Design, Synthesis, and Biological Evaluation of Tetrahydropyridine Analogues as Potent Antibiofilm Agents against S. aureus: In Vitro and In Silico Studies

Abstract

An ecofriendly approach has been developed for the highly efficient atom-economical one-pot multicomponent synthesis of substituted tetrahydropyridine scaffolds as potent antibiofilm agents via using 1,2,4,5-Benzenetetracarboxylic acid (H₄BTCA) as a novel, mild organocatalyst. This is the first-ever report on the use of 1,2,4,5-Benzenetetracarboxylic acid (H₄BTCA) as a catalyst in organic synthesis. This protocol’s appealing features include column chromatography-free purification, resulting in high-purity products and compatibility with a broad range of substrates. Moreover, this method afforded good results to excellent yields (i.e., 86–93%) of compounds (4a–4r). The biological utility of the tetrahydropyridine derivatives as antibiofilm agents has been demonstrated. The synthesized compounds were screened against Staphylococcus aureus and P. aeruginosa with in vitro and in silico approaches. It is noteworthy to mention that some of the tetrahydropyridine derivatives exhibited potent antibiofilm activity with minimum or no toxicity in the in vitro study. On the basis of antibiofilm activity, 3 compounds (4k, 4l, and 4n) were formulated into nanoparticles (4k NPs, 4l NPs, 4n NPs) have shown appreciable antibiofilm activity with a maximum disruption, 87.74 ± 2.26%, shown by 4l NPs and toxicity further reduced below 2% when the compound was loaded into nanoparticles. The docking studies of the active 3 compounds (4k, 4l, and 4n) have been tested against different biofilm target proteins like sarA, crtM, fnbA, PBP2, clfA, clfB, AgrA, and AgrC associated with the S. aureus biofilm. Our in silico study results demonstrate the high affinity of the molecules with lower (more negative) docking scores ranging from ∼−10 to −6 kcal/mol. The highest affinities were observed with 4k and 4b against the 1n67 protein (clumping factor protein) compared to other studied antibiofilm-related proteins.

1. Introduction

Biofilms are complex communities of bacteria that attach to surfaces and form a protective extracellular matrix. These formations can develop on a wide range of surfaces, including medical devices, natural tissues, and industrial equipment. They present significant obstacles in healthcare and industry due to their resistance to traditional therapies. , Combating biofilm-related issues are challenging since the bacteria inside a biofilms are highly resistant to traditional treatment. This resistance is caused by several reasons, including the protective matrix that shelters the microorganisms and the changes microenvironment within the biofilm, which can reduce the efficiency of treatment. Therefore, new broad-spectrum antibiofilm drugs/agents are required to treat the cases of antibiotic resistance due to biofilm formation.

Certain substances have shown the potential to enter and disrupt existing biofilms, decreasing their biomass and survival. Tetrahydropyridine derivatives are a prominent pharmacophore that exhibit substantial antibacterial action. Many studies have found that antibiotics having a piperidine subunit are efficient against Gram-positive bacteria like Staphylococcus aureus and Streptococcus pneumonia. − Tetrahydropyridine derivatives can disrupt bacterial early adherence to surfaces, which is necessary for biofilm formation. They may interfere with quorum sensing, which bacteria use to coordinate biofilm formation.

Tetrahydropyridine is an important N-heterocycle integral to numerous synthetic bioactive compounds, drug candidates, and some natural products. , Some representative drugs and natural products of tetrahydropyridine-containing compounds are depicted in Figure .

1.

Tetrahydropyridine core containing natural products and drugs.

Various interesting pharmacological and biological properties of tetrahydropyridine derivatives, such as antimalarial, anti-HIV agents, antimicrobial, analgesic, anti-inflammatory, hyperglycaemic, neurotoxic activity, HIV protease inhibitor as well as inhibitors of farnesyl transferase have been extensively investigated. The compounds bearing tetrahydropyridine frameworks have been reported in the literature for the treatment of cognitive dysfunctional diseases such as Alzheimer’s, schizophrenia, and migraine headaches. Considering their broad spectrum of medicinal, pharmacological, and biological activities, a plethora of methods have been reported in the literature for the synthesis of tetrahydropyridine. ^a–c

The most frequent approach for synthesizing tetrahydropyridine derivatives is a one-pot multicomponent reaction (MCR) of two molecules of aromatic aldehydes, two molecules of anilines, and one molecule of β-keto-esters, catalyzed by a variety of catalysts such as Bronsted acids, , metal catalysts, ,, supported catalysts, , and ionic liquids. , Many of the approaches mentioned above are restricted by inherent limitations, including harsh reaction conditions, side product generation, need for excess reagent, low or moderate yields, using hazardous or costly chemicals and solvents, prolonged reaction times, and laborious workup procedures. Hence, investigations are still required to develop more efficient and mild procedures to overcome these issues.

We were interested in developing mild, atom-economical, and metal-free protocols for the preparation of tetrahydropyridine analogues as antibiofilm agents. We present a 1,2,4,5-Benzenetetracarboxylic acid (H₄BTCA)-catalyzed simple and efficient one-pot synthesis via the direct condensation of a varieties of anilines with aryl aldehydes and β-keto esters at room temperature (Scheme ). 1,2,4,5-Benzenetetracarboxylic acids as novel organocatalysts have many advantages, including being readily available, having ease of handling, greater stability, economic viability, environmental friendliness, nontoxic, and being stable in air and moisture. They may be helpful to the pharmaceutical industry as organocatalysts because they prevent the production of metallic waste and metal traces in the reactions.

1. 1,2,4,5-Benzenetetracarboxylic Acid (H₄BTCA)-Catalyzed Synthesis of Tetrahydropyridine Derivatives as Antibiofilm Agents.

To the best of our knowledge, the application of 1,2,4,5-Benzenetetracarboxylic acid (H₄BTCA) as a catalyst in organic synthesis has not been reported earlier. So far, the applications of 1,2,4,5-Benzenetetracarboxylic acid included (a) formation of high-dimensional lanthanide-based coordination polymers, supramolecular architectures with several transition metals ions, (b) construction of two-dimensional metal–organic networks with chelate ligands, (c) engagement in crystal engineering for supramolecular assemblies, (d) functionalization of Fe₃O₄ nanoparticles for selective and recyclable Congo Red dye adsorption, and (e) construction of photoluminescent lanthanide-bta-flexible metal–organic frameworks.

In the present study, we report the first-time use of 1,2,4,5-Benzenetetracarboxylic acid (H₄BTCA) as an organocatalyst for the synthesis of tetrahydropyridine derivatives and the evaluation of the antibiofilm potential of tetrahydropyridine-loaded nanoparticles. Molecular docking studies of the antibiofilm active compounds with different proteins involved in the biofilm formation were also performed to understand the mode of action of the drug to disrupt S. aureus and P. aeruginosa biofilms.

2. Results and Discussion

Herein, we describe a simple, efficient, and original protocol for the one-pot synthesis of highly substituted tetrahydropyridine derivatives using 1,2,4,5-Benzenetetracarboxylic acid (H₄BTCA) as a novel organocatalyst via a multicomponent coupling reaction involving a variety of substituted aromatic aldehydes, β-keto esters, and substituted anilines at room temperature in good to excellent yields (Scheme ).

For the reaction optimization, we initiated our studies by selecting benzaldehyde, aniline, and methyl-acetoacetate as the model substrates in an ethanol solvent with various amounts of catalyst (H₄BTCA) loadings at room temperature (Table , entry 1–4). It was noticed that 15 mol % H₄BTCA was the optimum amount of catalyst needed, and lowering the amount of H₄BTCA had a negative effect on the yield and reaction time of this reaction (Table , entries 1–3). Further, raising the amount of catalyst did not result in any noteworthy time or yield improvement (Table , entry 4). Next, we examined the influence of various solvent systems such as MeOH, acetone, CH₃CN, THF, H₂O, DCM, DMF, t-BuOH, Dioxane, EtOH–H₂O under similar reaction conditions and found that none of them were giving satisfactory results in terms of time and yield (Table , entries 5–14). A control experiment conducted in the absence of catalyst H₄BTCA provided a trace amount of conversion even after prolonged reaction time (Table , entries 15). This result illustrates that the catalyst 1,2,4,5-Benzenetetracarboxylic acid (H₄BTCA) is essential for this reaction. Furthermore, we have tested various catalytic systems like benzoic acid, phthalic acid (1,2-benzenedicarboxylic acid), isophthalic acid (1,3-benzenedicarboxylic acid), and trimesic acid (1,3,5-benzenetricarboxylic acid) under similar reaction conditions (Table , entries 16–19). However, in all cases, the reactions afforded poor yields compared to the 1,2,4,5-benzenetetracarboxylic acid (H₄BTCA) catalyst and required longer reaction times to reach completion. These observations clearly suggest that 15 mol % of H₄BTCA catalyst with ethanol solvent at room temperature is the best optimal condition for carrying out the current protocol in terms of yield and reaction time (Table , entry 3).

1. Optimization of the Reaction Conditions for Synthesizing Tetrahydropyridine Derivatives (4a)­ .

S.No.	Catalyst (mol %)	Solvent	Time (h)	Yield (%)
1	H4BTCA (5)	EtOH	24	31
2	H4BTCA (10)	EtOH	24	71
3	H4BTCA (15)	EtOH	8	91
4	H4BTCA (20)	EtOH	8	92
5	H4BTCA (15)	MeOH	12	88
6	H4BTCA (15)	Acetone	24	22
7	H4BTCA (15)	CH3CN	20	45
8	H4BTCA (15)	THF	24	35
9	H4BTCA (15)	H2O	24	trace
10	H4BTCA (15)	DCM	20	42
11	H4BTCA (15)	DMF	20	37
12	H4BTCA (15)	t-BuOH	24	18
13	H4BTCA (15)	dioxane	24	25
14	H4BTCA (15)	EtOH/H2O	24	30
15	_	EtOH	38	trace
16	benzoic acid (15)	EtOH	24	20
17	phthalic acid (15)	EtOH	24	55
18	isophthalic acid (15)	EtOH	24	48
19	trimesic acid (15)	EtOH	24	60

^aReaction conditions: benzaldehyde (0.5 mmol), aniline (0.5 mmol), methyl-acetoacetate (0.25 mmol), room temperature.

^bIsolated yield after purification by recrystallization.

With the optimized reaction conditions in hand (Table , entry 3), we set out to examine the substrate scope of the developed synthetic strategy by reacting differently substituted aryl aldehydes and a variety of anilines with β-ketoesters to synthesize various highly substituted tetrahydropyridine derivatives 4a–4r in good to excellent yields (86–93%) (Scheme ). Irrespective of the electronic nature, the present strategy can be applied effectively to a diverse array of aldehydes and aniline derivatives having electroneutral, electron-withdrawing and electron-donating nature of aryl groups (Scheme , 4a–4r). The substituent effect did not influence the yields of the tetrahydropyridines to any appreciable level. Polycyclic and heterocyclic aldehydes are tolerated well under the same reaction conditions, and the corresponding tetrahydropyridine derivatives were obtained in good yields (86–89%) (Scheme , 4o–4r). In contrast, aliphatic aldehydes such as propionaldehyde and isovaleraldehyde did not produce the desired products. It was found that aromatic amines were effective substrates that produced good to excellent yields of the corresponding tetrahydropyridine derivatives (Scheme , 4a–4r). However, aliphatic amines did not afford any detectable products under the optimized conditions. Also, in our investigations, the reaction did not proceed efficiently when acetylacetones were employed as the substrate. Importantly, our catalyst also effectively synthesizes new, unreported tetrahydropyridine derivatives (Scheme , 4r). The current protocol’s scalability (10 mmol scales) has been successfully investigated without any considerable drop in the product yield. This revealed the excellent practical utility of the present protocol. The products were characterized by ¹H NMR and ¹³C NMR spectroscopic techniques.

2. Substrate Scope for the Synthesis of Various Tetrahydropyridine Derivatives 4 Using 1,2,4,5-Benzenetetracarboxylic Acid (H₄BTCA) as an Organocatalyst .

^a Reaction condition: aromatic aldehydes (1.0 mmol), anilines (1.0 mmol), β-Keto-esters (0.5 mmol), H₄BTCA (15 mol %) in 5 mL EtOH at room temperature.

In this study, no chiral catalyst was employed; therefore, the catalytic products were obtained without defined absolute stereochemistry. However, the relative stereochemical orientation of the substituents at positions 2 and 6 of the tetrahydropyridine ring was elucidated through detailed ¹H NMR investigations, including NOESY analysis. The absence of any observable NOESY correlation between protons H₂ and H₆ clearly indicates that these protons occupy trans positions (see Figure S39 in the Supporting Information at page 32). −

2.1. Mechanism for the Synthesis of Functionalized Tetrahydropyridine

The plausible mechanism for the 1,2,4,5-Benzenetetracarboxylic acid (H₄BTCA)-catalyzed synthesis of tetrahydropyridine derivatives is depicted in Scheme .

3. Plausible Reaction Mechanism for 1,2,4,5-Benzenetetracarboxylic Acid (H₄BTCA)-Catalyzed Synthesis of Tetrahydropyridines.

A catalytic amount of H₄BTCA initiates the reaction through proton donation to form enamine (E) and imine (F) from the condensation of aryl anilines (A), β-ketoester (B) and aryl aldehyde (C). Then, an intermolecular Mannich-type reaction occurs between enamine (E) and imine (F) to yield the intermediate (G). The intermediate (G) undergoes a condensation reaction with a second molecule of aromatic aldehyde, giving the intermediate (H). The intermediate (H) after tautomerization generates intermediate (I) followed by an intramolecular Mannich-type reaction to produce the desired substituted tetrahydropyridine derivatives (D).

2.2. Screening of Synthesized Compounds

After synthesis, screening of 18 synthesized compounds 4a-4r was done on an S. aureus biofilm. In brief, 100 μg/mL of treatment was given to the 48 h-grown biofilm of compounds 4a-4r for 24 h. The result was recorded in the form of absorbance (Figure ). It was found that compounds 4k, 4l, and 4n have shown better disruption than other compounds.

2.

Screening of synthesized compounds (100 μg/mL) on an S. aureus biofilm. The values are shown here as the means ± standard deviation (n = 3).

2.3. Size, Polydispersity Index, and Zeta Potential

Zetasizer was used to examine the size, zeta potential, and polydispersity index of NPs loaded with chemicals (Table ). It was found that NPs formulations ranged in size from 159.4 ± 1.24 to 315.7 ± 2.35 nm. The size of the NPs increased after the chemicals were loaded (4k, 4l, and 4n) with the size of blank NPs having a minimum size of 159.43 ± 1.24 nm. The homogeneity of the NPs produced by this method was confirmed by the PDI value of the NPs, which was in the 0.2–0.313 range. The nanoparticle exhibited a negative zeta potential. The particles negative charge increased when the compounds were loaded.

2. Size, Polydispersity Index, and Zeta Potential of Nanoparticles .

Groups	Size (nm)	PDI	Zeta potential (mV)
Blank NPs	159.4 ± 1.24	0.313	–7.66 ± 1.7
4k NPs	291.4 ± 4.06	0.26	–18.9 ± 0.7
4l NPs	287.13 ± 3.69	0.2	–11.3 ± 0.95
4n NPs	315.7 ± 2.35	0.241	–22.6 ± 0.264

^aThe values (size and zeta potential) are shown here as the means ± standard deviation (n = 3).

2.4. Cell Cytotoxicity Assay

A resazurin assay was used to determine the compatibility of the compound and compound loaded SLNs in vitro (Figure ). We accessed the cytotoxicity of the compound and compound-loaded nanoparticles by exposing AGS cell lines to the compound, blank nanoparticles, or equivalent amount of compounds (100 μg/mL)-loaded nanoparticles for 24 or 48 h. Free blank SLNs, compounds, and compound-loaded SLNs show cell viabilities of 84.9 ± 2.57%, 91.95 ± 2.45% to 94.48 ± 3.59%, and 81.05 ± 3.01% to 94.10 ± 4.85%, respectively.

3.

Cell cytotoxicity induced after exposure to the compound or compound-loaded nanoparticles (100 μg/mL) after treatment for 24 h. (hollow bar) and 48 h. (cross bar) in the AGS cell line. The values are shown here as the means ± standard deviation (n = 3).

In This study, it was found that compound and compound-loaded SLN formulations show very low cytotoxicity after exposure to cells.

2.5. Hemolysis Assay

A hemolysis assay was performed to evaluate the compatibility of the novel compound and equal amounts of compound-loaded nanoparticles with the RBC cells. The compatibility of compound and nanoparticles was studied in terms of cellular viability, blood cell compatibility, and plasma membrane integrity of the RBC cells. Hemocompatibility is a crucial aspect in determining the biological safety of a compound and its nanoparticle formulation. In the study, compound-loaded nanoparticles were treated in two concentrations (200 or 400 μg/mL) and showed very little or no cytotoxicity in the hemolysis assay (Figure ). In this study it was also observed that compound encapsulated into nanoparticles further reduced the toxicity of the compound. This is a promising indication of their potential application in drug delivery without affecting RBCs. This research represents an important first step in evaluating the biological safety of a compound and its nanoparticle formulation and may pave the way for further studies and potential clinical applications.

4.

RBCs lysis after exposure to a compound (200 and 400 μg/mL) (hollow bar) and equal amount of compound-loaded nanoparticles (cross bar) by a hemolysis Assay (***p value < 0.001). The values are shown here as the means ± standard deviation (n = 3).

2.6. Evaluation of Minimum Biofilm Eradication Concentration of Compounds 4k, 4l, and 4n

The MBEC study of compounds 4k, 4l, and 4n was performed on S. aureus and P. aeruginosa bacteria at different concentrations (100–5 μg/mL). These compounds did not show antimicrobial activity; therefore, 100% eradication of biofilm was not possible. In S. aureus and P. aeruginosa, compounds have shown dose-dependent eradication of biofilms. In the S. aureus study, a maximum of 75.08 ± 0.84% eradication was performed by the 4n compound, followed by 4l (69.69 ± 4.23%). Minimum eradication was shown by the 4k compound (68.85 ± 3.75%) (Figure a).

5.

[a] Minimum biofilm eradication concentration of compounds on an S. aureus biofilm: 4k (blue bar), 4l (red bar), and 4n (green bar). [b] Minimum biofilm eradication concentration of compounds on P. aeruginosa biofilm: 4k (blue bar), 4l (red bar), and 4n (green bar). The values are shown here as the means ± standard deviation (n = 3).

In the P. aeruginosa study, a maximum eradication of 85.99 ± 3.16% was achieved by the 4l compound followed by 4k (72.70 ± 8.48). Minimum eradication was done by 4n (58.65 ± 12.17), as shown in Figure b.

2.7. Quantitative Analysis of S. aureus and P. aeruginosa Biofilms’ Disruption after Exposure to NPs

In this study, we used 48 h-grown biofilms for treatments with different concentrations (5 μg/mL), and (15 μg/mL) of NPs for 24 h. It was found that S. aureus biofilm disruption was dose-dependent. 5 μg/mL had minimum disruption, and 15 μg/mL had maximum disruption. DMSO had less disruption. Maximum disruption was shown in 4n NPs (15 μg/mL), whereas the minimum disruption was shown by 4k NPs (5 μg/mL). In all concentrations, the maximum disruption was reported in 15 μg/mL of NPs. The percentage S. aureus disruption is shown in Table .

3. Percentage Disruption of an S. aureus Biofilm with NPs .

Groups	5 μg/mL (% disruption) ± SD	15 μg/mL (% disruption) ± SD
Control	0 ± 0	0 ± 0
DMSO	22.47 ± 10.38	22.47 ± 10.38
Rifampicin	91.51 ± 2	91.51 ± 2
4k NPs	50.23 ± 4.53	80.24 ± 1.39
4l NPs	81.89 ± 5.87	87.74 ± 2.26
4n NPs	80.70 ± 2.61	87.29 ± 2.46

^aThe values are shown here as the means ± standard deviation (n = 3).

^b p value <0.001.

^c p value <0.01, and.

^d p value <0.05. (rifampicin vs compounds).

In P. aeruginosa biofilm disruption, maximum disruption was shown by 4n NPs (15 μg/mL) (Figure ). The percentage P. aeruginosa disruption is shown in Table .

6.

Quantification of S. aureus (blue bar) and P. aeruginosa (red bar) biofilm disruption in the form of absorbance; ***p value < 0.001 **p value < 0.01, and *p value < 0.05. Data were recorded in triplicate and expressed as mean ± SD. Data were analyzed with MS Office Excel by an unpaired two tailed t-test.

4. Percentage Disruption of a P. aeruginosa Biofilm with NPs ^{, , ,}

Groups	5 μg/mL (% disruption) ± SD	15 μg/mL (% disruption) ± SD
Control	0 ± 0	0 ± 0
DMSO	8.45 ± 2.65	8.45 ± 2.65
Rifampicin	78.39 ± 1.63	78.39 ± 1.63
4k NPs	75.08 ± 4.3	74.40 ± 3.22
4l NPs	73.19 ± 2.53	64 ± 0.84
4n NPs	71.66 ± 3.56	84.63 ± 3.57

^aThe values are shown here as the means ± standard deviation (n = 3).

^b p value <0.001.

^c p value <0.01, and.

^d p value <0.05. (rifampicin vs compounds).

2.8. Scanning Electron Microscopy of Biofilm after Treatment with NP Formulations

A scanning electron microscopy (SEM) study was performed to evaluate the qualitative study of biofilm disruption (Figure ). In the control, where no treatment was given, a fully mature biofilm was observed (Figure A). The DMSO treatment group showed less disruption (22.47 ± 10.38%) compared to other formulations (Figure B). 4k and 4n NP formulations have shown 80.24 ± 1.39% and 87.74 ± 2.26% (Figure C,D) disruption, respectively. The 4l NPs-treated group showed the maximum result with 87.74 ± 2.26% disruption (Figure E).

7.

SEM study of biofilm disruption after treatments with different NPs (15 μg/mL) (A) control, (B) DMSO, (C) 4k NPs (D) 4n NPs, (E) 4l NPs.

2.9. Docking Study

As the binding sites of all eight considered target proteins are uncertain, we considered the entire protein surface as the search space to locate potential ligand-binding pockets and thus perform blind docking. Blind docking also creates the possibilities of identifying novel binding sites fairly. Since all eight proteins have different structures, grid box (search space) parameters were calculated for each target proteins through ADT (auto dock tool) independently (Table ). For all the proteins, we used constant exhaustiveness value, i.e., 16.

5. Considered Search Space Parameter for All Eight Target Proteins.

PDB_ID	center_X	center_Y	center_Z	size_X	size_Y	size_Z
1p4x	–30.13	16.067	–10.293	62.835	56.067	80.794
2zco	55.491	12.678	51.894	64.899	63.832	68.936
2rky	7.747	19.284	6.462	30.118	17.471	63.486
5m18	8.629	–12.583	–56.845	61.006	29.138	86.94
3bs1	–3.671	27.87	16.177	35.921	48.803	27.679
1n67	22.828	53.982	78.754	15.616	62.886	17.368
3at0	–18.724	16.391	–9.186	86.872	60.407	56.191
6E52	34.215	25.631	24.355	86.8	63.459	122.993

To evaluate the therapeutic potential of the synthesized compounds, targets were screened using molecular docking. Affinity analysis between compounds and proteins was based on the Gibbs free energy, which was used to assess the affinity. Literature shows that molecular complexes with Gibbs free energies of less than 6 kcal/mol significantly affect stability. , Docking results revealed all six compounds had substantial affinities with target proteins. Affinities ranged from −6 to –10 kcal/mol, with the highest between 4k and 4b with 1n67, a Clumping Factor protein (ClfA). Ligand 4b showed good affinity for nearly all targets but this result was not complemented by the in vitro study. Protein 2rky was the only protein that did not show an affinity below −7 kcal/mol with any compound (Table ). It lies at the lower margin of stability of complexes; therefore, its druggability is not significant.

6. Binding Affinity (kcal/mol) of the Considered Ligand with Potential Therapeutic Targets.

S.No.	ligands	target proteins
		1p4x	2zco	2rky	5m18	3bs1	1n67	3at0	6E52
1	4a	–7.6	–7.6	–6.5	–8	–7.9	–9.5	–7.2	–7.2
2	4b	–8.1	–7.5	–6.6	–8.3	–8.3	–10.1	–8.2	–7.5
3	4c	–7.1	–7.7	–6.6	–8.1	–8.2	–9.9	–8	–7.8
4	4k	–7	–7.8	–6.3	–7.6	–8.2	–10	–7.4	–7.5
5	4l	–7.1	–7.8	–6.5	–8.7	–8.5	–9.2	–7.7	–7.4
6	4n	–7.7	–8.1	–6.3	–7.5	–8.2	–9.5	–7.2	–7.6

Interaction forces and Gibbs free energy determine ligand binding specificity; their presence defines complex stability and specificity. These forces must be considered with affinity score when selecting a reliable complex. Two-dimensional (2D) and three-dimensional (3D) visualizations of the best-docked complex for each target protein were performed to observe interactions and forces in complex formation and define affinity (Figure ). The interaction visualization shows six major forces: (i) hydrogen bond, (ii) Pi-Sigma, (iii) alkyl, (iv) Carbon Hydrogen Bond, (v) amide–Pi stacked, and (vi) i-anion, which establish complex stability. All five types of interaction contribute to the formation of protein ligand complexes, but with respect to the specificity, hydrogen bonds provide directional contacts, which help ligands to recognize and fit properly in binding pockets. Hydrogen bonds determine interaction affinity and specificity; they influence enzymatic activity and ligand selectivity. Hydrogen bonds between a ligand and a protein can also significantly affect the protein’s activity by modulating binding affinity, specificity, and the structural environment of the active site. Since drug–target protein interactions are specific, hydrogen bonds are decisive for compound selection against target proteins. In Figure , the green residue and lines indicate hydrogen bonds. Among eight complexes, only five complexes, 1n67-4b, 1p4x-4b, 2zco-4n, 3bs1-4l, and 5m18-4l, show hydrogen bonds, qualifying them for further study. As the six compounds are similar, they share analogous features. Affinity is on the factors mainly responsible for selective binding, it does not always directly correlate with efficacy (biological activity) of the compounds. Drug’s pharmacokinetics is another factor which helps to determine drug-likeness of the compounds in terms of ADME. Lipinski’s rule of five assessments showed these pharmacokinetic features: (i) mass = 312.000000 Da, (ii) hydrogen bond donor (HBD) = 5, (iii) hydrogen bond acceptors (HBA) = 6, (iv) LOGP = −0.053101, and (v) molar reactivity = 77.145782. All features fulfill Thumb’s rule of five for drug-likeness of oral drugs, supporting their potential as oral drug compounds.

8.

2D and 3D visualization of the highly interacted complex (complex with ←8.00 kcal/mol affinity).

3. Conclusions

In brief, we have established a simple, highly atom-economical, and straightforward method for the synthesis of tetrahydropyridine derivatives as antibiofilm agents, using 1,2,4,5-Benzenetetracarboxylic acid (H₄BTCA) as an effective organocatalyst via a one-pot multicomponent reaction of easily accessible aryl aldehydes, anilines, and β-ketoesters. The attractive features of this procedure are the mild reaction conditions, high atom efficiency, clean reaction profiles, inexpensive starting materials, easy isolation, and purification of the product. Notably, this is the first report in which 1,2,4,5-benzenetetracarboxylic acid (H₄BTCA) has been used as a catalyst for organic synthesis.

The use of H₄BTCA as a metal-free catalyst provides a sustainable alternative by eliminating the reliance on toxic and expensive metals, thereby reducing both environmental impact and purification requirements. In combination with green solvents such as ethanol, it further minimizes the use of hazardous solvents, making the process eco-friendlier. Owing to its strong acidity, H₄BTCA enhances the reaction efficiency, suppresses side-product formation, and improves yields under mild conditions (e.g., room temperature). Collectively, these features establish it as a greener, cost-effective, and scalable option compared with conventional metal-catalyzed methods.

Further, several synthesized compounds exhibit good antibiofilm activity against S. aureus biofilms. Among these, the most active compounds, 4k, 4l, and 4n, featured methyl (-Me) substituents on the aromatic aldehydes. In this study, tetrahydropyridine derivatives 4k-, 4l-, and 4n-loaded NPs were successfully formulated. Further, S. aureus and P. aeruginosa biofilm disruption studies have shown excellent results. NP formulations were found nontoxic for human blood cells and showed less cytotoxicity on AGS cells. Additionally, docking studies were performed to rationalize the interaction of antibiofilm active compounds with proteins. These studies revealed hydrogen bond interactions in the complexes, i.e., 1n67-4b, 1p4x-4b, 2zco-4n, 3bs1-4l, and 5m18-4l, highlighting the involvement of hydrogen bonds. In the docking study, 4k, 4l, 4n, and 4b compound results showed a very potent drug-like molecule property but similar results were not obtained for compound 4b, which is the limitation of the study. These findings suggest that the compounds 4k, 4l, and 4n can be considered for further studies on different bacterial biofilms of ESKAPE pathogen to assess whether the antibiofilm properties of these compounds are bacterial strain-specific or can disrupt all bacterial biofilms. Biofilms are complex in nature, and the interactions between 4k-, 4l-, and 4n-loaded solid lipid nanoparticles (SLNs) and biofilms are still an area of further investigation, so we will focus on further interaction of compound nanoparticles with different bacterial biofilms. Nevertheless, at this initial stage, these compounds have shown a ray of hope toward the eradication of biofilms.

4. Materials and Methods

4.1. Materials

All chemicals were of reagent grade and purchased from Aldrich, Merck, Spectrochem, and Loba and were used without further purification. The reactions were monitored using precoated aluminum TLC plates of silica gel G/UV-254 of 0.25 mm thickness. IR spectra were recorded in KBr on a PerkinElmer FT-IR RXI spectrophotometer. NMR spectra were recorded with Bruker 400 and 500 MHz spectrometers for (¹H) and 100 and 125 MHz­(¹³C) in CDCl₃ using TMS as an internal reference with the chemical shift value being reported in ppm. All coupling constants (J) were reported in Hertz (Hz). HR mass spectra were recorded on a LCMS Q-TOF micro mass spectrometer. Melting points were determined by an open glass capillary method.

The following materials and instruments were used: S. aureus, Luria–Bertani (LB) broth (HiMedia), Luria–Bertani agar (LA) (HiMedia), crystal violet dye (Rankem), NaCl (HiMedia), KCl (HiMedia), Na₂HPO₄ (HiMedia), KH₂PO₄ (HiMedia), Glacial acetic acid (Rankem), Ethanol (Molychem), Methanol (SRL), Aqueous (Milli-Q), Ethyl acetate (Thermo-fisher), glutaraldehyde solution (Sigma-Aldrich), DMSO (Loba chemie AR), cacodylate buffer (Sigma-Aldrich), synergy multiplate reader (HTX, BioTek Instruments, USA), round coverslips (Blue Star), culture tubes (Borosil), 96-well culture plate (Tarsons, India), fluorescence microscope (CK40, Olympus, Japan), orbital shaker incubator (Murhopye Science Ltd., India), SEM (NovaNanosem-450, Thermo-Fisher), phase-contrast inverted microscope (Olympus, Japan, CK-41), Soxhlet apparatus (BEZIF-1000 mL), rotatory evaporator (IKA*RV10 Digital, IKA*HB 10 Digital).

4.2. Procedure for the Synthesis of Functionalized Tetrahydropyridine

A solution of aromatic aldehydes (1.0 mmol), aromatic amines (1.0 mmol), β-ketoesters (0.5 mmol), and 1,2,4,5-Benzenetetracarboxylic acid (H₄BTCA) (15 mol %) as catalyst in 5 mL of EtOH was stirred at room temperature until the completion of the reaction. After completion of the reaction as indicated by TLC, the thick precipitate that had formed was filtered off and washed with water, followed by ice-cold ethanol (3 × 2 mL), and subsequently dried to afford the pure product. The pure product was characterized by spectroscopic methods.

4.3. Screening of Synthesized Compounds

For the screening of 18 synthesized compounds, we used a 48 h-grown biofilm in a 96 microtiter plate. Treatment was given for 24 h. After 24 h, nonadherent bacterial cells were removed by careful washing with 1X PBS followed by 70% isopropanol for fixing. After that staining was done by 0.1% crystal violet followed by addition of 30% glacial acetic acid. The biofilm was quantified by a multimode reader at 595 nm.

4.4. Preparation of Solid-Lipid Nanoparticle Formulations

The solid lipid nanoparticles (SLNs) was prepaired by double emulsion and solvent evaporation method. In brief, dichloromethane (DCM) was used to dissolve 50 mg of lipid Witepsol. 500 mL of distilled water was added, and the mixture was then sonicated for 4 min to create blank Witepsol nanoparticles. This primary emulsion (W1/O) was put dropwise into 1% Tween-80 and homogenized for 15 min at 12500 rpm to create a secondary emulsion. For the purpose of allowing DCM to evaporate, the secondary emulsion was left on a magnetic stirrer set at 680 rpm for 8 to 12 h. Centrifugation was used to separate the nanoparticles for 1 h at 4 °C and 24,000 rpm. 1 mg of each chemical was added with 500 mL of distilled water to create the compound 4k-, 4l-, and 4n-loaded Witepsol nanoparticles. The remaining steps were the same as those of the blank NPs’ procedure. The supernatant was decanted, and the pellet was redispersed in distilled water, followed by addition of mannitol as a cryoprotectant and kept into −20 °C. After freezing, nanoparticle formulations were lyophilized.

4.5. Size, Polydispersity Index, and Zeta Potential

The size distribution, polydispersity index, and zeta potential of solid lipid nanoparticles was dtermined by Zetasizer (Nano-ZS90 Malvern, UK). In brief, 1 mL of distilled water was mixed with 1 mg of lyophilized NPs. Aggregates of NPs were sonicated for 1 min and then spun for 2 min. Single-use, disposable polystyrene cuvettes were used to transfer the nanoparticles for examination. All nanoparticle compositions’ sizes and surface charges were evaluated using the laser diffraction method in triplicate. ,

4.6. Cell Cytotoxicity Assay

The cell cytotoxicity of drug-loaded SLNs in different formulations or free drugs was studied in the AGS cell lines. Cytotoxicity was measured using a resazurin dye assay. One × 10⁴ cells per well was plated in a 96-well cell culture plate, allowing cells to reach up to 80% confluency. Cells were treated with 100 μg/mL of free compounds or an equivalent compound in the SLN formulations and incubated for 48 h. At the end of the incubation time, 10 μL of resazurin dye was added to each well and incubated for 6 h. Cell viability was determined by recording the fluorescence intensity of each well (excitation, 530 nm; emission, 590 nm). Untreated cells were used as controls. The percentage of viable cells was calculated using the following formula

$$\mathrm{C}\mathrm{e}\mathrm{l}\mathrm{l}\mathrm{v}\mathrm{i}\mathrm{a}\mathrm{b}\mathrm{i}\mathrm{l}\mathrm{i}\mathrm{t}\mathrm{y}(\%)=\frac{\mathrm{t}\mathrm{r}\mathrm{e}\mathrm{a}\mathrm{t}\mathrm{e}\mathrm{d}\mathrm{c}\mathrm{e}\mathrm{l}\mathrm{l}\mathrm{s}}{\mathrm{u}\mathrm{n}\mathrm{t}\mathrm{r}\mathrm{e}\mathrm{a}\mathrm{t}\mathrm{e}\mathrm{d}\mathrm{c}\mathrm{e}\mathrm{l}\mathrm{l}\mathrm{s}}\times 100$$

4.7. Hemolysis Assay

The compatibility of the antibiofilm agent loaded SLNs with human blood cells was evaluated by hemolysis assay. In brief, the human blood cells (RBCs) were isolated by centrifugation at 4000g at 4 °C for 10 min. RBCs were washed with 1X PBS and resuspended into the PBS to obtain a final 1 × 10⁸ erythrocyte/mL. This suspension was treated with equal volumes of SLNs at different concentrations (200 and 400 μg/mL) and incubated in a rotary shaker incubator for 1 h at 37 °C. After 1 h, the treated samples were subsequently centrifuged at 900g for 10 min at 4 °C, and the OD of the supernatants was measured at 540 nm to determine whether RBC lysis took place. 2% Tween-80 was taken as the positive control, where 100% RBCs were lysed, and 1× PBS buffer was used as the negative control, where no RBC lysis was found. The percentage of hemolysis was calculated using the following formula

$$\%\mathrm{o}\mathrm{f}\mathrm{h}\mathrm{e}\mathrm{m}\mathrm{o}\mathrm{l}\mathrm{y}\mathrm{s}\mathrm{i}\mathrm{s}\mathrm{a}\mathrm{t}540\mathrm{n}\mathrm{m}=\frac{(\mathrm{O}\mathrm{D}\mathrm{o}\mathrm{f}\mathrm{s}\mathrm{a}\mathrm{m}\mathrm{p}\mathrm{l}\mathrm{e}-\mathrm{O}\mathrm{D}\mathrm{o}\mathrm{f}-\mathrm{v}\mathrm{e}\mathrm{c}\mathrm{o}\mathrm{n}\mathrm{t}\mathrm{r}\mathrm{o}\mathrm{l})}{(\mathrm{O}\mathrm{D}\mathrm{o}\mathrm{f}+\mathrm{v}\mathrm{e}\mathrm{c}\mathrm{o}\mathrm{n}\mathrm{t}\mathrm{r}\mathrm{o}\mathrm{l}-\mathrm{O}\mathrm{D}\mathrm{o}\mathrm{f}-\mathrm{v}\mathrm{e}\mathrm{c}\mathrm{o}\mathrm{n}\mathrm{t}\mathrm{r}\mathrm{o}\mathrm{l})}\times 100$$

4.8. Evaluation of MBEC of Compounds 4k, 4l, and 4n

To determine the minimum concentration of compounds (4k, 4l, and 4n) to eradicate the biofilm, an MBEC experiment was performed. In brief, S. aureus and P. aeruginosa biofilms were grown for 24 h. Biofilms were treated with 100 μL of different concentrations of compounds (100–5 μg/mL) in triplicate. After 24 h, the MBEC was determine by using the following formula

$$\mathrm{E}\mathrm{r}\mathrm{a}\mathrm{d}\mathrm{i}\mathrm{c}\mathrm{a}\mathrm{t}\mathrm{i}\mathrm{o}\mathrm{n}\mathrm{o}\mathrm{f}\mathrm{b}\mathrm{i}\mathrm{o}\mathrm{f}\mathrm{i}\mathrm{l}\mathrm{m}(\%)=\frac{ODincontrol-ODintreatment}{ODincontrol}$$

4.9. Quantitative Analysis of S. aureus and P. aeruginosa Biofilm Disruption after Exposure to NPs

The quantitiative analysis of bacterial biofilm disruption study was done by crystal violet assay. For this study, a 96-well plate containing 200 μL of bacterial culture containing approximately 10⁸ CFU was cultured for 48 h to develop a mature biofilm. After 48 h, the biofilm was treated with four different nanoparticle formulations to evaluate their ability to disrupt S. aureus and P. aeruginosa biofilms, i.e., 4k-loaded nanoparticles (4k-NPs), 4l-loaded nanoparticles (4l-NPs), and 4n-loaded nanoparticles (4n-NPs). Antibiotic rifampicin was used as the positive control while DMSO-treated wells were taken as the negative control The treatment was given in 2 doses, 5 μg/mL and 15 μg/mL. After treatment, the media was discarded, the wells were washed with 1X PBS, and then 70% isopropyl alcohol was used to fix the biofilm. After fixing, the biofilm was stained for 15 min with 0.1% Crystal Violet, then 30% glacial acetic acid was added, and the absorbance was taken as 595 nm by a multiplate reader.

4.10. SEM of the Biofilm after Treatment with NP Formulations

The qualitative analysis of bacterial biofilm disruption by NP formulations was done by scaning electron microscopy (SEM) study. It was performed in a 24-well microtiter plate and on a 12 mm coverslip. The 48 h-grown S. aureus biofilm was treated with NP formulations (15 μg/mL) for 24 h. After treatment, the biofilm was fixed with 2.5% glutaraldehyde in 50 mM cacodylate buffer for 15 min at room temperature followed by keeping overnight at 4 °C. Next day, after fixing, dehydration was done by the series of ethanol series (30%, 50%, 70%, 90%, and 100%) for 10 min each followed by drying of coverslips (NOVA NANOSEM 450, Thermo Fisher, The Netherlands).

4.11. Molecular Docking

To determine the interaction between different proteins and potential compounds, molecular docking has been performed through Autodock-vina. The target for the docking study was chosen based on different proteins associated with or involved in the biofilm formation of S. aureus. In total, eight proteins from four different protein classes, i.e., (i) cell adhesive proteins, (ii) cell wall proteins, (iii) clumping factor protein, and (iv) QS proteins were considered as potential therapeutic targets against the considered molecules based on the screening results of the compound (Figure ). Experimentally predicted structures of all eight proteins were retrieved from the PDB database in pdb format and later converted into the pdbqt format, using Autodock vina Tool, for molecular docking. 3D structures of all compounds were converted into the pdbqt format and docked against each target protein. Details of the protein classes, proteins and their 3D structure ID (PDB ID), and comprehensive protein structure information are provided in the Supporting Information in tables S1 and S2 (page S34). A blind docking study was performed with the chosen protein with 4l, 4n, 4k, and 4b compounds.

4.12. Statistical Analysis

The data were recorded in triplicate and expressed as mean ± standard deviation The value of p < 0.05 was regarded as statistically significant and marked as * data presented in the manuscript. Data analysis was performed in MS office excel program by t-test analysis (two samples assuming equal variances) with a preferred alpha range of 0.05.

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acsomega.5c05577.

• General experimental details, characterization data (¹H NMR and ¹³C NMR, NOESY), and computational studies and molecular docking results of all compounds (PDF)

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Department of Paraclinical Studies, School of Veterinary Medicine, University of Zambia, P O Box 32379, Lusaka, Zambia

The authors declare no competing financial interest.