Synthesis , Characterization , and Antibacterial Activity of Diethyl 1-( ( 4-Methyl-2-phenyl-4 , 5-dihydrooxazol-4-yl ) methyl )-1 H-1 , 2 , 3-triazole-4 , 5-dicarboxylate

1Doctoral Training “Bioactive Molecules, Health and Biotechnology”, Center of Doctoral Studies “Sciences and Technology”, LCO, Faculty of Sciences Dhar Mahraz, Sidi Mohamed Ben Abdellah University, Fez, Morocco 2Organic Chemistry Laboratory (LCO), Faculty of Sciences Dhar Mahraz, Sidi Mohamed Ben Abdellah University, Fez, Morocco 3Laboratory of Biotechnologies (LB), Faculty of Sciences Dhar Mahraz, Sidi Mohamed Ben Abdellah University, Fez, Morocco


Introduction
Heterocycles having five-membered rings such as those containing the 1,2,3-triazole moiety play an important role in various biochemical processes.1,2,3-Triazoles are an important class of heterocyclic compounds due to their wide usage as synthetic intermediates and pharmaceuticals [1][2][3].Many triazole derivatives are found to exhibit various pharmacological properties such as antimicrobial [4], antiepileptic [5], antitubercular [6], and antibacterial [7], and a large number of 1,2,3-triazoles have also been reported with significant anticancer activities [8][9][10].Since they are nontoxic, highly stable compounds and mostly water soluble, the 1,2,3-triazole derivatives could be ideal drug candidate and they could participate actively in molecular interactions by hydrogen bond formation [11] which implies their facility to binding with the biological targets [12].

Results and Discussions
2.1.Chemistry.The starting 2-phenyl-4-methyl-4-(azidomethyl)oxazoline 1 was prepared from oxazoline derivative by reaction with sodium azide in reflux of DMF, using El Hajji's method [19].This intermediate azide compound was obtained pure with a 92% yield, as colorless oil after Scheme 1: Synthesis of compound 2 with good yield using the 1,3-dipolar cycloaddition reaction of 4-(azidomethyl)-4-methyl-2-phenyl-4,5dihydrooxazole and diethyl but-2-ynedioate in the absence of a solvent.chromatography on silica gel column.In the second time, compound 1 was submitted to 1,3-dipolar cycloaddition reaction at room temperature with diethyl but-2-ynedioate in the absence of a solvent.The reaction was monitored by thin-layer chromatography and after consumption of the starting material, stirring was stopped.The recrystallization of the crude mixture in ether/hexane (v/v) led to the cycloadduct 2 with a 75% yield (Scheme 1).
The structure of compound 2 was established on the basis of NMR spectroscopy ( 1 H and 13 C) (Figures 1 and 2), X-ray crystallography (Figure 3), and MS data.The definite assignment of the chemical shifts of protons and carbons is shown in Table 1.

Biological Activity.
The synthesized compound was tested for its in vitro antibacterial activity against the Gram-positive and the Gram-negative bacteria: Staphylococcus aureus ATCC 29213 (S. aureus) and Escherichia coli ATCC 25922 (E.coli) using the liquid serial dilutions method [20] for determination of MIC.The latter is defined according to the Antibiogram Committee of the French Society for Microbiology (CA-SFM) as being the lowest concentration that results in the inhibition of visible bacterial growth [21].
The determination of the minimum inhibitory concentration (MIC) was realized by the preparation of a series of dilutions of 1/2 of the synthetic product to test on liquid medium (microdilution).The minimum bactericidal concentration (MBC) was regarded as being the lowest concentration, in product tested, having shown an absence of growth.
According to our study, compound 2 has an inhibitory activity on the S. aureus and E. coli strains (MIC = 1.25 mg/mL).The results of the minimum bactericidal concentration, having shown a maximum number of 5 colonies on can, are MBC = 2.5 mg/mL for ATCC strains of S. aureus, E. coli, and B. subtilis.
The calculation of ratio MBC/MIC showed that this triazole derivative has a bactericidal effect on the 2 strains tested.

Experimental Protocols
3.1.Chemistry.Melting point was determined with an electrothermal melting point apparatus and was uncorrected.NMR spectra ( 1 H and 13 C) were recorded on a Bruker AM 300 (operating at 300.13 MHz for 1 H, at 75.47 MHz for 13 C) spectrometer (City of Innovation, USMBA-Fez).NMR data are listed in ppm and are reported relative to tetramethylsilane ( 1 H, 13 C); residual solvent peaks are used as internal standard.All reactions were followed by TLC.TLC analyses were carried out on 0.25 mm thick precoated silica gel plates (Merck Fertigplatten Kieselgel 60F 254 ) and spots were visualized under UV light or by exposure to vaporized iodine.Mass spectra were recorded on a PolarisQ Ion Trap GC/MSn Mass Spectrometer (City of Innovation, USMBA-Fez).ORTEP of compound 2 was obtained on a Bruker APEXII CCD detector diffractometer (CNRST-Rabat).
100 L of the BHI medium (Brain Heart Infusion) was distributed in all the wells except for those of the first line.Then, 200 L of the stock solution of product 2 was added in the first line.The realization of the dilution series was made by taking 100 L first well of the first column and by adding it in the second well pertaining to the same column and so on until the penultimate well.The same stages were repeated for the other columns.
The wells were inoculated by 100 L of the bacterial suspension with a concentration of 106 UFC/mL.The wells of the last line of the microplate contain only the inoculum (control).The microplate was incubated at 37 ∘ C for 24 hours.
The MIC corresponds to the well, containing the lowest concentration of the product tested, which showed no visible bacterial growth.
The structure of the obtained compound was confirmed by NMR spectroscopy ( 1 H, 13 C), X-ray crystallography, and MS data.
The biological tests, carried out on the synthesized compound, showed that this triazole derivative has a bactericidal effect against Gram-positive and the Gram-negative bacteria: Staphylococcus aureus ATCC 29213 (S. aureus) and Escherichia coli ATCC 25922 (E.coli).

Figure 3 :
Figure 3: Molecular structure of compound showing the atom numbering scheme.H atoms are represented as small spheres of arbitrary radii.

Table 1 :
1 H (300.13 MHz) and 13 C (75.47 MHz) NMR spectral data for compound 2 in CDCl 3 , including results obtained by homonuclear 2D shift-correlated and heteronuclear 2D shift-correlated HSQC ( 1  CH ).Chemical shifts ( in ppm) and coupling constants ( in Hz).The definite assignment of the chemical shifts of protons and carbons of compound 2.