds to the formyl-peptide receptor (FPR) grouping, promoting reduction in the production of the storm of pro-inflammatory cytokines [81]. The direct correlation between glucocorticoids and Annexin A1 can be described in in vivo studies using mice deficient in Annexin A1, where it was possible to observe an absence of response to administered glucocorticoids [82,83].
Studies by Pupjalis et al., 2011 [84], using apoptotic immune cells, demonstrated that dexamethasone-mediated Annexin A1 expression contributed to TNF-α suppression by a mechanism involving formyl peptide receptors (FPR). These data corroborate in vitro studies by Gobbetti and Cooray, 2016 [85], involving fibroblasts present in chronic inflammatory lung diseases, where it was possible to identify that mimicking the actions of Annexin A1 may have positive therapeutic effects in these pathologies, through the regulation of the NFκ-B pathway was induced by TNF-α, a mediator that had its release suppressed by JMPR-01 in in vitro studies. The suppressive activity of TNF-α presented by JMPR-01 opens doors for further investigations addressing the possible role of the same in increasing the expression of Annexin A1 or in promoting activities similar to the protein itself.
This action in the reduction of mediators can be justified by Molecular Docking, in which the compound JMPR-1 established important interactions, proving a possible action in the pathways of COX-2, 5-Lox and leukotrienes, which can be useful for conducting studies futures, as well as proof of activities that cannot be experimentally validated. Our results suggest possible mechanisms of action of phenylacrylamide derivatives towards the inhibition of inflammatory mediators involved in inflammatory signaling, and allow for future development of promising drugs to treat inflammatory diseases.

6. Conclusions

In conclusion, this study presents for the first time the anti-inflammatory potential of a new phenylacrylamide derivative obtained through bioisosteric ring modification of a previously studied drug. Using in vitro and in vivo approaches, immunomodulatory and anti-inflammatory properties were demonstrated, suggesting that its mechanism (in part) may be related to decreased expression of inflammatory cytokines (TNF-α and IL-1β) and nitric oxide. In the in silico studies, satisfactory couplings were observed for targets such as iNOs, COX-2 and PDE-IV, suggesting potentially multi-target interactions involving the inflammatory response. This can be further investigated. These preliminary results demonstrate the therapeutic potential of JMPR-01, making it a potential drug for inflammatory conditions, with both viable synthesis and low production costs.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/pharmaceutics14010188/s1, Figure S1: FT-IR spectrum of JMPR-01; Figure S2: 1H NMR spectrum of JMPR-01 (500 MHz, DMSO-d6); Figure S3: 13C NMR spectrum of JMPR-01 (125 MHz, DMSO-d6); Figure S4 HRMS m/z [M+H] of JMPR-01 (MALDI-TOF); Figure S5 Differential Scanning Calorimetry (DSC) (A) and Thermogravimetric Analysis (B) of JMPR-01; Table S1: CC50, EC50 and Selectivity Index of JMPR-01 standardized by TNF-α inhibition in determined in cultures of J774 macrophages.

Author Contributions

Participated in research design: P.R.d.S., V.L.d.S., R.O.d.M. and C.F.V.; performed the experiments and the data analysis: P.R.d.S., R.F.d.E.S., C.d.O.M., F.E.P.C., T.B.C., Y.V.B., Y.M.S.d.M.e.S. and N.F.d.S.; wrote or contributed to the writing of the manuscript: P.R.d.S., V.L.d.S., R.O.d.M., C.F.V., F.E.P.C., T.B.C., Y.V.B. and Y.M.S.d.M.e.S. All authors have read and agreed to the published version of the manuscript.

Funding

This research received no external funding.

Institutional Review Board Statement

Animal use protocol 003/2021 was approved by the Ethics Committee on Animal from the State University of Paraíba and all procedures were performed in accordance with the NIH Guidelines for the Care and Use of Laboratory Animal.

Informed Consent Statement

Not applicable.

Data Availability Statement

Data can be requested by contacting the corresponding author.

Acknowledgments

This study was financed in part by the State University of Paraiba grant 001/2021, Fundação de Amparo à Pesquisa do Estado da Paraiba (FAPESQ), Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (Capes) with collaboration from the Universidade Federal da Bahia (UFBA).

Conflicts of Interest

The authors declare no conflict of interest. The founding sponsors had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, and in the decision to publish the results.

References

  1. Mortaz, E.; Pourdolat, G.; Varahram, M.; Tabarsi, P.; Farnia, P.; Garssen, J.; Adcock, I. Investigation of CD14 with toll like receptors expression in pleural effusion for detection of thoracic disorders. Eur. Respir. Soc. 2017, 50, PA1588. [Google Scholar]
  2. Chen, L.; Deng, H.; Cui, H.; Fang, J.; Zuo, Z.; Deng, J.; Li, Y.; Wang, X.; Zhao, L. Inflammatory responses and inflammation-associated diseases in organs. Oncotarget 2018, 9, 7204–7218. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  3. Grosser, T.; Smyth, E.; Fitzgerald, G. Anti-Inflammatory, Antipyretic, and Analgesic Agents; Pharmacotherapy of Gout; ResearchGate: Berlin, Germany, 2011. [Google Scholar]
  4. Murakami, M.; Hirano, T. The molecular mechanisms of chronic inflammation development. Front. Immunol. 2012, 3, 1–2. [Google Scholar] [CrossRef] [Green Version]
  5. Marmitt, D.J.; Rempel, C.; Goettert, M.I.; Silva, A.C. Plantas Medicinais da RENISUS Com Potencial Anti-inflamatório: Revisão Sistemática Em Três Bases de Dados Científicas. Rev. Fitos 2015, 9, 129–144. [Google Scholar] [CrossRef] [Green Version]
  6. Bordoni, A.; Danesi, F.; Dardevet, D.; Dupont, D.; Fernandez, A.S.; Gille, D.; Nunes dos Santos, C.; Pinto, P.; Re, R.; Rémond, D.; et al. Dairy products and inflammation: A review of the clinical evidence. Crit. Rev. Food Sci. Nutr. 2017, 57, 2497–2525. [Google Scholar] [CrossRef]
  7. Gene, E.; Calvet, X.; Moron, A.; Iglesias, M. Recommendations for the use of anti-inflammatory drugs and indications for gastrointestinal protection in emergency departments. Emergencias 2009, 21, 295–300. [Google Scholar]
  8. Silva, P.; de Almeida, M.; Silva, J.; Albino, S.; Espírito-Santo, R.; Lima, M.; Villarreal, C.; Moura, R.; Santos, V. (E)-2-Cyano-3-(1H-Indol-3-yl)-N-phenylacrylamide, a hybrid compound derived from indomethacin and paracetamol: Design, synthesis and evaluation of the anti-inflammatory potential. Int. J. Mol. Sci. 2020, 21, 2591. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  9. Eilenberger, C.; Kratz, S.R.A.; Rothbauer, M.; Ehmoser, E.K.; Ertl, P.; Küpcü, S. Optimized alamarBlue assay protocol for drug dose-response determination of 3D tumor spheroids. MethodsX 2018, 5, 781–787. [Google Scholar] [CrossRef]
  10. Espírito-Santo, R.F.; Meira, C.S.; Dos Santos Costa, R.; Filho, O.P.S.; Evangelista, A.F.; Trossini, G.H.G.; Ferreira, G.M.; Da Silva Velozo, E.; Villarreal, C.F.; Pereira Soares, M.B. The anti-inflammatory and immunomodulatory potential of braylin: Pharmacological properties and mechanisms by in silico, in vitro and in vivo approaches. PLoS ONE 2017, 12, e0179174. [Google Scholar] [CrossRef] [PubMed]
  11. Green, L.C.; Wagner, D.A.; Glogowski, J.; Skipper, P.L.; Wishnok, J.S.; Tannenbaum, S.R. Analysis of nitrate, nitrite, and [15N] nitrate in biological fluids. Anal. Biochem. 1982, 126, 131–138. [Google Scholar] [CrossRef]
  12. de Morais Oliveira-Tintino, C.D.; Pessoa, R.T.; Fernandes, M.N.M.; Alcântara, I.S.; da Silva, B.A.F.; de Oliveira, M.R.C.; Martins, A.O.B.P.B.; da Silva, M.d.S.; Tintino, S.R.; Rodrigues, F.F.G.; et al. Anti-inflammatory and anti-edematogenic action of the Croton campestris A. St.-Hil (Euphorbiaceae) essential oil and the compound β-caryophyllene in in vivo models. Phytomedicine 2018, 41, 82–95. [Google Scholar] [CrossRef]
  13. Vinegar, R.; Truax, J.F.; Selph, J.L. Some Quantitative Temporal Characteristics of Carrageenin-Induced Pleurisy in the Rat. Proc. Soc. Exp. Biol. Med. 1973, 143, 711–714. [Google Scholar] [CrossRef] [PubMed]
  14. da Lima, M.S.; Quintans-Júnior, L.J.; de Santana, W.A.; Martins Kaneto, C.; Pereira Soares, M.B.; Villarreal, C.F. Anti-inflammatory effects of carvacrol: Evidence for a key role of interleukin-10. Eur. J. Pharmacol. 2013, 699, 112–117. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  15. Moraes, A.D.T.d.O.; de Miranda, M.D.S.; Jacob, Í.T.T.; Amorim, C.A.d.C.; de Moura, R.O.; da Silva, S.Â.S.; Soares, M.B.P.; de Almeida, S.M.V.; Souza, T.R.C.d.L.; de Oliveira, J.F.; et al. Synthesis, in vitro and in vivo biological evaluation, COX-1/2 inhibition and molecular docking study of indole-N-acylhydrazone derivatives. Bioorg. Med. Chem. 2018, 26, 5388–5396. [Google Scholar] [CrossRef] [PubMed]
  16. Chandak, N.; Kumar, P.; Kaushik, P.; Varshney, P.; Sharma, C.; Kaushik, D.; Jain, S.; Aneja, K.R.; Sharma, P.K. Dual evaluation of some novel 2-amino-substituted coumarinylthiazoles as anti-inflammatory-antimicrobial agents and their docking studies with COX-1/COX-2 active sites. J. Enzym. Inhib. Med. Chem. 2014, 29, 476–484. [Google Scholar] [CrossRef]
  17. Appaturi, J.N.; Selvaraj, M.; Abdul Hamid, S.B.; Bin Johan, M.R. Synthesis of 3-(2-furylmethylene)-2,4-pentanedione using DL-Alanine functionalized MCM-41 catalyst via Knoevenagel condensation reaction. Microporous Mesoporous Mater. 2018, 260, 260–269. [Google Scholar] [CrossRef]
  18. Wach, A.; Drozdek, M.; Dudek, B.; Szneler, E.; Kuśtrowski, P. Control of amine functionality distribution in polyvinylamine/SBA-15 hybrid catalysts for Knoevenagel condensation. Catal. Commun. 2015, 64, 52–57. [Google Scholar] [CrossRef]
  19. Li, J.P.H.; Adesina, A.A.; Kennedy, E.M.; Stockenhuber, M. A mechanistic study of the Knoevenagel condensation reaction: New insights into the influence of acid and base properties of mixed metal oxide catalysts on the catalytic activity. Phys. Chem. Chem. Phys. 2017, 19, 26630–26644. [Google Scholar] [CrossRef] [PubMed]
  20. Rampersad, S.N. Multiple applications of alamar blue as an indicator of metabolic function and cellular health in cell viability bioassays. Sensors 2012, 12, 12347–12360. [Google Scholar] [CrossRef] [PubMed]
  21. Feng, L.; Sun, Y.; Song, P.; Xu, L.; Wu, X.; Wu, X.; Shen, Y.; Sun, Y.; Kong, L.; Wu, X.; et al. Seselin ameliorates inflammation via targeting Jak2 to suppress the proinflammatory phenotype of macrophages. Br. J. Pharmacol. 2019, 176, 317–333. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  22. Lambden, S. Bench to bedside review: Therapeutic modulation of nitric oxide in sepsis—An update. Intensive Care Med. Exp. 2019, 7, 64. [Google Scholar] [CrossRef] [Green Version]
  23. Rahat, M.A.; Hemmerlein, B. Macrophage-tumor cell interactions regulate the function of nitric oxide. Front. Physiol. 2013, 4, 144. [Google Scholar] [CrossRef] [Green Version]
  24. De Moraes Pultrini, A.; Almeida Galindo, L.; Costa, M. Effects of the essential oil from Citrus aurantium L. in experimental anxiety models in mice. Life Sci. 2006, 78, 1720–1725. [Google Scholar] [CrossRef]
  25. De Freitas Silva, M.; Pruccoli, L.; Morroni, F.; Sita, G.; Seghetti, F.; Viegas, C.; Tarozzi, A. The Keap1/Nrf2-ARE pathway as a pharmacological target for chalcones. Molecules 2018, 23, 1803. [Google Scholar] [CrossRef] [Green Version]
  26. Gu, X.; Chen, J.; Zhang, Y.; Guan, M.; Li, X.; Zhou, Q.; Song, Q.; Qiu, J. Synthesis and assessment of phenylacrylamide derivatives as potential anti-oxidant and anti-inflammatory agents. Eur. J. Med. Chem. 2019, 180, 62–71. [Google Scholar] [CrossRef]
  27. Rudziak, P.; Ellis, C.G.; Kowalewska, P.M. Role and molecular mechanisms of pericytes in regulation of leukocyte diapedesis in inflamed tissues. Mediat. Inflamm. 2019, 2019, 4123605. [Google Scholar] [CrossRef]
  28. Nourshargh, S.; Alon, R. Leukocyte Migration into Inflamed Tissues. Immunity 2014, 41, 694–707. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  29. Cash, J.L.; White, G.E.; Greaves, D.R. Chapter 17 Zymosan-Induced Peritonitis as a Simple Experimental System for the Study of Inflammation, 1st ed.; Elsevier Inc.: Amsterdam, The Netherlands, 2009; Volume 461, ISBN 9780123749079. [Google Scholar]
  30. Volman, T.J.H.; Hendriks, T.; Goris, R.J.A. Zymosan-induced generalized inflammation: Experimental studies into mechanisms leading to multiple organ dysfunction syndrome. Shock 2005, 23, 291–297. [Google Scholar] [CrossRef] [PubMed]
  31. Underhill, D.M. Macrophage recognition of zymosan particles. J. Endotoxin Res. 2003, 9, 176–180. [Google Scholar] [CrossRef] [PubMed]
  32. Deng, X.; Wang, X.; Andersson, R. Alterations in endothelial barrier permeability in multiple organs during overactivation of macrophages in rats 1996. Shock 1996, 6, 126–133. [Google Scholar] [CrossRef]
  33. Ley, K. Molecular mechanisms of leukocyte recruitment in the inflammatory process. Cardiovasc. Res. 1996, 32, 733–742. [Google Scholar] [CrossRef] [Green Version]
  34. Wong, C.H.Y.; Heit, B.; Kubes, P. Molecular regulators of leucocyte chemotaxis during inflammation. Cardiovasc. Res. 2010, 86, 183–191. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  35. Ebisawa, M.; Bochner, B.S.; Georas, S.N.; Schleimer, R.P. Eosinophil transendothelial migration induced by cytokines. I. Role of endothelial and eosinophil adhesion molecules in IL-1 beta-induced transendothelial migration. J. Immunol. 1992, 149, 4021–4028. [Google Scholar]
  36. Alessandri, A.L.; Sousa, L.P.; Lucas, C.D.; Rossi, A.G.; Pinho, V.; Teixeira, M.M. Resolution of inflammation: Mechanisms and opportunity for drug development. Pharmacol. Ther. 2013, 139, 189–212. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  37. Saldanha, A.A.; Vieira, L.; Ribeiro, R.I.M.d.A.; Thomé, R.G.; dos Santos, H.B.; Silva, D.B.; Carollo, C.A.; de Oliveira, F.M.; de Oliveira Lopes, D.; de Siqueira, J.M.; et al. Chemical composition and evaluation of the anti-inflammatory and antinociceptive activities of Duguetia furfuracea essential oil: Effect on edema, leukocyte recruitment, tumor necrosis factor alpha production, iNOS expression, and adenosinergic and opioid. J. Ethnopharmacol. 2019, 231, 325–336. [Google Scholar] [CrossRef] [PubMed]
  38. Zhao, X.H.; Zhang, T.; Li, Y.Q. The up-regulation of spinal Toll-like receptor 4 in rats with inflammatory pain induced by complete Freund’s adjuvant. Brain Res. Bull. 2015, 111, 97–103. [Google Scholar] [CrossRef]
  39. Nisar, A.; Akhter, N.; Singh, G.; Masood, A.; Malik, A.; Banday, B.; Zargar, M.A. Modulation of T-helper cytokines and inflammatory mediators by Atropa accuminata. Royle in adjuvant induced arthritic tissues. J. Ethnopharmacol. 2015, 162, 215–224. [Google Scholar] [CrossRef]
  40. Ben, I.O.; Woode, E.; Koffuor, G.A.; Boakye-Gyasi, E.; Titiloye, N.A. Effect of trichilia monadelpha (Meliaceae) extracts on bone histomorphology in complete freund’s adjuvant-induced arthritis. J. Intercult. Ethnopharmacol. 2017, 6, 177–185. [Google Scholar] [CrossRef]
  41. Gris, G.; Merlos, M.; Vela, J.M.; Zamanillo, D.; Portillo-Salido, E. S1RA, a selective sigma-1 receptor antagonist, inhibits inflammatory pain in the carrageenan and complete Freund’s adjuvant models in mice. Behav. Pharmacol. 2014, 25, 226–235. [Google Scholar] [CrossRef] [PubMed]
  42. Xiang, H.C.; Lin, L.X.; Hu, X.F.; Zhu, H.; Li, H.P.; Zhang, R.Y.; Hu, L.; Liu, W.T.; Zhao, Y.L.; Shu, Y.; et al. AMPK activation attenuates inflammatory pain through inhibiting NF-κB activation and IL-1β expression. J. Neuroinflamm. 2019, 16, 34. [Google Scholar] [CrossRef]
  43. Cui, L.; Feng, L.; Zhang, Z.H.; Jia, X. Bin The anti-inflammation effect of baicalin on experimental colitis through inhibiting TLR4/NF-κB pathway activation. Int. Immunopharmacol. 2014, 23, 294–303. [Google Scholar] [CrossRef] [PubMed]
  44. Ghasemi, M. Nitric Oxide: Antidepressant Mechanisms and Inflammation, 1st ed.; Elsevier Inc.: Amsterdam, The Netherlands, 2019; Volume 86, ISBN 9780128166680. [Google Scholar]
  45. Meng, X.-Y.; Zhang, H.-X.; Mezei, M.; Cui, M. Molecular Docking: A Powerful Approach for Structure-Based Drug Discovery. Curr. Comput. Aided-Drug Des. 2012, 7, 146–157. [Google Scholar] [CrossRef]
  46. Pinzi, L.; Rastelli, G. Molecular docking: Shifting paradigms in drug discovery. Int. J. Mol. Sci. 2019, 20, 4331. [Google Scholar] [CrossRef] [Green Version]
  47. Gupta, M.; Sharma, R.; Kumar, A. Docking techniques in pharmacology: How much promising? Comput. Biol. Chem. 2018, 76, 210–217. [Google Scholar] [CrossRef] [PubMed]
  48. Westermaier, Y.; Barril, X.; Scapozza, L. Virtual screening: An in silico tool for interlacing the chemical universe with the proteome. Methods 2015, 71, 44–57. [Google Scholar] [CrossRef]
  49. Singh, M.; Padhy, G.; Vats, P.; Bhargava, K.; Sethy, N.K. Hypobaric hypoxia induced arginase expression limits nitric oxide availability and signaling in rodent heart. Biochim. Biophys. Acta -Gen. Subj. 2014, 1840, 1817–1824. [Google Scholar] [CrossRef]
  50. Lee, W.; Yang, S.; Lee, C.; Park, E.K.; Kim, K.M.; Ku, S.K.; Bae, J.S. Aloin reduces inflammatory gene iNOS via inhibition activity and p-STAT-1 and NF-κB. Food Chem. Toxicol. 2019, 126, 67–71. [Google Scholar] [CrossRef]
  51. Garcin, E.D.; Arvai, A.S.; Rosenfeld, R.J.; Kroeger, M.D.; Crane, B.R.; Andersson, G.; Andrews, G.; Hamley, P.J.; Mallinder, P.R.; Nicholls, D.J.; et al. Anchored plasticity opens doors for selective inhibitor design in nitric oxide synthase. Nat. Chem. Biol. 2008, 4, 700–707. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  52. Chiricozzi, A.; Caposiena, D.; Garofalo, V.; Cannizzaro, M.V.; Chimenti, S.; Saraceno, R. A new therapeutic for the treatment of moderate-to-severe plaque psoriasis: Apremilast. Expert Rev. Clin. Immunol. 2016, 12, 237–249. [Google Scholar] [CrossRef]
  53. Li, H.; Zuo, J.; Tang, W. Phosphodiesterase-4 inhibitors for the treatment of inflammatory diseases. Front. Pharmacol. 2018, 9, 1048. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  54. Hatzelmann, A.; Schudt, C. Anti-inflammatory and immunomodulatory potential of the novel PDE4 inhibitor roflumilast in vitro. J. Pharmacol. Exp. Ther. 2001, 297, 267–279. [Google Scholar]
  55. Mendes, R.T.; Stanczyk, C.P.; Sordi, R.; Otuki, M.F.; dos Santos, F.A.; Fernandes, D. Inibição seletiva da ciclo-oxigenase-2: Riscos e benefícios. Rev. Bras. Reumatol. 2012, 52, 774–782. [Google Scholar] [CrossRef] [Green Version]
  56. Moreira, V.; Gutiérrez, J.M.; Lomonte, B.; Vinolo, M.A.R.; Curi, R.; Lambeau, G.; Teixeira, C. 12-HETE is a regulator of PGE2 production via COX-2 expression induced by a snake venom group IIA phospholipase A2 in isolated peritoneal macrophages. Chem. Biol. Interact. 2020, 317, 108903. [Google Scholar] [CrossRef] [PubMed]
  57. Meshram, M.A.; Bhise, U.O.; Makhal, P.N.; Kaki, V.R. Synthetically-tailored and nature-derived dual COX-2/5-LOX inhibitors: Structural aspects and SAR. Eur. J. Med. Chem. 2021, 225, 113804. [Google Scholar] [CrossRef] [PubMed]
  58. Sağlık, B.N.; Osmaniye, D.; Levent, S.; Çevik, U.A.; Çavuşoğlu, B.K.; Özkay, Y.; Kaplancıklı, Z.A. Design, synthesis and biological assessment of new selective COX-2 inhibitors including methyl sulfonyl moiety. Eur. J. Med. Chem. 2021, 209, 112918. [Google Scholar] [CrossRef]
  59. Wang, X.; Bey, A.L.; Katz, B.M.; Badea, A.; Kim, N.; David, L.K.; Duffney, L.J.; Kumar, S.; Mague, S.D.; Hulbert, S.W.; et al. Altered mGluR5-Homer scaffolds and corticostriatal connectivity in a Shank3 complete knockout model of autism. Nat. Commun. 2016, 7, 11459. [Google Scholar] [CrossRef] [Green Version]
  60. Saeki, K.; Yokomizo, T. Identification, signaling, and functions of LTB4 receptors. Semin. Immunol. 2017, 33, 30–36. [Google Scholar] [CrossRef]
  61. Sailer, E.R.; Subramanian, L.R.; Rall, B.; Hoernlein, R.F.; Ammon, H.P.T.; Safayhi, H. Acetyl-11-keto-β-boswellic acid (AKBA): Structure requirements for binding and 5-lipoxygenase inhibitory activity. Br. J. Pharmacol. 1996, 117, 615–618. [Google Scholar] [CrossRef] [PubMed]
  62. Gilbert, N.C.; Gerstmeier, J.; Schexnaydre, E.E.; Börner, F.; Garscha, U.; Neau, D.B.; Werz, O.; Newcomer, M.E. Structural and mechanistic insights into 5-lipoxygenase inhibition by natural products. Nat. Chem. Biol. 2020, 16, 783–790. [Google Scholar] [CrossRef]
  63. Orning, L.; Krivi, G.; Fitzpatrick, F.A. Leukotriene A4 hydrolase. Inhibition by bestatin and intrinsic aminopeptidase activity establish its functional resemblance to metallohydrolase enzymes. J. Biol. Chem. 1991, 266, 1375–1378. [Google Scholar] [CrossRef]
  64. Thunnissen, M.M.G.M.; Nordlund, P.; Haeggström, J.Z. Crystal structure of human leukotriene A4 hydrolase, a bifunctional enzyme in inflammation. Nat. Struct. Biol. 2001, 8, 131–135. [Google Scholar] [CrossRef] [PubMed]
  65. Tarleton, M.; Dyson, L.; Gilbert, J.; Sakoff, J.A.; McCluskey, A. Focused library development of 2-phenylacrylamides as broad spectrum cytotoxic agents. Bioorg. Med. Chem. 2013, 21, 333–347. [Google Scholar] [CrossRef] [PubMed]
  66. Dias Quintão, J.L.; Reis Gonzaga, A.C.; Galdino, G.; Lima Romero, T.R.; Silva, J.F.; Lemos, V.S.; Campolina-Silva, G.H.; Aparecida de Oliveira, C.; Bohórquez Mahecha, G.A.; Gama Duarte, I.D. TNF-α, CXCL-1 and IL-1 β as activators of the opioid system involved in peripheral analgesic control in mice. Eur. J. Pharmacol. 2021, 896, 173900. [Google Scholar] [CrossRef]
  67. Le, K.T.T.; Chu, X.; Jaeger, M.; Plantinga, J.A.; Matzaraki, V.; Withoff, S.; Joosten, L.A.B.; Netea, M.G.; Wijmenga, C.; Li, Y.; et al. Leukocyte-released mediators in response to both bacterial and fungal infections trigger ifn pathways, independent of il-1 and tnf-α, in endothelial cells. Front. Immunol. 2019, 10, 1–13. [Google Scholar] [CrossRef] [Green Version]
  68. Pandey, S.; Kawai, T.; Akira, S. Microbial sensing by toll-like receptors and intracellular nucleic acid sensors. Cold Spring Harb. Perspect. Biol. 2015, 7, 1–18. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  69. Brenner, D.; Blaser, H.; Mak, T.W. Regulation of tumour necrosis factor signalling: Live or let die. Nat. Rev. Immunol. 2015, 15, 362–374. [Google Scholar] [CrossRef]
  70. Shamroukh, A.H.; Hegab, M.I. A review on synthesis, therapeutic, and computational studies of substituted 1, 3, 4 thiadiazole derivatives. Egypt. J. Chem. 2020, 63, 4387–4408. [Google Scholar]
  71. Albensi, B.C. What is nuclear factor kappa B (NF-κB) doing in and to the mitochondrion? Front. Cell Dev. Biol. 2019, 7, 1–7. [Google Scholar] [CrossRef] [PubMed]
  72. Rex, J.; Lutz, A.; Faletti, L.E.; Albrecht, U.; Thomas, M.; Bode, J.G.; Borner, C.; Sawodny, O.; Merfort, I. IL-1β and TNFα differentially influence NF-κB activity and FasL-induced apoptosis in primary murine hepatocytes during LPS-induced inflammation. Front. Physiol. 2019, 10, 1–15. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  73. Mussbacher, M.; Salzmann, M.; Brostjan, C.; Hoesel, B.; Schoergenhofer, C.; Datler, H.; Hohensinner, P.; Basílio, J.; Petzelbauer, P.; Assinger, A. Cell type-specific roles of NF-κB linking inflammation and thrombosis. Front. Immunol. 2019, 10, 85. [Google Scholar] [CrossRef] [Green Version]
  74. Lee, C.-L.; Chiang, L.-C.; Cheng, L.-H.; Liaw, C.-C.; Abd El-Razek, M.H.; Chang, F.-R.; Wu, Y.-C. Influenza A (H1N1) antiviral and cytotoxic agents from Ferula assa-foetida. J. Nat. Prod. 2009, 72, 1568–1572. [Google Scholar] [CrossRef]
  75. Yokomizo, T.; Izumi, T.; Shimizu, T. Leukotriene B4: Metabolism and signal transduction. Arch. Biochem. Biophys. 2001, 385, 231–241. [Google Scholar] [CrossRef]
  76. Tager, A.M.; Bromley, S.K.; Medoff, B.D.; Islam, S.A.; Bercury, S.D.; Friedrich, E.B.; Carafone, A.D.; Gerszten, R.E.; Luster, A.D. Leukotriene B4 receptor BLT1 mediates early effector T cell recruitment. Nat. Immunol. 2003, 4, 982–990. [Google Scholar] [CrossRef] [PubMed]
  77. Ott, V.L.; Cambier, J.C.; Kappler, J.; Marrack, P.; Swanson, B.J. Mast cell-dependent migration of effector CD8+ T cells through production of leukotriene B4. Nat. Immunol. 2003, 4, 974–981. [Google Scholar] [CrossRef]
  78. Kubes, P.; Ward, P.A. Leukocyte recruitment and the acute inflammatory response. Brain Pathol. 2000, 10, 127–135. [Google Scholar] [CrossRef] [Green Version]
  79. Verma, V.; Sheikh, Z.; Ahmed, A.S. Nociception and role of immune system in pain. Acta Neurol. Belg. 2015, 115, 213–220. [Google Scholar] [CrossRef]
  80. Andreakos, E.; Papadaki, M.; Serhan, C.N. Dexamethasone, pro-resolving lipid mediators and resolution of inflammation in COVID-19. Allergy Eur. J. Allergy Clin. Immunol. 2021, 76, 626–628. [Google Scholar] [CrossRef] [PubMed]
  81. Schloer, S.; Pajonczyk, D.; Rescher, U. Annexins in translational research: Hidden treasures to be found. Int. J. Mol. Sci. 2018, 19, 1781. [Google Scholar] [CrossRef] [Green Version]
  82. Yang, Y.H.; Morand, E.F.; Getting, S.J.; Paul-Clark, M.; Liu, D.L.; Yona, S.; Hannon, R.; Buckingham, J.C.; Perretti, M.; Flower, R.J. Modulation of Inflammation and Response to Dexamethasone by Annexin 1 in Antigen-Induced Arthritis. Arthritis Rheum. 2004, 50, 976–984. [Google Scholar] [CrossRef] [PubMed]
  83. Hannon, R.; Croxtall, J.D.; Getting, S.J.; Roviezzo, F.; Yona, S.; Paul-Clark, M.J.; Gavins, F.N.E.; Perretti, M.; Morris, J.F.; Buckingham, J.C.; et al. Aberrant inflammation and resistance to glucocorticoids in annexin 1−/− mouse. FASEB J. 2003, 17, 253–255. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  84. Pupjalis, D.; Goetsch, J.; Kottas, D.J.; Gerke, V.; Rescher, U. Annexin A1 released from apoptotic cells acts through formyl peptide receptors to dampen inflammatory monocyte activation via JAK/STAT/SOCS signalling. EMBO Mol. Med. 2011, 3, 102–114. [Google Scholar] [CrossRef] [PubMed]
  85. Gobbetti, T.; Cooray, S.N. Annexin A1 and resolution of inflammation: Tissue repairing properties and signalling signature. Biol. Chem. 2016, 397, 981–993. [Google Scholar] [CrossRef] [PubMed]
Pharmaceutics 14 00188 sch001 550
Scheme 1. Synthesis of (E)-2-cyano-N,3-diphenylacrylamide—JMPR-01.
Scheme 1. Synthesis of (E)-2-cyano-N,3-diphenylacrylamide—JMPR-01.
Pharmaceutics 14 00188 sch001
Pharmaceutics 14 00188 g001 550
Figure 1. In vitro cytotoxicity of J774 macrophage cell treated with JMPR-01 (6.25–100 μM). The graph representes cell viability at 72 h determined by Alamar Blue assay. Data representes mean standard erro of the mean ± SEMs; n = 4 determinations per group. * Significantly different from the vehicle treated cultures (p < 0.05); ANOVA followed by Tukey’s multiple comparison test.
Figure 1. In vitro cytotoxicity of J774 macrophage cell treated with JMPR-01 (6.25–100 μM). The graph representes cell viability at 72 h determined by Alamar Blue assay. Data representes mean standard erro of the mean ± SEMs; n = 4 determinations per group. * Significantly different from the vehicle treated cultures (p < 0.05); ANOVA followed by Tukey’s multiple comparison test.
Pharmaceutics 14 00188 g001
Pharmaceutics 14 00188 g002 550
Figure 2. Modulation of nitric oxide (NO) (nitrite in μM) in activated J774 macrophage cells treated with JMPR-01 (3.125-50 μM) by the Griess method. Data representes mean standard error of the mean ± SEMs; n = 6 determinations per group. * Significantly different from the vehicle treated cultures stimulated with LPS + IFN-γ (p < 0.05). ANOVA followed by Tukey’s multiple comparison test.
Figure 2. Modulation of nitric oxide (NO) (nitrite in μM) in activated J774 macrophage cells treated with JMPR-01 (3.125-50 μM) by the Griess method. Data representes mean standard error of the mean ± SEMs; n = 6 determinations per group. * Significantly different from the vehicle treated cultures stimulated with LPS + IFN-γ (p < 0.05). ANOVA followed by Tukey’s multiple comparison test.
Pharmaceutics 14 00188 g002
Pharmaceutics 14 00188 g003 550
Figure 3. Inhibition of cytokines TNF-α (A) and IL-1β (B) in activated J774 macrophage cells treated with JMPR-01 (3.125–50 μM) by enzyme-linked immunosorbent assay (ELISA). Data representes mean standard error of the mean ± SEMs; n = 4 for TNF-α and n = 6 IL-1β determinations per group. * Significantly different from the vehicle treated cultures stimulated with LPS + IFN-γ (p < 0.05). ANOVA followed by Tukey´s multiple comparison test.
Figure 3. Inhibition of cytokines TNF-α (A) and IL-1β (B) in activated J774 macrophage cells treated with JMPR-01 (3.125–50 μM) by enzyme-linked immunosorbent assay (ELISA). Data representes mean standard error of the mean ± SEMs; n = 4 for TNF-α and n = 6 IL-1β determinations per group. * Significantly different from the vehicle treated cultures stimulated with LPS + IFN-γ (p < 0.05). ANOVA followed by Tukey´s multiple comparison test.
Pharmaceutics 14 00188 g003
Pharmaceutics 14 00188 g004 550
Figure 4. Influence of JMPR-01 (5, 10 and 50 mg/kg) treatment on total leukocyte count in the peritonitis induced by zymosan. Data are expressed as means ± SEMs; n = 6 mice per group. Two-way ANOVA followed by the Bonferroni’s test. **** (p < 0.0001) significantly different from the control group.
Figure 4. Influence of JMPR-01 (5, 10 and 50 mg/kg) treatment on total leukocyte count in the peritonitis induced by zymosan. Data are expressed as means ± SEMs; n = 6 mice per group. Two-way ANOVA followed by the Bonferroni’s test. **** (p < 0.0001) significantly different from the control group.
Pharmaceutics 14 00188 g004
Pharmaceutics 14 00188 g005 550
Figure 5. Influence of JMPR-01 (50 and 100 mg/kg) in the CFA-induced paw edema. Paw edema was measured 2, 4, 6 and 24 h after CFA, represented as paw volume variation. Data are expressed as means ± SEMs; n = 6 mice per group. Two-way ANOVA followed by the Bonferroni’s test. * (p < 0.05), ** (p < 0.01) and *** (p < 0.001) significantly different from the control group.
Figure 5. Influence of JMPR-01 (50 and 100 mg/kg) in the CFA-induced paw edema. Paw edema was measured 2, 4, 6 and 24 h after CFA, represented as paw volume variation. Data are expressed as means ± SEMs; n = 6 mice per group. Two-way ANOVA followed by the Bonferroni’s test. * (p < 0.05), ** (p < 0.01) and *** (p < 0.001) significantly different from the control group.
Pharmaceutics 14 00188 g005
Pharmaceutics 14 00188 g006 550
Figure 6. Representations of the poses assumed by JMPR-01 at the target binding site: (A) Inducible nitric oxide synthase (PDB ID: 3E7G), (B) Phosphodiesterase 4B (PDB ID: 1XMU), (C) Leukotriene A4 hydrolase (PDB ID: 1HS6), (D) 5-Lipoxygenase (PDB ID: 6NCF) and (E) Cyclooxygenase-2 (PDB ID: 3LN1).
Figure 6. Representations of the poses assumed by JMPR-01 at the target binding site: (A) Inducible nitric oxide synthase (PDB ID: 3E7G), (B) Phosphodiesterase 4B (PDB ID: 1XMU), (C) Leukotriene A4 hydrolase (PDB ID: 1HS6), (D) 5-Lipoxygenase (PDB ID: 6NCF) and (E) Cyclooxygenase-2 (PDB ID: 3LN1).
Pharmaceutics 14 00188 g006
Pharmaceutics 14 00188 g007 550
Figure 7. Ligand AR-C95791 (A) and JMPR-01 (B) in iNOS target (PDB ID: 3E7G).
Figure 7. Ligand AR-C95791 (A) and JMPR-01 (B) in iNOS target (PDB ID: 3E7G).
Pharmaceutics 14 00188 g007
Pharmaceutics 14 00188 g008 550
Figure 8. Ligand Roflumilast (A) and JMPR-01 (B) in PDE4B target (PDB ID: 1XMU).
Figure 8. Ligand Roflumilast (A) and JMPR-01 (B) in PDE4B target (PDB ID: 1XMU).
Pharmaceutics 14 00188 g008
Pharmaceutics 14 00188 g009 550
Figure 9. Ligand Celecoxib (A) and JMPR-01 (B) in COX-2 target (PDB ID: 3LN1).
Figure 9. Ligand Celecoxib (A) and JMPR-01 (B) in COX-2 target (PDB ID: 3LN1).
Pharmaceutics 14 00188 g009
Pharmaceutics 14 00188 g010 550
Figure 10. Ligand AKBA (A) and JMPR-01 (B) in 5-LOX target (PDB ID: 6NCF).
Figure 10. Ligand AKBA (A) and JMPR-01 (B) in 5-LOX target (PDB ID: 6NCF).
Pharmaceutics 14 00188 g010
Pharmaceutics 14 00188 g011 550
Figure 11. Ligand Bestatin (A) and JMPR-01 (B) in LTA4 Hidrolase target (PDB ID: 1HS6).
Figure 11. Ligand Bestatin (A) and JMPR-01 (B) in LTA4 Hidrolase target (PDB ID: 1HS6).
Pharmaceutics 14 00188 g011
Table
Table 1. Physicochemical characteristic of the phenylacrylamide derivative JMPR-01.
Table 1. Physicochemical characteristic of the phenylacrylamide derivative JMPR-01.
MoleculeAppearenceMM (g/mol)Yield (%)Rf 1FM *Log (P)
JMPR-01Gray powder248.0972.370.60 1C16H12N2O2.43
* MF = Molecular formula; 1 Rf: hexane/AcOEt 7:3 system.
Table
Table 2. Binding free energy and inhibition constant values of target-ligand complexes.
Table 2. Binding free energy and inhibition constant values of target-ligand complexes.
Molecular TargetsBindersFree Energy of Biding (Kcal/mol)Inhibition Constant
(nM) 1 (μM) 2		RMSD (Å)
iNOSAR-C95791
JMPR-01		−8.12
−8.67		1.12 2
440.93 1		0.77
PDE 4BRoflumilast
JMPR-01		−7.7
−8.7		2.25 2
417.13 1		0.25
COX-2Celecoxib
JMPR-01		−11.18
−8.98		6.41 1
262.99 1		0.93
5-LOXAKBA
JMPR-01		−10.28
−8.31		29.3 1
816.3 1		1.71
LT-A4-HBestatin
JMPR-01		−9.44
−9.1		120.44 1
213.16 1		0.85
Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Share and Cite

MDPI and ACS Style

da Silva, P.R.; do Espírito Santo, R.F.; Melo, C.d.O.; Pachú Cavalcante, F.E.; Costa, T.B.; Barbosa, Y.V.; e Silva, Y.M.S.d.M.; de Sousa, N.F.; Villarreal, C.F.; de Moura, R.O.; et al. The Compound (E)-2-Cyano-N,3-diphenylacrylamide (JMPR-01): A Potential Drug for Treatment of Inflammatory Diseases. Pharmaceutics 2022, 14, 188. https://doi.org/10.3390/pharmaceutics14010188

AMA Style

da Silva PR, do Espírito Santo RF, Melo CdO, Pachú Cavalcante FE, Costa TB, Barbosa YV, e Silva YMSdM, de Sousa NF, Villarreal CF, de Moura RO, et al. The Compound (E)-2-Cyano-N,3-diphenylacrylamide (JMPR-01): A Potential Drug for Treatment of Inflammatory Diseases. Pharmaceutics. 2022; 14(1):188. https://doi.org/10.3390/pharmaceutics14010188

Chicago/Turabian Style

da Silva, Pablo Rayff, Renan Fernandes do Espírito Santo, Camila de Oliveira Melo, Fábio Emanuel Pachú Cavalcante, Thássia Borges Costa, Yasmim Vilarim Barbosa, Yvnni M. S. de Medeiros e Silva, Natália Ferreira de Sousa, Cristiane Flora Villarreal, Ricardo Olímpio de Moura, and et al. 2022. "The Compound (E)-2-Cyano-N,3-diphenylacrylamide (JMPR-01): A Potential Drug for Treatment of Inflammatory Diseases" Pharmaceutics 14, no. 1: 188. https://doi.org/10.3390/pharmaceutics14010188

Note that from the first issue of 2016, this journal uses article numbers instead of page numbers. See further details here.

Article Metrics

Back to TopTop