Synthesis and Biological Evaluation of Pyrimidine-oxazolidin-2-arylimino Hybrid Molecules as Antibacterial Agents

Pyrimidine-1,3-oxazolidin-2-arylimino hybrids have been synthesized as a new class of antibacterial agents. The synthetic approach exploits a Cu(II)-catalyzed intramolecular halkoxyhalogenation of alkynyl ureas, followed by a Suzuki coupling reaction with 2,4-dimethoxypyrimidin-5-boronic acid. Biological screenings revealed that most of the compounds showed moderate to good activity against two Gram-positive (B. subtilis, S. aureus) and three Gram-negative (P. aeruginosa, S. typhi, K. pneumonia) pathogenic strains. A molecular docking study, performed in the crystal structure of 50S ribosomal unit of Haloarcula marismortui, indicated that pyrimidine-oxazolidin-2-arylimino hybrids 8c and 8h exhibited a high binding affinity (−9.65 and −10.74 kcal/mol), which was in agreement with their good antibacterial activity. The obtained results suggest that the combination of pyrimidine and oxazolidone moieties can be considered as a valid basis to develop new further modifications towards more efficacious antibacterial compounds.


General
All chemicals were purchased from Sigma-Aldrich Chemical Co. The solvent was removed at aspirator pressure using a rotary evaporator. TLC was performed with Merck precoated TLC plates, and the compounds were made visible using a fluorescent inspection lamp and iodine vapor. Gravity chromatography was done with Merck silica gel 60 (mesh size 63-200 m). Nuclear magnetic resonance spectra were recorded on a Varian Inova instrument, operating at 500 MHz for 1 H NMR and 75 MHz for 13 C NMR. Chemical shifts ( ) for 1 H NMR spectra are reported in ppm downfield relative to the center line of CDCl3 triplet at 7.26 ppm. Chemical shifts for 13 C NMR spectra are reported in ppm downfield relative to the center line of CDCl3 triplet at 77.23 ppm. The abbreviations s, d, t, and m stand for the resonance multiplicities singlet, doublet, triplet, and multiplet, respectively. 13 C spectra, are 1 H decoupled, and multiciplities were determined by APT pulse sequence. The melting points were recorded on a Boëtius hot plate microscope. FT-IR spectra were recorded on FT-IR Shimadzu spectrometer (4000-400 cm -1. EI-MS and HRMS were performed with Finnigan MAT 95, EI: 70 eV, R:10000.

General procedure for syntheses of 5-[(iodomethylidene)-2-arylimino]oxazolidines (7a-i).
A solution of alkynyl amide (6) (1 mmol), CuI2 (0.05 mmol) and NIS (1 mmol) in MeCN (15 ml) was stirred under nitrogen at 70 °C for 7 h and then left at room temperature overnight. The reaction mixture turns red-brown and a precipitate appears. After the completion of the reaction, the mixture was filtered on a pad of silica gel, and washed with EtOAc until the silica appeared uncolored. The solvent was removed under reduced pressure and the crude product was purified by silica gel column chromatography to yield the final desired compounds 7a-i.

Antimicrobial al activity. MIC testing
The MIC values were tested using the agar dilution method according to the National Committee for Clinical Laboratory Standards (NCCLS, 4th ed). 24 Briefly, compounds were dissolved in 25% DMSO to a solution with concentration of 1280 μg/mL. Ciprofloxacin and fluconazol, used as reference drugs, were directly dissolved in 10 mL distilled water. All the solutions were then diluted two-fold with distilled water to provide 11 dilutions (final concentration is 0.625 μg/mL). A 2 mL volume of the 2-fold serial dilution of each test compound/drug was incorporated into 18 mL hot MuellereHinton agar medium, which resulted in the final concentration of each dilutions decreasing ten fold. The studied microorganisms were maintained at 4°C. The Gram positive bacteria studied were Bacillus subtilis MRCC121 and Staphylococcus aureus MTCC96. Gram negative bacteria were Pseudomonas aeruginosa MTCC741, Salmonella typhi MTT537, Klebsiella pneumoniae MTCC3384 and fungal strains were Candida albicans MTCC3017 and Candida tropicalis MTCC184. A 10 μL amount of bacterial suspension was spotted onto Muellere-Hinton agar plates containing serial dilutions of the compounds/drug. The plates were incubated at 36.5 °C for 24-48 h. The same procedure was repeated in triplicate.

MTT Bioassay
To monitor cell viability, HDF cell line was set up 60×10 4 cells/well of a 96 multiwell flat bottomed 200 μl microplates pre-coated with 1% Poly-L-lysine. Cells were incubated at 37°C in a humidified 5% CO2-95 % air mixture. A lot of cell line was treated with different concentrations (1-100 µM) of 8c or 8h for 24h. At the end of treatment time, 20 μl MTT 0.5 %, in PBS was added to each multiwell. After 1 h of incubation with the reagent, the supernatant was removed and replaced with 100 μl DMSO. The optical density of each well sample was measured with a microplate spectrophotometer reader (TitertekMultiskan; Flow Laboratories, Helsinki, Finland) at λ 570 nm. The concentration of compound required to cause 50 % inhibition of cell proliferation (IC50) was calculated from concentration-effect curves using Prism 5.0 (GrafPAD Software for Science). For all compounds, a non-linear regression analysis was used.

Receptor and Ligands Preparation
The X-ray crystal structure of 50S ribosomal unit of Haloarculamarismortui(PDB code 3CPW)enzyme conjugated with linezolid (PDB code 3CPW), with resolution of 2.7 Å, was retrieved from the protein data bank database (http://www.rcsb.org/pdb). The Accelrys Discovery Studio software has been used to decresing complexity of protein structure.All the atoms within30 Å distance from bound linezolid were selected and the relevantresidues were expanded. The selected construct was employed to molecular docking.The AutoDock Tools software has been used to remove ligands and water molecules, adding all the hydrogen atoms, and to calculate Gaisteiger charges for each atom of the macromolecules. The chemical structures of compounds were generated using ChemOffice v12.0 Ultra software package and optimized with ADT 4.2 for docking studies.

Docking Protocol
Molecular Docking was carried out using AutoDock 4.2. Grid Maps were centered on the coordinates of the co-cristallized ligands with 50 points per dimension and a step size of 0.375. The Lamarckian genetic algorithm implemented in AutoDock has been employed to dock compounds 8a-i into the50S ribosomal unit. We have carried out comparative docking experiments of compounds 8a-i. Each docking experiment consisted of 100 docking runs with 150 individuals and 2.5 x 10 5 energy evaluations. Other parameters were left to their default values. The search was conducted in a grid of 50 points per dimension and a step size of 0.375 centered on the binding site of enzyme. 100 highestscoring docking poses were saved and binding affinity of the best mode was selected.The cluster with the lowest free energy of binding was visually analyzed using Python Molecular Viewer 1.4.3 (PMV).The Autodock docking parameters were validated to ensure that the ligand orientation and the position obtained from the docking studies represent valid reasonable binding modes of inhibitors. The ligand linezolid was extracted and docked back into the corresponding binding pocket. The results of docking simulation predicted the binding conformation of linezolid with a root mean square deviation (RMSD) of 0.76 Å, compared with conformation of co-crystallized structure, thus indicating a valid docking protocol.