Synthesis and Biological Evaluation of Novel 5 , 7-Dichloro-1 , 3-benzoxazole Derivatives

A new class of 5,7-dichloro-1,3-benzoxazole derivatives 4–11 were synthesized by fusing 5,7-dichloro-2-hydrazino-1,3benzoxazole 3 nucleus with aliphatic acids, active methylene compounds, and with selected esters to form heterocyclic ring systems like 1,2,4-triazoles, pyrazoles, and triazine moieties. e compound 3 on diazotization reaction affords the tetrazole compound. e synthesized compounds were characterized by H NMR, IR, Mass, and C NMR spectral data and screened for cytotoxic, antimicrobial, antioxidant, and antilipase activities. e compounds 4, 5, and 8 have shown signi�cant antimicrobial activities, whereas compounds 6 and 8 have been emerged as leading cytotoxic agents.e compounds 9, 10, and 11 were found to be strong enzyme inhibitors.


Introduction
Wide-spread interest in the chemistry of benzoxazole derivatives in large number of products has attracted the researchers because of their potential applications in medicinal �eld.Benzoxazole derivative constitutes an important class of heterocyclic compounds and shows various types of biological properties such as antiviral, antineoplastic, anti-HIV-1, antitubercular, anthelmintic, antimicrobial, and antifungal activities [1,2].In the last few years, 2-substituted benzoxazole derivatives have been studied extensively for their biological importance.

Experimental Section
2.1.Chemistry.Melting points were recorded on electrothermal melting point apparatus and are uncorrected. 1H NMR and 13 C NMR spectra were recorded on Bruker 400 MHz spectrometer in Indian Institute of Science, Bangalore, Karnataka, India.e chemical shis are shown in  values (ppm) with tetramethylsilane (TMS) as an internal standard.LC-MS were obtained using C 18 column on Shimadzu, LCMS 2010A, Japan.e Fourier Transform Infrared (FT-IR) spectra of the compounds were taken in KBr pellet (100 mg) using Shimadzu (FT-IR) spectrophotometer.e elemental analysis was carried out using VarioMICROV1.7.0 (Elemental Analysersysteme GmbH) instrument.e column chromatography was performed using silica gel (230-400 mesh).Silica gel GF254 plates from Merck were used for TLC and spots located either by UV or dipping in potassium permanganate solution.e chemicals were purchased from Sigma-Aldrich Co. and from SD Fine Chemicals.e solvents for column chromatography were of reagent grade and were purchased from commercial source.e yields are of puri�ed compounds and are not optimized.e yield, melting point, molecular formula, and molecular weight of the compounds were recorded in Table 1.

Preparation of 5-Amino
Light brown color solid; IR (KBr) cm  (9).e compound 3 (10 mmol) and acetyl acetone (10 mmol) mixture in absolute ethanol (30 mL) was stirred under re�ux for 5 hr.e reaction mixture was allowed to cool at room temperature and poured onto water.e precipitate was �ltered off, dried, and crystallized from methanol.

Minimum Inhibitory Concentration (MIC).
MIC was determined by micro-dilution method using serially diluted compounds (0 to 20000 g/mL) in nutrient broth tubes.e standardized 0.1 mL of inoculum (10 7 CFU/mL) of test bacteria was added to each tube and incubated at 37 ∘ C for 24 hr.Two control tubes were maintained for each organism.e lowest concentration (highest dilution) of the compounds that produced no visible growth when compared with the control tubes was regarded as MIC [20].

Antioxidant Activity. DPPH Assay
. e radical scavenging ability of synthesized compounds and the ascorbic acid (standard) was tested on the basis of radical scavenging effect on DPPH free radical.Different concentrations (25,50,100,200, and 400 g/mL) of compounds and standard were prepared in methanol.In clean and labeled test tubes, 2 mL of DPPH solution (0.002% in methanol) was mixed with 2 mL of different concentrations of compounds and standard separately.e tubes were incubated at room temperature in dark for 30 minutes and the optical density was measured at 517 nm using UV-visible spectrophotometer.e absorbance of the DPPH control was also noted.e scavenging activity was calculated using the formula: Scavenging activity (%) =     , where  is the absorbance of DPPH and  is the absorbance of DPPH in standard combination [21].
Ferric Reducing Assay.Different concentrations (25, 50, 100, 200 and 400 g/mL) of compounds and standard (tannic acid) in 1 mL of methanol were mixed in separate tubes with 2.5 mL of phosphate buffer (200 mM, pH 6.6) and 2.5 mL of 1% potassium ferricyanide.e tubes were placed in water bath for 20 minutes at 50 ∘ C, cooled rapidly, and mixed with 2.5 mL of 10% trichloroacetic acid and 0.5 mL of 0.1% ferric chloride.e amount of iron (II)-ferricyanide complex formed was determined by measuring the formation of Perl's Prussian blue at 700 nm aer 10 minutes.e increase in absorbance of the reaction mixture indicated the increased reducing power [22].

Extraction of Lipase from Chicken (Gallus domesticus)
Pancreas.Pancreas of freshly slaughtered chicken were collected, washed, and placed in ice cold sucrose solution (0.01 M). e pancreas was homogenized in 0.01 M sucrose, centrifuged.e supernatant liquid was separated and subjected to ammonium sulphate precipitation (50% saturation).e pellet obtained aer centrifugation was dissolved in sucrose solution and again saturated with 50% ammonium sulphate and centrifuged.e pellet obtained was dissolved in phosphate buffer and used as enzyme source [23].
Determination of Chicken Pancreatic Lipase Activity.e activity of lipase was determined by incubating an emulsion containing 8 mL of olive oil, 0.4 mL of phosphate buffer, and 1 mL of chicken pancreatic lipase for 1 hr in rotary shaker, followed by stopping the reaction by the addition of 1.5 mL of a solution containing acetone and 95% ethanol (1 : 1).e liberated fatty acids were determined by titrating the solution against 0.02 M NaOH (standardized by 0.01 M oxalic acid) using phenolphthalein as an indicator [24,25].
Lipase Inhibitory Activity.Lipase inhibitory activity of different concentrations of synthesized compounds (2.5, 5, 10, and 20 mg/mL) was tested by mixing 100 L of each compound, 8 mL of oil emulsion, and 1 mL of chicken pancreatic lipase followed by incubation for 60 minutes.e reaction was stopped by adding 1.5 mL of a solution containing acetone and 95% ethanol (1 : 1).e liberated fatty acids were determined by titrating the solution against 0.02 M NaOH using phenolphthalein as an indicator [26].e orlistat was used as standard lipase inhibitor, and the percentage inhibition of lipase activity was calculated using the formula: Lipase inhibition =     , where  is lipase activity, and  is activity of lipase when incubated with the compounds.
2.2.5.Cytotoxic Activity.e brine shrimp lethality test was conducted to determine cytotoxic activity of compounds [27].Brine shrimp Artemianauplii eggs (Nihon Animal Pharmaceutical Inc., Tokyo, Japan) were hatched in a container �lled with air-bubbled arti�cial sea water, which was prepared with 10 gm of a commercial salt mixture (GEX Inc., Osaka, Japan) and 500 mL of distilled water.Aer 36-48 hr, the phototropic shrimps were collected by pipette for bioassay.Twenty-�ve shrimps were transferred to each vial containing different concentrations of compounds.e arti�cial sea water was added to make up to 5 mL and incubated at 25 ∘ C, and survived shrimps were counted microscopically in the stem of the pipette against a lightened background aer 24 hr.
Minimum inhibitory concentration of synthesized compounds against test bacteria ranged from 250 g/mL to 7500 g/mL.e compounds 4, 5, and 8 caused high inhibition at low concentration and are reported in Table 3.
Figure 1 explains antioxidant activity at different concentrations of compounds 4-11 in methanol and ascorbic acid in terms of free radical scavenging ability which was evaluated using DPPH free radical assay.e compounds exhibited marked antioxidant activity by scavenging DPPH * (free radical) and converting into DPPH, and the activity was found to be dose dependent.e compound 5 was shown to be more potent than ascorbic acid, and results were tabulated in Table 4. e result of reducing power at different concentrations of compounds 4-11 and tannic acid is represented in Table 5.In this study, the absorbance was found to be increased with the dose of compounds and standard, which is suggestive reducing power (Figure 2).e compounds 4, 6, 7, and 11 showed remarkable reducing power.
Inhibitory activity on chicken pancreatic lipase at different concentrations of compounds in DMSO was determined using olive oil as the substrate.It was found that, the activity of lipase was affected when incubated with the compounds.e inhibitory activity was found to be dose dependent that is, higher inhibition of enzyme was observed on increasing the concentrations of compounds (Figure 3).Lipase inhibition was more in case of compound 11.e results are tabulated in Table 6.e degree of lethality was found to be directly proportional to the concentration of the compounds.e highest mortality was observed at 1000 g/mL concentration of compounds.e most potent activity was observed in case of compounds 6 and 8, whereas the mortality was 100% at the lowest concentration of compounds (Figure 4).LC 50

Compounds
Inhibition of lipase activity (%) 20 mg/mL 10 mg/mL 5 mg/mL 2.5 mg/mL 0 mg/mL  of compound 5, 10, and 11 was found to be 896.05,22.85, and 31.99 g/mL, respectively, and the LC 50 values were determined by linear regression using Origin 6.0 soware.e results are mentioned in Table 7.

Conclusion
is study reports the successful synthesis of the title compounds via different routes in good yield.e target molecules were characterized and con�rmed by 1 H NMR, IR, Mass, and 13 C NMR spectral analysis and screened for selected biological activities.e compounds 4, 5, and 8 showed marked antibacterial activity, and compounds 10 and 11 exhibited potent pancreas lipase inhibitors, whereas compound 5 acted as scavengers and reducing agents.e compounds 6 and 8 showed potent cytotoxic activity.So, the derivatives of 5,7-dichloro-2-hydrazino-1,3-benzoxazole 3 were found to be biologically potent-fused heterocycles.

3. 2 .
Biological Evaluation.e results of antibacterial activity of compounds 4-11 were shown in Table