Preparation, characterization, antioxidant activity of 1-(2-furoyl) thiourea derivatives and study the molecular docking of them as potent inhibitors of Urease enzyme

and examined them


Introduction
Addition to thiourea derivatives' important role in many biological aspects, acyl thiourea occupied a special position in these biological activities in the literatures due to its unique characteristics 1 . Novel trifluoromethyl pyrazole acyl thiourea derivatives showed a promising fungicidal activity in vivo and could become in the future lead compounds in the development of this field 2 . Also, acetylphenol-based acyl thioureas compounds exhibit very good results as a Helicobacter pylori urease inhibitor in vitro comparing to standard drugs 3 . Acyl thiourea derivatives containing difluoromethyl pyrazole moiety showed good antibacterial activity addition to in vivo fungicidal activity against Botrytis cinerea and Fusarium oxysporum 4 . H. Aziz and his coworkers synthesized ten Nacyl-morpholine-4-carbothioamide derivatives, and their study showed that some of the derivatives possess a considerable antioxidant activity as well as have highly hemocompatible that make them biosafe and the molecular docking study showed two of them have good binding mode 5 .
Thiourea derivatives are well-known family of ligands that can form complexes with transition metals and post-transition metals like Ni(II), Cu(I/II), Co(III), Zn(II), Ag(I), Pb((II) etc. Acyl thioureas can act as bidentate chelators ligand by coordination to the central atom through S and O atoms, K. Ghazal and his coworkers successfully synthesized two acyl thioureas and coordinated them to nickel(II) and copper(II) and achieve good activities in antibacterial and antifungal evaluation 6 . K. Jeyalakshmi, et al synthesized copper(I) bromide complexes of aroyl thiourea ligands (L) [CuBr(L)3] and examined them as anticancer agents in vitro 7 . D. Mitrea and V. Cîrcu synthesized new series of acyl thiourea and testing their ion recognition and sensing properties by UV-VIS spectroscopy and the results concluded that these novel thioureas have the ability to sense biological important ions like floride and copper(II) 8 .
The role of thiourea in the preparation of nanoparticles is unforgettable. Hakeem, et al, used thiourea as a source of S element in synthesizing Pb1-xCdxS nanoparticles and using them in solar cell applications 9 . Moreover, Shanan, et al, synthesized copper sulfide nanoparticles by the reaction between different concentrations of thiourea and copper nitrate 10 .
Al-Amily, et al, successfully synthesized Nadipoyl / N-pimeloyl monoanilide thiourea and performed molecular docking study for these two compounds as histone deacetylases inhibitors 11 .
In our case, we synthesized thirteen 1-(2furoyl)thiourea derivatives in a simple reaction that consists of two steps in one pot , characterized them spectroscopically, then evaluated them as antioxidant agents in vitro, and finally studied them theoretically as inhibitors of Bacillus pasteurii urease enzyme through molecular docking study.

Chemicals and Methods
Gallenkamp capillary melting point apparatus was used to measure melting points in open glass capillaries and were uncorrected. The 1 H NMR and 13 C NMR spectra were recorded in DMSO-d6 as a solvent on Bruker Avance Neo 400 MHz spectrometer and tetramethyl silane as an internal standard. Shimadzu FT-IR spectroscopy (with KBr disc technique) was used to record IR spectra. TLC Sheets (Silica gel coated Aluminum sheet) made be Merk were used to observe the reaction progression and the Eluent was used is a mixture of Ethyl acetate: Petroleum Ether (2:1), Iodine vapor in a closed was used as a TLC visualizing agent.

General procedure for synthesis of thiourea derivatives 1-13
A mixture of 2-furoyl chloride 0.662 g, 0.5 ml, 5.07 mmol and Potassium thiocyanate 0.4928g, 5.07 mmol in 10 ml of dry Acetone was stirred for 1 hr. at room temperature to give 2-furoyl isothiocyanate then, at the same pot and without separating the reaction components, 5.07 mmol of 2-aminobenzothiazole for compound 1, 4aminoacetophenone for compound 2, benzyl amine for compound 3, 4-hydroxyaniline for compound 4, naphthyl amine for compound 5, 4aminobenzenesulfonamide for compound 6, 2aminobenzothiazole for compound 7, dibenzylamine for compound 8, diphenylamine for compound 9, N-ethylaniline for compound 10, or 2.535 mmol of p-phenylenediamine for compound 11, m-phenylenediamine for compound 12, o-phenylenediamine for compound 13 dissolved in dry acetone was added dropwise and the mixture refluxed for 3 to 5 hrs. (depending on TLC results) in order to give thiourea derivatives 12,13 as shown in scheme 1. After the reaction was completed, the mixture was poured into crushed ice. The product precipitated, filtered, washed and dried. Physical properties of compounds 1-13 are listed in Table 1.

Antioxidant activity
Preparation of the solutions of DPPH and the samples 50 ppm of DPPH (1,1-Diphenyl-2-picrylhydrazyl) was prepared by dissolving 5 mg in 100 ml of methanol and kept away from light ( a test tube covered by aluminum foil). A stock solution of some prepared compound 1-13 was prepared by dissolving 1 mg of the sample in 10 ml of methanol to get 100 ppm then diluted them in twice to achieve 50 and 25 ppm, moreover, Ascorbic acid (Vitamin C) was also prepared in the same concentration to use it as a standard solution.

Spectrophotometric measurement method
In a test tube, 1 ml of each of the concentrations 100, 50 and 25 ppm of the compounds was mixed with 1 ml of DPPH solution and incubated for 1 hr. at 37℃ in a dark area. The blank solution was only contained 1 ml of DPPH. After finishing the incubation, the absorbance of each solution was measured in a spectrophotometer at 517nm wavelength.

Molecular docking study Preparation of ligands
All compounds 1-13 were drawn as 3D structures by Chemdraw, then they were minimized their energy by Chem3D via MMFF94 minimization and saved in sdf format then converted to pdbqt format by Open Babel GUI 14 which that format is suitable to use in Autodock software.

Preparation of receptor protein
Crystal structure of Urease (pdb id: 4ubp) was downloaded from protein data bank 15 . Water molecules and other HET atoms (KCX, HAE, Ni and ACE) were removed from pdb file, then polar hydrogens were added by PyMol 16 .

Molecular docking method
Autodock 4.2.6 was used for molecular docking 17 and each of compounds 1-13 was docked separately into the active site of the receptor using default setting. A grid box with coordination of X: 29.143, Y: 72.792, Z: 71.848 Å with 0.5 Å of grid spacing and number of points X, Y, Z dimensions were all each equal to 60. For each of the docked compounds the best conformation was selected with the lowest binding energy among 50 conformations that were generated by Autodock. Finally, the Ligand-Receptor complex with the lowest binding energy was saved as pdbqt format then converted to pdb format by Open Babel GUI 14 for further analysis.

Docking results visualization
The pdb files for each Ligand-Receptor complex transformed into PILP website 18 and to ProteinsPlus website 19,20 to visualize the ligandreceptor interaction in 3D and in 2D respectively to https://dx.doi.org/10.21123/bsj.2023.7745 P-ISSN: 2078-8665 -E-ISSN: 2411-7986 Baghdad Science Journal show the details of the interactions between the ligand and the protein residues.

Chemistry
The reaction between 2-furoyl chloride and potassium thiocyanate undergoes (nucleophilic addition-elimination) mechanism by attacking the isothiocyanate group on carbonyl group in acyl chloride then followed by leaving the chloride ion to form the acyl isothiocyanate 21 , this mechanism takes the SN2 path. The condensation reaction between the in situ generated furoyl isothiocyanate and the amine can be understood by the nucleophilic addition of the amine by attacking its lone-pair of electrons on the carbon of isothiocyanate group 22 .

Spectral data
Spectral data for these set of compounds indicate the synthesis of thiourea group by presence of new groups as thiocarbonyl group, The FT-IR spectra of compounds 1-13 confirms the synthesis of thiourea by the presence of N-H absorption in the region of 3404-3164 cm -1 indicating the presence of secondary amine groups and at the same time, disappearance of NH2 absorption that was already existed in the starting materials (amines). Moreover, the absorption in the region of 1278-1178 cm -1 clearly indicates to the thiocarbonyl group of the thiourea and ν(C=O) was shifted from acid chloride region 1775 and 1746 cm -1 for the starting material (2-furoyl chloride) to the amide carbonyl region 1697-1656 cm -1 , these results were agreed with the data presented in literatures [23][24][25][26] in δ (ppm) spectrum of some compounds 1-13 also proved the synthesis of thioureas by the presence of the following chemical shifts ranges 14.04-10.02 (1H, s, CSNH), 12.06-9.83 (1H, s, CONHCS), these results were agreed with the data presented in literatures 23,27 . The 13 C NMR (DMSO d6) in δ (ppm) spectrum of some compounds 1-13 also proved the synthesis of thioureas by the presence of the following chemical shifts ranges in 180-149 (C=S) and 157-145 (C=O) 27 .

Antioxidant activity / DPPH Radical Scavenging Activity
The antioxidant activity measurement in this method can be identified as the ability of a compound to capture the free radicals that are generated by DPPH. Several concentrations of the compounds were prepared in order to examine their inhibition ability and to find out the (IC50) value. The Vitamin C was used as a standard solution because of its good-known ability as antioxidant due to its hydroxyl groups stable the free radicals which lead to owning high inhibition ability. Keeping the solution under the test away from light is very important because the negative effect of the light on the free radicals by duplicates them and affects the reading. and according to the literature 28 , if the IC50 value >250 (µg/mL) means inactive; > 100-250 (µg/mL) weakly active; > 50-100 (µg/mL) moderately active; 10-50 (µg/mL) strongly active;<10 (µg/mL) very strongly active. In general, the higher concentration of the compounds, the higher antioxidant activity due to the increasing the capacity of capturing of the free radicals which agreed with the literature 29 . In these synthesized compounds 1-13, and as illustrated in Table 2, and Fig. 7, the best antioxidant activity compound is compound 4 which reflects very clearly that the hydroxyl group it has play very crucial role in antioxidant which resemble in such a way the Ascorbic acid which has known a great antioxidant because of its many hydroxyl group it has, the compound 4 in this case exceeds the Ascorbic acid in terms of (IC50) value due to its high activity in lowest concentration 25 ppm as it is illustrated in Table 2. The second-best antioxidant compound is compound 1 which has fused aromatic ring (benzothiazole) that may give its highly antioxidant activity comparing to Ascorbic acid especially in the lowest concentration 25 ppm which that lead finally to better IC50.

Molecular docking study
All the synthesized compounds 1-13 were docked into the active site of Urease (pdb id:4ubp) Fig. 8 to study the interactions between these compounds and the receptor (urease) in order to estimate the inhibition activity of these compounds and the free energy of binding ΔG⁰, kcal/mol which represents hydrogen bonding, π-π interaction, Van  11 Ascorbic acid der Waals forces and other type of interactions. As clearly described in Table 3, compounds 13 and 12 have the best docking scores then it is followed by compound 6. compounds 13 and 12 have two groups of thiourea moiety and compound 6 has sulfone group, for all compounds in this series, only N and O atoms within the compounds make the hydrogen bonding with the target protein but not the S atoms. From these results, it can be suggested that the bulky ligands with more than one thiourea moiety could give better inhibition activity with urease protein (4ubp) see Figs. 9 and 10.

Conclusion
A series of 1-(2-furoyl)thiourea derivatives were prepared successfully by reaction of isothiocyanate, that were prepared in situ, with different amines. Spectroscopic techniques with yield percentage ranges from 87.5 to 33.4 (FT-IR, 1 H NMR and 13 C NMR) proved the synthesis process. The antioxidant activity testes showed that compound 4 has the best antioxidant activity among the group due to possession a hydroxyl group in its structure and it also exceeds the activity of the ascorbic acid in the lowest concentrations that why it has better IC50 than the ascorbic acid, also, the compound 1 exhibits good antioxidant activity especially in low concentration. The molecular docking study showed that compounds 13 and 12 exhibit better inhibition activity that implied bisthiourea compounds have better chance to bind to protein amino acid residues and then better inhibition activity.

Author's Contribution Statement
All the chemical aspects including compounds synthesis, characterization and antioxidant activity measurements are performed by O. H. R. A. and A. Q. O.