Exploring Urease Inhibition by Coumarin Derivatives through in silico and in vitro Methods

Abstract

In this report, we designed and synthesized ten N-(R-phenyl)-3-carboxamide-coumarin derivatives (2a-2j), exploring the coumarin nucleus, and an R-phenyl group as a structural scaffold and a peptide bond as a linker between them. The structure-activity relationships were investigated with different R-substituents (H, Br, NO₂, Cl) in ortho, meta, or para positions from the phenyl group. Coumarins were obtained in good yields (72-95%), and in vitro screening against Canavalia ensiformis urease showed potential inhibitory percentages ranging from 42 to 65%. Half-maximal inhibitory concentration (IC₅₀) values were determined for the best compounds 2b (R = 2-Br) and 2d (R = 4-Br). After conducting molecular docking and molecular dynamics on urease from C. ensiformis and Helicobacter pylori, potential binding modes for the most effective compounds 2b and 2d showed that these derivatives are able to interact with the crucial residue Cys592, thereby blocking the access of the urea substrate to the active site. According to density functional theory (DFT) calculations, 4-Br substitution on H. pylori urease was necessary for interacting with catalytic amino acids on the active site.

Keywords:
Canavalia ensiformis ; Helicobacter pylori ; molecular docking; DFT; molecular dynamics

Introduction

Ureases (EC 3.5.1.5, urea amidohydrolases) are nickel-dependent metalloenzymes that catalyze the hydrolysis of urea, producing ammonium bicarbonate after spontaneous decomposition, generating carbon dioxide, ammonia, and water.¹ This enzyme can be present in large quantities in plants such as Canavalia ensiformis, animal tissues, and many microorganisms such as fungi and bacteria.²,³,⁴ Due to its catalytic activity, ureases are related to several health problems, such as stomach cancer, peptic ulceration, pyelonephritis, or cryptococcosis, caused by pathogenic ureolytic microorganisms.⁵ For example, the Gram-negative bacterium Helicobacter pylori uses urease as a virulence factor since the products from catalytic hydrolyses, mainly ammonia and carbamate, increase the pH of the stomach, and then this neutralizing effect supports its survival and colonization of gastric mucosa.⁶ Therefore, urease inhibition is considered a promising strategy for searching for new and effective inhibitors to treat diseases caused by pathogenic microorganisms.

Several urease inhibitors have been reported in the literature due to their potential to inhibit urease in vitro,⁷,⁸,⁹,¹⁰ including compounds containing coumarin scaffold. Among them are bis-coumarins,¹¹,¹² coumarinyl pyrazolinyl thioamide derivatives,¹³ and hybrids featuring coumarin and thiazolotriazole¹⁴ that showed excellent results against urease from Canavalia ensiformis (CEU) when compared to the standard thiourea inhibitor. Based on this potential exhibited by coumarin derivatives as urease inhibitors, we decided to synthesize a series of ten N-(R-phenyl)-3-carboxamide-coumarin derivatives (2a-2j, R = H, Br, NO₂, Cl) and evaluate them in vitro against CEU urease.

Our series involves a peptide bond as a linker (shown in blue in Figure 1) to a phenyl group. The phenyl group has substituents at ortho-, meta-, or para-positions with varying steric and electronic properties, such as Cl, Br, or NO₂, aiming to explore the structure-activity relationship (SAR). Furthermore, our goal is to explore the potential pharmacophore role of the coumarin nucleus, which is highlighted in red in Figure 1. This is because these particular coumarins have yet to be recognized in the literature as inhibitors of CEU compounds. Another hypothesis being investigated is the potential connection between the coumarin core that contains a Michael acceptor group (indicated with dashed lines in Figure 1) and its susceptibility to a nucleophilic attack from the free thiol group of Cys592. For this purpose, we applied in silico methods, such as density functional theory (DFT), molecular docking, and molecular dynamics studies, to explore putative binding modes on CEU and Helicobacter pylori ureases (HPU) (Figure 1).

Figure 1
Structural design, experimental, and in silico approaches were applied to evaluate N-(R-phenyl)-3-carboxamide-coumarin derivatives as potential urease inhibitors (2a-2j).

Experimental

General procedures

Solvents were used without further purification, except for acetone which was dried on a 3 Å molecular sieve for 4-6 h before use. Melting points were recorded on a hot plate apparatus (MQAPF-302, Microquímica Equipamentos Ltda, Palhoça, Brazil) and are uncorrected. The mass spectra were obtained in gas chromatography coupled to a mass spectrometer (GC-MS) Shimadzu (Kyoto, Japan) model GCMS-QP5000, 99604. ¹H nuclear magnetic resonance (NMR) spectra were recorded on a Bruker Avance III (Billerica, USA) operating at 400 MHz using a 5 mm broadband probe and CDCl₃ as solvent. Chemical shifts (d in ppm) were referenced to the residual solvent signal (CHCl₃ in CDCl₃ at δ 7.26). Abbreviations used in the description of the splitting of proton resonances: s (singlet), d (doublet), t (triplet), q (quartet), m (multiplet), Ar (aromatic). Coupling constants (J) are reported in Hz.

Synthesis of coumarin-3-carboxylic acid (1)

Starting material was synthesized according to the method described by Deshmukh et al.¹⁵ Briefly, 2-hydroxybenzaldehyde (7.00 mmol) was dissolved in 25 mL of distilled water, followed by the addition of Meldrum’s acid (7.01 mmol). The reaction mixture remained under stirring and heating at 75 °C for 4 h. Then, the reaction mixture was cooled to room temperature, and the white precipitate was filtered under a vacuum and washed with ice-cold distilled water (3 × 5 mL).

White solid; 92% yield; mp 191 °C; lit. 191-192 °C; MS m/z, 190 (35%), 146 (100%); ¹H NMR (400.1 MHz, CDCl₃) δ 12.21 (1H, s, OH), 8.95 (1H, s, H4), 7.82-7.76 (2H, m, H5 and H7), 7.52-7.47 (2H, m, H6 and H8);^{15 13}C NMR (100.6 MHz, CDCl₃) δ 164.20 (C), 162.5 (C), 154.7 (C), 151.6 (C), 135.9 (C), 130.6 (CH), 126.4 (CH), 118.6 (CH), 117.3 (CH), 115.0 (CH).

Synthesis of N-phenyl-3-carboxamide-coumarin derivatives (2a-2j)

Derivatives 2a-2j were synthesized using the method described by Bispo et al.,¹⁶ with modifications. Firstly, coumarin-3-carboxylic acid (1, 0.792 mmol) was suspended in 1.20 mL of thionyl chloride (SOCl₂, 20 eq.). This mixture was heated under reflux for 2 h 30 min. Then, the SOCl₂ excess was removed under reduced pressure, and in the chloride in situ produced, a solution of the corresponding aniline (0.792 mmol, 1.0 eq) in 10 mL of dry acetone was added into the flask. The reaction remained under reflux for 1 h. Subsequently, the solution was poured into crushed ice, and the precipitate was filtered under vacuum and washed with cold distilled water and ethyl ether (both 3 × 5 mL). Thus, derivatives 2a-2j were obtained in 75-92% yields.

N-Phenyl-3-carboxamide-coumarin (2a)

Yellow solid; 89% yield; mp 266 °C; lit.¹⁷ 253.5-255 °C; ¹H NMR (400.1 MHz, CDCl₃) δ 10.84 (1H, s, NH), 9.03 (1H, s, H4), 7.77-7.68 (4H, m, H5, H7, H15 and H17), 7.47-7.37 (4H, m, H6, H8, H14 and H18), 7.19-7.15 (1H, m, J₁ 8.5, J₂ 1.0 Hz, H16);¹⁷ HSQC (400.1/100.6 MHz, DMSO-d₆) δ 8.93→147.8 (H4→C4), 8.0-8.02→130.84 (H8→C8), 7.77-7.80→134.7 (H7→C7), 7.72-7.74→120.4 (H5→C5), 7.55-7.57→116.8 (H15/H17→C15/C17), 7.46-7.49→125.7 (H6→C6), 7.38-7.41→128.48 (H14/H18→C14/C18), 7.14-7.17→124.7 (H16/C16); MS m/z, 265 (9%), 173 (100%).

N-(2-Bromophenyl)-3-carboxamide-coumarin (2b)

White solid; 88% yield; mp 236 °C; lit.¹⁸ 218-219 °C; ¹H NMR (400.1 MHz, CDCl₃) δ 11.23 (1H, s, NH), 9.02 (1H, s, H4), 8.52 (1H, dd, J₁ 8.3, J₂ 1.5 Hz, H15), 7.77-7.68 (2H, m, H5 and H7), 7.61 (1H, dd, J₁ 8.0, J₂ 1.4 Hz, H18), 7.4-7.33 (3H, m, H6, H8 and H17), 7.06-7.01 (1H, m, H16);¹⁸ HSQC (400.1/100.6 MHz, DMSO-d₆) δ 9.11→149.6 (H4→C4), 8.46-8.48→122.8 (H15→C15), 8.07-8.09→131.1 (H8→C8), 7.79-7.83→135.3 (H5→C5), 7.72-7.74→133.3 (H7→C7), 7.57-7.58→116.7 (H18→C18), 7.47-7.51→125.8 (H6→C6), 7.44-7.48→128.9 (H17→C17), 7.11-7.15→126.6 (H16→C16); MS m/z, 264 (21%), 173 (67%), 89 (100%).

N-(3-Bromophenyl)-3-carboxamide-coumarin (2c)

Yellow solid; 92% yield; mp 246 °C; lit.¹⁸ 232-233 °C; ¹H NMR (400.1 MHz, CDCl₃) δ 10.89 (1H, s, NH), 9.02 (1H, s, H4), 8.04 (1H, t, J₁ 1.9 Hz, H14), 7.78-7.69 (2H, m, H5 and H7), 7.62 (1H, ddd, J₁ 8.0, J₂ 1.9, J₃ 1.2 Hz, H18), 7.49-7.40 (2H, m, H6 and H8), 7.32-7.28 (1H, m, H16), 7.23 (1H, d, J₁ 8.0 Hz, H17);¹⁸ HSQC (400.1/100.6 MHz, DMSO-d₆) δ 8.90→147.9 (H4→C4), 8.11→122.7 (H14→C14), 8.00-8.02→130.9 (H16→C16), 7.77-7.80→134.9 (H8→C8), 7.55-7.56→116.8 (H5→C5), 7.62-7.64→119.4 (H7→C7), 7.46-7.49→125.9 (H6→C6), 7.34-7.35 →127.7 (H17/C17), 7.34-7.35→134.5 (H18→C18); MS m/z, 343 (1%), 173 (100%), 89 (97%).

N-(4-Bromophenyl)-3-carboxamide-coumarin (2d)

Yellow solid; 83% yield; mp 268 °C; lit.¹⁸ 247-248 °C; ¹H NMR (400.1 MHz, CDCl₃) δ 10.88 (1H, s, NH), 9.02 (1H, s, H4), 7.77-7.69 (2H, m, H5 and H7), 7.68-7.63 (1H, m, H15 and H17), 7.52-7.48 (2H, m, H14 and H18), 7.48-7.40 (2H, m, H6 and H8);¹⁸ HSQC (400.1/100.6 MHz, DMSO-d₆) δ 8.91→147.9 (H4→C4), 8.00-8.02→130.7 (H15/H17→C15/C17), 7.77-7.80→134.8 (H8→C8), 7.71-7.73→122.3 (H5/H7→C5/C7), 7.56-7.58→132.4 (H14→C14), 7.54-7.56→132.4 (H18→C18), 7.46-7.49→126.0 (H6/H8→C6/C8); MS m/z, 343 (1%), 173 (96%), 89 (100%).

N-(2-Nitrophenyl)-3-carboxamide-coumarin (2e)

Yellow solid; 80% yield; mp 243 °C; lit.¹⁹ 226-227 °C; ¹H NMR (400.1 MHz, CDCl₃) δ 12.56 (1H, s, NH), 9.00 (1H, s, H4), 8.75 (1H, dd, J₁ 8.5, J₂ 1.2 Hz, H15), 8.22 (1H, dd, J₁ 8.4, J₂ 1.4 Hz, H18), 7.77-7.65 (3H, m, H5, H7 and H17), 7.49-7.38 (2H, m, H6 and H8), 7.29 (1H, dd, J₁ 8.4, J₂ 1.1 Hz, H16);¹⁹ HSQC (400.1/100.6 MHz, DMSO-d₆) δ 9.09→150.0 (H4→C4), 8.56-8.58→124.4 (H15→C15), 8.18-8.20→126.1 (H18→C18), 8.06-8.08→131.2 (H5→C5), 7.80-7.84→135.5 (H16/H17→C16/C17), 7.56-7.58→116.8 (H7→C7), 7.47-7.50→125.8 (H8→C8), 7.38-7.42→125.2 (H6→C6); MS m/z, 264 (11%), 173 (93%), 89 (100%).

N-(3-Nitrophenyl)-3-carboxamide-coumarin (2f)

White solid; 82% yield; mp 292 °C; ¹H NMR (400.1 MHz, CDCl₃) δ 11.14 (1H, s, NH), 9.06 (1H, s, H4), 8.73 (1H, t, J₁ 2.1 Hz, H14), 8.01-8.05 (2H, m, H16 and H18), 7.80-7.72 (2H, m, H5 and H7), 7.57-7.51 (1H, dd, J₁ 14.9, J₂ 6.8 Hz, H17), 7.49-7.43 (2H, m, H6 and H8);²⁰ HSQC (400.1/100.6 MHz, DMSO-d₆) δ 8.93→148.2 (H4→C4), 8.81→114.7 (H14→C14), 8.05→126.7 (H16→C16), 8.02→119.5 (H17→C17), 8.01→130.8 (H18→C18), 7.78-7.71→134.9 (H5→C5), 7.77-7.70→130.9 (H7→C7), 7.56-7.58→116.8 (H8→C8), 7.47-7.50→125.7 (H6→C6); MS m/z, 310 (2%), 173 (100%), 89 (84%).

N-(4-Nitrophenyl)-3-carboxamide-coumarin (2g)

White solid; 79% yield; mp 366 °C; ¹H NMR (400.1 MHz, CDCl₃) δ 11.27 (1H, s, NH), 9.05 (1H, s, H4), 8.28 (2H, d, J₁ 9.1 Hz, H15 and H17), 7.94 (2H, d, J₁ 9.0 Hz, H14 and H18), 7.80-7.73 (2H, m, H5 and H7), 7.52-7.44 (2H, m, H6 and H8);²⁰ HSQC (400.1/100.6 MHz, DMSO-d₆) δ 8.93→148.2 (H4→C4), 8.28-8.30→125.6 (H15/H17→C15/C17), 8.03→130.92 (H14→C14), 8.01-7.99→120.43 (H18→C18), 7.78-7.82→135.1 (H5/H7→C5/C7), 7.56-7.58→116.9 (H6→C6), 7.47-7.50→126.0 (H8→C8); MS m/z, 310 (3%), 173 (100%), 89 (84%).

N-(2-Chlorophenyl)-3-carboxamide-coumarin (2h)

White solid; 92% yield; mp 238 °C, lit.¹⁸ 220-221 °C; ¹H NMR (400.1 MHz, CDCl₃) δ 11.34 (1H, s, NH), 9.02 (1H, s, H4), 8.56 (1H, dd, J₁ 8.2, J₂ 1.0 Hz, H15), 7.77-7.68 (2H, m, H5 and H7), 7.49-7.39 (3H, m, H6, H8 and H17), 7.33 (1H, m, H18), 7.14-7.07 (1H, m, H16);¹⁸ HSQC (400.1/100.6 MHz, DMSO-d₆) δ 9.10→149.5 (H4→C4), 8.52-8.54→122.2 (H15→C15), 8.09-8.07→131.1 (H18→C18), 7.80-7.83→135.3 (H17→C17), 7.57-7.59→116.8 (H7→C7), 7.57-7.59→129.90 (H5→C5), 7.51-7.48→125.9 (H6→C6), 7.44-7.40→128.3 (H8→C8); MS m/z, 299 (5.3%), 173 (100%), 264 (61%).

N-(3-Chlorophenyl)-3-carboxamide-coumarin (2i)

Yellow solid; 84% yield; mp 248 °C, lit.¹⁸ 217-218 °C; ¹H NMR (400.1 MHz, CDCl₃) δ 10.90 (1H, s, NH), 9.02 (1H, s, H4), 7.90 (1H, t, J₁ 1.8 Hz, H14), 7.78-7.69 (2H, m, H5 and H7), 7.56 (1H, dd, J₁ 8.2, J₂ 1.0 Hz, H18), 7.47-7.41 (2H, dd, J₁ 16.3, J₂ 8.4 Hz, H6 and H8), 7.30 (1H, t, J₁ 8.2 Hz, H16), 7.14 (1H, dd, J₁ 8.0, J₁ 0.9 Hz, H17);¹⁸ HSQC (400.1/100.6 MHz, DMSO-d₆) δ 8.91→148.0 (H4→C4), 8.00-8.03→130.9 (H14→C14), 7.98→119.9 (H16→C16), 7.77-7.81→134.9 (H17→C17), 7.58-7.59→118.9 (H18→C18), 7.55-7.57→116.8 (H5→C5), 7.46-7.49→125.8 (H7→C7), 7.42→131.2 (H8→C8), 7.20-7.23→124.6 (H6→C6); MS m/z, 299 (14%), 173 (100%), 89 (34%).

N-(4-Chlorophenyl)-3-carboxamide-coumarin (2j)

Yellow solid; 75% yield; mp 260 °C; lit.¹⁸ 264-265 °C; ¹H NMR (400.1 MHz, CDCl₃) δ 10.88 (1H, s, NH), 9.02 (1H, s, H4), 7.76-7.69 (4H, m, H5, H7, H15 and H17), 7.47-7.41 (2H, dd, J₁ 16.2, J₂ 8.2 Hz, H6 and H8), 7.34 (2H, d, J₁ 8.8 Hz, H14 and H18);¹⁸ HSQC (400.1/100.6 MHz, DMSO-d₆) δ 8.91→147.9 (H4→C4), 7.80→134.7 (H14→C14), 7.79-7.76→122.0 (H15/H17→C15/C17), 7.57-7.55→116.7 (H18→C18), 7.49-7.46→125.8 (H5/H7→C5/C7), 7.46-7.44→129.3 (H6/H8→C6/C8). MS m/z 299 (16%), 173 (100%), 89 (32%).

Urease inhibition assay and determination of half of the maximum inhibitory concentration (IC₅₀)

The urease inhibition screening was performed as described by our research group.²¹ In 96-well plates, the reaction mixture was prepared by addition of 55 μL phosphate buffer solution (100 mM, 7.4 pH) with 1 mM ethylenediaminetetraacetic acid (EDTA), 100 μL of the substrate (10 mM urea), 25 μL of CEU type III (Sigma-Aldrich, Saint Louis, USA) enzyme solution (0.035 mM) and 10 μL of test compound (0.5 mM). After incubation for 30 min at 45 °C, the reaction was cooled down for 10 min. Sequentially, 40 μL of phenol reagent (a mixture of 1% phenol and 0.05% of sodium nitroprusside) and 40 μL of alkaline reagents (0.5% NaOH and 0.1% sodium hypochlorite) were added to each of the wells. After 20 min, the final absorbance of the reaction mixture was recorded at 630 nm using a microplate reader (Synergy HT, BioTek-Agilent, Santa Clara, USA). Urease inhibition (I) was calculated after measuring absorbance values using the equation 1.

(1) $\text{I}\left(\%\right)=100-\left(\left(\frac{\text{Abs}\text{a}}{\text{Abs}\text{c}}\right)\times 100\right)$

where Abs a is the observed absorbance for the samples, and Abs c is the observed absorbance for the control. Thiourea was used as a reference to urease inhibitors.

For the determination of the half-maximal inhibitory concentration (IC₅₀), compounds 2b, 2d, and 2e were evaluated in different concentrations (5, 10, 20, 30, and 50 µM) using the same protocol described above. The assay was performed in triplicate, and IC₅₀ values were calculated by nonlinear regression using GraphPad Prism 8.0.2.²² Statistical analyses were performed using SISVAR software (version 5.70).²³ Distinct letters indicate a significant difference between treatments by Scott-Knott (P < 0.05) test.

Molecular modeling studies

DFT calculations

The DFT calculations were performed with the Gaussian 09 package²⁴ using the functional B3LYP exchange-correlation hybrid²⁵,²⁶ and the 6-311G(d,p) basis-set.²⁷,²⁸ The Polarized Continuum Model (PCM, solvent = water) also was adopted, in agreement with other studies involving molecules with biological activity.²⁹

Protein and ligands preparation for molecular docking study

The X-ray crystal structure of CEU (PDB ID: 3LA4 resolution: 2.05 ų⁰) and HPU (PDB ID: 6ZJA, resolution: 2.00 ų¹) was obtained from the Protein Data Bank.³⁰ All ions (except the Ni^II ions) and waters were removed. To the 3LA4 structure, the KCX490 (Lys490), CME59 (Cys59), CME207 (Cys207), and CME592 (Cys592) were modified since the molecular docking programs did not recognize these residues. To 6ZJA, the B subunit was considered since there is active binding. The center of the binding site to both enzymes was considered the middle between the Ni^II ions: CEU defined as X = -39.959, Y = -44.679, and Z = 74.986; HPU defined as X = 231.5970, Y = 239.1290, and Z = 195.9730. The 2D structures of 2b, 2d, and 2e were constructed using ChemDraw,³² and 3D structures were constructed in the Discovery Studio Visualizer program (v.19.1.0.18287),³³ followed by geometry optimization using the MMFF94 force field implemented in the Avogadro program (v. 1.2.0).³⁴

Molecular docking procedure

To CEU, a consensus molecular docking study was performed using AutoDock³⁵ AutoDock Vina³⁶ and GOLD.³⁷ The AutoDock Tools interface (v 1.5.6) was used for protein and ligand preparation for the molecular docking study with AutoDock 4.2 and AutoDock Vina software. After adding the polar hydrogen atoms, the Kollman and Gasteiger charge methods were applied to the protein and ligand. The grid box dimension considered was 60 × 60 × 60 ų with a grid spacing of 0.375 Å for AutoDock 4.2 and 30 × 30 × 30 ų with a grid spacing of 1 Å for AutoDock Vina. The additional parameters were considered as default. The Genetic Algorithm (GA) and the Lamarckian Genetic Algorithm (LGA) scoring functions were implemented in AutoDock 4.2, and the Vina scoring function was implemented in AutoDock Vina carried out with 10 interactive runs. Then, the lowest energy conformation was used for docking analysis.

The consensus molecular docking using the GOLD program (v. 2020.1) was performed to CEU considering the following scoring functions: GoldScore,³⁷ ChemScore,³⁸ ChemPLP,³⁹ and Astex Statistical Potential (ASP).⁴⁰ About HPU, the molecular docking was validated by redocking procedure considering the crystallized ligand (PDB ID: DJM), using the scoring function ASP. For both enzymes CEU and HPU, the hydrogen atoms were added to the protein supported on ionization inferred by the software, considering pH = 7, the number of genetic operations, and another parameter was set as default in each run. The binding site was defined within a 10 Å radius to CEU and 15 Å to HPU, with the ligands subjected to 50 interactive runs. The highest score was used to determine the best conformation for docking analysis. The poses resulting from the molecular docking studies were selected from the root-mean-square deviation (RMSD) values < 2.0 Å, and the analysis of the intermolecular interactions were carried out using the Discovery Studio Visualizer program (v.19.1.0.18287),³³ and figures were built in the Pymol program (version 3.8).⁴¹

Molecular dynamics simulation

The poses obtained in the docking studies were used for molecular dynamics simulations with the CHARMM36 force field⁴² employing the GROMACS program.⁴³ The ligands parameters were generated through the Cgenff server.⁴⁴,⁴⁵ To CEU and HPU targets, the protonation states were determined using the H⁺⁺ server,⁴⁶,⁴⁷ considering the pH 7.4 and 8.0, respectively. After that, the protein topology was obtained using the pdb2gmx module. Each protein-ligand system was inserted and centered in a triclinic box (dimensions: 10.772 × 7.312 × 7.653 nm; volume: 602.79 nm³) with periodic conditions. The water model considered was TIP3P,⁴⁸ and the complex (target-ligand-water) was neutralized with 19 atoms of Na⁺ ions. The energy minimization, equilibration, and production steps were executed according to our previous work.⁴⁹,⁵⁰ The energy minimization step was realized using the steepest-descent algorithm followed by the gradient conjugate algorithm with a convergence criterion of 1000 and 100 kJ mol^-1 nm^-1, respectively. Next, the equilibration step was realized considering 300 K and 1 bar with position restraint to the whole system, except for ions and water molecules. In the first step (1 ns), the NVT (constant-volume ensemble) ensemble was considered constant, and in the second step (1 ns), the system was viewed as isothermal-isobaric (NPT ensemble). The above equilibration allows the system to reach atom speeds compatible with the target temperature and pressure. Furthermore, the position restraint of the protein-ligand atoms allows ions and water molecules to organize optimally along the protein surface, forming more structured solvation layers. Temperature control was achieved with the V-rescale thermostat⁵¹ and pressure control through the Parrinello-Rahman barostat.⁵² All bonds to hydrogen atoms in the complex were constrained using the linear constrained solver (LINCS) algorithm.⁵³ The long-distance electrostatic interactions were treated using the Particle-Mesh Ewald (PME) algorithm⁵⁴ and the cut-off radius applied to the van der Waals and Coulomb interactions was 1 nm. After equilibration, molecular dynamics (MD) simulations were carried out during 100 ns, considering the NPT ensemble without any position restraint, using 2 fs integration time and 10 Å of cut-off radius to the longdistance interactions.

MD simulation analysis

All complexes were evaluated regarding RMSD, RMSF (root-mean-square fluctuation), and hydrogen bonds (cutoff radius of 4.0 Å and cutoff angle of 30 Å). We used the HbMap2Grace software⁵⁵ to calculate the frequency of the hydrogen bonds and the VMD software⁵⁶ to visualize the trajectories of the simulations. RMSD, RMSF, and hydrogen bonds were plotted with the xmgrace tool.⁵⁷ The binding free energies (ΔG_bind) were calculated using the modified method of molecular mechanics Poisson-Boltzmann surface area (MMPBSA) through the module added to GROMACS 5.1.4 program package.⁵⁸

Results and Discussion

The synthesis of the series of ten N-(R-phenyl)-3-carboxamide-coumarin derivatives (2a-2j) was efficiently reproduced according to Figure 2. Firstly, coumarin-3-carboxylic acid (1) was prepared through a Knovenagel condensation between 2-hydroxybenzaldehyde with Meldrum’s acid, using water as a solvent.¹⁵ Then, acid 1 was converted in situ into the corresponding acyl¹⁶ and condensate with the corresponding aniline (R-C₆H₄-NH₂), leading to the N-(R-phenyl)-3-carboxamide-coumarin derivatives (2a-2j) in 75-92% yields (Figure 2).

Figure 2
Reagents and conditions for the synthesis of N-(R-phenyl)-3-carboxamide-coumarin derivatives (2a-2j): (i) H₂O, 75 °C, 4 h, 92%; (ii) SOCl₂ (20 eq), reflux, 2 h 30 min; (iii) corresponding aniline (R-C₆H₄-NH₂), acetone, reflux, 1 h, 75-92%.

All the N-(R-phenyl)-3-carboxamide-coumarin structures (2a-2j) were confirmed by spectral data, which agree with previous data reported.¹⁷,¹⁸,¹⁹ Since the compounds are already described in the literature and are poorly soluble in common deuterated solvents, we characterized them by mass spectroscopy, ¹H NMR, and heteronuclear single quantum correlation (HSQC) experiments.

The mass spectra showed the molecular ion peaks with the m/z ratio compatible with the substances molar mass (MM), ranging from 265 to 344 g mol^-1. Besides, ¹H NMR spectra showed characteristic signals of the amides 2a-2j, such as broad singlets at 10.84-12.56 ppm relative to the NH proton and the singlet at 9.06-9.00 ppm corresponding to H4 of the coumarin ring. Moreover, the benzoyl group was confirmed by the expected signals for its aromatic protons between 8.75 and 7.01 ppm. The HSQC spectra generally showed the characteristic carbon signals from aromatic rings between 116 at 130 ppm and CH from the Michael acceptor group between 134 at 149 ppm (Figures S1-S35, Supplementary Information (SI) section).

All ten compounds (2a-2j) were assayed against CEU in vitro using the colorimetric indophenol method.⁵⁹ The results of the in vitro screening of 2a-2j (0.5 mM) against the enzyme are shown in Table 1 and were expressed as a percentage of urease inhibition (I), using thiourea as a standard inhibitor. Besides, the IC₅₀ values were determined for derivatives with a urease inhibition above 60% (Table 1).

Table 1
In vitro inhibitory activity against jack bean (Canavalia ensiformis) urease of coumarin derivatives 2a-2j, in reactions containing 10 mM urea, expressed as the percentage of urease inhibition (I) and the half-maximal inhibitory concentration (IC₅₀)

Compound	R	I / %	IC50 / µM
2a	H	55.97 ± 4.98a	Nd
2b	2-Br	64.18 ± 0.00b	35.29 ± 3.86e
2c	3-Br	59.17 ± 1.65a	Nd
2d	4-Br	65.24 ± 0.60b	22.86 ± 4.84e
2e	2-NO2	60.23 ± 0.76a	Nd
2f	3-NO2	56.18 ± 3.17a	Nd
2g	4-NO2	56.29 ± 5.43a	Nd
2h	2-Cl	59.27 ± 0.30a	Nd
2i	3-Cl	47.65 ± 2.86c	Nd
2j	4-Cl	42.43 ± 1.81c	Nd
Thiourea	-	88.59 ± 0.45d	28.89 ± 1.52e

• I: percentage of urease inhibition (%I) ± SEM (standard error of the mean, n = 2); IC₅₀: half maximal inhibitory concentration ± SEM (n = 2); Nd: not determined; Different letters indicate statistical difference by Scott-Knott test (P < 0.05). In bold are highlighted the best compounds of the series.

The in vitro screening results showed that all derivatives are potential urease inhibitors, with an inhibitory percentage ranging from 42 to 65%. In addition, a preliminary SAR study based on the I indicated that compounds presenting bromo at ortho (2b) and para (2d) positions showed the highest I when compared to meta (2c) position, suggesting that stereo and/or electronic effects could influence on the inhibitory activity. Besides, all bromine derivatives (2b-2d) have better I than the corresponding nitro (2e-2g) and chloro (2h-2i) derivatives at the same position in the ring. However, nitro-substituted compounds (2e-2g) and 2h (R = 2-Cl) presented around 60% of urease inhibition, indicating that the presence of chloro at meta (2i) and para (2j) positions seemed to be prejudicial to the inhibitory effect since the values observed were around 47%.

The IC₅₀ was determined for the best compounds 2b and 2d (I = 65%), presenting values of approximately 35 and 23 µM, respectively. In addition, these compounds did not show a significant statistical difference compared to standard inhibitor thiourea, showing that the coumarin class can be considered a promising scaffold for developing urease inhibitors.

Quantum calculations were used to evaluate the electronic structure of compounds 2a, 2b, and 2d. The graphical representation of the frontier molecular orbitals highest occupied molecular orbital (HOMO) and lowest unoccupied molecular orbital (LUMO) in Figure 3 showed that in 2a, HOMO is delocalized by the complete molecular structure while LUMO was localized in coumarin-nucleus. The addition of the Br-group kept the LUMO localized in the coumarin-nucleus region while making the HOMO localized in the region of the phenyl-group, independent of the position of the substituent. These results show that the smaller electron-withdrawing nature of the substituent (Br-group) localized the frontier orbitals in different molecule regions.

Figure 3
Graphical representation of 2a, 2b, and 2d. The color difference is related to the signal of the wave function. Atoms: carbon (black), hydrogen (white), oxygen (red), nitrogen (blue), and bromine (brown).

The energies of frontier molecular orbitals can indicate tendencies in the abilities to withdraw and donating-electron. For example, the high energy of the HOMO indicates the excellent ability of the molecule to donate electrons, and the low energy of the LUMO indicates its ability to receive electrons.⁶⁰

Comparing the results of the Br-substituent in distinct positions (Table 2), the presence of the Br in the 4-position increased the E_HOMO, suggesting the improvement of the coordination ability of the Ni. In the case of the LUMO, the position of the Br-group did not significantly change the energy of this orbital. In general, the increase in E_HOMO and the decrease in the E_LUMO caused by the Br-group in the 4-position improve the coordination abilities of Ni and suffer a nucleophilic attack on carbon B (C9, Figure S2, and Table S1, SI section) in comparison to 2a and 2b.

Table 2
Energies of the frontier molecular orbitals calculated with the theory level B3LYP/6-311G/PCM (solvent = water) compared with IC₅₀ obtained

R	IC50 / µM	EHOMO / eV	ELUMO / eV	Gap / eV
H	Nd	-6.68	-2.34	4.34
2Br	35.29	-6.72	-2.51	4.21
4Br	22.86	-6.54	-2.49	4.05

• IC₅₀: half maximal inhibitory concentration; Nd: not determined.

The gap value is a difference between the energies of LUMO and HOMO, and is an index of chemical reactivity: a smaller gap indicates high reactivity and low kinetic stability.⁶¹ For 2a, 2b, and 2d, it was observed that the addition of the Br-group and their change to the 4-position (compared to the 2-position) decreased the gap value. These results indicate that the position of the Br-group in the 4-position makes the coumarin-derivative more reactive.

The participation percentage of every atom on the HOMO and LUMO of 2a, 2b, and 2d was calculated (Table S1). The results of the HOMOs showed that, for all three molecules, the atoms with a significant tendency to coordinate the Ni of the active site were O18 and N19 (Figure S2, SI section), with a contribution of around 5.00-7.50 and 7.00-12.00% to generating this orbital, respectively, indicating that the coordination to the Ni should occur by these atoms.

The results of the LUMOs showed that, for all three molecules, the atoms with a significant tendency to suffer a nucleophilic attack from S-atom of cysteine residue were C9 (carbon β) and C12 (Figure S2), with a contribution of around 7.00-14.50 and 4.00-12.00% to generating this orbital, respectively. Once C9 (carbon β) would be the atom that should react with a cysteine residue, high participation of this atom on LUMO was expected. Instead, it was observed that the Br-group increased the involvement of C9 on LUMO. The results of frontier orbitals suggest that the substituent group that favored the biological activity of the coumarin derivatives was the Br-group in the 4-position of the phenyl ring (2d).

To predict the potential binding modes of the two promising compounds (2b and 2d), we performed molecular docking studies using 3D structures of the CEU (PDB ID: 3LA4, 2.05 Å resolution)³⁰ and HPU (PDB ID: 6ZJA, 2.00 Å resolution).³¹ Although ureases from diverse microorganisms, such as fungal, bacterial, and plants, have different characteristics, such as the number of polypeptide chains, studies indicate that regardless of the urease source, they have the same ancestor and, thus, share an active site of high similarity.⁶² Therefore, it is common to carry out in vitro studies with the CEU since it is the most commercially accessible.

The CEU 3LA4 did not contain a co-crystalized inhibitor. Therefore, considering the importance of the docking validation process and the reliability of the pose obtained in several studies, we choose to apply herein the consensus docking.⁶³,⁶⁴,⁶⁵ The RMSD between the different poses are shown in Figures 3 4 for compounds 2b (R = 2-Br) and 2d (R = 4-Br), respectively.

Figure 4
(a) Overlapping conformations and (b) RMSD values obtained in molecular docking of compound 2b (R = 2-Br) considering seven scoring functions (LGA, GA, Vina, GoldScore, ChemPLP, ChemScore, and ASP). Main interactions of 2b with amino acid residues from consensus docking by the (c) LGA and GA and (d) ASP and ChemPLP scoring functions. The dashed yellow lines represent hydrogen bonds, and the green ones the distance between atoms. Atom colors: oxygen (red); nitrogen (blue); sulfur (yellow); hydrogen (white), nickel (green).

By overlapping the structures of 2b (R = 2-Br) shown in Figure 4a, we can see the similarity between the conformations obtained by LGA and GA scoring functions, confirmed by the RMSD value of 0.47 Å. Besides, the RMSD (Figure 4b) shows that there is also a consensus between the poses generated by ASP and ChemPLP, with an RMSD of 1.49 Å. Among the consensual amino acid residues, for GA and LGA, Arg439 and Ala436 stand out, responsible for the hydrogen bond with the oxygen atoms of the lactone in the coumarin ring, as well as the Ala440, Met558, Cys592 (flap), Gln635, Ala636 and Met637 residues that interact hydrophobically with the aromatic rings of derivative 2b (Figure 4c). Besides, the distance (4.92 Å) between the thiol group of the Cys592 residue with the β carbon, Michael acceptor group of coumarin, does not allow the nucleophilic attack from the thiol group. Figure 4d reveals the consensual residues for the poses generated by ASP and ChemPLP. The compound only performed hydrophobic interactions in this case, with Ala436, Arg439, Ala440, His492, Asp494, His519, Cys592, His593, His594, Arg609, and Ala636 residues.

Figure 5a shows that compound 2d (R = 4-Br) displays similar poses generated in at least three scoring functions. This result is confirmed by the analysis of the RMSD (Figure 5b), in which LGA, GA, and ChemPLP are consensual, with RMSD between poses below 1.2 Å. Also, ASP and ChemScore demonstrate RMSD < 2.0 Å, but the better consensus obtained was among the first highlighted poses. Figure 5c illustrates the main consensual interactions for compound 2d. We emphasize the hydrogen interaction of Arg439 with the oxygen atom of the coumarin ring, while the other highlighted amino acids Ala436, Ala440, Met588, Cys592, and Asn635, Ala636 and Met637 perform hydrophobic interactions with the derivative. As noted for the 2b derivative, the thiol group of Cys592 is located at 4.70 Å from the carbon β of coumarin of compound 2d, making it difficult for the coumarin to suffer a nucleophilic attack from the group.

Figure 5
(a) Overlapping conformations and (b) RMSD values obtained in molecular docking of compound 2d (R = 4-Br) considering seven scoring functions (LGA, GA, Vina, GoldScore, ChemPLP, ChemScore, and ASP). (c) Main interactions of 2d with amino acid residues from consensus docking by the LGA, GA, and ChemPLP scoring functions. The dashed yellow lines represent hydrogen bonds, and the green ones the distance between atoms. Atom colors: oxygen (red); nitrogen (blue); sulfur (yellow); hydrogen (white), nickel (green).

Overall, these results indicate a consensus for the two compounds between LGA and GA function scoring, with RMSD < 1.0 Å. It is interesting to note that due to the size of the evaluated molecules, the bis-coumarins assessed by Alomari et al.¹¹ have shown the same tendency discussed here since they do not reach the center of the enzyme active site, fitting only on the surface of the urease active site. Furthermore, we investigated the interactions by coumarins derivates 2b and 2d on active sites from bacterial HPU (6ZJA). The docking protocol to the ASP function score was successfully validated by redocking the presented RMSD value of 1.05 Å (Figure S1, SI section).

The compounds 2b and 2d demonstrated binding affinity to catalytic subunit B (Figure 6a). However, differently from what has been observed for CEU, the substituent position drastically altered the observed interactions since the compound 2b was located on the surface of the protein in a region interacting with Met262, Glu292, Asn294, and Gly627 residues, far from the catalytic amino acids and nickel ions (Figure 6b). In contrast, the 2d binding mode was observed close to the active site. In addition, one hydrogen bond between oxygen from the amide scaffold of 2b was observed with His293 (Figure 6b). On the other hand, as mentioned for 2d, the binding mode into the active site from HPU presented hydrophobic interactions mainly with His221 and His248, described by its coordination with Ni²⁺ ion; catalytic amino acids Asp362 and Met366; His322 at the top of the flap region,³¹ and other residues as Gln142, Ala169, Glu222, Asp223, Phe334, and Ala365. In addition, one hydrogen bond between oxygen from the amide scaffold also was observed with Arg338 (Figure 6c).

Figure 6
(a) Overlapping conformations of 2b and 2d coumarin derivatives in the catalytic subunit B of H. pylori urease 6ZJA. (b) Main interactions of 2b (R = 2-Br) and (c) 2d (R = 4-Br) with amino acid residues from molecular docking studies. Atom colors: oxygen (red); nitrogen (blue); sulfur (yellow); hydrogen (white), nickel (green).

We performed MD simulations with CEU and HPU-complexes of compounds 2b and 2d to complement data from molecular docking results. Initially, we investigated the RMSD profiles for both α-carbon atoms of the enzymes. The results of CEU suggested stability compared to the docking and similar behavior between complexes with values of 2.36 ± 0.27 and 2.32 ± 0.37 Å to CEU-2b and CEU-2d, respectively (Figure 7a). The same behavior was observed in HPU results, where the RMSD profiles values were 2.54 ± 0.22 and 3.29 ± 0.36 Å for HPU-2b and HPU-2d complexes, respectively (Figure 7b). In both targets, it is possible that the low standard deviation (SD) indicated α-carbon atoms were stable between the frames during the 100 ns simulation.⁶⁶,⁶⁷

Figure 7
The RMSD analysis of (a) CEU and (b) HPU α-carbon atoms of the enzymes in the presence of the compounds 2b (black) and 2d (orange).

We observed a displacement of 2d from the active site in the first ten ns of the CEU simulations for the RMSD profile of ligands. However, it remained stable in another region with an RMSD value of 8.54 ± 1.30 Å and an SD greater than 1.00 (Figure 7c). On the other hand, ligand 2b showed stability from the beginning of the simulation with smaller SD values and an RMSD value of 7.32 ± 0.46 Å (Figure 7c). During the HPU simulations, we noticed distinct RMSD values among the ligands. Specifically, the RMSD value for 2d (8.57 ± 0.45 Å) was twice as high as 2b (4.22 ± 0.59 Å) but with a lower standard deviation. However, both ligands demonstrated stability from the initial nanoseconds of the MD simulations (Figure 7d).

In addition, the hydrogen bond analysis of the CEU and HPU complexes with compounds 2b and 2d were performed to understand structural changes and predict the binding mode with molecular targets. The examination for CEU complexes demonstrated direct hydrogen bonds of 2b only with His594 with a short 9% lifetime, proving unable to reach the center of the active site of the enzyme (Figure 8a). Interestingly, during MD simulations, 2d displayed a slightly longer lifetime for hydrogen-bond interactions with Ala636 (15.8%), Arg609 (9.6%), and His593 (7.1%) (Figure 8b). It is worth noting that the binding mode observed for 2d in the MD simulations was in the internal region of the active site, as opposed to the docking pose. This displacement of the initial bind location was also confirmed by the RMSD calculation discussed previously.

Figure 8
The predicted binding modes and the lifetime of compounds 2b and 2d hydrogen bonding interactions at the CEU active site during 100 ns MD. The red circles on 2D structures indicate which atoms involve amino acid bonds. Atom colors: oxygen (red); nitrogen (blue); bromine (salmon); nickel (green).

During the analysis of HPU complexes, it was observed that there were intermittent hydrogen bonds between 2b and amino acids His322 and Arg338 and short lifetime. Additionally, a stronger hydrogen bond with up to 55% lifetime was observed with Ala278, which lasted until the final stages of MD simulation (Figure 9a). The amino acids His332, Arg338, and Ala278 were on the HPU active site mobile flap. It is well-known that this flap strictly controls substrate entry and product exit from the urease active site.⁶⁸,⁶⁹ These findings are according to the low RMSD value observed since the flap region is close to the active site of HPU. On the other hand, the RMSD value to the HPU-2d complex was twice more significant, and MD simulations revealed weak interactions with His274 responsible for Ni²⁺ coordination. In contrast, persistent interactions occurred with His314 (55%), showing movement from the surface into the active site (Figure 9b). Thus, continuous interaction with these residues could inhibit the urease.

Figure 9
The predicted binding modes and the lifetime of compounds 2b and 2d hydrogen bonding interactions at the HPU active site during 100 ns MD. The red circles on 2D structures indicate which atoms involve amino acid bonds. Atom colors: oxygen (red); nitrogen (blue); bromine (salmon); nickel (green).

The RMSF evaluated the residue mobility. We analyzed especially the residues that form the active site of both ureases, regions between 136-362 to HPU and 409-633 to CEU (Figure 10). Based on previous observations, the RMSF results showed significant fluctuations of around 3 Å in the amino acids within the CEU active site. These fluctuations are particularly noticeable between 593-606, where hydrogen bond interactions occurred. This suggests that the presence of 2b and 2d could potentially affect the stability of the enzyme (Figure 10a). Additionally, there was considerable fluctuation in residues on flexible loops such as Pro332 (4 Å) due to 2b binding and high fluctuation of 7 Å to Ser666 by 2d. The HPU complexes displayed even higher fluctuations in the amino acids from the active site, with values ranging from 4 to 5 Å. This was observed for both 2b and 2d compounds, particularly in the region of the mobile flap (residues 310-342), according to MD simulation findings (Figure 10b).

Figure 10
The RMSF analysis of (a) CEU and (b) HPU α-carbon atoms of the enzymes in the presence of the compounds 2b (black) and 2d (orange). The colored square in 3D structures indicates highlighted fluctuations of the active site (red) and flexible loops (green and pink).

The G_bind values were evaluated using the frames from the most representative cluster from the last 50 ns of the MD simulation. Compound 2d had a better affinity than 2b for both urease enzymes, presenting G = 21.8 kcal mol¹ to CEU-complex and G = 12.1 kcal mol¹ to HPU-complex (Table 3). The G values for CEU-complexes are more representative ( 15.9 and 21.8 kcal mol¹) than HPU complexes ( 7.08 and 12.1 kcal mol¹), but this fact could be related to the active site characteristics of both enzymes. CEU has a smaller active site (106.95 Ų) and can accommodate smaller ligands. On the other hand, HPU has a hydrophobic and more extensive active area (238.29 Ų)⁷⁰ where larger substances such as these coumarin derivatives can interact more strongly.

Table 3
Binding free energy (mean ± standard deviation) of CEU and HPU-ligands complexes

Energy / (kcal mol-1)	CEU		HPU
	Compound 2b	Compound 2d	Compound 2b	Compound 2d
ΔEvdw	-31.5 ± 1.67	-34.5 ± 2.49	-36.6 ± 1.21	-40.2 ± 2.27
ΔEelect	-0.53 ± 0.89	-2.39 ± 1.22	-3.99 ± 0.61	-7.87 ± 0.60
ΔEsolv	19.5 ± 4.38	18.4 ± 5.19	37.3 ± 4.42	40.0 ± 3.02
ΔEsasa	-3.35 ± 0.15	-3.36 ± 0.22	-3.82 ± 0.18	-4.06 ± 0.16
ΔGbind	-15.9 ± 3.48	-21.8 ± 3.97	-7.08 ± 4.06	-12.1 ± 2.81

• HPU: Helicobacter pylori ureases; CEU: Canavalia ensiformis ureases; ΔEvdw: Van der Walls energy; ΔEelect: eletrostatic energy; Esolv: solvatation energy; Esasa: SASA energy; ΔG_bind: binding free energy.

The solvation energy analysis indicates a higher energy cost for ligands to enter the HPU active site due to its more hydrophobic environment than CEU.⁷¹ Consequently, the entropic cost for ligands to access the site is elevated. Although our molecular dynamics study showed a tendency for the compounds to be more selective for HPU, the G values were lower than for CEU. This is due to the energy values of van der Waals being compensated by the solvation energy, leading to a lower G value for the HPU complexes (Table 3). As previously discussed, this is attributed to the hydrophobicity of the active site. In contrast, for CEU, the ligands tended to bind to the surface of the active site instead of entering the cavity, resulting in a relatively lower energetic cost of solvation and reflecting a higher G observed (Table 3).

In summary, the results of molecular dynamics indicate that the amide and lactone scaffold were essential to the main interactions of both 2b and 2d compounds. The para-position of the bromine atom in the phenyl ring is believed to heighten the compounds affinity, which supports the DFT analyses. Moreover, the molecular dynamics study showed that these coumarin derivatives have a higher chance of selectivity by the bacterial enzyme than the vegetable one. The in vitro tests with CEU confirmed this observation, as derivatives 2b and 2d showed similar inhibition values. Our findings suggest that coumarins hold great potential for future enzymatic and phenotypic assays against H. pylori.

Conclusions

This research outlines a method for efficiently synthesizing ten N-(R-phenyl)-3-carboxamide-coumarin derivatives with high yields. All the compounds were effective inhibitors of CEU enzymatic activity in vitro. A study of structure-activity relationships by DFT discovered that the presence of bromo, particularly in the ortho and para positions, enhanced the compounds inhibitory activity. At the same time, a similar effect was not observed in the meta position. This finding was supported by molecular docking analysis, which indicated that the 4-Br substituent position is crucial for interacting with catalytic amino acids on the active site of both enzymes. Molecular dynamics simulations revealed that these coumarin derivatives may have selectivity for HPU, making them promising candidates for future enzymatic and phenotypic assays against H. pylori.

Supplementary Information

Supplementary information about DFT calculations and RMN spectra is free of charge athttp://jbcs.sbq.org.br as PDF file.

Acknowledgments

This work was supported by the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior Brasil (CAPES) under grant (number 001 and 88887.507109/2020-00) and Fundação de Amparo à Pesquisa do Estado do Rio de Janeiro (FAPERJ) (E-26/010.210.513/2019, E-26/205.966/2022, SEI-260003/009792/2021, SEI-260003/007043/2022 and SEI-260003/003788/2022). The authors wish to thank the Laboratory of Spectroscopy (ESPEC-UEL), Multiuser Laboratory of Nuclear Magnetic Resonance in Liquids (LABRMN-L/UFRJ), and Prof Dr Alviclér Magalhães for 2D-NMR analysis. Finally, the Centro Nacional de Processamento de Alto Desempenho em São Paulo (CENAPAD-SP) for the resources for the Molecular Dynamics simulations.

References

• ¹
  Mobley, H. L. T.; Island, M. D.; Hausinger, R. P.; Microbiol. Re v 1995, 59, 451. [Crossref]
  » Crossref
• ²
  Amtul, Z.; Kausar, N.; Follmer, C.; Rozmahel, R. F.; Atta-Ur-Rahman; Kazmi, S. A.; Shekhani, M. S.; Eriksen, J. L.; Khan, K. M.; Choudhary, M. I.; Bioorg. Med. Chem 2006, 14, 6737. [Crossref]
  » Crossref
• ³
  Krajewska, B.; J. Mol. Catal. B: Enzym 2009, 59, 9. [Crossref]
  » Crossref
• ⁴
  Modolo, L. V. ; da-Silva, C. J.; Brandão, D. S.; Chaves, I. S.; J. Adv. Res 2018, 13, 29. [Crossref]
  » Crossref
• ⁵
  Ito, Y.; Shibata, K.; Hongo, A.; Kinoshita, M.; Eur. J. Pharmacol 1998, 345, 193. [Crossref]
  » Crossref
• ⁶
  Kuehne, M. E.; He, L.; Jokiel, P. A.; Pace, C. J.; Fleck, M. W.; Maisonneuve, I. M.; Glick, S. D.; Bidlack, J. M.; J. Med. Chem 2003, 46, 2716. [Crossref]
  » Crossref
• ⁷
  Kot, M.; Zaborska, W.; J. Enzyme Inhib. Med. Chem 2003, 18, 413. [Crossref]
  » Crossref
• ⁸
  Kot, M.; Zaborska, W.; J. Enzyme Inhib. Med. Chem 2006, 21, 537. [Crossref]
  » Crossref
• ⁹
  Brito, T. O.; Souza, A. X.; Mota, Y. C. C.; Morais, V. S. S.; de Souza, L. T.; de Fátima, Â.; Macedo, F.; Modolo, L. V. ; RSC Adv 2015, 55, 44507. [Crossref]
  » Crossref
• ¹⁰
  Kalfa, A.; Ayazoglu, E.; Colak, A.; Yasar, A.; Bekircan, O.; Indian J. Chem 2019, 58B, 720. [Crossref]
  » Crossref
• ¹¹
  Alomari, M.; Taha, M.; Imran, S.; Jamil, W.; Selvaraj, M.; Uddin, N.; Rahim, F.; Bioorg. Chem 2019, 92, 103235. [Crossref]
  » Crossref
• ¹²
  Salar, U.; Nizamani, A.; Arshad, F.; Khan, K. M.; Fakhri, M. I.; Perveen, S.; Ahmed, N.; Choudhary, M. I.; Bioorg. Chem 2019, 91, 103170. [Crossref]
  » Crossref
• ¹³
  Saeed, A.; Mahesar, P. A.; Channar, P. A.; Larik, F. A.; Abbas, Q.; Hassan, M.; Raza, H.; Seo, S. Y. ; Chem. Biodiversity 2017, 14, e1700035. [Crossref]
  » Crossref
• ¹⁴
  Khan, I.; Khan, A.; Ahsan Halim, S.; Saeed, A.; Mehsud, S.; Csuk, R.; Al-Harrasi, A.; Ibrar, A.; Int. J. Biol. Macromol 2020, 142, 345. [Crossref]
  » Crossref
• ¹⁵
  Deshmukh, M. N.; Burud, R.; Baldino, C.; Chan, P. C. M.; Liu, J.; Synth. Commun 2003, 33, 3299. [Crossref]
  » Crossref
• ¹⁶
  Bispo, M. L. F.; Gonçalves, R. S. B.; Lima, C. H. S.; Cardoso, L. N. F.; Lourenço, M. C. S.; de Souza, M. V. N.; J. Heterocycl. Chem 2012, 49, 1317. [Crossref]
  » Crossref
• ¹⁷
  Sard, H.; Meltzer, P. C.; Razdan, R. K.; J. Heterocycl. Chem 1985, 22, 257. [Crossref]
  » Crossref
• ¹⁸
  Fonseca, A.; Matos, M. J.; Vilar, S.; Kachler, S.; Klotz, K. N.; Uriarte, E.; Borges, F.; Chem. Biol. Drug Des 2018, 91, 245. [Crossref]
  » Crossref
• ¹⁹
  Matos, M. J.; Vilar, S.; Vazquez-Rodriguez, S.; Kachler, S.; Klotz, K.-N.; Buccioni, M.; Delogu, G.; Santana, L.; Uriarte, E.; Borges, F.; J. Med. Chem 2020, 63, 2577. [Crossref]
  » Crossref
• ²⁰
  Payra, S.; Saha, A.; Banerjee, S.; ChemistrySelect 2018, 3, 7535. [Crossref]
  » Crossref
• ²¹
  Camargo, P. G.; Fabris, M.; Nakamae, M. Y. T.; Oliveira, B. G. F.; Lima, C. H. S.; de Fátima, Â.; Bispo, M. L. F.; Macedo Jr., F.; Chem.-Biol. Interact 2022, 365, 110045. [Crossref]
  » Crossref
• ²²
  GraphPad Prism, 8.0.2; Dotmatics, Boston, U.S.A., 2019.
• ²³
  Ferreira, D. F.; Ciênc. Agrotec 2014, 38, 109. [Crossref]
  » Crossref
• ²⁴
  Frisch, M. J.; Trucks, G. W.; Schlegel, H. B.; Scuseria, G. E.; Robb, M. A.; Cheeseman, J. R.; Scalmani, G.; Barone, V. ; Petersson, G. A.; Nakatsuji, H.; Li, X.; Caricato, M.; Marenich, A.; Bloino, J.; Janesko, B. G.; Gomperts, R.; Mennucci, B.; Hratchian, H. P.; Ortiz, J. V.; Izmaylov, A. F.; Sonnenberg, J. L.; Williams-Young, D.; Ding, F.; Lipparini, F.; Egidi, F.; Goings, J.; Peng, B.; Petrone, A.; Henderson, T.; Ranasinghe, D.; Zakrzewski, V. G.; Gao, J.; Rega, N.; Zheng, G.; Liang, W.; Hada, M.; Ehara, M.; Toyota, K.; Fukuda, R.; Hasegawa, J.; Ishida, M.; Nakajima, T.; Honda, Y. ; Kitao, O.; Nakai, H.; Vreven, T.; Throssell, K.; Jr., J. A. M.; Peralta, J. E.; Ogliaro, F.; Bearpark, M.; Heyd, J. J.; Brothers, E.; Kudin, K. N.; Staroverov, V. N.; Keith, T.; Kobayashi, R.; Normand, J.; Raghavachari, K.; Rendell, A.; Burant, J. C.; Iyengar, S. S.; Tomasi, J.; Cossi, M.; Millam, J. M.; Klene, M.; Adamo, C.; Cammi, R.; Ochterski, J. W.; Martin, R. L.; Morokuma, K.; Farkas, O.; Foresman, J. B.; Fox, D. J.; Gaussian, v. 0.9, Inc., Wallingford CT, 2016.
• ²⁵
  Becke, A. D.; J. Chem. Phys 1993, 98, 5648. [Crossref]
  » Crossref
• ²⁶
  Lee, C.; Yang, W.; Parr, R. G.; Phys. Rev. B 1988, 37, 785. [Crossref]
  » Crossref
• ²⁷
  Krishnan, R.; Binkley, J. S.; Seeger, R.; Pople, J. A.; J. Chem. Phys 1980, 72, 650. [Crossref]
  » Crossref
• ²⁸
  McLean, A. D.; Chandler, G. S.; J. Chem. Phys 1980, 72, 5639. [Crossref]
  » Crossref
• ²⁹
  da Silva, T. U.; da Silva, E. T.; Pougy, K. C.; Lima, C. H. S.; Machado, S. P.; J. Inorg. Biochem 2021, 2017, 111359. [Crossref]
  » Crossref
• ³⁰
  Balasubramanian, A.; Ponnuraj, K.; J. Mol. Biol 2010, 400, 274. [Crossref]
  » Crossref
• ³¹
  Cunha, E. S.; Chen, X.; Sanz-Gaitero, M.; Mills, D. J.; Luecke, H.; Nat. Commun 2021, 12, 230. [Crossref]
  » Crossref
• ³²
  Evans, D. A.; Angew. Chem., Int. Ed 2014, 53, 11140. [Crossref]
  » Crossref
• ³³
  Systèmes, D.; Biovia Discovery Studio, Dassault Systèmes, France, 2016.
• ³⁴
  Hanwell, M. D.; Curtis, D. E.; Lonie, D. C.; Vandermeersch, T.; Zurek, E.; Hutchison, G. R.; J. Cheminform 2012, 4, 17. [Crossref]
  » Crossref
• ³⁵
  Morris, G. M.; Huey, R.; Lindstrom, W.; Sanner, M. F.; Belew, R. K.; Goodsell, Olson, A. J. D. S.; J. Comput. Chem 2009, 30, 2785. [Crossref]
  » Crossref
• ³⁶
  Trott, O.; Olson, A. J.; J. Comput. Chem 2009, 31, 455. [Crossref]
  » Crossref
• ³⁷
  Verdonk, M. L.; Cole, J. C.; Hartshorn, M. J.; Murray, C. W.; Taylor, R. D.; Proteins: Struct., Funct., Bioinf 2003, 52, 609. [Crossref]
  » Crossref
• ³⁸
  Eldridge, M. D.; Murray, C. W.; Auton, T. R.; Paolini, G. V; Mee, R. P.; J. Comput.-Aided Mol. Des 1997, 11, 425. [Crossref]
  » Crossref
• ³⁹
  Korb, O.; Stützle, T.; Exner, T. E.; Swarm Intell 2007, 1, 115. [Crossref]
  » Crossref
• ⁴⁰
  Mooij, W. T. M.; Verdonk, M. L.; Proteins: Struct., Funct., Bioinf 2005, 61, 272. [Crossref]
  » Crossref
• ⁴¹
  DeLano, W. L.; CCP4 Newsl. Protein Crystallogr 2002, 40, 82. [Link] accessed in August 2023
  » Link
• ⁴²
  Vanommeslaeghe, K.; Hatcher, E.; Acharya, C.; Kundu, S.; Zhong, S.; Shim, J.; Darian, E.; Guvench, O.; Lopes, P.; Vorobyov, I.; Mackerell, A. D.; J. Comput. Chem 2009, 31, 671. [Crossref]
  » Crossref
• ⁴³
  Abraham, M. J.; Murtola, T.; Schulz, R.; Páll, S.; Smith, J. C.; Hess, B.; Lindahl, E.; SoftwareX 2015, 1-2, 19. [Crossref]
  » Crossref
• ⁴⁴
  Huang, J.; MacKerell Jr., A. D.; J. Comput. Chem 2013, 34, 2135. [Crossref]
  » Crossref
• ⁴⁵
  CGenFF, https://cgenff.umaryland.edu/, accessed in August 2023.
  » https://cgenff.umaryland.edu/,
• ⁴⁶
  Gordon, J. C.; Myers, J. B.; Folta, T.; Shoja, V.; Heath, L. S.; Onufriev, A.; Nucleic Acids Res 2005, 33, W368. [Crossref]
  » Crossref
• ⁴⁷
  H++, http://newbiophysics.cs.vt.edu/H++/, accessed in August 2023.
  » http://newbiophysics.cs.vt.edu/H++/,
• ⁴⁸
  Mark, P.; Nilsson, L.; J. Phys. Chem. A 2001, 105, 9954. [Crossref]
  » Crossref
• ⁴⁹
  de Santiago-Silva, K. M.; Camargo, P.; Gomes, G. F. S.; Sotero, A. P.; Orsato, A.; Perez, C. C.; Nakazato, G.; da Silva Lima, C. H.; Bispo, M.; J. Biomol. Struct. Dyn 2022, 1. [Crossref]
  » Crossref
• ⁵⁰
  da Silva, T. U.; Pougy, K. C.; Albuquerque, M. G.; da Silva Lima, C. H.; Machado, S. P.; J. Biomol. Struct. Dyn 2022, 40, 3481. [Crossref]
  » Crossref
• ⁵¹
  Bussi, G.; Donadio, D.; Parrinello, M.; J. Chem. Phys 2007, 126, 014101. [Crossref]
  » Crossref
• ⁵²
  Parrinello, M.; Rahman, A.; J. Appl. Phys 1981, 52, 7182. [Crossref]
  » Crossref
• ⁵³
  Hess, B.; Bekker, H.; Berendsen, H. J. C.; Fraaije, J. G. E. M.; J. Comput. Chem 1997, 18, 1463. [Crossref]
  » Crossref
• ⁵⁴
  Darden, T.; York, D.; Pedersen, L.; J. Chem. Phys 1993, 98, 10089. [Crossref]
  » Crossref
• ⁵⁵
  Gomes, D. E. B.; da Silva, A. W.; Lins, R. D.; Pascutti, P. G.; A., S.; HbMap2Grace software, Universidade Federal do Rio de Janeiro, Rio de Janeiro, Brazil, 2009.
• ⁵⁶
  Humphrey, W.; Dalke, A.; Schulten, K.; J. Mol. Graph 1996, 14, 33. [Crossref]
  » Crossref
• ⁵⁷
  XMGRACE, 5.1.19; Center for Coastal and Land-Margin Research, Oregon Graduate Institute of Science and Technology, U.S.A., 2005.
• ⁵⁸
  Kumari, R.; Kumar, R.; Lynn, A.; J. Chem. Inf. Model 2014, 54, 1951. [Crossref]
  » Crossref
• ⁵⁹
  Weatherburn, M. W.; Anal. Chem 1967, 39, 971. [Crossref]
  » Crossref
• ⁶⁰
  Ngo Nyobe, J. C.; Andiga, L. G. E.; Mama, D. B.; Amana, B. A.; Mfomo, J. Z.; Alfred, T. F. A.; Ndom, J. C.; J. Chem 2019, 2019, ID 7684941. [Crossref]
  » Crossref
• ⁶¹
  Miar, M.; Shiroudi, A.; Pourshamsian, K.; Oliaey, A. R.; Hatamjafari, F.; J. Chem. Res 2020, 174751982093209. [Crossref]
  » Crossref
• ⁶²
  Kappaun, K.; Piovesan, A. R.; Carlini, C. R.; Ligabue-Braun, R.; J. Adv. Res 2018, 13, 3. [Crossref]
  » Crossref
• ⁶³
  Tuccinardi, T.; Poli, G.; Romboli, V.; Giordano, A.; Martinelli, A.; J. Chem. Inf. Model 2014, 54, 2980. [Crossref]
  » Crossref
• ⁶⁴
  Gimeno, A.; Mestres-Truyol, J.; Ojeda-Montes, M. J.; Macip, G.; Saldivar-Espinoza, B.; Cereto-Massagué, A.; Pujadas, G.; Garcia-Vallvé, S.; Int. J. Mol. Sci 2020, 21, 3793. [Crossref]
  » Crossref
• ⁶⁵
  Palacio-Rodríguez, K.; Lans, I.; Cavasotto, C. N.; Cossio, P.; Sci. Rep 2019, 9, 5142. [Crossref]
  » Crossref
• ⁶⁶
  Albuquerque, S. O.; Barros, T. G.; Dias, L. R. S.; Lima, C. H. S.; Azevedo, P. H. R. A.; Flores-Junior, L. A. P.; dos Santos, E. G.; Loponte, H. F.; Pinheiro, S.; Dias, W. B.; Muri, E. M. F.; Todeschini, A. R.; Eur. J. Pharm. Sci 2020, 154, 105510. [Crossref]
  » Crossref
• ⁶⁷
  da Silva, T. U.; Pougy, K. C.; Albuquerque, M. G.; Lima, C. H. S.; Machado, S. P.; J. Biomol. Struct. Dyn 2023, 41, 2466. [Crossref]
  » Crossref
• ⁶⁸
  Roberts, B. P.; Miller, B. R.; Roitberg, A. E.; Merz, K. M.; J. Am. Chem. Soc 2012, 134, 9934. [Crossref]
  » Crossref
• ⁶⁹
  Zhou, J.-T.; Li, C.-L.; Tan, L.-H.; Xu, Y.-F.; Liu, Y.-H.; Mo, Z.-Z.; Dou, Y.-X.; Su, R.; Su, Z.-R.; Huang, P.; Xie, J.-H.; PLoS One 2017, 12, e0168944. [Crossref]
  » Crossref
• ⁷⁰
  Fabris, M.; Nascimento-Júnior, N. M.; Bispo, M. L. F.; Camargo, P. G.; 2023, 29, 777. [Crossref]
  » Crossref
• ⁷¹
  Arora, R.; Issar, U.; Kakkar, R.; J. Mol. Graph. Modell 2018, 83, 64. [Crossref]
  » Crossref

Edited by

Editor handled this article: Brenno A. D. Neto

Publication Dates

• Publication in this collection
  01 Mar 2024
• Date of issue
  2024

History

• Received
  30 June 2023
• Accepted
  05 Sept 2023