Synthesis of new 2-amino-1,3,4-oxadiazole derivatives with anti-salmonella typhi activity evaluation.

Reaction of phenyl acetic acid derivatives with thiosemicarbazide in the presence of POCl3 afforded 5-(4-bromobenzyl)-1,3,4-oxadiazole-2-amine 1 and 5-(3-nitrophenyl)-1,3,4-oxadiazole -2-amine 2. Acylation of the amino group of oxadiazoles 1 and 2 with some acid chlorides such as methyl 4-(chlorocarbonyl) benzoate, 3-nitrobenzoyl chloride, 4-methoxy-benzoyl chloride, 4-isobutylbenzoyl chloride and chloroacetyl chloride yielded the acylated compounds 3–8. Cyclization of acetamides 7 and 8 by reaction with ammonium thiocyanate gave the thiazolidinones 9 and 10. Coupling of chloroacetamide 7 with two mercaptothiazoles gave coupled heterocyclic derivatives 11 and 12. Coupling of amino-oxadiazole 1 with N-Boc-glycine and N-Boc-phenylalanine lead to the formation of 16 and 17 respectively. All compounds were screened for their antibacterial activity against Salmonella typhi where compounds 3, 4, 10, 11 and 15 showed significant activity. Structures of the new synthesized compounds were confirmed using the spectral analysis such as IR, 1H NMR and 13C NMR and mass spectrometry.

Typhoid is actually an infection as a result of Salmonella typhi which causes symptoms [18]. Symptoms can vary from gentle to extreme and in most cases, start 6 to 30 days soon after exposure. Frequently there is a progressive beginning of a very high fever more than several days. Weaknesses, abdominal pain, constipation, and migraines also commonly happen [19]. Diarrhea is uncommon, and vomiting is not usually severe. Some people develop a skin rash with rose-colored spots [20,21].
Salmonella enterica subsp. enterica is a subspecies of Salmonella enterica, the rod-shaped, flagellated, aerobic, Gram-negative bacterium. Many of the pathogenic serovars of the S. enterica species are in this subspecies, including that responsible for typhoid [22].
Herein, we synthesized about seventeen new oxadiazole derivatives and screen them against Salmonella typhi to find new leads.

Structure confirmation
Structure 1 has confirmed by infrared spectra which showed well defined bands attributable for ν C=N at 1610 cm −1 and ν NH2 at 3310-3400 cm −1 . The 4-bromophenyl ring revealed two doublets at d 7.215 and 7.497 ppm. Characteristic singlet of methylene group appeared at 4.097 ppm and the amino group was found as singlet at 7.006 ppm. 13 C-NMR of 1 revealed the presence two carbon of oxadiazole ring around 169.0 and 157.3 ppm, carbons of 4-bromophenyl appeared around 137.9 and 120.5 ppm whereas, the methylene carbon appeared at 35.2 ppm. The 1 H-NMR of 5-(3-nitrophenyl)-1,3,4-oxadiazole-2-amine 2 amino group at 7.622 ppm. 13 C NMR spectrum revealed the two oxadiazole carbons at 169.0 and 164.7 ppm. 1 H-NMR spectrum for compounds 3-6 showed NH signal appeared around 12.00 ppm. Infrared spectra showed well-defined bands attributed to ν NH at 3200-3400 cm −1 . 1 H NMR of 7 and 8 showed new signal for CH 2 around 4.00 ppm. 13  Structure of compound 13 was assigned from the characteristic two singlet's for two NH groups at 9.52 and 12.23 ppm. The methylene protons found at 4.26 ppm. Infrared spectra showed well-defined bands attributable for ν C=O

Antibacterial activity
The novel seventeen compounds were screened for their antibacterial activity against gram negative bacteria Salmonella typhi at three concentrations i.e. 1000, 100 and 10 ppm using ditch dilution method. The test organism was a 2-h culture of Salmonella typhi incubated and grown in peptone-water medium (temperature 37 °C). DMF was used as solvent control which did not show any zone of inhibition. Muller-Hilton agar medium was used as culture medium. The culture plates were incubated at 37 °C for 24 h. Antibacterial activity was determined by measuring the diameter of the inhibition zone. The results are given in Table 1. Compounds 3, 4, 10, 11 and 15 displayed greater antibacterial activity against Salmonella typhi. Especially Compounds 10 and 11 exhibited the broadest spectrum activity in this series due to the heterocyclic ring of the imine and sulfide. Whereas, compounds 2, 5, 6, 8, 9, 12 and 16 showed moderately activity. Resistance of bacteria to these synthesized compounds could be associated to alteration of the bacterial protein targeted by compounds, enzymatic degradation of the synthesized compounds, or change in the membrane permeability to them.

Experimental
All melting points were uncorrected, performed on a MEL-TEMP II. Melting point apparatus. Microanalysis was performed by micro analytical laboratory, Cairo University, Egypt. Infrared spectra were recorded (ν in cm −1 ) with pye Unicam SP 1200 spectrophotometer and using KBr Wafer technique. Mass spectra were measured with a Thermo Scientific LTQ Linear Ion Trap. Nuclear magnetic resonance spectra ( 1 H NMR, 13 C NMR) were recorded (δ in ppm) on Bruker (300 MH z ) spectrometer. The purity of the synthesized compounds was checked by TLC on glass coated plates in the laboratory with silica gel GF 254 type, 60 mesh, size 50-250.

Synthesis of thiazolidin-4-ones 9 and 10
Compound 7 and/or 8 (7 mmol) and ammonium thiocyanate (15 mmol) in ethanol 35 mL were refluxed for 3 h, the reaction mixture was left overnight. The obtained precipitate was filtered off, dried and recrystallised from ethanol-water to yield compounds 9 and 10.

Deprotection of N-protected group in compound 14 and 15
Protected compounds 14 and 15 (1 mol) in methylene chloride (3.75 mL) was stirred under nitrogen followed by cooling in an ice bath then trifluoroacetic acid (1.25 mL) was added dropwise for 10 min followed by 0.05 mL of anisole. The reaction mixture was stirred for 2 h. Then it evaporated under vaccum. The oil product was crushed by ether (30 mL) and formed solid was recrystallised from acetone.

Conclusion
Seventeen new functionalized oxadiazole hits were synthesized and characterized. The new hits were evaluated for their biological activity against gram-negative bacteria Salmonella typhi, among synthesized 3, 4, 10, 11 and 15 demonstrated strong activities which recommends them for further studies to be future leads.