Synthesis, Docking and Antimicrobial Activity of Some New Coumarin Incorporated Thiazole Derivatives

Synthesis and screening of a series of new coumarin derivatives coupled with thiazole are performed for their antimicrobial properties. A series of new thiazolyl coumarin derivatives were synthesized upon refluxing 3-bromoaceytl coumarin, substituted benzaldehyde and thiosemicarbazide in the presence of glacial acetic acid. Substituted 3-acetyl coumarin undergoes bromination in the presence of bromine and chloroform to form 3-Bromoaceytl coumarin. The thiazolyl coumarin derivatives were characterized based on IR, 1 H NMR, and Mass spectral data. The docking studies have been carried out against the enzyme DNA gyrase (1KZN). Compound SCT 2 showed the highest docking score -5.662 compared to other compounds. The final synthesized compounds were screened for their antibacterial activity by tube dilution method. Compound SCT 1 and SCT 2 showed significant antibacterial activity with minimum inhibitory concentration of 12.5μg/ml and 6.25μg/ml, respectively, compared to standard Cephalosporin. The MIC results suggest that compounds SCT 1 and SCT 2 showed promising antibacterial activity. So these compounds are interesting lead molecules for further synthesis as antimicrobial agents. Original Research Article Kumar et al.; JPRI, 33(57B): 332-340, 2021; Article no.JPRI.78027 333


INTRODUCTION
Exposure of resistance by fungal and bacterial strains for already existing antimicrobial agents is one of the crucial problems and also a motive to synthesize a new class of antimicrobial agents having potential activity compared to the commonly used therapy. The structurally attractive compounds for synthesizing antimicrobial agents are coumarin derivatives. The issue is more critical than that can be imagined because of the frequent genetic development of pathogenic bacterial strains by accomplishing resistance to antibiotics/ drugs that were often not used for treatment. Sadly, newer resistant bacteria develop continually with new antibiotics or freshly launched drugs. Around 17 million people die from infectious diseases every year, and approximately 50,000 people are infected [1]. As a result, the evolution of new antibacterial drug candidates remains the future scope.
Small ring heterocycles, including sulfur and nitrogen, have been investigated for a long time because of their therapeutic relevance and synthetic diversity. Amongst the extensive range of heterocycles inspected by elite prospects in the discovery of drugs, it is identified that thiazoles play a vital role in medicinal chemistry. [11] Thiazole ring is a structural fragment of natural compounds such as carboxylase, epothilones, thiamine pyrophosphate (TPP, a coenzyme essential in respiration in the Krebs cycle), thiamine (vitamin B1), and the large family of macrocyclic thiopeptide antibiotics, micrococcin P1 and thiostrepton. [12] Thiazole derivatives are related with a broad spectrum of biological properties, including hypnotics, HIV infections, schizophrenia, inflammation, hypertension, anticancer, antimalarial, antiviral, antituberculous, antimicrobial, anticonvulsant, bacteriostatic activities, and very recently for the treatment of pain, as fibrinogen receptor antagonists with anti-thrombotic activity, as new inhibitors of bacterial DNA gyrase B. [13][14][15] They are also used in the drug development application for allergy treatment. [16] It is reported in the literature that when the thiazole ring is coupled with coumarins, the biological activity gets enhanced manifold. The challenges of antibacterial research are significant, and a good start towards developing a new class of hybrid antimicrobials and the aid of computeraided drug design may deliver new antimicrobials to the clinic. Based on the above observation, it is worthwhile to prepare newer compounds for their antimicrobial activity.

Materials
All chemicals used are of analytical grade: Salicylaldehyde, ethyl acetoacetate, piperidine, ammonium acetate, ethanol, substituted benzaldehyde, and sodium hydroxide. Determinations of melting points was done by the open capillary method and are uncorrected. The purity of the final compounds and intermediates was checked by thin layer chromatography (TLC) using silica gel G plates. The spots were made visible under UV light. n-hexane: Ethylacetate (5:5) was used as a solvent for running the TLC of these compounds. All IR spectra were registered using the ATR method in Alpha Bruker. 1 H NMR spectra were recorded at 400 MHz Bruker Avance II NMR Spectrometer. The mass spectrum was recorded on GC-MS Perkin Elmer Clarus 680 Spectrometer obtained by electron impact ionization method.

General Procedure for the Synthesis of 3-Acetyl Coumarin
A mixture of salicylaldehyde (0.05 mol) and ethylacetoacetate was added to 250ml conical flask. It was then condensed by adding sufficient piperidine dropwise with stirring in ice-cold condition. The reaction mixture was then kept overnight in the refrigerator. The solid lumps were broken in cold ethanol. The resulting yellow-colored solid mass was filtered and washed with cold ethanol to remove the excess piperidine. It was then recrystallized from ethanol to give white needle-shaped crystals.

General Procedure for the Synthesis of 3-Bromo Acetyl Coumarin
3-acetyl-2H-chromene-2-one (0.01 mol) was dissolved in chloroform (0.1mol), and a solution of Bromine (0.01 mol) in chloroform was added drop-wise with continuous stirring, and mixture was kept in the water bath at 70 o C. The progression of the reaction was observed by thin layer chromatography. After the completion of the reaction, judged by TLC, the reaction mixture was washed with diethyl ether and recrystallized using ethanol to provide 3-(2-bromoacetyl)-2Hchromen-2-one.

General Procedure for the Synthesis of Thiazolyl Coumarin Derivatives (SCT1-SCT12)
An equimolar mixture of 3-Bromo Acetyl Coumarin (0.05 mol), thiosemicarbazide (0.05 mol), and various substituted benzaldehyde (0.05 mol) was refluxed in the presence of glacial acetic acid (2 ml) for 4-5 hours. The reaction mixture was transferred into crushed ice and stirred well until the product was formed. It was filtered, dried, and recrystallized using ethanol.

Minimum Inhibitory Concentration
The broth dilution test is one of the standard method for determining the level of resistance to an antibiotic. Serial dilutions of the antibiotic are made in a liquid medium inoculated with a standardized number of organisms and incubated for a prescribed time. The lowest concentration of antibiotic preventing the appearance of turbidity is considered to be the minimal inhibitory concentration (MIC). After preparing different concentrations of the test compound in nutrient broth (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. The method gives information on the storage of standard antibiotic powder, preparation of the stock antibiotic solution, media, preparation of inocula, incubation condition, and reading and interpretation of results [17].

Procedure
Double concentration of the nutrient broth was prepared. Distribute each 2.5 ml into 8 test tubes and label them A1 to A8. Distribute 2.5 ml in two test tubes and label them as positive control and negative control. Prepare a drug stock solution of 2000 μg/ml by dissolving the drug in water. From this stock solution, the following dilutions were prepared; 2.5 ml of the stock solution diluted to 25 ml with water to give 200 μg/ml. Serial dilution of the same was performed to give 100 µg/ml, 50 µg/ml, 25 µg/ml, 12.5 µg/ml and 6.25 µg/ml respectively. Add 2.5 ml of each double concentration nutrient broth to 2.5 ml of the above dilutions so that the concentration further gets halved. i.e., 100 µg/ml, 50 µg/ml, 25 µg/ml, 12.5 µg/ml, 6.25 µg/ml and 3.12 µg/ml respectively. 2.5 ml of water was added to positive control and negative control tube and well mixed. Mix all the tubes well close with nonabsorbent cotton plugs and sterilize by autoclaving 15 lbs./sq. in (121°C) for 15 min.
Cooled the tubes to room temperature and inoculated all the tubes with one loopful of the test organism Escherichia coli , except in the negative control tube. Incubated all the tube at 37°C for 48 hrs and observed the turbidity.

Scheme 1. Synthesis of Thiazolyl Coumarin Derivatives
Negative control: In this, no growth is expected. It confirms that the medium is sterile.
Positive control: In this the growth of the inoculated organism is expected. This indicates that (a) The nutrient of the medium supports the growth of organism that has been inoculated. (b) Inoculation of live organisms.

Molecular Docking Study
Molecular docking study was done to understand the interconnections between receptor and ligand (synthesized compounds). In silico analysis was performed on Schrodinger 2018-3 suite device Maestro 11.7.012, (Ligprep, Glide XP docking, QikProp), this software package programmed on DELL Inc.27" workstation machine running on Intel Core i7-7700 CPU @ 3.60 GHz x8, the processor with 8 GB RAM and 1 TB hard disk with Linux -X6_64 as the operating system. QikProp was used to predict the ADME properties of synthesized compounds. For docking calculation, the protein (PDB code: 1KZN) was downloaded from the protein data bank and refined using the protein preparation wizard. Assessment of binding affinity was done in terms of binding free energies (S-score, kcal/mol). The synthesized compounds were all docked in the groove of the binding site present in DNA Gyrase 1KZN.      Final synthesized compounds SCT1-SCT12 interacted with receptor DNA Gyrase (PDB code: 1KZN), and their binding energy and interaction pattern was studied. Compounds SCT1-SCT12 showed the binding energy in the range of -3.806 to -5.662 kcal/mol. Compound SCT2 showed the best interaction with the receptor and binding energy of -5.662 kcal/mol. These compounds interacted with forces such as hydrogen bond, hydrophobic interaction, charged negative, charged positively with the various amino acid of the receptor. Best docked compound SCT2 forms one hydrogen bond with amino acid VAL-43, hydrophobic interaction with amino acid ALA-47, ILE-78, PRO-79, charged negatives ARG-76, LYS-21, MET-166, and polar interaction ASP-73 and THR-165 of the receptor DNA Gyrase.

Antibacterial Activity
The different thiazolyl coumarin derivatives were evaluated for their antibacterial activity by Tube dilution method. Compound SCT1 and SCT2 showed significant antibacterial activity with MIC of 12.5µg/ml and 6.25µg/ml respectively compared to standard Cephalosporin as given in Table 3 and 4. The presence of electron donating groups like hydroxyl and electron withdrawing group like chloro resulted in increased antibacterial activity. Compound SCT1 and SCT2 resulted in increased antibacterial activity due to the unsubstituted coumarin moiety and the presence of electron donating hydroxyl group and electron withdrawing chloro group in the phenyl ring.

CONCLUSION
The study reports the successful synthesis of thiazolyl coumarin derivatives from cyclization of 3-Bromoacetyl coumarin with moderate yields. Thiazole incorporated coumarin ring was synthesized by the new synthetic route, and few compounds have shown significant antibacterial activity by tube dilution method. Compound SCT2 showed the highest docking score compared to other compounds, which also showed significant antibacterial activity with MIC of 6.25µg/ml. Compounds SCT1 and SCT2 might be useful as a lead molecule for pharmaceutical industries. So the current work requires further structural modification to get better antimicrobial actions.

CONSENT
It is not applicable.

ETHICAL APPROVAL
It is not applicable.