Mosquito-Larvicidal, Anti-Bacterial and Anti-Fungal Properties of Novel 2, 4-Disubstituted-[1, 3] Thiazoles

A series of novel hydrazones of 2, 4-disubstituted-[1, 3] thiazole (5) were prepared by the reaction of thio- semicabazone (3) with different substituted phenacyl halides (4) in the presence of dimethylformamide as a solvent. The requisite thiosemicarbazone (3) was synthesized by the treatment of thiosemicabazide (1) with 2, 4-dichloro- 5-fluroacetophenone (2) in presence of catalytic amount of mineral acid. All the novel compounds were confirmed and characterized by elemental analysis, FT-IR, 1 H-NMR, 13 C-NMR and Mass spectra and were subjected to anti-bacterial, anti- fungal activity studies and also screened for mosquito-larvicidal activitiy studies against two pathogenic vectors namely; Anopheles stephenesis (malaria vector) and C. tritaeniorhynchus (Japanese encephalitis). Among the novel 2, 4-disubstituted [1, 3]- thiazoles (5a- 5o), the compounds 2-{2-[1-(2, 4-dichloro-5-fluorophenyl) ethyl- idene] hydrazinyl}-4-(4-fluorophenyl)-[1, 3]-thiazole (5j) and 2-{2-[1-(2, 4-dichloro-5-fluorophenyl) ethylidene] hydrazinyl}-4-(4-nitrophenyl) –[1, 3] -thiazole (5m) exhibit anti-bacterial and anti-fungal properties. Though the mosquito-larvicidal activ- ity is low among the novel derivatives, yet, 4-(4-bromophenyl)-2-{2-[1-(2, 4-dichloro-5-fluorophenyl) ethylidene] hydrazinyl} –[1, 3]-thiazole (5i) and 2-{2-[1-(2, 4-dichloro-5-fluorophenyl) ethylidene] hydrazinyl}-4-(4-fluorophenyl)[1, 3]- thiazole (5j) may be recommended as mosquito larvicidal agents for further studies.


Anti-bacterial activity
All the synthesized compounds were evaluated for their antibacterial properties by Disc diffusion method [Bauer et al., 1966;Vardar-Unlu et al., 2003]. The microorganisms used in study of anti-bacterial properties were collected from Institute of Microbial Technology (IMTECH), Chandigarh, India. Two Gram +ve bacteria namely Staphylococcus aureus MTCC-7443 and Bacillus subtilis MTCC-441; two Gram -ve bacteria namely Escherichia coli MTCC-725, Aeromons hydrophila MTCC-1739 were used.
The bacterial strains were inoculated on Nutrient Agar (NA) and incubated for 24h at 37°C. Sterile empty discs (6mm diameter) (Himedia Laboratories Pvt. Ltd. Mumbai). The test compounds were dissolved in 5mL of DMSO taken as the solvent; from the stock solution 100μL of respective compound in the selected concentration (500μg/disc) was loaded on the disc individually and aseptically, dried and were used for screening the anti-bacterial assay.
Sterile discs were saturated with 100μL of the test solution, dried under laminar air flow and placed on the Nutrient Agar (NA) plate for bacteria, which was inoculated with a lawn ReseaRch PaPeR of the test microorganisms. Plates were incubated at 37°C, for 18 to 24h for bacteria. The compounds that produced distinct circular zones of inhibition around the discs and the diameters of clear zones were determined and used as an indication of anti-bacterial activity. Streptomycin, an antibiotic drug at a dose of 10μg/disc was used as the reference standard.

Anti fungal activity
All the synthesized compounds were evaluated for their antifungal properties by Disc diffusion method [Bauer et al., 1966;Vardar-Unlu et al., 2003]. The microorganisms used for the study were procured from Institute of Microbial Technology (IMTECH), Chandigarh, India. Four fungal species namely, Aspergillus niger MTCC-281, Aspergillus flavus MTCC-871, Candida albicans MTCC 183 and Alternaria alternata MTCC -149 were used.
The fungal strains were inoculated on on Potato Dextrose Agar (PDA) for 48h at 27 o C, then suspended in saline solution (0.85 and adjusted to yield approximately 1.0x10 7 -1.0x10 8 cfu/ ml by using spectrophotometer (25% transmittance at 530nm) as per the guidelines given in Indian Pharmacopoeia (2007). Sterile empty discs (6mm diameter) were purchased from Himedia Laboratories Pvt. Ltd. Mumbai. 50mg of each test compound was dissolved in 5ml of DMSO (Dimethyl Sulphoxide) to prepare the stock solution. From the stock solution 100μL of respective each test compound was loaded on the disc individually and aseptically, dried under laminar air flow and placed on the Potato Dextrose Agar (PDA) plate, which had been inoculated with a lawn of the test microorganisms. Plates were incubated at 27°C for 48h for fungi. Discs treated with 100 μL DMSO were used as negative controls. Commercial anti-fungal drug such Nystatin was used (10 μg/mL) as reference standard for anti-fungal activities. Distinct circular area around the discs representing the zone of inhibition was measured to determine the potentiality of anti-fungal activities.

Mosquito-Larvicidal activity
All the synthesized compounds were tested for their mosquito-larvicidal activity using two pathogenic vectors namely; Anopheles stephensi (malaria vector) and C. tritaeniorhynchus (Japanese encephalitis) as per the standard WHO guidelines [WHO, 1981]. In 500mL beakers containing 250mL of water and 25 numbers of late III or early IV instar mosquito larvae for various concentrations of the extracts. A negative control was kept with each set of experiment and mortality was recorded after 24h of exposure. Malathion, the commercial insecticide (Hindustan Insecticides Ltd, New Delhi, India) was used as the reference standard. Experiments were performed in triplicates for each sample. Median lethal concentration (LC 50 ) with 95% confidence limit (CI) was calculated using Abbott's formula and Log probit analysis [Raymond et al., 1993] and results are expressed as mg/mL. Relative potency was determined for comparison with the reference standard using the formula.

Pharmacological Screening
Anti-bacterial Activity All the novel 2, 4-disubstituted-[1, 3]-thiazoles (5a-5o) (Ta-ble 2, Figure 1) exhibited anti bacterial activity at the tested concentrations either in Gram +ve or Gram -ve or in both bacterial species under study. Among the tested compounds, (5m) exhibited the maximum activity against S. aureus, followed by (5l) and (5j) with zone of inhibition values, 26.7, 23.6 and 20.6 mm respectively. B. subtilis was found to be more sensitive to (5m) (18.9mm), (5j) (18.6) and (5c) (17.6mm) as indicated by the relatively maximum values for zone of inhibition. With reference to Gram -ve bacteria, (5j) was more effective against P. aeruginosa (19.2 mm) and K. pneumonia (17.5mm). P. aeruginosa was also found to be sensitive to (5a) with the zone of inhibition 18.2mm. Overall, (5m) compound is more effective against Gram +ve bacteria and (5j) is more effective against Gram -ve bacteria. Considering antibacterial activity against both Gram +ve and Gram -ve bacteria, (5j) exhibited relatively good activity with the zone of inhibition values ranging from 17.5 to 20.6mm. Therefore, 2-{2-[1- (2, 4- (5j) can be regarded as an agent with broad spectrum anti-bacterial activity, and recommended as drug candidate for further studies.

Antifungal Activity
All the tested 2, 4-disubstituted-[1, 3] thiazoles (5a-5o) (Table 3, Figure 2) exhibited a little or more fungicidal activity depending upon the type of fungi. Among all the tested compounds, (5j) and (5c) were found to be more effective against the fungus, C. albicans against which they showed relatively higher activity. (5c) and (5j) compounds are more effective against A. niger. The fungus, A. flavus is more sensitive to (5j) and (5c). The fungus, A. alternate did not show very good response against any of the the tested compounds. However, based on the zone of inhibition values, (5m) (12.8mm), (5c) (12.5mm) and (5j) (12.2mm) compounds can be considered to possess moderate activity against A. alternate. Overall, among the test compounds, (5j) can be considered as an agent with broad spectrum antifungal property showing the zone of inhibition more than 12 against all the tested fungal species.Thus, 2-{2-[1-(2, 4-dichloro-5-fluorophenyl) ethylidene] hydrazinyl}-4-(4-fluorophenyl)-[1, 3]-thiazole (5j) may be considered as an anti-fungal drug candidate for further toxicity and clinical studies.

Mosquito-Larvicidal Activity
Novel 2, 4-disubstituted-[1, 3] thiazoles (5a -5o)( Table 4, Figure 3) exhibited mosquito-larvicidal activity against two pathogenic vectors with LC 50 values ranging between 99.79 and 151 µg/mL against C. tritaeniorhynchus, while for A. stephensi, between 100.32 and 147.92 µg/mL. Among the tested compounds (5i) and (5j) were more potential against both the types of mosquito larvae. Comparatively (5h) also showed moderate activity against both the larvae. Results indicate that both C. tritaeniorhynchus and A. stephensi larvae are equally sensitive to the type of novel 2, 4-disubstituted [1, 3]-thiazoles. Relative potency determined comparing with the reference standard (malathion) indicate that the synthesized novel 2, 4-disubstituted-[1, 3]-thiazoles are not as potential as that of malathion, a commonly used commercial insecticidal/larvicidal agent. However, taken into consideration of various other factors such as persistence, half life, toxicity to non-target organisms, development of resistance, new agents need to be tested for their larvicidal activity to control disease vectors. In this context, among the new thiazole derivative in the present study, 4-(4-bromophe- The presence of halogens, that is bromo group in (5i) and fluoro group in (5j) respectively, may be plausible reason for higher activity.
Heterocyclic compounds with these moieties have been reported to exhibit insecticidal/mosquito-larvicidal activity.

Experimental Section
The chemicals used in the work were of standard quality and obtained from Sigma Aldrich (India), Alfaaesar (U. K.) S. D. Fine (Mumbai) and Hi-media (Mumbai). The novel synthesized 2, 4-disubstituted 1, 3-thiazoles (5a -5o) were confirmed by the spectral data. Melting points of novel compounds were determined in open capillary tubes and are uncorrected. The purity of synthesized compounds was checked by TLC observing single spot on Merck silica gel 60 F 254 coated alumina plates. The IR spectra (cm -1 ) were recorded on a Shimadzu-FTIR 577 Infrared spectrophotometer in KBr pellets. The 1 H-NMR and 13 C-NMR spectra were recorded on a Brucker AMX-400(400 MHz) spectrometer using recorded using CDCl 3 -d/ DMSO-d 6 as solvent and TMS as the internal standard. All the chemical shift values are reported in δ scale downfield from TMS. The splitting patterns are designated as follows; s, singlet; d, doublet; t, triplet; m, multiplet. The Mass spectra were recorded on Perkin-Elmer 018444 -Y, Triple Quadrupole LC/MS Spectrometer. The molecular ion peaks (m/z) were equivalent to the molecular weights of the newly synthesized compounds. The elemental analysis (C, H, N) was carried out on a Elementar Vario EL III analyzer.