Synthesis Characterisation in silico Studies and Anti-bacterial Evaluation of Acetaminophen Chalcone Derivatives

The present work is prompt disclosure of new experimental in many fields of drug design, discovery and elucidation of biological active compounds. Which focus relationship in chemical and biological evaluation on anti-bacterial activity. In silico studies will welcome the increase of your understanding of drug molecules. Acetaminophen also commonly known as Tylenol is the most commonly taken analgesic world-wide and is recommended as first line therapy in pain conditions by WHO (World Health Organisation). Acetaminophen drug is commonly used non-narcotic analgesic-antipyretic agents and is a component of many over-the-counter proprietary preparations.

USFDA has been approved this drug in 1951. It is available in varied forms like syrups, tablets and effervescent tablets, injections, suppositories and other forms.¹

Chalcones are the natural pigments found in nature which form the central core for a variety of significant biological compound. They are the aromatic ketones and enones. They are important constituents of many natural sources.^2–4 They are widely distributed in plants and are thought to serve as a starting point for a number of biosynthetic pathways that result in the synthesis of isoflavonoids and flavonoids. Chalcones shows activity mainly because of an enone pharmacophore in their structures.^5,6 The chalcone core is made up of two aromatic rings joined by a three-carbon aliphatic chain.⁷ These ketones are α,β-unsaturated and comprise of two aromatic rings with distinct substituents. The electrophonic three carbon α,β -unsaturated carbonyl system, which has a ketoethylenic group in its structure and a planar structure, connects these two aromatic rings.^8,9

Chalcones derivatives exhibit wide range of pharmacological actions such as antibacterial, anti-inflammatory, analgesic, anticholinergic, antiplatelet, antiulcer, antioxidant, antimalarial, anticancer, antiviral, anti-leishmanial, anti-diabetic, immunomodulatory, aldose reductase inhibition and as non-purine xanthine oxidase inhibitors.¹⁰ Chalcones are widely used because of their simple structure, open chain model, ease of construction, the feat and promising biological applications. Chalcones have potential research class of compounds with important uses against various diseases.^11,12

Drug resistance is the reduction in effectiveness of a drug in curing a disease or reducing a patient’s symptom.¹³ Antibacterial resistances happen when the bacteria develop the ability to defeat the drugs, which are designed to kill them. Due to severe side effects, toxicities and bacterial resistance lead to complications in treatment; so many researchers are working to find a new antibacterial agent that has potency against drug resistant strains.^14,15

The paracetamol used are Analytical grade procured from institution. All chemicals used are synthetic grade; different aldehydes were used mentioned in Table 1. All the compound structures were given in Table 2. We determined the melting points of synthesised organic derivative compounds using the open capillary tube method. One of the physical parameters, the melting point, indicates the purity of the organic derivative in the form of pure crystals with sharp and definite melting points. The melting points of the derivatives were performed using melting point apparatus. Solubility: The solubility of the synthesised derivatives was tested using different solvents. The solubility characters were listed. TLC solvent is chloroform: ethyl acetate ratio 9:1. IR spectra were recorded FTIR spectrophotometer and ¹H NMR spectra taken from Bruker 600 MHz instrument. Thin-layer chromatography is a chromatography technique used to separate and identify the components in a mixture based on their different physical and chemical properties. TLC is a quick, easy and inexpensive method that requires small amounts of sample.^16–18

Table 1:
List of aldehydes and codes.

Table 2:
Structures of synthesized compounds.

Synthetic Scheme

Scheme 1

Scheme 1:
synthetic route of chalcone derivatives.

All the synthesised compounds given were derived from scheme 1. Solubility studies were given in Table 4. Physico-chemical parameters were noted in Table 5. By measuring inhibition zones at different drug concentrations, the study aims to elucidate the drugs’ spectrum of activity and concentration-dependent efficacy in Table 6.

Table 4:
Solubility studies of synthesized compounds.

Table 5:
Physico chemical parameters of synthesized compounds.

Table 6:
Anti-bacterial activity of compounds.

Bacillus subtilis strain bacteria tested AS1, AV2 and APC4 shows a good inhibition across all concentrations and the MIC was found to be at 15.625μg showing zone of inhibition of 15 mm, 16 mm and 14 mm respectively shown in Figures 1–3. This bacterium is sensitive to AA3 drug at only two concentrations 250 μg and 125 μg exhibiting zone of inhibition at 12mm respectively. And it is resistant to other concentration. APN5 Shows a good zone of inhibition at 250 μg, 125 μg, 62.5 μg and 31.250 μg concentration.

Staphylococcus aureus strain bacteria exhibits potent inhibition at higher concentration of AS1 but loses effectiveness at lower concentrations. This strain is sensitive to APN5 at only 250μg concentration showing 12 mm of zone of inhibition and is resistant to all other concentration of drug including MIC.

Escherichia coli strain shows effective sensitivity to APN5 drug and MIC at 15.625 μg of 14 mm zone of inhibition shown in Figure 4. AS1 displays moderate inhibition, with effectiveness being resistant at lower concentration of drug. It shows resistance across all the concentration of AV2, AA3, APC4 compounds.

Pseudomonas aeruginosa strain is maximum sensitive to APN5 drug at all the concentration and shows MIC at 15.625 μg of 12 mm shown in Figure 5. AS1 and AA3 show moderate sensitivity at 250 and 125 μg concentrations. This bacteria show resistance across all the concentration of AV2, APC4 drugs.

Molinspiration results describe that compounds show the log p-value indicating partition coefficient. All compounds show TPSA less than 150A° indicating good drug permeability in the plasma membrane. Percentage absorption (%ABS) should be in the range of 79.96-89.88% signifying good absorption in the intestine shown in Table 7.

Table 7:
Calculation of molecular properties using Molinspiration.

Bioactivity scores calculation for organic compounds says, ‘the probability is that if bioactivity scores are greater than 0’, then it is active. If it is between -5 and 0 then moderately active, If less than -5 then inactive. All Compounds 1-5 are moderately active as GPCR ligands, Ion channel modulator, Kinase inhibitor, nuclear receptor ligand, Protease inhibitor, Enzyme inhibitor shown in Table 8.

Table 8:
Bioactivity scores prediction using Molinspiration.

Protox result analysis for all compounds, the dose predicted LD₅₀ is 2000 mg/kg. All compounds come under predicted toxicity is class 4. From the toxicity model report Table 9, all compounds toxicity are inactive towards cytotoxicity, carcinogenicity, mutagenicity, all the compounds showed toxicity active towards hepatotoxicity, immunogenicity. It is in silico toxicity prediction very useful for toxicologist, medicinal chemist, computational chemist and regulatory agencies. It saves the time, need of animal testing and cost associated with it.

Table 9:
Toxicity model report.

From the Swiss ADME search, we conclude that all the compounds follow Lipinski rule, all compounds show TPSA less than 150A° indicating good drug permeability in the plasma membrane, all the compounds shows bioavailability score. All the compounds shows high GI absorption AS1, AA, APC4 shows BBB permeant, all the compounds shows no to P-gp substrate, AS1does not inhibit all the inhibitor, AV2 does not inhibit all the inhibitor except CYP2C9 inhibitor, AA3 inhibits to all the inhibitor except CYP2D6, CYP3A4, APC4 inhibits to all the inhibitor except CYP2D6, APN5 inhibits to all the inhibitor except CYP2D6, CYP3A4 were reported in Table 10.

Table 10:
Swiss ADME results.

The starting material for synthesis is paracetamol, it is treated with acetic anhydride and few drops of sulphuric acid on slight heating gives a reaction mixture, further on cooling gives Acetylated paracetamol. An equimolar mixture of Acetylated paracetamol and different aromatic aldehydes were treated with 10% NaOH and stirred for 3 hr, solid precipitates, filtered and dried gives Acetaminophen-Chalcone derivatives. TLC were confirmed by using silica gel sheets which are pre-coated Aluminium sheets and melting points were analysed and by using different solvents solubility tested. An FTIR spectrum has been recorded in instrument and interpreted the functional groups. Proton NMR is analysed the number of protons, solvent used is deuterated chloroform, using 600 MHz instrument. An alkene has been formed at 3094 cm^-1 range reveals that chalcones are formed. Proton NMR 6.39 and 7.34 alkenes have single protons and a chemical shift was recorded.^27,28 This study examines the effectiveness of these synthesized derivatives against four distinct bacterial strains: ‘Bacillus subtilis, Staphylococcus aureus, Escherichia coli and Pseudomonas aeruginosa’ using Agar well diffusion method. Bacillus subtilis strain bacteria tested AS1, AV2 and APC4 shown a good zone of inhibition at all concentrations. E. coli strain bacteria tested APN5 shown effective at all concentrations. Pseudomonas aeruginosa strain bacteria tested APN5 shown sensitivity at all concentrations. All compounds Molinspiration properties were predicted and calculated the percentage absorption in the gut at range of 79.72-89.88. From the molinspiration results, all synthesized compounds satisfy the Lipinski rule of five properties, stating that compounds were under the limits. Bioactive scores were predicted all compounds which are in negative sign are moderately active at GPCR ligands, ion channel modulator, kinase inhibitor, nuclear receptor ligand, protease inhibitor, enzyme inhibitor.²⁹ From the Protox report we conclude that the predicted toxicity of synthesized compounds is free from toxicity carcinogenicity, mutagenicity, cytotoxicity. All compounds found active toxicity of Hepatotoxicity, Immunogenicity.³⁰ All the compounds smiles format were uploaded in Swiss ADME showed good GI absorption is high and bioavailability score is 0.55, permeability to BBB (AS1, AA3, APC4) and some enzyme inhibitor parameters have been noted. From this in silico studies we predict the possibilities of the therapeutic potential of new drug molecules more effectively.

Figure 1:
Bacillus subtilis is sensitive to AS1 at 15.625 μg concentration.

Figure 2:
Bacillus subtilis is sensitive to AV2 at 15.625 μg concentrations.

Figure 3:
Bacillus subtilis is sensitive to APC4 at 15.625 μg concentration

Figure 4:
Escherichia coli are sensitive to APN5 at 15.625 μg concentrations.

Figure 5:
Pseudomonas aeruginosa is sensitive to APN5 at 15.625 μg concentrations.

In the present study, a series of new five 4-acetamidophenyl 3-(4-aryl acrylates) derivatives were synthesized and screened for antibacterial activity and evaluation was carried out by using a standard drug. In silico studies results reveal that the entire compound showed characteristic properties using all these softwares such as Molinspiration, Protox, Swiss ADME, respectively. The compound APN5 shows better antibacterial activity against E.coli, Pseudomonas aeruginosa, Staphylococcus aureus, Bacillus subtilis. Further studies are required to perform in vitro, in vivo and molecular dynamic studies to know the stability of the drug.

We are thankful to SAIF-DST supported institute NMR facility at IISc for NMR spectra. We acknowledge Maratha Mandal’s Central Research Laboratory, Belagavi for doing Anti-bacterial activity.

The authors declare that there is no conflict of interest.