Synthesis , toxicity towards brine shrimp ( Artemia salina Leach ) and antimicrobial activity evaluation of 3 , 5-diphenylchlorinated-1 , 2 , 4-oxadiazoles

The known oxadiazoles 3,5-bis-(phenyl)-1,2,4-oxadiazole (3a); the 3-(4chlorophenyl)-5-phenyl-1,2,4-oxadiazole (3b); and the new 3,5-diphenylchlorinated-1,2,4oxadiazoles 3c-e were synthesized from the reaction of benzamidoximes with an appropriated acid chloride and cyclisation of the resulting O-acylbenzamidoxime intermediate. The compounds synthesized were characterized on the basis of their IR, NMR (1D and 2D) and mass spectral data. Compounds 3a-e were evaluated for their antimicrobial activity and for their toxicity towards brine shrimp (Artemia salina Leach).


Material and methods
Equipments.IR spectra were recorded on a Bomem spectrophotometer model MB 100. 1 H (300 MHz), 13 C (75.5 MHz) and 2D (COSY, HMQC, HMBC) NMR spectra were recorded in a Varian spectrometer model Mercury plus BB 300MHz, using CDCl 3 as solvent and TMS as internal standard.MS spectra were obtained in Hewllet Packard-GC/MS spectrometer model 5972, at 70 eV.Melting points were determined with a Microquimica-MQ APF-301 apparatus and are uncorrected.The purity of the compounds was confirmed by GC/MS analysis.
Antimicrobial bioassay.The antimicrobial activity and the minimum inhibitory concentration (MIC) of the compounds 3a-e were evaluated by the dilution method using Mueller-Hinton and RPMI-1640 medium for antibacterial and antifungal assays, respectively (NCCLS, 1997;2000).The stock solution of each compound in DMSO was diluted to serial twofold dilutions, which were added to each medium resulting in concentrations ranging 10 to 100 µg/mL.The following microorganisms were used for detecting antimicrobial activities: Escherichia coli ATCC 25922, Staphylococcus aureus ATCC 25923, Pseudomonas aeruginosa ATCC 15442, Bacillus subtilis ATCC 6623, Candida albicans, C. parapsilosis, C. krusei and C. tropicalis.Micostatin, vancomycin, tetracycline and penicillin were used as reference antibiotics.
Brine shrimp bioassay.Brine shrimps (Artemia salina) eggs were placed, for hatching, in a side of a tank divided by a net and containing a 3% (w/v) sodium chloride solution.A light source was placed in the other side of the tank to attract the nauplii.After 48h ten nauplii were added to a series of vials containing different concentrations of the test compounds.The vials were maintained under light and after 24h the number of survivors was counted.The bioassays were carried out in triplicate and expressed with 95% confidence limit (Mc Laughlin, 1991).

Results an Results an Results an Results and discussion d discussion d discussion d discussion
Synthesis and characterization of the oxadiazoles 3a Synthesis and characterization of the oxadiazoles 3a Synthesis and characterization of the oxadiazoles 3a Synthesis and characterization of the oxadiazoles 3a----e e e e Although several methodologies have been reported for the synthesis of 1,2,4-oxadiazoles, the reaction of an amidoxime with an acid carboxylic derivative, a carbonic acid derivative or related species remains the most commonly employed methodology for the synthesis of 1,2,4-oxadiazoles (Hemming, 2001).
In this work, 1,2,4-oxadiazoles (3a-e) were prepared by O-acylation of the benzamidoximes (2a-c) with appropriated carboxylic acid chlorides, in pyridine, and cyclisation of the resulting Oacylbenzamidoximes as shown in Scheme 1.The benzamidoximes 2a-c were available from the reaction of the nitriles 1a-c with hydroxylamine hydrochloride, in triethyl amine (Deegan et al., 1999).Nitriles 1b and 1c were obtained from the reaction of 4-chloro-benzotrichlorides or 4chlorobenzoic acid or 3,4-dichlorobenzoyl chlorides with ammonium chloride.
The IR spectra of the compounds 3a-e showed absorptions bands at 1606,1555,1490,1410,1360,1090 and 740 cm -1 , consistent with the presence of an oxadiazole moiety and phenyl chlorinated groups (Ryu et al., 2001).Molecular ion peaks (M +• ) consistent with the expected structures and fragmentation characteristics for aryl-1, 2, 4oxadiazoles were observed for all compounds in the mass spectra (Tyrkov et al., 2004).The formation of the 1,2,4-oxadiazole systems was confirmed by the signals at δ 167.2-168.7 and δ 173.9-176.1 in the 13 C NMR spectra, which were assigned to C-3 and C-5, respectively (Table 1).The 1 H and 13 C NMR data showed typical signals for di and tri-substituted aromatic groups containing the atom of chlorine.The signals for the carbons bearing chlorine atoms appear in the region of δ 132.8 -137.6.The NMR data for the new compounds 3c-e are presented in Table 1.The chemical shifts assignments of the oxadiazoles 3a-e were based on analysis of the correlations observed in the 1H-1H-COSY, HMQC and HMBC spectrum.From the correlations in 1H-1H-COSY spectra it was possible to confirm the connectivity between the aromatic hydrogens.The HMBC spectra showed correlations of the signals at δ 167.2 -168.7 (C-3) and at δ 173.9 -176.1 (C-5) with those of H-2'/ H-6' and H-2"/ H-6", respectively.From the correlations of the signals for C-1' and C-1" with those of aromatic hydrogens it was possible to confirm the positions of the hydrogens and, consequently, of the chlorine atoms in the aromatic rings.These data, together with the 1 H-13 C correlations in the HMQC spectra permitted the chemical shifts assignments of the hydrogens and carbons, and to confirm the chlorine atoms positions in the aromatic rings for the synthesized oxadiazoles.

Biological assays Biological assays Biological assays Biological assays
The antimicrobial bioassays showed that all compounds were inactive against the microorganisms tested with a MIC > 100 µg/mL.
The toxicities of the compounds 3a-e were tested in Artemia salina larvae.The lethal dose (LD 50 ), which corresponds to the minimum concentration that causes 50% of the larvae mortality, was determined for all compounds.The results indicated that the oxadiazoles 3a (LD 50 = 2.13 µg/mL; 1.59-2.66µg/mL, 95% confidence limits) and 3b (LD 50 = 4.57 µg/mL; 3.62-6.52µg/mL, 95% confidence limits) exhibited significant toxicity.On the other hand, compounds 3c-e were inactive (LD 50 > 500 µ/mL), indicating that the increasing of the number of chlorine atoms did not result in increasing of the toxicity.The brine shrimp assay has been used as a convenient and rapid assay to discover new cytotoxic compounds.Thus, the oxadiazoles 3a and 3b are potential cytoxoxic agents and their activity on cancer cell lines will be further tested.

Conclusion Conclusion Conclusion Conclusion
In the present work we have synthesized and evaluated the toxicity towards Artemia salina and the antimicrobial activity of 1,2,4-oxadiazoles 3a-e containing phenylchlorinated substituents attached at C-3 and C-5.The oxadiazoles 3a and 3b exhibited significant toxicity towards Artemia salina with LD 50 of 2.13 and 4.57 µg/mL, respectively.The antimicrobial bioassays showed that all compounds were inactive against the microorganisms tested with a MIC > 100 µg/mL.