Introduction

As the practice of medicinal chemistry has evolved over time, it has dedicated its entire existence to discovering and developing new remedies for diseases ¹. Furthermore, medicinal chemistry has always emphasized on re-establishing a connection between chemical structure and pharmacological activity. Besides heterocyclic compounds contributed the most to the invention of new medications and were extensively studied in clinical aspects. Benzoxazole derivatives being an integral part of the heterocycle family, have momentous pharmacological potentialities in the field of medicinal chemistry.

In research, benzoxazole finds its uses as a starting material for the synthesis of larger bioactive molecules. It has been found within the chemical structures of pharmaceutical medicines, like Flunoxaprofen. Despite the fact that as a heterocycle, its aromatic character makes it moderately stable, it possesses reactive sites, which allow functionalization ². The basic aim of the synthetic and medicinal chemistry was to synthesize the compounds that results in high yields with greatest purity and show excellent activity as therapeutic agents with minimal toxicity. Eminent among these are anti-histaminic³,  antifungal⁴, cyclooxygenase Inhibiting⁵, anti-tumor⁶, anti-ulcer⁷, anticonvulsant⁸, hypoglycemic⁹, anti-inflammatory^10,11, anti-tubercular activity¹², anti-parasitics¹³, herbicidal¹⁴, antiviral¹⁵, anti-allergic and anthelmintic activities¹⁶. Also, they have a number of optical applications such as photo luminescents, whitening agents and in dye lasers ¹⁷ and are also used as organic brightening agents and organic plastic scintillators ¹⁸.

The quest for new antimicrobial and antioxidant agents lacking side effects persists to be an active area of research in medicinal chemistry. Despite the development of new and important drugs, their cost was out of the reach of commoners. As a result, these changes have accentuated the urgent need for new, increasingly powerful, less expensive and safe antimicrobial agents. The current effort is intended for the design, synthesis, and investigation of novel benzoxazoles derivatives, with hydrazide serving as the parent molecule, based on the aforementioned facts. The synthesised derivatives of 2-[(4-chloro-1,3-benzoxazol-2-yl)sulfanyl]-N’-[phenylmethylidene]acetohydrazides 5(a-j) were tested for their antioxidant and cytotoxic activities as well as antibacterial activity against a number of chosen bacteria and fungi.  To understand the binding affinity of produced derivatives with the active receptor sites, a molecular docking research was conducted.

Experimental

Materials and Instrumentation

An electrically heated apparatus was used to measure melting points that were uncorrected by placing the sample in a glass capillary sealed at one end. The ¹H NMR and ¹³C NMR measurements were conducted via a Bruker at 400MHz at MIT, Manipal, Karnataka, India, with tetramethylsilane (TMS) as an internal standard and chemical shifts are expressed as 𝛿values (ppm). Analysis of elements like C, H, and N were performed by a Perkin-Elmer 2400 Series analyzer. At Centralized Instrumentation Facility of Mysore University, Karnataka, India, molecular weights of unknown compounds were characterized using LC-MS spectroscopy. A Shimadzu Fourier Transform Infrared (FT-IR Nicolet-5700) spectrometer was used to procure the FT-IR spectra of the compounds. A thin layer chromatography (TLC) method was used to examine the completion of the reaction using silica gel coated on aluminium sheets (silica gel 60 F254). Solvents and reagents of commercial grade were employed for synthesis purpose and Table 1 enlists the yields, melting points, molecular formula and molecular weight of the compounds.

Table 1: Physical data of synthesized compounds 5(a-j) comprising of molecular formula, molecular weight, percentage of carbon, hydrogen, nitogen, melting point and percentage of yield

 Results and discussion

Design and synthesis of novel 4-chloro-1,3-benzoxazole derivatives

Preparation of 4-chloro-1,3-benzoxazole-2-thiol (2)

Methanol (50ml) and potassium hydroxide (1.1 eq) were combined and agitated for 10 minutes to start the reaction. Next, a measured amount of carbon di sulphide (1.1 eq) was slowly added at room temperature. As the aforementioned reaction mass was still being stirred, 4-chloro-2-aminophenol was added and simultaneously refluxed for 6 hours on a water bath. TLC was used to monitor the reaction till it was finished. On purpose, reaction mass was added to ice-cold water, which was then acidified with glacial aceticacid. Finally the procured solid was further filtered, dried and recrystallized ¹⁹. Yield (95%), M.P.1980C -199⁰C. MS:m/z = 185.93 and (M+2) = 187.93.

Preparation of ethyl [(4-chloro-1,3-benzoxazol-2-yl)sulfanyl]acetate (3):

Ethyl chloroacetate was added drop wise in the presence of K₂CO₃ after completely dissolving the 2-mercaptothiozole in acetone upon continuous stirring in a reaction flask. For nearly 4-5 hours the resultant mixture was refluxed and poured over freezing water. The obtained semisolid was washed repeatedly with water. The formed crystals after filtration were washed completely with water and dried which was further recrystallized from ethanol ²⁰. Yield (95%), M.P. 198⁰C -199⁰C.

Synthesis of 2-[(4-chloro-1,3-benzoxazol-2-yl)sulfanyl]acetohydrazide (4):

The flask containing 20 ml of methanol along with the compound 3 were stirred continuously for 15 min.  The ester was added upon continuous by stirring for nearly 15 min. The hydrazine hydrate was added slowly to the above mentioned mixture which was agitated for 3 hours to get the desired product. The obtained semisolid compound was filtered and washed with pet ether. Finally the compound was collected after drying ²¹. ¹HNMR (DMSO-d₆,δppm): 7.347-7.725 (m,3H,Ar-H), 4.342 (d,2H,S-CH₂), 4.080 (s,2H,NH₂), 9.414(s,1H,NH); MS: m/z = 257.96.

General procedure for the synthesis of2-[(4-chloro-1,3-benzoxazol-2-yl)sulfanyl]-N’-[phenylmethylidene]acetohydrazides 5(a-j):

To an ethanolic solution (20ml), the hydrazide compound (1eq) and aromatic aldehyde (1.1eq) was added and stirred for 2-3 mins. To this mixture 2-3 drops of glacial acetic acid was added and refluxed on water bath for about 6 hours. After the completion of reaction the resultant product was added to the ice cold water and filtered, dried and recrystallized from ethanol to obtain pure product ²².

2-[(4-chloro-1,3-benzoxazol-2-yl)sulfanyl]-N’-[(4-chlorophenyl)methylidene]acetohydrazide (5a):

IR (KBr,cm^-1): 3282 (N-H), 2362 (Ar-CH), 1681 (O=C-NH), 1450 (C=C), 1250 (C=N), 746 (C-S), 681 (C-Cl); ¹HNMR (DMSO-d₆,δppm): 7.14-7.6 (m,7H,Ar-H), 3.82 (d,2H,S-CH₂), 8.0 (bs,1H,CH), 8.0(bs,1H,NH); ¹³C-NMR(DMSO-d₆,δppm): 173, 165.0, 151.4 143.0, 140.5, 136.6, 131.9, 130.6, 130.6, 129.0, 129.0, 125.8, 125.3, 123.8, 108.8, 40.9;  MS: m/z = 380.2.

2-[(4-chloro-1,3-benzoxazol-2-yl)sulfanyl]-N’-[(4-nitrophenyl)methylidene]acetohydrazide (5b):

IR(KBr,cm^-1): 3280(N-H), 2367(Ar-CH), 1688(O=C-NH), 1452(C=C), 1328(C-NO₂), 1252(C=N), 745(C-S), 680(C-Cl,Ar-H) ); 7.14-8.2 (m,7H), 3.82(d,2H,S-CH₂), 8.0(s,1H,CH), 8.1(bs,1H,NH); ¹³C-NMR(DMSO-d₆,δppm): 173, 165.0, 151.4,   148.2, 143.0, 140.5, 135.0, 131.9, 132.0, 130.1, 126.3, 125.8, 125.3, 123.8,121.2, 108.8, 40.9; MS: m/z=390.8.

2-[(4-chloro-1,3-benzoxazol-2-yl)sulfanyl]-N’-[(3-trimethylphenyl)methylidene] acetohydrazide (5c):

IR(KBr,cm^-1): 3284(N-H), 2809(N-CH₃), 2360(Ar-CH),  1692(O=C-NH), 1449(C=C), 1261(C=N), 744(C-S), 682(C-Cl); ¹HNMR(DMSO-d₆,δppm):6.6-7.27(m,7H,Ar-H), 3.82(d,2H,S-), 2.85(t,3H,CH₃), 8.0(bs,1H,CH), 8.1(s,1H,NH); ¹³CNMR(DMSOd₆,δppm):173, 165, 151.4, 149.7, 143.0, 140.5, 134.7, 129.8, 125.3, 123.8, 118.7, 116.6, 111.6, 108.8, 40.9,40.3; MS: m/z=388.8.

2-[(4-chloro-1,3-benzoxazol-2-yl)sulfanyl]-N’-[(3-methoxy,4-hydroxyphenylmethylidene] acetohydrazide (5d):

IR(KBr,cm^-1): 3280(N-H), 2367(Ar-CH),1688(O=C-NH), 1452(C=C), 1328(C-NO₂), 1252(C=N), 745(C-S), 680(C-Cl); ¹HNMR(DMSO-d₆,δppm):12.071(bs,1H,-NH), 9.531(s,1H,OH), 8.076(s,1H,-CH), 7.504-6.816(m,6H,Ar-H), 4.006(d,2H,S-CH₂), 3.812(s,3H,-OCH₃); ¹³CNMR(DMSOd₆,δppm):161.05, 149.20, 148.43, 147.31, 146.41, 128.69, 125.90, 121.86, 121.11, 116.44, 115.97, 110.78, 109.56, 56.04 ;MS: m/z=391.27, (M+1)=392.14.

2-[(4-chloro-1,3-benzoxazol-2-yl)sulfanyl]-N’-[(4-nitrophenyl)methylidene]acetohydrazide (5e):

IR(KBr,cm^-1): 3280(N-H), 2367(Ar-CH), 1688(O=C-NH), 1452(C=C), 1328(C-NO₂), 1252(C=N), 745(C-S), 680(C-Cl); 7.14-8.2(m,7H,Ar-H), 3.82(d,2H,S-CH₂), 8.0(s,1H,CH), 8.1(bs,1H,NH); ¹³C-NMR(DMSO-d₆,δppm):173, 165.0, 151.4,    148.2,    143.0, 140.5, 135.0, 131.9, 132.0, 130.1, 126.3, 125.8, 125.3, 123.8, 121.2, 108.8, 40.9; MS: m/z =390.8

2-[(4-chloro-1,3-benzoxazol-2-yl)sulfanyl]-N’-[(2-bromophenyl)methylidene] acetohydrazide (5f):

IR (KBr,cm^-1): 3285 (N-H), 2366 (Ar–CH), 1683 (O=C-NH), 1450 (C=C), 1252 (C=N), 744(C-S), 685 (C-Cl), 601 (C-Br); ¹HNMR (DMSO-d₆,δppm): 11.837 (bs,1H,-NH), 8.697 (s,1H,-CH), 8.197-7.349 (m,7H,Ar-H), 4.687 (d,2H,S-CH₂),3.812;¹³C-NMR (DMSOd₆,δppm): 168.52, 166.68, 163.37, 150.73, 146.76, 143.60, 143.15, 133.91, 132.61, 130.79, 129.54, 124.91, 123.88, 118.67, 112.07, 35.35; MS:m/z = 423.86,(M+2) = 425.85.

2-[(4-chloro-1,3-benzoxazol-2-yl)sulfanyl]-N’-[(4-hydroxyphenyl)methylidene] acetohydrazide(5g):

IR(KBr,cm^-1): 3445(O-H), 3287(N-H), 2368(Ar-CH), 1686(O=C-NH), 1455(C=C),   1253(C=N), 749(C-S),  682(C-Cl); ¹HNMR(DMSO-d₆,δppm): 12.071(bs,1H,-NH), 9.531(s,1H,OH), 8.1 (s,1H,-CH), 6.82-7.27(m,7H,Ar-H), 3.82(d,2H,S-CH₂); ¹³C-NMR(DMSO-d₆,δppm): 173, 160.8, 165.0, 151.4, 143.0, 140.5, 130.6, 126.4, 125.8, 125.3, 123.8, 116, 108.8, 40.9; MS:m/z =361.8.

2-[(4-chloro-1,3-benzoxazol-2-yl)sulfanyl]-N’-[(3-methoxyphenyl)methylidene] acetohydrazide (5h):

IR(KBr,cm^-1):3289(N-H), 2864(-OCH₃), 2366(Ar-CH), 1684(O=C-NH), 1454(C=C), 1259(C=N), 751(C-S), 688(C-Cl); ¹HNMR(DMSO-d₆,δppm): 12.071(bs,1H,-NH), 9.531(s,1H,OH), 8.1(s,1H,-CH), 6.82-7.27 (m,7H,Ar-H), 3.82 (d,2H,S-CH₂), 3.73(3H,-OCH₃); ¹³C NMR(DMSO-d₆,δppm): 173, 163, 145.8, 143.0, 134.3, 130.2, 130.2, 126.6, 126.1, 122.3, 121.6, 114.8, 114.4, 114.4, 53.9, 34.4; MS: m/z=375.4.

2-[(4-chloro-1,3-benzoxazol-2-yl)sulfanyl]-N’-[(4methoxyphenyl)methylidene]acetohydrazide (5i):

IR(KBr,cm^-1): 3286(N-H), 2866(-OCH₃), 2369(Ar-CH), 1683(O=C-NH), 1450(C=C), 1255(C=N), 752(C-S), 680(C-Cl); ¹HNMR(DMSO-d₆,δppm): 11.771(bs,1H,-NH), 8.163(s,1H,-CH), 8.137-6.979(m,7H,Ar-H), 4.670(d,2H,S-CH₂), 3.776(3H,-OCH₃); ¹³CNMR(DMSO-d₆,δppm): 168.32, 166.25, 159.98, 150.57, 144.41, 135.74, 130.40, 129.35, 124.64, 120.0, 118.51, 116.33, 113.18, 112.22, 111.91, 55.642; MS: m/z=375.95, (M+2)=377.95. 

2-[(4-chloro-1,3-benzoxazol-2-yl) sulfanyl]- N’-[(3,4,5-trimethoxy phenyl) methylidene] acetohydrazide (5j):

IR(KBr,cm^-1):3278(N-H), 2860(-OCH₃), 2371(Ar-CH), 1677(O=C-NH), 1457(C=C), 1249(C=N), 750(C-S), 686(C-Cl); ¹HNMR(DMSO-d₆,δppm):11.771(bs,1H,-NH), 8.163(s,1H,-CH), 7.27-6.6(m,5H,Ar-H), 3.82(2H,S-CH₂), 3.73(9H,-OCH₃); ¹³CNMR(DMSO-d₆,δppm):173, 165.0, 150.9, 150.9, 151.4, 143.0, 141.5, 140.5, 128.1, 125.8, 125.3, 123.8, 108.8, 106. 7, 106.7, 56.5, 56.2, 40.9 ; MS: m/z=435.88

Scheme 1: Synthesis of substituted of 2-[(4-chloro-1,3-benzoxazol-2-yl)sulfanyl]-N’-[phenylmethylidene] acetohydrazide derivatives. Click here to view Scheme

Biological Activities of novel 4-chloro-1,3-benzoxazole derivatives

Antibacterial Activity of compounds 5(a-j):

Novel benzoxazole derivatives were synthesized and tested for antibacterial activity by using the agar well diffusion method against Gram-positive bacteria, specifically Staphylococcus aureus and Bacillus subtillus, and Gram-negative bacteria Pseudomonas aeruginosa and Klebsiella pneumonia ^[23]. The 24 hour old Mueller-Hinton broth culture of test bacteria was swabbed on sterile Mueller-Hint on agar plates with the help of sterile cotton swab, which was continued by punching wells of 6mm with the aid of sterile cork borer. To the corresponding specified wells, the standard drug (Chloramphenicol, 1mg/mL of sterile distilled water), compounds 5(a-j) (250 µg/ml in 10% DMSO) and control (10% DMSO) were added. The plates were left to stand for nearly 30 minutes and incubated for 24 hour at 37⁰C in upright position and the zone of inhibition was observed and enlisted in Table 2 and represented in Figure1.

 Table 2: Antibacterial activity of synthesized compounds 5(a-j) using the agar well diffusion method against Gram-positive bacteria, specifically Staphylococcus aureus and Bacillus subtillus, and Gram-negative bacteria Pseudomonas aeruginosa and Klebsiella pneumonia

*STD=Chloramphenicol compound =250 𝜇g/ml

*Each value is expressed as the mean ± SD of three replicates for the zone of inhibition.

Figure 1: Antibacterial activity bar graph representing the zone of inhibition of synthesized compounds 5(a-j). Click here to view figure

Antifungal activity of compounds 5(a-j):

Antifungal activity of the compounds 5(a-j) were evaluated against fungal strains Gram positive fungi Candida albicans, Cryptococcus neoformans and Gram negative fungus Aspergillus niger, Pencillium using the sabouraud dextrose agar diffusion method²³. Wells were prepared (9 mm diameter) with a sterile cork borer. The standard medication (fluconazole, 100 g/mL of sterile distilled water) and control (10% DMSO) were added to the individually labelled wells. To these wells, compounds 5(a-j) (250 µg/mL of 10% DMSO) and control (10% DMSO) were added and the plates were permitted to cool for an hour to facilitate the diffusion. At 37 ºC, the plates were then incubated for 48 hours. At the final of the incubation period, the diameter of the zone of inhibition around the wells was estimated using vernier callipers and observed data are indexed in Table 3 and shown in Figure 2.

Table 3: Antifungal activity of synthesized compounds 5(a-j) using the sabouraud dextrose agar diffusion method against fungal strains Gram positive fungi Candida albicans, Cryptococcus neoformans and Gram negative fungus Aspergillus niger, Pencillium

*STD=Chloramphenicol compound

*Each value is expressed as the mean ± SD of three replicates for the zone of inhibition.

Figure 2: Antifungal activity bar graph representing the zone of inhibition of synthesized compounds 5(a-j). Click here to view figure

Minimum Inhibitory Concentration(MIC)

All the synthesized compounds have undergone testing for antibacterial and antifungal activity. Using the serial dilution technique, the Minimum inhibitory concentration (MIC) of the synthesized compounds 5(a-j) were calculated. The data of minimum inhibitory concentration for antibacterial and antifungal are presented in Table 4 and Table 5. Synthesized compounds were tested for their ability to inhibit the growth of bacterial and fungal strains at different concentrations that is 100, 50, 25, and 12.5 g/mL. The MIC zone of inhibition for antibacterial and antifungal activity of the compounds 5(a-j) are displayed in Figure 3 and Figure 4. . All of the synthesized compounds had promising MIC values against bacterial and fungal strains ²⁴. 

Table 4: MIC of synthesized Compounds 5(a-j) using serial dilution technique at different concentrations (100mg/ml, 50mg/ml, 25mg/ml and 12.5mg/ml) against two bacterial strains K.pneumoniae and B.subtilis

*Std = Ascorbic acid

*Each value is expressed as the mean ± SD of three replicates for the zone of inhibition. 

Figure 3: MIC of antibacterial activity bar graph representing the zone of inhibition of synthesized compounds 5(a-j). Click here to view figure

Table 5: MIC of synthesized Compounds 5(a-j) using serial dilution technique at different concentrations (100mg/ml, 50mg/ml, 25mg/ml and 12.5mg/ml) against two fungal strains C.albicans and A.niger

*STD=Fluconazole Compound

*Each value is expressed as the mean ± SD of three replicates for the zone of inhibition. 

Figure 4: MIC of antifungal activity bar graph representing the zone of inhibition of synthesized compounds 5(a-j). Click here to view figure

Antioxidant Activity (DPPH Assay)

The ability of synthetic compounds 5(a-j) and ascorbic acid(standard) to scavenge free radicals was assessed based on their ability to do so with regard to the DPPH free radical. Different concentrations of the compounds as well as the standard (5, 10, 15, 20 and 25 mg/ml) were prepared in methanol. In clean and clearly labeled test tubes, 3 ml of DPPH solution (0.002% in methanol) was blended with 05, 10, 15, 20 and 25 mg/mL of different concentrations of synthesized compounds and standard individually. Methanol was added to the solution to bring it up to 4 mL. A UV-Visible Spectrophotometer was used to measure the optical density at 517 nm after the tubes had been incubated at room temperature in the dark for 30 minutes. We measured the absorbance of the DPPH control. The Results are graphically represented in Figure 5 and summarised in Table 6. Using the formula, the scavenging activity was determined.

Scavenging activity (%) = A-B/A×100,

Where A is the absorbance of DPPH and B is the absorbance of DPPH in standard combination ^[25].

Table 6: Antioxidant activity of synthesized compounds 5(a-j) using DPPH methods at different concentrations (400µg/ml, 200µg/ml, 100 µg/ml, 50µg/ml, and 25µg/ml)

*Std = Ascorbic acid

*Each value is expressed as the mean ± SD of three replicates for the zone of inhibition.

Figure 5: Antioxidant activity bar graph representing the percentage of antioxidant potency of synthesized compounds 5(a-j). Click here to view figure

Molecular docking study

The reported approach was used to complete the molecular docking study ^{26, 27}. The In-Silco molecular docking procedure was used on the anticancer receptor, PDB code 2A91, the Protein Data Bank (PDB; http://www.rcsb.org/pdb) provided the receptor’s crystal structure. Prior to screening, the water molecules and heteroatoms were eliminated. Utilizing the protein preparation module of the HEX modelling package 8.0, the receptor structure was built before being used in the docking investigation. During the protein preparation, all hetero and water molecules were removed from the crystal structure except water molecules within 5Å from the ligand. The 3D structure of each ligand together with the receptor binding interactions were visualised to optimise quality by discovery studio 3.2. The results of the In-silico molecular docking provide important information on the capacity of recently synthesised drugs to attach to the receptor active sites. Thus, we performed a wet study of anticancer activity using the acquired docking values as a reference. The findings of binding scores of synthesized compounds 5(a-j) are indexed in Table 7 and the 2D and 3D binding orientation of prepared compounds 5(a-j) with receptor 2A91is displayed in Figure 6 to Figure 15.

Table 7: Binding energies and types of binding interaction of synthesized compounds 5(a-j) on the anticancer receptor, PDB code 2A91

Figure 6: 2D and 3D bonding interactions of receptor 2A91with compound 5a Click here to view figure

Figure 7: 2D and 3D bonding interactions of receptor 2A91 with compound 5b Click here to view figure

Figure 8: 2D and 3D bonding interactions of receptor 2A91 with compound 5c Click here to view figure

Figure 9: 2D and 3D bonding interactions of receptor 2A91 with compound 5d Click here to view figure

Figure 10: 2D and 3D bonding interactions of receptor 2A91 with compound 5e. Click here to view figure

Figure 11: 2D and 3D bonding interactions of receptor 2A91 with compound 5f. Click here to view figure

Figure 12: 2D and 3D bonding interactions of receptor 2A91 with compound 5g Click here to view figure

Figure 13: 2D and 3D bonding interactions of receptor 2A91 with compound 5h Click here to view figure

Figure 14: 2D and 3D bonding interactions of receptor 2A91 with compound 5i Click here to view figure

Figure 15: 2D and 3D bonding interactions of receptor 2A91 with compound 5j. Click here to view figure

Anticancer activity [Cell preparation and cell viability]

The in-vitro anticancer activity of the synthesized compounds 5(a-j) were assessed using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) against an human cancer cell line MCF-7 (cancer breast)^[28]. The assay was observed to be entirely relying on the decrease of the tetrazolium salt via mitochondrial dehydrogenase of viable cells in order to produce a blue formazan product dissolved in DMSO, which was measured at 570nm. With the aid of graph Pad Prism Version 5.1, IC₅₀ (µM) data of synthesized compounds were estimated and Paclitaxel was utilized as positive control. The human cancer cell lines were procured from National Centre for Cell Science, Pune, India and Dulbecco’s Modified Eagle Medium(DMEM) with low glucose (Cat No-11965-092, Gibco, Invitrogen) was used to culture the cell lines enhanced with 10% fetal bovine serum(CatNo-10270106,Gibco,Invitrogen) and 1% antimycotic (Cat No-15240062, Thermo fisher Scientific) was used for cell culture. Untreated cells were considered as control. The results of the anti-cancer screening are indexed in Table 8 and represented in Figure 16. In addition to this, IC₅₀ values of synthesized compounds were also estimated, which are indexed in Table 9 and depicted in Figure 17.

The cells were cultivated in a 96-well flat-bottom microplate and stored overnight at        37ºC in 95% humidity and 5% CO₂. Different sample concentrations (400, 200, 100, 50, 25, 12.5μg/ml) were treated. For an additional 48 hours, the cells were incubated and the wells were washed twice with PBS. Further, 20μL of the MTT staining solution was introduced to individual well and plates were incubated at 37ºC. After 4 hours, 100 mL of DMSO was added to each well to dissolve the formazan crystals, and using a microplate reader, the absorbance at 570 nm was measured. The following formulae were used to calculate the cytotoxicity:

Table 8: In-vitro cytotoxicity of synthesized compounds (5a, 5b, 5d, 5g, 5i) against MCF-7 cell lines

*Each value is expressed as mean ± SD of three replicates for the zone of inhibition

Figure 16: In-vitro cytotoxic potency of synthesized compounds Click here to view figure

Table 9: IC₅₀ values of synthesized compounds (5a, 5b, 5d, 5g, 5i) against MCF-7 cell lines

Figure 17: IC50 values of synthesized compounds against MCF-7 cell line in comparison with Paclitaxel (Positive control). Click here to view figure

Discussions

A cyclized product of chloro substituted 1, 3-benzoxazole-2-thiol 2 compound has been prepared from 4-Chloro-2-amino-phenol by treating it with carbon disulphide and potassium hydroxide in the presence of methanol ^[19].The SH group which is present in the compound 2 was undergo substitution reaction with ethyl chloroacetate with the addition of acetone to produce thio ether product ethyl [(4-chloro-1, 3-benzoxazol-2-yl) sulfanyl] acetate 3 ^[20]. A further treatment of compound 3 with hydrazine hydrate led to the formation of peptide or amide bond formation by the elimination of ethyl alcohol to ptoduce an  intermediate 2-[(4-chloro-1,3-benzoxazol-2-yl)sulfanyl]acetohydrazide 4 ^[21]. ¹H NMR characterized compound 4 as having two singlets at δ 4.490 and δ 9.414 ppm due to the presence of -NH₂ and -NH protons respectively. As a result of reacting intermediate 4 with varied aromatic aldehydes, the NH₂ group in the product 4 reacts with aldehyde to produce imine(C=N) bond through condensation reaction, derivatives 2-[(4-chloro-1, 3-benzoxazol-2-yl) sulfanyl]-N’-[-phenylmethylidene] acetohydrazides 5(a-j) have been obtained ^[22]. The newly synthesized molecules displayed intense absorbance band at 1660cm^-1 for -NH and 1692 cm^-1 for -C=O groups in IR spectrum and the ¹H NMR revealed a peak at δ 11.836 (bs, -NH) justifying the disappearance of NH₂ proton and the formation of new ring by insertion reaction [29]. In addition, the mass peak also correlated with the molecular weight of the synthesized molecules.

Studies have also been performed on the synthesized molecules 5(a-j) for their antibacterial, antifungal, MIC, antioxidant and cytotoxic activity. Based on the results of antibacterial and antifungal studies, few compounds have demonstrated potent zone of inhibitions, as shown in Table 2 and Table 3 and Figures 1 and 2. Comparatively to standard drugs Chloramphenicol and Fluconazole, compounds 5a, 5b, 5d, 5e, 5g and 5h displayed marked zones of inhibition against bacteria and fungi. At different concentrations, the compounds were explored for their Minimum Inhibitory Concentration (MIC) to determine their distinct zones of inhibition against bacteria and fungi and Tables 4 and 5 and Figures 3 and 4 illustrate the results of this analysis. A marked zone of inhibition was noted for compounds 5a, 5b, 5d, 5e, 5g and 5h against gram positive and gram negative bacteria at four various concentrations (100g/ml, 50g/ml, 25g/ml and 12.5g/ml). In spite of concentration differences, chloro, nitro, methoxy and hydroxy substituted benzoxazole derivatives showed significant efficacies. This observation is favoured by antioxidant activity, which was done with effective free radical scavenge as outlined in Table 6 and Figure 5 respectively. The derivatives 5(a-j) exhibited powerful free radical scavenging properties.

In order to become better acquainted with the binding energies and types of binding interactions of the prepared compounds, molecular docking was performed on the synthesized compounds. Compared to the rest of the prepared compounds, the synthesized compound 5i possessed admirable binding scores (-322.59 kcal/mol). The binding score obtained from the molecular docking study and also by considering the similar structures of newly prepared compounds, where only the position of substituent differs, few of the selected compounds were screened for their cytotoxic activity against MCF-7 cell line and the observations are tabulated in Table 8 and represented in Figure 16 [30]. Following the binding scores of docking study and considering that the only difference between newly prepared compounds is the position of the substituents, few compounds were selected for cytotoxic testing against MCF-7 cells. At the least concentration of 12.5 g/mL, both compounds 5g and 5i displayed impressive inhibitory activity of 45% and 51%, respectively. Also, compound 5i demonstrated potential activity for MCF-7 cell line with an IC₅₀ value of 8.77µg/mL

Conclusion

Current work comprises of series of the synthesis of novel 2-[(4-chloro-1,3-benzoxazol-2-yl)sulfanyl]-N’-[(-phenylmethylidene]acetohydrazide 5(a-j) derivatives. The expected target molecules were prepared, structurally confirmed by using IR, ¹H NMR, ¹³C NMR and mass spectral analysis. They were also subjected to various biological activities, which includes antimicrobial, antioxidant and in-vitro cytotoxic activity. Among the synthesized compounds 5g and 5i were found to exhibit increased potency and considered as potential molecules for further toxicological development of drugs.

Acknowledgement

The authors are thankful to the Directorate of minorities, Bangalore, Karnataka, India, for financial support. The authors are grateful to the Principal, Sahyadri Science College, Shivamogga for providing the necessary research facilities. We are also grateful to Sophisticated Analytical Instruments Facility, Mysore University, Karnataka India, MIT Manipal for providing ¹HNMR, ¹³C NMR and Mass spectral facilities.

Conflict of interest

There is no conflict of interest.

Funding Sources

There is no funding sources

References

1. Seetharama D, Satyanarayanjois and Ronald A Hill. Medicinal chemistry for 2020. Future Med Chem. 2011; 3(14): 1765-1786.
   CrossRef
2. Maruthamuthu, Shameela Rajam, Christina Ruby Stella P., Bharathi Dileepan A. G. and R. Ranjith. The chemistry and biological significance of imidazole, benzimidazole, benzoxazole, tetrazole and quinazolinone nucleus. Journal of Chemical and Pharmaceutical Research. 2016. 8(5): 505-526.
3. E Susithra, S. Rajkumar, S. Komal Walmik Pansare, S. Praveena , PV. Parvati Sai Arun, Rajasekhar Chekkara, Gangarapu Kiran. Design, Synthesis, Antimicrobial and Anticancer Activity of some Novel Benzoxazole-Isatin Conjugates. Biointerace Research in Applied Chemistry. 2022; 12(2): 2392-2403.
   CrossRef
4. Lingling Fan, Zhongfu Luo, Changfei Yang, Bing Guo, Jing Miao, Yang Chen, Lei Tang and Yong Li. Design and Synthesis of small molecular 2-aminobenzoxzoles as potential antifungal agents against phytopathogenic fungi. Molecular Diversity. 2022; 26: 981-992.
   CrossRef
5. Ryu C.K, Lee R.Y, Kim N.Y, Kim Y.H and Song A.L. Synthesis and antifungal activity of benzo[d]oxazole-4,7-diones. Bioorg Med ChemLett. 2009; 19(20): 5924-5926.
   CrossRef
6. Paramashivappa R, P. Phani Kumar, P. V. Subba Rao and A. Srinivasa Rao. Design, Synthesis and Biological Evaluation of Benzimidazole/Benzothiazole and Benzoxazole Derivatives as Cyclooxygenase Inhibitors. Bioorganic & Medicinal Chemistry Letters. 2003; 13: 657-660.
   CrossRef
7. PuXiang, Tian Zhou, Liang Wang, Chang-Yan Sun, Jing Hu and et al., Novel Benzothiazole, Benzimidazole and Benzoxazole Derivatives as Potential Antitumor Agents: Synthesis and Preliminary in Vitro Biological Evaluation. 2012; 17: 873-883.
   CrossRef
8. Sarafroz M, Mumtaz Alam M, Waquar Ahsan and Nadeem Siddiqui. Synthesis, Anticonvulsant and Neurotoxicity Evaluation of 5-Carbomethoxy benzoxazole Derivatives.  Acta Poloniae Pharmaceutica Drug Research.  2008; 65(4): 449-455.
9. Aiello S, Wells G, Stone E.L, Kadri H, Bazzi R, Bell D.R, Stevens M.F.G, Matthews C.S.T, Bradshaw D and Westwell A.D. Synthesis and biological properties of benzothiazole, benzoxazole, and chromen-4-one analogues of the potent antitumor agent 2-(3,4-dimethoxyphenyl)-5- fluorobenzothiazol. J Med Chem. 2008; 51(16): 5135-5139.
   CrossRef
10. Sondhi S.M, Singh N, Kumar A, Lozach O and Meijer L. Synthesis, anti-inflammatory, analgesic and kinase (CDK-1, CDK-5 and GSK-3) inhibition activity evaluation of benzimidazole/benzoxazole derivatives and some Schiff’s bases. Bioorg Med Chem. 2006; 14(11): 3758-3765.
    CrossRef
11. Veronika S lachtova and Lucie Brulı´kova. Benzoxazole Derivatives as Promising Antitubercular Agents. Chemistry Select 2018; 3: 4653-4662.
    CrossRef
12. Davidson J.P and Corey E.J. First enantiospecific total synthesis of the anti-tubercular marine natural product pseudopteroxazole revision of assigned stereochemistry. J Am ChemSoc. 2003; 125(44): 13486-13489.
    CrossRef
13. Benazzouz A, Boraud T, Dubedat P, Boireau A, Stutzmann J.M and Gross Riluzole prevents MPTP-induced parkinsonism in the rhesus monkey: a pilot study. Eur J Pharmacol. 1995; 284(3): 299–307.
    CrossRef
14. Yasuo S, Megumi Y, Satoshi Y, Tomoko Midori I, Tetsutaro N, Kokichi S and et al. Benzoxazole derivatives as novel 5-HT3 receptor partial agonists in the Gut. J Med Chem.1998; 41(16): 3015–3021.
    CrossRef
15. Razavi H, Palaninathan S.K, Powers E.T, Wiseman R.L, Purkey H.E and et al. Benzoxazoles as transthyretin amyloid fibril inhibitors: synthesis, evaluation, and mechanism of action. Chem.Int. Ed. 2003; 42(24): 2758–2761.
    CrossRef
16. Sessions E.H, Yin Y, Bannister T.D, Weiser A, Griffin E, Pocas J and et al.,Benzimidazole and benzoxazole-based inhibitors of Rho kinase. Bioorg Med Chem Lett. 2008; 18(24): 6390.
    CrossRef
17. Hangirgekar S. Phenyl-Trimethyl-Ammonium Tribromide: Facile Catalyst for the One Pot Synthesis of Substituted Benzoxazoles. Res.J.of Pharm. Bio and Chem. Sci. 2012; 3: 83-88.
18. Guzow K, Szabelski M, Malicka J, Karolczak J and Wiczk W. Synthesis and Photo physical Properties of 3-[2(pyridyl)Benzoxazole-5-yl]-L-Alanine Derivatives.   Tetrahedron . 2002; 58: 2201-2209.
    CrossRef
19. Mohammed, O.A.; Dahham, O.S. Synthesis, Characterization, and Study of Antibacterial Activity of Some New Formazan Dyes Derivatives, Derived from 2-Mercapto Benzoxazole. IOP Conf. Series: Materials Science and Engineering. 2018; 454: 1-11.
    CrossRef
20. Kakkar, S.; Tahlna, S.; Lim, S.M.; Ramasamy, K.; Mani, V.; Shah, S.A.A.; Narasimhan, B. Benzoxazole derivatives: design, synthesis and biological evaluation. Chem Cent J. 2018: 12: 1-16.
    CrossRef
21. Lubna Afroz, Moodgere Habeebulla Moinuddin Khan, Hosadu Manjappa Vagdevi, Mohammed Shafeeulla Rasheed, Malathesh Pari, Anjaiah Subbaraju. Synthesis, Characterization and Electrochemical Detection of Glucose and H2O2, Molecular Docking and Biological Inspection of Transition Metal Complexes of Novel Ligand 2-[(5-methyl-1,3-benzoxazol-2-yl)sulfanyl]acetohydrazide. Biointerace Research in Applied Chemistry. 2022; 13(4):1-34.
    CrossRef
22. Parvathy N.G, Manju Prathap, Mukesh M and Leena Thomas. Design, synthesis and molecular docking studies of benzothiazole derivatives as anti-microbial agents. International Journal of Pharmacy and Pharmaceutical Sciences. 2013; 5(2):101-106.
    CrossRef
23. Padmini T.R, Vagdevi H.M and Usha Jinendra. Synthesis of Benzoxazole Associated Benzothiazine-4-ones and their in-vitro and in-silico Antimicrobial, Antioxidant Activities. Asian Journal of Chemistry. 2021; 33(1): 137-150.
    CrossRef
24. Hasan, Shah A.A and Hameed A. Methods for detection and characterization of lipases: acomprehensive review. Biotechnology Advances. 2009; 27(6): 782-798.
    CrossRef
25. Lubna Afroz, Moinuddin Khan M.H, Vagdevi H.M, Malathesh Pari, Mohammed Shafeeualla.R and Mussuvir Pasha K.M. Emergent materials. 2021; 23.
26. Padmini T.R, Vagdevi H.M, Usha Jinendra and Ravikiran B. Synthesis of benzoxazole derivatives by Mannich reaction and invitrocytotoxic, antimicrobial and docking studies. Chemical Data Collections. 2021;31:100628.
    CrossRef
27. Shreedhara S.H, Vagdevi H.M, Jayanna N.D, Raghavendra R, Kiranmayee P and Prabhu Das. In-vitrocytotoxic, Antimicrobial and Antioxidant activity of 6-Chloro-2,3-dihydro[1,2,4]triazolo[3,4-b][1,3]benzoxazole Derivatives. Research Journal of Pharmaceutical, Biological and Chemical Sciences. 2017; 8(4): 835.
28. Kumbar V.M, Peram M.R, Kugaji M.S, Shah T, Patil S.P, Muddapur U.M and et al. Effect of curcumin on growth, biofilm formation and virulence factor gene expression of Porphyromonasgingivalis. Odontology. 2021; 109(1):18-28.
    CrossRef
29. Mohammad R Ahmad and Ali A. Mohsen. Synthesis and Characterization of Some New Derivatives from 2-Mercaptobenzoxazole. Iraqi Journal of Science. 2015; 56: 303-315.
30. Saloni Kakkar, Sumit Tahlan, Siong Meng Lim, Kalavathy Ramasamy, Vasudevan Mani, Syed Adnan Ali Shah, and Balasubramanian Narasimhan. Benzoxazole derivatives: design, synthesis and biological evaluation. Chemistry Central Journal. 2018; 12(92): 1-16.
    CrossRef