Synthesis and Biological Evaluation of Nitro-substituted chalcones as potent Antibacterial and Antifungal agents

 

Kamya Goyal^1,2, Anju Goyal², Rajendra Awasthi³, Nidhi Rani², Rajwinder Kaur²*

¹Department of Pharmaceutical Chemistry and Analysis, Laureate Institute of Pharmacy,

Jawalamukhi, Himachal Pradesh, India.

²Department of Pharmaceutical Chemistry, Chitkara College of Pharmacy, Chitkara University, Punjab, India.

³Department of Pharmaceutical Sciences, School of Health Sciences and Technology,

University of Petroleum and Energy Studies (UPES), Energy Acres, Dehradun, Uttarakhand, India.

 

ABSTRACT:

In recent years, complications of micro-organisms, which becomes drug-resistant, have extended dreadful level across the globe. One of the significant challenges in drug discovery to overcome the increased development of drug resistance is developing novel chemical moieties that advantageously comprise chemical features different from existing chemical entities. This communication presents the synthesis, docking, and antibacterial and antifungal evaluation of nitro chalcones. New series of nitro substituted chalcone derivatives (1-12) were synthesized via Claisen-Schmidt condensation using substituted acetophenone and various substituted benzaldehydes in the presence of base and ethanol. Spectral analysis of the synthesized compounds was carried out using ¹H-NMR, IR, ¹³C-NMR and HRMS methods. Synthesized derivatives were evaluated for antimicrobial potency via the microdilution method against two Gram-positive and two Gram-negative bacterial strains and two fungal strains. All compounds showed promising antimicrobial potency. However, compound 6 was the most potent derivative of the series against all the tested antibacterial strains. Compounds 10 and 12 were found to be the most potent analogues of the series against all the tested antifungal strains. All the synthesized derivatives had good antifungal potency in comparison to the antibacterial potency. Based on the preliminary study results, the study concluded that the synthesized chalcones derivatives promise antimicrobial activity that may be further investigated to achieve antimicrobial lead.

 

 

 

INTRODUCTION: 

Microbial infection-related diseases pose a severe threat to human health and are a dominant cause of death in developing and developed countries. In recent years, drug-resistant complications of micro-organisms have reached a dreadful level in several countries across the globe^1,2. A recent clinical report illustrated the elevating chances of MRSA (Methicillin-Resistant S. aureus) and various other antibiotic-resistant microbes pathogenic for humans³. The increased mortality rate of Gram-negative multidrug-resistant enteric bacteria is a severe problem⁴.

 

A major hurdle in drug discovery to overcome drug resistance is the development of new chemical moieties that are different from traditional antimicrobials.

 

There has been an increasing interest in natural and synthetic chalcones as antimicrobial agents in the last few years. Chalcones have received immense attention as potent candidates for antimicrobial activity due to their seemingly negligible side effects, high therapeutic index, and ease of synthesis Chalcone has basic moiety of 1,3-diaryl-2-propen-1-one in which the two aromatic rings are joined by a three-carbon α, β-unsaturated carbonyl system, representing a class of flavonoids that occur naturally in fruits and vegetables⁵. Chalcones comprise open-chain flavonoids that are eminent intermediates for synthesising several heterocyclic analogues such as pyrazole, pyrazoline, pyridine, pyrrole etc.⁶, related to an impressive assortment of biological activities viz. antimicrobial, antioxidant, anti-inflammatory, antimalarial, anticancer, antileishmanial activities, etc.^7-20. Other important uses of chalcones include synthesis of UV absorption filters in polymers, photosynthesis polymers, photosensitizer in color films, sweetener in food technology and in holographic recording technology²¹.

 

Various chalcones are present in market and are potentially used in the treatment of various serious illness such as metochalcone increase bile secretion by stimulating the liver²² and sofalcone as an antiulcer agent, which increases Prostaglandins concentration from the mucosa causing a gastroprotection from the ulcers induced by Helicobacter pylori²³. It is also found via clinical trials that hesperidin methylchalcone was tested and found effective for chronic peripheral venous lymphatic insufficiency and hesperidin trimethylchalcone was found effective for trunk or branch varicosis²⁴.

 

In chalcones, the presence of highly reactive α, β-unsaturated keto function is known to be responsible for their antimicrobial activity. This study presents continuing efforts for more potent antimicrobials and the synthesis, characterisation, antimicrobial evaluation, and molecular docking simulations of various nitro chalcones.

 

MATERIALS AND METHODS:

Chemistry:

Melting points of synthesized analogues were determined by the open capillary method. Reaction monitoring for the synthesis of pure analogs was carried out by thin layer chromatography using silica gel G. The IR spectra of the synthesized derivatives were recorded by KBr pellets method (λ_max in cm^-1) using Perkin-Elmer Spectrum-2 using software version 10.5.4. ¹H- Nuclear Magnetic Resonance (NMR) spectra and ¹³C-NMR spectra were obtained using Jeol JNM ECX-500 (¹H-NMR at 500 MHz and ¹³C-NMR 125 MHz) spectrometer (Jeol, India). CDCl₃ with tetramethylsilane was used as an internal standard. The assigned splitting pattern was - singlet, s; doublet, d; doublet of doublets, dd; triplet, t and multiplet, m. The chemical shifts (δ) were recorded in ppm. Mass spectra (HRMS) were acquired using Bruker Impact HD spectrometer.

 

General procedure for the synthesis of the compounds (III) (1-12):

An aqueous solution of sodium hydroxide (5%) was added to the solution of substituted acetophenone (I) (1 mmol) and substituted aromatic aldehyde (II) (1mmol) in 10ml of ethanol. The reaction mixture was left to stir at room temperature for an adequate time until all reactants disappeared completely. Upon completion of the reaction, the resulting mixture was quenched on ice containing water. The solid product was precipitated, filtered, washed with ice-cold distilled water, and dried to yield the crude compound. The obtained compound was recrystallised with ethanol^11,17,25.

 

In-vitro biological assays for Antibacterial and antifungal activity:

MIC values of synthesized chalcone derivatives were determined using broth microdilution method^11,26. DMSO was used for preparing dilutions to get the required concentrations of test compounds. Antibacterial activity of the test compounds was evaluated against two Gram-positive bacteria S. aureus (MTCC 96) and S. pyogenes (MTCC 443), and two Gram-negative bacteria E. coli (MTCC 442) and P. aeruginosa (MTCC 2488), against ampicillin. Antifungal activity was evaluated against two fungal strains C. albicans (MTCC 227), A. fumigatus (MTCC 343), and fluconazole was used as a standard antifungal drug. All the MTCC cultures were collected from the Institute of Microbial Technology, Chandigarh, India. Mueller-Hinton broth and Sabouraud’s broth were utilized as a nutrient medium for the growth of bacteria and fungi, respectively. By comparing the turbidity, the inoculum size for the test strain was regulated to 10⁶ colony-forming unit (CFU) per millilitre.

 

Serial dilutions were prepared in primary and secondary screening. The control tube (no antibiotic) was subcultured instantly. The subculturing was done using a sterile inoculation loop on a plate containing suitable growth medium for the test organism. The plates were incubated at 37°C temperature for bacteria and 22°C temperature for fungi overnight. Minimum inhibitory concentration – MIC (the lowest concentration which inhibited the growth of micro-organism) of the control organism was calculated to assess the accuracy of drug concentrations. Each test derivative was diluted to a 2000 μg/ml concentration as a stock solution. The test analogues were taken up at 125, 250, 500, and 1000 μg/ml concentrations for primary screening. Furthermore, the synthesized compounds that showed activity in primary screening were tested against all micro-organisms in a secondary set of dilutions. The active derivatives were diluted to the 6.25, 12.5, 25, 50, and 100 μg/ml concentrations. MIC was recorded as the highest dilution which showed a minimum of 99% inhibition.

 

Docking Study:

Twelve nitro-substituted chalcones were synthesized to screen and explore their antimicrobial potency. The molecular docking studies were performed to investigate the possible interactions of the synthesized derivatives with enzyme transpeptidase from S. aureus, S. pyogenes, E. coli and P. aeruginosa and 14α-demethylase from C. albicans and A. fumigatus.

 

Ligand preparation:

2-Dimensional structure of various nitro-substituted chalcones and reference to the standard compounds, i.e., Ampicillin and Fluconazole, were generated utilizing Chem Draw Ultra (Version 12.0). The generated structures were converted into 3-Dimensional structures, energetically reduced, and later saved as MDL MolFile.

 

Docking procedure:

Molecular docking study of compounds was performed through Molegro Virtual Docker (MVD) program^27-30. The antibacterial potency of chalcones was evaluated using enzyme transpeptidase from S. aureus, S. pyogenes, E. coli and P. aeruginosa, and antifungal potency was evaluated using enzyme 14α-demethylase from C. albicans and A. fumigatus.

 

RESULTS AND DISCUSSION:

Chemistry:

In the present work, twelve nitrochalcones (1-12) were synthesized via Claisen-Schmidt condensation (Scheme 1). The reaction of various nitro substituted acetophenone (I) with a different substituted aromatic aldehyde (II) in the presence of 5% aqueous sodium hydroxide as base and ethanol as solvent, yielded chalcone (1,3-diaryl-2-propen-1-one) analogues (III) (1-12). Various features of the synthesized chalcone derivatives are represented in Table 1.

 

The structures of the synthesized derivatives 1-12 were confirmed by ¹H-NMR, IR, ¹³C-NMR and HRMS spectral analysis. The IR spectra of chalcones (1-12) showed an absorption band at 1665-1690 cm^-1 due to C=O stretching. Nitro substituted analogues showed characteristic absorption bands at 1345-1525 cm^-1 due to the typical NO₂ stretching vibrations. In the ¹H-NMR spectra of all derivatives, protons of α and β appeared in the 7.03-8.20 ppm region. Aromatic protons appeared in the 7.29-8.80 ppm region. Mass spectra of synthesized derivatives displayed M+Na peak according to their molecular formula.

 

 

Scheme 1. Synthetic route for the chalcone analogues 1-12

 

 

Table 1. Physical details of the synthesized analogues (1-12)

 

(2E,4E)-1-(3-nitrophenyl)-5-phenylpenta-2,4-dien-1-one (1):

IR (KBr) λ_max (cm^-1): 3042 (=C-H aromatic); 1669 (C=O); 1595 (C=C); 1520 & 1352 (NO₂). ¹H-NMR (500 MHz, CDCl₃) δ ppm: 8.80-8.79 (m, 1H, H-2’), 8.43-8.41 (d, 1H, J= 8.3 Hz, H-4’), 8.32-8.30 (d, 1H, J= 8.1 Hz, H-6’), 7.71-7.66 (m, 2H, H-β & H-5’), 7.53-7.51 (d, 2H, J= 7.5 Hz, H-2” & 6”), 7.41-7.35 (m, 3H, H-3”, 4”, 5”), 7.12-7.07 (m, 3H, H-α, 4, 5). ¹³C NMR (125 MHz, CDCl₃) δ ppm: 187.85 (C=O), 148.3 (C-3’), 146.6 (C-β OR C-2), 143.5 (C-5), 135.7 (C-6’), 133.9 (C-5’), 129.8 (C-4’), 129.6 (C-α), (139.4, 128.9, 127.4, 126.9, phenyl), 126.4 (C-4), 123.1 (C-2’). MS (ESI) m/z= 302.08 [M+Na].

 

(2E,4E)-1-(4-nitrophenyl)-5-phenylpenta-2,4-dien-1-one (2):

IR (KBr) λ_max (cm^-1): 3010 (=C-H aromatic); 1686 (C=O); 1593 (C=C); 1515 & 1360 (NO₂). ¹H-NMR (500 MHz, CDCl₃) δ ppm: 8.34-8.32 (d, 2H, J= 8.3 Hz, H-3’ & 5’), 8.10-8.09 (d, 2H, J= 8.4 Hz, H-2’ & 6’), 7.66-7.62 (dd, 1H, J= 14.8 Hz, 9.4 Hz, H-β), 7.52-7.51 (d, 2H, J= 7.7 Hz, H-2” & 6”), 7.41-7.35 (m, 3H, H-3”, 4”, 5”), 7.10-7.03 (m, 3H, H-α, 4, 5). ¹³C NMR (125 MHz, CDCl₃) δ ppm: 188.8 (C=O), 149.9 (C-4’), 146.7 (C-1’), 143.5 (C-β), 143.0 (C-5), 129.6 (C-2’ & 6’), 129.2 (C-α), (135.6, 128.9, 127.4, 126.4, phenyl), 124.4 (C-4), 123.7 (C-3’ &5’). MS (ESI) m/z= 302.08 [M+Na].

 

(2E,4E)-1-(2-nitrophenyl)-5-phenylpenta-2,4-dien-1-one (3):

IR (KBr) λ_max (cm^-1): 3086 (=C-H aromatic); 1668 (C=O); 1593 (C=C); 1505 & 1352 (NO₂). ¹H-NMR (500 MHz, CDCl₃) δ ppm: 8.15-8.14 (d, 1H, J= 8.4 Hz, H-3’), 7.75-7.72 (m, 1H, H-4’), 7.64-7.61 (m, 2H, H-5’ & H-β), 7.48-7.42 (m, 3H, H-6’, 2”, 6”), 7.36-7.29 (m, 3H, H-3”, 4”, 5”), 7.04-6.99 (m, 1H, H-α), 6.95-6.83 (m, 1H, H-5), 6.56-6.53 (d, 1H, J= 15.2 Hz, H-4). ¹³C NMR (125 MHz, CDCl₃) δ ppm: 192.7 (C=O), 146.6 (C-2’), 146.2 (C-β), 142.6 (C-5), 136.2 (C-4’), 135.5 (C-5’), 130.4 (C-1’), 129.4 (C-6’), 129.2 (C-α), (133.9, 128.8, 128.7, 127.3, phenyl), 126.1 (C-4), 124.4 (C-3’). MS (ESI) m/z= 302.08 [M+Na].

 

(E)-3-(naphthalen-1-yl)-1-(3-nitrophenyl)prop-2-en-1-one (4):

IR (KBr) λ_max (cm^-1): 3088 (=C-H aromatic); 1685 (C=O); 1598 (C=C); 1525 & 1347 (NO₂). ¹H-NMR (500 MHz, CDCl₃) δ ppm: 8.85 (s, 1H, H-2’), 8.41-8.35 (m, 2H, H-4’ & 6’), 8.22-8.19 (d, 1H, J= 8.6 Hz, H-β), 7.96-7.87 (m, 4H, H-5”, 4”, 8”, 5’), 7.70-7.51 (m, 5H, H-2”, α, 6”, 7”, 3”). ¹³C NMR (125 MHz, CDCl₃) δ ppm: 187.6 (C=O), 148.2 (C-3’), 139.2 (C-1’), 135.2 (C-β), 134.0 (C-6’), 131.4 (C-5’), 129.8 (C-4’), (133.6, 131.6, 131.5, 128.8, 128.4, 127.0, 126.8, 126.3, 124.7, 123.1, naphthyl), 123.2 (C-2’), 122.8 (C-α). MS (ESI) m/z= 326.08 [M+Na].

 

(E)-3-(naphthalen-1-yl)-1-(4-nitrophenyl)prop-2-en-1-one (5):

IR (KBr) λ_max (cm^-1): 3090 (=C-H aromatic); 1690 (C=O); 1595 (C=C); 1515 & 1352 (NO₂). ¹H-NMR (500 MHz, CDCl₃) δ ppm: 8.72-8.69 (d, 2H, J= 15.4 Hz, H-3’ & 5’), 8.34-8.32 (d, 2H, J= 8.9 Hz, H-2’ & 6’), 8.21-8.16 (m, 2H, H-5”, β), 7.96-7.88 (m, 3H, H-4’, 8”, 2”), 7.61-7.51 (m, 4H, H-α, 6”, 7”, 3”). ¹³C NMR (125 MHz, CDCl₃) δ ppm: 188.5 (C=O), 149.9 (C-4’), 143.4 (C-1’), 133.6 (C-β), 129.0 (C-2’ & 6’), (131.6, 131.5, 129.3, 128.8, 127.2, 126.4, 125.3, 125.3, 123.7, 123.4, naphthyl), 123.8 (C-3’ & 5’), 123.1 (C-α). MS (ESI) m/z= 326.08 [M+Na].

 

(E)-3-(naphthalen-1-yl)-1-(2-nitrophenyl)prop-2-en-1-one (6):

IR (KBr) λ_max (cm^-1): 3096 (=C-H aromatic); 1688 (C=O); 1595 (C=C); 1520 & 1347 (NO₂). ¹H-NMR (500 MHz, CDCl₃) δ ppm: 8.20-8.19 (d, 1H, J= 8.2 Hz, H-3’), 8.15-8.12 (d, 1H, J= 15.8 Hz, H-β), 7.93-7.78 (m, 5H, H-4’, 5’, 5”, 8”, 4”), 7.69-7.66 (m, 1H, H-2”), 7.60-7.47 (m, 4H, H-6’, 6”, 7”, 3”), 7.10-7.07 (d, 1H, J= 15.8 Hz, H-α). ¹³C NMR (125 MHz, CDCl₃) δ ppm: 192.7 (C=O), 146.8 (C-2’), 142.8 (C-β), 136.3 (C-5’), 134.0 (C-4’), 131.1 (C-1’), 130.7 (C-6’), (133.6, 131.3, 131.2, 128.8, 128.4, 127.0, 126.3, 125.5, 124.5, naphthyl), 125.4 (C-3’), 122.8 (C-α). MS (ESI) m/z= 326.08 [M+Na].

 

(E)-3-(naphthalen-2-yl)-1-(3-nitrophenyl)prop-2-en-1-one (7):

IR (KBr) λ_max (cm^-1): 3085 (=C-H aromatic); 1688 (C=O); 1595 (C=C); 1520 & 1350 (NO₂). ¹H-NMR (500 MHz, CDCl₃) δ ppm: 8.80 (s, 1H, H-2’), 8.49 (d, 1H, J= 8.0 Hz, H-4’), 8.30 (d, 1H, J= 7.8 Hz, H-6’), 8.08 (d, 1H, J= 15.6 Hz, H-β), 8.01 (s, 1H, H-1”), 7.92-7.81 (m, 2H, H-5” & 8”), 7.79-7.73 (m, 2H, H-3” & 4”), 7.70 (m, 1H, H-5’), 7.64 (d, 1H, J= 15.6 Hz, H-α), 7.55-7.53 (m, 2H, H-6” & 7”). ¹³C NMR (125 MHz, CDCl₃) δ ppm: 187.7 (C=O), 148.3 (C-3’), 144.2 (C-β), 138.6 (C-1’), 133.8 (C-6’), 131.2 (C-5’), 129.8 (C-4’), (133.4, 133.2, 133.0, 128.6, 128.0, 127.6, 127.4, 126.6, 126.2, 125.3, naphthyl), 123.3 (C-2’), 121.0 (C-α). MS (ESI) m/z= 326.08 [M+Na].

 

(E)-3-(naphthalen-2-yl)-1-(4-nitrophenyl)prop-2-en-1-one (8):

IR (KBr) λ_max (cm^-1): 3089 (=C-H aromatic); 1683 (C=O); 1596 (C=C); 1519 & 1352 (NO₂). ¹H-NMR (500 MHz, CDCl₃) δ ppm: 8.52 (d, 2H, J=8.4 Hz, H-3’ & 5’), 8.25-8.23 (m, 2H, H-2’ & 6’), 8.09 (d, 1H, J= 15.6 Hz, H-β), 8.01 (s, 1H, H-1”), 7.96-7.94 (m, 2H, H-5” & 8”), 7.82-7.79 (m, 2H, H-3” & 4”), 7.66 (d, 1H, J= 15.6 Hz, H-α), 7.56-7.54 (m, 2H, H-6” & 7”). ¹³C NMR (125 MHz, CDCl₃) δ ppm: 188.8 (C=O), 151.7 (C-4’), 144.3 (C-β), 142.7 (C-1’), 129.8 (C-2’& 6’), (133.4, 133.3, 133.0, 128.0, 127.8, 126.8, 126.6, 126.1, 125.8, 125.2, naphthyl), 124.0 (C-3’& 5’), 121.0 (C-α). MS (ESI) m/z= 326.08 [M+Na].

 

(E)-3-(naphthalen-2-yl)-1-(2-nitrophenyl)prop-2-en-1-one (9):

IR (KBr) λ_max (cm^-1): 3086 (=C-H aromatic); 1685 (C=O); 1590 (C=C); 1521 & 1349 (NO₂). ¹H-NMR (500 MHz, CDCl₃) δ ppm: 8.49-8.45 (m, 2H, H-3” & 4”), 8.12 (d, 1H, J= 15.6 Hz, H-β), 8.06-8.04 (m, 1H, H-5’), 8.02 (s, 1H, H-1”), 7.95-7.93 (m, 2H, H-5” & 8”), 7.78-7.73 (m, 2H, H-3” & 4”), 7.71 (d, 1H, J= 8.2 Hz, H-6”), 7.63 (d, 1H, J= 15.6 Hz, H-α), 7.55-7.52 (m, 2H, H-6” & 7”). ¹³C NMR (125 MHz, CDCl₃) δ ppm: 189.4 (C=O), 148.0 (C-2’), 144.0 (C-β), 135.4 (C-5’), 135.3 (C-4’), 131.2 (C-1’), 130.7 (C-6’), (133.4, 133.3, 133.0, 128.2, 128.0, 127.7, 127.6, 126.5, 126.2, 125.9, naphthyl), 125.2 (C-3’), 121.8 (C-α). MS (ESI) m/z= 326.08 [M+Na].

 

(E)-methyl 4-(3-(3-nitrophenyl)-3-oxoprop-1-en-1-yl)benzoate (10):

IR (KBr) λ_max (cm^-1): 3082 (=C-H aromatic); 1680 (C=O); 1590 (C=C); 1515 & 1351 (NO₂), 1735 (Ester C=O). ¹H-NMR (500 MHz, CDCl₃) δ ppm: 8.78 (s, 1H, H-2’), 8.42 (d, 1H, J= 8.1 Hz, H-4’), 8.35 (d, 1H, J= 7.8 Hz, H-6’), 8.08 (d, 1H, J= 15.6 Hz, H-β), 7.78-7.75 (m, 3H, H-5’, 3” & 5”), 7.65 (d, 1H, J= 15.6 Hz, H-α), 7.33-7.32 (m, 2H, H-2” & 6”), 3.88 (s, 3H, OCH₃). ¹³C NMR (125 MHz, CDCl₃) δ ppm: 188.7 (C=O), 165.2 (Ester C=O), 148.3 (C-3’), 144.7 (C-β), 138.9 (C-1”), 137.4 (C-1’), 133.8 (C-6’), 130.0 (C-5’), 129.7 (C-3” & 5”), 129.5 (C-4’), 129.2 (C-4”), 128.8 (C-2” & 6”), 121.2 (C- α), 52.7 (OCH₃). MS (ESI) m/z= 334.07 [M+Na].

 

(E)-methyl 4-(3-(4-nitrophenyl)-3-oxoprop-1-en-1-yl)benzoate (11):

IR (KBr) λ_max (cm^-1): 3085 (=C-H aromatic); 1678 (C=O); 1593 (C=C); 1520 & 1355 (NO₂) 1730 (Ester C=O). ¹H-NMR (500 MHz, CDCl₃) δ ppm: 8.54 (d, 2H, J= 8.2 Hz, H-3’ & 5’), 8.24-8.23 (m, 2H, H-2’ & 6’), 8.092 (d, 1H, J= 15.6 Hz, H-β), 7.75-7.74 (m, 2H, H-3” & 5”), 7.62 (d, 1H, J= 15.6 Hz, H-α), 7.38-7.36 (m, 2H, H-2” & 6”), 3.87 (s, 3H, OCH₃). ¹³C NMR (125 MHz, CDCl₃) δ ppm: 188.4 (C=O), 165.0 (Ester C=O), 151.7 (C-4’), 144.8 (C-β), 143.4 (C-1’), 133.4 (C-1”), 131.0 (C-2’ & 6’), 129.9 (C-3” & 5”), 129.4 (C-4”), 129.0 (C-2” & 6”), 123.8 (C-3’ & 5’), 122.1 (C-α), 52.3 (OCH₃). MS (ESI) m/z= 334.07 [M+Na].

 

(E)-methyl 4-(3-(2-nitrophenyl)-3-oxoprop-1-en-1-yl)benzoate (12):

IR (KBr) λ_max (cm^-1): 3087 (=C-H aromatic); 1673 (C=O); 1597 (C=C); 1515 & 1352 (NO₂) 1726 (Ester C=O). ¹H-NMR (500 MHz, CDCl₃) δ ppm: 8.48-8.46 (m, 2H, H-3’ & 4’), 8.11 (d, 1H, J= 15.6 Hz, H-β), 8.07-8.06 (m, 1H, H-5’), 7.75-7.73 (m,3H, H-6’, 3” & 5”), 7.69 (d, 1H, J= 15.6 Hz, H-α), 7.40-7.38 (m, 2H, H-2” & 6”), 3.88 (s, 3H, OCH₃). ¹³C NMR (125 MHz, CDCl₃) δ ppm: 188.3 (C=O), 164.7 (Ester C=O), 148.3 (C-2’), 144.3 (C-β), 138.4 (C-1”), 134.7 (C-4’), 134.5 (C-5’), 131.4 (C-1’), 130.5 (C-6’), 129.7 (C-3” & 5”), 129.3 (C-4”), 129.0 (2” & 6”), 123.7 (C-3’), 121.0 (C-α), 51.3 (OCH₃). MS (ESI) m/z= 334.07 [M+Na].

 

In vitro antimicrobial potency:

Antibacterial potency against Gram-positive (S. pyogenes and S. aureus) and Gram-negative (P. aeruginosa and E. coli) bacteria and the antifungal effectiveness against C. albicans and A. fumigatus strains, was tested on the newly synthesized compounds 1-12. The synthesized derivatives' antibacterial activity and antifungal activity were compared with Ampicillin and Fluconazole as standard antibacterial and antifungal drugs, respectively.

 

The antimicrobial potency results showed that most of the synthesized chalcone derivatives exhibited good to moderate inhibitory activity, as shown in Table 2. Antibacterial activity results showed compound 6 as the most active derivative against all bacterial strains with MIC values ranging from 50-100 μg/ml (Fig. 1). Compounds 1, 3, 4, 7, 9, and 12 were active against S. aureus with MIC 125 μg/ml, i.e., two times higher than standard drug. Compound 2 was found equally active against S. aureus with MIC 250 μg/ml compared to the standard drug. Compounds 4, 7, 9, and 12 were found equally active against S. pyogenes with a MIC value of 100 μg/ml compared to the standard drug. Compounds 1, 2, and 3 showed moderate activity against S. pyogenes with a MIC value of 125 μg/ml. Compounds 4, 6, 7, 9, and 12 showed good to moderate activity against E. coli with MIC values ranging between 100-125 μg/ml. Compounds 6, 9, and 12 were found equal active against P. aeruginosa with a MIC value of 100 μg/ml.

 

The antifungal activity results showed compounds 10, and 12 being the most active compounds against all fungal strains with a MIC value of 25 μg/ml, which was four times higher than the standard drug (Fig. 2). Compounds 1, 3, 4, 5, 9, and 11 were most active against C. albicans with a MIC value of 50 μg/ml, two folds higher than the standard drug used. Compounds 2, 6, 7, and 8 were found equally active against C. albicans with a MIC value of 100 μg/ml. Derivatives 1, 3, 4, 5, 7, 8, 9, and 11 were equally active against A. fumigatus with a MIC value of 100 μg/ml compared with the standard drug. Compounds 2 and 6 showed moderate activity against A. fumigatus with a MIC value of 125 μg/ml.

 

In terms of antibacterial and antifungal activity, newly synthesized chalcone analogues showed a broad-spectrum antimicrobial effectiveness against the pathogenic microbial strains indicated above.

 

 

Table 2. In vitro antibacterial and antifungal potency (MIC values) of the tested derivatives

Analogue	MIC (μg/ml)
	Gram-positive bacteria		Gram-negative bacteria		Fungi
	S. aureus MTCC-96	S. pyogenes MTCC-443	E. coli MTCC-442	P. aeruginosa MTCC-2488	C. albicans MTCC-227	A. fumigatus MTCC-343
1	125	125	250	125	50	100
2	250	125	500	125	100	125
3	125	125	500	125	50	100
4	125	100	125	125	50	100
5	500	500	1000	500	50	100
6	100	50	100	100	100	125
7	125	100	125	125	100	100
8	500	500	500	500	100	100
9	125	100	100	100	50	100
10	500	500	500	500	25	25
11	500	250	250	250	50	100
12	125	100	100	100	25	25
Ampicillin	250	100	100	100	-	-
Fluconazole	-	-	-	-	100	100

 

 

Fig. 1. Antibacterial screening results: S.A.- S. aureus, S.P.- S. pyogenes, E.C.- E. coli, P.A.- P. aeruginosa

 

Fig. 2. Antifungal screening results: C.A.- C. albicans, A.F.- A. fumigatus

 

Molecular Modelling:

The chimeric enzyme was created, and its structure was simulated, followed by energy reduction to investigate the antibacterial and antifungal potency of nitro-substituted chalcone derivatives. Molegro Virtual Docker (MVD) was used to do a molecular docking study on the modelled structure.

 

The docking score function, i.e., Mol Dock score of the programme, was used to study the protein-ligand interaction. Table 3 and Fig. 3 show the expected binding energy and other docking data of nitro-substituted chalcone derivatives for antibacterial activity. Table 4 and Fig. 4 show the expected binding energy and other docking data of nitro-substituted chalcone counterparts for antifungal activity.

 

Table 3. Antibacterial compounds showing Docking Score, Interaction Data along with Distance between the protein residues

Compound	Mol Dock Score	Interaction Data
		Protein Residue	Ligand atom	Distance (A°)
1	-85.3848	Arg 285 Arg 285 Tyr 157 Tyr 157 Tyr 159 Arg 285	O1 O1 O1 N O2 O2	2.91 2.88 2.64 3.11 3.24 3.21
2	-83.1911	Thr 299 Ser 62 Tyr 157	O2 O2 O1	3.22 3.10 3.10
3	-85.7822	Asn 161 Thr 116 Thr 116	O2 O2 O1	2.91 3.22 3.32
4	-88.7482	Asn 161 Thr 116 Thr 116 Asn 117	O2 N O1 O1	3.06 3.09 2.58 3.35
5	-77.8818	Tyr 159 Ser 62 Asn 117 Asn 117 Asn 117 Thr 116 Thr 116	O2 O2 O1 N O3 N O3	3.10 2.55 2.58 3.10 2.80 3.22 2.60
6	-95.2905	Asn 161 Asn 161 Thr 116 Thr 116	O2 O1 O1 N	3.40 2.61 2.60 3.45
7	-89.1903	Tyr 159 Ser 62 Arg 285	O2 O2 O1	2.99 2.92 3.30
8	-79.2664	Thr 299 Thr 301 Tyr 157 Arg 285 Arg 285	O2 O2 O1 O1 N	3.26 3.56 3.00 3.14 3.52
9	-90.7906	Ser 62 Asn 161 Asn 161 Thr 116 Asn 161 Thr 116	O2 O2 O1 O1 N N	3.43 3.56 2.00 2.64 3.19 3.10
10	-74.0932	Thr 116 Ser 62 Lys 65 Ser 62 Tyr 157 Arg 285 Arg 285	O2 O1 O1 N O3 O3 O3	2.83 2.60 2.93 3.29 2.72 2.63 3.20
11	-79.9286	Arg 285 Arg 285 Tyr 157 Tyr 157	O1 O1 O1 N	3.43 2.60 2.60 3.29
12	-90.7495	Arg 285 Arg 285 Thr 299 Thr 299 Thr 301 Thr 301	O2 O2 O2 O3 N O1	2.80 3.58 2.86 3.52 3.10 2.54
Ampicillin	-93.7757	Arg 285 Thr 299 Tyr 159 Ser 62 Ser 62 Tyr 159 Lys 65 Asn 161	O1 O1 O2 O2 N O3 O3 O4	2.90 2.11 2.23 2.86 2.83 3.29 3.38 2.93

 

 

Fig. 3. Interaction of compound 6 with the active site of transpeptidase of bacterial strains

 

Table 4. Analogues with Docking Score, Interaction Data along with the Distance between the protein residues for antifungal activity

Compound	Mol Dock Score	Interaction Data
		Protein Residue	Ligand atom	Distance (A°)
1	-131.587	Thr 260 Gln 72 Arg 96	NO2(O) CO(O) CO(O)	3.18 3.18 2.89
2	-109.064	Thr 260	NO2(N)	2.95
3	-126.022	Arg 96 Arg 96 Arg 96 Arg 96 Aln 72	CO(O) NO2(N) NO2(O) NO2(O) NO2(O)	2.83 3.20 2.64 3.09 3.09
4	-133.887	Arg 96 Gln 72	CO(O) CO(O)	3.10 3.26
5	-133.383	Ile 323 Ile 323 Ile 323	NO2(N) NO2(O) NO2(O)	2.87 2.62 3.08
6	-129.195	Arg 96 Arg 96 Arg 96 Gln 72	CO(O) NO2(N) NO2(O) NO2(O)	2.87 3.23 2.70 2.84
7	-125.71	Arg 96	CO(O)	3.33
8	-124.718	Thr 260	NO2(O)	3.12
9	-132.933	Arg 96 Arg 96 Arg 96 Arg 96 Gln 72	CO(O) NO2(N) NO2(O) NO2(O) NO2(O)	3.22 3.32 2.60 3.12 3.13
10	-139.004	Thr 260 Arg 96 Gln 72	NO2(O) CO(O) CO(O)	3.17 2.89 3.45
11	-126.598	Ile 322	NO2(O)	2.65
12	-139.127	Arg 96 Arg 96 Arg 96 Arg 96 Gln 72	CO(O) NO2(N) NO2(O) NO2(O) NO2(O)	2.85 3.17 2.67 3.05 3.12
Fluconazole	-97.6403	Ala 256 Heme N	N(Ring 1) N (Ring 1)	3.20 3.50

 

 

Fig. 4(A) Interaction of compound 10 and (B) 12 with the active site of 14α-demethylase of fungal strains

 

Docking scores of the synthesized chalcone derivatives indicated that the derivatives could be a potential inhibitor for the microbial strains by competently binding to the transpeptidase and 14α-demethylase structure.

 

SAR (Structure-Activity Relationship):

From the outcomes mentioned above of antimicrobial activity of synthesized chalcone analogues, the succeeding SAR can be derived as presented in Fig. 5 and discussed below:

i.      In compounds 3, 6, 9, and 12, the presence of NO₂ group at o-position on benzylidene portion of acetophenone enhances the antimicrobial activity against S. aureus, S. pyogenes, C. albicans and A. fumigatus.

ii.    In compounds 1, 4, and 7, the presence of NO₂ group (EWG) at m-position on benzylidene portion of acetophenone imparts 2-folds antimicrobial activity against S. aureus, C. albicans and A. fumigatus.

iii.  Replacement of phenyl ring by α-naphthyl or β-naphthyl as in the case of derivatives 4-9 imparts better activity against fungal strains.

iv.   Cinnamaldehyde (compounds 1, 2, and 3) improves the antifungal potency against C. albicans and A. fumigatus.

v.     In analogues 10, 11, and 12, the presence of ester group on phenyl ring of aromatic aldehyde portion improves antifungal potency against C. albicans and A. fumigatus by 2-4 folds.

vi.   The presence of NO₂ group on benzylidene portion at o-, m- and p- positions of acetophenone plays a pivotal role in improving the antibacterial as well as antifungal potency against micro-organisms³¹.

 

 

Fig. 5. SAR studies of synthesized analogues

 

CONCLUSION:

Under this study, twelve new chalcone derivatives were synthesized by Claisen-Schmidt condensation. The synthesized derivatives were characterised by spectral analysis. Chalcones are known for their antibacterial and antifungal properties. Therefore, consisting of the same skeleton, the synthesized derivatives showed various levels of antibacterial and antifungal activities against S. pyogenes, S. aureus, P. aeruginosa, E. coli, C. albicans, and A. fumigatus. Some of the synthesized chalcone analogs assessed for activity had an antibacterial and antifungal activity higher than that of the standard drugs used (Ampicillin and Fluconazole). Compound 6 was demonstrated to be the most active analogue against all the bacterial strains. Compounds 10 and 12 were demonstrated to be the most active analogues against all the fungal strains. Most of the synthesized chalcone analogues showed good activity against all the bacterial and fungal strains.

 

ACKNOWLEDGEMENTS:

The authors are thankful to Dr. Ran Singh and Prof. (Dr.) M.S. Ashawat, Laureate Institute of Pharmacy, Jawalamukhi, HP, India for furnishing facilities for research work. Authors are also grateful to IIT, Mandi, HP, India for providing analytical services, to Chitkara College of Pharmacy, Chitkara University, Punjab, India, for providing the facilities for valuable software like Turnitin for plagiarism check and Molegro Virtual Docker for docking studies.

 

CONFLICT OF INTEREST:

There is no conflict of interest of any stakeholder.

 

ABBREVIATIONS:

MIC                       Minimum Inhibitory Concentration µg

                                Microgram

S. aureus               Staphylococcus aureus

S. pyogenes           Streptococcus pyogenes

E. coli                    Escherichia coli

P. aeruginosa      Pseudomonas aeruginosa

C. albicans           Candida albicans

A. fumigatus         Aspergillus fumigatus

IR                           Infrared spectroscopy

NMR                      Nuclear Magnetic Resonance

CDCl₃                    Deuterated chloroform

HRMS                   High Resolution Mass Spectroscopy

 

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Accepted on 12.07.2022           © RJPT All right reserved

Research J. Pharm. and Tech 2023; 16(4):1931-1939.