Novel 5-aminosalicylic derivatives as anti-inflammatories and myeloperoxidase inhibitors evaluated in silico, in vitro and ex vivo

2.2.1 Synthesis of 5-{[(2E)-3-bromo-3-carboxyprop-2-enoyl]amino}-2-hydroxybenzoic acid (C1)

To a solution of 1.0 g of 5-ASA (6.2 mmol) in 80 mL of ethyl acetate (AcOEt) was added 0.6 mL of bromomaleic anhydride (6.2 mmol), and the reaction mixture was kept under constant stirring and reflux for 5 h. The resulting solid was filtered under vacuum, re-suspended in 10 mL of cold ethanol (EtOH) and stirred for 3 min at room temperature. Finally, the solid was filtered under vacuum, washed with 10 mL of cold EtOH and air-dried to afford 1.53 g (4.4 mmol) of a beige solid at a 71% yield. Mp: 218 ± 2 °C. Soluble (1 mg/mL) in: acetone, dimethylformamide (DMF), DMSO, methanol (MeOH), and tetrahydrofuran (THF). Partially soluble (1 mg/mL) in: chloroform (CHCl₃) and EtOH. Insoluble (1 mg/mL) in: AcOEt, acetonitrile (CH₃CN), hexane, toluene. The specific water (H₂O) solubility was 0.5 mg/mL. FT IR (ATR) ν_max (cm⁻¹): 3311 (NH), 3300–2500 (OH), 1709, 1663 (C⚌O). ¹H NMR (300 MHz, DMSO-d₆): δ 11.20 (bs, 1H, CO₂H), 10.31 (s, 1H, NH), 8.12 (d, J = 2.7 Hz, 1H, H-6), 7.60 (dd, J = 9.0, 2.9 Hz, 1H, H-4), 6.92 (d, J = 9.1 Hz, 1H, H-3), 6.75 (s, 1H, H-9). ¹³C NMR (75.4 MHz, DMSO-d₆): δ 172.0 and 160.8 (COOH), 165.3 (C-8), 157.9 (C-2), 130.5 (C-5), 129.5 (C-9), 127.8 (C-4), 125.0 (C-10), 121.1 (C-6), 117.9 (C-3), 113.1 (C-1). HRMS (ESI) Calcd. for C₁₁H₈BrNO₆ [M+Na]⁺: 351.9433. Found 351.9427.

2.2.2 Synthesis of 5-[(4-carboxybutanoyl)amino]-2-hydroxybenzoic acid (C2)

To a solution of 5.0 g of 5-ASA (31.0 mmol) in 50 mL of glacial acetic acid was added 4.47 g of glutaric anhydride (37.2 mmol), and the reaction mixture was kept under reflux and constant stirring for 5 h. Finally, 100 mL of cold distilled water was added, and the resulting solid was filtered under vacuum, washed with 100 mL of cold distilled water and air-dried to afford 6.1 g (21.7 mmol) of a beige solid at a 70% yield. Mp: 232 ± 2 °C. Soluble (1 mg/mL) in: acetone, DMF, DMSO, EtOH, MeOH, and THF. Partially soluble (1 mg/mL) in: AcOEt, CHCl₃, hexane, toluene. Insoluble (1 mg/mL) in: CH₃CN. The specific H₂O solubility was 0.125 mg/mL. FT IR (ATR) ν_max (cm⁻¹): 3286 (NH), 3500–2500 (OH), 1705, 1657 (C⚌O). ¹H NMR (300 MHz, DMSO-d₆): δ 11.0 (bs, 1H, CO₂H), 9.85 (s, 1H, NH), 8.08 (d, J = 2.1 Hz, 1H, H-6), 7.62 (dd, J = 9.2, 2.1 Hz, 1H, H-4), 6.87 (dd, J = 6.8, 2.5 Hz, 1H, H-3), 2.28 and 2.24 (2t, J = 7.1 Hz, 4H, H-9, H-11), 1.76 (quintet, J = 7.2 Hz, 2H, H-10). ¹³C NMR (75.4 MHz, DMSO-d₆): δ 174.6 and 172.2 (COOH), 170.9 (CONH), 157.3 (C-2), 131.5 (C-5), 127.8 (C-6), 120.8 (C-4), 117.5 (C-3), 112.8 (C-1), 35.6 (C-9), 33.4 (C-11), 20.9 (C-10). HRMS (ESI) Calcd. for C₁₂H₁₃NO₆ [M]⁺: 268.0821. Found: 268.0816.

2.2.3 Synthesis of 2-((3-carboxy-4-hydroxyphenyl)carbamoyl)-4-fluorobenzoic acid (C3)

C3 was synthesized as described for C1, starting with mixing 0.5 g of 5-ASA (3.13 mmol) and 0.54 g of 4-fluoro phthalic anhydride (3.13 mmol) in 100 mL of AcOEt under reflux for 7 h. The resulting solid was filtered at vacuum and recrystallized from EtOH/hexane to afford 0.861 g (2.7 mmol) of a beige solid at an 83% yield. Soluble (1 mg/mL) in: DMF and DMSO. Partially soluble (1 mg/mL) in: acetone, MeOH, and THF. Insoluble (1 mg/mL) in: AcOEt, CH₃CN, CHCl₃, EtOH, hexane, toluene. The specific H₂O solubility was 0.02 mg/mL. FT IR (ATR) ν_max (cm⁻¹): 3312 (NH), 3400–2500 (OH), 1709, 1656 (C⚌O). ¹H NMR (400 MHz, DMSO-d₆): δ 12.29 (bs, 2H, CO₂H), 10.30 (s, 1H, NH), 8.22 (d, J = 4.0 Hz, 1H, H-6), 7.95 (dd, J = 8.0, 4.0 Hz, 1H, H-3′), 7.69 (dd, J = 8.0, 4.0 Hz, 1H, H-6′), 7.45 (dd, J = 8.0, 4.0 Hz, 1H, H-4), 7.41 (ddd, J = 8.0, 8.0, 4.0 Hz, 1H, H-5′), 6.94 (d, J = 8.0 Hz, 1H, H-3). ¹³C NMR (100 MHz, DMSO-d₆): δ 172.2 and 171.6 (CO₂H), 166.9 (CONH), 157.9 (C-2), 163.7 (C-4′, ¹J_C-F = 292.5 Hz), 142.0 (C-2′, ³J_C-F = 7.5 Hz), 131.4 (C-5′), 129.7 (C-5), 128.3 (C-6′, ²J_C-F = 40 Hz), 121.6 (C-6), 121.5 (C-1′), 117.6 (C-3), 116.7 (C-5′, ²J_C-F = 40 Hz), 115.4 (C-4), 113.1 (C-1), 111.6 (C-3′, ²J_C-F = 40 Hz). HRMS (ESI) Calcd. for C₁₅H₁₀FNO₆ [M+H]⁺ 320.0570. Found: 320.0569.

2.2.4 Synthesis of 5-(2-carboxybenzamido)-2-hydroxybenzoic acid (C4)

C4 was synthesized and isolated as described for C3, starting with mixing 0.54 g of 5-ASA (3.32 mmol) and 0.5 g of phthalic anhydride (3.32 mmol) at reflux for 5 h to afford 0.752 g (2.5 mmol) of a beige solid at a 74% yield. Mp: 268 ± 2 °C. Soluble (1 mg/mL) in: DMF, DMSO and THF. Partially soluble (1 mg/mL) in: acetone and MeOH. Insoluble (1 mg/mL) in: AcOEt, CH₃CN, CHCl₃, EtOH, hexane, toluene. The specific H₂O solubility was 0.02 mg/mL. FT IR (ATR) ν_max (cm⁻¹): 3298 (NH), 3400–2500 (OH), 1698, 1654 (C⚌O). ¹H NMR (400 MHz, DMSO-d₆): δ 12.60 (bs, 2H, CO₂H), 10.26 (s, 1H, NH), 8.25 (d, J = 4.0 Hz, 1H, H-6), 7.87 (dd, J = 8.0, 4.0 Hz, 1H, H-6′), 7.71 (dd, J = 8.0, 4.0 Hz, 1H, H-4), 7.66 (ddd, J = 8.0, 8.0, 4.0 Hz, 1H, H-4′), 7.58 (dd, J = 8.0, 4.0 Hz, 1H, H-3′), 7.56 (ddt, J = 8.0, 8.0, 4.0 Hz, 1H, H-5′), 6.94 (d, J = 8.0 Hz, 1H, H-3). ¹³C NMR (100 MHz, DMSO-d₆): δ 172.2 and 168.0 (CO₂H), 167.6 (CONH), 157.7 (C-2), 139.2 (C-5), 132.1 (C-4′), 131.7 (C-2′), 130.5 (C-1′), 130.0 (C-6′), 129.9 (C-3′), 128.3 (C-4), 128.2 (C-5′), 121.5 (C-6), 117.5 (C-3), 113.0 (C-1). HRMS (ESI) Calcd. for C₁₅H₁₁NO₆ [M+Na]⁺: 324.0484 Found: 324.0473.

2.3.1 Animals and treatments

Forty-two male CD-1 mice (weighing 25–30 g) were purchased from Universidad Autónoma Metropolitana Unidad Xochimilco (http://correo.xoc.uam.mx/bioterio/), Mexico City, Mexico. The animals were housed in standard polypropylene cages and maintained on a 12 h light/dark cycle at a constant temperature (22 ± 2 °C) and food and water ad libitum. All animal treatments and procedures were approved by the Mexican Official Standard (NOM-062-ZOO-1999) and the ethics committee for the Care and Use of Laboratory Animals (CICUAL, Spanish acronym) of the Escuela Superior de Medicina-IPN (Approval number: ESM.CICUAL-02/27-07-2015). After a 1-week acclimatization period, the anti-inflammatory activity and MPO peroxidation activity were determined using the mouse ear edema model and the O-dianisidine dihydrochloride method, respectively.

The animals used in the anti-inflammatory model (ear edema and thickness) and estimation of the MPO peroxidation activity were divided into three control groups and four experimental groups with a population of three mice per group (ear number per group = 6). The first group of ears was used as a control (acetone, vehicle), the second group of ears was treated with TPA, and a third group of ears was used as a positive control (indomethacin, IND). The remaining four groups of ears were assigned to each tested compound (C1–C4).

The control group only received vehicle, and the remaining groups received TPA (0.25 μg/μL) (Sigma Aldrich Co., USA) dissolved in acetone (99%). After 10 min, IND (0.46 mg/ear) diluted in acetone:ethanol (1:1) was administered to one group, and C1–C4 were administered at an equimolar ratio to IND in their respective groups. All the treatments were administered topically in the inner and outer ear surface using a volume of 5 μL after the anesthetic administration of sodium pentobarbital (60 mg/kg, i.p.).

2.3.2 Anti-inflammatory effect

The anti-inflammatory effect was evaluated using the topic TPA-induced mouse ear edema as previously described (Salazar et al., 2011) with some modifications. Four hours after the treatment, the animals were sacrificed by ether inhalation (Eddouks et al., 2012), and the ear punch biopsies (7 mm diameter) were obtained. The ear thickness was measured using a micrometer (Mitutoyo Corporation, Japan) (Zhang et al., 2014), and then the samples were weighed and dried at 50 °C for 24 h. The skin punches were then reweighed, and the ear thickness was determined by the differences between the punch weights. Edema was estimated as the change in weight of the ear punch using the expression: edema = [(TPA only) − (tested compound plus TPA)]/[(TPA only) − (vehicle)] (Afaq et al., 2005; Khan et al., 2012; Mizushina et al., 2011).

2.3.3 Estimation of MPO peroxidation activity from the ear samples

MPO peroxidation activity was measured as has been reported elsewhere (Kettle et al., 1997; Khan et al., 2012), with some modifications, using biopsies taken from ears 4 h after the administration of the treatments. MPO was extracted from the homogenates (maceration) of ear using hexadecyltrimethylammonium bromide (HTAB) (Sigma-Aldrich Co., USA) (0.5%) in 300 μL of assay buffer (potassium phosphate buffer; 50 mM, pH = 6.0). Then, the homogenate was frozen and sonicated 3 times in Eppendorf tubes. The final suspension was centrifuged at 13,500 rpm at 4 °C for 15 min.

For the assay, 0.1 mL of the supernatant was mixed with O-dianisidine dihydrochloride (0.167 mg/mL) and H₂O₂ (Sigma-Aldrich Co., México) (0.005%) dissolved in 2.9 mL of assay buffer. After the samples were incubated in darkness at room temperature for 15 min, MPO activity was measured at 490 nm in a UV–Vis PerkinElmer Lambda 25 spectrophotometer (PerkinElmer, Waltham, Massachusetts 02451, USA). The MPO units (U) were determined using a standard curve, which was done using different units of pure MPO (0.0039-0.25 U of MPO) and the same reference concentrations of O-dianisidine dihydrochloride and H₂O₂.

2.4.1 MPO activity

The peroxidase activity of pure MPO (Sigma-Aldrich Co., USA) (2.5 μL; 0.0625 Units (U)) was assayed by the oxidation of O-dianisidine dihydrochloride (0.167 mg/mL) in 2.9 mL of potassium phosphate buffer (50 mM, pH = 6) at 490 nm in a UV–Vis PerkinElmer Lambda 25 spectrophotometer (PerkinElmer, Waltham, Massachusetts 02451, USA) (Bradley et al., 1982; Kettle et al., 1997). The MPO inhibitory activity of IND and the compounds was evaluated by the addition of IND (97.5 μL) in various concentrations (0.01, 0.1, 1, 10, 100 and 1000 μM). Compounds C1–C4 were evaluated in an equimolar ratio to IND. The reaction was started by adding 8.77 μL of H₂O₂ (0.005%). The reactions were performed in darkness at room temperature for 15 min. The change in absorbance was measured, and the percentage of inhibition was calculated in comparison with the control without inhibitors, taking into account the absorbance of light by the tested compounds.

2.4.2 Antioxidant activity

The antioxidant activity was analyzed by the 2,2′-diphenyl-1-picrylhydrazyl (DPPH) and 2,2′-azino-bis(3-ethylbenzothiazoline)-6-sulfonic acid (ABTS) methods.

The DPPH assay was carried out as previously described (Backhouse et al., 2008; Brand-Williams et al., 1995). Briefly, 1 mL of different solutions of 5-ASA (0.408, 0.204, 0.102, 0.051, 0.025 and 0.012 mM) was dissolved in ethanol and mixed with 1 mL of DPPH (Sigma–Aldrich Co., USA) solution dissolved in DMSO at a final concentration of 3 × 10⁻⁵ M at room temperature in total darkness. After 60 min of incubation, the absorbance was measured at 517 nm in a UV–Vis PerkinElmer Lambda 25 spectrophotometer (PerkinElmer, Waltham, Massachusetts 02451, USA). The scavenging of DPPH radicals of C1–C4 was evaluated in an equimolar ratio to 5-ASA, and the antioxidant activity was compared to the control sample (1 mL DPPH solution + 1 mL compound solution). DMSO was used as a blank, and the antioxidant activity was determined by the mathematical expression: (1 − (Ai − Aj)/Ac) ∗ 100, where Ai = tested compound solution + DPPH solution, Aj = tested compound solution + DMSO and Ac = DPPH solution + DMSO.

The ABTS assay was performed as previously described with some modifications (Gliszczyńska-Świgło, 2006; Thaipong et al., 2006). Aqueous radical cation ABTS (Sigma-Aldrich Co., USA) and diammonium salt solution (7 mM) were mixed with potassium persulfate (2.42 mM) and allowed to stand in darkness at room temperature for 16 h. After the incubation, the absorbance was measured at 734 nm, and the solution was diluted with water until a final absorbance of 0.70 ± 0.02 was reached. A calibration curve with Trolox (2.5 mM) was constructed in a range of 0.0125–0.2 mM in DMSO by mixing 200 μL of each concentration of Trolox with 2000 mL of ABTS radical solution. The mixture was kept in darkness at room temperature for 6 min, and the absorbance was determined at 734 nm in a UV–Vis PerkinElmer Lambda 25 spectrophotometer (PerkinElmer, Waltham, Massachusetts 02451, USA). The samples of 5-ASA and C1–C4 were evaluated under the same conditions, and the percentage of antioxidant effectiveness was obtained by applying the formula: ((Ia − Fa)/Ia) ∗ 100, where Ia = initial absorbance and Fa = final absorbance.

2.5.1 Molecular docking

The ligands (5-ASA, IND, C1, C2, C3 and C4) were drawn using ChemBioDraw Ultra 12.0, and their geometry was pre-optimized using Hyperchem (Version 6.0, Hypercube, USA, http://www.hyper.com) at the molecular mechanic’s level. Afterward, the full optimization was performed using the Gaussian 03 program at the semi-empirical (AM1) level of theory.

Docking simulations were performed on the human MPO 3-D protein structure (PDB: 1DNU) using several snapshots obtained every 1 ns from 20 ns of molecular dynamics (MD) simulations of MPO that were previously reported (Ramírez-Durán et al., 2013). Prior to docking, ligands and water co-crystalized with MPO were removed from the protein structure, maintaining the heme group. Then, all possible flexible bonds were identified, and the partial atomic charges of the ligands (Gasteiger–Marsili formalism) were calculated using AutoDock Tools 1.5.2 (Morris et al., 1998, 2009).

The Kollman charges for all atoms in MPO were calculated, and the hydrogens were placed at the polar atoms on amino acids. All other parameters were kept at their default values. The protein binding site and ligand structure definitions were prepared using a GRID-based procedure (Ramírez-Durán et al., 2013) in AutoDock Tools version 3.4. A 60 × 60 × 60 Å point grid with 0.375 Å spacing centered on the Fe⁺² heme group was employed. All docking simulations used the hybrid Lamarckian Genetic Algorithm, with an initial population of 100 randomly placed individuals and 1 × 10⁷ evaluations using AutoDock version 4.2.0. Docked ligands with geometry orientations within a root-mean square deviation (RMSD) of 0.5 Å were clustered together. The lowest free energy cluster determined for each compound was used for further analysis. The docking studies were validated by predicting the binding mode of the pattern ligand (5-ASA). The interactions of the ligands with MPO were visualized using AutoDock Tools 1.5.6, and the figures were created using Pymol 1.0 and VMD v1.8.7 (Humphrey et al., 1996).

2.5.2 Physicochemical and toxicological parameters

The five Lipinski’s rule (Log p values, molecular weight, hydrogen bond donation/acceptation) provided drug properties related to biological bioavailability characteristics (Veber et al., 2002), which were calculated using the software Osiris Property Explorer available in http://www.organic-chemistry.org/chemicals/. In addition, the toxicity (mutagenicity, tumorigenicity, and reproductive effects) properties, as well as drug likeness, were evaluated by the Osiris Property Explorer software (http://www.organic-chemistry.org/chemicals/) (Nalini et al., 2011). Finally, ΔG values and binding pose (near the catalytic site) obtained from the docking studies sampling different protein snapshots were taken into account. This procedure helped to determine a score (Table 1) to select the best compounds.

3.2.1 Anti-inflammatory effect

Fig. 3 shows that the topical administration of TPA on the ear induced an edema that was diminished by the administration of IND and C1, when both are administered at an equimolar ratio. Although C2, C3, and C4 also diminished the edema approximately 10 mg/punch, C1 was the compound that showed the best anti-inflammatory effect, and this result correlated with the reduction in thickness (Fig. 4).

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Figure 3 TPA-induced ear edema in mice and the effect on its reduction by IND and each compound C1–C4. The data were expressed as the mean ± S.E. (n = 6). (a) (p < 0.001) compared with the TPA-treated group, (b) (p < 0.001) compared with the TPA + IND group, and (c) (p < 0.005) compared with the TPA + C1 group.

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Figure 4 TPA-induced ear thickness in mice and the effect on its reduction by IND and each compound C1–C4. The data were expressed as the mean ± S.E. (n = 6). (a) (p < 0.001) compared with the TPA-treated group, (b) (p < 0.001) compared with the TPA + IND group, (c) (p < 0.001) compared with the TPA + C1 group, and (d) and (e) (p < 0.005) compared with the TPA + C2 group.

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The topical application of TPA (0.25 μg/mL) induced a marked increase in ear thickness (169.33 ± 0.33 μm) (Fig. 4). Topical application of the vehicle (acetone, 96%) did not alter the ear thickness (data not shown); however, the ear thickness induced by TPA was inhibited by IND (positive control) with significant difference as well as by compounds C1, C2, C3, and C4. Further, the effect of C1 was comparable to IND, which demonstrates that C1 has a stronger inhibitory effect than the other compounds related to edema formation by reducing the thickness and weight of ears.

3.2.2 Estimation of MPO peroxidation activity from the ear samples

In Fig. 5, it is possible to observe that the group treated with TPA showed a significant increase in MPO activity with respect to the control group. A significant decrease in the units of MPO was observed for groups C1–C4 that were previously exposed to the topical administration of TPA in the ears of mice. Treatment with C4 was comparable to the IND group, and both compounds were administered in an equimolar dose.

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Figure 5 Units of MPO/mg tissue from homogenates of mouse ears (n = 6 ears for each group) treated topically with TPA and those treated with TPA and IND or each compound C1–C4. (a) (p < 0.001) compared with the control group (Ac) and (b) (p < 0.001) compared with the TPA group.

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3.3.1 MPO activity

The MPO inhibition by compounds C1–C4 was corroborated in vitro. The IC₅₀ values were obtained from the graphic of% of inhibition vs. log concentration (μM) (Fig. 6). Compounds C2, C3, and C4 showed IC₅₀ values that were better than those of IND (13, 85, 46, and 122 μM, respectively), and the IC₅₀ value for C1 (301 μM) was the highest.

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Figure 6 Inhibition percentage of MPO vs. the log of concentration of IND and compounds C1–C4 (n = 3).

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3.3.2 Antioxidant activity

The reduction in free radical DPPH was followed by the decrease in its absorbance, using C1–C4 and 5-ASA as a reference compound (Backhouse et al., 2008; Brand-Williams et al., 1995). Table 2 shows the concentration-dependent reduction percentage values of DPPH radical by 5-ASA and C1–C4 at six different concentrations (0.012, 0.025, 0.051, 0.102, 0.204, and 0.408 mM). Compounds C1, C2, and C4 presented a lower percentage of reduction in DPPH at concentrations below to 0.408 mM against 5-ASA. In contrast, C3 showed higher free radical (FR) scavenging activity than 5-ASA, except at 0.012 mM. Additionally, C1 shows better FR scavenging than C2 at the same concentrations (0.012–0.204 mM). Further, compound C1 showed better FR scavenging than C4 at lower concentrations of 0.204 mM, and at higher concentrations, the antioxidant effect of both compounds was comparable. On the other hand, C3 showed an increment in the percentage of reduction in the DPPH radical (93%) in relation to 5-ASA (85%) at 0.408 mM.

Furthermore, the ability of compounds C1–C4 to stabilize FR was evaluated by ABTS assay using Trolox as a reference compound (Gliszczyńska-Świgło, 2006; Thaipong et al., 2006). Table 3 shows that 5-ASA and compounds C3 and C4 produced almost a 100% reduction of ABTS radical at concentrations above 0.102 mM. In contrast, C1 and C2 were capable of reducing the ABTS radical to approximately 60% at the same concentrations. Therefore, compounds C3 and C4 were better FR scavengers than C1 and C2. Moreover, compound C3 was better than C4 since from the lower concentration (0.012 mM), C3 exhibited a higher percentage of reduction of ABTS radical, reaching 99% from 0.102 mM, while C4 reached this percentage at 0.204 mM. Hence, these results indicate that C3 and C4 have good antioxidant capacities.

3.4.1 Molecular docking

The docking studies of the target compounds (C1–C4) were performed using several MPO snapshots as were mentioned previously. One conformer was obtained from the Protein Data Bank (PDB: 1DNU), and 20 conformers were retrieved by MD simulations, as previously reported (Ramírez-Durán et al., 2013). As seen in Table 4, the average of the ΔG values showed that IND has a high affinity for MPO, while the ΔG values for 5-ASA reflect lower affinity for MPO when compared with IND. In addition, C4 showed the lowest ΔG values followed by C3, whereas C1 and C2 showed very similar ΔG values. These results correlate with those results obtained in vitro for C3 and C4 with respect to their behavior as MPO inhibitors.

To validate the docking studies, the binding mode of 5-ASA with MPO was analyzed. Due to 5-ASA having the lowest ΔG values on the conformer obtained at 17 ns in the docking studies, this conformer was analyzed. As seen in Fig. 7A, 5-ASA establishes interactions with the residues Gln91, Asp94, Arg239, Met243, Leu246, and His95 and the heme group 605, primarily by the formation of hydrogen bonds, π-cation and hydrophobic interactions.

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Figure 7 Molecular interactions between the amino acid residues from the MPO active site and each of the evaluated compounds (C1–C4). (A) MPO structure from 17 ns and 5-ASA. MPO structure from 7 ns and (B) IND, (C) C1, (D) C2, (E) C3, and (F) C4.

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IND showed better affinity on the conformer obtained at 7 ns (ΔG = −8.98 kcal/mol; Table 4), interacting with Arg239, the heme group 605, Phe407, Glu242, Gln91, Asp94, Phe365, Met243, Leu246, Val410, Leu406, Met411, and Val113 by π-cation and hydrophobic interactions (Fig. 7B).

The docking results showed that all compounds (C1–C4) evaluated in this work had better affinity also in the conformer obtained at 7 ns from MD simulations; for this reason, the binding mode was evaluated with this conformer. For instance, C1 (−7.37 kcal/mol) establishes interactions with Arg239, Asp94, Gln91, Phe407, Met243, Leu246, and the heme group 605 (Fig. 7C), whereas C2 (−8.23 kcal/mol) with Arg239, Asp94, Gln91, Glu242, Phe365, Met243, and Leu246 and the heme group 605; in both cases, the principal interactions were π-cation and hydrogen bonds (Fig. 7D).

Compounds C3 and C4 had the best affinity, with ΔG = −10.3 and −10.51 kcal/mol, respectively, at 7 ns, inclusive in the ΔG average (Table 4). Both compounds (C3 and C4) showed similar interactions with those exhibited by C1 and C2, with the principal interactions being the hydrogen bonds and π-cation (Fig. 7E and F, respectively). Meanwhile, C3 and C4 showed a similar binding mode, interacting by the formation of π-cation interactions with Arg239, hydrogen bonds with Asp94, Gln91 and Glu242, and hydrophobic interactions with Leu246, Phe365, Val410 and Leu406.

Interestingly, during the recognition between MPO and all other compounds, the principal interactions were the π-cation with Arg239 and hydrogen bonds with Gln91 and Asp94. Furthermore, C3 and C4 make π-π interactions with Phe407 and Phe365. Then, all compounds tend to interact with amino acid residues reported in the principal binding site of MPO, such as Gln91, Asp94, His95, Phe99, Arg239, Glu242, Arg333, Phe365 and Phe407; this place is where the halide and some inhibitors such as ABAH and acetylsalicylic acid are recognized (Ramírez-Durán et al., 2013).

3.4.2 Physicochemical and toxicological parameters

All compounds were evaluated using the Osiris Property Explorer server in order to describe their physicochemical and toxicological characteristics. Table 5 shows the score calculation results considering only the ΔG from 3 ns of MD simulations for each compound, despite that the final score was obtained taking into account the ΔG values at 20 ns. Then, the final score resulted from the sum of scores of each parameter, giving the maximum value to those parameters that are in accordance with Lipinski′s rule, such as molecular weight < 500 g/mol, Log p < 5, hydrogen bond donators (H bond donors) < 5 and hydrogen bond acceptors (H bond acceptors) < 10. If a compound meets these characteristics, it may exhibit good molecular properties that are important for a new drug's pharmacokinetics (Lipinski et al., 1997). It is important to observe in Table 5 that all compounds have a molecular weight less than 500 g/mol and a Log P lower than 5, whereas the H bond donors and H bond acceptors are below to 10 and 5, respectively. Only 5-ASA showed a risk of carcinogenesis and mutagenesis but not IND and the designed compounds (C1–C4). The sum of the scores of each parameter, taking into account the ΔG values at 20 ns, was greatest for C4 (644), followed by C1 (634), C2 (631), C3 (618), IND (492), and 5-ASA (414). Therefore, the physicochemical and toxicological properties evaluated indicate that the 5-ASA derivatives (C1–C4) are better than IND and 5-ASA.