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. 2019 Sep 12;10(11):1916–1925. doi: 10.1039/c9md00276f

Evaluation of anti-inflammatory activity and molecular docking study of new aza-bicyclic isoxazoline acylhydrazone derivatives†

Fernanda Virginia Barreto Mota a, Marlene Saraiva de Araújo Neta b, Eryvelton de Souza Franco c, Isla Vanessa Gomes Alves Bastos a, Larissa Cardoso Correia da Araújo a, Sandra Cabral da Silva a, Tatiane Bezerra de Oliveira a, Eduarda Karynne Souza b, Valderes Moraes de Almeida b, Rafael Matos Ximenes a, Maria Bernadete de Sousa Maia c, Francisco Jaime Bezerra Mendonça Junior d, Pascal Marchand e, Antônio Rodolfo de Faria b,✉, Teresinha Gonçalves da Silva a,✉
PMCID: PMC6977463  PMID: 32133104

graphic file with name c9md00276f-ga.jpgThe aim of this study was to investigate the anti-inflammatory effects of two new isoxazoline-acylhydrazone derivatives.

Abstract

The aim of this study was to investigate the anti-inflammatory effects of two new isoxazoline-acylhydrazone derivatives: N′-(4-methoxybenzylidene)-6-(4-nitro-benzoyl)-3a,5,6,6a-tetrahydro-4H-pyrrolo[3,2-d]isoxazole-3-carbohydrazide (R-123) and N′-(4-chlorobenzylidene)-6-(4-chlorobenzoyl)-3a,5,6,6a-tetrahydro-4H-pyrrolo[3,2-d]isoxazole-3-carbohydrazide (R-99). An air pouch induced by carrageenan was used for screening the best dose of R-99 and R-123. Using this mouse model, leukocyte migration and cytokine levels (TNF-α and IL-1β) were determined. Paw edema induced by several phlogistic agents and vascular permeability induced by acetic acid were employed to investigate the mechanism of action of the isoxazoline-acylhydrazone derivatives. A docking study was performed with the human histamine H1 receptor to investigate potential antihistaminic activity. Treatment with the compounds reduced leukocyte migration in the air pouch at all doses tested. TNF-α and IL-1β levels were similarly reduced by the two compounds. Vasoactive amines were inhibited in models of paw edema induced by several agents and vascular permeability induced by acetic acid. The docking study suggests that R-99 and R-123 may be inhibitors of the histamine H1 receptor. In conclusion, the results indicate that R-99 and R-123 exhibit promising anti-inflammatory activity related to their ability to inhibit TNF-α, IL-1β, and vasoactive amine production, as well as reduce leukocyte migration and inhibit mast cell degranulation.

1. Introduction

Inflammation is a protective and complex immune response mounted by the evolutionarily conserved innate immune system in response to harmful stimuli, such as pathogens, ischemia, and toxins or autoimmune injuries, dead cells or irritants, and is tightly regulated by the host.1,2 It plays a central role in cardiovascular and rheumatic diseases, and other inflammatory conditions.3 Insufficient inflammation can lead to persistent infection by pathogens, while excessive inflammation can cause chronic or systemic inflammatory diseases, like cancer, cardiovascular and rheumatic diseases among others. Inflammation is mediated by a variety of soluble factors, including a group of secreted polypeptides known as cytokines. Inflammatory cytokines can be divided into two groups: those involved in acute inflammation and those responsible for chronic inflammation. Some cytokines can have either pro- (Th1) or anti-inflammatory (Th2) actions according to the cellular microenvironment. Among proinflammatory cytokines, interleukins (IL) 1, 2, 6, 7, and TNF (tumor necrosis factor) are noteworthy. Anti-inflammatory cytokines include IL-4, IL-10, IL-13, and TGFβ (transforming growth factor).4,5

The controlled production of proinflammatory cytokines, such as interleukins (ILs) and TNF-α, triggers beneficial inflammatory responses that promote local coagulation to confine infection and tissue damage.5 However, when these pro-inflammatory cytokines are released in excess, they contribute to exacerbation of the inflammation. The regulation of the inflammatory process is essential to maintain or restore homeostasis in damaged biological compartments, and alteration of this regulation is associated with different human diseases.6,7

Various steroidal and non-steroidal anti-inflammatory drugs (NSAIDs) are under current clinical usage for the treatment of inflammation. Non-steroidal anti-inflammatory drugs are the oldest and most widely used medicines. However, their untoward effects, especially gastrointestinal toxicity, remain the main obstacle to their application. Major protective mechanisms of the gastrointestinal system are suppressed and deregulated by NSAIDs because of their mechanism of action, cyclooxygenase (COX) inhibition, in combination with the weakly acidic character of most of them.8 NSAIDs are associated with major side effects of gastrointestinal disorders like dyspepsia, gastric ulcers, due to a decrease in the production of prostaglandins in the tissues, which limit their clinical application.9 Efforts, aiming to the development of safe non-steroidal anti-inflammatory drugs are evolving, however there are still several problems concerning gastroprotection and cardiovascular side effects to be efficiently solved, thus the development of effective and safe agents for the treatment of inflammatory conditions remains a great challenge.

Molecules with pentagonal rings containing two heteroatoms, such as nitrogen and oxygen, in their structures play an important role in the synthesis of new drugs, and can exhibit different activities due to the heteroatom that causes changes to the structural and electronic properties of the molecule.10 Among the heterocyclic systems, isoxazolines can be highlighted due to their numerous biological activities, such as antibacterial,11 antifungal,12 antioxidant,13 antithrombotic,14 anticancer,15 hypoglycemic,16 antiasthmatic,17,18 anti-inflammatory,19,20 and antinociceptive21,22 activities. Isoxazolines are an important class of nitrogen- and oxygen-containing heterocycles, which belong to the azole family, known in the field of medicinal chemistry as anti-inflammatory and antinociceptive agents.23–26

Besides the isoxazoline ring, the compounds studied in this work have acylhydrazone moiety. Its functionality has been widely studied for having analgesic, anti-inflammatory, and antithrombotic properties.27 The mechanisms of action of acylhydrazone compounds may involve inhibition of proinflammatory enzymes such as cyclooxygenase (COX) and 5-lipoxygenase (5-LOX),28,29 while isoxazoline derivatives inhibit the formation of prostaglandins.30

Therefore, this study aimed to evaluate the anti-inflammatory activity of two isoxazoline-acylhydrazone molecules N′-(4-methoxybenzylidene)-6-(4-nitro-benzoyl)-3a,5,6,6a tetrahydro-4H-pyrrolo[3,2-d]isoxazole-3-carbohydrazide (R-123) and N′-(4-chlorobenzylidene)-6-(4-chlorobenzoyl)-3a,5,6,6a-tetrahydro-4H-pyrrolo[3,2-d]isoxazole-3-carbohydrazide (R-99) (Fig. 1), and to corroborate the potential antihistaminic results in vivo using molecular docking to the human histamine H1 receptor.

Fig. 1. Synthesis of the isoxazoline acylhydrazone derivatives R-99 and R-123.

Fig. 1

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2. Material and methods

2.1. Animals

Male Swiss mice provided by the Animal Facility of the Antibiotics Department of the Federal University of Pernambuco (UFPE), Recife, Brazil, were used for the evaluation of the anti-inflammatory activity of the compounds. The mice, weighing between 25–30 g, were kept in a room at a temperature of 23 ± 2 °C, with 12/12 h cycles of light and darkness and access to standard feed (Purina®) and water ad libitum. Before initiation of the experiments, the animals were acclimated to the laboratory environment for at least 60 min. All animals used in the determination of anti-inflammatory activity were maintained under fast for 8 h prior to experimentation. The Animal Studies Committee of the Federal University of Pernambuco approved the experimental protocols (no. 23076.018987/2012-82). The animals were treated according to the ethical principles of animal experimentation of the Brazilian Society of Laboratory Animal Science (SBCAL) and the norms of the National Institute of Health Guide for the Care and Use of Laboratory Animals.

2.2. Drugs and chemicals

Morphine was purchased from the União Química Farmacêutica Nacional (Minas Gerais, Brazil), ketamine and xylazine were purchased from Rhobifarma Indústria Farmacêutica Ltda (São Paulo, Brazil), glacial acetic acid was purchased from VETEC (São Paulo, Brazil), heparin was purchased from Cristália (São Paulo, Brazil) and carrageenan, cyproheptadine, histamine, dextran, serotonin hydrochloride, compound 48/80, bradykinin, Griess' reagent, EDTA, Evans blue, dexamethasone and indomethacin were purchased from Sigma. Bothropstoxin-II was kindly donated by Prof. Dr. Marcos H. Toyama from UNESP (São Vicente, Brazil). Mouse anti-TNF-α, mouse anti-IL-1β ELISA kits were purchased from eBioscience (San Diego, California, USA). All drugs were dissolved in saline, except for R-99 and R-123, which were dissolved in saline containing 5% DMSO. The doses used in this study were determined according to pilot tests.

2.3. Chemical procedure

The racemic isoxazoline acylhydrazones R-99 and R-123 were synthesized in two steps from the previously obtained aza-bicyclic isoxazoline esters 1a–b (Fig. 1).31 Briefly, isoxazoline hydrazides 2a–b were synthesized from the respective esters with 80% hydrazine. Subsequently, the isoxazoline acylhydrazones R-99 and R-123 were obtained by condensation of the hydrazides with the respective aromatic aldehydes.22 The structures of new isoxazoline acylhydrazones R-99 and R-123 were fully elucidated by 1H and 13C NMR, FTIR, and HRMS. Although NMR spectra at room temperature are complex, due to the presence of rotamers, some diagnostic signals, that confirm the aza-bicyclic isoxazoline and hydrazone, are present and clear in 1H NMR spectra, such as a broad signal at 6.26 ppm (6.72 rotamer) for H6a (R-99) and a broad doublet at 6.18 ppm (6.71 rotamer) for H6a (R-123). The signals that confirm the hydrazone function appear at 12.21 ppm (R-99) and 11.99 ppm (R-123) for N–H hydrogens, and 8.43 ppm (R-99) and 8.39 ppm (R-123) for methyne hydrogens (N Created by potrace 1.16, written by Peter Selinger 2001-2019 CH). The signals of the respective carbon C6a in 13C NMR spectra appear at 94.9 ppm (92.3 rotamer) for R-99 and 94.3 ppm (92.1 rotamer) for R-123. The other signals of hydrogens and carbons of the aza-bicyclic isoxazoline, aromatic rings, carbonyls and imine moieties appear at expected chemical shifts and are designated in detail in the general procedure for synthesis. Some of these signals, close to benzoyl amide, appear duplicate as rotamers.

The rotamer signals, due to the high rotational barrier of the amide bonds of the molecules R-99 and R-123 (benzoyl amides in this case), undergo coalescence when NMR spectra are obtained at high temperature (see example in ESI†). This phenomenon was verified in all N-benzoyl-aza-bicyclic 2-isoxazoline derivatives previously described.22,31 Another peculiarity of the 13C NMR and 1H NMR spectra was the low signal resolution of the bicyclic ring carbons and exocyclic hydrogens of isoxazolines, resulting in broad signals, probably due to the high ring tension of aza-bicyclic isoxazolines.

2.4. General experimental procedures

All melting points were determined using a POLAX WRS-1 apparatus and are uncorrected. FTIR spectra were obtained with a Bruker IFS 66 spectrometer using KBr pellets. NMR spectra were recorded on a Varian Unity 300 spectrometer (1H NMR at 300 MHz and 13C NMR at 75 MHz) or Bruker Avance III (1H NMR at 600 MHz); the remnant signal of solvent or TMS was used as an internal reference. Chemical shift (δ) values are expressed in parts per million (ppm) and coupling constant (J) values are given in Hz. Abbreviations: s = singlet; d = doublet; t = triplet; m = multiplet; br = broad. Thin-layer chromatography (TLC) was carried out on silica gel plates with a fluorescence indicator of F254 (0.2 mm, E. Merck). HRMS was performed with a Shimadzu LC-MS-IT-TOF spectrometer. All reagents used in the present study were of analytical grade and were purchased from Aldrich or Fluka (both: São Paulo, Brazil) and were used without additional purification.

2.4.1. General procedure for synthesis of hydrazides 2a–b

An 80% hydrazine solution (10.6 mL) was added dropwise to a solution of isoxazoline ester 1a–b (3.43 g, 10.64 mmol) in 30 mL of absolute ethanol. The reaction mixture was stirred at room temperature for about 2 h, when the end of reaction was checked by TLC. After removal of the solvent, crude solid hydrazides 2a–b were obtained. Recrystallization from ethanol provided pure hydrazides 2a–b with good yields.

Spectra data for hydrazides: (2a) yield 87%, mp 148–150 °C, 1H NMR (DMSO-d6, 300 MHz): 2.18 (br s, 2H), 3.05 and 3.49 (br s, 1H, rotamer), 4.11 (br s, 1H), 4.25 (m, 1H), 4.51 (br s, 2H), 6.16 and 6.63 (br s, 1H, rotamer), 7.57 (br s, 4H), 9.82 (s, 1H). 13C NMR (DMSO-d6, 75 MHz): 26.7 and 27.5 (CH2, rotamer), 43.5 and 47.4 (CH; rotamer), 50.4 and 52.3 (CH2, rotamer), 91.5 and 94.1 (CH, rotamer), 128.4 (CH), 129.5 (CH), 134.2 (C), 135.3 (C), 154.5 (C Created by potrace 1.16, written by Peter Selinger 2001-2019 N), 157.9 (C Created by potrace 1.16, written by Peter Selinger 2001-2019 O), 167.9 (C Created by potrace 1.16, written by Peter Selinger 2001-2019 O). FTIR (KBr) main bands: 3325, 3277, 2992, 2966, 2901, 1678, 1655, 1589, 1405, 1273, 1021, 931, 837, 760, 601.

(2b) yield 82%, mp 175–177 °C, 1H NMR (DMSO-d6, 300 MHz): 2.21 (br s, 2H), 3.08 and 3.42 (m, 1H; rotamer), 4.11 (br s, 1H), 4.27 (br s, 1H), 4.55 (br s, 2H), 6.10 and 6.63 (d, J = 7.8 Hz, 1H, rotamer), 7.82 (m, 2H), 8.34 (m, 2H), 9.88 (s, 1H). 13C NMR (DMSO-d6, 75 MHz): 26.6 and 28.1 (CH2, rotamer), 43.6 and 47.1 (CH), 50.6 and 52.4 (CH2, rotamer), 91.4 and 93.8 (CH, rotamer), 123.7 (CH), 129.0 (CH), 141.4 (C), 148.4 (C), 154.5 (C Created by potrace 1.16, written by Peter Selinger 2001-2019 N), 157.8 (C Created by potrace 1.16, written by Peter Selinger 2001-2019 O), 167.3 (C Created by potrace 1.16, written by Peter Selinger 2001-2019 O); FTIR (KBr) main bands: 3334, 3286, 3190, 2957, 2284, 1662, 1598, 1398, 1351, 1112, 922, 833, 724, 701.

2.4.2. General procedure for synthesis of the hydrazones R-99 and R-123

To a solution of 4.31 mmol of the hydrazide 2a–b were added 40 mL of absolute ethanol, two drops of concentrated HCl and 4.52 mmol of the respective aromatic aldehyde (p-chloro-benzaldehyde for R-99 and p-nitro-benzaldehyde for R-123). The reaction mixture was stirred for about 30 min, when extensive precipitation occurred. The reaction mixture was placed in an ice bath and neutralized with 10% aqueous NaHCO3 solution. The precipitate was filtered and dried in a drying oven at 40 °C. Recrystallization from methanol afforded the isolation of pure isoxazoline acylhydrazone derivatives.

N′-(4-Chlorobenzylidene)-6-(4-chlorobenzoyl)-3a,5,6,6a-tetrahydro-4H-pyrrolo[3,2-d]isoxazole-3-carbohydrazide R-99; yield 83%, mp 215 °C carbonization, 1H NMR (DMSO-d6, 300 MHz): 2.26 (m, 2H), 3.08 and 3.34 (m, 1H, rotamer), 3.52 and 4.14 (br m, 1H rotamer), 4.33 (t, J = 9 Hz, 1H), 6.25 and 6.72 (br s, 1H, rotamer), 7.51–7.73 (br m; 8H), 8.43 (s, 1H), 12.21 (s, 1H). 13C NMR (DMSO-d6, 75 MHz): 26.7 and 28.2 (CH2, rotamer), 43.7 and 47.4 (CH, rotamer), 50.0 and 51.9 (CH2, rotamer), 92.3 and 94.9 (CH, rotamer), 128.5 (CH), 128.9 (CH), 129.5 (CH) 132.9 (C), 134.1 (C), 134.9 (C), 135.4 (C), 148.5 (N Created by potrace 1.16, written by Peter Selinger 2001-2019 CH), 154.6 (C Created by potrace 1.16, written by Peter Selinger 2001-2019 O), 155.4 (C Created by potrace 1.16, written by Peter Selinger 2001-2019 N), 168.9 (C Created by potrace 1.16, written by Peter Selinger 2001-2019 O). FTIR (KBr): main bands: 3266, 3009, 2947, 2885, 1669, 1598, 1543, 1412, 1264, 1088, 1021, 931, 835, 751. HRMS (IT-TOF-ES): calcd for [C20H17Cl2N4O3]+ 431.0677, found 431.0700.

N′-(4-Methoxybenzylidene)-6-(4-nitrobenzoyl)-3a,5,6,6a tetrahydro-4H-pyrrolo[3,2-d]isoxazole-3-carbohydrazide R-123; yield 84%, mp 240 °C carbonization. 1H NMR (DMSO-d6, 600 MHz): 2.24 (m, 1H), 2.32 (m, 1H), 3.15 and 3.40 (m, 1H, rotamer), 3.80 (s, 3H), 3.43 and 4.13 (m, 1H, rotamer), 4.36 (m, 1H), 6.18 and 6.71 (br d, J = 7 Hz, 1H, rotamer), 7.01 (d, J = 8 Hz, 2H), 7.64 (d, J = 8 Hz, 2H), 7.83 (m, 2H), 8.33 (m, 2H), 8.39 (s, 1H), 11.99 (s, 1H). 13C NMR (DMSO-d6, 75 MHz): 26.6 and 28.1 (CH2, rotamer), 43.9 and 46.5 (CH, rotamer), 50.4 and 52.3 (CH2, rotamer), 55.3 (CH3), 92.1 and 94.3 (CH, rotamer), 114.4 (CH), 123.7 (CH), 126.5 (C), 128.7 (CH), 128.9 (CH), 141.4 (C), 148.4 (HC Created by potrace 1.16, written by Peter Selinger 2001-2019 N), 149.4 (C), 154.1 (C Created by potrace 1.16, written by Peter Selinger 2001-2019 O), 154.7 (C Created by potrace 1.16, written by Peter Selinger 2001-2019 N), 161.1 (C), 167.4 (C Created by potrace 1.16, written by Peter Selinger 2001-2019 O). FTIR (KBr): main bands: 3273, 3073, 2973, 2839, 1676, 1598, 1507, 1417, 1351, 1256, 1169, 1021, 922, 859, 832, 704, 616. HRMS (IT-TOF-ES): calcd for [C21H20N5O6]+ 438.1413, found 438.1509.

2.5. Anti-inflammatory activity

For the evaluation of the anti-inflammatory activity of the derivatives R-99 and R-123, three models were used: the subcutaneous air pouch model (as screening, with determination of IL-1β and TNF-α levels), paw edema induced by different phlogistic agents (carrageenan, dextran, histamine, serotonin, bradykinin, compound 48/80 and BthTX-II) and vascular permeability induced by acetic acid.

2.5.1. Carrageenan-induced subcutaneous air pouch model

The anti-inflammatory effects of the compounds were tested on the induction of inflammation by the introduction of carrageenan into air pouches in the dorsal cervical region of mice. A subcutaneous injection of 3.0 mL of sterile air on day 0 followed by a second injection of 3.0 mL of sterile air 3 days later resulted in an air pouch. On day six, the mice received, by oral route, vehicle (saline + 5% DMSO), R-99 (15, 30, 60 and 120 mg kg–1), R-123 (15, 30, 60 and 120 mg kg–1), or indomethacin (10 mg kg–1). One hour after drug administration, inflammation was induced by injecting 1 mL of 1% carrageenan in saline solution into the air pouch. After 6 h, the animals were euthanized in a CO2 chamber, and the pouches were washed with 3 mL of saline solution containing 3 μM of EDTA. White blood cells were counted using an ABX Micros 60 haematology analyzer. The average number of leukocytes from each treated group was normalized by the control group.32 The exudates were centrifuged, and the supernatant stored at –80 °C for analysis of IL-1β and TNF-α.

2.5.1.1. Measurement of cytokine levels

The concentrations of TNF-α and IL-1β were measured using sandwich ELISA kits specific for mice (eBioscience, San Diego, California, USA) according to the manufacturer's instructions. The results were expressed as pg mL–1.

2.5.2. Paw edema induced by different phlogistic agents

Paw edema was induced by injecting 50 μL of carrageenan (1%), dextran (0.2%), histamine (100 μg), 5-HT (100 μg), bradykinin (10 nmol), compound 48/80 (100 ng) or BthTX-II (50 μg) into the right hind paw of the mice. The treatment animals had received R-99 or R-123 (15 mg kg–1, p.o.) one hour before induction of edema. Indomethacin (10 mg kg–1, p.o.), cyproheptadine (10 mg kg–1, p.o.), or dexamethasone (0.5 mg kg–1, p.o.) were used as reference drugs. The control group received vehicle (saline + 5% DMSO, p.o.). Paw volume was measured immediately after induction of edema and at different times, according to the phlogistic agent. Captopril (5 mg kg–1) was used 1 h prior to bradykinin challenge in order to prevent the action of kininases.33 The paw volume was measured before introduction of the phlogistic agent and at 2, 4 and 6 h after carrageenan administration34,35 and every 60 minutes up to the 4th h after administration of dextran36 15, 30, 60 and 90 minutes after the administration of histamine,37 serotonin,38 bradykinin39 or compound 48/80;40 or 15, 30, 45 and 60 minutes after the administration of BthTX-II,41 using a plethysmometer (Ugo Basile, Italy). The results were expressed as the difference in volume (mL) of the paw that received carrageenan and the contralateral paw that received saline.

2.5.3. Acetic acid-induced vascular permeability in mice

The effects of test substances on increased vascular permeability induced by acetic acid in mice were determined according to the method described by Whittle42 (1964), with modifications. The animals received R-99 or R-123 (15 mg kg–1, p.o.). The control group received the vehicle (saline + 5% DMSO). One hour later, the animals were anesthetized with an associated solution of ketamine and xylazine (8:2, v/v, i.p.). Mice were injected with Evans blue 1% (0.2 mL per mouse) by the retro-orbital plexus. Then, 0.5 mL of acetic acid solution (1%) was administered to the peritoneal cavity. After 30 minutes, the mice were euthanized in a CO2 chamber, and the peritoneal cavity was washed with saline. 2.0 mL of exudate was collected and centrifuged at 2000 rpm for 10 min. The absorbance of the supernatant was read at 610 nm with a microplate reader.

2.6. Statistical analysis

The results were expressed as mean values ± standard deviation for each experimental group, using GraphPad Prism software (version 6.0). Statistical analysis of vascular permeability, carrageenan-induced subcutaneous air pouch model, and the measurement of IL-1β, and TNF-α levels were performed by one-way analysis of variance (ANOVA), followed by Tukey's test. The paw edema induced by various phlogistic agents was analyzed by two-way analysis of variance (ANOVA) followed by Tukey's test. With a confidence interval of 99%, p values less than 0.01 (p < 0.01) were considered as indicative of statistical significance.

2.7. Molecular docking

Molecular docking was performed as described in Scotti et al.43 (2013). Using the program Hyperchem v. 8.0.3,44 the chemical structures of the compounds (ligands) were drawn, and their geometry was optimized using MM+ force field.45 Afterwards, new geometry optimization based on the semi-empirical AM1 (Austin Model 1) method was performed.46–48 The optimized structures were subjected to conformational analyses using the software Spartan for Windows 10.0 [Spartan model homepage for Windows].49 We selected the Monte Carlo search method with 1000 interactions, 100 optimization cycles, and 10 conformers of lowest minimum energy. The dihedrals were evaluated by rotation in accordance with the standard (default) conditions of the program, in which the number of simultaneous variations was 1 to 8, acyclic chains were submitted to rotations from 60 to 180° and torsion rings were in the range of 30 to 120°. The conformers of lowest minimum energy were selected and saved in .sdf format.

The macromolecule human histamine H1 receptor in complex with doxepin (PDB id: 3RZE) was analyzed using the Virtual Molegro program 6.0 Docker. To start the docking, we created a template with the ligands from the Protein Data Bank. The directory of the compounds was then selected. The results for each calculation were analyzed to obtain the affinity binding energy (kcal mol–1) values for each ligand conformation in its respective complex; probable structure inaccuracies were ignored in the calculations. To verify the number of hydrogen bonds and non-covalent interactions between each ligand conformation and the catalytic residues of the human histamine H1 receptor enzyme, the Molegro Molecular Viewer 2.5 program was employed.43

3. Results

3.1. Carrageenan-induced air pouch

As shown in Table 1, both R-99 and R-123 presented anti-inflammatory activity indicated by a significant decrease in cell migration when compared to the control group. There was no dose-dependent effect for either compound at the doses tested.

Table 1. Effects of isoxazoline-acylhydrazone derivatives on carrageenan-induced air pouch.

Compound Dose (mg kg–1) No of PMNL/mL (×106) Inhibition (%)
Control — 13.76 ± 0.55 —
R-99 15 3.33 ± 0.80 a 76
30 4.97 ± 0.71 a 64
60 5.94 ± 0.82 a 57
120 6.10 ± 0.79 a 56
R-123 15 4.60 ± 0.65 a 67
30 6.72 ± 0.46 a 51
60 8.98 ± 0.74 a 35
120 12.72 ± 0.59 8
Indomethacin 10 4.84 ± 0.45 a 65
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a P < 0.01. Significance was determined with ANOVA one way followed by Tukey's post hoc when compared with control group. PMNL: Polymorphonuclear leucocytes.

3.1.1. Analysis of cytokine levels

The compounds R-99 and R-123 at 15 mg kg–1 (p.o.) caused a decrease in TNF-α concentration in the air pouch exudate (271.60 and 305.88 pg mL–1, respectively), when compared to the control (425.17 pg mL–1). Indomethacin presented a TNF-α concentration of 458.97 pg mL–1 (Fig. 2A).

Fig. 2. Effect of isoxazoline acylhydrazone derivatives R-99 and R-123 on levels of (A) TNF-α and (B) IL-1β in exudate of air pouch cavity. *p < 0.01. Significance was determined with one-way ANOVA followed by Tukey's post-hoc test when compared to the control group.

Fig. 2

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R-99 and R-123 derivatives (323.11 and 358.28 pg mL–1, respectively) inhibited IL-1β production compared to the control (564.06 pg mL–1). Indomethacin showed an IL-1β concentration of up to 329.81 pg mL–1 (Fig. 2B).

3.2. Carrageenan-induced paw edema

The intraplantar injection of carrageenan increased the paw volume. The animals of the groups pretreated with derivatives R-99 and R-123, or indomethacin, had reduced edema when compared to the control at all observation times (Fig. 3).

Fig. 3. Effect of isoxazoline acylhydrazone derivatives R-99 and R-123 on the paw edema induced by carrageenan in mice. *p < 0.01. Significance was determined with two-way ANOVA followed by Tukey's post-hoc test when compared with the control group.

Fig. 3

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3.3. Acetic acid-induced vascular permeability in mice

The derivatives R-99 and R-123 inhibited the increase of vascular permeability induced by acetic acid when compared to the control group. The derivatives demonstrated no significant difference between them (Fig. 4). Vascular permeability was inhibited by R-99 and R-123, and this inhibition probably involves a decrease in the release of vasoactive amines, with a consequent reduction of edema.

Fig. 4. Effect of isoxazoline acylhydrazone derivatives R-99 and R-123 on the increase of vascular permeability induced by acetic acid. *p < 0.01. Significance was determined with one-way ANOVA followed by Tukey's post-hoc test when compared with the control group.

Fig. 4

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3.4. Paw edema induced by dextran, histamine, 5-HT, bradykinin, compound 48/80 and BthTX-II

Paw edema was induced by the intraplantar injection of different phlogistic agents. In the dextran-induced paw edema, pretreatment with R-99 and cyproheptadine reduced the edema for all time periods. The R-123 inhibited the development of the edema only in the 3rd and 4th h (Fig. 5A). The groups pretreated with R-99 and R-123 derivatives inhibited the histamine-induced paw edema in the first fifteen minutes when compared to control, demonstrating that these compounds are effective in reducing the paw edema induced by histamine (Fig. 5B). The intraplantar injection of 5-HT induced the increase in paw volume. The R-123 compound reduced edema volume at all times observed when compared to control, while R-99 compound inhibited the edema 30 min after the phlogiston agent administration (Fig. 5C). Histamine and serotonin play an important role in changing the tone and vascular permeability, contributing to the extravasation of fluids. R-99 derivative showed inhibition on the histamine-induced edema but not significantly reduced the edema induced by 5-HT, whereas R-123 inhibited the edema induced by both agents. The R-99 and R-123 derivatives decreased the paw edema induced by bradykinin 15 and 30 minutes after injection of bradykinin (Fig. 5D). Dexamethasone inhibited the edema at all times measured. The intraplantar injection of compound 48/80 induced the increase in paw volume. The derivatives R-99 and R-123 decreased the paw edema 15 and 30 minutes after injection of compound 48/80 (Fig. 5E). The intraplantar injection of BthTX-II induced the increase in paw volume. R-99 and R-123 derivatives decreased the paw edema 15, 30 and 45 minutes after injection of BthTX-II (Fig. 5F).

Fig. 5. Effect of the derivatives R-99 and R-123 on paw edema in mice. A: induced by dextran; B: induced by histamine; C: induced by serotonin (5-HT); D: induced by bradykinin; E: induced by compound 48/80; F: induced by BthTX-II; *p < 0.01. Significance was determined with two-way ANOVA followed by Tukey's post-hoc test when compared with the control group.

Fig. 5

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3.5. Molecular docking to the human histamine H1 receptor

In order to evaluate whether the derivatives R-99 and R-123 could act as antihistaminic agents, since both compounds inhibited histamine edema at 15 min, docking studies were performed with the human histamine H1 receptor in complex with doxepin (PDB id: 3RZE), and the binding constant values (MolDock score) were compared with the reference drug cyproheptadine. The binding energy values of the binders in kcal mol–1, and the lower energy conformations with the histamine H1-binding site are shown in Table 2 and Fig. 6.

Table 2. Binding energies (MolDock scores in kcal mol–1) of ligands in the active site pocket of the human histamine H1 receptor (PDB id: ; 3RZE).

Ligand MolDock score (kcal mol–1)
R-123 –100.316
R-99 –99.8097
Cyproheptadine –69.7195
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Fig. 6. 3D (above) and 2D (below) representations of ligands binding to the active site of the human histamine H1 receptor (PDB id: 3RZE). A: R-123; B: R-99; C: cyproheptadine. Dashed lines represent the steric interactions which stabilize the complexes.

Fig. 6

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Docking analysis of the ligands into the histamine H1-binding site revealed that the isoxazoline-acylhydrazone derivatives R-99 and R-123 bind better than the reference drug cyproheptadine into the active site pocket. This can be seen from the lower formation values of the ligand-macromolecule complexes (Table 2).

4. Discussion

Isoxazoline derivatives are an important class of anti-inflammatory agents. Habeeb et al.30 showed that isoxazolines exhibit a potent and selective inhibitory activity on COX-2 and analgesic activity. On the other hand, Hernández et al.29 showed that acylhydrazone derivatives inhibit the production of NFκB and IL-8 in vitro, and LOX, COX-1, COX-2 and IL-8 in in vivo assays. In this work, we used as synthesis strategy the molecular hybridization to design molecules containing the oxazoline nucleus and the acylhydrazone function.

Since these substances (R-99 and R-123) are novel, leukocyte migration induced by carrageenan into the dorsal air pouch was chosen as the initial test, because it is an important test to study acute inflammation. This model mimics rheumatoid arthritis and is usually used as a screening test to select the best dose or to select drugs with anti-inflammatory and anti-arthritic activity. The subcutaneous injection of carrageenan into the air pouch induces a rapid inflammatory response characterized by high levels of prostaglandins and leukotrienes. Carrageenan is widely used to induce neutrophil migration due to the release of various inflammatory mediators, such as histamine, serotonin, kinins, prostaglandins, and thromboxanes.50 Both compounds significantly inhibited cell migration at all doses tested (15, 30, 60, and 120 mg kg–1). The effects observed were not dose-dependent, having been observed a decrease in activity at the highest dose. This effect can be attributed, at least in part, to the low solubility of the compounds, however, this was a limitation of the study. According to theoretical data calculated through the free software Swissadme, R-99 has presented poor solubility (log S = –5.33 and log P = 3.24; while R-123 showed moderate solubility (log S = –4.98 and log P = 1.63) (; www.swissadme.ch).51 Despite corroborating the biological results found, these data are only predictive and further pharmacokinetic studies are needed.

There are multiple cell types with direct roles in the pathogenesis of inflammation, including macrophages, epithelial, and endothelial cells. However, neutrophils are well established in the literature as playing a central role in driving the inflammatory state.52 Therefore, one of the parameters observed in our study was leukocyte (mainly neutrophil) migration.

The subsequent cell migration during the innate and adaptive immune response is orchestrated and controlled by cytokines and chemokines.53 Therefore, one of our objectives was to measure concentrations of the cytokines TNF-α and IL-1β in the exudate removed from the air pouches of animals treated with R-99 and R-123. These compounds inhibited the two major proinflammatory cytokines, TNFα and IL-1β. TNF-α is a potent activator of neutrophils, mediating adhesion, chemotaxis, and degranulation. This cytokine induces the production of autacoids in endothelial cells, including prostanoids, PAF, and nitric oxide.54 IL-1β is produced by neutrophils and monocytes/macrophages and acts as an important trigger for leukocyte migration,55 besides acting synergistically with TNF-α to stimulate COX-2 expression resulting in production and release of prostanoids.56–59 The results found in our study for R-99 and R-123 suggest that the reduction in pro-inflammatory cytokines decreases cell migration, and consequently reduces inflammation.

In the carrageenan-induced paw edema test, the 15 mg kg–1 dose was chosen because it was the lowest dose that showed the greatest inhibition of cell migration, as the lower the dose, the lower the probability of adverse effects. The derivatives R-99 and R-123 reduced the volume of edema and there was no statistically significant difference between them at the times of 2, 4 and 6 h. The development of edema induced by carrageenan is usually associated with the exudative phase at the onset of inflammation. The paw edema induced by carrageenan in mice is carried out in two phases: a 1st phase (0–24 h) and 2nd phase (after 24 h). During the first phase, the predominant cells are neutrophils and they are potentially capable of releasing inflammatory agents, such as histamine, serotonin, kinins, and prostaglandins (Henriques et al., 1987),35 while the second phase is related to the release of prostaglandin and kinins.59,60 So, the effect of the R-99 and R-123 against inflammation produced by these individual mediators (histamine, serotonin and bradykinin) or other agents (dextran, compound 48/80 and BthTX-II) was studied. The compounds R-99 and R-123 effectively reduced edema induced by dextran, histamine, serotonin, bradykinin, compound 48/80 and BthTX-II, but at different times (Fig. 5).

The inflammatory response is a physiological characteristic of vascularized tissues.61 Exudation, which is a consequence of increased vascular permeability, is considered a major feature of acute inflammation. In the inflammatory response, increased vascular permeability leads to exudation of fluid rich in plasma proteins including immunoglobulins, coagulation factors, and cells into the injured tissues, with subsequent edema formation in the injured tissue.62 Therefore, inhibition of increased vascular permeability can modulate the extent and magnitude of the inflammatory reaction.63 Chemically induced vascular permeability causes an immediate sustained reaction that is prolonged over 24 h and its inhibition by R-99 and R-123 suggests that they effectively suppress the exudative phase of acute inflammation, confirming the data obtained in carrageenan-induced paw edema.

The paw edema induced by dextran involves mast cell degranulation, and consequently the release of histamine and serotonin, which contribute to increased vascular permeability and extravasation of fluid.36,64 The inhibition of paw edema demonstrated by R-99 and R-123 is probably associated with interference in the synthesis/release or blocking of histamine and 5-HT, since both compounds inhibited the edema induced by these mediators.

The R-99 and R-123 derivatives significantly inhibited carrageenan- and dextran-induced paw edemas in mice. The paw edema induced by dextran, also mediated by histamine and serotonin, does not involve leukocyte migration.65 This suggests that both compounds act on vasoactive amines and other mediators, such as cytokines.

It is known that kinins are involved in many physiological processes such as the control of blood pressure, contraction or relaxation of smooth muscle, increased vascular permeability, stimulation of sensory neurons, and release of several pro-inflammatory substances such as prostanoids, neuropeptides, cytokines, and NO.66 Bradykinin may play an important physiological role as a mediator of inflammation, inflammatory pain and hyperalgesia, and exerts various effects such as release of several inflammation mediators like prostanoids in many cell types, cytokines (IL-1β and TNF-α) in macrophages, and NO release from vascular endothelial cells.67,68 Both compounds inhibit the edema induced by bradykinin.

Compound 48/80 is a polymer produced by the condensation of p-methoxy-N-methyl phenylethylamine with formaldehyde and widely used for non-IgE-dependent stimulation of mast cells, causing massive mast cell degranulation.69 Mast cell degranulation results in the release of histamine and serotonin, which contributes to increased capillary permeability and the extravasation of fluid, culminating in the formation of edema. In the paw edema model induced by compound 48/80, the derivatives R-99 and R-123 reduced the edema, corroborating the findings of the dextran-, histamine-, and serotonin-induced paw edemas.

Phospholipases are enzymes that hydrolyze phospholipids into fatty acids and other lipophilic substances. Viperidae snake venoms contain group II phospholipase A2 (PLA2), which shares structural characteristics with the secretory phospholipase A2 (PLA2s) present in inflammatory exudates of mammals.70 Phospholipases trigger an inflammatory response with the formation of edema, infiltration of inflammatory cells, and activation of mast cells.71 Bothropstoxin (BthTX-II) is a catalytically active Asp49 PLA2 homolog isolated from the venom of Bothrops jararacussu. The intraplantar injection of BthTX-II induced an increase in paw volume. The compounds R-99 and R-123 decreased the paw edema 15, 30 and 45 minutes after injection of BthTX-II (Fig. 5F). The present study showed that BthTX-II induces paw edema with a peak 15 min after its injection. This result corroborates previous studies showing the capacity of Bothrops venom to induce edema.72–74 In the paw edema model induced by BthTX-II, R-99 and R-123 significantly reduced the edema and inflammation, probably due to at least the partial inhibition of mast cells, kininogens and phospholipids. Our data corroborate also the findings of Basappa et al.23 and Park et al.,19 who demonstrated that isoxazolines may be potent phospholipase A2 (PLA2) inhibitors, presenting a good enzyme inhibitory response in vitro and in vivo in a paw edema model.

Since the derivatives R-99 and R-123 inhibited histamine-induced edema, and the literature reports the effects of acylhydrazone derivatives on allergic inflammation, we hypothesized that these compounds may act on histamine H1 receptors. In docking studies with H1 receptors, the analysis of the interactions with residues at the active side also revealed that isoxazoline-acylhydrazone derivatives form larger numbers of steric interactions (dashed lines) than cyproheptadine (Fig. 6). No hydrogen bonds or electrostatic interactions are observed in the stabilization of such complexes. The main residues involved in the isoxazoline-acylhydrazone derivatives host-guest complexes were Ala211, Lys215, Arg218, and Met421. Additionally, interactions were observed between R-123 and the apolar residue Gly418; and between R-99 with the polar residues Try210 and Try214, and the basic residue Lys212. Finally, formation of the cyproheptadine complex was stabilized by the interactions with the residues Try210, Ala 211, and Try214.

Conclusions

The evaluated isoxazoline-acylhydrazone derivatives R-99 and R-123 exhibit promising anti-inflammatory properties. This activity involves decreased cell migration, reduction on TNF-α and IL-1-β levels, and probably inhibition of the release of vasoactive amines, kinins, phospholipase A2 and mast cell degranulation. The molecular docking studies confirmed the antihistaminic effects observed in vivo.

Author contributions

R. M. X. and M. B. S. M. and E. S. F. reviewed the draft manuscript. F. J. B. M. J. performed the docking study. F. V. B. M., T. B. O., L. C. C. A., S. C. S. and I. V. B. G. A. B. performed the biological evaluation and participated in the analysis and interpretation of the data. M. S. A. N., V. M. A., E. K. S. performed the research in relation to the synthesis and characterization of novel compounds and interpretation of the data. T. S., P. M. and A. R. F. coordinated the research and drafted the manuscript. All the authors read and approved the final manuscript.

Conflicts of interest

The authors have no conflict of interests in this paper.

Supplementary Material

Click here for additional data file. (12.2MB, pdf)

Acknowledgments

This work was supported by the Fundação de Amparo à Ciência e Tecnologia de Pernambuco (FACEPE) and Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) (470901/2014-4). The authors would like to acknowledge Capes/Cofecub Program no. 865-15 for financial support.

Footnotes

†Electronic supplementary information (ESI) available. See DOI: 10.1039/c9md00276f

References

  1. Széles L., Töröcsik D., Nagy L. Biochim. Biophys. Acta. 2017;1771:1014–1030. doi: 10.1016/j.bbalip.2007.02.005. [DOI] [PubMed] [Google Scholar]
  2. Guo H., Callaway J. B., Ting J. P. Nat. Med. 2015;21:677–687. doi: 10.1038/nm.3893. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Hu C., Ma S. MedChemComm. 2017;9:212–225. doi: 10.1039/c7md00390k. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Zhang J. M., An J. Int. Anesthesiol. Clin. 2007;45:27–37. doi: 10.1097/AIA.0b013e318034194e. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Ulloa L., Tracey K. J. Trends Mol. Med. 2005;2:56–63. doi: 10.1016/j.molmed.2004.12.007. [DOI] [PubMed] [Google Scholar]
  6. Wu Y., Antony S., Meitzler J. L., Doroshow J. H. Cancer Lett. 2014;345:164–173. doi: 10.1016/j.canlet.2013.08.014. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Okin D., Medzhitov R. Curr. Biol. 2012;22:R733–R740. doi: 10.1016/j.cub.2012.07.029. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Tziona P., Theodosis-Nobelos P., Rekka E. A. Med. Chem. 2017;13:408–420. doi: 10.2174/1573406413666170209123433. [DOI] [PubMed] [Google Scholar]
  9. Chandel P., Kumar A., Singla N., Kumar A., Singh G., Gill R. K. MedChemComm. 2019;10:421–430. doi: 10.1039/c8md00457a. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Denisko O. V. and Katritzky A. R., Encyclopaedia Britannica, 2014, https://www.britannica.com/science/heterocyclic-compound/Major-classes-of-heterocyclic-compounds. [Google Scholar]
  11. Patel D., Kumari P., Patel N. B. Arabian J. Chem. 2017;10(suppl 2) doi: 10.1016/j.arabjc.2014.06.010. [DOI] [Google Scholar]
  12. Basappa, Sadashiva M. P., Mantelingu K., Swamy S. N., Rangappa K. S. Bioorg. Med. Chem. 2003;2003(11):4539–4544. doi: 10.1016/j.bmc.2003.08.007. [DOI] [PubMed] [Google Scholar]
  13. Kalirajan R., Rafick M. H. M., Sankar S., Jubie S. Sci. World J. 2012:165258. doi: 10.1100/2012/165258. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Quan M. L., Ellis C. D., He M. Y., Liauw A. Y., Lam P. Y. S., Rossi Wong K. A. P. C., Wexler R. R. Bioorg. Med. Chem. 2003;13:1023–1028. doi: 10.1016/s0960-894x(03)00080-5. [DOI] [PubMed] [Google Scholar]
  15. Khazir J., Singh P. P., Reddy D. M., Hyder I., Shafi S., Sawant S. D., Chashoo G., Mahajan A., Alam M. S., Saxena A. K., Arvinda S., Gupta B. D., Kumar H. M. S. Eur. J. Med. Chem. 2013;63:279–289. doi: 10.1016/j.ejmech.2013.01.003. [DOI] [PubMed] [Google Scholar]
  16. Goyard D., Konya B., Chajistamatiou A. S., Chrysina E. D., Leroy J., Balzarin S., Tournier M., Tousch D., Petit P., Duret C., Maurel P., Somsak L., Docsa T., Gergely P., Praly J., Azaymilhau J., Vidal S. Eur. J. Med. Chem. 2016;108:444–454. doi: 10.1016/j.ejmech.2015.12.004. [DOI] [PubMed] [Google Scholar]
  17. Dallanoce C., Meroni G., De Amici M., Hoffmann C., Klotz K. N., De Micheli C. Bioorg. Med. Chem. Lett. 2006;14:4393–4401. doi: 10.1016/j.bmc.2006.02.038. [DOI] [PubMed] [Google Scholar]
  18. Ghidini E., Capelli A. M., Carnini C., Cenacchi V., Marchini G., Virdis A., Italia A., Facchinetti F. Steroids. 2015;95:88–95. doi: 10.1016/j.steroids.2014.12.016. [DOI] [PubMed] [Google Scholar]
  19. Park K. K., Ko D. H., You Z., Khan M. O., Lee H. J. Steroids. 2006;71:183–188. doi: 10.1016/j.steroids.2005.10.003. [DOI] [PubMed] [Google Scholar]
  20. Alam A., Pal C., Goyal M., Kundu M. K., Kumar R., Iqbal M. S., Dey S., Bindu S., Sarkar S., Pal U., Maiti N. C., Adhikari S., Bandyopadhyay U. Bioorg. Med. Chem. 2011;19:7365–7373. doi: 10.1016/j.bmc.2011.10.056. [DOI] [PubMed] [Google Scholar]
  21. Dallanoce C., Frigerio F., Grazioso G., Matera C., Visconti G. L., De Amici M., Pucci L., Pistillo F., Fucile S., Gotti S., Clementi F., De Micheli C. Eur. J. Med. Chem. 2011;46:5790–5799. doi: 10.1016/j.ejmech.2011.09.028. [DOI] [PubMed] [Google Scholar]
  22. Reis S. L. G. B., Almeida V. M., Almeida G. C., Boaviagem J. M., Mendes C. C. D. B., Faria A. R., Góes A. J. S., Magalhães L. R., Silva T. G. Quim. Nova. 2011;34:76–81. [Google Scholar]
  23. Basappa, Kumar M. S., Swamy S. N., Mahendra M., Prasad J. S., Viswanath B., Rangappa K. Bioorg. Med. Chem. Lett. 2004;14:3679–3681. doi: 10.1016/j.bmcl.2004.05.012. [DOI] [PubMed] [Google Scholar]
  24. Hwang I. T., Kim H. R., Jeon D. J., Hong K. S., Song J. H., Cho K. Y. J. Agric. Food Chem. 2005;53:8639–8643. doi: 10.1021/jf051284f. [DOI] [PubMed] [Google Scholar]
  25. Kamal A., Reddy J. S., Ramaiah M. J., Dastagiri D., Bharathi E. V., Azhar M. A., Sultana F., Pushpavalli S. N., Pal-Bhadra M., Muvekar A., Sen S., Zingde S. Eur. J. Med. Chem. 2011;45:3924–3937. doi: 10.1016/j.ejmech.2010.05.047. [DOI] [PubMed] [Google Scholar]
  26. Rajanarendar E., Nagi Reddy M., Rama Krishna S., Rama Murthy K., Reddy Y. N., Rajam M. V. Eur. J. Med. Chem. 2012;55:273–283. doi: 10.1016/j.ejmech.2012.07.029. [DOI] [PubMed] [Google Scholar]
  27. Barreiro E. J., Fraga C. A. M., Rodrigues A. L. P. C. R. M. Quim. Nova. 2002;25:129–148. [Google Scholar]
  28. Higgs G. A., Vane J. R. Br. Med. Bull. 1983;39:265–270. doi: 10.1093/oxfordjournals.bmb.a071831. [DOI] [PubMed] [Google Scholar]
  29. Hernández P., Cabrera M., Lavaggi M. L., Celano L., Tiscornia I., Costa T. R., Thomson L., Bollati-Fogolin M., Miranda A. L. P., Lima L. M., Barreiro E. J., Gonzalez M., Cerecetto H. Bioorg. Med. Chem. 2012;20:2158–2171. doi: 10.1016/j.bmc.2012.01.034. [DOI] [PubMed] [Google Scholar]
  30. Habeeb A. G., Praveen Rao P. N., Knaus E. E. J. Med. Chem. 2001;44:2921–2927. doi: 10.1021/jm0101287. [DOI] [PubMed] [Google Scholar]
  31. de Almeida V. M., dos Santos R. J., da Silva Góes A. J., de Lima J. G., Duarte Correia C. R., de Faria A. R. Tetrahedron Lett. 2009;50:684–687. [Google Scholar]
  32. da Silva Guerra A. S. H., do Nascimento Malta D. J., Morais Laranjeira L. P., Souza Maia M. B., Cavalcanti Colaço N., do Carmo Alves de Lima M., Galdino S. L., da Rocha Pitta I., Gonçalves-Silva T. Int. Immunopharmacol. 2011;11:1816–1822. doi: 10.1016/j.intimp.2011.07.010. [DOI] [PubMed] [Google Scholar]
  33. Corrêa C. R., Calixto J. B. Br. J. Pharmacol. 1993;110:193–198. doi: 10.1111/j.1476-5381.1993.tb13791.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Winter C. A., Risley E. A., Nuss G. W. Proc. Soc. Exp. Biol. Med. 1962;111:544–547. doi: 10.3181/00379727-111-27849. [DOI] [PubMed] [Google Scholar]
  35. Henriques M. G., Silva P. M., Martins M. A., Flores C. A., Cunha F. Q., Assereuy-Filho J., Cordeiro R. S. Braz. J. Med. Biol. Res. 1987;20:243–249. [PubMed] [Google Scholar]
  36. Parrat J. R., West G. B. Br. J. Pharmacol. Chemother. 1958;3(1):65–70. doi: 10.1111/j.1476-5381.1958.tb00192.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Ribeiro A. E. A. S., Soares J. M. D., Silva H. A. L., Wanderley C. W. S., Moura C. A., de Oliveira-Junior R. G., de Oliveira A. P., Rolim L. A., Costa E. V., Almeida J. R. G. D. S., de Oliveira H. P., Palheta-Junior R. C. Biomed. Pharmacother. 2019;111:1046–1056. doi: 10.1016/j.biopha.2019.01.011. [DOI] [PubMed] [Google Scholar]
  38. Henriques M. G., Rae G. A., Cordeiro R. S., Williams T. J. Br. J. Pharmacol. 1992;106:579–582. doi: 10.1111/j.1476-5381.1992.tb14378.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Busnardo T. C. P. M., Padoani C., Mora T. C., Biavatti M. W., Fröde T. S., Bürger C., Claudino V. D., Dalmarco E. M., de Souza M. M. J. Ethnopharmacol. 2010;128:519–525. doi: 10.1016/j.jep.2009.12.017. [DOI] [PubMed] [Google Scholar]
  40. Castardo J. C., Prudente A. S., Ferreira J., Guimarães C. L., Monache F. D., Filho V. C., Otuki M. F., Cabrini D. A. J. Ethnopharmacol. 2008;118:405–411. doi: 10.1016/j.jep.2008.05.002. [DOI] [PubMed] [Google Scholar]
  41. Ximenes R. M., Rabello M. M., Araújo R. M., Silveira E. R., Fagundes F. H. R., Diz-Filho E. B. S., Buzzo S. C., Soares V. C. G., Toyama D. de O., Gaeta H. H., Hernandes M. Z., Monteiro H. S. A., Toyama M. H. J. Evidence-Based Complementary Altern. Med. 2012;2012:1–9. doi: 10.1155/2012/987517. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Whittle B. A. Br. J. Pharmacol. Chemother. 1964;22:246–253. doi: 10.1111/j.1476-5381.1964.tb02030.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Scotti L., Lima E. O., Silva M. S., Ishiki H., Lima I. O., Pereira F. O., Mendonça Junior F. J. B., Scotti M. T. Curr. Top. Med. Chem. 2013;14:64–80. [Google Scholar]
  44. HyperChem, Version 8.0, Hybercube Inc., Gainesville, FL, USA, 2009.
  45. Allinger N. L. J. Am. Chem. Soc. 1977;99:8127–8134. [Google Scholar]
  46. Dewar M. J. S., Zoebisch E. G., Healy E. F., Stewart J. J. P. J. Am. Chem. Soc. 1985;107:3902–3909. [Google Scholar]
  47. Cohen N. C., Guidebook on Molecular Modeling in Drug Design, Academic Press, San Diego, CA, USA, 1996. [Google Scholar]
  48. Leach A. R., Molecular Modeling: Principles and Applications, Prentice Hall, London, UK, 2001. [Google Scholar]
  49. Spartan model homepage for windows, available at http://www.wavefun.com/products/windows/SpartanModel/win_model.html, access in 4/4/2014.
  50. Silva K. A. B. S., Klein-Junior L. C., Cruz S. M., Cáceres A., Quintão N. L. M., Monache F. D., Cechinel-Filho V. Food Chem. 2012;132:1980–1986. doi: 10.1016/j.foodchem.2011.12.036. [DOI] [Google Scholar]
  51. Https://www.swissadme.ch. Researched on 08/16/2019.
  52. Williams A. E., Chambers R. C. Am. J. Physiol. 2014;306:L217–L230. doi: 10.1152/ajplung.00311.2013. [DOI] [PMC free article] [PubMed] [Google Scholar]
  53. Phillipson M., Kubes P. Nat. Med. 2011;17:1381–1390. doi: 10.1038/nm.2514. [DOI] [PMC free article] [PubMed] [Google Scholar]
  54. Mantovani A., Sozzani S., Introna M. Ann. N. Y. Acad. Sci. 1997;832:93–116. doi: 10.1111/j.1749-6632.1997.tb46240.x. [DOI] [PubMed] [Google Scholar]
  55. Benincá J. P., Montanher A. B., Zucolotto S. M., Schenkel E. P., Fröde T. S. Food Chem. 2007;104:1097–1105. [Google Scholar]
  56. LeMay L. G., Vander A. J., Kluger M. J. Am. J. Physiol. 1990;258:R798–R803. doi: 10.1152/ajpregu.1990.258.3.R798. [DOI] [PubMed] [Google Scholar]
  57. Oliveira S. H. P., Canetti C., Ribeiro R. A., Cunha F. Q. Inflammation. 2008;31:36–46. doi: 10.1007/s10753-007-9047-x. [DOI] [PubMed] [Google Scholar]
  58. Cunha T., Verri W. Eur. J. Pharmacol. 2007;573:221–229. doi: 10.1016/j.ejphar.2007.07.007. [DOI] [PubMed] [Google Scholar]
  59. Castro J. A., Sasame H. A., Sussman H., Gillette J. R. Life Sci. 1968;7:129–136. doi: 10.1016/0024-3205(68)90326-3. [DOI] [PubMed] [Google Scholar]
  60. Vane J., Botting R. FASEB J. 1987;1:89–96. [PubMed] [Google Scholar]
  61. Wu C.-R., Hseu Y.-C., Lien J.-C., Lin L.-W., Lin Y.-T., Ching H. Molecules. 2010;16:1–15. doi: 10.3390/molecules16010001. [DOI] [PMC free article] [PubMed] [Google Scholar]
  62. Okoli C. O., Akah P. A., Nwafor S. V., Anisiobi A. I., Ibegbunam I. N., Erojikwe O. J. Ethnopharmacol. 2007;109:219–225. doi: 10.1016/j.jep.2006.07.037. [DOI] [PubMed] [Google Scholar]
  63. Rogerio A. P., Fontanari C., Melo M. C. C., Ambrosio S. R., de Souza G. E. P., Pereira P. S., França S. C., da Costa F. B., Albuquerque D. A., Faccioli L. H. J. Pharm. Pharmacol. 2006;58:1265–1273. doi: 10.1211/jpp.58.9.0014. [DOI] [PubMed] [Google Scholar]
  64. Bastos J. K., Carvalho J. C. T., de Souza G. H. ., Pedrazzi A. H. P., Sarti S. J. J. Ethnopharmacol. 2001;75:279–282. doi: 10.1016/s0378-8741(01)00171-4. [DOI] [PubMed] [Google Scholar]
  65. Lucetti D. L., Lucetti E. C., Bandeira M., Veras H. N., Silva A. H., Leal L., Lopes A. A., Alves V. C., Silva G. S., Brito G., Viana G. B. J. Inflammation. 2010;7:60. doi: 10.1186/1476-9255-7-60. [DOI] [PMC free article] [PubMed] [Google Scholar]
  66. Campos M. M., Henriques M. G. M. O., Calixto J. B. Br. J. Pharmacol. 1997;120:502–508. doi: 10.1038/sj.bjp.0700914. [DOI] [PMC free article] [PubMed] [Google Scholar]
  67. Schmaier A. H. Curr. Opin. Hematol. 2000;7:261–265. doi: 10.1097/00062752-200009000-00001. [DOI] [PubMed] [Google Scholar]
  68. Ellis K. M., Fozard J. R. Auton. Autacoid Pharmacol. 2002;22:3–16. doi: 10.1046/j.1474-8673.2002.00230.x. [DOI] [PubMed] [Google Scholar]
  69. Schemann M., Kugler E. M., Buhner S., Eastwood C., Donovan J., Jiang W., Grundy D. PLoS One. 2012;7:e52104. doi: 10.1371/journal.pone.0052104. [DOI] [PMC free article] [PubMed] [Google Scholar]
  70. Zuliani J. P., Fernandes C. M., Zamuner S. R., Gutiérrez J. M., Teixeira C. F. P. Toxicon. 2005;45:335–346. doi: 10.1016/j.toxicon.2004.11.004. [DOI] [PubMed] [Google Scholar]
  71. Wei J.-F., Wei X.-L., Mo Y.-Z., Yang H., He S. BioMed Res. Int. 2013;2013:1–5. [Google Scholar]
  72. Ticli F. K., Hage L. I. S., Cambraia R. S., Pereira P. S., Magro Â. J., Fontes M. R. M., Stábeli R. G., Giglio J. R., França S. C., Soares A. M., Sampaio S. V. Toxicon. 2005;46:318–327. doi: 10.1016/j.toxicon.2005.04.023. [DOI] [PubMed] [Google Scholar]
  73. Picolo G., Chacur M., Gutiérrez J. M., Teixeira C. F. P., Cury Y. Braz. J. Med. Biol. Res. 2002;35:1221–1228. doi: 10.1590/s0100-879x2002001000016. [DOI] [PubMed] [Google Scholar]
  74. Olivo R. d. A., Teixeira C. F. P., Wallace J. L., Gutierrez J. M., Zamuner S. R. Toxicon. 2007;49:670–677. doi: 10.1016/j.toxicon.2006.11.006. [DOI] [PubMed] [Google Scholar]

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