Synthesis and Evaluation of Coumarin Chalcone Derivatives as DNA Gyrase Inhibitors

Introduction

Microbial resistance is becoming a big concern and a difficult challenge for researchers worldwide since it poses a substantial threat to human health. The WHO estimates, 50,000 population including men, women and also children dying per day due to infection of microbes.¹ Due to microbial resistance, a high level of toxicity, inadequate antimicrobial action, the current target is creation of antimicrobial agents has failed to reach expectations, prompting a quest for new antimicrobial agents.²According to the reports, drug resistance is more likely to occur with single (solo) targeting agent which defeats the expected successful drug compound.³It is commonly accepted that, drugs which affect many sites of single target or many more targets are thought to be more potent and less resistant than single targeting drugs.⁴,⁵ ,⁶ Pharmacologically significant heterocyclics are essential in the fight against illness that impact living things, including animals, humans and plants. They also give fresh findings on novel molecules that may have biological effects.⁷ The lack of new antifungal medications, the rise of infectious diseases, various infection’s resurgence, growing fungal resistance to present chemotherapeutic drugs are main problems associated in drug design and development. Due to this, analysts are searching for new affiliates that can fight against organisms which are resistant to multidrug therapy.⁸ Because the field of chemistry is developing steadily, novel compounds are created in laboratories to find leads with target-specific action.⁹

The molecular hybridization approach is the only way to get this. Molecular hybridization is a drug development technique to develop new drug entities by joining two distinct active pharmacophores with or without a linker. Nowadays, this technique is most widely used in drug development.⁷,¹⁰,¹¹ Thus, hybrid compounds may help human, fight against microbial resistance by decreasing both drug-drug interaction and multiple drug resistance. Chalcones, coumarins, heterocyclic molecules, and their derivatives can be found in both natural and synthetic sources. Because of their diverse range of pharmacological effects, these compounds have attracted the most attention in current research in drug development. Coumarins are classified as heterocyclic compounds belonging to the benzopyrone family, which has a 6-membered α-pyrone ring fused with a benzene ring. As a benzo derivative these compounds found in natural substances.¹² Coumarins have variety of therapeutic uses including anti-inflammatory¹³, antifungal¹⁴, antibacterial¹⁵, antiviral¹⁶and antioxidant.¹⁷ Coumarins also possess anticancer properties against various malignancies.^18-20

Throughout the entire kingdom of plants, chalcones are one of the most prominent types of flavonoids.²¹ ‘Chalcone’ word comes from “chalcos” a Greek word which means “bronze.” Chemistry of chalcones has developed extensive scientific research throughout the world.²² As benzyl acetophenone, chalcones are also known. Chalcones are α and β unsaturated ketones with two aromatic rings and various substituents arrangement. In chalcones three carbon aliphatic chain act as linker between two aromatic rings.²³

We attempted to create new substituted coumarinyl chalcone as heterocyclic compounds with heteroatom oxygen, which has been noted as a common denominator of pharmacological and biochemical activities. We then examined them like antimicrobials.²⁴ There is more literature on hybrid molecules based on coumarin moiety showed their antimicrobial potential such as coumarin-benzimidazole hybrid (1), coumarin-thiazolyl hybrids (2), sulfonamide-coumarin hybrids (3), hybrids of coumarin-imidazole (4), coumarin-chalcone hybrids (5,6), coumarin-thiosemicarbazones^25-30 (Figure 1).

Figure 1: Hybrid compounds as antibacterial agents25-30 (Recently reported) (Chem Draw Pro 12.0). Click here to View table

Additionally, the findings were consistent with another published studies which demonstrated that antimicrobial potential of coumarin chalcone hybrids are summarised in vast area of pharmaceutical chemistry. Study by Wei et al, 2016 coumarin chalcone hybrids assessed for antibacterial activity and displayed significant inhibition zones.³¹ A study carried by Vazquez-Rodriguez et al.,2015 found that methoxy bearing coumarin moiety of chalcone coumarin hybrids exhibited zones of inhibition ranges 16.1 to 41.4mm as compared to reference drug enrofloxacin.³² A study conducted by Hamdi et. al evaluated that presence of electron withdrawing and releasing groups on chalcone moiety of hybrids possess moderate antibacterial activity S. aureus with 8mm to 16mm zone of inhibition by disk diffusion method while gentamycin as reference has 15-20mm.³³ A study conducted by Wang et al ,2021 observed design, synthesis, characterization, remarkable antibacterial profile of chalcone derivatives bearing coumarin moiety with EC₅₀ value ranges from 49.77µg/ml to 162.48µg/ml.³⁴

These important findings offer a potential favour of combining two moieties into a single molecular structure that may have substantial antibacterial action and low toxicity. The aim of current study to synthesize coumarin chalcone hybrids using a click chemistry approach (figure 2) and evaluate them against different fungal and bacterial strains, keeping in mind the issue of antimicrobial resistance as a major limitation of currently available antimicrobial drugs. After the determination of zone of inhibition for each derivative, their MIC (minimum inhibitory concentration) were calculated for selection of potent compound. Additionally, using molecular modelling technique different binding interactions of the most potent compound was find out. Synthetic route shown in scheme 1 (figure 3).

Figure 2: Design of hybrids6(Chem Draw Pro software 12.0).Click here to View Figure

Figure 3: Synthetic route for derivatives35-37 (Chem Draw Pro 12.0 software).Click here to View Figure

Experimental Section

Methodology

We used all the reagent chemicals that were procured from Loba Chemie Pvt. India, sigma Aldrich, Qualigens, Thermo-Fisher Scientific India Pvt. Ltd. and CDH. Aromatic benzaldehydes, acetic acid, dioxane etc. These chemicals included sodium hydroxide, 4-hydroxy coumarin, piperidine, dimethylamine, chloroform, methanol. Using spectroscopic methods, such as ¹H NMR and ¹³CNMR compounds were characterized. Bruker FTIR and JNM-ECZ600R/S1 600MHz instruments were used to capture IR and NMR spectra respectively. Spectra obtained after dissolving in DMSO-d6 and CDCl3 in relation with TMS. In Nuclear Magnetic Resonance spectra chemical shifts shown as ppm values using the internal standard tetramithysilane (TMS) with a number of protons, coupling constants (J) in hertz (Hz), multiplicities (singlet, doublet, triplicate). Open capillaries were used to measure melting point and were uncorrected.

General procedure for synthesis of 4‑hydroxy-2‑oxo‑2H‑chromene‑3‑carbaldehyde (2)

In 250ml RBF, 4-hydroxy coumarin (20g) was added alongwith 80 ml of chloroform and aqueous sodium hydroxide to create alkaline condition. Obtained mixture was mixed with continuous shaking at room temperature for 3 hours, complete reaction was tracked/assessed by thin layer chromatography technique using solvent system in ratio 2:3 (chloroform: acetone). When reaction completed 40ml ice water added into solution. Resulting carbaldehyde coumarin was filtered, rinsed with water and allowed to air dry.³⁵

Synthesis of (E)- 3-(3-(4-aminophenyl)-3-oxoprop-1-enyl)-4-hydoxy-2H-chromen-one (3)

In small amount of obtained carbaldehyde (2) 4-aminoacetophenone (5g) was added with 40ml of chloroform. Piperidine used as catalyst and reaction mixture refluxed for 4 hours. Resultant solid rinsed with chloroform after removal of chloroform and obtained pure chalcone (3).³⁶

General method of synthesis of 3-((E)-3-(4-((Z)- substituted benzylidene amino) phenyl)-3-(oxoprop-1-enyl)-4-hydroxy-2H-chromen-2-one (4)

Obtained chalcone in 2^nd step was poured into RBF with methanol (40ml). Separately, aromatic substituted benzaldehyde mixed with methanol. Solution of benzaldehyde added dropwise in above solution. Refluxed the mixture for 4 hours. Excess of solvent removed under pressure from resultant mixture and recrystallization done with methanol.³⁵

General method of synthesis of 3-((E)-3-(4-((Z)- benzylideneamino) phenyl)-3-(dimethylimino) prop-1-enyl)-4-hydroxy-2H-chromen-2-one (5a-j)

Mixes of compound 4 and acetic acid (20ml) was added with dimethylamine (10ml) and continuous shaking was done for 30 minutes at room temperature. Resultant mixture was poured onto ice cold water. Obtained product was filtered, rinsed with aqueous acetic acid in three portion and kept dried. Recrystallization done with methanol to get pure desired product (5a-j).³⁶

3-((1E,3E)-3-(4-(((Z)-4-hydroxybenzylidene) amino) phenyl)-3-(dimethylimino) prop-1-enyl)-4-hydroxy-2H-chromen-2-one, 5a

Yield: 69; m pt: 179⁰C-182⁰C; R_f value: 0.89; Colour: Pale yellow; FTIR (KBr, cm-1): 3422 cm^-1, 3424cm^-1(OH), 1654cm^-1(C=O), 1716cm^-1 (C=C), 1468cm^-1 (C-C, Ar), 3069cm^-1 (C-H, Ar), 1638cm^-1 (C=C), 1359cm^-1(CH₃);  ¹H NMR (CDCl3, 600 MHz, ppm, TMS=0): 6.8-7.99 (s,1H, Ar-H), 8.01 (s, 1H, Imine), 3.76 (s, 1H, OH), 1.25-1.30 (d,3H, CH₃); ¹³CNMR (CDCl₃, 100 MHz, ppm): 29.7(CH₃), 37.1(CH₃), 67.1(C-O-C), 76.8-77.2(CO), 115.7, 119.9-127.5(C=C), 146.1-149.1, 132.2, 152.8, 168.0 and 171.62(CO, coumarin). Mass (ESI) m/z: 440.49; found: 442.24 (M+2).

3-((1E,3E)-3-(4-(((Z)-4-nitrobenzylidene) amino) phenyl)-3-(dimethylimino) prop-1-enyl)-4-hydroxy-2H-chromen-2-one, 5b

Yield: 61; m pt:185⁰C-189⁰C; R_f:0.84; Colour: Yellow; FTIR (KBr, cm-1): 3422cm^-1 (OH), 1656cm^-1(C=O), 1714cm^-1 (C=C), 1468cm^-1 (C-C), 3114cm^-1(C-H, Ar), 868cm^-1 (NO_2, str), 1685cm^-1 (C=C, alkenyl); ¹H NMR (CDCl3, 600MHz, ppm, TMA=0): 6.7-7.9 (s,1H, Ar -H), 8.01 (s, H, imine), 9.18 (No₂), 4.9 (s,1H, OH), 7.4-7.7 (m, 12H, Ar), 1.7-1.9 (s, CH₃); ¹³C NMR (CDCl3,100 MHz, ppm, TMS=0): 77.2-77.6(CO), 123.6(C=C), 130.4, 136.8, 151.2(C, aromatic),178.60(CO, coumarin), 129.3(C=C), 25.9(CH₃), 26.01(CH₃). Mass (ESI) m/z: 469.49 found 471.08 (M+2).

3-((1E,3E)-3-(4-(((Z)-3-chlorobenzylidene) amino) phenyl)-3-(dimethylimino) prop-1-enyl)-4-hydroxy-2H-chromen-2-one, 5c

Yield: 59; m pt:159⁰C-161⁰C; Rf: 0.79; Colour: Pale yellow; FTIR (KBr, cm^-1): 3420cm^-1 (OH), 1657cm^-1,1649cm^-1 (C=O), 1727cm^-1 and 1465cm^-1 (C=C, Ar) 3068cm^-1 (C-H, Ar), 1359cm^-1 (CH₃), 1688cm^-1 (C=C, alkenyl), 1219cm^-1 (C-O-C) and 755cm^-1 (C-Cl) respectively; ¹HNMR (CDCl₃, 600MHz, ppm, TMS=0): 7.15-7.19 (s,1H,Ar), 8.06 (s, imine), 3.78 (s, OH), 7.2-7.9 (s, 12H, Ar), 2.09 (s,CH₃); ¹³C NMR (CDCl3, 100MHz, ppm, TMS=0): 20.8, 24.8, 26.1 (CH₃), 35.8-36.3 (CH₃),171.6(CO, coumarin), 103.1 (C, aromatic), 115.7-125.4 (C, aromatic),128.1-133.04 (C, aromatic), 67.3 (C-O-C), 152.7 and 168.5 (C=C). Mass (ESI) m/z: 458.94 found: 460.21 (M+2).

3-((1E,3E)-3-(4-(((Z)-2-chlorobenzylidene) amino) phenyl)-3-(dimethylimino) prop-1-enyl)-4-hydroxy-2H-chromen-2-one, 5d

Yield: 71; m pt:149⁰C-152⁰C; R_f: 0.78; Colour: Dark red; FTIR (KBr, cm^-1): 3420cm^-1(OH), 1656cm^-1 (C=O), 1464cm^-1, 1685cm^-1, and 1717cm^-1 (C=C), 3114cm^-1 (C-H, Ar), 1356cm^-1 (CH₃),  763cm^-1 (C-Cl); ¹H NMR (CDCl_3, 600MHz, ppm, TMS=0): 7.1-7.4(m,1H,Ar), 8.06(d,H,imine), 3.78(s,H, OH), 7.6-7.9(s,12H,Ar), 1.98(s,CH₃); ¹³C NMR (CDCl₃, ppm, 100 MHz): 22.4(CH₃), 22.5(CH₃), 26.4(CH₃), 34.8, 76.9-77.3(CO), 103.1(C,aromatic), 126.7-129.0(C, aromatic), 168.5(CO, coumarin), 151.4(C=C). Mass (ESI) m/z: 458.94 found 460.29 (M+2).

3-((1E,3E)-3-(4-(((Z)-2,5-dimethoxybenzylidene) amino) phenyl)-3-(dimethylimino) prop-1-enyl)-4-hydroxy-2H-chromen-2-one, 5e

Yield: 66; m pt.:191⁰C-196⁰C; R_f: 0.76; Colour: Dark yellow; FTIR (KBr, cm^-1): 3424cm^-1 (OH), 1654cm^-1 (C=O), 1714cm^-1(C=C), 1469cm^-1(C-C), 3066cm^-1 (C-H, Ar, str), 1688cm^-1  (C=C, alkenyl), 1134 cm^-1 (OCH₃); ¹H NMR (CDCl₃, 600MHz, ppm, TMS=0): 7.2-7.4(m,1H,Ar), 8.08-8.09(s, H, imine), 3.70(H,OH), 7.10-7.46(m,12H,Ar) 2.84(s,OCH₃), 3.4-3.7(s,OCH₃), 1.38(s,CH₃); ¹³C NMR (CDCl₃, ppm, 100 MHz, TMs=0): 55.5(OCH₃), 56.5(OCH₃), 115.4(CH), 164.7-169.2(CO, coumarin), 171.2(CO, coumarin), 103.7 and 105.2(C, aromatic), 110.8-116.9(C, aromatic), 120.8 and 128.4(C, aromatic), 131.7 and 133.1(C, aromatic)), 33.3(CH₃), 35.9(CH₃). Mass (ESI) m/z: 480.54 found 481.74 (M+1).

3-((1E,3E)-3-(4-(((Z)-benzylidene) amino) phenyl)-3-(dimethylimino) prop-1-enyl)-4-hydroxy-2H-chromen-2-one, 5f

Yield: 52; m pt: 167⁰C-171⁰C; R_f: 0.69; Colour: Yellow; FTIR (KBr, cm^-1): 3426cm^-1 (O-H), 1654cm^-1(C=O), C=C and alkenyl C=C observed at 1712cm^-1, 1468cm^-1 and 1632cm^-1 respectively. A stretching peak for aromatic C-H was found to be at 3068cm^-1. At 1356cm^-1 peak showed in spectrum due to CH_3.; ¹ H NMR (CDCl_3, 600MHz, ppm, TMS=0): 3.15(s, H, OH), 1.6-1.8(s, CH₃), 7.3-7.4(m, 12H, Ar), 10.18(H, imine); ¹³C NMR (CDCL₃, 100MHz, ppm, TMS=0): 22.4(CH₃), 22.5(CH₃), 44.6, 196.5(CO, coumarin), 113.7-130.8(C, aromatic), 147.4(C=-phenyl). Mass (ESI) m/z: 424.49 found: 426.44 (M+2).

3-((1E,3E)-3-(4-(((Z)-3-bromoxybenzylidene) amino) phenyl)-3-(dimethylimino) prop-1-enyl)-4-hydroxy-2H-chromen-2-one, 5g

Yield: 80; m pt:163⁰C-168⁰C; R_f: 0.62; Colour: Pale yellow; FTIR (KBr, cm^-1): 3422cm^-1 (OH), 1641 cm^-1 (C=O), 1712cm^-1 and 1454cm^-1 (C=C, Ar), 3065cm^-1 (CH), 1628cm^-1 (C=C, alkenyl), and 1358cm^-1 (CH₃) respectively, 753cm^-1 (C-Br, str); ¹H NMR (CDCl3, 600MHz, ppm, TMS=0): 1.67, 3.17, 7.3, 9.30; ¹³C NMR (CDCl₃, 100MHz, ppm, TMS=0): 22.4(CH₃), 26.5(CH₃), 119.9-133.2(C, aromatic), 141.8(CH), 196.7(CO, coumarin). Mass (ESI) m/z: 503.39 found: 503.42 (M+H).

3-((1E,3E)-3-(4-(((Z)-4-dimethylaminebenzylidene) amino) phenyl)-3-(dimethylimino) prop-1-enyl)-4-hydroxy-2H-chromen-2-one, 5h

Yield: 73; m pt.:173⁰C-178⁰C; R_f: 0.89; Colour: Red; FTIR (KBr, cm^-1): 3424cm^-1(OH), 1460cm^-1 (C=C), 1716cm^-1 and 3048cm^-1 (CH, str) cm^-1, 1674cm^-1 (C=O), 1346cm^-1 (CH₃), 1602cm^-1 (C=C, alkenyl); ¹ H NMR (CDCl₃, 600MHz, ppm, TMS=0): 7.2-7.9(m,1H,aromatic), 3.82(H,OH),1.25-1.60(d,3H,CH₃), 2.81-2.90(m, CH₃), 8.05(H,imine); ¹³C NMR (CDCl₃, 100 MHz, ppm, TMS=0): 168.52(CO, coumarin), 112.8(C, aromatic), 123.7-127.3(C, aromatic), 131.3, 132.5(C, aromatic), 67.18(C-O-C), 115.6(CH), 116.6(CH), 152.6(C=C), 168.5(C=C).; Mass (ESI) m/z: 425.09 found: 425.13 (M+1).

3-((1E,3E)-3-(4-(((Z)-4-hydroxy-3-methoxybenzylidene) amino) phenyl)-3-(dimethylimino) prop-1-enyl)-4-hydroxy-2H-chromen-2-one, 5i

Yield: 66; m. pt:175⁰C-178⁰C; R_f: 0.76; Colour: Pale yellow; FTIR (KBr, cm^-1): 3424cm^-1, 3426cm^-1 (OH), 1656cm^-1 (C=O), 1718cm^-1 (C=C), 1456cm^-1 and 3068cm^-1 (CH, Ar), 1354cm^-1 (CH₃) and 1688cm^-1 (C=C, alkenyl) respectively.; ¹ H NMR (CDCl_3, 600MHz, ppm, TMS=0): 4.05(H, OH), 2.45(s, CH₃), 3.15(s, OCH₃), 7.2-7.6(H, Ar), 11.03(H, imine); ¹³C NMR (CDCl₃, 100 MHz, ppm, TMS=0): 55.8(OCH₃), 27.4(CH₃), 27.7(CH₃), 114.6-119.6(C, aromatic), 122.01-122.8(C, aromatic), 131.6-133.4(C, aromatic), 167.2 (CO, coumarin). Mass (ESI) m/z: 470.52 found: 471.81 (M+1).

3-((1E,3E)-3-(4-(((Z)-2-hydroxybenzylidene) amino) phenyl)-3-(dimethylimino) prop-1-enyl)-4-hydroxy-2H-chromen-2-one, 5j

Yield: 66; m pt.:153⁰C-155⁰C; R_f:0.87; Colour: Dark yellow; FTIR (KBr, cm^-1): 3420cm^-1 and 3424cm^-1 (OH), 1654cm^-1 (C=O), 1465cm^-1 and 1712cm^-1 (C=C, Ar), 3066cm^-1 (CH, Ar) , 1648cm^-1 (C=C, alkenyl), 1359cm^-1 9CH₃); ¹ H NMR (CDCl₃, 600MHz, ppm, TMS=0): 4.25(H, OH), 2.45(s,CH₃), 7.24-7.79(12H, Ar), 9.82(H, imine), 3.70(s, H, OH); ¹³C NMR (CDCl₃, 100MHz, ppm, TMS=0): 131.4, 133.04, 141.4(C, aromatic), 26.8(CH₃), 24.7(CH₃), 33.3, 35.7(CH₃), 55.5(OCH₃), 56.5(OCH₃), 110.8(C, aromatic), 151.4-153.6(C=C), 178.4(CO, coumarin). Mass (ESI) m/z: 440.49 found: 442.53 (M+2).

In silico studies

Auto Dock software was used for computational studies like molecular docking. 3D structures were drawn by Chem Sketch 12.0 software for both ligand (compounds) and reference. From RSCB protein data bank 3D Protein DNA gyrase (PDB ID: 6m1j) pdb file was downloaded and saved as pdbqt. Input configuration files were prepared in PDBQT format prior to docking.³⁸ Run the docking procedure after removal of water molecules and heteroatoms. Added polar hydrogens. Results and images of docking were analysed by Biovia Discovery studio and docking score expressed in terms of Kcal/mol. The potential of the ligands as DNA gyrase inhibitors was assessed by the interactions between the gyrase and ligands and identified the main residues involved in interactions. Physicochemical properties (in silico) of designed compounds were analysed by Swiss ADME web tool to evaluate their drug likenes³⁹ (https:/www.swissadme.ch/). SMILES formats of designed compounds were used for prediction.

Antimicrobial activity (in vitro)

The agar well diffusion method was used to check antimicrobial activity of synthesised hybrids against various strains such as E. coli, P. aeruginosa, S. aureus, B. subtills, and C. albicans in accordance with the NCCLS, 2002.⁴⁰ Target compound’s zone of inhibition was calculated and compared with standard drugs like ciprofloxacin and fluconazole. For their antifungal and antibacterial, the microbial strains were sub cultured in potato dextrose agar and pre-sterilised nutrient agar media, respectively. After that, pre-inoculated media was aseptically moved into 4-inch diameter sterilised petri plates. Once the medium solidified, well was made with a sterile cork borer and labelled it. DMSO was used to create the standard compound’s solution of 50µg/ml concentration, while DMSO was used to dissolve the test compounds and dilute them to dose level of 100µg/ml. After that, under sterilized conditions these concentrations were added to the petri plate bores then incubated for 48 hours. As a negative control DMSO has no effect. For every test and standard compound, zone of inhibition was measured and also compared against negative control. Almost all of the hybrids were effective against the tested microorganisms.⁴¹

Results and Discussion

Chemistry

Synthesis of coumarin chalcone hybrids was carried out by following scheme 1. (Figure 3) First, 4-hydroxy coumarin was mixed with CHCl₃ by using solution of sodium hydroxide at room temperature, resultant mixer was stirred continuously on magnetic stirrer for 3hrs. After 3 hours of stirring, ice cold water was added to the solution, filtered, rinsed with water twice then allowed to dry. Methanol was used for recrystallization and get carbaldehyde (2). Obtained product mixed with 4-aminoacetophenone by using chloroform as solvent and added piperidine as catalyst, refluxed mixture for 4hrs to get chalcone (3). Obtained residue recrystalised with methanol after removal of chloroform.

Separately, aromatic substituted benzaldehyde dissolved in methanol and this solution poured into RBF containing compound 3. Refluxed the mixture for 3hrs at 50⁰-70⁰-C. Recrystallization of obtained solid was carried with methanol (4).

Mixture of compound 4 further reacted with dimethylamine in the presence of CH₃COOH for 30 minutes at room temperature, shaken, filtered and rinsed with aqueous CH₃COOH thrice then kept dried. 1,4-dioxane used for recrystallization to obtain desired hybrid compounds (5(a-j)). Spectroscopic technique (1H NMR, 13C NMR and GC-MS(ESI)) were used for characterization of hybrids and spectral data were found accordingly as assumed structures. Characterization already discussed under experimental section.

Molecular docking

Autodock vina was used to analyse the targeted compounds’ inclusive docking interaction and evaluate their binding activity relationship with protein. Docking demonstrated the interaction of targeted molecules (ligand) with binding sites of receptor (protein), termed as binding affinity and which observed through various interactions like Van der waal interactions, hydrogen bonding, carbon hydrogen bonding, pi sigma, amide-pi stacked, halogen bonding and hydrophobic π alkyl, π-π interactions as well as electrostatic pi-anion, pi-cation interactions etc. Therefore, the strongest inhibitory action of compounds is due to hydrogen bonds and hydrophobic interactions. Whereas salt bridge also enhances inhibitory action.⁴² DNA gyrase protein (PDB ID:6m1j) was selected as receptor to find binding interactions. The DNA binding protein gyrase is a potential target for the ligands in our study.⁴³ Molecular docking results observed that coumarin chalcone hybrids had high binding affinity towards DNA gyrase as compared to ciprofloxacin and ranges between -8.4 kcal/mol to -8.9 kcal/mol. Generally, binding affinity showed the intensity of interaction and high ligand binding affinity resulting from a stronger intermolecular force between ligand and receptor. Binding pose with main active residues ILE A:80, ASP A:75, ILE A:96, ASN A:48 and GLU A:52 showed hydrogen bonding as well as hydrophobic interactions. These solid interactions suggested the effective inhibitory activity against DNA gyrase enzyme. The molecular docking interactions and binding affinities of derivatives with targeted protein displayed in table 1 and figure 4.

Table 1: Binding interactions and docking score of compounds. 

Figure 4: 2D and 3D images of docking interactions. (Obtained from Auto Dock Vina software).Click here to View Figure

ADMET parameters

The Swiss ADME web-based program was used to assess the ADMET profile of designed compounds 5(a-j) and predict their physicochemical properties. All the properties fall within desired pink area of bioavailability radar (figure 5), indicating that the derivatives have favourable drug-like properties. ADMET profile provided in table 2. All the compounds have reasonable ADMET properties.⁴⁴

Table 2: ADMET properties of compounds as per LRO5.⁴⁴

MS: moderately soluble, RB: rotatable bond, MR: molar refractivity, TPSA: topological polar surface area.

Figure 5: Bioavailability radar for compounds (5a-j) and ciprofloxacin (designed by Swiss ADME Swiss drug design software).Click here to View Figure

Antimicrobial Evaluation (In vitro)

Using agar well diffusion method,⁴⁵ the antimicrobial properties of synthesised derivatives assessed against P. aeruginosa, E. coli, B. subtills, S. aureus and C. albicans. MHA (Muller Hinton Agar) was used to culture the inoculum bacteria overnight at 37⁰C at 200 rpm with constant shaking. To create an even lawn, sterilised cotton buds were used to inoculate the cultured bacteria on MHA surface. A sterile paper disc was placed on the agar plate’s surface using sterile forceps after being impregnated with compound (100 ppm) in DMSO. The inhibitory zones were measured in mm after incubation of plates 24 hours at 37⁰C to estimate efficacy of tested derivatives.^46,47 Compounds 5c, 5e, 5g and 5h demonstrated significant inhibitory effect against the targeted strains of bacteria and fungi, according to the results whereas other compounds showed reduced activity. For the potent compounds zones of inhibition were observed as follows: 5.56±0.179, 5.58±0.449, 4.94±0.811, 4.82±0.378 against B. subtills; 7.25±0.191, 6.11±0.496, 5.55±0.496, 5.41±0.421 against S. aureus; 6.36±0.024, 6.27±0.029, 5.99±0.666, 6.04±0.432 against E. coli; 5.93±0.118, 4.94±0.016, 6.58±0.079, 5.94±0.119 against P. aeruginosa; 7.92±0.389, 7.12±0.401, 7.10±0.171, 6.96±0.331mm against C. albicans, respectively. Compound 5c exhibited strong potential as both antibacterial and antifungal against S. aureus and C. albicans more than reference drugs ciprofloxacin and fluconazole respectively. The obtained results of the selected compounds may be effective agents for antimicrobial efficacy. Table 3 and figure 6 and 7 lists the results of both antibacterial and antifungal activities.

Table 3: Antimicrobial profile of derivatives (5a-j).

** test not performed, #mean±SD

Figure 6: Graphical representation of antimicrobial activity of synthesised derivatives and reference drugs against different strains. (created by raw data obtained by well diffusion assay).Click here to View Figure

Figure 7: Antimicrobial activity of potential compounds 5c, 5e, 5g and 5h against various microbial strains such as B. subtills, S. aureus, E. coli, P. aeruginosa and C. albicans.Click here to View Figure

Conclusion

In medicinal chemistry, chalcones are useful lead compounds for development of novel candidates together with coumarin. These derivatives designed by in silico technique particularly molecular docking. All derivatives exhibited greater binding energy than ciprofloxacin (standard) in range -8.3 kcal/mol to -8.9 kcal/mol. In this study, chalcone coumarin hybrids were synthesised by base catalysed condensation reaction and synthesis performed by click chemistry approach. Characteristically the response was observed by TLC for purity determination of derivatives. Spectral analysis done for characterization of compounds and subjected for antimicrobial activity against DNA gyrase for the survivability of different strains such as B. subtills, S. aureus, E. coli, P. aeruginosa and C. albicans as potential antibacterial agents. However, in silico ADME analysis found that most of compounds fits in Lipinski’s rule of five of drug likeness and showed moderate water solubility that indicates synthesised compounds could be applicable for orally administration. Results found that derivatives 5c, 5e, 5g and 5h possess promising antimicrobial activity. Therefore, it is most evident that hybrids of coumarin and chalcone are most prominent candidate for antimicrobial action. The results of present study showed that coumarin chalcone derivatives provide an additional option for antimicrobial action.

Thus, this work opens the door for further research focussed on creating novel therapeutic candidates against microbial resistance.

Acknowledgment

The authors are highly thankful to Department of Pharmacy, Faculty of Medical/Paramedical & Allied Health Sciences, Jagannath University, Jaipur, Rajasthan-302022 and SGT University, Gurugram, Haryana-122005, for providing the necessary facilities to conduct the study.

Funding Sources

The author(s) received no financial support for the research, authorship, and/or publication of this article.

Conflict of Interest

Authors have no conflict of interest.

Data Availability Statement

This statement does not apply to this article.

Ethics Statement

This research did not involve human participants, animal subjects, or any material that requires ethical approval.

Author Contributions

All authors contributed to the design, conception, data collection, analysis of results of study. Sumita Kumari has written the manuscript, evaluation and supervision done by Dr. Amit Sharma and Dr. Sonia Yadav. 

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Abbreviation 

NCCLS: National Committee for Clinical Laboratory Standards;

TPSA: Topological polar surface area; TMS: Tetramethyl silane.