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Abstract

Antimicrobial resistance is on the rise, constituting a public health problem which accounts for millions of global deaths annually. Thus, there is an urgent need for new antimicrobial compounds that can target virulence factors of pathogens. Supramolecules and macromolecules are attractive antimicrobial candidates since they exhibit antibiotic effects sustained for longer periods. This study reports the synthesis of a phthalimide-triazole-based p-tert-butylcalix[4]arene compound (AT-2b) and its analogue from p-tert-butyl phenol (AT-1b) and their characterization using the ¹³C NMR and ¹H NMR data. There was observed agreement between experimental and theoretical chemical shifts (¹³C and ¹H NMR) determined through DFT (density functional theory) calculations. DFT was used to obtain optimized geometries and molecular electrostatic potentials of the compounds, indicating their nucleophilic and electrophilic reactive sites. The compounds exhibited good antimicrobial activity against Pseudomonas aeruginosa, Escherichia coli, Chromobacterium violaceum, Staphylococcus aureus, and Candida albicans with MIC (minimal inhibitory concentration) values ranging from 4.88 to 312 µg/mL. The compounds inhibited violacein production against C. violaceum CV12472 as well as quorum-sensing (QS) against C. violaceum CV026, at MIC and sub-MIC concentrations, suggesting that they can disrupt intercellular signaling in bacteria. Both compounds showed concentration-dependent inhibition of swimming and swarming motilities against flagellated P. aeruginosa PA01, indicating their bacteriostatic potential. The compounds reduced biofilm formation against C. albicans, E. coli, and S. aureus, with S. aureus biofilms being the most susceptible. Structurally, AT-1b is a subunit of AT-2b and was less active than the latter. Molecular docking predictions showed negative binding energies and adequate interactions between the compounds and the receptor proteins of the pathogens, substantiating the observed antimicrobial effects.

Keywords

antimicrobial activity biofilm inhibition calixarene DFT molecular docking phthalimide triazole

Graphical abstract

Introduction

Macromolecular chemistry studies fascinating chemical architectures from natural and synthetic origin composed of building blocks (monomers) that are linked together by covalent interactions into complex molecules with a high molecular weight. Antibacterial materials based on macrocycles and macromolecules are effective against drug-resistant fungi and bacteria with sustained antimicrobial effects.^1,2 Calix[n]arenes consist of a class of macromolecular cyclic compounds having multiple numbers of phenolic units bonded together through methylene bridges at ortho positions with the most prevalent systems possessing four to eight phenolic units.^3,4 Aromatic rings in calixarenes are arranged in three-dimensional form with an ancient calix crater vase shape, to which it owes its name.⁵ The calixarene structure is made up of a narrow lower rim and a wider upper rim, while the middle conic section is called the annulus.⁶ Depending on the amount of aromatic units, the cavities of calixarenes vary in their molecular dimensions. The calix[4]arenes, for instance, can adopt the 1,3-alternate (partial cone) or 1,2-alternate (cone) conformers which are stable and useful for studying their structural aspects.⁷ Derivatives with varying recognition, selectivity, solubility, and hydrophobicity can be created by functionalizing the calixarenes on the upper and/or lower rim using functional groups of different kinds, including imines, amides, sulfur, semicarbazone, azo, and alkyl groups.^7,8

Calixarenes are among the most extensively researched supramolecules, as they provide infinite structures through modification at the lower and/or upper rim and application options because of their ease of modification, which enables the establishment of sizable molecular libraries as a material basis for a variety of biomedical applications.^9–12 Upper and/or lower rims can be modified or functionalized, and small ions and neutral compounds can be hosted within the cavity or annulus of calix[n]arenes to confer biological activities.^13,14 Various derivatives of calix[4]arene continue to attract special attention from researchers due to their unique structural features, synthetic accessibility, and ease of chemical modification for different applications.^15,16 Although they have upper and lower rims, functionalization with groups including alcohols, amines, amides, aldehydes, alkyl, amino acids, and ester derivatives opens the door to their potential application as prodrugs or as biochemical process inhibitors, with the release of active substances usually triggered by external stimuli.^17–19 Specifically, the calix[4]arene gives researchers the ability to precisely tune the shape of their structures and shows numerous possibilities for regioselective poly-functionalization on both upper and lower rims.²⁰

Numerous biological activities including antiviral, antifungal, and antibacterial properties have been reported for various calix[4]arene derivatives.²⁰^–²² Since they have versatile molecular structures compared to conventional antibacterial agents, calixarene derivatives offer tremendous potential as antibacterial agents and may be able to prevent cross-resistance with them.²³ Resistance is a situation whereby pathogenic microorganisms become non-susceptible to the effects of antibiotics previously used to treat their infections, and it creates a serious health issue that needs urgent and immediate intervention.^24,25 Antibiotic resistance occurs through different mechanisms and constitutes a global health problem since it is detected for almost all antibiotics presently used in conventional medicine and only small amounts of new antimicrobial therapies are getting into the market.²⁶ Resistance is often aided by microbial virulence factors most especially biofilms, a QS (quorum-sensing)-dependent phenomenon in bacteria. QS consists of a bacterial cell-to-cell communication system, while biofilms are self-produced protective polymeric matrix that keeps sessile communities safe from antibiotics, host immune system, starvation, and drought.²⁷ About eighty percent of chronic and recurring infectious diseases in humans involve microbial biofilms, and the microbial cells that reside within these biofilms may be more resistant to conventional antibiotics about 10–1000 times more than the same bacterial species living within planktonic colonies.^28,29 Antimicrobial compounds of natural and synthetic origin which are able to exert biocidal effects on pathogens through mechanisms such as QS and biofilm disruption are necessary since they address the evolution and development of resistance in microbial pathogens.^30–32

Various calix[4]arenes and their derivatives are known to be suitable scaffolds for synthesis of antimicrobial agents.³³ Calix[4]arene derivatives are potential therapeutic agents and particularly, azo-based calix[4]arene derivatives are under-exploited, meanwhile they may be prospective antiviral and antibacterial agents.³⁴ Because calixarene synthesis is inherently challenging, particularly with regard to purification process, there are still few calixarenic platforms available despite the field’s ongoing attention.³⁵

In this study, phthalimide-based p-tert-butylcalix[4]arene derivative and its analogue are synthesized and their antimicrobial, biofilm, and QS inhibitory effects are investigated. Computational predictions were applied to investigate structural features of the compounds as well as molecular docking to understand their structure–activity relationships.

Experimental

Chemicals and reagents

Chemical reagents from Sigma-Aldrich including anhydrous sodium hydroxide (NaOH, ⩾99.95%), formaldehyde (37 wt. % in H₂O), potassium carbonate (K₂CO₃, ⩾99%), sodium chloride (NaCl, ⩾99%), cupper(II)sulfate pentahydrate (CuSO₄.5H₂O, ⩾99.99%), and anhydrous magnesium sulfate (MgSO₄, ⩾99.5%) were obtained. Solvents without further purification, such as dichloromethane (DCM, ⩾99.8%, Merck), N-N-dimethylformamide (DMF, ⩾99.8%, Merck), hexane (⩾99.8%, Merck), ethyl acetate (EtOAc, ⩾99.5%, Merck), diphenyl ether (⩾99%, Sigma-Aldrich), acetone (⩾99.8%, Merck), toluene (⩾99.8%, Merck), butanol (⩾99.8%, Merck), anhydrous methanol (MeOH, ⩾99.8%, Sigma-Aldrich) of analytical grade together with propargyl bromide (80 wt.% solution in toluene, Thermo Scientific Chemicals) and p-tert-butyl phenol (99%, Sigma-Aldrich) were used. TLC (thin-layer chromatography) cards (Merck) and silica gel (Merck) were used for chromatography. Luria-Bertani Broth, Tryptic Soy Broth, Nutrient Broth, Mueller Hinton Broth, Sabouraud Dextrose Broth, and corresponding agars were procured from Merck. For antibiofilm assays, crystal violet (Merck), ethanol (Merck), D-(+)-glucose (Merck), and glacial acetic acid (Merck) were used. N-hexanoyl-DL-homoserine lactone (C6-HSL, ⩾97%, Sigma-Aldrich), NaCl (Sigma-Aldrich), tryptone (Sigma-Aldrich), D-(+)-glucose (⩾99.5%, Sigma-Aldrich), and kanamycin sulfate (Sigma-Aldrich) were used for anti-QS and antimotility assays.

Synthesis of compound 2

Compound 2 (p-tert-butylcalix[4]arene) was obtained from the reaction of p-tert-butyl phenol (1) with HCOH (formaldehyde) in basic conditions as shown in Figure 1. Summarily, 100 g (1 equiv.) of p-tert-butylphenol (1) was reacted with 62.3 mL (1.25 equiv.) of aqueous formaldehyde in the presence of 1.2 g (0.03 equiv.) of NaOH at 110–120 °C for 2 hours. The obtained viscous product was suspended in diphenyl ether and heated at 220 °C under N₂ gas atmosphere. It was subsequently refluxed for 1 h, and the resulting colorless solid was precipitated with ethyl acetate, dissolved in hot toluene and crystallized from toluene to obtain p-tert-butylcalix[4]arene (2) in moderate yield as very pure white crystals.

Figure 1.

Synthesis of phthalimide-triazole based p-tert-butylcalix[4]arene and its p-tert-phenol derivative.

Synthesis of AT-1a and AT-2a

Propargyl bromide and p-tert-butylcalix[4]arene (2) were mixed to afford compound AT-2a as described elsewhere.³⁶ Under the nitrogen atmosphere, p-tert-butylcalix[4]arene (2) (5.0 g, 7.72 mmol) with K₂CO₃ (2.59 g, 18.67 mmol) was dissolved in dry acetone (150 mL) followed by stirring for 1 h. Propargyl bromide (3.17 g, 26.54 mmol) in 50 mL of acetone from a syringe was introduced in a dropwise manner to the reaction mixture for 30 min with continuous stirring and then refluxed for 24 h while monitoring with TLC. The reaction was topped, and the solvent evaporated under vacuum. A solution of 2 M HCl (50 mL) was added to the residue and re-extracted three times with DCM (50 mL) to afford an organic phase which was washed with brine (50 mL) and water (50 mL) successively and dried over MgSO₄. The solvent was evaporated with a rotavapor to give a solid crude product which was triturated in methanol and filtered and dried under vacuum to give the pure product with 76% yield. For Compound AT-1a, p-tert-butyl phenol (1.8 g, 12 mmol) was reacted with K₂CO₃ (4 g, 29.2 mmol) and propargyl bromide (1.53 g, 12.8 mmol) in the same procedure as above to yield Compound AT-1a (85%). The synthetic route is summarized in Figure 1.

Compound AT-1a: Yield 85%; ¹H NMR (400 MHz, CDCl₃) δ_H 7.41-7.26 (m, 2H, ArH), 7.02-6.81 (m, 2H, ArH), 4.66 (d, 2H, J = 2.4 Hz, OCH₂), 1.30 (s, 9H, CH₃). ¹³C NMR (100 MHz, CDCl₃) δ_C 155.31, 144.25, 126.25, 114.29, 78.86, 75.29, 55.81, 34.11, 31.49.

Compound AT-2a: Yield 76%; ¹H NMR (400 MHz, CDCl₃) δ_H 7.07 (s, 4H, ArH), 6.72 (s, 4H, ArH), 6.51 (s, 2H, ArOH), 4.75 (d, J = 2.4 Hz, 4H, OCH₂), 4.37 (d, J = 13.3 Hz, 4H, ArCH₂Ar), 3.34 (d, J = 13.4 Hz, 4H, ArCH₂Ar), 2.53 (m, 2H, CH), 1.31 (s, 18H, CCH₃), 0.90 (s, 18H, C CH₃). ¹³C NMR (100 MHz, CDCl₃) δ_C 150.3, 149.5, 147.2, 141.6, 132.5, 128.0, 125.5, 125.1, 78.7, 76.8, 63.2, 33.9, 33.8, 32.0, 31.7, 30.9.

Synthesis of AT-1b and AT-2b

Azidopropyl phthalimide 3 was prepared according to the described method.³⁷ Compounds AT-1b and AT-2b were prepared according to the described method.^38,39 A mixture of Compound AT-1a or AT-2a (4 mmol) and Compound 3 (0.58 g, 2.5 mmol for AT-1a or 3.68 g, 16 mmol for AT-2a) was put in 20 mL of water, tert-butanol, and DCM (2:1:1). CuSO₄.5H₂O (0.75 g, 3 mmol) was added, followed by sodium ascorbate (0.99 g, 5 mmol). The mixture was stirred at 60 °C for 12 h and checked by TLC till all starting material was totally consumed. The reaction mixture was cooled, and 20 mL of distilled water added and extracted with DCM three times with 50 mL dichloromethane. The organic phase was combined and washed with 50 mL saturated brine, dried over anhydrous MgSO₄, and the solvent evaporated. Column chromatography was used to purify the crude products using EtOAc/hexane (20:80 for AT-1b and 10:90 for AT-2b) with 54% (for AT-1b) or 62% (for AT-2b) yield. The synthesis of AT-1b and AT-2b is summarized in Figure 1. The NMR spectra of AT-1b and AT-2b are provided in the supplemental material.

Compound AT-1b: Yield 54%; ¹H NMR (400 MHz, CDCl₃) δ_H 7.92 (s, 1H, CH), 7.82 (dd, J = 5.1, 3.2 Hz, 2H, CH-pht), 7.71 (dd, J = 5.0, 3.1 Hz, 2H, CH-pht), 7.27–7.21 (m, 2H, CH-phenol), 6.86–6.80 (m, 2H, CH-phenol), 5.21 (s, 2H, OCH₂), 4.26 (t, J = 6.1 Hz, 2H, CH₂), 3.66 (t, J = 6.1 Hz, 2H, CH₂), 2.25 (p, J = 6.1 Hz, 2H, CH₂), 1.33 (s, 9H, CCH₃). ¹³C NMR (100 MHz, CDCl₃) δ_C 168.32, 156.06, 143.93, 134.24, 131.92, 126.31, 123.42, 123.19, 114.39, 62.15, 47.94, 35.07, 34.10, 31.68, 29.46.

Compound AT-2b: Yield 62%; ¹H NMR (CDCl₃, 400 MHz) δ_H 8.15 (s, 2H, CH), 7.80 (q, 4H, ArH), 7.72 (q, 4H, ArH), 7.48 (s, 2H, ArOH), 7.03 (s, 4H, ArH), 6.81 (s, 4H, ArH), 5.25 (s, 4H, OCH₂), 4.54 (t, 4H, CH₂), 4.33 (d, 4H, ArCH₂Ar, J = 13.2), 4.11 (t, 4H, CH₂), 3.70 (t, 4H, CH₂), 3.33 (d, 4H, ArCH₂Ar, J = 13.2), 2.34 (q, 4H, CH₂), 1.30 (s, 18H, C(CH₃)₃), 0.99 (s, 18H, C(CH₃)₃). ¹³C NMR (100 MHz, CDCl₃) δ_C 168.25, 150.50, 149.73, 147.31, 144.21, 141.70, 134.07, 134.02, 132.73, 132.03, 127.75, 125.73, 125.14, 123.84, 123.33, 123.31, 69.93, 49.09, 47.98, 35.42, 35.15, 33.99, 33.84, 31.92, 31.70, 31.02, 29.48, 28.08.

Strains of microbes

Staphylococcus aureus ATCC 25923, Candida albicans ATCC 10239, Escherichia coli ATCC 25922, Chromo-bacterium violaceum CV026, C. violaceum CV12472, and Pseudomonas aeruginosa PA01 were used in the different microbial studies.

Measurement of antimicrobial activity

A microtiter broth dilution method as described previously was used to measure the minimal inhibitory concentration (MIC).⁴⁰ MIC was the lowest concentration of compound at which no visible growth was observed. The Mueller–Hinton broth and a bacteria concentration of 5 × 10⁵ colony-forming units (CFU)/mL were used. Into wells of 96-well microtiter plates, 100 μL of fresh overnight microbial suspensions were inoculated with or without compounds at serial final concentrations (5, 2.5, 1.25, 0.625, 0.3125, 0.15625, 0.078125, 0.0390625, 0.01953, 0.009765 mg/mL) and incubated for 24 hours at 37 °C after which MIC values were measured.

Biofilm inhibition assay

Biofilm-forming inhibition of the compounds at sub-MIC and MIC concentrations was evaluated using the microplate antibiofilm assay.⁴¹ Summarily, 1% of overnight cultures of bacteria were seeded in 200 μL of Tryptose-Soy Broth (TSB) supplemented with 0.25% glucose and inoculated in the presence or absence of the compounds at 37 °C for 48 h. The wells were rinsed with distilled water to remove planktonic cells, and biofilm cells were stained with crystal violet (0.1% solution) at room temperature for 10 min. Unabsorbed crystal violet solution was removed, and 200 μL ethanol (for Gram-negative and Candida) or 33% glacial acetic acid (for Gram-positive) was added to the wells. In total, 125 μL were pipetted from each well after shaking into sterile tubes, and the volume was adjusted with distilled water to 1 mL. A Thermo Scientific Multiskan FC spectrophotometer at 550 nm was used to read optical density (OD) which was used in calculating biofilm inhibition percentages using the.

$\begin{array}{l} Biofilm inhibition (%) = \\ \frac{O D 550_{C o n t r o l} - O D 550_{S a m p l e}}{O D 550_{C o n t r o l}} x 100 \end{array}$

Quorum-sensing inhibition assay against C. violaceum CV026

Quorum-sensing inhibition (QSI) was determined as explained previously⁴² with little modifications. 5 mm of lukewarm molten Soft Top Agar (2.0 g tryptone, 200 mL deionized H₂O, 1.3 g agar, 1.0 g NaCl) was mixed with an overnight culture of CV026 (100 µL) in LB (Luria-Bertani) broth and treated with 20 µL of external acylhomoserine lactone (AHL), that is, C₆HSL (100 µg/mL) and kanamycin solution (10 µL). The mixture was immediately poured and spread as an overlay on the surface of solidified LB agar plates. In total, 5 mm diameter wells were carved after solidification and filled with 50 µL MIC and sub-MIC concentrations of compounds and incubated at 30 °C for 3 days. Cream or white halo zones around each well over the purple lawn of growing CV026 bacteria indicated areas of QS inhibition, and their diameters were measured in mm. Each assay was repeated three times.

Violacein inhibition assay against C. violaceum CV12472

Qualitative analyses of violacein inhibition by the compounds were measured against C. violaceum ATCC 12472.⁴³ In total, 180 µL of Luria–Bertani broth and 10 µL of overnight culture (0.4 OD at 600 nm) of C. violaceum were filled into each well of sterilized 96-well microtiter plates. In total, 10 µL of MIC and sub-MIC amounts of compounds were added in order of decreasing concentration, and well without compounds served as controls. Plates were incubated for 24 h at 30 °C and monitored for the prevention of violacein production. The absorbance of each well was measured at 585 nm, and the percentage inhibition of violacein production is calculated as shown as follows.

$\begin{array}{l} Violacein inhibition (%) = \\ \frac{O D 585 c o n t r o l - O D 585 s a m p l e}{O D 585 c o n t r o l} x 100 \end{array}$

Swimming and swarming inhibitions against P. aeruginosa PA01

Swarming inhibition was performed as described elsewhere with slight modifications.⁴⁴ Swarming plates were prepared by mixing 0.5% agar, 0.5% of D-glucose, 1% peptone, and 0.5% NaCl in distilled water together with test compounds (MIC, ½ MIC, ¼ MIC) and allowing them to solidify. Overnight cultures of P. aeruginosa PA01 were point inoculated at the center of each plate. Plates without compounds served as controls. The plates were incubated for 18 h in an upright position, after which the swarm migration zone diameters were measured and used to calculate percentage inhibition in relation to the control plates. Swimming agar consisted of 1.5% agar, 0.5% D-glucose, 1% peptone, and 0.5% NaCl, and the process was similar as for swarming inhibition assay. Saran papers were used in wrapping the plates to avoid dehydration. The plates were incubated in an upright position for 18 h at 37 °C. The percentage reduction in swimming movement by compounds was calculated.

Density functional theory calculations

The frequency calculations and geometry optimization of the ground state geometries of AT-1b and AT-2b have been carried out at the B3LYP/6-31G(d)⁴⁵ as implemented in Gaussian 16 software.⁴⁶ The B3LYP hybrid function is well-known for reproducing experimental data such as geometrical and spectroscopic data.^47–49 This can be considered so because of the hybrid nature of B3LYP which combines the Hartree–Fock exchange with density functional theory (DFT) exchange-correlation and also has three empirical parameters fitted to experimental data. A fair compromise between accuracy and computational cost for different molecular properties is achieved via B3LYP’s empirical parameterization. Earlier investigations reported that the chosen functional is crucial for better accuracy, while the basis set may slightly influence the calculated properties.^50,51 All imaginary frequencies are found positive, which confirms that all optimized geometries of AT1b and AT2b are true minima. The ¹H- and ¹³C-NMR chemical shifts were predicted using the Gauge-Independent Atomic Orbital method.⁵² The solvent effects were taken into account using the implicit polarizable continuum model (IEPCM) formalism, in which the substrate is embedded into a cavity encircled by a dielectric continuum described by its dielectric constant (ε_DMSO = 46.826).⁵³ The solvent effects are well modeled by the polarizable continuum model (PCM).⁵⁴

Molecular docking details

The predicted binding affinities of AT-1b and AT-2b into the binding sites of C. albicans, E. coli, and S. aureus were identified using the Autodock4 package.⁵⁵ The structural X-ray geometries of the targets and original ligands were downloaded from the RCSB data bank website of PDB codes 1JIJ, 5FGN, and 1EAG, respectively.^56–58 Structural geometries of AT-1b and AT-2b were minimized at the B3LYP/6-31G(d),⁴⁵ and the outputs were saved as PDB files. More details on molecular docking stepwise may be found in our previously reported studies.^59,60 The binding interactions of the docked at the B3LYP/6-31G(d)⁴⁵ into the binding sites of the targets were visualized using the Discovery Studio Client (Discovery Studio Client is A Product of Accelrys Inc., San Diego, CA, USA).

Statistical analyses

Analysis of variance (ANOVA) was used in determining the differences in activities among the compounds. Experiments were done in triplicates, and the values represent mean ± standard error of the mean of three measurements. The data were analyzed using the Microsoft Excel, and differences were considered to be statistically significant at p < 0.05.

Results and discussion

Inhibition of violacein production, QS, and motilities

The results of violacein inhibition against C. violaceum CV12472 are presented in Figure 2. MIC was 0.156 mg/mL for both AT-1b and AT-2b compounds against C. violaceum CV12472. The violacein pigment inhibition in chromobacterium was evaluated at MIC and below-MIC concentrations so as to avoid high unselective pressure on bacteria cells and the effect of cell death. Both compounds AT-1b and AT-2b had violacein inhibitions of 100% at MIC and ½ MIC. Violacein inhibitions decreased with a decrease in concentration to 17.4 ± 0.8% and 5.8 ± 0.3% at MIC/16 for AT-2b and AT-1b, respectively.

Figure 2.

Violacein inhibition against C. violaceum CV12472 by compounds.

The results of QS inhibition against C. violaceum CV026 are presented in Figure 3. MICs against C. violaceum CV12472 were 0.312 mg/mL for both AT-1b and AT-2b compounds. Diameters of QS inhibition zones are 14.0 ± 0.62 mm and 13.5 ± 0.41 mm for AT-1b and AT-2b, respectively, at MIC and 10.0 ± 0.20 mm and 9.5 ± 0.05 mm for AT-1b and AT-2b at MIC/2, respectively. No QS inhibition was observed below MIC/2.

Figure 3.

Quorum-sensing inhibition against C. violaceum CV026 by compounds.

Inhibition of swimming and swarming motilities against flagellated P. aeruginosa PA01 was evaluated at 100, 75, and 50 µg/mL and presented in Figure 4. In the swarming motility assay, at the highest test concentration (100 µg/mL), inhibitions were 47.1 ± 0.55% (AT-2b) and 46.9 ± 0.13% (AT-1b) and reduced to 29.1 ± 0.21% (AT-2b) and 18.5 ± 0.11% (AT-1b) at 50 µg/mL. A similar trend was observed for swarming motility where inhibitions were 52.6 ± 0.50% (AT-1b) and 45.3 ± 0.71% (AT-2b) at 100 µg/mL and decreased to 20.1 ± 0.34% (AT-1b) and 10.2 ± 0.18% (AT-2b) at 50 µg/mL.

Figure 4.

Motility inhibition against P. aeruginosa PA01.

The compounds were investigated for their ability to disrupt virulence factors including violacein production, motilities, biofilm, and QS against the microorganisms. Antivirulence compounds are a class of drugs that can prevent pathogens from attacking the host by targeting virulence factors instead of killing or stopping their growth.⁶¹ Antivirulence therapy is a suitable strategy for weakening pathogens and blocking their pathogenicity, thereby reducing the administration of broad-spectrum antimicrobials and the prevalence of resistant strains.⁶² The goal of any given antimicrobial technique is to control virulence factors and microbial survival, and these chemicals accomplish this via disrupting signaling pathways known as QS. Violacein produced by C. violaceum is an easily measurable means for evaluating QS. The compounds inhibited the production of C. violaceum CV12472, which produces this pigment in a QS-mediated process while growing normally. AT-2b was more active than AT-1b which is a subunit of the AT-2b. The C. violaceum CV026 strain fails to produce violacein except when an external source of AHL is supplied to it. The compounds inhibited the production of violacein by this strain CV026, even in the presence of the supplied AHL. Suppression of violacein production in both C. violaceum CV12472 and CV026 indicates that the compounds disrupt both signal molecule emission and reception.⁶³ QS inhibitors are molecules that can stop bacterial communication, which in turn controls pathogenicity, by inhibiting QS-regulated processes like enzyme activity, fluorescence, bioluminescence, pigment production, biofilm formation, and dispersal.^64,65 P. aeruginosa moves using different forms of motilities aided by type 4 pili and polar flagellum, enabling the bacteria to get attached and colonize surfaces.⁶⁶ These swimming, swarming, and twitching motilities facilitate proliferation and colonization in different environments as well as the passage from planktonic to sessile colonies of P. aeruginosa and are a key step that precedes biofilm formation.^67,68 Both compounds AT-1b and AT-2b exhibited concentration-dependent swarming and swimming inhibitions, indicating that they can be suitable remedies in limiting bacterial spread, surface contamination, and colonization. Swimming and swarming motility contribute to biofilm formation which is a crucial environmental and clinical problem since it helps to increase resistance to antibiotics.

Antimicrobial and antibiofilm activities

The antimicrobial activity of the compounds is presented in Figure 5. MIC was determined for fungus (C. albicans), Gram-negative (E. coli), and Gram-positive (S. aureus) bacteria. MIC values against S. aureus (Gram-positive) were 39.06 µg/mL (AT-1b) and 9.77 µg/mL (AT-2b), while against E. coli (Gram-negative), MIC values were 312.5 µg/mL (AT-1b) and 156.25 µg/mL (AT-2b). Against C. albicans, MIC values were 19.53 µg/mL (AT-1b) and 4.88 µg/mL (AT-2b).

Figure 5.

Antimicrobial activity of the compounds.

The percentage inhibition of biofilm formation for each bacterium was determined at sub-MIC and MIC, and the results are presented in Figure 6. Biofilm inhibition against S. aureus, varied from 45.15 ± 0.55% (MIC) and 4.22 ± 0.06% (MIC/4) for AT-1b and from 63.15 ± 2.25% (MIC) to 8.65 ± 0.06% (AT-2b) for AT-2b. Biofilm inhibition against E. coli at MIC was 19.75 ± 0.85% (AT-1b) and 28.55 ± 0.52% (AT-2b), and at MIC/2, the inhibition of biofilm was 8.30 ± 0.34% (AT-1b) and 11.25 ± 0.55% (AT-2b). C. albicans biofilm formation was inhibited by the compounds with percentage inhibitions of 35.26 ± 0.65% (AT-1b) and 50.28 ± 1.45% (AT-2b) at MIC and 5.78 ± 0.13% (AT-1b) and 15.90 ± 0.11% (AT-2b) at MIC/4.

Figure 6.

Biofilm inhibition activity of the compounds.

The results suggest that C. albicans-established biofilms are less susceptible to the compounds than S. aureus-established biofilms, although planktonic C. albicans is more susceptible than planktonic S. aureus. This may depend on the biofilm architecture of both pathogens. The rising antibiotic resistance to available antibiotics requires the synthesis and evaluation of the effects of new antimicrobial.^69,70 The compounds showed antimicrobial activity against C. albicans, S. aureus, P. aeruginosa, E. coli, and C. violaceum. The compounds showed biofilm inhibition effects against C. albicans, S. aureus, and E. coli. As shown in Figure 6, S. aureus biofilm was most susceptible to the compounds, while E. coli biofilms were least susceptible. Compound AT-2b was more active than compound AT-1b. Compounds that can prevent the formation of biofilms are of great interest.⁷¹ Since there is existence of a protective barrier for biofilm colonies of bacteria, it is difficult for antibiotics to get to them. Some antibiotics will destroy planktonic colonies, while the biofilm communities remain safe and will continue to attack the patients when the antibiotics effects subside. It is for such reasons that bacteria in a planktonic state are less resistant to antibiotics that those in sessile colonies encased within biofilms.⁷² Because of the recalcitrance of biofilms, diseases resulting from them pose serious problems to the host immune system and cause persistent and recurrent clinical infections.⁷³ The results in this study indicate great antimicrobial potential of the synthesized calixarene (AT-2b) and its analogue (AT-1b).

DFT calculation results

The optimized geometries and electrostatic potentials (ESPs) of AT-1b and AT-2b are displayed in Figure 7. In AT-1b, 2-propyl-2H-indene-1,3-dione and 1-tert-butyl-4-methoxybenzene moieties are out of the molecular plane of 1,4-dimethyl-1H-1,2,3-triazole, and they form torsion angles of τ1 and τ2 of 90 degrees (Figure 7). In AT-2b, these angles are varied by 6.00 and 3.00 degrees compared to AT-1b, which probably returns the steric effects. The molecular electrostatic potential (MEP) is defined as the interaction energy between the charge distribution of a molecule and a unit positive charge. It is generated by the electrical charge cloud of electrons and nuclei in a molecule and is represented on the molecular structure using different colors to indicate different regions of electrostatic potential. The green region indicates neutral sites, the red region indicates the nucleophilic sites (donor of electrons), while the blue ones are electrophilic sites (acceptor of electrons). The MEP was calculated at a B3LYP-optimized geometry to predict reactive sites for electrophilic and nucleophilic attack. The positive regions (blue) as shown in Figure 7 are prone to nucleophilic reactivity, and the negative regions (red) to electrophilic reactivity. The electron-rich sites are around the N atoms, and the averagely electron-rich areas are around the aromatic ring and the oxygen atoms. The electron-poor sites are around the H and C atoms as well as the aromatic ring of the pthalimide moiety.

Figure 7.

Optimized geometries (left) and ESPs (right) of AT-2b (a) and AT-1b (b).

Tables 1 and 2 summarize the predicted and experimental ¹³C and ¹H NMR chemical shifts of AT-1b and AT-2b, respectively. The experimental chemical shifts are relatively well reproduced with deviations correlation coefficients higher than 96%. For ¹H-NMR chemical shifts, the variation between the experimental and estimated chemical shifts are in the ranges of 0.05–1.80 and 0.02–1.30 ppm, respectively. While for ¹³C-NMR chemical shifts, the variation between the experimental and estimated chemical shifts are in the ranges of 5.00–15.00 and 3.00–8.00 ppm, respectively.

Table 1.

Experimental and predicted ¹³C-NMR chemical shifts (ppm) in DMSO-d₆ of AT-1b and AT-2b.

AT-1b			AT-2b
δ_pred	δ_Exp	\|δ_Exp-Pred.\|	δ_pred	δ_Exp	\|δ_Exp-Pred.\|
175.68	168.32	7.36	172.56	168.25	4.31
161.85	156.06	5.79	155.73	150.50	5.23
149.91	143.93	5.98	154.41	149.73	4.68
146.97	134.24	12.73	149.45	144.21	5.24
139.95	131.92	8.03	147.14	141.70	5.44
137.64	126.31	11.33	140.95	134.07	6.88
131.16	123.42	7.74	137.42	134.02	3.40
131.12	123.19	7.93	137.40	132.73	4.67
129.47	114.39	15.08	135.12	132.03	3.09
68.01	62.15	5.86	131.04	127.75	3.29
55.95	47.94	8.01	130.14	125.73	4.41
43.63	35.07	8.56	128.26	123.84	4.42
43.30	34.1	9.20	128.26	123.33	4.93
40.48	31.68	8.80	127.03	123.31	3.72
39.22	29.46	9.76	75.70	69.93	5.77
-	-	-	55.00	49.09	5.91
-	-	-	42.93	47.98	5.05
-	-	-	42.77	35.42	7.35
-	-	-	42.30	35.15	7.15
-	-	-	40.95	33.99	6.96
-	-	-	39.77	33.84	5.93
-	-	-	37.55	31.92	5.63
-	-	-	34.72	31.70	3.02

δpred: DFT-predicted chemical shifts; δExp: experimental chemical shifts.

Table 2.

Experimental and predicted ¹H-NMR chemical shifts (ppm) in DMSO-d₆ of AT-1b and AT-2b.

AT-1b			AT-2b
δ_pred	δ_Exp	\|δ_Exp-Pred.\|	δ_pred	δ_Exp	\|δ_Exp-Pred.\|
7.52	7.57	0.05	8.13	8.15	0.02
7.46	7.34	0.12	8.10	7.80	0.30
7.30	7.35	0.05	7.83	7.72	0.11
7.26	8.21	0.95	7.67	7.48	0.19
6.60	8.40	1.80	7.42	7.03	0.39
4.95	3.94	1.01	5.53	6.81	1.28
4.78	3.94	0.84	4.92	5.25	0.33
3.89	3.68	0.21	4.40	4.54	0.14
2.71	3.68	0.97	4.26	4.11	0.15
2.26	2.34	0.08	3.18	3.33	0.15
-	-	-	2.65	2.34	0.31
-	-	-	1.76	1.30	0.46

δpred: DFT-predicted chemical shifts; δExp: experimental chemical shifts.

The phthalimide p-tert-butylcalix[4]arene derivative (AT-2b) and its analogue from p-tert-butyl phenol (AT-1b) were synthesized and characterized using ¹H NMR and ¹³C NMR. Optimized geometries and electrostatic potentials of both compounds were evaluated using DFT. DFT gives precise and trustworthy information about the compounds’ shape, rotational barrier, vibrational frequency, and electronic properties.⁷⁴ This was done using the B3LYP which is a key function for drug design because it can capture magnetic and electronic properties of drug molecules including electron affinities, ionization potentials, spin densities, and NMR chemical shifts.⁷⁵ The structure and bonding of AT-1b and AT-2b were geometrically optimized in the ground state. The optimized geometries of AT-1b and AT-2b are provided in Figure 7, and this was realized by moving the atoms in the molecules to get the most stable structure with the lowest possible ground state energy. MEP of organic compounds is a highly helpful tool for analyzing electronic density sites by analyzing electrophilic and nucleophilic reaction sites, and it can be computed using DFT calculations. In general, it offers crucial details on an organic molecule’s chemical stability and reactivity in order to comprehend its electrophilic and nucleophilic characteristics.⁷⁶ As indicated in Figure 7, nucleophilic and electrophilic sites of both AT-1b and AT-2b are represented. The electrostatic potential shows static distributions of charge on the molecules AT-1b and AT-2b. MEP is a useful tool for analyzing and predicting molecular reactivity, as it indicates sites or regions of a molecule where an approaching electrophile/nucleophile will be attracted to. It has also been successfully used to explain the three-dimensional orientation of molecules in a crystal. Nucleophilic potential sites are indicated in red color, while electrophilic potential sites are indicated in blue color. Nucleophilic regions are mainly presented around the N and O atoms, and the electrophilic potentials are around the H and C atoms. Red indicates the most negative region, blue indicates the most positive region, and green indicates a neutral region. The ability of the compounds to bind to charged systems and receptor sites in living organisms is made possible by these reactivity centers, confirming their bioactivities.⁷⁷ DFT calculations B3LYP can be used to obtain theoretical values of the ¹³C NMR and ¹H NMR chemical shifts with a high degree of accuracy compared to the experimental data.⁷⁸ Computer prediction of NMR chemical shifts plays an increasingly important role in molecular structure assignment and elucidation for organic molecule studies. The experimental and theoretical NMR shifts provided in Table 1 indicates proper agreement for both compounds AT-1b and AT-2b. This further confirms the successful synthesis and structural assignment of the compounds. The in silico prediction of NMR chemical shifts is helpful in organic structural assignments because spectra can be computed for molecular structures and then compared with experimental values to find the best possible match.⁷⁹ The overall results here are in agreement with previous studies which demonstrated the antivirulence efficacy of p-tert-butylcalix[4]arenes and their derivatives and their possible applications as novel antimicrobial agents.⁸⁰

Molecular docking results

The antimicrobial activity of AT-1b and AT-2b toward the targets S. aureus, E. coli, and C. albicans presented in terms of MIC is summarized in Figure 5. AT-1b is a structural subunit of AT-2b (Figure 1). Antimicrobial tests indicated that AT-2b has a significant antibacterial activity compared to AT-1b. To explain the observed antimicrobial tests, binding modes between AT-1b and AT-2b from one side and the active residues of S. aureus, E. coli, and C. albicans on the other side have been explored using molecular docking. Table 2 summarizes the free binding energies, the number of hydrogen bonds, and the number of interactions in the complexes formed between AT-1b and the binding site amino acids of S. aureus, E. coli, and C. albicans. Computationally, AT-2b does not bind to S. aureus, E. coli, and C. albicans, which may probably be due to its big molecular structure.

Figures 8 and 9 show the binding modes of AT-1b into the binding site of S. aureus, E. coli, and C. albicans. AT-1b is relatively well fit into the binding site S. aureus, E. coli, and C. albicans, and it forms stable complexes with their amino acids of negative bending energies of −8.91, −8.42, and −6.96 kcal mol⁻¹, respectively (Table 3). The negative binding energies are higher than −5.00 kcalmol⁻¹, which may indicate that the inhibition processes are thermodynamically favorable (Table 3). The binding modes of AT-1b differ in each binding site of S. aureus, E. coli, and C. albicans, which may explain the difference in its antibacterial activities. In AT-1b-E. coli complex, the keto and 1-tert-butyl-4-methoxybenzene moieties of AT-1b form three hydrogen binding with amino acids TYR A123, GLN A91, and HIS A465 at distances of 2.67, 2.94, and 3.28 Å, respectively (Figures 8 and 9). Also, a strong π-sulfur interaction forms between the π-bonds of the triazole ring of AT-1b and the methanthiol moiety of the amino acid MET A103 at a distance of 4.41 Å (Figures 8 and 9).

Figure 8.

2D binding of AT-1b into the binding site of Staphylococcus aureus, Escherichia coli, and Candida albicans.

Figure 9.

3D binding of AT-1b into the binding site of Staphylococcus aureus, Escherichia coli, and Candida albicans.

Table 3.

Free binding energies, hydrogen bonding, and number of closest residues to the docked AT1b into the binding sites of Staphylococcus aureus, Escherichia coli, and Candida albicans along with their corresponding MIC values.

Bacteria	Free binding energy (kcal/mol)	H-bonds (HBs)	Number of interactions of closest residues to the docked ligand in the bonding site	MIC (µg/mL)
S. aureus	-8.91	0	8	39.06
E. coli	-8.42	3	13	312.5
C. albicans	-6.96	1	11	19.53

Molecular docking was used to predict the interactions between the synthesized compounds and receptor proteins of the S. aureus, E. coli, and C. albicans to identify the most effective method of bonding to create stable complexes. Docking studies substantiated antimicrobial studies, as the compounds had negative free binding energies, an appreciable number of H-bondings, as well as several interactions of the closest residues to the docked ligand in the bonding site. Due to its large size, AT-2b could not fit into the receptor sites of the bacteria.

Conclusion

The present study focused on the synthesis of phthalimide-triazol-based p-tert-butyl calix[4]arene derivative and its p-tert-butylphenol analogue and the evaluation of their antimicrobial, antibiotilm, and QS inhibitions. DFT calculations were used to predict optimized geometries and MEPs as well as ¹H NMR and ¹³C NMR chemical shifts. The compounds (AT-1b and AT-2b) were synthesized and characterized using NMR data, and the experimental chemical shifts agreed with theoretical ones. The compounds showed interesting antimicrobial effects against S. aureus, E. coli, C. albicans, P. aeruginosa, and C. violaceum. Both compounds inhibited virulence factors such as violacein production, QS, biofilm formation, swarming, and swimming motilities. Molecular docking was used to provide insights about structure–activity relationship. AT-1b had negative binding energies with an appreciable number of interactions with the receptor proteins, showing that stable complexes were formed. The results in this study can be further exploited to provide information regarding the mechanism of action of the synthesized antimicrobial compounds against resistant pathogens. In vivo antimicrobial effects will be necessary in future works to determine the therapeutic doses of the compounds.

Supplemental Material

sj-docx-1-chl-10.1177_17475198251368414 – Supplemental material for Synthesis, DFT calculations, and molecular docking of phthalimide-triazole based p-tert-butylcalix[4]arene derivative and its analogue with antimicrobial, antiquorum-sensing, and antibiofilm properties

Supplemental material, sj-docx-1-chl-10.1177_17475198251368414 for Synthesis, DFT calculations, and molecular docking of phthalimide-triazole based p-tert-butylcalix[4]arene derivative and its analogue with antimicrobial, antiquorum-sensing, and antibiofilm properties by Alfred Ngenge Tamfu, Selahattin Bozkurt, Ozgur Ceylan and El Hassane Anouar in Journal of Chemical Research

Footnotes

The Scientific and Technological Research Institution of Turkey (TUBITAK) is greatly acknowledged. The authors are grateful to the participating institutions,which are Usak University,Mugla Sitki Kocman University,Prince Sattam bin Abdulaziz University,and the University of Ngaoundere.

ORCID iD

Alfred Ngenge Tamfu

Ethical considerations

Ethical approval is not applicable for the article.

Author contributions

A.N.T.: Conceptualization;Methodology;Investigation;Writing – original draft;Writing – review and editing;Formal analysis;Data curation;Software;Funding acquisition;Resources. S.B.: Conceptualization;Methodology;Investigation;Writing – original draft;Formal analysis;Funding acquisition;Resources;Project administration. O.C.: Methodology;Writing – review & editing;Visualization;Resources. E.H.A.: Methodology;Writing – review & editing;Formal analysis;Data curation;Software;Resources.

Funding

The author(s) disclosed receipt of the following financial support for the research,authorship,and/or publication of this article: This work was supported by The Scientific and Technological Research Council of Turkey (TUBITAK) under the project 1059B212200092.

Declaration of conflicting interests

The author(s) declared no potential conflicts of interest with respect to the research,authorship,and/or publication of this article.

Data availability statement

The data generated in this study are available within the article and its supplementary data files or upon request from the corresponding author.

Statement of informed consent

There are no human subjects in this article,and informed consent is not applicable.

Supplemental material

Supplemental material for this article is available online.

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Supplementary Material

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Synthesis,DFT calculations,and molecular docking of phthalimide-triazole based p-tert -butylcalix[4]arene derivative and its analogue with antimicrobial,antiquorum-sensing,and antibiofilm properties

Abstract

Keywords

Graphical abstract

Introduction

Experimental

Chemicals and reagents

Synthesis of compound 2

Synthesis of AT-1a and AT-2a

Synthesis of AT-1b and AT-2b

Strains of microbes

Measurement of antimicrobial activity

Biofilm inhibition assay

Quorum-sensing inhibition assay against C. violaceum CV026

Violacein inhibition assay against C. violaceum CV12472

Swimming and swarming inhibitions against P. aeruginosa PA01

Density functional theory calculations

Molecular docking details

Statistical analyses

Results and discussion

Inhibition of violacein production, QS, and motilities

Antimicrobial and antibiofilm activities

DFT calculation results

Molecular docking results

Conclusion

Supplemental Material

sj-docx-1-chl-10.1177_17475198251368414 – Supplemental material for Synthesis, DFT calculations, and molecular docking of phthalimide-triazole based p-tert-butylcalix[4]arene derivative and its analogue with antimicrobial, antiquorum-sensing, and antibiofilm properties

Footnotes

ORCID iD

Ethical considerations

Author contributions

Funding

Declaration of conflicting interests

Data availability statement

Statement of informed consent

Supplemental material

References

Supplementary Material