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

 

Kamya Goyal1,2, 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.

*Corresponding Author E-mail: rajwinder.kaur@chitkara.edu.in

 

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.

 

KEYWORDS: Nitrochalcones, Biological evaluation, Antibacterial, Antifungal, Claisen-Schmidt condensation.

 

 


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 globe1,2. 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 (λmax 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 ethanol11,17,25.

 

In-vitro biological assays for Antibacterial and antifungal activity:

MIC values of synthesized chalcone derivatives were determined using broth microdilution method11,26. 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) program27-30. 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)

Analogue

R

Molecular Formulae

Mol. Wt.

(g/mol)

m.pt. (oC)

Yield (%)

1

3-NO2

 

C17H13NO3

279.29

139-140

91

2

4-NO2

 

C17H13NO3

279.29

141-142

82

3

2-NO2

 

C17H13NO3

279.29

140-141

84

4

3-NO2

 

C19H13NO3

303.31

136-137

97

5

4-NO2

 

C19H13NO3

303.31

138-139

76

6

2-NO2

 

C19H13NO3

303.31

139-140

85

7

3-NO2

 

C19H13NO3

303.31

139-140

83

8

4-NO2

 

C19H13NO3

303.31

136-137

79

9

2-NO2

 

C19H13NO3

303.31

138-139

84

10

3-NO2

 

C17H13NO5

311.29

136-137

82

11

4-NO2

 

C17H13NO5

311.29

139-140

78

12

2-NO2

 

C17H13NO5

311.29

140-141

81

 


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

IR (KBr) λmax (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) λmax (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) λmax (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) λmax (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) λmax (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) λmax (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) λmax (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) λmax (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) λmax (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) λmax (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) λmax (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) λmax (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 ΅g

                                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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Received on 16.03.2022            Modified on 04.05.2022

Accepted on 12.07.2022           © RJPT All right reserved

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

DOI: 10.52711/0974-360X.2023.00317