In-silico Studies, Synthesis, and Antibacterial Evaluation of Thiophene Linked Isoxazole Derivatives

In this work a series of thiophene linked isoxazole derivatives (LK1-LK8) was synthesized by cyclization of different substituted thienyl chalcones (PL1-PL8). The structures of the synthesized compounds were characterized by IR, 1H NMR and mass spectral data. These derivatives were evaluated for antibacterial activities. Compounds LK7 showed excellent antibacterial activities amongst the synthesized compounds with MIC value 6.75 μg/ml. Molecular docking of these linked isoxazole derivatives (LK1-LK8) was also performed with crystal structure of staph gyrase B 24kDa (PDB code: 5 4URM). All the isoxazole derivatives (LK1-LK8) were docked into same groove of the binding site of native co-crystallized (1R,4aS,5S,6S,8aR)-5-{[(5S)-1-(3-O-acetyl-4-O-carbamoyl-6deoxy-2-O-methyl-alpha-L-talopyra nosy l)-4 hydroxy 2-oxo-5-(propan-2-yl)-2,5-dihydro-1H-pyrrol3-yl]carbonyl}-6-methyl-4 1, 2, 3, 4 methylid-ene,4a,5,6,8a-octahydronaphthalen-1-yl2,6-dideoxy-3C-[(1S)-1-{[(3,4-dichloro-5-methyl-1 H-pyrrol-2-yl) carbonyl]amino}ethyl]-beta-D-ribohexopyranoside) ligand for activity explanation and exhibited good ligand interaction and binding affinity were of range -2.04 to -4.34 kcal/mol. Original Research Article Crasta et al.; JPRI, 33(43A): 256-264, 2021; Article no.JPRI.72934 257


INTRODUCTION
Treatment of bacterial infection remain important and challenging task due to its multiple factors such developing infectious disease, increase in number of multiple drug resistance bacteria and side of the drug presently used in the treatment [1][2][3]. These factors have enormously increased the importance specially in hospitalised and immune compromised patient. Although large number of antibiotics and chemotherapeutics are present in the market but due development of resistance to these drugs there is always a need of new class of compound to overcome this problem. However, many studies have been carried out to overcome this problem, but desirable result is still a challenge. In regard we have planned to synthesise a new class of compounds having thiophene and Isoxazole ring and evaluate its antimicrobial properties.
Thiophene is five membered heterocyclic rings, its derivatives show multiple activities such as antimicrobial [4], analgesic and anti-inflammatory [5], antihypertensive [6], and antitumor activity [7]. Isoxazole is an azole with an oxygen atom next to the nitrogen. Isoxazole and its derivatives have a broad range of chemotherapeutic action such as antibacterial activity [8], anti-tubercular activity [9], anti-depressant [10], anti-convulsant [11], anti-hyperglycaemic, anti-alzheimers, [12], anti-obesity or hypolipidemic activity, antiinflammatory [13], antioxidant, and anti-aging activity [14]. Some of the drugs that contain Isoxazole rings are Valdecoxib, Leflunomide, Flucloxacillin, Cloxacillin and Oxacillin. Hence owing to the wide range of activities of these heterocyclic compounds it was planned to synthesise derivatives containing these two heterocyclic rings and to compare the affinities of these compounds to the target protein crystal structure of staph gyrase B 24kDa PDB Code 4URM by molecular docking studies and evaluate its antibacterial properties by tube dilution method [15,16,17].

MATERIALS AND METHODS
All the reactions have been carried out under the prescribed laboratory conditions. All the synthetic work has been done by procuring the available analytical grade solvents and laboratory grade reagents . For the synthesis of the thiophene  linked Isoxazole derivatives the reagents used  are  substituted  benzaldehyde,  2-acetyl   thiophene,  sodium  hydroxide, ethanol, hydroxylamine hydrochloride, sodium acetate, glacial acetic acid which were procured from Sigma-Aldrich.
Purity of the intermediates and final compounds were monitored by thin layer chromatography (TLC) using silica gel G plates. The spots were visualized under UV light and solvent. n-hexane: ethyl acetate (7:3) mixture was used as solvent for running the TLC of these compounds. All IR spectra were recorded in Alpha Bruker using ATR method. 1 H NMR spectra were recorded at 400 MHz Bruker Avance II NMR Spectrometer. Mass spectrum was recorded on GC-MS Perkin Elmer Clarus 680 Spectrometer obtained by electro impact ionization method.

Preparation of Thienyl Chalcones: PL1-PL8
In a 250 ml conical flask 5mmol of substituted aldehyde and ethanol were added. When all the contents were dissolved 5 mmol substituted 2acetyl thiophene was added followed by 3-4 drops of aqueous sodium hydroxide. The mixture was kept for overnight stirring. The flask was then kept in the refrigerator for 2 hours. The solid was filtered and was then recrystallized using an ethanol as solvent. [18] The physiochemical properties of these thienyl chalcones are mentioned in Table 1.

Preparation of Isoxazole Derivatives: LK1-LK8
The mixture of previously prepared thienyl chalcone derivatives (0.01mole) along with hydroxylamine hydrochloride (0.01mole) was added to a 250ml conical flask. To this sodium acetate and glacial acetic acid were added along with 50ml ethanol and was stirred. The mixture was then refluxed for 8 to 10 hours. The reaction mixture was cooled and poured into ice-cold water. It is then filtered and dried to get the final product. The product was recrystallized using ethanol as the solvent [19]. The physiochemical properties of these isoxazole derivatives are mentioned in Table 2.

Minimum Inhibitory Concentration
The broth dilution test is one of the standard methods for determining the level of resistance to an antibiotic. Serial dilutions of the antibiotic are made in a liquid medium which is inoculated with a standardized number of organisms and incubated for a prescribed time. The lowest concentration of antibiotic preventing appearance of turbidity is the minimal inhibitory concentration (MIC). After preparation of different concentrations of the test compound in nutrient broth (by using the broth dilution method), we inoculate them with the test organism. The MIC is determined after incubation by choosing the lowest concentration in which no growth occurs. The MIC and the zone of inhibition are inversely correlated. In other words, the more susceptible the microorganism is to the antimicrobial agent, the lower the MIC and the larger the zone of inhibition. Conversely, the more resistant the microorganism, the higher the MIC and the smaller the zone of inhibition [20].

DISCUSSION
In this present work different substituted novel thiophene linked isoxazole derivatives were synthesized using two step reactions. In step one thienyl chalcones (PL1-PL8) were synthesized from substituted 2-acetyl thiophene and different substituted aldehyde. The formation of thienyl chalcones was confirmed with IR peak 1647 cm-1, shows the presence of enones (=C-C=O). In step two thiophene linked isoxazole derivatives (LK1-LK8) were synthesized from thienyl chalcones by treating with hydroxylamine hydrochloride and confirmed from spectral analysis. The compound LK-5 showed IR peak at 1240cm-

CONCLUSION
The study reports the successful synthesis of thiophene linked isoxazole derivatives from cyclisation of thienyl chalcones with moderate yields and most synthesized compounds have shown significant antibacterial activity. The docking studies shows good interaction with various amino acid of the receptor and the ligand through different bonds. The antibacterial properties of compound having electronegative substitution on the ring have shown excellent results thus favour the antibacterial properties whereas electropositive substituent on the ring shows less activity thus decrease the antibacterial properties. Although, these compounds have diverse biological properties, and their structures allow wide substitutions thus draw more interest to synthesize new antimicrobial compounds.

DISCLAIMER
The products used for this research are commonly and predominantly use products in our area of research and country. There is absolutely no conflict of interest between the authors and producers of the products because we do not intend to use these products as an avenue for any litigation but for the advancement of knowledge. Also, the research was not funded by the producing company rather it was funded by the personal efforts of the authors.

CONSENT
It is not applicable.

ETHICAL APPROVAL
It is not applicable .